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Biooxidation of methyl group: Part 2. Evidences for the involvement of cytochromes P450 in microbial multistep oxidation of terfenadine

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Biooxidation of methyl group: Part 2. Evidences for the involvement of cytochromes P450 in microbial multistep oxidation of terfenadine
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy  Journal of Molecular Catalysis B: Enzymatic 67 (2010) 172–178 Contents lists available at ScienceDirect  JournalofMolecularCatalysisB:Enzymatic  journal homepage: www.elsevier.com/locate/molcatb Biooxidation of methyl group: Part 2. Evidences for the involvement of cytochromes P450 in microbial multistep oxidation of terfenadine Amane El Ouarradi, Murielle Lombard, Didier Buisson ∗ Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris Descartes 5, 45, rue des Saints-Pères, 75270 Paris Cedex 06, France a r t i c l e i n f o  Article history: Received 24 March 2010Received in revised form 2 July 2010Accepted 27 July 2010 Available online 6 August 2010 Keywords:Streptomyces platensis BiotransformationHydroxylationAldehydeLabeling studyP450 inhibitors a b s t r a c t The actinomycete  Streptomyces platensis  grown in culture medium containing soybean peptones cantransformterfenadine,anantihistaminedrug,intoitsactivemetabolitefexofenadine.Themicrobialoxi-dation of methyl group of terfenadine into carboxylic acid could be an alternative to chemical ways toproduce fexofenadine. This bioconversion requires three oxidation steps: a hydroxylation of one methylgroup followed by the oxidation of the corresponding alcohol into the aldehyde and finally its oxidationinto the carboxylic acid. The oxidation reaction of each step has been studied. Terfenadine and inter-mediates incubated with whole cells were not oxidized under argon whereas their biotransformationunder  18 O 2 -enriched atmosphere gave labeled fexofenadine. P450 inhibitors, such as clotrimazole orfluconazole, inhibited oxidation activity of each step. While the two last steps could be catalyzed bydehydrogenasesoroxidases,thisstudystronglydemonstratestheroleofatleastone,orpossiblyseveralcytochromes P450, in the oxidation of terfenadine into fexofenadine by  S. platensis  cells. To our knowl-edge,thisisoneofthefewexamplesofinvolvementofP450sinsuchthreestepsoxidationofaxenobiotic. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Biocatalysis and biotransformation have become a commonlyused tool in organic chemical synthesis, in particular to producesynthons and metabolites of xenobiotics. They are used for thesynthesis of chiral molecules but also of achiral products wherea chemical process would not be possible. For example, chem-ical oxidations of heteroaromatic molecules and non-activatedcarbon-atom give side-products and are generally unspecific. Onthe contrary, some examples describe the specific hydroxyla-tion of methyl and methylene groups by microorganisms [1]. Themost appropriate activity could be obtained by screening isolatedenzymes or microorganisms. While some enzymes are commer-cially available (hydrolases, reductases), oxygenases are generallyprovidedbymicroorganismsandusedinwholecells[2].However,for preparative scale, an improvement of the biocatalyst is oftennecessary [3].We are interested in the chemoenzymatic synthesis of fexofe-nadine, an antihistamine drug devoid of cardiotoxicity [4,5] which ∗ Corresponding author. Present address: Muséum National d’Histoire Naturelle,Centre National de la Recherche Scientifique, Unité Molécules de Communicationet Adaptation des Microorganismes, CP54, 57 rue Cuvier, 75005 Paris, France.Tel.: +33 0 1 40 79 81 24; fax: +33 0 1 40 79 31 35. E-mail address:  buisson@mnhn.fr (D. Buisson). is the main mammalian metabolite of terfenadine. Because fexofe-nadine chemical synthesis is laborious, microbial hydroxylation of the easily accessible terfenadine has been considered as the firststep. Moreover, according to Microbial