Syntheses and conformational analyses of glutamate analogs: 2-(2-carboxy-3-substituted-cyclopropyl)glycines as useful probes for excitatory amino acid receptors.

An hypothesis that each subtype of glutamate receptors requires a specific conformation of L-glutamate for its selective activation was examined using the conformationally constrained analogs of L-glutamate, L-2-(2-carboxycyclopropyl)glycines (CCGs), and L-2-[2-carboxy-3-(methoxymethyl)cyclopropyl)glycines (MCGs). All MCG isomers were newly synthesized in a stereoselective manner via the common synthetic intermediate 5a starting with the oxazolidine aldehyde 1. The synthesis of the four MCG isomers was characterized by a stereoselective inversion of alpha-cyclopropyl acyl anion (e.g., from 10 to 11). The spectroscopic studies, in particular, pH vs J correlation experiments of CCGs and MCGs using 1H NMR and their molecular mechanics calculations, revealed that these analogs possessed an antiperiplanar conformation regarding the H-C2-C1'-H bond as a majority among the other possible rotamers in aqueous solution. The fact that each CCG and MCG exhibited potent and selective activities to the distinct types of glutamate receptors allowed us to extract an active conformation of L-glutamate. Thus, the conformational requirement of metabotropic glutamate receptors was speculated to be the anti-anti conformation (aa-A) because the conformations of CCG-1 and cis and trans-MCG-I, selective agonists of the receptors, closely mimicked the rotamer A of L-glutamate. On the other hand, N-methyl-D-aspartate and kainate receptors, representative ionotropic glutamate receptors, would require glutamate g+g+ rotamer E which was deduced from the conformation-activity relationship studies of the selective agonists CCG-IV, cis-MCG-IV, and trans-MCG-IV and the related analogs.

Potent NMDA-like actions and potentiation of glutamate responses by conformational variants of a glutamate analogue in the rat spinal cord.

1. Neuropharmacological actions of all possible-state isomers of alpha-(carboxycyclopropyl)glycine (CCG), conformationally restricted analogues of glutamate, were examined for electrophysiological effects in the isolated spinal cord of the newborn rat. 2. Eight CCG stereoisomers demonstrated a large variety of depolarizing activities. Among them, the (2R, 3S, 4S) isomers of CCG (D-CCG-II) showed the most potent depolarizing activity, followed by the (2S, 3R, 4S) isomer (L-CCG-IV). 3. The depolarization evoked by L-CCG-IV, D-CCG-II and other D-CCG isomers was effectively depressed by N-methyl-D-aspartate (NMDA) antagonists. D-CCG-II was about 5 times more potent than NMDA in causing a depolarization. 4. The (2S, 3S, 4S) isomer of CCG (L-CCG-I) was more potent than L-glutamate in causing a depolarization of spinal motoneurones. The depolarization was slightly depressed by NMDA antagonists, but residual amplitudes of responses to L-CCG-I in the presence of NMDA antagonists We almost insensitive to 6,7-dinitro-quinoxaline-2,3-dione (DNQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that L-CCG-I might be a novel potent agonist. 5. After application of the (2S, 3S, 4R) isomer of CCG (L-CCG-III), responses to L-glutamate, D- and L-aspartate were markedly enhanced. The enhancement lasted for a period of several hours without a further application of L-CCG-III. 6. L-CCG-III also caused a depolarization, but it seemed unlikely that the potentiation of the glutamate response was directly related to the depolarization evoked by L-CCG-III. 7. The potentiation might be due to inhibition of uptake processes, but L-CCG-III was superior to L-(-)-threo-3-hydroxyaspartate, a potent uptake inhibitor of L-glutamate and L-aspartate, in enhancing the response to L-glutamate in terms of amplitude and duration of responses. 8. CCG isomers should provide useful pharmacological tools for analysis of glutamate neurotransmitter systems.

A novel metabotropic glutamate receptor agonist: marked depression of monosynaptic excitation in the newborn rat isolated spinal cord.

