molecules Article Chemoenzymatic Synthesis of Indole-Containing Acyloin Derivatives Saad Alrashdi 1,2, Federica Casolari 1,†, Aziz Alabed 1,†, Kwaku Kyeremeh 3 and Hai Deng 1,* 1 Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, UK 2 College of Science and Arts in Gurayat, Jouf University, King Khaled Road, Aljouf 42421, Saudi Arabia 3 Marine and Plant Research Laboratory of Ghana, Department of Chemistry, University of Ghana, Legon, Accra P.O. Box LG56, Ghana * Correspondence: author: h.deng@abdn.ac.uk † These authors contributed equally to this work. Abstract: Indole-containing acyloins are either key intermediates of many antimicrobial/antiviral natural products or building blocks in the synthesis of biologically active molecules. As such, access to structurally diverse indole-containing acyloins has attracted considerable attention. In this report, we present a pilot study of using biotransformation to provide acyloins that contain various indole substituents. The biotransformation system contains the tryptophan synthase standalone β-subunit variant, Pf TrpB6, generated from directed evolution in the literature; a commercially available L-amino acid oxidase (LAAO); and the thiamine-diphosphate (ThDP)-dependent enzyme NzsH, encoded in the biosynthetic gene cluster (nzs) of the bacterial carbazole alkaloid natural product named neocarazostatin A. The utilization of the first two enzymes, the Pf TrpB variant and LAAO, is designed to provide structurally diverse indole 3-pyruvate derivatives as donor substrates for NzsH-catalysed biotransformation to provide acyloin derivatives. Our results demonstrate that NzsH displays a considerable substrate profile toward donor substrates for production of acyloins with different indole ring systems, suggesting that NzsH could be further explored as a potential biocatalyst via directed evolution to improve the catalytic efficiency in the future. Keywords: indole; acyloin; chemoenzymatic synthesis; thiamine-diphosphate dependent enzymes; tryptophan; indole-3-pyruvate Citation: Alrashdi, S.; Casolari, F.; Alabed, A.; Kyeremeh, K.; Deng, H. Chemoenzymatic Synthesis of Indole-Containing Acyloin 1. Introduction Derivatives. Molecules 2023, 28, 354. https://doi.org/10.3390/ Indole-containing acyloins are key precursor of antimicrobial/antiviral agents, such molecules28010354 as sattazolins 1–5 (Figure 1A) [1–3]. They are also useful intermediates in synthesis of biologically active molecules such as carbazoles, β-carbolines and other indole-containing Academic Editor: Khalid molecules [4]. However, access to structurally diverse indole-containing acyloins has Mohammed Khan presented a synthetic challenge. Received: 25 November 2022 Thiamine diphosphate (ThDP)-dependent enzymes are widely found in the biological Revised: 28 December 2022 systems and have been demonstrated to be involved in diverse biotransformation, including Accepted: 29 December 2022 C–C, C–N, C–S, and C–O bond cleavage and formation [5]. The general mechanism Published: 1 January 2023 underlying all these reactions is that the enzyme’s active site-mediated dissociation of the C2–H proton from the thiazolium ring of ThDP will generate the C2 anion ylid which is covalently bound to the donor substrate (Figure 1B). Subsequent decarboxylation results in the ThDP-bound enolate intermediate with nucleophilic reactivity which readily react with Copyright: © 2023 by the authors. electrophilic acceptors, such as aldehydes, ketones or α-ketoacids to form the α-hydroxyl Licensee MDPI, Basel, Switzerland. ketone (an acyloin) [6]. This article is an open access article In many cases, pyruvate is the donor in the ThDP-dependent reactions. However, distributed under the terms and some of ThDP-dependent enzymes utilise a range of α-ketoacids to initiate carboliga- conditions of the Creative Commons tion reactions. For example, the ThDP dependent enzyme, ScyA, was found to couple Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ p-hydroxyphenylpyruvate 6 as the donor and indole-3-pyruvate 7 as the acceptor to afford 4.0/). the β-ketoacid product 8, the key intermediate during the biosynthesis of scytonemin 9, a Molecules 2023, 28, 354. https://doi.org/10.3390/molecules28010354 https://www.mdpi.com/journal/molecules Molecules 2023, 28, 354 2 of 10 pigment produced by cyanobacteria [7]. During our biosynthetic studies of the bacterial carbazole alkaloid, neocarazostatin A 10 [8–11], we found that the ThDP-dependent en- zyme NzsH catalyses an unusual carboligation between indole-3-pyruvate 7 as the donor and pyruvate as the acceptor to generate an indole-containing acyloin intermediate 11 [9], followed by further modifications to finally decorate 10 [10,11], consistent with a parallel study in the biosynthetic pathway of the bacterial carbazole analogue, carbazomycin [12]. NzsH only accepts 2-oxobutyrate, an analogue of pyruvate, as the acceptor, unlike other ThDP enzymes that normally display a considerable range of aldehyde and ketones [9]. Phylogenetic analysis demonstrated that NzsH formed a distinct clade with other ThDP- dependent enzymes, suggesting that NzsH represents a unique group of ThDP-dependent enzymes that utilises indole-3-pyruvate as the donor for the carboligation reaction [9]. However, the donor substrate plasticity of NzsH has remain elusive. More recently, two NzsH homologues, CbeiHKI805_0381 and Cbei2730, were shown to be responsible to produce the indole-containing acyloin core of antimicrobial/antiviral agents, sattazolins 1-5 and its derivatives, isolated from various strains of Clostridium beijerinckii, a bacterium used for industrial solvent production [13]. Interestingly, the enzymes can accept branched chain α-ketoacids as acceptor substrates and both pyruvate and indole-3-pyruvate as donor substrates [13]. Molecules 2023, 28, 354 3 of 10 Molecules 2023, 28, x FOR PEER REVIEW 2 of 10 FiguFrigeu1re. 1I.n idnodloel-ec-oconnttaaiinniing acyloins generated from ThDP-dependent enzymes. (A). indole-containing antimicrobial/nagntiavciryallo iancsylgoeinn enraattuedralf ropmrodTuchtDs. P(-Bd)e. pean dgeennetriecn zmyemcheasn. ism(A )ofi ndole- contcaaibnoilniggaatinotni mcaitcarloybseiadl /bayn TtihvDirPa-ldaecpyelnodinennt aetnuzryaml perso. d(Cu)c. tTs.he(B k)eay gbeionterraincsmforemchaatinoins mto ogfecnaebraotlei gation catailnydsoelde-bcyonTtahiDniPn-gd eapcyelnodine nint teenrmzyemdieaste. (dCu)rTinhge tkheey biioostyrnatnhsefsoirs moaf tsiocyntotonegmenine r9a.t e(Din).d oTlhee- ckoenyt aining acylboiiontrianntsefromrmeadtiiaotne tdou greinngertahtee ibnidoosylen-ctohnetsaiisnoinfgs cayctyolonienm ininte9rm. (eDdi)aTteh edukreinygb itohter abniossfyonrmtheastiiso noft o gen- eratneeioncdaroalzeo-cstoantitna i1n0i. n(Eg).a Tchyelo biinotirnatnesrfmormedaitaioten dtou greinnegrathtee sbtriuocstyunratlhlye sdiisvoerfsne eaocyclaorianzs oins ttahtiisn st1u0d.y(.E ) The biotransfIonr mmaatnioyn ctaosegse, npeyrarutevsattreu icst utrhael ldyodniovre risne tahcey lTohinDsPin-dtehpisenstduednyt. reactions. However, soHmeer oef, TwheDPre-dpeopretnadepnitl oetnzsytumdeys uotfilinsee wa rtahnrgeee o-ef nαz-kyemtoeacids to initiate carboligation reactions. For example, the ThDP dependent enzyme, ScyAc,o uwpalse dfoubniodt rtao ncsofuoprmle apt-ion to accehsysdirnodxyopleh-ecnoynltpayinruinvgatea c6y alos