The migration and invasion of tumor cells are major contributors to mortality in malignant tumors. Studies have shown that matrix metalloproteinase 14 (MMP14) plays a crucial role in promoting tumor cell metastasis. Its activity can be suppressed by tissue inhibitor of metalloproteinases 2 (TIMP2), which acts as a natural inhibitor. However, the development of MMP14 inhibitors for clinical use has been unsuccessful, partly due to the unclear mechanism of the interaction between TIMP2 and MMP14. In this work, we successfully obtained the N-terminal domain of TIMP2 (N-TIMP2) protein through a four-segment-three-ligation total chemical synthesis strategy and confirmed its correct refolding, thus providing a novel tool for elucidating the specific interaction mechanisms between N-TIMP2 and MMP14.

The design and semi-synthesis of azurin-based artificial metalloenzymes incorporating a histidine brace-mimicking DPA ligand tailored for oxidative carbohydrate cleavage.

Herein, we report the semisynthesis of site-specifically sulfated triabin─a 142-amino-acid lipocalin protein─at Tyr124. Functional assays reveal that sulfation enhances anticoagulant activity by ∼5-fold, demonstrating that even rigid scaffolds can benefit from this modification. Modeling studies uncover a synergistic mechanism wherein the sulfate group engages in a hydrogen-bond network, electrostatic bridging, and charge-complementary interactions with thrombin exosite I, while the hydrophobic core (notably Phe106 and Val126) remains the primary driving force.

This chapter describes a straightforward strategy for obtaining peptide salicylaldehyde (SAL) ester surrogates from synthetic or recombinant peptide hydrazides via nitrite oxidation and phenolysis using 3-(1,3-dithian-2-yl)-4-hydroxybenzoic acid (SAL(-COOH)PDT). The process involves the oxidation of peptide hydrazides to peptide azides, followed by phenolysis to form peptide SAL(-COOH)PDT esters, which can then be activated to yield reactive peptide SAL(-COOH) esters. Detailed procedures for the oxidation, phenolysis, and activation steps are provided, along with troubleshooting tips and considerations to ensure successful implementation of the method for serine/threonine ligation (STL) and cysteine/penicillamine ligation (CPL).

We present a mild, highly efficient, and selective method for the aminolysis of acyl pyrazole at protein C termini. The protocol demonstrates a broad substrate scope, operational simplicity, and aqueous-compatible conditions. Its successful application to site-specific C-terminal modification and dual functionalization underscores its utility as a versatile platform for protein engineering.

The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes, the study of which necessitates advanced methods to produce proteins with precision and versatility. Chemical protein synthesis, a powerful approach utilizing chemical reactions for the de novo construction of structurally accurate proteins, has emerged as a transformative tool for studying proteins and generating protein derivatives/mimics inaccessible by natural biological machinery, including post-translationally modified proteins, proteins comprised of unnatural amino acids, as well as mirror-image proteins. This review summarizes recent strides in synthetic method developments for chemical protein synthesis, including innovative techniques in solid-phase peptide synthesis, the challenges presented by difficult sequences in either synthesis or folding and the exploration of novel ligation reactions using both chemical and enzymatic methods. Furthermore, the review also delves into newly developed protocols for site-selective protein modifications and the generation of stapled or macrocyclized peptides/mini-proteins, highlighting the power of chemical methods to make structurally diverse proteins. Recent applications of synthetic proteins in investigating post-translational modifications (phosphorylation, lipidation, glycosylation, ubiquitination, etc. ), mirror-image biological processes and drug development are further discussed. Together, these topics provide a comprehensive overview of the current landscape of chemical protein synthesis.

Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS), more efficient ligation methods that circumvent the requirement of peptidyl thioester and thiol additive—which allow the following desulfurization or refolding in one pot—are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl N-acylpyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester and thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin via an N-to-C sequential ligation-MFD strategy, the semi-synthesis of a copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.

The incorporation of non-canonical amino acids (ncAAs) into the metal coordination environments of proteins has endowed metalloproteins with enhanced properties and novel activities, particularly in hemoproteins. In this work, we disclose a scalable synthetic strategy that enables the production of myoglobin (Mb) variants with non-canonical heme ligands, i.e., HoCys and f4Tyr. The ncAA-containing Mb* variants (with H64V/V68A mutations) were obtained through two consecutive native chemical ligations and a subsequent desulfurization step, with overall isolated yield up to 28.6 % in over 10-milligram scales. After refolding and heme b cofactor reconstitution, the synthetic Mb* variants showed typical electronic absorption bands. When subjected to the catalysis of the cyclopropanation of styrene, both synthetic variants, however, were not as competent as the His-ligated Mb*. We envisioned that the synthetic method reported herein would be useful for incorporating a variety of ncAAs with diverse structures and properties into Mb for varied purposes.

