Crosslinkers

Homobifunctional and Heterofunctional linkers

Our homobifunctional linkers and heterofunctional linkers contain diverse functional groups, such as primary amines, sulfhydryls, acids, alcohols and bromides. Many of our linkers are functionalized with maleimide (sulfhydral reactive) and succinimidyl ester (NHS) or isothiocyanate (ITC) groups that react with amines. We also offer a broad selection of mono-protected (Boc, Fmoc, and Cbz) linkers.

Crosslinking Reagents

Crosslinking reagents are molecules containing two or more reactive ends that are ′activated′ to attach themselves to certain functional groups (e.g., amines and sulfhydryls) via a covalent bond. The usefulness of crosslinking chemistry is realized in applications such as:

• protein structure and/or function determination
• protein or other biomolecule immobilization
• general biomolecule-biomolecule conjugation
• antibody-drug conjugates

Some of the most common crosslinkers contain maleimide, sulfhydryl reactive groups, or succinimidyl esters (often referred to as NHS esters), which react with amines. Our portfolio comprises all functional groups with a variety of linker lengths and solubilities. Sulfosuccinimidyl esters allow for more water-soluble crosslinkers which can be useful when working with large biomolecules that are not amenable to organic solvents. Our crosslinkers with cleavable linkers (e.g., disulfides) are optimal for applications where a permanent linkage is not desired.

Selecting crosslinkers

When selecting a crosslinker for your application it is important to consider several factors, such as the reagent’s chemical and physical properties, the functional groups it targets for coupling, its length, its molecular size, its water solubility, and its cleavability:

Chemical Specificity: One of the most fundamental aspects of crosslinker design is whether the reagent is homobifunctional or heterobifunctional. Homobifunctional compounds will react at both ends with the same target functional group, thus forming a covalent crosslink between two molecules using the same type of bond. They are used in single-step reactions for polymerization of like functional groups, in the creation of intramolecular crosslinks, and in the evaluation of protein interactions. Heterobifunctional reagents have two different end groups, allowing for each end to react with a different functional group. They are used for controlled two-step reactions to avoid undesirable cross-reactions and polymerization.

Functional Groups Targeted: Amines, thiols, and hydroxyls are the main nucleophilic groups, and under the right conditions, they react directly with the electrophilic reactive groups present on many bioconjugation reagents. In contrast, functional groups consisting of carboxylates, aldehydes, organic phosphates, and reactive hydrogen sites require special activation agents or secondary coupling agents before they form covalent bonds with another functional group.

Crosslinker Length: The dimensions or overall linear length of the target molecule before and after conjugation should be considered when choosing a crosslinker. The spacer arm or cross-bridge of the reagent mainly determines the molecular length of the resulting compound. This length can be determined by use of certain molecular modeling software programs.

Cleavable vs. Noncleavable Crosslinkers: If interacting biomolecules that have been captured by crosslinking subsequently need to be isolated and analyzed, it is important for the spacer arm of the crosslinker to be cleavable. Additionally, cleavable linkers are used as transfer reagents for the study of interacting proteins. Our disulfides are some of the most common cleavable crosslinkers. Our portfolio also offers cleavable esters and sulfones.

Hydrophobic vs Hydrophilic Crosslinkers: In some applications, reagent hydrophobicity can be an advantage, especially when an application involves penetration of cell membranes. Hydrophobic reagents without strongly polar groups can quickly pass through membranes and crosslink or label internal cell proteins. However, hydrophobic compounds that contain one or more negatively charged sulfo-NHS groups will be restricted to reacting with the proteins on the outer membrane surface of cells due to their negative charge. The ability to switch between cell surface labeling and internal cellular labeling by choosing charged or uncharged reagents is one benefit of using hydrophobic crosslinkers.