WO2018173068A1 - A novel synthetic neisseria meningitidis serogroup a oligomer and process for synthesizing thereof - Google Patents

Abstract

The present invention relates to novel synthetic Neisseria meningitidis Serogroup A (hereinafter MenA) capsular polysaccharide repeating unit oligomer and a process for synthesizing said synthetic MenA capsular polysaccharide repeating unit oligomer. More specifically, the present invention relates to the chemical synthesis of the tetramer of MenA capsular polysaccharide repeating unit capable of being used as a candidate in the development of semisynthetic or fully synthetic conjugate vaccines as monovalent or as a part of combination vaccines against Meningococcal serogroup A bacterial infection.

Description

TITLE OF THE INVENTION

A Novel Synthetic Neisseria meningitidis Serogroup A oligomer And Process For Synthesizing Thereof

FIELD OF THE INVENTION

The present invention relates to novel synthetic Neisseria meningitidis Serogroup A (hereinafter MenA) capsular polysaccharide repeating unit oligomer and a process for synthesizing said synthetic MenA oligomer. More specifically, the present invention relates to the chemical synthesis of a tetramer of MenA capsular polysaccharide repeating unit capable of being used as a candidate in the development of semisynthetic or fully synthetic conjugate vaccines as monovalent or as a part of combination vaccines against meningococcal serogroup A bacterial infection.

BACKGROUND OF THE INVENTION

N. meningitidis (Meningococcus) is an aerobic gram-negative bacterium that has been serologically classified into 13 groups namely A, B, C, D, 29E, H, I, K, L, W135, X, Y and Z, but about 90% of the infections are due to serogroups A, B, C, Y and W135. The grouping system is based on the capsular polysaccharides of the organism.

N. meningitidis is transmitted by aerosol or direct contact with respiratory secretions of patients or healthy human carriers. As a rule, endemic disease occurs primarily in children and adolescents, with highest attack rates in infants aged 3-12 months, whereas in epidemics older children and young adults may be more involved. However, the rapid progression of meningococcal disease frequently results in death within 1-2 days after onset. Meningococcal disease is a medical emergency requiring immediate diagnosis and treatment. WHO official website mentions that N. meningitidis (Men) is one of the most common causes of bacterial meningitis in the world and the only bacterium capable of generating large epidemics of meningitis. Explosive epidemics with incidence rates of up to 1000 cases per 100,000 inhabitants have been reported, particularly in sub-Saharan Africa. Immunization is the only rational approach to the control of meningococcal disease.

Meningococcal vaccines containing unconjugated purified capsular polysaccharides (A, C, Y and W) have been available since 1970s and are still used to immunise travellers and at risk individuals. Conjugated vaccine containing polysaccharide chemically conjugated to a protein carrier such as the non-toxic diphtheria toxin, CRM 197 or tetanus toxoid are also now available. Vaccines can be formulated as monovalent (group A or C), bivalent (groups A and C) or tetravalent (groups A, C, Y, and W135) vaccines. These vaccines have been used successfully in populations during the last decade to prevent major meningitis epidemics in many parts of the world. However, there is a need to improve the conventional bacterial polysaccharide based vaccines because of risk of handling pathogenic bacteria for production of capsular polysaccharides, impurities from culture harvests and 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 many polysaccharide or conjugate batches.

In the view of the above said need of improvement and advancement in organic chemistry, the focus has increased 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 MenA oligosaccharide are advantageous over conventional vaccines. One of the main advantages with synthetic antigens is synthetic antigens are uniform in size and well characterized which reduce the heterogeneity of the conjugates produced with less batch to batch variation in the final conjugates. Another advantage with synthetic antigens is that these can be engineered to have desired in-built linker for simplifying conjugation. The synthetic antigens also result into better yields with chemical stability. The existing state of art discloses the MenA trimer synthesis process which is a completely deacetylated in the literature by Stefan Osc arson "Synthesis of structures corresponding to the capsular polysaccharide of Neisseria meningitidis group A", Organic & Biomolecular Chemistry 2005, 3, 3782-3787 and patent no. US8062641. The deacetylated synthetic MenA trimer antigen is prone to have low immunogenicity as compared to the acetylated synthetic antigens which mimic the natural bacterial polysaccharide structure. The natural bacterial MenA capsular polysaccharide is a repeating polymer of N-acetyl mannosamine with O- acetyl group on C-3 or C-4 hydroxyl group. The extent of O-acetyl group and its position varies in different bacterial isolates. According to the World Health Organization Technical Report Series 962 Annex 2, MenA polysaccharide should typically contain >61.5% O-acetyl groups per monomeric repeating unit. Thus in view of the above stated low immunogenicity status, there is need to develop the acelylated MenA synthetic antigens with higher oligomers for the development of the conjugated vaccines. OBTECT OF THE INVENTION

To obviate the drawbacks in the prior art, the main object of present invention is to provide a novel synthetic MenA capsular polysaccharide repeating unit oligomer. Another object of the present invention is to provide a novel synthetic MenA capsular polysaccharide repeating unit tetramer.

Yet another object of the present invention is to provide a novel synthetic MenA capsular polysaccharide repeating unit oligomer or derivative thereof which is capable of being used in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection.

Yet another object of the present invention is to obtain a novel synthetic MenA capsular polysaccharide repeating unit tetramer or derivative thereof which is capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection. Yet another object of present invention is to provide a process for synthesizing novel synthetic MenA capsular polysaccharide repeating unit oligomer.

Yet another object of the present invention is to provide a process of synthesizing synthetic MenA tetramer.

Yet another object of the present invention is to provide a process of synthesizing synthetic MenA oligomer capable of being used as a candidate in semisynthetic or synthetic MenA conjugate vaccine wherein said MenA tetramer displays homogeneity, uniformity, high chemical stability, reproducibility and repeatability.

Yet another object of the present invention is to provide a synthetic MenA tetramer capable of being used as a candidate in semisynthetic or synthetic MenA conjugate vaccine wherein said MenA tetramer displays homogeneity, uniformity, high chemical stability, reproducibility and repeatability. Yet another object of the present invention is to provide a synthetic MenA oligomer with improved antigenicity and increased efficacy.

SUMMARY OF THE INVENTION Accordingly, the present invention discloses novel synthetic N. meningitidis serogroup A oligomer and process for synthesizing thereof. More specifically, the present invention relates to the chemical synthesis of tetramer of MenA capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection.

The synthetic MenA oligomer of the present invention displays homogeneity, uniformity, high chemical stability, reproducibility and repeatability. The synthetic MenA oligomer of the present invention also provides improved antigenicity, increased efficacy and is capable of being used in the development of immunogenic monovalent or part of a multivalent conjugate vaccine against meningococcal serogroup A infections. The present invention also discloses a process of synthesizing synthetic MenA oligomer with uniformity in size, chemical stability, reproducibility, repeatability. The oligomer obtained from the process of present invention are capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection with uniformity in size, chemical stability, reproducibility, repeatability.

