A Dissertation Entitled Design, Synthesis, and Inhibition Studies Of

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A Dissertation

entitled

Design, Synthesis, and Inhibition Studies of Compounds Targeting the Enzymes in the

Trehalose Utilization Pathway of Mycobacterium Tuberculosis

Sunayana Kapil

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Chemistry

Dr. Steven J. Sucheck, Committee Chair

Dr. Donald R. Ronning, Committee Member

Dr. Peter R. Andreana, Committee Member

Dr. James T. Slama, Committee Member

Dr. Cyndee Gruden, Dean College of Graduate Studies

The University of Toledo August 2019

This document is copyrighted material. Under copyright law, no parts of this document

may be reproduced without the expressed permission of the author. An Abstract of Design, Synthesis and Inhibition Studies of Compounds Targeting the Enzymes in the Trehalose Utilization Pathway of Mycobacterium tuberculosis by Sunayana Kapil Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry

The University of Toledo August 2019 Tuberculosis (TB) is an infectious disease that is responsible for more deaths worldwide than any other contagious disease. TB treatment is challenging, which often results in poor adherence to medications, leading to the development of drug-resistant strains of

Mycobacterium tuberculosis (Mtb), the bacteria responsible for the disease. The increasing drug-resistance of Mtb has made it necessary to develop new therapeutics. Thus, this dissertation involves the design, synthesis, and inhibition studies of compounds targeting the enzymes involved in the trehalose utilization pathway (TUP) of Mtb.

The first chapter introduces the history of TB, the Mtb cell wall, and the role of trehalose dimycolate (TDM) in pathogenesis. The second chapter details the synthesis of a library of

2-aminothiophenes as potential inhibitors of polyketide synthase 13 (Pks13)1 and also introduces the synthesis of mycolic acids in Mtb. The thiophene cores were synthesized by the Gewald reaction and the cores were subsequently treated with different acid chlorides and alkyl amines to yield 2-alkylamido and 3-carboxamide compounds, respectively.

These compounds were evaluated for their inhibitory activity against Mtb H37Rv, and one of the compounds containing a perfluorobenzamido group showed excellent inhibition with a minimum inhibitory concentration (MIC) of 0.69 µM. Other literature reports have iii indicated that related compounds containing a perfluorobenzamido group act as potential

Pks13 inhibitors.2 Hence, it is assumed that the 2-aminothiophenes prepared in this work can also inhibit Pks13 or target enzymes involved in the biosynthesis of mycolic acids.

The third chapter discusses the design and synthesis of mimetic trehalose-6-phosphate

(T6P) substrates as inhibitors for Mtb trehalose phosphate phosphatase, which is found in

TUP.3 The 6-position of T6P was modified to produce 6-phosphonic acid- (TMP, 2), 6-

(methylene) phosphonic acid- (TEP, 3), 6-N-phosphonamide- (TNP, 4) and 6-oxirane trehalose as non-hydrolyzable inhibitors. The obtained compounds were evaluated for their inhibitory activities against homologs of TPP from Mtb, M. lentiflavum (Mlt) and M. triplex (Mtx). The results showed that all compounds strongly inhibited Mtx with IC50 values of 288 ± 32 µM, 421 ± 24 µM, and 1959 ± 261 µM for TMP, TNP, and TEP respectively. The results also indicated that the presence of an atom bearing a lone pair of electrons at the 6-position of trehalose played an important role in binding to TPP.

The fourth chapter discusses glycoside hydrolase (GH) enzymes and the targeting of the

GH-like enzyme, Mtb GlgE. The section also reviews the classification of GHs into subfamilies based on their mechanistic actions, as well as the importance of targeting Mtb

GlgE, as it is a genetically validated drug target. Three different classes of inhibitors were synthesized for targeting Mtb GlgE. First, glycoconjugate-based inhibitors were synthesized via the glycosylation of thioglycoside donor with phenols and cyclohexanols with different substituents. Second, pyrrolidine-based inhibitors were synthesized which mimicked the charge of the substrate in the transition state (TS)-catalyzed reaction with

Mtb GlgE. Third, cyclitol-based inhibitors were synthesized which were anticipated to form covalent adducts with GlgE. iv

The compounds were tested for their inhibitory activity against Streptomyces coelicolor

(Sco) GlgE1-V279S, a GlgE homolog. The results indicated that the glycoconjugate in which the first glucose unit was replaced with a phenyl group bearing a carboxylic acid and amine at the ortho position showed a Ki of 659 µM, and the pyrrolidine-based compound with a propyl group containing a benzyl chloroformate-protected amine showed a Ki of 132.6 ± 8 µM. In contrast, the cyclitol-based inhibitor containing a cyclopropyl ring did not show any inhibition, possibly due to its Michalis conformation which placed the aglycone moiety in an equatorial position. It can thus be concluded that in addition to mimicking the TS, a similar Michaelis conformation is necessary to develop inhibitors for

GH enzymes.

Dedicated to my mother. Acknowledgments

First and foremost, I express my immense gratitude to my advisor Dr. Steve Sucheck for giving me this opportunity to conduct research in his lab. I am thankful for his invaluable guidance and constant support throughout my PhD. His constant motivation to carry out work in the lab is the most driving force for me to move ahead. I deeply thank our collaborator and committee member Dr. Donald Ronning for his valuable inputs and contribution in various projects. I would like to thank Dr. Richard A. Slayden and Dr. Mary

Jackson for their contribution to one of the projects. I sincerely thank my graduate committee members Dr. Peter Andreana and Dr. James Slama for their constant support and insightful comments.

My sincere thanks to Dr. Sandeep Thanna, Dr. Cecil Petit, Victoria N. drago and Dr.

Christopher M. Goins for their contribution in accomplishing the projects. I thank my fellow labmates past and present for maintaining a cordial atmosphere in the lab especially,

Dr. Sandeep Thanna and Dr. Sri Kumar Veleti for guiding me initially through lab techniques. I am grateful to Dr. Yong Wah Kim for training me on NMR and MS. I thank my friends Shriraj, Shivani, Abhishek, Vidhisha, Abhijeet, Yousaf and Sarmad for always cheering me up.

Finally, I express my profound gratitude to my family: my mother for her unconditional love, support and motivation, my brother for always supporting me. Last but not least, I would like to thank my husband for his patience and encouragement throughout my PhD.

vii

Contents

Abstract ...……………………………………………………………………………….. iii Acknowledgment ………………………………………………………………………. vii Contents ……………………………………………………………………………….. viii List of Tables …………………….………………………………………………………. x List of Figures …………………………………………………………………………… xi 1. Introduction to Tuberculosis ……………………………………………………….…. 1 1.1 History of Tuberculosis ………………………………………………….…...... 1 1.1.1 Treatment of TB …………………………………………………………. 2 1.2 Mtb cell wall ……………………………………………………………………. 3 1.3 Role of TDM …………………………………………………………………… 6 1.4 Importance of Trehalose ……………………………………………………...... 7 2. Synthesis and evaluation of 2-aminothiophenes against Mtb ………………………..... 9 2.1 Introduction ……………….……………………………………………………. 9 2.2 Mycolic acid synthesis …….……………………………………………………. 9 2.2.1 Mechanistic action of Pks13 …………….……………………………... 10 2.3 Background ….………………………………………………………………… 13 2.4 Results ….……………………………………………………………………... 15 2.4.1 Chemistry ……………………………………………………………….. 15 2.4.2 Inhibition studies ……………………………………………………….. 17 2.5 Experimental …………………………………………………………………... 19 3. Synthesis and invitro characterization of trehalose-based inhibitors of mycobacterial trehalose-6-phosphate phosphatase …………………………………………………….. 29 3.1 Introduction ….………………………………………………………………... 30

viii

3.2. Results and discussion …………….…………………………………………. 32 3.2.1 Chemistry studies ……………………………………………………… 32 3.2.2 Discussion ……….……..……………………………………………… 36 3.3 Experimental …………………….…………..………………………………… 40 4. Targeting Glycoside Hydrolase enzyme GlgE ……………………………….………. 51 4.1 Introduction …………………………...………………………………………. 52 4.2 Classification of GH …………………………………………………………… 53 4.2.1 Based on substrate specificity …………………………...……………… 53 4.2.2 Based on mechanistic action ……………………………...…………….. 53 4.2.3 Based on mode of action ………………………………...……………… 54 4.2.4 Based on amino acid sequence ………………………………………….. 54 4.3 Mechanism for GH …………………………………………………….……… 55 4.4 Targeting GH enzyme GlgE ………………………………………….……….. 57 4.5 Glycoside Hydrolase inhibitors …………………………….…………………. 60 4.5.1 Glyco-conjugate as inhibitors ………………………...…………………. 60 4.5.1.1 Chemistry …………….…………………………………………… 62 4.5.1.2 Experimental for glyco-conjugates ….………………...………….. 65 4.5.2 Iminosugars as Inhibitors ……….………………………………………. 71 4.5.2.1 Experimental for iminosugar-based inhibitors ………...…………. 73 4.5.3 Inhibition studies ………….…………………………………………….. 74 4.5.4 Result and discussion ………….………………………………………... 75 4.5.5 Carbasugars as inhibitors …………….…………………………………. 76 4.5.5.1 Chemistry studies …………………………………………………. 81 4.5.5.2 Results ……………………………………………………………. 85 4.5.5.3 Discussion ………………………………………………………… 86 4.5.5.4 Experimental ……………………………………………………… 88 References …………………………………………………………………………….. 103 ix

Appendix A Supporting Information-Chapter 2 …..…………………………………... 125 Appendix B Supporting Information-Chapter 3 ……..………………………………... 161 Appendix C Supporting Information-Chapter 4 ……...…………..……………………. 197 Appendix D Journal Permission to reproduce material …………..……………………. 254

List of tables

2.1 MICs and IC50s (μM) of the 2AT Library Mtb H37Rv…………………………. 18-19

4.1 Entries of different drug-like compounds designed and their respective docking scores ……………………………………………………………………… 61-62

4.2 Reaction results of glycosylation of donor 1 with different alcohols (2a-d) to get compounds 3a-d with their respective yields………………………………………..…... 63

4.3 Reaction results of deprotection of compounds 3a-d to get glycol-conjugates 4a-e with their respective yields …………………………………………………………………... 64

4.4 Comparing the NMR chemical shift values of compounds 32A & 32B with C and D ……………………………………………………………………………. 82

4.5 Reaction condition optimization for Ring Closing Metathesis for compound 32A …………………………………………………………………………. 83

4.6 Comparing the NMR shift values of compounds 33A & 33B with E and F …………………………………………………………………………….. 84

List of Figures

1.1 Mycobacterium cell wall; Structure of TDM ……………………………………….. 5

1.2 Routes for the synthesis and recycling of trehalose, as well as its conversion to

TMM/TDM in Mtb ………………………………………………………………….. 8

2.1 The synthesis of mycolic acid by FAS-I and FAS-II further these substrates are

utilized by different domains of enzyme Pks 13 to produce mycolic acid ………… 12

2.2 Reported inhibitors for Ag85C and Pks13. In the present work synthesized FRT

(fused ring thiophene) and 5PT (5-phenyl thiophene) ……………………………... 15

2.3 Library of 2-aminothiophenes with yields …………………………………………. 18

3.1 6-deoxy-α,α'-trehalose derivatives prepared from heptabenzyl α,α'-trehalose

derivative 3 and Validamycin A (8) which shows topological similarities with

trehalose …………………………………………………………………………….. 32

3.2 TPP Inhibition studies ……………………………………………………………… 38

4.1 Different mechanisms of GH: a) Retaining mechanism b) Inverting mechanism c)

Neighboring group participation mechanism d) Mechanism for GlvA ……………... 57

4.2 Mechanism for GlgE1 ………………………………………………………………. 59

4.3 2a-2d structure of different acceptors; 3a-3d structure of glycosylated products …. 63

4.4 4a-4e structure of different glycol-conjugates ……………………………………… 64

4.5 Iminosugars-based inhibitors for GH enzymes …………………………………….. 72

4.6 Ki determination of compound 4a with Sco GlgEI-V279. Vo’/Vo are steady-state rates with and without inhibitor ………………………………………………………………. 75

xii

4.7 Conformations attained by Transition State (TS) ….……………………………….. 77

4.8 Carbasugars compounds as GH inhibitors ………………………………………….. 78

4.9 Cyclitol-based compounds C1 and C2 based on M1P (substrate of GlgE) ………… 79

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Chapter 1 Introduction to Tuberculosis

1.1 History of Tuberculosis

Tuberculosis (TB), also known as the “Great White Plague,” is an infectious disease that has victimized humanity for centuries. It is caused by the Mycobacterium tuberculosis

(Mtb) bacteria, which first originated more than 150 million years ago.4 However, evidence from skeletal remains revealed that the first instance of TB infection in humans dates back to 5000 BCE. TB is an airborne disease which became an epidemic due to rapid urbanization.5 It was believed to be a genetic disease until 1790 when Benjamin Marten first suggested that it was transmissible.6 A greater understanding of the disease was developed in the 19th century with the invention of the stethoscope by Theophile Laennec, which enabled pulmonary and extra-pulmonary TB to be distinguished from one another by their different physical symptoms.7 In 1882, Robert Koch discovered that Mtb was the etiological agent of TB.8

Since its discovery, there has been a continuous effort to eradicate TB, but it remains one of the leading causes of death worldwide. In 2017, the World Health Organization (WHO) reported that approximately 10 million people developed TB, and 1.6 million died due to the disease.9 Approximately one-quarter of the world’s population is infected by latent TB, meaning they are infected but display no symptoms and cannot transfer the infection, but

1 they are at risk for developing TB later in life. Efforts to treat TB have been hindered due to the development of multi-drug resistant TB (MDR-TB).

1.1.1 Treatment of TB

The search to prevent TB began in 1921 with the introduction of the Bacille Calmette-

Guérin (BCG) vaccine, but its usage was discontinued in some countries due to contraindication and its interference with TB tests. Streptomycin was the first drug introduced against TB by Waksman in 1944.10 However, prolonged use of streptomycin caused the development of resistance in patients, and it was discontinued as a first-line drug, though it is still administered in combination with other drugs. In 1952, Isoniazid

(INH) was discovered as an anti-TB drug, and it is still one of the most potent medications.

In 1957, rifampin (RIF) was introduced, and soon after its introduction, drug-resistant cases emerged.11 It was observed that when medications were administered in combination, the drug resistance of TB was suppressed, which introduced the multidrug treatment regimen.12

The primary treatment for TB involves an intensive phase of two months of four first-line drugs i.e., INH, RIF, ethambutol (EMB), and pyrazinamide (PZA), accompanied by a continuation phase of four to seven months of INH and RIF.13 The drugs used to treat TB have several targets: a) Cell wall synthesis: INH, EMB, Thioamides, Nitroimidazoles,

Cycloserine; b) RNA synthesis: RIF; c) DNA synthesis: Para-aminosalicylic acid (PAS), fluoroquinolones; e) Protein synthesis: Streptomycin; f) ATP synthase: Bendaquiline. Pill burden and side effects result in poor adherence to treatments, which leads to the development of drug-resistant TB.14 MDR-TB is classified as TB which is resistant to at least two first-line drugs, INH and RIF, and extensively drug-resistant TB (XDR-TB) is

2 resistant to two first-line drugs and one second-line drug. Some recent cases have emerged with total drug-resistant TB, which is resistant to all first-line and second-line drugs

(ofloxacin, moxifloxacin, kanamycin, amikacin, capreomycin, para-aminosalicylic acid, and ethionamide).15 The alarming emergence of drug resistance and the growing number of global TB cases each year has propelled researchers to explore new drug-targets for chemotherapy.

1.2 Mtb cell wall

Mtb cell walls are comprised of three main layers: peptidoglycan, arbinogalactan, and mycolic acids. Between the mycolic acid layers are glycophospholipids and waxes which comprise the outer membrane.16 The complex Mtb cell wall is essential to its virulence and acts as a barrier to antibiotics, thus the biosynthetic pathways of the cell wall serve as potential targets for therapeutic agents.

The peptidoglycan layer consists of a polymer containing alternating units of N- acetylglucosamine and N-acetylmuramic acid linked via β(1→4) with amino acid side chains cross-linked by transpeptide bridges.17, 18 The peptidoglycan layer is found in both

Gram-positive and Gram-negative bacteria and provides shape and rigidity to the cell to help it withstand osmotic pressure.19 The muramic acid in the peptidoglycan layer contains

N-acetyl and N-glycolyl derivatives, which form strong hydrogen bonds that help fortify the cell wall.17 The amino acid side chains consist of L-alaninyl-D-isoglutaminyl-meso- diaminopimelyl-D-alanine moieties which are linked to other peptide chains from adjacent glycans.20 This intensive cross-linking in Mtb is much higher compared with the peptidoglycan layer of E. coli.21 Arabinogalactan is a polysaccharide of galactan and

3 arabinose in the furanose form in which galactan consists of 30 alternating β(1→5) and

β(1→6) linked β-D-Gal units attached to highly-branched arabinose residues.18 The non- reducing end of galactan is covalently attached to a muramic acid residue of the peptidogalactan layer via the disaccharide linker, diglycosyl-P bridge, α-L-Rhap-(1→3)-

D-GlcNAc-(1→P).22 Three units of arbinan, each consisting of 23 residues, are attached to the 8th, 10th, and, 12th residues of the C-5 hydroxyl of galactan.23 The highly-branched arbinan is linked via α(1→5) and α(1→3) linkages.24 The α(1→5) arbinan linkage is attached to the non-reducing end of the β(1→2) residue, and two-thirds of the reducing end is attached to mycolic acid and the remaining one-third to succinyl and galactosamine.25, 26

Mycolic acids are long chains of α-alkyl-β-hydroxy fatty acids that are aligned perpendicularly to the membrane with long mermycolates and short chains, and typical keto-, methoxy-, and hydroxy-mycolic acid chain lengths range from C84-C88. Mycolic acid forms the lipids present in the outer membrane, such as trehalose monomycolate

(TMM), trehalose dimycolate (TDM), and glucose monomycolate, and they are important for cell survival and are essential to the virulence of Mtb. Mycolyl-arbinogalactan- peptidoglycan forms the core cell wall, above which are lipids containing longer and shorter fatty acid chains which are intercalated with cell wall proteins and lipids, such as phosphatidylinositol mannosides (PIMs), lipamannan (LM), and lipoarabinomannan

(LAM).27 More than 60% of the cell wall in Mtb consist of lipids, primarily mycolic acids, cord factor, and wax-D.

Figure 1.1 Mycobacterium cell wall; Structure of TDM (Adapted).28 The Mtb cell wall is a thick bastion acting as a barrier to the antibiotics. The cytoplasmic compartment is protected with plasma membrane covered with peptidoglycan layer which is covalently attached to the arbinogalactan layer. Mycolic acids are linked to arbinangalactan layer and firm the inner leaflet of the mycomembrane. The outerleaflet consists of several lipids as TDM (in blue), TMM (in yellow), Glycolipids (GL in red) and phospholipids (PL in pink).28, 29

Mycolic acids are the major components of the Mtb cell wall. Corynebacterium glutamicum has a cell envelope similar to that of Mtb and contains a cell wall primarily composed of mycolic acids.30 Asselineau and Lederer first described mycolic acids in 1950 as α-alkyl β-hydroxy long-chain fatty acids with chain lengths ranging from C60 to C90 with

2R and 3R configurations.31 They are present in cell walls as either free chains or esters of trehalose or glycerol or the pentarbinofuranosyl unit of arbinogalactan. In Mtb, mycolic 5 acids have different chain lengths and contain various double bonds, cyclopropyl groups, and oxygen functionalities, such as keto, methoxy, epoxy ester, and hydroxyl groups.32

Since they are the main constituent of the Mtb cell wall, and their related products are responsible for its pathogenesis, the synthesis of mycolic acids has intrigued many researchers. For example, Isoniazid, the most potent first-line drug, has been shown to inhibit the synthesis of mycolic acids.

1.3 Role of TDM

The outer membrane of Mtb is rich in TDM, which is also known as cord factor33 and is the most easily extracted lipid on the Mtb surface.33 It is present in high amounts in virulent

Mtb compared with avirulent Mtb, suggesting it plays an important role in the pathogenesis of TB.34 It is known that removing TDM from the surface of TB allows the organism to be rapidly killed by macrophages within three days, and adding TDM back reinstates its ability to survive macrophages.35 TDM also protects the cell from phagosome fusion, acidification, and macrophages.36, 37 Structural alteration of mycolic acids in TDM reduces the virulence of TB.38 TDM is an amphiphilic molecule which contains hydrophilic trehalose and lipophilic mycolic acid chains, and when it is intercalated with LAM, it forms a bilayer, and the interlinked short and long mycolic acid chains give TDM its high stability and impermeability. Free monolayer TDM is highly toxic and kills macrophages in as little as five minutes37 and is also responsible for the production of granulomas, which suggests that TDM plays a role in Mtb pathogenesis.

1.4 Importance of Trehalose

α,α'-Trehalose is an essential non-reducing disaccharide containing an α(1→1) glycosidic linkage between two glucose units. It is present in plants,39 some bacteria,40 fungi,41 parasitic nematodes,42 and insects,43 but it is absent in mammals. The glycoconjugate formed by trehalose and lipids forms an integral part of the Mtb cell wall and is essential to the virulence and viability of Mtb and also serves as an energy source, metabolic regulator, and anti-desiccant.40 The lack of mammalian trehalose synthesis, import, or processing pathways makes these pathways potential targets for Mtb chemotherapy.44, 45

The three major production pathways of trehalose in Mtb are OtaA-OtsB, TreS, and TreY-

TreZ (Figure 1.2).45

The first pathway, the OtsA-OtsB, or TPS-TPP2 pathway (shown in red in Figure 1.2), involves two steps. In the first step, glucose is transferred from UDP-glucose to glucose-

6-phosphate catalyzed by enzyme TPS (trehalose phosphate synthase). In the next step, trehalose-6-phosphate is catalytically dephosphorylated by enzyme TPP2 (trehalose phosphate phosphatase) to produce free trehalose and phosphate. The TreS pathway

(shown in blue in Figure 1.2) involves the isomerization of maltose with an α-(1→4) linkage to trehalose and an α-(1→1) linkage catalyzed by enzyme TreS (trehalose synthase). The TreY-TreZ pathway (shown in pink in Figure 1.2) utilizes a maltooligosaccharide to enzymatically convert the maltose at the reducing end to trehalose by maltooligosyltrehalose synthase (TreY). In the next step, maltooligosyltrehalose is enzymatically hydrolyzed to release free trehalose by maltooligosyltrehalose trehydrolase

(TreZ).46

Figure 1.2 Routes for the synthesis and recycling of trehalose, as well as its conversion to TMM/TDM in Mtb; a) OtsA–OtsB pathway (in red). b) TreS pathway (in blue) c) TreY–TreZ pathway (in pink). Following enzymes: Antigen85 complex (Ag85s), mycobacterial membrane protein large 3 (MmpL3), trehalose phosphate synthase (TPS), trehalose phosphate phosphatase 2 (TPP2), trehalose synthase (TreS), maltokinase (Mak), maltooligosyltrehalose synthase (TreY), maltooligosyltrehalose trehalohydrolase (TreZ). (Adapted)29

Chapter 2

Synthesis and evaluation of 2-aminothiophenes against Mycobacterium tuberculosis.

2.1 Introduction

As discussed in the first chapter, mycolic acids are one of the major components of the Mtb cell wall. The glycolipids associated with them, such as TMM, TDM (cord factor), and mAG (mycolyl-arbonigalactan), are the most abundant lipids in the Mtb cell wall and play structural and functional roles which are essential for the viability of Mtb. They are also responsible for the impermeability of some antibiotics and are highly toxic when released into their host cell. Thus, the structure and biosynthetic pathways for the production of these lipids and mycolic acids have motivated researchers to target them for the treatment of TB.

2.2 Mycolic acid synthesis

Mycolic acids are a long-chain fatty acids which typically range from C60-C90 in

Mycobacterium. The precursors for mycolic acids are fatty acids, which are synthesized by fatty acid synthase I (FAS-I) and fatty acid synthase II (FAS-II) which have different

9 substrates and carrier specificities, but similar reaction pathways. FAS-I synthesizes fatty

28 acids with C16-C18 and C24-C26 chain lengths, which serve as α-chains in mycolic acids, whereas FAS-II elongates the fatty acid (FA) chain length of mycolic acids. FAS-I has seven different domains which perform catalytic reactions in the cycle and is encoded by a single gene (fas, Rv2524c) and is crucial for the transposon mutagenesis of Mtb.47 FAS-

I produces C24-C26 acyl-CoAs which are further carboxylated by ACCase and participate

48 in the synthesis of mycolic acids. FAS-II first elongates C12-C16 chains to C18-C30 chains to acyl carrier proteins (ACPs) by covalently linking the intermediates to a mycobacterial acyl carrier protein (AcpM).49 The malonyl-ACP is produced by transferring a malonate group from malonylCoA:ACP transacylase to ACP which participates in Claisen-type condensation with acyl-CoA to form β-ketoacyl-ACP, which is catalyzed by β-ketoacyl-

ACP synthase III.28 Chain elongation continues until acyl-ACP attains the required chain length, and four enzymes (InhA, MabA, HadB, and KasA) catalyze each elongation cycle.

The final Claisen-type condensation of the two activated fatty acid synthases leads to the final mycolic acid, but it is unclear which condensate is responsible for mycolic acid synthesis.50 In C. glutamicum and M. smegmatis, enzyme Pks13, which belongs to the polyketide synthase family 1, is responsible for the final condensation step.

2.2.1 Mechanistic action of enzyme Pks13

In Mtb, more than 18 Pks-1 genes are present, Pks13 is encoded by fad32-pks13-accD4 and is essential for the synthesis of mycolic acids. It contains five different domains responsible for the catalytic activity of the condensation of two activated fatty acids. Pks13 is activated by 4’phophopantetheinyl transferase Ppt, leading to post-translational

10 modification that activates the acyl-carrier protein (ACP) domain and peptidyl carrier protein (PCP) domain. Claisen-type condensation occurs between the two substrates – mermycoloyl on the ketosynthase domain and carboxyacyl on the ACP domain – to yield an α-alkyl-β-keto thioester (Figure 2.1). This product forms an ester bond with S1533 that is then cleaved by the thioesterase domain and transfers the α-alkyl-β-ketoacyl chain to trehalose to form TMMk (trehalose monomycolate keto form). The keto group is then reduced by CmrA (corynebacterineae mycolate reductase A) to form TMM which is translocated by the transmembrane protein MmpL3 (mycobacterium membrane protein large 3), to an antigen 85 (Ag85) complex that transfers the mycoloyl chain from TMM to form TDM and mAG.

