Uncovering the Cryptic Gene Cluster ahb for 3-amino-4-hydroxybenzoate Derived Ahbamycins, by Searching SARP Regulator Encoding Genes in the Streptomyces argillaceus Genome.

Genome mining using standard bioinformatics tools has allowed for the uncovering of hidden biosynthesis gene clusters for specialized metabolites in Streptomyces genomes. In this work, we have used an alternative approach consisting in seeking "Streptomyces Antibiotic Regulatory Proteins" (SARP) encoding genes and analyzing their surrounding DNA region to unearth cryptic gene clusters that cannot be identified using standard bioinformatics tools. This strategy has allowed the unveiling of the new ahb cluster in Streptomyces argillaceus, which had not been retrieved before using antiSMASH. The ahb cluster is highly preserved in other Streptomyces strains, which suggests a role for their encoding compounds in specific environmental conditions. By combining overexpression of three regulatory genes and generation of different mutants, we were able to activate the ahb cluster, and to identify and chemically characterize the encoded compounds that we have named ahbamycins (AHBs). These constitute a new family of metabolites derived from 3-amino-4-hydroxybenzoate (3,4-AHBA) known for having antibiotic and antitumor activity. Additionally, by overexpressing three genes of the cluster (ahbH, ahbI, and ahbL2) for the synthesis and activation of 3,4-AHBA, a new hybrid compound, AHB18, was identified which had been produced from a metabolic crosstalk between the AHB and the argimycin P pathways. The identification of this new BGC opens the possibility to generate new compounds by combinatorial biosynthesis.

Bioprospecting Fluorescent Plant Growth Regulators from Arabidopsis to Vegetable Crops.

The phytohormone auxin is involved in almost every process of a plant's life, from germination to plant development. Nowadays, auxin research connects synthetic chemistry, plant biology and computational chemistry in order to develop innovative and safe compounds to be used in sustainable agricultural practice. In this framework, we developed new fluorescent compounds, ethanolammonium p-aminobenzoate (HEA-pABA) and p-nitrobenzoate (HEA-pNBA), and investigated their auxin-like behavior on two main commercial vegetables cultivated in Europe, cucumber (Cucumis sativus) and tomato (Solanumlycopersicum), in comparison to the model plant Arabidopsis (Arabidopsis thaliana). Moreover, the binding modes and affinities of two organic salts in relation to the natural auxin indole-3-acetic acid (IAA) into TIR1 auxin receptor were investigated by computational approaches (homology modeling and molecular docking). Both experimental and theoretical results highlight HEA-pABA as a fluorescent compound with auxin-like activity both in Arabidopsis and the commercial cucumber and tomato. Therefore, alkanolammonium benzoates have a great potential as promising sustainable plant growth stimulators to be efficiently used in vegetable crops.

An Unusual Route for p-Aminobenzoate Biosynthesis in Chlamydia trachomatis Involves a Probable Self-Sacrificing Diiron Oxygenase.

Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p-aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis, the CT610 gene, that rescues Escherichia coli ΔpabA, ΔpabB, and ΔpabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli ΔpabA cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor.IMPORTANCEChlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p-aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis We further provide evidence that CT610 is a self-sacrificing or "suicide" enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections.

Novel Gene Encoding 5-Aminosalicylate 1,2-Dioxygenase from Comamonas sp. Strain QT12 and Catalytic Properties of the Purified Enzyme.