The process of present invention also provides synthetic MenA oligomer with improved antigenicity, increased efficacy and improved shelf-life and capable of being used in the development of immunogenic monovalent or part of a multivalent conjugate vaccine against meningococcal serogroup A infections. The present invention also discloses the process of synthesizing a cost effective synthetic MenA oligomer.

The production of synthetic MenA oligomer starts with pre-defined starting material which is selected from a group of hexose sugars such as p-D-(+)-Glucose pentaacetate, glucose, any glucose derivative with different protecting groups like benzyl, allyl, chloroacetyl and pivaloyl. The starting material undergoes series of chemical processes resulting into several intermediate compounds to obtain an initiation unit and a propagation unit. Said pre-defined starting material is determined based the scheme of synthesis. The initiation unit and the propagation unit are coupled together at predetermined reaction conditions using coupling reagent to provide a protected dimeric unit.

The protected dimeric unit is treated with at least one deprotecting reagent at predetermined conditions followed by addition of another propagation unit to obtain a protected trimer unit. The trimer undergoes iterative reactions under similar conditions to get protected higher oligomers (A) including tetramer, pentamer, hexamer, heptamer etc. The protected higher oligomers so obtained are subjected to sequential transformation and deprotection of protecting groups resulting in synthetic MenA higher oligomers.

The synthetic higher oligomers of MenA 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 MenA infections.

The synthetic MenA oligomer of the present invention or derivative thereof are capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 depicts HPLC-SEC Chromatogram of MenA tetramer (Compound 20)

Figure 2 depicts Ή-NMR spectrum of Men A tetramer (compound 20)

Figure 3 depicts ¹³C-NMR spectrum of Men A tetramer (compound 20) Figure 4 depicts HPLC-SEC Chromatogram for MenA tetramer (compound 20)- carrier protein conjugate and unconjugated carrier protein.

Figure 5 depicts graphical representation of percentage inhibition of binding of anti-Men-A antibodies to bacterial polysaccharide in an Inhibition ELISA with synthetic Men-A Tetramer (compound 20) and synthetic Men-A Tetramer-TT conjugates (compound 20-TT)

Figure 6 Post 3 dose anti-MenA total IgG GMC and rSBA titers of against Synthetic MenA-CRM conjugate (compound 20-CRM)

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention discloses novel synthetic N. meningitidis serogroup A oligomer and process for synthesizing thereof. More specifically, the present invention relates to the chemical synthesis of tetramer of MenA capsular polysaccharide capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection.

The synthetic MenA oligomer of the present invention or derivative thereof is capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection.

The synthetic MenA oligomer of the present invention comprises of the following structure:

wherein "n" is the number of capsular polysaccharide repeating monomer units ranging from 1 to 9.

The synthetic MenA oligomer of the present invention is preferably a tetramer of compound 20 of structure:

The production of synthetic MenA oligomer starts with pre-defined starting material which is selected from a group of hexose sugar such as β- D-(+)-Glucose pentaacetate, glucose and glucose with protecting groups. The starting material undergoes series of chemical processes resulting into several intermediate compounds to obtain an initiation unit (compound 12) and a propagation unit (compound 10). The intermediate compounds are compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9 and compound 11. The initiation unit and the propagation unit are coupled together at predetermined reaction conditions using coupling reagent to provide a protected dimeric unit.

The protected dimeric unit is treated with atleast one deprotecting reagent at predetermined conditions followed by addition of another propagation unit to obtain a protected trimer unit. The trimer undergoes iterative reactions under similar conditions to get protected higher oligomers (A) including tetramer, pentamer, hexamer, heptamer, octamer, nonamers and further higher oligomers. The protected higher oligomers so obtained are subjected to sequential deprotection of protecting groups resulting in synthetic MenA higher oligomers.

The present invention also provides a process to obtain the synthetic MenA oligomer which is described herein with the help of non-limiting embodiments and examples.

The process to obtain the synthetic MenA oligomer comprises of following steps:

(a) selecting of a pre-defined starting material from a group of Hexose sugar, preferably p-D-(+)-Glucose pentaacetate ,

(b) synthesis of the propagation unit (compound 10) and initiation unit (compound 12) from said selected pre-defined starting material (compound 1) by carrying out a series of chemical processes,

(c) coupling of said initiation unit (compound 12) with said propagation unit (compound 10) to synthesize a protected dimer (compound 13),

(d) deprotection of protecting group of said dimer (compound 13) in presence of at least one deprotecting reagent resulting in a deprotected MenA dimer (compound 14), (e) sequential addition of further propagation units to said dimer via iterating the process of step (c) to obtain protected higher synthetic oligomers,

(f) sequential iterating of reaction of step (d) followed by reaction of step (e) to obtain a desired protected higher synthetic oligomer, and

(g) deprotecting said desired protected higher synthetic oligomer to obtain a desired deprotected higher synthetic oligomer.

The synthetic MenA oligomer of the present invention displays homogeneity, uniformity, high chemical stability, reproducibility and repeatability. The synthetic MenA oligomer of the present invention also provides improved antigenicity, increased efficacy and is capable of being used in the development of immunogenic monovalent or part of a multivalent meningococcal conjugate vaccine(s).

The present invention also discloses a process of producing synthetic MenA oligomer with uniformity in size, chemical stability, reproducibility, repeatability. The oligomer obtained from the process of present invention are capable of being used as a candidate in the development of semisynthetic and synthetic conjugate vaccines against meningococcal serogroup A bacterial infection with uniformity in size, chemical stability, reproducibility, repeatability.

The process of present invention also provides synthetic MenA oligomer with improved antigenicity, increased efficacy and improved shelf-life and capable of being used in the development of MenA polysaccharide-carrier protein conjugates which are capable of being used as a candidate in semi synthetic and/ or synthetic MenA conjugate vaccine. Said MenA conjugate vaccine are immunogenic monovalent or part of a multivalent meningococcal conjugate vaccine. Said MenA polysaccharide-carrier protein conjugates show IgG titre of 125 fold higher or more as compared to those achieved with the vehicle control and show Serum Bactericidal Assay (SBA) titre of 80 fold higher or more as compared to those achieved with the vehicle control.

The novel synthetic MenA capsular polysaccharide repeating unit oligomer so obtained shows high antigenicity, increased efficacy and improved shelf-life. In a preferred embodiment, the synthesis of MenA oligomer with a predefined starting material is disclosed.

The synthesis of MenA tetramer starts with use of p-D-(+)-Glucose pentaacetate as starting material (1). The starting material undergoes series of chemical reactions. The starting material preferably the β-ϋ-(+)- Glucose pentaacetate on glycosylation with ethane thiol provides the α: β mixture of the thioglycoside (2) in a ratio of 50:50. The α : β mixture of the thioglycoside (2) is separated by column chromatography and the desired a-thioglycoside (2) obtained in pure form is used for further steps.

