The Study of &Cyciodextrin Interactions Sugars Using "Inhibition Kinetics" And The Brornination of Pyrimidine Derivatives

Isabelle Turner

A Thesis

in

The Department

of

Chemistry and Biochemistry

Presenïed in Parüal Fuifilment of the Requirements for the Degree of Master of Scienœ at

Concordia University Montréal, Québec, Canada

August 1 999

O Isabelle Turner, 1999

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ABSTRACT

The Study of fbCyclodextrin Interactions with Sugars Using Ynhibiüon Kinetics"

And The Bromination of Pyrimidine Derivatives

Isabelle Turner

PART 1: The binding of &cyclodexîrin @CD) with sugars was studied using

"inhibition kinetics". The rates of hydroiysis of trimethyl orthobenzoate and

benzaldehyde dimethyl acetal in acidic solution were monitoreâ using a single

beam stopped-flow spectrophotometer. Both substrates exhiba saturation kinetics

and 1:1 binding with &CD, and the rates of hydrolysis are retarded by the

cyclodextn'n, in agreement with earlier studies. The retarded rates of hydrolysis in

the presence of PCD are modulated by the addition of guests that bind to the CD,

and "in hibition constants" (KI) may be determined. Several aldoses were studied.

Pentoses were found to bind modestly to PCD (K, = 100 - 1 O00 mM) and hexoses

hardly bind at al1 (K, > 500 mM). The results obtained with the two probe reacüons

agree well with each other, and the K, values are compared to the disparate values

in literature.

PART II: The aqueous bromination of 2-aminopyrimidine (2-AP) involves two

major steps. and the first step fons an addition product, an intemediate that was

previously observed by NMR. The second step, which is quite slow. involves

iii

elimination of water from the intermediate to yield the Sbromo derivath The focus

of this investigation was on the initial fast step to try to discover how the

in termediate is formed. The kinetics of the bromination of 2-aminopwmidine and

several related derivathes have been studied using a stopped-flw apparatus. The

mechanisms were investigated by constnicting pH-rate profiles for each mpound.

The pH-rate profiles for 2-amino pyrimidine and 1.2-d ihydro-2-imino-1 -methyl-

pyrimidine (M2A) dernonstrate the complexity of these reactions and the evidenœ

that more than one mechanism is invoived. This statement is especial true for the

latter compound where mixed orders of readions were found. For comparative

purposes the kinetics of bromination of 2-amino4,6dimethyl-pyrimidine (2ADP).

2-aminopyridine (2-APy). 2dimethylaminopyridine (2-DAPy), aniline and N, N-

dimethylaniline were also sstdied.

I would like to thank my supeMsor Professor Oswald S. Tee for his patience

and his support. I am grateful for his teachings of both life and science. Thank you

for being such a good pedagogue and above al1 a patient English teacher.

I am appreciative of Dr. Timothy A Gadosy and Dr. Joanne Tumbull for

readily accepting to be on my cornmittee, even if they knew what they were getting

into.

I am pleased to have met and exchanged with a large number of fellow

students in the Department of Chemistry and Biochernistiy. Several have become

friends and I h o p to keep in touch. Special thanks to Alexei A. Fedortchenko,

Delna Ghadiali, Michael John Boyd, Lee Fadet, Araz Jakalian, Michael Harvey and

Donald Paquette. Good luck to Paul Loncke, Ogaritte Jennifer Yazbeck, and Dons

Badaan in reaching their acadernic goals.

Finally I could not have cornpleted this thesis without the support and love

of rny family and friands.

=Tout ce que l'homme pouvait gagner au jeu de la peste et de la vie, c'&ait la connaissance et la mémoire..

Albert Camus La Peste

vii

Table of Contenîs

................................................... List of Figures xi

................................................ List of Schemes xiv

ListofTables .................................................. xvi

............................................. List of Abbreviations xix

PART I The Stuw of ~Cyclodextn'n lnteracions wrah Sugats Using "hhibifrbn

Nnetrcs"

4-Introduction ................................................... 2

1 . 1 Supramolecular Chemistry and Molecular Recognition ........... 2

........................................... 1.2 Cyclodextrins 4

1.2.1 Stmcture, Chernical and Physical Properties ............ 5

1.2.2 Inclusion Complexes and Kinetics .................... 6

1.2.3 Effect of CDS on reactions ......................... 11

1.3 Probe Reactions, Hydrolysis of TM06 and BDMA ............. 11

1.4 Alcohols as Guests ..................................... 17

1.5Sugarsasguests ....................................... 17

2.Results&Discussion .......................................... 19

2.1 Effects of guests ....................................... 19

........................................ 2.2 Sugars structure 28

3. Conclusion .................................................. 37

................................................. 4.Expenmental 38

4.1Materiats ............................................. 38

viii

4.2 Kinetic Experiments ..................................... 38

4.3 Calculation of Rate Constants ............................. 39

....................... 4.4 Calculation of Dissociation Constants 39

PART N The Bmmination of Pyrimidine Derivatives

5.lntroduction .................................................. 42

5.1 Pyrimidine Structure and Properbies ........................ 42

................ 5.1.1 Bromination of Pyrimidine Derivatives -44

............ 5.1 -1 -1 Brornination of 2-Aminopyrimidine 44

5.f . 1 -2 Bromination of 1.2dihydro-2-imino-1-

methylpyrimidine ................................ 45

5.1 -1 -3 Bromination of 2-Amino4.6-dimethylpfimidine . . 45

5.2 Pyridine Structure and Properties .......................... 46

.................. 5.2.1 Bromination of Pyridine Derivatives 47

.............................. 5.2.1.1 Bromination of 2-Arninopyridine 47

....... 5.2.1.2 Brornination of 2-Dirnethylaminopyridine 48

5.3 Aniline Structure and Properties ........................... 49

5.3. f Bromination of Aniline ............................ 50

.................. 5.3.2 Bromination of N, KDirnethylaniline 51

........................................ 5.4 Simple Kinetics 51

.......................................... 6.Results&Discussion 54

................................... 6.1 Bromination of Aniline 54

6.2 Bromination of Arninopyridine ............................. 57

.......................... 6.3 Bromination of Aminopyridimidine 61

ix

7.Conclusion .................................................. 80

8.mperimental ................................................. 81

8.1 Materials ............................................. 81

8.2 Kinetic Experiments ..................................... 81

............................ 8.3 Calculations of Rate Constants 83

......................................... 8.4p&€stimation 84

8.5 Observation of the Bromination of 2AP Intemediate by 'H NMR . . 84

9. References .................................................. 87

10.AppendixA ................................................. 93

1tAppendixB ................................................. 98

List of Figures

Figure 1.1

Figure 1 -2

Figure 1.3

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Catalysis of the Cleavage of nNitrophenyl Alkanoate by B-CD. 12

.. Retardation of the Hydrolysis of TMOB in 0.1 M HCI by $40. 12

Retardation of the Hydrolysis of TM08 and BDMA in 0.1 M HCI

byf3GD. ........................................... 14

pK, estimated values from this work yg literature pK, values from

~ o y a m a et al.'' ...................................... 25

pK, estimated values from this work literature pKl values from

Hacket et al." ....................................... 25

pK, estimated values from this work for benzaldehyde dimethyl acetal

vs pKl estimated values for Acetophenone dimethyl acetal. .... 26 - pK, estimated values from this work for benzaldehyde dimethyl acetal

vs pK, estimated values from this work for Tnmethyl orthobenzoate. - .................................................. 26

pK, estimated values for Acetophenone dimethyl acetal 1 ~ s pK,

. . estimated values from this work for Trimeth yl orthobenzoate. 27

AG0 values from the literature AG0 averaged values of BDMA and

TMOB from this work. ................................. 27

bb. scaled according to the k, value for the hydrolysis of TM06 in the

presence of 1 mM of B-CD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

bb scaled according to the value for the hydrolysis of BDMA in the

presence of 5 mM of BGD. ............................. 30

xi

Figure 2.9

Figure 2.1 0

Figure 2.1 1

t

Figure 2.1 2

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Figure 6.8

scaled according to the k, value for the hydroiysis of TM06 in the

presence of 1 mM of B-CD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

scaled acwrding to the k,, value for the hydrolysis of BDMA in the

presenceof5 mM of P-CD. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 31 scaled according to the value for the hydrolysis of TM08 in the

presence of 1 mM of p-CD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

scaled according to the Id value for the hydrolysis of BDMA in the

presence of 5 mM of PGD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

pH rate profile for the bromination of N,Mimethylaniline and aniline.

............................. . . . . . W . . . . . . . . . . . . . . . 54

Hammett plot for the bromination of parasubstituted anilines.

Substihients: F, Br, CI. Me, NHAc, NH;. . . . . . . . . . . . . . . . . . . 56

pH rate profile for the bromination of 2-aminopyridine and 2-dimethyf-

aminopyridine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Hammett plot for the bromination of rnonosubstituted 2-Arninopyridine

and 2-Dimethylaminopyridine at 20°C. Substituents: Br, CI, NO,. 59

pH rate profile for the bromination of 2-amino4.6-dimethylpynmidine.

.................................................. 61

pH rate profile for the bromination of 2-aminopyrimidine. . . . . . 63

pH rate profile for the bromination of 1,2-dihydro-1 -imino-1 methyl

pyrimidine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Traces of (a) first-order kinetics, (b) rnixed-order kinetics and

xii

Figure 81.1.1

Figure 81.3.1

Figure B i .3.2

Figure B I .4.1

Figure 81.5.1

Figure 81.6.1

Figure 81.6.2

Figure 81.7.1

(c) zerwrder kinetics ............................ 71

The Dependence of k,* on the aniline concentration at a pH

of 1.14 for experiment IT-196M and a pH of 0.852 for

........... ............... experiment IT-197M.. ; 100

Dependence of kt* on the Paminopyndine concentration at

............. pH4.43 .................... .... 102

Dependence of the amplitude on the brornine concentration at

..... pH4.407 for the bromination of 2-aminopyndine 103

Dependence of k,Oh on the 2-dimethylaminopyridine

concentration al pHd.865 ....................... 1 05

Dependence of klob on the 2-amino4,6-dimethylpyrirnidine

concentration minus the bromine concentration at pH=1.429.

............................................ 106

Dependence of k,& on the 2-aminopyrimidine concentration

at pHd.852 for experiment IT-93M and a pH of 4.34 for

experiment lT-94M ............................. 108

Dependence of the amplitude on the bromine concentration at

pH=0.852 for the bromination of Barninopyrimidine . . . 109

Dependence of klobs on the 1 ,BdihydroQ-imino-l -

methylpyrimidine concentration minus the bromine

concentration at pH4.465 for experiment IT-124M and a pH

of 5.56 for expenment IT-125M ................... 1 1 1

xiii

Ust of Schemes

Scheme 1.1 Schematic Representation of a Molecular Complex . The Host molecule is

................... convergent and the Ouest molecule is divergent 3

Scheme 1.2 The Molecular dimensions of the Three Different Cyclodextrins (a.. p- and

y-CD) .................................................... 5

Scheme 1.3 Structure of an a-CD Molecule and a Schernatic Representation of the

Binding site of a CD Molecule .................................. 7

Scheme 5.1 Structure of Pyrimidine Molecule .............................. 42

Scheme 5.2 Equilibrium between Tautomeric forms of 2-AP i.e . the Amino and the lmino

. . . form and between the Hydroxy and the Ketone form of Pyrimidone 43

Scheme 5.3 Bromination of 2-AP in D,O to observe the intemiediate by NMR

spectroscopy . ............................................ 44

Scheme 5.4 The Bromination of M2A in D, O to observe a similar intermediate by NMR

............................................. spectroscopy 45

Scheme 5.5 Structure of 2.Amin04,6~dimethyIpyrimidine ..................... 46

Scheme 5.6 The structure of Pyridine and the partial and overall charge distribution . 47

Scheme 5.7 The bromination of 2.Aminopyridine ........................... 48

Scheme 5.8 The structura of 2.dimethylaminopyridine . . . . . . . . . . . . . . . . . . . . . . . 48

Scheme 5.9 Resonnance structures of Aniline . . ........................... 49

Scheme 5.10 Resonnace structures of an aryiamrnoniurn ion . . . . . . . . . . . . . . . . . . 50

Scheme 5 . 1 1 The bromination of Aniline ................................... 51

xiv

........................ Scheme 5.1 2 The bromination of N. N4imethyl aniline 51

....... Scheme 6.1 Reacüon of both fom of 2-AP with bromine in acidic solution. 64

