NOVEL NEISSERIA MENINGITIDIS SEROGROUP Y OLIGOMERS AND PROCESS FOR SYNTHESIZING THEREOF FIELD OF THE INVENTION

The present invention relates to novel oligomers of Neisseria meningitidis serogroup Y capsular polysaccharide repeating unit ('hereinafter Men-Y oligomer). The present invention also relates to a process for synthesizing novel N. meningitidis serogroup Y oligomers. More specifically, the present invention relates to the chemical synthesis of the tetramer of N. meningitidis serogroup Y ('hereinafter Men-Y tetramer) capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup Y bacterial infection. BACKGROUND OF THE INVENTION

Bacterial meningitis causes approximately 1, 70, 000 annual deaths, with at least 5-10% of case fatality in industrialized countries and a 20% case fatality in the developing world. Streptococcus pneumoniae, Haemophilus influenzae type b (Hib) and N. meningitidis are responsible for most of the cases of bacterial meningitis worldwide. However, N. meningitidis still remains a major cause of bacterial meningitis and other invasive bacterial infections despite the availability of modern antibiotics.

Meningococcal disease is a medical emergency requiring immediate diagnosis and treatment.

In total 13 different serogroups namely A, B, C, D, 29E, H, I, K, L, W135, X, Y and Z of N. meningitidis have so far been identified, but about 90% of the infections are due to serogroups A, B, C, Y and W135. The following types of vaccines are available for meningococcal infection:

• Polysaccharide vaccines which are available for over 30 years to prevent the disease. Meningococcal polysaccharide vaccines are available in various combinations e.g. either bivalent (groups A and C), trivalent (groups A, C and W), or tetravalent (groups A, C, Y and W) forms to control the disease.

• Meningococcal conjugate vaccines against group C have been available since 1999. Tetravalent A, C, Y and W conjugate vaccines are widely used and have been licensed since 2005 for use in children and adults in different parts of world including Canada, the United States of America, Europe and India. Other combinations of the serogroups are also available in licensed conjugate vaccines. The currently available meningococcal conjugate vaccines except monovalent group A conjugate vaccine are out of reach for needy people in low resource countries due to their high cost.

Presently, vaccines available for Men-Y utilize the polysaccharide isolated from bacterial source which are associated with various risks like danger in handling the live bacterial culture. Also, many a times the isolated bacterial polysaccharide is so heterogeneous that it fails to pass the desired homogeneity criteria. The heterogeneous nature of the bacterial polysaccharide provides the heterogeneity in the conjugates prepared using it leading to large variations and hence, disqualification of the conjugate batches.

During the last decade there has been an increasing focus on organic synthesis of bacterial antigens including analogues of capsular oligosaccharides. In view of the existing state of art, the meningococcal conjugate vaccines containing synthetic Men-Y oligosaccharide are advantageous over conventional vaccines. The synthetic antigens are uniform in size and well characterised which reduce the heterogeneity of the conjugates produced with less batch to batch variation in the final conjugates. One of the main advantages with synthetic antigens is that these can be engineered with desired linker for simplifying conjugation as well as resulting into better yields.

Presently there is no prior art disclosing the production and purification procedures of organic synthesis of Men-Y. The International patent application no. PCT/US2013/ 042428 "Multivalent meningococcal conjugates and methods for preparing conjugates" relates to immunogenic conjugates including at least one polysaccharide or protein conjugated to a Neisseria surface protein, which can elicit immune responses against meningococcal polysaccharides (PS) from groups A, C, W-135, and Y. The said International patent application discloses only general statement on preparation of Men Y oligomers but there is no enabling disclosure on the preparation of Men-Y oligomer. Moreover, the existing art available for other serogroups of N. meningitidis are either time consuming or give rise to a mixture of different sizes of oligomers.

OBTECT OF THE INVENTION

The main object of present invention is to provide novel oligomers of Neisseria meningitidis serogroup Y capsular polysaccharide repeating unit.

Another object of present invention is to provide novel oligomers of Neisseria meningitidis serogroup Y capsular polysaccharide repeating unit capable of being used as candidates in the development of synthetic and semisynthetic conjugate vaccines against meningococcal bacterial infection.

Yet another object of the present invention is to provide novel Men-Y tetramer capable of being used as a candidate in the development of semisynthetic or fully synthetic vaccine against meningococcal serogroup Y bacterial infection.

Yet another object of the present invention is to provide a chemical process of synthesizing Men-Y oligomers using purified saccharides with specific chain length.

Yet another object of the present invention is to provide a chemical process of synthesizing Men-Y oligomers with improved antigenicity capable of producing immunogenic conjugate vaccine.

Yet another object of the present invention is to provide a process for the preparation of synthetic Men-Y capsular oligomers which meet the physico-chemical quality standards for the purity.

