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. 2025 Aug 19;147(35):31811–31820. doi: 10.1021/jacs.5c08936

Late-Stage Aromatic C–H Bond Functionalization for Cysteine/Selenocysteine Bioconjugation

Zhenguang Zhao †,‡, Jian Huang †,§, Yao Cai †, Tai-Ping Zhou ∥, Fatina Khatib †, Daphna Shimon †,*, Binju Wang ∥,*, Norman Metanis †,⊥,#,*
PMCID: PMC12412152  PMID: 40828607

Abstract

Bioconjugation of peptides and proteins has become an indispensable tool in fundamental biological research and drug development. Herein, we report a copper-mediated efficient cysteine/selenocysteine-specific bioconjugation through direct C–H functionalization of electron-rich arenes under biocompatible reaction conditions. In this method, a series of commercial electron-rich arenes, including natural products and drug molecules, are conjugated to cysteine/selenocysteine-containing peptides and proteins. Furthermore, we show that this new bioconjugation method allows the efficient stapling of peptides, as well as the cross-linking of different peptides to a single arene, all in high yields. The tunable electron density of small molecules enables the selective modification of selenocysteine in the presence of cysteine residues. Finally, mechanistic studies suggest that the conjugation proceeds via a proton-coupled electron transfer (PCET) process and substrate radical binding to the copper for C–Se/S bond formation. This approach provides an efficient strategy for the late-stage functionalization of complex small molecules to generate peptide/protein conjugates.

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Introduction

Peptide and protein bioconjugation has significantly advanced our understanding of biological processes, such as the actions of complex biomolecules, the development of therapeutics e.g., antibody-drug conjugates (ADCs), and the construction of new functional materials. However, achieving efficient and chemoselective functionalization of peptides and proteins remains challenging due to the presence of diverse reactive functional groupsincluding thiols, acids, alcohols, and aminesespecially under mild biocompatible conditions. To address these challenges, chemists have developed chemo- and regioselective reactions for modifying naturally occurring amino acids in peptides and proteins. Cysteine (Cys, C) is frequently targeted due to its high intrinsic reactivity and relatively low abundance in proteins. Similarly, selenocysteine (Sec, U), the 21st encoded amino acid, presents an attractive alternative for chemoselective functionalization , to Cys and its even lower abundance. Nevertheless, many existing strategies require the preinstallation of reactive functional groups on the target amino acids (Figure a) or payload reagents (Figure b,c) if not commercially available.

1.

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Representative strategies of Sec/Cys-specific bioconjugation. (a) Premodification of Cys/Sec to create a reactive site in target proteins. (b) An additional step to install a reactive functional group on target molecules for Cys/Sec bioconjugation. (c) Electrochemical and photocatalyzed diselenide contraction for Sec modification. (d) Direct aromatic C–H bond functionalization with free Cys/Sec for protein diversification.

The direct coupling of an aromatic C–H bond in a complex molecule with the S/Se–H bond of Cys/Sec represents a straightforward and ideal approach for protein bioconjugation. Direct C–H bond thiolation of arenes with thiols and selenylation with diaryl selenides has been demonstrated. Unfortunately, these techniques are typically performed between small molecules and under harsh conditions, which are incompatible for proteins. A more recent method involves the direct replacement of an aromatic C–H bond in electron-rich arenes with the electrophilic Sec, enabling the chemoselective modification of peptides and proteins (Figure a). However, to induce the electrophilic character in Sec, it must be prefunctionalized with 2-thiol-5-nitropyridine (TNP), which carries the risk of disrupting other Cys residues in the protein.

We previously reported the chemoselective modification of Sec in peptides and proteins using various hydrazine compounds (Figure b). This efficient transformation was enabled by copper-mediated in situ generation of alkyl/aryl radicals from hydrazine substrates. Despite the broad scope and biocompatible conditions of this approach, it required the presence of a hydrazine moiety to facilitate the radical intermediate formation, followed by coupling with Sec. However, the need to preinstalled hydrazine on target molecules may reduce the overall efficiency of protein modification. Additionally, further modification of hydrazine molecules is challenging due to their sensitivity to oxidation. Encouragingly, conjugating N-heterocycles to Sec in oligopeptides has been achieved via a photocatalytic reaction, although it was implemented in acetonitrile. A direct coupling of a C–H bond in target molecules with a S/Se–H bond in Cys/Sec under mild conditions is highly desired (Figure d), which would provide an efficient solution for constructing peptide/protein-natural products or drug conjugates, eliminating the need for substrate prefunctionalization.

Here we report an efficient chemoselective modification of peptides and proteins through Cys/Sec-bioconjugation with a series of electron-donating arenes (Figure d). This method leverages a copper-mediated coupling reaction between an aromatic C–H bond and the S/Se–H bond under biocompatible conditions, enabling the bioconjugation of peptides and proteins.

