Chapter-II, Section-B 2.2.0.

Introduction to saponins:Saponins are steroid or triterpenoid glycosides common in a large number of plants and plant products that play an important role in human and animal nutrition. Several biological effects have been ascribed to saponins (Sparg et al., 2004; Lacaille-Dubois 2005) and they are also believed to form the main constituents of many plant drugs and folk medicines and are considered responsible for numerous pharmacological properties (Liu and Henkel 2002). Saponins consist of a sugar moiety usually containing glucose, galactose, glucuronic acid, xylose, rhamnose or methylpentose, glycosidically linked to a hydrophobic aglycone (sapogenin) which may be triterpenoid or steroid in nature (Bruneton 1995; Oleszek and Bialy 2006). The aglycone may contain one or more unsaturated CC bonds. The oligosaccharide chain is normally attached at the C-3 position (monodesmosidic), but many saponins have an additional sugar moiety at the C-26 or C-28 position (bidesmosidic). The great complexity of saponin structure arises from the variability of the aglycone structure, the nature of the side chains and the position of attachment of these moieties on the aglycone (Vincken et al., 2007). Experiments demonstrating the physiological, immunological and pharmacological properties of saponins have provoked considerable clinical interest in these substances. Extensive research has been carried out into the membrane-permeabilising, cytotoxic and antitumor activity of saponins (Lacaille-Dubois 2000; Rao and Gurfinkel 2000; Sparg et al., 2004; Sun and Fang 2006; Bachran et al., 2008) and so far, thousands of homogeneous saponins have been isolated and characterized which display a tremendous structural diversity. Although saponins having potential biological activity, but they never become potential clinical candidate because of their hemolytic nature and furthermore still mechanism of action of this hemolytic cascade remains unclear. Hemolytic property of saponins discourages the development of the various therapeutic potentials of this type of abundant natural product (Francis et al., 2002). Despite of this side effects, saponins are also well known for activating mammalian immune system which has lead to significant interest in their potential as vaccine adjuvants (Skene and Sutton 2006). Saponins such as QuilA and QS-21 are potential immunostimulatory molecules (Kensil and Kammer 1998). Among all the saponins, QS-21 is the only candidate which has been tested in different clinical trials in various vaccine formulations (Bomford et al., 1992; 92

Chapter-II, Section-B Chapman et al., 2000; Moreno et al., 2000; Hancock et al., 1995; Britt et al., 1995). QS-21 stimulate both the Th1 immune response and the production of cytotoxic T-lymphocyte (CTL) against exogenous antigens and make them ideal as immune adjuvant for use in subunit vaccines and vaccines directed against intracellular pathogens as well as for therapeutic cancer vaccines (Takahashi et al., 1990; Soltysik et al., 1995). Quil A and others having similar structure with QS-21 are also explored by several researchers as ideal candidate for immune adjuvant. Meanwhile, reports during last decades (Barr et al., 1998; Press et al., 2000; Rivera et al., 2003; Sun et al., 2005; Xie et al., 2008; Sun et al., 2009) show that many saponins from other natural sources possess the adjuvant activity. Literature survey reaveled that the adjuvanticity of saponins depends on its amphipathic structure comprised of hydrophilic sugar side chain and lipophilic aglycone backbone (Oda et al., 2000; Oda et al., 2003). However, relatively little is known about the minimum critical structure of saponins required for these adjuvant functions. Recently, Sun et al studied the relationship between haemolytic and adjuvant activity and structure of protopanaxadiol and protopanaxatriol-type saponins from the roots of P. notoginseng. It was found that the number, length and position of sugar side chains as well as type and the linkage of glucosyl group in the structure of these saponins could not only affect the adjuvant potentials, but have significant effects on the nature of the immune responses (Sun et al., 2005; Sun et al., 2006). It has recently been reported that the number of sugar residues in the glycidic chains attached to C-3 of aglycone could affect the adjuvant activities of platycodigenin-type saponins and the adjuvant activity decreased with the increased number of monosaccharides of the glycidic moieties at the C-3 of the aglycone (Xie et al., 2008) and type and number of sugar units also effect the undesired hemolytic effect. There is no specific report in the literature regarding the correlations between the hemolytic and adjuvant activity of saponins, therefore the correlations between the hemolytic and adjuvant activity require further exploration through the generation of focused library of simplified saponins structures. In this direction, our interest is to explore the minimum structural requirements for saponins to become immune adjuvants with possible reduced hemolytic property. Among saponins, QS-21 is a well-known immune-adjuvant with complicated chemical structure different from that of general saponins (Press et al., 2000). Such special saponins (QS-21) cannot necessarily explain adjuvanticity or other immunological activities of general saponin. 93

Chapter-II, Section-B Like saponin many other immune adjuvants such as muramyl dipeptides, non-ionic block copolymers, and carbohydrate polymers have amphipathic structures and contain both hydrophilic and lipophilic parts responsible for physicochemical features and immune adjuvant activities. In addition, most of the saponins are isolated from natural source and most of the studies have often used these structurally unspecified samples. Literature report shows that the purity of a saponin sample affected the immune response. Generally, the structure activity relationship (SAR) studies on saponins have been hampered by the limited availability of single molecule saponin in each laboratory. These glycosides exist in such a microheterogeneous manner in plants that separation of each component in an appreciable amount is always a formidable task (Hostettmann and Marston 1995). In this direction, the developments of synthetic saponins are, therefore, well suited to the study of structure/function relationship.

2.2.1. Rationale behind the design and synthesis of saponins as immune-adjuvants:Saponins are well known for activating the mammalian immune system and generated significant interest due to their potential as vaccine adjuvants. The purpose of this study is to synthesize mono- and di-saccharides based saponins by joining chemical determinants of adjuvants within a single molecule and study their role for adjuvanticity. The strategies involved herein are based on the following features: Chemical synthesis is a realistic alternative to determine the availability of homogeneous saponins and also affording new opportunities for SAR studies (Wang and Bertozzi 2001). These novel saponins synthesized would encompass triterpenoids and steroids as aglycan backbone in order to explore the role of aglycan moiety in adjuvanticity. These novel designer saponins synthesized would encompass sugars like mannose because their receptors (Stahl and Ezekowitzt 1998; Leteux et al., 2000) are well established on immune cells which are responsible for inducing various types of protective immune responses. In this study, conjugates would have chemical determinants such as carbonyl group or specific sugar units (Bomford et al., 1992) probably involved in adjuvanticity. These novel saponin candidates encompassing various sugar units (shown in Scheme 1 and 6) assembled to aglycan moieties viz., dehydroepiendrosterone 1, cholesterol 2, oleonolic acid 3, 94

Chapter-II, Section-B lupeol 4, betulin 5, following trichloroacetimidate (TCA) method. This section deals with the studies directed towards the development of saponins as immune adjuvants.
O

OH Dehydroepiendrosterone 1

OH Cholesterol 2

HO O OH Oleonolic acid 3 Lupeol 4 OH Betulin 5 HO OH

Fig 1: Structure of selected steroids and triterpenoids

2.2.2. Present work:- Synthesis of mono- and di-saccharide saponins: In order to study the SAR and to develop simple saponin as potent vaccine-adjuvant, a number of triterpenoids and steroids based saponins have been synthesized. In this section, we describe the synthesis of saponins containing either a monosaccharide residue or a disaccharide residue and some of the conjugates contain functionality/chemical determinants such as carbonyl group and/or sugars (Rhodes 1989; Palatnik et al., 2004; Kensil 1996; Bomford 1992; Soltysik 1995) which further help in interaction with receptors present in the immune systems and thus improve the adjuvanticity of these saponin analogues. Initially, we have synthesized steroid based saponins analogs containing

monosaccharide residues viz., glucose, galactose and mannose by coupling of steroids viz., dehydroepiendrosterone 1 and cholesterol 2 as glycosyl acceptors with various trichloroacetimidates as glycosyl donor. Trichloroacetimidates 9a-c were easily prepared from their respective monosaccharides viz., glucose 6a, galactose 6b and mannose 6c following literature procedures (Scheme 1) for instance by treating commercially available glucose 6a (Fernndez-Herrera et al., 2009) with acetic anhydride in the presence of HClO4 and then selective deprotection of anomeric acetate by means of NH4OAc (Chittabonia et al., 2006) gave 2,3,4,6-tetra-O-acetyl--D-glucopyranose 8a. Finally, the formation of the corresponding 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl trichloroacetimidate 9a was performed by the treatment of 8a with trichloroacetonitrile in the presence of DBU (Schmidt 1986). Other glycosyl donors viz., 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl trichloroacetimidate 9b

95

Chapter-II, Section-B (Schmidt 1986; Eichler et al., 1999) and 2,3,4,6-tetra-O-acetyl--D-mannopyranosyl trichloroacetimidate 9c were also prepared in similar manner.
O HO
4

a OH AcO
4

b AcO OAc
4

O OH

c AcO
4

O O HN 9a -c CCl3

6a-c

7a-c

8a-c

Scheme 1: Synthesis of glycosyl donors 9a-c Reagent and conditions: a) AC2O, HClO4, 0 oC - rt, 95%; b) NH4OAc, rt, 90%; c) CCl3CN, DBU, CH2Cl2, rt, 90%.

Glycosylation of 1 with trichloroacetimidates 9a in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as the catalyst at -20 oC afforded -glycoside 10a (Fernndez-Herrera et al., 2009) in 65% yield (Scheme 2). The presence of the 2-O-acetyl group on donor 9a ensured the exclusive formation of the -anomer, which was confirmed by
1

H NMR. Finally, deacetylation with NaOMe in MeOH was performed to give the

corresponding saponins analogs 11a. The similar strategy has been followed for the coupling of 1 with 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl trichloroacetimidate 9b gave -glycoside 10b but the coupling of 1 with 2,3,4,6-tetra-O-acetyl-D-mannopyranosyl trichloroacetimidate 9c gave glycoside 10c with -linkage. The formation of -linkage in the synthesis of compound 10c is due to the presence of axial acetyl group at 2-position of donor 9c. Thereafter, de-protection of acetyl group of 10b and 10c with NaOMe and MeOH affording dehydroepiandrosterone saponin 11b and 11c respectively in good yield. Similar approach has been applied for the synthesis of structurally related cholesterol based saponins 11d-f. The linkages of the synthesized saponin analogs were confirmed by 1H NMR and furthermore, the structures of the synthesized saponin analogues were confirmed by
1 13

H NMR,

C NMR and mass spectroscopic data. In order to know the role of aglycan and

functional group on aglycon of saponin on adjuvant activity, we have chosen three triterpenoids viz, oleanolic acid 3, lupeol 4 and betulin 5 which are having functionality at C28 position which can be converted either into carbonyl group or utilized for the preparation of bidesmosidic saponins by functional group manipulation to explore the effect of position of functionality on immune adjuvant activity. Furthermore, exploiting the chemistry developed for the synthesis of the steroid based saponins, we could synthesize focused library of structurally related triterpenoids based saponin analogs 15 and 17. For the synthesis of oleanolic acid based saponins 15a-b, C-28 96

Chapter-II, Section-B carboxylic acid function of oleanolic acid 3 was first protected with benzyl ester 12 on treatment with BnBr and Et3N in dry THF, which was easily removed through catalytic hydrogenation because the double bond between C-12 and C-13 of oleanolic acid is inert to catalytic hydrogenation (Winterstein and Stein 1931). Thereafter, the glycosylation of 2,3,4,6tetra-O-acetyl-D-glucopyranosyl trichloroacetimidate 9a with benzyl ester 12 in the presence of TMSOTf lead to the formation of protected glycoside 14a with predominant -linkage in excellent yields. De-protection of acetyl group followed by debenzyaltion gave desired product 15a with 70% yield. The glycosylation of benzyl ester 12 with 2,3,4,6-tetra-O-acetylD-mannopyranosyl trichloroacetimidate 9c gave -glycoside 14b in good yield which on deprotection of acetyl and benzyl group gave required compound 15b. Likewise, lupeol 4 was also coupled with TCA 9b and 9c under standard glycosylation condition at room temperature followed by de-acetylation to afford -glycoside 17a and -glycoside 17b respectively in good yields.

