Experimental Study On The Preparation Of Mitomycin Analogues By Enzyme Catalysis And Its Reaction Kinetics

Mitomycin is an antibiotic with anti-tumor activity.Mitomycin C?MMC?is an important member of mitomycin.It has been widely used in the treatment of gastric cancer,pancreatic cancer,colorectal cancer,breast cancer,lung cancer and other cancers^[1].Nevertheless,the high toxicity of MMC can lead to serious side effects,and its clinical application is limited.Therefore,people are devoted to finding mitomycin derivatives or analogues with high anti-tumor activity.Although chemical synthesis can form molecular skeleton with essential pharmacological activity,it has some disadvantages,such as poor selectivity,complicated reaction steps and drastic reaction conditions.Enzyme biocatalysis can overcome these problems and show superior catalytic properties.Lipase with high selectivity and specificity,resistance to organic solvents and extreme temperature,pH,which is the most widely used multi-functional enzyme in organic synthesis,has been widely used in industrial production^[2].In this experiment,the optimal conditions for the reaction of 2-methyl-1,4-benzoquinone with n-butylamine catalyzed by immobilized Trichosporon laibacchii?T.laibacchii?CBS5791 lipase were investigated.Under this condition,the yield was over 90%.In the experiment,the PEG 4000/K₂HPO₄ aqueous two-phase system was used to realize the coupling of the purification and immobilization process of T.laibacchii lipase.The diatomaceous earth was added to realize the in situ fixation of lipase by the interface activation on the hydrophobic carrier.An immobilized enzyme having a diameter of 0.5 mm.In phosphate-citrate buffer solution?pH7.0?,immobilized T.laibacchii lipase catalyzed the cross-coupling reaction of 2-methyl-1,4-benzoquinone with n-butylamine to form 2-methyl-3-n-butylamino-1-hydrogen-4-quinone.In order to solve the problem of substrate solubility,2%?v/v?methanol was introduced as cosolvent in a shaker at 50 and 200 rpm.The limitation of mass transfer was studied and it was found that both internal and external mass transfer could be neglected.In addition,the kinetic model of enzymatic Michael addition of n-butylamine in 2-methyl-1,4-benzoquinone was studied in this paper.Firstly,the modified ordered and random Bi-Bi mechanisms were proposed,and King-Altman graphs were drawn to obtain the differential equations representing the reaction rate.Secondly,the differential equations were solved by combining numerical integration toolbox ode45,and the kinetic parameters were obtained.The objective function error is minimized by using the non-linear optimization toolbox fmincon.The results showed that the simulated values based on the random Bi-Bi mechanism were highly consistent with the experimental data,which implied that the enzymatic Michael addition reaction followed the modified random Bi-Bi mechanism.Through the experimental study,the kinetic model of the Michael addition reaction of n-butylamine and 2-methyl-1,4-benzoquinone catalyzed by immobilized T.laibacchii lipase was obtained,which has Far-reaching practical significance.