Development of Biocatalysts for Production of Fine Chemicals (Asymmetric Bioreduction Systems)

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1 Development of Biocatalysts for Production of Fine Chemicals (Asymmetric Bioreduction ystems) umitomo Chemical Co., Ltd. rganic ynthesis Research Laboratory Hiroyuki AAK Biocatalysis has matured into the standard technology for synthesizing industrially-important chemicals such as pharmaceuticals and agrochemicals. The principal advantage of biocatalysts is their ability to catalyze reactions with high specificity (often enantio- or regio-selectively). Furthermore, biocatalysts have the advantage of operating under mild conditions, typically at ambient temperature and pressure. Here, I present recent industrial applications of biocatalysts to perform asymmetric reduction synthesis. 1) 2) 22ECD 3) BINAP 4) β 5) NADH NADPH 212

2 NAD NADP Fig. 1Table 1 CR-1 β CR-2 4 α CR-3 4 CR-5 6 β R1 Fig. 1 glucono lactone or acetone etc. H H CNH2 N R reduced form N + R oxidized form Asymmetric reduction and recycling of the coenzyme H R1 CNH2 H glucose or 2-propanol etc. CR-1 Fig. 2 9 Table 1 CR-1 CR-2 CR-3 CR-4 CR-5 CR-6 Reductase libraries ubstrate X C2R C2R1 C2R1 R1 R1 99%e.e. () 99%e.e. (R) > 99%e.e. (R) > 99%e.e. (R) > 99%e.e. () Microbe screening Fig. 2 Improvement of catalytic activity Cloning Expression Engineering Recombinant DNA technique Development of the biocatalyst manufacturing process Biocatalyst libraries trategy for biocatalyst development CR

3 Table 2 Biotransformation of BAM by acetone-dried microorganisms Microorganisms Molar yield (%) Arthrobacter paraffineus 4.2 Bacillus alvei 44.1 Rhodotorula minuta 1.1 Cryptococcus humicolus 12.9 Penicillium citrinum 36. 1) : not tested, 2) N.D.: not detected. NADPH tereo- selectivity R Coenzyme availability e.e. (%) Molar yield (%) N.D. 2) NADH tereo- selectivity R 1) e.e. (%) H Microbe Br CMe Br CMe BAM NAD(P)H NAD(P)+ ()-BHBM Fig. 3 Asymmetric reduction of BAM to BHBM BAM 4 3 BHBM Fig. 3 Penicillium citrinumr Bacillus alvei Table 2 BHBM 98.1 e.e e.e. NADPH NADH BAM CR-1 CR-1 6) 85 AKR AKR AKR3E1 CR-1 CR-1 CR-1 CR-1 CR-137kDa BAM NADPH ph 6.5pH6 1 Fig ) 4 3 1,1-1 1 Table 3 Activity (%) CR-1 activity (solid lines) and stability (dashed lines) as a function of ph. The activity was measured in the following buffers (.1 M): KPB (ph ; open squares), Tris-HCl buffer (ph 7. 9.; open triangles), and Tris-glycine buffer(ph 9. 1.; open diamonds). The remaining activity of CR-1 (filled circles) was also measured after incubation in the following buffers at 2 C for 1 h: citrate-k2hp4 buffer (ph 4. 5.), KPB (ph 6. 8.), Tris-HCl buffer (ph 8. 9.) and Tris-glycine buffer (ph 1.). Fig ph ptimum ph and ph stability of CR-1 212

4 Table 3 ubstrate specificity of CR-1 ubstrate Relative activity (%) Aldehydes Acetaldehyde 2.2 n-butylaldehyde 3.4 n-valeraldehyde 2.8 DL-glyceraldehyde 3.2 Pyridine-3-aldehyde 2. Ketones Ethyl 4-Chloro-3-oxobutyrate 1 BAM 139 Ethyl 4-bromo-3-oxobutyrate 667 Isopropyl 4-bromo-3-oxobutyrate 125 ctyl 4-bromo-3-oxobutyrate 24 1,1-Dichloroacetone 42 Chloroacetone Chloro-2-butanone 2.4 Methyl 3-oxopentanoate Bromo-1-indanone 2 Dihydroxyacetone 56 CR-1 GDH CR-1 GDH CR-1 GDH DNA DNA DNAGDH CR-1 DNA Table 4 a)cr-1gdh DNA Table 4 b)gdhcr-1 GDHA CR-1A DNA Table 4 c) BAM GDH Table 4 c) DNA Table 4 Table 4 c)dna BAM ph6.5 2 NADP 3 ph ()-BHBM e.e. CR-1 CR CR-1 A3-49 A8-39 T Table 5A K245RA N271DT L54Q14 Table 4 Activities of CR-1 and GDH of recombinant E. coli cells Plasmid a) P D D b) P D D c) P D GDH CR-1 CGCGGTA ATGTCTAACGG CR-1 activity GDH activity (units/ml of culture) (units/ml of culture) P: Promoter, D: hine-dalgarno sequence Table 5 Clone CR-1 A3-49 A8-39 T1-99 Thermostability and enantioselectivity of CR-1 mutants generated by random mutagenesis Mutation site(s) None K245R N271D L54Q/R14C Remaining activity at 4 C for 2 h (%) ()-BHBM e.e. (%)

