Biocatalytic Redox Approaches For Asymmetric Synthesis Of Chiral Carboxyl-substituted Tetrahydroisoquinolines

Optically pure 1,2,3,4-tetrahydroisoquinolines(THIQs),especially 1-substituted THIQs,constitute a class of privileged structural moieties that exist in a variety of drugs and natural isoquinoline alkaloids.Due to the advantages,such as high efficiency,excellent enantioselectivity,and mild reaction conditions,the preparation of chiral THIQs by oxidoreductase-mediated biocatalysis has attracted widespread attention in recent years.However,these approaches mainly focused on the preparation of non-carboxyl substituted THIQs,such as 1-alkyl-,phenyl-,benzyl-or aryl-substituted THIQs.To date,few general comparable biocatalytic approach for the synthesis of chiral carboxyl-substituted THIQs has been reported for the lack of suitable oxidoreductases.In this study,the enzymes with excellent enantioselectivity toward the model substrate 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid(1-TIC)were exploited through genome mining.These enzymes were applied for the construction of kinetic resolution,chemo-enzymatic deracemization,and multi-enzymatic deracemization system,thus providing effective methods for the synthesis of chiral 1,2,3,4-tetrahydroisoquinolines carboxylic acids(TICs).According to the structural characteristics of model substrate,an enzyme library containing D-amino acid oxidases(DAAOs),L-amino acid oxidases(LAAOs),L-amino acid deaminases(LAADs),amino acid dehydrogenases(AADHs),and L-pipecolate oxidases(LPOs)was constructed.The genes were obtained through polymerase chain reaction or chemical synthesis and the corresponding recombinant plasmids were transformed into E.coli BL21(DE3)for overexpression.At least 42 enzymes were obtained with activities toward the natural substrates.Among them,four DAAOs showed different activities toward racemic 1-TIC with strict(R)-configuration enantioselectivity while ten LPOs exhibited opposite enantioselectivity.The FsDAAO from F.solani M-0718 with(R)-configuration enantioselectivity and highest activity was applied for the kinetic resolution of a number of racemic carboxyl-substituted THIQs,yielding the corresponding(S)-enantiomers with conversions up to 50%and excellent enantiomeric excess(ee)values(up to>99%).NMR and HRMS analysis confirmed that the oxidative dehydrogenation product of(R)-1-TIC was 3,4-dihydroisoquinoline-1-carboxylic acid,which could be transformed into racemic 1-TIC by ammonia-borane(NH3·BH3).By combining FsDAAO with NH3·BH3 in one pot,deracemization of these racemic carboxyl-substituted THIQs was successfully achieved to obtain(S)-enantiomers with conversions up to>98%and>99%ee.Preparative-scale deracemization was demonstrated with good isolated yields(up to 82%)and ee>99%.To explore an alternative method with multi-enzyme cascade,a panel of ?1-piperidine-2-carboxylate/?1-pyrrolidine-2-carboxylate reductases(DpkAs)for the reduction of compound 3,4-dihydroisoquinoline-1-carboxylic acid was cloned and evaluated.The PpDpkA from P.putida KT2440 was selected with the highest activity and good(S)-configuration enantioselectivity.Based on the feasibility and compatibility evaluation,a biocatalytic approach for deracemization of racemic carboxyl-substituted THIQs was established by combining FsDAAO-catalyzed enantioselective oxidative dehydrogenation and PpDpkA-mediated asymmetric reduction in one pot,producing the corresponding(S)-enantiomers with>99%conversions and ee.Preparative-scale biotransformation gave the(S)-1-TICs with good isolated yield(up to 91%)and>99%ee.In comparison with the chemo-enzymatic deracemization,this multi-enzymatic cascade was more efficient.To demonstrate a bio-oxidative kinetic resolution method for the preparation of(R)-1-TICs,the gene dadA in E.coli BL21(DE3)was deleted by using CRISPR-Cas9,thus providing a host without any activity toward(R)-1-TIC.The putative SoLPO from S.octosporus yFS286 was mined with the highest activity and good(S)-configuration enantioselectivity.A panel of racemic 1-carboxyl-substituted THIQs were resolved,giving the unreacted(R)-enantiomers with 50%conversions and>99%ee.Further analysis revealed that the oxidative dehydrogenation product of(S)-1-TIC was(S)-1,4-dihydroisoquinoline-1-carboxylic acid,which hampers the construction of deracemization.Preparative-scale biotransformation was further performed,and up to 40%isolated yields of(R)-1-TICs were obtained with>99% ee.