Results and Discussion

In our previous efforts to study chemoselective radical-mediated arylation of Sec, we explored the use of electron-rich resorcinol (2a) as an alternative for the hydrazine substrates to generate the appropriate aromatic radical for Sec bioconjugation. Although resorcinol remained inert toward the Sec-containing peptide (TF U GK) in a neutral aqueous buffer, even after a prolonged reaction time, intriguingly, we observed a moderate conjugation of resorcinol to Sec in the presence of CuSO4 at pH 7 (Figures S1 and S2). To systematically optimize Sec bioconjugation with resorcinol, we prepared a Sec-containing model peptide, LG U ALG-NH2 (1, isolated as a diselenide dimer under atmospheric conditions due to the lower pK a and redox potential of Sec), , to better assess the regioselectivity in its conjugation with resorcinol. We tested various metal ion additives for the conjugation of 1 (1 mM) and 2a (5 mM) at 37 °C in phosphate buffer (PB) (Figure S5), and an appreciable 93% conjugation was achieved using Cu­(OTf)2 (1 mM) in only 15 min (Figure a, entry 1). After characterization by LC-MS, 1D and 2D NMR experiments, including H–H correlation spectroscopy (COSY, Figure b) and heteronuclear single quantum correlation (HSQC) (Figures b and S110–S114), it was indicated that the C–H bond at C-4 of resorcinol was substituted by a C–Se bond in the primary monosubstituted conjugate 3a 1 . Additionally, a disubstituted conjugate 3a 2 was produced in 6% conversion (Figure S10). A pH screening study revealed the Sec conjugation with resorcinol proceeded sluggishly under acidic conditions (Figure S3). When excess peptide 1 reacted with 2a, disubstituted conjugates predominated, and a trisubstituted conjugate 3a 3 was also observed (Figure S7), suggesting the potential for peptide stapling, dimerization, and cross-linking different peptides and proteins (see below). Given the challenges associated with incorporating Sec into proteins, we expected that a Cys bioconjugation might also be feasible using the same strategy. To this end, Sec was replaced with Cys in the model peptide 4 (LG C ALG-NH2), and its reaction with 2a (3 mM) under optimized conditions resulted in the formation of a single monosubstituted conjugate 5a with 89% conversion within 1 h (Figures c and S24).

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Development of direct aromatic C–H bond functionalization with Cys/Sec for peptide diversification. (a) Optimization of reaction conditions with Sec-containing peptide. (b) The characterization of conjugate 3a 1 by NMR. Further details can be found in the Supporting Information. aYields were calculated based on the integrated areas of HPLC peaks (at 220 nm). (c) The optimal reaction system is transferable to Cys-containing peptide functionalization.

To establish a general method for Cys/Sec-bioconjugation via aromatic C–H functionalization, we evaluated the scope of electron-rich arenes using peptides 1 (LG U ALG-NH2) and 4 (LG C ALG-NH2) in parallel. As shown in Figure , a series of resorcinol derivatives (3b–3f, Figures S11–S15 and S25–S29) demonstrated broad compatibility in modifying both Cys and Sec residues. For Sec, occasional disubstituted conjugates were observed, while Cys yielded exclusively monosubstituted conjugates. Phenol (2m) and 1,3,5-trimethoxybenzene (2n) were found to be inert to both Cys and Sec under the optimized conditions (Figures S22 and S23). Strong electron-donating groups (2g–2i) enhanced the reactivity to both Cys/Sec (Figures S16–S18 and S30–S32). However, m-cresol (2j) and 3,5-dimethylphenol (2k) were ineffective for Cys conjugation (Figures S33 and S34). Additionally, m-phenylenediamine (2l), a diamino analogue of resorcinol (2a), exhibited no reactivity toward Cys under the same conditions (Figure S35).

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(a) Sec/Cys-specific conjugation with a series of small molecules. The conversions shown in parentheses were calculated based on the integrated areas of HPLC peaks. Each conjugate is shown with a red bond highlighting the new linkage formed in the reaction and a gray ball as the Sec or Cys peptide. Standard conditions for Sec-peptide 1: 1 mM peptide 1 with respect to the selenol monomer, 1 equiv copper, and 5 equiv small molecule, 15 min; standard conditions for Cys-peptide 4: 1 mM peptide 4, 1 equiv copper, and 3 equiv small molecule, 1 h. a1 equiv bipyridine was used. b0.5 equiv copper and bipyridine were used. c1 equiv bipyridine was used, and the reaction was conducted in a degassed solution. d3 equiv copper, bipyridine, and 10 equiv small molecule were used. e1 equiv copper, bipyridine, and 10 equiv small molecule were used, and the reaction was conducted in a degassed solution. f5 equiv copper, bipyridine, and 10 equiv 6f were used, and the reaction was conducted in a degassed solution. g1 equiv copper and bipyridine, and 1.5 equiv 9d were used. h1 mM peptide 1, 1 equiv copper and bipyridine, and 1.5 equiv 9g were used. (b) ROESY for vancomycin in the peptide–vancomycin conjugate 10d.

We also explored the compatibility of the modification reaction with various heteroaromatic compounds with different scaffolds, including pyrrole (6a-b), indole (6c-e), quinolinediol (6f), coumarin (6g) and pyridine (6h), and found the reaction to be with wide scope of substrates (Figures and S36–S50).

Intriguingly, all the products were singly monosubstituted conjugates, with the exception of 7e. These findings demonstrate that the reactivity of electron-rich arenes toward Cys and Sec can be tuned by incorporating different electron-donating groups, enabling selective modification of Sec over Cys in peptides, or even sequential modifications of these residues.