The a-thioglycoside (2) is deacetylated on treatment with sodium methoxide (NaOMe) base in methanol solvent to provide the alcohol

Compound 3. Said Compound 3 is protected with benzylidine protecting group by reacting with benzaldehyde dimethyl acetal in presence of acidic catalyst camphorsulfoninc acid in acetonitrile solvent to provide benzylidene protected compound 4. The C2-OH in compound 4 is converted to its O-triflate on treatment with trifluoromethanesulfonic anhydride and pyridine base in dichloro methane ragioselectively. Then the O-triflate is replaced with azide by reacting with sodium azide to give azide compound 5. Said Compound 5 is subjected to acetylation on treatment with acetic anhydride in basic solvent pyridine to give acetylated compound 6. The benzylidene acetate compound 6 is subjected to regio selective benzylidene ring opening by reacting with dibutylboryl trifluoromethanesulfonate and borane-THF complex at low temperature ranging from -60 °C to -15 °C to give alcohol compound 7. The OTBS tert- butyldimethylsilyl ether protection of compound 7 is carried out by reaction with tert-butyldimethylsilyl chloride (TBDMS-Cl) and base pyridine provided compound 8. Finally, thioethyl deprotection of said compound 8 by reacting with N-bromosuccinimide in acetone provided hemiacetal compound 9. Said Hemiacetal compound 9 on phosphorylation by reacting with diphenyl phosphite gives propagation unit phosphate 10.

The thioglycoside compound 8 is a common starting material for the preparation of the propagation unit 10 as well as the initiation unit compound 12.

Said Compound 8 is subjected to glycosylation with 6-(Z-amino)-l- hexanol in presence of reagents N-iodosuccinimide (NTS) and silver triflate (AfOTf) in dichloromethane at -20°C to give compound 11. Said compound 11 further subjected to tert-butyldimethylsilyl ether (TBDMS) deprotection by reacting with acidic catalyst, camphorsulfoninc acid to provide the initiation unit compound 12. The synthetic scheme is schematically presented as below (Scheme 1):

, pyridine, (1 : 1), 2 h

The phosphonate 10 and the initiation compound 12 is coupled with each other using the pivaloyl chloride reagent followed by oxidation of resulting H-diphosphonate using iodine in single pot to give the dimeric compound 13. The dimeric compound 13 is then subjected to OTBS deprotection using triethylamine trihydrofluoride (TREAT-HF) to free up the 6-OH to give hydroxyl dimer compound 14. The compound 14 is then subjected to the pivaloyl chloride coupling again followed by oxidation using iodine to provide trimer compound 15. The compound 15 is then subjected to the OTBS deprotection by treatment with TREAT-HF in tetrahydrofuran (THF) to provide the hydroxyl trimer compound 16. The hydroxyl trimer compound 16 is subjected to coupling with propagation unit 10 using pivaloyl chloride as a coupling reagent followed by oxidation using iodine to provide tetramer compound 17. The iteration of the pivaloyl chloride coupling and the OTBS deprotection leads to the preparation of higher protected oligomers for MenA. The tetrameric compound 17 is subjected for the OTBS deprotection by reaction with TREAT-HF to give hydroxyl tetramer compound 18, compound 18 is further subjected to azide (N₃) to N-acetyl (NHAc) conversion by reaction with thioacetic acid to provide compound 19. Finally, the O-benzyl (OBn) and the N-carbobenzyloxy (NHCBZ) groups in compound 19 are deprotected by hydrogenation using Palladium hydroxide Pd(OH)₂ catalyst and H₂ gas pressure ranging from 20 to 100 psi followed by Na Exchange in place of NHEt3 salt to give MenA tetramer 20 in pure form.

Said compound 20 is capable of being conjugated with at least one carrier protein (PR) to obtain polysaccharide-protein conjugates with high immunogenicity. The synthetic scheme is schematically presented as below (Scheme 2):

Examples:

Example V. Step 1-Preparation of Ethyl 2,3,4,6-tetra-O-acetyl-l-thio-a-D- glucopyranoside (compound 2)

The 200 g (0.5 mol) of β-D-Glucose pentaacetate has been dissolved in 1L of anhydrous DCM. Ethanethiol (39 mL, 0.53 mol) has been added at once at room temperature under stirring. BF3.OEt₂ (131 mL, 1 mol) has been added slowly at room temperature and the stirring has been continued for 36 h at room temperature. After completion, reaction mixture has been quenched by careful addition of the saturated aqueous NaHC03 solution till effervescences stopped. The organic layer has been separated and the aqueous layer has been washed with 500 mL of DCM. The collected organic layer has been dried over Na₂S0₄ and concentrated in rotavapour. The crude product has been purified by column chromatography (100-200 silica gel, 15% EtOAc in pet ether) to get compound 2 (80 g, 40%) as a white solid. R_f = 0.5 (25% EtOAc in pet ether).

Example 2: Step 2-Preparation of compound 3 and Ethyl 4,6-0- benzylidene-l-thio- a-D-glucopyranoside (compound 4)

To a solution of compound 2 (130 g, 331 mmol) in MeOH (1.2 L) MeONa (25 g, 464 mmol) has been added portion wise at room temperature under nitrogen atmosphere. The reaction mixture has been allowed to stir for 5h at the same condition. Then the reaction mixture has been neutralized with IR-120 Amberlite resin, filtered and concentrated in the reduced pressure to remove MeOH completely. The crude product has been purified by column chromatography (100-200 silica gel, 5-15% MeOH in EtOAc) to get compound 3 (90 g, 87%) as a white solid R_f = 0.3 (15% MeOH in CH2CI2). Without characterization compound 3 has been protected with benzylidene.

To a solution of compound 3 (90g, 401 mmol) in CH3CN (750 mL) PhCH(OMe)₂ (120 mL, 802 mmol) has been added at room temperature under nitrogen atmosphere. Then catalytic amount of camphor sulfonic acid (18.6g, 80 mmol) has been added to the reaction mixture and stirred for about 12 h. After completion of the reaction, the reaction mixture has been concentrated; the residue has been diluted with ethyl acetate (EtOAc) and quenched with saturated sodium bicarbonate solution. The organic layer has been extracted three times with EtOAc. The combined organic layers have been washed with brine, dried over Na₂SO4, concentrated in rotavapour, and crystalized with 5-7% of EtOAc in pet ether (each 500 mL) and filtered through Buckner funnel using vacuum to get compound 4 (115 g, 92% ) as a white solid. Rf = 0.3 (60% EtOAc in pet ether).

Example 3: Step 3-Preparation of Ethyl 2-azido-4,6-0-benzylidene-2- deoxy-l-thio-a-D-mannopyranoside (compound 5)

To a solution of the diol compound 4 (56.1 g, 179.6 mmol) in CH2CI2 (570 mL) pyridine (29 mL, 359.2 mmol) has been added followed by dropwise addition of Tf20 (33 mL, 197.5 mmol) at -20 °C and nitrogen atmosphere. The reaction mixture has been allowed to stir for 3 h and then quenched with saturated sodium bicarbonate solution. The organic layer has been separated three times in CH2Q2, dried over Na2S0_4/ concentrated in reduced pressure and used directly for the next reaction.

