Synthesis, Characterization, and Antimicrobial Activity of
Water-soluble, Tri-carboxylato Amphiphiles
Eko Winny Sugandhi
Dissertation submitted to the Faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
Chemistry
Richard D. Gandour, Chair Paul R. Carlier
Brian E. Hanson David G. I. Kingston
James M. Tanko
January 31, 2007 Blacksburg, Virginia
Keywords: multi-tailed, multi-headed, tri-carboxylato, dendritic, amphiphile, solubility, microorganism, biological and intrinsic activity
Copyright 2007, Eko W. Sugandhi
Synthesis, Characterization, and Antimicrobial Activity of Water-soluble, Tri-carboxylato Amphiphiles
Eko Winny Sugandhi
ABSTRACT
Many previous studies of biological activity in a homologous series of
amphiphiles have shown a cut-off effect, where the biological activity increases with an
increase in chain length, after which the activity plateaus or weakens. One factor
suspected to cause this problem is solubility issues. We have designed several series of
very hydrophobic, water-soluble amphiphiles to overcome this problem. Three
homologous series containing mobile hydrophobic moieties and two series of epimers
containing rigid cholestane moieties have been synthesized; the hydrophobic moiety is
connected to the first-generation, Newkome-type dendron via a ureido linker. We have
demonstrated that as tris(triethanolammonium) salts, these amphiphiles show excellent
solubility in water. The solubilities in aqueous solution of the three series containing
mobile hydrophobic moieties are 19,500 to 25,700 µM depending on the formula weight
of the homolog, while those containing rigid cholestane moieties are 18,900 and 17,400
μM.
Having eliminated the solubility issue, the antimicrobial activity against a broad
spectrum of microorganisms has been screened. We have demonstrated that the
antimicrobial activity depends on the amphiphile-series, species, chain-length, or epimer
specificities, as well as hydrophobicity. The one-tailed, tri-carboxylato amphiphiles are
iii
generally better than the other series, with two exceptions. First, the two-tailed tri-
carboxylato amphiphiles, 3CUr1(11)2 and 3CUr1(12)2, are more active against
Cryptococcus neoformans; in fact, both amphiphiles (MICs are 6.9 and 7.2 μM,
respectively) are considered to display good antifungal activity. Second, amphiphile
3CUr-β-cholestane, whose MIC is 27 μM, is more active against Staphylococcus
aureus. Overall, these new tri-carboxylato amphiphiles only exhibit moderate activity
with two promising leads.
Furthermore, we have demonstrated the intrinsic activity (MIC0) of the one-tailed,
tri-carboxylato amphiphile series (3CUrn) against Mycobacterium smegmatis. All the
MIC0‘s observed are at least 8-fold lower than the corresponding CMCs. Amphiphile
3CUr16 is the most active; the MIC0 is 100-fold smaller than the CMC. With this
consideration, we have suggested that the mechanism of action of the antimycobacterial
activity in amphiphile 3CUr16 is not related to detergency.
iv
ACKNOWLEDGMENTS
I would like to express my deepest appreciation to my research advisor, Dr.
Richard D. Gandour for his guidance, ideas, encouragement, and continual patience
throughout the duration of my graduate years. His method of gentle guidance made this
very difficult time in my life a little less stressful. I also acknowledge my committee
members⎯Dr. Paul Carlier, Dr. Brian Hanson, Dr. David Kingston, and Dr. James
Tanko⎯for their advice and assistance.
I thank Dr. Joseph Falkinham, III, and Ms. Myra Williams for their generous
assistance in doing biological assays. Beginning with no experience in handling
microorganisms, I learned a great deal in preparing the media, growing the culture, safe
handling, storing, running the assays, and finally interpreting the results.
I thank Mr. Tom Glass and Mr. William Bebout for their help and support in the
analytical services, and Mrs. Carla Slebodnick for her help in the X-ray crystallography
services. Many thanks go to Mrs. Kay Castagnoli and Mrs. Janice McGinty for their
special friendship. Special thanks also to Dr. Yang Yanyan, and current Gandour group
members, especially André William, Richard Macri, Brett Kite, Shauntrece Hardrict, for
making my stay a most enjoyable one.
Finally I would like to thank my husband Ernest for his unconditional love and
sacrifices in order to make this work a reality. Also, my parents and the rest of my
family, who are always loving and supporting me in their characteristic style, deserve
more gratitude than I can ever express.
v
DEDICATION
To my parents,
my husband Ernest, and my little Stanley.
vi
Table of Contents
List of Figures……………………………………………………………………………ix List of Schemes…………………………………………………………………………..xi List of Tables…………………………………………………………………………….xii Chapter I Background of Amphiphiles and Their Antimicrobial Activity .................... 1
I.1 Introduction to Amphiphiles ................................................................................ 1 I.2 Designing Amphiphiles as Antimicrobial Agents ............................................... 2
I.2.1 Detergency ..................................................................................................... 3 I.2.2 Hydrophilic Moiety of an Amphiphile .......................................................... 4 I.2.3 Hydrophobic Moiety of an Amphiphile......................................................... 5 I.2.4 Amphiphiles of Interest.................................................................................. 6
I.3 Antimicrobial Properties of Amphiphiles............................................................ 7 I.3.1 Overview on Previous Studies in Gandour’s Laboratory .............................. 7 I.3.2 The Cut-off Effect........................................................................................ 10
I.4 Literature Review in Designing Amphiphiles of Interest .................................. 11 I.4.1 Multi-headed Amphiphiles .......................................................................... 12 I.4.2 Newkome Dendrons..................................................................................... 13 I.4.3 Ureido Linker............................................................................................... 15 I.4.4 One-tailed Tri-carboxylato Amphiphile ...................................................... 18 I.4.5 Two-tailed Tri-carboxylato Amphiphile...................................................... 19
I.5 Cholestane-based Polyanionic Compounds Derived from Natural Product...... 22 I.6 Tri-carboxylato Amphiphile Containing Rigid Cholestane-based Hydrophobic
Moiety ................................................................................................................ 26 I.7 Microorganisms in Antimicrobial Screening..................................................... 26
I.7.1 Bacteria ........................................................................................................ 27 I.7.2 Fungi ............................................................................................................ 28
I.8 Summary ............................................................................................................ 29 References for Chapter 1 .................................................................................................. 29 Chapter II Synthesis of One-Tailed, Tri-headed Amphiphiles (3CUrn) ...................... 39
II.1 Preparation of WeisocyanateTM (3).................................................................... 40 II.2 Preparation of Alkan-1-amines .......................................................................... 41
II.2.1 Literature Review on Syntheses of Long-chain Alkan-1-amines (4) .......... 41 II.2.2 Literature Review on Syntheses of 1-Azidoalkanes (8) .............................. 43 II.2.3 Literature Review on Syntheses of 1-Methanesulfonyloxyalkanes (9) ....... 46 II.2.4 Literature Review on Syntheses of 1-Bromoalkanes (11) ........................... 46 II.2.5 Preferred Starting Material⎯Alkan-1-ols (12) or 1-Bromoalkanes (11) .... 47 II.2.6 Synthesis of Long-chain Alkan-1-amines (4) .............................................. 47
II.3 Synthesis of the 3EUrn Series (2a−e) ............................................................... 50 II.4 Synthesis of the 3CUrn Series (1a−e)............................................................... 51 II.5 X-ray Crystal Structure of Tri-tert-butyl Ester 3EUr16.................................... 51 II.6 CMCs (Critical Micelle Concentration) of Amphiphiles 3CUrn...................... 53 II.7 Experimental Procedures ................................................................................... 55
References for Chapter II.................................................................................................. 65 Chapter III Synthesis of Two-Tailed, Tri-headed Amphiphiles (3CUr(n)2 and
3CUr1(n)2) .................................................................................................. 73
vii
III.1 The 3CUr(n)2 Homologous Series .................................................................... 74 III.1.1 Preparation of Secondary Amines ............................................................... 75
III.1.1.1 Literature Review on Syntheses of N-Alkylalkan-1-amines (4a, 4c, and 4e) ....................................................................................................... 75
III.1.1.2 Literature Review on Syntheses of N-Alkylalkanamides (5) ............. 77 III.1.2 Synthesis of N-Alkylalkan-1-amines (4a, 4c, and 4e) ................................. 78 III.1.3 Synthesis of Two-tailed, Tri-headed 3CUr(n) 2 Homologous Series.......... 79
III.2 The 3CUr1(n)2 Homologous Series .................................................................. 81 III.2.1 Preparation of Primary Amines (10a−f) ...................................................... 82
III.2.1.1 Literature Review on Syntheses of 1-Alkylalkan-1-amines (10a−f) .. 82 III.2.1.2 Literature Review on Syntheses of Azidoalkanes .............................. 85 III.2.1.3 Literature Review on Syntheses of Methanesulfonyloxyalkanes ....... 86 III.2.1.4 Literature Review on Syntheses of Secondary Alkanols.................... 87
III.2.2 Synthesis of Amines (10a−f) ....................................................................... 89 III.2.3 Synthesis of Two-tailed, Tri-headed 3CUr1(n)2 Homologous Series......... 91
III.3 Comparison of Trends in Melting Temperatures (Tri-tert-butyl Esters vs Tri-carboxylic Acids) ............................................................................................... 93
III.4 Comparison of NMR Spectra of 3EUr(n)2 vs 3EUr1(n)2................................. 94 III.5 Experimental Procedures ................................................................................... 97
References for Chapter III .............................................................................................. 115 Chapter IV Synthesis of Tri-headed Amphiphiles Containing Cholestane-based
Structure on the Hydrophobic Moiety ....................................................... 122 IV.1 The 3CUr-z-cholestane Series........................................................................ 123
IV.1.1 Preparation of 5α-Cholestan-3-amine (4a−b) ........................................... 125 IV.1.1.1 Literature Review on Syntheses of Amines (4a−b).......................... 125 IV.1.1.2 Literature Review on Syntheses of 3-Azidocholestane (5a−b) ........ 127 IV.1.1.3 Literature Review on Syntheses of 3-Methanesulfonyloxy-5α-
cholestane (6a–b).............................................................................. 129 IV.1.1.4 Literature Review on Synthesis of 3α-Bromo-5α-cholestane (7) .... 129
IV.1.2 Synthesis of 5α-Cholestan-3-amines (4a−b) ............................................. 131 IV.1.3 Synthesis of 3CUr-z-cholestane (1a−b) ................................................... 133 IV.1.4 X-ray Structure of 3EUr-α-cholestane (2a) ............................................. 134
IV.2 The 3CUrnEs-z-cholestane Homologous Series............................................ 137 IV.2.1 Preparation of 3α- and 3β- Epimer of 5α-Cholestan-3-yl Aminoethanoate
(11a−b) ...................................................................................................... 138 IV.2.1.1 Literature Review on the Synthesis of 5α-Cholestan-3α-yl
Aminoethanoates (11a−b) ................................................................ 139 IV.2.1.2 Literature Review on the Synthesis of 5α-Cholestan-3α-yl
Azidoethanoate (12a−b) ................................................................... 140 IV.2.1.3 Literature Review on the Synthesis of 5α-Cholestan-3α-yl
Chloroethanoate (13a−b) .................................................................. 140 IV.2.2 Synthesis of 5α-Cholestan-3α-yl Aminoethanoate (11a−b) ..................... 141 IV.2.3 Synthesis of 3CUr1Es-z-cholestane (9a−b)............................................... 143
IV.3 Experimental Procedures ................................................................................. 144 References for Chapter IV .............................................................................................. 162
viii
Chapter V Antimicrobial Activity of Various Tri-headed Amphiphiles..................... 168 V.1 Introduction to Biological Assays.................................................................... 168
V.1.1 Measurement of Antimicrobial Activity.................................................... 168 V.1.2 Goals .......................................................................................................... 169
V.2 Recent Studies of Solubility of One-tailed Tri-carboxylato Amphiphiles....... 170 V.3 Results of Biological Assays ........................................................................... 171
V.3.1 Range of Concentration of Active Amphiphiles........................................ 174 V.3.2 The Most Active Amphiphiles within Each Homologous Series .............. 175 V.3.3 Various Specificities Patterns of Antimicrobial Activity .......................... 177
V.3.3.1 One- and Two-tailed Amphiphiles................................................... 177 V.3.3.2 Cholestane-based Amphiphiles........................................................ 179
V.3.4 Comparing Hydrophobicity and Antimicrobial Activity........................... 180 V.3.4.1 One- and Two-tailed Amphiphiles.................................................... 180 V.3.4.2 Cholestane-based Amphiphiles.......................................................... 182
V.4 Comparison with Prior Work........................................................................... 182 V.5 Conclusions...................................................................................................... 184 V.6 Experimental Procedures ................................................................................. 185
V.6.1 Preparation of Tri-carboxylato Amphiphiles Solutions in Aqueous Triethanolamine ......................................................................................... 185
V.6.2 Microbial Strains, Culture Conditions, and Preparations of Inocula for Susceptibility Testing................................................................................. 185
V.6.3 Measurement of MICs ............................................................................... 187 References for Chapter V................................................................................................ 188 Chapter VI Intrinsic Activity of the 3CUrn Homologus Series Against Mycobacterium
smegmatis................................................................................................... 192 VI.1 The “inoculum effect”...................................................................................... 194 VI.2 Intrinsic Activity of Homologous Amphiphiles .............................................. 194 VI.3 Results and Discussions................................................................................... 197
VI.3.1 MICs of the 3CUrn Series......................................................................... 197 VI.3.2 Comparison with Previous Work............................................................... 199 VI.3.3 Comparison of MICs and CMCs ............................................................... 199
VI.4 Conclusions...................................................................................................... 201 VI.5 Experimental Procedures ................................................................................. 201
VI.5.1 Microbial Strain, Culture Conditions, and Preparations of Inoculum for Susceptibility Testing................................................................................. 201
VI.5.2 Measurement of MICs ............................................................................... 202 References for Chapter VI .............................................................................................. 203 Chapter VII Accomplishments, Conclusions, and Future Work.................................... 205
VII.1 Accomplishments............................................................................................. 205 VII.2 Conclusions...................................................................................................... 206 VII.3 Future Work ..................................................................................................... 207
References for Chapter VII............................................................................................. 208 APPENDICES ................................................................................................................ 209 VITA............................................................................................................................... 354
ix
List of Figures Figure I.1 Common representations of an individual amphiphile ............................... 1 Figure I.2 Representation of amphiphile aggregates ................................................... 2 Figure I.3 Mono-, di-, and tri-ionic amphiphiles ......................................................... 5 Figure I.4 Schematic representations of proposed multi-headed amphiphiles ............ 7 Figure I.5 Plots of log MEC (M) vs chain length in spermicidal (left) and log MIC
(M) vs chain length in antifungal (right) assays of Z-n.............................. 8 Figure I.6 Plots of log MEC vs chain length in spermicidal assays of 1(p) and 2(p) 10 Figure I.7 Various multi-headed amphiphiles with anti-HIV properties................... 12 Figure I.8 Various Newkome-type dendrons (first and second generations) associated
with ferrocene, dansyl, pyrene, and viologen ........................................... 14 Figure I.9 Structures of one-tailed tri-carboxylato amphiphiles having amido and
carbamato linkers ...................................................................................... 15 Figure I.10 Various compounds containing ureido functional groups ........................ 17 Figure I.11 General representation and structure of one-tailed tri-carboxylato
amphiphiles ............................................................................................... 18 Figure I.12 Various fatty acids tested for skin penetration rates and skin irritation.... 21 Figure I.13 General representations of two-tailed, tri-headed amphiphile series........ 22 Figure I.14 Polyol and polyamine dendrimers associated with cholestane moieties .. 24 Figure I.15 General structures of cholestane-based, tri-headed amphiphile series ..... 26 Figure II.1 X-ray crystal structure of tri-tert-butyl ester 3EUr16, showing the packing
diagram and the intermolecular hydrogen bonding to water and to a neighboring molecule. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(8)···N(2), 2.863(3) Å and 159.1°; O(15)···O(1), 2.718(4) Å and 170(4)°; O(15)···O(3), 2.843(4) Å and 169(4)°; N(4)···O(15), 2.832(3) Å and 153.4°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(1)···O(8), 3.201(3) Å and 145.6°; N(3)···O(15), 3.074(3) Å and 141.6°................................................................................................................... 53
Figure III.1 General representation of two-tailed, tri-headed amphiphiles.................. 73 Figure IV.1 Structural representations of 5α- and 5β-cholestane .............................. 122 Figure IV.2 General structures of tri-headed amphiphiles series containing cholestane
moiety ..................................................................................................... 123 Figure IV.3 X-ray crystal structure of 3EUr-α-cholestane (2a), showing the packing
and the intermolecular hydrogen bonding to ethanol. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(16)···O(1), 2.621(7) Å and 158.1°; O(15)···O(8), 2.625(6) Å and 161.0°; O(4)···O(16), 2.865(7) Å and 155(7)°; N(2)···O(15), 2.849(8) Å and 159.6°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(3)···O(16), 3.035(7) Å and 141.6°; N(1)···O(15), 3.084(8) Å and 146.0°............................................................................. 135
Figure V.1 Relationship between log MIC of the 3CUrn series vs chain length for E. coli, M. smegmatis (left) and C. albicans, C. neoformans, and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides............................................................................... 175
x
Figure V.2 Relationship between log MIC of the 3CUr(n)2 and 3CUr1(n)2 series vs chain length for C. neoformans (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides................................................................................................................. 176
Figure V.3 Relationship between log MIC of one- and two-tailed amphiphiles vs logD for M. smegmatis (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3...................................................................................... 181
Figure V.4 Relationship between log MIC and logD for C. neoformans (left) and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3............... 182
Figure VI.1 Effect of initial cell density on inhibition of growth of M. smegmatis for the 3CUrn series. Combined data of two separate assays⎯3.2 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108 CFU/mL. Error bars (not shown for clarity) are ± 0.3................................................................................................... 197
Figure VI.2 Effect of chain length on inhibition of growth of M. smegmatis of the 3CUrn series as function of the initial cell density. Error bars (not shown for clarity) are ± 0.3................................................................................. 198
Figure VI.3 Comparison of log CMC and log MIC of the 3CUrn series at the highest and lowest initial cell densities of M. smegmatis.................................... 200
xi
List of Schemes Scheme I.1 Synthesis of isocyanate monomer of Newkome-type dendron................. 18 Scheme II.1 Retrosynthesis of one-tailed, tri-headed amphiphiles (3CUrn) ............... 39 Scheme II.2 Synthetic scheme of the synthesis of isocyanate 3 ................................... 40 Scheme II.3 Retrosynthesis of alkan-1-amines ............................................................. 41 Scheme II.4 Synthetic scheme of amines 4d and 4e ..................................................... 49 Scheme II.5 The synthetic scheme of one-tailed, tri-headed amphiphiles (3CUrn) .... 50 Scheme III.1 Retrosynthesis of the 3CUr(n)2 homologous series ................................. 74 Scheme III.2 Preparation of N-heptylheptan-1-amines from heptan-1-amine3 and N-
heptylheptanamide14 ................................................................................. 76 Scheme III.3 Synthetic scheme of N-alkylalkan-1-amines 4a, 4c, and 4e ..................... 79 Scheme III.4 Synthetic scheme of the 3CUr(n)2 homologous series............................. 80 Scheme III.5 Retrosynthesis of 3CUr1(n)2 series.......................................................... 82 Scheme III.6 Retrosynthesis of amines 10a−f................................................................ 84 Scheme III.7 Synthetic scheme of the alkanamines 10a−f............................................. 90 Scheme III.8 Synthetic scheme of the preparation of 3CUr1(n)2 .................................. 92 Scheme IV.1 Retrosynthesis of 3CUr-z-cholestane .................................................... 124 Scheme IV.2 Retrosynthesis of epimer 5α-cholestan-3-amines................................... 125 Scheme IV.3 Synthetic scheme of the preparation of epimeric amines (4a−b) ........... 133 Scheme IV.4 Retrosynthesis of the 3CUrnEs-z-cholestane homologous series......... 138 Scheme IV.5 Retrosynthesis of 3α- and 3β- epimer of 5α-cholestan-3-yl
aminoethanoate ....................................................................................... 139 Scheme IV.6 Synthetic scheme of the preparation of amines 11a−b ........................... 143 Scheme V.1 Structures of dendritic tri-carboxylato amphiphiles ............................... 172 Scheme VI.1 Simplified kinetic model of inhibition of cell growth ............................ 195
xii
List of Tables Table I.1 Physical differences between Gram-negative and Gram-positive bacteria
................................................................................................................... 27 Table II.1 Preparations of amines 4 from different precursors.................................. 43 Table II.2 Preparations of azides 8 from different precursors ................................... 45 Table II.3 Preparation of bromoalkanes 11 from alcohols 12 ................................... 47 Table II.4 Isolated yields and melting ranges of the tri-tert-butyl esters 3EUrn ...... 50 Table II.5 Isolated yields and melting ranges of the triacids 3CUrn ........................ 51 Table II.6 CMCs of the 3CUrn homologous series .................................................. 54 Table II.7 Crystal data and structure refinement of 3EUr16..................................... 61 Table III.1 Melting ranges of the 3EUr(n)2 and 3CUr(n)2 homologous series ......... 81 Table III.2 Preparations of 1-alkylalkan-1-amines from different precursors ............ 83 Table III.3 Preparation of secondary azidoalkanes from secondary
methanesulfonyloxyalkanes...................................................................... 86 Table III.4 Preparation of secondary methanesulfonyloxyalkanes from secondary
alcohols ..................................................................................................... 87 Table III.5 Preparations of secondary alcohols 13a−f from different precursors....... 88 Table III.6 Isolated yields and melting ranges of intermediates and alkanamines 10a−f
................................................................................................................... 90 Table III.7 Melting ranges of the 3EUr1(n)2 and 3CUr1(n)2 .................................... 93 Table III.8 Comparison of trends in melting temperatures (tri-tert-butyl esters vs tri-
carboxylic acids) ....................................................................................... 93 Table IV.1 Preparation of amines 4a−b from different precursors........................... 126 Table IV.2 Preparation of azides 5a and 5b from various precursors....................... 128 Table IV.3 Preparations of 6a−b from 8a−b............................................................. 129 Table IV.4 Preparations of bromoalkane 7 from 8b and 8b’s derivatives................ 130 Table IV.5 Preparations of 5α-cholestan-3α-yl chloroethanoate from 5α-cholestan-
3β-ol (13a−b).......................................................................................... 141 Table IV.6 Crystal data and structure refinement for 3EUr-α-cholestane (2a)....... 152 Table V.1 MICs of tri-carboxylato amphiphiles against bacteria, yeasts, and a
filamentous fungus.................................................................................. 173
1
Chapter I Background of Amphiphiles and Their Antimicrobial Activity
I.1 Introduction to Amphiphiles
An amphiphile contains two distinct parts, which are in an opposite nature.1 The
word is translated from the Greek, amphi means ‘on both sides’ and phileein means ‘to
love’. Consequently, amphiphiles preferentially absorb at an interface of two immiscible
phases.
Depending on the solvent system used, the two opposite parts of an amphiphile
are referred to as solvent-loving (solvophilic) and solvent-hating (solvophobic). In
aqueous solutions, these two parts are known as hydrophilic (water-loving) and
hydrophobic (water-hating) moieties, respectively; the character of the former is polar,
which generally is depicted as a circle, and that of the latter is nonpolar, which generally
is depicted as a straight or wavy line(s) (Figure I.1).1
Figure I.1 Common representations of an individual amphiphile
Because of this unique property of absoption in the interface of two immiscible
phases, amphiphiles will distribute themselves so that the nonpolar moiety associates
with the nonpolar phase, and vice versa. When surrounded in one phase, for example in
an aqueous environment, amphiphiles tend to form the ‘other phase’ by assemblying their
nonpolar moieties. In other words, the hydrophobic tails cluster together to create a
nonpolar environment, while the polar moieties are still interacting with the polar
environment. At a given concentration and above, the amphiphiles associate with
a b
a. polar hydrophilic head; b. nonpolar hydrophobic tail
a b
2
themselves to form aggregates (Figure I.2); these aggregates are named based on the
shape of the aggregates form. One-tailed amphiphiles tend to form micelles, while two-
tailed amphiphiles tend to form bilayers.1 Higher concentrations of amphiphiles
eventually cause aggregation (crystalization or precipitation) where amphiphiles are no
longer soluble. The concentration at which micellar aggregates start to form is defined as
the critical micelle concentration (CMC).
micelle bilayer Figure I.2 Representation of amphiphile aggregates
Variation in temperatures affects the aggregation of an amphiphile. When a
solution of amphiphiles is cooled, the amphiphiles reach the point where they are not
soluble; this temperature is known as the Kraft temperature. Above the Kraft
temperature, the solubility of the amphiphile monomers increases. When the amphiphile
solution is heated up, it eventually reaches to a point where aggregation begins.1,2
I.2 Designing Amphiphiles as Antimicrobial Agents
Amphiphiles have been used as antimicrobial agents.3 Some advantages of using
amphiphiles as microbicides have been presented;4 they are inexpensive, fast-acting, and
have a broad spectrum of biological activity. However, there are also some drawbacks,
especially with those that can function as detergents.
3
I.2.1 Detergency
Often, the word amphiphile and surfactant are used synonymously. The word
surfactant itself comes from the phrase SURFace ACTive AgeNT, whose main traditional
application is as a soap and a detergent in various cleaning processes. The word
detergent is generally reserved for cleaning products containing surfactants and other
materials; these other materials function by improving the detergency or by adding
appealing properties, such as color (dye) or aroma (perfume). As cleaning agents,
surfactants remove the unwanted material, such as oils, greases, along with dust and clay,
from the surface of a solid. Based on the various physicochemical and mechanical
methods, this process is known as detergency.1 Detergency involves the micellar solution
of a surfactant, where the unwanted nonpolar materials interact with the hydrocarbon
core of the micelles. This interaction basically causes the unwanted material to be
solubilized within the core of the micelles. As micellar formation is needed, good
detergents must contain surfactants that have a low CMC. In other words, detergency
occurs at concentrations close to the CMC.1,5
In a recent study,6 detergency is claimed to cause irritation, which is a
disadvantage in any topical antimicrobial agent. As detergency occurs at concentrations
close to the CMC,5 controlling the CMC is necessary. Recently, it has been pointed out
that the active form of a good non-irritating amphiphile as an antimicrobial agent should
be in the monomeric form, not the micellar form.4 Consequently, it becomes our goal to
design amphiphiles that are not detergents, but antimicrobial agents. This means that the
amphiphiles of our interest should have high CMCs.
4
I.2.2 Hydrophilic Moiety of an Amphiphile
A hydrophilic moiety of a amphiphile is usually called the headgroup, and it is
either polar or charged. Depending on the type of their headgroup, amphiphiles are
usually classified as anionic, cationic, zwitterionic, and nonionic. The largest type of
amphiphiles in general use today falls in the anionic class, which constitutes 70−75% of
total amphiphile consumption.2 Anionic amphiphiles are mostly found in soaps,
detergents, toiletries, while cationic amphiphiles are often found in bactericides and hair
conditioners. Zwitterionic amphiphiles, which are also referred as amphoteric
amphiphiles, are generally milder on the skin than anionic or cationic; their main
applications are in shampoo and cosmetics. Nonionic amphiphiles are often found as
foam enhancers, detergents, and emulsifiers.1
The solubility and CMC of an amphiphile are affected by the type and number of
headgroups. In aqueous solutions, ionic amphiphiles are more soluble than the
corresponding nonionic amphiphiles. In studies of the effect of multi-headed ionic
amphiphiles on the CMCs, cationic7-9 and anionic10,11 amphiphiles, where chain length
are maintained equal, have been utilized. In these studies, the CMCs of mono-, di-, and
tri-ionic amphiphiles are measured (Figure I.3); compared to their mono-ionic
counterparts (1, 4), di- and tri-ionic amphiphiles (2, 3, 5, 6) display higher CMCs. In
other words, an increase in number of headgroups causes an increase in the CMCs. It is
suggested that higher CMCs are achieved because there are more dissociable group.7-9
5
ON
O
Br
O
ON
N
O
O
Br
Br
O
O N
O O
N
Br
Br
Br
1313
13
O N
O
O
O Kn
O
O Kn
O O K
O
O
K
O O K
O OK
nn = 7, 9, 11, 13, 15
1
2
3
4 5 6
Figure I.3 Mono-, di-, and tri-ionic amphiphiles
I.2.3 Hydrophobic Moiety of an Amphiphile
A hydrophobic moiety of an amphiphile is typically called the tail. This tail is
most commonly represented by a linear hydrocarbon. The chain length of the tail affects
the solubility and the CMC of an amphiphile. In the one-tailed amphiphile, an increase in
chain length causes a linear decrease in the log CMC, following the so-called Klevens
equation.2,12 In this equation, the relationship of log CMC vs the number of carbons in
the chain (nc), is stated as log10 CMC = A−Bnc. In this mathematical equation, the
intercept A and slope B are constants specific to the homologous series under constant
temperature and pressure.2 The intercept A depends on the ionic strength and type of
headgroups, while the slope B depends on the number of hydrophilic headgroups and the
number of hydrophobic alkyl chains.
The linear relationship of log CMC vs chain length has been observed
6
previously.13-15 A slope of –0.3 is observed with potassium salts of natural saturated fatty
acids.11 The CMCs of triethanolammonium salts of natural saturated fatty acids show a
dependence on chain length;16 the slopes have values of –0.06 for octanoate to
tetradecanoate and –0.6 for tetradecanoate to octadecanoate. Generally, one-headed
amphiphiles have slopes ranging from –0.26 to –0.33 for anionic and cationic
amphiphiles, and –0.45 to –0.52 for nonionic and zwitterionic amphiphiles.17
In different studies,10,11 log CMC of homologous series with different number of
headgroups are investigated. In these studies, the log CMC also decreases linearly as the
chain length increases in every homologous series. Among the different homologous
series compared, the slope of the log CMC vs chain length also changes with the different
number of headgroup attached.10 However, only slight changes in slope are observed in
the di- and tri-headed amphiphiles; diheaded amphiphiles RCH(COOK)2 and tri-headed
amphiphiles RCH(COOK)CH(COOK)2 have slopes of –0.22 to –0.23, respectively.10,11
I.2.4 Amphiphiles of Interest
We have discussed that the ideal active form of amphiphiles as antimicrobial
agents is the monomeric form; thus, the amphiphiles should have high CMCs.
Furthermore, CMCs have been shown to be affected by the hydrophilic and hydrophobic
moieties. Based on the previous discussion, we have designed multi-headed amphiphiles.
In addition, another modification made is to add a linker between the hydrophilic and
hydrophobic moieties.
In order to enrich the multi-headed amphiphiles series, we plan on attaching
different hydrophobic moieties (Figure I.4). Two natural sources of hydrophobic
moieties, such as one-tail hydrocarbons (a) and a steroidal backbone (d, e), will be
7
employed; a spacer will be introduced between the linker and the steroidal moiety. As
for the unnatural source of hydrophobic moiety, two symmetrical hydrocarbon chains
will be used in the design (b, c); furthermore, modification will be made on the chain
topology.
linker
head
head
head
tail
linker
head
head
head
tail
tail
linker
head
head
head
tail
tail
steroidalmoiety
spacerlinker
head
head
head
steroidalmoiety linker
head
head
head
a
b c
d e
Figure I.4 Schematic representations of proposed multi-headed amphiphiles
I.3 Antimicrobial Properties of Amphiphiles I.3.1 Overview on Previous Studies in Gandour’s Laboratory
A previous study18 in Gandour’s laboratory have shown that hydrocarbon chain
length directly affects biological activity. Homologous long-chain zwitterionic
amphipiles that are derived from acylcarnitine have been synthesized; these Z-n
amphiphiles display spermicidal and antifungal activity.18
8
N
OCO2
ROH
Z-nR = n-CnH2n+1, n = 10−18
NO
O O
acylcarnitine
OR
In the spermicidal assays (Figure I.5, left), the plot of the activity is represented as
log MEC (Minimum Effective Concentration) vs chain length. MEC is defined as the
lowest concentration of the amphiphile required to stop all sperm motility in 20 seconds.
In general, as the chain length increases, the activity of these amphiphiles increases,
represented by lower MECs. However, a breaking point is observed in the amphiphiles
with longer chains; in the spermicidal assays, the activity of Z-14 and Z-15 is similar. In
a recent report,4 MECs of Z-16−Z-18 cannot be determined because of solubility issues.
Figure I.5 Plots of log MEC (M) vs chain length in spermicidal (left) and log MIC (M) vs chain length in antifungal (right) assays of Z-n
In the antifungal assays (Figure I.5, right), the plot of the activity is represented as
log MIC vs chain length, where MIC is defined as the lowest concentration of amphiphile
-4
-3.5
-3
-2.5
-2
9 10 11 12 13 14 15
chain length
log
[MEC
] (M
)
-5.5
-5
-4.5
-4
-3.5
11 12 13 14 15
chain length
log
[MIC
] (M
)
9
required to inhibit the growth of Candida albicans. MICs of Z-10 and Z-11 are greater
than 3 × 10-4 M (data not shown); the exact MICs are not measured.4 Clearly, among Z-
12−Z-15, Z-15 shows the best antifungal activity. Within the plotted data, it is apparent
that as the chain becomes longer, the activity is better. No breaks or plateaus in activity
are observed within the homologs tested. It will be interesting to see how homologs with
longer chain lengths affect the antifungal activity. Unfortunately, under the assays
condition, Z-16−Z-18 are not soluble enough to give reliable MICs.4
In more recent studies,19,20 homologous series of di- and trihydroxylated cationic
amphiphiles, namely 2-hydroxy-N-(2-hydroxyethyl)-N,N,-dimethyl-1-alkaminium
bromide (1(p)) and 2-hydroxy-N,N-bis(2-hydroxyethyl)-N-methyl-1-alkaminium bromide
(2(p)), have been synthesized and investigated for spermicidal activity.
N
HOHO R
N
HOHO R
OHBr Br
1(p) 2(p)
R = n-CnH2n+1, p = n+2
Spermicidal assays of these series show similar behavior (Figure I.6). Initially,
the activity of these amphiphiles increases as the chain length increases. After a certain
point, the activity plateaus or weakens. Comparison of the two longest homologs 1(p)
show that the activity of 1(18) is slightly stronger than that of 1(17); however, both have
very similar activity. On the other homologous series, the longest homolog, 2(18), is
weaker than 2(17). MICs of 1(p) and 2(p), except 2(18), are observed to be lower than
their CMCs.
10
These behaviors observed in both spermicidal assays represent a condition where
a cut-off effect21 is observed. As the chain length becomes longer, solubility of the
amphiphiles may encounter a problem, as it is pointed out later.4 When such a problem
occurs, it can be suggested that the cut-off effect is actually due to the physicochemical
factor, not the biological factor.
-4
-3.5
-3
-2.5
-2
11 12 13 14 15 16 17
chain length
log
[MEC
] (M
)
1(p)2(p)
Figure I.6 Plots of log MEC vs chain length in spermicidal assays of 1(p) and 2(p)
I.3.2 The Cut-off Effect
In a homologous series of compounds, antimicrobial activity often increases up to
an optimal point as chain length increases; after that, the activity decreases;21,22 such a
phenomenon is referred to as the cut-off effect.21 Several factors are suggested to be
responsible for such a phenomenon. These factors include solubility (monomer
concentration), size discrimination, kinetic effect, and free volume.21 In order to
eliminate solubility issues, the amphiphiles must be completely soluble and in the
monomeric form. For this reason, we attempted to design series of homologous water-
11
soluble amphiphiles.
Theoretically, micelles are easier to form as chain length increase. The
concentration of monomer decreases when micelles form. Supposedly, the monomer is
the active form of the amphiphile, a decrease in monomer concentration certainly
weakens the activity of an amphiphile. Size discrimination involves the partitioning of
the compound into the membrane. Increasing the chain length should increase
partitioning into the membrane; however, there are evidences that increasing the chain
length will eventually restrict partitioning into the membrane. Kinetic effect is related to
the rate of an amphiphile partitioning itself into the membrane, where increasing the
chain length reduces the rate. The free volume changes when an amphiphile is inserted
into the membrane. Insertion of an amphiphile into the membrane increases the free
volume. The maximum biological effect is achieved when the total free volume is
maximized without causing the membrane to solubilize.
I.4 Literature Review in Designing Amphiphiles of Interest
In a recent review,4 it is suggested that amphiphiles can inactivate pathogens by
acting as micelles, or monomers via several mechanisms. However, because irritation
results from detergency,6 and detergency itself occurs at the concentrations close to the
CMC,5 amphiphiles as topical microbicides should be in their active form as monomers,
but not micelles. As is mentioned above, one goal in this project is to design amphiphiles
that have excellent activity that is not related to detergency, consequently, amphiphiles of
interest should have high CMCs.
12
I.4.1 Multi-headed Amphiphiles
Multi-headed amphiphiles, such as alkyl malonates11 and alkane-1,1,2-
tricarboxylic acid,10 have been studied. With regards to biological matters,
polycarboxylate amphiphiles (8, 9) anti-HIV activity are synthesized based on
aurintricarboxylic acid (Figure I.7).23,24 Cushman et al.25 have shown how the
carboxylate groups play an important role in inhibiting the attachment of the virus to a
cell. Polymerizable monomers with multiple headgroups (10−14) have also display anti-
N
O OR
O O
ClCl
OHO OHO
8, R = Me, Et
O
OH
OHO OHO
OH
OH O
OH
HO
O
HO
OH
O OH
O9
NH8
OO OH
O
OHn
NH8
OO
O
O
OH
O
OH
O
OH
O NH8
ON
OH
OHN
OH
OO OH
OO OH
O
H
NH8
O OSO3H
OH
O
9
10 11, n = 1, 2 12
13 14
Figure I.7 Various multi-headed amphiphiles with anti-HIV properties
13
HIV activity (Figure I.7).26 In addition to anionic amphiphiles, incorporation of multiple
cationic headgroups in amphiphiles has been shown to lead to impressive antibacterial
activity.27
We have mentioned how multiple headgroups affect solubility; multiple
headgroup increases the solubility. In addition, previous studies (see Section I.3.1) in our
laboratory have shown how insolubility has become a problem in exploring the biological
activity of compounds with very hydrophobic moiety. With all the advantages offered by
having multiple headgroups in the compounds, we have designed and synthesized multi-
headed amphiphiles that can possibly overcome the insolubility issues of longer chains.
This will ultimately give us a chance to explore biological activity of new classes of
amphiphiles with very long chains and very hydrophobic moieties.
I.4.2 Newkome Dendrons
The presence of Newkome-type, 1→3 C-branched dendrons28,29 in amphiphiles
with very hydrophobic groups have been shown to improve amphiphile solubility in
aqueous solutions. As shown by Kaifer and collaborators, Newkome-type dendrons
(first, second, and third generation) that terminate with carboxylate groups impart water
solubility on hydrophobic groups—those containing a ferrocene (15, 16),30 dansyl (17),31
pyrene (18),32 and viologen (19) (Figure I.8).33 In the case of the dansyl group, the first
generation dendron provides the most polar microenvironment.31 Novel of multi-headed,
multi-tailed amphiphiles containing two second-generation Newkome-type dendritic
heads and lipophilic tails attached to calixarene34 and fullerene35 have also been
established. In a study36 of froth flotation, amphiphiles containing first- and second-
14
generation Newkome-type dendrons have been synthesized and shown to improve the
packing density of the adsorption layer on the mineral particle.
Fe
NH
OHO
O
OH
OH
O
HOFe
NH
HN
O
O
NH
O
HO
OH
O
OH
HN
OH
O
3
3
3O
NH
OH
O
OH
OH
O
HO
SO
O
N
NH
OHO
O
OH
OH
O
HO
NH
OHO
O
OH
OH
O
HO
NN
15 16
17 18
19
Figure I.8 Various Newkome-type dendrons (first and second generations) associated with ferrocene, dansyl, pyrene, and viologen
With all the examples above, Newkome-type, 1→3 C-branched dendrons are
potential candidates as hydrophilic moieties in designing water-soluble amphiphiles that
have a very long chain in the hydrophobic moiety. Current group members André
Williams37,38 and Richard Macri37 have made a homologous series of water-soluble
amphiphiles containing the Newkome-type first-generation dendron and linear alkyl
15
chains with twenty or more carbons (Figure I.9)—ultra-long chains,39 which have been
rarely studied in amphiphile chemistry.40-45
NH
O
n-1 NH
O
n-1O
n = 14, 16, 18, 20, 22n = 13−17, 19, 21
O OH
OH
O
OHO
O OH
O
OH
OHO
Figure I.9 Structures of one-tailed tri-carboxylato amphiphiles having amido and carbamato linkers I.4.3 Ureido Linker
The term linker is mostly found in combinatorial chemistry. In combinatorial
chemistry,46 a linker is a bifunctional chemical moiety that functions to attach a
compound to a solid support. This linker is always attached during a multistep synthesis;
in the final step, the linker is cleaved to release the product. The linker utilized in this
project is different from those utilized in combinatorial chemistry. We apply the term
linker as a functional chemical moiety to connect the hydrophobic and hydrophilic
moieties.
Having investigated the chemistry associated with the Newkome-type first-
generation dendron, several homologous series of water-soluble amphiphiles, each of
which containing a particular linker, can be designed. Three different linkers, namely,
amido, carbamato, and ureido, have been employed in our laboratory. One linker
proposed in this project is a ureido (−HNCONH−) linker. The origin of the name is
derived from a urea; compounds containing such a linker are disubstituted ureas.
16
NH
O
NH
O
O NH
O
NH
amido carbamato ureido
A recent review47 points out that ureides, compounds that incorporate urea as a
substructural component, are one among the oldest classes of bioactive compounds. Urea
and its derivatives have displayed antibacterial activity against Gram-negative bacteria,
and to the less extent to Gram-positive bacteria.48 A number of compounds (20−22)
containing a ureido linker, even though they are not amphiphiles, have been shown to
have antifungal and antibacterial activity; most of them contains heterocyclic moieties. A
study49 of a gemini amphiphile (23) containing a ureido linker and two carboxylate
groups has been conducted in the field of cosmetics. In another study,50 mono- and bis-
urea substituted ferrocene receptors, containing a ureido linker and the first-generation
Newkome-type dendron, have been synthesized for an investigation of electrochemical
recognition and sensing of anionic guest (24, 25) (Figure I.10).
17
NH
N N NH
OO O
OOOHHO
OO
1212
NH
NH
OO
O
O
O
O
O
Fe
NH
NH
OO
O
O
O
O
O
Fe
HN
HN
OO
O
O
O
O
O
HN
HN
O
ClClCl
H2N NH
NH
HNO
20 21
23 24
25
NH
NH
O
Cl22
NN
Figure I.10 Various compounds containing ureido functional groups
In a recent study,51 alkylureas, which are monosubstituted ureas, have displayed
unique properties that render them as potential candidates for microbicide formulations.
Furthermore, the author suggests that alkylureas with longer alkyl chains may have
greater selective microbicidal activity.
Having presented all this evidence on how a ureido functional group may give
some advantages in topical microbicides, we were inspired to utilize the ureido linker in
our project.
18
I.4.4 One-tailed Tri-carboxylato Amphiphile
Based on the background in designing multi-headed amphiphiles derived from the
first generation of Newkome-type dendron, a straight hydrocarbon as the hydrophobic
moiety can be attached via a ureido linker (Figure I.11). Formation of the ureido
functional groups have recently been reviewed;52 one commonly applied method is the
reaction of amines and isocyanates. The synthesis of the isocyanates derived from tri-
headed first-generation Newkome-type dendron has been well developed via a three-step
synthesis.29,53 Such a synthesis begins with commercially available nitromethane and
tert-butyl acrylate (Scheme I.1).
linker
head
head
head
tail NH
NH
COOH
COOH
COOH
R
O
R = n-CnH2n+1
Figure I.11 General representation and structure of one-tailed tri-carboxylato amphiphiles
Scheme I.1 Synthesis of isocyanate monomer of Newkome-type dendron
O2NOtBu
O 3
CH3O2N H2NOtBu
O 3
OCNOtBu
O 3
i ii iii
i. tert-butyl acrylate, Triton B, dimethoxyethane; b. Raney Ni, H2, EtOH; c. BOC2(O), DMAP, CH2Cl2
As shown by previous studies,54-59 the isocyanate monomer reacts readily with a
variety of primary amines. Based on this chemistry, a hydrophobic moiety⎯a long chain
hydrocarbon⎯can be attached to the hydrophilic moiety by combining an isocyanate and
long chain alkan-1-amines, which in turn will afford a ureido linker. This strategy will
allow us to develop a rapid synthesis to generate homologous series of a new class of
19
amphiphiles with a common headgroup. Having these multiple headgroups in
amphiphiles with very long hydrocarbon chain should increase the solubility in aqueous
solutions, which in turn allow us to explore the biological activity of the new class of the
amphiphiles.
I.4.5 Two-tailed Tri-carboxylato Amphiphile
As the solubility of an amphiphile in aqueous solutions is often a problem,
extensive attempts have been made to design amphiphiles that are more stable in aqueous
solution. In addition, when amphiphiles are intended as topical microbicides, they must
be non-irritating. The level of irritancy of amphiphiles is affected by the chemical
structure, steric factors, and relative polarity.60 Structural modification, such as
branching, can be used as tool to improve solubilitation and minimize the level of
irritancy.
The concept of branching does not necessarily mean insertions of an alkyl group
to the backbone.61 Rather, branching can be considered as indications of the amphiphile
bulkiness or reduced packing density.62 These include introduction of unsaturation,
addition of alkyl groups, modification on the attachment to the hydrophilic and
hydrophobic moieties, or even substitutions of hydrogen atoms in the alkyl group with
other functional groups.61
As suggested by Wormuth and Zushma,63 branching can be classified as methyl
branching, double-tail branching⎯equal and unequal tails, and multiple branching.
Studies64-68 of double-tail branched amphiphiles indicate when the tails are designed
more equal, Kraft temperature decreases,64 the CMC increases,67 solubilization in
cyclohexane increases,66,68 and the amphiphiles is more effective at reducing the air-water
20
surface tension.65
Langhals et al.69-71 have investigated and proven that solubility of perylene
bisimide dyes is improved by attaching a long chain through the middle carbon. A
compound containing this kind of tail is also referred to as a “swallowtail”.72 In this
study, a compound containing a “swallowtail” improve the solubility and has the ability
to form a Langmuir−Blodgett monolayer.72 Derivatives of dioctadecylamine are
functioning as precursors to liposomes,73 lipid anchors for ion channel mimics,74 artificial
lipid clusters,75 and organic gelators.76 These suggest that compounds with two equal
tails, as well as those with a single tail, are also able to pack themselves or associate with
a bilayer membrane that might cause biological activities.
Aungst77 has demonstrated how the structure of fatty acid isomers⎯branched vs
unbranched saturated fatty acids (26−39) and cis- vs trans- unsaturated fatty acids (40,
41)⎯act as skin penetration enhancher (Figure I.12). No significant difference exists
between cis- vs trans- unsaturated fatty acids in the skin penetration rates; however,
compared to saturated fatty acids, unsaturated fatty acids with the same chain length
increased the skin penetration rates. Neodecanoic acid (35) and elaidic acid (41) showed
less skin irritation compared to lauric acid (37) and stearic acid (39), respectively.
Several other studies78-81 also reported that structure modification by having alkyl
branched compounds results in less or no irritative effect when applied to the animal and
human skin.
21
O
OH16OH
O
14
8 OH
O
5
O
OH7
O
OH5
O
OH
O
OH
O
OHO
OH
O
OH
O
OH8
O
OH4 OH
O
OH
O
7 77 OH
O
7
26 27 28 29
30 31 32 33
5
34 35 36 37
38 39 40
O
OH10
41
Figure I.12 Various fatty acids tested for skin penetration rates and skin irritation
We are interested in constructing two-tailed tri-headed amphiphiples with a ureido
group and a Newkome-type dendron as the linker and the hydrophilic headgroup,
respectively. Long, straight alkyl chains are employed as the hydrophobic moieties.
More specifically, both chain lengths of the alkyl on each tail are the same. In this
project, we present double-tail branching in two different series with different geometry
on how the hydrophobic tails are attached to the linker. One is where a nitrogen on the
linker is connected to two chains that are equal in length, and the other is where a
nitrogen on the linker is connected to a long chain through the middle carbon.
22
linker
head
head
head
tail
tail
linker
head
head
head
tail
tail
Figure I.13 General representations of two-tailed, tri-headed amphiphile series I.5 Cholestane-based Polyanionic Compounds Derived from Natural Product
Natural products are often used as a source in molecular diversity to drug
discovery and development. Approximately 60% of the currently available antitumor and
anti-infective agents are of natural product origin.82 Before investigations were focused
on biological targets, natural product-based drugs were originally discovered by the
traditional in-vitro assays, including antibacterial, antifungal, antiviral, antiparasitic, and
cytotoxic assays.83
Several natural products contain the steroidal skeleton. Steroids are a class of
lipids that have the same chemical skeleton of four fused rings⎯three cyclohexanes and
a cyclopentane. These four rings are designated as A, B, C, and D. The large number of
C−H bonds in a steroid makes this molecule nonpolar. Compounds containing a steroidal
skeleton have many advantages for studies in biology because the skeleton contains
unique properties, such as chirality and structural rigidity.84 A couple examples of
steroids found in the human body include cholesterol and sex hormones, such as
testosterone and estrogen. Because cholesterol is a building block of cell membranes,
cholesterol derivatives are often synthesized for studies as cell membrane models.84
23
A B
C D
HO
H
HH
H
21
43
6
5
8
7
10
9
HO
H
HH
H
21
43
6
5
8
7
10
9
steroidal skeleton cholesterol 5α-cholestane
H
The C3 hydroxy group of cholesterol has been the key portion for structural
modification, where different functional groups such as esters or ethers are introduced to
the steroidal moiety. These modified compounds are widely found in the fields of
toiletries, cosmetics, pharmaceuticals, and in the chemical industry.79 Recently, another
structural modification has been studied. When hydrazone derivatives85 and a hydroxyl
group86 are introduced to C6 or C7, new compounds with antifungal properties are
generated.
Synthetic steroids are often found in medical drugs. Compounds with steroidal
skeletons, such as a cholestane moiety, are synthesized, modified, and investigated for
any biological activity. Introduction of a heterocyclic ring to the 2,3-position of various
steroids generates new compounds with anabolic,87 anti-inflamatory,88,89
antiprogestational,90 contraceptive,91 anticancer,92 and antimicrobial93,94 properties.
Modification on the steroidal skeleton itself has been explored. Compounds with
antimicrobial properties are generated when a carbon (C4) is substituted by nitrogen.95,96
Many sites in the steroidal skeleton can be explored; the steroidal skeleton is a potential
building block in structural modification.
Cholestane moiety has been involved in the syntheses of different types of
dendrimers (Figure I.14). In a study97 of transmembrane ion transport, a dendrimer of
24
amphiphilic polyamine (42) anchored to a hydrophobic cholestane moiety is involved.
Dendrimers of polyol (43, 44) anchored to cholestane moiety are synthesized and
employed in the study of the interaction of liposome with a blood complement system.98
H
HH
H
21
43
6
5
8
7
10
9
HNN
N
H3N
N
H3N
H3N
H3N
H
H
H H H
8TFA
42
H
HH
H
21
43
6
5
8
7
10
9
HO
HOO
O
HO
43
H
HH
H
21
43
6
5
8
7
10
9
HO
OO
O
HO
HO
HO
HO
44
O
Figure I.14 Polyol and polyamine dendrimers associated with cholestane moieties
Cosalane, a synthetic anti-HIV (Human Immunodeficiency Virus) agent derived
from aurintricarboxylic acid, contains a steroidal cholestane moiety. Because of its
25
HOOC
HO
ClOH
HOOC
Cl
HOOC
HO
ClOH
HOOC
Cl
cosalane dichlorinated disalicylmethane fragment
cholestane moiety
H
H
ability to inhibit multiple sites in the retroviral replication, it is considered as a potent
inhibitor in the treatment of AIDS.99 In cosalane, a cholestane moiety is connected
through a three-carbon linker chain to a dichlorinated disalicylmethane fragment of the
polymeric anti-HIV agent aurintricarboxylic acid.
A number of cosalane analogs have been synthesized to improve its potency as an
anti-HIV agent. A study100 has investigated the role of the steroidal cholestane; this
moiety serves as a lipophilic accessory appendage to escort the dichlorodisalicylmethane
pharmacophore to a lipid environment. In other words, the steroidal cholestane functions
as nonpolar hydrophobic moiety as any hydrocarbon chain in most amphiphiles.
Maintaining the steroidal cholestane moiety, most modifications are made on the three-
carbon linker, carboxylic acid, and hydroxyl groups. A hypothetical model is proposed
by Cushman et al.25 that the two negatively charged carboxylate group of cosalane
interact with the positively charged Arg-58 and Arg-59 residues of gp120, a large
glycoprotein molecule. Consequently, this blocks the virus attachment to the primary
binding site, CD4, of the cell. This hypothetical model shows the crucial factor of having
the carboxylic acid groups.
26
I.6 Tri-carboxylato Amphiphile Containing Rigid Cholestane-based Hydrophobic Moiety
In our tri-headed amphiphile series, the triheadgroups are tricarboxylic acids. So
far, the different series are presented based on the modification of the alkyl chain on the
hydrophobic moiety. Hydrophobic moieties are not limited to alkyl chains. Any moiety
containing nonpolar hydrocarbon, such as steroidal skeleton, may serve as a hydrophobic
moiety as well. It becomes our interest to incorporate steroidal skeleton as a hydrophobic
moiety into our tri-headed amphiphiles.
