Tadehaginoside modulates lipogenesis and glucose consumption in HepG2 cells

Tadehaginoside (TS) is a phenylpropanoid glycoside found in Tadehagi triquetrum, a medicinal plant with multiple biological activities. This study investigated the effect of TS on lipogenesis and glucose consumption in HepG2 cells. Treatment with TS inhibited lipid accumulation in a dose-dependent manner. This inhibition was closely associated with the downregulation of lipogenic genes such as SREBP-1a, SREBP-2 and their downstream targets (FAS, ACC, HMGR) and the upregulation of lipolytic gene PPARα as revealed by real-time quantitative PCR. Further investigation showed that TS significantly stimulated glucose consumption by HepG2 cells and glucose uptake by C2C12 myotubes, which could be partially explained by the upregulation of PPARγ. Collectively, these results clearly indicate that TS is an effective regulator of lipogenesis and glucose consumption, which could be useful in treatment of obesity and diabetes.     

Identification and validation of tetracyclic benzothiazepines as Plasmodium falciparum cytochrome bc1 inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc₁ complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc₁ activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

Deorphaning Pyrrolopyrazines as Potent Multi‐Target Antimalarial Agents

The discovery of pyrrolopyrazines as potent antimalarial agents is presented, with the most effective compounds exhibiting EC₅₀ values in the low nanomolar range against asexual blood stages of Plasmodium falciparum in human red blood cells, and Plasmodium berghei liver schizonts, with negligible HepG2 cytotoxicity. Their potential mode of action is uncovered by predicting macromolecular targets through avant‐garde computer modeling. The consensus prediction method suggested a functional resemblance between ligand binding sites in non‐homologous target proteins, linking the observed parasite elimination to IspD, an enzyme from the non‐mevalonate pathway of isoprenoid biosynthesis, and multi‐kinase inhibition. Further computational analysis suggested essential P. falciparum kinases as likely targets of our lead compound. The results obtained validate our methodology for ligand‐ and structure‐based target prediction, expand the bioinformatics toolbox for proteome mining, and provide unique access to deciphering polypharmacological effects of bioactive chemical agents.     

New HIV-protease assays applying self-quenching peptide substrates in combination with time-resolved fluorescence single-molecule spectroscopy

This work describes the optimization and adoption of an assay system for the Human Immunodeficiency Virus (HIV)-protease, whose inhibition plays a central role in HIV therapy. The HIV-protease, which is an essential enzyme during viral maturation, has a specific cleavage site of eight amino acid residues (SQNY*PIV). Adding two amino acid residues at the N-terminus and enclosing the resulting sequence by a dye-labelled lysine residue and a tryptophan residue leads to the substrate (K(dye)CGSQNY*PIVW) in which the fluorescence of the fluorophore is efficiently quenched by the intrinsic tryptophan due to a photoinduced electron transfer reaction. After cleavage of the substrate by the target enzyme, the dye and the tryptophan residue are separated, effecting a significant increase in fluorescence intensity. Measuring the fluorescence versus time enables an online-monitoring of the enzyme activity. With this method, a HIV-PR concentration of 10^?9?M is detectable within minutes, which is comparable with commercially available assays using doubly labelled substrates based on a fluorescence resonance energy transfer. We were able to further increase the sensitivity to the subnanomolar range by using confocal single-molecule spectroscopy.     

