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 (Galβ1–3GlcNAcβProN3), type 2 structures (Galβ1–4GlcNAcβProN3 and 6-sulfo-Galβ1–4GlcNAcβProN3), type 4 structure (Galβ1–3GalNAcβProN3), type 3 or core 1 structure (Galβ1–3GalNAcαProN3) and human milk oligosaccharide or lipooligosaccharide lacto-N-tetraose (LNT) (Galβ1–3GlcNAcβ1–3Galβ1–4GlcβProN3), 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

Sequential two-step multienzyme synthesis of tumor-associated sialyl T-antigens and derivatives

“…2, PmST1 exhibited promiscuous donor substrate specificity and catalyzed the transfer of sialic acids from in situ generated CMP-sialic acids to Galβ1–3GalNAcαProN 3 3 synthesized by the Step I to produce sialyltrisaccharides 10 – 15 containing different terminal sialic acid forms in good to excellent (85–97%) yields. The sialosides containing three natural occurring sialic acid forms, Neu5Acα2–3Galβ1–3GalNAcαProN 3 10 , 15 Neu5Gcα2–3Galβ1–3GalNAcαProN 3 11 , 15 and Kdnα2–3Galβ1–3GalNAcαProN 3 12 were obtained in 87%, 94%, and 89% yields, respectively, from ManNAc 4 , ManNGc 5 , and mannose 6 as six-carbon sialic acid precursors. Neu5Gc is a non-human sialic acid which is overexpressed in certain cancer cells.…”

“…8 These β1–3-linked galactodisaccharides 16–22 were then used as sialyltransferase acceptors in the Step II one-pot three-enzyme sialylation process for the synthesis of α2–3-linked sialosides containing Neu5Ac formed from ManNAc 4 as a sialic acid precursor. As shown in Table 1, preparative-scale sialylation of galacto- N -biose (GNB) Galβ1–3GalNAc 16 and Galβ1–3GalNAcβProN 3 17 by Neu5Ac formed from ManNAc and pyruvate successfully produced the sialylated products 3′-sialyl galacto- N -biose (GNB) Neu5Acα2–3Galβ1–3GalNAc 23 16 and Neu5cα2–3Galβ1–3GalNAcβProN 3 24 15 in 90% and 92% yields, respectively. The one-pot three-enzyme system was also applied in sialylation of biologically important T antigens Galβ1–3GalNAcα1- O -Ser and Galβ1–3GalNAcα1- O -Thr to produce the desired trisaccharide products 25 and 26 in excellent 86% and 89% yields, respectively.…”

Converting Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) to a regioselective α2–6-sialyltransferase by saturation mutagenesis and regioselective screening

“…While the CAZy GT80 family α2–3-sialyltransferases (α2–3STs), such as multifunctional Pasteurella multocida sialyltransferase 1 (PmST1), 15 are quite specific in catalyzing α2–3-sialylation to only the terminal galactose residues in galactosides, 10, 17–19 the α2–6-sialyltransferase (α2–6ST) activity of multifunctional Photobacterium damselae α2–6ST (Pd2,6ST) 20 can catalyze α2–6-sialylation to both terminal and internal galactose or GalNAc residues. 10, 12, 16, 17, 21 …”

“…15, 31, 32 Additionally, PmST1 has been shown to be promiscuous towards a wide range of CMP-sialic acid donors and galactoside acceptors. 15, 19, 46–49 PmST1 has multiple functions (Figure 1) including (1) α2–3ST, (2) α2–6ST, (3) α2–3-sialidase, (4) α2–3-trans-sialidase, (5) donor hydrolysis, 15, 45 and (6) α2–6-trans-sialidase 50 activities activity. As the α2–6ST activity of PmST1 is much weaker than its α2–3ST activity especially at pH≥7.0, 15 PmST1 has been broadly used in highly efficient synthesis of α2–3-sialosides with in situ generation of CMP-sialic acids by a CMP-sialic acid synthetase with or without a sialic acid aldolase.…”

Chemoenzymatic synthesis of lacto-N-tetrasaccharide and sialyl lacto-N-tetrasaccharides

“…In recent years, several elegant chemical syntheses, [4][5][6][7] microbial and coupled enzymatic approaches, 8,9 and chemoenzymatic synthesis 10 have been developed for the synthesis of LNT and its derivatives. The reactivity-based one-pot synthesis has been demonstrated as a valuable way to prepare LNT and fucosyl LNT in a few steps from several specifically designed building blocks, 5 however, this strategy may not be amenable to preparation of other structurally similar HMOs.…”