Embed Size (px)
Citation preview
Synthesis of pyrrolidine and
5a-carbaglycosylamine derivatives as potential
bacterial transglycosylase inhibitors
Éva Papp Promotor: Prof. Dr. Johan Van der Eycken Supervisor: Dr. Jurgen Caroen A dissertation submitted to Ghent University in partial fulfilment of the requirements for the
degree of Master of Science in Chemistry A DISSERTATION SUBMITTED TO GHENT UNIVERSITY IN PARTIAL
FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF
SCIENCE IN CHEMISTRY
ACADEMIC YEAR 2016-2107
Synthesis of pyrrolidine and 5a-
carbaglycosylamine derivatives as potential
bacterial transglycosylase inhibitors
Éva Papp Student number: 01513164
Promotor: Prof. Dr. Johan Van der Eycken
Supervisor: Dr. Jurgen Caroen
A dissertation submitted to Ghent University in partial fulfilment of the requirements for the
degree of Master of Science in Chemistry
Academic year: 2017 – 2018
This document may contain confidential information proprietary to Ghent University. It is
therefore strictly forbidden to publish, cite or make public in any way this document or any part
thereof without the express written permission by Ghent University. Under no circumstance
this document may be communicated to or put at the disposal for third parties; photocopying or
duplicating it in any other way is strictly prohibited. Disregarding the confidential nature of this
document may cause irremediable damage to Ghent University.
Acknowledgement
First of all, I would like to thank my promotor prof. dr. Johan Van der Eycken for
welcoming me in the Laboratory of Organic and Bioorganic Synthesis at the University of
Ghent. The time that I have spent there – not only my master thesis, but my Erasmus internship
prior to my master program – was unquestionably a very useful and productive part of my life.
Particular thanks to my supervisor, dr. Jurgen Caroen. His daily guidance and advices
helped me tremendously to deepen my knowledge and to gain my practical experiences and on
the other hand, his enthusiasm for this project showed me a really good example which is worth
to follow.
I would like to thank Timothy De Cleyn that he made the environment in the VDE
laboratory so unique to come back and work there also on my thesis after my Erasmus
internship.
I thank Ing. Jan Goeman for the analytic work and the NMR team for all the provided
NMR data.
I am grateful that I had so helpful lab mates during this academic year, and especially I
would like to mention Tom De Smet, who was always open to discuss and to tackle problems
together.
1
Synthesis of Pyrrolidine and 5a-carbaglycosylamine Derivatives as Potential Bacterial
Transglycosylase Inhibitors
E. Pappa*, J. Caroena, and J. Van der Eyckena
a Department of Organic and Macromolecular Chemistry, Laboratory for Organic and
Bioorganic Synthesis, Ghent University, Krijgslaan 281 (S.4), B-9000 Ghent (Belgium)
*Corresponding author: [email protected]
Up till now, there is no marketed drug targeting the enzymes which
catalyze the transglycosylation reaction in bacterial cell wall synthesis;
in fact the small family of moenomycins is the only naturally occuring
known inhibitor. Using the hypothesized transition state of the
transglycosylation reaction, moenomycin-like analogues based on a
pyrrolidine scaffold were envisaged and synthesized. Their screening
against bacterial transglycosylase will aid in understanding the SAR of
these type of structures. With the aim to diversify the library of
transglycosylase inhibitors, lipid IV-like 5a-carbaglycosylamine
structures were also designed whose synthesis is based on the Schöllkopf
approach to obtain a crucial appropriately protected α,α-
cyclohexylamino acid scaffold. Using the commercially available chiral
Schöllkopf auxiliary, diverse alkylating reactions were investigated in
order to form the basic skeleton from which further research towards the
desired scaffold can be performed.
Keywords: Asymmetric synthesis, Moenomycin, Lipid IV, pyrrolidine,
5a-carbaglycosylamine, transglycosylase inhibitor, antibiotics
Introduction
Although numerous antibiotics have already landed on the market, it is very clear that
bacteria have a worrying ability to easily gain resistance to newly introduced drugs. (1) Dealing
with resistant and multiresistant phenotypes remains a major issue, which demands a
continuous development of new antibiotics, either as improved versions of prior compounds or
based on new bacterial targets.
The key feature of a successful antibacterial agent is its ability to act selectively against
bacterial cells rather than human/animal cells. (1) An outstanding difference between
eukaryotic and prokaryotic cells from the resistance point of view is the presence of a cell wall
in case of bacteria and the absence of this in prokaryotic cells.
Generally, the main component of the bacterial cell wall is peptidoglycan (PG) which forms
a net-like layer protecting the bacteria against the high osmotic pressure and providing integrity
and stability to the bacteria. (2) (3)
The two key components in the peptidoglycan biosynthesis are glycosyltransferase (GT)
and transpeptidase (TP) enzymes. Each step in the PG biosynthesis could serve as potential
target for antibacterial agents. However, the most attractive targets are situated on the outside
of the bacterial membrane (periplasm) thus potential drug molecule do not need to penetrate
the cell to exert their activity in the cytoplasm.
2
The most extensively targeted feature is the final cross-linking step catalyzed by TPs which
bind to the outer face of the cell membrane. Besides the popular use of the TP inhibitors, the
GT can also be considered to be an interesting target, as it is also situated in the periplasm and
actually often is part of the same enzymes possessing TP-activity.
As such, the direct inhibition of bacterial GTs could provide a mechanism to develop a
novel class of antibiotics. Up till now, there is no marketed drug targeting GTs, in fact the
(small) family of moenomycins is the only naturally occurring known inhibitor. (1) (2)
Moenomycin mimics the growing PG chain (also refered to as „lipid IV”), in which
interactions with the catalytic amino acid residues are crucial. The mechanism is considered to
be an SN2-type substitution at the anomeric position of the growing PG chain (lipid IV) by the
incoming nucleophilic lipid II monomer. The glutamate side chain acts as general base catalyst
to enhance the nucleophilicity of the attacking alcohol, while the lysine- and arginine-
containing pocket is responsible for the stabilisation of the pyrophosphate leaving group.
Figure 1: Transition state of glycosyl transfer with crucial catalytic residues (left), Moenomycin
and its interactions in the enzymatic pocket (right)
Moenomycins are not suitable for systemic administration due to their unfavorable
pharmacokinetic profile, however they do serve as a good template for potential derivatives.
With the aim of decreasing the molecular complexity of the moenomycin and lipid IV
templates, amine containing cyclic scaffolds were chosen, which are appropriately
functionalized to selectively introduce diverse sets of substituents to optimize binding
interactions. (2) (3)
It is expected that the amine moiety (protonated at physiological pH) would provide a
strong electrostatic interaction with the Glu residue. As such, pyrrolidine, piperidine and 5a-
carbaglycosylamine skeletons are currently investigated as potential candidates for GT
inhibition.
Figure 2: General scaffolds, studied at our laboratory and their intended GT interactions
3
Experimental
Materials
Dry solvents were dried in continuous distillation. (CaH2 for DCM and Et3N, Na and
benzophenone for THF).
1H-tetrazole (Sigma-Aldrich), n-BuLi (Sigma-Aldrich, 2.5 M in hexane), (R)-2,5-Dihydro-3,6-
dimethoxy-2-isopropylpyrazine (Sigma-Aldrich, ≥97.0%), (S)-2,5-Dihydro-3,6-dimethoxy-2-
isopropylpyrazine (Sigma-Aldrich, ≥97.0%).
Instrumentation
The 1H/31P/13C- -NMR spectra were measured at 400 MHz (Bruker Avance 400 spectrometer);
the chemical shifts are expressed in ppm. As solvents chloroform-d (7.26 ppm (1H), 77.00 ppm
(13C)) and methanol-d4 (3.31 ppm (1H)), are used. Coupling constants are given in Hz.
All LC-MS-analysis were recorded on Agilent 1100 Series HPLC with G1946-MSD equipped
with ESI-source and DAD. Column: Phenomenex Kinetex C18, 150mm x 4.6mm, particule
size = 5 μm. T = 35 , injection = 15 μm, flow = 1.5 mL/min.
Representative procedure for phosphorylation. Synthesis of 13
To a cooled (0°C) solution of 11 (80 mg, 0,164 mmol, 1,9 eq.) in dry DCM (0,5 ml), 1H-
tetrazole (0,45 M in acetonitrile, 290 µl, 0,129 mmol, 1,5 eq) and 9 (24 mg, 0,086 mmol, 1 eq)
dissolved in DCM (1,0 ml) are added. The reaction mixture is under argon atmosphere. The ice
bath is removed and the reaction mixture is stirred at room temperature for 1,5 hours. H2O2 is
added (80 µl, 30 % in H2O solution; used in excess to oxidize the phosphorus atom). After 1,5
hours of stirring time Na2S2O3 solution (1,0 ml, 10 %, aqueous solution) is added. The reaction
mixture is transferred to a separation funnel containing distilled H2O (10 ml) and extraction is
carried out with DCM (3x8 ml). Organic fractions are collected and dried on MgSO4. The
drying agent is filtered and the filtrate is concentrated under reduced pressure. The crude
mixture is purified by column chromatography (DCM: Acetone, 100% DCM →60% DCM).
Compound 13 is obtained as an oil (19 mg, 32 % yield).
Molecular formula: C37H57N2O7P, Molecular weight: 672,83 g/mol
1H NMR (400 MHz, CDCl3) – mixture of diastereoisomers and rotamers: 7,30-7,25 (m, 10H),
5,08-4,86 (m, 5H), 4,33-4,25 (m, 1H), 3,92-3,78 (m, 3H), 3,43 (d, J = 8,0 Hz, 1H), 2,73 (s, 2H),
2,57-2,14 (m, 3H), 1,81-1,36 (m, 4H), 1,18-1,10 (m, 26H), 0,80 (t, J = 5,6 Hz, 3H) ppm; 13C
NMR (100 MHz, CDCl3) – mixture of diastereoisomers: 171,1 (Cq), 136,0 (Cq), 135,7 (Cq),
135,7 (Cq), 128,7 (CH), 128,6 (CH), 128,3 (CH), 128,0 (CH), 127,9 (CH), 69,5 (CH2), 69,4
(CH2), 68,3 (CH2), 67,7 (CH2), 59,3 (CH), 58,7 (CH), 53,3 (CH2), 35,3 (CH2), 31,9 (CH2), 30,2
(CH2), 29,7 (CH2), 29,7 (CH2), 29,6 (CH2), 29,5 (CH2), 29,4 (CH2), 29,1 (CH2), 26,4 (CH3),
25,4 (CH2), 22,7 (CH2), 14,1 (CH3) ppm
31P NMR (100 MHz, CDCl3): -1,61 ppm
4
Representative procedure for the alkylation of Schöllkopf-type intermediates. Synthesis of 37
To a cooled (-78 °C) solution of the (S)-Schöllkopf-auxiliary (S)-15 (375 µl, 2,1 mmol, 1 eq)
in THF (dried, freshly distilled; 8 ml) n-BuLi in 2,5 M hexane solution (1 ml, 2,51 mmol, 1,2
eq) is added dropwise, under Ar atmosphere. The reaction mixture is stirred for 1 hour after
which a solution of 33 (805 mg, 3,14 mmol, 1,5 eq) in THF (5 ml) is added. The reaction
mixture is stirred overnight allowing to slowly warm to room temperature. A saturated aqueous
NH4Cl (15 ml) solution is added after which the reaction mixture is stirred for 3-5 minutes and
transferred to a separation funnel. Extraction is performed using EtOAc (3* 25 ml). The
combined organic layers are dried on MgSO4, the drying agent is filtered and the filtrate is
concentrated under reduced pressure. Further purification is accomplished by column
chromatography (hexane: ethylacetate 100:0 → 70:30). Compound 37 is obtained as
colorless/slightly yellow oil in 34 % yield (227 mg).
Molecular formula: C20H28N2O3, Molecular weight: 344,44 g/mol
1H NMR (400 MHz, CDCl3): 7,29-7,25 (m, 4H), 7,24-7,16 (m, 1H), 5,08 (s, 1H), 4,91 (s, 1H),
4,42 (s, 2H), 4,10-4,06 (m, 2H), 3,93 (d, J=5.3 Hz, A part of AB system, 1H), 3,87 (d, J=13.0
Hz, B part of AB system, 1H), 3,83-3,82 (t, J=11.5 Hz, 1H), 3,60 (s, 3H), 3,54 (s, 3H), 2,60-
2,56 (dd, J=14.4 Hz, 1H), 2,37-2,32 (dd, J = 14.0, 7.2 Hz, 1H), 2,22-2,12 (m, 1H), 0,96 (d, J=6.9
Hz), 0,60 ppm (d, J=6.8 Hz) ppm
13C NMR (100 MHz, CDCl3):163,5 (Cq), 163,3 (Cq), 142,6 (Cq), 138,5 (Cq), 128,4 (CH), 128,3
(CH), 127,7 (CH), 127,5 (CH), 127,7 (CH), 127,6 (CH), 127,5 (CH), 114,3 (CH2), 73,5 (CH2),
71,9 (CH2), 60,7 (CH), 55,5 (CH), 52,3 (CH3), 52,2 (CH3), 37,4 (CH2), 31,6 (CH), 19,1 (CH3),
16,6 (CH3) ppm
Results and Discussion
Moenomycin-inspired pyrrolidine based analogues
Earlier work at the laboratory has revealed that structure 1 possesses transglycosylase
inhibitory activity (70 % inhibition, 500 µM). This is rather remarkable, as the compound has
a relatively simple structure, although the necessary features expected for TG binding are
present (amine, amide and phospholipid characteristics). As such, we are interested in the
synthesis of further analogues to determine the SAR and to improve the binding capacity.
Figure 3: Lead structure 1 and newly-synthesized pyrrolidine derivatives 2-5
The total synthesis of the pyrrolidine analogues is based on the coupling of two main
building blocks: N-diisopropyl,O-benzyl,O-n-hexadecylphosphoramidite 12 and a proline
5
derivative 8 or 9 obtained from the commercially available trans-4-hydroxy-D-proline (Scheme
1). The proline-based building block is obtained via the protection of trans-4-hydroxy-D-
proline 6 as benzyl carbamate using benzyl chloroformate. Next, the carboxylic acid moiety
can be activated using EDC and HOAt, and coupled to commercially available MeNH2.HCl
and isopropyl amine, respectively. Crucial benzyl bisamidite 12 was prepared via treatment of
PCl3 with diisopropylamine to give chlorophosphine 10, which was converted to 11 using
benzyl alcohol. The phosphoramidite 11 is then reacted with 1-hexadecanol to successfully
obtain building block 12. In a one-pot reaction sequence, the decoration of the hydroxyproline
scaffold is done by the phophorylation of the available hydroxyl function.
Scheme 1: Synthesis route towards intermediates 13 and 14
The last step of the synthesis strategy of the pyrrolidine-based analogues is the
simultaneous deprotection of the amine and phosphate functionalities by removing the Z- and
the Bn-groups (Scheme 2). This step is performed by hydrogenolysis using heterogeneous
catalysis applying Pd/C under hydrogen atmosphere for 1,5 to 4,5 hours. In this way, target
structures 2 and 3 were obtained with 77 % and 64 % yield, respectively.
To obtain the corresponding isopropylated target compounds 4 and 5, we additionally
performed the hydrogenation in the presence of acetone. Reaction were run for 2-5 hours to
ensure complete conversion (monitoring by TLC and 1H-NMR, NMR purity is >95%).
Compound 4 was produced with 88 %, compound 5 with 74 % yield.
Scheme 2: Hydrogenation reactions to obtain final compounds 2-5
6
Synthesis strategy towards 5a-carbaglycosylamine scaffolds
In addition to the synthesis of moenomycin-inspired analogues, our laboratory is also
interested in the synthesis of lipid IV-resembling aza-analogues. Next to the classic iminosugar-
based derivatives, we wish to explore the 5a-carbaglycosylamine scaffold 25 for analogue
synthesis. Our retro-synthetic approach to obtain this 5a-carbaglycosylamine scaffold is
depicted in Scheme 3. The focus of this work is on a double stereoselective alkylation of the
Schöllkopf chiral auxiliary in order to obtain the desired amino acid intermediate 22. The
synthesis of uncommon α,α-cyclohexylamino acids is known to be challenging as consequence
of the central quaternary center. Our approach is based on the use of a heterocyclic chiral
auxiliary in asymmetric synthesis, combined with the efficiency of ring-closing metathesis.