Simultaneous Determination of Concentration and Enantiomeric Composition of Amino Acids in Aqueous Solution by Using a Tetrabromobinaphthyl Dialdehyde Probe

3,3′-Diformyl-1,1′-bi-2-naphthol or its methoxymethyl-protected derivative is found to undergo a highly selective reaction with excess bromine in CH₂Cl₂ at reflux to give the novel 5,5′,6,6′-tetrabrominated product (S)- or (R)- 2 . The observed electrophilic substitution at the 5,5′-positons of an optically active binaphthyl compound is unprecedented. Unlike unbrominated 3,3′-diformyl-1,1′-bi-2-naphthol, which is not suitable for fluorescent recognition in water, compound (S)- 2 , in combination with Zn²⁺, exhibits a highly enantioselective fluorescent response toward amino acids in aqueous solution (HEPES buffer, pH 7.4). It is further found that the condensation product of (R)- 2 with tryptophan, (R)- 3 , shows dual-responsive emissions toward amino acids; the short wavelength (λ₁=350 nm) emission is sensitive to the concentration of the substrate regardless of the chiral configuration and the long wavelength (λ₂>500 nm) emission is highly enantioselective. Thus, the use of (R)- 3 allows the simultaneous determination of the concentration and enantiomeric composition of an amino acid sample from one fluorescence measurement.     

Cell‐ and Tissue‐Based Proteome Profiling and Bioimaging with Probes Derived from a Potent AXL Kinase Inhibitor

AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Recently, our group has developed a lead compound, 9im, capable of displaying potent and specific inhibition of AXL. To further identify the cellular on/off targets, in this study, competitive affinity‐based proteome profiling was carried out, leading to the discovery of several unknown cellular targets such as BCAP31, LPCAT3, POR, TM9SF3, SCCPDH and CANX. In addition, trans‐cyclooctene (TCO) and acedan‐containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of AXL in various biosystems.     

SPORCalc: A development of a database analysis that provides putative metabolic enzyme reactions for ligand-based drug design

Understanding both the enzyme reactions that contribute to intermediate metabolism and the biochemical fate of candidate therapeutic and toxic agents are essential for drug design. Traditional metabolic databases indicate whether reactions have been observed but do not provide the likelihoods of reactions occurring, for example those of mixed function oxygenases and oxidases, during phase I metabolism. The desire for more quantitative predictions motivated the development of the recently introduced Substrate Product Occurrence Ratio Calculator (SPORCalc) that identifies metabolically labile atom positions in candidate compounds. This paper describes a further development and provides a clearer explanation of SPORCalc for the computational pharmacology, medicinal chemistry and drug design communities interested in metabolic prediction of xenobiotics using chemical databases of biotransformations.Examples of reaction centre detection in Metabolite? are described followed by a demonstration of almokalant, an anti-arrhythmic agent, undergoing phase I metabolism. In general, occurrence ratio (OR) values are calculated throughout a compound and its transformed metabolites to give propensity (p) values at each atom position. The OR values from substrates and products in the database are essential for addition and elimination reactions. For almokalant, the resulting p values ranged from 10^?1 to 10^?5 and their order of magnitude reflected the known and experimentally observed metabolites.SPORCalc depends entirely on the level of detail from isoform- or species-specific reaction classes in Metabolite?. Labile atom positions (sites of metabolism) are identified in both the candidate compound and its metabolites. In general, the likelihood of one enzyme isoform-dependent reaction occurring relative to another and the putative metabolic routes from different isoforms can be investigated. SPORCalc can be developed further to include suitable three-dimensional, structure–activity and physiochemical information.     

Stereostructure of oligomeric polyisoprenyllithium in tetrahydrofuran

The oligomer of polyisoprenyllithium, 2,5,5-trimethylhexen-2-yllithium, formed from tert-butyllithium and one molecule of isoprene in benzene has a structure similar to that of higher members of the series and also similar properties. In THF only one stereoisomer is stable, and an attempt is made to determine its stereostructure. The lithium compound was converted at low temperature to the acid, and this by a series of steps in which the structure was monitored to the acid's parent hydrocarbon 3,6,6-trimethyl-3-heptene. This was then compared with a synthesized sample of this hydrocarbon of known structure. In this way it was shown that the original lithium compound had a cis configuration in tetrahydrofuran solution.     

