Studies On Stereoselective Metabolism And The Mechanism Of Chiral Inversion Of Mandelic Acid

Mandelic acid(MA,also named phenylglycolic acid,α-hydroxyphenylacetic acid), is used as an intermediate for the synthesis of target molecules,such as urinary tract bactericide hexamine mandelate,peripheral vasodilator hacosan,eyedrops hydrobenzole and spasmolytic agent.It is also one of the major urinary metabolites of styrene in mammal.Early investigation on MA metabolism showed that it can partly dehydrogenate to PGA in human and dogs.In 1990s,besides stereoselectively metabolized to PGA,MA was also found could undergo chiral inversion(S-MA to R-MA) in Wistar rats by Drummond et al for the first time.Our laboratory study indicated that a strong stereo-selective metabolism of MA had occurred in SD rats after a single oral administration of 100 mg·kg^-1 MA.The mechanism of stereoselective metabolism and chiral inversion of MA remains unknown,and whether this phenomenon occurred in human also needs explornation.Our study focused on the stereoselective metabolism and chiral inversion of MA to explain the internal mechanism by molecular cloning technology and in vitro metabolic models.Aim To clone,express human,rat alcohol dehydrogenase and aldo-keto reductase and study the enantioselective metabolism of mandelic acid(MA).At the same time, study the interactionm between MA enantiomers and human ADH2 ligand binding domain using a docking approach.To study the location,coenzyme dependency and other properties of enzyme responsible for the stereoselective metabolism of MA,using many different types of in vitro metabolic models.To chemically synthesize S-MA-CoA thioester,investigate its hydrolysis in SD liver homogenate,study the mechanism of chiral inversion of MA.Methods 1) Human alcohol dehydrogenase 2,rat alcohol dehydrogenase 1,human and rat aldo-keto reductase 1A1 were amplified using RT-PCR from human and rat liver samples.Then subcloned into pET-28a(+) and expressed in E.coli BL21(DE3) stably.The proteins were induced with IPTG and purified by affinity chromatography. Then the enzyme activities were measured.MA enantiomers were incubated with rat, human ADH and phenylglyoxylic acid(PGA) with AKR1A1,respectively.Molecular docking of MA enantiomers and human ADH2 ligand binding domain was performed using the proper docking model and analyzed through energy and binding mode.The metabolism of chloromadelicacid and styrene glycol in huamn,rat ADH were also studied.HPLC was used to determine the metabolism.2) Human,rat,mouse microsomes and SD rat liver S9,mitochondria,cytoplasm protein were prepared by calcium precipitation method.MA enantiomers were incubated with these tissue fractions.The stereoselective metabolism was determined by high-performance liquid chromatography.Coenzymes NADPH,NADP,NADH,NAD and inhibitors of malate dehydrogenase oxaloacetic acid,adenosine-5-monophosphate were co-incubated with MA to investigate their effects on MA metabolism.Malate dehydrogenase from pig heart was purchased and used to study the metabolism of MA.3) S-MA-CoA thioester was chemically synthetized and purified by semi-preparative column.The purified thioester was freeze-dehydrated,and incubated with rat liver homogenate.Normal HPLC and precolumn derivatization chromatograhpy were used to determine the metabolism.Results 1) The proper genes were cloned and purified proteins were obtained.All of the proteins obtained showed good activity.Stereoselective metabolism of MA was observed in human ADH2,which favors for S-MA metabolism.No metabolism was detected of MA incubating in rat ADH1 or PGA in human,rat AKR.Chloromadelicacid and styrene glycol were not metabolized in human,rat ADH.Docking results showed that S-MA can form five hydrogen bonds with human ADH2 protein while the R isomer only can form two.Analyzed through energy and binding mode,the binding of S-MA and human ADH2 protein is much more stable,as a result,human ADH2 metabolized S-MA more easily.2) Both enantiomer of MA were stable in rat,mouse,human microsomes.There was a rind enantioselective biotransformation of S-MA to PGA in SD rat liver S9,mitochondria and cytoplasm protein.The amount of PGA produced by mitochondria proein was comparable with that by cytoplasm protein.The metabolism of MA in mitochondria and cytoplasm protein was decreased when co-incubated with cofactors,and was inhibited when oxaloacetic acid,adenosine-5-monophosphate were added.Catalytic inhibition of the reaction by the two inhibitors increased as a function of ion concentration.When the concentration of oxaloacetic acid reached 10mmol·L^-1, the enzyme activity was totally inhibited.When the concentration of adenosine-5-monophosphate reached 7.5 mmol·L^-1,80%activity of the enzyme was inhibited.MA enantiomers were not metabolized in malate dehydrogenase from pig heart.None of the cofactors had effect on MA metabolism.3) The synthesized yellow solid was identified to have the parent ion fragmentation of m/z 900([MA-CoA-H]^-) with MS.MS/MS data obtained showed the individual daughter ions of parent ion fragmentation of m/z 900.It showed that except the fragmentation of m/z 766 ([CoA-H]^-),the other daughter ions of m/z 686,408,338,159,134 were the same with that of CoA.So it was confirmed that the synthesized and purified chemical was the target chemical.Achirality HPLC showed S-MA-CoA thioester was hydrolyzed to MA, and precolumn derivatization chromatography confirmed it was R antipode.Conclusion The expression plasmids are constructed and the recombinant proteins are expressed successfully.The recombinant alcohol dehydrogenase and aldo-keto reductase have been employed to study MA metabolism.The enzyme responsible for MA metabolism resided in cytosol and mitochondrion,not in microsome.Oxaloacetic acid,adenosine-5-monophosphate showed great inhibition in mitochondria and cytoplasm protein,it indicated that the enzyme responsible for MA metabolism may be malate dehydrogenase.S-MA-CoA thioester was successfully synthetized and was hydrolyzed to R-MA in rat liver homogenate.Thus we conclude that the mechanism of MA chiral inversion might be the same with that of 2-Arylpropionic acid(2-APA) drugs. Thioesterification of S-MA to S-MA-CoA was priorityly taken place,then it isomerized to R-MA-CoA thioester via an enolate intermediatetic,at last nonstereoselective hydrolyzed of the R-MA-CoA thioesters to yield free R-MA.