In the first of our three part series on Bioconjugation, we’ll be exploring what’s involved, how it enables life science research, the challenges associated with it, and the excitement in future work.  

What is Bioconjugation? 

Bioconjugation can be described as the chemical process of forming a stable covalent bond between two molecules. The establishment of the covalent bond modifies the structures of the biomolecules and grants them novel functions that they could not exhibit in their standalone states.  

At least one member of bioconjugation is a biomolecule, such as a protein, a carbohydrate, a lipid, or a nucleic acid. Small molecules are often coupled with biomolecules in biomedical and pharmaceutical applications. In particular, the conjugation of drug molecules to proteins is instrumental in producing functional biomaterials. Antibody-drug conjugates (ADC), protein nanoparticles as drug carriers, and antibody-fluorescent dye are typical examples widely encountered in biomedical applications [1]. 

Basics of Bioconjugation Techniques 

A wide range of organic chemistry techniques can be exploited to create functional bioconjugates. Several factors, including the choice of method, can significantly impact the success of bioconjugation. To start with, the purity and quality of the starting compounds are crucial, as impurities can cause side reactions that interfere with the native functions of both molecules.  

An efficient bioconjugation method has high levels of reactivity, meaning that it can proceed to completion with minimum energy input, minimum byproduct formation, and maximum yield. It must also be site-specific, which is critical for the reproducibility of the bioconjugation process. Furthermore, functional groups must have the correct stereochemistry to be accessible, and appropriate linkers must be used to bring the two components closer if necessary.  

In many cases, the bioconjugation must occur in the presence of living cells without interfering with the essential cellular processes. This demand is called bioorthogonality and may be achieved by choosing the right conjugation chemistry. 

Below is a high-level summary of commonly used chemical reactions in bioconjugation. 

The amide bond is natively found in many biomolecules, where it connects building blocks, for example amino acids in case of formation of peptides or proteins. It is also a common bioconjugation technique used in pharmaceuticals as well as nanoparticle-mediated drug delivery, owing to its high stability under a wide pH and temperature range [2]. Here, an amine group (e.g., in the nucleophilic center of a lysine residue) reacts with a carboxylic acid in the presence of coupling agents. Prior to the main interaction, the carboxylic acid is usually converted to more active electrophiles, such as esters, anhydrides, or acyl chlorides. However, other formation protocols have been proposed, such as coupling amines with alcohol and hydrolysis of nitriles [3]. 

Thiol-maleimide reaction, also known as the Michael addition reaction, is a very popular technique commonly used to modify protein structures, mainly the thiol group of cysteine residues. This bioconjugation technique is the most popular way of labeling proteins to produce functional therapeutics, the so-called Antibody-Drug Conjugates (ADC). The method has several advantages, such as the rapid reaction speed at neutral pH in aqueous buffers. Furthermore, the low relative abundance of available cysteine residues makes this reaction more selective and homogenous [4]. On the other hand, the instability of the conjugates leaves room for side reactions, such as thiazine rearrangement especially beyond pH 6.5-7.5, and non-selective labeling of primary amines (mainly lysine residues) at pH above 7.5.  

The emergence of click chemistry methods gave rise to robust and simple bioconjugation methods. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) is one such reaction, where an alkyne and an azide undergo 1,3-dipolar cycloaddition to form a 1,2,3-triazole ring. The copper catalyst is key to the progress of this reaction, as it forms copper-acetylide complexes with both compounds and brings them closer before the main reaction step [5]. Cycloaddition has a wide range of applications, from polymer synthesis to protein-small drug conjugates, owing to its high selectivity, which prevents side reactions with nearby functional groups. A big disadvantage of CuAAC is its inapplicability for bioconjugation in living tissues and organisms due to high toxicity of Copper. To overcome this limitation, the totally bioorthogonal copper free click chemistry was developed. The most commonly used functional group for copper free click chemistry is DBCO (Dibenzocyclooctyne) which was used in many in vivo studies. [15-17].   

Bioconjugation is a powerful and versatile technique that can merge a biomolecule with various types of small molecules to unlock functions that neither standalone component could exhibit. Detailed explanations of our bioconjugation products can be found in the Vector Laboratories Bioconjugation Resource Guide and Bioconjugation Application Page. In part 2, we’ll be looking at the various applications and how they can contribute to your research.   

1.        Algar, W.R., A brief introduction to traditional bioconjugate chemistry. Chemoselective and bioorthogonal ligation reactions: concepts and applications, 2017. 1: p. 1-36. 

