Study on the Synthesis of ZnO Nanoparticles Using Azadirachta indica Extracts for the Fabrication of a Gas Sensor.

This paper compared the effects of A. indica plant proteins over chemical methods in the morphology of zinc oxide nanoparticles (ZnO NPs) prepared by a co-precipitation method, and ethanol sensing performance of prepared thin films deposited over a fluorene-doped tin oxide (FTO) bind glass substrate using spray pyrolysis technique. The average crystallite sizes and diameters of the grain-sized cluster ZnO NPs were 25 and (701.79 ± 176.21) nm for an undoped sample and 20 and (489.99 ± 112.96) nm for A. india dye-doped sample. The fourier transform infrared spectroscopy (FTIR) analysis confirmed the formation of the Zn-O bond at 450 cm-1, and also showed the presence of plant proteins due to A. indica dye extracts. ZnO NPs films exhibited good response (up to 51 and 72% for without and with A. indica dye-doped extracts, respectively) toward ethanol vapors with quick response-recovery characteristics at a temperature of 250 °C for undoped and 225 °C for A. indica dye-doped ZnO thin films. The interaction of A. indica dye extracts helps to decrease the operating temperature and increased the response and recovery rates of the sensor, which may be due to an increase in the specific surface area, resulting in adsorption of more oxygen and hence high response results.

Circular Aqueous Fmoc/t-Bu Solid-Phase Peptide Synthesis.

Circular economy and aqueous synthesis are attractive concepts for sustainable chemistry. Here it is reported that the two can be combined in the universal method for peptide chemistry, fluorenylmethoxycarbonyl(Fmoc)/t-Bu solid-phase peptide synthesis (SPPS). It was demonstrated that Fmoc/t-Bu SPPS could be performed under aqueous conditions using standard Fmoc amino acids (AAs) employing TentaGel S as resin and 4 : 1 mixture of water with inexpensive green solvent PolarClean. This resin/solvent combination played a crucial dual role by virtue of improving resin swelling and solubility of starting materials. In a model coupling, TCFH and 2,4,6-collidine afforded a full conversion at only 1.3 equiv. AA, and these conditions were used in SPPS of Leu enkephaline amide affording the model peptide in 85 % yield and 86 % purity. A method to recycle the waste by filtration through a mixed ion exchange resin was developed, allowing reusing the waste without affecting quality of the peptide. The method herein obviates the use of unconventional or processed AAs in aqueous SPPS while using lower amounts of starting materials. By recycling/reusing SPPS waste the hazardous dipolar aprotic solvents used in SPPS were not only replaced with an aqueous medium, solvent use was also significantly reduced. This opens up a new direction in aqueous peptide chemistry in which efficient use of inexpensive starting materials and waste minimization is coupled with the universal Fmoc/t-Bu SPPS.

Investigation of biosurfactants produced by three indigenous bacterial strains, their growth kinetics and their anthracene and fluorene tolerance.

The study explored the polycyclic aromatic hydrocarbon tolerance of indigenous biosurfactant producing microorganisms. Three bacterial species were isolated from crude oil contaminated sites of Haldia, West Bengal. The three species were screened for biosurfactant production and identified by 16S rRNA sequencing as Brevundimonas sp. IITISM 11, Pseudomonas sp. IITISM 19 and Pseudomonas sp. IITISM 24. The strains showed emulsification activities of 51%, 57% and 63%, respectively. The purified biosurfactants were characterised using FT-IR, GC-MS and NMR spectroscopy and found to have structural similarities to glycolipopeptides, cyclic lipopeptides and glycolipids. The biosurfactants produced were found to be stable under a wide range of temperature (0-100 °C), pH (4-12) and salinity (up to 20% NaCl). Moreover, the strains displayed tolerance to high concentrations (275 mg/L) of anthracene and fluorene and showed a good amount of cell surface hydrophobicity with different hydrocarbons. The study reports the production and characterisation of biosurfactant by Brevundimonas sp. for the first time. Additionally, the kinetic parameters of the bacterial strains grown on up to 300 mg/L concentration of anthracene and fluorene, ranged between 0.0131 and 0.0156 µmax (h-1), while the Ks(mg/L) ranged between 59.28 and 102.66 for Monod's Model. For Haldane-Andrew's model, µmax (h-1) varied between 0.0168 and 0.0198. The inhibition constant was highest for Pseudomonas sp. IITISM 19 on anthracene and Brevundimonas sp. IITISM 11 on fluorene. The findings of the study suggest that indigenous biosurfactant producing strains have tolerance to high PAH concentrations and can be exploited for bioremediation purposes.

