Intermediates in the metabolism of m-carboxy-substituted aromatic amino acids in plants: Phenylpyruvic acids,mandelic acids,and phenylglyoxylic acids

Tracer experiments with ¹⁴C-labelled precursors in Iris × hollandica cv. Wedgwood, Roseda lutea L. and Reseda Odorata L. have demonstrated that 3-(3-carboxyphenyl)alanine and 3-(3-carboxy-4-hydroxyphenyl)alanine can be derived from the corresponding pyruvic acids, presumably by unspecific trans-aminations, and that (3-carboxyphenyl)glycine and (3-carboxy-4-hydroxy-phenyl)glycine can be derived from the corresponding phenylglyoxylic acids The glycine derivatives are derived from the alanine derivatives, and the corresponding mandelic acids are intermediates in these transformations. The corresponding phenylacetic acids are incorporated only slightly into the glycine derivatives, indicating that oxidation at the benzylic position in the C₆–C₃ compounds takes place early in the transformation. The corresponding cinnamics acids are not metabolized at all in the plants.     

Biosynthesis of phenylalanine,tyrosine, 3-(3-carboxyphenyl)alanine and 3-(3-carboxy-4-hydroxyphenyl)alanine in higher plants: Examples of the transformation possibilities for chorismic acid

¹⁴C-labelled shikimic acid and double labelled shikimic acid tritiated stereospecifically at C-6 are incorporated into 3-(3-carboxyphenyl)alanine, 3-(3-carboxyl-4-hydroxyphenyl)alanine, phenylalanine, and tyrosine in Resda lutea L., Reseda odoratta L., Iris x Hollandica cv. Prof. Blauw, and Iris x hollandica cv. Wedgwood. The experiments with ¹⁴C-labelled shikimic acid confirm that the aromatic carboxyl groups and rings in 3-(3-carboxyphenyl)-alanine and 3-(3-carboxy-4-hydroxyphenyl)alanine derive from the carboxyl group and ring in shikimic acid whereas the experiments with double labelled shikimic acid demonstrate that the pro-6S-hydrogen atom is retained and the pro-6R-hydrogen atom lost in the biosynthesis of 3-(3-carboxyphenyl)alanine, phenylalanine, and tyrosine in the plants used. ³H was located in the ortho-position in the aromatic rings of phenylalanine and tyrosine but in a position para to the alanine side chain of 3-(3-cabroxyphenyl)alanine. No ³H was found in 3-(3-carboxy-4-hydroxyphenyl)alanine. This supports a derivation of the last two compounds from chorismic acidvia isochorismic acid, isoprephenic acid, and 3′-carboxyphenylpyruvic acid and 3′-carboxy-4′-hydroxyphenylpyruvic acid. The ³H/¹⁴ C ratio in 3-(3-carboxyphenyl)alanine was found higher than in the precursor used. This isotope effect must operate by competition between the pathways from isoprephenic acid to 3′-carboxyphenylpyruvic acid and to 3′-carboxy-4′-hydroxyphenylpyruvic acid. The proposed biosynthetic pathways for the two carboxy-substituted amino acids are in agreement with their distribution patterns in the plant kingdom and suggest that they may derive from minor changes of enzymes involved in the general pathways of aromatic biosynthesis.     

On the absolute configuration of 19′-hexanoyloxyfucoxanthin

The esterifying C₆-acid in 19′-hexanoyloxyfucoxanthin has been identified as n-hexanoic acid by GLC of the methyl ester. Ozonolysis of 19′-n-hexanoyloxyfucoxanthin 3-benzoate provided the n-hexanoyloxy derivative of the allenic ketone produced from fucoxanthin 3-benzoate. NMR and CD correlation of the ozonolysis products and NMR of the native carotenoids provided the basis for assignment of the same absolute configuration of the 19′-n-hexanoyloxy derivative (3S, 5R, 6S, 3′S, 5′R, 6′S) as for fucoxanthin. Biosynthetic implications are considered. CD data for 19′-n-hexanoyloxyfucoxanthin, fucoxanthin and some derivatives thereof are reported. Previously unreported minor carotenoids in Coccolithus huxleyi were diadinoxanthin and 3′-desacetyl 19′-n-hexanoyloxy-fucoxanthin.     