Model of Mammalian DrugMetabolism it was expected to biotransform terfenadine into fex-ofenadine.It was found that several microorganisms are able to catalyzethemultistepoxidationofterfenadine(Fig.1)[6,7]andanalogs[8]. Among them, we showed [9] that the fungus  Absidia corymbifera and the bacterium  Streptomyces platensis  are the most efficient infexofenadine synthesis. However, the activity per gram of cells istoo low for a scale-up process and fexofenadine production mustbe optimized by proteins engineering. This scale-up requires theprecise knowledge of the enzymatic activity(ies) involved in thismultistep oxidation and the cloning of the proteins correspondinggenes [10].While whole microbial cells contain a wealth of enzymesholding different redox activities, various enzymatic systems cancatalyze these three reactions: the first step, hydroxylation of amethyl group can only be catalyzed by monooxygenases, whereasthe two following oxidation steps could be catalyzed by eitherdehydrogenases, oxidases or monooxygenases (a hem and non-hemenzymes)[11–13].Dehydrogenasesarethemostoftencitedinbiotechnologicalapplications.Forexample,arecentworkdescribes[14] an efficient oxidation of alcohol into the corresponding acidusing whole cells of   Brevibacterium  sp. or a three enzymes system, 1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2010.07.017  Author's personal copy  A. El Ouarradi et al. / Journal of Molecular Catalysis B: Enzymatic  67 (2010) 172–178 173 Fig. 1.  Biotransformation of terfenadine. 2-phenylethanoldehydrogenase,phenylacetaldehydedehydroge-nase and NADH oxidase to regenerate the NAD + . In an industrialprocess, the oxidation of methyl groups on aromatic heterocyclesto the corresponding carboxylic acids were achieved by enzymesfromwildtype Pseudomonasputida ,xylenemonooxygenase(XMO),benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase[15,16]. However, Schmid and coworkers showed [17] that XMO has an alcohol and an aldehyde oxidizing activities, and usedit in synthesis of aryl carboxylic acids [18]. There are only fewexamples of involvement of microbial cytochrome P450 in the for-mation of carboxylic acid, including oxidation of 2-ethylhexanolby CYP101 from  P. putida  [19], and formation of dicarboxylic acidfrom alkanes in  Candida tropicalis  [20]. However, three fatty alco-hol oxidases from the strain  C. tropicalis , which could be involved,were also characterized [21]. Some aldehyde oxidases are charac-terizedincluding onefroma Streptomyces strain[22]and onefroma  Brevibacillus  sp. that is used to remove glutaraldehyde, a poten-tialenvironmentalpollutant[23].Finally,somefungalextracellularhem thiolate peroxidases are capable of performing oxidation of methyl groups leading to the corresponding acids [24].In some cases, there is a lack of knowledge of the enzymesinvolvedinthepreparationofacidsbymicrobialoxidation[25–30].Schwartzetal.reportedtheoxidationofebastinetocarebastineby C. blabesleena  and suggested that the two alcohol oxidation stepswere catalyzed by oxido-reductases [31].Contrary to Schwartz suggestion, we obtained some results inagreement with a monooxygenase-dependent mechanism for thethree steps oxidation involved in the fexofenadine formation by S. platensis . Firstly, oxidation of hydroxyterfenadine to fexofena-dineoccurswithcellsgrowninculturemediumcontainingsoybeanpeptone, which are known to induce cytochrome P450 in  Strepto-myces griseus  [32]. Secondly, oxidation of hydroxyterfenadine in 18 O 2 -enriched atmosphere lead to labeled fexofenadine, resultingto incorporation of one atom of dioxygen [33].Then, prior to endeavor to purify the enzymes involved in themultistep oxidation of terfenadine by  S. platensis  cells, it was nec-essary to identify the precise class of these oxidizing enzymes. Wedescribe here the investigations performed to inquire the type of enzyme implicated in each oxidation step. Alcohol dehydrogenaseand monooxygenase-dependent oxidative activities were