Ester Hydrolysis Products Research Articles

Polyurethane degradation by extracellular urethanase producing bacterial isolate Moraxella catarrhalis strain BMPPS3

Plastic waste has become a global issue and a threat to the ecosystem. The present study isolated polyurethane (PU) degrading bacterial species from soil dumped with plastic wastes. Four bacterial isolates, RS1, RS6, RS9 and RS13 were obtained and their ability to degrade PU in a synthetic medium with PU as a sole source of carbon was assessed individually. After thirty days of incubation, the highest PU weight loss of 67.36 ± 0.32% was recorded in the medium containing RS13 isolate. The results of FTIR revealed the occurrence of carbonyl peaks. The putative isolate RS13 confirmed with the genus Moraxella according to 16S rRNA gene sequencing and the isolate was specified as Moraxella catarrhalis strain BMPPS3. The restriction analysis of Moraxella catarrhalis strain BMPPS3 revealed that the GCAT content to 51% and 49% correspondingly. Moraxella catarrhalis strain BMPPS3 was able to colonize on PU surface and form a biofilm as revealed by SEM investigation. Fatty acids and alkanes were found to be the degradation products by GC-MS analysis. The presence of these metabolites facilitated the growth of strain RS13 and suggested that ester hydrolysis products had been mineralized into CO2 and H2O. Extracellular biosurfactant synthesis has also been found in Moraxella catarrhalis strain BMPPS13 inoculated with synthetic media and mineral salt media containing PU and glucose as carbon sources, respectively with a significant level of cell-surface hydrophobicity (32%). The production and activity of extracellular esterase showed consistent increase from day 1–15 which peaked (1.029 mM/min/mg) on day 24 significantly at P < 0.001. Crude biosurfactants were lipopeptide-based, according to the characteristic investigation. According to this study findings, Moraxella catarrhalis produces biosurfactants of the esterase, urethanase and lipase (lipopeptide) types when carbon source PU is present.

Human phase-I metabolism and prevalence of two synthetic cannabinoids bearing an ethyl ester moiety: 5F-EDMB-PICA and EDMB-PINACA.

Around 2017, with the appearance of 5F-EDMB-PINACA, synthetic cannabinoids (SCs) carrying an ethyl ester moiety at the linked group started spreading on the market of new psychoactive substances (NPS). In 2020 and 2021, the indole analog of 5F-EDMB-PINACA (5F-EDMB-PICA) and the non-fluorinated analog of this compound (EDMB-PINACA) were analytically characterized. Here, we present suitable urinary markers to prove the consumption of these two ethyl analogs. Ten authentic urine samples for each compound were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qToF-MS). Anticipated phase-I metabolites detected in urine samples were confirmed in vitro by applying a pooled human liver microsomes (pHLM) assay. Prevalence data were obtained from urines collected for abstinence control and submitted to a screening method for SC metabolites. Ten phase-I metabolites of 5F-EDMB-PICA and 18 of EDMB-PINACA were detected by LC-qToF-MS analysis of authentic urine specimens. The main in-vivo metabolites were built by ester hydrolysis, often coupled to further metabolic processes. Investigation of phase-I biotransformation led to the identification of ester hydrolysis, monohydroxylation, and defluorination products as the most suitable urinary biomarkers for 5F-EDMB-PICA. Metabolites formed by ester hydrolysis coupled to ketone formation and by monohydroxylation are suggested for the detection of EDMB-PINACA. From October 1, 2020 to February 1, 2022, among positive urine samples, 5.4% and 10.1% tested positive 5F-EDMB-PICA and EDMB-PINACA, respectively. Due to common metabolites shared among structurally related SCs, the unequivocal detection of their consumption remains challenging for forensic laboratories and requires sensitive methods to monitor multiple metabolites, ideally including highly specific species.

In vitro metabolic fate of the synthetic cannabinoid receptor agonists (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB]) including isozyme mapping and carboxylesterases activity testing.

