The ability of carbon nanoparticles to increase transmembrane current of cations coincides with impaired synaptic neurotransmission.

Here, carbon nanodots synthesized from ß-alanine (Ala-CDs) and detonation nanodiamonds (NDs) were assessed using (1) radiolabeled excitatory neurotransmitters L-[14C]glutamate, D-[2,33H]aspartate, and inhibitory ones [3H]GABA, [3H]glycine for registration of their extracellular concentrations in rat cortex nerve terminals; (2) the fluorescent ratiometric probe NR12S and pH-sensitive probe acridine orange for registration of the membrane lipid order and synaptic vesicle acidification, respectively; (3) suspended bilayer lipid membrane (BLM) to monitor changes in transmembrane current. In nerve terminals, Ala-CDs and NDs increased the extracellular concentrations of neurotransmitters and decreased acidification of synaptic vesicles, whereas have not changed sufficiently the lipid order of membrane. Both nanoparticles, Ala-CDs and NDs, were capable of increasing the conductance of the BLM by inducing stable potential-dependent cation-selective pores. Introduction of divalent cations, Zn2+ or Cd2+ on the particles` application side (cis-side) increased the rate of Ala-CDs pore-formation in the BLM. The application of positive potential (+100 mV) to the cis-chamber with Ala-CDs or NDs also activated the insertion as compared with the negative potential (-100 mV). The Ala-CD pores exhibited a wide-range distribution of conductances between 10 and 60 pS and consecutive increase in conductance of each major peak by ~10 pS, which suggest the clustering of the same basic ion-conductive structure. NDs also formed ion-conductive pores ranging from 6 pS to 60 pS with the major peak of conductance at ~12 pS in cholesterol-containing membrane. Observed Ala-CDs and NDs-induced increase in transmembrane current coincides with disturbance of excitatory and inhibitory neurotransmitter transport in nerve terminals.

Preparation and preliminary quality evaluation of aspirin/L-glutamate compound pellets.

L-glutamate is an important component of protein. It can prevent gastrointestinal damage caused by NSAIDs. We constructed two-phase enteric-coated granules of aspirin and L-glutamate compound by extrusion spheronization method and fluidized bed coating. The subliminal effective dose of L-glutamate is 100 mg/kg tested by model of gastric ulcer of rats induced by aspirin and drug administration. HPLC-UV and UV-Vis methods were adopted to determine content and cumulative release of aspirin and L-glutamate as quality analysis method indexes. The prescription and process optimization were carried out with yield, sphericity and dissolution. The two-phase compound granules have good sphericity of 0.93 ± 0.05 (aspirin pellets) and 0.94 ± 0.02 (L-glutamate pellets), content of salicylic acid (0.24 ± 0.03)%, dissolution of aspirin (2.36 ± 0.11)%. Quality evaluation and preliminary stability meet the commercial requirements. The stored environment of compound preparation should be sealed in a cool and dark place.

Poly(L-Glutamic Acid)-Based Brush Copolymers: Fabrication, Self-assembly, and Evaluation as Efficient Nanocarriers for Cationic Protein Drug Delivery.

Protein drugs were considered to be the first choice to treat many human diseases, but their clinical application was usually limited by their short half-life and lack of validated targeted therapy. Here, a series of folate-functionalized poly(ethylene glycol)-b-(poly(2-aminoethyl-L-glutamate)-g-poly(L-glutamic acid))s (FA-PEG-b-(PELG-g-PLGA)s) were designed as tumor-targeted carriers for cationic protein delivery. Compared with traditional copolymers consisting of PEG and linear charged hydrophilic blocks, FA-PEG-b-(PELG-g-PLGA) with brush-like polyelectrolyte segments were beneficial to improving their electrostatic interactions with loading protein molecules, thus increasing drug-loading stability and protecting encapsulated proteins from degradation. The designed polymer brushes could efficiently encapsulate cytochrome C (CytC), a cationic model protein, to form polyion complex (PIC) micelles with an average particle size of approximately 200 nm. An in vitro drug release study showed that the drug-loading stability of the formed PIC micelles was largely improved. The functionalization of the block copolymer carriers with a targeting folate group enhanced the tumor cell growth inhibition and total apoptotic rates induced by CytC. Our results shed light on the unique advantages of brush-like polymer carriers in delivering cationic proteins, and the poly(L-glutamic acid)-based linear-brush diblock copolymers could be applied as a versatile delivery platform for molecular targeting in cancer therapy.

