TBHP Mediated C-N Bond Cleavage of Tertiary Amines toward the Synthesis of Oxalamides and α,ß-Epoxy Amides.

An efficient and convenient method for the synthesis of oxalamides by the reaction of ß-ketoamides with tertiary amines and TBHP was developed. A variety of ß-ketoamides and tertiary amines substrates were well-tolerated in this transformation. Based on the control experiments, a plausible mechanism for this reaction was proposed that involved the tandem oxidation/amination process. In addition, α,ß-epoxy amides could be obtained by adjusting the reaction conditions.

In situ monitoring of toxic effects of algal toxin on cells by a novel microfluidic flow cytometry instrument.

Algal toxins produced by microalgae, such as domoic acid (DA)1, have toxic effects on humans. However, toxicity tests using mice only yield lethal doses of algal toxins without providing insights into the mechanism of action on cells. In this study, a fast segmentation of microfluidic flow cytometry cell images based on the bidirectional background subtraction (BBS)2 method was developed to get the visual evidence of apoptosis in both bright-field and fluorescence images. This approach enables mapping of changes in cell morphology and activity under algal toxins, allowing for fast (within 60 s) and automated biological detection. By combining microfluidics with flow cytometry, the intricate cellular-level reaction process can be observed in micro samples of 293 T cells and mouse spleen cells, offering potential for future in vitro experiments.

Interbacterial Chemical Communication-Triggered Nascent Proteomics.

Metabolic labeling with clickable noncanonical amino acids has enabled nascent proteome profiling, which can be performed in a cell-type-specific manner. However, nascent proteomics in an intercellular communication-dependent manner remains challenging. Here we develop communication-activated profiling of protein expression (CAPPEX), which integrates the LuxI/LuxR quorum sensing circuit with the cell-type-specific nascent proteomics method to enable selective click-labeling of newly synthesized proteins in a specific bacterium upon receiving chemical signals from another reporter bacterium. CAPPEX reveals that E. coli competes with Salmonella for tryptophan as the precursor for indole, and the resulting indole suppressed the expression of virulence factors in Salmonella. This tryptophan-indole axis confers attenuation of Salmonella invasion in host cells and living mice. The CAPPEX strategy should be widely applicable for investigating various interbacterial communication processes.

GlnR-mediated regulation of KstR controls cholesterol catabolism in Mycobacterium smegmatis.

Tuberculosis, caused by mycobacteria, continues to pose a substantial public health threat. Mycobacteria typically use cholesterol from the membranes of host macrophages as a carbon and energy source. Most genes that control cholesterol degradation are regulated by KstR, which is highly conserved in Mycobacterium tuberculosis and Mycobacterium smegmatis. Through bioinformatic analysis, we found a typical global nitrogen regulator (GlnR)-binding motif (CCGAC-AACAGT-GACAC) in the promoter region of kstR of M. smegmatis, and we determined its binding activity in vitro using electrophoretic mobility shift assays. Using RT-qPCR, we found that nine genes involved in side-chain or sterol-ring oxidation were upregulated in a ΔglnR M. smegmatis strain compared to the WT strain and glnR-complemented strains under nitrogen limitation. ATP assays in macrophages revealed that coordinated GlnR-KstR regulation significantly reduced the viability of M. smegmatis in macrophages. Thus, we found that various genes involved in cholesterol catabolism are regulated by GlnR via KstR in response to environmental nitrogen, and that they further affect the invasive ability of M. smegmatis. These findings revealed a novel regulatory mechanism of cholesterol catabolism, which may be useful in the development of new strategies for controlling tuberculosis.

Low-dose lipopolysaccharide protects nerve cells against spinal cord injury via regulating the PI3K-AKT-Nrf2 signaling pathway.

