The known methyl 2-O-acetyl-3,4-di-O-benzyl-1-thio-α-L-rhamnopyranoside (3) was converted to the corresponding 5-methoxycarbonylpentyl glycoside 4 which was deacetylated. The product 5 was used as the initial glycosyl acceptor to construct two trirhamnoside glycosyl acceptors having HO-3(III) flanked by either benzoyl or benzyl groups, compounds 10 and 29, respectively [fully protected, except HO-3(III), α-L-Rha-(1→3)-α-L-Rha-(1→2)-α-L-Rha-1-O-(CH2)5COOCH3]. When these were glycosylated with ethyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-bromoacetyl-1-thio-β-D-glucopyranosi de (18), only the benzylated glycosyl acceptor 29 gave good yield of the desired tetrasaccharide 30. The α- and β-linked products, together with the corresponding orthoester 23, were formed in almost equal amount when glycosylation of 10 was performed with the glycosyl donor carrying the 2-O-bromoacetyl protecting group. Deprotection at O-2 of 30, followed by further functionalization of the molecule and global deprotection, gave the 5-methoxycarbonylpentyl glycoside of the title tetrasaccharide, β-Ant-(1→3)-α-L-Rha-(1→3)-α-L-Rha-(1→2)-α-L-Rha (35). Except for differences due to presence of the anomeric 5-methoxycarbonylpentyl group, the fully assigned NMR spectra of glycoside 35 were found to be virtually identical to those reported for the parent tetrasaccharide isolated from Bacillus anthracis exosporium, thus proving the correct structure assigned to the naturally occurring substance. All theoretically possible structural fragments of 35, as well as analog of 35 lacking the 2-O-methyl group at the terminal 4,6-dideoxyglucosyl residue, compound 40, were also synthesized. Tetrasaccharide 35, its β-linked and non-methylated analogs 2 and 40, respectively, as well as the trirhamnoside fragment of 35, glycoside 12, were further functionalized and conjugated to BSA using squaric acid chemistry, to give neoglycoconjugates with a predetermined carbohydrate-protein ratio of approximately 3 and approximately 6

carbohydrates, Oligosaccharides, glycoconjugates, Synthetic carbohydrate antigens, conjugation by squaric acid chemistry

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
2,3,3,4	lX3HOiVl
2,3,3,2	Me
2,3,3	bDQuip4N	98.62	76.66	70.56	56.42	68.58	18.02
2,3	aLRhap	101.76	69.97	76.28	71.37	69.04	17.81
2	aLRhap	101.71	69.67	79.48	71.65	68.31	17.73
	aLRhap	103.50	84.36	72.77	72.22	70.34	18.18


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,3,3,4	lX3HOiVl
2,3,3,2	Me
2,3,3	bDQuip4N	4.577	2.845	3.281	3.402	3.275	1.074
2,3	aLRhap	4.857	3.872	3.745	3.402	3.636	1.32
2	aLRhap	4.842	3.775	3.572	3.325	3.487	1.101
	aLRhap	4.566	3.634	3.530	3.155	3.373	1.134


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
2,3,3,4	lX3HOiVl
2,3,3,2	Me
2,3,3	bDQuip4N	98.62/4.577	76.66/2.845	70.56/3.281	56.42/3.402	68.58/3.275	18.02/1.074
2,3	aLRhap	101.76/4.857	69.97/3.872	76.28/3.745	71.37/3.402	69.04/3.636	17.81/1.32
2	aLRhap	101.71/4.842	69.67/3.775	79.48/3.572	71.65/3.325	68.31/3.487	17.73/1.101
	aLRhap	103.50/4.566	84.36/3.634	72.77/3.530	72.22/3.155	70.34/3.373	18.18/1.134

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CO[C@H]1[C@H](O[C@@H]2[C@@H](O)[C@H](C)O[C@@H](O[C@@H]3[C@@H](O)[C@H](C)O[C@@H](O[C@H]4[C@H](O)O[C@@H](C)[C@H](O)[C@H]4O)[C@@H]3O)[C@@H]2O)O[C@H](C)[C@@H](NC(=O)CC(C)(C)O)[C@@H]1O

715.743 g/mol (C₃₀H₅₃NO₁₈)