Cyanobenzothiazole (CBT) Derivatives for “Click” Conjugation

In nature, the CBT-Cys click reaction is reported as part of the D-luciferin regeneration pathway in bioluminescent animals, e.g., fireflies, where 5-hydroxy-2-cyanobenzotriazole (2-CBT) and predominantly D-cysteine are forming D-luciferin, a light generating substrate for the enzyme luciferase.

Biosynthesis of luciferin from hydroxy-2-cyanobezotriazole and D-cysteine.

In the biomimetic CBT click reaction, the nitrile function of 2-cyanobenzothiazole (2-CBT) reacts with a beta-aminothiol (1,2 aminothiol) and forms a 4,5-dihydro-1,3 thiazole. The beta-aminothiol may originate from, e.g., an N-terminal D- or L-cysteine residue, which can be easily introduced recombinantly or during SPPS.

Mechanism of the CBT-Click reaction: The electron pair of the sulfur attacks the carbon of the cyano group, the nitrogen forms an enamine. The cysteine’s nitrogen then attacks the carbon of the enamine, and a cyclic intermediate is formed. Finally, the lone electron pair of the amino nitrogen of the cysteine attacks the positively charged carbon to yield the product. The nitrogen which came from the nitrile is leaving the reaction as ammonia.

Especially for biochemical and biomedical applications, the CBT-Cys click reaction bears several benefits: it is highly efficient at physiological conditions, about 120 times faster than the copper mediated azide-alkyne click reaction (CuAAC), and does not require catalysis by potentially toxic heavy metal ions (copper or ruthenium). The reaction may be used, e.g., to functionalize biomolecules, to add payloads, to fuse peptide fragments, or to generate sophisticated probes for in vitro and in vivo imaging.

Besides utilizing an N-terminal cysteine, a beta-aminothiol can also be introduced within the peptide sequence via a side chain modification, e.g., via a deliberately protected lysine. An economical way is to incorporate Lys as Fmoc-L-Lys(Mmt)-OH (FAA1622), which may be deprotected with 1% TFA as final step of the peptide synthesis. For other lysins, Boc (which requires higher TFA concentrations for removal) is reported for the side chain protection (FAA1125). Then, Fmoc-Cys(Mmt)-OH - available as L- (FAA1030) or D-isomer (FAA1614) - can be attached to the side chain of that lysine. The Fmoc is removed from Cys with piperidine in the final deprotection step, together with the N‑terminal Fmoc, and the Mmt residue is removed during the recovery of the peptide from the resin with TFA in DCM.

Schematic example for the introduction of an internal 1,2-aminothiol by SPPS into a pentapeptide (XXKXX). An internal lysine, which has been protected with an acid labile monomethoxytrityl (Mmt) group at the side chain amino group, is coupled with a Fmoc/Mmt protected cysteine as the last step of the peptide synthesis.

Besides, 2-cyanobenzothiazoles themselves have a further interesting property: They can react with the active site of cysteine proteases and inactivate these enzymes, although at rather high IC₅₀ values in the µM range.

To make the versatile CBT click reaction accessible to peptide chemists, Iris Biotech has expanded its portfolio with a collection of 2-cyanobenzothiazoles, equipped with various functionalities, e.g., biotin, additional clickable handles (azide, alkyne, DBCO), fluorescent dyes (fluoresceine, indocyanine green), or as single building block (carboxy-and amino-2-CBT). For structures and order codes, please refer to the related products at the bottom of this blog.

→ You are looking for a specifically functionalized 2-CBT derivative not listed in our portfolio? Get in contact for a custom synthesis. 

→ Interested in Click Chemistry? Check out our website or download our brochure! 

→ You want to know more about ligation technologies? See our booklet! 

References:

CBT-Cys click reaction for optical bioimaging in vivo. X. Hu, R. Tang, L. Bai, S. Liu, G. Liang, X. Sun; View. 2023; 20220065. https://doi.org/10.1002/VIW.20220065

Computational study on the mechanism of CBT-Cys click reaction. Y. Xu, C. Qi, C. Wang; CompTheorChem. 2020; 1185: 112874. https://doi.org/10.1016/j.comptc.2020.112874

Biosynthesis of Firefly Luciferin in Adult Lantern: Decarboxylation of ʟ-Cysteine is a Key Step for Benzothiazole Ring Formation in Firefly Luciferin Synthesis; Y. Oba, N. Yoshida, S. Kanie, M. Ojika, S. Inouye; Plos One. 2013; 8: e84023. https://doi.org/10.1371%2Fjournal.pone.0084023

Discovery of selective fragment-sized immunoproteasome inhibitors; L. Kollár, M. Gobec, B. Szilágyi, M. Proj, D. Knez, P. Ábrányi-Balogh, L. Petri, T. Imre, D. Bajusz, G. Ferenczy, S. Gobec, G. Keserű, I. Sosič; Eur J Med Chem. 2021; 219: 113455. https://doi.org/10.1016/j.ejmech.2021.113455

Novel regioselective approach to cyclize phage-displayed peptides in combination with epitope-directed selection to identify a potent neutralizing macrocyclic peptide for SARS-CoV-2; J. Hampton, T. Lalonde, J. Tharp, Y. Kurra, Y. Alugubelli, C. Roundy, G. Hamer, S. Xu, W. Liu; ACS Chem. Biol. 2022; 17: 2911-2922. https://doi.org/10.1021/acschembio.2c00565

Deciphering the cellular targets of bioactive compounds using a chloroalkane capture tag; Ohana, R. Ohana, T. Kirkland, C. Woodroofe, S. Levin, H. Uyeda, P. Otto, R. Hurst, M. Robers, K. Zimmerman, L. Encell, K. Wood. ACS Chem. Biol. 2015; 10: 2316-2324. https://doi.org/10.1021/acschembio.5b00351

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Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid; Z. Zheng, P. Chen, G. Li, Y. Zhu, Z. Shi, Y. Luo, C. Zhao, Z. Fu, X. Cui, C. Ji, F Wang, G. Huang, G. Liang; Chem. Sci. 2017; 8: 214-222. https://doi.org/10.1039/C6SC01461E

A biocompatible condensation reaction for the labeling of terminal cysteine residues on proteins; H. Ren, F. Xiao, K. Zhen, Y.P. Kim, H. Xie, Z. Xia, J. Rao; Angew. Chem. Int. Ed. 2009; 48: 9658-9662. https://doi.org/10.1002/anie.200903627

Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation; D. P. Nguyen, T. Elliott, M. Holt, T. W. Muir, J. W. Chin; J. Am. Chem. Soc. 2011; 133: 11418–11421. https://doi.org/10.1021/ja203111c

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Peptide cleavage induced assembly enables highly sensitive electrochemiluminescence detection of protease activity; M. Cheng, J. Zhuo, X. Zhou, D. Xing; Sens. Actuators B Chem. 2018; 262: 516-21. https://doi.org/10.1016/j.snb.2018.01.191

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