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

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.