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Selection of Natural Peptide Ligands for Copper-Catalyzed Azide− Alkyne Cycloaddition Catalysis Allison G. Aioub,† Lindsay Dahora,† Kelly Gamble,† and M. G. Finn*,† †

School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332, United States S Supporting Information *

ABSTRACT: The copper-catalyzed azide−alkyne cycloaddition (CuAAC) reaction is a powerful tool for making connections in both organic reactions and biological systems. However, the use of this ligation process in living cells is limited by the toxicity associated with unbound copper ions. As an initial attempt to create peptide-based accelerating ligands capable of cellular expression, we performed synthesis and selection for such species on solid-phase synthesis beads bearing both candidate ligand and alkyne substrate. A simple histidinecontaining motif (HXXH) was identified, and found after solution-phase optimization to produce single-turnover systems showing moderate rate acceleration over the ligand-free reaction. CuAAC reaction rates and yields for different alkynes were found to respond to the peptide ligands, demonstrating a substrate scope beyond what was used for the selection steps, but also illustrating the potential difficulty in evolving a general CuAAC catalyst.

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INTRODUCTION The copper-catalyzed azide−alkyne cycloaddition (CuAAC) reaction provides 1,4-disubstituted 1,2,3-triazole products with excellent rate and selectivity.1,2 The small size, easy installation, and unobtrusive nature of the reacting azide and terminal alkyne groups provide for relatively easy incorporation into cells through metabolic labeling and other methods.3−6 Biological molecules so labeled are often addressed by biocompatible strain-promoted alkyne−azide reactions. However, these alkynes are larger, induce greater perturbation, and can suffer from relatively low ligation rates and greater side reaction rates relative to simple azides and alkynes and the CuAAC process that joins them.7−9 CuAAC is a highly reliable reaction without special ligands for the Cu(I) center, but such accelerating ligands are usually helpful at low reactant concentrations and mild temperatures, such as for bioconjugation. The development of these ligands has been rapid and diverse, beginning with the tris(triazolylmethyl)amine family10 and the identification of other Cu-binding motifs.11 While electron-rich Cu centers generally provide faster reactions, they are more air-sensitive, so a balance between this parameter and catalytic activity is necessary for practical catalysts on the benchtop. 12 Furthermore, catalysts optimal in organic media13−16 are not usually well suited for CuAAC reactions under aqueous conditions. The tris(heterocycle-methyl)amine family of ligands has allowed for facile ligations on cell surfaces and in cell lysates; however, its use inside living cells is severely limited by the toxicity of unbound copper ions.17 Reaction improvements, © XXXX American Chemical Society

such as the use of picolyl azide and similar moieties by the Ting laboratory18,19 and others20,21 to increase the local concentration of reactants, cannot be easily translated into living cells because the requisite concentration of copper is still too high. However, additional progress is being made using Cu-chelate complexes,21,22 heterogeneous catalysts,23,24 or standard ligands25 introduced into cells. The ultimate biocompatible catalyst would be one that is expressed by the cell, and is therefore composed of natural building blocks such as amino acids, nucleotides, lipids, or metabolites. Here we describe our initial efforts to develop oligopeptide-based CuAAC catalysts using only natural amino acids. We previously showed that free histidine in solution had measurable but modest accelerating activity in the CuAAC reaction.26 Therefore, we started with His-rich sequences, and employed a tethered substrate screening strategy that allowed for the convenient surveying of bead-based libraries of potential catalysts. In this effort, the N-termini of candidate peptides were usually left unprotected because N-terminal amino groups can be helpful in metal binding.27 Initial leads were further improved by rational design and testing, resulting in robust, if still sluggish, peptide-based catalysts. Finally, we confirmed a broad substrate scope for the CuAAC reaction. Received: March 25, 2017 Revised: May 1, 2017

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DOI: 10.1021/acs.bioconjchem.7b00161 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 1. (A) Schematic of the OBOC resin construction for ligand libraries, gray spheres representing resin beads. (B) Positive control resin beads reacted with 1 under standard conditions. (C) Beads from a representative peptide library screen. The arrow marks a positive hit. Images are contrast-enhanced for easier viewing here; under normal conditions, only the highlighted bead in panel C was easily visible.