The 1,125-bp mabB gene encoding 5-aminosalicylate (5ASA) 1,2-dioxygenase, a nonheme iron dioxygenase in the bicupin family that catalyzes the cleavage of the 5ASA aromatic ring to form cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate in the biodegradation of 3-aminobenzoate, was cloned from Comamonas sp. strain QT12 and characterized. The deduced amino acid sequence of the enzyme has low sequence identity with that of other reported ring-cleaving dioxygenases. MabB was heterologously expressed in Escherichia coli cells and purified as a His-tagged enzyme. The optimum pH and temperature for MabB are 8.0 and 10°C, respectively. FeII is required for the catalytic activity of the purified enzyme. The apparent Km and Vmax values of MabB for 5ASA are 52.0 ± 5.6 µM and 850 ± 33.2 U/mg, respectively. The two oxygen atoms incorporated into the product of the MabB-catalyzed reaction are both from the dioxygen molecule. Both 5ASA and gentisate could be converted by MabB; however, the catalytic efficiency of MabB for 5ASA was much higher (∼70-fold) than that for gentisate. The mabB-disrupted mutant lost the ability to grow on 3-aminobenzoate, and mabB expression was higher when strain QT12 was cultivated in the presence of 3-aminobenzoate. Thus, 5ASA is the physiological substrate of MabB.IMPORTANCE For several decades, 5-aminosalicylate (5ASA) has been advocated as the drug mesalazine to treat human inflammatory bowel disease and considered the key intermediate in the xenobiotic degradation of many aromatic organic pollutants. 5ASA biotransformation research will help us elucidate the microbial degradation of these pollutants. Most studies have reported that gentisate 1,2-dioxygenases (GDOs) can convert 5ASA with significantly high activity; however, the catalytic efficiency of these enzymes for gentisate is much higher than that for 5ASA. This study showed that MabB can convert 5ASA to cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, incorporating two oxygen atoms from the dioxygen molecule into the product. Unlike GDOs, MabB uses 5ASA instead of gentisate as the primary substrate. mabB is the first reported 5-aminosalicylate 1,2-dioxygenase gene.

A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp. strain QT12.

The biodegradation pathway of 3-aminobenzoate has been documented, but little is known about the sequence and biochemical properties of the proteins involved. In the present study, a 10,083-bp DNA fragment involved in 3-aminobenzoate degradation was identified in 3-aminobenzoate-degrading Comamonas sp. strain QT12. The mabA gene, whose encoded protein shares 39% amino acid sequence identity with 3-hydroxybenzoate 6-hydroxylase of Polaromonas naphthalenivorans CJ2, was identified on this DNA fragment, and the mabA-disrupted mutant was unable to grow on and convert 3-aminobenzoate. MabA was heterologously expressed in Escherichia coli and purified to homogeneity as an approximately ~ 48-kDa His-tagged protein. It was characterized as 3-aminobenzoate 6-hydroxylase capable of catalyzing the conversion of 3-aminobenzoate to 5-aminosalicylate, incorporating one oxygen atom from dioxygen into the product. It contains a non-covalent but tightly bound FAD as the prosthetic group and NADH as an external electron donor. 5-Aminosalicylate was produced with equimolar consumption of NADH. The apparent Km and kcat values of the purified enzyme for 3-aminobenzoate were 158.51 ± 4.74 µM and 6.49 ± 0.17 s-1, respectively, and those for NADH were 189.85 ± 55.70 µM and 7.41 ± 1.39 s-1, respectively. The results suggest that mabA is essential for 3-aminobenzoate degradation in strain QT12, and that 3-aminobenzoate is the primary and physiological substrate of MabA.

A Facile Semi-Synthetic Approach towards Halogen-Substituted Aminobenzoic Acid Analogues of Platensimycin.

Platensimycin (PTM), produced by several strains of Streptomyces platensis, is a promising drug lead for infectious diseases and diabetes. The recent pilot-scale production of PTM from S. platensis SB12026 has set the stage for the facile semi-synthesis of a focused library of PTM analogues. In this study, gram-quantity of platensic acid (PTMA) was prepared by the sulfuric acid-catalyzed ethanolysis of PTM, followed by a mild hydrolysis in aqueous lithium hydroxide. Three PTMA esters were also obtained in near quantitative yields in a single step, suggesting a facile route to make PTMA aliphatic esters. 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU)-catalyzed coupling of PTMA and 33 aminobenzoates resulted in the synthesis of 28 substituted aminobenzoate analogues of PTM, among which 26 of them were reported for the first time. Several of the PTM analogues showed weak antibacterial activity against methicillin-resistant Staphylococcus aureus. Our study supported the potential utility to integrate natural product biosynthetic and semi-synthetic approaches for structure diversification.

Degradation of Methyl 2-Aminobenzoate (Methyl Anthranilate) by H2O2/UV: Effect of Inorganic Anions and Derived Radicals.

