Metabolic pathway monitoring of phenalinolactone biosynthesis from Streptomyces sp. Tü6071 by liquid chromatography/mass spectrometry coupling.

RATIONALE: A rapid and precise analytical method for the investigation of natural products is required for pathway monitoring of the biosynthesis of secondary metabolites. Phenalinolactones, used in antibiotic research, are produced by Streptomyces sp. Tü6071. For the analysis of those compounds, prior to mass spectrometric analysis, an efficient separation technique is required. METHODS: For the identification of phenalinolactones from liquid cultures of Streptomyces sp. Tü6071, a new method comprising the combination of solid-phase extraction (SPE) prior to liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was established. MS/MS product ion scans were applied for phenalinolactone detection and structure elucidation, performed in negative mode and optimized for sensitivity and specificity. For the discovery of new intermediates, a MS/MS precursor ion scan was applied. RESULTS: Analysis of the extracts revealed that the Oasis® MAX cartridge, containing a quaternary amine functionality, is the most efficient SPE material for purification of phenalinolactones, since it allowed sufficient enrichment and detection of intermediates from the biosynthetic pathway by LC/ESI-MS/MS. Using the precursor ion scan technique, two new secondary metabolites, PL IM1 with m/z 672.6 and PL IM2 with m/z 433.3, have been detected. The structures of the new intermediates are postulated and arranged into the biosynthetic pathway of phenalinolactones. CONCLUSIONS: A precise analytical method was established for the identification of phenalinolactones by combining purification from Streptomyces using SPE prior to LC/ESI-MS/MS. By optimising LC/ESI-MS/MS settings, this method has been successfully applied for pathway monitoring of secondary metabolites. Application of a precursor ion scan allowed for the identification of unknown intermediates in biosynthetic pathways.

Metallophilic bonding and agostic interactions in gold(I) and silver(I) complexes bearing a thiotetrazole unit.

Gold(I) and silver(I) complexes of 1-methyl-5-thio-tetrazole (1) have been prepared and the coordination chemistry of this ligand toward metal-phosphine frameworks has been explored. As indicated by IR and Raman data, ligand 1 is deprotonated and the resulted anion acts as a bidentate (S,N)-tetrazole-5-thiolato unit in the new gold(I) complexes, [Au(SCN(4)Me)(PPh(3))] (2), [{Au(SCN(4)Me)}(2)(µ-dppm)] (3), and [{Au(SCN(4)Me)}(2)(µ-dppe)] (4), while it is coordinated only through the sulfur atom as its neutral tetrazole-5-thione form in the silver(I) derivative, [Ag(HSCN(4)Me)(PPh(3))](2)(OTf)(2) (5). Further characterization of the new compounds was performed using multinuclear ((1)H, (13)C, (31)P, (19)F) NMR spectroscopy, mass spectrometry, and DSC measurements. Single-crystal X-ray diffraction studies revealed basically linear P-M-S arrangements in complexes 3-5. The bidentate (S,N) coordination pattern results in a T-shaped (S,N)PAu core in 3 and 4, whereas, in 5, a similar coordination geometry is achieved in the dimer association based on S-bridging ligand 1. Herein, weak (C)H···Au and (C)H···Ag agostic interactions were observed. An intramolecular Au···Au contact occurs in 3, while in 4 intermolecular aurophilic bonds lead to formation of a chain polymer. An intermolecular Ag···Ag contact is also present in the dimer unit of 5. Low-temperature (31)P NMR data for 5 evidenced the presence of monomer and dimer units in solution. Theoretical calculations on model of the complexes 2 and 4 are consistent with the geometries found by X-ray diffraction studies.

Absolute Configurations and Sensory Properties of the Stereoisomers of a Homologous Series (C6-C10) of 2-Mercapto-4-alkanols.

A homologous series (C6-C10) of 2-mercapto-4-alkanols was obtained by the addition of thioacetic acid to the respective alkenones and subsequent reduction with LiAlH4. Gas chromatographic separation of the stereoisomers was achieved using chiral stationary phases. Their absolute configurations were assigned by the correlation of 1H NMR data and enzyme-catalyzed kinetic resolutions. Odor thresholds and odor qualities were determined by capillary gas chromatography/olfactometry. Compared to the odor qualities reported for the isomeric 4-mercapto-2-alkanols, the homologous series of 2-mercapto-4-alkanols lacked fruity, tropical notes. There was no consistent correlation between the configurations and the odor qualities. However, the observed odor thresholds indicated the importance of the configuration of the asymmetric center bearing the hydroxyl group and the alkyl substituent. The length of this alkyl chain is a main driver for the odor properties, ranging from pungent, vegetable to earthy, mushroom notes.