Peptide-drug conjugates (PDCs) have emerged as a promising direction for cancer therapy due to their small molecular weight and low immunogenicity. A critical aspect of PDC development is the selective conjugation of drug molecules to a targeted peptide. To this end, we investigated the potential of aromatic C–H functionalization of Cys/Sec residues to facilitate the construction of PDCs with complex functional molecules. Natural products like resveratrol (9a), genistein (9b), serotonin (9e) and β-carboline harmol (9f), and drug molecules like vancomycin (9d) and phentolamine (9g), were all readily linked to peptide 1 via Sec conjugation (Figures S52–S53 and S55–S58), and minimal disubstitutions were observed in the resulting conjugates (10a–10b, 10f and 10d). Additionally, the conjugation of biotin (9c) to peptide 1, achieved through coupling with phloroglucinol and Sec, demonstrated the feasibility of Sec functionalization for nonaromatic molecules (Figure S54). Meanwhile, resveratrol (9a) and serotonin (9e) were also smoothly conjugated to Cys residues with excellent yields (Figures S59 and S63). These findings highlight the potential of Cys/Sec-based aromatic C–H functionalization as a versatile strategy for generating PDCs with diverse functional molecules.

To assess the applicability of this conjugation approach to complex target molecules, we aimed to evaluate the chemoselectivity and the impact of the chemical environment on conjugation reactivity. First, two negative controls of Sec/Cys-free peptides, 12 and 13, while containing aromatic residues Trp and Tyr, were found to remain inert toward 2e under the standard conditions (Figures S66 and S67), even after extended reaction time. Two additional peptides containing Sec (14) or Cys (15), respectively, were reacted with 2a or 2e. Both reactions yielded single monosubstituted conjugates with good conversion, despite the presence of other reactive amino acids in their sequences (Figures a, S68, and S69). MS/MS analysis of 15e confirmed conjugation at the Cys residue (Figure a). Given the complex chemical environment in most biomolecules, we evaluated its impact on our conjugation efficiency. As shown in Figure b, disubstituted products (two peptides linked to one small molecule) were observed in both Sec- and Cys-conjugations when Sec/Cys was located at the N-terminus or adjacent to amino acids with small steric hindrance. In contrast, only single monosubstituted products were obtained when Sec/Cys in the middle sequence was buried by amino acids with larger steric hindrance. These data demonstrate a significant impact of the chemical environment on conjugation reactivity, providing insights into the design of controllable conjugate production.

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Sec/Cys-specific modification in the presence of other reactive residues. (a) Chemoselectivity study with MS/MS spectra of 15e. aPeptide (0.1 mM), 2e (1 mM), and Cu­(OTf)2 (2 equiv) were used. (b) Evaluate the influence of chemical environment on conjugation reactivity. bCu­(OTf)2 (0.3 equiv) was used. cCu­(OTf)2 (8 equiv) was used. dPeptide (0.1 mM), 2e (1 mM), and Cu­(OTf)2 (3 equiv) were used. *Refers to disubstituted conjugates: two peptides conjugated with the identical molecule. The conversions were calculated based on the integrated areas of HPLC peaks.

Peptide cyclization is a useful strategy for stabilizing short and flexible peptides into well-defined bioactive stapled peptides, which can have improved biological properties, including increased resistance to proteolytic degradation, enhanced cell permeability and higher-affinity binding toward its intended biological target. To evaluate the applicability of our method for constructing stapled peptides, we used phloroglucinol 2e as the cross-linking agent to staple Sec and/or Cys at various positions within the peptides with the following sequences: G U ANKHTWYL U A-NH2 (20), G U ALNKFQEKSRMKYRWKHR C G-NH2 (21), and G C ANKHTWYL C A-NH2 (22). As shown in Figure a, the cyclization of peptides 20 and 22 was achieved through the stapling of Sec-Sec (Figure S75) and Cys-Cys (Figure S79), respectively, with the yield of 90% and 89%, although 25% oxidation (+32 Da) in the case of stapled peptide 22e was observed. Staple Sec-Cys in peptide 21 was also successfully constructed with 94% conversion (Figure S77). To confirm the stapled residues in the peptides, stapled peptides 20e, 21e, and 22e were digested by trypsin and subjected to HPLC-ESI MS analysis (Figures S76, S78, and S80). The analysis indicated conjugation exclusively at the Sec/Cys over the rest of the amino acids.

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(a) Peptides stapling with Sec/Cys conjugation. The conversions shown in parentheses were calculated based on the integrated areas of HPLC peaks. aReaction conditions: 0.5 mM peptide, 1 equiv Cu­(OTf)2 and 5 equiv 2e were used. bReaction condition: 0.1 mM peptide, 10 equiv Cu­(OTf)2 and bipyridine, and 20 equiv 2e were used, and the reaction was conducted in a degassed solution, 25% is attributed to the oxidized conjugates (+32 Da). (b) Representative crude HPLC and ESI-MS spectrum for the stapled peptide 20e. (c) One-pot stepwise cross-linking of different peptides. (d) Representative HPLC and ESI-MS spectrum of cross-linked conjugates 25 prepared using purified 5f. (e) Representative HPLC and ESI-MS spectrum of the cross-linked conjugates 28 in one pot.

In addition, ICP-MS analysis showed that less than 1 ppm copper remained in purified peptide conjugates (Table S6).