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RESULTS AND DISCUSSION In order to screen the large chemical space that can be achieved using amino acids, we reprised an approach described by Wennemers for organocatalytic reactions28,29 and Romesberg for phage display evolution of DNA polymerases,30 in which immobilized catalyst candidates are tasked with operating on co-immobilized substrates. Thus, using well-established methods for the split-pool one-bead-one-compound (OBOC) synthesis of oligopeptides,31,32 we modified the library design to include both the substrate and ligand (Figure 1A) on each resin bead. Amine-functionalized NovaSyn TG amino resin was addressed first with an HBTU activated undecynoic acid (0.5 equiv) to occupy ∼50% of the accessible sites with a terminal alkyne. This resin was then reacted with HBTU activated alanine (3 equiv) to prepare the remaining accessible sites for peptide synthesis using standard Fmoc reagents and methods. A methionine residue was then installed, and the resulting -AlaMet modified beads were used for split-pool synthesis. The starting dipeptide provided a convenient site for high-yield cleavage by cyanogen bromide. Proper functionalization of the alkyne-bearing beads was verified by attachment of the BimPy2 ligand in place of the -AM sequence, as BimPy2 was previously identified as a CuAAC ligand compatible with resin attachment.33 As shown in Figure 1B, the vast majority of beads decorated with this ligand mediated enough CuAAC ligation with fluorogenic 7-hydroxy coumarin azide34 to fluoresce strongly when illuminated at 404 nm. Negative controls (no ligand, and with scrambled peptides described below) showed no such activity. We were able to optimize CuAAC conditions (33 μM CuSO4, 11 mM NaAsc, 1:4 H2O:DMF, 0.4 μmol 7-hydroxy coumarin azide) to give

clear evidence of ligand-accelerated catalysis with BimPy2, but without background reactivity in the absence of ligand, important because nonligated CuI centers are of course able to mediate the reaction as well. Application of the same reaction conditions to the peptide libraries gave rise to a heterogeneous distribution in which the majority of beads showed no evidence of reactivity (darker spots in Figure 1C) while a few others were quite fluorescent (marked with an arrow). The fluorescent beads were manually separated and the tethered peptides cleaved by cyanogen bromide at the anchoring Met position and identified by LCMS. Initial small libraries (32 possible peptides per library) lacked enough variation to provide a meaningful number of leads. Larger library sizes were limited by the problem of mass redundancy among peptide candidates using convenient onedimensional mass spectrometry techniques. Therefore, library sizes of several hundred peptides were chosen to provide a good balance between potential diversity and analytical convenience. Several different initial libraries were explored, as summarized in Figure 2. Group 1 consisted of peptide sequences biased toward the motifs HXXH, CXXC, and HXH, common in CuI proteins such as Atx1 and in amino-terminal CuII- and NiIIbinding peptide sequences.35−37 A similar His- and Cys-rich library was also prepared to include proline in the middle of the peptide to bias the sequences toward turns (Figure 2, group 2). Among these first two classes, Cys-containing sequences were found to have significantly lower activity, providing no fluorescent beads, suggesting that thiol groups served as poisons rather than as components of productive ligand structures. Therefore, Group 3 libraries were created to further B

DOI: 10.1021/acs.bioconjchem.7b00161 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. OBOC libraries constructed and tested, using one-letter amino acid codes to list the split and pool at each position. The bracketed number is the total number of sequences in the library.

