Biocatalysts to generate novel aminoglycosides and hydroxylate inactivated C-H bonds

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Biocatalysts are enantio-, chemo-, and regio-selective across a wide range of reactions under mild conditions. Their use in synthesis is however limited in part by chemists' apprehension to handle enzymes. We propose to develop simple biocatalytic methods to generate novel aminoglycosides and hydroxylate inactivated C-H bonds. Aminoglycosides are clinically important broad spectrum antibiotics. Unfortunately, the emergence of resistance is increasingly restricting their use. A better understanding of resistance-causing mechanisms is critical to conquer this health threat. The synthesis of aminoglycoside derivatives is challenging because of solubility issues and of the complex protection schemes required. We have developed a novel methodology for selective N-6' acylation of aminoglycosides. This has allowed the one-pot synthesis of the first nanomolar inhibitors of resistance-causing aminoglycoside 6'-N-acetyltransferase Ii. Our next goal is to prepare aminoglycosides able to escape resistance-causing enzymes while maintaining their antibacterial activity. To this end, we propose to develop new chemoenzymatic methodologies for the regioselective derivatization of aminoglycosides. Commercial enzymes and acetyltransferases will be used. In parallel, we propose to study P450 enzymes for their impressive ability to catalyze the insertion of oxygen into non-activated C-H bonds. Very few methods exist that directly hydroxylate aliphatic C-H bonds, and most of them are of limited scope. P450s represent a promising alternative; however, a number of limitations have restricted their use. These include substrate specificity, the need for 2 or 3 cofactors, incompatibility with organic solvents, low activity and poor stability. The very promiscuous P450s 3A4 and 2D6 were selected to overcome the substrate specificity issue. We have reported that some cheap chemicals can efficiently replace the natural cofactors. and recently found conditions to use these enzymes in the presence of organic solvents. Next we propose to develop a system of substrate tagging to allow prediction of the regio- and stereo-selectivity of P450s.