P450-Mediated Coupling of Indole Fragments To Forge Communesin

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P450-Mediated Coupling of Indole Fragments to Forge Communesin and Unnatural Isomers Hsiao-Ching Lin, Travis C. McMahon, Ashay Patel, Michael Corsello, Adam Simon, Wei Xu, Muxun Zhao, K. N. Houk, Neil K. Garg, and Yi Tang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b01413 • Publication Date (Web): 10 Mar 2016 Downloaded from http://pubs.acs.org on March 10, 2016

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Journal of the American Chemical Society

P P450-Me ediated Couplin C g of Indo ole Fragments to o Forge Commu unesin a and Unn natural Issomers H Hsiao-Ching Lin,1,3 Traviss C. McMahon,2 Ashay Pattel,2 Michael Corsello,2 Addam Simon,2 W Wei Xu,1 Muuxun 2 Z Zhao,1 K. N. Houk, H Neil K. K Garg,2 Yi Tang T 1,2 1

Department of Chemical and Biomolecular B En ngineering, 2Deppartment of Chhemistry and Bioochemistry, Uniiversity of Califoornia, Los 3 A Angeles, CA 900095, USA; Instiitute of Biologiccal Chemistry, Academia A Sinicaa, Taipei 115, Taaiwan

SSupporting Infoormation Placehholder A ABSTRACT: Diimeric indole alkkaloids are structturally diverse naatuural products th hat have attracteed significant atttention from th he ssynthetic and biiosynthetic com mmunities. Heree we describe th he ccharacterization of a P450 monooxygenase CnsC C from Penicilliuum tthat catalyzes th he heterodimericc coupling betw ween two differeent indole moieties, tryptamine and aurantioclavine,, to construct viccinnal quaternary stereocenters and yield the heptaccyclic communessin sscaffold. We show, using bioch hemical characteerization, substraate aanalogs, and com mputational meth hods, that CnsC not only catalyzzes tthe C3-C3 carbo on-carbon bond d formation, butt also controls th he rregioselectivities of the pair of suubsequent aminaal bond formatioons tto yield the comm munesin core. Th he use of -N-meethyltryptamine an nd ttryptophol in plaace to tryptaminee led to the enzyymatic synthesis of isocommunesin co ompounds, which h have not been issolated to date.

Dimeric indolee alkaloids are a large subset off plant and funggal nnatural productss that have a wide w range of biiological activitiees. E Examples includee the tryptamine-derived calycan nthaceous alkaloiids ssuch as (+)-calyycanthine, epipolythiodiketopipperazines such as cchaetocin, and th he perophoramiidine and comm munesin alkaloid ds.1 A defining featurre of these natural products is th he presence of viccinnal quaternary carbon c stereocen nters, which are particularly inttrigguing from both synthetic and biiosynthetic persppectives.2 Syntheetic approaches to the vicinal quateernary stereocen nters of these natturral products havve been develop ped and includee Heck cyclizatioon m methodology, biis(alkylation) ch hemistry, pericycclic reactions, an nd rradical couplingss.2 The biosyn nthetic pathwayss to these naturral pproducts, howevver, have been much m less well-studied and the ene zzymes responsib ble for vicinal quaternary q stereoocenter formatioon hhave not been reevealed. Identifyying the biocatallysts that form th he C C3-C3’ linkage is an importantt objective towaards better undeersstanding of indo ole dimer biosyn nthesis and how w Nature can effficciently construct complex scaffold ds. We have speciffically targeted th he biosynthesis of o the communessin aalkaloids, given their t remarkable structures and the t vast interest in tthese compound ds from the synth hetic communityy.3 The core struuctture of the comm munesins, as rep presented by thee simplest isolated m member, commuunesin K (3), consists of seveen interconnected rrings, two aminall linkages, and fo our contiguous stereocenters. Ottheer family membeers include comm munesins A (1) and B (2), which ppresent additional synthetic challlenges (Figure 1 and S1). Severral ttotal syntheses have h been reportted.4 With regarrd to communessin

