Mycobacterial infection is a major health concern. High drug resistivity of the mycobacterium is due to its multi-layered, thick hydrophobic waxy cell wall components, consisting of cross-linked peptidoglycan (PG), mycolyl arabinogalactan (mAG) and lipoarabinomannan (LAM) polysaccharides. These polysaccharides are composed of arabinose and galactose in the furanose form and mannose in the pyranose form. The high waxy hydrophobic components of the mycobacterial cell wall acts as a barrier for most hydrophilic antibacterial agents. Enzymes responsible for the biosynthesis of polysaccharides of mAG and LAM are arabinosyl transferase (AraT), galactosyl transferase (GlfT) and mannosyl transferase (ManT). In the absence of furanoside derivatives of D-arabinose and D-galactose in mammalian systems, inhibitors based on these sugars arise an interest. Upon realizing structural characteristics of cell wall polysaccharides, the chemical syntheses of such polysaccharides were reported. Biological studies of synthetic arabinomannan and arabinogalactan oligosaccharides were performed, in order to identify their effects in enzymatic, as well as, mycobacterial growth assays. Chapter 1 of the thesis describes the structural features of mAG and LAM polysaccharides. Chemical synthesis of oligosaccharides related to mycobacterial cell wall components and their effects of mycobacterial growth and enzymatic assays are discussed. In my research program, synthesis and studies of oligosaccharides pertaining to mycobacterial cell wall components were undertaken. Monovalent and bivalent glycolipids 1 and 2 (Figure 1), containing arabinofuranoside trisaccharide as the sugar head group, were synthesized and their effects on the growth of M. smegmatis strain were studied. In the presence of arabinan glycolipids, retardation of the growth of M. smegmatis was observed and the inhibitory activity was found to be specific with glycolipids containing arabinofuranoside head groups. Glycolipid with maltosyl sugar and arabinofuranoside trisaccharide without lipid chains, did not affect the mycobacterial growth. Continuing the effort, tri- and tetrasaccharide of arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that 3 was inhibiting the growth of the mycobacterium, whereas in the case of 4, inhibition was found to be less when compared to 3. Relative inhibitions of mycobacterial growth by synthetic glycolipids 1-4, at a concentration 200 µg/mL, were found to be in a varying degrees, ranging from 16 % in the case of 4 and 65 % in the case of 3. Figure 1. Molecular structures of arabinan and arabinomannan oligosaccharides 1-7. Following mycobacterial growth inhibition studies, surface plasmon resonance studies of synthetic oligosaccharides were performed, in order to identify their interactions with mycobacterial cell lysates. Amine tethered glycosides 5-7 (Figure 1) were synthesized and immobilized onto SPR sensor chip through amine coupling methodology. From SPR studies, it was found that the binding affinity was higher with cell lysates from motile strains than non-motile strain. Among various arabinomannans, glycoside 5, presenting two mannose units showed higher affinity than 6 and 7, having no or one mannose unit, respectively. Chapter 2 of the thesis provides details of synthesis, biological and biophysical studies of arabinan and arabinomannan glycolipids. Continuing the synthesis and studies with arabinose oligosaccharides, a linear tetra-, hexa and octasaccharide glycolipids, containing α-(1→5) linkages (10-12), as well as, a branched heptasaccharide containing α-(1→2) and α-(1→5) linkages (14) between the arabinofuranoside units (Figure 2) were synthesized. In addition to glycolipids, oligosaccharides without alkyl chains (8, 9 and 13) were also prepared. Synthesis was performed using trichloroacetimidate and Figure 2. Molecular structures of linear and branched arabinan derivatives 8-14. thioglycosides as glycosyl donors. Synthesis of linear oligosaccharide derivatives 8-12 was achieved by iterative glycosylation and deprotection strategies. Branched heptasaccharide derivatives 13 and 14 were synthesized by using block glycosylation method, wherein two fold excess of arabinose disaccharide was reacted with a suitably protected arabinose trisaccharide. Upon synthesis, molecular modeling studies were performed to identify the conformational behavior of arabinan glycolipids. Conformational studies were performed in three steps, namely, (i) dihedral scan (ii) conformational search and (iii) molecular