Synthesis of neoglycoproteins containing 5-O-phosphorylated Kdomonosaccharide, 4-O- and 5-O-phosphorylated α-Kd↬(2 6)-2-acetamido-2-deoxy- -D-glucopyranosyl disaccharide residues

Abstract

Radical addition of cysteamine to anomeric allyl glycosides of Kdo 5-phosphates 1 and 4 afforded good yields of the corresponding 3-(2-aminoethylthio)propyl glycosides. Similar reactions with the α-allyl glycoside of Kdo 4-phosphate 7 led to substantial formation of the dephosphorylated product 10 through intramolecular hydrolysis of the 4-O-phosphomonoester by the terminal amino function of the spacer group. Similar side reactions occurred upon deblocking of the 4- O-phosphorylated 6-aminohexyl-α-glycoside lactone derivative 18. Allyl glycosides of 4'- O- and 5'- O-phosphorylated Kd↬ (2 6)-glucosamine derivatives were prepared in good yields via phosphorylation using the amidite procedure. The 4'-phosphate moiety was introduced following intramolecular protection of the 5'-OH group as a lactone. Subsequent deprotection gave the allyl glycosides 25 and 33 which were transformed into the corresponding stable spacer derivatives 26 and 34 by coupling with cysteamine. The ligands were activated with thiophosgene and reacted with bovine serum albumin to give the neoglycoconjugates 3, 6, 27 and 35 to be used in immunochemical studies of monoclonal antibodies directed against Haemophilus influenzae Re-mutant and related lipopolysaccharides.

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I‐69 Rd−/b+ of Haemophilus influenzae

“…However, definition of ␣-Kdo-4P as the minimal structure could not be achieved by direct binding assays since the attempt to synthesize ␣-Kdo-4P-BSA failed. When the addition reaction of the allyl glycoside of ␣-Kdo-4P with cysteamine was performed, the phosphate group became eliminated (Sekljic et al, 1996). However, this antibody bound equally to ␣-Kdo-4P-GlcNAc-BSA and LPS deac -BSA, and both bindings could be inhibited with mono-(␣-Kdo-4P-All) and polyvalent (␣-Kdo-4P-PA) inhibitors, clearly indicating the Kdo-4P-specificity.…”

“…To answer the question of whether this antibody required the GlcNAc residue for binding, and to confirm the binding behaviour of the other antibodies, inhibition experiments were performed. It should be noted that the straightforward experiment, namely testing the binding to ␣-Kdo-4P-BSA, could not be done since the introduction of the cysteamine spacer arm during the synthesis resulted in the elimination of the phosphate group (Sekljic et al, 1996). a. EIA plates were coated with neoglycoconjugates at a concentration of 400 pmol of ligand per ml.…”

“…1→allyl were synthesized as described (Sekljic et al, 1996). The allyl glycosides R-CH 2 -CH¼CH 2 , wherein R represents the glycosyl residue, were conjugated with cysteamine yielding R-(CH 2 ) 3 -S-(CH 2 ) 2 -NH 3 þ (Lee and Lee, 1974), which was activated with thiophosgen into the isothiocyanate derivatives R-(CH 2 ) 3 -S-(CH 2 ) 2 -N¼C¼S and then conjugated to bovine serum albumin (BSA) yielding R-(CH 2 ) 3 -S-(CH 2 ) 2 -NH-CS-NH-BSA (Sekljic et al, 1996).…”

Monoclonal antibodies against 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) and D-glycero-α-D-talo-oct-2-ulosonic acid (Ko)

“…), aKdo(2® 4)aKdo(2® allyl, aKo(2®4)aKdo(2®allyl and aKdo(2®4)aKo(2®allyl) were synthesized as described. [8][9][10][11][12][13] The allyl glycosides R-OCH 2 -CH=CH 2 were reacted with cysteamine 14 yielding R-O(CH 2 ) 3 -S-(CH 2 ) 2 -NH 3 + ligands (it is noted that conjugation was not achieved with Kdo-4P, 7epiKdo and Kdh), which were activated with thiophosgene into the isothiocyanate derivatives R-O(CH 2 ) 3 -S-(CH 2 ) 2 -N=C=S and then conjugated to bovine serum albumin (BSA) yielding R-O(CH 2 ) 3 -S-(CH 2 ) 2 -NH-CS-NH-BSA, where R represents the glycosyl residue. The latter compounds are abbreviated as R-BSA.…”

Anomeric O-acylation of Kdo using alkyl and aryl isocyanates

“…Several groups have examined the a-selective glycosidation of Kdo to introduce a functional spacer, which can be potentially utilized for further coupling to other macromolecules. [1][2][3][4][5][6] Up to date, two groups have accomplished the synthesis of an a-Kdo derivative carrying a spacerarm in high yields. [5][6][7] However, these methods have not yet been fully developed as a general conjugation method for chemically synthesized OS containing Kdo at the reducing end.…”