Publications (59) View all
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Article: Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation.
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ABSTRACT: The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.International Journal of Peptide Research and Therapeutics 03/2013; 19(1):43-54. · 0.99 Impact Factor -
Article: The traditional medical uses and cytotoxic activities of sixty-one Egyptian plants: Discovery of an active cardiac glycoside from Urginea maritima.
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ABSTRACT: ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants from the Sinai desert are widely used in traditional Bedouin medicine to treat a range of conditions including cancers, and may thus be useful sources of novel anti-tumor compounds. Information on plants used in this way was obtained through collaboration with Bedouin herbalists. AIM OF THE STUDY: To document the traditional uses of 61 species from 29 families of Egyptian medicinal plants and to investigate their biological activity using a cytotoxicity assay. MATERIAL AND METHODS: MeOH extracts of the 61 plant species investigated were dissolved in 10% DMSO and their cytotoxic activity was evaluated. The extracts were tested in duplicate on three separate occasions at three different concentrations (1, 10 and 100µg/ml) against human lymphoma U-937 GTB. The most active extract was subjected to bioassay-guided fractionation using HPLC and LC/ESI-MS to isolate and identify its active components. RESULTS AND DISCUSSION: The most potent extracts were those from Asclepias sinaica, U. maritima, Nerium oleander and Catharanthus roseus, followed by those from Cichorium endivia, Pulicaria undulate and Melia azedarach. Literature reports indicate that several of these plants produce cardiac glycosides. Bioassay-guided fractionation of alcoholic U. maritima extracts led to the isolation of a bioactive bufadienolide that was subsequently shown to be proscillaridin A, as determined by 1D and 2D NMR spectroscopy. This result demonstrates the value of plants used in traditional medicine as sources of medicinally interesting cytotoxic compounds. The paper concludes with a brief discussion on the integration of traditional medical knowledge into the existing literature.Journal of ethnopharmacology 12/2012; · 2.32 Impact Factor -
Article: Bioassay-guided Supercritical Fluid Extraction of Cyclooxygenase-2 Inhibiting Substances in Plantago major L.
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ABSTRACT: INTRODUCTION: Selective extraction of plant materials is advantageous for obtaining extracts enriched with desired constituents, thereby reducing the need for subsequent chromatography purification. Such compounds include three cyclooxygenase-2 (COX-2) inhibitory substances in Plantago major L. targeted in this investigation: α-linolenic acid (α-LNA) (18:3 ω-3) and the triterpenic acids ursolic acid and oleanolic acid. OBJECTIVE: To investigate the scope for tuning the selectivity of supercritical fluid extraction (SFE) using bioassay guidance, and Soxhlet extraction with dichloromethane as solvent as a reference technique, to optimise yields of these substances. METHOD: Extraction parameters were varied to optimise extracts' COX-2/COX-1 inhibitory effect ratios. The crude extracts were purified initially using a solid phase extraction (SPE) clean-up procedure and the target compounds were identified with GC-MS, LC-ESI-MS and LC-ESI-MS(2) using GC-FID for quantification. RESULTS: α-LNA was preferentially extracted in dynamic mode using unmodified carbon dioxide at 40°C and 172 bar, at a 0.04% (w/w) yield with a COX-2/COX-1 inhibitory effect ratio of 1.5. Ursolic and oleanolic acids were dynamically extracted at 0.25% and 0.06% yields, respectively, with no traces of (α-LNA) and a COX-2/COX-1-inhibitory effect ratio of 1.1 using 10% (v/v) ethanol as polar modifier at 75°C and 483 bar. The Soxhlet extracts had ursolic acid, oleanolic acid and αLNA yields up to 1.36%, 0.34% and 0.15%, respectively, with a COX-2/COX-1 inhibitory effect ratio of 1.2. CONCLUSION: The target substances can be extracted selectively by bioassay guided optimisation of SFE conditions. Copyright © 2012 John Wiley & Sons, Ltd.Phytochemical Analysis 09/2012; · 2.63 Impact Factor -
Article: Chemical composition and repellency of essential oils from four medicinal plants against Ixodes ricinus nymphs (Acari: Ixodidae).
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ABSTRACT: In our search for effective tick repellents from plant origin, we investigated the effect of essential oils of four medicinal and culinary plants belonging to the family Lamiaceae on nymphs of the tick Ixodes ricinus (L.). The essential oils of the dry leaves of Rosmarinus officinalis (Rosemary) (L.), Mentha spicata (Spearmint) (L.), Origanum majorana (Majoram) (L.), and Ocimum basilicum (Basil) (L.) were isolated by steam distillation and 15 microg/cm2 concentration of oils was tested against ticks in a laboratory bioassay. The oils of R. officinalis, M. spicata, and O. majorana showed strong repellency against the ticks 100, 93.2, and 84.3%, respectively, whereas O. basilicum only showed 64.5% repellency. When tested in the field, the oils of R. officinalis and M. spicata showed 68.3 and 59.4% repellency at a concentration of 6.5 microg/cm2 on the test cloths. The oils were analyzed by gas chromatography mass spectrometry and the major compounds from the most repellent oils were 1,8-cineole, camphor, linalool, 4-terpineol, borneol, and carvone.Journal of Medical Entomology 09/2012; 49(5):1067-75. · 1.76 Impact Factor -
Article: An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS
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ABSTRACT: Cyclotides are mini-proteins of approximately 30 amino acid residues that have a unique structure consisting of a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. This unique cyclotide structure provides exceptional stability to chemical, enzymatic and thermal treatments and has been implicated as an ideal drug scaffold for the development into agricultural and biotechnological agents. In the current work, we present the first method for microwave assisted Fmoc-SPPS of cyclotides. This protocol adopts a strategy that combines optimized microwave assisted chemical reactions for Fmoc-SPPS of the peptide backbone, the cleavage of the protected peptide and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. To exemplify the utility of this protocol in the synthesis of a wide array of different cyclotide sequences we synthesized representative members from the three cyclotide subfamilies—the Möbius kalata B1, the bracelet cycloviolacin O2 and the trypsin inhibitory MCoTI-II. In addition, a “one pot” reaction promoting both cyclization and oxidative folding of crude peptide thioester was adapted for kalata B1 and MCoTI-II. KeywordsCyclotides-Microwave chemistry-Fmoc-SPPS-Circular proteins-Cystine knot-Native chemical ligationInternational Journal of Peptide Research and Therapeutics 04/2012; 16(3):167-176. · 0.99 Impact Factor
