α-Vinyl azide–cysteine click coupling reaction enabled bioorthogonal peptide/protein modification

Abstract

α-Alkyl and α-aryl vinyl azides were found to be able to couple with cysteine-derived alkyl thiols chemoselectively under mild conditions, providing the corresponding β-ketosulfides with simultaneous extrusion of N₂ and ammonia. This reaction was developed into an effective chemical platform for peptide and protein modification, which is completely cysteine selective and highly bioorthogonal, that avoids the interference from other native residues. The in situ formed ketone group can react specifically with alkoxyamines or hydrazines and therefore can serve as a versatile handle for a second modification. The modification of bovine serum albumin (BSA) with a dansyl fluorescent probe and the labeling of the genetically encoded fluorescent protein YPet-ECFP with biotin have been accomplished successfully through this platform.

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Chemical modifications of peptides or proteins, or alternatively bioconjugations, have been proved and continue to be a powerful means to construct bioconjugates with improved or desired properties and have thus been widely used not only in chemical biology to study cellular processes but also in the medical industry to prepare biopharmaceuticals.¹ Current trends in chemical modification emphasize on chemoselectivity, site-specificity and bioorthogonality.² Cysteine, a proteogenic amino acid that is relatively less abundant yet ubiquitously distributed among mammalian proteins, has a number of distinct chemical properties related to the characteristic sulfhydryl group. Collectively, these merits make cysteine arguably the most popular endogenous residue for protein bioconjugation, and a myriad of methods for selective cysteine ligation have been developed over the years.³ Conventional cysteine modification reactions include disulfide bond formation,⁴ S_N2 alkylation,⁵ S_NAr (hetero)arylation⁶ and the most practiced conjugate addition to an array of electrophilic alkenes and alkynes.⁷ Recently, a number of creative methods have appeared, such as transition metal mediated cysteine arylation, alkenation, allyation and borylation,⁸ hypervalent iodine and alkynyl dibenzothiophenium reagent mediated cysteine functionalizations,⁹ light, strain or proximity promoted thiol–ene/yne reactions,¹⁰ and others.¹¹ Overall, from the mechanistic perspective, the majority of the aforementioned modification methods involve the coupling of the nucleophilic cysteine thiol group with a particular electrophile under certain conditions, which can potentially be interfered by other biological nucleophiles (Fig. 1a). Disulfide exchange and retro-Michael addition also do harm to the stability, homogeneity and structural integrity of the relevant bioconjugates. On the other hand, thiol ene/yne reactions of inactivated alkenes/alkynes usually proceed with exclusive chemical or site selectivity to give robust products, but their applications are quite restricted probably because of the slow kinetics or the requirement for radical initiators (Fig. 1b).¹⁰ Therefore, there is an unmet need for a chemoselective and bioorthogonal chemical cysteine-modifying protocol that can deliver robust bioconjugates. To this end, we report such a protocol based on an α-vinyl azide–cysteine click coupling reaction that affords stable β-ketosulfide adducts through a radical C–S bond forming event (Fig. 1c).

Fig. 1 Chemical modification of a peptide or protein at the cysteine residue.

The reaction of aryl thiol with α-aryl vinyl azide to give a β-ketosulfide derivative was initially noted by Montevecchi and co-workers in 1997.¹² Recently, our group reinvestigated this coupling process and expanded the scope of azides to α-alkyl vinyl azides.¹³ This thioether forming transformation takes place smoothly under benign conditions, without the need for extra additives and releasing ammonia and nitrogen as the only by-products. In addition, an aryl thiyl radical initiated radical-chain process was proposed and this process should tolerate aqueous medium due to its radical nature. These features prompted us to envisage that this reaction could evolve into an ideal platform for cysteine modification, based on the assumption that alkyl thiol is also able to participate in this radical event (Fig. 1c). To examine this premise, cysteine-derived alkyl mercaptan 2a was reacted with α-phenyl vinyl azide 1a (Table 1). To our delight, when mixed and stirred with 10 fold of 1a in THF at 40 °C under air, the cysteine derivative 2a disappeared in 30 min, as shown by TLC, and the desired β-ketosulfide 3aa was isolated in 77% yield (entry 1). An additional compound was obtained and identified as 4a, an oxidative homo-coupling product of 2a. Cysteine containing dipeptide 2b reacted similarly with 1a to deliver conjugate 3ab in 61% yield (entry 2). Furthermore, thioethers 3ac–an were obtained successfully from the coupling of 1a with the corresponding dipeptides 2c–n, indicating the good compatibility of this reaction with various proteogenic side chains such as thioether, hydroxyl, phenol, indole, and primary amide groups (entries 3–14). Tripeptide 2o with a cysteine residue in the middle was also a good substrate for this coupling reaction, affording adduct 3ao in 61% isolated yield (entry 15). Vinyl azide 1b, carrying a strong electron donating pivalamido group at the para position of the phenyl ring, reacted quickly with protected dipeptides 2b and 2l and tripeptide 2p to give 3bb, 3bl and 3bp, respectively. The relatively lower yields (30–40%) observed for these three reactions might be attributed to the use of a much less amount (2.0 vs. 10 equiv.) of vinyl azide (entries 16–18). With the restoration of the amount of vinyl azide to 10 equiv., the recovery in yields (50% for 3bj and 61% for 3bk) was observed for the reactions of 1b with 2j and 2k (entries 19 and 20). Notably, although the reaction time varied broadly from less than 1 h to as long as 8 h, the yields were constantly moderate to good, and in all cases, the dimerization of the related alkyl thiols via S–S bond formation was more or less detected, which is a detrimental sideway that competes with the desired C–S bond forming event for the cysteine sulfhydryl group.

