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.
Click Peptide Services
CPC Scientific can provide click chemistry services for modifications 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, fluorophores, and sugars.
- Bioconjugation, ligation, stapled peptides, and macrocyclization
Chart 1. Cyclo[RGD-DPhe-Lys(Azido-PEG4)] (RGDP-011) is clickable peptide containing an azide moiety connected to a PEG4 linker and cyclic hexa RGD sequence that selectively binds to αvβ3 receptors on neovascular blood vessel sections of different major human cancers. RDGP-011 is available from stock from CPC Scientific.
Click Chemistry
The CuAAC click reactions work by “clicking” an alkyne-modified peptide with an azide-modified molecule, forming a triazole link connecting two units (Figure 1). The click reaction is 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).
Figure 1. Click reaction between alkyne and azide peptide side chains.
Figure 2. 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.
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 (Figure 3). 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.
Figure 3. Click chemistry provides an alternative to hydrocarbon stapling by way of triazole-stapled peptides.
Copper-Free Click Chemistry
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.
Figure 4. Copper-free click chemistry linkers: (1) monofluorinated cyclooctyne (MOFO), (2) dibenzoannulated cyclooctyne (DIBO), and (3) dibenzoazacyclooctyne (DIBAC).
Singh, S.S., Calvo, R., Kumari, A., Sable, R.V., Fang, Y., Tao, D., Hu, X., Castle, S.G., Nahar, S., Li, D. and Major, E. Molecular Cancer Therapeutics (2024).
Zhu, Shu, et al. Blood 126.12 (2015): 1494-1502.
Click Peptide Citations
The synthesis of the linear RP-182 analog, bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl-PEG2-Lys-Phe-Arg-Lys-Ala-Phe-Lys-Arg-Phe-Phe-Lys(azido-PEG)-NH2, was achieved using standard solid-phase peptide synthesis (SPPS) protocols. After cleaving the linear peptide from the resin, macrocyclization was performed in the liquid phase through a strain-promoted click reaction. BCN introduces extra ring strain due to its fused cyclopropane structure. The combined effect of ring strain, the selection of BCN, and copper catalysis significantly increases the macrocyclization efficiency of longer peptides like RP-182.
Singh, S.S., Calvo, R., Kumari, A., Sable, R.V., Fang, Y., Tao, D., Hu, X., Castle, S.G., Nahar, S., Li, D. and Major, E. Molecular Cancer Therapeutics (2024).
- CPC Scientific Inc., 160E Tasman Dr., Suite 200, San Jose, CA 95134
[..] assembling the peptide on the Rink Amide resin and attaching the PEG azide moiety to the N-terminal Lys, the Dde group was removed as previously shown and coupled to the Fmoc-PEG2-acid. Removal of the Fmoc followed by simultaneously click/coupling to bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate gave 1c which was deprotected and cleaved from the resin to give 1c.
Liu, J., Heddleston, J., Perkins, D.R., Chen, J.J.H., Ghanbarpour, A., Smith, B.W., Miles, R., Aihara, E. and Afshar, S. Scientific Reports 14, no. 1 (2024): 13437.
- Protein Engineering, Lilly Biotechnology Center, Eli Lilly and Company, San Diego, CA, 92121, USA
- Biotechnology Research, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN, 46221, USA
- Genetic Medicine, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN, 46221, USA
Synthetic peptides were ordered from CPC scientific with > 95% purity. NNJA peptides in the formats of monomer or dendrimer were conjugated by click chemistry to siRNA targeting hypoxanthine–guanine phosphoribosyltransferase (HPRT) gene [..]
Hao, Liangliang, Natalie Boehnke, Susanna K. Elledge, Nour-Saïda Harzallah, Renee T. Zhao, Eva Cai, Yu-Xiong Feng et al. Proceedings of the National Academy of Sciences 121, no. 11 (2024): e2307802121.
The azido-modified biosensor peptide (sequence B(biotin)-eGvndneeGffsarK-(FAM)dGGPLGVRGKK-(N3)) was synthesized by CPC Scientific with >90% purity.
Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.
Chandramohan, A., Josien, H., Yuen, T.Y., Duggal, R., Spiegelberg, D., Yan, L., Juang, Y.C.A., Ge, L., Aronica, P.G., Kaan, H.Y.K. and Lim, Y.H. Nature Communications 15, no. 1 (2024): 489.
- Merck & Co., Inc., Kenilworth, NJ 07033, USA.