........... Scheme 6.2 Formation of the intermediate in the bromination of 2.AP 65

Scheme 6.3 Bromine attack follawed by water attack and addition .............. 65

...... Scheme 6.4 Proposed mechanism for the bromination of 2.aminopyrimidine 67

Scheme 6.5 Suggested pathways for the bromination of 2-AP and M2A ......... 69

Scheme6.6 ........................................................ 72

Scheme6.7 ........................................................ 73

Scheme6.8 ........................................................ 75

Scheme 6.9 Proposed mechanism for the bromination of M2A ................ 79

Scheme B I Lotus 1-2-3 spreadsheet for the brornination of aniline ............ 113

List of Tables

. . ......... Table 1 0 Dissociation Constant (Ks) of PCD-guest Complexes 8

Table 1.1 Constants for the Hydrolysis of Acetals in the presenœ of

Cyclodextn'ns ........................................ 16

............ Table 1 -2 @CD host-guest dissociation constants. K, (mM) 18

Table 2.1 Dissociation constants for the @CD.guest} complexes ....... 19

Table 2.2 Correlation coefficients for the comparison of the pK, values -mated

using dafierant probe reactions .......................... 23

Table 2.3 Correlation coefficients for the comparison of the pK, values estimated

ta the pK, values from the literature ....................... 24

............................... Table 6.1 Bromination Parameters 55

Table 6.2 Hammett Equation Parameten for Aniline ................. 57

............................... Table 6.3 Bromination Parameters 58

. . . . . . . . Table 6.4 Hammett Equation Parameten for 2APy and 2.DAPy 59

Table 6.5 Bromination Parameters ............................... 62

............................... Table 6.6 Bromination Parameters 64

............................ Table 8.1 Parameters for Bromination 82

Table 8.2 H NMR Shifts ....................................... 86

Table 81 . 1 Raw Data for the Bromination ([Br& = 0.0250 mM) of Aniline (1 . 00

........................................ mM) at 25OC 99

Table 81.1 -1 Raw Data for the Dependenœ of k, * on [Aniline] using [Br& =

0.0250 r n ~ at 25OC ................................... 99

xvi

Table 81 .2 Raw Data for the Bromination ([Br& = 0.0250 mM) of N,Ndimethyl-

............................ aniline (0.50 mM) at 25OC 1 O0

Table 81.3 Raw Data for the Bromination of 2-Arninopyridine (1 .O0 mM) at 25OC

................................................. 101

Table 81.3.1 Raw Data for the Dependence of k,Ob on [2-Aminopyndine] using

[Br& = 0.05W mM at 25OC ............................ 102

Table 61 3.2 Raw Data for the dependence of the Amplitude on the [Br& at 25OC,

for the bromination of 2-Aminopyridine ................... 103

Table 81.4 Raw Data for the Bromination ( [ & J O = 0.0250 mM) of 2-dimethyl-

aminopyridine (1 -00 mM) a1 25% ....................... 104

Table 81.4.1 Raw Data for the Dependence of k,* on [2-dimethylaminopyridine]

using [BrJo = 0.0500 mM at 2S°C ....................... 104

Table 81.5 Raw Data for the Bromination of 2-Amino4,6-dimethylpyrimidine

(1.00 mM) at 25OC ................................... 1 05

Table 8 1 5 .1 Raw Data for the Dependence of ktobt on (2-amino-4,6-dimethyl-

pyrimidine] using [BrJ, = 0.0500 mM at 25OC .............. 1 06

Table 81.6 Raw data for the Bromination of 2-Aminopyrirndine (1 -00 mM at 25OC

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 7

Table 81.6.1 Raw Data for-the Dependence of k,Ob on [2-aminopyrimidine] using

[Br& = 0.0500 mM at 25OC ............................ 1 08

Table 81.6.2 Raw Data for the Dependence of the Amplitude on the [Br&, for the

Bromination of 2-aminopyrimidine at 25OC ...... .- ......... 109

Table BI .7 Raw Data for the bromination ([Br& = 0.0250 mM) of 1,2-dihydro-2-

xvii

imino-lniethyipyrimidine (0.250 mM) at 2S°C . . . . . . . . . . . . . 1 10

Table B 1.7.1 Raw Data for the Dependenœ of - kob. on [f ,2dihydro-2-irnino-1-

meti~yi~rnidine] using [Br&, = 0.0500 mM at 2S°C . . . . . . . . . 11 1

Table B i .8 Raw Data for the Bromination ([Br$ = 0.0250 mM) of Synthesued

1,2dihydro-2-imino-1-methylpyndmaine (0.250 mM) at 2S°C . 1 12

PART 1

The Stud of

1 In-

1.1 Supramolecukr Chemistry and Molecular Recognition

During the late sixües a new field was developed. an explosion in the

investigations of hghG structured complexes mth the goal of mimicking enzymes.

Supramolecular chemistry is the study of the fundons and the structures of

superniolecules which are formed during the associations of substrates and

recepton.' Many biological processes, such as substrate binding to an enzyme.

and enzymic cataiysis, exploit supramolecular properties.

Following the growing interest in supramolecular chemistry Cam and

coworkem in 1977, defined the ternis to be used in host-guest complexation.

Suprarnolecular complexes were defined as 'cornposed of two or more molecules

or ions held together in unique structural relationships by electrostatic forces other

than those of full covalent bonds."' The forces that are usualiy involved include Van

der Waals, hydrophobic, dipolar and polar interactions. Covalent bonds are

excluded since they lead to a molecule instead of a complex. A molecular wmplex

requires a minimum of one host and one guest component. The host is known as

'an organic molecule or ion whose binding sites converge in the complex."' The

guest is therefore 'any molecule or ion whose binding sites diverge in the

cornplex."' A simple representation of a molecular complex can be seen in Scheme

1.1.

Host Guest Complex

Scheme 1.1 Schematic Represenation of a Molecular Cornplex- The Host molecuîe is convergent and the Guest mofecule is divergent-

For a guest to bind to a potential host severaf requirements must be met:

both the host and the guest must camplement each other i-e., the geometry, size

and shape must allow appropriate interactions to occur; the host should have a

degree of flexibility and of rigidity to allow the access of the guest to the binding

site.' When interaction is made possible. the function of the supiamolecular

complex can be investigated. Funaional properties of a supermolecule include

catalysis, transport and rnolecular recognition.'

The main requirement of molecular recognition is that the receptor and its

substrates be in contact over a large area? In order to satisfy this requirernent

several factors play a key role. The first factor involves diffusion. Encounters

between a host and a guest occur through diffusion. If a complex is fomed upon

contact, the rate of cornplex formation is diffusioncontrolled. On the other hand. if

some adjustments to structure of the host andlor the guest are needed

(preorganisation), the rate of complexation will be ~ lower.~ When the surfaces of

both mofecules corne in contact. multiple weak bonds are fomed and w-ll hold until

random thermal motions lead to the dissociation of the mole~ules.~

3

Therefore the second fadar involves thermal motions. A general correlation

behveen binding Wngth and the rate of dissociation is the stronger the binding the

slower the rate of dissociation- In the case of a fast rate of dissociation, the total

energy of the bonds formed is negligible in contrast with that of thermal motion, the

molecules dissociate very rapidly. If the binding is strong. the bond energy is high

resulting in a slow rate of dissociation.' Preorganisation will play a major role in

detennining the binding powr,' this is because by adjusting their structures the

malecules enhance the fit and the number of noncovalent interactions.

Corn plementa rïty wïil be invohred in structural recognition ,' not only does it increase

the number of noncovalent bonds fomed, it also increases the area of contact,

satisfying the main requirement of molecular recognition.

The third and last factor involves molecular motion. All three kinds of

molecular motions are significant in bnnging the surfaces of interacting molecules

together. These motions are: translational, vibrational and rotationaL6

Many scientists have contributed to the studies of host-guest chemistry and

have elaborated potential applications. some of which are drug design, new

analyücal and diagnostic procedures, enzyme-analogous catalysis in water, etc ... 7

Other researchers focussed on designing and constnicting hosts. adding to the Iist

of existing hosts. Cornmon hosts incfude crown ethers, cryptands and cyclodextrins.

1.2 Cyclodextrins

Cyclodextrins were first diswvered from a culture of Bacillus amylobacter

4

grown on a starch medium by Villiers in 1891 ? Following this discovery, Schardinger

was able to isolate a bacillus (Bacillus macerans) which yields cyclodextrins upon

treatrnent with starch? Through years of extensive work by various researchers

secrets of the cyclodextnns have unfolded revealing their structures and chernical

and physical properties.

The reaction of the enzyme cyclodextrinase on starch is relatively speufic,

fornting only a few byproducts? The main products are three different cyclodex&ins,

a-, p, and y-, varying in the number of glucose units as follow: 6 units, 7 units and

8 units (Scheme 1.2). Several methods can be used to isolate them including: gel

chromatography, column chromatograp hy, HPLC, etc.'#'

Scheme 1.2 The Molecular dimensions of the Three Different Cyclodextrins (a-, f3- and y-CD).

1.2.1 Structure, Chemical and Physical Properties

The doughnut-shape of CDS is one of their interesting features (Scheme

1.3). This parücular shape is allowed because the undistorted D(+)-glucopyranose

units in the C l conformation are attached by a-1.4-linkages?** A wnsequenœ of

the Cl wnformation is that the sewndary hydroxy groups. which are situated on

the C-2 and C-3 positions of the glucose un*. are located on the larger opening of

the CD, and the primary hydroxy groups are located on the smalier side of the CD?

The seaindary hydroxy groups of adjacent glucose un& (G2 and C-3) are

involved in hydrogen bonding, resulting in stabilkation of the shape of the molecuk

and affecüng its solubility in ~ a t e r . ~ Secondary hydroxyl groups are fairly rigid and

do not rotate whereas free rotation of the primaiy hydroxyl groups is allowed and

they can partially block the cavity by rotating.'

The pnmary characteristic of this toms shape is its cavity. The cavity of CDS

are mainly wmposed of C-H groups and glucosidic oxygens which makes ït

relatively apolar? The fact that the electmn pairs of the glycosidic oxygen bridges

are directed towards the inside of the cavity produces a high eledron density and

gives some Lewis-base charader?

1.2.2 Inclusion Complexes and Kinetics

The CD cavity can accommodate a variety of organic guests,* however,

strong interactions are usually associated with hydrophobic molecules. The only

requirement for inclusion of guests appean to be spatial and therefore the dïfFerent

cavity sites allow a-CD to include benzene derivatives, for P-CD to include

naphthalene and for y-CD to include anthracene?

Inclusion complex formation is usually associateci with unfavouraMe entropy

changes and favourable enthalpy changes.' The favourable enthalpy change is

thought to be caused by: the Van der Waals interactions between the CD and its

guest (London dispersion attraction and dipole-dipole intera~tions)~, hydrogen

bonding between the hydroxyl groups of the CD and the guest, high energy water

molecules released upon complex formation, and strain energy being released in O

the macromolecular ring of the CD.'

Secondary Hydroxy \

Hydropho ic Cavity P

Primary Hydroxy

Scheme 1.3 Structure of an a-CD Molecule and a Schematic Representation of the Binding site of a CD Molecule. !

The study of the complexes formed by CDS with their guests can be undertaken by

various methods depending on the type of information sought. When essential

information about factors involved in complexation are needed. dissociation

constants are easily measured. The dissociation constant of a complex is an

equilibnum constant (i$) for the dissociation of two or more molecules that form this

cornplex? Ks relates to the stabilii of the wmplex. " and from equation 1 -0 the

Gibbs fke energy can be obtained.

LW = -RT In K (1 -0)

The stronger a guest binds to a host the lower the Ks value. As previousfy

mentioned, alis is because the binding energy is higher and usually results in a

slower rate of dissociation.' Several factors influence the binding strength and

hence the dissociation constant value. These factors were enumerated and

explained in Section 1.1.

Some exam pies of K, values for binding to PCD are presented in Table 1 -0.

The larger the Ks value is the lesser interaction there is between the host and the

guest Note how the values depend on the guest size and shape.

