Determination of D-aspartate N-methyltransferase activity in the starfish by direct analysis of N-methyl-D-aspartate with high-performance liquid chromatography

We describe a method for the detection and quantification of D-aspartate N-methyltransferase activity. The enzyme catalyzes the S-adenosyl-L-methionine-dependent N-methylation of D-aspartate to form N-methyl-D-aspartate (NMDA). NMDA is detected directly by high-performance liquid chromatography (HPLC) of their (+)- and/or (-)-1-(9-fluorenyl)ethyl chloroformate fluorescent derivatives. The NMDA production in the assay mixture is linearly proportional to the incubation time and the amount of tissue homogenate. Using a 10 min incubation time, the method allows detection of the enzyme activity below 10 fmol/min. It can be used to analyze kinetic behavior and to quantify the enzyme from a wide variety of organisms.     

Occurrence of N-methyl- -aspartate in bivalves and its distribution compared with that of N-methyl- -aspartate and , -aspartate

The presence of N-methyl- -aspartate (NMLA) was demonstrated in bivalves, Corbicula sandai and Tapes japonica. To our knowledge, this is the first report on the occurrence of NMLA in animal tissues. NMLA in bivalve tissues was identified according to the following findings; (a) its derivatives with (+)- and (−)- 1-(9-fluorenyl)ethyl chloroformate (FLEC) behaved identically with those of authentic NMLA, respectively, on high-performance liquid chromatography (HPLC), (b) its derivatives with (+)- and (−)- FLEC behaved identically with (−)- and (+)-FLEC derivatives of authentic N-methyl- -aspartate (NMDA), respectively, on HPLC and (c) its behavior on thin-layer chromatography was the same as those of authentic NMLA. We also describe the distribution of NMDA, and - and -aspartate, to which N-methylaspartate enantiomers are structurally related. NMDA was more widely dirtributed than NMLA in bivalves. These bivalves containing NMLA showed lower -aspartate contents and /( + ) ratios of aspartate, than the bivalves containing NMDA.     

N-methyl-D-glutamate and N-methyl-L-glutamate in Scapharca broughtonii (Mollusca) and other invertebrates

The presence of N-methyl-D-glutamate (NMDG) and N-methyl-L-glutamate (NMLG) has been demonstrated in the tissues of Scapharca broughtonii, which are known to contain N-methyl-D-aspartate (NMDA). To our knowledge, this is the first report on the natural occurrence of NMDG and the occurrence of NMLG in eukaryotes. These compounds were identified according to the following findings; (a) their derivatives with (+)- and (-)-l-(9-fluorenyl)ethyl chloroformate (FLEC) showed identical behaviors with those of authentic NMDG and NMLA, respectively, on high-performance liquid chromatography (HPLC), (b) the HPLC peak of NMDG disappeared when the extract, as well as the authentic compound, was treated with D-aspartate oxidase before derivatization, (c) they behaved identically with authentic compounds on thin-layer chromatography and differently from NMDA. Both or either of NMDG and NMLG were also detected in several mollusks and other animals. Concentrations of the enantiomers were comparable in the tissues of S. broughtonii and a few other species.     

Liquid chromatographic analysis of propranolol enantiomers in human blood using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate

A method for the determination of the R-(+) and S-(−) enantiomers of propranolol in blood was developed. After extraction with heptane—isopentanol and derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, excess reagent was removed using solid-phase extraction. The enantiomers were separated on an achiral, reversed-phase, radially compressed column, and detected by fluorescence with excitation and emission wavelengths of 260 and 340 nm, respectively. The limit of quantification was 0.5 ng/ml. This method was used for pharmacokinetic analysis of propranolol enantiomers after administration of immediate-release (80 mg) or sustained-release (160 mg) racemic propranolol.     

2,7-Di-tert-butyl-Fmoc-P-OSu: a new polymer-supported reagent for the protection of the amino group

The polymer-supported (2,7-di-tert-butyl-9-fluorenyl)methyl succinimidyl carbonate (Dtb-Fmoc-P-OSu), derived from (2,7-di-tert-butyl-9-fluorenyl)methyl chloroformate (Fmoc-Cl) and a polymeric N-hydroxysuccinimide (P-HOSu), has been used for the preparation of Dtb-Fmoc-protected amines and amino acids. After the N-protection reaction, the liberated P-HOSu can be recovered and reused. This Dtb-Fmoc-protection improves the solubility of the Fmoc-protected analogues.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.     

