Marfey's Reagent Research Articles

Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S.aureus varied from 1.4μM for UDP-GlcNAc-Enolpyruvyate to 1200μM for UDP-MurNAc. Levels of amine intermediates (L-Ala, D-Ala, D-Ala-D-Ala, L-Glu, D-Glu, and L-Lys) varied over a range of from 860μM for D-Ala-D-Ala to 30-260mM for the others. Total PG was determined from the D-Glu content of isolated PG, and used to estimate the rate of PG synthesis (in terms of cytoplasmic metabolite flux) as 690μM/min. The total UDP-linked intermediates pool (2490μM) is therefore sufficient to sustain growth for 3.6min. Comparison of UDP-linked metabolite levels with published pathway enzyme characteristics demonstrates that enzymes on the UDP-branch range from >80% saturation for MurA, Z, and C, to <5% saturation for MurB. Metabolite levels were compared with literature values for Escherichia coli, with the major difference in UDP-intermediates being the level of UDP-MurNAc, which was high in S.aureus (1200μM) and low in E.coli (45μM).

Synthesis of variants of Marfey's reagent having d-amino acids as chiral auxiliaries and liquid-chromatographic enantioseparation of (RS)-Mexiletine in spiked plasma: Assessment and comparison with l-amino acid analogs

Five d-amino acids have been used for the first time to synthesize chiral derivatizing reagents (as variants of Marfey's reagent) by nucleophilic displacement of one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene as against the literature reports on application of only l-amino acids or their amides as chiral auxiliaries in dinitrobenzene (DNB) moiety. Five other DNB based reagents were also prepared by nucleophilic substitution of fluorine atom with the set of the same amino acids in l-configuration, as chiral auxiliaries. These reagents were characterized and used for synthesis of diastereomers of (RS)-Mexiletine spiked in human plasma. Diastereomers were synthesized employing microwave irradiation and were separated on reversed-phase C18 column. Performance of the two types of chiral derivatizing reagents was compared. The reagents containing d-amino acids provided enhanced separation of diastereomers than those containing l-amino acids. The best resolution was obtained using mobile phase consisting of acetonitrile and 0.1% trifluoroacetic acid in gradient mode. The detection was carried out at 340nm. The method so developed was validated for linearity, accuracy and precision. The limit of quantitation was found to be approximately 25.2ngmL−1 in human plasma.

Enantiomeric analysis of theanine in different teas (Camellia sinensis) using Marfey's reagent

A fast and simple method for the enantioseparation of l- and d-theanine using Marfey's reagent was developed. The method was validated for linearity, specificity, accuracy, stability, detection and quantification limits. The separation was achieved on RP columns with a gradient of 1 % acetic acid and acetonitrile. d-Theanine was synthesized with a purity of 96.5 %, and the enantiomeric composition of theanine was determined in 56 tea samples including instant teas. No detectable d-theanine was found in the fresh leaves and in the most of white teas. It was observed that d-theanine arises initially at the wither process and mounts up according to the strength of this step and to further handling or storage procedures. A quantitative relationship between the smallness of tea grades and the enantiomeric composition was observed just in Darjeeling teas. Higher proportions of d-theanine were found in the five cold water-soluble instant black teas gained after alkaline treatment (5.56–51.80 % of total theanine).

ChemInform Abstract: An Efficient Synthesis of 4(5)‐Benzyl‐l‐histidines Employing Catalytic Transfer Hydrogenolysis at Elevated Temperatures.

An efficient two-step synthesis of 4(5)-benzyl- l -histidine from l -histidine was developed. A Pictet–Spengler reaction between l -histidine and benzaldehyde in the presence of excess strong base yielded 4- l -phenylspinacine within one hour. Catalytic transfer hydrogenolysis in methanol at reflux using ammonium formate rapidly converted 4- l -phenylspinacine to 4(5)-benzyl- l -histidine within five minutes. No racemization of the final product 4(5)-benzyl- l -histidine was observed using the Marfey reagent. To show the utility of this method, a series of fluorinated benzylhistidines were prepared.