explore the value of short (4−5 amino acids) HXXHcontaining sequences, followed by extended versions (8−10 residues, Group 4) to probe the length of the peptides as a variable. Additionally, random library sequences biased toward the amino acids bearing metal-binding side chains (His, Cys, Asp, Glu) and of varying sequence length were tested to enhance sequence diversity (Group 5).38−40 Overall, Met use was limited to diminish the multiplicity of fragments produced by CNBr cleavage, thereby simplifying the identification process.41 Isolation and peptide identification of fluorescent beads in initial library screening was followed in a subset of cases by repeat synthesis and testing on-resin using the same design (alkyne and peptide on the same bead). While all such positive signals were confirmed, their intensity on repeat trials varied quite a bit (Figure 3A). To assess solution-phase performance, select sequences were again made using traditional Fmoc techniques with a Rink amide linker, cleaved from the resin, and tested under similar CuAAC conditions as the on-resin library screen (Figure 3B, with the use of DMSO as cosolvent

rather than DMF). Here, a comparison was made to the tris(hydroxypropyltriazolylmethyl)amine (THPTA) accelerating ligand, which can be used in excess in the presence of minor amounts of DMSO without compromising CuAAC rate very much.42 Relative activities on the bead were found to be somewhat, but not completely, predictive of solution-phase activity. For example, HDSDPGAMA-Resin (peptide A) and HILHPGAMA-Resin (peptide B) showed roughly equivalent behavior, much more active than QCKPHFMA-Resin (peptide C). The sluggishness of CuAAC reactions involving peptide C was confirmed in solution, but under those conditions peptide B proved to be a significantly better accelerating ligand than peptide A, which was not anticipated based on the resin-bound results. Factors such as peptide clustering (presumably easier on the resin than in solution) could well contribute to such differences. Another interesting aspect of these experiments was revealed in testing of different ligand:metal ratios. Thus, the CuAAC reaction tolerated the presence of peptide B (HILHPGAMA) in 3:1 excess relative to Cu, whereas the same excess of peptide C

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content, was responsible for accelerated triazole formation. With rare exceptions such as shown in Figure 3, these peptides behaved as standard coordinating ligands, in which an excess of ligand slowed the rate, presumably by inhibiting access of azide and alkyne to the metal center. Figure 4B shows a representative case (a peptide containing a repeat HAAH sequence) in which a 0.5:1 peptide:Cu ratio was the most active. In addition, the peptides behaved as weakly coordinating ligands, as did THPTA here and in previous tests, with respect to lack of resistance to a more strongly coordinating medium.43 Thus, in 80% DMSO, several peptides that performed reasonably well in 95:5 H2O:DMSO gave no rate acceleration relative to the ligand-free control in 20:80 H2O:DMSO (Figure S7). Furthermore, the addition of aminoguanidine (5 μM), designed to intercept dehydroascorbate, gave rise to more sluggish reactions and decreased conversion with peptide-based ligands (Figure S8), in contrast to THPTA.42 Most peptide-based reactions in solution did not provide more than an equimolar amount of product relative to the copper concentration. This is illustrated, along with the determination of initial rate constants, in Figure 5 and Table 1. The stoichiometric nature of the reaction with these ligands is not surprising in that catalyst turnover was not required in the on-resin screen, since the number of peptide and alkyne units was roughly equivalent on each bead. The magnitude of rate acceleration relative to the no-peptide control was approximately 80-fold for THPTA at the relatively low copper concentrations used. Hexahistidine (Table 1, entry 3), and alternating hisitine sequences [(HA)x repeats, entries 4−6] gave poor reactivity compared to an active sequence HALHAAMA (entry 2, 38-fold rate acceleration relative to no ligand). Met-for-His substitutions were also made to adjust Cu binding affinity44,45 (entries 10−16), mostly without good effect. However, HAAHAAMAMA (entry 12) provided a good stoichiometric Cu complex (approximate 50-fold rate enhancement). We suspect that the N-terminal HXXH motif drives most of the reactivity, but the methionine residues may be responsible for a more restricted coordination environment, disfavoring catalyst turnover.44 Previous explorations of small-molecule ligands revealed that the sensitivity of catalyst activity to ligand:Cu ratio depends on the Cu-binding ability of the constituent heterocycles of the ligand, with tighter-binding systems shown to diminish reactivity by sequestering the metal in nonactive form(s).26,43 This parameter also proved to be important for these peptidebased systems, as shown with (HAA)-repeat sequences in Figure 4B,C. Here, methionine proved to be an active Cubinding component, inferred by the observation that the most active complexes arose from shorter sequences terminated in HAMA (Table 1, entries 2 and 7) or a longer sequence in which the terminal Met was replaced with His (entry 16). In all of these cases, the most active formulations were of 1:1 or 0.5:1 ligand:Cu stoichiometry. Note also the striking difference in CuAAC activity of (HAA)3HAMA at this ligand:Cu ratio (entry 15, very little activity) vs (HAA)3HAHA (entry 16, the highest activity observed, 58-fold faster than the no-ligand reaction and more than one turnover). Furthermore, while the addition of more HAA tripeptides onto the sequence did not enhance the reaction rate or yield significantly (Supporting Information, Figure S3), it was also not inhibitory. Thus, the eventual goal of in vivo expression and function of a peptide-based CuAAC ligand might be aided by the use of longer HXX repeats, allowing for effective reactions at low Cu-ligand concentrations.