Yet another object of the invention is to provide cost effective Men-Y oligomers with increased efficacy and improved shelf-life when conjugated to a carrier protein. SUMMARY OF THE INVENTION

Accordingly, the present invention discloses N. meningitidis serogroup Y oligomers and synthetic process to obtain thereof. Said Men-Y oligomers are capable of being used as candidates in the development of semisynthetic and synthetic conjugate vaccine against meningococcal serogroup Y bacterial infection after conjugation to a suitable carrier protein. Said vaccine can be administered by parental routes.

The Men-Y oligomers of the present invention possess improved antigenicity and improved shelf-life. Said Men-Y oligomers also meet the physico-chemical quality standards for the purity and are cost effective.

The process of present invention discloses synthesis of Men-Y oligomers comprising of the two main building blocks; an initiation unit and a propagation unit. The initiation unit is prepared by the glycosidation of suitably protected monosaccharide more particularly but not limited to neuraminic acid with another suitably protected monosaccharide. Said another suitably protected monosaccharide is selected from hexose, more particularly but not limited to glucose. The propagation unit is prepared by glycosidation of suitably protected monosaccharide more particularly but not limited to neuraminic acid with another suitably protected monosaccharide more particularly hexose, more particularly but not limited to glucose. The initiation unit and the propagation unit are coupled at predetermined temperature together using catalyst to provide the tetrasaccharide or dimeric unit. The catalyst used in the said coupling is glycosilation reagent/ Lewis acid catalyst selected from but not limited to Kilo do succinimide (ΝΓ3), Trifluoromethanesulfonic acid (TfOH), Trimethylsilyltrifluoromethanesulfonate (TMSOTf), Silver trifluoromethanesulfonate (CFsSOsAg). The dimeric unit is reacted with a basic reagent such as but not limited to sodium methoxide to facilitate ring opening. The dimeric unit so obtained is again reacted with propagation unit resulting in the formation of trimer. The trimer undergoes iterative reactions under similar conditions to get protected higher oligomers (Y) including tetramer, pentamer, hexamer, heptamer etc.

The protected higher oligomers so obtained are subjected to sequential deprotection of protecting groups resulting in Men-Y higher oligomers.

The higher oligomers of N. meningitidis serogroup Y so obtained have improved yields, high efficacy and are capable of being used as a candidate for development of conjugate vaccine which confers protection against disease due to Men-Y infections.

All the illustrative steps devised for the synthesis of Men-Y oligomers using novel approach result in better yield of tetramer, purity of >95% shown in Figure 8 and enhanced antigenicity as shown in Figure 9.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 depicts Ή-NMR of Men-Y tetramer (compound 26)

Figure 2 depicts ¹³ C-NMR of Men-Y tetramer

Figure 3 depicts COSY NMR spectrum of Men-Y tetramer

Figure 4 depicts HMBC NMR spectrum of Men-Y tetramer

Figure 5 depicts HSQC NMR spectrum of Men-Y tetramer

Figure 6 depicts TOCSY NMR spectrum of Men-Y tetramer

Figure 7 depicts DEPT NMR spectrum of Men-Y tetramer

Figure 8 depicts HPSEC analysis for purity of Men-Y tetramer

Figure 9 depicts antigenicity analysis of Men-Y tetramer and Men-Y

tetramer-tetanus toxoid (TT) conjugate by inhibition ELBA against anti-Men-Y polyclonal sera

DETAILED DESCRIPTION OF THE INVENTION Accordingly, the present invention relates to novel Men-Y oligomers. The present invention further relates to a process for synthesizing novel Men-Y oligomers. More specifically, the present invention relates to the chemical synthesis of Men-Y tetramer capable of being used as a candidate in the development of semisynthetic and synthetic vaccine against meningococcal bacterial infection. Said vaccine can be administered with suitable mode of administration more particularly via parental route. Said synthesis being accomplished in the following steps:

Step 1: Synthesis of Propagation Unit (Compound 18 as shown in Scheme I) Step 2: Synthesis of Initiation Unit (Compound 20 as shown in Scheme II)

Step 3: Synthesis of Higher Oligomers (Compound 21, 22, 23, 24, 25, d26 as shown in Scheme III)

Before the preferred embodiment of the present invention is described, it is understood that this invention is not limited to the particular materials described, as they may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiment only, and is not intended to limit the scope of the invention in any way. It must be noted that as used herein, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise.

