Konstantina Vougogiannopoulou

Recent advances and new strategies in the NMR-based identification of natural products.

Nature comprises an untapped pool of unique compounds with high structural uniqueness and exceptional properties. At the core of natural products (NPs) discovery is the identification procedure and NMR remains the most efficient method. Technical improvements such as miniaturized and crycogenic NMR probes along with hyphenation capabilities and computational support are at the center of evolution. Concepts such as dereplication and metabolomics are increasingly adopted in NPs using the power of databases, currently fragmented. The introduction and utilization of these technical and computational implements could lead NPs research to more comprehensive structure identification and new holistic perspectives.

Identification of the Coloring Constituents of Four Natural Indigoid Dyes

Abstract A high performance liquid chromatography (HPLC) method combined with spectrophotometric UV‐Vis detection is developed for the separation and identification of seven indigoid coloring compounds: indigotin, which is commercially available, and indirubin, 6‐bromoindigotin, 6′‐bromoindirubin, 6‐bromoindirubin, 6,6′‐dibromoindigotin, and 6,6′‐dibromoindirubin, which are synthesized to be used as reference compounds in the HPLC analysis. The chromatographic method is employed for the identification of the blue/purple coloring compounds in samples extracted from four mollusks, which have been used for the production of Tyrian Purple since antiquity: Hexaplex trunculus, Murex brandaris, Nucella lapillus, and Thais haemastoma. The composition of the analyzed samples, with respect to the reference materials, is compared and discussed. All seven indigoids are identified in Hexaplex trunculus and some of them are identified in the other three purple extracts.

One-step semisynthesis of oleacein and the determination as a 5-lipoxygenase inhibitor.

The dialdehydes oleacein (2) and oleocanthal (4) are closely related to oleuropein (1) and ligstroside (3), the two latter compounds being abundant iridoids of Olea europaea. By exploiting oleuropein isolated from the plant leaf extract, an efficient procedure has been developed for a one-step semisynthesis of oleacein under Krapcho decarbomethoxylation conditions. Highlighted is the fact that 5-lipoxygenase is a direct target for oleacein with an inhibitory potential (IC50: 2 μM) more potent than oleocanthal (4) and oleuropein (1). This enzyme catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes. Taken together, the methodology presented here offers an alternative solution to isolation or total synthesis for the procurement of oleacein, thus facilitating the further development as a potential anti-inflammatory agent.

The raputindoles: novel cyclopentyl bisindole alkaloids from Raputia simulans.

A novel class of bisindole alkaloids is established by the isolation and structural determination of raputindoles A-D (1-4) from the Amazonian plant Raputia simulans Kallunki (Rutaceae). Complete spectroscopic characterization was accomplished by means of NMR spectroscopy and APCI (+) HRMS. Raputindoles A-D possess a cyclopentyl moiety fused on the benzene part of the indole ring, originating from the combination of prenylated indole monomers.

From drug screening to target deconvolution: A target-based drug discovery pipeline using Leishmania casein kinase 1 isoform 2 to identify compounds with anti-leishmanial activity.

ABSTRACT Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.

Indirubin derivatives: a patent review (2010 – present)

Introduction: Indirubins are bisindole alkaloids naturally occurring in indigo-bearing plants or in mollusks from the Muricidae family. They belong to the rather small family of indigoids, which has nevertheless found an extreme importance in the fields of dyes and medicinal chemistry. Indirubin has been found to be the active ingredient of a traditional Chinese Medicine used to treat the symptoms of leukemia. Further biological explorations revealed the ability of indirubin to bind cyclin-dependent kinases and 6-bromoindirubin, extracted from mollusks, to bind glycogen synthase kinase-3. The high affinity displayed by the two natural products has opened a vast field of research and triggered the development of hundred of derivatives with biological activities. Areas covered: The traditional use of indirubin for the treatment of leukemia has prompted different research groups to study the cytotoxic effect of indirubin derivatives on both solid tumors and leukemia. Moreover, the affinity of indirubins for kinases also allowed the exploration of their activity towards stem cells. Expert opinion: The derivatives presented are in accordance with first discoveries and establish the close relation between activity and kinase inhibition. New derivatives have been patented and new interferences in signaling pathways are described. However, few in vivo studies have been performed and more efficient solutions are needed to unravel the major issue of solubility.

Chemical and Biological Investigation of Olive Mill Waste Water - OMWW Secoiridoid Lactones.

Olive mill waste water is the major byproduct of the olive oil industry containing a range of compounds related to Olea europaea and olive oil constituents. Olive mill waste water comprises an important environmental problem in olive oil producing countries, but it is also a valuable material for the isolation of high added value compounds. In this study, an attempt to investigate the secoiridoid content of olive mill waste water is described with the aid of ultrahigh-performance liquid chromatography-electrospray ionization (±)-high-resolution mass spectrometry and centrifugal partition chromatography methods. In total, seven secoiridoid lactones were isolated, four of which are new natural products. This is the first time that a conjugate of hydroxytyrosol and a secoiridoid lactone has been isolated from olive mill waste water and structurally characterized. Furthermore, the range of isolated compounds allowed for the proposal of a hypothesis for the biotransformation of olive secoiridoids during the production of olive mill waste water. Finally, the ability of the representative compounds to reduce the intracellular reactive oxygen species was assessed with the dichlorofluorescein assay in conjunction with the known antioxidant agent hydroxytyrosol.

Phytochemical Composition of the Decoctions of Greek Edible Greens (Chórta) and Evaluation of Antioxidant and Cytotoxic Properties

Wild or semi-wild edible greens (chórta) are an integral part of the traditional Greek Mediterranean diet due to their nutritional value, containing various phytonutrients beneficial to human health. Water-based decoctions of chórta are widely consumed in Greek alternative medicine as health promoting agents. This study examined the chemical profile of the decoctions of eight edible plants, Cichorium intybus, C. endivia, C. spinosum, Crepis sancta, Sonchus asper, Carthamus lanatus, Centaurea raphanina, and Amaranthus blitum, by UPLC-ESI-HRMS and HRMS/MS analysis, to determine possibly bioactive constituents. The profiles of the plants from the Asteraceae family are dominated by the presence of phenolic acids and flavonoid derivatives, whereas the A. blitum decoction is rich in triterpene saponins. Interestingly, the Centaurea raphanina decoction was found to be extremely rich in flavanones, particularly in the aglycone pinocembrin. Further phytochemical investigation and fractionation of this extract resulted in the isolation and identification of five compounds: phlorin (1), syringin (2), pinocembrin (3), pinocembroside (4), and pinocembrin-7-O-neohesperidoside (5). The extracts were also tested for their antioxidant and differential cytotoxic activity against tumor cells. C. raphanina was found to be differentially toxic against metastatic tumor cells. In conclusion, we found that Greek edible greens are a rich source of bioactive secondary metabolites and their consumption could contribute to the maintenance of overall health.

Rare bisindole alkaloids from the Amazonian tree Raputia simulans.

The stem bark of Raputia simulans (Rutaceae) has been reported to contain simple and dimeric indole alkaloids. Further phytochemical investigation of R. simulans stem bark resulted in the isolation of three new alkaloids. These compounds represent a relatively new category of dimeric indole alkaloids with a cyclohexene moiety in their core. Their structure elucidations were based on NMR and HR‐MS techniques, while structural aspects concerning their relative configuration were investigated using molecular mechanics calculations and NOESY experiments.