Table 1 Coupling reaction of cysteine derivatives with α-aryl vinyl azides^a

Entry	1 (equiv.)	Time	Adduct (yield)	Entry	1 (equiv.)	Time	Adduct (yield)
a Conditions: 2 (0.2 mmol, 1.0 equiv.), 1 (2.0 or 10 equiv.), THF (3 mL), 40 °C, 0.5–8 h in air; isolated yields are reported.
1	1a (10)	30 min		11	1a (10)	8 h
2	1a (10)	50 min		12	1a (10)	40 min
3	1a (10)	6 h		13	1a (10)	40 min
4	1a (2.0)	40 min		14	1a (10)	8 h
5	1a (10)	5 h		15	1a (10)	8 h
6	1a (10)	4 h		16	1b (2.0)	1.0 h
7	1a (10)	3 h		17	1b (2.0)	30 min
8	1a (10)	8 h		18	1b (2.0)	50 min
9	1a (10)	8 h		19	1b (10)	1.0 h
10	1a (10)	3 h		20	1b (10)	30 min

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After realizing and evaluating the ligation of cysteine derivatives with α-aryl vinyl azides, we then turned to the reaction of the more stable and hence less reactive α-alkyl vinyl azides. (Benzo)imidazole substituted vinyl azides 1c–j were readily obtained and reacted with 2a under the above conditions. The reactions did occur, but we encountered considerable difficulty in separating the thioether products from the corresponding vinyl azides through silica gel column chromatography. To avoid this annoying problem, an excess of cysteine 2a was employed to ensure complete consumption of vinyl azides, and the outcomes are presented in Table 2. Vinyl azides 1c–f, each carrying an imidazole moiety, all coupled successfully with alkyl thiol 2a to give rise to the related conjugates 3ca–3fa in high yields (entries 1–4). The lower reactivity of alkyl vinyl azides was evidenced by the much longer reaction time needed for their full conversions (20–24 h). With their benzoimidazole congeners 1g–j, even their corresponding adducts 3g–j were collected in quantitative yields (entries 5–8, 90–99% yield). Two equivalents of 2a were found to be capable of consuming the alkyl vinyl azide in all instances.

Table 2 Coupling reaction of BocNHCysOMe with α-alkyl vinyl azides^a

Entry	Vinyl azide	Time	Adduct, yield	Entry	Vinyl azide	Time	Adduct, yield
a Conditions: 2a (0.4 mmol, 2.0 equiv.), 1 (0.2 mmol, 1.0 equiv.), THF (3 mL), 40 °C, 6–24 h in air; isolated yields are reported.
1		24 h		5		6 h
2		20 h		6		24 h
3		20 h		7		24 h
4		24 h		8		8 h

