Metabolomics for Natural Products: Fast screening and Discovery Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller- Universität Jena von Mayuri Tharanga Napagoda, M.Phil geboren am 21. November 1976 in Negombo, Sri Lanka Beginn der Promotion : 03. März 2010 Eingereicht am : 14. Oktober 2013 Tag der Verteidigung : 16. September 2014 Gutachter: Dr. Aleš Svatoš, Max Planck Institute for Chemical Ecology, Jena, Germany Prof. Dr. Georg Pohnert, Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University of Jena, Germany Prof. Dr. Wolfram Weckwerth, University of Vienna, Austria CONTENTS Chapter 1. General Introduction…………………………………........................... 1 Chapter 2. Azahelicene Superbases as MAILD Matrices for acidic analytes ……. …………………………………………………………. 21 Chapter 3. Inhibition of 5-lipoxygenase as anti-inflammatory mode of action of Plectranthus zeylanicus Benth and chemical characterization of ingredients by mass spectrometric approach ……………………………………………………………….. 63 Chapter 4. Munronia pinnata (Wall.) Theob: Unveiling phytochemistry and dual inhibition of 5-lipoxygenase and microsomal prostaglandin E2 synthase (mPGES)-1……….……………………….... 80 Chapter 5. Identification of female specific fatty acid derivatives in Drosophila melanogaster surface lipid extracts…………………… ..... 94 Chapter 6. Discussion …………………………………………………………….. 118 Chapter 7. Summary………………………………………………………………. 131 Chapter 8. References …………………………………………………………….. 137 Appendices ……………………………………………………………………......... 147 Eigenständigkeitserklärung ..................................................................................... (i) Erklärung über laufende und frühere Promotionsverfahren .................................... (i) Curriculum Vitae ..................................................................................................... (ii) Acknowledgements.................................................................................................. (vi) Chapter 1 General Introduction General Introduction 1.1 Natural products : Small molecules with big roles Natural products, the remarkable collection of low molecular weight compounds made by living organisms have been exploited for human use for thousands of years and have made important contributions to organic chemistry, biology, medicine, agriculture and numerous other fields. Throughout history, humans have displayed a great enthusiasm and interest in naturally occurring compounds from microbial, plants and animals sources. Early man realized that many plants contain components with powerful biological effects and thus utilized them as a major resource for treating and preventing diseases. This formed the basis of sophisticated traditional medicinal practices that are still popular among many communities around the world (Clardy and Fischbach, 2008). After centuries of empirical use of herbal preparations, the first isolation of the isoquinoline alkaloid, morphine in the early 19th century marked a new era in the use of medicinal plants and the beginning of modern medicinal plant research (Samuelsson, 2004). Subsequently, many valuable drugs like atropine, cocaine, codeine, digitoxin, quinine, etc. came into use through the study of indigenous remedies. These discoveries made significant contribution to the development of organic and medicinal chemistry (Sneader, 2005). T he discovery and the development of penicillin as a microbial metabolite was another breakthrough in this field and opened up t he era of antibiotics, sav ing countless lives in the last century. Isolation, concentration, purification and mass production of penicillin was followed by the development of streptomycin, tetracycline, chloramphenicol and many other antibacterial agents w hose origin in most cases could be traced to naturally occurring sources (Dax, 1997 ). Furthermore, t he isolation and synthesis of steroid hormones as well as the discovery of bioactive lipids such as prostaglandins and leukatrienes ha ve made a big impact on modern medicine and also on the development of novel concepts in molecular biology ( Ogura, 1997 ). Despite the recent interest in other drug discovery approaches such as molecular modeling, combinatorial chemistry, and other synthetic chemistry methods, natural products are still providing their fair share of new clinical candidates and drugs (Butler, 2004). About half of the drugs currently in clinical use are based on natural product scaffolds (Newman and Cragg, 2007; Harvey, 2008). These compounds are derived directly, by use of semi-synthetic natural product analogs, or indirectly- through the use of synthetic compounds based upon natural product pharmacophores. Therefore natural-product-derived compounds are still proving to be an invaluable source of medicines for humans. Natural products often have an ecological role in regulating the interactions between plants, micro-organisms, insects and animals and act as defensive substances, anti- feedants, attractants, pheromones etc. The concept of “natural pesticides” arose early in the development of agriculture (Dayan, et al., 2009) and the use of natural product and natural product–derived insecticides continue to increase nowadays. These compounds be touted as attractive alternatives to synthetic chemical insecticides for pest management due to their minimum threat to the environment and to the human health (Isman, 2006). The discovery of botanical insecticidal powders from Chrysanthemum cinearifolium flower heads and Derris root which contain pyrethrum and rotenone, respectively formed the basis of commercial insecticides (Dayan, et al., 2009). Azadirachtin isolated from the Indian neem tree, Azadirachta indica is another well known potent antifeedant to many insects, which could block the synthesis and release of molting hormones leading to incomplete ecdysis in immature insects. (Isman, 2006). Today azadirachtin based products are well established commercially and extensively used as organic insecticides against wide range of insects. The idea of using naturally-occurring compounds as herbicides has ancient origins, however, natural herbicides have not gained much popularity mainly because of their little or no selectivity as well as the need of being applied in large quantities. Nevertheless the quest for such compounds has taken on renewed vigor in recent years with the concept of organic farming. Several essential oils such as lemon grass oil, clove oil, citronella oil have been commercialized and act as non-selective, contact herbicides. Although “allelopathy” does not involve the direct application of natural products for weed management, specific allelochemicals for example Sorgolene, Momilactone, Benzoxazinoids, etc. have been identified as being the primary molecules involved in weed control by crops (Dayan, et al., 2009). Biological control of plant diseases and plant pathogens is of great significance in forestry and agriculture. In crop production, over half of potential crop yield is lost due to plant pathogens, and in storage, up to one third of the harvest product can be lost due to post-harvest diseases, mostly as a result of activities of fungi (Prescott, et al, 1996). The excessive use of synthetic chemicals to combat such situations has been counterproductive, causing damage to the environment and humans while the continuing development of fungicide resistance in plant pathogens necessitates the discovery and development of new fungicides which are eco-friendly (Martinez, 2012). Many natural compounds and preparations have been described with activity against bacterial or fungal plant pathogens and some are available in the market for the management of plant diseases in organic agriculture. Several plant essential oils for example jojoba oil, rosemary oil, thyme oil have been marketed as fungicides for organic farmers while an extract of the giant knotweed (Reynourtria sachalinensis) is used in Europe for the control of a broad spectrum of both fungal and bacterial plant diseases in both organic and non-organic agriculture. In addition, fermentation secondary products from actinomycetes (mostly Streptomyces spp.) have been commercialized and used extensively as agricultural fungicides in several countries (Dayan, et al., 2009). Natural products often serve central roles as biological signaling agents, usually referred to as “semiochemicals”, and have been widely considered within various integrated pest management (IPM) strategies. These compounds are now recognized as performing a multitude of vital functions including serving as quorum sensing agents among bacteria, as algal and fungal gamete attractants, as sex attractants and alarm pheromones in many insect species, as attractants to plant pollinating organisms, as plant and animal defensive chemicals, etc. (Meinwald, 2011). Among the diverse forms of semiochemicals, pheromones, the compounds involved in intra-specific communications, have been subjected to extensive studies in the recent past. The first isolation and identification of an insect pheromone, bombykol ((10E,12Z)-10,12-hexadecadien-1-ol) from the female silk worm moth Bombyx mori occurred in 1959. However the existence of insect pheromones has been known for centuries, apparently originating in observations of mass bee stinging in response to a chemical released by the sting of a single bee (Flint and Doane, 1996). Since then, hundreds or perhaps thousands of insect pheromones might have been identified. Use of pheromones for pest control promises to be an important component of the ongoing challenge to develop alternatives that could help to solve problems associated with chemical pesticides (Kirsch, 1988). Therefore, understanding Nature’s chemical language has a great potential in agriculture. Taking all the above mentioned aspects together, Nature has created secondary metabolites for a plethora of roles and humans have always inquisitively attempted to harness these benefits. Thus, applications of natural products chemistry have become all-pervasive in modern society and the continuous quest to identify the myriad secondary metabolites demands the availability of highly selective, sensitive, precise, accurate, reproducible and efficient analytical techniques. 1.2. Old tools in natural products chemistry The complex nature of natural products usually requires maximum performance from the sample preparation, separation and identification methods and these tedious procedures are the main bottle-neck in natural products chemistry (Casas, 2009). In order to obtain secondary metabolites from biogenic materials, the first step is to release them from the matrix by means of extraction (Cannell, 1998). The choice of extraction method is of great importance as an incorrect approach would not allow the release of all of the desired components. The conventional extraction methods involve the use of solvents, either organic or aqueous while supercritical fluid extraction (SFE) is a more recent method whose application has steadily increased. However, the current trends in extraction techniques have largely focused on finding solutions that minimize the use of solvents (Casas, 2009). Isolation of natural products generally combines various separation techniques depending on the solubility, volatility and stability of the compounds to be separated. The choice of different separation steps and an analytical-scale optimization of the separation parameters are of great importance for the maximum outcome (Sticher, 2008). The conventional natural product isolation strategies consist of extremely time consuming and technically demanding multi-step purifications. These involve techniques like preparative thin layer chromatography, conventional column chromatography, flash chromatography, medium pressure liquid chromatography, vacuum liquid chromatography, high performance liquid chromatography etc. often performed with the purpose of obtaining pure compounds for structure elucidation. Once the purification is completed, several spectroscopic techniques such as nuclear magnetic resonance (NMR), ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), and X-ray crystallography are utilized in the identification and characterization of natural products. As these conventional approaches require substantial amount of purified compound to solve the puzzle of the compounds’ structural identity, the structural characterization of minor components in a bioactive fraction appears unfeasible. In bio-activity guided fractionation approaches, the major disadvantage is the inherent risk of re-isolating known or trivial constituents. This necessitates the development of new methodologies that could provide detailed structural information about particular constituents directly from the extract before investing in isolation and purification (Jarosewski, 2004; Hostettmann, et al., 2001). If the structures of the extract constituents were known in advance, the isolation efforts could be focused only on novel and interesting compounds and this would increase the efficiency of the whole process. Since scarcity of the plant materials restricts large scale extraction and isolation procedures, novel techniques that could provide detailed structural information directly from the crude extract or less purified fractions of the crude extracts are essential for the phytochemical studies of rare or endangered medicinal plants. However, with the outstanding developments in the areas of separation methods, spectrometric techniques, and sensitive bioassays, natural products research has gained momentum in recent years (Sticher, 2008). These novel approaches afford for a rapid identification and characterization of secondary metabolites without the necessity of isolation and purification while the detailed information about their metabolic profiles can be obtained with a minimal amount of material. A detailed discussion on novel approaches in natural products chemistry is beyond the scope of this thesis, therefore only a brief discussion on several mass spectrometric approaches that have demonstrated great potential in fast and efficient exploration of secondary metabolites are presented here. 1.3 Mass spectrometry in natural product analysis Recent advancements in mass spectrometry and novel separation techniques have remarkably widened their applications to the analysis of complex biomaterials and established a new paradigm in natural products research. With the availability of a number of modern sophisticated hyphenated techniques, such as gas chromatography–mass spectrometry (GC-MS), liquid chromatography–mass spectrometry (LC-MS), liquid chromatography with parallel nuclear magnetic resonance spectroscopy and mass spectrometry (LC-NMR-MS), and capillary electrophoresis-mass spectrometry (CE-MS), the pre-isolation analysis of crude extracts or