The synthetic cannabinoid receptor agonists (SCRAs) (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB], also known as SGT-46) are based on the structure of quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) that has been identified on seized plant material in 2011. In clinical toxicology, knowledge of the metabolic fate is important for their identification in biosamples. Therefore, the aim of this study was the identification of in vitro Phase I and II metabolites of QMMSB and QMiPSB in pooled human liver S9 fraction (pHLS9) incubations for use as screening targets. In addition, the involvement of human monooxygenases and human carboxylesterases (hCES) was examined. Analyses were performed by liquid chromatography coupled with high-resolution tandem mass spectrometry. Ester hydrolysis was found to be an important step in the Phase I metabolism of both SCRAs, with the carboxylic acid product being found only in negative ionization mode. Monohydroxy and N-dealkyl metabolites of the ester hydrolysis products were detected as well as glucuronides. CYP2C8, CYP2C9, CYP3A4, and CYP3A5 were involved in hydroxylation. Whereas enzymatic ester hydrolysis of QMiPSB was mainly catalyzed by hCES1 isoforms, nonenzymatic ester hydrolysis was also observed. The results suggest that ester hydrolysis products of QMMSB and QMiPSB and their glucuronides are suitable targets for toxicological screenings. The additional use of the negative ionization mode is recommended to increase detectability of analytes. Different cytochrome P450 (CYP) isozymes were involved in the metabolism; thus, the probability of drug-drug interactions due to CYP inhibition can be assessed as low.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists 2F-QMPSB and SGT-233 Including Isozyme Mapping and Carboxylesterases Activity Testing.

Quinolin-8-yl 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methylbenzoate (2F-QMPSB) and 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methyl-N-(2-phenylpropan-2-yl)benzamide (SGT-233) belong to a new group of synthetic cannabinoid receptor agonists containing a sulfamoyl benzoate or sulfamoyl benzamide core structure. 2F-QMPSB was identified in herbal material seized in Europe in 2018. The aims of this study were the identification of in vitro Phase I and II metabolites of 2F-QMPSB and SGT-233 to find analytical targets for toxicological screenings. Furthermore, the contribution of different monooxygenases and human carboxylesterases to Phase I metabolism was investigated. Liquid chromatography coupled to high-resolution tandem mass spectrometry was used for analysis. Ester hydrolysis was found to be an important step in the metabolism of 2F-QMPSB, which was catalyzed mainly by human carboxylesterases (hCES)1 isoforms. Additionally, nonenzymatic ester hydrolysis was observed in case of 2F-QMPSB. Notably, the carboxylic acid product derived from ester hydrolysis and metabolites thereof were only detectable in negative ionization mode. In case of SGT-233, mono- and dihydroxy metabolites were identified, as well as glucuronides. The cytochrome P450 (CYP) isozymes CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 were found to be involved in the hydroxylation of both compounds. The results of these in vitro experiments suggest that the ester hydrolysis products of 2F-QMPSB and their glucuronides are suitable targets for toxicological screenings. In the case of SGT-233, the mono- and dihydroxy metabolites were identified as suitable screening targets. The involvement of various CYP isoforms in the metabolism of both substances reduces the likelihood of drug-drug interactions due to CYP inhibition.

Synthetic cannabinoids carrying an ethyl ester moiety: Human phase I metabolism and prevalence of 5F-EDMB-PICA and EDMB-PINACA