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With this robust thioether-forming protocol in hand, our efforts were directed to evaluate its feasibility in selective cysteine modification employing phosphate buffered saline (PBS) as the solvent or cosolvent. Commercially available glutathione (GSH, 2q), a cysteine-containing natural peptide antioxidant, was used as the model substrate (Table 3). In an open vial, a solution of vinyl azide 1a (2.0 equiv.) in THF and a solution of glutathione (1.0 equiv.) in PBS (pH 7.4) were mixed and stirred at 40 °C. GSH 2q disappeared in 4 h and the coupling product 3aq was isolated in 75% yield through reverse phase column chromatography (entry 1). Without exception, α-aryl vinyl azides 1k–m were all proven to be competent agents for cysteine ligation, furnishing the corresponding conjugates 3kq–mq efficiently in longer reaction times (entries 2–4). Aliphatic vinyl azides 1n and 1i also demonstrated substantial potential in cysteine ligation (entries 5 and 6). For vinyl azides 1e, 1f and 1o–q that can dissolve well in water, the coupling reactions were conducted in PBS buffer without the use of THF as the co-solvent. For these reactions, the yields were determined from their LC-mass spectra (entries 7–12). In PBS buffer, both imidazolyl vinyl azides 1e and 1f underwent this thiyl radical initiated process faster than parallel events in THF–PBS cosolvents, affording 3eq and 3fq in 71% and 88% LC-MS yields, respectively (entries 7 and 8). The benzoimidazolium functionalized homologues 1o and 1p also efficiently modified GSH at cysteine with high yields in PBS (entries 9 and 10). Glycopeptide 3qq was constructed via the modification of GSH with 1q, a glucoside functionalized with a vinyl azide moiety at the anomeric position (entry 11). As shown in entry 12, the use of water to replace PBS as the reaction medium is feasible as well, albeit with a slight decrease in the yield. Masking the thiol group with trityl protection resulted in no reaction with 1a under the standard conditions even if stirred in air for 24 hours, indicating the inertness of the free amino and carboxyl groups toward this vinyl azide. At this point, the effectiveness of this reaction for selective chemical modification at cysteine has been firmly established. It is worth noting that in 2017, Chiba and colleagues realized site-specific modification at the cysteine residue in peptides and proteins using 2-azidoacrylates.¹⁴ Although 2-azidoacrylates also fall in the class of vinyl azides, they serve as Michael acceptors and undergo 1,4-addition with free cysteinyl thiols, a mechanism totally different from the radical chain process for the present method.

Table 3 Bioconjugation of glutathione with α-vinyl azides^a

Entry	Vinyl azide	Solvent	Adduct, yield	Entry	Vinyl azide	Solvent	Adduct, yield
		Time				Time
a Unless otherwise noted, all reactions were carried out with 0.2 mmol GSH 2q (1.0 equiv.) and 0.4 mmol vinyl azide 1 (2.0 equiv.) at 40 °C, using 2.0 mL PBS (pH 7.4) as the solvent or cosolvent. b Isolated yields are reported. c LC-MS yields are reported.
1^b		PBS/THF (1/1, v/v)		7^c		PBS
		4.0 h				8.0 h
2^b		PBS/THF (1/1, v/v)		8^c		PBS
		24 h				8.0 h
3^b		PBS/THF (1/1, v/v)		9^c		PBS
		24 h				8.0 h
4^b		PBS/THF (1/1, v/v)		10^c		PBS
		24 h				8.0 h
5^b		PBS/THF (1/1, v/v)		11^c		PBS
		24 h				8.0 h
6^b		PBS/THF (1/1, v/v)		12^c		H2O (pH 7.0)
		8.0 h				30 h

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To further investigate the applicability of the above system in protein chemical ligation, dansylated vinyl azide 1r was obtained and incubated with bovine serum albumin (BSA, 2r, 68 kDa) at 40 °C for 24 in air. In two solvent mixtures, i.e. 1/1 and 1/3 EtOH/PBS (v/v), two parallel experiments were conducted, respectively (Fig. 2a). SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of these reactions was carried out and the results are shown in Fig. 2b. For each reaction sample, a strong fluorescent-emission band appears around 70 kDa in the fluorescence image, which can also be visualized through Coomassie blue staining (lanes R1 and R2). As a comparison, no such band is observed in the fluorescence image but showed up upon staining for the control experiment for BSA (lane BSA). As expected, no protein band appears for the control with vinyl azide 1r (lane 1r). The fluorescent bands at the bottom correspond to the dansylated reagent 1r. These experimental outcomes clearly demonstrate that BSA has been efficiently labeled by the dansyl fluorescent vinyl azide. And we claim with confidence that the current methodology can serve as an effective chemical tool for selective protein modification.

Fig. 2 Labeling of BSA with a dansyl fluorescent probe: (a) coupling reaction of BSA with 1r; and (b) SDS-PAGE analysis (M: protein marker, R1: reaction in 1/1 EtOH/PBS (v/v), R2: reaction in 1/3 EtOH/PBS (v/v), BSA: control experiment without 1r, 1r: control experiment without BSA.