fractions from different natural matrices, isolation, online detection and dereplication of natural products, studies on chemotaxonomy and biosynthesis, chemical finger-printing, quality control of herbal products, and metabolomic studies have now become much easier than ever before (Sarker and Nahar, 2012; Patel, et al., 2010). In traditional mass spectrometric approaches in small molecule investigations, purified samples were subjected to high-energy (70 eV) electron ionization (EI) under high vacuum conditions (Hoffmann and Stroobant, 2007). Although this technique is applicable only for the analysis of thermally stable, low molecular weight volatile compounds, it produces consistent and fragment rich mass spectra which can be easily used for a mass spectral library search (Hocart, 2010). However, the low abundance or absence of molecular ion in the EI spectra in most of the situations appears to be problematic in the calculation of elemental composition. Therefore, a chemical ionization (CI) technique is employed to obtain molecular ion information though electron ionization is widely used in GC-MS setups (Kind and Fiehn, 2010). In spite of the power in online separation and identification, the penetration of GC- MS into the field of natural products chemistry has been restricted by the polar and ionic character as well as low volatility of majority of the natural products (Colegate and Molyneux, 2007). Therefore, the development of electrospray ionization (ESI) MS has marked a milestone in the analysis of natural products, as diverse classes of secondary metabolites are amenable to this method. As it can be directly coupled to a high performance liquid chromatography, ESI has turned in to be the ionization of choice for LC-MS in the identification and isolation of secondary metabolites from complex extracts (Cech and Enke, 2001; Xing, et al., 2007; Lim and Lord, 2002). The sample is sprayed into the ion source as a solution, where it is evaporated under atmospheric pressure in the presence of an electric field, charged ions so generated to be separated by the mass analyzer. In contrast to “hard” ionization techniques, ESI rarely generates fragments and the molecules are ionized by protonation, cationization, or deprotonation. ESI-MS experiments provide reliable evidence for the molecular weight of a compound and depending on the mass analyzer used, high accuracy in the determination of the molecular mass can be achieved from which the chemical formula and the number of double bonds, rings or heteroatoms can be inferred (Kind and Fiehn, 2010). Atmospheric pressure chemical ionization (APCI) is another “soft ionization” technique and is considered as an ideal method of ionization for low- to medium- polar compounds. In contrast to the strong electric field desolvation in ESI, in APCI the conversion of the solvent into an aerosol is brought about by thermal energy. Continuous vaporization of the aerosol gives rise to gas phase molecules before ionization is initiated. This mixture of gas phase analytes, solvent and atomizing gas is then subjected to a discharge current produced from the corona discharge needle due to the application of high voltage. This leads to the generation of charged plasma through a combination of collisions and charge transfer reactions. Ultimately protonation or deprotonation reactions would take place and usually some degree of fragmentation that is useful for structural characterization could also occur (Byrdwell, 2001). The “soft ionization” in the above mentioned atmospheric pressure ionization processes, specially in ESI, leads to little or no fragmentation, thus making it inconvenient for structural elucidation studies based on fragment ions. This issue was addressed by the emergence of tandem mass spectrometry (MS/MS), in which an ion (called precursor ion) from the first stage of MS is selected and activated, to produce fragment ions, which are then analyzed in the second stage of MS. Collision-induced dissociation (CID), which consists in promoting the energy-controlled collision of a chemically inert gas, with the precursor ion is the most widely employed ion activation method. In order to optimize the MS/MS spectrum, the collision energy may be chosen, since low collision energy values promote soft fragmentation and produce few fragments, whereas high collision energy values prompt extensive fragmentation (Dias, et al., 2012). The resulted fragment ions allow individual components of complex mixtures to be characterized, thus indicating the potential of LC-MS/MS in natural products chemistry. This technique reduces the need for laborious separation procedures since structural elucidation of the compounds can often be performed directly from crude plant extracts (Pachuta, et al., 1988). Although High Performance Liquid chromatography (HPLC) is an efficient analytical chromatographic technique for the separation of natural products in complex crude extracts, the recent introduction of ultra-HPLC (UHPLC) has provided new possibilities in liquid chromatography and demonstrated that it can advantageously replace existing HPLC methods for many applications, including quality control, profiling and fingerprinting, dereplication, and metabolomics (Eugster, et al., 2011). UHPLC results in a better separation while decreasing time and solvent consumption (Nováková, et al., 2006; Swartz, 2005) and UHPLC-MS/MS is considered as a high throughput analysis method in drug discovery compared to the normal HPLC-MS approaches (Chesnut and Salisbury, 2007). Unlike the fragmentation by electron ionization in GC-MS, the fragmentation resulted from LC-MS/MS is less reproducible hence spectral library searches are problematic. Furthermore, the manual interpretation of CID spectra is cumbersome and requires expert knowledge, thus development of computational methods for fully automated analysis of such data was highly in demand (Rasche, et al., 2011). The recently introduced automated method for annotating tandem MS data using a hypothetical fragmentation trees has opened a way to fast classification and identification of secondary metabolites. This approach resulted in hypothetical fragmentation trees in which nodes are annotated with molecular formulas of the fragments while the arcs represent fragmentation events. The automated comparison of fragmentation trees enables the automated analysis of large MS data sets for identifying unknown compounds and facilitates the understanding of secondary metabolism as well as the discovery of botanical therapeutics, biomarkers, signaling molecules etc. (Rasche, et al, 2012) Another soft ionization technique, matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF) has drawn much attention over the recent years as a successful approach in natural products analysis. The sample is premixed with a UV absorbing matrix compound, and exposed to laser pulses which leads to desorption and ionization of the analytes at low pressure. MALDI is considerably tolerant to salts, does not frequently require pretreatment and allows for high throughput analysis, thus, reinforce a logical rationale to utilize in the studies of secondary metabolites (Cohen, et al., 2007). With the various developments in the field of MALDI, an atmospheric pressure interface, AP-MALDI, has also been presented allowing for much experimental flexibility, as it could be coupled with any mass spectrometer equipped with atmospheric pressure ionization. (Hoffmann and Stroobant, 2007) Since its inception, MALDI - TOF has been considered to be a powerful tool in the analysis of large biomolecules like proteins, peptides and oligosaccharides. However, appli cation of MALDI - MS towards small molecule analysis lagged behind due to saturation by matrix ions signals below 500 Da . i n the spectrum. With the d evelopment of 1,8 - bis(dimethylamino)naphthalene (DMAN), a super - base belong ing to a class of compounds call ed “Proton s ponges ” as a novel matrix for the negative mode MALDI - MS analysis, the problem of low mass region interferences w as overcome and the potential of MALDI - TOF – MS technique for the analysis of low - molecular - weight compounds was well demonstrated (Shroff and Svatoš, 2009). Furthermore, there are several reports on successful application of MALDI-TOF-MS in identification of secondary metabolites in intact microorganisms without any extraction procedures. This approach allowed to speed up the process of dereplication of secondary metabolites and to gain the information on the distribution of secondary metabolites within an organism (Grube, et al., 2007). Thus, MALDI profiling offers exceptionally high-throughput and a strategy for accelerating research on natural products. Although only a few mass spectrometric approaches that facilitate rapid and efficient analysis of secondary metabolites have been summarized in this section, it is worthwhile to mention that there is an enormous number of other mass spectrometric techniques that have been successfully utilized in the field of natural products chemistry to reveal the mysteries of Nature. 1.4 Motivation for the thesis The recent developments in mass spectrometry has tremendously expanded the understanding of the chemistry as well as the biological functions of secondary metabolites, which is crucial in diverse fields from the pharmaceutical industry to agriculture, where there is an immense number of potential applications. Therefore, the overall aim of this thesis is to develop and optimize mass spectrometric approaches for fast screening and discovery of natural products in a wide range of samples of biological origin, thus to broaden the horizons in metabolomics. This overall aim is achieved under three themes and a brief explanation of the rationale for each theme is given below. Theme 1 - Development of novel MALDI matrices for metabolomic analysis Since conventional MALDI matrices produce a forest of interfering peaks at low mass region in the spectrum, 1,8 - bis(dimethylamino)naphthalene (DMAN) was developed as a novel matrix for the analysis of low molecular weight compounds (Shroff and Svatoš, 2009). DMAN is a super base belongs to a class of compounds called “Proton sponges”. This name comes from the ability of these compounds to take up any available protons. The presence of two basic nitrogen centers in the molecule, having an orientation that allows the uptake of one proton to yield a stabilized [N----H----N] + intra molecular hydrogen bond is considered as a general feature of all proton sponges (Raab, et al., 2002). Beside these classical “proton sponges”, a new class of proton sponges with exceptional basicities have also drawn the attention during the last two decades (Staab and Saupe, 1988). This group of compounds known as “Azahelicenes”, have prospective applications in fields like optoelectronics, catalysis, sensors, etc. (Caronna, et al., 2012), however, remained rather unexplored. As some of the azahelicenes posses high proton affinities comparable to classical proton sponges (Roithová, et al., 2007), it would be rather intersting to investigate the effectiveness of this group of compounds as novel MALDI matrices in terms of achieved sensitivity, ion-lessness and suitability for the analysis of anions. Thereby the applicability of MALDI-TOF-MS could be enhanced for the analysis of low-molecular weight acidic metabolites in wide range of biological samples. Theme 2 - Identification of bioactive secondary metabolites from medicinal plants in Sri Lanka Sri Lanka has a high biodiversity among its flora which comprises over 3700 angiosperms and over 350 ferns, of which over 28% of flowering plants and 18% of ferns are endemic to the island. Plants and their products are the main components of the indigenous medicinal system in the country which has been practiced for over thousand years and is still popular among people, even though modern health care facilities are readily available in most part of the country. It is reported that over 1400 plants are used in indigenous medicine in Sri Lanka and herbal drugs have gained much attention and popularity in recent years because of their safety, efficacy and cost effectiveness (Wijesundera, 2004). Despite the rich biodiversity and the vital role of plants in traditional medicine, Sri Lankan flora has not yet been adequately studied phytochemically or pharmacologically. The requirement for large scale extractions as well as laborious isolation and purification methods which are highly technically demanding have hindered the chemical profiling of medicinal plants, hence the validation of their uses in traditional medicine. However, with the emergence of novel hyphenated techniques that facilitate online characterization of secondary metabolites in crude natural product extracts, together with the newly developed computer algorithms for the de novo identification of organic compounds based on their tandem mass spectra, invaluable information regarding the presence of bioactive metabolites could be obtained. Thereby, the extensive use of plants in Sri Lankan traditional medicine could be rationalized. Theme 3 - Identification of sex dependent lipids in Drosophila melanogaster The investigations on surface lipids of Drosophila melanogaster were initiated several decades ago, for the characterization of the components and later extended towards the determination of quantitative and qualitative differences between cuticular lipids in male and female flies ( Jackson, et al., 1981). Thereafter , cuticular hydrocarbons in D. melanogaste r have been subjected to intensive studies and several of these compounds have displayed a marked sexual dimorphism, thus pheromonal functions (Antony and Jallon, 1982; Jallon, 1984; Ferveur and Sureau, 1996; Ferveur, 2005; Foley, et al., 2007). Apart fr om the cuticular hydrocarbons, the understanding o n other types of cuticular substances, e specially fatty acids and their derivatives in D. melanogaster is not adequate, despite over a quarter century of investigations. Previous studies conducted on cuticu lar fatty acid s ha ve only revealed quantitative differences between fatty acid composition in male and female flies . No qualitative differences were observed however ( Jackson, et al., 1981). To explore this sparsely known area and conduct in - depth studies on the cuticular non - hydro carbo n components that exist in minor quantities, advanced analytical approaches are desirable . N ovel mass spectrometric techniques specially the UHPLC - APCI - MS , facilitate the detect ion and identif ication of even minor components in a complex surface lipid extract thus enabl ing the conduction of comprehensive studies on sex specific fatty acids and their derivatives . This c ould lead the field of insect cuticular chemistry to a new dimension . To be concise, under the above mentioned themes, this thesis addresses different aspects in the field of natural products chemistry and demonstrates the capability of mass spectrometry to create a powerful platform which could move metabolomics to new heights. 