1. Neuropharmacological actions of a novel metabotropic glutamate receptor agonist, (2S,1'R,2'R,3'R)-2(2,3-dicarboxycyclopropyl)glycine (DCG-IV), were examined in the isolated spinal cord of the newborn rat, and compared with those of the established agonists of (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD). 2. At concentrations higher than 10 microM, DCG-IV caused a depolarization which was completely blocked by selective N-methyl-D-aspartate (NMDA) antagonists. The depolarization was pharmacologically quite different from that caused by L-CCG-I and (1S,3R)-ACPD. 3. DCG-IV reduced the monosynaptic excitation of motoneurones rather than polysynaptic discharges in the nanomolar range without causing postsynaptic depolarization of motoneurones. DCG-IV was more effective than L-CCG-I, (1S,3R)-ACPD or L-2-amino-4-phosphonobutanoic acid (L-AP4) in reducing the monosynaptic excitation of motoneurones. 4. DCG-IV (30 nM-1 microM) did not depress the depolarization induced by known excitatory amino acids in the newborn rat motoneurones, but depressed the baseline fluctuation of the potential derived from ventral roots. Therefore, DCG-IV seems to reduce preferentially transmitter release from primary afferent nerve terminals. 5. Depression of monosynaptic excitation caused by DCG-IV was not affected by any known pharmacological agents, including 2-amino-3-phosphonopropanoic acid (AP3), diazepam, 2-hydroxysaclofen, picrotoxin and strychnine. 6. DCG-IV has the potential of providing further useful information on the physiological function of metabotropic glutamate receptors.

Agonist analysis of 2-(carboxycyclopropyl)glycine isomers for cloned metabotropic glutamate receptor subtypes expressed in Chinese hamster ovary cells.

1. 2-(Carboxycyclopropyl)glycines (CCGs) are conformationally restricted glutamate analogues and consist of eight isomers including L- and D-forms. The agonist potencies and selectivities of these compounds for metabotropic glutamate receptors (mGluRs) were studied by examining their effects on the signal transduction of representative mGluR1, mGluR2 and mGluR4 subtypes in Chinese hamster ovary cells expressing the individual cloned receptors. 2. Two extended isomers of L-CCG, L-CCG-I and L-CCG-II, effectively stimulated phosphatidylinositol hydrolysis in mGluR1-expressing cells. The rank order of potencies of these compounds was L-glutamate > L-CCG-I > L-CCG-II. 3. L-CCG-I and L-CCG-II were effective in inhibiting the forskolin-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in mGluR2-expressing cells. Particularly, L-CCG-I was a potent agonist for mGluR2 with an EC50 value of 3 x 10(-7) M, which was more than an order of potency greater than that of L-glutamate. 4. L-CCG-I evoked an inhibition of the forskolin-stimulated cyclic AMP production characteristic of mGluR4 with a potency comparable to L-glutamate. 5. In contrast to the above compounds, the other CCG isomers showed no appreciable effects on the signal transduction involved in the three mGluR subtypes. 6. This investigation demonstrates not only the importance of a particular isomeric structure of CCGs in the interaction with the mGluRs but also a clear receptor subtype specificity for the CCG-receptor interaction, and indicates that the CCG isomers would serve as useful agonists for investigation of functions of the mGluR family.

Effects of glutamic acid analogues on identifiable giant neurones, sensitive to beta-hydroxy-L-glutamic acid, of an African giant snail (Achatina fulica Férussac).

The effects of the seven glutamic acid analogues, alpha-kainic acid, alpha-allo-kainic acid, domoic acid, erythro-L-tricholomic acid, DL-ibotenic acid, L-quisqualic acid and allo-gamma-hydroxy-L-glutamic acid were examined on six identifiable giant neurones of an African giant snail (Achatina fulica Férussac). The neurones studied were: PON (periodically oscillating neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone), RAPN (right anterior pallial neurone), FAN (frequently autoactive neurone) and v-RCDN (ventral-right cerebral distinct neurone). Of these, d-RPLN and RAPN were excited by the two isomers (erythro- and threo-) of beta-hydroxy-L-glutamic acid (L-BHGA), whereas PON, VIN, FAN and v-RCDN were inhibited. L-Glutamic acid (L-Glu) had virtually no effect on these neurones. alpha-Kainic acid and domoic acid showed marked excitatory effects, similar to those of L-BHGA, on d-RPLN and RAPN. Their effective potency quotients (EPQs), relative to the more effective isomer of L-BHGA were: 0.3 for both substances on d-RPLN, and 1 for alpha-kainic acid and 3-1 for domoic acid on RAPN. alpha-Kainic acid also had excitatory effects on FAN and v-RCDN (EPQ for both: 0.3), which were inhibited by L-BHGA but excited by gamma-aminobutyric acid (GABA). Erythro-L-tricholomic acid showed marked effects, similar to those of L-BHGA, on VIN (EPQ: 0.3) and RAPN (EPQ: 3-1), but produced weaker effects on PON and d-RPLN (EPQ: 0.1). DL-Ibotenic acid produced marked effects, similar to those of L-BHGA, on PON, VIN (EPQ for both: 1) and RAPN (EPQ: 1-0.3), but had weak effects on d-RPLN (EPQ: less than 0.1) and FAN (EPQ: 0.1). It had excitatory effects on v-RCDN (EPQ: 0.1). This neurone was inhibited by L-BHGA but excited by GABA. L-Quisqualic acid showed the same effects as L-BHGA on all of the neurones examined (EPQ range 30-0.1). It was the most potent of the compounds tested on RAPN (EPQ: 30-10), FAN (EPQ: 30) and v-RCDN (EPQ: 3). alpha-Allo-kainic acid and allo-gamma-hydroxy-L-glutamic acid had no obvious effect on any of the neurones examined. As described above, the responses of the neurones examined to these substances varied widely. However, L-quisqualic acid generally had effects on the neurones similar to those of L-BHGA; the L-BHGA-excited neurones were also excited by alpha-kainic acid and domoic acid.