biosyntthesis, Stoltz et aal proposed that the communesinn core could be deriived from the hhetero-dimerizattion of tryptamiine (5) and aurantiooclavine (6), thrrough either dirrect C3-C3’ indoole coupling followeed by aminal bonnd formation orr via an exo inveerse electron demandd Diels-Alder reaction between 6 and the quinoone methide imine dderivative of 5.22b We recently identified the bbiosynthetic pathway ay of 1 and 2 in Penicillium exppansum and verified that indeed, 5 and 6 are biosyynthetic precursors to the core sstructure 4.5 We fouund that a cytochhrome P450 monnooxygenase, CnnsC, may be involveed in the oxidativve coupling betw ween 5 and 6, ass deletion of Pe-cnsC C in P. expansuum abolished coommunesin prodduction and insteadd accumulated 6. Sequence anallysis showed thaat CnsC displays loow sequence hoomology to knoown P450s in thhe database (Figuree S2). While CnnsC must play a key role in the selective dimerizattion and generattion of the core sstructure, numerrous mechanistic qquestions remainn to be answered:: 1) are there othher enzymes involveed in the biosyntthesis of 4, whicch requires the fformation of three b onds (C3-C3’, C C2-N1’ and C2’-N10) between 5 and 6? 2) How arre the sequencess and regioselecttivities of the boond-forming steps coontrolled to arrivve at 4; and 3) w what is the substraate specificity of CnnsC towards twoo different indolee building blockss in order to achievee the hetero-couppling?

Figure 11. Formation of ccommunesin coree. A P450 (Pe-C CnsC from P. expansuum) has been impplicated in the couupling of 5 and 6 into the core 4. Com mmunesin K (3) iss the simplest, stabble communesin isolated, and is formeed upon N-methyylation of 4 by thee methyltransferasse Pe-CnsE.

To innvestigate the acttivity of CnsC, tthe intron-free cn cnsC and the P450 reedox partner cyytochrome P4500 oxidoreductasee (Pe-CPR) from P P. expansum werre cloned underr the ADH2 proomoter and transforrmed into Sacchharomyces cerevvisiae strain BJ55464-NpgA.6 Howevver, western blootting analysis of the membraane fraction showedd very weak exppression of the P P450, and no nnew product