1.5 Structure of the thesis The thesis will be introduced in 8 chapters as follows: Chapter 1 : General Introduction Chapter 2 : Manuscript-I Azahelicene superbases as MAILD matrices for acidic analytes Chapter 3 : Manuscript-II Inhibition of 5-lipoxygenase as anti-inflammatory mode of action of Plectranthus zeylanicus Benth and chemical characterization of ingredients by a mass spectrometric approach Chapter 4 : Manuscript-III Munronia pinnata (Wall.) Theob.: Unveiling phytochemistry and dual inhibition of 5-lipoxygenase and microsomal prostaglandin E2 synthase (mPGES)-1 Chapter 5 : Manuscript-IV Identific ation of female specific fatty acid derivatives in Drosophila Melanogaster surface lipid extracts Chapter 6 : Discussion Chapter 7 : Summary Chapter 8 : References OVERVIEW OF MANUSCRIPTS Manuscript-I Azahelicene superbases as MAILD matrices for acidic analytes Mayuri Napagoda, Lubomír Rulíšek, Andrej Jancarík, Jirí Klívar, Michal Šámal, Irena G. Stará, Ivo Starý, Veronika Šolínová, Václav Kašicka, and Aleš Svatoš Published in ChemPlusChem 2013 (78), 937-942. In this manuscript, we attempted to explore the suitability of several azahelicenes as MALDI or MAILD matrices for the analysis of fatty acids and organic acids in wide range of samples and explain the differences in efficiency of individual azahelicenes using methods of theoretical chemistry. Out of the tested matrices, 1,14- diaza[5]helicene performed exceptionally well and resulted in clear deprotonated signals of the acid analytes without any matrix related peaks or alkali adducts formation. The higher gas phase proton affinity, higher pKa , higher pKb for deprotonation and the UV absorbance maximum in the frequency close to that of the lasers used, strongly favor a Matrix Assisted Ionization/Laser Desorption (MAILD) type of ionization, when this matrix is mixed with an acidic analyte. Development of this novel MAILD matrix for small molecule analysis demonstrates that MALDI-MS is no longer meant only for the high-molecular weight compound analysis. Mayuri Napagoda designed and carried out all experiments on MALDI-MS, analyzed data and drafted the manuscript. Lubomír Rulíšek, Andrej Jancarík, Jirí Klívar, Michal Šámal, Irena G. Stará, Ivo Starý, Veronika Šolínová, Václav Kašicka, involved in the synthesis of azahelicenes, calculation and measurement of the physiochemical parameters. Aleš Svatoš designed the study, analyzed the data and wrote the manuscript. Manuscript-II Inhibition of 5-lipoxygenase as anti-inflammatory mode of action of Plectranthus zeylanicus Benth and chemical characterization of ingredients by a mass spectrometric approach Mayuri Napagoda, Jana Gerstmeier, Sandra Wesely, Sven Popella, Sybille Lorenz, Kerstin Scheubert, Aleš Svatoš and Oliver Werz Published in Journal of Ethnopharmacology 2014 (151), 800-809 The herb Plectranthus zeylanicus is extensively used in traditional medicine in Sri Lanka and South India for the treatment of inflammatory disorders, however the pharmacological features of this plant are unexplored. This manuscript showed that n- hexane and dichloromethane extracts of P. zeylanicus potently suppress the activity of human 5-lipoxygenase (IC50 = 0.7-12 µg/ml) in cell-free and cell-based assays without significant radical scavenging activity or suppression of ROS formation. By means of UHPLC/ESI-MS and GC-MS analysis and also with the analysis of hypothetical fragmentation trees computed from the CID spectra, we identified coleone P, cinncassiol A / C, and callistric acid as uncommon constituents in the most active fractions of the separated extracts. In addition to the above compounds for which the knowledge regarding bioactivities are rare, the presence of compounds such as a- and ß-amyrin with reported anti-inflammatory properties were also detected. Mayuri Napagoda designed the experiments, collected the plants, carried out the experiments on extraction, fractionation and LC-MS measurements, analyzed the data and wrote the manuscript. Jana Gerstmeier, Sandra Wesely and Sven Popella conducted the bioassays. Sybille Lorenz carried out GC-MS measurements. Kerstin Scheubert computed the fragmentation trees. Aleš Svatoš and Oliver Werz designed the experiments, analyzed the data and wrote the manuscript. Manuscript-III Munronia pinnata (Wall.) Theob.: Unveiling phytochemistry and dual inhibition of 5-lipoxygenase & microsomal prostaglandin E2 synthase-1 Mayuri Napagoda, Jana Gerstmeier, Andreas Koeberle, Sandra Wesely, Sven Popella, Sybille Lorenz, Kerstin Scheubert, Sebastian Boecker, Aleš Svatoš, and Oliver Werz Published in Journal of Ethnopharmacology 2014 (151), 882-890 Preparations from Munronia pinnata (Wall.) Theob is extensively used in traditional medicine in Sri Lanka for treating inflammatory conditions, however the pharmacological features or the phytochemistry of this plant are hardly explored in order to rationalize the reported ethnobotanical significance. Therefore in this manuscript, we explored the chemical profile as well as inhibition of 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase (mPGES)-1 inhibition of this important medicinal plant. n-Hexane extract of M. pinnata efficiently suppressed 5- LO activity in stimulated human neutrophils (IC50 = 8.7 µg/ml) and potently inhibited isolated human recombinant 5-LO (IC50 = 0.48 µg/ml) and mPGES-1 (IC50 = 1.0 µg/ml). The phytochemistry of the plant was unveiled for the first time with the detection of phytosterols, fatty acids, sesquiterpenes and several other types of secondary metabolites. The chemical profiling was carried out solely by UHPLC/ESI- MS, UHPLC/APCI-MS and GC-MS analysis, without any extensive isolation and purification procedures. Mayuri Napagoda designed the experiments, collected the plants, carried out the experiments on extraction, fractionation and LC-MS measurements, analyzed the data and wrote the manuscript. Jana Gerstmeier, Andreas Koeberle, Sandra Wesely, and Sven Popella conducted the bioassays. Sybille Lorenz carried out GC-MS measurements. Kerstin Scheubert computed the fragmentation trees under supervision of Sebastian Boecker. Aleš Svatoš and Oliver Werz designed the experiments, analyzed the data and wrote the manuscript. Manuscript-IV Ide ntification of female specific fatty acid derivatives in Drosophila melanogaster surface lipid extracts Mayuri Napagoda, Jerrit Weißflog, Sybille Lorenz and Aleš Svatoš In preparation for the submission to ChemBioChem Since the investigations carried out so far on sex dependent differences in composition of cuticular lipids in Drosophila melanogaster have been exclusively focused on cuticular hydrocarbons, the understanding on non-hydrocarbon components in the surface lipid extracts of male and female flies is rather primitive. In order explore this untouched area in the D. melanogaster’s cuticular chemistry, we have focused on identification of sex specific cuticular fatty acid and fatty acid derivatives and our achievements are summarized in this manuscript. A female specific 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid and its triglyceride was identified by UHPLC - APCI - MS and GC - MS methods and o ur finding challenges the decades - old concept of the absence of qualitative differences between cuticular fatty acid profiles in male and female flies . Mayuri Napagoda designed and carried out experiments on preparation of flies for extraction, fractionation, LC-MS measurements, analyzed data and wrote the manuscript. Jerrit Weißflog carried out the synthesis of the fatty acids, NMR analysis and wrote the manuscript. Sybille Lorenz carried out GC-MS measurements. Aleš Svatoš designed the study, analyzed the data and wrote the manuscript. Chapter 2 Manuscript-I Azahelicene Superbases as MAILD Matrices for Acidic Analytes Mayuri Napagoda, Lubomír Rulíšek, Andrej Jancarík, Jirí Klívar, Michal Šámal, Irena G. Stará, Ivo Starý, Veronika Šolínová, Václav Kašicka, and Aleš Svatoš Published in ChemPlusChem 2013, (78), 937-942 Abstract A superbasic 1,14-diaza[5]helicene can serve as an efficient, ionless matrix for matrix-assisted ionization/laser desorption (MAILD) spectrometry. The matrix outperforms other bases by acting as a kinetically active proton sponge and is highly suitable for high-throughput metabolomics analysis. There is a correlation between the basicity (and proton-sponge character) of matrices and their efficacy in MAILD- MS. Keywords: Helicenes, laser spectroscopy, mass spectrometry, matrix isolation, metabolomics Description: C:\Users\Asus\Desktop\Azafinalpaper\1.png Chapter 3 Manuscript-II Inhibition of 5-lipoxygenase as Anti-inflammatory Mode of Action of Plectranthus zeylanicus Benth & Chemical Characterization of Ingredients by a Mass Spectrometric Approach Mayuri Napagoda, Jana Gerstmeier, Sandra Wesely, Sven Popella, Sybille Lorenz, Kerstin Scheubert, Aleš Svatoš, Oliver Werz Published in Journal of Ethnopharmacology 2014, (151), 800-809 Abstract Ethnopharmacological relevance: The perennial herb Plectranthus zeylanicus Benth is extensively used in traditional medicine in Sri Lanka and South India for treating inflammatory conditions, but pharmacological features of P. zeylanicus are hardly explored in order to understand and rationalize its use in ethnomedicine. As 5- lipoxygenase (5-LO) is a key enzyme in inflammatory disorders, we investigated 5- LO inhibition by P. zeylanicus extracts and analyzed relevant constituents. Materials and Methods: We applied cell-free and cell-based assays to investigate suppression of 5-LO activity. Cell viability, radical scavenger activities, and inhibition of reactive oxygen species formation (ROS) in neutrophils were analysed to exclude unspecific cytotoxic or antioxidant effects. Constituents of the extracts were characterized by bioassay-guided fractionation and by analysis using gas or liquid chromatography coupled to mass spectrometric (Orbitrap) analysis. Results: Extracts (n-hexane or dichloromethane) of P. zeylanicus potently suppressed 5-LO activity in stimulated human neutrophils (IC50 = 6.6-12 µg/ml) and inhibited isolated human recombinant 5-LO (IC50 = 0.7-1.2 µg/ml). In contrast, no significant radical scavenging activity or suppression of ROS formation was observed, and neutrophil viability was unaffected. Besides ubiquitously occurring ingredients, coleone P, cinncassiol A and C, and callistric acid were identified as constituents in the most active fraction. Conclusions: Together, potent inhibition of 5-LO activity, without concomitant anti- oxidant activity and cytotoxic effects, rationalizes the ethnopharmacological use of P. zeylanicus as anti-inflammatory remedy. Modern chromatographic/mass spectrometric analysis reveals discrete chemical structures of relevant constituents. Keywords: Plectranthus zeylanicus, inflammation, 5-lipoxygenase, neutrophils, radical scavenger, mass spectrometry. Supplementary material – 1 Fig. S-1: Collision-induced spectra of the protonated adduct of Coleone P at different collision energies, (A) 2 eV , (B) 4 eV, (C) 8 eV, (D) 20 eV and (E) 55 eV Supplementary material -2 Description: C:\Users\Asus\Downloads\C22H31O6_1_C22H30O6 (1).png Fig. S-2: Hypothetical fragmentation tree for the protonated adduct of Coleone P Supplementary material -3 Description: C:\Users\Asus\Downloads\C22H30O6Na_1_C22H30O6.png Fig. S-3: Hypothetical fragmentation tree for the sodium adduct of Coleone P Supplementary material -4 Fig. S-4: Hypothetical fragmentation tree for the protonated adduct of Cinncassiol A / Cinncassiol C3 Description: C:\Users\Asus\Downloads\C20H31O7_1_C20H30O7.png Chapter 4 Manuscript-III Munronia pinnata (Wall.) Theob.: Unveiling Phytochemistry & Dual Inhibition of 5-lipoxygenase and Microsomal Prostaglandin E2 Synthase (mPGES)-1 Mayuri Napagoda, Jana Gerstmeier, Andreas Koeberle, Sandra Wesely, Sven Popella, Sybille Lorenz, Kerstin Scheubert, Sebastian Boecker, Aleš Svatoš & Oliver Werz Published in Journal of Ethnopharmacology 2014,(151), 882-890 Abstract Ethnopharmacological relevance: Preparations from Munronia pinnata (Wall.) Theob are extensively used in traditional medicine in Sri Lanka for the treatment of inflammatory conditions. However, neither the pharmacological features nor the phytochemistry of this plant are explored in order to understand and rationalize the reported ethnobotanical significance. As 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase (mPGES)-1 are crucial enzymes in inflammatory disorders, we evaluated their inhibition by M. pinnata extracts and studied the chemical profile of the plant for the identification of relevant constituents. Materials and Methods: Cell-free and cell-based assays were employed in order to investigate the suppression of 5-LO and mPGES-1 activity. Cell viability, radical scavenger activities, and inhibition of reactive oxygen species formation (ROS) in neutrophils were studied to assess cytotoxic or antioxidant effects. Gas or liquid chromatography coupled to mass spectrometric analysis enabled the characterization of secondary metabolites. Results: The n-hexane extract of M. pinnata efficiently suppressed 5-LO activity in stimulated human neutrophils (IC50 = 8.7 µg/ml) and potently inhibited isolated human recombinant 5-LO (IC50 = 0.48 µg/ml) and mPGES-1 (IC50 = 1.0 µg/ml). In contrast, no significant radical scavenging activity or suppression of ROS formation was observed, and neutrophil viability was unaffected. The phytochemistry of the plant was unveiled for the first time and phytosterols, fatty acids, sesquiterpenes and several other types of secondary metabolites were identified. Conclusions: Together, potent inhibition of 5-LO and mPGES-1 activity, without concomitant antioxidant activity and cytotoxic effects, rationalizes the ethnopharmacological use of M. pinnata as anti-inflammatory remedy. Modern chromatographic/mass spectrometric analysis reveals discrete chemical structures of relevant constituents. Key words: Munronia pinnata, inflammation, 5-lipoxygenase, radical scavenger, microsomal prostaglandin E2 synthase-1, mass spectrometry. Supplementary -1 Fig. S-1: Collision induced dissociation spectra of putatively identified ganoderiol F at (A) 4 eV and (B) 17 eV Supplementary -2 C30H47O3_1_C30H46O3 Fig. S-2: Hypothetical fragmentation tree for putatively identified ganoderiol F (protonated adduct) Supplementary -3 Fig. S-2: Collision induced dissociation spectra of putatively identified triterpenoid at (A) 12 eV and (B) 15 eV Chapter 5 Manuscript-IV Identification of Female Specific Fatty acid Derivatives in Drosophila melanogaster Surface