Electrostatic interactions regulate the release of small molecules from supramolecular hydrogels.

Supramolecular hydrogels have great potential as biomaterials for sustained delivery of therapeutics. While peptide-based supramolecular hydrogels have been developed that show promise for drug delivery applications, the high cost of production has limited their widespread adoption. Low molecular weight (LMW) supramolecular hydrogels are emerging as attractive and inexpensive alternatives to peptide-based hydrogels. We recently reported novel cationic fluorenylmethyloxycarbonyl-modified phenylalanine (Fmoc-Phe) hydrogels for localized and sustained in vivo release of an anti-inflammatory agent for functional pain remediation. In an effort to further elucidate design principles to optimize these materials for delivery of a variety of molecular agents, we herein report a systematic examination of electrostatic effects on the release of cargo molecules from Fmoc-Phe derived hydrogels. Specifically, we interrogate the release of cationic, anionic, and neutral cargo molecules from a series of cationic and anionic Fmoc-Phe derived hydrogels. We observed that cargo was readily released from the hydrogels except when the cargo and hydrogel network had complementary charges, in which case the cargo was highly retained in the network. These results demonstrate that the electrostatic characteristics of both the hydrogel network and the specific cargo are critical design parameters in the formulation of LMW supramolecular hydrogel systems in the development of next-generation materials for drug delivery applications.

Enantioselective Separation Using Chiral Amino Acid Functionalized Polyfluorene Coated on Mesoporous Anodic Aluminum Oxide Membranes.

Homochiral mesoporous anodic aluminum oxide membranes (AAO) were prepared by coating protected chiral D/L aspartic acid appended polyfluorene in the pores. These chiral AAO membranes successfully demonstrated enantioselective recognition and separation of a range of amino acids from their aqueous racemic mixture by simple filtration. Enantioselective separation was achieved by selective adsorption of one enantiomer from the aqueous racemic mixture into the chiral pores of the AAO membrane leaving the filtrate enriched with the other enantiomer. Extraction and quantification of the adsorbed amino acid (glutamic acid) demonstrated that 1 mg of homochiral polyfluorene could effectively extract about 3.5 mg of glutamic acid with 95% enantiomeric excess in 24 h. This is one of the highest enantiomeric excesses (ee %) and yields reported so far in the literature for a racemic mixture of glutamic acid. The pore size of the AAO membrane influenced the efficiency of separation with a reduction in pore size from 200 to 20 nm leading to reduced ee % (∼95% to ∼28%). These results raise the possibility for a facile method to carry out enantioselective separation.

Influence of metal ions on glyphosate detection by FMOC-Cl.

