Biocatalysis Of Nitrogen Acetylation Of Small Nitrogen-Containing Heterocyclic Substrates

Background: Nitrogen-containing heterocyclic compounds have important biological and pharmacological properties.The nitrogen atoms in heterocyclic could be modified by nitrogen amidation.At present,many small molecule drugs have nitrogencontaining heterocyclic structures which modified by N-amidation.Mostly synthesis method utilized chemical synthesis method.There are some report about enzymes synthesis method about nitrogen-containing heterocyclic N-amidation heterocyclic,however,related enzymes tools still lacked.Target: Screening enzymes tools with biocatalytic activity for nitrogen-containing heterocyclic nitrogen acetyl,then design mutation to improve the catalytic efficiency to target substrates.Method: This article selected three commercial lipases(Novozymes: TL-IM,RM,435)and one commercial cutinase(Novozymes: 51032)to verification feasibility for piperidine N-acetyl catalytic reaction.Then this article researched the influence of temperature and solvent,then preliminarily established optimized reaction system.Then this article recombination and expression Aspergillus oryzae cutinase A1 and its mutations(A1＿M1 and A1＿M2),Burkholderia cenocepacia cutinase B1 and B2,Pseudomonas putida cutinase P1.Finally,this article verified N-acetyl biocatalytic activity for piperazine substrate.(1)The result of feasibility experiments based on commercial enzymes indicat that lipase 435 and cutinase 51032 could N-acetyl biocatalytic for piperidine.Lipase suitable for higher temperature(45 ℃)while cutinase suitable for lower temperature(35 ℃).N,N-dimethyl formamide as the optimum reagent for this reaction.Under the condition of piperidine(1.5 g/L),mole ratio of piperidine to vinyl acetate(1.5:2),50 ℃,24 h,the maximum conversion rate up to 91.64% while use lipase 435 in reaction of product Acetylpiperidine.While the maximum conversion rate up to 64.02% while use cutinase51032 in same conditions.(2)This article recombination and expression A1、A1＿M1、A1＿M2 to verify Nacetyl on piperazine.The results show that,the yield of 1-acetylpiperazine more than50% with A1,A1＿M1 and A1＿M2,however,the yield of 1,4-diacetyl-piperazine all very low.Under the best reaction conditions(0.5 g/L enzymes,1.5 g/L piperidine,1.5:2 mole ratio of piperidine to vinyl acetate,30 ℃,12 h),the yield of 1-acetylpiperazine is 50.86%with A1,the yield of 1,4-diacetyl-piperazine is 9.61% with A1.When this article use A1＿M1,the yield of 1-acetylpiperazine and 1,4-diacetyl-piperazine is 51.03% and11.93%,respectively.While use A1＿M2,the yield of 1-acetylpiperazine and 1,4-diacetyl-piperazine is 51.32% and 13.76%.Though the yield of 1,4-diacetyl-piperazine was increased by mutation,no significant improvement effect.(3)This article recombination and expression cutinase B1,B2 and P1 to verify Nacetyl on piperazine.B1 shows no obvious enzyme activity,B2 could not express.In the process of P1 N-acetyl modification on piperazine to biosynthesis 1-acetylpiperazine,the yield relatively low,while in the process of P1 N-acetyl modification on piperazine to biosynthesis 1,4-diacetyl-piperazine,the yield significant improvement compared to chapter 3.Under the best reaction conditions,the yield of 1-acetylpiperazine is 16.02%with P1,the yield of 1,4-diacetyl-piperazine is 29.28% with P1.Conclusion and perspective: This article screening and validation enzyme tools to N-acetyl modified to piperidine and piperazine.The research result shows that the feasibility of mono N-acetyl modified on piperidine and piperazine,however,the multiN-acetyl modified on piperazine is difficult to achieve.This article screening and design recombinant enzymes thus increased yield of double N-acetyl biocatalysis.However,there is a large gap with practical applications.In follow-up work,it is necessary that to rational design on enzyme tools,to achieve the target of efficient yield of muti-N-acetyl on nitrogen heterocyclic.