Lipid Extracts Mayuri Napagoda, Jerrit Weißflog, Sybille Lorenz, Aleš Svatoš In preparation for the submission to ChemBioChem Abstract Investigations carried out so far on sex dependent differences in composition of cuticular lipids in Drosophila melanogaster have been exclusively focused on cuticular hydrocarbons. As a result, the understanding on non-hydocarbon components particularly fatty acid and fatty acid derivatives in the surface lipid extracts of male and female flies is rather primitive. Therefore, the identification of female specific 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid and its TAG by UHPLC - APCI - MS and GC - MS methods directs the field of insect cuticular chemistry in to new dimensions. Our finding not only contradicts the decades - old concept of the absence of qu alitative differences between cuticular fatty acid profiles in male and female flies but also highlights the necessity of a detailed study on the biosynthesis and physiological functions of this female specific fatty acid. Key words : cuticle, fatty acid derivatives, Drosophila melanogaster, 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid 1. Introduction The knowledge on surface chemistry of insects is crucial for a better understanding of the physiological functions performed by the insect cuticle. Particularly the cuticular chemistry of insects has drawn much attention with the recognition of its involvement in various types of chemical communications (Howard, 1993). The advent of analytical methods enabled the determination of structures and the composition of the cuticular lipids in many insect species. Hydrocarbons were identified as the major component while esters, free alcohols, fatty acids, ketones, etc. were also detected in minor quantities in many species. After the observation of sex dependent differences in surface lipid composition in several Diptera species, the investigations of surface lipids in Drosophila melanogaster were initiated few decades ago. The preliminary evaluation of D. melanogaster surface lipids indicated the presence of hydrocarbons; alkanes, alkenes and alkadienes in considerable amount. The study was then extended towards the determination of quantitative and qualitative differences in cuticular lipids in males and female flies (Jackson, et al., 1981) which revealed quantitative differences in hydrocarbons, acylglycerol, and free fatty acids in hexane extracts of both sexes. Since the commencement of studies on surface lipids of D. melanogaster, the hydrocarbon components have undergone intensive investigations. This led to the identification of several compounds displaying marked sexual dimorphism hence performing an important role as species-specific signals (pheromones). For example, CHs with two double bonds (often 7,11-dienes) are produced only in females and stimulate male courtship while monoenes (such as 7-Tricosene) are mostly found in males (Jallon, 1984; Antony and Jallon, 1982; Foley, et al., 2007). However, the non- hydrocarbon components of surface lipids in D. melanogaster have not been extensively studied so far. A previous study on non-hydrocarbon lipid components revealed the presence of myristic acid, palmitic acid, palmitoleic acid, oleic acid, and linoleic acid as major fatty acids from both the acylglycerol and free fatty acid fractions of both sexes (Jackson, et al., 1981). Despite the quantitative differences, any qualitative differences between the fatty acid compositions of either free fatty acids or acylglycerol fatty acids from male and female flies have not been observed in this study. Interestingly, any evidences to disprove this observation have not been reported yet neither. Nowadays, mass spectrometry has turned a new era in the analysis of lipids and several mass spectrometry based techniques are widely employed in the analysis of insect surface lipids. GC-MS is a well-established tool and plays a predominant role in the detection and structure elucidation of sufficiently volatile nonpolar cuticular hydrocarbons. However, the high probability of missing larger and more polar cuticular compounds by this method appears as a major drawback in such an analysis. Thus the conventional approach involves hydrolysis of lipids followed by derivatization in to fatty acid methyl esters (FAME) and subsequent GC-MS analysis of released fatty acid derivatives. The analysis of FAMEs by GC-MS does not provide complete information about the original lipid molecule, particularly, the arrangement of constituent FAs to the glycerol back-bone. As a result, there was a demand for the development of novel approaches that facilitate the detection and identification of more polar and high molecular weight cutitular components. With the emergence of HPLC/APCI-MS, the analysis of complex lipid mixtures has redeem a momentum as it allows for a direct analysis of triacylglyerols of higher molecular weight (Kofronová, et al., 2009; Cvacka, et al., 2006). Depending on the mass analyzer used, high accuracy in the determination of the molecular mass can be achieved where as extrem sensitivity in mass spectrometric approaches enable the identification of even minor components in a crude lipid extract without extensive purifications. In this study, we are attempting to investigate the presence of sex dependent fatty acids and fatty acid derivatives in the surface lipids extracts of D. melanogaster by means of novel mass spectrometric approaches. Thereby, we hope to rectify the validity of the decades’ old concept of the absence of qualitative differences in fatty acid profiles of male and female flies. 2. Experimental 2.1 Rearing flies Each mature pupae of D. melanogaster (Canton S) was reared on corn meal sugar agar diet in an individual Eppendorf tube for 3 days at 25 °C. Thereafter, the flies of each sex were sorted out and transferred into separate vials containing 2 mL of 2% sucrose and incubated at 25 °C for 2 days. The flies were then freezed at -20 °C and the extraction of surface lipids was carried out as mentioned below. 2.2 Extraction Two thousand and five hundred flies of each sex were superficially washed with dichloromethane : methanol (2:1) (7.5 mL) mixture. Each extract was concentrated to nearly dryness with the use of rotary evaporator (R-114, BÜCHI, Switzerland). 100 µL of the crude extract were taken out and stored at -20 °C for future references. The remaining portion of the extract was dissolved in methanol (about 100 µL) and was adsorbed into silica gel (about 100 mg) and completely evaporated the solvents. This was loaded into the column packed with silica gel (1 g) and eluted with hexane, 2% EtOAc in hexane, 5% EtOAc in hexane, 8% EtOAc in hexane, 10% EtOAc in hexane, 12% EtOAc in hexane, 15% EtOAc in hexane, 18% EtOAc in hexane, 20% EtOAc in hexane, 25% EtOAc in hexane, 30% EtOAc and methanol successively. These 12 fractions from male and female flies were subjected to spectrometric analysis. 2.3 Ultra-High Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry (UHPLC-APCI-MS) Non-aqueous reversed phase UHPLC separation was carried out by Dionex –Acclaim ® RSLC 120 C18 column (2.1 . 150 mm packed with 2.2 µm, 120 Å) using acetonitrile (A) and propan-2-ol (B) as mobile phases. The gradient program was set as: 0 min - 100% A 0.5 mL/min, 58 min - 30% A, 70% B, 0.5 mL/min, 70 min - 10% A, 90% B, 0.3 mL/min, 100 min - 100% A, 0.5 mL/min. The APCI source was operated at 400 °C, the heated capillary temperature was 220 °C and the corona discharge current was set to 4.5 µA. the full scan mass spectra were recorded in the m/z range 150 -1400. The full scan and collision-induced dissociation (CID) mass spectra were generated using 30 000 and 7500 full width at half maximum (fwhm) resolutions respectively. 2.4 LC-MS fractionation The above-mentioned solvent gradient was used for the collection of TAG of interest. The sub-fraction eluted at 20-24 min in the LC run fraction was collected, concentrated and half of it was trans-methanolyzed and converted into a methyl ester of a fatty acid (FAME) while the rest is subjected to different chemical reactions. 2.5 Trans-methanolysis and GC-MS analysis of the TAG The collected fraction was evaporated and dissolved in ACN (about 1 mL) into which MSTFA (N-methyl-N-trimethylsilyl trifluoroacetamide) (20 µL) was added. The reaction mixture was heated at 65 °C for 1 hour. Thereafter, the solvents were completely evaporated. In to the residue, 0.05 M KOH in MeOH (50 µL) was added and incubated at 4 °C for 20 min. Thereafter, 1M KH2PO4 + KHPO4 buffer (pH 5-6) (75 µL ) and hexane (500 µL ) was added and vortexed for 5 min. Hexane layer was taken out and any water droplets were removed by addition of Na2SO4. Thereafter, the sample was methylated with CH2N2 and injected into GC-MS. The GC-MS measurements were executed on a gas chromatograph HP6890 (Agilent, CA, USA) connected to a MS02 mass spectrometer from Micromass (Waters, UK) with EI 70 eV using ZB5ms column (30 m × 0.25mm, 0.25-µm film thickness; Phenomenex, CA, USA). The carrier gas was helium at the flow rate of 1mL/min. The injector temperature was kept at 220 °C and the temperature program was set as 40 °C (2 min), 15°C / min to 300 °C (3 min) 2.6 Hydrogenation Small amount of 10% Pd/C in acetone which was pre-washed with ethyl acetate was mixed with TAG sample (50 µL). The mixture was treated with H2 for about 2 hours while stirring. Thereafter, the reaction mixture was concentrated and analyzed by LC- APCI-MS. 2.7 Oxime formation Pentaflurobenzyl hydroxylamine hydrochloride in CH2Cl2 (10 µL) and one bead of molecular sieve (3A) was added to TAG sample (50 µL) and shaken for about 2 hours. Thereafter, the mixture was evaporated and finally dissolved in ACN: propan- 2-ol (1:1) (50 µL) and analyzed by LC-APCI-MS. Following the same procedure, fatty acid methyl ester sample (50 µL) was treated with pentaflurobenzyl hydroxylamine hydrochloride in CH2Cl2 (10 µL) and the resulting mixture was analyzed by GC-MS. 2.8 Acetylation TAG sample (50 µL) was reacted with 10 µL of acetic anhydride and 10 µL of lutidin in CH2Cl2. The reaction mixture was kept for about 2 hours for the completion of the reaction. Thereafter it was evaporated and finally dissolved in 50 µL of ACN: propan- 2-ol (1:1) and analyzed by LC-APCI-MS. 2.9 DMDS derivatization Using an argon stream, any air present in the TAG sample/ FAME sample was removed and 5% I2 in diethyl ether (1 drop) followed by DMDS (1 drop) was added to it. Then the reaction mixture was concentrated and kept in dark. The reaction was quenched by the addition of 10% Na2S2O3 in water after 1 hour. The experiment was repeated following the same procedure, however the reaction was quenched after 6 hours. Then the organic layer was taken out, concentrated and analyzed by GC-MS. 2.10 Synthesis of Fatty Acids Scheme1: Synthesis of 4-Methyl-2-pentylfuran (3a) Scheme2: Synthesis of 3-Methyl-2-pentylfuran (3b) a) POCl3, DMF, 0°C to r.t. b) 10% H2SO4, c) Ph3P, reflux, in Xylene, d) NaOMe, 0°C to r.t., in DMF, e)H2, cat. Pd/C, in MeOH, f) 1M NaOH, 60°C, in MeOH/THF Scheme 3: Synthesis of 9-(3-Methyl-5-pentyl-2-furyl)-nonanoic acid and 9-(4- Methyl-5-pentyl-2-furyl)-nonanoic Acid All chemicals were purchased from Sigma-Aldrich. NMR-spectra were obtained using a Bruker Avance DRX 500 NMR Spectrometer. MS-data were recorded on MS02 mass spectrometer from Micromass (Waters, UK). 2.10.1 Synthesis of 9-(3-Methyl-5-pentyl-2-furyl)-nonanoic Acid (14a) 9-(3-Methyl-5-pentyl-2-furyl)-nonanoic acid was synthesized from 4-methyl-2- pentylfuran (3a) and methyl-6-bromooctanoate (10) in 4 steps using the protocol of Tsukasa (1993). All spectral data were in accordance with the data in the literature. 2.10.1.a Synthesis of 4-Methyl-2-pentylfuran (3a) 25 mL (0.18 mmol) of hexanoyl chloride (1) and 17 mL (0.17 mmol) 3-chloro-2- methyl-1-propene (2) were added dropwise to a suspension of 24 g (0.18 mmol) AlCl3 in 35 mL dichloromethane while maintaining the temperature at -10 to -15 °C. The mixture was stirred at the same temperature for 30 min before it was poured on crushed ice. After the separation of the phases, the aqueous layer was extracted with ethylacetate (3 . 100 mL). The combined organic phase was washed with water, sat. Na2CO3-solution and brine, before being dried with Na2SO4 and concentrated in vacuo. The residue was distilled (bp. 75-76 °C at 33 mbar) to give 21 g (81% yield) of 4-methyl-2-pentylfuran (3a) as colorless oil. Spectral data were in accordance with those found in literature. 2.10.1.b Synthesis of Methyl-8-bromooctanoate (10) Methyl-8-bromooctanoate (10) was synthesized from 8-bromooctanoic acid (9) using the protocol of Savariar, et al., (2006). All spectral data were in accordance with the data in the literature. 2.10.2 Synthesis of 9-(4-Methyl-5-pentyl-2-furyl)-nonanoic Acid (14 b) 2.10.2.a Synthesis of 3-Methylnon-1-en-4-yn-3-ol (6) Hexyne (4) (12.6 mL 110 mmol) was dissolved in THF (200 mL) under an atmosphere of argon and cooled to -78 °C. A 1.6 M solution of n-BuLi in hexane (68.75 mL, 110 mmol) was added dropwise over a period of 15 min. After stirring at - 78 °C for 10 min, 8.1 mL (100 mmol) of 3-buten-2-one (5) were added dropwise over a period of 5 min. The solution was stirred at the same temperature for further 20 min, before being allowed to warm to room temperature. The reaction was then quenched with 100 mL water. After the separation of the phases, the aqueous layer was extracted with diethylether (3 . 50 mL). The combined organic phase was washed with water and brine, before being dried over Na2SO4 and concentrated in vacuo. The residue was distilled (bp. 92-93 °C at 20 mbar) to give 13 g (86% yield) of 3- methylnon-1-en-4-yn-3-ol (6) as colorless oil. 1H NMR (500 MHz, CDCl3) . 5.97 (dd, J = 17.0, 10.2 Hz, 1H), 5.48 (d, J = 17.0 Hz, 1H), 5.08 (d, J = 10.1 Hz, 1H), 2.23 (d, J = 7.1 Hz, 2H), 1.52 (s, 3H), 1.49 (m, 2H), 1.41 (p, J = 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 2H) ppm; 13C NMR (125 MHz, CDCl3) ..142.6, 113.1, 86.0, 82.2, 67.9, 30.7, 30.2, 21.9, 18.3, 13.6. 2.10.2.b Synthesis of 3-Methyl-2-pentylfuran (3b) 3-Methyl-2-pentylfuran (3b) was synthesized from 3-methylnon-1-en-4-yn-3-ol (6) in 2 steps using the protocol of Gabriele, et al., (1999). The spectral data were in accordance with the data in the literature. 2.10.2.c Synthesis of 4-Methyl-5-pentylfuran-2-carbaldehyde (8b) Phosphorous oxychloride (1.21 g, 7.9 mmol) was added dropwise to N,N- dimethylformamide (2.5 mL) while stirring at 0 °C. After stirring for 1 h at the same temperature, 3-Methyl-2-pentylfuran (3b) (1g, 6.6 mmol) dissolved in 2.5 mL of N,N- dimethylformamide was added dropwise while keeping the temperature at 0 °C. After the addition, the reaction was stirred for 1h at room temperature, after which TLC showed complete conversion of the furan. The deep red solution was poured into 5% aqueous NaOH (50 mL) and extracted with diethylether (3 . 