HO O OH 3

BnBr, Et3N Bu4NI, THF, rt, 90%

BnO O 12 OH

Ac2O, Pyridine HO OH 5 13 DMAP, rt, 70% AcO OH

Scheme 3: Protection of C-28 of oleanolic acid and betulin

For the synthesis of betulin based saponins 17c and 17d, betulin 5 was first acetylated in order to avoid multiple glycosylation reactions at the C-28 position with Ac2O in pyridine to give betulin acetate 13. Betulin acetate 13 was coupled with the TCA 9a and 9c under the catalytic promotion of the TMSOTf in dry DCM to afford predominant protected -glycoside 16c and -glycoside 16d in good yield. Thereafter, de-protection of the acetyl groups with NaOMe and MeOH affording betulin based saponins 17c and 17d in isolable yields. For the synthesis of saponin analogs containing a disaccharide residue, in order to explore the role of number of sugar unit for adjuvant activity, we first tried the direct coupling of disaccharide donor 21 with select steroid (1, 2) and triterpenoid (3, 4, 5) based aglycans. The disaccharide donor 21 was prepared from commercially available disaccharide viz., 97

Chapter-II, Section-B lactose 18a and cellobiose 18b following literature procedure, in which disaccharide 18 was first treated with acetic anhydride in pyridine leading to synthesis of peracetyalted lactose (19, 90%), which is followed by selective deprotection of acetyl group at anomeric position with NH4OAc/DMF.
R R1
HO OH OH O O OH HO O OH OH

R a R1
AcO

OAc OAc O O OAc AcO O OAc OAc

18a R = OH, R1 = H 18b R = H, R1 = OH

19 b
OAc OAc O O OAc AcO O OH OAc

R R1
AcO

OAc OAc O O OAc AcO O O OAc

R R1
AcO

CCl3

21a R = OAc, R1 = H 21b R = H, R1 = OAc

HN

20

Scheme 6: Synthesis of disaccharide based glycosyl donor 18 Reagent and conditions: a) AC2O, HClO4, 0 oC - rt, 95%; b) NH4OAc, rt, 90%; c) CCl3CN, DBU, CH2Cl2, rt, 90%.

Finally,

trichloroacetimidate

derivatives

viz.,

2,3,4,6-tetra-O-acetyl--D21a

galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-2,2,2-trichloroacetimidate and

2,3,4,6-tetra-O-acetyl--D-glucopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-

2,2,2-trichloroacetimidate 21b were synthesized from the corresponding 1-OH sugars 20a and 20b respectively using trichloroacetonitrile (CCl3CN) in the presence of DBU in CH2Cl2 according to Schmidts procedure. Firstly, glycosylation of steroids 1 with 2,3,4,6-tetra-Oacetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-2,2,2trichloroacetimidate 21a as glycosyl donor under TMSOTF mediated standard condition affording predominantly protected -glycoside 22a. The formation of -glycosidic bond was again confirmed by 1H NMR. Deprotection of acetyl group of 22a with NaOMe and MeOH gave desired product 23a in good isolable yield. In a similar manner, the glycosylation of 1 and 2 with 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl-(14)-2,3,6-tri-O-acetyl-D21b and 2,3,4,6-tetra-O-acetyl--D-

glucopyranosyl-2,2,2-trichloroacetimidate

galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl -2,2,2-trichloroacetimidate 21a resspectively under standard Schmidt glycosylation approach followed by deprotection of acetyl group gave cholesterol based saponin 23b and dehydroepiendrosterone based saponin 98

Chapter-II, Section-B 23c in good yields. Each oleanane 12 and lupane type 4 triterpenoids were similarly coupled with 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl2,2,2-trichloroacetimidate 21a under standard Schmidt glycosylation approach resulting in the synthesis of peracetylated triterpenoids saponin analogs 24a and 24b respectively. The peracetylated triterpenoid saponin analogs 24a were first deacetylated followed by debenzylation to afford oleanolic acid based saponin 25a. Similarly, peracetylated triterpenoid saponin analogs 24b were deacetylated with NaOMe/MeOH which afforded lupeol based saponin 25b in good yields. Moreover, several researchers prepared steroids and triterpenoids based saponins analogs to study their effect on cytotoxic activity and hemolytic activity. However, no detailed immune adjuvant studies due to these saponin analogs reported. In this direction, triterpenoids based saponin containing disaccharide residue were also prepared in order to know the effect on adjuvant activity as well as hemolytic activity of triterpenoid-disaccharide saponins analogs in comparision with the steroid-disaccharide and steroid/triterpenoid-monosaccharide saponin analogs.

Scheme 2: Synthesis of steroid based saponins Reagent and conditions: a) TMSOTf, Molecular sieves 4 oA, Dry CH2Cl2, rt; b) NaOAc, MeOH, rt.

{
+ HN 1 or 2 O

O OAc

O O OAc

b O 11

O OH

CCl3 9a-c 10

99

Chapter-II, Section-B
Entry
O O

Reactant 1 or 2

Reactant 9
OAc OAc OAc CCl3 AcO O OAc O OAc O

Product (11)

a
O OH HN

OH O O OH OH OH

b
HN

HO O CCl3 OAc OAc O OH OAc OAc OAc CCl3 O O

OH

OH

OAc

c
O HN

O O

OH

OH OH OH

d O OH HN O CCl3 AcO O e O HN CCl3

OAc OAc OAc OAc O O OH OAc HO OAc OAc O OH OAc O OAc OAc OAc CCl3 O O OH OH OH OH OH O OH OH OH

OH

f HN

Table 1: Synthesis of dehydroepiendrosterone and cholesterol based saponins

Scheme 4: Synthesis of triterpenoid based saponins Reagent and conditions: a) TMSOTf, Molecular sieves 4 oA, Dry CH2Cl2, rt; b) (i) NaOAc, MeOH, rt (ii) H2/Pd(OH)2, MeOH, CH2Cl2, H2O, rt.

{
+ OH 4 or 12 or 13 HN O

O OAc

O O OAc O 15

O OH

CCl3 9 14

100

Chapter-II, Section-B
Reactant 12 Reactant (9) Product (15)

Entry

OAc a BnO O OH HN O CCl3 O OAc OAc OAc HO OH O O OH OH O OH

OAc b O HN CCl3 O

OAc OAc OAc HO O OH O

OH OH OH

Table 2: Synthesis of oleanolic acid based saponins

Scheme 5: Synthesis of triterpenoid based saponins Reagent and conditions: a) TMSOTf, Molecular sieves 4 oA, Dry CH2Cl2, rt; b) NaOAc, MeOH, rt.
Entry Reactant 4 or13 Reactant (9) Product (17)

{
+ OH 4 or 13 HN O
a 4 b c AcO 13 d

O OAc

O O OAc O 17

O OH

CCl3 9 16

AcO O O HN OH CCl3

OAc HO

OAc

OAc O

OH

O OH OH

OAc O O HN CCl3

OAc OAc OAc O OH OH OH OH

OAc O O HN OH OAc OAc OAc CCl3 HO O OH O OH OH OH CCl3 OAc OAc OAc HO O OH OAc O O HN OH OH O OH

Table 2: Synthesis of lupeol and betulin based saponins

101

Chapter-II, Section-B
AcO O O OAc AcO

AcO

R R1 OAc AcO O O O OAc AcO AcO

Scheme 7: Synthesis of steroid based saponins Reagent and conditions: a) TMSOTf, Molecular sieves 4 oA, Dry CH2Cl2, rt; b) NaOAc, MeOH, rt.
Glycosyl acceptor
O

c
O

Scheme 8: Synthesis of triterpenoid based saponins Reagent and conditions: a) TMSOTf, Molecular sieves 4 oA, Dry CH2Cl2, rt; b) (i) NaOAc, MeOH, rt; for 21a (ii) H2/Pd(OH)2, MeOH, CH2Cl2, H2O, rt.

{
+ OH 1 or 2
Cl3C O NH

O AcO

R R1 OAc

O
AcO

21

22

HO O O O OH HO

HO

{
O
HO

R R1 OH

23

Glycosyl donor
AcO O O OAc AcO OAc

Steroid based saponin analogs

O
OAc HO O O O OH HO OH HO OH

AcO O O AcO Cl3C NH

a
OH 1

O 21a
AcO AcO O O OAc AcO OAc OAc O HO

23a

O
HO O O OH HO HO OH OH

1
Cl3C

O AcO NH

O 21b
HO

23b
AcO O AcO O O OAc AcO HO OAc O O OH HO O OH AcO OAc HO OH

OH

Cl3C

NH

O 21a

HO

23c

Table 4: Synthesis of dehydroepiendrosterone and cholesterol based saponins

AcO AcO O O OAc AcO R R1 OAc AcO O O O OAc AcO AcO

{
+ OH 12 or 4 or 13
Cl3C O NH

O AcO

R R1 OAc

O
AcO

21

24

HO O O O OH HO

HO

{
O
HO

R R1 OH

25

102

Chapter-II, Section-B
Glycosyl acceptor Glycosyl donor
AcO O O OAc AcO OAc

Triterpenoid based saponin analogs

AcO

OAc HO HO O O O HO OH O HO O OH HO OH

a BnO
O 12 OH
Cl3C O

O AcO NH

21a
AcO O O O OAc AcO OAc O AcO OAc

25a

b
OH 4
Cl3C

O AcO NH

HO O O OH HO

HO

OH

21a

O HO

OH

25b

Table 5: Synthesis of triterpenoid based saponins

2.2.3. Biological activity:- Evaluation of immune adjuvant activity of synthetic saponins Considering the importance of saponins such as QS-21 and Quil A as potent adjuvants and to study the structureactivity relationship and to develop simple saponin as potent vaccine-adjuvant, a number of triterpenoids and steroids based saponins have been synthesized. Furthermore, these synthetic saponins also contain either a monosaccharide residue or a disaccharide residue and some of the conjugates contain functionality/ chemical determinants such as carbonyl group and/or sugars which further help in interaction with receptors present in the immune system and thus improve the adjuvanticity of these saponin analogues. In order to evaluate these monosaccharide and disaccharide based saponins (11a-11f, 15a-15b, 17a-17d, 23a-23c and 25a-25b) on immunomodulatory activity, preliminary splenocyte proliferation ex vivo studies were performed. Based on the preliminary results, detailed immune adjuvant activity of active analogs were studied in the presence of weak antigen ovalbumin by stimulating anti OVA IgG titre, neutralizing antibody (IgG1 and IgG2a) titer as well as the production of soluble mediators of a Th1 response (IL-2 and IFN-) and Th2 response (IL-4) and proliferation of T lymphocytes sub-sets (CD4/CD8). In all 16 monoand disaccharide based saponins for their possible immunomodulatory activity were tested. Among these, only monosacchaide based saponin such as dehydroepiendrosterone 3-O-Dmannopyranoside 11c exhibited potential immune adjuvant activity.

