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Cite this: Chem. Commun., 2014, 50, 12680 Received 28th June 2013, Accepted 22nd August 2014
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Enzymatic synthesis of chiral c-amino acids using x-transaminase† Minsu Shon,‡a Ramachandran Shanmugavel,‡b Giyoung Shin,a Sam Mathew,a Sang-Hyeup Lee*b and Hyungdon Yun*c
DOI: 10.1039/c3cc44864a www.rsc.org/chemcomm
In this study, we successfully synthesized enantiomerically pure (R)- and (S)-c-amino acids (499% ee) using x-transaminase (x-TA) through kinetic resolution and asymmetric synthesis respectively. The present study demonstrates the high potentiality of x-TA reaction for the production of chiral c-amino acids.
Non-natural g-amino acids have attracted a great deal of attention because of their important role in the design and synthesis of bioactive molecules and in the application of hybrid peptides.1 For example, g-amino acids are key components of natural products such as hapalosin,2 dolastatin,3 caliculins4 and of various enzyme inhibitor g-aminobutyric acid analogues.1 Given the significance of g-amino acids, their efficient synthesis in optically pure form has become an attractive challenge to organic chemists and biologists. Lately, a number of strategies have been developed for the chemical synthesis of enantiomerically pure g-amino acids.5 Recently, peptidecatalyzed asymmetric Michael addition of nitromethane to b-disubstituted a,b-unsaturated aldehydes5a and enantioselective g-amination of a,b-unsaturated acyl chlorides with azodicarboxylates5b were developed to synthesize chiral g-amino acid derivatives. Enzyme-catalyzed chemical transformations are now widely recognized as practical alternatives to traditional organic synthesis.6 A number of biocatalytic routes were developed for the synthesis of chiral a- and b-amino acids using enantioselective enzymes such as transaminase (TA), hydantoinase, dehydrogenase, aminoacylase, penicillinG amidase, b-aminopeptidase, lipase, phenylalanine aminomutase, and Baeyer–Villiger monooxygenase.7,8 However, biocatalytic routes for the synthesis
of chiral g-amino acids have not been studied well. Recently, o-TA catalyzed reaction has gained a lot of attention due to its ability to generate enantiomerically pure amines and unnatural amino acids.9 Desirable characteristics like broad substrate specificity, high enantioselectivity, high turnover number and non-requirement for regeneration of external cofactors have made o-TA an attractive biocatalyst.9 Some o-TAs such as a o-TA from Polaromonas sp. (o-TAPO) showed a broad substrate specificity toward amines, L-amino acids and b-amino acids, suggesting that those enzymes may also be reactive towards g-amino acids.10,11 Here, we report an alternative method for the synthesis of (R)- and (S)-g-amino acids via kinetic resolution and asymmetric synthesis using o-TAs, respectively, (Scheme 1). The genes of o-TAPO11 and a newly identified o-TA from Burkholderia graminis C4D1M (accession #; ZP_02885261, o-TABG) were cloned into the vector pET24ma with a C-terminal His6-tag and effectively expressed in Escherichia coli BL21 (DE3). The enzymes were then purified on a Ni-NTA affinity column (Fig. S1, ESI†). The purified enzymes were then subjected to activity assay which was performed with 10 mM (S)-b-phenylalanine and 20 mM pyruvate (a typical amino acceptor) in 200 mM Tris/HCl buffer (pH 7.0) at 37 1C. The specific activities of o-TAPO and o-TABG were 5.6 and 13.6 U mg 1, respectively (Fig. 1). rac-g-Amino acids (1a–g) were prepared from their corresponding, commercially available g-keto acids via a two step
a
School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, South Korea b Department of Life Chemistry, Catholic University of Daegu, Gyeongbuk 700-443, Korea. E-mail: [email protected] c Department of Bioscience & Biotechnology, Konkuk University, Seoul 143-701, Korea. E-mail: [email protected]; Fax: +82-2-450-0686; Tel: +82-2-450-0496 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc44864a ‡ These authors contributed equally to this work.
12680 | Chem. Commun., 2014, 50, 12680--12683
Scheme 1 Synthesis of enantiomerically pure g-amino acid by using o-TA. (a) Kinetic resolution of g-amino acids. (b) Asymmetric synthesis of g-amino acids.
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ChemComm Table 1
Kinetic resolution of g-amino acidsa
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o-TAPO
Fig. 1 Substrate scope for deamination of o-TAPO and o-TABG. Enzyme reactions were carried out in 1 mL of 200 mM Tris/HCl buffer (pH 7.0) containing 10 mM racemic substrate (1a–g), 20 mM pyruvate, 0.1 mM PLP and o-TA (0.02 mg mL 1) for 30 min at 37 1C. One unit is defined as the amount of enzyme that catalyzes depletion of 1 mmol of pyruvate, betaPhe and (S)-b-phenylalanine.