itnhed deorinvoart iavneds iwnditohles-3tr-upyctruurvaalted 7iv aesr tshitey .acTchepetroera tcot iaofnfomrdi xture incltuhde eβs-kaentoeancgidin pereordeudctt r8y, pthtoe pkheya ninetesrymntehdaiastee βd-usruinbgu tnhiet, baiocsoymntmheesricsi oafl lsycyatvoanielambilne 9L, -aa mino acidpiogmxiednats pero(LdAucAedO b),y acnydanNobzascHte.riTa h[7is]. cDouurpinlge dourre abciotsioynnthisetiinc isttiuadteieds forfo tmhe cboamctemriearl cially avacialarbalezoinled aollkeaoloridin, dnoeloecadrearziovsattaitvine sAw 1it0h [L8-–S1e1r], cwatea lfyosuenddb tyhatht etheen gTihnDeePr-eddepternydpetnotp hane synethnzeytamse tNozpsrHo vciadtealtyrsyeps taonp huannuesudaelr icvaarbtiovleigsa, tfiolnlo bweetwdebeyn aidndoitlieo-3n-poyf rLuAvaAteO 7f oars otxhied ation reacdtoionnosr atondg pivyeruthvaetec oarsr tehsep aocncedpintogr itno dgoenlee-r3a-tpe yarnu ivnadtoelea-ncoanlotagiuniensg. Iancyclouisni ointeorfmNedzsiaHte in the reac1t1i o[9n]s, fifonllaolwlyedal lboyw fuarcthcers smoofdaifniceawtiornasn tgoe fionfailnlyd doeleco-croatnet a1i0n [i1n0g,1a1c],y cloninsidsternitv watiitvhe as . This newplayrailnletlr osdtuudceyd ifnu ntchtei onbiaolsiysendthientidc olpea-t3h-wpayyr uvofa tethaen ablaocgteureiasl sucabrsbtaaznotliea llaynablroogaudee, n the doncoarrbsauzobmstyracitne [r1a2n].g eNzosfHth oenTlyh aDcPce-pdtesp 2e-noxdoebnuttyNraztseH, aenn aznyamloeg,uteh oufs phyorludviantge, paos ttehnet ial to genaecrcaetpetonre, wunalnikteiv oirtahle/r aTnhtiDmPi cernozbyimaleasc ythloati nnsowrmitahllyd idffiesrpelanyt ian dcoonlesirdienrgabslyes treamngse. of aldehyde and ketones [9]. Phylogenetic analysis demonstrated that NzsH formed a 2. Results and Discussion To generate structurally diverse indole-containing acyloin derivatives, indole-3-pyruvate derivatives with structural diversity to evaluate the substrate plasticity of NzsH is key. However, most of indole-3-pyruvate derivatives are not commercially available. In nature, Molecules 2023, 28, 354 4 of 10 indole-3-pyruvate is directly descended from L-tryptophane via deamination reactions. There has been a considerable interest in the application of tryptophane synthase to gener- ate structurally diverse tryptophane derivatives due to its importance as a key building block for many bioactive molecules [14]. Tryptophan synthase is a heterodimeric complex consisting of two subunits, TrpA (α-subunit) catalysing the cleavage of indole glycerol phosphate to indole, and TrpB (β-subunit) mediating the coupling between indole and L-Ser to provide L-Tryptophan using pyridoxal phosphate (PLP) as cofactors (Supplemen- tary Scheme S1A). The activities of both subunits are required for wild type tryptophan synthases, although only the reactivity of TrpB, coupling L-Ser and indole is necessary and desirable for L-tryptophan synthesis. Recent studies reported an engineered β-subunit of tryptophan synthase (Pf TrpB) from Pyrococcus furiosus as standalone function that restore the catalytic efficiency and surpass the activity of the native complex (Supplementary Scheme S1B) [15–17]. One variant (Pf TrpB6) with six amino acid site mutations displayed the best kinetics towards L-Ser and indole and a wide range of indole derivatives. To this end, we synthesized the gene coding Pf TrpB6 for overexpression in E. coli. Pf TrpB6 was ex- pressed as an N-terminal pHis6 recombinant protein and was purified to near homogenesis by Ni-NTA chromatography, giving estimated molecular weight of ~45 kDa as previously reported (Supplementary Figure S1A). Incubation of Pf TrpB6 with indole, L-Ser and PLP resulted in the accumulation of L-tryptophan 13 as evidenced in our LC-MS and tandem MS analyses (Figure 2 and Supplementary Figure S2). To further in situ generate indole-3-pyruvate from L-tryptophan, we decided to use commercially available L-amino acid oxidase (LAAO) from snake venom (Sigma Aldrich catalogue number: A5147) because this enzyme displays broad substrate tolerance toward amino acids and does not require any additional cofactors (Supplementary Scheme S1C). It also can efficiently shift the reaction equilibrium during the coupled reaction as observed in our previous report when the fluorination enzyme, FlA, from Streptomyces cattleya, was investigated for a chlorination reaction [18]. Addition of LAAO into the aforementioned enzyme system led to the formation of indole-3-pyruvate 14 as evidenced in our MS and tandem MS analysis (Figure 2 and Supplementary Figure S3). We next overexpressed the synthetic construct containing the gene nzsH in E coli BL-21 (DE3). NzsH was expressed as an N-terminal pHis6 recombinant protein and was purified to near homogenesis by Ni-NTA chromatography, giving estimated molecular weight of ~64 kDa as previously reported [9] (Supplementary Figure S1B). Inclusion of NzsH into the coupled reaction of Pf TrpB6 and LAAO together with TPP and Mg2+ resulted in the formation of the indole- containing acyloin scaffold. However, this molecule is not stable and is subjected to facile decarboxylation in the LC-MS analysis [9]. We used NaBH4 to reduce the β-ketone of the molecule to generate two diastereomeric diols, 15 and 15′, as evidenced in our LC-MS and tandem MS analyses (Figure 2 and Supplementary Figure S4). Molecules 2023, 28, x FOR PEER REVIEW 4 of 10 aforementioned enzyme system led to the formation of indole-3-pyruvate 14 as evidenced in our MS and tandem MS analysis (Figure 2 and Supplementary Figure S3). We next overexpressed the synthetic construct containing the gene nzsH in E coli BL-21 (DE3). NzsH was expressed as an N-terminal pHis6 recombinant protein and was purified to near homogenesis by Ni-NTA chromatography, giving estimated molecular weight of ~64 kDa as previously reported [9] (Supplementary Figure S1B). Inclusion of NzsH into the coupled reaction of PfTrpB6 and LAAO together with TPP and Mg2+ resulted in the formation of the indole-containing acyloin scaffold. However, this molecule is not stable and is subjected to facile decarboxylation in the LC-MS analysis [9]. We used NaBH4 to reduce the β-ketone of the molecule to generate two diastereomeric diols, 15 and 15′, as Molecules 2023, 28, 354 evidenced in our LC-MS and tandem MS analyses (Figure 2 and Supplementary F5igouf r1e0 S4). 