Synthesis of 4-epi-2-deoxy-2-Heq-N-acetylneuraminic acid and 2,4-dideoxy-2-Heq N-acetylneuraminic acid

We have continued our work to develop novel analogues of sialic acid [1–4] that may specifically modulate the interaction between endogenous sialic acid and influenza virus haemagglutinin [3,5,6]. Functional groups of sialic acid that have been implicated for this virus-host recongnition are the glycerol side chain, N-acetyl group and the axially oriented carboxylic acid function In this report we describe the synthesis of two analogues, namely, 4-epi-2-deoxy-2-H_eq-N-acetylneuraminic acid (4-epi-2-d-2-H_eq-Neu5Ac) and 2,4-dideoxy-2-H_eq-N-acetylneuraminic acid (2,4-d₂-2-H_eq-Neu5Ac).     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

Inhibitors of phenylalanine ammonia-lyase: substituted derivatives of 2-aminoindane-2-phosphonic acid and 1-aminobenzylphosphonic acid

Six derivatives of 2-aminoindane-2-phosphonic acid and 1-aminobenzylphosphonic acid were synthesized. The compounds were tested both as inhibitors of buckwheat phenylalanine ammonia-lyase (in vitro) and as inhibitors of anthocyanin biosynthesis (in vivo). (+/-)-2-Amino-4-bromoindane-2-phosphonic acid was found to be the strongest inhibitor from investigated compounds. The results obtained are a basis for design of phenylalanine ammonia-lyase inhibitors useful in the enzyme structure studies in photo labelling experiments.     

2-fluorourocanic acid, a potent reversible inhibitor of urocanase.

The K_i for the interaction of 2-fluorourocanic acid with urocanase (from $\text{Pseudomonas fluorescens}$) is 1000 times as great as K_m for the natural substrate, urocanic acid, whereas enzymatic hydration of the fluoro analog occurs $\text{ca}$. 100 times more slowly. Inhibition is competive and is eventually overcome by utilization of the analog. By contrast, 4-fluoro- and 2-amino-urocanic acid are neither significant inhibitors nor substrates for the enzyme. 2-Fluorourocanic acid may prove a useful tool for blocking the utilization of histidine as a one-carbon source in metabolism.     

Stimulation of insulin biosynthesis in isolated mouse islets by L-leucine, 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid

An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [³H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine and D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.     

Decomposition of 2-chloroethylphosphonic acid in stems and leaves of Hevea brasiliensis

The plant growth regulator 2-chloroethylphosphonic acid is converted to at least 13 compounds other than ethylene in leaf and stem tissue of Hevea brasiliensis. A minor component of the products is probably 2-hydroxyethylphosphonic acid.     

Bioconversion of 2-amino acids to 2-hydroxy acids by Clostridium butyricum

Analysis by gas chromatography-mass spectrometry (GC-MS) of 24-h cultures of Clostridium butyricum type strain in synthetic BMG medium supplemented with various 2-amino acids (10 mM) revealed the presence of the corresponding 2-hydroxy acids. C. butyricum was able to bioconvert l-valine, dl-norvaline, l-leucine, dl-norleucine, l-methionine and l-phenylalanine as well as unusual 2-amino acids, i.e., l-2-aminobutyric acid, l-2-amino-4-pentenoic acid, dl-2-aminooctanoic acid, and dl-2-amino-4-phenylbutanoic acid. l-Isoleucine and cycloleucine were not converted into their corresponding 2-hydroxy acids. The bioconversion rate was maximal with dl-norvaline (6.2%). Chiral GC analysis demonstrated that only d-2-hydroxy-4-methylpentanoic acid is formed from l-leucine, indicating that the bioconversion is stereospecific, with inversion of configuration. d-Leucine and d-methionine were also converted to the corresponding 2-hydroxy acids. This observation opens new aspects in the study of C. butyricum and raises questions about the amino acid metabolism by this species.     

Quantitation of 2-chlorovinylarsonous acid in human urine by automated solid-phase microextraction--gas chromatography--mass spectrometry

Lewisite [dichloro(2-chlorovinyl)arsine] is a highly toxic chemical warfare agent with vesicant properties. The accidental exposure to lewisite or its intentional use as a chemical terrorism weapon are a public health threat and warrant investigations for the development of analytical methods to detect biomarkers of exposure to lewisite. Under aqueous conditions, lewisite rapidly hydrolyzes to the non-volatile 2-chlorovinylarsonous acid (CVAA). We have developed a sensitive, simple, and automated method for measuring CVAA in human urine. The assay is based on the use of solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) after derivatization of the CVAA with 1,3-propanedithiol (PDT). The volatile CVAA-PDT is adsorbed onto a SPME fiber and analyzed by GC-MS. The assay was validated on human urine samples spiked with CVAA to determine the accuracy, precision, and limit of detection (LOD). The LOD was 7.4 pg in 1 ml of urine.     

