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 a biomolecule's properties 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), sulfhydryls (found in Cys), and amines (present at the N-terminus and in Lys). Other groups, such as histidine's imidazole nitrogen and arginine's guanidino group, can also be targeted, although they are less commonly used. Non-natural amino acids with different functional groups are available commercially from companies like Novabiochem (now part of EMD Biosciences).
Unlike proteins, DNA does not naturally contain many functional groups for bioconjugation, so DNAs are typically modified during or after synthesis to introduce 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 a formyl group, which are excellent 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 is distinct from typical organic reaction performed in anhydrous solvent. Common bioconjugation reactions performed by CellMosaic includes:
- Amide bond formation through preactivated carboxylate, such as NHS ester with amine.
- Thioether formation through maleimide/alkyl halide with sulfhydryl.
- Hydrazone/oxime formation through ketone/aldehyde and hydrazine/aminooxy.
- Reductive amination to conjugate aldehydes and amines.
- Click chemistry
Other less common bioconjugation reactions include Diels–Alder reaction and photochemical reactions involving azide.
What are the key concerns when performing a bioconjugation reaction?
1. Accessibility of the functional groups: Biopolymers are generally large molecules with complex structures, making some functional groups inaccessible. Careful manipulation of conditions such as detergent, salt, or pH can expose these functional groups, but it is crucial not to denature the biopolymer.
2. Molar ratio of the reactants: In a conventional chemical reaction, the molar ratio of the reactants typically reflects the stoichiometry of the reaction. Thus, in a simple combinatorial reaction in which two compounds are to be covalently coupled, approximately equimolar amounts of starting reagents would be used. However, in a biopolymer conjugation, the molar ratio of reactants depends largely on the availability of the starting materials and the desired degree of conjugation. For example, in a reaction to modify a biopolymer by conjugating it with a small molecule, the starting biopolymer is usually limiting. You can use a large excess of the small molecule to drive a reaction to completion.
3. Concentration of the reactants: Most biomolecules are typically present in low concentration, necessitating higher reaction rate constants for effective conjugation. Concentrating the biomolecules before the reaction can improve the rate.
4. Characterization of the reaction: Unlike conventional organic reactions, standard characterization methods (e.g., TLC, IR, NMR, C18 HPLC) may not work for monitoring bioconjugation. Alternative techniques such as HPLC/LC-MS, gel electrophoresis, or size exclusion column chromatography are often used.
5. Complexities of the reactions: In contrast to a conventional chemical reaction in which the yield is high, the product is simple and the reaction is reproducible, in a bioconjugation reaction, the yield is often low, products are complex (and usually contain various products and isoforms including poly conjugated products). A particular reaction condition which works for one biopolymer may not work for another similar biopolymer.
6. Characterization of bioconjugates: Gel electrophoresis is commonly used to assess purity, and mass spectroscopy may be used to determine molecular weight if the MW is not too big. However, unless a crystal structure is available or a single unique functional group was used for conjugation, it may be difficult to know the exact conjugation site and the molar ratio of the reactants. Because of this complexity and difficulty, the effort required assess the composition and purity of a bioconjugate depends largely on the requirements and rigor of the downstream research.
How to choose a crosslinker for a bioconjugation?
Several factors need to be considered when selecting a crosslinker, including the reactive groups at the termini, the spacer length (zero-length, length by carbon atom number), and the physical properties of the spacer such as whether it is hydrophobic (such as an alkyl chain), or hydrophilic (such as polyethylene glycol), or cleavable after conjugation. If heterogeneity of the conjugate is not a concern, a homobifunctional crosslinker is an easy and quick approach to generate a conjugate. If you want to specifically conjugate one biopolymer to another, the heterobifunctional crosslinker approach is the better choice. Pierce (now part of Thermo Scientific) has a large selection of crosslinkers.
How to determine the molar ratio of the reacting biopolymers?