This study shows that methyl 2-aminobenzoate (also known as methyl anthranilate, hereafter MA) undergoes direct photolysis under UVC and UVB irradiation and that its photodegradation is further accelerated in the presence of H2O2. Hydrogen peroxide acts as a source of hydroxyl radicals (·OH) under photochemical conditions and yields MA hydroxyderivatives. The trend of MA photodegradation rate vs. H2O2 concentration reaches a plateau because of the combined effects of H2O2 absorption saturation and ·OH scavenging by H2O2. The addition of chloride ions causes scavenging of ·OH, yielding Cl2·- as the most likely reactive species, and it increases the MA photodegradation rate at high H2O2 concentration values. The reaction between Cl2·- and MA, which has second-order rate constant k C l 2 • - + M A = (4.0 ± 0.3) × 108 M-1·s-1 (determined by laser flash photolysis), appears to be more selective than the ·OH process in the presence of H2O2, because Cl2·- undergoes more limited scavenging by H2O2 compared to ·OH. While the addition of carbonate causes ·OH scavenging to produce CO3·- ( k C O 3 • - + M A = (3.1 ± 0.2) × 108 M-1·s-1), carbonate considerably inhibits the photodegradation of MA. A possible explanation is that the elevated pH values of the carbonate solutions make H2O2 to partially occur as HO2-, which reacts very quickly with either ·OH or CO3·- to produce O2·-. The superoxide anion could reduce partially oxidised MA back to the initial substrate, with consequent inhibition of MA photodegradation. Fast MA photodegradation is also observed in the presence of persulphate/UV, which yields SO4·- that reacts effectively with MA ( k S O 4 • - + M A = (5.6 ± 0.4) × 108 M-1·s-1). Irradiated H2O2 is effective in photodegrading MA, but the resulting MA hydroxyderivatives are predicted to be about as toxic as the parent compound for aquatic organisms (most notably, fish and crustaceans).

Identification and characterization of a pab gene cluster responsible for the 4-aminobenzoate degradation pathway, including its involvement in the formation of a γ-glutamylated intermediate in Paraburkholderia terrae strain KU-15.

Paraburkholderia terrae strain KU-15 grows on 2- and 4-nitrobenzoate and 2- and 4-aminobenzoate (ABA) as the sole nitrogen and carbon sources. The genes responsible for the potential degradation of 2- and 4-nitrobenzoate and 2-ABA have been predicted from its genome sequence. In this study, we identified the pab operon in P. terrae strain KU-15. This operon is responsible for the 4-ABA degradation pathway, which involves the formation of a γ-glutamylated intermediate. Reverse transcription-polymerase chain reaction revealed that the pab operon was induced by 4-ABA. Herein, studying the deletion of pabA and pabB1 in strain KU-15 and the examining of Escherichia coli expressing the pab operon revealed the involvement of the operon in 4-ABA degradation. The first step of the degradation pathway is the formation of a γ-glutamylated intermediate, whereby 4-ABA is converted to γ-glutamyl-4-carboxyanilide (γ-GCA). Subsequently, γ-GCA is oxidized to protocatechuate. Overexpression of various genes in E. coli and purification of recombinant proteins permitted the functional characterization of relevant pathway proteins: PabA is a γ-GCA synthetase, PabB1-B3 functions in a multicomponent dioxygenase system responsible for γ-GCA dioxygenation, and PabC is a γ-GCA hydrolase that reverses the formation of γ-GCA by PabA.

A single PLP-dependent enzyme PctV catalyzes the transformation of 3-dehydroshikimate into 3-aminobenzoate in the biosynthesis of pactamycin.

Natural amino donation: A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature.

Enhancing pterin and para-aminobenzoate content is not sufficient to successfully biofortify potato tubers and Arabidopsis thaliana plants with folate.

Folates are important cofactors in one-carbon metabolism in all living organisms. Since only plants and micro- organisms are capable of biosynthesizing folates, humans depend entirely on their diet as a folate source. Given the low folate content of several staple crop products, folate deficiency affects regions all over the world. Folate biofortification of staple crops through enhancement of pterin and para-aminobenzoate levels, precursors of the folate biosynthesis pathway, was reported to be successful in tomato and rice. This study shows that the same strategy is not sufficient to enhance folate content in potato tubers and Arabidopsis thaliana plants and concludes that other steps in folate biosynthesis and/or metabolism need to be engineered to result in substantial folate accumulation. The findings provide a plausible explanation why, more than half a decade after the proof of concept in rice and tomato, successful folate biofortification of other food crops through enhancement of para-aminobenzoate and pterin content has not been reported thus far. A better understanding of the folate pathway is required in order to determine an engineering strategy that can be generalized to most staple crops.