Glyphosate (GLP, N-(phosphonomethyl)glycine) is the most important broadband herbicide in the world, but discussions are controversial regarding its environmental behaviour and distribution. Residue analyses in a variety of environmental samples are commonly conducted by HPLC-MS where GLP needs to be derivatised with 9-fluoromethoxycarnonyl chloride (FMOC-Cl). Since this derivatisation reaction was suspected to be inhibited by metal ions in the sample matrix, the present study provides a comprehensive experimental study of the effect of metal ions (Al3+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, Mg2+, Mn2+, Zn2+) on derivatisation and GLP recovery. Results show that some metals (Cd2+, Co2+, Cu2+, Mn2+ and Zn2+) decreased the GLP recovery down to 19 to 59%. Complementary, quantum chemical modelling of 1:1 GLP-metal complexes as well as their reactivity with respect to FMOC-Cl was performed. Here, a decrease in reactivity of FMOC-Cl towards GLP-metal complexes is observed; i.e. the reaction is non-spontaneous in contrast to the free GLP case. The present results are in accord with previous studies and provide an explanation that full GLP recovery in different matrices was never reached. Remedy strategies to compensate for the inhibition effect are explored such as pH adjustment to acidic or alkaline conditions or addition of ethylenediaminetetraacetic acid (EDTA). In general, our results question the use of internal isotopic labelled standards (ILS) since this presupposes the presence of the analyte and the ILS in the same (free) form.

Determination of 1-Deoxynojirimycin by a developed and validated HPLC-FLD method and assessment of In-vitro antioxidant, α-Amylase and α-Glucosidase inhibitory activity in mulberry varieties from Turkey.

BACKGROUND: Morus alba and Morus nigra leaves which have been widely used as herbal teas in Anatolian region of Turkey, were extracted twice by 50 mM HCI solution, derivatized with 9-fluorenylmethyl chloroformate and analyzed by reversed phase HPLC equipped with a fluorescence detector. HYPOTHESIS/PURPOSE: This study was performed to determine the main antidiabetic active compounds 1-deoxynojirimycin by HPLC method and evaluate the in-vitro antioxidant and antidiabetic activity of ethanol extracts prepared from Morus alba L. and Morus nigra leaves. STUDY DESIGN: A reliable simple, and rapid high-performance liquid chromatographic (HPLC) method for the determination of 1-deoxynojirimycin in M. alba L. and M. nigra leaves with fluorimetric detection after pre-column derivatization with 9-fluorenylmethyl chloroformate was developed. In addition, the chemical composition of ethanol extract of mulberry leaves was analyzed with GC-MS. METHODS: Separation and quantitation were performed on C18, 250 × 4.6 mm, 5 µm analytical column. Mobile phase consisted of acetonitrile and 0.1% acetic acid solution (1:1, v/v) was performed applied to the column 1.0 ml/min flow rate at 26 °C. Potential antioxidant activity of ethanol extract of different mulberry varieties were evaluated by DPPH, and ABTS radical scavenging assay as well as total phenol and flavonoid content were determined. In addition, α-amylase and α-glucosidase activity was determined by 96-well plate method to evaluate the probable antidiabetic potential use of Turkish mulberry leaves. RESULTS: The isocratic HPLC method showed excellent correlation coefficient (r2 = 0.9985) between 0.3 and 30 µg/ml calibration points. The method was specific and sensitive with detection and quantification limits of 1.07 and 3.27 ng/ml, respectively. Intraday and interday method precision (n = 5) were < 7.3 (RSD%). Intraday and interday method accuracy (n = 5) were between 3.77 and (-8.35) (RE%). The average method recovery (n = 3) was 102.5%. The results showed that the content of 1-deoxynojirimycin in leaves of Morus alba L. was 0.103% (n = 3), and in leaves of M. nigra L. was 0.102%. 2-hexadecen-1-ol, oleamide, 2-propenoic acid, and cyclododecane were identified as the major compounds by GC-MS in the ethanol extract of mulberry leaves. CONCLUSION: The obtained robustness values from emission and excitation detection, mobile phase ingredients and flow rates changes showed that method was very strong. This work contributes to the knowledge of antioxidant and antidiabetic properties of Morus species, thus may be provide useful data in evaluation of food products and pharmaceutical preparations produced from Morus species.

Melatonin protects embryonic development and maintains sleep/wake behaviors from the deleterious effects of fluorene-9-bisphenol in zebrafish (Danio rerio).