2.        Mahesh, S., K.-C. Tang, and M. Raj, Amide bond activation of biological molecules. Molecules, 2018. 23(10): p. 2615. 

3.        Wenner, W., Phenylacetamide. Org Synth, 1952. 4: p. 760. 

4.        Stenzel, M.H., Bioconjugation using thiols: old chemistry rediscovered to connect polymers with nature’s building blocks. 2013, ACS Publications. 

5.        Presolski, S.I., V.P. Hong, and M. Finn, Copper‐catalyzed azide–alkyne click chemistry for bioconjugation. Current protocols in chemical biology, 2011. 3(4): p. 153-162. 

6.        Brassard, J., et al., Antibody-drug conjugates targeting tumor-specific mucin glycoepitopes. Frontiers in Bioscience-Landmark, 2022. 27(11): p. 301. 

7.        Parakh, S., et al., Radiolabeled antibodies for cancer imaging and therapy. Cancers, 2022. 14(6): p. 1454. 

8.        Shevtsov, M.A., et al., Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION–EGF) for targeting brain tumors. International journal of nanomedicine, 2014: p. 273-287. 

9.        Huang, Y., et al., Superparamagnetic iron oxide nanoparticles conjugated with folic acid for dual target-specific drug delivery and MRI in cancer theranostics. Materials Science and Engineering: C, 2017. 70: p. 763-771. 

10.     Ren, X., X. Meng, and F. Tang, Preparation of Ag–Au nanoparticle and its application to glucose biosensor. Sensors and Actuators B: Chemical, 2005. 110(2): p. 358-363. 

11.     Lieser, R.M., et al., Site-specific bioconjugation approaches for enhanced delivery of protein therapeutics and protein drug carriers. Bioconjugate chemistry, 2020. 31(10): p. 2272-2282. 

12.     Travis, C.R., et al., Genetic encoding of a bioconjugation handle for [2+ 2+ 2] cycloaddition reactions. ChemBioChem, 2020. 21(3): p. 310-314. 

13.     Yamazoe, S., et al., High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody–Drug Conjugate Libraries. Bioconjugate Chemistry, 2020. 31(4): p. 1199-1208. 

14.     Wan, C., et al., The thiol-sulfoxonium ylide photo-click reaction for bioconjugation. Chemical Science, 2023. 14(3): p. 604-612. 

15. Lee, S., et al. (2014). Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages. Theranostics, 4 (4), 420-31. 

16. Murrey, H. E, et al. (2015). Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging. J Am Chem Soc., 137 (35), 11461-75. 

17. Kim, K., et al. (2016). Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes In Vivo. Bioconjug Chem., 27 (4), 927-36. 

In the previous section, we discussed the methods of bioconjugation. And the marriage of a biomolecule and a small molecule has given rise to various modalities commonly used in biomedical research. Here, we focus on the applications in the fields of therapeutics, diagnostics, protein-protein interactions, and nanotechnology. 

Therapeutics 

The enhanced functionality unlocked by bioconjugation is an asset to targeted cancer therapies. Traditional chemotherapeutics lack the selectivity to distinguish between healthy and diseased cells, hence very high doses are administered to achieve potency, with severe negative ramifications on healthy cells. To overcome this, drug innovators began to use bioconjugation to attach the therapeutic to an antibody that can specifically target tumor-associated antigens on cancer cells. Thus, the bioconjugate is selectively taken up by only tumor cells, where the cytotoxin can exert its effects, and the healthy cells are exempted from cytotoxicity. Glycans are ideal targets for ADCs, as cancer cells display different glycan epitopes from their healthy counterparts. For example, mucin – an important surface peptide that is aberrantly O-glycosylated in various cancers (e.g., through extensive sialylation and fucosylation), is often a viable target for ADCs (monoclonal antibody (mAb) conjugated drugs) [6].  

The successful targeting of ADCs has been complemented with the radiolabeling of antibodies to detect tumor cells, ensure targeted delivery, and monitor drug mechanisms of action. Today, various radiolabeled antibodies are undergoing clinical trials for diagnostic and therapeutic purposes against breast cancer, lymphoma, lung cancer, prostate cancer, and gastric tumors [7]. 

Diagnostics 

Identifying and detecting disease-associated proteins requires cutting-edge diagnostics assays, where bioconjugation finds a central role. One example is enzyme-linked immunosorbent assays (ELISA) that investigate antigen-antibody interactions often using a secondary antibody for detection and quantification. This is achieved by conjugating the secondary antibody to an enzyme, such as horseradish peroxidase (HRP) or a fluorophore to generate detectable signals. The same protocol is applied to flow cytometry and imaging methods by conjugating antibodies or probes with fluorophores. This allows researchers to quantify and visualize differences between healthy and diseased cells. 