seekedinvitrowithcell-freeextracts.However,asP450-monooxygenasesdependent activities that result from a multi-component systemswitharelativelylownaturallevelofexpression[34],arerarelypre-served during preparation of cell-free extract, indirect approachesare often used to decipher the precise type of enzymes involved inoxidation activities [35–39]. Using this strategy, we performed thebiotransformation of terfenadine, and of the corresponding alco-hol and aldehyde intermediates under  18 O 2  atmosphere or in thepresence of P450 inhibitors. Our results suggest that the overallbiotransformation arise from three cytochrome P450 dependentoxidation steps. 2. Materials and methods  2.1. Chemicals Terfenadine was purchased from Sigma, hydroxyterfenadinewas prepared as previously reported [33] and the correspond-ing aldehyde was obtained as described in this work. Dioxygen 18 O 2  (99% atom  18 O) and H 218 O was purchased from Cortecnet(Paris) and Euriso-top, respectively. Clotrimazole is obtained fromSigmachemicals(Lyon,France)andfluconazolefromPfizer(Orsay,France).  2.2. Preparation of aldehyde  3 Hydroxyterfenadine  2  (96.6mg, 0.2mmol), TEMPO (31.2mg,0.2mmol)andtetrabutylammoniumchloride(22.2mg,0.08mmol)were dissolved in CH 2 Cl 2  and buffer (NaHCO 3 /K 2 CO 3  pH 8.3,2ml). The mixture was stirred at room temperature and N-chlorosuccinimide (53.4mg, 0.4mmol) was added in four portions(2h). After 1h, aqueous layer was extracted with CH 2 Cl 2  (twotimes). The combined organic layers were washed with brine,dried over anhydrous sodium sulfate and evaporated. The residuewas chromatographed on silica gel (dichloromethane/methanol:97/3) to afford  3  in 45% yield.  1 H RMN (CDCl 3 , 500MHz):  ı =9.50(s, 1 H, C H  O), 7.53–7.20 (m, 14 H,  H  ar), 4.67 (m, 1H, C H  OH),3.26–2.97 (m, 2H, C H  NC H  ′ ), 2.72 (m, 2H, C H  2 (CH 2 ) 2 CHOH), 2.56(t,H,  J  =5.00Hz,C H  ),2.51–2.34(m,2H,C H  NC H  ′ ),2.03–1.99(m,2H,C H  CH 2 NCH 2 C H  ′ ), 1.91–1.76 (m, 4H, C H  2 C H  2 CHOH), 1.60–1.49 (m,2H, C H  CH 2 NCH 2 C H  ′ ), 1.47 (s, 6H, 2 C H  3 ). RMN (CDCl 3 , 500MHz) ı =203.7(CHO),146.9,145.2,141.4,129.8,128.2,127.7,127.0,80.5,74.1,58.2,54.5,51.6,44.2,38.3,25.4,23.9,23.1.HRMS m /  z  calcdforC 32 H 40 NO 3  [M+H] + 486.3008, Found. 486.3010.  2.3. Bacteria and culture conditionsS. platensis  NRRL 2364 cultures were maintained on agar slants(ISP medium 2) and stored at 4 ◦ C. Liquid culture media containing(per l) glucose 16g, yeast extract (DIFCO) 4g, malt extract (DIFCO)10g (YM medium), and glucose 16g, yeast extract 4g, malt extract10g and soybean peptones (Organotechnie) 5g (YMS medium)were sterilized without glucose at 120 ◦ C for 20min. Microorgan-ism was cultivated at 30 ◦ C for 48h in an orbital shaker (200rpm).Celldryweight(CDW)wasobtainedbycentrifugationof100mlculture medium and biomass was dried at 100 ◦ C for 24h.  Author's personal copy 174  A. El Ouarradi et al. / Journal of Molecular Catalysis B: Enzymatic  67 (2010) 172–178  2.4. Incubation with cell-free extractsS.platensis cells(500ml)werecultivatedinYMSmediumina2-lErlenmeyerflask.After48h,cellswerecollectedbycentrifugation.ThebacterialpelletsweresuspendedinTEGbuffer(50mMTris–HClpH7.6,EDTA1mM,Glycerol10%)containingchickenegglysozymeandDnaseI.After2hofincubationat30 ◦ C,cellsweresubsequentlydisruptedbyadditionofdetergent-basedBugBusterreagent,inthepresence of protease inhibitors. The cells were then sonicated andcentrifuged (7000 ×  g  ) at 4 ◦ C to remove cells debris. The cloudysupernatant,whichcontainedbothcytosolicandmembranemate-rial, was fractionated by a centrifugation at 100000 ×  g   for 1h, at4 ◦ C. The supernatant fraction, which contained the soluble pro-teins,wasdecanted.Thepelletcontainingmembraneproteinswasresuspended in TEG buffer. Protein concentration of both cytosolicand membrane fractions was estimated using Bradfod method.Substratehydroxyterfenadine(10–50  M)andsolubleormem-brane fractions (100–400  g) were incubated in the presence of NAD + or NADP + (1mM) in TEG buffer, in a total reaction volumeof 