The crude triflate has been dissolved in DMF (620 mL) and to it NaN3 (35g, 538.8 mmol) has been added. The reaction mixture has been heated at 85 °C for 2.5 h followed by quenching slowly with ice water in an ice bath. The organic layer has been extracted in EtOAc four times from aqueous layer. The combined organic layers have been washed with brine, dried over Na2S0₄ and concentrated in rotavapour under reduced pressure. The crude product has been purified by column chromatography (100-200 silica gel, 10% EtOAc in pet ether) to get compound 5 (26 g, 43%) as a viscous liquid. Rf = 0.5 (15% EtOAc in pet ether).

Example 4: Step 4-Preparation of Ethyl 3-O-acetyl-2-azido-4,6-O- benzylidene-2-deoxy-l-thio- a -D-mannopyranoside (compound 6)

To a solution of alcohol compound 5 (26 g, 77.1 mmol) in pyridine (100 mL) acetic anhydride (14.6 mL, 154.1 mmol) has been added at room temperature and nitrogen atmosphere followed by catalytic DMAP (471 mg, 3.8 mmol). After 3 h, the pyridine and acetic anhydride have been removed in rotavapour. The reaction mixture has been diluted with water- CH2CI2 and the aqueous layer has been extracted three times with CH2CI2. The combined organic layer has been dried over Na2S0₄ and concentrated under reduced pressure. The crude product has been purified by column chromatography (100-200 silica gel, 10% EtOAc in pet ether) to get compound 6 (24 g, 82%) as a viscous liquid. R_f = 0.3 (10% EtOAc in pet ether).

Example 5: Step 5-Preparation of Ethyl 3-0-acetyl-2-azido-4-0-benzyl-2- deoxy-l-thio-a-D-mannopyranoside (compound 7):

At -60 °C, BH3.THF solution (1 M in THF, 116 mL, 116 mmol) has been added to a solution of benzylidene derivative compound 6 (22 g, 58 mmol) in CH2CI2 (220 mL) under nitrogen atmosphere. After 5 minutes, Bu2BOTf (1 M in CH2CI2, 64 mL, 64 mmol) has been added drop by drop with the help of a syringe and allowed to stir at -15 °C for about 3 h. Then the reaction mixture has been quenched with Et3N (20 mL) followed by MeOH (100 mL) and concentrated under vacuum. The residue has been again diluted with MeOH (100 mL) and concentrated in vacuum. The crude product (48 g) has been directly used in the next reaction (TBS protection). For characterization purpose, a portion of crude sample has been purified by column chromatography (100-200 silica gel, 20% EtOAc in pet ether) to obtain compound 7 as a viscous liquid. Rf = 0.3 (30% EtOAc in pet ether). Example 6: Step 6-Preparation of Ethyl 3-0-acetyl-2-azido-4-0-benzyl-2- deoxy-6-O-tert-butyldimethylsilyl-l-thio-a-D-mannopyranoside

(compound 8):

To a solution of crude alcohol compound 7 (48 g, 125.8 mmol) in CH2CI2 (300 mL) has been added imidazole (17.1 g, 251.7 mmol) at 0 °C followed by TBSC1 (19 g, 125.8 mmol) and DMAP (770 mg, 6.3 mmol). The reaction mixture has been then warmed to room temperature and stirred for about 5 h. After completion, the reaction mixture has been quenched with water. The organic layer has been extracted three times with CH2CI2. The combined organic layer has been dried over Na2S0₄ and concentrated. The purification of the crude residue by column chromatography (100-200, 10% EtOAc in pet ether) gave compound 8 (24 g, 83% in two-steps) as a little viscous colorless liquid. Rf = 0.6 (15% EtOAc in pet ether).

Example 7: Step 7-Preparation of 3-0-Acetyl-2-azido-4-0-benzyl-2- deoxy-6-0-tert-butyldimethylsilyl-( P-D-mannopyranose (compound 9):

To a solution of thiol derivative compound 8 (8.8 g, 17.7 mmol) in acetone (180 mL), water (20 mL), and pyridine (17.2 mL, 213 mmol) has been added a solution of NBS (22.1 g, 124.3 mmol) in acetone (300 mL) slowly at 0 °C. The reaction mixture has been allowed to stir at the same temperature for about 15 minutes. After completion of reaction, it has been quenched with saturated solution of sodium thiosulfate solution. The organic layer has been separated three times in CH2CI2 from aqueous layer. The combined organic layer has been dried over Na2S0₄, concentrated in rotavapor and loaded in the column for purification (100- 200 silica gel, 30% EtOAc in pet ether) to get compound 9 (7 g, 87%) as a viscous liquid. Rf = 0.1 (30% EtOAc in pet ether).

Example 8: Step 8-Preparation of 3-0-Acetyl-2-azido-4-0-benzyl-2- deoxy-6-O-tert-butyldimethylsilyl-a-D-mannopyranosyl

hydrogenphosphonate triethylammonium salt (compound 10):

To a solution of compound 9 (7 g, 15.5 mmol) in pyridine (70 mL) has been added diphenyl phosphite (12 mL, 62.0 mmol) at room temperature under nitrogen atmosphere. After 2 h, a suspension of Et3N (35 mL) and water (35 mL) has been added drop by drop to the reaction mixture at 0 °C and then warmed to room temperature and stirred for 2 h. The reaction mixture has been diluted in toluene and concentrated in the rotavapor twice. The reaction mixture has been again diluted with water-CL C and the aqueous layer has been extracted three times with CH2CI2. The combined organic layer has been dried over Na2S0₄ and concentrated under reduced pressure. The crude product has been purified by column chromatography (230-400 silica gel, 4% methanol in CH2CI2) to get phosphonate compound 10 (7.5 g, 78%) as a viscous liquid. Rf = 0.3 (10% methanol in CH2CI2). Example 9: Step 9-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl 3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-6-0-tert-butyldimethylsilyl-a-D- mannopyranoside (Compound 11):

To a solution of compound 8 (5 g, 10.1 mmol) and 6-(Z-Amino)-l-hexanol (2.79 g, 11.1 mmol) in anhydrous CH2CI2 (50 mL), NIS has been added (3.4 g, 15.1 mmol) followed by AgOTf (259 mg, 1.0 mmol) at temperature ranging from 0 to -20 °C in nitrogen atmosphere. After 1 h, the reaction mixture has been quenched with saturated aqueous solution of Na2S₂03. The reaction mixture has been diluted with water-C!H Cb and the aqueous layer has been extracted three times with CH2CI2. The combined organic layer has been dried over Na2S0₄ and concentrated in rotavapor. The crude product compound 11 (8 g) has been directly used in the next reaction (TBS deprotection). For characterization purpose, a portion of crude sample has been purified by column chromatography (100-200 silica gel, 15% EtOAc in pet ether) to get compound 11 as a dense liquid. Rf = 0.6 (30% EtOAc in pet ether).