Table 1.0 Dissociation Constants (KJ of PCD-Guest Complexes

Guest Ks, mM Reference

CH3-CH2-CH2-OH

n-propanol

n-butanol

Cyclo hexanol

C yclohexanone

Acetophenone 8.1 1 I 0.55 14

One of the methods that w e have ernployed to calculate dissociation constant

values invoives the use of 'inhibition kineti~s''.'~-'* Using saturation kinetics several

equations can be defined:

The first equation (1 -1) involves the conversion of the substrate (S) to the produd

(P) without the involvement of a 'catalyst". The rate constant of the reaction is

therefore written as k,, for 'uncataiyzed". Equation (1 -2) describes the conversion of

S to P in the presenœ of CD. The substrate can form a 1 :1 complex with CD (S-CD)

and then be converted to P. The dissociation constant of this complex is expressed

as Ks (equation 1.3). k, represents the rate constant of the readion from the S.CD

wmplex to P (c is used for 'mtalyzed") From equations (1.1, 1.2 and 1.3). equation

(1.4) is derived, where k, is the observed (measurable) rate constant. Experiments

can be camed out where oniy the [CD] is varied and k, is measured. Analysis of

the results using equation (1.4) provides calculated values for the rate constant k,

and the dissociation constant Ks.

Depending on the reaction, CDS cm have one of two effedo. The first case

is catalysis, which implies that with increasing [CD] k, will increase as seen in

Figure 1.1. This is because the 'uncataiyred' rate constant (k,,) in equation (1 -4) is

srnaller than the ucatalyzed" rate constant (k& CDS have been known to catalyre

a large number of react i~ns.~* '~ Catalysis of a reaction by CDS, or a catalyst,

resides in their ability to lower the fke energy of the transition statemS This can be

done via nonavaient or covalent interactions. Nonco~ len t catalysis may involve . one of Wo possible effects, microsoivent effect or conformational effect? Covalent

catalysis involves the formation of covalent inter me dia te^.^^^ The second case is

retardation, where &< k. Therefore k, decreases with increasing [CD] (Figure

1.2)-

1.3 Probe Reactions, Hydrolysis of TM06 and BDMA

Of the reactions that were found to be retarded by CDS, two were of

parD'cular interest, the hydrolysis of trimethyl orthobenzoate and the hydrolysis of

benzaldehyde dimethyl acetal. Both readions were well known and had previously

been ~tudied.""~ In their own nght these reactions were interesting because they

are relatively fast and their products are chromophores which made them suitable

for stopped-flow experiments.

Figure 1.1 Catalysis of the Cleavage of m-Nitrophenyl Alkanoate by a- CD?

Figure 1.2 Retardation of the Hydrolysis of TMOB in 0.1 M HCI by B-CD.

In the case of acid catalyzed hydrolysis of TMOB, two major steps are

involved :

acid PhC(OH)(OMe), - PhC(0)OMe + MeOH (1 -6)

or base

Where the first step (1.5) invohres general-acid cataiysis and is rate-lirniting.qq

Appearanœ of the product, methyl benzoate, can be monitored by meawring the

absorbanœ increase at 228 nm.

The acid cataiyzed hydroiysis of BDMA follows a similar course.

H+ PhCH(OMe), + H,O - PhC(OH)(OMe) + MeOH (1 -7)

acid PhCH(OH)(OMe) - PhCHO + MeOH (1 -8)

or base

Step (1 -7) is the acid catalysed conversion of the acetal to the hemiaœtal, followed

by step (1 -8) where a molecule of methanol is eliminated by acid or base ~atalysis.'~

Benzaldehyde appearance can be monitored by measuring the absorbanœ

increase at 252 nm.

TM06 0 BDMA

Figure 1.3 Retardation of the Hydroiysis of TMOB and BDMA in 0.1 M HCI by 8 CD.

Discovery of the effect that PCD has on these rea~t ions '~ '~ lead to the

conclusion that they could be used as probe reactions. By making use of inhibition

kinetics, and carrying several expenrnents under the same conditions with the

exception of the [CD], which was varied (Figure 1.3). pertinent information (k, and

Ks) could be estimated. Analysis of the results yielded the parameten presented in

Table 1.1

To exploit inhibition kinetics as probes. equations have to be elaborated. By

adding an 'inhibitof or a guest. Le. a molecule that binds to CD. to the reaction

mixture, with increasing concentration, an increase in the observed rate of the

reaction is expected. The guest would compete with the substrate for the CD.

therefore increasing the amount of free substrate available for hydrolysis.

The guest can fom a cornplex with the CD and the dissociation constant can

be expressed as equation (2.0).

An estimation of the [CD] compared to the [CD],, is obtained as follows:

[CD], = [CD] + [CD-Il (2-1 )

[Ilo = [l] + [CD-Il (2-2)

The [CD] is estimated using equation (1 -4) where [CD] may be isolateci ta yield:

AI1 the parameten are known with the exception of K, which can therefore be

calculated.

Table 1.1 Constants for the Hydroiysis of Acetals in the Presenœ of Cyclodextrins-

Substrate k,,,s-' &, mM k, s" uk

a In 0.1 0 M HCI, at 25 O C . Values of &, K, and k, were obtained by non-linear fming of equation (1 -4).

See ref. 12 ' Value indistinguishable from zero. See ref. 48 Acetophenone dimethyl acetal in 0.010 M HCI, at 25 OC. See ref. 46 Benzaldehyde diethyl acetal in 0.1 M Chloroacetate buffer. See ref. 49 See ref. 50

1.4 Alcohols as Guests

The use of inhibition kinetics to calculate the dissociation constants of

different CD cornpiexes was proven to yield satïsfactory r e ~ u l t s . ~ * ~ ~ * * ~ particulaily

when alcohols were used as gue~ts.'*'~ Table 1.2 illustrates the results obtained

with TMOB and BDMA hydrolysis as probe reacbons in determining the dissociation

constant of alcohols to PCD.

Comparing & values with Iiterature values and values obtained with a

different probe madion demonstrated strong agreement.1213 The validity of this

method established, a new goal couiâ be undertaken: the study of the interactions

of &CD with sugars.

1.5 Sugars as guests

In previous yean, several publications presented the interactions of CDS with

s u g a r ~ . ' ~ ' ~ Surpnsingly, the resuits of some groups seem to disagree strongly with

the results of other groups. Due to their size and properties, it was believed that

sugan wouM interact weakly with CDS. Hirsh et al. estimated with a blood glucose

meter a dissociation constant (4) for {$-CD.D-glucose} complex of 2.38 mM.' In

contrast, Hacket et al. determined by Ruorometric cornpetition titrations in water a

K, value of 1667 mM for the same complex.17 From the wnflicting results cornes the

interest to study the interaction of cycloheptaamylose, P-CD, with several sugars

using a difierent method: kinetics measurernents.

Table 1.2 PCD host-guest dissociation constants. K, (mM)

1-Hexanol 4.07kO. 18

1 -Heptano1 1.27I0.01

3-Hexanol 16.2k0.6

tert-Butanol 20.311 .O

Cyclohexanol 1 -51iû.09

' Acetophenone dimethyl acetal, in 0.01 M HCI at 25 OC. Product increase monitored at 244 nrn. See ref- 46

From the effect of guests on the retardation of the hydrolysis of trimethyl orthobenzoate by CDS in 0.10 M aqueous HCI.12

From the effect of guests on the retardation of the hydrolysis of benzaldehyde dimethyl acetal by CDS in 0.1 0 M aqueous HCI.12

From displaœment of a fluorescent probe. at pH 11.6 (0.2 M phosphate buffer)." Mainly ref. 15.

' Other literature values.52

2.4 Effects of guests

As seen in the introduction. W D retards the hydroiysis of TMOB and

BDMA. By adding a guest that binds to N D , and that campetes with the substrate

for the CD, an incmase in the obsenred rate should be observed. An increase in

the k, is expcded because binding of the guest will lower the amount of free CD

available, increase the fkee subotrate, resulting in faster hydroiysis. The

dissociation constant for the @-CD-guest} complex (6) can be estimated by

exploiting the same methodokgy as in 'inhibition kineticsn. In this case, the reaction

is slowed dom instead of cataiysed and the inhibitor (sugar) increases the

observed rate. Equations 1.4 and 2.4 derived in the Introduction and the h, k, and

Ks values from Table 1.1 are needed to estimate the K, values. The estimation is

done by obtaining k, for a reacüon inciuding a guest (sugar) at several known initial

concentrations. The K, values estimated in this work are shown in Table 2.1.

Table 2.1 Dissociation constants for the {&CD.guest) complexes

Guest Lit8 Litb TMOB BDMA ADMA'

Pentoses

D-ara binose 1430 667 1060*80 1500e20 1 07Oî45

L-arabinose 1430 949190 1 05Oi50 1 23e54

D-2deoxyribose 106 63i16 145k3 1 34î1 2

D-l yxose 233. 394S4 456e1 4Mk11

0-ribose 389 159 209I16 252i11 21 018

D-xyiose 1 O00 625 573i20 - 757k13 834î75

L-xylose 480k32 9581124 775î74

DL-xylose 427S6 1020i85 786i30

Hexoses

D-fructose 7 170186 18OOi90 1670*100

Dgalactose 2000 3920M90 2960i300 3500î185

D-gluwse 1670 2630ï130 47201400 5390k730

0-mannitol 3650I21 O 8720i2020 4250Q60

D-mannose 1000 1460*190 2790Q35 2540i180

L-rhamnose 503i76 1640&140 1 1 90î58

D-sorbitol 120011 50 3420k180 41 1 Oi290

L-sorbose 665146 829176 969i27

Others

P-D-iactose 2200182 1 040011 700 5550i1760

sucrose 22001380 31 90k290 6030I2060

D-glucosamine.HCI 81 3148 847I27 1040*110

Reference 18. Reference 17. Hydrolysis of acetophenone dimethyl acetal in 0.01 M HCI (monitored for

acetophenone at 244 nm). Work perfomied by Samer s us sain.^

The "inhibition kinetic" method can oniy be used if the guest binds in the

cavity of the CD. This is because the probe substrate is bound inside the CD cavity.

The interior of the CD is relatively hydrophobic which prevents solvated protons

from reaching the substrate and supresses its hydrolysis. If the gwst interads with

the exterior of the PCD, this will not be monitored. It is possible that the sugars

associate outside of the cavity, a point that Hacket et al. have raised. "and that has

been demonstrated by NMR to occur between protons on the outside the CD host

cavity and those of cyclohexanecar6oxyiate in unpublished work by Gadre et a/.,

cited by Hacket et al. l7 If the sugars interact with the exterior of the &CD cavity,

the actual binding constant of the (sugar.&CD) cornplex may be different than the

estimated K, value. As an example, if a sugar is bound on the exterior of the cavity

is blocking the opening of the CD, then the substrate is "lockedn in the CD cavity

and cannot be hydrolysed. The rate of hydrolysis would be lower than if the sugar

was bound in the CD cavity, leading to a lower estimated concentration of bound

sugar, resulting in a larger dissociation constant for the {sugar.&CD} cornplex.

One cannot condude that the sugan are actually interacting on the exterior of the

CD cavity since there is a lack of prwf, nevertheless. many indications suggest that

they are. In fad this might explain the observed enhancement in solubility of the &

CD in the presence of glucose noticed by Hacket et al."

The resutts obtained by Hirsch et al? may refled a similar ambiguity: their

dissociation constants for {glucose.PCD) are very small which implies strong

binding, and the method they used only monitored the free glucose, it could not

differentiate between glucose interading in the CD cavity or on the exterior of the

CD cavity. If the glucose is binding to the exterior of the CD cavïity. kss fke glucose

will be detected, and a lower K, values will be estimated. This could be s possible

explanation for the estimatecl K, value of 2.38 mM for (glucose.gCD} cornplex. More

over, Hi- et al. found that the dissociation constants for the @lucose.a-CD} and

{gluwse.&CD} complexes were indistinguishable m i n experimental enor, which

is not usually the case for large molecules like glucose. The a-CD cavity is of

smaller diameter (0.6 nm) than &CD (0.8 nm) and the general binding pattern is

that PCD can accommodate branched and cyclic compounds better. as seen in

several publications and s t ~ d i e s . ~ ~ * ~ ~ If the glucose is binding to the exterior of the

CD cavity, 1 is possible that the K, values are relatively the same for a-CD and &

CD, if not, the dissociation constants are unlikely to be so similar.