The reaction of phloroglucinol 2e with an equimolar mixture of peptides 1 and 4 yielded a single Sec-conjugate 3e, with no Cys-conjugation observed. The distinct reactivity of Sec and Cys toward electron-rich arenes suggests that cross-linking different peptides can be achieved through a stepwise conjugation of “Se-aryl-S” in one pot. We initially performed the reaction of Cys-containing peptide 4 with phloroglucinol 2e, and LC-MS indicated quantitative conversion within 1 h. Subsequently, an additional Sec/Cys-containing peptide and fresh copper were added to complete the cross-linking reaction in one pot (Figure c). Using this strategy, we produced a series of cross-linked peptides (24–28) in good yields (Figures c–e and S81–S85). Notably, purifying the conjugates produced in the initial step and then using these purified conjugates for the subsequent cross-linking reaction significantly enhances the conversion of the final cross-linked products compared to performing the cross-linking reaction in one pot. For example, up to 90% conversion could be achieved in the production of cross-linked products 25 using peptide 1 and purified conjugate 5f (Figure d), compared to 67% conversion observed when conducted in a one-pot manner without an intermediate purification step (Figure S82).

To further examine our approach’s robustness, we turned our attention to functional modification of proteins (Figure ). To achieve this goal, the protein ubiquitin(2–76)­(Q2U) (29) and ubiquitin(2–76)­(Q2C) (30) were prepared by chemical protein synthesis, including Fmoc-SPPS, native chemical ligation (NCL) Fmoc-protected selenazolidine, and deselenization processes (Figures S89 and S90). Also, the Sec-containing (37) and the Cys-containing (38) variants of the trihelical affibody protein ZHER2 were synthesized using Fmoc-SPPS. In addition to these, we utilized protein expression to obtain ubiquitin(1–76)­(M1S, Q2C) (34) and wildtype (WT) ubiquitin(1–76)­(M1S) (35) (Figures S93 and S94). The treatment of ubiquitin(2–76)­(Q2U) 29 with 20 equiv of serotonin 9e, Cu­(OTf)2, and bipyridine for 3 h yielded the conjugated product 31 with a 93% yield (Figure ). The remaining 7% was attributed to the deselenization of 29, likely due to the extended reaction time in a basic solution (Figure S95). Using Vancomycin 9d, a potent pharmaceutical molecule, as a labeling reagent for 29, a 79% conversion was achieved within 10 h (Figures and S96). Subsequently, the functionalization of ubiquitin(2–76)­(Q2C) 30 with 9e resulted in the conjugate 33 with a 50% conversion within 3 h (Figures and S97), which was validated by the 50% conjugation of expressed ubiquitin(1–76)­(M1S, Q2C) 34 with 9e (Figure S98). WT ubiquitin(1–76)­(M1S) 35, used as a negative control, remained inert toward 9e under the same conditions (Figure S99). These results demonstrate the excellent chemoselectivity of our developed conjugation chemistry for Sec and Cys. Furthermore, ZHER2, a 58-amino-acid affibody molecule, exhibits selective binding and high affinity to HER2, consequently, its bioconjugates hold promising applications in diagnosing and treating various types of cancers. Hence, ZHER2 containing a C-terminal Sec (37) or Cys (38) was used as alternative protein model to further exemplify the biocompatibility of our approach. 9e was successfully installed on the proteins 37 and 38 with 80% (39) and 70% (41) conversion (Figures , S100, and S102), respectively. While 40% conjugation of 37 with 9d (Figures and S101), the reaction of 37 with 9d was significantly more sluggish.

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Protein modification. (a) General scheme for protein modifications. (b) ESI-MS spectra for both unmodified and modified proteins.

To obtain insight into the mechanism of copper-mediated Sec/Cys-specific conjugation with electron-rich arenes, we conducted extensive investigations combining experimental studies and performed density functional theory (DFT)-based calculation. Resorcinol can readily form free radicals in the presence of Cu­(II) and O2, however, radical scavengers such as TEMPO, DMPO, BHT, and 1,1-diphenylethylene, did not inhibit the reaction under standard conditions (Figure S108). While the reaction was significantly inhibited when conducted in DMSO with DMPO as a radical scavenger, we did not observe the formation of DMPO-resorcinol adduct (Figure S108). Yet, using electron paramagnetic resonance (EPR) spectroscopy, we observed a signal highly indicative of an aryl carbon radical in the presence of both Cu­(II) and phloroglucinol 2e, which diminished by exposing to peptides containing either Cys or Sec (Figures S103–S107), but not Ala. These results may suggest an aromatic carbon-centered radical intermediate is involved during the reaction mechanism.

We next investigated the process of aromatic radical reaction with Sec. Free phenyl radicals can be readily captured by Sec to form Se–C bonds, as confirmed by EPR study in previous work. To determine whether Sec conjugation with electron-rich aromatic radicals occurs through a substrate radical binding to copper or direct capture by Sec, we performed DFT calculations (see Supporting Information for details). The Sec-peptide-Cu­(II) complex 1a is reduced by 2a via an exergonic proton-coupled electron transfer (PCET, Scheme S8a) process facilitated by a water cluster (the reaction becomes more favorable as the number of water molecules in the cluster increases, see Scheme S9), generating the Cu­(I)-coordinated Sec-peptide (1a 1 ) and the radical of 2a (2a·). Next, 1a 1 and 2a· form the reaction complex (RC) in the substrate radical binding route, which is exergonic by 7.1 kcal/mol (Figure ). Analysis of the spin population of RC reveals that the spin populations on Cu and C1 are 0.43 and 0.13, respectively (Figure ). The radical properties of 2a· are delocalized to Cu­(I) ions and aromatic ring, as indicated by the spin population results, explaining why radical signals were difficult to detect experimentally. Subsequently, the Se1–C1 bond forms via TS1 with a free energy barrier of 19.5 kcal/mol, which is significantly favorable compared to the substrate radical-free route (directly captured by Sec, see Scheme S8), leading to intermediate Int1. The path from RC to Int1 is a concerted process through TS1, which corresponds to Se1–Se2 bond homolytic and Se1–C1 bond formation. Finally, Int1 undergoes tautomerization, yielding the product Int2 that is 9.1 kcal/mol lower than 1a 1 and 2a·, indicating thermodynamically favorable process for the entire reaction (Figure ).