Example 10: Step 10-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl 3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D-mannopyranoside

(compound 12):

To a solution of crude TBS compound 11 (8 g, 11.4 mmol) in MeOH (50 mL), a catalytic amount of camphor sulfonic acid has been added (1.33 g, 5.7 mmol) at room temperature and stirred for about 2 h. The reaction mixture has been then concentrated in reduced pressure and diluted with CH2CI2 followed by quenching with saturated sodium bicarbonate solution. The organic layer has been separated and the aqueous layer extracted with CH2CI2 for two to three times. The combined organic layer has been dried over Na2S0₄ and concentrated in rotavapor. The residue has been purified by column chromatography (100-200 silica gel, 15% EtOAc in pet ether) to have alcohol compound 12 (4 g, 95%, over two- steps) as a viscous liquid. Rf = 0.3 (20% EtOAc in pet ether).

Example 11: Step 11-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl ()-( 3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-6-0-tert-butyldimethylsilyl- a-D-mannopyranosyl phosphate)-0-(l-»6)-3-0-acetyl-2-azido-4-0- benzyl-2-deoxy-a-D-mannopyranoside, triethylammonium salt (compound 13):

¾ NHCbz

A solution of alcohol compound 12 (2 g, 3.4 mmol) and phosphite compound 10 (4.2 g, 6.8 mmol) in anhydrous pyridine (10 mL) has been concentrated in rotavapor thrice and the residue has been kept in vacuum for about 1 h. The residue has been then dissolved in anhydrous pyridine (30 mL) and pivaloyl chloride (1.3 mL, 10.2 mmol) has been added to it drop by drop at room temperature and allowed to stir for 1.5 h. After that, the reaction mixture has been cooled to -40 °C and a solution of iodine (4.3 g, 17.0 mmol) in pyridine: water (20 mL: 1 mL) has been added to it drop by drop with the help of syringe. The stirring has been continued for about 2.5 h and then 0.5 h at 0 °C. The reaction mixture has been then quenched with Na2S₂03 solution and diluted with water-CLbCb and the aqueous layer has been extracted four times with CH2CI2. The combined organic layer has been dried over Na2S0₄ and concentrated under reduced pressure. The crude product has been purified by column chromatography (230-400 silica gel, Acetone: water: Et3N = 97:2:1) to get dimer compound 13 (3.7 g, 92%) as a white amorphous solid. Rf = 0.5 (in Acetone: water: Et₃N = 98:1:1).

Example 12: Step 12-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl 0-(3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D-mannopyranosyl phosphate)-0-(l->6)-3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D- mannopyranoside, triethylammonium salt (compound 14):

To a solution of dimeric TBS compound 13 (5.2 g, 4.4 mmol) in THF (50 mL) has been added TREAT-HF (3.6 mL, 21.9 mmol) at room temperature under nitrogen atmosphere and stirred for about 20 h. The reaction mixture has been then quenched with saturated sodium bicarbonate solution at 0 °C and diluted with CH2CI2. The organic layer has been extracted from the aqueous layer with CH2CI2 for four times. The combined organic layer has been dried over Na₂S0₄ and concentrated in rotavapour. The residue has been purified by column chromatography (230-400 silica gel, Acetone: water: Et3N = 95:4:1) to get alcohol compound 14 (2.6 g, 55%) as a white amorphous solid. Rf = 0.5 (in Acetone: water: Et₃N = 97:2:1).

Example 13: Step 13-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl ()-( 3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-6-0-tert-butyldimethylsilyl- a-D-mannopyranosyl phosphate)-0-(l- 6)-(3-0-acetyl-2-azido-4-0- benzyl-2-deoxy-a-D-mannopyranosyl phosphate)-(l-»6)-3-0-acetyl-2- azido-4-0-benzyl-2-deoxy-a-D-mannopyranoside, bis- triethylammonium salt (compound 15):

-BpNHCbz

The procedure used for the synthesis of compound 13 has been followed here. Alcohol compound 14 (1.5 g, 1.4 mmol), phosphite compound 10 (2.6 g, 4.2 mmol), pivaloyl chloride (0.9 mL, 7 mmol) in anhydrous pyridine (20 mL) followed by iodine (1.4 g, 5.6 mmol) in pyridine: water (15 mL: 1 mL) has been used. The crude product has been purified by column chromatography (230-400 silica gel, Acetone: water: Et3N = 93:6:1) to get compound 15 (2.1 g, 89%) as a white amorphous solid. Rf = 0.3 (in

Acetone: water: Et₃N = 95:4:1).

Example 14: Step 14-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl 0-(3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D-mannopyranosyl phosphate)-0-(l→6)-(3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D- mannopyranosyl phosphate)-(l— »6)-3-0-acetyl-2-azido-4-0-benzyl-2- deoxy-a-D-mannopyranoside, bis-triethylammonium salt (compound

16):

To a solution of TBS compound 15 (1.6 g, 0.95 mmol) in THF (20 mL) has been added TREAT-HF (1.6 mL, 9.5 mmol) at room temperature under nitrogen atmosphere and stirred for about 15 h. The reaction mixture has been then quenched with saturated sodium bicarbonate solution at 0 °C and diluted with CH2CI2. The organic layer has been extracted from the aqueous layer with CH2CI2 for four times. The combined organic layer has been dried over Na2S0₄ and concentrated in rotavapor. The residue has been purified by column chromatography (230-400 silica gel, Acetone: water: Et3N = 93:6:1) to get alcohol compound 16 (1.16 g, 78%) as a white amorphous solid. Rf = 0.25 (in Acetone: water: Et3N = 95:4:1). Example 15: Step 15-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl 0-(3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-6-0-tert-butyldimethylsilyl- a-D-mannopyranosyl phosphate)-0-(l-»6)-(3-0-acetyl-2-azido-4-0- benzyl-2-deoxy-a-D-mannopyranosyl phosphate)-0-(l-»6)-(3-0-acetyl- 2-azido-4-0-benzyl-2-deoxy-a-D-mannopyranosyl phosphate)-(l-»6)-3- 0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D-mannopyranoside, tris- triethylammonium salt (compound 17):

The procedure used for the synthesis of compound 13 has been followed here. Alcohol compound 16 (1.16 g, 0.74 mmol), phosphite compound 10 (1.37 g, 2.21 mmol), pivaloyl chloride (0.5 mL, 3.7 mmol) in anhydrous pyridine (15 mL) followed by iodine (750 mg, 2.95 mmol) in pyridine: water (10 mL: 1 mL) has been used. The crude product has been purified by column chromatography (230-400 silica gel, Acetone: water: Et3N = 92:7:1) to get compound 17 (1.22 g, 76%) as a white amorphous solid. Rf = 0.4 (in Acetone: water: Et3N = 94:5:1).

