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Protein Substrates for Reaction Discovery: Site-Selective Modification with Boronic Acid Reagents Published as part of the Accounts of Chemical Research special issue “Artificial Metalloenzymes and Abiological Catalysis of Metalloenzymes”. Zachary T. Ball*
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Department of Chemistry, Rice University, Houston, Texas 77005, United States CONSPECTUS: Chemical modification of natural proteins must navigate difficult selectivity questions in a complex polyfunctional aqueous environment, within a narrow window of acceptable conditions. Limits on solvent mixtures, pH, and temperature create challenges for most synthetic methods. While a protein’s complex polyfunctional environment undoubtedly creates challenges for traditional reactions, we wondered if it also might create opportunities for pursuing new bioconjugation reactivity directly on protein substrates. This Account describes our efforts to date to discover and develop new and useful reactivity for protein modification by starting from an openended screen of potential transition-metal catalysts for boronic acid reactivity with a model protein substrate. By starting from a broad screen, we were hoping to take advantage of the very many potential reactive sites on even a small model protein. And perhaps more importantly, whole proteins as reaction screening substrates might exhibit uniquely reactive local environments, the results of a dense combination of functional groups that would be nearly impossible to mimic in a small-molecule context. This effort has resulted in the discovery of four new protein modification reactions with boronic acid reagents, including a remarkable modification of specific backbone N−H bonds. This histidine-directed Chan−Lam coupling, based on specific proximity of an imidazole and two amide groups, is one important example of powerful reactivity that depends on a combination of functional groups that proteins make possible. Other bioconjugation reactions uncovered include a three-component tyrosine metalation with rhodium(III), a nickelcatalyzed cysteine arylation, and an unusual ascorbate-mediated oxidative process for N-terminal modification. The remarkably broad scope of reactivity types encountered in this work is a testament to the breadth of boronic acid reactivity. It is also a demonstration of the diverse reactivities that are possible by the combined alteration of boronic acid structure and metal promoter. The discovery of specific backbone modification chemistry has been a broadly empowering reactivity. Pyroglutamate, a naturally occurring posttranslational modification, exhibits remarkably high reactivity in histidine-directed backbone modification, which allows us to treat pyroglutamate as a reactive bioorthogonal handle that is readily incorporated into proteins of interest by natural machinery. In another research direction, the development of a vinylogous photocleavage system has allowed us to view backbone modification as a photocaging modification which is released by exposure to light.
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INTRODUCTION
pursued many strategies for chemical protein manipulation that might address diverse needs. Examples of the creative and diverse approaches to protein modification include unnatural amino acid incorporation,1,2 reactive peptide tags,3,4 and residue-selective chemical reactions.5 We6−8 ourselves have contributed to broader efforts9−18 to develop general approaches for site-selective modification of natural proteins. Most of these efforts begin from known synthetic methods applied to protein substrates. Against this backdrop, we recently began to think about how we might uncover new chemical reactions that would exhibit interesting selectivity
Proteins contain a straightforward primary sequence, yet present a bewildering array of structures, functions, and reactivities. Secondary and tertiary protein structure, built from the sum of weak, local interactions, creates local motifs that differ significantly from isolated functional groups. Bioconjugation methods must succeed in this complex environment, where unique functional groups are rare or nonexistent. Beyond fundamental reactivity design motivations, chemical manipulation of proteins plays an increasingly important role in biologic drug development and in other translational pursuits. The goals and constraints of protein modification processes can be quite varied in different contexts, and so chemists have © XXXX American Chemical Society
Received: December 7, 2018
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DOI: 10.1021/acs.accounts.8b00626 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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We chose to screen a few model proteins for incorporation of an arylboronic acid reagent in the presence of metal salts as potential catalysts. An arylboronic acid with a bioorthogonal tag, such as an alkyne (Figure 2b, 1), allowed us to visualize
with protein substrates. Mechanism-based design of new bioconjugation reactions can be powerful, especially when applying known small-molecule methods. But alternative solutions are needed for reactivity without small-molecule precedent. We wondered if empirical screening concepts, which are used extensively for reaction optimization such as in asymmetric catalysis19,20 and cross-coupling reactions,21−23 could help uncover new reactivity for bioconjugation. Conceptually, the approach relies on treating a protein molecule as a small “library” of diverse functional groups, local environments, and potentially reactive sites. This modest exercise has been remarkably fruitful, leading, directly or indirectly, to at least four new reactions of boronic acids for protein modification (Figure 1). These efforts have also
Figure 1. Suite of new reactions utilizing boronic acid reagents for protein modification.
