Designing Selectivity in Dirhodium Metallopeptide Catalysts for

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Designing Selectivity in Dirhodium Metallopeptide Catalysts for Protein Modification Samuel C. Martin, Farrukh Vohidov, Haopei Wang, Sarah E. Knudsen, Alex A. Marzec, and Zachary T. Ball* Department of Chemistry, Rice University, Houston, Texas 77005, United States S Supporting Information *

ABSTRACT: The ability to chemically alter proteins is important for broad areas of chemical biology, biophysics, and medicine. Chemical catalysts for protein modification, and particularly rhodium(II) conjugates, represent an important new approach to protein modification that develops novel functionalization approaches while shedding light on the development of selective chemistries in complex environments. Here, we elucidate the reaction parameters that allow selective catalysis and even discrimination among highly similar proteins. Furthermore, we show that quantifying modification allows the measurement of competitive ligand affinity, permitting straightforward measurement of protein−peptide interactions and inhibitors thereof. Taken as a whole, rhodium(II) conjugates replicate many features of enzymes in an entirely chemical construct.

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INTRODUCTION Selective modification, functionalization, or conjugation of natural proteins remains a daunting challenge. Like related efforts to perform selective chemistry on nucleic acids1−5 or secondary metabolites,6−9 modified proteins are broadly useful in chemical biology, as therapeutics, and as building blocks for biomaterials. Among other challenges, biopolymers lack unique reactive handles, and so new selectivity concepts are required. Ideal methods should enable the modification of specific individual sites on a target protein and should discriminate among even highly similar proteins. Finally, simple and versatile reagents should allow flexible modification with various functional handles under biologically relevant conditions. While protein modification has often focused on engineered substrates or residue-specific chemistry, ligand−catalyst conjugates are an emerging approach to site-specific protein modification.10−15 Proximity-driven modification by transitionmetal catalysts or organocatalysts has numerous advantages, including access to unique, bioorthogonal chemistries. In contrast to reactive probes, a catalytic approach separates the molecular recognition motif (“ligand”) on the catalyst from the stoichiometric reactive group and functional tag (Figure 1a). The concentration of ligand in the ligand−catalyst conjugate can be kept to substoichiometric, biologically relevant levels. In preliminary efforts, we discovered rhodium(II) conjugates that act as efficient catalysts for modification at specific side chains based on molecular recognition.15 Single-protein modification was possible even in complex environments, such as cell lysate. In this manuscript, we describe studies on the effects of several reaction parameters on reaction selectivity. We elucidate, in turn, the effects of diazo structure, additives, and reaction pH. We discovered an unanticipated effect of the © XXXX American Chemical Society

tetrahydrothiophene group of biotin that explains previous contradictory results and use this discovery to design improved diazo reagents and improved selectivity in protein modification reactions. The several effects are combined to demonstrate orthogonal modification of individual proteins among highly similar SH3 folds. In addition, we show that quantifying modification efficiency can be a fast, efficient, and useful way to measure the potency of inhibitors against protein−protein interactions.

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FUNCTIONALIZED DIAZO REAGENTS For the accommodation of a variety of modification and functionalization needs, broadly effective reactivity that tolerates a variety of functionalized reagents is desirable, and we have developed a set of simple diazoacetate reagents for protein modification (Figure 1, 1−5). The highly stabilized nature of α-styryl-α-diazoacetate derivatives makes them optimal for protein modification,16−18 in which stabilization of the metallocarbene intermediate improves reaction efficiency and minimizes the nonproductive O−H insertion of water, a common pathway for rhodium metallocarbene intermediates.19 The Francis lab showed,20 and we have subsequently verified, that some simpler diazo esters (α-phenyl-α-diazoacetates or even ethyl α-diazoacetate) succeed to some extent in polypeptide modification, but α-styryl-α-diazoacetates are significantly more efficient. However, changes to the ester −OR group are fairly broadly tolerated. Due to the hydroReceived: December 12, 2016 Revised: December 29, 2016 Published: December 30, 2016 A

DOI: 10.1021/acs.bioconjchem.6b00716 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

known to provide optimal reactivity and (2) that the functional handle (alkyne) is linked via a stable amine linker rather than via the ester linkage. As an added benefit, the newly installed amine would be charged under physiological conditions, aiding probe solubility. A new synthetic route was required to produce a probe with a site of functionalization that would remain intact after ester cleavage (Scheme 1). We assumed access to diazo 9 Scheme 1. Synthetic Approaches to Alkyne-diazo 9