Environmental endocrine chemicals have various adverse effects on the development of vertebrates. Fluorene-9-bisphenol (BHPF), a substitute of bisphenol A (BPA), is widely used in commercial production. The effects of BHPF on development and behavior are unclear. Melatonin plays a protective role under many unfavorable conditions. In this study, we investigated the effects of BHPF on the development and behaviors of zebrafish and whether melatonin reverses effects induced by BHPF. Zebrafish embryos were exposed to 0.1, 10, or 1000 nmol/L BHPF with or without 1 µmol/L melatonin from 2 hours postfertilization to 6 days postfertilization. The results showed that 0.1 and 10 nmol/L BHPF had little effect on development. High-dose BHPF (1000 nmol/L) delayed the development, increased mortality and surface tension of embryonic chorions, caused aberrant expression of the key genes (ntl, shh, krox20, pax2, cmlc2) in early development detected by in situ hybridization, and damaged the CaP motor neurons, which were associated with locomotion ability detected by immunofluorescence. Melatonin addition reversed or weakened these adverse effects of BHPF on development, and melatonin alone increased surface tension as the effects of high-dose BHPF. However, all groups of BHPF exposure triggered insomnia-like behaviors, with increased waking activity and decreased rest behaviors. BHPF acted on the hypocretin (hcrt) system and upregulated the expression of sleep/wake regulators such as hcrt, hcrt receptor (hcrtr), arylalkylamine N-acetyltransferase-2 (aanat2). Melatonin recovered the alternation of sleep/wake behaviors induced by BHPF and restored abnormal gene expression to normal levels. This study showed that high-dose BHPF had adverse effects on early development and induced behavioral alternations. However, melatonin prevented BHPF-induced aberrant development and sleep/wake behaviors.

Reductive chemical release of N-glycans as 1-amino-alditols and subsequent 9-fluorenylmethyloxycarbonyl labeling for MS and LC/MS analysis.

Glycoproteins play pivotal roles in a series of biological processes and their glycosylation patterns need to be structurally and functionally characterized. However, the lack of versatile methods to release N-glycans as functionalized forms has been undermining glycomics studies. Here a novel method is developed for dissociation of N-linked glycans from glycoproteins for analysis by MS and online LC/MS. This new method employs aqueous ammonia solution containing NaBH3CN as the reaction medium to release glycans from glycoproteins as 1-amino-alditol forms. The released glycans are conveniently labeled with 9-fluorenylmethyloxycarbonyl (Fmoc) and analyzed by ESI-MS and online LC/MS. Using the method, the neutral and acidic N-glycans were successfully released without peeling degradation of the core α-1,3-fucosylated structure or detectable de-N-acetylation, revealing its general applicability to various types of N-glycans. The Fmoc-derivatized N-glycans derived from chicken ovalbumin, Fagopyrum esculentum Moench Pollen and FBS were successfully analyzed by online LC/MS to distinguish isomers. The 1-amino-alditols were also permethylated to form quaternary ammonium cations at the reducing end, which enhance the MS sensitivity and are compatible with sequential multi-stage mass spectrometry (MSn) fragmentation for glycan sequencing. The Fmoc-labeled N-glycans were further permethylated to produce methylated carbamates for determination of branches and linkages by sequential MSn fragmentation. SIGNIFICANCE OF THE STUDY: N-Glycosylation represents one of the most common post-translational modification forms and plays pivotal roles in the structural and functional regulation of proteins in various biological activities, relating closely to human health and diseases. As a type of informational molecule, the N-glycans of glycoproteins participate directly in the molecular interactions between glycan epitopes and their corresponding protein receptors. Detailed structural and functional characterization of different types of N-glycans is essential for understanding the functional mechanisms of many biological activities and the pathologies of many diseases. Here we describe a simple, versatile method to indistinguishably release all types of N-glycans as functionalized forms without remarkable side reactions, enabling convenient, rapid analysis and preparation of released N-glycans from various complex biological samples. It is very valuable for studies on the complicated structure-function relationship of N-glycans, as well as for the search of N-glycan biomarkers of some major diseases and N-glycan related targets of some drugs.