In one of the non-limiting example of said process, N-acetyl neuraminic acid which is compound 1 undergoes methylation in presence of catalyst, such as Dowex 50W X8 (H ⁺ ) resin to obtain compound 2. The compound 2 so obtained undergoes acylation in presence of acylating agents such as acetic anhydride in presence of pyridine to give compound 3. This process helps in shielding of hydroxyl groups. The compound 3 so obtained undergoes selective halogenation more particularly chlorination in presence of halogenating agents such as acetylcholride and HC1 to obtain compound 4. Compound 4 reacts with thiolating reagent Such as p-methylphenylthiol (TolSH) in presence of DIPEA also known as Hunig's base (poor nucleophile) resulting in compound 5 with -Stol group attached to 2 ^nd position. The compound 5 so obtained undergoes deacylation in presence of MeOH, methanesulfonic acid (MsOH) to give compound 6. The compound 6 so obtained reacts with carbonylation reagent such as Triphosgene, NaHC03 in presence of MeCN/H ₂ O and undergoes oxazolidinones ring formation to yield compound 7. The compound 7 so obtained is subjected to protection of hydroxyl groups present at terminal position in presence of protecting reagents such as ferf-butyldiphenylsilyl chloride TBDPSC1/DMF, and then for acetonide protection using 2,2-DMP, followed by acylation using AcCl to yield compound 8. The compound 8 so obtained is reacted with reacting agents such as dibutyl phosphate (Bu ₂ P0 ₄ H) in presence of NIS, TfOH, DCM to obtain a phosphate leaving group at terminal position which increases alpha selectivity as compound 9.

Commercially purchased compound 10 β-D-glucose pentaacetate is reacted with thiocresol and (catalyst) BF3:OEt ₂ in DCM (solvent) to protect alpha position yielding compound 11. Compound 11 undergoes deacetylation in presence of MeONa in MeOH resulting in compound 12. The compound 12 so obtained reacts with silyl protecting group such as ferf-butyldiphenylsilyl chloride (TBDPS-C1) thereby protecting -OH group at 6 ^th position of compound 12 gives compound 13 with 80% yield. The compound 13 so obtained undergoes alkylation and protection of specific position with benzylation to yield compound 14 with 76% yield of reaction at 0°C to room temperature. The above mentioned process is a known process.

Compound 15 is then obtained with yield in the range of 85% to 95% by recovering one of the protected hydroxyl group of Compound 14 by reacting compound 14 with deprotecting reagents such as Tetra-n- butylammoniumfluoride (TBAF) in Tetrahydrofuran (THF). The compound 14 acts as an intermediate in the synthesis of Propagation Unit 18.

Remaining amount of compound 14 so obtained undergoes glycosidation with 5-azido-hexanol to yield compound 16. The yield of compound 16 so obtained is in the range of 65% to 75%, more particularly 70% . The compound 16 is subjected to silyl group deprotection reagent such as TBAF in THF solvent resulting in the removal of sterically hindering - OTBDPS group to yield compound 17. The above reaction yield compound 17 with an overall increase in Alpha:Beta ratio (95:5) along with yield of reaction in the range of 55% to 65% more particularly 60% The compound 17 so obtained acts as an intermediate in the synthesis of Initiation Unit 20.

One part of Compound 9 is then reacted with compound 15 at low temperatures, more particularly in the range of -80°C to -60°C in presence of catalyst such as TMSOTf to yield Propagation unit (compound 18).

Other remaining part of Compound 9 is then reacted with compound 17 at low temperatures, more particularly in the range of -80°C to -60°C in presence of catalyst such as TMSOTf to yield compound 19. Compound 19 so obtained is reacted with a basic reagent such as NaOMe in presence of Methanol (MeOH)/ Dichloromethane (DCM) to yield Initiation Unit (compound 20). The Initiation Unit (20) and the Propagation Unit (18) are coupled together using catalyst at low temperatures, more particularly in the range of -50°C to— 30°C to provide the tetrasaccharide or dimeric unit (21). The catalyst used in said coupling is but not limited to NB/TfOH /Trimethylsilyltrifluoromethanesulfonate (TMSOTf)/ Silver trifluoromethanesulfonate (CFsSOsAg) in presence of solvent. The catalyst activates thiotolyl (Stol) group of compound 18. After activation it is prone to removal and then the Stol group is replaced by free hydroxy group of Initiation Unit (20). The dimeric unit (21) is reacted with a basic reagent, such as but not limited to Sodium methoxide, which facilitate ring opening resulting into protected dimeric unit (22). Thereafter the dimer so obtained is subjected to iterative reactions under similar conditions in presence of said catalyst and said basic reagent to yield a higher synthetic oligomers including trimer, tetramer, pentamer, hexamer, heptamer, 24, 26, 28....n) etc.

