Structure elucidation of chlorophyll catabolites (phyllobilins) by ESI-mass spectrometry—Pseudo-molecular ions and fragmentation analysis of a nonfluorescent chlorophyll catabolite (NCC)

AbstractThe hyphenation of high performance chromatography with modern mass spectrometric techniques providing high-resolution data as well as structural information from MS/MS experiments has become a versatile tool for rapid natural product identification and characterization. A recent application of this methodology concerned the investigation of the annually occurring degradation of green plant pigments. Since the first structural elucidation of a breakdown product in the early 1990s, a number of similarly structured, tetrapyrrolic catabolites have been discovered with the help of chromatographic, spectroscopic and spectrometric methods. A prerequisite for a satisfactory, manually operated or database supported analysis of mass spectrometric fragmentation patterns is a deeper knowledge of the underlying gas phase chemistry. Still, a thorough investigation of the common fragmentation behavior of these ubiquitous, naturally occurring chlorophyll breakdown products is lacking. This study closes the gap and gives a comprehensive overview of collision-induced fragmentation reactions of a tetrapyrrolic nonfluorescent chlorophyll catabolite, which is intended to serve as a model compound for the substance class of phyllobilins

Ortsaufgelöster massenspektrometrischer Nachweis und Strukturaufklärung biologischer Metaboliten in Pflanzen und Pilzen

Schwerpunkt dieser Arbeit lag auf der Entwicklung und Optimierung von massenspektrometrischen Verfahren zur zweidimensionalen Darstellung von organischen Naturstoffen und der Strukturaufklärung unbekannter biologischer Kataboliten mittels Massenspektrometrie. Die Methoden wurden an zwei Systemen mit unterschiedlichen Anforderungen erprobt. Im ersten Teil dieser Arbeit wurde ein neues MALDI-Matrix Beschichtungsverfahren für stark wasserhaltige Proben entwickelt. Dieses basierte auf der Anwendung einer Suspensionssprühmethode in Kombination mit einer optimierten Probenpräparation. Die dadurch erzielte Verbesserung der Matrixbedeckung führte zu einer relativen Signalverstärkung in MALDI-MSI Ionenbilder von mindestens 300 Prozent im Vergleich zu herkömmlichen Beschichtungsverfahren. Gleichzeitig konnte das Auftreten probenschädigender Prozessen („sample flaking“) deutlich minimiert werden. Das Problem der vergleichsweise hohen Nachweisgrenze sowie des geringen lateralen Auflösungsvermögens bei herkömmlichen mikrobiellen MALDI-MSI Verfahren [33] konnte somit erfolgreich gelöst werden. Das Verfahren erlaubte die sensitive Detektion des Siderophors desferri-Triacetylfusarinin (dfTAFC) in Agarosegel-Proben aus Schimmelpilzkulturen (Aspergillus fumigatus) mit hohem lateralen Auflösungsvermögen. Der zweite Teil dieser Arbeit befasste sich mit dem ortsaufgelösten, massenspektrometrischen Nachweis (MALDI-TOF-MSI, Abdruckverfahren) sowie der Strukturaufklärung von Chlorophyllabbaustoffen in pflanzlichen Proben am Beispiel des heimischen Adlerfarns (Pteridium aquilinum). Es ist gelungen, drei bisher unbekannte, nichtfluoreszierende Chlorophyllabbauprodukte aus dem Adlerfarn (Pteridium aquilinum) zu isolieren (Pa-NCC-1 bis Pa-NCC-3). Alle Kataboliten zeigten ein von bisher beschriebenen NCCs (z. B. Cj-NCC-1) abweichendes Verhalten in spektroskopischen sowie spektrometrischen Experimenten. Die Kombination der erhaltenen Daten ermöglichte die Erstellung vorläufiger Strukturvorschläge aus 2D-NMR, Msn, UV/Vis- und CD-Experimenten. Die Untersuchungen ergaben, dass alle Kataboliten (mindestens) zwei bisher nicht beobachtete Struktureigenschaften besitzen: (i) an Position C82 ist die in anderen NCCs beschriebene Methylester-Funktionalität durch ein Wasserstoffatom ersetzt (ii) (Poly-)hydroxylierungen(en) an den Kohlenstoffatomen im Ringsystem B/E und C (mono-Hydroxylierung an Position C6 im Fall von Pa-NCC-1 und Pa-NCC-3; dreifach-Hydroxylierung an undefinierten Positionen im Fall von Pa-NCC-2 (IG) Die experimentellen Befunde (UV/Vis, CD) für die vorgeschlagenen Strukturen von Pa-NCC-1 und Pa-NCC-3 stehen im Einklang mit Resultaten aus DFT-basierten Simulationen. Darüber hinaus zeigten HPLC-ESI-MSn Experimente neuartige Fragmentierungsmuster für alle Pa-NCCs (collision induced dissociation, CID), welche vom Fragmentierungsmuster bekannter, (linearer) tetrapyrrolischer Chlorophyllkataboliten abweichen.main objective of this work was the development and optimization of mass spectrometry based methodology for spatially resolved detection and structure elucidation of catabolites in plants and fungi. In the first part of this work the problem of MALDI-matrix application onto samples with high water content is addressed. Therefore a novel coating technique based on a matrix suspension-spray was developed. In combination with an improved sample-preparation process a drastic improvement concerning sensitivity and lateral resolution in MALDI-MSI images could be accomplished. Common sample degradation processes (sample flaking) were thereby minimized and the sensitive and reliable detection of the siderophore desferri-tricacetylfusarinine (dfTAFC) in agar-grown samples of Aspergillus fumigatus was shown. In the second part of this work 2D resolved mass-spectrometry (MALDI-MSI) was utilized to identify chlorophyll catabolites in fern (Pteridium aquilinum). HPLC-MSn investigations of plant extracts were performed and three novel nonfluorescent chlorophyll catabolites (NCCs) were found (Pa-NCC-1, Pa-NCC-2 (IG), Pa-NCC-3). It was possible to elucidate chemical structures for Pa-NCC-1 and Pa-NCC-3 by implementing data from 2D-NMR, MSn and UV/Vis- and CD-experiments. The results indicated two novel structural features: (i) at position C82 the methylester-functionality is substituted by a proton (ii) (Poly-)hydroxylation(s) at carbon atom(s) in ring B/E and ring C (mono-hydroxylation at C6 in case of Pa-NCC-1/Pa-NCC-3; triple-hydroxylation at undefined positions in case of Pa-NCC-2 (IG)) The experimental findings (UV/Vis, CD) for the proposed structural features of Pa-NCC-1 and Pa-NCC-3 were in accordance with results from DFT-based simulations. Furthermore, some HPLC-ESI-MSn experiments revealed novel fragmentation patterns for collision induced dissociation of Pa-NCCs compared to that of known linear tetrapyrrolic chlorophyll catabolites [47].vorgelegt von Mag. rer. nat. Stefan VergeinerAbweichender Titel laut Übersetzung der VerfassersZusammenfassung in englischer SpracheKumulative Dissertation aus Fünf ArtikelnUniversität Innsbruck, Dissertation, 2016OeBB(VLID)89373

