Synthetic oligomers of
The present invention relates to synthesis of novel higher oligomers and process of preparing the same. In particular the present invention relates to the chemical synthesis of oligomers of Neisseria meningitidis serogroup X (‘hereinafter Men-X), more particularly tetramer. The present invention provides Men-X capsular oligomers obtained from synthetic pathway using purified saccharides of specific chain length and provides said novel oligomers as candidates for the development of conjugate vaccine against bacterial meningitis caused due to Men-X infections.
1. Novel process of synthesizing oligomers of Neisseria meningitidis serogroup X of chemical formula
where n is 3 to 5,
said process comprising the steps of:
synthesizing hemiacetal compound (10), wherein compound (10) is 4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α,β-D-glucopyranose
subjecting said hemiacetal compound (10) to phosphitylating reagent to obtain compound (11) which is subjected to anomerization to obtain propagation unit (12), wherein compound (11) is
and propagation unit (12) is 4-O-Acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl hydrogenphosphonate
synthesizing terminal unit(s) (14) and (14A),
wherein terminal unit (compound 14) is 6-(Benzyloxycarbonyl)aminohexyl 2-deoxy-α-D-glucopyranoside and wherein terminal unit (compound 14A) is 6-(Benzyloxycarbonyl)aminohexyl 2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside
coupling said propagation unit (12) with said terminal unit (14) and coupling said propagation unit (12) with said terminal unit (14A), wherein a molar ratio of compound (12) to compound (14) and compound (14A) is 1:2, in the presence of coupling reagents to obtain a yield in the range of 86% to 90% of compound (15) and (15A) respectively, wherein compound (15) is 6-(Carbobenzyloxy)aminohexyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside, triethylammonium salt and compound (15A) is 6-(Carbobenzyloxy)aminohexyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside, triethylammonium salt
reacting said compounds (15) and (15A) with deacetylating reagents to obtain dimeric compounds (16) and (16A), wherein compound (16) is 6-(Carbobenzyloxy)aminohexyl (2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside, triethylammonium salt and compound (16A) is 6-(Carbobenzyloxy)aminohexyl (2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside, triethylammonium salt
coupling compound (16) to said propagation unit (12) and compound (16A) to said propagation unit (12), wherein a molar ratio of compound (12) to compound (16) and (16A) is 1:3, using said coupling reagents to obtain trimer compounds (17) and (17A) with a yield in the range of 60% to 70%,
wherein compound (17) is 6-(Carbobenzyloxy)aminohexyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside, bis-triethylammonium salt and compound (17A) is 6-(Carbobenzyloxy)aminohexyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside, bis-triethylammonium salt
reacting said compounds (17) and (17A) with deacetylating reagents to obtain triimeric compounds (18) and (18A), wherein compound (18) is 6-(Carbobenzyloxy)aminohexyl (2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside, bis-triethylammonium salt and oligomer (18A) is 6-(Carbobenzyloxy)aminohexyl (2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside, bis-triethylammonium salt
coupling said compound (18) to said propagation unit (12) and said compound (18A) to said propagation unit (12), wherein a molar ratio of compound (12) to compound (18) and compound (18A) is 1:3, using said coupling reagents to obtain tetramer compounds (19) and (19A), with yield in the range of 53% to 58% wherein compound (19) is 6-(Carbobenzyloxy)aminohexyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside, tris-triethylammonium salt and oligomer (19A) is 6-(Carbobenzyloxy)aminohexyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside, tris-triethylammonium salt
optionally performing iterative reactions in the presence of said deacetylating reagents and said coupling reagents to yield a pentamer or hexamer,
performing one step reduction by reductive N-acetylating reagent, deacetylation using said deacetylating reagents and final deprotection of benzyl and Cbz by hydrogenation,
wherein said process results in novel higher synthetic oligomers with high yield capable of being used for the development of a conjugate vaccine against meningococcal serogroup X infection.
2. The novel process of synthesizing oligomers as claimed in claim 1 wherein said novel higher synthetic oligomers are tetramers.
3. The novel process of synthesizing oligomers as claimed in claim 1 wherein the overall reaction time taken to synthesize said oligomers is in the range of 225 hours to 276 hours.
