Biosynthesis and utilization of aromatic compounds by Mycobacterium smegmatis with particular reference to the origin of salicylic acid

1. Although Mycobacterium smegmatis could utilize a number of aromatic compounds as sole sources of carbon for growth, it did not appear to be able to use salicylic acid for growth or to metabolize it to any great extent. 2. When M. smegmatis was grown on shikimic acid as sole source of carbon, salicylic acid, anthranilic acid and 3,4-dihydroxybenzoic acid were released into the medium. When it was grown on quinic acid these compounds, together with p-hydroxybenzoic acid, p-hydroxyphenylacetic acid and a number of unidentified compounds, were formed. When it was grown on glucose only small amounts of salicylic acid could be detected. 3. When a washed suspension of cells with a normal iron content was incubated with shikimic acid, only small amounts of aromatic compounds were formed in the medium. When the cells were iron-deficient, substantial amounts of salicylic acid, 3,4-dihydroxybenzoic acid and catechol were formed, together with several other compounds not definitely identified. 4. When washed suspensions of cells, whether iron-sufficient or iron-deficient, were incubated with tryptophan no evidence of formation of salicylic acid, anthranilic acid or phenolic compounds was obtained. Washed suspensions did not convert anthranilic acid into salicylic acid. 5. When cell-free extracts of M. smegmatis were incubated with shikimic acid, or shikimic acid 5-phosphate, traces of anthranilic acid were formed under certain conditions. No formation of salicylic acid or other phenolic compound was observed even when a number of combinations of cofactors and coenzymes were tried.     

Tetrahydro anthranilic acid as a surrogate for anthranilic acid: application to the discovery of potent niacin receptor agonists

The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described. This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were identified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and CYP2C9, and improved oral exposure in mice.     

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.     

Neutral, acidic, and basic derivatives of anthranilamide that confer different formal charge to reducing oligosaccharides

To facilitate the use of oligosaccharides as analytical tools in biological studies, we have designed, synthesized, and conjugated to maltosaccharides a novel series of homologous small fluorescent moieties that differ in formal charge. These moieties are amide derivatives of anthranilic acid: uncharged N-(2-aminobenzoyl)glycinamide (ABGlyAmide; 2), acidic N,N-dimethyl-N(')-(2-aminobenzoyl)ethylenediamine (ABGlyDIMED; 3), and basic N-(2-aminobenzoyl)glycine (ABGly; 1). Routes for synthesis and optimal reaction conditions for glycoconjugation by conventional reductive amination are presented, as is the compatibility of these adducts with common analytical and preparative chromatographic methods, including RP-HPLC and HPAEC-PAD. These novel anthranilic acid derivatives confer both fluorescence and defined charge to oligosaccharides, and so enhance the repertoire of chromatographic and analytical methods for which anthranilic acid can be used. Furthermore, because glucosaccharides have rigid solution structure, these small fluorescent adducts with different formal charge are ideal tools for molecular sizing studies of membrane pores.     

An anthranilic acid-responsive transcriptional regulator controls the physiology and pathogenicity of Ralstonia solanacearum

Quorum sensing (QS) is widely employed by bacterial cells to control gene expression in a cell density-dependent manner. A previous study revealed that anthranilic acid from Ralstonia solanacearum plays a vital role in regulating the physiology and pathogenicity of R. solanacearum. We reported here that anthranilic acid controls the important biological functions and virulence of R. solanacearum through the receptor protein RaaR, which contains helix-turn-helix (HTH) and LysR substrate binding (LysR_substrate) domains. RaaR regulates the same processes as anthranilic acid, and both are present in various bacterial species. In addition, anthranilic acid-deficient mutant phenotypes were rescued by in trans expression of RaaR. Intriguingly, we found that anthranilic acid binds to the LysR_substrate domain of RaaR with high affinity, induces allosteric conformational changes, and then enhances the binding of RaaR to the promoter DNA regions of target genes. These findings indicate that the components of the anthranilic acid signaling system are distinguished from those of the typical QS systems. Together, our work presents a unique and widely conserved signaling system that might be an important new type of cell-to-cell communication system in bacteria.     