Figure 2.1 The synthesis of Mycolic acid by FAS-I and FAS-II substrates. These substrates are utilized by different domains of enzyme Pks13 to produce mycolic acid. The FAS-I produces C26 chain which constitutes as α-branch and C16 acyl-CoA chain acts as a substrate for FAS II to produce mermycolic acid. These two substrate produced by FAS-I and FAS-II utilized by different domains of Pks13 as acyl carrier protein (N-ACP and C-ACP) having P-pant, ketosynthase (KS), acyltransferase (AT) and final condensation domain thioesterase (TE) which releases α-alkyl β-ketoacyl chain with R1 = mermycolic chain (C48 – C64); R2 = α-branch (C24 – C26).

2.3 Background

Thiophenes are compounds found in a variety of natural and synthetic products which show a many pharmacological properties,51, 52 as well as anti-tuberculosis activity. For example,

2-aminothiophenes have been reported to inhibit the Mtb enzymes Pks13 and Ag85 (Figure

2.2).2, 53 Due to the inhibition shown by thiophene (XI3-AG85) A2 against Ag85s, Ibrahim et al. designed and synthesized thiophenyl-arabinoside conjugate A1 as an Ag85C inhibitor.53 Studies from 1113 previously-screened compounds by Wilson et al. showed that 2-aminothiophenes P1 and P2 containing a perfluorobenzamido group showed MIC values of 0.5-1.0 µM against Mtb (Figure 2.2).2

Figure 2.2 Reported inhibitors for Ag85C and Pks13. In the present we work synthesized FRT (fused ring thiophene) and 5PT (5-phenyl thiophene).

Based on the literature, a library of 2-aminothiophenes were synthesized and tested for their inhibitory activity against Mtb H37Rv.

2.4 Results

2.4.1 Chemistry

A library of 2-aminothiophenes with different derivatives at R1, R2 and R3 positions were synthesized. Two different 2-aminothiophene cores were synthesized in which one contains a fused-ring thiophene (FRT) and the other contains 5-phenylthiophene (5PT).

FRT cores were synthesized by performing Gewald reactions, beginning with four different ketones i.e., benzyl 4-oxopiperidine-1-carboxylate (2), tert-butyl 4-oxopiperidine-1- carboxylate (3), 1-ethylpiperidine-4-one (4), and cyclohexanone (5) (Scheme 2.1). The

Gewald reaction utilized a ketone or aldehyde, an activated nitrile (ethyl-2-cyanoacetate), sulfur (S8), and diethylamine to produce the thiophene cores. The FRT cores were treated with different acid chlorides in the presence of DMAP (dimethyl amino pyridine) and Et3N

(triethylamine) to produce 2-alkylamido compounds 12-19 and 21-24. The different acid chlorides used were: acetyl chloride, benzoyl chloride, 2,3,4,5,6-pentafluorobenzoyl chloride, and 3,5-bis(trifluoromethyl)benzoyl chloride. The 3-carboxamide compounds

(29-38) were synthesized by first treating the FRT cores with LiOH for ester deprotection to produce acids (25-28) which were coupled with different alkyl amines in presence of coupling reagents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC.HCl), hydroxybenzotriazole (HOBt), and DMAP to produce 3-carboxamide. The following alkyl amines were used: butylamine, benzylamine, 4-methylbenzylamine, 2- methoxybenzylamine, and adamantylamine. The synthesis of 5PT core is not discussed here and can be found in the literature.1

Scheme 2.1 Reagents and Conditions: (i) S8, Et2NH, EtOH, 7 (86%), 8 (63%), 9 (75%), 10 (83%), 11

(61%); (ii) Acid chloride (AC), Et3N, DCM, DMAP (15–82%); (iii) LiOH, THF : H2O: MeOH (3 : 1 :

1) (44–89%); (iv) Alkylamine (AA), EDC. HCl, HOBt, DMAP, dry CH2Cl2 (21–61%).

Figure 2.3 Library of 2-aminothiophenes with yields. Synthesized collectively by Dr. Sandeep Thanna and Sunayana Kapil.

2.4.2 Inhibition studies

A library of 2-aminothiophenes was prepared and tested to evaluate their inhibitory activities against Ag85C. Unfortunately, no compound showed significant inhibition towards Ag85C. However, when these were subjected to growth inhibition studies using a modified 96-well microplate Alamar blue assay (MABA),1 compounds containing a perfluorobenzamido group showed significant inhibition with MIC values < 50 µM. All 17 other compounds displayed MIC values > 200 µM, which indicates that the perfluorobenzamido group is essential for the anti-Mtb activity. The active compounds 18 and 23 have similar structures to the Pks13 inhibitory compounds P1 and P2 reported by

Wilson et al. Hence, it was anticipated that the compounds prepared here would inhibit

Pks13 or would target enzymes involved in the biosynthesis of mycolic acids. Compound

18 showed an MIC of 0.69 µM and was synthesized by Dr. Sandeep Thanna and was further evaluated for its potency using the National Institute of Health screening contract which is discussed elsewhere.1

Comp No. NV/CLogP MIC IC50 Comp No NV/CLogP MIC IC50

7 0/2.80 > 200 N/A 28 0/1.88 507 N/A

8 0/2.39 > 300 N/A 29 0/2.15 > 300 > 300

9 0/1.73 > 300 N/A 30 0/2.11 > 300 N/A

10 0/2.52 > 400 N/A 31 0/2.56 > 300 N/A

11 0/3.26 > 400 N/A 32 0/2.12 > 200 N/A

12 0/2.12 > 200 N/A 33 0/3.48 > 200 N/A

13 0/3.79 > 200 N/A 34 0/2.94 > 300 N/A

14 1/4.33 > 100 N/A 35 0/2.90 > 300 N/A

15 2/5.51 > 100 N/A 36 0/3.35 333 N/A

16 0/1.46 > 300 N/A 37 0/2.91 316 N/A

17 0/3.50 279 N/A 38 0/4.27 > 300 N/A

18 0/4.03 0.69 0.65 39 0/3.00 > 300 N/A

19 1/5.22 > 200 N/A 40 0/4.67 > 200 N/A

20 2/5.69 300 N/A 41 1/5.20 > 200 N/A

21 0/2.25 > 300 N/A 42 1/6.39 > 200 N/A

22 0/3.95 > 600 N/A 43 0/2.62 228 129

23 0/4.45 30 13 44 0/3.68 > 300 N/A

24 1/5.64 > 200 N/A 45 0/3.64 324.5 N/A

25 0/2.16 > 300 N/A 46 0/4.09 > 300 N/A

26 0/1.75 > 300 N/A 47 0/3.65 > 200 N/A

27 0/1.09 > 400 N/A 48 1/5.01 > 200 N/A

Table 2.1 MICs and IC50s (μM) of the 2AT Library Mtb H37Rv; NV = Number of violations for Lipinski rule of five, Clog P = Calculated log P value. The inhibition studies data was provided by Dr. Slayden group.

2.5 Experimental

General procedure for Gewald synthesis (7-10)

A carbonyl 4 (2.15 mL, 15.72 mmol, 1 eq), ethyl cyanoacetate (1.84 mL, 17.30 mmol, 1.1 eq), and sulfur (0.60 g, 18.86 mmol, 1.2 eq) were added to a round-bottom flask containing

19 ethanol (30 mL). Diethylamine (3.23 mL, 31.44 mmol, 2 eq) was added to this suspension, and the reaction was heated at 65 oC for 3 hours. After reaction completion, solvent was evaporated under reduced pressure, and DCM (20 mL) was added. The organic layer was washed with NH4Cl solution, NaCl solution, and water, while the organic layer was dried over Na2SO4 and filtered. DCM was evaporated under reduced pressure, and the crude product was crystallized with cold MeOH to obtain a yellow product 9.

General procedure for ethyl ester to acid (25-28)

LiOH (74.25 mg, 1.77 mmol, 3 eq) was added to ethyl ester compound 9 (150 mg, 0.59 mmol, 1 eq) dissolved in THF/H2O/MeOH (3:1:1), and the reaction was stirred at 70-80 oC overnight. After reaction completion, everything was evaporated under reduced pressure and water was added to the reaction mixture. Organic layer was extracted with

Diethyl ether, to which 6 N HCl was added to give the aqueous layer a pH of 2, which resulted in the immediate formation of a precipitate. Ethyl acetate (EtOAc) 20 mL was used to extract the organic layer three times, and then the organic layer was evaporated under reduced pressure to obtain a viscous liquid which was re-dissolved in a minimal amount of DCM. Afterwards, hexane was added which formed a precipitate that was then filtered to obtain the carboxylic acid 27.

General procedure for 3-carboxamide thiophene (29-38)

A solution of benzyl amine (51.5 µL, 0.44 mmol, 1 eq), EDC.HCl (84.48 mg, 0.44 mmol,

1 eq), DMAP (81.0 mg, 0.663 mmol, 1.5 eq), and HOBt (67.68 mg, 0.44 mmol, 1 eq) dissolved in 1.5 ml of DCM was added dropwise at 10 oC to the carboxylic acid 27 dissolved in 1.5 mL of DCM. The reaction mixture was stirred for 18 h at room temperature

20 and then diluted with 10 mL of DCM after reaction completion and washed with brine. The organic layer was collected and dried over Na2SO4 and then evaporated under reduced pressure to give a crude oil which was purified by silica gel flash column chromatography.

General Procedure for 2-amido thiophene (12-19, 21-24)

The thiophene amine 9 was dissolved in 2 mL of DCM and cooled to 0 oC, to which DMAP

(25.96 mg, 0.212 mmol, 0.4 eq) and Et3N (148 µL, 1.06 mmol, 2 eq) were added, followed by the dropwise addition of an acetyl chloride (45 µL, 0.637 mmol, 1.2 eq). The reaction mixture was allowed to stir for 12 h at room temperature. After reaction completion, all liquids were evaporated under reduced pressure and subjected to silica gel flash column chromatography.

Ethyl 2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate (9)

1 Yield: 58%; silica gel TLC Rf = 0.50 (40 % EtOAc in Hexane ); H-NMR (600 MHz,

3 CDCl3): δ 5.96 (s, 2H, H-10), 4.25 (q, 2H, J15,16 7.2 Hz, H-15), 3.43 (s, 2H, H-8), 2.84 (t,

3 3 3 2H, J5,6 8 Hz H-5), 2.72 (t, 2H, J6,5 8 Hz, H-6), 2.58 (q, 2H, J13,14 7.2 Hz H-13), 1.33 (t,

3 3 13 3H, J16,15 7.2 Hz H-16), 1.17 (t, 3H, J14,13 7.2 H-14) ; C-NMR (600 MHz, CDCl3): δ

165.96 (C-2), 162.01 (C-11), 131.14 (C-4), 114.82 (C-9), 105.36 (C-3), 59.41 (C-15), 51.57

(C-12), 51.09 (C-8), 50.23 (C-6), 27.43 (C-5), 14.43 (C-16), 12.58 (C-14) ; mass spectrum,

+ + m/z = 277.10 (M+23) C12H18N2O2S requires 277.10 (M+23)

Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (10)

1 Yield: 40% (60 mg); silica gel TLC Rf = 0.50 (40 % EtOAc in Hexane); H-NMR (600

3 3 MHz, CDCl3): δ 5.94 (s, 12H, H-10), 4.26 (q, 2H, J13,14 7.2 Hz, H-13), 2.71 (t, 2H, J8,7

3 5.01 Hz H-8), 2.51 (t, 2H, J5,6 5.01 Hz, H-5), 1.80-1.73 (m, 4H, 2 × CH2, H-6,7), 1.34 (t, 21

3 13 3H, J14,13 7.2 H-14) ; C-NMR (600 MHz, CDCl3): δ 166.13 (C-2), 161.63 (C-11), 132.46

(C-4), 117.63 (C-9), 105.76 (C-3), 59.36 (C-13), 26.93 (C-8), 24.52 (C-5), 23.23 (C-7),

+ 22.81 (C-6), 14.46 (C-14) ; mass spectrum, m/z = 248.07 (M+23) C11H15NO2S requires

248.07 (M+23)+.

Ethyl 2-acetamido-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate

(16)

1 Yield: 33% (78.0 mg); silica gel TLC Rf = 0.30 (10 % Methanol in CH2Cl2 ); H-NMR

3 (600 MHz, CDCl3): δ 11.22 (s, 1H, H-10), 4.34 (q, 2H, J17,18 7.15 Hz, H-17), 3.59 (s, 2H,

3 3 H-8), 2.92 (t, 2H, J5,6 5.50 Hz, H-5), 2.79 (t, 2H, H-6), 2.65 (q, 2H, J15,16 6.97 Hz, H-15),

3 3 2.27 (s, 3H, H-19), 1.38 (t, 3H, J18,17 6.97 Hz, H-18), 1.20 (t, 3H, J16,15 7.15 Hz, H-16);

13 C-NMR (600 MHz, CDCl3): δ 167.13 (C-2), 166.50 (C-11), 148.27 (C-13), 129.39 (C-

4), 128.36 (C-9), 110.90 (C-3), 60.72 (C-17), 51.66 (C-15), 51.01 (C-8), 50.24 (C-6), 26.87

(C-5), 23.82 (19), 14.43 (C-18), 50.24 (C-6), 26.87 (C-5), 23.82 (C-19), 14.43 (C-18),

+ 12.53 (C-16); mass spectrum, m/z = 319.10 (M+23) C14H20N2O3S requires 319.10

(M+23)+.

Ethyl 2-acetamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (21)

1 Yield: 59% (140.0 mg); silica gel TLC Rf = 0.42 (10 % Methanol in CH2Cl2 ); H-NMR

3 (600 MHz, CDCl3): δ 11.23 (s, 1H, H-10), 4.29 (q, 2H, J15,16 7.14 Hz, H-15), 2.73 (t, 2H,

3 3 J8,7 5.94 H-8), 2.60 (t, 2H, J5,6 5.76 Hz, H-5), 2.23 (s, 3H, H-17), 1.78-1.73 (m, 4H, 2 ×

3 13 CH2, H-6,7), 1.36 (t, 3H, J16,15 7.14 H-16) ; C-NMR (600 MHz, CDCl3): δ 166.88 (C-2),

166.66 (C-11), 147.65 (C-13), 130.65 (C-4), 126.59 (C-9), 111.23 (C-3), 60.46 (C-15),

26.39 (C-8), 24.36 (C-5), 23.74 (C-17), 23.00 (C-7), 22.88 (C-6), 14.34 (C-16) ; mass

+ + spectrum, m/z = 290.08 (M+23) C13H17NO3S requires 290.08 (M+23) .

Ethyl-2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (22)

1 Yield: 57.2% (167.0 mg); silica gel TLC Rf = 0.54 (10 % EtOAc in hexanes); H-NMR

(600 MHz, CDCl3): δ 12.32 (s, 1H, H-10), 8.01 (m, 2H, H-18,22), 7.56 (m, 1H, H-20), 7.50

3 3 (m, 2H, H-19.21), 4.35 (q, 2H, J15,16 7.14 Hz, H-15), 2.78 (t, 2H, J8,7 6 Hz H-8), 2.67 (t,

3 3 2H, J5,6 5.82 Hz, H-5), 1.83-1.76 (m, 4H, 2 × CH2, H-6,7), 1.39 (t, 3H, J16,15 7.14 H-16) ;

13 C-NMR (600 MHz, CDCl3): δ 167.06 (C-2), 163.39 (C-11), 148.08 (C-13), 132.53 (C-

17), 132.45 (C-20), 131.08 (C-4), 128.94 (C-19,21), 127.47 (C-18,22), 127.06 (C-9),

111.97 (C-3), 60.65 (C-15), 26.45 (C-8), 24.46 (C-5), 23.03 (C-7), 22.89 (C-6), 14.39 (C-

+ + 16) ; mass spectrum, m/z = 352.10 (M+23) C18H19NO3S requires 352.10 (M+23) .

Ethyl 2-(perfluorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(23)

1 Yield: 50.5% (188.0 mg); silica gel TLC Rf = 0.62 (10 % Methanol in CH2Cl2 ); H-NMR

3 (600 MHz, CDCl3): δ 12.05 (s, 1H, H-10), 4.35 (q, 2H, J15,16 7.14 Hz, H-15), 2.80 (t, 2H,

3 3 J8,7 6.06 Hz H-8), 2.70 (t, 2H, J5,6 5.94 Hz, H-5), 1.85-1.78 (m, 4H, 2 × CH2, H-6,7), 1.80

3 13 (t, 3H, J16,15 7.14 H-16) ; C-NMR (600 MHz, CDCl3): δ 166.76 (C-2), 153.97 (C-11),

145.94 (C-20), 145.80 (C-13), 144.16 (C-21), 138.82 (C-19), 137.13 (C-22,18) 131.57 (C-

4), 128.43 (C-9), 113.47 (C-3), 110.10 (C-17), 61.03 (C-15), 26.48 (C-8), 24.56 (C-5),

23.04(C-7), 22.88 (C-6), 14.39 (C-16) ; mass spectrum, m/z = 442.05 (M+23)+

+ C18H14F5NO3S requires 442.05 (M+23) .

Ethyl-2-(3,5-bis(trifluoromethyl)benzaimdo)-4,5,6,7-tetrahydrobenzo[b]thiophene-

3-carboxylate (24)

1 Yield: 14.5% (60.0 mg); silica gel TLC Rf = 0.86 (10 % Methanol in CH2Cl2 ); H-NMR

(600 MHz, CDCl3): δ 12.58 (s, 1H, H-10), 8.44 (s, 2H, H-18,22), 8.08 (s, 1H, H-20), 4.40

3 3 3 (q, 2H, J15,16 7.14 Hz, H-15), 2.82 (t, 2H, J8,7 6.06 Hz H-8), 2.71 (t, 2H, J5,6 5.82 Hz, H-

3 13 5), 1.84-1.81 (m, 4H, 2 × CH2, H-6,7), 1.42 (t, 3H, J16,15 7.14 H-16) ; C-NMR (150 MHz,

1 CDCl3): δ 167.11 (C-2), 160.52 (C-11), 146.88 (C-13), 135.03 (C-17), 132.71 (q, 2C, JC,F

3 132 C-26,27), 131.60 (C-4), 128.23 (C-9), 127.50 (d, 2C, JC,F 12.06 C-22,18), 125.89 (m,

C-20), 123.87 ( C-19), 122.06 (C-21), 113.21 (C-3), 61.10 (C-15), 26.49 (C-8), 24.60 (C-

5), 23.06 (C-7), 22.88 (C-6), 14.44 (C16) ; mass spectrum (HRMS), m/z = 488.07 (M+23)+

+ C20H17F6NO3S requires 488.07 (M+23) .

2-amino-N-butyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (29)

Yield: 36% (67 mg); silica gel TLC Rf = 0.59 (7 % methanol in CH2Cl2); 1 H-NMR (600

MHz, CDCl3): δ 5.96 (s, 2H, H-10), 5.59 (s, 1H, H-15), 3.51 (s, 2H, H-8), 3.36 (q, 2H, H-

16), 2.81 (m, 4H, H-5, 6), 2.65 (q, 2H, 3 J13,14 7.14 Hz, H-13), 1.54 (quin, 2H, 3 J17,16,18

7.14 Hz, H-17), 1.38 (sex, 2H, 3 J18,17,19 7.5 Hz H-18), 1.20 (t, 3H, 3 J14,13 7.14 Hz H-

14), 0.94 (t, 3H, 3 J19,18 7.5 Hz, H-19); 13C-NMR (150 MHz, CDCl3): δ 166.88 (C-2),

166.66 (C-11), 147.65 (C-13), 130.65 (C-4), 126.59 (C-9), 111.23 (C-3), 60.46 (C-15),

26.39 (C-8), 24.36 (C-5), 23.74 (C-17), 23.00 (C-7), 22.88 (C-6), 14.34 (C-16); mass spectrum (HRMS), m/z = 282.1647 (M + H)+ C14H24N3O3S requires 282.1635 (M + H)+

2-amino-N-benzyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide

(30)

Yield: 24.78% (86 mg); silica gel TLC Rf = 0.57 (10 % Methanol in CH2Cl2 with 1% NEt3

1 ); H-NMR (600 MHz, CDCl3): δ 7.36-7.28 (m, 5H, H-18,19,20,21,22), 6.04 (s, 2H, H-

3 10), 5.91 (s, 1H, H-15), 4.59 (d, 2H, J16,15 558 Hz, H-16), 3.46 (s, 2H, H-8), 2.74 (s, 4H,

3 3 13 H-6,5), 2.58 (q, 2H, J13,14 7.14 H-13), 1.16 (t, 3H, J14,13 7.14 H-14) ; C-NMR (600 MHz,

CDCl3): δ 166.26 (C-2), 159.70 (C-11), 138.69 (C-9), 128.76 (C-21,19), 127.45 (C-

18,20,22,4), 116.34 (C-17), 108.52 (C-3), 51.56 (C-16), 51.27 (C-8), 50.08 (C-6), 43.20

+ (C-13), 27.73 (C-5), 12.58 (C-14) ; mass spectrum, m/z = 338.13 (M+23) C17H21N3OS requires 338.13 (M+23)+.

2-amino-6-ethyl-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (32)

1 Yield: 32% (100 mg); silica gel TLC Rf = 0.55 (7 % Methanol in CH2Cl2 ); H-NMR (600

3 MHz, CDCl3): δ 7.30-7.25 (m, 2H, H-20,19), 6.92 (t, 1H, J21,20 7.44 Hz, H-21), 6.87 (d,

3 3 1H, J22,21 8.22 H-22), 6.29 (s, 1H, H-15), 6.01 (s, 2H, H-10), 4.54 (d, 2H, J16,15 5.88 Hz,

3 H-16), 3.87 (s, 3H, H-24), 3.47 (s, 2H, H-8), 2.76 (s, 4H, H-6,5), 2.61 (q, 2H, J13,14 7.14

3 13 H-13), 1.18 (t, 3H, J14,13 7.14 H-14) ; C-NMR (600 MHz, CDCl3): δ 165.96 (C-2), 159.32

(C-11), 157.55 (C-13), 129.66 (C-4), 128.76 (C-9), 127.60 (C-22), 126.66 (C-20), 120.76

(C-21), 110.20 (C-3), 108.52 (C-19), 55.28 (C-24), 51.59 (C-16), 51.07 (C-8), 50.20 (C-

6), 39.34 (C-13), 27.54 (C-5), 12.38 (C-14) ; mass spectrum (HRMS), m/z = 368.14

+ + (M+23) C18H23N3O2S requires 368.14 (M+23) .

N-(adamantan-1-yl)-2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (33)

1 Yield: 29% (140.0 mg); silica gel TLC Rf = 0.70 (10 % Methanol in CH2Cl2 ); H-NMR

(600 MHz, CDCl3): δ 5.84 (s, 2H, H-10), 5.37 (s, 1H, H-15), 3.50 (s, 2H, H-8), 2.78 (d,

3 3 3 2H, J6,5 4.8 Hz, H-6), 2.75 (d, 2H, J5,6 4.8 Hz H-5), 2.63 (q, 2H, J13,14 7.14 Hz, H-13),

2.09-2.06 (m, 10H, H-17,22,21,18,23,20), 1.70-1.68 (m, 5H, H-19,24,25), 1.86 (t, 3H,

3 13 J14,13 7.14 H-14) ; C-NMR (600 MHz, CDCl3): δ 165.61 (C-2), 158.42 (C-11), 127.51

(C-4), 115.70 (C-9), 109.95 (C-3), 51.99 (C-13), 51.50 (C-8), 51.09 (C-6), 50.08 (C-16),

42.18 (C-17,21,22), 36.43 (C-19,24,25), 29.49 (C-18,20,23), 27.41 (C-5), 27.41 (C-5),

+ 12.34 (C-14) ; mass spectrum (HRMS), m/z = 382.19 (M+23) C20H29N3OS requires

382.19 (M+23)+.

2-amino-N-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (34)

1 Yield: 25% (80 mg); silica gel TLC Rf = 0.41 (30% EtOAc in hexanes); H-NMR (600

MHz, CDCl3): δ 5.66 (s, 1H, H-13), 3.36 (m, 2H, H-14), 2.60 (m, 2H, H-8), 2.53 (m, 2H,

3 H-5), 1.80 (m, 4H, H-6,7), 1.54 (m, 2H, H-15), 1.38 (sextet, 2H, J16,17 7.38 Hz H-16),

3 13 0.94 (t, 3H, J17,16 7.38 H-17) ; C-NMR (600 MHz, CDCl3): δ 166.53 (C-2), 158.53 (C-

11), 128.80 (C-9), 118.90 (C-4), 108.95 (C-3), 38.92 (C-14), 31.88 (C-15), 27.18 (C-8),

24.53 (C-5), 22.99 (C-7), 22.90 (C-6), 20.29 (C-16), 13.81 (C-17); mass spectrum, m/z =

+ + 252.13 (M+23) C13H20N2OS requires 252.13 (M+23) .

2-amino-N-benzyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (35)

1 Yield: 24% (86 mg); silica gel TLC Rf = 0.33(30 % EtOAc in hexanes); H-NMR (600

MHz, CDCl3): δ 7.35-7.33 (m, 5H, H-16,17,18,19,20), 6.00 (s, 2H, H-10), 4.59 (d, 2H,

3 J14,15 5.58 Hz, H-14), 2.60-2.59 (m, 2H, H-8), 2.55-2.54 (m, 2H, H-5), 1.78 (m, 4H, H- 26

13 6,7) ; C-NMR (600 MHz, CDCl3): δ 166.43 (C-2), 158.66 (C-11), 138.75 (C-9), 128.80

(C-15), 128.74 (C-17,19), 127.54 (C-16,20), 127.36 (C-18), 119.31 (C-4), 108.88 (C-3),

43.25 (C-14), 27.24 (C-8), 24.56 (C-5), 22.93 (C-7), 22.85 (C-6) ; mass spectrum, m/z =

+ + 286.11 (M+23) C16H18N2OS requires 286.11 (M+23) .

2-amino-N-(4-methylbenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(36)

1 Yield: 25% (83.0 mg); silica gel TLC Rf = 0.52 (30 % EtOAc in hexanes); H-NMR (600

3 3 MHz, CDCl3): δ 7.22 (d, 2H, J17,16 7.98 Hz H-17,19), 7.15 (d, 2H, J16,17 7.86 Hz H-16,20),

3 5.95 (s, 1H, H-13), 4.54 (d, 2H, J14,13 5.46 Hz H-14), 2.58 (m, 2H, H-8), 2.54 (m, 2H, H-

13 5), 2.34 (s 3H, H-21), 1.77 (m 4H, H-6,7) ; C-NMR (600 MHz, CDCl3): δ 166.38 (C-2),

158.62 (C-11), 137.03 (C-9), 135.65 (C-18), 129.40 (C-19,17), 128.82 (C-5), 127.56 (C-

16,20), 119.21 (C-4), 108.90 (C-3), 43.04 (C-14), 27.21 (C-8), 24.55 (C-5), 22.92

(C-7), 22.85 (C-6), 21.12 (C-21); mass spectrum (HRMS), m/z = 300.13 (M+23)+

+ C17H20N2OS requires 300.13 (M+23) .

