Norbornenes – Will You Click Me?

Norbornene (also named norborylene, norcamphene, IUPAC: Bicyclo[2.2.1]hept-2-ene) may be regarded as cyclohexene with a methylene bridge between the carbons 1 and 4. The double bond is inducing a ring strain, which leads to significant reactivity. Molecules bearing a norbornene moiety may be coupled quickly and under mild conditions in click reactions, e.g., with azides (copper(I) catalyzed, CuAAC) or with tetrazines in Diels-Alder-reactions ([3+2] cycloadditions) with inverse electron demand (IEDDA). The latter one makes norbornene derivatives ideal for the modification of biomolecules and pharmaceutical compounds, e.g. to produce antibody-drug-conjugates (ADCs), as the use of the heavy metal copper as catalyst can be avoided. Norbornene can also be used to label proteins for further analysis or purification with tetrazine-conjugated probes or affinity matrices, or to modify surfaces in material science. The fabrication of functionalized hydrogels is another frequently reported application of norbornene-tetrazine click systems.

Diels-Alder reaction between a norbornyl moiety (green) and a methyl tetrazine (red/blue), followed by a proton shift to yield the final product. The liberation of nitrogen is driving the reaction to completion and makes it irreversible.

Besides, norbornyl residues may also be coupled with thiols under oxidative conditions with hydrogen peroxide, which may be generated in situ with horse-radish peroxidase (HRP).

There are different options how to introduce norbornene to your peptide:
In the case of biosynthetic proteins, a respective non-canonical amino acid needs to be introduced into the peptide chain. This can be realized by feeding the derivative to suitable bacterial hosts or cell-free systems followed by incorporation into the protein utilizing biorthogonal tRNA/tRNA-synthase pairs. At Iris Biotech, we are offering a Norbornene sidechain-derivatized Lysine (HAA9235). Both wildtype and specifically mutated pyrrolysine synthetases (PyIRSs) are reported to load this noncanonical amino acid onto the cognate pyrrolysine tRNA.

For the derivatization of synthetic peptides, we offer bifunctional building blocks bearing an amino-reactive activated ester moiety and a click-reactive, i.e., a tetrazine-reactive norbornene (see related products RL-2080, RL-2090). This combination allows to introduce the norbornyl group to your molecule of interest via a stable amide bond.

→ For more information about Click Chemistry or available tetrazine derivatives, download our brochure!  

→ You are looking for a special norbornene derivative not listed in our catalogue?
Please inquire for a custom synthesis! 

References:

Structural Insights into Incorporation of Norbornene Amino Acids for Click Modification of Proteins; S. Schneider, M. J. Gattner, M. Vrabel, V. Flügel, V. López-Carrillo, S. Prill, T. Carell; ChemBioChem 2013; 14(16): 2114-2118. https://doi.org/10.1002/cbic.201300435

A Need for Speed: Genetic Encoding of Rapid Cycloaddition Chemistries for Protein Labelling in Living Cells; M. J. Schmidt, D. Summerer; ChemBioChem 2012, 13(11): 1553-1557. https://doi.org/10.1002/cbic.201200321

A Genetically Encoded Norbornene Amino Acid for the Mild and Selective Modification of Proteins in a Copper-Free Click Reaction; E. Kaya, M. Vrabel, C. Deiml, S. Prill, V. S. Fluxa, T. Carell; Angew. Chem. Int. Ed. 2012; 51(18): 4466-4469. https://doi.org/10.1002/anie.201109252

Norbornenes in Inverse Electron-Demand Diels–Alder Reactions; M. Vrabel, P. Kölle, K. M. Brunner, M. J. Gattner, V. López-Carrillo, R. de Vivie-Riedle, T. Carell; Chem Europe 2013; 19(40): 13309-13312. https://doi.org/10.1002/chem.201301838

Hydrolytically Degradable PEG-Based Inverse Electron Demand Diels-Alder Click Hydrogels: N. H. Dimmitt, M. R. Arkenberg, M. Moraes de Lima Perini, J. Li, C-C. Lin; ACS Biomater Sci Eng 2022; 8(10): 4262-427. https://doi.org/10.1021/acsbiomaterials.2c00714

Fabrication of hydrogel microspheres via microfluidics using inverse electron demand Diels-Alder click chemistry-based tetrazine-norbornene for drug delivery and cell encapsulation applications; R. Pareja Tello, S. Wang, F. Fontana, A. Correia, G. Molinaro, S. López Cerdà, S. Hietala, J. Hirvonen, G. Barreto, H. A. Santos; Biomater Sci 2023; 11(14): 4972-4984. https://doi.org/10.1039/d3bm00292f

Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels; H. D. Nguyen, H.-Y. Liu, B. N. Hudson, C.-C. Lin; ACS Biomater Sci Eng 2019; 5(3): 1247–1256. https://doi.org/10.1021/acsbiomaterials.8b01607

Red Light Activation of Tetrazine–Norbornene Conjugation for Bioorthogonal Polymer Cross-Linking across Tissue; V. X. Truong, K. M. Tsang, F. Ercole, J. S. Forsythe; Chem. Mater. 2017; 29(8): 3678–3685. https://doi.org/10.1021/acs.chemmater.7b00561

Tetrazine-Norbornene Click Reactions to Functionalize Degradable Polymers Derived from Lactide; I. A. Barker, D. J. Hall, C. F. Hansell, F. E. Du Prez, R. K. O'Reilly, A. P. Dove; Macromol Rapid Commun 2011; 32(17): 1362-66. https://doi.org/10.1002/marc.201100324

A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein–Protein Conjugates; M. Baalmann, L. Neises, S. Bitsch, H. Schneider, L. Deweid, P. Werther, N. Ilkenhans, M. Wolfring, M. J. Ziegler, J. Wilhelm, H. Kolmar, R. Wombacher; Angew. Chem. Int. Ed. 59(31): 12885-12893. https://doi.org/10.1002/anie.201915079

Ligating Catalytically Active Peptides onto Microporous Polymers: A General Route Toward Specifically-Functional High Surface Area Platforms; S. A. Busche, M. Traxler, A. Thomas, H. G. Börner; ChemSusChem 2024; 17(2): e202301045. https://doi.org/10.1002/cssc.202301045

Heavy metal on stage: Making ion-exchange resin selective by peptide tetrazine-norbornene ligation; S. A. Busche, G. Vigarani, A. Retzmann, B. Meermann, H. G. Börner; Polymer 2024; 291, 126608. https://doi.org/10.1016/j.polymer.2023.126608