Mobilize a Proton to Transform the Collision-Induced Dissociation Spectral Pattern of a Cyclic Peptide.

The collision-induced dissociation (CID) behaviors of protonated molecules of anabaenopeptins, a group of cyanobacterial cyclic peptides, were investigated in detail using liquid chromatography-tandem mass spectrometry. Although anabaenopeptin A and B share a macrocyclic peptide structure, they give strikingly different fragmentation patterns; the former gives a variety of product ions including cleavages in the cyclic peptide structure, which is useful for structural analysis; whereas the latter gives far fewer product ions and no fragmentation in the cyclic moiety. Energy-resolved CID experiments clarified the mechanism behind the striking difference attributable to the difference in exocyclic amino acid residues, Tyr or Arg. The guanidino group in Arg-containing analogue, anabaenopeptin B, should be by far the most preferred protonation site; the proton would be sequestered at the guanidino group in the protonated molecule, with the lack of proton mobility prohibiting opening of the charge-directed fragmentation channels in the cyclic moiety. Enzymatic hydrolysis of the guanidino group to give citrullinated-anabaenopeptin B restored proton mobility. The fragmentation pattern of the citrullinated peptide became almost identical to that of anabaenopeptin A. The observed fragmentation behaviors of these cyclic peptides were consistent with those of linear peptides, which have been well understood based on the mobile proton model.

An ex vivo screening using mouse brain mitochondria identified seco-cycline D as an inhibitor of mitochondrial permeability transition pore.

Mitochondrial dysfunction is implicated in neuropsychiatric disorders. Inhibition of mitochondrial permeability transition pore (mPTP) and thereby enhancement of mitochondrial Ca2+ retention capacity (CRC) is a promising treatment strategy. Here, we screened 1718 compounds to search for drug candidates inhibiting mPTP by measuring their effects on CRC in mitochondria isolated from mouse brains. We identified seco-cycline D (SCD) as an active compound. SCD and its derivative were more potent than a known mPTP inhibitor, cyclosporine A (CsA). The mechanism of action of SCD was suggested likely to be different from CsA that acts on cyclophilin D. Repeated administration of SCD decreased ischemic area in a middle cerebral artery occlusion model in mice. These results suggest that SCD is a useful probe to explore mPTP function.

Structure and biosynthesis of the ribosomal lipopeptide antibiotic albopeptins.

Albopeptins produced by Streptomyces albofaciens JC-82-120 were isolated as effective antibiotics for plant pathogenetic disease in 1986. However, their unusual physicochemical properties hampered the determination of their chemical structures. In this report, we describe our efforts to elucidate their structures. Initially, the structure of an unusual C13-fatty acid with an N-hydroxyguanidyl group was determined using degradation and chemical synthesis. After the linear portion of the octapeptide core was constructed based on the 2D-NMR data, the final assembly of the unusual structure, including the sulfoxide bridge, was achieved through the analysis of detailed NMR data. The proposed structure of albopeptin B was supported by MS/MS data, which also enabled us to determine the structure of 5 albopeptin family members. Bioinformatics analysis of the genomic data of the producer strain further led us to propose that their biosynthetic pathway is similar to the ribosomally derived lanthipeptides possessing a long-chain fatty acid.

A new peptaibol, RK-026A, from the soil fungus Trichoderma sp. RK10-F026 by culture condition-dependent screening.

A new peptaibol, RK-026A (1) was isolated from a fungus, Trichoderma sp. RK10-F026, along with atroviridin B (2), alamethicin II (3), and polysporin B (4) as a cytotoxic compound, which was selected by principal component analysis of the MS data from 5 different culture conditions. The structure of 1 was determined as a new atroviridin B derivative containing Glu at the 18th residue instead of Gln by NMR and HR-MS analyses including the investigation of detailed MS/MS fragmentations. 1 showed cytotoxicity toward K562 leukemia cells at an IC50 value of 4.1 µm.

An integrated screening system for the selection of exemplary substrates for natural and engineered cytochrome P450s.

Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of "exemplary" substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acquired an ability to hydroxylate non-natural substrate steroids regio- and stereoselectively by two amino acid mutations. The distinct transition of exemplary substrates due to each single amino acid mutation was revealed, demonstrating the utility of the established system.

Isolation, Identification, and Synthesis of a New Prenylated Cinnamic Acid Derivative from Brazilian Green Propolis and Simultaneous Quantification of Bioactive Components by LC-MS/MS.