Among novel psychoactive substances (NPS), synthetic cannabinoids (SCs) represent one of the largest subgroups. Around 2017, with the appearance of 5F-EDMB-PINACA, the ethyl analog of 5F-MDMB-PINACA (5F-ADB), the first synthetic cannabinoid carrying an ethyl ester moiety at the linked group started to circulate on the market. The unambiguous detection of the use of these substances remains challenging for forensic laboratories, especially because they tend to form common metabolites with structurally related SCs. In 2020 and 2021, further SCs with an ethyl ester moiety at the linked group emerged, among them 5F-EDMB-PICA and EDMB-PINACA, which were distributed on the German drug market. Here we present data on suitable urinary markers to prove the consumption of the ethyl ester compounds 5F-EDMB-PICA and EDMB-PINACA, facilitating differentiation from structurally similar compounds like ADB-PINACA, 5F-MDMB-PICA and 5F-EDMB-PINACA. Twenty authentic urine samples (10 for each substance) collected for abstinence control were analyzed by means of liquid chromatography time-of-flight mass spectrometry (LC-qToF-MS) after enzymatic cleavage of glucuronides. Anticipated phase I metabolites detected in authentic urine samples were confirmed in vitro by applying a pooled human liver microsomes (pHLM) assay. The prevalence of the compounds among German users between 2020 and 2022 was estimated by analyzing forensic urine samples collected for abstinence control. Ten phase I metabolites of 5F-EDMB-PICA and 18 metabolites of EDMB-PINACA were detected by LC-qToF-MS analysis of authentic human urine specimens. Not all of these metabolites could be confirmed in vitro using a pHLM assay. This was particularly seen for the products of multiple metabolic reactions. The first urine samples positive for 5F-EDMB-PICA were detected in October 2020, and for EDMB-PINACA in April 2021. From October 2020 to February 2022, a total of 1232 urine samples tested positive for SCs in the Institute of Forensic Medicine Freiburg, of which 66 (5.4%) were positive for 5F-EDMB-PICA and 125 (10.1%) for EDMB-PINACA. For both substances, the main in vivo metabolites were built by ester hydrolysis, coupled to further metabolic processes. However, these metabolites could be formed from other SCs as well and are, thus, non-specific. Investigation of phase-I biotransformation led to the identification of ester hydrolysis, defluorination and mono-hydroxylation products as the most suitable urinary biomarkers for 5F-EDMB-PICA. Suggested biomarkers for the use of EDMB-PINACA are represented by the products of ester hydrolysis coupled to ketone formation and mono-hydroxylation. Due to several common metabolites among structurally related SCs, the unequivocal detection of their consumption remains challenging for forensic laboratories and requires sensitive methods to monitor multiple targets, sometimes including minor metabolites. 5F-EDMB-PICA and EDMB-PINACA, two SCs carrying an ethyl ester moiety, peaked in 2021 and seem to have vanished afterwards from the German market.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists QMPSB and QMPCB (SGT-11) Including Isozyme Mapping and Esterase Activity.

Quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) and quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11) are synthetic cannabinoid receptor agonists (SCRAs). Knowing their metabolic fate is crucial for the identification of toxicological screening targets and to predict possible drug interactions. The presented study aimed to identify the in vitro phase I/II metabolites of QMPSB and QMPCB and to study the contribution of different monooxygenases and human carboxylesterases by using pooled human liver S9 fraction (pHLS9), recombinant human monooxygenases, three recombinant human carboxylesterases, and pooled human liver microsomes. Analyses were carried out by liquid chromatography high-resolution tandem mass spectrometry. QMPSB and QMPCB showed ester hydrolysis, and hydroxy and carboxylic acid products were detected in both cases. Mono/dihydroxy metabolites were formed, as were corresponding glucuronides and sulfates. Most of the metabolites could be detected in positive ionization mode with the exception of some QMPSB metabolites, which could only be found in negative mode. Monooxygenase activity screening revealed that CYP2B6/CYP2C8/CYP2C9/CYP2C19/CYP3A4/CYP3A5 were involved in hydroxylations. Esterase screening showed the involvement of all investigated isoforms. Additionally, extensive non-enzymatic ester hydrolysis was observed. Considering the results of the in vitro experiments, inclusion of the ester hydrolysis products and their glucuronides and monohydroxy metabolites into toxicological screening procedures is recommended.

Synthesis, Anticancer Activity, and Preliminary Pharmacokinetic Evaluation of 4,4-Disubstituted Curcuminoid 2,2-bis(Hydroxymethyl)Propionate Derivatives.