Characterization and regulation of anthranilate synthetase from a chloramphenicol-producing streptomycete.

In Streptomyces sp. 3022a, anthranilate synthetase is composed of two non-identical subunits. The major subunit (molecular weight, 72,000) converts chorismic acid to anthranilic acid, using ammonia as the source of the amino group. The smaller subunit (molecular weight 28,000 to 29,000) confers on the enzyme the ability to use glutamine instead of ammonia as a substrate. In this study, reactivity with glutamine reached its maximum at pH 7.2 to 7.6, whereas that with ammonia increased linearly through pH 9.0 without reaching a maximum. Activity was increased and stabilized by adding glutamine and magnesium chloride to the buffer system. Both activities of the enzyme were inhibited by anthranilic acid and by tryptophan. Synthesis was repressed by histidine, anthranilic acid, tryptophan, and p-aminobenzoic acid. When activity was repressed by anthranilic acid and by tryptophan, there was a concomitant increase in the activity of arylamine synthetase, an enzyme involved in chloramphenicol production. Stimulating arylamine synthetase, however, did not increase antibiotic synthesis.     

Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Synthesis of some newer derivatives of 2-amino benzoic acid as potent anti-inflammatory and analgesic agents

Diazotization of N-benzylidene anthranilic acids 1a-1n at pH 9 yielded N-[alpha-(phenylazo) benzylidene] anthranilic acids 2a-2n and at pH 3 yielded N-benzylidene-5-(phenylazo) anthranilic acids 3a-3n. When compounds 3a-3n were treated with thioglycolic/thiolactic acid in the presence of anhydrous ZnCl(2), 2-(4-oxo-2-phenylthiazolidin-3-yl)-5-(phenylazo) benzoic acids 4a-4n were afforded. The newly synthesized compounds were screened for their anti-inflammatory and analgesic activities and were compared with standard drugs, aspirin and phenylbutazone. Out of the compounds studied, the most active compound 4n showed more potent activity than the standard drugs at all doses tested.     

Enzymatic synthesis of cyclopeptine intermediates in Penicillium cyclopium

Cyclopeptine, a benzodiazepine alkaloid of Penicillium cyclopium, is formed from anthranilic acid, L-phenylalanine and the methyl group of L-methionine by cyclopeptine synthetase. The following partial activities of this enzyme system were determined in vitro: anthranilic acid and L-phenylalanine adenylyltransferase activity, binding of anthranilic acid and L-phenylalanine as thioesters to proteins, formation of thioester-bound N-methyl-L-phenylalanine and N-methyl-L-phenylalanylanthranilic acid. The obtained results indicate that cyclopeptine is formed via enzyme-bound intermediates by the thiotemplate mechanism of peptide biosynthesis.     

Interactions of a beta-dipeptide with monovalent metal cations: crystal structures of (anthranoyl)anthranilic acid and its lithium, sodium and thallium salts

X-ray crystal structure analyses have been performed on the beta-dipeptide (anthranoyl)anthranilic acid [HAnthAnthOH] and its lithium, sodium and thallium salts [HAnthAnthOM] to give a first set of data for this representative model ligand. Crystals of the beta-dipeptide are orthorhombic, space group Pca2(1). The unit cell contains two molecules of (anthranoyl)anthranilic acid which form a dimer via hydrogen bonds. The components of the beta-dipeptide are rotated into the trans-conformation which allows for internal hydrogen bonds. The pKS value of (anthranoyl)anthranilic acid (9.80+/-0.14) shows a slight decrease as compared to anthranilic acid; the metal salts can therefore be prepared by direct neutralization of the beta-dipeptide with metal hydroxides or carbonates. The alkali compounds crystallize as the trihydrates [HAnthAnthOM(H2O)3, M=Li, Na] in the triclinic space group p1. Both metal ions show a clear preference for water molecules over the (anthranoyl)anthranilate anions as ligands in their coordination spheres. As a consequence, the [HAnthAnthO]- anions are only partially involved in metal complexation. The cell plots of both compounds exhibit a stacking with an alternation of oppositely charged layers. The negatively charged layers are composed exclusively of (anthranoyl)anthranilate anions. The thallium compound crystallizes as the hemihydrate [HAnthAnthOTl(H2O)0.5] in the monoclinic space group C2/c. In the dinuclear units, the thallium ions accommodate one nitrogen and four oxygen atoms of the anions in their coordination sphere and in addition entertain weak Tl-arene contacts. In contrast to the alkali compounds, the water molecules are not involved in metal complexation, but contribute to a network of hydrogen bonding.     

