Gamma-diaminobutyric Acid Research Articles

Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (α-,γ-diaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms

We aimed to determine concentrations of the neurotoxic amino acids beta-N-methylamino-L-alanine (BMAA) and alpha-,gamma-diaminobutyric acid (DAB) in mixed species scum material from Dutch urban waters that suffer from cyanobacterial blooms. BMAA and DAB were analysed in scum material without derivatization by LC-MSMS (liquid chromatography coupled to tandem mass spectrometry) using hydrophilic interaction chromatography (HILIC). Our method showed high selectivity, good recovery of added compounds after sample extraction (86% for BMAA and 85% for DAB), acceptable recovery after sample hydrolysation (70% for BMAA and 56% for DAB) and acceptable precision. BMAA and DAB could be detected at an injected amount of 0.34 pmol. Free BMAA was detected in nine of the 21 sampled locations with a maximum concentration of 42 microg/g DW. Free DAB was detected in two locations with a maximum concentration of 4 microg/g DW. No protein-associated forms were detected. This study is the first to detect underivatized BMAA in cyanobacterial scum material using LC-MSMS. Ubiquity of BMAA in cyanobacteria scums of Dutch urban waters could not be confirmed, where BMAA and DAB concentrations were relatively low; however, co-occurrence with other cyanobacterial neurotoxins might pose a serious health risk including chronic effects from low-level doses.

Determination of the neurotoxin BMAA (β-N-methylamino-l-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry)

A highly specific method for the analysis of beta-N-methylamino-L-alanine (BMAA) by LC-MS/MS (liquid chromatography tandem mass spectrometry) has been developed and applied for cycad seeds and cyanobacteria. BMAA was analysed as a free fraction or as total BMAA after acidic hydrolysis to release any protein-bound BMAA. Deuterium labelled BMAA was synthesised and used as internal standard. The method comprises HILIC (hydrophilic interaction chromatography) and positive electrospray ionisation of the native compound, i.e. no derivatisation was used. For safe identification five specific product ions (m/z 102, 88, 76, 73 and 44), all derived from a precursor ion of m/z 119 and originating from different parts of the molecule, were detected (typical relative abundance 100%, 16%, 14%, 12% and 22% respectively). Cyanobacteria or muscle tissue was spiked with BMAA (10 to 1000 microg g(-1)) to validate the method (accuracy 95% to 109%, relative standard deviation 1% to 6%). The detection limit for free and total BMAA in tissue was <1 microg g(-1) and <4 microg g(-1) respectively. BMAA was successfully identified and quantified in cycad seeds, whereas previously reported findings of BMAA in samples of cyanobacteria could not be confirmed. Instead, the presence of alpha-,gamma-diamino butyric acid (DAB), an isomer of BMAA, was confirmed in one sample. The possible implications of this finding are discussed.

Gram-Negative Outer and Inner Membrane Models: Insertion of Cyclic Cationic Lipopeptides

Most Gram-negative bacteria are susceptible to polymyxin B (PxB), and development of resistance to this cationic lipopeptide is very rare. PxB mechanism of action involves interaction with both the outer membrane (OM) and the inner membrane (IM) of bacteria. For the design of new antibiotics based on the structure of PxB and with improved therapeutic indexes, it is essential to establish the key features of PxB that are important for activity. We have used an approach based on mimicking the outer layers of the OM and the IM of Gram-negative bacteria using monolayers of lipopolysaccharide (LPS) or anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), respectively, and using a combination of penetration assay, analysis of pressure/area curves, and Brewster angle microscopy to monitor surface morphology changes. Synthetic analogue sp-B maintains the basic structural characteristics of the natural compound and interacts with the OM and the IM in a similar way. Analogue sp-C, with a mutation of the sequence [d-Phe6-Leu7] into [d-Phe6-Dab7], shows that this hydrophobic domain is involved in LPS binding. The significant role of the positive charges is demonstrated with sp-Dap analogue, where l-alpha,gamma-diaminobutyric acid residues Dab1 and Dab8 are replaced by l-alpha,gamma-diaminopropionic acid (Dap), resulting in lower degrees of insertion in both LPS and PG monolayers. The importance of the N-terminal acyl chain is demonstrated with polymyxin B nonapeptide (PxB-np). PxB-np shows lower affinity for LPS compared to PxB, sp-B, or sp-C, but it does not insert into PG monolayers, although it binds superficially to the anionic film. Since PxB microbial killing appears to be mediated by osmotic instability due to OM-IM phospholipid exchange, the ability of the different peptides to induce membrane-membrane lipid exchange has been studied by use of phospholipid unilamellar vesicles. Results indicate that cationic amphipathicity determines peptide activity.