Functionalization of ZnO Nanoparticles by Glutamic Acid and Conjugation with Thiosemicarbazide Alters Expression of Efflux Pump Genes in Multiple Drug-Resistant Staphylococcus aureus Strains.

Efflux-mediated drug resistance in bacterial strains is regarded as a major cause of drug resistance. In this study, we aimed to evaluate the expression of some major facilitator superfamily class efflux pump genes (EPGs) in the presence of ZnO nanoparticles (NPs) conjugated to thiosemicarbazide (TSC) under amine functionalization by glutamic acid (ZnO@Glu-TSC) as well as ciprofloxacin (CIP) among multiple drug-resistant Staphylococcus aureus. Synthesized NPs were characterized by ultraviolet-visible spectroscopy, X-ray diffraction pattern, and transmission electron microscopy. Antibiogram and ethidium bromide agar cartwheel method were used to determine the efflux-mediated multidrug-resistant phenotype of clinical strains. Then, expression of EPGs, including norA, norB, norC, and tet38 among the strains, exposed to ZnO@Glu-TSC and CIP was evaluated using quantitative real-time PCR (qPCR). According to the results, the strains resistant to CIP showed minimum inhibitory concentration (MIC) values ranging from 256 to 1,024 µg/mL, while ZnO@Glu-TSC NPs showed MICs from 8 to 256 µg/mL against bacterial strains, which indicates stronger antibacterial activity of NPs (2-8-fold) compared to CIP. ZnO@Glu-TSC NPs showed a good bacterial inhibitory potential with average inhibition zones of 11, 15, and 20 mm for concentrations of 50, 100, and 150 µg/mL, respectively. Moreover, simultaneous use of ZnO@Glu-TSC NPs (1/2 MIC) in combination with CIP (1/2 MIC) significantly reduced the expression of norA, norB, norC, and tet38 by 5.4-, 3.8-, 2.1-, and 3.4-fold, respectively, compared to the CIP alone. Therefore, ZnO@Glu-TSC NPs with their potent antimicrobial effects could be used as an antimicrobial agent against S. aureus for preventive and/or therapeutic approaches.

Exploring Ferredoxin-Dependent Glutamate Synthase as an Enzymatic Bioelectrocatalyst.

Ferredoxin-dependent glutamate synthase (Fd-GltS) is reported as an enzymatic bioelectrocatalyst for the first time. By configuring mediated electrochemical interfaces with mediators of different redox potentials, we realize bioelectrosynthesis or bioelectrooxidation of glutamate with recombinant Fd-GltS from cyanobacteria. Particularly, bioelectrocatalytic oxidation of glutamate by Fd-GltS is demonstrated to be oxygen independent. This study reinforces a new catalytic option for developing enzymatic bioelectronic devices for powering, sensing or synthesis.

Improved Synthesis of Caged Glutamate and Caging Each Functional Group.

Glutamate is an excitatory neurotransmitter that controls numerous pathways in the brain. Neuroscientists make use of photoremovable protecting groups, also known as cages, to release glutamate with precise spatial and temporal control. Various cage designs have been developed and among the most effective has been the nitroindolinyl caging of glutamate. We, hereby, report an improved synthesis of one of the current leading molecules of caged glutamate, 4-carboxymethoxy-5,7-dinitroindolinyl glutamate (CDNI-Glu), which possesses efficiencies with the highest reported quantum yield of at least 0.5. We present the shortest route, to date, for the synthesis of CDNI-Glu in 4 steps, with a total reaction time of 40 h and an overall yield of 20%. We also caged glutamate at the other two functional groups, thereby, introducing two new cage designs: α-CDNI-Glu and N-CDNI-Glu. We included a study of their photocleavage properties using UV-vis, NMR, as well as a physiology experiment of a two-photon uncaging of CDNI-Glu in acute hippocampal brain slices. The newly introduced cage designs may have the potential to minimize the interference that CDNI-Glu has with the GABAA receptor. We are broadly disseminating this to enable neuroscientists to use these photoactivatable tools.