- Merck & Co., Inc., Boston, MA 02115, USA
- Merck & Co., Inc., West Point, PA 19486, USA
- Genentech, South San Francisco, CA 94080, USA
We thank Evans (Chen) Ge, Mike (Dixin) Xue, and Simon (Junhua) Li at Chinese Peptide Company (CPC) for peptide synthesis support.
Paul S. Marinec, Kyle E. Landgraf, Maruti Uppalapati, Gang Chen, Daniel Xie,‡ Qiyang Jiang, Yanlong Zhao, Annalise Petriello, Kurt Deshayes, Stephen B. H. Kent, Dana Ault-Riche*, and Sachdev S. Sidhu* ACS Chem. Biol. 2021, 16, 3, 548–556.
- Chinese Peptide Company, Hangzhou Economic and Technical Development Zone, China, 310018.
"The D-VEGF-A polypeptide chain (COOH acid, residues 8-109 (1)) was chemically synthesized using solid phase peptide synthesis (SPPS) and native chemical ligation, and folded to form the protein covalent homodimer, using methods adapted from our previous work [..]"
Hao, Liangliang, Renee T. Zhao, Chayanon Ngambenjawong, Heather E. Fleming, and Sangeeta Bhatia. bioRxiv (2020).
All peptides were chemically synthesized by CPC Scientific, Inc. [..] K(N3)-ANP-GPVPLSLVMGGC [..] 5FAM-GGf-Pip-KSGGGK(CPQ2)-PEG2-GC
Vickers, Timothy A., Meghdad Rahdar, Thazha P. Prakash, and Stanley T. Crooke. Nucleic Acids Research (2019).
A solution of 1.5 umol SmBiT peptide (Val-Thr-Gly-Tyr-Arg-Leu-Phe-Glu-Glu-Ile-Leu-Gly-Gly-Ser-Gly-Gly-Lys(N3)-NH2) containing an azide group at the C-terminus (CPC Scientific, 1245 Reamwood Ave, Sunnyvale, CA, USA) in DMSO (0.5 ml) was added and the reaction mixture was stirred at room temperature for 5 h.
Puthenveetil, Sujiet, et al. PloS One 12.5 (2017): e0178452.
- Worldwide Medicinal Chemistry, Pfizer Global R&D, Groton, Connecticut, United States of America.
- Pfizer Oncology Research, Pearl River, NY, United States of America.
"Peptide-payload conjugate (7) was prepared by reacting 2mM aizoacetyl-Ser-Lys-Gly-Ser-Lys (CPC scientific, inc. Sunnywale, CA) with 8 mM NHS-ester payload [19] in 50% Dimethyl sulfoxide (DMSO), 50 mM borate buffer pH 8.5 for 2 h at 37°C."
Novel theranostic nanoporphyrins for photodynamic diagnosis and trimodal therapy for bladder cancer.
Lin, Tzu-Yin, et al. Biomaterials 104 (2016): 339-351.
"Our previously reported PLZ4-PEG 5k -CA 8 telodendrimer was synthesized by the conjugation of alkyne-derivatized bladder cancer targeting ligand PLZ4 (CPC Scientific, Sunnyvale, CA)"
References
- Laughlin, Scott T., Nicholas J. Agard, Jeremy M. Baskin, Isaac S. Carrico, Pamela V. Chang, Anjali S. Ganguli, Matthew J. Hangauer, Anderson Lo, Jennifer A. Prescher, and Carolyn R. Bertozzi. “Metabolic labeling of glycans with azido sugars for visualization and glycoproteomics.” Methods in Enzymology 415 (2006): 230-250.
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Poloukhtine, Andrei A., Ngalle Eric Mbua, Margreet A. Wolfert, Geert-Jan Boons, and Vladimir V. Popik. “Selective labeling of living cells by a photo-triggered click reaction.” Journal of the American Chemical Society 131, no. 43 (2009): 15769-15776.
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Debets, Marjoke F., Christianus WJ van der Doelen, Floris PJT Rutjes, and Floris L. van Delft. “Azide: a unique dipole for metal‐free bioorthogonal ligations.” ChemBioChem 11, no. 9 (2010): 1168-1184.
- Zhu, Shu, Richard J. Travers, James H. Morrissey, and Scott L. Diamond. “FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow.” Blood, The Journal of the American Society of Hematology 126, no. 12 (2015): 1494-1502.