Mass Spectrometric Analysis of Cucurbitacins and Dihydrocucurbitacins from the Tuber of Citrullus naudinianus.

The vast pool of structurally and functionally distinct secondary metabolites (i.e., natural products (NPs)) is constantly being expanded, a process also driven by the rapid progress in the development of analytical techniques. Such NPs often show potent biological activities and are therefore prime candidates for drug development and medical applications. The ethyl acetate extract of the tuber of Citrullus naudinianus (C. naudinianus), an African melon with edible fruits and seeds, shows in vitro immunomodulatory activity presumably elicited by cucurbitacins that are known major constituents of this plant. Further potentially immunomodulatory cucurbitacins or cucurbitacin derivatives were assumed to be in the tuber. Given the typically high content of cucurbitacins with similar physicochemical features but often distinct bioactivities, an efficient and reliable separation process is a prerequisite for their detailed characterization and assessment in terms of bioactivity. We therefore developed a detection method to screen and differentiate cucurbitacins via high-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS). In order to confirm the identification, the fragmentation patterns of two cucurbitacins and one 23,24-dihydrocucurbitacin were also investigated. Six characteristic fragments were identified and three of them were employed for the identification of cucurbitacins and 23,24-dihydrocucurbitacins in the extract. As a result, in addition to eight previously reported cucurbitacins from this plant four distinct 23,24-dihydrocucurbitacins (B, D, E, and I) were putatively identified and newly found in the ethyl acetate extract of the tuber of C. naudinianus. The established methodology enables rapid and efficient LC-MS-based analysis and identification of cucurbitacins and 23,24-dihydrocucurbitacins in plant extracts.

Integrated Metabolomic and Transcriptomic Analysis of Modified Nucleosides for Biomarker Discovery in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers. The biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is the truncal driver mutation of most cases of ccRCC. Cancer cells are metabolically reprogrammed and excrete modified nucleosides in larger amounts due to their increased RNA turnover. Modified nucleosides occur in RNAs and cannot be recycled by salvage pathways. Their potential as biomarkers has been demonstrated for breast or pancreatic cancer. To assess their suitability as biomarkers in ccRCC, we used an established murine ccRCC model, harboring Vhl, Trp53 and Rb1 (VPR) knockouts. Cell culture media of this ccRCC model and primary murine proximal tubular epithelial cells (PECs) were investigated by HPLC coupled to triple-quadrupole mass spectrometry using multiple-reaction monitoring. VPR cell lines were significantly distinguishable from PEC cell lines and excreted higher amounts of modified nucleosides such as pseudouridine, 5-methylcytidine or 2'-O-methylcytidine. The method's reliability was confirmed in serum-starved VPR cells. RNA-sequencing revealed the upregulation of specific enzymes responsible for the formation of those modified nucleosides in the ccRCC model. These enzymes included Nsun2, Nsun5, Pus1, Pus7, Naf1 and Fbl. In this study, we identified potential biomarkers for ccRCC for validation in clinical trials.

Mitochondrial Metabolomics of Sym1-Depleted Yeast Cells Revealed Them to Be Lysine Auxotroph.

Metabolomics has expanded from cellular to subcellular level to elucidate subcellular compartmentalization. By applying isolated mitochondria to metabolome analysis, the hallmark of mitochondrial metabolites has been unraveled, showing compartment-specific distribution and regulation of metabolites. This method was employed in this work to study a mitochondrial inner membrane protein Sym1, whose human ortholog MPV17 is related to mitochondria DNA depletion syndrome. Gas chromatography-mass spectrometry-based metabolic profiling was combined with targeted liquid chromatography-mass spectrometry analysis to cover more metabolites. Furthermore, we applied a workflow employing ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry with a powerful chemometrics platform, focusing on only significantly changed metabolites. This workflow highly reduced the complexity of acquired data without losing metabolites of interest. Consequently, forty-one novel metabolites were identified in addition to the combined method, of which two metabolites, 4-guanidinobutanal and 4-guanidinobutanoate, were identified for the first time in Saccharomyces cerevisiae. With compartment-specific metabolomics, we identified sym1Δ cells as lysine auxotroph. The highly reduced carbamoyl-aspartate and orotic acid indicate a potential role of the mitochondrial inner membrane protein Sym1 in pyrimidine metabolism.

