Maleimide linkers are an essential tool in bioconjugation, allowing for the covalent attachment of biomolecules such as antibodies, peptides, and proteins to various compounds like drugs, fluorophores, or other macromolecules. These linkers play a vital role in a range of biomedical applications, including the development of antibody-drug conjugates (ADCs), imaging agents, and targeted therapies. Their unique chemical reactivity and stability have made them one of the most widely used linkers in the field of targeted drug delivery.

What is Maleimide Linker?

Maleimide (MC) is a functional group characterized by a five-membered cyclic imide structure. Its most notable feature is its ability to form stable thioether bonds with thiol groups (-SH) on cysteine residues in proteins or peptides through Michael addition reactions. This reaction occurs under mild physiological conditions, making maleimide linkers ideal for bioconjugation applications that require maintaining the biological activity of the molecule of interest. The core maleimide group is often attached to other functional moieties, such as polyethylene glycol (PEG), in order to enhance solubility, biocompatibility, and reduce immunogenicity. Maleimide linkers are versatile and can be further modified with other reactive groups like NHS esters or azides to create multifunctional linkers.

Fig. 1. MC linker in antibody-drug conjugates.

Reactivity of Maleimides

The double bond of maleimides can undergo alkylation reactions with thiols, forming stable thioether bonds. Maleimide reactions exhibit specificity for thiols in the pH range of 6.5 to 7.5. At pH 7.0, the reaction rate of maleimide with thiols is 1000 times faster than that with amines. At higher pH levels, some cross-reactivity with amino groups may occur (Brewer and Riehm, 1967). One of the carbons adjacent to the maleimide double bond is nucleophilically attacked by the thiolate anion, resulting in an addition product (Reaction 3.19). When a sufficient amount of -SH groups has been alkylated, the reaction can be tracked spectrophotometrically by a decrease in absorbance at 300 nm as the double bond reacts and disappears. Maleimide groups can also hydrolyze to form the open-ring maleamic acid, which does not react with thiols. Hydrolysis may also occur after the coupling of thiols with maleimides. This open-ring reaction typically accelerates with increasing pH. Hydrolysis is also dependent on the type of chemical groups adjacent to the maleimide functional group. For example, the cyclohexane ring in SMCC provides greater stability against maleimide hydrolysis, possibly due to its steric effects and lack of aromaticity. However, the adjacent phenyl ring in MBS can significantly increase the rate of hydrolysis on the maleimide ring.

Types of Maleimide Linkers

Maleimide linkers play a crucial role in bioconjugation, enabling the selective attachment of a wide range of functional groups. The variety of linkers, including Mal-PEG-alkyne, Mal-PEG-amine, and Mal-PEG-biotin, allows researchers and developers to tailor their approaches for specific applications in drug development, diagnostics, and biomolecular research.

Maleimide Linkers from BOC Sciences

What are Maleimide Linkers Used For?

Maleimide linkers are versatile chemical entities widely used in bioconjugation, drug delivery, and diagnostics due to their unique ability to form stable covalent bonds with thiol-containing compounds. Their primary function is to facilitate the selective attachment of various biomolecules, such as proteins, peptides, and small molecules, thereby enhancing the efficacy and specificity of therapeutic applications.

• Protein Labeling and Imaging

Maleimide linkers are extensively used to label proteins with fluorescent dyes, biotin, or other detectable markers for imaging or analytical applications. By targeting cysteine residues on proteins, maleimide linkers enable the stable attachment of these probes, ensuring that the label remains intact during downstream analysis. For example, maleimide-based biotinylation reagents are used to attach biotin to proteins, facilitating their detection or purification using streptavidin-based methods.

• PEGylation

PEGylation involves attaching PEG chains to proteins or peptides to improve their pharmacokinetic properties. Maleimide linkers can be used to conjugate PEG to thiol groups on biomolecules, enhancing the solubility, stability, and half-life of therapeutic proteins. This strategy has been widely adopted to improve the performance of protein-based drugs and reduce their immunogenicity.

