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PubMed1995

Blockade of both epileptogenesis and glutamate release by (1S,3S)-ACPD, a presynaptic glutamate receptor agonist.

The influence of intracerebrally focally administered doses of a presynaptic metabotropic glutamate receptor agonist, (1S,3S)-ACPD, and of the post-synaptically targeted competitive NMDA receptor antagonist, D-CPPene (SDZ EAA 494), was tested on the development of amygdaloid kindling. The actions of these drugs, compared to that of D-CPP, was also tested on fully developed stage 5 amygdala kindled seizures. Both (1S,3S)-ACPD and D-CPPene dose-dependently increased the generalised seizure threshold in fully kindled animals. They showed a similar potency, with (1S,3S)-ACPD acting presynaptically and D-CPPene postsynaptically. Both drugs reversibly inhibited epileptogenesis at 10 nmol in 0.5 microliter of injection vehicle, keeping the kindling stage at or below stage 2. All animals reached stage 5 after withdrawal of the 2 drugs. Whereas (1S,3S)-ACPD inhibited depolarisation-induced release of [3H]L-glutamate and [3H]D-aspartate from cortical synaptosomes (IC50 63 microM and 50 microM, respectively), D-CPPene (postsynaptically active) was without effect. These findings suggest a new approach to the development of clinically effective anticonvulsants through the development of presynaptic glutamate receptor agonists which could be administered systemically to control the extent of synaptic release of glutamate.

Animals↗
PubMed1989

Stimulation of peripheral cholinergic nerves by glutamate indicates a new peripheral glutamate receptor.

The bronchial smooth muscle of the rat was examined for contractile responses to excitatory amino acids. The nerve-mediated contraction induced by electrical field stimulation was enhanced by exogenous L-glutamate (L-Glu). The apparent affinity (ED50) of L-Glu was 3.5 +/- 0.1 mM. Both tetrodotoxin and hemicholinium-3 completely abolished the electrical field-induced contraction and therefore the potentiation by L-Glu, which indicates that L-Glu has a prejunctional effect. Concentrations of L-Glu higher than 22 mM inhibited the electrical field-induced contractions and enhanced the tonus of the smooth muscle by postjunctional stimulation. The ED50 of exogenous ACh was not altered by L-Glu. High concentrations (62 mM) of L-Glu increased the intrinsic activity (alpha) of ACh, indicating a postjunctional potentiation of ACh-induced contractions. L-Glu did not inhibit the activity of acetylcholinesterase, therefore the postjunctional potentiation was not due to ACh accumulation. Inhibition of the electrical field-induced contraction was seen with high concentrations of D-Glu, L-aspartate (L-Asp), L-alpha-amino adipate and ibotenate. Neither glutamate diethyl ester nor 2-amino-5-phosphonovalerate had any inhibitory effects on the L-Glu- and L-Asp-induced alterations of the electrical field-stimulated contraction or on the L-Glu-enhanced tonus of the bronchial smooth muscle. Kainate, N-methyl-D-aspartate, quisqualate and N-acetyl-aspartyl-glutamate had only minor transient potentiating effects on the electrical field-induced contraction. The results provide evidence for a L-Glu receptor in rat bronchi that has a different specificity for glutamate agonists and antagonists than the L-Glu receptor described in the CNS. The receptor seems to be located prejunctionally and enhances nerve-mediated responses and thereby stimulates the bronchial smooth muscle to contract. The possible involvement of this type of receptor in the 'Chinese restaurant syndrome' is discussed.

Amino Acids↗
PubMed1995

L-glutamate-induced changes in intracellular calcium oscillation frequency through non-classical glutamate receptor binding in cultured rat myocardial cells.