2 4 min Figure 2. tThhee rereaacctitoionn sscchheemmee oof ftthhee sstteepp--wiissee ccoouupplleedd rreeaaccttiioonnss lleeaaddiinngg tthhee fforrmaattiioonn off indolle-- containing aaccyylloiinss.. ((AA))t. htehex terxatcrtaecdteido niocnh rochmroatmogatroagprhayphoyf Lo-tfr yLp-troypphtaonphcanta lcyasteadlybsyedt hbeye ntghie- neneegriendeePrfeTdr pPBfT6.rp(BB)6.t h(Be)e. xtthraec etexdtriaocntecdh rioomn acthorgormapahtoygorafpinhdyo loef- 3i-npdyorluev-3a-tpeycrautvalaytsee cdabtaylyasceodu bpyle da rceoaucptiloedn orefatchteioenn gofin teheer ee 6dngPinf TerepreBd6 aPnfTdrpLB-A AanOd. L(C-A) AthOe .e (xCtr)a. cthteed eixotnracchterdom ioanto cghrraopmhaytoogf rraepdhuyc eodf irneddoulcee-dco nitnadinoilneg-coacnytaloininindge riavcaytliovienc adtaelryivseadtivbey actahtarelye-seendz ybmye rae atchtiroene-oefntzhyemeen grineaeectrieodnP foTfr ptBh6e, engineered PfTrpB6, L-AAO and NzsH, followed by NaBH4 reduction. L-AAO and NzsH, followed by NaBH4 reduction. EEnnccoouurraaggeedd bbyy tthhee rreessuullttss ooff tthhiiss tthhrreeee--eennzzyymmee ccoouupplliinngg ssyysstteemm,, wwee tthheenn aaccqquuiirreedd ffoouurr ccoommmmeerrcciiaallllyy aavvaaiillaabbllee iinnddoollee ddeerriivvaattiivveess iinncclluuddiinngg 44--fflluuoorrooiinnddoollee 1166,, 55--fflluuoorrooiinnddoollee 1177,, 44--bbrroommooiinnddoollee 1188 aanndd 66--hhyyddrrooxxyyll--iinnddoollee 1199 ((TTaabbllee 11)).. OOuurr LLCC--MMSS aanndd ttaannddeemm MMSS aannaallyyssiiss ddeemmoonnssttrraatteedd tthhaatt aallll ooff tthheessee iinnddoollee ddeerriivvaattiivveess ccaann bbee eefffificciieennttllyy ttrraannssffoorrmmeedd iinnttoo tthhee ccoorrrreessppoonnddiinngg iinnddoollee--33--ppyyrruuvvaattee ddeerriivvaattiivveess,, 2266––2299 aass weellll aass tthhee ccoorrrreessppoonnddiinngg ddiioollss,, 3311––3355,, ((TTaabbllee 11)),, iinn tthhee ccoorrrreessppoonnddiinngg ccoouupplleedd cchheemooeennzzyymaattiiccs syysstteemss.. We also monitored the biotransformation of the formation of fluorinated indole- containing acyloins in 19F NMR. As shown in Figure 3A,E, the reaction was initiated by addition of 4-fluoroindole 16 (−122.8 ppm) and 5-fluoroindole 17 (−125.2 ppm), respec- tively, in the Pf TrpB6 -mediated systems to provide 4-fluoro-tryptophan 21 (−124.3 ppm) and 5-fluoro-tryptophan 22 (−125.4 ppm), respectively, in 19FNMR spectrum (Figure 3B,F). When LAAO was added to the reactions, 4-fluoro-indole-3-pyruvate 22 (−124.3 ppm) and 5-fluoroindole-3-pyruvate 27 (−124.8 ppm), respectively, appeared (Figure 3C,G). Fi- nally, after inclusion of NzsH, the corresponding 4-fluoro-acyloin 31 (−124.4 ppm) and 5-fluoro-acyloin 32 (−125.6 ppm) was formed (Figure 3D,H), respectively. Next, we acquired three commercially available indole derivatives, 2-methyl-indole 36, indoline 37, and indazole 38 that contain modified indole rings (Figure 4). Consistent with the previous report [15–17], the biotransformation catalysed by Pf TrpB6 gave the corresponding tryptophan derivatives, 39–41, respectively. In all three cases, addition of LAAO provided the corresponding indole-pyruvate derivatives, 42–44, respectively, further demonstrating the considerable substrate promiscuity of LAAO. To our surprise, these indole-pyruvate derivatives can be further utilized as the donor substrates in the NzsH-mediated enzymatic reaction to provide the final indole-containing acyloins, 45–47, respectively, as observed in our LC-MS and tandem MS analyses of the corresponding reduced forms (Supplementary Figure S20–S28). Taken together, our studies demonstrated that NzsH, unlike its activities towards acceptor substrates, displays considerable substrate tolerances toward its donor substrates. However, further studies are required to improve Molecules 2023, 28, 354 6 of 10 the reactivities of NzsH enzyme toward the unnatural indole-3-pyruvate derivatives. This can be achieved via either identification of new NzsH homologues through comparative genomics or directed evolution to provide an engineered NzsH with the aim of finding a MoMlecoulMelecsou l2le0csu2 23le0,s 2M2 2M38 0,o, l2xe83c F,,u xO2le8 sFR, 2x0 Folecules O2 P0RE 2 O3, 22E3P,RE 2 E 8RPR,E x VER FREIOE VRWRIE PVWEIE WR R EVIEsuWi table biocatalyst with better kinetics in order to efficiently generate struMo lMecoulleecsu 2le0s2 30, 238, 2x8 F, Ox RFO PE E8P,R x E RFREO VRIE EPVWEIE RW R EVIEW 5 o5fc to1u 5f0 r1oa0fl l1y0 d5i voef r1s0e Mo lecMuleosl 2023, 28, x FOR P 5 of 10 M oMl ecoul elecsu 2leec0su2 2l3e0,s 2 2M2380,,o 2 2lx3e8 c,F, u 2xOl8 eFR,s O x 2P 0RF EOER REVIEW 5 o5f o15f0 1o0f 10 E2 EP3R,E 2 E PR8RE,E xEVR RFEIE OVRWRIEE VPWIEE EWR REVIiEnWd ole-containing acyloin derivatives. 5 o5f o15f0 1o0f 10 5 of 10 MoMlecoulelecsu l2e0sM2 230o, 2l2e3c8,u, 2lxe8 sF, 2xO0 FR2O3 P,R E2 E8P,RE xE RFREO RVREI EPVWEIE WR REVIEW 5 o5f o1f0 10 5 of 10 MoM leoclM uelceousll e2ecs0u 2le30s,2 M2308,o ,2l 3ex8c, , Fu 2xOl8 eF,sR O x2 P0RFE2O P3ER,ER 2 EP 8RE, E xERV RFEIO VERWRIEE VP W I EE EWR REVIEW 5 o5f o15f0 1o0f 10 5 of 10 TMao bTlecu laebT ll1aee.sb 2S1l0et. r2 Su31t,cT. Tr 2tS8t, rxTable 1. Straauucbbcrtelue sFl cr Oet11ous. R .f r S Soei Pnttsf Err d u E uionoc Rfldt REVIEWcteuuion rrdldeeess ol d erivativesT aabnlde r1e.laStterdu citnudroesleo-cfointdaoinleindge prirvoadtuivcetss agnenderrea tures of indole r d diooveeffra r iiitvnvnivadadtetooisivvll eeae snsdd daan rrnriiddvev la aarrtteteiielvvladaeett sseei dnda dn niinonldderro-oeecllleoeaa-n-ttcceetooddanni ntiitannainididnngooii nlnlpeegg--r cc oppodrnnruoottdcaadtiuiusnnc ciigtnntssegg ng gepprnrnaooeetddreraudauttc ceefttdrds lto aefgmtrdeeo dnmfterhior neatm dhtseo tdte lhee-c ostnetpa-iwniinseg cporuopdlue5c dto sfr eg1ae0cn teiorant.e d fromTableTAT a1dab.bd lSlieett ir 1 s gfreonmer athteed sp s tfft-errewopopmmi--swwe i ttishchseoee u ccspootteueluepppd--llw weerddeiiss a rrce etcacia upled reaction. Addition o1u.Anf. c SNtSdouttdfrzr sNieHcsiczto tu osuntHrofr eogei stfnseo tdNooghfofze ltisreinh Hn wed roiteo tolwrhlegie v ie Ttdadthete iDrTerviirhvPev wsaD aa ttaPniitvnv dhaeed nsMTst 2 hd rh+aa gee nDMnl2ad+sdP tpgt e e2arrdp+eoen pl -vldaiwarnit oteMdievsdedoeigd d li2eic+n- ocduoopnlletea-dcinoirnnetgaac inptiironongd. upAcrtodsd dguietcintosen rgaoetefndeN rfazrtsoeHmd tftorhogeme tsh tteehpre- wstiietshpe- TwcohiuDsepP lceoadnu dprelaeMdctg i oocrocnteui2+tnia.opo .c nlnte.i. do n r.e action. ATAaddTbAddlaideitbtiA diol1oeidn.tn id S1oo oit.nf r SuNotAcrfTzt usNudacHbrdzteuli sstteHroi eg1 snft.e o tSoighnftfe erd Niturnoh cwzldetesruoi H tdlrhe provided the TaTblaeb l1e w e tsiTrodt ihhgoev eDrfaT ittvihPinvaD edateriPno svw dleaa isn nMt hddaeg TMrr2ihe+vpg Dlra2re+Pto ielp vda2rti+no de siv enai d Mni noetdghld e2e or net-+ e hlpdpceleorar -nt oroctlchrtevvoaed-niirc isdd cnrtpoiaeoinonisddrdtnprg aiettdo nhihslpnigpeenird- ongoccpigoo dnrid rrnguoprritd ceeniartnosusigspnpldc eigiotoun-snencl cdgenodgdt -onseicipl nnrtoegraggn-eoetic rnetdiioanndiuetnidd nerctgfoodatir nstllioa e engfcmgd--ri yccnoea oolnfmcgtornnhye ioa ttnlermataoca hiiydistn netleteioid hrnndspii etnggve-fe w r apsdoittvcc-iemiswyvyarpeiltel i -vooitswcvhiai,eonn et 3iusi c 1dvd,ee cs,o u1p– the corresponding indole-contai ing acyloin derivativesspo, – 3tu3l3erre11p5iid–v–v l-3( e3aawTr5d5tte iaii l (v3avsebTr5cdeela t ses a(icb,,r oTc o1el33ntae)uai11,. bo cp1––lnt)33lei,e.o55 d1 n()T., Aasd eviit (Table 1), able 1), as eviden.tif ic S1eNena.tTn drs zS cauo oseitbfcnHrfdv t luN Nuei o cidt rznuozt1eesu srg.nsoH Hre LuSceot e Ctstrhtfrod o e-Luogi MgrnicCfen twdtS-iu thMnooh iraeutdlenheSrrrso d walTdLwoe ntheCi fiatdr dtDh-ni Menv tPd TraTad eiSnhatvhom idnDavalDedne tePM iPmds vd M a eSa atnMsn grcn dinoddnavS rd nM aa pMrareltderneyimgsog vlasp2rave+ eleoMt yispselpdn a srd(SaredetoSo n edsiavuivnd n(iptig S dhadipruenleoiylndp dledcesda pmoo et-ttohlclersheelor edeemn(-en -Sc ctsccioteuapnooannrpondiryrtntrpntoaae eiaFldrislnseineyipnipgmg-in oicn Foungpeongirg nrdei donutpta iiaSanddrcnrr5iegyouogy– ldclS iioSeiFnntu5ni1s-ignd–ccg d9 oStgo)uodsp 1nel rleer9netgero-a)-e ic dcSivrnono5uaein–ttcnrteiSttaavgads1tie in e9ansgfd)ic,rien yno 3ngflg1mreo – oaria 3nacmtc5yht yde le(ltdoeTo hir sainefintbvr e ldospadetmet-eei1rwrvp i)iv,te-ivhwsasaae,st ti i 3sievcsv1evotee –iuscs3dp,op, 5 e-3u3lw n1e(1pTc–di–les3a e3de5rbd5 e l ic( ea(nTroT ce1auotaa)ibpub,oc llrltneei oL.d1 1 ( )T,r eaabcleti o1n),. aAaTssd ae ATadebvsavdili tbiTdeaid ovlesai1et