When the ureido linker is located between the hydrophilic moiety and C3 of
cholestane moiety, only two compounds that are epimeric are generated. To expand our
study on this new cholestane-based amphiphiles, we are challenged to make
modifications so that a number of compounds are generated within a series. In order to
fulfill this need, we put a spacer between the nitrogen on the ureido linker and C3 of
cholestane moiety (Figure I.15).
linker
head
head
head
head
head
head
C3cholestane moiety
linker spacerC3cholestane moiety
Figure I.15 General structures of cholestane-based, tri-headed amphiphile series I.7 Microorganisms in Antimicrobial Screening
Our very goal in this project is to design amphiphiles that are not detergents, but
antimicrobial agents. As we have synthesized the amphiphiles, we need to screen them
for any antimicrobial activity. What microorganisms are targeted?
27
Microorganisms or microbes, which are considered neither as plant nor animal,
are defined organisms that in general are too small to be seen with an aided eye. They
can be both useful in human life and also responsible for many infectious diseases.
Microorganisms that can cause diseases are known as pathogens. Based on their physical
appearance of their cells microorganisms can be classified into six major
categories⎯protozoa, microscopic algae, fungi, bacteria, cyanobacteria, and viruses.
Protozoa, microscopic algae, and fungi are classified as eucaryotes, while bacteria and
cyanobacteria are classified as prokaryotes. Viruses are considered neither. Eucaryotic
cells contains a nucleus surrounded by a membrane, on the other hand, procaryotic cells
contains no nucleus, thus lacks of nuclear membrane.101 In this project we narrow our
scope to employ just bacteria and fungi.
I.7.1 Bacteria
In general, bacteria are classifed based on their responses to Gram-staining, which
basically are due to differences in the physical structure of their cell walls.101 This results
in Gram-negative and Gram-positive bacteria (Table I.1). An obvious difference between
the two is the presence of an outer membrane in the cell wall. Gram-negative bacteria
posses an outer membrane, on the other hand, Gram-positive bacteria lack an outer
membrane.
Table I.1 Physical differences between Gram-negative and Gram-positive bacteria Gram-negative Gram-positive thinner cell walls ( ~ 100−150 Å) thicker cell walls ( ~ 200 Å) surrounded by an outer membrane lack of an outer membrane low amino sugar content ( ≤ 10%) high amino sugar content (10−30%) high lipid content (10−20%) low lipid content ( ≤ 2%)
Another class of bacteria is mycobacteria, which is not classified as neither Gram-
28
negative nor Gram-positive bacteria.101 As found in Gram-negative bacteria, an outer
membrane is found in mycobacteria, however, the outer membrane in mycobacteria is
thicker than that in Gram-negative bacteria. The outer membrane of mycobacteria
contains mycolic acids, which are α-alkyl, β-hydroxy fatty acids; mycolic acids contain
60 to 90 carbons, and α-branches contain 20 to 24 carbons.102 In fact, it is suggested that
the resistance of mycobacteria to any lethal environment is caused by the low
permeability of the mycolic acids in the mycobacterial cell wall.103-105
OH
OOH
R'R"
general structure of mycolic acids
Two Gram-negative bacteria employed include Escherichia coli and Klebsiella
pneumoniae, and Gram-positive bacteria employed include Lactobacillus plantarum,
Micrococcus luteus, Staphyllococcus aureus, and methicillin-resistant Staphyllococcus
aureus. Mycobacterium smegmatis will be used as a model of mycobacteria.
I.7.2 Fungi
Fungi are characterized by the presence of cell walls, the lack of motility, and the
absence of photosynthesis.101 Some fungi develop as single cells, or produce networks of
filaments. The former is called yeast, and the later is called molds. Even though some
yeasts have been known for their useful action in life, some yeasts are pathogens. We
include three different yeasts, namely Saccharomyces cerevisiae, Candida albicans, and
Cryptococcus neoformans. The first is non-pathogenic yeast; S. cerevisiae is known as
brewer’s yeast and it is used in bread-making process. The last two yeasts are classified
29
as pathogenic yeasts. In addition, we also include Aspergillus niger, which is a
filamentous fungus.
I.8 Summary
Finally, we intend to achieve two goals in this project. First, we will synthesize
several homologous series of tri-headed amphiphiles with different type of hydrophobic
moieties, namely mobile and rigid hydrocarbon chain. In the series containing mobile
hydrocarbon moieties, two different series, in this case one- vs two-tail, will be presented
while the numbers of carbons on the chain are maintained equal. In addition, the two-tail
will be modified based on the topology of the tails. Second, we will screen the
antimicrobial activity of the tri-headed amphiphiles against a broad array of
microorganisms to explore the structure-relationship activity. We hope to discover leads
for development as topical microbicides.
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39
Chapter II Synthesis of One-Tailed, Tri-headed Amphiphiles (3CUrn)
To construct a homologous series of one-tailed, tri-headed amphiphiles, we have
chosen to use amines as the hydrophobic moiety and a tricarboxylic dendron as the
hydrophilic moiety. The hydrophobic moiety is attached to the hydrophilic moiety by
forming a ureido linker, which is attached to the Newkome-type dendron by combining
an isocyanate and a variety of amines.1-6 This homologous series is abbreviated as
3CUrn (Scheme II.1), where “3C” represents three carboxylic groups on the hydrophilic
moiety, “Ur” represents a ureido linker (b) between the hydrophobic (c) and hydrophilic
(a) moieties, and “n” represents the number of carbons of the n-alkyl on the hydrophobic
moiety. Based on this chemistry, the synthesis of 3CUrn requires two steps⎯addition
and removal of tert-butyl groups⎯and begins with WeisocyanateTM (3) and alkan-1-
amines (4a−e) (Scheme II.1). Similar to the naming of the 3CUrn series, the
Scheme II.1 Retrosynthesis of one-tailed, tri-headed amphiphiles (3CUrn)
HN
HN
O
OCNO
O3
R NH2
R
O
O
O
O
O
O
H
H
HN
HN
O
R
O
O
O
O
O
O
+
4a-e
3
3CUrn 3EUrn
R = n-CnH2n+1
1 2
c b a
a. a hydrophilic moiety; b. a ureido linker, c. a hydrophobic moiety
a, n=14b, n=16c, n=18d, n=20e, n=22
H
40
homologous series in which the tert-butyl groups will be removed, is abbreviated as
3EUrn, where “3E” represents three tert-butyl esters on the dendron.
II.1 Preparation of WeisocyanateTM (3)
WeisocyanateTM (3) was prepared according to the published
procedure7⎯combining Behera’s amine (5, 0.25M), DMAP, and (Boc)2O in
dichloromethane. Newkome et al.7 reported that the reaction was run for 30 minutes,
95% crude yield, and no purification yield. These conditions produced side products in
our laboratory.
Scheme II.2 Synthetic scheme of the synthesis of isocyanate 3
DMAP, (Boc)2O
CH2Cl2rt
3 +
7
NH
O
O 3
NH
OO
O 3
NH
O
O 3
O
O
+
6
H2NO
O 35
Based on the mechanistic study8 of the reaction of various amines and (Boc)2O in
the presence of DMAP, the desired isocyanate 3 was obtained along with the side
products⎯the t-Boc-protected amine 6 and urea 7. We speculated from the 1H NMR
spectrum that the only side product was urea 7. When the reaction solution was run at a
more dilute concentration (i.e., 0.12 M of amine 5 for two hours), isocyanate 3 and urea 7
based on its 1H NMR spectrum, were obtained in 76% and 24% yields, respectively.
When the reaction was run for a shorter reaction time, the side products 6 and 7 (as
41
shown by the 1H NMR spectrum) were not observed. The optimal reaction time was 15
minutes. The crude product was recrystallized from hexane to give isocyanate 3 (84%
yield).
II.2 Preparation of Alkan-1-amines
We needed five even-numbered long-chain alkan-1-amines (4a−e)⎯tetradecan-,
hexadecan-, octadecan-, icosan-, and docosanamine⎯to construct a homologous series.
However, amines 4d−e are not commercially available. Thus, the synthesis of both was
necessary. We sought to design the synthesis beginning with inexpensive commercially
available materials.
II.2.1 Literature Review on Syntheses of Long-chain Alkan-1-amines (4)
There are few literature procedures describing the synthesis of long-chain
alkan-1-amines (n > 18). Retrosynthesis (Scheme II.3) shows different commercially
available materials can be employed as precursors.
Scheme II.3 Retrosynthesis of alkan-1-amines
4 8 9
10 11 12
R NH2 R N3 R OMs
R OHR Br
R = n-CnH2n+1
4d, n = 204e, n = 22
n = 14, 16, 18, 20, 22
letters "d" and "e" refer to the corresponding amines
NH2
O
n-2
42
Amines 4 can be simply obtained from reduction of the corresponding amides 10
or azides 8 (Table II.1). In the literature,9-17 there are a variety of reagents reported to
reduce azides 8 to the corresponding amines 4. The reducing agents may have some
limitations such as selectivity, ready availability, and convenience of operation. In our
case, reagent selectivity is not considered because there is no other functional group in
the precursors being reduced.
Various reducing agents of 1-azidoalkanes have been documented. These include
lithium aluminum hydride,9,10,17 and different forms of borohydride,11,13,18-23 tributyltin
hydride,12 tributyltin hydride in the presence of nickel dichloride
diphenylphosphinoethane,24 tin dichloride and aluminum trichloride in methanol,25 tin(II)
complex,26 catalytic hydrogenation,16,27 N,N-dimethylhydrazine/ferric chloride in
acetonitrile,15 magnesium in methanol,28 metallic zinc in ethanol,29 methanol,30,31
tetrahydrofuran in the presence of cobaltdichloride hexahydrate,32 powdery zinc in
tetrahydrofuran in the presence of nickeldichloride hexahydrate,33 tetracarbonylhydrido-
ferrate,34 borane or dichloroborane-dimethylsulfide,35 aluminum triiodide,36 ferric
chloride and sodium iodide in acetonitrile,14 metallic samarium and samarium iodide in
various alcohols,37-41 indium in aqueous hydrochloric acid,42 and tellurium in water.43
43
Table II.1 Preparations of amines 4 from different precursors Precursor Reagent, Solvent Yield (%) Reference
18NH2
O
LiAlH4, Et2O
not reported 9
N319
LiAlH4
not reported 10
N315
NaBH4, H2O, C16H33PBu3Br, PhCH3
88
11
N315
Te, H2O
91 43
N314
LiAlH4, Et2O
not reported 17
N311
AIBN, Bu3SnH, PhSiH3, n-PrOH AIBN, Bu3SnH, PhMS, n-PrOH
94 95
12
N311
(Sn(SPh)3)(Et3NH), PhH
98 26
N311
BHCl2.SMe2, THF or Et2O
78 35
N310
FeCl3/NaI, CH3CN
85 14
N310
Zn(BH4)2, silica gel, DME
92 13
N310
AlI3, PhH
75 36
N39
SnCl2, AlCl3, MeOH
88 25
N39
In, aq. HCl, THF
92
42
N39
Bu3SnH, NidppeCl2, THF dpee = diphenylphosphinoethane
80 24
N39
Mg, MeOH
95 28
II.2.2 Literature Review on Syntheses of 1-Azidoalkanes (8)
Azides 8 are usually synthesized from the corresponding alcohols 12 or
44
bromoalkanes 11. Starting with alcohols 12, the first step involves the conversion of the
hydroxyl group into a better leaving group. This leaving group is typically a bromide or
a sulfonate ester. In some cases, bromoalkanes 11 are commercially available. The next
step involves a direct nucleophilic substitution (SN2) with an azide acting as the
nucleophile.
Several examples of the synthesis of azides 8 from the corresponding
bromoalkanes 11 and sulfonate esters are presented in Table II.2.
45
Table II.2 Preparations of azides 8 from different precursors Precursor Reagent, solvent Yield (%) Reference
Br19
azide not specified
not reported 10
Brn-1
n = 8, 16
(C8H17)3NCH3Cl, NaN3, H2O, 80 °C
89 (n=16) 85 (n=8)
11
Br10
NaN3, dry DMF, 80 °C
75 13
Br7
NaN3, DMSO, rt
99 40
OSO2CH3n-1
n = 8, 10, 15
NaN3, DMF, 90 °C
not reported (n=15)
n = 15,17 10,24 8.24
OSO2CH3n-1
n = 6, 8
[hexmim][N3], various solvents, 60 °C solvents: PhCl, DMSO, MeOH, [hexmim][ClO4], and [hexmim][PF6] [hexmim] = [1-hexyl-3-methylimidazolium]
not reported 44
OSO2CH37
(C8H17)3NCH3Cl, NaN3, H2O, 80 °C
89 11
OSO2CH37
[cis-Dicyclohexane-18-crown-6][K]+[N3]- or C16H33NBu3N3 in PhCl, H2O, 60 °C
not reported 45
OTs11
Me3SiN3, Bu4NF, THF, rt
96 46
OTs7
NaN3 and [bmim][PF6] NaN3 and [bmim][N(Tf)2] NaN3 and [hpyr][N(Tf)2] [bmim] = [1-butyl-3-methylimidazolium] [hpyr] = [1-hexylpyridinium]
>90 >90 >90
47
OTs7
NaN3, CH3CN, heating
not reported 48
OTs7
NaN3, H2O, 90 °C NaN3, DMF, 90 °C, microwave Irradiation
92 94
49
OTs7
C16H33PBu3N3, MeOH, 60 °C
not reported 50
OTs7
n = 5−7
NaN3, DMF, 80 °C
not reported 51
46
II.2.3 Literature Review on Syntheses of 1-Methanesulfonyloxyalkanes (9)
As mentioned above, a sulfonate ester is one of several typical leaving groups in
many SN2 reactions. When SN2 reactions are performed with alcohols as precursors,
transformation of the alcohols into the corresponding sulfonate esters is often required.
As sulfonate esters⎯mesylate and tosylate⎯are good leaving groups, such a
transformation is almost routine in organic synthesis. In addition, not only is the reaction
run under mild conditions, but it also gives excellent yield. The transformation of many
alcohols into the corresponding 1-methanesulfonyloxyalkanes,24,52-59 several of which are
long-chain alcohols 12 (dodecan-,52 tetradecan-,53 octadecan-,58 and icosan-56), has been
reported to give good to excellent yields.
II.2.4 Literature Review on Syntheses of 1-Bromoalkanes (11)
In general, bromoalkanes 11 are commercially available. However, should
synthesis be required, the most common precursor is the corresponding alcohols 12.
Alcohols 12 are mostly commercially available at low cost. One-step syntheses of
bromoalkanes 11 have been well documented. Many of them employ triphenylphosphine
in combination with carbon tetrabromide,60-62 bromine,63,64 N-bromosuccinimide,65
2,4,4,6-tetrabromo-2,5-cyclohexadien-1-one,66 and 1,2-dibromotetrachloroethane.67
Several ultra long-chain bromoalkanes 11,68 such as -tetracosane, -octacosane, and -
pentatriacontane, are obtained in 56–63% from the corresponding alcohols 12 in the
presence of 48% aqueous hydrobromic acid and concentrated sulfuric acid; these
procedures are typically run by heating at high temperature (≥ 80 °C). On the other hand,
bromoalkanes 11 can also be obtained under mild conditions in several steps (Table II.3).
47
Table II.3 Preparation of bromoalkanes 11 from alcohols 12 Precursor Reagent Yield (%) Reference
OH21
1.ClCH2CH2SO2Cl (or CH2=CHSO2Cl), Me3N; 2. Me2NH; 3. CH3SO3F; 4. KBr
67
69,70
OH15
1.ClCH2CH2SO2Cl, Me3N; 2. Me2NH; 3. MeBr
83 69,70
OH19
1. Et3N, MsCl, CH2Cl2; 2. LiBr, THF
77 56
OH15
1. MsCl; 2. NaBr
not reported 71
II.2.5 Preferred Starting Material⎯Alkan-1-ols (12) or 1-Bromoalkanes (11)
Both bromoalkanes 11 and alcohols 12 can be employed as the precursors for the
synthesis of azides 8 via SN2 reactions. Beginning with bromoalkanes 11, the synthesis
of azides 8 is only a one-step reaction. On the other hand, should alcohols 12 be utilized,
the synthesis of azides 8 requires a two-step reaction⎯transformation to the
corresponding sulfonate esters, such as 1-methanesulfonyloxyalkanes, followed by SN2
reactions. As sulfonate esters are better leaving groups than bromide,72 theoretically, the
SN2 reaction of 1-methanesulfonyloxyalkanes that leads to the formation of the
corresponding azides 8 should be easier than that of bromoalkanes 11.
II.2.6 Synthesis of Long-chain Alkan-1-amines (4)
At the beginning, we repeated the reduction procedure9 with lithium aluminum
hydride and technical grade of docosanamide (amide 10e) as the starting material, which
was commercially available at a low cost. Attempts to purify the technical grade amide
10e were not successful because of low solubility in many organic solvents. As the
reaction yield was low and there were several unidentified side products based on the 1H
NMR spectrum, no attempt was made to isolate the desired product.
48
We then switched our strategy by utilizing bromoalkane 11d−e as the precursors.
Initially, we followed the procedure 11 for phase-transfer catalysis reaction. Some
modifications were made; cetyltrimethylammonium bromide (CTAB) was used as the
catalyst instead of trioctylmethylammonium chloride, and the reaction was sonicated
prior to reflux. When the reaction was conducted without sonication in a variety of high
boiling-point solvents, such as tert-butyl alcohol and toluene, we observed no reaction.
We found out that sonication for three hours prior to reflux was very critical for the
transformation. Compared to the published procedure11 for 1-azidohexadecane (8c) and
1-azidooctane, in which the reaction time was only eight hours, the reactions for the
syntheses of 1-azidoicosane (8d) and 1-azidodocosane (8e) were refluxed for three days
to get complete conversions. The overall yields of azides 8d and 8e from the
corresponding bromoalkanes 11 were 87% and 75%, respectively.
In order to overcome the long reaction time above, we decided to use alcohols as
our precursors. Alcohols 12d10 and 12e73 were transformed into the corresponding
methanesulfonyloxyalkanes 9 in excellent yields. Methanesulfonyloxyalkane 9e was
characterized by comparing its 1H NMR spectrum to published values,73 and used
without further purification. Again, the resulting methanesulfonyloxyalkane 9e could be
transformed into bromoalkane 11e as we wished. However, we attempted to directly
convert them into azide 8e according to the published procedure.13 Because
methanesulfonate was a better leaving group than bromide, we expected that the reaction
would be completed in less time than that in the procedure13 followed. On the other
hand, because we employed a longer chain, the reaction could require a longer time. We
monitored the reactions by thin layer chromatography, and after several trials on our scale
49
(7.82 mmol of methanesulfonylalkanes 9), we concluded the reaction was completed
within 3–4 hours. The crude azides 8 were purified by flash column chromatography in
hexane. Melting temperatures of the desired products were not taken because we
obtained “glue-like” solids. The desired products were characterized by comparing their
IR and NMR to published data.69
Because of convenience (ease of work-up and fewer side products), the desired
azides 8 were reduced via hydrogenation with 10% palladium on carbon as a catalyst.16
The resulting amines 4 were purified by recrystallization from ethyl acetate. Amines 4
were characterized by comparing the mp, IR, and NMR spectra to published data.10,74,75
The general synthesis and the yield of 1-alkanamines (4d and 4e) are presented in
Scheme II.4. The overall yields of 4d and 4e obtained from the corresponding
bromoalkanes 11 in two steps were 65% and 53%, respectively, while those from the
corresponding alcohols 12 in three steps were about 60% and 57%, respectively. Amine
4d was obtained in a higher yield from bromoalkane 11d, while amine 4e was obtained in
a higher yield from alcohol 12e.
Scheme II.4 Synthetic scheme of amines 4d and 4e
i. MsCl, Et3N, CH2Cl2, rt, 3h (9d−e, 92–95%)ii. NaN3, DMF, reflux, 4h (8d, 86%; 8e, 87%)iii. Pd/C, H2, hexane, rt, 3h (4d, 75%; 4e, 70%)iv. LiBr, dry THF, rt, overnight (11d, 84%; 11e, 65%)v. CTAB, H2O, , for 3 h; NaN3, 80–90 °C, 3 d (8d, 87%; 8e, 75%)
N3OSO2CH3
) ))
NH2OH
Br
i ii iii
ivv
R R R R
R
4
R = n-CnH2n+1d, n = 20e, n = 22
8912
11
50
II.3 Synthesis of the 3EUrn Series (2a−e)
The tri-tert-butyl esters 3EUrn were generated by combining amines 4 and
isocyanate 3 in dichloromethane at ambient temperature (Scheme II.5). Purification was
carried out by either recrystallization (from ethanol−water) or flash column
chromatography. Both purifications gave pure compounds in good to excellent yields
(Table II.4). The trend (Table II.4) observed in the melting ranges data was expected; the
longer the chain, the higher the melting ranges.
Scheme II.5 The synthetic scheme of one-tailed, tri-headed amphiphiles (3CUrn)
HN
HN
O
OHO
n-13
HCOOHrt, 9 h
HN
HN
O
OtBuO
n-1+
CH2Cl2
rt, overnight
33EUrn 3CUrn
4 3
Table II.4 Isolated yields and melting ranges of the tri-tert-butyl esters 3EUrn
n Isolated yield (%) Melting range (°C) 14 86 55.0–55.6 16 72 59.1–59.8 18 92 65.0–65.8 20 69 66.5–67.9 22 84 67.9–68.8
Interestingly, the recrystallized tri-tert-butyl ester 3EUr16 contained one water
molecule for two molecules of 3EUr16 as shown by the X-ray crystal structure. The
presence of water in the recrystallized compounds was supported by both elemental and
thermogravimetric analyses. Elemental analysis showed there was a water molecule for
every two ester molecules, and thermogravimetric analysis showed 1.3% weight loss
from 40–80 °C corresponds to loss of ½H2O. We presumed that ½H2O would be present
in the other 3EUrn since X-ray crystal structure showed that it was trapped between two
of 3EUr16 molecules. Indeed, the elemental analyses from tri-tert-butyl esters 3EUr18,
51
3EUr20, and 3EUr22 supported this hypothesis. However, elemental analysis of tri-tert-
butyl ester 3EUr14 did not support the hypothesis. Unlike the other 3EUrn, 3EUr14
was purified by flash column chromatography because attempts to recrystallize 3EUr14
were not successful. In this case, elemental analysis showed there was no water molecule
in the purified 3EUr14.
II.4 Synthesis of the 3CUrn Series (1a−e)
Removal of the tert-butyl group is a routine procedure in organic synthesis.
Newkome et al.76-82 employed formic acid to remove such a group; the reaction condition
was mild and the yield was excellent. Formolysis of the tri-tert-butyl esters 3EUrn
produced the triacids, 3CUrn, in good yields of recrystallized products (Table II.5). In
contrast to the melting range trend observed in the tri-tert-butyl esters 3EUrn series,
there was no particular trend in the melting range of the triacids 3CUrn series (Table
II.5). Triacid 3CUr16 was observed to have the highest melting range, after which the
melting range slightly decreases for longer chain lengths. All tri-tert-butyl esters and
triacids—3EUrn and 3CUrn— were fully characterized.
Table II.5 Isolated yields and melting ranges of the triacids 3CUrn n Isolated yield (%) Melting range (°C) 14 84 159.0–159.5 16 83 162.4–162.8 18 85 160.7–161.5 20 82 160.3–161.2 22 74 157.3–158.3
II.5 X-ray Crystal Structure of Tri-tert-butyl Ester 3EUr16
The crystal structure of tri-tert-butyl ester 3EUr16 (Figure II.1) reveals how these
molecules that have bulky head groups assemble in the solid state. The molecule can be
considered in three parts: a head group (the three esters), a linker (ureido), and a tail
52
(alkyl chain). Figure II.1 illustrates the closely packed alkyl chains and hydrogen bonds,
which are essential for self-assembly. The molecules are arranged in the lattice in the
typical manner that is seen for compounds with polar head groups and long alkyl chains.
There are two crystallographically independent molecules arranged in parallel;
inversion symmetry generates the antiparallel chains. The hydrogen bonding among the
headgroups and the extensive overlap of the interdigitated alkyl chains suggest that both
are essential for self assembly. Antiparallel chains, which are separated by carbon-to-
carbon distances ranging from 3.9 to 4.4 Å, pack more closely than parallel chains, which
are separated by carbon-to-carbon distances ranging from 4.9 to 5.4 Å. To achieve this
close packing of antiparallel chains, the C(3)–C(4)–C(5)–C(6) and the C(42)–C(43)–
C(44)–C(45) torsion angles adopt gauche conformations, ±67.4(3)° and ±60.9(3)°,
respectively. The hydrogen bonds involve primarily the linker atoms on neighboring
molecules; a water molecule connects alternate pairs of neighbors. In one hydrogen
bond, the carbonyl oxygen [O(8)] on the ureido linker accepts a hydrogen bond from the
amido nitrogen [N(2)] connected to the long alkyl chain. In the other hydrogen bond, the
carbonyl oxygen [O(1)] of the ureido linker accepts a hydrogen bond from a water
[O(15)], which in turn accepts a hydrogen bond from the amido nitrogen [N(4)]
connected to the long alkyl chain. The water also donates a hydrogen bond to an ester-
carbonyl oxygen [O(3)]. The hydrogen bond between the carbonyl oxygen [O(1)] of the
ureido linker and the water [O(15)] is the shortest. The other amido nitrogens in the
ureido linkers, N(1) and N(3), form weak hydrogen bonds (donor–acceptor distances >
3.0 Å) with O(8) and O(15), respectively.
53
Figure II.1 X-ray crystal structure of tri-tert-butyl ester 3EUr16, showing the packing diagram and the intermolecular hydrogen bonding to water and to a neighboring molecule. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(8)···N(2), 2.863(3) Å and 159.1°; O(15)···O(1), 2.718(4) Å and 170(4)°; O(15)···O(3), 2.843(4) Å and 169(4)°; N(4)···O(15), 2.832(3) Å and 153.4°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(1)···O(8), 3.201(3) Å and 145.6°; N(3)···O(15), 3.074(3) Å and 141.6° II.6 CMCs (Critical Micelle Concentration) of Amphiphiles 3CUrn
One of our current goals in designing the new series of amphiphiles, 3CUrn, is to
have a more water-soluble amphiphile. In the case of amphiphiles, the concept of
solubility is often involved with critical micelle concentration (CMC); CMC is defined as
the concentration in which micellar aggregates start to form. The number of headgroups
and carbons in the chain lengths affect the CMCs.83,84 The number of headgroups affects
CMCs in a proportional manner.83,85-87 On the other hand, the number of carbons in the
hydrophobic moiety is inversely proportional to the CMCs.84 Based on that concept, we
present a series of long-chain multi-headed (tri-headed) amphiphiles, 3CUrn.
54
The choice of the counterion followed from a study88 of N-lauroyl-L-glutamate in
water; the triethanolammonium salt dissolves to a much greater concentration than did
the potassium salt. As chain length can affect the pKa of fatty acids because of
aggregation,89 we explored different conditions to achieve the maximum solubility in
aqueous solutions of triethanolamine (pKa 7.76).90 The 3CUrn homologus series
dissolved readily in a solution of triethanolamine/water [~ 5% (wt/vol)]; the final solution
contained ≥ 9:1 molar equivalents of triethanolamine : 3CUrn.
The CMCs measurement of the homologous 3CUrn series was performed by
Richard V. Macri (Table II.6). Using a pendant-drop analyzer to measure surface
tensions, the CMCs of 3CUr16–3CUr22 were measured in triethanolamine/water [~ 6%
(wt/vol)]. The pH of the solutions ranged from 9.3 to 10.0 depending on the
concentration of the amphiphiles.
Table II.6 CMCs of the 3CUrn homologous series Compound CMCs (mM)
3CUr14 N/A 3CUr16 3.4 ± 0.17 3CUr18 2.2 ± 0.11 3CUr20 1.4 ± 0.070 3CUr22 0.92 ± 0.046
The slope of the plot of log CMC vs chain length is ~ 0.1; this value contrasts
with a value of 0.3 for a similar plot of the CMCs of potassium salts of fatty acids.83
Slopes of similar plots for di- and tri-carboxylato amphiphiles83,87 have values of 0.22.
Unlike the others, amphiphile 3CUr14 did not show a leveling off of the surface tension
with increasing concentrations up to 10 mM suggesting that it failed to form micelles.
The CMC for the longest homolog, 3CUr22, is 1 mM, which is quite high for an ultra-
long chain.
55
II.7 Experimental Procedures Materials and Methods.
Chemicals were obtained from Aldrich, Acros and TCI; they were used without further
purification. Solvents were reagent grade or HPLC grade; they were used as received
unless otherwise specified. THF was distilled from sodium/benzophenone ketyl.
WeisocynateTM was prepared as described7 with a shorter reaction time as mentioned
above. Analytical thin layer chromatography was performed by polyester-coated silica
gel 60 Å and detected by treating with 10% ethanolic phosphomolybdic acid reagent (20
wt. % solution in ethanol) followed by heating. Flash column chromatography was
carried out on silica gel (60 Å); column diameter × height (13/4 × 6 in), eluted samples
varied between ~ 2.00−4.00 g, flow rate (~ 1.5−2 in/min) was controlled by air pressure.
Solutions were concentrated by rotary evaporation. Melting ranges, determined in open
capillary tubes, were uncorrected. NMR spectra were recorded on an INOVA at 400 and
100 MHz for 1H and 13C, respectively, and reported in ppm relative to the known solvent
residual peak. Resonances were reported in the order of chemical shift (δ), followed by
the splitting pattern, and the number of protons. Abbreviations used in the splitting
pattern were as the following: s = singlet, d = doublet, t = triplet, q = quartet, quin =
quintet, m = multiplet, and b = broad. IR spectra were recorded on neat samples with an
FTIR equipped with a diamond ATR system, and reported in cm-1. HRMS data were
obtained on a dual-sector mass spectrometer in FAB mode with 2-nitrobenzylalcohol as
the proton donor. Elemental analyses were performed by Atlantic Microlabs, Inc. in
Norcross, GA.
56
Preparation of alkyl mesylate (9d−e)56
OMsn-1
OHn-1 MsClEt3N+ +
DCMrt, 3h
To a solution of alcohol 12 (16.2 mmol) and Et3N (5.23 mL, 37.3 mmol) in CH2Cl2 was
added MsCl (2.00 mL, 25.7 mmol) through a syringe. The resulting solution was stirred
for 3 h. The resulting yellow reaction was washed successively with water (14 mL), HCl
(2 M, 14 mL), water (14 mL), satd NaHCO3 (14 mL), and water (14 mL). The organic
solution was dried with MgSO4 and concentrated to give a white-yellow solid (92–95%).
The 1H NMR spectra of the respective crude products—icosyl mesylate and docosyl
mesylate73—suggested that the material was suitable for the next step without further
purification.
Preparation of 1-bromoicosane56
Br19
OMs19
+ LiBrdry THF
rt, overnight
To a solution of 9d (6.10 g, 16.2 mmol) in dry THF (50 mL) was slowly added LiBr
(3.57 g, 40.7 mmol). The cloudy yellow suspension was stirred at rt for 14 h, after which
the solution was concentrated to dryness to give an off-white solid. This solid was
treated with hexane (60 mL); most of the solid dissolved. After filtration, the filtrate was
concentrated to dryness to leave a light yellow solid, which was recrystallized from hot
EtOH to give a white solid (60%); mp 36.0–36.4 °C (lit.91 mp 36.2–37.0 °C); 1H NMR
(CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 32H), 1.42 (m, 2H), 1.86 (quin, 2H), 3.41 (t, 2H)
(lit.92 200 MHz); 13C NMR (CDCl3) δ 14.3, 22.9, 28.4, 29.0, 29.6, 29.7, 29.82, 29.86,
29.89, 32.1, 33.0, 34.21; IR 2915, 2847, 1472, 1462, 730, 719 (lit.92 in CHCl3).
57
General procedure for the preparation of 1-azidoalkane (8d−e)
Method A.11
N3n-1
Brn-1 + NaN3
CTAB, H2O))) , 4h
80–90 °C, 3 d
A mixture of bromoalkane 11 (6.64 mmol), CTAB (243 mg, 0.661 mmol), NaN3 (1.12 g,
17.0 mmol), and water (3 mL), was sonicated for 4 h at 60 °C. The resulting emulsion
was refluxed (80–90 °C) for 3 d. The milky reaction mixture was treated with hexane
and some solid formed. After gravity filtration, the filtrate was concentrated to give a
sticky white solid (75–87%).
Method B.
N3n-1
OMsn-1
+ NaN3
85 °C, 4hDMF
NaN3 (1.28 g, 19.5 mmol) was added to a solution of methanesulfonyloxyalkane 9 (7.82
mmol) in DMF (52 mL). The mixture was stirred at rt for 30 min, and then refluxed at 85
°C for 4 h. Afterwards, the reaction was cooled before portions of hexane (104 mL) and
water (10.5 mL) were added. The organic layer was separated and washed successively
with satd NaHCO3 (10.5 mL) and satd NaCl (10.5 mL). The organic layer was dried with
Na2SO4 and concentrated to give a crude product, which was purified by flash column
chromatography in hexane to give a sticky white solid (82–86%).
1-Azidoicosane (8d)
The procedures described above afforded a sticky white solid (1.87 g, 87%, Method A;
2.37 g, 86%, Method B); 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.40 (bm, 34H), 1.60
(quin, 2H), 3.25 (t, 2H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.9, 29.0, 29.4, 29.56, 29.68,
58
29.74, 29.83, 29.89, 32.1, 51.7; IR 2915, 2848, 2093, 1467, 1260.
1-Azidodocosane (8e)
The procedures described above afforded a sticky white solid (lit.69 mp 41–42 °C); 1H
NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.40 (bm, 38H), 1.60 (p, 2H), 3.26 (t, 2H) (lit.69 60
MHz); 13C NMR (CDCl3) δ 14.3, 22.9, 26.9, 29.0, 29.4, 29.6, 29.69, 29.75, 29.83, 29.86,
29.90, 32.1, 51.7; IR 2915, 2848, 2093, 1466, 1253 (lit.69 in CHCl3).
General procedure for the preparation of alkan-1-amine (4d−e):16
NH2n-1
N3n-1
Pd/C, H2
hexane, rt, 3 h
To a solution of azide 8 (5.38 mmol) in hexane (25 mL) was added 10% Pd/C (3% w/w
of azide 8). The resulting mixture was hydrogenated at 62 psi at rt for 3 h. Following
gravity filtration, the filtrate was concentrated to give off-white solid, which was
recrystallized from ethyl acetate (70–75%).
Icosan-1-amine (4d)
The procedures described above afforded a white solid (1.20 g, 75%); mp 58.3–59.0 °C
(lit.74 mp 58.5 °C); 1H NMR (CDCl3) δ 0.89 (t, 3H), 1.20–1.35 (bm, 36H; 2H exchange
with D2O), 1.44 (m, 2H), 2.69 (t, 3H) (lit.10); 13C NMR (CDCl3) δ 14.3, 22.9, 27.1, 29.55,
29.72, 29.85, 29.89, 32.1, 34.1, 42.5; IR 3328, 2915, 2849, 1486, 1472.
Docosan-1-amine (4e)
The procedures described above afforded a white solid (1.23g, 70%); mp 62.9–63.8 °C
(lit.75 mp 62.7 °C); 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 40H; 2H exchange
with D2O), 1.44 (m, 2H), 2.68 (t, 3H); 13C NMR (CDCl3) δ 14.3, 22.9, 27.1, 29.55, 29.71,
29.83, 29.85, 29.89, 32.1, 34.1, 42.5; IR 3300, 2915, 2848, 1472, 1462.
59
General procedure for the preparation of long chain tri tert-butyl esters 3EUrn (2a−e)
HN
HN
O
OtBuO
n-1OCN
OtBu
O 3
NH2n-1 +
DCMrt, overnight
3
An amine 4 (5.43 mmol) was added slowly to a solution of isocyanate 3 (5.43 mmol) in
CH2Cl2 (45 mL). The resulting colorless solution was stirred at rt. After stirring
overnight, the solvent was removed to leave a crude yellow oil (n = 14) or a crude white
solid (n = 16, 18, 20, 22). The crude yellow oil was purified by flash chromatography
(hexane:ethyl acetate = 5:1), while the crude white solid was purified by recrystalization
with EtOH–H2O. Both purification methods gave a pure white solid (69–92%).
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-tetradecylureido)heptanedioate,
3EUr14
The general procedure described above afforded a white solid (3.07 g, 86%); mp 55.0–
55.6 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 22H), 1.35–1.50 (bm, 29H),
1.94 (t, 6H), 2.24 (t, 6H), 3.14 (q, 2H), 4.08 (m, 1H), 4.57 (s, 1H); 13C NMR (CDCl3) δ
14.1, 22.7, 26.9, 28.1, 29.3, 29.58, 29.60, 29.64, 29.65, 29.9, 30.2, 30.6, 31.9, 40.6, 56.4,
80.5, 156.8, 173.1; IR 3327, 2919, 2850, 1721, 1673, 1646, 1559, 1148; HRMS: for
C37H71N2O7 (M + H)+calcd 655.5246, found 655.5261. Anal. Calcd for C37H70N2O7: C,
67.85; H, 10.77; N, 4.28. Found: C, 68.01; H, 10.92; N, 4.27.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-hexadecylureido)heptanedioate,
3EUr16
The general procedure described above afforded a white solid (2.68 g, 72%); mp 59.1–
59.8 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 26H), 1.35–1.50 (bm, 29H),
1.94 (t, 6H), 2.24 (t, 6H), 3.07 (q, 2H), 4.10 (m, 1H), 4.59 (s, 1H); 13C NMR (CDCl3) δ
60
14.1, 22.7, 26.9, 28.1, 29.3, 29.58, 29.60, 29.64, 29.7, 29.9, 30.2, 30.6, 31.9, 40.6, 56.5,
80.5, 156.8, 173.1; IR 3310, 2917, 2851, 1719, 1700, 1645, 1563, 1147; HRMS: for
C39H75N2O7 (M + H)+ calcd 683.5574, found 683.5540. Anal. Calcd for
C39H74N2O7·½H2O: C, 67.69; H, 10.92; N, 4.05. Found: C, 67.92; H, 11.03; N, 4.04.
Thermogravimetric analysis: ~ 1.3 % weight loss from 40–80 °C corresponds to loss of
½H2O.
X-ray analysis of amphiphile 3EUr16
Colorless needles (~3 × 0.2 × 0.1 mm3) were crystallized from EtOH–H2O at rt. The
chosen crystal was cut (0.37 x 0.12 x 0.07 mm3) and mounted on a nylon CryoLoop™
(Hampton Research) with Krytox® Oil (DuPont) and centered on the goniometer of a
Oxford Diffraction XCalibur2™ diffractometer equipped with a Sapphire 2™ CCD
detector. The data collection routine, unit cell refinement, and data processing were
carried out with the program CrysAlis.93 The Laue symmetry was consistent with the
triclinic space group P1̄. The structure was solved by direct methods and refined with the
SHELXTL NT program package.94 The asymmetric unit of the structure comprises two
crystallographically independent molecule of amphiphile 3EUr16 and one water
molecule. One tert-butyl group of molecule two showed evidence of rotational disorder
and the methyl groups were modeled as occupying two conformations with relative
occupancies of 0.728 and 0.272. The final refinement model involved anisotropic
displacement parameters for non-hydrogen atoms. A riding model was used for the
aromatic and alkyl hydrogens. Hydrogen atom positions for the water molecule were
located from the electron density map and subsequently restrained to be 0.84 Å from the
61
oxygen. The program package SHELXTL NT94 was used for molecular graphics
generation.
Table II.7 Crystal data and structure refinement of 3EUr16 Category Crystal data and structure refinement Empirical formula C39H74N2O7 • 0.50H2O Formula weight 692.01 Temperature 100(2) K Wavelength 0.71073 Å Crystal system triclinic Space group P1̄ Unit cell dimension a = 11.362(3) Å; b = 12.2865(19); c = 32.335(3) Å;
α = 88.141(12)°; β = 59.584(9)°; γ = 67.804(19)° Volume 4177.1(13) Å3 Z 4 Density (calculated) 1.100 Mg/m
3
Absorption coefficient 0.075 mm-1 F(000) 1532 Crystal size 0.37 × 0.12 × 0.07 mm3 Theta range for data collection 2.78 to 24.13° Index ranges −13 ≤ h ≤ 10, −14 ≤ k ≤ 14, −36 ≤ l ≤ 37 Reflections collected 21062 Independent reflections 13197 [R(int) = 0.0378] Completeness to theta = 25.07° 99.2 % Absorption correction None Refinement method Full-matrix least-squares on F
2
Data / restraints / parameters 13197/2/932 Goodness-of-fit on F2 0.949 Final R indices [I>2σ(I)] R1 = 0.0517, wR2 = 0.1207 R indices (all data) R1 = 0.0956, wR2 = 0.1392 Largest diff. peak and hole 0.719 and −0.523 e.Å
-3
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-octadecylureido)heptanedioate,
3EUr18
The general procedure described above afforded a white solid (3.54 g, 92%); mp 65.0–
65.8 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 30H), 1.35–1.50 (bm, 29H),
1.94 (t, 6H), 2.24 (t, 6H), 3.14 (q, 2H), 4.03 (m, 1H), 4.55 (s, 1H); 13C NMR (CDCl3) δ
14.1, 22.7, 26.9, 28.0, 29.3, 29.58, 29.62, 29.66, 29.9, 30.2, 30.6, 31.9, 40.5, 56.4, 80.5,
156.9, 173.1; IR 3310, 2917, 2850, 1721, 1700, 1646, 1565, 1149; HRMS: for
62
C41H79N2O7 (M + H)+ calcd 711.5887, found 711.5893. Anal. Calcd for
C41H78N2O7·½H2O: C, 68.39; H, 11.06; N, 3.89. Found: C, 68.46; H, 11.03; N, 3.89.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-icosylureido)heptanedioate,
3EUr20
The general procedure described above afforded a white solid (2.79 g, 69%); mp 66.5–
67.3 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 34H), 1.35–1.50 (bm, 29H),
1.94 (t, 6H), 2.23 (t, 6H), 3.07 (q, 2H), 4.01 (m, 1H), 4.54 (s, 1H); 13C NMR (CDCl3) δ
14.2, 22.7, 26.9, 28.1, 29.4, 29.63, 29.65, 29.69, 29.73, 29.9, 30.2, 30.6, 32.0, 40.6, 56.5,
80.5, 156.8, 173.2; IR 3310, 2917, 2851, 1721, 1701, 1646, 1567, 1150; HRMS: for
C43H83N2O7 (M + H)+ calcd 739.6200, found 739.6199. Anal. Calcd for
C43H82N2O7·½H2O: C, 69.03; H, 11.18; N, 3.74. Found: C, 69.07; H, 11.26; N, 3.73.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3-docosylureido)heptanedioate,
3EUr22
The general procedure described above afforded a white solid (3.49g, 84%); mp 67.9–
68.8 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 38H), 1.35–1.50 (bm, 29H),
1.94 (t, 6H), 2.24 (t, 6H), 3.07 (q, 2H), 4.12 (m, 1H), 4.60 (s, 1H); 13C NMR (CDCl3) δ
14.1, 22.7, 26.9, 28.1, 29.3, 29.59, 29.61, 29.7, 29.9, 30.2, 30.6, 31.9, 40.6, 56.5, 80.5,
156.8, 173.1; IR 3310, 2916, 2850, 1723, 1701, 1646, 1565, 1152; HRMS: for
C45H87N2O7 (M + H)+ calcd 767.6513, found 767.6532. Anal. Calcd for
C45H86N2O7·½H2O: C, 69.63; H, 11.30; N, 3.61. Found: C, 69.99; H, 11.56; N, 3.60.
General procedures for preparation of long-chain triacids, 3CUrn (1a−e)
HN
HN
O
OHO
n-1HCO2Hrt, 9 h
HN
HN
O
OtBuO
n-1
3 3
63
A tri tert-butyl ester 2 (5.02 mmol) was added to HCOOH (20 mL). The resulting
mixture was stirred to give a transparent solution. The mixture might need to be warmed
slightly to get all the 3CUrn to dissolve. The transparent solution was stirred at rt for 9
h; the complete reaction was identified by the formation of milky solution. The solution
was concentrated to yield a white solid, which was recrystallized with HOAc−hexane to
give a white solid (74–85%).
4-(2-Carboxyethyl)-4-(3-tetradecylureido)heptanedioic acid, 3CUr14
The general procedure described above afforded a white solid (2.06 g, 84%); 159.0–159.5
°C; 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 22H), 1.44 (bm, 2H), 1.95 (t, 6H),
2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.0, 22.1, 26.4, 28.2, 28.7, 28.8,
29.04, 29.09, 30.0, 31.3, 38.8, 55.0, 157.0, 174.5; IR 3395, 3352, 2916, 2849, 1709,
1693, 1610, 1559; HRMS: for C25H47N2O7 (M + H)+ calcd 487.3383, found 487.3384.
Anal. Calcd for C25H46N2O7: C, 61.70; H, 9.53; N, 5.76. Found: C, 61.94; H, 9.55; N,
5.70.
4-(2-Carboxyethyl)-4-(3-hexadecylureido)heptanedioic acid, 3CUr16
The general procedure described above afforded a white solid (2.14 g, 83%); mp 162.4–
162.8 °C; 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 26H), 1.44 (bm, 2H), 1.95 (t,
6H), 2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.4, 22.6, 26.9, 28.6, 29.2, 29.3,
29.5, 30.47, 30.54, 31.8, 39.3, 55.5, 157.5, 175.0; IR 3395, 3355, 2916, 2849, 1701,
1692, 1610, 1560; HRMS for C27H51N2O7 (M + H)+ calcd 515.3696, found 515.3669.
Anal. Calcd for C27H50N2O7: C, 63.01; H, 9.79; N, 5.44. Found: C, 62.91; H, 9.79; N,
5.33.
64
4-(2-Carboxyethyl)-4-(3-octadecylureido)heptanedioic acid, 3CUr18
The general procedure described above afforded a white solid (2.33 g, 85%); mp 160.7–
161.5 °C; 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 30H), 1.44 (bm, 2H), 1.95 (t,
6H), 2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.6, 22.8, 27.1, 28.8, 29.4, 29.5,
29.71, 29.74, 30.7, 32.0, 39.5, 55.7, 157.7, 175.2; IR 3396, 3348, 2914, 2849, 1709,
1692, 1607, 1558; HRMS: for C29H55N2O7 (M + H)+ calcd 543.4009, found 487.3384.
Anal. Calcd for C29H54N2O7: C, 64.18; H, 10.03; N, 5.16. Found: C, 64.17; H, 10.12; N,
5.12.
4-(2-Carboxyethyl)-4-(3-icosylureido)heptanedioic acid, 3CUr20
The general procedure described above afforded a white solid (2.36 g, 82%); mp 160.3–
161.2 °C; 1H NMR (CD3OD) 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm 34H),
1.44 (bm, 2H), 1.95 (t, 6H), 2.28 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 14.4, 22.6,
26.9, 28.6, 29.2, 29.3, 29.5, 30.48, 30.54, 31.8, 39.3, 55.5, 157.5, 175.0; IR 3394, 3348,
2914, 2848, 1708, 1691, 1608, 1560; HRMS: for C31H59N2O7 (M + H)+ calcd 571.4322,
found 571.4290. Anal. Calcd for C31H58N2O7: C, 65.23; H, 10.24; N, 4.91. Found: C,
65.08; H, 10.28; N, 4.80.
4-(2-Carboxyethyl)-4-(3-docosylureido)heptanedioic acid, 3CUr22
The general procedure described above afforded a white solid (2.24 g, 74%); mp 157.3–
158.3 °C; 1H NMR (CD3OD) 1H NMR (CD3OD) δ 0.90 (t, 3H), 1.25–1.35 (bm, 38H),
1.44 (bm, 2H), 1.95 (t, 6H), 2.27 (t, 6H), 3.05 (t, 2H); 13C NMR (DMSO-d6) δ 13.9, 22.1,
26.4, 27.7, 28.1, 28.7, 28.8, 29.0, 30.0, 31.3, 38.8, 55.0, 157.0, 174.5; IR 3395, 3347,
2914, 2849, 1708, 16921, 1609, 1560; HRMS: for C33H63N2O7 (M + H)+ calcd 599.4635,
found 599.4636. Anal. Calcd for C33H62N2O7: C, 66.19; H, 10.44; N, 4.68. Found: C,
65
66.44; H, 10.60; N, 4.57.
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quantitative contribution of an aryl group on C(2) of 8-azaadenines to binding with adenosine deaminase: a new syntesis of 8-azaadenosine. Farmaco 1992, 47.
52 Arpicco, S.; Canevari, S.; Ceruti, M.; Galmozzi, E.; Rocco, F.; Cattel, L.
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53 Pijper, D.; Bulten, E.; Smisterova, J.; Wagenaar, A.; Hoekstra, D.; Engberts, J. B.
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54 Hong, Y.-R.; Gorman, C. B. Synthetic approaches to an isostructural series of
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55 Adam, G. C.; Cravatt, B. F.; Sorensen, E. J. Profiling the specific reactivity of the
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Djerassi, C. J. Biosynthetic studies of marine lipids. 17. The course of chain elongation and desaturation in long-chain fatty acids of marine sponges. J. Am. Chem. Soc. 1988, 110, 8117-8124.
57 Robinson, P. L.; Barry, C. N.; Kelly, J. W.; Evans, S. A.
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58 Prinz, H.; Six, L.; Ruess, K.-P.; Lieflaender, M. Stereoselective synthesis of long-
chain 1-O-(β-D-maltosyl)-3-O-alkyl-sn-glycerols (alkyl glyceryl ether lysoglycolipids). Liebigs Ann. Chem. 1985, 2, 217-225.
59 Huffman, J. W.; Desai, R. C. A procedure for alcohol inversion using cesium
acetate. Synth. Commun. 1983, 13, 553-558. 60 Kim, H. S.; Lee, S. M.; Ha, K.; Jung, C.; Lee, Y.-J.; Chun, Y. S.; Kim, D.; Rhee,
B. K.; Yoon, K. B. Aligned inclusion of hemicyanine dyes into silica zeolite films for second harmonic generation. J. Am. Chem. Soc. 2004, 126, 673-682.
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61 Easton, C. J.; Xia, L.; Pitt, M. J.; Ferrante, A.; Poulos, A.; Rathjen, D. A.
Polyunsaturated nitroalkanes and nitro-substituted fatty acids. Synthesis 2001, 3, 451-457.
62 Keyes, R. F.; Golebiewski, W. M.; Cushman, M. Correlation of anti-HIV potency
with lipophilicity in a series of cosalane analogs having normal alkenyl and phosphodiester chains as cholestane replacements. J. Med. Chem. 1996, 39, 508-514.
63 Pissas, D.; Dais, P.; Mikros, E. Quantitative treatment of the rotational dynamics
of flexible-chain molecules. 13C NMR relaxation study of hydrocarbon chains attached to the fluorene anchor. Magn. Reson. Chem. 1994, 32, 263-275.
64 Kocian, O.; Stransky, K.; Zavada, J. Unsaturated analogs of 1-triacontanol.
Collect. Czech. Chem. Commun. 1982, 47, 1346-1355. 65 Dulayymi, J. a. R. A.; Baird, M. S.; Roberts, E. The synthesis of a single
enantiomer of a major a-mycolic acid of M. tuberculosis. Tetrahedron 2005, 61, 11939-11951.
66 Tanaka, A.; Oritani, T. A mild and efficient method for converting alcohols and
tetrahydropyranyl ethers to bromides with inversion of configuration. Tetrahedron Lett. 1997, 38, 1955-1956.
67 Bringmann, G.; Schneider, S. Improved methods for dehydration and
hydroxy/halogen exchange using novel combinations of triphenylphosphine and halogenated ethanes. Synthesis 1983, 2, 139-141.
68 Menger, F. M.; Yamasaki, Y. Hyperextended amphiphiles. Bilayer formation
from single-tailed compounds. J. Am. Chem. Soc. 1993, 115, 3840-3841. 69 King, J. F.; Loosmore, S. M.; Aslam, M.; Lock, J. D.; McGarrity, M. J. Betylates.
3. Preparative nucleophilic substitution by way of [2]-, [3]-, and [4]betylates. Stoichiometric phase transfer and substrate-reagent ion-pair (SRIP) reactions of betylates. J. Am. Chem. Soc. 1982, 104, 7108-7122.
70 King, J. F.; Loosmore, S. M.; Lock, J. D.; Aslam, M. Mixed phenazine-N-
methylphenazinium7,7,8,8-tetracyano-p-quinodimethanide. A quasi-one-dimensional "metal-like" system with variable band filling. J. Am. Chem. Soc. 1978, 100, 1639-1641.
71 Jeong, T.-S.; Kim, E. E.; Lee, C.-H.; Oh, J.-H.; Moon, S.-S.; Lee, W. S.; Oh, G.-
T.; Lee, S.; Bok, S.-H. Hypocholesterolemic activity of hesperetin derivatives. Bioorg. Med. Chem. Lett. 2003, 13, 2663-2665.
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72 Smith, M. B.; March, J. Advanced organic chemistry: reactions, mechanisms, and structure; John Wiley & Sons, Inc:, 2001.
73 Deckert, A. A.; Farrell, C.; Ross, J.; Waddell, R.; Stubna, A. Binding of
myoglobin to thin films. Langmuir 1999, 15, 5578-5583. 74 Watanabe, A.; Ishizawa, A.; Kosaka, M.; Goto, R. Synthesis and physical
properties of long chain compounds. X. Studies of the x-ray diffraction patterns of normal higher primary amines. Bull. Chem. Soc. Jpn. 1969, 42, 1360-1363.