Rapid quantitative chiral amphetamines liquid chromatography–tandem mass spectrometry: Method in plasma and oral fluid with a cost-effective chiral derivatizing reagent

Methamphetamine is a widely abused psychostimulant containing a chiral center. Consumption of over-the-counter and prescription medications may yield positive amphetamines results, but chiral separation of l- and d-methamphetamine and its metabolite amphetamine can help determine whether the source was licit or illicit. We present the first LC–MS/MS method with precolumn derivatization for methamphetamine and amphetamine chiral resolution in plasma and oral fluid collected with the Oral-Eze® and Quantisal™ devices. To 0.5mL plasma, 0.75mL Oral-Eze, or 1mL Quantisal specimen racemic d11-methamphetamine and amphetamine internal standards were added, followed by protein precipitation. Samples were centrifuged and supernatants loaded onto pre-conditioned Phenomenex® Strata™-XC Polymeric Strong Cation solid phase extraction columns. After washing, analytes were eluted with 5% ammonium hydroxide in methanol. The eluate was evaporated to dryness and reconstituted in water. Derivatization was performed with 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide (Marfey's reagent) and heating at 45°C for 1h. Derivatized enantiomer separations were performed under isocratic conditions (methanol:water, 60:40) with a Phenomenex® Kinetex® 2.6μm C18 column. Analytes were identified and quantified by two MRM transitions and their ratio on a 3200 QTrap (AB Sciex) mass spectrometer in ESI negative mode. In all three matrices, the method was linear for all enantiomers from 1 to 500μg/L, with imprecision and accuracy of ≤11.3% and 85.3–108%, respectively. Extraction efficiencies ranged from 67.4 to 117% and matrix effects from −17.0 to 468%, with variation always ≤19.1%. Authentic plasma and OF specimens were collected from an IRB-approved study that included controlled Vicks® VapoInhaler™ administration. The present method is sensitive, selective, economic and rapid (separations accomplished in <10min), and improves methamphetamine result interpretation.

Effects of pregabalin on D‐serine content and NMDA receptor‐mediated synaptic transmission in the mouse spinal cord (1129.9)

The anticonvulsant pregabalin (PGB) is used to relieve neuropathic pain. It has been shown that PGB binds to presynaptic calcium channels and subsequently inhibits transmitter release. Additionally, a preponderance of the evidence raises the possibility of other mechanisms independent of binding to calcium channels. In the present experiments, we have studied the effects of PGB on D‐serine content and NMDA receptor‐mediated synaptic transmission in the spinal cord dorsal horn.Experiments were performed on adult ICR mice. The sciatic nerve was partially ligated under halothane anesthesia. Mechanical allodynia was assessed by von Frey filaments. PGB (30‐50 mg/kg body weight) was intraperitonealy injected daily for 5‐7 days. The lumbar spinal cord was removed, homogenized, and ultrafiltrated. Supernatant samples were treated with Marfey's reagent and analyzed with liquid chromatography‐mass spectrometry for D‐serine concentration. Tight‐seal whole‐cell recordings were made from neurons in the superficial dorsal horn of spinal cord slices. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation.D‐serine content was 9.9±1.0 nM/g of wet tissue in sciatic nerve‐ligated mice (n=6), which was significantly larger than that in sham‐operated mice (7.9±0.8 nM/g, p &lt; 0.05). PGB considerably decreased D‐serine content to 6.0±0.9 nM/g (n=6, p &lt; 0.05, vs. sciatic nerve‐ligated mice). PGB decreased the NMDA/non‐NMDA ratio of the EPSC amplitude, and shortened the decay phase of the NMDA component of the EPSCs. These observations suggest that PGB decreases D‐serine concentration and thereby affects synaptic plasticity.Grant Funding Source: Supported by a grant from the Japan Society for the Promotion of Science (23500468 to Y.H.).

Isolation and Structures of Pipecolidepsins A and B, Cytotoxic Cyclic Depsipeptides from the Madagascan Sponge Homophymia lamellosa

Two new cyclic depsipeptides, pipecolidepsins A and B (1 and 2), have been isolated from the sponge Homophymia lamellosa collected off the coast of Madagascar. Their structures were determined by a combination of NMR experiments and by LC-MS analysis of the amino acid fragments obtained by hydrolysis and derivatization using Marfey's reagent. In addition to several common amino acids, these peptides contain unusual residues, including 2-amino-3-hydroxy-4,5-dimethylhexanoic acid, 3-ethoxyasparagine, 3,4-dimethylglutamine, 4,7-diamino-2,3-dihydroxy-7-oxoheptanoic acid, and 3-hydroxyaspartic acid as well as a terminal 3-hydroxy-2,4,6-trimethylheptanoic acid residue. Pipecolidepsins A and B displayed cytotoxic activity against a panel of different human tumor cell lines.