103

Chapter-II, Section-B 2.2.4. Results and Discussion:- Results Effect of monsaccharide and disaccharide based saponins on cell proliferation ex vivo To observe the effect on cell proliferation, splenocytes isolated from OVA sensitized mice were cultured with OVA and monsaccharide and disaccharide based saponins (0.01, 0.1, 1 and 10 g/ml) as shown in Table 6. Three anolgs viz., 11c, 11e and 25a significantly enhanced OVA-stimulated splenocyte proliferation compared to OVA treated group and Ova/alum was taken as positive control. Furthermore, among these three analogs, only monosaccharide based saponin such as dehydroepiendrosterone 3-O-D-mannopyranoside 11c were taken for detailed immune-adjuvant activity along with weak antigen ovalbumin. Effect of dehydroepiendrosterone 3-O-D-mannopyranoside 11c on OVA-specific antibody titre The OVA-specific IgG, IgG1, and IgG2a antibody titers in the serum were measured by indirect Elisa as shown in Fig 2 (A-C). The serum IgG titer in OVA-immunized mice was significantly enhanced by 11c at a dose of 100 g when compared with OVA only treated group. Compound 11c at a dose 100 g significantly enhanced the serum IgG1 titers in OVA immunized mice. Significant enhancements in OVA-specific serum IgG2a titers were also observed with 11c immunized mice at a dose of 100 g as compared with OVA only treated group. These findings indicated that dehydroepiendrosterone 3-O-D-mannopyranoside 11c significantly enhanced serum OVA-specific antibody production in mice immunized with OVA. Estimation of Th1 (IL-2 and IFN-) and Th2 (IL-4) cytokines in serum The effect of dehydroepiendrosterone 3-O-D-mannopyranoside 11c at a dose range of 1, 10 and 100 g on the release of Th1 (IFN-, IL-2) and Th2 (IL-4) cytokines in serum were shown in Fig 3 (A-C). These results indicate that compound dehydroepiendrosterone 3-O-Dmannopyranoside 11c significantly stimulated Th1 (IFN-, IL-2) cytokine release in a doserelated manner. Dehydroepiendrosterone 3-O-D-mannopyranoside 11c enhanced the IFN- and IL-2 cytokines at a dose of 100 g and 100 g respectively. These finding indicates that dehydroepiendrosterone 3-O-D-mannopyranoside 11c enhance Th1 type of immune response. Immunophenotyping of lymphocytes by flow cytometry The overall percentage of T-cell subsets CD4 and CD8 in the splenic lymphocyte population was estimated in the presence of dehydroepiendrosterone 3-O-D-mannopyranoside 104

Chapter-II, Section-B 11c (Fig 4). The effects of dehydroepiendrosterone 3-O-D-mannopyranoside 11c treated subpopulations were dose dependently increased compared to untreated group. The presence of dehydroepiendrosterone 3-O-D-mannopyranoside 11c significantly enhanced percentage of T-cell populations at the dose of 100 g. Discussion: A number of triterpenoids and steroids based saponins have been synthesized and screened for immune adjuvant potential in order to know the minimum structural requirement for saponins to act as immune adjuvant and to develop a potent simple saponin as immune adjuvant. Here, we also tried to correlate the effect of functionality/ chemical determinants such as carbonyl group and/or sugars on saponins and their immune responses. In order to evaluate the immune-adjuvant activity of steroid and triterpenoid based saponin analogs, splenocyte proliferation in ex vivo was evaluated. The results shown in Table 6 indicate that three anolgs viz., 11c, 11e and 25a enhanced the OVA-stimulated splenocyte proliferation at a dose of 10, 1 and 1 g respectively as compared to all other molecules. Among these analogs, only dehydroepiendrosterone 3-O-D-mannopyranoside 11c shown significant enhancement of OVA-stimulated splenocyte proliferation as compared to 11e and 25a. Carefully structure investigation revealed that 11c and 11e contain monosaccharide sugar unit and 25a contain disaccharide sugar unit and furthermore, recent finding of Xie et al. 2009 suggested that saponin contain monosccahride are less hemolytic than saponin contain disaccharide sugar unit. In our preliminary screening two saponin analogs 11c and 11e contain monosaccharide unit. Moreover, saponin analogs 11c contain mannose and carbonyl group which might help these analog to interact with the receptor present in the immune system. Based on the preliminary results and literature findings, dehydroepiendrosterone 3-O-Dthe

mannopyranoside 11c was taken for detailed in vivo experiments. In in-vivo experiments, dehydroepiendrosterone 3-O-D-mannopyranoside 11c was evaluated in the presence of OVA with or without aluminum hydroxide in BALB/c mice. Compound 11c strongly induces Th1 responses shown in Fig 2-4. We determined the possible effect of 11c on humoral immune response by measuring OVA-specific IgG titre and neutralizing antibody titers (IgG1 and IgG2a). The OVA-specific serum IgG, IgG1 and IgG2a antibody titers in the OVA-immunized mice were shown in Fig 2 (A-C). This compound 11c significantly increased the production of IgG titre IgG1 and IgG2a titre in the OVA immunized mice at a dose of 100 g. 105

Chapter-II, Section-B The addition of 11c at a suitable dose to the OVA immunized group increased the potency of the soluble mediators of Th1 (IL-2 and IFN-) response only when compared to the OVA only treated group. As shown in Fig 3 (A-C), 11c significantly increased the production of Th1 cytokines (IL-2 and IFN-) in the OVA immunized mice at a dose of 100 g. Furthermore, we evaluated the effect of dehydroepiendrosterone 3-O-D-mannopyranoside 11c on the population of cell surface marker like CD4/CD8. As shown in Fig 4, the 11c significantly enhanced the population of CD4/CD8, but the population of CD4 increased more as compared to CD8. In conclusion, our present study revealed that synthetic saponin having monosaccharide unit are more potent than saponin bearing a disaccharide unit. Moreover, saponins having receptor based functionally such as carbonyl and sugar such as mannose are more potent than any other saponins devoid 11c of these structural the Th1 features. response-

Dehydroepiendrosterone

3-O-D-mannopyranoside

enhanced

(characterized by the production of IgG2a, IFN- and IL-2) more significantly than Th2 responses (characterized by the production of IgG) and against OVA in mice. Furthermore, these analogs also significantly enhanced the population of cell surface marker CD4/CD8. Thus, dehydroepiendrosterone 3-O-D-mannopyranoside 11c is potent enhancers of Th1 responses. The present study opens a new direction for the development of designer saponins as potent vaccine adjuvants.

106

Chapter-II, Section-B

Treatment

Dose g/ml Control 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 N.T.

Cells + OVA Stimulation Mean SE Index 0.4430.031 0.5350.026 0.6430.031 0.6850.003 0.6620.013 0.5770.025 0.6740.034 0.6850.012 0.6770.002 0.5750.036 0.6510.033 0.7150.027 0.7910.026 N.T. 1.207 1.451 1.546 1.494 1.302 1.521 1.546 1.528 1.297 1.469 1.613 1.789 N.T. 10

Treatment

Dose g/ml

Cells + OVA Stimulation Mean SE Index

11a

17b

N.T.

N.T.

N.T.

11b

11

17c

N.T.

N.T.

N.T.

11c

12

17d

11d

13

23a

11e

11f

15a

15b

0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10

0.5770.025 0.6340.034 0.7050.012 0.6770.002 0.5820.017 0.6500.035 0.6820.046 0.6610.020 0.6900.011 0.5820.022 0.5220.040 0.5090.035 0.5840.035 0.6480.046 0.6950.020 0.6640.029

1.521 1.431 1.591 1.187 1.467 1.539 1.492 1.297 1.313 1.178 1.148 1.318 1.462 1.568 1.498 1.564

14

23b

0.01 0.1 1 10 0.01 0.1 1 10 N.T.

0.5260.038 0.6670.007 0.6890.040 0.6350.052 0.6100.033 0.6690.005 0.6950.040 0.6700.052 N.T.

1.505 1.555 1.433 1.557 1.376 1.510 1.568 1.512 N.T.

15

23c

16

25a

17

25b

0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10

0.5690.033 0.6720.006 0.6920.041 0.6650.050 0.5820.017 0.6500.035 0.7820.046 0.6610.020 0.5900.035 0.6550.046 0.6990.020 0.6700.029

1.284 1.516 1.562 1.501 1.313 1.467 1.765 1.492 1.331 1.478 1.577 1.512

17a

0.6930.005 0.686 0.01 0.1 0.6790.008 1.465 1 0.6490.009 1.320 10 0.5850.02 1.313 Table 6: Effect of saponin analogs on OVA-stimulated cell proliferation ex-vivo

107

Chapter-II, Section-B

Fig 2: Effect of dehydroepiendrosterone 3-O-D-mannopyranoside 11c on OVA-specific IgG, IgG1 and IgG2a antibody. Groups of ten Balb/C mice were immunized subcutaneously with OVA (100 g) on days 1 and 15 along with 11c (1, 10 and 100 g). Group of animals treated with Alum (200 g) along with Ova served as positive control. Sera were collected on day 15, 28 and 60 respectively after 1st immunization to observe the effect on IgG, IgG1 and IgG2a antibodies by ELISA. The values are presented as mean S.E. (n = 10). The difference between the ova only and drug treated groups is determined by Bonferroni multiple comparison test. * P < 0.05, **P < 0.01.

108

Chapter-II, Section-B

Fig 3: Effect of dehydroepiendrosterone 3-O-D-mannopyranoside 11c on concentration of Th1 (IFN-, IL2) and Th2 (IL-4) cytokines in mouse serum. The effect of 11c on the expression of IFN-, IL-2 and IL-4 were determined on 28 days after 1st immunization. Cytokine estimation was done by ELISA. Values are means S.E. of ten mice; *P < 0.05, **P < 0.01 when compared with ova only group determined by one-way ANOVA (Bonferroni correction multiple comparison test).

109

Chapter-II, Section-B

Fig 4: Effect of dehydroepiendrosterone 3-O-D-mannopyranoside 11c on T cell sub population CD4 and CD8 in the spleen cells. Mice immunized with ova were given graded doses of 11c along with Ova as described in materials and methods. Splenocyte isolated on 28 days after 1st immunization from the PK treated and untreated mice were analyzed by flow cytometry for the expression of various T cell sub populations. A typical bivariate analysis of splenocytes for the expression of cell surface markers and the percent gated population corresponding to each phenotype marker CD4 and CD8 are shown. Cells incubated with anti-mouse FITClabeled CD8 and PE conjugated CD4 monoclonal antibodies for 30 mints, were washed with PBS before FACScan analysis. Data are representative of one of the two experiments.

110

Chapter-II, Section-B 2.2.5. Conclusion:In conclusion, a facile and expedient synthesis of biologically important steroidal and triterpenoid based saponins has been presented in this section. The synthesis is based on easily available starting materials and involves high yielding and economical synthetic steps resulting in the quantitative overall yields of the products. All the saponin anaologs, thus, generated are being screened for immune-adjuvant activity. The preliminary results are very encouraging and saponin particularly dehydroepiendrosterone 3-O-D-mannopyranoside 11c is potent enhancers of Th1 responses.

