Molecular Engineering and Characterization of Imine Reductases for the Synthesis of 1-Phenyl-1,2,3,4-tetrahydro-isoquinoline
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摘要: 对亚胺还原酶酶库的筛选获得了性能优良的AdIR1，基于蛋白结构分析对其进行分子改造获得催化效率更优的突变体酶F172Y，并对其进行动力学与热稳定性表征；最后利用所开发的突变酶实现了(S)-1-Ph-THIQ克级制备，证明了该酶法合成路线的实用性。Abstract: 1,2,3,4-Tetrahydro-isoquinolines are a significant class of building blocks used in the pharmaceutical and agrochemical industries, and existed widely in a variety of chiral amine drugs. Among them, (S)-1-Phenyl-1,2,3,4-tetrahydro-isoquinoline ((S)-1-Ph-THIQ) is the key precursor for the synthesis of Solifenacin, a drug for the treatment of overactive bladder. Imine reductase (IRED)-catalyzed asymmetric reduction of 1-phenyl-3,4-dihydroisoquinoline (1-Ph-DHIQ) is a green and promising route towards chiral 1-Ph-THIQ. However, currently there is only a limited number of reported IREDs that could catalyze the synthesis of chiral 1-Ph-THIQ from 1-Ph-DHIQ, and they may suffer from issues including low activity, poor stereoselectivity, and substrate inhibition. In this study, we first discovered an IRED AdIR1 with considerable properties by screening a panel of IREDs and identified key residues which may affect the activity via homo-modelling and structure comparison. Protein engineering was performed to generate mutant F172Y with elevated catalytic efficiency, which was then characterized in terms of kinetic parameters and thermostability. Finally, preparative synthesis of (S)-1-Ph-THIQ on gram-scale was achieved employing mutant F172Y, demonstrating the considerable applicability of this biocatalytic route in the synthesis of (S)-1-Ph-THIQ.
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Key words:
- tetrahydro-isoquinolines /
- chiral amines /
- imine reductases /
- asymmetric synthesis /
- biocatalysis
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图 3 pH对母本(A)与F172Y(B)酶活力的影响
Figure 3. Effect of pH on the activity of WT(A) and F172Y(B). Activity-pH data was measured using the following buffer: (i) citrate buffer (●, pH 3.0-6.0); (ii) potassium phosphate buffer (♦, pH 6.0-8.0); (iii) Tris-HCl buffer (▲, pH 8.0-9.0); (iv) Gly-NaOH buffer (■, pH 9.0-11.0). Relative activity was expressed as a percentage of the maximum activity.
图 5 母本与突变体F172Y的动力学特征曲线
Figure 5. Kinetic characteristic curves of the wild type and F172Y of AdIR1
图 6 母本(黄色)与突变体F172Y(绿色)的反应进程曲线
Figure 6. Reaction process curves of Wild Type (yellow) and F172Y (blue) of AdIR1. Conversion (◆); enantiomer excess (■).
图 7 不同助溶剂浓度下的反应进程曲线
Figure 7. Reaction process curves with different cosolvent concentrations.
图 8 克级制备反应的反应进程曲线
Figure 8. Reaction process curve of gram-scale preparative reaction.
表 1 目的基因克隆所用的引物
Table 1. Primers used in gene cloning.
Entry Enzyme Primers (5’ to 3’) Restriction site 1 AdIR1 GGAATTCCATATGATCACACTGATCGGGCTCGGT
CCGGAATTCCTACTTGCGGTCCGCCTTGATGNde I
EcoR I2 SeIR2 GGGAATTCCATATGAGCACGCCGCTGACGCTGAT
CCGGAATTCCTACTTCGCGGGCTTGATCNde I
EcoR I
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表 2 目的基因定点突变所用PCR引物
Table 2. Primers used for site-directed mutagenesis.
Entry Name Primers (5’ to 3’) Length 1 118_FP GGCGTGATGNDTCCTGCCGAGTTGGTGGGCAAGGAG 36 2 118_RP CTCGGCAGGAHNCATCACGCCGCCCGCGACGAACCG 36 3 172_FP CTGGACATCNDTCTGACCTCGCTTTCGGTGTTCATG 36 4 172_RP CGAGGTCAGAHNGATGTCCAGCTGCGCCTGGTAGAA 36 5 216_FP GCCGAGCAGNDTGAAAAGGGCGAGCACCCCGGCGAC 36 6 216_RP GCCCTTTTCAHNCTGCTCGGCCGCGGCGTCCAGGTA 36
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表 3 亚胺还原酶不对称还原1-苯基-3,4-二氢异喹啉的结果
Table 3. Asymmetric reduction of 1-phenyl-3,4-dihydroisoquinoline by AdIR1and SeIR2.
Entry Enzyme Time/h C/% ee/% 1 AdIR1 24 98 98 (S) 2 SeIR2 24 54 94 (S)
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表 4 亚胺还原酶的关键位点比对结果
Table 4. Alignment of key residues of the imine reductases.
Entry Enzyme 1st site 2nd site 3rd site 1 SnIR T123 F178 G228 2 AdIR1 V118 F172 I216
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表 5 AdIR1突变体的测活结果
Table 5. Activity assay results of AdIR1 mutants.
Entry Enzyme Mutation site Specific activity/(mU·mg−1) Fold 1 WT − 7.90 1.0 2 M1 118 8.81 1.1 3 M2 118 5.92 0.7 4 M3 118 8.88 1.2 5 M4 172 15.3 1.9
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表 6 突变体F172Y与母本的半衰期对比
Table 6. The half-lives of WT and F172Y at different temperatures.
Enzyme t1/2/h 30 ℃ 40 ℃ 50 ℃ WT 70.0 50.4 1.0 F172Y 324.0 314.0 3.9
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表 7 母本与F172Y的催化动力学参数
Table 7. Kinetic parameters of WT and F172Y.
Enzyme Temperature/
(℃)Km/
(mmol/L)kcat/ (s−1) kcat/Km/
(s−1·mmol/L−1)WT 30 0.05 ± 0.01 0.005 ± 0.000 0.10 F172Y 30 0.05 ± 0.01 0.017 ± 0.000 0.34 F172Y 40 0.07 ± 0.01 0.027 ± 0.000 0.38
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表 8 亚胺还原酶突变体F172Y在30 ℃与40 ℃下的反应转化结果
Table 8. Reaction conversion of F172Y under 30 and 40 ℃.
T/(℃) Time/h Conversion/ (%) ee/(%) 30 6 69 99 12 84 99 40 6 84 99 12 91 99
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