Selective Peptide Cysteine Manipulation on Demand and Difficult Protein Chemical Synthesis Enabled by Controllable Acidolysis of N,S-Benzylidene Thioacetals.

Although solid-phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S-benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V-like (PD-L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.

N,O-Benzylidene Acetal Dipeptides (NBDs) Enable the Synthesis of Difficult Peptides via a Kinked Backbone Strategy.

Proteins with highly hydrophobic regions or aggregation-prone sequences are typically difficult targets for chemical synthesis at the current stage, as obtaining such type of peptides via solid-phase peptide synthesis requires sophisticated operations. Herein, we report N,O-benzylidene acetal dipeptides (NBDs) as robust and effective building blocks to allow the direct synthesis of difficult peptides and proteins via a kinked backbone strategy. The effectiveness and easy accessibility of NBDs have been well demonstrated in our chemical syntheses of various challenging peptides and proteins, including chemokine, therapeutic hormones, histone, and glycosylated erythropoietin.

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 (NaBEt4) 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. This protocol can be applied to synthesize proteins of interest with homogenous post-translational modifications. For complete information on the generation and use of this protocol, please refer to Sun et al. (2022).1.

Effects of L-Chg10-Teixobactin on Viability, Proliferation, and Osteo/Odontogenic Differentiation of Stem Cells from Apical Papilla.

INTRODUCTION: Intracanal medicament is one of the essential steps for ensuring success in regenerative endodontic procedures. L-Chg10-teixobactin is a novel antimicrobial agent that exhibited potent antibacterial and antibiofilm effects against Enterococcusfaecalis at low concentrations compared with ampicillin. At the same time, its cytotoxicity on dental stem cells has not been studied. This study aimed to investigate the effects of L-Chg10-teixobactin on the viability, proliferation, migration, and osteo/odontogenic differentiation of stem cells from apical papilla (SCAPs). MATERIALS AND METHODS: SCAPs isolated from immature human third molars were treated with various concentrations of L-Chg10-teixobactin, calcium hydroxide, and dimethyl sulfoxide. The viability and proliferation of SCAPs were assessed using the LIVE/DEAD Viability/Cytotoxicity Kit and Cell Counting Kit-8. A scratch wound healing test was used to evaluate the lateral migration capacity of SCAPs. Alkaline phosphatase (ALP) activity, calcium mineralization ability tests -ie, ALP staining and alizarin red S staining, and quantitative real-time polymerase chain reaction were performed to assess the osteo /odontogenic differentiation of SCAPs. RESULTS: The tested concentrations of L-Chg10-teixobactin (0.01, 0.02, and 0.03 mg/mL), 1 mg/mL calcium hydroxide, and 0.03% dimethyl sulfoxide had no significant cytotoxic effect on SCAPs at any time point (P > .05). Besides, there were no significant differences between the control and experimental groups in SCAPs' viability, proliferation, and migration. L-Chg10-teixobactin upregulated the gene expression of osteo/odontogenic markers in SCAPs, while no significant difference was found in the ALP activity and alizarin red S staining. CONCLUSIONS: L-Chg10-teixobactin demonstrated excellent biocompatibility on SCAPs at concentrations from 0.01 to 0.03 mg/mL and potentially enhance the osteo/odontogenic differentiation of SCAPs; suggesting its promising role as root canal medicament for regenerative endodontic procedures.

Antimicrobial Effects of L-Chg10-Teixobactin against Enterococcus faecalis In Vitro.

Objective: Teixobactin and its analogues are a new class of antibiotics that have no detectable bacterial resistance. This study was designed to determine the antibacterial and antibiofilm activities of a novel teixobactin analogue, L-Chg10-teixobactin, against two strains of Enterococcus faecalis (E. faecalis). Materials and Methods: The efficacy of L-Chg10-teixobactin against two strains of E. faecalis (ATCC 29212 and 47077) was determined using Clinical and Laboratory Standards Institute methods. L-Chg10-teixobactin was prepared at a stock concentration of 1 mg/mL in 5% DMSO. The minimum inhibitory concentration (MIC) was calculated using a two-fold serial broth dilution method, utilizing a 96-well plate. The minimum bactericidal concentration (MBC) was determined by plating the bacteria onto agar to define the concentration that resulted in 99.9% of bacterial death. Ampicillin was used as the control. The effect of L-Chg10-teixobactin on the inhibition of ATCC 47077 strain biofilm formation was determined by measuring the minimum biofilm inhibitory concentration (MBIC) using the safranin assay, while the eradication of the preformed biofilm was determined by measuring the minimum biofilm eradication concentration (MBEC) using the XTT assay. For nonlinear data, the log dose-response curve was plotted to calculate the optimum concentration using Excel (version 16.51, Microsoft® excel. 2021, Microsoft Corporation, Reymond, WA, USA). The data are presented as mean ± standard deviation (SD). Results: The MIC and MBC values of L-Chg10-teixobactin against both strains of E. faecalis were 0.8 µg/mL. The MIC of ampicillin was 1.25 µg/mL for ATCC 29212 and ranged from 1.25 to 5 µg/mL for ATCC 47077. The MBC of ampicillin for ATCC 29212 and ATCC 47077 was 10 and 20 µg/mL, respectively. The MIC and MBC of ampicillin were much higher compared with those of L-Chg10-teixobactin. The MBEC80 of L-Chg10-teixobactin was 4.60 µg/mL for ATCC 47077, which was much lower than that of ampicillin (20 µg/mL). Conclusions:L-Chg10-teixobactin demonstrated potent antibacterial and antibiofilm effects against E. faecalis, suggesting its potential role an effective antibacterial and antibiofilm agent in endodontic treatment.