Facile synthesis of Nalpha-protected-L-alpha,gamma-diaminobutyric acids mediated by polymer-supported hypervalent iodine reagent in water.

Hofmann rearrangement of Nalpha-Boc-L-Gln-OH mediated by a polymer-supported hypervalent iodine reagent poly[(4-diacetoxyiodo)styrene] (PSDIB) in water afforded Nalpha-Boc-L-alpha,gamma-diaminobutyric acid (Boc-Dab-OH, 1) in 87% yield. Nalpha-Z-derivative (Z-Dab-OH, 2) was prepared with PSDIB in 83% yield. Since the reaction of Nalpha-Fmoc-Gln-OH by this procedure did not proceed because of the insolubility of Fmoc-Gln-OH in aqueous media, we synthesized Fmoc-Dab(Boc)-OH (5) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution-phase synthesis.

Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata.

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.

N-acyl-(alpha, gamma diaminobutyric acid)n hydrazide as an efficient gene transfer vector in mammalian cells in culture.

This study investigates the structure/activity relationship of a series of N-acyl-peptides (lipopeptides) for the transfection of mammalian cells. Lipopeptides comprising 1 to 3 basic amino-acids and a single fatty acid chain were synthesized. Transfecting complexes between lipopeptide, plasmid DNA and dioleoyl phosphatidylethanolamine were prepared and applied on cells in culture. Transfection efficiency was evaluated by measuring beta-galactosidase activity 48 h post-transfection. Lipopeptide-DNA binding was also investigated by physical means and molecular modelling. Besides the length of the fatty acid chain, the nature of the basic amino-acid and the C-terminal group were crucial parameters for high transfection efficiency. The N-acyl-(diaminobutyric acid)n derivatives were the most potent transfecting agents among those tested and induced a beta-galactosidase activity 2 to 20 times higher than the N-acyl-lysine, -ornithine or -diaminopropionic acid derivatives. Furthermore, a hydrazide C-terminal modification greatly enhanced transfection efficiency for all compounds tested. The reason why alpha, gamma-diaminobutyric acid hydrazide-based lipopeptides were the most potent in transfection is not fully understood but could be related to their high DNA binding. Poly- or oligo-diaminobutyric acid containing or not a hydrazide C-terminus could advantageously be used in peptide-based gene delivery systems.

Peptide nucleic acid (PNA): a model structure for the primordial genetic material?

It is proposed that the primordial genetic material could have been peptide nucleic acids, i.e., DNA analogues having a peptide backbone. PNA monomers based on the amino acid, alpha, gamma-diaminobutyric acid or ornithine are suggested as compounds that could have been formed in the prebiotic soup. Finally, the possibility of a PNA/RNA world is presented, in which PNA constitutes the stable genetic material, while RNA which may be polymerized using the PNA as template accounts for enzymatic activities including PNA replication.

