title: 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 mumol/min g-lipase, respectively, vs 0.13 mumol/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 mumol/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 mumol/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 muM). The novel derivative 14-(2-thiophene acetate) DOX was relatively potent against both cell lines (IC50 of 65 nM and 8 muM, 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.
authors.affiliation: Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Altreuter
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dordick
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000138:chemical synthesis* / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy; D002384:Catalysis; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D008049:Lipase / Q000737:chemistry / Q000378:metabolism; D009089:Mucor / Q000201:enzymology; D011189:Potassium Chloride / Q000737:chemistry; D012995:Solubility; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000903:Antibiotics, Antineoplastic; D011189:Potassium Chloride; D004317:Doxorubicin; D008049:Lipase; D020860:Subtilisin