Biocatalytic Synthesis Of Chiral Five-membered Carbasugars Intermediates

Five-membered carbasugars have been widely used in antiviral and anti-tumordrugsandcandidatedrugs.（S）-4-（hydroxymethyl）cyclopent-2-enone and（3R）-3-（hydroxymethyl）cyclopentanone,as key intermediates of five-membered carbasugars,can be used to synthesize antitumor compound didemnenone C and anti hepatitis B drug carbocyclic dd A,respectively.This article investigates a new pathway for the synthesis of chiral five-membered carbasugars intermediates using biological enzymatic methods based on enzyme promiscuity.CV2025ω-transaminase from Chromobacterium violaceum was selected based on substrate similarity.The enzyme was cloned,expressed in E.coli and purified.The results showed that the molecular weight of the protein was about 51 k Da,which was consistent with the expected molecular weight.CV2025 can be used to convert（（1S,4R）-4-aminocyclopent-2-enyl）methanol to（S）-4-（hydroxymethyl）cyclopent-2-enone.It has R configuration preference,in contrast with the conventional S preference.The characterization of CV2025 was studied.The highest activity was obtained below 60°C and at p H 7.5.Cations Ca²⁺and K⁺enhanced activity by 21%and 13%,respectively.The conversion rate reached 72.4%within 60 min at 50°C,p H 7.5,using 0.5m M PLP,0.6μM CV2025,and 10 m M substrate.CrS chromate reductase from Thermos scoteductus SA-01 was selected based on substrate similarity.The enzyme was cloned and expressed in E.coli,and the protein was purified.The results showed that the molecular weight of the protein was about 38 k Da,which was consistent with the expected molecular weight.The protein of formate dehydrogenase FDH from Candida boidinii was purify and used to construct a NADH coenzyme regeneration system.With the help of NADH coenzyme regeneration system,CrS catalyzes the reduction of substrate（S）-4-（hydroxymethyl）cyclopent-2-enone to obtain the target product（3R）-3-（hydroxymethyl）cyclopentanone.The characterization of CrS was studied,and its reaction activity was highest at45°C and p H 8.0.The reaction conditions were optimized.The conversion rate reached 100%within 45 min at 35°C,p H 7.0,using 5 m M（S）-4-（hydroxymethyl）cyclopent-2-enone,0.05 m M FMN,0.2 m M NADH,10μM CrS,40μM FDH,and 40 m M sodium formate.Immobilized enzyme TLIM from Thermomyces lanuginosus was selected based on enzyme promiscuity.During the Michael addition reaction of2-cyclopentene-1-one and its analogues with nitro compounds,TLIM showed good substrate promiscuity and reaction type promiscuity.Utilizing three enzymes including CV2025ω-transaminase,CrS enoate reductase,immobilized enzyme TLIM to catalyze the synthesis of chiral five-membered carbasugars intermediates,providing a promising new method for the synthesis of chiral five-membered carbasugars.