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

生物催化剂具有对映性、化学性和区域选择性，可在温和条件下进行广泛的反应。然而，它们在合成中的应用受到限制，部分原因是化学家对处理酶的顾虑。我们建议开发简单的生物催化方法来生成新的氨基糖苷和羟基化失活的C-H键。氨基糖苷类抗生素是临床上重要的广谱抗生素。不幸的是，耐药性的出现日益限制了它们的使用。更好地了解耐药性产生机制对于克服这一健康威胁至关重要。氨基糖苷衍生物的合成是具有挑战性的，因为溶解度问题和复杂的保护方案所需要的。我们已经开发了一种新的方法选择性N-6'酰化氨基糖苷。这使得第一个纳米摩尔耐药氨基糖苷6′- n -乙酰转移酶Ii抑制剂的一锅合成成为可能。我们的下一个目标是制备能够在保持抗菌活性的同时逃避引起耐药性的酶的氨基糖苷。为此，我们建议开发新的化学酶方法用于氨基糖苷的区域选择性衍生化。将使用商业酶和乙酰转移酶。同时，我们建议研究P450酶，因为它们具有催化氧插入非活化的C-H键的令人印象深刻的能力。直接羟基化脂肪碳-氢键的方法很少，而且大多数方法的应用范围有限。p450是一个很有前途的选择；然而，一些限制限制了它们的使用。这些因素包括底物特异性，需要2或3个辅因子，与有机溶剂不相容，活性低，稳定性差。选择非常混杂的P450s 3A4和2D6来克服底物特异性问题。我们已经报道了一些廉价的化学品可以有效地取代天然辅因子。最近发现了在有机溶剂存在下使用这些酶的条件。接下来，我们建议开发一个底物标记系统，以预测p450的区域选择性和立体选择性。

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.