Protein Research 

Besides therapeutic and diagnostic applications, we also encounter bioconjugation in biochemistry research in general. Biotin is perhaps the most widely used detection label for protein detection and isolation studies. The conjugation of biotin to antibodies and cell surface receptors can be used to highly enhance the detection signal since streptavidin has a high affinity towards biotin and can be used as a replacement of secondary detection antibody or as a tertiary detection reagent. In addition, bioconjugation plays a central role in creating fusion proteins that allow us to study protein-protein interactions. Several strategies, such as genetic fusion, enzyme-mediated conjugation, and chemical ligation e.g. click chemistry, tetrazine ligation, oxime/hydrazone ligation, etc., are often employed to covalently link protein domains that enable two proteins to function as a single unit. 

Nanotechnology 

Nanotechnology is an emerging field in biomedical research aiming to harness the unique properties of nanoparticles to improve the selectivity of therapeutics and imaging agents. For example, superparamagnetic iron oxide nanoparticles (SPION) are frequently studied as magnetic resonance imaging (MRI) agents of brain tumors due to their small diameter and ability to cross the blood-brain barrier. Shevtsov et al. used SPION-EGF nanoconjugates to selectively target EGFR-overexpressing C6 glioma cells, achieving a robust MRI contrast of tumor cells in mouse models [8]. Another study by Huang et al. developed a multifunctional folic acid-modified SPION conjugate that not only enabled selective MRI imaging of tumors but also mediated the targeted delivery of the anticancer drug Doxorubicin to the mouse tumor cells with high potency [9]. 

Another noteworthy example is a nanoparticle-based biosensor used to quantify a biomolecular interaction or a cellular process. This method is a crucial part of medical diagnostics, such as glucose monitoring for diabetic patients [10] and biomarker detection as well as drug screening methods to evaluate potency, dosage, and pharmacokinetics. Unsurprisingly, bioconjugation is involved in the development of biosensors when attaching the biomolecule used in recognition to the nanosurface. 

In summary, bioconjugation gives biochemical modalities that can serve as promising tools in targeted therapies and diagnostics and can be integrated with nanotechnology. Detailed explanations of our bioconjugation products can be found in the Vector Laboratories Bioconjugation Resource Guide and Bioconjugation Application Page. In Part 3, we’’ll be diving into the numerous advantages and drawbacks to bioconjugation.  

1.        Algar, W.R., A brief introduction to traditional bioconjugate chemistry. Chemoselective and bioorthogonal ligation reactions: concepts and applications, 2017. 1: p. 1-36. 

2.        Mahesh, S., K.-C. Tang, and M. Raj, Amide bond activation of biological molecules. Molecules, 2018. 23(10): p. 2615. 

3.        Wenner, W., Phenylacetamide. Org Synth, 1952. 4: p. 760. 

4.        Stenzel, M.H., Bioconjugation using thiols: old chemistry rediscovered to connect polymers with nature’s building blocks. 2013, ACS Publications. 

5.        Presolski, S.I., V.P. Hong, and M. Finn, Copper‐catalyzed azide–alkyne click chemistry for bioconjugation. Current protocols in chemical biology, 2011. 3(4): p. 153-162. 

6.        Brassard, J., et al., Antibody-drug conjugates targeting tumor-specific mucin glycoepitopes. Frontiers in Bioscience-Landmark, 2022. 27(11): p. 301. 

7.        Parakh, S., et al., Radiolabeled antibodies for cancer imaging and therapy. Cancers, 2022. 14(6): p. 1454. 

8.        Shevtsov, M.A., et al., Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION–EGF) for targeting brain tumors. International journal of nanomedicine, 2014: p. 273-287. 

9.        Huang, Y., et al., Superparamagnetic iron oxide nanoparticles conjugated with folic acid for dual target-specific drug delivery and MRI in cancer theranostics. Materials Science and Engineering: C, 2017. 70: p. 763-771. 

10.     Ren, X., X. Meng, and F. Tang, Preparation of Ag–Au nanoparticle and its application to glucose biosensor. Sensors and Actuators B: Chemical, 2005. 110(2): p. 358-363. 

11.     Lieser, R.M., et al., Site-specific bioconjugation approaches for enhanced delivery of protein therapeutics and protein drug carriers. Bioconjugate chemistry, 2020. 31(10): p. 2272-2282. 

