Zanamivir Enzymatic Synthesis of A-Acetylneuraminic Acid

An elegant continuous process for N-acetylneuraminic acid synthesis was developed by introducing the N-acetylneuraminic acid-2-epimerase for epimerization of N-acetyl glucosamine 119 and integrating the epimerization with synthesis in an enzyme-membrane reactor [151]. Although this process has an excellent space-time yield, the epimerase enz)une is not freely available and the product stream is very dilute and chromatography is required for purification, making scale-up difficult. 

Subsequently, the enzyme from E. coli has been overexpressed in an inducible system at very high expression levels, and homogenized bacterial cells were directly used to immobilize the enz3une from crude extracts onto Eupergit C beads without any clarification, and the immobilized enzyme was used for the synthesis of N-acetylneuraminic acid 117 at 155 g/1 concentration. At the manufacturing scale, the same batch of enzymes was reused in 2000 cycles in batch column reactors, without any significant loss of activity, to produce multi-tonne quantities of N-acetylneuraminic acid [152], 

The antiviral activity of nucleoside analogs mainly resides in the natural 6-d-isomer. However the unnatural 6-l-(-)-120 isomer (Epivir) is substantially less cytotoxic and very effective than its corresponding natural B-D-(+)-isomer [154]. 

One of the initial routes considered for the preparation of isomers was the enzymatic resolution using 5 -nucleotidase and alkaline phosphatase that would allow access to both enantiomers, racemic compound. The chemically synthesized monophosphate derivative ( )-121 was resolved using 5 -nucleotidase from Crotalus atrox venom, and the resulting mixture was separated by chromatography and purified on silica gel to give (+)-123 with a 90% ee. Hydrolysis of the remaining monophosphate (-)-122 with alkaline phosphatase from E. coli afforded Epivir [155]. 

Epivir enzymatic deamination process for the synthesis of (2 / -c s)-2 -deoxy-3-thiacytidine. 

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