The metabolism of α-ionylidene compounds by Cercospora rosicola

α-Ionylidene ethanol was converted to 4′hydroxy-α -ionylidene acetic acid, 1′-deoxy-ABA and ABA by Cercospora rosicola. Both the 4′-(R) and 4′-(S-epimers of 4′-hydroxy-α-ionylidene acetic acid were detected but the configuration of the 1′-position was not established. Both epimers were metabolized to 1′-deoxy-ABA and ABA. Both the cis- and trans 1′,4′diols of ABA were also converted to ABA. 1′-Deoxy-ABA was stereospecifically hydroxylated to form ABA. 1′-Hydroxy-α-ionylidene derivatives inhibited ABA production and were only oxidised to ABA in low yield. α-Ionylidene ethanol, α-ionylidene acetic acid and both epimers of 4′-hydroxy-α-ionylidene acetic acid were identified as endogenous compounds.     

Free amino acids in some sponges

Most invertebrates, particularly those of marine origin, have relatively high concentrations of free amino acids which are considered an important constituent of their osmoregulatory mechanisms [1]. Very little information is available on the free amino acid distribution in Porifera [2,3]. Common amino acids in some sponges were recognised by paper chromatography by Inskip and Cassidy [4] and Ackermann et al. [5,6] included a few sponges in their survey of the occurence of nitrogen compounds in marine invertebrates. More recently Bergquist and Hartman [7] surveyed semiquantitatively the distribution of free amino acids in several sponges. In the present paper we report on the amino acid composition of 12 species of sponges belonging to the class Demospongiae as a part of a study on the metabolites of Porifera [8]. Fresh sponges were extracted with aqueous ethanol. The organic solvent was removed and the aqueous solution, after removal of the ether soluble compounds, was separated into cationic, anionic and neutral fractions by ion-exchange chromatography. The cation fraction was analysed for amino acids using an automatic amino acid analyser. The results, which are presented in Table 1, show that all species of sponges examined have a similar composition in common amino acids. Glycine almost always appears as the dominant protein amino acid, followed by high concentrations of alanine and glutamic acid, whereas relatively lower concentrations of basic amino acids are present. In Axinella cannabina, Chondrosia reniformis, Chondrilla nucula, Cliona viridis and Hymeniacidon sanguinea, glycine represents more than 77% of the total amino acids. The high percentage of free glycine (90.4%) in Chondrosia reniformis is noteworthy. The anionic and the neutral fractions were examined for sulfur-containing amino acids using PC. Taurine (Table 2) was detected in all the Porifera examined; this is in agreement with previous observations [5–7]. N-Methyltaurine was identified in some of the species examined, whereas neither N,N-dimethyltaurine nor N,N,N-trimethyltaurine were found.     

4-Carboxy-4-hydroxy-2-aminoadipic acid and other acidic amino acids in Caylusea abyssinica

Two diastereoisomers of 4-carboxy-4-hydroxy-2-aminoadipic acid have been isolated from leaves and inflorescences of Caylusea abyssinica. Green parts of the plant also contain appreciable amounts of the two diastereoisomers of 4-hydroxy-4-methylglutamic acid, 3-(3-carboxyphenyl)alanine, (3-carboxyphenyl)glycine, 3-(3-carboxy-4-hydroxyphenyl)alanine, (3-carboxy-4-hydroxyphenyl)glycine and in low concentration 2-aminoadipic acid, saccharopine [(2S, 2′S)-N⁶-(2-glutaryl)lysine] and some γ-glutamyl peptides. The acidic amino acids were separated from other amino acids on an Ecteola ion exchange column with M pyridine as eluant.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [¹²⁵I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptor'sα, β, γ, orδ subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.     

