Maleimide Conjugation

Maleimide conjugation is a term employed in biochemistry and molecular biology that describes the covalent entanglement of maleimide functional groups onto a protein, peptide or other biomolecule. This conjugation technology can be applied to drug delivery systems, diagnostic reagents and therapeutic drug discovery. Maleimide group is ideal for stable covalent bonds with thiol groups so it has been a valuable tool for biomolecular modification.

What is Maleimide Conjugation?

Maleimide conjugation is a chemical procedure in biochemistry and molecular biology wherein the maleimide functional groups are attached to a target protein, peptide or other biomolecule. It is used for drug delivery, diagnostic reagents and therapeutic drugs. Maleimide group can best bond to thiol group in stable covalent bonds and hence, it is a powerful tool in biomolecular engineering.

Maleimide Structure

Maleimide a cyclic compound, consists of double bonds and amide groups. It is chemically a C₄H₃NO₂ with a nitrogen atom and carbonyl atom inside the ring. This was maleimide's most important feature – it is electronegative at the carbon end and therefore easily subjected to the nucleophilic reaction of sulfhydryl groups. This chemical reactivity renders the maleimide group excellent for reacted with sulfhydryl groups to form stable covalent bonds (maleimide conjugating technology is based on this).

Fig.1 A schematic of the structure of maleimide.

What is the Maleimide Reaction?

Maleimide reaction is a nucleophilic reaction of the maleimide functional group with the sulfhydryl group to form a covalent bond. This reaction is very specific, and generally possible to do so on an asymptotic scale so that the modification of proteins or other biomolecules does not affect their structure or function.

The carbon of maleimide group is electronegative and can be attacked by sulfhydryl groups. When the reaction is complete, the sulfhydryl group will be in stable thioether bond to the maleimide ring's carbon.

This conjugating reaction is typically quick and efficient which is why maleimide groups are extensively used in protein labeling, antibody conjugation and drug conjugation. Because it is a selective reaction, the maleimide-sulfhydryl reaction can modify only the molecule in question without any disruption of the other functional groups of the molecule.

Fig.2 Mercapto-maleimide click reaction. (Wu, Dongdong, et al., 2019)

Table.1 Custom Bioconjugation Services at BOC Sciences.

Maleimide Conjugation Mechanism

Maleimide conjugation is a relatively simple chemical reaction process that involves the following basic steps:

Activation of the maleimide functional cluster: The maleimide functional group has a high degree of electronegativity, which makes it vulnerable to nucleophilic attack under the right conditions. In the maleimide conjugating reaction, the β carbon of the maleimide group is susceptible to nucleophile attack due to its electronegativity, especially the sulfhydryl group (-SH). Under specific reaction conditions, these maleimide groups can be "activated" and ready to react with sulfhydryl groups.

Nucleophilic attack of sulfhydryl groups: Sulfhydryl groups (-SH) are usually present in cysteine (Cys) residues in proteins. The sulfhydryl group is strongly nucleophilic and is able to attack the β carbon on the maleimide ring to initiate a nucleophilic reaction. This reaction usually occurs rapidly, resulting in a stable intermediate in which the sulfhydryl group forms a covalent bond with maleimide. Due to the very strong nucleophilic nature of the sulfhydryl group, this reaction is highly selective and will only react with the maleimide group without interfering with other functional groups.

Formation of stable covalent conjugates: As the reaction progresses, the sulfhydryl group is linked to the maleimide group by a thioether bond (–S–C–) to form a stable covalent conjugate. Thioether bonds are very stable and are not easily hydrolyzed or degraded by other chemical agents under conventional conditions. This makes the maleimide conjugate stable over long periods of time, making it suitable for subsequent experiments or applications. Because of this stability, maleimide conjugation technology is commonly used in targeted drug delivery, antibody-drug conjugate (ADC) development, and other biomolecular modifications.

This conjugating reaction is not only fast and efficient, but also highly selective. The nucleophilic reaction of maleimide groups with sulfhydryl groups makes it an ideal biomolecular modifier for precise modification of proteins, peptides, and other biomolecules, which are widely used in drug delivery systems, antibody conjugation, targeted therapy, and other fields.

Fig.3 Maleimide-based drug conjugation. (Akkapeddi, Padma, et al., 2016)

Cysteine Maleimide Conjugation

There are sulfhydryl strands in the cysteine residue gene which is a popular maleimide conjugate target. Cysteine is one of 20 typical amino acids whose side chain has a sulfhydryl (-SH) group capable of joining nucleophilic reactions with the maleimide group.

The cysteine residues typically sit at active locations or domains in proteins and these areas must be occupied for protein to function. Such cysteine residues can be targeted or truncated with maleimide conjugation technology without affecting the protein structure or function. So maleimide conjugation is popular in targeted delivery systems where the drug is conjugated to an antibody or peptide by cysteine maleimide.

For instance, when producing antibody-drug conjugates, maleimide is bonded to an antibody with a cytotoxic drug through a cysteine residue to deliver targeted drugs without damaging healthy tissue.

Maleimide Antibody Conjugation

Maleimide conjugation is essential to antibody-drug conjugates. ADCs are a type of targeted therapy that takes cytotoxic drugs right to cancer cells. Using maleimide conjugation technology, a drug is linked to an antibody and resulting in targeted drugs. The main procedures of maleimide antibody conjugation are as follows:

Change of the antibody: First, the antibody is modified so that a cysteine amino acid is added (usually at some point in the heavy or light chain). This was the location selected to make the best use of the affinity and stability of the antibody.

Maleimide -functionalized linker: After that, the antibody reacts with a linker that functionalizes the maleimide group. The maleimide group of this linker bonds to the cysteine on the antibody to create a stable thioether bond.

Conjugation of drugs: The linker has an active group for interaction with cytotoxic drugs too. When the antibody interacts with the linker, the drug is then attached to the antibody to create an entire ADC that could attack cancer cells and disperse the drug.

What is different about maleimide conjugation is that you can control precisely the way the drug binds to the antibody and this improves the therapeutic potential and toxicity of the ADC.

Table.2 Antibody Conjugation Services at BOC Sciences.

Thiol Maleimide Conjugation

Sulfhydryl maleimide conjugating, the nucleophilic reaction of the sulfhydryl (-SH) and maleimide to create a thioether bond. The reaction is central to most biomolecule conjugation protocols and widely employed in protein engineering, peptide modification and drug design. Advantages of mercapto-maleimide conjugation include:

High selectivity: The maleimide group is extremely selective, reacting only with sulfhydryl groups, so non-specific reaction with other functional groups is very low.

High stability: The thioether bonds obtained by conjugating of sulfhydryl maleimide are very stable and not subject to hydrolysis or other chemistry degradation.

Multiple applications: With thiol maleimide conjugation, you can bond all kinds of molecules: small molecules, peptides, proteins and large biomolecules like antibodies.

This conjugation technology is the heart of designing drugs for specific therapy, delivering medications or diagnostic molecules to precise cells or tissues. It is a crucial building block for antibody-drug conjugates, biotherapeutics and other molecular probes.

Summary

Maleimide conjugation is a very effective and flexible instrument that has a broad range of uses in molecular biology and biochemistry. Maleimide group is able to create stable thioether bonds by nucleophilic interactions with sulfhydryl groups (usually cysteine residues), which can be used for protein labeling, antibody-drug conjugates, targeted therapy drugs, etc.