Bioconjugation refers to the chemical technique used for coupling together two molecules, at least one of which is the biomolecule, such as a protein, nucleic acid, or carbohydrate. Proteins are particularly diverse biomolecules because of the various amino acids available and are therefore key substrates in bioconjugation reactions.
The bioconjugation reactions play a particularly important role in protein synthesis. Due to recent advances in the study of biomolecules, it is now possible to modify proteins to perform various functions, which include imaging biomarkers such as streptavidin conjugates, cellular tracking, as well as target drug delivery.
Synthesis and Strategy
The chemistry behind conjugation might be simple, but its execution certainly isn’t. Efficient bioconjugation reactions can be hindered by several obstacles. For instance, since certain residues of amino acids tend to be more prevalent compared to others, some reactions cannot be selective and are thus inefficient. The other key obstacle is that polar molecules on the surface of proteins may interfere with reactions. Selectively modifying multiple sites on a protein also present considerable challenges.
Classical approaches to the modification of proteins are usually second-order reactions targeting side chains of specific amino acids such as lysine and cysteine. Lysine and cysteine side chains contain amino and thiol groups, respectively, and this allows them to undergo modifications with a wide range of reagents. With the advancements in biochemistry and the advent of new technologies, strategies have been developed to increase the efficiency of bioconjugation reactions.
Strategy selection largely depends on the target protein. If the protein is present in a mixture where isolation isn’t possible, alternative approaches will have to be used. If the protein is present in the purified form, the next question that should be considered is whether or not site specificity is required.
Bio-orthogonal reactions are a subset of bioconjugation reactions that has grown increasingly important and they are reactions in living systems that don’t interfere with the native processes. Rather, they provide a mechanism for the targeting and modification of a specific site on a protein. Applications include enhanced fluorescence, live cell labelling, as well as photochemical switching behaviour in proteins.
Common examples of bio-orthogonal reactions are ketone and aldehyde modification reactions. These functional groups tend to be effective since they aren’t present on cell surfaces, which means that they can be uniquely identified after attachment. In such a scenario, a ketone or aldehyde functional group on a biomolecule is coupled to a protein using hydrazide or aminooxy compounds thus forming stable hydrazine or oxime linkages between the protein and biomolecule.
N-and C- Termini Modification
Natural amino acid residues are prevalent in proteins, which is why it is difficult to target a single residue selectively. Techniques have thus been developed for targeting residues on the N- and C- termini due to enhanced site selectivity in these locations.
A good N-terminal modification example is the oxidation of the threonine or serine residues thus forming an N-terminal Aldehyde that can undergo bio-orthogonal reactions that are similar to those previously described.
A good example of a C-terminal modification is the native chemical litigation (NLC) where a thioester residue on the C-terminal is coupled to a thiol group on a cysteine residue on another protein’s N-terminal. This technique makes the construction of large polypeptides possible through the assembly of several smaller ones. NCL is particularly powerful since the first step, which is the interaction between thiol and thioester groups is reversible, while the second step that forms an and amide is irreversible. This leads to high yields of the final ligation product.
Selective modification of a protein site is done through the alkylation of cysteine residues. The technique is usually effective for naturally expressed proteins and depends on the low abundance of cysteine residues. Still, many reagents used in the alkylation process, such as vinyl sulfones and iodoacetamides, have been shown to modify other amino acids in the proteins thus lowering selectivity.
The vast majority of bioconjugation reactions don’t usually go to full completion, which is why excess reagent is usually added. This makes it harder to extract the product out of the resultant mixture. In addition, only a handful of techniques have been developed for purifying products of bioconjugation reactions.
A simple technique such as size-exclusion chromatography can be used for isolating bioconjugated molecules if the target product is large enough to elute faster compared to the rest of the molecules present in the mixture. Purification methods unique to the bioconjugation reaction have to be developed in other instances.