The validity of this biocompatible chemical tool in protein modification was further confirmed through the successful decoration of nickel NTA agarose beads (Ni-NTA) with a fluorescent protein (FP) carrying three fluorophores (Fig. 3a). Genetically encoded FP YPet-ECFP (2s),¹⁵ a 6×His tagged bioconjugate comprising a yellow fluorescent protein (YPet) and an enhanced cyan fluorescent protein (ECFP) linked via a peptide CPKESCNLFVLKD that bears two cysteine residues, was incubated with excess biotin-derived vinyl azide ^Biotin1s at 37 °C under physiological reaction conditions for 40 h. This reaction mixture, presumably containing the biotinylated FP 3ss, was then shaken with the Ni-NTA resin for 1 h and the biotin-grafted nickel particles Ni-3ss were obtained through subsequent centrifugation and washing. A 30 min treatment of these freshly made biotin-modified nickel beads with the dye Alexa Fluor® 568 streptavidin (STAV AF568) in PBS completed the installment of a third chromophore. After the removal of the unbound streptavidin dye molecules, the thus prepared nickel complex Ni-YPet-ECFP-AF568 was suspended in PBS and visualized through fluorescence microscopy (Fig. 3b). The bright annuli in both ECFP and YPet emission images suggest the successful attachment of YPet-ECFP (2s) onto the surface of the Ni-coated NTA resin beads; the identical but red annuli in the Alex Flor 568 emission microscopy spectrum indicate an efficient labeling of the Ni-NTA resin surface with the streptavidin dye STAV AF568 (Fig. 3b row I). These observations confirmed the successful installation of biotin segments on the nickel beads, which was accomplished via the coupling reaction of YPet-ECFP (2s) with biotin-conjugated vinyl azide ^Biotin1s. Following the same protocol but omitting the azide agent ^Biotin1s, a control sample was prepared and subjected to fluorescence imaging too (Fig. 3b row II). The green and yellow rings were present in its ECFP and YPet emissions, just like those in row I for Ni-YPet-ECFP-AF568. In contrast, in its Alex Fluor 568 emission spectrum, the otherwise red rings now become invisible but with even distribution of the red fluorescence in the beads. This sharp difference might be attributed to the staining of the whole Ni-NTA resin beads with the dye STAV AF568 simply via kinetic diffusion when no specific biotin–streptavidin conjugation took place.

Fig. 3 (a) The labeling of Ni-NTA resin with 6×His-tagged FP conjugate YPet-ECFP and streptavidin-Alex Flor 568 dye STAV AF568; and (b) fluorescence imaging of labeled, unlabeled Ni-NTA resin and control samples.

Conclusions

In summary, we have disclosed a coupling reaction of α-vinyl azides with cysteine-based alkyl mercaptans which gives stable β-ketothioethers. This reaction possesses several distinct merits that potentially make it a magnificent chemical platform for protein bioconjugation: (1) the intrinsic radical nature makes it completely feasible for use in aqueous solvent systems; (2) the non-electrophilic vinyl azide used prevents the attack from biological nucleophiles, especially other native amino acid side chains; (3) there is no need for extra reagents and the harmlessness of the by-products formed show its excellent biocompatibility; and moreover (4) the in situ formed stable β-ketothioether linkage might serve as a second ligating site specific for primary amine, hydrazine and alkoxylamine. These merits meet the characteristics of click reactions and we therefore would like to name this C–S bond forming process as vinyl azide–cysteine (VA–Cys) click coupling.¹⁶ Using this click reaction, the chemical installation of β-ketothioether on glutathione at cysteine has been realized with an array of α-vinyl azides under (quasi)physiological conditions. The effective fluorescent labelling of BSA with a dansylated α-vinyl azide shows its effectiveness in protein modification. Its avidity is confirmed unequivocally through the successful introduction of a biotin functionality to an engineered FP YPet-ECFP. Based on these solid experimental data, we can claim confidently that a useful chemical platform for selective and bioorthogonal protein modification at the cysteine residue has been established. Further studies aiming to extend its application scenarios are ongoing in our laboratory.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors thank the National Natural Science Foundation of China (22177015), the Natural Science Foundation of Jiangsu Province (BK20211334) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX21-2860) for financial support. This work is also supported by the State Key Laboratory of Organic Electronics and Information Displays, Institute of Advanced Materials (IAM), Nanjing University of Posts & Telecommunications. We are grateful to Dr Aijun Cui and Dr Li Liu, Analysis and Testing Centre, NERC Biomass of Changzhou University, for their help with the NMR analysis.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experiment details, characterization of compounds, copies of compound NMR spectra. See DOI: https://doi.org/10.1039/d2qo00736c

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