2-amino-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(37)

1 Yield: 23% (32 mg); silica gel TLC Rf = 0.35 (30 % EtOAc in hexanes); H-NMR (600

MHz, CDCl3): δ 7.31-7.25 (m, 2H, H-17,20), 6.94-6.88 (m, 2H, H-19,18), 6.36 (s, 1H, H-

3 13), 4.55 (d, 2H, J14,13 5.58 Hz, H-14), 3.88 (s, 3H, H-22), 2.60 (m, 2H, H-5), 2.53 (m,

13 2H, H-8), 1.79 (m, 4H, H-6,7) ; C-NMR (600 MHz, CDCl3): δ 166.19 (C-2), 158.75 (C-

11), 157.53 (C-16), 129.67 (C-9), 128.95 (C-20), 128.71 (C-18), 126.75 (C-15), 120.77 (C-

4), 118.73 (C-19), 110.17 (C-17), 108.92 (C-3), 55.20 (C-22), 39.25 (C-14), 27.01 (C-8),

24.54 (C-5), 23.04 (C-7), 22.92 (C-6) ; mass spectrum (HRMS), m/z = 316.12 (M+23)+

+ C17H20N2O2S requires 316.12 (M+23) .

N-(adamantan-1-yl)-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(38)

1 Yield: 21 % (30 mg); silica gel TLC Rf = 0.53 (30% EtOAc in hexanes); H-NMR (600

MHz, CDCl3): δ 5.43 (s, 1H, H-13), 2.58 (m, 2H, H-8), 2.53 (m, 2H, H-5), 2.05 (m, 9H,

H-15, 16, 18, 19, 20, 21), 1.78 (m, 4H, H-6,7), 1.70 (m, 6H, H-17, 22, 23), 13C-NMR (600

MHz, CDCl3): δ 165.89 (C-2), 157.87 (C-11), 128.84 (C-9), 118.95 (C-4), 110.31 (C-3),

51.94 (C-14), 42.19 (C-15,19,20), 36.47 (17,23,22), 29.52 (C-16,18,21), 27.32 (C-8), 24.57

(C-5), 23.09 (C-7), 22.94 (C-6); mass spectrum (HRMS), m/z = 330.18 (M+23)+

+ C19H26N2OS requires 330.18 (M+23) .

2-Amino-6-ethyl-N-(4-methylbenzyl)-4,5,6,7-tetrahydrothieno [2,3-c]pyridine-3- carboxamide (31)

1 Yield: 40% (235.6 mg); silica gel TLC Rf = 0.52 (30% EtOAc in hexanes); H-NMR (600

MHz, CDCl3): δ 7.20 (m, 2H, H-21,19), 7.16 (m, 2H, H-22,18), 6.03 (s, 2H, H-10), 5.87

(s, 1H, H-15), 4.53 (d, 2H, J16,15 6 Hz, H-16), 3.50 (s, 2H, H8), 2.75 (m, 4H, H-5,6), 2.61

(q, 2H, J13,14 6 Hz H-13), 2.35 (s, 3H, H-23), 1.17 (t, 2H, J14,13 6 Hz H-14); 13C-NMR

(150 MHz, CDCl3): δ 166.19 (C-11), 159.63 (C-2), 137.08 (C-20, 9), 135.57 (C-17), 129.43

(C-21, 19), 127.49 (C-22, 18), 116.01 (C-4), 51.52 (C-8), 51.17(C-6), 50.02 (C-13), 43.03

(C-16), 27.54 (C-23), 21.13(C-5), 12.47 (C-14); mass spectrum (HRMS), m/z = 352.1460

(M+23)+ C18H23N3OS requires 352.1460 (M + 23)+

Chapter 3

Synthesis and in vitro characterization of trehalose-based inhibitors of mycobacterial trehalose 6-phosphate phosphatases

α,α’-Trehalose plays roles in the synthesis of several cell wall components involved in pathogenic mycobacteria virulence. Its absence in mammalian biochemistry makes trehalose-related biochemical processes potential targets for chemotherapy. The trehalose-

6-phosphate synthase (TPS)/trehalose-6-phosphate phosphatase (TPP) pathway, also referred to as the OtsA/OtsB2 pathway, is the major pathway involved in the production of trehalose in Mycobacterium tuberculosis (Mtb). In addition, TPP is essential for Mtb survival. We describe the synthesis of 6-phosphonic acid 4 (TMP), 6-

(methylene)phosphonic acid 5 (TEP) , and 6-N-phosphonamide 6 (TNP) derivatives of

α,α’-trehalose (Figure 3.1). These non-hydrolyzable substrate analogs of TPP were examined as inhibitors of Mtb, M. lentiflavum (Mlt), and M. triplex (Mtx) TPP. In all cases the compounds inhibited Mtx TPP most strongly, with TMP (IC50 = 288 ± 32 μM) inhibiting most strongly, followed by TNP (IC50 = 421 ± 24 μM) and TEP (IC50 = 1959 ±

261 μM). The results also indicate significant differences in the analog binding profile when comparing Mtb TPP, Mlx TPP, and Mtx TPP homologs.

3.1 Introduction

Trehalose is an important metabolite in the cell wall of Mtb, its absence from mammilian cell makes its biosynthetic pathway an important target. In Mtb the OtsA/OtsB pathway is considered most prevalent in replicating Mtb.54 OtsA catalyzes the formation of trehalose-

6-phosphate (T6P) from UDP-glucose or ADP-glucose and glucose-6-phosphate.55 OtsB has two homologues, OtsB1 and OtsB2, and only OtsB2 has phosphatase activity and is essential for Mtb growth.54, 56 The phosphate moiety of T6P is removed by OtsB2 to yield trehalose. The OtsA-OtsB pathway is also referred to as the trehalose-6-phosphate synthase

(TPS)/trehalose-6-phosphate phosphatase (TPP) pathway and TPP is used in place of

OtsB2 hereafter.

Trehalose has several fates related to virulence and cell wall synthesis. These include conversion into intracellular α-glucan, sulfolipid-1, diacyltrehalose, and polyacyltrehalose.

57-61 Trehalose is also modified with mycolic acids by polyketide synthase 13 (Pks13) to form mono-α-alkyl β-ketoacyl trehalose (TMMk).62 TMMk is reduced to trehalose monomycolate (TMM) by CmrA63, 64 which is transported to the cell wall by MmpL3

(Figure 1).65 The Antigen 85 Complex (Ag85) transfers66, 67 the mycolic acids to the cell wall arabinogalactan (AG) or to a second molecule of TMM to form trehalose dimycolate

(TDM) a key virulence factor.68 Free trehalose produced by the action of Ag85s on TMM is salvaged by the action of the ABC transporter LpqY-SugABC.44 It is also known that

Mtb can enter an antibiotic resistant state under the stress of low oxygen.69 Intriguingly, trehalose has been linked to the ability of Mtb to adapt its metabolism under these

30 conditions.70 Thus, compounds interfering with the various trehalose-producing pathways may offer new approaches for more effective treatments against Mtb.

Recently, 6-sulfate, 6-(methylene)phosphonate, and 6-(fluoromethylene) ﬂuorophosphonate

71 derivatives of α,α'-trehalose were reported active (Ki =130-480 μM) against Mtb TPP while aryl-D-glucopyranoside 6-sulfates were also reported as mimics of T6P with activity against TPP.72 Based on the activity of these α,α'-trehalose derivatives against Mtb TPP we developed heptabenzyl α,α'-trehalose derivative 3 and synthetic routes to access 6- phosphonic acid 4 (TMP), 6-(methylene)phosphonic acid 5 (TEP), 6-N-phosphonamide 6

(TNP), and a 6-oxirane 7 derivatives of 6-deoxy-α,α'-trehalose (Figure 2). The library of

6-deoxy-α,α'-trehalose derivatives was evaluated for inhibition against TPP homologs encoded by Mtb, Mlt and Mtx. Validamycin A (8) was included due to topological similarity with α,α'-trehalose and for known inhibitory activity against E. coli TPP.73 The

Mlt and Mtx TPP homologs were included in this study because of the high sequence identity with the Mtb TPP, 72% and 71%, respectively, and the 100% identity in catalytic residues. Additionally, the relative paucity of available Mtb TPP structural information suggests protein structural dynamics that hinder crystallization. The Mlt and Mtx TPP homologs lack two large loops indicated in the Mtb TPP sequence and are being pursued as TPP surrogates to afford structural determination of mycobacterial TPP enzymes.

54 74 Finally, the Mtb, Mlt, and Mtx TPP enzymes in this study exhibit KM values of 640, 130, and 82 μM,74 respectively.

Figure 3.1 6-deoxy-α,α'-trehalose derivatives prepared from heptabenzyl α,α'-trehalose derivative 3 and Validamycin A (8) which shows topological similarities with trehalose.

3.2 Results and Discussion

3.2.1 Chemistry studies.

Prior research has shown that 6-sulfate, 6-(methylene)phosphonate TEP, and 6-

(fluoromethylene)phosphonate derivatives of α,α'-trehalose possessed inhibitory activity against Mtb TPP;71 however, no microbiological evaluation was reported. That data, in addition to the know growth inhibition data of 6-modified derivatives of α,α'-trehalose against mycobacteria,67, 75-79 prompted us to investigate the synthesis of additional 6- modified derivatives. In the current work, we focused on synthesis and study of 6- phosphonate α,α'-trehalose TMP, an alternate route to 6-(methylene)phosphonate analogue

TEP, a 6-N-phosphonamide analogue TNP, and an 6-oxirane analogue 7 due to similarity with reported Mtb TPP inhibitors (Figure 2).71

The target 6-phosphonate analog TMP was accessible through a heptabenzyl α,α'-trehalose intermediate 377 prepared by our reported route.78 The route allows gram scale access to unsymmetrical 6-modified α,α'-trehalose analogues. In order to access 6-phosphonate

TMP, heptabenzyl α,α'-trehalose derivative 3 was converted to the 6-iodo-6-deoxy-α,α'- trehalose derivative 9 in 70% yield by treatment with triphenylphosphine in the presence of iodine and imidazole (Scheme 1). Iodide 9 was subjected to Michaelis–Arbuzov conditions by treatment with trimethyl phosphite to afford 6-phosphonate derivative 10 in

58% yield. The phosphonate ester 10 was deprotected with bromotrimethylsilane (TMSBr) to afford the 6-phosphonic acid derivative 11. The latter was debenzylated by hydrogenolysis with 20% Pd(OH)2 on carbon under 1 atm. of H2 to afford 6-phosphonate

TMP.

Scheme 3.1 Synthesis of 6-phosphonic acid-α,α'-trehalose derivative TMP. 33

The target 6-(methylene)phosphonic acid analog TEP was also accessible through intermediate 3. Intermediate 3 was first subjected to a Swern oxidation to afford aldehyde

12 in 62% yield (Scheme 2).71 Aldehyde 12 was converted to the phosphonate 13 in 49% yield via a Horner–Wadsworth–Emmons reaction utilizing tetramethyl methylenediphosphonate and sodium hydride. The phosphonate ester 13 was deprotected with bromotrimethylsilane (TMSBr) to afford the 6-(vinylphosphonic acid) derivative 14.

The latter was debenzylated by hydrogenolysis with 20% Pd(OH)2 on carbon under 1 atm. of H2 to afford 6-(methylene)phosphonic acid TEP. The route is a minor modification of reported work71 which used tetraethyl methylenediphosphonate in the Horner–

Wadsworth–Emmons reaction along with other modifications in reagents and reaction sequence.

Scheme 3.2 Synthesis of 6-(methylenephosphonic acid)-α,α'-trehalose derivative TEP.

The target 6-phosphoramidic acid analog TNP was accessible through iodide intermediate

9. Iodide 9 was subjected nucleophilic substitution with sodium azide to afford azide 15 34 in 82% (Scheme 3). Azide 15 was reduced to amine 16 via a Staudinger reaction reduction in good yield. The 6-amino-α,α'-trehalose derivative 16 was treated with dibenzylphosphoryl chloride to afford dibenyzl phosphoramidate 17. Global deprotection of the benzyl groups with 10% Pd on carbon under 1 atm. of H2 yielded 6-phosphoramidic acid analog TNP.

Scheme 3.3 Synthesis of 6-(phosphoramidic acid)-α,α'-trehalose derivative TNP.

The target oxirane analog 7 was likewise accessible through iodide intermediate 9. Iodide

9 was subjected to nucleophilic substitution with trimethylsilylacetylene followed by deprotection of the silyl group with tetrabutylammonium fluoride (TBAF) to afford compound 18 in 48% yield over 2 steps (Scheme 4). Compound 18 was subjected to Birch reduction to reduce the benzyl groups and convert the alkyne to an alkene. The resulting hydroxyl groups were protected by acetylation to afford compound 19 in 40% yield.80 The alkene 19 was treated with meta-chloroperoxybenzoic acid (mCPBA) to afford epoxide 20

35 as a mixture of diastereomers in 59% yield. Deprotection of the acetyl groups of 20 was

81 achieved with NEt3/MeOH/H2O (1:3:1) to afford oxirane analog 7.

Scheme 3.4 Synthesis of 6-oxiranyl-α,α'-trehalose derivative 7.

3.2.2 Discussion

These non-hydrolysable substrate analogs of trehalose TEP, TMP, TNP, Oxirane (7) and

Validamycin A (8) were tested for their inhibitory activity against different homologs of

TPP encoded by Mtb, Mlx, and Mtx. Fig 3 shows the IC50 values for all the tested compounds. Results showed TEP compound exhibited the weakest inhibition among the three substrate analogs which is justifable due to the fact that it lacks the coordination of the phosphonate and the trehalose moiety with Mg2+ due to the presence of the large ethyl moiety. This study is in agreement with the mechanism of TPP. The most potent inhibitor for Mtb TPP was TNP whereas TMP showed lower IC50 values for Mlt and Mtx TPP.

Compound 7 showed no inhibition for the TPP. Validamycin A which has previously

73 shown inhibiton for E. coli TPP at a concentration of 25 μM showed a higher IC50 when studied for its inhibitory activity against the homologs of TPP (Mtb, Mtl, Mtx). PDB shows no structure for either E. coli TPP or Mtb TPP so it is difficult to give a rational explaination for higher IC50 values for Validamycin A. The inactive Cryptococcus neoformans TPP shows a coordination of Mg2+ with both Aspartate residues and the phosphate moiety with Histidine and Lysine residues in the active site.82 There are some structural changes which takes place in C. neoformans when T6P binds in the active site and not all the catalytic residue in C. neoformans are conserved with TPP homologs.

Hence, the difference in the sequence and structural dynamics could be the reason for unexpected high IC50 value for Validamycin A.

Figure 3.2 TPP Inhibition studies. A, B and C; TPP inhibition curves for TNP. D. E, F; TPP inhibition curves for TEP. G, H, and I; TPP inhibition curves for TMP. J, K, L; TPP inhibition curves for

Validamycin A. M; IC50 values of TNP, TEP, TMP and Validamycin A against Mtb TPP, Mlx TPP and Mtx TPP. The inhibition studies were performed by Dr. Cecil Petit.

The mechanism for TPP involves two steps in which the first step involves the attack of

Aspartate (Asp153/120 corresponds to Mtb TPP/MltTPP) residue acting as a nucleophile on the phosphoryl group of T6P forming a penta-coordinated T6P enzyme-bound intermediate. The second Aspartic acid (Asp153/120) acts as an acid and protonates the trehalose which facilitates the formation of phosphoaspartyl intermediate and releases the free trehalose. The Magnesium cofactor is essential for the reaction to take place as it

38 enables the correct orientation of substrate phosphoryl group and residues to compensate for the negative charge developed during the reaction. The TMP showed 1.8 to 2.8 fold worse inhibitory activity compared to T6P whereas TEP showed 3.4-135 fold worse inhibition for Mtx TPP and Mtb TPP. This data suggest that increasing the length of the linker between trehalose and the phosphate group has a negative impact on the inhibition.

TNP showed similar IC50 to that of T6P for Mtb TPP suggesting the importance of hydrogen bond acceptor and donor at the 6th position. Hence, more potent inhibitors can be designed for the enzyme with α-hydrodroxy- or α-amino-phosphonates groups which can compensate for the loss of putative H-bond acceptor as present in substrate T6P and which is missing in the present series of substrate analogs. Further, these compounds can be imported by LpqY-SugABC into growing bacilli as it recognizes trehalose and some of its derivatives e.g fluorescein-containing trehlaose probe.67, 75-79 Some of the trehalose derivatives with the replacement of 6th position of trehalose with a halide (6-fluoro-6- deoxy and 6-bromo-6-deoxy α,α'-trehalose) have MIC 200 μg/mL against Mtb.75

For future studies, it would interesting to investigate how these new compounds might effect trehalose production, Mtb growth or recovery from dormancy, Ag85 activity,

TMM/TDM production, or biofilm formation. Related compounds, like 6-azido-6-deoxy-

α,α'-trehalose, are known to inhibit growth of M. aurum (MIC 200 μg/mL) as well as inhibit

Ag85C activity, synthesis of TMM, TDM, and reduced cell wall–bound mycolic acids and

TMM export. 67 While a library of N,N-dialkylamino and 6,6-bis(sulfonamido) analogs of

76 α,α'-trehalose was shown active (MIC 16-128 μg/mL) against Mtb H37Ra. A second 6,6- bisalkyl library of α,α'-trehalose derivatives was found active against M. smegmatis.77 In

39 addition, 6,6-bis(α-ketoesters/amides) analogs of α,α'-trehalose have been reported to be active against Ag85C78 and 6-deoxy-, 6-fluoro-6-deoxy-, and 6-azido-6-deoxy-α,α'- trehalose were shown to inhibit (MIC 50-100 μM) M. smegmatis bioﬁlm formation.79

Collectively, these studies suggest several possible applications of the target compounds.

3.3 Experimental

2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-iodo-6-deoxy-α-D-glucopyranosyl-(1→1)-α-D- glucopyranoside (9). To a solution of 3 (0.950 g, 0.977 mmol, 1eq.) in tetrahydrofuran (10 mL) at 0 °C was added triphenylphosphine (0.307 g, 1.17 mmol, 1.2 eq.), imidazole (0.166 g, 2.44 mmol, 2.5 eq.) and iodine (0.297 g, 1.17 mmol, 1.2 eq.). The reaction was refluxed for 3 h. After completion, water (10 mL) was added to the reaction flask and organic layer was extracted with ethyl acetate (15 mL). The organic layer was washed successively with

Na2S2O3 (10 mL X 3) and water (10 mL X 3). The ethyl acetate was dried over anhydrous

Na2SO4 and filtered. Purification was performed by flash column chromatography on silica gel (5% ethyl acetate-hexanes) to afford a colorless viscous liquid (0.768 g, 70% yield): Rf

1 = 0.35 (20% ethyl acetate-hexanes); H NMR (600 MHz, CDCl3): δ 7.38-7.24 (m, 33H, aromatic), 7.13 (dd, 2H, J = 7.4, 1.9 Hz, aromatic), 5.27 (d, 1H, J = 3.5 Hz, H-1), 5.23 (d,

1H, J = 3.5 Hz, H-1’), 4.98 (m, 3H, benzylic), 4.85 (m, 3H, benzylic), 4.72 (m, 5H, benzylic), 4.56 (d, 1H, J = 12.1 Hz, benzylic), 4.47 (d, 1H, J = 10.8 Hz, benzylic), 4.40 (d,

1H, J = 12.1 Hz, benzylic), 4.14 (m, 1H, H-5’), 4.09 (t, 1H, J = 9.3 Hz, H-3), 4.03 (t, 1H,

J = 9.4 Hz, H-3’), 3.69 (m, 2H, H-4’, H-5), 3.62 (dd, 1H, J = 9.6, 3.6 Hz, H-2’), 3.52 (m,

2H, H-6a’, H-4), 3.41 (m, 2H, H-6b’, H-4), 3.25 (dd, 1H, J = 10.9, 4.5 Hz, H-6a), 3.12 (dd,

13 1H, J = 10.8, 2.9 Hz, H-6b); C NMR (150 MHz, CDCl3): δ 139.03, 138.81, 138.52,

138.44, 138.35, 138.30, 138.02, 128.74, 128.68, 128.63, 128.62, 128.62, 128.60, 128.22, 40

128.20, 128.14, 128.14, 128.11, 127.95, 127.89, 127.86, 127.80, 127.60, 94.61, 94.24,

82.06, 81.89, 81.40, 79.81, 79.68,77.92, 75.90, 75.85, 75.68, 75.35, 73.74, 73.34, 73.01,

70.97, 68.92, 68.30, 66.12, 9.59; HRMS (ESI): calculated for C61H63IO10, 1105.3364

[M+Na]+; observed, m/z = 1105.3331 [M+Na]+.

2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-α-D-glucopyranosyl-

(1→1)-α-D-glucopyranoside (10). Trimethyl phosphite (10 mL) was added to compound

9 (0.500 g, 2.16 mmol) and the reaction was refluxed for 36 h. After the completion, excess trimethyl phosphite was evaporated under reduced pressure. The product was purified by flash column chromatography on silica gel (5% ethanol-choroform) to afford the product

1 as a colorless solid (0.202 g, yield 58%): Rf = 0.30 (8% ethanol-chloroform); H NMR

(600 MHz, CDCl3): δ 7.38-7.14 (m, 35H, aromatic), 5.47 (d, 1H, J = 3.5 Hz, H-1), 5.26 (d,

1H, J = 3.5 Hz, H-1’), 4.97 (m, 4H, benzylic), 4.84 (d, 3H, J = 10.8 Hz, benzylic), 4.71 (m,

3H, benzylic), 4.65 (d, 1H, J = 11.2 Hz, benzylic), 4.55 (d, 1H, J = 12.3 Hz, benzylic), 4.49

(d, 1H, J = 11 Hz, benzylic), 4.43 (d, 1H, J = 12.1 Hz, benzylic), 4.30 (m, 1H, H-5), 4.11

(m, 3H, H-3, H-3’, H-5’), 3.66 (m, 2H, H-2, H-2’), 3.61 (m, 6H, 2X-OCH3), 3.55 (m, 1H,

H-4’), 3.42 (t, 1H, H-4), 2.12 (m, 1H, H-6a), 1.88 (m 1H, H-6b); 13C NMR (150 MHz,

CDCl3): δ 139.11, 138.87, 138.84, 138.68, 138.58, 138.33, 138.14, 128.62, 128.61, 128.58,

128.55, 12853, 128.48, 128.17, 128.10, 128.01, 127.76, 127.73, 92.55, 92.14, 82.18,

82.09,82.03, 81.50, 81.49, 79.81, 79.73, 78.01, 75.82, 75.71, 75.17, 75.15, 73.70, 73.27,

72.92, 70.83, 68.66, 66.92, 66.88, 66.11, 52.46, 52.41, 52.32, 52.28, 15.52; 31P NMR (162

+ MHz, CDCl3): δ 32.13 (s, 1-P); LRMS (ESI): calculated for C63H69PO13, 1087.4 [M+Na] ; observed, m/z = 1087.5 [M+Na]+.

6-Deoxy-6-(phosphonic acid)-α,α'-trehalose (4, TMP). To a solution of 10 (0.080 g,

0.075 mmol) in dichloromethane (4 mL) was added bromotrimethylsilane (129 µL, 0.977 mmol, 13 eq.) dropwise and the resulting solution stirred for 2 h at room temperature. A solution of methanol-water (1.1:1.8 mL) was added followed by concentration to dryness to afford phosphonicacid 11. The residue was dissolved in 3 mL of tetrahydrofuran-ethanol

(1:4) and 20% Pd(OH)2/C (80 mg) was added. The suspension was stirred overnight at room temperature under 1 atm. of hydrogen. The catalyst was filtered away through a plug of Celite® 545 that was washed with 20% methanol-dichloromethane. The filtrate and washings were concentrated to dryness to afford product TMP as a colorless solid (0.030

1 g, quantitative yield): H NMR (600 MHz, D2O): δ 5.24 (d, 1H, J = 3.5 Hz, H-1), 5.02 (d,

1H, J = 3.8 Hz, H-1’), 3.94 (m, 1H, H-5), 3.75-3.53 (m, 7H, H-2, H-2’, H-4, H-4’, H-5’,

H-6a’, H-6b’), 3.33 (t, 1H, J = 9.4 Hz, H-3’), 3.15 (t, 1H, J = 9.4 Hz, H-3), 2.12 (m, 1H,

13 H-6a), 1.75 (m, 1H, H-6b); C NMR (150 MHz, D2O): δ 92.93, 92.88, 74.32, 74.22, 72.25,

31 72.05, 71.11, 70.91, 69.59, 68.03, 34.82; P NMR (162 MHz, D2O): δ 30.78 (s, 1-P);

+ HRMS (ESI): calculated for C12H23PO13, 429.0774 [M+Na] ; observed, m/z = 429.0774

[M+Na]+.

2,2',3,3',4,4',6'-Hepta-O-benzyl-6-carbaldehyde-α-D-glucopyranosyl-(1→1)-α-D- glucopyranoside (12). Oxalyl chloride (0.07 mL, 0.80 mmol, 4 eq.) was slowly added to a solution of dimethyl sulfoxide (0.11 mL, 1.60 mmol, 8 eq.) in dichloromethane (5 mL) maintained at -78 °C. The solution was allowed to stir at that temperature for 20 min. To this solution was added 3 (0.20 g, 0.020 mmol, 1eq.) dissolved in 2 mL of dichloromethane.

This solution was allowed to stir for 1 hour at -78 °C. Triethylamine (0.45 mL, 3.2 mmol,

16 eq.) was added and the solution was stirred and additional 20 min. The reaction was 42 allowed to warm to room temperature over 30 min. dichloromethane (5 mL) was added to the solution followed by successive washings with saturated NH4Cl solution (10 mL X 3) and saturated NaCl solution (10 mL X 3). The organic layer was dried over anhydrous

Na2SO4 and concentrating under reduced pressure. The residue was purified by flash column chromatography on silica gel (40% ethyl acetate-hexanes) to afford the product as

1 a viscous liquid (0.12 g, 62% yield): Rf = 0.37 (30% ethyl acetate-hexanes); H NMR (600

MHz, CDCl3): δ 9.36 (s, 1H, H-6), 7.40-7.13 (m, 35H, aromatic), 5.23 (d, 1H, J = 3.5 Hz,

H-1), 5.15 (d, 1H, J = 3.7 Hz, H’-1), 5.02 (dd, 2H, J = 10.9, 5.6 Hz, benzylic), 4.87 (m,

5H, benzylic), 4.73 (d, 1H, J = 11.7 Hz, benzylic), 4.67 (m, 3, H-5, benzylic), 4.57 (m, 3H, benzylic), 4.48 (d, 1H, J = 10.6 Hz, benzylic), 4.41 (d, 1H, J = 12.1 Hz, benzylic), 4.15 (m,

1H, H-5’), 4.10 (t, 1H, J = 9.6 Hz, H-3), 4.02 (t, 1H, J = 9.4 Hz, H-3’), 3.70 (t, 1H, J = 9.6

Hz, H-4’), 3.60 (dd, 1H, J = 9.7, 3.7 Hz, H-2’), 3.57-3.51 (m, 3H, H-2, H-4, H-6a’), 3.40

13 (dd, 1H, J = 10.6, 1.8 Hz, H-6b’); C NMR (150 MHz, CDCl3): δ 197.98, 138.93, 138.60,

138.35, 137.99, 137.95, 137.91, 137.55, 128.71, 128.64, 128.61, 128.58, 128.54, 128.51,

128.38, 128.32, 138.23, 128.20, 128.10, 128.05, 127.92, 127.87, 127.83, 127.78, 127.66,

95.29, 94.66, 81.97, 81.73, 79.50, 78.85, 78.51, 77.78, 76.00, 75.81, 75.38, 74.85, 73.70,

+ 73.40, 72.90, 71.05, 68.20; LRMS (ESI): calculated for C61H62O11, 993.4 [M+Na] ; observed, m/z = 993.4 [M+Na]+.

