Synthesis Of β-hydroxyketones By Dual Enzyme/chemoenzymatic Cascade Reactio

β-Hydroxyketone is a kind of compounds with a wide range of biological activities,which plays an important role in the synthesis of natural products and drug molecules.β-Hydroxyketone can be conveniently obtained from aldehydes conveniently,but there are some practical problems such as poor water solubility when using aldehydes as substrates.Therefore,it is of great significance to develop the research on its synthetic methods.Based on activated alkenes and alcohols as substrates,a multi-step cascade reaction combining chemical and biological catalysis was proposed to obtain the target compoundβ-hydroxyone.In the first part,the preparation ofβ-hydroxyketones by a chemically enzymatic cascade reaction using Galactose Oxidase variant(GOase M_3-5)and 1,4-Diazabicyclo[2.2.2]octane（DABCO）as catalyst under mild conditions was studied.Firstly,benzyl alcohol 1m andα-cyclohexene ketone 4 were used as substrates to carry out the model reaction.The experimental results showed that under the"green"condition of biocatalysis compatibility,benzyl alcohol 1m was oxidized to the corresponding aldehyde 2m,and then the target compound can be effectively obtained by the reaction cascade with Morita-Baylis-Hillman（MBH）.Then,by optimizing the concentrations of substrate,enzyme and catalyst,the optimum reaction conditions were determined(the concentration of alcohol compound was 50 mmol/L,the concentration of GOase M_3-5 was 10μmol/L,and the concentration of 2m/4/DABCO was 1:4:4).On this basis,the applicability of the substrate for the reaction was further investigated,and19 achiral target compounds were successfully separated by semi-preparative scale reaction.Finally,the practical study of the obtainedβ-hydroxy ketone was carried out,namely,the key intermediate of benflumetol with anti-malaria and novel coronavirus infection activity was synthesized by intramolecular Heck reaction.All the target compounds were confirmed by ¹H NMR and ¹³C NMR.In the second part,the enantioselectiveβ-hydroxyketone was prepared by double-enzyme cascade reaction using GOase M_3-5 and the calculated enzyme MBHase variant（BH32.14）as catalysts.There were two ways of this reaction,one was a double-enzyme cascade reaction using purified enzymes of GOase M_3-5 and MBHase（BH32.14）obtained by affinity chromatography as catalysts;the other was the whole cell obtained by constructing the above two enzymes in the same vector,that is,the target molecules were prepared by the cascade reaction of co-expression whole cell double enzyme method.The experimental results show that both reaction strategies can effectively obtain target compounds with optical purity under mild conditions.Firstly,4-nitrobenzyl alcohol 1a andα-cyclohexene ketone 4 were used as substrates to carry out the model reaction.Then,the application ranges of the two reaction strategies were investigated,and 12β-hydroxyketones with enantioselectivity were obtained by semi-preparative scale purification double-enzyme cascade reaction.All the target compounds were characterized by ¹H NMR and ¹³C NMR,and their enantioselectivity was analyzed by normal phase liquid chromatography.In conclusion,this study provides three sustainable strategies for the efficient synthesis ofβ-hydroxyketones by chemical-enzymatic and dual-enzymatic cascades using GOase M_3-5,DABCO or MBHase BH32.14 as catalysts.This strategy can be used to synthesize chiral or achiralβ-hydroxyketones.In the absence of chiral target compounds,chemo-enzymatic cascade reaction is both efficient and labor saving,while double-enzyme cascade strategy can effectively obtain target compounds with enantioselectivity,providing a new idea for green chemical synthesis.