Figure 2. Discovery of metal-catalyzed protein modification reactions with boronic acid reagents. (a) Metal salts were screened as mediators of bioconjugation reactions for lysozyme and boronic acid 1. Modification was assessed by quantification of alkyne incorporation by chemical blotting. Note: Rh2+ was screened initially, but it was later determined that Rh3+ is the active species. (b) Functionalized boronic acids used in this study. (c,d) Modification of neuromedin by an alkenylboronic acid. Crude analysis by MALDI−MS. (e) Single-site modification of the protein lysozyme under copper-catalyzed modification with boronic acid 2, after avidin-based affinity purification.
created new opportunities to develop stimulus-responsive modifications based on a new photo-uncaging design, and have allowed us to discovery a heretofore unknown bioorthogonal reactive handle.
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AN APPROACH TO REACTION DISCOVERY Metal-catalyzed reactions of boronic acids seemed a reasonable place to start a reaction-screening endeavor with protein substrates. Boronic acids are ubiquitous in synthetic organic chemistry. Both organic and transition-metal catalysis provide ample evidence that numerous reactivity manifolds might be possible. Boron is an exotic element in biology, but organoboron compounds have a rich and valuable history in biological chemistry. Organoboron compounds are a classical approach to protease inhibitors. Boronic acids are well established in protein chemistry and chemical biology,24 including as fragments for glycoside binding,24−27 as reagents for Suzuki coupling with aryl halides,28 and as reagents for ultrafast amine−aldehyde conjugation.29−32 The metal-catalyzed reactivity of boronic acids for bioconjugation has typically focused on reaction with non-natural, bioorthogonal functional groups. With relatively few footholds onto reactivity with canonical sequences, we decided that boronic acids represented an interesting initial study.
even low conversion to covalent modification products by standard SDS-PAGE gel and subsequent blotting by chemical33 or other means. Lysozyme was one such model protein, and in that screen two initial leads appeared: modified protein was observed with both copper and rhodium salts (Figure 2a). When we investigated further, it became clear that these hits were each unique and unrelated reactions.
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UNIQUE SELECTIVITIES FOR BACKBONE N−H FUNCTIONALIZATION To understand the observed copper reactivity, we next screened our conditions against a larger set (∼20) of readily available proteins and peptides. Characterizing product structure is no simple matter in an open-ended screen such B
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Accounts of Chemical Research as this, and analytical challenges may well represent a significant limitation of this approach. Nonetheless, we eventually established that, for Cu2+-mediated reactions, only histidine-containing sequences exhibited covalent modification. Even cysteine residues were unreactive. In the end, a simple tripeptide (thyrotropin releasing hormone, pyroglutamatehistidine-proline) was found to be a suitable substrate that allowed complete characterization of the product structure. The product contained an N-aryl structure, with the aryl moiety found not on the histidine imidazole side chain, or anywhere on the histidine residue, but rather on the backbone amide nitrogen of the residue immediately preceding the histidine (i.e., i − 1) (Figure 1A). We established the product structure through NMR analysis of the simple tripeptide. But we soon found that rather complex peptides, such as neuromedin B (Figure 2c,d), exhibited conversion to a single backbone-modified product at the residue immediately preceding a histidine, in this instance with an alkenylboronic acid reagent. The reactivity can be understood as a coppercatalyzed coupling between an amide N−H and a boronic acid reagent. In that sense, the reactivity is a histidine-directed variant of the well-known Chan−Lam reaction.34 The reactivity is compatible with complex boronic acids bearing a range of useful affinity tags or reactive handles. Using the desthiobiotin−boronic acid conjugate 2, lysozyme with a single modification was produced and isolated (Figure 2e). In terms of boronic acid structure, both alkenyl- and aryl-boronic acids are appropriate reagents. The reaction is sterically sensitive, so trans-alkenylboronic acids, such as trans-2-cyclohexylvinylboronic acid (Figure 2c), typically give the most efficient