The titration microcalorimetry used by de Namor et al? is another technique

that cannot distinguish between the sugar interacting with the exterior or the interior

of the CD cavity. And the same thinking as for the Hirsch et al. values may apply

here, since the K, values reported by de Namor et al. are significantly lower than the

K, values by Hacket et al., Aoyama et al. or this work (see Figure 2.6). One has to

keep in mind that at the moment, al1 this thinking is purely speculative and that

further studies and experiments have to be perfomed in order ta confirrn or reject

it .

The TM06 and BOMA values were obtained under the same acidic

conditions and using the same sugar solutions, therefore cornparing them is one

way of detennining if the estimated K, values are reasonable. Figures 2.3 and 2.4

22

presents the correlation of Ge BDMA values with the ADMA (obtained in l o w r

[HCI]) values as well as with the TM06 vakies. Figure 2.5 shows the conelation of

the ADMA values with the TMOB values. The conelation coefficient values are

shown in Table 2.2,

Table 2.2 Correlation coefficients for the cornparison of the pK, values estirnateci osing different probe readions

Correlation Pentoses Hexoses Othe-

- - -

BDMA ADMA 0-977 0.880 0.885

BDMA vs TM06 0.937 0.81 9 0.894

ADMA TMOB 0.949 0.855 0.965

In general. the values seem to agree, which is significant since most of the

K, values obtained were large (explained further). Within experimental error the

results show appreciable constancy, the difference in the K, values are most likely

due ta the distinct probe reactions. A low K, value is estimated more precisely due

to the strong binding: the effed of a guest is greater when it interacts strongly with

its host, the increase in k, is larger therefore the range of the values used in the

calculations is bigger, resulting in less enor. The fact most sugan interact weakly

with P-CD is a major source of enor, as one can notice in Table 2.1, the lower K,

values have usually smaller relative error values. It is also important to note that the

pentoses values agree more closely than the hexoses or the othen sugars values,

23

which is fùrther proof that estimation of a binding constant for weakfy interacüng

host and guest is difficutt. When cornparing the estirnated values with the literature

values the correlation coefficient values obtained are presented in Table 2.3.

Table 2.3 Correlation coefficients for the cornparison of the pK, values estimated to the pK, values from the literature

Correlation TM06 BDMA ADMA

Referenœ 1 8. Referenœ 17.

Figure 2.1 shows the values from this work plotted against the Aoyama1'

values and Figure 2.2 shows the estimated K, values compared to the Hacket17

values. The K, values obtained in our work tend to agree well with the literature

values from these two publications, even though the rnethods used were very

ditferent In contrast, when looking at Figure 2.6, one c m obviously see that the AG0

values, and therefore the K, values, from de Namor et al. and Hirsch et al. do not

correlate with our values. This may be due to the fad mentioned previousiy (sugars

interacting with the exterior of the cavity) or the difference in the techniques used.

pKl Estimated YS pKl Aoyama

+ pK (BDMA) pK(ADMA) PK (-1

pK, Aoyama values

Figure 2.1 pK, estimated values from this worù yg literature pK, values from Aoyama et al. .la

pKi Estimated us pKI Hacket

pK ( W B ) pK (BDMA) pK (ADMA)

-1 J O

pK( Hacket values

Figure 2.2 pK, estimateci values from this work literature pK, values from Hacket et al-.''

Figure 2.3 pK, estimated values from this work for benzaldehyde dimethyi acetal pK, estimated values for Acetophenone dirnethyl aœtal.

pK Pentoses pK Hexoses pK Ohers

Figure 2.4 pK, estimated values from this work for benzaldehyde dimethyl acetal .

pK, estimated values from this work for Trimethyl orthobenzoate.

Pentoses Hemses Others

Figure 2.5 pK, esümated values for Acetophenone dimethyl acetal pK, estimated values from this work for Trimethyl orthobenzoate.

Aoyama x de Narnor 0' Hacket

Hirsch

Figure 2.6 AG0 values from the iiterature AG0 averaged values of BDMA and TMOB from this work.

2.2 Sugars structure

The structure of most of the sugars used in this work are shown in Appendix

A. The majonty of hem are found to be in the pyranose form in so~ution."~ In

general, there is an equilibrium between the a and f a n of the sugars. At this

point, the anomer or anomen present in solution and mutarotation is not the main

concem. This is because mutarotation is promoted by acids and bases and

temperature dependent, the rate of the reaction may double or triple every

increment of 10 OC? ln this case. the reactions were carrïed at room temperature

and a low acid concentration. The important factor is which cyclic fonn the sugars

adopt For some sugan, only one cyclic form is found. as an example, a-D-

glucopyranose exhibits a simple mutarotation suggesting that only the a- and

pyranoses exist in ~olution.~ For other sugars, both cyclic forms are present, and

in the D-ribose case. no chair conformation is preferred ('C, or 'C,)? Which makes

it dinicult to determine what type of structure is interacting with PCD.

In general, the pentoses bind more tightly to W D than the hexoses. Figures

2.7,2.8, 2.9 and 2.10 present the k, y the sugar mncentrations for the pentoses

and hexoses. By looking at those graphics one can see the effect that adding

sugan has. The tighter the sugar binds. the more effect can be seen, as an

example in figures 2.7 and 2.8. 2deoxy-D-ribose (2deoxy-D-erythm-pentose)=

demonstrates the larger increase in k, with increasing concentration. Using TMOB

as a probe reaction, the dissociation complex of Z-deoxy-D-ribose was estirnated

to be 63 i 16 mM- In the case of BDMA, it was estimated to be 145 I 3 mM, Which

explains wh y a larger effect for 2deoxy-Dribose is seen in Figure 2.7 than in Figure

28

2-8,

The lowest dissociatÏon constant was found to be for the cornplex (2deoxy-

D-ribose.wD). The reason for thf most Iikeiy stands in thc stnich~re of this sugar.

Pdeoxy-0-ribose is the most important 2deory-aidose sinœ it is a component of

deoxyrïbonucleic acids (DNA). The fom and conformation of this sugar in solution

was not studied and therefore is not knomi with certainty. In DNA, 2 d e o x y - & n i

exists in the furanose f ~ n n , ~ s o this migM be the case in solution. The sugar could

also be found in an acyciic form. Both these forms could explain why this sugar

binds the most tightly, sinœ no omet sugars adopt them ni solution (with the

exception of the alcohols).

0 Larabinose 0-deoxyribose L-xybse DL-)sr bse

[Pentoses],, mM

Figure 2.7 scaled y~ [Pentoses], for the hydrolysis of TtdOB in the presence of 1 mM of PCD.

Figure 2.8 scaled [Pentoses], for the hydrolysis of BDMA in the presenœ of 5 mM of &CD.

t I I I I 1 1

O 100 200 300 400 500 600

[Hexoses],, mM

Figure 2.9 scaled [Hexoses], for the hydrolysis of TM08 in the presence of 1 mM of &CD.

30

Figure 2.10 k, scaled [Hexosesl, for the hydroiysis of BDMA in the presence of 5 mM of N D .

The xyloses al1 show relatively the same effect with both probe readons.

The configuration of these sugars does not seem to affect their binding to W D . In

the case of the arabinoses one can see the same pattern: the configuration does

not seem to affect the binding. By contrast, the conformation seems to be slightly

more important with respect to the binding. D-arabinose adopts the 'C,

conformation in solution and the D-xylose adopts the 'C, in solution. Even though

both those sugan have three hydroxyl groups in the equatorial position and one in

the axial position. their dissociation constants differ only by approximately a factor

of 2. The anomer also seems to be a factor in the case of D-arabinose and 0-

xylose. The different anomers indicate the "stenc arrangement, with respect to the

conformational disposition."' This explaim why both sugars have the same number

of axial and equatorial groups while they have different conformations.

a-D-Lyxose has a %, prefened conformation and two axial hydroxyl groups

31

and two equatorial hydroxy groups. a-û-xylose adopts the same conformation but

diiffer in the number of axial hydroxyi groups shce those hiuo sugars are bpirners.

The effect of adding those hno sugars are similar when using TMOB. but differ

slightly when using BDMA. This may be caused by the difkrent probe reacüons.

a-0-Ribose does not have a preferred conformation in solutionu. and it can

be found in the pyranose form (76-ûû%) and the furanose fom (2420%). Therefore

several structures of ribose wïll be in solution, this rnay explain why it binds the

tightest after 2deory-0-ribose. Dribose is the tnie epimer (2-epimer) of D-

arabinoseY, but those epimen do not have similar binding constants (they differ by

a factor of approximately 5).

The hexoses show les of an effect on the kinetics and this is reflecîed in the

dissociation constants estimated. The hexoses are larger molecules and might not

be as well accommodated by the PCD as the pentoses. D-mannitol is one of the

sixcarbon carbohydrates that did not bind tightly to the cyclodextrin. This compound

is found in an acyclic fom in solution and was thought of having easy access to

P-CD cavity. The other alcohol studied, D-sorbitol (D-glucÏtol), did bind more tightly

to PCD. This sugar is also found in an acydic fomi in solution but with a bentehain

arrangement,- due to an unfavouable 1,3 interaction between two oxygen atoms.

The bent-chain arrangement rnay facilitate the accommodation of D-sorbitol by &

CD. resulting in a lower dissociation constant.

D-glucose and D-galactose are 4-epimers and behave in the same rnanner

for both probe reactions. D-glucose and O-mannose are true epimers and differ in

their effed with TMOB and BDMA. The position of the hydroxyl group on C-2 seems

32

an important one in the binding of the sugars to FCD sinœ all of the true epimers

studied were found to behave difFerently and have large variation in their binding

constants.

The sugan that have the lowest dissociation constant are L-rhamnose and

L-sorbose. L-rhamnose is a deoxy sugar (6-deoxy-L-mannose) and L-sorbose is a

ketose (D-xylo-henlose) wïth a carbonyl group on C-2.2-Deoxy-0-ribose was the

pentose with the lowest dissociation constant and here again we have a deoxy

sugar for the hexoses. More over, both 2deoxy-D-ribose and L-rhamnose contain

carbonyl groups (not on C-2). It seems that the sugars that posses a carbonyi group

are better accomrnodated by &CD.

a-D-Fructose is .an hexose that can be found both in the pyranose form

(68.4-76.0%) and the furanose form (31 -624%). a-D-Ribose is a pentose that is

also found in both forms, and it is interesting to see that they both bind the tightest

after the deoxy sugars (L-rhamnose and 2deoxy-D-ribose). Again. this may be

explained by the different structures found in solution. It is important to note that

fructose (D-arabino-hexulose) is a ketose with a carbonyl group at carbon atom no.

2.

Figures 2.1 1 and 2.1 2 present k, the sugars concentrations for various

monosaccharides, both pentoses and hexoses. When sugars adopt the pyranose

form, some of the pentoses and hexoses have the sarne structure. that differs only

by the R group on carbon 5. It is interesting to compare them and see if they behave

in the same manner.

0-ribose - Pm- D-ybse L-aiabinose L-rhamnose

Figure 2.1 1 k, scaled [sugars], for the hydrolysis of TMOB in the presence of 1 mM of PCD.

Figure 2.12 k, scaled y~ [sugars], for the hydrolysis of BDMA in the presence of 5 mM of PCD.

The firsthno sugars to be compared are Dglucose and 0-xyiose. They adopt

the same confornation. but do not have the same anomer le. that the hydroxyl

group on C-4 is in the a position for O-xylose and in the position for Dglucose. By

looking at Figures 2.1 1 and 2.12 one can see that Dglucose does not bind as tighüy

to W D as 0-xybse. Two hdors could be involved: The R group on G 5 an& the

position of the hydroxyl group on C-1. a-D-Iyxose and PD-mannose are similar to

a-0-xylose and @Dglucose, they both have the same conformation and difFer by

their anomer. Hem again, the a-aidopentose binds more tightly to &CD than the

aldohexose. Now. FD-galactose and a-L-anbinose do not have the same

conformation or anomer. These two aldoses also have different dissociation

constants.

The resuits in Table 2.1 are clear, the pentoses have lower dissociation

constants than the hexoses, therefore the R group on C-5 is a major factor in the

binding of the carbohydrates to W D , which was also observed by Aoyama et al.''

It also becomes evident that the a anomers bind more tightly to PCD than the P

anomers. This leads to the conclusion that both the R group on C-5 and the position

of the hydroxyl group on the Cl are important factors involved in the interaction of

PCD to monosaccharides.

