Introduction

Bioorthogonal chemistry rendered a general and practical approach to access the selective modification of biological species under mild physiological conditions. Due to its widespread use and impact, bioorthogonal chemistry, together with click chemistry was recognized with the awarding of the 2022 Nobel Prize in chemistry1,2,3,4,5,6,7,8,9. However, clickable handles such as alkynyl, azide, tetrazine, and alkenyl, are barely found in native biomolecules. Introducing these structures is a challenging task due to the vast complexity of biomolecules. Thus, bioconjugations leveraging the in situ orthogonal reactivities of biomolecules that are structurally and functionally diverse and compatible with various functional groups are of great significance and highly desirable.

Proteins and peptides are extremely important biological species that participate in vast biological processes and have fascinated the chemical, biological, pharmaceutical, and medicinal communities. Focusing on 20 proteinogenic amino acids, orthogonal chemical modifications of peptides and proteins have recently showcased an enormous potential in drug discovery, proteomic profiling, and clinical diagnostics, and flourished the development of peptide and/or protein engineering10,11,12. Particularly, late-stage modification over de novo synthesis of peptides and/or proteins is wonderfully beneficial for the ideal bioconjugation (Fig. 1A). However, endogenous amino acid residues are mostly nucleophilic residues that pose a formidable task of selectivity when a chemical reaction is introduced to engineer complex peptides and proteins. In this regard, achieving excellent and exquisite selectivity is important for the generation of homogeneous products that neither interact nor interfere with the other residues. Moreover, the inherent sensitive structure of complex biomolecules is extremely susceptible to harsh conditions, such as high temperatures, acidic or basic pH, and metal catalysts. To selectively and compatibly modify peptides and proteins, an orthogonal chemical reaction must be (1) selective toward other potential reactive functional groups, (2) proceed with very fast reaction rates, (3) under physiological pH, and (4) at room temperature or 37 °C. Therefore, with the most ideal chemical bioconjugations, catalysts or additives are not needed, and the reaction components are just mixed into physiological media (Fig. 1B).

Fig. 1: Ideal chemical bioconjugation.
figure 1

A Strategies for bioconjugations of peptide/protein. B The condition preference for late-stage bioconjugation. C Orthogonal bioconjugation targeting cysteine-containing peptide/protein using thianthrenium salts (this work).

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Among the canonical amino acids, cysteine (Cys) sharing lower abundance and possessing a nucleophilic thiol group, catches most of the eyes for peptide/protein engineering. Cysteine can undergo alkylation with alkyl halides, alkynylation and alkenylation with ethynyl benziodoxolone, arylation with aryl palladium(II) complexes, or perfluoroaromatic species, Michael addition with α,β-unsaturated carbonyl compounds, and disulfide exchange to form mixed disulfides13,14,15,16,17,18,19,20,21,22. The kaleidoscope of reactivity enabled cysteine as the most common target of interest for diverse applications in protein labeling23, antibody-drug conjugates24,25, peptide-drug conjugates26, and native chemical ligation27,28. Despite significant progress within this field, challenges persist in terms of chemoselectivity, wide scope, fast timescale, and mild reaction conditions, as a result of the inherent structural complexity of peptides and proteins. As part of our ongoing interest in peptide engineering29,30,31,32,33,34,35,36,37, we have been searching for a suitable couple-partner to meet the fast, selective, and compatible bioconjugation requirements. Through massive screening, we found that the synthetically useful thianthrenium salts (TT+) might undergo this selective cross-coupling reaction. Thianthrenium salts have recently attracted considerable attention due to their high reactivity and diversity. Ritter and coworkers reported a highly selective C–H thianthrenation reaction to access aryl and alkenyl thianthrenium salts, which could engage in different transformations and showed large potential in biomolecular modifications38,39,40,41,42,43,44,45,46. Shi and coworkers also developed alkyl thianthrenium salts for diverse cross-coupling reactions47,48. To modify biomolecules, during our submission of this work, Ritter and coworkers described a chemoselective umpolung of thiol to episulfoniums for cysteine bioconjugation through conjugate addition between thiol and vinyl thianthrenium tetrafluoroborate or vinyl tetrafluorothianthrenium tetrafluoroborate followed by a nucleophilic ring opening49.

In this work, we disclose that thianthrenium salts could directly and selectively react with cysteine residues via an SN2 reaction to achieve the orthogonal chemical modification under mild conditions (Fig. 1C). The method proceeds with high chemoselectivity and compatibility, introducing various functional structures, including PEG chains, fluorescent molecules, drugs, carboranes, carbohydrates, and peptides. Additionally, multifaceted bioconjugation is achieved through clickable handles to incorporate structurally diverse functional molecules. Our method is also successfully applied to label bovine serum albumin (BSA).