Efficient Synthesis of Novel N-[4-Methyl-3-(4-(5-phenyl-1,3,4-oxadiazol-2- yl)phenyl)thiazol-2(3H)-ylidene]benzamide Hybrid Ring System as Potential Antibacterial Agents.

BACKGROUND: Heterocyclic compounds display versatile biological applications, so the aim of this paper was to prepare biologically important heterocycles with enhanced bacterial resistance and to evaluate for their various structural features that are responsible for their biological properties. OBJECTIVE: The objective was to synthesize bacterial resistance compounds with enhanced antibacterial properties. METHODS: Ester moiety containing thiazole ring was converted into its hydrazide derivatives. These heterocyclic derivatives were cyclized into another ring oxadiazole; hence a hybrid ring system of two biologically active rings was prepared. RESULTS: All the synthesized compounds were characterized by spectroscopic techniques and were screened for their antibacterial potential; they possess significant antibacterial activities. CONCLUSION: New hybrid heterocyclic ring systems were synthesized by cyclization of hydrazide derivatives by adopting two step strategy in good yields. All the synthesized compounds were evaluated for their antioxidant activities; they showed moderate to significant activities. QSAR and Molecular docking studies were performed to determine the mode of interaction. Experimental and computational data is in accordance with the determined antibacterial activities.

Case report: Kinetics of human leukocyte antigen receptor HLA-DR during liver injury induced by potassium para-aminobenzoate as assessed for causality using the updated RUCAM.

Potassium para-aminobenzoate (POTABA) is used to treat Peyronie's disease by decreasing fibrosis and plaque size progression. Among potential side effects, drug-induced liver injury (DILI) attributed to POTABA administration has been reported in a few cases and inferred to immune hypersensitivity. In the present case, we investigated clinical, biochemical, and serological features as well as searched for non-drug-related causes, and applied the updated Roussel Uclaf Causality Assessment Method (RUCAM) confirming a highly probable causality of POTABA-induced liver injury. Moreover, we here observed specific activated CD3+ T lymphocytes during the acute phase of liver injury by monitoring of human leukocyte antigen receptor (HLA-DR) expression. Furthermore, improvement of biochemical markers of liver injury after POTABA withdrawal was associated with a rapid decline of CD3+ HLA-DR+ immune cells. In contrast, CD14+ monocytes expressing HLA-DR remained stable during recovery from liver injury. These observations implicate a specific involvement of activated T lymphocytes in liver injury mediated by POTABA. Clinicians should be aware of POTABA-induced liver injury, and measurement of activated immune cells by assessment of HLA-DR could provide pathomechanistic insights enabling biomonitoring of recovery from DILI.

Synthesis, Structure and Cytotoxicity of N,N and N,O-Coordinated RuII Complexes of 3-Aminobenzoate Schiff Bases against Triple-negative Breast Cancer.

Half-sandwich RuII complexes, [(YZ)RuII (η6 -arene)(X)]+, (YZ=chelating bidentate ligand, X=halide), with N,N and N,O coordination (1-9) show significant antiproliferative activity against the metastatic triple-negative breast carcinoma (MDA-MB-231). 3-aminobenzoic acid or its methyl ester is used in all the ligands while varying the aldehyde for N,N and N,O coordination. In the N,N coordinated complex the coordinated halide(X) is varied for enhancing stability in solution (X=Cl, I). Rapid aquation and halide exchange of the pyridine analogues, 2 and 3, in solution are a major bane towards their antiproliferative activity. Presence of free -COOH group (1 and 4) make complexes hydrophilic and reduces toxicity. The imidazolyl 3-aminobenzoate based N,N coordinated 5 and 6 display better solution stability and efficient antiproliferative activity (IC50 ca. 2.3-2.5 µM) compared to the pyridine based 2 and 3 (IC50 >100 µM) or the N,O coordinated complexes (7-9) (IC50 ca. 7-10 µM). The iodido coordinated, 6, is resistant towards aquation and halide exchange. The N,O coordinated 7-9 underwent instantaneous aquation at pH 7.4 generating monoaquated complexes stable for at least 6 h. Complexes 5 and 6, bind to 9-ethylguanine (9-EtG) showing propensity to interact with DNA bases. The complexes may kill via apoptosis as displayed from the study of 8. The change in coordination mode and the aldehyde affected the solution stability, antiproliferative activity and mechanistic pathways. The N,N coordinated (5 and 6) exhibit arrest in the G2/M phase while the N,O coordinated 8 showed arrest in the G0/G1 phase.