5 R14C2 T1-99 BAM 4 CR-1 3 L54Q K245R N271DTable 6 Table 6 Mutation site(s) Thermostability and enantioselectivity of CR-1 mutants generated by site-directed mutagenesis None (wild-type) L54Q R14C L54Q/R14C N271D L54Q/N271D L54Q/R14C/N271D K245R L54Q/K245R L54Q/K245R/N271D L54Q/R14C/K245R/N271D Remaining activity at 45 C for 7 h (%) ()-BHBM e.e. (%) L54Q KR HD E BAM 97.1 e.e. 98 e.e. 4 3 CAE 63.1 e.e. 9 e.e. L54 L54A L54D L54ETable 7 CR-1 L54Q Km453 CR-1 L54Q Fig. 5CR-1 54 Fig Table 7 Thermostability and enantioselectivity for BAM and CAE of CR-1(L54) mutants generated by saturation mutagenesis Mutation None (wild-type) L54G L54 L54T L54C L54Y L54N L54Q L54A L54V L54I L54M L54P L54F L54W L54K L54R L54H L54D L54E Remaining activity at 45 C for 2 h (%) BAM Relative activity (%) ()-BHBM e.e. (%) CAE Relative activity (%) ()-CHBE e.e. (%)

Relative activity (%) 16 14 12 1 8 6 4 2 1 2 3 4 5 6 Temperature ( C) GDH BAMCR-1 L54Q 5 198 e.e. ()- BHBMCR-18 BAM 93 97 e.e. Fig.

6 Relative activity (%) Temperature ( C) GDH BAMCR-1 L54Q e.e. ()- BHBMCR-18 BAM e.e. Fig. 7CR-1 L54Q CR-1 8) Purified enzyme was incubated for 3 min at the indicated temperature, and activity was determined Fig. 5 Effect of temperature on the stability of CR-1 (L54Q) (open circle) and wild-type CR-1 (filled circle) L54Q Active center Concentration (mm) Time (hr) BAM (filled square), ()-BHBM (filled triangle) Fig. 7 Time-course of BAM reduction to ()-BHBM by recombinant E. coli expressing wild-type CR-1 (dashed line) and CR-1 (L54Q) (solid line) This model based on the crystal structure of AKR4C9 from Arabidopsis thaliana (PDB code: 3h7u) Fig. 6 Homology modeling of CR-1 Table 4 c) DNA CR-1 CR-1 L54Q DNA CR-1 GDH BAM CR-1Table 1CR-5 BAM CR-5 CR-52 NADPH NADH Fig. 8 9) CR-6 BAM R CR-5 2 NADH Fig. 8 1) Br CMe Glucono lactone Glucose GDH NADPH NADP + CR-1 BAM H CR-5 or 6 NADH NAD + CR-5 or 6 Acetone 2-propanol Br * Chiral BHBM CMe Fig. 8 Asymmetric reduction of BAM and recycling of the coenzyme 212

7 Table 8 Biocatalytic reduction of BAM CR-1(L54Q)+GDH CR-5 CR-6(HAR1) Coenzyme NADPH NADH NADH Wet cells (w/w-bam) Reaction time (h) Final concn. of BHBM (mm) Yield (%) e.e. (%) > 99 () > 99 () > 99 (R) CR-1BAM CR-1 CR-1 L54Q CR-6CR-6 HAR1 11) CR-1 L54Q ph 6.5 CR-5 CR-6 HAR1 2 CR-1 L54QGDH mM99 e.e. ()- BHBM Table 8CR mM99 e.e. ()- BHBMBAM 53CR-6 HAR1 1 BAM mM99 e.e. (R)-BHBM BAM CR-1 L54Q CR-1 L54QCR-5 CR-6 HAR1BAM 2 BHBM Table 9 12) CR-5 CR-6 HAR1 ph NADPH NADH BAM Table 9 CR-1(L54Q) +GDH CR-5 CR-6(HAR1) Properties of BAM reduction enzymes pecific activity Activity in for BAM E. coli cells (units/mg protein) (units/g wet cells) Half-life in the two-phase system (h) ).1 14) DNA DNA 212

1),,,,, 21-@, 4 (21). 2),,,,,,,,, 24-!, 4 (24). 3). Mitsuda, T. Umemura and H. Hirohara, Appl Microbiol. Biotechnol., 29, 31 (1988). 4) M. Mikami, T. Korenaga, T. hkuma and R. Noyori, Angew. Chem.

8 1),,,,, 4 (21). 2),,,,,,,,, 24-!, 4 (24). 3). Mitsuda, T. Umemura and H. Hirohara, Appl Microbiol. Biotechnol., 29, 31 (1988). 4) M. Mikami, T. Korenaga, T. hkuma and R. Noyori, Angew. Chem. Int. Ed. Engl. 39, 377 (2). 5). ervi, ynthesis, 1, 1 (199). 6) H. Asako, R. Wakita, K. Matsumura, M. himizu, J. akai and N. Itoh, Appl. Environ. Microbiol., 71, 111 (25). 7) N, Itoh, H. Asako, K. Banno, Y. Makino, M. hinohara, T. Dairi, R. Wakita and M. himizu, Appl. Microbiol. Biotechnol., 66, 53 (24). 8) H. Asako, M. himizu and N. Itoh, Appl. Microbiol. Biotechnol., 8, 85 (28). 9) H. Asako, M. himizu and N. Itoh, Appl. Microbiol. Biotechnol., 84, 397 (29). 1) N. Itoh, M. Matsuda, M. Mabuchi, T. Dairi and J. Wang, Eur. J. Biochem., 269, 2394 (22). 11) Y. Makino, T. Dairi and N. Itoh, Appl. Microbiol. Biotechnol., 77, 833 (27). 12) H. Asako, M. himizu, Y. Makino and N. Itoh, Tetrahedron Lett., 51, 2664 (21). 13) B. M. Matute, M. Edin, K. Bogár, F. B. Kaynak and J. E. Bäckvall, J. Am. Chem. oc., 127 (24), 8817 (25). 14),,, 38 (4), 23 (2). PRFILE Hiroyuki AAK 212

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