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Deracemisation of Mandelic Acid to Optically Pure Non?Natural L COMMUNICATIONS DOI: 10.1002/adsc.200900891 Deracemisation of Mandelic Acid to Optically Pure Non-Natural l-Phenylglycine via a Redox-Neutral Biocatalytic Cascade Verena Resch,a Walter M. F. Fabian,a and Wolfgang Kroutila,* a Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria Fax : (+43)-316-380-9840; e-mail: [email protected] Received: December 22, 2009; Published online: April 7, 2010 Abstract: A biocatalytic redox-neutral reaction cas- enzymes might be the perfect catalysts to set up a cade was designed for the deracemisation of race- similar system for synthetic applications. Combina- mic mandelic acid to yield optically pure l-phenyl- tions of simultaneous biocatalytic reduction and oxi- glycine employing three enzymes. The cascade con- dation have already been reported for the deracemi- sisted of three steps: a racemisation, an enantiose- sation of sec-alcohols.[8,9] lective oxidation and a stereoselective reductive Enantiomeric pure a-amino acids are becoming amination. The enantioselective oxidation of d- more and more important as building blocks and mandelic acid to the corresponding oxo acid was starting materials, especially in the pharmaceutical coupled with the stereoselective reductive amina- and fine chemicals industry.[10] Particularly non-natu- tion of the latter; thus the oxidation as well as the ral a-amino acids are in the focus of interest. For ex- reduction reactions were performed simultaneously. ample, d-phenylglycine or d-4-hydroxyphenylglycine The formal hydrogen abstracted in the first step – are used as building blocks for semi-synthetic broad- the oxidation – was consumed in the reductive ami- spectrum antibiotics like Ampicillin and Amoxicil- nation allowing a redox-neutral cascade due to a lin.[11] Other non-natural amino acids are needed as cascade-internal cofactor recycling. The enantio- templates in asymmetric synthesis.[12] Various methods mers of the starting material were interconverted for the preparation of enantiopure non-natural a- by a racemase (mandelate racemase) ensuring that amino acids have been reported.[11] in theory 100% of the starting material can be The aim of the concept presented in this paper is to transformed. Using this set-up racemic mandelic transform the a-hydroxy acid mandelic acid 1 into the acid was transformed to optically pure l-phenylgly- corresponding optically pure non-natural a-amino cine (ee > 97%) at 94% conversion without the re- acid. In a first step the a-hydroxy acid should be oxi- quirement of any additional redox reagents in stoi- dised to the corresponding a-oxo acid (Scheme 1); chiometric amounts. the latter should concurrently be transformed to the corresponding a-amino acid via an asymmetric reduc- Keywords: amino acids; deracemisation; domino re- tive amination, whereby the formal hydrogen ab- action; enzyme catalysis; oxidation; reduction stracted in the oxidation is used in the reduction; therefore both reactions – the enantioselective oxida- tion as well as the stereoselective reduction – have to run simultaneously. Such a concept of re-using the hydrogen has been Catalytic tandem reactions[1–5] are processes where referred to as borrowing hydrogen in metal cataly- different catalysts operate concurrently in one pot to overcome the limitations and disadvantages of a mul- tistep synthesis such as time-consuming or yield-re- ducing isolation and purification of the intermedi- ates.[6] Although the concept of cascade reactions is very elegant, there are still a lot of challenges that need to be addressed. Especially a cascade involving a concurrent reduction and an oxidation step renders the system even more complex due to the diverging Scheme 1. Concurrent redox-neutral cascade for the trans- [7] reaction conditions. Since in living cells oxidation formation of an a-hydroxy acid to the corresponding a- and reduction processes are working simultaneously, amino acid. l-AADH: l-amino acid dehydrogenase. Adv. Synth. Catal. 2010, 352, 993 – 997 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 993 COMMUNICATIONS Verena Resch et al. sis.[13] In biocatalysis only few related examples of Table 1. Reductive amination of phenyl glyoxylate 2 yielding redox-neutral cascades with internal cofactor recy- 3. cling have been mentioned,[14–16] where the nicotin- Entry AADH[a] Conv.[b,c] [%] ee[d] [%] amideACHTUNGRE cofactors NAD(P)+ are reduced in the first step of a cascade and the NAD(P)H formed is then 1 l-AADH-101 >97 >97 (l) consumed in a second step, whereby oxidation and re- 2 l-AADH-102 <1 n.d.[e] duction mediated by the NAD(P)-cofactor occur at 3 l-AADH-103 5 52 (l) l l the same molecule. This approach of internal cofactor 4 -AADH-104 >97 >97 ( ) l l recycling avoids the additional use of other enzymes 5 -AADH-105 >97 >97 ( ) 6 l-AADH-106 >97 >97 (l) as well as reagents for the recycling of the cofactors, 7 l-AADH-107 >97 >97 (l) therefore leading to a redox-neutral cascade. 