2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6E-(dimethyl vinylphosphonate)-α,α’- trehalose (13). Sodium hydride, 60% dispersion in oil, (7 mg, 0.30 mmol, 3 eq.) was added to a solution of tetramethyl methylenediphosphonate (0.03 mL, 0.15 mM, 1.5 eq.) in tetrahydrofuran (3 mL) maintained at 0 °C. The mixture was stirred for 30 min. A solution of aldehyde 12 (0.10 g, 0.10 mmol, 1eq.) in 1 mL of tetrahydrofuran was added dropwise 43 to the anion. The solution was allowed to warm to room temperature and stirred for another

2 h. The solvent was evaporated and the residue purified by flash chromatography on silica gel (5% methanol-dichloromethane) to afford 13 as a colorless viscous liquid (0.054 g,

1 49% yeild): Rf = 0.48 (10% methanol-dichloromethane); H NMR (600 MHz, CDCl3): δ

7.38-7.23 (m, 33H, aromatic), 7.24-7.13 (m, 2H, aromatic), 6.96-6.88 (m, 1H, H-6), 5.94

(m, 1H, H-7), 5.23 (d, 1H, J = 3.7 Hz, H-1), 5.1 (d, 1H, J = 3.7 Hz, H-1’), 5.0 (dd, 2H, J =

10.8, 2.2 Hz, benzylic), 4.86 (m, 4H, benzylic), 4.75 (m, 1H, H-5), 4.72-4.76 (m, 2H, benzylic), 4.57 (m, 4H, benzylic), 4.47 (d, 1H, J = 10.8 Hz, benzylic), 4.39 (d, 1H, J = 12.1

Hz, benzylic), 4.15 (m, 1H, H-5’), 4.09 (t, 1H, J = 9.06 Hz, H-3), 4.03 (t, 1H, J = 9.06 Hz,

H-3’), 3.66 (m, 6H, H-4’, 2X-OCH3), 4.56 (m, 2H, H-2 H-2’), 3.51 (m, 1H, H-6a’), 3.38

(dd, 1H, J = 10.6, 1.8 Hz, H-6b’), 3.25 (dd, 1H, J = 10.1, 9.2 Hz, H-4); 13C NMR (150

MHz, CDCl3): δ 149.17, 138.57, 138.35, 138.01, 137.76, 137.67, 137.50, 137.36, 128.28,

128.18, 127.75, 127.39, 127.15, 116.68, 115.42, 94.30, 93.63, 81.64, 81.58, 81.19, 78.98,

78.60, 77.41, 75.48, 75.40, 75.31, 74.87, 73.27, 72.71, 72.59, 70.45, 70.31, 70.17, 67.86,

31 52.10; P NMR (162 MHz, CDCl3): δ 32.42 (s, 1-P); HRMS (ESI): calculated for

+ + C64H69PO13, 1099.4373 [M+Na] ; observed, m/z = 1099.4370 [M+Na] .

6-Deoxy-6-(methylenephosphonic acid)-α,α’-trehalose (5, TEP). Bromotrimethylsilane

(83.4 µL, 0.652 mmol, 13 eq.) was added dropwise to a solution of 13 (0.09 g, 0.05 mmol) in dichloromethane (4 mL) and the resulting solution was stirred for 2 h at room temperature. The solution was quenched with 2 mL of methanol-water (1.1:1.8) followed by concentration to dryness. To the solution of benzylated disaccharide 14 was taken up in

3 mL of tetrahydrofuran-ethanol (1:4) and 20% Pd(OH)2/C (50 mg) was added. The mixture was stirred overnight under hydrogen (1 atm.). The catalyst was filtered off 44 through Celite® 545 and washed with 20% methanol-dichloromethane. The combined filtrate and washings were concentrated to dryness to afford TEP as viscous syrup (0.019

1 g, quantitative yield): H NMR (600 MHz, D2O): δ 5.04 (s, 2H, H-1, H-1’), 3.74-3.64 (m,

6H, H-6a’, H-6b’, H-5, H-5’, H-3, H-3’), 3.55-3.50 (m, 2H, H-2, H-2’), 3.32 (t, 1H, J = 9.6

Hz, H-4’), 3.16 (t, 1H, J = 9.4 Hz, H-4), 1.98 (m, 1H, H-6a), 1.84 (m, 1H, H-6b), 1.59 (m,

13 2H, H-7a, H-7b); C NMR (600 MHz, D2O): δ 93.39, 93.11, 73.14, 72.43, 72.36, 72.08,

31 71.61, 71.50, 71.11, 70.88, 69.60, 60.41, 24.33; P NMR (162 MHz, D2O): δ 27.29 (s, 1-

+ P); HRMS (ESI): calculated for C13H29PO13, 443.0930 [M+Na] ; observed, m/z = 443.0946

[M+Na]+.

2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-azido-α,α’-trehalose (15). Sodium azide

(0.114 g, 1.76 mol, 5 eq.) was added to a solution of iodide 9 (0.380 g, 351 mmol, 1eq.) in

DMF (8 mL). The mixture was heated to reflux for 24 h. The solution was diluted with ethyl acetate and washed with saturated NaCl solution (10 mL X 3). The organic layer was dried over anhydrous Na2SO4, filtered, and the solvent evaporated under reduced pressure.

The residue was purified by flash column chromatography on silica gel (8% ethyl acetate- hexanes) to afford 15 as a colorless viscous syrup (0.300 g, 86% yield): Rf = 0.33 (20%

1 1 ethyl acetate:hexanes); H NMR (600 MHz, CDCl3): δ H NMR (600 MHz, CDCl3): 7.32

(m, 33H, aromatic), 7.12 (m, 2H, aromatic), 5.22 (d, 2H, J = 3.5 Hz, H-1, H-1’), 4.99 (dd,

J = 10.8, 7.3 Hz, 2H, benzylic), 4.85 (m, 4H, benzylic), 4.71 (m, 4H, benzylic), 4.62 (s,

1H, benzylic), 4.55 (m, 2H, benzylic), 4.46 (d, 1H, J = 10.6 Hz,, benzylic), 4.38 (d, 1H, J

= 12.1 Hz, benzylic), 4.16 (m, 1H, H-5’, H-5), 4.02 (m, 2H, H-3’, H-3), 3.68 (m, 1H, H-

4’), 3.61 (m, 1H, H-2), 3.56 (m, 1H, H-2’), 3.49 (m, 2H, H-6a’, H-6b’), 3.37 (m, 1H, H-4),

13 3.18 (m, 2H, H-6a, H-6b); C NMR (150 MHz, CDCl3): δ 138.83, 138.66, 138.29, 138.16, 45

138.06, 137.79, 128.49, 128.43, 128.38, 128.37, 128.03, 128.01, 127.93, 127.90, 127.89,

127.73, 127.66, 127.62, 127.57, 127.47, 127.38, 94.50, 94.01, 81.82, 81.46, 79.50, 79.36,

78.30, 77.69, 77.26, 77.04, 76.83, 75.62, 75.21, 75.13, 73.53, 72.96, 72.78, 70.74, 70.35,

+ 68.10, 51.08; HRMS (ESI): calculated for C61H63IO10, 1020.4411 [M+Na] ; observed, m/z

= 1020.4424 [M+Na]+.

2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-amino-α,α’-trehalose (16).

Triphenylphosphine (0.236 g, 0.900 mmoL, 3 eq.) and water (0.027 g, 1.50 mmoL, 5 eq.) were added to compound 15 (0.300 g, 0.300 mmol, 1 eq.) dissolved in tetrahydrofuran (8 mL). The solution was stirred for 12 h and concentrated under reduced pressure. Ethyl acetate (10 mL) and water (10 mL) were added to the residue and the organic layer separated and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure and the residue subjected to flash column chromatography on silica gel (10% acetone-hexanes) to afford 16 as a white solid (0.290 g, quantitative yield): Rf = 0.21 (30%

1 acetone:hexanes); H NMR (600 MHz, CDCl3): δ 7.67 (m, 9H, aromatic), 7.52 (m, 4H, aromatic), 7.44 (m, 9H, aromatic), 7.37-7.29 (12H, aromatic), 7.14 (dd, 2H, J = 7.7, 1.5

Hz, aromatic), 5.22 (d, 1H, J = 3.7 Hz, H-1), 5.20 (d, 1H, J = 3.7 Hz, H-1’), 5.01 (t, 2H, J

= 10 Hz, benzylic), 4.88 (m, 3H, benzylic), 4.83 (d, 1H, J = 10.8 Hz, benzylic), 4.72 (m,

5H, benzylic), 4.64 (m, 1H, J = 11.2 Hz, benzylic), 4.55 (d, 1H, J = 12.3 Hz, benzylic),

4.48 (d, 1H, J = 10.8 Hz, benzylic), 4.39 (d, 1H, J = 12.1 Hz, benzylic), 4.18 (dd, 1H, J =

12.1, 2.2 Hz, H-5’), 4.08 (td, 2H, J = 9.3, 7.1 Hz, H-3, H-3’), 4.00 (m, 1H, H-5), 3.7 (t, 1H,

J = 9.7 Hz, H-4’), 3.63 (dd, 1H, J = 9.7,3.7 Hz, H-2’), 3.53 (m, 2H, H-6a’, H-4), 3.41 (m,

2H, H-6b’, H-4), 2.81 (dd, 1H, J = 13.7, 2.8 Hz, H-6a), 2.66 (dd, 1H, J = 13.8, 5.3 Hz, H-

13 6b); C NMR (150 MHz, CDCl3): δ 138.66, 138.63, 138.16, 138.04, 138.01, 137.62, 46

132.67, 131.98, 131.93, 131.87, 131.79, 131.76, 128.37, 128.29, 128. 21, 128.16, 127.97,

127.81, 127.77, 127.77, 127.70, 127.51, 127.47, 127.36, 127.33, 127.29, 127.27, 127.19,

93.94, 93.57, 81.67, 81.56, 79.54, 79.28, 78.08, 77.58, 75.41, 75.34, 74.87, 74.70, 73.32,

72.81, 72.60, 71.71, 70.46, 67.97, 42.34; LRMS (ESI): calculated for C61H63IO10, 972.5

[M+H]+; observed, m/z = 972.9 [M+H]+.

2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-(dibenyzl phosphoramidate)-α,α’- trehalose (17). Dibenzylphosphoryl chloride (0.045 g, 0.154 mmol, 3 eq.) dissolved in 2 mL of dichloromethane was added to compound 16 (0.050 g, 0.051 mmoL, 1eq.) dissolved in 5 mL of dichloromethane. Triethylamine (0.051 g, 0.51 mmoL, 10 eq.) was added and the solution stirred for 6 h. The solution was concentrated under reduced pressure and the residue subjected to flash column chromatography on silica gel (8% acetone-hexanes) to

1 afford a 17 as a viscous syrup (0.033 g, 52% yield): Rf =0.32 (30% acetone:hexanes); H

NMR (600 MHz, CDCl3): δ 7.3 (m, 43H, aromatic), 7.14 (m, 2H, aromatic), 5.12 (d, 1H,

J = 3.7 Hz, H-1), 5.10 (d, 1H, J = 3.7 Hz, H-1’), 4.97 (m, 6H, benzylic), 4.81 (m, 5H, benzylic), 4.63 (m, 6H, benzylic), 4.53 (m, 1H, benzylic), 4.45 (m, 1H, benzylic), 4.38 (m,

1H, benzylic), 4.12 (m, 1H, H-5’), 4.01 (m, 3H, H-3, H-3’, H-5’), 3.65 (m, 1H, H-4’), 3.56

(m, 1H, H-2’), 3.50 (m, 2H, H-5, H-6a’), 3.44 (m, 2H, H-2), 3.37 (m, 1H, H-6b’), 3.08 (m,

13 1H, H-4), 2.93 (m, 1H, H-6a), 2.70 (m, 1H, H-6b); C NMR (150 MHz, CDCl3): δ 138.96,

138.91, 138.46, 138.35, 138.23, 138.21, 137.96, 128.70, 128.65, 128.62, 128.56, 128.54,

128.51, 128.43, 128.40, 128.23, 128.14, 128.06, 128, 127.96, 127.91, 127.86, 127.72,

127.59, 127.56, 127.52, 94.38, 93.91, 79.55, 79.32, 77.87, 77.83, 75.73, 75.66, 75.25,

75.03, 73.66, 72.97, 72.93, 70.83, 68.30, 68.24, 68.20, 68.14, 68.11, 41.53; 31P NMR (162

+ MHz, D2O): δ 10.89 (s, 1-P); HRMS (ESI): calculated for C61H63IO10, 1232.5289 [M+H] ; observed, m/z = 1232.5276 [M+H]+.

6-(phosphoramidic acid)-α,α’-trehalose (6, TNP). A catalytic amount of 20%

Pd(OH)2/C was added to a solution of benzylated disaccharide derivative 17 (0.020 g,

0.016 mmol) dissolved in 3 mL of tetrahydrofuran-ethanol (1:4). The mixture was stirred overnight under hydrogen (1 atm.). The catalyst was filtered away through a plug of

Celite® 545 and washed with 20% methanol-dichloromethane. The combined filtrate and washings were concentrated to dryness afford TNP as a viscous syrup (0.006 g,

1 quantitative yield): H NMR (600 MHz, D2O): δ 5.12 (d, 1H, J = 3.5 Hz, H-1), 5.07 (d, 1H,

J = 3.5 Hz, H-1’), 3.87 (m, 1H, H-5), 3.82 (m, 1H, H-6a’), 3.75 (m, 4H, H-3, H-3’, H-5’,

H-6b’), 3.64 (m, 1H, H-4’), 3.55 (m, 2H, H-2, H-2’), 3.33 (m, 1H, H-4), 3.25 (m, 1H, H-

13 6a), 3.03 (m, 1H, H-6b); C NMR (150 MHz, D2O): δ 93 .52, 93.35, 72.46, 72.14, 71.40,

31 70.99, 70.66, 69.53, 68.09, 60.36, 40.44; P NMR (162 MHz, D2O): δ 4.62 (s, 1-P); HRMS

+ + (ESI): calculated for C12H23PO13, 422.1064 [M+H] ; observed, m/z = 422.1052 [M+H] .

2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-ethynyl-α,α’-trehalose (18). n-Butyllithium

(0.415 mL, 2 M) was slowly added to a solution of trimethylsilylacetylene (118 µL, 0.831 mmol, 3 eq.) in tetrahydrofuran (3 mL) at -78 °C. The solution was allowed to warm to 0

°C and was stirred for 30 min. Afterward, the solution was allowed to warm to room temperature over 50 min. The solution of anion was added to another round bottom flask containing iodide 9 (0.300 g, 0.277 mmol, 1 eq). To the resulting solution was added hexamethylphosphoramide (0.768 mL, 4.43 mmol, 16 eq.). The solution changed to brown in color and was stirred for 12 h at room temperature. The solution was concentrated under reduced pressure and the residue subjected to flash column chromatography on silica gel 48

(5% ethyl acetate-hexanes) to afford 18 as a colorless viscous syrup (0.095 g, 35% yield):

1 Rf = 0.73 (30% ethyl acetate-hexanes); H NMR (600 MHz, CDCl3): δ 7.26-7.15 (m, 33H, aromatic), 7.06 (dd, 2H, J = 7.5 Hz, 1.8, aromatic), 5.17 (dd, 2H, J = 8.9, 3.6 Hz, H-1, H-

1’), 4.93 (dd, 2H, J = 10.8, 6.2 Hz, benzylic), 4.8 (m, 4H, benzylic), 4.63 (m, 5H, benzylic),

4.48 (d, 1H, J = 12.1 Hz, benzylic), 4.39 (d, 1H, J = 10.6 Hz, benzylic), 4.32 (d, 1H, J =

12.1 Hz, benzylic), 4.09 (m, 2H, H-5, H-5’), 3.97 (td, 2H, J = 9.3, 7.7 Hz, H-3, H-3’), 3.62

(t, 1H, J = 9.7 Hz, H-3), 3.53 (ddd, 3H, J = 9.5, 7.6, 3.6 Hz, H-4’, H-2, H-2’), 3.45 (dd,

1H, J = 10.7, 3.2 Hz, H-6a’), 3.31 (dd, 1H, J = 10.6, 1.8 Hz, H-6b’), 2.37 (m, 1H, H-6a),

2.25 (dt, 1H, J = 17, 3.3 Hz, H-6b), 1.95 (t, 1H, J = 2.6 Hz, H-7 alkyne); 13C NMR (150

MHz, CDCl3): δ 139.03, 138.90, 138.49, 138.45, 138.30, 137.97, 128.59, 128.53, 128.17,

128.13, 127.59, 127.54, 94.54 (anomeric), 94.46 (anomeric), 81.96, 81.67, 80.46, 80.20,

79.72, 79.52, 77.82, 75.83, 75.80, 75.59, 75.28, 73.66, 72.95, 72.87, 71.05, 70.82, 68.63,

+ 68.24, 21.24; LRMS (ESI): calculated for C63H64O10, 1003.4 [M+Na] ; observed, m/z =

1003.9 [M+Na]+.

2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-deoxy-6-vinyl-α,α’-trehalose (19). Compound 18

(0.245 g, 0.250 mmol) was dissolved in 2 mL of tetrahydrofuran in a three-neck flask equipped with a dry ice condenser. The flask was then set in a dry ice-acetone bath.

Ammonia gas at was passed through the condenser until several milliliters of ammonia collected. A small piece of sodium metal was added to the solution, which immediately turned turquois in color. The ammonia was refluxed at -33 °C for 15 minutes. The reaction was quenched with 5 mL of methanol. The ammonia was evaporated, and the remaining liquids removed under reduced pressure and the residue further dried under high vacuum for 12 h. To the dried residue was added pyridine (3 mL), acetic anhydride (3 mL) and a 49 catalytic amount of DMAP. The solution was stirred for 12 h and then the solvent was concentrated under reduced pressure. The residue was subjected to flash column chromatography on silica gel (5% ethyl acetate-hexanes) to afford 19 as an amorphous

1 solid (0.095 g, 59% yield): Rf = 0.33 (30% ethyl acetate:hexanes); H NMR (600 MHz,

CDCl3): δ 5.72 (m, 1H, H-7), 5.48 (m, 2H, H-3, H-3’), 5.27 (d, 2H, J = 3.7 Hz, H-1, H-1’),

5.01 (m, 7H, H-2, H-2’, H-4, H6a,b, H8a,b), 4.23 (m, 1H, H-6a’), 4.04 (m, 3H, H-4’, H-5’,

13 H-6b’), 3.38 (m, 1H, H-5), 2.06 (m, 21H, 7XCH3); C NMR (150 MHz, CDCl3): δ 170.67,

170.06, 169.95, 169.70, 169.69, 169.65, 169.61, 132.39, 118.48, 91.94, 71.78, 70.10,

69.74, 69.10, 68.56, 68.07, 61.78, 35.45, 29.72, 20.75, 20.64; HRMS (ESI): calculated for

+ + C61H63IO10, 647.2187 [M+H] ; observed, m/z = 647.2193 [M+H] .

2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-deoxy-6-oxiranyl-α,α’-trehalose (20). Compound 19

(0.050 g, 0.077 mmol, 1 eq.) was dissolved in dichloromethane (3 mL) and 3- chloroperoxybenzoic acid (0.066 g, 0.387 mmol, 5 eq.) was added. The solution was stirred for an hour at room temperature and the solvent was evaporated under reduced pressure.

The reside was subjected to flash column chromatography on silica gel (10% ethyl acetate- hexanes) to afford 20 as an amorphous solid (0.030 g, 59% yield): Rf = 0.42 (30% ethyl

1 acetate:hexanes); H NMR (600 MHz, CDCl3): δ 5.51 (m, 3H), 5.32 (m, 4H), 5.08 (m, 7H),

4.92 (t, 1H, J = 9.7 Hz), 4.23 (dt, 2H, J = 12.6, 6.3 Hz), 4.06 (m, 5H), 2.98 (m, 1H), 2.78

13 (m, 2H) 2.43 (td, 2H, J = 5.5, 2.7 Hz), 2.17 (m, 6H, 2XCH3), 2.08 (m, 36H, 12XCH3); C

NMR (150 MHz, CDCl3): δ 170.69, 170.11, 170.10, 170.02, 169.98, 169.95, 169.94,

169.87, 169.87, 169.79, 196.73, 169.65, 169.64, 169.62, 92.29 (anomeric), 92.11

(anomeric), 91.75 (anomeric), 91.34 (anomeric), 72.20, 71.85, 70.24, 70.13, 70.05, 70.00,

69.95, 69.92, 69.40, 69.22, 68.62, 68.17,68.09, 67.49, 67.38, 61.85, 48.27, 48.18, 20.65;

+ + LRMS (ESI): calculated for C61H63IO10, 685.2 [M+Na] ; observed, m/z = 685.3 [M+Na] .

6-deoxy-6-oxiranyl-α,α’-trehalose (7). Compound 20 (0.044 g, 0.066 mmol) was dissolved in 9 mL of a mixture of triethylamine-methanol-water (2:6:1). The solution was stirred for an hour in dark. The solvent was evaporated under reduced pressure and subjected to flash column chromatography on silica gel using ethyl acetate-isopropanol- water (6:3:1) as an eluent to afford 7 as an viscous syrup (0.012 g, 50% yield): Rf = 0.45

1 (6:3:1 ethyl acetate-isopropanol-water); H NMR (600 MHz, D2O): δ 5.05 (m, 2H, anomeric), 3.87 (m, 1H), 3.72 (m, 5H), 3.54 (m, 2H), 3.33 (m, 2H), 3.19 (m, 1H), 3.09 (m,

13 2H), 1.79 (m, 2H). C NMR (150 MHz, D2O): δ 93.54, 93.38, 93.21, 93.04, 73.24, 73.19,

72.53, 72.50, 72.41, 72.11, 71.97, 71.38, 71.07, 71.05, 70.92, 70.29, 69.62, 69.59, 69.52,

69.16, 60.42, 51.44, 50.44, 48.28, 46.71, 46.59, 33.63, 23.19, 20.40, 8.15; HRMS (ESI):

+ + calculated for C61H63IO10, 369.1397 [M+H] ; observed, m/z = 369.1219 [M+H] .

Chapter 4

Targeting Glycoside hydrolase enzyme GlgE

4.1 Introduction

Carbohydrates (saccharides and glycoconjugates) are molecules that play various fundamental roles in living organisms, from serving as energy providers to structural components. The enzymes involved in the modification or degradation of carbohydrates are of great interest and are known as carbohydrate-active enzymes (CAZy). Glycoside hydrolases (GHs) are part of the CAZy family and perform the hydrolytic cleavage of glycosidic bonds in carbohydrates and encode for almost 1% of the genome in any organism.83 GHs are involved in the function and dysfunction of many diseases, including

HIV,84 diabetes,85 and infectious diseases,86 which has motivated researchers to study and inhibit these enzymes for therapeutics. Several drugs have been introduced to the market as GH inhibitors to treat some diseases, such as Miglitol87 and Acarbose88 which are used to treat type II diabetes by inhibiting the intestinal α-glucosidase (α-amylase) to delay glucose absorption in the blood stream. In addition, Zanamivir and Oseltamivir prevent the infection from further spreading to the respiratory tract by inhibiting neuraminidase, which is responsible for replication of the virus.89

4.2 Classification of GH

Over the past two decades, new GH enzymes and their 3D structures have been continually reported, and it is necessary to classify them into specific categories. Several methods have been adopted to classify GH enzymes based on their amino acid sequences, substrate specificity, the stereochemical outcome (such as retaining or inverting), and regioselectivity.90

4.2.1 Based on substrate specificity

In this category, GH enzymes are classified based on their catalytic activity, and are indicated by an EC (enzyme commission) number. Specific GH enzymes are given particular codes e.g., EC 3.2.1.x represents an enzyme that catalyzes O-, S-glycosidic linkages via hydrolysis (where x refers to substrate specificity or the molecular mechanism).91 This method was introduced by the International Union of Biochemistry and Molecular Biology (IUBMB)92 and is a widely used standard to describe GH enzymes.

However, there are several drawbacks to this method, as it fails to account for enzymes acting on more than one substrate or enzymes acting on polysaccharides. Some enzymes in separate categories have similar molecular mechanisms and 3D structures, while others that are structurally different are classified together.

4.2.2 Based on stereochemical outcome

GH enzymes have two stereochemical outcomes: either a net retention or net inversion of their configuration at the anomeric position. This classification is based on whether the hydrolyzed bond is equatorial or axial and whether the stereochemical outcome is retention

53 or inversion, of which there are four possible permutations: equatorial-equatorial, equatorial-axial, axial-axial, and axial-equatorial.90 However, the stereochemical result alone cannot be the basis of classification, though it provides crucial information for the mechanistic action of GH enzymes.

4.2.3 Based on regioselectivity

This method is based on the GH enzyme acting on polysaccharides either at their terminal ends or their intermediate positions. This enzyme activity is classified as either exo or endo.93 In exo enzymes, the active catalytic site is located inside a pocket and in an open cleft for endo enzymes. This classification is essential, but it fails to account for enzymes which exhibit both exo and endo properties, or enzymes which perform multiple glycosidic cleavages after they are coupled with a polysaccharide, e.g., cellobiohydrolases.90

4.2.4 Based on amino acid sequence

This classification is based on similarities in amino acid sequences, which can alone account for structural and mechanistic information of GH enzymes and is therefore highly specific.94 Similar sequences account for similar enzyme folding patterns and are highly important for homology modeling. This method complements the other methods for a better classification, but GH enzymes that are poly-specific i.e., acting on more than one substrate, are difficult to classify. CAZy provides a database of GH enzymes based on their amino acid sequence,95 and GH enzymes belonging to GH5, GH13, GH30, and GH43 families are further categorized into subfamilies.

4.3 Mechanism for GH

The GH enzymes that are categorized based on sequence similarity follow the same catalytic mechanism for the cleavage of glycosidic bonds, making it essential to study the cleavage mechanism of GHs. In general, GHs have two catalytic mechanisms for the cleavage of glycosidic bonds with net retention and inversion of configuration at the anomeric position.96 Both mechanisms utilize two acidic residues from the enzymes and form an oxacarbenium ion-like transition state (TS) to perform the cleavage.97 However, there are other mechanisms for GHs, as discussed below.