The effects of L-glutamate, N-methyl-D-aspartate (NMDA), kainate (KA) and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) on intracellular Ca2+ oscillation frequency were studied in cultured rat myocardial cells. Ca2+ oscillations per minute were increased as compared to control by L-glutamate (100 microM) from 3.8 +/- 2.2 to 25.7 +/- 4.3 (p < 0.001) and the NMDA-receptor agonist, NMDA (100 microM), from 1.2 +/- 0.8 to 34.8 +/- 10.1 (p < 0.011). Increases over control frequency were also seen in response to the non-NMDA receptor agonists KA (100 microM) from 5.8 +/- 2.3 to 25.6 +/- 3.2 (p < 0.001) and AMPA (10 microM) from 3.8 +/- 1.2 to 13.3 +/- 1.8 (p < 0.001). The non-competitive NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801) (10 microM), decreased the Ca2+ oscillation frequency induced by NMDA (100 microM from 36.8 +/- 12.2 to 7.2 +/- 7.2 (p < 0.05). (+/-)-2-Amino-7-phosphonoheptanoic acid (AP7), a competitive inhibitor at the NMDA receptor inhibited the increase in frequency induced by KA (100 microM) at all concentrations tested (0.1, 1.0, 10 and 100 microM). 6,7-Dinitroquinoxaline-2,3-dione (DNQX), a competitive inhibitor at non-NMDA receptors, also decreased the oscillation frequency elicited by KA (100 microM) from 35.4 +/- 9.4 to 28.2 +/- 9.8, 24.8 +/- 9.8 and 11 +/- 9.5 at concentrations of 0.1, 1.0 and 10 microM respectively. The peak amount of intracellular Ca2+ as expressed as the fluo 3 ratio, F/Frcst, was not increased by L-glutamate, NMDA or KA. These results suggest the presence of a novel glutamate receptor composed of both non-NMDA and NMDA subunits on cultured rat myocardial cells, and receptor stimulation leads to an increase in intracellular Ca2+ oscillation frequency.

Animals↗
PubMed1989

Extracellular taurine increase in rat hippocampus evoked by specific glutamate receptor activation is related to the excitatory potency of glutamate agonists.

Taurine increases in brain extracellular space due to glutamate agonists were studied in vivo in the rat hippocampus using a dialysis technique, both in the absence and in the presence of glutamate receptor antagonists. Extracellular taurine levels increased during perfusions of agonists, listed in descending order of potency: kainate (KA), N-methyl-D-aspartate (NMDA), and quisqualate (QA). While taurine increases due to KA or QA perfusions were inhibited by 6,7-dinitro-quinoxaline-2,3-dione (DNQX), those induced by NMDA were abolished in the presence of 3-(carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). These results indicate that increases in extracellular taurine levels evoked by NMDA, KA or QA in the rat hippocampus are caused by activation of their specific receptors. Field potentials, concomitantly recorded, were quickly abolished during NMDA or KA perfusions (0.1 mM), while QA (0.25 mM) induced the appearance of bicuculline-like evoked responses. Since taurine has been proposed as an osmoregulatory substance in the rat brain, and cell swelling is known to be an early component of glutamate agonists neurotoxicity, the increases in extracellular taurine reported here could be due to taurine released through an osmoregulatory process, counteracting the neurotoxic cellular oedema induced by glutamate agonists.

Action Potentials↗
PubMed1994

Role of arachidonic acid in the facilitation of glutamate release from rat cerebrocortical synaptosomes independent of metabotropic glutamate receptor responses.

The role of arachidonic acid in the facilitation of glutamate release by the metabotropic glutamate receptor, mGluR, agonist (1S,3R)-1-amino-cyclo-pentane-1,3-dicarboxylate ((1S,3R)-ACP), was investigated in cerebrocortical nerve terminals from adult rats. mGluR activation with the agonist (1S,3R)-ACPD involved a transient increase in the intrasynaptosomal concentration of diacylglycerol that was not altered by the presence of arachidonic acid either in polarized synaptosomes, or in synaptosomes depolarized with 4-aminopyridine. These results suggest that the locus of the arachidonic acid action in the facilitation of glutamate release is downstream of the metabotropic glutamate receptor in the signal transduction pathway.

4-Aminopyridine↗
PubMed1996

The synergism between metabotropic glutamate receptor activation and arachidonic acid on glutamate release is occluded by induction of long-term potentiation in the dentate gyrus.