111

Chapter-II, Section-B 2.2.6. Experimental:- General methods Solvents were purified according to the standard procedures and reagents used were of highest purity available. All reactions were performed in flame-dried glass apparatus under argon/nitrogen atmosphere unless otherwise mentioned. Anhydrous solvents like CH2Cl2, Et2O, THF, CH3OH, pyridine, Et3N were freshly dried using standard methods. NMR spectra were recorded at 200 MHz and 500 MHz on Bruker DPx200 instrument in CD3OD/DMSO/CDCl3 with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values were mentioned in (ppm) and coupling constants were given in Hz. Mass spectra were recorded on ESI-esquire 3000 Bruker Daltonics instrument. The progress of all reactions was monitored by pre-coated silica gel TLC. The chromatograms were visualized under UV 254-366 nm and iodine and charred with 20% H2SO4 in EtOH. All the synthetic saponins were characterized by 1H NMR and Mass spectroscopy and by comparing their chemical shift value with literature value. 1. Synthesis of C-28-benzyl oleanolate 12:

BnO O OH

A solution of oleanolic acid 3 (0.5 g), BnBr (0.2 ml, 1.5 eq), Et3N (0.22 ml, 1.5 eq) and Bu4NI (40 mg, 1 eq) in dry THF (10 ml) was stirred at room temperature overnight. The solvent was evaporated in vacuum and the residue was purified through flash chromatography (n-hexane : EtOAc) to give 12 (0.53 g, 90%) as a white amorphous solid.
1

H NMR (CDCl3, 200 MHz): 7.33 (m, 5H), 5.29-5.20 (m 3H), 3.21-3.19 (m 1H), 2.91 (dd,

1H, J = 14.0 and 4.2 Hz), 2.02-1.75 (m, 3H), 1.74-1.23 (m, 18H), 1.22-1.08 (m, 4H), 1.050.76 (s, 4 x 3H), 0.75 (s, 3H), 0.67 (s, 3H). MS (EI, 70eV): 546 (M+).

112

Chapter-II, Section-B 2. Betulin 28-acetate 13:

AcO OH

Compound 5 (1.0 g) dissolved in 4 mL of CH2Cl2 and added acetic anhydride (3 ml). After stirring at room temperature overnight, the mixture was washed with saturated NaHCO3 solution and brine and then extracted three times with CH2Cl2 (20 mL) and combined organic phases were dried over Na2SO4, filtered and evaporated under reduced pressure. The crude product was purified by flash chromatography using n-hexane:EtOAc as eluent. Betulin 28acetate (13) (0.80 g, 73% yield) was obtained as white solid along with minor quantity of betulin 3,28-diacetate. NMR and mass spectral data of 13 were in agreement with those published in the literature. Mp: 206-208 oC (lit. 210-212 oC, Gauthier et al., 2006);
1

H NMR (500 MHz, CDCl3): 4.68 (s, 1H), 4.61 (s, 1H), 4.24 (d, 1H, J = 11.04 Hz), 3.85 (d,

1H, J = 11.3 Hz), 3.20-3.16 (m, 1H), 2.45-2.44 (m, 1H), 2.07 (s, 3H), 1.93-1.88 (m, 1H), 1.861.85 (m, 1H), 1.79-1.70 (m, 1H), 1.69-145 (m, 12 H), 1.42-1.33 (m, 6H), 1.29-1.20 (m, 3H), 1.11-0.98 (m, 12 H), 0.82 (s, 3H), 0.75 (s, 3H). Maldi TOF-TOF-MS: 484 (M+), 483 (M+-1).

3. Synthesis of monosaccharide based glycosyl donor 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl trichloroacetimidate 9a:

OAc O O HN CCl3 OAc OAc OAc

To a cold solution of acetic anhydride (20 ml), 60% perchloric acid (100 l) was added followed by slow addition of 5 g of powdered glucose 6a over a time of 30 min. Allowed the reaction to stirred for 3-4 h at room temperature and then poured the liquid into a mixture of 113

Chapter-II, Section-B ice and water. Crystalline solid which was separated on vigorous stirring was filtered off and washed thoroughly with cold water. After recrystalisation with industrial spirit afford 9.7 g of peracetylated glucose 7a with 90% yield. Peracetylated glucose 7a (9 g) was dissolved in DMF (19 mL) and added NH4OAc (1.50 g, 5.44 mmol) portionwise at 0oC. Allowed the reaction mixture to stir for 2-4 h at room temperature, then the mixture was filtered and then poured into ice cold water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford 2,3,4,6-tetra-O-acetyl--D-glucopyranose 8a (7.0 g, 88%). This compound was immediately used in the next step without further purification. This compound 8a (5 g) was again dissolve in dry CH2Cl2 and then added DBU (catalytic) followed by CCl3CN (1.5 ml, 2 equivalent wrt 8a), and the reaction mixture was stirred for 4 h at room temperature. The mixture was then filtered off, concentrated under reduced pressure and the residue was purified by flash chromatography (hexane:ethyl-acetate) to give 2,3,4,6tetra-O-acetyl--D-glucopyranosyl trichloroacetimidate 9a (4.8 g, 70 %).
1

H NMR (CDCl3, 200 MHz): 2.05-2.10 (m, 12H), 4.18-4.28 (m, 2H), 4.39-4.42 (m, 1H),

4.84-4.86 (m, 1H), 5.21-5.24 (m, 1H), 5.33-5.35 (m, 1H), 6.46 (d, 1H, J = 1.2 Hz), 8.79 (brs, 1H). MS (EI, 70eV): 491 (M+).

2,3,4,6-Tetra-O-acetyl--D-galactopyranosyl trichloroacetimidate 9b:

AcO O O HN CCl3 OAc OAc OAc

This compound was prepared from galactose 6b by similar methods as described above.
1

H NMR (CDCl3, 200 MHz): 199-2.20 (m, 12H), 4.06-4.30 (m, 3H), 5.39-5.47 (m, 2H),

5.49-5.51 (m, 1H), 6.28 (d, 1H, J = 1.6 Hz), 8.74 (s, 1H). MS (EI, 70eV): 491 (M+).

114

Chapter-II, Section-B 2,3,4,6-Tetra-O-acetyl--D-mannopyranosyl trichloroacetimidate 9c:

OAc O O HN CCl3 OAc OAc OAc

The tittle compound 9c was prepared from mannose 6c by similar methods as described above.
1

H NMR (CDCl3, 200 MHz): 199-2.05 (m, 9H), 2.07 (m, 3H), 4.03-4.18 (m, 2H), 4.41-4.47

(m, 1H), 5.37-5.41 (m, 2H), 5.56-5.57 (m, 1H), 6.60 (d, 1H, J = 3.0 Hz), 8.67 (s, 1H). MS (EI, 70eV): 491 (M+).

4. General procedure for the synthesis of steroid based saponin containing monosaccharide residue 11a-f: A mixture of one equivalent of the appropriate acceptor (1 or 2) and 1.5 equivalents of the appropriate donor (9) and freshly activated powdered 4 Ao molecular sieves were dried by azeotropic removal of residual moisture through toluene. The mixture was dissolved in anhyd CH2Cl2, stirred under nitrogen at room temperature for 30 min, and then cooled to 0 oC. To the above was added a solution of TMSOTf (0.05 equivalent) drop-wise and the mixture was stirred further for 1-4 h at room tempperature. After completion, the reaction mixture was neutralized with triethylamine, filtered through celite and concentrated. The flash column chromatography (hexane/ethyl acetate) provided the product 10. The protected glycosides 10 again dissolved in a MeOH and then treated with NaOMe. The reaction was stirred for 8-10 h at room temperature. After completion, the reaction was neutralized with cation-exchange resin, filtered and concentrated. The residue was purified on a silica column using MeOH in CHCl3 (2-15%) as eluent providing desired compound 11. All the synthetic saponins were characterized by 1H NMR and Mass spectroscopy as well as by comparing their chemical shift value with literature value.

115

Chapter-II, Section-B Dehydroepiendrosterone 3-O-D-glucopyranoside 11a:

O OH O O OH OH OH

As described above, dehydroepiendrosterone 1 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-glucopyranosyl trichloroacetimidate 9a (51 mg) gave 34 mg of 10a with 80% yield. Dehydroepiendrosterone 3-O-2,3,4,6-tetraacetyl--D-glucopyranoside 10a: 1H NMR (400 MHz, CDCl3): 5.30 (d, 1H, J = 5.18 Hz), 5.10-4.88 (m, 3H), 4.54 (d, 1H, J = 7.9 Hz), 4.17 (dd, 1H, J = 5.6 and 11.2 Hz), 4.09 (dd, 1H, J = 2.01 and 12.01 Hz), 3.72-3.66 (m, 1H), 3.453.42 (m, 1H), 2.49-2.48 (m, 1H), 2.23-2.11 (m, 12H), 2.09-1.99 (m, 9H), 1.90-1.80 (m, 2H), 1.74-1.54 (m, 4H), 1.49-1.31 (m, 2H), 1.17-1.11 (m 1H), 1.04 (s, 3H), 0.93 (s, 3H). ESI-MS: 641 (M++Na). Deacetylation of 10a (34 mg) afforded 22 mg of dehydroepiendrosterone 3-O--Dglucopyranoside 11a with 90% yield. 1H NMR (200 MHz, CD3OD): 5.34 (d, 1H, J = 5.3 Hz), 4.54 (d, 1H, J = 7.9 Hz), 3.94-3.68 (m, 2H), 3.54-3.50 (m, 1H), 3.30-3.07 (m, 4H, signals obscured with solvents peak), 2.49-2.46 (m, 1H), 2.09-1.85 (m, 9H), 1.90-1.80 (m, 2H), 1.741.54 (m, 4H), 1.49-1.31 (m, 2H), 1.17-1.11 (m 1H), 1.03 (s, 3H), 0.91 (s, 3H). ESI-MS: 450 (M+).

Dehydroepiendrosterone 3-O-D-galactopyranoside 11b:

O HO O O OH OH OH

As described above, dehydroepiendrosterone 1 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-galactopyranosyl trichloroacetimidate 9b (51 mg) gave 36 mg of 10b with 85% yield. 116

Chapter-II, Section-B Dehydroepiendrosterone 3-O-2,3,4,6-tetraacetyl--D-galactopyranoside 10b: 1H NMR (400 MHz, CDCl3): 5.34 (d, 1H, J = 5.21 Hz), 5.10-4.88 (m, 3H), 4.56 (d, 1H, J = 7.87 Hz), 4.18 (dd, 1H, J = 5.69 and 11.21 Hz), 4.10 (dd, 1H, J = 2.01 and 12.01 Hz), 3.72-3.66 (m, 1H), 3.44-3.42 (m, 1H), 2.49-2.48 (m, 1H), 2.23-2.11 (m, 12H), 2.09-1.99 (m, 9H), 1.90-1.80 (m, 2H), 1.74-1.54 (m, 4H), 1.49-1.31 (m, 2H), 1.18-1.10 (m 1H), 1.05 (s, 3H), 0.91 (s, 3H); ESI-MS: 641 (M++Na). Deacetylation of 10b (36 mg) afforded 22 mg of dehydroepiendrosterone 3-O--Dgalactopyranoside 11b with 87% yield. 1H NMR (200 MHz, CD3OD): 5.29 (d, 1H, J = 5.23 Hz), 4.55 (d, 1H, J = 7.98 Hz), 3.90-3.65 (m, 2H), 3.54-3.50 (m, 1H), 3.33-3.07 (m, 4H, signals obscured with solvent peak), 2.50-2.46 (m, 1H), 2.09-1.85 (m, 9H), 1.90-1.80 (m, 2H), 1.74-1.54 (m, 4H), 1.49-1.31 (m, 2H), 1.17-1.11 (m 1H), 1.03 (s, 3H), 0.91 (s, 3H). ESI-MS: 450 (M+).

