Potential of Ramalin and Its Derivatives for the Treatment of Alzheimer's Disease.

The pathogenesis of Alzheimer's disease (AD) is still unclear, and presently there is no cure for the disease that can be used for its treatment or to stop its progression. Here, we investigated the therapeutic potential of ramalin (isolated from the Antarctic lichen, Ramalina terebrata), which exhibits various physiological activities, in AD. Specifically, derivatives were synthesized based on the structure of ramalin, which has a strong antioxidant effect, BACE-1 inhibition activity, and anti-inflammatory effects. Therefore, ramalin and its derivatives exhibit activity against multiple targets associated with AD and can serve as potential therapeutic agents for the disease.

Highly efficient and enantioselective syntheses of (2S,3R)-3-alkyl- and alkenylglutamates from Fmoc-protected Garner's aldehyde.

A 6-step enantioselective synthesis of (2S,3R)-3-alkyl/alkenylglutamates, including the biologically significant amino acid, (2S,3R)-3-methylglutamate, protected for Fmoc SPPS, is reported. Overall yields range from 52-65%. Key to the success of these syntheses was the development of a high-yielding 2-step synthesis of Fmoc Garner's aldehyde followed by a Horner-Wadsworth-Emmons reaction to give the corresponding Fmoc Garner's enoate in a 94% yield. The diastereoselective 1,4-addition of lithium dialkylcuprates to the Fmoc Garner's enoate was explored. Significant decomposition occurred when using lithium diethylcuprate and conditions previously reported for the 1,4-addition of lithium dialkylcuprates to Boc or Cbz-protected Garner's enoate. An optimization study of this reaction resulted in a robust set of conditions that addressed the shortcomings of previously reported conditions. Under these conditions, highly diastereoselective (> 20:1 in most cases) 1,4-addition reactions of lithium dialkyl/dialkenylcuprates to the Fmoc Garner's enoate were achieved in 76-99% yield. The resulting 1,4-addition products were easily converted into the Fmoc-(2S,3R)-3-alkyl/alkenylglutamates in two steps.

Ratiometric Detection of γ-Glutamyltransferase in Human Colon Cancer Tissues Using a Two-Photon Probe.

γ-Glutamyltransferase (GGT) plays a role in cleaving the γ-glutamyl bond of glutathione. The GGT is known to be overexpressed in some tumors and has been recognized as a potential biomarker for malignant tumors. Colon cancer is one of the most common cancers worldwide; however, there is no quantitative method for detecting cancer cells in human colon tissues. In this study, we report a ratiometric two-photon probe for GGT that can be applied in human colon tissues. The probe (Probe 2) showed high fluorescence efficiency, marked fluorescence changes, excellent kinetics, and selectivity for the GGT in live colon cells. Additionally, we obtained ratiometric two-photon microscopy images of GGT activity in human colon tissue. We used this method to compare normal and cancer tissues based on their ratio values; the ratio value was higher in cancer tissue than in normal tissue. This study provides a method for quantitative analysis of GGT, particularly in human colon cancer, which will be useful for studying GGT-related diseases and diagnosing colon cancer.

A novel "AIE + ESIPT" near-infrared nanoprobe for the imaging of γ-glutamyl transpeptidase in living cells and the application in precision medicine.

γ-Glutamyl transpeptidase (GGT) has been reported as a biomarker of hepatocellular carcinoma (HCC), and its imaging is of great benefit for early detection in precise medicine as well as intraoperative navigation. Herein, we have designed and synthesized a novel near-infrared fluorescent probe coupled aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) effect for the detection of GGT. Thanks to conjugated glutamate acid, this probe could be dispersed in aqueous solution and showed barely any fluorescence emission. Through a GGT-mediated enzymatic reaction, the aggregation state of the probe in aqueous solution was changed and an intramolecular hydrogen bond was formed, resulting in an enhanced fluorescence emission. An excellent linear relationship was observed and the concentration of GGT measured was in the range of 10-90 U L-1 with a limit of detection calculated at 2.9 U L-1. Its feasibility has been confirmed by detecting GGT in HepG2 cells with high specificity and long-term sustainability, satisfying clinical need. Moreover, this nanoprobe showed great potential for precise medicine guided surgery by realizing fluorescence imaging in human liver tumour tissue and distinguishing it from normal tissue. Thus, we supposed that our AIE coupled ESIPT fluorescent nanoprobe has great potential in the early detection of HCC, the selective fluorescence imaging of GGT positive cells during surgery and application in precision medicine.

