Nonaqueous Biocatalytic Synthesis of New Cytotoxic Doxorubicin Derivatives:  Exploiting Unexpected Differences in the Regioselectivity of Salt-Activated and Solubilized Subtilisin

Abstract

Two enzymes, Mucor javanicus lipase and subtilisin Carlsberg (SC), catalyzed the nonaqueous acylation of doxorubicin (DOX). Compared to the untreated enzyme the rate of DOX acylation at the C-14 position with vinyl butyrate in toluene was 25-fold higher by lipase ion-paired with Aerosol OT (AOT) and 5-fold higher by lipase activated by 98% (w/w) KCl co-lyophilization (3.21 and 0.67 μmol/min g-lipase, respectively, vs 0.13 μmol/min g-lipase). Particulate subtilisin Carlsberg (SC) was nearly incapable of DOX acylation, but ion-paired SC (AOT-SC) catalyzed acylation at a rate of 2.85 μmol/min g-protease. The M. javanicus formulations, AOT-SC, and SC exclusively acylated the C14 primary hydroxyl group of DOX. Co-lyophilization of SC with 98% (w/w) KCl expanded the enzyme's regiospecificity such that KCl-SC additionally acylated the C4‘ hydroxyl and C3‘ amine groups. The total rate of DOX conversion with KCl-SC was 56.7 μmol/min g-protease. The altered specificity of KCl-SC is a new property of the enzyme imparted by the salt activation, and represents the first report of unnatural regioselectivity exhibited by a salt-activated enzyme. Using AOT-SC catalysis, four unique selectively acylated DOX analogues were generated, and KCl-SC was used to prepare DOX derivatives acylated at the alternative sites. Cytotoxicities of select derivatives were evaluated against the MCF7 breast cancer cell line (DOX IC50 = 27 nM) and its multidrug-resistant sub-line, MCF7-ADR (DOX IC50 = 27 μM). The novel derivative 14-(2-thiophene acetate) DOX was relatively potent against both cell lines (IC50 of 65 nM and 8 μM, respectively) and the 14-(benzyl carbonate) DOX analogue was as potent as DOX against the MCF7 line (25 nM). Activated biocatalysts and their novel regioselectivity differences thus enabled single-step reaction pathways to an effective collection of doxorubicin analogues