Thiol–maleimide conjugation is currently one of the most widely used conjugation technologies in antibody-drug conjugate (ADC) development. However, the thiosuccinimide linkage formed by this chemistry undergoes two competing reactions in vivo, which directly determine the fate of an ADC:
1) Retro-Michael reaction: causes the Drug-Linker to deconjugate from the antibody and is a primary cause of off-target toxicity and reduced therapeutic efficacy of ADCs.
2) Succinimide ring-opening hydrolysis: an irreversible stabilization reaction. Once the succinimide ring has undergone hydrolysis, the resulting product can no longer undergo deconjugation, making this process essential for maintaining the structural stability of ADCs (Figure 1). [1]
Figure 1. Stability Challenges of Thiol–Maleimide Protein Conjugates
To address the above challenges, multiple research groups have reported different strategies to overcome this limitation. [2]
In 2013, Toda et al. [3] first reported that methylsulfonyl phenyloxadiazole compounds exhibited excellent chemoselectivity toward cysteine under a variety of buffer conditions. In addition, the resulting protein conjugates displayed significantly greater stability in human plasma than conventional cysteine–maleimide conjugates. This novel thiol-click chemistry provides a new approach for the preparation of stable protein conjugates and PEGylated proteins (Figure 2).
Figure 2. Protein Conjugates Based on Methylsulfonyl Phenyloxadiazole Compounds
In 2014, Lyon et al. [4] first prepared a drug-linker using diaminopropionic acid (DPR), in which a basic amino group was introduced adjacent to the maleimide moiety. This basic amino group provides intramolecular catalysis for the hydrolysis of the thiosuccinimide ring. This basic amino group promotes the rapid hydrolysis of the thiosuccinimide ring under neutral pH and at room temperature. Once hydrolysis is complete, the drug-linker no longer undergoes the maleimide elimination reaction, thereby preventing nonspecific deconjugation (Figure 3). In vivo studies demonstrated that the improved stability enhanced the antitumor activity of ADCs while reducing the incidence of neutropenia.
Figure 3. Rapid Succinimide Hydrolysis Significantly Delays Cleavage of Thioether Adducts
In the same year, Abbas et al. [5] established an extremely simple and straightforward method for the selective labeling of target cysteine residues in completely unprotected peptides and proteins. This method is based on the reaction between allenamides and cysteine thiol groups. The reaction proceeds rapidly in aqueous media, exhibiting excellent selectivity and quantitative conversion efficiency, and forms stable, irreversible conjugates.
The method is simple to perform and requires mild reaction conditions, demonstrating the advantages of allenamides as highly efficient and versatile reactive groups for cysteine targeting, with significant potential for applications in biological systems (Figure 4).
Figure 4. Construction of Peptide/Protein Conjugates Based on Allenamide Linkers
In 2015, Christie et al. [6] reported that N-aryl maleimides can form stable antibody conjugates under exceptionally mild reaction conditions while maintaining high conjugation efficiency. Introduction of an N-phenyl or N-fluorophenyl substituent onto the maleimide ring nitrogen accelerates the thiol–maleimide conjugation reaction and stabilizes ADCs through thiosuccinimide hydrolysis (Figure 5). The N-phenyl maleimide-based ADC carrying the anticancer drug monomethyl auristatin E (MMAE) retained high cytotoxicity after prolonged exposure in serum. In contrast, the activity of the N-alkyl maleimide-MMAE conjugate gradually decreased over time. These results demonstrate that structural modification of the substituent attached to the maleimide ring nitrogen provides a simple and flexible strategy. N-aryl maleimides therefore represent an effective platform for improving the stability of ADCs.
Figure 5. Rapid Hydrolysis of N-Aryl Succinimides Significantly Delays Cleavage of Thioether Adducts
In the same year, Kolodych et al. [7] developed a heterobifunctional reagent for amine–thiol conjugation, sodium 4-((4-cyanoethynyl)benzoyl)oxy-2,3,5,6-tetrafluorobenzenesulfonate (CBTF). This reagent replaces the maleimide moiety with the recently reported 3-arylpropionitrile (APN) structure, enabling the preparation of conjugates with significantly improved stability. Using this reagent, the authors synthesized a series of antibody–fluorophore conjugates that exhibited greater stability in human plasma than conventional maleimide-based conjugates (Figure 6).
Figure 6. Novel CBTF Conjugates
In the same year, Kalia et al. [8] prepared a series of exocyclic olefinic maleimides. The study demonstrated that these scaffolds react selectively with thiol groups under physiological conditions to form linkages that resist thiol exchange-mediated degradation, thereby highlighting their potential applications in the preparation of stable thiol bioconjugates (Figure 7).
Figure 7. Novel Exocyclic Olefinic Maleimide Conjugates
In 2017, Nunes et al. [9] conjugated a next-generation maleimide (NGM) with the engineered cysteine residues of THIOMAB™ antibodies, affording a THIOMAB™ antibody-drug conjugate (TDC) with a drug-to-antibody ratio (DAR) of approximately 2. The resulting TDC exhibited excellent stability under serum conditions and demonstrated high selectivity and potent cytotoxicity against human epidermal growth factor receptor 2 (HER2)-positive cell lines, meeting the performance criteria of optimized antibody-drug conjugates (ADCs) (Figure 8).
