The reaction of maleimides with thiol-containing biomolecules is a commonly used conjugation method. This conjugation reaction allows adding a label or cargo molecule to a protein, a peptide, or an oligonucleotide containing a linker with a thiol group.
Maleimide activated molecules modify thiol group-containing biomolecules at physiological pH conditions. An example is the conjugation of fluorescein-5-maleimide to a molecule with a thiol group.
Fluorescein-5-maleimide is a fluorophore with a sulfhydryl-reactive maleimide group on its lower-ring structure. This conjugation reaction results in a stable thioether bond.
Figure 1: Fluorescein-5-maleimide allows the modification of sulfhydryl groups to form thioester bonds.
Figure 2: The general reaction scheme of maleimides with thiol group used for bioconjugation and labeling of biomolecules.
Maleimides are electrophilic compounds with high selectivity towards thiols. Natural DNA does not contain thiols; therefore, linkers with thiol groups can be added to synthetic oligonucleotides. In proteins and peptides, thiol groups are very abundant as cysteine residues. Thiols can form disulfide bonds via oxidative dimerization. Cysteine residues in proteins form cystine bridges which stabilize tertiary structures. Disulfides do not react with maleimides. Therefore, it is necessary to reduce disulfides before conjugation. Conjugation protocols depend on the solubility of the starting components. For the conjugation of compounds with low solubility in water, organic solvents such as DMSO or DMF are essential.
Martínez-Jothar et al. (2018) found that the reaction between maleimide groups present in nanoparticles (NPs) and the cyclic peptide cRGDfK was optimal at a maleimide to thiol molar ratio of 2:1, reaching a conjugation efficiency of 84 ± 4% after 30 min at room temperature in 10 mM HEPES pH 7.0. However, for the 11A4 nanobody, the optimal reaction efficiency, 58 ± 12%, was achieved after 2 h of incubation at room temperature in PBS pH 7.4 using a 5:1 maleimide to protein molar ratio.
Maleimide conjugation protocol
The following is a general protocol to conjugate fluorescent dye maleimides to proteins, peptides, and other thiolated biomolecules.
1. Dissolve the biomolecule containing a thiol group to be labeled in a degassed buffer (PBS, Tris, HEPES are good, although others buffers containing no thiols can be used) at pH 7-7.5 in a reaction vial. The buffer can be degassed by applying a vacuum on it for several minutes, or by bubbling inert gas through it (nitrogen, argon, or helium may be used). For proteins, a good concentration range is between 1to10 mg/mL.
2. Add an excess of TCEP (tris-carboxyethylphosphine) reagent to reduce disulfide bonds and flush with inert gas. Close the reaction vial. A 50 to 100x molar excess of TCEP will work well. Incubate the mixture for 20 minutes at room temperature.
3. Dissolve the maleimide dye in DMSO or fresh DMF. Use 1 to10 mg in a volume of 100 uL.
4. Add the dye solution to the thiol solution (If possible us a 10 to 20x fold excess of dye), flush vial with inert gas, and close the reaction vial tightly.
5. Mix thoroughly, and keep overnight at room temperature, or 4 Celsius.
6. Purify the conjugate using gel filteration, HPLC, FPLC, or electrophoresis.
For maleimides with poor solubility in water, use a co-solvent such as DMF or DMSO. Maleimides with good aqueous solubility such as sulfo-Cyano maleimides can be dissolved in water. If a precipitation occurs, increase the content of organic solvent in the mixture to achieve better labeling.
Please note: Dialysis is only useful for purification of maleimides with good water solubility.
Reference
Abdolvahab, M.H., Karimi, P., Mohajeri, N. et al. Targeted drug delivery using nanobodies to deliver effective molecules to breast cancer cells: the most attractive application of nanobodies. Cancer Cell Int 24, 67 (2024). [BMC]
Brewer, C.F. and Riehmn, J.P. (1967). Evidence for possible nonspecific reactions between N-ethymaleimide and proteins. Anal Biochem 18:248-55. [Sciencedirect]
Gorin G, Martic PA, Doughty G. Kinetics of the reaction of N-ethylmaleimide with cysteine and some congeners. Arch Biochem Biophys. 1966 Sep 9;115(3):593-7.[Sciencedirect] "The half-reaction time at pH 7 and a 10−3 M concentration was found to be around 0.7 second."
Haugaard N, Cutler J, Ruggieri MR. Use of N-ethylmaleimide to prevent interference by sulfhydryl reagents with the glucose oxidase assay for glucose. Anal Biochem. 1981 Sep 15;116(2):341-3. [Sciencedirect]
Heitz JR, Anderson CD, Anderson BM. Inactivation of yeast alcohol dehydrogenase by N-alkylmaleimides. Arch Biochem Biophys. 1968 Sep 20;127(1):627-36. [Pubmed]
Hermanson, G.T., Bioconjugate Techniques. 2nd Edition, Elsevier 2008
Martínez-Jothar L, Doulkeridou S, Schiffelers RM, Sastre Torano J, Oliveira S, van Nostrum CF, Hennink WE. Insights into maleimide-thiol conjugation chemistry: Conditions for efficient surface functionalization of nanoparticles for receptor targeting. J Control Release. 2018 Jul 28;282:101-109. [Sciencedirect]
Partis, M.D., Griffuths, D.G., Roberts, G.C. and Beechey, R.B.; (1983). Cross-linking of protein by ω-maleimido alkanoyl N-hydroxysuccinimido esters. J Protein Chem 2:263-77. [Springer]
Smyth, D.G., Nagamatsu, A., and Fruton, J.S.; (1960). Some Reactions of N-ethylmaleimide. J Am Chem Soc 82:4600-4604. [ACS]
Smyth DG, Blumenfeld OO, Konigsberg W.; Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J. 1964 Jun;91(3):589-95. [PMC]
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