20 mL). The extract was washed with water and brine, before it was dried over Na2SO4. After evaporating the solvent, the aldehyde was purified by column chromatography (silica gel, 5% EtOAc in hexane) giving 910 mg (77% yield) of 4-methyl-5-pentylfuran-2-carbaldehyde (8b). 1H NMR (500 MHz, CDCl3) . 9.46 (s, 1H), 7.04 (s, 1H), 2.65 (t, J = 7.1 Hz, 2H), 2.03 (s, 3H), 1.67 (m, 2H), 1.31 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H) ppm; 13C NMR (125 MHz, CDCl3) ..176.8, 160.0, 150.6, 119.0, 118.0, 31.4, 27.6, 26.4, 22.3, 13.9, 9.7. 2.10.2.d Synthesis of (E/Z)-Methyl 9-(4-methyl-5-pentylfuran-2-yl)non-8-enoate (12b) (7-Carbomethoxyheptyl)triphenylphosphonium bromide (11), (3.6g 6.9 mmol) which was synthesized from methyl-8-bromooctanoate (10) using the protocol of Tsukasa (1993), was dissolved in 12 mL N,N-dimethylformamide under an atmosphere of argon and cooled to 0 °C. Sodium methoxide (470 mg, 8.7 mmol) were added, after which the reaction was stirred for 10 min at the same temperature. After the addition of 900 mg (1.67 mmol) of 4-methyl-5-pentylfuran-2-carbaldehyde (8b) the reaction was stirred for 1h at room temperature and then quenched by pouring the mixture on ice. The products were extracted with diethylether (3 . 20 mL) washed with brine and dried over Na2SO4. After evaporating the solvent the product was purified by column chromatography (silica gel, 5% EtOAc in hexane) giving 977 mg (61% yield) of a 10:1 mixture of (E)- and (Z)-methyl 9-(4-methyl-5-pentylfuran-2-yl)non-8-enoate (12b). 1H NMR (500 MHz, CDCl3) . 6.07 (dd, J = 11.6, 1.6 Hz, 1H), 6.02 (s, 1H), 5.42 (dt, J = 11.9, 7.3 Hz, 1H), 3.66 (s, 3H), 2.53 (t, J = 7.4 Hz, 2H), 2.39 (qd, J = 12.2, 1.5 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 1.93 (s, 3H), 1.62 (m, 4H), 1.40 (m, 10H), 0.89 (t, J = 7.0, 3H)ppm; 13C NMR (125 MHz, CDCl3) . 174.2, 150.7, 150.4, 129.2, 117.5, 115.2, 112.3, 51.4, 34.1, 31.4, 29.4, 29.2, 29.0, 28.1, 25.9, 24.9, 22.4, 14.0, 9.8. MS (EI, 70eV): m/z 320 (M+ 21), 263 (24), 191 (81), 147 (25), 135 (47), 134 (16), 121 (95), 119 (15), 109 (34), 107 (19), 105 (20), 95 (26), 93 (51), 91 (61), 81 (26), 79 (32), 77 (28), 69 (24), 67, 24), 59 (24), 55 (100), 53 (17), 43 (93), 41 (75). 2.10.2.e Synthesis of Methyl 9-(4-methyl-5-pentyl-2-furyl)-nonanoate (13b) The mixture of (E)- and (Z)-methyl 9-(4-methyl-5-pentylfuran-2-yl)non-8-enoate (12b) (400 mg, 1.25 mmol) was dissolved in 4 mL of methanol and hydrogenated over 10 mg of 10% palladium on activated charcoal at atmospheric pressure. After 1 h the catalyst was filtered off and the solution concentrated in vacuo. Column chromatography (silica gel, 5% EtOAc in hexane) gave 371 mg (92 % yield) of methyl 9-(4-methyl-5-pentyl-2-furyl)-nonanoate (13b). 1H NMR (500 MHz,CDCl3)... 5.72 (s, 1H), 3.66 (s, 3H), 2.51 (t, J = 7.7 Hz, 2H), 2.49 (t, J = 7.5 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.89 (s, 3H), 1.58 (m, 4H), 1.30 (m, 12H), 0.89 (t, J = 7.0, 3H) ppm; 13C NMR (125 MHz, CDCl3) . 174.3, 153.4, 149.9, 113.8, 107.7, 51.4, 34.1, 31.4, 29.2, 29.15, 29.1, 28.4, 28.1, 28.0, 25.9, 25.0, 22.4, 14.0, 9.9. MS (EI, 70eV): m/z 322 (M+ 61), 291 (17), 265 (100), 179 (13), 165 (87), 135 (6), 121 (19), 109 (42), 108 (10), 95 (12), 55 (7). 2.10.2.f Synthesis of 9-(4-Methyl-5-pentyl-2-furyl)-nonanoic Acid (14b) The mixture of (E)- and (Z)-Methyl 9-(4-methyl-5-pentylfuran-2-yl)non-8-enoate (12b) (200 mg, 0.62 mmol) was dissolved in a mixture of methanol (1 mL) and THF (2 mL) and NaOH solution (1.9 mL,1M) were added. After the reaction mixture was stirred for 1.5 h at 60 °C, HCl (2 mL, 1 M ) was added and the mixture was extracted with ethyl acetate (3 . 10 mL). The combined organic phase was washed with brine and dried over Na2SO4. After evaporating the solvent the acid was purified by column chromatography (silica gel, 20% EtOAc in hexane) giving 175 mg (92% yield) of 9- (4-Methyl-5-pentylfuran-2-yl)nonanoic acid (14b). 1H NMR (500 MHz, CDCl3) ... 5.72 (s, 1H), 2.51 (t, J = 7.6 Hz, 2H), 2.49 (t, J = 7.5 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.89 (s, 3H), 1.60 (m, 4H), 1.30 (m, 10H), 0.88 (t, J = 7.0, 3H)ppm; 13C NMR (125 MHz, CDCl3) ... 179.9, 153.4, 149.8, 113.8, 107.7, 33.8, 31.4, 29.2, 29.1, 29.0, 28.4, 28.1, 28.0, 25.9, 24.7, 22.4, 14.0, 9.9. MS (EI, 70eV): m/z 308 (M+ 30), 252 (17), 251 (100), 166 (7), 165 (48), 134 (16), 121 (10), 109 (17), 108 (5), 107 (19), 95 (5). 3. Results and Discussion Although the literature does not reveal any evidences so far for the existence of qualitative differences in cuticular fatty acid composition between male and female D. melanogaster, our observations on the total ion chromatograms (TIC) of male and female surface lipid extracts which are not identical to each other (Supplementary-1) suggest for the presence of sex dependent nonhydrocarbon components such as fatty acids or fatty acid derivatives. Still many scientists doubt that the nonhydrocarbon components, particularly triglycerides are not considered as surface lipid components and supposed to be appear when the extraction has been so extensive as to extract internal lipids. It is usually believed that the extraction solvents and the exposure time have been crucial factors that would led to the rupture of cuticle, thus extraction of the internal components (Jackson, et al., 1981). Therefore, extreme care has been taken during the handling and only a superficial rinse of the flies was performed in order to avoid the extraction of any internal lipids. Since we are interested in investigating the nonhydrocarbon components in the insect surface, our focus was on the medium polar fractions that have been eluted from the silica gel column chromatography. Thus the male and female fractions eluted with 15% EtOAc in hexane (Fra.7) was selected for a in-depth study (Fig.1). Fig. 1 – UHPLC-APCI Total Ion chromatograms of male (A) and female (B) fraction eluted with 15% EtOAc in hexane Among the detected peaks, several compounds have exclusively been detected in the female fraction whereas only one compound was detected as male specific. The accurate mass measurements on LTQ Orbitrap XL has been used to determine molecular composition of these unknown compounds. A list of sex specific compounds detected in corresponding female and male fractions eluted with 15% EtOAc in hexane (Fra.7) is given in Supplementary- 2. Out of the identified compounds, our attention was focused on the compound at m/z 963.7275 with the molecular composition of C60H99O9 which presumed to be a TAG. This peak was virtually absent in the corresponding male fraction and it appears interesting as it showed unexpected 19 carbon atoms in the fatty acid chain and nine oxygen atoms. To get more structural insights, CID/HCD experiments have been performed which give rise to one diacylglycerol ion [M+H-RiCOOH]+ (m/z 655.4918), one monoacylglycerol ion [M+H-RiCOO-Ri’CO]+ (m/z 365.2665) and one acyl ion [RiCO]+ (m/z 291.23) (Fig. 2 ). It helped to rationalize the presence of three identical fatty acids with 19 carbon atoms, three double bonds/cycles and additional oxygen in the fatty acid chain. Fig. 2 HCD/CID spectra of the female specific compound of m/z 963.7275 In order to confirm the number of double bonds in this molecule, hydrogenation of the intact TAG was performed in which a peak at m/z 975.8189 was observed (Fig. 3). This corresponds to the hydrogenation of two double bonds in each of the three constituent fatty acids. However the double bond positions were not able to be deduced from the DMDS derivatization, which thus suggests that the double bonds might have not been arranged in a simple aliphatic chain and rather could be in a cyclic form. image description Fig. 3 Peak corresponding to the hydrogenated TAG In order to determine whether the additional oxygen atom in the fatty acid chain exists as a hydroxyl group or a ketone group, acetylation and oxime formation reactions were performed respectively. However, neither the acetylated product nor the oxime product were detected suggesting the absence of hydroxyl or ketone groups in fatty acid chain. The accurate mass GC-MS analysis of the trans-methanolyzed TAG provided invaluable details towards the identification of this unusual fatty acid. It has not only supported the previously determined molecular composition but also provided data for partial localization of the desaturation and oxidation. The subsequent data base search in AOCS Lipid Library (William W. Christie, Dundee, Scotland) allowed the identification of this constituent fatty acid as 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid. The identity of the compound was confirmed by the comparison of GC - MS spectra of the trans - methanoly z ed TAG sample with the synthesized fatty acid methyl esters , 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoate ( 13 a ) and its’ isomer 9- (4-Methyl-5-pentyl-2-furyl)-nonanoate (13b) (Fig.4). Fig . 4 - GC spectra of (A) -trans methanolyzed TAG, (B)- synthetic 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoate and (C) 9-(4-Methyl-5-pentyl-2-furyl)-nonanoate The retention time and the fragmentation pattern of synthesized 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoate was identical with the trans - methanolyzed TAG , thus confirmed the presence of this complex fatty acid and its TAG as a female specific compound in D. melanogaster surface lipid extract. 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid has been reported in some fish oils and vegetable oils ( Vetter, et al., 2012; Guth and Grosch, 1991) previously, however it has never been detected in any insect species to the best of our knowledge. There are reports suggesting that the enteric bacteria in fish are responsible for its synthesis and a puta tive biosynthetic pathway has been proposed ( Shirasaka , et al., 1997) which starts from cis - vaccenic acid, a common precursor of the pheromones in D. melanogaster (Jallon, 1997). Therefore, t he identification 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonaoic acid and its TAG form in D. melanogaster female flies is of great interest in terms of its biosynthesis as well as the biological significance . Although , we have carried out preliminary experiments with 30 flies of each to trac k its presence in different development stages (2 day old, 3 day old , 6 day old and 6 day old after mating) , the low abundance of the compound in crude extract has hindered its det ection. Therefore a large scale extraction of flies of different developmental stages might help to solve this puzzle and hope to be carried out in the future. Nevertheless, our investigation on sex dependent fatty acids and fatty acid derivatives in D. melanogaster led to the findings that contradicts decade’s old observations on the absence of qualitative differences between cuticular fatty acid profiles in male and female flies. Further more, it would inspire the scientific community for a detailed study on the biosynthesis and physiological functions of the unusual female specific fatty acid that has been reported for the first time in an insect species. 4. Conclusion By demonstrating the presence of female specific 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)non anoic acid and its TAG , the present investigation reveals the existence of qualitative differences between the composition of fatty acid and fatty acid derivatives in male and female Drosophila melanogaster flies. This observation contradicts the widely accepted concept of the absence of sex-dependent qualitative differences in the non-hydrocarbon components in the cuticle of Drosophila melanogaster and inspired for further study on the biosynthesis and physiological functions of these sex specific compounds. Acknowledgements M.N is supported by the International Max Planck Research School, Jena. We thank Dr. Kathrin Steck and Dr. Markus Knaden (MPI, Jena) for their assistance in providing the flies for the experiments, Dr. Marco Kai (MPI, Jena) and Dr. Josef Cvacka (Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic) for their assistance in the analysis of LC-MS data. References Antony. C., Jallon, J.M., 1982. The chemical basis for sex recognition in Drosophila melanogaster. Journal of Insect Physiology 28,873–880 Cvacka, J., Hovorka, O., Jiroš, P., Kindl, J., Stránský, K., Valterová, I., 2006 a. Analysis of triacylglycerols in fat body of bumblebees by chromatographic methods, Journal of Chromatography A, 1101 ,226–237 Guth, H., Grosch, W., 1991. Detection of Furanoid Fatty Acids in Soya-Bean Oil – Cause for the Light-Induced Off-Flavour. European Journal of Lipid Science and Technology 93 (7), 249–255 for discovering drugs from previously unexplored natural products Jackson, L., Arnold, M.T., Blomquist, G.J., 1981. Surface lipids of Drosophila melanogaster ; comparison of the lipids from female and male wild type and sex linked yellow mutant, Insect Biochemistry 11, 87-91 Jallon, J.M., 1984. A few chemical words exchanged by Drosophila during courtship and mating. Behavior Genetics 14, 441–478 Jallon, J., Kunesch, G., Bricard, L., Pennanec'h, M., 1997. Incorporation of fatty acids into cuticular hydrocarbons of male and female Drosophila melanogaster. Journal of Insect Physiology 43(12),1111-1116 Foley, B., Chenoweth, S.F., Nuzhdin, S.V., Blows, M.W., 2007.Natural genetic variation in cuticular hydrocarbon expression in male and female Drosophila melanogaster. Genetics 175: 1465–1477 Gabriele, B., Salerno, G., Lauria, E., (1999) A General and Facile Synthesis of Substituted Furans by Palladium-Catalyzed Cycloisomerization of (Z)-2-En-4- yn-1-ols. Journal of Organic Chemistry 64 (21), 7687 – 7692 Howard, R.W., 1993. Cuticular hydrocarbons and chemical communication. In: Insect lipids: chemistry, biochemistry and biology. Stanley-Samuelson, D.W., Nelson, D.R., (eds) pp 179–226. University of Nebraska Press, Lincoln Kofronová, E., Cvacka, J., Jiroš, P., Sýkora, D., Valterová, I., 2009. Analysis of insect triacylglycerols using liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. European Journal of Lipid Science and Technology 111, 519–525 Savariar, E. N., Aathimanikandan, S.V., Thayumanavan, S., 2006. Supramolecular Assemblies from Amphiphilic Homopolymers: Testing the Scope. Journal of the American Chemical Society 128 (50), 16224 – 16230 Shirasaka, N., Nishi, K., Shimizu, S., 1997 , Biosynthesis of furan fatty acids (F- acids) by a marine bacterium Shewanella putrefaciens . Biochimica et Biophysica Acta 1346, 253–260 Tsukasa, H., 1993. Synthesis of 9-(3-Methyl-5-pentyl-2-furyl)nonanoic Acid Bioscience Biotechnology and Biochemistry 57 (3), 511 – 512 Vetter, W., Laure, S., Wendlinger, C., Mattes, A., Smith, A.W.T., David W. Knight, D.W., 2012, Determination of Furan