Current Methods for Determination of L-Carnitine and Acylcarnitines

L-Carnitine as endogenous compound plays an important role within several metabolic pathways and a deficiency of L-carnitine can cause adverse effects in physiological and/or mental state of health and disease. The prevention of diseases related to carnitine deficiency requires, first of all, the exact determination of L-carnitine and its esters in biological material at pmol/cm³ level. A series of analytical procedures based on biochemical assays as well as on physical methods are available today. Determination of free and total carnitine is sometimes sufficient for a clinical diagnosis, but in most cases, such as in newborn screening for genetic disorders, detailed qualitative and quantitative L-carnitine/acylcarnitine profiling is needed. Technological progress has also revolutionized the determination of carnitines. Today, comprehensive and diagnostically relevant information can be obtained by mass spectrometry. An overview is given of the technical and methodological developments in carnitine analysis and some applications, such as in neonatal screening, diabetes mellitus, and cardiomyopathy.     

Chromatographic determination of harmalans in the urine of autistic children

This paper presents a new approach to autism – a complex and still enigmatic condition. We present the results of our preliminary research which was based on the detection of the hallucinogenic substance 6‐ (or 10‐)methoxyharmalan in the urine samples of autistic children with the use of chromatographic methods. Additionally, we aim to describe the relationship between the level of tryptophan and harmalan, and the influence of supplementation on the level of this compound. We applied HPLC‐UV /v is, HPLC‐DAD and LC–MS in order to determine McIsaac's compound in the urine samples obtained from autistic children (n = 132) and healthy individuals (n = 10). The level of tryptophan was quantified with the use of GC–MS. Our research shows the presence of the McIsaac's compound in 110 samples of ASD children contrary to healthy children, where it was not found. No relationship between the level of tryptophan and 6‐methoxyharmalan was noticed. The study shows a strong influence of melatonin supplementation on the presence of the McIsaac's compound. We believe that the results of our research can contribute to a better understanding of autism spectrum disorders. Moreover, our findings can form the basis for other studies focused on autism, eventually making it possible to understand its etiology.     

Total Synthesis and Configurational Assignment of Chondramide A

The first total synthesis of the cyclodepsipeptide chondramide A ( 2 b ) is described. This depsipeptide is composed of four subunits, namely L ‐alanine, N‐Me‐D ‐tryptophan, 3‐amino‐2‐methoxy‐propionic acid (β‐tyrosine derivative), and a 7‐hydroxy‐alkenoic acid. While the configuration of the stereogenic centers in the 7‐hydroxy‐alkenoic acid were known, the configuration of the tyrosine derivative required clarification and turned out to be (2S,3R) or (2L ,3L ), respectively. The synthesis of the 3‐amino‐2‐methoxy‐3‐arylpropanoic ester 20 b relied on an asymmetric dihydroxylation yielding diol ent‐ 15 a followed by a regioselective Mitsunobu substitution leading to 3‐azido‐2‐hydroxypropanoate 18 b . We could also show that the ester bond in the seco compound 26 b can be fashioned by a Mitsunobu esterification by using hydroxy ester (7S)‐ 7 and the tripeptide acid 25 b . This synthesis should allow for the preparation of various analogues.     

Metabolomics reveals hippocampal metabolic fluctuations of postoperative fatigue syndrome and anti‐fatigue effect of Carthamus tinctorius L. extract in rat model