Figure 3. (A) On-resin assay of identified peptides from OBOC libraries. [CuSO4] = 33 μM; [1] = 330 μM; [Na ascorbate] = 5 mM; 3 mg resin (0.4 μmol peptide + 0.4 μmol alkyne); solvent = 200 μL, 20:80 H2O:DMF. (B) In solution assay of three synthesized peptides, identified in panel A. [CuSO4] = 33 μM, [propargyl alcohol] = 100 μM, [1] = 80 μM, [Na ascorbate] = 5 mM, solvent = 95:5 H2O:DMSO.

A (HDSDPGAMA) and peptide C (QCKPHFMA) gave rise to little or no reaction. This was somewhat surprising, given the presence of only one strongly coordinating residue (His) in peptide A, compared to two His residues in peptide B. While we cannot assign a reason for this variation in metal:ligand ratio tolerance, it may be worth noting that some highly active tris(heterocyclic amine) based ligands such as THPTA show a similar, and unusual, tolerance of high ligand:Cu ratios;42 peptide B seems to follow suit. Most of the active ligands identified in these initial library screens contained the HXXH sequence, where X is an amino acid other than the Cu-binding His, Met, or Cys. Further investigation therefore focused on this motif using peptidebased catalysts in solution, since solid-phase results were not completely predictive of solution-phase behavior. Varying the sequence of the key tetrad (HXXH) among HAAH, HILH, HALH, or HSDH had little effect, but the HXXH motif was essential. Thus, scrambled versions of HALHRDKAMA (Figure 4A) and HAAHAMA (Figure S6) provided very sluggish reactivity, barely faster than the nonliganded CuAAC reaction, showing that the sequence, and not merely the histidine D

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Figure 4. (A) Effect of scrambling amino acid sequence on CuAAC reactivity. (B) Representative screen of ligand:Cu ratio on the solution-phase reaction of 1 + 2. [CuSO4] = 33 μM, [propargyl alcohol] = 100 μM, [1] = 80 μM, [Na ascorbate] = 11 mM, solvent = 95:5 H2O:DMSO. (C) Plot of data in Table 1, entries 7, 14, 15.

Figure 5. Representative kinetic measurements for peptide-based CuAAC catalysts. Apparent rate constants given in the figure are for the initial stages of the reaction (in most cases, over the first 30%, representing one turnover).

Expression of long peptides would not be an inconvenience in the cell, as it is for molecular synthesis in the laboratory. In addition to exploring how varied the sequence could be, we also tested the tolerance of sequences at the N- and Cterminal ends and the nature of the XX identity in the sequence. It was found that installation of other amino acids at the N-terminus abrogated activity (Figure S4), highlighting the importance of N-terminal histidine, while substitution at the Cterminus was tolerated (Figure S4). As with on-resin screening, the identity of the XX dipeptide between the key His residues was not of critical importance in solution-phase reactions. For example, HAAH was modestly more active than HADX (Figure S5), but the ability to manipulate properties such as charge without sacrificing reactivity to a great degree may prove useful. The tolerance of the HAAH-based peptide CuAAC complexes toward different alkyne substrates was probed in preliminary fashion as shown in Figure 6. In the presence of peptide ligands several alkynes (propargylamine, dimethyl propargyl malonate, propargyl acetate, 3-butyn-2-ol) underwent CuAAC reaction with coumarin azide 1 to a greater degree than the no-ligand control, but others (homopropargyl glucoside, propargyl glycine, and undecynoic acid) did not. Detailed exploration of this selectivity must await the development of