Chemical protein (semi-)synthesis is a powerful technique allowing the incorporation of unnatural functionalities at any desired protein site. Herein we describe a facile one-pot semi-synthetic strategy for the construction of a type 2 copper center in the active site of azurin, which is achieved by substitution of Met121 with unnatural amino acid residues bearing a strong ligand N,N-bis(pyridylmethyl)amine (DPA) to mimic the function of typical histidine brace-bearing copper monooxygenases, such as lytic polysaccharide monooxygenases (LPMOs) involved in polysaccharide breakdown. The semi-synthetic proteins were routinely obtained in over 10-mg scales to allow for spectroscopic measurements (UV–Vis, CD, and EPR), which provides structural evidences for the CuII–DPA-modified azurins. 4-nitrophenyl-β-D-glucopyranoside (PNPG) was used as a model substrate for the H2O2-driven oxidative cleavage reaction facilitated by semi-synthetic azurins, and the CuII–6 complex showed a highest activity (TTN 253). Interestingly, our semi-synthetic azurins were able to tolerate high H2O2 concentrations (up to 4000-fold of the enzyme), making them promising for practical applications. Collectively, we establish that chemical protein synthesis can be exploited as a reliable technology in affording large quantities of artificial metalloproteins to facilitate the transformation of challenging chemical reactions.

Triabin, a lipocalin-like thrombin inhibitor from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, exhibits effective inhibition comparable to hirudin despite binding exclusively at exosite I. Interestingly, it was reported that higher triabin doses would not inhibit thrombin completely, which makes it a promising antithrombotic candidate agent with a larger therapeutic window. However, few structural and functional studies about triabin have been reported in the past three decades, mostly due to the lack of a reliable and practicable recombinant expression technology for this seemingly small protein. In this work, we have adopted the SUMO fusion technology for the expression of triabin in E. coli cells-with facile refolding and purification procedures-and the bioactive triabin was produced in ∼12 mg/L culture medium. Subsequently, the structure-function studies through extensive site-directed mutagenesis reveal that triabin's Phe-106 involved in the hydrophobic contacts plays a surprisingly important role in the thrombin inhibition, in contrast to the negatively charged residues Asp-135 or Glu-128 involved in the salt-bridge interaction. As such, this study complements our understanding of the interaction mechanism of natural thrombin inhibitors, which should facilitate the development of anticoagulant drugs with a novel mode of action against thrombin.

Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin. Herein, we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales, after a single HPLC purification step. Through these synthetic proteins, a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed, which would help to delineate the structure–function relationships in other sulfated proteins and more importantly, to serve as a basis for the development of related antithrombotic agents.

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.

Glioblastoma(GBM)is the most common and lethal malig-nancy in the central nervous system.1 One of the major difficulties in treatment is that the initial clinical diagnosis of GBM is already WHO grade Ⅳ,without recognizable lower-grade precursor lesions.Copy number variations(CNVs)were found to appear in malignant cells several years before the initial diagnosis of GBM.2 Less differenti-ation and more aggressive phenotypes were observed in GBM cells with a higher degree of CNVs.3 Additionally,CNVs provide more accurate stratifiication of clinical outcomes than does the WHO grade system.

Herein, we report a semi-synthetic strategy affording a nitrophorin 2 (NP2) variant with a N,N′-bis(2-pyridylmethyl)amine (Dpa) ligand as sidechain selectively installed at position 27, which was assembled from a synthetic peptide thioester bearing the Dpa ligand and an expressed protein segment via native chemical ligation. The semi-synthetic NP2 was able to accept the natural heme b cofactor and the Dpa ligand was able to bind Cu(II)/Fe(III) ions, leading to heteronuclear active site.

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.

A highly efficient one-pot solid-phase synthesis of peptides with azopyridine is reported. When conjugated with the Mn–CO moiety, the resulting peptide- photoCORMs were able to achieve targeted intracellular CO release upon red light illumination.

A growing number of heme proteins have recently been repurposed for catalyzing abiological carbene transfer reactions. Herein, we rationally designed an engineered variant of nitrophorin 2 (NP2)-a nitric oxide transport hemoprotein-that catalyzes olefin cyclopropanation with high activity and stereoselectivity. Being a beta-barrel protein, the engineered NP2 variant showed a unique substrate preference, in contrast to the mainstream alpha-helical carbene-transfer heme enzymes like cytochrome P450 enzymes and myoglobin. The catalytic reactions can be carried out on a preparative scale while maintaining the stereoselectivity. The stereoselectivity of the NP2-catalyzed styrene cyclopropanation was further supported by quantum chemical calculations, and the significance of key residues was elucidated. As such, this work establishes NP2 as a robust lipocalin scaffold amenable for carbene-transferase development, complementing the current biocatalytic toolbox.

Selenoprotein F (SelF) is an endoplasmic reticulum-residing eukaryotic protein that contains a selenocysteine (Sec) residue. It has been suggested to be involved in a number of physiological processes by acting as a thiol-disulfide oxidoreductase, but the exact role has remained unclear due to the lack of a reliable production method. We document herein a robust synthesis of the human SelF through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are the use of a mild desulfurization process to protect the side-chain of the Sec residue from being affected and the simultaneous removal of acetamidomethyl and p-methoxybenzyl protection groups by PdCl2, thus facilitating the synthesis of multi-milligram of homogenous SelF. The reduction potential of SelF was determined and the thiol-disulfide oxidoreductase activity was further supported by its ability to catalyze the reduction and isomerization of disulfide bonds.

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.