Conformation‐Activity relationships of cyclic dermorphin analogues

A theoretical conformational analysis (molecular mechanics study) of nine cyclic tetrapeptides, structurally related to the highly mu-receptor-selective dermorphin analogue H-Tyr-D-Orn-Phe-Asp-NH2, was performed. These compounds display considerable diversity in their mu-receptor affinity and selectivity. A systematic search and subsequent energy minimization in absence of the exocyclic Tyr1 residue and Phe3 side chain revealed the constrained nature of the 11-13-membered ring structures contained in these analogues. No more than four low-energy conformers (within 2 kcal/mol of the lowest energy conformation) were found in each case. After attachment of the Tyr1 moiety and Phe3 side chain to the "bare" low-energy ring structures, a systematic search and energy minimization of these exocyclic moieties resulted in a limited number of low-energy conformational families for all compounds. Five analogues with high mu-receptor affinity--H-Tyr-D-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-Phe-D-Asp-NH2, H-Tyr-D-Asp-Phe-Orn-NH2, H-Tyr-D-Asp-Phe-A2bu-NH2 (A2 bu: alpha, gamma-diaminobutyric acid) and H-Tyr-D-Cys-Phe-Cys-NH2--all showed a tilted stacking interaction between the Tyr1 and Phe3 aromatic rings in the lowest or second lowest energy conformation found. The same kind of stacking was not possible in low-energy conformers of the four analogues with poor affinity for the mu-receptor [H-Tyr-L-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-D-Phe-Asp-NH2, H-Tyr-D-Orn-Phe(NMe)-Asp-NH2 [Phe(NMe): N alpha-methylphenylalanine], and H-Tyr-D-Orn-Phg-Asp-NH2 (Phg: phenylglycine)].(ABSTRACT TRUNCATED AT 250 WORDS)

Enkephalin analogues containing β‐naphthylalanine at the fourth position

To examine the importance of the aromatic side chains of enkephalin on opiate activity, we report the synthesis and conformational analysis of a series of analogues related to enkephalin with beta-naphthylalanine in place of phenylalanine at the fourth position. Three linear analogues (Tyr-D-Ala-Gly-(L and D)-beta Nal(1)-Leu-NH2 and Tyr-D-Ala-Gly-beta Nal(2)-Leu-NH2) were initially synthesized to examine the effect of the substitution on biological activity. The increased activity of these peptides at the mu-opiate receptor, compared to native Leu-enkephalin, prompted us to examine the more conformational constrained analogues, Tyr-c[D-A2bu-Gly-(L and D)-beta Nal(1)-Leu], incorporating a alpha, gamma-diaminobutyric acid at the second position and cyclization to the carboxylic end of the leucine. These two cyclic analogues provide insight into the necessity for the L chirality of the aromatic residue at position 4. The Tyr-c[D-A2bu-Gly-L-beta Nal(1)-Leu] analogue is highly potent and displays a slight preference for the mu receptor. The conformational analysis indicates that despite the high flexibility of the tyrosine side chain, the aromatic rings of the tyrosine and naphthylalanine are relatively distant from each other. The presence of two intramolecular hydrogen bonds help maintain the conformation of the 14-membered backbone ring that keeps the side chains directed away from each other. These findings are in agreement with our model of an extended structure required for mu selectivity and a folded form with close aromatic ring placement for delta selectivity.

ChemInform Abstract: SYNTHESIS AND PHARMACOLOGICAL CHARACTERIZATION IN VITRO OF CYCLIC ENKEPHALIN ANALOGS: EFFECT OF CONFORMATIONAL CONSTRAINTS ON OPIATE RECEPTOR SELECTIVITY

AbstractGemäß Formelschema werden die Verbindungen (IV) dargestellt.

Synthesis and pharmacological characterization in vitro of cyclic enkephalin analogues: effect of conformational constraints on opiate receptor selectivity.

Using a combination of solid-phase and solution methods, we synthesized a series of cyclic [Leu5]enkephalin analogues by substitution of D-alpha, omega-diamino acids in position 2 of the enkephalin sequence and cyclization of the omega-amino group to the C-terminal carboxy group of leucine. Cyclic analogues containing D-alpha, beta-diaminopropionic acid (1), D-alpha, gamma-diaminobutyric acid (2), D-ornithine (3), or D-lysine (4) in position 2 and the [D-Leu5] and [des-Leu5] analogues of 4 (5 and 6) showed, in general, high potency in the guinea pig ileum (GPI) assay and low potency in the mouse vas deferens (MVD) assay. IC50 (MVD)/IC50 (GPI) ratios ranging from 3.1 to 29.4 were obtained, indicating the preference of the cyclic analogues for mu receptors over delta receptors. With two exceptions, preferential affinity for mu receptors is reflected in the Ki ratios determined in parallel binding assays using [3H]naloxone and [3H] [D-Ala2, D-Leu5]enkephalin as mu and delta receptor selective radioligands, respectively. Comparison of the pharmacological profiles of the cyclic analogues 1-4 with those of their corresponding open-chain analogues, [D-Ala2, Leu5]enkephalinamide (1a), [D-Abu2, Leu5]enkephalinamide (2a), [D-Nva2, Leu5]enkephalinamide (3a), and [D-Nle2, Leu5]enkephalinamide (4a), revealed that the pronounced mu character of compounds 1-4 is a direct consequence of the conformational constraints introduced by cyclization. This finding is in agreement with the concept of different conformational requirements of mu- and delta-opiate receptors and raises the possibility of manipulating opiate receptor selectivity by varying the type and degree of conformational restriction.