D-glucosamine hydrochloride has the same structure as 0-glucose, with the

exception of having an NH, group on G2. The glucosamine has a lower dissociation

constant than D-glucose. This agrees well with the previous statement that "the

position of the hydroxyl group on C-2 seems an important one in the binding of the

sugars to &CD since al1 of the tnie epimers studied were found to behave

35

differently and have large variation in their binding constants:

Two oligosaccharides were studied, &0-bctose and sucrose. lt is interestng

to note that both these sugars have, within experimental eror, approximately the

same dissociation constants as D-glucose. For sucrose, this might indicate that not

al1 the sugar is accommodated in the &CD cavity and that either the glucose or

fructose part of the moiecule binds to the cyclodextrin. The same couki appîy to

D-lactose, where either the Dgalactose or Oglucose part of the molecule could

interact with the W D - Sinœ the values for those two sugars a n similar, it is most

likely the glucose part of the molecule that binds to the cyclodextrin.

Since the K, values from the (B-CD.oligosaccharides) complexes are so hrge

(over a 1000 mM) they indicate that very little interactions are ocairring betiiineen the

P-CD and the sugars. A value over a 1000 mM is almost meaningless and hardly

reproducible. It is possible that the values for PD-lactose and D-glucose are similar

because of a large deviation rather than an indication of what part of the molecule

is involved in binding .

The rate of hydrdysis of trimethg orthobenroate and benzaldehyde dimethyl acetal

were confirmed to be slowed dom by P-CD; By exploiting the saturation type

kinetics involved and using 'inhibition kinetics', the dissociation constants (K,) for

several (B-CD.sugar} complexes were esh'mated. These K, values were found to

conelate well with the values obtained using the acidic hydrolysis of acetophenone

dirnethyl acetal as a probe reactim." It was al- demonstrateâ that the results

obtained in this work agreed tolerabiy with the results of Hacket et al." and of

Aoyama et al.,'' but aiere are strong disagreements between ouf results and those

of de Namor et al?' and of Hirsch et al.'' These conflicting results do not seem to

indicate flaws in the technique used. It could possibly mean that the sugars are

binding with the exterior of the cyclodextrin, as well as in the cavity. The viability of

the acidic hydrolysis of TM06 and BDMA as probe reactions in estimating

dissociation constants for the binding of guests to CDS had been established

before'l'' and this work provides further evidence to confinn this statement. This

work did not provide astonishing results as it was expected that B-CD would interact

only slightly with sugars. Pentoses were found to bind modestly to PGD (K, = 100 - 1 000 mM) and hexoses hardly bind at al1 (K, w 500 mM).

4.11 Materials

P-Cyclodextrin was purchased from Wacker C hernie (Munich. Gemany).

Trimethyl orthobenzoate (TMOB), benzalâehyde dimethyi acetal (BDMA) and al1 the

sugan were obtained from Aldrich Chernical Co. Standard HCI solutions were

purchased from A & C Chernicals (Montrkal).

4.2 Kinetic Experiments

The kinetic experirnents were camed out by using a single beam stopped-

flow spectrophotometer by Applied Photophysics modal SXI 7MV. In the stopped-

flow apparatus the mixing is 1:1 and so the concentrations of the fwo solutions

before and after mixing dHer by a factor of 2. The observation cell was kept at 25.0

r 0.1 O C . The sugar solutions were prepared in 0.10 M HCI. A concentration after

mixing of 1 mM of FCD was used for the hydrolysis of TMOB. The concentration

of TMOB and BDMA after mixing was 5 mM (50 pl of 0.1 M solution in 50 ml flask

before mixing) The concentration afler mixing of &CD for the hydrolysis of BDMA

was 5 mM.

The hydrolysis of TMOB in 0.10 M HCI was monitored for changes in

absorbance at 228 nm for the appearance of the product, methyl benzoate. The

hydrolysis of BDMA under the same conditions was monitored for changes in

absorbance at 252 nm for the appearance of its product, benzaldehyde.

4.3 Calculation of Rate Constants

Using the instrument manufacturer's software, the observed rate constants

(kod were calwlated by fitting the absorbanœ increase with a single exponential

function. The absorbance traces were composeci of 400 points. For the purpose of

this work, the first 15 points were not taken into account when calculating b, to

allow for the small induction p e r i d 2 Each recorded k, value is derived from the

average of 5 - 10 absoibance traces. Absorbanœ traces extended over 7 half-ives

for the hydroiysis of TMOB and 8 haClives for the hydroiysis of BDMA.

4.4 Calculation of Dissociation Constants

The calculation of K, values was carried out following the procedure

explained in the Introduction. In a spread sheet (see Scheme 4.1) the [CD] was

estimated using equation 2.5:

and this was used to estirnate K,. through equation 2.4. A dissociation constant was

calculated for each concentration of guest or "inhibitor" added. The K, values were

then averaged and a graph of k, vs b , was plotted to verify the linear

relationship. A sample calculation is shown in Scheme 4.1, overleaf.

The k, values are entered in the second column of the spread sheet. and

these values are scaled according to the Ig,. The calculated rate constant (163

was detenined using the known parameters presented in Table 1.1. In Scheme

4.1, b, was calculated for the hydrolysis of TMOB in 0.1 0 M HCI in the presence

of 1 rnM of &CD in solution and was found to be 2.188 s-'. Different factors may

influence the k, obtainad for a [CD], = 1 mM and [guestb = O mM: the precïse [CD]

and the [HCI]. By scalirig the obsenred rate constants to a calculated rate constant

cornparison by gnphic representation is facilitateci, sinœ the initial point for al1

inhibition analysis are the same.

Scheme 4.1 Calculation of the dissociation constant of D-glucosamine to N D using a Lotus 1-2-3 spread sheet.

PART Il

The Bromination of Pwimidine Derivatives

5JQQd-

5.1 Pyrimidine Sbutaii. and Properüm

Pyrimidine derivatives are important heterocydes since they are essential

components of nudei aads as well as some coenzymes and d r ~ g s . ~ Pyrimidine,

*rtsetf, is composed of a six-membered anniatic ring containing two ni&ogen atoms,

as seen in Scheme 5.1. The pyrimidine mokcuie is symmetrical with two planes of

symmetry, with one plane and axis of symmetry going through carbons 2 and 5.

which makes positions on carbons 4 and 6 equivaient, as well as positions 1 and

3 equivalent Yïelding an overall C, syrnmetry.

Scheme 5.1 Structure of Pyrimidine Molecule

The nitrogens are powemil electron-withdrawing atoms resulting in electron-

deficient positions 2.4. and 6? Their separate effects are reinforcd because the

nitrogen. atoms are meta to each other? The reaeüvity of substituent g roups found

in positions 2,4 and 6 will reflect the electron deficiency. Therefore, if a halogen

atorn is found in one of these positions, the halogen would be easily attacked by a

nucleophiie.

The Iposition is much less eledrondeficient due to the general inductive

effectn Consequenüy. eiectrophiles would attack at this point Nitration, nitrosation,

halogenation may easily be carried at this position.

When eiectron-releasing substituents are introduced. the pyrimidine reacts

more as an aromatic ring because the rr-electrons are restored. This facilitates

electrophilic reacüons at the 5-position. Some of these substituents are hydroxy and

amino groups. As seen in Scheme 5.2, pyrimidines with these substituents in the

2,4 and 6 positions may be in equilibrium with their tautomeric forms. As an

example, 2-aminopyrimidine will be tautomeric mai its ûimino fom. Of course. the

equilibrium will lie towards the more energetically favoured structure, and in the

case of 2-aminopyrimidine. Brown and coworken. found that in solution the major

tautomeric fom is the amino fomP

Arnino

H ydroxy

Scheme 5.2 Equilibrium between Tautomeric forms of 2-AP 'e. the Arnino and the lmino fom and between the Hydroxy and the Ketone form of . Pyrimidone.

5.1.1 Brominrtion of Pyrimidine Derivatives

5.1 A.1 Bromination of 2-Aminopyrimidine

The brornination of 2-aminopyrimidine (Z-AP) has been largely studied through the

yearqna and it invohres two major steps. First a fast $tep where addition of water

and attack by bromine occur. The second step is slow elimination of water to yield

the 5-bromo produd. Further investigation into this reaction provided key

information about the intemediate. This intermediate was isolated and observed by

NMR by ~anerjee," who also had obsenred similar intermediates for the

bromination of pyn'midone and uracil derivatives. Barbien' et al. had observed

analogous intermediates in the bromination of pyrimidine sulphonamides in

methanol solution?

2-aminopyrimidine intenned iate 5-bromo produd

Scheme 5.3 Bromination of 2-AP in D,0 to observe the intermediate by NMR spectroswpy.

Following the observation of the intermediate in the bromination of 2-AP, an

interesting question anses, how is this intemediate formed? Does it first involve the

attack of bromine then foifowed by addition of M e r or the inverse? This question

was addressed by an undergraduate student in a CHEM 450 research project and

thesis in 1980 but with no conaete conclu~ion.~

5.1 .1.2 Bromination of 1,2-dihydm-2-imino4-rnethylpyrimidine

The addition of bromine to 1,2dihydro-2-imino-1 -methylpyrimidine was

studied in 1980, here again. a similar intermediate was isolated and observed by

NMR? In this case, can the same mechanism as for 2-AP by applied? If it c m . do

al1 the pyrimidine derivatives read with brornine in the same manner? More

derivatives need to be studied.

020 DCI

Scheme 5.4 The Bromination of M2A in D,O to observe a sirniiar intermediate by NMR spectroswpy.

5.1 -1 -3 Bromination of 2-Amino4,6dimethylpyrimidine

The literature contains very little information about the bromination of 2-

amino4,6dimethy(Wnmidine (2ADP). The articles usually involve product studies.

Studying the bromination of 2-ADP could give insghts about the mechanism

involved, furthemore, it could be compareci with other pyrimidine derivatives.

Scherne 5.5 Structure of 2-Amin04.6dimethylpyrïmidine.

5.2 Pyridine Structure and Properties

The broad interest in pyridine and its derivatives has produced a lot of

literature? Pyridine is similar to pyrimidine, but it only contains one nitrogen atom.

The molecufe is relatively flat with bond angles close to 120'. The nitrogen atom is

sp2 hybridized and will contribute one s electron to the aromatic sextet. The other

TI electrOns are provided by the carbon atorns.

Since the nitrogen atom is electron withdrawing it will have some negative

charge. Partial positive charges are found at the 2,4 and 6 positions. creating an

overall charge distribution of negative for the nitrogen atom and posiüve for the ring

(Scheme 5.6). The properties of pyridine are parallel to b e n ~ e n e , ~ but with

differences due to the presence of the nitrogen atom. The electronegative nitrogen

causes the pyridine to be more polar than benzene. Eledrophilic substitution

reacüons can ocair but with more difficulty than with benzene, and primarily at

positions 3 and 5.

Scheme 5.6 The structure of Pyridine and the partial and overall charge distribution.

Electrondonating groups are ortho and para direding groups. Therefore. if

a substituent is at the 2 position it further activates position 3 and 5 for electrophilic

attack.

5.2.1 Bromination of Pyridine Derivatives

5.2.9.1 Bromination of 2-Aminopyridine

Frorn the literature, it is known that the bromination of 2-aminopyridine (2-

APy) yields a mixîure of products. Wth excess brornine, the major products are 33-

dibromo and 5-bromo-2-aminopyridine. To a lesser extent, the 3-bromo derivative

is obtained. Occasionally. one gels Sbromo and 3.5.6-tribromo derivatives. 27-29

CH3,

Scheme 5.7 The bromination of 2-Aminopyridine.

No study on the mechanisms involved have been undertaken. The interest in this

type of study is for comparative reasons.

5.2.1 -2 Bromination of 2-Dimethykminopyridine

In the case of the bromination of 24imethylaminopyridine (2-DAPy), the

literature states that with a limiting amount of bromine a mixîure of the mono (5-

brorno-2-dimethyl aminopyridine and dibrorno (3,5-dibromo-2-

dimethylaminopyridine) derivatives is obtained?

Scheme 5.8 The structure of 2-dimethylaminopyndina.

5.3 Aniline Structure and Propertks

Aniline is an important compound in organic chemistry, not only for its

chemical and physical properties. but for its historical background. In 1856. Henry

Perkin oxidized impure aniline with potassium dichromate, yielding a purple

pigment. which gave hirn the idea of using it as a dye. Following this discovery,

Perkin resigned his post with Hoffinan and created his own chemical Company. To

produce his dye on a brge-scak he had to elaborne a methd for prepanng aniline,

the early beginnings of organic synthesis and of the synthetic dye industry?

