Effect of 1,4-Dioxane Solvent on ß-Glucuronidation Using Methyl 1,2,3,4-Tetra-O-acetyl-ß-D-glucuronate as the Glycosyl Donor.

A facile and selective ß-D-glucuronidation of alcohols, such as (-)-menthol, cholestanol, (+)- and (-)-borneols, and 2-adamantanol, using commercially available methyl 1,2,3,4-tetra-O-acetyl-ß-D-glucuronate as the glycosyl donor and trimethylsilyl bis(trifluoromethanesulfonyl)imide (Tf2NTMS) (0.5 equivalent) as the activator in 1,4-dioxane at 60 °C gave products in moderate yields. The addition of MS4A increased the ß : α ratios of D-glucuronides when cholestanol, (+)-borneol, and 2-adamantanol were used as the acceptor substrate.

Resveratrol and its metabolites elicit neuroprotection via high-affinity binding to the laminin receptor at low nanomolar concentrations.

Resveratrol prevents various neurodegenerative diseases in animal models despite reaching only low nanomolar concentrations in the brain after oral administration. In this study, based on the quenching of intrinsic tryptophan fluorescence and molecular docking, we found that trans-resveratrol, its conjugates (glucuronide and sulfate), and dihydro-resveratrol (intestinal microbial metabolite) bind with high affinities (Kd, 0.2-2 nm) to the peptide G palindromic sequence (near glycosaminoglycan-binding motif) of the 67-kDa laminin receptor (67LR). Preconditioning with low concentrations (0.01-10 nm) of these polyphenols, especially resveratrol-glucuronide, protected neuronal cells from death induced by serum withdrawal via activation of cAMP-mediated signaling pathways. This protection was prevented by a 67LR-blocking antibody, suggesting a role for this cell-surface receptor in neuroprotection by resveratrol metabolites.

Pharmacokinetics, tissue distribution, and excretion study of GL-V9 and its glucuronide metabolite 5-O-glucuronide GL-V9 in Sprague-Dawley rats.

The objective of this study is to explore the pharmacokinetics, tissue distribution, and excretion patterns of GL-V9 and its glucuronide metabolite, 5-O-glucuronide GL-V9, following the administration of GL-V9 to Sprague-Dawley (SD) rats. In this research, we developed and validated rapid, sensitive, and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for quantifying GL-V9 and 5-O-glucuronide GL-V9 in various biological samples, including SD rat plasma, tissue homogenate, bile, urine, and feces. Quantification of GL-V9 and 5-O-glucuronide GL-V9 in plasma, tissue homogenate, bile, urine, and feces was performed using the validated LC-MS/MS methods. The bioavailability of GL-V9 in SD rats ranged from 6.23% to 7.08%, and both GL-V9 and 5-O-glucuronide GL-V9 exhibited wide distribution and rapid elimination from tissues. The primary distribution tissues for GL-V9 and 5-O-glucuronide GL-V9 in rats were the duodenum, liver, and lung. GL-V9 was predominantly excreted in urine, while 5-O-glucuronide GL-V9 was primarily excreted in bile. GL-V9 exhibited easy absorption and rapid conversion to its glucuronide metabolite, 5-O-glucuronide GL-V9, following administration.

Tethered Indoxyl-Glucuronides for Enzymatically Triggered Cross-Linking.

Indoxyl-glucuronides, upon treatment with ß-glucuronidase under physiological conditions, are well known to afford the corresponding indigoid dye via oxidative dimerization. Here, seven indoxyl-glucuronide target compounds have been prepared along with 22 intermediates. Of the target compounds, four contain a conjugatable handle (azido-PEG, hydroxy-PEG, or BCN) attached to the indoxyl moiety, while three are isomers that include a PEG-ethynyl group at the 5-, 6-, or 7-position. All seven target compounds have been examined in indigoid-forming reactions upon treatment with ß-glucuronidase from two different sources and rat liver tritosomes. Taken together, the results suggest the utility of tethered indoxyl-glucuronides for use in bioconjugation chemistry with a chromogenic readout under physiological conditions.

Evaluation of In Vivo Prepared Albumin-Drug Conjugate Using Immunoprecipitation Linked LC-MS Assay and Its Application to Mouse Pharmacokinetic Study.

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.