Synthesis and Structure Reassignment of Malylglutamate, a Recently Discovered Earthworm Metabolite.

Malylglutamate, a newly identified metabolite in earthworms, was synthesized using a traditional peptide coupling approach for assembling the amide from protected malate and glutamate precursors. The proposed structure (1) and a diastereomer were synthesized, but their NMR spectra did not match the natural sample. Further analysis of the natural sample using HMBC spectroscopy suggested an alternative attachment of the malyl moiety, and ß-malylglutamate (2) diastereomers were synthesized, L,L-2 and D,D-2. NMR spectra were an excellent match with the natural sample, and chiral-phase chromatography was employed to identify (-)-ß-l-malyl-l-glutamate (2) as the isomer native to Eisenia fetida.

Isolation of γ-Glutamyl-Transferase Rich-Bacteria from Mouse Gut by a Near-Infrared Fluorescent Probe with Large Stokes Shift.

Bacterial γ-glutamyltranspeptidases (γ-GT) is a well-known metabolic enzyme, which could cleave the γ-glutamyl amide bond of γ-glutamyl analogues. As a key metabolic enzyme of bacteria and a virulence factor for the host, bacterial γ-GT was determined to be a novel pharmaceutical target for new antibiotics development. However, there is no efficient method for the sensing of γ-GT activity in bacteria and the recognition of γ-glutamyltransferase rich-bacteria. In the present work, a dicyanoisophorone derivative (ADMG) has been designed and developed to be a sensitive and selective near-infrared fluorescent probe for the sensing of bacterial γ-GT. ADMG not only sensed bacterial γ-GT in vitro, but also imaged intestinal bacteria in vivo. More interesting, the intestinal bacteria existed in the duodenum section of mouse displayed significant fluorescence emission. Under the guidance of the sensing of γ-GT using ADMG, three intestinal bacteria strains K. pneumoniae CAV1042, K. pneumoniae XJRML-1, and E. faecalis were isolated successfully, which expressed the bacterial γ-GT. Therefore, the fluorescent probe ADMG not only sensed the endogenous bacterial γ-GT and imaged the intestinal bacteria but also guided the isolation of intestinal bacteria possessing γ-GT efficiently, which suggested a novel biological tool for the rapid isolation of special bacteria from a mixed sample.

Development of a Ga-68 labeled PET tracer with short linker for prostate-specific membrane antigen (PSMA) targeting.

Glu-Urea-Lys (GUL) derivatives have been reported as prostate-specific membrane antigen (PSMA) agent. We developed derivatives of GUL conjugated with NOTA or DOTA via a thiourea linker and tested their feasibility as PSMA imaging agents after labeling with 68Ga. NOTA-GUL and DOTA-GUL were synthesized and labeled with 68Ga using generator-eluted 68GaCl3 in 0.1 M HCl in the presence of 1 M NaOAc at pH 5.5. The stabilities of 68Ga-labeled compounds in human serum were tested at 37.5 °C. A competitive binding assay was performed using the PSMA-positive prostate cancer cell line 22Rv1 and [125I]MIP-1072 (PSMA-specific binding agent) as a tracer. Biodistribution and micro-PET studies were performed using 22Rv1-xenograft BALB/c nude mice. The radiolabeling efficiency of NOTA-GUL (>99%) was higher than that of DOTA-GUL (92%). The IC50 of Ga-NOTA-GUL was 18.3 nM. In the biodistribution study, tumor uptake of 68Ga-NOTA-GUL (5.40% ID/g) was higher than that of 68Ga-DOTA-GUL (4.66% ID/g) at 1 h. Tumor/muscle and tumor/blood uptake ratios of 68Ga-NOTA-GUL (31.8 and 135, respectively) were significantly higher than those of 68Ga-DOTA-GUL (16.1 and 31.1, respectively). The tumor/kidney uptake ratio of 68Ga-NOTA-GUL was 3.4-fold higher than that of 68Ga-DOTA-GUL. 68Ga-NOTA-GUL showed specific uptake to PSMA positive tumor xenograft and was blocked by co-injection of the cold ligand. In conclusion, we successfully synthesized 68Ga-NOTA-GUL and 68Ga-DOTA-GUL for prostate cancer imaging. 68Ga-NOTA-GUL showed better radiochemical and biodistribution results. 68Ga-NOTA-GUL may be a promising PSMA targeting radiopharmaceutical.