75 Zvejnieks, A. Preparation and properties of some pure fatty and rosin amines.
Sven. Kem. Tidskr 1954, 66, 316-322. 76 Newkome, G. R.; He, E.; Godinez, L. A.; Baker, G. R. Electroactive
metallomacromolecules via tetrabis(2,2':6',2''-terpyridine)ruthenium(II) complexes: dendritic nanonetworks toward constitutional isomers and neutral species without external counterions. J. Am. Chem. Soc. 2000, 122, 9993-19996.
77 Newkome, G. R.; Patri, A. K.; Godinez, L. A. Design, syntheses, complexation,
and electrochemistry of polynuclear metallodendrimers possessing internal metal binding loci. Chem. ___Eur. J. 1999, 5, 1445-1451.
78 Newkome, G. R.; Gross, J.; Moorefield, C. N.; Woosley, B. D. Approaches
towards specifically functionalized cascade macromolecules: dendrimers with incorporated metal binding sites and their palladium(II) and copper(II) complexes. Chem. Commun. 1997, 6, 515-516.
79 Newkome, G. R.; Woosley, B. D.; He, E.; Moorefield, C. N.; Guether, R.; Baker,
G. R.; Escamilla, G. H.; Merrill, J.; Luftmann, H. Supramolecular chemistry of flexible, dendritic-based structures employing molecular recognition. Chemical Communications. Chem. Commun. 1996, 24, 2737-2738.
80 Newkome, G. R.; Guther, R.; Moorefield, C. N.; Cardullo, F.; Echegoyen, L.;
Perez-Cordero, E.; Luftmann, H. Chemistry of micelles. Routes to dendritic networks: bis-dendrimers by coupling of cascade macromolecules through metal centers. Angew. Chem., Int. Ed. Engl. 1995, 34, 2023-2036.
81 Newkome, G. R.; Nayak, A.; Behera, R. K.; Moorefield, C. N.; Baker, G. R.
Chemistry of micelles series. 22. Cascade polymers: synthesis and characterization of four-directional spherical dendritic macromolecules based on adamantane. J. Org. Chem. 1992, 57, 358-362.
82 Newkome, G. R.; Behera, R. K.; Moorefield, C. N.; Baker, G. R. Chemistry of
micelles. 18. Cascade polymers: syntheses and characterization of one-directional arborols based on adamantane. J. Org. Chem. 1991, 56, 7162-7167.
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84 Shinoda, K. The effect of alcohols on the critical micelle concentrations of fatty
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Madison, MI, 2001.
73
Chapter III Synthesis of Two-Tailed, Tri-headed Amphiphiles (3CUr(n)2 and 3CUr1(n)2)
Two new series of two-tailed, tri-headed amphiphiles have been designed. These
series share common parts with the previous 3CUrn series (Chapter II), where a ureido
and a tricarboxylic Newkome-type dendron are still applied as the linker and the
hydrophilic moeity, respectively. Long, straight alkyl chains are still employed as the
hydrophobic moieties. Instead of one tail, the new amphiphiles contain two tails.
Another modification is made based on how the alkyl chains on the hydrophobic moiety
are connected to the linker. In order to correspond with the previous single-tailed, tri-
headed amphiphiles 3CUrn series, the total number of the carbons on the hydrophobic
moiety is equal as that in the 3CUrn series. Both alkyl chain lengths on each tail are also
equal. By doing this, two homologous series, which have different geometries of
hydrophobic moieties, are designed.
Based on how the tails are connected to the linker, we present these two different
series. How do these two differ? One is where two tails are directly connected to the
same nitrogen on the ureido linker; the other is where two tails are attached to one carbon
that is bonded to a nitrogen on the ureido linker (Figure III.1). In other words, the middle
linker
head
head
head
tail
tail
linker
head
head
head
tail
tail
Figure III.1 General representation of two-tailed, tri-headed amphiphiles
74
carbon of a long alkyl chain is connected to the nitrogen on the linker. This kind of tail is
later referred to a “swallowtail”.1
III.1 The 3CUr(n)2 Homologous Series
A Newkome-type dendron has been generated from the reaction of
WeisocyanateTM (3) and the cyclic amine piperidine.2 Based on the same chemistry,
acyclic secondary amines and 3 should react readily. In the first homologous series
(Scheme III.1), two tails are directly attached to the same nitrogen on the ureido linker.
This homologous series is abbreviated as 3CUr(n)2 (Scheme III.1), where “3C”
represents three carboxylic groups on the hydrophilic moiety, “Ur” represents a ureido
linker between the hydrophobic and hydrophilic moieties, “n” represents the number of
carbons of the alkyl group on the symmetrical tail, and subscript “2” represents the two
tails attached to the linker. In the same manner as the 3EUrn naming, the homologous
series of tri-tert-butyl esters is abbreviated as 3EUr(n)2. Retrosynthesis (Scheme III.1)
Scheme III.1 Retrosynthesis of the 3CUr(n)2 homologous series
NHN
O
OCNO
O3
O
O
O
O
O
O
H
H
H
NHN
O
O
O
O
O
O
O
+
4a−e
3
3CUr(n)2 3EUr(n)2
R = CnH2n+1
1 2 a, n=7b, n=8c, n=9d, n=10e, n=11
R
R
R
RHN
R R
75
shows that beginning with 3 and necessary secondary amines, the addition reaction takes
place to give the 3EUr(n)2 series. The tert-butyl groups of the 3EUr(n)2 series are later
removed to generate the 3CUr(n)2 series.
III.1.1 Preparation of Secondary Amines
Five secondary amines containing two symmetrical alkyl chains (4a−e)⎯N-
heptylheptan-, N-octyloctan-, N-nonylnonan-, N-decyldecan-, and N-undecylundecan-1-
amine⎯ were utilized to construct the 3CUr(n)2 series. Amines 4a−e were
commercially available. The secondary amines containing even number of carbons on
each tail were available at low cost. On the other hand, those containing an odd number
of carbons on each tail⎯4a, 4c, and 4e⎯ were commercially available only in milligram
quantities and expensive. This motivated us to develop our own synthesis of such
compounds.
III.1.1.1 Literature Review on Syntheses of N-Alkylalkan-1-amines (4a, 4c, and 4e)
Studies documented that compounds 4a, 4c, and 4e could be generated from the
corresponding primary amines,3-6 oximes,7-9 1-aminoalcohol,9 aldehyde,10 nitrile,11,12
ester,13 and amides.14-16 In most of these reported procedures, secondary amines were
obtained as side products when generating primary amines. Only the work of Murahashi
et al.3 and Djedovič et al.14 (Scheme III.2) reported the desired secondary amines were
obtained in excellent yields. These two procedures utilized ruthenium3 complex catalyst
and lithium aluminum hydride,14 respectively.
Alkylation of a primary amine is not the best way to generate a secondary amine,
because the resulting secondary amine is more reactive than the primary amine; the
76
reaction proceeds to give the tertiary amine as a major side product. Alkylation of
heptan-1-amine6 and decan-1-amine5 with the corresponding bromoalkanes produced a
low yield of the N-heptylheptan-1-amine and N-decyldecan-1-amine (yield not reported),
respectively.
Scheme III.2 Preparation of N-heptylheptan-1-amines from heptan-1-amine3 and N-heptylheptanamide14
NH2RuH2(PPh3)4, EtOH, H2
HN
HN
O5 6
LiAlH4, THF
yield 98%
yield 99%
+OH
The ruthenium complex, RuH2(PPh3)4, is selective for the oxidation of the
alcohol. When a reaction of RNH2 and R′OH is performed in the presence of
RuH2(PPh3)4, the N-alkyl iminium ion complex is formed, and the secondary amine
RR′NH is formed selectively.3 Murahashi et al.3 have shown that hydrogenation of
heptanal oxime in the presence of RuH2(PPh3)4 leads to the formation of heptan-1-amine
and the corresponding imine. These latter two also react to release ammonia gas and the
corresponding secondary imine. Hydrogenation of N-alkyl imine gives the desired N-
heptylheptan-1-amine in an excellent yield. Besides hydrogenation, reduction of heptanal
oxime is also performed with a mild reducing agent (sodium borohydride in the presence
of copper (II) sulfate) where an imine is reported to be the key intermediate.7 An imine is
also reported as the key intermediate when heptanal10 and heptanitrile12 are employed as
77
precursors, in which the presence of ammonia minimizes the formation of N-
heptylheptanamine to less than 5%.
Hydrogenation of heptanamide15 and N,N-diethylheptanamide 16 in the presence
of copper-chromium catalyst gives N-heptylheptan-1-amine as the side product, which is
isolated in 25−50% and 58% yields, respectively. On the other hand, N-
heptylheptanamide is reduced with lithium aluminum hydride to give N-heptylheptan-1-
in an excellent yield.14
We developed our procedure on the reduction of the N-alkylalkanamide with
lithium aluminum hydride based on the published procedure17 for the synthesis of
unsymmetrical secondary amines and N-undecylundecanamide. Hence, we needed to
synthesize the necessary N-alkylalkanamides. Later, Djedovič et al.14 reported an
excellent yield of N-alkylalkanamine from a reduction of N-alkylalkanamide with the
same reducing agent.
III.1.1.2 Literature Review on Syntheses of N-Alkylalkanamides (5)
Amide bonds are usually made from the reaction of amines and carbonyl
compounds, such as carboxylic acids, acid halides, and anhydrides. In the literature, a
number of preparations of long-chain N-alkylalkanamide beginning with primary amines
have been documented.14,17-32 However, only few contain the synthesis of our interest
(5), where the alkyl attached to the nitrogen atom contains one more carbon than the alkyl
attached to the carbonyl carbon.
78
RHN R'
O
5
R = CnH2n+1, n>6R' = CnH2n+1, n>6
5a,R = C7H15, R' = C6H135c, R = C9H19, R' = C8H175e, R = C11H23, R' = C10H21
letter "a", "c", and "e" refer to the corresponding amines
Most procedures for generating N-alkylalkanamides in the literature are
performed by standard methods. Long-chain carboxylic acid,21,28 acid
chloride,14,17,19,23,26,27,29,31,32 nitrile,30 methyl ester,24,25 ethyl ester,18 4-nitrophenyl ester,20
and 1-(5,7-dinitro-2,3-dihydroindol-1yl)-dodecanone22 react with necessary primary
amines to give amides 5.
III.1.2 Synthesis of N-Alkylalkan-1-amines (4a, 4c, and 4e)
Following the published procedures,26,27 acid chlorides, which are very reactive
and commercially available at low cost, were employed as acylating reagents of the
required primary amines with triethylamine as a base (Scheme III.3). The formation of
amide 5a was confirmed by comparing the 1H NMR and IR spectra to published values.31
Djedovič et al.14 later reported that amide 5a can be obtained in excellent yield from the
reaction of heptan-1-amine, heptanoyl chloride, triethylamine, and N,N-dimethyl-4-
aminopyridine as a catalyst in dichloromethane. The 1H NMR chemical shifts of amides
5c and 5e were compared to those of 5a, and necessary adjustments were made for the
proton integration of the higher homologs.
79
Scheme III.3 Synthetic scheme of N-alkylalkan-1-amines 4a, 4c, and 4e
R NH2R' Cl
O
R' NH
O
R R' NH
R+ i ii
6a, R = C7H156c, R = C9H196e, R = C11H23
6 7 45
7a, R' = C6H137c, R' = C8H177e, R' = C10H21
i. Et3N, dry THF, rt, 15 min (5a, 85%; 5c, 83%; 5e, 71%)ii. LiAlH4, Et2O, reflux, overnight (4a, 84%; 4c, 83% 4e, 80%)
RHN
R
Amides 5a, 5c, and 5e were reduced to the corresponding amines 4 with lithium
aluminum hydride in good yields. The reduction was confirmed by the disappearance of
the amide I and amide II bands absorption (1640 cm-1 and 1555 cm-1, respectively) in the
IR spectra. The 1H NMR spectrum of amine 4a was compared to that of published
values7 before being used in the subsequent step. Similar chemical shifts were observed
in the 1H NMR spectra of the other homologs 4c and 4e. Amines 4a and 4c were
obtained as colorless liquids, in which some portions started solidifying. Later, Djedovič
et al.14 reported amine 4a as a solid with a low melting temperature.
III.1.3 Synthesis of Two-tailed, Tri-headed 3CUr(n) 2 Homologous Series
Similar to the synthesis of the 3CUrn series, the synthesis of 3CUr(n)2 begins
with 3 and amines 4. The reactions were run in dichloromethane at room temperature
overnight (Scheme III.4). In the first step, the tri-tert-butyl ester 3EUr(n)2 series is
obtained. After purifications performed by flash column chromatography (hexane:ethyl
acetate, 5:1), the 3EUr(n)2 homologs were obtained in good yields.
80
Scheme III.4 Synthetic scheme of the 3CUr(n)2 homologous series
NHN
O
O tBuO
OCNO tBu
O 3
RHN
+
3
3EUr(n)2
3
R
NHN
O
OHO
3CUr(n)2
3R
R R
R
R = CnH2n+1
4a−e
a, n=7b, n=8c, n=9d, n=10e, n=11
i ii
i. CH2Cl2, rt, overnight; a, 79%; b, 60%; c, 70%; d, 85%; e, 73%ii. HCO2H, rt, 9 h; a, 90%; b, 86%; c, 88%; d, 86%; e, 77%
Removal of the tert-butyl groups on the 3EUr(n)2 series to generate 3CUr(n)2
series was accomplished by stirring the tri-tert-butyl esters in formic acid at room
temperature for nine hours. The resulting 3CUr(n)2 series was then recrystallized from
ethyl acetate to give good yields of pure materials. All compounds, 3EUr(n)2 and
3CUr(n)2, were fully characterized.
The melting temperatures of the 3EUr(n)2 homologous series increased up to
3EUr(9)2, then the melting temperatures decreased for longer chain (Table III.1). This
trend was different from that found in the 3CUrn series, where homologs with longer
chain melted at higher temperature. Conversion of the tri-tert-butyl esters into the
corresponding tricarboxylic acids caused a big difference in melting temperatures; in
general, melting temperatures of the 3CUr(n)2 series are higher than those of the
3EUr(n)2 series. In addition, the trend observed in the 3CUr(n)2 series suggested that
melting temperatures appeared to be determined by the interactions of the carboxylic
acids and not the chain length.33
81
Table III.1 Melting ranges of the 3EUr(n)2 and 3CUr(n)2 homologous series n Melting range (°C)
3EUr(n)2 3CUr(n)2 7 53.5–54.1 125.3–125.9 8 56.8–57.5 120.4–120.8 9 62.6–63.4 121.3–121.7
10 49.7–50.4 120.9–121.2 11 42.9–43.5 121.4–122.1
III.2 The 3CUr1(n)2 Homologous Series
The second homologous series is abbreviated as 3CUr1(n)2 (Scheme III.5), where
“3C” represents three carboxylic groups on the hydrophilic moiety, “Ur” represents a
ureido linker between the hydrophobic and hydrophilic moieties, “1” represents there is
one carbon separating the linker and the two symmetrical tails, “n” represents the number
of carbons of the alkyl group on the symmetrical tail, and subscript “2” represents the two
tails attached to the carbon next to the linker. In other words, a long tail is connected to a
nitrogen on the ureido linker via the midcarbon (R2CH−). The series with the tri-tert-
butyl esters is abbreviated as 3EUr1(n)2.
Retrosynthesis (Scheme III.5) shows that beginning with WeisocyanateTM (3) and
amines 10a−f, the addition reaction takes place to give 3EUr1(n)2 series. The chemistry
of this series is very similar to that of the 3CUrn series, where primary alkylamines were
allowed to react with isocyanate 3. The tert-butyl groups of the 3EUr1(n)2 series are
later removed to generate 3CUr1(n)2 series.
82
Scheme III.5 Retrosynthesis of 3CUr1(n)2 series
HN
HN
O
OCNO
O3
O
O
O
O
O
H
H
H
HN
HN
O
O
O
O
O
O+
10a−f
3
3CUr1(n)2 3EUr1(n)2
R = CnH2n+18 9a, n = 7b, n = 8c, n = 9d, n = 10e, n =11f, n = 12
R R
R
R
R
R
O ONH2
a long tail connected to a nitrogen on the linker via the midcarbon
III.2.1 Preparation of Primary Amines (10a−f)
We utilized six primary amines where two symmetrical carbon chains are
connected to the carbon attached to the nitrogen atom. These are amines 10a−f
⎯pentadecan-8-, heptadecan-9-, nonadecan-10-, henicosan-11, tricosan-12-, and
pentacosan-13-amine. As they were not commercially available, syntheses were
required.
III.2.1.1 Literature Review on Syntheses of 1-Alkylalkan-1-amines (10a−f)
There are several procedures1,5,34-38 for the synthesis of compounds 10a−f
reported in the literature (Table III.2). Most precursors used in the synthesis of these
amines are ketones1,5,35-38 and azidoalkanes.34 Ketones, such as pentadecan-8-one, react
with an amine to give an imine, which then is converted into the corresponding amine in
moderate yield. Another way is to transform ketones to other intermediates, such as the
corresponding N-alkylformamide and oxime derivatives, following which are converted
83
into alkanols of interest in low to moderate yields. The reaction of ketones and
formamide is performed at 160−170 °C for 15h,37 and 180−185 °C for 6−8h.36 The N-
alkylformamides are hydrolyzed with hydrochloric acid and the free amines are released
after reaction with aqueous sodium hydroxide. The yields are improved when
nitrobenzene is added to increase the solubility of ketones.36 Reduction of ketoximes
with sodium in ethanol5 gave a very low yield (≤ 5%), on the other hand, reduction of
ketoximes with RedAl1 improve the yield to 84%.
Table III.2 Preparations of 1-alkylalkan-1-amines from different precursors Precursors Reagents Remarks References
O
6 6
H2O, EtOH, Raney nickel, NH3, H2
150 °C, 56% yield 5
O
6 6
1. NH2CHO 2. concd. HCl 3. aq. NaOH
Step 1 reaction condition not specified, yield not reported Step 2 was run at 160−170 °C, 15h, 40%
37
O
n-1 n-1
n = 7, 8, 9, 11
1. NH2CHO, PhNO2 2. concd. HCl 3. aq. NaOH
Step 1 was run at 180−185 C, 4h Step 2 was run by heating for 6−8 h. 65% (n = 7), 60% (n = 8), 63% (n = 9), 50% (n = 11)
36
O
6 6
1. HO−NH2, ROH5,38 2. Na, EtOH5
Step 1 was run at rt for 2 d, no yield reported Step 2. 5%5
5,38
O
8 8
1. EtOH, HO−NH2
.HCl,1,352. PhCH3, RedAl, aq. HCl, concd. HCl1
84%1 1,35
N3
3 3
Bu3SnH, NidppeCl2 95% 34
Based on the starting materials employed (Table III.2), both azidoalkane and
oxime derivatives of ketone can be utilized as precursors for the synthesis. Thus, two
84
different retrosynthesis pathways are constructed (Scheme III.6). The first pathway of
retrosynthesis of amines 10a−f shows that the necessary ketones 16a−f can be
transformed to amines 10a−f via the oxime derivatives 15a−f. The second, where one of
the intermediates is the corresponding azidoalkanes 11a−f, shows that the synthesis can
begin with commercially available alcohols 13a−f.
Scheme III.6 Retrosynthesis of amines 10a−f
R R
NH2
R R
N3
R R
OMs
R R
OHRBr
H OEt
O+R = n-CnH2n+1
10a−f
11a−f
12a−f
13a−f 14a−f10a, n = 710b, n = 810c, n = 910d, n = 1010e, n = 1110f, n = 12
R R
O
16a−f
R R
N
15a−f
OH
CO+n-3
Most ketones (16a−e) and alcohols (13a−e) are commercially available, but they
are very expensive. Thus, we are required to synthesize the required precursor⎯either
ketones or alcohols. In general, ketones are generated from alkenes, and the reactions are
run in special conditions, such as extremely high pressures and temperatures. Because of
the harsh conditions required for generating ketones, these reactions are not easily
performed in our laboratory. Thus, we consider the second alternative route, where
alcohols are utilized as the precursors.
Alcohols 13a−f can be easily synthesized via Grignard reaction starting with
85
bromoalkanes 14a−f and ethyl formate. Based on the consideration on cost-per mole
basis, bromoalkanes 14a−f are one-tenth the price of ketones 16a−e, and the reactions for
generating alcohol 13a−f are easily performed with standard laboratory glasswares.
Thus, we have chosen this second alternative route in order to synthesize amines 10a−f.
Following this route, bromoalkaness 14a−f are converted into amines 10a−f in at least
three steps⎯Grignard reaction, azidation, and reduction.
III.2.1.2 Literature Review on Syntheses of Azidoalkanes
Very few procedures for generating long chain azidoalkanes are documented in
the literature. The syntheses of 10-azidoicosane39 and 11-azidodocosane40 are achieved
from the corresponding alcohols via Mitsunobu reaction utilizing diphenylphosphorazide
and triphenylphosphine in the presence of diisopropylazodicarboxylate (DIAD),39
diethylazodicarboxylate (DEAD).40
R R'
OH
R R'
N3(PhO)2PN3, PPh3
DIAD or DEAD
R = C9H19, R' = C10H21, 86−93%R = C10H21, R' = C11H23
O
Unfortunately, the synthesis of compounds 11a−f has never been reported.
Despite of the advantages offered by the Mitsunobu reaction, including the fact that it is a
one-pot reaction and thus a shorter route, and the products are obtained in good to
excellent yield on the example above, all the reagents needed for such a transformation
are only commercially available at high cost. Because of this reason, a different
alternative route is presented. Considering low-cost commercially available
86
methanesulfonyl chloride, and the success of the synthesis of both 10-azidoicosane and
11-azidodocosane utilizing sodium azide in N,N-dimethylformamide, we elected to apply
the same procedure for the transformation of alcohols 13a−f into azides 11a−f. Even
though that this means the synthesis is one-step longer, the reagents needed for the
transformations are available in much lower costs. In addition, the side
products⎯triethylamine hydrochloride and sodium methanesulfonyloxide⎯are easier to
handle, and yet the yields of the reactions are just as high.
Based on examples found in the literature,34,40 two secondary alcohols are
converted into the corresponding methanesulfonyloxyalkanes. These
methanesulfonyloxyalkanes are then allowed to react with sodium azide in two different
solvent, dimethylsulfoxide41 and N,N-dimethylformamide,34 to produce the corresponding
azides (Table III.3). Following this procedure, methanesulfonyloxyalkanes as precursors
are required.
R R'
OMs NaN3
R R'
N3
various solvent
Table III.3 Preparation of secondary azidoalkanes from secondary methanesulfonyloxyalkanes
R R' solvent condition yields (%) C2H5 C3H7 DMSO 34−40 °C, 6 h not reported41 C2H5 C4H9 DMF 60 °C, 6 h 8034
III.2.1.3 Literature Review on Syntheses of Methanesulfonyloxyalkanes
Transformation of secondary alcohols into the corresponding
methanesulfonyloxyalkanes is routinely performed in organic synthesis. Utilizing low-
cost reagents, the transformation is simply performed in a mild condition and short
87
reaction time. Moreover, the products are obtained in quantitative yields. However,
there were not many examples involving symmetrical straight chain alkyls. Based on
examples found in the literature,42-44 three symmetrical secondary alcohols are converted
into the corresponding methanesulfonyloxyalkanes (Table III.4).
R R
OH MsCl
base R R
OMs
Table III.4 Preparation of secondary methanesulfonyloxyalkanes from secondary alcohols
R base condition yield (%) C3H3 pyridine 0−5 °C, 3.5 h 4344 C4H9 Et3N 0 °C→rt, 12 h not reported34
C16H33 Et3N 15−60 min 6642 III.2.1.4 Literature Review on Syntheses of Secondary Alkanols
Secondary alkanols 13a−f are synthesized via different ways (Table III.5). Most
of them are routinely generated from ketones or combination of haloalkanes⎯mostly
bromoalkanes⎯and carbonyl compounds, such as aldehydes and ethyl formate. Less
common precursors are alkenes and primary alcohols.
Ketones are reduced by hydrogenation in the presence of Raney nickel,43,45 Raney
nickel with alcohol as a solvent serving as the hydrogen source,46 sodium,38,47
hydrides,48,49 and aluminum isopropoxide,43 which is known as the Meerwein-Pondorff-
Varley method. Haloalkanes are typically converted into the corresponding Grignard
reagents prior to the reaction with the carbonyl compound of interest.
When primary alkanols are employed as precursors, two mechanisms of the
transformation into the corresponding secondary alkanols in the presence of calcium
hydroxide have been proposed. The first involves dehydrogenation, aldol condensation,
88
Table III.5 Preparations of secondary alcohols 13a−f from different precursors Precursors Reagents Yield (%) Reference
O
n-1n-1n = 7−9
H2, Ni or Raney nickel
No reported yield 43,45
11 11
O
PhCH3, iPrOH, Raney nickel
80 46
O
n-1 n-1n = 7, 8
Na, EtOH
No yield reported 38
7 7
O
Na, TiCl3, THF
5 47
O
77
Li+iPrO-
No reported yield 43
7 7
O
LiAlH4, THF
90 48
6 6
O
iBu2AlH
No yield reported 49
Brn-1
n = 7−11
H
O
n-1
Mg ,
No reported yield 50,51
Xn-1
X = Br, n = 7, 8 ,10, 1252,53 X = Cl, n = 7, 8
H
OMg ,
9053, 8452 No reported yield
X = Br50-54 X = Cl43
5 1. LiAlH(OCH3)3, THF, CO 2. HCl 3. NaOH/H2O2
78 55
5 1. borane−THF; 1,3-dithiane, n-BuLi; HgCl2 2. NaOH, H2O2
74
56
7OH
Ca(OH)2 55−67 57
89
decarbonylation, and neutralization. The second involves oxidation by water, ketonic
decomposition, hydrogenation, and neutralization. With this method, the transformation
of oct-1-ene into heptadecan-9-ol is obtained in a moderate yield.57
Beginning with oct-1-ene, carbonylation of the corresponding trialkylborane
using trimethoxyaluminohydride produced an intermediate; this intermediate is
successively treated with concentrated hydrochloric acid and peroxide to give the desired
heptadecan-9-ol in a good yield.55 Another way is to treat the corresponding
trialkylborane with lithiated 1,3-dithiane in the presence of mercury chloride, followed by
treatment with peroxide.56
III.2.2 Synthesis of Amines (10a−f)
We began the synthesis with Grignard reaction of bromoalkanes 14a−f and ethyl
formate, which Mr. Richard V. Macri and Ms. Erika Bechtold optimized. In order to get
the optimal result, the reaction was run for three days (Scheme III.7). After
recrystallization from ethyl acetate, 13C NMR spectra of pentadecan-9-ol and heneicosan-
11-ol were compared to published values.58 The chemical shifts on the 1H NMR of
compounds 13a−f were compared to those42 of another homolog, namely tritriacontan-
17-ol. In addition, the melting temperature data were compared to published values58-61
before being used for the following step.
Alkanols 13a−f were then converted into the corresponding
methanesulfonyloxyalkanes 12a−f. It is to our advantage that the reaction time was short
and the products were obtained in near quantitative yields. The formation of products
was verified by 1H NMR spectra. The chemical shifts of the hydrogen on the carbon
bonded to a new electron-withdrawing group (−OSO2CH3) were shifted from δ 3.58 ppm
90
to 4.60 ppm; this confirmed that the corresponding methanesulfonylalkanes (12a−f) were
formed. As 1H NMR spectra of 12a−f were not in the literature, we compared the
chemical shifts of 12a−f to those42 of corresponding longer-chain homolog tritriacontan-
17-ol. Necessary adjustment of the proton integration was made depending on the chain
length of the homolog. Based on that analysis, 12a−f were used without purification for
the next step.
Azides 11a−f were generated via an SN2 reaction with sodium azide. The reactive
site on this SN2 reaction was a secondary carbon. Thus, compared to the SN2 reaction for
the formation of 1-azidoalkanes (Chapter II), whose reactive site was a primary carbon,
we expected this reaction would require a longer time. However, after several trials, we
Scheme III.7 Synthetic scheme of the alkanamines 10a−f
R R
NH2
R R
N3
R R
OMs
R R
OHRBr
H OEt
O+
R = n-CnH2n+1
10a−f
11a−f12a−f13a−f14a−f
14a, n = 714b, n = 814c, n = 914d, n = 1014e, n = 1114f, n = 12
i ii iii
ivi. Mg, dry THF, 60 °C, 3 dii. MsCl, Et3N, dry THF, rt, 15 miniii. NaN3, DMF, 80 °C, 4 hiv. Pd/C, H2, EtOH, rt, 3 h
Table III.6 Isolated yields and melting ranges of intermediates and alkanamines 10a−f i ii iii iv n
yield (%)
melting range (°C)
yield (%)
yield (%)
melting range (°C)
yield (%) melting range (°C)
7 49 53.5−54.3 90−95 78 oil 66 oil 8 52 61.7−62.2 92−96 83 oil 75 oil 9 54 67.0−67.4 90−96 92 oil 84 oil
10 55 71.1−72.0 94−98 97 oil 88 oil 11 52 76.8−77.6 91−96 84 oil 81 65.5−64.3 12 56 80.2−81.0 91−94 90 36.8−37.4 86 79.7−80.4
91
concluded the reaction was completed in 4 hours, the same reaction time as that in the
synthesis of 1-azidoalkanes. Thin layer chromatography (in hexane) and the absorption
(~ 2100 cm-1) in the infrared spectra verified the presence of new products containing
azido groups. Azides 11a−f were obtained in good to excellent yields after purification
by flash column chromatography with hexane as the eluent. The 1H NMR chemical
shifts of the hydrogen on the carbons bonded to the azido groups were shifted upfield (δ
~ 3.22 ppm). Azides 11a−f were fully characterized.
Finally, conversion of azides 11a−f into amines 10a−f was performed by
hydrogenation in the presence of 10% palladium/carbon catalyst (Scheme III.7, Table
III.6). The absorption at ~ 2100 cm-1 in the infrared spectrum disappeared; this
confirmed that azido groups had been reduced. Compounds 10a−f were obtained in good
yields and used without purification.
III.2.3 Synthesis of Two-tailed, Tri-headed 3CUr1(n)2 Homologous Series
The 3EUr1(n)2 series was obtained by stirring isocyanate 3 and amines 10a−f in
dichloromethane at room temperature overnight (Scheme III.8). Purification by flash
column chromatography (hexane:ethyl acetate, 5:1) generated white solids in good yields.
Repeated attempts to recrystallize 3EUr1(n)2 series from acetonitrille in order to generate
a crystal for X-ray analysis were not successful.
92
Scheme III.8 Synthetic scheme of the preparation of 3CUr1(n)2
HN
HN
O
OtBuO
+
3EUr1(n)2
3HN
HN
O
OHO
3CUr1(n)2
3
R = n-CnH2n+1
10a−f
a, n = 7b, n = 8c, n = 9d, n = 10e, n = 11 f, n = 12
,
i iiR
R
R
R
i. CH2Cl2, rt, overnight; a, 60%; b, 70%, c, 85%, d, 77%; e, 86%; f, 87%ii. HCO2H, rt, 9 h; a, 85%; b, 86%, c, 83%, d, 80%; e, 85%; f, 87%
3
Removal of the tert-butyl groups on the 3EUr1(n)2 was performed by stirring in
formic acid at room temperature for about 9 hours. The crude material was recrystallized
from acetic acid-hexane to give good yields of pure 3CUr1(n)2 homologs.
In 3EUr1(n)2 homologs, homologs containing longer alkyl chain melted at lower
temperature. Melting temperatures of 3EUr1(n)2 homologs decreased irregularly as
chain lengths increased (Table III.7). These melting temperatures were lower compared
to those of 3CUr1(n)2 homologs, suggesting that the presence of the carboxylic acid
groups caused the raise in the melting temperatures. The observed changes in melting
temperatures do show a particular trend in compounds containing either odd or even alkyl
chains. Compounds containing odd-numbered alkyl chain have lower melting
temperatures as the chain length increases (Table III.7). The same trend was observed for
those containing even-numbered alkyl chain. In both cases, it appeared that the longer
the chain length, the less significant is the difference in the melting temperatures.
93
Table III.7 Melting ranges of the 3EUr1(n)2 and 3CUr1(n)2 homologous series
n Melting range (°C) 3EUr1(n)2 3CUr1(n)2
7 109.0–109.8 168.5–169.2 8 108.5–109.3 164.3–165.1 9 100.4–101.2 165.1–165.7
10 87.0–87.6 163.8–164.3 11 86.1–86.9 164.6–165.1 12 73.8–74.3 163.3–163.9
III.3 Comparison of Trends in Melting Temperatures (Tri-tert-butyl Esters vs Tri-carboxylic Acids)
Each tri-tert-butyl esters and tricarboxylic acids presents unique characteristic
(Table III.8). Melting temperatures of 3EUrn homologs (Chapter II) were affected by
the chain length; the longer the chain, the higher the melting temperatures. This trend
differed from those observed in both 3EUr(n)2 and 3EUr1(n)2 homologs. Melting
temperatures of 3EUr(n)2 homologs appeared to fit a parabolic figure, where they
increased up to 3EUr(9)2 and decreased for a longer homologs. The trend observed in
the 3EUr1(n)2 homologs⎯melting temperatures decrease as the chain length
increase⎯was in contrast to that observed in the 3CUrn homologs. In addition,
compared to both 3EUrn and 3EUr(n)2 homologs, 3EUr1(n)2 homologs melted at higher
temperature.
Table III.8 Comparison of trends in melting temperatures (tri-tert-butyl esters vs tri-carboxylic acids)
Trends in melting temperatures tri-tert-butyl esters tricarboxylic acids
3EUrn: increases as chain lengths increases 3CUrn: increases and then decreases, parabolic figure; 3CUr16 melts at the highest temperature
3EUr(n)2: increases and then decreases, parabolic figure; 3EUr(9)2 melts at the highest temperature
3CUr(n)2: irregular trend, within 120-126 °C
3EUr1(n)2: decreases as chain length increases 3CUr1(n)2: decreases as chain length increases in compounds containing odd- or even-numbered alkyl chains
94
All the tricarboxylic acid homologs melt at significantly higher temperature than
the corresponding tri-tert-butyl esters. The high melting temperatures accompanied by
small and irregular changes with chain length suggested that interaction of the carboxyl
groups were dominant than the chain length.
III.4 Comparison of NMR Spectra of 3EUr(n)2 vs 3EUr1(n)2
In the 1H NMR spectra of the 3EUr(n)2 homologous series, symmetrical protons
on the hydrophobic moieties showed to have identical chemical shifts. Protons Has and
Hbs appeared at ~ δ 3.1 and 1.5 ppm, respectively, and both set showed as multiplet.
Chemical shifts of protons on the rest of the methylene groups, δ ~ 1.2−1.35 ppm, were
clearly differentiated from those on tert-butyl groups, δ ~ 1.4 ppm.
Fourteen different chemical shifts appeared in the 13C NMR spectrum of
3EUr(7)2. This suggested that symmetrical carbons on the alkyl chains had identical
chemical shifts. Thus the rotation of the C−N bond, formed between the carbonyl carbon
and the nitrogen bonded to the two alkyl chain, was fast on an NMR time scale compared
to that of an N,N-dialkylamide.62,63 The significant difference of restricted rotational
barrier of carbon−nitrogen in compounds containing amido and ureido functional groups,
which are within 60−100 kJ/mol and < 40 kJ/mol, respectively, caused different
observation in the NMR spectroscopy.64 Two factors contributed to lower the rotational
N
O HN
n-3 n-3
3
n = 7−11
O
O
H HH H
H H H H
a aaa
b bbb
95
barrier on C−N bonds in a ureido group were cross-conjugation, which is the competition
between both nitrogen for conjugation with the carbonyl group, and steric hindrance of
the substituents on nitrogens, which prevents the planarity for conjugation of the
N−CO−N group.65
As expected, when alkyl chain on each tail was longer by one methylene, an
additional peak was observed in the 13C NMR spectra. This behavior was observed in the
spectra of tri-tert-butyl ester 3EUr(7)2−3EUr(10)2. The chemical shifts of each
individual carbon in different environment were observed. However, the homolog with
the longest chain⎯3EUr(11)2 ⎯did not follow this observation. Eighteen different
chemical shifts were expected in the 13C NMR spectra of tri-tert-butyl ester 3EUr(11)2,
and yet only seventeen different chemical shifts were observed.
Unlike 1H NMR spectra of tri-tert-butyl esters 3EUr(n)2, protons bonded to the
symmetrical carbons on the hydrophobic moieties were not completely in the same
environment. Each set⎯Has, Hbs, Hcs, Hds, etc⎯of symmetrical protons on the two
symmetrical tails were in the same environment. The methylenes showed two distinct
chemical shifts⎯1.43 and 1.20–1.35ppm.
HN HH H
HHH H
H
HN
O
H
a ab b
c cdd
n-3n-3
x
O
O
3
96
From proton integrations of all 3EUr1(n)2 homologs, we noticed that two protons
were buried along with the protons of the tert-butyl groups at δ 1.43 ppm, raising the
proton integration to 29 protons. Protons Has, as well as Hbs, are in the same
environment, as only a set of two protons was observed at δ 1.43 ppm. Thus, the
chemical shifts at δ 1.43 ppm must have arisen from either Has or Hbs. The other two
protons⎯either Has or Hbs⎯appeared at δ ~ 1.20−1.35 ppm along with the rest of the
methylene groups. So, geminal protons Ha and Hb appeared to be diastereotopic. This
evidence was supported by a further experiment on the two-dimensional 1H−1H COSY
NMR. Proton Hx appeared at δ ~ 3.5 ppm. The two-dimensional 1H−1H COSY NMR
spectrum showed that proton Hx appeared to couple to other protons at 1.20–1.35 and
1.43 ppm. It was obvious that Hx coupled to protons on the neighboring carbon, Has and
Hbs. This suggested that the geminal protons Ha and Hb were diastereotopic, thus found
in different chemical shifts.
The 13C NMR spectrum of tri-tert-butyl ester 3EUr1(7)2 showed fifteen different
chemical shifts. This suggested each symmetrical carbons on the alkyl chains was in the
same environment, thus they appeared to have identical chemical shifts. As each tail was
longer by one methylene, an additional chemical shift was observed in the 13C NMR
spectrum. This behavior was observed in the first four 3EUr1(n)2 homologs. As the
chain became longer, carbons on the symmetrical chain began overlapping, which caused
fewer peaks observed in the 13C NMR spectra. Thus, the two highest homologs
⎯3EUr1(11)2 and 3EUr1(12)2⎯did not appear to follow the pattern.
The chemical shifts in 1H NMR spectra of the 3CUr(n)2 series followed the same
pattern as those of the 3EUr(n)2 series. Symmetrical carbons on both tails appeared to
97
have identical chemical shifts. Protons Has and Hbs appeared at δ ~ 3.1 and 1.38 ppm,
respectively.
As shown in the 1H NMR spectra of the 3CUr1(n)2 homologs, geminal protons
Has and Hbs remained diastereotopic; the chemical shift appeared at δ ~ 1.20−1.40, along
with the rest of the methylene protons, and 1.44 ppm. Unlike 3EUr1(7)2−3EUr1(10)2,
where all carbons in different environment were observed in the 13C NMR spectra, fewer
carbons with different chemical shift were observed in the 3CUr1(n)2 series.
III.5 Experimental Procedures Materials and Methods. Chemicals were obtained from Aldrich, Acros, Lancaster, and TCI; they were used
without further purification. Solvents were reagent grade or HPLC grade; they were used
as received unless otherwise specified. THF was distilled from sodium/benzophenone
ketyl. WeisocyanateTM was prepared as described66 with a shorter reaction time as
mentioned above. Analytical thin layer chromatography was performed by polyester-
coated silica gel 60 Å and detected by treating with 10% ethanolic phosphomolybdic acid
reagent (20 wt. % solution in ethanol) followed by heating. Flash column
chromatography was carried out on silica gel (60 Å); samples were loaded as a
concentrated solution in the solvent system needed; column diameter × height (13/4 × 6
inches), eluted sample varied between ~ 2.00−4.00 g, flow rate (~ 1.5−2 inches/min) was
controlled by air pressure. Solutions were concentrated by rotary evaporation. Melting
ranges, determined in open capillary tubes, were uncorrected. NMR spectra were
recorded on an INOVA at 400 and 100 MHz for 1H and 13C, respectively, and reported in
ppm relative to the known solvent residual peak. Resonances were reported in the order
98
of chemical shift (δ), followed by the splitting pattern, and the number of protons.
Abbreviations used in the splitting pattern were as the following: s = singlet, d = doublet,
t = triplet, q = quartet, q = quin, m = multiplet, and b = broad. IR spectra were recorded
on neat samples with an FTIR equipped with a diamond ATR system, and reported in
cm-1. HRMS data were obtained on a dual-sector mass spectrometer in FAB mode with
2-nitrobenzylalcohol as the proton donor. Elemental analyses were performed by
Atlantic Microlabs, Inc. in Norcross, GA.
General procedure for the preparation of N-alkylalkanamides (5a, 5c, 5e)
NH
O
n-2 n-1
O
n-2 ClNH2
n-1+
NEt3, dry THF
rt, 15 min
An amine 6 (13.0 mmol) and Et3N (15.6 mmol) were combined and dissolved in dry THF
(38 mL), and stirred in the ice bath. An acid chloride 7 (14.3 mmol) was added dropwise
to the cold solution. After the addition, the ice bath was removed, and the reaction was
stirred for another 15 min at rt. The resulting reaction was washed successively with 1M
HCl (7.5 mL), satd aq NaHCO3 (12 mL), and water (12 mL). The organic layer was
dried with Na2SO4 and concentrated to dryness to give a white solid, which was purified
by flash column chromatography to give a pure white solid, which gave a single spot on
TLC in 2%MeOH in CH2Cl2 (Rf = 0.23−0.31).
N-Heptylheptanamide (5a) The general procedure described above afforded a white solid (2.50 g, 85%); mp 45.0–
45.7 °C (lit.14 mp 44–46 °C); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 14H), 1.50
(m, 2H), 1.62 (m, 2H), 2.17 (t, 2H), 3.24 (m, 2H), 5.69 (bs, 1H) (lit.14,31 300 MHz14); 13C
NMR (CDCl3) δ 14.19, 14.20, 22.69, 22.75, 26.0, 27.1, 29.15, 29.17, 29.9, 31.7, 31.9,
99
37.1, 39.7, 173.3 (lit.14 75 MHz); IR 3287, 2955, 2921, 2851, 1640, 1555, 1467 (lit.31
neat). HRMS: for C14H30NO (M + H)+ calcd 228.2327, found 228.2328.
N-Nonylnonamide (5c) The general procedure described above afforded a white solid (3.05 g, 83%); mp 62.7–
63.3.9 °C (lit.67 mp 52−55 °C); 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.20–1.35 (bm, 22H),
1.48 (m, 2H), 1.62 (m, 2H), 2.15 (t, 2H), 3.25 (m, 2H), 5.36 (bs, 1H); 13C NMR (CDCl3)
δ 14.1, 22.66, 22.68, 25.9, 26.9, 29.18, 29.25, 29.32, 29.35, 29.5, 29.7, 31.84, 31.87, 37.0,
39.5, 173.03; IR 3287, 2955, 2918, 2849, 1638, 1551, 1467; HRMS: for C18H38NO (M +
H)+ calcd 284.2953, found 284.2940.
N-Undecylundecanamide (5e)
The general procedure described above afforded a white solid (3.12 g, 71%); mp 73.9–
74.7 °C (lit.68 mp 73 °C); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 32H), 1.49 (m,
2H), 1.62 (m, 2H), 2.15 (t, 2H), 3.24 (m, 2H), 5.36 (bs, 1H); 13C NMR (CDCl3) δ 14.3,
22.9, 26.1, 27.2, 29.53, 29.54, 29.59, 29.73, 29.77, 29.79, 29.80, 29.9, 32.1, 37.2, 39.7,
173.3; IR 3312, 2953, 2914, 2849, 1634, 1544, 1471 (lit.19 bands); HRMS: for C22H46NO
(M + H)+ calcd 340.3579, found 340.3584.
General procedure for the preparation of N-alkylalkan-1-amines (4a, 4c, and 4e)
NH
O
n-2 n-1+
reflux, overnightLiAlH4
Et2O HN
n-1 n-1
An N-alkylalkanamide 5 (9.58 mmol) was added slowly to a stirred suspension of LiAlH4
(574 mg, 14.7 mmol) in Et2O (50 mL). Addition took place in an ice bath. After the
addition, the reaction was warmed to rt and then heated to reflux. After being refluxed
overnight, the resulting reaction was cooled to rt. Successive addition of water (2 mL),
100
NaOH (10 M, 2.5 mL), and water (4 mL) yields a sticky white solid. The ether layer was
decanted, and the sticky white solid was washed once with ether (10 mL). The ether
layers were combined, dried with Na2SO4, and concentrated to give a clear liquid (n = 7,
9), and a white solid (n = 11) in of 80–85% yield. The resulting N-alkylalkan-1-amine
(4a, 4c, 4e) was used for the next step as it was without purification.
N-Heptylheptan-1-amine (4a)
The general procedure described above afforded a clear liquid (1.72 g, 84%) (lit.14 mp
30−31 °C); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 16H), 1.48 (m, 4H), 2.58 (t,
4H) (lit.14 300 MHz); 13C NMR (CDCl3) δ 14.3, 22.8, 27.6, 29.5, 30.4, 32.0, 50.4 (lit.7,14
60 MHz,7 75 MHz14); IR 2955, 2922, 2853, 1457, 1377, 1129 (lit.7 neat); HRMS: for
C14H32N (M + H)+ calcd 214.2535, found 214.2538.
N-Nonylnonan-1-amine (4c)
The general procedure described above afforded a clear liquid (2.14 g, 83%); 1H NMR
(CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 24H), 1.47 (m, 4H), 2.58 (t, 4H); 13C NMR
(CDCl3) δ 14.3, 22.9, 27.7, 29.5, 29.79, 29.82, 30.5, 32.1, 50.4; IR 2954, 2921, 2852,
1466, 1377, 1130; HRMS: for C18H40N (M + H)+ calcd 270.3161, found 270.3148.
N-Undecylundecan-1-amine (4e)
The general procedure described above afforded a white solid (2.50 g, 80%); mp 47.5–
48.3 °C (lit.11 51.5–52.5); 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 32H), 1.47
(m, 4H), 2.58 (t, 4H); 13C NMR (CDCl3) δ 14.3, 22.9, 27.7, 29.6, 29.8, 30.5, 32.1, 50.4;
IR 2955, 2913, 2827, 1469, 1375, 1127; HRMS: for C22H48N (M + H)+ calcd 326.3787,
found 326.3775.
101
General procedure for the preparation of long chain tri-tert-butyl esters, 3EUr(n)2
NHN
O
OtBuO
OCNOtBu
O 3R
HN +R R
RCH2Cl2, rt
overnight3
A dialkylamine 4 (4.95 mmol) was added slowly to a solution of isocyanate 3 (4.71
mmol) in CH2Cl2 (20 mL). The resulting transparent solution was stirred at rt. After
stirring overnight, the solution was concentrated to leave a crude white solid. The crude
solid was purified by flash column chromatography with a mixture of hexane:EtOAc to
give a white solid (60–85%), which gave a single spot on TLC (hexane:EtOAc, 5:1, Rf =
0.18−0.25)
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-diheptylureido)heptanedioate,
3EUr(7)2
The general procedure described above afforded a white solid (2.44 g, 79%); mp 53.5–
54.1 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 16H), 1.43 (bs, 27H), 1.51 (m,
4H), 1.96 (m, 6H), 2.22 (m, 6H), 3.12 (m, 4H), 4.56(s, 1H); 13C NMR (CDCl3) δ 14.0,
22.5, 27.0, 28.0, 28.7, 29.1, 29.9, 30.6, 31.8, 47.2, 56.6, 80.3, 156.1, 173.1; IR 3368,
2925, 1733, 1725, 1615, 1365, 1143; HRMS: for C37H71N2O7 (M + H)+ calcd 655.5261,
found 655.5242. Anal. Calcd for C37H70N2O7: C, 67.85; H, 10.77; N, 4.28. Found: C,
67.89; H, 10.84; N, 4.30.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-dioctylureido)heptanedioate,
3EUr(8)2
The general procedure described above afforded a white solid (1.93 g, 60%); mp 56.8–
57.5 °C; 1H NMR (CDCl3) δ 0.89 (t, 6H), 1.20−1.35 (bm, 20H), 1.43 (s, 27H), 1.50 (m,
4H), 1.96 (m, 6H), 2.21 (m, 6H), 3.11 (m, 4H), 4.55 (s, 1H); 13C NMR (CDCl3) δ 14.1,
102
22.6, 27.0, 28.1, 28.7, 29.27, 29.44, 29.9, 30.6, 31.8, 47.3, 56.6, 80.4, 156.1, 173.2; IR
3366, 2923, 1732, 1724, 1615, 1365, 1144; HRMS: for C39H75N2O7 (M + H)+ calcd
638.5574, found 638.5540. Anal. Calcd for C39H74N2O7: C, 68.58; H, 10.92; N, 4.10.
Found: C, 68.55; H, 11.07; N, 4.10.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-dinonylureido)heptanedioate,
3EUr(9)2
The general procedure described above afforded a white solid (2.34 g, 70%); mp 62.6–
63.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 24H), 1.44 (bs, 27H), 1.51 (bm,
4H), 1.97 (m, 6H), 2.22 (m, 6H), 3.12 (m, 4H), 4.57 (s, 1H); 13C NMR (CDCl3) δ 14.1,
22.6, 27.0, 28.1, 28.7, 29.2, 29.49, 29.58, 29.9, 30.6, 31.8, 47.3, 56.6, 80.4, 156.1,
173.15; IR 3377, 2922, 1732, 1725, 1617, 1365, 1144; HRMS: for C41H79N2O7 (M + H)+
calcd 711.5887, found 711.5893. Anal. Calcd for C41H78N2O7: C, 69.25; H, 11.06; N,
3.94. Found: C, 69.34; H, 11.27; N, 3.96.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-didecylureido)heptanedioate,
3EUr(10)2
The general procedure described above afforded a white solid (2.96 g, 85%); mp 49.7–
50.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.35 (bs, 28H), 1.43 (s, 27H), 1.50 (m,
4H), 1.96 (m, 6H), 2.21 (m, 6H), 3.11 (m, 4H), 4.55(s, 1H); 13C NMR (CDCl3) δ 14.1,
22.7, 27.0, 28.1, 28.7, 29.3, 29.49, 29.55, 29.63, 29.9, 30.6, 31.9, 47.3, 56.6, 80.4, 156.1,
173.2; IR 3389, 2925, 1729, 1719, 1618, 1365, 1143; HRMS: for C43H83N2O7 (M + H)+
calcd 739.6200, found 739.6213. Anal. Calcd for C43H82N2O7: C, 69.88; H, 11.18; N,
3.79. Found: C, 70.02; H, 11.41; N, 3.80.
103
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-(3,3-diundecylureido)heptanedioate,
3EUr(11)2
The general procedure described above afforded a white solid (2.64 g, 73%); mp 42.9–
43.5 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 32H), 1.43 (bs, 27H), 1.50 (bm,
4H), 1.96(m, 6H), 2.21 (m, 6H), 3.11 (m, 4H), 4.56(s, 1H); 13C NMR (CDCl3) δ 14.1,
22.65, 27.03, 28.1, 28.7, 29.3, 29.49, 29.59, 29.62, 29.9, 30.6, 31.9, 47.3, 56.6, 80.35,
156.1, 173.1; IR 3372, 2923, 1734, 1725, 1616, 1365, 1143; HRMS: for C45H87N2O7 (M
+ H)+ calcd 767.6513, found 767.6498. Anal. Calcd for C45H86N2O7: C, 70.45; H, 11.30;
N, 3.65. Found: C, 70.45; H, 11.46; N, 3.71.
General procedure for the preparation of long chain triacids, 3CUr(n)2
NHN
O
OO 3
R
R
rt, 9hN
HN
O
OtBuO 3
R
RHCOOH H
A tri-tert-butyl ester 3EUr(n)2 (3.33 mmol) were dissolved in 99% HCOOH so that its
concentration is 0.1 M. The mixture might need to be warmed slightly to get all the
3EUr(n)2 to dissolve. Once dissolved to give a transparent solution, the solution was
stirred at rt. After 9 h, the resulting milky white solution was concentrated and the
resulting residue was recrystallized from EtOAc to yield a white solid (77–90%).
4-(2-Carboxyethyl)-4-(3,3-diheptylureido)heptanedioic acid, 3CUr(7)2
The general procedure described above afforded a white solid (1.46 g, 90%); mp 125.3–
125.9 °C; 1H NMR (DMSO-d6) δ 0.83 (t, 6H), 1.10–1.30 (bm, 16H), 1.38 (m, 4H), 1.81
(m, 6H), 2.09 (m, 6H), 3.09 (m, 4H), 5.07 (s, 1H), 12.00 (bs, 3H); 13C NMR (DMSO-d6)
δ 14.4, 22.5, 26.7, 28.5, 28.7, 29.0, 30.2, 31.7, 46.3, 56.4, 156.3, 175.1; IR 2926, 1735,
1701, 1590, 1527, 1285, 1174; HRMS: for C25H47N2O7 (M + H)+ calcd 487.3383 found
104
487.3391. HRMS: for C39H74N2O7 calcd 683.5574, found 683.5540. Anal. Calcd for
C25H46N2O7: C, 61.70; H, 9.53; N, 5.76. Found: C, 61.61; H, 9.50; N, 5.75.