process, oximation followed by catalytic hydrogenation of the resultant oximes12 (ESI†). The amino donor specificities of enzymes toward 1a–g were examined in the presence of 10 mM rac-g-amino acids and 20 mM pyruvate (Fig. 1). Among 1a–g, o-TAPO and o-TABG showed the highest reactivity for 1d (5.5 U mg 1) and 1e (3.9 U mg 1), respectively. Both o-TAPO (1.5 U mg 1) and o-TABG (0.36 U mg 1) showed the least reactivity towards 1f. The lower activity of 1f and 1g towards o-TAs may be due to the presence of bulky side chains which cause steric hindrance in the active sites of the enzymes. o-TABG showed about 2.4-fold higher specific activity towards (S)-bphenylalanine, while o-TABG showed only about 36% specific activity towards 1a than that of o-TAPO. In addition, (R)-selective o-TAs from Mycobacterium vanbaalenii13 and Neosartorya fischeri,13 and (S)-selective o-TAs from Vibrio fluviallis JS1714 were expressed in E. coli and purified. To examine the suitability of these enzymes for the production of g-amino acids, enzyme activities were measured using 1a as a representative g-amino acid, and these enzymes did not show any reactivity towards 1a (data not shown). The kinetic resolution of 1a was carried out using o-TAPO and o-TABG, and both enzymes converted only (S)-1a into 2a, and enantiomerically pure (R)-1a (499% ee) was obtained using both the enzymes in the presence of 10 mM rac-1a and 20 mM pyruvate (Fig. S2, ESI†). o-TAPO showed a faster reaction profile than o-TABG. Subsequently, the kinetic resolution of various g-amino acids (1a–g) using o-TAs was carried out under similar conditions as described above (Table S3, ESI†). All racemic substrates except 1f and 1g were successfully resolved into (R)-g-amino acids (499% ee) with about 50% conversion using o-TAPO (0.02 mg mL 1) and o-TABG (0.04 mg mL 1). In the case of 1f and 1g, higher concentration of enzymes (0.2 mg mL 1) was required to reach 499% ee due to its lower reactivity towards these substrates. The enantioselectivities (Es) of the enzymes were calculated using the equation E = ln[(1 c)(1 ees)]/ln[(1 c)(l + ees)]15 and were found to be 4100 for all substrates. This result indicates that o-TAPO and o-TABG are suitable biocatalysts for the production of highly enantiomerically pure g-amino acids.
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o-TABG
Sub.
Time (h)
Conv. (%)
eeR (%)
Time (h)
Conv. (%)
eeR (%)
1a 1b 1c 1d 1e 1f b,c 1g 1g c
24 24 24 24 24 24 36 24
50.1 50.3 50.1 n.c.d 50.1 49.6 46.5 50.1
499 499 499 — 499 98 87 499
24 24 24 24 24 24 36 24
50.0 50.5 50.2 n.c. 50.2 50.2 45.6 50.2
499 499 499 — 499 499 84 499
a Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/HCl buffer (pH 7.0) containing 100 mM g-amino acid, 200 mM pyruvate, DMSO (10%), and 1 mM PLP by using o-TAPO (0.2 mg mL 1) or o-TABG (0.4 mg mL 1); conv. and ee were determined by HPLC. b 50 mM 1f. c 0.68 mg mL 1 of o-TA was used. d No conversion.
To carry out the enzyme reaction at higher concentration, dimethylsulfoxide (DMSO) was added into the reaction mixture for increasing the solubility of the substrates. The kinetic resolution of g-amino acids was carried out in 1 mL of 10% DMSO (v/v) solution containing 100 mM g-amino acids (Table 1). In the case of 1f, 50 mM 1f was used for the reaction due to its low solubility. Except for 1d, all aromatic g-amino acids were successfully resolved into the (R)-form by both enzymes with a high ee value and ca. 50% conversion. In the case of 1d, 10 mM rac-1d was successfully resolved into a (R)-gamino acid (499%) by both enzymes (Table S3, ESI†), however, both enzymes did not show any reactivity in the presence of 100 mM 1d which may be due to severe substrate inhibition. (S)-g-Amino acids were also produced using g-keto acids (2a–g) as initial substrates via asymmetric synthesis (Scheme 1b). Asymmetric synthesis has been of keen interest among researchers, as it can theoretically generate two-fold higher yield (100%) than that of the kinetic resolution. To identify the best amino donor for o-TAs, the asymmetric syntheses of 1a from 10 mM 2a were carried out with L-Ala (with or without a pyruvate removal system), b-Ala, L-glutamic acid (2-ketoglutarate is a good amino acceptor for o-TAPO),11 and (S)-a-methylbenzylamine (MBA, a typical substrate
Fig. 2 Asymmetric synthesis of 1a from 2a using various amino donors. Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/HCl buffer (pH 7.0) containing 100 mM amino donor, 10 mM 2a, 0.1 mM PLP by using o-TA (0.34 mg mL 1) for 16 h. Conversion was determined by HPLC. In the case of L-Ala/LDH, 200 mM glucose, 1 mM NADH, lactate dehydrogenase (LDH, 55 U), and glucose dehydrogenase (2.7 U) were added in the L-Ala reaction solution for removal of pyruvate.