Zur biosynthese des graveolins bei Ruta angustifolia

The carbon skeleton of the quinoline alkaloid graveoline is built up from the aromatic ring and probably the carboxylic group of anthranilic acid and the ring and the C-atoms 2′ and 3′ of a phenylpropane. The nitrogen atom of the alkaloid is derived from that of anthranilic acid. It seems that a benzoylacetic acid derivative which is formed from phenylalanine via cinnamic acids reacts with anthranilic acid with loss of its carboxylic group. The two oxygen atoms of the methylenedioxy group of graveoline are introduced by mixed function oxygenation. The value of the NIH-shift which occurs during this reaction shows that the oxygen in the p-position is introduced before that in the m-position. A monomethylated o-dihydroxy group seems to be the direct precursor of the methylenedioxy structure. The introduction of the oxygen atoms, the methylation and the formation of the methylenedioxy group can proceed at different stages in the pathway of graveoline biosynthesis.     

Kynurenines and the respiratory parameters on rat heart mitochondria

It has been shown recently that the L-kynurenine metabolite kynurenic acid lowers the efficacy of mitochondria ATP synthesis by significantly increasing state IV, and reducing respiratory control index and ADP/oxygen ratio of glutamate/malate-consuming heart mitochondria. In the present study we investigated the effect of L-tryptophan (1.25 microM to 5 mM) and other metabolites of L-kynurenine as 3-hydroxykynurenine (1.25 microM to 2.5 mM), anthranilic acid (1.25 microM to 5 mM) and 3-hydroxyanthranilic acid (1.25 microM to 5 mM) on the heart mitochondria function. Mitochondria were incubated with saturating concentrations of respiratory substrates glutamate/malate (5 mM), succinate (10 mM) or NADH (1 mM) in the presence or absence of L-tryptophan metabolites. Among tested substances, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and anthranilic acid but not tryptophan affected the respiratory parameters dose-dependently, however at a high concentration, of a micro molar range. 3-Hydroxykynurenine and 3-hydroxyanthranilic acid lowered respiratory control index and ADP/oxygen ratio in the presence of glutamate/malate and succinate but not with NADH. While, anthranilic acid reduced state III oxygen consumption rate and lowered the respiratory control index only of glutamate/malate-consuming heart mitochondria. Co-application of anthranilic acid and kynurenic acid (125 or 625 microM each) to glutamate/malate-consuming heart mitochondria caused a non-additive deterioration of the respiratory parameters determined predominantly by kynurenic acid. Accumulated data indicate that within L-tryptophan metabolites kynurenic acid is the most effective, followed by anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid to influence the respiratory parameters of heart mitochondria. Present data allow to speculate that changes of kynurenic acid and/or anthranilic acid formation in heart tissue mitochondria due to fluctuation of L-kynurenine metabolism may be of functional importance for cardiovascular processes. On the other hand, beside the effect of 3-hydroxyanthranilic acid and 3-hydroxykynurenine on respiratory parameters, their oxidative reactivity may contribute to impairment of mitochondria function, too.     

