Use of transaminase enzymes for the synthesis of pharmaceutical intermediates

酶的使用和合成生物学策略在药物中间体的合成方面具有巨大的潜力，由于70%的药物是手性胺的衍生物，手性胺的可持续合成受到了极大的追捧。到目前为止，手性胺的生成主要是通过生物催化策略，主要是通过外消旋混合物与水解酶的动力学拆分，在这种情况下只能获得50%的最高产率。还描述了一种去消旋生物催化策略[1，2]，然而，最近人们的兴趣集中在使用转氨酶()在真正的不对称转化中生成手性胺[3]。这有可能开发一种使用转氨酶来产生100%不对称产物的方法，从而减少浪费，并为这些重要的合成子提供更低的成本策略。TAMs催化氨基从供体(如氨基酸)转移到受体酮或醛部分。虽然α-TAMs强烈倾向于以α-酮酸为受体，并且首选供体通常是20种α-氨基酸中的一种，但omega-转氨酶可以将氨基转移到醛或酮，并且通常不需要α-酮酸部分[4]。他们也有更广泛的氨基供体可以使用。在Shin和他的同事以前的工作中[5]，我们从河流弧菌中分离出一种用于包括芳香族底物在内的多种酮和醛的胺化反应的omega-，我们使用河流弧菌JS17 omega-的蛋白质序列来筛选相关酶的基因组数据库。这种生物信息学方法产生了几个omega-TAMs，包括来自紫色铬杆菌DSM30191的一个，它可以转化酮和醛，包括脂肪族和芳香族1，3-二羟基酮，导致产品((S)-胺)[6]具有非常高的立体选择性。在目前为期12个月的EPSRC后续基金奖(EP/G005834/1)中，正在采用这一生物信息学战略来确定新的TAMs，以寻找更多容易转化一系列脂肪、环状和芳香酮和醛的TAMs。BBSRC工业案例项目的目标是将生物信息学筛选扩展到新的TAMs组，然后使用EPSRC项目中的几个TAMs，以及将被克隆和表征为项目一部分的新TAMs(例如，糖特定的TAMs和omega、beta和Gamma-TAMs)，用于合作公司Chirotech Technology Limited感兴趣的手性胺的合成。这将使生物转化战略能够在工业环境中应用，并确立在更大范围内翻译这一方法的关键优势和问题。参考文献：[1]Turner，N.J.Curr.奥平。《生物技术》，2003，14,401。[2]P？mies，O.；Bäck vall，J.E.趋势生物技术，2004，22,130。[3]Koszelewski，D.；Clay，D.；Rozzell，D.；Kroutil，W.J.组织化学，2009,2289。[4]Hwang，B.Y.；Byung-kwan，C.B.K.；Yun，H.；Kinera，K.；Kim，J.Mol.卡塔。B：恩兹，2005，37，47。[5]Sin，J.S.；Kim，B.G.Biosi生物技术。生物化学，2001，65,1782[6]；Smithies，K.；Smith，M.E.B.；Hailes，H.C.；Ward，J.M.；酵素微生物。Technol，2007年，41628。

The use of enzymes and synthetic biology strategies hold significant potential for the synthesis of pharmaceutical intermediates, and sustainable syntheses of chiral amines are highly sought after since 70% of all pharmaceutical are derivatives of chiral amines. To date chiral amines have been generated using biocatalytic strategies predominantly via the kinetic resolution of racemic mixtures with hydrolytic enzymes, where only a 50% maximum yield can be achieved. A deracemization biocatalytic strategy has also been described [1,2], however, recently interest has focused of the use of transaminases (TAm) to generate chiral amines in a genuinely asymmetric transformation [3]. This has the potential for a process to be developed using transaminases that would yield 100% of the asymmetric product, resulting in less waste and a lower cost strategy to these important synthons. TAms catalyse the transfer of an amino group from a donor such as an amino acid, to an acceptor ketone or aldehyde moiety. While the alpha-TAms have a strong preference for an alpha-keto acid as the acceptor and the preferred donor is usually one of the 20 alpha-amino acids, the omega-transaminases can transfer an amino group to an aldehyde or a ketone and do not frequently have a requirement for the alpha-keto acid moiety [4]. They also have a broader range of amino donors that they can use. In previous work by Shin and co-workers [5] an omega-TAm used for the amination of a wide range of ketones and aldehydes, including aromatic substrates, was isolated from Vibrio fluvialis and we used the protein sequence of V. fluvialis JS17 omega-TAm to screen the genome databases for related enzymes [6]. This bioinformatics approach yielded several omega-TAms including one from Chromobacterium violaceum DSM30191 that can convert of a range of ketones and aldehydes, including aliphatic and aromatic 1,3-dihydroxy ketones, resulting in very high stereoselectivities in the product ((S)-amine) [6]. In a current, 12 month EPSRC Follow-on-Fund award (EP/G005834/1), this bioinformatics strategy to identify new TAms is being pursued to find further TAms that readily convert a range of aliphatic, cyclic and aromatic ketones and aldehydes. Our aim with a BBSRC industrial CASE project is to extend the bioinformatics screen to new groups of TAms and then use several of the TAms from the EPSRC project, and new TAms (including for example sugar-specific TAms and the omega, beta and gamma-TAms) which will be cloned and characterised as part of the project, for the synthesis of chiral amines of interest to the collaborating company Chirotech Technology Limited. This will enable application of the TAm biotransformation strategy in an industrial environment and establish key advantages and problems of translating this approach at a larger scale. References: [1] Turner, N.J. Curr. Opin. Biotechnol., 2003, 14, 401. [2] Pàmies, O.; Bäckvall, J.E. Trends Biotechnol., 2004, 22, 130. [3] Koszelewski, D.; Clay, D.; Rozzell, D.; Kroutil, W. Eur. J. Org. Chem., 2009, 2289. [4] Hwang, B.Y.; Byung-Kwan, C. B. K.; Yun, H.; Kinera, K. K.; Kim, J. Mol. Catal. B: Enz., 2005, 37, 47. [5] Shin, J.S.; Kim, B.G. Biosci. Biotechnol. Biochem., 2001, 65, 1782 [6] Kaulmann, U.; Smithies, K.; Smith, M.E.B.; Hailes, H.C.; Ward, J.M.; Enzyme Microb. Technol., 2007, 41, 628.