dynamics. Dihedral scan was performed to assess favorable torsion angles at each glycosidic linkage with respect to overall conformation of the molecule. Monte-Carlo conformational search was performed to obtain the lowest energy structure of arabinan glycolipids. Relative orientations of lipidic portions and sugar portions were identified for linear and branched arabinan glycolipids. The least energy conformations of 10, 11, 12 and 14 are shown in Figure 3. In the case of linear molecules 10, 11 and 12, alkyl chains and arabinofuranoside portion did not phase segregate, whereas in the case of branched glycolipid 14, the alkyl chains were observed to move away from the sugar moieties. Molecular dynamic calculations were performed for the lowest energy structure, in order to evaluate the torsion angles in the trajectory. Following the synthesis and conformational analysis of the arabinan glycolipids, surface plasmon resonance studies were performed to assess their interactions with a host protein, namely, pulmonary surfactant protein-A (SP-A). For the interaction studies, SP-A was immobilized on to the CM-5 sensor chip using amine coupling method. Varying concentrations of arabinan glycolipids 10, 11, 12 and 14 and oligosaccharides 8, 9 and 13 were used as analytes. Responses from the surface of SP-A were subtracted from that of ethanolamine to eliminate the non-specific interactions. Primary sensorgrams were fitted using 1:1 Langmuir model to obtain the kinetic parameters of the interactions. Specificities and relative binding affinities of arabinan oligosaccharides interacting with SP-A are presented in Table 1. The affinities between Figure 3. Lowest energy structures of glycolipids 10, 11, 12 and 14 derived from molecular modeling studies. arabinan oligosaccharides and SP-A were found in the range of 4.9-47x103 M-1. Among the series, branched arabinan oligosaccharides 13 and 14 showed higher Ka values than the linear arabinan glycolipids. The association rate constants (kon) were generally higher for the oligosaccharides without lipidic chain, whereas, the dissociation rate constants (koff) were slower with oligosaccharides having lipidic chains. Faster kon was also associated with a faster koff for oligosaccharides without the lipidic chains. For the glycolipids, a relatively slower koff was found to be the trend. In the case of branched heptasaccharide derivatives, glycolipid 14 showed higher binding constant than heptasaccharide with a thiocresyl group at the reducing end 13. Chapter 3 of the thesis presents the synthesis, conformational analysis and SPR studies of linear and branched arabinan glycolipids. Table 1. Kinetic parameters of the interactions between arabinose derivatives 8-14 and SP-A. Compound kon (M-1s-1) kd (s-1) (104) Ka (M-1) (10-3) χ2 12 3.9 7.91 4.9 8.3 11 1.5 3.98 3.77 2.9 10 0.384 0.22 17.5 6.7 14 27.3 5.79 47.2 4.5 8 11.3 6.14 18.4 2.3 9 23.3 11.6 20.1 2.4 13 53.6 17.9 29.9 5.4 Upon assessing the biophysical studies of the α-arabinofuranoside glycolipids, an effort was undertaken to prepare glycolipids containing β-arabinofuranoside linkages and to study their conformational and biophysical properties. Arabinan glycolipids 15 and 16 (Figure 4), containing β-(1→2), β-(1→3) and β-(1→5) linkages between furanoside units were synthesized to compare the properties with the corresponding synthetic α-arabinan glycolipids. Incorporation of β-arabinofuranoside linkages in 15 and 16 was achieved using low temperature activation of silyl substituted glycosyl donor 17 (Figure 4), with NIS and AgOTf. The configurations in 15 and 16 were confirmed through 1H-1H COSY, 1H-13C HMQC NMR techniques. During the synthesis of 15 and 16, stereoselective incorporation of two β-Araf linkages on a single furanoside unit was achieved for the first time. Conformational studies of 15 and 16 were conducted similar to α-arabinan glycolipids, as above, to identify most favorable conformations of inter-ring, as well as, overall conformation of the molecule. The interactions between the SP-A and β-arabinofuranoside glycolipids 15 and 16 were also assessed with the aid of SPR technique. The analysis showed that the affinities of glycolipids 15 and 16 to SP-A were found to be relatively lower when compared to α-arabinofuranoside glycolipids. Synthesis and studies of β-arabinofuranoside glycolipids are described in chapter 4 of the thesis. Figure 4. Molecular structures of β-arabinofuranoside glycolipids 15 and 16. In summary, the present thesis describes synthesis, conformational and biophysical