Compound 1 is a curcumin di-O-2,2-bis(hydroxymethyl)propionate that shows significant in vitro and in vivo inhibitory activity against MDA-MB-231 cells with eight to ten-fold higher potency than curcumin. Here, we modified the α-position (C-4 position) of the central 1,3-diketone moiety of 1 with polar or nonpolar functional groups to afford a series of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)propionate derivatives and evaluated their anticancer activities. A clear structure–activity relationship of compound 1 derivatives focusing on the functional groups at the C-4 position was established based on their anti-proliferative effects against the MDA-MB-231 and HCT-116 cell lines. Compounds 2–6 are 4,4-dimethylated, 4,4-diethylated, 4,4-dibenzylated, 4,4-dipropargylated and 4,4-diallylated compound 1, respectively. Compounds 2m–6m, the ester hydrolysis products of compounds 2–6, respectively, were synthesized and assessed for anticancer activity. Among all compound 1 derivatives, compound 2 emerged as a potential chemotherapeutic agent for colon cancer due to the promising in vivo anti-proliferative activities of 2 (IC50 = 3.10 ± 0.29 μM) and its ester hydrolysis product 2m (IC50 = 2.17 ± 0.16 μM) against HCT-116. The preliminary pharmacokinetic evaluation of 2 implied that 2 and 2m are main contributors to the in vivo efficacy. Compound 2 was further evaluated in an animal study using HCT-116 colon tumor xenograft bearing nude mice. The results revealed a dose-dependent efficacy that led to tumor volume reductions of 27%, 45%, and 60% at 50, 100, and 150 mg/kg doses, respectively. The established structure–activity relationship and pharmacokinetic outcomes of 2 is the guidance for future development of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)- propionate derivatives as anticancer drug candidates.

Identification of Azoxystrobin Glutathione Conjugate Metabolites in Maize Roots by LC-MS.

Xenobiotic detoxification in plant as well as in animals has mostly been studied in relationship to the deactivation of the toxic residues of the compound that, surely for azoxystrobin, is represented by its β-methoxyacrylate portion. In maize roots treated for 96 h with azoxystrobin, the fungicide accumulated over time and detoxification compounds or conjugates appeared timewise. The main detoxified compound was the methyl ester hydrolysis product (azoxystrobin free acid, 390.14 m/z) thought to be inactive followed by the glutathione conjugated compounds identified as glutathione conjugate (711.21 m/z) and its derivative lacking the glycine residue from the GSH (654.19 m/z). The glycosylated form of azoxystrobin was also found (552.19 m/z) in a minor amount. The identification of these analytes was done by differential untargeted metabolomics analysis using Progenesis QI for label free spectral counting quantification and MS/MS confirmation of the compounds was carried out by either Data Independent Acquisition (DIA) and Data Dependent Acquisition (DDA) using high resolution LC-MS methods. Neutral loss scanning and comparison with MS/MS spectra of azoxystrobin by DDA and MSe confirmed the structures of these new azoxystrobin GSH conjugates.

Urinary metabolites of the UV filter octocrylene in humans as biomarkers of exposure.

Octocrylene (OC) is a UV filter used in sun screens and other personal care products, but also in polymers and food contact materials for stabilization. In this study, we investigate human OC metabolism and urinary excretion after oral dosage of approx. 5mg OC [≙ 61.8-89.5µg/(kg body weight)] in three male volunteers. In a screening approach, we tentatively identified six urinary OC metabolites. For three, renal elimination kinetics was quantitatively investigated using authentic standards: the sidechain oxidation product 2-ethyl-5-hydroxyhexyl 2-cyano-3,3-diphenyl acrylate (5OH-OC), the beta-oxidation product 2-(carboxymethyl)butyl 2-cyano-3,3-diphenyl acrylate (dinor OC carboxylic acid; DOCCA), and the ester hydrolysis product 2-cyano-3,3-diphenylacrylic acid (CPAA). CPAA was the major urinary metabolite, representing 45% (range 40-50%) of the OC dose. 5OH-OC and DOCCA were only minor metabolites with low, but highly consistent renal conversion factors of 0.008% (0.005-0.011%) and 0.13% (0.11-0.16%), respectively. Peak urinary metabolite concentrations were observed between 3.2h and 4.2h postdose. All three metabolites were excreted with biphasic elimination kinetics, with considerably longer elimination half-lives for DOCCA (1st phase: 3.0h; 2nd phase: 16h) and CPAA (5.7h and 16h) compared to 5OH-OC (1.3h and 6.4h). 99% of all 5OH-OC was excreted within 24h compared to 82% of DOCCA and 77% of CPAA. After dermal exposure, we detected the same metabolites with similar ratios in urine, however, at much lower concentrations and with considerably delayed elimination.