inbe.id n oc1lvoSece.iefnt d d rSd1Nocu etei. afArTni ncz dsnSuNtcs a duo tcHeiobrznurtvdusle iirte Hoctrdsi oi nL t uoeLg1u os Ctrno.Ceorf c -eL touSg-oMhiesfMCtrnefd er tdSoiL-uNr hSnMi foCcna we dzaltinrS- eusnoiMd Htwlraduhe nSt roi estatT dloar Lhnehog dTfDe 2+ 2+ ehtiPhmnD da PrMno wdlaSen i Mtaddhne gaTr2il+hvyg pDsretoPpisvv ra(ioSdnsvu deiapd nMp etdldhge emrt ehcpeleoanr rtocteroavedris ydrpie neoFsddnigodotuhlnenred-e gcci o Snoin5ngr–rtd aieSnois1nldp9eio-)n clneod -ncpitnoangoindt iainuinndcgtio snal gecg- yeaclncoyneinrltoaa ditned irdni evgfrra oiatvmcivayte litosvh,i ene3s 1,ds –t3ee31r5–iv -3(waT5t aii(svbTeela escb ,1 l3eu),1 p1–)3, 5 nC ) (,r.T- eMacStiaonnd. AdAdditdioitnio onAf odN 2+ led re aabcltei o1n),. aTAsa daeAbvdsld iieadtd viseo1i itend.ni voceS oienntddfr c oeuN einfacAnd zctsN ude fddz Nis tiHinzos notHo uo grtf eo LNtghCeze-tsrMhH ewS rti oawthgie nCvt i ertadTaih-tn vehM tt tidmarTDeavinSterhnvei P mdMvDdsaw eante PaSiimMsnmdvtn hadae dSt M nns MaTt aadndrhaSnlnSe dg ynD M aldra2esdel+nPenemgyt lpmae sar2sr dl+tel(Myn oeyplS Mssadvdis ruSen(teio SSep sMddisavu dnpa e(no(igSp dndlSlaieupoanu elmt-ypllddhpcyespoe rep-msetocnl helsceovtes eoma-(mni cS(rdrciStoernuyeoaeunin prdrsFnpttrytpp aigeaitp gnrloihFsr elnyupgpyneiemig r m odFnFcepuoeniigo iegnrdnSgd rnoeug5tpuri da–ScrruSo5teys1c– d St9SFgus5)1i ec– g9gntSus)e1 rnrg9eae)e tS rnea5edt–er Sadf1rt o9fdrm) o fmrtho emth s et hespete -pwst-eiwspei- swceo iscueop uclepodlue pdrel eardec atircoetnaioc. nti.o n. Additi n os oifenHzrd uvNes iro iHst dziu oLoos ergtCnHnf oeL - cgit oMCtnehefod e-tSgNMhr o i aenzlwtSnersh o idHawedtunh r teir d atwr oThLni gtvh idCaTteaDhneh-ttMh mPdiDTve eh SaePrMmsn D wa ad SnaPMni ndt aMdahSdn nt Mg aTdr2lnh+eyg MaplDs2al+ery tPgposes2 rvda+(e o Sipns dvui dr(neiopS dd Mvupaeo iptldgleheap 2met-l+lhdyc epceoes mornt enhocrtsetoraev ane( rriicStsdrynaope eirFrsnodyrpineg otFd sluhnpeipigrendroe uocgi nenSrdo dei5grun itr– tSen raaiSe5srngr1 p–yy dS9iSo 5nFo)F1n –dlii9egdSgo)-1iu cln9erorge-)en c SiotSna55ndi––ntoSaSii1ln1ne99g-i)cn .aogcn yatlacoyiinlnoi nidnge rdaicevyrailtvoivaintei svd,e 3sr1,i –v33a15t–i 3v(T5e as(,bT 3lae1b –1l3e), 5 1 (),T able 1), as aesv iedveindceeandscI n einvdI inoidnduleI eonr udlcLeereCo drLl- iMCievn rdI-a SiMnovte auidraSvnriot ev daiLlaeCt di-vMeerSsi v aantdiv taeTns dryempt oMpTShr yapnat ldoypesrehisav (naSt udivpeeprsliev mateiInvnte d cts generated from svn t edasn t danemdTe rmMyT SpMr ytaoSpn patolhnypaslhneysa sd en(Ses u dr(iSevupraiplvtpimavlteimsv easnr tyIa nrFydiIg noFudilgreoeu- l3anrdSieesrn-eog5so- yp d3p– lSl iyeeFSo-n5p--r1iln–dIc3geu9ynSod--u)v1crnpdli renou9atyeo-a)ngvt c rlSiet eoanuia5 -ntiv–3nedtSaa- gio1pnti lne9agey)ic- ) n acy Addition of NzIsnHdI ntodlIgenoe ltdehoe edrlreie vw rIdaniievttdhiravoi tvTeilveahs teDdisvPe e rasinv daT trMiTyvgprey2Tst+op rpptyroohppvathoindpTa ednhrdey a drtpnhietv eroda icpevtoirhavritarveienvass tepdisove nerIdsinv idInangotdi lIvienoe-ld3seo- -pl3leey---pcIr3onuy-ndrpvtuoyaviltrneua-i tnv3eg-a p taeyc rryu gocltoyv nhailtencoa y isdnltoee idnirpnieg-v rw daiveticisrayveitlvei ovcsai,oetn is3uv ,d1p e3–els13er, –i5d3v3 1(a5rT–te i3(aavT5bcea lt(sebiT,o l1a3en)b1 .,1 A –l)e3, 51 )(,T able 1), as aesv eaidvs eiednvecineddcee naidncs e ieondvu oiirdnu IL ernoC uLc-rCeMo L-lMeS Ci nad-SM n eoadruSni r tvd aL antCtadin-v etMdameensS m dM ae nMSmd aS Mnt aaTSnlry dayslenypemast leo (yMsSps u(ehSps au p(npSle apudmllpyeepesmres (Suppleme tary Figure S5–S19) ulovAi aancAAytt deelcc oeAyAyrilinlcvocoy yaidinltnloeio v dridieninAAeves r,dradcc i3ivtyecyloin derivayev1i rallrva–ooitivit3e vtviei5svnn avas e t ( tedd iTissvv eae eerbrsisilvv e aa1tt)ii,vv eess as evidenced inI noduInor ldlLeeoC DdlIndol- e d eMe rdiS ivee aa rIaIirntn nvt iivd ivddav etotoeasilvlsatinve e ed ddse esem rivTa r iM vaS rtt Tyiaivvrnpryeeyatpsoslp yt potsoheppsah h(naSna TuTdnrDpre yypdrepiplrevetitrmvoaoiavtpTryptophan deripve itavhnai nlhivtveetaimeanasvnsrt teeya indsvr F yteaIi rsgnFri yuivdIg rnaFouedtil IIrgiSenneovu5- ddleS3–reose5o-S-p l–13lSeS951)– 9S)1 9) Indole derivatives Tryptophan derivttaaianvrty eid vsFe edirgsieu vdrIrainevt rdiIdSavino5vteeid–lravseSiot ev-1il3sv ea9y-- -)etp3r d3iIsIuy-p-v - nnyP e vpredrdyusa yiorov vtu r lela u eave t-t va-ie3vta3-e -e te ppDsyy erruruiAvvaacAtytelco A yilncoy idlnoe idrniev rdaicevtyiravliotveiiavstive ruvate Atievc eysloin dAAecrcyiyvlloaoitniin n e ds vD edsee r s ririvivvaataittvievses e-d3-epryivrautvivaetes Acyloin derivatives ives InIdnodIlnoe dldeo edlreei vrdIianevtrdiaivtvoielvasete idsv ersi vTartTiyvrpeyTtspor ytpophptaohnpa Tdnhr eaydrnpei vtrdoiaevptriahivtvaieavnste ds ddiveIrneievdI d rrs nia ieeovvtd rdliaovieevrivt-lerir3e aivsi-vtv iveatie3pavas-ytep tii sv es Iantdivoelse -3rdsIvyu-n eprvdrusyaio vrtluea tv-ei3derivativav -tepesy ruAvacAtyeclA yoilcnoyi ldno eidrneiA vrdiacevytrialivotvieivanste idsv ersi vatives Indole derivatives Tryptophan derivatives Indoedlreiev-r3ai-vtpiavyterivsu evsa te es Acyloin derivatives dedreivrdiaevtriaivtvievaste idsv ersi vatives derivatives COOCOH OCHO OH COOH CO COOH OCHO OH CO OH CO OCOHO CHOOH COOH HOHO HO HO NH2N CHO2NCOHOH2OH NCHO2OH HOHO HO HO NCHON2COHOH2NH NNOCHHO2OH C HO2 OH HOHO HO N HNO N NCHO2NOHH2N 2 NH2 HO HO HN H2N4 2HN4 24NH2HNH224 NH2 NH HHO24NH24 24 H 24 HOHOHO HN2O4 H24 N24HN2H2NNH2 24 NH2 H HN N H HO H H24N2H4 N 2 H 24 N N N N H H24H24 24 H 24N H 24 W We WeWWe aeaeWl l ssaao oell ssmaoaolso m l osmomWWon oimet We also e no iora containing aWcn yomimetlnoo oi aottelonlnossdriiroote teo otddmmhr eairnelisdto o i ntrrmhe e et 1e o dot ddh h nnb o9F ebe titi th itohboob eteitrri o We closon tminoinigto arecydl othinet s hN tbioenMi eoeabbtddtnriiro a soatftntnhhorrsaseareffnm n oobbsrsrafimifmotoirttaroarrmmtntaaiionn aoation of the formation of fluorinated indole- We lso mo it red the biotr or abeatRndirnFoa. s tfntAfNrhosarsefMrnm m sbrRhfamaiot.t iriwaAotmortsnaiao n onsstnoiff fo o otnrrtfhfmmh etoeth aahf ftt efeoiito o hrfrfnmnoemor raoomfamtoffti airoaottmtnthhinio eoea on tnoiff fo o ofnrrfflfmmlu ufofolaaolfurutt riiofionolnrruanniainto n teoaoeradidtfftne e fifdadinlltnu u edidioodnonorrd ldiileioeno-a-alldeetto-ee-ddle -iinnddoollee-- c coonccnototan 19 19 19 ancitintonaainWiinntgaienWgi in an gagecci clo nys ayanoglcclylot o l syiamoWiInli ocnoi nsyomsie i nnl ci oinssaing it lnio isn1anicsro9F tclFe o u i1yd nN F rm9sNlFe oi M 1otNMi9NhnFMd RetsRiM , tN .o. biw R RAnMrAi.oe . As he bs 1sdtR A9idr sFo s.ahs etthnAN hov s rso h asewshMwfn olosnbwnRrwhfpi mo i.oie n tion of t nAw sonin Frtnad i mr Ftia nsFni ani oig s F tgsiF shhunff iufi on gico or g t norFer wuwrfhuigure mfm ip r e3grnnt3eeh afieton otlaAfuh e A ti 3re3d,n ie EAheAor f e i,oE ,nmFF 3on b,3, ,E tiAr ghfa,mo ,turEiAg, ue,tEfiEth ao r m rh, t ahtio rn of fluorinated indole- containing ac tohtfe rt,etern heeihe t oa 3eh3rearonce AA ectffafnsf tai o,,r eacti icoEEcforetrflnt,,minau mi o ofttc owhlahnwatnurma itee t oa ioiowai w s nonrrs wa neaein aitiawnsa sen aocciody iiattfsntfin sei s i i aooiaif diftnitlnli n ten uiuae a d iiowwdtntotem e ieri rb al ndaibdie toysts -ey e b al ba di iedtyntc uny- e ibedi bddtt yi a itne iiy nai gtneddoo bblleeyco a ge y-- - aaddnacdadodtdiaitnd a conctdiaW iiotn didoiatntW diediino o iignetatWinoini loagan ofn