Results and discussion

To verify our assumption, glutathione and thianthrenium salt 2a were initially chosen as model substrates to evaluate and optimize the reaction conditions (Table 1). The reaction was carried out in a mixed solvent without the need for a catalyst or additive. Organic solvents were used to enhance the solubility of thianthrenium salt. Compared with other solvents, DMSO showed higher conversion and isolated yield (Table 1, entries 1–6), albeit with potential disulfide formation by DMSO oxidation50. Different buffers and diverse pH values were also examined to increase the isolated yield (Table 1, entries 7–19). It was found that Tris-HCl buffer (pH 9.0) generated the desired product 3a in excellent yield and excellent chemoselectivity, even in the presence of free –NH2 and –COOH (Table 1, entry 18). Notably, this method shows great superiority to its previous cysteine alkylation counterparts, such as alkyl halides, by milder reaction conditions, higher yields, and chemoselectivities13,14.

Table. 1 Initial investigation of the model reactiona
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Under the optimized conditions, we explored the methodology to modify the bioactive peptides and further investigated the chemoselectivity when more free active residues were exposed, such as His, Tyr, Trp, Lys, Ser, Arg, Thr, Glu/Asp, and Gln/Asn. Several bioactive peptides were prepared in which cysteine was installed at random positions in the sequences. Linear and cyclic peptides were introduced with different sequence lengths. As shown in Fig. 2, these complex and diverse bioactive peptides suffered from site-selective modifications at cysteine residues in excellent yields (3a–3w). Thianthrenium salts bearing various functional structures, such as alkyne, azide, deuteromethyl, trifluoromethyl, and amino acid, specifically decorated cysteine smoothly even when other polar residues were unmasked under the same spatiotemporal conditions, which showed more versatile modification in comparison with Ritter’s work49. These meaningful results demonstrated possible applications of orthogonal bioconjugation and modification toward cysteine-containing peptides and proteins. Crucially, the mild and highly selective nature of this methodology, as well as its compatibility with biological conditions, rendered it an ideal platform for bioconjugation.

Fig. 2: Orthogonal bioconjugation targeting bioactive peptides using thianthrenium saltsa.
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aUnless otherwise stated, the reaction was carried out with peptides 1 (1.0 eq.) and thianthrenium salts 2 (1.0 eq.) in DMSO/Tris-HCl buffer (v/v = 3:1) at room temperature for 1 h. bExtension the reaction to 6 h. Isolated yield after semipreparative HPLC was given. For details, see the Supplementary Information.

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Having efficiently and successfully modified bioactive peptides, we further drove the method to construct structurally complex stapled and bridged peptides based on two cysteine residues, considering that cyclization and dimerization peptides exhibited a positive effect on biological properties, including binding affinity and proteolytic stability, as well as pharmacokinetic property (Fig. 3). Four peptide drugs, which were derived from octreotide, argipressin, terlipressin and somatostatin, were synthesized in their reduced forms. Then, the peptides underwent selective cysteine-specific conjugation with a flexible alkyl chain tethered two free thiols together, forging the novel stapled peptides (4a–4e). As expected, bridged peptides were also obtained by coupling two bioactive peptides with one hinge, generating the homologous (4f–4h) and heterologous dimerized hybrid peptides (4i). These stapling and bridging peptides could not be available by dihalogenated alkanes51, thereby rendering this methodology tremendous potential in peptide drug discovery and molecular splicing.

Fig. 3: Orthogonal construction of stapled and bridged peptidesa.
figure 3

aThe reaction was carried out with peptide 1 (1.0 eq.) and thianthrenium salt 2 (1.0 eq.) in DMSO/Tris-HCl buffer (v/v = 3:1) at room temperature for 1 h. bThe reaction was carried out with peptide 1 (2.0 eq.) and thianthrenium salt 2 (1.0 eq.) in DMSO/Tris-HCl buffer (v/v = 3:1) at room temperature for 1 h. Isolated yield after semipreparative HPLC was given. For details, see the Supplementary Information.

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Based on the integration of bioorthogonally useful clickable handles, we harnessed alkynes and azides to achieve multifaceted bioconjugation (Fig. 4). For instance, Cyclo(RGDfC), a sensitive integrin αvβ3 receptor ligand52,53, was selectively installed alkyne and azide at cysteine residues, respectively, generating masked products 3b and 3c. Subsequent classical copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry could enable the diversification of 3b by introducing the PEG chain (7a), fluorescent molecules (7b and 7f), drugs (7d and 7e), and carborane (7c)54,55. Similarly, peptide 3c also proceeded smoothly to access these diverse fluorescence labeling (8b), glycopeptide (8e), peptide-drug, and peptide-peptide conjugations (8a, 8c, 8d, and 8f). This multifaceted bioconjugation is a brilliant method to further expand orthogonal modification.

Fig. 4: Multifaceted bioconjugation through the click reactiona.
figure 4

aUnless otherwise stated, the reaction was carried out with peptide 3b or 3c (1.0 eq.), alkyne 5 or azide 6 (1.0 eq.), THPTA (2.0 eq.) CuSO4•5H2O (1.0 eq.) and sodium ascorbate (2.0 eq.) in DMF/H2O (v/v = 4:1, 1 mL) at room temperature for 1 h. Isolated yield after semipreparative HPLC was given. For details, see the supplementary information.

