Synthesis Of 5-aminoindazole Podophyllotoxin By Mining And Engineering Imine Reductase

The antitumor activity of 4β-NH-(5-aminoindazole)-podophyllotoxin(5-ID-PTOX)reaches nanomolar,while the antitumor activity of the clinical antitumor drug etoposide(VP-16)is micromolar,thus,5-ID-PTOX has great pharmaceutical potential.However,the chemical synthesis route of 5-ID-PTOX is not environment friendly.Therefore,it is necessary to develop a biosynthetic route with mild reaction conditions and green catalyst.In this study,imine reductase was mined and engineered to apply for synthesis of 5-ID-PTOX.5-ID-PTOX is a chiral amine compound,which is produced by introducing 5-aminoindazole at the C4 position of podophyllotoxin via C-NH.The biosynthesis of chiral amine compounds is mainly though the reductive amination of ketone and amine catalyzed by imine reductases.Therefore,we design a biosynthetic route with podophyllotoxone and5-aminoindazole as starting substrates and imine reductases as biocatalysts.However,through the characterization of the enzyme library containing 222 imine reductases previously established in the laboratory,it was found that no imine reductases in the enzyme library can catalyze the reductive amination reaction of large arylketones and arylamines,and they tended to conversion of a small substrate that derived from the resolution of podophyllotoxone by substrate walking.In order to obtain the enzyme with initial activity,5-aminoindazole-podophyllotoxin imine intermediate was synthesized by chemical method and was as the substrate for screening enzyme library.Though mass spectrometry identification,nuclear magnetic resonance identification and small molecule crystal structure analysis,it was found that imine reductase I16 can reduce 5-aminoindazole-podophyllotoxin imine intermediate to 4α-NH-(5-aminoindazole)-podophyllotoxin.In order to obtain the complex structure of I16 and substrate for semi rational design of I16 to improve its activity.The culture conditions of protein crystals were screened.I16 single crystal with resolution of 6 ? was obtained,this lays a foundation for resolving of the crystal structure of I16 and complex structure of I16 and substrate.I16 was semi rationally designed through two rounds by molecular docking and key residues prediction via Auto Duck Vina and Hot Spot Wizard.The variants library that contains 1023 I16 variants constructed in first round and 736 I16-M1 variants constructed in second round were screened by using the 5-aminoindazole-podophyllotoxin imine intermediate as the substrate,the optimal variant I16-M2(I16-L180T/W215L/A219L/F223W)was obtained,and its conversion rate in reducing imine intermediate to 4α-NH-(5-aminoindazole)-podophyllotoxin was improved from wild-type13.6% to 41.0%.In this study,the chemoenzymatic synthesis route of antitumor candidate drug 4α-NH-(5-aminoindazole)-podophyllotoxin was built via imine reductase I16-M2,this provides technical route support for the establishment of 5-ID-PTOX biosynthesis.