Synthesis and gelation of copolypept(o)ides with random and block structure.

Copolypept(o)ides of polysarcosine (PSar) and poly(N-isopropyl-L-glutamine) (PIGA) with random and block sequence structures were synthesized by ring-opening polymerization (ROP) of sarcosine N-carboxyanhydrides (Sar-NCA) and γ-benzyl-l-glutamate N-carboxyanhydrides (BLG-NCA) and post modification. With different distribution of Sar along the main chain, H-bonding pattern and secondary structure of polypeptides were turned, as well as aggregation and gelation behavior. Both copolypept(o)ides formed hydrogels above their critical gelation concentrations (CGCs) without thermo-sensitivity, which was normally reserved for PEG copolypeptides (eg, PEG-b-PIGA). In particular, a different mechanism from previously reported micellar percolation or fibrillar entanglement was suggested for gelation of the random copolypept(o)ide. Therefore, hydrogels from copolymers of PSar and PIGA represented a new approach to construct easy-handling, biocompatible, biodegradable and thermo-stable gels that could potentially be applied in biomedical fields.

Automated synthesis of N-(2-[18 F]Fluoropropionyl)-l-glutamic acid as an amino acid tracer for tumor imaging on a modified [18 F]FDG synthesis module.

N-(2-[18 F]Fluoropropionyl)-l-glutamic acid ([18 F]FPGLU) is a potential amino acid tracer for tumor imaging with positron emission tomography. However, due to the complicated multistep synthesis, the routine production of [18 F]FPGLU presents many challenging laboratory requirements. To simplify the synthesis process of this interesting radiopharmaceutical, an efficient automated synthesis of [18 F]FPGLU was performed on a modified commercial fluorodeoxyglucose synthesizer via a 2-step on-column hydrolysis procedure, including 18 F-fluorination and on-column hydrolysis reaction. [18 F]FPGLU was synthesized in 12 ± 2% (n = 10, uncorrected) radiochemical yield based on [18 F]fluoride using the tosylated precursor 2. The radiochemical purity was ≥98%, and the overall synthesis time was 35 minutes. To further optimize the radiosynthesis conditions of [18 F]FPGLU, a brominated precursor 3 was also used for the preparation of [18 F]FPGLU, and the improved radiochemical yield was up to 20 ± 3% (n = 10, uncorrected) in 35 minutes. Moreover, all these results were achieved using the similar on-column hydrolysis procedure on the modified fluorodeoxyglucose synthesis module.

Synthesis and Kinetic Characterisation of Water-Soluble Fluorogenic Acyl Donors for Transglutaminase†2.

Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

Development of Anionically Decorated Caged Neurotransmitters: In Vitro Comparison of 7-Nitroindolinyl- and 2-(p-Phenyl-o-nitrophenyl)propyl-Based Photochemical Probes.

Neurotransmitter uncaging, especially that of glutamate, has been used to study synaptic function for over 30 years. One limitation of caged glutamate probes is the blockade of γ-aminobutyric acid (GABA)-A receptor function. This problem comes to the fore when the probes are applied at the high concentrations required for effective two-photon photolysis. To mitigate such problems one could improve the photochemical properties of caging chromophores and/or remove receptor blockade. We show that addition of a dicarboxylate unit to the widely used 4-methoxy-7-nitroindolinyl-Glu (MNI-Glu) system reduced the off-target effects by about 50-70 %. When the same strategy was applied to an electron-rich 2-(p-Phenyl-o-nitrophenyl)propyl (PNPP) caging group, the pharmacological improvements were not as significant as in the MNI case. Finally, we used very extensive biological testing of the PNPP-caged Glu (more than 250 uncaging currents at single dendritic spines) to show that nitro-biphenyl caging chromophores have two-photon uncaging efficacies similar to that of MNI-Glu.