Optimization of <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacteria culturing for obtaining myristoleic acid

Plankton filament cyanobacteria Prochlorothrix hollandica is characterized by a very high content of C₁₄ and C₁₆ fatty acids (FA) in the lipid membranes. Depending on culturing conditions of the cyanobacteria, total concentrations of myristic and myristoleic acids can reach 35% and those of palmitic and palmitoleic acids can reach 60% of all esterified FA cells. In P. hollandica, a variety of monounsaturated FA is represented by myristoleic and palmitic acids, and by hexadecenoic (C_16:1) acid with olefin bond of cis-configuration, located in the Δ4 position. The process of intensive culturing for P. hollandica cells to yield a maximal biomass in order to isolate the pure drug of myristoleic acid derivative has been optimized. The use of a threestage purification gives 30 mg of chromatographically pure myristoleic acid methyl ester from 17 g of P. hollandica raw biomass (dry mass is 3 g), which is 1% of dry cell mass.     

Isolation and Characterization of a Free Gibberellin and Glucosyl Esters of Gibberellins in Mature Seeds of Phaseolus vulgaris

Treatment of N⁶,N⁶,5′-O-tribenzoyl-2′,3′-O-isopropylidenetubercidin (VI) with aqueous acetic acid afforded N⁶,5′-O-dibenzoyltubercidin (V), which was mesylated to yield the dimesylate X. On treatment of X with sodium iodide and zinc dust, the 2′,3′-unsaturated derivatives of tubercidin XI and XIII were obtained.

N⁶,5′-O-Dibenzoyltubercidin 2′,3′-thionocarbonate (XIV), prepared from V by treatment with Corey-Winter reagent, was converted to the 1-methyl-2′,3′-unsaturated derivative XV in refluxing trimethyl phosphite.     

Confirmation of color determination factors for Ser286 derivatives of firefly luciferase from Luciola cruciata (LUC-G)

The bioluminescence color of firefly luciferase including its mutants ranges from green to red (530–640 nm) and is affected by the species of firefly, the reaction conditions, and by the substitution of amino acids. Although there is a general agreement that the microenvironment mechanism is the dominant model for the color determination of firefly luciferase, a complete mechanism has not been shown, partially due to the lack of comprehensive data on which amino acid positions alter the bioluminescent color. In this paper, a mutant library of position serine 286 (S286) in Luciola cruciata luciferase (LUC-G) was constructed and characterized. The substitution of S286 resulted in a drastic red shift in bioluminescence color (>600 nm), and only glycine (G) and alanine (A) mutants remained yellow-green. To explain this color difference, molecular dynamics (MD) calculations of 3 S286 derivatives (S286G, S286N, and S286I), in addition to wild type (WT), were performed. The active site rigidity and active site hydrogen bonding networks were compared to WT for each derivative. The results suggested that both factors affected the active site environment and affected the difference in bioluminescence colors in LUC-G S286 derivatives.     

Novel Ring Cleavage Products in the Biotransformation of Biphenyl by the Yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4′-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-Trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

A new amine,stizolamine, from Stizolobium hassjoo

A new pyrazine derivative, stizolamine (1-methyl-3-guanidino-6-hydroxymethylpyrazin-2-one), has been isolated from seeds of Stizolobium hassjoo. This amine, which has a blue fluorescence, gives guanidine, N-methyl-alanine, oxalic acid, alanine and glycine on treatment with 6 N HCl. The permanganate oxidation product of stizolamine is 4-amino-6-methylcarbamoyl-1,3,5-triazine-2-carboxylic acid.     

Metabolism of Indole-3-Acetic Acid: III. Identification of Metabolites Isolated from Crown Gall Callus Tissue

The metabolism of labeled indole-3-acetic acid (IAA-2-¹⁴C) was investigated in Parthenocissus tricuspidata crown gall callus tissue. After 48 hours incubation, 85 to 90% of the supplied IAA was taken up by the tissue, and of that taken up, about 45% was conjugated with five amino acids. The conjugates found were aspartic and glutamic acid (minor ones) as well as glycine, alanine, and valine (major ones). The last four are being reported for the first time as metabolites of IAA. These conjugates were identified through their chromatographic properties, hydrolysis products, and their mass spectra. The possible significance of these amino acid conjugates is discussed.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

l-3-(3′-Carboxy-4′-hydroxyphenyl)alanine (3-carboxytyrosine) in seeds of Neonotonia wightii (Glycine wightii) and its possible systematic significance

l-3-(3′-Carboxy-4′-hydroxyphenyl)alanine (3-carboxytyrosine) constitutes 3% of the seeds of Neonotonia wightii (Glycine wightii); it has also been detected in the seeds of 3 species belonging to 2 other genera of the Glycineae. The systematic significance of these findings is discussed.     