In synaptosomes prepared from dentate gyrus, activation of the metabotropic glutamate receptor by the specific agonist, trans-1-amino-cyclopentyl-1,3-dicarboxylate, increases release of glutamate in the presence of a low concentration of arachidonic acid. A similar interaction between trans-1-amino-cyclopentyl-1,3-dicarboxylate and arachidonic acid is observed on inositol phospholipid turnover and on protein kinase C activity. We report here that when long-term potentiation is induced in the dentate gyrus by high frequency tetanic stimulation to the perforant path, the synergism between arachidonic acid and trans-1-amino-cyclopentyl-1,3-dicarboxylate is occluded. The occlusion of the synergistic action between arachidonic acid and trans-1-amino-cyclopentyl-1,3-dicarboxylate on glutamate release extended to occlusion of the effect in inositol phospholipid turnover and protein kinase C activation in synaptosomes prepared from dentate gyrus in which long-term potentiation was induced in vivo. One interpretation of the results presented here is that tetanic stimulation is followed by stimulation of metabotropic glutamate receptors at a time when arachidonic acid concentration in the synaptic region is elevated, and that this interaction triggers the presynaptic changes required for expression of long-term potentiation.

Animals↗
PubMed1999

Localization of L-glutamate and glutamate-like receptors at the squid giant synapse.

HPLC analysis of the amino acid contents of the second- and third-order giant fibres at the giant synapse in the stellate ganglion of the squid Loligo vulgaris shows that there are significantly higher amounts of L-glutamate and L-aspartate in the second-order (presynaptic) fibre than in the third-order (postsynaptic) fibre. Immunocytochemical staining of sections of the ganglion with an antibody raised against L-glutamate produces specific positive staining in the synaptic region of the second-order fibre. In contrast, staining with antibodies raised against glutamate-receptors (mammalian GluR1 with GluR2/3) produces positive staining in the third-order fibre at the postsynaptic region. These data provide further evidence for the hypothesis that L-glutamate is an excitatory transmitter at the giant synapse.

Alanine↗
PubMed1999

Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis.

Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313-318 K in a flow injection analysis (FIA) system. The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions. The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD (n = 10) for 5 mM L-glutamate was 7.6%. The response to some potential interferents has been quantified. Addition of finely ground hexaammineruthenium (III) trichloride ([Ru(NH3)6]Cl3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current. The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO.

Biosensing Techniques↗
PubMed1979

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state.

Ammonia is known to inhibit the steady-state rate of oxidation of L-glutamate catalyzed by glutamate dehydrogenase. We reported previously [Brown, A., Colen, A. H., & Fisher, H. F. (1978) Biochemistry 17, 2031] kinetic evidence supporting the formation in the initial rapid phase of a complex which is composed of enzyme, reduced coenzyme, alpha-ketoglutarate, and ammonia. We show here that the effects of ammonia on the steady-state reaction can be correlated with transient-state kinetic effects related to the concentration of that ammonia-containing complex. These results indicate the existence of alternate reaction pathways which become important at high ammonia concentrations. These new pathways provide an additional route for the release of NADPH from the enzyme surface. The expanded mechanism shows that the noncompetitive product inhibition by ammonia can occur without the simultaneous presence of ammonia and L-glutamate on the enzyme. This mechanism also accommodates the observed substrate inhibition by L-glutamate.

Ammonia↗
PubMed1969

Kinetic studies of glutamate dehydrogenase with glutamate and norvaline as substrates. Coenzyme activation and negative homotropic interactions in allosteric enzymes.

1. Kinetic studies of glutamate dehydrogenase were made with wide concentration ranges of the coenzymes NAD(+) and NADP(+) and the substrates glutamate and norvaline. Initial-rate parameters were evaluated. 2. Deviations from Michaelis-Menten behaviour towards higher activity were observed with increasing concentrations of either coenzyme with glutamate as substrate, but not with norvaline as substrate. 3. In phosphate buffer, pH7.0, Lineweaver-Burk plots with either coenzyme as variable and a constant, large glutamate concentration showed three or four linear regions of different slope with relatively sharp discontinuities. Maximum rates obtained by extrapolation and Michaelis constants for the coenzymes increased in steps with increase of coenzyme concentration. 4. In the absence of evidence of heterogeneity of the enzyme and coenzyme preparations, the results are interpreted in terms of negative homotropic interactions between the enzyme subunits. It is suggested that sharp discontinuities in Lineweaver-Burk plots or reciprocal binding plots may be characteristic of this new type of interaction, which can be explained in terms of an Adair-Koshland model, but not by the model of Monod, Wyman & Changeux.