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for o-TA)9 (Fig. 2). o-TAs gave the highest conversion with (S)-a-MBA, and o-TAPO gave higher conversion (56.1%) than that of o-TABG (25.3%). Thus, (S)-a-MBA was selected as the best amino donor for the asymmetric synthesis. When asymmetric synthesis was carried out with 100 mM 2a, 100 mM (S)-a-MBA and o-TAPO (1 mg mL 1), the conversion was o3% after 24 h of reaction. Therefore, the substrate inhibition of o-TA by 2a was examined with 50 mM (S)-a-MBA and various concentrations of 2a (0–100 mM) (Fig. S3, ESI†). The initial reaction rates of o-TAPO and o-TABG were the highest at 5 mM 2a, and thereafter decreased with an increase in the concentration of 2a. In the presence of 10 mM 2a, o-TAPO and o-TABG showed, respectively, 74 and 87% activities of those with 5 mM 2a. Next, the asymmetric syntheses were carried out with 10 mM 2a due to severe substrate inhibition at higher concentration of 2a (410 mM). Here, (S)-a-MBA was used as the amino donor for the asymmetric synthesis of g-amino acids. The product inhibition by acetophenone (deaminated product of a-MBA) is well reported.9 To overcome the product inhibition by acetophenone, a biphasic reaction system with iso-octane was introduced (Fig. S4, ESI†).14,16 In the biphasic system, g-amino acid (1a), g-keto acid (2a), and (S)-a-MBA existed in the aqueous solution due to their electric charges at neutral pH while the inhibitory acetophenone gets transferred to the iso-octane layer. In the biphasic reaction system, the biphasic mixture was gently shaken in a shaking incubator with 250 rpm at 37 1C. As expected, the biphasic reaction system gave higher conversion of the product (97%) than the monophasic reaction system (80%). Subsequently, asymmetric syntheses of various g-amino acids were carried out in monophasic and biphasic systems (Table 2). Both systems gave enantiomerically pure (S)-g-amino acids (499% ee). For all the substrates, o-TAPO gave higher conversion than o-TABG, which indicates that o-TAPO is a more suitable catalyst than o-TABG for the asymmetric synthesis of g-amino acids. In most of the cases, the biphasic system gave a higher conversion. It is notable that 1f and 1g were less reactive substrates for the kinetic resolution, but in asymmetric synthesis, the conversion of 2f and 2g with o-TAPO was higher than other substrates. Finally a preparative scale reaction was carried out at 37 1C in Table 2
Asymmetric synthesis of g-amino acids
o-TAPO
o-TABG
Monophasic
a
S
b
Biphasic
Monophasic S
Biphasic
Sub.
Conv. [%]
ee [%]
Conv. [%]
ee [%]
Conv. [%]
ee [%]
Conv. [%]
eeS [%]
2a 2b 2c 2d 2e 2f 2g
77.6 75.1 72.1 72.9 52.7 85.3 99.9
499 499 499 499 499 499 499
95.8 94.3 91.0 88.5 67.5 99.0 99.9
499 499 499 499 499 499 499
14.0 17.3 14.7 3.21 5.10 1.02 48.9
499 499 499 499 499 499 499
23.0 17.2 27.7 14.5 8.45 23.4 57.6
499 499 499 499 499 499 499
a
S
Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/ HCl buffer (pH 7.0) containing 10 mM substrate, 20 mM (S)-a-MBA, 0.1 mM PLP for 36 h by using o-TAPO (0.34 mg mL 1) or o-TABG (0.68 mg mL 1); conv. and ee were determined by HPLC (see the ESI). b 1 mL of iso-octane was added to 1 mL of the aqueous reaction mixture.
12682 | Chem. Commun., 2014, 50, 12680--12683
80 mL of 20 mM phosphate buffer (pH 7.0) containing 10 mM 2a, 100 mM (S)-a-MBA, and 0.1 mM PLP using purified o-TAPO (8 mg), and carefully 40 mL of iso-octane was added to 80 mL of the aqueous reaction mixture. After 24 h of reaction, HPLC analysis showed 93% conversion with 499% ee. 1a was isolated from this reaction with 38% yield (ESI†). In conclusion, enantiomerically pure (R)- and (S)-g-amino acids (499% ee) were produced using o-TAs through kinetic resolution and asymmetric synthesis respectively. The present study demonstrates the high potentiality of o-TA reaction for the production of chiral g-amino acids. We are currently developing an asymmetric synthesis method to overcome product inhibition by using a coupling reaction. This research was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A2A2A01068013). And this research was partially supported by a grant (A10301712131560200) of Global Cosmetics R&D Project, Ministry of Health & Welfare, Korea.