Example 16: Step 16-Preparation of 6-(N-Benzyloxycarbonyl)aminohexyl

0-(3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D-mannopyranosyl phosphate)-0-(l-»6)-(3-0-acetyl-2-azido-4-0-benzyl-2-deoxy-a-D- mannopyranosyl phosphate)-0-(l-»6)-(3-0-acetyl-2-azido-4-0-benzyl-2- deoxy-a-D-mannopyranosyl phosphate)-(l-»6)-3-0-acetyl-2-azido-4-0- benzyl-2-deoxy-a-D-mannopyranoside, tris-triethylammonium salt (compound 18):

To a solution of TBS compound 17 (550 mg, 0.25 mmol) in THF (6 mL) TREAT-HF has been added (2 mL, 12 mmol, 48 eq.) slowly at room temperature under nitrogen atmosphere and heated at 55 °C for 3.5 h. The reaction mixture has been then quenched with saturated sodium bicarbonate solution at 0 °C and diluted with CH2CI2. The organic layer has been extracted from the aqueous layer with CH2CI2 for four times. The combined organic layer has been dried over Na2S0₄ and concentrated in rotavapor. The residue has been purified by column chromatography (230-400 silica gel, Acetone: water: Et3N = 91:8:1) to get alcohol compound 18 (435 mg, 83%) as a white amorphous solid. Rf = 0.4 (in Acetone: water: Et₃N = 93:6:1).

Example 17: Step 17-Preparation of 6-Aminohexyl-0-(2-acetamido-3-0- acetyl-2-deoxy-a-D-mannopyranosyl phosphate)-(l— »6)-(2-acetamido-3- O-acetyl-2-deoxy-a-D-mannopyranosyl phosphate)-(l- 6)-(2-acetamido- 3-0-acetyl-2-deoxy-a-D-mannopyranosyl phosphate)-(l -»6)-2- acetamido-3-O-acetyl-2-deoxy-a-D-mannopyranoside, trisodium salt (compound 20):

To a solution of alcohol compound 18 (220 mg, 0.1 mmol) in pyridine (4 mL), thioacetic acid has been added (2.4 mL) slowly at room temperature in nitrogen atmosphere and heated at 42 °C for 4.5 h. The reaction mixture has been then cooled to room temperature followed by directly loading on the column for purification (230-400 silica gel, Acetone: water: Et3N = 90:9:1) to get amide compound 19 (190 mg, 84%) as a white amorphous solid. R_f = 0.3 (in Acetone: water: Et₃N = 93:6:1).

To a solution of amide compound 19 (100 mg, 0.47mmol) in MeOH: Water (10 mL: 10 mL) Pd(OH)₂-C ( 25 mg) has been added. The reaction mixture has been then stirred vigorously in a round bottom flask at 25 °C under a hydrogen atmosphere in baloon. After nearly 15 h, the reaction mixture has been filtered through celite and concentrated in rotavapor (by keeping the water bath temperature less than 20 °C). The white solid residue thus obtained has been then dissolved in water (20 mL) and Na exchange resin Dowex 50W X8(500 mg) has been added to it. The mixture has been stirred for about 3 h at 2-4 °C. The reaction mixture has been then filtered through Whatman filter paper and solution has been concentrated in rotavapor (by keeping the water bath temperature less than 20 °C). The white solid residue thus obtained has been then purified in GPC to get final product compound 20 (40 mg, 61%) as a white solid.

Analytical characterization of Men A tetramer (Compound 20):

The MenA tetramer has been checked for its purity by HPLC-size exclusion chromatography (HPLC-SEC). The analysis has been carried out using Waters HPLC system with RI detector. 0.1 m Nitrate buffer mobile phase has been used for analysis. The chromatogram for said MenA tetramer shows single peak covering 100% area indicating homogeneity and purity of greater than 95% (Fig. 1). The structural identity of the synthetic MenA tetramer (compound 20) of the present invention with the MenA tetramer obtained from naturally occurring Neisseria meningitidis Serogroup A has been established by NMR characterization. NMR characterization conducted by ^aHNMR for Men A tetramer (Compound 20) has been recorded over 400 MhZ NMR instrument from Bruker. The dry Men A tetramer samples have been dissolved in D₂0 for NMR recording and the spectra is shown in figure 2. NMR characterization conducted by ¹³C-NMR spectra for compound 20 has been recorded in D₂0 on 400 MhZ NMR instrument from Bruker and the spectra is shown in figure 3. Both the Figure 2 and Figure 3 show desired NMR peaks and confirm the structural identity of the synthetic MenA tetramer (compound 20) of the present invention with the structure of repeating units of polysaccharide obtained from naturally occurring Neisseria meningitidis Serogroup A. Preparation of MenA tetrasaccharide-tetanus toxoid (TT) and CRM (compound 20-TT/CRM) conjugates

The conjugate of the synthesized MenA tetrasaccharide (compound 20) with tetanus toxoid (TT) and CRM has been prepared through the amino linker present at the reducing end of compound 20. The free amino group of compound 20 has been reacted with S-acetylthioglycolic acid-N- hydroxysuccinimide ester (SATA) followed by treatment with hydroxylamine hydrochloride to furnish glycan derivative attached to a linker with free thiol (-SH) group. The oligosaccharide (OS) and SH content have been analyzed by the methods of Chen and Ellman, respectively. In another experiment, the TT protein has been modified by reacting with 3-(maleimido) propionic acid-N-hydroxysuccinimide ester (BMPS) to generate TT-maleimide conjugate derivative. Protein content is determined by Lowry method and maleimide labelling is estimated by Ellman method. Thiolated compound 20 and maleimide linked TT have been coupled together to furnish compound 20-TT conjugate. The saccharide content in the compound 20-TT conjugate has been calculated by Chen's assay and protein content has been calculated by Lowry assay. The glycan-protein w/w ratio has been theoretically calculated by dividing saccharide content by protein content and the ratio has been found to be in the range of 0.3 to 0.35. The yield for the conjugation process has been observed in the range from 35% to 45% . Similarly, the Men tetramer (compound 20) -CRM conjugates (compound 20-CRM) have been prepared by using identical protocol.

Analytical characterization of MenA tetramer (compound 20)-carrier protein conjugates and the unconjugated carrier protein: The MenA (compound 20)-carrier protein conjugates and unconjugated carrier protein have been tested for the total oligosaccharide carrier protein ratio and free oligosaccharide in the purified conjugates. The HP- SEC analysis of the conjugates has been carried out using Water's HPLC system with RI detector. The 0.1 molar nitrate buffer has been used as mobile phase for the analysis The chromatogram for MenA tetramer and its conjugate with CRM is shown in figure 4. The peak A in chromatogram indicates the MenA (compound 20)-carrier protein conjugates and the peak B indicates the unconjugated carrier protein.

