Structures of New Alkaloids from Rain Forest Trees Galbulimima belgraveana and Galbulimima baccata in Papua New Guinea, Indonesia, and Northern Australia.

Following on our 60-year research on the chemical constituents of the rain forest trees Galbulimima belgraveana and Galbulimima baccata, we report the isolation of seven new alkaloids: GB14 (14), GB22 (15), GB25 (16), GB21 (17), GB23 (18), GB24 (19), and GB26 (20). Their structures were elucidated by a combination of spectroscopic analyses and single-crystal X-ray crystallography, as well as structure degradation and interconversion. The newly isolated alkaloids are precursors or derivatives of the known family members from our early studies and could be intermediates in the biosynthesis of the Galbulimima alkaloids. Therefore, the present study has expanded the range of structures in this family of alkaloids and provided some missing links in the biosynthetic sequences.

Improving a natural enzyme activity through incorporation of unnatural amino acids.

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

Using a genetically encoded fluorescent amino acid as a site-specific probe to detect binding of low-molecular-weight compounds.

Development of enzyme inhibitors requires an activity assay for the identification of hits and lead compounds. To determine dissociation constants in a straightforward manner, we explored the use of a genetically encoded fluorescent amino acid for site-specific tagging of the target protein. The unnatural amino acid 7-(hydroxy-coumarin-4-yl) ethylglycine (Hco) was site-specifically incorporated in the target protein by cell-free protein synthesis using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair. Using the West Nile virus nonstructural protein 2B-nonstructural protein 3 protease as the target protein, the fluorescence of Hco-tagged samples proved to be exquisitely sensitive to the presence of inhibitors and small ligand molecules if they bind in the vicinity of the Hco residue. No significant change in fluorescence was observed when the ligand-binding site was far from the Hco residue. Hco-tagged proteins thus combine outstanding sensitivity with accurate information on the site of binding, making Hco labeling an attractive tool in drug discovery.

4-Alkoxycarbonyl- and aminocarbonyl-substituted isoxazoles as masked acrylates and acrylamides in the asymmetric synthesis of delta2-isoxazolines.

4-Alkoxycarbonyl and aminocarbonyl-substituted isoxazoles undergo conjugate reduction to give delta2-isoxazolines on treatment with sodium borohydride and sodium trifluoroacetoxyborohydride, respectively. They are also alkylated at C5 through sonication with secondary and tertiary alkyl iodides in the presence of zinc dust and copper(I) iodide. These reactions are analogous to those observed with acrylates and acrylamides. The behavior is characteristic of the 4-substituted isoxazoles but not the 5-substituted regioisomers. The reductions of 4,5-disubstituted isoxazoles and the C5 alkylations of 4-substituted isoxazoles generally afford trans-4,5-disubstituted isoxazolines. Incorporating chiral auxiliaries into the alkoxycarbonyl group maintains this relative stereoselectivity. It does not provide significant levels of asymmetric induction in the reductions, but the alkylations occur with good levels of stereocontrol at both C4 and C5. Because both enantiomers of the auxiliaries are available, this provides access to either enantiomer of the products, in 93 to > or = 98% de. The methodology, therefore, provides a complementary approach to nitrile oxide cycloadditions to alkenes for the asymmetric synthesis of delta2-isoxazolines.

Quantification of sideroxylonals in Eucalyptus foliage by high-performance liquid chromatography.

This paper describes the extraction and quantification of sideroxylonals, a group of formylated phloroglucinol compounds found in the foliage of some eucalypt species. Samples of dry, ground foliage were Soxhlet-extracted with light petroleum spirit:acetone (4:1) and the resultant extract analysed (in the presence of internal standard) by reversed-phase HPLC without further purification. The yield of sideroxylonals was exponential with time and showed an inflection at ca. 4 h of extraction. It is recommended that samples be extracted for 6 h, giving a 92% recovery of the sideroxylonals. The title compounds deteriorate under various conditions, e.g. 10% are lost when foliage is oven-dried at 40 degrees C compared to freeze-drying. Storing samples in mobile phase led to a slow deterioration of sideroxylonals with a 7% loss after 4 days, while 22% of these compounds were lost from dry, ground eucalypt leaf stored at room temperature for 20 months.

Two major saponins from seeds of Barringtonia asiatica: putative antifeedants toward Epilachna sp. larvae.

Two major saponins have been isolated from a methanol extract of the seeds of Barringtonia asiatica, and their structures elucidated (mainly by two-dimensional NMR spectroscopy) as 3-O-[[beta-D-galactopyranosyl(1-->3)-beta-D-glucopyranosyl(1-->2)]-beta-D-glucuronopyranosyloxy]-22-O-(2-methylbutyroyloxy)-15,16,28-trihydroxy-(3beta,15alpha,16alpha,22alpha)-olean-12-ene (3) and 3-O-[[beta-D-galactopyranosyl(1-->3)-beta-D-glucopyranosyl(1-->2)]-beta-D-glucuronopyranosyloxy]-22-O-[2(E)-methyl-2-butenyloyloxy]-15,16,28-trihydroxy-(3beta,15alpha,16alpha,22alpha)-olean-12-ene (4). The antifeedant properties of 3 and 4 toward Epilachna larvae are discussed.