• Nanoparticle and Liposome Functionalization

Maleimide linkers are widely used in functionalizing nanoparticles and liposomes for targeted drug delivery. By conjugating targeting ligands, such as antibodies or peptides, to the surface of these carriers via maleimide linkers, selective binding to diseased cells is achieved, enhancing drug accumulation at the target site and minimizing off-target effects. For example, maleimide-functionalized PEG linkers are commonly employed to attach antibodies to liposomes for cancer therapy, allowing the drug to preferentially localize to tumor tissues. Additionally, maleimide linkers facilitate the attachment of imaging agents to nanoparticles, enabling theranostic applications where drug delivery is combined with real-time imaging.

• Enzyme-Activated Drug Release

Maleimide linkers are crucial in enzyme-activated drug release systems, where drug activation is triggered by specific enzymes overexpressed in diseased tissues, such as tumors. These linkers remain stable during circulation and are cleaved by tumor-associated enzymes, such as matrix metalloproteinases (MMPs) or cathepsins, to release the active drug precisely at the tumor site. This strategy improves the therapeutic window by reducing systemic toxicity and enhancing drug efficacy. For instance, maleimide linkers can incorporate peptide sequences that are cleaved by enzymes like cathepsin B, releasing the drug only in the tumor microenvironment.

• Antibody-Drug Conjugates

In ADCs, maleimide linkers are used to attach potent cytotoxic drugs to monoclonal antibodies. This enables targeted drug delivery, as the antibody can bind specifically to cancer cells while minimizing off-target effects. Maleimide-thiol chemistry is particularly suited for ADCs because cysteine residues on antibodies can be selectively modified, ensuring precise conjugation of the drug payload. Maleimide linkers also allow for the controlled release of the drug in the tumor microenvironment. For instance, cleavable maleimide linkers can be designed to release the drug under specific conditions, such as in the presence of reducing agents like glutathione, which is elevated in tumor cells.

Maleimide Linkers for ADCs

Of the 12 FDA-approved ADCs, 10 utilize the maleimide-thiol reaction in their construction. Cysteine-coupled ADC drugs are formed by coupling MC with cysteine on the antibody. It is noteworthy that Kadcyla utilizes lysine conjugation, but its MC reacts with the thiol group on DM1 through an addition reaction (Fig. 2). Nevertheless, the thioester bonds generated in these ADCs are unstable in the presence of thiol-containing substances, as they can break through reverse Michael reactions or exchange with endogenous thiols such as HSA and GSH, leading to suboptimal pharmacodynamics, pharmacokinetics, and safety. Within 7-14 days, the payload drop-off rate of ADCs containing thioester bonds in plasma can be as high as 50-75%.

Fig. 2. Structures of FDA-approved ADCs (Protein Cell. 2018, 9(1): 33-46).

The ideal approach would be to prepare open-ring MC (t1/2 > 2 years); however, the reactivity of the MC group is relatively weak and challenging to achieve. Reported methods for preparing stable open-ring products mainly involve the maleimide-thiol reaction to form thioester bonds, followed by inducing hydrolysis. However, these indirect strategies often require the introduction of additional catalytic groups near the maleimide or hydrolysis of ADCs containing thioester bonds in specific alkaline buffers, leading to further clinical validation and increasing the risk of contamination and denaturation of the final product. Consequently, researchers have attempted to develop linkers based on the coupling of thiols with maleic acid methyl ester (MMAE). Subsequent antibody coupling reactions indicate that maleic acid methyl ester-based linkers possess similar reactivity characteristics to traditional maleimides and can effectively couple with thiol groups of antibodies. Under mild reaction conditions, the maleic acid methyl ester group can undergo addition reactions with different types of thiols, directly yielding structurally defined open-ring products. This approach allows for the direct acquisition of open-ring thiol-linked ADCs through a one-step coupling reaction while retaining the advantages of traditional maleimide-based ADCs.

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