Table 1: Oligosaccharide-carrier protein (TT) ratio, free oligosaccharide present in the purified conjugate and conjugate yield percentage for MenA (compound 20)-CRM/TT conjugates

Determination of antigenic properties of synthetic Men-A tetramer and Men-A tetramer-TT conjugate

The antigenicity of synthetic MenA tetramer and semi-synthetic MenA tetramer-TT conjugate has been estimated in relation to no-antigen control in a competitive enzyme-linked immune-sorbent assay (Inhibition-ELISA) experiment. The compound 20 (MenA tetramer) and compound 20-TT conjugate have been able to neutralize the specific antiserum against N. meningitidis serogroup A significantly and inhibit the binding of antibodies to the bacterial MenA polysaccharide coated on the plate. Unconjugated synthetic MenA tetramer has showed lower inhibition (up to 59% inhibition) compared to its conjugate (up to 75% inhibition) at all different concentrations tested i.e. 12.5-400 μg/ ml. Data obtained from the inhibition ELISA experiment demonstrated that both synthetic MenA tetramer (compound 20) and compound 20-TT conjugate are antigenic and able to neutralize the rabbit antiserum against N. meningitidis serogroup A. The inhibition increased with increase in concentration of antigen used in the assay. Both MenA tetramer as well as the MenA tetramer-TT conjugates has showed observable inhibitions from the starting concentration of 25 μg/ ml itself (Fig. 5). These findings potentially target towards development of an immunogenic semi-synthetic glycoconjugate prepared using synthetic MenA tetramer and TT.

Evaluation of immunogenicity of MenA-CRM conjugates in mice

Groups of 8 female BALB/ c mice (5-8 weeks old) have been immunized at 2 week interval with 1-3 μg of MenA-CRM conjugates via subcutaneous route. Normal saline alone has been used for negative (vehicle) control group. Sera have been collected at 7-14 days after each injection. Specific anti-OS IgG antibody titers have been estimated by indirect ELISA and anti-meningococcal serogroup A functional antibodies have been titrated using serum bactericidal assay.

Determination of serum anti-meningococcal serogroup A antibodies against Synthetic MenA-CRM conjugates by Indirect ELISA Ninety-six-well plates (Nunc Maxisorp) have been coated with mixture of a 5 μg/ ml PS and m-HSA in PBS buffer, pH 7.4 by adding 100 μΐ per well. Plates have been incubated overnight at 4 °C, and then washed three times with assay wash buffer containing Brij 35 in PBS, pH 7.4 and blocked with blocking solution containing FBS and Brij 35 in PBS, pH 7.4. Each incubation step has been followed by three PBS buffer wash. Reference and test sera samples have been diluted in blocking buffer (0.1% Brij 35, 5% FBS in PBS, pH 7.4, transferred into coated-blocked plates, and serially twofold diluted followed by overnight incubation at 4 °C. Then 100 μΐ per well of 1:1000 diluted peroxidase conjugated anti-mouse IgG has been added and left for 1 hour at 25 °C. Further substrate, 3, 3', 5, 5' - tetramethylbenzidine-H2O2 has been added for color development. After 10 minutes of development at 25 °C, reaction has been stopped by adding 50 μΐ of 2 M H2SO4, and Optical density of each well has been measured at 450 nm on micro plate reader. Using a standard curve generated from different dilutions of reference serum Anti-MenA polysaccharide IgG concentration (in terms of ELISA Units/ml) for each formulation has been evaluated at different time points using Combistat software and the geometric mean concentrations (IgG GMC) (Fig 6).

Serum Bactericidal Assay (SBA) for the functional antibody titration against synthetic MenA-CRM Conjugates

N. meningitidis serogroup A bacterial stock (ATCC® 13102™) have been grown overnight on sheep blood agar plate at 37°C with 5% CO2. Isolated colonies have been picked and incubated on the surface of another sheep blood agar plate at 37°C with 5% CO2. A loopful bacteria have been scraped and suspended in of assay buffer containing bovine serum albumin in Hank's balanced salt solution. The optical density (OD₆5o) of the suspension has been adjusted to achieve the working dilution of ~1 x 10⁵ colony-forming units per ml. Quality control (QC) sera and test sera samples have been heat inactivated for 30 min at 56 °C. In microwell plate, 20 μΐ of serial two fold dilutions of test serum have been mixed with 10 μΐ of bacteria at the working dilution and 10 μΐ of baby rabbit complement. Negative control wells had everything except the test serum. The well contents have been mixed by gently tapping the assay plate and incubated the plates for 1 hour at 37°C with 5% CO2. 10 μΐ, sample from each well has been plated on blood agar plate and the plates have been incubated overnight and colonies have been counted. The highest serum dilution showing > 50% decrease in colony-forming units per ml after incubation of bacteria with reaction mixture, as compared to respective active complement control has been considered as the SBA titer.

Immunogenicity comparison with vehicle control:

The IgG-ELISA and serum bactericidal assay results reveals that the MenA tetramer-CRM conjugates of the present invention result in high total IgG antibody titres. The OS-PR conjugates after 3 doses at 1 and 3 μg dose display more than 4 fold higher and up to 125 fold higher IgG titres than the vehicle control titers for meningitis A oligosaccharide conjugate. The functional antibody titers (also referred to as SBA titers) have been observed 80 fold higher than the vehicle control (Fig 6).

Claims

claim:

l. A novel synthetic Neisseria meningitidis serogroup A (MenA) capsular polysaccharide repeating unit oligomer or derivative thereof comprising of formula :

wherein "n" is the number of capsular polysaccharide repeating monomer units ranging from 1 to 9.

The novel synthetic MenA oligomer as claimed in claim 1, wherein said oligomer is a tetramer (Compound 20) or derivative thereof

The novel synthetic MenA oligomer as claimed in claim 1, wherein said novel synthetic MenA oligomer is obtained from at least one predefined starting material (compound 1) selected from a group of hexose sugars with protecting groups preferably β-D-Glucose pentaacetate, 2-0,3-0,4-0,5-0,6-O-Pentabenzyl-D-glucose.

The novel synthetic MenA oligomer as claimed in claim 1, wherein said oligomer comprises of:

- an initiation unit (compound 12); and

-a propagation unit (compound 10).

The novel synthetic MenA oligomer as claimed in claim 4, wherein said initiation unit (compound 12) comprises of formula:

The novel synthetic MenA oligomer as claimed in claim 4, wherein said propagation unit (compound 10) comprises of formula:

The novel synthetic MenA oligomer as claimed in claim 1, wherein said oligomer or derivative thereof has homogeneity, uniformity, high chemical stability, reproducibility and high immunogenicity.

The novel synthetic MenA oligomer as claimed in claim 7, wherein said oligomer has homogeneity and purity of greater than 95% .

The novel synthetic MenA oligomer as claimed in claim 1, wherein said oligomer mimics the structure of natural polysaccharide as evident by binding to anti-MenA polysaccharide antibodies in an inhibition immunoassay.