o oc dyofa dnlcyi4ttia- lniofi taitnaiin ofsaf egacoin lon ods 4e faa4ognmd- tcl-ofi tsifcly4amfotlua Wi-ulcino fo ylns4inur ln-ioio fnsliirg uonn io1 nofsd9ai F r noci1o 4ndy9lNFi-e lonf1o Ml9lNduiFe1no R M6sNlr .1e oRi6 MA(ni− .n1 sR11Ad6( 92−s.F os 2h1A l(.2Nose8−sh2w 1M .os281pnhw2 6R o. ip8.n w (A p −iFnmps1i p2gi)Fsn2m uhi. gro8aF)eu nwi grd3paneuA np r3idtm5,enEAh - ff,e3)F,o l5 E Atuir-hfg,amfo, elEnutrruh ado,mro teit irn haaoro35dtcenAii-t ooanf irl,colndeEuetnfa oi , oc lfrtw1etfnlhoi u7 o aeif wolur iownraai se idtn itn no ia o4lminuientr-o fooil 17 (−1 5.2 gatriruonoeln ofsaoirdd tioecrno o dy4trdmlh-i eenoftedl ohildu ne ne1bot s i6hilr tboe1 iooen it6 i(orn b−e1a1tdd19i6r(nFo−a2 os t1n2tflNr(he2o.sa−8 e2fMrn1 o m.21s8brpR n srd( aei−onsa1 lic2ietnti5 iaio.21tie 7ad twpe (bpd ia−tnympo es1bd ldp2ei)yon5 m,- bl.ie2ty)-i , a ptepdm b)y, adadrWeditsdeipo itaniclo strnioave dfsem podly4feio t,-c nifilty4lniiuity-nlvn oft elshm d 1i9fi1e9d β-subunit o raedsrdpeiestpcioteincv teilvoyef, l yi4n,- iftnlhu etoh Preof TriiPurl nnyoreifsn inF NF MN6 RM. RA. sA ssh sohw f26 mapfioop. ot8 mpntrwiamAtr( o prt−y)iains)pmn 1oa n 2p stniato)Fsainh2om no fin. opog 8dnFa)dwtrfhu nih m goarnptdea5euhn5fa np - -ierfft3tdnfmsleo5i hluAoy fu-re)F3ofnonm, o5l AEirr utgfr-maoah,ofo, unEtlifatriruina rhmdn,soote odedinht i orahon 3 eo(r5tnledPAo eile-i o enffoa r,o l nETcledufr1etr a1,lo mi7 upoc7oftlr lfhtoBe1 anoui (7reot6 fi(o−in wl)−1onru1rd1f7 (enia2aorwn−2 oat 5sr15eaclo .a(ime2.i2dttn−f2snei 5 1ao di.fiPi1tt2plnn peiu57y a piidp.w rnot2mpioo emrda( cipldi−nt)oeos)pme,1 -cd, labd 2peicon)ty5u- m,elbi .sedt2y)i-f a, u itpnreipddomos bule)ys,-, rceoscranpoedtsneacadrpcoitedinatesntdiicipvtnoitaetiingivinlcon ytegarnaii,cloevc n dyn oiayfseng,d ncpo ll iyoyittfa4entaah li,icc- oniofetiyc4nlinhsi ouln-viP o efn mitoselfn ihguT Plorni myeo nr 1fsdpfTa i,9 r ,T P n PFcoBriii1rnf t ndy9p 4 6 flpT ihFTNe n -loB-ed Bttr1o f rmh9 6 hlMNpp6iFeu 1ne ob-B e M6oRm lsieo tr1a6n sf(6o−r122a.8t eB- mNPdP 1r . Ri-e oiff-6Me(nTmaATmdi− .dt nrrRi1seAi(ppa9a 2d−.FstBB to21e hiAe is6a.l2Ndas d8 oeyst-h-2t wem mMs e.os8dt1ydp1yenhw R6 s6 psmsd odst n.ptiymey iwin onpiesA paasm(mi −)tntmtFs ee1os p s imd i2as)F g otmdm npt h 2 f noiuoss gr .oa d) Fyyr8 opt ho pirenoB4rdic-6n foi-ladlmuelolo yelr6ed1oa 6iivan 1ta6de(i −dloa1(l bse2−y l21s.28t2Le .- m8pa (ps−p m1tipon2) m2o p.8r n )aupt ow ei s ncsvorgr tdaert o3in5eeup nd Av -mfd rm5-afltui nr oinf dfoluleo ri17a te(d−1 in5d.2o lepioem pvrr3feioi5eolinm,Ad ssudEx- 4v f3eioF)e,t-tliEd Aofoud rit4l4g, ho uae- ,pp-Esutfien rfohrnlr4elo ,rudroou r-editof(ev v nhLol-ra3ruiirt5lAeddocAroeo -at-yeerf -rAi,tclte pEoroeu4t4r1-Oayint ,y7o cotfpf tr)p 1tlnlwphtuiyuoft7(oo heopinro−wa onp ap1srrtr( hadnom he2−wisap -oa-5n1a2 ttnsac.lh2nrir12ennts y5ya i2 2in1 271 a(ao.ppit12−1pkntitt 1ea ooe(pi (2dt−w−ppmei41 va1phdh(ba.−e2t2)3ma eys4, - 1bpn d .i2)3ym n ,542 bi.1)ty23,i a(−tpe1pd2m4 b.3)y, prcaepodspramnpdedptesi)ardampt ceaiidiptonesn)tidm icnvpiadonti eenitn )gvl5ci odya t-eafipf ,nnrl5oa vlcyiepdu -dfnye4f,s m o ldol 5-piyoturf4n-ih)el,oi tf- unicelafit-ru4loht snouPirno 19 -revdfy-tifo rlt nh e PFf T6NrMp6 BR 6 .- mAse an4no.3 2 1 a(n−1d2t4h.3e p pppmp)m an) dapTn 5pdh-fmD 5lu-P)f al-rudnood-rpeTt p Purr oio55rleonyrty-f-fnoTtifpdftp,r rnlrl pBdrtuoyuitydp4onhoeilpop n-oepB ap-frrtt mdlhnohoeu o1apt -- paNe2omttl6n hndrh2reP1r z yya e oi2a6f2(sap(pnTd−2in 21H−tnt1 trie 62(1(o2a pds2tpBhie (ahsd6o−y ta- w1esnmd2tyn e52 ess .2dti4yen i(sspa − tFtpe1osi2mg dt5puos ).sr4, r otyper ovser va pd3tp osipendApp dvmrrdmoe,5odE c v-v4)tf,i5i il-dvtdu-fhafleeoulne lu r4yo 4dor-,r-f e irfoilnlaonu5-ucti -1onrtf9irylrFdluoop2−51.245 p.4p pmp)m, r)e, srpeespcteicvteilv 19 19 eNon- o- ttl torMrewr1oyp y7iRpahp1n 1at7d(on −op1 (l2he2−151a .2n(2−5 7 12. 21 (4 p(−.p−3m11p 22)m5,4 .2.)3, ppm), 6 6 o(d− −2epo112 n(h6(l2−2.e−z8a215 1y .281p2 6.p8 pm 9sto ipnh n.24m 25 p2.e4p ( p(fpm−−rpm)1po1 )p2m2,)a m5 r2n.)e.a4),8ds spethn r p edaepps5n cpbmp-dtf5iimlo )) v-scy4u,,fes te)-5,rlrotiyl fuloeiev-y4rlynnfsuas -eo,l,p tfpnu riloiihl1nyronenuedr9Foe, cicdo r Nnt1vitt-9oinriFid5iFtcvvoMildr Nn-oNe1e-yefp9e tdlRFllMpr Meauyy4oN1y tts,o,-Rol7 h RpfieiM1r pnnl wt o es7uso h (pRci 1poa−pn9ate F7Fye1(rshdnc−ou cNN2pato 1ot-5m2r(erntMl i2−u.c1ret2 aMcu5 it1y t2m ( a mR Rr( n.rFp12−u t1 u p5i e 2d1 s1ts(7mg((mo.Fppp2F−2 pupi4em e1i g( bF−yi1 g2u4r.3e a f ne ca pr((ghc.c2−Fe3upm)utt4a1 i,rrpz rg.nu2u)e3eom,u 5 mm 2s.r)12te ,a(( (FFt−ipiin1ggp2suum4rrt.eeo)3, 3p3rBp dpprmdmepis)ptr3m)ie aBpoase)n,nmnpFcadd te)n) i. 5 cvo 5dat-Wfe-n3i frp f5vlleBdlyu-phue4sf, ,oFpl5e-ml oyuifrn-)enrlf,o.)uoc l i-auLtrW-othiotnrAvr re-ytodhryethA opire Plp5ney-tnOft-t od,TpoP fr plioLytrpfunwopThlpAhe opBartta rAhpohnsno 1e pBa O-- 2a 62mnhtP2d r 2 a- fyw2e (mT(n(2−pd−ear 1e2i1(pdsa2d−22 B t5p1 2ia5e(a.2o.−d.d4 8t4-51 a ed m p.ts2pnd4ehyp5ep d2ps.mdm4 2typmiert )sap(m)e,−t,t )pa re 1r)tsecm,deh2 atsrts5i epn)soeop., yes4 dnterrppco se csprtt eta,siop5 ivpcv4-rmteef-ieoiilvlfcsdlu)yly te,nuti oi,rl,o vd so iyre4in ,eonr, ps-l f4ipi y4rln-oue-F,ifdF fnc1viloNlN9nuotudFridl oMov1M3,FB),.F 9-rt oR4r-y- tsfprlpyuteocptrtorhu-atmhnrya 2(npF1 t 2io(g1−pu 1h(r2−ae41n .23 42 .13 (−124.3 eBrs3p,epFBps)erp,3pm. Fec BW )et)sW.)i.,cp FvahtWWhi)enev.cel hen LAAO was6 pm) adnythenWi3pa rl,ve5 BydLcepinLeh-nc,s, Afm l F5Aepli yLunt-)nAse),hAf.so lcaitOeuWLOnrthsAn io PAt evd trh-Owr fhwetuA PoTe5rl eac-ynyfar-Ow TtstfpPs,p ru l r LayuBifatprwnaTospAdadB ra alt-d 19 aAp6lhmsd ydeB- eOdmea 6Pe d de-iewtdfmidvoTda ae triettreatsdopdohs dt Be eiaet tatd6sdh oh( tyr- eedeemstzd yahtrr2 edsces)e2dttyai aeintricoscmsaedttnt t ieatioseostoomc hd,lnttn epoie ss4s-os ,rc-,yt pnr ofo4els4rsvun -tao,-pfie ftocvdl4lrautruioe-iodnfs vl-4er niruiito-n dnoosf4o-gl,-de -ir uipnf oon4a4lordu-ldc-orefifoynloov-lulr3-ldieoeto-doN e-rp-r3eery oi-rl F n3tloRoyeM-elr N17M R(−1 91s -py4p-yrd -p-is3it-3uR,ynpnoye pfr--ivlr pydtdnprpesuoiaupypo cohyvopvttvrtllraearrFeoahuc po n2u-Ntv35emra-.cMu2ptt m eryRFu pr i2 musgpFpuv m ((F−)i,1g 2prdhe-atdnry 2tpo2t o(t−ph1he2 a5rn.e4 a pctp i(om−n1)2s, ,5r .e4s-pflepucmotri)vo, e-rilenysd,p ionelc et-iF3v-NeplMyr, uinv a19tFeuat iegarceutter R specN- tpait2vt-3e 2en2rth2M- a2r ue1p a2ty2 2s(m eRn2y(2(p−.1 −− r 1 ts2T2(−124.3 eu24 r.e3 3rBe3s,FB)e3,p.F cBpW)t,.imF vhW)e).e lyhnWap,3en pLBinhdnmA ,e F Lnt5A)hA .- eLfOaAWl AuP OwofhATr eaOorw5nspi- nafBLwalsdu6dA ao-tadomsAlrde po-Oaedhi3dne -atipdwnoa eyot a2derltose2uhd - tev a(3tos−h ad-y r1petdtse2 hytae 5rrecde2.um 4ta7 irv cto pseoat (napit t−ocsemt1t,nh i2p)2ose4,r,7 n- 19 19 .orfr8 4selve (u,-sa −ifppod4lc1pure-te2foimc 4ol4t-ur.nii-)8ovnf,s -lerd,uir olnoey4p-dsl-ir,menpfoil-ndlu-3)et ,c-or -pty3lriFevoe-pN-prse-3tiuyloM-nypverd,uRcay hotvr aiesluvapnp tev-p2 e32la2ec y-1t a2tp,e r( r2(y −ue−a 2r1dm1 u1((o2−121−hv p4 p((u2p 2−F22 p v4 1e 1 i1444(( e(ag.(h2−3.t3c4a 1e o.n 232 4(e.a 2 2cFF−..−3a33ut4tii 411reg g .r .2u332 euu4d24m 2m22..31b3 (i( (Fn−−F−i11ai1gg22t2uiu44o4r..r.3n3ee3 pppppppppm3mppBp)pm),p)m F paap)a)))nn . m n aaddaWdn))n n5d ad5hda-5np3 op-f 5en -fBdlpf5-fpnlu5dfl,u mP-5F-u lo fufoLf-l)or5l)ufT.)ru Ao rl- oraurfaoW-pAliotnrnirunroBn-odhOrdyodtid6oir epn ro5n y-ona5dtw-ldpolnef r-ioenlfoyLta-pdul-lo3ldspuA3eheo-p L-o-tpa-raA3poh3lrnoyd-epya-oOp- rA-pd2nhtri3uynr uy2e Oa-y2vwrd prvn(u2pau−oya vt2tv1(ltsreo−oale2ua op1- ta5t(2v3ewt2he2−d.7h- a45 71aped 2te .2 2pned4y(7 7(5− p rdr −2p g.12eum(412 (p2ea7−124. re 3B,F). When L AO was ad ed to the r−v 2t pn4(cm e)o14a−,t.