Studying the Potential of Calcite Dissolution on Oil Liberation from Rock Surfaces during Single-Well-Chemical-Tracer Tests by Coupling a Multiphase Flow Simulator to the Geochemical Package

Calcite mineral dissolution has been considered to be an important mechanism for dampening the considerable pH-variation during Single-Well-Chemical-Tracer (SWCT) tests by improving the buffer capacity of the aqueous solution. Other parameters that also could have a great effect on the geochemistry of the reservoir during the SWCT tests are water buffer capacity, soluble hydrocarbon components, and temperature. Additionally, calcite mineral dissolution has been also presented over the last decade as an underlying mechanism for liberation of the adsorbed oil from the surface by modified salinity water injection (MSWI) in carbonate reservoirs. This contradiction of effects of calcite dissolution might pose a challenge for the accuracy of the SWCT tests in carbonates. This concern motivated us to highlight the potential of the calcite dissolution on the oil liberation from the carbonate rock surfaces during the SWCT tests by coupling a multiphase flow simulator to the geochemistry package PHREEQC. The results show that although the calcite dissolution is marginal during injection time, it might be substantial during shut-in time which is much longer. During shut-in time, the results show that the potential of calcite dissolution on the oil liberation from the rock surfaces could be more significant at higher reservoir temperatures although initial solid calcite concentration and buffer capacity also could have an effect. It is also clear that the pH of the system reaches the lowest level when the shut-in time reaches the transient time (i.e., injection and production times) and it is not changed significantly afterwards. At longer shut-in times, the additional ester hydrolysis and acid product is neutralized by the calcite dissolution and the buffer capacity of water. Therefore, the probability of the liberation of the adsorbed oil from the rock surface is higher at larger shut-in times so that test designs with shorter shut-in times and even as short as the transient time for the carbonate reservoirs is highly recommended. We hope that this study can be used to minimize the uncertainties of the SWCT tests and improve the reliability of the Sor measurements.

Determination of Urinary Metabolites of the Emerging UV Filter Octocrylene by Online-SPE-LC-MS/MS.

Octocrylene (OC) is an emerging UV filter, which is used in the majority of sunscreens as well as other personal care products (PCP) and consumer products. Its presence in various environmental matrices has been reported. However, information on the internal OC exposure in humans is not available, due to the lack of appropriate biomarkers of exposure and analytical methods. Here, we describe a rugged, precise, and accurate analytical method for the determination of three OC metabolites (ester hydrolysis and alkyl chain oxidation products) in human urine by stable isotope dilution analysis. Urine samples are incubated with β-glucuronidase (E. coli K12) and then analyzed by liquid chromatography-electrospray ionization-triple quadrupole-tandem mass spectrometry with online turbulent flow chromatography for sample cleanup and analyte enrichment (online-SPE-LC-MS/MS). Syntheses of analytical standards, including deuterium-labeled internal standards, are also described. In a pilot study, we investigated the applicability of the metabolites as biomarkers of exposure in urine samples from the general population (n = 35). OC metabolites were detected in 91% of the samples, with the highest concentrations for three individuals having used sunscreen within 5 days prior to sample collection. We will apply the method in future human biomonitoring studies for OC exposure and risk assessment.

Phase I metabolism of the highly potent synthetic cannabinoid MDMB-CHMICA and detection in human urine samples.

Among the recently emerged synthetic cannabinoids, MDMB-CHMICA (methyl N-{[1-(cyclohexylmethyl)-1H-indol-3-yl]carbonyl}-3-methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB-CHMICA was investigated using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-Q ToF-MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC-ESI-MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB-CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB-CHMICA at the CB1 receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright © 2016 John Wiley & Sons, Ltd.