Chemical Analysis of Counterfeit Hepatitis C Drug Found in Japan.

In January 2017, counterfeits of the hepatitis C drug 'HARVONI® Combination Tablets' (HARVONI®) were found at a pharmacy chain through unlicensed suppliers in Japan. A total of five lots of counterfeit HARVONI® (samples 1-5) bottles were found, and the ingredients of the bottles were all in tablet form. Among them, two differently shaped tablets were present in two of the bottles (categorized as samples 2A, 2B, 4A, and 4B). We analyzed the total of seven samples by high-resolution LC-MS, GC-MS and NMR. In samples 2A, 3 and 4B, sofosbuvir, the active component of another hepatitis C drug, SOVALDI® Tablets 400 mg (SOVALDI®), was detected. In sample 4A, sofosbuvir and ledipasvir, the active components of HARVONI®, were found. A direct comparison of the four samples and genuine products showed that three samples (2A, 3, 4B) are apparently SOVALDI® and that sample 2A is HARVONI®. In samples 1 and 5, several vitamins but none of the active compounds usually found in HARVONI® (i.e., sofosbuvir and ledipasvir) were detected. Our additional investigation indicates that these two samples are likely to be a commercial vitamin supplement distributed in Japan. Sample 2B, looked entirely different from HARVONI® and contained several herbal constitutents (such as ephedrine and glycyrrhizin) that are used in Japanese Kampo formulations. A further analysis indicated that sample 2B is likely to be a Kampo extract tablet of Shoseiryuto which is distributed in Japan. Considering this case, it is important to be vigilant to prevent a recurrence of distribution of counterfeit drugs.

Hollow Au-Cu Nanocomposite for Real-Time Tracing Photothermal/Antiangiogenic Therapy.

High absorption in the near-infrared (NIR) region is essential for a photoabsorbing agents to realize efficient photothermal therapy (PTT) for cancer. Here, a novel hollow Au-Cu nanocomposite (HGCNs) is developed, which displays a significantly enhanced NIR surface plasmon resonance absorption and photothermal transduction efficiency. Besides, fluorescent polymer dots poly(9,9-dioctylfluorene-2,7-diyl-co-benzothiadiazole) (PFBT) and chemotherapeutic mammalian target of rapamycin (mTOR) inhibitor agent rapamycin (RAPA) are attached onto the HGCNs (RAPA/PFBT-HGCNs) for real-time NIR fluorescence tracing and combined PTT/antiangiogenesis therapy. In particular, due to the fluorescence resonance energy transfer effect, RAPA/PFBT-HGCNs can act as NIR-activatable on/off probe system for real-time tracing of tumor tissues. A standard in vitro cellular uptake study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, dual-staining study, and flow cytometry assay reveal that the RAPA/PFBT-HGCNs combined with NIR laser exhibit higher drug accumulation and cytotoxicity in both tumor cells and epithelial cells. Moreover, the margins of tumor and normal tissue can be accurately indicated by NIR-stimulated dequenched PFBT after 24 h intravenous administration. Further, tumor growth can be considerably hampered by the optimal formulation plus laser treatment with relatively lower side effects. Consequently, the work highlights the real-time tracing and enhanced PTT/antiangiogenesis therapy prospects of the established HGCNs with tremendous potential for treatment of cancer.

A dietary strategy for the management of artemether-lumefantrine-induced cardiovascular and renal toxicity.