studies of synthetic arabinan and arabinomannan glycolipids. Monovalent and bivalent arabinan, tri- and tetrasaccharide arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that arabinan and arabinomannan glycolipids inhibited the growth of the mycobacterium. The inhibitory activity is specific with the arabinan and arabinomannan glycolipids and the glycolipids with higher arabinose composition were found to be better inhibitors for mycobacterial growth. The interactions of mycobacterial cell lysates with arabinomannan compounds were evaluated through SPR technique. Linear tetra-, hexa-, octa- and branched heptasaccahride arabinan glycolipids containing α-Araf linkages between furanoside units were synthesized. Molecular modeling studies of arabinan glycolipids were performed, in order to identify their lowest energy conformations. Biophysical studies of linear and branched arabinan glycolipids were conducted to assess their interactions with pulmonary surfactant protein-A (SP-A) through surface plasmon resonance technique. Syntheses, conformational and biophysical studies were extended further to β-arabinofuranoside glycolipids. Overall, the thesis provides synthesis, conformational, biological and biophysical studies of a series of lipoarabinomannan oligosaccharides. The results provide a possibility to evolve newer types of glycolipids that can act as inhibitors of mycobacterial growth. (For structural formula pl see the hard copy)

Pathogenesis originating from mycobacterial invasion on host cells is prevalent and is a major challenge in efforts towards overcoming the burden of mycobacterial diseases. Complex architecture of mycobacterium cell wall includes an assortment of glycolipids, phospholipids, glycopeptidolipids (GPLs), peptidoglycans, arabinogalactans, lipoarabinomannans and mycolic acid. Aided by thick cell wall envelope, mycobacteria are known to survive in hostile environment. As most antibiotics target the log phase of the bacteria, bacterial survival is also largely dependent on its stationary phase. Mycobacteria have evolved colonization by means of biofilm formation in the stationary phase, so as to survive under stress and hostile conditions. Biofilms are the specialized form of phenotype which makes bacteria several fold resistant to antibiotics. Development of inhibitors against biofilms remains a challenge due to the poor permeability of molecules and coordination among cells. The first part of Chapter 1 of the thesis describes the details of formation of biofilm in the stationary phase of bacteria and understanding the molecular level details for making the strategies to overcome antidrug resistance of mycobacteria. Among the cyclic hosts, cyclodextrins are prominent. Due to their unique structural and physical properties, cyclodextrins can form inclusion complexes with a wide range of guest molecules. Although synthetic modifications of cyclodextrins through hydroxy groups are very common, modifications at backbone continue to be a challenge. Backbone modified cyclodextrins using different organic moieties were developed and their altered cavity properties were explored in many instances. Chemical synthesis of cyclic oligosaccharides is, in general, involved (i) a cyclo-oligomerization of linear oligosaccharide precursor and (ii) an one-pot polycondensation of appropriately designed monomer under suitable reaction conditions. The second part of Chapter 1 deals with a literature survey of skeletal modification of cyclodextrins, their synthesis and binding abilities with different guest molecules. In my research programme, synthesis and studies of oligosaccharide glycolipids relevant to mycobacterial cell wall were undertaken. Arabinofuranoside trisaccharide glycolipids, containing β-anomeric linkages at the non-reducing ends and double hexadecyloxy lipid moieties, interconnected to the sugar moiety through a glycerol core, were synthesized (Figure 1). Arabinan trisaccharides 1 with lipidic chain and 3 without lipidic chain comprise β-(1→2), β-(1→3) anomeric linkages at the non-reducing end, whereas in the case of arabinan trisaccharides 2 and 4, β-(1→2), β-(1→5) linkages are present between the furanoside units. In the scheme of synthesis of trisaccharide glycolipids, monosaccharide derivative and lipidic portions were individually prepared first and were assembled subsequently to secure the target glycolipids. Incorporation of β-arabinofuranoside linkages in trisaccharide arabinofuranosides 1-4 was achieved by low temperature activation of silyl