Improved Synthesis and Isolation of 2′‐O‐Methyladenosine: Effective and Scalable Enzymatic Separation of 2′/3′‐O‐Methyladenosine Regioisomers

An efficient separation of a mixture of 2′/3′‐O‐methyladenosine regioisomers (1 + 2; 1:1) has been developed by selective enzymatic acylation using immobilized Pseudomonas cepacia lipase (PSL‐C) in combination with acetonoxime levulinate as acyl donor. The 3′‐hydroxy group of 2′‐O‐methyladenosine (1) was acylated with high selectivity (ca. 70 %), whereas an equal amount of 3′‐O‐methyladenosine (2) in the same solution resulted in minor acylation of 5′‐hydroxy group (ca. 8 %). The differential behavior of both regioisomers towards enzymatic acylation allowed to develop a separation protocol. Upon extraction of the acylated products, the 3′‐O‐methyladenosine was isolated in 81 % yield and 97 % purity from the aqueous layer. Hydrolysis of acylated products in organic layer furnished 2′‐O‐methyladenosine in 67 % yield and 99 % purity. The separation process was successfully applied to the crude reaction mixture of methylated products (ca. 3:1 of 1/2) on 5‐g scale. We also report on the use of methyl p‐toluenesulfonate as a safe reagent for 2′‐O‐methylation of adenosine.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) Direct alkylation of adenosine with methyl p‐toluenesulfonate furnishes a mixture2′/3′‐O‐methyladenosine regioisomers which upon enzymatic acylation with Pseudomonas cepacia lipase selectivelyyields acylated 2′‐O‐methyladenosine. Chromatographic separation and hydrolysis of the latter offers easy access to 2′‐O‐methyladenosine.