Immobilization Of Transaminase And Its Catalytic Synthesis Of Chiral Intermediates For Niraparib In Continuous Flow

Niraparib is an oral poly-adenosine diphosphate（ADP）ribose polymerase（PAPR）inhibitor,which is mainly used for the maintenance treatment of advanced recurrent ovarian cancer.It can significantly prolong the survival period of patients with advanced ovarian cancer and reduce the recurrence of ovarian cancer.The most important part for the synthesis of niraparib is to construct the intermediate（S）-5-（4-bromophenyl）piperidin-2-one with high chiral purity,but the current chemical synthesis method has many shortcomings,such as low yield,poor selectivity and high reaction cost.Therefore,in this experiment,through the pre-screening of fourω-transaminase constructed and preserved in the laboratory,the high-activity and high enantioselective S-typeω-transaminase ATA-301 was obtained,which can efficiently catalyze the synthesis of niraparib intermediate.On this basis,a multi-point covalent immobilization method was used to immobilize it on an epoxy resin carrier,and the immobilization conditions and immobilized enzyme catalytic conditions were optimized,and the final immobilized enzyme specific enzyme activity was 72.11 U/g,the enzyme activity recovery rate reached 53.38%.Finally,the immobilized enzyme ATA301@ES103B was applied to a continuous flow reaction system and successfully catalyzed the chiral intermediate of niraparib.The main research contents of this paper are as follows:（1）Preparation of niraparib precursor ketone.Using succinic anhydride as starting material,through Friedel-Crafts acylation reaction,DCC condensation reaction,Corey-Chaykovsky epoxidation reaction and ethylene oxide rearrangement reaction to synthesize niraparib precursor ketone 4-（4-bromobenzene）)-5-oxobutyrate isopropyl.Among these reactions,according to the description in the literature,with concentrated sulfuric acid as the catalyst,the actual yield of the esterification product was only 37%.In this experiment,DMAP was used as the catalyst and DCC as the condensing agent for the reaction,and the product yield was significantly increased to98.8%.At the same time,it was found in the experimental operation that using KOt-Bu as a strong alkali to deprotonate trimethylsulfoxide iodide to obtain dimethylsulfoxide methylene ylide was not good,and the epoxidation reaction failed,while after using stronger alkali,Na H,the product yield was increased to 80%.（2）Screening of transaminase.Using the niraparib precursor ketone prepared above as a substrate,pyridoxal-5-phosphate（PLP）as a cofactor,and i-Pr NH₂as an amine donor,four transaminase enzymes constructed and preserved in the laboratory were screened.Finally,the S-typeω-transaminase ATA-301 with high activity and high enantioselectivity was obtained.The yield of the chiral intermediate of the target product niraparib was 87%,and the ee value was over 99%.（3）Optimization of immobilization conditions and research on the properties of immobilized enzymes.Six different resin carriers were screened based on protein adsorption rate and enzyme activity recovery rate,and the best immobilized carrier was determined to be epoxy resin ES-103B.By optimizing the immobilization process conditions of the transaminase ATA-301 immobilized on the epoxy resin carrier ES-103B,the optimal immobilization process conditions are finally determined as follows:the mass ratio of the carrier and the transaminase is 10:1,and the immobilization time is 8 h,Phosphate buffer solution 500 mmol/L p H 7.0,temperature 30℃.The reaction conditions of the immobilized enzyme ATA301@ES103B catalyzed to synthesize the chiral intermediate of niraparib were optimized.The results showed that the enzymatic activity of the immobilized enzyme reached the highest at a temperature of 50℃in 100 mmol/L p H 9.0 phosphate buffer.The storage stability of the immobilized enzyme and the stability of repeated batch operations were investigated,and it was found that 89.83%of the residual enzymatic activity was retained after 15 days of storage in the refrigerator at 4℃,and 80.41%of the initial enzymatic activity was retained after repeated use for 10 times,which demonstrate good storage stability and operational stability of the immobilized enzyme.（4）Preparation of chiral intermediate of niraparib by continuous flow method and its process optimization.The immobilized enzyme ATA301@ES103B obtained above was packed into a reaction column（inner diameter 5 mm,length 10 cm）,the continuous flow process conditions were optimized,and the optimal process conditions were screened out:2.0 g immobilized enzyme filler;flow rate:0.3 m L/min;the reaction solution pumped into the reaction column from top to bottom.Under these conditions,the continuous asymmetric synthesis of niraparib chiral intermediates can achieve a space-time yield of 968.2 g/（L·d）.After 24 hours of continuous operation,the reaction conversion rate is still above 90%,indicating that this method has very high feasibility for efficient and continuous production of（S）-5-（4-bromophenyl）piperidin-2-one.