A conformationally restricted analogue of L-glutamate, the (2S,3R,4S) isomer of L-alpha-(carboxycyclopropyl)glycine, activates the NMDA-type receptor more markedly than NMDA in the isolated rat spinal cord.

Depolarizing actions of 4 conformationally restricted L-glutamate analogues, (2S,3S,4S) isomer (L-CCG-I), (2S,3R,4R) isomer (L-CCG-II), (2S,3S,4R) isomer (L-CCG-III) and (2S,3R,4S) isomer (L-CCG-IV) of L-alpha-(carboxycyclopropyl)-glycine (L-CCG), were investigated in the isolated rat spinal cord by extracellular recordings of potential changes of motoneurones from the ventral roots, in order to study the interaction between the conformation of glutamate and its receptor subtype. The order of the depolarizing activity was quisqualate greater than L-CCG-IV = kainate greater than NMDA greater than L-CCG-I greater than L-CCG-III greater than L-CCG-II. The depolarization caused by L-CCG-IV was effectively blocked by the NMDA antagonists and Mg2+ ions, while the L-CCG-I response was not affected by these blockers. These results suggest that the NMDA-type receptor is activated by a folded form of L-glutamate.

Changes in preference for receptor subtypes of configurational variants of a glutamate analog: conversion from the NMDA-type to the non-NMDA type.

The (2S,3R,4S) isomer of 2-(carboxycyclopropyl)glycines (CCG) (L-CCG-IV) is a potent NMDA-type agonist in the mammalian central nervous system. L-CCG-IV is a conformationally restricted glutamate analogue in which the cyclopropyl group fixes the glutamate chain, and closely mimics the folded conformation of L-glutamate. (6R)-Substituted L-CCG-IV, however, demonstrated pharmacological properties of non-NMDA type agonists in the newborn rat spinal motoneuron while (6S)-CCG derivatives showed similar properties to the parent compound, L-CCG-IV. In the dorsal root fiber of newborn rats, (6R)-methoxymethyl and benzyloxymethyl substituted L-CCG-IV caused kainate-like depolarization. The depolarizing potency of (6R)-benzyloxymethyl substituted L-CCG-IV was slightly lower than that of kainate, demonstrating a relatively high potency.

A potent metabotropic glutamate receptor agonist: electrophysiological actions of a conformationally restricted glutamate analogue in the rat spinal cord and Xenopus oocytes.

The (2S,3S,4S) isomer of alpha-(carboxycyclopropyl)glycine (L-CCG-I), a conformationally restricted glutamate analogue, caused a marked depolarization of motoneurons in the isolated rat spinal cord, which was almost insensitive to CPP and CNQX. Depolarizing responses to L-CCG-I were markedly decreased by reducing the temperature of the bathing fluid. Similar results were obtained in the case of trans-ACPD, which is a metabotropic glutamate receptor agonist, but the depolarizing action of L-CCG-I was more potent than that of trans-ACPD. In Xenopus oocytes injected with poly(A)+ mRNA extracted from the rat brain, L-CCG-I induced significant oscillatory chloride currents, suggesting that L-CCG-I is a potent agonist for metabotropic-type glutamate receptors.

2-(Carboxycyclopropyl)glycines: binding, neurotoxicity and induction of intracellular free Ca2+ increase.

