Click chemistry is a popular technique for generating complex molecules rapidly and reliably by joining small units together. This technique has tremendous potential to modify peptides and proteins such as attaching the following: ligands, lipophilic or lipophobic groups, or hydrophilic and hydrophobic linkers. CPC Scientific can help you achieve your click chemistry goals quickly and efficiently.
The CuAAC click reactions work by “clicking” an alkyne-modified peptide with an azide-modified molecule, forming a triazole link connecting two units.
The click reactions are highly efficient, wide in scope, stereospecific, and simple to perform using inexpensive reagents. In addition, they can be conducted in benign solvents such as water and they have final products that are easy to isolate. Most click reactions have a high energy content that make the reactions irreversible and involve carbon-heteroatom bonding processes. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) between an alkyne and an azide, under mild conditions to form a rigid five-membered triazole ring, fits the concept well and is one of the most popular prototype click reactions to date. As for functionality, the azides are easy to introduce, stable to water and oxidative conditions, orthogonal to many commonly used functional groups, and vigorously reactive. For applications in vitro and in vivo, azides are virtually absent from any naturally occurring species (bioorthogonal).
Due to its relative planarity, strong dipole moment (~5 D), and hydrogen bonding ability, the 1, 2, 3-triazole function formed by a click reaction between an azide and alkyne bears a physicochemical resemblance to the amide bond.
The simplicity and reliability of CuAAC, as well as the bioorthogonality of starting reactants, has contributed to a wide range of peptide science applications. The most important applications of click chemistry in peptide science include cyclization, chemical ligation, and conjugation to biomolecules, nanoparticles, polymers, and other chemical entities. Peptide modification for a variety of applications utilizing click chemistry can be performed in different ways. For example, peptides can be converted post-synthetically to an azido derivative, which can be clicked with an appropriate substrate containing a clickable alkynyl group or vice versa. Peptides can also be made by inter- and intramolecular click reactions using azide or alkyne containing amino acids or building blocks during peptide synthesis.
Copper-free click reactions
The cytotoxicity of copper remains a concern and a limiting factor for widespread in vivo applications of CuAAC click reactions. The presence of copper and/or reducing agents can cause degradation or aggregation of the targeted biomolecules. Fortunately, these challenges can be overcome by using copper-free ‘click’ chemistry. This technique is based on the reaction of cyclooctynes (such as DIBAC and MOFO) with azides in the absence of a copper catalyst at ambient temperature. A recent peptide application is the synthesis of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptide conjugate prepared by the attachment of DOTA to monofluoro-cyclooctyne (MOFO) followed by bioconjugation to an azide-modified peptide.
Cyclo (-RGDfK) Azido-PEG4 (RGDP-011). This RGD-based peptide contains a Click-ready azide group and PEG4 spacer. It binds specifically and with high affinity to αvβ3 receptors on neovascular blood vessel sections of different major human cancers. The integrin α(IIb)β(3)-specific cyclic hexapeptide contains an Arg-Gly-Asp (RGD) sequence.
Custom Click Peptide Citations
1. Puthenveetil, Sujiet, et al. "Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate." PloS One 12.5 (2017): e0178452.Learn More »
2. Graaf, Matthew D., et al. "New Methods for the Site-Selective Placement of Peptides on a Microelectrode Array: Probing VEGF–v107 Binding as Proof of Concept." ACS Chemical Biology (2016).Learn More »
3. Chen, Long, et al. "Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency." Scientific Reports 6 (2016).Learn More »
4. Lin, Tzu-Yin, et al. "Novel theranostic nanoporphyrins for photodynamic diagnosis and trimodal therapy for bladder cancer." Biomaterials 104 (2016): 339-351.Learn More »
5. Lo, Justin Han Je. Targeting nucleic acids for pancreatic cancer: disease modeling and therapy. Diss. Massachusetts Institute of Technology, 2015." Science China Life Sciences 57.1 (2014): 117-127.Learn More »
6. Zhu, Shu, et al. "FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow." Blood 126.12 (2015): 1494-1502.Learn More »
7. Zhu, S., et al. "Platelet-targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow." Journal of Thrombosis and Haemostasis (2016), 14: 1070–81.Learn More »
Click Peptide Modifcations and Applications
CPC Scientific is an expert in click chemistry reactions for peptide modifications and applications that include:
- Synthesis of clickable peptides containing alkyne or azide functionalities
- Synthesis of clickable amino acids for incorporation into peptides
- Synthesis of building blocks for peptide-click chemistry
- Design and synthesis of substituted cyclooctyne-modified peptides for copper-free click reactions
- Conjugation to small molecules, PEG chains, surfaces, metal-chelates, and fluorophores
- Bioconjugation, ligation, stapled peptides, and macrocyclization
Click chemistry provides a relatively easy approach to peptide glycosylation.
Stapled Peptides by Click Chemistry
The high efficiency and mild conditions of “click” reaction (Copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction) combined with the ease of synthesis of the necessary unnatural amino acids, allows for facile synthesis of triazole-stapled peptides. For example, a combination of L- Nle (εN3) and D-Pra (D-propargylalanine) substituted at the i and i+4 positions, can be used for the generation of single triazole-stapled peptides.