Liquid chromatography coupled with tandem mass spectrometry for simultaneous quantification of valproate, valproate-glucuronide and lamotrigine in various biological matrices of rat.

Valproate and lamotrigine are commonly used as antiepileptic drugs even in pregnant and breastfeeding women. The extent and effects of drug exposure on the developing brain of the offspring are not well understood. Animal models can be utilised to investigate the transfer of substances into fetal brain with the ultimate aim of providing insights to aid clinical decisions. In the present study, an LC-MS/MS method was developed and validated for quantification of valproate (VPA), valproate-glucuronide (VPA-Gluc, a major metabolite of valproate) and lamotrigine (LTG) in rat blood plasma, cerebrospinal fluid and brain tissue. A 10 µl sample was spiked with stable isotope-labelled internal standards and extracted by methanol. An Agilent RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm) was used. The MS/MS transitions were 143.1016-143.1016 (VPA), 319.1392-143.0978 (VPA-Gluc) and 256.0157-210.9826 (LTG). The linear ranges of VPA, VPA-Gluc and LTG were 30-250, 10-140 and 0.3-1 µg/ml, respectively. The intra- and inter-day accuracy and precision, carryover, sensitivity and recovery were evaluated according to the US Food and Drug Administration guidance for bioanalytical method validation. Finally, the validated method was applied to a set of experimental animal samples and produced results highly comparable with those from an orthogonal analytical method.

Effects of structural characteristics of (un)conjugated steroid metabolites in their collision cross section value.

In this work, the collision cross section (CCS) value of 103 steroids (including unconjugated metabolites and phase II metabolites conjugated with sulfate and glucuronide groups) was determined by liquid chromatography coupled to traveling wave ion mobility spectrometry (LC-TWIMS). A time of flight (QTOF) mass analyzer was used to perform the analytes determination at high-resolution mass spectrometry. An electrospray ionization source (ESI) was used to generate [M+H]+, [M + NH4]+ and/or [M - H]- ions. High reproducibility was observed for the CCS determination in both urine and standard solutions, obtaining RSD lower than 0.3% and 0.5% in all cases respectively. CCS determination in matrix was in accordance with the CCS measured in standards solution showing deviations below 2%. In general, CCS values were directly correlated with the ion mass and allowed differentiating between glucuronides, sulfates and free steroids although differences among steroids of the same group were less significant. However, more specific information was obtained for phase II metabolites observing differences in the CCS value of isomeric pairs concerning the conjugation position or the α/ß configuration, which could be useful in the structural elucidation of new steroid metabolites in the anti-doping field. Finally, the potential of IMS reducing interferences from the sample matrix was also tested for the analysis of a glucuronide metabolite of bolasterone (5ß-androstan-7α,17α-dimethyl-3α,17ß-diol-3-glucuronide) in urine samples.

Characterization of Clofazimine Metabolism in Human Liver Microsomal Incubation In Vitro.

Clofazimine [N,5-bis(4-chlorophenyl)-3-[(propane-2-yl)rimino]-3,5-dihydrophenazin-2-amine] is an antimycobacterial agent used as a second-line antituberculosis (anti-TB) drug. Nonetheless, little information is known about the metabolic routes of clofazimine, and the enzymes involved in metabolism. This study aimed to characterize the metabolic pathways and enzymes responsible for the metabolism of clofazimine in human liver microsomes. Eight metabolites, including four oxidative metabolites, three glucuronide conjugates, and one sulfate conjugate were identified, and their structures were deduced based on tandem mass spectrometry (MS/MS) spectra. Hydroxylated clofazimine and hydrated clofazimine was generated even in the absence of the NADPH generating system presumably via a nonenzymatic pathway. Hydrolytic-dehalogenated clofazimine was catalyzed mainly by CYP1A2 whereas hydrolytic-deaminated clofazimine was formed by CYP3A4/A5. In case of glucuronide conjugates, UGT1A1, UGT1A3, and UGT1A9 showed catalytic activity toward hydroxylated and hydrated clofazimine glucuronide whereas hydrolytic-deaminated clofazimine glucuronide was catalyzed by UGT1A4, UGT1A9, UGT1A3, and UGT2B4. Our results suggested that CYP1A2 and CYP3A are involved in the formation of oxidative metabolites while UGT1A1, 1A3, 1A4, 1A9, and 2B4 are involved in the formation of glucuronide conjugates of oxidative metabolites of clofazimine.

