Bioconjugation involves ligating molecules together in which at least one of the reactants is a biomolecule, often an antibody, protein, or oligonucleotide. Bioconjugates are used for detection, assay, or targeting and tracking of biomolecules in the fields of biotechnology, medicine, and nanotechnology. Applications include attaching fluorescent probes to antibodies for flow cytometry and microscopy imaging, attaching antibodies to beads for immunoprecipitations, attaching antibodies to drugs for therapeutic development, and crosslinking of proteins to detect their biological interactions. Antibody-drug conjugation (ADC) technology uses monoclonal antibodies or other biologics to deliver highly active or potent pharmaceutical ingredients (HPAPIs) to targeted cells. In conjugated form, the HPAPIs exhibits more selective therapeutic activity, sparing non-target cells. This bioconjugation technique is utilized for targeted and safer drug delivery. The chemical and physical properties, length, molecular size, water miscibility, and cleavability of the reagent, the application criteria and the functional groups targeted for coupling determine which crosslinking reagents and reaction method are chosen for optimal bioconjugation.
One of the most fundamental aspects of crosslinker design is whether the reagent is homobifunctional or heterobifunctional. The overwhelming majority of bioconjugate reagents are bifunctional, with two reactive groups usually located at the outer ends of an organic spacer. In a homobifunctional compound, the two reactive groups are identical, whereas in a heterobifunctional compound they are different. Heterobifunctional reagents have major advantages over homobifunctional ones when forming bioconjugates, since one reactive end group couples with only a specific functional group, while the other reactive end group reacts with a different functional group.
The dimensions or overall linear length of the target molecule before and after conjugation should be considered when choosing a crosslinker or modification reagent for the conjugation reaction. The spacer arm or cross-bridge of the reagent mainly determines the molecular length of the resulting compound. Crosslinkers of different sizes thus become molecular rulers for measuring the distances between functional groups in biomolecules.
It is important for the crosslinker to be cleavable if the interacting biomolecules need to be isolated and analyzed, e.g. a crosslinker used to detect protein-protein interactions. Alternatively, a non-cleavable linker could be used where stability is required, e.g. an antibody attached to a resin for protein capture.
In some applications, reagent hydrophobicity can be an advantage, especially when an application involves the penetration of cell membranes. Hydrophobic reagents without any strongly polar groups quickly pass through cell membranes to crosslink or label internal cell proteins. On the other hand, hydrophilic crosslinkers do not cause aggregation or precipitation of the interacting molecule and can lead to water solubility of antibodies and proteins modified by them. The use of hydrophilic bioconjugation reagents also results in greater biocompatibility.
The most reactive functional groups in biomolecules are associated with the heteroatoms N, O, and S, which are nucleophilic due to an unshared pair of electrons and can spontaneously react with the compatible and electrophilic active groups on crosslinkers and modification reagents. In many cases, the nucleophilic functional groups in biomolecules are free and accessible. However, in some instances they are created to allow reactivity and coupling to take place. There are several specialized reagents available that facilitate the creation of an appropriate functional group for bioconjugation if the desired one is not available. Naturally occurring functional groups on biomolecules also exist and may consist of any combination of amines, thiols, hydroxyls, carboxylates, aldehydes, organic phosphates, and reactive hydrogens on certain activated carbon atoms.