Aniline is a benzene molecule with an arnino substituent It is less basic than

simple alkylamines, because the electron lone-pair of the nitrogen atom is

delocalized by orbital overlap with the B electron system of the aromatic ring and

therefore less available for bonding?

Scheme 5.9 Resonanœ structures of Aniline.

The protonation of the amino group of aniline is less favoured, because the

resonance stabilization present in aniline is lost. due to the fact Bat only two

principal resonance structures are possible for the arylammoniurn ion:

Scheme 5.10 Resonanœ structures of an arylammonium ion.

Subslituents which decrease the reactiv'i of aromatics towards eiectrophilic

attack also decrease the basicity of anilines. As an example, the pK,, of aniline is

equal to 4.58 and that of the pbromoaniline is equal to 3.86? The basicity is

affected by deadivating groups (eledron-withdrawing groups) because they make

the arornatic ring electron-poor. If the amino group is protonated. a deactivating

substituent will demase the stability of the posiovely charged ion, yielding a smaller

AGO for protonation and a lower p&.

5.3.1 Bromination of Aniline

It is known that the bromination of aniline takes place rapidly and yields the

2,4,6-tribrorninated product The amino group being so strongly acüvating, and

ortho- and para- directing, it is hard to stop at the monosubstituted producta

Scheme 5.1 1 The bromination of Aniline.

Similarly, the bromination of N,N,-dimethylaniline is rapid and with exœss

bromine the ûibromo aniline is obtained.

Scheme 5.12 The brornination of N,N-dimethyl aniline.

For the purposes of wmparison with that of aminopyrimidines and aminopyridines.

the study of the bromination of aniline and its derivative was considered important.

5.4 Simple Kinetics

To further understand the reaction of bromine with the aminopyrimidines,

aminopyridines and anilines previously mentioned, kinetic equations can be derived.

51

These equations date to the rate of the reaction and express the dependance of

the rate on different factors such as the concentration. The kineüc equations can

help resohre the mechanism puzzle.

Bromination is usualiy camed out under acidic conditions and the substrate

can be found in the protonated or unprotonated form. Assuming that the bromine

only attacks the unprotonated form. equation 2.7 would represent the reaction

equation where S is the substrate. P is the product, K, is the equilibrium constant

between the protonated and unprotonated fonn of the substrate (equation 3.0) and

k, is the second order rate constant for the brominaüon of the substrate. From the

type of traces obtained, it can be established that the rate of the reaction will be

equal to the disappearance of bromine. as shown in equation (2.8a). Sinœ the

disappearance of bromine is what is measured, k2ObS is used for the obsenrable

second order rate constant The rate of the reaction should also be equal to that

required by the scherne (eq. 2.7) of the reaction as seen in equation (2.8b).

rate = -- = k20bl [SIO [BrJ

rate = k, [SI [BrJ

Knowing that both equations (2.8a) and (2.8b) are equal, one may isolate k,& and

obtain equation (2.9). Where [SI and [SI, differ due to the protonation of some

52

substrate. The substrate concentration may be substihited using equation (3.0) to

yield equation (3.1).

A low pH, Le. at a pH lower than the pK, value, equation 3.1 may be simplifieci to

equation (3.2). The logarithmic version of this equatîon is shown in (3.3).

log k2- = log k, Y + pH (3-3)

A plot of log k2ObS ys pH should yield a Iinear graphic where the intercept is equal to

the log k,. This situation is applicable to the following substrates. where the pH of

the experiments was always much lower than the pK, values of the protonated fonn

of each substrate: 24Py, 2-DAPy, 2-ADP, aniline and N,Ndimethylaniline.

6.1 Bromination of Aniline

As seen in the Introduction, equation 3.3 can be useâ to express pH

dependence of the the second order rate constant k2. The requirement of this

equation is that a dope of approximately 1 is obtained when log kZ* is plotted

against pH. The pH rate profiles obtained for aniline and N,Mimethylaniline (F~ure

6.1) meet the requirements of equation 3.3 and the calculated resub are tabulatecl

in Table 6.1.

log k,pb = log k2K, + pH (3-3)

N, Wdimethy bniline

Aniline

Figure 6.1 pH rate profile for the Bromination of N, Ndimethylaniline and aniline. The points with open symbols (o and 0) were excluded from the calculations. This is mostly due to the high ionic strength at low pH.

Table 6.1 Bromination Panmeters

N, Ndimeth ylanilinea Aniline8

log k? 8.54

r 0-999

a For an ionic strength of 0.1 M. Reference 38

The value bapg is the value before any corrections for the tribromide ion has been

made in Table 6.1. The reason for estimating this value is that a value for N,N-

dimethylaniline was available from the Merature. Bell and Ramsden detennined an

apparent secondorder rate constant of 3.16 x 10' M1 se' for an ionic strength of

0 .O5 M ," whereas. the value for this work is 3.46 x 1 OB iW1 S-' for an ionic strength

of 0.10 M. These values are the same within experimental error, given the different

ionic strengths. The simple Iinear relationship between the log of k2- and pH leads

to the conclusion that direct bromine attack on the unprotonated substrates is

involved .

Aniline was expeded to be less reactive towards bromine than N,N-

dimethylaniline. This is because aniline is less basic than N.Ndimethyianiline as

stated in the Introduction. T& effect of two methyl groups on the nitrogen atom is

moderate, and k, was expeded to be slghtly higher for N,Mimethyianiline. In order

to compare the value obtained for aniline with the Iiterature. a Hammett plot. Figure

6.2, was wnstructed using the values for substituted anilines taken from Katritzky

et al?'

Figure 6.2 Hammett plot for the bromination of psubstituted aniline^.^ Subsütuents: F, Br, CI. Me, NHAc, NH,'.

From the Hammett equationYb (3.4), the rate constant for the unsubsMuted

compound can be estimated-

log k = log k, + po (3-4)

In this equation p is the reaction constant and o depends only on the substituent.

From the plot of log k, a,', the intercept is equal to log k, for the unsubstiuted

aniline.

The results from this plot are shown in Table 6.2.

Table 6.2 Hammett Equation Parameten for aniline8

Aniline

r 0.989

S ~ O P ~ (P) 4-40 i 0.33

log k2 8.97 i 0.1 3

k2 9.33 x I O a M" s"

a From the literature datan in Figure 6.1.

There seems to be a discrepancy between the value of 9 x I O 8 M-' S'. estimated

from the Hammett plot, and the value of 5 x I O 8 M-' s-' measured in this work. This

may have arisen because the literature results3' were obtained in fairly strong acid

and had to be extrapolated to aqueous solution.

6.2 Bromination of Arninopyrïdine

In the case of the pyridines, similar pH rate profiles were obtained, as seen

in Figure 6.3. To compare the values of k, obtained in this work for the

aminopyridines (Table 6.3). to eariier results. Two Hammett plots were constructed

from the values for six derivatives studied by Katntzky et aLn, these plots gave the

parameters presented in Table 6.4, according to which the values of k, for 2APy

and 2-DAPy should be 2 x 107 M-' s-' and 2 x 10' M" s-'. whereas our measured

values are 9.4 x 106 M-' s-' and 1.5 x 10' M-' s-' respecüvely.

Figure 6.3 pH rate profile for the bromination of 2-aminopyndine and 2dimethyC aminopyridine. The points with open syrnbols (n and O ) were excluded from the c@culations-

Table 6.3 Bromination Parameters

. -- -~

pK,

Slope

log k2 K,

k2

log k2

r

a For an ionic strength of 0.1 M. Reference 38 This work

Figure 6.4 Hammett plot for the bromination of monosubstituted 2-Arninopyridine and 2-Dimethylaminopyridine at 20°C. Substihients: Br, CI, NO,.

Table 6.4 Hammett Equation Parameters for 2-APy and 2-DAPÿ

S ~ O P ~ (P) -6.78 * 0-43 -5.80 2 0.88

log k2 7.35 i 0.22 8.40 i 0.45

k2 2.24 x 1 O7 M-l S-' 2-51 x 1 0' M-' S-'

' From the data3' in Figure 6.4.

The major source of the discrepancy for 2-APy is probably due to the limited

number of points in the Hammett plot. Only three data points were available, and

so any slight deviation in the slope may result in a large variation in the y intercept

(log ka value. It is interesting to note that the ratios of the second order rate

constants (2-DAPy12-APy) are quite sirnilar. The ratio for our work is 15.8 whereas

the ratio for the values estimated fmm the literahire is 1 1.2.

The object of stud ying aniline, 2-aminopyridine (2-APy), and their N, N-

dirnethyl derivatives, was to observe the effect of an ka-nitrogen (=N-) in a

benzene ring. From our value of k, for aniline and 2-APy, and the p+ of 4.4 obtained

by Katritrky et al?. we estimate a value of

a,' = log (9.35 x IO6) - log (5.11 x 108)14.4 = 0.4

Assuming the effects of two aza-nitrogens in a ring are additive, then for bromine

attack on 2-aminopyrimidine:

log k 2 = l ~ ( 5 . 1 1 ~103-4-4(2~0.4)=5.19

And so an estimate of k2 is 1.5 x I O 5 M-l s-'.

Altematively, we can use a value of a,* - 0.55 for aza-nitrogen, derived

earlier by Kanitzky's group.= Using this value and the same p+ gives an estimated

k, of 7.4 x 1 O3 M-' s-'. So, we expected 2-aminopynmidine (2-AP) to react with

bromine with a rate constant in the vicinity of 7400 to 150000 M-l s-'. As shown in

the next section, we actually found a value of 2.5 x I O 4 M-' se', which is within the

range mentioned above.

In a similar way, we can estimate a value of k, to be expected for bromine

attack on 2-amino4,6-dïmethylpytimïdine (2-ADP). Proceeding as above, and using

O,' = O,' = -0.31",

log k2 = 8.71 - 4.4 (2 x 0.55 + 2(-0.31)) = 6.60

60

and k, 4 x 1 O6 M1 s-l. which is very close to the value achially recorded in the next

section.

6.3 Bromination of 2-A~ninopyridirnidine (2dP)

. Before we discuss the resuîts obtained for the bromination of 2-AP, let us

brïefly look at the bromination of 2-amino-4.6dimethylpyrhidine (2-ADP). No

Iiterature values are available for this particular campound. Sinœ the studies of

aniline derivatives and aminopyndine derivatives yielded satisfactory resuits, the

assumption is that the results obtained for 2-ADP are also satisfactory. The kinetic

parameters are presented in Table 6.5.

Figure 6.5 pH rate profile for the bromination of 2-amino4.6dimethylpyrimidine.

Table 6.5 Brominatian Parameters

-- -

P& 4.8Sb

Slope 1.17 I 0-01

109 kz K, 1.92 i 0-07

k2 5.87 x 1 O6 M-l

log b 6-77

r 0.999

a For an ionic strength of O. 1 M. Reference 38

The value of k, = 5.9 x 106 M' s-' in Table 6.5 is very dose to that estimateci

above (4 x I O 6 M-' s-'). Accordingly, it is concluded that 2-ADP reads simply by

direct attack of bromine on its free base fom-

The pH rate profile of 2-aminopyrimidine is not straightfomard. as seen in

Figure 6.6. In fact, the 's" type curve indicates that more than one mechanism is

involved ,

Figure 6.6 pH rate profile for the bromination of 2-aminopyrimidine.

At low pH (O - 2) one mechanism is involved and at higher pH (2.5 - 6)

another is taking part. Thinking in ternis of kinetics, one can derive an equation,

which would correspond to the curve in Figure 6.6. Using this equation. the pK, of

2-AP could be estimated.

In order to derive a kinetic equation, one must consider which species are

reacting. The reactions were camed out under acidic conditions, therefore both the

protonated and unprotonated foms of the substrate are present in solution. If the

bromine can react with both foms, with k, as the rate constant for the bromine

reacting with the protonated form and k,' for the bromine reacting with the

unprotonated fom (Scheme 6.1). then, equation 3.5 can be derived.

Scheme 6.1 Reacüon of both forms of 2-AP with btomine in acidic solution.

The results obtained by fitüng this equation are shown in Tabie 6.6. Note, in

particular. that a pK, of 3.66 was estimated, whereas the literature pK, for 2AP is

3.54 at 20 OC? The agreement between these two vaiues is obvious and it is an

indication that the equation derived is appropriate. at least in part. Confirmation is

also brought about by the fad that the data points are lying close to the curve.