Determination of methyl-, 2-hydroxyethyl- and 2-cyanoethylmercapturic acids as biomarkers of exposure to alkylating agents in cigarette smoke

Alkylating agents occur in the environment and are formed endogenously. Tobacco smoke contains a variety of alkylating agents or precursors including, among others, N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acrylonitrile and ethylene oxide. We developed and validated a method for the simultaneous determination of methylmercapturic acid (MMA, biomarker for methylating agents such as NDMA and NNK), 2-hydroxyethylmercapturic acid (HEMA, biomarker for ethylene oxide) and 2-cyanoethylmercapturic acid (CEMA, biomarker for acrylonitrile) in human urine using deuterated internal standards of each compound. The method involves liquid/liquid extraction of the urine sample, solid phase extraction on anion exchange cartridges, derivatization with pentafluorobenzyl bromide (PFBBr), liquid/liquid extraction of the reaction mixture and LC–MS/MS analysis with positive electrospray ionization. The method was linear in the ranges of 5.00–600, 1.00–50.0 and 1.50–900 ng/ml for MMA, HEMA and CEMA, respectively. The method was applied to two clinical studies in adult smokers of conventional cigarettes who either continued smoking conventional cigarettes, were switched to test cigarettes consisting of either an electrically heated cigarette smoking system (EHCSS) or having a highly activated carbon granule filter that were shown to have reduced exposure to specific smoke constituents, or stopped smoking. Urinary excretion of MMA was found to be unaffected by switching to the test cigarettes or stop smoking. Urinary HEMA excretion decreased by 46 to 54% after switching to test cigarettes and by approximately 74% when stopping smoking. Urinary CEMA excretion decreased by 74–77% when switching to test cigarettes and by approximately 90% when stopping smoking. This validated method for urinary alkylmercapturic acids is suitable to distinguish differences in exposure not only between smokers and nonsmokers but also between smoking of conventional and the two test cigarettes investigated in this study.     

Auxin activity and molecular structure of 2-alkylindole-3-acetic acids

2-Methylindole-3-acetic acid (2-Me-IAA) is a known auxin, but its 2-ethyl homologue has been considered inactive. Here we show that the compound previously bioassayed as 2-ethylindole-3-acetic acid (2-Et-IAA) was, in fact, 3-(3-methylindol-2-yl)propionic acid. The proper 2-Et-IAA and its 2-(n-propyl) homologue (2-Pr-IAA) are prepared, unambiguously characterized, and their auxin activity is demonstrated in the Avena coleoptile-section straight-growth test. Their half-optimal concentrations are approximately the same as for 2-Me-IAA (2 × 10^–5mol L^–1), and hence about ten times larger than for unsubstituted indole-3-acetic acid (IAA) and its derivatives alkylated in positions 4, 5, 6 or 7. The optimal response elicited by 2-Et-IAA and 2-Pr-IAA is about half that observed for 2-Me-IAA. These characteristics place the three 2-alkyl-IAAs along the borderline between the classes of strong and weak auxins, thus corroborating the results of interaction similarity analysis, a mathematical approach based on the capability of auxin molecules to participate in non-bonding interactions with a generalized receptor protein. X-ray diffraction analysis shows no explicit structural features to be blamed for the decrease in auxin activity caused by attaching a 2-alkyl substituent to the IAA molecule; sterical interference of the 3-CH₂COOH group and the 2-alkyl moiety is barely recognizable in the crystalline state. Quantum-chemical calculations and molecular dynamics simulations suggest that 2-alkyl-IAAs, in the absence of crystal-packing restraints, prefer conformations with the CH₂-COOH bond tilted to the heterocyclic ring system. Substantially higher conformational energy (and hence lower abundance) is predicted for planar conformers which were previously shown to prevail for IAA and many of its derivatives substituted in the benzene moiety of the indole nucleus. This shift in the rotational preferences of the -CH₂COOH moiety may be one of the reasons for the reduced plant-growth promoting activity of 2-alkyl-IAAs.     

Effect of dielectric constant on protonation equilibria of 2, 3-dihydroxybenzoic acid and malonic acid in 1, 2-propanediol-water mixtures

Abstract

The protonation constants of 2,3-diydroxybenzoic acid (2, 3-DHBA) and malonic acid (MA) at 303.0 ± 0.1 K and 0.16 mol L^-1 ionic strength in various concentrations (0–60% v/v) of 1,2-propanediol–water-mixtures were determined by pH-metric method. The protonation constants were calculated with MINIQUAD75 computer program. Selection of the best fit chemical models of the acid–base equilibria was based on statistical parameters. The log K values were found to increase with the increase in percentage of 1,2-propanediol and vary linearly with the reciprocal of the dielectric constant of the medium. This has been attributed to the dominance of electrostatic forces. Distributions of species and effect of influential parameters on the protonation constants are also presented.     