Notes and references ´n ˜ez and C. Cativiela, Tetrahedron: Asymmetry, 1 For a review, see: M. Ordo 2007, 18, 3–99. 2 K. Stratmann, D. L. Burgoyne, R. E. Moore and G. M. L. Patterson, J. Org. Chem., 1994, 59, 7219–7226. 3 G. R. Pettit, Y. Kamano, C. L. Herald, A. A. Tuinman, F. E. Boettner, H. Kizu, J. M. Schmidt, L. Baczynskyj, K. B. Tomer and R. J. Bontems, J. Am. Chem. Soc., 1987, 109, 6883–6885. 4 Y. Kato, N. Kusetani, S. Matsunaga, K. Hashimoto and T. Furuya, J. Am. Chem. Soc., 1986, 108, 2780–2781. 5 For selected examples, see: (a) K. Akagawa and K. Kudo, Angew. Chem., 2012, 124, 12958–12961 (Angew. Chem., Int. Ed., 2012, 51, 12786–12789); (b) L.-T. Shen, L.-H. Sun and S. Ye, J. Am. Chem. Soc., 2011, 133, 15894–15897; (c) H. Y. Bae, S. Some, J. H. Lee, J.-Y. Kim, M. J. Song, S. Lee, Y. J. Zhang and C. E. Song, Adv. Synth. Catal., 2011, 353, 3196–3202; (d) Y. Zhu, S. Khumsubdee, A. Schaefer and K. Burgess, J. Org. Chem., 2011, 76, 7449–7457; (e) C. Shao, H.-J. Yu, N.-Y. Wu, P. Tian, R. Wang, C.-G. Feng and G.-Q. Lin, Org. Lett., 2011, 13, 788–791; ( f ) S. E. Park, E. H. Nam, H. B. Jang, J. S. Oh, S. Some, Y. S. Lee and C. E. Song, Adv. Synth. Catal., 2010, 352, 2211–2217; ( g) M. Furutachi, S. Mouri, S. Matsunaga and M. Shibasaki, Chem. – Asian J., 2010, 5, 2351–2354. 6 K. M Koeller and C. H. Wong, Nature, 2001, 409, 232–240. 7 For selected Reviews see: (a) W. Leuchtenberger, K. Huthmacher and K. Drauz, Appl. Microbiol. Biotechnol., 2005, 69, 1–8; (b) J. M. ´nez, S. Martı´nez-Rodrı´guez, F. Rodrı´guez-Vico and Clemente-Jime ´zquez, Recent Pat. Biotechnol., 2008, 2, 35–46. F. J. Heras-Va ¨ger, H. Trauthwein, 8 For selected examples, see: (a) H. Gro S. Buchholz, K. Drauz, C. Sacherer, S. Godfrin and H. Werner, Org. Biomol. Chem., 2004, 2, 1977–1978; (b) G. Cardillo, A. Tolomelli and C. Tomasini, Eur. J. Org. Chem., 1999, 155–161; (c) D. Li, S. Cheng, D. Wei, Y. Ren and D. Zhang, Biotechnol. Lett., 2007, 29, 1825–1830; (d) T. Heck, D. Seebach, S. Osswald, M. K. J. ter Wiel, H.-P. E. Kohler and B. Geueke, ChemBioChem, 2009, 10, 1558–1561; (e) A. Liljeblad and L. T. Kanerva, Tetrahedron, 2006, 62, 5831–5854; ( f ) W. Szymanski, B. Wu, B. Weiner, S. de Wildeman, B. L. Feringa and D. B. Janssen, J. Org. Chem., 2009, 74, 9152–9157; (g) J. Rehdorf, M. D. Mihovilovic and U. T. Bornscheuer, Angew. Chem., Int. Ed., 2010, 49, 4506–4508. 9 For selected reviews, see: (a) D. Koszelewski, K. Tauber, K. Faber and W. Kroutil, Trends Biotechnol., 2010, 28, 324–332; (b) M. Hohne and U. T. Bornscheuer, ChemCatChem, 2009, 1, 42–51; (c) M. S. Malik, E. S. Park and J. S. Shin, Appl. Microbiol. Biotechnol., 2012, 94, 1163–1171; (d) S. Mathew and H. Yun, ACS Catal., 2012, 2, 993–1001; (e) W. Kroutil, E. M. Fischereder, C. S. Fuchs, H. Lechner, F. G. Mutti, D. Pressnitz, A. Rajagopalan, J. H. Sattler, R. C. Simon and E. Siirola, Org. Process Res. Dev., 2013, 17, 751–759.
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13 (a) M. Hohne, S. Schatzle, H. Jochens, K. Robins and U. T. Bornscheuer, Nat. Chem. Biol., 2010, 6, 807–813; (b) S. Schatzle, F. Steffan-Munsberg, A. Thontowi, M. Hohne, K. Robins and U. T. Bornscheuer, Adv. Synth. Catal., 2011, 353, 2439–2445. 14 Y. M. Seo, S. Mathew, H. S. Bea, Y. H. Khang, S. H. Lee, B. G. Kim and H. Yun, Org. Biomol. Chem., 2012, 10, 2482–2485. 15 C. S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Shi, J. Am. Chem. Soc., 1982, 104, 7294–7299. 16 (a) J.-S. Shin, B.-G. Kim, A. Liese and C. Wandrey, Biotechnol. Bioeng., 2001, 73, 179–187; (b) J.-S. Shin and B.-G. Kim, Biotechnol. Bioeng., 1997, 55, 348–358; (c) J.-S. Shin and B.-G. Kim, Biotechnol. Lett., 2009, 31, 1595–1599.
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10 (a) H. Yun, S. Lim, B.-K. Cho and B.-G. Kim, Appl. Environ. Microbiol., 2004, 70, 2529–2534; (b) J. Kim, D. Kyung, H. Yun, B.-K. Cho, J.-H. Seo, M. Cha and B.-G. Kim, Appl. Environ. Microbiol., 2007, 73, 1772–1782; (c) J. Rudat, B. R. Brucher and C. Syldatk, AMB Express, 2012, 2, 11, DOI: 10.1186/21910855-2-11. 11 H.-S. Bea, H.-J. Park, S.-H. Lee and H. Yun, Chem. Commun., 2011, 47, 5894–5896. 12 D. G. Allen, D. M. Coe, C. M. Cook, M. D. Dowle, C. D. Edlin, J. N. Hamblin, M. R. Johnson, P. S. Jones, M. K. Lindvall, C. J. Mitchell, A. J. Redgrave, J. E. Robinson and N. Trivedi, Chem. Abstr., 2005, 143, 97353. PCT Int. Appl., WO/2005/058892.