Table 6.6 Bromination Parameters

P& 3.66 i 0.05

k2 1200 i 60 M-' s-'

k2' 25200 * 1200 M-' s-'

r 0-996

a For an ionic strength of 0.1 M at 25 O C .

Any viable mechanism for the bromination of 2-AP must be in accordanœ

with the observed kinetics, the pH dependenœ of the rate. and the formation of the

observed intemiediate. As mentioned in the Introduction, the reaction of 2-AP with

bromine in water ieads inioally to the fornation of an addition intermediate, 6, rather

than a substitution product (Scheme 6.2). This formation has been confimed by

new NMR experiments describecl in the Experimental section. Conceivably, the

intermediate 6 could arise from bromine attack followed by water attack and

addition, as in Scheme 6.3.

Scheme 6.2 Formation of the intermediate in the bromination of 2-AP.

Scheme 6.3 Bromine attack followed by water attack and addition.

65

Altemativeiy. there could be the addition of water to 2-AP. followed by bromine

attack and water attack (Scheme 6.4). A simibr mechanism has been shami for the

bromination of 2- and ~pyrimidone." Part of the object of this work was to try to

differentiate between the two pathways in Scheme 6.3 and 6.4. - The mechanism in Scheme 6.4 could follow the pathway described below.

Addition of water to the fke base form 1, probabiy via the cation 2, can give 4 or 5.

Of the three hydrated species (3,4, and 5), the cation 3 would be dominant at a low

pH. However, structures 4 and 5 are strong guanidine-type bases and should be

very reactive towards bromine. Structure 5 is in gray to illustrate that the paths

leading from structures 4 and 5 are kinetically equivalent In contrast, the path 6om

the protonated fom 3 would have a different dependence on the pH. Attack of

bromine on 3.4 or 5, fdlowed by addition of a second water molecule. would yield

the observed intemediate 6.

The k', value of 2.5 x I O 4 M" s-' obtained for the bromination of 2-AP at pH

3 - 6 agrees with the range of values predicted for bromine attack on 2-AP in the

previous section. Therefore, the reaction of 2-AP seems to occur, at least in part,

in the same fashion as the bromination of aniline and 2-APy, by direct attack of

brornine, as in Scheme 6.3.

H@ CK~~ - - Ka"

Q Strong base

. . -9 9

Ka' - c~~~ 7 H H

H2

al Dominant fom

at low pH

Scheme 6.4 Proposed mechanism for the bromination of 2-aminopyrimidine.

At lover pH values (pH c 2). another mechanism is taking place and it is

harder to explain what is happening. The mechanism in Scheme 6.4 is complex

because there are sevenl equilibria involved. Of course, there is an equilibrium

between the N o fomis of the substrate. 1 and 2. but there may be smali amounts

of the hydrated cation 3 and its flee base foms 4 and 5. Since the pH rate profile

for the bromination is Rat at pH 2 (Figure 6.6) the reactive species must be the

protonated fom 2 or some other prolonateci fom in equiIibrium wwi fn. most

probably the hydrated fom 3 (Scheme 6.4). Support for the idea that the reactike

fom is the d i o n 3, in equilibrium wiai the cation 2, cornes frorn observations made

for the &on 7 (Scherne 6.5) which reacts with an observed second order rate

constant of 1 O00 M" s-', very close to the value of 1200 M-l s-' for 2. So. t is

proposed that 2 reacts via the hydrated fomi 3, and 7 reacts via its hydrated fom

10 (Scheme 6.5).

Because it is a guanidine, 1.2-dihydro-2-irnino-1 -meth yl pyrimidine (M2A) is

very basic. with a p h of 10.75~, and it exists as its cation 7 at al1 pHs studied. We

chose to study this system because the cation 7 could serve as a mode1 of the

protonated f o m of 2-AP, 2.

Scheme 6.5 Suggested pathways for the bromination of 2-AP and M2A.

The pH rate profile for the bromination of M2A is very interesüng and quite

cornplex, as seen in Figure 6.7. Assuming that this pyrimidine reacts with bromine

in a similar way as 2-AP, at least two mechanisms must be involved. one at low pH

( O to 3.5) and one or more at higher pH (4 to 7). At low pH, the pH rate profile for

7 is almost identical to the pH rate profile for the bromination of 2-AP, therefore we

propose a similar mechanism as for 2AP, as shown in Scheme 6.5. Since M2A is

very basicw, the cation 7 of the substrate is the predominant fom in solution, but

structures 8 and 1 0 could be available due to pre-equilibrium hydration, which

leaves the rate determining step to be the attack by bromine. Bromine attack on the

cation 10 could explain the pH-rate profile (Figure 6.7) at low pH.

- 1st order 0 mi>aed order

fast raie slow rate

Figure 6.7 pH rate profile for the bromination of 1.2dihydro-1 imino-1 -methyl pyrimidine.

Around pH 2. we observe a transioon from first-order kinetics (figure 6.8 (a))

through mixed order (b) to zero-order (c). Again. sirnilar behaviour was observed

previously for the bromination of 2- and 4-pyrimidone~.~ The change in order is

consistent with a change of rate-limiting step from bromine attack to hydrate

formation. To understand where these dïfterent orders arise h m , kinetic equations

(a) pH = 0.465 .

(b) pH = 2.12

Figure 6.8 Traces of (a) first-order kinetics, (b) mixedorder kinetics and (c) zero- order kinetics

We have considered the following posibili.

Scheme 6.6

The total amount of 8 will be found in two forms, namely, 8 and 10. but since

8 is a very strong base, we can make the following assumption.

and

We followed the reaction by monitoring the disappearance of bromine and

according to Scherne 6.6 the rate is given by:

rate = = k, [IO] [Bq dt

As long as 8 and 10 are in rapid equilibrium,

and substituting for [8] from (3.7)

Using the steady state assumption on ( 8 * 10) equation 4.1 = 0, and so we can

isoiate [l O].

SubstiMing for [fol in equation 3.8 yields

The above rate equation cannot explain a change of rate-lirnibing step near

pH 2 with pH because it has no term in [Hq (or [OH']) in the denominator.

Accordingly, the mechanism in Scheme 6.6 is not viable.

Consider now, another possibility.

Scheme 6.7

In this mechanism the cation 90 is formed in a pre-equilibriurn from 7, and the ftee

base fom 8 is fomied from f O. However, 8 is a strong, guanidine-type base which

would form slowly from 10 and it is an enamine so it should react rapidly with

73

bromine. We will consider 8 as a steady-state intemediate:

and so,

But K,, = [10]Im, and [IO] = K,, m

Thus, according to Scheme 6.7 the rate of bromination is

For the rate equation in (4.7) there are two exheme situations.

(i) k, [Hq >> k, [BrJ, in which case

This situation corresponds to rate-limiting attack of bromine on 8, and it requires

first order kinetics for high m and fixed [H+l. The corresponding pseudo-first

order rate constant (= k, K,, k2 m / k, [Hl) should increase with increasing pH.

(ii) k, [BrJ >> k, [Hl. In this case. the acidity is low enough that reprotonation of

8 to 10 cannot compete with fast bromine attack and

-d[BrJ/dt=k,K,J] (4.9)

In other words, the formation of 8 is the rate-limiting step and the kinetics are

pseudo-zero order.

ûverall, Scheme 6.7 and its rate equation (4.7) require the pseuddirst order

rate constant to Vary wiüi lI[H) at high [Hl whereas at low [H? there should be

pseudo-zerwrder behaviour and no pH dependence. Unfominateiy, such

behaviour was not observeci.

The third and final possbility tha will be considered is the mechanisrn shown

in Scheme 6.8.

Scheme 6.8

For this scheme,

rate = k, [ lO] [BrJ

Using the steady state assumption on 10:

d [IO] 1 dt = k, [7m - k, [IO] [Hq - k, [BrJ [IO] = O (5- 1

However, [ I H ~ = m [WK. (5-3)

and. substituting for [IO] and r H 7

This rate equation, which is appropriate for the mechanisrn in Scheme 6.8, can

explain the pH-rate profile for the bromination of 7 in the pH range O - 3.

Equation (5.6) has no dependence on the [Hl, and it corresponds to rate-limiting

attack of bromine on the hydrated cation 10, formed in a preequilibrium from the

cation 7. This mechanism is exactly that proposed earlier in Scheme 6.5.

At higher pH, if k, [BrJ >> k-, [H7 then

Equation (5.7) has a [H l term in the numerator and therefore the rate would

decrease with increasing pH. Since this equation is zero-order with respect to

bromine, the rate-limiting step involves the formation of 10 by water attack on 7H'.

Thus, the mechanism presented in Scheme 6.8 and the rate equation (5.4)

can explain the observed change in rate-limiting step. At firot glane, the dication

7H' is not easy to conceive. but it is not far-fetched since double protonation has

been observed ni the case of Barnina-2,4dimethyipyrÏmidine (6ADP) (pK, = 6.90;

pK2 = -0.14).*

By obseMng the pH rate profile for the bromination of 2-AP (Figure 6.6). one

can see that two points (O) were not taken into account to estimate the p K value.

These points were not taken into account because mixeâ order kinetics were

observed at a pH of about 2.3 - 2.5. If we consider applying a similar mechanism as

in Scheme 6.8 to 2-AP, we rnust consider the possibility of a dication. A dication

could be viable ifwe think in ternis of pK, and pK2. By camparison with 6-ADP. 2-

AP should have a pK, = 3.50 and pK, - -2 or -3. which is not far from the pHs at

which we performed the experiments. It seems likely that there is also a change of

ratelimiting step in the bromination of the cation of 2-AP, near pH 2.5, but the zero

order process never bewmes truly established because attack of bromine on the

free base form of 2AP takes over at pH > 2.5 (Figure 6.6).

Returning to the subject of the bromination of 7, at higher pH (4 - 7). the pH

rate profile is cornplex. in addition to the various orders obtained two unexpected

results were noticeâ. First, in the pH region of 4.5 to 6 for log k2- vs pH a dope of

- 1.7 was calculated, which suggests that two protons are rernoved in the reacüon.

in the case of the bromination of 2-AP a slope of - 1 had been calculated. The

second surprise was that two difFerent rates could be observed at the highest pHs.

A very rapid rate and a slow rate were seen. For the moment, we cannot explain

these various observations, and further experiments, with the same compound and

with similar derivathes. will have to be camed out to resolve this enigma.

The only problem with performing other experiments is the reproducibilïfy

factor. Whik working with the various substrates (aniline, N,N-dimethylaniline, 2-

APy, PDAPy, 2-ADP and 2-AP), the results obtained were easily reproduced. 1

whereas in the case of M2A reproducibility was very hard to attain. In a science

where precision is a key factor, substrates like M2A can rnake Our work tediow and

interesting at the same tirne.

Scheme 6.9 Proposed mechanism for the bromination of M2A.

79

Direct bromination seems to be involved for the bromination of aniline, N,N-

dimethylaniline, 2-DAPy. 2-APy and 2-ADP. These substrates exhibit a simpk linear

relationship beîween kg be and pH. The pH rate profile of the bromination of 2-AP

is slig htfy more cornplex as it is a sigrnoidal w rve. A mechanism for the bromination

of 2AP was pmposed that agrees with the data coliected. A similar mechanism was

proposed forthe bromination of M2A Further experiments will have to be perfomed

to detemine whether the mechanism is proper. The pH rate profile of the

bromination of M2A was unusual. ieading to mixed observations. New substrates

should be studied to further investigate the mechanism involved. As an example,

2-am in&methylpp*midine is available in the la boratory . It would also be

interesting to perfom substituent effect studies to evaluate the effect of the

presence or absence of a singk nitrogen group or both nitrogen groups in the

aromatic ring.

8.1 Materials

2-Aminopyrimidine, 2-amino4,6dimethylpyrirnidine. 2-aminopyrÏdine, 2-

dimethyl aminopyridine. aniline, N.Ndimethylaniline and the bromine were

purchased from Aldrich Chemical Co. 2-Aminopyrimidine and 2-amino4,6-

dimethylpyrimidine were recrystallïzed previous to their use. Standard HCI solutions

were obtained from A & C Chernicals (Montréal).