4-(2-Carboxyethyl)-4-(3,3-dioctylureido)heptanedioic acid, 3CUr(8)2
The general procedure described above afforded a white solid (1.47 g, 86%); mp 120.4–
120.8 °C; 1H NMR (DMSO-d6) δ 0.80 (t, 6H), 1.10–1.30 (bm, 20H), 1.38 (m, 4H),
1.81(m, 6H), 2.08 (m, 6H), 3.09 (m, 4H), 5.06 (s, 1H), 12.00 (bs, 3H); 13C NMR (DMSO-
d6) δ 13.9, 22.0, 26.2, 28.0, 28.2, 28.7, 28.8, 29.7, 31.2, 45.8, 55.9, 155.9, 174.7; IR 2921,
1713, 1694, 1607, 1533, 1293, 1175; HRMS: for C27H51N2O7 (M + H)+ calcd 515.3702,
found 515.3696. Anal. Calcd for C27H50N2O7: C, 63.01; H, 9.79; N, 5.40. Found: C,
62.98; H, 9.79; N, 5.40.
4-(2-Carboxyethyl)-4-(3,3-dinonylureido)heptanedioic acid, 3CUr(9)2
The general procedure described above afforded a white solid (1.59 g, 88%); mp 121.3–
121.7 °C; 1H NMR (DMSO-d6) δ 0.83 (t, 6H), 1.10–1.30 (bm, 24H), 1.38 (m, 4H), 1.82
(m, 6H), 2.09 (m, 6H), 3.09 (m, 4H), 5.07 (s, 1H), 11.98 (bs, 3H); 13C NMR (DMSO-d6)
δ 14.6, 22.8, 27.0, 28.7, 29.0, 29.3, 29.58, 29.66, 30.4, 32.0, 46.6, 56.6, 156.6, 175.4; IR
3446, 2919, 1712, 1694, 1608, 1534, 1293, 1175; HRMS: for C29H55N2O7 (M + H)+ calcd
543.4009, found 543.3997. Anal. Calcd for C29H54N2O7: C, 64.18; H, 10.03; N, 5.16.
Found: C, 63.93; H, 10.02; N, 5.15.
4-(2-Carboxyethyl)-4-(3,3-didecylureido)heptanedioic acid, 3CUr(10)2
The general procedure described above afforded a white solid (1.63 g, 88%); mp 120.9–
121.2 °C. 1H NMR (DMSO-d6) δ 0.83 (t, 6H), 1.10–1.30 (bm, 28H), 1.37 (m, 4H), 1.81
(m, 6H), 2.08 (m, 6H), 3.09 (m, 4H), 5.08 (s, 1H), 11.99 (bs, 3H); 13C NMR (DMSO-d6)
δ 14.4, 22.5, 26.7, 28.5, 28.7, 29.1, 29.32, 29.40, 29.46, 30.2, 31.7, 46.3, 56.4, 156.3,
105
175.2; IR 2919, 1712, 1694, 1608, 1534, 1293, 1175; HRMS: for C31H59N2O7 (M + H)+
calcd 571.4322, found 571.4315. Anal. Calcd for C31H58N2O7: C, 65.23; H, 10.24; N,
4.91. Found: C, 65.33; H, 10.34; N, 4.93.
4-(2-Carboxyethyl)-4-(3,3-diundecylureido)heptanedioic acid, 3CUr(11)2
The general procedure described above afforded a white solid (1.53 g, 77%); mp 121.4–
122.1 °C; 1H NMR (DMSO-d6) δ 0.83 (t, 6H), 1.10–1.30 (bm, 32H), 1.38 (m, 4H), 1.81
(m, 6H), 2.08 (m, 6H), 3.09 (m, 4H), 5.07 (s, 1H), 12.00 (bs, 3H); 13C NMR (DMSO-d6)
δ 14.4, 22.6, 26.7, 28.5, 28.7, 29.17, 29.33, 29.46, 30.2, 31.8, 46.4, 56.4,156.3, 175.2; IR
2917, 1713, 1694, 1608, 1535, 1295, 1185; HRMS: for C33H63N2O7 (M + H)+ calcd
599.4635, found 599.4620. Anal. Calcd for C33H62N2O7: C, 66.19; H, 10.44; N, 4.68.
Found: C, 66.17; H, 10.40; N, 4.69.
General procedure for the preparation of dialkylcarbinols (13a−f)
Br 1. Mg, catalytic I2, dry THF
2. HCO2Et, dry THF, 65−70 °Cn-1n-1 n-1
OH
To a three-neck round bottom flask containing Mg turnings (2.34 g, 100 mmol) was
added several chips of I2. After the flask was flushed with N2, the mixture was stirred for
a couple minutes until the I2 sublimed. A portion of THF (12 mL) was added, following
a solution of bromoalkanes (48.1 mmol) in THF (36 mL) in a dropwise (1 drop / 3
seconds) manner through a dropping funnel. After the addition, ethyl formate (1.43 g,
19.0 mmol) in dry THF (24 mL) was added in the same rate through a dropping funnel.
The reaction was stirred heated (65−70 °C) for 3 d. The reaction mixture was diluted
with THF (36 mL), followed by successive addition of MeOH (4 mL) and satd NH4Cl
(30 mL). The organic layer was separated and washed once with satd NaCl (65 mL).
106
The organic layer was dried and concentrated to give a light yellow solid, which was
recrystallized from EtOAc to give a pure white solid in moderate yields (60–75%); 1H
NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.50 (bm, various number of Hs), 3.58 (bm, 1H); mp
53.5−54.3 °C (n = 7), lit.61 52−52.6 °C; 61.7−62.2 °C (n = 8), lit.61 60.8−61.2 °C; 67.0−
67.4 °C (n = 9), lit.59 65.9−66.1; 71.1−72.0 °C (n = 10), lit.58,61 71.3−72.5 °C; 76.8−77.6
°C (n = 11), lit.61 75.5−75.7; 80.2−81.0 °C (n = 12), lit.60 79.5−80.5.
General procedure for the preparation of methanesulfonyloxyalkanes (12a−f)
+OH
n-1 n-1MsCl
dry THF
rt, 15 min
OMs
n-1 n-1Et3N +
To a solution of dialkylcarbinol 13 (8.96 mmol) and Et3N (9.86 mmol) in THF (59 mL)
was added mesyl chloride (25.7 mmol) dropwise rate through a syringe at rt. In the
middle of the addition, the clear solution started getting cloudy. After the addition of
mesyl chloride, the reaction was stirred for another 15 min. The resulting suspension was
filtered; the filtrate was washed with water (8 mL) and satd NaCl (2 mL). The organic
layer was dried with Na2SO4 and concentrated to give a yellowish liquid (90–98%). The
product was used for the next step without further purification; 1H NMR (CDCl3) δ 0.88
(t, 6H), 1.20−1.50 (bm, 24−44Hs), 1.60−1.75 (m, 4H), 3.00 (s, 3H), 4.70 (quin, 1H).
General procedure for the preparation of azidoalkanes (11a−f)
+OMs
n-1 n-1NaN3
DMF85 °C, 4h
N3
n-1 n-1
To a stirred solution of methanesulfonyloxylalkane 12 (7.41 mmol) in DMF (49 mL) at rt
was slowly added NaN3 (36.3 mmol). The suspension was heated to 85 °C for 4 h. After
the resulting yellow solution was cooled to rt, hexane (98 mL) and water (16 mL) were
107
added. The organic layer was separated and washed successively with satd NaHCO3 (8
mL) and satd NaCl (8 mL). The organic layer was dried with Na2SO4 and concentrated
to give a colorless oil, which was purified by flash column chromatography with hexane
to give a pure colorless oil (n = 6–10) and a white solid (n = 11), which gave a single spot
on TLC with hexane (Rf = 0.41), in 78–97% yield.
8-Azidopentadecane (11a)
The general procedure described above afforded a colorless liquid (1.46 g, 78%); 1H
NMR (CDCl3) δ 0.89 (m, 6H), 1.20–1.53 (bm, 24H), 3.22 (quin, 1H); 13C NMR (CDCl3)
δ 14.3, 22.9, 26.4, 29.4, 29.6, 32.0, 34.6, 63.4; IR 2924, 2855, 2093; HRMS: for
C15H32N3 (M + H)+ calcd 254.2596, found 254.2597. Anal. Calcd for C15H31N3: C,
71.09; H, 12.33; N, 16.58. Found: C, 71.16; H, 12.44; N, 16.44.
9-Azidoheptadecane (11b)
The general procedure described above afforded a colorless liquid (1.73 g, 83%); 1H
NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.53 (bm, 28H), 3.22 (quin, 1H); 13C NMR (CDCl3)
δ14.3, 22.9, 26.4, 29.5, 29.68, 29.70, 32.1, 34.6, 63.4; IR 2923, 2854, 2091; HRMS: for
C17H36N3 (M + H)+ calcd 254.2848, found 254.2847. Anal. Calcd for C17H35N3: C,
72.54; H, 12.53; N, 14.93. Found: C, 72.83; H, 12.68; N, 14.86.
10-Azidononadecane (11c)
The general procedure described above afforded a colorless liquid (2.11 g, 92%); 1H
NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.54 (bm, 32H), 3.22 (quin, 1H); 13C NMR (CDCl3) δ
14.3, 22.9, 26.4, 29.5, 29.65, 29.72, 32.1, 34.6, 63.3; IR 2922, 2853, 2093; HRMS: for
C19H40N3 (M + H)+ calcd 282.3161, found 282.3149. Anal. Calcd for C19H39N3: C,
73.73; H, 12.70; N, 13.57. Found: C, 73.91; H, 12.77; N, 13.42.
108
11-Azidohenicosane (11d)
The general procedure described above afforded a colorless liquid (2.47 g, 97%); 1H
NMR (CDCl3) δ 0.89 (t, 6H), 1.24–1.55 (bm, 36H), 3.22 (quin, 1H); 13C NMR (CDCl3) δ
14.3, 22.9, 26.3, 29.5, 29.65, 29.71, 29.76, 29.78, 32.1, 34.6, 63.4; IR 2922, 2853, 2093;
HRMS: for C21H44N3 (M + H)+ calcd 310.3474, found 310.3467. Anal. Calcd for
C21H43N3: C, 74.71; H, 12.84; N, 12.45. Found: C, 74.86; H, 12.98; N, 12.44.
12-Azidotricosane (11e)
The general procedure described above afforded a colorless liquid (2.28 g, 84%); 1H
NMR (CDCl3) δ 0.89 (t, 6H), 1.20–1.54 (bm, 40H), 3.23 (quin, 1H); 13C NMR (CDCl3) δ
14.3, 22.9, 26.4, 29.6, 29.7, 29.75, 29.78, 29.85, 29.86, 32.1, 34.6, 63.4; IR 2921, 2852,
2094; HRMS: for C23H48N3 (M + H)+ calcd 338.3787, found 338.3794. Anal. Calcd for
C23H47N3: C, 75.55; H, 12.96; N, 11.49. Found: C, 75.78; H, 13.10; N, 11.49.
13-Azidopentacosane (11f)
The general procedure described above afforded a white solid (2.63 g, 90%); mp 36.8−
37.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.26–1.52 (bm, 44H), 3.22 (quin, 1H); 13C
NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.6, 29.67, 29.75, 29.8, 29.86, 29.88, 32.1, 34.6, 63.4;
IR 2913, 2848, 2085; HRMS: for C25H52N3 (M + H)+ calcd 366.4100, found 366.4090.
Anal. Calcd for C25H51N3: C, 72.67; H, 13.06; N, 10.67. Found: C, 76.35; H, 13.14; N,
10.63.
General procedure for the preparation of alkanamines (10a−f)
+N3
n-1 n-1H2
Pd/C, hexane
rt, 3 h
NH2
n-1 n-1
To a solution of azidoalkane 11 (7.03 mmol) in hexane (35 mL) was added 10% Pd/C
109
(3% weight of azidoalkane). The resulting suspension was shaken and hydrogenated
under 62 psi at rt for 3 h. After sitting overnight, the suspension was filtered. The filtrate
was concentrated to give a colorless liquid (n = 6–10) and a white solid (n = 11, 12) in
75–88% yield. The product was used in the next step without purification.
Pentadecan-8-amine (10a)
The general procedure described above afforded a colorless liquid (1.22 g, 76%); 1H
NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.45 (bm, 26H; 2Hs exchange with D2O), 2.67 (bm,
1H); 13C NMR (CDCl3) δ 14.3, 22.8, 26.4, 29.5, 30.0, 32.0, 38.4, 51.40; IR 2955, 2921,
2852, 1464, 797.
Heptadecan-9-amine (10b)
The general procedure described above afforded a colorless liquid (1.35 g, 75%); 1H
NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.45 (bm, 30H), 2.67 (bm, 1H); 13C NMR (CDCl3) δ
14.3, 22.9, 26.4, 29.5, 29.8, 30.0, 32.1, 38.2, 51.4; IR 2955, 2921, 2852, 1464, 800.
Nonadecan-10-amine (10c)
The general procedure described above afforded a colorless liquid (1.67 g, 84%); 1H
NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.45 (bm, 34H; 2Hs exchange with D2O), 2.68 (bm,
1H) (lit.1 500 MHz); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.5, 29.8, 29.9, 30.0, 32.1,
38.4, 51.4; IR 2955, 2921, 2852, 1464, 797 (lit.1 KBr pellet).
Henicosan-11-amine (10d)
The general procedure described above afforded a colorless liquid (1.93 g, 88%); 1H
NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.45 (bm, 38H; 2Hs exchange with D2O), 2.67 (bm,
1H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.5, 29.83, 29.85, 29.87, 30.0, 32.1, 38.3,
51.4; IR 2955, 2920, 2852, 1464, 792.
110
Tricosan-12-amine (10e)
The general procedure described above afforded a white solid (1.93 g, 81%); mp 65.5−
66.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.13 (s, 2H), 1.20–1.45 (bm, 42H; 2Hs
exchange with D2O), 2.67 (bm, 1H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.6, 29.86,
29.89, 30.1, 32.1, 33.3, 51.4; IR 2953, 2913, 2848, 1468, 720.
Pentacosan-13-amine (10f)
The general procedure described above afforded a white solid (2.15 g, 86%); mp 79.7−
80.4 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.45 (m br, 46H; 2Hs exchange with
D2O), 2.67 (bm, 1H); 13C NMR (CDCl3) δ 14.3, 22.9, 26.4, 29.6, 29.87, 29.89, 29.91,
30.1, 32.1, 38.4, 51.4; IR 2952, 2914, 2848, 1467, 720.
General procedure for the preparation of long chain tri-tert-butyl esters, 3EUr1(n)2
3OCN
OtBu
O
n-1 n-1
CH2Cl2rt, overnight
+NH2
n-1 n-1
HN NH
OtBuO
O3
An amine 10 (4.95 mmol) was added slowly to a stirred solution of isocyanate 3 (4.71
mmol) in CH2Cl2 (20 mL). After stirring overnight at rt, the solution was concentrated to
afford a crude white solid. This crude was purified by flash column chromatography,
which gave a single spot on TLC (hexane:EtOAc, 5:1, Rf = 0.17−0.30) to give a white
solid. The isolated solid was then recrystallized from CH3CN (60–87%).
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-heptyloctyl)ureido]-
heptanedioate, 3EUr1(7)2
The general procedure described above afforded a white solid (1.89 g, 60%); mp 109.0–
109.8 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20−1.35 (bm, 22H), 1.43 (bs, 29H), 1.94
111
(m, 6H), 2.23 (m, 6H), 3.52 (bm, 1H), 3.76 (d, 1H), 4.41 (s, 1H); 13C NMR (CDCl3) δ
14.0, 22.6, 25.8, 28.0, 29.2, 29.6, 29.8, 30.6, 31.8, 35.7, 50.2, 56.4, 80.4, 156.4, 173.0; IR
3329, 2922, 2852, 1726, 1650, 1151; HRMS: for C38H73N2O7 (M + H)+ calcd 669.5418,
found 669.5419. Anal. Calcd for C38H72N2O7: C, 68.22; H, 10.85; N, 4.19. Found: C,
67.96; H, 10.91; N, 4.26.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-octylnonyl)ureido]-
heptanedioate, 3EUr1(8)2
The general procedure described above afforded a white solid (2.30 g, 70%); mp 108.5–
109.3 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.35 (bm, 26H), 1.43 (bs, 29H), 1.94 (m,
6H), 2.23 (m, 6H), 3.51 (bm, 1H), 3.75 (d, 1H), 4.41 (s, 1H); 13C NMR (CDCl3) δ 14.0,
22.6, 25.8, 28.0, 29.2, 29.5, 29.7, 29.8, 30.6, 31.8, 35.7, 50.2, 56.4, 80.4, 156.4, 173.0; IR
3317, 2925, 2855, 1730, 1630, 1147; HRMS: for C40H77N2O7 (M + H)+ calcd 697.5731,
found 697.5731. Anal. Calcd for C40H76N2O7: C, 68.92; H, 10.99; N, 4.02. Found: C,
69.06; H, 11.17; N, 4.06.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-nonyldecyl)ureido]-
heptanedioate, 3EUr1(9)2
The general procedure described above afforded a white solid (2.90 g, 85%); mp 89.6–
90.3 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.40 (bm, 30H), 1.43 (bs, 29H), 1.93 (m,
6H), 2.23 (m, 6H), 3.51 (bm, 1H), 3.76 (d, 1H), 4.41 (s, 1H); 13C NMR (CDCl3) δ 14.0,
22.6, 25.9, 28.0, 29.3, 29.55, 29.56, 29.7, 29.9, 30.7, 31.8, 35.7, 50.2, 56.4, 80.4, 156.4,
173.0; IR 3328, 2922, 2852, 1726, 1651, 1148; HRMS: for C42H81N2O7 (M + H)+ calcd
725.6044, found 725.6028. Anal. Calcd for C42H80N2O7: C, 69.57; H, 11.12; N, 3.86.
Found: C, 69.60; H, 11.22; N, 3.85.
112
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-decylundecyl)ureido]-
heptandioate, 3EUr1(10)2
The general procedure described above afforded a white solid (2.73 g, 77%); mp 87.0–
87.6 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.40 (bm, 34H), 1.44 (bs, 29H), 1.93 (m,
6H), 2.23 (m, 6H), 3.52 (bm, 1H), 3.86 (d, 1H), 4.47 (s, 1H); 13C NMR (CDCl3) δ 14.1,
22.6, 25.9, 28.02, 28.04, 29.3, 29.57, 29.61, 29.68, 29.9, 30.6, 31.9, 35.7, 50.2, 56.4, 80.4,
156.4, 173.0; IR 3376, 2919, 2850, 1730, 1675, 1146; HRMS: for C44H85N2O7 (M + H)+
calcd 753.6357, found 753.6376. Anal. Calcd for C44H84N2O7: C, 70.17; H, 11.24; N,
3.72. Found: C, 69.89; H, 11.33; N, 3.70.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(1-undecyldodecyl)ureido]-
heptanedioate, 3EUr1(11)2
The general procedure described above afforded a white solid (3.16 g, 86%); mp 92.9–
93.8 °C; 1H NMR (CDCl3) δ 0.88 (t, 6H), 1.20–1.40 (bm, 38H), 1.44 (bs, 29H), 1.94 (m,
6H), 2.24 (m, 6H), 3.52 (bm, 1H), 3.83 (m, 1H), 4.45 (s, 1H); 13C NMR (CDCl3) δ 14.0,
22.6, 25.9, 28.0, 29.3, 29.58, 29.60, 29.7, 29.9, 30.7, 31.9, 35.7, 50.2, 56.4, 80.4, 156.4,
173.0; IR 3374, 2920, 2850, 1722, 1676, 1148; HRMS: for C46H89N2O7 (M + H)+ calcd
781.6670, found 781.6657. Anal. Calcd for C46H88N2O7: C, 70.72; H, 11.35; N, 3.59.
Found: C, 70.49; H, 11.37; N, 3.67.
Di-tert-butyl 4-(2-tert-butoxycarbonyl-ethyl)-4-[3-(1-dodecyltridecyl)-ureido]-
heptanedioate, 3EUr1(12)2
The general procedure described above afforded a white solid (3.49 g, 87%); mp 73.8–
74.3 °C; 1H NMR (CDCl3) δ 0.89 (t, 6H), 1.20–1.40 (bm, 42H), 1.44 (bs, 29H), 1.94 (m,
6H), 2.24 (m, 6H), 3.51 (bm, 1H), 3.80 (d, 1H), 4.44 (s, 1H); 13C NMR (CDCl3) δ 14.1,
113
22.7, 25.9, 28.0, 29.3, 29.61, 29.63, 29.66, 29.70, 29.85, 30.6, 31.9, 35.7, 50.2, 56.4, 80.4,
156.4, 173.1; IR 3319, 2917, 2851, 1730, 1654, 1147; HRMS: for C48H93N2O7 (M + H)+
calcd 809.6983, found 809.7014. Anal. Calcd for C48H92N2O7: C, 71.24; H, 11.46; N,
3.46. Found: C, 71.43; H, 11.58; N, 3.50.
General procedure for the preparation of long chain triacids, 3CUr1(n)2
OtBuNHN
O
O
n-1
HCO2H
rt, 9 h
n-1
HOH
NHNO
O
n-1 n-1
H3 3
A tri-tert-butyl ester 3EUr1(n)2 (3.00 mmol) were dissolved in 99% HCOOH so that the
concentration was 0.1 M. Some mixtures needed warming to completely dissolve the
3EUr1(n)2. Once dissolved to give a transparent solution, the mixture was stirred at rt.
After stirring 9 h, the resulting milky white solution was concentrated in vacuo. The
white solid was recrystallized from HOAc−hexane to yield a white solid (77–90%).
4-(2-Carboxyethyl)-4-[3-(1-heptyloctyl)ureido]heptanedioic acid, 3CUr1(7)2
The general procedure described above afforded a white solid (1.28 g, 85%); mp 168.5–
169.2 °C; 1H NMR (CD3OD) δ 0.89 (t, 6H), 1.20−1.40 (bm, 24H), 1.44 (m, 2H), 1.95 (m,
6H), 2.28 (m, 6H), 3.55 (bm, 1H); 13C NMR (DMSO-d6) δ 14.4, 22.5, 25.8, 28.6, 29.2,
29.4, 30.5, 31.7, 35.8, 48.3, 55.4, 157.4, 175.0; IR 3405, 2924, 2855, 1732, 1700, 1585;
HRMS: for C26H49N2O7 (M + H)+ calcd 501.3540, found 501.3562. Anal. Calcd for
C26H48N2O7: C, 62.37; H, 9.66; N, 5.60. Found: C, 62.28; H, 9.57; N 5.52.
4-(2-Carboxyethyl)-4-[3-(1-octylnonyl)ureido]heptanedioic acid, 3CUr1(8)2
The general procedure described above afforded a white solid (1.36g, 86%); mp 164.3–
165.1 °C; 1H NMR (CD3OD) δ 0.89 (t, 6H), 1.20–1.40 (bm, 28H), 1.44 (m, 2H), 1.95 (m,
6H), 2.28 (m, 6H), 3.54 (bm, 1H); 13C NMR (DMSO-d6) δ 14.7, 22.8, 26.1, 28.8, 29.4,
114
29.69, 29.73, 30.8, 31.9, 36.0, 48.5, 55.6, 157.6, 175.2; IR 3404, 2921, 2852, 1730, 1699,
1556; HRMS: for C28H53N2O7 (M + H)+ calcd 529.3853, found 529.3837. Anal. Calcd
for C28H52N2O7: C, 63.61; H, 9.91; N, 5.30. Found: C, 63.65; H, 9.94; N 5.20.
4-(2-Carboxyethyl)-4-[3-(1-nonyldecyl)ureido]heptanedioic acid, 3CUr1(9)2
The general procedure described above afforded a white solid (1.39g, 83%); mp 165.1–
165.7 °C; 1H NMR (CD3OD) δ 0.86 (t, 6H), 1.20–1.35 (bm, 30H), 1.41 (m, 2H), 1.92 (m,
6H), 2.25 (m, 6H), 3.51 (bm, 1H); 13C NMR (DMSO-d6) δ 14.4, 22.5, 25.8, 28.6, 29.1,
29.36, 29.43, 30.5, 31.7, 35.7, 48.3, 55.4, 157.3, 174.9; IR 3405, 2921, 2852, 1730, 1699,
1554; HRMS: for C30H57N2O7 (M + H)+ calcd 557.4166, found 557.4172. Anal. Calcd
for C30H56N2O7: C, 64.72; H, 10.14; N, 5.03. Found: C, 64.73; H, 10.20; N 5.01.
4-(2-Carboxy-ethyl)-4-[3-(1-decylundecyl)ureido]heptanedioic acid, 3CUr1(10)2
The general procedure described above afforded a white solid (1.42, 81%); mp 162.8–
163.5 °C; 1H NMR (CD3OD) δ 0.86 (t, 6H), 1.20–1.35 (bm, 34H), 1.41 (m, 2H), 1.87 (t,
6H), 2.25 (t, 6H), 3.51 (bm, 1H); 13C NMR (DMSO-d6) δ 14.4, 22.5, 25.8, 28.5, 29.1,
29.41, 29.43, 30.5, 31.7, 35.6, 48.3, 55.4, 157.3, 174.9; IR 3404, 2921, 2852, 1731, 1699,
1557; HRMS: for C32H61N2O7 (M + H)+ calcd 585.4479, found 585.4501. Anal. Calcd
for C32H60N2O7: C, 65.72; H, 10.34; N, 4.79. Found: C, 65.52; H, 10.31; N 4.78.
4-(2-Carboxyethyl)-4-[3-(1-undecyldodecyl)ureido]heptanedioic acid, 3CUr1(11)2
The general procedure described above afforded a white solid (1.56 g, 85%); mp 164.6–
165.1 °C; 1H NMR (CD3OD) δ 0.86 (t, 6H), 1.20–1.35 (bm, 38H), 1.41 (m, 2H), 1.92 (m,
6H), 2.25 (m, 6H), 3.51 (bm, 1H); 13C NMR (DMSO-d6) δ 14.4, 22.5, 25.7, 28.5, 29.1,
29.41, 29.44, 30.5, 31.7, 35.6, 48.2, 55.3, 157.3, 174.9; IR 3404, 2921, 2852, 1732, 1699,
1557; HRMS: for C34H65N2O7 (M + H)+ calcd 613.4792, found 613.4792. Anal. Calcd
115
for C34H64N2O7: C, 66.63; H, 10.53; N, 4.57. Found: C, 66.77; H, 10.64; N 4.56.
4-(2-Carboxyethyl)-4-[3-(1-dodecyltridecyl)ureido]heptanedioic acid, 3CUr1(12)2
The general procedure described above afforded a white solid (1.67 g, 87%); mp 163.3–
163.9 °C. 1H NMR (CD3OD) δ 0.86 (t, 6H), 1.20–1.35 (bm, 42H), 1.41 (m, 2H), 1.95 (m,
6H), 2.25 (m, 6H), 3.51 (bm, 1H); 13C NMR (DMSO-d6) δ 14.6, 22.8, 26.0, 28.8, 29.4,
29.67, 29.71, 29.75, 30.8, 32.0, 35.9, 48.5, 55.6, 157.6, 175.2; IR 3403, 2920, 2851,
1731, 1700, 1559; HRMS: for C36H69N2O7 (M + H)+ calcd 641.5105, found 641.5095.
Anal. Calcd for C36H68N2O7: C, 67.46; H, 10.69; N, 4.37. Found: C, 67.31; H, 10.62, N
4.34.
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Publisher, Inc.: Deerfield, 1985. 63 Stewart, W. E.; Siddall, I., T. H. Nuclear magnetic resonance studies of amides.
Chem. Rev. 1970, 70, 517-551. 64 Hanson, P.; Williams, D. A. R. Restricted carbon-nitrogen bond rotation in some
ureas, thioureas, and thiuronium salts. J. Chem. Soc. Perkin Trans. II 1973, 2162-2165.
65 Siddall, I., T. H.; Stewart, W. E. Slow rotation in a thiourea detected by proton
magnetic resonance. J. Org. Chem. 1967, 32, 3261. 66 Newkome, G. R.; Weis, C. D.; Childs, B. J. Syntheses of 1→3 branched
isocyanate monomers for dendritic construcion. Des. Monomers. Polym. 1998, 1, 3-14.
121
67 Gall, R.; Bosies, E. Preparation of diphosphonic acid derivatives for treating calcium metabolic disorders.1988, DE 3623397 A1, 12pp.
68 Froger, C.; Parisot, A. The melting points of the N-monosubstituted amides.
Compt. rend 1954, 238, 1589-1591.
122
Chapter IV Synthesis of Tri-headed Amphiphiles Containing Cholestane-based Structure on the Hydrophobic Moiety
Two new series of tri-headed amphiphiles containing cholestane moiety have
been designed. Both series still contains a ureido linker and a tricarboxylic Newkome-
type dendron. Instead of a long, straight alkyl chains, these new series contains a
nonpolar cholestane moiety as the hydrophobic tail. With the consideration of
commercial availability, only the 5α-cholestane moiety is utilized as a starting material.
In one series, C3 of the cholestane moiety is directly connected to the linker. In the other
series, a spacer −(CH2)nCOO−, where n = 1, is constructed between C3 of the cholestane
moiety and the linker (Figure IV.2).
H
5α-cholestane 5β-cholestane
H
H
H
H1
2
34
5
H
H
H
H1
2
34
5A A
B
C D C D
B
H H
H H
Figure IV.1 Structural representations of 5α- and 5β-cholestane
123
linker
head
head
head
head
head
head
C3cholestane moiety
linker spacerC3cholestane moiety
Figure IV.2 General structures of tri-headed amphiphiles series containing cholestane moiety
The linker and spacer can be attached through two different configurations⎯
axial and equatorial at C3 of cholestane. These configurations are referred as α- and β-
stereochemistry, respectively. Based on how either the linker or spacer is connected to
C3 of cholestane, each series contains a pair of epimers.
IV.1 The 3CUr-z-cholestane Series
The first series is abbreviated as 3CUr-z-cholestane, where “3C” represents three
carboxylic groups on the hydrophilic moiety, “Ur” represents a ureido linker between the
hydrophobic and hydrophilic moieties, and “z-cholestane” represents the
configuration⎯α or β⎯ on how a nitrogen on the linker is attached to the C3 of 5α-
cholestane. When a nitrogen on the ureido linker is connected to C3 of 5α-cholestane via
the α-configuration, the amphiphile is abbreviated as 3CUr-α-cholestane. In a similar
manner, when connection is made via the β-configuration, the abbreviation is 3CUr-β-
cholestane. In a similar manner to the one-tail and two-tail series, the series containing
the tert-butyl esters is abbreviated as 3EUr-z-cholestane.
The chemistry on the 3CUr-z-cholestane was very similar to that of 3CUr1(n)2
(Chapter III). In a similar manner as how the previous series had been synthesized,
retrosynthesis of 3CUr-z-cholestane shows the synthesis begins with WeisocyanateTM
124
(3) and the cholestan-3-amines (4) (Scheme IV.1).
Scheme IV.1 Retrosynthesis of 3CUr-z-cholestane
NH
NH
OH
O
OH
OH
O
O
O
NH
NH
OO
O
OO
O
O
NCOO
O 3+
H
H
H
H1
23
45
H2N
1a, 3CUr-α-cholestane1b, 3CUr-β-cholestane
3
4
4a, 5α-cholestan-3α-amine4b, 5α-cholestan-3β-amine
a b c
a. hydrophilic moiety, b. a ureido linker,c. a hydrophobic 5α-cholestane moiety
2a, 3EUr-α-cholestane2b, 3EUr-β-cholestane
H
H
H
H1
2
34
5
A
C D
B
H
H
When 5α-cholestane moiety is drawn in the chair conformation for the A ring, α-
and β-configurations of C3 correspond to axial and equatorial positions, respectively. As
the only difference is the C3 configuration on the cholestane moiety, 3CUr-z-cholestane
and 3EUr-z-cholestane are a pair of epimers.
HX
Y
X is in axial or 3α-positionY is in equatorial or 3β-position
H
125
IV.1.1 Preparation of 5α-Cholestan-3-amine (4a−b)
In order to synthesize the epimeric pair 1a and 1b, we required amines 4a and 4b,
respectively, to react with 3. As both amines are not commercially available, syntheses
of such were necessary.
IV.1.1.1 Literature Review on Syntheses of Amines (4a−b)
Retrosynthesis (Scheme IV.2) of amines 4a−b shows the synthesis can begin with
cholestanol 8a−b as precursors. However, as only 5α-cholestan-3β-ol (8b) is
commercially available, we sought to design the synthesis beginning with 8b.
Scheme IV.2 Retrosynthesis of epimer 5α-cholestan-3-amines
3
H2N
3
N3
3
MsO3
HO
3
H2N3
N3
3
Y3
HO
4a5a 6b 8b
4b 5b 8a6a, Y = OMs7, Y = Br
H
H
H H
H3
Most reported syntheses of cholestanamine 4a−b begin with the corresponding 3-
azidocholestane (5a−b),1-7 5α-cholestan-3-one oxime, 8-12 5α-cholestan-3-one, 13,
sulfonate ester derivatives of 5α-cholestan-3-ols (6a−b) (Table IV.1).14,15 Lithium
aluminum hydride1,2,4-7 and catalytic hydrogenation in the presence of ammonia3 were
employed to convert azides 5a−b into amines 4a−b with retention of stereochemistry at
126
C3 in good yields. When a particular epimer 5 (5a or 5b), 8 (8a or 8b), or sulfonate ester
derivatives of 8a−b is employed as precursors, the corresponding cholestanamine, either
4a or 4b, is obtained exclusively via an SN2 reaction. However, when 5α-cholestan-3-
Table IV.1 Preparation of amines 4a−b from different precursors Precursors Reagents Product(s); remarks References
N3
LiAlH4, Et2O
H2N
Reaction run under reflux1,2,6 and rt5,7 Yield: 65%1, 77% 5
1,2,4-7
N3
LiAlH4, Et2O
H2N68%
1
N3
H2, NH3, Pd/C
H2N89%
No solvent reported
3
O
H2, NH3, M black M = Ru, Pd
H2N H2N+
For M = Ru, ratio = 26:54 (3α:3β); addition of NH4Cl yields ratio = 16:82 (3α:3β) For M = Pd, ratio= 19:81(3α:3β) addition of NH4Cl yields ratio = 21:79 (3α:3β)
13
NHO
LiAlH4, Et2O, reflux
H2N H2N+
10,11
NHO
n-C5H11OH, Na
H2N
8,9,12
NHO
1. H2, PtO, HOAc 2. HCl
H2N H2N+
9
FG FG = −OTs,14,15 −OMs15
Anhydrous NH3
H2N
FG = −OTs, 70%;15 56%14 FG = −OMs, 63%
14,15
127
one oxime and 5α- cholestan-3-one are utilized, a mixture of epimers, 4a and 4b, is
obtained in various ratios. Reduction of 5α-cholestan-3-one oxime performed with
lithium aluminum hydride results in a mixture of the same epimers, in almost equal
amounts.10,11 Interestingly, the reduction performed with sodium in pentan-1-ol yields
amine 4b exclusively.8,9,12 When reduction of cholestan-3-one oxime is carried out by
hydrogenation in acetic acid followed by hydrolysis of the N-acetyl derivative with
concentrated hydrochloric acid, amine 4a is obtained as the major product.9 Sodium
borohydride and hydrogenation in dioxane are ineffective.9
IV.1.1.2 Literature Review on Syntheses of 3-Azidocholestane (5a−b)
Both azides 5a−b are usually synthesized starting from the corresponding alcohol
by using derivatives such as a sulfonate ester, 6a−b, and a haloalkane (Table IV.2). The
syntheses involve SN2 reactions with different azide reagents, and the corresponding
azides are mostly obtained in good to excellent yields. Azides 5b and 5a are prepared
directly from alcohols 8b and 8a, respectively, via a Mitsunobu-type substitution. The
traditional approach is where alcohols are converted into the corresponding sulfonate or
halide derivative prior to a nucleophilic displacement of the corresponding sulfonate ester
or halide by azide. In any case, conversion of alcohol 8 into azide 5 results in inversion
of the stereochemistry at C3.
Compared to the traditional approach, a Mitsunobu-type substitution is more
convenient because such a reaction can be performed in one-pot; it is a shorter route. In
addition, most of these Mitsunobu-type reactions give good to excellent yields. Only one
procedure1 following a Mitsunobu-type reaction reported that azide 5a was obtained in
slightly low yield.
128
Table IV.2 Preparation of azides 5a and 5b from various precursors Precursors Reagents Product(s); remarks References
HO
PPh3, DEAD, THF, various azides Azides: nicotinoyl azide, benzoyl azide N3
94%
16
HO
2,4,4,6-tetrabromo-2,5-cyclohexadienone, PPh3, Zn(N3)2
.2Py, PhCH3, CH3CN N3 94%
17
HO
PPh3, DEAD, (PhO)2PON3, THF
N3 51%1
1,18
MsO
Dry DMPU, NaN3
N3 75%
19
HO
PPh3, DEAD, THF, various azides Azides: nicotinoyl azide, benzoyl azide N3
nicotinoyl azide (96%) benzoyl azide (91%)
16
HO
PPh3, DEAD, HN3, PhH
N3 77%
20
Z
Z = I, 1 OMs21
NaN3, DMSO
N3 Z = I (81%);1 OMs (75%)21
1,21
Z
Z = OMs,22 OTs,2,6,23 p-oxopyridinium tosylate5
NaN32,6,22,23 or LiN3
5 ,22 Solvents: HMPT,2 DMA, 6 DMF22,23 N3
95% without purification; 2 75%23
2,5,6,22,23
129
IV.1.1.3 Literature Review on Syntheses of 3-Methanesulfonyloxy-5α-cholestane (6a–b)
Conversion of alcohols 8a−b into the corresponding sulfonate esters, such as a
methanesulfonyloxy- and toluenesulfonyloxyalkane, is widely utilized in organic
synthesis. Even though methanesulfonyloxy is a better leaving group than
toluenesulfonyloxy, toluenesulfonyloxy derivatives are more widely found in the
literature. The conversion of 8 into 6 is usually achieved by the standard method utilizing
toluenesulfonyl or methanesulfonyl chloride15 ,21,22,24,25 and pyridine as a base; the
corresponding product is obtained with retention of stereochemistry (Table IV.3). On the
other hand, when the conversion into methanesulfonyloxy derivative is prepared with a
Mitsunobu-type reaction, the product is obtained with inversion of stereochemistry.
Table IV.3 Preparations of 6a−b from 8a−b Precursors Reagents Remarks References
HO
MsCl, pyridine
MsO 100%,24 60%25
21,22,24,25
HO
PPh3, DEAD, MsOH, THF
MsO no yield reported
19
HO
MsCl, pyridine
MsO 60%,25 50%15
15,25
IV.1.1.4 Literature Review on Synthesis of 3α-Bromo-5α-cholestane (7)
As depicted on the retrosynthesis of amine 4b (Scheme IV.3), the synthesis of
bromoalkane 7 is required. Several procedures, most of which involve phosphine,26-29
have been reported to give 7 in good to excellent yields. Employing the phosphine-free
130
method, 30,31 the traditional catalyst cuprous chloride (CuCl) works as well as cupric
triflate (Cu(OTf)2) in the conversion of the primary alcohols. However, when secondary
alcohols are employed, Cu(OTf)2 is superior to the traditional catalyst CuCl. When CuCl
is employed for such a conversion, the reaction takes longer time and the isolated product
is identified as a complex mixture. Acetyl bromide, N-bromosuccinimide (NBS), lithium
bromide, and 2,4,4,6-tetrabromocyclohexanone are commonly used brominating agents
in such a conversion (Table IV.4). Lithium bromide as a bromide source needs to be
added in large excess (10 equiv) in order to obtain the product in excellent yield.28
Table IV.4 Preparations of bromoalkane 7 from 8b and 8b’s derivatives Precursors Reagents Remarks References
HO
Cu(OTf)2, DIC, brominating agents (AcBr, 30 NBS31)
80%30,31 solvent : THF,30dioxane31 minor elimination products observed
30,31
HO
PPh3Br2, DMF 80%
29
HO
DEAD, PPh3, LiBr, THF 96%
28
RO
R = H, THP
PPh3,Br
Br
Br
Br
O , CH3CN
86% (R = H), 91% (R = THP)
26
OEt3Si PPh3,
Br
Br
Br
Br
O , CH3CN
CaCO3, THF
86% 27
131
IV.1.2 Synthesis of 5α-Cholestan-3-amines (4a−b)
We began our synthesis with commercially available alcohol 8b. In order to
synthesize the epimeric pair 4a−b, the first step was to convert alcohol 8b into the
corresponding sulfonate ester derivatives 6a−b or bromoalkane 7 (Scheme IV.3).
Considering availability, better reactivity, and successful conversion into the intermediate
sulfonate ester in the previous homologous series (Chapter II and III), we decided to
transform alcohol 8b into the corresponding methanesulfonyloxy derivative (6b). Both
cholestanol 8a and bromoalkane 7 could be employed as an intermediate in the synthesis
of amine 4b. As cholestanol 8b was easily converted into bromoalkane 7 via 6b, we
chose to synthesize bromoalkane 7 instead of cholestanol 8a as an intermediate to
generate amine 4b.
As expected, the conversion of alcohol 8b into the corresponding derivative 6b
retained the stereochemistry on C3. The reaction was completed in a relatively short time
(15 minutes), and the product was obtained in quantitative yield. Completion of the
reaction was indicated by the appearance of a singlet at δ 3.00 ppm (3H on the CH3SO2−)
in the 1H NMR spectrum and a multiplet at 4.62 ppm (H-α). These chemical shifts
compared favorably to published values.25 After comparing the proton integration of the
axial hydrogen on C3, the methyl group on sulfur (CH3SO2−), and the rest of the
cholestanyl hydrogens in the 1H NMR spectrum of the crude product, we decided that the
crude product was suitable for use without purification for the next step.
The crude derivative 6b was allowed to react with lithium bromide (2.5 mol eq.)
via an SN2 reaction to give 3α-bromo-5α-cholestane 7. The reaction was refluxed in
tetrahydrofuran, and completion of the reaction was shown by the 1H NMR spectrum.
132
After purification, the product 7 was obtained in 74% yield based on 6b. In the 1H NMR
spectrum, the chemical shifts of the methyl group (CH3SO2−, s, δ 3.00 ppm) and the α-H
(m, δ 4.62 ppm) on C3 of the starting 6b were no longer observed. An SN2 reaction took
place at C3 to give a β-H, which was observed at δ 4.73 ppm (lit. 60 MHz32 δ 4.63 ppm,
60 MHz33 δ 4.71 ppm) as a triplet. It was intriguing in the beginning to find that the
reported32 chemical shift of the β-H on C3 of bromocholestane 7 was actually much
closer to that of the starting 6b. However, we noticed the change in the splitting pattern
at the reactive C3, caused by the change of the proton position⎯axial H (α-H) to
equatorial H (β-H). Finally, comparison of the 1H NMR spectrum to another published
value33 verified that we did obtain 3α-bromo-5α-cholestane (7).
Azides 5a and 5b were obtained via an SN2 reaction of 6b and 7, respectively,
with sodium azide as the nucleophile. Purification of the resulting azides was carried out
by flash column chromatography in hexane in good yields. The absorption at 2100 cm-1
in the IR spectra showed the presence of azido groups. The configuration⎯α or β⎯of
the proton bonded to C3 on the products was confirmed by change in both splitting
pattern and chemical shift in the 1H NMR spectra.
The last step was reduction of azides 5. Reduction was performed via two
different ways⎯catalytic hydrogenation in hexane and application of reducing agent
lithium aluminum hydride. Both methods gave amines 4a−b. The absorptions at 2100
cm-1 in the IR spectra were no longer present. Both chemical shifts of the α- or β-H on
C3 were shifted downfield ~ 0.7 ppm, however, their splitting patterns were not changed,
indicating the stereochemistry at C3 was retained in both compounds.
The former method, catalytic hydrogenation, was more convenient⎯ease of
133
work-up⎯than the latter. Even though their NMR spectra were the same, the product
generated from the latter method was obtained in higher yields, higher melting
temperatures, and whiter in color. Multiple gravity filtrations on the products generated
from the former method, namely hydrogenation, were attempted. However, the color of
the products obtained did not improve much. Even worse, multiple gravity filtrations
caused material loss during transfers. Other attempts to improve the quality of the color
included filtering through a Celite® pad in vacuo and adding charcoal in the beginning of
the recrystallization process met with no success. Regardless of the convenience,
considering all the advantages offered by lithium aluminum hydride, we decided to carry
out the reduction of azides 5 with such a reducing agent in future experiments.
Scheme IV.3 Synthetic scheme of the preparation of epimeric amines (4a−b)
MsOHO N3 H2N
Br N3 H2N
i ii iii
iv
ii iii
i. MsCl, Et3N, dry DCM; quantitative yieldii. NaN3, DMF, 90 °C, 4h; 3a, 85%; 3b 79%iii. Pd/C, hexane; 3a, 68%; 3b, 65% or LiAlH4, THF, reflux; 3a, 80%; 3b, 86%iv. LiBr, THF, 70 °C, 14h; 74%
8b
7
6b 5a
5b
4a
4b
IV.1.3 Synthesis of 3CUr-z-cholestane (1a−b)
The tri-tert-butyl esters 2a−b were generated by combining 3 and amines 4 in
dichloromethane at ambient temperature (Scheme IV.1). Purification was carried out by
134
flash column chromatography (hexane:THF:MeOH=1:1:0.04) to give a white solid. Tri-
tert-butyl esters 2a and 2b were obtained in 78% and 74% yields, respectively, and the
melting temperatures were 179.9−180.5 °C and 187.4−187.8 °C, respectively. Both 2a
and 2b were fully characterized.
Recrystallization from ethanol was performed in order to obtain a crystal for X-
ray analysis; an X-ray crystal structure of 2a was obtained. Ethanol was observed in the
crystal lattice (see below). On the other hand, repeated attempts to recrystallize 2b in
order to generate a crystal for X-ray analysis were not successful.
Formolysis of the compounds 2a and 2b at room temperature produced the
triacids 1a and 1b, respectively. Triacids 1a and 1b were obtained in 73% and 78%
yields, respectively. As for the melting temperature observation, decomposition of both
compounds was observed at 199.0 °C and 194.3 °C, respectively. Both 1a and 1b were
fully characterized.
IV.1.4 X-ray Structure of 3EUr-α-cholestane (2a)
The crystal structure of 3EUr-α-cholestane (2a) shows how the molecules with a
bulky dendritic polar moiety and extremely rigid hydrophobic moiety assemble in the
solid state. Two different crystallographic independent molecules are found in the X-ray
crystal structure arranged in parallel; inversion symmetry generates the antiparallel
packing. In general, molecules that are antiparallel are packed more closely than
molecules that are parallel. The closest interaction in the antiparallel orientation ranges
from 3.8 to 4.1 Å involving the side chain of one cholestane moiety and both ring B and
C, including the methyl substituents at the junction of ring A-B and ring C-D on the other
cholestane moiety. The closes interaction in the parallel orientation ranges from 4.0 to
135
4.1 Å involving the side chain of one cholestane moiety and ring B, C, D, including the
methyl substituents at the junction of ring A-B of the other cholestane moiety. Ring A of
this cholestane moiety displays short intermolecular contacts to the headgroup of the
other cholestane moiety, ranging from 3.4 to 4.1 Å.
Symmetrically equivalent molecules are separated by 12.12 Å from each other. In
the two independent molecules, all six-membered rings are found in full chair
conformations, while all five-membered rings are found in twisted envelope
conformations. The torsion angles in two headgroups of each independent molecule
Figure IV.3 X-ray crystal structure of 3EUr-α-cholestane (2a), showing the packing and the intermolecular hydrogen bonding to ethanol. Hydrogen-bond Donor(D)–Acceptor(A) distances and approximate ∠DHA: O(16)···O(1), 2.621(7) Å and 158.1°; O(15)···O(8), 2.625(6) Å and 161.0°; O(4)···O(16), 2.865(7) Å and 155(7)°; N(2)···O(15), 2.849(8) Å and 159.6°. The other nitrogens on the ureido linker are just outside the range for significant hydrogen bonding. N(3)···O(16), 3.035(7) Å and 141.6°; N(1)···O(15), 3.084(8) Å and 146.0°
136
adopt anti conformations, while the torsion angle of the other headgroup adopts a gauche
conformation. Torsion angles of the two headgroups in the first molecule are –170.1(6)°
[C(1)−C(37)−C(38)−C(39) and +177.2(6)° [C(1)−C(44)−C(45)−C(46)], and those in the
second molecule are –170.5(6)° [C(51)−C(80)−C(81)−C(82) and +166.8(7)°
[C(51)−C(94)−C(95)−C(96)]. Torsion angles of the third headgroup adopt gauche
conformations, however, in different direction; those in the first and second molecules are
−96.9(5)° [C(1)−C(30)−C(31)−C(32)] and +91.6(7)° [C(51)−C(87)−C(88)−C89)],
respectively.
The presence of ethanol molecules is very important in the packing. Each 3EUr-
α-cholestane (2a) molecule is close to an ethanol molecule; they are arranged in alternate
manner and connected by hydrogen bonds; the short contacts within any different
molecules range from 1.823 Å [O(1)−H(16)] to 3.171 Å [O(1)−C(103)]. Each ethanol
molecule serves as both donor and acceptor for the hydrogen bonds formed; it serves as a
hydrogen bond donor to one crystallographic molecule of 3EUr-α-cholestane (2a) and a
hydrogen bond acceptor to the other. As hydrogen bond donors, they donate hydrogen
bonds, from namely O(16) and O(15), to the carbonyl-oxygen on the linker, namely O(1)
and O(8), respectively (Figure IV.1). As hydrogen bond acceptors, ethanol molecules
[O(16) and O(15)] accept hydrogen bonds from the amido nitrogens [N(4) and N(2),
respectively] connected to the cholestane moiety. In addition, the other amido nitrogens
[N(3) and N(1)] in the linker form weak hydrogen bonds with the same ethanol molecules
(donor−acceptor distance > 3.0 Å). The hydrogen bond between the solvent ethanol and
the carbonyl oxygen of the linker is the shortest. These last two interactions are similar
to those previously observed in the X-ray crystal structure of 3EUr16 (Chapter II) where
137
water, instead of ethanol, is used as the solvent in recrystallization. The formation of
weak hydrogen bonds between the solvent and the amido nitrogen attached to the
headgroup suggests that the bulkiness of the headgroups can possibly obstruct the solvent
to get close enough to the amido nitrogen attached to the headgroup, resulting weak
hydrogen bonds. Compared to the X-ray crystal structure of 3EUr16, there is
unfortunately no hydrogen bond involving the participation of the headgroups.
Intramolecular hydrogen bonds are observed in the X-ray crystal structure of 3EUr16
where the carbonyl oxygen of the linker and an ester carbonyl oxygen accept hydrogen
bonds from water; this is possible as each water molecule contains two hydrogens.
IV.2 The 3CUrnEs-z-cholestane Homologous Series
The second homologous series is abbreviated as 3CUrnEs-z-cholestane (Scheme
IV.5), where “3C” represents three carboxylic groups on the hydrophilic moiety, “Ur”
represents a ureido linker between the hydrophobic and hydrophilic moieties, “n”
represent the number of methylene between the ureido linker and the carbonyl carbon of
the ester functional group, which is represented as “Es”, and finally “z-cholestane”
represents the configuration⎯α- or β-configuration⎯ on how the oxygen on the ester
group is attached to the C3 of 5α-cholestane. As the 3CUrnEs-z-cholestane
homologous series is generated from the corresponding tri-tert-butyl esters, the
homologous series containing the tert-butyl groups is abbreviated as 3EUrnEs-z-
cholestane.
Retrosynthesis of 3CUrnEs-z-cholestane shows that the synthesis can begin with
Weisocyanate (3) and 5α-cholestan-3-yl ω-aminoalkanoates (Scheme IV.4). In this
preliminary study, only the shortest homologs (9, n = 1)⎯3CUr1Es-z-cholestane⎯are
138
synthesized. Thus, a synthesis of both 3α- and 3β- epimers of 5α-cholestan-3-yl
aminoethanoate (11) is necessary.
Scheme IV.4 Retrosynthesis of the 3CUrnEs-z-cholestane homologous series
NCOO
O 3
H
H
H
H12
34
5
O 3
OHN
HN
O
H
H
H
H
3
9, n = 19a, 3CUr1Es-α-cholestane9b , 3CUr1Es-β-cholestane
3
OHO
HO
HO
O
O
O
a b d c
n
a. a hydrophilic moiety, b. a ureido linker,c. a −(CH2)nCO2− spacer, d. a hydrophobic linker
H2NO
+
OHN
HN
O
10, n = 110a, 3EUr1Es-α-cholestane10b, 3EUr1Es-β-cholestane
3
OO
O
O
O
O
O
n
11, n = 111a, 5α-cholestan-3α-yl aminoethanoate11b, 5α-cholestan-3β-yl aminoethanoate
n
3CUrnEs-z-cholestane 3EUrnEs-z-cholestane
5α-cholestan-3-yl ω-aminoalkanoate
H
H
A
B
C D
IV.2.1 Preparation of 3α- and 3β- Epimer of 5α-Cholestan-3-yl Aminoethanoate (11a−b)
In order to synthesize 11a−b (Scheme IV.5), we employed retrosynthesis
following our strategy for the synthesis of amines in the previous 3CUrn (Chapter II) and
3CUr1(n)2 (Chapter III) series. We chose a route so that amines 11 were generated from
the corresponding azides 12. As we sought to construct the synthesis beginning with
139
commercially available cholestanol 8b, syntheses of intermediates 13a−b and 8a would
be required.