BACKGROUND: Unsweetened natural cocoa has antimalarial properties. Unsweetened natural cocoa powder (UNCP), obtained as a result of the removal of cocoa butter from a cocoa bean protects against malaria episodes. Cocoa powder, which is prepared after removal of the cocoa butter, contains about 1.9 % theobromine and 0.21 % caffeine. Concomitant consumption of cocoa and artemether/lumefantrine (A/L) is a common practice in Ghana, West Africa. This study seeks to determine the elemental composition of UNCP and its protective effect on the heart and kidney against (A/L) administration. METHODS: Energy dispersive x-ray fluorescence spectroscopy was used to detect the quality and quantity of the elemental composition in UNCP. Thereafter, 30 nonmalarious male guinea pigs were divided into five groups of six animals each. One group was administered with 75 mg/kg body weight A/L only and another group distilled water (control group). The rest received 300 mg/kg, 900 mg/kg and 1500 mg/kg body weight UNCP for 14 days orally and A/L for the last 3 days (ie day 11 to day 14). Biochemical and histopathological examinations were carried out after euthanisation of the animals. RESULTS: A total of thirty-eight (38) micro and macro elements were detected with the ED-XRF. Macro elements like sodium (Na), magnesium (Mg), aluminium (Al), phosphorus (P), chlorine (Cl), potassium (K), calcium (Ca), manganese (Mn) and iron (Fe) and micro elements like chromium (Cr), copper (Cu), zinc (Zn), arsenic (As), and lead (Pb) were identified and evaluated. Biochemical analysis revealed increases in HDL levels (p>0.05) while there were decreases in LDL levels (p>0.05), creatine kinase and AST levels (P<0.05) in animals that received UNCP compared to A/L only administered group. Urea levels reduced significantly by 53 % (p<0.05) in group that received 1500 mg/kg UNCP. Histopathological examinations of the heart and kidney buttressed the protective effects of cocoa administration. CONCLUSION: The percentage of recommended daily allowance of UNCP for chromium is 3750 % for men and 5250 % for women while % RDA for copper corresponds to 103.6 % in both sexes. UNCP proved to possess cardioprotective and renoprotective potential during artemether-lumefantrine administration.

Selenazolidine: a selenium containing proline surrogate in peptide science.

In the search for new peptide ligands containing selenium in their sequences, we investigated l-4-selenazolidine-carboxylic acid (selenazolidine, Sez) as a proline analog with the chalcogen atom in the γ-position of the ring. In contrast to proteinogenic selenocysteine (Sec) and selenomethionine (SeMet), the incorporation within a peptide sequence of such a non-natural amino acid has never been studied. There is thus a great interest in increasing the possibility of selenium insertion within peptides, especially for sequences that do not possess a sulfur containing amino acid (Cys or Met), by offering other selenated residues suitable for peptide synthesis protocols. Herein, we have evaluated selenazolidine in Boc/Bzl and Fmoc/tBu strategies through the synthesis of a model tripeptide, both in solution and on a solid support. Special attention was paid to the stability of the Sez residue in basic conditions. Thus, generic protocols have been optimized to synthesize Sez-containing peptides, through the use of an Fmoc-Xxx-Sez-OH dipeptide unit. As an example, a new analog of the vasopressin receptor-1A antagonist was prepared, in which Pro was replaced with Sez [3-(4-hydroxyphenyl)-propionyl-d-Tyr(Me)-Phe-Gln-Asn-Arg-Sez-Arg-NH2]. Both proline and such pseudo-proline containing peptides exhibited similar pharmacological properties and endopeptidase stabilities indicating that the presence of the selenium atom has minimal functional effects. Taking into account the straightforward handling of Sez as a dipeptide building block in a conventional Fmoc/tBu SPPS strategy, this result suggested a wide range of potential uses of the Sez amino acid in peptide chemistry, for instance as a viable proline surrogate as well as a selenium probe, complementary to Sec and SeMet, for NMR and mass spectrometry analytical purposes.

Effect of contact time and the use of hydroxypropyl-ß-cyclodextrin in the removal of fluorene and fluoranthene from contaminated soils.