PENGARUH KONSENTRASI BASA TERHADAP PRODUK HIDROLISIS ESTER

This study aims to determine the effect of concentration of base to ester hydrolysis products. Ester can dihidroisis with an alkaline catalyst at different concentrations. In this study, the concentration of metal acetate used was 1 M NaOH while coud concentration was 0.5 M, 0.75 M, 1 M, 1.25 M. The hydrolysis reaction using ethanol and refluxed for 2 hours at room temperature. Hydrolysis of methyl acetate ester compounds produced methanol with purity varies by different concentrations of methyl acetate and NaOH. At concentrations of 0.5 M NaOH acquired 79.5068% purity methanol, 0.75 M NaOH concentration 85.6665% purity methanol, 1 M NaOH concentration 99.6311% purity methanol and NaOH concentration of 1.25 M purity methanol 100,0000 %. Based on the results of the hydrolysis product purity shows the influence of the concentration of NaOH to the product ester hydrolysis. Methanol compound structure was analyzed by an infrared spectrometer and a mass spectrometer.

Elucidation of the upper pathway of alicyclic musk Romandolide® degradation in OECD screening tests with activated sludge

The degradation of Romandolide ([1-(3',3'-dimethyl-1'-cyclohexyl)ethoxycarbonyl] methyl propanoate), a synthetic alicyclic musk, by activated sludge inocula was investigated using both the manometric respirometry test OECD 301F and the CO₂ evolution test. In addition to measuring its biodegradability, key steps of the upper part of the metabolic pathway responsible for Romandolide degradation were identified using extracts at different time points of incubation. Early metabolism of Romandolide yielded ester hydrolysis products, including Cyclademol (1-(3,3-dimethylcyclohexyl)ethanol). The principal metabolites after 31 days were identified as 3,3-dimethyl cyclohexanone and 3,3-dimethyl cyclohexyl acetate. Formation of 3,3-dimethyl cyclohexanone from Cyclademol by sludge was confirmed in subsequent experiments using Cyclademol as a substrate, indicating the involvement of an oxygen insertion reminiscent of a Baeyer-Villiger oxidation. Further mineralization of 3,3-dimethyl cyclohexanone was also confirmed in subsequent studies. Three steps were thus required for complete biodegradation of the alicyclic musk: (1) successive ester hydrolyses leading to the formation of Cyclademol with concomitant degradation of the resulting acids, (2) conversion of Cyclademol into 3,3-dimethyl cyclohexanone, and (3) further mineralization via ring cleavage.

Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75

A polyurethane (PU) degrading bacterial strain MZA-75 was isolated from soil through enrichment technique. The bacterium was identified through 16S rRNA gene sequencing, the phylogenetic analysis indicated the strain MZA-75 belonged to genus Bacillus having maximum similarity with Bacillus subtilis strain JBE0016. The degradation of PU films by strain MZA-75 in mineral salt medium (MSM) was analyzed by scanning electron microscopy (SEM), fourier transform infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM revealed the appearance of widespread cracks on the surface. FTIR spectrum showed decrease in ester functional group. Increase in polydispersity index was observed in GPC, which indicates chain scission as a result of microbial treatment. CO2 evolution and cell growth increased when PU was used as carbon source in MSM in Sturm test. Increase in both cell associated and extracellular esterases was observed in the presence of PU indicated by p-Nitrophenyl acetate (pNPA) hydrolysis assay. Analysis of cell free supernatant by gas chromatography-mass spectrometry (GC-MS) revealed that 1,4-butanediol and adipic acid monomers were produced. Bacillus subtilis strain MZA-75 can degrade the soft segment of polyester polyurethane, unfortunately no information about the fate of hard segment could be obtained. Growth of strain MZA-75 in the presence of these metabolites indicated mineralization of ester hydrolysis products into CO2 and H2O.