(ape 2.8ti1−r)8ctroem4, e21hat n.rps 8 i52e8tp)2oepsi .p4, o74sp ,ne prr. mpp 8nmesec4 pp(e ,tsa-)p−o m i)cppmf,cv41, lfmt tpu ), -ier2i)(rvfceml,o)4a le yte,usn.rz r isr8r)lp,vesap ye,e i es,e)nse,s ppirc lpp4ic nyetp1net-ei9es,ifcmvF ccpv1ilot9NentutieF)ililv v,evlcy1MN ye9-tree,rF3il, oMllRevy Ny-yasape, i,pR, sn Mpl yipaypa ednprspe,Repcup oe1ctpap 9valeitsrFeevrpercape-aNuetapt3derredlm .3 r-ceMuye ep tdad(,m dr( (yFRre uFF aeri (i(mgi(spduiFgFgFvpup viuiu iage(gr(gaerFrFtecuuauteieit ievrg r rg eeeuu2 d s mrr2r2, eee s( ( ( (F(tF−F−aii1ig1rggt2uuiu4nrrr.eeg3e 3p3CpB3p33,m,GCBFp p) C)),33,m3, FG..GBC p Ca)WF),. m ),n ,.F G.iG ) aWdn)hFF) 5. o-rion-d 3C,G). Fin.n) a eia.ih dan5lW-n3lFpf3ne- ay ClfiBnph5du o5ro-f-lturyrptoin n3lpfFlraCypiolL, nlnl, ,uml -,emy GGAyFafaLona, lf,)l) )uAlfr lt. y, c.. at yeo a LoeOaAWfFrF,firAnrtm n tiioe a OeadninhdAwr f iinmrfna a t n e to cO eawiel5lcndi5elnl rnsrle-uyy -r o auf c p -fcLws,i dhn l,sail3s luieinuA ud- oaoaa- acapsof3lclsfdsnAl n llid etyru -oe - 23tiyuoprsnruiyi 2 -n opvi ro(dnua−yfco iorn v t 1rloelN2aue f5tv-s z2e3.iNas47 o-tH p2nze(7y s ,p− Ho2r1mt(u7fh2−, v )e41N,t(a . 2h−8rtcz41e os.s2 p8Hrpc42 rpo.e7e,p8m rcs p trtp(h)piem−,voes p1ne p)r2m,cledo 4yosirn).,prn8, edi r esgneripc nes1t4eps9giiF-cvpnmo oftNeel4dnilu)lvce-Mod,yft -elli,i3rlue vnRy-oe-aeopg ,3s-sp lra yp-yaop4cre,p- yueycacfpaaltvrtcoiuprureayvieuoapvntledeomreal eo yia3tn2d e-,r(1F a2 Fe aci3 2di(gy(pgF12− −ul ip1(o1gr((2Fre2−−ieue4nai411gr .r .224eu3344 d r1..e43 (−F1ig2 2u44.r.43e p33CpBp3mp,,GFCpp))3mpp), m.G.aC p p)WnF)),mm .G adi han)F) )5n ae.dia -anldp nFfl35nal y piduC-Ldln5,flm o,Aly-5 aGaufr5-l,)lfAo lf )u-tyal.-feruO oa,lnfor Futoc d a-eoiyriawf rnoir5ltc aoeoc-yiadfilrnislnuln cy oocuiy sadl,il3no iuneldo2ocar- sdl oln33fii(eunteo2-- eeo-O dapods3rinoaden iyf - vfo dipwdrt oNanou yeNofcai t dvr zlosoztaeNua sh sf-btHvstat 3eHzoelihadN- eos, pet, rHdtn 2ez(te hytaeh7 sra,hre zdo eH2ecu etaa(7f thri)ctceoeNitoai n roczdctsrtnhso,ei Hosresl4r,e pn ,-er f4osdsetl,-npuha f4dlcoeoru-it inrvfincoldaourg-niotir ninsr-4vr,gied -one sf4o-sdlp4-iul,nf-eoowlfo-dlnl3eruhodo-p3liri-ecrnoa-yh-opc-g3ri-yyu w-naplr4vcdoueyyaiofrvrnlteluoea itosv3-ne23ur1a 2-ob t2p3 e-(s21(a−y e− c12rq1((yu22−−u2l4 v11o4(.a224−.in3n4t4 1te . .2l3 4y342 1.2a3 c ((c−−e11s22s44e..d43 ppppppmmppp)p)m papa))n m nadad)n)n 5 dada-5p3bn pnf 5-Cplydpf5du-lm,f -uN Go5lfu5lor-))u)zf -o.r alfs o-rulnHFauorinodiocn--nrdya imdro5nac lo-olyd5eafliliyldn-eocnuof-,dyli i3oeau3nloa-tro2o 3pfleo3i etr-d(ny−- e2-oep−a sd a o13nrr3(i iycu2-nf −o 2yp rt5iov1dn uoNl(y.2fao o6− vc r5tzoi tl1lNueanp.husf26 tv - Hpezs5e3 32Npai sm .2-,7o6rtH p2zesaHct r 1r3c(e−ya1 l(co2− pp 2i5c.6tli51iun .op26 sn3p5 ipo2..m6 p ne On(t() 7my)p− (hm− , w −u 2ro11wpeot)1(u)r722fhm ,f −v,i 5 coec1taN)(. t o onh62 − ht erwcres ez41rpor, se.2 a e8rp2cH4c4srs o7mo. -p,8fr rsoolr )p(rtpure−npep nmms1sdppr2p)eimo,4nod -.ng)nri8 ,(de dFsd4irpiinpe-nogpfgsegllumpeuc rt-4eo4e)i3-c,vr- - f3tfopelirluDv-uleyyaeso,rc,Hrplu ryooaev)l,-,c-po a atraiptcicenpvyeys pelapl32olore1yeia ,ndr ( e− a3d1(p1F2 p i4(ge(F.− a−2wv rs14(ea 2a−f.st8o4s1eu .r2 f8lmpo4t2 spr.7pe8md dp ( 2inc (t i−3v11e2 l4y(−..3 a4ui11g r22eu4d4 r..4 e4 Figure 3Cp3), GCpam,)nG. d))F . ai 5nnF-adifnll lu5ay-ol,fl ya,f taefrt eirn cinlucsluiosnio nof oNf zNsHzs,H tm,h a w5eNst.)h 6 a fzwc esp oso rafHprcmosrom,r e fremsotr)dph e ehpr m−)wew (omed,(eFdp1 Fn )aai 2imr oe,n(cidgs(sc e4FgdFong ouris).ffiunri8 ,se ogopr(g rt 4rsgFerruerueep-mm ieacsrf 3srg4ep3tslpetpeDeiu-ecpD mov fd3dt3or,line,HDneu D)rvHc((l,td odFytF e,)3h,Hi)i-,Hrirl,iDv, naa gnyge or)ertcga)eu,,sgu-,eH, ly Npas y rsarpl4cep4e)p,eopzy -,es -ces efi33pfaprlctnloDon ing 4-fluoro-acyplacHDieupte uerv teiiosaoipecv ,3,nocepvHHrtdr1eetlr ni eeoiiy o lav3 ))sv3ldcy ,-,(,r(-1yep 1 tea −.Fa reri l1eesponding 4-flu ro-a yloin . a l3lcev y(cdi(ay(p1Fg s2s−ye.y . ppli4up1 ll(ogsoy.e2Fere4iui.ec4n t.i4− 1(2−cani41 gort r .2i e4un vv33 4 drs1 ee1l y(..4ei ldy ((−eF. − ri1ga2bu4lr.y4e p3pCp3m,CpG)3m, )GaC. )n), F.Gad inFn )5 124.4 pppmp)m a)n . dia -np lgFfp35lalup-fomlur)oadn 5dopyiCln-ol, mf, 5yalGdau-l,) flf)s rl yat.-aurou ea,fon Firbtcna-e dirsyanfidrot lntc 5rao-eyaic-alinrlfltlnce lyuoecuy-i l,is3nt lunoioo2-caor sp li3foien(untyo2−r-e sr an1a3ro(iucn2−o 2fvyi5o1 c n(lNae.2f− 6oc ,t5o1 lezNisp.un2f 6su pz5 sH2 3Ngpis.m76o2g,Hpz ne)p( p and 5-f uoro-acyloin 32 ((mts−, s− h wH1to1t)ei m 2h an2f, w 5es5cg 4t)N. .oha .w8 6f6tcosrezh opr pr s afapermcHopsptrs po mrept fe,mrh osdor)tp)i r)m eehn sw(wo ,sded Ftpnah eraii(odgcendrsFoensui g ifnpe(rgdro Fr-geu4ireein c -mgrn3sf4tgeuplDiz-eu v fyro3d,4loeHmDnu- l r(fy3d,o)FelHD,,uir- i ncorag)a,oegHc-,urp asuy rorp)ce4pl-,yo es-al rf3eplicacelondDturyeses form d (Figu 3Dii cv p ,lno,s3 de HoHteyr 1iiolv c)y(t,eF i.rl viyege.s ulpyre.e c tively. 3C,G). Finally, afoltueror -riaonc-cyallcuoysilinoo in3n2 o 3(2f− 1(2−51.265 p.6p pmp)m w) aws afos rfmoremde (dF i(gFuigreu r3eD 3,DH,)H, r)e, srpesepctei 3)csnv ,-1tt( eeair− 3vlemc1(y1es−y2. pl1 lh4y(o2e−.o.i4c 1nl t.d24 ivs43 e.1p4l o y(t.−e 1n2t4ia.4l pppmppm) pa)mn adn) d5a-na pf5sdlp-uf am5lou-rm)fo laure-onaoc-dnrayo sc5l-yo-aflicflonuyni l3noeo2r w3io n(2-− ab31(c−i2oy 1 5(Ntl2−.ro65a1zi .n2sp6H5 s p3p.fm62,op rtp(m)h− pw1e)ma 2 watc5si)oo.a 6wfrsno r pafertopmsso rpfmepood)rne mo wd(ddF iea(inudsFggc iuf(eg oFr4urei-nmgr f3edluuDe or3doe,DlH re (3o,-F)HDc-,iao gr),cHn,ue yrstr)laep,o si erni3pneciDetsn icp3,vgHt1ievca )lyt(,ec i−ryl.v y1ele2o.s lp4iyn.e.4 sc ,tiivmelpyo. rtant ppm) and 5-pflrueocurors-aocrysloofinm 3a2n (y−1b2io5l.o6g picpamll)y wrealse vfoarnmt medo l(eFciguulerse. 3D,H), respectively. MoleculeMs o2l0ec2u3l,es2 820, 2335,4 28, x FOR PEER REVIEW 6 of 10 7 of 10 Figure 3. 13F NMR results for the coupled reaction leading to the formation of 4-fluoroindole (left Figucorelu3m.n1)3 aFnNd M5-fRluroersouinldtsolfeo (rritghhet ccooulupmlend) croenatcatiinoinngl eaacydlioningst. o(At)h. e4-ffoluromroaitniodnoleo f(−41-2fl2u.8o prpomin)d aosl e (left colusmtarnti)nagn dma5t-eflriuaol.r o(Bin).d o4-lfelu(orirgoh-Lt-tcroylputmopnh)anc o(n−t1a2i4n.3in gppamcy) loyinelsd.ed(A f)ro4m-fl utohero iPnfTdroplBe6 (−an1d2 24.8- ppm) as sftluaortrioningdmolea steyrsitaeml. . (C(B).) 4-4f-luflouroorinod-Lol-etr-3y-pptyorupvhaatne (−(−12152.04 p.3pmp)p ombt)aiynieedl dwehdenf rLoAmAOth weasP afdTdrpedB 6 and 4-flutoo rtohien dreoalcetiosny.s t(eDm).. 4(-fClu)o4ro-fl-aucoylrooiinn d(−o1l2e4-.34- ppyprmu)v agenerated by three-enzyme system. (E). 5-fluoroindole (−125.2 ppm) as starting material. (F). 