Total Synthesis of Mannopeptimycin ß via ß-Hydroxyenduracididine Ligation.

Nonribosomal peptide synthesis in bacteria has endowed cyclic peptides with fascinating structural complexity via incorporating nonproteinogenic amino acids. These bioactive cyclic peptides provide interesting structural motifs for exploring total synthesis and medicinal chemistry studies. Cyclic glycopeptide mannopeptimycins exhibit antibacterial activity against antibiotic-resistant Gram-positive pathogens and act as the lipid II binder to stop bacterial cell wall biosynthesis. Here, we report a strategy streamlining solution phase-solid phase synthesis and chemical ligation-mediated peptide cyclization for the total synthesis of mannopeptimycin ß.

Total Synthesis of Malacidin A by ß-Hydroxyaspartic Acid Ligation-Mediated Cyclization and Absolute Structure Establishment.

The development of novel antibiotics is critical to combating the growing emergence of drug-resistant pathogens. Malacidin A is a new member of the calcium-dependent antibiotic (CDAs) family with activity against antibiotic-resistant pathogens. Its mode of action is distinct from classical CDAs. However, the absolute structure of malacidin A has not been established. Herein, the total syntheses of malacidin A and its analogues are reported by a combination of Fmoc-based solid-phase peptide synthesis (SPPS) and ß-hydroxyaspartic acid ligation-mediated peptide cyclization. The total synthesis enabled us to establish the absolute configuration of malacidin A, which is in agreement with those for natural malacidin A confirmed by advanced Marfey's analysis in our study.

Establishing the Structure-Activity Relationship of Daptomycin.

Daptomycin is effective in treating infections caused by antibiotic-resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and vancomycin-resistant S. aureus (VRSA). Due to its distinct mechanism of action toward multidrug-resistant bacteria, daptomycin provides an attractive structural motif to generate new daptomycin-based antibiotics to combat the problem of bacterial resistance. In this study, we used the total synthesis method to produce daptomycin analogues with a variety in terms of types and sites of modifications. Five classes of daptomycin analogues were synthesized, and the antimicrobial activities of the analogues were analyzed by several biological assays. From this study, we established a comprehensive structure-activity relationship of daptomycin which will lay the foundation for the further development of daptomycin-based antibiotics.

Coetaneous catalytic kinetic resolution of alkynes and azides through asymmetric triazole formation.

A non-enzymatic simultaneous (coined coetaneous) kinetic resolution of a racemic alkyne and racemic azide, utilising an asymmetric CuAAC reaction is reported. The use of a CuCl (R,R)-Ph-Pybox catalyst system effects a simultaneous kinetic resolution of two racemic starting materials to give one major triazolic diastereoisomer in the ratio 74:12:4:10 (dr 84:16, 90% ee maj). The corresponding control reaction using an achiral copper catalyst gives the four possible diastereoisomers in a 23:27:23:27 ratio, demonstrating minimal inherent substrate control.

Total Synthesis and Structural Establishment/Revision of Antibiotics A54145.

A54145 is a family of antibacterial cyclic lipodepsipeptides structurally resembling daptomycin. Since its discovery in 1990, only the ambiguous structures of the methoxy-aspartic acid (MeO-Asp) and the hydroxy-asparagine (HO-Asn) have been reported. We have developed efficient routes to obtain the fully protected l-MeO-Asp and l-HO-Asn building blocks compatible with Fmoc-SPPS, and a total synthesis of A54145 that enabled us to establish its structure, consisting of l-3S-HO-Asn and l-3R-MeO-Asp, revising the wrongly proposed structure of l-3S-MeO-Asp.

Oxidative capacities of size-segregated haze particles in a residential area of Beijing.

