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Biological Matching of Chemical Reactivity: Pairing Indole Nucleophilicity with Electrophilic Isoprenoids Christopher T. Walsh* ChEM-H Institute and Department of Chemistry, Stanford University, Stanford, California 94305, United States ABSTRACT: The indole side chain of tryptophan has latent nucleophilic reactivity at both N1 and all six (nonbridgehead) carbons, which is not generally manifested in post-translational reactions of proteins. On the other hand, all seven positions can be prenylated by the primary metabolite Δ2-isopentenyl diphosphate by dimethyallyl transferase (DMATs) family members as initial steps in biosynthetic pathways to bioactive fungal alkaloids including ergots and tremorgens. These are formulated as regioselective capture of isopentenyl allylic cationic transition states by the indole side chain as a nucleophile. The balance of regiospecificity and promiscuity among these indole prenyltransferases continues to raise questions about possible Cope and azaCope rearrangements of nascent products. In addition to these two electron reaction manifolds, there is evidence for one electron reaction manifolds in indole ring biosynthetic functionalization.

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chain of Trp is not sampled in these tens of thousands of common post-translational modifications. On the other hand, there is evidence that indole rings are involved in long-range transfer of electrons via transient radical intermediates in certain enzymes, from iron to Cys-SH side chains, for example in ribonucleotide reductase catalytic cycles,3 indicating that one electron manifolds are accessible. There is a rich area of small molecule fungal biology in which the intrinsic nucleophilicity of the indole moiety of Trp is fully manifested, in the formation of a plethora of indole alkaloids. Not only is the indole nitrogen (N1) alkylated by C5 prenyl groups, but the C5 electrophilic prenyl group can be added to all six of the nonbridgehead (non-ring-fusion carbons) carbons (C2,3,4,5,6,7) of the indole bicyclic framework, singly or iteratively to give, for example, bis- and tris-prenylated indole ring products.15 Notoamide D (Figure 1b) reflects bis prenylation at carbons 2 and 7 of brevianamide F.4 Δ2-IDP, with its trianionic diphosphate tail, is stable in intracellular milieus. But in the active site of many dimethylallyl (prenyl) transferases Δ2-IDP becomes a kinetically accessible source of the isoprenyl cation, perhaps as a discrete intermediate in the ion pair with the nascent inorganic pyrophosphate in some cases or as a transition state in others, from SN1-like C1−OPP heterolytic bond cleavage5 (Figure 2a). This is an allyl cation, with the charge deficiency delocalized over both primary carbon C1 and tertiary carbon C3. The stabilizing charge delocalization both accounts for its kinetic accessibility to biological catalysts and is relevant to capture of cosubstrate nucleophiles both via C1 (“normal” regiochemistry) or C3 (“reverse” regiochemistry) (Figure 2b).6,7 Thus, the isoprenyl group along with the methyl group in S-adenosylmethionine

wo of nature’s primary metabolites, used widely as biosynthetic blocks in parallel spheres of oligomerization activity, are the indole-containing proteinogenic amino acid tryptophan (Trp) and the five carbon biological isoprene Δ2-isopentenyl diphosphate (Figure 1a). There are metabolic intersections combining these building blocks. In addition to its central role as one of the 20 amino acids essential for protein biosynthesis in all organisms, L-Trp is also the starting point for elaboration of thousands of indole alkaloids as conditional metabolites in microbes (e.g., fungi) and plants. The Δ2-IDP along with its isomeric Δ3-IDP are the two building blocks in a set of iterative prenyl chain transfers to generate some 50 000 isoprenoid scaffolds, perhaps the largest known class of natural products.1 Prenylation of other types of cosubstrate nucleophiles also occurs. Although N, O, and S atoms as biological nucleophiles are common, it is the nucleophilic carbon centers on the indole side chain of tryptophan and derivatives that are examined here.