12.     Travis, C.R., et al., Genetic encoding of a bioconjugation handle for [2+ 2+ 2] cycloaddition reactions. ChemBioChem, 2020. 21(3): p. 310-314. 

13.     Yamazoe, S., et al., High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody–Drug Conjugate Libraries. Bioconjugate Chemistry, 2020. 31(4): p. 1199-1208. 

14.     Wan, C., et al., The thiol-sulfoxonium ylide photo-click reaction for bioconjugation. Chemical Science, 2023. 14(3): p. 604-612. 

15. Lee, S., et al. (2014). Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages. Theranostics, 4 (4), 420-31. 

16. Murrey, H. E, et al. (2015). Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging. J Am Chem Soc., 137 (35), 11461-75. 

17. Kim, K., et al. (2016). Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes In Vivo. Bioconjug Chem., 27 (4), 927-36. 

With knowledge of the applications that bioconjugation can play into, we finish by highlighting its key advantages and critical uses. While the applications are much broader than what we summarize here, we can deduce common threads between these methods.  

Advantages of Bioconjugation 

One of the highlights of bioconjugation is its contribution to targeting cells and tissues. In particular, the conjugation of proteins and peptides to nanoparticles, drugs, and imaging agents often helps to enhance the signal, minimize off-target interactions, which remains one of the most insurmountable bottlenecks in therapeutics and diagnostics, and label cells and tissues in vivo. 

Efficient targeting is not the only advantage. In some cases, bioconjugation serves several other purposes to improve drug efficacy. For instance, drug molecules can be coupled with biomolecules to keep them inactive until they are taken up by the target host cells. Thus, they protect drugs from enzymatic degradation, extending their half-life and reducing the required dosage. Furthermore, the attachment of hydrophilic biomolecules can increase the solubility of the drug to enhance the circulation and absorption of injectable or orally administered drugs. 

Perhaps the most important and overlooked aspect of bioconjugation is its biocompatibility. In other words, the involvement of biomolecules in functional enhancement ensures that the targeted therapeutic or diagnostic method is not cytotoxic to live and healthy cells. Therefore, bioconjugation provides an opportunity to visualize and track molecular interactions in live cells in real-time. 

Challenges and Considerations in Bioconjugation 

Despite the promising outcomes, bioconjugation methods can pose a few challenges, especially in the face of therapeutic protein manufacturing. That said, novel techniques are emerging to address these challenges and upgrade bioconjugation mechanisms. 

Specificity is one of the main bottlenecks in successful bioconjugation. The components of a bioconjugate are coupled via a linkage group involving reactive functional groups of these molecules. For proteins, lysine and cysteine are reactive sites ideal for conjugation. However, their high relative abundance in a protein can lead to heterogeneity in the number and the location of conjugation sites and disrupt the native function of the biomolecule, which can lead to undesired effects or weakened efficacy in the body.  

With the emergence of site-specific bioconjugation methods, researchers can gain a much higher level of control over the structure and function of protein conjugates. By ensuring that the covalent attachment only occurs at a specific site, they can prevent unprecedented structural alterations that might disrupt the function of the biomolecule and trigger immunogenicity.  

Furthermore, site-specific conjugation makes fine-tuning much easier when investigating the optimum stoichiometry and ligand-to-target ratio [11]. 

The concept of multivalency is another exciting upgrade in bioconjugation, where a central molecule can be attached to multiple copies of a biomolecule, resulting in enhanced target affinity and enhanced therapeutic effect. However, the technique brings on additional problems in synthesis, such as stoichiometric control, the risk of steric hindrance, and difficulty of intracellular delivery due to increased size. Furthermore, tracking the structural integrity and detecting batch-to-batch variability can be challenging, which hinders their regulatory approval and translation into clinical settings. 

Future Directions 

Bioconjugation technologies continue to evolve thanks to novel chemical, enzymatic, genomic, and computational methods. Among the most influential upgrades are click chemistry methods for improving selectivity and maximizing yield and bioorthogonal methods enabling bioconjugation of molecules in living cells. With the involvement of genomics, automation, and computational science, more improvements are on the horizon. 

As mentioned in the previous section, site-specific labeling has been a pain point in bioconjugation. Genetically encoded bioconjugation overcomes this through genetic modification of the protein to give rise to a predetermined bioconjugation site. The incorporation of unnatural amino acids into the gene sequence gives rise to a modified protein containing a functional group designed for bioconjugation [12]. 

Additionally, high-throughput screening methods can be implemented for identifying favorable conjugation sites. For example, Yamazoe et al. used mass spectrometry to analyze antibody-drug conjugate libraries to rapidly discover novel conjugation sites [13]. 