reactivity, and ortho-substituted arylboronic acids are uniformly unreactive. Histidine-driven activation of local amide N−H bonds to form amidate−copper and other amidate−metal species is well-known. Deprotonated amidate ligands form key parts of metalloprotein ligand spheres, and some metalloproteins contain both histidine and backbone amidate metal coordination. Well-studied examples include nitrile hydratase35,36 iron centers (Cys2 amidate2), nickel superoxide dismutase37,38 (Cys2 His1 amidate1 N-terminus1), and acetyl CoA synthase (Cys2 amidate2).39 One common metal-binding motif, the amino terminal Cu2+- and Ni2+-binding motif40,41 (ATCUN, a His1 amidate2 N-terminus1 motif), bears a strong resemblance to the presumed bisamidate intermediate observed in backbone arylation/alkenylation (Figure 3a). The bisamidate structure of ATCUN motifs engenders unique properties. Copper-bound ATCUN motifs stabilize the high-valent Cu3+ oxidation state, and a quasi-reversible Cu2+/Cu3+ redox couple is observed by cyclic voltametry.40,41 A mechanistic hypothesis for backbone N−H modification considers reactivity through a similar amidate complex (His1 amidate2, A in Figure 3b). While natural ATCUN motifs are coordinatively saturated, tetradentate complexes, nonterminal sequences lack an N-terminal amine group, and thus would form a tridentate complex (A) with a free site suitable for transmetalation to an organocopper intermediate (B). The ability of ATCUN-like motifs to stabilize high-valent CuIII may be crucial to facilitate formation of a key CuIII intermediate B, from which reductive elimination to form product is possible. This facile redox chemistry of natural ATCUN motifs has allowed applications including sequence-specific oxidative DNA cleavage,42−46 directed oxidative protein cleavage,47 and oxidative protein cross-linking.48,49 Similar cobalt ATCUN
Figure 3. (a) N-Terminal ATCUN motif occurs naturally in proteins and exhibits a quasi-reversible Cu2+/3+ redox couple. (b) Proposed mechanism to account for backbone arylation.
complexes are efficient electrocatalysts for hydrogen evolution.50 However, useful bond-forming reactivity of ATCUNlike motifs has been absent. In this respect, the Cu-catalyzed backbone modification is an independent discovery of a metalloprotein mimic for completely new and important reactivity. In addition to copper, initial screening of simple arylboronic acid reagents turned up a different modification in the presence of simple rhodium salts. Again, it was necessary to examine smaller peptide substrates to characterize the observed reactivity. This study allowed a determination that rhodium mediates stoichiometric metalation of tyrosine residues, in a three-component process that forms an arylrhodium η6-arene complex (Figures 1b and 4).51 Optimization of the reactivity identified arylboronic acids with ortho-carboxamide substituents as optimal reagents, allowing formation of a chelated arylrhodium product. As with the copper-catalyzed discovery, our discovery of rhodium-mediated bioconjugation has important precedent, this time in wholly synthetic inorganic chemistry. Preformed cyclopentadienyl complexes have been studied by several laboratories in the context of side-chain metalation of aromatic side chains, and in some ways the observed reactivity is closely related.52,53 Like the coppercatalyzed backbone reactivity, Rh3+ tyrosine metalation also has remarkable and unique features that would have been challenging to design from known reactivity. The local polyamide environment is essential for the observed reactivity: we have been completely unable to observe similar organometallic arene complexes with simple phenols, such as p-cresol. The three-component nature of this process forms an unexpected arylrhodium complex that is completely stable in air and water and under a variety of biologically relevant conditions. C
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Figure 4. (a) Three-component process to form organometallic arylrhodium arene complexes at tyrosine residues. (b) Previously reported method produced ruthenium52 or rhodium53 arene complexes at aromatic side chains (tryptophan is shown) from a preformed cyclopentadienyl (Cp) complex by arene exchange.
Figure 5. (a) Fast nickel-catalyzed cysteine arylation. (b) Scope of metals capable of catalyzing the reaction. (c) Structure of a B domain protein, containing one free thiol, which was arylated with Ni2+. (d) The effect of ligand structure on reaction conversion for modification of the BSA protein with Ni2+ and the alkyne-functionalized reagent shown (e).
