Late‐Stage Functionalization of Histidine in Unprotected Peptides

Abstract

The late-stage functionalization (LSF) of peptides represents av aluable strategy for the design of potent peptide pharmaceuticals by enabling rapid exploration of chemical diversity and offering novel opportunities for peptide conjugation. While the C(sp 2 )ÀHa ctivation of tryptophan (Trp) is well documented, the resurgence of radical chemistry is opening new avenues for the C À Hf unctionalization of other aromatic side-chains.H erein, we report the first example of LSF at C2 of histidine (His) utilizing abroad scope of aliphatic sulfinate salts as radical precursors.Inthis work, the exquisite selectivity for histidine functionalization was demonstrated through the alkylation of complex unprotected peptides in aqueous media. Finally,this methodology was extended for the installation of ak etone handle,p roviding an unprecedented anchor for selective oxime/hydrazone conjugation at histidine.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis

“…These generated instead acyl fluorides due to their ready pre initiation hydrolysis under oxidative conditions ( Supplementary Figure S1 ), leading to confounding unwanted mixed amidation and acylation that we examined in detail by proteolytic-MS/MS analyses ( Supplementary Figure S2 ). 25 , 18 …”

“…Given our strong, observed Trp selectivity, we were intrigued by the recent disclosure of Minisci-type methods applied to peptides at low pH (2–3) that appeared to favor His modification. 18 While such acidic conditions have not been applied to proteins, we explored pH dependence down to 5. Consistent with increased reactivity for protonated His imidazoliums (and so reaction at His), difluoromethylation started to occur in higher copy numbers ( Supplementary Figure S30 ), to levels exceeding the total number of Trp residues.…”

“…Here, during the course of our investigation into other difluoroalkyl radicals with proteins, we have discovered not only the sufficient reactivity of HCF 2 · toward native residues when generated under mild, reductive conditions but also a reactivity that is coupled through their inherent chemoselectivity with a Reimer-Tiemann hydrolytic manifold in heteroaromatic residue (X = Trp > His, Figure 2 c) adducts. 17 , 18 This now allows effective use of difluoromethyl radicals as masked “HC(O)· equivalents” for protein C-formylation ( Figure 2 c). The resulting, nonproteinogenic residue C -formyl-Trp (Cfw) thus bears a minimally sized reactive group at C2 that we show here proves useful for selective, electrophilic carbonyl-mediated modification in proteins as well acting as an enhanced fluorophore.…”

Exploring Chemical Modifications of Aromatic Amino Acid Residues in Peptides

“…Installation of the keto group in a site-specific manner enabled further modification of the carbonyl functionality by oxide and hydrazone chemistries to facilitate the incorporation of different tags. 93…”

A Brief Guide to Preparing a Peptide–Drug Conjugate

“…Carbonyl groups can easily condense with oxyamino group, yielding oximes, or with hydrazines, giving hydrazone functionalities. Thus, although ketones and aldehyde usually are not typical functional group of peptides, many procedures have been developed to install this group on peptidic structures [56,57,58] . Generally, oxime and hydrazone formation can be considered as a single type of conjugation reaction, as they are very similar functional groups; however, oximes have been demonstrated to be more stable than hydrazones [59] …”