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DFT-calculated energy profile on Cu­(II)-mediated modification reaction of Sec-peptide. Distances are given in Å. The spin population on key atoms is shown in red italics. The imaginary frequencies for transition states are also shown.

In short, the DFT calculations support our conclusion that substrate 2a undergoes PCET first, followed by binding to the Cu ion center, and subsequent Se–C bond formation. Overall, the reaction is exergonic by 9.5 kcal/mol with a rate-determining barrier of 19.5 kcal/mol, indicating that the reaction could proceed facilely. Beyond the radical-based mechanism, we also explored two additional pathways (B and C, see Scheme S8) for the reaction process, inspired by Buchwald and Pentelute’s work , (Scheme S8), but were found to be less feasible compared to our proposed mechanism (Figure S109), and therefore were excluded. While this mechanism focuses on a monomeric copper catalyst, alternative pathways involving multicopper clusters may also be possible. In the case of Cys bioconjugation, comparable efficiency was observed when the Cys disulfide dimer, but not reduced Cys, especially under N2 atmosphere (Figure S110), was used as the starting material (Figure S24b), suggesting that the reaction proceeds via the same mechanistic pathway as proposed for the Sec. This was further supported by DFT calculations (see Schemes S9 and S10).

Conclusions

In conclusion, we have described a method that is both efficient and broadly applicable for the chemoselective modification of Sec and/or Cys-containing peptides and proteins under mild conditions. This method exhibits excellent selectivity and compatibility, as demonstrated by the use of a series of commercial small molecules with different scaffolds and different peptide and protein sequences. The selective functionalization of Sec can also be achieved by tuning the electron density of aromatic molecules in the presence of Cys, allowing for targeted biochemical reactions. EPR and radical trapping experiments, as well as DFT calculations, support that the conjugation approach is driven by the radical-mediated arylation of Sec and/or Cys with electron-rich (hetero)­arenes in the presence of Cu­(II) ions. Given these results, we believe our chemical method will be widely applied in the future for modifying proteins and generating therapeutically valuable protein conjugates. Ongoing research in our group is focused on investigating the labeling and adapting of more intricate biological molecules both in vivo and in vitro using this technique.

Supplementary Material

ja5c08936_si_001.pdf (11.7MB, pdf)

Acknowledgments

We thank the members of our respective laboratories for the fruitful discussions. We would like to thank Dr. Tsafi Danieli, Mrs. Yael Nir Keren, and Dr. Hadar Amartely for their assistance with the expression and purification of proteins. We also thank Prof. Doron Pappo for the valuable discussions. Y.C. is grateful for a CSC Ph.D. fellowship. N.M. acknowledges the financial support of the Israel Science Foundation (1388/22).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c08936.

  • Materials and methods, characterization data, and synthesis procedures (PDF)

∇.

Z.Z., J.H., Y.C., and T.-P.Z. contributed equally to this work.

The authors declare no competing financial interest.