more active peptide-based catalysts. No difference was noted in the reactivity of the two enantiomers of 3-butyn-2-ol, although it is rather difficult to transmit chirality from Cu-binding ligands to the substrates.46−48 In a preliminary probe of CuAAC activity of a selected peptide as an appended motif, we attached sequence HSDHADAMA-Aza (where Aza = β-azidoalanine) to the Qβ viruslike particle42 with an average of two peptides per coat protein (360 per particle). These particles were then used as the ligand for the CuAAC reaction at low concentrations of peptide (to reflect potential application conditions; 2.5 μM particledisplayed peptide, 50 μM CuSO4, 5 mM NaAsc, 80 μM coumarin-azide, 100 μM propargyl alcohol). Unfortunately, no rate enhancement and minimal increase in conversion relative to peptide-free conditions were observed. Similar attachment of azide-functionalized HALHAAMA, HAAHAMA, and HAAHAAHAAHAMA sequences resulted in irreversible aggregation of the resulting particles, even in the presence of EDTA solution to sequester copper ions thought to be responsible for particle crosslinking. The CuAAC-accelerating effectiveness of these peptides was therefore not assessed. E

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Table 1. Representative Initial Rate Constants and Extent of Reaction for CuAAC Reactions of Coumarin Azide (1) + Propargyl Alcohol (2) in the Presence of the Indicated Peptides ([Cu] = 33 μM, [2] = 100 μM, [1] = 80 μM)a

a

Each peptide had a free amino group at the N-terminus and primary amide at the C-terminus. Highlighted in green are those reactions giving more than one turnover. Reactions providing 95% purity by HPLC, the peptides were stored as lyophilized powders until use. Test solution-phase reactions were performed in microtiter plates, beginning with the charging of each well with aqueous CuSO4 (0.3 mM, 22 μL, final concentration 33 μM). Stock solutions of peptide ligands were added to establish 0.5:1, 1:1, and 2:1 ligand:copper ratios, each condition in triplicate. The solubility of the ligand and reagents was ensured by the use of 10 μL DMSO in a final reaction volume of 200 μL. The CuSO4 solution and ligand solution was incubated for 5 min at room temperature. Stock solutions of propargyl alcohol and coumarin azide were added in that order to final concentrations of 100 μM and 80 μM, respectively. Each reaction was initiated by addition of freshly prepared aqueous sodium ascorbate (final concentration 5 μM) after the plate was in place on the reader tray to minimize the time required for the start of data acquisition.

CONCLUSIONS An on-bead synthesis and screening protocol was used to identify short oligopeptides having the ability to accelerate the copper-catalyzed azide−alkyne cycloaddition reaction. A simple histidine-based tetrapeptide motif was identified that mediated CuAAC ligation up to approximately 50-fold faster than the ligand-free process in the presence of CuI at a concentration (33 μM) modestly lower than the 50 μM threshold identified earlier as a cutoff for reliable active catalyst formation in water.49 The importance of amino acid sequence, rather than just histidine content, was illustrated by the observation that scrambled or oligo-hisitidine sequences were ineffective. This initial effort did not yield ligands with robust substrate tolerance or high Cu-binding affinity, properties necessary for a functional in vivo catalyst, but the combinatorial platform demonstrated here can be used for selections under a wide variety of conditions, including those that more closely mimic the intracellular environment.