10. The novel synthetic MenA oligomer as claimed in claim 1, wherein said oligomer or derivative thereof is capable of being used as a candidate in semisynthetic or synthetic MenA conjugate vaccine monovalent or as a part of combination vaccines.

11. The novel synthetic MenA oligomer as claimed in claim 1, wherein said oligomer or derivative thereof is capable of being conjugated with at least one carrier protein and/ or at least one synthetic polypeptide to obtain a semi-synthetic and/ or synthetic MenA polysaccharide-carrier protein conjugate, wherein said at least one carrier protein is selected from, but not limited to, TT (tetanus toxoid), DT (Diphtheria toxoid), CRM197 (non-toxic mutant of DT), OMPV (Outer membrane protein vesicles).

12. The novel synthetic MenA oligomer as claimed in claim 11, wherein said semi-synthetic and/ or synthetic MenA polysaccharide-carrier protein conjugate is capable of being used as a candidate in semisynthetic or synthetic monovalent MenA conjugate vaccine or as a part of combination vaccines.

13. The novel synthetic MenA oligomer as claimed in claim 11, wherein said semi-synthetic and/ or synthetic MenA polysaccharide -carrier protein conjugate shows IgG titre exceeding 125 fold as compared to those achieved with the vehicle control and shows Serum Bactericidal Assay (SBA) titre exceeding 80 fold as compared to those achieved with the vehicle control.

14. The novel synthetic MenA oligomer as claimed in claim 11, wherein said semi-synthetic and/ or synthetic MenA polysaccharide -carrier protein conjugate provides conjugation yield in the range of 40±5 % . A process for synthesizing said novel synthetic MenA oligomer as claimed in claim 1, wherein the process comprising the steps of:

(a) selecting at least one pre-defined starting material (compound 1), wherein said pre-defined starting material is determined on the basis of scheme of synthesis;

(b) conducting a series of chemical processes with said predefined starting material of step (a) to obtain an initiation unit (compound 12) and a propagation unit (compound 10);

(c) coupling of said initiation unit with said propagation unit of step (b) at predetermined reaction conditions to obtain a protected dimeric compound (compound 13);

(d) treating said protected dimeric compound of step (c) with at least one deprotecting reagent including but not limited to TREAT-HF resulting in a deprotected MenA dimer (compound 14);

(e) adding another said propagation unit of step (b) to said deprotected MenA dimer of step (d) to obtain a protected trimer compound

(compound 15);

(f) sequential iterating of reaction of step (d) followed by reaction of step (e) to obtain a desired protected higher synthetic oligomer; and

(g) deprotecting said desired protected higher synthetic oligomer to obtain a desired deprotected higher synthetic oligomer; wherein said desired deprotected higher synthetic oligomer is capable of being used as a candidate in semisynthetic or synthetic MenA conjugate vaccine.

16. The process for synthesizing said novel synthetic MenA oligomer as claimed in claim 15, wherein said at least one pre-defined starting material (compound 1) is selected from a group of hexose sugars with protecting groups preferably β-D-Glucose pentaacetate, 2-0,3- 0,4-0,5-0,6-0-Pentabenzyl-D-glucose.

The process for synthesizing said novel synthetic MenA oligomer as claimed in claim 15, wherein said series of chemical processes to obtain said initiation unit (compound 12) comprises the steps of:

(a) performing glycosylation of said at least one pre-defined starting material (compound 1) in the presence of glycosyl acceptor including but not limited to ethane thiol to obtain a compound 2 followed by deacetylation in presence of organic solvent to obtain a compound 3;

(b) protecting said compound 3 of step (a) with a protecting group preferably benzylidene protecting group including but not limited tobenzaldehyde dimethyl acetal in presence of acidic catalyst including but not limited to camphorsulfonic acid in acetonitrile solvent to obtain a benzylidene protected compound 4, followed by converting C2-OH of said compound 4 to its O-triflate on treatment with triflate forming reagent including but not limited to trifluoromethanesulfonic anhydride and pyridine base in dichloromethane region- selectively followed by replacement of O-triflate with azide by reacting with sodium azide to obtain a compound 5;

(c) acetylating said compound 5 of step (b) with acetic anhydride in basic solvent pyridine to obtain a acetylated compound 6;

(d) subjecting said compound 6 to regio selective benzylidene ring opening by reacting with regioselective ring opening reagent including but not limited to dibutylboryl trifluoromethanesulfonate and borane-THF complex at low temperature ranging from -60 °C to -15 °C to obtain a compound 7;

(e) protecting said compound 7 with a protecting group including but not limited to OTBS to obtain a compound 8;

(f) subjecting said compound 8 to glycosylation with glycosyl acceptor including but not limited to 6-(Z-amino)-l-hexanol in presence of reagents N-iodosuccinimide (NIS) and silver triflate (AgOTf) in dichloromethane at temperature ranging from 0 to -20°C to obtain a compound 11; and

(g) deprotecting said compound 11 with acidic catalyst to obtain said initiation unit (compound 12).

The process for synthesizing said novel synthetic MenA oligomer as claimed in claim 15, wherein said series of chemical processes to obtain said propagation unit (compound 10) comprises the steps of:

(a) deprotecting said compound 8 with at least one deprotecting group to obtain a compound 9; and

(b) phosphorylating said compound 9 with at least one phosphorylating reagent including but not limited to diphenyl phosphite to obtain said propagation unit (compound 10).

The process for synthesizing said novel synthetic MenA oligomer as claimed in claim 15, wherein said predetermined reaction conditions comprise of presence of at least one coupling reagent including but not limited to pivaloyl chloride and Pyridine-water ratio of 9.75:0.25 to 9.95:0.05.

The process for synthesizing said novel synthetic MenA oligomer as claimed in claim 19, wherein said predetermined reaction conditions further comprises of temperature wherein said temperature is maintained from 0°C to room temperature for 90±10 min and then dropped up to -40°C or below.

21. The process for synthesizing said novel synthetic MenA oligomer as claimed in claim 15, wherein said deprotecting of said desired protected higher synthetic oligomer comprises the steps of:

(a) OTBS deprotection of said desired protected higher synthetic oligomer in the presence of deprotecting group including but not limited to triethylamine trihydrofluoride (TREAT-HF) to obtain a hydroxylated higher synthetic oligomer;

(b) Amide formation of azides of said hydroxylated higher synthetic oligomer with thio acids preferably thioacetic acid to obtain a N-acetylated product;

(c) deprotecting said protecting groups by hydrogenation under predefined reaction conditions followed by Na Exchange in place of NHEt3 salt wherein said reaction conditions comprises of presence of catalyst preferably but not limited to Palladium hydroxide, H₂ gas pressure ranging from 20 to 100 psi.

The process for synthesizing said novel synthetic MenA oligomer as claimed in claim 15, wherein said desired higher oligomer is preferably a tetramer.