Binding Sites↗
PubMed1999

Reversal of glutamate excitotoxicity by activation of PKC-associated metabotropic glutamate receptors in cerebellar granule cells relies on NR2C subunit expression.

Stimulation of metabotropic glutamate receptors (mGluRs) belonging to group I has been found to reduce N-methyl-D-aspartate (NMDA) receptor function in terms of both intracellular calcium concentration ([Ca2+]i) rise and neurotoxicity in cultured cerebellar granule cells. In the present study, we investigated whether the mGluR-elicited modulation of glutamate responses might rely on the heteromeric composition of NMDA receptor channel. NMDA receptors consist of two distinct groups of subunits: NR1, that is ubiquitously in the receptor complexes; and NR2A-D, that differentiate and potentiate NMDA receptor responses by assembling with NR1. Among NR2 subunits, only NR2A and NR2C mRNAs and relative proteins are detected in cerebellar granule cells at 10 days in vitro. To dissect the involvement of the two different subunits in making the NMDA receptor channel sensitive to modulation by group I mGluR agonists, expression of the NR2C subunit was prevented by treating the cells with specific antisense oligodeoxynucleotide (ODN). The capability of the mGluR agonists, trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD, 100 microM) or 3 hydroxyphenylglycine (3HPG, 100 microM), and the protein kinase C (PKC) activator, 4beta-phorbol-12,13-dibutyrate (PDBu, 1 microM), to inhibit the function of resultant NMDA receptors was then evaluated. We found that depletion of the NR2C subunit abolished the inhibitory effect of group I mGluR stimulation on glutamate-induced [Ca2+]i rise and neurotoxicity. The antisense ODN treatment also prevented the inhibitory effect of PDBu on glutamate responses. Conversely, in NR2C-lacking neurons, both group I mGluRs and PKC stimulation enhanced NMDA receptor-mediated effects. The present findings indicate that the capability of PKC-associated mGluRs to modulate native NMDA receptor function relies on the heteromeric configuration of the receptor-channel complex. Particularly, expression of the NR2C subunit is required to make the NMDA receptor sensitive to inhibitory modulation by mGluRs or PKC activation.

Animals↗
PubMed1996

Interaction between A1 adenosine and class II metabotropic glutamate receptors in the regulation of purine and glutamate release from rat hippocampal slices.

Electrical stimulation of rat hippocampal slices evoked the release of excitatory amino acids and purines, as reflected by a time-dependent increase in the extracellular levels of glutamate and adenosine, as well as by the increased efflux of radioactivity in slices preloaded with both [14C]glutamate and [3H]adenosine. The evoked release of excitatory amino acids and purines was amplified when slices were exposed to 8-cyclopentyl-1,3-dipropylxanthine (a selective A1 adenosine receptor antagonist), (+)-alpha-methyl-4-carboxyphenylglycine [a mixed antagonist of metabotropic glutamate receptors (mGluRs)], or (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (a selective antagonist of class II mGluRs). In contrast, 2-chloro-N6-cyclopentyladenosine (CCPA; a selective A1 receptor agonist) or (2S,1R,2R,3R)-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; a selective agonist of class II mGluRs) reduced the evoked release of excitatory amino acids and purines. CCPA and DCG-IV also reduced the increase in cyclic AMP formation induced by either forskolin or electrical stimulation in hippocampal slices. The inhibitory effect of CCPA and DCG-IV on release or cyclic AMP formation was less than additive. We conclude that the evoked release of excitatory amino acids and purines is under an inhibitory control by A1 receptors and class II mGluRs, i.e., mGluR2 or 3, which appear to operate through a common transduction pathway. In addition, although these receptors are activated by endogenous adenosine and glutamate, they can still respond to pharmacological agonists. This provides a rationale for the use of A1 or class II mGluR agonists as neuroprotective agents in experimental models of excitotoxic neuronal degeneration.

Adenosine↗
PubMed2001

Alpha 1-adrenergic modulation of metabotropic glutamate receptor-induced calcium oscillations and glutamate release in astrocytes.