Sorption-desorption experiments of fluorene (FLU) and fluoranthene (FLT) in soils were carried out and correlated to their removal from aged contaminated soils using aqueous solutions in the absence and in the presence of hydroxypropyl-ß-cyclodextrin (HPBCD) as the extraction agent. FLU became more resistant to extraction in aged contaminated soils due to its initial adsorption onto the mineral and amorphous soil organic matter (SOM) domains, sites of lower binding energy from which, due to its small size, it could spread towards the condensed SOM as the contact time increased. Therefore, FLU will not be easily desorbed from aged contaminated soils due to physical entrapment mechanisms, even when using HPBCD as extractant, presenting FLU low risks to the environment. On the contrary, FLT was extracted from aged soils in the presence of HPBCD in solutions to a much greater extent than in its absence. Due to its more hydrophobic character FLT sorption in soils was relatively quicker, remaining more or less fixed on hydrophobic sites of the organic matter (OM) with different energies, and therefore the amount of FLT extracted was almost constant for different ageing times. During extraction experiments, the influence of the OM quality of the soils was also highlighted because an inverse proportionality between OM content of soil and extractability of sorbed FLT was observed. It was concluded that soils with lower OM content that had more diagenetically processed OM could block the extraction of FLT more effectively than soils with higher OM content that are less humified. This indicates the need to use not only adsorption-desorption data in contaminant fate and transport models, but also extraction studies in aged contaminated soils and other complementary analytical approaches when assessing soil contamination-related risks.

Study of the matrix effects and sample dilution influence on the LC-ESI-MS/MS analysis using four derivatization reagents.

For liquid chromatographic analysis of amino acids involving derivatization and mass-spectrometric detection, it becomes more important to evaluate the presence of matrix effects in complex samples. This is somewhat complicated for amino acid analysis where analyte free sample matrix is often unavailable. In this work, matrix effects were investigated using post-column infusion method for 9-fluorenylmethyl chloroformate (FMOC-Cl) derivatives of ß-Ala, Gly and Phe and diethyl ethoxymethylenemalonate (DEEMM) derivative of ß-Ala. While for DEEMM derivatives, the main signal suppression was due to the borate buffer, in case of FMOC-Cl, other FMOC-derivatives caused signal suppression. Analysis of amino acids in tea and honey with DEEMM, FMOC-Cl, p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS) and dansyl chloride (DNS) showed that amino acid concentrations found with different reagents do not agree well. Sample dilution experiments indicated that the sample matrix affected the analysis results obtained with DEEMM the least, but with FMOC-Cl, TAHS and DNS, sample dilution had an influence on the results. When sample dilution and extrapolative dilution approach were applied on the latter results, an agreement of amino acid concentrations measured with different reagents was achieved within relative standard deviation (RSD) of 22% for most cases.

Approaches to a multitargeting drug development: first profiled 3- ethoxycarbonyl-1-aza-9-oxafluorenes representing a perspective compound class targeting Alzheimer disease relevant kinases CDK1, CDK5 and GSK-3ß.

Since Alzheimer disease (AD) is a multifactorial disease, recent therapeutical approaches concentrate on the development of a multitargeting drug. Various protein kinases are known to be involved in the progression of AD. A first series of 3-ethoxycarbonyl-1-aza-9-oxafluorenes has been synthesized and biologically evaluated as AD-relevant protein kinase inhibitors. A concentration-dependent inhibition of important AD-relevant kinases has been characterized after the selectivity of kinase inhibition had been demonstrated. Structure-activity relationships of protein kinase inhibition are discussed and first multitargeting inhibitors have been identified.

Multitargeted drug development: Discovery and profiling of dihydroxy substituted 1-aza-9-oxafluorenes as lead compounds targeting Alzheimer disease relevant kinases.