In the retaining mechanism, two acidic residues, usually Asp or Glu, are positioned 5.5 Å apart on opposite faces.98 In the first step, a glycosyl-enzyme intermediate is formed when one acidic residue protonates aglycone, while the second residue acts as a nucleophile and attacks the anomeric carbon formed in the oxacarbenium ion-like TS.99 In the second step, this glycosyl-enzyme intermediate undergoes hydrolytic cleavage. The residue that served as an acid in the first step now serves as a base which deprotonates the incoming nucleophile, which attacks the anomeric carbon via another oxacarbenium ion-like TS. In this way, this two-step mechanism forms two oxacarbenium ion-like TS.97

In the inverting mechanism, the two acidic residues are 6 Å apart, and the first acts as a base and deprotonates the incoming nucleophile, which attacks the anomeric carbon. The second residue acts as an acid and protonates the aglycone to facilitate its departure. The reaction follows a concerted mechanism, i.e., bond making and breaking occurs in a single step. Hence, only one oxacarbenium ion-like TS is formed, unlike the retaining glycosidases. The two residues in inverting glycosidases are little apart compared with

55 retaining glycosidases to accommodate the attack of the incoming nucleophile in a single step.

Another GH mechanism involves the participation of neighboring groups such as β- hexosaminidases. The first step involves the protonation of the aglycone by an acidic residue, followed by the attack of a 2-acetamido group at the anomeric carbon to form a bicyclic oxazolinium intermediate. In the next step, the same acidic residue from the enzyme acts as a general base that deprotonates the incoming water molecule to open up the oxazilinium ring, regaining the acetamido group which performs hydrolysis.83

There is another unusual mechanism for GH, which involves NAD+ and a divalent ion to perform glycosidic cleavage100 in which the C3 hydroxyl is oxidized by the abstraction of hydride by NAD+. This acidifies the C2 proton followed by its elimination to form an α-β unsaturated ketone which is attacked by a water molecule, and the ketone at C3 is reduced to a hydroxyl group by NADH. The reaction involves an anionic intermediate, which differs from the other discussed mechanisms which have only cationic oxacarbenium ion- like TS. This mechanism is usually followed by GH4 family 6-phospho-α-glycosidase

GlvA, which cleaves both α-β-glucosidase with the same catalytic site.

Figure 4.1 Different mechanism of GH101; a) Retaining mechanism b) Inverting mechanism c) Neighboring group participation mechanism d) Mechanism for GlvA.100, 102

4.4 Targeting GH enzyme GlgE

The α-glucans are polysaccharides with α-1,4 and α-1,6 linkages that encapsulate Mtb cells and are responsible for carbon storage and evading the immune response.103, 104, 105 In

57 bacteria, these are synthesized by enzyme GlgA which is a glycogen synthase enzyme that uses ADP glucose as a substrate, and further branches are introduced by enzyme GlgB.

There is also an alternate four-step pathway for the production of α-glucan, known as the

GlgE pathway, which uses maltose-1-phosphate (M1P) as the substrate.106 The first step involves the isomerization of trehalose to maltose catalyzed by the TreS enzyme,107 which is strictly an intramolecular mechanism because the enzyme’s active site forbids the escape of the glucose unit.108 The inactivation of TreS does not affect the growth in vitro or in vivo, hence, it is not crucial for the viability of Mtb.109 The second steps involves the production of M1P catalyzed by the Mak enzyme, and since the gene which encodes Mak is essential, it could serve as a potential drug target. The third step involves the transfer of maltose from M1P to glycogen catalyzed by maltosyltransfer enzyme GlgE to give an α-

1,4-linkage to form a linear polymeric chain. The fourth step involves the α-1,6 branching of linear polymeric chains catalyzed by enzyme GlgB.

The inactivation of GlgE results in the accumulation of its M1P substrate, which induces a stress response in cells that causes the up-regulation of the enzymes involved in the production of trehalose, as it protects the cell from stress. The increased production of trehalose increases the production of M1P, which disturbs the cell respiration instead of alleviating its effect by generating reactive oxygen species (ROS),109 which damage DNA and cause rapid cell death. The inactivation of GlgB results in oligosaccharide accumulation, rendering it insoluble and hence, unavailable for GlgE.109 This eventually disrupts the function of GlgE and accumulates M1P. Thus, GlgE is considered to be a genetically validated drug target and has the potential to be exploited for chemotherapy for the treatment of TB.110 58

Syson et al. reported the X-ray structure of GlgE isoform 1 from Streptomyces coelicolor

(Sco) and referred to it as (1→4)-α-D-glucan:phosphate-α-D-maltoyltransferase enzyme that belongs to the GH13_3 CAZy subfamily.111 The GlgE isoform 1 from Sco has a catalytic site homologous to that of GlgE from Mtb which allowed it to catalyze the reaction by a double displacement mechanism similar to that of the GH retaining mechanism. In the first step of the GlgE1 mechanism, the phosphate moiety (leaving group) is protonated by a Glu residue, and a second Asp residue attacks at the anomeric carbon to form a β- maltosyl-enzyme intermediate via the oxacarbenium ion-like TS. In the next step, the Glu residue deprotonates the glycogen, which acts as a nucleophile and attacks the anomeric carbon to which maltose is transferred.

Figure 4.2 Mechanism for Sco GlgE1111 (homolog of GlgE)

4.5 Glycoside Inhibitors

The main characteristic involved in the development of inhibitors for GH enzymes is TS mimicking. TS analogs/inhibitors are important compounds used for chemical biology to understand the structure of enzymes and are also used for therapeutics. The three main measures taken into account for mimicking the TS are: configuration, conformation, and charge. Based on these characteristics, we designed inhibitors to mimic the TS of enzyme

GlgE in terms of its shape and charge112 and also designed and synthesized inhibitors based on the results received from docking studies.

4.5.1 Glyco-conjugates as inhibitors

Computational chemistry is a growing field which uses computer modeling to study the properties of molecules. Glide (grid-based ligand docking with energetics) is a methodology used to dock ligands into receptor (enzyme) active sites. The binding affinity for a ligand is determined by the docking scores, which depend on ligand-receptor interactions and the ligand’s correct docked position. The ligand interacts with a receptor via different interactions (hydrogen bonding, electrostatic interactions, etc.) by displacing a water molecule in the protein cavity which results in a net increase in the entropy.113 The binding interaction of the ligand with the receptor is only accurate to a few cal/mol. Hence, molecular modeling is an important tool for screening virtually designed libraries of compounds, and Glide docking has gradually become a key technique for drug design and is actively used to distinguish between active and non-active ligands for a particular enzyme.113, 114

Drug-like glycoconjugates were designed as inhibitors for the enzyme GlgE, and ligands were docked for enzyme GlgE. Proteins were prepared using a protein preparation wizard to assign protons, atomic charges, and for the removal of water molecules. Ligands were prepared using a ligand preparation wizard to obtain the lowest-energy conformer, and receptor grids were generated which depicted the active sites for glide ligand docking. The affinity of a ligand was determined by the negative value of the docking scores received, where a more negative value represented a better affinity.

Entry Docking score Entry Docking score

-7.312 -6.863

1. 2.

-6.783 -9.036

4. 3.

-7.629 -6.413

5. 6.

-6.102 -6.786

8. 7.

-9.107 -6.689

9. 10.

-6.971 -8.112

11. 12.

-6.104 -7.321

13. 14.

-8.205

15.

Table 4.1 Entries of different drug-like compounds designed and their respective Docking scores.

Based on Table 4.1, the glycoconjugates (Entries-1, 4, 6, 7, 9, and 15) containing substituted phenols, cyclohexanols, and pyrrolidines that received good docking scores were envisioned as potential inhibitors for enzyme GlgE and were selected for synthesis.

4.5.1.1 Chemistry

Scheme 4.1 Synthesis of compound 3a-d (39-52%); Reagents and conditions: i) Donor 1 (1.5 eq), ROH (1 eq), NIS (2 eq), TfOH (cat. amount), -40 oC, DCM/Ether (8/2)

Entry ROH Gly co-conjugates Yield

1 2a 3a (α only) 40%

2 2b 3b (α only) 39%

3 2c 3c (α only) 55%

4 2d 3d (α only) 50%

Table 4.2 Reaction results of glycosylation of donor 1 with different alcohols (2a-d) to get compounds 3a-d with their respective yields.

Figure 4.3 2a-2d structure of different acceptors; 3a-3d structure of glycosylated products

To synthesize drug-like inhibitors for enzyme Sco-GlgEI-V279S, compounds were prepared by performing glycosylations. Donor 1 was reacted with phenols and cyclohexanols in the presence of NIS/TfOH at -40 oC to afford glycoconjugates 3a-d (39-

55%).115,116 The ester deprotection by NaOH and benzyl group deprotection by

117 hydrogenation with Pd(OH)2/C under a hydrogen atmosphere yielded inhibitors 4a-e.

Scheme 4.2 Deprotection of compounds (3a-d) to produce glycol-conjugates 4a-d. Compounds 3a and 3c were first subjected to ester deprotection with NaOH in MeOH followed by hydrogenation; i)

10% Pd(OH)2/C, H2.

Entry Compounds Glyco - conjugates Yield

1 3a* 4a 40%

2 3a 4b 80%

3 3b 4c Quantitative

4 3c* 4d 54%

5 3d 4e 76%

Table 4.3 Reaction results of deprotection of compounds 3a-d to get glycol-conjugates 4a-e with their respective yields. Compounds 3a* and 3c* were first subjected to ester deprotection with NaOH in MeOH followed by hydrogenation

Figure 4.4 4a-4e structure of different glycol-conjugates.

4.5.1.2 Experimental

Molecular Modeling

Three-dimensional ligand structures were created using Chem3D (Cambridge soft) and were imported into Maestro V 9.0 (Schrodinger, Inc.) to compute their docking scores using MacroModel (Schrödinger, Inc.). The ligands were then prepared to obtain the lowest energy conformer by conformational analysis and minimization method. The X-ray crystal structure for Sco GlgEI-V279S with maltose-c-phosphonate as native ligand complex

(PDB code: 4U31)2 was obtained from the Protein Data Bank (www.rcsb.org) and protein was prepared by removing solvents and adding hydrogen atoms and minimal minimization in the presence of bound ligand. Further Grid was generated by Glide V 6.0 (Schrodinger,

Inc) to obtain protein without any constraints to which ligands were docked in extra precision mode with 2 poses per ligand.

Glycosylation (3a-3d)

Donor 1 (1.5 eq) and acceptors (1 eq) were placed in a round-bottom flask and stored under high vacuum overnight. Afterwards, a mixture of dichloromethane: ether (8:2) was added to the flask, along with powdered 4 Å molecular sieves and the mixture was stirred for 30 minutes at room temperature. The temperature of the reaction was then reduced to -40 oC, and NIS (2 eq) was added to the reaction mixture, followed by the addition of a catalytic amount of triflic acid. The resulting reaction was allowed to stir at -40 oC under a nitrogen atmosphere for 1 h before it was quenched with triethylamine, and the organic layer was successively washed with a saturated Na2S2O3 solution and brine. The organic layers were

65 combined, dried over sodium sulfate, concentrated under reduced pressure, and purified via silica gel column chromatography (EtOAc/Hexane).

Hydrogenation (4a-4e)

Protected glycosides were placed in a round-bottom flask, and EtOH:THF (1.8:0.2) was added, followed by Pd(OH)2/C. The reaction was allowed to proceed at room temperature under a hydrogen atmosphere for 18 h. After the completion of the reaction, 10%

Pd(OH)2/C was filtered through Celite® 545 and washed with CH2Cl2:MeOH (8:2), and the solvent was evaporated under reduced pressure at high vacuum.

Methyl-2-(((benzyloxy)carbonyl)amino)-5-hydroxybenzoate (2a)

Methyl 2-amino-5-hydroxybenzoate (100 mg, 0.59 mmol) was dissolved in DMF (5 mL), followed by the addition of DIPEA (117 µL, 0.70 mmol) and benzyl chloroformate (171

µL, 1.2 mmol). The resulting reaction mixture was allowed to stir at room temperature for

8 h. The reaction was monitored by TLC, and after completion, the solution was diluted by

EtOAc (8 mL), and the organic layer was washed with water (10 mL). The organic layer was collected and dried over sodium sulfate, concentrated to dryness, and purified via silica gel column chromatography (acetone: hexane, 3:7) to afford as 2a white solid (120 mg,

1 66% yield); Rf = 0.3 (acetone/ hexane 30%); H NMR (600 MHz, CDCl3): 7.48 (m, 1H, aromatic), 7.44-7.35 (m, 6H, aromatic), 7.06 (dd, 1H, J = 3, 9, aromatic), 5.22 (s, 2H,

13 benzylic), 3.91 (s, 2H, OCH3); C NMR (150 MHz, CDCl3 ): 167.97, 153.66, 149.89,

136.21, 135.23, 1288.60, 128.57 128.55, 128.28, 128.26, 128.23, 128.20, 122.07, 116.57,

+ + 66.91, 52.38. HRMS, m/z = 324.0849 (M+23) C77H82O11Si requires 324.0848 (M+23) .

Methyl-2-(((benzyloxy)carbonyl)amino)-5-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)benzoate(3a)

Purified by chromatography on silica gel (hexane/ethyl acetate = 7/3) to afford 3a as

1 viscous liquid (72 mg, 40% yield); Rf = 0.94 (EtOAc:hexane 30%); H NMR (600 MHz,

CDCl3): 10.42 (s, 1H, aromatic), 8.43 (d, 1H, J=9.2, aromatic), 7.77 (d, 1H, J=2.9 aromatic), 7.19 (m, 2H, aromatic), 5.45 (d, 1H, J = 3.5, H-1), 5.27 (s, 2H, benzylic), 5.11

(d, 1H, J = 10.6, benzylic), 4.92 (m, 3H, benzylic), 4.72 (d, 1H, J = 11.9, benzylic), 4.64

(d, 1H, J = 11.9, benzylic), 4.54 (d, 1H, J = 10.6, benzylic), 4.46 (d, 1H, J = 12.1, benzylic),

4.24 (t, 1H, J = 9.3, H-2), 3.91 (m, 4H, H-6a, OCH3), 3.85 (m, 1H, H-3), 3.77 (m, 2H, H-

13 4, H-6b), 3.62 (m, 1H, H-5); C NMR (150 MHz, CDCl3 ): 168.07,153.60, 150.74, 138.74,

138.14, 137.96, 137.74, 136.70, 136.26, 128.60, 128.52, 128.46, 128.43, 128.30, 128.26,

128.16, 128.08, 128.03, 127.95, 127.83, 127.82, 127.76, 123.60, 120.36, 118.48, 115.54,

95.91, 81.97, 79.70, 75.93, 73.60, 73.47, 71.00, 68.13, 66.90, 52.37. HRMS, m/z =

+ + 846.3240 (M+23) C77H82O11Si requires 846.3254 (M+23) .

Benzyl 4-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)phenyl)carbamate (3b)

Purified by chromatography on silica gel (hexane/ethyl acetate = 7/3) to afford 3b as

1 viscous liquid (100 mg, 39% yield); Rf = 0.55 (EtOAc/hexane 30%); H NMR (600 MHz,

CDCl3): 7.34(m, 25H, aromatic), 7.18 (m 2H, aromatic), 7.07 (m, 2H, aromatic), 5.43 (d,

1H, J = 3.5 H-1), 5.08 (m, 1H, benzylic), 4.88 (m, 4H, benzylic), 4.70 (d, 1H, J = 11.9, benzylic), 4.59 (m, 3H, benzylic), 4.44 (m, 1H, benzylic), 4.23 (t, J = 9.3, 1H, H-3), 3.9

(m, 1H, H-5), 3.82 (m, 1H, H-4), 3.76 (m, 2H, H-2, H-6a), 3.6 (m, 1H, H-6b).; 13C NMR

(150 MHz, CDCl3 ): 153.39, 152.93, 138.81, 138.52, 138.21, 138.19, 138.21, 138.19,

138.00, 137.79, 136.15, 128.67, 128.48, 128.40, 128.00, 127.77, 127.65, 120.28, 117.70,

117.46, 102.22, 95.87, 84.68, 82.00, 79.71, 77.67, 75.86, 75.11, 73.46, 73.39, 70.81, 68.17,

+ + 67.03. mass spectrum, m/z = 788.5 (M+23) C77H82O11Si requires 788.32 (M+23) .

Methyl-3-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)benzoate (3c)

Purified by chromatography on silica gel (hexane/ethyl acetate = 7/3) to afford 3c as

1 viscous liquid (104 mg, 55% yield); Rf = 0.92 (EtOAc/hexane 30%); H NMR (600 MHz,

CDCl3): 7.79(m, 2H, aromatic), 7.2 (m, 2H, aromatic), 7.37 (m, 20H, aromatic), 5.56 (d,

1H, J = 3.3 anomeric), 5.12 (m, 1H, benzylic), 4.92 (m, 3H, benzylic), 4.73 (d, 1H, J =

11.9, benzylic), 4.64 (m, 1H, benzylic), 4.55 (d, 1H, J = 10.6, benzylic), 4.46 (d, 1H, J =

12.1, benzylic), 4.26 (m, 1H, H-2), 3.95 (s, 3H, OCH3), 3.85 (m, 4H, H-3, H-4, H-6a, H-

13 6b), 3.62 (dd, 1H, J = 10.8, 1.5, H-5); C NMR (150 MHz, CDCl3 ): 166.76, 156.57,

138.75, 138.16, 137.94, 137.76, 131.58, 129.62, 129.53, 128.57, 128.45, 128.03, 127.98,

127.92, 127.79, 127.75, 127.66, 123.67, 123.67, 121.60, 117.61, 101.49, 95.53, 84.66,

81.96, 79.66, 75.91, 75.23, 73.52, 71.10, 68.14, 52.24 mass spectrum, m/z = 697.7 (M+23)+

+ C77H82O11Si requires 697.28 (M+23) .

Benzyl 4-(3’,4’,5’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohexylcarbamate (3d)

Purified by chromatography on silica gel (hexane/ethyl acetate = 7/3) to afford 3d as

1 viscous liquid (180 mg, 50% yield); Rf = 0.46 (EtOAc/hexane 30%); H NMR (600 MHz,

CDCl3): δ 7.34(m, 25H, aromatic), 5.13 (m, 2H, benzylic, Cbz group), 4.97 (dd, 2H, J =

10.1, 6.1, benzylic), 4.84 (dd, 2H, J = 19.7, 10.7, benzylic), 4.74 (d, 1H, J = 10.8, benzylic),

4.63 (m, 3H, benzylic), 4.51 (d, 1H, J = 7.91, H-1), 3,77 (m, 1H, H-6a), 3.67 (m, 2H, H-

3, H-6b), 3.58 (t, 1H, J = 9.4), 3.48 (m, 2H, H-2, H-5), 2.10 (m, 4H, cyclohexane), 1.6 (m

13 2H, cyclohexane), 1.23 (m, 2H, cyclohexane); C NMR (150 MHz, CDCl3): δ 155.53,

138.55, 138.33, 138.15, 138.0, 136.49, 128.51, 128.37, 128.32, 127.84, 127.65, 127.57,

102.16, 84.74, 82.18, 77.87, 75.67, 74.99, 74.74, 73.37, 69.07, 66.58, 49.15, 31.90, 30.90,

+ + 30.78, 30.31. HRMS, m/z = 794.3651 (M+23) C77H82O11Si requires 794.3669 (M+23) .

2-amino-5-(α-D-glucopyranosyl)benzoic acid (4a)

Compound 3a (100 mg, 0.12 mmol) was dissolved in 8 mL of MeOH:THF:H2O (1:3:1), followed by the addition of NaOH (8.73 mg, 0.36 mmol), and the reaction was allowed to stir at room temperature for 1 h. After reaction completion, as monitored by TLC in which the product had Rf of 0.11 in hexane/ethyl acetate (7:3), all liquids were evaporated under reduced pressure and subjected to flash column chromatography. Yield: 40%, 40 mg. The purified material was subjected to hydrogenation under standard conditions listed previously to afford 4a as white solid (14 mg, quantitative); 1H NMR (600 MHz, MeOD):

δ 7.75 (m, 1H, aromatic), 7.53 (m, 1H, aromatic), 7.41 (m, 1H, aromatic), 5.64 (m, 1H, H-

1), 3.83 (m, 1H, H-3), 3.65 (m, 2H, H-2, H-4), 3.40 (m, 2H, H-6a, H-6b), 3.08 (m, 1H, H-

5); 13C NMR (150 MHz, MeOD): δ 156.18, 133.01, 122.62, 122.43, 118.72, 118.54, 96.31,

+ 72.74, 70.97, 60.22, 53.36. HRMS, m/z = 316.1472 (M+H) C77H82O11Si requires

316.1032 (M+H)+.

Methy-2-amino-5-(α-D-glucopyranosyl)benzoate (4b)

1 Yield: 80%, 14 mg. H NMR (600 MHz, D2O): δ 7.81 (d, 1H, J = 2.8, aromatic), 7.40 (dd,

1H, J = 8.9, 2.8, aromatic), 7.31 (d, 1H, J = 9, aromatic), 5.58 (d, 1H, J = 3.7, H-1), 3.86

(s, 3H, OCH3), 3.81 (m, 1H, H-3), 3.62 (m, 2H, H-2, H-4), 3.40 (m, 1H, H-6a), 3.33 (m,

13 2H, H-5, H-6b); C NMR (150 MHz, D2O ): δ 167.23, 154.20, 123.73, 123.39, 120.13,

97.24, 72.82, 72.67, 70.89, 69.22, 60.22, 53.02, 46.04. HRMS mass spectrum, m/z =

+ + 330.1193 (M+H) C77H82O11Si requires 330.1189 (M+H) .

(4-aminophenyl)-α-D-glucopyranoside (4c)

1 Yield: quantitative, 8 mg. H NMR (600 MHz, D2O): δ 7.37 (m, 2H, J = 9, aromatic), 7.26

(m, 2H, J = 9, aromatic), 5.60 (d, 1H, J = 3.4, H-1), 3.82 (m, 1H, H-3), 3.65 (m, 4H, H-2,

13 H-4, H-6a, H-6b), 3.41 (m, 1H, H-5); C NMR (150 MHz, D2O ): δ 156.85, 130.82,

123.63, 118.45, 96.84, 72.86, 72.65, 70.91, 69.16, 60.18. mass spectrum, m/z = 294.2

+ + (M+23) C77H82O11Si requires 294.10 (M+23) .

3-(α-D-glucopyranosyl)benzoic acid (4d)

Compound 3c (100 mg, 0.14 mmol) was dissolved in 8 mL of MeOH:THF:H2O (1:3:1), followed by the addition of NaOH (10.6 mg, 0.44 mmol), and the reaction was allowed to stir at room temperature for 1 h. The reaction was completed as monitored by TLC in which the product had Rf of 0.11 in hexane:ethyl acetate (7:3), and all liquids were evaporated under reduced pressure and subjected to flash column chromatography. Yield: 55%, 54 mg. The purified material was subjected to hydrogenation under standard conditions to

1 afford 4d as white solid (22mg, quantitative yield). H NMR (600 MHz, D2O): δ 7.53 (m,

2H, aromatic), 7.33 (m, 1H, aromatic), 7.20 (m, 1H, aromatic), 5.58 (d, 1H, J = 3.7, H-1),

3.83 (m, 1H, H-3), 3.63 (m, 4H, H-2, H-5, H-6a, H-6b), 3.40 (m, 1H, H-4); 13C NMR (150

MHz, D2O): δ 155.51, 136.62, 129.85, 123.04, 119.34, 116.92, 96.98, 72.94, 72.32, 71.82,

+ 70.91, 62.41, 59.97, 46.37. mass spectrum, m/z = 323.4 (M+23) C77H82O11Si requires

323.07 (M+23)+.

(4-aminocyclohexyl)-α-D-glucopyranoside (4e)

1 Yield: 76%, 50mg. H NMR (600 MHz, D2O): δ 4.46 (d, 1H, J = 8.1, H-1), 3.78 (dd, 1H,

J = 12.3, 2.2, H-5), 3.59 (m, 1H, H-6a), 3.36 (m, 2H, H-3, H-6b), 3.26 (d, 1H, J = 9.5, H-

4), 3.1(dd, 1H, J = 9.3, 8.2, H-2),2.04 (m, 5H, cyclohexane), 1.36 (m, 5H, cyclohexane).;

13 C NMR (150 MHz, D2O ): δ 100.54, 76.73, 75.85, 75.68, 72.99, 89.56, 60.66, 49.02,

+ 30.40, 29.00, 28.09, 27.93. HRMS, m/z = 274.1595 (M+H) C77H82O11Si requires

274.1604 (M+H)+.

4.5.2 Iminosugars as Inhibitors

Iminosugars are known to be potent inhibitors for GHs as they are substrate mimics of

GH.118 In iminosugars, the endocyclic oxygen is replaced by a nitrogen atom which can be protonated at physiological pH to form an ammonium cation.119 This cation strongly mimics the TS i.e., the oxacarbenium ion which contains a positive charge on its oxygen, and hence strongly interacts with the catalytic residues at the enzyme active site.120

There are several natural and synthetic iminosugars (piperidines, pyrrolidines, and indolizidines) that are known to inhibit glycosidases.121 The most notable of these is the deoxynojirimycin (DNJ) iminosugar mimic of glucose extracted from the roots of mulberry trees.122 It has been reported to inhibit glucosidase from human lysosomes and yeast, and the alkylation of the nitrogen in the ring increases its consumption in the mammalian cell.123, 124 Hence, the miglitol derivative of DNJ in which the nitrogen is ethylated is a commonly-used drug to treat postprandial blood glucose. The N-alkyl substituted

71 derivatives of DNJ are also known to act as pharmacological chaperones for the treatment of Gaucher disease.125

Figure 4.5 Iminosugars-based inhibitors for GH enzymes.

Polyhydroxypyrrolidines display a wide range of inhibitory activities against various GH enzymes, including α-glucosidase126, α-mannosidase127, α-L-rhamnosidase128 and many others. The 5-membered aza sugars are considered to be better inhibitors of glucosidase hydrolase than 6-membered aza sugars since they mimic both the charge generated in the

TS and its shape i.e. flattened half-chair.129 Hence, Sri. et al reported the synthesis of disaccharide-related pyrrolidine-based compounds (11-13) as inhibitors for enzyme

GlgE1-V279S which has an active site completely identical to that of Mtb GlgE.115, 130

Based on previously synthesized pyrrolidines and the docking scores calculated for the pyrrolidine-based compounds from Table 4.1 (entries 1 and 7), compounds 14 and 15 were

72 synthesized by following the synthesis procedure reported by Veleti et al. beginning with commercially-available fructose and donor 1.115

Scheme 4.2 Synthesis of pyrrolidine-based inhibitor 14 and 15.

