| A biocatalytic approach to combinatorial chemistry was demonstrated through the generation of a library from the lead compound doxorubicin (DOX), a potent chemotherapeutic and complex natural product. Two enzymes, Mucor javanicus lipase (MJ) and subtilisin Carlsberg (SC), catalyzed nonaqueous acylation of DOX. Aerosol OT ion-paired solubilization of MJ (AOT-MJ) and SC (AOT-SC) in organic solvents led to enhanced rates over particulate enzymes and maintained selectivity for the primary hydroxyl of DOX. Co-lyophilization of SC with 98% (w/w) KCl led to increased reactivity and expanded regiospecificity such that KCl-SC additionally acylated the 4′ hydroxyl and 3′ amine groups of DOX. A library of unique DOX analogs was generated using a combination of AOT-SC and KCl-SC. The cytotoxicities of select compounds were evaluated against MCF7 breast cancer cells and the multidrug-resistant sub-line, MCF7-ADR. Two derivatives had potencies equal to, or greater than, DOX with one of the cell lines.; The ion-pairing solubilization technique required adaptation for optimal use with Mucor javanicus lipase, the first ion-paired lipase. The impact of ionic strength and pH of the aqueous phase during solubilization was characterized by response surface analysis of extraction efficiency and specific activity. Fatty acid esterification provided a rapid and low-cost measure for optimizing activity, and the results were transferable to the acylation of DOX.; Solid-phase synthesis of dipeptides in nonaqueous media was achieved using AOT-α-chymotrypsin. For both solid-phase and solution-phase reactions, multiple solvents and systematic variation of water activity were used to establish correlations for the rate of coupling N-α-benzyloxycarbonyl- L-phenylalanine methyl ester (Z-Phe-OMe) to leucine as a function of the reaction medium. In solution, the maximum reaction rate varied in a direct fashion with solvent hydrophobicity. For solid-phase synthesis, the highest synthetic rates were confined to environments that fell inside abruptly defined regions of thermodynamic parameter space (e.g., log P > 2.3). Correlation of the reaction rate with the activity of the reactants and products was also observed. Optimized conditions were used to synthesize seven different N-protected solid-supported dipeptides with yields of 74 to 98%. |
