Directed Evolution Of Vitreoscilla Hemoglobin For The Synthesis Of Indolizines

Indolizines,a class of heteroaromatic compounds containing two condensed rings and a bridging nitrogen atom,which have been widely applied in natural products and synthetic pharmaceuticals.Most of the reported traditional synthetic routes of indolizines have some disadvantages,such as the formation of conjugate-acid waste,harsh reaction conditions,narrow substrate scope,the utilization of toxic transition-metal catalysts and harmful organic solvents.Therefore,it is important to develop an environment-friendly and efficient method to synthesize indolizines.Enzymes are more and more widely used as biocatalysts along with the developement of enzymology.Recently,hemoproteins are widely used in organic synthesis due to their catalytic promiscuity,mild reaction conditions and environmental friendliness.Vitreoscilla hemoglobin(VHb)is a soluble protein expressed by Vitreoscilla under hypoxia.It is a homodimer composed of two heme-containing identical subunits.Previous studies have proved that hemoglobin can present carbene transferase activity.In this paper,VHb was used to catalyze carbene transfer reaction for the first time.Under the optimal condition,VHb could catalyze the synthesis of indolyzine from pyridine,methyl propionate and ethyl diazoacetate.VHb mutant with higher catalytic activity was obtained by directed evolution.The main results are as follows:(1)VHb and VHb _Q53Hcould catalyze the model reaction:pyridine(0.5 mmol),methyl propionate(0.5 mmol),ethyl diazoacetate(0.6 mmol)and 5mg VHb in ethanol(0.5m L)stirring for 2h at 25^oC(product yield 59%-64%).(2)A UV prescreening method was established to improve the screening efficiency.The mutants with higher catalytic activity were obtained by HPLC.Both UV prescreening and HPLC rescreening were carried out under whole cells.(3)After two rounds of random mutation and screening,the mutant strain VHb^8-B5was obtained.Compared with VHb _Q53H,four amino acid mutation siteswere added:T23A,Y29H,I118T and V132A.The reaction was catalyzed by whole cells.(4)The enzymatic properties of mutant enzyme VHb^8-B5were explored.The effects of reaction medium and temperature on the reaction yield were explored.The optimal conditions were:pyridine(0.5 mmol),methyl propionate(0.5 mmol),ethyl diazoacetate(0.6 mmol)and 5mg VHb^8-B5in ethanol(0.5m L)stirring for 2h at 25^oC.(5)We explored the substrate scope of VHb^8-B5.The six kinds of pyridine analogues couldreact with methyl propionate and ethyl diazoacetateto produce the corresponding products in the presence of VHb^8-B5.Compared with meta-and para-substituted pyridines(83%-95%),the yield of ortho-substituted counterpart is disminished(68%),which is due to steric effects.All of theelectron-deficient alkynes could also react with pyridine and ethyl diazoacetate in the presence of VHb^8-B5(69%-81%).This work not only explores the potential of VHb and its mutants to synthesize indolizine,but also expands the application of enzyme catalytic promiscuity as well.