5-fluotreo-(L−-t1r2y5p.t0opphpamn ()−1o2b5t.a4 ipnpemd) wyihelednedL fAroAmO was addtehde PtfoTrtphBe6 arnedac 4t-ifoluno.ro(iDnd)o4le- flsyusotermo-.a (cGy)l. o5i-nflu(o−ro1i2n4d.o4lep-3p-pmy)rugveantee r(−a1t2e4d.8b pypmth) roebet-aeinnezdy mwheens ystem. (E) 5L-AflAuoOr owiansd aodlede(d− t1o2 t5h.e2 rpepacmtio) na.s (sHta).r 5t-inflguomroa-taecryilaoli.n( F(−)152-5fl.6u poprom-)L g-ternyepratotepdh bayn t(h−e 1th2r5e.e4-epnpzmym) ye ielded fromsytshteemP.Af TzrapinBd6oalensd ar4e- flsturuocrtouirnadlloy lbeiosiyssotsetmeri.c( cGhe)m5-iflcaul ostrrouicntduroelse -t3o -ipnydroulevsa inte b(i−ol1og2i4c.a8l pmpamter)iaolbs tained whe[1n9L]. AThAeOy dwisapslaayd ddisetdinctot pthhyesrioecahcetmioinca. l( pHr)op5e-flrtuieosr, oaq-auceyoluosi nso(lu−b1il2it5y. 6anpdp tmot)alg peonlearr asuterdfacbey atrheae three- due to the presence of extra nitrogen atom within the six membered ring in comparison to indoles. enzAyms seucshy,s atezamin.Adozlaei-ncodnotaleinsianrge bsutirludicntug rbalollcyksb hioaivseo asttterraicctecdh memedicicailnsatl rcuhcetmuirsetss ttoo dienrdivoel eas niunmbbieorl ogical matoefr ipahlsar[m19a]c.oTlohgeiycadl iasgpelnatys d[i2s0t]i.n Actmpohnygs itohcehse,m 7i-caazlapinrdoople rt2i0e sh, asq uapeopueasresdo luinb iali tpyleatnhdortao toafl polar surfbaicoeloagriecaalldyu aecttiovet hmeoplerceusleens c[2e1o],f seuxchtr aasn piotrteongte anntaitcoamncewr nitahtiunratlh perosidxumctse,m vabreiorelidnsr [i2n2g] aindc othmeipr arison to insdynotlheest.icA dsesruivcaht,ivaezsa, imndeoriloel-icnos n[2ta3i]n. iTnog thbiusi lednidn,g wbel otecsktsedh acovme matetrracicatlelyd amveadilaicbilne a7l-cahzaeimndisotlse t2o0 derive as the starting material in our biotransformation system. Again, the formation of aza-tryptophan 25, a nuamzabinedroolfe-p3h-payrrmuvacaotel o3g0i caanlda agzeanintsdo[2le0-]c.oAntmaionningg tahceysleo,in7 -3a5z awinerdeo olbes2e0rvheads inap thpee acroerdreisnpoanpdlientgh ora of biolPofgTircpaBll6y, PafcTtrivpBe6m + oLlAecAuOle san[2d1 P],fTsurpcBh6 a+s LpAoAteOn t+aNnztsiHca nsycsetremnast, ureraspl epcrtoivdeulyc,t sa,sv eavriidoelnincesd[ 2i2n] oaunrd their syntLhCe-tMicSd aenrdiv MatSiv teasn,dmemer iaonlainlyss[e2s3 (]T.aTbolet h1 iasnedn dSu, pwpeletmesetnetdarcyo Fmigmuerer cSia9,lSly14a.Sv1a9i)l.a Tbaleke7n-a tzoagienthdeorl,e 20 as the NstzasrHti ndgispmlaayte croianlsiidneroaubrleb siuobtrsatrnastefo tromleraatniocen tsoywsaterdm m. Aodgiafiiend, bthenezfeonrem raintgiosn ofo ifnadzoale-t mryopietotipesh an 25, azaiwnhdeonl ein-3d-opley-r3u-pvyartuev3a0tea dnedrivaaztaivineds owleer-ec ounsetda iansi substratesMolecules 2023, 28, x FOR PEER REVIEW ng acyloin . 35 were observed in the c7o orrf e1s0p onding Pf TrpB6,NPefxTtr, p 6 6 wBe a+cqLuAirAedO thanrede Pcof TmrpmBerc+iaLllAy AavOail+aNblzes iHndsoylset edmersi,vraetisvpeesc,t 2iv-melye,thaysl-eivniddoelne ced in our3L6C, i-nMdSolainned 3M7,S atnadn dinedmazaonlael y38se tsh(aTta cbolnet1aiann md oSduipfipelde minednotlaer yrinFgigsu (rFeigsuSr9e, S41).4 CaonndsiSs1te9n).t Taken teonggewitnhiteeher ,rteNhdez sNpHrzesdvHiiso puwlsai tyrhec pothonerst i ad[i1em5ra– ob17fle ]f,si ntuhdbeis ntbrgia otate rsatuonlisetfarobarnlmec aebttioocwna atcaraldtyasmlty osweddiitfi hbe ydb ebPtetfeTnrrz pekBnin6e egrtiaincvsge s intoh fei ndole morodiceotrir ertsoes wepfhofiencndieintgdl yot rlgeye-p3nt-eoprpyahrtuea nvs atdrtuercditveuarritavilvalyetis vd, ei3vs9ew–r4se1er, e irnuedspoedleec-atcisovsneultyba.is ntIrnian tagel sla .tchyrleoein c adseersi,v adtidvietsio. n of LAAO provided the corresponding indole-pyruvate derivatives, 42–44, respectively, further demonstrating the considerable substrate promiscuity of LAAO. To ouHOr surprise, COOH COOH COOH these indole-pyruvate derivatives can be further utilized as the donor substrates in the NzsH-mediated enzymatic reNaHc2tion to provide the finalO indole-containing acyloiOnHs, 45–47, respeNctively, as observeNd in our LC-MS and tanNdem MS analyses of theNH H H H corresponding 2-methylindole 36 45reduced forms (Supp3le9mentary Figure S20–4S228). Tak1.eNzsHpnyru vatetogether, our studies demonstratedPfT rptBh6 at NzsH, unlike itsL- AAaOctivities towards acMcge 2p+ tor substrateHsO, displays N COOH COOH ThDP N COOH consNHiderable substrate tolerances toward its dono Nr substrates. However, further studies iandroeli nree3q7uired LPt-oSerLP improve4 t0he rNeHa2ctivities of NzsH4 3enzymO e tow2.aNradBH 4the unna4t6ural OiHndole-3- pyruvate derivatives. This can be achieved via either identification of new NzsH hom N NNologues through coNmparative genomics or N directed evolution to N pHOrovide an H COOH N NCOOH COOH indazole 38 41 NH 472 44 O OH FFiigguurree 44.. CCoouuplpelde drearcetaiocntiso yniselydiienlgd 2in-mge2th-myl-eitnhdyoll-ei,n inddooleli,nien adnodl iinndeaaznodle-icnodnatazionlien-gc aocnytlaoiinnisn 4g5-acyloins 47, respectively, starting from three commercially available indole derivatives 36-38. 45–47, respectively, starting from three commercially available indole derivatives 36–38. In conclusion, we developed a coupled biotransformation system including a genetically modified β-subunit of tryptophan synthase (PfTrpB6) from Pyrococcus furiosus, and a commercially available L-amino acid oxidase (LAAO) from snake venom and the ThDP-dependent NzsH enzyme from the biosynthetic pathway of neocarazostatins to access structurally diverse (aza)indole-containing acyloin derivatives. The combination of PfTrpB6 and LAAO allowed generation of (aza)indole-3-pyruvate derivatives, starting from commercially available (aza)indole derivatives, which were subsequently accessed by NzsH-mediated reaction. Our results demonstrated that NzsH displays considerably good substrate tolerance, suggesting that this three-enzyme coupled system holds potential as a means of new biotransformation to produce indole-containing acyloins, important precursors of many biologically relevant molecules. 3. Methods and Materials 3.1. General chemicals, reagents, and analytical methods. All starting materials and reagents were bought from commercial sources and used as received. All biochemical reactions apart from where noted were carried out in triplicate. Before every set of measurements, triplicate control reactions were performed to ensure that the assay were functioning correctly. All flash column chromatography was carried out using silica purchased from Sigma Aldrich using the solvent system noted. 19F NMR spectra were recorded at 298 K on Bruker Avance III 400 using CFCl3 as an external reference. Chemical shifts are reported in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). Enzymatic assays were analyzed on a Bruker MaXis II ESI-Q-TOF-MS connected to an Agilent 1290 Infinity II UHPLC fitted with a Phenomenex Kinetex XB-C18 (2.6 μM, 100 × 2.1 mm) column. The column was eluted with a linear gradient of 5–100% MeCN containing 0.1% formic acid over 15 min. The mass spectrometer was operated in positive ion mode with a scan range of 200–3000 m/z. Source conditions were: end plate offset at −500 V; capillary at −4500 V; nebulizer gas (N2) at 4.0 bar; dry gas (N2) at 9.0 L min−1; dry temperature at 200 °C. Ion transfer conditions were: ion funnel RF at 400 Vpp; multiple RF at 200 Vpp; quadrupole low mass at 200 m/z; collision energy at 8.0 eV; collision RF at 2000 Vpp; transfer time at 110.0 μs; pre-pulse storage time at 10.0 μs. MS data were analysed using Bruker DataAnalysis or Thermo Xcalibur. 3.2. General methods of protein expression and purification. Molecules 2023, 28, 354 8 of 10 3. Methods and Materials 3.1. General Chemicals, Reagents, and Analytical Methods All starting materials and reagents were bought from commercial sources and used as received. All biochemical reactions apart from where noted were carried out in triplicate. Before every set of measurements, triplicate control reactions were performed to ensure that the assay were functioning correctly. All flash column chromatography was carried out using silica purchased from Sigma Aldrich using the solvent system noted. 