4.5.2.1 Experimental for Iminosugar-based inhibitors

Benzyl(3-(3-hydroxy-2,5-bis(hydroxymethyl)-4(-O-α-D-glucopyranosyl)pyrrolidin-

1-yl)propyl)carbamate (14)

Compound 11 (23 mg, 0.07 mmol) was dissolved in 2 mL of MeOH to which 3-

[(Benzyloxycarbonyl)amino]propionaldehyde (14.6 mg, 0.07 mmol) was added, followed by NaCNBH3 (8.89 mg, 0.14 mmol). The reaction was allowed to proceed at room temperature for 12 h, and the reaction completion was determined by ESI-MS. Purification was performed by gel permeation chromatography using Sephadex LH-20 (MeOH:H2O =

2:8) to obtain compound 14 as a colorless solid (15 mg, 41.6% yield). 1H NMR (600 MHz,

MeOD): 7.37 (m, 5H, aromatic), 5.09 (m, 2H, H-1, H-1’), 4.13 (m, 2H), 4.00 (m, 1H), 3.81

(m, 14H), 3.42 (m, 2H, aliphatic), 1.41 (m, 2H); 13C NMR (150 MHz, MeOD ): 158.95,

138.57, 129.60, 129.10, 128.94, 128.91, 104.53, 80.11, 71.37, 67.49, 60.74, 60.50, 59.54,

+ 53.76, 46.08, 39.92, 37.85, 34.08. HRMS, m/z = 517.2406 (M+H) C77H82O11Si requires

517.2397 (M+H)+.

2-((-1-(3-aminopropyl)-4-hydroxy-2,5-bis(hydroxymethyl)pyrrolidin-3-yl)-α-D- glucopyranoside (15)

Compound 14 (9 mg, 0.017 mmol) was dissolved in EtOH (3 mL), followed by the addition of 10% Pd(OH)2 on carbon. The reaction was allowed to proceed at room temperature under a hydrogen atmosphere for 12 h. After the completion of the reaction, 10%

Pd(OH)2/C was filtered through Celite® 545 and washed with CH2Cl2:MeOH (8:2).

Solvent was evaporated, and the crude residue was dissolved in a minimal amount of water and loaded onto a C18 column and eluted with water. Water was removed to obtain

1 compound 15 as colorless solid (4 mg, 66% yield); H NMR (600 MHz, D2O) : 5.07 (m,

1H, H-1), 3.97-3.37 (m, 15H), 2.60 (s, 1H, aliphatic), 2.46 (s, 2H, aliphatic), 2.11 (s, 2H,

13 aliphatic), 2.02 (s, 1H, aliphatic); C NMR (150 MHz, D2O): 95.47, 91.38, 71.62, 70.43,

69.04, 68.37, 67.26, 66.00, 62.34, 61.51, 57.89, 34.40, 24.48, 20.08, 19.93. HRMS, m/z =

+ + 383.2031 (M+H) C77H82O11Si requires 383.2030 (M+H) .

4.5.3 Inhibition studies

Inhibition of the glycoconjugates (4a-4e) and pyrrolidine-based inhibitors (14 & 15) was performed using an Enzcheck phosphate assay kit131 which utilizes the released phosphate from M1P to give an absorbance value at 360 nm. The inhibition studies of the compounds were tested for the Sco GlgEI-V279S variant. Assays were performed in a 96 well plate at atmospheric pressure and room temperature on a SpectraMax 340PC Microplate Reader.

Reactions were performed in triplicates, and each reaction contained 1 mM MESG (2- amino-6-mercapto—7-methylpurine riboside), 0.2 U PNP (purine nucleoside phosphorylase), 50 nM Sco GlgE1-V279 enzyme, glycogen, and 250 µM of M1P in buffer

(1.0 M Tris-HCl, 20 mM MgCl2, 2 mM NaN3, pH 7.5), with different inhibitor concentrations. Inhibitory activity was determined by comparing steady rates with and without inhibitor (Vo’ & Vo respectively). Glycoconjugate compound 4a showed a Ki value of 659 µM, and none of the other compounds (4b-e) showed any notable inhibition.

Pyrrolidine-based compound 14 showed a Ki value of 132.6 ± 8 µM, while compound 15 did not show any significant inhibition.

Figure 4.6 Ki determination of compound 4a with Sco GlgEI-V279. Vo’/Vo are steady-state rates with and without inhibitor. 4.5.4 Results and discussion

The glycoconjugates were designed as a drug-like inhibitors for enzyme GlgE. From the designed library of potential inhibitors, the compounds that received good docking scores

4a-e (Table 4.1; Entries- 4, 6, 9, 15) were synthesized by treating thioglucoside donor 1 with different alcohols (2a-d). The glycoconjugates in which the first glucose unit of M1P was replaced with substituted a phenyl/cyclohexyl ring showed significantly reduced

75 binding affinities compared with M1P. The loss of extensive hydrogen bonding compared with the first glucose unit could possibly explain the weak affinity of these glycoconjugates.

Compound 4a containing a carboxylic acid and an amine group at the ortho position showed a Ki value of a 659 µM, which suggests that the presence of a significant number of hydrogen bond donors is essential for binding in the first unit.

The pyrrolidine-based compounds 14 and 15 were synthesized from known compound 11.

Compound 14 containing a propyl group with a benzyl chloroformate (Cbz)-protected amine on the pyrrolidine ring showed a Ki value of 132.6 ± 8 µM, whereas the compound with a free amine on the pyrrolidine showed no affinity. This suggests that the Cbz group on the amine interacts with the active site of the enzyme. The size of the pyrrolidine containing a Cbz group is larger than the M1P substrate, suggesting that the Cbz group interacted with a deeper pocket. This Cbz group interaction can be utilized to understand the deeper pocket of the enzyme and develop additional potent inhibitors.

In addition, the molecular modeling data was useful for comparing the affinity of ligands within the same class. After examining the molecular modeling results, it was found that compound 4a showed better docking scores than all other glycoconjugates in which the first unit was replaced with a substituted phenyl/cyclohexyl ring. In addition, pyrrolidine- based compound 14 showed better docking results than compound 15.

4.5.5 Carbasugars as Inhibitors

Carbasugars are an important class of compounds where the ring oxygen is replaced by a carbon atom which mimics the molecular shape of the TS.132, 133, 134 ,135 The pyranosylium 76 ion gives the region between O5 and C1 double bond character which flattens the system at C2-C1-O5-C5 and allows for motion at C3 and C4.136 Thus, the possible TS

3 4 2,5 conformations are H4, H3, B, B2,5 and the envelope conformations (4E, 3E, E4, E3), which are slightly higher in energy. The compounds, which are conformationally restricted to attain the TS or closely-related conformations, have great potential to act as GH inhibitors.137

Figure 4.7 Conformations attained by TS138

There have been numerous reports of carbasugars containing a cyclopropyl group acting as GH inhibitors.139 Recently, Bennet et al. synthesized carbasugar analogs of galactose compounds 16 and 17 with a cyclopropyl group as mechanism-based inactivators for GH

α-galactosidase. These compounds were evaluated for their inhibitory activity towards α- galactosidase from coffee beans and Thermotoga maritima (TmGa1A) (Figure 4.7).140

Compound 16, which contains an above-plane cyclopropyl ring, inactivated TmGa1A, while compound 17 did not show any inactivation. The importance of the cyclopropyl group orientation was further demonstrated by examining the inhibition of both α- galactosidase and α-glucosidase, as inhibitor 18 showed better inhibition than 19 against

α-glucosidase.141 Due to the inclusion of a cyclopropyl ring, the C5 and C6 positions are confined, and the only possible conformations of compound 16 to catalyze the inactivation

3 1,4 of TmGa1A are either H2 or B. Later, compounds 20, 23, and 24 were synthesized as

TmGa1A inactivators,142 and all of these compounds form a complex with the enzyme and

2 143 bind in the H3 TS.

In 2017, Bennet’s group reported compounds 21 and 22 as part of a new class of reversible covalent inhibitors for enzyme GH13, and their activities were tested against α-glucosidase from yeast (Figure 4.7).144 The two compounds bound to the enzyme via allylic and bicyclobutonium ion TS, respectively, and the reactivation of the enzyme was faster in compound 21 than 22. The different activities of these two compounds was explained by their Michaelis complexes with which they bound to the enzyme, and it was reasoned that

2 1,4 the Michaelis complex formed by compound 21 was H3, whereas it was the B conformation for 22. The conformation itinerary for GH13 was described as

1 4 # 4 1,4 1 2 S3→[ H3] → C1, and it was noted that B was more closely related to S3 than H3.

Figure 4.8 Carbasugars compounds as GH inhibitors

In 2017, carba-cyclophellitol inhibitors (25-27) were designed and synthesized, in which the epoxide oxygen of cyclophellitol was replaced by a carbon atom.145 The conformational constraints at the C-1(anomeric) and O-5 positions in carba-cyclophellitol inhibitors

4 enables them to bind in the H3 conformation, which makes them potent glycosidase inhibitors. In 2018, 1,6-cyclophellitol cyclosulfates (28 & 29) were introduced as a new class of irreversible glycosidase inhibitors.146 These compounds were tested for their inhibitory activity against α-glucosidase GAA, GANAB (which both belong to GH31), β- glucosidase GBA1 (GH30), and GBA2 (GH116). The results showed that α-cyclosulfate

(28) was a much more potent inhibitor for α-glucosidase with an IC50 = 82 nM against

GAA and 29 nM against GANAB than β-cyclosulfate (29) was for β-glucosidase. The

4 4 # 1 conformational itinerary for α-glucosidase was described as C1→[ H3] → S3 and for β-

1 4 # 4 glucosidase as S3→[ H3] → C1. The difference in the inhibition of the two cyclosulfates

4 was explained based on their C1 Michalis conformation, which is favorable with α-

1 glucosidase but unfavorable with β-glucosidase which adopts the S3 Michaelis conformation. Thus, β-cyclosulfate did not strongly inhibit β-glucosidase because it failed to mimic the Michaelis conformation, but both α- & β-cyclosulfate mimicked the TS for their respective glucosidase enzymes. This indicates the importance of the Michaelis complex conformation when designing GH inhibitors.

Figure 4.9 Cyclitol-based compounds C1 and C2 based on M1P (substrate of GlgE).

The above literature results show that the inhibitors designed and synthesized were closely related to the TS or Michaelis complex of the substrate for the enzymes. Based on this, compound C1 (Figure 4.8) was designed and synthesized as cyclitol-based inhibitor for enzyme GlgE. Since compound 22 binds to yeast α-glucosidase from the GH13 family, compound C1 was designed and synthesized in which the second sugar moiety of the maltose, i.e. the substrate for GlgE, is replaced by a cyclopropyl-based carbasugar.144 The second cyclitol compound C2 (Figure 4.8) containing 1,2-cis cyclic sulfate was designed by considering the inhibition shown by α-cyclosulfate 28 in the nanomolar range for α- glucosidase.146 It was anticipated that these compounds would form a covalent adduct with

GlgE.

Scheme 4.3 Synthesis of cyclopropyl-based cyclitol C1.

4.5.5.1 Chemistry studies

The synthesis of the cyclopropyl-containing pseudo disaccharide-based cyclitol C1 began from a disaccharide maltose. Veleti et al. reported the synthesis of compound 30131 in which the alcohol at the 6-position of 30 was oxidized using Moffatt oxidation by it treating with DCC (dicyclohexyl carbodiimide), DMSO (dimethyl sulfoxide), pyridine, and TFA

(trifluoroacetic acid) in benzene to afford ketone 31 in 90% yield.147 Ketone 31 was further subjected to a stereoselective addition reaction using vinyl magnesium bromide in dry THF to afford diastereomeric dienes 32A and 32B in 9:1 respectively. The NMR shifts of the two epimers 32A and 32B were compared with the values of epimers C and D, which were derived from glucose, but the comparison did not provide a clear indication as to which stereochemical configuration was preferred (shown in Table 3).148

32A 32B C D

H-C(1) 5.27 5.30 5.30 5.26

H-C(1’) 5.27 5.30 5.20 5.27

H-C(2) 5.79 5.81 5.83 5.94

H-C(3) 4.41 4.24 4.07 4.09

H-C(4) 3.92 3.91 3.88-3.84 3.74-3.68

H-C(5) 4.03 4.04 3.88-3.84 3.99

H-C(8a) 5.55 5.58 5.47 5.45

H-C(8b) 5.36 5.30 5.19 5.23

Table 4.4 Comparing the NMR shift values of compound 32A & 32B with C and D

Structural determination of the desired epimer was achieved by further subjecting dienes

32A and 32B to ring-closing metathesis using Grubb’s 2nd generation catalyst which failed to yield product. Following this, Grubb’s 1st generation catalyst was used under optimized reaction conditions (Table 4.4).

Catalyst (10 mol%) Temperature Solvent Yield

Grubb’s 2nd gen rt DCM NR

Grubb’s 2nd gen 40 oC DCM NR

Hoveyda Grubb’s rt DCM NR

Grubb’s 1st gen rt DCM 10-15%

Grubb’s 1st gen 40 oC DCM 20%

Grubb’s 1st gen rt Toluene 14%

Grubb’s 1st gen 80 oC Toluene 15%

Table 4.5 Reaction condition optimization for ring closing metathesis for compound 32A. Starting material recovered in all the reactions based on which yield is 70-80%. NR = no reaction, rt = room temperature

The NMR shifts of 33A and 33B were compared with the gluco-carbasugar cyclitols E and

F. The alkene protons of compounds 33A and 33B exactly matched with E and F (Table

4.5), clearly indicating that the desired product 33A was obtained.148 The free hydroxyl of

33A was protected with an acetyl group by treating it with KHMDS (potassium hexamethyl disilyl amide) and acetyl chloride in THF. Compound 34 was subjected to palladium- catalyzed C=C isomerization using the catalyst bis(benzonitrile)Pd(II)Cl, followed by

Zemplén deacetylation to remove the acetyl group and obtain compound 35 in 60% yield.144 The alkene in compound 35 was subjected to Furukawa-Simmons-Smith reaction by treating it with diethyl zinc, diiodomethane, and TFA in toluene to incorporate a cyclopropyl group which afforded compound 36.149 This product was further subjected to aromatic nucleophilic substitution using 1,3,5-triflurobenzene, followed by global

83 deprotection of the benzyl groups using 5% Pd/C under an H2 atmosphere to obtain the target inhibitor C1.

33A 33B E F

H-C(1) 4.23 4.24 4.20 4.20

H-C(2) 3.95 4.25 4.02 3.87

H-C(3) 3.67 3.92 3.76 3.76

H-C(4) 3.88 3.93 - -

H-C(5) 5.69 5.74 5.69 5.74

H-C(6) 5.92 5.74 5.92 5.74

H-C(7) 3.38 3.15 3.38 3.83

H’-C(7) 3.65 3.48 3.30 3.63

J(5,6) 10.14 10.38 10.3 0

J(1,6) 1.9 2.1 1.9 0

Table 4.6 Comparing the NMR shift values of compound 33A & 33B with E and F.

To synthesize the cyclosulfate-based cyclitol, the free hydroxyl of 33A was protected with a benzyl group by treating it with KHMDS and benzyl bromide to obtain the product in

65% yield. The alkene of compound 38 was treated with NaIO4 and Ru(III)Cl to obtain a single diastereomer β-cis-diol in 75% yield. The β-cis-diol 39 was treated with thionyl chloride in the presence of Et3N to give a cyclic sulfite which was further oxidized to give 84 the benzylated cyclosulfate 40.146 The global deprotection of benzyl groups in compound

40 gave the target compound 41.

Scheme 4.4 Synthesis of cyclosulfate-based cyclitol 41 compound

4.5.5.2 Results

Compound C1 was tested for its inhibitory activity for enzyme Sco GlgE1-V279S and α- amylase (pancreatic). Unfortunately, the compound did not show any activity against these two enzymes which belonged to the GH13 family, possibly due to the Michaelis conformation formed by compound C1. As per the literature, the Michaelis conformation attained by a cyclopropyl-based carbasugar is the skew conformation. This places the aglycone moiety in a pseudo equatorial position for σ-bond participation to cleave the C—

O bond similar to that of the oxacarbenium ion in which the ᴨ-molecular orbital aids the cleavage.113 The substrate for the enzyme GlgE, M1P, orients the aglycone (phosphate moiety) in the axial position to perform the cleavage, and the enzyme is inactive towards the cleavage of β-M1P in which the phosphate moiety is in the equatorial position.110 This inactivation of the enzyme towards the substrate containing the aglycone in the equatorial position could be why C1 did not show any activity. Another reason for the inactivity of

85 compound C1 could be explained by the X-ray structures of enzyme-bound intermediates of α-M1P and β-2-deoxy-2-fluoromaltosyl with muteins D394A and E423A of Sco GlgE isoform I, as reported by Syson et al.111 The structure revealed the conformational itinerary for the first step in the double displacement mechanism of the enzyme to be

4 4 # 4 1 C1→[ H3] → C1. However, the Michaelis conformation adopted by compound C1 is S3, which does not agree with the conformational itinerary of the enzyme. However, there are some examples in the literature which show different conformations of the complex with the mutein compared with wild type (WT) enzyme. For example, in GH38 Golgi α-

150 4 mannosidase, the D204A mutant exhibits a C1 conformation, whereas the WT enzymes

151 adopt a B2,5 conformation. This reveals that enzyme GlgE1 shows the same conformational itinerary for the WT and its mutant.

Synthesized compound 41 has a different configuration than compound C2 which was

4 designed. As discussed above, both α- & β-cyclosulfate mimic the TS and form a C1

Michaelis complex which is the same as the GlgE enzyme. Currently, compound 41 is being evaluated for its activity and to determine if mimicking the TS alone can result in any activity.

4.5.5.3 Discussion

Carbohydrates exhibit a large number of conformations and can transform from one conformation to another.152 The exact knowledge of the conformation of a carbohydrate or related compounds is extremely important for developing inhibitors for a particular GH enzyme, especially since there are about 113 known GH families which have a common catalytic mechanism (Figure 4.1). TS mimics have attracted significant attention in the 86 development of inhibitors for each GH e.g., Tamiflu and Relenza inhibit neuraminidase in its boat TS conformation.153 However, the TS conformations of each family are unique and are mostly conserved for enzymes within the same family or on the stereochemical outcome of the substrates with few exceptions.151

In a Michaelis complex, the distortion of a sugar molecule occurs when binding to a GH enzyme and also during the substrate’s pre-activation process to obtain the TS.154, 155 This distortion minimizes steric interactions and places the aglycone in a position which facilitates the nucleophilic attack of the enzyme at the anomeric center, as verified by

QM/MM (quantum mechanics/molecular mechanics).156, 157 Hence, the conformation of the Michalis complex is an important factor to consider during the development of inhibitors for GH enzymes as it prepares the substrate for the catalytic activity of the enzyme, allowing it to more easily form the TS. It has also been observed that not all enzymes within the same family exhibit the same conformational itinerary or Michalis complex158 e.g., the conformational itinerary proposed for GH13 yeast α-glucosidase is

1 4 # 4 4 4 # 4 S3→[ H3] → C1, whereas it is C1→[ H3] → C1 for GH13_3 GlgE. Thus, it can be concluded that although mimicking the TS is one of the prime factors to consider when designing inhibitors for specific glycosidase enzymes, the resemblance to the Michaelis conformation should also be taken into account.

4.5.5.4 Experimental

General Methods

All chemicals and solvents were purchased from Fisher Scientific, Acros Organics, Alfa

Aesar, or Sigma-Aldrich. Solvents were dried by through a solvent purification system by passing them through activated alumina and copper catalyst columns. All reactions were carried out at room temperature under a nitrogen atmosphere using a nitrogen balloon, unless otherwise mentioned. Reactions were monitored by TLC (silica gel, f254) under UV light or by charring (5% H2SO4-MeOH), and purification was performed by column chromatography with silica gel (230-400 mesh) using the solvent system specified, and solvents were used without purification for chromatography. 1H NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer using CDCl3 and D2O as internal references. 13C spectra were recorded on Bruker Avance III 600 MHz spectrometer using

CDCl3 and D2O as internal references. High-resolution mass spectrometry was performed on a TOF MS-ES+ instrument, and low-resolution mass spectrometry was performed on an ESquire-LC-MS.

Metathesis procedure

A dialkene solution in DCM was degassed by passing argon through it for 20 min.

Afterwards, Grubb’s catalyst (10 mol%) was added to the solution, and the reaction was kept under an argon atmosphere using an argon balloon for 7 days, or until the catalyst turned dark brown-black. Afterwards, solvent was evaporated under reduced pressure, and purification was performed by silica gel flash column chromatography.

3,4,7-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D-gluchept-

1-enone (31)

Compound 30 (0.800 g, 0.823 mmol) was dissolved by gently warming it in anhydrous benzene (2.5 mL), and then dry DMSO (2.5 mL) was added. To the clear solution were added, in order, anhydrous pyridine (66.42 µL, 0.823 mmol, 1 eq.), TFA (31.51 µL, 0.411 mmol, 0.5 eq.), and N,N'-Dicyclohexylcarbodiimide (0.509 g, 2.47 mmol, 3 eq.). The reaction was left at room temperature for 18 h. After the completion of the reaction, as determined by TLC, benzene (5 mL) was added, and crystalline dicyclohexylurea was removed by filtration and then washed with benzene. The combined filtrates and washings were extracted with water three times (10 mL) to remove DMSO. The organic layer was dried over anhydrous Na2SO4, evaporated under reduced pressure, and subjected to silica gel flash column chromatography to obtain the product as a colorless viscous liquid (700

1 mg, 88% yield); Rf = 0.72 (30 % EtOAc : Hexane); H NMR (600 MHz, CDCl3): δ 7.33-

7.23 (m, 33 H, aromatic), 7.17-7.15 (m, 2H, aromatic), 5.97 (m, 1H, H-1’), 5.24 (m, 2H,

=CH2), 4.93 (d, 1H, J = 11, benzylic), 4.82 (m, 4H, benzylic and H-1), 4.68 (dd, 2H, J =

12, 4.1, benzylic), 4.56 (m, 2H, benzylic), 4.48 (d, 1H, J = 10.8, benzylic), 4.35 (m, 4H, benzylic and H-6a’), 4,26 (d, 1H, J = 6.2, H-4’), 4.18 (m, 3H, benzylic and H-6b’), 4.02

(m, 2H, H-3, H-2’), 3.93 (m, 1H, H-5), 3.85 (dd, 1H, J = 6.2, 4.4, H-3’), 3.72 (m, 2H, H-

4, H-6a), 3.54 (dd, 1H, J = 9.7, 3.7, H-2), 3.45 (dd, 1H, J = 10.8, 1.8, H-6b); 13C NMR (150

MHz, CDCl3): δ 204.40, 138.77, 138.53, 138.28, 138.19, 137.92, 137.91, 137.64, 134.92,

128.41, 128.34, 127.70, 127.63, 118.92, 99.72, 81.73, 80.64, 79.71, 79.71, 79.23, 75.64,

74.90, 74.55, 73.48, 73.20, 73.06, 70.74, 68.06. mass spectrum (HRMS), m/z= 969.4601

+ + (M+H) C62H64O10 requires 969.4578 (M+H) . 89

3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D-gluco- octa-1,7-dienitol (32A)

To a cooled (-78 oC) solution of 31 (0.346 g, 0.357 mmol) in THF (5 mL), 0.7 M of vinyl magnesium bromide (652 µL, 1.072 mmol, 3 eq.) was added dropwise, and the reaction mixture was stirred for 1 h at the same temperature. The reaction mixture was warmed to room temperature, and Et2O (10 mL) and aq. NH4Cl (10 mL) were added. The organic layer was separated, washed twice with brine (10 mL), and dried over anhydrous Na2SO4.

The solvent was evaporated under reduced pressure, and purification was performed by silica gel flash column chromatography to afford the product as a colorless viscous liquid.

Yield: 32A (0.250 g, 70.2%), 32B (0.30 g, 8.2 %) Rf = 0.65 and 0.72 (30 % EtOAc :

1 Hexane) respectively. H NMR (600 MHz, CDCl3): δ 7.34-7.23 (m, 31H, aromatic), 7.19-

7.16 (m, 4H, aromatic), 6.26 (dd, 1H, J = 17.3, 10.9, H-7’), 5.75 (m, 1H, H-1’), 5.55 (dd,

1H, J = 17.3, 1.7, H-8a’), 5.32-5.23 (m, 4H, H-1, H-8b’, =CH2), 4.48 (m, 2H, benzylic),

4.85 (d, 2H, J = 11.6, benzylic), 4.61 (m, 2H, benzylic), 4.54 (m, 3H, benzylic), 4.47 (t,

2H, J = 11.6X2, benzylic), 4.41 (dd, 3H, J = 12.1, 5.3, benzylic, H-2’), 4.26 (d, 1H, J =

11.7, benzylic), 4.03 (m, 2H, H-4’, H-3), 3.95 (m, 1H, H-5), 3.92 (m, 1H, H-3’), 3.68 (m,

2H, H-4, H-6a’), 3.6 (dd, 1H, J = 9.7, 3.59, H-2), 3.51 (dd, 1H, J = 10.8, 2.8, H-6a), 3.41

13 (dd, 1H, J = 10.7, 1.6, H-6b), 3.34 (d, 1H, J = 9, H-6b’). C NMR (150 MHz, CDCl3): δ

139.92, 138.86, 138.71, 138.60, 1338.38, 137.99, 137.97, 137.71, 135.78, 128.35, 128.30,

128.18, 128.11, 127.92, 127.75, 127.65, 127.58, 127.48, 127.43, 127.35, 127.24, 119.35,

1155.67, 97.01, 82.97, 81.77, 79.74, 79.37, 77.61, 77.49, 75.47, 74.93, 74.78, 74.32, 73.44,

73.02, 72.54, 70.77, 70.48, 67.81. mass spectrum (HRMS), m/z= 997.4899 (M+H)+

+ C64H68O10 requires 997.4891 (M+23) .

