Study On The Chemical And Enzymatic Synthesis Of Pregabalin Intermediate(R)-(-)-3-Carbamoymethyl-5-Methylhexanoic Acid

Pregabalin(PGB)is an analog of the neurotransmitter γ-aminobutyric acid,which can be used to treat neuropathic pain and epilepsy.The current mainstream process for synthesizing PGB is as follows:(R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid undergoes Hofmann degradation and rearrangement reaction in sodium hypochlorite and sodium hydroxide solution to generate.The chiral intermediate(R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid is the key to the synthesis of PGB during the progress of during synthesis.At present,synthesis processof(R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid exists some disadvantages such as low yield,involving highly toxic chemicals,and high environmental pressure,etc.Therefore,this article intends to construct a synthetic system to produce(R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid catalyzed by lactam hydrolase.Firstly,isovaleraldehyde and cyanoacetamide are used for Knoevenagel condensation and Michael addition reaction,and then hydrolysis and decarboxylation are carried out to obtain 3-Isobutylglutarimid(IBI)by optimizing the chemical synthesis process.In this article,by optimizing the conditions,changing the reaction parameters,and adjusting the materials,using morpholine as the basic catalyst,the reaction efficiency is greatly improved.And the phase transfer catalyst tetrabutylammonium bromide is introduced to make the conversion rate more than99%.The whole synthesis process does not involve organic solvents,the yield of IBI reaches 90%,and the purity is more than 99.8%.It provides a better-quality substrate for enzyme catalysis.In this paper,a recombinant strain of lactam hydrolase(E.Coli BL21(DE3)-prga-1)was constructed.By optimizing key fermentation parameters such as carbon source,nitrogen source,induction temperature,etc.,cells with high hydrolysis activity were obtained by culturing them.Then,by investigating the influence of transformation time,temperature,p H,substrate concentration,bacterial volume and other factors on the transformation rate,the synthesis of(R)-(-)-3-(carbamoylmethyl)-5 catalyzed by lactam hydrolase was obtained.The optimum reaction conditions for methylhexanoic acid: substrate IBI concentration of100g/L,bacterial volume of 5%,reaction temperature of 35-37°C,reaction time of10 h,p H 7-7.5.The catalytic efficiency of the system is over 99.9%,and the extraction yield of the product is over 90%.The lactam hydrolase was also used for the first time in the synthesis of this project,and it has not been reported in the literature,which is extremely innovative.