Fatty Acids in Food Samples Journal of the American Oil Chemists' Society 89 (8), 1501-1508 Supplementary – 1 Fig S-1. UHPLC-APCI Total Ion Chromatograms of male (A) and female (B) surface lipid crude extracts Supplementary – 2 Sex dependent compounds in corresponding male and female fractions eluted with 15%EtOAC in hexane (Fra.7) in the silica gel column chromatography Chapter 6 Discussion Discussion A few decades ago, the task of mass spectrometry was limited to providing information on elemental composition of a molecule and its partial structural insights and has been seldom used for the de novo structural characterization of small molecules (Kind and Fiehn, 2010). However, with the recent advancements in instrumentation, the roles of mass spectrometry have been widely expanded and need to be re-defined. Today it is considered as an indispensable tool for structural characterization of secondary metabolites in the field of natural products chemistry. The present study, which has been undertaken, under three themes as mentioned in Section 1.4, is an attempt to exploit mass spectrometry in the analysis of diverse forms of secondary metabolites. This is achieved by optimizing the existing mass spectrometric tools while developing high throughput analytic methods for studies of natural products and metabolomics. The efforts we have made in this thesis to explain the development and optimization of mass spectrometric approaches for the analysis of secondary metabolites in a wide array of biological samples clearly reflects the expediency of mass spectrometry in “small molecule research”. In addition, the chemistry and potential bioactivities of the secondary metabolites that have been characterized in this study will undoubtedly be topics of interests for many researchers in the future. The significance of our mass spectrometric approaches and the contribution of our findings on secondary metabolites for prospective applications in diverse fields are discussed below. Novel MALDI matrices for metabolomics studies Until recently, MALDI-MS has been extensively used in the analysis of large biomolecules like proteins, peptides, oligosaccharides etc. (Karas and Hillenkamp, 1988) and pigeonholed as a “high-molecular mass tool only”. The high sensitivity and high throughput nature of this technique made it to be a good choice for such analysis (Karas, et al., 1987). The success in the analysis of a wide variety of molecules lies in its ability to generate intact ions of thermally labile molecules using UV absorbing matrices. A good matrix should be able to absorb light at the wavelength of the laser used and several molecules with this property have been employed in the analysis of large biomolecules (Cohen, et al., 2007). As most of these conventional matrices have a molecular weight below 500 Da, the presence of matrix related ions in the low mass region of the spectrum was a common phenomenon. Since all analytes of interest are in the high-mass range, this forest of interfering peaks produced by the conventional matrices in the low mass region was not an issue of concern. During the last decade, MALDI-MS has gained much attention in the arena of small molecule research due its incredible tolerance to salts and buffers, minimal requirements for sample preparation or pretreatment and the relative ease of spectral interpretation. However, the application has been suppressed mainly due to the unavailability of suitable matrices (Cohen, et al., 2007). A careful selection of matrix is a crucial factor in successful application of MALDI to small molecule analysis and a compound that could provide an efficient ionization, minimal or controllable fragmentation and lack of mass interferences is considered to be an ideal matrix. However, due to the lack of knowledge on overall mechanisms of the processes of desorption and ionization, the choice of MALDI matrices has remained empirical. To address this issue, a novel tactic for the selection of “ionless” matrices was introduced by our laboratory (Shroff, et al., 2009). It is based on the Brønstead-Lowry acid-base theory. Two novel matrices 1,8 - bis(dimethylamino)naphthalene ( DMAN) and 2-naphthylsulfonic acid were developed for the negative and positive mode MALDI analysis respectively, which give rise to spectra completely devoid of any matrix-related ions. A new mode of ionization, Matrix Assisted Ionization/Laser Desorption (MAILD) was suggested as the mode of ionization when either a strong base (DMAN) or a strong acid (2-naphthylsulfonic acid) is used as a matrix in the above situations. In the analysis of acidic metabolites in the negative ion mode, UV absorbing super base matrix DMAN is premixed with an acidic analyte. The ionization takes place in the solution during the mixing of a weak to moderate acidic analyte with the strong base matrix. Proton exchange from analyte to matrix results in the formation of salt/ion pair on the MALDI target plate that is desorbed by the laser pulses and separated into “naked ions” in the gas phase. However, it was recently realized that under experimental conditions, DMAN is volatile and may desorb deposits in MALDI/TOF-MS instruments’ ion sources, thus possibly yielding interfering peaks in the mass spectra (Eibisch, et al., 2012). In order to eliminate this problem and to enhance the capability of MALDI-TOF-MS in low molecular weight compound analysis, we have been extensively searching for alternate MAILD matrices either by modification of DMAN or looking in other classes of super bases. Manuscript-I summarizes our success in this endeavor. A new class of “proton sponges”, named azahelicenes were tested in this study. Some of these azahelicenes have displayed exceptional basicities (Roithová, et al., 2007: Staab, et al., 1994) even though these compounds lack hydrophobic shielding in their basic centers and N---H---N hydrogen bridges, which is considered as the characteristic feature of all classical proton sponges (Staab and Saupe, 1988). Not surprisingly, out of the tested azahelicenes, an exceptional performance by 1,14- diaza[5]helicene as a MAILD matrix was observed, which was further supported by the measured/calculated physiochemical parameters. The higher gas phase proton affinity, higher pKa and higher pKb for deprotonation values observed for 1,14-diaza[5]helicene, as well as the absorbance maximum in the frequency close to that of the lasers used, strongly favor a MAILD type of ionization when it is mixed with an acidic analyte and leads to the formation of a conjugated acid/base ion pair. Upon UV laser irradiation, the ion pair absorbs light energy and effectively desorbed and a clear spectrum without any matrix related peaks could be visible for the deprotonated analyte. A schematic illustration of the MAILD ionization is given in Fig.3.1 Gas phase “naked ion” A- is detected in negative mode MALDI/TOF-MS analyte matrix “ion pair” formation Fig. 6.1 Suggested mechanism of MAILD type ionization for 1,14-diaza[5]helicene The successful application of 1,14-diaza[5]helicene in the analysis of organic acids in fruit juice and wine samples ( Manuscript-I, fig.1 and Suppl. fig.3) further suggests the feasibility of using of this matrix in quantification of organic acids with the use of stable isotopes, which is one of the current topics of interest in our laboratory. This would open up a relatively untouched area in mass spectrometry where MALDI-MS is rarely employed in quantification of small molecules, and thus address the ever- present demand for a high throughput analytical tool for quantification in the pharmaceutical arena. Therefore, our findings on novel MALDI matrices for small molecule analysis not only demonstrate the versatility of MALDI-MS beyond its conventional uses in high- molecular weight compound analysis, but also open up new venues for high throughput metabolomics. Identification of bioactive secondary metabolites from medicinal plants in Sri Lanka Although, the flora of Sri Lanka is enriched with a vast number of biologically active secondary metabolites which have been investigated for antimicrobial, insecticidal, piscicidal, immunomodulatory etc. properties (Hewage, et al., 1997; Hewage, et al., 1998), a large portion of plants still remains unexplored. Plants and their products have been extensively used in indigenous medicine and most of the domestic supply of plants comes from the wild, causing overharvesting of populations from their natural habitats. In addition, increased demand for agricultural land and unsustainable cultivation practices are destroying the natural habitats of medicinal plants and as a result some of the medicinal plants (eg. Munronia pinnata) are in the threat of being endangered (Wijesundera, 2004). Even though, isolation and structural elucidation of new natural products from these medicinal plants and investigation of their bioactivities is rewarding, the collection of plant material for conventional natural product isolation strategies is becoming unfeasible due to the scarcity of plants. In addition, putting more efforts into isolating each compound in pure form by performing tedious multi-step separation and purification processes are becoming less attractive since these approaches are no longer cost-effective and time-effective. Thus, the emergence of online characterization methodologies of secondary metabolites in crude natural product extracts or fractions has provided a solution to these long persisting problems and made life easier for natural product chemists. Online characterization of secondary metabolites in crude natural product extracts or fractions demands high degree of sophistication, richness of structural information, sensitivity, and selectivity. The remarkable improvements in hyphenated analytical methods offer shorter analysis time, higher degree of automation, higher sample throughput, better reproducibility, enhanced selectivity and therefore a higher degree of information (Joshi, et al., 2012). In particular, the addition of Orbital trap mass analyzer to the arsenal of mass spectrometric analyzers supports a wide range of applications from routine compound identification to the analysis of trace-level components in complex mixtures, permitting the characterization of natural products directly from the crude extract with a minimal amount of material (Perry, et al., 2008; Hu, et al., 2005). Manuscripts-II and III summarize our achievements in the application of MS-based rapid screening strategies for characterization of bioactive metabolites in two popular medicinal plants in Sri Lanka that are neither phytochemically nor pharmacologically explored yet. Twelve medicinal plants were initially selected for this study based on their use in the traditional system of medicine in Sri Lanka and only a few grams (maximum 20 g) of plant materials were used in the preparation of crude extracts which were then subjected to several bio-assays such as 5-lipoxygenase inhibition, microsomal prostaglandin E2 synthase-1 inhibition, cell viability of human leukemic cells, antioxidant and reactive oxidant scavenging assays. Out of the tested crude extracts, the most active four extracts were subjected to partial fractionation by silica gel column chromatography and the resulted fractions were tested for the 5-lipoxygenase inhibitory activity. The most active fractions that exhibited 5-lipoxygenase inhibition at a concentration of 1µg/mL were thoroughly analyzed to identify the presence of possible bioactive compounds. In this investigation, UHPLC system coupled to LTQ Orbitrap XL instrument and GC-MS have been employed as sole techniques in structural characterization of secondary metabolites. The accurate mass measurements of adduct ions by the Orbitrap instrument enabled the determination of molecular composition within 1-5 ppm mass errors and database searching of exact masses for possible relevant secondary metabolites. MS/MS data has provided powerful tool to dereplicate possible structures. To obtain more clear overview, the fragments resulted from MS/MS experiments were further analyzed by computer assisted algorithms to yield hypothetical fragmentation trees which allow the assignment of specific relevant fragments and fragmentation pathways. As emphasized in Manuscript-II, the traditional use of the Sri Lankan endemic plant Plectranthus zeylanicus for the treatment of inflammatory conditions could be rationalized with our phytochemical findings. In addition to the identification of several known compounds that have been already studied for their anti-inflammatory activities, the detection of coleone P in the active fraction has immense significance. This is not only because of its occurrence in this plant is reported for the first time, but also due to the high possibility of this compound to exhibit anti-inflammatory or anti-proliferative properties. Since coleon variety of diterpenoids which are characteristic to genus Plectranthus have already demonstrated several bioactivities including antiproliferative activity (Marques, et al., 2002; Xing et al., 2008), a detailed study on the bioactivities of coleone P would definitely inspire the pharmacological community. Manuscript-III describes our scientific investigations on one of the most valued and rare medicinal plants in Sri Lanka, which has never undergone thorough phytochemical analysis, despite the numerous efforts made over the years. This first ever report on the chemical profile of popular Sri Lankan folk medicinal plant Munronia pinnata, would not have been possible without the advanced hyphenated techniques, as its phytochemical investigations have been severely hampered by the dearth of plant material for traditional natural product extraction strategies. The conventional use of M. pinnata in Sri Lankan indigenous medicine for the treatment of fever and several other inflammatory conditions (Department of Ayurveda, 1979; Jayaweera, 1982) was supported by our chemical profiling. It has revealed the presence of several secondary metabolites with already known anti-inflammatory activities. Although the emphasis in this phase of the study was mainly on the identification of known bioactive metabolites, so as to validate the long-established use of plants in Sri Lankan folk medicine, there’s a great possibility to extend this study towards the identification of unknown compounds in the active extracts and fractions. Even though these compounds are not in any database, the recently introduced automated alignment of fragmentation trees (FT-BLAST, fragmentation tree basic local alignment search tool) could be utilized for the identification process (Rasche, et al., 2012). The computed fragmentation trees which have already been employed in this study for the compound identification provided a good explanation of our