Figure 1. (a) Protein modification reactions proceed via metallocarbene and subsequent formation of functionalized protein. (b) αStyryl-α-diazoacetate derivatives are most common for aqueous protein modification reactions. (c) Preparation of the desthiobiotin− diazo reagent 5. For full details, see the Supporting Information.

from acid 10. Initially, we planned access to acid 10 by the Wittig olefination of aldehyde 11, which is readily available by the alkylation of bromoaldehyde 12, but in our hands, the subsequent Wittig transformation with the unique phosphonium salt 1328 was not effective. Alternatively, desymmetrizing olefination with phosphonium 1429,30 and the dialdehyde 15 afforded an aldehyde intermediate, and the alkyne group was subsequently introduced by reductive amination, providing alkyne 16. However, we were unsuccessful at late-stage oxidation to the potentially unstable β,γ-unsaturated carboxylic acid 10 in spite of some limited precedent for similar transformations.31−34 In the end, a new approach (Scheme 2) produced the desired target compound by late-stage onecarbon homologation via cyanide alkylation to alkyne 17 followed by hydrolysis to the styrylacetic acid derivative, which obviated the need for late-stage oxidation. The route takes advantage of a recent preparation of aldehyde 18 by DIBAL-H reduction of cyano-ester 19.35

phobicity of the α-styryl-α-diazoacetate moiety, an oligoethylene glycol linker has been included for solubility in water. For affinity pull-down and related tasks, biotin is perhaps the most commonly used group in biological applications.21 For applications with rhodium(II) catalysts, biotin−diazo conjugate 3 is readily prepared from styrylacetic acid.13 However, unique (and at times deleterious) reactivity was observed with biotin− diazo 3, especially in more-demanding modification reactions with full-length protein substrates (see below for details). Limitations of the biotin conjugate, together with an interest in developing more flexible and diverse functionalized diazo reagents, led to the preparation and study of other diazo reagents.13,22−24 The synthesis of desthiobiotin-containing compound 5 avoids reactivity problems of the biotin probe 3 (see below) and also allows for improved release of bound protein in affinity purification applications.25−27 The synthesis of desthiobiotin−diazo 5 (Figure 1c) is illustrative of many αstyryl-α-diazoacetate derivatives, requiring late-stage esterification of a functionalized alcohol with styrylacetic acid, followed by diazo transfer with p-ABSA (see the Supporting Information for information on the preparation of analogous compounds 1−4.). A significant weakness of first-generation α-styryl-α-diazoacetate derivatives is the ester linkage in reagents 1−5, which is susceptible to chemical or enzymatic hydrolysis. We encountered some applications and conditions for which this was a substantial limitation. Thus, probe 9 was designed to abrogate these concerns. The most important design features of this probe are (1) that it preserves the α-styryl-α-diazoester core

Scheme 2. Preparation of Alkyne-diazo 9

B


Article

Bioconjugate Chemistry The new linkage in diazo 9 does prevent hydrolysis-based loss of the functional handle (Figure 2a). For example, under

Table 1. Influence of Biotin Additive on Protein Modification Efficiencya

Figure 2. (a) Protein−mod construct formed by transesterification with TBHA buffer or by hydrolysis. In either case, any functionality incorporated into the diazo reagent through an ester linkage is removed, preventing further manipulation. (b,c) Reaction conditions: Fyn SH3 (2 μM), R5DRh (1 μM), diazo (100 mM), N-tertbutylhydroxylamine (TBHA) buffer (100 mM, pH = 6.2), and room temperature for 4 h. Matrix-assisted laser desorption/ionization−mass spectrometry (MALDI−MS) data demonstrates the tendency of the ester linkage to transesterify. Protein modified by diazo 2 no longer features the alkyne handle; protein modified by diazo 6 retains its incorporated alkyne. R5DRh: Ac−VSLADRhRPLPPLPP−NH2.15

entry

protein

conditions

residue

conversion (%)

1 2 3 4 5 6 7

Fyn SH3 Fyn SH3 Fyn SH3 Lck SH3 Lck SH3 Hck SH3 Hck SH3

R5DRh Rh2(OAc)4 Rh2(OAc)4, saturated biotin R5DRh R5DRh, saturated biotin R6ERh R6ERh, saturated biotin