Scheme 3: Retrosynthetic approach of the 5a-carbaglycosylamine derivatives using central
scaffold 25
Using the so-called Schöllkopf-bislactim ether 15, regioselective metalation by n-BuLi
results in a delocalized planar anion which then reacts diastereoselectively with alkyl halides as
shown in Figure 4. (4) Because of sterical reasons, the alkyl residue enters trans to the bulky
isopropyl group on C5. Stereoselectivities are usually excellent (>95%) when R≠H. (5) The
new optically active amino acid derivative can be liberated from the auxiliary after a hydrolysis
step.(5)
Figure 4: Structure of the deprotonated (R)-Schöllkopf auxiliary and principle of asymmetric
induction (left), Liberation of the optically active amino acids via acid hydrolysis (right)
7
The synthesis strategy we propose, involves the use of homochiral hydroxyl butenyl
fragments 17, for the one step alkylation of Schöllkopf auxiliary 15. Actually, a prior test
reaction using the iodide 27 delivered the desired compound 18 in reasonable yield. Encouraged
by this preliminary result, we wanted to optimize and complete the reaction sequence to
intended scaffold 25. To this aim, compounds 28-31 were prepared and tested in the first
alkylation step.
Scheme 4: Synthesis strategy towards the protected 3-hydroxy-5a-carbaglycosylamine scaffold
25
Route starting from (R)-Schöllkopf auxiliary
First alkylation step:
After deprotonation of (R)-15 using n-BuLi at -78°C, different derivatives of building
block 17 (27-31) were added, the reaction mixture was stirred overnight allowing to slowly
warm to room temperature.
To our surprise, none of these reagents were found to be more effective for this
transformation than 27 (used for a trial reaction). Only in case of tosylate 29 could the formation
of product 18 be observed, but the isolated yield was very low (14 %). In all other instances,
the desired compound was not detected (TLC, LC-MS); instead complex mixtures were
observed.
TABLE I. Summary of the first alkylation step on (R)-Schöllkopf auxiliary
Entry X Yield Remark
27 I 43 % Fast degradation of the alkylating agent
28 OMs - Complex mixture containing mainly starting material and some degradation products, no product detected
29 OTs 14 % Complex mixture, difficult separation, several side products
30 ONs - Complex mixture containing mainly starting material and some degradation products, no product detected
31 Br - Very complex mixture, no product detected
8
Taking all these observations into account, the best outcome was achieved using freshly
made batches of 27 for this first alkylation step on the (R)-Schöllkopf auxiliary. Interestingly,
according to NMR and LC-analysis, only one diastereoisomer could be detected. We assume
the formation of the trans diastereoisomer.
Second alkylation step
Prior research in our laboratory has shown that alkylation of 32 using mesylate 33 is
feasible (Scheme 5, left). Therefore, we decided to also use this alkylating condition for the
alkylation of 18 (Scheme 5, right).
Scheme 5: The extra OMEM-substituent has a detrimental influence on the second Schöllkopf
alkylation step
After deprotonation of 18 using n-BuLi at -50°C, mesylate 33 was added at -78°C and
reaction mixture was stirred overnight while slowly raising the temperature to room
temperature. The outcome of this reaction was highly disappointing as only trace amounts of
the desired product could be detected within the complex reaction mixture (LCMS-analysis of
the crude reaction mixture). We thus decided to synthesize the corresponding bromide 35 for
alkylation of 18, however no desired product could be detected. Instead, a highly complex
mixture was observed.
From these test reactions, it is clear that the additional presence of the protected alcohol
moiety is detrimental for its reactivity. Most likely steric hindrance factors are to be held
responsible for this observation. If indeed, the MEM group causes the low outcome of this
specific step, one solution could be the removal of this protecting group and the replacement
by another, smaller one could improve the success rate of the reaction.
Importantly, the observed low yields of first and second alkylation of (R)-15 could be the
result of a mismatch situation between the stereo centers present. Therefore, we also wished to
investigate the reversed order of alkylation of the corresponding enantiomeric (S)-15 as starting
Schöllkopf auxiliary, which after the removal of the auxiliary should deliver the same target
amino acid (Scheme 6).
9
Scheme 6: Investigated approaches towards 24
Route starting from (S)-Schöllkopf auxiliary
First alkylation step:
Using the available mesylate 33, alkylation of the commercially available (S)-Schöllkopf
compound (S)-15 was performed under the previously established conditions. Interestingly, the
uniform formation of only one diastereoisomer was observed. However, the isolated yield was
disappointingly low (34 %), partly because of difficult purification from the complex reaction
mixture.
Scheme 7: Double alkylation of (S)-Schöllkopf auxiliary to obtain intermediate 38
Second alkylation step on (S)-Schöllkopf auxiliary:
Because of the extensive prior investigation of the alkylation of (R)-15 with different
alkylating agents, we choose iodide 27 for this transformation. As such, we were pleased to
observe the successful isolation of double-alkylated compound 38 (49 %) using the conditions
developed before (deprotonation at -50°C, alkylation at -78°C). However, LCMS analysis
showed the presence of a side product as a shoulder on the main peak displaying an identical
mass spectrum as to the main product. NMR analysis also showed two sets of highly similar
signals (ratio 87/13). These data could be interpreted as the formation of two epimeric structures
as consequence of poor diastereoselective alkylation.
However, this seems highly unlikely considering the previously obtained high
diastereoselectivities. Of course, a possible mismatched situation could be occurring, leading
to the observed diastereoisomeric ratio. Alternatively, it may be hypothesized that because of
sterical reasons, n-BuLi treatment could have led to deprotonation at the valine position,
causing epimerization at this center.
Repeating the reaction on large scale, however, delivered impure 38 containing only one
diastereoisomer according to LCMS analysis. Because of time constrains, further purification
10
and more detailed analytical investigation of that specific batch and of subsequent synthetic
steps could not be pursued.
Conclusion
As part of an ongoing SAR study on the TG inhibitory activity of moenomycin inspired
pyrrolidine derivatives, four additional analogues (2, 3, 4 and 5) were successfully synthetised
and their screening is in progress. The determination of their TGase inhibition properties will
contribute to the understanding of the Structure-Activity Relationship necessary for potential
future antibiotic development.
Regarding the synthetic route to obtain 5a-carbaglycosylamine derivatives, the crucial
ingredient to success seems to be the choice of Schöllkopf starting enantiomer and the order of
alkylation, probably because of mismatch reasons. Thus, the change to the (S)-Schöllkopf chiral
auxiliary as starting material was an important step forward in this synthesis route. However,
there is definitely room for improvement as the current yields are not optimal. One of the
problems which has to be tackled is in connection with the alkylating reagents, either looking
at iodide 27 (fast degradation) or talking about mesylate 33 (non-identified persistent impurity).
Another possible solution could be the replacement of the MEM protection of the hydroxyl
group to determine its influence in these alkylation steps.
Acknowledgments
The authors would like to thank Ing. Jan Goeman for the LC-MS analyses and NMR department
for all the provided NMR measurements.
Bibliography
1. Patrick G.L. An introduction to medicinal chemsitry. 5th ed. 2013.
2. Vollmer W, Blanot D, De Pedro MA. Peptidoglycan structure and architecture. FEMS
Microbiol Rev. 2008;32(2):149–67.
3. Zuegg J, Muldoon C, Adamson G, McKeveney D, Le Thanh G, Premraj R, et al.
Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity. Nat
Commun [Internet]. 2015;6:1–11. Available from: http://dx.doi.org/10.1038/ncomms8719
4. Schollkopf BU, Groth U, Hiinig S. Enantioselective Synthesis of (R)-Amino Acids Using L-
Valine as Chiral Agent[**]. 1981;1187(9):798–9.
5. Ulrich Schöllkopf. Enantioselective synthesis of non-proteogenic amino acids via metallated
bis-lactim ethers of 2,5-diketopiperazines. Tetrahedron. 1983;39:2085–91.
1
TABLE OF CONTENT
TABLE OF CONTENT ..................................................................................................................... 1
LIST OF ABBREVIATIONS .............................................................................................................. 3
LIST OF FIGURES .......................................................................................................................... 4
LIST OF SCHEMES ........................................................................................................................ 4
I. INTRODUCTION .................................................................................................................... 5
I.1. Brief historic overview of antibiotics and the rise of antimicrobial resistance .......................................... 5
I.2. Cell wall synthesis as a potential target ........................................................................................................ 7
I.3. Glycosyltransferase inhibition as potential antibacterial strategy ............................................................. 9
II. GOALS ................................................................................................................................ 13
III. RESULTS AND DISCUSSION ............................................................................................. 16
III.1. Synthesis of moenomycin-inspired derivatives ....................................................................................... 16 III.1. 1. General synthesis route of moenomycin inspired pyrrolidine-based analogues .................................. 16 III.1. 2. Synthesis of scaffold 15 ....................................................................................................................... 17 III.1. 3. Synthesis of benzylhexadecyldiisopropylphosphoramidite 24 ............................................................ 18 III.1. 4. Coupling of building blocks ................................................................................................................ 19 III.1. 5. Removal of the protecting-groups and N-alkylation ............................................................................ 20 III.1. 6. Conclusion and future perspectives of the pyrrolidine-based family ................................................... 21
III.2. Synthesis of lipid IV inspired 5a-carbaglycosylamine derivatives ........................................................ 22 III.2 1. Synthesis strategy towards 5a-carbaglycosylamine scaffolds ............................................................... 22 III.2. 2. Previous work on a simplified structure .............................................................................................. 24 III.2. 3. Literature precedent ............................................................................................................................. 26 III.2. 4. Current work: route starting from (R)-Schöllkopf auxiliary ................................................................ 28
III.2. 4.1. First alkylation step ...................................................................................................................... 29 III.2. 4.2. Second alkylation step .................................................................................................................. 33
III.2. 5. Route starting from (S)-Schöllkopf auxiliary ...................................................................................... 36 III.2. 5.1. First alkylation step ...................................................................................................................... 36 III.2 5.2. Second alkylation step ................................................................................................................... 37
III. 2. 6. Conclusion .......................................................................................................................................... 39
IV. EXPERIMENTAL PROCEDURES ........................................................................................ 40
IV.1. Pyrrolidine-based compounds .................................................................................................................. 41 IV.1. 1. Synthesis of iPr2N-P(OBn)-OC16H33 (24) ............................................................................................ 41 IV.1. 2. Synthesis of Z-D-Hyp(OPO(OBn)OC16H33)-methylamine (27) ....................................................... 42 IV.1. 3. Synthesis of Z-D-Hyp(OPO(OBn)OC16H33)-isopropyl amine (28) ..................................................... 44 IV.1. 4. Synthesis of 5H-D-Hyp(OPO(OH)OC16H33)-methylamine (11) ...................................................... 45
2
IV.1. 5. Synthesis of iPr-D-Hyp(OPO(OH)OC16H33)-methylamine (13) ....................................................... 46 IV.1. 6. Synthesis of 5H-D-Hyp(OPO(OH)OC16H33)-isopropyl amine (12) ................................................. 48 IV.1. 7. Synthesis of iPr-D-Hyp(OPO(OH)OC16H33)-isopropyl amine (14).................................................. 49
IV.2 Synthesis of compounds in order to obtain 5a-carbaglycosylamine scaffolds ....................................... 51 IV.2. 1. Synthesis of (S)-2-((2-methoxyethoxy)methoxy)but-3-en-1-yl 4-methanesulfonate (65) ................... 51 IV.2. 2. Synthesis of (S)-2-((2-methoxyethoxy)methoxy)but-3-en-1-yl 4-methylbenzensulfonate (66) .......... 52 IV.2. 3. Synthesis of (S)-2-((2-methoxyethoxy)methoxy)but-3-en-1-yl 4 nitrobenzene sulfonate (67) ........... 53 IV.2. 4. Synthesis of (S)-4-bromo-3-((2-methoxyethoxy)methoxy)but-1-ene (68) .......................................... 54 IV.2. 5 Synthesis of (S)-4-bromo-3-((2-methoxyethoxy)methoxy)but-1-ene (60) ........................................... 55 IV.2. 6. Synthesis of (2R)-2-isopropyl-3,6-dimethoxy-5-((R)-2-((2-methoxyethoxy)methoxy)but-3-en-1-yl)-
2,5-dihydropyrazine (31) .................................................................................................................................. 56 IV.2. 7. Synthesis of 2-((benzyloxy)methyl)prop-2-en-1-ol (40) ..................................................................... 57 IV.2. 8. Synthesis of 2-((benzyloxy)methyl)allyl methanesulfonate (65) ......................................................... 58 IV.2. 9. Synthesis of (((2-bromomethyl)allyl)oxy)methyl)benzene (68) .......................................................... 59 IV.2. 10. Synthesis of (5S)-2-(2((benzyloxy)methyl)allyl)-5-isopropyl-3,6-dimethoxy-2,5-dihydripyrazine (73)
.......................................................................................................................................................................... 60 IV.2. 11. Synthesis of (2S,5S)-2-(2-((benzyloxy)methyl)allyl)-2-(but-3-en-1-yl)-5-isopropyl-3,6-dimethoxy-
2,5-dihydropyrazine (74) .................................................................................................................................. 61 IV.2. 12. Synthesis of (2S,5S)-2-(2-((benzyloxy)methyl)allyl)-5-isopropyl-3,6-dimethoxy-2-(2-((2-
methoxyethoxy)methoxy)-but-3-en-1-yl)2,5-dihydropyrazine (72) ................................................................. 63
V. REFERENCES ....................................................................................................................... 65
3
List of abbreviations ACN Acetonitrile
APCI Atmospheric Pressure Chemical Ionization
Ar Argon
Bn Benzyl
Cbz Carboxybenzyl
DCM Dichloromethane
DMAP 4-dimethylaminopyridine
EDC Ethyl-3-dimethylaminopropylcarbodiimide
ESI Electrospray Ionization
EtOAC Ethyl acetate
HPLC High-performance liquid chromatography
HOAt 1-Hydroxy-7-azabenzotriazole
IR Infrared spectroscopy
NAG ß-1,4-Linked-N-acetylglucosamine
NAM N-acetyl-muramic acid
MEM 2-methoxyethoxymethyl ether
MHz Mega hertz
Ms Mesyl
n-BuLi n-Butyl lithium
NEt3 triethylamine
NMR Nuclear magnetic resonance
Ns Nosyl
PBP Penicillin Binding Protein
(P)GT (Peptidoglycan-)glycosyltransferase
Pd Palladium
ppm Parts per million
TG Transglycosylase
Ts Tosyl
TLC Thin Layer Chromatography
TP Transpeptidase
Z Carboxybenzyl
4
List of figures
1. figure Salvarsan, Penicillin G, Tetracycline, Valinomycin and Cephalosporin ..................... 5
2. figure: The last two steps of bacterial cell wall synthesis7 ..................................................... 7
3. figure: Lactam hydrolysis by ß-lactamases ............................................................................ 8
4. figure. Structure of vancomycin bound to terminal D-Ala-D-Ala units9 ............................... 9
5. figure: Steps of bacterial cell wall biosynthesis and structure of the GT ligands. (A) Structure
of moenomycin A, an analogue of the growing glycan chain which binds to the donor site (B)14
.................................................................................................................................................. 10
6. figure: Left – Transition state of glycosyl transfer with crucial catalytic residues, Right –
Moenomycin and its most important interactions in the enzymatic pocket ............................. 11
7. figure: Molecular structure of TS30663 ............................................................................... 11
8. figure: Active compounds discovered by other groups ........................................................ 12
9. figure: General scaffolds and their intended interactions within the GT-enzyme target ..... 13
10. figure: Active compounds synthetized by the group of Wong18,19 ..................................... 14
11. Figure: Template compound 2 and pyrrolidine based targets ............................................ 16
12. figure: Synthesis route to obtain 14 and 15 ........................................................................ 17
13. figure: Reaction steps to obtain compound 24 ................................................................... 18
14. figure: Phosphorylation reaction scheme ........................................................................... 19
15. figure: Mechanism of the phosphorylation reaction .......................................................... 19
16. figure: Hydrogenation reactions ......................................................................................... 20
17. figure: Left: (R)-Schöllkopf auxiliary and its vulnerable positions, Right: Structure of the
deprotonated (R)-Schöllkopf auxiliary and the principle of stereoselective induction ............ 23
18. figure: Hydrolysis of the chiral auxiliary ........................................................................... 24
19. figure: Outcome of alkylation of 42 with vinyloxirane ...................................................... 27
20. figure: Non-diastereoselective formation of 57 and 58 via oxirane-alkylation, oxidation and
vinylmagnesium bromide addition ........................................................................................... 28
21. figure: Investigation in different alkylating agents ............................................................ 31
22. figure: Mechanism of the Appel reaction ........................................................................... 32
23. figure: First alkylation step of (R)-6 .................................................................................. 32
24. Figure: Second alkylation step on (R)-Schöllkopf intermediates ...................................... 34
25. figure: Second alkylation step using 69 ............................................................................. 35
26. Figure: First alkylation step on (S)-Schöllkopf auxiliary using mesyate 41 ...................... 37
27. figure: Second alkylation step on (S)-Schöllkopf auxiliary using iodide 60 ..................... 37
28. figure: Second alkylation step on (S)-Schöllkopf auxiliary using 4-bromobutene ............ 38
List of schemes 1. Scheme: Retrosynthetic approaches for this thesis work ..................................................... 15
2. Scheme: Retrosynthetic approach of the pyrrolidine based target compounds ................... 17
3. Scheme: Retrosynthetic approach of the 5a-carbaglycosylamine derivatives ..................... 22
5. Scheme: Synthesis strategy towards the 3-deoxy-5a-carbaglycosylamine scaffold ............ 25
5. Scheme: Synthesis strategy towards the 3-hydroxy-5a-carbaglycosylamine scaffold ......... 29
6. Scheme30:Synthesis of iodide 60 from (R)-glycidol ............................................................ 30
7. Scheme: Synthesis of 41 ...................................................................................................... 33
8. Scheme: Replacement of the MEM-protection .................................................................... 35
9. Scheme: Two different approaches to achieve 37 starting from (R)- and (S)-Schöllkopf
auxiliary .................................................................................................................................... 36
5
I. Introduction
I.1. Brief historic overview of antibiotics and the rise of antimicrobial resistance
Among many other success stories of medicinal chemistry, the fight against bacterial
infections has a history that started more than 100 years ago when Ehrlich successfully
synthetized the very first antimicrobial drug, Salvarsan1.