References

  1. Zhang G., Zheng S., Liu H., Chen P. R.. Illuminating biological processes through site-specific protein labeling. Chem. Soc. Rev. 2015;44(11):3405–3417. doi: 10.1039/C4CS00393D. [DOI] [PubMed] [Google Scholar]
  2. Takaoka Y., Ojida A., Hamachi I.. Protein Organic Chemistry and Applications for Labeling and Engineering in Live-Cell Systems. Angew. Chem., Int. Ed. 2013;52(15):4088–4106. doi: 10.1002/anie.201207089. [DOI] [PubMed] [Google Scholar]
  3. a Krall N., da Cruz F. P., Boutureira O., Bernardes G. J.. Site-selective protein-modification chemistry for basic biology and drug development. Nat. Chem. 2016;8(2):103–113. doi: 10.1038/nchem.2393. [DOI] [PubMed] [Google Scholar]; b Qian L., Lin X., Gao X., Khan R. U., Liao J. Y., Du S., Ge J., Zeng S., Yao S. Q.. The Dawn of a New Era: Targeting the “Undruggables” with Antibody-Based Therapeutics. Chem. Rev. 2023;123(12):7782–7853. doi: 10.1021/acs.chemrev.2c00915. [DOI] [PubMed] [Google Scholar]
  4. Spicer C. D., Pashuck E. T., Stevens M. M.. Achieving Controlled Biomolecule-Biomaterial Conjugation. Chem. Rev. 2018;118(16):7702–7743. doi: 10.1021/acs.chemrev.8b00253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. a Chauhan P., Ragendu V., Kumar M., Molla R., Mishra S. D., Basa S., Rai V.. Chemical technology principles for selective bioconjugation of proteins and antibodies. Chem. Soc. Rev. 2024;53(1):380–449. doi: 10.1039/D3CS00715D. [DOI] [PubMed] [Google Scholar]; b Xie X., Moon P. J., Crossley S. W. M., Bischoff A. J., He D., Li G., Dao N., Gonzalez-Valero A., Reeves A. G., McKenna J. M., Elledge S. K., Wells J. A., Toste F. D., Chang C. J.. Oxidative cyclization reagents reveal tryptophan cation−π interactions. Nature. 2024;627(8004):680–687. doi: 10.1038/s41586-024-07140-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. a Zuo Q., Li Y., Lai X., Bao G., Chen L., He Z., Song X., Ruiyao E., Wang P., Shi Y.. et al. Cysteine-Specific Multifaceted Bioconjugation of Peptides and Proteins Using 5-Substituted 1,2,3-Triazines. Adv. Sci. 2024;11(21):e2308491. doi: 10.1002/advs.202308491. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Zhang Z., Li L., Xu H., Lee C. K., Jia Z., Loh T. P.. Silicon-Containing Thiol-Specific Bioconjugating Reagent. J. Am. Chem. Soc. 2024;146(3):1776–1782. doi: 10.1021/jacs.3c12050. [DOI] [PubMed] [Google Scholar]; c Wan C., Zhang Y., Wang J., Xing Y., Yang D., Luo Q., Liu J., Ye Y., Liu Z., Yin F., Wang R., Li Z.. Traceless Peptide and Protein Modification via Rational Tuning of Pyridiniums. J. Am. Chem. Soc. 2024;146(4):2624–2633. doi: 10.1021/jacs.3c11864. [DOI] [PubMed] [Google Scholar]; d Patel M., Forte N., Bishop C. R., Porter M. J., Dagwell M., Karu K., Chudasama V., Baker J. R.. The Nitrile Bis-Thiol Bioconjugation Reaction. J. Am. Chem. Soc. 2024;146(1):274–280. doi: 10.1021/jacs.3c08762. [DOI] [PMC free article] [PubMed] [Google Scholar]; e Hartmann P., Bohdan K., Hommrich M., Julia F., Vogelsang L., Eirich J., Zangl R., Fares C., Jacobs J. B., Mukhopadhyay D., Mengeler J. M., Vetere A., Sterling M. S., Hinrichs H., Becker S., Morgner N., Schrader W., Finkemeier I., Dietz K. J., Griesinger C., Ritter T.. Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation. Nat. Chem. 2024;16(3):380–388. doi: 10.1038/s41557-023-01388-7. [DOI] [PMC free article] [PubMed] [Google Scholar]; f Doud E. A., Tilden J. A. R., Treacy J. W., Chao E. Y., Montgomery H. R., Kunkel G. E., Olivares E. J., Adhami N., Kerr T. A., Chen Y., Rheingold A. L., Loo J. A., Frost C. G., Houk K. N., Maynard H. D., Spokoyny A. M.. Ultrafast Au­(III)-Mediated Arylation of Cysteine. J. Am. Chem. Soc. 2024;146(18):12365–12374. doi: 10.1021/jacs.3c12170. [DOI] [PMC free article] [PubMed] [Google Scholar]; g Lin X., Harel O., Jbara M.. Chemical Engineering of Artificial Transcription Factors by Orthogonal Palladium­(II)-Mediated S-Arylation Reactions. Angew. Chem., Int. Ed. 2024;63(5):e202317511. doi: 10.1002/anie.202317511. [DOI] [PubMed] [Google Scholar]; h Wang C., Zhao Z., Ghadir R., Yang D., Zhang Z., Ding Z., Cao Y., Li Y., Fassler R., Reichmann D., Zhang Y., Zhao Y., Liu C., Bi X., Metanis N., Zhao J.. Peptide and Protein Cysteine Modification Enabled by Hydrosulfuration of Ynamide. ACS Cent. Sci. 2024;10(9):1742–1754. doi: 10.1021/acscentsci.4c01148. [DOI] [PMC free article] [PubMed] [Google Scholar]; i Wan L. Q., Zhang X., Zou Y., Shi R., Cao J. G., Xu S. Y., Deng L. F., Zhou L., Gong Y., Shu X., Lee G. Y., Ren H., Dai L., Qi S., Houk K. N., Niu D.. Nonenzymatic Stereoselective S-Glycosylation of Polypeptides and Proteins. J. Am. Chem. Soc. 2021;143(31):11919–11926. doi: 10.1021/jacs.1c05156. [DOI] [PubMed] [Google Scholar]; j Chen F.-J., Gao J.. Fast Cysteine Bioconjugation Chemistry. Chem. - Eur. J. 2022;28(66):e202201843. doi: 10.1002/chem.202201843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Weerapana E., Wang C., Simon G. M., Richter F., Khare S., Dillon M. B., Bachovchin D. A., Mowen K., Baker D., Cravatt B. F.. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature. 2010;468(7325):790–795. doi: 10.1038/nature09472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Siemion I. Z.. Compositional frequencies of amino acids in the proteins and the genetic code. Biosystems. 1994;32(3):163–170. doi: 10.1016/0303-2647(94)90039-6. [DOI] [PubMed] [Google Scholar]
