Semi-rational Engineering Aldo-keto Reductases And Its Efficient Preparation Of Chiral Αα-hydroxy Amides

Highly optically pureα-hydroxy amides are crucial intermediates in the synthesis of chiral drugs such as bradykininβ₁ selective antagonists and inverse agonists.Currently,chemical methods dominate the synthesis of chiralα-hydroxy amides,the asymmetric reduction ofα-keto amides by metal catalysts provide a direct way for the preparation of such chiral compounds.However,the usage of metal catalysts is limited due to strict regulatory on the heavy metal and high costs.The enzymatic approach is expected to become the best choice for the synthesis of chiralα-hydroxy amides due to the advantages of mild conditions,high selectivity,and environmental friendliness.However,there is limited research on asymmetric reduction ofα-keto amides by biological methods,the reported biocatalysts show poor stereoselectivity and low catalytic activity.Therefore,the development of efficient carbonyl reductases for the asymmetric synthesis of chiralα-hydroxy amides is of great significance.In this paper,aldo-keto reductases（AKR）stored in the laboratory were used as the research object to construct the asymmetric reduction reaction catalyzed by AKRs（iol S and yhd N）towardα-keto amides.The key residues of iol S and yhd N were analyzed and remodeled by semi-rational engineering.Finally,the efficient preparation of chiralα-hydroxy amide compounds was realized.The main contents are as follows:（1）The asymmetric reduction of 2-oxo-N,2-diphenylacetamide（ONDPA）catalyzed by aldo-keto reductases iol S and yhd N was constructed.The synthesis of N-phenyl-2-hydroxy-2-phenylacetamide（NPHPA）was achieved successfully.AKRs could catalyze ONDPA in the presence of different cofactors,and NADPH has better catalytic performance as a coenzyme.The reaction time of ONDPA catalyzed by iol S and yhd N was 12 hours,the ee value of 76.0%（S）and 3.4%（S）toward ONDPA,respectively,and the conversion rates reached 40.8%and 38.3%,respectively.To avoid the additional cost associated with cofactor addition,recombinant E.coli containing aldo-keto reductases and glucose dehydrogenase（GDH）were constructed for the preparation of NPHPA.The asymmetric reduction of ONDPA in iol S-GDH and yhd N-GDH could be completed within 3 h without the addition of exogenous coenzymes,it indicatied that whole-cell catalysis could significantly improve the reaction rate.In addition,the reaction conditions were optimized.The optimal p H and temperature for AKRs-GDH were 7.0 and 30℃,respectively.Under the optimal conditions,the conversion rate of 5 m M substrate catalysed by iol S-GDH and yhd N-GDH was improved to 68.1%and 48.4%,respectively,and the ee value of the product（S）-NPHPA was increased to 80.3%and 4.5%,respectively.（2）To enhance the catalytic performance of iol S in the asymmetric reduction of ONDPA,we conducted an analysis of the structure and key residues of iol S.Alanine scanning was performed on 12 amino acid positions on the substrate-binding pocket,identifying six mutation sites（N21,L28,Y29,I57,H87,and F126）that significantly influenced the stereoselectivity of iol S.Subsequently,based on the screening and analysis of saturation mutagenesis at these sites,the combinational mutagenesis strategy was used to obtain the two stereocomplementary variants（I57F/F126L and N21A/F126A）with ee value of 97.6%（S）and 99.9%（R）toward ONDPA,respectively,delivering chiralα-hydroxy amide with>98%conversions.（3）Based on molecular docking and molecular dynamics simulation,the stereoselective mechanism of iol S was studied.The probability of the prereaction state formation was calculated using the ratio of configurations with distances d(O_sub-OH_Y58)≤3.4（?）and d(C_sub-H4_NADPH)≤4.5（?）.The results showed that I57F/F126L and ONDPA_{pro S} and N21A/F126A and ONDPA_{pro R} were more likely to form prereaction state,which proved that I57F/F126L and N21A/F126A were conducive to the synthesis of corresponding（S）-and（R）-NPHPA.Moreover,the binding free energy of key residues of iol S and its mutants was calculated and analyzed,it suggested that the 21and 126 sites were used as the"molecular switch"of iol S to control the stereoscopic preference of enzymes.Finally,the catalytic activity and stereoselectivity of iol S and its mutants for asymmetric reduction ofα-keto amides with different structures were investigated.The results showed that steric and electronic effects of substrate substituents changed the stereorecognition of enzymes,thus affected the enantioselectivity of iol S and mutants.（4）The molecular modification of yhd N was carried out based on iol S semi-rational engineering strategy.The saturation mutagenesis library was established of key residues.Through iterative saturation mutagenesis,the optimal mutants W21N/W126F/L278I/G204I（91.4%ee,S）and W21A/W126A（60.4%ee,R）were successfully constructed.Molecular docking results indicated that the cavity volume of the yhd N substrate-binding pocket and the hydrophobic interactions formed between the substrate and the enzyme played a crucial role in regulating stereopreference.In summary,this study successfully constructed the asymmetric reduction ofα-keto amide catalyzed by AKRs,optimized the catalytic performance of iol S and yhd N by semi-rational engineering,explored the stereoselective recognition of enzymes based on molecular simulation,and deepened the understanding of the correlation between their structure and catalytic reaction.Our results provide an effective method to prepare chiralα-hydroxyl amides with high optical purity.