This study explored the molecular mechanism behind the protective effects from low-dose lipopolysaccharide (LPS) on an in-vitro model of spinal cord injury (SCI). For this, PC12 cells were treated with different concentrations of LPS and the cell counting kit-8 assay was used to measure the toxicity of LPS to the cells. Next, we used immunofluorescence to measure nuclear translocation of Nrf2 in PC12 cells. PC12 cells were then treated with IGF-1 (PI3K agonist) and LY294002 (PI3K inhibitor). An in-vitro model of SCI was then established via oxygen-glucose deprivation/reoxygenation. Rates of apoptosis were measured using flow cytometry and the TUNEL assay. Low-dose LPS increased the expression levels of Nrf2, p-PI3K/PI3K, and p-AKT/AKT, and facilitated nuclear translocation of Nrf2. The activation of PI3K-AKT signaling by IGF-1 significantly increased the expression of Nrf2, whereas inhibition of PI3K-AKT signaling significantly decreased the expression of Nrf2. Low-dose LPS reduced the apoptotic ratio of PC12 cells, decreased the expression levels of caspase 3 and caspase 9, and increased the expression levels of HO-1, NQO1, and γ-GCS. Low-dose LPS also reduced the rate of apoptosis and oxidative stress by activating the PI3K-AKT-Nrf2 signaling pathway. Collectively, the results indicate that PI3K-AKT-Nrf2 signaling participates in the protective effects from low-dose LPS in an in-vitro PC12 cell model of SCI.

Synthetic Route to Enaminones via Metal-Free Four-Component Sequential Reactions of Aryl Olefins with CHCl3, Et3N, and TBHP.

An efficient and modular strategy was used to obtain enaminones with a wide range of functional groups via a four-component sequential reaction. This reaction proceeded under mild conditions without a catalyst in one pot. Furthermore, the products could be transformed into thiadiazoles.

Highly Selective Synthesis of 2-tert-Butoxy-1-Arylethanones via Copper(I)-Catalyzed Oxidation/tert-Butoxylation of Aryl Olefins with TBHP.

A practical and environmentally friendly protocol for the selective oxidation of aryl olefins to arylethanone derivatives by using a Cu(I) catalyst and tert-butyl hydroperoxide (TBHP) has been developed. A series of 2-tert-butoxy-1-arylethanones were obtained in moderate to good yields under mild conditions with high selectivity. In this method, TBHP acts not only as an oxidant but also as the tert-butoxy and carbonyl oxygen sources. This enables one-step oxidation/tert-butoxylation. Various allyl peroxides were also synthesized from allyl substrates.

The Nitrogen Regulator GlnR Directly Controls Transcription of the prpDBC Operon Involved in Methylcitrate Cycle in Mycobacterium smegmatis.

Mycobacterium tuberculosis utilizes fatty acids of the host as the carbon source. Metabolism of odd-chain fatty acids by Mycobacterium tuberculosis produces propionyl coenzyme A (propionyl-CoA). The methylcitrate cycle is essential for mycobacteria to utilize the propionyl-CoA to persist and grow on these fatty acids. In M. smegmatis, methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in the methylcitrate cycle are encoded by prpC, prpD, and prpB, respectively, in operon prpDBC In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of the prpDBC operon and inhibits its transcription. The binding motif of GlnR was identified by bioinformatic analysis and validated using DNase I footprinting and electrophoretic mobility shift assays. The GlnR-binding motif is separated by a 164-bp sequence from the binding site of PrpR, a pathway-specific transcriptional activator of methylcitrate cycle, but the binding affinity of GlnR to prpDBC is much stronger than that of PrpR. Deletion of glnR resulted in faster growth in propionate or cholesterol medium compared with the wild-type strain. The ΔglnR mutant strain also showed a higher survival rate in macrophages. These results illustrated that the nitrogen regulator GlnR regulates the methylcitrate cycle through direct repression of the transcription of the prpDBC operon. This finding not only suggests an unprecedented link between nitrogen metabolism and the methylcitrate pathway but also reveals a potential target for controlling the growth of pathogenic mycobacteria.IMPORTANCE The success of mycobacteria survival in macrophage depends on its ability to assimilate fatty acids and cholesterol from the host. The cholesterol and fatty acids are catabolized via ß-oxidation to generate propionyl coenzyme A (propionyl-CoA), which is then primarily metabolized via the methylcitrate cycle. Here, we found a typical GlnR binding box in the prp operon, and the affinity is much stronger than that of PrpR, a transcriptional activator of methylcitrate cycle. Furthermore, GlnR repressed the transcription of the prp operon. Deletion of glnR significantly enhanced the growth of Mycobacterium tuberculosis in propionate or cholesterol medium, as well as viability in macrophages. These findings provide new insights into the regulatory mechanisms underlying the cross talk of nitrogen and carbon metabolisms in mycobacteria.