Enantioselective analysis of glufosinate using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography

We have developed a new analytical method to quantify the DL-homoalanine-4-yl(methyl)phosphinate (DL-GLUF) enantiomers in biological specimens using a reversed-phase high-performance liquid chromatography system with a fluorescence detection system. The derivatization of DL-GLUF enantiomers with (+)-1-(9-fluorenyl)ethyl chloroformate was carried out under mild conditions (40 degrees C for 30 min) without inducing racemization. The lower limit of quantitation was 0.01 microg/ml for both D-GLUF and L-GLUF, and the detection limit was 5 ng/ml. When DL-GLUF enantiomers were added to serum to produce concentrations between 0.1 and 100 microg/ml, the mean recovery rate was at least 93.8%. The recovery rate from urine was also satisfactory.     

Determination of the geometrical diarylpropenamine isomers in feces by high-performance liquid chromatography

Diarylpropenamine derivatives are a class of compounds which have been evaluated as potential drug candidates. Here a specific and reproducible HPLC method for the determination of cis- and trans-isomers of the unsubstituted derivative, 3-(4′-bromo-[1,1′-biphenyl]-4-yl)-3-(4-X-phenyl-N,N-dimethyl-2-propen-1-amine (I, where X=H) in feces is described. The analyte I and internal standard, nitro derivative (II, where X=NO₂), were isolated from the basified biological matrix using a liquid–liquid extraction with ethyl acetate followed by a solid-phase procedure performed on a silica cartridge. The organic phase was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The analytes were eluted with ethyl acetate–hexane–triethylamine (59:40:1) in HPLC column (silica) and detected by UV spectrophotometry at 272 nm. Linearity, precision and accuracy data for feces standards after extraction were acceptable. The method has been applied to analyses of feces samples from rats dosed with I, in which it could be anticipated that fecal excretion is quantitatively the major route for I elimination.     

Determination of peptides and amino acids from wool and beer with sensitive fluorescent reagent 2-(9-carbazole)-ethyl chloroformate by reverse phase high-performance liquid chromotography and liquid chromotography mass spectrometry

A new method for the sensitive determination of amino acids and peptides using the tagging reagent 2-(9-carbazole)-ethyl chloroformate (CEOC) with fluorescence (FL) detection has been developed. Identification of derivatives was carried out by liquid chromotography mass spectrometry. The chromophore in the 2-(9-fluorenyl)-ethyl chloroformate (FMOC) reagent was replaced by carbazole, which resulted in a sensitive fluorescence lerivatizing agent CEOC. CEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Studies on derivatization demonstrate excellent derivative yields over the pH range 8.8-10.0. Maximal yields close to 100% are observed with three- to fourfold molar reagent excess. Derivatives exhibit strong fluorescence and allow direct injection of the reaction mixture with no significant disturbance from the major fluorescent reagent degradation by-products, such as 2-(9-carbazole)-ethanol and bis-(2-(9-carbazole)-ethyl) carbonate. In addition, the detection responses for CEOC derivatives are compared to those obtained with FMOC. The ratios AC(CEOC)/AC(FMOC) = 1.00-1.82 for fluorescence (FL) response and AC'(CEOC)/AC'(FMOC) = 1.00-1.21 for ultraviolet (UV) response are observed (here, AC and AC' are, respectively, FL and UV response). Separation of the derivatized peptides and amino acids has been optimized on a Hypersil BDS C18 column. Excellent linear responses are observed. This method was used successfully to analyze protein hydrolysates from wool and from direct-derivatized beer.     

Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

The synthesis of peptides employing 9-fluorenylmethyl chloroformate(Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtainedin good yield (70–95%). Furthermore, both the ¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

Summary The synthesis of peptides employing 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtained in good yield (70–95%). Furthermore, both the¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

High-performance liquid chromatography analysis of naturally occurring D-amino acids in sake