3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-L-ido-octa-

1,7-dienitol (32B)

1 H NMR (600 MHz, CDCl3): δ 7.33-7.13 (m, 33H, aromatic), 7.04 (m, 2H, aromatic), 6.27

(dd, 1H, J = 17.3, 10.9, H-7’), 5.81 (ddd, 1H, J = 17.3, 10.2, 8.3, H-1’), 5.58 (dd, 1H, J =

17.3, 1.9, H-8a’), 5.44 (d, 1H, J = 3.3, H-1), 5.3 (m, 3H, =CH2, H-8b’), 4.94 (m, 2H, benzylic), 4.8 (m, 2H, benzylic), 4.59 (m, 2H, benzylic), 4.4 (m, 8H, benzylic), 4.24 (t, 1H,

J = 8.3X2, H-2’), 4.04 (d, J = 1.8, H-4’), 3.91 (m, 2H, H-3’, H-3), 3.83 (d, 1H, J = 9.2, H-

6a’), 3.75 (m, 1H, H-5), 3.65 (m, 1H, H-4), 3.52 (dd, 1H, J = =9.6, 3.4, H-2) 3.37 (m, 1H,

13 H-6a), 3.3 (m, 2H, H-6b, H-6b’); C NMR (150 MHz, CDCl3): δ 139.20, 138.81, 138.68,

138.59, 138.35, 137.93, 137.77, 137.20, 135.86, 128.28, 127.63, 127.54, 127.13, 120.06,

115.69, 94.33, 84.60, 81.93, 79.01, 77.90, 77.30, 78.15, 75.46, 74.46, 74.86, 74.67, 73.43,

+ 72.78, 71.15, 70.44, 67.86. mass spectrum, m/z= 997.4 (M+H) C64H68O10 requires 997.4

(M+23)

(1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-O- benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33A)

1 Rf = 0.32 (30% EtOAc/Hexane); H NMR (600 MHz, CDCl3): δ 7.30-7.20 (m, 33H, aromatic), 7.19-7.11 (mm, 2H, aromatic), 5.92 (dd, 1H, J = 10.2, 1.9, H-6’), 5.66 (dd, 1H,

J = 10.3, 1.8, H-5’), 5.63 (d, 1H, J = 3.7, H-1), 4.93 (d, 1H, J = 11.7, benzylic), 4.83 (m,

2H, benzylic), 4.73 (d, 1H, J = 11, benzylic), 4.64 (m, 2H, benzylic), 4.54 (d, 1H, J = 12.3, benzylic), 4.22 (dd, 1H, J = 4.9, 2.1, H-1’), 4.17 (m, 1H, H-2’), 3.94 (m, 1H, H-3), 3.86

(m, 1H, H-5), 3.63 (m, 2H, H-4, H-7a’), 3.53 (ddd, 2H, J = 18.9, 10.1, 3.7, H-2, H-6a),

3.46 (dd, 1H, J = 10.5, 1.8, H-6b), 3.35 (d, 1H, J = 9.2, H-7b’); 13C NMR (150 MHz,

CDCl3): δ 139.10, 138.66, 138.34, 138.31, 138.10, 137.96, 137.78, 130.75, 130.31, 128.35,

128.33, 128.27, 128.17, 128.08, 127.92, 127.84, 127.77, 127.65, 127.55, 127.41, 127.66,

126.92, 97.02, 81.77, 80.82, 80.10, 79.60, 77.54, 75.48, 74.95, 74.12, 73.49, 73.10, 72.98.

+ 72.74, 71.50, 68.10). mass spectrum (HRMS), m/z= 991.4418 (M+Na) C62H64O10 requires

991.4397 (M+Na)+.

(1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-O- benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33B)

1 Rf = 0.38 (30% EtOAc/Hexane); H NMR (600 MHz, CDCl3): δ 7.35-7.15 (m, 35H, aromatic), 5.77 (ddd, J = 22.2, 10.4, 1.9 Hz, 2H, H-5’, H-6’), 5.32 (d, J = 3.7 Hz, 1H, H-

1), 5.04 (d, J = 12.0 Hz, 1H, benzylic), 4.93 (d, J = 10.9 Hz, 1H, benzylic), 4.87 – 4.76 (m,

3H, benzylic), 4.42-4.64 (m, 9H, benzylic), 4.27 (m, 1H, H-1’), 4.21 – 4.12 (m, 3H, H-2’,

H-7a’, H-7b’), 3.97 – 3.88 (m, 2H, H-3, H-3’), 3.70 (d, J = 9.4 Hz, 1H, H-6a), 3.66 – 3.51

13 (m, 4H, H-2, H-4, H-6b). C NMR (150 MHz, CDCl3): δ 139.37, 138.72, 136.64, 136.21,

138.13, 137.99, 137.80, 132,14, 128.41, 128.38, 128.37, 128.32, 128.17, 128.10, 128.06,

128.03, 127.90, 127.80,, 127.76, 127.73, 127.62, 127.56, 127.47, 126.99, 126.95, 126.66,

99.37, 84.31, 81.67, 80.95, 80.46, 79.24, 77.24, 75.52, 75.12, 75.05, 74.37, 73.62, 73.58,

73.53, 72.41, 71.33, 71.11, 68.51, 29.73. mass spectrum HRMS m/z= 991.396 (M+Na)+

+ C62H64O10 requires 991.4397 (M+Na) .

(1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-4-O-acetyl-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (34)

To a cooled (0 oC) solution of 33A (0.200 g, 0.206 mmol) in THF (5 mL), 200 µL of

NaHMDS (2 M in THF) was added dropwise, and the reaction mixture was stirred for 30 min at the same temperature. Afterwards, AcCl (19 µL, 0.268 mmol, 1.3 eq) was added to the reaction, which was allowed to proceed for 24 h at room temperature. After reaction completion, as determined by TLC, EtOAc (6 mL) was added to the reaction mixture. The organic layer was washed twice with both NaHCO3 solution (10 mL) and brine (10 mL), and then dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and then purified by silica gel flash column chromatography to afford the product as a

1 colorless viscous liquid (107 mg, 51.4 % yield); Rf = 0.79 (30 % EtOAc : Hexane); H

NMR (600 MHz, CDCl3): δ 7.33-7.22 (m, 33H, aromatic), 7.18-7.16 (m, 2H, aromatic),

6.24 (dd, 1H, J = 10.3, 1.3, H-5’), 5.96 (dd, 1H, J = 10.3, 1.8, H-6’), 5.42 (d, 1H, J = 3.5,

H-1), 5.1 (m, 1H, benzylic), 4.87 (m, 3H, benzylic), 4.76 (m, 1H, benzylic), 4.66 (s, 2H, benzylic), 4.53 (m, 5H, benzylic), 4.45 (m, 1H, benzylic), 4.38 (d, 1H, J = 12.1, benzylic),

4.2 (m, 3H, H-1’, H-2’), 4.11 (s, 2H, H-7a’, 7-b’), 4.06 (m, 2H, H-3, H-5), 3.63 (m, 2H, H-

13 4, H-6a), 3.52 (m, 2H, H-2, H-6b), 2.03 (s, 3H, CH3); C NMR (150 MHz, CDCl3): δ

169.85, 139.42, 138.80, 138.58, 138.38, 138.21, 138.05, 137.91, 130.95, 129.42, 128.41,

128.38, 128.33, 128.19, 128.08, 127.98, 127.97, 127.71, 127.67, 127.60, 127.52, 127.44,

127.42, 127.06, 126.98, 98.40, 81.75, 81.26, 81.18, 80.01, 79.67, 77.74, 77.37, 75.47,

75.11, 74.74, 73.47, 73.09, 72.12, 70.91, 69.51, 68.68, 21.90. mass spectrum (HRMS),

+ + m/z= 1033.4484 (M+Na) C64H66O11 requires 1033.4503 (M+Na) .

(1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-O- benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol (35)

To a solution of 34 (100 mg, 0.099 mmol) in EtOAc (4 mL), bis(benzonitrile)palladium

(II) chloride (5 mg, 0.013 mmol, 10 mol %) was added, and the reaction was allowed to stir under refluxing conditions for 36 h. The solvent was evaporated under reduced pressure, and purification was performed by silica gel flash column chromatography

(eluent: 7% EtOAc:Hexane) to afford the product as a colorless viscous liquid. Rf = 0.78

1 (30 % EtOAc : Hexane); H NMR (600 MHz, CDCl3): δ 7.34-7.2 (m, 33H, aromatic), 7.15

(m, 2H, aromatic), 5.98 (m, 1H, H-5’), 5.65 (t, 1H, J = 4.5X2, H-6’), 5.63 (d, 1H, J = 3.7,

H-1), 4.96 (d, 1H, J = 11.6, benzylic), 4.91 (d, 1H, J = 11.1, benzylic), 4.82 (d, 2H, J = 9.4, benzylic), 4.73 (d, 1H, J = 11.7, benzylic), 4.59 (m, 6H, benzylic, H-3’), 4.45 (m, 2H, benzylic), 4.37 (d, 1H, J = 12.1, benzylic), 4.29 (d, 1H, J = 12.7, H-7a’), 4.23 (dd, 1H, J =

9.2, 6.2, H-2’), 3.98 (m, 2H, H-3, H-7b’), 3.91 (m, 1H, H-5), 3.73 (dd, 1H, J = 9.3, 3.8, H-

1’), 3.69 (t, 1H, J = 9.5, H-4), 3.57 (td, 2H, J = 9.4, 3.3, H-2’, H-6a), 3.42 (dd, 1H, J = 10.8,

13 1.7, H-6b), 2.13 (s, 3H, CH3); C NMR (150 MHz, CDCl3): δ 170.84, 140.45, 138.88,

138.76, 138.62, 138.16, 138.12, 138.00, 128.49, 128.46, 128.43, 128.23, 128.16, 127.98,

127.93, 127.89, 127.86, 127.82, 127.79, 127.76, 127.74, 127.68, 127.41, 127.01, 122.92,

97.06, 82.27, 79.79, 79.57, 77.96, 75.65, 75.07, 74.64, 73.74, 73.66, 73.10, 72.45, 72.45,

72.13, 71.27, 70.45, 68.35, 66.15, 21.40 mass spectrum (HRMS), m/z= 1033..499 (M+Na)+

+ C64H66O11 requires 1033.450 (M+Na) .

A small piece of sodium was added to this viscous product in methanol (8 mL), and the reaction was allowed to run for 40 min at room temperature under an N2 atmosphere. The reaction completion was monitored by TLC, and the reaction mixture was neutralized to a

94 pH of approximately 7 with an Amberlite resin. The resin was filtered off, followed by solvent evaporation under reduced pressure, and purification was performed by silica gel flash column chromatography (eluent: 20% EtOAc:Hexane) to afford product 35 as a colorless viscous liquid. Yield: 58 mg, 60% over two steps; Rf = 0.38 (30 % EtOAc :

1 Hexane); H NMR (600 MHz, CDCl3): δ 7.35-7.12 (m, 35H, aromatic) 5.98 (d, J = 2.8 Hz,

1H, H-5’), 5.36 (d, J = 3.7 Hz, 1H, H-1), 4.80 (dd, J = 11.3, 2.8 Hz, 2H, benzylic), 4.75

(dd, J = 11.5, 9.2 Hz, 2H, benzylic), 4.71 – 4.64 (m, 3H, benzylic), 4.60 – 4.52 (m, 2H, benzylic), 4.43 (m, 8H, H-3’,benzylic), 4.33 – 4.28 (m, 1H, H-7a’), 4.18 (dd, J = 6.9, 4.0

Hz, 1H, H-2’), 3.95 (m, 3H, H-3, H-5, H-7b’), 3.70 (m, 2H, H-1’, H-4), 3.60 (dd, J = 10.7,

3.0 Hz, 1H, H-6a), 3.57 (dd, J = 9.9, 3.7 Hz, 1H, H-2), 3.45 (dd, J = 10.6, 1.9 Hz, 1H, H-

13 6b); C NMR (150 MHz, CDCl3): δ 138.70, 138.46, 138.35, 138.23, 138.03, 137.94,

137.32, 136.09, 128.44, 128.40, 128.35, 128.32, 128.28, 128.02, 127.91, 127.86, 127.75,

127.73, 127.70, 127.69, 127.57, 127.52, 127.02, 97.75, 82.00, 79.61, 76.81, 75.37, 74.89,

74.00, 73.83, 73.49, 71.96, 71.77, 71.19, 64.79, 29.30 mass spectrum (HRMS), m/z=

+ + 991.418 (M+23) C62H64O10 requires 991.439 (M+23) .

3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)bicyclo[4.1.0]heptane-2-ol (36)

Under N2 atmosphere, ZnEt2 (0.431 mL, 0.51 mmol, 15 eq.) 15% by wt. in Toluene solution was added to a cooled dry Toluene (3 mL). The mixture was stirred at -15 oC for 10 min and then CH2I2 (26 µL, 0.68 mmol, 20 eq.) was added dropwise to the reaction mixture.

After 10 min, TFA (4.3 µL,0.0578mmol,1.7eq.), was added dropwise to the cooled solution following which the cooling bath was removed and the reaction mixture was stirred at room temperature for 5 min. To the resultant mixture, a solution of compound 35 (35 mg, 0.034 95 mmol) in dry Toluene (3 mL) was added and the reaction mixture was stirred at room temperature for 15 h. The reaction was quenched by the addition of aq. HCl (10%) and then diluted with EtOAc (10 mL). After separation, the organic layer was washed with

NaHCO3 solution (2X 10 mL) and brine (2X 10 mL), dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and purification was performed by flash column chromatography on silica gel (15% EtOAc:Hexane) to afford product as colorless

1 viscous liquid (0.22 mg, 61.9% yield); Rf = 0.38 (30 % EtOAc : Hexane); H NMR (600

MHz, CDCl3) δ 7.35 – 7.29 (m, 19H, aromatic), 7.28 – 7.21 (m, 10H, aromatic), 7.16 (m,

6H, aromatic), 5.13 (d, J = 3.6 Hz, 1H, H-1), 4.79 (ddd, J = 35.7, 18.5, 10.8 Hz, 4H), 4.61

(ddd, J = 21.5, 19.1, 9.7 Hz, 4H), 4.52 – 4.39 (m, 7H), 4.29 (d, J = 11.3 Hz, 1H), 4.05 (d,

J = 10.3 Hz, 1H, H-7a’), 4.00 – 3.95 (m, 1H, H-5), 3.93 (t, J = 9.4 Hz, 1H, H-3), 3.78 (dd,

J = 6.8, 4.6 Hz, 1H), 3.74 (dd, J = 10.5, 3.2 Hz, 1H, H-6a), 3.68 (t, J = 12 Hz, 1H, H-4),

3.62 (dd, J = 6.8, 4.5 Hz, 1H), 3.54 (ddd, J = 9.8, 7.5, 5.1 Hz, 2H, H-2, H-6b), 2.68 (d, J =

10.3 Hz, 1H, H-7b’), 2.47 (s, 1H), 1.57 (s, 2H), 1.40 – 1.34 (m, 2H), 0.93 – 0.88 (m, 1H),

13 0.44 (q, J = 7.7 Hz, 1H). C NMR (151 MHz, CDCl3) δ 138.81, 138.65, 138.58, 138.29,

138.19, 137.93, 128.38, 128.36, 128.33, 128.30, 128.02, 128.00, 127.85, 127.78, 127.72,

127.63, 127.47, 127.43, 127.39, 127.32, 98.77, 81.88, 80.06, 78.92, 78.01, 77.83, 77.24,

77.03, 76.82, 76.62, 76.10, 75.38, 75.14, 73.53, 73.16, 72.81, 72.37, 72.26, 70.96, 68.48,

64.40, 29.73, 27.16, 24.36, 9.43 mass spectrum (HRMS), m/z= 1005.492 (M+23)+

+ C63H66O10 requires 1005.455 (M+23) .

3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)bicyclo[4.1.0]heptane-2-(3,5-difluorophenoxy) (37)

After a suspension of NaH (6.11 mg, 0.152 mmol, 4.5 eq.) in mineral oil (60 %) was washed twice with 5 mL hexane, it was transferred into dry DMF (6 mL) in a round-bottom flask. To this mixture, a solution of compound 36 (30 mg, 0.030 mmol) in 2 mL of DMF was added dropwise, and the resulting mixture was stirred for 30 min. Potassium benzoate

(9.6 mg, 0.06 mmol, 2 eq.) was then added, and stirring was continued for 30 min, during which time 1,3,5 trifluorobenzene was slowly added. After 2 h, the reaction was quenched by the addition of NH4Cl. Following the addition of brine (8 mL), the organic layer was extracted with diethyl ether (10 mL), and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and purification was performed by silica gel flash column chromatography (eluent: 7% EtOAc:Hexane) to afford the product as a colorless

1 viscous liquid (17 mg, 51.5% yield); Rf = 0.8 (30 % EtOAc : Hexane); H NMR (600 MHz,

CDCl3) δ 7.37 – 7.29 (m, 17H, aromatic), 7.28 – 7.21 (m, 16H, aromatic), 7.14 – 7.12 (m,

2H, aromatic), 6.49 (dd, J = 9.0, 2.1 Hz, 2H, ), 6.43 (tt, J = 8.9, 2.1 Hz, 1H), 5.24 (d, J =

3.6 Hz, 1H, H-1), 4.94 (d, J = 10.9 Hz, 1H), 4.88 – 4.78 (m, 5H), 4.72 (d, J = 12.0 Hz, 1H),

4.67 – 4.62 (m, 1H), 4.62 – 4.38 (m, 8H), 4.07 – 3.99 (m, 2H, H-3, H-7a’), 3.98 – 3.93 (m,

1H, H-5), 3.83 (dd, J = 9.3, 6.4 Hz, 1H), 3.73 – 3.61 (m, 3H, H-4, H-6a), 3.54 (dd, J = 9.8,

3.6 Hz, 1H , H-2), 3.49 (dd, J = 10.4, 1.9 Hz, 1H, H-6b), 2.61 (d, J = 10.1 Hz, 1H, H-7b’),

1.40 – 1.34 (m, 2H), 1.09 (t, J = 5.7 Hz, 2H), 0.93 – 0.88 (m, 3H), 0.48 (dd, J = 9.3, 5.4

13 Hz, 1H); C NMR (151 MHz, CDCl3) δ 138.95, 138.77, 138.43, 138.26, 137.84, 128.41,

128.38, 128.29, 128.24, 128.20, 128.07, 128.01, 127.88, 127.79, 127.75, 127.72, 127.64,

127.59, 127.55, 127.50, 127.45, 127.39, 127.23, 99.56, 98.73, 81.79, 80.52, 79.87, 77.88,

77.24, 77.03, 76.82, 75.60, 75.54, 75.22, 73.95, 73.54, 72.98, 72.74, 72.58, 71.80, 70.88,

68.44, 29.72, 28.35, 22.75, 14.15, 10.42. mass spectrum (HRMS), m/z= 1095.4851

+ + (M+23) C69H68F2O10 requires 1095.4814 (M+23) .

2-(3,5-difluorophenoxy)-6-(hydroxymethyl)-5-O-(2’,3’,4’,6’-tetra-ol-α-D- glucopyranosyl) bicyclo[4.1.0]heptane-3,4-diol (C1)

Compound 37 (10 mg, 0.009 mmol) was dissolved in 3 mL of MeOH, and 5% Pd/C on carbon (8 mg) was added. The mixture was stirred for 4 h under a hydrogen atmosphere (1 atm). The catalyst was filtered through a plug of Celite® 545 and washed with methanol

(5 mL). The combined filtrate and washings were concentrated to dryness afford C1 as a highly viscous liquid (3 mg, quantitative yield); 1H NMR (600 MHz, MeOD) δ 6.66 (dd, J

= 9.2, 2.2 Hz, 2H), 6.52 (tt, J = 9.2, 2.3 Hz, 1H), 5.22 (d, J = 3.8 Hz, 1H), 4.21 – 4.11 (m,

2H), 3.84 – 3.65 (m, 4H), 3.56 – 3.52 (m, 1H), 3.51 – 3.47 (m, 1H), 3.06 (q, J = 7.3 Hz,

1H), 1.34 – 1.30 (m, 1H), 1.28 – 1.22 (m, 1H), 1.04 – 0.99 (m, 3H), 0.49 (dd, 1H, J = 9.4,

5.4 Hz), 13C NMR (150 MHz, MeOD): δ 161.90, 161.66, 161.44, 101.48, 99.17, 98.97,

73.80, 73.71, 72.94, 72.85, 71.14, 70.21, 30..36, 21.26, 13.03, 8.77; mass spectrum

+ + (HRMS), m/z= 487.1401 (M+23) C20H26F2O10 requires 487.1392 (M+23) .

(1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-O- benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (38)

To a cooled (0 oC) solution of 33A (0.100 g, 0.103 mmol) in THF (4 mL), 150 µL of

KHMDS (1 M in THF) was added dropwise, and the reaction mixture was stirred for 1 h at the same temperature. Afterwards, BnBr (25 µL, 0.206 mmol, 2 eq) was added to the 98 reaction and allowed to proceed for 2 h at room temperature. After reaction completion, as determined by TLC, EtOAc (6 mL) was added to the reaction mixture. The organic layer was washed twice with NaHCO3 solution (10 mL) and twice with brine (10 mL), and then dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and purification was performed by silica gel flash column chromatography to afford the product

1 as a colorless viscous liquid (71 mg, 65% yield); Rf = 0.81 (30 % EtOAc : Hexane); H

NMR (600 MHz, CDCl3) δ 7.33 – 7.26 (m, 11H), 7.24 – 7.17 (m, 21H), 7.15 (d, J = 6.3

Hz, 6H), 7.10 (dd, J = 7.5, 1.9 Hz, 2H), 6.04 (dd, J = 10.2, 1.9 Hz, 1H, H-1’), 5.68 (dd, J

= 10.3, 1.9 Hz, 1H, H-7’), 5.39 (d, J = 3.5 Hz, 1H, H-1), 5.07 (d, J = 11.8 Hz, 1H), 4.89 –

4.75 (m, 3H), 4.73 – 4.68 (m, 1H), 4.68 – 4.60 (m, 4H), 4.54 – 4.37 (m, 6H), 4.30 (dd, J =

12.1, 5.7 Hz, 1H), 4.21 – 4.15 (m, 1H), 4.13 (dd, J = 7.4, 4.2 Hz, 2H), 4.11 – 3.99 (m, 2H,

H-3, H-5), 3.84 (d, J = 9.1 Hz, 1H, H-6a’), 3.64 (dd, J = 17.8, 8.6 Hz, 1H, H-4), 3.53 –

3.45 (m, 3H, H-2, H-6a, H-6b’), 3.37 (dd, J = 10.6, 1.9 Hz, 1H, H-6b). 13C NMR (151

MHz, CDCl3) δ 139.55, 139.07, 138.75, 138.62, 138.25, 137.95, 131.84, 130.04, 128.39,

128.31, 128.23, 128.12, 128.05, 127.93, 127.77, 127.66, 127.52, 127.44, 127.40, 127.23,

126.84, 99.20, 82.09, 81.99, 80.31, 79.78, 78.74, 77.88, 77.64, 77.25, 77.03, 76.82, 75.54,

74.71, 74.62, 73.39, 73.10, 72.73, 72.45, 72.17, 70.75, 68.26, 67.03. mass spectrum

+ + (HRMS), m/z= 1059.5044 (M+H) C69H70O10 requires 1059.5042 (M+H) .

(1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-O- benzyl-α-D-glucopyranosyl)cyclohexane-1,2,3,4-tetrol-β-(5,6)-diol (39)

To a cooled (0 oC) solution of 38 (60 mg, 0.056 mmol) in EtOAc:acetonitrile (1:1), 2 mL of a solution containing NaIO4 (16 mg, 0.078 mmol, 1.4 eq.) and a catalytic amount of

Ru(III) chloride dissolved in 2 mL of water were added dropwise. The reaction was stirred at 0 oC for 2 h, and reaction progress was monitored by TLC. After completion, the reaction was quenched with 6 mL of 10% Na2S2O3 solution. The organic layer was extracted 3 times with 10 mL with EtOAc, and then collected and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by silica gel flash

1 column chromatography (47 mg, 75% yield); Rf = 0.32 (30 % EtOAc : Hexane); H NMR

(600 MHz, CDCl3) δ 7.38 – 7.00 (m, 40H), 5.83 (d, J = 3.9 Hz, 1H, H-1), 5.06 (d, J = 11.0

Hz, 1H), 4.85 – 4.27 (m, 16H), 4.27 – 4.22 (t, J = 2.46 Hz, 1H, H-7), 4.22 – 3.91 (m, 3H,

H-1’, H-3’), 3.87 (m, 2H, H-3, H-4’), 3.80 (m, 1H), 3.77 (q, H-2’, J = 9.6 Hz, 1H), 3.64

(m, 2H, H-4, H-6a), 3.53 (dd, J = 10.4, 1.8 Hz, 1H, H-5), 3.48 (dd, J = 9.7, 3.9 Hz, 1H, H-

13 2), , 2.72 (d, J = 2.1 Hz, 1H), 2.23 – 2.15 (m, 1H); C NMR (151 MHz, CDCl3) δ 139.05,

138.54, 138.40, 138.33, 137.94, 137.47, 128.54, 128.50, 128.42, 128.35, 128.31, 128.19,

128.13, 128.06, 128.00, 127.94, 127.86, 127.79, 127.75, 127.59, 127.30, 127.10, 126.80,

126.61, 125.89, 97.36, 83.41, 82.84, 81.89, 79.96, 79.36, 77.68, 77.24, 77.03, 76.82, 76.03,

75.64, 75.33, 75.01, 73.87, 73.63, 73.44, 73.07, 71.57, 71.25, 68.65, 68.44, 66.76. mass

+ + spectrum (HRMS), m/z= 1093.5121 (M+H) C69H72O12 requires 1093.5102 (M+H) .

2,3,5-tri-O-benzyl-6-C-[(benzyloxy)methyl]-4-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)hexahydro-1,3,2-β-dioxathiole 2,2-dioxide (40)

Compound 39 (45 mg, 0.041 mmol) was dissolved in DCM (3 mL), and triethylamine (23

µL, 0.164 mmol, 4 eq.) was added at 0 oC, followed by the addition of thionyl chloride (3 eq.) over a 5-minute period. After reaction completion, diethyl ether and cold water were added to the reaction mixture. The organic layer was washed with 10 mL brine, collected,

100 and then dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure and then the crude product was subjected to a high vacuum to remove residual triethylamine.

o This oil was dissolved in 2 mL of CCl4:acetonitrile (1:1) and cooled to 0 C. To this cooled solution, NaIO4 (1.4 eq) and Ru(III)chloride (0.07 eq) dissolved in 2 mL water were added dropwise, and the reaction was stirred at 0 oC for 3 h. Diethyl ether (8 mL) and water (10 mL) were added to the reaction, and the organic layer was extracted 3 times with 10 mL diethyl ether. The combined organic extracts were dried over anhydrous Na2SO4, and solvent was evaporated under reduced pressure, and the product was purified by silica flash

1 column chromatography (27 mg, 60% yield); Rf = 0.83 (30 % EtOAc : Hexane); H NMR

(600 MHz, CDCl3) δ 7.39 – 7.06 (m, 40H), 5.36 (d, J = 5.5 Hz, 1H), 5.20 – 5.03 (m, 1H),

4.95 – 4.85 (m, 1H), 4.78 (ddd, J = 23.8, 16.9, 8.7 Hz, 4H), 4.66 (dd, J = 26.1, 14.2 Hz,

3H), 4.62 – 4.50 (m, 3H), 4.47 (dd, J = 11.1, 8.1 Hz, 3H), 4.38 – 4.30 (m, 64H), 4.29 –

4.17 (m, 2H), 4.17 – 4.07 (m, 1H), 3.99 (s, 1H), 3.83 – 3.69 (m, 2H), 3.66 – 3.55 (m, 2H),

13 3.50 (dt, J = 27.3, 13.7 Hz, 2H), 0.94 – 0.87 (m, 1H). C NMR (151 MHz, CDCl3) δ 138.24,

138.20, 138.04, 137.77, 137.64, 137.61, 137.19, 137.05, 128.52, 128.49, 128.45, 128.39,

128.34, 128.27, 128.22, 128.13, 128.09, 128.00, 127.98, 127.87, 127.81, 127.68, 127.37,

127.27, 126.26, 97.04, 81.62, 80.07, 78.99, 78.81, 77.64, 75.38, 75.27, 75.00, 74.75, 73.63,

+ 73.44. mass spectrum (HRMS), m/z= 1155.5177 (M+H) C69H70O14S requires 1155.4559

(M+H)+.

