Preparative‐enzymatic formation of cytidine 5′‐monophosphosialate by integrated cytidine 5′‐triphosphate regeneration

Abstract

The preparative formation of cytidine 5′‐triphosphate by an enzymatic process with adenylate kinase and pyruvate kinase is described. The enzymes may either be used free, in the “MEEC” technique, or immobilized on a synthetic copolymer of vinyl acetate and N,N′‐divinylethyleneurea (“VA‐Epoxy”). Cytidine 5′‐monophosphosialate synthase (preferably isolated from calf brain) and inorganic pyrophosphatase were used to prepare the activated neuraminic acid cytidine 5′‐monophosphosialate (CMP‐Neu5Ac) from CTP and neuraminic acid in good yield. Finally, both processes could be integrated and led to an in situ approach which, owing to moderate yields, requires further development.

Chemoenzymatic synthesis of CMP-N-acetyl-7-fluoro-7-deoxy-neuraminic acid

“…2,3 The enzymatic synthesis of N-acetyl-neuraminic acid (sialic acid, 1) from N-acetyl-D D-mannosamine or N-acetyl-D D-glucosamine followed by activation as CMP-ester is well established. [4][5][6] Multienzyme systems have been optimized for this sequence, [7][8][9] which includes the regeneration of CMP to CTP. 10 To access new derivatives, every position of sialic acid was addressed during the past years [1][2][3] and several variations at position 7 of sialic acid were synthesized.…”

ChemInform Abstract: Enzymes in Organic Synthesis. Part 18. CMP‐Sialate Synthetase from Neisseria meningitidis — Overexpression and Application to the Synthesis of Oligosaccharides Containing Modified Sialic Acids.

“…The enzyme catalyzes a nucleophilic attack of the anomeric oxygen of b-Neu5Ac on the a-phosphate of CTP [19] and requires Mg 2+ or Mn 2+ for activity. [17,20] So far, enzymatic procedures for the synthesis of 2 (Equation 1) have been developed to routine application, [21,22,23,24,25,26,27,28] however, these are not generally amenable to the preparative nucleotide activation of modified sialic acids or their structural analogs because of the narrow substrate specificity of known CMP-sialate synthetases, and an often poor access to larger protein quantities. [25,29] CMP-sialate synthetases have been isolated from various eukaryotic and prokaryotic sources.…”

“…[25,29] CMP-sialate synthetases have been isolated from various eukaryotic and prokaryotic sources. [17,21,24,27,30,31,32,33] Synthetases purified from different vertebrate tissues are rather unstable and offer only low specific activities (ca. 0.2 U/mg) which 1 significantly restricts their use for preparative syntheses.…”

“…0.2 U/mg) which 1 significantly restricts their use for preparative syntheses. [21,23,24,32,34] Several microorganisms have also been shown to contain CMP-sialate synthetase but are unsuited as possible enzyme sources because sialic acid metabolism parallels pathogenic virulence. [17,27,31,33] Therefore, some bacterial CMP-sialate synthetases have been produced from nonpathogenic clones and a few have been tested for their synthetic utility.…”

Applications of enzymes in synthetic carbohydrate chemistry

“…CTP with Neu5Ac and sialate-cytidyltransferase (viii) led to the formation of activated neuraminic acid (CMPNeu5Ac), and the resulting pyrophate (PPi) was again reacted to inorganic phosphate (Pi) with inorganic pyrophosphatase (iii) [66,67]. These processes employing the four enzymes (iii, vi, viii, and x) could be combined [68] and thus it was of interest to incorporate the sialyltransferase step as well. This was recently published with the synthesis of the derivative e [64] (Fig.…”