Dehydroepiendrosterone 3-O-D-mannopyranoside 11c:

O O OH OH OH O

OH

As described above, dehydroepiendrosterone 1 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-mannopyranosyl trichloroacetimidate 9c (51 mg) gave 29 mg of 10c with 70% yield. Dehydroepiendrosterone 3-O-2,3,4,6-tetraacetyl--D-mannopyranoside 10c: 1H NMR (400 MHz, CDCl3): 5.38 (d, 1H, J = 5.20 Hz), 5.11-5.03 (m, 1H), 5.03-4.85 (m, 2H), 4.65 (d, 1H, J = 2.09 Hz), 4.25-3.99 (m 3H), 3.70-3.66 (m, 1H), 2.49-2.48 (m, 1H), 2.34-2.32 (m, 5H), 2.23-2.11 (m, 12H), 2.38-2.22 (m, 5H), 2.09-1.99 (m, 4H), 1.89-1.80 (m, 2H), 1.74-1.55 (m, 4H), 1.49-1.30 (m, 2H), 1.19-1.10 (m 1H), 1.07 (s, 3H), 0.90 (s, 3H). ESI-MS: 641 (M++Na). Deacetylation of 10c (29 mg) afforded 16 mg of dehydroepiendrosterone 3-O--Dmannopyranoside 11c with 85% yield. 1H NMR (200 MHz, CD3OD): 5.30 (d, 1H, J = 5.22 Hz), 4.44 (d, 1H, J = 2.99 Hz), 3.95-3.68 (m, 2H), 3.56-3.52 (m, 1H), 3.48-3.38 (m, 1H), 3.30117

Chapter-II, Section-B 3.07 (m, 3H, signals obscured with solvent peak), 2.49-2.46 (m, 1H), 2.36-2.30 (m, 5H), 2.001.86 (m, 4H), 1.91-1.81 (m, 2H), 1.74-1.54 (m, 4H), 1.47-1.30 (m, 2H), 1.16-1.12 (m, 1H), 1.00 (s, 3H), 0.90 (s, 3H); ESI-MS: 450 (M+).

Cholesterol 3-O-D-glucopyranoside 11d

OH O O OH OH OH

As described above, cholesterol 2 (20 mg) on coupling with 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl trichloroacetimidate 9a (38 mg) gave 31 mg of 10d with 85% yield. Cholesterol 3-O-2,3,4,6-tetraacetyl--D-glucopyranoside 10d:
1

H NMR (200 MHz,

CDCl3): 5.36-5.34 (m, 1H), 5.20-4.90 (m, 3H), 4.58 (d, 1H, J = 7.87 Hz), 4.25 (dd, 1H, J = 4.69 and 11.01 Hz), 4.20 (dd, 1H, J = 2.22 and 11.91 Hz), 3.74 (m, 1H), 3-46-3.50 (m, 1H), 2.26-2.20 (m, 2H), 2.19-1.74 (m, 15H), 1.73-1.24 (m, 15H), 1.23-0.75 (m, 20H), 0.70 (s, 3H). ESI-MS: 734 (M++H2O), 739 (M++Na). Deacetylation of 10d (31 mg) afforded 21 mg of cholesterol 3-O--D-glucopyranoside 11d with 90% yield. 1H NMR (200 MHz, CD3OD): 5.34-5.33 (m, 1H), 4.32-4.30 (d, 1H, J = 7.78 Hz), 3.82-3.70 (m, 2H), 3.68-3.65 (m, 1H), 3.30-3.10 (m, 4H, signals obscured with solvent peak), 2.25-2.21 (m, 2H), 2.00-1.70 (m, 3H), 1.70-1.31 (m, 15H), 1.20-0.70 (m, 20H), 0.65 (s, 3H). ESI-MS: 548 (M+) Cholesterol 3-O-D-galactopyranoside 11e

HO O O OH

OH

OH

118

Chapter-II, Section-B As described above, cholesterol 2 (20 mg) on coupling with 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl trichloroacetimidate 9b (38 mg) gave 29.6 mg of 10e with 80% yield. Cholesterol 3-O-2,3,4,6-tetraacetyl--D-galactopyranoside 10e: 1H NMR (200 MHz, CDCl3): 5.35-5.33 (m, 1H), 5.18-4.90 (m, 3H), 4.62 (d, 1H, J = 7.87 Hz), 4.23 (dd, 1H, J = 4.69 and 11.01 Hz), 4.19 (dd, 1H, J = 2.22 and 11.91 Hz), 3.84-3.82 (m, 1H), 3.74-3.72 (m, 1H), 2.23-2.19 (m, 2H), 2.20-1.73 (m, 15H), 1.73-1.25 (m, 15H), 1.23-0.74 (m, 20H), 0.72 (s, 3H). ESI-MS: 739 (M++Na). Deacetylation of 10e (29.6 mg) afforded 20.3 mg of cholesterol 3-O--D-galactopyranoside 11e with 90% yield. 1H NMR (200 MHz, CD3OD): 5.28-5.25 (m, 1H), 4.20-4.18 (d, 1H, J = 7.4 Hz), 3.95-3.84 (m, 2H), 3.62-3.59 (m, 1H), 3.56-3.54 (m, 1H), 3.30-3.10 (m, 3H, signals obscured with solvents peak), 2.24-2.21 (m, 2H), 1.99-1.70 (m, 3H), 1.71-1.31 (m, 15H), 1.200.70 (m, 20H), 0.64 (s, 3H). ESI-MS: 548 (M+).

Cholesterol 3-O-D-mannopyranoside 11f

OH OH O OH OH O

As described above, cholesterol 2 (20 mg) on coupling with 2,3,4,6-tetra-O-acetyl-D-mannopyranosyl trichloroacetimidate 9c (38 mg) gave 27.7 mg of 10f with 75% yield. Cholesterol 3-O-2,3,4,6-tetraacetyl--D-mannopyranoside 10f: 1H NMR (200 MHz, CDCl3): 5.21-5.19 (m, 1H), 5.15-4.88 (m, 3H), 4.58 (d, 1H, J = 3.7 Hz), 4.20 (dd, 1H, J = 4.69 and 11.01 Hz), 4.17 (dd, 1H, J = 2.22 and 11.91 Hz), 3.80-3.78 (m, 1H), 3.76-3.73 (m, 1H), 2.22-2.19 (m, 2H), 2.14-1.73 (m, 15H), 1.70-1.25 (m, 15H), 1.22-0.74 (m, 20H), 0.68 (s, 3H). ESI-MS: 739 (M++Na). Deacetylation of 10f (27.7 mg) afford 18 mg of cholesterol 3-O--D-mannopyranoside 11f with 85% yield. 1H NMR (200 MHz, CD3OD): 5.20-5.19 (m, 1H), 4.32 (d, 1H, J = 3.5 Hz), 119

Chapter-II, Section-B 3.90-3.80 (m, 2H), 3.62-3.60 (m, 1H), 3.55-3.53 (m, 1H), 3.30-3.25 (m, 3H, signals obscured with solvent peak), 2.24-2.19 (m, 2H), 1.90-1.66 (m, 3H), 1.71-1.30 (m, 15H), 1.20-0.71 (m, 20H), 0.64 (s, 3H). ESI-MS: 548 (M+).

5. Synthesis of oleanolic acid based saponin 15: Oleanolic acid 3-O-D-glucopyranoside 15a

HO OH O O OH OH O OH

As described above, C-28-benzyl oleanolate 12 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-glucopyranosyl trichloroacetimidate 9a (27 mg) gave 25.6 mg of 14a with 80% yield. Deacetylation of 14a (25.6 mg) afforded 18.6 mg of benzyl oleanolate 3-O--Dglucopyranoside with 90% yield. Benzyl oleanolate 3-O-2,3,4,6-tetraacetyl--D-glucopyranoside 14a: 1H NMR (200 MHz, CDCl3): 7.33-7.28 (m, 5H), 5.28-5.22 (m, 2H), 5.12-4.90 (m, 4H), 4.44 (d, 1H, J = 7.9 Hz), 4.21 (dd, 1H, J = 6.03 and 11.77 Hz), 4.15 (dd, 1H, J = 2.01 and 11.33 Hz), 3.70-3.68 (m, 1H), 3.58-3.56 (m, 1H), 2.73 (dd, J = 3.04 and 12.91, 1H), 2.25-2.05 (m, 12H), 2.02-1.75 (m, 3H), 1.74-1.23 (m, 17H), 1.22-1.08 (m, 5H), 1.05-0.76 (s, 4 x 3H), 0.75 (s, 3H), 0.67 (s, 3H). Maldi TOF-TOF-MS: 876 (M+). Benzyl oleanolate 3-O--D-glucopyranoside (10 mg and the catalyst 20% Pd(OH)2 (10 mg) were dissolved in a solvent mixture of MeOH (1 ml), CH2Cl2 (1 ml) and H2O (1 ml). The residual and the dissolved air from the flask were removed by repeated evacuations and reaction mixture was stirred under hydrogen atmosphere overnight. After completion of reaction, the mixture was filtered through a small celite pad, and concentrated under reduced pressure. The product was purified by a quick filtration through a silica column using MeOH:CH2Cl2 to provide the 7.8 mg, 90% of compound 15a (oleanolic acid 3-O--Dglucopyranoside). 120

Chapter-II, Section-B
1

H NMR (200 MHz, CD3OD): 5.24-5.22 (m, 1H), 4.46 (d, 1H, J = 7.80 Hz), 3.90-3.80 (m,

2H), 3.62-3.60 (m, 1H), 3.45-3.25 (m, 4H, signals obscured with solvent peak), 2.78 (dd, J=3.04 and 12.91, 1H), 2.02-1.74 (m, 3H), 1.74-1.20 (m, 17H), 1.22-1.08 (m, 5H), 1.05-0.76 (s, 4 x 3H), 0.75 (s, 3H), 0.67 (s, 3H). Maldi TOF-TOF-MS: 618 (M+).

Oleanolic acid 3-O-D-mannopyranoside 15b:

HO O

OH O

OH OH OH

As described above, C-28-benzyl oleanolate 12 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-mannopyranosyl trichloroacetimidate 9c (27 mg) gave 22.4 mg of 14b with 70% yield. Benzyl oleanolate 3-O-2,3,4,6-tetraacetyl--D-mannopyranoside 14b: 1H NMR (200 MHz, CDCl3): 7.33-7.28 (m, 5H), 5.30-5.29 (m, 2H), 5.12-4.90 (m, 4H), 4.01 (d, 1H, J = 3.32 Hz), 4.21 (dd, 1H, J = 6.03 and 11.77 Hz), 4.15 (dd, 1H, J = 2.01 and 11.33 Hz), 3.70-3.68 (m, 1H), 3.58-3.56 (m, 1H), 2.56-2.54 (m, 1H), 2.25-2.01 (m, 12H), 1.90-1.68 (m, 3H), 1.68-1.45 (m, 17H), 1.40-1.32 (m, 5H), 0.93-0.83 (m, 12H), 0.78 (s, 3H), 0.66 (s, 3H). Maldi TOF-TOF-MS: 876 (M+). Deacetylation of 14b (22.4 mg) afford 15.7 mg of benzyl oleanolate 3-O--D-glucopyranoside with 87% yield. Benzyl oleanolate 3-O--D-mannopyranoside (10 mg and the catalyst 20% Pd(OH)2 (10 mg) were dissolved in a solvent mixture of MeOH (1 ml), CH2Cl2 (1 ml) and H2O (1 ml). The residual and the dissolved air from the flask were removed by repeated evacuations and reaction mixture was stirred under hydrogen atmosphere overnight. After completion of reaction, the mixture was filtered through a small celite pad, and concentrated under reduced pressure. The product was purified by a quick filtration through a silica column using MeOH:CH2Cl2 to provide the compound 15b (7.5 mg, 89%). 1H NMR (200 MHz, CD3OD): 5.23-5.21 (m, 1H), 4.68 (d, 1H, J = 3.01 Hz), 3.82-3.78 (m, 2H), 3.60-3.58 (m, 121

Chapter-II, Section-B 1H), 3.55-3.53 (m, 1H), 3.30-3.25 (m, 3H, signals obscured with solvent peak), 2.66-2.64 (m, 1H), 1.90-1.68 (m, 3H), 1.68-1.45 (m, 17H), 1.40-1.30 (m, 5H), 0.90-0.84 (m, 12H), 0.77 (s, 3H), 0.69 (s, 3H). Maldi TOF-TOF-MS: 618 (M+).