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Cite this: Chem. Commun., 2014, 50, 12680 Received 28th June 2013, Accepted 22nd August 2014
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Enzymatic synthesis of chiral c-amino acids using x-transaminase† Minsu Shon,‡a Ramachandran Shanmugavel,‡b Giyoung Shin,a Sam Mathew,a Sang-Hyeup Lee*b and Hyungdon Yun*c
DOI: 10.1039/c3cc44864a www.rsc.org/chemcomm
In this study, we successfully synthesized enantiomerically pure (R)- and (S)-c-amino acids (499% ee) using x-transaminase (x-TA) through kinetic resolution and asymmetric synthesis respectively. The present study demonstrates the high potentiality of x-TA reaction for the production of chiral c-amino acids.
Non-natural g-amino acids have attracted a great deal of attention because of their important role in the design and synthesis of bioactive molecules and in the application of hybrid peptides.1 For example, g-amino acids are key components of natural products such as hapalosin,2 dolastatin,3 caliculins4 and of various enzyme inhibitor g-aminobutyric acid analogues.1 Given the significance of g-amino acids, their efficient synthesis in optically pure form has become an attractive challenge to organic chemists and biologists. Lately, a number of strategies have been developed for the chemical synthesis of enantiomerically pure g-amino acids.5 Recently, peptidecatalyzed asymmetric Michael addition of nitromethane to b-disubstituted a,b-unsaturated aldehydes5a and enantioselective g-amination of a,b-unsaturated acyl chlorides with azodicarboxylates5b were developed to synthesize chiral g-amino acid derivatives. Enzyme-catalyzed chemical transformations are now widely recognized as practical alternatives to traditional organic synthesis.6 A number of biocatalytic routes were developed for the synthesis of chiral a- and b-amino acids using enantioselective enzymes such as transaminase (TA), hydantoinase, dehydrogenase, aminoacylase, penicillinG amidase, b-aminopeptidase, lipase, phenylalanine aminomutase, and Baeyer–Villiger monooxygenase.7,8 However, biocatalytic routes for the synthesis
of chiral g-amino acids have not been studied well. Recently, o-TA catalyzed reaction has gained a lot of attention due to its ability to generate enantiomerically pure amines and unnatural amino acids.9 Desirable characteristics like broad substrate specificity, high enantioselectivity, high turnover number and non-requirement for regeneration of external cofactors have made o-TA an attractive biocatalyst.9 Some o-TAs such as a o-TA from Polaromonas sp. (o-TAPO) showed a broad substrate specificity toward amines, L-amino acids and b-amino acids, suggesting that those enzymes may also be reactive towards g-amino acids.10,11 Here, we report an alternative method for the synthesis of (R)- and (S)-g-amino acids via kinetic resolution and asymmetric synthesis using o-TAs, respectively, (Scheme 1). The genes of o-TAPO11 and a newly identified o-TA from Burkholderia graminis C4D1M (accession #; ZP_02885261, o-TABG) were cloned into the vector pET24ma with a C-terminal His6-tag and effectively expressed in Escherichia coli BL21 (DE3). The enzymes were then purified on a Ni-NTA affinity column (Fig. S1, ESI†). The purified enzymes were then subjected to activity assay which was performed with 10 mM (S)-b-phenylalanine and 20 mM pyruvate (a typical amino acceptor) in 200 mM Tris/HCl buffer (pH 7.0) at 37 1C. The specific activities of o-TAPO and o-TABG were 5.6 and 13.6 U mg 1, respectively (Fig. 1). rac-g-Amino acids (1a–g) were prepared from their corresponding, commercially available g-keto acids via a two step
a
School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, South Korea b Department of Life Chemistry, Catholic University of Daegu, Gyeongbuk 700-443, Korea. E-mail: [email protected] c Department of Bioscience & Biotechnology, Konkuk University, Seoul 143-701, Korea. E-mail: [email protected]; Fax: +82-2-450-0686; Tel: +82-2-450-0496 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc44864a ‡ These authors contributed equally to this work.
12680 | Chem. Commun., 2014, 50, 12680--12683
Scheme 1 Synthesis of enantiomerically pure g-amino acid by using o-TA. (a) Kinetic resolution of g-amino acids. (b) Asymmetric synthesis of g-amino acids.
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Kinetic resolution of g-amino acidsa
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o-TAPO
Fig. 1 Substrate scope for deamination of o-TAPO and o-TABG. Enzyme reactions were carried out in 1 mL of 200 mM Tris/HCl buffer (pH 7.0) containing 10 mM racemic substrate (1a–g), 20 mM pyruvate, 0.1 mM PLP and o-TA (0.02 mg mL 1) for 30 min at 37 1C. One unit is defined as the amount of enzyme that catalyzes depletion of 1 mmol of pyruvate, betaPhe and (S)-b-phenylalanine.