Sitosterol as an antioxidant in frying oils

The antioxidative effect of sitosterol at 1, 2 and 5% levels, in triolein, refined canola, high oleic sunflower and flaxseed oils, continuously heated for a period of up to 72h at frying temperature of 180°C, was studied. High Pressure Size Exclusion Chromatography (HPSEC) was used to monitor changes in peak areas of triacylglycerol (TG) polymer, monomer and ester hydrolysis products. The presence of enhanced levels of sitosterol was found to significantly decrease TG polymer formation in triolein and the vegetable oil samples after heating at 180°C for a period of 72h. A corresponding increase in the level of intact TG monomer and the extent of TG ester hydrolysis was observed in all samples with enhanced levels of sitosterol. Conversion of sterol to steradiene, by the 1, 2 elimination of water, may be responsible for the antioxidative effect of sitosterol at frying temperatures.

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates’ bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure–activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.

Effects of cell stress protectant glutathione on the whole-cell biotransformation of ethyl 2-chloro-acetoacetate with Saccharomyces cerevisiae

The whole-cell biotransformation of the β-keto ester ethyl 2-chloro-acetoacetate to ethyl (2R,3S)-2-chloro-3-hydroxybutyrate with Saccharomyces cerevisiae was investigated. Due to the xenobiotic stress exerted by the alkylating substrate, several competing reactions (up to 54%) were encountered at the activated positions of the substrate molecule which are mediated by cytosolic glutathione (GSH): reductive dehalogenation, ester hydrolysis and cleavage of the C4-backbone (retro-Claisen condensation). The catalytic effect of a glutathione S-transferase accelerates these processes, but is not necessarily required for the reactions to occur. The ester hydrolysis product chloro-acetone and retro-Claisen product ethyl chloro-acetate significantly affect the stereoselectivity of the whole-cell reduction. However, for the dehalogenation product ethyl (S)-3-hydroxybutyrate no effects on the biotransformation were observed.

The 'apparent' hydrolysis of alkyl esters during electrospray ionization.

Electrospray ionization (ESI) mass spectrometry was employed to obtain both molecular weight confirmation and structural information for a series of novel alkenyldiarylmethane (ADAM) analogs. The mass spectral data were intended for use during the structure elucidation of ester hydrolysis products formed during an in vitro metabolism study of a series of novel ADAM analogs. The data on the precursor molecules show the presence of the molecular ion peak, [M+H](+), as well as a peak consistent with the hydrolysis product of the original ester ([MH-ROH+H(2)O](+)). However, chemical ionization mass spectrometry, elemental analysis and (1)H NMR data indicated the presence of only the intact diester compounds, suggesting that the formation of the hydrolysis product was an instrumental artifact, i.e., in-beam hydrolysis during ESI or a result of longer ion residence times of the ion trap mass analyzer.

Remifentanil in the Horse: Identification and Detection of its Major Urinary Metabolite*

Remifentanil (4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic acid methyl ester) is a mu-opioid receptor agonist with considerable abuse potential in racing horses. The identification of its major equine urinary metabolite, 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid, an ester hydrolysis product of remifentanil is reported. Administration of remifentanil HCl (5 mg, intravenous) produced clear-cut locomotor responses, establishing the clinical efficacy of this dose. ELISA analysis of postadministration urine samples readily detected fentanyl equivalents in these samples. Mass spectrometric analysis, using solid-phase extraction and trimethylsilyl (TMS) derivatization, showed the urine samples contained parent remifentanil in low concentrations, peaking at 1 h. More significantly, a major peak was identified as representing 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid, arising from ester hydrolysis of remifentanil. This metabolite reached its maximal urinary concentrations at 1 h and was present at up to 10-fold greater concentrations than parent remifentanil. Base hydrolysis of remifentanil yielded a carboxylic acid with the same mass spectral characteristics as those of the equine metabolite. In summary, these data indicate that remifentanil administration results in the appearance of readily detectable amounts of 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid in urine. On this basis, screening and confirmation tests for this equine urinary metabolite should be optimized for forensic control of remifentanil.