Scheme IV.5 Retrosynthesis of 3α- and 3β- epimer of 5α-cholestan-3-yl aminoethanoate
OCl
O
ON3
O
OH2N
O
OCl
O
ON3
O
OH2N
O
HO
HO
11b
11a 12a
12b
13a
13b 8b
8a
IV.2.1.1 Literature Review on the Synthesis of 5α-Cholestan-3α-yl Aminoethanoates (11a−b)
Only one procedure34 on the synthesis of 5α-cholestan-3α-yl aminoethanoate has
been reported. Matsumoto et al.34 generated cholestanol (8b) esters of a various amino
acids, including the simplest amino acid glycine. Beginning with glycine, fusion of such
an amino acid and cholestanol (8b) was achieved by passing hydrogen chloride into the
two reactants. Free amine 11b was then released from the resulting hydrochloride ester.
H2NO
HO
+ 8b 11b HClHCl, CHCl3:MeOH (10:1)
.160 °C, 1.5 h
11banhydrous Na2CO3
With this procedure, a couple intermediates (Scheme IV.6) would not be needed
because 11b could be synthesized in one-step reaction from 8b. However, one drawback
on this procedure was that the products from various amino acids were obtained in
140
20−44% yield; 11b was obtained in 44%. Regardless it was a one-step reaction, and
necessary reagents were commercially available, we did not attempt to follow the
reported procedure because of the resulting low yield. We then followed our original
strategy (Scheme IV.6) to synthesize azides 12a−b.
IV.2.1.2 Literature Review on the Synthesis of 5α-Cholestan-3α-yl Azidoethanoate (12a−b)
The procedure on the synthesis of 5α-cholestan-3α-yl azidoethanoate has never
been documented. However, we have demonstrated that the transformation of
haloalkanes into the corresponding azidoalkanes (in the previous 3CUrn homologous
series) performed by heating the haloalkanes and sodium azide in N, N-
dimethylformamide, gives good yields of azidoalkanes.
It is well documented that several alkyl azidoethanoates,35-40 where the alkyls
contain carbacyclic moieties including cholestane moiety,35 are transformed into the
corresponding amines in good to excellent yields. In these reactions, sodium azide is
again utilized; most reactions are carried out in various solvents⎯ N, N-
dimethylformamide,35,36,40 acetone,37 acetone−water39 and dimethylsulfoxide38⎯ by
heating35-37,39 or stirring at ambient temperature.40 In any case, the ester functional
groups are not affected. With this consideration, we decide to adopt our previous
procedure for the transformation of 5α-cholestan-3α-yl chloroethanoate (13) into 5α-
cholestan-3α-yl azidoethanoate (12).
IV.2.1.3 Literature Review on the Synthesis of 5α-Cholestan-3α-yl Chloroethanoate (13a−b)
Compounds 13a−b are commercially available, however, in milligram quantities,
141
thus syntheses are required. Several procedures41-45 on the syntheses of 13a−b have been
reported (Table VI.5). All syntheses begin with commercially available 8b. Both
chloroacetic acid and chloroacetyl chloride were utilized as the acylating agents.
Beginning with chloroacetic acid, the inverted product is obtained in the presence
of Mitsunobu reagents.43 Chloroacetyl chloride is mostly combined with a base to give
products with retention of stereochemistry. 41,42,44,45
Table IV.5 Preparations of 5α-cholestan-3α-yl chloroethanoate from 5α-cholestan-3β-ol (13a−b) Precursors Reagents Remarks References
HO
ClCH2CO2H, DEAD, PPh3, THF
rt, 24 h, inverted product, 85%, inversion ratio >99%
43
HO
ClCH2CO2H, TMAD, Bu3P, PhH
43%, mixture of α:β=28:72
43
HO
ClCH2COCl, Et3N, PhH reflux at 80 °C, overnight41 or 24 h,44 retention product, 65%
41,44
HO
ClCH2COCl, DMAP, Et3N, PhH
rt, overnight, retention product, 78%
42
HO
ClCH2COCl, CHCl3 retention product, no yield reported
45
IV.2.2 Synthesis of 5α-Cholestan-3α-yl Aminoethanoate (11a−b)
We began our synthesis with cholestanol 8b as it was commercially available
(Scheme IV.6). Conversion of 8b into 8a was carried out following the literature
procedure;46 8a was obtained in 80% yield. Cholestanol 8a and 8b were converted into
13a and 13b, respectively. The formation of the products was confirmed by the
appearance of a singlet at δ ~4.0 ppm (ClCH2CO2−) in 1H NMR spectra. No change in
142
the splitting pattern on the proton bonded to C3 confirmed that the products were
obtained with retention of configuration at C3.
5α-Cholestan-3-yl chloroethanoates (13a−b) were converted into the
corresponding 5α-cholestan-3-yl azidoethanoate (12a−b) via an SN2 reaction. The
reaction was carried out by heating sodium azide in a solution of 13 in N,N-
dimethylformamide. Substitutions of the halogen by azide were confirmed by 1H NMR
spectra; the chemical shifts of the methylene protons (−CH2CO2−) shifted from δ ~4.0 to
δ ~ 3.8 ppm. The absorption at ~ 2100 cm-1 in the IR spectra also confirmed the presence
of an azido group. Compounds 12a−b were obtained in 47−49% yield.
In the synthesis of the amine intermediates of 3CUr-z-cholestane (see Section
IV.1.3), reduction of the azides was carried out with both lithium aluminum hydride and
hydrogenation. From the product characteristics, such as higher melting temperatures
and higher isolated yields, we concluded reduction with lithium aluminum hydride is the
better way to perform such a transformation
Choices of reducing agents become limited when other functional groups present.
The epimers of 3CUr1Es-z-cholestane contain both azido and ester functional groups.
The powerful reducing agent lithium aluminum hydride reduces azido as well as ester
functional groups.47-49 Thus, we performed the conversion of the azides into the
corresponding amines by hydrogenation. Hydrogenation reduces azido groups but does
not affect ester groups.50-54
With this consideration, hydrogenation reduced azides 12a−b to form amines
11a−b, which were identified by the appearance of weak absorptions at ~ 3400 cm-1 in
the IR spectra. The transformation of azides 12a−b into amines 11a−b was also
143
supported by the disappearance of the absorptions of the azido group (~ 2100 cm-1) in the
IR spectra. In the 1H NMR spectra, protons on the methylene (−CH2CO2−) shifted from
~ 3.8 ppm to ~ 3.4 ppm. The products 11a and 11b were obtained in 88 and 76% yields,
respectively.
Scheme IV.6 Synthetic scheme of the preparation of amines 11a−b
OCl
O
ON3
O
OH2N
O
OCl
O
ON3
O
OH2N
O
HO
HO
11b
11a12a
12b
13a
13b8b
8a
i
i
ii
ii
iii
iii
iv
i. ClCH2COCl, Et3N, PhH, rt, 24 h; 13a, 68%; 13b, 57%ii. NaN3, DMF, ref lux; 12a, 47%; 12b, 49%iii. Pd/C, CH2Cl2, H2, 69 °C, 4 h; 11a, 88%; 11b, 76%iv. DIAC, Et3N, TFA, PPh3, dry THF, rt, overnight, then MeOH 50 °C, overnight; 8a, 80% IV.2.3 Synthesis of 3CUr1Es-z-cholestane (9a−b)
The tri-tert-butyl esters 10a−b were generated by combining 3 and amines 11 in
dichloromethane at ambient temperature (Scheme IV.4). Purification was carried out by
flash column chromatography (hexane:ethyl acetate = 2.5:1) to give a white solid, which
gave a single spot on TLC (Rf = 0.33−0.35) in the solvent system above. The product was
further purified by recrystallization from ethanol. The tri-tert-butyl esters 10a and 10b
were obtained in 64% and 61% yields, respectively, and the melting temperatures were
82.9−83.3 °C and 195.8−196.2 °C, respectively. Assignments in the 1H NMR spectra
144
were supported by the two-dimensional 1H−1H COSY NMR.
In 10a and 10b, the geminal protons on the methylene of the ethanoates coupled
to the neighboring N−H (δ 4.65 and δ 4.62 ppm, respectively). In 10a, these methylene
protons were not interchangeable, thus observed to be diastereotopic protons. These
diasterotopic protons (δ 3.94 and 3.89 ppm) were not equivalent; each showed a doublet
of doublets. The bigger group at C3 was on axial position, which possibly restricted the
free rotation about CH2−CO bond. Consequently, the methylene protons were in
different environment, thus giving different chemical shifts. On the other hand, this
behavior was not observed in 10b, where the bigger group at C3 was in equatorial
position; the methylene protons (δ 3.84 ppm) showed a doublet, which suggested that the
free rotation about CH2−CO bond was fast on an NMR time scale. The axial hydrogen at
C3 of 10a and the equatorial hydrogen at C3 of 10b (δ 5.08 and 4.70 ppm, respectively)
coupled to some of the cholestanyl protons (δ 0.60−2.00 ppm).
Formolysis of the compounds 10a−b at room temperature produced the triacids
9a−b, respectively. Triacids 9a−b were obtained in 51% and 57% yields, respectively.
As for the melting temperature observation, compound 9a melted at 192.5−192.9 °C,
while 9b decomposed at 194.2 °C; the large difference in melting temperature observed
between 10a and 10b disappeared when the tert-butyl groups were removed. Triesters
10a−b and triacids 9a−b were fully characterized.
IV.3 Experimental Procedures Material and Methods.
Chemicals were obtained from Aldrich, Acros and TCI; they were used without further
145
purification. Solvents were reagent grade or HPLC grade; they were used as received
unless otherwise specified. THF was distilled from sodium/benzophenone ketyl.
WeisocyanateTM was prepared as described55 with a shorter reaction time as mentioned
above. Analytical thin layer chromatography was performed by polyester-coated silica
gel 60 Å and detected by treating with 10% ethanolic phosphomolybdic acid reagent (20
wt. % solution in ethanol) followed by heating. Flash column chromatography was
carried out on silica gel (60 Å); samples were loaded as concentrated solutions in the
solvent system needed; column diameter × height (13/4 × 6 in), eluted samples varied
between ~ 2.00−4.00 g, flow rate (~ 1.5−2 in/min) was controlled by air pressure.
Solutions were concentrated by rotary evaporation. Melting ranges, determined in open
capillary tubes, were uncorrected. NMR spectra were recorded on an INOVA at 400 and
100 MHz for 1H and 13C, respectively, and reported in ppm relative to the known solvent
residual peak. Resonances were reported in the order of the chemical shifts (δ), followed
by the splitting pattern, and the number of protons. Abbreviations used in the splitting
pattern were as the following: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, and b = broad. IR spectra were recorded on neat samples with an FTIR
equipped with a diamond ATR system, and reported in cm-1. Optical rotations were
measured at the sodium line by using a Perkin-Elmer 241 polarimeter in a 1-dm tube.
HRMS data were obtained on a dual-sector mass spectrometer in FAB mode with 2-
nitrobenzylalcohol as the proton donor. Elemental analyses were performed by Atlantic
Microlabs, Inc. in Norcross, GA.
146
Preparation of 3α-methanesulfonyloxy-5α-cholestane (6b)
MsOHO+ MsCl
Et3N, dry DCM
15 min 0 °C
A 100-mL round-bottomed flask containing a stirred solution of 8b (5.12 g, 12.9 mmol)
and Et3N (2.55 mL, 18.2 mmol) in dry CH2Cl2 was cooled in an ice bath. When the
temperature reached 0 °C, MsCl (1.2 mL, 2.96 g, 15.4 mmol) was added slowly through a
syringe. The resulted reaction was stirred for another 15 min while maintaining the low
temperature. The resulting reaction was quenched by washing successively with water (5
mL), HCl (2 M, 5 mL), water (5 mL), satd NaHCO3 (5 mL), and water (5 mL). The
organic solution was dried and concentrated to give an off-white solid (5.71 g, 12.2
mmol, 94%), which gave a single spot on TLC in CH2Cl2 (Rf = 0.70). The 1H NMR
spectrum of the respective crude product suggested that the material was suitable for the
next step without further purification. 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 3.00 (s,
3H), 4.62 (m, 1H) (lit.25 200 MHz); 13C NMR (CDCl3) δ 12.1, 12.2, 18.7, 21.2, 22.6,
22.8, 23.8, 24.2, 28.0, 28.2, 28.5, 28.7, 31.9, 35.2, 35.3, 35.4, 35.8, 36.2, 36.8, 38.9, 39.5,
39.9, 42.6, 44.9, 54.1, 56.3, 56.4, 82.3.
Preparation of 3α-bromo-5α-cholestane (7)
BrMsOLiBr, dry THF
70 °C, 14 h
To a stirred solution of crude mesylated product 6b (4.37 g, 9.36 mmol) in dry THF (40
mL) was added LiBr (2.06 g, 23.5 mmol) slowly. The suspension was refluxed at 70 °C
for 14 h. The resulting reaction was concentrated, and the residue was extracted into
hexane (50 mL); the undissolved solid was filtered off and the filtrate was concentrated to
147
give a white solid, which was recrystallized from EtOH to give product 7 (3.15 g, 74%);
mp 103.5–103.9 °C (lit.29 mp 100–102 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 4.74
(m, 1H) (lit.33 60 MHz); 13C NMR (CDCl3) δ 12.1, 13.3, 18.7, 20.8, 22.6, 22.8, 23.9,
24.2, 27.9, 28.0, 28.2, 31.0, 31.8, 32.9, 35.4, 35.8, 36.17, 36.23, 37.3, 39.5, 39.95, 40.13,
42.6, 53.9, 56.16, 56.24, 56.4; IR 2942, 2923, 2844, 694; [α]25D +29.4° (c 1.62, CHCl3)
(lit.29 [α]D +26° (c 1.2, CHCl3); HRMS: for C27H47Br calcd 450.2861, found 450.2851.
Preparation of 3-azido-5α-cholestanes (5a−b)
a. Preparation of 3α-azido-5α-cholestane (5a)
N3MsONaN3, dry DMF
90 ºC, 4 h
To a 250-mL round-bottomed flask containing solution of crude 6a (4.97 g, 10.65 mmol)
in DMF (75 mL) was slowly added NaN3 (3.49 mg, 53.2 mmol). The resulted suspension
was heated at 90 ºC for 4 h. Then, the reaction was cooled to rt before hexane (150 mL)
and water (25 mL) were added. The organic layer was separated and washed
successively with satd NaHCO3 (25 mL) and satd NaCl (25 mL). The organic layer was
dried with Na2SO4 and concentrated to give a crude product, which is purified by flash
chromatography in hexane to give a white solid (3.73 g, 85%); mp 64.4–64.8 °C (lit.6
62.5−63.5 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 3.88 (bs, 1H) (lit.1 300 MHz);
13C NMR (CDCl3) δ 11.6, 12.1, 18.7, 20.8, 22.6, 22.9, 23.9, 24.2, 25.7, 28.0, 28.3, 28.4,
31.9, 32.6, 32.9, 35.5, 35.8, 35.9, 36.2, 39.5, 40.0, 40.1, 42.6, 54.2, 56.3, 56.5, 58.3; IR
2931, 2865, 2852, 2084 (lit.1 film); [α]26D +18.6° (c 1.52, CHCl3) (lit.6 [α]26
D +18.6° (c
1.01, CHCl3)).
148
b. Preparation of 3β-azido-5α-cholestane (6b)
Br N390 ºC, 4 h
NaN3, DMF
To a 250-mL round-bottomed flask containing solution of 3α-bromocholestane 7 (2.36 g,
5.22 mmol) in DMF (36 mL) was slowly added NaN3 (1.72 mg, 26.1 mmol). The
resulted suspension was heated at 90 °C for 5 hours. The reaction was cooled to rt before
hexane (72 mL) and water (15 mL) were added. The organic layer was separated and
washed successively with satd NaHCO3 (15 mL) and satd NaCl (15 mL). The organic
layer was dried with Na2SO4 and concentrated to give a crude product, which is purified
by flash chromatography in hexane to give a white solid (1.70 g, 79%); mp 66.3–66.8 °C
(lit.16 mp 71.5–72 °C, lit.21 65–66 °C); 1H NMR (CDCl3) δ 0.60–2.00 (t, 47H), 3.25 (m,
1H) (lit.1 300 MHz); 13C NMR (CDCl3) δ 12.1, 12.4, 18.7, 21.2, 22.6, 22.8, 23.8, 24.2,
28.0, 28.3, 38.8, 32.1, 32.7, 35.55, 35.56, 35.8, 36.2, 37.7, 39.51, 39.53, 40.1, 42.6, 45.6,
51.2, 54.5, 56.3, 56.6; IR 2965, 2929, 2848, 2090, 1463, 1256 (lit.1 film); [α]25D +25.6° (c
1.84, CHCl3) (lit.21 [α]20D +26° (c 1.00, CHCl3), lit.20 [α]20
D +25.5° (c 1.00, CHCl3)).
General procedure for the preparation of epimeric 5α-cholestan-3-amines (4)
a. Preparation of 5α-cholestan-3α-amine (4a)
Method A.
H2NN3
Pd/C, hexane
rt, 6 h
To a 250-mL hydrogenation bottle was added an azide 5, 10% Pd/C (4% weight of
azide), and hexane so that the concentration of the azide was ~ 0.13 M. The resulting
suspension was shaken and hydrogenated at 60 psi at rt for 6 h. After sitting overnight,
149
the resulting suspension was filtered; the filtrate was concentrated to give an off-white
solid.
Method B.
H2NN3 reflux, 4h
LiAlH4, THF
A 250mL round-bottomed-flask containing a suspension of LiAlH4 in dry THF was put
in an ice bath. When the temperature reached 0 °C, an azide 5 was added slowly to the
flask, so that its concentration was ~ 0.80 M. The resulting suspension was then refluxed
for 4 hours. After being refluxed, the mixture was diluted with THF so that the volume
doubled, and cooled in an ice bath once again. Water (one-eighth of THF volume),
NaOH (10 M, one-fifth of THF volume), and water (one-eighth of THF volume) were
added successively to the resulting mixture. The solid that formed was filtered; the
filtrate was dried with Na2SO4, filtered, and concentrated to give a white solid.
5α-Cholestan-3α-amine (4a)
Method A. The general procedure for the synthesis of 4 above was followed: Azide 5a
(1.84 g, 4.46 mmol), 10% Pd/C (82.8 mg), and hexane (35 mL) were combined and
treated as above. Amine 4a was obtained as an off-white solid (1.18 g, 3.06 mmol; 68%);
mp 84.6−85.4 °C (lit.11 88−89 °C).
Method B. The general procedure for the synthesis of 4 above was followed: LiAlH4
(320 mg, 8.01 mmol), dry THF (10 mL), and azide 5a (1.00 g, 2.42 mmol) were
combined and treated as above. Amine 4a was obtained as a white solid (755 mg, 1.95
mmol; 80%); 88.4–89.2 °C (lit.11 88−89 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m, 47H),
3.18 (bs, 1 H) (lit.1 300 MHz); 13C NMR (CDCl3) δ 11.4, 12.1, 18.7, 20.8, 22.6, 22.8,
150
23.6, 24.2, 28.0, 28.3, 28.8, 29.3, 32.1, 32.2, 35.6, 35.8, 36.2, 36.37, 36.41, 39.2, 39.5,
40.1, 42.6, 45.8, 54.6, 56.3, 56.6; IR 2930, 2849, 1465, 1380 (lit.1 film); [α]25D +26.4° (c
1.81, CHCl3) (lit.4 [α]28D +28° (c 1.1, CHCl3)).
5α-Cholestan-3β-amine (4b)
Method A. The general procedure for the synthesis of 4 above was followed: azide 5b
(1.38 g, 3.35 mmol), 10% Pd/C (54.3 mg), and hexane (26 mL) were combined and
treated as above. Amine 4b was obtained as an off-white solid (849 mg, 2.19 mmol;
65%); 85.8–86.4 °C (lit.1 104–106 °C).
Method B. The general procedure for the synthesis of 4 above was followed: LiAlH4
(851 mg, 21.3 mmol), dry THF (26 mL), and azide 5b (2.94 g, 7.10 mmol) were
combined and treated as above. Amine 4b was obtained as a white solid (2.36 g, 6.09
mmol; 85.8 %); mp 91.6–92.1 °C (lit.1 104–106 °C); 1H NMR (CDCl3) δ 0.60–2.00 (m,
47H), 2.64 (m, 1 H) (lit.1 300 MHz); 13C NMR (CDCl3) δ 12.1, 12.4, 18.7, 21.2, 22.6,
22.8, 23.8, 24.2, 28.0, 28.3, 28.8, 32.1, 32.6, 35.53, 35.55, 35.8, 36.2, 37.7, 39.4, 39.5,
40.1, 42.6, 45.6, 51.2, 54.5, 56.3, 56.5; IR 2931, 2849, 1465, 1381(lit.1 film); [α]25D
+24.6° (c 1.80, CHCl3) (lit.10 [α]D +29° (c 0.9, CHCl3)).
General procedure for the preparation of 3EUr-z-cholestane (2)
NH
NH
OO
O
H
H
H
H12
34
5 3
+
3
DCM
rt, 24hOCN
OtBu
O 3
H
H2N
To a 50-mL round-bottomed flask containing a solution of 3 in CH2Cl2 was added an
amine 4 slowly and stirred at rt for 24 h. The reaction was concentrated to give colorless
151
oil that was solidified under vacuum. Purification was carried out via flash column
chromatography (hexane:THF:MeOH=1:1:0.04) to give a white solid, which TLC gave a
single spot (Rf = 0.56−0.63), in 74−78% yield.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3α-
yl)ureido]heptanedioate, 3EUr-α-cholestane (2a)
The general procedure for the synthesis of tri-tert-butyl ester 2 above was followed: 3
(1.15 g, 2.61 mmol), CH2Cl2 (10 mL), and amine 4a (1.01 g, 2.61 mmol) were combined
and treated as above. The tri-tert-butyl ester 2a was obtained in a white solid (2.04 g,
1.69 mmol, 78%); mp 179.9–180.6 °C; 1H NMR (CDCl3) δ 0.60–2.00 (m, 46H), 1.44 (s,
27H), 1.95 (m, 6H), 2.23 (m, 6H), 3.67 (bs, 1H), 4.38–4.42 (m, 2H); 13C NMR (CDCl3) δ
11.4, 12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.6, 28.0, 28.1, 28.3, 28.6, 30.0, 30.7, 32.0,
33.1, 33.5, 35.5, 35.8, 36.0, 36.2, 39.5, 40.1, 40.7, 42.6, 45.4, 54.5, 56.3, 56.5, 56.6, 80.5,
156.2, 173.1; IR 2929, 2864, 1726, 1628, 1551, 1456, 1366, 1247, 1146; [α]27D +15.6° (c
1.41, CHCl3); HRMS: for C50H89N2O7 (M + H)+ calcd 829.6670, found 829.6675. Anal.
Calcd for C50H88N2O7: C, 72.42; H, 10.70; N, 3.38. Found: C, 72.18; H, 10.79; N, 3.38.
X-ray analysis of 3EUr-α-cholestane (2a)
Colorless plates (0.055 x 0.47 x 0.55 mm3) were crystallized from ethanol. The chosen
crystal was mounted on a nylon CryoLoop™ (Hampton Research) with Krytox® Oil
(DuPont) and centered on the goniometer of an Oxford Diffraction Xcalibur™
diffractometer equipped with a Sapphire 2™ CCD. The data collection routine, unit cell
refinement, and data processing were carried out with the program CrysAlis.56 The Laue
symmetry and systematic absences were consistent with the monoclinic space groups P21
and P21/m. As the sample was known to be enantiomerically pure from the synthetic
152
procedure, the chiral space group P21 was chosen. Because there are no heavy atoms, the
absolute configuration could not be determined from the Friedel pairs; the Friedel pairs
were therefore merged for the final refinement. The structure was solved by direct
methods and refined using the SHELXTL NT program.57 The asymmetric unit of the
structure comprises two crystallographically independent cholesterol derivatives and two
ethanol molecules. The final refinement model involved anisotropic displacement
parameters for non-hydrogen atoms and a riding model for all hydrogen atoms. The
program SHELXTL NT was used for molecular graphics generation.57
Table IV.6 Crystal data and structure refinement for 3EUr-α-cholestane (2a) Category Crystal data and structure refinement
Empirical formula C50H88N2O7•CH3CH2OH Formula weight 875.29 Temperature 100(2) K Wavelength 0.71073 Å Crystal system monoclinic Space group P21 Unit cell dimension a = 12.1220(16) Å; b = 12.8097(16); c = 35.589(3) Å;
α = 90°; β = 99.166(9)°; γ = 90° Volume 5455.7(11) Å3 Z 4 Density (calculated) 1.066 Mg/m
3
Absorption coefficient 0.070 mm-1 F(000) 1936 Crystal size 0.550 x 0.465 x 0.055 mm
3
Theta range for data collection 2.81 to 25.07° Index ranges −14 ≤ h ≤ 14, −14 ≤ k ≤ 15, −42 ≤ l ≤ 41 Reflections collected 25907 Independent reflections 10121 [R(int) = 0.0573] Completeness to theta = 25.07° 99.6 % Absorption correction None Refinement method Full-matrix least-squares on F
2
Data / restraints / parameters 10121 / 1 / 1119 Goodness-of-fit on F2 0.967 Final R indices [I>2sigma(I)] R1 = 0.0734, wR2 = 0.1820 R indices (all data) R1 = 0.1180, wR2 = 0.2131 Absolute structure parameter −2.9(17) Largest diff. peak and hole 0.686 and -0.273 e.Å
-3
153
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3β-
yl)ureido]heptanedioate, 3EUr-β-cholestane (2b)
The general procedure for the synthesis of tri-tert-butyl ester 2 above was followed: 3
(1.39 mg, 3.15 mmol), CH2Cl2 (14 mL), and 4b (1.22 mg, 3.16 mmol) were combined
and treated as above. Tri-tert-butyl ester 2b was obtained as a white solid (2.35 g, 1.94
mmol, 74%); mp 187.4–187.8 °C; 1H NMR (CDCl3) δ 0.60–2.0 (m, 46H), 1.43 (s, 27H),
1.93 (m, 6H), 2.23 (m, 6H), 3.87 (sb, 1H), 3.88 (d, 1H), 4.46 (s, 1H); 13C NMR (CDCl3) δ
12.1, 12.3, 18.7, 21.2, 22.6, 22.8, 23.9, 24.2, 28.0, 28.1, 28.3, 28.6, 29.6, 30.0, 30.7, 32.0,
35.4, 35.5, 35.8, 36.2, 36.3, 37.6, 39.5, 40.0, 42.6, 45.1, 49.8, 54.4, 56.3, 56.5, 80.5,
156.2, 173.2; IR 2929, 2864, 1724, 1625, 1558, 1457, 1365, 1308, 1148; [α]27D +9.6° (c
1.49, CHCl3); HRMS: for C50H89N2O7 (M + H)+ calcd 829.6670, found 829.6638. Anal.
Calcd for C50H88N2O7: C, 72.42; H, 10.70; N, 3.38. Found: C, 72.67; H, 10.77; N, 3.35.
General procedure for the preparation of 3CUr-z-cholestane (1)
NH
NH
OO
O 3
HCOOH
rt, 24hNH
NH
OH
O
O 3
To a 50-mL round-bottomed flask containing the tri-tert-butyl ester 2 was added
HCOOH; the flask was warmed in a hot-water bath until a transparent solution appeared.
The resulting solution was stirred overnight at rt. The complete reaction was identified
by the formation of milky solution. The solvent was evaporated to leave a white solid,
which was recrystallized with AcOH/hexane successively to give a white solid.
4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3α-yl)ureido]heptanedioic acid, 3CUr-α-
cholestane (1a)
The general procedure for the synthesis of the triacid 1 above was followed: 2a (1.53 g,
154
1.90 mmol) and HCOOH (30 mL) were combined and treated as above. Triacid 1a was
obtained as a white solid (922 mg, 1.39 mmol; 73%); dec. at 199.0 °C; 1H NMR
(DMSO-d6) δ 0.60–2.00 (m, 51H), 2.09 (m, 6H), 3.67 (bs, 1H), 5.50 (s, 1H), 5.97 (d, 1H),
12.00 (s, 3H); 13C NMR (DMSO-d6) δ 11.6, 12.3, 18.9, 20.8, 22.8, 23.1, 22.8, 23.1, 23.7,
24.2, 26.7, 27.8, 28.2, 28.6, 30.6, 32.2, 33.0, 33.7, 35.4, 35.7, 36.0, 36.1, 39.4, 42.6, 44.0,
54.5, 55.5, 56.2, 56.7, 157.0, 175.0; IR 3373, 1288, 2925, 2865, 1705, 1635, 1559;
[α]27D +19.9° (c 0.87, EtOH); HRMS: for C38H65N2O7 (M + H)+ calcd 661.4792, found
661.4780. Anal. Calcd for C38H64N2O7: C, 69.06; H, 9.76; N, 4.24. Found: C, 69.10; H,
9.80; N, 4.22.
4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3β-yl)ureido]heptanedioic acid, 3CUr-β-
cholestane (1b)
The general procedure for the synthesis of triacid 1 above was followed: 2b (907 mg,
1.09 mmol) and HCOOH (26 mL) were combined and treated as above. Triacid 1b was
obtained as a white solid (560 mg, 0.85 mmol; 78%); dec. at 194.3 °C; 1H NMR (DMSO-
d6) δ 0.60–2.00 (m, 51H), 2.08 (m, 6H), 3.23 (bs, 1H), 5.38 (s, 1H), 5.50 (d, 1H), 12.02
(s, 3H); 13C NMR (DMSO-d6) δ 12.3, 12.4, 19.0, 21.2, 22.8, 23.1, 23.7, 24.2, 27.8, 28.3,
28.6, 28.7, 29.5, 30.5, 32.1, 35.5, 35.7, 36.1, 36.2, 37.7, 42.6, 45.6, 48.9, 54.2, 55.5, 56.2,
56.4, 156.9, 175.0; IR 3405, 2930, 2847, 1704, 1613, 1559, 1295; [α]27D +14.8° (c 0.83,
EtOH); HRMS: for C38H65N2O7 (M + H)+ calcd 661.4792, found 661.4794. Anal. Calcd
for C38H64N2O7: C, 69.06; H, 9.76; N, 4.24. Found: C, 68.85; H, 9.71; N, 4.20.
155
Preparation of 5α-cholestan-3α-ol46 (8a)
HOHO+ DIAD + TFA + PPh3 +
1. dry THF, rt, overnight
2. MeOH, 50 C, overnight
O
O Na
To a 100-mL round bottom flask containing a colorless solution of 10b (4.90 g, 12.4
mmol) in dry THF (24 mL) was added DIAD (2.45 mL, 15.6 mmol). The resulting
solution turned from colorless to orange. Addition was continued with trifluoroacetic
acid (1.20 mL, 15.6 mmol), PPh3 (4.09 g, 15.5 mmol), and sodium benzoate (2.25 g, 15.5
mmol), successively. The resulting colorless solution was stirred at rt overnight. The
solution was concentrated to give an off-white solid. After diluted with MeOH (24 mL),
the resulting solution was heated at 50 °C overnight. The solvent was concentrated; the
residue was partitioned between CH2Cl2 (150 mL) and water (15 mL). The organic layer
was washed once again with water (15 mL) and dried with MgSO4. Concentration of the
solution gave a white solid, which was purified by flash column chromatography in
CH2Cl2 to yield a white solid (3.84 g, 80%); mp 185.2−185.7 °C (lit.46 mp 182−185 °C);
1H NMR (CDCl3) δ 0.60−2.00 (m, 46H), 4.04 (bs, 1H) (lit.46 300 MHz); 13C NMR
(CDCl3) δ 11.2, 12.1, 18.7, 20.8, 22.6, 22.8, 23.8, 24.2, 28.0, 28.3, 28.6, 29.0, 32.0, 32.2,
35.5, 35.8, 35.9, 36.1, 36.2, 39.2, 39.5, 40.1, 42.6, 54.3, 56.2, 56.6, 66.7; IR 3375, 2928,
2848, 1002 (lit.58 bands); [α]27D +25.5° (c 1.78, CHCl3) (lit.59 [α]26.5
D +27° (c 1.92,
CHCl3)).
General preparation of 5α-cholestan-3-yl chloroethanoate (13)
HO
ClO
ODMAP, Et3N, PhHCl
O
Cl+
rt, overnight
156
Chloroacetyl chloride was added dropwise to a 100-mL round bottom flask containing a
solution of a cholestanol (8), Et3N, and DMAP in benzene. The resulting solution was
stirred at rt overnight. The reaction was filtered; the filtrate was concentrated to give the
crude product, which was purified by flash column chromatography to give a white solid,
which gave a single spot on TLC in CH2Cl2:hexane (1:1, Rf = 0.33−0.41), in 57−68%
yield.
5α-Cholestan-3α-yl chloroethanoate (13a)
The general procedure for the synthesis 13 was followed: 8a (3.80 g, 9.78mmol), Et3N
(1.50 mL, 10.7 mmol), DMAP (60.0 mg, 0.49 mmol), benzene (58 mL) and chloroacetyl
chloride (1.80 g, 15.6mmol) were combined and treated as above. The compound above
was obtained as a white solid (2.59 mg, 8.56 mmol, 57%); mp 134.7–135.2 °C; 1H NMR
(CDCl3) δ 0.65–2.00 (m, 46H), 4.07 (s, 2 H), 5.12 (m, 1H); 13C NMR (CDCl3) δ 11.3,
12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.0, 28.0, 28.25, 28.32, 31.9, 32.7, 32.8, 35.5,
35.76, 35.82, 36.2, 39.5, 40.0, 41.4, 42.6, 54.2, 56.3, 56.5, 72.7, 166.8; IR 2932, 2864,
2849, 1754, 1194, 1185; [α]27D +24.5° (c 1.18, CHCl3).
5α-Cholestan-3β-yl chloroethanoate (13b)
The general procedure for the synthesis 13 was followed: 8b (5.00 g, 12.6 mmol), Et3N
(1.90 mL, 13.5 mmol), DMAP (77.3 mg, 0.63 mmol), benzene (94 mL) and chloroacetyl
chloride (1.804 g, 15.6 mmol) were combined and treated as above. The compound
above was obtained as a white solid (3.98 mg, 8.56 mmol, 68%), mp 176.1–176.9 °C (lit.
mp 178−179 °C,45 180−181 °C41); 1H NMR (CDCl3) δ 0.60–2.00 (t, 46H), 4.02 (s, 2H),
4.79 (m, 1H); 13C NMR (CDCl3) δ 12.1,12.2, 18.7, 21.2, 22.6, 22.8, 23.8, 24.0, 27.3,
28.0, 28.2, 28.6, 31.9, 33.8, 35.43, 35.45, 35.8, 36.2, 36.7, 39.5, 40.0, 41.3, 42.6, 44.6,
157
54.2, 56.3, 56.4, 76.1, 166.9; IR 2935, 2917, 2848, 1747, 1205, 1180; [α]24D +11.3° (c
0.36, CHCl3).
General procedure for the preparation of 5α-cholestan-3-yl azidoethanoate (12)
To a 0.20-M solution of 13 in DMF was added slowly NaN3. The mixture was heated at
85−90 °C for 10 h. The resulting mixture was yellow. After it cooled to rt, hexane (as
much as the volume of DMF) and water (one-third volume of DMF) were added. Two
layers formed; the organic layer was separated. The organic layer was washed with satd
NaHCO3 and satd NaCl successively. Each aqueous solution added was the same as the
volume of the water added. The light yellow organic layer was dried and concentrated to
give a light yellow solid. Purification was carried out by flash column chromatography
to give a white solid, which gave a single spot on TLC in hexane (Rf = 0.15−0.21).
5α-Cholestan-3α-yl azidoethanoate (12a)
The general procedure for the synthesis 12 was followed: NaN3 (1.24 g, 18.9 mmol), 13a
(1.76 g, 3.79 mmol), DMF (19 mL) were combined and treated as above. Compound 12a
was obtained as a white solid (846 mg, 1.80 mmol, 47%), mp 114.9–115.6 °C; 1H NMR
(CDCl3) δ 0.65–2.00 (m, 46H), 3.86 (s, 2H), 5.16 (m, 1H); 13C NMR (CDCl3) δ 11.4,
12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.1, 28.0, 28.2, 28.3, 31.9, 32.81, 32.83, 35.5,
35.76, 35.81, 36.2, 39.5, 39.99, 40.04, 42.6, 50.7, 54.2, 56.3, 56.5, 72.6, 167.8; IR 2926,
2865, 2104, 1733, 1218, 1159, 975; [α]27D +29.8° (c 1.14, CHCl3); HRMS: for
C29H49N3O2 (M+) calcd 471.3825, found 471.3812. Anal. Calcd for C29H49N3O2: C,
N3 O
OCl
O
O NaN3, DMF
85−90 °C, 10 h
158
73.84; H, 10.47; N, 8.91. Found: C, 73.89; H, 10.53; N, 8.91.
5α-Cholestan-3β-yl azidoethanoate (12b)
The general procedure for the synthesis 12 was followed: NaN3 (798 mg, 12.2 mmol),
13b (1.85 g, 3.98 mmol), DMF (20 mL) were combined and treated as above. Compound
12b was obtained as a white solid (915 mg, 1.94 mmol, 49%), mp 124.2–124.9 °C; 1H
NMR (CDCl3) δ 0.60–2.00 (m, 46H), 3.83 (s, 2H), 4.81 (m, 1H); 13C NMR (CDCl3) δ
12.3, 12.5, 18.9, 21.4, 22.8, 23.1, 26.1, 24.4, 27.6, 28.2, 28.5, 28.8, 32.2, 34.1, 35.67,
35.68, 36.0, 36.4, 36.9, 39.7, 40.2, 42.8, 44.9, 50.8, 54.4, 56.5, 56.6, 76.0, 168.1; IR
2934, 2849, 2108, 1738, 1218; [α]27D +14.5° (c 1.00, CHCl3); HRMS: for C29H49N3O2
(M+) calcd 471.3825, found 471.3809. Anal. Calcd for C29H49N3O2: C, 73.84; H, 10.47;
N, 8.91. Found: C, 73.90; H, 10.60; N, 8.92.
General procedure for the preparation of 5α-cholestan-3-yl aminoethanoate (11)
A cholestanyl azidoethanoate (12) was added to a hydrogenator vessel containing a
suspension of 10% Pd/C (4% weight of azide 12) in CH2Cl2 so that the concentration of
the ethanoate was ~ 0.11 M. The mixture was shaken and hydrogenated at 60 psi at low
temperature (~ 60 °C) for 4 h. The reaction was cooled to rt and filtered through a pad of
Celite® in vacuo; the filtrate was concentrated to give an off-white solid.
5α-Cholestan-3-yl aminoethanoate (11a)
The general procedure for the synthesis 11 was followed: 12a (736 mg, 1.56 mmol), 10%
Pd/C (29.7 mg), CH2Cl2 (15 mL) were combined and treated as above. Compound 11a
was obtained as a white solid (615 mg, 1.38 mmol, 88%), mp 109.2–110.1 °C; 1H NMR
N3 O
OH2N O
OPd/C, DCM
60 °C, 4h
159
(CDCl3) δ 0.65–2.00 (t, 49H), 3.43 (s, 2H), 5.08 (m, 1H); 13C NMR (CDCl3) δ 11.3, 12.1,
18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.2, 28.0, 28.3, 28.4, 31.9, 32.87, 32.92, 35.5, 35.79,
35.82, 36.2, 39.5, 40.03, 40.05, 42.6, 44.3, 54.2, 56.3, 56.5, 70.9, 173.8; IR 3400, 2931,
2865, 1728, 1206; [α]27D +28.2° (c 0.94, CHCl3); HRMS: for C29H52NO2 (M + H)+ calcd
446.3998, found 446.4001. Anal. Calcd for C29H51NO2: C, 78.15; H, 11.53; N, 3.14.
Found: C, 78.17; H, 11.67; N, 3.12.
5α-Cholestan-3β-yl aminoethanoate (11b)
The general procedure for the synthesis 11 was followed: 12b (2.70 g, 5.72 mmol), 10%
Pd/C (110 mg), CH2Cl2 (50 mL) were combined and treated as above. Compound 11b
was obtained as an off-white solid (1.93 mg, 4.34 mmol, 76%); mp 157.5−158.3 °C (lit.34
178−180 °C); 1H NMR (CDCl3) δ 0.65–2.00 (m, 49H), 3.38 (s, 2H), 4.75 (m, 1H); 13C
NMR (CDCl3) δ 12.0, 12.2, 18.6, 21.2, 22.5, 22.8, 23.8, 24.2, 27.4, 27.97, 28.03, 28.2,
28.6, 31.9, 34.0, 35.4, 35.8, 36.1, 36.7, 39.5, 39.9, 42.5, 44.0, 44.6, 54.2, 56.2, 56.4, 74.4,
173.8; IR 3400, 2933, 2849, 1725, 1220; [α]24D + 12.2° (c 0.43, CHCl3); HRMS: for
C29H52NO2 (M + H)+ calcd 446.3998, found 446.4003. Anal. Calcd for C29H51NO2: C,
78.15; H, 11.53; N, 3.14. Found: C, 78.18; H, 11.57; N, 3.10.
General procedure for the preparation of 3EUr1Es-3α-cholestane (10a−b)
OHN
HN
O
O
3O
ODCMrt, 5 hH2N
O
O
OtBuOCN
O
3
+
An amine 11 was added to a solution containing 3 in CH2Cl2. The solution was stirred at
rt for 5 h. The resulting transparent solution was concentrated to give a white solid.
Flash column chromatography was performed to purify the crude product to give a white
solid, which gave a single spot on TLC (hexane:EtOAc, 2.5:1, Rf = 0.33−0.35) in
160
61−64% yield.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3α-
yloxycarbonylmethyl)ureido]heptanedioate, 3EUr1Es-α-cholestane (10a)
The general procedure for the synthesis of 10 above was followed: 11a (542 mg, 1.22
mmol), isocyanate 3 (534 mg, 1.21 mmol), CH2Cl2 (6 mL) were combined and treated as
above. Compound 10b was obtained as a white solid (691 mg, 0.78 mmol, 64.0%): mp
82.9–83.3 °C; 1H NMR (CDCl3) δ 0.60–2.00 (m, 79H), 2.24 (m, 6H), 3.94 (dd, 1H), 3.89
(dd, 1H), 4.65 (m, 1H; exchange with D2O), 4.79 (s, 1H; exchange with D2O), 5.08 (m,
1H); 13C NMR (CDCl3) δ 11.3, 12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.1, 28.0, 28.1,
28.2, 28.3, 29.9, 30.6, 31.9, 32.8, 35.5, 35.7, 35.8, 36.2, 39.5, 39.9, 40.0, 42.3, 42.6, 54.1,
56.3, 56.5, 56.7, 71.6, 80.5, 156.1, 170.5, 173.0; IR 2928, 1727, 1365, 1203, 1149, 1100;
[α]27D +6.3° (c 0.80, CHCl3); HRMS: for C52H91N2O9 (M + H)+ calcd 887.6725, found
887.6702. Anal. Calcd for C52H90N2O9: C, 70.39; H, 10.22; N, 3.16. Found: C, 70.43; H,
10.30; N, 3.17.
Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5α-cholestan-3β-
yloxycarbonylmethyl)ureido]heptanedioate, 3EUr1Es-β-cholestane (10b)
The general procedure for the synthesis of 10 above was followed: 11b (1.09 g, 2.44
mmol), 3 (989 mg, 2.24 mmol), CH2Cl2 (20 mL) were combined and treated as above.
Compound 10b was obtained as a white solid (1.21g, mmol, 61%); mp 195.8–196.2 °C;
1H NMR (CDCl3) δ 0.60–2.00 (m, 79H), 2.20 (m, 6H), 3.84 (d, 2H), 4.62 (t, 1H;
exchanges with D2O), 4.70 (s, 1H; exchanges with D2O), 4.75 (m, 1H); 13C NMR
(CDCl3) δ 11.7, 11.8, 18.3, 20.8, 22.2, 22.4, 23.5, 23.8, 27.0, 27.6, 27.7, 27.8, 28.2, 29.4,
30.1, 31.6, 33.5, 35.0, 35.1, 35.4, 35.8, 36.3, 39.1, 39.6, 41.8, 42.2, 44.2, 53.8, 55.9, 56.0,
161
56.1, 74.4, 80.0, 156.0, 170.4, 172.6; IR 2927, 1726, 1365, 1147, 1199, 1097; [α]27D
+11.6° (c 1.50, CHCl3); HRMS: for C52H91N2O9 (M + H)+ calcd 887.6725, found
887.6744. Anal. Calcd for C52H90N2O9: C, 70.39; H, 10.22; N, 3.16. Found: C, 70.42; H,
10.31; N, 3.16.
General procedure for the preparation of 3CUr1Es-cholestane (9)
OHN
HN
O
O
3O
O
HOHN
HN
O
O
3O
OHCOOHrt, 9 h
To a 50-mL round-bottomed flask containing a tri-tert-butyl ester 10 was added HCOOH;
the flask was warmed in a hot-water bath until a transparent solution appeared. The
resulting solution was removed from the hot-water bath and stirred at rt for 5 h. The
resulting reaction was concentrated to dryness to leave a white solid. Recrystallization in
HOAc−hexane was carried out to purify the crude white solid in 50−57% yield.
4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3α-yloxycarbonylmethyl)ureido]heptanedioic
acid, 3CUr1Es-α-cholestane (9a)
The general procedure for the synthesis of 9 above was followed: HCOOH (6 mL) and
10a (891 mg, 0.472 mmol) were combined and treated as above. The triacid 9a was
obtained as a white solid (174 mg, 0.242 mmol, 51%); mp 192.5–192.9 °C; 1H NMR
(DMSO-d6) δ 0.60–2.10 (m, 53H), 3.66 (d, 2H), 4.87 (s, 1H), 5.86 (s, 1H), 5.91 (t, 1H),
11.97 (bs, 1H); 13C NMR (DMSO-d6) δ 11.7, 12.5, 19.2, 21.0, 23.1, 23.3, 23.9, 24.4,
26.2, 28.0, 28.5, 28.6, 28.8, 30.7, 32.1, 33.0, 33.1, 35.6, 35.9, 36.0, 36.3, 39.6, 42.1, 42.8,
54.2, 55.9, 56.4, 56.6, 70.5, 157.5, 171.2, 175.1; IR 3409, 2933, 1751, 1702, 1559, 1204,
1158; [α]27D +15.7° (c 0.65, EtOH); HRMS: for C40H67N2O9 (M + H)+ calcd 719.4847,
found 719.4851. Anal. Calcd for C40H66N2O9: C, 66.82; H, 9.25; N, 3.90. Found: C,
162
66.96; H, 9.37; N, 3.89.
4-(2-Carboxyethyl)-4-[3-(5α-cholestan-3β-yloxycarbonylmethyl)ureido]heptanedioic
acid, 3CUr1Es-β-cholestane (9b)
The general procedure for the synthesis of 9 above was followed: HCOOH (10 mL) and
10b (891.4 mg, 1.00 mmol) were combined and treated as above. The triacid 9b was
obtained as a white solid (409 mg, 0.569 mmol, 57%); dec at 194.2 °C; 1H NMR
(DMSO-d6) δ 0.50–2.10 (m, 59H), 3.62 (d, 2H), 4.54 (h, 1H), 5.84 (s, 1H), 5.91 (t, 1H),
11.96 (bs, 1H); 13C NMR (DMSO-d6) δ 12.5, 12.6, 19.2, 21.4, 23.0, 23.3, 23.9, 24.5,
27.7, 28.0, 28.4, 28.7, 30.6, 32.2, 34.3, 35.6, 35.9, 36.3, 36.8, 42.1, 42.8, 44.6, 54.2, 55.9,
56.4, 56.6, 74.1, 157.4, 171.2, 175.1; IR 3410, 2933, 1728, 1704, 1564, 1221, 1187;
[α]27D +9.8° (c 0.58, EtOH); HRMS: for C40H67N2O9 (M + H)+ calcd 719.4847, found
719.4851. Anal. Calcd for C40H66N2O9: C, 66.82; H, 9.25; N, 3.90. Found: C, 66,72; H,
9.41; N, 3.87.
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168
Chapter V Antimicrobial Activity of Various Tri-headed Amphiphiles
V.1 Introduction to Biological Assays
As several homologous tri-carboxylato amphiphiles had been synthesized, we
aimed to fulfill our next goal, which was to explore the structure-activity relationship of
these amphiphiles. However, our laboratory was not equipped to do any biological
assays. In doing so, we performed the biological assays in collaboration with the
Department of Biological Sciences, Virginia Tech, under Dr. Joseph O. Falkinham’s
supervision.
V.1.1 Measurement of Antimicrobial Activity
In these assays, the antimicrobial activity is measured as MIC (minimum
inhibitory concentration), defined as the lowest minimum concentration of a drug, an
amphiphile in this case, required to inhibit the growth of a microorganism. MICs of
amphiphiles dissolved in aqueous triethanolamine (see Section V.2) are measured by
broth microdilution in 96-well microtiter plates,1 where the concentration of an individual
amphiphile is varied through a series of two-fold dilutions. A suspension of
microorganisms is added to each well of the microtiter plates. These assays require
tedious attention to details; the suspension of the organisms must not be contaminated
and any spattering to any other well should be avoided. After the plates are incubated at
a specific temperature for 4−7 days, the plates are screened visually for any inhibition,
indicated by turbidity, compared to the positive (drug-free) control of growth; this
positive control only contains broth and microorganism, where microorganisms should
grow. Theoretically, growth of microorganisms causes the well to look turbid, on the
other hand, transparent wells means no growth of microorganisms or complete inhibition.
169
It gets more complicated when turbidity with decreasing intensity is observed on all wells
for a specific amphiphile. In this case, we define the result as incomplete inhibition. As
these assays are designed by varying the amphiphile concentration in a series of two-fold
dilutions, we have no ability to determine our reading (MIC interpretation) as the exact
MICs. With this limitation, the experimental error in reading the MICs is within ±1 well
of the recorded wells.
The homologous amphiphile series are screened for their general antimicrobial
activity against a broad spectrum of microorganisms. These included Gram-negative
bacteria (Escherichia coli and Klebsiella pneumoniae), Gram-positive bacteria
(Micrococcus luteus, Lactobacillus plantarum, Staphylococcus aureus, and methicillin-
resistant Staphylococcus aureus (MRSA)), Mycobacterium smegmatis, yeasts
(Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans), and
filamentous fungus (Aspergillus niger).
V.1.2 Goals
For this study, as we have no previous knowledge whether the amphiphiles
display any antimicrobial activity, firstly, we intend to find a range of concentration at
which the amphiphiles are active against a broad spectrum of microorganisms. Secondly,
we identify the most active amphiphile in the homologous series. And thirdly, as we
have designed several homologous series having similar hydrophobicities, but different
tail topology, we will explore if the biological activity is affected by hydrophobicity or
the tail topology.
170
V.2 Recent Studies of Solubility of One-tailed Tri-carboxylato Amphiphiles
As it was initially investigated by a group member, André Williams,2 the 3CAmn
homologous series was dissolved in phosphate buffer (pH = 7.2). Comparison of the
resulting solubilities of the 3CAmn series to those of long chain fatty acids (pH = 7.4)
reveals that the presence of multiple headgroups did improve the solubilities. The
solubilities of 3CAm13, 3CAm15, and 3CAm17 were 6900, 3400 and 1700 μM,
respectively; the solubility of the corresponding long chain fatty acids3⎯myristic (n =
13), palmitic (n = 15), and stearic (n = 17) acids⎯ are 20−30, ~1, and <<1 μM,
respectively. However, as the chains become longer, 3CAm19 and 3CAm21, the
solubilities fell to 140 and 79 μM, respectively. Because of these low solubilities, a
different aqueous solution was considered.2
NH
COOH
COOH
COOH
O
n-1
3CAmn
OH
O
n-1
long-chain fatty acid
The choice of the counterion followed from a study4 of N-lauroyl-L-glutamate in
water; the triethanolammonium salt dissolved to a much greater concentration than did
the potassium salt. Another group member, Richard Macri, explored different conditions
to achieve the maximum solubility of the amphiphile in aqueous triethanolamine.
We initiated this biological study by solubilizing 25 mg of 3CAm13 in 2 mL of
5% (w/v) aqueous triethanolamine. The molar ratio of 3CAm13:triethanolamine was
1:12. Based on this result, we followed the procedure, where an individual amphiphile
(25 mg) was dissolved in 5% (w/v) aqueous triethanolamine solution (2 mL).
171
We successfully dissolved the amphiphiles into aqueous triethanolamine
solutions. These aqueous solutions of the single- and two-tailed amphiphiles (3CUrn,
3CUr(n)2, and 3CUr1(n)2) ranged from 19,500 to 25,700 µM depending on the formula
weight of the homolog, while those of the 3CUr-z-cholestane, and 3CUr1Es-z-
cholestane were 18,900 and 17,400 μM, respectively. We did not pursue the maximum
solubilities of these amphiphiles in the aqueous triethanolamine solution because any
compound with an MIC at this concentration is not worth developing as a lead. The
molar ratio of the one- and two-tailed amphiphiles:triethanolamine ranged from 1:13 to
1:17 depending on the formula weight of the amphiphile, while those of 3CUr-z-
cholestane:triethanolamine and 3CUr1Es-z-cholestane:triethanolamine were 1:17 and
1:19, respectively.
V.3 Results of Biological Assays
Table V.1 presents the MICs for all the amphiphiles. The MICs are recorded as
complete and incomplete inhibitions. As indicated, incomplete inhibitions are observed
in most cases. The MICs are presented in mg/L, following the standard representation of
MICs. However, the MICs will be discussed in term of μM as the formula weights of the
amphiphiles in a series differ by 28 amu between adjacent homologs.