Figure 8. Next-Generation Maleimide ADC Conjugates
In the same year, Kemp et al. [10] reported a novel iodoacetamide-based antibody-drug conjugate (ADC) payload, SG3227. Using a site-specific conjugation strategy, the authors constructed a trastuzumab-based ADC. In vitro evaluation demonstrated that the ADC exhibited excellent stability under serum conditions and showed high target selectivity and HER2-positive tumor cell lines, confirming the potential of SG3227 as an effective payload for the development of antitumor ADCs (Figure 9).
Figure 9. Construction of an Iodoacetamide-Based Antibody-Drug Conjugate (ADC)
In 2019, Hackenberger et al. [11] reported cysteine-selective ethynylphosphonamidate reagents as a new class of conjugation reagents. Through a simple one-pot reduction and alkylation process, these reagents enable the rapid preparation of highly effective ADCs directly from native, non-engineered monoclonal antibodies. Ethynylphosphonamidates readily accommodate hydrophilic substituents, allowing the preparation of electrophilic labeling reagents with tunable aqueous solubility. The study demonstrated that ADCs prepared using ethynylphosphonamidate conjugation exhibited excellent serum stability and in vivo antitumor activity, representing the performance expected of next-generation antibody therapeutics (Figure 10).
Figure 10. Novel Ethynylphosphonamidate Conjugates
In the same year, Matos et al. [12] developed quaternized vinyl pyridine and alkynyl pyridine reagents that react with various cysteine-tagged proteins under near-stoichiometric conditions quickly with excellent selectivity, enabling the efficient preparation of homogeneous antibody-drug conjugates (ADCs) with a precisely controlled DAR of 2. These conjugates remained stable in human plasma while retaining their specific targeting capability toward HER2-positive cells.
In addition, the designed reactive moiety introduces one additional positive charge to the overall net charge of the protein after conjugation. This feature indicates that simply attaching a quaternized vinyl pyridinium reagent to a cysteine residue provides a convenient method for modulating the physicochemical properties of proteins. (Figure 11).
Figure 11. Novel Quaternary Ammonium ADC Conjugates
In 2022, Wang et al. [13] designed a series of maleamic acid methyl ester-based linkers and conjugated the widely used MMAE to an anti-HER2 antibody to prepare the target ADCs. This strategy improves conjugate stability without altering the structure of the desired stable metabolite produced by conventional ADCs. In vivo studies demonstrated that the optimized molecule not only exhibited superior therapeutic efficacy compared with conventional ADCs, but also showed an improved safety profile in mice (Figure 12).
Figure 12. ADCs Based on Maleamic Acid Methyl Ester Linkers
In 2023, Cao et al. [14] demonstrated that tyrosinase can oxidize readily accessible phenolic compounds to generate highly reactive o-quinone intermediates, thereby enabling efficient cysteine conjugation for the site-specific modification of antibody surfaces with phenolic functional molecules. Experimental validation showed that, compared with conventional maleimide linkages, the chemical bonds formed through thiol–o-quinone conjugation exhibited significantly greater resistance to deconjugation under physiological conditions. Based on this strategy, the authors successfully constructed antibody conjugates carrying cytotoxic payloads, which demonstrated excellent targeted tumor cell-killing activity.
Furthermore, this method is broadly applicable to the conjugation of various functional molecules to antibodies, including fluorophores, oligonucleotides, and other functional entities, demonstrating excellent versatility (Figure 13).
Figure 13. Tyrosinase-Mediated Synthesis of ADCs
In the same year, Lang et al. [15] reported a bottom-up construction strategy based on an aqueous nickel-catalyzed cross-coupling reaction, enabling chemoselective arylation of cysteine residues in peptides and proteins. The authors further applied this technology to the preparation of antibody-drug conjugates. The study demonstrated a variety of aromatic linker architectures that enable the site-specific introduction of small molecules, molecular probes, and cytotoxic payloads. Using this bioconjugation platform, the authors prepared novel antibody-drug conjugates that exhibited target-dependent cytotoxic activity in vitro, fully demonstrating the potential value of this technology for drug development (Figure 14).
Figure 14. Antibody-Drug Conjugates Constructed by Aqueous Nickel-Catalyzed Cross-Coupling
In 2026, Dang et al. [16] reported a programmable pyridine-based sequential conjugation platform that integrates sulfonium and fluorinated reaction sites, enabling the sequential chemoselective conjugation of alkylamines and cysteine (Cys) under mild reaction conditions. This system generates achiral and highly stable aryl–cysteine linkages, making it suitable for late-stage peptide modification, peptide stapling, and protein functionalization, while also enabling the rapid construction of serum-stable ADCs (Figure 15).