Astrocytic responses to activation of metabotropic glutamate receptors group I (mGluRs I) and alpha(1)-adrenoreceptors in cultured cells have been assessed using spectral analyzes and calcium imaging. Concentration-dependent changes were observed after stimulation with the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG). These responses changed from a regular low frequency signal with sharp peaks at 1 microm to a pronounced stage of irregularity at 10 microm. After stimulation with 100 microm the signal was again homogenous in shape and regularity but occurred at a higher frequency. In contrast, the spectral properties after stimulation with the alpha(1)-adrenoreceptor agonist phenylephrine, exhibited considerable variation for all investigated concentrations. DHPG-induced increases in [Ca(2+)](i) were also associated with astroglial glutamate release, whereas no release was observed after noradrenergic stimulation. Both DHPG-mediated calcium signaling and glutamate release were inhibited by preincubation with 10 or 100 microm phenylephrine. Collectively, the present investigation provides new information about the spatial-temporal characteristics of astroglial intracellular calcium responses and demonstrates distinct differences between noradrenergic and glutamatergic receptors regarding intracellular calcium signaling and coupling to glutamate release. The noradrenergic modulation of DHPG-induced responses indicates that intracellular astroglial processes can be regulated in a bi-directional feedback loop between closely connected astrocytes and neurons in the central nervous system.

Animals↗
PubMed2003

Divergent evolution in metabotropic glutamate receptors. A new receptor activated by an endogenous ligand different from glutamate in insects.

The metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors involved in the regulation of glutamatergic synapses. Surprisingly, the evolution-arily distant Drosophila mGluR shares a very similar pharmacological profile with its mammalian orthologues (mGlu2R and mGlu3R). Such a conservation in ligand recognition indicates a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs and suggests that structural constraints within the ligand binding pocket (LBP) would hinder divergent evolution. Here we report the identification of a new receptor homologous to mGluRs found in Anopheles gambiae, Apis mellifera, and Drosophila melanogaster genomes and called AmXR, HBmXR, and DmXR, respectively (the mXRs group). Sequence comparison associated with three-dimensional modeling of the LBP revealed that the residues contacting the amino acid moiety of glutamate (the alpha-COO(-) and NH(3)(+) groups) were conserved in mXRs, whereas the residues interacting with the gamma-carboxylic group were not. This suggested that the mXRs evolved to recognize an amino acid different from glutamate. The Drosophila cDNA encoding DmXR was isolated and found to be insensitive to glutamate or any other standard amino acid. However, a chimeric receptor with the heptahelical and intracellular domains of DmXR coupled to G-protein. We found that the DmX receptor was activated by a ligand containing an amino group, which was extracted from Drosophila head and from other insects (Anopheles and Schistocerca). No orthologue of mXR could be detected in Caenorhabditis elegans or human genomes. These data indicate that the LBP of the mGluRs has diverged in insects to recognize a new ligand.

Animals↗
PubMedDate not supplied

Glutamate concentration in whole saliva and taste responses to monosodium glutamate in humans.

It is universally accepted that saliva plays an important role in taste sensations. However, interactions between constituents of whole saliva and the five basic taste modalities are still poorly understood. The aim of the present study was to evaluate possible relationship between endogenous glutamate (Glu) levels in whole saliva and taste responses to a prototypic umami substance, monosodium glutamate (MSG; 0.03-10.0%). Rated intensity and pleasantness of MSG taste was studied in healthy volunteers divided into a high glutamate (HG) in saliva (HG; n = 19) and low glutamate in saliva (LG; n = 18) group based on the median split level of salivary Glu. The HG and LG group did not differ in terms of electrogustometric thresholds, rated intensity of the MSG samples and pleasantness of distilled water and the lower MSG concentrations (0.03-1.0%). Perceived intensity of water taste was significantly (P < 0.05) higher in the LG subjects. The LG group rated the higher MSG concentrations (3.0-10.0%) as more unpleasant (P < 0.01). The difference remained significant after controlling for a between-group difference in age. The present results suggest that individual differences in salivary Glu levels may alter hedonic responses to suprathreshold MSG concentrations.