L-Aspartic and l-glutamic acid ester-based ProTides of anticancer nucleosides: Synthesis and antitumoral evaluation.

A series of novel aryloxyphosphoramidate nucleoside prodrugs based on l-aspartic acid and l-glutamic acid as amino acid motif has been synthesized and evaluated for antitumoral activity. Depending on the cancer cell line studied and on the nature of the parent nucleoside compound (gemcitabine, 5-iodo-2'-deoxy-uridine, floxuridine or brivudin), the corresponding ProTides are endowed with an improved or decreased cytotoxic activity.

Robust design of some selective matrix metalloproteinase-2 inhibitors over matrix metalloproteinase-9 through in silico/fragment-based lead identification and de novo lead modification: Syntheses and biological assays.

Broad range of selectivity possesses serious limitation for the development of matrix metalloproteinase-2 (MMP-2) inhibitors for clinical purposes. To develop potent and selective MMP-2 inhibitors, initially multiple molecular modeling techniques were adopted for robust design. Predictive and validated regression models (2D and 3D QSAR and ligand-based pharmacophore mapping studies) were utilized for estimating the potency whereas classification models (Bayesian and recursive partitioning analyses) were used for determining the selectivity of MMP-2 inhibitors over MMP-9. Bayesian model fingerprints were used to design selective lead molecule which was modified using structure-based de novo technique. A series of designed molecules were prepared and screened initially for inhibitions of MMP-2 and MMP-9, respectively, as these are designed followed by other MMPs to observe the broader selectivity. The best active MMP-2 inhibitor had IC50 value of 24nM whereas the best selective inhibitor (IC50=51nM) showed at least 4 times selectivity to MMP-2 against all tested MMPs. Active derivatives were non-cytotoxic against human lung carcinoma cell line-A549. At non-cytotoxic concentrations, these inhibitors reduced intracellular MMP-2 expression up to 78% and also exhibited satisfactory anti-migration and anti-invasive properties against A549 cells. Some of these active compounds may be used as adjuvant therapeutic agents in lung cancer after detailed study.

Polypeptide-b-Poly(Phenyl Isocyanide) Hybrid Rod-Rod Copolymers: One-Pot Synthesis, Self-Assembly, and Cell Imaging.

Hybrid rod-rod diblock copolymers, poly(γ-benzyl L-glutamate)-poly(4-cyano-benzoic acid 2-isopropyl-5-methyl-cyclohexyl ester) (PBLG-PPI), with determined chirality are facilely synthesized through sequential copolymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) and phenyl isocyanide monomers bearing chiral menthyl pendants using a Ni(cod)(bpy) complex as the catalyst in one-pot. Circular dichroism and absorption spectra reveal that each block of the block copolymers possesses a stable helical conformation with controlled helicity in solution due to the induction of chiral pendants. The two diastereomeric polymers self-assemble into helical nanofibrils with opposite handedness due to the different chiral induction of the L- and D-menthyl pendants, confirmed by transmission electron microscopy (TEM). Deprotection of the benzyl groups of the PBLG segment affords biocompatible amphiphilic diblock copolymers, poly(L-glutamic acid)-poly(4-cyano-benzoic acid 2-isopropyl-5-methyl-cyclohexyl ester) (PLGA-PPI), that can self-assemble into well-defined micelles by cosolvent induced aggregation. Very interestingly, a chiral rhodamine chromophores RhB(D) can be selectively encapsulated into the chiral polymeric micelles, which is efficiently internalized into living cells when directly monitored with a confocal microscope. This contribution will be useful for developing novel rod-rod biocompatible hybrid block copolymers with a controlled helicity, and may also provide unique chiral materials for potential bio-medical applications.

[Improved synthesis process of diethyl N-[4-[(2,4-diaminopyrido[3,2-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamate].