Trp119 Trp119 Trp119 Trp97 Trp97 His93 His93

100 0 25 2 50 90 20

a Reaction conditions: protein (10 μM), dirhodium catalyst (5 μM), diazo 2 (500 μM), and room temperature for 5 h. For Trp mod: TBHA buffer (100 mM, pH of 6.2; for His mod: Tris buffer (100 mM, pH of 7.4). Conversion values determined by MALDI−MS integration. R5DRh: Ac−VSLADRhRPLPPLPP−NH2.15 R6ERh: Ac− VSLARERhPLPPLPP−NH2.24 In the generic reaction scheme at top, the native peptide VSL12 is shown bound to the Lck SH3. Residue numbering is based on the full Lck protein. Lck Trp97 is analogous to Fyn Trp119; Lck His76 is analogous to Hck His93. PDB files: 2IIM and 1QWF.

some modification and purification conditions, we see extensive transesterification of the modification product by the buffer Ntert-butylhydroxylamine (TBHA), which is often optimal for protein modification.16,17 In the case of diazo 2, this results in the loss of the alkyne handle, while transesterification of the product derived from diazo 9 retains the alkyne handle (Figure 2b,c).

modified product (Figure 3, entry 1). However, further investigation has revealed that a biotin moiety is essential for effective reactivity. The alkyne-diazo 2 gives no conversion at all (entry 2), which is in line with results from proximity-driven modification, in which low background reactivity was observed. Simple unfunctionalized diazo compounds such as 1 are similarly unreactive.

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ADDITIVES AND BIOTIN Additives, in the form of coordinating buffers, can significantly affect protein modification reactions with rhodium(II) catalysts.16,17 In the course of our investigations, we recognized that biotin also had significant effects. Typical of our experience with SH3 domain modification, the Trp119 residue of the Fyn SH3 domain (see Table 1) could be uniquely modified upon treatment with a suitable Fyn-binding metallopeptide catalyst (R5DRh),15 while using the negative control, Rh2(OAc)4, gave no product (Table 1, entries 1 and 2). In the whole-cell lysate reaction, we sometimes experienced nonselective background reaction with biotin−diazo 3 but not with other functionalized diazo compounds.15 To better understand this result, we added biotin to protein-modification reactions and observed Fyn modification, even in negative-control experiments with Rh2(OAc)4, albeit in decreased yield (entry 3). The increased tryptophan modification in the presence of biotin is general. Indeed, including biotin as an additive could be employed to facilitate modification of a specific tryptophan residue of Lck (Trp97) that was quite sluggish under normal conditions (entries 4 and 5). The unique effects of biotin on the efficiency of rhodium(II)catalyzed diazo reactions extends to a completely different amino-acid substrate case. We examined cysteine alkylation with diazo compounds that were catalyzed by Rh2(OAc)4. In that study, the biotin−diazo reagent, 3, was used to facilitate analysis. Reagent 3 delivered high conversion to the cysteine-

Figure 3. Protein modification at cysteine using proceeds efficiently using even Rh2(OAc)4 if a biotin moiety (within the diazo reagent or exogenous) is present in the reaction mixture. Reaction conditions: E3g2C peptide (100 μM), Rh2(OAc)4 (10 μM), diazo (2 mM), TBHA buffer (10 mM, pH of 6.2), and room temperature for 5 h. Conversion values determined by MALDI−MS integration. E3g2C: EISALEKCISALEQEISALEK.23 C



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SELECTIVITY Taken together, the effects of catalyst design, of reaction pH, and of a biotin additive provide impressive and exquisite control over the design of orthogonal protein reactivity. We examined a representative collection of Src-family SH3 domains: Hck, Yes, and Lck. These three domains exhibit significant homology and have 77% sequence similarity, providing a stringent test for the design of orthogonal catalysts. Gratifyingly, sequence and structure analysis and optimization of reaction conditions according to the discoveries outlined above did indeed allow highly selective single-domain modification (see Figures 5 and 6). Structural analysis (Figure 5) shows the presence of reactive residues (histidine and