Design, synthesis and biological evaluation of glutamic acid derivatives as anti-oxidant and anti-inflammatory agents.

A series of glutamic acid derivatives was synthesized and evaluated for their antioxidant activity and stability. We found several potent and stable glutamic acid derivatives. Among them, compound 12b exhibited good in vitro activity, chemical stability and cytotoxicity. A prototype compound 12b showed an anti-inflammatory effect in LPS-stimulated RAW 264.7 cell lines and in a zebrafish model.

Selective killing of Helicobacter pylori with pH-responsive helix-coil conformation transitionable antimicrobial polypeptides.

Current clinical treatment of Helicobacter pylori infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill H. pylori under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix-coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)m-r-(PHLG-MHH)n, bearing randomly distributed negatively charged glutamic acid and positively charged poly(γ-6-N-(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against H. pylori, including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable H. pylori killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill H. pylori in the stomach while not harming the commensal bacteria/normal tissues.

Catalytic Stereoselective 1,4-Addition Reactions Using CsF on Alumina as a Solid Base: Continuous-Flow Synthesis of Glutamic Acid Derivatives.

A novel methodology using CsF⋅Al2 O3 as a highly efficient, environmentally benign, and reusable solid-base catalyst was developed to synthesize glutamic acid derivatives by stereoselective 1,4-addition of glycine derivatives to α,ß-unsaturated esters. CsF⋅Al2 O3 showed not only great selectivity toward 1,4-addtion reactions by suppressing the undesired formation of pyrrolidine derivations by [3+2] cycloadditions, but also offered high yields for the 1,4-adduct with excellent anti diastereoselectivities. The catalyst was well characterized by using XRD, 19 F MAS-NMR and 19 F NMR spectroscopy, FT-IR, CO2 -TPD, and XPS. And highly basic F from Cs3 AlF6 was identified as the most probable active basic site for the 1,4-addition reactions. Continuous-flow synthesis of 3-methyl glutamic acid derivative was successfully demonstrated by using this solid-base catalysis.

Simple and rapid radiosynthesis of N-18F-labeled glutamic acid as a hepatocellular carcinoma PET tracer.

INTRODUCTION: We have reported that N-(2-18F-fluoropropionyl)-L-glutamate (18F-FPGLU) showed good tumor-to-background contrast and 18F-FPGLU was prepared via complex multi-step reaction sequence; here, it is synthesized by a facile two-step reaction sequence. The objectives of this study are to synthesize 18F-FPGLU via a two-step reaction sequence and to evaluate the value of 18F-FPGLU in nude mice bearing human hepatocellular carcinoma SMCC-7721 (HCC SMCC-7721). METHODS: 18F-FPGLU was synthetized from the precursor (2S)-dimethyl 2-(2-bromopropanamido)pentanedioate via the two-step on-column hydrolysis using a modified commercial FDG synthesizer. To investigate the transport mechanism of 18F-FPGLU, we conducted a series of competitive inhibition experiments on HCC SMCC-7721 cells in the absence or presence of Na+ and various types of inhibitors. Small-animal PET-CT imaging was performed on tumor-bearing nude mice using 18F-FPGLU and 2-18F-2-deoxy-D-glucose (18F-FDG). RESULTS: The radiochemical yield of 18F-FPGLU was up to 15±5% (EOS, n=10) in 35min with the two-step procedure and the radiochemical purity was higher than 95% with a specific activity of 30-40GBq/µmol. In vitro cell experiments show that 18F-FPGLU is primarily transported through the Na+-dependent system XAG- and Na+-independent system XC-. PET imaging in a tumor model indicates that 18F-FPGLU may be superior to 18F-FDG for hepatocellular carcinoma (HCC) imaging. CONCLUSION: An optimized route to prepare 18F-FPGLU was developed and 18F-FPGLU was synthetized from the precursor ((2S)-dimethyl 2-(2-bromopropanamido)pentanedioate) via the two-step on-column hydrolysis. 18F-FPGLU was a potential novel PET tracer for HCC imaging.

MechanoAPI-ILs: Pharmaceutical Ionic Liquids Obtained through Mechanochemical Synthesis.

