Chemoenzymatic Synthesis of Sialosides Containing 7- N- or 7,9-Di- N-acetyl Sialic Acid as Stable O-Acetyl Analogues for Probing Sialic Acid-Binding Proteins

Abstract

A novel chemoenzymatic synthon strategy has been developed to construct a comprehensive library of α2-3- and α2-6-linked sialosides containing 7-N- or 7,9-di-N-acetyl sialic acid, the stable analogue of naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl-sialic acid. Diazido and triazido-mannose derivatives that were readily synthesized chemically from inexpensive galactose were shown to be effective chemoenzymatic synthons. Together with bacterial sialoside biosynthetic enzymes with remarkable substrate promiscuity, they were successfully used in one-pot multienzyme (OPME) sialylation systems for highly efficient synthesis of sialosides containing multiple azido groups. Conversion of the azido groups to N-acetyl groups generated the desired sialosides. The hydrophobic and UV-detectable benzyloxycarbonyl (Cbz) group introduced in the synthetic acceptors of sialyltransferases was used as a removable protecting group for the propylamine aglycon of the target sialosides. The resulting N-acetyl sialosides were novel stable probes for sialic acid-binding proteins such as plant lectin MAL II, which bond strongly to sialyl T antigens with or without an N-acetyl at C7 or at both C7 and C9 in the sialic acid.

Figure 1.

Structures of N-acetylneuraminic acid (Neu5Ac, 1), the most abundant natural sialic acid form, and its naturally occurring O-acetylated forms 7-O-acetyl-N-acetylneuraminic acid (Neu5,7Ac₂, 2) and 7,9-di-O-acetyl-N-acetylneuraminic acid (Neu5,7,9Ac₃, 3).

Figure 2.

Structures of N-acetyl analogue Neu5Ac7NAc (4) and azido derivative Neu5,7diN₃ (5) of Neu5,7Ac₂ (2); N-acetyl analogue Neu5Ac7,9diNAc (6) and azido derivative Neu5,7,9triN₃ (7) of Neu5,7,9Ac₃ (3); as well as their corresponding hexose precursors Man2,4diNAc (8), Man2,4diN₃ (9), Man2,4,6triNAc (10), and Man2,4,6triN₃ (11), respectively.

Figure 3.

Glycan microarray study results for sialoside binding by hSiglec 7, hSiglec 9, SNA, and MAL II (Numeric data are shown in Table S1). Asialoglycans (R’OH) corresponding to the internal glycans in the sialosides are negative controls shown in white bars. R1 = ProNH₂.

Scheme 1.

Chemical synthesis of Man2,4diNAc (8) and Man2,4diN₃ (9) from galactose (12). Ac = acetyl, pMP = para-methoxyphenyl, Bz = benzoyl.

Scheme 2.

Synthesis of Man2,4,6triNAc (10) and Man2,4,6triN₃ (11) from galactoside 14.

Scheme 3.

PmAldolase-catalyzed preparative-scale synthesis of sialic acid derivatives Neu5Ac7NAc (4), Neu5,7diN₃ (5), and Neu5,7,9triN₃ (7) from the corresponding mannose derivatives Man2,4diNAc (8), Man2,4diN₃ (9), and Man2,4,6triN₃ (11), respectively. Man2,4,6triNAc (10) was not a suitable substrate for PmAldolase-catalyzed synthesis of Neu5Ac7,9diNAc (6).