During the last century, several compound families were discovered and used later on to
treat bacterial infections. Not only the world-wide famous penicillin – and then the derivatives
based on its structure - but also the sulfonamide-types antibacterial agents served as a good base
for drug development studies in the second part of the 20th century.1,2
The second World War also initiated the extension of the antibiotic agent arsenal and
research provided effective compounds such as the tetracycline-based derivatives, peptide
antibiotics (e.g. valinomycin) and the discovery of additional ß-lactam-type antibiotics (such as
cephalosporin C)2.
1. figure Salvarsan, Penicillin G, Tetracycline, Valinomycin and Cephalosporin
Although numerous compounds have landed on the market, it is very clear that bacteria
have a worrying ability to easily gain resistance to newly introduced drugs. The positive effect
of antibacterial agents in the last few decades is unquestionable, which is manifested in the
notable drop in death cases caused by bacterial infection. However, dealing with resistant and
6
multiresistant phenotypes remains a major issue, which demands a continuous development of
new antibiotics, either as improved versions of prior compounds or based on new bacterial
targets.
Basically, two groups of antibiotics can be distinguished: bactericidal drugs induce cell
death while bacteriostatic drugs aim to inhibit bacterial growth and division. Our interest is
focused on the first group, the bactericidal drugs, which can be further divided according to
their mode of action1:
a) Inhibition of cell metabolism
b) Inhibition of nucleic acid transcription and replication
c) Disruption of protein synthesis
d) Interaction with the plasma membrane
e) Inhibition of bacterial cell wall synthesis
The key feature of a successful antibacterial agent is its ability to act selectively against
bacterial cells rather than human/animal cells.1 Distinguishing between eukaryotic and
prokaryotic cells can be done in several ways, but an outstanding difference from the resistance
point of view is the presence of a cell wall in case of bacteria and the absence of this in
prokaryotic cells. The cell wall has a very important role in the organism’s survival rate. This
layer is responsible for the protection of the cell against various environmental conditions, such
as different pH or changing osmotic pressure.1 In absence of the cell wall, osmotic pressure
would cause the continuous entry of water into the cell resulting in swelling and eventual
bursting of the cell.
Bacteria fall into two subtypes based on their cell wall thickness: Gram-positive (20-40
nm) and Gram-negative (2-7 nm) bacteria.1 Gram-negative bacteria also have an additional
outer membrane built up by lipopolysaccharides - opposite to Gram-positives where this extra
membrane layer is lacking. Due to their different structures, their vulnerable points which can
be targeted by drugs also have to be distinguished.1 In general, more resistant strains are
observed in the family of the Gram-negative bacteria in comparison with the Gram-positive
bacterial species.
7
I.2. Cell wall synthesis as a potential target
Generally, the main component of the bacterial cell wall is peptidoglycan (PG) which
forms a net-like layer protecting the bacteria against the high osmotic pressure and providing
integrity and stability to the bacteria3,4. Peptidoglycan is made up of peptide and sugar units and
is essentially a polymer, formed from the monomeric building block lipid II.
These lipid II units are also called glycan units, containing ß-1,4-linked N-acetyl
glucosamine (NAG) and N-acetylmuramic acid (NAM) moieties in an alternating pattern. The
PG is built up of glycan units4,5. (Figure 2)
Short pentapeptide chains are bound to the MurNAc sugar units which contain D-Ala
units. Looking at the general biochemical composition of compounds playing role in human
biochemical regulatory steps, only L-amino acids are present. In contrast, bacteria can have
racemase enzymes which can convert L-amino acids to D-amino acids.1
The two key components in the peptidoglycan biosynthesis are glycosyltransferase (GT)
and transpeptidase (TP) enzymes. The role of the GT is to catalyze the carbohydrate
polymerization step of the lipid II while TPs are responsible for the cross-linking of the
pentapeptide chains by the displacement of D-Ala causing the final mesh-like network of PG.
Both catalytic steps are often performed within the same protein complex, as these so-called
penicillin-binding proteins (PBPs) contain both TP and GT domains. 6
2. figure: The last two steps of bacterial cell wall synthesis7
PGT= peptidoglycan glycosyl transferase, MmA= moenomycin A
8
Each step in the PG biosynthesis could be a vulnerable point and could serve as potential
target for antibacterial agents. However, the most attractive targets are situated on the outside
of the bacterial membrane (periplasm); thus potential drug molecules do not need to penetrate
the cell to exert their activity in the cytoplasm.
As such, targeting the extracellular part of the bacterial cell wall has been the subject of
lots of research. The most extensively targeted feature is the final cross-linking step catalyzed
by TPs which bind to the outer face of the cell membrane. As a result of TP-inhibition, the cell
wall framework is no longer interlinked and becomes fragile, leading to swelling of the bacterial
cell because the cell wall is no longer capable to prevent the water in-flow. 1
Penicillin derivatives as well as other ß-lactam antibiotics are acting as a mimic for the
D-AlaD-Ala moiety of the lipid II pentapeptide chain, which can serve as a good explanation
about their lack of toxicity.1 Because neither human nor animal protein segments contain D-
amino acids, it is unlikely that any of the human serine protease enzymes would recognize that
specific segment.1
However, the efficacy of the ß-lactam derivatives is limited due to the widespread
resistance to this type of antibiotics, as expressed ß-lactamases destroy the active compound by
lactam hydrolysis. This problem can be tackled by using a combination therapy with ß-
lactamase inhibitors, for example combined treatment with clavulanic acid (Figure 3).1,5,8
3. figure: Lactam hydrolysis by ß-lactamases
Besides ß-lactams, other kinds of antibiotics can inhibit the last stage of peptidoglycan
polymerization (GT and TP) such as glycopeptides and lantibiotics. Their cell wall synthesis
inhibition is based on the binding to the lipid II monomer or the unfinished growing PG chain
preventing the GT/TP enzymes to complete the PG synthesis (due to sterical hindrance).5
As an example, the glycopeptide vancomycin is a powerful antibiotic which acts by
binding to the D-AlaD-Ala sequence of the PG substrate and it is therefore used as a last-resort
treatment for drug-resistant infections.1,9
9
4. figure. Structure of vancomycin bound to terminal D-Ala-D-Ala units9
I.3. Glycosyltransferase inhibition as potential antibacterial strategy
Besides the popular use of the TP inhibitors as described above, the GT can also be
considered to be an interesting target, as it is also situated in the periplasm and actually often is
part of the same enzymes possessing TP-activity. Instead of binding to the substrate (as for
example vancomycin does), an alternative way of inhibition of the glycosylation process would
be the direct interaction with the enzyme itself. Up till now, there is no marketed drug targeting
GTs, in fact the (small) family of moenomycins is the only naturally occurring known inhibitor
(Figure 5-B). The most relevant compound of this family is moenomycin A which was first
described in 196510. Moenomycins are phosphoglycolipids containing the unique structural
element of 3-phosphoglyceric acid (3-PG)11,10. This compound is produced by Streptomyces
ghanensis, S. bambergiensis, S ederensis and S. geysiriensis. 12
Their common acting mechanism is based on the inhibition of peptidoglycan
glycosyltransferases involved in the cell wall synthesis.12 Despite their promising minimal
inhibitory concentration (1 ng/ ml to 100 ng/ml), moenomycins were neglected for long time
due to their suboptimal pharmacokinetic properties. However, they became successfully
commercialized and widely used as animal growth promoter (also known as flavomycin).
During the last decades, their usage was debated and there were contradictory opinions
regarding their use by humans. In the European Union, they were banned in contrast to the
10
United States where these compounds have been widely and successfully used for decades.
However, a promising feature in this case is that no animal microflorae have been shown to be
significantly resistant to moenomycin.12,13
5. figure: Steps of bacterial cell wall biosynthesis and structure of the GT ligands. (A) Structure of moenomycin A, an analogue
of the growing glycan chain which binds to the donor site (B)14
In Figure 5, the schematic representation of the GT domain catalyzing the glycan chain
elongation is represented. The elongating glycan chain binds to the donor site, while the lipid
II occupies the acceptor site. Catalysis occurs by deprotonation of the 4-OH group of lipid II
by a basic residue (E233 in PBP1b) followed by nucleophilic attack on the C1 of the growing
chain and departure of the undecaprenylpyrophosphate.14
The interaction of moenomycin with bacterial GTs has been vital to the understanding of
binding modes and mechanism of the glycosyl transfer reaction. From co-crystallisation
experiments and subsequent X-ray structural analysis, it has been demonstrated that
moenomycin mimics the growing PG chain (also refered to as „lipid IV”), in which interactions
with the catalytic amino acid residues are crucial. The glycosyl transfer mechanism is now
considered to be an SN2-type substitution at the anomeric position of the growing PG chain
(lipid IV) by the incoming nucleophilic lipid II monomer. The glutamate side chain acts as
general base catalyst to enhance the nucleophilicity of the attacking alcohol, while the lysine-
11
and arginine-containing pocket is responsible for the stabilisation of the pyrophosphate leaving
group. Both the transition state and the binding mode of moenomycin are shown in Figure 6.
6. figure: Left – Transition state of glycosyl transfer with crucial catalytic residues, Right – Moenomycin and its most
important interactions in the enzymatic pocket
Moenomycins are not suitable for systemic administration due to their unfavorable
pharmacokinetic profile arisen from their lipophilic characteristic4, however they do serve as a
good template for potential derivatives.
As such, SAR has identified the central E-F disaccharide as the pharmacophore, along
with the phosphoglycerate moiety and a lipophilic tail. This has triggered the synthesis of
compound libraries, identifying TS30663 as active inhibitor.15
7. figure: Molecular structure of TS30663
12
Additionally, other moenomycin-inspired saccharide analogues have been reported as GT
inhibitors (e.g. ACL20215 and ACL20964 are two active monosaccharide derivatives4,
ACL19273 containing disaccharide unit16), other weak inhibitors are clear analogues of lipid
II (e.g. Compound 2114 and Compound 517).
8. figure: Active compounds discovered by other groups
13
II. Goals
Using the hypothesized transition state of the transglycosylation reaction (Figure 6-left),
the Laboratory for Organic and Bio-Organic Synthesis (LOBOS) is engaged in the creation of
molecular libraries based on Moenomycin- and Lipid IV-inspired structures to obtain potential
inhibitors without the pharmacokinetic limitations of Moenomycin A.
Inspired by the well-known mode of action of iminosugar derivatives in the inhibition of
glycosidases, the use of an appropriately oriented amine (present as ammonium at physiological
pH) would be expected to engage in a strong electrostatic binding interaction with the catalytic
carboxylate residue.
9. figure: General scaffolds and their intended interactions within the GT-enzyme target
For the purpose of decreasing the molecular complexity of the moenomycin and lipid IV
templates, simplifications in the overall substitution patterns are necessary. We therefore aim
to use amine containing cyclic scaffolds, which are appropriately functionalized and
differentiated to selectively introduce diverse sets of substituents to optimize binding
interactions. As such, pyrrolidine, piperidine and 5a-carbaglycosylamine skeletons are
currently investigated as potential candidates for GT inhibition (Figure 9).
Actually, this idea of iminosugar-type GT inhibition has recently been explored by the
group of Wong leading to active compounds such as Compound 3118 and Compound 119, but
their synthetic strategy is not compatible with the introduction of diverse iminosugar moieties.
14
10. figure: Active compounds synthetized by the group of Wong18,19
As a result of previous research at our laboratory, a series of analogues of the pyrrolidine
and piperidine scaffolds A-B-C (Figure 9) have already been tested showing promising
biological activity (see Table 1).
Among those are 1 and 2, which are the lead structures for further research.
This thesis thus forms a part of a larger research project aiming to provide new
antibacterial compounds with transglycosylase inhibitory activity based on five- and six-
membered iminosugar-type structures decorated with moenomycin-like and lipid-IV-like
substitution patterns.
The first goal of this thesis project is to synthesize additional analogues as a part of a
SAR study on the moenomycin-inspired pyrrolidine scaffold. To this end, a number of
compounds is envisaged differing at the amide site. Both NH and N-iPr derivatives are targeted.
The second goal of this master thesis is the contribution to the asymmetric synthesis of
lipid IV-inspired 5a-carbaglycosylamine derivatives by exploring synthetic routes to the central
scaffold for subsequent decoration.
1. Table
15
1. Scheme: Retrosynthetic approaches for this thesis work
16
III. Results and discussion
As mentioned in part II (Goals), this thesis project will specifically focus on two target
structures. On one hand, the synthesis of moenomycin-inspired pyrrolidine derivatives are
envisaged as analogues of an earlier identified active compound. On the other hand, the
synthesis route towards novel lipid IV-inspired 5a-carbaglycosylamine scaffold will be
investigated.
In the first part of this chapter, pyrrolidine-based structures will be discussed while the
second part will focus on the 5a-carbaglycosylamine scaffolds.
III.1. Synthesis of moenomycin-inspired derivatives
III.1. 1. General synthesis route of moenomycin inspired pyrrolidine-based analogues
Earlier work at the laboratory has revealed that structure 2 possesses transglycosylase
inhibitory activity (70 % inhibition, 500 µM). This is rather remarkable, as the compound has
a relatively simple structure, although the necessary features expected for TG binding are
present (amine, amide and phospholipid characteristics). As such, we are interested in the
synthesis of further analogues to determine the SAR and to improve the binding capacity.
11. Figure: Template compound 2 and pyrrolidine based targets
In this thesis work, we envisage the synthesis of compound 11, 12, 13 and 14 using a
synthesis route previously developed at our laboratory. The retrosynthesis is depicted in Scheme
2. Final stage alkylation (R3 introduction) would give the target molecules 19. The general
convergent synthesis strategy is based on the coupling of two advanced intermediers 15 and 16
17
containing the amide (R1 and R2) and phosphate substituents, respectively. In the following
paragraphs, the forward synthesis of the target compounds is discussed.