I2-DMSO-H2O: A Metal-Free Combination System for the Oxidative Addition of Alkynes to Access (E)-α-Iodo-ß-methylsulfonylalkenes.

A simple and green reaction was discovered for iodization-methylsulfoxidation of alkynes to access (E)-α-iodo-ß-methylsulfonylalkenes. This is the first report for the synthesis of iodovinyl methylsulfones by employing alkynes to react with molecular iodine (I2), dimethyl sulfoxide (DMSO), and H2O. Additionally, this protocol represents a new avenue for utilizing DMSO as the source of the -SO2Me group and H2O as the "O" source for the construction of the -SO2Me group from DMSO, which is a valuable finding.

Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes.

The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.

N,N-Dimethylformamide (DMF) as a Source of Oxygen To Access α-Hydroxy Arones via the α-Hydroxylation of Arones.

An unprecedented α-hydroxylation strategy was developed for the synthesis of α-hydroxy arones using N,N-dimethylformamide (DMF) as an oxygen source. Control experiments demonstrated that the oxygen atom of the hydroxy group in the α-hydroxy arones produced in this reaction was derived from DMF. This new reaction therefore not only provides an alternative strategy for the α-hydroxylation of arones but also highlights the possibility of using the inexpensive common solvent DMF as a source of oxygen in organic synthesis.

Treatment of oral lichen planus using 308-nm excimer laser.

Oral lichen planus (OLP) is a chronic inflammatory disease, has prolonged courses, repeated attacks and resistance to treatment. The traditional narrow spectrum UVB treatment has an established efficacy on skin lichen planus, and high safety. However, most of ultraviolet phototherapy devices have a huge volume, thereby cannot be used in the treatment of OLP. Lymphocytic infiltration is evident in the lesions of lichen planus, and the direct irradiation of 308-nm excimer laser can induce apoptosis of the T lymphocytes in skin lesions, thereby has a unique therapeutic effect on the diseases involving T lymphocytes. This study aims to investigate the efficacy of 308-nm excimer laser in the treatment of OLP. A total of six OLP patients were enrolled into this study, and further pathological diagnosis was conducted, then 308-nm excimer laser was used in the treatment. The efficacy of 308-nm excimer laser in the treatment of OLP was satisfactory. The clinical symptoms of five patients were significantly improved. In two patients, the erosion surface based on congestion and the surrounding white spots completely disappeared, and clinical recovery was achieved. Three patients achieved partial remission, that is, the erosion surface healed, congestion and white spot area shrunk by more than 1/2 of the primary skin lesions. In the remaining one patient, the erosion surface had not completely healed after treatment, and congestion and white spot area shrunk by less than 1/2 of the primary skin lesions. Only one patients had developed mild pain during the treatment, and this symptom alleviated by itself. The 308-nm excimer laser therapy can serve as a safe and effective treatment for OLP.

α-Acetoxyarone synthesis via iodine-catalyzed and tert-butyl hydroperoxide-mediateded self-intermolecular oxidative coupling of aryl ketones.

We present a metal-free method for α-acetoxyarone synthesis by self-intermolecular oxidative coupling of aryl ketones using I2-tert-butyl hydroperoxide (TBHP). Under the optimum conditions, various aryl ketones gave the corresponding products in moderate to excellent yields. A series of control experiments were performed; the results suggest the involvement of radical pathways. Multiple radical intermediates were generated in situ and the overall process involved several different reactions, which proceeded self-sequentially in a single reactor. A labeling experiment using 18O-labeled H2O confirmed that the oxygen in the product was derived from TBHP, not from H2O in the TBHP solvent.

Difunctionalization of alkenes with iodine and tert-butyl hydroperoxide (TBHP) at room temperature for the synthesis of 1-(tert-butylperoxy)-2-iodoethanes.

We developed a direct vicinal difunctionalization of alkenes with iodine and TBHP at room temperature. This iodination and peroxidation in a one-pot synthesis produces 1-(tert-butylperoxy)-2-iodoethanes, which are inaccessible through conventional synthetic methods. This method generates multiple radical intermediates in situ and has excellent regioselectivity, a broad substrate scope and mild conditions. The iodine and peroxide groups of 1-(tert-butylperoxy)-2-iodoethanes have several potential applications and allow further chemical modifications, enabling the preparation of synthetically valuable molecules.