6. Synthesis of lupeol based saponin 17 Lupeol 3-O-D-galactopyranoside 17a:

HO O O OH

OH

OH

As described above, lupeol 4 (20 mg) on coupling with 2,3,4,6-tetra-O-acetyl--Dgalactopyranosyl trichloroacetimidate 9b (34.5 mg) gave 25.6 mg of 16a with 72% yield. Lupeol 3-O-2,3,4,6-tetraacetyl--D-galactopyranoside 16a: 1H NMR (200 MHz, CDCl3): 5.31-5.29 (m, 1H), 5.22-4.93 (m, 2H), 4.60 (s, 1H), 4.57 (s, 1H), 4.51 (d, 1H, J = 7.8 Hz), 4.24-4.07 (m 2H), 3.70-3.68 (m, 1H), 3.53-3.52 (m, 1H), 2.38-2.36 (m, 1H), 2.20-2.00 (m, 12H), 1.90-1.68 (m, 4H), 1.70-1.45 (m, 12H), 1.40-1.32 (m, 6H), 1.29-120 (m, 3H), 0.93-0.83 (m, 14H), 0.79 (s, 3 H), 0.71 (s, 3H). Maldi TOF-TOF-MS: 756 (M+). Deacetylation of 16a (25.6 mg) afforded 16.8 mg of lupeol 3-O--D-galactopyranoside 17a with 85% yield. 1H NMR (200 MHz, CD3OD): 4.63 (s, 1H), 4.54 (s, 1H), 4.30 (d, 1H, J = 7.6 Hz), 3.70-3.67 (m, 2H), 3.44-3.43 (m, 1H), 3.30-3.11 (m, 4H, signal obscured with solvent peak), 2.37-2.35 (m, 1H), 1.90-1.68 (m, 4H), 1.70-1.45 (m, 12H), 1.40-1.31 (m, 6H), 1.30-120 (m, 3H), 0.93-0.85 (m, 14H), 0.71 (s, 3 H), 0.68 (s, 3H). Maldi TOF-TOF-MS: 588 (M+).

122

Chapter-II, Section-B Lupeol 3-O-D-mannopyranoside 17b:

OH O

OH OH OH

As described above, lupeol 4 (20 mg) on coupling with 2,3,4,6-tetra-O-acetyl--Dmannopyranosyl trichloroacetimidate 9c (34.5 mg) gave 24.2 mg of 16b with 68% yield. Lupeol 3-O-2,3,4,6-tetraacetyl--D-mannopyranoside 16b: 1H NMR (200 MHz, CDCl3): 5.30-5.28 (m, 1H), 5.22-4.93 (m, 2H), 4.60 (s, 1H), 4.58 (s, 1H), 4.48 (d, 1H, J = 3.8 Hz), 4.25-4.08 (m 2H), 3.70-3.68 (m, 1H), 3.53-3.51 (m, 1H), 2.37-2.35 (m, 1H), 2.21-204 (m, 12H), 1.90-1.68 (m, 4H), 1.70-1.45 (m, 12H), 1.40-1.32 (m, 6H), 1.29-120 (m, 3H), 0.93-0.83 (m, 14H), 0.78 (s, 3H), 0.72 (s, 3H). Maldi TOF-TOF-MS: 756 (M+). Deacetylation of 16b (24.2 mg) afforded 16.3 mg of lupeol 3-O--D-mannopyranoside 17b with 87% yield. 1H NMR (200 MHz, CD3OD): 4.64 (s, 1H), 4.55 (s, 1H), 4.34 (d, 1H, J = 3.2 Hz), 3.70-3.68 (m, 2H), 3.44-3.43 (m, 1H), 3.30-3.11 (m, 4H, signal obscured with solvent peak), 2.37-2.35 (m, 1H), 1.90-1.68 (m, 4H), 1.70-1.45 (m, 12H), 1.40-1.32 (m, 6H), 1.29-120 (m, 3H), 0.93-0.83 (m, 14H), 0.78 (s, 3H), 0.72 (s, 3H). Maldi TOF-TOF-MS: 588 (M+). 7. Synthesis of betulin based saponin 17 Betulin 3-O-D-glucopyranoside 17c:

HO O

OH O OH OH OH

123

Chapter-II, Section-B As described above, betulin 28-acetate 13 (20 mg) on coupling with 2,3,4,6-tetra-Oacetyl--D-glucopyranosyl trichloroacetimidate 9a (30.5 mg) gave 22.5 mg of 16c with 67% yield. 28-Acetyl betulinate 3-O-2,3,4,6-tetraacetyl--D-glucopyranoside 16c: 1H NMR (500 MHz, CDCl3): 5.23-4.93 (m, 3H), 4.68 (s, 1H), 4.61 (s, 1H), 4.51 (d, J = 7.24, 1H), 4.24 (m, 2H), 4.20-4.22 (dd, 1H, J = 2.1 and 12.2 Hz), 3.85 (d, 1H, J = 11.3 Hz), 3.70 (m, 1H), 3.533.52 (m, 1H), 2.46-2.43 (m, 1H), 2.18-1.98 (m, 15H), 1.93-1.88 (m, 1H), 1.86-1.85 (m, 1H), 1.79-1.70 (m, 1H), 1.69-145 (m, 12H), 1.42-1.33 (m, 6H), 1.28-1.20 (m, 3H), 1.09-0.95 (m, 12H), 0.83 (s, 3H), 0.75 (s, 3H). Maldi TOF-TOF-MS: 814 (M+). Deacetylation of 16c (22.5 mg) afforded 15.2 mg of betulin 3-O--D-glucopyranoside 17c with 91% yield. 1H NMR (200 MHz, CD3OD): 4.67 (s, 1H), 4.60 (s, 1H), 4.44 (d, J = 7.24, 1H), 4.01 (d, 1H J=7.2 Hz), 3.80-3.70 (m, 3H), 3.68-3.65 (m, 1H), 3.30-3.11 (m, 4H, signal obscured with solvent peaks), 2.46-2.43 (m, 1H), 1.90-1.87 (m, 1H), 1.83-1.80 (m, 1H), 1.791.70 (m, 1H), 1.69-145 (m, 12 H), 1.42-1.33 (m, 6H), 1.30-1.20 (m, 3H), 1.05-0.96 (m, 12H), 0.89 (s, 3H), 0.69 (s, 3H). Maldi TOF-TOF-MS: 604 (M+).

Betulin 3-O-D-mannopyranoside 17c:

OH O HO O

OH OH OH

As described above, betulin 28-acetate 13 (20 mg) on coupling with 2,3,4,6-tetra-Oacetyl--D-mannopyranosyl trichloroacetimidate 9c (30.5 mg) gave 21.8 mg of 16d with 65% yield. 28-Acetyl betulinate 3-O-2,3,4,6-tetraacetyl--D-mannopyranoside 16d: 1H NMR (500 MHz, CDCl3): 5.22-4.93 (m, 3H), 4.68 (s, 1H), 4.61 (s, 1H), 4.52 (d, 1H, J = 3.2 Hz), 4.244.22 (m 2H), 4.20 (dd, 1H, J = 2.1 and 12.2 Hz), 3.85 (d, 1H, J = 11.3 Hz), 3.70 (m, 1H), 124

Chapter-II, Section-B 3.52-3.50 (m, 1H), 2.46-2.43 (m, 1H), 2.19-1.98 (m, 15H), 1.93-1.88 (m, 1H), 1.86-1.85 (m, 1H), 1.79-1.70 (m, 1H), 1.69-145 (m, 12H), 1.43-1.33 (m, 6H), 1.28-1.21 (m, 3H), 1.09-0.95 (m, 12H), 0.82 (s, 3H), 0.74 (s, 3H). Maldi TOF-TOF-MS: 814 (M+). Deacetylation of 16d (21.8 mg) afforded 14.6 mg of betulin 3-O--D-mannopyranoside 17d with 90% yield. 1H NMR (200 MHz, CD3OD): 4.65 (s, 1H), 4.61 (s, 1H), 4.43 (d, 1H, J = 3.5 Hz), 4.01 (d, 1H, J = 7.2 Hz), 3.80-3.70 (m, 3H), 3.68-3.65 (m, 1H), 3.30-3.11 (m, 4H, signal obscured with solvent peaks), 2.44-2.42 (m, 1H), 1.93-1.89 (m, 1H), 1.86-1.85 (m, 1H), 1.791.70 (m, 1H), 1.69-145 (m, 12H), 1.42-1.33 (m, 6H), 1.28-1.20 (m, 3H), 1.09-0.95 (m, 12H), 0.81 (s, 3H), 0.73 (s, 3H). Maldi TOF-TOF-MS: 604 (M+).

8. Synthesis of disaccharide based glycosyl donor 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl2,2,2-trichloroacetimidate 21a:

AcO O O AcO Cl3C NH O O OAc AcO OAc AcO OAc

To a cold solution of acetic anhydride (10 ml) and pyridine (25 ml) add 5 g of powdered lactose 18a. Allow the reaction to stirred for overnight at rt and then pour the liquid into a mixture of ice and water. The compound was extracted with ethyl acetate (2x50 ml) and then washed with diluted 2 N HCl. The organic layer was dried over anhy Na2SO4 and then evaporated in vaccum afford 9.2 g of peracetylated lactose 19a with 93% yield. Peracetylated lactose 19a (9 g) was dissolved in DMF (20 mL) and added NH4OAc (3 g) portion wise at 0 oC. Allowed the reaction mixture to stir for 2-4 h at room temperature, then the mixture was filtered and then poured into ice cold water and then extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-Dglucopyranose 20a. This compound was immediately used in the next step without further purification. This compound 20a (5 g) was again dissolve in dry CH2Cl2 and then added DBU 125

Chapter-II, Section-B (catalytic) followed by CCl3CN (1.5 ml, 2 equivalent wrt 20a), and the reaction mixture was stirred for 4 h at room temperature. The mixture was then filtered off, concentrated under reduced pressure and the residue was purified by flash chromatography (hexane:ethyl-acetate) to
1

give

2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-gluco-

pyranosyl-2,2,2-trichloroacetimidate 21a (4.0 g, 65 %). H NMR (200 MHz, CDCl3): 8.74 (s, 1H), 6.49 (d, 1H, J = 3.8 Hz), 5.56-5.49 (t, 1H, J = 9.4

Hz), 5.27-5.26 (m, 1H), 5.24-4.90 (m, 3H), 4.62-4.40 (m, 2H), 4.25-4.00 (m, 4H), 3.95-3.74 (m, 2H), 2.25-2.00 (m, 21H). Maldi TOF-TOF-MS: 803 (M++Na) and 819 (M++K).

2,3,4,6-Tetra-O-acetyl--D-glucopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl2,2,2-trichloroacetimidate 21b:
AcO O O AcO Cl3C NH O O OAc AcO AcO OAc OAc

2,3,4,6-Tetra-O-acetyl--D-glucopyranosyl-(14)-2,3,6-tri-O-acetyl-Dglucopyranosyl-2,2,2-trichloroacetimidate 21b was prepared from cellobiose 18b by similar methods as described above.
1

H NMR (500 MHz, CDCl3): 8.73 (s, 1H), 6.49 (d, 1H, J = 3.7 Hz), 5.55-5.49 (t, 1H, J = 9.3

Hz), 5.27-5.26 (m, 1H), 5.24-4.95 (m, 3H), 4.52-4.44 (m, 2H), 4.25-4.00 (m, 4H), 3.95-3.73 (m, 2H), 2.25-2.01 (m, 21H). Maldi TOF-TOF-MS: 803 (M++Na) and 819 (M++K).