Spodoptera littoralis detoxifies neurotoxic 3-nitropropanoic acid by conjugation with amino acids

Spodoptera littoralis is a phytophagous generalist. Its host range includes more than 40 plant species, some of which produce 3-nitropropanoic acid (3-NPA), an irreversible inhibitor of mitochondrial succinate dehydrogenase. Growth in larvae fed an artificial diet with a sublethal admixture of 3-NPA (4.2 μmol per g) was slowed significantly, but larvae experienced no increase in mortality. In contrast, larvae injected with 25.2 μmol/g (bodyweight) 3-NPA experienced acute toxicity and death. To study the detoxification mechanism of 3-NPA in S. littoralis, the insect frass was analyzed by HPLC-MS. Comparative analysis of 3-NPA-treated and -untreated control samples using HR-MS² revealed a group of differential signals that were identified as amino acid amides of 3-NPA with glycine, alanine, serine, and threonine. When sublethal amounts of stable isotope-labeled 3-NPA were injected into a larva's hemolymph, 3-NPA amino acid conjugates were identified as putative detoxification products. Bioassays with synthetic standards confirmed that the toxicity of the amides was negligible in comparison to the toxicity of free 3-NPA, demonstrating that amino acid conjugation in S. littoralis represents an efficient way to detoxify 3-NPA. Furthermore, biosynthetic studies using crude fractions of the gut tissue indicated that conjugation of 3-NPA with amino acids occurs in epithelial cells of the insect's gut. Taken together, these results suggest that the detoxification of 3-NPA in S. littoralis proceeds via conjugation to specific amino acids within the epithelial cells followed by export of the nontoxic amino acid conjugates to the hemolymph via as yet uncharacterized mechanisms, most likely involving the Malpighian tubules.     

A labdane derivative from Chromolaena collina and a p-hydroxyacetophenone derivative. From Stomatanthes corumbensis

A new labdane derivative, 7α-acetoxy-trans-communic acid was isolated from Chromolaena collina. Extraction of Stomatanthes corumbensis yielded a new p-hydroxyacetophenone derivative which was identified as 4-methoxy-3- [3′-methyl-4′-angeloyloxy-but-2-en- 1′-yl ]-acetophenone.     

Systematic characters of some polychaetes (Annelida) at the level of the chemical composition of the jaws

The mandibles and maxillae of the buccal ventral organ of 2 species of Eunicidae (Marphysa sanguinea and Eunice torquata) are highly calcified, in contrast to the jaws of 4 species of other families of ‘errant’ predacious Polychaetes (Nereidae, Nephthyidae, Aphroditidae and Glyceridae) with axial proboscis. The amino acid composition of the structural proteins of these buccal pieces is also different in the two groups. The structural proteins of the jaws of Glycera convoluta (Glyceridae) are essentially made up of glycine and histidine (up to 86 residues per 100 residues). These chemical characters confirm the phyletic relationships proposed by Dales.¹     

Ascorbalamic acid: An ascorbigen-like plant constituent yielding in hot acid 3-(2-furoyl)alanine

Ascorbalamic acid (C₉H₁₃NO₈) was isolated from Brassica olerocea L. MS study of various methylated derivatives suggested a structure (Ia) derivable by CC coupling of C-3 of alanine with C-2 of ascorbic acid, followed by lactone → lactam rearrangement. Other derivatives provided supporting evidence, as did study of the reaction of L-3-chloroalanine with L-ascorbic acid in vitro. On treatment with hot 6 M HCl, ascorbalamic acid yielded L-aspartic acid and 3-(2-furoyl)alanine. For identification of the latter, DL-3-(2-furoyl)alanine and its N-2,4-dinitrophenyl and N-acetyl methyl ester derivatives were synthesized. Unlike ascorbigens, ascorbalamic acid is probably present in the living plant. It seemed to be present in all crucifers examined, but to have a capricious distribution in other orders. During permethylation, rearrangements of ester groups were observed, both with ascorbalamic acid and with pyrrolidonecarboxylic acid as a model.