The dimeric unit (22) so obtained is again reacted with propagation unit under similar conditions of temperature and atmospheric pressure resulting in the formation of trimer (23, 24). The trimer so obtained is again reacted with propagation unit under similar conditions to obtain tetramer unit (25). The tetramer undergoes iterative reactions under similar conditions to get higher oligomers (Y) including tetramer, pentamer, hexamer, heptamer, etc. The higher oligomers so obtained are subjected to final deprotection of protecting groups in presence of deprotecting reagents such as BF3:OEt ₂ ACN, NaOH, MeOH, H ₂ /Pd(OH) ₂ . The sequential deprotection of protecting groups in presence of a deprotecting reagent resulting in deprotected Men-Y higher oligomers (d22, d24, d26, d28.... dn)

The total time taken for said process is in the range of 330 hours to 400 hours and more particularly is 370 hours, resulting in significantly low cost of production. The higher oligomers so obtained are linked to carrier proteins by conjugation through in-built linker to obtain a semisynthetic conjugate vaccine against meningococcal serogroup Y bacterial infection. This chemical process of synthesis can also be employed for the preparation of synthetic protein/ peptide to prepare fully synthetic conjugate vaccine as well. It is noted that such conjugation with carrier proteins result in vaccines with good yield and enhanced antigenicity.

In one of the embodiment Men-Y tetramer is tested for antigenicity by conducting inhibition enzyme-linked immunosorbent assay (Inhibition ELISA) and the tetramer and its conjugate with tetanus toxoid is found to give neutralization of specific antibodies against Men-Y capsular polysaccharide.

Complete scheme for MenY tetramer synthesis:

Scheme I: Synthesis of propagation unit 18 Synthesis of the sialic acid phosphate donor:

Synthesis of Glucose acceptors 15 & 17

Scheme II: Synthesis of Initiation unit 20

Scheme III: Synthesis of dimer trimer and tetramer

Synthesis of the dimer trimer and tetrameric units

The above detailed description of process is illustrated by non-limiting examples as follows:

Examples:

Example V. Step 1-Preparation of compound 2

Procedure: To a stirred solution of N-acetyl Neuraminic acid (2.0 kg, 6.472 mol) in methanol (32 lit); Dowex 50W X8 resin is added. Reaction is stirred at RT for 24h. Reaction is monitored by TLC (20% methanol; DCM). After completion, reaction mixture was filtered, resin was washed with methanol (2 lit). The filtrate was concentrated to give required product as white solid (2.0 kg; 95%).

Example 2: Step 2-Preparation of compound 3

Procedure: To a mechanically stirred solution of compound 2 (1 kg, 3.095 mol) in pyridine (10 lit), DMAP (38 g, 0.0309 mol) was added at 0°C followed by acetic anhydride (11.7 lit). Reaction was stirred at RT for 24 h. After completion reaction mixture was concentrated under vacuum (10 mbar). Crude compound was purified by flash column chromatography over silica-gel (230-400 mesh) eluting with 30% ethyl acetate-hexanes to 100% ethyl acetate. Compound-3 was obtained as white foamy solid (1.4 kg; 87%).

Example 3: Step 3-Preparation of compound 4

4

Procedure: A stirred solution of compound-3 (1.4 kg, 2.626 mol) in acetyl chloride (7.5 lit) was cooled to 0°C; and a solution of freshly prepared HC1 in acetyl chloride (4.5 lit) was added. The resulting reaction mixture was warmed to RT and stirred for 16h. After completion, reaction mixture was concentrated under reduced pressure. Obtained compound was co- distilled with toluene (2*2.5 lit), and the crude product was forwarded to the next step (1.4 kg, 99%).

Example 4: Step 4: Preparation of compound 5

5

Procedure: To a mechanically stirred solution of compound-4 (1.35 kg, 2.652 mol) in DCM (13.5 lit) p-thiocresol (324 g, 2.652 mol) was added. The solution was cooled to 0°C and diisopropylethyl amine (548 ml, 3.135 mol) was added slowly with dropping funnel for 30 mins under nitrogen atmosphere. The resulting reaction mixture was warmed to RT and stirred for 18 h. Reaction was monitored by TLC, after completion; reaction mixture was cooled to 0°C and diluted with DCM (5.0 lit), washed with water (2*5 lit). Organic layer was separated, washed with brine solution, dried over sodium sulphate and concentrated. Obtained compound was treated with 25% ethyl acetate/ petroleum ethers. Resulting white solid was filtered and dried (900 g, 60%).