The frequent haze days around the Chinese capital of Beijing in recent years have aroused great attention owing to the detrimental effects on visibility and public health. To discover the potential health effects of the haze, oxidative capacities of airborne particles collected in Beijing during haze and clear days were comparably assessed by a plasmid scission assay. Eleven water-soluble trace elements (As, Cd, Cr, Cu, Mn, Ni, Pb, V, Se, Tl, and Zn) in the size-segregated airborne particles were quantitatively analyzed by inductively coupled plasma mass spectrometry, and most of the water-soluble trace elements were found to mainly concentrate in the fine particle size of 0.56-1.0 microm. In comparison with clear days, the mass concentrations of 11 analyzed water-soluble trace elements remarkably increased during haze days, and the oxidative capacities determined by the plasmid scission assay were markedly elevated accordingly during the haze days under the same dosage of particles as for those during clear days. Water-soluble trace elements in airborne particles, such as Cu, V, and particularly Zn, were found to have significantly positive correlations with the plasmid DNA damage rates. Because Cu, V, and Zn have been considered as bioavailable elements, the evident increase of these elements during haze days may be greatly harmful to human health.

A comparison study on airborne particles during haze days and non-haze days in Beijing.

Airborne particles in Beijing during haze days and non-haze days were collected by an eleven-stage cascade impactor (MOUDI 110, MSP, USA), and the mass concentrations and water soluble inorganic ions of the size segregated airborne particles were quantitatively analyzed. PM10 concentrations during haze days ranged from 250.5 to 519.4 µgm(-3) which were about 3-8 times greater than those (ranged from 67.6 to 94.0 µgm(-3)) during non-haze days, and PM1.8 concentrations during haze periods were in the range of 117.6-378.6 µgm(-3) which were 3-14 times higher than those (27.0 to 36.8 µgm(-3)) during non-haze days. In comparison with non-haze days, all water soluble inorganic ions investigated in the airborne particles greatly enhanced during haze days. NH4(+), NO3(-) and SO4(2-) were found to be the dominant water soluble inorganic ions, accounting for 91-95% of the total inorganic ions in PM1.8 during haze days, and 73-81% during non-haze days. The size distributions of SO4(2-), NO3(-), Cl(-), K(+) and Na(+) exhibited bimodal types, while single mode was found for NH4(+), Ca(2+) and Mg(2+). Only with exception of Ca(2+) and Mg(2+), all ions were concentrated in fine particles around 0.56-1.0 µm of "droplet mode" during haze days, while 0.32-0.56 µm of "condensation mode" during non-haze days. The extremely high mole ratio (>2) of [NH4(+)]/[SO4(2-)] during haze days implied that the main form of ammonium in PM1.8 might be (NH4)2SO4 and NH4NO3. The mass ratio of NO3(-)/SO4(2-) was >1 in PM1.8 during haze days and ~1 during non-haze days, indicating that NOx from the vehicle exhaust in Beijing is playing more and more important role on fine particle formation.

TLR4 signaling is involved in the protective effect of propofol in BV2 microglia against OGD/reoxygenation.

Propofol exhibits neuroprotective effects against hypoxic-ischemic brain injury, but the underlying mechanisms are still not clear. Toll-like receptor 4 (TLR4) plays a considerable role in the induction of innate immune and inflammatory responses. The purposes of this study are to investigate the effect of propofol on the oxygen and glucose deprivation (OGD)/reoxygenation (OGD/R) BV2 microglia and to explore the role of TLR4/myeloid differentiation protein 88 (MyD88)/nuclear factor-kappa B (NF-κB) pathway in the neuroprotective effects of propofol. BV2 microglia were placed into an airtight chamber and in glucose-free medium for OGD/reoxygenation. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. TLR4 and its downstream signaling molecules, MyD88 and NF-κB expressions were detected by Western blotting. Level of tumor necrosis factor alpha (TNF-α) in culture medium was determined with enzyme-linked immunosorbent assay. BV2 microglia apoptosis was determined by flow cytometry. We found that pretreatment with propofol significantly alleviated the hypoxic injury in BV2 microglia. Propofol inhibited upregulation of TLR4, MyD88, and NF-κB expressions in BV2 microglia exposed to OGD/reoxygenation. Propofol pretreatment also significantly reduced the production of TNF-α and apoptosis in OGD/reoxygenation BV2 microglia. The results indicated that TLR4 and its downstream MyD88-dependent signaling pathway contributed to neuroprotection of propofol to microglia exposed to OGD/reoxygenation.