We measured all of the D- and L-amino acids in 141 bottles of sakes using HPLC. We used two precolumn derivatization methods of amino acid enantiomer detection with o-phthalaldehyde and N-acetyl-L-cysteine, as well as (+)-1-(9-fluorenyl)ethyl chloroformate/1-aminoadamantane and one postcolumn derivatization method with o-phthalaldehyde and N-acetyl-L-cysteine. We found that the sakes contained the D-amino acids forms of Ala, Asn, Asp, Arg, Glu, Gln, His, Ile, Leu, Lys, Ser, Tyr, Val, Phe, and Pro. We were not able to detect D-Met, D-Thr D-Trp in any of the sakes analyzed. The most abundant D-Ala, D-Asp, and D-Glu ranged from 66.9 to 524.3 μM corresponding to relative 34.4, 12.0, and 14.6% D-enantiomer. The basic parameters that generally determine the taste of sake such as the sake meter value (SMV; "Nihonshudo"), acidity ("Sando"), amino acid value ("Aminosando"), alcohol content by volume, and rice species of raw material show no significant relationship to the D-amino acid content of sake. The brewing water ("Shikomimizu") and brewing process had effects on the D-amino acid content of the sakes: the D-amino acid contents of the sakes brewed with deep-sea water "Kaiyoushinosousui", "Kimoto yeast starter", "Yamahaimoto", and the long aging process "Choukijukusei" are high compared with those of other sakes analyzed. Additionally, the D-amino acid content of sakes that were brewed with the adenine auxotroph of sake yeast ("Sekishoku seishu kobo", Saccharomyces cerevisiae) without pasteurization ("Hiire") increased after storage at 25 °C for three months.     

X-ray crystal structure and activity of fluorenyl-based compounds as transthyretin fibrillogenesis inhibitors

Transthyretin (TTR) is a 54?kDa homotetrameric protein that transports thyroxine (T4) and retinol (vitamin A), through its association with retinol binding protein (RBP). Under unknown conditions, it aggregates to form fibrils associated with TTR amyloidosis. Ligands able to inhibit fibril formation have been studied by X-ray crystallography. The use of polyethylene glycol (PEG) instead of ammonium sulphate or citrate has been evaluated as an alternative to obtain new TTR complexes with (R)-3-(9-fluoren-9-ylideneaminooxy)-2-methyl-N-(methylsulfonyl) propionamide (48R(1)) and 2-(9H-fluoren-9-ylideneaminooxy) acetic acid (ES8(2)). The previously described fluorenyl based inhibitors (S)-3-((9H-fluoren-9-ylideneamino)oxy)-2-methylpropanoic acid (6BD) and 3-((9H-fluoren-9-ylideneamino)oxy)propanoic acid (7BD) have been re-evaluated with the changed crystallization method. The new TTR complexes with compounds of the same family show that the 9-fluorenyl motif can occupy alternative hydrophobic binding sites. This augments the potential use of this scaffold to yield a large variety of differently substituted mono-aryl compounds able to inhibit TTR fibril formation.     

Synthesis and NMDA receptor affinity of fluorinated dioxadrol analogues

A series of dioxadrol analogues with fluorine substituents in position 4 of the piperidine ring has been synthesized and pharmacologically evaluated. The key step in the synthesis was the fluorination of diastereomeric piperidones 6a and 6c as well as diastereomeric alcohols 9a and 9c with DAST. The reaction of the alcohols 9a and 9c took place with inversion of configuration. After removal of the Cbz-protective group, the NMDA receptor affinities of the resulting secondary amines 8a, 8c, 12b, and 12d were investigated in receptor binding studies. It was shown that the like-configuration of the ring junction was crucial for high NMDA receptor affinity. An axially oriented fluorine atom in position 4 led to 2-(2,2-diphenyl-1,3-dioxolan-4-yl)-4-fluoropiperidine (12d, WMS-2517) with a K_i-value of 27 nM. The NMDA receptor affinity of 8c (WMS-2513) with an additional fluorine atom in equatorial 4-position was slightly reduced (K_i = 81 nM). Both fluorinated dioxadrol derivatives 8c and 12d showed high selectivity against σ₁ and σ₂ receptors as well as the polyamine binding site of NR2B receptors.     

Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: An ultra-large metazoan mitochondrial genome

The complete mitochondrial (mt) genome of the ark shell Scapharca broughtonii was determined using long PCR and a genome walking sequencing strategy with genus-specific primers. The S. broughtonii mt genome (GenBank accession number AB729113) contained 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 2 ribosomal RNA genes, and 42 transfer tRNA genes, in a length of 46,985 nucleotides for the size of mtDNA with only one copy of the heteroplasmic tandem repeat (HTR) unit. Moreover the S. broughtonii mt genome shows size variation; these genomes ranged in size from about 47 kb to about 50 kb because of variation in the number of repeat sequences in the non-coding region. The mt-genome of S. broughtonii is, to date, the longest reported metazoan mtDNA sequence. Sequence duplication in non-coding region and the formation of HTR arrays were two of the factors responsible for the ultra-large size of this mt genome. All the tRNA genes were found within the S. broughtonii mt genome, unlike the other bivalves usually lacking one or more tRNA genes. Twelve additional specimens were used to analyze the patterns of tandem repeat arrays by PCR amplification and agarose electrophoresis. Each of the 12 specimens displayed extensive heteroplasmy and had 8–10 length variants. The motifs of the HTR arrays are about 353–362 bp and the number of repeats ranges from 1 to 11.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