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CHEMICAL CONSIDERATIONS FOR THE REACTIVITY OF INDOLE AND Δ2-IPP The indole side chain of Trp, like free indole itself, is a versatile nucleophile in chemical syntheses and can be functionalized at every position, including by alkylation with carbon electrophiles. Yet, the electron-rich indole ring in L-Trp residues in proteins is very rarely modified post-translationally, suggesting that the intrinsic nucleophilicity of the indole side chain is not often plumbed by the large array of PTM enzyme catalysts acting by two electron manifolds.2 By contrast to side chain alcohols and phenols, of Ser, Thr, and Tyr residues, which can be activated as nucleophiles to undergo phosphorylations and then dephosphorylations, and Lys and Arg side chains that, when deprotonated, are sites of electrophilic methylations and acetylations, the intrinsic nucleophilicity of the indole side © XXXX American Chemical Society

Received: September 8, 2014 Accepted: October 10, 2014

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Figure 1. (a) Structure of L-tryptophan (Trp) and Δ2-isopentenyl diphosphate, also known as dimethyllallyl diphosphate. (b) Notamide D arises from bisprenylation of the Trp−Pro diketopiperazine known as brevianamide F at positions 2 (reverse regiochemistry) and 7 of the indole ring.

Figure 2. (a) SN1-type cleavage of Δ2-isopentenyl diphosphate to the allyl cation inorganic pyrophosphate ion pair. (b) The charge deficiency at both C1 and C3 of the allyl cation leads to capture by cosubstrate nucleophiles both at C1 (deemed “normal” regiochemistry) and at C3 (“reverse” regiochemistry).

Figure 3. (a) Trp-X diketopiperazine metabolites as substrates for prenylation, termed cyclo-Trp-X. Shown are cyclo-Trp-Pro, cyclo Trp-Leu, cyclo Trp-Trp, cyclo Trp-Ala. (b) Formation of brevianamide F (cyclo-Trp-Pro) by intramolecular release from a bimodular NRPS assembly line.

cyclo-Trp-Pro, cyclo-Trp-Leu, -Trp-Trp, -Trp-Ala (Figure 3a)) as starting points for short efficient biosynthetic pathways to alkaloids of substantial architectural complexity and biologic activities. One such dipeptide is the Trp-Pro-diketopiperazine (cyclo-Trp-Pro), a tricyclic framework that presumably arises from intramolecular capture of a Trp-Pro-S-enzyme biosynthetic intermediate via attack of the Trp-NH2 group on the thioester carbonyl on a bimodular nonribosomal peptide synthetase assembly line.12 The released diketopiperazine is known as brevianamide F (Figure 3b) and is the first committed step to a set of architecturally more complex brevianamides, trypostatin and fumitremorgin indole alkaloid scaffolds.13,14 Assays of the purified DMAT proteins for enzymatic activity against Trp and brevianamide F and congeners, largely from systematic studies by Li,8,14,15 reveal that distinct DMATs show regiochemical preferences for indole alkylation with the C5-prenyl moiety of the Δ2-IPP (DMAPP) cosubstrate. The anticipated nucleophilic N1 of the indole side chain of Trp is prenylated by the enzyme CymD from the Salinospora arenicola cyclomarin biosynthetic pathway.16 We will return below to the

comprise the two most widely used carbon electrophiles in metabolism. Prenylation of the indole ring of Trp can be quite pervasive in microbial metabolism while Trp C-methylation is by comparison much rarer, as though there has been a matching of the indole and isopentenyl reactivity manifolds but not the methylation manifold by enzyme catalysts.

