Midazolam and theN-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP-7) attenuate stress-induced expression of c-fos mRNA in the dentate gyrus

Summary 1. The effects of restraint stress on c-fos mRNA expression in the dentate gyrus were investigated byin situ hybridization.2. Confirming previous findings, c-fos mRNA expression increased after 30 min of forced restraint.3. This effect was attenuated by a previous i.c.v. injection of the anxiolytic benzodiazepine midazolam (20 nmol/2 µl) or theN-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP-7; 5 nmol/2 µl).4. These results suggest that the dentate gyrus is activated during restraint stress and that this activation may be modulated by benzodiazepine -aminobutyric acid_A (GABA_A) or NMDA receptors.     

Enantioselective actions of 4-amino-3-hydroxybutanoic acid and (3-amino-2-hydroxypropyl)methylphosphinic acid at recombinant GABA(C) receptors

The R- and S-enantiomers of 4-amino-3-hydroxybutanoic acid (GABOB) were full agonists at human recombinant rho1 GABA(C) receptors. Their enantioselectivity (R>S) matched that reported for their agonist actions at GABA(B) receptors, but was the opposite to that reported at GABA(A) receptors (S>R). The corresponding methylphosphinic acid analogues proved to be rho1 GABA(C) receptor antagonists with R(+)-CGP44533 being more potent than S(-)-CGP44532, thus showing the opposite enantioselectivity to the agonists R(-)- and S(+)-GABOB. These studies highlight the different stereochemical requirements for the hydroxy group in these analogues at GABA(A), GABA(B) and GABA(C) receptors.     

Distribution of ciliatine (2-aminoethylphosphonic acid) and phosphonoalanine (2-amino-3-phosphonopropionic acid) in human tissues

Ciliatine (2-aminoethylphosphonic acid) was detected in the human brain, heart, kidney, liver, intestine, spleen, adrenal glands, and aorta. Phosphonoalanine (2-amino-3-phosphonopropionic acid) was found in the human liver, intestine and spleen. Tissue homogenates were extracted with trichloroacetic acid and a chloroform-methanol mixture. After hydrolysis, each fraction was subfractionated by ion-exchange chromatography and examined by paper chromatography and electrophoresis using a specific ninhydrin-molybdate staining procedure to detect the phosphonic acids. The acids were found bound either to lipid or to protein; no free phosphonic acid was detected.     

2-amino-4-acetylaminobutyric acid, 2,4-diaminobutyric acid and 2-amino-6N-oxalylureidopropionic acid (oxalylalbizziine) in seeds of acacia angustissima

A new amino acid previously detected in 17 species of Acacia has been isolated from seeds of Acacia angustissima and identified as oxalylalbizziine. These seeds also contain more than 6% dry weight of 2-amino-4-acetylaminobutyric acid, which has not been reported previously in a legume, and lower concentrations of 2,4-diaminobutyric acid.     

Poly(2-amino-8-methyladenylic acid). Competing structural and energetic effects of substituents

The ribopolynucleotide poly(2-amino-8-methyladenylic acid), (r2NH2(8)MeA)n, has been synthesized, and its physical and chemical properties have been examined. The study reveals competing effects on these properties of the 2-NH2 and 8-Me substituents. In marked contrast to the analogous (r8MeA)n, the new polymer readily interacts to form double helixes with complementary pyrimidine polynucleotides. Triple helixes are not formed. The 8-Me group is strongly destabilizing for helix formation (delta Tm approximately 65 degrees C), presumably by favoring a syn conformation, which blocks heteroduplex formation with ribohomopolymers. The 2-NH2 substituent stabilizes helixes in the ribo series by about 30 degrees C in Tm by forming a third interbase hydrogen bond. We suggest that the free energy from the 2-NH2 interaction drives the syn-anti equilibrium to the purine polymer to the anti form present in the double helix. CD spectra of the homopolymers (r2NH2A)n and (r2NH2(8)MeA)n are completely different, reflecting major differences of conformation. The double helixes formed by these polymers with (rT)n and (rBrU)n, on the other hand, have closely similar CD spectra, supporting our proposal of a major change in conformation of (2NH2(8)MeA)n on going from single strand to double helix.     

