Biosynthesis of meteloidine from 3α-tigloyloxytropane in Datura innoxia

The administration of 3α-tigloyl-[1-¹⁴C]-oxytropane-[3β-³H] (³H/¹⁴C = 11·0 to Datura innoxia plants for 7 days led to the formation of radioactive meteloidine (³H/¹⁴C = 11·6). Degradation of the meteloidine indicated that the alkaloid was labeled specifically with ³H at C-3 of its teloidine moiety, and on the carbonyl group of its tigloyl residue with ¹⁴C. These results strongly favor the hypothesis that hydroxylation of tropine occurs after formation of its tigloyl ester.     

Competitive feeding experiments with tropine in Datura

Datura meteloides plants were fed with tropine-[N-¹⁴Me] and the same compound together with the competitive inhibitors 3α-tigloyloxytropane: tropan-3α,6β-diol: 6β-hydroxy-3α-tigloyloxytropane: 3α,6β-ditigloyloxytropane: teloidine: meteloidine and 3α,6β-ditigloyloxytropan-7β-ol. The results obtained favour two distinct routes for the biosynthesis of the tigloyl esters of tropan-3α,6β-diol and teloidine (tropan-3α,6β,7β-triol); the first, either tropine → tropan-3α,6β-diol-3α,6β-ditigloyloxytropane or more probably, tropine→3α-tigloyloxytropane→6β-hydroxy-3α-tigloyloxytropane→3α,6β-ditigloyloxytropane; and second, tropine→3α,-tigloyloxytropane→“7β-hydroxy-3α-tigloyloxytropane”→meteloidine→3α,6β-ditigloyloxytropan-7β-ol.     

Biosynthesis of tropic acid: Feeding experiments with cinnamoyltropine and littorine

The administration of cinnamoyl-[2-¹⁴C]-tropine-[N-methyl-¹⁴C] to Datura stramonium plants resulted in the formation of labeled atropine and scopolamine. However the atropine was found to have almost all its radioactivity located on the N-methyl group of the alkaloid, indicating that the administered ester had undergone hydrolysis in the plant affording tropine and cinnamic acid, the latter not being utilized for the biosynthesis of tropic acid. Dual labeled RS-littorine (3β-(2-hydroxy-3-phenylpropionyloxy-[1-¹⁴C]-tropane-[3β-³H]) was also fed to D. stramonium and radioactive atropine was obtained. However the drastic change in the ³H:¹⁴C ratio found in the atropine indicated that the littorine was not converted directly to the alkaloid, and it is suggested that the littorine is hydrolysed _{in vivo} to tropine and phenyl-lactic acid, the latter undergoing rearrangement to tropic acid prior to esterification with tropine.     

Biosynthesis of 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol in Datura

Datura meteloides; plants were fed with tiglic acid-[-¹⁴C] via the roots and after 2 days the percentage incorporation into the alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, meteloidine and 3α,6β-ditigloyloxytropan-7β-ol were 15·2, 1·82, 2·2 and 1·8 respectively. 3α,6β-Ditigloyloxytropane was partially hydrolysed to 6β-hydroxy-3α-tigloyloxytropane which contained 58·1% of the radioactivity of the original base, whereas 3α,6β-ditigloyloxytropan-7β-ol gave meteloidine containing only 9·2% of the original activity. The results suggest that the di- and tri-hydroxytropanes may be formed by different routes.     

Alkaloids of Datura suaveolens

The alkaloid mixture of Datura suaveolens shows distinct differences compared with that of other tree daturas. Aerial parts contain in addition to hyoscine, apohyoscine, norhyoscine, atropine and noratropine, a relatively high proportion of tigloyl esters—3α,6β-ditigloyloxytropan-7β-ol, 6β-tigloyloxytropan-3α,7β-diol,3α-tigloyloxytropan-6β,7β-diol (meteloidine) and (—)- and (±)-3α-tigloyloxytropan-6β-ol. The roots contain hyoscine, meteloidine, atropine, littorine, 3α-acetoxytropane, 6β-(α-methylbutyryloxy)-3α-tigloyloxytropane, 3α,6β-ditigloyloxytropan-7β-ol, 3α-tigloyloxytropan-6β-ol, tropine and cuscohygrine. Other, as yet uncharacterized, bases are present in the plant. Norhyoscine is a principal alkaloid of the corollas.     