process, oximation followed by catalytic hydrogenation of the resultant oximes12 (ESI†). The amino donor specificities of enzymes toward 1a–g were examined in the presence of 10 mM rac-g-amino acids and 20 mM pyruvate (Fig. 1). Among 1a–g, o-TAPO and o-TABG showed the highest reactivity for 1d (5.5 U mg 1) and 1e (3.9 U mg 1), respectively. Both o-TAPO (1.5 U mg 1) and o-TABG (0.36 U mg 1) showed the least reactivity towards 1f. The lower activity of 1f and 1g towards o-TAs may be due to the presence of bulky side chains which cause steric hindrance in the active sites of the enzymes. o-TABG showed about 2.4-fold higher specific activity towards (S)-bphenylalanine, while o-TABG showed only about 36% specific activity towards 1a than that of o-TAPO. In addition, (R)-selective o-TAs from Mycobacterium vanbaalenii13 and Neosartorya fischeri,13 and (S)-selective o-TAs from Vibrio fluviallis JS1714 were expressed in E. coli and purified. To examine the suitability of these enzymes for the production of g-amino acids, enzyme activities were measured using 1a as a representative g-amino acid, and these enzymes did not show any reactivity towards 1a (data not shown). The kinetic resolution of 1a was carried out using o-TAPO and o-TABG, and both enzymes converted only (S)-1a into 2a, and enantiomerically pure (R)-1a (499% ee) was obtained using both the enzymes in the presence of 10 mM rac-1a and 20 mM pyruvate (Fig. S2, ESI†). o-TAPO showed a faster reaction profile than o-TABG. Subsequently, the kinetic resolution of various g-amino acids (1a–g) using o-TAs was carried out under similar conditions as described above (Table S3, ESI†). All racemic substrates except 1f and 1g were successfully resolved into (R)-g-amino acids (499% ee) with about 50% conversion using o-TAPO (0.02 mg mL 1) and o-TABG (0.04 mg mL 1). In the case of 1f and 1g, higher concentration of enzymes (0.2 mg mL 1) was required to reach 499% ee due to its lower reactivity towards these substrates. The enantioselectivities (Es) of the enzymes were calculated using the equation E = ln[(1 c)(1 ees)]/ln[(1 c)(l + ees)]15 and were found to be 4100 for all substrates. This result indicates that o-TAPO and o-TABG are suitable biocatalysts for the production of highly enantiomerically pure g-amino acids.
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o-TABG
Sub.
Time (h)
Conv. (%)
eeR (%)
Time (h)
Conv. (%)
eeR (%)
1a 1b 1c 1d 1e 1f b,c 1g 1g c
24 24 24 24 24 24 36 24
50.1 50.3 50.1 n.c.d 50.1 49.6 46.5 50.1
499 499 499 — 499 98 87 499
24 24 24 24 24 24 36 24
50.0 50.5 50.2 n.c. 50.2 50.2 45.6 50.2
499 499 499 — 499 499 84 499
a Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/HCl buffer (pH 7.0) containing 100 mM g-amino acid, 200 mM pyruvate, DMSO (10%), and 1 mM PLP by using o-TAPO (0.2 mg mL 1) or o-TABG (0.4 mg mL 1); conv. and ee were determined by HPLC. b 50 mM 1f. c 0.68 mg mL 1 of o-TA was used. d No conversion.
To carry out the enzyme reaction at higher concentration, dimethylsulfoxide (DMSO) was added into the reaction mixture for increasing the solubility of the substrates. The kinetic resolution of g-amino acids was carried out in 1 mL of 10% DMSO (v/v) solution containing 100 mM g-amino acids (Table 1). In the case of 1f, 50 mM 1f was used for the reaction due to its low solubility. Except for 1d, all aromatic g-amino acids were successfully resolved into the (R)-form by both enzymes with a high ee value and ca. 50% conversion. In the case of 1d, 10 mM rac-1d was successfully resolved into a (R)-gamino acid (499%) by both enzymes (Table S3, ESI†), however, both enzymes did not show any reactivity in the presence of 100 mM 1d which may be due to severe substrate inhibition. (S)-g-Amino acids were also produced using g-keto acids (2a–g) as initial substrates via asymmetric synthesis (Scheme 1b). Asymmetric synthesis has been of keen interest among researchers, as it can theoretically generate two-fold higher yield (100%) than that of the kinetic resolution. To identify the best amino donor for o-TAs, the asymmetric syntheses of 1a from 10 mM 2a were carried out with L-Ala (with or without a pyruvate removal system), b-Ala, L-glutamic acid (2-ketoglutarate is a good amino acceptor for o-TAPO),11 and (S)-a-methylbenzylamine (MBA, a typical substrate
Fig. 2 Asymmetric synthesis of 1a from 2a using various amino donors. Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/HCl buffer (pH 7.0) containing 100 mM amino donor, 10 mM 2a, 0.1 mM PLP by using o-TA (0.34 mg mL 1) for 16 h. Conversion was determined by HPLC. In the case of L-Ala/LDH, 200 mM glucose, 1 mM NADH, lactate dehydrogenase (LDH, 55 U), and glucose dehydrogenase (2.7 U) were added in the L-Ala reaction solution for removal of pyruvate.