An alternative, efficient, and green synthetic strategy for the preparation of pharmaceutical ionic liquids using mechanochemistry (MechanoAPI-ILs) is reported. Six new API-ILs based on gabapentin and l-glutamic acid were successfully synthesized and characterized, demonstrating that mechanochemistry is a very promising synthetic strategy. Results compare both the new and the classical approach and clearly show the advantages of the new method. This new technique is faster, solvent free, reproducible, selective, and leads to higher yields.

Synthesis and pharmacological evaluation of conformationally constrained glutamic acid higher homologues.

Homologation of glutamic acid chain together with conformational constraint is a commonly used strategy to achieve selectivity towards different types of glutamate receptors. In the present work, starting from two potent and selective unnatural amino acids previously developed by us, we investigated the effects on the activity/selectivity profile produced by a further increase in the distance between the amino acidic moiety and the distal carboxylate group. Interestingly, the insertion of an aromatic ring as a spacer produced a low micromolar affinity NMDA ligand that might represent a lead for the development of a new class of NMDA antagonists.

Preparation and Characterization of Four Stereoisomers of Monatin.

Monatin is a naturally occurring, sweet amino acid comprising four stereoisomers due to its two asymmetric centers at C2 and C4. However, the characteristics of each stereoisomer have not yet been fully investigated. To obtain a sufficient amount of racemic monatin for optical resolution, a synthetic method was developed by modifying a possible biosynthetic pathway, i.e., a cross-aldol reaction and subsequent transamination. The key intermediate, 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid, was obtained via the cross-aldol reaction of pyruvic acid and indole-3-pyruvic acid. Subsequently, the carbonyl group was converted to a hydroxyimino group through reaction with hydroxylamine and then to an amino group via hydrogenation to produce monatin. Next, the racemic monatin was divided into mixtures of two pairs of enantiomers through recrystallization. Finally, both enantiomers of the N-carbobenzoxy-γ-lactone derivatives of monatin were separated by preparative HPLC and deprotected. It was found that all optically pure stereoisomers exhibited a sweet taste. The isomer that displayed the most intense sweetness was the (2R,4R)-isomer, as determined by single crystal X-ray structure analysis of the monatin potassium salt, whereas the least sweet isomer was the (2S,4S)-isomer, which demonstrated a far lower sweetness than was previously reported.

Concise Synthesis of (2R,4R)-Monatin.

Monatin, 4-hydroxy-4-(3-indolylmethyl)-glutamic acid, is a naturally occurring sweet amino acid. The (2R,4R)-monatin isomer has been found to be the sweetest among its four stereoisomers. A concise and efficient synthesis of (2R,4R)-monatin was accomplished by the alkylation of (4R)-N-tert-butoxycarbonyl (tBoc)-4-tert-butyldimethylsilyoxy-D-pyroglutamic acid methyl ester with tert-butyl 3-(bromomethyl)-1H-indole-1-carboxylate to give (4R)-N-tBoc-4-tert-butyldimethylsilyloxy-4-(N-tBoc-3-indolylmethyl)-D-pyroglutamic acid methyl ester, i.e., the lactam form of (2R,4R)-monatin with protecting groups. This was followed by the hydrolysis of the lactam ring and deprotection. The 4-hydroxyl D-pyroglutamic acid derivative was demonstrated to be a suitable precursor for the efficient preparation of (2R,4R)-monatin in high optical purity because the alkylation proceeded in regioselective and stereoselective manners at C4 to form appropriate asymmetric tetra-substituted carbon center; the resulting alkylated pyroglutamic acid derivative was then easily converted into the linear form of monatin.

Three-Component, Diastereoselective Prins-Ritter Reaction for cis-Fused 4-Amidotetrahydropyrans toward a Precursor for Possible Neuronal Receptor Ligands.

Here, we report an unprecedented, highly diastereoselective Prins-Ritter reaction of aldehydes, homoallylic alcohols, and nitriles in a three-component coupling reaction for the synthesis of tetra-cis-substituted 4-amidotetrahydropyrans. In this study, the reaction was not only applied for carbohydrate-based heterobicycles but also for more complex heterotricycles, showing acceptable levels of conversion yield (42-97% BRSM) and exclusive diastereoselectivity. Furthermore, the latter heterotricycles were converted to nine analogues of our neuronal receptor ligands IKM-159 and MC-27. An in vivo assay by intracerebroventricular injection in mice suggested that the substituent at C9 of the novel analogues interferes with the molecular interactions with the AMPA receptor, which was originally observed in the complex of IKM-159 and the GluA2 ligand binding domain. Our research has thus shown the power of a multicomponent coupling reaction for the preparation of a structurally diverse compound collection to study structure-activity relationships of biologically active small molecules.