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THE CATALYTIC INVENTORY OF DIMETHYLALLYL TRANSFERASES IN FUNGI: LEAVING NO SITE ON INDOLE UNALKYLATED Genomic sequences of fungi in genera such as Claviceps, Penicillium, and Aspergilli reveal almost two dozen orfs that are members of the aromatic dimethylallyl transferase (DMAT) superfamily.8,9 These are metal independent prenyltransferases that differ from the classic magnesium-dependent prenyl transferase superfamily prevalent in the head to tail condensations so pervasive in terpenoid biogenesis.5,10,11 These microbes use both free tryptophan and a variety of Trp-X-dipeptide diketopiperazine metabolites (most notably B

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Figure 4. Diverse modes of prenylation of the indole moiety of Trp and cyclo-Trp-X metabolites: (a) reverse prenylation at N1; (b) prenylations at N1, C2,C3, C4, C5, C6, C7 of Trp; (c) resonance contributors showing lone pair electron density at four carbon sites of the indole ring; (d) rearomatization of an initial prenylated indole, shown for the C4 regioisomer.

fact that the new N1−C bond is to C3 rather than to C1 of the transferred prenyl group, the so-called “reverse” prenylation mode (Figure 4a), more properly denoted as capture of a tertiary (C3) vs primary locus (C1) of the allylic carbocation.17 Four distinct carbon positions of Trp are prenylated by four distinct DMATs: 4-dimethylallyl-Trp arises by action of FgaPT2, 5-dimethylallyl-Trp from a 5-DMAT from Aspergillus clavulatus,7 the 6-dimetylallyl Trp product from the IptA enzyme from a soil streptomycete18 and homologues,19 and 7-dimethylallylTrp from the 7-DMATs enzyme (ref 20, reviewed in ref 7; Figure 4b). One can readily show that the lone pair nitrogens on the indole N1 of Trp can be delocalized to generate carbanionic resonance contributors where the lone pairs are stored at C4, C5, C6, and C7 (Figure 4c), respectively. These carbanionic contributors account for the observed nucleophilic reactivity at each of these carbon sites toward the five carbon electrophilic allyl cation equivalent generated in the active sites of these four regiospecific C-prenyl transferases. The

initial alkylated products are arenium ions, and enzyme-assisted abstraction of the carbon-bound proton (as shown at C4 for the 4-DMAT) rearomatizes the indole, now regiospecifically prenylated (Figure 4d). It has been presumed that the specific orientation of the indole side chain with respect to the electrophilic allyl cation in each enzyme’s active site determines product regiospecificity. The 4-prenyl-Trp is the first step in ergot alkaloid biosynthesis,21 while C5, C6, and C7 alkylations are on pathways to flustramines,22 6-prenyl indole carboxylic acid,23 and mellamide,24 respectively (Figure 5a). Hundreds of even more more fully elaborated indole alkaloid scaffolds have been isolated and characterized.25,26 The two additional nonbridgehead carbons of the indole moiety of tryptophan, carbons 2 and 3 (in the pyrrole ring), can likewise be shown to have nucleophilic character. Those carbons do undergo regioselective enzyme-mediated prenylation from Δ2-IPP, not at the stage of the free amino acid but one biosynthetic step further along, as cyclo-Trp-Pro and C

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Figure 5. (a) Examples of (relatively simple) metabolites derived from C4, C5, C6, and C7 prenylated Trps; (b) C2 prenylation of brevianamide F to trypostatin D; C3 prenylation of cyclo-Trp-Leu creates a tricyclic pyrroloindole scaffold in the tetracyclic product; AnaPT carries out an anti addition across the 2,3-double bond of the pyrrole ring of its substrate benzodiazepinedione to yield 2R,3S stereochemistry at the new ring junction while CdpC3PT yields the 2S,3R stereochemistry, indicating reverse orientation of the indole and prenyl cosubstrates in the two enzymes’ active sites.

