Discovery of Thioether-Cyclized Macrocyclic Covalent Inhibitors by mRNA Display.

Macrocyclic peptides are promising scaffolds for the covalent ligand discovery. However, platforms enabling the direct identification of covalent macrocyclic ligands in a high-throughput manner are limited. In this study, we present an mRNA display platform allowing selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS).

Discovery of a Monoclonal Antibody That Targets Cell-Surface Pseudaminic Acid of with Direct Bactericidal Effect.

The therapeutic effects of antibodies include neutralization of pathogens, activation of the host complement system, and facilitation of phagocytosis of pathogens. However, antibody alone has never been shown to exhibit bactericidal activity. In this study, we developed a monoclonal antibody that targets the bacterial cell surface component Pseudaminic acid (Pse).

Revealing the extracellular function of HMGB1 N-terminal region acetylation assisted by a protein semi-synthesis approach.

HMGB1 (high-mobility group box 1) is a non-histone chromatin-associated protein that has been widely reported as a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in the proinflammatory process once it is in an extracellular location. Accumulating evidence has shown that HMGB1 undergoes extensive post-translational modifications (PTMs) that actively regulate its conformation, localization, and intermolecular interactions. However, fully characterizing the functional implications of these PTMs has been challenging due to the difficulty in accessing homogeneous HMGB1 with site-specific PTMs of interest.

Protocol for semisynthesis of serotonylated histone H3 by rapid protein desulfurization in tandem with native chemical ligation.

Here, we present a protocol of rapid protein desulfurization in tandem with native chemical ligation for facile syntheses of proteins with site-specific modifications. We describe using sodium tetraethylborate (NaBEt) to carry out this desulfurization in an add-and-done manner under ambient conditions without requirement of inert atmosphere protection, UV irradiation, heating, or exogenous thiol additives. Specifically, we detail the semisynthesis of serotonylated histone H3(H3Q5ser) via one-pot ligation desulfurization.

Guanidine Additive Enabled Intermolecular ortho-Phthalaldehyde-Amine-Thiol Three-Component Reactions for Modular Constructions.

Recently, ortho-phthalaldehyde (OPA) is experiencing a renascence for the modification of proteins and peptides through OPA-amine two-component reactions for bioconjugation and intramolecular OPA-amine-thiol three-component reactions for cyclization. Historically, small thiol molecules were used in large excess to allow for the intermolecular OPA-amine-thiol reaction forming 1-thio-isoindole derivatives. In this study, we discovered that guanidine could serve as an effective additive to switch the intermolecular OPA-amine-thiol three-component reaction to a stoichiometric process and enable the modular construction of peptide-peptide, and peptide-drug conjugate structures.

Chemical Synthesis of Proteins with Base-Labile Posttranslational Modifications Enabled by a Boc-SPPS Based General Strategy Towards Peptide C-Terminal Salicylaldehyde Esters.

Chemical synthesis of proteins bearing base-labile post-translational modifications (PTMs) is a challenging task. For instance, O-acetylation and S-palmitoylation PTMs cannot survive Fmoc removal conditions during Fmoc-solid phase peptide synthesis (SPPS). In this work, we developed a new Boc-SPPS-based strategy for the synthesis of peptide C-terminal salicylaldehyde (SAL) esters, which are the key reaction partner in Ser/Thr ligation and Cys/Pen ligation.

Thiophene-2,3-Dialdehyde Enables Chemoselective Cyclization on Unprotected Peptides, Proteins, and Phage Displayed Peptides.

Ortho-phthalaldehyde has recently found wide potentials for protein bioconjugation and peptide cyclization. Herein, the second-generation dialdehyde-based peptide cyclization method is reported. The thiophene-2,3-dialdehyde (TDA) reacts specifically with the primary amine (from Lys side chain or peptide N-terminus) and thiol (from Cys side chain) within unprotected peptides to generate a highly stable thieno[2,3-c]pyrrole-bridged cyclic structure, while it does not react with primary amine alone.

Ligation Embedding Aggregation Disruptor Strategy Enables the Chemical Synthesis of PD-1 Immunoglobulin and Extracellular Domains.

Chemical synthesis of proteins with aggregable or colloidal peptide segments presents a formidable task, as such peptides prove to be difficult for both solid-phase peptide synthesis and peptide ligation. To address this issue, we have developed ligation embedding aggregation disruptor (LEAD) as an effective strategy for the chemical synthesis of difficult-to-obtain proteins. The ,/-benzylidene acetals generated from Ser/Thr ligation and Cys/Pen ligation are found to effectively disrupt peptide aggregation, and they can be carried for sequential ligations toward protein synthesis.

Enabling chemical protein (semi)synthesis reducible solubilizing tags (RSTs).

Chemical synthesis of proteins with poor solubility presents a challenging task. The existing solubilizing tag strategies are not suitable for the expressed protein segment. To address this issue, we report herein that solubilizing tags could be introduced at the side chain of the peptide and C-terminal peptide salicylaldehyde esters a disulfide linker.

Synthetic Pseudaminic-Acid-Based Antibacterial Vaccine Confers Effective Protection against Infection.

exhibits resistance to most first-line antibiotics; thus, development of new antibacterial agents is urgently required. Pseudaminic acid exists as the surface glycan of . In this study, we chemically synthesized pseudaminic acid, conjugated it to carrier protein CRM197 using the OPA (-phthalaldehyde) chemistry, and obtained three Pse-CRM197 conjugates with different Pse loadings.

Serine/threonine ligation-assisted chemical synthesis of HMGA1a protein with site-specific post-translational modifications.

Dissecting the function of proteins' post-translational modifications (PTMs) is seriously hindered by the difficulty in obtaining the homogeneous protein with the PTMs of interest. Chemical protein synthesis offers a great potential to overcome this limitation. Here, a detailed protocol is introduced for chemical synthesis of HMGA1a protein with site-specific modifications via Ser/Thr ligation strategy, by which we can systematically study the function of the triple phosphorylation (3pSer) in the HMGA1a acidic tail.

Phosphorylation-regulated HMGA1a-P53 interaction unveils the function of HMGA1a acidic tail phosphorylations via synthetic proteins.

As a typical member of intrinsically disordered proteins (IDPs), HMGA1a carries many post-translational modifications (PTMs). To study the undefined function of acidic tail phosphorylations, seven HMGA1a proteins with site-specific modification(s) were chemically synthesized via Ser/Thr ligation. We found that the phosphorylations significantly inhibit HMGA1a-P53 interaction and the phosphorylations can induce conformational change of HMGA1a from an "open state" to a "close state.

The Antidepressant Effects of Resveratrol are Accompanied by the Attenuation of Dendrite/Dendritic Spine Loss and the Upregulation of BDNF/p-cofilin1 Levels in Chronic Restraint Mice.

Depression afflicts more than 300 million people worldwide, but there is currently no universally effective drug in clinical practice. In this study, chronic restraint stress (CRS)-induced mice depression model was used to study the antidepressant effects of resveratrol and its mechanism. Our results showed that resveratrol significantly attenuated depression-like behavior in mice.

Chemical Protein Synthesis: Advances, Challenges, and Outlooks.

Contemporary chemical protein synthesis has been dramatically advanced over the past few decades, which has enabled chemists to reach the landscape of synthetic biomacromolecules. Chemical synthesis can produce synthetic proteins with precisely controlled structures which are difficult or impossible to obtain via gene expression systems. Herein, we summarize the key enabling ligation technologies, major strategic developments, and some selected representative applications of synthetic proteins and provide an outlook for future development.