101

2,3,5-tri-hydroxy-6-C-[(hydroxy)methyl]-6-O-(α-D-glucopyranosyl)hexahydro-1,3,2-

β-dioxathiole 2,2-dioxide (41)

Compound 40 (10 mg, 0.008 mmol) was dissolved in 3 mL of MeOH, and then 5% Pd/C on carbon (8 mg) was added, and the mixture was stirred for 16 h under a hydrogen atmosphere (1 atm). The catalyst was filtered through a plug of Celite® 545 and washed with methanol (3 mL). The combined filtrate and washings were concentrated to dryness to afford 41 as a white solid (2.5 mg, quantitative yield); 1H NMR (600 MHz, MeOD) δ

5.23 (d, J = 3.89 Hz, 1H), 4.83 (dt, J = 11.5, 3.6 Hz, 2H), 3.98 – 3.92 (m, 2H), 3.88 – 3.81

(m, 4H), 3.73 – 3.68 (m, 3H), 3.46 – 3.43 (m, 2H). 13C NMR (151 MHz, MeOD) δ 101.56,

80.95, 77.51, 73.70, 73.30, 73.06, 72.88, 70.63, 70.18, 63.02, 61.32, 60.57, 58.43, 48.16.

+ + mass spectrum (HRMS), m/z= 457.0628 (M+23) C13H22O14S requires 457.0645 (M+23) .

102

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124

Appendix A

Supporting information-Chapter 2

1H NMR Ethyl 2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate

(9)……………………………………………………………………………………… 129

13C NMR Ethyl 2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate

(9)……………………………………………………………………………………… 130

1H NMR Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(10)…………………………………………………………………………………….. 131

13C NMR Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(10)…………………………………………………………………………………….. 132

1H NMR Ethyl 2-acetamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(21)…………………………………………………………………………………….. 133

13C NMR Ethyl 2-acetamido-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylate

(21)…………………………………………………………………………………….. 134

1H NMR Ethyl-2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(22)……………………………………………………………………………………. 135

125

13C NMR Ethyl-2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(22)………………………………………………………………………………….…. 136

1H NMR Ethyl 2-(perfluorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxylate (23)……………………………………………………………………….. 137

13C NMR Ethyl 2-(perfluorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxylate (23)………………………...... 138

1H NMR Ethyl-2-(3,5-bis(trifluoromethyl)benzaimdo)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate (24)…………………………………..…. 139

13C NMR Ethyl-2-(3,5-bis(trifluoromethyl)benzaimdo)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate (24)……………………………………… 140

1H NMR 2-amino-N-butyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (29)………………………………………………………………………. 141

13C NMR 2-amino-N-butyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (29)………………………………………………………………………. 142

1H NMR 2-amino-N-benzyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (30) ..…………………………………………………………………….. 143

13C NMR 2-amino-N-benzyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (30) ……………………………………………………………………… 144

1H NMR 2-amino-6-ethyl-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-

3-carboxamide (32)………………………………………………………………….… 145

126

13C NMR 2-amino-6-ethyl-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-

3-carboxamide (32)…………………………………………………………………… 146

1H NMR N-(adamantan-1-yl)-2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (33)……………………………………………………………………… 147

13C NMR N-(adamantan-1-yl)-2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-

3-carboxamide (33)……………………………………………………………………. 148

1H NMR 2-amino-N-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(34)…………………………………………………………………………………….. 149

13C NMR 2-amino-N-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(34)…………………………………………………………………………………….. 150

1H NMR 2-amino-N-benzyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(35)…………………………………………………………………………………….. 151

13C NMR 2-amino-N-benzyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(35)…………………………………………………………………………………….. 152

1H NMR 2-amino-N-(4-methylbenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (36)……………………………………………………………………… 153

13C NMR 2-amino-N-(4-methylbenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (36)……………………………………………………………………… 154

1H NMR 2-amino-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (37)………………………………………………………………………. 155

13C NMR 2-amino-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (37)………………………………………………………………………. 156 127

1H NMR N-(adamantan-1-yl)-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (C1)………………………………...……………………………………. 157

13C NMR N-(adamantan-1-yl)-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (C1)……………………………………………………………………… 158

1H NMR 2-Amino-6-ethyl-N-(4-methylbenzyl)-4,5,6,7-tetrahydrothieno [2,3-c]pyridine-

3-carboxamide (31)……………………………………………………………………. 159

13C NMR 2-Amino-6-ethyl-N-(4-methylbenzyl)-4,5,6,7-tetrahydrothieno [2,3-c]pyridine-

3-carboxamide (31)……………………………………………………………………. 160

128

1H NMR Ethyl 2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxylate (9)

129

13C NMR Ethyl 2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxylate (9)

130

1H NMR Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (10)

131

13C NMR Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (10)

132

1H NMR Ethyl 2-acetamido-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylate (21)

133

13C NMR Ethyl 2-acetamido-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylate

(21)

134

1H NMR Ethyl-2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(22)

135

13C NMR Ethyl-2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

(22)

136

1H NMR Ethyl 2-(perfluorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxylate (23)

137

13C NMR Ethyl 2-(perfluorobenzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxylate (23)

138

1H NMR Ethyl2-(3,5-bis(trifluoromethyl)benzaimdo)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate (24)

139

13C NMR Ethyl2-(3,5-bis(trifluoromethyl)benzaimdo)-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate (24)

140

1H NMR 2-amino-N-butyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (29)

141

13C NMR 2-amino-N-butyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (29)

142

1H NMR 2-amino-N-benzyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (30)

143

13C NMR 2-amino-N-benzyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide (30)

144

1H NMR 2-amino-6-ethyl-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3- c]pyridine-3-carboxamide (32)

145

13C NMR 2-amino-6-ethyl-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrothieno[2,3- c]pyridine-3-carboxamide (32)

146

1H NMR N-(adamantan-1-yl)-2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3- c]pyridine-3-carboxamide (33)

147

13C NMR N-(adamantan-1-yl)-2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3- c]pyridine-3-carboxamide (33)

148

1H NMR 2-amino-N-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (34)

149

13C NMR 2-amino-N-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (34)

150

1H NMR 2-amino-N-benzyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(35)

151

13C NMR 2-amino-N-benzyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

(35)

152

1H NMR 2-amino-N-(4-methylbenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (36)

153

13C NMR 2-amino-N-(4-methylbenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (36)

154

1H NMR 2-amino-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (37)

155

13C NMR 2-amino-N-(2-methoxybenzyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (37)

156

1H NMR N-(adamantan-1-yl)-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (38)

157

13C NMR N-(adamantan-1-yl)-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxamide (38)

158

1H NMR 2-Amino-6-ethyl-N-(4-methylbenzyl)-4,5,6,7-tetrahydrothieno [2,3- c]pyridine-3-carboxamide (31)

159

13C NMR 2-Amino-6-ethyl-N-(4-methylbenzyl)-4,5,6,7-tetrahydrothieno [2,3- c]pyridine-3-carboxamide (31)

160

Appendix B

Supporting information-Chapter 3

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-iodo-6-deoxy-α-D-glucopyranosyl-(1→1)-α-

D-glucopyranoside (9)…………………………………………………………………. 164

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-iodo-6-deoxy-α-D-glucopyranosyl-(1→1)-

α-D-glucopyranoside (9)………………………………………………………………. 165

1H NMR 2 ,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-α-D- glucopyranosyl-(1→1)-α-D-glucopyranoside (10)……………………………………. 166

13C NMR 2 ,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-α-D- glucopyranosyl-(1→1)-α-D-glucopyranoside (10)……………………………………. 167

31P NMR 2 ,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-α-D- glucopyranosyl-(1→1)-α-D-glucopyranoside (10)………………………...... 168

1H NMR 6-(phosphonic acid)-6-deoxy-α,α’-trehalose (4)…………………………….. 169

13C NMR 6-(phosphonic acid)-6-deoxy-α,α’-trehalose (4)……………………………. 170

31P NMR 6-(phosphonic acid)-6-deoxy-α,α’-trehalose (4)……………………………. 171

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-carbaldehyde-α-D-glucopyranosyl-(1→1)-α-

D-glucopyranoside (12)……………………………………………………………….. 172

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-carbaldehyde-α-D-glucopyranosyl-(1→1)-

α-D-glucopyranoside (12)…………………………………………………………….. 173

161

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-α-D-gluco-6-Deoxy-6- dimethoxyphosphinylmethylene-α,α’-trehalose (13)……………………….…………. 174

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-α-D-gluco-6-Deoxy-6- dimethoxyphosphinylmethylene-α,α’-trehalose (13)………………………………….. 175

31P NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-α-D-gluco-6-Deoxy-6- dimethoxyphosphinylmethylene-α,α’-trehalose (11)………………………………….. 176

1H NMR 6-(methylenephosphonic acid)-α,α’-trehalose (5)…………………………… 177

13C NMR 6-(methylenephosphonic acid)-α,α’-trehalose (5)…………………………... 178

31P NMR 6-(methylenephosphonic acid)-α,α’-trehalose (5)…………………………… 179

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-azido-α,α’-trehalose

(15)…………………………………………………………………………………….. 180

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-azido-α,α’-trehalose

(15)…………………………………………………………………………………….. 181

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-amino-α,α’-trehalose

(16)…………………………………………………………………………………….. 182

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-amino-α,α’-trehalose

(16)…………………………………………………………………………………….. 183

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-dibenzylphosphoramidate)-α,α’- trehalose (17)………………………………………………………………………….. 184

162

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-dibenzylphosphoramidate)-α,α’- trehalose (17)………………………………………………………………………….. 185

1H NMR 6-(phosphoramidic acid)-α,α’-trehalose (6)…………………………………. 186

13C NMR 6-(phosphoramidic acid)-α,α’-trehalose (6)………………………………… 187

31P NMR 6-(phosphoramidic acid)-α,α’-trehalose (6)…………………………………. 188

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-ethynyl-α,α’-trehalose

(18)…………………………………………………………………………………….. 189

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-ethynyl-α,α’-trehalose

(18)…………………………………………………………………………………….. 190

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-vinyl-α,α’-trehalose

(19)…………………………………………………………………………………….. 191

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-vinyl-α,α’-trehalose

(19)…………………………………………………………………………………….. 192

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-oxiranyl-α,α’-trehalose

(20)…………………………………………………………………………………….. 193

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-oxiranyl-α,α’-trehalose

(20)…………………………………………………………………………………….. 194

1H NMR 6-Deoxy-6-oxiranyl-α,α’-trehalose (7)……………………………………… 195

13C NMR 6-Deoxy-6-oxiranyl-α,α’-trehalose (7)…………………………………….. 196

163

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-iodo-6-deoxy-α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside

(9)

164

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-iodo-6-deoxy-α-D-glucopyranosyl-(1→1)-α-D- glucopyranoside (9)

165

1H NMR 2 ,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-α-D-glucopyranosyl-(1→1)-α-D- glucopyranoside (10).

166

13C NMR 2 ,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-α-D-glucopyranosyl-(1→1)- α-D-glucopyranoside (10)

167

31P NMR 2 ,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-methylphosphonate-6-deoxy-α-D-glucopyranosyl-(1→1)- α-D-glucopyranoside (10).

168

1H NMR 6-(phosphonic acid)-6-deoxy-α,α’-trehalose (4)

169

13C NMR 6-(phosphonic acid)-6-deoxy-α,α’-trehalose (4)

170

31P NMR NMR 6-(phosphonic acid)-6-deoxy-α,α’-trehalose (4)

171

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-carbaldehyde-α-D-glucopyranosyl-(1→1)-α-D- glucopyranoside (12).

172

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-carbaldehyde-α-D-glucopyranosyl-(1→1)-α-D- glucopyranoside (12).

173

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-α-D-gluco-6-Deoxy-6-dimethoxyphosphinylmethylene-α,α’- trehalose (13)

174

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-α-D-gluco-6-Deoxy-6-dimethoxyphosphinylmethylene-α,α’- trehalose (13)

175

31P NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-α-D-gluco-6-Deoxy-6-dimethoxyphosphinylmethylene-α,α’- trehalose (11).

176

1H NMR 6-(methylenephosphonic acid)-α,α’-trehalose (5)

177

13C NMR 6-(methylenephosphonic acid)-α,α’-trehalose (5)

178

31P NMR 6-(methylenephosphonic acid)-α,α’-trehalose (5)

179

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-azido-α,α’-trehalose (15).

180

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-azido-α,α’-trehalose (15). s

181

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-amino-α,α’-trehalose (16).

182

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-amino-α,α’-trehalose (16).

183

1H 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-dibenzylphosphoramidate)-α,α’-trehalose (17).

184

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-Deoxy-6-dibenzylphosphoramidate)-α,α’-trehalose (17).

185

1H NMR 6-(phosphoramidic acid)-α,α’-trehalose (6)

186

13C NMR 6-(phosphoramidic acid)-α,α’-trehalose (6)

187

31P NMR 6-(phosphoramidic acid)-α,α’-trehalose (6)

188

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-ethynyl-α,α’-trehalose (18)

189

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-benzyl-6-deoxy-6-ethynyl-α,α’-trehalose (18)

190

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-vinyl-α,α’-trehalose (19)

191

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-vinyl-α,α’-trehalose (19)

192

1H NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-oxiranyl-α,α’-trehalose (20)

193

13C NMR 2,2’,3,3’,4,4’,6’-Hepta-O-acetyl-6-Deoxy-6-oxiranyl-α,α’-trehalose (20)

194

1H NMR 6-Deoxy-6-oxiranyl-α,α’-trehalose (7)

195

13C NMR 6-Deoxy-6-oxiranyl-α,α’-trehalose (7)

196

Appendix C

Supporting information-Chapter 4

1H NMR Methyl-2-(((benzyloxy)carbonyl)amino)-5-hydroxybenzoate

(2a)…………………………………………………………………………………….. 202

13C NMR Methyl-2-(((benzyloxy)carbonyl)amino)-5-hydroxybenzoate

(2a)…………………………………………………………………………………….. 203

1H NMR Methyl 2-(((benzyloxy)carbonyl)amino)-5-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)benzoate (3a)…………………………………………………………. 204

13C NMR Methyl 2-(((benzyloxy)carbonyl)amino)-5-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)benzoate (3a)…………………………………………………………. 205

1H NMR Benzyl 4-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)phenyl)carbamate

(3b)…………………………………………………………………………………….. 206

13C NMR Benzyl 4-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)phenyl)carbamate

(3b)…………………………………………………………………………………….. 207

1H NMR Benzyl 4-(3’,4’,5’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohexylcarbamate

(3d)……………………………………………. 208

13C NMR Benzyl 4-(3’,4’,5’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohexylcarbamate

(3d)……………………………………………. 209

197

1H NMR 2-amino-5-(α-D-glucopyranosyl)benzoic acid

(4a)…………………………………………………………………………………….. 210

13C NMR 2-amino-5-(α-D-glucopyranosyl)benzoic acid

(4a)…………………………………………………………………………………….. 211

1H NMR Methyl-2-amino-5-(α-D-glucopyranosyl)benzoate

(4b)…………………………………………………………………………………….. 212

13C NMR Methyl 2-amino-5-(α-D-glucopyranosyl)benzoate

(4b)…………………………………………………………………………………….. 213

1H NMR (4-aminophenyl)-α-D-glucopyranoside (4c)……………………………. 214

13C NMR (4-aminophenyl)-α-D-glucopyranoside (4c)…………………………… 215

1H NMR (α-D-glucopyranosyl)benzoic acid (4d)………………………………… 216

13C NMR (α-D-glucopyranosyl)benzoic acid (4d)……………………………….. 217

1H NMR (4-aminocyclohexyl)-α-D-glucopyranoside (4e)………………………….. 218

13C NMR (4-aminocyclohexyl)-α-D-glucopyranoside (4e)………………………… 219

1H NMR Benzyl(3-(3-hydroxy-2,5-bis(hydroxymethyl)-4(-O-α-D- glucopyranosyl)pyrrolidin-1-yl)propyl)carbamate (14)……………………………….. 220

13C NMR Benzyl(3-(3-hydroxy-2,5-bis(hydroxymethyl)-4(-O-α-D- glucopyranosyl)pyrrolidin-1-yl)propyl)carbamate (14)……………………………….. 221

1H NMR 2-((-1-(3-aminopropyl)-4-hydroxy-2,5-bis(hydroxymethyl)pyrrolidin-3-yl)-α-D- glucopyranoside (15)………………………………………………………………….. 222

198

13C NMR 2-((-1-(3-aminopropyl)-4-hydroxy-2,5-bis(hydroxymethyl)pyrrolidin-3-yl)-α-

D-glucopyranoside (15)……………………………………………………………….. 223

1H NMR 3,4,7-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluchept-1-enone (31)…………………………………………………………………. 224

13C NMR 3,4,7-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluchept-1-enone (31)…………………………………………………………………. 225

1H NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluco-octa-1,7-dienitol (32A)…………………………….…………………………… 226

13C NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluco-octa-1,7-dienitol (32A)………………………………………………………….. 227

1H NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-L-ido- octa-1,7-dienitol (32B)………………………………………………………………… 228

13C NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-L- ido-octa-1,7-dienitol (32B)……………………………………………………………. 229

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-

O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33A)………………….. 230

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33A)……………. 231

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-

O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33B)………………….. 232

199

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33B)…………….. 233

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-4-O-acetyl-3-O-

(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol

(34)…………………………………………………………………………………….. 234

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-4-O-acetyl-3-O-

(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol

(34)…………………………………………………………………………………..… 235

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-6-O-acetyl-3-O-

(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol………. 236

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-6-O-acetyl-3-O-

(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol..…….. 237

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’-tetra-

O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol (35)………………….… 238

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol (35)………...……. 239

1H NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)bicyclo[4.1.0]heptane-2-ol (36)………………..…………………….. 240

13C NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)bicyclo[4.1.0]heptane-2-ol (36)………………………………………. 241

1H NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)bicyclo[4.1.0]heptane-2-(3,5-difluorophenoxy) (37)………………… 242

200

13C NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)bicyclo[4.1.0]heptane-2-(3,5-difluorophenoxy) (37)………………… 243

1H NMR 2-(3,5-difluorophenoxy)-6-(hydroxymethyl)-5-O-(2’,3’,4’,6’-tetra-ol-α-D- glucopyranosyl) bicyclo[4.1.0]heptane-3,4-diol (C1)…………………………………. 244

13C NMR 2-(3,5-difluorophenoxy)-6-(hydroxymethyl)-5-O-(2’,3’,4’,6’-tetra-ol-α-D- glucopyranosyl) bicyclo[4.1.0]heptane-3,4-diol (C1)…………………………………. 245

1H NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (38)……………… 246

13C NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (38)……………… 247

1H NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohexane-1,2,3,4-tetrol-β-(5,6)-diol (39)…… 248

13C NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohexane-1,2,3,4-tetrol-β-(5,6)-diol (39)…..... 249

1H NMR 2,3,5-tri-O-benzyl-6-C-[(benzyloxy)methyl]-4-O-(2’,3’,4’,6’-tetra-O-benzyl-α-

D-glucopyranosyl)hexahydro-1,3,2-β-dioxathiole-2,2-dioxide (40)………………….. 250

13C NMR 2,3,5-tri-O-benzyl-6-C-[(benzyloxy)methyl]-4-O-(2’,3’,4’,6’-tetra-O-benzyl-α-

D-glucopyranosyl)hexahydro-1,3,2-β-dioxathiole-2,2-dioxide (40)….……………….. 251

1H NMR 2,3,5-tri-hydroxy-6-C-[(hydroxy)methyl]-4-O-(α-D-glucopyranosyl)hexahydro-

1,3,2-β-dioxathiole-2,2-dioxide(41)…………………………………….……………... 252

13C NMR 2,3,5-tri-hydroxy-6-C-[(hydroxy)methyl]-4-O-(α-D- glucopyranosyl)hexahydro-1,3,2-β-dioxathiole-2,2-dioxide(41)……………………... 253

201

1H NMR Methyl-2-(((benzyloxy)carbonyl)amino)-5-hydroxybenzoate (2a)

202

13C NMR Methyl-2-(((benzyloxy)carbonyl)amino)-5-hydroxybenzoate (2a)

203

1H NMR Methyl 2-(((benzyloxy)carbonyl)amino)-5-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)benzoate(3a)

204

13C NMR Methyl 2-(((benzyloxy)carbonyl)amino)-5-(2’,3’,4’,6’-tetra-O-benzyl-α-D- glucopyranosyl)benzoate(3a)

205

1H NMR Benzyl 4-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)phenyl)carbamate (3b)

206

13C NMR Benzyl 4-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)phenyl)carbamate (3b)

207

1H NMR Benzyl 4-(3’,4’,5’,6’-tetra-O-benzyl-α-D- glucopyranosyl)cyclohexylcarbamate (3d)

208

13C NMR Benzyl 4-(3’,4’,5’,6’-tetra-O-benzyl-α-D- glucopyranosyl)cyclohexylcarbamate (3d)

209

2-amino-5-(α-D-glucopyranosyl)benzoic acid (4a)

210

2-amino-5-(α-D-glucopyranosyl)benzoic acid (4a)

211

1H NMR Methyl-2-amino-5-(α-D-glucopyranosyl)benzoate (4b)

212

13C NMR Methyl 2-amino-5-(α-D-glucopyranosyl)benzoate (4b)

213

1H NMR (4-aminophenyl)-α-D-glucopyranoside (4c)

214

13C NMR (4-aminophenyl)-α-D-glucopyranoside (4c)

215

1H NMR 3-(α-D-glucopyranosyl)benzoic acid (4d)

216

13C NMR 3-(α-D-glucopyranosyl)benzoic acid (4d)

217

1H NMR (4-aminocyclohexyl)-α-D-glucopyranoside (4e)

218

13C NMR (4-aminocyclohexyl)-α-D-glucopyranoside (4e)

219

1H NMR Benzyl(3-(3-hydroxy-2,5-bis(hydroxymethyl)-4(-O-α-D- glucopyranosyl)pyrrolidin-1-yl)propyl)carbamate (14)

220

13C NMR Benzyl(3-(3-hydroxy-2,5-bis(hydroxymethyl)-4(-O-α-D- glucopyranosyl)pyrrolidin-1-yl)propyl)carbamate (14)

221

1H NMR 2-((-1-(3-aminopropyl)-4-hydroxy-2,5-bis(hydroxymethyl)pyrrolidin-3-yl)- α-D-glucopyranoside (15)

+ [M+H]

222

13C NMR 2-((-1-(3-aminopropyl)-4-hydroxy-2,5-bis(hydroxymethyl)pyrrolidin-3-yl)- α-D-glucopyranoside (15)

223

1H NMR 3,4,7-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluchept-1-enone (31)

224

13C NMR 3,4,7-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluchept-1-enone (31)

225

1H NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluco-octa-1,7-dienitol (32A)

226

13C NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-D- gluco-octa-1,7-dienitol (32A)

227

1H NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-L- ido-octa-1,7-dienitol (32B)

228

13C NMR 3,4,9-Tri-O-benzyl-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)-L- ido-octa-1,7-dienitol (32B)

229

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33A)

230

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33A)

231

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33B)

232

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (33B)

233

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-4-O-acetyl-3-O- (2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (34)

234

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-4-O-acetyl-3-O- (2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (34)

235

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-6-O-acetyl-3-O- (2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol

236

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-6-O-acetyl-3-O- (2’,3’,4’,6’-tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol

237

1H NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol (35)

238

13C NMR (1D)-(1,3,4/2)-1,2-Di-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-4-ene-1,2,3,6-tetrol (35)

239

1H NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α- D-glucopyranosyl)bicyclo[4.1.0]heptane-2-ol (36)

240

13C NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α- D-glucopyranosyl)bicyclo[4.1.0]heptane-2-ol (36)

241

1H NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α- D-glucopyranosyl)bicyclo[4.1.0]heptane-2-(3,5-difluorophenoxy) (37)

242

13C NMR 3,4-Di(benzyloxy)-6-((benzyloxy)methyl)-5-O-(2’,3’,4’,6’-tetra-O-benzyl-α- D-glucopyranosyl)bicyclo[4.1.0]heptane-2-(3,5-difluorophenoxy) (37)

243

1H NMR 2-(3,5-difluorophenoxy)-6-(hydroxymethyl)-5-O-(2’,3’,4’,6’-tetra-ol-α-D- glucopyranosyl) bicyclo[4.1.0]heptane-3,4-diol (C1)

244

13C NMR 2-(3,5-difluorophenoxy)-6-(hydroxymethyl)-5-O-(2’,3’,4’,6’-tetra-ol-α-D- glucopyranosyl) bicyclo[4.1.0]heptane-3,4-diol (C1)

245

1H NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (38)

246

13C NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohex-5-ene-1,2,3,4-tetrol (38)

247

1H NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohexane-1,2,3,4-tetrol-β-(5,6)-diol (39)

248

13C NMR (1D)-(1,3,4/2)-1,2,4-tri-O-benzyl-4-C-[(benzyloxy)methyl]-3-O-(2’,3’,4’,6’- tetra-O-benzyl-α-D-glucopyranosyl)cyclohexane-1,2,3,4-tetrol-β-(5,6)-diol (39)

249

1H NMR 2,3,5-tri-O-benzyl-6-C-[(benzyloxy)methyl]-4-O-(2’,3’,4’,6’-tetra-O-benzyl-

α-D-glucopyranosyl)hexahydro-1,3,2-β-dioxathiole-2,2-dioxide (40)

250

13C NMR 2,3,5-tri-O-benzyl-6-C-[(benzyloxy)methyl]-4-O-(2’,3’,4’,6’-tetra-O- benzyl-α-D-glucopyranosyl)hexahydro-1,3,2-β-dioxathiole-2,2-dioxide (40)

251

1H NMR 2,3,5-tri-hydroxy-6-C-[(hydroxy)methyl]-4-O-(α-D- glucopyranosyl)hexahydro-1,3,2-β-dioxathiole-2,2-dioxide(41)

252

13C NMR 2,3,5-tri-hydroxy-6-C-[(hydroxy)methyl]-4-O-(α-D- glucopyranosyl)hexahydro-1,3,2-β-dioxathiole-2,2-dioxide(41)

253

Appendix D

Journal Permission to reproduce material

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