cyclo-Trp-Leu, respectively.8,14,15 As shown in Figure 5b, brevianamide F is prenylated at C2 by FtmP1 to yield trypostatin D. The cyclo- L -Trp- L -Leu diketopiperazine is prenylated at C3 attendant with a new N−C bond at C2 of the indole ring (Figure 5c). This C3 prenylation/C2 cyclization creates a tricyclic pyrroloindole framework that is characteristic

of other Trp-dipeptidyl DKP derivatives prenylated at C3. The C3 prenylation can be formulated as yielding an initial imine tautomer that can be captured intramolecularly by the amide nitrogen of the diketopiperazine ring. Although a poor nucleophile, the amide nitrogen is in high local concentration, and N−C bond formation converts the bicyclic indole to the D

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tricyclic pyrroloindole scaffold. An analogous transformation occurs in the production of the aszonalenin framework.27 Fan and Li28 have made use of the collection of regioselective DMAT enzyme family members with cyclo-Trp-Leu to produce the corresponding N1, C2, C4, C5, C6, and C7 monprenylated derivatives. The C3 reverse prenyl adduct, as noted above, has undergone intramolecular cyclization to the pyrroloindole scaffold. The prenyltransferase AnaPT from the aszonalenin pathway produces the bis-5,6-prenylated cyclo-Trp-Leu as a product7 (Figure 1b). When the stereochemistry of addition to the C2 and C3 positions of cyclo-L-Trp-L-Leu during pyrroloindole formation was examined by Li and co-workers, one prenyltransferase, Ana PT, carried out a net trans or anti addition, with capture of the prenyl cation from below as the intramolecular amide formation occurs from above7 (Figure 5d). This yields the 2R,3S stereochemistry at the new ring junction. Surprisingly, the structurally related CdpC3Pt from the same fungus N. f ischeri, instead, yields the 2S,3R stereochemistry where the reacting partners act at the opposite face of carbons 2−3 of the pyrrole moiety of the cylo-L-Trp-L-Leu substrate.7

the amino group of the Trp side chain had added intramolecularly to C2 to yield the tricyclic pyrroloindole framework, akin to the intramolecular capture in the cyclo-Trp-X dipeptides noted above. As discussed in the next section, a comparable suite of alternate products was seen by Tanner and Mahmoodi30 using the C2-specific FtmPT1 from A. f umigatus that catalyzes the cyclo-Trp-Pro conversion to brevianamide F noted in Figure 3b. Further electron-releasing perturbation of the indole side chain in Trp, proceeding from 4-methyl to 4-methoxy- to 4-amino-Trp, with the purified 4-DMAT yielded additional variant prenylated products.29 The 4-MeO-Trp gave prenylation at N1 and C5, while use of 4-amino-Trp gave ortho- and para-directed prenylation at C5 and C7 (Figure 6b). Also the K174A mutant form of the enzyme30 when presented with its normal substrate L-Trp gave C3 prenylation where the amino group of Trp had then closed on C2 to give the tricyclic pyrroloindole.

REDIRECTING PRENYLATION REGIOCHEMISTRY Incomplete regiospecificity can be detected from some DMAT family members if one looks hard enough for the minor products. One indication of how flexible the relative orientation of the nucleophilic indole partner and the electrophilic prenyl donor cosubstrate can be comes from recent studies by Poulter and colleagues.29 Assay of the C4- specific DMAT enzyme that catalyzes the first committed step in ergot alkaloid biosynthesis was conducted with the surrogate substrate 4-Me-Trp where the preferred locus of prenylation was blocked by that methyl substituent. At a catalytic rate of about 1% compared to the physiologic substrate L-Trp, three alternative 4-methyl products were detected and characterized. Two were adducts at C3, reflecting both normal and reverse regiochemistry of prenylation, and the third was the N1-prenyl adduct (Figure 6a). The two C3 adducts qualify as indolines rather than indoles as