An Efficient Synthesis of 4(5)-Benzyl-L-Histidines Employing Catalytic Transfer Hydrogenolysis at Elevated Temperatures.

An efficient two-step synthesis of 4(5)-benzyl-L-histidine from L-histidine was developed. A Pictet-Spengler reaction between L-histidine and benzaldehyde in the presence of excess strong base yielded 4-phenylspinacine within one hour. Catalytic transfer hydrogenolysis in methanol at reflux using ammonium formate rapidly converted 4-L-phenylspinacine to 4(5)-benzyl-L-histidine within five minutes. No racemization of the final product 4(5)-benzyl-L-histidine was observed using the Marfey reagent. To show the utility of this methodology, a series of fluorinated benzylhistidines is presented.

Euryjanicins E–G, poly-phenylalanine, and poly-proline cyclic heptapeptides from the Caribbean sponge Prosuberites laughlini

A new investigation of the active sponge extracts of Prosuberites laughlini collected off the West coast of Puerto Rico has yielded three new cyclic heptapeptides, namely euryjanicins E (1)–G (3), containing multiple phenylalanine and proline residues. In CDCl3 solution, each euryjanicin F (2) and G (3) exists as an inseparable complex mixture of conformational isomers. The molecular structures of 1–3 were elucidated by a combination of chemical degradation, extensive ESI-MS/MSn analyses, and 2D NMR methods. The elucidation of the absolute configuration was achieved by HPLC following analysis of the acid hydrolysates after derivatization with Marfey's reagent. When assayed against the National Cancer Institute 60 tumor cell line panel, the new cyclic peptides did not display significant in vitro cytotoxicity.

Isomerization and epimerization of the aspartyl tetrapeptide Ala‐Phe‐Asp‐GlyOH at pH 10—A CE study

Isomerization and enantiomerization of Asp in the tetrapeptide Ala-Phe-Asp-GlyOH are studied at pH 10 and 80°C as well as 25°C. CE-MS allowed the distinction between α-Asp and β-Asp linkages in degradation products based on the ratio of the b and y fragment ions. Besides isomerization and enantiomerization of Asp, enantiomerization of Ala and Phe was also observed at both temperatures by chiral amino acid HPLC analysis using Marfey's reagent for derivatization. The rate of enantiomerization of the amino acids proceeded in the order Asp > Ala > Phe. The CE assay was validated with respect to linearity, LOQ, LOD, and precision and employed to characterize the time course of the degradation of the tetrapeptide upon incubation in borate buffer, pH 10. Isomerization to β-Asp peptides was identified as the major degradation reaction. The configuration of Asp or Ala affected the half-life of the starting peptide to a minor extent but did not influence the distribution of the individual products under equilibrium conditions at 80°C. Degradation at 25°C proceeded very slowly so that the equilibrium was not reached after 245 days.

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography–tandem mass spectrometry with chiral derivatization

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey's reagent, N(α)-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide, without extraction. The diastereomers of the N(α)-(2,4-dinitrophenyl)-D-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03 μg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20 μg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.

Indirect enantioresolution of (R,S)‐mexiletine by reversed‐phase high‐performance liquid chromatography via diastereomerization with [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride], (S)‐naproxen and nine chiral reagents synthesized as variants of Marfey's reagent

Eleven chiral derivatizing reagents (CDRs) were used for preparation of diastereomers of (R,S)-mexiletine containing a primary amino group in close proximity to the stereogenic center. One anhydride, namely [(S,S)-O,O'-di-p-toluoyl tartaric acid anhydride] was synthesized and (S)-naproxen was used as such as the chiral derivatizing reagent. The other nine CDRs were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene with six amino acid amides and three amino acids. The diastereomers were separated by reversed-phase high-performance liquid chromatography. The method was validated for linearity, accuracy, limit of detection and limit of quantification. The limit of detection was found in the range of 10-30 pmol.