Iron requirement of Rhizobium leguminosarum and secretion of anthranilic acid during growth on an iron-deficient medium

Rhizobium leguminosarum GF160 required iron for growth under aerobic conditions in a chemically defined medium. Maximal growth of bacteria previously depleted in iron was obtained with approximately 50 microM unchelated ferric iron and with glucose as the only carbon source. Growth under iron deficiency did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate types. Growing cells released a Fe3+-reducing agent that was identified as anthranilic acid by paper and thin-layer chromatography, ultraviolet and nuclear magnetic resonance spectroscopy, and mass spectrometry. The amount of anthranilic acid secreted per unit of cell growth was inversely related to the iron concentration in the culture medium and reached concentrations up to 1 mM. Ferric but not ferrous ions were solubilized in the growth medium by anthranilic acid.     

Lead optimization of methionine aminopeptidase-2 (MetAP2) inhibitors containing sulfonamides of 5,6-disubstituted anthranilic acids

A series of aryl sulfonamides of 5,6-disubstituted anthranilic acids were identified as potent inhibitors of methionine aminopeptidase-2 (MetAP2). Small alkyl groups and 3-furyl were tolerated at the 5-position of anthranilic acid, while -OCH(3), CH(3), and Cl were found optimal for the 6-position. Placement of 2-aminoethoxy group at the 6-position enabled interaction with the second Mn(2+) but did not result in enhancement in potency. Introduction of a tertiary amino moiety at the ortho-position of the sulfonyl phenyl ring gave reduced protein binding and improved cellular activity, but led to lower oral bioavailability.     

Production of Substituted l-Tryptophans by Fermentation

Claviceps purpurea has been shown to produce extracellular l-tryptophan from indole in stirred fermentors. The substrate specificity of this conversion was investigated by using substituted indoles, anthranilic acid, and 4-chloro-anthranilic acid. Addition of 2-, 4-, 5-, 6-, and 7-methyl indole or 6-chloroindole to C. purpurea C1M produced the corresponding substituted l-tryptophan. In contrast, addition of l-methyl, 6-trifluoromethyl, 6-nitro-, or 4-benzyloxy-substituted indoles, or anthranilic and 4-chloroanthranilic acids did not produce detectable amounts of the corresponding tryptophan.     

Interferon-gamma-induced degradation of tryptophan by human cells in vitro

Several human cells were investigated for their ability to degrade tryptophan and to synthesize neopterin upon induction by interferon-gamma (500 units/ml for 48 h). Concentrations of tryptophan, kynurenine, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, 7,8-dihydroneopterin and neopterin were assessed in the culture supernatants by HPLC. Fibroblasts, A-22 arachnoidea, HK-2351 scalp, T-2346 meningeom and HeLa cervical carcinoma cells but not HL-60 promyelocytic leukaemia cells were found to degrade tryptophan upon induction by interferon-gamma. Tryptophan is converted to kynurenine by fibroblasts, A-22 arachnoidea and HK-2351 scalp cells and to kynurenine and anthranilic acid by HeLa cervical carcinoma and T-2346 meningeom cells. Kynurenine and anthranilic acid always make up more than 82% of the tryptophan degraded. None of these cells synthesizes 3-hydroxyanthranilic acid, 3-hydroxykynurenine, 7,8-dihydroneopterin or neopterin. Human macrophages form 3-hydroxyanthranilic acid and neopterin, but not 3-hydroxykynurenine, beside kynurenine and anthranilic acid upon activation by interferon-gamma. These data indicate that several human cells can be induced by interferon-gamma to degrade tryptophan. The interferon-gamma induced synthesis of 3-hydroxyanthranilic acid and neopterin, however, appears to be restricted to human macrophages. A hypothesis explaining these findings is presented.     

Anthranilic acid based CCK1 receptor antagonists: Blocking the receptor with the same ‘words’ of the endogenous ligand