400  l. Reaction mixtures were incubated at 30 ◦ C for up to2h, and methanol (1 volume) was added to quench the reaction.Theprecipitatedproteinswereremovedbycentrifugation(10min,10000 ×  g  ) and the supernatants were analyzed by HPLC.  2.5. Incubations with whole cells The biotransformations were performed in culture mediumor in citrate buffer pH 5 at 30 ◦ C in an orbital shaker (200rpm)and substrate was added in DMF. Biotransformation was moni-tored as followed. Samples (800  l) were diluted with methanol(700  l), mixed vigorously and centrifuged at 10000 ×  g   for 5min.Theresultingsupernatantsweremicro-filtered(0.45  m)andana-lyzed by HPLC.  2.5.1. Incubations of hydroxyterfenadine in presence of terfenadineS. platensis  was grown in YM medium (500ml), harvested, andresuspended in citrate buffer pH 5 (125ml). Cells suspension wasseparate in 5 flasks and terfenadine was added in DMF (25  l) toobtain final concentrations: 0, 0.01, 0.025, 0.05, 0.1 and 0.2gl − 1 .After30min,hydroxyterfenadinewasaddedinDMF(finalconcen-tration0.15gl − 1 ).Biotransformationweremonitoringasdescribedabove. After 20h, samples (5ml) of cells suspensions were takenand1mlofmethanolwasadded.Mixtureswerevigorouslystirred,centrifuged and analyzed by HPLC.  2.5.2. Biotransformation under   18 O  2  atmosphereS.platensis wasgrowninYMorYMSmedium(50ml),harvested,and resuspended in citrate buffer pH 5 (50ml) in which nitrogenwasbubbledfor5min.2mlofcellsuspensionweretransferredina10-mlvial,whichwassealedbyturnoffflangestopped.Theremain-ing air was removed by vacuum using needle and replaced bynitrogenthreetimes.Finally,theheadspaceofvialswereevacuatedandreplacedwithpureoxygenenrichedwith 18 O 2  (99atom% 18 O),substrateswereaddedbyinjectionof4  lofsolutioninDMF(finalconcentration 0.2gl − 1 , 0.42mM) and vials were placed in rotaryshaker at 200rpm and 30 ◦ C. After 48h (hydroxyterfenadine andaldehyde) or 120h (terfenadine) of incubation, 400  l of reactionmixtures were withdrawn, 350  l of methanol was added. Aftervigorousagitationandcentrifugation,supernatantswereanalyzedby LC–MS.  2.5.3. Effects of cytochrome P450 inhibitors on oxidationS. platensis  was grown in flask (500ml) containing YM or YMSculture medium (250ml). Culture was separated in 24 ×  10ml in25-mlflask.InhibitorswereaddedinsolutioninDMF(clotrimazole20  l, fluconazole 40  l) to obtain final concentration (clotrima-zole:0.2,0.3,0.4,0.5and0.7mM;fluconazole:10and15mM).DMFwas added in controls without inhibitor. Substrates were addedin solution in DMF (20  l) to obtain final concentration (0.2gl − 1 ).Incubations were monitored by HPLC throughout 48h. Experi-ments in presence of both inhibitors were conducted with finalconcentrationsclotrimazole(0.5mM)andfluconazole(15mM)andhydroxyterfenadine (0.2gl − 1 ) or 2-phenylpropionaldehyde.  2.6. Analyses of metabolites Supernatants were analyzed by HPLC performed on a GilsonHPLC interfaced to computer using the Gilson Unipoint software.The system involved pump 305 and 306, gradient dynamic mixer811B and autoinjector 234 and the detector was Shimadzu-SDP6Amodel.The column (Agilent, C18, 5  m (250 ×  4.6)) was in oven (Shi-madzu CTO-10A model) at 40 ◦ C and eluted (flow rate 1ml/min)with a gradient solvent system: isocratic 70% (NH 4 OAc 0.1M/30%CH 3 CN) for 7min followed by gradient to 100% (40% NH 4 OAc0.1M/60% CH 3 CN) in 5min and held 18min. The detection was atUV 230nm and sample volumes were 20  l.LC–MS data were performed with a Surveyor-LCQ Advantagemass spectrometer, using the same conditions except a flow rateof 0.2ml/min and UV. The mass spectrometer was in ESI-positivemode, using a 4kV capillary tube voltage and an inlet temperatureof 275 ◦ C. 3. Results and discussion The main goal of our study is to obtain an effective method of fexofenadine production from terfenadine. This objective needs toconfirmthechemicalnatureoftheintermediatesandtodeterminethe type of enzyme catalyzing each reaction step.  3.1. Incubation with intermediate as substrate The putative