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for o-TA)9 (Fig. 2). o-TAs gave the highest conversion with (S)-a-MBA, and o-TAPO gave higher conversion (56.1%) than that of o-TABG (25.3%). Thus, (S)-a-MBA was selected as the best amino donor for the asymmetric synthesis. When asymmetric synthesis was carried out with 100 mM 2a, 100 mM (S)-a-MBA and o-TAPO (1 mg mL 1), the conversion was o3% after 24 h of reaction. Therefore, the substrate inhibition of o-TA by 2a was examined with 50 mM (S)-a-MBA and various concentrations of 2a (0–100 mM) (Fig. S3, ESI†). The initial reaction rates of o-TAPO and o-TABG were the highest at 5 mM 2a, and thereafter decreased with an increase in the concentration of 2a. In the presence of 10 mM 2a, o-TAPO and o-TABG showed, respectively, 74 and 87% activities of those with 5 mM 2a. Next, the asymmetric syntheses were carried out with 10 mM 2a due to severe substrate inhibition at higher concentration of 2a (410 mM). Here, (S)-a-MBA was used as the amino donor for the asymmetric synthesis of g-amino acids. The product inhibition by acetophenone (deaminated product of a-MBA) is well reported.9 To overcome the product inhibition by acetophenone, a biphasic reaction system with iso-octane was introduced (Fig. S4, ESI†).14,16 In the biphasic system, g-amino acid (1a), g-keto acid (2a), and (S)-a-MBA existed in the aqueous solution due to their electric charges at neutral pH while the inhibitory acetophenone gets transferred to the iso-octane layer. In the biphasic reaction system, the biphasic mixture was gently shaken in a shaking incubator with 250 rpm at 37 1C. As expected, the biphasic reaction system gave higher conversion of the product (97%) than the monophasic reaction system (80%). Subsequently, asymmetric syntheses of various g-amino acids were carried out in monophasic and biphasic systems (Table 2). Both systems gave enantiomerically pure (S)-g-amino acids (499% ee). For all the substrates, o-TAPO gave higher conversion than o-TABG, which indicates that o-TAPO is a more suitable catalyst than o-TABG for the asymmetric synthesis of g-amino acids. In most of the cases, the biphasic system gave a higher conversion. It is notable that 1f and 1g were less reactive substrates for the kinetic resolution, but in asymmetric synthesis, the conversion of 2f and 2g with o-TAPO was higher than other substrates. Finally a preparative scale reaction was carried out at 37 1C in Table 2
Asymmetric synthesis of g-amino acids
o-TAPO
o-TABG
Monophasic
a
S
b
Biphasic
Monophasic S
Biphasic
Sub.
Conv. [%]
ee [%]
Conv. [%]
ee [%]
Conv. [%]
ee [%]
Conv. [%]
eeS [%]
2a 2b 2c 2d 2e 2f 2g
77.6 75.1 72.1 72.9 52.7 85.3 99.9
499 499 499 499 499 499 499
95.8 94.3 91.0 88.5 67.5 99.0 99.9
499 499 499 499 499 499 499
14.0 17.3 14.7 3.21 5.10 1.02 48.9
499 499 499 499 499 499 499
23.0 17.2 27.7 14.5 8.45 23.4 57.6
499 499 499 499 499 499 499
a
S
Enzyme reactions were carried out at 37 1C in 1 mL of 200 mM Tris/ HCl buffer (pH 7.0) containing 10 mM substrate, 20 mM (S)-a-MBA, 0.1 mM PLP for 36 h by using o-TAPO (0.34 mg mL 1) or o-TABG (0.68 mg mL 1); conv. and ee were determined by HPLC (see the ESI). b 1 mL of iso-octane was added to 1 mL of the aqueous reaction mixture.
12682 | Chem. Commun., 2014, 50, 12680--12683
80 mL of 20 mM phosphate buffer (pH 7.0) containing 10 mM 2a, 100 mM (S)-a-MBA, and 0.1 mM PLP using purified o-TAPO (8 mg), and carefully 40 mL of iso-octane was added to 80 mL of the aqueous reaction mixture. After 24 h of reaction, HPLC analysis showed 93% conversion with 499% ee. 1a was isolated from this reaction with 38% yield (ESI†). In conclusion, enantiomerically pure (R)- and (S)-g-amino acids (499% ee) were produced using o-TAs through kinetic resolution and asymmetric synthesis respectively. The present study demonstrates the high potentiality of o-TA reaction for the production of chiral g-amino acids. We are currently developing an asymmetric synthesis method to overcome product inhibition by using a coupling reaction. This research was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A2A2A01068013). And this research was partially supported by a grant (A10301712131560200) of Global Cosmetics R&D Project, Ministry of Health & Welfare, Korea.