Crystal structures of 2-amino-2-oxoethyl 4-bromo-benzoate, 2-amino-2-oxoethyl 4-nitro-benzoate and 2-amino-2-oxoethyl 4-amino-benzoate monohydrate.

The title mol-ecules were synthesized by the reaction of 4-substituted sodium benzoates with chloro-acetic acid amide in the presence of di-methyl-formamide. The yields of 2-amino-2-oxoethyl 4-bromo-benzoate, C9H8BrNO3, I, 2-amino-2-oxoethyl 4-nitro-benzoate, C9H8N2O5, II, and 2-amino-2-oxoethyl 4-amino-benzoate monohydrate, C9H10N2O3·H2O, III, are 86, 78 and 88%, respectively. The low yield of II is explained by the reduced reactivity of the mol-ecule in a nucleophilic exchange reaction because of the negative induction and negative mesomeric effects of the nitro group on the benzene ring. Single crystals were obtained from the products under the same (temperature and solvent) conditions. In the case of III, the crystals formed as a monohydrate. In all three crystal structures, the same type of inter-molecular N-H⋯O hydrogen bonds are observed, but the mol-ecules differ in some torsion angles as well as in the dihedral angles between the mean planes of the benzene rings and the amide groups.

[Improving the production of 4-aminobenzoic in engineered Escherichia coli by combinatorial regulation].

Para-aminobenzoate (PABA) is an important chemical for organic synthesis and extensively used in pharmaceutical and dye industry. In recent years, PABA has received increasing attention as a potential component of high-strength polymer. In Escherichia coli, three genes of pabA, pabB and pabC are responsible for PABA production from chorismate in folate synthetic pathway. However, E. coli does not accumulate or accumulates very few amounts of PABA under normal growth condition. In this study, the tyrosine-producing E. coli TYR002 constructed previously was used as the starting strain for developing PABA-producing strain. First, the activity of bifunctional chorismate mutase/prephenate dehydrogenase TyrA in E. coli TYR002 was weakened to reduce the production of tyrosine. Then, three different constitutive promoters were used to regulate the expression of pabA, pabB and pabC in recombinant plasmid which was transformed into E. coli for improving PABA production. The shake-flask fermentation showed that the different combination of constitutive promoters significantly affected the production of PABA, and the highest shake-flask fermentation titer was 0.67 g/L. After further condition optimization, the engineered E. coli produced 6.4 g/L PABA under 5 L fed-batch fermentation. This study could be a good reference for improving microbial production of PABA.

Rare Incidence of Acute Liver Injury with Potassium Para-Aminobenzoate Introduction.

Acute liver injury is an alarming condition, as it may lead to a devastating outcome. Of the many causes of acute liver injury, review of medications is crucial to identifying the cause of the injury. Some commonly used medications may unpredictably be the underlying cause of liver injury. We present a case of Peyronie's disease treated with potassium para-aminobenzoate that developed acute liver injury. After starting the new treatment, the patient complained of right upper quadrant pain. He was found to have elevated liver enzymes. The condition resolved after stopping potassium para-aminobenzoate use. We report a potassium para-aminobenzoate side effect of liver injury that can be managed conservatively.

Environmental behavior of 12 UV filters and photocatalytic profile of ethyl-4-aminobenzoate.