Cryptic chlorophyll breakdown in non-senescent green Arabidopsis thaliana leaves

Chlorophyll (Chl) breakdown is a diagnostic visual process of leaf senescence, which furnishes phyllobilins (PBs) by the PAO/phyllobilin pathway. As Chl breakdown disables photosynthesis, it appears to have no role in photoactive green leaves. Here, colorless PBs were detected in green, non-senescent leaves of Arabidopsis thaliana. The PBs from the green leaves had structures entirely consistent with the PAO/phyllobilin pathway and the mutation of a single Chl catabolic enzyme completely abolished PBs with the particular modification. Hence, the PAO/phyllobilin pathway was active in the absence of visible senescence and expression of genes encoding Chl catabolic enzymes was observed in green Arabidopsis leaves. PBs accumulated to only sub-% amounts compared to the Chls present in the green leaves, excluding a substantial contribution of Chl breakdown from rapid Chl turnover associated with photosystem II repair. Indeed, Chl turnover was shown to involve a Chl a dephytylation and Chl a reconstitution cycle. However, non-recyclable pheophytin a is also liberated in the course of photosystem II repair, and is proposed here to be scavenged and degraded to the observed PBs. Hence, a cryptic form of the established pathway of Chl breakdown is indicated to play a constitutive role in photoactive leaves

Air Pollution Transport in an Alpine Valley: Results From Airborne and Ground-Based Observations

An observational dataset from a wintertime field campaign in the Inn Valley, Austria, is analysed in order to study mechanisms of air pollution transport in an Alpine valley. The results illustrate three types of mechanisms: transport by a density current, back-and-forth transport by valley winds, and transport by slope winds. The first type is associated with an air mass difference along the valley. Cooler air located in the lower part of the valley behaves like a density current and produces the advection of pollutants by upvalley winds. In the second type, strong horizontal gradients in pollution concentrations exist close to ground. Multiple wind reversals result in a back-and-forth transport of pollutants by weak valley winds. In the third type, upslope winds during daytime decrease low-level pollution concentrations and cause the formation of elevated pollution layers

2-Azidoimidazolium Ions Captured by N-Heterocyclic Carbenes: Azole-Substituted Triazatrimethine Cyanines

1,3-Disubstituted 2-azidoimidazolium salts (substituents = methyl, methoxy; anion = PF6) reacted with N-heterocyclic carbenes to yield yellow 2-(1-(azolinylidene)triazen-3-yl)-1,3-R2-imidazolium salts (azole = 1,3-dimethylimidazole, 1,3-dimethoxyimidazole, 4-dimethylamino-1-methyl-1,2,4-triazole; R = methyl, methoxy; anion = PF6). Crystal structures of three cationic triazenes were determined. Numerous interionic C–H···F contacts were observed. Solvatochromism of the triazenes in polar solvents was investigated by UV-Vis spectroscopy, involving the dipolarity π* and hydrogen-bond donor acidity α of the solvent. Cyclovoltammetry showed irreversible reduction of the cations to uncharged radicals. Thermoanalysis showed exothermal decomposition