With the growing popularity of serine/threonineligation(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 obtainingexpressed protein SAL esters. Herein, we report the access of SALester surrogates from peptide hydrazides (obtained either syntheticallyor recombinantly) via nitrite oxidation and phenolysis by 3-(1,3-dithian-2-yl)-4-hydroxybenzoicacid (SAL(-COOH)(PDT)). The resulting peptide SAL(-COOH)(PDT) esters can be activated to afford the reactive peptideSAL(-COOH) esters for subsequent STL/CPL. While being operationallysimple for both synthetic peptides and expressed proteins, the currentstrategy facilitates convergent protein synthesis and combined applicationof STL with NCL. The generality of the strategy is showcased by theN-terminal ubiquitination of the growth arrest and DNA damage-inducibleprotein (Gadd45a), the efficient synthesis of ubiquitin-like protein5 (UBL-5) via a combined N-to-C NCL-STL strategy, and the C-to-N semisynthesisof a myoglobin (Mb) variant.

The hydrazinolysis of S-cyanylated peptide provides an alternative way to afford protein α-hydrazide, 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 S-cyanylation 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 semi-synthesis 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.

Rubredoxins (Rds), like those from Pyrococcus furious (Pf), have largely been found to be expressed in Escherichia coli (E. coli) as a mixture of different N-terminal forms, which may affect the properties of the protein. The typical procedures for the purification of Rds are cumbersome and usually with low yield. We present herein a streamlined purification strategy based on the reversed-phase high performance liquid chromatography (RP-HPLC), which offers high yield and high resolution after simply one-step purification following pre-treatment. We also show that RP-HPLC can be a valuable tool to gain information related to the thermal decomposition pathway of Pf-Rds.

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.

Recent years have seen an ever increasing number of enzyme mediated protein/peptide modification reactions, which contribute significantly to the elucidation of related biological functions. The many available enzymes have, however, caused difficulties for practitioners in choosing the most appropriate enzyme for a certain purpose. This review surveyed the widely used enzymes（i.e., sortases, butelase 1,subtiligase, formylglycine generating enzyme and farnesyltransferase） in the manipulation of proteins/peptides, and the application fields of these enzymes as well as the advantages and limitations of each enzyme are summarized.