2. Scheme: Retrosynthetic approach of the pyrrolidine based target compounds
III.1. 2. Synthesis of scaffold 15
12. figure: Synthesis route to obtain 14 and 15
Starting from the commercially available trans-4-hydroxy-D-proline (3), the amino group
was protected as benzyl carbamate using benzyl chloroformate. Next, the carboxylic acid
moiety can be activated using EDC and HOAt, and coupled to commercially available primary
and secondary amines. As such, compounds 20 and 21 were obtained using MeNH2.HCl and
isopropyl amine (prepared before by Alejandro Lumbreras Teijeiro).20
18
III.1. 3. Synthesis of benzylhexadecyldiisopropylphosphoramidite 24
Crucial benzyl bisamidite 23 was prepared by Vanessa Nozal (2015)21 via treatment of
PCl3 with diisopropylamine to give chlorophosphine 22, which was converted to 23 using
benzyl alcohol. The benzyl group serves as a protecting group for the latent phosphate
functionality.
Due to the susceptibility to decomposition, compound 23 was stored at -20 °C, under
argon as exposure to humidity of the air has to be carefully avoided. However, purification of
the material was necessary before usage. To this end, an extraction using hexane and acetonitrile
is performed after which concentration of the hexane phase is carried out under argon
atmosphere. To verify the structure and purity 1H and 31P NMR analysis are performed.
13. figure: Reaction steps to obtain compound 24
The purified phosphoramidite 23 can be then reacted with different alcohols R2OH to
obtain phosphoramidite building block 16 containing the desired sidechain R2. As we were
focusing on target compounds 11, 12, 13 and 14, we reacted 23 with 1-hexadecanol to
successfully obtain building block 24 which was purified using a hexane/CH3CN extraction.
Purity and identity were confirmed using NMR spectroscopy. Like 23, compound 24 needs to
be stored at -20°C but is best freshly prepared before use.
19
III.1. 4. Coupling of building blocks
The next step in the decoration of the hydroxyproline scaffold is the phophorylation of
the available hydroxyl function.
14. figure: Phosphorylation reaction scheme
In this one-pot reaction sequence, first an intermediate phosphite (25 and 26) is formed
upon tetrazole mediated reaction between 24 and 21 or 22, respectively, which is immediately
afterwards oxidized to the phosphate by treatment with hydrogen-peroxide. Reactions are
monitored by TLC and products can be readily purified using column chromatography. As such,
compounds 27 and 28 were obtained in 32 % and 41 % yield, respectively.
The first step includes the usage of 1H-tetrazole which initially protonates 24, which is
then more prone to undergo attack by the alcohol nucleophile The mechanism is shown in
Figure 15.
15. figure: Mechanism of the phosphorylation reaction
20
III.1. 5. Removal of the protecting-groups and N-alkylation
16. figure: Hydrogenation reactions
The last step of the synthesis strategy of the pyrrolidine based analogues is the
simultaneous deprotection of the amine and phosphate functionalities by removing the Z- and
the Bn-groups. This step is performed by hydrogenolysis using heterogeneous catalysis
applying Pd on carbon surface under hydrogen athmosphere for 1,5 to 4,5 hours.
In this way, target structures 11 and 12 were obtained with 77 % and 64 % yield,
respectively. After filtration of the catalyst, the product was found to be sufficiently pure
(according to NMR analysis: purity >95%), so no further purification was performed.
To obtain the corresponding isopropylated target compounds 13 and 14, we additionally
performed the hydrogenation in the presence of acetone. Reaction were run for 2-5 hours to
ensure complete conversion (monitoring by TLC and 1H-NMR, NMR purity is >95%).
Compound 13 was produced with 88 % , compound 14 with 74 % yield.
All four compounds have been submitted for assessment of their GT inhibitory activity
by our collaborating partner (Dr. M. Terrak, University of Liège).
21
III.1. 6. Conclusion and future perspectives of the pyrrolidine-based family
As part of an ongoing SAR study on the TG-inhibitory activity of moenomycin-inspired
pyrrolidine derivatives, the goal of this part of the thesis work was the synthesis of four
additional analogues using a previously developed synthetic strategy (see Scheme 2). The
desired compounds were successfully synthesized and their screening is in progress. The
determination of their TGase inhibition properties will contribute to the understanding of the
Structure-Activity Relationship necessary for potential future antibiotic development.
22
III.2. Synthesis of lipid IV inspired 5a-carbaglycosylamine derivatives
III.2 1. Synthesis strategy towards 5a-carbaglycosylamine scaffolds
In addition to the synthesis of moenomycin-inspired analogues, our laboratory is also
interested in the synthesis of lipid IV-resembling aza-analogues. Next to the classic iminosugar-
based derivatives, we wish to explore the 5a-carbaglycosylamine scaffold for analogue
synthesis. In these structures, the exocyclic amine moiety mimics the attack of the incoming
nucleophile, which should provide a suitable-positioned H-bond to the Glu catalytic residue.
Our retro-synthetic approach to obtain these 5a-carbaglycosylamine scaffold is depicted in
Scheme 3.
3. Scheme: Retrosynthetic approach of the 5a-carbaglycosylamine derivatives
23
The strategy involves the access to an appropriately protected α,α-cyclohexylamino acid
38, which would not only serve as scaffold for the synthesis of GT inhibitors but could be used
as cyclitol amino acid building block for alternative research purposes.
Targeting such more densely functionalized complex, optically active structures
containing several chiral centers is synthetically challenging. To direct the synthesis route to
the desired stereoisomers, a design of an asymmetric synthetic pathway is necessary taking into
account economic and time aspects.
The synthesis of uncommon α,α-cyclohexylamino acids is known to be challenging as
consequence of the quaternary center.22 Our approach is based on the use of a heterocyclic
chiral auxiliary as a „chiral glycine equivalent”, combined with the efficiency of ring-closing
metathesis.
The retrosynthesis is depicted in Scheme 3 and involves the final stage hydroboration23
of cyclohexane derivative 37, which would be obtained from diene 36 via ring-closing
metathesis (RCM).
This cyclic quaternary α,α-amino acid would the arise from double stereoselective
alkylation of the bislactim ether 6 using alkylating agents 30 and 33, to be synthesized from
commercially available glycidol derivative 29 and chloromethylchloropropene 32, respectively.
17. figure: Left: (R)-Schöllkopf auxiliary and its vulnerable positions, Right: Structure of the deprotonated (R)-Schöllkopf
auxiliary and the principle of stereoselective induction
Using the so-called Schöllkopf-bislactim ether 6 (a cyclo[D-Val-Gly] derivative),
regioselective metalation by n-BuLi results in a delocalized planar anion which then reacts
diastereoselectively with alkyl halides as shown in Figure 17.24 Because of sterical reasons, the
24
alkyl residue enters position 5 trans to the bulky isopropyl group on C2. Stereoselectivities are
usually excellent (>95%) when R≠H. The principle of the Schöllkopf-strategy is thus the
alkylation of a cyclic, rigid glycine anion equivalent. 25
Diastereoselective introduction of various electrophiles can be carried out via the anion
of the heterocyclic system. 26 The obtained latent optically active amino acid derivative can
subsequently be liberated from the auxiliary after a hydrolysis step (Figure 18).25
18. figure: Hydrolysis of the chiral auxiliary
III.2. 2. Previous work on a simplified structure
This Schöllkopf-based strategy has already been investigated in our laboratory for the
synthesis of a closely related cyclohexenyl amino acid 47.
Starting from the (R)-Schöllkopf chiral auxiliary (R)-6, the introduction of the first alkyl
group is carried out by using a commercially available reagent, 4-bromobutene. 24,27,28
The stereochemical outcome of the first alkylation step is influenced by the chiral
auxiliary inducing the (S)-configuration on position C5 (42), albeit in moderate excess (~70%
d.e.). However, this stereogenic center is destroyed in the following deprotonation step, and the
mixture can thus be used as such. During the following second alkylation step using mesylate
41, greater steric effects are at play and complete diastereoselective formation of 43 is observed.
Mesylate 41 is synthesized from commercially available chloromethylchloropropane 32 via
desymmetrization of diol 39.
25
4. Scheme: Synthesis strategy towards the 3-deoxy-5a-carbaglycosylamine scaffold
After the double diastereoselective alkylation, the removal of the chiral auxiliary is the
following step using carefully chosen acidic hydrolysis conditions (0.1 M TFA in CH3CN/H2O
1/1).27 The obtained diene intermediate is then protected at the N-terminus and transformed
using ring closing metathesis to the cyclohexenyl derivative 4727. Importantly, the alternative
acidic auxiliary removal of cyclohexene derivative 44 (formed by prior RCM) does not proceed,
as observed by others in the synthesis of cycloalkyl-aminoacids.29
26
The choice of either (R)- or (S)-form of the chiral auxiliary starting material is in
principle possible, since the order of alkylation can be switched. However, the availability and
the non-recyclability of the chiral auxiliary are also very important issues which have to be
taken into account. In this specific case, the cost of the (S)-enantiomer would be higher
compared to the used (R)-enantiomer. This is especially important as the auxiliary is fully
destroyed after the alkylation and hydrolysis, so no recyclisation is possible.
Based on this successful previous experience, we envisaged the synthesis of our target
3-hydroxy-5a-carbaglycosylamine scaffold 38 using a highly similar synthetic sequence.
III.2. 3. Literature precedent
A literature study revealed earlier attempts to obtain our target skeleton via the
Schöllkopf approach.
Undheim et al.26,29 investigated the direct alkylation of 42 by racemic vinyloxirane, and
as it is depicted on Figure 19, the general conditions ((i) n-Buli, THF, -50°C, (ii) electrophile -
78°C) only provided 50, arising from SN2’ reaction. This is explained by the observation that
only “hard” nucleophiles attack the epoxide position, while “soft” nucleophiles (as the
stabilized Schöllkopf anion) prefer the conjugate addition pathway.29
As an alternative, the use of Lewis acids was investigated to enforce epoxide attack. Upon
the use of BF3.Et2O at -78°C, the conjugate addition was completely subdued, however product
53 was only obtained in 23% yield as the nucleophilic attack can happen at different positions
on the oxirane derivative leading to other products (51, 52).
27
19. figure: Outcome of alkylation of 42 with vinyloxirane
Alternatively, the alkylation of 42 with oxirane was accomplished to give 55, which was
subsequently oxidized to the corresponding aldehyde 56. Addition of vinylmagnesium-bromide
then delivered both diastereoisomers 57 and 58 in equimolar ratio.
28
20. figure: Non-diastereoselective formation of 57 and 58 via oxirane-alkylation, oxidation and vinylmagnesium bromide
addition
Although successful, clearly this synthesis is not ideal as it delivers both epimeric forms
57 and 58. Further research on the diastereoselective vinyl addition would be necessary to
optimize this reaction sequence.
III.2. 4. Current work: route starting from (R)-Schöllkopf auxiliary
As an alternative and more convergent approach, we propose the use of homochiral
hydroxybutenyl fragments, such as iodide 60, for the one step alkylation of Schöllkopf
intermediate 31 (Scheme 5).
29
5. Scheme: Synthesis strategy towards the 3-hydroxy-5a-carbaglycosylamine scaffold
Actually, a prior test reaction using this alkylating agent delivered the desired compound
31 in reasonable yield. Encouraged by this preliminary result, we wanted to optimize and
complete the reaction sequence to intended scaffold 38.
III.2. 4.1. First alkylation step
The synthesis of iodide 60 can be achieved starting from (R)-glycidol 59. Protection of
the primary alcohol as TBS ether, followed by epoxide opening and chain homologation using
sulfurylide gave allylic alcohol 62. Subsequent MEM-protection and TBS removal using tetra-
n-butyl ammonium fluoride (TBAF) delivered primary alcohol 64. This sequence was
performed before in the laboratory on large scale, giving an available quantity of 20 g of 64.
From this alcohol the iodide derivative 60 was previously obtained via reaction with Ph3P
and I2 in the presence of imidazole, albeit in moderate yield (65%), probably as a consequence
of difficult purification.
30
6. Scheme30:Synthesis of iodide 60 from (R)-glycidol
We therefore decided to synthesize the iodide in a two step procedure using a Finkelstein
approach via mesylate 65. To this end alcohol 64 was first treated with MsCl, delivering
mesylate 65 in excellent yield after purification. Substitution of the leaving group using sodium
iodide gave the desired iodide derivative 60 in 86% yield. However, this compound is highly
sensitive and quickly degrades. Moreover, the product is rather volatile, so care has to be taken
when removing solvent under vacuum conditions.
Using the iodide 60 the alkylation of Schöllkopf auxiliary (R)-6 was undertaken.
Obviously, all reactions need to be carefully performed under inert conditions.
After deprotonation of (R)-6 using n-BuLi at -78°C, the iodide 60 was added dropwise at
the same temperature and after stirring overnight (temperature was slowly raised to room
temperature), the desired product could be isolated after column chromatography.
However, the reaction outcome of the alkylation reaction is highly dependent on the „life-
time” of 60. Batches stored for only few days in the freezer (-18 °C, under Ar) showed already
some degradation and impurities.
Further purification was therefore done to remove the degradation product(s), however it
did not lead to improved purity. Probably the purification step itself, the contact with the silica
gel could also initiate some degradation process. Taking all these observations into account, the
best outcome was achieved using freshly made batches of 60 for this first alkylation step on the
(R)-Schöllkopf auxiliary.
31
As such, the best yield we obtained was 43 %. Interestingly, according to NMR and LC-
analysis, only one diastereoisomer could be detected. Clearly the presence of the extra MEM-
ether moiety has an influence on the cause of the reaction. We assume the formation of the
trans-diastereoisomer.
Considering the rather moderate alkylation yield and the high instability of the iodide 60,
we decided to investigate in the use of a series of alternative alkylating agents.
21. figure: Investigation in different alkylating agents
Starting from available alcohol 64, we prepared the corresponding mesylate 65, tosylate
66, nosylate 67 using standard conditions, in 94 %, 77 % and 61 % yield respectively. In
addition, we obtained the bromide 68 using an Appel reaction (Ph3P, CBr4), Although the
reaction seemed to show clear conversion on TLC, only 66 % yield was achieved, as
consequence of the volatility of the compound. The mechanism of the Appel reaction is shown
in Figure 22.
32
22. figure: Mechanism of the Appel reaction
With these compounds in hand, the alkylation of (R)-6 was further investigated.
23. figure: First alkylation step of (R)-6
Entry X Yield Remark
65 OMs - Complex mixture containing mainly starting material
and some degradation products, no product detected
66 OTs 14 % Complex mixture, difficult separation, several side
products
67 ONs - Complex mixture containing mainly starting material
and some degradation products, no product detected
68 Br - Very complex mixture, no product detected
60 I 43 % Iodide degradation
4-bromobutene Br 88 %30 Clear conversion
2. Table
33
To our surprise, none of these reagents were found to be effective for this transformation.
Only in case of tosylate 66 could the formation of product 31 be observed, but the isolated yield
was very low (14 %). In all other instances, the desired compound was not detected (TLC, LC-
MS); instead complex mixtures were observed.
In conclusion, this alkylation step clearly is a difficult transformation and should be
further optimized if possible. Nevertheless, with the available product at hand, we already
decided to investigate the following second alkylation step.
III.2. 4.2. Second alkylation step
As mentioned before, prior research in our laboratory has shown that alkylation of 42
using mesylate 41 is feasible. Therefore, we decided to also use this alkylating condition for the
alkylation of 31. This compound can be prepared from commercially available
chloromethylchloropropane 32.
7. Scheme: Synthesis of 41
In a first double substitution of the chlorine atoms delivers diol 39, which was synthesized
before on a large scale (10 g) in our laboratory. From this diol, we synthesized the mesylate via
direct monobenzylation in the presence of Ag2O, followed by treatment of 40 with
methanesulfonyl chloride.
Compound 41 was prepared on 8 g scale, but was found to contain a minor unidentified
impurity that could not be separated.
34
24. Figure: Second alkylation step on (R)-Schöllkopf intermediates
After deprotonation of 31 using n-BuLi at -50°C, mesylate 41 was added at -78°C and
reaction mixture was stirred overnight while slowly raising the temperature to room
temperature. Unfortunately, the outcome of this reaction was highly disappointing as only trace
amounts of the desired product could be detected within the complex reaction mixture (LCMS-
analysis of the crude reaction mixture).
We thus decided to synthesize the corresponding bromide 68 for alkylation of 31.
