The general definition of bioconjugation refers to a chemical reaction where 2+ biomolecules link together and produce new molecules. This idea of bioconjugation is built around understanding labelling, conjugation, immobilisation, and covalent modification of the biomolecule’s properties.
What are the primary issues associated with a bioconjugation reaction?
A few things are going to pop up when it comes to completing the bioconjugation reaction.
1) Availability of the biopolymer’s functional groups – Since biopolymers are bigger, this means there are additional structures to think about as well. When certain structures become unavailable in a biopolymer, it makes it harder to spot the lyophilisation of the agents. The only way to do it then is to start using salt, detergent, and/or playing around with the pH level. While doing this, it’s also important not to ruin the integrity of the biopolymer.
2) Molar ratio of the reactants – With a traditional chemical reaction, the stoichiometry of the reaction is going to line up with the molar ratio of the reactants. This means the reaction with two covalently coupled compounds will lead to the use of an equal amount of starting reagents. On the other hand, a biopolymer conjugation is going to have a differing amount of starting materials, which then impact the molar ratio of the reactants. A good example of this would be the starting biopolymer being limited during a biopolymer conjugating with a small molecule. The only way to do this is then to increase the amount of that small molecule to create a stronger reaction.
3) Reactants’ concentration – Since the average biomolecule is going to be set at a low concentration, this means commercial biomolecules are going to be the same. As a result, it’s important to find higher reaction constants to make sure the reaction is in line with what’s required for organic synthesis. This is key when it is time to concentrate the biomolecules for the bioconjugation reaction.
4) Identification of a bioconjugation reaction – In comparison to a more traditional organic reaction while assessing a small-sized molecule, methods including IR, TLC, NMR, and HPLC are just not going to cut it for a biopolymer conjugation reaction. The only time this is possible would involve biopolymers that are smaller in size such as oligonucleotides and/or peptides. These can be monitored using LC-MS and HPLC. On the other hand, large-sized biopolymers (proteins) will require size exclusion column chromatography or gel electrophoresis.
5) Complex reactions – With a standard chemical reaction, the yield is commonly high, which means the product is straightforward and the reaction can be reproduced. On the other hand, a biopolymer conjugation has a lower yield that is difficult to manage due to the inclusion of poly conjugated products. As a result, if something is working for one type of biopolymer, it is not going to lead to the same types of results for another biopolymer.
6) Analysing bioconjugates – When using gel electrophoresis, the goal is to analyse the bioconjugates’ purity. If the size of the biopolymer is not as large, it is best to use Mass Spectroscopy as a way to determine its overall weight. It is not always easy to pinpoint what the precise conjugation site or molar ratio of the reactants is because the crystal structure may not be available. Due to this reason alone, it is challenging to pinpoint what the purity of a bioconjugate is, which can put another hurdle in the research process.