Postoperative fatigue syndrome (POFS) is a common clinical complication following almost every major abdominal surgery. There is not a full explanation of the etiology of POFS, especially its central mechanism. Carthamus tinctorius L. is a classic traditional Chinese medicine (TCM) which could exert ananti‐fatigue effect on POFS. However, its mechanism is still lacking. Here, ultra‐high performance liquid chromatography coupled with a quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOFMS) based metabonomic approach was used to characterize hippocampal metabolic fluctuations of POFS in a rat model induced by partial hepatectomy, and to evaluate the anti‐fatigue effect of C. tinctorius L. extract (CTLE). With partial least‐squares discriminant analysis for classification and selection of biomarkers, 15 hippocampal metabolites related to POFS were identified, primarily involving alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine degradation, purine metabolism, phenylalanine metabolism, tryptophan metabolism, phospholipid metabolism and fatty acid metabolism. With these altered metabolic pathways as possible drug targets, we systematically analyzed the protective effect of CTLE, which showed that CTLE could provide an anti‐fatigue effect on POFS through partially regulating the perturbed metabolic pathways. This study indicated that UHPLC‐Q‐TOFMS‐based metabolomics provided a powerful tool to reveal hippocampal metabolic fluctuations of POFS and study the mechanism of TCM. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and biological properties of 3-methyl-10-propargyl-5,8-dideazafolic acid

The synthesis of N³-methyl-10-propargyl-5,8-dideazafolic acid ( 1b ) is described. Ring closure of methyl-5-methylanthranilate with chloroformamidine hydrochloride gave a high yield of pure 2-amino-4-hydroxy-6-methylquinazoline treatment of which with iodomethane/sodium hydroxide provided the corresponding 3-methylquinazoline (6) which was converted to its 2-pivaloylamino derivative. This synthetic approach, next involving functionalisation of the 6-methyl group, was not further pursued because of difficulty encountered in removing the pivaloyl group. Methyl 5-methylanthranilate was treated with p-toluenesulfonyl chloride and the product then N-methylated. The tosyl group was cleaved with hydrogen bromide/phenol and the resulting methylamine ring-closed with chloroformamidine hydrochloride to provide 2-amino-1,4-dihydro-1,6-dimethyl-4-oxoquinazoline ( 11 ). The 2-pivaloylamino derivative of 11 was prone to hydrolytic deamination when attempts were made to remove the pivaloyl group and further elaboration of this heterocycle, with the intention of obtaining N¹-methyl-10-propargyl-5,8-dideazafolic acid was, too, not attempted. Di-t-butyl N-(4-propargylamino)benzoyl)-L-glutamate was therefore prepared and coupled with 2-amino-6-bromomethyl-4-hydroxyquinazoline hydrobromide. The resulting antifolate diester was N-monomethylated. Removal of the t-butyl groups with trifluoracetic acid afforded the target compound 1b and its structure was proved by degradation to the quinazoline 6 . Its IC₅₀ for L1210 thymidylate synthase (TS) was 26 μM; the control value for 10-propargyl-5,8-dideazafolic acid ( 1a ) was 0.02 μM. Thus the substitution of the lactam hydrogen in 1a by a methyl group reduced the TS inhibition by 1300-fold. Compound 1b was poorly cytotoxic to L1210 cells in culture (ID₅₀ > 100 μM). An unperturbed lactam group in this class of antifolate is important for binding to TS.     

丁二酸脱水制备丁二酸酐的微观反应机理研究

丁二酸脱水法是工业生产丁二酸酐的方法之一. 用量子化学密度泛函理论(DFT)对该反应的微观机理进行了详细研究, 得到了该反应的微观过程. 根据计算和分析可知: 丁二酸脱水制备丁二酸酐的微观反应途径为IM1→TS1→IM2→TS2→IM3→TS3→P＋H₂O, 在反应过程中IM3为氢键复合物, 整个反应的速控步为IM3→TS3→P＋H₂O, 其所需活化能为167.17 kJ/mol.     

Current Methods for Determination of <Emphasis Type="Italic">L</Emphasis>-Carnitine and Acylcarnitines

Summary. L-Carnitine as endogenous compound plays an important role within several metabolic pathways and a deficiency of L-carnitine can cause adverse effects in physiological and/or mental state of health and disease. The prevention of diseases related to carnitine deficiency requires, first of all, the exact determination of L-carnitine and its esters in biological material at pmol/cm³ level. A series of analytical procedures based on biochemical assays as well as on physical methods are available today. Determination of free and total carnitine is sometimes sufficient for a clinical diagnosis, but in most cases, such as in newborn screening for genetic disorders, detailed qualitative and quantitative L-carnitine/acylcarnitine profiling is needed. Technological progress has also revolutionized the determination of carnitines. Today, comprehensive and diagnostically relevant information can be obtained by mass spectrometry. An overview is given of the technical and methodological developments in carnitine analysis and some applications, such as in neonatal screening, diabetes mellitus, and cardiomyopathy.     