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VARIATION OF BORONIC ACID STRUCTURE AND A NICKEL-PROMOTED REACTION In the course of our investigations, we discovered that other transition metals, beyond copper and rhodium, can exhibit productive, interesting, and novel bioconjugation reactivity by alteration of boronic acid structure. Indeed, it is now clear that boronic acid structural modification provides an alternative approach for entering new reactivity and selectivity paradigms. In a first reported example, we found that arylboronic acids with electron-withdrawing ortho substituents participate in metal-catalyzed arylation of cysteine thiol groups, yet are unreactive toward N−H modification (Figures 1d and 5). The histidine-directed backbone modification is quite sterically sensitive; cis-alkenylboronic acids react only sluggishly, and ortho-substituted arylboronic acids are completely unreactive. Against this general structure−reactivity relationship, we noted that electron-withdrawing ortho substituents gave anomalous metal-catalyzed reactivity with cysteine-containing peptides, even in the absence of histidine (Figure 5a,b). Although cysteine is perhaps the most well-studied target for residueselective bioconjugation, metal-catalyzed cysteine arylation remains a challenge.54−56 Successful catalytic arylation has been elusive enough that the community has recently pursued the alternative approach of stoichiometric organometallic reagents, prepared and isolated in a separate step, for reliable and versatile cysteine arylation.57−60 With small peptide substrates, several divalent transition metals (Cu2+, Ni2+, Co2+) promoted this reaction (Figure 5a,b). With larger protein substrates, Ni2+ proved to be a superior catalyst, allowing cysteine arylation within minutes at micromolar protein concentrations.61 In contrast to coppermediated reactions, an important ligand effect was noted in this nickel-catalyzed reactivity, especially in reactions of protein, rather than peptide, substrates. We noted significantly slower conversion under ligand-free conditions when we moved from peptide to protein substrates. Fast reaction rates
were restored upon addition of the ligand 6,6′-dimethyl-2,2′dipyridyl. Interestingly, added ligand does not improve catalyst activity with simple peptide substrates, and in some cases ligand has a modest negative effect on reaction efficiency. But with protein substrates, the chelating nitrogen ligand has a significant and general effect on reaction efficiency. The divergent effects of ligand with peptide and protein substrates leads to a postulate that the added ligand binds Ni2+ and limits nonselective metal binding by the protein. Nickel halide salts are known to form 4-coordinate (bpy)NiX2 complexes in the presence of bipyridine and its derivatives.62 In this context, the steric bulk of the 6,6′-dimethyl groups in the coordination sphere may further affect interactions of the bulky protein surface with the metal center. This ligand effect further differentiated the cysteine-selective chemistry from histidinedirected backbone modification. We propose a coordinatively saturated 4-coordinate Cu2+ intermediate in histidine-directed N−H modification, and consistent with this proposal, we have found no beneficial ligand effect in this chemistry. In contrast, the ligand effect for cysteine arylation is consistent with a lower coordination number with substrate at the transition state. The reactivity trends of ortho-nitro substituted arylboronic acids differ from established Chan−Lam reactivity, in which electron-rich arylboronic acids react more efficiently. Together with the rare instance of Ni2+ catalysis in Chan−Lam-like reactions, these observations may point to a new mechanistic pathway for this reaction, possibly including single-electrontransfer steps. D
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A METAL-FREE COUPLING One final reactivity from this discovery program so far came from a negative control experiment. The addition of sodium ascorbate, a common reductant for Cu2+, resulted in drastically reduced N−H alkenylation. Surprisingly, reactivity was observed in the presence of ascorbate and in the absence of Cu2+, an observation that led to the discovery of an organocatalytic N-terminal modification as a heterocyclic aminal linkage (Figures 1c and 6).63 The reaction formally
Figure 6. Ascorbic acid mediates N-terminal modification with alkenylboronic acids via formation of heterocyclic aminal. The process can be used for N-terminal fluorescent labeling with the boronic acid shown.
