Applied Microbiology and Biotechnology

, Volume 66, Issue 5, pp 512–519 | Cite as

Chemo-enzymatic synthesis of 2′-O-methoxyethyl ribonucleosides using a phosphodiesterase from Serratia marcescens

Biotechnological Products and Process Engineering
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Abstract

An enzyme able to cleave the 3′,5′-phosphate ring of 2′-methoxyethyl cyclic nucleotides (3′,5′-cyclic nucleotide phosphodiesterase, EC 3.1.4.17) from Serratia marcescens DSM 30121 was used to deprotect the cyclic phosphate nucleotides after chemical alkylation. The process yielded 2′-O-alkylated nucleosides used as building blocks of antisense oligonucleotides for subsequent potential applications in therapeutics (antisense oligonucleotide synthesis) and diagnostics. The phosphodiesterase from the Gram-negative enteric bacterium S. marcescens was selected on account of the broad substrate range and high activity of the enzyme. The protein was purified by heat-treatment of the crude cell-free extract, followed by column chromatography (gel filtration). It was characterised and showed optimal activity at a broad pH range (pH 6.8–9.4, with a peak at ca. pH 8.5) and at a temperature of 60–65°C. No metal ions were required for activity, although Ba2+ was an activator. Conversion of 2′-O-methoxyethyl cAMP into the corresponding nucleoside derivative on a multi-gram scale was successfully performed in two steps, using the S. marcescens enzyme in conjunction with a commercially available alkaline phosphatase from Escherichia coli.

Keywords

Phosphodiesterase Cyclic Nucleotide Adenosine Deaminase Serratia Marcescens Phosphodiesterase Activity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Novartis Pharma AG, Novartis Institutes of Biomedical ResearchDiscovery TechnologiesBaselSwitzerland

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