Enzyme-Catalyzed Synthesis Of Aziridine Ternary Rings Mediated By Sulfotransferase

Aziridine ternary ring as an annular structure is one of the important functional group of many natural active compounds,which is made up of two carbon atoms and a nitrogen atom.Due to its compact structure,it has great ring tension and strong chemical reactivity,and plays an important role in the function of natural active products and as drug intermediates.In addition,aziridine compounds obtained by in vitro synthesis also have broad application prospects in bacteriostasis and anticancer.At present,the research on aziridine ring mainly focuses on the activity analysis and chemical synthesis of related natural active products,while the research on biosynthesis progresses slowly.Previous studies have found that sulfotransferase plays an important role in the synthesis of aziridine functional groups by analyzing the fisilomycin biosynthesis gene cluster from Streptomyces’s.Ficellus.In this study,substrate screening experiments of different sources of sulfotransferase were carried out,and the sulfotransferase activity detection system by enzyme-coupled method was optimized,so as to explore the synthesis of aziridine ternary ring catalyzed by sulfoyltransferase in vitro.Through the screening of phosphatase,gene mutation and the optimization of reaction conditions of mutants,a phosphatase mutant with strong specificity of PAP substrate and its optimum reaction conditions were obtained.The G236D mutant of YND was treated with 50m M Hepes buffer containing 0.1 mg/m L bovine serum albumin（BSA）,50 m M KCl,2 m M Mg Cl₂ and p H 7.5 in water bath at 20℃.Application feasibility analysis of the optimized phosphatase and the optimum reaction conditions in the enzyme linked method sulfotransferase activity determination was verified by the enzyme catalytic experiment applied to ST1A1,ST1A3 and substrate 1-naphthol.The sulfotransferases from different sources were expressed by escherichia coli expression system,and the activity of the purified protein was detected,the results showed that the sulfotransferases obtained by this method had good catalytic activity.The optimized sulfoyltransferase activity detection system was used to screen the substrate of heteroexpressed sulfoyltransferase.The results showed that the expressed sulfoyltransferase could transfer the sulfoyltransferase of PAPS to the correspondingβ-amino alcohol substrate to form O-sulfate.It was proved by mass spectrometry that the structure spontaneously underwent intramolecular nucleophilic attack under alkaline conditions and was successfully transformed into azproidine ring compounds.This study further verified the mechanism of sulfotransferase in the process of aziridine terring biocatalytic synthesis,and provided a more flexible detection method for the activity detection of sulfotransferase substrate screening,which facilitated the study of functional diversity of sulfotransferase.At the same time,it provides a new route of biocatalytic synthesis of aziridine compounds with good prospects for patent medicine,and advances the research progress of synthesis of active aziridine derivatives in an environmentally friendly biocatalytic way.