Bioconjugation involves chemical processes that link biomolecules together (two or more) to create other or new molecules. In this article, we will be using “bioconjugation” as a description for any of the chemical processes that entails changing the properties of biomolecules through conjugation, covalent modification, immobilisation, or labelling.
The Primary Concerns For Performing Bioconjugation Reactions
There are several things to consider while performing bioconjugation reactions.
1. Accessibility Of Functional Groups In A Biopolymer
Biopolymers are typically large molecules with secondary, or in some cases tertiary or quaternary structures. There are also a Fleet Bioprocessing few functional groups within biopolymers that cannot be accessed by certain types of modification agents. Manipulations that involve adding salt, detergent, or altering the pH, are often used to expose the functional groups. However, it becomes important to ensure that the biopolymer does not become denatured when exposed to those conditions.
2. Molar Ratio Of The Reactants
In conventional chemical reactions, the “molar ratio” of reactants usually reveals the “stoichiometry” of a reaction. For this reason, in simple combinatorial reactions, where two compounds will be covalently joined, roughly equimolar measurements of any starting reagents will be used. However, when it comes to biopolymer conjugation, molar ratios of reactants that are used in reactions will mainly depend on the available starting materials, along with the conjugation degree required. For instance, in reactions that modify biopolymers in the way of conjugating them with smaller molecules, the starting biopolymers are typically limiting. It is possible to use the larger excess of small molecules to force the reaction into completion.
3. The Concentration Of The Reactants
Many of the biomolecules naturally exist at lower concentrations, while commercial biomolecules are also usually only made available in lower concentrations. Due to this, reaction-rate constants that are much higher are often needed to achieve effective reactions when compared to traditional organic-synthesis. It is often recommended to concentrate biomolecules before proceeding with bioconjugation reactions to intensify and speed up reaction rates.
4. Characterization Of Bioconjugation Reactions
Unlike traditional organic reactions that involve examining the small molecules. The usual characterization methods including HPLC, NMR, IR, and TLC, might not work with biopolymer conjugation reactions. LC-MS and HPLC can be used to monitor some of the smaller biopolymers like oligonucleotides and peptides. Size exclusion-column chromatography or gel electrophoresis are often used to monitor larger biopolymers like proteins.
5. Complexities Of Reactions
When compared to conventional chemical reactions, where the yield is usually high, while the reaction can be reproduced, and the products are simple when it comes to biopolymer conjugation the yields are usually low, while the product is complex (usually containing several isoforms and products which include poly-conjugated products). Certain types of reaction conditions that work for 1 biopolymer might not work with other similar biopolymers.
6. Characterization Of Bioconjugates
In most cases, gel-electrophoresis will be used for assessing the “purity” of a bioconjugate. In some cases, Mass Spectroscopy is used to determine the molecular weight in biopolymers that are not too large. However, unless a unique and single functional group or a crystal-structure was available for conjugation, then it can become harder to find the precise conjugation site or the molar-ratio of any of the reactants. Due to this difficulty and complexity, the energy required for assessing the purity and composition of bioconjugates will mainly depend on the rigour and requirements of the actual downstream research.