The addition of external biotin could recover some cysteinemodification activity. Biotin itself resulted in some minimal observed reactivity, presumably due to poor solubility under the reaction conditions (Figure 3, entry 3). However, the biotin− PEG 22, produced by thermal decomposition of the biotin− diazo reagent, 3,36,37has improved aqueous solubility and resulted in significantly increased cysteine modification when used as an additive in reactions with alkyne-diazo 2 (entry 4). Thus, our initial success with cysteine alkylation was a serendipitous discovery that relied on the unique reactivity of the biotin−diazo functionality, which we chose for convenience in the initial study. We have since verified that the tetrahydrothiophene ring is necessary for this anomalous biotin-mediated reactivity. The use of desthiobiotin as an affinity handle produces diazo compounds such as 5 that react normally. Furthermore, simple water-soluble thioether compounds, such as 2-methylthioethanol, do not increase diazo reactivity as does the biotin tetrahydrothiophene functionality. The reasons for altered reactivity in the presence of biotin− diazo 3 and the pyrazole control 22 are challenging to assess. Biotin and other thioether moieties bind RhII axial sites with modest affinity (Kd ≈ 100 μM in aqueous media).38−40 In spite of early evidence to the contrary, there is now convincing evidence that axial ligands can influence the reactivity and selectivity of rhodium metallocarbene intermediates.41−44 Whatever the exact nature of the effect, biotin additives provide a convenient way to improve reaction conversion in preparative applications. In sharp contrast, modification at histidine residues in the SH3 fold differs significantly from modification at tryptophan or cysteine. Biotin strongly inhibits histidine modification, in contrast to the opposite effect observed with tryptophan or cysteine (Table 1, entries 6−7). However, histidine modification is strongly correlated with increasing reaction pH (Figure 4). For the illustrative example of Lck modification (at

Figure 5. Schematic of the SH3 domain of Lck with the parent peptide VSL12 bound. Multiple reactive Trp and His residues are conserved among Hck, Yes, and Lck. The arrows point to the site of attachment of the catalytic dirhodium core in the various metallopeptides. R5DRh: Ac−VSLADRhRPLPPLPP−NH2.15 R6ERh: Ac−VSLARERhPLPPLPP− NH2.24 11LRh: Ac−VSLARRPLPPLRh.15 PDB files: 1BU1, 2HDA, 2IIM, and 1QWF.

tryptophan) on the ligand-binding surface of the SH3 domains, and labels indicate potential substrate residues the three SH3 domains (Hck, Yes, and Lck). The Yes SH3 domain, uniquely among the three proteins, has no histidine residues at the ligand-binding interface, the presence of which we previously demonstrated leads to slower modification.45 Thus, the catalyst R5DRh cleanly modifies Yes SH3 at this tryptophan only. Importantly, this tryptophan residue is conserved across all Src family SH3 domains, but modification there is not observed in Hck or Lck (Figure 6, middle row). Because Hck and Lck possess a conserved histidine residue (His93 and His 76, respectively), which is close in space to the reactive tryptophan, neither protein is modified by R5DRh. However, the Lck domain contains a unique histidine residue, His70, which is located at the bottom of the binding face. Thus, the catalyst 11LRh, which moves the dirhodium core in the vicinity of this histidine side chain, produces only single modification at the His70 of Lck and does not modify either Hck or Yes (Figure 6, bottom row). Selective Hck modification represents perhaps the most daunting challenge of these three SH3 domains. The Hck

Figure 4. Increasing reaction pH increases efficiency of protein modification at His residues. The metallopeptide 11LRh was used to modify Lck SH3. Reaction conditions: Lck SH3 (10 μM), 11LRh (5 μM), diazo 1 (500 μM), Tris buffer (20 mM), and room temperature for 5 h. 11LRh: Ac−VSLARRPLPPLRh.15

His70) with the 11LRh catalyst,15 histidine modification could be suppressed below pH 6, and conversion reached quantitative levels at pH 7.4. In contrast, tryptophan modification displays slightly decreasing reaction conversion with increasing pH, consistent with previous studies.16,17 The pH effect with histidine is consistent with increased imidazole protonation to an imidazolium at lower pH, thus decreasing the concentration of available nucleophilic imidazole groups. D


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Figure 6. Orthogonal catalysts for the modification of Hck, Yes, and Lck SH3 domains, demonstrated by MALDI−MS analysis. Reaction conditions: protein (10 μM), metallopeptide (5 μM), diazo 2 (1 mM), and room temperature for 5 h. For Trp mod: TBHA buffer (100 mM, pH of 6.2; for His mod: Tris buffer (100 mM, pH of 7.4). PDB file: 1KIK.

sequence contains two adjacent histidine residues in the ligandbinding pocket, His93 and His94. Although both are potential targets for modification, proximate histidine residues can block modification by simultaneous coordination to both reactive rhodium atoms.24 Additionally His93 is conserved in Lck, so selective reaction there is unlikely. Nevertheless, we found that the R6ERh catalyst24 delivered selective modification of His93 on Hck but did not target the structurally similar His76 on the Lck domain (Figure 6, top row). Elevated pH (7.4) also enforces histidine modification and suppresses modification of Hck, Yes, or Lck at tryptophan. Thus, three different metallopeptide catalysts allow the individual and selective modification of three SH3 domains (Hck, Yes, and Lck) without appreciable cross-reactivity.

