Preperation Research On The Hydroxylation Of Androst-4-ene-3,17-dione By Biotransformation

Microbial transformation of steroids has the advantages of small pollution,high efficiency and specificity,of which hydroxylation is the most important reaction.Androst-4-ene-3,17-dione?1,referred to as androstenedione or 4-AD?is an important intermediate for the synthesis of diuretics,contraceptives and androgens and other steroid drugs.The resulting 11?-hydroxyandrostenedione?11?-OH-4-AD?is a key intermediate for the synthesis of eplerenone,a drug used in the treatment of cardiovascular disease.Obtaining 11?-OH-4-AD by microbial transformation is not only the primary target of semi-chemical synthesis of the corresponding steroid drugs,but also one of the most crucial steps in semi-synthesis.In this paper,4-AD as a substrate for screening of bacteria and transformation process optimization,in order to get a certain industrial value of the species.In addition,the thesis also studied the transformation of Aspergillus awamori to andrnenedione?ADD?.The detailed research contents are as follows:1.Three strains with strong ability to transform 4-AD were obtained from 15 soil samples collected from different parts of the country and numbered as MH-14,MH-18,and MH-19.Among them,MH-18 was able to transform 4-AD to obtain11?-OH-4-AD with a basic yield of 48.50%.Through morphological observation and comparison of gene sequences in strain molecular biology,the strain was finally identified as Aspergillus awamori.2.The transformation of the steroidal substrate andadienone?ADD?by Aspergillus awamori was studied.3.The growth curve of Aspergillus awamori was determined,and the optimum input time of substrate 4-AD was 48 h.The effects of different culture conditions and conversion conditions on the conversion of target product 11?-OH-4-AD were finally determined.The optimum culture conditions were as follows:dextrin 33.6 mg/mL,corn flour 10.5 mg/mL,KH₂PO₄ 1.2 mg/mL,initial pH 4.5.The optimum conditions were as follows:temperature 32?,rotation speed 200 r/min,liquid volume 100mL/500 mL Erlenmeyer flask,the substrate solvent was chloroform,the substrate concentration was 1 g/L and the reaction time was 96 h.Based on the above process optimization conditions,the conversion rate of 11?-OH-4-AD can reach 84.02%,which is 35.52%higher than that before optimization,which is 7.54%higher than the76.48%reported in the literature.In this dissertation,the transformation of 4-AD and ADD by MH-14,Aspergillus awamori MH-18 and MH-19 was studied for the first time.A total of 8 transformation products were obtained.By analyzing the IR,¹H NMR,¹³C NMR and HRMS spectra of each transformation product and comparing the relevant literature reports,the structure of the transformation product was finally confirmed.In the meantime,this study found that the strain Aspergillus awamori MH-18 which has a high ability of11?-hydroxylation 4-AD not only enriches the research content of 11?-OH-4-AD,a key intermediate for the synthesis of eplerenone by microbial transformation,but also laid the foundation for the development of other steroid drugs.