Most biological applications don't require the use of a homogeneous bioconjugate, which means that the specific site of the conjugation, and the degree of the modification may vary a little among conjugates without negatively affecting the functional properties of the bioconjugate. To determine the average molar ratio of the reacting biopolymers, UV or fluorescence spectroscopy can be used if the compound is ultraviolet- or fluorescent-active. Any remaining unreacted fluorescent compound (or dye) must be removed before taking such measurements. For conjugates that have no fluorophore, gel electrophoresis can be performed to estimate the extent of conjugation. However, it may be difficult to determine the average molar ratio through this method. Other methods that can be used are HPLC (biopolymer can be denatured first), FPLC (size exclusion column), and mass spectrometry.
How to generate a homogeneous bioconjugate?
In some applications, site-specific labeling of a biopolymer may be required. In order to achieve this, it is necessary to make sure that there is only one functional group in each biopolymer that will react. Most site-specific reactions are done by targeting sulfhydryl groups. Sulfhydryl is a very popular functional group due to its reaction specificity and its easy introduction through Cysteine mutagenesis in vitro. Sulfhydryl also plays special roles in specific antibody modification and conjugation. Other functional groups that can be targeted are post-modified aldehyde, hydrazine, and azide functional groups.
What are the advantages of bioconjugation in pharmaceutical chemistry?
Advantages (Reference 5):
1) Stabilization of substances in circulation.
2) Protection from proteolytic degradation (such as polypeptide).
3) Reduction of immunogenicity.
4) Decreased antibody recognition.
5) Increased body residence time.
6) Modification of organ disposition.
7) Drug penetration by endocytosis.
8) New possibilities of drug targeting.
How to label a membrane protein?
To label a membrane protein, it is important to pay attention to a number of details:
1) Are the functional groups in the membrane protein located in a site that is accessible to the labeling agent? This is usually determined empirically through experimental testing, because structures of most membrane proteins are not fully known.
2) Is the labeling agent compatible with the detergent to be employed? A non-ionic detergent should be used if possible.
3) Can the unreacted labeling agent be easily separated from membrane protein? Depending on the application, if you don't care about the secondary and tertiary structural integrity of the membrane protein, such as in proteomic studies, it is often possible to easily recover a membrane protein by precipitating it out of solution (for example, by lowering detergent concentration). However, if maintaining the stability of your membrane protein is important, care must be taken throughout the process. Simple size exclusion, ultracentrifugation, or dialysis can be used to separate unreacted labeling agents.
Selected References
Books for Bioconjugation
1) Greg T. Hermanson "Bioconjugate Techniques", Copyright© 2008 Elsevier Inc. ISBN: 978-0-12-370501-3.
2) Christof M. Niemeyer "Bioconjugation Protocols: Strategies and Methods", Copyright© 2004 Humana Press Inc. ISBN: 978-1-58-829098-4.
Review papers for chemistry
1) Jennifer A. Prescher; Carolyn R. Bertozzi "Chemistry in living systems" Nature Chemical Biology, 2005, 1, 13-21.
2) Muctarr Ayoub Sesay "Monoclonal antibody conjugation via chemical modification" Biopharm International, 2003, 16(12), 32-39.
3) Matthew B. Francis "New methods for protein bioconjugation" 593-634 in "Chemical Biology. From Small Molecules to System Biology and Drug Design." Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess, Copyright© 2007 Wiley-VCH, Verlag GmbH & Co. KGaA, Weinheim, ISBN: 978-3-527-3115D-7.
4) Christof M. Niemeyer "Semi-synthetic nucleic acid-protein conjugates: applications in life sciences and nanobiotechnology" Reviews in Molecular Biotechnology 2001, 82, 47-66.
5) F.M. Veronese, M. Morpurgo "Bioconjugation in pharmaceutical chemistry" IL Farmaco 1999, 54, 497-516.
6) Hardy, R. R. "Purification and coupling of fluorescent proteins for use in flow cytometry". In: Handbook of Experimental Immunology, 4th ed. DM Weir, LA Herzenberg, C Blackwell, and LA Herzenberg, editors. Blackwell Scientific Publications, Boston, 1986, pp. 31.1-31.12.
There are numerous references for specific biopolymer bioconjugation in "Methods in Enzymology", Academic Press (now part of Elsevier).