observed data, thus, we expect FT-BLAST approach to be a promising tool in the identification of unknowns in the active extracts and fractions. Therefore, our MS based rapid screening of plant extracts not only substantiates the conventional use of medicinal plants in Sri Lanka but also proves that phytochemical screening is no longer as cumbersome as it was a few years before. Identification of sex dependent lipids in Drosophila melanogaster The cuticular chemistry of insects has become a topic of interest with the realization of its involvement in various types of chemical communications (Howard, 1993). Normally, the insect cuticle is covered with complex mixtures of nonpolar and polar compounds out of which hydrocarbons comprise the majority while short-chain unsaturated aldehydes, ketones, fatty acids, and acetate esters of short-chain unsaturated alcohols are also present as minor components (Blomquist and Jackson, 1979). There are several MS based techniques widely employed in the analysis of insect surface lipids. GC-MS is a well-established tool for the detection and structure elucidation of sufficiently volatile nonpolar cuticular hydrocarbons, however larger and more polar cuticular compounds are likely to be missed by this method. Therefore the conventional approach generally relied on hydrolysis of lipids followed by derivatization in to fatty acid methyl esters (FAME) and subsequent GC-MS analysis of released fatty acid derivatives. However, the analysis of FAMEs by GC- MS does not provide complete information about the original lipid molecule, especially how the constituent FAs were bonded to the glycerol backbone. In the case of unsaturated FAs, the double bonds tend to migrate along the aliphatic hydrocarbon chains in the electron ionization mode, challenging the localization of double bond (Mossoba, et al. 1994). Therefore, different methods like analysis of dimethyl disulfide (DMDS) adducts (Dunkelblum, et al., 1985), positive-ion chemical ionization using acetonitrile as reactant gas in GC-ion trap (Moneti, et al., 1996; Oldham and Svatoš, 1999; Van Pelt and Brenna, 1999; Kroiss, et al., 2011) were introduced for the analysis of unsaturated lipids and localization of their double bonds. The emergence of HPLC/APCI-MS as a novel tool offers advantages over GC-MS in the analysis of complex lipid mixtures. The analysis of triacylglycerols (TAG) of higher molecular weight (high equivalent carbon number-ECN) was achieved by optimizing non-aqueous reversed phase HPLC on octadecyl-modified silica (Kofronová, et al., 2009) and successfully applied in the analysis of insect TAG (Cvacka, et al., 2006 a). APCI affords structure related fragment ions in the first MS step. The interpretation of the spectral data has greatly sped up with the development of “TriglyAPCI”, an algorithm for automatic APCI mass spectra interpretation, which uses diacylglycerol fragments and molecular adducts to suggest the structures of TAG while precluding possible mistakes (Cvacka, et al., 2006 b). All these developments significantly contribute in the analysis of even trace components in complex surface lipid extracts, thus uncovering the hidden chemistry of the insect cuticle. Manuscript-IV describes our achievements in studying the sex-dependent surface lipids in Drosophila melanogaster with the help of UHPLC-APCI-MS and GC-MS techniques that shed new light onto the field of Drosophila melanogaster’s cuticular chemistry. The observations on sex dependent differences in composition of cuticular lipids in several Diptera species, have prompted the investigations of surface lipids in D. melanogaster and since then several cuticular hydrocarbons that display sexual dimorphism and perform pheromonal functions have been identified (Jallon, 1984 ; Ferveur, 2005; Everaerts, et al., 2010). However, the attention paid to other types of cuticular substances is not sufficient and until now only a handful of investigations have been conducted to study the non-hydrocarbon components especially fatty acids and fatty acid derivatives. With quantitative differences existing between them, myristic acid, palmitic acid, palmitoleic acid, oleic acid and linoleic acid were identified as major fatty acids from both the acylglycerol and free fatty acid fractions of both sexes. It was observed that there were no significant differences between the fatty acid compositions of either free fatty acids or acylglycerol fatty acids from male and female flies (Jackson, et al., 1981) and no evidence has been reported so far to disprove this observation. However, t his concept was challenged with the identification of female specific 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid and its TAG form, as described in the Manuscript - IV. Even though this triglyceride is present in trace amount in female surface l ipid extract, the LC - APCI - MS enabled its detection and even purification with UHPLC system with a gradient of acetonitrile and propan-2-ol as mobile phases (Manuscript-IV, fig.1 and fig.S1). This peak was virtually absent in samples from males of the same age. Since purification yielded only a low amount of the compound, the NMR data was not sufficient to get the structural details of this complex molecule. Therefore GC-MS analysis has played the key role in characterization of its structure. The accurate MS and MS/MS spectra of the intact TAG obtained by APCI ionization on LTQ Orbitrap XL has been used to determine molecular composition of the unknown TAG. It showed unexpected 19 carbon atoms in the fatty acid chain and nine oxygen atoms. CID experiment has given rise to one diacylglycerol ion [M+H- RiCOOH]+ , one monoacylglycerol ion [M+H-RiCOO-Ri’CO]+ and one acyl ion [RiCO]+ (Manuscript-IV, fig.2 ). It was rationalized that the three identical fatty acids with 19 carbon atoms, three double bonds/cycles and an additional oxygen in the fatty acid chain. The accurate mass GC-MS analysis of the trans-methanolyzed TAG has supported previously determined molecular compositions and provide data for partial localization of the desaturation and oxidation. Subsequent database search has allowed the identification of the constituent fatty acid and thereby well demonstrated the capacity of mass spectrometry in structural characterization of complex molecules that are present in trace amounts. Although, 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid has been reported in some fish oils and vegetable oils ( Vetter, et al., 2012 ; Guth and Grosch, 1991) it has never been detected in any insec t species to the best of our knowledge. There are reports suggesting that the enteric bacteria in fish are responsible for its synthesis and a putative biosynthetic pathway has been proposed ( Shirasaka , et al., 1997) which starts from cis - vaccenic acid, a common precursor of the pheromones in D. melanogaster (Jallon,et al., 1997). Therefore, t he identification 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)non anoic acid and its TAG form in D. melanogaster female flies would instigate the scientific community to conduct investigations on its biosynthesis as well as its biological significance. Therefore, our mass spectrometric approach in identification of sex dependent surface lipids in D. melanogaster not only reveals the presence of female specific 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid and its TAG and hence the existence of qualitative differences in cuticular fatty acid profiles in male and female fli es, but also highlights the necessity of a detailed study on the biosynthesis and physiological functions of this female specific fatty acid. Conclusions This thesis reflects the capacity of mass spectrometry in the analysis of secondary metabolites in complex mixtures with the development and optimization of high throughput analytic methods. The success in development of novel MALDI matrices facilitates the study of low molecular weight metabolites by MALDI-MS and directs towards the exploration its possibilities to employ in high throughput quantifications. The characterization of secondary metabolites in two Sri Lankan medicinal plants which are still regarded as phytochemically unexplored, by UHPLC-Orbitrap in combination with the fragmentation trees as well as by GC-MS analysis contributes to rationalizing the traditional use of these plants, while promising to pharmacologists much room for more detailed bioactivity studies. The identification of the female specific fatty acid and the TAG in Drosophila melanogaster by UHPLC-APCI-MS and GC-MS analysis contradicts the decades-old observations on the absence of qualitative differences in cuticular profiles of male and female flies and has inspired a detailed study of its biosynthesis and physiological functions. In short, the development and optimization of high throughput and efficient approaches turns mass spectrometry into a powerful workhorse for the analysis of nature’s secrets. Chapter 7 Summary Summary Over the years, mass spectrometry has increasingly become an analytical tool of choice in the field of natural products chemistry owing to its high throughput nature, quantitative capability and the facility to integrate with chromatographic separation methods. This study enhances the capacity of mass spectrometry in the analysis of wide variety of complex mixtures with the development of novel MALDI matrices as well as optimizing the existing hyphenated techniques, and thereby cast new light on some pharmacological and ecological aspects that have not been successfully addressed yet. The initial success in development of 1,14-diaza[5]helicene as a novel MALDI matrix for the analysis of acidic analytes demonstrates the applicability of MALDI-MS in small molecule analysis and challenges the perception of MALDI-MS to be a “high molecular mass tool only”. Furthermore, it confirms the recently introduced strategy for the selection of “ionless” matrices based on the Brønsted- Lowry acid-base theory and signifies the importance of rational matrix design. These findings indicate a tremendous potential for studying biological systems and opens up new venues for high throughput targeted metabolomics work. Apart from MALDI- MS, the thesis demonstrates the immense contribution of hyphenated techniques in the study of secondary metabolites as indicated in the chemical profiling of two phytochemically unexplored medicinal plants in Sri Lanka. In comparison to the extracts of other medicinal plants reported in literature, the lipophilic extracts of Plectranthus zeylanicus and Munronia pinnata have exhibited extremely potent 5- lipoxygenase inhibitory activity suggesting a high pharmacological potential of these plants for intervention with 5-LO related disorders. This observation was reinforced by UHPLC-ESI-MS and GC-MS analysis of the active extracts and fractions, which have revealed the presence of several bioactive secondary metabolites together with some compounds for which the knowledge regarding bioactivities are rare. In particular, the identification of coleone P for the first time in P. zeylanicus, would spotlight the pharmacological community for future comprehensive studies, due to the high possibility of this compound to exhibit anti-inflammatory or anti- proliferative properties. The hidden phytochemistry in M. pinnata has been unveiled for the first time and the platform laid by this study will be indispensable for further phytochemical and bioactivity research on this precious and rare medicinal plant in future. Therefore, the MS-based rapid screening of medicinal plants substantiates the traditional use of these plants as anti-inflammatory remedy, while ensures that phytochemical screening is no longer as cumbrous as it was a few years ago. The applications of novel hyphenated techniques could be expanded beyond the discovery of pharmaceuticals, as reflected by the study on complex surface lipids extracts in Drosophila melanogaster . The identification of female specific 9 - (3 - methyl - 5 - pentylfuran - 2 - yl)nonanoic acid and i ts TAG by UHPLC - APCI - MS and GC - MS analysis contradicts the decades - old concept of the absence of qualitative differences between cuticular fatty acid profiles in male and female flies. Despite its occurrence in several other biological sources, the biosyn thesis of this fatty acid is not clear yet, however, it was suggested that the putative biosynthetic pathway starts from cis - vaccenic acid, a common precursor of the pheromones in D. melanogaster. Therefore, the detection of this female specific fatty aci d for first time in an insect would have a great significance and inspire the biologists for detailed study of its biosynthesis and physiological functions. In conclusion, the thesis breaks new grounds in several aspects of natural product chemistry with the development and optimization of high throughput, efficient and robust mass spectrometric approaches. Zusammenfassung In den letzten Jahren wurde die Massenspektrometrie zunehmend zum analytischen Werkzeug der Wahl im Bereich der Naturstoffchemie, dank ihres hohen Potentials in Bezug auf Probendurchsatz und Quantifizierung, sowie die einfache Integrierbarkeit in chromatografische Trennmethoden. Diese Arbeit erweitert die Möglichkeiten der Massenspektrometrie in der Analyse einer Vielzahl von komplexen Stoffgemischen einerseits durch die Entwicklung neuartiger MALDI-Matrixverbindungen und andererseits durch die Optimierung bereits bestehender Techniken. Sie beleuchtet dabei einige pharmakologische und ökologische Sachverhalte, die bisher nicht erfolgreich aufgeklärt wurden. Der anfängliche Erfolg bei der Entwicklung der 1,14- Diaza[5]helicene als einer neuen MALDI-Matrix zur Analyse acider Verbindungen, zeigt die Anwendbarkeit von MALDI-MS in der Untersuchung von kleinen Molekülen und stellt somit die vorherrschende Ansicht, dass MALDI-MS nur „ein Werkzeug für hochmolekulare Verbindungen“ sei, in Frage. Desweiteren bestätigt er die kürzlich eingeführte Auswahlstrategie für „ionenlose“ Matrices gemäß der Säure- Base-Theorie nach Brønsted und Lowry und unterstreicht die Wichtigkeit eines rationellen Vorgehens bei der Entwicklung von Matrices. Diese Ergebnisse deuten ein gewaltiges Potential zur Untersuchung biologischer Systeme an und eröffnen neue Wege für metabolomische Studien mit hohem Durchsatz. Neben der MALDI-MS veranschaulicht die vorliegende Arbeit den immensen Beitrag, den gekoppelte Analysemethoden zur Aufklärung von Sekundärmetaboliten liefern. Dies wird im Erstellen des chemischen Profils von zwei phytochemisch unerforschten Heilpflanzen aus Sri Lanka gezeigt. Im Vergleich zu Extrakten aus anderen dokumentierten Heilpflanzen weisen die lipophilen Extrakte aus Plectranthus zeylanicus und Munronia pinnata extreme Aktivität zur Inhibierung der 5- Lipoxygenase auf und legen