Treatment of allylic alcohol 40 under Appel conditions gave a clean conversion to the
desired bromide (TLC-analysis). However, the product turned out to be very volatile, and
significant loss of product was observed during concentration under vacuum. After column
chromatographic separation the isolated yield was 47 %.
Treatment of 31 with n-BuLi is followed by the addition of allylic bromide 69, however
the reaction outcome was also disappointing as no desired product could be detected. Instead,
a highly complex mixture was observed.
35
25. figure: Second alkylation step using 69
From these test reactions, it is clear that the additional presence of the protected alcohol
moiety is detrimental for its reactivity. Most likely steric hindrance factors are to be held
responsible for this observation.
If indeed, the MEM group causes the low outcome of this specific step, one solution could
be the removal of this protecting group and the replacement by another, smaller one could
improve the success rate of the reaction. However, this is challenging considering the
compatibility with the following reaction steps.
To verify this idea, an option would be to investigate the alkylation potential of the
methylated derivative as this would be the smallest sterical group imaginable. One hand, this
should involve the synthesis of an alternative alkylating agent, starting from (R)-glycidol.
However, considering the available time, we decided to deprotect the MEM-group on 31
and to convert the alcohol to the methyl ether 71.
8. Scheme: Replacement of the MEM-protection
Unfortunately, the deprotection of 31 did not proceed as planned, as no conversion could
be observed. Further research will have to be conducted to verify this idea.
36
Importantly, the observed low yields of first and second alkylations of (R)-6 could be the
result of a mismatch situation between the stereocenters present.
Therefore, we also wished to investigate the reversed order of alkylation of the
corresponding enantiomeric (S)-6 as starting Schöllkopf auxiliary, which after the removal of
the auxiliary should deliver the same target amino acid.
9. Scheme: Two different approaches to achieve 37 starting from (R)- and (S)-Schöllkopf auxiliary
III.2. 5. Route starting from (S)-Schöllkopf auxiliary
III.2. 5.1. First alkylation step
Using the available mesylate 41, alkylation of the commercially available (S)-Schöllkopf
compound (S)-6 was performed under the previously established conditions. Interestingly, the
uniform formation of only one diastereoisomer was observed. Again, NMR analysis could not
prove the relative stereochemistry.
Moreover, as stated before, the mesylated agent 41 contained an unknown impurity,
which could also have been influential on the reaction outcome.
However, the isolated yield was disappointingly low (34 %), partly because of difficult
purification from the complex reaction mixture.
37
26. Figure: First alkylation step on (S)-Schöllkopf auxiliary using mesyate 41
III.2 5.2. Second alkylation step
Because of the extensive prior investigation of the alkylation of (R)-6 with different
alkylating agents, we choose iodide 60 for this transformation. As such, we were pleased to
observe the successful isolation of double-alkylated compound 72 (49 %) using the conditions
developed before (deprotonation at -50°C, alkylation at -78°C).
27. figure: Second alkylation step on (S)-Schöllkopf auxiliary using iodide 60
However, LCMS analysis showed the presence of a side product as a shoulder on the
main peak displaying an identical mass spectrum as to the main product. NMR analysis also
showed two sets of highly similar signals (ratio 87/13). These data could be interpreted as the
formation of two epimeric structures as consequence of poor diastereoselective alkylation.
However, this seems highly unlikely considering the previously obtained high
diastereoselectivities. Of course, a possible mismatched situation could be occurring leading to
the observed diastereoisomeric ratio. Alternatively, it may be hypothesized that because of
sterical reasons, n-BuLi treatment could have led to deprotonation at the valine position,
causing epimerization at this center.
38
Repeating the reaction on larger scale, however, delivered impure 72 containing only one
diastereoisomer according to LCMS analysis. Because of time constraints, further purification
and more detailed analytical investigation of that specific batch and of subsequent synthetic
steps could not be pursued.
In order to compare the influence of the extra MEM-ether functionality on the alkylation
outcome, the corresponding reaction with 4-bromobutene was also performed.
28. figure: Second alkylation step on (S)-Schöllkopf auxiliary using 4-bromobutene
The reaction mixture was clearly much less complex, and the product could be isolated
in 60 % yield. Although slightly higher than the obtained yield of 72 (49 %), it can be deduced
that there is only a marginal steric effect of the MEM-group in this case. As such, the difficulties
observed in the route starting from (R)-6 could be attributed to a mismatched situation for the
alkylations.
39
III. 2. 6. Conclusion
In conclusion, it is clear that the Schöllkopf alkylation strategy is a challenging endeavor
for the synthesis of the target compounds. The crucial ingredient to success seems to be the
choice of Schöllkopf starting enantiomer and the order of alkylation, probably because of
mismatch reasons.
Thus, the change to the (S)-Schöllkopf chiral auxiliary as starting material was an
important step forward in this synthesis route.
The upscaling of these reaction steps were done and provided the same outcome as was
shown during trial reactions, confirming the reproducibility of the alkylation reactions.
However, there is definitely room for improvement as the current yields are not optimal.
One of the problems which has to be tackled is in connection with the alkylating reagents,
either looking at iodide 60 (fast degradation) or talking about mesylate 41 (non-identified
persistent impurity).
Another possible solution could be the replacement of the MEM protection of the
hydroxyl group to determine its influence in these alkylation steps. Once optimized, 71 could
be used as intermediate for the further synthesis of intended scaffold 38.
40
IV. Experimental procedures
The 1H/31P/13C- -NMR spectra were measured at 400 MHz (Bruker Avance 400
spectrometer); the chemical shifts are expressed in ppm. As solvents chloroform-d (7.26
ppm (1H), 77.00 ppm (13C)), methanol-d4 (3.31 ppm (1H), 49.15 ppm (13C)), benzene-
d6 (7.16 ppm (1H), 128.39 ppm (13C)), acetone-d6 (2.05 ppm (1H), 29.92 ppm (13C)) are
used. Coupling constants are given in Hz. Multiplicities are indicated by: app =
apparent, br = broadened signal, s = singlet, d = doublet, dd= doublet of doublet, ddd =
doublet of doublet of doublet, t = triplet, q = quadruplet, m = multiplet.
Molecular weights and molecular formulas are based on calculation made by
ChemDraw Ultra v.8.0
All LCMS-analysis were recorded on Agilent 1100 Series HPLC with G1946-MSD
equipped with ESI-source and DAD. Column: Phenomenex Kinetex C18, 150mm x
4.6mm, particule size = 5 μm. T = 35 , injection = 15 μm, flow = 1.5 mL/min.
All IR were recorded on a Perkin-Elmer SPECTRUM 1000 FTIR spectrometer with
HATR module. The following abbreviations were used: very weak (vw), weak (w),
medium (m), strong (s), very strong (vs).
For TLC-analysis Machery-Nagel SIL G-25 UV254 0.25 mm was used. Detection: UV-
lamp (254 nm and 360 nm). Mo7O24/Ce(SO4)2/H2SO4, KMnO4/NaOH/H2O and
ninhydrin/n-butanol/AcOH were used as staining agents.
Flash chromatography was performed on and Rocc Silicagel (0,040 – 0,063 mm).
Dry solvents were dried on continuous distillation. (CaH2 for DCM and Et3N, Na and
benzophenone for THF).
Analytical HPLC grade solvents (hexane, EtOAc, acetone, diethyl ether,
dichloromethane, chloroform, ethanol and methanol) were used without any further
purification.
41
IV.1. Pyrrolidine-based compounds
IV.1. 1. Synthesis of iPr2N-P(OBn)-OC16H33 (24)
Important: this reaction has to be performed in dry conditions, so all the used lab material
has to be previously dried and flushed with inert gas (nitrogen or argon); every time an
evaporation under reduced pressure is performed, or any product is added, Ar atmosphere must
be applied.20
Purification of 23 is carried out first by extraction. Compound 23 (m=197 mg) is dissolved
in hexane (15 ml) and transfered to a separation funnel, extracted with CH3CN (2x8 ml). Hexane
layer is concentrated under reduced pressure and flushed with Ar (obtained: 137 mg). NMR
analysis confirmed the purity of 23.
Compound 23 (132 mg, 0,389 mmol, 1 equivalent) is dissolved in dry, freshly distilled
DCM (2 ml) under Ar, at room temperature. The solution (solution A) is cooled down to 0°C.
1-Hexadecanol (78 mg, 0,322 mmol, 0.8 equivalents) is dissolved in dry, freshly distilled
DCM (1,4 ml), (solution B).
Tetrazole (780 µl, 0,351 mmol, 0,9 eq) is added to solution A at 0°C, and immediately
solution B is transfered to that flask, then the cooling bath is removed. After 30 minutes of
reaction time, extra tetrazole (260 µl , 0,117 mmol, 0,3 eq) is added.
After 55 minutes of stirring the reaction mixture is transfered to a separation funnel
containing hexane (25 ml) and acetonitrile (15 ml). The hexane phase is washed with
acetonitrile (2x15 ml). The hexane phase is collected and then evaporated under reduced
pressure, under Ar atmosphere. Compound 24 (122 mg, 65 % yield) is obtained and used as
such in the next reaction.
42
1H NMR 23 (400 MHz, benzene-d6):
7,53 (d, J=7.6 Hz, 2H), 7,32 (t, J=7.6 Hz, 2H), 7,21 (t, J=7.6 Hz, 1H), 4,82 (d, J=7.5 Hz, 2H),
3,74-3,61 (m, 4H), 1,35 (d, J=6.6 Hz, 12H), 1,32 (d, J=7.0 Hz, 12H) ppm
31P NMR 23 (400 MHz, benzene-d6): 136,1 ppm
Molecular formula 24: C29H54NO2P
Molecular weight 24: 479,72 g/mol
1H NMR 24 (300 MHz, benzene-d6):
7,39 (m, 2H), 7,23-7,04 (m, 3H), 4,85 (A-part of AB-spin system, dd, J=12.8 Hz, 7.9 Hz, 2H),
4,76 (B-part of AB-spin system, dd, J=12.6 Hz, 8,3 Hz, 2H), 3,87-3,60 (m, 4H), 1,72-1,57 (m,
2H), 1,50-1,27 (m, 23H), 1,23 (d, J=6.8, 6H), 1,19 (d, J=6.8 Hz, 6H), 0,91 (t, J=6.7 Hz, 3H)
ppm21
31P NMR 24 (400 MHz, benzene-d6): 160,9 ppm
IV.1. 2. Synthesis of Z-D-Hyp(OPO(OBn)OC16H33)-methylamine (27)
To a cooled (0°C) solution of 24 (80 mg, 0,164 mmol, 1,9 eq.) in dry DCM (0,5 ml),
tetrazole (0,45 M in acetonitrile, 290 µl, 0,129 mmol, 1,5 eq) and 20 (24 mg, 0,086 mmol, 1 eq)
dissolved in DCM (1,0 ml) are added. The reaction mixture is under argon atmosphere. The ice
bath is removed and the reaction mixture is stirred at room temperature for 1,5 hours.
H2O2 is added (80 µl, 30 % in H2O solution; used in excess to oxidize the phosphorus
atom).
43
After 1,5 hours of stirring time Na2S2O3 solution (1,0 ml, 10 %, aqueous solution) is
added.
The reaction mixture is transferred to a separation funnel containing distilled H2O (10 ml)
and extraction is carried out with DCM (3x8 ml). Organic fractions are collected and dried on
MgSO4. The drying agent is filtered and the filtrate is concentrated under reduced pressure. The
crude mixture is purified by column chromatography (DCM: Acetone, 100% DCM →60%
DCM). Compound 27 is obtained as an oil (19 mg, 32 % yield).
Molecular formula: C37H57N2O7P
Molecular weight: 672,83 g/mol
LC-MS: tr=6,93 min (75% → 100 % acetonitrile in 6 minutes)
1H NMR (400 MHz, CDCl3) – mixture of diastereoisomers and rotamers:
7,30-7,25 (m, 10H), 5,08-4,86 (m, 5H), 4,33-4,25 (m, 1H), 3,92-3,78 (m, 3H), 3,43 (d, J = 8,0
Hz, 1H), 2,73 (s, 2H), 2,57-2,14 (m, 3H), 1,81-1,36 (m, 4H), 1,18-1,10 (m, 26H), 0,80 (t, J =
5,6 Hz, 3H) ppm
13C NMR (100 MHz, CDCl3) – mixture of diastereoisomers:
171,1 (Cq), 136,0 (Cq), 135,7 (Cq), 135,7 (Cq), 128,7 (CH), 128,6 (CH), 128,3 (CH), 128,0 (CH),
127,9 (CH), 69,5 (CH2), 69,4 (CH2), 68,3 (CH2), 67,7 (CH2), 59,3 (CH), 58,7 (CH), 53,3 (CH2),
35,3 (CH2), 31,9 (CH2), 30,2 (CH2), 29,7 (CH2), 29,7 (CH2), 29,6 (CH2), 29,5 (CH2), 29,4
(CH2), 29,1 (CH2), 26,4 (CH3), 25,4 (CH2), 22,7 (CH2), 14,1 (CH3) ppm
31P NMR (100 MHz, CDCl3): -1,61 ppm
IR:
3306 (w), 3065 (vw), 3033 (vw), 2922 (m), 2852 (m), 1708 (m), 1674 (m), 1556 (w), 1498
(vw), 1455 (w), 1415 (m), 1354 (m), 1316 (vw), 1264 (m), 1211 (w), 1172 (w), 1122 (w), 999
(s), 940 (w), 918 (vw), 872 (vw), 822 (vw), 768 (w), 734 (m), 696 (m) cm-1
Rf: 0,17 (DCM: Acetone 8:2)
44
IV.1. 3. Synthesis of Z-D-Hyp(OPO(OBn)OC16H33)-isopropyl amine (28)
To a cooled (0°C) solution of 24 (133 mg, 0,279 mmol, 1,9 eq.) in dry DCM (1,0 ml),
tetrazole (0,45 M in acetonitrile, 490 µl, 0,219 mmol, 1,5 eq) and 21 (45 mg, 0,147 mmol, 1 eq)
dissolved in DCM (1,0 ml) are added. The reaction mixture is under argon atmosphere. The ice
bath is removed and the reaction mixture was stirred at room temperature for 2,5 hours.
H2O2 is added (150 µl, 30 % in H2O solution; used in excess to oxidize the phosphorus
atom).
After 1,5 hour of stirring time Na2S2O3 solution (1,5 ml, 10 %, aqueous solution) is added.
The reaction mixture is transferred to a separation funnel containing distilled H2O (15 ml)
and extraction is carried with DCM (3 x 12 ml). Organic fractions are collected and dried on
MgSO4. The drying agent is filtrated and the filtrate is concentrated under reduced pressure.
The crude mixture is purified by column chromatography (DCM: Acetone, 100% DCM →60%
DCM). Compound 28 is obtained as an oil (42 mg, 41 % yield).
Molecular formula: C39H61N2O7P
Molecular weight: 700,89 g/mol
LC-MS: tr= 7,50 min (75% → 100 % acetonitrile in 6 minutes)
31P NMR (100 MHz, CDCl3): -1,91 ppm
Rf: 0,33 (DCM:Acetone 8:2)
45
IV.1. 4. Synthesis of 5H-D-Hyp(OPO(OH)OC16H33)-methylamine (11)
To the solution of 27 (21 mg, 0,033 mmol, 1 eq) in MeOH (1 ml), Pd/C (10 % Pd on
carbon, 5,3 mg, 5*10-3 mmol, 0,15 eq) is added. A H2 balloon is applied.
After 2,5 h stirring, the reaction mixture is filtered with a syringe filter and the filtrate is
concentrated under reduced pressure.
NMR analysis shows that the reaction is not complete (aromatic region was still present,
the debenzylation was not fully done at that point).
Therefore 27 is redissolved in MeOH (0,5 ml) Pd/C (0,07 mg, 6,57*10-4 mmol, 0,02 eq)
is added and hydrogen atmosphere is provided for extra 3 hours. The reaction mixture is filtered
with a syringe filter and the filtrate is concentrated under reduced pressure. Compound 11 is
obtained as a white solid (11,4 mg, 77 % yield).