  9. Zhao Z., Laps S., Gichtin J. S., Metanis N.. Selenium chemistry for spatio-selective peptide and protein functionalization. Nat. Rev. Chem. 2024;8(3):211–229. doi: 10.1038/s41570-024-00579-1. [DOI] [PubMed] [Google Scholar]
  10. Hondal R. J., Marino S. M., Gladyshev V. N.. Selenocysteine in thiol/disulfide-like exchange reactions. Antioxid. Redox Signal. 2013;18(13):1675–1689. doi: 10.1089/ars.2012.5013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kryukov G. V., Castellano S., Novoselov S. V., Lobanov A. V., Zehtab O., Guigo R., Gladyshev V. N.. Characterization of mammalian selenoproteomes. Science. 2003;300(5624):1439–1443. doi: 10.1126/science.1083516. [DOI] [PubMed] [Google Scholar]
  12. a Cohen D. T., Zhang C., Fadzen C. M., Mijalis A. J., Hie L., Johnson K. D., Shriver Z., Plante O., Miller S. J., Buchwald S. L., Pentelute B. L.. A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins. Nat. Chem. 2019;11(1):78–85. doi: 10.1038/s41557-018-0154-0. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Cohen D. T., Zhang C., Pentelute B. L., Buchwald S. L.. An Umpolung Approach for the Chemoselective Arylation of Selenocysteine in Unprotected Peptides. J. Am. Chem. Soc. 2015;137(31):9784–9787. doi: 10.1021/jacs.5b05447. [DOI] [PMC free article] [PubMed] [Google Scholar]; c Bogart J. W., Bowers A. A.. Dehydroamino acids: chemical multi-tools for late-stage diversification. Org. Biomol. Chem. 2019;17(15):3653–3669. doi: 10.1039/C8OB03155J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. a Zhang C., Vinogradova E. V., Spokoyny A. M., Buchwald S. L., Pentelute B. L.. Arylation Chemistry for Bioconjugation. Angew. Chem., Int. Ed. 2019;58(15):4810–4839. doi: 10.1002/anie.201806009. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Lin X., Mandal S., Nithun R. V., Kolla R., Bouri B., Lashuel H. A., Jbara M.. A Versatile Method for Site-Specific Chemical Installation of Aromatic Posttranslational Modification Analogs into Proteins. J. Am. Chem. Soc. 2024;146(37):25788–25798. doi: 10.1021/jacs.4c08416. [DOI] [PMC free article] [PubMed] [Google Scholar]; c Mackay A. S., Maxwell J. W. C., Bedding M. J., Kulkarni S. S., Byrne S. A., Kambanis L., Popescu M. V., Paton R. S., Malins L. R., Ashhurst A. S.. et al. Electrochemical Modification of Polypeptides at Selenocysteine. Angew. Chem. Int. Ed. 2023;62(50):e202313037. doi: 10.1002/anie.202313037. [DOI] [PubMed] [Google Scholar]; d Dowman L. J., Kulkarni S. S., Alegre-Requena J. V., Giltrap A. M., Norman A. R., Sharma A., Gallegos L. C., Mackay A. S., Welegedara A. P., Watson E. E.. et al. Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine. Nat. Commun. 2022;13(1):6885. doi: 10.1038/s41467-022-34530-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. a Peng K., Dong Z.-B.. Recent Advances in Cross-Dehydrogenative Couplings (CDC) of C–H Bond in Aqueous Media. Adv. Synth. Catal. 2021;363(5):1185–1201. doi: 10.1002/adsc.202001358. [DOI] [Google Scholar]; b Ranjit S., Lee R., Heryadi D., Shen C., Wu J. E., Zhang P., Huang K.-W., Liu X.. Copper-Mediated C–H Activation/C–S Cross-Coupling of Heterocycles with Thiols. J. Org. Chem. 2011;76(21):8999–9007. doi: 10.1021/jo2017444. [DOI] [PubMed] [Google Scholar]
  15. a Fang X.-L., Tang R.-Y., Zhong P., Li J.-H.. Iron-Catalyzed Sulfenylation of Indoles with Disulfides Promoted by a Catalytic Amount of Iodine. Synthesis. 2009;2009(24):4183–4189. doi: 10.1055/s-0029-1217037. [DOI] [Google Scholar]; b Zhang S., Qian P., Zhang M., Hu M., Cheng J.. Copper-catalyzed thiolation of the di- or trimethoxybenzene arene C-H bond with disulfides. J. Org. Chem. 2010;75(19):6732–6735. doi: 10.1021/jo1014849. [DOI] [PubMed] [Google Scholar]
  16. Zhao Z., Shimon D., Metanis N.. Chemoselective Copper-Mediated Modification of Selenocysteines in Peptides and Proteins. J. Am. Chem. Soc. 2021;143(32):12817–12824. doi: 10.1021/jacs.1c06101. [DOI] [PubMed] [Google Scholar]
  17. Lapcinska S., Dimitrijevs P., Lapcinskis L., Arsenyan P.. Visible Light-Mediated Functionalization of Selenocystine-Containing Peptides. Adv. Synth. Catal. 2021;363(13):3318–3328. doi: 10.1002/adsc.202100373. [DOI] [Google Scholar]
  18. a Labunskyy V. M., Hatfield D. L., Gladyshev V. N.. Selenoproteins: molecular pathways and physiological roles. Physiol. Rev. 2014;94(3):739–777. doi: 10.1152/physrev.00039.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Wright D. E., O’Donoghue P.. Biosynthesis, Engineering, and Delivery of Selenoproteins. Int. J. Mol. Sci. 2024;25(1):223. doi: 10.3390/ijms25010223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. He H., Deng X., Wang Z., Chen J.. Recent progress in the development of peptide-drug conjugates (PDCs) for cancer therapy. Eur. J. Med. Chem. 2025;284:117204. doi: 10.1016/j.ejmech.2024.117204. [DOI] [PubMed] [Google Scholar]
