Maleimide Crosslinkers

Maleimides readily react with thiols in a Michael type addition to form succinimidyl thioethers. This reaction may be carried out in buffered aqueous solutions at pH 6.8 (useful range: 6.5-7.0) and usually completes within a few minutes. However, this reaction and linkage is reversible.

Schematic representation of the thioether formation between a thiol and a maleimide.

If increased stability of the conjugate is required, the resulting succinimide ring may be deliberately hydrolyzed. To this end, we are providing self-stabilizing crosslinkers with Boc-protected amines, which, after deprotection, may act as intramolecular nucleophiles to open the succinimide ring.

Hydrolysis of thiosuccinimides via an intrinsic catalytic nucleophilic amine. P = Payload.

Besides, we offer linkers with aryl moieties, which directly destabilize the succinimide after it has been formed, so that the ring then is quickly and irreversibly hydrolyzed.

Resonance promoted hydrolysis of thiosuccinimides. P = Payload.

Within our maleimido-crosslinker portfolio, we offer several kinds of additional reactive functions with various properties: reactivity towards amino groups, for use in Click Chemistry, for photoactivation, or to introduce new functional groups into your structure or biomolecule.

In the following, we are highlighting those additional functionalizations:

Succinimidyl esters (abbreviated as OSu or NHS) are a frequently used tool for coupling small molecules to peptides and proteins. These activated carboxyl groups react readily with amino groups in buffered aqueous solutions at pH 7.5 (useful range: 6.5-8.5). Iris Biotech is offering a wide selection of ready to use maleimide-succinimide crosslinkers with spacers of different properties and lengths between 6 and 16 atoms.

Pentafluorophenyl (PFP-) esters work like succinimidyl esters but are slightly less sensitive to undesired hydrolysis during the coupling process. As an example, Mal-L-Dap(Boc)-OPfp (MAA1080) introduces a spacer of five atoms.

Regarding Click Chemistry, we offer DBCO (dibenzoazacyclooctyne) and MeTz (methyltetrazine) functionalized maleimide crosslinkers suitable for copper-free conjugation in biological environments, as well as alkyne-functionalized ones for classical Click chemistry (see related products).

As photoactivatable crosslinker, we provide 4-(N-Maleimido)benzophenone (LS-3350), which can be activated upon irradiation with light of 350 nm.

Further functional groups may be introduced using maleimide-crosslinkers bearing amino, hydroxyl and carboxyl functions.

Besides “permanent” crosslinkers, our portfolio also includes cleavable linkers with a predetermined breaking point for targeted payload release, e.g. upon enzymatic activity, changes in pH or redox potential.

→ Browse our Brochure Linkerology® to discover the panoply of available linker technologies.
→ For more details about Click Chemistry or Photochemistry, download our booklets!
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References:

Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase; R. A. Bednar; Biochemistry 1990; 29: 3684-3690. https://doi.org/10.1021/bi00467a014

Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides; R. J. Christie, R. Fleming, B. Bezabeh, R. Woods, S. Mao, J. Harper, A. Joseph, Q. Wang, Z. Q. Xu, H. Wu, C. Gao,N. Dimasi; J Control Release 2015; 220: 660-70. https://doi.org/10.1016/j.jconrel.2015.09.032

A versatile acid-labile linker for antibody–drug conjugates; M. C. Finniss, K. S. Chu, C. J. Bowerman, J. C. Luft, Z. A. Haroon,J. M. DeSimone; Med. Chem. Commun. 2014; 5: 1355-1358. https://doi.org/10.1039/c4md00150h

Long-term stabilization of maleimide-thiol conjugates; S. D. Fontaine, R. Reid, L. Robinson, G. W. Ashley,D. V. Santi; Bioconjug Chem 2015; 26: 145-52. https://doi.org/10.1021/bc5005262

Bioconjugation - A versatile and essential research tool; G. Hermanson; Aldrichimica Acta 2017; 50: 43-57.

Chapter 3 - The Reactions of Bioconjugation; G. T. Hermanson; in Bioconjugate Techniques (Third Edition); edited by G. T. Hermanson; Academic Press; Boston 2013: 229-258 https://doi.org/10.1016/B978-0-12-382239-0.00003-0

Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C; J. Leszyk, J. H. Collins, P. C. Leavis,T. Tao; Biochemistry 1987; 26: 7042-7. https://doi.org/10.1021/bi00396a028

Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates; R. P. Lyon, J. R. Setter, T. D. Bovee, S. O. Doronina, J. H. Hunter, M. E. Anderson, C. L. Balasubramanian, S. M. Duniho, C. I. Leiske, F. Li,P. D. Senter; Nature biotechnology 2014; 32: 1059-62. https://doi.org/10.1038/nbt.2968

pH Dependence of Succinimide-Ester-Based Protein Cross-Linking for Structural Mass Spectrometry Applications; E. Trabjerg, A. Keller,A. Leitner; ACS Measurement Science Au 2022; 2: 132-138. https://doi.org/10.1021/acsmeasuresciau.1c00032

Novel Silyl Ether-Based Acid-Cleavable Antibody-MMAE Conjugates with Appropriate Stability and Efficacy; Y. Wang, S. Fan, D. Xiao, F. Xie, W. Li, W. Zhong,X. Zhou; Cancers (Basel) 2019; 11: 957. https://doi.org/10.3390/cancers11070957

Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity; B. Wei, J. Gunzner-Toste, H. Yao, T. Wang, J. Wang, Z. Xu, J. Chen, J. Wai, J. Nonomiya, S. P. Tsai, J. Chuh, K. R. Kozak, Y. Liu, S. F. Yu, J. Lau, G. Li, G. D. Phillips, D. Leipold, A. Kamath, D. Su, K. Xu, C. Eigenbrot, S. Steinbacher, R. Ohri, H. Raab, L. R. Staben, G. Zhao, J. A. Flygare, T. H. Pillow, V. Verma, L. A. Masterson, P. W. Howard,B. Safina; J Med Chem 2018; 61: 989-1000. https://doi.org/10.1021/acs.jmedchem.7b01430

Characterization of Ring-Opening Reaction of Succinimide Linkers in ADCs; K. Zheng, Y. Chen, J. Wang, L. Zheng, M. Hutchinson, J. Persson,J. Ji; J Pharm Sci 2019; 108: 133-141. https://doi.org/10.1016/j.xphs.2018.10.063