The excitatory actions of the eight stereoisomers of 2-(carboxycyclopropyl)glycine (CCG), conformationally rigid glutamate analogues, were analyzed for the glutamate receptor subtypes by means of binding assays with rat brain membranes. All CCG isomers inhibited the binding of [3H]3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid ([3H]CPP) to N-methyl-D-aspartate (NMDA) receptors. The (2S,3R,4S) isomer (L-CCG-IV) was the most potent agonist for the NMDA receptor and its binding potency was 17- and 790-fold higher than that of L-glutamate and NMDA, respectively. The (2S,3S,4R) isomer (L-CCG-III) showed a potent inhibitory activity for [3H]D-aspartate uptake. Further, L-CCG-IV caused a marked increase of intracellular free Ca2+ concentration [( Ca2+]i) and potent neurotoxicity in the single rat cerebral cortical neurons in vitro, and both were blocked effectively by the NMDA antagonists. Significant correlations were observed between neurotoxicity and the increase of [Ca2+]i and [3H]CPP binding affinity to the NMDA receptor.

Classification of GABA receptors in snail neurones.

The present study aimed to elucidate the pharmacological features of GABA receptors on identifiable neurones of Achatina fulica Férussac by testing the effects of GABA analogues, muscimol, (+/-)-baclofen, (-)-beta-hydroxy GABA and those conformationally fixed in either the extended or folded form of carbon chain, such as trans- and cis-isomers of (+/-)-2-(aminomethyl)cyclopropane-1-carboxylic acid [+/-)-cyclo-GABA-extended and (+/-)-cyclo-GABA-folded) and trans-4-amino-crotonic acid (GABA-extended). The giant neurones used were TAN, d-LPeLN, v-VNAN, v-LCDN and RPeNLN. The minimum effective concentrations (MEC) of these compounds to produce hyper- or depolarization of the membrane potentials of the neurones were determined, and the effective potency quotient (EPQ) of each compound vis-à-vis that of GABA was calculated for each neurone. The GABA receptors in these neurones were classified into the muscimol I, muscimol II and baclofen types. The muscimol I (TAN and d-LPeLN) and muscimol II (v-VNAN and v-LCDN) receptors were respectively hyperpolarized and depolarized by GABA and muscimol but were insensitive to (+/-)-baclofen. These muscimol receptors are inferred to accept GABA in an extended form of its carbon chain, since muscimol, conformationally fixed in this form from C-1 to C-4, was quite effective. Muscimol was more potent on the muscimol II receptors (MEC: 3 X 10(-7)-3 X 10(-6) M; EPQ: 30-10) than on the muscimol I type (MEC: 3 X 10(-5)-10(-4) M; EPQ: 1-0.3).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of alpha-kainic acid, domoic acid and their derivatives on a molluscan giant neuron sensitive to beta-hydroxy-L-glutamic acid.

No identifiable giant neuron of an African giant snail (Achatina fulica Férussac) was found to be sensitive to L-glutamic acid (L-Glu). However, some of them, including the RAPN (right anterior pallial neuron), were sensitive to beta-hydroxy-L-glutamic acid (L-BHGA). The effects on RAPN of alpha-kainic acid (alpha-KA), domoic acid (DMA) and their derivatives, which are structurally related to L-Glu and L-BHGA, were examined in order to elucidate the chemical structures necessary to produce the effects under study. Erythro- and threo-L-BHGA showed similar excitatory effects on RAPN (the minimal effective concentration (M.E.C.): 10(-4)M). alpha-KA, alpha-KA methylketone and DMA also showed marked excitatory effects on the same neuron (M.E.C.: 3 X 10(-5)-10(-4)M). The two derivatives of alpha-KA, (2S, 3S, 4S)-2-carboxy-4-(1-methyl-5(R)-hydroxymethyl-l(Z),3(Z)-hexadienyl) pyrrolidine-3-acetic acid and (2S,3S,4S)-2-carboxy-4-(1-methyl-5(R)-hydroxymethyl-1(Z),3(E)-hexadie nyl) pyrrolidine-3-acetic acid, also had excitatory effects (M.E.C.: about 3 X 10(-4)M) which were somewhat less potent than those of L-BHGA, alpha-KA etc. However, alpha-allo-KA, alpha-allo-KA methylketone and dihydro-alpha-KA even at a high concentration of 4.7 X 10(-4)M, had no effect on RAPN. From the results obtained, it appears likely that the seven excitatory compounds mentioned above act on the common receptors of the RAPN neuromembrane, due to similarities in their structures and in the type of excitation caused by these compounds.