Determination of the Absolute Configurations and Sensory Properties of the Enantiomers of a Homologous Series (C6-C10) of 2-Mercapto-4-alkanones.

The enantiomers of a homologous series (C6-C10) of 2-mercapto-4-alkanones were obtained by lipase-catalyzed kinetic resolution of the corresponding racemic 2-acetylthio-4-alkanones. Their configurations were assigned via vibrational circular dichroism and 1H NMR anisotropy based methods. Odor thresholds and odor qualities were determined by capillary gas chromatography-olfactometry using chiral stationary phases. There were minima of the odor thresholds for the chain lengths C7 and C8. Except for chain length C8, the enantiomers of the other homologues showed similar odor thresholds. The odor qualities ranged from pungent (C5) to mushroom (C9 and C10) and were similar to those known for the corresponding 1-alken-3-ones with one less C atom. In contrast to their positional isomers (4-mercapto-2-alkanones), the investigated 2-mercapto-4-alkanones do not meet the requirements of a "tropical olfactophore" (i.e., compounds possessing a 1,3-oxygen-sulfur functionality and specific arrangements of the substituents).

Distributions of the Stereoisomers of ß-Mercaptoheptanones and ß-Mercaptoheptanols in Cooked Bell Pepper (Capsicum annuum).

2-Mercapto-4-heptanone, 4-mercapto-2-heptanone, and the corresponding mercaptoalcohols, previously identified in cooked red bell pepper (Capsicum annuum), were used as examples to determine the distributions of stereoisomers of naturally occurring polyfunctional thiols. The thiols were isolated using simultaneous distillation-extraction and enriched by affinity chromatography. Enantioselective analysis was performed via multidimensional gas chromatography. For the studied cultivar California Wonder, the investigation of different batches of cooked red bell pepper revealed consistent ratios of the stereoisomers independent of origin and date of purchase. Quantitative estimations showed that the stereoisomers were present in cooked red bell peppers at concentrations in the range of 0.04-10.2 µg/kg. Lower concentrations were observed in cooked green bell peppers. The change from green to red color was also accompanied by shifts in the proportions of stereoisomers in favor of the (S)-enantiomers of the mercaptoheptanones and of the (4S)-configured stereoisomers of 4-mercapto-2-heptanol.

Analysis and Sensory Evaluation of the Stereoisomers of a Homologous Series (C5-C10) of 4-Mercapto-2-alkanols.

A homologous series of 4-mercapto-2-alkanols (C5-C10) was used to investigate the impact of the stereochemistry on the sensory properties of a class of naturally occurring polyfunctional thiols having a 1,3-oxygen-sulfur functionality. Stereoisomers were obtained via syntheses of racemic mixtures and subsequent lipase-catalyzed kinetic resolutions. Analytical separations of the stereoisomers were achieved by capillary gas chromatography (GC) using chiral stationary phases. The absolute configurations were assigned via NMR analysis. Sensory evaluations by means of GC/olfactometry revealed odor threshold minima for the medium-chain homologues (C7-C9) of the 4-mercapto-2-alkanol stereoisomers. Except for the C5 homologue, the lowest odor thresholds were determined for the (2R,4R)-configured stereoisomers. The variability in odor qualities was mainly determined by the chain length. None of the 4-mercapto-2-alkanol stereoisomers showed consistent odor qualities for all homologues.

Reinvestigation of the Absolute Configurations of Chiral ß-Mercaptoalkanones Using Vibrational Circular Dichroism and 1H NMR Analysis.

The absolute configurations of chiral ß-mercaptoalkanones were previously assigned on the basis of the 1H NMR anisotropy method using (S)-2-methoxy-2-(1-naphthyl)propionic acid ((S)-MαNP) as the chiral auxiliary. This study presents a reinvestigation of the configurations of 4-mercapto-2-pentanone 1, 4-mercapto-2-heptanone 2, and 2-mercapto-4-heptanone 3. Enantiomers of 1, 2, and 3 were obtained by lipase-catalyzed hydrolyses of the respective acetylthioalkanones. Upon derivatization with (S)-MαNP, the configurations of the reaction products were deduced based on the order of the HPLC elution of the diastereoisomeric thioesters, assuming that the sector rule previously developed for secondary alcohols is also valid for thiols. In addition, the configurations were experimentally determined by vibrational circular dichroism (VCD) and 1H NMR analyses after esterification with (R)-hydratropic acid (HTA) and 2-methoxy-2-phenylacetic acid (MPA). The assignments of the configurations using VCD and NMR analyses of HTA- and MPA-thioesters were in agreement. However, they were opposite to those deduced for (S)-MαNP thioesters via the sector rule. Consequently, the formerly assigned configurations of ß-mercaptoalkanones deduced via investigation of (S)-MαNP-derivatives have to be revised.