intermediates, alcohol  2  and aldehyde  3 , respec-tively were obtained by microbial hydroxylation as described [33]and oxidation of alcohol  2  as depicted in Section 2 (Section 2.2). We showed that both intermediates were oxidized into fex-ofenadine by  S. platensis  NRRL 2364 cells cultured either in YMor YMS culture medium, and this result confirmed the oxidationpathway of terfenadine into fexofenadine (Fig. 1). The hydrox-yterfenadine oxidation activity of YM-cells was surprising becauseincubations of terfenadine with cells obtained in these condi-tions afforded hydroxyterfenadine as the main product and a lowamount of fexofenadine. To explain these results, we supposedan influence of terfenadine in the hydroxyterfenadine oxida-tion. To verify this hypothesis, cells produced in YM culturemedium were incubated with hydroxyterfenadine (0.15gl − 1 ) inthe presence of terfenadine at various concentrations (0–0.2gl − 1 ).Incubations were performed with 14g of CDWl − 1 in citrate bufferpH 5 at 30 ◦ C for 20h (Fig. 2). At low concentrations of terfe-nadine (0.01–0.025gl − 1 ), fexofenadine formation increased withterfenadine concentration, while concentration of hydroxyterfe-nadine was lower than initial concentration. This is the result of hydroxylation of terfenadine in concentration-dependant mannerand of oxidation of hydroxyterfenadine in concomitant reactions.For higher concentration of terfenadine (0.025–0.2gl − 1 ), hydrox-yterfenadine formation increased with terfenadine concentration,whereasfexofenadineformationdecreasedundertheseconditions.Therefore, at 0.2gl − 1 of terfenadine, no formation of fexofenadinewas observed and the concentration of hydroxyterfenadine washigher than its initial concentration.  Author's personal copy  A. El Ouarradi et al. / Journal of Molecular Catalysis B: Enzymatic  67 (2010) 172–178 175  Table 1 Comparison of oxidative activity catalyzed by whole cells grown in YMS and YM culture medium.Substrate Terfenadine  1a  Hydroxyterfenadine  1b  Aldehyde  1d Product Hydroxyterfenadine  1b  Fexofenadine  1c  Fexofenadine  1cR  a 1,12 1,46 1,02 a Ratio of specific activity (YMS/YM). Thus, cells grown in YM culture medium were able to oxi-dizehydroxyterfenadinetoproducefexofenadineintwoconditionseither when hydroxyterfenadine is used as substrate or when ter-fenadine has disappeared more or less completely. We stronglysuggest that this phenomenon is explained by terfenadine inhi-bition phenomenon. The same observation was described in theoxidationoftolueneandpseudocumenebyxylenemonooxygenase[18].The best oxidizing activities were observed with cells grownin YMS culture medium. On the one hand, these cells are able toproduce fexofenadine in the presence of terfenadine. On the otherhand, hydroxyterfenadine oxidation was approx. 1.5-fold higherthanoxidationbycellsgrowninYMculturemedium,whilenosig-nificant difference between YM and YMS-cells was observed forterfenadinehydroxylationandaldehydeoxidation(Table1).Theseresults are in agreement with the soybean peptone-induction of alcohol-oxidative activity not inhibited by terfenadine.  3.2. Incubation with cell-free extracts Experiments were conducted with cell-free extracts on ter-fenadine metabolism, in order to determine if flavin or P450dependent monooxygenases were involved in the oxidation of themethyl group of the t-butyl moiety.  S. platensis  was cultivatedin YMS culture medium and cells were disrupted as describedin Section 2 (Section 2.6). Terfenadine was incubated with solu- ble or membrane fractions or a mixture of them, in the presenceof the reduced form of the nicotinamide cofactor, NADH, H + or NADPH, H + . No oxidation of terfenadine was observed witheither compartment cells, in these conditions. Indeed, such aloss of enzymatic activity is often observed in the case of multi-protein complexes, as P450-dependent monooxygenases usuallyare.Therefore,thisresultindicatedthatP450scouldberesponsiblefor the first step of terfenadine oxidation, i.e. its hydroxyla-tion.In the same manner, experiments were conducted on hydrox-yterfenadine metabolism, in order to determine if an alcoholdehydrogenasewasinvolvedintheoxidationofthealcoholintothecorresponding aldehyde. Hydroxyterfenadine was incubated withsoluble or membrane fractions or a mixture of them, in the pres- Fig. 2.  