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 degrees 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.
Marais
Guy
G
Novartis Pharma AG, Novartis Institutes of Biomedical Research, Discovery Technologies, 4002 Basel, Switzerland.
Ghisalba
Oreste
O
D006151:Guanosine / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012263:Ribonucleosides / Q000138:chemical synthesis* / Q000737:chemistry; D012266:Ribose / Q000031:analogs & derivatives*; D012706:Serratia marcescens / Q000201:enzymology* / Q000378:metabolism
D009841:Oligonucleotides; D012263:Ribonucleosides; D006151:Guanosine; D012266:Ribose; D010727:Phosphoric Diester Hydrolases