A new cinnamic acid derivative, (E)-3-[4-hydroxy-3-((E)-3-formyl-2-butenyl)phenyl]-2- propenoic acid (20) has been isolated from the ethanol extract of Brazilian green propolis along with three known cinnamic acid derivatives, 3,4-dihydroxy-5-prenyl-(E)-cinnamic acid (4), capillartemisin A (6), and 2,2-dimethylchromene-6-(E)-propenoic acid (8), and a flavonoid, dihydrokaempferide (16) by liquid-liquid participation, a series of column chromatography and preparative HPLC. Their structures have been determined by spectroscopic analyses and chemical synthesis of compound 20. The simultaneous quantification of 20 constituents, including 10 cinnamic acid derivatives, 7 flavonoids, and 3 caffeoylquinic acid derivatives, has also been developed and validated using LC-MS/MS. The new compound 20 was shown to activate PPAR α but not PPAR ß or γ.

Plasma Nervonic Acid Is a Potential Biomarker for Major Depressive Disorder: A Pilot Study.

Background: Diagnostic biomarkers of major depressive disorder, bipolar disorder, and schizophrenia are urgently needed, because none are currently available. Methods: We performed a comprehensive metabolome analysis of plasma samples from drug-free patients with major depressive disorder (n=9), bipolar disorder (n=6), schizophrenia (n=17), and matched healthy controls (n=19) (cohort 1) using liquid chromatography time-of-flight mass spectrometry. A significant effect of diagnosis was found for 2 metabolites: nervonic acid and cortisone, with nervonic acid being the most significantly altered. The reproducibility of the results and effects of psychotropic medication on nervonic acid were verified in cohort 2, an independent sample set of medicated patients [major depressive disorder (n=45), bipolar disorder (n=71), schizophrenia (n=115)], and controls (n=90) using gas chromatography time-of-flight mass spectrometry. Results: The increased levels of nervonic acid in patients with major depressive disorder compared with controls and patients with bipolar disorder in cohort 1 were replicated in the independent sample set (cohort 2). In cohort 2, plasma nervonic acid levels were also increased in the patients with major depressive disorder compared with the patients with schizophrenia. In cohort 2, nervonic acid levels were increased in the depressive state in patients with major depressive disorder compared with the levels in the remission state in patients with major depressive disorder and the depressive state in patients with bipolar disorder. Conclusion: These results suggested that plasma nervonic acid is a good candidate biomarker for the depressive state of major depressive disorder.

Identification of a gene cluster for telomestatin biosynthesis and heterologous expression using a specific promoter in a clean host.

Telomestatin, a strong telomerase inhibitor with G-quadruplex stabilizing activity, is a potential therapeutic agent for treating cancers. Difficulties in isolating telomestatin from microbial cultures and in chemical synthesis are bottlenecks impeding the wider use. Therefore, improvement in telomestatin production and structural diversification are required for further utilization and application. Here, we discovered the gene cluster responsible for telomestatin biosynthesis, and achieved production of telomestatin by heterologous expression of this cluster in the engineered Streptomyces avermitilis SUKA strain. Utilization of an optimal promoter was essential for successful production. Gene disruption studies revealed that the tlsB, tlsC, and tlsO-T genes play key roles in telomestatin biosynthesis. Moreover, exchanging TlsC core peptide sequences resulted in the production of novel telomestatin derivatives. This study sheds light on the expansion of chemical diversity of natural peptide products for drug development.

Total Synthesis of Kehokorins A-E, Cytotoxic p-Terphenyls.

This paper describes a general method for the synthesis of kehokorins A-E, novel cytotoxic p-terphenyls. 2,4,6-Trihydroxybenzaldehyde served as a common building block for preparation of the central aromatic ring. Construction of their p-terphenyl skeletons was achieved by a stepwise Suzuki-Miyaura coupling, whereas the phenyldibenzofuran moiety was built up by an intramolecular Ullmann reaction. Introduction of an l-rhamnose residue into partly protected kehokorin B was performed by the trichloroacetimidate method.

Analysis of Metabolites in Stem Parasitic Plant Interactions: Interaction of Cuscuta-Momordica versus Cassytha-Ipomoea.

Cuscuta and Cassytha are two well-known stem parasitic plant genera with reduced leaves and roots, inducing haustoria in their stems. Their similar appearance in the field has been recognized, but few comparative studies on their respective plant interactions are available. To compare their interactions, we conducted a metabolite analysis of both the Cassytha-Ipomoea and the Cuscuta-Momordica interaction. We investigated the energy charge of the metabolites by UFLC (ultra-high performance liquid chromatography), and conducted GC-MS (gas chromatography-mass spectrometry) analysis for polar metabolites (e.g., saccharides, polyols) and steroids. The energy charge after parasitization changed considerably in Cassytha but not in Cusucta. Cuscuta changed its steroid pattern during the plant interaction, whereas Cassytha did not. In the polar metabolite analysis, the laminaribiose increase after parasitization was conspicuous in Cuscuta, but not in Cassytha. This metabolite profile difference points to different lifestyles and parasitic strategies.