Example 5: Step 5-Pre aration of compound 6

Procedure: To a stirred solution of compound-5 (500 g, 0.8375 mol) in methanol (5.0 lit), methane sulphonic acid (322 g, 3.35 mol) was added slowly at 0°C. The resulting reaction mixture was heated to reflux for 36 h. The reaction mixture was cooled to 0 °C and quenched with triethyl amine (790.0ml). The mixture was concentrated and the residue was taken to next step without any further purification. LC MS purity 96 % . (Crude weight:

650 g) Example 6: Step 6-Preparation of compound 7

7

Procedure: Compound-6 was dissolved in a mechanically stirred mixture of acetonitrile (4.0 lit), water (5.0 lit) and NaHCO ₃ (2.352 kg, 46.51 moles), (1.8 kg, 4.651 moles) at room temperature. Reaction mixture was cooled with ice-bath to 0°C and a solution of triphosgene (0.895 kg, 4.651 moles) in acetonitrile (1.0 lit) was added drop wise over a period of 60 mins. Reaction was stirred at 0°C for additional 3 h. After completion, the reaction mixture was neutralized with 10% HC1 (3 lit). Product was extracted with ethyl acetate (2*4.0 lit). Organic layer was washed with water, brine, dried over Na ₂ SO ₄ and concentrated. Crude compound was purified by flash column chromatography, eluting with 2-5 % methanol; DCM. Compound-6 was obtained as off-white solid (700 g, 56% for 2 steps).

Example 7: Ste 7-Preparation of compound 7A

Procedure: To a stirred solution of compound-7 (700 g, 1.694 mol) in DMF (6.0 lit), imidazole (288 g, 4.237 mol) was added. t-ButylDiphenylsilyl chloride (557 g, 2.033 mol) was added slowly at 0°C for 10 mins. Reaction temperature was raised to RT and stirred for 16h. The progress of the reaction was monitored by TLC. After completion, RM was diluted with water (10 lit). Product was extracted with ethyl acetate (2*4 lit). Organic layer was separated, washed with brine, dried over Na ₂ SO ₄ and concentrated. Product was purified by flash column chromatography over silica-gel using 60% ethyl acetate-hexanes as eluent to yield off-white solid (1000 g, 90%).

Example 8:

Procedure: To a stirred solution of Compound-7A (1050 g, 1.697 mol) in 2, 2 -dimethoxy propane (7 lit) camphorsulphonic acid (470 g, 2.036 mol) was added. The mixture was stirred at RT for 3h. The progress of the reaction was monitored by TLC. After completion by TLC; reaction mixture was quenched with triethyl amine (350 mL; 2.515 mol). Reaction mixture was concentrated. Crude compound was purified by flash column chromatography over silica gel. Compound-7B was obtained as off-white foamy solid (1000 g, 92%).

Example 9: St -Preparation of compound 8

Procedure: To a stirred solution of compound-7B (590g, 0.8538 mol) in DCM (5.0 lit) diisopropylethyl amine (1500 ml, 8.538 mol) was added. The solution was cooled to 0°C, acetyl chloride (490 ml, 6.83 mol) was added slowly with dropping funnel for 10 mins under nitrogen atmosphere. The resulting reaction mixture was warmed to RT and stirred for 18 h. Reaction was monitored by TLC, after completion; reaction mixture was cooled to 0°C and diluted with DCM (3 lit), washed with water (3 lit). Organic layer was separated, washed with brine solution, dried over sodium sulphate and concentrated. Product was purified on silica column chromatography; 10-20% ethyl acetate; hexanes as eluent (Yield: 500 g, 80%).

Example 10: Step 1 -Preparation of compound 9

Procedure: To a stirred mixture of Compound-8 (100 g, 0.1364 mol) and di butyl phosphate (54 mL; 0. 2728 mol) in DCM (1 Lit.) under nitrogen atmosphere, 4A° MS (Activated Under high vacuum at 140°C for 2h; 100 g, wt/ wt) was added. The mixture was stirred at RT for 2h then cooled to 0°C and ΝΓ3 (61.11 g, 0.2728 mol) followed by triflic acid (3.62 mL, 0.0409 mol) was added. Reaction temperature was maintained at 0°C for 5h. The progress of the reaction was monitored by TLC. After completion, reaction mixture was quenched with saturated hypo solution (250 ml). It was filtered through celite, and extracted. Organic layer was separated and then washed with water, dried using Na ₂ SO ₄ and concentrated. Crude compound was purified by flash column chromatography over silicagel. Compound-9 was obtained as sticky material (80.0 g, 70%).

Example 11: Step 11-Preparation of compound 11

Procedure: β-D-Glucose pentaacetate 10 (500 g, 1.2816 mol), p-thiocresol (255 g, 2.050mol) and 4A° Molecular sieves powder (500 g) were added in dichloromethane (5 lit) under argon atmosphere in a flame dried flask. The solution was cooled to 0 °C and BF ₃ Et ₂ 0 (49%; 1000 ml, 3.5886 mol) was added. The mixture was stirred for 16 h. The reaction mixture was diluted with dichloromethane (2 lit), washed with sodium hydrogen carbonate (2x2.5 lit of a saturated aqueous solution) and then with water (3 lit). The organic layer dried over Na ₂ S0 ₄ filtered and concentrated in vacuum. The residue was purified by flash column chromatography to afford compound-ll(417 g, 72% ) as a white solid.

