Site-selective functionalization of complex molecules, e.g. proteins, is essential for the generation of materials with a defined composition and remains a major challenge in chemistry.

For the modification of proteins, typically cysteine is the first choice for bioconjugation because of the unique reactivity of its thiol group as well as the low abundance of Cys residues in the majority of naturally occurring proteins (ca. 1.7%). Nevertheless, traditional cysteine-based conjugation techniques are not site specific and often multiple equal labeling sites are present which would result in a random labeling mixture. To circumvent this, previous approaches described the use of sub-stoichiometric labeling, which leads to a large degree of batch-to-batch variability. Another strategy for the selective modification of a single site among many competing ones is the use of orthogonal protecting groups or catalysts.

In nature, selective reactions in proteins are triggered by the formation of certain microenvironments by three-dimensional secondary structures from polypeptides.

Inspired by this, Pentelute and co-workers envisioned a new strategy for site-selective chemistry on proteins by fine-tuning the local environment around an amino acid residue in a small peptide sequence. The so called “π-clamp” is a four-amino-acid sequence (Phe-Cys-Pro-Phe) which tunes the reactivity of the embedded cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. Thus, the π-clamp allows the selective modification of one cysteine site in a protein containing multiple endogenous cysteine residues.

A cysteine residue inside the π-clamp selectively reacts with perfluoroaromatic probes in the presence of other competing cysteine residues or thiols.

The reported reaction is site-specific, operational under physiological conditions, enzyme-free, and comparably efficient as the commonly used azide-alkyne click chemistry. Furthermore, the π-clamp works as part of the N-terminus, the C-terminus, as well as in the middle of a polypeptide chain. Besides, its small size hardly perturbs the target protein’s native structure.

In addition to the possibility for site-selective cysteine labeling, in principle, in any linear polypeptide, the π-clamp approach should allow for macrocyclization between cysteine thiolates as a last, high-yielding synthetic step either via an exogenously added perfluoroaryl-based linker, or by incorporating non-crosslinked perfluoroaryl-based moieties first, followed by their macrocyclization with dithiol reagents.

References:

Enzymatic "click" ligation: selective cysteine modification in polypeptides enabled by promiscuous glutathione S-transferase; C. Zhang, A. M. Spokoyny, Y. Zou, M. D. Simon, B. L. Pentelute; Angew Chem Int Ed Engl. 2013; 52(52): 14001-5. https://doi.org/10.1002/anie.201306430

Convergent diversity-oriented side-chain macrocyclization scan for unprotected polypeptides; Y. Zou, A. M. Spokoyny, C. Zhang, M. D. Simon, H. Yu, Y.-S. Lin, B. L. Pentelute; Org. Biomol. Chem. 2014; 12: 566-573. https://doi.org/10.1039/C3OB42168F

π-Clamp-mediated cysteine conjugation; C. Zhang, M. Welborn, T. Zhu, N. J. Yang, M. S. Santos, T. Van Voorhis, B. L. Pentelute; Nat Chem. 2016; 8: 120-128. https://doi.org/10.1038/nchem.2413

Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation; J. A. Baccile, P. J. Voorhees, A. J. Chillo, M. Berry, R. Morgenstern, T. J. Schwertfeger, F. M. Rossi, C. D. S. Nelson; Chembiochem 22(21): 3037-3041. https://doi.org/10.1002/cbic.202100188