9. General procedure for the synthesis of steroid based saponin containing disaccharide residue 11a-f: A mixture of one equivalent of the appropriate acceptor (1 or 2 or 12 or 4 or 13) and 2.0 equivalents of the appropriate donor (21a or 21b) and freshly activated powdered 4 Ao molecular sieves were dried by azeotropic removal of residual moisture through toluene. The mixture was dissolved in anhyd CH2Cl2, stirred under argon at room temperature for 30 min, and then cooled to 0 oC. To the above was added a solution of TMSOTf (0.05 equiv) drop126

Chapter-II, Section-B wise and the mixture was stirred further for 1-4 h at room temperature. After completion, the reaction mixture was neutralized with triethyamine, filtered through celite and concentrated. The flash column chromatography (hexane/ethyl acetate) provided the product 10. The protected glycosides 10 again dissolved in a MeOH and then treated with NaOMe. The reaction was stirred for 8-10 h at room temperature. After completion, the reaction was neutralized with cation-exchange resin, filtered and concentrated. The residue was purified on a silica column using MeOH in CHCl3 (2-15%) as eluent providing desired compound 11. All the synthetic saponins were characterized by 1H NMR and Mass spectroscopy as well as by comparing their chemical shift value with literature value.

Dehydroepiendrosterone 3-O--D-galactopyranosyl-(1-4)-O-D-glucopyranoside 23a:

O
HO O O O OH HO OH HO OH

O
HO

As described above, dehydroepiendrosterone 1 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-2,2,2trichloroacetimidate 21a (108 mg) gave 44 mg of 22a with 70% yield. Dehydroepiendrosterone 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-Oacetyl--D-glucopyranoside 22a: 1H NMR (200 MHz, CDCl3): 5.64 (d, 1H, J = 5.1 Hz), 5.54-5.52 (m, 1H), 5.38-5.35 (m, 2H), 5.20-5.15 (m, 1H), 5.04-4.94 (m, 1H), 4.64 (d, 1H, J = 7.8 Hz), 4.61-4.52 (m, 1H), 4.36-4.16 (m, 2H), 4.12-4.09 (m, 3H), 3.94-3.84 (m, 2H), 3.743.72 (m, 1H), 2.53-2.50 (m, 1H), 2.40-2.35 (m, 5H), 2.23-2.05 (m, 21H), 2.02-1.99 (m, 4H), 1.89-1.80 (m, 2H), 1.74-1.55 (m, 4H), 1.50-1.30 (m, 2H), 1.19-1.11 (m, 1H), 1.04 (s, 3H), 0.88 (s, 3H); Maldi TOF-TOF-MS: 929 (M++Na) and 945 (M++K). Deacetylation of 22a (44 mg) afforded 24.6 mg of dehydroepiendrosterone 3-O--Dgalactopyranosyl-(1-4)-O-D-glucopyranoside 23a with 90% yield. 1H NMR (200 MHz, CD3OD): 5.53 (d, 1H, J = 5.1 Hz), 4.26 (d, 1H, J=7.1 Hz), 4.24-4.09 (m, 3H), 3.78-3.34 (m, 6H), 3.30-3.01 (m, 5H, signal obscured with solvent peak), 2.53-2.50 (m, 1H), 2.43-2.45 (m, 127

Chapter-II, Section-B 5H), 2.02-1.93 (m, 4H), 1.89-1.80 (m, 2H), 1.74-1.56 (m, 4H), 1.50-1.32 (m, 2H), 1.19-1.11 (m, 1H), 0.98 (s, 3H), 0.99 (s, 3H). Maldi TOF-TOF-MS: 635 (M+ + Na) and 651 (M+ + K). Dehydroepiendrosterone 3-O--D-glucopyranosyl-(1-4)-O-D-glucopyranoside 23b:
O
HO O O O OH HO HO OH OH

O
HO

As described above, dehydroepiendrosterone 1 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-glucopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-2,2,2trichloroacetimidate 21b (108 mg) gave 42.7 mg of 22b with 68% yield. Dehydroepiendrosterone 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl-(14)-2,3,6-tri-Oacetyl--D-glucopyranoside 22b: 1H NMR (200 MHz, CDCl3): 5.64 (d, 1H, J = 5.1 Hz), 5.54 (m, 1H), 5.39-5.36 (m, 2H), 5.20-5.15 (m, 1H), 5.04-4.96 (m, 1H), 4.62 (d, 1H, J = 7.8 Hz), 4.61-4.52 (m, 1H), 4.36-4.16 (m, 2H), 4.12-4.09 (m, 3H), 3.94-3.84 (m, 2H), 3.68-3.63 (m, 1H), 2.50-2.48 (m, 1H), 2.23-2.05 (m, 21H), 2.02-1.99 (m, 9H), 1.89-1.80 (m, 2H), 1.741.55 (m, 4H), 1.49-1.30 (m, 2H), 1.19-1.10 (m 1H), 1.04 (s, 3H), 0.90 (s, 3H). Maldi TOF-TOF-MS: 929 (M++Na) and 945 (M++K). Deacetylation of 22b (42.7 mg) afforded 23.7 mg of dehydroepiendrosterone 3-O--Dglucopyranosyl-(1-4)-O-D-glucopyranoside 23b with 80% yield.
1

H NMR (200 MHz,

DMSO): 5.53 (d, 1H, J = 5.1 Hz), 4.34 (d, 1H, J = 7.8 Hz), 4.26-4.05 (m, 3H), 3.78-3.34 (m, 6H), 3.30-3.01 (m, 5H, signal obscured with solvent peak), 2.53-2.50 (m, 1H), 2.00-1.90 (m, 9H), 1.89-1.79 (m, 2H), 1.70-1.54 (m, 4H), 1.51-1.31 (m, 2H), 1.19-1.11 (m 1H), 0.99 (s, 3H), 0.95 (s, 3H). Maldi TOF-TOF-MS: 635 (M++Na) and 651 (M++K).

128

Chapter-II, Section-B Cholesterol 3-O--D-galactopyranosyl-(1-4)-O-D-glucopyranoside 23c:

HO O O O OH HO

HO OH OH

HO

As described above, cholesterol 2 (20 mg) on coupling with 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-2,2,2-trichloroacetimidate 21a (80 mg) gave 35.4 mg of 22c with 67% yield. Cholesterol 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl--Dglucopyranoside 22c: 1H NMR (200 MHz, CDCl3): 5.73 (d, 1H, J = 5.1 Hz), 5.53 (m, 1H), 5.39-5.38 (m, 2H), 5.20-5.15 (m, 1H), 5.04-4.96 (m, 1H), 4.62 (d, 1H, J = 7.8 Hz), 4.61-4.52 (m, 1H), 4.36-4.16 (m, 2H), 4.12-4.09 (m, 3H), 3.94-3.84 (m, 2H), 3.68-3.63 (m, 1H), 2.251.97 (m, 22H), 1.80-1.74 (m, 4H), 1.58-1.43 (m, 15H), 1.30-1.74 (m, 20H), 0.68 (s, 3H). Maldi TOF-TOF-MS: 1027 (M++Na) and 1043 (M++K). Deacetylation of 22c (44 mg) afforded 21.5 mg of cholesterol 3-O--D-galactopyranosyl-(12)-O-D-glucopyranoside 23c with 86% yield. 1H NMR (200 MHz, DMSO): 5.54 (d, 1H, J = 5.0 Hz), 5.26 (m, 1H), 4.26 (d, 1H, J = 7.0 Hz), 4.24-4.09 (m, 3H), 3.78-3.25 (m, 10H), 2.252.24 (m, 1H), 2.23-1.74 (m, 4H), 1.58-1.43 (m, 15H), 1.30-1.74 (m, 20H), 0.68 (s, 3H). Maldi TOF-TOF-MS: 710 (M+).

10. Synthesis of oleanolic acid 3-O--D-galactopyranosyl-(1-4)-O-D-glucopyranoside 25a:

HO HO O O O HO OH O HO O

HO

OH

OH

As described above, C-28-benzyl oleanolate 12 (20 mg) on coupling with 2,3,4,6-tetraO-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-2,2,2trichloroacetimidate 21a (57 mg) gave 26.8 mg of 24a with 64% yield. 129

Chapter-II, Section-B Benzyl oleanolate 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranoside 24a: 1H NMR (200 MHz, CDCl3): 7.34-32 (m, 2H), 7.30-7.25 (m, 3H), 5.73 (d, J = 5.09, 1H), 5.53 (m, 1H), 5.39-5.38 (m, 2H), 5.20-5.15 (m, 3H), 5.04-4.96 (m, 1H), 4.62 (d, J = 7.83, 1H), 4.61-4.52 (m, 1H), 4.36-4.16 (m, 2H), 4.12-4.09 (m, 3H), 3.943.84 (m, 2H), 3.68-3.63 (m, 1H), 2.73 (dd, 1H, J = 3.1 and 12.9 Hz), 2.18-2.05 (m, 21H), 2.02-1.75 (m, 3H), 1.74-1.23 (m, 17H), 1.22-1.08 (m, 5H), 1.05-0.76 (s, 4 x 3H), 0.75 (s, 3H), 0.67 (s, 3H). Maldi TOF-TOF-MS: 1105 (M+). Deacetylation of 24a (26.8 mg) afford 17.6 mg of benzyl oleanolate 3-O--Dgalactopyranosyl-(1-2)-O-D-glucopyranoside with 88% yield. Benzyl oleanolate 3-O--Dgalactopyranosyl-(1-2)-O-D-glucopyranoside (10 mg and the catalyst 20% Pd(OH)2 (10 mg) were dissolved in a solvent mixture of MeOH (1 ml), CH2Cl2 (1 ml) and H2O (1 ml). The residual and the dissolved air from the flask were removed by repeated evacuations and reaction mixture was stirred under hydrogen atmosphere overnight. After completion of reaction, the mixture was filtered through a small celite pad, and concentrated under reduced pressure. The product was purified by a quick filtration through a silica column using MeOH:CH2Cl2 to provide the compound 25a (8 mg, 90%). 1H NMR (200 MHz, DMSO): 5.16 (brs, 1H), 4.23-4.21 (m, 2H), 3.73-3.69 (m, 1H), 3.61-3.44 (m, 5H), 3.33-3.25 (m, 5H), 3.04-3.01 (m, 2H), 2.73 (dd, 1H, J = 3.4 and 12.8 Hz), 2.00-1.75 (m, 3H), 1.74-1.23 (m, 17H), 1.22-1.08 (m, 5H), 1.00-0.75 (s, 4 x 3H), 0.74 (s, 3H), 0.68 (s, 3H). Maldi TOF-TOF-MS: 780 (M+).