172
Scheme V.1 Structures of dendritic tri-carboxylato amphiphiles
FG NH
COOH
COOH
COOH
O
+ NOH
33 FG N
HCOO
COO
COO
O
HNOH
3
HNOH
3
HNOH
3
3CUrn, FG = −NHR, R = CnH2n+1, n = 14, 16, 18, 20, 223CUr(n)2, FG = −NR2, R = CnH2n+1, n = 7−113CUr1(n)2, FG = −CHNR2, R = CnH2n+1, n = 7−12
NH O
HN
O3CUr-z-cholestane , FG = 3CUr1Es-z-cholestane , FG =
The five series of dendritic tri-carboxylato amphiphiles did not display a uniform
pattern of activity against the broad spectrum of microorganisms tested, but displayed
amphiphile-series-, species-, chain-length-, or epimer-specific patterns. Although the
MIC values obtained were not in the range of 1 mg/L or lower (e.g., ≤ 1 µM), some MICs
were below 10 mg/L (Table V.1).
In general, L. plantarum, S. aureus, M. luteus and A. niger were simply not
inhibited to any degree by any of the amphiphiles (Table V.1). Lack of antimicrobial
activity against L. plantarum is advantageous because lactobacilli, members of the skin
microflora (e.g., vagina), provide some protection to acquisition of infections (e.g.,
sexually transmitted pathogens).5 Some exceptions are observed for several
microorganisms. MRSA is not inhibited by the one- and two-tailed series, however it is
by the amphiphiles with cholestane moieties. None of the yeasts are inhibited to any
degree by the amphiphiles with cholestane moieties. K. pneumoniae also display an
exception; it is weakly inhibited only by the 3CUrn series.
173
Table V.1 MICs of tri-carboxylato amphiphiles against bacteria, yeasts, and a filamentous fungus MIC (mg/L) Amphiphile E
coli K. pneumoniae
L. plantarum
S. aureus
MRSA M. luteus
M. smegmatis
S. cerevisiae
C. albicans
C. neoformans
A. niger
3CUr14b 140 140a 3100a 3100a 3100a >6300 280a 280 780a 780a 3100 3CUr16b 71 140a 3100a 390a 3100a >6300 71a 280 24a 24a 1600 3CUr18b 71 140a 780a 780a 390a >6300 18a 71 780a 49a 780 3CUr20b 36 140a 780a 780a 780a >6300 36a 71 1600a 1600a 390 3CUr22b 71 280a 560a 780 780a >6300 71a 18 1600a 3100a 3100 3CUr(7)2 1600a >560 3100a >6300 1600a 3100a 1600a 390a 780a 570a 390a 3CUr(8) 2 390a >560 780a >6300 1600a 1600a 780a 200a 98a 280a 200a 3CUr(9) 2 780a >560 6300a >6300 6300a 6300a 780a 390a >6300 140a 3100a 3CUr(10) 2 1600a >560 6300a >6300 6300a 6300a 390a 1600a >6300 36a 6300a 3CUr(11) 2 1600a >560 6300a >6300 6300a 6300a 390a 3100a >6300 >560 6300a 3CUr1(7) 2 1600a >6300 3100a >6300 >6300 >6300 1600a 3100a 1600a 560a 3100 3CUr1(8) 2 780a >6300 3100a >6300 >6300 >6300 780a 1600a 390a 140a 3100 3CUr1(9) 2 200a >6300 3100a >6300 >6300 >6300 200a 1600a >6300 36a 1600 3CUr1(10) 2 1600a >6300 3100a >6300 >6300 >6300 200a 1600a >6300 8.9a 6300 3CUr1(11) 2 1600 >6300 6300a >6300 >6300 >6300 390a 780a >6300 4.4a 6300 3CUr1(12) 2 1600 >6300 6300a >6300 >6300 >6300 780a 390a >6300 4.4 a 6300 A 140 560 1600a 6300a 200a 3100 390a >6300a >6300a >6300a 1600 B 140 560 1600a 390a 18a 780 390a >6300a >6300a >6300a 1600 C 560 560 3100a 6300a 200a 6300 390a >6300a >6300a >6300a 6300 D c 560 3100a 160a 780a 6300 390a >6300a >6300a >6300a 6300 aIncomplete inhibition bMIC data from reference 2 cnot measured
A, 3CUr-α-cholestane; B, 3CUr-β-cholestane; C, 3CUr1Es-α-cholestane; D, 3CUr1Es-β-cholestane
174
V.3.1 Range of Concentration of Active Amphiphiles
In general, the 3CUrn series are more active than the rest of the series (3CUr(n)2,
3CUr1(n)2, 3CUr-z-cholestane and 3CUr1Es-z-cholestane). The 3CUrn homologs are
active against a Gram-negative bacterium E. coli and M. smegmatis within 62−290 μM
and 33−580 μM, respectively. The former is observed for complete inhibition, while the
latter is for incomplete inhibition. Against the three yeasts, the concentration of the
3CUrn homologs vary; 30−580, 47−2700, and 47−5200 μM for S. cerevisiae, C.
albicans, and C. neoformans, respectively. Complete inhibitions are observed against S.
cerevisiae, but not observed against C. albicans and C. neoformans. For the rest of the
microorganisms, the 3CUrn homologs are active at concentrations above 250 μM.
Based on the MICs reported for the two-tailed series, the only microorganism that
attracts our attention in these series is C. neoformans. These results showed that the two-
tailed series are very specific against a particular microorganism, i.e., C. neoformans.
The range of the concentration of the 3CUr(n)2 and 3CUr1(n)2 series are 62−1200 and
6.9−1100 μM, respectively. The wide ranges of concentration are observed, especially
in the 3CUr1(n)2.series To us, this can simply mean that particular homologs can be
much more active than other homologs. For the rest of the microorganisms, the
3CUr(n)2 and 3CUr1(n)2 homologs are active at concentrations above 190 μM.
Compared to the one- and two-tailed series, the two last series, 3CUr-z-
cholestane and 3CUr1Es-z-cholestane, are not as active against the microorganisms
tested, with an exception to MRSA. All homologs but 3CUr1Es-β-cholestane are more
active than any homolog in the first three series. The active concentration ranges of the
3CUr-z-cholestane and 3CUr1Es-z-cholestane series are 27−300 and 270−1100 μM,
175
respectively. It is apparent that 3CUr-z-cholestane displays more activity than the
3CUr1Es-z-cholestane; the more active amphiphile in the epimeric pair of 3CUr-z-
cholestane is tenfold more potent than that of the 3CUr1Es-z-cholestane against MRSA.
For the rest of the microorganisms, the active concentrations of all homologs within these
series are above 210 μM.
V.3.2 The Most Active Amphiphiles within Each Homologous Series
From Table V.1, the most active amphiphile within a homologous series can be
identified. Plots of log MIC vs chain length in the 3CUrn series (Figure V.1) clearly
show parabolic figures, which simply means that cut-off effects are reached against E.
coli, M. smegmatis, C. albicans, and C. neoformans. Amphiphiles 3CUr20 and 3CUr18
are the most active against E. coli and M. smegmatis, respectively (Figure V.1, left), and
3CUr16 is the most active against both C. albicans, and C. neoformans (Figure V.1,
Figure V.1 Relationship between log MIC of the 3CUrn series vs chain length for E. coli, M. smegmatis (left) and C. albicans, C. neoformans, and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides
-4.8
-4.3
-3.8
-3.3
12 14 16 18 20 22
chain length
log
[MIC
] (M
)
E. coli M. smegmatis
-5-4.5
-4-3.5
-3-2.5
-2
12 14 16 18 20 22chain length
log
[MIC
] (M
)
C. albican C. neoformansS.cerevisiae
176
right). Against S. cerevisiae, the amphiphiles with longer hydrocarbon chain display
better activity (Figure V.1, right). Within this series, the cut-off effect has not yet been
reached.
In the two-tailed series against C. neoformans, plots of log MIC vs chain length display
two different patterns (Figure V.2). A cut-off effect has been reached in the 3CUr(n)2
series, but not in the 3CUr1(n)2 series (Figure V.2, left). Amphiphile 3CUr(10)2 appears
to be the most active amphiphile. Within the chain length tested, the longest homolog of
3CUr1(n)2 is the most active amphiphile. Amphiphile 3CUr1(12)2 is even more active
than the most active homolog in the 3CUrn series; 3CUr1(12)2 is sevenfold more active
than 3CUr16. An ideal cut-off effect is shown on these two-tailed series against C.
albicans (Figure V.2, right), where the activity of the amphiphiles increases up to a point,
then the activity ceased within the highest concentrated tested.
Figure V.2 Relationship between log MIC of the 3CUr(n)2 and 3CUr1(n)2 series vs chain length for C. neoformans (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3. Lines connecting points are eye guides
-5.5
-5
-4.5
-4
-3.5
-3
-2.5
12 14 16 18 20 22 24
chain length
log
[MIC
] (M
)
3CUr(n)2 3CUr1(n)2
-4
-3.5
-3
-2.5
-2
-1.5
12 14 16 18 20 22 24
chain length
log
[MIC
] (M
)
3CUr(n)2 3CUr1(n)2
177
As the last two series contains a pair of epimeric compounds in each series, there
is no cut-off effect for this discussion. However, a particular epimer is found to be more
active than the other. Amphiphiles 3CUr-β-cholestane and 3CUr1Es-α-cholestane are
more active against MRSA compared to their epimeric pairs.
V.3.3 Various Specificities Patterns of Antimicrobial Activity
V.3.3.1 One- and Two-tailed Amphiphiles
C. neoformans was unique among the microorganisms tested by virtue of its
susceptibility to members of both series of two-tailed amphiphiles and the one-tailed tri-
carboxylato dendritic amphiphiles (Table V.1). M. smegmatis, E. coli, K. pneumoniae,
and S. cerevisiae were distinguished from the other microorganisms by their
susceptibility to all five members of the 3CUrn series tested. (Although the three
bacteria have an outer membrane, S. cerevisiae and the remaining microorganisms lack
an outer membrane, so that is apparently not the common structure responsible for
susceptibility.)
These results with long chains contrasted with those studies6,7 that have shown
that short chain free fatty acids (C8–C12) exhibit highest antibiotic activity against a
variety of mycobacteria, including M. smegmatis. C. neoformans was unique by its
susceptibility to all five members of the 3CUr1(n)2 series (Table V.1). With the
exception of C. neoformans (e.g., 3CUr(10)2), the panel of microorganisms, including
the other pathogenic yeast C. albicans, were resistant to the 3CUr(n)2 series (Table V.1).
Amphiphile-series specificity is shown when a particular class of
amphiphile⎯3CUrn, 3CUr(n)2 or 3CUr1(n)2⎯is specifically active against a tested
microorganism. Amphiphile-series-specific antimicrobial activities were displayed by
178
the 3CUrn series against E. coli, K. pneumoniae, M. smegmatis, and Saccharomyces
cerevisiae (Table V.1). Members of the 3CUrn series were significantly more active
against those microorganisms than the corresponding members of the 3CUr(n)2 and
3CUr1(n)2 series. The one exception to that statement was activity against S. cerevisiae
by 3CUr(8)2 (MIC = 380 µM) and 3CUr16 (MIC = 550 µM); 3CUr(8)2 showed
incomplete inhibition but 3CUr16 showed complete inhibition.
Species specificity is displayed when a particular microbial species is uniquely
susceptible to a particular amphiphile. The 3CUrn homologs are active against the two
Gram-negative bacteria, a mycobacterium, and S. cerevisiae; they are not active against
Gram-positive bacteria, the pathogenic yeast, and a filamentous fungus (Table V.1).
Two-tailed amphiphiles were only active against the yeast, but not the bacteria,
mycobacterium, or filamentous fungus. For the yeasts tested, some homologs of the
3CUr(n)2 series had antimicrobial activity against both C. albicans and C. neoformans,
while all but one of the 3CUr1(n)2 amphiphile series had strong antimicrobial activity
against C. neoformans.
As only particular members of the homologous series with specific chain length
show distinct activities, these amphiphiles also display another type of specificity,
namely chain-length specificity. Chain-length specificity is displayed when amphiphiles
in a homologous series with a particular chain length are exceptionally active compared
to those with other chain lengths. Chain-length specificity was displayed by members of
the 3CUrn series against M. smegmatis, S. cerevisiae, C. albicans, and C. neoformans,
by those of the 3CUr(n)2 series against C. albicans and C. neoformans, and by those of
the 3CUr1(n)2 series against C. neoformans (Table V.1).
179
In the 3CUrn series, the MIC of 3CUr16 against both C. albicans and C.
neoformans is 47 μM, the MIC of 3CUr18 against M. smegmatis is 33 μM, and the MIC
of 3CUr22 against S. cerevisiae was 32 μM. This chain-length specificity was not found
in the 3CUrn series against the rest of the microorganisms tested and the homologs of
3CUrn were not particularly active against those microorganisms. Among the two-tailed
amphiphiles, the three homologs 3CUr1(10)2, 3CUr1(11)2, and 3CUr1(12)2 showed
unique and promising MICs of 15 µM, 7.2 µM, and 6.9 µM, respectively, against C.
neoformans.
Chain-length specificity was particularly demonstrated by 3CUr(8)2 against C.
albicans (MIC = 190 μM) and 3CUr(10)2 against C. neoformans (MIC = 62 μM) (Table
V.1). As the strain of C. albicans was grown at 37 °C, it had low cell surface
hydrophobicity,8 which likely contributed to relative resistance of the cells to
amphiphiles.
V.3.3.2 Cholestane-based Amphiphiles
As seen in the previous series of amphiphiles, there was no general pattern
observed in the cholestane-based amphiphiles; the amphiphiles behaved differently
against the tested microorganisms. The only common pattern observed was that these
cholestane-based amphiphiles were not active against yeast; none of the yeasts tested
responded to the cholestane-based amphiphiles to any degree under given concentration.
Epimer specificity was displayed by both series against S. aureus and MRSA, and
by 3CUr-z-cholestane series against M. luteus. Epimer specificity was not displayed
against both Gram-negative K. pneumoniae and E. coli (3CUr-z-cholestane) for both
Gram-positive L. plantarum and M. luteus (for 3CUr1Es-z-cholestane), M. smegmatis,
180
and filamentous A. niger.
V.3.4 Comparing Hydrophobicity and Antimicrobial Activity
It was our intention next to explore how amphiphiles with similar hydrophobicity,
but different tail topology will affect their antimicrobial activity. The amphiphiles
maintain common head groups and linker. However, the tails were arranged in different
geometry. As the number of carbon on the tail(s) is maintained equal, amphiphiles with
the same number of carbon in their tails will have similar hydrophobicity.
V.3.4.1 One- and Two-tailed Amphiphiles
Partitioning of these amphiphiles into both outer and cytoplasmic membranes
should be a major contributor to their antimicrobial activity. Based on simple
approximations of hydrophobicity, the distribution coefficient increases as the chain
length increases. Calculations9 of logD at pH 7.4 give values that range from –5.1 for
3CUr14 to –1.9 for 3CUr22 in increments of 0.8 for each additional –CH2–CH2– unit,
from –5.2 for 3CUr(7)2 to –2.0 for 3CUr(11)2 in increments of 0.8 for each additional –
CH2– in each chain, and from –5.0 for 3CUr1(7)2 to –1.0 for 3CUr1(12)2 in increments
of 0.8 for an additional –CH2– in each chain. Because of the multiple ionization states of
the headgroup, logD is the appropriate choice. These calculations suggest that the tri-
headed amphiphiles are substantially more likely to favor being in a hydrophilic phase
than in a hydrophobic phase.
Comparing logD vs MIC for each member of the three series reveals the
similarities with respect to the antimicrobial activity against M. smegmatis (Figure V.1,
left) and C. albicans (Figure V.1, right). For M. smegmatis, all series show a parabolic
181
dependence on logD; the 3CUrn series gives the best activities. The 3CUr(n)2 and
3CUr1(n)2 series overlap each other in activity against M. smegmatis such that a logD of
–2 to –3 predicts the best activity. All series show similar pattern of activity against C.
albicans, a microorganism that shows chain-length specificity. The one-tailed
amphiphile 3CUr16 (logD = –4.3) has better activity than 3CUr(8)2 (logD = –4.4) and
3CUr1(8)2 (logD = –4.2).
Figure V.3 Relationship between log MIC of one- and two-tailed amphiphiles vs logD for M. smegmatis (left) and C. albicans (right). Error bars (not shown for clarity) are ± 0.3
Comparing logD vs MIC for each member of the three series reveals the
differences with respect to the antimicrobial activity against C. neoformans (Figure V.2,
left) and S. cerevisiae (Figure V.2, right). For C. neoformans, there is no similarity in the
patterns among the three series. Amphiphiles 3CUr16 (logD = –4.3), 3CUr(10)2 (logD =
–2.8), and 3CUr1(12)2 (logD = –1.0) are the most active members of the respective
series. Clearly, the 3CUr1(n)2 series is the best among these three series. For S.
cerevisiae, the 3CUrn and 3CUr1(n)2 series show similar patterns of improving activity
with increasing hydrophobicity, although the 3CUrn series is significantly more active.
-5
-4.5
-4
-3.5
-3
-2.5
-2
-6 -4 -2 0
logD
log
[MIC
] (M
)
3CUrn 3CUr(n)23CUr1(n)2
-5
-4
-3
-2
-1
-6 -4 -2 0
logD
log
[MIC
] (M
)
3CUrn 3CUr(n)23CUr1(n)2
182
The 3CUr(n)2 series has a parabolic dependence with the best activity at 3CUr(8)2 (logD
= –4.4).
Figure V.4 Relationship between log MIC and logD for C. neoformans (left) and S. cerevisiae (right). Error bars (not shown for clarity) are ± 0.3
V.3.4.2 Cholestane-based Amphiphiles
The values9 of logD at pH 7.4 for 3CUr-z-cholestane and 3CUr1Es-z-
cholestane were –2.62 and –3.68, respectively. Based on simple approximations of
hydrophobicity, addition of a spacer –CH2COO− simply decreased the distribution
coefficient by 1.06.
In each pair of epimeric amphiphiles, the distribution coefficients were exactly the
same, yet the activity, represented by MICs, against several organisms were different.
This evidence suggests that geometry of the molecules affect their biological activities.
V.4 Comparison with Prior Work
A number of compounds containing a ureido group, where both nitrogen atoms
are monosubstituted, have been synthesized. In those studies, a nitrogen is attached to a
-5.5
-5-4.5
-4-3.5
-3-2.5
-2
-6 -4 -2 0
logD
log
[MIC
] (M
)
3CUrn 3CUr(n)23CUr1(n)2
-5
-4.5
-4
-3.5
-3
-2.5
-2
-6 -4 -2 0
logDlo
g [M
IC] (
M)
3CUrn 3CUr(n)23CUr1(n)2
183
long single alkyl chain while the other nitrogen is attached to a various group, which can
be classified as depsipeptide,10 pyrimidine nucleoside,11 glucosamine,12 crown ether,13
and penicillin14 substituents. All the compounds above have displayed antibacterial10,12-14
and antifungal11,12 activity.
Only one study15 evidences that urea derivatives where one of the nitrogen on the
ureido functional group is attached to two symmetrical n-alkyl chain have shown utility
as agricultural fungicides. On the other hand, many compounds containing two
symmetrical alkyl chains bonded to a nitrogen atom, not necessarily associated with a
ureido functional group, have been synthesized and studied for their antibacterial and
antifungal activity. To our knowledge, none of these compounds have anionic
headgroups as reported in this study. In these previous studies, the compounds are in the
neutral16,17 or charged form.18-21 The charged forms involve quarternary ammonium
with chloride counterions,18-20 or intercalation complexes21 with montmorillonite or
saponite. In a study20 of antibacterial properties against S. aureus, several homologous
tertiary amines are employed to quarternize crosslinked chloromethyl polystyerenes;
antibacterial efficiency increases as the alkyl chain length increases.
To the best of our knowledge, no urea derivative, where one of the nitrogen on the
ureido functional group attached to a swallowtail, is found to show antibacterial or
antifungal properties in the open literature. However, there are several compounds
containing a nitrogen atom bonded to a swallowtail, a long fatty chain bonded through
the middle carbon atom, have been synthesized. Unlike compounds containing a nitrogen
atom directly bonded to two symmetrical fatty chains, there are only few studies on
compounds containing a nitrogen atom bonded to a swallowtail. These compounds have
184
been investigated for antibacterial22,23 and antifungal23 activity. In the studies, these
compounds have cationic22,23 and neutral23 headgroups.
Several compounds where cholestane moiety is attached to a ureido linker have
been synthesized. No study of antifungal or antibacterial properties in such compounds
was found. Nevertheless, two studies pointed out that these compounds in their neutral
forms were actually display anticancer activity.24,25 Apart from that, a number of
compounds containing cholestane moiety indeed have been synthesized26-28 or even
isolated from natural product;29-32 these compounds exhibit antibacterial26-31 and
antifungal26,30-32 properties. To the best of our knowledge, no compound with the general
structure of linker-spacer-hydrophobic moiety, such as that in 3CUr1Es-z-cholestane,
were found in the open literature. However, compounds containing a cholestane moiety
bonded to an ethanoate moiety have been synthesized and investigated for their
antibacterial,33 antimycobacterial,34 and antifungal33,35 properties.
V.5 Conclusions
Overall, we concluded that the antimicrobial activity displayed is modest with a
couple of promising leads. However, we could say that the one-tailed series is more
active than the others. In general, the five series of dendritic tri-carboxylato amphiphiles
did not display a uniform pattern of activity against the broad spectrum of
microorganisms tested, but displayed amphiphile-series-, species-, chain-length-, or
epimer-specific patterns. It would be interesting to further the investigation on the
antimicrobial activity where the cut-off effect has not been reached.
185
V.6 Experimental Procedures V.6.1 Preparation of Tri-carboxylato Amphiphiles Solutions in Aqueous Triethanolamine
An aqueous triethanolamine was prepared by the following manner.
Triethanolamine (4.900 g) was added to a 100-mL volumetric flask, and then diluted with
water until the mark. The stock (12,500 mg/L) solutions for all homologues were easily
prepared by simply vortexing the tricarboxylic acid in the aqueous triethanolamine
solution. Each tri-headed amphiphile⎯3CUrn, 3CUr(n)2, 3CUr1(n)2, 3CUr-z-
cholestane, and 3CUr1Es-z-cholestane⎯(25 mg) was dissolved into aqueous
triethanolamine (2 mL). An aliquot (200 μL) of the resulting solution was diluted with
aqueous triethanolamine (2 mL) to give a more dilute solution (568 mg/L). These
original and the diluted solutions were called the stock and trial solutions, respectively.
V.6.2 Microbial Strains, Culture Conditions, and Preparations of Inocula for Susceptibility Testing
Strains of E. coli strain C (ATCC strain 13706), K. pneumoniae (ATCC strain
4352), L. plantarum (ATCC strain 14917), S. aureus (ATCC strain 6538), and M.
smegmatis (ATCC strain 607) were obtained from the American Type Culture Collection.
A methicillin-resistant isolate of S. aureus (MRSA) was obtained from the Microbiology
Laboratory, Danville Community Hospital (Virginia) and S. cerevisiae, C. albicans, C.
neoformans, A. niger, and M. luteus strains were obtained from the Virginia Tech
Microbiology teaching culture collection.
Colonies of E. coli, K. pneumoniae, S. aureus, MRSA, M. luteus, S. cerevisiae, C.
albicans, and C. neoformans were grown on 1/10-strength Brain Heart Infusion Broth
(BBL Microbiology Systems, Cockeysville, MD) containing 0.2% (wt/vol) sucrose
186
(BHIB+S) and 1.5% (wt/vol) agar. L. plantarum was grown on ¼-strength Tryptic Soy
Broth (TSB, BBL Microbiology Systems, Cockeysville, MD) containing 0.2% glucose
(TSB+G) and 1.5% (wt/vol) agar. M. smegmatis was grown on Middlebrook 7H10 agar
(BBL Microbiology Systems, Cockeysville, MD) and A. niger on Potato Dextrose Agar
(PDA, BBL Microbiology Systems, Cockeysville, MD). Streaked plates were incubated
at 37 °C for 3–7 days, except for that of A. niger, which was incubated in the dark at 30
°C. A single colony for each microbe except A. niger was used to inoculate 5 mL of
1/10-strength BHIB+S (E. coli, K. pneumoniae, M. luteus, S. aureus, and MRSA), full
strength TSB (L. plantarum), Middlebrook 7H9 broth (M. smegmatis), or Yeast Extract
Peptone Maltose broth (S. cerevisiae, C. albicans and C. neoformans) and incubated at
37°C (S. cerevisiae and M. luteus at 30 °C) for 4–7 days. After growth, the resulting
broth cultures were diluted with buffered saline gelatin [BSG, gelatin (0.1 g/L), NaCl (8.5
g/L), KH2PO4 (0.3 g/L), Na2HPO4 (0.6 g/L)] to equal the turbidity of a No. 1 McFarland
Standard. Spores of A. niger were scraped from the surface of PDA and suspended in 5
mL of 1/10-strength BHIB+S and that suspension transferred to a sterile test tube. The
turbidity was adjusted to a No. 1 McFarland Standard by dilution with BSG.
To check for viability and contamination, broth cultures of all organisms, except
A. niger, were streaked on Plate Count Agar (BBL Microbiology Systems, Cockeysville,
MD), and the plates incubated at 37 °C for 3–4 days, except those for M. luteus and S.
cerevisiae grown at 30 °C. To check for viability and contamination on A. niger, the
spore suspensions were streaked on PDA and incubated at 37 °C for 3–4 days.
For the work reported here, all cultures and suspensions that were used as inocula
were uncontaminated and the colonies had the expected morphologies. All viable,
187
uncontaminated inocula were stored up to 14 days at 4 °C until used without any
differences in susceptibility to antimicrobial compounds.
V.6.3 Measurement of MICs
MICs of compounds dissolved in aqueous triethanolamine were measured by
broth microdilution in 96-well microtitre plates.2 Preliminary experiments demonstrated
that 5% (wt/vol) triethanolamine/water did not inhibit the growth of any microorganism
tested. A two-fold dilution series of tri-headed amphiphiles⎯3CUrn, 3CUr(n)2,
3CUr1(n)2, 3CUr-z-cholestane, and 3CUr1Es-z-cholestane⎯was prepared in 96-well
microtitre plates (96 wells: rows A–H, columns 1–12) in the following manner. Aliquots
(50 μL) of 1/10-strength BHIB+S (pH 7.4) were placed in all the wells except for those in
column 1. Stock solutions (100 μM of 25 mg amphiphile 5% triethanolamine/water),
were placed in column 1. An aliquot (50 μL) was removed from a well in column 1 and
mixed with the BHIB+S in the well of column 2. Then, an aliquot (50 μL) of this mixture
was removed and mixed with BHIB+S in column 3. This process was repeated through
column 11, at which point an aliquot (50 μL) was removed and discarded. Column 12
was the blank (positive growth) control, BHIB+S only, for each row. An aliquot (50 μL)
of the suspension of the organism adjusted to No.1 McFarland Standard was added to all
wells in a row. Aqueous triethanolamine without any amphiphile was also tested for
antimicrobial activity by using the same protocol; no antimicrobial activity was found.
For E. coli and K. pneumoniae, the volumes of the medium and the inoculum were
doubled. The resulting inoculated dilution series were incubated at 30 °C (37 °C for E.
coli and K. pneumoniae) and growth, as turbidity, scored visually and recorded on the
188
fourth day (the seventh day for E. coli and K. pneumoniae). The MIC of each compound
was measured in triplicate and was defined as the lowest concentration of drug resulting
in a prominent visible decrease in turbidity (incomplete) or an absence of visible turbidity
(complete) compared to the drug-free control. In many cases (identified in Table V.1 by
superscript a), inhibition was incomplete.
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Chapter VI Intrinsic Activity of the 3CUrn Homologus Series Against Mycobacterium smegmatis
It had been discussed (Chapter I), that our goal is to design amphiphiles that have
good antimicrobial activity and does not involve detergency as the mechanism of action.
In order to fulfill the goal, we needed to explore solubility and CMC. In a homologous
series, antimicrobial activity can show a cut-off effect,1 known earlier as the parabolic
case,2 where antimicrobial activity increases up to an optimal point as chain length
increases; after that, the activity decreases. To conclude that the cut-off effect is due to
the intrinsic activities3,4 (i.e., the maximal response that a compound induces) or the
decreased solubility of the homologous amphiphiles, the amphiphiles must fully dissolve
in aqueous media.
We had designed amphiphiles that are water-soluble. This should allow the
biological assays of all amphiphiles to take place without encountering any solubility
problem. Following this, all homologous tri-carboxylato amphiphiles were screened for
the antimicrobial activity, which was represented as the MIC. As the solubility was no
longer an issue, it could be eliminated as the cause of a cut-off effect. Consequently, we
intended to explore if a cut-off effect was due to the intrinsic activity. How would we do
this?
In the previous biological assays (Chapter V), the suspension of the
microorganisms used was adjusted to equal the turbidity of a No. 1 McFarland Standard,
which has a cell density of 3 × 108 colony forming unit (CFU). For this number, it was
possibly too many of microorganisms added to the amphiphiles. Under this condition,
there might not be enough amphiphiles to inhibit the microorganisms. In other words, we
could not determine if the amphiphiles gave their maximum response. As we observed
193
some amphiphiles showed complete inhibition, and some others showed incomplete
inhibition. In order to overcome this problem, we intended to explore this antimicrobial
activity in more detail, where the MICs were measured based on the maximum response
of the amphiphiles. Under this condition, we should only observe the complete
inhibition, where we had to be certain that the amphiphiles should saturate the
microorganims. One way to satisfy this was to control the cell density of the
microorganisms used, that it should be less than that of a No. 1 McFarland Standard.
In this new protocol, MICs of the series were measured at several different initial
cell densities, expressed as colony-forming units (CFU/mL). We will vary the density of
the suspension of the microorganism by ten-fold dilution starting with supposedly that
where the turbidity has been adjusted to equal the turbidity of a No. 1 McFarland
Standard. In this dilution series, the more diluted suspension contains fewer
microorganisms. The least concentrated suspension should allow us to count the number
of the colony grown in term of CFU. This dilution series are used in the biological assays
following the previous protocol (Chapter V); both amphiphile concentration and cell
density of the suspension of the microorganism are varied. Under a certain concentration
(of suspension of the microorganism) and below, we expect they become saturated with
the amphiphile. In other words, the observed MIC should not change, instead, it should
be a constant; this is the MIC threshold, which is caused by the intrinsic activity.
In a homologous series, if the cut-off effect is observed under this protocol, we
can conclude that such a phenomenon is due to intrinsic activity. Following this, we
should compare these MICs to the CMC to determine if detergency is involved in the
mechanism of action of the antimicrobial activity. In this chapter, we would only address
194
the intrinsic activity of the 3CUrn homologous series against M. smegmatis.
VI.1 The “inoculum effect”
In studies of antimicrobial activity, the MIC is often observed to increase as initial
cell density increases. This phenomenon is referred to as the “inoculum effect”.5-7 Thus
patients with higher microbial loads often require higher drug doses.6 Gehrt et al.5 have
proposed that the “inoculum effect” reflects the increased demand for a given drug as the
number of cells and targets increases. From a bioorganic mechanistic perspective, the
“inoculum effect” may provide information about a change in the mechanism of action.
A mathematical model (Equation 1) of the “inoculum effect”, developed by Li
and Ma8 from a study of four aminoglycosides against Escherichia coli and
Staphylococcus aureus, shows the dependence of MIC on initial cell density (i.e.,
inoculum). Equation 1 describes the independence of log MIC at low inoculum by
log MIC = log MIC0 + (e − 1) (1)k (log I - log Itr ).
defining log MIC0 as the intrinsic activity, log I as the inoculum, log Itr as the inoculum at
the threshold immediately before the rise in MIC, and k is a constant that decribes the rate
of increase in MIC. Equation 1 is flat at low inoculum and the rises exponentially after
Itr, which varies by drug (see Figure VI.1 below).
VI.2 Intrinsic Activity of Homologous Amphiphiles
Our study addresses Gruber’s thesis that “many [reported] chain length profiles of
apparent potencies…produce an unclear, or misleading impression of the role [of] alkyl
chain length”.4 Gruber suggests that increasing the number of measurements by varying
195
the drug–cell ratios provides at least a qualitative measure of how membrane partitioning
affects activity. Gruber’s suggestion should lead to measurements where the amphiphiles
saturate the cells; hence the activity of the amphiphiles should be at an optimal value.
Our studies of measuring MICs as a function of initial cell density follow Gruber’s
suggestion. The results provide measurements of the intrinsic activity, which is equal to
log MIC0 in Equation 1.
Scheme VI.1 Simplified kinetic model of inhibition of cell growth
n drug + cell drugn . cell
k grow k inhibit
Keq
In our study, the MIC to prevent bacterial growth within a given time can be
described as the minimal concentration of drug needed to achieve the following
equality—kinhibit[drugn·cell] = kgrow[cell]—as described in Scheme VI.1. In this simplified
model, the drug diffuses into or onto a cell. Undoubtedly, more than one drug molecule
is needed to inhibit the cell. Hence, n indicates the number of drug molecules needed to
inhibit the cell. This n is probably not a specific number, but some average number,
especially, because the sizes of the cells vary. This means that Keq is actually a product
of several equilibria as successive drug molecules are absorbed by the cell. In addition to
the partitioning equilibria, there is the added complication of the microbe developing
drug-destruction and drug-export pathways,9 which drive the equilibrium to the left.
Further, kinhibit is also a composite of many different rate constants and equilibrium
constants.
The results of these experiments—measuring MIC as a function of initial cell
196
density—enable identification of the intrinsic activities. Varying the initial cell density
also provides a measure of how changing the number of cells (and as a result the amount
of cell wall, membranes, and proteins) affects MIC. If partitioning of these amphiphiles
into both outer and cytoplasmic mycobacterial membranes10 is a significant component of
the activity, then estimates of the partitioning coefficients would be useful in measuring
intrinsic activities. At a very low cell density, the cells are so few that we presume that
they are saturated with amphiphile. At the highest cell density, saturation might depend
on the partition coefficient of the amphiphile. For a homologous series, partition
coefficients increase as the chain length increases. For compounds with ionizable groups,
the distribution coefficient, which is comprised of the partition coefficient and the pKa’s,
provides a better estimate of the partitioning between octan-1-ol and water at given pH.
Calculations11 of log D at pH 7.4 give values that range from –5.1 for 3CUr14 to
–1.9 for 3CUr22 in increments of 0.8 for each additional –CH2–CH2– unit. Calculations
of log D at pH 7.4 for natural saturated fatty acids give values that range from 2.4 for
tetradecanoic acid to 5.6 for docosanoic acid. These calculations suggest that the 3CUrn
series substantially favors the hydrophilic phase so that low initial cell densities are
needed to saturate the cell with amphiphile.
To probe the intrinsic activities of these amphiphiles, we measured the MICs of
3Ur14–3Ur22 at several different initial cell densities, expressed as colony-forming units
(CFU)/mL, of M. smegmatis. Experiments were performed in duplicate at two
overlapping ranges of initial inocula—32 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108
CFU/mL. The MIC was defined as the lowest concentration at which an amphiphile
completely inhibited the growth after four days at 37 °C by successive twofold dilution in
197
96-well microtiter plates.
VI.3 Results and Discussions VI.3.1 MICs of the 3CUrn Series
All amphiphiles show an inoculum effect and follow Equation 1. The
antimycobacterial activity of these amphiphiles is constant as the initial cell density
increases until a threshold inoculum (Figure VI.1). These plateaus equal log MIC0, the
intrinsic activity. For these amphiphiles, 3CUr16 has the lowest intrinsic activity. The
log Itr, inoculum at the threshold immediately before the rise in MIC, varies among the
amphiphiles. The lowest log Itr (4.7) is for 3CUr16; the highest log Itr (6.7) occurs for
both 3CUr14 and 3CUr20. If partitioning into a hydrophobic part of the cell were a
dominant mechanism, the log Itr should be lowest for 3CUr14. After the threshold, the
increases in log MICs vs log initial cell densities are approximately the same for all the
amphiphiles.
Figure VI.1 Effect of initial cell density on inhibition of growth of M. smegmatis for the 3CUrn series. Combined data of two separate assays⎯3.2 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108 CFU/mL. Error bars (not shown for clarity) are ± 0.3
-5
-4.5
-4
-3.5
-3
-2.5
0 1 2 3 4 5 6 7 8 9 10log initial cell density (CFU/mL)
log
[MIC
] (M
)
3CUr14 3CUr16 3CUr20
-4.5
-4
-3.5
-3
-2.5
0 1 2 3 4 5 6 7 8 9 10log initial cell density (CFU/mL)
log
[MIC
] (M
)
3CUr18 3CUr22
198
Figure VI.2 shows plots of log MIC vs. chain length at four different initial cell
densities to better illustrate the effect of chain length on activity. Figure VI.2 also
illustrates Gruber’s thesis that at cell densities above the lowest threshold, one is
measuring apparent values and can be misled as to which chain length gives the best
intrinsic activity. At the initial cell densities of 3.2 × 104 CFU/mL and lower, the profile
is the same and the intrinsic activity is revealed (Figure VI.2 left, circles). The most
active compound is 3CUr16. One might have reached a similar conclusion at an initial
cell density of 5 × 108 CFU/mL (Figure VI.2 left, diamonds). The data at that inoculum
show almost no variation among the amphiphiles, except that the MIC for 3CUr16 is
only one dilution lower than the rest. On the other hand, at initial cell densities of 5 × 106
and 3.2 × 105 (Figure IV.2, right), one would conclude that both 3CUr16 and 3CUr18
are the most active compounds.
Figure VI.2 Effect of chain length on inhibition of growth of M. smegmatis of the 3CUrn series as function of the initial cell density. Error bars (not shown for clarity) are ± 0.3
-5
-4.5
-4
-3.5
-3
-2.5
-2
12 14 16 18 20 22 24
chain length
log
[MIC
] (M
)
5.0E+08 3.20E+04
-4.5
-4
-3.5
-3
-2.5
12 14 16 18 20 22 24
chain length
log
[MIC
] (M
)
5.0E+06 3.2E+05
199
VI.3.2 Comparison with Previous Work
Although the mechanism of action of these amphiphiles remains to be determined,
we expect that these amphiphiles exert their antimycobacterial activity by interacting
with the mycobacterial membrane. Excluding detergency (disrupting cell walls and
solubilizing membranes), these amphiphiles can inactivate microorganisms by several
mechanisms: (1) as monomers, changing fluidity and porosity of membranes; (2) as
monomers, altering membrane-associated pathways; (3) as monomers, changing the flow
of constituents and nutrients between a cell and a medium; and (4) as monomers,
inhibiting specific membrane-associated enzymes. The discovery that the homolog with
the hexadecyl chain gives the best activity contrasts with the results for natural saturated
fatty acids. In an early study12 of several mycobacteria, tetradecanoic acid is slightly
more active than dodecanoic acid in inhibiting the growth of M. smegmatis. In a very
recent study13 dodecanoic acid is the most active of the even-numbered natural saturated
fatty acids up to twenty-six carbons for several mycobacteria, including M. smegmatis.
VI.3.3 Comparison of MICs and CMCs
The MICs at the lowest and highest initial cell densities with the CMCs are
compared (Figure VI.3). The values of MIC at the lowest initial cell density illustrate the
intrinsic activity of the series. As all MICs of the series are lower than the CMCs, we
conclude that detergency is not the likely mechanism of action. In this comparison, the
MIC of 3CUr16 is 100-fold smaller than the CMC, the MIC of 3CUr22, 8-fold smaller
than the CMC. The data at the highest initial cell density illustrate conditions where the
amphiphiles may not be saturating the cells. In this comparison, the MIC of 3CUr16 at
the highest initial cell density is 5-fold smaller than the CMC, the MIC of 3CUr22 at the
200
Figure VI.3 Comparison of log CMC and log MIC of the 3CUrn series at the highest and lowest initial cell densities of M. smegmatis
highest initial cell density equals the CMC. We tentatively conclude that detergency is
the likely mechanism of action at the highest initial cell density for 3CUr22 and 3CUr20.
For the other members of the series detergency is likely a significant contributor to the
mechanism of action.
We initiated this project to design long chain amphiphiles with high CMCs. The
underlying thesis is that the higher the CMC, the less likely detergency will contribute to
the mechanism of action. Even though these amphiphiles do not have MICs low enough
to be considered as leads for drug development, one should always remember that the
therapeutic index (ratio of cytotoxicity to potency) is the ultimate predictor for potential
applications. In this regard, a potential application of these amphiphiles is as affordable
topical anti-infectives. If cytotoxicity is only related to detergency (hence CMC), then a
therapeutic index of 100 looks more promising. Although it is unlikely that cytotoxicity
-5
-4.5
-4
-3.5
-3
-2.5
-2
12 14 16 18 20 22 24
chain length
log
MIC
or C
MC
(M)
high inoculum low inoculum CMCs
201
will be that simple, the strategy of designing amphiphiles with high CMCs is essential to
developing non-detergent amphiphilic microbicides.
VI.4 Conclusions
Water-soluble amphiphiles that have ultra-long chains can be made readily from
first-generation, Newkome-type dendrons. As tris(triethanolammonium) salts, these
amphiphiles show excellent solubility in water. This homologous series of alkyl chains
that extends to docosyl reveals that the cut-off for antimycobacterial activity is the
hexadecyl chain. As the intrinsic activity is well below the CMC, a mechanism of action
that does not involve detergency is suggested.intrinsic activity of the series.
VI.5 Experimental Procedures
To probe the intrinsic activities of these amphiphiles, we measured the MICs of
3Ur14–3Ur22 at several different initial cell densities, expressed as colony-forming units
(CFU)/mL, of M. smegmatis. Experiments were performed in duplicate at two
overlapping ranges of initial inocula—32 to 3.2 × 107 CFU/mL and 500 to 5.0 × 108
CFU/mL. The MIC was defined as the lowest concentration at which an amphiphile
completely inhibited the growth after four days at 37 °C by successive twofold dilution in
96-well microtiter plates.
VI.5.1 Microbial Strain, Culture Conditions, and Preparations of Inoculum for Susceptibility Testing
Mycobacterium smegmatis was obtained from the Virginia Tech Microbiology
culture collection. Two separate assays with initial cell densities ranging from 32 to
3.2 × 107 CFU/mL and 500 to 5.0 × 108 CFU/mL were performed. The different initial
202
cell densities were prepared in the following manner. A single isolated colony of M.
smegmatis on M7H10 agar was used to inoculate sterile M7H9 broth (2 mL) in a screw
capped tube (16 ×125 mm). After incubation without shaking at 37 °C for 5 d, 2 mL was
used to inoculate M7H9 broth (18 mL) in a 125-mL flask and then incubated with
aeration (120 rpm) at 30 °C for 5 d. The culture was transferred to a sterile 50-mL screw
capped centrifuge tube; centrifugation (5,000 × g) for 20 min was performed. The
supernatant-spent medium was discarded, and the cells were suspended in 1/10-strength
BHIB medium (2 mL). A tenfold dilution series was prepared in 1/10-strength BHIB by
transferring the concd cell suspension (0.5 mL) into 4.5 mL of 1/10-strength BHIB to
yield a 10-fold to 107-fold dilution series. The concd cell suspension, measured in
CFU/mL, was measured by spreading different dilutions (0.1 mL each) on M7H10 agar.
The agar plates were incubated at 37 °C and colonies were counted after 4 d. The average
of the colony counts was calculated. MIC measurements were performed within 24 h
after the dilutions were performed.
VI.5.2 Measurement of MICs
Measurement of MICs was carried out in a similar manner is the measurement of
MICs for the preliminary screening (Chapter V). Instead of a suspension of the organism
adjusted to McFarland standard No.1, suspensions of M. smegmatis in different cell
density prepared as above were employed. An aliquot (50 μL) of the microbial
inoculum, M. smegmatis, at a given cell density was added to all wells in a row. Aqueous
triethanolamine without amphiphile 3CUrn was also tested for antimicrobial activity by
using the same protocol; no antimicrobial activity was found. The concentration of the
3CUrn series ranged from 6.25 mg/mL to 5.5 μg/mL. After the plates were incubated at
203
37 °C for 4 d, MICs results were read by comparing the turbidity (due to growth of
microbes) of each test well to the positive control wells. MIC was defined as the lowest
concentration completely inhibiting the growth. The experiments were run in duplicate
for the initial cell densities of 32 to 3.2 × 107 CFU/mL and in single for the initial cell
densities of 500 to 5.0 × 108 CFU/mL.
References for Chapter VI 1 Balgavý, P.; Devínsky, F. Cut-off effects in biological activities of surfactants.
Adv. Coll. Interface Sci. 1996, 66, 23-63. 2 Hansch, C.; Clayton, J. M. Lipophilic character and biological activity of drugs.
II. The parabolic case. J. Pharm. Sci. 1973, 62, 1-21. 3 Gruber, H. J.; Low, P. S. Interaction of amphiphiles with integral membrane
proteins. I. Structural destabilization of the anion transport protein of the erythrocyte membrane by fatty acids, fatty alcohols, and fatty amines. Biochim. Biophys. Acta 1988, 944, 414-424.
4 Gruber, H. J. Interaction of amphiphiles with integral membrane proteins. II. A
simple, minimal model for the nonspecific interaction of amphiphiles with the anion exchanger of the erythrocyte membrane. Biochim. Biophys. Acta 1988, 944, 425-436.
5 Gehrt, A.; Peter, J.; Pizzo, P. A.; Walsh, T. J. Effect of increasing inoculum sizes
of pathogenic filamentous fungi on MICs of antifungal agents by broth microdilution method. J. Clin. Microbiol. 1995, 33, 1302-1307.
6 Soriano, F. Optimal dosage of β-lactam antibiotics: time above the MIC and
inoculum effect. J. Antimicrob. Chemother. 1992, 30, 566-569. 7 Brook, I. Inoculum effect. Rev. Infect. Dis. 1989, 11, 361-368. 8 Li, R. C.; Ma, H. H. Parameterization of inoculum effect via mathematical
modeling: aminoglycosides against Staphylococcus aureus and Escherichia coli. J. Chemother. 1998, 10, 203-207.
204
9 Thrupp, L. D. Antibiotics in laboratory medicine; 2nd ed.; Williams & Wilkins: Baltimore, MD, 1986.
10 Brennan, P. J.; Nikaido, H. The envelope of mycobacteria. Annu. Rev. Biochem.
1995, 64, 29-63. 11 Csizmadia, F. logP and logD calculator, http://intro.bio.umb.edu/111-
112/OLLM/111F98/newclogp.html; September 26 2006. 12 Kondo, E.; Kanai, K. Lethal effect of long-chain fatty acids on mycobacteria. Jpn.
J. Med. Sci. Biol. 1972, 25, 1-13. 13 Seidel, V.; Taylor, P. W. In vitro activity of extracts and constituents of
Pelagonium against rapidly growing mycobacteria. Int. J. Antimicrob. Agents 2004, 23, 613-619.
205
Chapter VII Accomplishments, Conclusions, and Future Work
VII.1 Accomplishments
We have initiated a project designing amphiphiles with antimicrobial properties
that do not involve detergency as the mechanism of action. We have established a route
to synthesize homologous, water-soluble amphiphiles with very hydrophobic tails
utilizing the first-generation, tri-carboxylato Newkome-type dendron attached to a ureido
linker. With this route, three homologous series containing mobile hydrophobic moieties
and two series of epimers containing a rigid cholestane moiety have been generated
easily within a relatively short period of time. The tri-carboxylato amphiphiles have been
obtained in moderate to excellent yields.
Ten new compounds, which were tri-tert-butyl esters and triacids, were generated
in the synthesis of one-tailed, tri-carboxylato amphiphiles. All these new compounds
were fully characterized. A new synthetic route to long-chain alkan-1-amines (n = 20,
22), which are rarely found in the literature, was established. Beginning with low-cost
commercially available alcohols, the three-step synthesis afforded long-chain amines in
moderate yields.
Twenty-two new compounds, which are tri-tert-butyl esters and triacids, were
generated in the synthesis of two-tailed, tri-carboxylato amphiphiles. A new synthetic
route to amines attached to a “swallowtail” was established. Most documented
procedures for the preparation of such amines, which begins with costly ketones, required
extreme conditions. Beginning with relatively low-cost commercially available
bromoalkanes and ethyl formate, this newly established four-step synthesis could be
easily performed with standard laboratory glassware. Six new secondary azides were
206
generated during this process. All compounds⎯eleven tri-tert-butyl esters, eleven
triacids, and six azidoalkanes with a swallowtail⎯were fully characterized.
Eight new compounds, which are epimeric pairs of tri-tert-butyl esters and
triacids, were generated in the synthesis of tri-carboxylato amphiphiles containing the
rigid cholestane moiety. Two new 5α-cholestan-3-yl azidoethanoates, which are a pair of
epimers, had been synthesized during this process. All the ten new compounds are fully
characterized.
We screened all these amphiphiles against a broad spectrum of eleven
microorganisms. During this screening process, more than one hundred biological assays
were performed.
VII.2 Conclusions
The Newkome-type dendron provides a unique headgroup for amphiphiles with a
very hydrophobic moieties. As tris(triethanolammonium) salts, these amphiphiles show
excellent solubility in water. The solubilities of the first three series in aqueous solution
are 19,500 to 25,700 µM depending on the formula weight of the homolog, while those
of the last two are 18,900 and 17,400 μM.
We screened these amphiphiles against a broad spectrum of microorganisms by
broth microdilution in 96-well microtiter plates, where the antimicrobial activity is
represented by MICs. Plots of log MIC vs chain length show the cut-off effect is
observed in the first three series (3CUrn, 3CUr(n)2, and 3CUr1(n)2) against most of the
microorganisms tested. As all amphiphiles are soluble, solubility issues can be ruled out
as the cause of the cut-off effect. Based on the plots of log MIC vs hydrophobicity, we
cannot make the generalization that hydrophobicity is the only factor affecting
207
antimicrobial activity; both hydrophobicity and tail topology affect the antimicrobial
activity.
In general, the five series of dendritic tri-carboxylato amphiphiles show modest
antimicrobial properties with a couple promising leads, and they do not display a uniform
pattern of activity against the broad spectrum of microorganisms tested, but display
amphiphile-series-, species-, chain-length-, or epimer-specific patterns.
Finally, to determine if detergency is the mechanism of action in the antimicrobial
activity, we have compared the MICs to CMCs. For this intention, we have developed a
more detail biological assays to investigate the intrinsic activity (MIC0) of the 3CUrn
series against Mycobacterium smegmatis. As the resulting MIC0’s are below the CMCs,
we conclude that detergency is not the mechanism of action. Amphiphile 3CUr16 is the
most active with the MIC0 100-fold smaller than the CMC.
VII.3 Future Work
As two of the amphiphile series have not reached the cut-off effects, we are
interested in generating longer amphiphiles of the corresponding series to explore the
more active amphiphile. Especially against Cryptococcus neoformans, a longer
3CUr1(n)2 is worth synthesizing and exploring for antimicrobial activity.
A very recent study1 documented that ultra-long chain unsaturated fatty acids⎯2-
hexadecynoic and 2-octadecynoic acids⎯posses antimycobacterial against M.
smegmatis. Thus, synthesis of the tri-headed amphiphiles containing ultra-long,
unsaturated, linear alkyl chains, followed by investigation on antimycobacterial activity is
encouraged.
208
References for Chapter VII 1 Morbidoni, H. R.; Vilcheze, C.; Kremer, L.; Bittman, R.; Sacchettini, J. C.;
Jacobs, W. R. Dual inhibition of mycobacterial fatty acid biosynthesis and degradation by 2-alkynoic acids. Chem. Biol. 2006, 13, 297-307.