Pharmacokinetic study of the novel phosphocholine derivative 3-dibutylaminopropylphosphonic acid by LC-MS coupling.

CRP is an important mediator of the inflammatory response. Pro-inflammatory CRP effects are mediated by pCRP* and mCRP, dissociation products of the native pCRP. The concentration of pCRP during inflammation may rise up to concentrations 1000-fold from baseline. By prevention of the conformational change from pCRP to pCRP*, pro-inflammatory immune responses can be inhibited and local tissue damage reduced. 3-(Dibutylamino)propylphosphonic acid (C10m) is a new substance that can suppress ischemic-reperfusion injury by targeting CRP in the complement cascade. It hampers dissociation of pCRP into its monomers, thus preventing exacerbation of tissue inflammation subsequent to reperfusion injury. In this study, the pharmacokinetics and metabolism of the new drug candidate C10m was investigated. A sensitive and selective method for detection of C10m and its metabolites from plasma and urine was developed with LC-MS and LC-MS/MS coupling. The LLOQ is at 0.1 µg mL-1 and recovery at 87.4% ± 2.8%. Accuracy and precision were within 15% coefficient of variation and nominal concentrations, respectively. Concentration time profile after i.v. bolus injection of C10m was analyzed by LC-MS/MS. Bioavailability has shown to be below 30%. Most likely due to the compounds' very polar chemical properties, no phase-I or phase-II metabolism could be observed. Absence of phase-I metabolism was cross-checked by performing microsomal incubations. Our study revealed that C10m is rapidly eliminated via urine excretion and that half-times appear to be increased with coadministration of the target pCRP.

Metabolic and Lipidomic Assessment of Kidney Cells Exposed to Nephrotoxic Vancomycin Dosages.

Vancomycin is a glycopeptide antibiotic used against multi-drug resistant gram-positive bacteria such as Staphylococcus aureus (MRSA). Although invaluable against resistant bacteria, vancomycin harbors adverse drug reactions including cytopenia, ototoxicity, as well as nephrotoxicity. Since nephrotoxicity is a rarely occurring side effect, its mechanism is incompletely understood. Only recently, the actual clinically relevant concentration the in kidneys of patients receiving vancomycin was investigated and were found to exceed plasma concentrations by far. We applied these clinically relevant vancomycin concentrations to murine and canine renal epithelial cell lines and assessed metabolic and lipidomic alterations by untargeted and targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses. Despite marked differences in the lipidome, both cell lines increased anabolic glucose reactions, resulting in higher sorbitol and lactate levels. To the best of our knowledge, this is the first endometabolic profiling of kidney cells exposed to clinically relevant vancomycin concentrations. The presented study will provide a valuable dataset to nephrotoxicity researchers and might add to unveiling the nephrotoxic mechanism of vancomycin.

A comparative structural analysis of the surface properties of asco-laccases.

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function-in particular towards large substrates.

Thermodynamic Integration Networks and Their Application to Charge Transfer Reactions within the AauDyPI Fungal Peroxidase.

We present a computer simulation study of the thermodynamics and kinetics of charge transfer reactions within the fungal peroxidase AauDyPI from Auricularia auriculae-judae. Driving forces and reorganization energies are obtained from a thermodynamic integration scheme based upon molecular dynamics simulations. To enhance the numerical accuracy, the free energies are analyzed within a least-squares scheme of a closely knit thermodynamic network. We identify Tyr147, Tyr229, and Trp105 as oxidative agents, and find Trp377 to be a long-lived reaction intermediate. The results are compared to recent experimental findings.

Mechanism of the Ti(III)-Catalyzed Acyloin-Type Umpolung: A Catalyst-Controlled Radical Reaction.