HPLC purification and re‐evaluation of chemical identity of two circular bacteriocins,gassericin A and reutericin 6

Aim: The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Methods and Results: Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse‐phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2‐propanol. Mass analysis, N‐terminal sequencing and bacteriocin assay of the HPLC‐purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H]⁺ and both had the same specific activity. d/l‐ amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)‐1‐(9‐fluorenyl)ethyl chloroformate and o‐phthalaldehyde/N‐acetyl‐l ‐cystein revealed neither gassericin A nor reutericin 6 contained d ‐alanine residues contrary to our previous results. Conclusion: Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no d ‐alanine residues. Significance and Impact of the Study: The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.     

Stereospecific high-performance liquid chromatographic assay of ibuprofen: improved sensitivity and sample processing efficiency

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of the non-steroidal anti-inflammatory drug ibuprofen (IB) in plasma was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), samples are acidified and extracted with a mixture of isooctane—isopropanol (95:5, v/v). After evaporation of the organic layer, the drug and I.S. are derivatized with S-(−)-1(1-naphthyl)ethylamine (S-NEA) after addition of ethyl chloroformate as the coupling reagent. Ethanolamine is added 3 min after the addition of S-NEA to react with the excessive ethyl chloroformate. The resultant diastereomers corresponding to IB and I.S. were chromatographed at ambient temperature on a 100 mm × 4.6 mm I.D. C₁₈ reversed-phase column using acetonitrile—water—acetic acid—triethylamine (60:40:0.1:0.02) as the mobile phase pumped at a flow-rate of 1.2 ml/min. Detection of the fluorescent chromophore was at 280 and 320 nm for excitation and emission, respectively. The suitability of the assay for clinical pharmacokinetic studies of IB was determined by the analysis of plasma samples obtained from a healthy volunteer, following administration of a single 400-mg oral dose of racemic IB.     

9-Fluorenylmethyl chloroformate (Fmoc-Cl) as a useful reagent for the synthesis of pentafluorophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl, p-nitrophenyl, o-nitrophenyl and succinimidyl esters of Nα-urethane protected amino acids

9-Fluorenylmethyl chloroformate has been demonstrated to be useful reagent for the synthesis of several commonly used activeesters of Fmoc-/Boc-/Z-amino acids. These include pentafluorophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl, p-nitrophenyl, o-nitrophenyl, and succinimidyl esters. The method is simple, rapid and efficient. All the compounds made have been isolated as crystaline solids in good yield and optical purity. They were fully characterized by IR, and ¹H NMR.     

The release of glutamate and aspartate from rat brain synaptosomes in response to domoic acid (amnesic shellfish toxin) and kainic acid

Kainic acid is known to stimulate the release of glutamate (GLU) and aspartate (ASP) from presynaptic neurons. It has been suggested that the enhanced release of these endogenous EAA's plays a significant role in the excitotoxic effects of KA. Domoic acid (DOM), a shellfish toxin, is structurally similar to KA, and has been shown to be 3–8 times more toxic than KA. In this study, effects of KA and DOM on the release of GLU and ASP from rat brain synaptosomes were investigated. Amino acid analysis was performed by the reversed phase HPLC, following derivatization with 9-fluorenylmethyl chloroformate (FMOC). Potassium chloride (40 mM) was used as a positive control, and stimulated GLU release from rat brain synaptosomes in presence or absence of Ca²⁺. DOM enhanced the release of GLU, whereas KA stimulated the release of both GLU and ASP from synaptosomes in the presence of Ca²⁺. However, their potency to stimulate GLU and ASP release was enhanced in absence of Ca²⁺. These results indicate that diferent mechanisms may be involved in the release of GLU and ASP in response to DOM and KA, and that neurotransmitter release appeared to be highly specific for these agonists. It would appear that DOM and KA may interact with different receptors on the presynaptic nerve terminal, and/or activate different subtypes of voltage-dependent Ca²⁺ channels to promote influx of Ca²⁺ which is targeted for different pools of neurotransmitters.Abbreviations ANOVA analysis of variance - ASP aspartate - DOM domoic acid - DHKA dihydrokainic acid - EAA excitatory amino acid - FMOC 9-fluorenylmethyl chloroformate - GLU glutamate - KA kainic acid