209
APPENDICES
210
APPENDIX A: 1H and 13C NMR spectra
211
1H NMR spectrum of 3EUr14
NH
NH
O
O O
3
212
1H NMR spectrum of 3EUr16
NH
NH
O
O O
3
213
1H NMR spectrum of 3EUr18
NH
NH
O
O O
3
214
1H NMR spectrum of 3EUr20
NH
NH
O
O O
3
215
1H NMR spectrum of 3EUr22
NH
NH
O
O O
3
216
1H NMR spectrum of 3EUr(7)2
N
NO O
O
3H
217
1H NMR spectrum of 3EUr(8)2
N
NO O
O
3H
218
1H NMR spectrum of 3EUr(9)2
N
NO O
O
3H
219
1H NMR spectrum of 3EUr(10)2
N
NO O
O
3H
220
1H NMR spectrum of 3EUr(11)2
N
NO O
O
3H
221
NH
ONH
O
3
222
1H NMR spectrum of 3EUr1(8)2
NH
ONH
O
3
223
1H NMR spectrum of 3EUr1(9)2
NH
ONH
O
3
224
1H NMR spectrum of 3EUr1(10)2
NH
ONH
O
3
225
1H NMR spectrum of 3EUr1(11)2
NH
ONH
O
3
226
1H NMR spectrum of 3EUr1(12)2
NH
ONH
O
3
227
1H NMR spectrum of 3EUr-α-cholestane
NH
NH
O
O
O
3
228
1H NMR spectrum of 3EUr-β-cholestane
NH
NH
O
O
O
3
229
1H NMR of 3EUr-β-cholestane (expansion)
230
1H NMR spectrum of 3EUr1Es-α-cholestane
ONH
NH
O
O
O
O
3
231
1H NMR spectrum of 3EUr1Es-β-cholestane
ONH
NH
O
O
O
O
3
232
1H NMR spectrum of 3CUr14
NH
O
NH
OH
O 3
233
1H NMR spectrum of 3CUr16
NH
O
NH
OH
O 3
234
1H NMR spectrum of 3CUr16 (expansion)
235
1H NMR spectrum of 3CUr18
NH
O
NH
OH
O 3
236
1H NMR spectrum of 3CUr20
NH
O
NH
OH
O 3
237
1H NMR spectrum of 3CUr20 (expansion)
238
1H NMR spectrum of 3CUr22
NH
O
NH
OH
O 3
239
1H NMR spectrum of 3CUr(7)2
N
NO OH
O
3H
240
1H NMR of 3CUr(8)2
N
NO OH
O
3H
241
1H NMR spectrum of 3CUr(9)2
N
NO OH
O
3H
242
1H NMR spectrum of 3CUr(10)2
N
NO OH
O
3H
243
1H NMR spectrum of 3CUr(11)2
N
NO OH
O
3H
244
1H NMR spectrum of 3CUr1(7)2
NH
ONH
H
O
3
245
1H NMR spectrum of 3CUr1(8)2
NH
ONH
H
O
3
246
1H NMR spectrum of 3CUr1(9)2
NH
ONH
H
O
3
247
1H NMR spectrum of 3CUr1(10)2
NH
ONH
H
O
3
248
1H NMR spectrum of 3CUr1(11)2
NH
ONH
O
H 3
249
1H NMR spectrum of 3CUr1(12)2
NH
ONH
O
H 3
250
1H NMR spectrum of 3CUr-α-cholestane
NH
NH
O
O
O
H
3
251
1H NMR spectrum of 3CUr-β-cholestane
NH
NH
O
O
O
H
3
252
1H NMR spectrum of 3CUr1Es-α-cholestane
ONH
NH
O
O
O
O
H
3
253
1H NMR spectrum of 3CUr1Es-β-cholestane O
NH
NH
O
O
O
O
H
3
254
1H NMR spectrum of 8-azidopentadecane
N3
255
1H NMR spectrum of 9-azidoheptadecane
N3
256
1H NMR spectrum of 10-azidononadecane
N3
257
1H NMR spectrum of 11-azidohenicosane
N3
258
1H NMR spectrum of 12-azidotricosane
N3
259
1H NMR spectrum of 13-azidopentacosane
N3
260
1H NMR spectrum of 5α-cholestan-3α-yl azidoethanoate
O
ON3
261
1H NMR spectrum of 5α-cholestan-3β-yl azidoethanoate
O
ON3
262
1H NMR spectrum of 5α-cholestan-3β-yl azidoethanoate
263
13C NMR spectrum of 3EUr14
NH
NH
O
O O
3
264
13C NMR spectrum of 3EUr16
NH
NH
O
O O
3
265
13C NMR spectrum of 3EUr18
NH
NH
O
O O
3
266
13C NMR spectrum of 3EUr20
NH
NH
O
O O
3
267
13C NMR spectrum of 3EUr22
NH
NH
O
O O
3
268
13C NMR spectrum of 3EUr(7)2
N
NO O
O
3H
269
13C NMR spectrum of 3EUr(8)2
N
NO O
O
3H
270
13C NMR spectrum of 3EUr(8)2
N
NO O
O
3H
271
13C NMR spectrum of 3EUr(9)2
N
NO O
O
3H
272
13C NMR spectrum of 3EUr(10)2
N
NO O
O
3H
273
13C NMR spectrum of 3EUr(11)2
N
NO O
O
3H
274
13C NMR spectrum of 3EUr1(7)2
NH
ONH
O
3
275
13C NMR spectrum of 3EUr(8)2
NH
ONH
O
3
276
13C NMR spectrum of 3EUr1(9)2
NH
ONH
O
3
277
13C NMR spectrum of 3EUr1(10)2
NH
ONH
O
3
278
13C NMR spectrum of 3EUr1(11)2
NH
ONH
O
3
279
13C NMR spectrum of 3EUr1(12)2
NH
ONH
O
3
280
13C NMR spectrum of 3EUr-α-cholestane
NH
NH
O
O
O
3
281
13C NMR spectrum of 3EUr-β-cholestane
NH
NH
O
O
O
3
282
13C NMR spectrum of 3EUr1Es-1-cholestane
ONH
NH
O
O
O
O
3
283
13C NMR spectrum of 3EUr1Es-β-cholestane
ONH
NH
O
O
O
O
3
284
13C NMR spectrum of 3CUr14
NH
O
NH
OH
O 3
285
13C NMR spectrum of 3CUr16
NH
O
NH
OH
O 3
286
13C NMR spectrum of 3CUr18
NH
O
NH
OH
O 3
287
13C NMR spectrum of 3CUr20
NH
O
NH
OH
O 3
288
13C NMR spectrum of 3CUr22
NH
O
NH
OH
O 3
289
13C NMR spectrum of 3CUr(7)2
N
NO OH
O
3H
290
13C NMR spectrum of 3CUr(8)2
N
NO OH
O
3H
291
13C NMR spectrum of 3CUr(9)2
N
NO OH
O
3H
292
13C NMR spectrum of 3CUr(10)2
N
NO OH
O
3H
293
13C NMR spectrum of 3CUr(11)2
N
NO OH
O
3H
294
13C NMR spectrum of 3CUr1(7)2
NH
ONH
OH
O
3
295
13C NMR spectrum of 3CUr1(8)2
NH
ONH
OH
O
3
296
13C NMR spectrum of 3CUr1(9)2
NH
ONH
OH
O
3
297
13C NMR spectrum of 3CUr1(10)2
NH
ONH
OH
O
3
298
13C NMR spectrum of 3CUr1(11)2
NH
ONH
O
OH 3
299
13C NMR spectrum of 3CUr1(12)2
NH
ONH
O
OH 3
300
13C NMR spectrum of 3CUr-α-cholestane
NH
NH
O
O
O
H
3
301
13C NMR spectrum of 3CUr-α-cholestane (expansion)
302
13C NMR spectrum of 3CUr-β-cholestane
NH
NH
O
O
O
H
3
303
13C NMR spectrum of 3CUr1Es-α-cholestane
ONH
NH
O
O
O
O
H
3
304
13C NMR spectrum of 3CUr1Es-β-cholestane
ONH
NH
O
O
O
O
H
3
305
13C NMR spectrum of 8-azidopentadecane
N3
306
13C NMR spectrum of 9-azidoheptadecane
N3
307
13C NMR spectrum of 10-azidononadecane
N3
308
13C NMR spectrum of 11-azidohenicosane
N3
309
13C NMR spectrum of 12-azidotricosane
N3
310
13C NMR spectrum of 13-azidopentacosane
N3
311
13C NMR spectrum of 5α-cholestan-3α-yl azidoethanoate
O
ON3
312
13C NMR spectrum of 5α-cholestan-3β-yl azidoethanoate
O
ON3
313
APPENDIX B: X-ray tables
314
Table I.1 Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) of 3EUr16.
U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
________________________________________________________________________________
x y z U(eq)
________________________________________________________________________________ O(1) 5406(2) 8565(2) 2493(1) 25(1)
O(2) 3665(2) 13670(2) 2412(1) 25(1)
O(3) 5437(2) 12118(2) 2578(1) 35(1)
O(4) 2010(2) 11480(1) 454(1) 21(1)
O(5) 901(2) 12338(2) 1018(1) 32(1)
O(6) 6393(2) 6243(1) 1283(1) 22(1)
O(7) 6191(2) 7741(2) 832(1) 32(1)
N(1) 3535(2) 9478(2) 2145(1) 19(1)
N(2) 3604(2) 8429(2) 2749(1) 23(1)
C(1) 3999(2) 10021(2) 1801(1) 17(1)
C(2) 4249(2) 8814(2) 2464(1) 19(1)
C(3) 4245(2) 7680(2) 3099(1) 24(1)
C(4) 4457(3) 8335(2) 3460(1) 28(1)
C(5) 5025(2) 7533(2) 3838(1) 29(1)
C(6) 6375(3) 6655(2) 3790(1) 31(1)
C(7) 6908(2) 5963(2) 4188(1) 28(1)
C(8) 8257(3) 5090(2) 4164(1) 32(1)
C(9) 8756(2) 4427(2) 4572(1) 26(1)
C(10) 10132(2) 3582(2) 4567(1) 28(1)
C(11) 10564(2) 2918(2) 4978(1) 25(1)
C(12) 11942(2) 2082(2) 5000(1) 24(1)
C(13) 12294(2) 1420(2) 5413(1) 25(1)
C(14) 13669(2) 579(2) 5455(1) 24(1)
C(15) 13966(2) -75(2) 5873(1) 26(1)
C(16) 15357(2) -845(2) 5943(1) 27(1)
C(17) 15615(2) -1517(2) 6356(1) 32(1)
C(18) 17019(3) -2196(3) 6447(1) 45(1)
C(19) 4525(2) 10922(2) 1956(1) 19(1)
C(20) 3690(2) 11798(2) 2262(1) 29(1)
C(21) 4365(3) 12529(2) 2435(1) 26(1)
C(22) 4152(2) 14561(2) 2553(1) 24(1)
315
C(23) 4425(3) 14388(2) 3010(1) 33(1)
C(24) 5301(3) 14505(3) 2304(1) 36(1)
C(25) 3038(3) 15704(2) 2458(1) 33(1)
C(26) 2830(2) 10610(2) 1519(1) 19(1)
C(27) 3081(2) 11027(2) 1093(1) 19(1)
C(28) 1863(2) 11689(2) 860(1) 21(1)
C(29) 965(2) 12062(2) 154(1) 22(1)
C(30) -179(2) 11757(3) 263(1) 34(1)
C(31) 660(2) 13374(2) 145(1) 26(1)
C(32) 1535(2) 11535(2) -252(1) 29(1)
C(33) 5084(2) 9091(2) 1571(1) 20(1)
C(34) 4782(2) 8079(2) 1405(1) 22(1)
C(35) 5852(2) 7355(2) 1137(1) 21(1)
C(36) 7510(2) 5392(2) 1076(1) 24(1)
C(37) 8616(2) 5802(2) 1098(1) 30(1)
C(38) 7765(3) 4286(2) 1337(1) 33(1)
C(39) 7181(2) 5226(2) 634(1) 31(1)
O(8) 8919(2) 1015(2) 7560(1) 27(1)
O(9) 8526(2) 3776(1) 8685(1) 22(1)
O(10) 8580(2) 2298(2) 9127(1) 37(1)
O(11) 13029(2) -1452(1) 9506(1) 22(1)
O(12) 14212(2) -2112(2) 8935(1) 38(1)
O(13) 11297(2) -3903(2) 7580(1) 31(1)
O(14) 9383(2) -2552(2) 7655(1) 101(1)
N(3) 10962(2) 406(2) 7807(1) 21(1)
N(4) 10457(2) 1275(2) 7163(1) 28(1)
C(40) 10773(2) -139(2) 8199(1) 17(1)
C(41) 10045(2) 897(2) 7511(1) 21(1)
C(42) 9608(2) 1810(2) 6815(1) 25(1)
C(43) 10244(2) 2344(2) 6510(1) 28(1)
C(44) 9465(2) 2836(2) 6119(1) 28(1)
C(45) 8179(2) 3815(2) 6182(1) 26(1)
C(46) 7533(2) 4379(2) 5780(1) 26(1)
C(47) 6214(2) 5299(2) 5814(1) 25(1)
C(48) 5698(2) 5915(2) 5403(1) 26(1)
C(49) 4340(2) 6803(2) 5406(1) 25(1)
316
C(50) 3946(2) 7470(2) 4995(1) 25(1)
C(51) 2580(2) 8320(2) 4961(1) 24(1)
C(52) 2286(2) 8983(2) 4547(1) 24(1)
C(53) 917(2) 9802(2) 4478(1) 25(1)
C(54) 705(2) 10437(2) 4056(1) 25(1)
C(55) -672(2) 11099(2) 3939(1) 31(1)
C(56) -858(3) 11738(3) 3521(1) 37(1)
C(57) -2245(3) 12373(3) 3400(1) 50(1)
C(58) 9741(2) 781(2) 8453(1) 18(1)
C(59) 10003(2) 1870(2) 8554(1) 23(1)
C(60) 8963(2) 2654(2) 8822(1) 21(1)
C(61) 7462(2) 4684(2) 8895(1) 23(1)
C(62) 7311(2) 5793(2) 8644(1) 29(1)
C(63) 6283(2) 4388(2) 8866(1) 28(1)
C(64) 7822(2) 4785(2) 9342(1) 31(1)
C(65) 12059(2) -582(2) 8431(1) 18(1)
C(66) 11964(2) -978(2) 8878(1) 20(1)
C(67) 13212(2) -1573(2) 9100(1) 22(1)
C(68) 14046(2) -2026(2) 9817(1) 21(1)
C(69) 15174(3) -1686(3) 9739(1) 44(1)
C(70) 14386(3) -3342(2) 9806(1) 37(1)
C(71) 13409(3) -1565(3) 10222(1) 33(1)
C(72) 10323(2) -1144(2) 8115(1) 20(1)
C(73) 11156(2) -2097(2) 7834(1) 24(1)
C(74) 10492(3) -2865(2) 7691(1) 31(1)
C(75) 10866(3) -4810(2) 7419(1) 33(1)
C(76A) 12077(4) -5897(4) 7437(3) 51(2)
C(77A) 9906(7) -5009(6) 7728(2) 40(2)
C(78A) 10332(8) -4435(7) 7008(2) 49(2)
C(76B) 12019(12) -5525(14) 7132(6) 43(5)
C(77B) 10644(18) -5523(15) 7733(4) 36(4)
C(78B) 9680(19) -4225(17) 7099(6) 37(4)
O(15) 3144(2) 10294(2) 7221(1) 36(1)
________________________________________________________________________________
317
Table I.2 Bond lengths [Å] and angles [°] of 3EUr16.
_____________________________________________________
O(1)-C(2) 1.236(3)
O(2)-C(21) 1.324(3)
O(2)-C(22) 1.487(3)
O(3)-C(21) 1.216(3)
O(4)-C(28) 1.343(3)
O(4)-C(29) 1.480(3)
O(5)-C(28) 1.205(3)
O(6)-C(35) 1.340(3)
O(6)-C(36) 1.478(3)
O(7)-C(35) 1.205(3)
N(1)-C(2) 1.360(3)
N(1)-C(1) 1.473(3)
N(2)-C(2) 1.352(3)
N(2)-C(3) 1.449(3)
C(1)-C(33) 1.535(3)
C(1)-C(26) 1.539(3)
C(1)-C(19) 1.541(3)
C(3)-C(4) 1.509(4)
C(4)-C(5) 1.528(3)
C(5)-C(6) 1.516(4)
C(6)-C(7) 1.516(3)
C(7)-C(8) 1.506(4)
C(8)-C(9) 1.521(4)
C(9)-C(10) 1.516(4)
C(10)-C(11) 1.519(3)
C(11)-C(12) 1.515(3)
C(12)-C(13) 1.515(3)
C(13)-C(14) 1.517(3)
C(14)-C(15) 1.523(3)
C(15)-C(16) 1.519(3)
C(16)-C(17) 1.518(4)
C(17)-C(18) 1.520(4)
C(19)-C(20) 1.523(3)
C(20)-C(21) 1.507(4)
C(22)-C(23) 1.503(4)
C(22)-C(24) 1.509(4)
C(22)-C(25) 1.518(3)
C(26)-C(27) 1.516(3)
C(27)-C(28) 1.507(3)
C(29)-C(32) 1.513(3)
C(29)-C(31) 1.515(3)
C(29)-C(30) 1.518(4)
C(33)-C(34) 1.523(3)
C(34)-C(35) 1.495(3)
C(36)-C(38) 1.508(4)
C(36)-C(39) 1.518(4)
C(36)-C(37) 1.523(4)
O(8)-C(41) 1.243(3)
O(9)-C(60) 1.338(3)
O(9)-C(61) 1.479(3)
O(10)-C(60) 1.211(3)
O(11)-C(67) 1.334(3)
O(11)-C(68) 1.483(3)
O(12)-C(67) 1.208(3)
O(13)-C(74) 1.317(3)
O(13)-C(75) 1.486(3)
O(14)-C(74) 1.176(3)
N(3)-C(41) 1.365(3)
N(3)-C(40) 1.468(3)
N(4)-C(41) 1.351(3)
N(4)-C(42) 1.454(3)
C(40)-C(72) 1.537(3)
C(40)-C(58) 1.539(3)
C(40)-C(65) 1.543(3)
C(42)-C(43) 1.496(3)
C(43)-C(44) 1.519(3)
C(44)-C(45) 1.518(3)
C(45)-C(46) 1.512(3)
318
C(46)-C(47) 1.504(3)
C(47)-C(48) 1.514(3)
C(48)-C(49) 1.517(3)
C(49)-C(50) 1.517(3)
C(50)-C(51) 1.510(3)
C(51)-C(52) 1.518(3)
C(52)-C(53) 1.513(3)
C(53)-C(54) 1.523(3)
C(54)-C(55) 1.510(3)
C(55)-C(56) 1.517(4)
C(56)-C(57) 1.518(4)
C(58)-C(59) 1.521(3)
C(59)-C(60) 1.501(3)
C(61)-C(62) 1.516(4)
C(61)-C(63) 1.519(3)
C(61)-C(64) 1.526(4)
C(65)-C(66) 1.528(3)
C(66)-C(67) 1.502(3)
C(68)-C(69) 1.508(4)
C(68)-C(71) 1.510(3)
C(68)-C(70) 1.516(4)
C(72)-C(73) 1.519(3)
C(73)-C(74) 1.498(4)
C(75)-C(77B) 1.403(13)
C(75)-C(78A) 1.452(8)
C(75)-C(76A) 1.513(5)
C(75)-C(77A) 1.554(6)
C(75)-C(76B) 1.586(12)
C(75)-C(78B) 1.625(19)
C(21)-O(2)-C(22) 121.8(2)
C(28)-O(4)-C(29) 121.49(19)
C(35)-O(6)-C(36) 120.59(19)
C(2)-N(1)-C(1) 125.9(2)
C(2)-N(2)-C(3) 121.5(2)
N(1)-C(1)-C(33) 111.03(19)
N(1)-C(1)-C(26) 104.45(18)
C(33)-C(1)-C(26) 111.4(2)
N(1)-C(1)-C(19) 111.56(19)
C(33)-C(1)-C(19) 106.92(18)
C(26)-C(1)-C(19) 111.6(2)
O(1)-C(2)-N(2) 122.0(2)
O(1)-C(2)-N(1) 122.8(2)
N(2)-C(2)-N(1) 115.2(2)
N(2)-C(3)-C(4) 114.2(2)
C(3)-C(4)-C(5) 112.9(2)
C(6)-C(5)-C(4) 115.7(2)
C(5)-C(6)-C(7) 112.5(2)
C(8)-C(7)-C(6) 115.2(2)
C(7)-C(8)-C(9) 113.3(2)
C(10)-C(9)-C(8) 115.2(2)
C(9)-C(10)-C(11) 112.8(2)
C(12)-C(11)-C(10) 115.7(2)
C(13)-C(12)-C(11) 112.8(2)
C(12)-C(13)-C(14) 115.2(2)
C(13)-C(14)-C(15) 113.0(2)
C(16)-C(15)-C(14) 114.7(2)
C(17)-C(16)-C(15) 113.4(2)
C(16)-C(17)-C(18) 113.8(2)
C(20)-C(19)-C(1) 115.81(19)
C(21)-C(20)-C(19) 111.3(2)
O(3)-C(21)-O(2) 123.9(2)
O(3)-C(21)-C(20) 123.7(2)
O(2)-C(21)-C(20) 112.4(2)
O(2)-C(22)-C(23) 109.8(2)
O(2)-C(22)-C(24) 110.2(2)
C(23)-C(22)-C(24) 112.6(2)
O(2)-C(22)-C(25) 102.0(2)
C(23)-C(22)-C(25) 111.2(2)
C(24)-C(22)-C(25) 110.5(2)
C(27)-C(26)-C(1) 116.22(19)
C(28)-C(27)-C(26) 111.6(2)
319
O(5)-C(28)-O(4) 125.0(2)
O(5)-C(28)-C(27) 124.1(2)
O(4)-C(28)-C(27) 110.8(2)
O(4)-C(29)-C(32) 102.99(19)
O(4)-C(29)-C(31) 108.9(2)
C(32)-C(29)-C(31) 110.9(2)
O(4)-C(29)-C(30) 110.3(2)
C(32)-C(29)-C(30) 110.7(2)
C(31)-C(29)-C(30) 112.7(2)
C(34)-C(33)-C(1) 115.69(19)
C(35)-C(34)-C(33) 109.1(2)
O(7)-C(35)-O(6) 124.7(2)
O(7)-C(35)-C(34) 123.5(2)
O(6)-C(35)-C(34) 111.8(2)
O(6)-C(36)-C(38) 102.0(2)
O(6)-C(36)-C(39) 110.61(19)
C(38)-C(36)-C(39) 110.8(2)
O(6)-C(36)-C(37) 109.4(2)
C(38)-C(36)-C(37) 111.2(2)
C(39)-C(36)-C(37) 112.3(2)
C(60)-O(9)-C(61) 120.78(19)
C(67)-O(11)-C(68) 123.16(19)
C(74)-O(13)-C(75) 122.2(2)
C(41)-N(3)-C(40) 124.8(2)
C(41)-N(4)-C(42) 121.6(2)
N(3)-C(40)-C(72) 109.91(19)
N(3)-C(40)-C(58) 109.55(19)
C(72)-C(40)-C(58) 108.18(19)
N(3)-C(40)-C(65) 106.60(18)
C(72)-C(40)-C(65) 112.0(2)
C(58)-C(40)-C(65) 110.59(19)
O(8)-C(41)-N(4) 122.4(2)
O(8)-C(41)-N(3) 123.1(2)
N(4)-C(41)-N(3) 114.4(2)
N(4)-C(42)-C(43) 109.9(2)
C(42)-C(43)-C(44) 113.4(2)
C(45)-C(44)-C(43) 115.6(2)
C(46)-C(45)-C(44) 112.8(2)
C(47)-C(46)-C(45) 116.1(2)
C(46)-C(47)-C(48) 112.6(2)
C(47)-C(48)-C(49) 115.9(2)
C(50)-C(49)-C(48) 112.4(2)
C(51)-C(50)-C(49) 116.0(2)
C(50)-C(51)-C(52) 112.4(2)
C(53)-C(52)-C(51) 115.8(2)
C(52)-C(53)-C(54) 112.4(2)
C(55)-C(54)-C(53) 114.8(2)
C(54)-C(55)-C(56) 113.8(2)
C(55)-C(56)-C(57) 113.4(2)
C(59)-C(58)-C(40) 115.6(2)
C(60)-C(59)-C(58) 109.9(2)
O(10)-C(60)-O(9) 124.7(2)
O(10)-C(60)-C(59) 123.1(2)
O(9)-C(60)-C(59) 112.2(2)
O(9)-C(61)-C(62) 102.6(2)
O(9)-C(61)-C(63) 109.1(2)
C(62)-C(61)-C(63) 111.5(2)
O(9)-C(61)-C(64) 110.40(19)
C(62)-C(61)-C(64) 110.6(2)
C(63)-C(61)-C(64) 112.2(2)
C(66)-C(65)-C(40) 112.96(19)
C(67)-C(66)-C(65) 115.2(2)
O(12)-C(67)-O(11) 125.7(2)
O(12)-C(67)-C(66) 125.0(2)
O(11)-C(67)-C(66) 109.3(2)
O(11)-C(68)-C(69) 111.0(2)
O(11)-C(68)-C(71) 103.27(19)
C(69)-C(68)-C(71) 111.5(2)
O(11)-C(68)-C(70) 107.9(2)
C(69)-C(68)-C(70) 112.7(2)
C(71)-C(68)-C(70) 110.0(2)
C(73)-C(72)-C(40) 116.5(2)
320
C(74)-C(73)-C(72) 112.0(2)
O(14)-C(74)-O(13) 123.7(3)
O(14)-C(74)-C(73) 124.0(3)
O(13)-C(74)-C(73) 112.2(2)
C(77B)-C(75)-C(78A) 133.2(7)
C(77B)-C(75)-O(13) 113.0(6)
C(78A)-C(75)-O(13) 108.7(4)
C(77B)-C(75)-C(76A) 75.9(7)
C(78A)-C(75)-C(76A) 115.3(4)
O(13)-C(75)-C(76A) 101.9(2)
C(77B)-C(75)-C(77A) 32.4(6)
C(78A)-C(75)-C(77A) 113.0(4)
O(13)-C(75)-C(77A) 109.2(3)
C(76A)-C(75)-C(77A) 108.0(3)
C(77B)-C(75)-C(76B) 112.3(8)
C(78A)-C(75)-C(76B) 78.0(7)
O(13)-C(75)-C(76B) 101.7(5)
C(76A)-C(75)-C(76B) 40.2(6)
C(77A)-C(75)-C(76B) 140.6(6)
C(77B)-C(75)-C(78B) 113.8(8)
C(78A)-C(75)-C(78B) 27.4(6)
O(13)-C(75)-C(78B) 111.0(7)
C(76A)-C(75)-C(78B) 136.6(7)
C(77A)-C(75)-C(78B) 87.3(6)
C(76B)-C(75)-C(78B) 104.0(8)
_____________________________________
321
Table I.3 Anisotropic displacement parameters (Å2x 103) of 3EUr16. The anisotropic
displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]
______________________________________________________________________________
U11 U22 U33 U23 U13 U12
______________________________________________________________________________
O(1) 17(1) 31(1) 26(1) 5(1) -5(1) -9(1)
O(2) 23(1) 22(1) 30(1) -5(1) -3(1) -8(1)
O(3) 29(1) 26(1) 45(1) -6(1) -15(1) -5(1)
O(4) 21(1) 27(1) 12(1) 1(1) -3(1) -6(1)
O(5) 22(1) 40(1) 23(1) -1(1) 1(1) 1(1)
O(6) 23(1) 20(1) 20(1) -3(1) 1(1) -4(1)
O(7) 35(1) 29(1) 23(1) 5(1) 6(1) -5(1)
N(1) 16(1) 21(1) 18(1) 6(1) -2(1) -5(1)
N(2) 18(1) 29(1) 21(1) 9(1) -4(1) -9(1)
C(1) 16(1) 20(1) 15(1) 2(1) 0(1) -7(1)
C(2) 22(2) 18(1) 16(1) 1(1) -2(1) -7(1)
C(3) 26(2) 23(2) 23(2) 5(1) -3(1) -10(1)
C(4) 34(2) 22(2) 27(2) 2(1) -4(1) -8(1)
C(5) 38(2) 27(2) 22(2) 4(1) -9(1) -14(1)
C(6) 39(2) 29(2) 26(2) 3(1) -9(1) -13(1)
C(7) 33(2) 26(2) 25(2) 5(1) -9(1) -11(1)
C(8) 38(2) 33(2) 23(2) 1(1) -9(1) -10(1)
C(9) 31(2) 26(2) 24(2) 6(1) -8(1) -12(1)
C(10) 36(2) 31(2) 20(2) 1(1) -7(1) -15(1)
C(11) 27(2) 26(2) 24(2) 4(1) -3(1) -13(1)
C(12) 27(2) 24(2) 24(2) 1(1) -3(1) -13(1)
C(13) 24(2) 26(2) 27(2) 0(1) -1(1) -11(1)
C(14) 24(2) 26(2) 23(2) 0(1) 0(1) -13(1)
C(15) 25(2) 24(2) 30(2) 2(1) -2(1) -11(1)
C(16) 24(2) 28(2) 32(2) 3(1) 0(1) -12(1)
C(17) 28(2) 25(2) 41(2) 5(1) -2(1) -10(1)
C(18) 34(2) 44(2) 55(2) 18(2) -7(2) -15(2)
C(19) 17(1) 21(1) 18(1) 1(1) -1(1) -7(1)
C(20) 23(2) 26(2) 37(2) -8(1) 0(1) -9(1)
C(21) 27(2) 26(2) 25(2) -7(1) 2(1) -9(1)
C(22) 29(2) 25(2) 21(2) -2(1) -3(1) -13(1)
322
C(23) 40(2) 29(2) 28(2) -6(1) -5(1) -11(1)
C(24) 35(2) 42(2) 36(2) -6(2) 3(1) -20(2)
C(25) 37(2) 23(2) 38(2) -4(1) -5(1) -9(1)
C(26) 20(1) 19(1) 18(1) 1(1) -1(1) -8(1)
C(27) 18(1) 23(1) 15(1) -1(1) -2(1) -8(1)
C(28) 21(2) 23(1) 19(2) 1(1) 1(1) -8(1)
C(29) 21(1) 24(2) 18(2) 3(1) -8(1) -7(1)
C(30) 32(2) 44(2) 30(2) 7(1) -9(1) -20(2)
C(31) 29(2) 24(2) 22(2) 4(1) -7(1) -7(1)
C(32) 32(2) 33(2) 17(2) -1(1) -7(1) -6(1)
C(33) 15(1) 24(1) 18(1) 2(1) -1(1) -6(1)
C(34) 22(1) 21(1) 24(2) -3(1) 2(1) -8(1)
C(35) 20(1) 23(2) 19(2) -2(1) -3(1) -8(1)
C(36) 16(1) 25(2) 26(2) -7(1) 2(1) -2(1)
C(37) 21(2) 32(2) 35(2) 0(1) -2(1) -7(1)
C(38) 29(2) 23(2) 42(2) -1(1) 2(1) -3(1)
C(39) 24(2) 35(2) 32(2) -14(1) 4(1) -9(1)
O(8) 17(1) 39(1) 23(1) 6(1) -3(1) -9(1)
O(9) 22(1) 17(1) 23(1) -3(1) 2(1) -4(1)
O(10) 43(1) 28(1) 32(1) 1(1) 16(1) -7(1)
O(11) 20(1) 29(1) 11(1) 0(1) -3(1) -3(1)
O(12) 21(1) 60(1) 19(1) -2(1) 1(1) 0(1)
O(13) 22(1) 26(1) 46(1) -14(1) 4(1) -8(1)
O(14) 24(1) 82(2) 200(3) -96(2) 5(2) -15(1)
N(3) 15(1) 29(1) 18(1) 5(1) -2(1) -9(1)
N(4) 19(1) 41(1) 22(1) 10(1) -3(1) -11(1)
C(40) 16(1) 21(1) 14(1) 4(1) -1(1) -6(1)
C(41) 22(2) 21(1) 19(2) 0(1) -2(1) -7(1)
C(42) 25(2) 28(2) 20(2) 3(1) -5(1) -8(1)
C(43) 23(2) 36(2) 22(2) 6(1) -3(1) -8(1)
C(44) 30(2) 31(2) 22(2) 8(1) -1(1) -10(1)
C(45) 27(2) 28(2) 23(2) 4(1) -3(1) -10(1)
C(46) 28(2) 23(2) 25(2) 5(1) -6(1) -10(1)
C(47) 30(2) 23(2) 22(2) 6(1) -6(1) -10(1)
C(48) 29(2) 26(2) 24(2) 5(1) -6(1) -11(1)
C(49) 28(2) 26(2) 24(2) 6(1) -6(1) -13(1)
323
C(50) 30(2) 23(2) 21(2) 7(1) -5(1) -10(1)
C(51) 28(2) 25(2) 21(2) 3(1) -3(1) -14(1)
C(52) 28(2) 24(2) 21(2) 3(1) -2(1) -12(1)
C(53) 26(2) 23(2) 26(2) 4(1) -4(1) -11(1)
C(54) 23(2) 23(2) 27(2) 3(1) -2(1) -8(1)
C(55) 29(2) 34(2) 31(2) 9(1) -5(1) -12(1)
C(56) 31(2) 39(2) 35(2) 11(2) -7(1) -7(1)
C(57) 43(2) 65(2) 36(2) 19(2) -11(2) -15(2)
C(58) 19(1) 21(1) 13(1) 1(1) -1(1) -7(1)
C(59) 20(1) 21(1) 25(2) -2(1) 4(1) -5(1)
C(60) 22(2) 21(2) 21(2) -1(1) -2(1) -8(1)
C(61) 18(1) 21(1) 26(2) -9(1) 1(1) -4(1)
C(62) 29(2) 20(2) 34(2) -2(1) 0(1) -5(1)
C(63) 22(2) 31(2) 31(2) -7(1) 2(1) -9(1)
C(64) 25(2) 38(2) 30(2) -14(1) 3(1) -11(1)
C(65) 18(1) 23(1) 13(1) -1(1) 0(1) -7(1)
C(66) 17(1) 26(2) 16(1) 1(1) -1(1) -6(1)
C(67) 22(2) 26(2) 16(2) 2(1) 0(1) -9(1)
C(68) 22(1) 27(2) 16(1) 4(1) -7(1) -9(1)
C(69) 42(2) 73(2) 31(2) 12(2) -9(2) -38(2)
C(70) 45(2) 33(2) 26(2) 1(1) -13(1) -5(1)
C(71) 34(2) 45(2) 16(2) -1(1) -3(1) -10(1)
C(72) 20(1) 24(1) 16(1) 0(1) -2(1) -7(1)
C(73) 21(1) 24(2) 26(2) -3(1) 0(1) -6(1)
C(74) 22(2) 33(2) 37(2) -17(1) 3(1) -8(1)
C(75) 28(2) 28(2) 43(2) -10(2) -2(1) -11(1)
C(76A) 38(3) 29(3) 82(5) -20(3) 1(3) -9(2)
C(77A) 42(4) 43(4) 41(3) 0(3) -4(3) -24(3)
C(78A) 71(6) 39(3) 40(4) -10(3) -6(4) -26(4)
C(76B) 37(7) 48(9) 57(12) -36(8) 18(8) -27(7)
C(77B) 36(10) 38(9) 32(7) 0(7) 5(7) -12(7)
C(78B) 56(12) 45(10) 22(9) -4(7) -10(8) -32(11)
O(15) 23(1) 32(1) 51(1) 1(1) -9(1) -9(1)
______________________________________________________________________________
324
Table I.4 Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) of 3EUr16.
________________________________________________________________________________
x y z U(eq)
________________________________________________________________________________
H(1A) 2721 9594 2143 23
H(2A) 2777 8638 2720 27
H(3A) 5079 7115 3007 28
H(3B) 3736 7221 3194 28
H(4A) 3636 8948 3538 34
H(4B) 5035 8733 3374 34
H(5A) 5004 8032 4075 34
H(5B) 4475 7093 3909 34
H(6A) 6923 7080 3700 38
H(6B) 6393 6102 3572 38
H(7A) 6863 6524 4407 34
H(7B) 6359 5535 4275 34
H(8A) 8812 5512 4076 39
H(8B) 8307 4517 3950 39
H(9A) 8658 5007 4788 32
H(9B) 8221 3979 4651 32
H(10A) 10678 4027 4499 34
H(10B) 10243 3009 4347 34
H(11A) 10416 3499 5197 30
H(11B) 10023 2464 5041 30
H(12A) 12106 1509 4778 29
H(12B) 12493 2534 4951 29
H(13A) 11742 969 5459 30
H(13B) 12110 2000 5634 30
H(14A) 14228 1027 5415 28
H(14B) 13863 -1 5234 28
H(15A) 13692 509 6094 31
H(15B) 13458 -574 5901 31
H(16A) 15643 -1413 5718 33
H(16B) 15866 -344 5927 33
H(17A) 15175 -2076 6360 38
325
H(17B) 15249 -955 6579 38
H(18A) 17399 -2731 6222 67
H(18B) 17113 -2651 6708 67
H(18C) 17448 -1643 6470 67
H(19A) 5362 10486 2088 22
H(19B) 4669 11367 1713 22
H(20A) 2898 12321 2121 34
H(20B) 3455 11367 2491 34
H(23A) 3663 14399 3157 49
H(23B) 4665 15023 3107 49
H(23C) 5123 13630 3064 49
H(24A) 6035 13807 2392 54
H(24B) 5481 15212 2349 54
H(24C) 5131 14461 2010 54
H(25A) 2833 15760 2162 49
H(25B) 3265 16369 2530 49
H(25C) 2299 15721 2619 49
H(26A) 2409 10045 1484 23
H(26B) 2223 11294 1662 23
H(27A) 3563 11545 1122 23
H(27B) 3609 10340 934 23
H(30A) 94 10904 307 51
H(30B) -798 12019 35 51
H(30C) -571 12152 516 51
H(31A) 321 13691 414 39
H(31B) 26 13758 -71 39
H(31C) 1435 13521 87 39
H(32A) 2309 11687 -306 44
H(32B) 920 11891 -476 44
H(32C) 1743 10684 -237 44
H(33A) 5342 9487 1336 23
H(33B) 5822 8760 1761 23
H(34A) 4668 7586 1638 27
H(34B) 3983 8392 1242 27
H(37A) 8451 6490 911 45
H(37B) 9400 5167 1014 45
326
H(37C) 8710 6014 1382 45
H(38A) 7848 4447 1628 50
H(38B) 8554 3671 1246 50
H(38C) 7058 4022 1306 50
H(39A) 6377 5104 631 46
H(39B) 7858 4538 523 46
H(39C) 7097 5927 464 46
H(3C) 11720 416 7758 25
H(4C) 11259 1195 7146 33
H(42A) 8818 2425 6916 31
H(42B) 9376 1207 6680 31
H(43A) 11072 1739 6433 33
H(43B) 10416 2984 6643 33
H(44A) 9967 3136 5928 34
H(44B) 9328 2185 5982 34
H(45A) 7624 3492 6337 31
H(45B) 8294 4425 6352 31
H(46A) 8072 4742 5636 31
H(46B) 7492 3751 5603 31
H(47A) 6226 5889 6013 30
H(47B) 5639 4923 5924 30
H(48A) 5754 5311 5201 31
H(48B) 6255 6323 5304 31
H(49A) 3762 6389 5473 30
H(49B) 4252 7371 5626 30
H(50A) 4503 7912 4939 30
H(50B) 4103 6890 4775 30
H(51A) 2404 8891 5185 28
H(51B) 2013 7881 5001 28
H(52A) 2828 9449 4517 29
H(52B) 2526 8402 4325 29
H(53A) 666 10391 4697 29
H(53B) 365 9344 4502 29
H(54A) 1141 10999 4053 30
H(54B) 1108 9852 3843 30
H(55A) -1081 11679 4153 38
327
H(55B) -1108 10537 3937 38
H(56A) -430 11162 3307 44
H(56B) -444 12316 3525 44
H(57A) -2654 11802 3383 75
H(57B) -2302 12779 3131 75
H(57C) -2675 12949 3609 75
H(58A) 8928 1035 8298 22
H(58B) 9627 398 8716 22
H(59A) 10829 1633 8700 28
H(59B) 10051 2302 8295 28
H(62A) 8071 5978 8678 44
H(62B) 6564 6447 8741 44
H(62C) 7200 5673 8351 44
H(63A) 6177 4189 8582 43
H(63B) 5536 5069 8946 43
H(63C) 6376 3716 9053 43
H(64A) 7866 4081 9503 46
H(64B) 7178 5482 9461 46
H(64C) 8651 4858 9348 46
H(65A) 12674 -1248 8281 22
H(65B) 12391 57 8429 22
H(66A) 11442 -282 9035 24
H(66B) 11510 -1525 8880 24
H(69A) 14883 -830 9701 66
H(69B) 15616 -2064 9490 66
H(69C) 15757 -1945 9977 66
H(70A) 14996 -3736 10028 56
H(70B) 14764 -3628 9538 56
H(70C) 13616 -3513 9845 56
H(71A) 13178 -712 10224 50
H(71B) 13994 -1941 10452 50
H(71C) 12641 -1743 10252 50
H(72A) 10247 -1522 8383 25
H(72B) 9462 -798 7990 25
H(73A) 11403 -1724 7590 29
H(73B) 11942 -2588 7985 29
328
H(76A) 12340 -6107 7726 76
H(76B) 12748 -5738 7285 76
H(76C) 11929 -6549 7311 76
H(77A) 9789 -5735 7663 59
H(77B) 9089 -4343 7704 59
H(77C) 10236 -5073 8010 59
H(78A) 10068 -5039 6895 73
H(78B) 10973 -4318 6827 73
H(78C) 9593 -3694 7024 73
H(76D) 12804 -5831 7296 65
H(76E) 12107 -5003 6908 65
H(76F) 11859 -6181 7014 65
H(77D) 10439 -6147 7609 54
H(77E) 9932 -5045 7904 54
H(77F) 11408 -5876 7906 54
H(78D) 9522 -4848 6956 55
H(78E) 9885 -3715 6896 55
H(78F) 8919 -3759 7252 55
H(15C) 3590(30) 9556(17) 7246(12) 90(10)
H(15D) 3530(30) 10730(30) 7307(11) 90(10)
________________________________________________________________________________
329
Table I.5 Torsion angles [°] of 3EUr16.
________________________________________________________________
C(2)-N(1)-C(1)-C(33) 57.0(3)
C(2)-N(1)-C(1)-C(26) 177.2(2)
C(2)-N(1)-C(1)-C(19) -62.1(3)
C(3)-N(2)-C(2)-O(1) -1.3(4)
C(3)-N(2)-C(2)-N(1) 177.9(2)
C(1)-N(1)-C(2)-O(1) -2.1(4)
C(1)-N(1)-C(2)-N(2) 178.7(2)
C(2)-N(2)-C(3)-C(4) 83.3(3)
N(2)-C(3)-C(4)-C(5) 174.8(2)
C(3)-C(4)-C(5)-C(6) 67.4(3)
C(4)-C(5)-C(6)-C(7) 175.3(2)
C(5)-C(6)-C(7)-C(8) -178.9(2)
C(6)-C(7)-C(8)-C(9) 179.4(2)
C(7)-C(8)-C(9)-C(10) -177.1(2)
C(8)-C(9)-C(10)-C(11) -178.2(2)
C(9)-C(10)-C(11)-C(12) -178.3(2)
C(10)-C(11)-C(12)-C(13) -178.0(2)
C(11)-C(12)-C(13)-C(14) -179.3(2)
C(12)-C(13)-C(14)-C(15) -179.2(2)
C(13)-C(14)-C(15)-C(16) -174.8(2)
C(14)-C(15)-C(16)-C(17) -178.0(2)
C(15)-C(16)-C(17)-C(18) -174.3(2)
N(1)-C(1)-C(19)-C(20) -47.4(3)
C(33)-C(1)-C(19)-C(20) -168.9(2)
C(26)-C(1)-C(19)-C(20) 69.0(3)
C(1)-C(19)-C(20)-C(21) 172.6(2)
C(22)-O(2)-C(21)-O(3) 1.2(4)
C(22)-O(2)-C(21)-C(20) -178.3(2)
C(19)-C(20)-C(21)-O(3) -49.4(4)
C(19)-C(20)-C(21)-O(2) 130.1(2)
C(21)-O(2)-C(22)-C(23) -62.9(3)
C(21)-O(2)-C(22)-C(24) 61.6(3)
C(21)-O(2)-C(22)-C(25) 179.0(2)
N(1)-C(1)-C(26)-C(27) -167.7(2)
C(33)-C(1)-C(26)-C(27) -47.8(3)
C(19)-C(1)-C(26)-C(27) 71.6(3)
C(1)-C(26)-C(27)-C(28) -174.6(2)
C(29)-O(4)-C(28)-O(5) 0.6(4)
C(29)-O(4)-C(28)-C(27) -177.68(19)
C(26)-C(27)-C(28)-O(5) 36.8(3)
C(26)-C(27)-C(28)-O(4) -144.8(2)
C(28)-O(4)-C(29)-C(32) -177.8(2)
C(28)-O(4)-C(29)-C(31) 64.5(3)
C(28)-O(4)-C(29)-C(30) -59.7(3)
N(1)-C(1)-C(33)-C(34) 55.7(3)
C(26)-C(1)-C(33)-C(34) -60.2(3)
C(19)-C(1)-C(33)-C(34) 177.6(2)
C(1)-C(33)-C(34)-C(35) 171.6(2)
C(36)-O(6)-C(35)-O(7) 1.7(4)
C(36)-O(6)-C(35)-C(34) -177.09(19)
C(33)-C(34)-C(35)-O(7) -59.6(3)
C(33)-C(34)-C(35)-O(6) 119.3(2)
C(35)-O(6)-C(36)-C(38) -179.0(2)
C(35)-O(6)-C(36)-C(39) -61.1(3)
C(35)-O(6)-C(36)-C(37) 63.1(3)
C(41)-N(3)-C(40)-C(72) 57.8(3)
C(41)-N(3)-C(40)-C(58) -60.9(3)
C(41)-N(3)-C(40)-C(65) 179.4(2)
C(42)-N(4)-C(41)-O(8) -2.1(4)
C(42)-N(4)-C(41)-N(3) 179.4(2)
C(40)-N(3)-C(41)-O(8) 5.8(4)
C(40)-N(3)-C(41)-N(4) -175.7(2)
C(41)-N(4)-C(42)-C(43) 169.4(2)
N(4)-C(42)-C(43)-C(44) 175.3(2)
C(42)-C(43)-C(44)-C(45) 60.9(3)
C(43)-C(44)-C(45)-C(46) 172.1(2)
C(44)-C(45)-C(46)-C(47) 175.8(2)
C(45)-C(46)-C(47)-C(48) 173.4(2)
330
C(46)-C(47)-C(48)-C(49) 176.3(2)
C(47)-C(48)-C(49)-C(50) 174.0(2)
C(48)-C(49)-C(50)-C(51) 176.3(2)
C(49)-C(50)-C(51)-C(52) 177.6(2)
C(50)-C(51)-C(52)-C(53) 176.6(2)
C(51)-C(52)-C(53)-C(54) 179.7(2)
C(52)-C(53)-C(54)-C(55) 169.7(2)
C(53)-C(54)-C(55)-C(56) 179.2(2)
C(54)-C(55)-C(56)-C(57) 178.4(3)
N(3)-C(40)-C(58)-C(59) -58.2(3)
C(72)-C(40)-C(58)-C(59) -178.0(2)
C(65)-C(40)-C(58)-C(59) 59.0(3)
C(40)-C(58)-C(59)-C(60) -176.7(2)
C(61)-O(9)-C(60)-O(10) -3.0(4)
C(61)-O(9)-C(60)-C(59) 177.42(19)
C(58)-C(59)-C(60)-O(10) 48.4(3)
C(58)-C(59)-C(60)-O(9) -132.0(2)
C(60)-O(9)-C(61)-C(62) 179.1(2)
C(60)-O(9)-C(61)-C(63) -62.5(3)
C(60)-O(9)-C(61)-C(64) 61.3(3)
N(3)-C(40)-C(65)-C(66) 169.8(2)
C(72)-C(40)-C(65)-C(66) -69.9(3)
C(58)-C(40)-C(65)-C(66) 50.8(3)
C(40)-C(65)-C(66)-C(67) 171.8(2)
C(68)-O(11)-C(67)-O(12) -2.9(4)
C(68)-O(11)-C(67)-C(66) 174.5(2)
C(65)-C(66)-C(67)-O(12) -27.3(4)
C(65)-C(66)-C(67)-O(11) 155.3(2)
C(67)-O(11)-C(68)-C(69) 56.7(3)
C(67)-O(11)-C(68)-C(71) 176.3(2)
C(67)-O(11)-C(68)-C(70) -67.2(3)
N(3)-C(40)-C(72)-C(73) 55.1(3)
C(58)-C(40)-C(72)-C(73) 174.6(2)
C(65)-C(40)-C(72)-C(73) -63.2(3)
C(40)-C(72)-C(73)-C(74) -166.4(2)
C(75)-O(13)-C(74)-O(14) -3.4(5)
C(75)-O(13)-C(74)-C(73) -178.7(2)
C(72)-C(73)-C(74)-O(14) 27.7(4)
C(72)-C(73)-C(74)-O(13) -157.1(2)
C(74)-O(13)-C(75)-C(77B) -87.3(9)
C(74)-O(13)-C(75)-C(78A) 70.9(5)
C(74)-O(13)-C(75)-C(76A) -166.9(4)
C(74)-O(13)-C(75)-C(77A) -52.7(4)
C(74)-O(13)-C(75)-C(76B) 152.1(9)
C(74)-O(13)-C(75)-C(78B) 41.9(8)
________________________________________
331
Table I.6 Hydrogen bonds [Å and °] of 3EUr16.
