π-Clamp-mediated cysteine conjugation

Abstract

Site-selective functionalization of complex molecules is a grand challenge in chemistry. Protecting groups or catalysts must be used to selectively modify one site among many that are similarly reactive. General strategies are rare such the local chemical environment around the target site is tuned for selective transformation. Here we show a four amino acid sequence (Phe-Cys-Pro-Phe), which we call the “π-clamp”, tunes the reactivity of its cysteine thiol for the site-selective conjugation with perfluoroaromatic reagents. We used the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues (e.g. antibodies and cysteine-based enzymes), which was impossible with prior cysteine modification methods. The modified π-clamp antibodies retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates (ADCs) for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach for site-selective chemistry and provides opportunities to modify biomolecules for research and therapeutics.

Site‐Selective Cysteine–Cyclooctyne Conjugation

“…To evaluate whether the protein function was impaired by the labelling reaction, we measured the binding affinity of the site-selectively biotinylated trastuzumab to HER2 protein. The labelled trastuzumab showed full binding affinity to recombinant HER2 protein in an Octet BioLayer Interferometry assay ( K d = 39 ± 2 pM, consistent with the previously measured binding affinity of native trastuzumab [31] , Figure 3b and S24). …”

“…We found DBCO-(PEG) 4 -biotin is inert to our previously reported π-clamp (Phe-Cys-Pro-Phe) [31] . This enables, for the first time, a site-selective labeling of three unprotected cysteine peptides in a single solution using three different reagents (Figure S33).…”

“…In particular, site-selective reactions enabled by unique amino acid sequences have been discovered, including tetracysteine tag that reacts with the biarsenical fluorescent dyes, [26,27] tetraserine tag that reacts with rhodamine-derived bisboronic acid, [28] sequence-specific 2-cyanobenzothiazole ligation, [29] p- (chloromethyl)benzamide-reactive peptide [30] and the the π-clamp-mediated cysteine perfluoroarylation. [31–33] In light of these findings, we envisioned that a designer peptide sequence could be used to achieve site-selective cysteine bioconjugation by thiol-yne reaction. We focused on the thiol-yne reaction because the important DBCO reagents are widely used and commercial available.…”

Self-assembling peptide-based building blocks in medical applications

“…A recent approach called “π-clamping” tailored biomolecules with the help of a natural small peptide sequence, and is providing to be a very exciting strategy that expands the ability of bio-conjugation chemistry [63,64]. Another approach is bioorthogonal ligand tethering (BOLT), in which a genetically encoded, unnatural amino acid on a protein is bioorthogonally reactive with an inhibitor conjugate to enable reversible regulation of protein activity in mammalian cells [65].…”

Site-Specific Incorporation of a Thioester Containing Amino Acid into Proteins

“…1–2 To this end, a number of methods have been developed including the selective modification of cysteine residues with electrophiles, 3–4 the fusion of peptide tags to proteins of interest which can be subsequently chemically or enzymatically modified, 5–8 and the incorporation of noncanonical amino acids (ncAAs) into proteins through semisynthetic 9–11 or recombinant methods. 12–13 In nature, the thioester group provides a means for selective protein conjugation; examples include protein ubiquitination, 14 intein splicing, 15 and the attachment of complement factors to pathogens.…”