Recent advances of thiol-selective bioconjugation reactions

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Abstract

Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes, fulfilling specific functions with unmatched precision. This unique specificity makes proteins an ideal scaffold to generate tools for the exploration of natural systems or for the construction of modern therapeutics. Thus, the chemoselective modification of proteins with functionalities that are not defined by the genetic code has become an indispensable approach for life science research and the development of therapeutics. Amongst site-selective strategies for protein modification, cysteine-selective approaches have long been used for the generation of functional protein conjugates and new reactions continue to emerge, offering solutions for diverse research questions. In this review, we are highlighting new strategies for the chemoselective modification of cysteine residues in peptides, proteins and antibodies with a particular focus on the most recent years. We lay special focus on new reagents for efficient cysteine conjugation that produce stable conjugation products with significant pharmaceutical application.

Introduction

The approval of three new antibody–drug conjugates (ADCs) in 2019 demonstrates the high value and relevance of bioconjugates as important modern therapeutics [1]. Strikingly, all of the recently approved ADCs are conjugated via cysteine residues in the polypeptide chain, highlighting the importance of thiol-selective reactions for the generation of functional bioconjugates. Cysteine qualifies as an outstanding target for bioconjugation because of its relative low abundance in proteins, the unique nucleophilicity of its thiolate and its ability to take part in various reaction types. Consequently, cysteine has been exploited extensively for bioconjugation with several reported thiol-selective modification strategies, as documented in reviews by Chalker et al. in 2009 [2] and Gunnoo and Madder in 2016 [3]. Despite having an array of thiol-selective conjugation techniques available, the go-to method for cysteine modification usually remains the use of maleimides. Maleimide reagents allow extraordinary fast labelling with bimolecular rate constants of 102–104 M−1 s−1, while showing acceptable cysteine selectivity [4,5]. Furthermore, a variety of maleimide derivatives, including dyes, affinity probes and crosslinkers, are commercially available, allowing easy access without the need of synthetic expertise. However, maleimides suffer from stability issues, thus rendering conjugation products unstable under certain conditions [6, 7, 8]. Tackling these problems, several strategies have been developed to improve the stability of maleimide-based bioconjugates, as summarized in a recent comprehensive review by Ravasco et al. [9]. Yet, there is an ongoing effort to develop new thiol-selective reagents beyond maleimides, thereby combining improved stabilities with similar labelling efficiencies. In this review, we want to highlight recent developments in the field and focus on strategies that stand out by means of efficient cysteine conjugation, stable conjugation products and significant pharmaceutical applications.

Section snippets

α,β-Unsaturated carbonyl reagents

Moving away from maleimides, Bernardim et al. [10] investigated a set of carbonylacrylic reagents (Figure 1a) to identify highly cysteine reactive probes that allowed bioconjugation reactions in aqueous conditions. With the aid of quantum mechanical calculations on the thiol-Michael addition, they designed three derivatives out of which benzoyl acrylamide was experimentally identified as the best candidate. The straightforward synthesis of fluorescent and PEGylated benzoyl acrylamides allowed

Unsaturated electrophiles

Electrophilic reagents displaying double-bonded or triple-bonded carbon–carbon moieties were traditionally explored for cysteine-selective conjugations as exemplified with alkynoic amides, esters and alkynones [24] or by exploitation of light-initiated radical thiol–ene [25] and thiol–yne reactions [26]. Alternative to the radical thiol–yne reaction, thiolates can also react with electrophilic alkynes through a nucleophilic addition process. Pentelute et al. [27] were able to increase the rate

Substituted electrophilic reagents

Halogen-substituted electrophiles like iodoacetamide are routinely used for cysteine alkylation through SN2 reactions. Following such a substitution approach, Baker et al. introduced dibromopyridazinediones (DBPD) as a compound class suitable for crosslinking reduced disulfide bridges to yield serum stable dithio-1,2-dihydro-pyridazine-3,6-diones (Figure 3a). These reagents were used for the construction of various bioconjugates including multi-functionalized Fab fragments and full antibodies [

Metal-based reagents

Initiated by the introduction of Pd(II)-complexes that allowed rapid selective arylation of cysteine residues in peptides and proteins by the Buchwald laboratory [47, 48, 49], metal-mediated arylation emerged as a complementary approach to SNAr. A representative selection of existing organometallic approaches was recently reviewed by Pentelute et al. [41]. A recent, rather unconventional approach of metal-mediated cysteine modification was introduced by Gupta et al. [50] who used Pt(II)-based

Conclusion

Cysteine-selective functionalization of peptides and proteins occupies a prominent place in the field of bioconjugation as exemplified by rising numbers of FDA approvals for cysteine-linked ADCs. Therefore, new strategies to complement the manifold toolbox of cysteine labelling are of high interest in contemporary research. The steady advances in the field furthermore document the aspiration to move away from the generic use of maleimide chemistry whenever cysteine labelling is desired and

Declaration of Competing Interest

Nothing declared.

Acknowledgements

The authors thank Alice Baumann for valuable comments on the manuscript and proofreading as well as Anselm Schneider for providing graphical elements. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) within the SPP1623, the CRC765 and the RTG2473, the Einstein Foundation Berlin (Leibniz–Humboldt Professorship) and the Leibniz Association with the Leibniz Wettbewerb.

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