Bioconjugation: What Is It?
It is a chemical technique that’s used to couple two different molecules together with one being a biomolecule like a protein, carbohydrate, or nucleic acid. Of these, proteins are more diverse biomolecules since they contain a wider variety of amino acids and are, therefore, vital substrates in most bioconjugation reactions. It is worth noting that this process plays a crucial role in how proteins are modified. However, due to recent advances in biomolecule studies, proteins can now be modified to perform several functions, including target drug delivery, cellular tracking, and imaging biomarkers.
Synthesis and Strategies
While the chemistry behind the process isn’t that complicated, execution isn’t. There are a couple of obstacles that hinder bioconjugation reactions. For instance, considering that some types of amino acid residues are more predominant, some reactions can’t be selective and, as a result, are inefficient. Another problem is the presence of polar molecules protein surfaces – their presence has been known to affect reactions. Furthermore, selectively modifying multiple sites on proteins has proven challenging.
Conventional protein modification approaches are mostly second-order reactions that generally target the side chains of particular amino acids like lysine and cysteine. Lysine and cysteine side chains contain amino and thiol groups, respectively. These are what make it easier for them to be modified using a wider variety of reagents. However, as technology has advanced and advances in biochemistry have been made, strategies have now been developed that make the bioconjugation process more efficient.
The protein of interest often decides which strategy is going to be used. If the protein being worked on is present in a mixture and can’t be isolated, alternate techniques will have to be employed. However, if its present as a purified protein, the next question to ask is if site-specificity is required.
A subsection of bioconjugation reactions that’s become quite vital in recent days is biorthogonal reactions. These are living system reactions that don’t affect natural processes and provide mechanisms for modifying and targeting specific sites on proteins and applications include photochemical-switching behavior in proteins, live-cell surface labeling, and enhanced fluorescence.
Aldehyde and ketone modification reactions are excellent examples of biorthogonal reactions. As functional groups, there are quite effective since they aren’t present on the surface of a cell and are, therefore, easily identifiable when attached. In such cases, aldehyde or ketone functional groups on biomolecules are coupled to proteins using hydrazide or aminooxy compounds to form stable hydrazine or oxime linkages between the protein and biomolecule.
N-and C-termini Modification
Since natural amino acid residues are quite widespread in proteins, it’s difficult to target single residues on a selective basis. As such, chemists have come up with techniques that allow them to target residue on N-termini and C-termini since these locations have enhanced site selectivity.
A good example of N-termini modifications involves the oxidation of threonine or serine residues to form N-termini aldehyde, which is capable of undergoing biorthogonal reactions very similar to those described earlier.
A good example of C-termini modifications is NCL or native chemical ligation. Here, thioester residues on C-termini are coupled to thiol groups on cysteine residues available on the N-termini of other proteins. This method allows for the creation of larger polypeptides by accumulating smaller ones. Native chemical ligation is particularly effective because its first step, which involves thioester and thiol group interaction, is reversible. However, the second step, which leads to the formation of an amide, is permanent. This leads to higher ligation product yields.
Cysteine residue alkylation is used to modify protein sites on a selective basis. The technique is generally effective for proteins that have been naturally expressed and depends on low cysteine residue levels. However, a majority of the reagents used during the alkylation process, such as vinyl sulfones and iodoacetamide, can modify other types of amino acids in proteins, lowering selectivity.
Since a majority of bioconjugation reactions don’t go to full completion, most chemists tend to add reagents as part of the protein purification services. This, in turn, makes it harder to extract the product from the resulting mixture. Furthermore, there are very few techniques that can be used to purify bioconjugation reaction products. However, methods like size-exclusion chromatography do a great job of isolating bioconjugated molecules if the final product is large enough to elute much faster than all the molecules inside the mixture. In some cases, purification methods that are unique to specific bioconjugation reactions have to be developed.