Another group of advancements aims to facilitate better stoichiometric control and integrate bioconjugation in high-throughput settings. For example, photo-click chemistry makes use of UV or visible light to trigger covalent bonds, allowing spatiotemporal control of bioconjugation reactions. Photoreactive groups are crucial to controlling the timing and site of covalent bond formation [14].   

Recently, microfluidic devices have been employed for precise and controlled bioconjugation. Microfluidic chips contain networks of channels and chambers that help control reaction volumes as well as temperature and pH. The precision bestowed by microfluidics greatly enhances reproducibility and can aid high-throughput bioconjugation.  

The advancements in stem cell research have positive implications for bioconjugation as well. 3D models derived from stem cells, such as organoids, are much better at recapitulating the physicochemical properties of tissues. Thus, using bioorthogonal conjugation methods in these models can help researchers gather biologically relevant data regarding structure and function. 

Conclusion 

With a series of three blog posts, we introduce Bioconjugation, which is a powerful and versatile technique that can merge a biomolecule with various types of small molecules to unlock functions that neither standalone component could exhibit. It can give biochemical modalities that can serve as promising tools in targeted therapies and diagnostics and can be integrated with nanotechnology. It is not without trials and tribulations; successful linkage between two molecules requires meticulous experimental procedures for selective and homogenous conjugation. However, constant improvements in click chemistry methods and cutting-edge analytical platforms will support biochemists in their quest to fulfill the criteria. Detailed explanations of our bioconjugation products can be found in the Vector Laboratories Bioconjugation Resource Guide and Bioconjugation Application Page. To learn more about bioconjugation and other ways to advance your scientific research, stay tuned! 

1.        Algar, W.R., A brief introduction to traditional bioconjugate chemistry. Chemoselective and bioorthogonal ligation reactions: concepts and applications, 2017. 1: p. 1-36. 

2.        Mahesh, S., K.-C. Tang, and M. Raj, Amide bond activation of biological molecules. Molecules, 2018. 23(10): p. 2615. 

3.        Wenner, W., Phenylacetamide. Org Synth, 1952. 4: p. 760. 

4.        Stenzel, M.H., Bioconjugation using thiols: old chemistry rediscovered to connect polymers with nature’s building blocks. 2013, ACS Publications. 

5.        Presolski, S.I., V.P. Hong, and M. Finn, Copper‐catalyzed azide–alkyne click chemistry for bioconjugation. Current protocols in chemical biology, 2011. 3(4): p. 153-162. 

6.        Brassard, J., et al., Antibody-drug conjugates targeting tumor-specific mucin glycoepitopes. Frontiers in Bioscience-Landmark, 2022. 27(11): p. 301. 

7.        Parakh, S., et al., Radiolabeled antibodies for cancer imaging and therapy. Cancers, 2022. 14(6): p. 1454. 

8.        Shevtsov, M.A., et al., Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION–EGF) for targeting brain tumors. International journal of nanomedicine, 2014: p. 273-287. 

9.        Huang, Y., et al., Superparamagnetic iron oxide nanoparticles conjugated with folic acid for dual target-specific drug delivery and MRI in cancer theranostics. Materials Science and Engineering: C, 2017. 70: p. 763-771. 

10.     Ren, X., X. Meng, and F. Tang, Preparation of Ag–Au nanoparticle and its application to glucose biosensor. Sensors and Actuators B: Chemical, 2005. 110(2): p. 358-363. 

11.     Lieser, R.M., et al., Site-specific bioconjugation approaches for enhanced delivery of protein therapeutics and protein drug carriers. Bioconjugate chemistry, 2020. 31(10): p. 2272-2282. 

12.     Travis, C.R., et al., Genetic encoding of a bioconjugation handle for [2+ 2+ 2] cycloaddition reactions. ChemBioChem, 2020. 21(3): p. 310-314. 

13.     Yamazoe, S., et al., High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody–Drug Conjugate Libraries. Bioconjugate Chemistry, 2020. 31(4): p. 1199-1208. 

14.     Wan, C., et al., The thiol-sulfoxonium ylide photo-click reaction for bioconjugation. Chemical Science, 2023. 14(3): p. 604-612. 

15. Lee, S., et al. (2014). Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages. Theranostics, 4 (4), 420-31. 

16. Murrey, H. E, et al. (2015). Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging. J Am Chem Soc., 137 (35), 11461-75. 

17. Kim, K., et al. (2016). Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes In Vivo. Bioconjug Chem., 27 (4), 927-36.