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ccould be detecteed when 5 and 6 were supplemeented to the yeaast cculture. To find a CnsC alternattive that may be better b expressed in yyeast, we sequen nced the genomee of Penicillium m rivulum, which is aalso a reported producer p of com mmunesin.3d Thee P. rivulum com mm munesin cluster is highly similar to that of the cns cn cluster in P. exe ppansum (Table S2), S including th he P450 of interest Pr-CnsC, which sshows 84% sequence homology to Pe-CnsC and d a different mem mbbrane anchoring region(Figures S3). S Upon feed ding 5 and 6 to th he yyeast strain exprressing Pr-CnsC C and Pe-CPR, we w observed coonssumption of both h substrates and production of trace t amounts off 4 (m/z 385 [M+H H]+). The low signal s of 4 is duue to its instabiliity ffrom the two lab bile aminal linkages, which wass also observed in kknockout studiess with P. expansuum.5 To further confirm c formatioon oof the core struccture, we coexpreessed the N-metthyltransferase PeP C CnsE (Figure 1) with Pr-CnsC an nd Pe-CPR. Metabolite analysis of tthe yeast culture supplied with 5 and 6 showed clear c accumulatioon oof a new producct that matches the t retention tim me and m/z of th he N N1-methylated communesin c K (3) (Figure S4A A, ii). In contraast, uuntransformed yeast (Figure S4A A, i) did not accumulate 3. Microsomal fraactions containin ng the overexpreessed Pr-CnsC an nd P Pe-CPR were puurified from three-day cultures of the yeast expreesssion strain. From m the in vitro asssay in which 0.55 mM 5, 0.5 mM M6 aand 2 mM NADPH were incubaated with microssomes (20 mg/m mL ttotal protein con ncentration), a cllear product thatt corresponds too 4 eemerged (Figuree S4B, ii), albeit with low converrsion. Introducin ng P Pe-CnsE purified from Escheric ichia coli and 2 mM S-adenosyylm methionine (SAM M) led to the formation f of thee expected 3, fullly cconsistent with th he yeast biotranssformation resultts (Figure S4B, iii). C Collectively, thesse results demon nstrate that the P450 CnsC alonee is ssufficient to oxid datively couple th he two different indole-containin ng ssubstrates togeth her to yield the heptacyclic h coree of communesin ns. IIn vitro assays using u either 5 orr 6 alone did noot lead to the foorm mation of homod dimeric productss, or any other deetectable produccts. C CnsC is thereforee a rare example of a P450 that catalyzes C-C bon nd fformation between two different substrates.7. It is particularly inttrigguing that CnsC C strategically un nifies 5 and 6 with w controlled foorm mation of four new stereocenterss and three new bonds. The moost liikely order of bond formation is i the C3-C3’ cooupling between n5 aand 6, followed by b the two aminaal bonds. It is alsso possible that th he P P450 active site orients the two substrates to promote aminal foorm mation first, follo owed by the more difficult C3-C33’ bond formation. To gain insightt into the sequen nce of the bond forming steps an nd ssubstrate specificcities of CnsC, we w tested enzymee activities towarrds ddifferent analogs of 5 and 6 usingg the in vitro reaaction. When N1N m methyl tryptamin ne or N1-methyll aurantioclavinee was used in plaace oof 5 or 6, respecttively, no producct formation wass detected, indicaating the N1 hydro ogens of both suubstrates are essential for the coouppling reaction (F Figure S7). In contrast, c when N10-methyl N trypttam mine 7 was used d, we observed the formation of o two oxidativeely ccoupled productts 8 and 9 with h the same masss as 3 (m/z 3999 [[M+H]+) at a reelative ratio of 5:1 (Figure 2A). To elucidate th he sstructures of 8 an nd 9, a 4L culturre of the yeast sttrain co-expressin ng P Pr-CnsC and Pe--CPR was grown n for three days then concentrated tten-fold and supp plemented with 5.0 5 mg each of 6 and 7. Cultivatioon ffor one additional day afforded sufficient quantiities of 8. The 1H aand 13C NMR spectra s of 8 sho owed characterisstic signals of tw wo aaminals at δ 5.01 (H2’)/δ 84.8 (C C2’) and δ 4.17 (H2)/δ 84.2 (C2) ((Figure S8-S13 and a Table S3), and with other keey HMBC correllattions, the skeleto on of 8 was established. The NOESY N spectruum sshowed the correlations of H2’ to t H8’ (δ 2.25),, H9 (δ 2.65) an nd H H8 (δ 3.42); H22 to H8’ and H9 (δ 2.08); H11 (δ ( 5.26) to H7’ (δ

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6.67); and no correlattion of H9 to H H11 confirming the relative stereochhemistry at the corresponding ccarbon atoms. B Based on the R confiiguration of C11 in 6,8 the absoluute stereochemisstry of 8 was confirm med to be 2’S, 3’S, 3R, 2S, 11R. We were not abble to isolate sufficiennt amount of 9 ffor full structuraal characterizatioon, however, the shifft in retention tiime and mass inncrease are indiccative of the N10’ m methylated form oof 4. Thhe structure of 8 is unexpected aand represents ann alternative way in w which the two inndole-containingg building blockss 6 and 7 can be couppled. Compounnd 8 remains a hheptacyclic alkalooid with the C3-C3’’ linkage and fouur contiguous sttereocenters, buut with both indolinne moieties remaaining intact. In the communesinn configuration, thhe two indole nittrogen atoms andd the two -nitrrogen atoms are eachh paired to form m the two aminal linkages (N1-C22-N1’, N10C2’-N110’). However, inn 8 two different C–N bonds (N N1-C2-N10’ and N11’-C2’-N10) are formed to give the bis(indolinne) “isocommunesiin” scaffold of 8,, which has not bbeen isolated in any natural productts to date. It is ttherefore intriguuing that N-methhylation in 7 can draastically alters thee products formeed by CnsC. Too learn more abbout these subsstituent effects, we coupled other trryptamine analoogs with 6 and m monitored formation of adducts uusing LC/MS annd selective ionn monitoring. SSurprisingly, CnsC ddisplayed significcant substrate tollerance towards analogs of 5; productt formation can be detected from m substrates rannging in size betweenn indole and -N-boc-tryptaminne (Figures S5). Other C3substituuted indoles ssuch as 3-metthyl-indole, -N-dimethyltryptam mine, 3-(2-azidoeethyl)-indole all yyielded productss with retention tim me suggestive off the isocommunnesin structures ((Figures S6S7). W We were able too detect a singlee new product 11 (m/z 386 [M+H]]+) when tryptopphol 10 was usedd as a substrate toogether with 6 (Figuure 2B). Subseqquent feeding of 6 and 10 the yeasst culture (6 mg eachh) led to the isollation of 11 (3 m mg) for structuraal characterization ((Figure S14-S18).