The titanium(III)-catalyzed cross-coupling between ketones and nitriles provides an efficient stereoselective synthesis of α-hydroxyketones. A detailed mechanistic investigation of this reaction is presented, which involves a combination of several methods such as EPR, ESI-MS, X-ray, in situ IR kinetics, and DFT calculations. Our findings reveal that C-C bond formation is turnover-limiting and occurs by a catalyst-controlled radical combination involving two titanium(III) species. The resting state is identified as a cationic titanocene-nitrile complex and the beneficial effect of added Et3N·HCl on yield and enantioselectivity is elucidated: chloride coordination initiates the radical coupling. The results are fundamental for the understanding of titanium(III)-catalysis and of relevance for other metal-catalyzed radical reactions. Our conclusions might apply to a number of reductive coupling reactions for which conventional mechanisms were proposed before.

Oxidation and nitration of mononitrophenols by a DyP-type peroxidase.

Substantial conversion of nitrophenols, typical high-redox potential phenolic substrates, by heme peroxidases has only been reported for lignin peroxidase (LiP) so far. But also a dye-decolorizing peroxidase of Auricularia auricula-judae (AauDyP) was found to be capable of acting on (i) ortho-nitrophenol (oNP), (ii) meta-nitrophenol (mNP) and (iii) para-nitrophenol (pNP). The pH dependency for pNP oxidation showed an optimum at pH 4.5, which is typical for phenol conversion by DyPs and other heme peroxidases. In the case of oNP and pNP conversion, dinitrophenols (2,4-DNP and 2,6-DNP) were identified as products and for pNP additionally p-benzoquinone. Moreover, indications were found for the formation of random polymerization products originating from initially formed phenoxy radical intermediates. Nitration was examined using (15)N-labeled pNP and Na(14)NO2 as an additional source of nitro-groups. Products were identified by HPLC-MS, and mass-to-charge ratios were evaluated to clarify the origin of nitro-groups. The additional nitrogen in DNPs formed during enzymatic conversion was found to originate both from (15)N-pNP and (14)NO2Na. Based on these results, a hypothetical reaction scheme and a catalytically responsible confine of the enzyme's active site are postulated.

The toolbox of Auricularia auricula-judae dye-decolorizing peroxidase - Identification of three new potential substrate-interaction sites.

Dye-decolorizing peroxidases (DyPs) such as AauDyPI from the fungus Auricularia auricula-judae are able to oxidize substrates of different kinds and sizes. A crystal structure of an AauDyPI-imidazole complex gives insight into the binding patterns of organic molecules within the heme cavity of a DyP. Several small N-containing heterocyclic aromatics are shown to bind in the AauDyPI heme cavity, hinting to susceptibility of DyPs to azole-based inhibitors similar to cytochromes P450. Imidazole is confirmed as a competitive inhibitor with regard to peroxide binding. In contrast, bulky substrates such as anthraquinone dyes are converted at the enzyme surface. In the crystal structure a substrate analog, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), binds to a tyrosine-rich hollow harboring Y25, Y147, and Y337. Spin trapping with a nitric oxide donor uncovers Y229 as an additional tyrosine-based radical center in AauDyPI. Multi-frequency EPR spectroscopy further reveals the presence of at least one intermediate tryptophanyl radical center in activated AauDyPI with W377 as the most likely candidate.

Mechanistic investigations of the rhodium catalyzed propargylic CH activation.

Previously we reported the redox-neutral atom economic rhodium catalyzed coupling of terminal alkynes with carboxylic acids using the DPEphos ligand. We herein present a thorough mechanistic investigation applying various spectroscopic and spectrometric methods (NMR, in situ-IR, ESI-MS) in combination with DFT calculations. Our findings show that in contrast to the originally proposed mechanism, the catalytic cycle involves an intramolecular protonation and not an oxidative insertion of rhodium in the OH bond of the carboxylic acid. A σ-allyl complex was identified as the resting state of the catalytic transformation and characterized by X-ray crystallographic analysis. By means of ESI-MS investigations we were able to detect a reactive intermediate of the catalytic cycle.

Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits.

Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.

Radical formation on a conserved tyrosine residue is crucial for DyP activity.