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EXPERIMENTAL SECTION

Library Construction and Screening. One-Bead-OneCompound Library Fmoc-Protected Synthesis. NovaSyn resin (90 μm average particle size, 0.26 mequiv/g amino groups, 0.20 g, 0.050 mmol) was allowed to swell in DMF (10 mL) for 30 min in a tared fritted syringe with gentle automatic rotation, followed by removal of the excess solvent by vacuum filtration. Undecynoic acid (0.5 equiv, 4.55 mg, 0.025 mmol) was added to a 0.4 M solution of HBTU in DMF (0.063 mL, 0.025 mmol, 0.5 equiv), followed by the addition of diisopropylethylamine (10 μL, 0.05 mmol, 1 equiv). The resulting solution was mixed by vortexing and pulled into the resin syringe. The reaction mixture was incubated for 1 h on a rotator. The solution was removed from the syringe by vacuum filtration, and the resin was flow washed with DMF (15 mL, 3 rounds of filling up the fritted syringe containing the resin with DMF, shaking/stirring it and then dispelling the DMF). The remaining resin linkage sites were then addressed by Fmoc-protected alanine (46.7 mg, 0.15 mmol, 3 equiv) using the same procedure as the alkyne coupling (0.4 M HBTU in DMF: 0.375 mL, 0.15 mmol, 3 equiv; iPr2EtNH2: 0.018 mL, 0.3 mmol, 6 equiv). The Fmoc group was deprotected by two sequential treatments with piperidine (20% v/v in DMF, 5 mL) for 5−7 min, followed by washing with DMF. Fmoc-methionine (55.7 mg, 0.15 mmol, 3 equiv) was then coupled and deprotected by the same procedure. The resin was then split into the appropriate number of fritted syringes according to the number of amino acids to be placed at position 1 of the desired peptide sequence. Each amino acid (0.15 mmol, 3 equiv) was coupled according to the same Fmoc-protected synthesis protocol. The resin from all syringes was recombined, Fmoc deprotected, split, and coupled as above until the desired library sequences were completed. Library Screening. The constructed libraries were screened for activity in the CuAAC reaction as follows: N-terminal and side chain deprotection was performed by incubation with a mixture of 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane (TIS) (total volume: 5 mL) for 1 h. The resin was then flow washed with dichloromethane (15 mL, 3 rounds of filling up the fritted syringe containing the resin with CH2Cl2, shaking/stirring it, and then dispelling the solvent) and then dried under vacuum. A sample of 3.0 mg, corresponding to 0.4 μmol of alkyne, was suspended in DMF

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00161.

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Details of on-bead and solution-phase peptide synthesis, characterization, and kinetic analysis (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: mgfi[email protected]. Phone: 404-385-0906. ORCID

