Click Chemistry in Biomedical Applications

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Click Chemistry in Biomedical Applications

The 2022 Nobel Prize in chemistry was awarded to scientists Carolyn R. Bertozzi, Morten
Meldal and K. Barry Sharpless for their development of click chemistry and bioorthogonal
chemistry. [1]

Figure 1. 2022 Nobel Prize in chemistry, soucce: reference [1]

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Barry Sharpless and Morten Meldal brought chemistry into the age of functionalism and
laid the foundation for click chemistry. While Carolyn Bertozzi took click chemistry into a
new dimension and began using it to map cells. Among many other applications, her
bioorthogonal chemistry contributes to more targeted cancer therapies, such as
antibody-drug conjugates (ADCs), fluorescent imaging, and targeted drug delivery, etc.

What Is Click Chemistry & Bioorthogonal
Chemistry?

Click chemistry: A chemical synthesis method for the rapid and efficient synthesis of
useful new molecules based on the heteroatom link (C-X-C). [1]

Bioorthogonal Chemistry: Chemical reactions that use the principles of click chemistry
to occur inside of living systems without interfering with native biochemical processes.

In 2001, an examination of nature's favorite molecules reveals a striking preference for
making carbon-heteroatom bonds over carbon-carbon bonds. The concept of "Click
Chemistry" was inspired by the fact that nucleic acids, proteins and polysaccharides are
condensed polymers held together by carbon-heteroatom bonds. Click chemistry is a
chemical synthesis method that allows for the rapid and efficient synthesis of useful new
molecules based on carbon-heteroatom link (C-X-C).

Prior to this, chemical synthesis was complicated and difficult but with low yields. Until the
first generation of click chemistry, the monovalent copper-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction was proposed, the complex reactions started to be
simplified by building functional molecules in a patterned reaction manner. However, the
cytotoxicity of copper catalysts limited the application of CuAAC reactions in vitro and in
vivo.

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Chemists have since discovered a strain-promoted alkyne-azide cycloaddition
(SPAAC) reaction that allows the azide-alkyne reaction to occur without the need for a
cytotoxic copper catalyst. This reaction has been used to label glycoproteins on the cell
surface in vitro and in vivo with no apparent cytotoxicity.

However, some chemists were not satisfied with the second-order reaction rate constants
of SPAAC. Therefore, Blackman et al. developed the inverse electron-demand
Diels-Alder (iEDDA) reaction between the cycloaddition reactions of s-tetrazine and
trans-cyclooctene (TCO) derivatives to produce faster copper-free click chemistry than the
SPAAC reaction.

Figure 2. Characteristics of currently used click chemistry reactions, source: reference [3]

Click Chemistry in Biomedical Applications

Click chemistry has made important advances in the field of biomedical research,
particularly copper-free click chemistry, including SPAAC and iEDDA reactions. In in vitro
studies, click chemistry allows for the specific labeling of cellular target proteins and the

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study of the interaction of drug targets with drug surrogates in living cells. In addition, cell
membrane lipids and proteins can be selectively labeled in vitro and cells can be linked
together by click chemistry. In vivo studies, click chemistry makes molecular imaging and
drug delivery for diagnosis and therapy efficient and effective. [3]

Following, we present several specific applications of click chemistry in biomedical
research.

Click Chemistry for Fluorescence Imaging

One of the most interesting applications of copper-free click chemistry might be the
uorescence imaging of target of interest (TOI) proteins inside cells.[3] Especially with
iEDDA reactions, innate TOI proteins in live cells could be successfullyvisualized with a
TCO–ligand conjugate and subsequent treatment of Tz containing uorophores (FLTz).

For example, the clinical drug AZD2281 was combined with TCO to develop a bioprobe
for the study of PARP1 protein (known to be an important cellular protein for DNA repair).
TCO was coupled to the anticancer agent Taxol and tubulin proteins inside cells were
successfully visualized with the Taxel-TCO/Tz-BODIPY FL combination. Later
multiligand-TCO conjugates such as BI2536, Foretinib, Dasatinib and Ibrutinib were also
used to develop targeting various TOI proteins such as polo-like kinase 1 (PLK1), MET,
and BTK proteins. [3]

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Figure 3. Copper-free click reaction between AZD2281-TCO and Texas Red-Tz in
MDA-MB436 cells. Source: reference [3]

Click Chemistry in Targeted Drug Delivery

Click chemistry has emerged as a powerful chemical tool for the targeted delivery of drugs
in the study of living organisms. The fast second-order reaction rate constants, simplicity
and orthogonality of click chemistry can be used for polymer synthesis or for site-specific
modification of bioligands during drug carrier development. For example, in 2012, Koo
and Lee et al. provides evidence, for the first time, that click chemistry in vivo could be
used for nanoparticle delivery. In the study, azide group labeling of tumor cells by
Ac4ManNAz-loaded nanoparticles and second tumor targeting by DBCO-modified
nanoparticles containing photosensitizers, were injected into mice sequentially, and
tumor-targeting was enhanced by SPAAC between azide groups and DBCO. [3]

