Use of transaminase enzymes for the synthesis of pharmaceutical intermediates

利用酶和合成生物学策略对药物中间体的合成具有巨大的潜力，手性胺的可持续合成受到高度追捧，因为70%的药物都是手性胺的衍生物。迄今为止，手性胺主要是通过水解酶的外消旋混合物的动力学分解来产生的，其中最大产率只能达到50%。去消酰基化生物催化策略也被描述[1,2]，然而，最近的兴趣集中在使用转氨酶（TAm）在真正的不对称转化bbb中产生手性胺。这有可能开发出一种利用转氨酶产生100%不对称产物的工艺，从而减少浪费，降低这些重要合成子的成本。tam催化氨基从供体（如氨基酸）转移到受体酮或醛部分。虽然- tam对-酮酸有强烈的受体偏好，而首选的供体通常是20种-氨基酸中的一种，但-转氨酶可以将氨基转移到醛或酮上，并且通常不需要-酮酸部分b[4]。它们也有更广泛的氨基供体供它们使用。在Shin及其同事[6]之前的工作中，从河流弧菌中分离出一种用于多种酮类和醛类（包括芳香底物）胺化的omega-TAm，我们使用河流弧菌JS17 omega-TAm的蛋白质序列筛选相关酶[6]的基因组数据库。这种生物信息学方法得到了几种omega- tam，其中一种来自紫色杆菌DSM30191，它可以转化一系列酮类和醛类，包括脂肪族和芳香的1,3-二羟基酮，导致产物（(S)-胺）[6]具有很高的立体选择性。在目前为期12个月的EPSRC后续基金奖励（EP/G005834/1）中，这种识别新tam的生物信息学策略正在进行中，以发现更多易于转化一系列脂肪族、环酮和芳香酮和醛的tam。我们在BBSRC工业案例项目中的目标是将生物信息学筛选扩展到新的tam组，然后使用EPSRC项目中的一些tam，以及将作为项目一部分克隆和表征的新tam（包括例如糖特异性tam和ω， β和γ - tam），用于合作公司Chirotech科技有限公司感兴趣的手性胺的合成。这将使TAm生物转化战略在工业环境中的应用成为可能，并确立在更大范围内转化这种方法的关键优势和问题。参考文献：[1]特纳，N.J. Curr。当今。Biotechnol。， 2003,14, 401。[2] Pàmies, o；Bäckvall， J.E.趋势生物技术。， 2004, 22, 130。[3] Koszelewski, d；粘土,d;Rozzell d;克鲁蒂尔，W. Eur。j . Org。化学。， 2009, 2289。[4] Hwang B.Y.；炳宽，c.b.k.；Yun, h;基内拉，k.k.；金姆，j·莫尔·卡塔尔。劳动部。， 2005, 37, 47。J.S. Shin；金B.G.生物科学。Biotechnol。物化学。[j] .中国农业科学，2001,6(1):1 - 2。•史密斯,k;史密斯M.E.B.;冰雹,H.C.;病房里,J.M.;酶活。抛光工艺。中文信息学报，2007,41,628。

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.