Bioconjugation General Questions
What is bioconjugation?
Strictly speaking, bioconjugation is a chemical process that links two or more biomolecules together to create new molecules. On this website, "bioconjugation" refers to any chemical process that modifies the properties of a biomolecule through covalent modification, labeling, conjugation, or immobilization.
What are the common functional groups in a natural biopolymer that can be used for bioconjugation?
The most common functional groups targeted in peptides or proteins for bioconjugation include hydroxyl groups (found in Thr, Ser, and the phenolic group of Tyr), carboxylic acids (present at the C-terminus and in Asp and Glu), sulfhydryl groups (found in Cys), and amines (present at the N-terminus and in Lys). Other groups, such as the imidazole nitrogen of histidine and the guanidino group of arginine, can also be targeted, although they are less commonly used. Non-natural amino acids containing different functional groups are commercially available from companies such as Novabiochem, now part of EMD Biosciences.
Unlike proteins, DNA does not naturally contain many functional groups suitable for bioconjugation. Therefore, DNA is typically modified during or after synthesis to introduce the required functional groups. Glen Research provides a variety of phosphoramidites for DNA modification. For polysaccharides, hydroxyl groups on adjacent carbon atoms are typically oxidized to generate formyl groups, which are highly suitable for conjugation.
What are common types of chemical reactions used in bioconjugation?
Most bioconjugation reactions require a high water content in the reaction medium, which distinguishes them from typical organic reactions performed in anhydrous solvents. Common bioconjugation reactions performed by CellMosaic include:
- Amide bond formation between a preactivated carboxylate, such as an NHS ester, and an amine.
- Thioether formation between a maleimide or alkyl halide and a sulfhydryl group.
- Hydrazone or oxime formation between a ketone or aldehyde and a hydrazine or aminooxy group.
- Reductive amination between aldehydes and amines.
- Click chemistry.
Other less common bioconjugation reactions include Diels–Alder reactions and photochemical reactions involving azides.
What are the key concerns when performing a bioconjugation reaction?
- Accessibility of the functional groups: Biopolymers are generally large molecules with complex structures, making some functional groups inaccessible. Careful adjustment of conditions such as detergent concentration, salt concentration, or pH may expose these functional groups, but care must be taken not to denature the biopolymer.
- Molar ratio of the reactants: In a conventional chemical reaction, the molar ratio of the reactants typically reflects the reaction stoichiometry. Thus, in a simple reaction in which two compounds are covalently coupled, approximately equimolar amounts of the starting reagents may be used. In a biopolymer conjugation reaction, however, the molar ratio of the reactants depends largely on the availability of the starting materials and the desired degree of conjugation. For example, when modifying a biopolymer with a small molecule, the biopolymer is usually the limiting reagent. A large excess of the small molecule may be used to drive the reaction toward completion.
- Concentration of the reactants: Most biomolecules are typically present at low concentrations, requiring reactions with sufficiently high rate constants for effective conjugation. Concentrating the biomolecules before the reaction may improve the reaction rate.
- Characterization of the reaction: Unlike conventional organic reactions, standard characterization methods such as TLC, IR, NMR, and C18 HPLC may not be suitable for monitoring bioconjugation reactions. Alternative techniques, such as HPLC, LC-MS, gel electrophoresis, or size-exclusion chromatography, are often used.
- Complexity of the reactions: In conventional chemical reactions, the yield is often high, the product is relatively simple, and the reaction is reproducible. In bioconjugation reactions, however, the yield may be low, and the products may be complex, often containing multiple conjugated species and isoforms, including products with different degrees of conjugation. A reaction condition that works for one biopolymer may not work for another similar biopolymer.
- Characterization of bioconjugates: Gel electrophoresis is commonly used to assess purity, and mass spectrometry may be used to determine molecular weight if the molecule is not too large. However, unless a crystal structure is available or a single unique functional group is used for conjugation, it may be difficult to determine the exact conjugation site and molar ratio. Because of this complexity, the effort required to assess the composition and purity of a bioconjugate depends largely on the requirements and rigor of the downstream application.
How do you choose a crosslinker for bioconjugation?
Several factors should be considered when selecting a crosslinker, including the reactive groups at its termini, the spacer length, and the physical properties of the spacer, such as whether it is hydrophobic, hydrophilic, or cleavable after conjugation. Spacer length may be described as zero-length or by the number of carbon atoms. Examples of hydrophobic and hydrophilic spacers include alkyl chains and polyethylene glycol, respectively.