8 l-AADH-108 >97 >97 (l) Since in such a sequence all transformations are re- 9 l-AADH-109 4 51 (l) versible the favoured product formed depends on 10 l-AADH-110 5 48 (l) thermodynamics. Calculation[17] of the Gibbs free 11 l-AADH-111 >97 >97 (l) energy DG for the interconversion of lactic acid and 12 l-AADH-112 4 >97 (l) alanine showed that the thermodynamic equilibrium 13 l-AADH-113 <1 n.d. is on the side of the amino acid (À5.7 kcalmolÀ1); 14 l-AADH-114 >97 >97 (l) l l thus an a-hydroxy acid should be preferentially con- 15 -AADH-115 >97 >97 ( ) l l verted to the amino acid, which encouraged us to per- 16 -AADH-116 4 >97 ( ) 17 l-AADH-117 5 >97 (l) form further investigations on this subject. 18 l-AADH-118 >97 >97 (l) Since the enantioselective oxidation of d-mandelic d l [18] 19 -AADH-101 4 54 ( ) acid by mandelate dehydrogenase is well described 20 d-AADH-102 5 51 (l) we tested first various commercial amino acid dehy- 21 d-AADH-103 3 >97 (l) drogenases (AADHs) exclusively for the reductive 22 d-AADH-104 4 >97 (l) amination of phenyl glyoxylate 2 employing glucose 23 d-AADH-105 4 >97 (l) and glucose dehydrogenase for the recycling of the 24 d-AADH-106 3 >97 (l) cofactor NADH (Table 1). [a] l d Amino acid dehydrogenase, the numbers refer to the Interestingly - as well as -AADHs led to the for- enzyme kit of Codexis. mation of the l-amino acid, although in case of d- [b] Reaction conditions: phenylglyoxylic acid 2 (10 mg, AADHs at rather low conversion. Probably these d- 0.07 mmol), amino acid dehydrogenase (2 mg, l-AADH- AADHs are only stereospecific for aliphatic amino 101–118, d-AADH-101–105), glucose (30 mg, acids as stated by the provider.[19] 0.17 mmol), glucose dehydrogenase (10 mL, 5 U), NADH Before the d-mandelate dehydrogenase (d-MDH) (1 mg, 0.002 mmol), buffer (1 mL, pH 9.5, sodium bicar- was coupled with an AADH the activity of the d- bonate 100 mM, NH4Cl 200 mM), shaking at 308C with l l 120 rpm for 22 h. MDH as well -AADH ( -AADH-101) was deter- [c] mined by a UV assay, following the formation/con- Conversions were measured by HPLC-DAD analysis on an achiral phase. The measured values were corrected sumption of NADH. The units of the enzymes em- using a calibration curve. ployed for the cascade were chosen in such a way that [d] Enantiomeric excess was determined by HPLC analysis the reductive amination step is six times faster than on a chiral phase. In brackets the isomer obtained in the oxidation step. excess is given. Employing this set-up it is ensured that NADH is [e] n.d. not determined due to too low conversion. quickly consumed in the second step providing NAD+ for the thermodynamically unfavoured first step, the asymmetric oxidation. itial idea for internal cofactor recycling to set up a Having now chosen possible suitable reaction con- redox-neutral cascade. Since the substrate possessed ditions, enantiopure d-mandelic acid was transformed the d-configuration and the product showed the l- in the presence of d-MDH, l-AADH, cofactor and configuration an inversion of absolute configuration ammonium to yield indeed the desired l-phenylgy- was achieved resembling a Mitsunobu stereoinver- cine. At a substrate concentration of 10 gLÀ1 a con- sion.[20–23] In contrast to the reagent intensive Mitsu- version of up to 76% was reached, whereby the nobu reaction (stoichiometric amount of, for example, amount of oxo acid intermediate was below 0.3%. In- PPh3 and DEAD), the here presented system requires terestingly, longer reaction times did not lead to just two biocatalysts, catalytic amounts of cofactor higher conversion. Additional experiments showed and ammonium ions. that the achieved concentration of l-phenylglycine In the first experiments the substrate was optically caused inhibition of d-MDH. Reducing the substrate pure d-mandelic acid. As a further challenge our aim concentration to 5 gLÀ1 circumvented inhibition. Nev- was to start from racemic mandelic acid and to con- ertheless, the successful transformation proved the in- vert the racemate to the optically pure l-amino acid, 994 asc.wiley-vch.de 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Synth. Catal. 2010, 352, 993 – 997 Deracemisation of Mandelic Acid to Optically Pure Non-Natural l-Phenylglycine Table 2. Transformation of racemic mandelic acid to optical- ly pure l-phenylglycine via a redox-neutral cascade (see Scheme 2).[a] [b] [c] [c] [c] [d] Entry AADH 1 [%] 2 [%] 3 [%] eeP [%] 1 l-AADH-101 14.0 0.2 85.8 > 97 2 l-AADH-104 15.7 0.2 84.1 > 97 3 l-AADH-105 14.9 0.2 84.9 > 97 4 l-AADH-106 16.1 0.2 83.7 > 97 5 l-AADH-107 16.7 0.2 83.1 > 97 6 l-AADH-108 19.4 0.2 80.3 > 97 7 l-AADH-111 14.8 0.2 85.0 > 97 Scheme 2.
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