No abstract

The hydrazinolysis of S-cyanylated peptide provides an alternative way to afford protein αhydrazide, a key reagent used in native chemical ligation (NCL), without the aid of any inteins or enzymes. The currently used non-selective S-cyanylation, however, allows no other cysteine in the protein besides the one at the cleavage site. Herein, we report a regioselective Scyanylation and hydrazinolysis strategy achieved via the fusion of a tetracysteine tag to the C-terminal of the protein of interest. We term it tetracysteine enabled protein ligation (TCEPL). While highly selective, the strategy is applicable for proteins expressed as inclusion bodies, and this was showcased by the efficient semisynthesis of an iron-sulfur protein rubredoxin and the catalytic and hinge domains of matrix metalloprotease-14 (MMP-14) containing 207 amino acid residues. Furthermore, the TCEPL strategy was exploited for protein C-terminal labeling with amino reagents bearing a variety of functional groups, demonstrating its versatility and generality.

Nitric oxide (NO) is an essential biological messenger, contributing a significant role in a diverse range of physiological processes. The light-controllable NO releasers are of great interest because of their potential as agents for NO-related research and therapeutics. Herein, we developed a pair of red-light-controllable NO releasers, pfRd-C9A-{FeNO}7 and pfRd-C42A-{FeNO}7 (pfRd = Pyrococcus furiosus rubredoxin), by constructing a nonheme {FeNO}7 center within the redesigned iron–sulfur protein scaffolds. While shown to be both air and thermally stable, these complexes are highly sensitive to red-light irradiation with temporal precision, which was confirmed by electron paramagnetic resonance spin trapping and Griess assay. The temporally controlled NO release from these complexes was also demonstrated in DNA cleavage assay. Overall, this study demonstrates that such a protein-based nonheme iron nitrosyl system could be a viable chemical tool for precise NO administration.

No abstract

With the growing popularity of serine/threonine ligation (STL) and cysteine/penicillamine ligation (CPL) in chemical protein synthesis, facile and general approaches for the preparation of peptide salicylaldehyde (SAL) esters are urgently needed, especially those viable for obtaining expressed protein SAL esters. Herein, we report the access of SAL ester surrogates from peptide hydrazides (obtained either synthetically or recombinantly) via nitrite oxidation and phenolysis by 3-(1,3-dithian-2-yl)-4-hydroxybenzoic acid (SAL(−COOH)PDT). The resulting peptide SAL(−COOH)PDT esters can be activated to afford the reactive peptide SAL(−COOH) esters for subsequent STL/CPL. While being operationally simple for both synthetic peptides and expressed proteins, the current strategy facilitates convergent protein synthesis and combined application of STL with NCL. The generality of the strategy is showcased by the N-terminal ubiquitination of the growth arrest and DNA damage-inducible protein (Gadd45a), the efficient synthesis of ubiquitin-like protein 5 (UBL-5) via a combined N-to-C NCL-STL strategy, and the C-to-N semisynthesis of a myoglobin (Mb) variant.