involves alkenylboronate oxidation to an aldehyde oxidation state. Similar bioconjugation structures are also accessible by direct condensation with heteroaryl aldehydes,64 Ascorbate (typically a reductant) mediates oxidation of an alkenylboronic acid under these conditions. The reaction is O2-dependent, leading to a mechanistic hypothesis involving ascorbate oxidation by O 2 to provide the active oxidant for alkenylboronic acids. The (transiently stable) dehydroascorbate (Figure 6), a product of two-electron ascorbate oxidation, is also a competent reagent in this process, implicating dehydroascorbate as the likely active oxidant in the ascorbate chemistry. The ascorbate-mediated oxidation is a curious instance of redox “umpolung” reactivity in tandem with O2. This aside from metal-mediated methods of boronic acids expands the scope of heterocyclic N-terminal modifications that are readily accessible.64
Figure 7. (a) Conceptual scheme depicting backbone modification as a photocaging approach to controlling structure and function. (b) Concept of a “vinylogous” ortho-nitrobenzyl approach to photodeprotection of an alkenyl−N bond. (c) Backbone modification and photocleavage (MALDI−MS, top graph) can be used to block and initiate triple-helix formation in a collagen-type peptide (bottom graph).
TOWARD PRODUCTIVE APPLICATIONS: PHOTOCLEAVABLE MODIFICATIONS In the course of fundamental reactivity investigations, we have also begun to explore what new capabilities and applications are made possible by reactivity discoveries. Alkylation of backbone N−H bonds has profound effects on secondary structure and biological activity of polypeptide structure, because N−H alkylation disrupts the hydrogen bonding networks essential for folding into α-helix, β-sheet, and other folding elements. The copper-catalyzed backbone N−H arylation/alkenylation produces products with similarly disrupted folding potential. Backbone arylation/alkenylation also perturbs electronic structure due to conjugation and amide cis/ trans equilibrium, which allows access to meaningful levels of the cis amide rotomer. Developing a system for modifications that are “cleavable” in response to external stimuli provides a new approach to stimulus-driven control over backbone structure, and thus of folding and function (Figure 7a). For an initial demonstration of this concept, we set our sights on photoreactive boronic acid
reagents that could provide “photocaged” backbone amide structures. Although conceptually simple, this idea presented a challenge: no general approaches existed for photocleavage of aryl−X or alkenyl−X bonds. In contrast, methods for photocleavage of alkyl−X and acyl−X bonds are well established.65 After considering several options, we proposed a vinylogous analogue of 2-nitrobenzyl protecting groups. Photoexcitation of nitrobenzyl protecting groups proceeds through benzylic C−H abstraction, and results in a formal oxidation of the benzylic carbon together with photocleavage.66 Cleavage of an alkenyl−N bond by a vinylogous strategy envisions similar C−H abstraction of a complex allylsubstituted aromatic core (Figure 7b). The approach hinges on regioselective trapping of the resulting zwitterionic, highly conjugated product by solvent water. Fortuitously, we observed just such a cleavage process on a small-molecule model, releasing the amide N−H backbone together with an aldehyde-containing aromatic compound. With this capability in hand, we demonstrated67 “uncaging” of peptides in this manner. A collagen-type triple-helix peptide
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provides a path to develop pyroglutamate as a tool in chemical biology and protein chemistry, and also an opportunity to better understand pyroglutamate biology. This extremely fast and efficient reactivity is similar to many common biorthogonal bioconjugation reactions, but pyroglutamate-histidine sequences differ from traditional biorthogonal reactions: pyroglutamate is a natural posttranslational modification, and pyroglutamate-histidine sequences are readily and site-specifically introduced into proteins of interest without non-natural machinery. Pyroglutamate occurs at the N-terminus of natural proteins and peptide hormones.68−71 Indeed, its prevalence in readily available peptide hormones was the only reason we investigated pyroglutamate-containing sequences at all. Pyroglutamate can form spontaneously in uncatalyzed cyclization of an N-terminal glutamine, but its formation is predominantly catalyzed in nature by a glutaminyl cyclase (QC) enzyme.68,72,73 But the role of pyroglutamate in living systems is not fully understood, presumably due in part to its subtle effect on local protein structure and to a dearth of methods to identify pyroglutamate residues. The uniquely reactive pyroglutamate−histidine sequence is not common in natural proteins. For example, we are unable to find any examples of a pyroglutamate−histidine protein sequence in the PDB database. By virtue of its unique reactivity and absence in natural proteins, the pyroglutamate−histidine sequence provides a rare example of a simple structural motif that both (i) reacts quickly and efficiently through reactivity that is orthogonal to other biological species and (ii) readily encoded via natural processes into specific proteins of interest. Importantly, simple and efficient expression of pyroglutamate-containing proteins in E. coli was recently described.74 Thus, combining pyroglutamate expression with the unique copper-catalyzed reactivity allowed straightforward modification of a protein of interestencoded with an N-terminal pyroglutamate−histidine dipeptidewith chemically diverse alkenylboronic acid reagents. Simple amino-acid substitution of the plasmid sequence in the reported pyroglutamate expression system74 allowed straightforward production of a protein of interest tagged with the pyroglutamate-histidine dipeptide on the N-terminus (Figure 8b). Protein expression levels are not affected by the posttranslational pyroglutamate incorporation. With an expression system in hand, we demonstrated selective modification of the protein-of-interest (POI) directly from E. coli lysate with functionalized boronic acid reagents bearing fluorescent tags, affinity handles, biorthogonal reactive groups, and/or saccharides. Subsequent (secondary) bioconjugation of an introduced azide with a 5 kDa PEG chain further confirmed, via gel-shift assay, that pyroglutamate is faithfully and completely incorporated the POI, and that Cu2+-catalyzed pyroglutamate N−H alkenylation proceeds to complete conversion, even in a demanding lysate environment (Figure 8b). This remarkable reactivity represents perhaps the smallest, simplest bioconjugation handle that is both orthogonal to canonical side chains and readily encoded by natural biological systems. While ATCUN motifs provide ample biological precedent for the idea of histidine-directed activation of amide N−H bonds with copper or nickel, the unique reactivity of pyroglutamate residues has no obvious biological precedent. In part due to limited analytical capabilities, relatively little is known about the function of pyroglutamate residues. Postulated roles for pyroglutamate include switching N-
(Figure 7c) could be cleanly modified (top, blue trace), and, once modified, no longer folded in solution (bottom, blue trace). Uncaging this peptide with UV light released the free peptide (top, red trace), which folded into the expected trimeric assembly (bottom, red trace). We further demonstrated that protease cleavage sites could also be effectively blocked by backbone N−H modification, and photoirradiation led to uncaging and immediate enzymatic processing.67 The project led to some interesting and challenging synthetic efforts to build analogous “photocaging” boronic acids with affinity purification handles,67 which were needed to demonstrate backbone N−H modification and photorelease on a natural protein substrate. Further development of whole protein photocaging will require that the issues of tractability, including aggregation, of backbone-modified proteins be addressed.
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PYROGLUTAMATE-HISTIDINE AS A USEFUL BIORTHOGONAL REACTIVE HANDLE One intriguing example of unique reactivity surfaced in the course of investigations into Cu2+‑catalyzed backbone N−H modification. While most amide backbone N−H modification reactions are slow, with half-lives of several hours even under optimized conditions, pyroglutamate-histidine sequences react many orders of magnitude more quickly (Figure 8a). The reaction half-life (t1/2) is ∼5 min, and an apparent secondorder rate constant could be calculated as kapp ∼ 4 M−1 s−1 (where kapp = k1/[boronate]), under pseudo-first-order conditions. At short reaction times under the conditions studied, no measurable modification was observed for other histidine-containing sequences. This curious observation
Figure 8. (a) Pyroglutamate−histidine sequences are uniquely reactive and can be selectively modified in lysate. An E. coli expression system incorporating the QC enzyme incorporates a pyroglutamate− histidine sequence at the protein N-terminus. (b) Structure of a trifunctional boronic acid reagent. (c) Total protein (CBB) stain of an SDS−PAGE gel. Left lane: lysate. Center lane: results from affinity purification of a POI (GFP) based on the chemically introduced desthiobiotin handle. Reaction of modified POI with a 5 kDa PEG via azide−alkyne cycloaddition. F
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substrates to work back to a system for which reactivity could be characterized with molecular precision. It is possible that “new” reactivity on the protein level could simply be a rediscovery of a known small-molecule reaction, similar to the challenges of rediscovering known structures in natural products isolation. In a sense, our discovery of an ascorbatemediated N-terminal modification with alkenylboronic acids bears some hallmarks of this possibility. But in the process of this discovery we also demonstrated an underappreciated oxidative reactivity for the common reductant, ascorbate. Studies of boronic-acid−protein reactivities uncovered through this study continue to unearth new reactivity and selectivity paradigms, currently under study in our laboratories. Judicious and creative choice of other protein-based reactivity searches seems like fertile grounds for discovery of even more interesting protein reactivity in the future.