Design and synthesis of N-benzoyl amino acid derivatives as DNA methylation inhibitors.

The inhibition of human DNA Methyl Transferases (DNMT) is a novel promising approach to address the epigenetic dysregulation of gene expression in different diseases. Inspired by the validated virtual screening hit NSC137546, a series of N-benzoyl amino acid analogues was synthesized and obtained compounds were assessed for their ability to inhibit DNMT-dependent DNA methylation in vitro. The biological screening allowed the definition of a set of preliminary structure-activity relationships and the identification of compounds promising for further development. Among the synthesized compounds, L-glutamic acid derivatives 22, 23, and 24 showed the highest ability to prevent DNA methylation in a total cell lysate. Compound 22 inhibited DNMT1 and DNMT3A activity in a concentration-dependent manner in the micromolar range. In addition, compound 22 proved to be stable in human serum and it was thus selected as a starting point for further biological studies.

Synthesis of hydrophobic cross-linked polyzwitterionic acid for simultaneous sorption of Eriochrome black T and chromium ions from binary hazardous waters.

Hydrophobic cross-linked polyzwitterionic acid (HCPZA) containing long chain (C18) hydrophobes and residues of a glutamic acid having unquenched nitrogen valency was synthesized. Exploiting the chelating ability of the amino acid residues to scavenge toxic metals and the hydrophobic surface to scoop up the organic contaminants, the resin HCPZA was evaluated for simultaneous removal of chromium and Eriochrome black T (EBT) from wastewaters. The structure and morphology of the polymer before and after sorption were characterized by using FTIR, TGA, EDX and SEM. The effect of various parameters such as contact time, pH and initial concentrations were investigated to arrive at optimum conditions. The adsorption of Eriochrome black T and Cr (III) on HCPZA reached equilibrium in 30 min. The mechanism of adsorption was investigated using kinetic, diffusion and isotherm models. The adsorption kinetic data were described well by the pseudo-second order model and by the Freundlich isotherm model. EDX analysis confirmed the adsorption of Cr (III) and EBT on the polymer. The hydrophobic resin exhibited a remarkable simultaneous adsorption capacity for EBT and Cr (III) and thus demonstrated its potential to be a promising adsorbent for removal of dyes and heavy metal ions from wastewaters.

New 4-Functionalized Glutamate Analogues Are Selective Agonists at Metabotropic Glutamate Receptor Subtype 2 or Selective Agonists at Metabotropic Glutamate Receptor Group III.

The metabotropic glutamate (Glu) receptors (mGluRs) play key roles in modulating excitatory neurotransmission in the brain. In all, eight subtypes have been identified and divided into three groups, group I (mGlu1,5), group II (mGlu2,3), and group III (mGlu4,6-8). In this article, we present a L-2,4-syn-substituted Glu analogue, 1d, which displays selective agonist activity at mGlu2 over the remaining mGluR subtypes. A modeling study and redesign of the core scaffold led to the stereoselective synthesis of four new conformationally restricted Glu analogues, 2a-d. Most interestingly, 2a retained a selective agonist activity profile at mGlu2 (EC50 in the micromolar range), whereas 2c/2d were both selective agonists at group III, subtypes mGlu4,6,8. In general, 2d was 20-fold more potent than 2c and potently activated mGlu4,6,8 in the low-mid nanomolar range.

Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.

A remarkable enhancement of selectivity towards versatile analytes by a strategically integrated H-bonding site containing phase.

A double ß-alanylated L-glutamide-derived organic phase has been newly designed and synthesized in such a way that integrated H-bonding (interaction) sites make it very suitable for the separation of versatile analytes, including shape-constrained isomers, and nonpolar, polar and basic compounds. The ß-alanine residues introduced into two long-chain alkyl group moieties provide ordered polar groups through H-bonding among the amide groups.