group protected conformationally locked thioglycoside donor 5 (Figure 1), in the presence of N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf). Figure 1. Molecular structures of trisaccharides 3, 4 and glycolipids 1, 2 with β-arabinofuranoside linkages at the non-reducing end and glycosyl donor 5. Following the synthesis, the efficacies of synthetic glycolipids to interact with surfactant protein A (SP-A) were assessed by using surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. SP-A, a lung innate immune system component, is known to bind with glycolipids present in the cell surface of a mycobacterial pathogen. From the analysis of SPR studies with glycolipids 1, 2 and SP-A, the association rate constants (ka) were found to be in the range of 0.3 to 0.85 M−1 s−1, whereas the dissociation rate constants (kd) were varied between 2.21 and 3.2×10−3 s−1. The equilibrium constants (Ka) values were in the range of 93 and 274 M−1. Trisaccharides 3 and 4, without lipidic chains, were also assessed for their efficacies to interact with SP-A. The association constants for 3 were found to be in the range of 2,470 to 9,430 M−1, whereas for the derivative 4, Ka values varied between 25,600 and 76,900 M−1. The association and equilibrium binding constants for 3 and 4 were found to be significantly higher when compared to glycolipids 1 and 2. In conjunction with our previous report, the present study shows that arabinofuranoside glycolipids, with β-anomeric linkages bind to SP-A with lesser extent as compared to α-anomers. Further, the studies of trisaccharides and glycolipids in mycobacterial growth and sliding motility assays were performed with model organism M. smegmatis and it was found that the synthetic compounds affected both growth and motility and the extent was lesser than that of α-anomeric glycosides and glycolipids. Chapter 2 of the thesis describes the details of synthesis, biophysical and biological studies of arabinan trisaccharide glycolipids, with β-anomeric linkages at the non-reducing end. Continuing the synthesis and studies of arabinan oligosaccharides, a linear arabinomannan pentasaccharide and heptasaccharide glycolipids 6 and 10, containing α-(1→2) and α-(1→3) linkages between core arabinofuranoside units, as well as, a branched arabinomannan pentasaccharide and heptasaccharide glycolipids 7 and 11, with α-(1→2) and α-(1→5) linkages between core arabinofuranoside units, were synthesized (Figures 2 and 3). Figure 2. Molecular structures of arabinomannan glycolipids 6 and 7 and the corresponding oligosaccharides 8 and 9. In addition to glycolipids, arabinomannan pentasaccharides without lipidic chain 8 and 9 and arabinomannan heptasaccharides without lipidic chain 12 and 13, were also synthesized. Synthesis was performed using trichloroacetimidate and thioglycosides as glycosyl donors. A block condensation methodology was adopted by which disaccharide donor and monosaccharide acceptor were chosen to assemble the pentasaccharide, by a two-fold glycosylation. Monosaccharide acceptors with and without lipidic chain were used in the glycosylations for the synthesis of glycolipids and pentasaccharides, respectively. Similarly, a trisaccharide thioglycoside donor and monosaccharide acceptors were chosen for the double glycosylation to synthesize heptasaccharides in the presence of NIS and AgOTf. Figure 3. Molecular structures of arabinomannan heptasaccharide glycolipids 10, 11 and corresponding heptasaccharides 12 and 13. Subsequent to synthesis, activities of pentasaccharide glycolipids were assayed on M. smegmatis bacterial growth, sliding motilities and also the effects on mycobacterial biofilms. Profound effects were observed with the synthetic compounds, to reduce the mycobacterial growth, sliding motilities and biofilm structures. Whereas reduction up to ~50% occurred on mycobacterial growth, as much as, 70% reduction in the motilities of the bacteria was observed in the presence of the synthetic glycolipids, at 100 µg mL-1 concentration. At the same concentration, 80–85% reduction in the biofilm was observed. These effects were more pronounced with branched glycolipids than linear analogues. Chapter 3 of the thesis presents the synthesis of linear and branched arabinomannan penta- and heptasaccharide glycolipids and biological studies of arabinomannan pentasaccharide glycolipids with M. smegmatis. Cyclodextrins, the most abundant naturally-occurring cyclic oligosaccharides, are valuable synthetic hosts, primarily as a result of their properties to form inclusion