terminal charge state and altered susceptibility to proteolytic degradation by aminopeptidases, and limited examples of pyroglutamate metal binding have surfaced.75 Methods to identify and analyze pyroglutamate or its derivatives remain quite limited.76−80 Against these analytical and functional questions, the fast-reacting and highly selective nature of pyroglutamate-histidine sequences could be employed to study QC enzymatic activity in a variety of contexts. But beyond questions about natural function, the pyroglutamate−histidine motif seems an ideal tool for chemical protein functionalization.
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CONCLUDING REMARKS Methods for protein modification and bioconjugation have traditionally been developed by design. Chemical intuition and careful study of the literature has helped chemists identify reactivity from small-molecule experiments that is ripe for application to the different demands of protein chemistry. In the subarea of transition-metal-mediated methods for modification of peptides and proteins, the need for air- and watertolerant processes severely limits methods that might be of use. Our own extensive studies with rhodium carboxylate catalysts for protein modification are but one example of this approach, in which an especially functional-group-tolerant metalcatalyzed reaction was recognized and applied to bioconjugation.81,82 While bioconjugation studies provide extensive opportunities for problem-solving, serendipitous discovery, and beautiful molecular design, most other valuable metalmediated bioconjugation methods fit this discovery paradigm, such as arylmetal species for stoichiometric arylation,57,59 tyrosine O-allylation,83 iridium-catalyzed reductive alkylation,82 and C−H activation approaches.84 The studies discussed here imply that polypeptides themselves are substrates that provide fertile ground for new reaction discovery. A protein molecule contains an extensive collection of functional groups, in congested proximity to one another. A protein substrate as reaction discovery platform presents a significant set of diverse functional groups, but also provides proximal environments of different functional groups that would be difficult to emulate in a small-molecule screen. One aspiration of this approach is to uncover new chemistries that would be impossible or challenging to discover by alternative means. The three-component RhIII metalation reaction (Figure 4) would be difficult to predict or discover by alternative means. Metalation products from small peptide substrates have negligible solubility in all solvents we have examined, which creates characterization challenges. Proteins contain unique local microenvironments with unique chemical reactivity. In the case of histidine-driven modification, the subtle combination of at least three functional groups, two amides and one imidazole, necessary for Cu2+-promoted backbone modification (Figure 2) make it unlikely that chemists could have designed this reactivity with simple model substrates. In the future, similar efforts may provide a path study, quantify, and predict reactivity differences of different accessible residues of a given type; our studies to date have not been up to the significant challenge of providing general insights into this important type of selectivity. While our efforts to date indicate the value of a protein-level reactivity search, the challenges are many. Observation of initial reactivity is difficult to correlate with specific reactions and product structures. In cases described here, we employed a combination of approaches and examined ever-simpler peptide
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Zachary T. Ball: 0000-0002-8681-0789 Notes
The author declares no competing financial interest. Biography Zachary T. Ball is an Associate Professor of Chemistry at Rice University, and serves as faculty director of the Institute for Biosciences & Bioengineering. He grew up in Columbus, OH, and earned an A.B. in Chemistry from Harvard University (1999), where he worked with Gregory Verdine. He moved to Stanford University to study transition-metal catalysis with Barry Trost. During this time, Zach worked extensively with organosilicon compounds, and developed an interest in organic metalloid chemistry that continues with the work described here. He then moved to UC−Berkeley as a Miller Fellow to study photo- and electroactive polymers in the lab of Jean Fréchet until 2006, when he moved to Houston to take up his current position at Rice. Zach’s current research interests center on protein chemistry and transition-metals in biological contexts.
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ACKNOWLEDGMENTS I am deeply indebted to the exceptional and hard-working coworkers who have been integral to the work described here. This work was supported by generous grants from NSF (CHE1609654 and CHE-1055569) and the Robert A. Welch Foundation research Grant C-1680.
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REFERENCES
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