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Figure 4. Application of click chemistry for tumor-targeted drug delivery. Source: reference
[3]

Click Chemistry-based ADC Synthesis

Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has great potential in the synthesis
of antibody-drug conjugates (ADCs) [4]. Researchers have now devised efficient and
cost-effective CuAAC-based ADC conjugation methods and demonstrated that ADCs can
be synthesized rapidly, which led to the development of the GlycoConnect coupling
technology. GlycoConnect enables targeted conjugation using native glycosylation sites
and can convert monoclonal antibodies into stable conjugated ADCs in just a few days.
The technology is based on two processes: first, enzymatic remodeling (modification and
tagging with azide), followed by ligation of the payload based on copper-free click
chemistry. Synaffix has partnered with several companies with its next-generation ADC
technology platform, including GlycoConnect.

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Figure 5. Next-generation ADCs being developed under a license agreement

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ADC Therapeutics was the earlier company to license Synaffix ADC platform technology
and is currently the representative with the largest number of products developed using
this technology, of which ADCT-601 is already in the clinical study phase. [5] Currently,
ADC Therapeutics' publicly available products in the solid tumor space (3/5) is using
Synaffix's ADC technology.

Figure 6. ADC Therapeutics Pipelines

Figure 7. Structure of ADCT-601

Click Chemistry-based PROTAC Synthesis

Click chemistry is often used in the linker of PROTAC molecules to connect the two ends
of the molecule due to milder reaction conditions and higher efficiency. Ryan P Wurz et
al. demonstrates the utility of this approach with the bromodomain and extraterminal

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domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von
Hippel–Lindau (VHL) proteins [6].

Figure 8. Click Chemistry-based PROTAC Synthesis, source: reference [6]

Click Chemistry-based Diagnosis

Click chemistry can also be used to develop molecular tools for understanding tissue
development, disease diagnosis and treatment monitoring. Many cancers release
membrane-bound microvesicles (MVs) into the peripheral circulation, and the analysis of
MVs such as glioblastomas (GBMs) is a promising method for disease diagnosis. For
example, Lee et al. reported a microfluidic system combining iEDDA-type click chemistry
and small micro-nuclear magnetic resonance (μNMR) for the analysis of MVs in the blood
of GBM patients [4].

​
Figure 9. Click Chemistry-based Diagnosis. source: reference [3]

Summary

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Click chemistry and non-copper bioorthogonal reactions have made important advances
in the field of biomedical research. Click chemistry allows for the specific labeling of
cellular target proteins and can be used to adhere cells together and also enables efficient
and effective molecular imaging and drug delivery for diagnostic and therapeutic purposes.
Click chemistry can also be used to develop molecular tools such as DNA nanocatalysts,
chemical synthesis of genomic DNA, assisted CRISPR-Cas gene editing, ADC and
PROTAC synthesis, etc. Overall, click chemistry has become a valuable tool in the
biomedical field and in organic chemistry.

Biopharma PEG proudly nurtures this energy by being a leading provider of click
chemistry reagents worldwide. We supply PEG products & reagents functionalized with
azide, alkyne, DBCO and other cyclooctyne. Contact us at [email protected] now.

References:
[1] https://www.nobelprize.org/prizes/lists/all-nobel-prizes-in-chemistry/
[2] Kolb HC, Finn MG, Sharpless KB. Click Chemistry: Diverse Chemical Function from a Few Good
Reactions. Angew Chem Int Ed Engl. 2001 Jun 1;40(11):2004-2021.
[3] Kim E, Koo H. Biomedical applications of copper-free click chemistry: in vitro, in vivo, and ex vivo.
Chem Sci. 2019 Aug 16;10(34):7835-7851.
[4] Vatansever EC, Kang J, Tuley A, Ward ES, Liu WR. An optimal "Click" formulation strategy for
antibody-drug conjugate synthesis. Bioorg Med Chem. 2020 Dec 15;28(24):115808. doi:
10.1016/j.bmc.2020.115808.
[5]. Zammarchi F, Havenith KE, Chivers S, Hogg P, Bertelli F, et al. Preclinical Development of
ADCT-601, a Novel Pyrrolobenzodiazepine Dimer-based Antibody-drug Conjugate Targeting
AXL-expressing Cancers. Mol Cancer Ther. 2022 Apr 1;21(4):582-593.
[6] Wurz RP, Dellamaggiore K, Dou H, Javier N, Lo MC, McCarter JD, Mohl D, Sastri C, Lipford JR, Cee
VJ. A "Click Chemistry Platform" for the Rapid Synthesis of Bispecific Molecules for Inducing Protein
Degradation. J Med Chem. 2018 Jan 25;61(2):453-461.

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