Investigation of Human in vivo Metabolism of SEP-227900 Using the Samples from First-in-Human Study by LC-HRMS/UV and NMR.

OBJECTIVE: The study aims to explore the human in vivo metabolism of SEP-227900 (4H-furo[3, 2-b] pyrrole-carboxylic acid, m.w 151.03), a D-amino-acid oxidase (DAAO) inhibitor, by using plasma and urine samples from first-in-human study. METHODS: The human plasma and urine samples were from a single dose cohort that consisted of 9 healthy male volunteers each received an 80- mg dose of SEP-227900 orally. The pooled pre-dose urine and the pooled 0-24 h urine sample were created across 9 subjects by equal volume. Plasma samples were pooled by equal volume across 9 subjects to obtain 0-12 h plasma for metabolite searching, and also pooled by timepoints across 9 subjects to obtain 0.5, 5, and 12-h plasma for semi-quantitation. The plasma was de-proteinized by acetonitrile (1:3 v/v plasma-acetonitrile), then the supernatant was dried down, reconstituted, and injected for LC-HRMS/UV analysis. The urine sample was just simply centrifuged before analysis. LC-HRMS/UV was utilized to search predictable and unknown metabolites and estimate their relative abundances. Accurate mass measurement by Orbitrap-MS and MS/MS was used for metabolite identification. Chromatographic separation was achieved on a MACMOD AQ C8 column (250 × 4.6 mm, 5-µm) with a gradient mobile phase (A: 10 mM NH4Ac; B: acetonitrile; flowrate: 0.700 ml/min) for a total run-time of 65 min. The definite position in the molecule for the glucuronidation metabolism was characterized by the detected migration phenomenon, methylation with diazomethane (CH2N2), and NMR. RESULTS: Unchanged parent drug and four metabolite peaks were detected in humans: M1 was a mono-oxidative metabolite of SEP-227900; M2 was a glucuronide conjugate of SEP-227900; M3 was a glycine conjugate of SEP-227900; M4 was a glycine conjugate of M1. The specific position of the oxidation in M1 solely based on the mass spectral (MS and MS/MS) data was not identified. However, for the major metabolite M2, the acyl glucuronidation was unambiguously determined through multiple pieces of experimental evidence such as the observation of a migration pattern, mono-methylation by diazomethane, and NMR measurement. This determination is of significance related to the safety evaluation of investigational new drug development. The glycine conjugate of SEP-227900, i.e., M3, was found to be the most abundant metabolite in human urine (approximately 3-fold higher level than the glucuronide level). All together (mainly glycine-conjugate and glucuronide), it resulted in greater than 80% of the dosed amount in urine excretion (a separate measurement showed 23% of the dosed amount in urine excretion as the glucuronide). CONCLUSION: Four metabolites were found in humans: SEP-227900-glycine conjugate, SEP- 227900-glucuronide, mono-oxidative metabolite, and its consequent glycine conjugate. The glucuronide metabolite was identified as acyl glucuronide. Greater than 80% of the dosed amount of SEP-227900 was excreted in the urine, mainly in the forms of glycine- and glucuronide- conjugates.

Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives.

Next generation ß-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices. Each enzyme shows distinct substrate preferences, pH optima, and variability across clinical specimens. These results demonstrate how reliance on a single glucuronidated substrate as the internal hydrolysis control cannot ensure performance across a broader panel of analytes. Moreover, sample specific urine properties compromise ß-glucuronidases to varying levels, more pronounced for some enzymes, and thereby lower the recovery of some drug analytes in an enzyme-specific manner. A minimum of 3-fold dilution of urine with buffer yields measurable improvements in achieving target pH and reducing the impact of endogenous compounds on enzyme performance. After subjecting the enzymes to pH extremes and compromising chemicals, one particular ß-glucuronidase was identified that addressed many of these challenges and greatly lower the risk of failed hydrolyses. In summary, we present strategies to evaluate glucuronidases that aid in higher accuracy urine drug tests with lower potential for false negatives.

Simultaneous determination of duloxetine and 4-hydroxy duloxetine glucuronide in human plasma and back-conversion study.