Adult↗
PubMed1999

Extracellular glutamate diffusion determines the occupancy of glutamate receptors at CA1 synapses in the hippocampus.

Following exocytosis at excitatory synapses in the brain, glutamate binds to several subtypes of postsynaptic receptors. The degree of occupancy of AMPA and NMDA receptors at hippocampal synapses is, however, not known. One approach to estimate receptor occupancy is to examine quantal amplitude fluctuations of postsynaptic signals in hippocampal neurons studied in vitro. The results of such experiments suggest that NMDA receptors at CA1 synapses are activated not only by glutamate released from the immediately apposed presynaptic terminals, but also by glutamate spillover from neighbouring terminals. Numerical simulations point to the extracellular diffusion coefficient as a critical parameter that determines the extent of activation of receptors positioned at different distances from the release site. We have shown that raising the viscosity of the extracellular medium can modulate the diffusion coefficient, providing an experimental tool to investigate the role of diffusion in activation of synaptic and extrasynaptic receptors. Whether intersynaptic cross-talk mediated by NMDA receptors occurs in vivo remains to be determined. The theoretical and experimental approaches described here also promise to shed light on the roles of metabotropic and kainate receptors, which often occur in an extrasynaptic distribution, and are therefore positioned to sense glutamate escaping from the synaptic cleft.

Animals↗
PubMed1988

Glutamate receptors of ganglion cells in the rabbit retina: evidence for glutamate as a bipolar cell transmitter.

1. Intracellular and extracellular recordings were obtained from ganglion cells in the rabbit retina. The effects of glutamate analogues and antagonists were studied using a perfusion method for drug application. 2. Kainate (KA) excited all ganglion cells directly and caused a large increase in firing rate. N-Methyl-DL-aspartate (NMDLA) also excited ganglion cells but it was less potent and caused burst firing. 3. Quisqualate (QQ) and (RS)-2-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) excited many ganglion cells and were approximately as potent as KA. Less frequently, QQ and AMPA had inhibitory effects possibly due to polysynaptic action. 4. General glutamate antagonists such as cis-2,3-piperidine dicarboxylic acid (PDA) and kynurenic acid blocked the light input to all ganglion cells. PDA and kynurenic acid blocked the effects of KA and NMDLA, but not carbachol, indicating that they act as glutamate antagonists in the rabbit retina. Kynurenic acid did not block the excitatory action of QQ, even though light responses were abolished. 5. Amacrine cells were depolarized by KA or QQ and less potently by NMDLA. Their light-evoked responses were blocked by PDA. 6. We conclude that the light input to ganglion cells in the rabbit retina is predominantly mediated by KA receptors. This is consistent with the idea that 'on' and 'off' bipolar cells are excitatory and release glutamate.

Action Potentials↗
PubMed1992

N-alkoxycarbonyl-glutamic and aspartic acids. Studies on the activation and cyclodehydration and side-reaction encountered in analysis of glutamic acid using Fmoc-chloride.

N-Alkoxycarbonylaminodicarboxylic acids were reacted in dichloromethane with N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride, and with methyl chloroformate in the presence of N-methylmorpholine. Removal of secondary products by washing the mixtures with aqueous solutions gave good yields of the pure crystalline internal anhydrides. Anhydrides of N-benzyloxycarbonyl- (Z) and N-9-fluorenylmethoxycarbonyl-(Fmoc) L-glutamic and L-aspartic acids and of N-tert.-butoxycarbonyl-L-aspartic acid were prepared in this way. The compounds were shown to be amenable to normal phase high-performance liquid chromatography (NP-HPLC) on a CN-column using tert.-butanol-hexane as solvent. The products of the reactions of Z- and Fmoc-glutamic acid with hot acetic anhydride were examined by nuclear magnetic resonance and NP-HPLC before and after methanolysis in an attempt to establish if any of the corresponding pyroglutamates were formed. The reaction of Fmoc-chloride with Fmoc-glutamate was examined for the same reason. It is concluded that the side product generated during the reaction of Fmoc-chloride with glutamic acid which is used for analysis of the latter is the N-protected internal anhydride and not the pyroglutamate as reported in the literature.

Amino Acids↗