4. The novel process of synthesizing oligomers as claimed in claim 1 wherein said hemiacetal compound (10) is synthesized by:
a. subjecting sugar of compound (2) to diazotransfer reagent and then acetylation in the presence of pyridine to obtain compound (3), wherein compound (2) is D-(+)-Glucosamine hydrochloride
and compound (3) is 1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose
b. reacting compound (3) with donor group at a temperature ranging from 0° C. to room temperature to obtain compound (4), wherein compound (4) is 4-Methylphenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-α,β-D-glucopyranoside
c. reacting compound (4) with deacetylating reagent in organic solvent at a temperature ranging from 0° C. to room temperature to obtain compound (5), wherein compound (5) is 4-Methylphenyl 2-azido-2-deoxy-1-thio-α,β-D-glucopyranoside
d. subjecting compound (5) to benzylidene protection at room temperature resulting in compound (6), wherein compound (6) is 4-Methylphenyl 2-azido-4,6-O-benzylidene-2-deoxy-1-thio-α,β-D-glucopyranoside
e. subjecting compound (6) to benzyl protection at room temperature to obtain compound (7), wherein compound (7) is 4-Methylphenyl 2-azido-4,6-O-benzylidene-3-O-benzyl-2-deoxy-1-thio-α,β-D-glucopyranoside
f. subjecting compound (7) to regioselective benzylidine ring opening reagents at 0° C. to obtain compound (8) which is subjected to acylating reagents at 0° C. to obtain compound (9), wherein compound (8) is 4-Methylphenyl 2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-α,β-D-glucopyranoside
and compound (9) is 4-Methylphenyl 4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-α,β-D-glucopyranoside
g. subjecting compound (9) to thiotolyl deprotection at a temperature ranging from 0° C. to room temperature to obtain hemiacetal compound (10).
5. The novel process of synthesizing oligomers as claimed in claim 1 wherein said termination unit (14, 14A) is synthesized by
subjecting compound 9 to glycosidation reaction resulting in attachment of linker to obtain compound 13, wherein compound 9 is 4-Methylphenyl 4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-α,β-D-glucopyranoside
and compound 13 is
reacting said compound 13 with deacetylating reagents and subjecting to purification by known process to obtain compound 14 and 14A.
6. The novel process of synthesizing oligomers as claimed in claim 1 wherein said coupling reagents are selected from Pivaloyl chloride in presence of pyridine and 1-Adamantanecarbonyl chloride.
7. The novel process of synthesizing oligomers as claimed in claim 1 wherein said deacetylating reagents are selected from Sodium Methoxide and Sodium ethoxide.
8. The novel process of synthesizing oligomers as claimed in claim 1 wherein said final deprotecting reagents are selected from Pd(OH) 2 —H 2 , Pd over carbon-H 2 .
9. The novel process of synthesizing oligomers as claimed in claim 4 wherein said diazotransfer reagent is selected from 1H-imidazole-1-sulfonyl azide and trifluoromethanesulfonyl azide (TfN3).
10. The novel process of synthesizing oligomers as claimed in claim 4 wherein said benzylidene protection is obtained by reacting with reagents selected from benzaldehyde dimethyl acetal [PhCH(OMe) 2 ] and benzaldehyde.
11. The novel process of synthesizing oligomers as claimed in claim 4 wherein said benzyl protection is obtained by reacting with reagents selected from benzyl bromide and sodium hydroxide.
12. The novel process of synthesizing oligomers as claimed in claim 4 wherein the said regioselective ring opening reagent is selected from combination of Triethyl Silane (Et 3 SiH), Boron trifluoride diethyl etherate BF 3 .OEt 2 ), Borane tetrahydrofuran complex (BH 3 .THF) and copper triflate Cu(OTf) 2 .
13. The novel process of synthesizing oligomers as claimed in claim 4 wherein said acylation reagent is Acetic Anhydride (Ac 2 O) in pyridine or 4-Dimethylaminopyridine (DMAP).
14. The novel process of synthesizing oligomers as claimed in claim 4 wherein said thiotolyl deprotection is obtained by reacting with reagents selected from N-Bromosuccinimide (NBS), N-Iodosuccinimide (NIS), and Silver triflate (AgOTf) in an equimolar ratio.
15. The novel process of synthesizing oligomers as claimed in claim 1 wherein said phosphitylating agent is selected from Di-phenyl phosphite pyridine, phosphorous trichloride, imidazole, salicylchlorophosphite, and phosphorous acid.
16. The novel process of synthesizing oligomers as claimed in claim 1 wherein said novel higher synthetic oligomers are selected from the group consisting of oligomers (18, 18A, 19, 19A, 1, and 1A),
wherein oligomer (1) is 6-Aminohexyl (2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-acetamido-2-deoxy-α-D-glucopyranoside, tris-sodium salt and oligomer (1A) is 6-Aminohexyl (2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-(2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate)-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside, tris-sodium salt
17. The novel process of synthesizing oligomers as claimed in claim 16 wherein time required for said final deprotection by hydrogenation for compound (1) is 10 hours to 15 hours and for compound (1A) is 6 hours to 8 hours.
18. The novel process of synthesizing oligomers as claimed in claim 16 wherein the overall reaction time taken to synthesize said oligomers (1) and (1A) is 257 hours.
19. The novel process of synthesizing oligomers as claimed in claim 1 , wherein the steps of coupling said propagation unit (12) with said terminal unit (14) and coupling said propagation unit (12) with said terminal unit (14A) is performed in the presence of coupling reagents at room temperature for 30 minutes followed by oxidation using iodine at −40° C. in pyridine:water at a ratio of 9.75:0.25 for 1.5 hours.