If conjugate heterogeneity is not a concern, a homobifunctional crosslinker provides a simple and rapid approach for generating a conjugate. If one biopolymer must be specifically conjugated to another, a heterobifunctional crosslinker is generally the better choice. Pierce, now part of Thermo Scientific, offers a large selection of crosslinkers.
How do you determine the molar ratio of the reacting biopolymers?
Most biological applications do not require a homogeneous bioconjugate. This means that the conjugation site and degree of modification may vary slightly among conjugates without negatively affecting the functional properties of the final bioconjugate.
To determine the average molar ratio of the reacting biopolymers, UV or fluorescence spectroscopy may be used if the attached compound is UV-active or fluorescent. Any remaining unreacted fluorescent compound or dye must be removed before these measurements are taken. For conjugates that do not contain a fluorophore, gel electrophoresis may be used to estimate the extent of conjugation, although it may be difficult to determine the average molar ratio using this method. Other methods include HPLC, which may require denaturation of the biopolymer, FPLC using a size-exclusion column, and mass spectrometry.
How do you generate a homogeneous bioconjugate?
For some applications, site-specific labeling of a biopolymer may be required. To achieve this, only one functional group on each biopolymer should be available for reaction. Many site-specific reactions target sulfhydryl groups because of their high reaction specificity and the relative ease with which they can be introduced through cysteine mutagenesis. Sulfhydryl groups also play important roles in specific antibody modification and conjugation. Other functional groups that may be targeted include introduced aldehyde, hydrazine, and azide groups.
What are the advantages of bioconjugation in pharmaceutical chemistry?
Advantages include the following (Reference 5):
- Stabilization of substances in circulation.
- Protection from proteolytic degradation, such as degradation of polypeptides.
- Reduction of immunogenicity.
- Decreased antibody recognition.
- Increased residence time in the body.
- Modification of organ distribution.
- Drug penetration through endocytosis.
- New possibilities for drug targeting.
How do you label a membrane protein?
When labeling a membrane protein, it is important to consider the following:
- Are the functional groups accessible? The functional groups in the membrane protein must be accessible to the labeling reagent. This is usually determined empirically because the structures of many membrane proteins are not fully known.
- Is the labeling reagent compatible with the detergent? A nonionic detergent should be used whenever possible.
- Can the unreacted labeling reagent be separated from the membrane protein? If preservation of the secondary and tertiary structure of the membrane protein is not required, as may be the case in proteomic studies, the protein may often be recovered by precipitation, for example, by lowering the detergent concentration. If maintaining protein stability is important, care must be taken throughout the process. Size-exclusion chromatography, ultracentrifugation, or dialysis may be used to separate unreacted labeling reagents.
Selected References
Books on Bioconjugation
- Hermanson, G. T. Bioconjugate Techniques. Elsevier Inc., 2008. ISBN: 978-0-12-370501-3.
- Niemeyer, C. M. Bioconjugation Protocols: Strategies and Methods. Humana Press Inc., 2004. ISBN: 978-1-58829-098-4.
Review Papers on Bioconjugation Chemistry
- Prescher, J. A.; Bertozzi, C. R. Chemistry in living systems. Nature Chemical Biology. 2005, 1, 13–21.
- Sesay, M. A. Monoclonal antibody conjugation via chemical modification. Biopharm International. 2003, 16(12), 32–39.
- Francis, M. B. New methods for protein bioconjugation. In Chemical Biology: From Small Molecules to Systems Biology and Drug Design; Schreiber, S. L.; Kapoor, T. M.; Wess, G., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2007; pp. 593–634. ISBN: 978-3-527-31150-7.
- Niemeyer, C. M. Semi-synthetic nucleic acid-protein conjugates: applications in life sciences and nanobiotechnology. Reviews in Molecular Biotechnology. 2001, 82, 47–66.
- Veronese, F. M.; Morpurgo, M. Bioconjugation in pharmaceutical chemistry. Il Farmaco. 1999, 54, 497–516.
- Hardy, R. R. Purification and coupling of fluorescent proteins for use in flow cytometry. In Handbook of Experimental Immunology, 4th ed.; Weir, D. M.; Herzenberg, L. A.; Blackwell, C.; Herzenberg, L. A., Eds.; Blackwell Scientific Publications: Boston, 1986; pp. 31.1–31.12.
Numerous references on specific biopolymer bioconjugation methods are also available in Methods in Enzymology, published by Academic Press, now part of Elsevier.