Figure 22. Formation of communesin andd isocommunesinn scaffolds in the pressence of tryptaminne analogs. (A) iin vitro assay usinng 6 and 7 led to the syynthesis of 8 andd 9; (B) assay using 6 and 10 led to the hemiaminal eether containing 111; (C) structurees of 8, 9 (putativee) and 11.


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The 13C NMR spectrum of 11 showed characteristic signals of one aminal at δ 83.0 (C2’), and one hemiaminal at δ 92.5 (C2). The HMBC correlations showed key correlations that confirmed the skeleton of 11 to be also of the isocommunesin scaffold. The absolute stereochemistries of the four contiguous stereocenters of 11 were confirmed to be analogous to 8 (2’S 3’S, 3R, 2S, 11R) based on NOESY data and the known R configuration of precursor 6 at C7. Interestingly, when only 6 was supplemented to the yeast culture expressing Pr-CnsC and Pe-CPR, 11 was also detected in the organic extracts. This is due to 10 being a known metabolite in S. cerevisiae that is involved in quorum sensing.9 We also assayed the activities of CnsC towards N10-methyl analogs of 6, however, no coupled products were detected, suggesting the tolerance is low towards aurantioclavine analogs (Figure S7). The reconstitution of CnsC activities and generation of either communesin or isocommunesin scaffolds provide significant insight into coupling between 5 and 6. The common C3-C3’ bond in 4, 8, and 11 suggests that this coupling step takes place first in the active site of CnsC and staples the heterodimers together. Because C3-C3’ indole coupling is widely observed among alkaloid natural products and only a few mechanistic proposals exist, we computationally examined four possible mechanisms of coupling that can afford 4 (Figure S23). These mechanisms include radical addition, radical cation addition, electrophilic aromatic addition, and radical combination. The radical addition mechanism involves the intermediacy of a tryptaminyl radical formed by CnsC-catalyzed N–H hemolysis, followed by addition to 6. Computations show that this mechanism has a 105-fold intrinsic preference for the formation of a C3-C2’ adduct, which was not observed experimentally (Figure S24). Similarly, reaction of a tryptaminyl radical cation (generated by CnsC-catalyzed single electron transfer) with 6 is predicted to also lead to facile formation of a C3-C2’ product (Figure S26). C– C bond formation via the capture a tryptaminyl cation, the product of the two-electron oxidation of 5, by 6 is predicted to be nonselective (Figure S25). None of these mechanisms are deemed likely given the exclusive C3-C3’ coupling observed here. Further discussion and details of this computational work are provided in the Supporting Information. In contrast, the combination of two indole C3 radicals 12 and 13 to form the vicinal quaternary stereocenters and yield the product 14 must exclusively form the C3-C3 regioproduct (Figure 3). Both radicals can be generated by CnsC-catalyzed abstraction of the indole N1 hydrogen followed by facile migration of the radical to C3 positions. Such one-electron oxidation that initiate at the indole NH has been implicated in the P450-catalyzed dimerization of ditryptophenalanine and chaetocin.10-11 This mechanism is supported by the observation that CnsC is inactive towards either N1methylated indole substrate. From 14 (or 14’), the two 3H-indoles in the two halves of the molecule can be subjected to intramolecular nucleophilic attack from the two amine groups and form the pair of aminal linkages. The regioselectivity of the aminal bond formation determines whether the communesin scaffold (as in 4) or the isocommunesin scaffold (as in 8) is formed. Our results indicate this regioselectivity is strongly influenced by the C3 substituent of 5. Energetics of intermediates and products of the reactions starting from 14 or 14’ were calculated to determine how the additional N-methyl substitution may influence the free energy of aminal C–N bond forming steps (Figure 3). Efforts to locate transition states for these reactions were unsuccessful. We calculated the energies with the Nprotonated analogues of these structures to model the protonation

