Recent advances of thiol-selective bioconjugation reactions
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.
References (51)
Thiol/disulfide exchange equilibria and disulfide bond stability
Methods Enzymol
(1995)- et al.
Site-specific dual functionalization of cysteine residue in peptides and proteins with 2-azidoacrylates
Bioconjugate Chem
(2017) - et al.
Fast, irreversible modification of cysteines through strain releasing conjugate additions of cyclopropenyl ketones
Org Biomol Chem
(2018) - et al.
Site-specific PEGylation of native disulfide bonds in therapeutic proteins
Nat Chem Biol
(2006) - et al.
Bis-sulfide bioconjugates for glutathione triggered tumor responsive drug release
Chem Commun
(2014) - et al.
Azabicyclic vinyl sulfones for residue-specific dual protein labelling
Chem Sci
(2019) - et al.
Stable pyrrole-linked bioconjugates through tetrazine-triggered azanorbornadiene fragmentation
Angew Chem
(2020) - et al.
Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization
Chem Sci
(2019) - et al.
N-Hydroxysuccinimide-modified ethynylphosphonamidates enable the synthesis of configurationally defined protein conjugates
ChemBioChem
(2020) - et al.
Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones
Org Biomol Chem
(2018)
Rapid biocompatible macrocyclization of peptides with decafluoro-diphenylsulfone
Chem Sci
“Doubly orthogonal” labeling of peptides and proteins
Chem
Development of a facile antibody-drug conjugate platform for increased stability and homogeneity
Chem Sci
2019 FDA drug approvals
Nat Rev Drug Discov
Chemical modification of proteins at cysteine: opportunities in chemistry and biology
Chem - Asian J
Chemical protein modification through cysteine
ChemBioChem
Ring substituent effects on the thiol addition and hydrolysis reactions of N-arylmaleimides
J Org Chem
Maleimidocysteineamido-DOTA derivatives: new reagents for radiometal chelate conjugation to antibody sulfhydryl groups undergo pH-dependent cleavage reactions
Bioconjugate Chem
Contribution of linker stability to the activities of anticancer immunoconjugates
Bioconjugate Chem
Long-term stabilization of maleimide-thiol conjugates
Bioconjugate Chem
Bioconjugation with maleimides: a useful tool for chemical biology
Chem Eur J
Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents
Nat Commun
Chemoselective and photocleavable cysteine modification of peptides and proteins using isoxazoliniums
Commun Chem
Highly reactive and tracelessly cleavable cysteine-specific modification of proteins via 4-substituted cyclopentenone
Angew Chem Int Ed
Thiol specific and tracelessly removable bioconjugation via Michael addition to 5-methylene pyrrolones
J Am Chem Soc
Cited by (91)
Phenylpropynones as Selective Disulfide Rebridging Bioconjugation Reagents
2024, Organic LettersHydrogel Cross-Linking via Thiol-Reactive Pyridazinediones
2023, BiomacromoleculesPeptide Bispecifics Inhibiting HIV-1 Infection by an Orthogonal Chemical and Supramolecular Strategy
2023, Bioconjugate ChemistryBioorthogonal Peptide Macrocyclization Using Oxime Ligation
2023, Organic LettersSite-specific dual encoding and labeling of proteins via genetic code expansion
2023, Cell Chemical Biology