Isolation and characterization of the female sex pheromone of the sugar beet cyst nematode, heterodera schachtii, and an analysis of male precopulatory behaviour /

The sugar beet cyst nematode, Heterodera schachtii, is a major agricultural pest. The disruption of the mating behaviour of this plant parasite in the field may provide a means of biological control, and a subsequent increase in crop yield. The H. schachtii female sex pheromone, which attracts homospecific males, was collected in an aqueous medium and isolated using high performance liquid chromatography. Characterization of the male-attractive material revealed that it was heat stable and water soluble. The aqueous medium conditioned by female H. schachtii was found to be biologically active and stimulated male behaviour in a concentration dependent manner. The activity of the crude pheromone was specific to males of H. schachtii and did not attract second stage juveniles. Results indicated that vanillic acid, a putative nematode pheromone, is not an active component of the H. schachtii sex pheromone. Male H. schachtii exhibited stylet thrusting, a poorly understood behaviour of the male, upon exposure to the female sex pheromone. This behaviour appeared to be associated with mate-finding and was used as a novel indicator of biological activity in bioassays. Serotonin, thought to be involved in the neural control of copulatory behaviour in nematodes, stimulated stylet thrusting. However, the relationship between stylet thrusting induced by the sex pheromone and stylet thrusting induced by serotonin is not clear. Extracellular electrical activity was recorded fi-om the anterior region of H. schachtii males during stylet thrusting, and appeared to be associated with this behaviour. The isolation of the female sex pheromone of H. schachtii may, ultimately, lead to the structural identification and synthesis of the active substance for use in a novel biological control strategy.

The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3

Total synthesis of (S)-4-hydroxy-[a]-Lapachone

(S)-4-Hydroxy-a-lapachone has been prepared for the first time. The commercially available compound 2-acetyl-1-naphthol was used as the starting material. The synthesis involved methylation, followed by Baeyer-Villiger oxidation, and hydrolysis of the acetate to give 1-methoxy-2-naphthol. After protecting of the hydroxyl group, t-BuLi was used to form 3-(3',3'-dimethyl-acryloyl)-1- meth oxy-2- (meth oxymethoxy)-naphthalen e. eycl izationand oxidation then gave 4-keto-a-lapachone. Finally enzymic biotransformation by Mortierella isabellina ATCC 42613 was used to yield the target compound. The enantiomeric excess of the product was determined to be ~98% by using 1H NMR chiral shift analysis. The overall yield is 80/0. The biological activity of (S)-4-hydroxy-alapachone and its acetate are under investigation.

The synthesis of a-Tocohexaenol, a new fluorescent analogue of a-Tocopherol

Since its discovery in 1922, vitamin E has been widely investigated for its role as a powerful, chain-breaking antioxidant that is required for human health. However, some basic issues still remain unclear, such as the mechanism and dynamics of the intracellular trafficking of a-tocopherol. To better understand tocopherol's biological activity at the cellular level, fluorescence spectroscopy and microscopy have been found to be valuable tools. This thesis reports the synthesis of a new fluorescent analogue of a-tocopherol, atocohexaenol, an intrinsically fluorescent analogue of a-tocopherol. Different methodologies of preparation have been attempted and a strategy using a preformed chromanol head plus ClO and Cs portion of the polyene side chain finally provided us the desired a-tocohexaenol. a-Tocohexaenol shows a strong fluorescence in both ethanol and hexanes with maximum Aab = 368 nm and maximum /...em = 521 nm. This compound is stable for a couple of weeks in ethanol or hexane solution if stored at 0 °C and protected form light. It decomposes slowly at room temperature and light will accelerate its decomposition (within 5 hours). Thus, a-Tocohexaenol may be a useful fluorescent probe to study the biochemistry and cell biology of vitamin E.

Stereoselective synthesis of 5-substituted pyrrolo[1,2-c]imidazol-3-ones. Access to aldosterone synthase inhibitors and chiral precatalysts

Compounds containing the pyrrolidine moiety are key substructures of compounds with biological activity and organocatalysts. In particular, annulated chiral pyrrolidines with alpha stereogenic centers have aldostereone synthase inhibition activity. In addition, 5-substituted pyrroloimidazol(in)ium salts precursors to N-heterocyclic carbene (NHC) precatalysts are rare due to a lack of convenient synthetic routes to access them. In this thesis is described a rapid synthesis of NHC precursors and a possible route to 5-substituted pyrroloimidazole biologically active compounds. The method involves the preparation of chiral saturated and achiral unsaturated pyrrolo[I,2- c]imidazol-3-ones from N-Cbz-protected t-Butyl proline carboxamide. The resulting starting materials may be used to prepare the target chiral annulated imidazol(in)ium products by a two-step sequence involving first stereoselective lithiation-substitution, followed by POCh induced salt formation.