Concentration of fexofenadine and hydroxyterfenadine after 20h of incu-bation of hydroxyterfenadine (0.15gl − 1 ) in presence of different concentrationsof terfenadine. Biotransformations by  S. platensis -whole cells grown in YM culturemedium (65h). Symbols:  square , hydroxyterfenadine;  triangle , fexofenadine. ence of the oxidized form of the nicotinamide cofactor, NAD + orNADP + . No oxidation of hydroxyterfenadine was observed in theseconditions.OnthecontrarytoflavinorP450-dependentmonooxy-genases which are known to be multi-protein systems, alcoholdehydrogenases do not require any other protein cofactor. Thusthis result suggested that no alcohol dehydrogenase was involvedin the transformation of the alcohol intermediate.  3.3. Biotransformations under controlled atmosphere These experiments were performed with  S. platensis  grown inYM or YMS culture media. Under argon, terfenadine was not oxi-dized into hydroxyterfenadine as expected when the reaction wascatalyzed by a monooxygenase. In these conditions, when hydrox-yterfenadine and aldehyde were added as substrates, there areno formations of aldehyde and/or acid, but the reduction of alde-hydewasobserved.Theseresultsshowedsurvivalofcellsoractiveenzymes in these conditions and suggested that the oxidations of alcohol and aldehyde were dioxygen-dependant.To investigate the dioxygen function in these reactions, ter-fenadine, hydroxyterfenadine and aldehyde were incubated withwhole cells of   S. platensis  under  18 O 2 -enriched atmosphere(99atom%  18 O). The reaction mixtures were analyzed by liquidchromatography–mass spectrometry in ESI-positive mode and the m /  z  valuesat488,486and502Daforstandardhydroxyterfenadine,aldehyde and fexofenadine correspond to [M+H] + molecular ions,respectively. The results are summarized in Table 2.In both culture conditions, mass spectrum of hydroxyterfena-dine formed from terfenadine showed incorporation of one atomof oxygen, with the normalized ratios of [M+H] + to [M+H] + +2molecular ion peaks of 10:90 and 20:80 for YM and YMS cultureconditions, respectively (entries 1 and 2).Further oxidations of hydroxyterfenadine afford fexofenadine,which is formed by incorporation of a second oxygen-atom.In monooxygenase-dependent reaction, this oxygen-atom isincorporatedfrommoleculardioxygenwhereasindehydrogenase-dependent reaction, fexofenadine is formed from aldehyde byincorporation of one oxygen atom from water. Then, when terfe-nadine was added as a substrate in an  18 O 2 -enriched atmosphere,fexofenadine formed via monooxygenase catalyzed oxidationwould give an [M+H] + ion at 506Da, 4Da greater than that of the standard, whereas fexofenadine formed via dehydrogenases(alcohol and aldehyde dehydrogenase) catalyzed oxidation wouldgive an [M+H] + ion at 504Da, 2Da greater than that of thestandard.Inthisstudy,fexofenadine(49%)wasobtainedafter120-h incubation of terfenadine with  S. platensis  whole cells grownin YMS-culture medium and mass spectrum showed a ratio of [M+H] + /[M+H] + +2/[M+H] + +4 molecular ion peaks of 7:64:29(entry 2). Thus, we observed an incorporation of two  18 O atomsinto the carboxylic acid with a [M+H] + +4 enrichment of 29%.Mass spectra of fexofenadine formed when hydroxyterfena-dine was added as substrate in incubations with  S. platensis  wholecells grown in YM and YMS-culture medium showed a ratio of [M+H] + /[M+H] + +2/[M+H] + +4 molecular ion peaks of 5:84:11and 6:80:14, respectively (entries 3 and 4). Fexofenadine obtainedin incubations performed with aldehyde as substrate, was entirelymono-labeled (entries 5 and 6). Obviously, oxidation of aldehydewas catalyzed by monooxygenase(s).
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