Exogenous administration of PACAP alleviates traumatic brain injury in rats through a mechanism involving the TLR4/MyD88/NF-κB pathway.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is effective in reducing axonal damage associated with traumatic brain injury (TBI), and has immunomodulatory properties. Toll-like receptor 4 (TLR4) is an important mediator of the innate immune response. It significantly contributes to neuroinflammation induced by brain injury. However, it remains unknown whether exogenous PACAP can modulate TBI through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. In this study, we investigated the potential neuroprotective mechanisms of PACAP pretreatment in a weight-drop model of TBI. PACAP38 was microinjected intracerebroventricularly before TBI. Brain samples were extracted from the pericontusional area in the cortex and hippocampus. We found that TBI induced significant upregulation of TLR4, with peak expression occurring 24 h post-trauma, and that pretreatment with PACAP significantly improved motor and cognitive dysfunction, attenuated neuronal apoptosis, and decreased brain edema. Pretreatment with PACAP inhibited upregulation of TLR4 and its downstream signaling molecules MyD88, p-IκB, and NF-κB, and suppressed increases in the levels of the downstream inflammatory agents interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), in the brain tissue around the injured cortex and in the hippocampus. Administration of PACAP both in vitro and in vivo attenuated the ability of the TLR4 agonist lipopolysaccharide (LPS) to increase TLR4 protein levels. Therefore, PACAP exerts a neuroprotective effect in this rat model of TBI, by inhibiting a secondary inflammatory response mediated by the TLR4/MyD88/NF-κB signaling pathway in microglia and neurons, thereby reducing neuronal death and improving the outcome following TBI.

Toll-like receptor 4 signaling is involved in PACAP-induced neuroprotection in BV2 microglial cells under OGD/reoxygenation.

OBJECT: The neuroprotective effects of pituitary adenylate cyclise-activating polypeptide (PACAP) have been well documented in vivo and in vitro. However, the mechanisms by which PACAP protected microglia from ischemic/hypoxic injury via inhibition of microglia activation remain unclear. Toll-like receptor 4 (TLR4) plays a considerable role in the induction of innate immune and inflammatory responses. The purpose of this study is to investigate the effect of PACAP on the oxygen and glucose deprivation (OGD)/reoxygenation BV2 microglia and to explore the role of TLR4/myeloid differentiation protein 88 (MyD88)/nuclear factor-kappa B (NF-kappaB) pathway in the neuroprotective effects of PACAP. METHODS: We conducted OGD/reoxygenation by placing BV2 microglia into an airtight chamber and in glucose-free medium. BV2 microglia cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Western blot was utilized to detect TLR4, MyD88 expression, inhibitory protein of NF-kappaB (IkappaB) phosphorylation/degradation, NF-kappaB activation. Level of tumor necrosis factor-alpha (TNF-alpha) in culture medium was measured with enzyme-linked immunosorbent assay (ELISA). Apoptosis was determined by flow cytometry. RESULTS: We found that pretreatment with PACAP to BV2 cells immediately before OGD/reoxygenation significantly alleviated microglia hypoxic injury. PACAP inhibited upregulation of TLR4, MyD88 and NF-kappaB in BV2 microglial cells exposed to OGD/reoxygenation. PACAP administration also significantly reduced the production of proinflammatory cytokines and apoptosis in BV2 microglia exposed to OGD/reoxygenation. DISCUSSION: Pretreatment with PACAP inhibited activation of the TLR4/MyD88/NF-kappaB signaling pathway and decreased inflammatory cytokine levels, as well as apoptosis in microglia, thereby attenuating microglia hypoxic injury. Our results suggested that TLR4-mediated MyD88-dependent signaling pathway contributed to neuroprotection of PACAP to microglia against OGD/reoxygenation.

Atmospheric levels of BTEX compounds during the 2008 Olympic Games in the urban area of Beijing.

The hourly concentrations of BTEX (Benzene, Toluene, Ethylbenzene, m,p-Xylene and o-Xylene) in the urban area of Beijing were measured during July-October 2008, covering the periods of the 2008 Olympic Games and Paralympic Games. The atmospheric BTEX were pre-concentrated on Tenax-TA tubes, and analyzed by GC-PID (Gas Chromatography with Photo Ionization Detector) after thermal desorption. During the games, the mean daytime concentrations of benzene, toluene, ethylbenzene, m,p-xylene and o-xylene were 2.37, 3.97, 1.92, 3.51 and 1.90 microg/m3, respectively, and were 52.8%, 63.9%, 56.4%, 56.8% and 46.9%, respectively lower than those after the games. The significantly positive correlation between BTEX and CO as well as the ratio of benzene/toluene suggested that the vehicle exhaust was the major source of BTEX during the whole investigated period. The extremely high ratios of ethylbenzene to m,p-xylene (E/X) were mainly observed at noontime in haze days, indicating that photochemical reactions were highly active under these typical days.