Synthesis of trifold-labeled versatile reagent [3,5-13 C2 ,4-15 N]4-phenyl-1,2,4-triazoline-3,5-dione.

Triazolinediones are an important class of derivatization agents that have found application in various research disciplines. Their unique reactivity often allows precise and selective tagging of relevant molecular scaffolds to facilitate structural elucidation, tracking in biological systems, and stabilization of labile compounds. Recent research efforts mainly focused on the development of novel fluorescent and ionizable or isotopically labeled tags improving the quantification and identification of the parent molecule by suitable analytical methods. However, these concepts often lack the ability to improve properties facilitating the analysis by nuclear magnetic resonance (NMR) spectroscopy. We herein describe the first synthesis of 13 C and 15 N labeled [3,5-13 C2 ,4-15  N]4-phenyl-1,2,4-triazoline-3,5-dione utilizing the Cookson/Zinner-Deucker synthesis of urazoles. The introduced isotopic labels are ideally suited to support the structural elucidation of unknown and complex derivatization mixtures by NMR, thereby exploiting the increased sensitivity of detecting long-range JHC and additional JCC and JCN couplings within the derivatized compounds of interest.

Lab-on-a-Chip Device for Rapid Measurement of Vitamin D Levels.

Lab-on-a-chip assays allow rapid analysis of one or more molecular analytes on an automated user-friendly platform. Here we describe a fully automated assay and readout for measurement of vitamin D levels in less than 15 min using the Fraunhofer in vitro diagnostics platform. Vitamin D (25-hydroxyvitamin D3 [25(OH)D3]) dilution series in buffer were successfully tested down to 2 ng/mL. This could be applied in the future as an inexpensive point-of-care analysis for patients suffering from a variety of conditions marked by vitamin D deficiencies.

Fungal bis-Naphthopyrones as Inhibitors of Botulinum Neurotoxin Serotype A.

An in silico screen of the NIH Molecular Library Small Molecule Repository (MLSMR) of ∼350000 compounds and confirmatory bioassays led to identification of chaetochromin A (1) as an inhibitor of botulinum neurotoxin serotype A (BoNT A). Subsequent acquisition and testing of analogues of 1 uncovered two compounds, talaroderxines A (2) and B (3), with improved activity. These are the first fungal metabolites reported to exhibit BoNT/A inhibitory activity.

Rapid determination of 25-hydroxy vitamin D3 in swine tissue using an isotope dilution HPLC-MS assay.

A rapid method for quantification of 25-hydroxy vitamin D3 in different swine tissues based on isotope dilution HPLC-MS has been developed and validated. Six times deuterated analyte is used as internal standard. The method is fast and can be performed with only 1g sample. Sample preparation for kidney, liver, muscle and spleen requires only homogenisation and extraction with methanol. An additional enzymatic digest is required for skin, and clean-up of the extract by solid-phase extraction (SPE) is used for adipose tissue and skin. The lower limit of detection varies from 1 ng/g (muscle) to 5 ng/g (adipose and skin). The method has been successfully applied to various tissue samples of pigs fed for 119 days either 2000 IU of vitamin D3 or 50 microg of 25-hydroxy vitamin D3 per kg feed. For animals ingesting 25-OH-D3 supplements the highest tissue contents were observed in the skin (24.8+/-3.5 ng/g), followed by kidney (14.2+/-1.5 ng/g), liver and muscle (5.7+/-0.6 ng/g). The 25-OH-D3 content in the skin was significantly higher in animals ingesting 2000 IU/kg of vitamin D3 (39.5+/-13.4 ng/g). Levels in selected tissues of some animals were below the lower limit of quantification. No measurable amounts of 25-OH-D3 were found in spleen, abdominal fat and subcutaneous fat of the animals of both groups as well as in the liver, kidney and muscle of the animals ingesting 2000 IU/kg of vitamin D3.

Quantification of biotin in feed, food, tablets, and premixes using HPLC-MS/MS.

Two sensitive and specific methods for quantification of biotin in feed, food, tablets, and premixes based on HPLC-MS/MS have been developed and validated. Depending on sample matrix and biotin content different extraction procedures and HPLC conditions were applied. Key steps in sample preparation were an alkaline extraction or a hydrolysis with sulphuric acid followed by enzymatic digest with papain. For many samples with low biotin content the latter combination of extraction steps was shown to be necessary for an optimal release of biotin from the matrix. The first time synthesis of deuterated biotin for use as internal standard allowed the compensation of losses during sample work-up and ion suppression during HPLC-MS/MS analysis. The new methods are faster than the commonly used microbiological assay using Lactobacillus plantarum. Additionally, they have a higher specificity as results for biotin are based on determination of a chemically defined compound, and not of a biological activity. Quantification is applicable to samples with a biotin content >100 microg/kg. Results obtained with the new methods have been compared with those of the microbiological assay, and were in good agreement.

Microwave-assisted rapid determination of vitamins a and e in beverages.