Example 12: Step 12-Preparation of compound 12

12 Procedure: Compound 11 (417 g, 0.9182mol) was dissolved in methanol (4.0 lit) under argon atmosphere in a flame dried RB flask. The solution of sodium methoxide in methanol (39.6 ml of a 25% solution) was added and the resulting mixture stirred for 2 h. After completion, amberlite IR-120 ⁺ ion exchange resin was added portion wise until the solution becomes neutral. The reaction mixture filtered to remove resin and the filtrate was concentrated in vacuum to afford compound 12 (254 g, 95%) as a white crystalline solid. Example 13: Step 13-

Procedure: To a stirred solution of compound-12 (200 g, 0.402 moles) in DMF (2.0 lit), imidazole (114 g, 1.678 moles) was added. To above solution t-butyl diphenylsilyl chloride (269 g, 0.979 moles) was added slowly at 0°C for 10 mins. Reaction temperature was raised to RT and stirred for 16 h. The progress of the reaction was monitored by TLC. After completion, reaction mixture was diluted with water (4 lit). Product was extracted with ethyl acetate (2*1 lit). Organic layer was separated, washed with brine, dried over Na ₂ SO ₄ and concentrated. Product was purified by flash column chromatography over silica-gel using 60% ethyl acetate-hexanes as eluent to yield off-white solid (260 g, 72%).

Example 14: Step 14-Preparation of compound 14

Procedure: A stirred mixture of compound 13 (260 g, 0.4962 mol) and benzyl bromide (273 ml, 2.2328 mol) in DMF (2.5 lit) was cooled to 0°C, sodium hydride (99 g, 2.480 mol) was added portion wise at 0°C. RM was stirred at RT for 16h. TLC was showing SM consumed, then RM was quenched with ice cold water (4 lit), and extracted with ethyl acetate (2Xllit). Organic layer was separated and concentrated under reduced pressure. Crude compound was purified by flash column chromatography, product was eluted with 5% ethyl acetate-hexanes gave Compound 14 as a colorless liquid (300 g, 76% yield).

Example 15: Step 15-Preparation of compound 15

15

Procedure: Compound 14 (145 g, 0.1826 mol) was dissolved in THF (1.4 lit), was cooled to 0°C and 1M TBAF in THF (219 mL, 0.2191 mol) was added. Cooling bath was removed and the reaction mixture was stirred for 16h at RT. Reaction was monitored by TLC, after completion reaction was diluted with water, extracted with ethyl acetate (2*500 ml). Organic layer was dried over Na2S0 _4/ filtered and concentrated. Crude product was purified by FCC, eluted with 20% EtOAc/hexane. Compound was obtained as viscous liquid (90 g, 89%). Example 16: Step 16-Preparation of compound 16

16

Procedure: To a stirred mixture of Compound-14 (50 g, 0.0629 mol) and 6- azidohexanol (18 g; 0.1259 mol) in diethyl ether (500 ml) under nitrogen atmosphere, 4A° MS (Activated under high vacuum at 140°C for 2h; 50 g, wt/wt) was added. The mixture was stirred at RT for 2h and then was cooled to -40°C. NIS (28.2 g, 0.1259 mol) followed by triflic acid (2.8 ml, 0.0314 mol) was added and stirring continued for 2h. After completion by TLC, reaction mixture was quenched with addition of saturated hypo solution (100 ml). The reaction mixture filtered through celite and the filtrate transferred to a separating funnel. Organic layer was separated, washed with water, dried over Na ₂ SO ₄ and concentrated. Crude compound was purified by flash column chromatography, eluted with 10% ethyl acetate and hexane. Compound-16 the anomeric mixture was obtained as sticky material (35 g, 70%).

Example 17: Step 17-Preparation of compound 17

Procedure: Compound 16 (100 g, 0.123 mol) was dissolved in THF (1.0 nt), cooled to 0°C and 1M TBAF in THF (148 mL, 0.1476 mol) was added. Cooling bath was removed and stirred for 16h at RT. Reaction was monitored by TLC. After completion, reaction was diluted with water, extracted with ethyl acetate (2*500ml). Organic layer was dried over Na ₂ S04, filtered and concentrated. Crude product was purified by flash column chromatography, eluted with 20% EtOAc/hexane. Compound was obtained as viscous liquid (55g, 78%).