Iodinated [A1-N gamma-(4-azidophenylacetyl)-D-alpha, gamma-diaminobutyric acid]insulin: its preparation and properties.

The synthesis of a photo-sensitive analogue of pork insulin (A1-N gamma-(4-azidophenylacetyl)-D-alpha, gamma-diaminobutyric acid]insulin is reported. It could be shown that this analogue forms specific cross-links to binding proteins on irradiation with UV light. Furthermore, it was shown that the photo-label itself was not iodinated, nor did it influence the normal distribution of iodination. It can be concluded that this derivative bears all of the characteristics necessary for specifically labeling membrane-bound receptors.

A cyclic enkephalin analog with high in vitro opiate activity.

A conformationally restricted analog of [Leu5]enkephalin was synthesized by cyclization of the COOH-terminal carboxyl group of leucine to the gamma-amino moiety of alpha, gamma-diaminobutyric acid (A2bu) substituted in position 2 of the peptide. Relative to [Leu5]enkephalin, the cyclic analog with D configuration in position 2, H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-), was 17.5 times more potent in the guinea pig ileum assay and twice as potent in the rat brain receptor binding assay, whereas its diastereomer H-Tyr-cyclo(-N gamma-L-A2bu-Gly-Phe-Leu-) showed low activity. The cyclic D isomer was also slightly more active than the open-chain reference compound [D-Nva2, Leu5]enkephalinamide in both assays, and it proved to be highly resistant to degradation by brain "enkephalinases." The steric constraints introduced in H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-) were shown to prevent the realization of most of the conformational features ascribed to linear enkephalin in solution or in the crystalline state and permitted an assessment of proposed models of the conformation of enkephalin when it is bound to the receptor.

Electric birefringence of ionized polypeptides in solution and the effect of high electric fields on the helix-coil transition

Electric birefringence and circular dichroism measurements have been made on solutions of two po!y (L-lysine) homologs. The specific Kerr constant and the molar ellipticity at 222 nm of poly (L-alpha, gamma-diaminobutyric acid hydrochloride) in methanol/water mixtures underwent an abrupt change between 75 and 80 vol% methanol at 25 degrees C, corresponding to a solvent-induced helix-coil transition. On the helix side of the transition region, i.e., between 78 and 80 vol% methanol, anomalous birefringence transients indicative of field-induced helix-to-coil transition were observed at high fields. In the case of poly (L-ornithine hydrobromide) in methanol/water mixtures, a helix-coil transition was induced between 93 and 98 vol% methanol and anomalous birefringence transients were observed between 96 and 98 vol% methanol. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for various solvent compositions and polymer concentrations could be superimposed on one another by horizontal and vertical shifts, except for the range where anomalous birefringence transients were observed. This enabled us to estimate the threshold field strength.

SYNTHESIS AND BIOLOGICAL ACTIVITIES OF TWO ACTH‐ANALOGUES CONTAINING L‐NORARGININE IN POSITION 8

Two new ACTH-analogues, an octadecapeptide amide and a tetracosapeptide containing the lower homologue of arginine (norarginine) in position 8, have been synthesized by the generally accepted method. Special attention was paid to the synthesis of the required tetrapeptide representing the 7-10 sequence, which was obtained either by direct introduction of L-nitronorarginine or by amidination of the gamma-amino function in a protected peptide containing alpha, gamma-diaminobutyric acid. Biological activity determination showed that the shortening e arginine side chain in position 8 results in the formation of active ACTH-analogues.

Interaction of a 5-trans-vinylcarboxylic acid analogue of pyridoxal 5'-phosphate with apoaspartate aminotransferase. Covalent labeling of the enzyme.