Molecular formula: C22H45N2O5P
Molecular weight: 448,58 g/mol
LC-MS: tr=6,61 min
HRMS: (APPI + dopant acetone), mass calculated for C22H46N2O5P [M+H+]: 449,3139
mass found: 449,3150
1H NMR (400 MHz, methanol-d4):
4,94-4,90 (m, 1H), 4,35 (dd, J=10.7 Hz, 7.3 Hz, 1H), 3,87 (app q, J=6.6 Hz, 2H), 3,56 (brdd,
J=12.7 Hz, 0.7 Hz, 1H), 3,45 (brdd, J=12.7 Hz, 3.2 Hz, 1H), 2,81 (s, 3H), 2,69 (app dd, J=13.7
Hz, 7.3 Hz, 1H), 2,05 (ddd, J= 14.3 Hz, 11.4 Hz, 3.8 Hz, 1H), 1,67-1,60 (quintet, J=6.6 Hz,
2H), 1,39-1,29 (m, 26H), 0,9 (t, J=6.7 Hz, 3H) ppm
46
13C NMR (100 MHz, methanol-d4):
169,8 (Cq), 75,7 (d, J=5.1 Hz, CH), 67,1 (d, J=5.9 Hz, CH2), 60,1 (CH), 54,3 (d, J=5.1 Hz, CH2),
38,9 (d, J=4.4 Hz, CH2), 33,2 (CH2), 32,0 (d, J=7.3 Hz, CH2), 30,9 (CH2), 30,9 (CH2), 30,9
(CH2), 30,7 (CH2), 30,6 (CH2), 27,1 (CH2), 26,8 (CH3), 23,9 (CH2), 14,6 (CH3)
31P NMR methanol-d4: -2,24 ppm
IR:
3235 (w), 3075 (w), 2919 (s), 2850 (m), 2360 (m), 2356 (s), 2076 (w), 1682 (m), 1582 (w),
1465 (w), 1450 (w), 1303 (vw), 1274 (vw), 1215 (m), 1049 (s), 985 (m), 928 (vw), 900 (vw),
838 (w), 719 (w), 675 (vw) cm-1
Optical rotation: 𝛼58925 = +8,6° ; 𝛼365
25 = +25° [c=5,7 mg/ml, MeOH]
Rf:0,1 (DCM: MeOH 8:2)
IV.1. 5. Synthesis of iPr-D-Hyp(OPO(OH)OC16H33)-methylamine (13)
To a solution of 27 (10 mg, 0,015 mmol, 1 eq) in MeoH (0,3 ml) is added acetone (0,4
ml) and Pd/C (1 mg, 10 % Pd on C, 8,92*10-4 mmol, 0,06 eq). A H2 balloon is placed and the
reaction mixture is stirred for 3 hours. Additional catalyst is added and the reaction mixture is
stirred for 2 hours (TLC shows full conversion).
The mixture is filtered using a syringe filter, after which the filtrate is concentrated under
reduced pressure. Compound 13 is obtained as a white solid (6,4 mg, 88 % yield).
Molecular formula: C25H51N2O5P
Molecular weight: 490,66 g/mol
LC-MS: tr=1, 77 min (75→100% ACN in 6 min)
47
HRMS: (APCI); mass calculated for C25H52N2O5P [M+H+]: 491,3608
mass found: 491,3615
1H NMR methanol-d4:
4,91-4,87 (m, 1H), 4,42 (dd, J=11.9 Hz, 6.3 Hz, 1H), 3,87 (app q, J=6.5 Hz, 2H), 3,80 (dd,
J=13.4 Hz, 4.5 Hz, 1H), 3,70-3,59 (m, 2H), 2,82 (s, 3H), 2,68-2,60 (m, 1H), 2,21-2,10 (m, 1H),
1,67-1,60 (quintet, J=6.3 Hz, 2H), 1,37-1,27 (m, 34H), 0,9 (t, J=6.7 Hz, 3H) ppm
13C NMR methanol-d4:
169,0 (Cq), 74,2 (d, J=5.1 Hz, CH), 67,2 (d, J=5.9 Hz, CH2), 65,3 (CH), 59,3 (d, J=5.1 Hz, CH2),
58,7 (d, J=5.1 Hz, CH2), 39,6 (d, J=4.4 Hz, CH2), 33,2 (CH2), 32,0 (d, J=7.3 Hz, CH2), 30,9
(CH2), 30,9 (CH2), 30,9 (CH2), 30,6 (CH2), 27,1 (CH2), 26,8 (CH3), 23,9 (CH2), 18,7 (CH3),
18,0 (CH3), 14,6 (CH3) ppm
31P NMR methanol-d4:-2,34 ppm
IR:
3227 (w), 3046 (w), 2918 (s), 2850 (m), 1742 (w), 1681 (m), 1583 (w), 1467 (w), 1386 (w),
1223 (s), 1156 (w), 1054 (s), 993 (m), 916 (w), 901 (w), 826 (w), 745 (vw), 720 (w) cm-1
Optical rotation: 𝛼58925 = +15° ; 𝛼365
25 = +43° [c=2,1 mg/ml,MeOH]
Rf: 0,33 (CH3Cl:MeOH 6:4)
48
IV.1. 6. Synthesis of 5H-D-Hyp(OPO(OH)OC16H33)-isopropyl amine (12)
To the solution of 28 (11 mg, 0,014 mmol, 1 eq) in MeOH (0,5 ml), Pd/C (10 % Pd on
carbon, 0,3 mg, 2,86*10-4 mmol, 0,02 eq) is added. A H2 balloon is applied.
The reaction is not complete in 1 hour (followed by TLC), additional Pd/C (0,3 mg,
2,86*10-4 mmol, 0,02 eq) is added which leads to the full conversion in total 1,5 hours.
The reaction mixture is filtered with a syringe filter and the filtrate is concentrated under
reduced pressure. NMR analysis confirms that the reaction is complete. Compound 12 is
obtained as a white solid (5 mg, 70 % yield).
Molecular formula: C24H49N2O5P
Molecular weight: 476,63 g/mol
LC-MS: tr= 2,85 min (75% → 100 % acetonitrile in 6 minutes)
1H NMR (400 MHz, methanol-d4):
4,96-4,90 (m, 1H), 4,35 (dd, J=10.8 Hz, 7.4 Hz, 1H), 4,01 (septet, J=6.6 Hz, 1H), 3,87 (app q,
J=6.5 Hz, 2H), 3,56 (brdd, J=12.3 Hz, 0.9 Hz, 1H), 3,45 (brdd, J=12.5 Hz, 3.4 Hz, 1H), 2,69
(app dd, J = 13.8 Hz, 7.2 Hz, 1H), 2,05 (ddd, J= 13.9 Hz, 10.6 Hz, 3.5 Hz, 1H), 1,67-1,60
(quintet, J=6.35 Hz, 2H), 1,42-1,29 (m, 26H), 1,19 (d, J=6.7 Hz, 3H), 1,16 (d, J = 6.6 Hz, 3H),
0,9 (t, J=6.6 Hz, 3H) ppm
49
13C NMR (100 MHz, methanol-d4):
168,3 (Cq), 75,8 (d, J=5.1 Hz, CH), 67,1 (d, J=6.6 Hz, CH2), 60,1 (CH), 54,4 (d, J=4.4 Hz, CH2),
43,4 (CH), 39,2 (d, J=3.7 Hz, CH2), 33,2 (CH2), 32,1 (d, J=6.4 Hz, CH2), 30,9 (CH2), 30,9
(CH2), 30,9 (CH2), 30,6 (CH2), 30,6 (CH2), 27,1 (CH2), 23,9 (CH2), 22,6 (CH3), 14,6 ppm (CH3)
31P NMR (400 MHz, methanol-d4): -2,19 ppm
IR:
3204 (w), 3054 (w), 2921 (s), 2852 (m), 2359 (s), 2336 (m), 1673 (m), 1571 (m), 1463 (m),
1390 (vw), 1365 (w), 1221 (s), 1066 (s), 990 (m), 931 (w), 907 (w), 845 (w), 719 (w) cm-1
Optical rotation: 𝛼58925 = +6,6° ; 𝛼365
25 = +24° [c=2,3 mg/ml, MeOH]
IV.1. 7. Synthesis of iPr-D-Hyp(OPO(OH)OC16H33)-isopropyl amine (14)
To a solution of compound 28 (10 mg, 0,014 mmol, 1 eq) in MeOH (0,3 ml) is added
acetone (0,4 ml) and Pd/C (0,9 mg, 10 % Pd on C, 8,56*10-4 mmol, 0,06 eq). A H2 balloon is
placed and the reaction mixture is stirred for 3 hours. The reaction is followed by TLC, and no
transformation is visible. Additional catalyst is added (not measured) and the reaction mixture
is stirred for other 2 hours.
The mixture is filtered using a syringe filter, after which the filtrate is concentrated under
reduced pressure. Compound 14 is obtained as a white solid (5,5 mg, 74 % yield).
Molecular formula: C27H55N2O5P
Molecular weight: 518,71 g/mol
LC-MS: tr= 2,16 min (75→100% ACN) [M-1]= 517,4
HRMS: (APCI) calculated for C27H56N2O5P [M+H+]: 519,3921
50
mass found: 519,3905
1H NMR (400 MHz, methanol-d4):
4,96-4,90 (m, 1H), 4,35 (dd, J = 11.8 Hz; 6.4 Hz, 1H), 4,01 (septet, J=6.6 Hz, 1H), 3,87 (app q,
J=6.5 Hz, 2H), 3,81 (dd, J= 13.3 Hz, 4.4 Hz, 2H), 3,62-3,57 (m, 2H), 2,69-2,62 (m, 1H), 2,11
(td, J= 13.3 Hz, 3.7 Hz, 1H), 1,64 (quintet, J=6.6 Hz, 2H), 1,42-1,29 (m, 33H), 1,19 (d, J=7.0
Hz, 3H), 1,16 (d, J=7.0 Hz, 3H), 0,9 (t, J=6.6 Hz, 3H) ppm
13C NMR (100 MHz, methanol-d4):
167,5 (Cq), 74,2 (d, J=5.1 Hz, CH), 67,1 (d, J=5.9 Hz, CH2), 65,3 (CH), 59,5 (CH), 59,2 (d,
J=5.1 Hz, CH2), 43,5 (CH), 39,8 (d, J=5.1 Hz, CH2), 33,2 (CH2), 32,0 (d, J=7.3 Hz, CH2), 30,9
(CH2), 30,9 (CH2), 30,9 (CH2), 30,6 (CH2), 27,1 (CH2), 23,9 (CH2), 22,5 (CH3), 18,6 (CH3),
18,3 (CH3), 14,6 ppm (CH3)
31P NMR (100 MHz, methanol d4): -2,33 ppm
IR:
3202(w), 2922 (s), 2852 (m), 2265 (w), 1674 (m), 1576 (m), 1466 (m), 1386 (w), 1370 (vw),
1228 (m), 1063 (s), 1012(m), 1001 (m), 923 (w), 903 (w), 831 (w), 768 (vw), 721 (vw) cm-1
Optical rotation: 𝛼58925 = +12° ; 𝛼365
25 = +34° [c=2,6 mg/ml, MeOH]
Rf: Rf: 0,35 (CH3Cl:MeOH 6:4)
51
IV.2 Synthesis of compounds in order to obtain 5a-carbaglycosylamine scaffolds
IV.2. 1. Synthesis of (S)-2-((2-methoxyethoxy)methoxy)but-3-en-1-yl 4-
methanesulfonate (65)
To a solution of 64 (1,010 g, 5,68 mmol, 1 eq) in DCM (50 ml), Et3N (1,19 ml, 8,51
mmol, 1,5 eq) is added. The mixture is cooled down to 0°C and MsCl (527 µl, 6,81 mmol, 1,2
eq) is added dropwise in ~5 minutes. The reaction mixture is allowed to slowly warm to room
temperature.
After 1,5 hours, the reaction mixture is transferred to a separation funnel containing
saturated aqueous NH4Cl solution (50 ml). The aqueous layer is separated and extracted with
DCM (2*50 ml). The combined organic layers are dried on MgSO4, the drying agent is filtered
and the filtrate is concentrated under reduced pressure.
The residue is purified by column chromatography (Hexane: EtOAc 100:0→60:40
gradient). Compound 65 is obtained as a colorless oil in 94 % yield (1,459 g).
Molecular formula: C9H18O6S
Molecular weight: 254,30 g/mol
LC-MS: tr= 4,77 min, ESMS 272,1 [M+NH4+], 255,1 [M+H+]
1H NMR (400 MHz, CDCl3):
5,79-5,69 (m, 1H), 5,46-5,36 (m, 2H), 4,80 (d, J=7.1 Hz, A part of AB system, 1H), 4,75 (d,
J=7.1 Hz, B part of AB system, 1H), 4,44-4,39 (m, 1H), 4,35-4,20 (m, 2H), 3,87-3,82 (m, 1H),
3,68-3,60 (m, 1H), 3,59-3,56 (m, 2H), 3,40 (s, 3H), 3,06 (s, 3H) ppm
13C NMR (100 MHz, CDCl3): 132,7 (CH), 120,5 (CH2), 93,2 (CH2), 74,6 (CH), 71,7 (CH2),
71,1 (CH2), 67,2 (CH2), 59,0 (CH3), 37,6 (CH3) ppm
52
IR:
3023 (vw), 2941 (w), 1890 (w), 2822 (vw), 1641 (vw), 1455 (w), 1414 (w), 1351 (s), 1251 (w),
1197 (w), 1172 (s), 1130 (m), 1104 (m), 1020 (s), 949 (s), 848 (m), 815 (s), 747 (w), 703 (vw),
678 (w), 636 (vw) cm-1
IV.2. 2. Synthesis of (S)-2-((2-methoxyethoxy)methoxy)but-3-en-1-yl 4-
methylbenzensulfonate (66)
To a solution of 64 (306 mg, 1,73 mmol, 1 eq) and DMAP (41,1 mg, 0,336 mmol, 0,2 eq)
in dried DCM (10 ml), NEt3 (360 µl, 2,55 mmol, 1,5 eq) is added. The mixture is cooled to 0
°C, after which a solution of TsCl (360 mg, 1,88 mmol, 1,1 eq) in DCM (17 ml) is added. After
~ 2 minutes, the ice-bath is removed and the reaction mixture is stirred overnight.
After transferring the reaction mixture to a separation funnel, distilled water (20 ml) is
added and the layers are separated. The aqueous layer is extracted with DCM (2*20 ml). The
combined organic layers are dried on MgSO4, the drying agent is filtered and the filtrate is
concentrated under reduced pressure. The residue is purified using column chromatography
(hexane: ethyl-acetate, 30 % EtOAc → 40 % EtOAc gradient). Compound 66 is obtained as
colorless-slightly yellow oil in 77 % yield (434 mg).
Molecular formula: C15H22O6S
Molecular weight: 330,40 g/mol
LC-MS: tr= 5,98 min, ESMS: 348,0 [M+NH4+]
1H NMR (400 MHz, CDCl3):
7,73-7,70 (m, 2H), 7,27-7,21 (m, 2H), 5,59-5,50 (m, 1H), 5,29-5,19 (m, 2H), 4,63 (d, J=7.0 Hz,
A part of AB system, 1H), 4,57 (d, J=7.0 Hz, B part of AB system, 1H), 4,28-4,23 (m, 1H),
3,97-3,91 (m, 2H), 3,72-3,67 (m, 1H), 3,54-3,41 (m, 2H), 3,31 (s, 3H), 2,38 (s, 3H) ppm
53
13C NMR (100 MHz, CDCl3):
144,9 (Cq), 133,0 (Cq), 132,9 (CH), 129,8 (CH), 128,0 (CH), 120,1 (CH2), 93,1 (CH2), 74,4
(CH), 71,7 (CH2), 71,3 (CH2), 67,0 (CH2), 59,0 (CH3), 21,7 (CH3) ppm
IR:
2930 (w), 1889 (w), 1598 (w), 1494 (vw), 1451 (w), 1403 (vw), 1360 (s), 1305 (vw), 1290
(vw), 1257 (vw), 1183 (m), 1175 (s), 1096 (s), 1019 (s), 976 (s), 949 (w), 894 (w), 845 (w), 813
(s), 790 (m), 724 (w), 664 (s) cm-1
IV.2. 3. Synthesis of (S)-2-((2-methoxyethoxy)methoxy)but-3-en-1-yl 4 nitrobenzene
sulfonate (67)
To a solution of 64 (305 mg, 1,73 mmol, 1 eq) and DMAP (42 mg, 0,341 mmol, 0,2 eq)
in dried DCM (10 ml), NEt3 (360 µl, 2,55 mmol, 1,5 eq) is added under Ar, at room temperature.