Example 18:

Procedure: A mixture of compound-15 (43.3 g, 0.0778 mol) and compound-9 (84 g; 0.1307 mol) was dissolved in DCM (430 ml) and was added MS 4A° (45 g). The reaction mixture was stirred at RT for 2h. It was then cooled to -78°C and TMSOTf (15.58 mL, 0.0855 mol) was added. Reaction was maintained at -78°C and stirred for lh. The progress of the reaction was monitored by TLC, after completion; reaction mixture was quenched with triethyl amine (1.2 eq.) filtered through celite. Organic layer was washed with saturated sodium bicarbonate solution (300 mL), organic layer was separated and washed with water and brine solution, dried over Na ₂ SO ₄ , filtered and concentrated. Crude compound was purified by flash column chromatography; eluted with 30% ethyl acetate and hexane gave compound-18 as a white solid (85 g, 93%).

Exa - Preparation of compound 19

19

Procedure: To the mixture of compound-17 (45 g, 0.0778 mol) and compound-9 (85 g; 0.1037 mol) in DCM (450 mL) was added MS 4A° (45 g) and the resulting reaction mixture stirred at RT for 2h. It was then cooled to -78°C, and TMSOTf (15.66 mL, 0.0855 mol) was added. Stirring continued for 2h at same temperature. The progress of the reaction was monitored by TLC. After completion, reaction mixture was quenched with triethyl amine (1.2 eq.) filtered through celite. Organic layer was washed with saturated sodium bicarbonate solution (500 mL), followed by water and brine solution, dried over Na ₂ SO4, filtered and concentrated. Crude compound was purified by flash column chromatography, eluted with 30% ethyl acetate and hexane to provide compound -19 as a white foamy solid (80 g, 86%).

Example 20: Step 20- Preparation of compound 20 TBDPSO

Procedure: Compound-19 (80 g, 0.0676 mol) was dissolved in methanol (533 mL) and DCM (267 mL) under argon atmosphere. Solution of sodium methoxide in methanol (14.6 mL, 25% NaOMe in methanol) was added and the mixture stirred at RT for 1 h. After completion of reaction, Ion exchange resin (IR 120+) was added portion wise until the solution was neutralized. The reaction mixture was filtered, the filtrate concentrated and the crude product purified by flash column chromatography (ethyl acetate: petroleum ether 2:1 as eluent) to provide compound-20 (55 g, 70%) as a white foamy solid.

Example 21: Step 21- Preparation of compound 21

Procedure: To a stirred solution of compound-18 (53 g, 0.0453 mol) and Compound-20 (35 g; 0.0302 mol) in diethyl ether (350 mL) 4A° molecular sieves (35 g) was added and stirred at room temperature for 2h. The mixture was cooled to 0°C. To this mixture NIS (13.54 g, 9.030 mmol) was added in one lot followed by triflic acid (1.34 mL, 0.0151 mol). Reaction temperature was raised to RT and maintained for 16h. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was quenched with saturated sodium thiosulfate solution (250 ml), diluted with ethyl acetate (300 ml). It was filtered through celite and the organic layer was separated. The organic layer was washed with water, dried over anhydrous Na ₂ S04, filtered and concentrated. Crude compound was purified by flash column chromatography eluted with 30% ethyl acetate and hexane. Compound-21 was obtained as white solid (32 g, 48%).

Exa — Preparation of compound 22

Procedure: Compound-21 (10 g, 4.5454 mmol) was dissolved in methanol (65 mL) and DCM (35 mL) under argon atmosphere in a flame dried flask. Solution of sodium methoxide in methanol (0.982 mL of a 25% in methanol) was added and the mixture stirred at RT for 1 h. After completion of reaction, the reaction mixture quenched using amberlite IR 120 was added portion wise until the solution was neutralized. The reaction mixture filtered, the filtrate was concentrated and purified by flash column chromatography (ethyl acetate: petroleum ether 2:1-3:1 as eluent) to afford compound-22 (6.1 g, 62%) as a white foamy solid.

Exa - Preparation of compound 23

Procedure: To a stirred solution of compound-22 (15 g, 6.899 mmol) and Compound-18 (12 g; 10.349 mmol) in dry diethyl ether (150 mL) 4A° molecular sieves (15 g) was added and the mixture stirred for 2h at RT. The mixture was cooled to 0°C. To this mixture NIS (3.1 g, 13.790 mmol) followed by triflic acid (0.306 ml, 3.450 mmol) was added. The resulting reaction mixture was stirred at RT for 16h. After completion by TLC, the reaction mixture was quenched with addition of saturated sodium thiosulfate solution (150 ml). The reaction mixture filtered through celite and the filtrate organic layer was washed with water and brine, dried over anhydrous Na ₂ S04, filtered and concentrated. Crude compound was purified by flash column chromatography eluting with 50% ethyl acetate and hexane. Compound-23 was obtained as white foamy solid (12 g, 41%).