Total Synthesis of the Proposed Structure for Aromin and Its Structural Revision.

This paper describes the first total synthesis of the proposed structure for aromin, an annonaceous acetogenin possessing an unusual bis-THF ring system, and its 4S,7R-isomer. The key steps involve an oxidative cyclization of a couple of terminal-diene alcohols and an intermolecular metathesis of an alkenyl tetrahydrofuran with an enone carrying a tetrahydrofuranyl lactone. The spectral data of both samples did not match those of aromin. Re-examination of the NMR data using the CAST/CNMR Structure Elucidator and chemical derivations suggested that the real structure of aromin should be revised to be a tetrahydropyran acetogenin, montanacin D. Cytotoxicities in human solid tumor cell lines for synthetic samples were also evaluated.

Biodiesel and poly-unsaturated fatty acids production from algae and crop plants - a rapid and comprehensive workflow for lipid analysis.

Fatty acid methyl ester analysis (FAME) by gas chromatography coupled to mass spectrometry (GC-MS) is a widely used technique in biodiesel/bioproduct (e.g. poly-unsaturated fatty acids, PUFA) research but typically does not allow distinguishing between bound and free fatty acids. To understand and optimize biosynthetic pathways, however, the origin of the fatty acid is an important information. Furthermore the annotation of PUFAs is compromised in classical GC-EI-MS because the precursor molecular ion is missing. In the present protocol an alkaline methyl esterification step with TMS derivatization enabling the simultaneous analysis of bound and free fatty acids but also further lipids such as sterols in one GC-MS chromatogram is combined. This protocol is applied to different lipid extracts from single cell algae to higher plants: Chlorella vulgaris, Chlamydomonas reinhardtii, Coffea arabica, Pisum sativum and Cuscuta japonica. Further, field ionization (GC-FI-MS) is introduced for a better annotation of fatty acids and exact determination of the number of double bonds in PUFAs. The proposed workflow provides a convenient strategy to analyze algae and other plant crop systems with respect to their capacity for third generation biodiesel and high-quality bioproducts for nutrition such as PUFAs.

Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

Fragmentation patterns of methyloxime-trimethylsilyl derivatives of constitutive mono- and disaccharide isomers analyzed by gas chromatography/field ionization mass spectrometry.

RATIONALE: In saccharide analysis by gas chromatography/mass spectrometry (GC/MS), electron ionization (EI) is used almost exclusively, whereas other gentler methods of ionization are rarely used. Field ionization (FI) is recognized as a GC/MS ionization method that causes fewer fragment ions, but only few studies are available on its use in saccharide analysis. METHODS: To evaluate the usefulness of FI in profiling isomeric saccharides by GC/MS and to explore its potential application in metabolome analysis, we compared EI, chemical ionization (CI), and FI spectral patterns of consecutive mono- and disaccharides derivatized with methoxamine-HCl and N-methyl-N-(trimethylsilyl)trifluoroacetamide. RESULTS: FI produced molecular ions and fragment ions characteristic of constitutive isomeric disaccharides. All of the derivatized saccharides that originally had free anomeric OH showed methyloxime-moiety fragment ions, attributable to the cleavage between C2 and C3. Some fragment ions in FI were indicative of the position of dihexose linkages. Although EI with lowered voltage (18 V, 130 °C) produced fewer fragment ions than conventional EI (70 V, 250 °C) did, fragmentation patterns were different from those of FI. CONCLUSIONS: Our data show that FI is useful for distinguishing isomeric saccharides in qualitative analyses.

Synthesis and structural revision of a brominated sesquiterpenoid, aldingenin C.

This paper describes a short step synthesis of the proposed structure for aldingenin C from trans-limonene oxide. The tetrahydropyran-fused 2-oxabicyclo[3.2.2]nonane skeleton as the structural feature was constructed by an intramolecular epoxide-opening reaction and a brominative cyclization. The spectral data of the synthetic compound did not match those of the natural product reported. Re-examination of the reported NMR data using new CAST/CNMR Structure Elucidator suggests that the structure of aldingenin C should be revised to that of known caespitol.

Detection of oxygen addition peaks for terpendole E and related indole-diterpene alkaloids in a positive-mode ESI-MS.