The 5-trans-vinylcarboxylic acid analogue of pyridoxal 5'-phosphate has been prepared. Its pKa values were determined as 3.08, 4.10, and 7.33. The third pKa, that of the pyridinium nitrogen, is considerably lower than that of 8.2 observed for the corresponding saturated compound, 5'-carboxymethyl-5'-deoxypyridoxal. Absorption spectra of individual ionic forms have been resolved into component bands using lognormal distribution curves. The vinylcarboxylic acid analogue inactivates apoaspartate aminotransferase slowly at pH 8.3. An initial product absorbs at 26 kK (385 nm) and is converted slowly to a species with a narrow absorption band at 24.0 kK (417 nm). Meanwhile, the circular dichroism in the same region changes from positive to negative. At pH 5.2 the product abosrbs at 25.2 kK (397 nm). The 24.0-kK (417 nm) form is not reducible with sodium borohydride and the tightly bound chromophore is not released from the protein during denaturation by acid, base, or heat. L-Glutamate and erythro-beta-hydroxyaspartate both facilitate the formation of the 24.0-kK form. The reaction of the analogue with apoenzyme in the presence of erythro-beta-hydroxyaspartate is also accompanied by transient peaks, presumably representing quinonoid forms, at 19.0 kK (526 nm) and 20.3 kK (492 nm). The analogue reacts at basic pH with arginine, alpha-amino-gamma-guanidinobutyric acid, ornithine, cysteine, alpha, gamma-diaminobutyric acid, eh narrow absorption bands centered in the 24.0-24.4-kK (417-410 nm) region and resembling the product formed with the apoenzyme. Nuclear magnetic resonance and absorption spectroscopy indicate that the reaction with alpha- gamma-diaminobutyric acid proceeds via a hexahydropyrimidine derivative to a substituted tetrahydropyrimidine (a cyclic Schiff base) which is the final product. A similar reaction sequence with the apoenzyme is postulated and a structure with an unknown X group from the enzyme replacing the gamma-amino group of alpha, gamma-diaminobutyric acid is proposed for the 24.0-kK (417 nm) chromophore obtained with the apoenzyme. The proposed reactions are closely related to enzymatic and nonenzymatic reactions of pyridoxal 5'-sulfate (Yang, I. -Y., Khomutov, R. M., and Metzler, D. E. (1974), Biochemistry 13, 3877).

Regulation of Betacyanin Efflux from Beet Root by Poly-L-Lysine, Ca-Ion and Other Substances

Poly-l-lysine, poly-alpha, gamma-diaminobutyric acid and basic proteins cause efflux of betacyanin from beet root tissues to varying degrees. Membrane activities fall in the order: polylysine > poly-alpha, gamma-diaminobutyric acid > polyarginine (protamine), suggesting the importance of steric factors in side-chain to backbone relations. It was also observed that homopolymer activity > heteropolymer activity, using ribonuclease and lysozyme as examples of the latter. Among polylysines, there appears to be an optimal chain length at a molecular weight equal to 50,000. Lowered activity of larger polymers is interpreted in terms of a diffusion barrier, the cell wall.Polylysine and Ca(++) exhibit competitive kinetics, and Ca(++) otherwise is far more active than other cations. It is assumed that polylysine displaces Ca(++) from anionic centers on the membrane, but cannot confer equivalent dimensional stability, rendering the membrane leaky. The possible role of cationic shielding in ionic stabilization of the membrane was also considered. The order of divalent ion activity against polylysine was Ca(++) > Sr(++) > Mg(++), suggesting again a specific size-fit relationship.

Isolation and identification of a neuroactive factor from Lathyrus latifolius.

A neuroactive principle of Lathyrus latifolius has been isolated in crystalline form as a monohydrochloride and has been identified as L-alpha,gamma-diaminobutyric acid. This amino acid has also been found in very high concentration in the toxic seed of Lathyrus sylvestris Wagneri. A structural relationship between this factor, the lathyrus factor from Lathyrus odoratus, and, beta-cyano-L-alanine, a new, synthetic neuroactive amino acid nitrile, is pointed out, and a possible biosynthetic pathway relating these is indicated.