Indirect reversed‐phase high‐performance liquid chromatographic and direct thin‐layer chromatographic enantioresolution of (R,S)‐Cinacalcet

Enantioresolution of the calcimimetic drug (R,S)-Cinacalcet was achieved using both indirect and direct approaches. Six chiral variants of Marfey's reagent having L-Ala-NH(2), L-Phe-NH(2), L-Val-NH(2), L-Leu-NH(2), L-Met-NH(2) and D-Phg-NH(2) as chiral auxiliaries were used as derivatizing reagents under microwave irradiation. Derivatization conditions were optimized. Reversed-phase high-performance liquid chromatography was successful using binary mixtures of aqueous trifluoroacetic acid and acetonitrile for separation of diastereomeric pairs with detection at 340 nm. Thin silica gel layers impregnated with optically pure L-histidine and L-arginine were used for direct resolution of enantiomers. The limit of detection was found to be 60 pmol in HPLC while in TLC it was found to be in the range of 0.26-0.28 µg for each enantiomers.

ChemInform Abstract: Total Structure of Hormothamnin A, a Toxic Cyclic Undecapeptide from the Tropical Marine Cyanobacterium Hormothamnion enteromorphoides.

Abstract The tropical marine cyanobacterium Hormothamnion enteromorphoides produces a suite of cytotoxic and antimicrobial cyclic peptides. The structure of the most lipophilic of these, hormothamnin A, was determined by interpretation of physical data, principally high NMR and FAB MS, in combination with chemical derivitization and degradation schemes. Isolation of a key pentapeptide fragment DPHE-D-LEU-L-ILE-D-allo-ILE-L-LEU, obtained under partial hydrolysis conditions, was instrumental to the final structure determination. β-D-aminooctanoic acid (D-BAOA) was characterized as a per-ester derivative following complete acid hydrolysis and Z-didehydrohomoalanine (DHHA) was spectroscopically in the intact peptide. The remaining residues (HYPRO, 2 × HSER, GLY) were evident from amino acid and spectroscopic analysis. Sequencing of these residues made use of knowledge from fragments, high field NMR (NOESY and ROESY) and FAB MS analysis of the intact peptide. Absolute stereochemistries of the α-amino residues were determined by HPLC analysis of the acid liberated residues derivatized with Marfey's reagent. The absolute stereochemistry of the β-amino residue was shown by circular dichoroism analysis, HPLC analysis of the Marfey derivative, and chiral synthesis of a homolog. By these techniques, hormothamnin A was demonstrated to possess a cyclo-[D-PHE-D-LEU-L-ILE-D-allo-ILE-L-LEU-GLY-D-BAOA-L-HSER-DHHAL-HYPRO-L-HSER] structure.

Uptake and conversion of d-amino acids in Arabidopsis thaliana

The D-enantiomers of proteinogenic amino acids fulfill essential functions in bacteria, fungi and animals. Just in the plant kingdom, the metabolism and role of D-amino acids (D-AAs) still remains unclear, although plants have to cope with significant amounts of these compounds from microbial decay in the rhizosphere. To fill this gap of knowledge, we tested the inhibitory effects of D-AAs on plant growth and established a method to quantitate 16 out of 19 proteinogenic amino acids and their D-enantiomers in plant tissue extracts. Therefore, the amino acids in the extracts were derivatized with Marfey's reagent and separated by HPLC-MS. We used two ecotypes (Col-0 and C24) and a mutant (lht1) of the model plant Arabidopsis thaliana to determine the influence and fate of exogenously applied D-AAs. All of them were found in high concentrations in the plant extracts after application, even in lht1, which points to additional transporters facilitating the import of D-AAs. The addition of particular amino acids (D-Trp, D-Phe, D-Met and D-His) led to the accumulation of the corresponding L-amino acid. In almost all cases, the application of a D-AA resulted in the accumulation of D-Ala and D-Glu. The presented results indicate that soil borne D-AAs can actively be taken up and metabolized via central metabolic routes.