The mixture is cooled to 0 °C after which the solution of 4-nitrobenzenesulfonyl chloride (NsCl)
(442 mg, 1,87 mmol, 1,1 eq) in DCM (17 ml) is added. After ~ 2 minutes, the ice-bath is
removed and the reaction mixture is stirred overnight.
After transferring the reaction mixture to a separation funnel, distilled water (20 ml) is
added and the layers are separated. The aqueous layer is extracted with DCM (2*20 ml). The
combined organic layers are dried on MgSO4, the drying agent is filtered and the filtrate is
concentrated under reduced pressure. The residues are purified using column chromatography
(hexane and ethyl-acetate 30 % EtOAc → 40 % EtOAc gradient). Compound 67 is obtained
as yellowish oil in 61 % yield (371 mg).
Molecular formula: C14H19NO8S
Molecular weight: 361,37 g/mol
LC-MS: tr= 5,90 min, ESMS: 379,0 [M+NH4+]
54
1H NMR (400 MHz, CDCl3):
8,36-8,32 (m, 2H), 8,07-8,04 (m, 2H), 5,59-5,50 (m, 1H), 5,31-5,23 (m, 2H), 4,62 (d, J=7.0 Hz,
A part of AB system, 1H), 4,56 (d, J=7.0 Hz, B part of AB system, 1H), 4,31-4,27 (m, 1H),
4,11-4,02 (m, 2H), 3,69-3,63 (m, 1H), 3,52-3,44 (m, 3H), 3,31 (s, 3H) ppm
13C NMR (100 MHz, CDCl3):
150,8 (Cq), 141,8 (Cq), 132,4 (CH), 129,4 (CH), 124,5 (CH), 120,7 (CH2), 93,1 (CH2), 74,2
(CH2), 72,4 (CH), 71,6 (CH2), 67,1 (CH2), 59,0 (CH3) ppm
IR:
3111 (vw), 3070 (vw), 2935 (w), 2889 (w), 2817 (w), 1607 (w), 1531 (s), 1476 (vw), 1452
(vw), 1404 (w), 1367 (m), 1350 (s), 1312 (m), 1292 (vw), 1243 (vw), 1184 (s), 1132 (m), 1094
(s), 1020 (s), 957 (s), 855 (m), 812 (m), 736 (m), 683 (m), 636 (w), 614 (m) cm-1
IV.2. 4. Synthesis of (S)-4-bromo-3-((2-methoxyethoxy)methoxy)but-1-ene (68)
To a cooled solution (0°C) of 64 (303 mg, 1,72 mmol, 1 eq) and PPh3 (496 mg, 1,89
mmol, 1,1 eq) in dried DCM (9 ml), a solution of CBr4 (624 mg, 1,88 mmol, 1,1 eq) in DCM
(11 ml) is added. After ~ 2 minutes, the ice-bath is removed and the reaction mixture is stirred
overnight.
After transferring the reaction mixture to a separation funnel, distilled water (20 ml) is
added and the layers are separated. The aqueous layer is extracted with DCM (2*20 ml). The
combined organic layers are dried on MgSO4, the drying agent is filtered and the filtrate is
concentrated under reduced pressure. The residue is purified using column chromatography
(due the volatility of the compound: Pentane: Et2O 100:0 → 60:40). Compound 68 is obtained
as yellowish oil in 66 % yield (269 mg).
Molecular formula: C8H15BrO3
Molecular weight: 239,11 g/mol
55
1H NMR (400 MHz, CDCl3):
5,73-5,64 (m, 1H), 5,34-5,26 (m, 2H), 4,75 (d, J=7.1 Hz, A part of AB system, 1H), 4,69 (d,
J=7.1 Hz, B part of AB system, 1H), 4,20-4,17 (m, 1H), 3,89-3,85 (m, 1H), 3,64-3,58 (m, 1H),
3,56-3,48 (m, 2H), 3,38 (m, 2H), 3,35 (s, 3H) ppm
13C NMR (400 MHz in CDCl3):
135,2 (CH), 119,7 (CH2), 95,4 (CH2), 76,3 (CH), 71,7 (CH2), 67,2 (CH2), 59,0 (CH3), 35,1
(CH2) ppm
IR:
2935 (w), 2887 (w), 2806 (vw), 1450 (w), 1421 (w), 1362 (vw), 1303 (vw), 1285 (vw), 1198
(w), 1104 (m), 1019 (s), 987 (m), 932 (m), 847 (w), 702 (w), 610 (vw) cm-1
IV.2. 5 Synthesis of (S)-4-bromo-3-((2-methoxyethoxy)methoxy)but-1-ene (60)
To the solution of 65 (447 mg, 1,54 mmol, 1 eq) in acetone (5 ml), NaI (1,62 g, 10,8
mmol, 6,5 eq) is added. The reaction mixture is refluxed overnight under Ar atmosphere.
The reaction mixture is filtered and washed with acetone. The filtrate is concentrated
under reduced pressure. After the solvent evaporation, the reaction mixture is re-dissolved in
DCM (7 ml) and extraction is carried out with saturated aqueous Na2S2O3 solution (7 ml).
Organic layer is dried on MgSO4, drying agent is filtered out and the filtrate is concentrated
under reduced pressure. Product is used as such in the following reaction.
Compound 60 is obtained as a brownish oil in 86 % yield (402 mg).
Molecular formula: C8H15IO3
Molecular weight: 286,11 g/mol
56
1H NMR (400 MHz, CDCl3):
5,75-5,65 (m, 1H), 5,29-5,22 (m, 2H), 4,72 (d, J=7.1 Hz, A part of AB system, 1H), 4,65 (d,
J=7.1 Hz, B part of AB system, 1H), 4,11-4,06 (m, 1H), 3,89-3,85 (m, 1H), 3,67-3,61 (m, 1H),
3,59-3,51 (m, 2H), 3,38 (s, 3H), 3,22-3,15 (m, 2H) ppm
13C NMR (100 MHz, CDCl3):
136,2 (CH), 119,4 (CH2), 93,1 (CH2), 76,4 (CH), 71,7 (CH2), 67,4 (CH2), 59,1 (CH3), 9,1 (CH2)
ppm
IR:
3088 (vw), 2952 (w), 2885 (w), 2819 (w), 1692 (w), 1453 (w), 1419 (w), 1366 (vw), 1297 (vw),
1279 (w), 1241 (w), 1186 (m), 1104 (m), 1061 (m), 1017 (s), 991 (m), 966 (m), 931 (m), 847
(m), 748 (vw), 691 (w) cm-1
IV.2. 6. Synthesis of (2R)-2-isopropyl-3,6-dimethoxy-5-((R)-2-((2-
methoxyethoxy)methoxy)but-3-en-1-yl)-2,5-dihydropyrazine (31)
To a cooled (-78 °C) solution of the Shöllkopf-auxiliary (R)-6 (50 µl, 0,278 mmol, 1 eq)
in THF (dried, freshly distilled; 1 ml), n-BuLi (135 µl, 0,334 mmol, 1,2 eq) is added dropwise,
under Ar atmosphere. The reaction mixture is stirred for 1 hour after which 60 (122 mg, 0,419
mmol, 1,5 eq) in THF (0,8 ml) is added. Reaction mixture is stirred overnight allowing to slowly
warm to room temperature.
A saturated aqueous NH4Cl (3 ml) solution is added after which the reaction mixture is
stirred for 3-5 minutes and transferred to a separation funnel. Extraction is performed using
EtOAc (3* 5 ml). The combined organic layers are dried on MgSO4, the drying agent is filtered
and the filtrate is concentrated under reduced pressure. Further purification is accomplished by
column chromatography (hexane: ethyl-acetate 100:0 → 70:30).
Compound 31 is obtained as a colorless oil in 43 % yield (53 mg).
57
Molecular formula: C17H30N2O5
Molecular weight: 342,43 g/mol
LC-MS: tr= 6,75 min, ESMS: 343,2 [M+H+]
HRMS: (ESI), mass calculated for C24H35N2O3 [M+H+]: 343,2228
mass found: 343,2226
1H NMR (400MHz, CDCl3):
5,71-5,62 (m, 1H), 5,21-5,16 (m, 1H), 5,14-5,12 (m, 1H), 4,68 (d, J=6.8 Hz, A part of AB
system, 1H), 4,55 (d, J=6.8 Hz, B part of AB system, 1H), 4,28 (q, J=7.5 Hz, 1H), 3,95-3,89
(m, 1H), 3,85 (t, J=3.5 Hz, 1H),3,72-3,64 (m, 1H), 3,63 (s, 3H), 3,60 (s, 3H), 3,57-3,52 (m, 1H),
3,46 (t, 2H), 3,30 (s, 3H), 2,22 (m, 1H), 2,01-1,95 (m, 1H), 1,91-1,83 (m, 1H), 0,96 (d, J=6.9
Hz, 3H), 0,62 (d, J=6.8 Hz, 3H) ppm
13C NMR (100 MHz, CDCl3):
163,9 (Cq), 163,4 (Cq), 138,0 (CH), 117,8 (CH2), 92,9 (CH2), 74,9 (CH2), 71,8 (CH2), 66,9
(CH2), 60,8 (CH3), 59,0 (CH3), 52,4 (CH3), 40,0 (CH2), 31,9 (CH), 19,0 (CH3), 16,7 (CH3) ppm
IR:
2944 (m), 2873 (m), 1692 (s), 1463 (w), 1436 (m), 1385 (vw), 1365 (w), 1307 (w), 1236 (s),
1195 (m), 1172 (m), 1120 (m), 1107 (s), 1034 (s), 1012 (s), 926 (m), 851 (w), 832 (w), 809
(vw), 770 (w), 743 (m), 704 (w), 672 (vw), 634 (vw) cm -1
IV.2. 7. Synthesis of 2-((benzyloxy)methyl)prop-2-en-1-ol (40)
To the suspension of 39 (8 g, 91 mmol, 1 eq) and Ag2O (21 g, 91 mmol, 1 eq) in dried
DCM (160 ml), BnBr (12 ml, 100 mmol, 1,1 eq) is added dropwise at room temperature, under
Ar atmosphere. Active stirring is provided and the flask is covered with aluminum foil. The
reaction mixture is stirred overnight.
The reaction mixture is filtered through a thick silica gel pad. Et2O is used to wash the
compound over the silica gel pad. Ethyl-acetate is used as a second washing step. The filtrate
58
is evaporated under reduced pressure. Purification is done with hexane - ethyl-acetate 90:10
→60:40, giving 9,36 g as a mixture fraction of the desired product and (probably) BnOH. Due
to its volatility, the compound is used as such for further reactions.
Molecular formula: C11H14O2
Molecular weight: 178,23 g/mol
IV.2. 8. Synthesis of 2-((benzyloxy)methyl)allyl methanesulfonate (65)
To a solution of 64 (9,25 g, 52 mmol, 1 eq) in DCM (300 ml), Et3N (22 ml, 156 mmol, 3
eq) is added. The mixture is cooled down to 0°C and MsCl (6 ml, 78 mmol, 1,5 eq) is added
dropwise in ~5 minutes. The ice-bath is removed and reaction mixture is allowed to warm to
room temperature.
After 10 minutes of stirring, no starting material is visible on TLC. The reaction mixture
is transferred to a separation funnel containing aqueous KHSO4 solution (1 M, 300 ml). The
aqueous layer is separated and then re-extracted with DCM (1*200 ml). The combined organic
layers are extracted with distilled water (500 ml) and the aqueous phase is re-extracted with
DCM (200 ml). The combined organic layers are extracted with brine (200 ml). This aqueous
layer is again re-extracted with DCM (200 ml). All the combined organic layers are dried on
MgSO4, the drying agent is filtered and the filtrate is concentrated under reduced pressure.
The residue is purified by column chromatography (Hexane: EtOAc 75:25 → 70:30
gradient). Compound 65 is obtained in 58 % yield (7,74 g) as a colorless oil.
Molecular formula: C12H16O4S
Molecular weight: 256,32 g/mol
LC-MS: tr=5,99 min, ESMS: 257,1 [M+H+]
HRMS: (ESI), mass calculated for C12H17O4S [M+H+]: 257,0842
mass found: 257,0852
59
1H NMR (400 MHz, CDCl3):
7,40-7,30 (m, 5H), 5,40 (s, 2H), 4,80 (s, 2H), 4,53 (s, 2H), 4,11 (s, 2H), 3,01 (s, 3H) ppm
13C NMR (100 MHz, CDCl3):
139,0 (Cq),137,7 (Cq), 128,5 (CH), 127,9 (CH), 127,8 (CH), 118,2 (CH2), 72,5 (CH2), 70,1
(CH2), 70,0 (CH2), 37,8 (CH3) ppm
IR:
3028 (w), 2935 (w), 2860 (w), 1731 (vw), 1654 (vw), 1496 (w), 1453 (w), 1414 (vw), 1350 (s),
1248 (vw), 1171 (s), 1091 (m), 1074 (m), 1024 (w), 968 (m), 926 (s), 829 (s), 739 (m), 698 (m),
610 (w) cm-1
IV.2. 9. Synthesis of (((2-bromomethyl)allyl)oxy)methyl)benzene (68)
To the cooled (0°C) solution of 64 (405 mg, 2,27 mmol, 1 eq) and PPh3 (656 mg, 2,50
mmol, 1,1 eq) in dried DCM (7 ml) is added a solution of CBr4 (829 mg, 2,50 mmol, 1,1 eq) in
DCM (7 ml). After ~ 2 minutes, the ice-bath is removed and the reaction mixture is stirred
overnight.
After transferring the reaction mixture to a separation funnel, distilled water (50 ml) is
added and the layers are separated. The aqueous layer is extracted with DCM (2*50 ml). The
combined organic layers are dried on MgSO4, the drying agent is filtered and the filtrate is
concentrated under reduced pressure (heating bath is set to 30°C due to the volatility of the
compound). The residue is purified using column chromatography (due the volatility of the
compound: Pentane: Et2O 100:0 →80:20). Compound 68 is obtained as yellowish oil in 47 %
yield (308 mg).
Molecular formula: C11H13BrO
Molecular weight: 240,12 g/mol
1H NMR (400 MHz, CDCl3):
60
7,27-7,19 (m, 5H), 5,26 (s, 1H), 5,18 (s, 1H), 4,44 (s, 2H), 4,06 (s, 2H), 3,96 (s, 2H) ppm
13C NMR (100 MHz, CDCl3):
142,4 (Cq), 138,0 (Cq), 128,5 (CH), 127,8 (CH), 117,3 (CH2), 72,5 (CH2), 70,4 (CH2), 33,1
(CH2) ppm
IR:
3085 (vw), 3070 (vw), 3028 (vw), 2855 (m), 1646 (w), 1494(w), 1452 (m), 1437 (w), 1393 (w),
1362 (w), 1305 (vw), 1259 (w), 1210 (s), 1092 (s), 1091 (s), 1028 (m), 919 (m), 884 (w), 820
(vw), 735 (s), 670 (s), 675 (m) cm -1
IV.2. 10. Synthesis of (5S)-2-(2((benzyloxy)methyl)allyl)-5-isopropyl-3,6-dimethoxy-2,5-
dihydripyrazine (73)
To a cooled (-78 °C) solution of the (S)-Shöllkopf-auxiliary (S)-6 (375 µl, 2,1 mmol, 1
eq) in THF (dried, freshly distilled; 8 ml) n-BuLi in 2,5 M hexane solution (1 ml, 2,51 mmol,
1,2 eq) is added dropwise, under Ar atmosphere. The reaction mixture is stirred for 1 hour after
which a solution of and 65 (805 mg, 3,14 mmol, 1,5 eq) in THF (5 ml) is added. The reaction
mixture is stirred overnight allowing to slowly warm to room temperature.
A saturated aqueous NH4Cl (15 ml) solution is added after which the reaction mixture is
stirred for 3-5 minutes and transferred to a separation funnel. Extraction is performed using
EtOAc (3* 25 ml). The combined organic layers are dried on MgSO4, the drying agent is filtered
and the filtrate is concentrated under reduced pressure. Further purification is accomplished by
column chromatography (hexane: ethyl-acetate 100:0 → 70:30).
