PAPER www.rsc.org/obc | Organic & Biomolecular Chemistry Rapid screening and scale-up of transaminase catalysed reactions†‡ Matthew D. Truppo,* a J. David Rozzell, b Jeffrey C. Moore c and Nicholas J. Turner* a Received 8th October 2008, Accepted 17th October 2008 First published as an Advance Article on the web 26th November 2008 DOI: 10.1039/b817730a A rapid, high-throughput screening methodology has been developed for the determination of transaminase activity. This pH based, colorimetric assay can also be used to scale reactions directly from 100 mL screening scale to 25 mL development scale. Additionally, three techniques have been developed to drive transamination reactions toward complete conversion. The first method uses lactate dehydrogenase to remove the inhibitory pyruvate keto acid by-product from the reaction and drive reaction equilibrium toward the desired amine. The second method is a single enzyme system, and uses a large excess of isopropylamine to drive the transamination. Method three requires only a catalytic amount of amine donor, as an amino acid dehydrogenase is employed to regenerate the amine donor in situ using ammonia. All three systems have been demonstrated for the production of optically pure methylbenzylamine from acetophenone. An enantiomeric excess of >99% was achieved for both the R- and S-methylbenzylamine products. Introduction Enantiomerically pure chiral amines are valuable building blocks for the preparation of pharmaceutical agents that span a range of therapeutic areas including antihypertensives, antibiotics, antide- pressants, antihistamines, and antidiabetics. 1 Although a number of enzymatic methods have been developed for the production of chiral amines with high enantiomeric excess, most processes are based upon kinetic resolution via enantioselective acylation using a lipase or acylase. 2–6 Attempts to develop dynamic kinetic resolutions (DKRs) using lipases and metal catalysts have met with partial success, although the reaction conditions required for racemization of the amine are quite harsh and hence incompatible with the requirements for the acylating enzyme. 7–9 A conceptu- ally different approach for deracemization of amines has been developed using an amine oxidase in combination with chemical reducing agents. 10 Transaminases represent an attractive option for chiral amine synthesis in that they catalyse the transfer of ammonia from an amine donor to a prochiral ketone. 11 Moreover, these enzymes typically possess high turnover rate, stability and a tightly bound pyridoxal cofactor. However, few examples of large-scale reactions have been reported 12,13 largely as a result of three drawbacks that hinder their application on a preparative scale; (i) the equilibrium constant for the reaction generally favours the ketone starting material, 14 (ii) screening for substrate acceptance largely a School of Chemistry, University of Manchester, Manchester Interdisci- plinary Biocentre, 131 Princess Street, Manchester, UK M17DN. E-mail: Nicholas.turner@manchester.ac.uk; Fax: (+44)161 306 5173 b Codexis Inc., 129 N. Hill Avenue, Pasadena, CA, 91106, USA c Department of Process Research, Merck Research Laboratories, Merck & Co., Inc., Rahway, NJ, 07065, USA †Dedicated to Professor Andrew B. Holmes on the occasion of his 65th birthday. ‡Electronic supplementary information (ESI) available: General experi- mental and HPLC assay conditions, rapid pH indicator based colorimetric transaminase activity assay, 25 mL scale transamination reactions, and HPLC chromatograms. See DOI: 10.1039/b817730a relies upon low-throughput methods such as HPLC and (iii) transaminases typically suffer significant product inhibition by the ketone or keto acid by-product. 14 Although previous researchers have separately addressed the issues of screening 15 and product inhibition, 16 no simple procedure exists for carrying out both processes under the same conditions to allow rapid progress from small to large-scale reactions. Results and discussion Scheme 1 shows a transaminase catalysed reaction for conversion of acetophenone 1 to 1-phenylethylamine 2 together with three different systems that can potentially provide the amino donor. As outlined above, we envisaged developing a high-throughput Scheme 1 Overview of transaminase catalysed reaction. This journal is © The Royal Society of Chemistry 2009 Org. Biomol. Chem., 2009, 7, 395–398 | 395