states of these species in aqueous solution (Figure S28-30), but calculations with neutral species yielded similar results (Figure S27). All intermediates leading to the communesin (4 and 9) and isocommunesin scaffolds (21 and 8) are thermodynamically accessible at room temperature. The formation of either scaffold is irreversible as scaffold formation is highly exergonic (ca. –25 kcal mol–1 for the formation 4 and 21, and ca. –28 kcal mol–1 for the formation of 9 and 8). The only energetically uphill step in Figure 3 is the ring opening of pyrroloindole 15 to 16 required for formation of the communesin scaffold. Assuming the activation free energies of each step shown in Figure 3 are proportional to energies for the formation of the intermediates, we would expect that of the elementary step shown in Figure 3, the conversion of 15 to 16 would occur most slowly. Thus, formation of the isocommunesin scaffold would be more facile nonenzymatically than formation of the communesin scaffold. Given that the reaction of 5 and 6 with CnsC yields only the communesin product 4, both the initial oxidative coupling that forms 14 and the subsequent steps involving C-N bond formations and scission must be enzyme controlled. When the methylated 7 is coupled with 6 to form 14’, CnsC may no longer be able to exert complete control over the orientation of the intermediate of 14’, leading to the nonenzymatic formation of 8 and small amount of 9. Computational analysis also provided explanation to the exclusive formation of the isocommunesin scaffold 11 when 10 was used as a coupling partner to 6, which is due to a prohibitively high energy intermediate in the reaction path required for the formation of the communesin-like product (Figure S30). In conclusion, we have identified a P450 monooxygenase CnsC that catalyzes the C3-C3’ coupling of two different indole substrates to introduce vicinal quaternary stereocenters. The enzyme also controls the regioselectivity of the subsequent aminal bond forming steps. The discovery of such a remarkable P450 further illustrates the biocatalytic power of fungal biosynthetic enzymes in the synthesis of complex molecular scaffolds.

ASSOCIATED CONTENT Supporting Information Experimental details, spectroscopic and computational data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected], [email protected], [email protected]

ACKNOWLEDGMENT This work was supported by the NIH (1DP1GM106413 to Y.T. and 1R56 AI101141 to Y.T. and N.K.G.) and the NSF (CHE-1361104 to K.N.H.) H-C. L. thanks the NSC of Taiwan (102-2917-I-564-008) and M.A.C. thanks the NSF for a graduate fellowship (DGE-1144087). Computational resources were provided by the UCLA Institute for Digital Research and Education (IDRE) and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the NSF (OCI-1053575). These studies were supported by shared instrumentation grants from the NSF (CHE-1048804) and the National Center for Research Resources (S10RR025631). We thank Prof. Frisvad for the P. rivulum strain (IBT 24420).



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F Figure 3. Propossed mechanism of CnsC catalyzed d C3-C3’ couplingg and formation oof both communeesin and isocomm munesin scaffoldss. 14 and 14’ aare the immediate products of thee enzymatic oxidative coupling beetween tryptaminne and aurantiocllavine. Assumingg N-protonation of substrate, inntermediates, and d products, the relative Gibbs free energies e (in kcal mol m -1) of the aminnal bond formingg steps starting from 14 and 14’ are shown.

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TOC Figure 73x36mm (600 x 600 DPI)


P450-Mediated Coupling of Indole Fragments To Forge Communesin

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