Figure 15. Pyridine-Based Chemoselective Sequential Conjugation Platform
In the same year, Rojas et al. [17] established a novel universal immunocapture method for enriching ADCs from complex biological matrices, followed by characterization using reversed-phase liquid chromatography–mass spectrometry (RPLC–MS), with particular emphasis on the quantitative analysis of deconjugation and succinimide ring-opening hydrolysis. Using this method, the authors found that replacing the industry-standard hydrophobic MC spacer with the more hydrophilic MT spacer is a key strategy for improving the in vivo stability of ADCs and reducing the risk of deconjugation.
Furthermore, selecting conjugation sites with greater solvent exposure and a more hydrophilic microenvironment further promotes succinimide ring-opening hydrolysis, thereby enhancing ADC stability (Figure 16).
Figure 16. Hydrophilic MT Spacer Significantly Reduces the Risk of ADC Deconjugation
Summary
Following more than a decade of research, scientists have successfully developed a variety of linker chemistries to address the inherent deconjugation problem associated with thiol–maleimide protein conjugates.
Precise PEG offers a broad portfolio of these linker building blocks, which are specifically designed to support the rapid development of site-specific thiol bioconjugates. Our ready-to-ship products are manufactured using high-purity, highly consistent production processes, enabling researchers to rapidly obtain stable and reliable reagents without lengthy lead times. Scalable synthetic processes have been established for selected products, facilitating the seamless transition from early-stage research to process development and large-scale manufacturing.
In addition, we can provide custom products based on these linker building blocks to meet specific customer requirements.
References:
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[2] Colombo, R.; Seredick, S.; Barnscher, S. D.; Rich, J. R. Re-Evaluating Antibody-Drug Conjugate Linker Stability: Assessment, Interpretation, and Clinical Translation. Ann. Oncol. 2026, 37, 902-919.
[3] Toda, N.; Asano, S.; Barbas III, C. F. Rapid, Stable, Chemoselective Labeling of Thiols with Julia-Kocieński-like Reagents: A Serum-Stable Alternative to Maleimide-Based Protein Conjugation. Angew. Chem. Int. Ed. 2013, 52, 12592-12596.
[4] Lyon, R. P.; Setter, J. R.; Bovee, T. D.; et al. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nat. Biotechnol. 2014, 32, 1059-1062.
[5] Abbas, A.; Xing, B.; Loh, T.-P. Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins. Angew. Chem. Int. Ed. 2014, 53, 7491-7494.
[6] Christie, R. J.; Fleming, R.; Bezabeh, B.; Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides. J. Control. Release 2015, 220, 660-670.
[7] Kolodych, S.; Koniev, O.; Baatarkhuu, Z.; et al. CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma Stability. Bioconjugate Chem. 2015, 26, 197-200.
[8] Kalia, D.; Malekar, P. V.; Parthasarathy, M. Exocyclic Olefinic Maleimides: Synthesis and Application for Stable and Thiol-Selective Bioconjugation. Angew. Chem. Int. Ed. 2015, 55, 1432-1435.
[9] Nunes, J. P. M.; Vassileva, V.; Robinson, E.; et al. Use of a next generation maleimide in combination with THIOMAB™ antibody technology delivers a highly stable, potent and near homogeneous THIOMABTM antibody-drug conjugate (TDC). RSC Adv. 2017, 7, 24828-24832.
[10] Kemp, G. C.; Tiberghien, A. C.; Patel, N. V.; et al. Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepinedimer antibody-drug conjugate payload based on sibiromycin. Bioorganc Med. Chem. Lett. 2017, 27, 1154-1158.
[11] Kasper, M.-A.; Stengl, A.; Ochtrop, P.; et al. Ethynylphosphonamidates for the Rapid and Cysteine-SelectiveGeneration of Efficacious Antibody-Drug Conjugates. Angew. Chem. Int. Ed. 2019, 58, 11631-11636.
[12] Matos, M. J.; Navo, C. D.; Hakala, T.; et al. Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast Cysteine-Selective Protein Modification and Charge Modulation. Angew. Chem. Int. Ed. 2019, 58, 6640-6644.
[13] Wang, Y.; Xie, F.; Liu, L.; et al. Development of applicable thiol-linked antibody-drug conjugates with improved stability and therapeutic index. Drug Deliv. 2022, 29, 754-766.
[14] Cao, W.; Maza, J. C.; Chernyak, N.; et al. Modification of Cysteine-Substituted Antibodies Using Enzymatic Oxidative Coupling Reactions. Bioconjugate Chem. 2023, 34, 510-517.
[15] Bacauanu, V.; Merz, Z. N.; Hua, Z. L.; et al. Nickel-Catalyzed Antibody Bioconjugation. J. Am. Chem. Soc. 2023, 145, 25842-25849.
[16] Dang, X.; Zhang, C.; Shang, J.; et al. Modular Assembly of Bioconjugates Enabled by a Pyridine-Based Chemoselective Sequential Conjugation Platform. Angew. Chem. Int. Ed. 2026, 65, e3286230.
[17] Rojas, A. H.; Wong, J.; Alonzo, D. A.; et al. Systematic Evaluation of Maleimide Spacer Impact on Drug-Linker Deconjugation in Antibody-Drug Conjugates Ex Vivo and In Vivo. Bioconjugate Chem. 2026, 37, 713-725.