1 :2-Oihyd ro-2-imino-1 -methylpyrimidine (M2A) hyd riod ide was synthesized

by methyiation of 2-aminopyrimidine following Brown et al.?A total yield of 64.7 %

was obtained. The conesponding perchlorate salt was synthesized following Tee

et al. procedure? The total yield obtained was 79.3%. The 'H NMR shifts are

shown in Table 8.2. The 'H NMR chernical shifts are almost identical for the

reference compound (M2A) as for the newly synthesized M2A.

8.2 Kinetic Expriment.

The kinetic experiments were carried out by using a single beam stopped-

flow spectrophotometer by Applied Photophysics model SX17MV. Bromination was

followed by monitoring absorbance decrease at 267 nm (tnbromide ion band) for the

disappearanœ of bromine. In the stopped-flow apparatus the mixing is 1:l and so

the concentrations before and after mixing differ by a factor of 2. The observation

cell was kept at 25.0 k 0.1 OC. Bromine solutions were prepared in 0.1 M KBr to

maintain the ionic strength.

Table 8.1 Parameters for Brornination

Compound

-

[Br& u=~M [substrate] used before mixing, M

2-AP 5 x l o 4

1 x 104

2-ADP 5 x lo4

MZA 5 x 104

i x 104

2-APy 1 x l o 4

2-DAPY 5~ 105

aniline 5 x lo4

N, Ndimethylaniline 5 x lo4

For the pH range of 0.1 5 to 2.12, HCI solutions were used. For the pH range

of 2.20 to 3.40 chloroacetic buffers were used. For pH between 4.10 to 5-30,

acetate buffers were used. And finally for pH between 6.40 to 7.70 phosphate

buffen were used. Succinic buffers were used for a pH range of 4-40 to 5-90.'

a Succinic buffer reacts slowly with bromine, and to prevent any problems the bMer was only added to the substmte.

82

With the instrument manufacturer's software, the obsewed first order rate

constants (w were calailated by fitting the absorbance trace with a single

exponential. The absorbanœ traces were composed of 400 points. The trace

analyzed was an average of 5 - 10 individual traces. In some cases the trace

exhibited different or mixed orden, when this occuned the absorbanœ trace was

fitted with two exponentiah or a linear portion followed by a single exponential.

From the observed rate constants, the second order rate constants were

calculated using a spreadsheet (see Appendbc B). A value of kZw is calculateci by

dividing k,* by ([substratel - [Br&) for reasons given earlier.m Then is obtained

by dividing k,* by the fraction of Br, present in solution. The free bromine in

solution is influenced by two equilibria: i) formation of tribromide ion (eq. 8.1); ii)

hyd rolysis to hypobrornous acid (eq. 8.2).

K,

Br, + H20 * H' + B f + HOBr K2

For these equilibria, the constantsaa are:

In fact, under the reaction conditions used, the co~~ection for HOBr is only signifiant

The fraction of Br, is calculated using these dissociation constants, the [Br] and

[Hq and equation (8.3).

In each case where the firotorder disappearance of bromine was observeci.

rate = = kIe [BrJ dt

and it was verified that the reactions were also first order in substrate:

rate = kIe [B rJ = ktobr [SI [B rJ (8-7)

In other words, k,& values Vary with [substrate]. The relevant data are given in the

Appendix B.

8.4 p K Estimation

The pK, of 2-DAPy was estimated by t i i n g a 0.05 M solution of 2-DAPy

with a 0.10 M HCI solution at 22 OC. The experiment was camed out by monitoring

the pH change using an Accumet mode1 25 piilion metre by Fisher Scientific. The

calculations were carried using a Lotus 1-2-3 spreadsheet, based on the method

described by Albert and Serjeant?

8.5 Observation of the Bromination of 2-AP intemediate by 'H NMR

Approximately 0.005 moles of 2-AP (- 0.47 g) was dissoived in 5 ml solution

of 2 M DCI in D,O. The DCI solution was prepared by adding - 1 g of 37 wt% DCI

(F.W. 37.47) in D20 and diluting to the 5 ml mark with D20. The brornine solution

was prepared by adding 0.005 moles (- 0.79 g) of bmmine to 5 ml of D,O.

Approximately 0.5 ml of the 2-AP solution was mixed in a NMR tube with 0.5 ml of

the bromine solution and a 'H NMR spectnim was nn. In addition to the two 2-AP

peaks found at 8.65 and 7.15 ppm. two peaks characteristic of the intemediate

appeared upfieid at 5.33 and 4.49 ppm (TaMe 8.2). By adding more bromine and

therefore saturaüng the mixture. the 'H NMR spedrum showed that the intemiediate

peaks increased and the starting material peaks disappeared.

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24. S. Banerjee, The Mechanism of Bromination of Mono- and Dioxo-

Pyrimidines, M.Sc. Thesis, Sir George Williams University, 1974.

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26. W. Barbien, L. Bernardi, G. Palamidessi, and M. T. Venturi, Tefrahdmn

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34. R. D. Guth rie and J. Honeyman, introduction to Cahohydrafe Chemistry.

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O.S. Tee and S. Banerjee, J. Org. Chem., 44,18,3256,1979.

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O.S. Tee, Carbohydr. Res., 192.181 1989.

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A. A. Fedortchenko, Ph. D., work h progress.

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1992-

53. (a) O. S. Tee, The Hydrogen-Deutenum Exchange of Some Simple

Pyrimidines, Ph-D. Thesis, University of East Anglia, 1967; (b) A-R. Katntd<y,

M. Kingsland and 0. S. Tee, J. Chem. Soc. (B), 1484,1968.

54. (a) T. H. Lowry and K. S. Richardson, Mechanism and Theory h Organic

Chemistry. Td Edition, Harper & Row, New York, 1981, p. 1 31, Table 2.2; (b)

Ibid, p. 132.

55. (a) O.S. Tee and S. Banerjee, Can. J. Chem., 52.45 1, 1974; (b) O.S. Tee

and S. Banerjee, J. Org. Chem., 44, 18, 3256, 1979; (c) O S Tee and M.

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60. J.M. Pink, Can. JI chem., 48, 1 169, 1970.

Pentoses:

&DAm binose Pyranose forrn 97 %. Preferred conformation 'C,

a u - L y x o ~ Pyranose forh 99.5 1, Preferred conformation %,

a-0-ri bose Pyranose fom 76-80 %, No preferred conformation

a-D-Xylose Pyranose fom 99.5 %. Preferred conformation 'Cl

Hexoses:

a-ûlfructose Pyranose fomi 68.4 - 76.0 %, Preferred conformation 'C,

B-D-Oalactore Pyranose fom 93 - 100%. Prefened conformation 4G,

~-DGlucose Pyranose fom 100%. Preferred conformation 'C,

B-D-Mannose Pyranose fom ? 00%. Preferred confornation 4C,

Others:

Alcohols:

CYOH I

H-C-OH I

HO-C-H I

H-C-OH I

H-C-OH I

CH20H

CH20H I

HO-C-H I

HO-C-H I

H-C-OH I

H-C-OH I

Sugars with uncertain configuration andlor conformation andlor anomec

L-Rhamnose monohydrate

D-Glucosamine hydrochloride

97

Table 61 -1. Raw Data for the Bromination ([Br& = 0.0250 mM) of Aniline . (1 -00 mM) at 25°C.

Table B1.1.1 Raw Data for the Dependence of k,-on [Aniline] using [Br& = 0.0250 mM at 25OC.

Exp. # [Aniline] - [Br$, mM kto., s-'

a The experiments were performed at a pH of 1.14. The experiments were performed at a pH of 0.852.

Figure B I -1.1 The Dependenœ of kq* on the aniline concentration at a pH of 1 -14 for experiment IT-196M and a pH of 0.852 for experiment IT-197M.

Table 61.2. Raw Data for the Bromination ([Br& =0.0250 mM) of N,K dimethylaniline (0.500 mM) at 25OC.

Table 81.3. Raw Data for the Bromination of 2-Aminopyridine (1 -00 mM) at 25OC. -

lT-1 O3M

IT-96M

IT-113M

IT-111 M

IT-1 i OM

TT-99M

IT-l02M

Il-101 M

IT-1 OOM

IT-1 OSM

IT-1 O4M

IT-l12M

IT-106M

IT-107M

4.60 x I O 4

1-30 x 1 O=

3.00 x 1 O4

3.80 x I O a

8.50 x I O 4

1.52 x IO-'

3.95 x 1 Oe2

9.29 x 10'~

2.86 x 1 O-'

1.04

2.68

9.06

22.5

71 -4

a The [Br& used was 0.0500 mM for al1 the experiments with the exception of IT- 11 0M through IT-113M where0.0250 mM was used instead.

Table B1.3.1 Raw Data for the ûependenœ of k,OD. on [2-aminopyndine] using [Br$ = 0.0500 mM a 25%.

a The expenments were performed at a pH of 4.43.

Figure 81.3.1 Dependenœ of kl* on the 2aminopyridine concentration at pH=4.43.

Table B1.3.2 Raw Data for the Dependence of the Amplitude on the [Br&, at 25OC. for the bromination of Zaminopyridine.

a The experiments were performed at a pH of 4.41.

Figure B I -3.2 Dependence of the amplitude on the brornine concentration at pHa.407 for the bromination of 2-aminopyridine.

Table 61 A. Raw Data for the Bromination ([Br&, = 0.0250 mM) of 24imethyC aminopyridine (1 -00 mM) at 25OC.

Table BI .4.1 Raw Data for the Dependence of k,-on [2- dimethylaminopyridineJ using [Br& = 0.0500 rnM at 2S°C.

a The expenments were performed at a pH of 0.865.

Figure 61 -4.1 Dependence of k1* on the 2dimethylaminopyridine concentration a pH= 0.865.

Table BI .S. Raw Data for the Bromination of 2-Amin~.6dimethylpynynmidine (1 -00 mM) at 25OC.

- --

a The [Br& used was 0.0250 mM for al1 the experiments with the exception of IT- 129M where 0,0126 mM was used instead.

Table B1.5.1 Raw Data for the Dependence of kqm on [2-arnino-4.6- dimethylpyrimidine] using [Br& = 0.0500 mM at 25%.

' The experiments were perforrned at a pH of 1.44.

Figure B I .5.1 Dependenœ of kte on the 2-amin04~6dimethylpyrimidine concentration minus the bromine concentration at pH4.429.

Table B1.6. Raw Data for the Bromination of 2-Aminopyrimidine (1 -00 mM) at 25°C-

a These experiments were not taken into amunt when calculating the pK, value.

107

Table 61.6.1 Raw Data for the ûependence of kfa on [2-aminopyrimidine] using [Br& = 0.0500 mM at 25OC.

a The experiments were peiformed at a pH of 0.852. The experiments were performed at a pH of 4.34.

O 0 0.0 0.5 1 -0 1.5 2.0 2.5

[Barninopyrimidine] - [Br&, mM

Figure B I .6.1 Dependence of kIh on the 2-aminopyrimidine concentration at pH=0.852 for expenment IT-93M and a pH of 4.34 for experiment IT-94M.

Table Bi -6.2 Raw Data for the Dependence of the Amplitude on the [Br$ for the Bromination of 2-aminopyrimidine at 2S°C.

-- -

Amplitude

a The experiments were performed at a pH of 1.44.

Figure 81.6.2 Dependence of the amplitude on the bromine concentration at pH=0.852 for the bromination of 2-aminopyrimidine.

Table 61.7. Raw Osta far the Bromination aBr& =0.0250 mM) of 1.24ihydro-2- imino-lmethyipyrimidine (0.250 rnM) at 25OC.

Table Bi.7.1 Raw Data for the Dependence of k,*on [1,2-dihydro-2-imino- 1-methylpyrimidine] using [Br&, = 0.0500 mM a 25OC.

a The experiments were performed at a pH of 0.465. The experiments were perfomed at a pH of 5.56.

Figure B1.1.1 Dependence of k,* on the 1.2dihydro-2-imino-1- methylpyrïmidine concentration minus the bromine concentration at pHz0.465 for experiment IT-124M and a pH of 5.56 for experiment IT-125M.

Table B I .8. Raw aata for the Bromination (IBrJ,, = 0.0250 mM) of Synthesized 1.2- dihydro-2mino-lmethflpflmidine (0.250 mM) at 25%.

1-42 x 10''

2.06 x 1 O-'

' Two different rates were obsenred, a fast rate and a slow rate.

112

[ m: oz- KI = 0.0562 M

Scheme B1 Lotus 1-2-3 spreadsheet for the bromination of aniline.