damit ein hohes pharmakologisches Potential zur Behandlung von Krankheiten nahe, die mit 5-LO in Zusammenhang stehen. Diese Beobachtungen wurden durch UHPLC-ESI-MS und GC-MS-Analysen der aktiven Extrakte und Fraktionen bekräftigt, welche das Vorhandensein von mehreren bioaktiven Sekundärmetaboliten enthüllte, zusammen mit einigen Verbindungen, über deren Bioaktivität kaum etwas bekannt ist. Insbesondere der erstmalige Nachweis von Coleon P in P.zeylanicus wird Pharmakologen zu umfassenden Studien bewegen, aufgrund der hohen Wahrscheinlichkeit, dass diese Verbindung anti-inflammatorische oder anti-proliferative Eigenschaften besitzt. Die verborgene Phytochemie in M. pinnata wurde erstmalig enthüllt und somit ein Grundstein gelegt, der für zukünftige phytochemische Untersuchungen bezüglich der Bioaktivität dieser seltenen und wertvollen Heilpflanze unverzichtbar sein wird. Das MS-basierte schnelle Screening dieser Heilpflanzen erklärt daher nicht nur ihre traditionelle Verwendung zur Entzündungshemmung, sondern zeigt auch, dass phytochemisches Screening nicht mehr so aufwendig und zeitraubend ist wie noch vor ein paar Jahren. Die Anwendbarkeit von neuartigen gekoppelten Methoden konnte über die Entdeckung von Arzneimitteln hinaus erweitert werden, wie die vorliegende Studie über komplexe lipidhaltige Oberflächenextrakte von Drosophila melanogaster zeigt. Mittels UHPLC-APCI-MS und GC-MS konnte 9-(3-Methyl-5-pentylfuran-2-yl)- nonansäure und ihr entsprechendes Triacylglycerid nachgewiesen werden, jedoch nur bei weiblichen Individuen. Damit konnte das über Jahrzehnte bestehende Konzept widerlegt werden, welchem zufolge es keine qualitativen Unterschiede zwischen der Zusammensetzung cutikularer Fettsäuren zwischen männlichen und weiblichen Fruchtfliegen gibt. Obwohl diese Verbindung auch aus anderen biologischen Quellen bekannt ist, ist deren Biosynthese nicht vollständig aufgeklärt. Jedoch wird angenommen, dass der Biosyntheseweg von cis-Vaccensäure ausgeht, einem typischen Pheromonvorläufer in D. melanogaster. Die erstmalige Entdeckung dieser weibchenspezifischen Fettsäure hat daher eine große Bedeutung und wird Biologen zu detaillierten Studien bezüglich ihrer Biosynthese und ihrer physiologischen Funktion anregen. Abschließend lässt sich sagen, dass die Arbeit durch die Entwicklung und Optimierung von effizienten und zuverlässigen massenspektrometrischen Methoden in einigen Bereichen der Naturstoffchemie neue Wege einschlägt. Chapter 8 References References (for Introduction and Discussion sections) Antony, C., Jallon, J.M., 1982. The chemical basis for sex recognition in Drosophila melanogaster. Journal of Insect Physiology 28, 873–880 Blomquist, G.J., Jackson, L.L., 1979. Chemistry and biochemistry of insect waxes. 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Personen, die mich bei der Auswahl und Auswertung des Materials sowie bei der Fertigstellung der Manuskripte unterstützt haben, sind am Beginn eines jeden Kapitels genannt. Es wurde weder die Hilfe eines Promotionsberaters in Anspruch genommen, noch haben Dritte für Arbeiten, welche im Zusammenhang mit dem Inhalt der vorliegenden Dissertation stehen, geldwerte Leistungen erhalten. Die vorgelegte Dissertation wurde außerdem weder als Prüfungsarbeit für eine staatliche oder andere wissenschaftliche Prüfung noch als Dissertation an einer anderen Hochschule eingereicht. Jena, den 16. September 2014 Mayuri Tharanga Napagoda Erklärung über laufende und frühere Promotionsverfahren Hiermit erkläre ich, dass ich keine weiteren Promotionsverfahren begonnen oder früher laufen hatte. Das Promotionsverfahren an der Biologisch- Pharmazeutischen Fakultät ist mein erstes Promotionsverfahren überhaupt. Jena, den 16. September 2014 Mayuri Tharanga Napagoda Curriculum Vitae 1. Personal Information Name : Mayuri Tharanga Napagoda Nationality : Sri Lankan Date of Birth : 21.11.1976 Place of Birth : Negombo, Sri Lanka Permanent Address : Kandahenawatte, Nittambuwa 11 880, Sri Lanka Email : mayurinapagoda@yahoo.com, mnapagoda@ice.mpg.de 2. Educational Background . March 2010 onwards: PhD student at Friedrich-Schiller-University and Max Planck Institute for Chemical Ecology, Jena, Germany. Dissertation Title: Metabolomics for Natural Products: Fast screening and Discovery . 2002- 2005 : Master of Philosophy - University of Peradeniya, Sri Lanka Dissertation Title: Bioactivity studies of some Sri Lankan flora and bioactive Xanthones from Calophyllum thwaitesii . 2003- 2004 : Postgraduate Certificate Course in Advanced Organic Chemistry, University of Peradeniya, Sri Lanka . 1997-2001 : Bachelor of Science (Special Degree -Botany)- First Class Honours, University of Colombo, Sri Lanka Dissertation Title: Investigation of characteristics of isolates of Rigidiporous microporous, the causative fungus of the white root disease 3. Work Experiences . June 2005- To date: Lecturer in Biochemistry, Faculty of Medicine, University of Ruhuna, Sri Lanka . July 2002- May 2005: Research Assistant, Institute of Fundamental Studies, Kandy, Sri Lanka. . December 2001- June 2002: Teaching Assistant in Botany, Department of Plant Sciences. University of Colombo, Sri Lanka 4. Language Proficiency English - Very Good (IELTS Overall Band Score of 7.5) German - Basic knowledge ( A1+ : 93% , A2 : 80 %) Sinhala - Mother tongue 5. Publications Scientific Articles . Napagoda, M., Rulíšek, L., Jancarík, A., Klívar, J., Šámal, M., Stará, I, G., Starý, I., Šolínová, V., Kašicka, V., Svatoš, A.(2013). Azahelicene Superbases as MAILD Matrices for Acidic Analytes. ChemPlusChem 78, 937-942 . Napagoda, M., Gerstmeier, J., Wesely, S., Popella, S., Lorenz, S., Scheubert, K., Svatoš, A., Werz, O. (2014). Inhibition of 5-lipoxygenase as anti-inflammatory mode of action of Plectranthus zeylanicus Benth and chemical characterization of ingredients by a mass spectrometric approach. Journal of Ethnopharmacology 151, 800-809 . Napagoda, M., Gerstmeier, J., Koeberle, A., Wesely, S., Popella, S., Lorenz, S., Scheubert, K., Boecker, S., Svatoš, A., Werz, O. (2014). Munronia pinnata (Wall.) Theob.: Unveiling phytochemistry and dual inhibition of 5- lipoxygenase and microsomal prostaglandin E2 synthase (mPGE)-1. Journal of Ethnopharmacology 151, 882-890 . Napagoda, M., Weißflog, J., Lorenz, S, Svatoš, A. Identification of female specific fatty acid derivatives in Drosophila melanogaster surface lipid extracts. To be submitted to ChemBioChem . Dharmaratne, H.R.W., Napagoda, M.T., Tennakoon, S.B. (2009); Xanthones from root bark of Calophyllum thwaitesii and their Bioactivity, Natural Products Research 23(6),539 - 545 Talk Presentations . Napagoda, M., Svatoš, A. (2012). Identification of Sex Dependent Surface Lipids in Drosophila melanogaster. 11th IMPRS Symposium, MPI for Chemical Ecology, Dornburg, Germany . Napagoda, M.T., Jayaweera, S., Thevanesam, V., Dharmaratne, H.R.W., (2007). Antimicrobial properties of some Sri Lankan plants. Abstracts for the 4th Academic Sessions, University of Ruhuna. Matara, Sri Lanka . Napagoda, M.T., Tennakoon, S.B., Thevanesam, V., Dharmaratne, H.R.W., (2006). Xanthones from roots of Calophyllum thwaitesii and their Bioactivity. 3rd Academic Sessions, University of Ruhuna, Matara, Sri Lanka . Haroon, M.H., Premaratne, S.R., Napagoda, M.T., Dharmaratne H.R.W., (2006). Chemistry and bioactivity studies of Ulva lactuca, 62nd Annual Sessions of Sri Lanka Association for Advancement of Science, Colombo, Sri Lanka . Napagoda, M.T., Balasuriya, B.M.G.K., Dharmaratne, H.R.W., (2005). Inhibitory activities of some plant extracts upon germination of lettuce, 61st Annual Sessions of Sri Lanka Association for Advancement of Science, Colombo, Sri Lanka . Napagoda, M.T., Dharmaratne, H.R.W., Tennakoon, S.B., (2004). Antifungal activity and free radical scavenging property of xanthones from Calophyllum thwaitesii. 60th Annual Sessions of Sri Lanka Association for Advancement of Science, Colombo, Sri Lanka . Napagoda, M.T., Dharmaratne, H.R.W., Tennakoon, S.B., (2003). Antifungal activity of xanthones from Calophyllum thwaitesii. 59th Annual Sessions of Sri Lanka Association for Advancement of Science, Colombo, Sri Lanka Poster Presentations . Napagoda, M., Stará, I., Svatoš, A., (2013) 1,14-diaza[5]helicene : A novel MALDI matrix for the analysis of acidic metabolites. 46. Jahrestagung der Deutschen Gesellschaft für Massenspektrometrie, Humboldt-Universität Berlin, Berlin, Germany . Napagoda, M., Svatoš, A., (2013) Exploration of low - molecular - weight metabolites by MALDI - MS: Development of novel MALDI matrices . IMPRS Evaluation Symposium 2013/ 12th IMPRS Symposium, MPI for Chemical Ecology, Jena, Germany . Weißflog, J., Napagoda, M., Svatoš, A.,(2012). New matrices for negative mode MALDI-MS in the low mass region. SAB Meeting 2012, MPI for Chemical Ecology, Jena, Germany . Napagoda, M., Stará, I., Svatoš, A., (2012). Azahelicenes as MALDI matrices for acidic analytes. Joint Conference of German and Polish Mass Spectrometry Societies, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland . Napagoda, M., Svatoš, A., (2011). Identification of Sex Dependent Lipids in Drosophila Fruit flies. ICE Symposium, MPI for Chemical Ecology, Jena, Germany . Napagoda, M., Svatoš, A., (2011) Development of fast screening and imaging methods for the analysis of natural products. 10th IMPRS Symposium, MPI for Chemical Ecology, Dornburg, Germany . Haroon, M.H., Premaratne, S.R., Napagoda, M.T. Dharmaratne H.R.W., (2006). Inhibitory activities of some Sea weed extracts upon germination of lettuce. Poster sessions for the 10th Anniversary celebrations of Post Graduate Institute of Science, University of Peradeniya, Sri Lanka 6. Awards - International Max Planck Research School(IMPRS) fellowship for PhD studies at Max Planck Institute for Chemical Ecology/ Friedrich-Schiller- University, Jena, Germany (2010-2013) - Hiran Tillekeratne Award for the Outstanding Postgraduate Research in Medicine -University Grants Commission, Sri Lanka (2008) - Kandiah Memorial Graduateship Award for the Outstanding Postgraduate Research in Chemistry – Institute of Chemistry Ceylon, Sri Lanka (2006) - Award for the Best Scientific Paper (Oral) in Medicine - Third Academic Sessions, University of Ruhuna, Sri Lanka (2006) - Swarna Senathirajah Memorial Award for Genetics and Plant Breeding- University of Colombo, Sri Lanka (2001) Jena, 16th September 2014 Mayuri Tharanga Napagoda Acknowledgements I acknowledge with deep gratitude and appreciation, the guidance, kindness and encouragement extended to me by all the helpful hands in making this venture a success. My most sincere gratitude goes to my principal supervisor Dr. Aleš Svatoš, Group Leader-Research group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology- Jena, who gave me the confidence to undertake a research project in mass spectrometry and help me with his excellent guidance, invaluable advice and constant encouragement throughout the study. I feel privileged having had opportunity to work with him and thereby acquire knowledge and experiences in a field which was completely new to me. I’m very grateful to my co-supervisors, Dr. Bernd Schneider, Group Leader- Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology- Jena and Prof. Dr. Hans-Peter Saluz, Department of Cell and Molecular Biology, Leibniz Institute for Natural Product Research and Infection biology- Jena, for their direction, constructive suggestions, enthusiastic encouragement and useful critiques that were indispensable towards the success of my research work. I am deeply indebted to Prof. Oliver Werz, Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University-Jena, for contributing his expertise in the field of pharmaceutical chemistry for a successful collaboration. His kind and willing help rendered to me in the bioactivity studies and the enormous support from his team, Jana Gerstmeier, Sandra Wesely and Sven Popella are sincerely appreciated. I would like to extend my deep gratitude to Dr. Irena Stará, Dr. Lubomír Rulíšek, Andrej Jancarík, Jirí Klívar, Michal Šámal, Dr. Ivo Starý, Dr.Veronika Šolínová, Dr. Václav Kašicka, Dr. Olivier Songis and Dr. Jirí Míšek at the Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic for an excellent collaboration which led to a fruitful outcome in my studies on novel MALDI matrices. I am grateful to Dr. Josef Cvacka at the Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic as well as Dr. Kathrin Steck , Dr. Markus Knaden, Dr. Marco Schubert, Tom Retzke and the members of the Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology - Jena for their invaluable support to make my research studies on Drosophila fruit flies a success. I would also like to thank Prof. Sebastian Boecker and Ms. Kerstin Scheubert, Chair for Bioinformatics, Friedrich-Schiller-University- Jena, for their enthusiastic cooperation for the bioinformatics component of my studies. I make use of this opportunity to convey my appreciation to all my colleagues past and present at the Research group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology – Jena, for their kind help, great patience and constant encouragement. My special thanks are extended to Dr. Jerrit Weißflog for his help in the German translation of the summary of this thesis as well as for sharing his chemistry knowledge with me whenever necessary, Ms. Sybille Lorenz for her endless assistance which was not restricted to the laboratory level only and Dr. Marco Kai for helping me in numerous ways during my research work. I would also thank Ms. Ellen Hascher, Max Planck Institute for Chemical Ecology - Jena for her kind help in proof reading the German translation of the summary of my thesis and also for her great patience and efforts in the process of improving my skills for a better communication in German language. Dr. Karin Groten, Scientific Coordinator, Max Planck Institute for Chemical Ecology – Jena, is gratefully appreciated for her invaluable assistance and advices which helped me to tackle the problems I came across during my studies in Jena. International Max Planck Research School and the Max Planck Society is sincerely acknowledged for providing me with a PhD fellowship and the Max Planck Institute for Chemical Ecology – Jena is also thanked for providing laboratory facilities for my research work. My life in Jena would not be nice without all the wonderful friends, I’ve met during the last three years. My special thanks are extended to Amol Fatangare, Mina Dost, Joy Michal Johnson, Ravi Kumar and Filip Kaftan who were with me all the time, for their sincere friendship and the tremendous support in numerous ways throughout my stay in Jena. Last but not least, I would like to thank all the staff members at the Max Planck Institute for Chemical Ecology – Jena for helping me in various ways to make my study a success.