Compound 73 is obtained as colorless/slightly yellow oil in 34 % yield (227 mg).
Molecular formula: C20H28N2O3
61
Molecular weight: 344,44 g/mol
LC-MS: tr=7,63 min, ESMS: 345,2 [M+H+]
1H NMR (400 MHz, CDCl3):
7,29-7,25 (m, 4H), 7,24-7,16 (m, 1H), 5,08 (s, 1H), 4,91 (s, 1H), 4,42 (s, 2H), 4,10-4,06 (m,
2H), 3,93 (d, J=5.3 Hz, A part of AB system, 1H), 3,87 (d, J=13.0 Hz, B part of AB system,
1H), 3,83-3,82 (t, J=11.5 Hz, 1H), 3,60 (s, 3H), 3,54 (s, 3H), 2,60-2,56 (dd, J=14. 4 Hz, 1H),
2,37-2,32 (dd, J = 14.0, 7.2 Hz, 1H), 2,22-2,12 (m, 1H), 0,96 (d, J=6.9 Hz), 0,60 ppm (d, J=6.8
Hz) ppm
13C NMR (100 MHz, CDCl3):
163,5 (Cq), 163,3 (Cq), 142,6 (Cq), 138,5 (Cq), 128,4 (CH), 128,3 (CH), 127,7 (CH), 127,5 (CH),
127,7 (CH), 127,6 (CH), 127,5 (CH), 114,3 (CH2), 73,5 (CH2), 71,9 (CH2), 60,7 (CH), 55,5
(CH), 52,3 (CH3), 52,2 (CH3), 37,4 (CH2), 31,6 (CH), 19,1 (CH3), 16,6 (CH3) ppm
IV.2. 11. Synthesis of (2S,5S)-2-(2-((benzyloxy)methyl)allyl)-2-(but-3-en-1-yl)-5-
isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (74)
To a cooled (-50°C) solution of compound 73 (55 mg, 0,16 mmol, 1 eq) in dried THF (1
ml), n-BuLi (77 µl, 0,19 mmol, 1,2 eq) is added dropwise, under Ar atmosphere. The reaction
mixture is stirred for 1 hour at -50°C after which the reaction mixture was cooled to -78°C and
4-bromobutene (24 µl, 0,24 mmol, 1,5 eq) is added. The reaction mixture is stirred overnight
allowing to slowly warm to room temperature.
A saturated aqueous NH4Cl (3 ml) solution is added after which the reaction mixture is
stirred 3-5 minutes and transferred to a separation funnel. Extraction is performed using EtOAc
(3* 5 ml). The combined organic layers are dried on MgSO4, the drying agent is filtered and
62
the filtrate is concentrated under reduced pressure. Further purification is accomplished by
column chromatography (hexane: ethyl-acetate 100:0 → 70:30).
Compound 74 is obtained as a colorless oil in 60 % yield (39 mg).
Molecular formula: C24H34N2O3
Molecular weight: 398,54 g/mol
LC-MS: tr=8,53 min, ESMS: 399,3 [M+H+]
HRMS: (ESI), mass calculated for C24H35N2O3 [M+H+]: 399,2642
mass found: 399,2633
1H NMR:
7,31-7,17 (m, 5H), 5,71-5,61 (m, 1H), 5,07-5,06 (m, 1H), 4,89-4,80 (m, 2H), 4,40 (s, 2H), 3,80-
3,75 (m, 2H), 3,57 (s, 3H), 3,52 (s, 3H), 2,55 (d, J=13.0 Hz, 1H, A part of AB system), 2,33 (d,
J=13.1 Hz, 1H, B part of AB-system), 2,23-2,12 (m, 1H), 1,82-1,54 (m, 4H), 0,98 (d, J=6.9 Hz,
3H), 0,54 (d, J=6.8 Hz, 3H) ppm
13C NMR (100 MHz, CDCl3):
163,4 (Cq), 162,3 (Cq), 141,9 (Cq), 138,6 (Cq), 138,3 (CH), 128,4 (CH), 128,3 (CH), 127,3 (CH),
127,7 (CH), 127,5 (CH), 127,3 (CH), 115,8 (CH2), 114,2 (CH2), 74,0 (CH2), 72,1 (CH2), 62,4
(Cq), 60,8 (CH), 52,2 (CH3), 51,9 (CH3), 43,0 (CH2) 40,0 (CH2), 30,5 (CH), 28,6 (CH2), 19,6
(CH3), 17,0 (CH3) ppm
IR:
3070 (vw),2942 (m), 2852 (w), 2361(w), 2348 (w), 1689 (s), 1641 (m), 1496 (vw), 1433 (m),
1435 (m), 1383 (w), 1364 (m), 1334 (w), 1308 (m), 1277 (w), 1236 (s), 1215 (s), 1195 (m),
1142 (m), 1100 (m), 1073 (m), 1029 (w), 1004 (m), 907 (m), 829 (w), 798 (w), 734 (m), 697
(m), 626 (w) cm-1
63
IV.2. 12. Synthesis of (2S,5S)-2-(2-((benzyloxy)methyl)allyl)-5-isopropyl-3,6-dimethoxy-2-
(2-((2-methoxyethoxy)methoxy)-but-3-en-1-yl)2,5-dihydropyrazine (72)
To a cooled (-50°C) solution of 73 (109 mg, 0,32 mmol, 1 eq) in dried THF (1 ml), n-
BuLi (152 µl, 0,38 mmol, 1,2 eq) is added dropwise. The reaction mixture is stirred for 1 hour
at -50°C after which the reaction mixture was cooled to -78°C and 60 (136 mg, 0,47 mmol, 1,5
eq) in dried THF (1 ml) is added. The reaction mixture is stirred overnight allowing to slowly
warm to room temperature.
A saturated aqueous NH4Cl (6 ml) solution is added after which the reaction mixture is
stirred for 3-5 minutes and transferred to a separation funnel. Extraction is performed using
EtOAc (3* 8 ml). The combined organic layers are dried on MgSO4, the drying agent is filtered
and the filtrate is concentrated under reduced pressure. Further purification is accomplished by
column chromatography (first purification step: hexane-ethyl acetate 100:0 → 70:30, second
purification step: DCM-Acetone 100:0 → 97:3).
Compound 72 is obtained as a colorless oil in 49 % yield (78 mg).
Molecular formula: C28H42N2O6
Molecular weight: 502,64 g/mol
LC-MS: tr= 4,60 min (75→100% ACN in 6 min), ESMS: 503,2 [M+H+]
HRMS: (ESI), mass calculated for C28H43N2O6 [M+H+]: 503,3116
mass found: 503,3108
64
1H NMR (400 MHz, CDCl3) –two sets of signals, ratio 87/13: signals mostly overlap, their
combined integration corresponds to the expected total proton number. Wherever possible,
signals are designated to the major and minor constituents, indicated in bold and in italic,
respectively:
7,30-7,17 (m, 5H), 5,56-5,50 (m, 1H), 5,50-5,41 (m, 1H), 5,13-4,87 (m, 4H), 4,62 (d, A part
of AB system, J= 6.9 Hz, 1H), 4,60 (d, A part of AB system, J= 6.8 Hz, 1H), 4,51 (d, B part
of AB system, J = 6.8 Hz, 1H), 4,45 (d, B part of AB system, J = 7.0 Hz, 1H), 4,40 (s, 2H),
3,88-3,79 (m, 2H), 3,78-3,71 (m, 1H), 3,71 (d, J = 3.5 Hz, 1H), 3,66-3,61 (m, 2H), 3,55 (s, 3H),
3,54 (s, 3H), 3,51 (s, 3H), 3,50 (s, 3H), 3,4-3,42 (m, 2H), 3,32 (s, 3H), 3,31 (s, 3H), 2,50 (d, A
part of AB system, J = 13.2 Hz, 1H), 2,40 (d, B part of AB system, J = 13.1 Hz, 1H), 2,22-1,93
(m, 3H), 0,99 (d, J = 6.8 Hz, 3H), 0,97 (d, J= 6.8 Hz, 3H), 0,58 (d, J = 6.8 Hz, 3H) and 0,57
(d, J =6.8 Hz, 3H) ppm
13C NMR (100 MHz, CDCl3) – two sets of signals:
Major constituent
162,89 (Cq), 162,33 (Cq), 141,46 (Cq), 138,55 (Cq), 137,40 (CH), 117,52 (CH2), 116,46 (CH2),
91,97 (CH2), 74,30 (CH), 73,98 (CH2), 72,03 (CH2), 71,75 (CH2), 66,74 (CH2), 60,77 (CH3),
60,33 (CH2), 58,97 (CH3), 52,18 (CH3), 51,57 (CH3), 45,26 (CH2), 44,02 (CH2), 30,64 (CH3),
19,63 (CH3), 17,41 (CH3) ppm
Minor constituent:
163,29 (Cq), 162,28 (Cq), 141,46 (Cq), 138,61 (CH), 138,55 (Cq), 116,60 (CH2), 116,37 (CH2),
92,74 (CH2), 75,10 (CH), 73,98 (CH2), 72,03 (CH2), 71,75 (CH2), 67,03 (CH2), 61,24 (CH3),
60,46 (CH2), 58,97 (CH3), 51,90 (CH3), 51,57 (CH3), 46,18 (CH2), 44,11 (CH2), 30,60 (CH3),
19,63 (CH3), 17,33 (CH3) ppm
IR:
3077 (vw), 2941 (m), 2870 (m), 1689 (s), 1648 (w), 1494 (vw), 1455 (m), 1435 (m), 1382 (w),
1364 (m), 1338 (w), 1307 (m), 1233 (s), 1196 (m), 1175 (m), 1139 (m), 1099 (s), 1004 (s), 923
(m), 859 (m), 788 (vw), 736 (m), 698 (m), 628 (w) cm-1
65
V. References 1. Patrick, G. L. An introduction to medicinal chemsitry. (OXFORD, 2013).
2. Ng, V. & Chan, W. C. New Found Hope for Antibiotic Discovery: Lipid II Inhibitors. Chem.
- Eur. J. 22, 12606–12616 (2016).
3. Vollmer, W., Blanot, D. & De Pedro, M. A. Peptidoglycan structure and architecture.
FEMS Microbiol. Rev. 32, 149–167 (2008).
4. Zuegg, J. et al. Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo
antibacterial activity. Nat. Commun. 6, 1–11 (2015).
5. Sauvage, E. & Terrak, M. Glycosyltransferases and Transpeptidases/Penicillin-Binding
Proteins: Valuable Targets for New Antibacterials. Antibiotics 5, 12 (2016).
6. Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A. & Charlier, P. The penicillin-binding
proteins: Structure and role in peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32,
234–258 (2008).
7. Yuan, Y. et al. Structural Analysis of the Contacs Anchoring Moenomycin to
Peptidoglycan Glycosyltransferases and Implications for Antibiotic Design. 3, 429–436
(2008).
8. Karen Bush. A resurgence of -lactamase inhibitor combinations effective against
multidrug-resistant Gram-negative pathogens. Intarnational J. Antimicrob. Agents 46,
483–9 (2015).
9. Vre, V. E. & Mitchell, M. O. Antibacterial Agents Against Methicillin-Resistant
Staphylococcus aureus. 243–247 (2007).
10. Wallhausser K. H, Nesemann G, Prave P, S. A. Moenomycin, a new antibiotic. I.
Fermentation and isolation. Antimicrob. Agents Chemother. 5, 734–736 (1965).
11. Edwin, H. F. Annual Reports in Medicinal Chemistry. 1, 109–117 (1966).
12. Ostash, B. & Walker, S. Moenomycin family antibiotics: chemical synthesis,
biosynthesis, and biological activity. Nat. Prod. Rep. 27, 1594 (2010).
13. Butaye, P., Devriese, L. A. & Haesebrouck, F. Antimicrobial Growth Promoters Used in
Animal Feed : Effects of Less Well Known Antibiotics on Gram-Positive Bacteria
Antimicrobial Growth Promoters Used in Animal Feed : Effects of Less Well Known
Antibiotics on Gram-Positive Bacteria. Clin. Microbiol. Rev. 16, 175–188 (2003).
14. Dumbre, S. et al. Synthesis of Modified Peptidoglycan Precursor Analogues for the
Inhibition of Glycosyltransferase. J. Am. Chem. Soc. 134, 9343–9351 (2012).
66
15. Sofia, M. J. et al. Discovery of novel disaccharide antibacterial agents using a
combinatorial library approach. J. Med. Chem. 42, 3193–3198 (1999).
16. Halliday, J., McKeveney, D., Muldoon, C., Rajaratnam, P. & Meutermans, W. Targeting
the forgotten transglycosylases. Biochem. Pharmacol. 71, 957–967 (2006).
17. Garneau, S., Qiao, L., Chen, L., Walker, S. & Vederas, J. C. Synthesis of mono- and
disaccharide analogs of moenomycin and lipid II for inhibition of transglycosylase
activity of penicillin-binding protein 1b. Bioorganic Med. Chem. 12, 6473–6494 (2004).
18. Shih, H.-W. et al. Combinatorial approach toward synthesis of small molecule libraries
as bacterial transglycosylase inhibitors. Org. Biomol. Chem. 8, 2586 (2010).
19. Hsu, C. H. et al. Iminosugar C-glycoside analogues of α-D-GlcNAc-1-phosphate:
Synthesis and bacterial transglycosylase inhibition. J. Org. Chem. 79, 8629–8637 (2014).
20. Teijeiro, A. L. Contribution to the synthesis of moenomycin-inspired pyrrolidine
scaffolds as potential bacterial transglycosylase inhibitors, Bachelor Thesis, Ghent
University (2017).
21. Nozal, V. Synthesis of Pyrrolidine-Based Lipid IV Analogues as Potential Bacterial
Transglycosylase Inhibitors. Bachelor thesis, Ghent University (2015).
22. Cativiela, C. & Ordóñez, M. Recent progress on the stereoselective synthesis of cyclic
quaternary α-amino acids. Tetrahedron Asymmetry 20, 1–63 (2009).
23. Espeel, P. E. R., Piens, K., Callewaert, N. & Van der Eycken, J. Synthesis of isofagomine
and a new C6 pyrrolidine azasugar with potential biological activity. Synlett 2321–2325
(2008).
24. Schollkopf, B. U., Groth, U. & Hiinig, S. Enantioselective Synthesis of (R)-Amino Acids
Using L-Valine as Chiral Agent. 20, 798–799 (1981).
25. Ulrich Schöllkopf. Enantioselective synthesis of non-proteogenic amino acids via
metallated bis-lactim ethers of 2,5-diketopiperazines. Tetrahedron 39, 2085–2091
(1983).
26. Undheim, K. The Schöllkopf chiron and transition metal mediated reactions, a powerful
combination for stereoselective construction of cyclic α-quaternary-α-amino acid
derivatives. Amino Acids 34, (2008).
27. Hammer, K. & Undheim, K. Ruthenium ( ll ) in Ring Closing Metathesis for The
Stereoselective Preparation of Cyclic 1-Amino-1-Carboxylic Acids. Tetrahedron 53,
2309–2322 (1997).
67
28. Cativiela, C. & Díaz-de-Villegas, M. D. Stereoselective synthesis of quaternary α-amino
acids. Tetrahedron Asymmetry 645–732 (2000).
29. Hammer, K. & Undheim, K. Hydroxylated cyclic α-amino acid dipeptides by ruthenium
ring closing metathesis. Tetrahedron Asymmetry 9, 2359–2368 (1998).
30. Unpublished results.
![Application Brochure A265 - Patriot Supply1].pdf · Electrical Essential Control Settings ... 115 V (ac) Class II Transformer L Do not apply power 12 13 Com – 5A 5A 5A 5A 5A 5A](https://img.dokumen.tips/doc/110x75/5eaeca02e603423ba506622e/application-brochure-a265-patriot-1pdf-electrical-essential-control-settings.jpg)
![Construction of Enantiopure Pyrrolidine Ring System via Asymmetric [3+2]-Cycloaddition of Azomethine Ylides Du Yu-liu 2014.5.5](https://img.dokumen.tips/doc/110x75/56649e9e5503460f94b9f844/construction-of-enantiopure-pyrrolidine-ring-system-via-asymmetric-32-cycloaddition.jpg)