High‐performance liquid chromatographic enantioseparation of (R,S)‐fluoxetine using Marfey's reagent and (S)‐N‐(4‐nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as chiral derivatizing reagents along with direct thin‐layer chromatographic resolution and isolation of enantiomers using l‐tartaric acid as mobile phase additive

Chiral assay of enantiomers of fluoxetine was achieved in pharmaceutical formulations using direct and indirect methods. L-tartaric acid was used as a mobile phase additive in thin-layer chromatography; the enantiomers were separated and isolated and were used to determine the elution order in HPLC. (R,S)-flouxetine was derivatized with (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester [(S)-NIFE], Marfey's reagent and 1-fluoro-2,4-dinitrophenyl-L-methionine amide (FDNP-L-Met-NH₂. The diastereomers were separated using RP-HPLC. The effect of flow rate and TFA concentration on resolution was studied. The diastereomers obtained by derivatization with FDNP-L-Met-NH₂ were also separated by RP-TLC.

Novel thiazole and oxazole containing cyclic hexapeptides from a waterbloom of the cyanobacterium Microcystis sp.

Eight novel thiazole and oxazole containing cyclic peptides, microcyclamides GL616, GL582, GL614A, GL614B, GL614C, GL546A, GL546B, and GL628, as well as the known microcyclamide A, were isolated from the hydrophylic extract of a Microcystis sp. water-bloom collected in Gilboa reservoir, Valley of Armageddon, Israel. The planar structure of the compounds was determined by homonuclear and inverse-heteronuclear 2D NMR techniques as well as high-resolution mass spectrometry. The absolute configuration of the asymmetric centers of the amino acids was studied using Marfey's method for HPLC following ozonolysis, hydrolysis and derivatization with Marfey's reagent. This is the first example where acidic and modified amino acids are incorporated in this group of ribosomally biosynthesized metabolites.

Fast chiral separation of drugs using columns packed with sub‐2 μm particles and ultra‐high pressure

The use of columns packed with sub-2 microm particles in liquid chromatography with very high pressure conditions (known as UHPLC) was investigated for the fast enantioseparation of drugs. Two different procedures were evaluated and compared using amphetamine derivatives and beta-blockers as model compounds. In one case, cyclodextrins (CD) were directly added to the mobile phase and chiral separations were carried out in less than 5 min. However, this strategy suffered from several drawbacks linked to column lifetime and low chromatographic efficiencies. In the other case, the analysis of enantiomers was carried out after a derivatization procedure using two different reagents, 2,3,4-tri-O-acetyl-alpha-D-arabinopyranosyl isothiocyanate (AITC) and N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent). Separation of several amphetamine derivatives contained within the same sample was achieved in 2-5 min with high efficiency and selectivity. The proposed approach was also successfully applied to the enantiomeric purity determination of (+)-(S)-amphetamine and (+)-(S)-methamphetamine. Similar results were obtained with beta-blockers, and the separation of 10 enantiomers was carried out in less than 3 min, whereas the individual separation of several beta-blocker enantiomers was performed in 1 min or less.

Torrubiellutins A–C, from insect pathogenic fungus Torrubiella luteorostrata BCC 12904

Investigation of the insect pathogenic fungus Torrubiella luteorostrata led to the isolation of three new macrocyclic torrubiellutins A–C ( 1– 3) and the known pyrone diterpene 4. Structures were elucidated by spectroscopic data including 1D, 2D NMR, and MS spectral data. The absolute stereochemistry was determined by chemical means using Mosher reactions and Marfey's reagent, together with NOESY spectral data. Torrubiellutin C showed biological activities against KB, MCF-7, NCI-H187, and Vero cell lines with IC 50 varying from 0.78 to 4.36 μg/mL, while compound 4 exhibited antimalaria and anti-inflammatory activity with IC 50 values of 3.49 and 1.21 μg/mL, respectively.

Reversed‐phase liquid chromatographic determination of enantiomers of atenolol in rat plasma using derivatization with Marfey's reagent

An HPLC method was established for enantioseparation of (R,S)-atenolol (ATE) and determination of enantiomers in rat plasma. Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDNP-L-Ala-NH(2), MR) was used as chiral derivatizing reagent with detection of diastereomers at 340 nm. It was shown that the R-isomer eluted before the S-isomer. The method was validated for linearity, repeatability, limits of detection and limit of quantification (LOQ). Recovery of ATE at LOQ was 92.8% for (R)-ATE and 92.6% for (S)-ATE.