complexes with guest molecules. In spite of voluminous literature on the application of cyclodextrins, through modifications of hydroxy groups, modifications at the backbone continue to be a challenge. Skeletal modifications using aromatic, triazole, diyne, thioether and disulfide moieties were developed, that helped to alter the cavity properties of cyclodextrins. A programme was undertaken to synthesize backbone modified cyclic oligosaccharide, which was achieved using a monomer wherein a one carbon insertion is conducted at C4 of a pyranose, such that the hydroxy moiety at C4 is replaced with a hydroxymethyl moiety. In an approach, a linear trisaccharide monomer was anticipated to provide cyclic oligosaccharides in multiples of such a monomer. In the event, a trisaccharide linear monomer 14 was found to afford a cyclic trisaccharide macrocycle 15, as the major cyclo-oligomer (Scheme 1). Subsequent solid state structural studies show that the molecule confers a perfect trigonal symmetry in the P3 space group, in a narrow cone shape and a brick-wall type arrangement of molecules, such a geometry is hither-to unknown to a cyclic oligosaccharide (Figure 4). Furthermore, binding abilities of cyclic trisaccharide with few organic bases, such as 1-aminoadamantane and hexamethylenetetramine, was evaluated by the means of isothermal titration calorimetry and it was found that such a cyclic trisaccharide exhibits strong binding affinities towards 1-aminoadamantane in aqueous solutions, as compared to the same with naturally-occurring β-cyclodextrin. Scheme 1 Apart from cyclic trisaccharide, synthesis of cyclic tetrasaccharide 17, containing alternative anomeric α-(1→4) and β-(1→4) linkages was also undertaken by one-pot cyclo-oligomerization in the suitable reaction condition, from an activated disaccharide thioglycoside monomer 16, having β-(1→4) linkage at the non-reducing end (Scheme 2). Chapter 4 describes the synthesis of cyclic oligosaccharides 15 and 17, as well as, the details of solid state structure and binding studies of cyclic trisaccharide 15. Scheme 2 Figure 4. (a) Stick model of the crystal structure of 15, as viewed along the crystallographic c-axis; (b) trigonal view from crystal packing; (c) packing diagram crystal lattice, as viewed along the crystallographic b-axis, and without solvent inclusion and (d) packing diagram included with methanol (grey) and water (red) solvents, as viewed along the crystallographic c-axis. Hydrogen atoms are omitted for clarity in (c and d). In summary, the thesis presents (i) synthesis, biophysical and biological studies of synthetic arabinan and arabinomannan glycolipids, and (ii) synthesis, solid-state structural analysis and binding studies of glycosidic bond expanded cyclic oligosaccharides. Synthetic trisaccharide arabinofuranoside glycolipids containing β-anomeric linkages at the non-reducing end showed binding affinity towards pulmonary surfactant protein A, as assessed by surface plasmon resonance technique, with comparatively lower extent as compared to synthetic glycolipids having α-anomeric linkages. Linear and branched arabinomannan penta- and heptasaccharide glycolipids, having α-anomeric linkages were synthesized and biological studies with non-pathogenic strain M. smegmatis were conducted with pentasaccharide glycolipids. It was found that arabinomannan glycolipids inhibited the growth and sliding motility of mycobacteria. Importantly, disruption of biofilm and significant reduction in biofilm formation was observed in the presence of the synthetic glycolipids. Glycosidic bond expanded cyclic trisaccharide with anomeric α-(1→4) linkages and cyclic tetrasaccharide with alternative anomeric α-(1→4) and β-(1→4) linkages were prepared from suitably designed trisaccharide and disaccharide monomer, respectively, by cyclo-oligomerization. Solid-state structural analysis and binding studies of cyclic trisaccharide in solution by isothermal titration calorimetry were also conducted. Cyclic trisaccharide possessed a bowl shape and brick-wall type of arrangement in the solid-state structure, whereas it exhibited stronger binding affinity towards 1-aminoadamantane as compared to β-cyclodextrin in aqueous solution. Overall, the results presented in the thesis provide a possibility to develop new types of synthetic glycolipids that can act as inhibitors of biofilm formation of mycobacteria, as well as, to develop newer types of cyclic oligosaccharide synthetic hosts that can modify binding abilities towards various guest compounds.