Compounds and methods for chemical and chemo-enzymatic synthesis of complex glycans
View Patent ↗The present invention provides chemical and chemo-enzymatic methods for the synthesis of a wide array of complex asymmetric multi-antennary glycans.
1. An orthogonally protected branched oligosaccharide having the formula (I):
wherein each of R 1 , R 4 , R 6 and R 8 is independently an orthogonal protecting group or a permanent protecting group, provided that at least two of R 1 , R 4 , R 6 and R 8 are orthogonal protecting groups;
each of R 2 , R 3 , R 5 , R 7 , and R 10 is independently a permanent protecting group;
R 9 is an orthogonal protecting group, a permanent protecting group, or a glucosamine moiety;
X is —OR 15 or —SR 16 ;
R 15 is H, alkyl, cycloalkyl, substituted alkyl or cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl or substituted aryl; a protecting group; an anomeric spacer or linker; a fluorous tag; or a leaving group; and
R 16 is H, alkyl, cycloalkyl, substituted alkyl or cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl.
2. The orthogonally protected oligosaccharide of claim 1 further comprising a glycosidically linked glucosamine moiety in place of X, wherein the ring hydroxyls on the glucosamine moiety are protected, wherein the amine at the C-2 position of the glucosamine moiety is protected, and wherein the anomeric position at the reducing end of the glucosamine moiety comprises X as in formula (I).
3. The orthogonally protected oligosaccharide of claim 1 further comprising a glycosidically linked glucosamine disaccharide in place of X, wherein the ring hydroxyls on the glucosamine disaccharide are protected, wherein the amines at both C-2 positions of the glucosamine disaccharide are protected, and wherein the anomeric position at the reducing end of the glucosamine disaccharide comprises X as in formula (I).
4. The orthogonally protected oligosaccharide of claim 2 comprising a glycosidically linked fucose moiety at position C-6 of the terminal reducing glucosamine.
5. The orthogonally protected oligosaccharide of claim 1 wherein the orthogonal protecting group is selected from the group consisting of levulinoyl (Lev); 9-fluorenylmethoxycarbonyl (Fmoc); allyloxycarbonyl (Alloc); 2-naphthylmethyl (Nap); 1-naphthylmethyl (1-Nap); benzoyl (Bz); difluorobenzoyl (dfBz); pivaloyl levulinoyl (PivLev); pivaloyl benzoyl (PivBz); para-methoxybenzyl ether (PMB); methoxy phenyl ether (MP); allyl ether (Allyl); chloroacetyl ester (ClAc); trichloroacetyl ester (Cl 3 Ac), trifluoroacetyl ester (F 3 Ac); and a silyl ether.
6. A method for making an oligosaccharide comprising:
deprotecting the orthogonally protected oligosaccharide of claim 1 by removing an orthogonal protecting group to yield a deprotected glycosyl acceptor; and
linking the deprotected glycosyl acceptor to a glycosyl donor to yield an extended oligosaccharide.
7. The method of claim 6 wherein the glycosyl donor is a protected or masked glucosamine or a lactosamine.
8. The method of claim 6 wherein the extended oligosaccharide comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 monosaccharide units.
9. The method of claim 6 wherein the extended oligosaccharide is a multi-antennary glycan comprising two, three, four or five branches.
10. The method of claim 6 comprising sequentially removing orthogonal protecting groups from the orthogonally protected oligosaccharide.
11. The method of claim 10 further comprising removing permanent protecting groups to yield a deprotected multi-antennary glycan.
12. The method of claim 11 wherein the multi-antennary glycan is asymmetric.
13. The method of claim 12 wherein the deprotected multi-antennary glycan is masked at at least one position.
14. The method of claim 12 further comprising contacting the asymmetric multi-antennary glycan with a first carbohydrate processing enzyme under conditions to yield an enzymatically extended multi-antennary glycan.
15. The method of claim 14 comprising unmasking the enzymatically extended multi-antennary glycan to yield an unmasked multi-antennary glycan, followed by contacting the unmasked multi-antennary glycan with a second carbohydrate processing enzyme under conditions to yield a multi-antennary glycan that is further extended.
16. The method of claim 15 further comprising sequentially contacting the extended multi-antennary glycan with at least one additional carbohydrate processing enzyme under conditions to further extend the multi-antennary glycan.
17. The orthogonally protected oligosaccharide of claim 3 comprising a glycosidically linked fucose moiety at position C-6 of the terminal reducing glucosamine.