There is one well-established and one putative precedent for prenylation of the indole side chain of a tryptophan residue during post-translational modification of a protein.2 The more well characterized is in the processing of the bacterial ComX protein (where Com stands for competence, the physiological state where bacteria such as strains of Bacillus subtilis, become competent to take up foreign DNA and undergo transformation).31 The nascent protein that emerges from the Bacillus ribosome in strain RO-E2 is a 58 residue pre-ComX (Figure 7a) where Trp56 is the specific residue that will become prenylated.32 The prenyl donor is not the usual C5 dimethylallyl-PP but instead the homologated C10 prenyl donor geranyl-PP. The prenyl transferase is ComQ.33 It transfers the C10 geranyl moiety with normal regiochemistry, making a new C−C bond between C1 of the geranyl unit and C3 of the indole ring of Trp56. The Gly57 amide nitrogen closes onto the indole ring at C2 to generate the tricyclic pyrroloindole framework (Figure 7b). At some point in the maturation process, the 58mer chain of the pre-ComX is proteolyzed between K52 and G53 to release the final mature geranylated hexapeptide ComX. This is the quorum sensing pheromone that binds to ComP and activates the downstream gene transcription program to render the Bacillus cells competent for transformation.34 In other bacillus species, the mature ComX peptide has been prenylated with a C15 farnesyl rather than a C10 geranyl group.32 A second example is found in the cyanobactin kawaguchipeptins from Microcystis aeruginosa.35 Kawaguchipeptin A is a cyclic undecapeptide with two Trp residues at positions 1 and 5. The B isomer has both Trps prenylated at C3 of the indole ring, and as now expected, the adjacent amide nitrogen has added in to form the tricyclic pyrroloindole scaffolds. While the producer genome has not been reported, it is likely that this cyanobactin is typical in arising from a ribosomal protein precursor.36,37 In other prenylated cyanobactins, O-prenylation of Ser, Thr, and Tyr residues is observed, and in some cases, a Claisen rearrangement occurs nonenzymatically under physiologic conditions to give the C-prenylated Tyr derivative.38

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Figure 6. Redirecting prenylation regiochemistry (a) action of C4-specific DMAT on 4-Me-Trp, (b) reactions of 4-MeO-Trp and 4-NH2-Trp. E

POSTTRANSLATIONAL PRENYLATION GENERATES A COMPETENCE PHEREMONE AND A CYANOBACTIN ANTIBIOTIC

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Figure 7. Posttranslational maturation of Bacillus competence pheremone: (a) 58 residue pre-ComX is the nascent ribosomal protein product that undergoes geranylation at Trp56 and proteolysis between K52 and G53 to yield the mature prenylated hexapeptide derivative that is the active ComX pheremone. (b) The tricyclic pyrroloindole scaffold results as the geranyl group is added to C3 and the nitrogen of E57 adds to C2 of the indole ring of W56.

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RUMINATIONS ON “FORWARD” AND “REVERSE” PRENYLATIONS From the early days of prenylated indole biosynthetic investigations, focused first on 4-DMT in ergot alkaloid biosynthesis, the question of prenylation mechanisms has been debated.29,17 The recent availability of X-ray structures of members of the DMAT superfamily (refs 11 and 39), with bound indole and Δ2-IPP reactant partners do not show apposition of C2 of indole to C1 of the prenyl substrate but rather to C3 of the indole ring.30 One possibility is substantial reorientation of the bound substrate pair during catalysis; the low rates of alternate regioisomeric products may reflect low probability captures by the different carbons and N1 of the indole ring with the allylic cation that is a proximate prenyl donor in the active sites. Alternatively, Wenkert and Sliwa back in 197740 proposed that the 4-prenyl-Trp could arise from a Cope rearrangement (perhaps accelerated in the enzyme active site) by way of an initial 3′-reverse prenyl adduct as a nascent product (Figure 8a).17 Poulter et al’s findings29 that the 4-aminoTrp gives 5-prenyl- and 7-prenylated products argues against Cope rearrangements in those manifolds. Those are abnormally slow substrates, and it is possible that the energy surfaces may be sufficiently close to each