M. G. Finn: 0000-0001-8247-3108 Notes

The authors declare no competing financial interest. G

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(17) Biaglow, J. E., Manevich, Y., Uckun, F., and Held, K. D. (1997) Quantitation of hydroxyl radicals produced by radiation and copperlinked oxidation of ascorbate by 2-deoxy-D-ribose method. Free Radical Biol. Med. 22, 1129−1138. (18) Uttamapinant, C., Tangpeerachaikul, A., Grecian, S., Clarke, S., Singh, U., Slade, P., Gee, K. R., and Ting, A. Y. (2012) Fast, cellcompatible click chemistry with copper-chelating azides for biomolecular labeling. Angew. Chem., Int. Ed. 51, 5852−5856. (19) Uttamapinant, C., Sanchez, M. I., Liu, D. S., Yao, J. Z., and Ting, A. Y. (2013) Site-specific protein labeling using PRIME and chelationassisted click chemistry. Nat. Protoc. 8, 1620−1634. (20) Bevilacqua, V., King, M., Chaumontet, M., Nothisen, M., Gabillet, S., Buisson, D., Puente, C., Wagner, A., and Taran, F. (2014) Copper-Chelating Azides for Efficient Click Conjugation Reactions in Complex Media. Angew. Chem., Int. Ed. 53, 5872−5876. (21) Su, Y., Li, L., Wang, H., Wang, X., and Zhang, Z. (2016) All-inOne azides: empowered click reaction for in vivo labeling and imaging of biomolecules. Chem. Commun. 52, 2185−2188. (22) Notni, J., and Wester, H. J. (2016) A Practical Guide on the Synthesis of Metal Chelates for Molecular Imaging and Therapy by Means of Click Chemistry. Chem. - Eur. J. 22, 11500−11508. (23) Clavadetscher, J., Hoffmann, S., Lilienkampf, A., Mackay, L., Yusop, R. M., Rider, S. A., Mullins, J. J., and Bradley, M. (2016) Copper Catalysis in Living Systems and In Situ Drug Synthesis. Angew. Chem., Int. Ed. 55, 15662−15666. (24) Sun, Y., Hong, S., Ma, X., Cheng, K., Wang, J., Zhang, Z., Yang, M., Jiang, Y., Hong, X., and Cheng, Z. (2016) Recyclable Cu(I)/ Melanin Dots for Cycloadditions, Bioconjugation and Cell Labeling. Chem. Sci. 7, 5888−5892. (25) Li, S., Wang, L., Zhu, Z., Shobaki, D., Chen, H., Wang, M., Wang, J., Qin, G., Erasquin, U. J., Ren, L., et al. (2017) Coppercatalyzed click reaction on/in live cells. Chem. Sci. 8, 2107−2114. (26) Rodionov, V. O., Presolski, S. I., Gardinier, S., Lim, Y. H., and Finn, M. G. (2007) Benzimidazole and related ligands for Cu-catalyzed azide-alkyne cycloaddition. J. Am. Chem. Soc. 129, 12696−12704. (27) Harford, C., and Sarkar, B. (1997) Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) Motif of Proteins and Peptides: Metal Binding, DNA Cleavage, and Other Properties†. Acc. Chem. Res. 30, 123−130. (28) Krattiger, P., McCarthy, C., Pfaltz, A., and Wennemers, H. (2003) Catalyst-Substrate Coimmobilization: A Strategy for Catalysts Discovery in Split-and-Mix Libraries. Angew. Chem., Int. Ed. 42, 1722− 1724. (29) Krattiger, P., Kovasy, R., Revell, J. D., Ivan, S., and Wennemers, H. (2005) Increased Structural Complexity Leads to Higher Activity: Peptides as Efficient and Versatile Catalysts for Asymmetric Aldol Reactions. Org. Lett. 7, 1101−1103. (30) Xia, G., Chen, L., Sera, T., Fa, M., Schultz, P. G., and Romesberg, F. E. (2002) Directed Evolution of Novel Polymerase Activities: Mutation of a DNA Polymerase into an Efficient RNA Polymerase. Proc. Natl. Acad. Sci. U. S. A. 99, 6597−6602. (31) Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T., and Cuervo, J. H. (1991) Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354, 84−86. (32) Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmierski, W. M., and Knapp, R. J. (1991) A new type of synthetic peptide library for identifying ligand-binding activity. Nature 354, 82−84. (33) Presolski, S. I., Mamidyala, S. K., Manzenrieder, F., and Finn, M. G. (2012) Resin-supported catalysts for CuAAC click reactions in aqueous or organic solvents. ACS Comb. Sci. 14, 527−530. (34) Sivakumar, K., Xie, F., Cash, B. M., Long, S., Barnhill, H. N., and Wang, Q. (2004) A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3-Azidocoumarins and Acetylenes. Org. Lett. 6, 4603−4606. (35) Alterio, V., Hilvo, M., Di Fiore, A., Supuran, C. T., Pan, P., Parkkila, S., Scaloni, A., Pastorek, J., Pastorekova, S., Pedone, C., et al. (2009) Crystal structure of the catalytic domain of the tumorassociated human carbonic anhydrase IX. Proc. Natl. Acad. Sci. U. S. A. 106, 16233−16238.

ACKNOWLEDGMENTS This work was supported by The Shurl and Kay Curci Foundation, the Georgia Institute of Technology, a Department of Education Graduate Assistance in Areas of National Need (GAANN) Molecular Biophysics and Biotechnology Fellowship (to A.G.A), and the Georgia Tech Undergraduate Research Opportunities Program (President’s Undergraduate Research Award to L.D.). We thank Ms. Erin Geoghan and Mr. Nick Daniel for developing the program used to determine peptide molecular weight for the OBOC libraries.

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DOI: 10.1021/acs.bioconjchem.7b00161 Bioconjugate Chem. XXXX, XXX, XXX−XXX