Example 24: Step 24- Preparation of compound 24

Procedure: Compound 23 (12 g, 3.732 mmol) was dissolved in methanol (80 mL) and DCM (40 mL) under argon atmosphere in a flame dried flask. Solution of sodium methoxide in methanol (0.806 mL of a 25%) was added and the mixture stirred at 0°C for 1 h. After completion by TLC, the reaction mixture quenched by portion wise addition of ion exchange resin (IR 120+) until the solution was neutralized. The reaction mixture filtered, concentrated and the crude product purified by flash column chromatography (ethyl acetate: petroleum ether 2:1 as eluent) to afford compound 24 (7 g, 60%) as a white foamy solid.

Example 25: S

Procedure: To a stirred solution of Compound-24 (4.1 g, 1.4733 mmol) and Compound-18 (3.1 g; 2.6505 mmol) in diethyl ether (40 mL) 4A° molecular sieves (4.1 g) was added and stirred at room temperature for 2h. NIS (0.34 g, 1.5175 mmol) was added at once followed by addition of triflic acid (0.13 mL, 1.4733 mmol) at RT. The stirring continued at RT for 60h. The progress of the reaction was monitored by TLC (35% ethyl acetate; hexanes). After completion, the reaction mixture was quenched with saturated sodium thiosulfate solution (40 mL). It was filtered through celite and the organic layer was separated. The organic layer was washed with water, dried over anhydrous Na ₂ S04, filtered and concentrated. Crude compound was purified by flash column chromatography eluting with 20-30% ethyl acetate and hexane. Compound-25 was obtained as white solid (2.06g, 38%).

Example 26: - Preparation of compound 25A

Procedure: Compound-25 (3 g, 0.7088 mmol) was dissolved in methanol (624 mL) and DCM (12 mL) under argon atmosphere. Solution of sodium methoxide in methanol (0.184 mL, 25%) was added and the mixture stirred at RT for 1 h. After completion by TLC reaction mixture was quenched using Ion exchange resin amberlite IR 120 until the solution was neutralized. The mixture was filtered and concentrated. The crude product purified by flash column chromatography (ethyl acetate: petroleum ether 2:1) to afford compound-25A (1.7 g, 58%) as a white solid.

Example 27: - Preparation of compound 25B

Procedure: Compound 25A (1.7 g, 0.4040 mmol) was dissolved in acetonitrile (45 mL) and cooled to 0°C. 45% BF ₃ ; OEt ₂ (7.65 mL, 24.2453 mmol) was added and the stirring continued for 3h. The reaction mixture was diluted with ethyl acetate (100 mL), washed with saturated aqueous NaHCOs (80 mL) and then with brine solution. The organic layer was then dried over Na ₂ S0 _4/ filtered and concentrated.

The residue washed with hexane and the resulting solid product obtained was taken in ethanol and water. Lithium hydroxide was added (0.17g, 4.0408 mmol) and the reaction was stirred at 80°C for overnight. The solvent was removed under reduced pressure; crude material was dissolved in water (20 mL), washed with diethyl ether (20 mL) and the aqueous layer separated. pH of the aqueous layer was adjusted to 5 using IN HC1 (0.4 mL) solution. Product was extracted with 10% methanol:DCM (2*60 mL). Organic layer dried over Na ₂ SO _4/ filtered and concentrated to yield the compound 25B (0.8 g; 80% for two steps). Example 28: Step 28- Preparation of compound d26

Procedure: Compound-25B (0.8 g, 0.2638 mmol) was dissolved in a mixture of methanol (50 mL) and water (12 m 1) in a 250 mL hydrogenation flask. 20% Pd (OH) ₂ /C (0.8 g) was added under nitrogen atmosphere. The flask was purged with hydrogen for three times and then the content was stirred under hydrogen atmosphere (100 psi) for 24 h. After the completion the reaction mixture was filtered through celite and concentrated under reduced pressure at 25°C. Product was purified by gel filtration through Sephadex-G-10 column, eluted with water. Compound fractions collected and lyophilized to obtain Men-Y tetramer (d26) as white solid (350 mg; 70%). Example 29: Antigenicity analysis of Men-Y tetramer and Men-Y tetramer-tetanus toxoid (TT) conjugate

Procedure: The ELISA plate was coated with bacterial polysaccharide which was reacted with polyclonal serum raised against anti-Men-Y bacterial capsular polysaccharide with or without inhibition with different concentrations (0.39-100 microgram/ ml) of Men-Y tetramer or Men-Y tetramer-TT conjugate prepared using thio-ether chemistry. When the reaction was developed using HRP labelled secondary antibody, both the Men-Y tetramer as well as its conjugate neutralized the Men-Y specific IgGs evident with reduction in the optical density (% inhibition) after adsorption of serum dilution with different antigen concentrations as compared to no antigen control as shown in Figure 9.