氨基茚磷酸(AIP)和氨基氧乙酸(AOA)对茉莉酸甲酯诱导的烟草一些酶活性的影响

用茉莉酸甲酯(MJ,1mg ml^-1)处理培养在含0．1mmol／L AIP(水杨酸合成抑制剂)和／或1mmol／L AOA(乙烯合成抑制剂)的MS培养基上的烟草愈伤组织,测定某些酶的活性。结果表明：MJ明显提高过氧化物酶(POD)、β1,3-葡聚糖苷酶和几丁质外切酶的活性,略微促进苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性,抑制几丁内切酶的活性,而AOA和AIP则明显抑制MJ对POD和β1,3-葡聚糖苷酶活性的诱导作用,但对MJ诱导的PAL和PPO的活性影响很小,AOA和AIP可能作为逆境因子促进PAL的活性。AOA能部分解除MJ对几丁内切酶的抑制作用,但对MJ诱导的几丁外切酶的影响较小,而AIP抑制几丁内切酶的活性,也抑制MJ对此酶的诱导作用。因此我们认为：MJ对POD、PPO和几丁内切酶的影响可能是通过乙烯途径,对β1,3-葡聚糖苷酶和几丁外切酶的影响可能是通过水杨酸(SA)途径,而对PAL的影响可能是通过其它途径。     

Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway

A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism. Received: 27 November 1997 / Accepted: 14 May 1998     

Fatty acid 2-Hydroxylation in mammalian sphingolipid biology

2-Hydroxy fatty acids (hFA) are important components of a subset of mammalian sphingolipids. The presence of hFA in sphingolipids is best described in the nervous system, epidermis, and kidney. However, the literature also indicates that various hFA-sphingolipids are present in additional tissues and cell types, as well as in tumors. Biosynthesis of hFA-sphingolipids requires fatty acid 2-hydroyxlase, and degradation of hFA-sphingolipids depends, at least in part, on lysosomal acid ceramidase and the peroxisomal fatty acid α-oxidation pathway. Mutations in the fatty acid 2-hydroxylase gene, FA2H, have been associated with leukodystrophy and spastic paraparesis in humans, underscoring the importance of hFA-sphingolipids in the nervous system. In the epidermis, hFA-ceramides are essential for the permeability barrier function. Physiological function of hFA-sphingolipids in other organs remains largely unknown. Recent evidence indicates that hFA-sphingolipids have specific roles in cell signaling.     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Rapid determination of chloroprocaine and its major metabolite, 2-chloroaminobenzoic acid, in plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic method for determination of the 2-chloroprocaine, local anesthetic of ester type, and its major metabolite 2-chloroaminobenzoic acid, has been developed and validated. A single-step extraction procedure is employed followed by high-performance liquid chromatographic separation using reversed-phase column and analysis using variable length UV detection. Lidocaine was used as internal standard for 2-chloroprocaine measurement and p-aminobenzoic acid was used as internal standard for 2-chloroaminobenzoic acid analysis. The analysis of spiked plasma demonstrated good accuracy and precision of the method with limit of detection 0.1 μg/ml for 2-chloroprocaine and 0.5 μg/ml for 2-chloroaminobenzoic acid. The method has been used for pharmacokinetic studies in laboratory animals.     

Role of phospholipase D2 in anti-apoptotic signaling through increased expressions of Bcl-2 and Bcl-xL

We have previously reported that Fas-resistant A20 cells (FasR) have phospholipase D (PLD) activity upregulated by endogenous PLD2 overexpression. In the present study, we investigated how overexpressed PLD2 in FasR could generate survival signals by regulating the protein levels of anti-apoptotic Bcl-2 and Bcl-xL. To confirm the effect of PLD2 on Bcl-2 protein levels, we transfected PLD2 into wild-type murine B lymphoma A20 cells. The transfected cells showed markedly the increases in Bcl-2 and Bcl-xL protein levels, and became resistant to Fas-induced apoptosis, similar to FasR. Treatment of wild-type A20 cells with phosphatidic acid (PA), the metabolic end product of PLD2 derived from phosphatidylcholin, markedly increased levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Moreover, PA-induced expressions of Bcl-2 and Bcl-xL were enhanced by propranolol, an inhibitor of PA phospholydrolase (PAP), whereas completely blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), suggesting that PLA(2) metabolite of PA is responsible for the increases in Bcl-2 and Bcl-xL protein levels. We further confirmed the involvement of arachidonic acid (AA) in PA-induced survival signals by showing that 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), PA without AA, was unable to increase Bcl-2 and Bcl-xL proteins. Moreover, PA notably increased cyclooxygenase (COX)-2 protein expression, and PA-induced expression of both Bcl-2 and Bcl-xL was inhibited by NS-398, a specific inhibitor of COX-2. Taken together, these findings demonstrate that PA generated by PLD2 plays an important role in cell survival during Fas-mediated apoptosis through the increased Bcl-2 and Bcl-xL protein levels which resulted from PLA(2) and AA-COX2 pathway.     

2-amino-4-acetylaminobutyric acid, 2,4-diaminobutyric acid and 2-amino-6N-oxalylureidopropionic acid (oxalylalbizziine) in seeds of acacia angustissima

A new amino acid previously detected in 17 species of Acacia has been isolated from seeds of Acacia angustissima and identified as oxalylalbizziine. These seeds also contain more than 6% dry weight of 2-amino-4-acetylaminobutyric acid, which has not been reported previously in a legume, and lower concentrations of 2,4-diaminobutyric acid.