Serine‐Selective Aerobic Cleavage of Peptides and a Protein Using a Water‐Soluble Copper–Organoradical Conjugate

Abstract

The site-specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine-selective peptide-cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water-soluble copperorganoradical conjugate. The method is applicable to the siteselective cleavage of polypeptides that possess various functional groups. Peptides comprising d-amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site-selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.Chemical modifications of proteins [1,2] have a great impact on a broad range of research fields, such as chemical biology, [3] chemical genetics, [4] protein engineering, [5] proteomics, [6] and drug discovery. [7] Site-specific cleaving reactions of peptide bond are among such important chemical modifications. The method is routinely used when structural determination or fragmentation of peptides plays a pivotal role, [8] and it is also regarded as a new strategy for modulating the physiological functions of proteins for medical applications. [9,10] Although peptidases hydrolyze peptide bonds at specific sites under mild conditions with high fidelity, [11] the scope of the scissile substrates is restricted in principle to genetically encoded amino acid sequences. Unnatural or structurally modified peptides and proteins are out of the scope of peptidase digestion. Metal-catalyzed site-selective hydrolysis of pep-tides [12] has been intensively studied as a potential alternative to the enzymatic method. The establishment of practical protocols and generalization to protein cleavage, however, remain challenging because of the chemical robustness of amides. [13] Herein we report a serine (Ser)-selective cleavage of peptides (including a native protein) through mild aerobic oxidation of the hydroxymethyl group of Ser, promoted by a water-soluble Cu/N-oxyl radical system.We envisioned a Ser-selective cleavage of the peptide bond initiated by aerobic chemoselective oxidation of its hydroxymethyl moiety (see Scheme 1). [8c,d] Oxidation of the hydroxymethyl moiety of a Ser residue (A) to a formyl group would produce an a-formylglycineamide intermediate B.Further oxidation of B would produce oxalimide C through oxidative deformylation. Hydrolysis of the latent precursor C under mild conditions should then be possible, because the carbonyl groups of the oxalimide are more electrophilic than those of simple amides, giving peptide bond cleaved fragments D and D'. Using molecular oxygen as a terminal oxidant, water and a C 1 molecule (possibly HCO 2 H) are stoichiometric side product...

Protein Modification at Tyrosine with Iminoxyl Radicals

“…Iminoxyl Radicals. Identifying orthogonal reactivity of aminoxyl radicals to water and various polar functional groups, [75][76][77][78] we previously developed a Trp-selective SPM using a sterically less-hindered and electronically activated persistent aminoxyl radical, keto-ABNO. 78 We are also interested in properties and reactivity of iminoxyl radicals, an alternative group of persistent N-oxyl radicals unexplored in SPM.…”

Chemical modifications of tryptophan residues in peptides and proteins

“…In 2014, Oisaki and Kanai's group found 9‐azabicyclo[3.3.1]nonane‐3‐one‐ N ‐oxyl (keto‐ABNO) adducts on Trp when exploring copper‐induced serine‐selective cleavage 40 . On the basis of this work, they established a Trp‐selective modification reaction using ABNO reagents with NaNO 2 in 0.1% acetic acid at room temperature (Figure 3).…”

A novel tyrosine hyperoxidation enables selective peptide cleavage

“…36,37 The use of chemical reagents to cleave peptide bonds, on the other hand, provides an inherent advantage over proteases when applied to chemically modied peptides. Nevertheless, their application to the cleavage of proteins/peptides is limited by low site-selectivity, 38,39 undesirable modications 40,41 (such as oxidation of Cys, Met, Tyr, and Trp) and the use of harsh reaction conditions. 42 The development of novel methods with improved selectivity and efficiency is therefore highly desirable to facilitate robust sequencing of chemically modied peptides, such as cyclic peptides.…”