Aim: To develop an LC-MS/MS method for simultaneous determination of duloxetine and its metabolite, 4-hydroxy duloxetine glucuronide (4HDG) in human plasma and to investigate the potential back-conversion of 4HDG to duloxetine using stability study. Materials & methods: The LC-MS/MS method was validated according to the EMA and USFDA Bioanalytical Method Validation Guidelines and applied to pilot bioequivalence study. Results & conclusion: The method validation results were within the acceptance limits. The stability study and incurred sample reanalysis results ruled out the occurrence of back-conversion. The study highlighted the conduct of back-conversion test and the advantages of LC-MS/MS method in terms of sensitivity, specificity and low consumption of organic solvents.

A rapid ultra high performance liquid chromatography-tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs.

Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein-7-O-glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one-step protein precipitation method with methanol added to 50 µL of plasma and were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1 mm, 2.6 µm) within only 2.6 min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0-1000 ng/mL for daidzein, and 8.0-4000 ng/mL for the prodrug and daidzein-7-O-glucuronide. The accuracy of the quality control samples was between 95.5 and 110.2% with satisfactory intra- and interday precision (relative standard deviation values < 10.85%), respectively. This sensitive, rapid, low-cost, and high-throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs.

A broad-spectrum substrate for the human UDP-glucuronosyltransferases and its use for investigating glucuronidation inhibitors.

Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drug/herb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived Km values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors.

Discovery of CYR715: A novel carboxylic acid-containing soluble guanylate cyclase stimulator.

Soluble guanylate cyclase (sGC) is a clinically validated therapeutic target in the treatment of pulmonary hypertension. Modulators of sGC have the potential to treat diseases that are affected by dysregulation of the NO-sGC-cGMP signal transduction pathway. This letter describes the SAR efforts that led to the discovery of CYR715, a novel carboxylic acid-containing sGC stimulator, with an improved metabolic profile relative to our previously described stimulator, IWP-051. CYR715 addressed potential idiosyncratic drug toxicity (IDT) liabilities associated with the formation of reactive, migrating acyl glucuronides (AG) found in related carboxylic acid-containing analogs and demonstrated high oral bioavailability in rat and dose-dependent hemodynamic pharmacology in normotensive Sprague-Dawley rats.

Metabolic products of European-type propolis. Synthesis and analysis of glucuronides and sulfates.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis is a bee-derived product used since antiquity for its general health-giving properties and is especially noted for its anti-bacterial activity. In more recent times, propolis has been employed against more specific targets such as antiproliferative effects vs cancer cells, wound healing and type-2 diabetes. AIM OF THE STUDY: European (poplar)-type propolis from New Zealand contains a number of hydroxy cinnamic acid esters and a set of aglycone flavonoid compounds, mainly chrysin, galangin, pinocembrin and pinobanksin. Propolis is usually taken orally and propolis metabolites quickly appear in the plasma of the ingested. In this work we aimed to identify the major flavonoid plasma metabolites by direct analysis of the plasma. MATERIALS AND METHODS: After consumption of a large dose of propolis in a single sitting, blood samples were taken and analysed using LCMS/MS. The major flavonoid metabolites identified were also synthesised using chemical (sulfates) or enzymatic methods (glucuronides). RESULTS: Both the sulfate and glucuronide conjugates of the four major propolis flavonoids are readily detected in human plasma after propolis ingestion. Preparation of the sulfates and glucuronides of the four major flavonoids allowed the relative proportions of the various metabolites to be determined. Although the sulfates are seen as large peaks in the LCMS/MS, the glucuronides are the dominant conjugate species. CONCLUSIONS: This study shows most of the flavonoids in the plasma are present as 7-O-glucuronides with only galangin showing some di-glucuronidation (3,7-O-diglucuronide). No evidence was found for hydroxy cinnamic acid type metabolites in the plasma samples.

Targeted LC-MS/MS analysis of steroid glucuronides in human urine.

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1-3 G), 17ß-estradiol 17-glucuronide (E2-17 G), 17ß-estradiol 3-glucuronide (E2-3 G), estriol 16-glucuronide (E3-16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F-G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F-G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC-MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC-MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC-MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15-20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0-1 month of age: 184.5 nmol/L; 15-20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.

Synthesis and toxicity profile in 293 human embryonic kidney cells of the ß D-glucuronide derivatives of ortho-, meta- and para-cresol.