19F NMR spectra were recorded at 298 K on Bruker Avance III 400 using CFCl3 as an external reference. Chemical shifts are reported in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). Enzymatic assays were analyzed on a Bruker MaXis II ESI-Q-TOF-MS connected to an Agilent 1290 Infinity II UHPLC fitted with a Phenomenex Kinetex XB-C18 (2.6 µM, 100 × 2.1 mm) column. The column was eluted with a linear gradient of 5–100% MeCN containing 0.1% formic acid over 15 min. The mass spectrometer was operated in positive ion mode with a scan range of 200–3000 m/z. Source conditions were: end plate offset at −500 V; capillary at −4500 V; nebulizer gas (N2) at 4.0 bar; dry gas (N2) at 9.0 L min−1; dry temperature at 200 ◦C. Ion transfer conditions were: ion funnel RF at 400 Vpp; multiple RF at 200 Vpp; quadrupole low mass at 200 m/z; collision energy at 8.0 eV; collision RF at 2000 Vpp; transfer time at 110.0 µs; pre-pulse storage time at 10.0 µs. MS data were analysed using Bruker DataAnalysis or Thermo Xcalibur. 3.2. General Methods of Protein Expression and Purification The synthetic constructs encoding Pf TrpB6 and NzsH proteins were purchased from Genscript Ltd. and were individually transformed into E. coli BL21 (DE3). Single colonies from each transformation were grown overnight in LB media (5 mL) containing kanamycin (50 µg/mL) and chloramphenicol (25 µg/mL). The overnight culture was transferred to fresh LB medium (500 mL) supplemented with kanamycin (50 µg/mL) and cultivated at 37 ◦C until the cell density reached an OD600 of 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce protein expression. Cells were grown for 16–20 h at 16 ◦C and then harvested by centrifugation at 4 ◦C. The cells pellets were resuspended in ice-cold lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), and further disrupted by Ultrasonic Homogenizer JY92-IIN. Then, the supernatant of cell debris was loaded onto Ni-NTA-affinity column. Bound proteins were eluted with the same Tris-HCl buffer containing different concentrations of imidazole. The desired elution fractions were combined and concentrated using a Centrifugal Filter Unit (Millipore). The final yields of Pf TrpB6 and NzsH were estimated to be 10 mg/100 mL culture and 5 mg/100 mL culture, respectively. 3.3. Biochemical Reactions A sample of Pf TrpB6 (20 µM) was incubated with indole or indole derivatives (1 mM), L-Ser (1 mM) and PLP (1 mM) in phosphate buffer (50 mM, pH 7.5) to the final volume of 100 µL at 28 ◦C for 3 h and then quenched by addition of 100 µL of acetonitrile. The mixture was centrifuged at 13,000 rpm for 10 min to remove protein precipitates. For the production of indole-3-pyruvate derivatives, a sample of Pf TrpB6 (20 µM) was incubated with indole or indole derivatives (1 mM), L-Ser (1 mM) and PLP (1 mM) in phosphate buffer (50 mM, pH 7.5) to a final volume of 50 µL at 28 ◦C for 3 h. To this mixture were added LAAO (20 µM) to a final volume of 100 µL at 28 ◦C for another 1 h. The reaction mix was then quenched by addition of 100 µL of acetonitrile. The mixture was centrifuged at 13,000 rpm for 10 min to remove protein precipitates. For the production of indole-containing acyloin derivatives, a sample of Pf TrpB6 (20 µM) was incubated with indole or indole derivatives (1 mM), L-Ser (1 mM) and PLP (1 mM) in phosphate buffer (50 mM, pH 7.5) to a final volume of 100 µL at 28 ◦C for 3 h. To this mixture were added LAAO (20 µM) to a volume of 100 µL at 28 ◦C for another 1 h. Inclusion of NzsH (25 µM), TPP (1 mM) and Mg2+ (1 mM) into the mixture was perform to Molecules 2023, 28, 354 9 of 10 the final volume of 100 µL at 28 ◦C for 3 h. Because of the instability of acyloins, overdose NaBH4 was treated to the three-enzyme systems. Finally, the reaction mix was quenched by addition of 100 µL of acetonitrile. The mixture was centrifuged at 13,000 rpm for 10 min to remove protein precipitates. All the supernatants from the above reaction systems were then analyzed by Bruker MaXis II QTOF in tandem with an Agilent 1290 Infinity UHPLC. Samples were separated on a Phenomenex Kinetex XB-C18 (2.6 µM, 100 × 2.1 mm) column with a mobile phase of 5% ACN + 0.1% formic acid to 100% ACN + 0.1% formic acid in 15 min. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28010354/s1, Figure S1 SDS Page electrophoresis analysis of purifies PfTrpB6(A) and NzsH(B); Figure S2. LC-MS (A) and MS/MS data (B) of L-tryptophan 13.;Figure S3. LC-MS (A) and MS/MS data (B) of indole-3-pyruvate 14. Figure S4. LC-MS (A) and MS/MS data(B) of indole-containing acyloin 15.Figure S5. LC-MS (A) and MS/MS data (B) of 4-fluoro- indole-tryptophan 21. Figure S6. LC-MS (A) and MS/MS data (B) of 5-fluoro-indole-tryptophan 22. Figure S7. LC-MS (A) and MS/MS data (B) of 4-bromo-indole-tryptophan 23. Figure S8. LC-MS (A) and MS/MS data (B) of 6-hydroxyl-tryptophan 24.Figure S9. LC-MS (A) and MS/MS data (B) of 7-azaindole-tryptophan 25.Figure S10. LC-MS (A) and MS/MS data (B) of 4-fluoro-3-indole pyruvate 26; Figure S11. LC-MS (A) and MS/MS data (B) of 5-fluoro-3-indole pyruvate 27. Figure S12. LC-MS (A) and MS/MS data (B) of 4-bromo-3-indole pyruvate 28. Figure S13. LC-MS (A) and MS/MS data (B) of 6-hydroxyl-3-indole pyruvate 29. Figure S14. LC-MS (A) and MS/MS data (B) of 7-azaindole- 3-pyruvate 30. Figure S15. LC-MS (A) and MS/MS data (B) of 4-fluoro-indole-containing acyloin 31. Figure S16. LC-MS (A) and MS/MS data (B) of 5-fluoro-indole-containing acyloin 32.Figure S17. LC-MS (A) and MS/MS data (B) of 4-bromo-indole-containing acyloin 33; Figure S18. LC-MS (A) and MS/MS data (B) of 6-hydroxyl-indole-containing acyloin 34.Figure S19. LC-MS (A) and MS/MS data (B) of 7-azaindole-containing-acyloin 35.Figure S20. LC-MS (A) and MS/MS data (B) of 2-methyl-indole-tryptophan 39.Figure S21. LC-MS (A) and MS/MS data (B) of indoline-tryptophan 40. Figure S22. LC-MS (A) and MS/MS data (B) of indazole-tryptophan 41. Figure S23. LC-MS (A) and MS/MS data (B) of 2-methyl-indole-3-pyruvate 42.Figure S24. LC-MS (A) and MS/MS data (B) of indoline-3-pyruvate 43.Figure S25. LC-MS (A) and MS/MS data (B) of indazole-3-pyruvate 44.Figure S26. LC-MS (A) and MS/MS data (B) of 2-methyl-indole-containing-acyloin 45.Figure S27. LC-MS (A) and MS/MS data (B) of indoline-containing-acyloin 46.Figure S28. LC-MS (A) and MS/MS data (B) of indazole-containing-acyloin 47. Author Contributions: Conceptualization, H.D.; formal analysis, S.A., F.C. and A.A.; investigation, S.A.; writing—review and editing, H.D. and K.K.; All authors have read and agreed to the published version of the manuscript. Funding: The research was funded by the College of Science and Arts, Jouf University, King Khaled Road, Kingdom of Saudi Arabia, and the Royal Embassy of Saudi Arabia Cultural Bureau in the UK for the PhD scholarship (S.A.); the PhD studentship funded by IBioIC (F.C.), thank the financial supports of Leverhulme Trust-Royal Society Africa award (AA090088) and the jointly funded UK Medical Research Council UK Department for International Development (MRC/DFID) Concordat agreement African Research Leaders Award (MR/S00520X/1) (H.D. and K.K.). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. 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