other that such electronic perturbations in substrates could cause reaction manifolds to leak from one channel to another. Tanner’s investigations of FtmPT1 involved both mutant forms of the enzyme and also alternate substrates.41,17,30 With 2-methyl cyclo-Trp-Pro as a substrate, blocking the preferred C2 site from prenylation, they observed the C3 prenylated species as a major product and the N1-prenylated form as a minor product. They generated the C3-reverse prenylated product with a different DMAT and detected facile nonenzymatic aza Cope rearrangement to the N1-prenyl adduct (Figure 8b). They then argued that it is reasonable to keep open the possibility that FtmPT1 may first make C3 prenyl adducts, both with reverse and normal prenylation regiochemistry, as the primary products. The C3 normal adduct could undergo a 1,2-alkyl shift (known in nonenzymatic indole chemistry42 to yield the physiologic 2-prenylated product (trypostatin B; Figure 8c). If the reverse 3-prenyl adduct forms it could either undergo a Cope rearrangement to the 4-dimethylallyl-indole or an aza Cope to the observed N1-prenylated indole scaffold. (Figure 8b). The postulate of these electrocyclic rearrangements minimizes any necessary reorientation of Trp and IPP in the enzyme active sites and yet would account for distinct product regioisomers. F

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Figure 8. Mechanistic proposals for reverse prenylations: (a) 1977 proposal40 for formation of 4-prenyl-Trp by a Cope rearrangement from an initial reverse-3-prenyl-Trp adduct. (b) Facile nonenzymatic conversion of 3-reverse prenyl-cyclo-Trp-Pro by an azaCope rerrangement to the N1-prenyl product. The initial adduct could also rearrange by the Cope pathway to the 4-prenyl product. (c) Possible formation of the observed 2-prenyl product via a 1,2 prenyl shift of an initially formed 3-prenyl adduct with normal regiochemistry.

It may be that (some of) the DMAT family members have reaction energetics poised such that distinct reaction channels could be utilized depending on subtle changes in substrate orientation and electronic demand in the enzyme active sites. This may be particularly valid for iterative prenylations by a single enzyme leading to bis- and tris-penylated indole scaffolds during biosynthesis, e.g., of fumotremorgin B and A.15 Whatever the details of specific enzyme mechanisms, it is increasingly clear that this fungal enzyme superfamily has been able to coax out a remarkably wide range of indole chemistry under ambient physiologic conditions.

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TWO ELECTRON VS ONE ELECTRON MANIFOLDS FOR INDOLE FUNCTIONALIZATION In addition to two electron reaction manifolds of the electronrich indole ring of tryptophan and derivatives, one electron manifolds are also available. Molecular oxygen provides one such opportunity. An oxygen atom from O2 is transferred as L-Trp is converted regiospecifically to 5-OH-Trp in biosynthesis of the neurotransmitter serotonin by tryptophan hydroxylase.43 This oxygen transfer can be formulated as homolytic cleavage of the C5−H bond in the indole ring by a high valent oxoiron species, followed by an OH• rebound44 (Figure 9A). Analogously, microbial chlorinations at carbons 5, 6, and 7 of tryptophan are known for FADH2-dependent regioselective halogenases,45 but those appear instead to proceed by a two electron pathway from an N-chloro-Lys in the halogenase active site46 (Figure 9B). The convergence of both two electron and one electron reaction manifolds in the indole ring of tryptophan occurs during biosynthesis of lyngbyatoxin A, the active component of “swimmers itch”47 (Figure 10). The dipeptide N-methylvalyl-tryptophanol is generated by reductive release from a two

Figure 9. (a) Oxygenation vs (b) chlorination of the indole ring of tryptophan: one electron vs two electron manifolds with rearomatization as late stage events.

module nonribosomal peptide synthetase assembly line.48 The N-methyl nitrogen is then coupled to C4 of the indole ring by G

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Figure 10. One electron and two electron manifolds for indole modification during lyngbyatoxin biosynthesis. (a) N−C4 bond formation by one electron pathway. (b) C−C7 bond formation via two electron capture of a geranyl unit by a C7 carbanion equivalent.