The formation of ß-glucuronides is a major route by which mammals detoxify and remove breakdown products, such as l-tyrosine, as well as many xenobiotics, from their systems. In humans, dietary l-tyrosine is broken down largely by the action of the anaerobic gut bacterium C. difficile to p-cresol, providing a competitive advantage in the gut microbiota. Ortho- (o-) and meta- (m-), cresols, also present in the environment, may share a common degradative pathway. Relatively little work has been done on cresyl glucuronides. Here, a direct synthesis of o-, m-, and p-cresyl ß-D-glucuronides from methyl 1,2,3,4 tetra-O-acetyl-ß-d-glucuronate and the respective cresol employing trimethylsilyltriflate as promoter is presented. The protected intermediates were hydrolysed using aqueous sodium carbonate to yield the cresyl ß-glucuronides. The toxicities of the o-, m- and p-cresyl ß-D-glucuronides were compared. All three were less toxic to HEK293 cells than their respective cresol precursors: toxicity followed the order o < m < p for Na+ salts and o < p < m for Ca2+ salts. The m-cresyl-glucuronide Ca2+ salt and p-cresyl-glucuronide Na+ salt reduced colony formation by 11% and 9% (v. 30% reduction from the aglycone) respectively, whereas o-cresyl-glucuronide (both Na+ and Ca2+ salts), mildly stimulated HEK293 cell growth.

Glucuronidation of Methylated Quercetin Derivatives: Chemical and Biochemical Approaches.

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'-O-methyl-quercetin-3-O-ß-d-glucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4'-O-methyl-quercetin-7-O-ß-d-glucuronide (4), 3-O-methyl-quercetin-3'-O-ß-d-glucuronide (5), and 4'-O-methyl-quercetin-3'-O-ß-d-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; 3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. A mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.

Aldosterone Glucuronide, an Important Biomarker: Synthesis and Structure Elucidation of Novel Isomers.

Aldosterone 1 is a mineralocorticoid, it has great influence on the blood pressure and its glucuronide is an important marker for the detection of several diseases. Here, we describe the chemical synthesis of different aldosterone-18- and 20-glucuronides. Reaction of trimethylsilyl 2,3,4-tri- acetyl-1-ß-glucuronic acid methyl ester 5 b and aldosterone diacetate 11 in the presence of TMSOTf gave the 18-α-glucuronide 9 a. The 18-ß-glucuronide 15 b and the 20-ß-glucuronide 16 b could be obtained by reaction of methyl 2,3,4-tri-O-isobutyryl-1α-glucuronate trichloroacetimidate 14 and aldosterone 21-acetate 8 in the presence of TMSOTf or BF3 ⋅OEt2 . Finally, reaction of aldosterone 21-acetate 8 and methyl 2,3,4-triacetyl-1α-glucuronate trichloroacetimidate 19 in the presence of TMSOTf gave the corresponding methyl 18-ß-triacetylglucuronate 9 b, which was transformed into the desired aldosterone-18-ß-glucuronide 3 by two enzyma- tic transformations.

Water Soldier Stratiotes aloides L.-Forgotten Famine Plant With Unique Composition and Antioxidant Properties.

Stratiotes aloides L. is common water plant in central Poland. Due to its expansive character, S. aloides L. can strongly affect the functioning of aquatic ecosystems. S. aloides L. was an important famine plant in central Poland. This plant was commonly collected and cooked until the turn of the 20th century. It has also been used to heal wounds, especially when these are made by an iron implement. The objective of the present work was to study the phenolic profile in the leaves and roots of S. aloides as well as their antioxidant potential and ability to inhibit lipoxygenase (LOX) in the light of their potential bioaccessibility. The dominant compound in its leaves was luteolin-7-O-hexoside-glucuronide (5.84 mg/g DW), whereas the dominant root component was chrysoeriol-7-O-hexoside-glucuronide (0.83 mg/g DW). Infusions from leaves, roots, and their 1:1 (v/v) mixture contained potentially bioaccessible antiradical compounds. S. aloides is a good source of water-extractable reductive compounds. Especially valuable are the leaves of this plant. The roots of S. aloides contained very active hydrophilic compounds able to chelate metal ions. However, their potential bioaccessibility was relatively low. The hydrophilic compounds from the leaves were the most effective XO inhibitors (EC50 = 9.91 mg DW/mL). The water-extractable compounds derived from the leaves and roots acted as uncompetitive LOX inhibitors.