Non-reversibility of the isoleucine-acetate pathway in Datura

Five-month-old Datura meteloides plants were fed via the roots with 3-hydroxy-2-methylbutanoic acid-[1-¹⁴C] and isoleucine-[U-¹⁴C] as a positive control. After 5 days the plants were collected and in each case the root alkaloids 3α,6β-ditigloyloxytropane, 3α,6β-ditigloyloxytropan-7β-ol, meteloidine, hyoscine and hyoscyamine were isolated. Whereas isoleucine served as a precursor for the tiglic acid moieties 3-hydroxy-2-methylbutanoic acid did not.     

Distribution of alkaloids in Anthocercis littorea and A. viscosa

The distribution of tropane alkaloids in organs of Anthocercis littorea and A. viscosa is reported. The following alkaloids have been isolated: atropine (hyoscyamine), apoatropine, noratropine (norhyoscyamine), littorine, hyoscine, norhyoscine, meteloidine, 3α, 6β-ditigloyloxytropan-7β-ol, 6β-tigloyloxytropan-3α-ol, 3α-tigloyloxytropane, tigloidine, tropine, ψ-tropine, (?)-tropan-3α-6β-diol, cuscohygrine and unknown bases.     

Biosynthetic conversion of α-methylbutyric acid to tiglic acid in Datura meteloides

The administration of RS-α-methylbutyric-[1-¹⁴C] acid to Datura meteloides plants resulted in the formation of radioactive meteloidine. A systematic degradation indicated that essentially all the activity was located at C-1 of the tiglic acid moiety of the alkaloid.     

A sensitive and specific tritium release assay for dopamine-beta-hydroxylase (DbetaH) in serum.

The release of tritium from [7-³H₂]dopamine was investigated as a possible procedure for the assay for dopamine-β-hydroxylase (DβH) in rat and human serum. The release was found to have the same characteristics as those deseribed previously for DβH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu²⁺ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DβH in serum. Tritium release was blocked by the DβH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of ¹⁴C-labeled dopamine along with [7-³H₂]dopamine revealed that, under the standard assay conditions, the formation of [¹⁴C]norepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DβH activity in serum.     

Biosynthesis of the tigloyl esters of Datura: Cis-trans isomerism

Datura innoxia plants were wick fed with angelic acid-[1-¹⁴C] and l-isoleucine-[U-¹⁴C] to act as a positive control. After 7 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, and 3α,6β-ditigloyloxytropan-7β-ol were isolated and it was determined that angelic acid is not a precursor for the tigloyl moiety of these alkaloids. Tiglic acid-[1-¹⁴C] which was fed via the roots to hydroponic cultures of Datura innoxia, was incorporated to a considerable degree after 8 days.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Synthesis and cytotoxic activity of the N-acetylglucosamine-bearing triterpenoid saponins

Fourteen ursolic acid and oleanolic acid saponins with N-acetyl-β-d-glucosamine, and (1→4)-linked and (1→6)-linked N-acetyl-β-d-glucosamine oligosaccharide residues were synthesized in a convergent manner. The structures of all compounds were confirmed by ¹H NMR and ¹³C NMR spectroscopy and by mass spectrometry, and their cytotoxic activities were assayed in three cancer cell lines. Only oleanolic acid-3-yl β-d-GluNAc showed significant cytotoxicity against HL-60 and BGC-823.     