11. Synthesis of lupeol 3-O--D-galactopyranosyl-(1-4)-O-D-glucopyranoside 25b:

HO O O HO OH O O HO

HO

OH

OH

130

Chapter-II, Section-B As described above, lupeol 4 (20 mg) on coupling with 2,3,4,6-tetra-O-acetyl--Dgalactopyranosyl-(14)-2,3,6-tri-O-acetyl-D-glucopyranosyl-2,2,2-trichloroacetimidate (73.1 mg) gave 35.4 mg of 24b with 67% yield. Lupeol 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(14)-2,3,6-tri-O-acetyl--Dglucopyranoside 24b: 1H NMR (200 MHz, CDCl3): 5.74 (d, 1H, J = 5.9 Hz), 5.53 (m, 1H), 5.37-5.35 (m, 2H), 5.20-5.15 (m, 1H), 5.04-4.95 (m, 1H), 4.74 (s, 1H), 4.64 (d, 1H, J = 7.9 Hz), 4.61-4.52 (m, 1H), 4.35-4.16 (m, 2H), 4.11-4.09 (m, 3H), 3.84-3.80 (m, 2H), 3.65-3.63 (m, 1H), 2.34-2.32 (m, 1H), 2.25-2.01 (m, 21H), 1.90-1.68 (m, 4H), 1.70-1.45 (m, 12H), 1.401.32 (m, 6H), 1.29-122 (m, 3 H), 0.93-0.84 (m, 14H), 0.75 (s, 3H), 0.65 (s, 3H). Maldi TOF-TOF-MS: 1044 (M+). Deacetylation of 24b (35.4 mg) afforded 21.6 mg of lupeol 3-O--D-galactopyranosyl-(1-2)O-D-glucopyranoside 25b with 93% yield. 1H NMR (200 MHz, DMSO): 5.36 (brs, 1H), 4.64 (s, 1H), 4.61 (s, 1H), 4.44 (d, 1H, J = 7.7 Hz), 4.23-4.21 (m, 1H), 3.73-3.69 (m, 1H), 3.613.44 (m, 4H), 3.33-3.25 (m, 5H), 3.04-3.01 (m, 2H), 2.34-2.32 (m, 1H), 1.96-1.80 (m, 4H), 1.70-1.45 (m, 12H), 1.40-1.32 (m, 6H), 1.27-120 (m, 3H), 0.90-0.81 (m, 14H), 0.89 (s, 3H), 0.60 (s, 3H). Maldi TOF-TOF-MS: 750 (M+). 21a

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Chapter-II, Section-B References: Bachran. C., Bachran, S., Sutherland, M., Bachran, D. and Fuchs, H. (2008). Saponins in tumor therapy. Mini-Reviews in Medicinal. Chemistry 8: 575-584. Barr, I.G., Sjolander, A. and Cox, J.C. (1998). ISCOMs and other saponin based adjuvants. Advanced Drug Delivery Reviews 32(3): 247271. Bomford, R., Stapleton, M., Winsor, S., Beesley, J.E., Jessup, E.A., and Price, K.R. (1992). Adjuvanticity and ISCOM formation by structurally diverse saponins. Vaccine 10(9): 572 577. Bomford, R., Stapleton, M., Winsor, S., Mc Night, A. and Andronova T. (1992). The control of the antibody isotype response to recombinant immunodeficiency virus gp120 antigen by adjuvants. AIDS Research Human Retroviruses 8: 17651771. Britt, W., Fay, J., Seals, J. and Kensil, C. (1995). Formulation of an immunogenic human cytomegalovirus vaccine: response in mice. The Journal of Infectious Diseases 171: 1825. Bruneton, J. (ed) (1995). In Pharmacognosie, Phytochimie, Plantes Mdicinales pp 1120. Technique & Documentation, Paris. Chapman, P.B., Morrissey, D.M., Panageas, K.S., Hamilton, W.B., Zhan, C. and Destro, A.N. (2000). Induction of antibodies against GM2 ganglioside by immunizing melanoma patients using GM2-keyhole limpet hemocyanin + QS-21 vaccine: a doseresponse study. Clinical Cancer Research 6: 874879. Chittaboina, S., Hodges, B. and Wang, Q. (2006). A Facile Route for the Regioselective Deacetylation of Peracetylated Carbohydrates at Anomeric Position. Letters in Organic Chemistry 3(1): 35-38. Eichler, E., Jennings, H.J., Gilbert, M. and Whitfield, D.M. (1999). Synthesis of a disialylated hexasaccharide of Type VIII Group B Streptococcus capsular polysaccharide. Carbohydrate Research 319: 1-16. Fernndez-Herrera, M.A., Sandoval-Ramrez, J., Lpez-Muoz, H. and Snchez-Snchez, L. (2009). Formation of the steroidal 3-hydroxy-6-oxo-moiety. Synthesis and cytotoxicity of glucolaxogenin. ARKIVOC xiii: 170-184. Francis, G., Kerem, Z., Makkar, H.P.S. and Becker, K. (2002). The biological action of saponins in animal systems: a review. British Journal of Nutrition 88: 587-605. Gauthier, C., Legault, J., Lebrun, M., Dufour, P. and Pichette, A. (2006). Glycosidation of lupine-type triterpenoids as potent in vitro cytotoxic agents. Bioorganic and Medicinal Chemistry 14: 6713-6725.

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Chapter-II, Section-B Hancock, G.E., Speeelman, D.J., Frenchick, P.J., Mineo-Kuhn, M.M., Baggs, R.B. and Hahn, D.J. (1995). Formulation of the purified fusion protein of respiratory syncytial virus with the saponin QS-21 induces protective immune response in Balb/c mice that are similar to those generated by experimental infection. Vaccine 13: 391400. Hostettmann, K. and Marston, A. (1995). Saponins. Cambridge University Press, Cambridge. Kensil, C.R. (1996). Saponins as vaccine adjuvants. Critical Reviews in Therapeutic Drug Carrier Systems 13: 155. Kensil, C.R. and Kammer, R. (1998). QS-21: a water-soluble triterpene glycoside adjuvant. Expert Opinion on Investigational Drugs 7(9): 14751482. Lacaille-Dubois, M. A. (2000). In Saponins in Food, Feedstuffs and Medicinal Plants pp. 205218. Kluwer Academic Publishers: Pays-Bas. Lacaille-Dubois, M.A. (2005). Bioactive saponins with cancer related and immunomodulatory activity: recent developments. Studies in Natural Products Chemistry 32(12): 209246. Leteux, C., Chai, W., Loveless, R.W., Yuen, C.T., Uhlin-Hansen, L., Combarnous, Y., Jankovic, M., Maric, S.C., Misulovin, Z., Nussenzweig, M.C. and Feizi, T. (2000). The Cysteine-rich Domain of the Macrophage Mannose Receptor Is a Multispecific Lectin That Recognizes Chondroitin Sulfates A and B and Sulfated Oligosaccharides of Blood Group Lewis a and Lewis x Types in Addition to the Sulfated N -Glycans of Lutropin Journal of Experimental Medicine 191: 11171126 Liu, J. and Henkel, T. (2002). Traditional Chinese medicine (TCM): are polyphenols and saponins the key ingredients triggering biological activities? Current Medicinal Chemistry 9: 14831485. Moreno, C.A., Rodriguez, R., Oliveira, G.A., Ferreira, V., Nussenzweig, R.S. and Castro Z.R.M. (2000). Preclinical evaluation of a synthetic Plasmodium falciparum MAP malaria vaccine in Aotus monkeys and mice. Vaccine 18: 8999. Oda, K., Matsuda, H.,Murakami, T., Katayama, S., Ohgitani, T. and Yoshikawa M. (2000). Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biological Chemistry 381(1): 6774. Oda, K., Matsuda, H., Murakami, T., Katayama, S., Ohgitani, T. and Yoshikawa, M. (2003). Relationship between adjuvant activity and amphipathic structure of soyasaponins. Vaccine 21(1718): 21452151. Oleszek, W. and Bialy, Z. (2006). Chromatographic determination of plant saponinsan update (20022005). Journal of Chromatography A 1112(12): 7891.

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Chapter-II, Section-B Palatnik de Sousa, C.B., Santos, W.R., Casas, C.P., Paraguai de Souza, E., Tinoco, L.W. and da Silva, B.P. (2004). Protective vaccination against murine visceral leishmaniasis using aldehyde-containing Quillaja saponaria sapogenins. Vaccine 22(19): 24702479. Press, J.B., Reynolds, R.C., May, R.D. and Marciani, D.J. (2000). Structure/function relationships of immunostimulating saponins. Studies in Natural Products Chemistry 24(5): 131174. Rao, A.V. and Gurfinkel, D.M. (2000). The bioactivity of saponins: Triterpenoid and steroidal glycosides. Drug Metabolism and Drug Interactactions 17: 211-235. Rhodes, J. (1989). Evidence for an intercellular covalent reaction essential in antigen specific T cell activation. The Journal of Immunology 143(5): 14821489. Rivera, E. ,Hu, S. and Concha, C. (2003). Gingseng and aluminum hydroxide act synergistically as vaccine adjuvants. Vaccine 21(1112): 11491157. Schmidt, R.R. (1986). New Methods for the Synthesis of Glycosides and Oligosaccharides Are There Alternatives to the Koenigs-Knorr Method? [New Synthetic Methods (56)]. Angewandte Chemie International Edition in English 25(3): 212235. Skene, C.D. and Sutton P. (2006). Saponin-adjuvanted particulate vaccines for clinical use. Methods 40(1): 5359. Soltysik, S., Wu, J.Y., Recchia, J., Wheeler, D.A., Newman, M.J. and Coughlin, R.T. (1995). Structure/ function studies of QS-21 adjuvant: assessment of triterpene aldehyde and glucuronic acid roles in adjuvant function. Vaccine 13(15): 14031410. Sparg, S.G., Light, M.E. and van Staden, J. (2004). Biological activities and distribution of plant saponins. Journal of Ethnopharmacology 94(23): 219243. Stahl, P.D. and Ezekowitzt, R.A.B. (1998). The mannose receptor is a pattern recognition receptor involved in host defense. Current Opinion in Immunology 10: 50-55. Sun, H. and Fang, W.-S. (2006). Structure-activity relationships of oleanane- and ursane- type triterpenoids. Botanical Studies 47: 339-368. Sun, H.X., Qin, F. and Ye, Y.P. (2005). Relationship between haemolytic and adjuvant activity and structure of protopanaxadiol-type saponins from the roots of Panax notoginseng. Vaccine 23(4849): 55335542. Sun, H.X., Yang, Z.G. and Ye, Y.P. (2006). Structure and biological activity of protopanaxatrioltype saponins from the roots of Panax notoginseng. International Immunopharmacology 6(1): 1425.

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Chapter-II, Section-B Sun, H.X., Xie, Y. and Ye, Y.-P. (2009). Advances in saponin-based adjuvants Vaccine 27: 17871796. Sun, H.X., Ye, Y.P. and Pan, Y.J. (2005). Immunological adjuvant saponins from the roots of Panax notoginseng. Chemistry and Biodiversity 2(4): 510515. Takahashi, H., Takeshita, T.,Morein, B., Putney, S., Germain, R.N. and Berzofsky, J.A. (1990). Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope proteins in ISCOMS. Nature 344(6269): 873875. Vincken, J.P., Heng, L. de Groot, A. and Gruppen, H. (2007). Saponins, classification and occurrence in the plant kingdom. Phytochemistry 68: 275-297. Wang, P. G. and Bertozzi, C. R. (ed) (2001). Glycochemistry: Principles Synthesis and Applications pp 167. Marcel Dekker, New York. Winterstein, A. and Stein, G. (1931). Double bond between C-12 and C-13 of oleanolic acid is inert to catalytic hydrogenation. Zeitschrift fr Physiologische Chemie 202: 222-231. Xie, Y., Deng, W., Sun, H.X. and Li, D. (2008). Platycodin D2 is a potential less hemolytic saponin adjuvant eliciting Th1 and Th2 immune responses. International Immunopharmacology 8(8): 11431150. Xie, Y., Ye, Y.P., Sun, H.X. and Li, D. (2008). Contribution of the glycidic moieties to the haemolytic and adjuvant activity of platycodigenin-type saponins from the root of Platycodon grandiflorum. Vaccine 26(2728): 34523460.

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