an iron-based enzyme that does homolytic coupling.49 Finally a C10 geranyl diphosphate unit is coupled, via the tertiary C3 of the allyl cation, to C7 of the indole ring to complete the biosynthesis of lyngbyatoxin.47 The biological oxidzability of the indole nucleus by one electron pathways is further revealed in tryptophan-containing diketopiperazines such as cyclo Trp-Phe and cyclo Trp-Pro. These cyclic dipeptides can undergo one-electron-mediated dimerizations by a fungal cytochrome P450-type heme protein dtpC, to yield 3,3′-dimers such as dibrevianamide F and ditryptophenalanine50,51 (Figure 11a). Comparable one electron redox pathways have been implicated in the biosynthesis of indole sesquiterpenes such as the dimeric bixiamycins.52 One electron oxidation at the indole NH populates carbon-centered radical contributors in addition to the nitrogen radical to yield the observed N1−N1 coupling regiochemistry as well as the N1−C6 coupling outcome. In this case, it is a flavoenzyme rather than an iron oxidase that sets the one electron manifolds in motion (Figure 11b).53 In principle, one might expect methyl tryptophan regioisomers to arise from transfer of the other widely used, metabolically available electrophile, the C1 fragment from S-adenosylmethionine

SAM.2 However, in the only characterized example, a 2-methylindole carboxylate residue is formed from Trp by NosL during assembly of the antibiotic thiostrepton, but not via delivery of an electrophilic [CH3+] equivalent. Instead, this occurs via agency of a radical-SAM enzyme in which one electron chemistry is involved, coupling a proposed 2-indole radical with a transferring [CH3•].54,55 The second case, in physostigmine biosynthesis, does appear to use SAM as an electrophilic methyl group as the pyrroloindole forms.56 Deeper probing may yet reveal electrophilic methylations of the indole ring in microbial metabolism, but so far it lags far behind prenylations and suggests a mismatch between cataysts offering the indole ring as a nucleophile and the methyl group of SAM as an electrophilic C1 fragment. The regiospecific 4-DMAT has been shown to accept benzyl diphosphate as an alternate electrophile to produce the 5-benzyl-L-trp product57 (Figure 12) and may augur for synthetic utility. The altered product regiochemistry (C5 rather than the anticipated C4) is hypothesized to be an access problem in an early transition state with benzyl cation features for the coupling electrophile. In contrast to the robust activity of the purified 4-DMATs, 5-DMATs and 7-DMATS enzymes gave detectable but only trace amounts of benzyl Trp products. H

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Figure 11. One electron-mediated oxidative dimerization of indole scaffolds: one electron oxidation of brevianamide F by the heme enzyme DtpC leads to radical coupling at C3 of the indoles to dibrevianamide; analogous oxidative cylization of cylo-(Trp-Phe) yields the corresponding ditryptophenaline.

electrophilic alkylation”5 by the C5 prenyl group, and how that reacts with and can build up a set of regiospecifically modified indoles as first or second (on cyclo-Trp-X-dipeptides) steps in pathways that go on to build complex architectures into indole alkaloids. These reactions reveal the intrinsic nucleophiliticity of the indole ring at many positions, not normally manifested in tryptophanyl residues in proteins.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 12. Regiospecific 4-DMATs showing acceptance of benzyl diphosphate as an alternate electrophile to produce the 5-benzyl-L-trp product.

REFERENCES

(1) Keasling, J. D. (2010) Manufacturing molecules through metabolic engineering. Science 330, 1355−1358. (2) Walsh, C. T., Garneau-Tsodikova, S., and Gatto, G. J., Jr. (2005) Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem., Int. Ed. Engl. 44, 7342−7372.

With the exception of the one electron manifolds just noted, this perspective has focused on one reaction type, “dissociative I

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ACS Chemical Biology

Reviews

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