The anthranilic acid diamides represent the more recent class of nonpeptide CCK₁ receptor antagonists. This class is characterized by the presence of anthranilic acid, used as a molecular scaffold, and two pharmacophores selected from the C-terminal tetrapeptide of CCK. The lead compound coded VL-0395, endowed with sub-micromolar affinity towards CCK₁ receptors, was characterized by the presence of Phe and 2-indole moiety at the C- and N-termini of anthranilic acid, respectively. Herein we describe the first step of the anthranilic acid C-terminal optimization using, instead of Phe, aminoacids belonging to the primary structure of CCK-8 and other not coded residues. Thus we demonstrate that the CCK₁ receptor affinity depends on the nature of the aminoacidic side chain as well as that the free carboxy group of the alpha-aminoacids is crucial for the binding. The R enantiomers of the most active compounds represent the eutomers of this class of antagonists confirming thus the stereo preference of the receptor. Moreover this SAR study demonstrates that the receptor binding pocket, that host the aminoacidic side chain, results much more tolerant respect to that accommodating the indole ring. As a result, an appropriate variation of the aminoacidic side chain could provide a better CCK₁ receptor affinity diorthosis.     

Anthranilic acid derivatives: a new class of non-peptide CCK1 receptor antagonists

Having successfully obtained new CCK(1) ligands holding appropriate groups on the anthranilic acid dimer used as molecular scaffold we were interested in increasing their micromolar affinity for the CCK(1) receptors by modifying the spatial relationship of the main pharmacophoric groups. Since, we have proposed simplified analogues reducing the anthranilic acid dimer to a monomer. In this stage of our research program we have prepared and tested on CCK receptors a series of N-substituted anthranilic acid derivatives keeping a Phe residue at the C-terminal site. The indole-2-carbonyl group imparts the best CCK(1) receptor binding affinity (compound 1: IC(50)=197.5 nM) while a sharp decrease in binding affinity is observed for the other indole containing derivatives. Moreover, in order to support the different binding behaviour observed for the synthesized compounds, a conformational investigation was carried out. Finally, on the basis of the main pharmacophoric groups of the obtained new lead compound (1) (coded VL-0395) a receptor binding hypothesis has been provided.     

Studies on some iridium(III) complexes with Schiff bases derived from amino carboxylic acids

The reactions of iridium(III) chloride with different Schiff bases gave complexes of types [Ir(SB)3], [Ir(SB')Cl(H2O)2], [Ir(SB')Cl2]n, [Ir(SB' ')Cl(H2O)]n (SBH = Schiff bases derived from anthranilic acid and benzaldehyde, acetophenone, vanillin, cinnamaldehyde or m-hydroxyacetophenone; SB'H2 = Schiff bases derived from anthranilic acid and salicylaldehyde or o-hydroxyacetophenone; SB'H = Schiff bases derived from p-aminobenzoic acid and benzaldehyde, acetophenone, vanillin, cinnamaldehyde, or m-hydroxyacetophenone; SB' 'H2 = Schiff bases derived from p-aminobenzoic acid and salicylaldehyde or o-hydroxyacetophenone). These complexes have been characterized on the basis of elemental analyses, conductance, magnetic moment, and spectral (electronic, i.r., and 1H n.m.r.) data. The electronic spectra reveals octahedral geometry for these complexes except for [Ir(SB')Cl2]n, which is trigonal bipyramidal. The thermal behavior of these complexes has also been studied by TG, DTG, and DSC techniques. The different kinetic parameters, viz., order of reaction, activation of energy, and heat of reaction were calculated. The antifungal and antiviral activities of the complexes with Schiff bases derived from anthranilic acid have also been investigated.     

Anthranilic acid release in adenosine-inhibited cultures of Saccharomyces cerevisiae and its inhibition by thiamin

Adenosine, at 1 mM concentrations or above, was found to have a fungistatic effect on Saccharomyces cerevisiae. A substance with amethyst fluorescence was detected in the medium of adenosine-inhibited cultures of S. cerevisiae. This compound was isolated and physicochemically identified as anthranilic acid. Both the inhibition of growth and release of anthranilic acid induced by adenosine were abrogated by thiamin or by the pyrimidine portion of thiamin, 2-methyl-4-amino-5-hdroxymethyl-pyrimidine (hydroxymethyl-pyrimidine); the latter was found to restore intracellular thiamin content that had been reduced by adenosine. It was demonstrated that effects of thiamin and hydroxymethylpyrimidine on S. cerevisiae cultured with adenosine resulted from their inhibition of adenosine uptake by growing yeast cells.