Alzheimer disease (AD) turned out to be a multifactorial process leading to neuronal decay. So far merely single target structures which attribute to the AD progression have been considered to develop specific drugs. However, such drug developments have been disappointing in clinical stages. Multitargeting of more than one target structure determines recent studies of developing novel lead compounds. Protein kinases have been identified to contribute to the neuronal decay with CDK1, GSK-3ß and CDK5/p25 being involved in a pathological tau protein hyperphosphorylation. We discovered novel lead structures of the dihydroxy-1-aza-9-oxafluorene type with nanomolar activities against CDK1, GSK-3ß and CDK5/p25. Structure-activity relationships (SAR) of the protein kinase inhibition are discussed within our first compound series. One nanomolar active compound profiled as selective protein kinase inhibitor. Bioanalysis of a harmless cellular toxicity and of the inhibition of tau protein phosphorylation qualifies the compound for further studies.

Discovery of a fluorene class of compounds as inhibitors of botulinum neurotoxin serotype E by virtual screening.

Botulinum neurotoxins are one of the most poisonous biological substances known to humans and present a potential bioterrorism threat. There are no therapeutic interventions developed so far. Here, we report the first small molecule non-peptide inhibitor for botulinum neurotoxin serotype E discovered by structure-based virtual screening and propose a mechanism for its inhibitory activity.

Determination of biogenic amines in infusions of tea (Camellia sinensis) by HPLC after derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl).

The reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for the pre-column derivatization of the biogenic amines (BAs) cadaverine (Cad), histamine (Him), octopamine (Ocp), phenylethylamine (Pea), putrescine (Put), spermidine (Spd), spermine (Spm), tyramine (Tym) and the internal standard 1,6-diaminohexane (Dhx). The resulting Fmoc-derivatives were resolved by high-performance liquid chromatography on a Superspher(©) C(18) column using a binary gradient generated from sodium acetate and acetonitrile. For quantification, the fluorescence of derivatives was used at 263 nm excitation and 313 nm emission wavelength. This approach was applied to free BAs extractable with boiling water from 14 black, 5 green, 1 Oolong, and 1 instant tea. Infusions were prepared by adding 35 ml boiling water to one gram of tea and extracted for 20 min. In the Oolong tea and two black teas, no BAs could be detected. Limits of detection were 0.07-1.0 pmol for BAs at signal-to-noise ratio 3:1. Besides most abundant Tym and Spm lower quantities of Pea, Put, and Spd were detected, albeit not in all teas. Quantities of Tym ranged from 16 to 431 µg Tym/L infusion (1.1-25.3 µg Tym/g tea) and 31 to 319 µg Spm/L infusion (1.5-16.9 µg Spm/g tea). In none of the teas, Him was detected. Owing to the low amounts of free BAs in tea infusions, no health risks are to be expected even on consumption of large quantities of tea as beverage.

Enantiomeric separation of FMOC-amino acids by nano-LC and CEC using a new chiral stationary phase, cellulose tris(3-chloro-4-methylphenylcarbamate).

A novel polysaccharide-based chiral stationary phase (CSP), cellulose tris(3-chloro-4-methylphenylcarbamate), also known as Sepapak-2 or Lux Cellulose-2, has been evaluated for the enantiomeric separation of FMOC derivatives of amino acids. After mobile-phase optimization in nano liquid chromatography (nano-LC) the column enabled the enantiomeric separation of 19 out of 23 amino acids tested, indicating the high chiral recognition power of this new CSP. Subsequently, a comparison of the driving force employed (pressure or voltage) was carried out comparing nano-LC and CEC under the same conditions. Better peak efficiencies and resolution were observed by using CEC experiments, which enabled the chiral discrimination of 20 out of 23 amino acids tested. Finally, in order to show the potential of this new CSP, the determination of the content and the enantiomeric purity of the non-protein amino acid citrulline in food supplements was performed. For that purpose, the method was optimized, evaluated and applied to different commercial samples.