Studies on the Chemical Modification of the Essential Groups of <Emphasis Type="Italic">N</Emphasis>-Acetyl-β-<Emphasis Type="SmallCaps">d</Emphasis>-Glucosaminidase from Viscera of Green Crab (<Emphasis Type="Italic">Scylla Serrata</Emphasis>)

The chemical modification of N-acetyl-β-d-glucosaminidase (EC3.2.1.30) from viscera of green crab (Scylla serrata) has been first studied. The modification of indole groups of tryptophan of the enzyme by N-bromosuccinimide can lead to complete inactivation, accompanying the absorption decreasing at 275 nm and the fluorescence intensity quenching at 338 nm, indicating that tryptophan is essential residue to the enzyme. The modification of histidine residue, the carboxyl groups, and lysine residue inactivates the enzyme completely or incompletely. The results show that imidazole groups of histidine residue or sulfhydryl residues, the carboxyl groups of acidic amino acid, amino groups of lysine residue, and indole groups of tryptophan were essential for the catalytic activity of enzyme, while the results demonstrate that the disulfide bonds and the carbamidine groups of arginine residues are not essential to the enzyme’s function.     

Singlet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation

Several studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A₂ (PLA₂) activity. Therefore, the regulation of PLA₂ catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA₂ due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA₂ inhibition. A more detailed analysis of the plasma-treated PLA₂ identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation.     

241. Geijerone (3-isopropenyl-4-methyl-4-vinylcyclohexanone), a new, naturally occurring C 12 terpenoid

In the essential oil of Juniperus communis L. (fruit), the title compound (trans- isomer) has been identified; its structure being confirmed by synthesis from elemol. The optical rotation of the natural compound was nearly the same as the synthetic sample, suggesting that it is not an artefact formed from a ten-membered ring precursor during the isolation.     

Pteridines. Part CVI. Isolation and characterization of limipterin (1-O-(L-erythro-biopterin-2′-yl)-β-N-acetylglucosamine) and its 5,6,7,8-tetrahydro derivative from green sulfur bacterium Chlorobium limicola f. thiosulfatophilum NCIB 8327

A new pteridine compound was isolated from green sulfur photosynthetic bacteria, Chlorobium limicola f. thiosulfatophilum NCIB 8327. The structure of this pterin derivative was established to be 1-O-(L -erythro-5,6,7,8- tetrahydropterin-2′-yl)-β-N-acetylglucosamine ( 1 ) from ¹H-NMR and CD spectra as well as from various mass spectrometric techniques and chemical-cleavage techniques. Upon acid hydrolysis of 1 , equimolar amounts of biopterin ( 2 ) and N-acetylglucosamine were produced. The structure of the hydrolysis product 2 was confirmed by comparing its NMR, UV, CD, and MS and its chromatographical behavior with those of an authentic specimen. N-Acetylglucosamine was identified by an enzymatic hydrolysis experiment as well as by NMR and thin layer chromatography. Electrospray (ES), fast-atom-bombardment (FAB), and thermospray (TS) mass spectrometry of 1 yielded an MH⁺ at m/z 441. Periodate-oxidation experiments of the intact molecule 1 and of its hydrolysis product 2 are consistent with the proposed structure. Differential I₂ oxidation experiments with the native compound showed that the in vivo oxidation state of this pterin is its tetrahydro form. We propose the trivial name ‘limipterin’ for this new compound.