Poly(2-amino-8-methyldeoxyadenylic acid): contrasting effects in deoxy- and ribopolynucleotides of 2-amino and 8-methyl substituents

Poly(2-amino-8-methyldeoxyadenylic acid) interacts readily with pyrimidine polynucleotides to form double helices only slightly less stable than those in which the purine polymer lacks the 8-Me group. In the ribo series, by contrast, complexes formed with poly(2-amino-8-methyladenylic acid) are very strongly destabilized by the 8-Me group, despite a larger stabilizing effect of the 2-NH2 group in the ribo series. These results are interpreted in terms of a smaller steric interference of the 8-Me group with 2'-CH2 than with 2'-CHOH, leading to a smaller population of syn structures in the deoxy chain and a consequent lower interference with homopolymer duplex formation. UV, circular dichroism (CD), and IR spectra of the new polymer and its complexes are reported and related to structural and energetic characteristics of the molecules. Since direct synthesis of 2-amino-8-methyldeoxyadenosine was not feasible, the corresponding riboside was prepared, the 3'- and 5'-positions were protected with a disilyloxy group, and a 2'-[(imidazol-1-yl)thiocarbonyl] group was introduced. Reduction with tributyltin hydride followed by deprotection gave the nucleoside, which was then converted to the triphosphate by standard methods. The homopolymer was prepared with terminal deoxynucleotidyl transferase.     

(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] but not (-)-3-PPP produces (+)-N-allylnormetazocine-like (SKF 10,047) discriminative stimuli

In rats trained to discriminate the prototypic sigma receptor agonist, (+)-N-Allylnormetazocine [(+)-N-Allylnormetazocine [(+)-NANM/SKF 10,047], from saline, the (+)- but not the (-)-isomer of 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) produced (+)-NANM-like discriminative stimuli. (+)-3-PPP binds stereo selectively to the (+)-NANM binding site, but not to the phencyclidine binding site. Additionally, phencyclidine was found to produce (+)-NANM-like discriminative stimuli. Although the 3-PPP isomers were shown to produce changes in central dopaminergic activity (Hjorth et al. Life Sci 37, 673, 1985), the discriminative stimulus properties of (+)-3-PPP are apparently not mediated via the dopaminergic system. This hypothesis is supported by the fact that apomorphine did not produce (+)-NANM-like discriminative stimuli. These stimuli are thus non-dopaminergic and may be due to the (+)-3-PPP actions at the sigma binding site. However, it is possible that (+)-NANM, PCP, and (+)-3-PPP may have common non-sigma pharmacologic properties that account for the similar discriminative stimulus properties of these compounds.     

Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

Synthesis of AZT analogues: 7-(3-azido-2-hydroxypropyl)-, 7-(3-amino-2-hydroxypropyl)-,7-(3-triazolyl-2-hydroxypropyl)theophylline

Nucleophilic displacement of the tosyloxy group in 7-(2-hydroxy-3-p-toluenesulfonyloxypropyl)theophylline (1) with azide anion afforded 7-(3-azido-2-hydroxypropyl)theophylline (2). Reduction of the 3-azido group in 2 with Ph3P/Py/NH4OH afforded the 3-amino derivative 4, alternatively obtained by regioselective amination of 7-(2,3-epoxypropyl)theophylline (3). Selective acetylation of 4 gave the N-acetyl derivative 5. 1,3-Dipolar cycloaddition of the azide group in 2 with N1-propargyl thymine (6) afforded the regioisomeric triazole 7.     

Synthesis of AZT analogues: 7-(3-azido-2hydroxypropyl)-, 7-(3-amino-2-hydroxypropyl)-, 7-(3-triazolyl-2-hydroxypropyl)theophyllines

Nucleophilic displacement of the tosyloxy group in 7-(2-hydroxy-3-p-toluenesulfonyloxypropyl)theophylline (1) with azide anion afforded 7-(3-azido-2-hydroxypropyl)theophylline (2). Reduction of the 3-azido group in 2 with Ph₃P/Py/NH₄OH afforded the 3-amino derivative 4, alternatively obtained by regioselective amination of 7-(2,3-epoxypropyl)theophylline (3). Selective acetylation of 4 gave the N-acetyl derivative 5. 1,3-Dipolar cycloaddition of the azide group in 2 with N¹-propargyl thymine (6) afforded the regioisomeric triazole 7.     

