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Bioconjugation General Questions

What is bioconjugation?

Strictly speaking, bioconjugation is a chemical process that links two or more biomolecules together to create new molecules. In this website, we use "bioconjugation" to describe any chemical process that involves changing 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 in a peptide or protein that are targeted for bioconjugation are hydroxyl (present on Thr, Ser, and the phenolic group in Tyr), carboxylic acid (present at the C-terminus, and on Asp, and Glu), sulfhydryl (present on Cys), and amine (present at the N-terminus, and Lys). Histidine's imidazolyl nitrogen and arginine's guanidinyl group can also be targeted, but are less commonly used. Novabiochem (now part of the EMD Biosciences) also sells non-natural amino acids that have different functional groups. Unlike proteins, DNA does not have many functional groups available for bioconjugation. Consequently, DNAs are generally modified during DNA synthesis or post synthesis to introduce extra functional groups before conjugation. Glen Research provides a variety of phosphoramidites for DNA modification. All polysaccharides, whether existing alone or attached to a protein, have sugar hydroxyl functional groups available for conjugation. The hydroxyl groups on adjacent carbon atoms of carbohydrates are generally oxidized to generate a formyl group, which is an excellent functional group for conjugation.

What are common types of chemical reactions employed in bioconjugation?

A typical organic reaction is performed in an anhydrous organic solvent under an inert atmosphere. Water is not a very good medium for an organic reaction because of its reactivity and the presence of absorbed oxygen. However, most bioconjugation reactions require a high water content in the reaction medium. Among typical conjugation reactions used by CellMosaic are 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, and reductive amination to conjugate aldehydes and amines. Other less common bioconjugation reactions include click chemistry (Cu(I)-promoted azide-alkyne [3+2] cycloaddition), the Diels–Alder reaction, and photochemical reactions involving azide.

What are the key concerns for performing a bioconjugation reaction?

There are a number of things to keep in mind while doing a bioconjugation reaction.

1) Accessibility of the functional groups in a biopolymer: Biopolymers are generally large molecules that have secondary, or even tertiary and quaternary structures. Some of the functional groups in a biopolymer may be inaccessible to the modification agents. Some manipulations such as adding detergent, salt, or changing the pH, can be done to expose functional groups. However, it is important to make sure that your biopolymer is not denatured under those conditions.

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 the reactants used in the reaction 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 exist naturally at a very low concentration and commercial biomolecules may be only available in low concentrations. Because of this, much higher reaction rate constants may be required for an effective reaction compared with traditional organic synthesis. It is often useful to concentrate your biomolecules before attempting a bioconjugation reaction to enhance the reaction rate.

4) Characterization of a bioconjugation reaction: Unlike a conventional organic reaction, which entails analysis of a small molecule, standard characterization methods such as TLC, IR, NMR, and HPLC may not work for a biopolymer conjugation reaction. Some relatively small biopolymers such as peptides and oligonucleotides, can be monitored by HPLC and LC-MS. Large biopolymers such as proteins can be monitored by gel electrophoresis or size exclusion column chromatography.

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 biopolymer conjugation 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: Typically gel electrophoresis is used to assess purity of bioconjugates. Sometimes, if the biopolymer is not too massive, Mass Spectroscopy can be used to determine its molecular weight. 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?

There are several things to consider when choosing a crosslinker: Reactive groups at the termini of the linker, length of the spacer (zero-length, length by carbon atom number), 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).