Figure 7. (a) MALDI−MS spectra of modification of the Lck SH3 domain in the presence of increasing amounts of S2E peptide,46 which competes with the 11LRh catalyst15 for the binding site. Reaction conditions: Lck SH3 (4 μM), 11LRh (2.5 μM), diazo 2 (2 mM), Tris buffer (100 mM, pH of 7.4), and room temperature for 2 h. (b) A general scheme of the interactions taking place during the assay; inhibitor (blue) and rhodium(II) catalyst (green) compete for the same binding site, with modification blocking further inhibitor-binding events. (c) Structures of metallopeptide catalyst 11LRh and peptide inhibitor S2E. (d) Data from (a) plotted with a logistic fit yielding an IC50 of 15 μM, which is in line with the reported value for VSL12derived peptides and Src-family SH3 domains.

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QUANTIFICATION Conceptually, proximity-driven protein modification correlates a noncovalent protein−ligand assembly with a covalently modified protein product. This correlation can be used to identify competitive ligand-binding behavior in the presence of another ligand.14 Extending this concept, quantitative information about protein−peptide interactions can be gleaned from modification reactions in the presence of varying inhibitor levels (Figure 7). Varied concentrations of a simple peptide inhibitor of Src-family SH3 interactions (S2E46) displayed characteristic sigmoidal plots of modification versus log[inhibitor]. The IC50 values from modification reactions, stopped at short reaction times to approximate initial rates, correlated well with Kd values derived from isothermal titration calorimetry. A similar analysis can be conducted to probe the affinity of a small-molecule inhibitor of Src-family SH3 interactions (Figure 8). As with the peptide inhibitors, quantification of modification by matrix-assisted laser desorption/ionization− mass spectrometry (MALDI−MS) as a function of inhibitor concentration allows a measurement of competitive inhibition. Relative modification levels are also sufficient to extract affinity measurements. Additionally, chemical blot visualization of modified proteins enabled us to extract IC50 values after

quantification of blot image intensity. Thus, proximity-driven modification provides a simple way to assess the binding affinity of ligands for protein−protein interactions.

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CONCLUSIONS The rhodium(II) conjugates described exhibit many properties of natural enzymatic systems despite being completely designed, nonbiological constructs. The system allows modification of residues, such as histidine or tryptophan, that are largely inaccessible to other chemical methods and provides a general strategy for tag-free chemical modification.14 The dose−response behavior in the presence of competing protein ligands provides a fundamentally new assay of inhibition potency, in analogy to enzyme-based assays. This assay format succeeds with small amounts of material (∼5 nmol/well) and is E



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experimentally simple. As such, it provides an alternative to more time-consuming and material-intensive assays that are sometimes required to assess protein−peptide binding.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00716. General experimental details, methods for protein modification and product analysis, and characterization of new compounds. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zachary T. Ball: 0000-0002-8681-0789 Notes

The authors declare no competing financial interest.

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REFERENCES

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Figure 8. (a) Reported SH3 domain inhibitor 23.47 (b) Addition of inhibitor 23 slows modification (red arrow) of Yes SH3−MBP fusion protein by metallopeptide R5DRh. Reaction conditions: protein (4 μM), R5DRh (2.5 μM), diazo 2 (2 mM), TBHA buffer (100 mM, pH of 6.2), and room temperature of 2 h. (c) Quantification of Fyn SH3 modification after 2 h by MALDI−MS. The observed IC50 value is consistent with the Kd measured independently by isothermal titration calorimetry (Kd = 43 μM). (d) As an alternative analytical method, modification reactions from (b) can be visualized with a fluorogenic probe (via chemical blotting protocol). 48 R5D Rh = Ac− VSLADRhRPLPPLPP−NH2.15

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ACKNOWLEDGMENTS

This work was supported by Welch Foundation under award no. C-1680 and by the National Science Foundation under award nos. CHE-1609654, CHE-1055569 and Graduate Research Fellowship no. 1450681. F


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Designing Selectivity in Dirhodium Metallopeptide Catalysts for

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