Ethyl-4-aminobenzoate (Et-PABA) is currently used as a substitute for 4-aminobenzoate (PABA) in sunscreens and anesthetic ointments. Despite its widespread use and hydrophilicity, Et-PABA has never been found in environmental waters. This study, probed the occurrence of Et-PABA in both seawater and drinking water sources in Hong Kong, and evaluated its transformation products (TPs) and environmental fate via cumulative potency and photocatalytic profile analyses. Another 11 UV filters used in skin-care products were also studied. Et-PABA was not detected in any water sample. Four other UV filters were dominant at ng/L level in both seawater and drinking water sources. UHPLC-QTOF-MS was used to elucidate the structure of TPs. With high resolution accurate mass data and fragment rationalization, 11 Et-PABA TPs were characterized, including seven intermediates firstly proposed as TPs; two compounds were reported for the first time. It is proposed that photocatalysis induces transformation pathways of (de)hydroxylation, demethylation and molecular rearrangement. Luminescent bacteria tests showed decreasing toxicity with increasing irradiation of Et-PABA, suggesting that irradiation TPs are less toxic than the parent compound. Transformation of Et-PABA appears to explain why Et-PABA has not been detected in the natural environment.

Exploration of ethyl anthranilate-loaded monolithic matrix-type prophylactic polymeric patch.

Compromised stability of pharmaceutical formulations loaded with volatiles is a serious problem associated with devices designed to deliver volatile compounds. The present study has been focused to evaluate the stability potential of matrix-type polymeric patches composed of volatile ethyl anthranilate for prophylaxis against vector-borne diseases. Ethyl anthranilate-loaded matrix-type polymeric patches were fabricated by solvent evaporation method on an impermeable backing membrane and attached to temporary release liners. Stability testing of the polymeric patches was performed as per the International Conference on Harmonization (ICH) guidelines for 6 months under accelerated conditions. In addition, the quantification of residual solvents was also performed as per the ICH guidelines. After conducting the stability studies for 6 months, the optimized patches showed the best possible results with respect to uniformity of drug content, physical appearance, and other analytical parameters. Furthermore, the amount of residual solvent was found well below the accepted limit. Thus, the present report outlined the analytical parameters to be evaluated to ensure the stability of a certain devices consisting of volatile compounds.

Aggregation of Thaumatomyia glabra (Diptera: Chloropidae) Males on Iris spp. Flowers Releasing Methyl Anthranilate.

Aggregations of Thaumatomyia glabra (Diptera: Chloropidae) were observed on flowers of Iris pallida Lamarck (Asparagales: Iridaceae), whereas no T. glabra (Meigen) were observed on nearby Iris germanica L. flowers. Sampling of T. glabra on I. pallida flowers revealed the presence of males only. In a previous study, T. glabra males were attracted to methyl anthranilate. We found methyl anthranilate in extracts of I. pallida flowers on which T. glabra aggregated, but not in extracts of I. germanica flowers. Applying methyl anthranilate to I. germanica flowers elicited attraction of T. glabra to the flowers. This study suggests that I. pallida flowers may attract T. glabra males to aggregate because they release the known attractant, methyl anthranilate, whereas I. germanica flowers may not be attractive because they do not release methyl anthranilate.

Competitive homolytic and heterolytic decomposition pathways of gas-phase negative ions generated from aminobenzoate esters.

An alkyl-radical loss and an alkene loss are two competitive fragmentation pathways that deprotonated aminobenzoate esters undergo upon activation under mass spectrometric conditions. For the meta and para isomers, the alkyl-radical loss by a homolytic cleavage of the alkyl-oxygen bond of the ester moiety is the predominant fragmentation pathway, while the contribution from the alkene elimination by a heterolytic pathway is less significant. In contrast, owing to a pronounced charge-mediated ortho effect, the alkene loss becomes the predominant pathway for the ortho isomers of ethyl and higher esters. Results from isotope-labeled compounds confirmed that the alkene loss proceeds by a specific γ-hydrogen transfer mechanism that resembles the McLafferty rearrangement for radical cations. Even for the para compounds, if the alkoxide moiety bears structural motifs required for the elimination of a more stable alkene molecule, the heterolytic pathway becomes the predominant pathway. For example, in the spectrum of deprotonated 2-phenylethyl 4-aminobenzoate, m/z 136 peak is the base peak because the alkene eliminated is styrene. Owing to the fact that all deprotonated aminobenzoate esters, irrespective of the size of the alkoxy group, upon activation fragment to form an m/z 135 ion, aminobenzoate esters in mixtures can be quantified by precursor ion discovery mass spectrometric experiments.