Dye-decolorizing peroxidases (DyPs) are able to cleave bulky anthraquinone dyes. The recently published crystal structure of AauDyPI reveals that a direct oxidation in the distal heme cavity can be excluded for most DyP substrates. It is shown that a surface-exposed tyrosine residue acts as a substrate interaction site for bulky substrates. This amino acid is conserved in eucaryotic DyPs but is missing in the structurally related chlorite dismutases (Clds). Dye-decolorizing peroxidases of procaryotic origin equally possess a conserved tyrosine in the same region of the polypeptide albeit not at the homologous position.

First crystal structure of a fungal high-redox potential dye-decolorizing peroxidase: substrate interaction sites and long-range electron transfer.

Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.1 Å resolution. The mostly helical structure also shows a ß-sheet motif typical for DyPs and Cld (chlorite dismutase)-related structures and includes the complete polypeptide chain. At the distal side of the heme molecule, a flexible aspartate residue (Asp-168) plays a key role in catalysis. It guides incoming hydrogen peroxide toward the heme iron and mediates proton rearrangement in the process of Compound I formation. Afterward, its side chain changes its conformation, now pointing toward the protein backbone. We propose an extended functionality of Asp-168, which acts like a gatekeeper by altering the width of the heme cavity access channel. Chemical modifications of potentially redox-active amino acids show that a tyrosine is involved in substrate interaction. Using spin-trapping experiments, a transient radical on the surface-exposed Tyr-337 was identified as the oxidation site for bulky substrates. A possible long-range electron transfer pathway from the surface of the enzyme to the redox cofactor (heme) is discussed.

Oxidative cleavage of α-aryl aldehydes using iodosylbenzene.

We found that α-aryl aldehydes can be cleaved to chain-shortened carbonyl compounds and formaldehyde by various iodosylbenzene complexes. A mechanistic scheme is presented that accounts for the loss of one carbon atom. Formaldehyde is further oxidized to CO and CO(2) under the reaction conditions.

[P9]+ [Al(OR(F))4)]-, the salt of a homopolyatomic phosphorus cation.

Positive at last: The first condensed-phase homopolyatomic phosphorus cation [P(9)](+) was prepared using a combination of the oxidant [NO](+) and weakly coordinating anion, [Al{OC(CF(3))(3)}(4)](-). [P(9)](+) consists of two P(5) cages linked by a phosphonium atom to give a D(2d)-symmetric Zintl cluster. NMR (see picture), Raman, and IR spectroscopy, mass spectrometry, and quantum-chemical calculations confirmed the structure.

Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron.

Some litter-decaying fungi secrete haem-thiolate peroxygenases that oxidize numerous organic compounds and therefore have a high potential for applications such as the detoxification of recalcitrant organic waste and chemical synthesis. Like P450 enzymes, they transfer oxygen functionalities to aromatic and aliphatic substrates. However, in contrast to this class of enzymes, they only require H(2)O(2) for activity. Furthermore, they exhibit halogenation activity, as in the well characterized fungal chloroperoxidase, and display ether-cleavage activity. The major form of a highly glycosylated peroxygenase was produced from Agrocybe aegerita culture media, purified to apparent SDS homogeneity and crystallized under three different pH conditions. One crystal form containing two molecules per asymmetric unit was solved at 2.2 A resolution by SAD using the anomalous signal of the haem iron. Subsequently, two other crystal forms with four molecules per asymmetric unit were determined at 2.3 and 2.6 A resolution by molecular replacement.

Online ESI-MS analysis of reactions under high pressure.

A new approach for real-time mass spectrometric analysis and identification of intermediates in confined, pressurized vessels is presented. The high-pressure reaction media is depressurized, mixed with a solvent, and fed directly to the atmospheric pressure electrospray ionization source of a tandem mass spectrometer. The method was applied to track product evolution for the dehydration of hydroxenin monoacetate, a commercially, important vitamin A precursor, and for the hydrogenation of 5-norbornene-2-carbonitrile with Pd/CaCO(3) as catalyst. Faster access but comparable results as by conventional off-line methods were obtained in every case.