Chemoenzymatic synthesis of α2-3-sialylated carbohydrate epitopes

Abstract

Sialic acids are common terminal carbohydrates on cell surface. Together with internal carbohydrate structures, they play important roles in many physiological and pathological processes. In order to obtain α2-3-sialylated oligosaccharides, a highly efficient one-pot three-enzyme synthetic approach was applied. The P. multocida α2-3-sialyltransferase (PmST1) involved in the synthesis was a multifunctional enzyme with extremely flexible donor and acceptor substrate specificities. Sialyltransferase acceptors, including type 1 structure (Galβ1-3GlcNAcβProN(3)), type 2 structures (Galβ1-4GlcNAcβProN(3) and 6-sulfo-Galβ1-4GlcNAcβProN(3)), type 4 structure (Galβ1-3GalNAcβProN(3)), type 3 or core 1 structure (Galβ1-3GalNAcαProN(3)) and human milk oligosaccharide or lipooligosaccharide lacto-N-tetraose (LNT) (Galβ1-3GlcNAcβ1-3Galβ1-4GlcβProN(3)), were chemically synthesized. They were then used in one-pot three-enzyme reactions with sialic acid precursor ManNAc or ManNGc, to synthesize a library of natural occurring α2-3-linked sialosides with different internal sugar units. The sialylated oligosaccharides obtained are valuable probes for their biological studies.

Figure 1

Common sialic acid-containing structures in nature.

Figure 2

One-pot three-enzyme synthesis of sialosides containing different sialic acid forms and various internal glycans. Abbreviations: ManNAc, N-acetylmannosamine; ManNGc, N-glycolylmannosamine; CTP, cytidine 5′-triphosphate. Enzymes: E. coli aldolase, Escherichia coli K12 sialic acid aldolase; NmCSS, Neisseria meningitidis CMP-sialic acid synthetase; PmST1, Pasteurella multocida sialyltransferase for the formation of α2–3-linked sialosides.

Scheme 1

Reagents and yields: (a) (i) NaOMe, MeOH; (ii) PhCH(OMe)₂, CSA, DMF, 87%; (b) TMSOTf, CH₂Cl₂, 53%; (c) (i) Pd/C, H₂; (ii) NaN₃, TBAI, DMF, 95%; (d) (i) N₂H₄-H₂O, EtOH; (ii) Ac₂O, pyridine; (iii) NaOMe, MeOH, 84%.

Scheme 2

Reagents and yields: (a) (i) NaOMe, MeOH (ii) TBSCl, imidazole, CH₂Cl₂, 84%; (b) 9, TMSOTf, CH₂Cl₂, 79%; (c) (i) TABF, THF; (ii) NaN3, TBAI, DMF, 76%; (d) (i) Ac₂O, pyridine; (ii) TABF, THF; (iii) NaN₃, TBAI, DMF, 85%; (e) (i)) N₂H₄-H₂O, EtOH; (ii) Ac₂O, pyridine; (iii) NaOMe/MeOH, 82%; (f) (i) Py-SO₃, pyridine; (ii) N₂H₄-H₂O, EtOH; (iii) Ac₂O, pyridine; (iv) NaOMe, MeOH, 66%.

Scheme 3

Reagents and yields: (a) (i) AcCl, 3-chloro-1-propanol; (ii) PhCH(OMe)₂, CSA, DMF, 17:21%; 18: 57%; (b) 9, TMSOTf, CH₂Cl₂, 19:62%; 20: 48%; (c) (i) NaN₃, TBAI, DMF; (ii) 80% HOAc, 21:94%; 22: 91%; (d) (i) N₂H₄-H₂O, EtOH; (ii) Ac₂O, pyridine; (iii) NaOMe, MeOH, 4:85%; 5: 76%.

Scheme 4

Reagents and yields: (a) (i) NaOMe, MeOH; (ii) PhCH(OMe)₂, CSA, 88%; (b) 9, TMSOTf, CH₂Cl₂, 77%; (c) NIS, TfOH, CH₂Cl₂, 64%; (d) (i) 80% HOAc; ii) N₂H₄-H₂O, EtOH; (iii) Ac₂O, pyridine; (iv) NaOMe, MeOH, 54%.