Application of Copper-free Click Chemistry: In vivo

Click Chemistry is a term that was introduced by K. B. Sharpless in 2001 to describe the reactions which can afford products in high yields with excellent selectivities by carbon-hetero bond forming reactions. Here, “Click” means joining of molecular pieces as easily as clicking together of two pieces of a seat belt buckle and “in vivo” means “within the living organisms”. Click chemistry has been broadly used for chemical reactions that have orthogonality, high yields, and fast kinetic second order reaction rate constants [1,2]. These kinds of orthogonal reactions are useful for organic synthesis containing multiple steps and various functional groups.

Particularly, Organic chemists have attempted to remove the toxic copper catalyst from the representative click reaction, copper catalyzed [3 + 2] azide–alkyne cycloaddition (CuAAC). Their trials have resulted in ‘copper-free’ click chemistry which is highly attractive to biological or biomedical researchers [3].

To reduce the cytotoxicity of the copper catalyst in CuAAC, researchers tried to use stabilizing ligands. Amo et al., utilized bis(tert-butyltriazoly) ligand which is water-soluble and effective in promoting CuAAC [4]. Cells were alive after CuAAC using the ligand and it was applied to in vivo imaging of zebrafish embryo. Bevilacqua et al. and Kennedy et al. also have demonstrated cell labelling by CuAAC using similar bis(tert-butyltriazoly) ligand and Cu(II)–bis-L-histidine complex, respectively [5]. To overcome this limitation and enhance our convenience, other chemists have increased the reactivity of alkynes using a rings strain which enables an azide–alkyne reaction without the need for a cytotoxic copper catalyst.

Eunha Kim et al., copper free click chemistry is successfully applied in the biomedical field of imaging, drug delivery, and diagnostic analysis [1]. They categorize this research based on the site at which the click reaction occurs, namely in vitro, in vivo, and ex vivo. But we discuss in this content copper free click Chemistry of in vivo imaging bio-medical application only. 

In vivo imaging 

Artificial chemical reactions in vivo have been challenging for the researchers because the environment in a living body has large amounts of different molecules including ions, small chemicals, nucleotides, and proteins. This means that bioorthogonality is required for reactions in vivo. Furthermore, the concentration and contact time of click molecules are limited in vivo, and the second order reaction rate constant is also important.

Encouraged by these promising results regarding click chemistry in vivo, researchers have attempted to utilize the click chemistry to obtain biological or biomedical information from mice. Recently, Xie et al. introduced in vivo labelling of brain sialoglycans using liposomes (Figure 1) [6]. 9-Azido sialic acid, a metabolic precursor used in their research paper, cannot cross the blood–brain-barrier (BBB), thus they have used a liposome carrier to deliver the molecules to the brain tissue. After i.v. injection of 9-azido sialic acid-loaded-liposomes, the molecule could reach the brain and participate in brain metabolic glycoengineering. As a result, the newly synthesized sialic acid in the brain tissue could be modified with azide groups, which could be further labeled after i.v. injection of DBCO-Cy5.5 by SPAAC (strain-promoted azide– alkyne cycloaddition) in vivo. Even though the fluorescence imaging has many advantages, including easy handling and high resolution, its application in medical imaging is restricted due to its short penetration depth [7]. Therefore, other imaging modalities including PET/SPECT, MRI, and ultrasound are, preferred in clinical settings. Researchers have also tried to apply click chemistry in vivo with these imaging techniques. Rossin et al. applied click chemistry in vivo to SPECT imaging for tumor imaging in mice [8].

Figure 1. In vivo fluorescence imaging of sialoglycans in mouse brain by copper-free click chemistry [6].

Zlitni et al. have introduced a targeting method based on click chemistry in vivo for ultrasound imaging [9]. Similar to the study by Rossin et al., they have used TCO-modified antivascular endothelial growth factor receptor 2 antibodies (TCO-antiVEGFR2). VEGFR2 is over expressed in tumor cells, thus TCO antiVEGFR2 (Vascular endothelial growth factor receptor 2) can bind SKOV-3 human adenocarcinoma tumor tissues in mice after i.v. injection. After that, they have injected Tz-modified microbubbles filled with gas for ultrasound contrast enhancement to mice via tail veins. These bubbles could bind TCO-antiVEGFR2 to tumor tissues by click chemistry in vivo, generating ultrasound signals in tumor tissues. Quantitative data showed that these pre-targeting methods provided approximately 4-fold increased signals in tumor tissues compared to non-targeted microbubbles. It showed an approximately 40% increase in value compared to that of the control microbubbles directly modified with antibodies. This type of approach using bioorthogonally modified microbubbles and pretargeting is useful to increase targeting efficacy. Using a similar strategy, Wang et al. performed successful ultrasound imaging of acute thrombus in rats [10]. The results discussed are promising and demonstrate the click chemistry which has a great potential in biomedical imaging in vivo. However, strategies and the click molecules used are need to be selected carefully because the required doses of probes and reaction times will vary according to the types of imaging modalities and disease models. 

References 

1. E. Kim and H. Koo, Chem. Sci., 10, 7835 (2019).
2. H. C. Kolb et al., Angew. Chem., Int. Ed., 40, 2004 (2001).
3. Y. Takayama et al., Molecules, 24, 172 (2019).
4. D. Soriano del Amo, et al., J. Am. Chem. Soc., 132, 16893 (2010).
5. V. Bevilacqua et al.,  Angew. Chem., 126, 5982 (2014).
6. R. Xie et al., Proc. Natl. Acad. Sci. U. S. A., 113, 5173 (2016).
7. S. Luo et al., Biomaterials, 32, 7127 (2011).
8. R. Rossin et al.,  Angew. Chem., Int. Ed., 49, 3375 (2010).
9. A. Zlitni et al., Angew. Chem., Int. Ed., 53, 6459 (2014).
10. T. Wang et al., ChemBioChem, 18, 1364 (2017).

Blog Written By

Dr. S. Thirumurugan

Assistant Professor

National College, Tiruchirappalli

Tamil Nadu, India

Editors

Dr. A. S. Ganeshraja

Dr. K. Rajkumar

Dr. S. Chandrasekar

Reviewers

Dr. Y. Sasikumar

Dr. K. Vaithinathan