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Encouraged by the superb specificity and selectivity at the peptide level and the power of multifaceted modifications, the strategy was utilized to decorate the more complex proteins. As a proof-of-concept study, we selected BSA, the most abundant carrier protein in blood that contains a single and solvent-exposed free cysteine (Cys-34). BSA was incubated with thianthrenium salt 2c in Tris-HCl/DMSO for 30 min. Then, a second bioconjugation was immediately performed with the crude mixtures through CuAAC click chemistry to introduce fluorescent BODIPY. As shown in Fig. 5, this one-pot multifaceted bioconjugation smoothly furnished fluorescently labeled BSA, which was verified via UPLC-HRMS analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore, this orthogonal modification together with click chemistry provides a valuable and versatile platform for selective cysteine bioconjugation under biologically ambient conditions.

Fig. 5: Multifaceted modifications of BSAa.
figure 5

aLabeling cystein residues on BSA (50 µM) with fluorescent BODIPY in the “two steps, one pot” method. bCoomassie blue staining. cIrradiation with a 460 nm wavelength laser.

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In summary, late-stage bioconjugation of peptides and proteins has exposed the need for efficient strategies to enable orthogonal modification and labeling with high selectivity and specificity. We herein presented a chemoselective, fast, mild, and compatible bioconjugation methodology to engineer cysteine residues using synthetically useful thianthrenium salts. The strategy can be used to functionalize bioactive peptides and proteins with various functional groups and further expand to construct structurally complex stapled and bridged peptides. Notably, by introducing clickable handles, the method can achieve multifaceted bioconjugation to invoke PEG chains, fluorescent molecules, drugs, carboranes, carbohydrates, and peptides. In addition, this “two steps, one pot” method was successfully applied to label BSA with BODIPY under mild conditions. We envision that this orthogonal modification will be a valuable tool for chemical biology, material sciences, and drug discovery.

Methods

Synthesis of 3a–3w and 4a–4j

In an oven-dried 10 mL vial with a magnetic stir bar was charged with cysteine-containing peptides (1.0 eq.), alkyl thianthrenium salts (1.0 eq. for 3a–3w and 4a–4d; 0.5 eq. for 4e–4j), DMSO (1.5 mL, 0.1 M) and Tris-HCl buffer (pH 9.0, 0.5 mL). The resulting mixture in the vial was stirred open to air at room temperature for 1 h. Then, the crude reaction mixture was directly purified by Semi preparative HPLC on a Waters 2996 using a Dubhe C18 (10 μm, 20× 250 mm) preparative column, linear gradients using A: MeCN (0.1% CF3COOH) and B: H2O (0.1% CF3COOH).

Synthesis of 7a–7f and 8a–8f

Under argon atmosphere, to a solution of 3b or 3c (0.01 mmol, 1.0 eq.), alkyne derivatives 5 or azide 6 (0.01 mmol, 1.0 eq.) and (THPTA) (0.01 mmol, 1.0 eq.) in DMF (0.8 mL), CuSO4•5H2O in 0.1 mL H2O and sodium ascorbate (0.02 mmol, 2.0 eq.) in 0.1 mL H2O were added successively. The resulting mixture was stirred at room temperature for 3 h. Then, the reaction was identified by LC-MS and the crude reaction mixture was directly purified by Semi preparative HPLC on a Waters 2996 using a Dubhe C18 (10 μm, 20× 250 mm) preparative column, linear gradients using A: MeCN (0.1% CF3COOH) and B: H2O (0.1% CF3COOH).

Labeling of BSA

A solution of alkyl thianthrenium salt 2c (4.2 mg, 10 μmol, 100 eq.) dissolved in DMSO (0.1 mL) was added to a solution of BSA (6.6 mg, 0.1 μmol, 1.0 eq.) in Tris-HCl buffer (pH 9.0, 1.8 mL). The reaction mixtures were stirred for 30 min at room temperature. Then, the mixture of fluorescent BODIPY (4.0 mg, 10 μmol, 100 eq.), THPTA (8.7 mg, 20 μmol, 200 eq.), CuSO4•5H2O (2.5 mg, 10 μmol, 100 eq.), sodium ascorbate (4.0 mg, 20 μmol, 200 eq.) in DMF (0.1 mL) was added to the reaction mixture. After 1 h, an aliquot of each sample (4 µL) was diluted with 5× SDS sample buffer (10 µL) and ddH2O (36 µL). Each sample (10 µL) was loaded onto a 12-well 12% SDS-PAGE gel. The gel was run at room temperature and at 160 V for 70 min. In-gel fluorescence was imaged with a Typhoon FLA 9500 (GE) at 460 nm. Protein modification reaction was monitored and evaluated on a ThermoFisher Orbitrap IQ-X mass spectrometer coupled to an Acquity UPLC Protein BEH C4 column (300 Å, 1.7 um, 2.1 mm × 100 mm). Total mass spectra were reconstructed from the ion series using the intact mass analysis of Thermo BioPharma Finder software according to the manufacturer’s instructions.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.