This report describes that a regular positive electrospray ionization mass spectrometry (MS) analysis of terpendoles often causes unexpected oxygen additions to form [M + H + O](+) and [M + H + 2O](+), which might be a troublesome in the characterization of new natural analogues. The intensities of [M + H + O](+) and [M + H + 2O](+) among terpendoles were unpredictable and fluctuated largely. Simple electrochemical oxidation in electrospray ionization was insufficient to explain the phenomenon. So we studied factors to form [M + H + O](+) and [M + H + 2O](+) using terpendole E and natural terpendoles together with some model indole alkaloids. Similar oxygen addition was observed for 1,2,3,4-tetrahydrocyclopent[b]indole, which is corresponding to the substructure of terpendole E. In tandem MS experiments, a major fragment ion at m/z 130 from protonated terpendole E was assigned to the substructure containing indole. When the [M + H + O](+) was selected as a precursor ion, the ion shifted to m/z 146. The same 16 Da shift of fragments was also observed for 1,2,3,4-tetrahydrocyclopent[b]indole, indicating that the oxygen addition of terpendole E took place at the indole portion. However, the oxygen addition was absent for some terpendoles, even whose structure resembles terpendole E. The breakdown curves characterized the tandem MS features of terpendoles. Preferential dissociation into m/z 130 suggested the protonation tendency at the indole site. Terpendoles that are preferentially protonated at indole tend to form oxygen addition peaks, suggesting that the protonation feature contributes to the oxygen additions in some degrees.

RK-1355A and B, novel quinomycin derivatives isolated from a microbial metabolites fraction library based on NPPlot screening.

Two novel quinomycin derivatives, RK-1355A (1) and B (2), and one known quinomycin derivative, UK-63,598 (3), were isolated from a microbial metabolites fraction library of Streptomyces sp. RK88-1355 based on Natural Products Plot screening. The structural elucidation of 1 and 2 was established through two-dimensional NMR and mass spectrometric measurements. They belong to a class of quinomycin antibiotics family having 3-hydroxyquinaldic acid and a sulfoxide moiety. They are the first examples for natural products as a quinoline type quinomycin having a sulfoxide on the intramolecular cross-linkage. They showed potent antiproliferative activities against various cancer cell lines and they were also found to exhibit moderate antibacterial activity.

Winners of CASMI2013: Automated Tools and Challenge Data.

CASMI (Critical Assessment of Small Molecule Identification) is a contest in which participants identify the molecular formula and chemical structure of challenging molecules using blind mass spectra as the challenge data. Seven research teams participated in CASMI2013. The winner of CASMI2013 was the team of Andrew Newsome and Dejan Nikolic, the University of Illinois at Chicago, IL, USA. The team identified 15 among 16 challenge molecules by manually interpreting the challenge data and by searching in-house and public mass spectral databases, and chemical substance and literature databases. MAGMa was selected as the best automated tool of CASMI2013. In some challenges, most of the automated tools successfully identified the challenge molecules, independent of the compound class and magnitude of the molecular mass. In these challenge data, all of the isotope peaks and the product ions essential for the identification were observed within the expected mass accuracy. In the other challenges, most of the automated tools failed, or identified solution candidates together with many false-positive candidates. We then analyzed these challenge data based on the quality of the mass spectra, the dissociation mechanisms, and the compound class and elemental composition of the challenge molecules.

Human myo-inositol monophosphatase 2 rescues the nematode thermotaxis mutant ttx-7 more efficiently than IMPA1: functional and evolutionary considerations of the two mammalian myo-inositol monophosphatase genes.

Mammals express two myo-inositol monophosphatase (IMPase) genes, IMPA1/Impa1 and IMPA2/Impa2. In this study, we compared the spatial expression patterns of the two IMPase gene transcripts and proteins in mouse tissues. Results indicated discrete expression of the two IMPase genes and their protein products in various organs, including the brain. In Caenorhabditis elegans, loss of the IMPase gene, ttx-7, disrupts cellular polarity in RIA neurons, eliciting abnormal thermotaxis behavior. We performed a rescue experiment in mutant nematodes using mammalian IMPases. Human IMPA2 rescued the abnormal behavioral phenotype in the ttx-7 mutants more efficiently than IMPA1. These results raise a question about the phylogenetic origin of IMPases and the biological roles of mammalian IMPase 2 in mammals. Impa2 knockout mice generated in our laboratory, exhibited neither behavioral abnormalities nor a significant reduction in myo-inositol content in the brain and other examined tissues. Given the ability of human IMPA2 to rescue the ttx-7 mutant, and its genetic association with multiple neuropsychiatric disorders, close scrutiny of IMPA2 function and the evolutionary origin of IMPase genes is warranted.

Novel angiotensin I-converting enzyme inhibitory peptides found in a thermolysin-treated elastin with antihypertensive activity.

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 µM against ACE and may have potential use as a functional food.