A new rapid procedure for the determination of vitamins A and E in beverages has been developed and validated. Key steps include a microwave-assisted saponification of the sample and a single-step extraction of the vitamins prior to HPLC analysis. All sample preparation steps are carried out consecutively in the same vial. The vitamins are determined using normal-phase (Si-60) HPLC with fluorescence detection. The method is applicable to beverages with a content of all-trans-retinol >0.14 mg/L and/or a content of alpha-tocopherol >1 mg/L. Recoveries determined by spiking experiments ranged from 91.3 to 106.3%. The precision of the method is characterized by relative standard deviations of <2% for alpha-tocopherol and <5% for all-trans-retinol.

Automated determination of selected water-soluble vitamins in tablets using a bench-top robotic system coupled to reversed-phase (RP-18) HPLC with UV detection.

The determination of folic acid, nicotinamide, nicotinic acid, riboflavin, riboflavin-5'-phosphate, pyridoxine and thiamine in tablets has been successfully automated using a bench-top robotic system (Zymark Tablet Processing Workstation II) coupled to reversed-phase (RP-18) HPLC with UV-detection. The new automated methods have been validated and were found to be applicable in routine analysis for all common types of multivitamin tablets. The precision is exemplified by relative standard deviations of 5.4% for folic acid, 2.1% for nicotinamide, 1.6% for pyridoxine, 0.7% for riboflavin and 1.1% for thiamine using film-coated tablets as sample. Mean recoveries obtained during spiking experiments were in the range of 95.2-103.9%.

New antioxidant hydroquinone derivatives from the algicolous marine fungus Acremonium sp.

A marine fungal isolate, identified as Acremonium sp., was mass cultivated and found to produce two novel hydroquinone derivatives, 7-isopropenylbicyclo[4.2.0]octa-1,3,5-triene-2,5-diol (1) and 7-isopropenylbicyclo[4.2.0]octa-1,3,5-triene-2,5-diol-5-beta-D-glucopyranoside (2). Compound 1 and its glucoside 2 possess a most unusual ring system. The new natural products (3R,4S)-3,4-dihydroxy-7-methyl-3,4-dihydro-1(2H)naphthalenone (3) and (3S,4S)-3,4-dihydroxy-7-methyl-3,4-dihydro-1(2H)-naphthalenone (4) were obtained as a 1:0.8 mixture. 2-(1-Methylethylidene)pentanedioic acid (5) was isolated for the first time as a natural product and its structure proven by X-ray analysis. In addition to these compounds an inseparable mixture of three new isomeric compounds, pentanedioic acid 2-(1-methylethylidene)-5-methyl ester (6), pentanedioic acid 2-(1-methylethylidene)-1-methyl ester (7), and pentanedioic acid 2-(1-methylethenyl)-5-methyl ester (8), was also obtained. Isolated together with the new compounds were three known hydroquinone derivatives, 9, 10, and 11. The structures of all compounds were determined by interpretation of their spectroscopic data (1D and 2D NMR, MS, UV, and IR). Each isolate was tested for its antioxidant properties, and compounds 1 and 9-11 were found to have significant activity.

Biologically active polyketide metabolites from an undetermined fungicolous hyphomycete resembling Cladosporium.

Eight new polyketide-derived metabolites [cladoacetals A and B (1 and 2), 3-(2-formyl-3-hydroxyphenyl)propionic acid (3), 3-deoxyisoochracinic acid (4), isoochracinol (5), 7-hydroxy-3-(2,3-dihydroxybutyl)-1(3H)-isobenzofuranone (6), (+)-cyclosordariolone (10), and altersolanol J (11)] and six known metabolites [two isomeric 1-(1,3-dihydro-4-hydroxy-1-isobenzofuranyl)butan-2,3-diols (7a/b), 7-hydroxy-1(3H)-isobenzofuranone (8), isoochracinic acid (9), altersolanol A (12), and macrosporin (13)] have been isolated from solid-substrate fermentation cultures of an undetermined fungicolous isolate (NRRL 29097) that resembles Cladosporium sp. All structures were assigned primarily by analysis of 1D and/or 2D NMR data. Five of the compounds showed antibacterial activity.

Rare sesquiterpenes from the algicolous fungus Drechslera dematioidea.

From the inner tissue of the marine red alga Liagora viscida the fungus Drechslera dematioidea was isolated. After mass cultivation, the fungus was investigated for its secondary metabolite content, and 10 new sesquiterpenoids [isosativenetriol (1), drechslerines A (2) and B (3), 9-hydroxyhelminthosporol (5), drechslerines C-G (6-10), and sativene epoxide (12)] were isolated. Compounds 8 and 10 exhibited antiplasmodial activity against Plasmodium falciparum strains K1 and NF54. The known compounds helminthosporol (4), cis-sativenediol (11), isocochlioquinone A (14), isocochlioquinone C (15), and cochlioquinone B (16) were also isolated. All structures were elucidated using spectroscopic methods, mainly 1D and 2D NMR and MS.