Failure of 3-hydroxy-3-phenylpropanoic acid and cinnamic acid to serve as precursors of tropic acid in Datura innoxia

Datura innoxia plants were fed the R- and S-isomers of [3-¹⁴C]-3-hydroxy-3-phenylpropanoic acid, and [3-¹⁴C]cinnamic acid along with dl-[4-³H]phenylalanine. The hyoscyamine and scopolamine isolated from the plants 7 days later were labeled with tritium, but devoid of ¹⁴C, indicating that 3-hydroxy-3-phenylpropanoic acid and cinnamic acid are not intermediates between phenylalanine and tropic acid. The [³H] tropic acid obtained by hydrolysis of the hyoscyamine was degraded and shown to have essentially all its tritium located at the para position of its phenyl group, a result consistent with previous work.     

Stereochemistry of tropane alkaloid formation in Datura

Five-month-old Datura innoxia plants were fed via the roots with either d(+)-hygrine-[2′-¹⁴C] or l(?)-hygrine-[2′-¹⁴C]. After 7 days the root alkaloids 3α,6β-ditigloyloxytropane, 3α,6β-ditigloyloxytropan-7β-ol, hyoscine, hyoscyamine and cuscohygrine were isolated from both groups of plants. d(+) but not l(?)-hygrine acts as a precursor for the tropane alkaloids whereas both enantiomers appeared to serve equally well in the biosynthesis of cuscohygrine.     

Biosynthese und Stoffwechsel der chinolizidinalkaloide von Sophora alopecuroides

Administration of matrine-U-³H and sophocarpine-U-³H to Sophora alopecuroides seedlings shows that these compounds were incorporated into quinolizidine alkaloids such as matrine, sophacarpine, and their N-oxides, but not into sophoridine. It is suggested that there is no stereochemical conversion of alkaloids of matrine configuration into sophoridine by the plant. The incorporation of cadaverine-1,5-¹⁴C was so low that it cannot be regarded with certainty as a physiological precursor of the alkaloids. The N-oxides of matrine and sophocarpine were isolated and identified by their chromatographic and chemical properties.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

Effects of Several Tunicamycin-like Antibiotics on Glycoprotein Biosynthesis in Mung Beans and Suspension-cultured Soybean Cells

The antibiotics Streptovirudin and 24010 were tested to determine their effects on the formation of lipid-linked saccharide intermediates associated with glycoprotein biosynthesis in mung bean (Vigna radiata) and suspension-cultured soybean cells (Glycine max cv. Mandarin). In vitro both compounds strongly inhibited the transfer of N-acetyl[³H]glucosamine from UDP-N-[³H]acetylglucosamine to N-acetylglucosaminyl-pyrophosphoryl-polyisoprenol and lipid-linked oligosaccharides, although they had no apparent effect on the incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol with a small inhibition into lipid-linked oligosaccharides. In vivo, Streptovirudin and tunicamycin dramatically inhibited the incorporation of N-[¹⁴C]acetylglucosamine and [³H]mannose into Pronase-released material (glycoproteins), whereas there was no effect on [³H]leucine incorporation into Pronase-released material (protein). Because the action of Streptovirudin and antibiotic 24010 in plants and other systems is similar to that for tunicamycin, these antibiotics are believed to be closely related. The use of tunicamycin is discussed with respect to its importance in studying glycoprotein biosynthesis and function in animal and plant systems.     

Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl-¹⁴C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [¹⁴C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-¹⁴C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.     

Biosynthesis of cardenolides in Digitalis lanata

[8-³H]-Cholesterol was synthesized. A doubly labelled sample of [8-³H, 4-¹⁴C]-cholesterol was administered to Digitalis lanata plants and the cardenolides were isolated. Biosynthesized digitoxigenin and digoxigenin retained all the tritium. Barring the migration of the tritium in biosynthesis the results are interpreted as indicative that neither intermediates with δ7, δ⁷ or δ⁸⁸⁽¹⁴⁾ are participating in the elaboration of cardenolides.     

Biosynthesis of azetidine-2-carboxylic acid in Convallaria majalis

Convallaria majalis plants were fed dl-methionine-[1-¹⁴C]. [1-¹⁴C, 4-³H], and [1-¹⁴C, 2-³H], S-adenosyl-l-methionine-[1-¹⁴C], and dl-homoserine-[1-¹⁴C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-¹⁴C, 4-³H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-¹⁴C, 2-³H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.