  20. Rizvi S. F. A., Zhang L., Zhang H., Fang Q.. Peptide-Drug Conjugates: Design, Chemistry, and Drug Delivery System as a Novel Cancer Theranostic. ACS Pharmacol. Transl. Sci. 2024;7(2):309–334. doi: 10.1021/acsptsci.3c00269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zhang L. X., Li C. X., Kakar M. U., Khan M. S., Wu P. F., Amir R. M., Dai D. F., Naveed M., Li Q. Y., Saeed M., Shen J. Q., Rajput S. A., Li J. H.. Resveratrol (RV): A pharmacological review and call for further research. Biomed. Pharmacother. 2021;143:112164. doi: 10.1016/j.biopha.2021.112164. [DOI] [PubMed] [Google Scholar]
  22. Coward L., Barnes N. C., Setchell K. D. R., Barnes S.. Genistein, daidzein, and their.beta.-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets. J. Agric. Food Chem. 1993;41(11):1961–1967. doi: 10.1021/jf00035a027. [DOI] [Google Scholar]
  23. Mohammad-Zadeh L. F., Moses L., Gwaltney-Brant S. M.. Serotonin: a review. J. Vet Pharmacol Ther. 2008;31(3):187–199. doi: 10.1111/j.1365-2885.2008.00944.x. [DOI] [PubMed] [Google Scholar]
  24. Rubinstein E., Keynan Y.. Vancomycin Revisited  60 Years Later. Front Public Health. 2014;2:217. doi: 10.3389/fpubh.2014.00217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Bhargava D.. Phentolamine mesylate. J. Stomat. Occ. Med. 2012;5(2):53–56. doi: 10.1007/s12548-012-0038-7. [DOI] [Google Scholar]
  26. a Walensky L. D., Kung A. L., Escher I., Malia T. J., Barbuto S., Wright R. D., Wagner G., Verdine G. L., Korsmeyer S. J.. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science. 2004;305(5689):1466–1470. doi: 10.1126/science.1099191. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Bluntzer M. T. J., O’Connell J., Baker T. S., Michel J., Hulme A. N.. Designing stapled peptides to inhibit protein-protein interactions: An analysis of successes in a rapidly changing field. Pept. Sci. 2021;113(1):e24191. doi: 10.1002/pep2.24191. [DOI] [Google Scholar]
  27. Merrifield R. B.. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963;85(14):2149–2154. doi: 10.1021/ja00897a025. [DOI] [Google Scholar]
  28. Dawson P. E., Muir T. W., Clark-Lewis I., Kent S. B.. Synthesis of proteins by native chemical ligation. Science. 1994;266(5186):776–779. doi: 10.1126/science.7973629. [DOI] [PubMed] [Google Scholar]
  29. Zhao Z., Mousa R., Metanis N.. One-Pot Chemical Protein Synthesis Utilizing Fmoc-Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F. Chem. - Eur. J. 2022;28(16):e202200279. doi: 10.1002/chem.202200279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Metanis N., Keinan E., Dawson P. E.. Traceless ligation of cysteine peptides using selective deselenization. Angew. Chem. Int. Ed. 2010;49(39):7049–7053. doi: 10.1002/anie.201001900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Eigenbrot C., Ultsch M., Dubnovitsky A., Abrahmsen L., Hard T.. Structural basis for high-affinity HER2 receptor binding by an engineered protein. Proc. Natl. Acad. Sci. U. S. A. 2010;107(34):15039–15044. doi: 10.1073/pnas.1005025107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. a Nazari M., Radmanesh R.. ZHER2 Affibody as a Good Candidate for Detection of Metastatic Prostate Cancer. Avicenna J. Med. Biotechnol. 2021;13(3):171. doi: 10.18502/ajmb.v13i3.6366. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Bragina O., Chernov V., Larkina M., Rybina A., Zelchan R., Garbukov E., Oroujeni M., Loftenius A., Orlova A., Sorensen J., Frejd F. Y., Tolmachev V.. Phase I clinical evaluation of (99m)­Tc-labeled Affibody molecule for imaging HER2 expression in breast cancer. Theranostics. 2023;13(14):4858–4871. doi: 10.7150/thno.86770. [DOI] [PMC free article] [PubMed] [Google Scholar]; c Orlova A., Magnusson M., Eriksson T. L., Nilsson M., Larsson B., Hoiden-Guthenberg I., Widstrom C., Carlsson J., Tolmachev V., Stahl S., Nilsson F. Y.. Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res. 2006;66(8):4339–4348. doi: 10.1158/0008-5472.CAN-05-3521. [DOI] [PubMed] [Google Scholar]
  33. a Ngamchuea K., Tharat B., Hirunsit P., Suthirakun S.. Electrochemical oxidation of resorcinol: mechanistic insights from experimental and computational studies. RSC Adv. 2020;10(47):28454–28463. doi: 10.1039/D0RA06111E. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Ling K. Q., Lee Y., Macikenas D., Protasiewicz J. D., Sayre L. M.. Copper­(II)-mediated autoxidation of tert-butylresorcinols. J. Org. Chem. 2003;68(4):1358–1366. doi: 10.1021/jo020582y. [DOI] [PubMed] [Google Scholar]
  34. Lukeš V., Kováčová A., Hartmann H.. On thermodynamics of electron, proton and PCET processes of catechol, hydroquinone and resorcinol – Consequences for redox properties of polyphenolic compounds. J. Mol. Liq. 2022;360:119356. doi: 10.1016/j.molliq.2022.119356. [DOI] [Google Scholar]

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