Notes and references ´n ˜ez and C. Cativiela, Tetrahedron: Asymmetry, 1 For a review, see: M. Ordo 2007, 18, 3–99. 2 K. Stratmann, D. L. Burgoyne, R. E. Moore and G. M. L. Patterson, J. Org. Chem., 1994, 59, 7219–7226. 3 G. R. Pettit, Y. Kamano, C. L. Herald, A. A. Tuinman, F. E. Boettner, H. Kizu, J. M. Schmidt, L. Baczynskyj, K. B. Tomer and R. J. Bontems, J. Am. Chem. Soc., 1987, 109, 6883–6885. 4 Y. Kato, N. Kusetani, S. Matsunaga, K. Hashimoto and T. Furuya, J. Am. Chem. Soc., 1986, 108, 2780–2781. 5 For selected examples, see: (a) K. Akagawa and K. Kudo, Angew. Chem., 2012, 124, 12958–12961 (Angew. Chem., Int. Ed., 2012, 51, 12786–12789); (b) L.-T. Shen, L.-H. Sun and S. Ye, J. Am. Chem. Soc., 2011, 133, 15894–15897; (c) H. Y. Bae, S. Some, J. H. Lee, J.-Y. Kim, M. J. Song, S. Lee, Y. J. Zhang and C. E. Song, Adv. Synth. Catal., 2011, 353, 3196–3202; (d) Y. Zhu, S. Khumsubdee, A. Schaefer and K. Burgess, J. Org. Chem., 2011, 76, 7449–7457; (e) C. Shao, H.-J. Yu, N.-Y. Wu, P. Tian, R. Wang, C.-G. Feng and G.-Q. Lin, Org. Lett., 2011, 13, 788–791; ( f ) S. E. Park, E. H. Nam, H. B. Jang, J. S. Oh, S. Some, Y. S. Lee and C. E. Song, Adv. Synth. Catal., 2010, 352, 2211–2217; ( g) M. Furutachi, S. Mouri, S. Matsunaga and M. Shibasaki, Chem. – Asian J., 2010, 5, 2351–2354. 6 K. M Koeller and C. H. Wong, Nature, 2001, 409, 232–240. 7 For selected Reviews see: (a) W. Leuchtenberger, K. Huthmacher and K. Drauz, Appl. Microbiol. Biotechnol., 2005, 69, 1–8; (b) J. M. ´nez, S. Martı´nez-Rodrı´guez, F. Rodrı´guez-Vico and Clemente-Jime ´zquez, Recent Pat. Biotechnol., 2008, 2, 35–46. F. J. Heras-Va ¨ger, H. Trauthwein, 8 For selected examples, see: (a) H. Gro S. Buchholz, K. Drauz, C. Sacherer, S. Godfrin and H. Werner, Org. Biomol. Chem., 2004, 2, 1977–1978; (b) G. Cardillo, A. Tolomelli and C. Tomasini, Eur. J. Org. Chem., 1999, 155–161; (c) D. Li, S. Cheng, D. Wei, Y. Ren and D. Zhang, Biotechnol. Lett., 2007, 29, 1825–1830; (d) T. Heck, D. Seebach, S. Osswald, M. K. J. ter Wiel, H.-P. E. Kohler and B. Geueke, ChemBioChem, 2009, 10, 1558–1561; (e) A. Liljeblad and L. T. Kanerva, Tetrahedron, 2006, 62, 5831–5854; ( f ) W. Szymanski, B. Wu, B. Weiner, S. de Wildeman, B. L. Feringa and D. B. Janssen, J. Org. Chem., 2009, 74, 9152–9157; (g) J. Rehdorf, M. D. Mihovilovic and U. T. Bornscheuer, Angew. Chem., Int. Ed., 2010, 49, 4506–4508. 9 For selected reviews, see: (a) D. Koszelewski, K. Tauber, K. Faber and W. Kroutil, Trends Biotechnol., 2010, 28, 324–332; (b) M. Hohne and U. T. Bornscheuer, ChemCatChem, 2009, 1, 42–51; (c) M. S. Malik, E. S. Park and J. S. Shin, Appl. Microbiol. Biotechnol., 2012, 94, 1163–1171; (d) S. Mathew and H. Yun, ACS Catal., 2012, 2, 993–1001; (e) W. Kroutil, E. M. Fischereder, C. S. Fuchs, H. Lechner, F. G. Mutti, D. Pressnitz, A. Rajagopalan, J. H. Sattler, R. C. Simon and E. Siirola, Org. Process Res. Dev., 2013, 17, 751–759.
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13 (a) M. Hohne, S. Schatzle, H. Jochens, K. Robins and U. T. Bornscheuer, Nat. Chem. Biol., 2010, 6, 807–813; (b) S. Schatzle, F. Steffan-Munsberg, A. Thontowi, M. Hohne, K. Robins and U. T. Bornscheuer, Adv. Synth. Catal., 2011, 353, 2439–2445. 14 Y. M. Seo, S. Mathew, H. S. Bea, Y. H. Khang, S. H. Lee, B. G. Kim and H. Yun, Org. Biomol. Chem., 2012, 10, 2482–2485. 15 C. S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Shi, J. Am. Chem. Soc., 1982, 104, 7294–7299. 16 (a) J.-S. Shin, B.-G. Kim, A. Liese and C. Wandrey, Biotechnol. Bioeng., 2001, 73, 179–187; (b) J.-S. Shin and B.-G. Kim, Biotechnol. Bioeng., 1997, 55, 348–358; (c) J.-S. Shin and B.-G. Kim, Biotechnol. Lett., 2009, 31, 1595–1599.
Published on 27 August 2014. Downloaded by Universidad de Oviedo on 30/10/2014 07:44:07.
10 (a) H. Yun, S. Lim, B.-K. Cho and B.-G. Kim, Appl. Environ. Microbiol., 2004, 70, 2529–2534; (b) J. Kim, D. Kyung, H. Yun, B.-K. Cho, J.-H. Seo, M. Cha and B.-G. Kim, Appl. Environ. Microbiol., 2007, 73, 1772–1782; (c) J. Rudat, B. R. Brucher and C. Syldatk, AMB Express, 2012, 2, 11, DOI: 10.1186/21910855-2-11. 11 H.-S. Bea, H.-J. Park, S.-H. Lee and H. Yun, Chem. Commun., 2011, 47, 5894–5896. 12 D. G. Allen, D. M. Coe, C. M. Cook, M. D. Dowle, C. D. Edlin, J. N. Hamblin, M. R. Johnson, P. S. Jones, M. K. Lindvall, C. J. Mitchell, A. J. Redgrave, J. E. Robinson and N. Trivedi, Chem. Abstr., 2005, 143, 97353. PCT Int. Appl., WO/2005/058892.
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