Effects of compounds related to beta-hydroxyglutamic acid (BHGA) on identifiable giant neurones of an African giant snail (Achatina fulica Férussac).

The present study aimed to further elucidate the pharmacological features, with respect to sensitivity to L-BHGA agonists, of the receptors sensitive to beta-hydroxy-L-glutamic acid (L-BHGA) in five Achatina giant neurones: PON (periodically oscillating neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone), RAPN (right anterior pallial neurone) and v-RCDN (ventral-right cerebral distinct neurone). Of these neurones, d-RPLN and RAPN were depolarized by L-BHGA, while PON, VIN and v-RCDN were inhibited. Threo-beta-hydroxy-DL-aspartic acid markedly depolarized d-RPLN and RAPN (effective potency quotient (EPQ) in relation to the more effective L-BHGA isomer: 1 for d-RPLN and 0.3 for RAPN). This compound produced only slight inhibitory effects on PON, VIN and v-RCDN with EPQs calculated to be less than 0.03, less than 0.03 and 0.03, respectively. On the other hand, erythro-beta-hydroxy-DL-aspartic acid at 10(-3) M was almost ineffective, except on v-RCDN where it elicited some slight inhibitory effects (EPQ: 0.01). L-Aspartic and D-aspartic acid at 10(-3) M, also had almost no effect except for slight effects of D-aspartic acid on d-RPLN (EPQ: 0.1). N-Methyl-L- and N-methyl-D-aspartic acid were slightly effective only on v-RCDN (EPQ: less than 0.01 and 0.01, respectively). The other compounds, including beta-hydroxypyrroglutamic acid (cyclic BHGA) and proline derivatives, were almost ineffective at 10(-3) M; very weak effects were occasionally observed on some neurones.(ABSTRACT TRUNCATED AT 250 WORDS)

Chirality transferring [3,3] sigmatropic rearrangement of (1-Acyloxy-2-alkenyl)trianlkylsilane synthesis of optically active vinylsilane-containing alpha-amino acid.

A vinylsilane-containing alpha-amino acid and alpha,alpha-disubstituted alpha-amino acid 2 having two contiguous asymmetric carbon centers at their alpha and beta positions were synthesized in an optically active form by ester-enolate Claisen rearrangement of the alpha-acyloxysilane 1 as the key step, where the chirality of an alpha-acyloxy-TBDMS group was completely transferred to the rearranged product.

Synthesis and paralytic activities of squaryl amino acid-containing polyamine toxins.

Eight analogs 4A-7A and 4B-7B of philanthotoxin (PhTX) from wasp venom and nephilatoxin-8 (NPTX-8) from spider venom whose tyrosine or asparagine linker is replaced by squaryl (sq) amino acid or 4-amino squaryl (4-asq) amino acid have been synthesized in an efficient manner via coupling of N-acyl squaryl amino acid intermediate 19 or 26 with the corresponding polyamine part. Preliminary bioassay using crickets revealed that the analogs substituted by glutamate-type squaryl amino acid-containing NPTX 7A and 7B showed more potent paralytic activities than that of NPTX-8.

Syntheses of potent Leu-enkephalin analogs possessing beta-hydroxy-alpha,alpha-disubstituted-alpha-amino acid and their characterization to opioid receptors.

Novel Leu-enkephalin (Leu-Enk) (1) analogs possessing various types of alpha-substituted serine instead of its glycine residue in the position 2 were synthesized via an efficient O,N-migration method. The binding characteristics of the synthetic analogs using Chinese hamster ovary (CHO) cells expressed cloned rat mu-, delta-, and kappa-receptors revealed that [(1R,2S)-Ahh2]Enk (7) was the most potent agonist of delta-opioid receptors among all the synthetic analogs tested, and was 10 times more potent than the native Leu-Enk.

Antimutagenic unusual amino acids from plants.

Five unusual amino acids were identified as antimutagens against spontaneous mutation of Salmonella typhimurium TA100: L-azetidine-2-carboxylic acid (1) from Liliaceae plants, alpha-(methylenecyclopropyl)glycine (2) from Litchi chinensis seeds, and 2-amino-4-methylhex-5-ynoic acid (3), hypoglycin A (4), and (2S,4R)-2-amino-4-hydroxyhept-6-ynoic acid (5) from Euphoria longana seeds. The absolute stereochemistry of 5 was determined by its chiral synthesis from L-allylglycine, proving that 5 is the C-4 epimer of the amino acid previously isolated from dried longan seeds.