Organosilane with Gemini-Type Structure as the Mesoporogen for the Synthesis of the Hierarchical Porous ZSM-5 Zeolite.

A new kind of organosilane (1,6-bis(diethyl(3-trimethoxysilylpropyl)ammonium) hexane bromide) with a gemini-type structure was prepared and used as a mesoporogen for the synthesis of hierarchical porous ZSM-5 zeolite. There are two quaternary ammonium centers along with double-hydrolyzable -RSi(OMe)3 fragments in the organosilane, which results in a strong interaction between this mesoporogen and silica-alumina gel. The organosilane can be easily incorporated into the ZSM-5 zeolite structure during the crystallization process, and it was finally removed by calcination, leading to secondary pores in ZSM-5. The synthesized ZSM-5 has been systematically studied by XRD, nitrogen adsorption, SEM, TEM, TG, and solid-state one-dimensional (1D) and two-dimensional (2D) NMR, which reveal information on its detailed structure. It has a hierarchical porosity system, which combines the intrinsic micropores coming from the crystalline structure and irregular mesopores created by the organosilane template. Moreover, the mesoposity including pore size and volume within ZSM-5 can be systematically tuned by changing the organosilane/TEOS ratio, which confirms that this organosilane has high flexibility of use as a template for the synthesis of hierarchical porous zeolite.

TBHP-mediated highly efficient dehydrogenative cross-oxidative coupling of methylarenes with acetanilides.

A TBHP-mediated dehydrogenative cross-oxidative-coupling approach has been developed for the synthesis of N-arylbenzamides from methylarenes and acetanilides. This cross-coupling method is free of transition metal catalysts and ligands, and no extra organic solvents are required, which make it an useful and attractive strategy for the straightforward construction of C-N bonds. Besides, this conversion is an important complement to the conventional C-N forming strategies.

Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP.

UNLABELLED: NADP(+) is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP(+) negatively regulates an acetyltransferase from Myxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations. MxKat possesses an NAD(P)-binding domain fused to the Gcn5-type N-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP(+) bound to MxKat and that the binding had strong effects on enzyme activity. The Gly11 residue of MxKat was confirmed to play an important role in NADP(+) binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated that MxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP(+) concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP(+) at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation. IMPORTANCE: Microorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP(+)-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation in M. xanthus. We found that NADP(+) specifically binds to the Rossmann fold of MxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP(+) metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs.

Dimethylamine as the key intermediate generated in situ from dimethylformamide (DMF) for the synthesis of thioamides.

An improved and efficient method for the synthesis of thioamides is presented. For this transformation, dimethylamine as the key intermediate is generated in situ from dimethylformamide (DMF). All the tested substrates produced the desired products with excellent isolated yields.

Direct construction of 5-methyl-2-phenylisoxazol-3(2H)-ones via hypervalent iodine mediated sequential tandem oxidative cyclization of 3-oxo-N-phenylbutanamides catalyzed by zinc oxide (ZnO).

A sequential oxidative tandem cyclization reaction mediated by a combination of (diacetoxyiodo)benzene (DIB) with zinc oxide (ZnO) is presented for the synthesis of 5-methyl-2-phenylisoxazol-3(2H)-ones from ß-ketobutylanilides. A variety of ß-ketobutylanilide compounds were used in this approach, and a wide range of functionalized 5-methylisoxazol-3(2H)-ones were obtained in good to excellent yields.

An efficient method for the construction of polysubstituted 4-pyridones via self-condensation of ß-keto amides mediated by P2O5 and catalyzed by zinc bromide.

A self-condensation cyclization reaction mediated by phosphorus pentoxide (P2O5) and catalyzed by zinc bromide (ZnBr2) is presented for the synthesis of polysubstituted 4-pyridones and 2-pyridones from ß-keto amides. A variety of ß-keto amides are used in this approach, and a wide range of functionalized 4-pyridones and 2-pyridones were obtained in good to excellent yields. When employing the N-aryl ß-keto amides as the substrates in this protocol, 4-pyridones are resulted, however, when using N-aliphatic-substituted ß-keto amides as the partners of N-aryl ß-keto amides under the same conditions, 2-pyridones are afforded.