Oxidative stress in vitiligo: photo-oxidation of pterins produces H(2)O(2) and pterin-6-carboxylic acid

Patients with vitiligo accumulate millimolar levels of H(2)O(2) in their epidermis. The recycling process of (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin in these patients is disrupted due to deactivation of 4a-OH-BH(4) dehydratase by H(2)O(2). The H(2)O(2) oxidation products 6- and 7-biopterin lead to the characteristic fluorescence of the affected skin upon Wood's light examination (UVA 351 nm). Here we report for the first time the presence and accumulation of pterin-6-carboxylic acid (P-6-COOH) in the epidermis of these patients. Exploring potential sources for P-6-COOH revealed that sepiapterin and 6-biopterin are readily photo-oxidised to P-6-COOH by UVA/UVB irradiation. Photolysis of sepiapterin and 6-biopterin produces stoichiometric H(2)O(2) under aerobic conditions, where O(2) is the electron acceptor, thus identifying an additional source for H(2)O(2) generation in vitiligo. A detailed analysis utilising UV/visible spectrophotometry, HPLC, TLC, and mass spectroscopy showed for sepiapterin direct oxidation to P-6-COOH, whereas 6-biopterin formed the intermediate 6-formylpterin (P-6-CHO) which is then further photo-oxidised to P-6-COOH.     

E-2(S)-amino-3-methyl-3-pentenoic acid from coniogramme intermedia

A new amino acid, E-2(S)-amino-3-methyl-3-pentenoic acid was isolated from Coniogramme intermedia. The structure was elucidated by elementary analysis, optical rotation, catalytic hydrogenation, ¹H and ¹³C NMR spectra.     

Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

Immunomodulating effects of tofizopam (Grandaxin) and diazepam in vitro

Benzodiazepines (BDZs) are known to act not only in the central nervous system, but on peripheral cells and tissues binding to the peripheral-type benzodiazepine receptors. In the present study, the influence of two different BDZs (diazepam (Dz) and tofizopam (Tof) on several immune functions has been examined in vitro. Some differences between Dz and Tof in their effects on human lymphocyte proliferative response, changes in glucocorticoid-induced suppression of cell proliferation and influence on cytokine production (tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2)) have been determined. Dz suppressed mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation, enhanced dexamethasone-induced inhibition of PBMC proliferative response, and suppressed lymphocyte production of TNF-alpha and IL-2. Tof usually enhanced PBMC proliferation and IL-2 production in low and moderate doses, but in high doses it suppressed both. Tof in all investigated doses enhanced dexamethasone-induced suppression of lymphocyte proliferation and depressed TNF-alpha production. Thus, both Dz and Tof are shown to have immunomodulating effects in vitro. Tof, opposite to Dz even in the therapeutic doses, is able to enhance in vitro mitogen-induced lymphocyte proliferation and IL-2 production.     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

2-O-(beta-D-glucuronyl)-7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose: a constituent of the Bordetella pertussis endotoxin.

The presence of bound D-glucuronic acid in the endotoxin of Bordetella pertussis was demonstrated. The branched chain trisaccharide named in the title was isolated after hydrolysis of the endotoxin with 3 M HCl for 2 h at 100 degrees C. Its structure was established by chemical and enzymic degradation.     

Cobalt(III) promoted ligand fusion reactions of thiobarbituric acid and 4,6-diamino-2-thiouracil (or 4-amino-2-thiouracil)

Novel cobalt(III) complexes containing three kinds of assembled ligands, L₁L₂=dapymt-tbba(2-), tbba-dapymt(1-) and apymt-tbba(1-) (H₃tbba=thiobarbituric acid; Hdapymt=4,6-diamino-2-thiouracil; Hapymt=4-amino-2-thiouracil), were prepared from the mixed ligand systems, where L₂ indicates the coordinated ligand to the Co(III) ion and L₁ is a pendant ligand bonded to L₂. These complexes were characterized by UV-Vis absorption spectra and NMR spectroscopy. The crystal structures of [Co(Htbba)(en)₂]ClO₄·2H₂O (2) (en=ethane-1,2-diamine), [Co{dapymt-tbba(2-)}(en)₂]ClO₄·3H₂O (3) and [Co{apymt-tbba(1-)}(en)₂](ClO₄)Cl·3H₂O (5′) revealed that coordination occurs through the S(1) and N(1) donors of tbba and the latter complexes 3 and 5′ have an assembled ligand; a new bond is formed between the C(5) atom of tbba and the S(2) atom of dapymt or apymt. An intramolecular hydrogen bond between O(1) of tbba and NH of en was found in all crystals. An interesting intermolecular π-π stacking interaction was found in 5′.