OBJECTIVE: To establish a new approach to synthesis of diethyl N-[4-[(2,4-diaminopyrido[3,2-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamate. METHODS: Target compound (5) was synthesized by the use of (2,4-dioxo-tetrahydropyridopyrimidin-6-yl)methyl acetate (1) as starting material via hydrolysis, chlorination, condensation with diethyl (p-aminobenzoyl)glutamate and aminolysis. RESULTS: A new approach to synthesis of diethyl N-[4-[(2,4-diaminopyrido[3,2-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamate was established. This synthetic route has hydrolysis reaction, chlorination, diethyl N-(p-aminobenzoyl)-L-glutamate condensation reaction and ammonolysis reaction. The total yield is 36.7%.The structures of those compounds have identified by 1H nuclear magnetic resonance, 13C nuclear magnetic resonance and mass spectrometry. This synthetic route avoid the unstable brominated reaction product and improves the harsh condition of ammonolysis reaction. CONCLUSION: The new synthetic route has improved the reaction condition and the stability of the intermediate, and increased the extent of the derivative compounds, which has great significance to anti-folic acid of anti-tumor inhibitor synthesis.

Organomediated Enantioselective (18)F†Fluorination for PET Applications.

The first organomediated asymmetric (18)F fluorination has been accomplished using a chiral imidazolidinone and [(18)F]N-fluorobenzenesulfonimide. The method provides access to enantioenriched (18)F-labeled α-fluoroaldehydes (>90% ee), which are versatile chiral (18)F synthons for the synthesis of radiotracers. The utility of this process is demonstrated with the synthesis of the PET (positron emission tomography) tracer (2S,4S)-4-[(18)F]fluoroglutamic acid.

Stereochemical assignment of the protein-protein interaction inhibitor JBIR-22 by total synthesis.

Recent reports have highlighted the biological activity associated with a subfamily of the tetramic acid class of natural products. Despite the fact that members of this subfamily act as protein-protein interaction inhibitors that are of relevance to proteasome assembly, no synthetic work has been reported. This may be due to the fact that this subfamily contains an unnatural 4,4-disubstitued glutamic acid, the synthesis of which provides a key challenge. A highly stereoselective route to a masked form of this unnatural amino acid now enabled the synthesis of two of the possible diastereomers of JBIR-22 and allowed the assignment of its relative and absolute stereochemistry.

Caged glutamates with π-extended 1,2-dihydronaphthalene chromophore: design, synthesis, two-photon absorption property, and photochemical reactivity.

Caging and photochemical uncaging of the excitatory neurotransmitter l-glutamate (glu) offers a potentially valuable tool for understanding the mechanisms of neuronal processes. Designing water-soluble caged glutamates with the appropriate two-photon absorption property is an attractive strategy to achieve this. This paper describes the design, synthesis, and photochemical reactivity of caged glutamates with π-extended 1,2-dihydronaphthalene structures, which possess a two-photon cross-section of ∼120 GM and an excellent buffer solubility (up to 115 mM). High yields up to 99% glutamate were observed in the photolysis of two caged glutamates. Suzuki-Miyaura cross-coupling and Buchwald-Hartwig amination were used as the key reactions to synthesize the caged compounds.

Peptide hydrogels assembled from nonionic alkyl-polypeptide amphiphiles prepared by ring-opening polymerization.

Three alkyl-polypeptide (AP) amphiphiles were prepared using ring-opening polymerization of α-amino acid N-carboxyanhydride. The polypeptide segment was composed of diethylene-glycol-monomethyl-ether-functionalized poly-L-glutamate (poly-L-EG(2)Glu). These AP amphiphiles can spontaneously self-assemble into transparent hydrogels in water. These hydrogels showed shear thinning properties, and their strength can be modulated by hydrophobic alkyl tails. CryoTEM and AFM characterizations suggested that these hydrogels were formed by nanoribbons arising from intermolecular interactions between nonionic poly-L-EG(2)Glu segments.

[The influence of calcium N-(5-hydroxynicotinoyl)-L-glutamate on reproductive function, prenatal and postnatal development of rats].

The safety of a new nootrope and neuroprotector--calcium N-(5-hydroxynicotinoyl)-L-glutamate (ampasse)--has been evaluated. It is shown that ampasse at a dose of 6.7 mg/kg (10 times the maximum therapeutic dose for humans) did not affect the reproductive function in experimental animals and did not produced any embryotoxic and teratogenic effects.