___________________________________________
D-H...A d(D-H) d(H...A) d(D...A) <(DHA)
____________________________________________________________________________
N(1)-H(1A)...O(8)#1 0.88 2.44 3.201(3) 145.6
N(2)-H(2A)...O(8)#1 0.88 2.02 2.863(3) 159.1
N(3)-H(3C)...O(15)#2 0.88 2.33 3.074(3) 141.6
N(4)-H(4C)...O(15)#2 0.88 2.02 2.832(3) 153.4
O(15)-H(15C)...O(3)#3 0.857(2) 2.00(2) 2.843(4) 169(4)
O(15)-H(15D)...O(1)#3 0.863(2) 1.864(2) 2.718(4) 170(4)
____________________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x+1,-y+1,-z+1 #2 x+1,y-1,z #3 -x+1,-y+2,-z+1
332
Table II.1 Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) of 3EUr-α-cholestane. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ________________________________________________________________________________ x y z U(eq) ________________________________________________________________________________ O(1) 4343(4) 7655(4) 1830(1) 50(1)
O(2) -620(4) 9402(4) 1567(1) 60(1)
O(3) -294(4) 10571(3) 1122(1) 44(1)
O(4) 3705(8) 6352(6) 399(2) 123(3)
O(5) 3589(5) 8131(4) 384(1) 64(2)
O(6) 2572(5) 11008(4) 2079(2) 72(2)
O(7) 2610(4) 10050(4) 2614(1) 48(1)
O(8) 9386(4) 5986(4) 1773(1) 51(1)
O(9) 7457(5) 2761(4) 2168(1) 63(2)
O(10) 7683(4) 3979(3) 2635(1) 41(1)
O(11) 4396(4) 4184(4) 1538(2) 66(2)
O(12) 4730(4) 3064(4) 1079(1) 50(1)
O(13) 9020(9) 7089(7) 437(2) 177(5)
O(14) 8637(4) 5388(4) 364(1) 65(2)
N(1) 2519(5) 7219(4) 1651(1) 41(1)
N(2) 3587(5) 6300(4) 2123(1) 41(1)
N(3) 7549(5) 6421(4) 1614(1) 38(1)
N(4) 8654(4) 7267(4) 2102(1) 36(1)
C(1) 2181(5) 8135(5) 1412(2) 38(2)
C(2) 3547(6) 7078(5) 1869(2) 38(2)
C(3) 4633(5) 6006(5) 2370(2) 41(2)
C(4) 4529(6) 4876(5) 2507(2) 45(2)
C(5) 3742(5) 4775(5) 2801(2) 40(2)
C(6) 4045(5) 5495(5) 3147(2) 37(2)
C(7) 3146(5) 5468(4) 3405(2) 33(1)
C(8) 2955(6) 4404(5) 3576(2) 40(2)
C(9) 2010(5) 4369(5) 3816(2) 34(1)
C(10) 2142(5) 5196(5) 4132(2) 37(1)
C(11) 1105(5) 5450(5) 4309(2) 36(2)
C(12) 1317(6) 6572(5) 4461(2) 47(2)
C(13) 2196(6) 7045(5) 4246(2) 44(2)
C(14) 2298(5) 6246(5) 3937(2) 37(2)
333
C(15) 3302(5) 6297(5) 3719(2) 36(2)
C(16) 3439(6) 7397(5) 3563(2) 45(2)
C(17) 4316(5) 7423(5) 3305(2) 47(2)
C(18) 4094(5) 6616(5) 2990(2) 37(2)
C(19) 4911(6) 6748(5) 2704(2) 43(2)
C(20) 5184(5) 5156(6) 3366(2) 44(2)
C(21) 3137(5) 4928(5) 4443(2) 44(2)
C(22) 693(5) 4736(5) 4609(2) 37(1)
C(23) 653(5) 3592(5) 4490(2) 41(2)
C(24) -488(5) 5095(5) 4663(2) 42(2)
C(25) -882(6) 4733(5) 5033(2) 46(2)
C(26) -578(6) 5472(5) 5360(2) 47(2)
C(27) -934(6) 5192(5) 5735(2) 46(2)
C(28) -2208(6) 5174(7) 5705(2) 61(2)
C(29) -441(7) 5942(6) 6040(2) 62(2)
C(30) 905(6) 7969(5) 1287(2) 46(2)
C(31) 307(6) 8856(5) 1054(2) 43(2)
C(32) -258(6) 9626(5) 1283(2) 43(2)
C(33) -875(6) 11457(6) 1286(2) 54(2)
C(34) -296(9) 11684(8) 1690(2) 93(3)
C(35) -2103(6) 11220(7) 1254(3) 73(3)
C(36) -663(7) 12334(6) 1021(3) 76(3)
C(37) 2808(6) 8169(5) 1072(2) 47(2)
C(38) 2643(8) 7246(6) 810(2) 66(2)
C(39) 3400(9) 7198(8) 518(2) 86(3)
C(40) 4261(7) 8274(9) 71(2) 77(3)
C(41) 5411(9) 7779(11) 197(3) 121(5)
C(42) 3660(7) 7772(7) -289(2) 71(2)
C(43) 4287(7) 9459(8) 40(2) 79(3)
C(44) 2433(5) 9162(5) 1637(2) 38(2)
C(45) 2110(6) 9183(5) 2038(2) 43(2)
C(46) 2447(5) 10198(6) 2235(2) 42(2)
C(47) 2866(6) 10954(6) 2872(2) 52(2)
C(48) 3957(7) 11426(7) 2824(2) 72(2)
C(49) 1940(8) 11731(9) 2799(4) 131(5)
C(50) 2972(9) 10412(8) 3259(2) 99(4)
334
C(51) 7172(5) 5491(5) 1390(2) 38(2)
C(52) 8596(6) 6522(5) 1829(2) 36(2)
C(53) 9725(5) 7573(5) 2326(2) 38(2)
C(54) 10123(5) 6803(5) 2640(2) 38(2)
C(55) 9367(5) 6783(5) 2950(2) 36(1)
C(56) 9214(5) 7854(5) 3125(2) 33(1)
C(57) 8298(5) 7797(5) 3382(2) 34(1)
C(58) 8505(5) 6959(5) 3693(2) 38(2)
C(59) 7595(5) 6919(5) 3942(2) 35(1)
C(60) 7416(5) 7974(4) 4127(2) 33(1)
C(61) 6320(5) 8118(5) 4305(2) 39(2)
C(62) 6151(6) 9302(5) 4299(2) 55(2)
C(63) 6783(7) 9743(5) 3981(2) 60(2)
C(64) 7159(6) 8765(5) 3793(2) 43(2)
C(65) 8065(6) 8877(5) 3549(2) 44(2)
C(66) 7747(7) 9651(5) 3232(2) 62(2)
C(67) 8566(7) 9716(5) 2944(2) 55(2)
C(68) 8785(5) 8614(5) 2794(2) 38(2)
C(69) 9568(5) 8650(5) 2498(2) 40(2)
C(70) 10323(5) 8217(6) 3351(2) 46(2)
C(71) 8434(5) 8260(6) 4412(2) 49(2)
C(72) 6289(5) 7618(5) 4700(2) 37(2)
C(73) 6407(6) 6434(5) 4701(2) 51(2)
C(74) 5184(5) 7950(5) 4832(2) 39(2)
C(75) 4965(5) 7467(5) 5198(2) 42(2)
C(76) 3916(6) 7920(5) 5326(2) 45(2)
C(77) 3550(5) 7355(5) 5665(2) 41(2)
C(78) 4441(6) 7395(7) 6023(2) 62(2)
C(79) 2452(6) 7790(7) 5746(2) 68(2)
C(80) 7405(6) 4483(5) 1633(2) 42(2)
C(81) 7000(6) 4556(5) 2017(2) 44(2)
C(82) 7403(6) 3650(5) 2271(2) 42(2)
C(83) 8062(6) 3243(5) 2948(2) 44(2)
C(84) 9094(6) 2682(6) 2878(2) 64(2)
C(85) 7097(6) 2501(6) 2992(2) 61(2)
C(86) 8295(7) 3970(6) 3287(2) 57(2)
335
C(87) 5927(5) 5680(5) 1263(2) 44(2)
C(88) 5291(6) 4815(5) 1025(2) 46(2)
C(89) 4770(6) 3997(6) 1248(2) 49(2)
C(90) 4205(7) 2144(6) 1232(2) 60(2)
C(91) 4798(7) 1891(7) 1629(2) 77(3)
C(92) 2965(6) 2345(7) 1206(2) 70(2)
C(93) 4405(8) 1296(6) 958(3) 74(3)
C(94) 7814(6) 5380(5) 1050(2) 43(2)
C(95) 7737(8) 6292(6) 789(2) 68(2)
C(96) 8540(10) 6312(8) 511(2) 83(3)
C(97) 9413(8) 5200(10) 82(2) 85(3)
C(98) 10556(11) 5511(19) 236(5) 236(12)
C(99) 8956(14) 5804(8) -275(3) 146(6)
C(100) 9309(8) 4087(8) 4(2) 82(3)
O(15) 1441(4) 5337(5) 1994(2) 64(2)
C(101) 1426(8) 4399(7) 1789(3) 88(3)
C(102) 1356(8) 4709(8) 1335(3) 97(3)
O(16) 6458(4) 8155(4) 2006(2) 61(1)
C(103) 6421(8) 9171(7) 1856(5) 142(6)
C(104) 5911(13) 9833(11) 2240(5) 172(6) ________________________________________________________________________________
336
Table II.2 Bond lengths [Å] and angles [°] of 3EUr-α-cholestane. _____________________________________________________
O(1)-C(2) 1.240(8)
O(2)-C(32) 1.200(7)
O(3)-C(32) 1.337(8)
O(3)-C(33) 1.500(8)
O(4)-C(39) 1.241(10)
O(5)-C(39) 1.320(11)
O(5)-C(40) 1.493(9)
O(6)-C(46) 1.197(8)
O(7)-C(46) 1.346(7)
O(7)-C(47) 1.478(8)
O(8)-C(52) 1.220(7)
O(9)-C(82) 1.201(8)
O(10)-C(82) 1.353(8)
O(10)-C(83) 1.476(7)
O(11)-C(89) 1.215(8)
O(12)-C(89) 1.335(8)
O(12)-C(90) 1.483(8)
O(13)-C(96) 1.203(10)
O(14)-C(96) 1.308(10)
O(14)-C(97) 1.499(9)
N(1)-C(2) 1.370(8)
N(1)-C(1) 1.469(8)
N(2)-C(2) 1.341(8)
N(2)-C(3) 1.471(8)
N(3)-C(52) 1.378(8)
N(3)-C(51) 1.466(8)
N(4)-C(52) 1.357(8)
N(4)-C(53) 1.465(8)
C(1)-C(37) 1.530(8)
C(1)-C(44) 1.544(9)
C(1)-C(30) 1.554(9)
C(3)-C(19) 1.519(9)
C(3)-C(4) 1.540(9)
C(4)-C(5) 1.529(8)
C(5)-C(6) 1.534(9)
C(6)-C(7) 1.533(8)
C(6)-C(20) 1.538(9)
C(6)-C(18) 1.545(9)
C(7)-C(8) 1.526(8)
C(7)-C(15) 1.533(8)
C(8)-C(9) 1.534(8)
C(9)-C(10) 1.534(8)
C(10)-C(11) 1.528(8)
C(10)-C(14) 1.538(8)
C(10)-C(21) 1.540(9)
C(11)-C(12) 1.544(9)
C(11)-C(22) 1.549(8)
C(12)-C(13) 1.532(9)
C(13)-C(14) 1.522(8)
C(14)-C(15) 1.545(8)
C(15)-C(16) 1.533(8)
C(16)-C(17) 1.513(9)
C(17)-C(18) 1.516(9)
C(18)-C(19) 1.538(8)
C(22)-C(23) 1.523(9)
C(22)-C(24) 1.545(8)
C(24)-C(25) 1.542(8)
C(25)-C(26) 1.498(9)
C(26)-C(27) 1.511(9)
C(27)-C(29) 1.500(10)
C(27)-C(28) 1.531(9)
C(30)-C(31) 1.522(9)
C(31)-C(32) 1.510(9)
C(33)-C(35) 1.505(11)
C(33)-C(36) 1.514(11)
C(33)-C(34) 1.526(11)
C(37)-C(38) 1.499(9)
C(38)-C(39) 1.494(11)
337
C(40)-C(42) 1.511(12)
C(40)-C(43) 1.522(14)
C(40)-C(41) 1.532(12)
C(44)-C(45) 1.539(8)
C(45)-C(46) 1.503(9)
C(47)-C(48) 1.489(10)
C(47)-C(49) 1.492(12)
C(47)-C(50) 1.530(11)
C(51)-C(87) 1.524(9)
C(51)-C(94) 1.548(8)
C(51)-C(80) 1.553(9)
C(53)-C(54) 1.509(9)
C(53)-C(69) 1.532(8)
C(54)-C(55) 1.543(8)
C(55)-C(56) 1.529(8)
C(56)-C(70) 1.527(9)
C(56)-C(57) 1.548(8)
C(56)-C(68) 1.551(8)
C(57)-C(58) 1.535(8)
C(57)-C(65) 1.550(8)
C(58)-C(59) 1.520(8)
C(59)-C(60) 1.533(8)
C(60)-C(71) 1.512(8)
C(60)-C(64) 1.555(9)
C(60)-C(61) 1.572(8)
C(61)-C(62) 1.529(9)
C(61)-C(72) 1.550(8)
C(62)-C(63) 1.570(9)
C(63)-C(64) 1.526(9)
C(64)-C(65) 1.511(8)
C(65)-C(66) 1.505(9)
C(66)-C(67) 1.539(8)
C(67)-C(68) 1.548(9)
C(68)-C(69) 1.527(8)
C(72)-C(73) 1.524(9)
C(72)-C(74) 1.547(8)
C(74)-C(75) 1.504(8)
C(75)-C(76) 1.531(9)
C(76)-C(77) 1.533(8)
C(77)-C(79) 1.512(9)
C(77)-C(78) 1.536(9)
C(80)-C(81) 1.528(8)
C(81)-C(82) 1.504(9)
C(83)-C(84) 1.497(9)
C(83)-C(86) 1.517(9)
C(83)-C(85) 1.534(10)
C(87)-C(88) 1.528(9)
C(88)-C(89) 1.513(9)
C(90)-C(93) 1.504(11)
C(90)-C(92) 1.514(10)
C(90)-C(91) 1.516(11)
C(94)-C(95) 1.486(10)
C(95)-C(96) 1.494(11)
C(97)-C(100) 1.455(14)
C(97)-C(98) 1.461(14)
C(97)-C(99) 1.517(15)
O(15)-C(101) 1.404(10)
C(101)-C(102) 1.652(14)
O(16)-C(103) 1.404(10)
C(103)-C(104) 1.80(2)
C(32)-O(3)-C(33) 120.3(5)
C(39)-O(5)-C(40) 121.9(6)
C(46)-O(7)-C(47) 119.6(5)
C(82)-O(10)-C(83) 121.6(5)
C(89)-O(12)-C(90) 122.3(5)
C(96)-O(14)-C(97) 121.3(7)
C(2)-N(1)-C(1) 125.3(5)
C(2)-N(2)-C(3) 121.8(6)
C(52)-N(3)-C(51) 123.9(5)
C(52)-N(4)-C(53) 121.3(5)
N(1)-C(1)-C(37) 110.6(5)
338
N(1)-C(1)-C(44) 111.5(5)
C(37)-C(1)-C(44) 107.7(5)
N(1)-C(1)-C(30) 103.3(5)
C(37)-C(1)-C(30) 111.9(5)
C(44)-C(1)-C(30) 111.8(5)
O(1)-C(2)-N(2) 125.0(6)
O(1)-C(2)-N(1) 120.7(6)
N(2)-C(2)-N(1) 114.3(6)
N(2)-C(3)-C(19) 111.8(5)
N(2)-C(3)-C(4) 108.9(6)
C(19)-C(3)-C(4) 110.9(5)
C(5)-C(4)-C(3) 112.9(5)
C(4)-C(5)-C(6) 113.7(5)
C(7)-C(6)-C(5) 111.4(5)
C(7)-C(6)-C(20) 110.8(5)
C(5)-C(6)-C(20) 108.6(5)
C(7)-C(6)-C(18) 108.0(5)
C(5)-C(6)-C(18) 106.7(5)
C(20)-C(6)-C(18) 111.2(5)
C(8)-C(7)-C(15) 109.6(5)
C(8)-C(7)-C(6) 115.3(5)
C(15)-C(7)-C(6) 113.9(5)
C(7)-C(8)-C(9) 115.0(5)
C(8)-C(9)-C(10) 112.8(5)
C(11)-C(10)-C(9) 116.9(5)
C(11)-C(10)-C(14) 99.7(5)
C(9)-C(10)-C(14) 106.2(5)
C(11)-C(10)-C(21) 110.7(5)
C(9)-C(10)-C(21) 110.6(5)
C(14)-C(10)-C(21) 112.3(5)
C(10)-C(11)-C(12) 103.9(5)
C(10)-C(11)-C(22) 122.4(5)
C(12)-C(11)-C(22) 111.1(5)
C(13)-C(12)-C(11) 106.4(5)
C(14)-C(13)-C(12) 103.8(5)
C(13)-C(14)-C(10) 103.4(5)
C(13)-C(14)-C(15) 119.7(5)
C(10)-C(14)-C(15) 115.3(5)
C(7)-C(15)-C(16) 112.3(5)
C(7)-C(15)-C(14) 108.8(5)
C(16)-C(15)-C(14) 111.1(5)
C(17)-C(16)-C(15) 111.6(5)
C(16)-C(17)-C(18) 112.0(5)
C(17)-C(18)-C(19) 110.9(5)
C(17)-C(18)-C(6) 112.4(5)
C(19)-C(18)-C(6) 114.0(5)
C(3)-C(19)-C(18) 111.4(5)
C(23)-C(22)-C(24) 109.4(5)
C(23)-C(22)-C(11) 112.0(5)
C(24)-C(22)-C(11) 108.5(5)
C(25)-C(24)-C(22) 115.8(5)
C(26)-C(25)-C(24) 113.7(5)
C(25)-C(26)-C(27) 117.8(6)
C(29)-C(27)-C(26) 110.5(6)
C(29)-C(27)-C(28) 110.0(6)
C(26)-C(27)-C(28) 111.6(6)
C(31)-C(30)-C(1) 114.9(5)
C(32)-C(31)-C(30) 114.2(5)
O(2)-C(32)-O(3) 125.8(6)
O(2)-C(32)-C(31) 123.9(6)
O(3)-C(32)-C(31) 110.4(5)
O(3)-C(33)-C(35) 109.9(6)
O(3)-C(33)-C(36) 100.5(5)
C(35)-C(33)-C(36) 111.6(7)
O(3)-C(33)-C(34) 109.6(6)
C(35)-C(33)-C(34) 114.2(7)
C(36)-C(33)-C(34) 110.3(8)
C(38)-C(37)-C(1) 115.8(6)
C(39)-C(38)-C(37) 115.4(8)
O(4)-C(39)-O(5) 126.0(8)
O(4)-C(39)-C(38) 121.5(9)
O(5)-C(39)-C(38) 112.1(7)
339
O(5)-C(40)-C(42) 109.4(7)
O(5)-C(40)-C(43) 101.3(7)
C(42)-C(40)-C(43) 112.1(7)
O(5)-C(40)-C(41) 108.0(7)
C(42)-C(40)-C(41) 111.4(8)
C(43)-C(40)-C(41) 113.9(10)
C(45)-C(44)-C(1) 115.9(5)
C(46)-C(45)-C(44) 111.0(5)
O(6)-C(46)-O(7) 125.3(6)
O(6)-C(46)-C(45) 125.3(6)
O(7)-C(46)-C(45) 109.4(6)
O(7)-C(47)-C(48) 110.6(5)
O(7)-C(47)-C(49) 109.7(6)
C(48)-C(47)-C(49) 111.5(8)
O(7)-C(47)-C(50) 100.5(6)
C(48)-C(47)-C(50) 109.7(7)
C(49)-C(47)-C(50) 114.2(8)
N(3)-C(51)-C(87) 103.9(5)
N(3)-C(51)-C(94) 110.4(5)
C(87)-C(51)-C(94) 112.3(5)
N(3)-C(51)-C(80) 111.1(5)
C(87)-C(51)-C(80) 112.5(5)
C(94)-C(51)-C(80) 106.8(5)
O(8)-C(52)-N(4) 124.0(6)
O(8)-C(52)-N(3) 122.4(6)
N(4)-C(52)-N(3) 113.7(5)
N(4)-C(53)-C(54) 112.2(5)
N(4)-C(53)-C(69) 107.6(5)
C(54)-C(53)-C(69) 109.9(5)
C(53)-C(54)-C(55) 112.6(5)
C(56)-C(55)-C(54) 113.6(5)
C(70)-C(56)-C(55) 109.5(5)
C(70)-C(56)-C(57) 110.9(5)
C(55)-C(56)-C(57) 110.1(5)
C(70)-C(56)-C(68) 111.6(5)
C(55)-C(56)-C(68) 107.6(5)
C(57)-C(56)-C(68) 107.0(5)
C(58)-C(57)-C(56) 114.2(5)
C(58)-C(57)-C(65) 111.5(5)
C(56)-C(57)-C(65) 112.2(5)
C(59)-C(58)-C(57) 113.2(5)
C(58)-C(59)-C(60) 112.7(5)
C(71)-C(60)-C(59) 110.2(5)
C(71)-C(60)-C(64) 113.4(5)
C(59)-C(60)-C(64) 105.7(4)
C(71)-C(60)-C(61) 110.6(5)
C(59)-C(60)-C(61) 117.6(5)
C(64)-C(60)-C(61) 98.8(5)
C(62)-C(61)-C(72) 113.5(5)
C(62)-C(61)-C(60) 103.4(5)
C(72)-C(61)-C(60) 117.5(5)
C(61)-C(62)-C(63) 106.7(5)
C(64)-C(63)-C(62) 103.6(5)
C(65)-C(64)-C(63) 118.0(5)
C(65)-C(64)-C(60) 115.2(5)
C(63)-C(64)-C(60) 103.7(5)
C(66)-C(65)-C(64) 111.7(5)
C(66)-C(65)-C(57) 109.9(5)
C(64)-C(65)-C(57) 109.2(5)
C(65)-C(66)-C(67) 114.8(6)
C(66)-C(67)-C(68) 110.2(5)
C(69)-C(68)-C(67) 111.8(5)
C(69)-C(68)-C(56) 111.9(5)
C(67)-C(68)-C(56) 111.4(5)
C(68)-C(69)-C(53) 112.2(5)
C(73)-C(72)-C(74) 111.1(5)
C(73)-C(72)-C(61) 113.4(5)
C(74)-C(72)-C(61) 107.9(5)
C(75)-C(74)-C(72) 115.4(5)
C(74)-C(75)-C(76) 111.8(5)
C(75)-C(76)-C(77) 114.5(5)
C(79)-C(77)-C(76) 110.6(6)
340
C(79)-C(77)-C(78) 110.6(6)
C(76)-C(77)-C(78) 112.7(5)
C(81)-C(80)-C(51) 113.0(5)
C(82)-C(81)-C(80) 111.7(5)
O(9)-C(82)-O(10) 124.7(6)
O(9)-C(82)-C(81) 125.3(6)
O(10)-C(82)-C(81) 110.0(5)
O(10)-C(83)-C(84) 110.5(5)
O(10)-C(83)-C(86) 101.9(5)
C(84)-C(83)-C(86) 111.9(6)
O(10)-C(83)-C(85) 108.8(5)
C(84)-C(83)-C(85) 113.0(6)
C(86)-C(83)-C(85) 110.3(6)
C(51)-C(87)-C(88) 116.0(5)
C(89)-C(88)-C(87) 115.3(6)
O(11)-C(89)-O(12) 124.5(7)
O(11)-C(89)-C(88) 123.6(7)
O(12)-C(89)-C(88) 111.9(5)
O(12)-C(90)-C(93) 102.3(5)
O(12)-C(90)-C(92) 108.9(6)
C(93)-C(90)-C(92) 110.2(7)
O(12)-C(90)-C(91) 110.1(6)
C(93)-C(90)-C(91) 110.4(7)
C(92)-C(90)-C(91) 114.2(6)
C(95)-C(94)-C(51) 115.3(6)
C(94)-C(95)-C(96) 116.4(7)
O(13)-C(96)-O(14) 125.7(9)
O(13)-C(96)-C(95) 123.2(9)
O(14)-C(96)-C(95) 111.0(7)
C(100)-C(97)-C(98) 112.8(13)
C(100)-C(97)-O(14) 103.9(7)
C(98)-C(97)-O(14) 111.4(8)
C(100)-C(97)-C(99) 109.3(8)
C(98)-C(97)-C(99) 111.9(12)
O(14)-C(97)-C(99) 107.1(9)
O(15)-C(101)-C(102) 107.1(8)
O(16)-C(103)-C(104) 98.3(10)
_______________________________________________________________
341
Table II.3 Anisotropic displacement parameters (Å2x 103) of 3EUr-α-cholestane. The anisotropic
displacement factor exponent takes the form: -2�2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]
______________________________________________________________________________
U11 U22 U33 U23 U13 U12
______________________________________________________________________________
O(1) 53(3) 58(3) 38(3) 10(2) 8(2) -11(2)
O(2) 70(3) 69(3) 47(3) 8(3) 28(3) 1(3)
O(3) 59(3) 29(2) 46(3) -1(2) 20(2) 4(2)
O(4) 226(9) 94(5) 59(4) 22(4) 56(5) 88(5)
O(5) 96(4) 71(4) 31(3) 5(3) 26(3) 30(3)
O(6) 117(5) 43(3) 49(3) 2(3) -5(3) -23(3)
O(7) 61(3) 53(3) 35(3) -16(2) 21(2) -18(2)
O(8) 49(3) 66(3) 41(3) -7(2) 12(2) 20(3)
O(9) 110(4) 40(3) 41(3) -1(2) 19(3) 20(3)
O(10) 55(3) 36(2) 34(2) 6(2) 14(2) 12(2)
O(11) 73(4) 77(4) 56(3) -12(3) 31(3) -5(3)
O(12) 58(3) 50(3) 48(3) 0(2) 24(2) -4(2)
O(13) 327(13) 145(7) 89(5) -82(5) 128(7) -156(9)
O(14) 85(4) 82(4) 31(3) 0(3) 22(3) -26(3)
N(1) 57(4) 37(3) 28(3) 2(2) 5(3) -1(3)
N(2) 53(3) 44(3) 29(3) 9(3) 15(3) 11(3)
N(3) 54(4) 29(3) 30(3) 3(2) 8(3) 13(2)
N(4) 44(3) 37(3) 31(3) -1(2) 17(2) 7(2)
C(1) 52(4) 35(3) 25(3) 4(3) 5(3) -1(3)
C(2) 55(4) 38(4) 24(3) -3(3) 11(3) 9(3)
C(3) 37(4) 48(4) 39(4) 10(3) 12(3) 8(3)
C(4) 55(4) 46(4) 40(4) 11(3) 24(3) 13(3)
C(5) 47(4) 37(4) 39(4) 5(3) 13(3) 10(3)
C(6) 41(4) 36(4) 35(3) 3(3) 11(3) 1(3)
C(7) 37(3) 32(3) 31(3) 0(3) 7(3) 3(3)
C(8) 61(4) 29(3) 35(4) 1(3) 24(3) 9(3)
C(9) 41(3) 29(3) 36(3) 2(3) 13(3) 4(3)
C(10) 46(4) 33(3) 33(3) -2(3) 12(3) -1(3)
C(11) 49(4) 34(4) 27(3) -5(3) 8(3) 6(3)
C(12) 60(4) 44(4) 39(4) -2(3) 18(3) -5(3)
C(13) 63(4) 39(4) 29(3) -7(3) 9(3) 0(3)
342
C(14) 47(4) 36(4) 29(3) 4(3) 12(3) -1(3)
C(15) 44(4) 39(4) 25(3) 1(3) 5(3) -3(3)
C(16) 57(4) 39(4) 42(4) -5(3) 16(3) -12(3)
C(17) 55(4) 44(4) 44(4) 9(3) 11(3) 1(3)
C(18) 41(4) 39(4) 32(3) 0(3) 10(3) 0(3)
C(19) 53(4) 45(4) 32(3) 13(3) 13(3) 6(3)
C(20) 44(4) 53(4) 38(4) 13(3) 14(3) 13(3)
C(21) 47(4) 56(4) 30(3) 5(3) 8(3) 2(3)
C(22) 46(4) 39(4) 28(3) 0(3) 12(3) 2(3)
C(23) 42(4) 41(4) 42(4) 6(3) 13(3) 6(3)
C(24) 54(4) 40(4) 34(3) 12(3) 14(3) 12(3)
C(25) 59(4) 38(4) 46(4) 11(3) 20(3) 6(3)
C(26) 59(4) 36(4) 50(4) 8(3) 17(4) 3(3)
C(27) 62(4) 41(4) 41(4) 8(3) 28(3) 7(3)
C(28) 74(5) 69(5) 43(4) 3(4) 21(4) 13(4)
C(29) 96(6) 46(4) 50(5) 3(4) 26(4) 14(4)
C(30) 61(4) 34(4) 40(4) -5(3) 0(3) -10(3)
C(31) 57(4) 41(4) 33(4) 0(3) 7(3) 1(3)
C(32) 46(4) 43(4) 41(4) -1(3) 13(3) -5(3)
C(33) 58(5) 43(4) 65(5) 4(4) 20(4) 18(4)
C(34) 128(8) 79(7) 65(6) -41(5) -5(6) 22(6)
C(35) 61(5) 77(6) 86(7) 13(5) 29(5) 16(4)
C(36) 78(6) 50(5) 108(7) 14(5) 38(5) 17(4)
C(37) 68(5) 46(4) 31(4) 2(3) 16(3) 20(4)
C(38) 117(7) 52(5) 32(4) -5(3) 24(4) 12(5)
C(39) 152(9) 85(7) 26(4) 9(4) 31(5) 52(7)
C(40) 73(6) 132(9) 30(4) 14(5) 18(4) 32(6)
C(41) 104(8) 196(13) 66(6) 4(8) 22(6) 73(9)
C(42) 90(6) 88(6) 33(4) -7(4) 8(4) 24(5)
C(43) 80(6) 110(8) 56(5) -13(5) 35(5) -5(6)
C(44) 48(4) 37(4) 31(3) 4(3) 12(3) -6(3)
C(45) 58(4) 37(4) 36(4) -2(3) 16(3) -9(3)
C(46) 46(4) 47(4) 35(4) -2(3) 13(3) -6(3)
C(47) 58(4) 54(4) 47(4) -22(4) 17(3) -23(4)
C(48) 65(5) 97(7) 56(5) -29(5) 11(4) -27(5)
C(49) 71(6) 127(9) 188(12) -129(9) -2(7) 11(6)
343
C(50) 162(10) 96(7) 43(5) -28(5) 32(6) -49(7)
C(51) 53(4) 34(4) 28(3) -1(3) 12(3) 12(3)
C(52) 48(4) 36(4) 29(3) 1(3) 18(3) 12(3)
C(53) 40(4) 41(4) 37(4) -12(3) 19(3) -3(3)
C(54) 40(4) 43(4) 33(3) -5(3) 12(3) 4(3)
C(55) 46(4) 35(3) 27(3) -4(3) 11(3) 9(3)
C(56) 41(4) 35(3) 27(3) -4(3) 16(3) 3(3)
C(57) 38(3) 30(3) 35(3) -6(3) 9(3) -2(3)
C(58) 48(4) 39(4) 28(3) 0(3) 9(3) 4(3)
C(59) 42(3) 34(3) 29(3) -2(3) 6(3) 3(3)
C(60) 36(3) 33(3) 31(3) -9(3) 12(3) -6(3)
C(61) 47(4) 44(4) 27(3) 5(3) 12(3) 0(3)
C(62) 73(5) 51(4) 51(4) 0(4) 37(4) 14(4)
C(63) 86(5) 42(4) 64(5) 6(4) 51(4) 9(4)
C(64) 57(4) 36(4) 41(4) -2(3) 25(3) 4(3)
C(65) 54(4) 36(4) 51(4) -1(3) 32(4) 1(3)
C(66) 103(6) 28(4) 73(5) 12(3) 65(5) 17(4)
C(67) 85(5) 35(4) 55(4) 6(3) 43(4) 6(4)
C(68) 47(4) 36(4) 36(4) 2(3) 22(3) 4(3)
C(69) 48(4) 39(4) 38(4) 2(3) 16(3) -7(3)
C(70) 41(4) 56(4) 43(4) -7(4) 12(3) -5(3)
C(71) 45(4) 65(5) 40(4) -19(4) 17(3) -11(4)
C(72) 40(3) 44(4) 30(3) 2(3) 14(3) -6(3)
C(73) 65(5) 49(4) 45(4) 10(3) 26(4) -2(4)
C(74) 44(4) 42(4) 34(3) -3(3) 14(3) -5(3)
C(75) 43(4) 46(4) 37(3) -3(3) 11(3) -6(3)
C(76) 57(4) 36(4) 43(4) 2(3) 15(3) -2(3)
C(77) 49(4) 47(4) 32(3) -4(3) 18(3) -14(3)
C(78) 77(5) 81(6) 28(4) -9(4) 11(4) -22(5)
C(79) 71(5) 81(6) 64(5) 17(4) 49(4) 6(4)
C(80) 61(4) 39(4) 30(4) 2(3) 16(3) 13(3)
C(81) 58(4) 47(4) 33(4) 9(3) 22(3) 16(3)
C(82) 55(4) 45(4) 31(4) -2(3) 15(3) 5(3)
C(83) 67(4) 38(4) 27(3) 13(3) 10(3) 15(3)
C(84) 67(5) 59(5) 68(5) 15(4) 15(4) 26(4)
C(85) 80(5) 53(5) 52(4) 8(4) 20(4) 3(4)
344
C(86) 85(5) 46(4) 40(4) 6(3) 5(4) 2(4)
C(87) 56(4) 43(4) 33(4) -2(3) 7(3) 1(3)
C(88) 58(4) 44(4) 39(4) 6(3) 12(3) 4(3)
C(89) 58(5) 50(5) 41(4) -2(4) 17(4) 5(4)
C(90) 67(5) 56(5) 60(5) 22(4) 25(4) -4(4)
C(91) 85(6) 74(6) 76(6) 32(5) 27(5) -10(5)
C(92) 62(5) 75(6) 81(6) -6(5) 37(5) -10(4)
C(93) 88(6) 46(5) 94(7) 2(5) 39(5) -12(4)
C(94) 57(4) 49(4) 26(3) -3(3) 12(3) -3(3)
C(95) 100(6) 69(6) 37(4) 9(4) 20(4) 0(5)
C(96) 154(9) 69(6) 32(4) -13(4) 34(5) -46(6)
C(97) 85(6) 136(9) 42(5) -33(6) 32(4) -57(6)
C(98) 101(10) 420(30) 201(17) -190(20) 54(10) -93(15)
C(99) 330(19) 72(7) 58(7) 7(5) 103(10) -23(9)
C(100) 98(7) 109(8) 47(5) 8(5) 36(5) 16(6)
O(15) 39(3) 87(4) 69(4) -24(3) 12(3) 10(3)
C(101) 63(5) 73(6) 135(9) -18(6) 36(6) 20(5)
C(102) 82(6) 77(6) 136(10) -32(7) 27(6) 8(5)
O(16) 46(3) 59(3) 84(4) 13(3) 26(3) 6(2)
C(103) 51(5) 59(6) 320(20) 88(10) 55(8) 19(5)
C(104) 191(15) 104(11) 197(16) -2(11) -39(13) 20(11)
______________________________________________________________________________
345
Table II.4 Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) of 3EUr-α-cholestane.
________________________________________________________________________________
x y z U(eq)
________________________________________________________________________________
H(1A) 2022 6720 1654 49
H(2A) 2971 5955 2142 49
H(3B) 7080 6946 1612 45
H(4C) 8037 7573 2145 44
H(3A) 5253 6035 2215 49
H(4A) 4252 4428 2286 54
H(4B) 5279 4620 2621 54
H(5A) 3752 4043 2890 48
H(5B) 2972 4936 2676 48
H(7A) 2429 5648 3237 39
H(8A) 3657 4182 3738 48
H(8B) 2787 3891 3366 48
H(9A) 1993 3669 3932 41
H(9B) 1287 4478 3647 41
H(11A) 471 5499 4092 44
H(12A) 619 6986 4415 56
H(12B) 1596 6561 4738 56
H(13A) 1945 7728 4134 52
H(13B) 2918 7138 4417 52
H(14A) 1618 6339 3741 44
H(15A) 3994 6123 3901 43
H(16A) 2715 7634 3419 54
H(16B) 3654 7885 3778 54
H(17A) 5060 7293 3458 56
H(17B) 4329 8126 3190 56
H(18A) 3333 6770 2847 44
H(19A) 4877 7476 2610 51
H(19B) 5682 6612 2834 51
H(20A) 5128 4445 3463 66
H(20B) 5407 5633 3580 66
H(20C) 5744 5175 3196 66
346
H(21A) 3795 4774 4324 66
H(21B) 2952 4317 4586 66
H(21C) 3298 5523 4616 66
H(22A) 1210 4810 4856 45
H(23A) 1405 3359 4460 61
H(23B) 150 3513 4247 61
H(23C) 379 3169 4685 61
H(24A) -509 5867 4655 50
H(24B) -1029 4837 4445 50
H(25A) -1704 4648 4984 55
H(25B) -549 4042 5106 55
H(26A) -902 6162 5281 57
H(26B) 245 5553 5404 57
H(27A) -644 4478 5809 55
H(28A) -2409 4990 5953 91
H(28B) -2521 4655 5515 91
H(28C) -2510 5865 5628 91
H(29A) 375 5941 6060 94
H(29B) -650 5730 6284 94
H(29C) -727 6646 5975 94
H(30A) 790 7319 1136 55
H(30B) 555 7871 1518 55
H(31A) 855 9237 926 52
H(31B) -263 8555 852 52
H(34A) 494 11841 1687 139
H(34B) -356 11073 1851 139
H(34C) -653 12285 1791 139
H(35A) -2416 11080 988 109
H(35B) -2486 11821 1346 109
H(35C) -2206 10606 1410 109
H(36A) -1036 12177 762 114
H(36B) 143 12404 1022 114
H(36C) -958 12988 1107 114
H(37A) 2573 8804 921 57
H(37B) 3616 8238 1169 57
H(38A) 2749 6602 966 79
347
H(38B) 1860 7249 677 79
H(41A) 5330 7020 213 182
H(41B) 5736 8054 447 182
H(41C) 5901 7947 11 182
H(42A) 3644 7014 -255 106
H(42B) 4053 7939 -502 106
H(42C) 2894 8039 -343 106
H(43A) 3525 9722 -37 119
H(43B) 4749 9661 -150 119
H(43C) 4603 9757 288 119
H(44A) 3244 9305 1660 45
H(44B) 2035 9737 1486 45
H(45A) 1292 9089 2018 51
H(45B) 2482 8599 2190 51
H(48A) 3886 11771 2576 108
H(48B) 4527 10879 2840 108
H(48C) 4177 11941 3026 108
H(49A) 1934 12051 2549 197
H(49B) 2053 12272 2996 197
H(49C) 1225 11379 2805 197
H(50A) 3607 9929 3288 148
H(50B) 2285 10024 3275 148
H(50C) 3092 10938 3462 148
H(53A) 10299 7626 2154 46
H(54A) 10893 6987 2758 46
H(54B) 10143 6096 2528 46
H(55A) 8625 6506 2838 43
H(55B) 9694 6299 3155 43
H(57A) 7594 7591 3212 41
H(58A) 8556 6269 3572 46
H(58B) 9231 7098 3856 46
H(59A) 7798 6390 4144 42
H(59B) 6886 6696 3785 42
H(61A) 5691 7809 4123 46
H(62A) 6460 9607 4550 66
H(62B) 5345 9473 4242 66
348
H(63A) 6278 10172 3796 72
H(63B) 7432 10173 4093 72
H(64A) 6485 8492 3622 52
H(65A) 8762 9126 3713 53
H(66A) 7687 10351 3345 75
H(66B) 6998 9464 3094 75
H(67A) 9279 10028 3068 66
H(67B) 8249 10170 2729 66
H(68A) 8052 8337 2664 45
H(69A) 10304 8922 2618 49
H(69B) 9259 9137 2291 49
H(70A) 10890 8243 3183 69
H(70B) 10562 7728 3560 69
H(70C) 10231 8914 3455 69
H(71A) 9108 8162 4296 73
H(71B) 8471 7813 4637 73
H(71C) 8380 8992 4487 73
H(72A) 6923 7911 4884 44
H(73A) 7121 6244 4623 77
H(73B) 5792 6129 4522 77
H(73C) 6386 6166 4957 77
H(74A) 5188 8719 4861 47
H(74B) 4557 7770 4628 47
H(75A) 5617 7589 5399 50
H(75B) 4873 6703 5163 50
H(76A) 3295 7894 5109 54
H(76B) 4056 8663 5394 54
H(77A) 3425 6605 5593 50
H(78A) 5145 7111 5965 93
H(78B) 4192 6979 6225 93
H(78C) 4554 8120 6109 93
H(79A) 1896 7762 5514 101
H(79B) 2558 8517 5831 101
H(79C) 2192 7375 5946 101
H(80A) 7031 3886 1488 51
H(80B) 8219 4344 1676 51
349
H(81A) 7274 5215 2144 53
H(81B) 6173 4573 1976 53
H(84A) 8909 2217 2658 96
H(84B) 9393 2270 3103 96
H(84C) 9655 3191 2827 96
H(85A) 6953 2038 2770 91
H(85B) 6424 2909 3010 91
H(85C) 7298 2083 3223 91
H(86A) 8919 4433 3257 86
H(86B) 8490 3557 3521 86
H(86C) 7628 4388 3304 86
H(87A) 5838 6335 1113 53
H(87B) 5575 5789 1492 53
H(88A) 4692 5139 840 56
H(88B) 5811 4463 877 56
H(91A) 5592 1767 1622 115
H(91B) 4467 1263 1723 115
H(91C) 4718 2478 1799 115
H(92A) 2640 2495 942 105
H(92B) 2848 2943 1367 105
H(92C) 2604 1726 1294 105
H(93A) 5209 1163 981 110
H(93B) 4114 1517 697 110
H(93C) 4024 657 1017 110
H(94A) 8612 5252 1151 52
H(94B) 7528 4757 901 52
H(95A) 7848 6934 945 81
H(95B) 6969 6317 644 81
H(98A) 10836 5078 458 354
H(98B) 10565 6246 313 354
H(98C) 11034 5417 41 354
H(99A) 8207 5547 -377 218
H(99B) 9450 5709 -465 218
H(99C) 8915 6548 -213 218
H(10A) 9664 3695 227 123
H(10B) 9676 3917 -215 123
350
H(10C) 8516 3899 -54 123
H(15B) 788 5572 1976 97
H(10D) 770 3973 1826 106
H(10E) 2112 3990 1877 106
H(10F) 1344 4071 1183 146
H(10G) 2009 5130 1302 146
H(10H) 673 5111 1252 146
H(16C) 5851 7851 1929 91
H(10I) 5897 9228 1614 170
H(10J) 7169 9424 1821 170
H(10K) 5879 10585 2190 257
H(10L) 6415 9698 2479 257
H(10M) 5161 9576 2260 257
________________________________________________________________________________
Table II.5 Torsion angles [°] of 3EUr-α-cholestane.
________________________________________________________________
C(2)-N(1)-C(1)-C(37) 69.1(7)
C(2)-N(1)-C(1)-C(44) -50.8(7)
C(2)-N(1)-C(1)-C(30) -171.0(5)
C(3)-N(2)-C(2)-O(1) -4.0(9)
C(3)-N(2)-C(2)-N(1) 177.3(5)
C(1)-N(1)-C(2)-O(1) -12.3(9)
C(1)-N(1)-C(2)-N(2) 166.4(5)
C(2)-N(2)-C(3)-C(19) 78.8(7)
C(2)-N(2)-C(3)-C(4) -158.2(5)
N(2)-C(3)-C(4)-C(5) -72.1(7)
C(19)-C(3)-C(4)-C(5) 51.4(8)
C(3)-C(4)-C(5)-C(6) -54.8(8)
C(4)-C(5)-C(6)-C(7) 172.5(5)
C(4)-C(5)-C(6)-C(20) -65.1(7)
C(4)-C(5)-C(6)-C(18) 54.8(7)
C(5)-C(6)-C(7)-C(8) 61.3(7)
C(20)-C(6)-C(7)-C(8) -59.8(7)
C(18)-C(6)-C(7)-C(8) 178.2(5)
C(5)-C(6)-C(7)-C(15) -170.7(5)
C(20)-C(6)-C(7)-C(15) 68.2(7)
C(18)-C(6)-C(7)-C(15) -53.8(7)
C(15)-C(7)-C(8)-C(9) 53.2(7)
C(6)-C(7)-C(8)-C(9) -176.7(5)
C(7)-C(8)-C(9)-C(10) -53.9(7)
C(8)-C(9)-C(10)-C(11) 162.5(5)
C(8)-C(9)-C(10)-C(14) 52.4(7)
C(8)-C(9)-C(10)-C(21) -69.7(6)
C(9)-C(10)-C(11)-C(12) -154.9(5)
C(14)-C(10)-C(11)-C(12) -41.1(6)
C(21)-C(10)-C(11)-C(12) 77.4(6)
C(9)-C(10)-C(11)-C(22) 78.5(7)
C(14)-C(10)-C(11)-C(22) -167.7(5)
C(21)-C(10)-C(11)-C(22) -49.3(8)
C(10)-C(11)-C(12)-C(13) 20.4(7)
C(22)-C(11)-C(12)-C(13) 153.8(5)
C(11)-C(12)-C(13)-C(14) 9.1(7)
351
C(12)-C(13)-C(14)-C(10) -35.2(6)
C(12)-C(13)-C(14)-C(15) -165.1(6)
C(11)-C(10)-C(14)-C(13) 47.5(6)
C(9)-C(10)-C(14)-C(13) 169.4(5)
C(21)-C(10)-C(14)-C(13) -69.7(6)
C(11)-C(10)-C(14)-C(15) -179.9(5)
C(9)-C(10)-C(14)-C(15) -58.1(7)
C(21)-C(10)-C(14)-C(15) 62.9(7)
C(8)-C(7)-C(15)-C(16) -177.1(5)
C(6)-C(7)-C(15)-C(16) 52.1(7)
C(8)-C(7)-C(15)-C(14) -53.7(6)
C(6)-C(7)-C(15)-C(14) 175.5(5)
C(13)-C(14)-C(15)-C(7) -175.2(5)
C(10)-C(14)-C(15)-C(7) 60.4(7)
C(13)-C(14)-C(15)-C(16) -51.1(7)
C(10)-C(14)-C(15)-C(16) -175.5(5)
C(7)-C(15)-C(16)-C(17) -50.2(7)
C(14)-C(15)-C(16)-C(17) -172.2(5)
C(15)-C(16)-C(17)-C(18) 53.5(7)
C(16)-C(17)-C(18)-C(19) 173.0(5)
C(16)-C(17)-C(18)-C(6) -58.1(7)
C(7)-C(6)-C(18)-C(17) 56.5(7)
C(5)-C(6)-C(18)-C(17) 176.4(5)
C(20)-C(6)-C(18)-C(17) -65.3(6)
C(7)-C(6)-C(18)-C(19) -176.3(5)
C(5)-C(6)-C(18)-C(19) -56.4(7)
C(20)-C(6)-C(18)-C(19) 62.0(7)
N(2)-C(3)-C(19)-C(18) 70.0(7)
C(4)-C(3)-C(19)-C(18) -51.8(7)
C(17)-C(18)-C(19)-C(3) -175.0(5)
C(6)-C(18)-C(19)-C(3) 57.0(7)
C(10)-C(11)-C(22)-C(23) -47.6(8)
C(12)-C(11)-C(22)-C(23) -171.0(5)
C(10)-C(11)-C(22)-C(24) -168.4(6)
C(12)-C(11)-C(22)-C(24) 68.2(7)
C(23)-C(22)-C(24)-C(25) 76.7(7)
C(11)-C(22)-C(24)-C(25) -160.9(5)
C(22)-C(24)-C(25)-C(26) 89.4(7)
C(24)-C(25)-C(26)-C(27) 179.6(6)
C(25)-C(26)-C(27)-C(29) 173.0(6)
C(25)-C(26)-C(27)-C(28) -64.3(8)
N(1)-C(1)-C(30)-C(31) 176.0(5)
C(37)-C(1)-C(30)-C(31) -64.9(7)
C(44)-C(1)-C(30)-C(31) 56.0(7)
C(1)-C(30)-C(31)-C(32) -96.9(7)
C(33)-O(3)-C(32)-O(2) -3.3(10)
C(33)-O(3)-C(32)-C(31) 176.5(6)
C(30)-C(31)-C(32)-O(2) -29.3(10)
C(30)-C(31)-C(32)-O(3) 150.9(6)
C(32)-O(3)-C(33)-C(35) -63.9(8)
C(32)-O(3)-C(33)-C(36) 178.4(6)
C(32)-O(3)-C(33)-C(34) 62.3(9)
N(1)-C(1)-C(37)-C(38) 59.5(8)
C(44)-C(1)-C(37)-C(38) -178.4(6)
C(30)-C(1)-C(37)-C(38) -55.1(8)
C(1)-C(37)-C(38)-C(39) -170.1(6)
C(40)-O(5)-C(39)-O(4) -2.7(16)
C(40)-O(5)-C(39)-C(38) -176.0(7)
C(37)-C(38)-C(39)-O(4) 148.9(9)
C(37)-C(38)-C(39)-O(5) -37.5(11)
C(39)-O(5)-C(40)-C(42) 64.7(10)
C(39)-O(5)-C(40)-C(43) -176.8(8)
C(39)-O(5)-C(40)-C(41) -56.8(12)
N(1)-C(1)-C(44)-C(45) -44.8(8)
C(37)-C(1)-C(44)-C(45) -166.4(6)
C(30)-C(1)-C(44)-C(45) 70.3(7)
C(1)-C(44)-C(45)-C(46) 177.2(6)
C(47)-O(7)-C(46)-O(6) 5.9(10)
C(47)-O(7)-C(46)-C(45) -175.4(5)
C(44)-C(45)-C(46)-O(6) 24.9(10)
C(44)-C(45)-C(46)-O(7) -153.8(5)
C(46)-O(7)-C(47)-C(48) -64.3(8)
352
C(46)-O(7)-C(47)-C(49) 59.1(9)
C(46)-O(7)-C(47)-C(50) 179.8(6)
C(52)-N(3)-C(51)-C(87) 170.9(5)
C(52)-N(3)-C(51)-C(94) -68.5(7)
C(52)-N(3)-C(51)-C(80) 49.7(7)
C(53)-N(4)-C(52)-O(8) 7.3(9)
C(53)-N(4)-C(52)-N(3) -172.4(5)
C(51)-N(3)-C(52)-O(8) 20.2(9)
C(51)-N(3)-C(52)-N(4) -160.0(5)
C(52)-N(4)-C(53)-C(54) -78.8(6)
C(52)-N(4)-C(53)-C(69) 160.2(5)
N(4)-C(53)-C(54)-C(55) -66.9(6)
C(69)-C(53)-C(54)-C(55) 52.7(7)
C(53)-C(54)-C(55)-C(56) -55.0(7)
C(54)-C(55)-C(56)-C(70) -67.2(6)
C(54)-C(55)-C(56)-C(57) 170.6(5)
C(54)-C(55)-C(56)-C(68) 54.3(7)
C(70)-C(56)-C(57)-C(58) -66.3(7)
C(55)-C(56)-C(57)-C(58) 55.0(7)
C(68)-C(56)-C(57)-C(58) 171.7(5)
C(70)-C(56)-C(57)-C(65) 61.7(7)
C(55)-C(56)-C(57)-C(65) -177.0(5)
C(68)-C(56)-C(57)-C(65) -60.3(7)
C(56)-C(57)-C(58)-C(59) -180.0(5)
C(65)-C(57)-C(58)-C(59) 51.6(7)
C(57)-C(58)-C(59)-C(60) -55.9(7)
C(58)-C(59)-C(60)-C(71) -66.8(6)
C(58)-C(59)-C(60)-C(64) 56.1(6)
C(58)-C(59)-C(60)-C(61) 165.2(5)
C(71)-C(60)-C(61)-C(62) 76.0(6)
C(59)-C(60)-C(61)-C(62) -156.1(6)
C(64)-C(60)-C(61)-C(62) -43.2(6)
C(71)-C(60)-C(61)-C(72) -49.9(7)
C(59)-C(60)-C(61)-C(72) 78.0(7)
C(64)-C(60)-C(61)-C(72) -169.1(6)
C(72)-C(61)-C(62)-C(63) 151.7(6)
C(60)-C(61)-C(62)-C(63) 23.3(7)
C(61)-C(62)-C(63)-C(64) 6.5(8)
C(62)-C(63)-C(64)-C(65) -163.0(6)
C(62)-C(63)-C(64)-C(60) -34.3(7)
C(71)-C(60)-C(64)-C(65) 61.5(7)
C(59)-C(60)-C(64)-C(65) -59.4(7)
C(61)-C(60)-C(64)-C(65) 178.5(6)
C(71)-C(60)-C(64)-C(63) -69.0(6)
C(59)-C(60)-C(64)-C(63) 170.1(5)
C(61)-C(60)-C(64)-C(63) 48.1(6)
C(63)-C(64)-C(65)-C(66) -57.2(9)
C(60)-C(64)-C(65)-C(66) 179.7(6)
C(63)-C(64)-C(65)-C(57) -179.0(6)
C(60)-C(64)-C(65)-C(57) 57.9(8)
C(58)-C(57)-C(65)-C(66) -174.0(6)
C(56)-C(57)-C(65)-C(66) 56.5(7)
C(58)-C(57)-C(65)-C(64) -51.1(7)
C(56)-C(57)-C(65)-C(64) 179.4(5)
C(64)-C(65)-C(66)-C(67) -173.3(6)
C(57)-C(65)-C(66)-C(67) -51.8(9)
C(65)-C(66)-C(67)-C(68) 52.0(9)
C(66)-C(67)-C(68)-C(69) 178.2(6)
C(66)-C(67)-C(68)-C(56) -55.7(8)
C(70)-C(56)-C(68)-C(69) 64.2(6)
C(55)-C(56)-C(68)-C(69) -56.0(7)
C(57)-C(56)-C(68)-C(69) -174.3(5)
C(70)-C(56)-C(68)-C(67) -61.7(6)
C(55)-C(56)-C(68)-C(67) 178.1(5)
C(57)-C(56)-C(68)-C(67) 59.8(7)
C(67)-C(68)-C(69)-C(53) -176.0(6)
C(56)-C(68)-C(69)-C(53) 58.3(7)
N(4)-C(53)-C(69)-C(68) 67.4(7)
C(54)-C(53)-C(69)-C(68) -55.1(7)
C(62)-C(61)-C(72)-C(73) 177.5(6)
C(60)-C(61)-C(72)-C(73) -61.7(8)
C(62)-C(61)-C(72)-C(74) 54.1(7)
353
C(60)-C(61)-C(72)-C(74) 174.8(5)
C(73)-C(72)-C(74)-C(75) 50.5(7)
C(61)-C(72)-C(74)-C(75) 175.4(5)
C(72)-C(74)-C(75)-C(76) 174.4(5)
C(74)-C(75)-C(76)-C(77) 171.7(5)
C(75)-C(76)-C(77)-C(79) -174.6(6)
C(75)-C(76)-C(77)-C(78) 61.0(8)
N(3)-C(51)-C(80)-C(81) 49.3(8)
C(87)-C(51)-C(80)-C(81) -66.7(7)
C(94)-C(51)-C(80)-C(81) 169.7(6)
C(51)-C(80)-C(81)-C(82) -170.5(6)
C(83)-O(10)-C(82)-O(9) -1.7(10)
C(83)-O(10)-C(82)-C(81) 177.4(5)
C(80)-C(81)-C(82)-O(9) -39.5(10)
C(80)-C(81)-C(82)-O(10) 141.4(6)
C(82)-O(10)-C(83)-C(84) 59.7(8)
C(82)-O(10)-C(83)-C(86) 178.7(5)
C(82)-O(10)-C(83)-C(85) -64.9(7)
N(3)-C(51)-C(87)-C(88) -179.1(5)
C(94)-C(51)-C(87)-C(88) 61.7(7)
C(80)-C(51)-C(87)-C(88) -58.8(7)
C(51)-C(87)-C(88)-C(89) 91.6(7)
C(90)-O(12)-C(89)-O(11) 0.4(11)
C(90)-O(12)-C(89)-C(88) -176.8(6)
C(87)-C(88)-C(89)-O(11) 33.7(10)
C(87)-C(88)-C(89)-O(12) -149.0(6)
C(89)-O(12)-C(90)-C(93) -178.0(7)
C(89)-O(12)-C(90)-C(92) 65.3(9)
C(89)-O(12)-C(90)-C(91) -60.6(9)
N(3)-C(51)-C(94)-C(95) -56.8(8)
C(87)-C(51)-C(94)-C(95) 58.7(8)
C(80)-C(51)-C(94)-C(95) -177.6(6)
C(51)-C(94)-C(95)-C(96) 166.8(7)
C(97)-O(14)-C(96)-O(13) 1.5(17)
C(97)-O(14)-C(96)-C(95) -178.7(7)
C(94)-C(95)-C(96)-O(13) -138.2(12)
C(94)-C(95)-C(96)-O(14) 42.0(11)
C(96)-O(14)-C(97)-C(100) 177.6(8)
C(96)-O(14)-C(97)-C(98) 55.9(16)
C(96)-O(14)-C(97)-C(99) -66.8(10)
______________________________________
Table II.6 Hydrogen bonds [Å and °] of 3EUr-α-cholestane.
____________________________________________________________________________
D-H...A d(D-H) d(H...A) d(D...A) <(DHA)
____________________________________________________________________________
O(16)-H(16C)...O(1) 0.84 1.82 2.621(7) 158.1
O(15)-H(15B)...O(8)#1 0.84 1.82 2.625(6) 161.0
N(4)-H(4C)...O(16) 0.88 2.04 2.865(7) 155.7
N(3)-H(3B)...O(16) 0.88 2.30 3.035(7) 141.6
N(2)-H(2A)...O(15) 0.88 2.01 2.849(8) 159.6
N(1)-H(1A)...O(15) 0.88 2.32 3.084(8) 146.0
____________________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 x-1,y,z
354
VITA
Eko Winny Sugandhi was born in Jepara, Jawa Tengah, Indonesia on Oct 8, 1973, to
Sugandhi and Henny Susanti. She attended Gadjah Mada University in Yogyakarta,
where she earned her B.S. equivalent in chemistry in 1996. She came to the US in 1997
to pursue her M.S. degree in chemistry with Dr. Arthur S. Howard at Eastern Michigan
University, Ypsilanti MI. This was the very first time she enjoyed teaching, and
eventually decided to pursue her career in academia. Upon completion of her M.S.
degree, she entered the chemistry graduate program at Virginia Polytechnic Institute and
State University in 2000. She finally obtained her M.S. degree in chemistry in 2002. At
Virginia Polytechnic Institute and State University, she joined the research group of Dr.
Richard D. Gandour. She received her Doctorate degree in chemistry in 2007. She has
been a wife of Ernest Lada since 2002 and a mother of Stanley Lada since 2006.