One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities

Abstract

Chemoenzymatic synthesis, which combines the flexibility of chemical synthesis and the high selectivity of enzymatic synthesis, is a powerful approach to obtain complex carbohydrates. It is a preferred method for synthesizing sialic acid-containing structures, including those with diverse naturally occurring and non-natural sialic acid forms, different sialyl linkages and different glycans that link to the sialic acid. Starting from N-acetylmannosamine, mannose or their chemically or enzymatically modified derivatives, sialic acid aldolase-catalyzed condensation reaction leads to the formation of sialic acids and their derivatives. These compounds are subsequently activated by a CMP-sialic acid synthetase and transferred to a wide range of suitable acceptors by a suitable sialyltransferase for the formation of sialosides containing natural and non-natural functionalities. The three-enzyme coupled synthesis of sialosides can be carried out in one pot without the isolation of intermediates. The time for synthesis is 4-18 h. Purification and characterization of the product can be completed within 2-3 d.

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

“…Preparation of Sialylated GAEABs-We chose a synthetic strategy that combined two earlier developments, the bifunctional fluorescent tag AEAB (27) and the one-pot three-enzyme sialylation reaction (19,20,25) (Fig. 1).…”

“…Sialyl glycans containing various natural modifications of sialic acid linked ␣2-3 or ␣2-6 to the three different fluorescent glycans described above were synthesized using a highly efficient one-pot three-enzyme system similar to that reported previously (19,20,25,28). The fluorescent glycan-AEAB conjugates (GAEABs) of LNT, LNnT, or NA2 (130 -200 g) were incubated with three enzymes, including Escherichia coli sialic acid aldolase (28) (10 -15 g), Neisseria meningitidis CMPsialic acid synthetase (28) (10 -15 g), and Photobacterium damselae ␣2-6-sialyltransferase (25) (10 -15 g; used for the synthesis of ␣2-6-linked sialosides) or Pasteurella multocida ␣2-3-sialyltransferase PmST1 (19) (3-6 g; used for the synthesis of ␣2-3-linked sialosides) in a reaction mixture of 25 l containing 100 mM Tris-HCl buffer, pH 8.5.…”

“…acetyl or lactyl group). The following were added to this mixture: (a) sialic acid precursors (20,25,28,29) (e.g. mannose, N-acetylmannosamine, or their derivatives) at 2.0 or 4.0 eq relative to LNT or LNnT and NA2 (biantennary-AEAB conjugate), respectively; (b) sodium pyruvate (5 eq for LNT or LNnT, 10 equiv.…”

“…To further explore the recognition and function of modified sialic acid residues, we exploited the development of new chemoenzymatic methods to synthesize modified sialic acids (5), along with recent developments of glycan microarray technology, which provide an innovative approach to studying glycan recognition and function by glycan-binding proteins. We adapted our recent development of natural glycan microarrays (21)(22)(23)(24) to the production of a diverse array of sialylated glycans using fluorescent derivatives of glycans that terminate with ␤-linked galactose, which are sialyltransferase acceptors, in a one-pot three-enzyme approach for the microscale chemoenzymatic synthesis of sialosides (19,20,25). The resultant microarray consists of 77 sialylated glycans incorporating 16 modified sialic acids in ␣2-3 and ␣2-6 linkages to different underlying structures.…”

Towards the Enzymatic Synthesis of Carbohydrates

“…During the past twenty years, a multi-enzyme one-pot reaction fashion with only one purification step has becoming very popular in carbohydrate synthesis since most of the key enzymes are proved to be active under similar reaction conditions [3][4]. In addition to enhanced efficiency, problems such as the availability of high cost sugar nucleotide donors and product inhibition of glycosyltransferases have also been solved by following the biosynthetic pathways: The one-pot reaction can be further conjugated to sugar Another subject to emerge over the past decade is that in vitro multi-enzyme carbohydrate synthesis is transferred onto solid beads or into whole cells.…”