Chemoenzymatic routes to enantiomerically pure 2-azatyrosine and 2-, 3- and 4-pyridylalanine derivatives.

Enantiomerically pure 2-, 3- or 4-pyridylalanine (pya) and 2-azatyrosine (azatyr) are known to present various biological activities. After incorporation into appropriate peptide sequences, these heterocyclic non natural α-amino acids could behave as new substrates or inhibitors of elastase from Pseudomonas aeruginosa. This enzyme is known to be involved in nosocomial infections and infections related to the cystic fibrosis disease. New efficient chemoenzymatic preparations of those compounds using α-chymotrypsin (α-CT) are presented.

Concomitant polymorphism in the stereoselective synthesis of a ß-benzyl-ß-hydroxyaspartate analogue.

Two concomitant polymorphs, (I) and (II), of a ß-benzyl-ß-hydroxyaspartate analogue [systematic name: dibenzyl 2-benzyl-2-hydroxy-3-(4-methylphenylsulfonamido)succinate], C(32)H(31)NO(7)S, crystallize from a mixture of ethyl acetate and cyclohexane at ambient temperature. The structure of (I) has triclinic (P-1) symmetry and that of (II) monoclinic (P2(1)/c) symmetry. Both crystal structures are made up of a stacking of homochiral racemic dimers (2S,3S and 2R,3R) which are internally connected by a similar R(2)(2)(9) hydrogen-bonding pattern consisting of intermolecular N-H...O and O-H...O hydrogen bonds. The centroid of the racemic dimer lies on an inversion centre. The main structural difference between the two polymorphs is the conformational orientation of two of the four aromatic rings present in the molecule. Polymorph (II) is found to be twinned by reticular merohedry with twin index 3 and twin fractions 0.854 (1) and 0.146 (1).

threo-Diethyl 2-ethyl-2-hy-droxy-3-(4-methyl-benzene-sulfonamido)-succinate.

The asymmetric unit of the title compound, C(17)H(25)NO(7)S, contains two independent mol-ecules, which are enanti-omers forming a hydrogen-bonded dimer associated with two R(2) (2)(7) patterns. In each mol-ecule, one ethyl group from the two available ethyl ester functional groups is disordered. In one mol-ecule, the ethyl group of the ester function from an α-carb-oxy-lic acid is positionally disordered over two sets of sites with occupancies of 0.66:0.34. In the second mol-ecule, it is the ethyl group in the γ-ester function that is disordered over two sets of sites with occupancies of 0.58:0.42.

The Glutathione Metabolite γ-Glutamyl-Glutamate Partially Activates Glutamate NMDA Receptors in Central Neurons With Higher Efficacy for GluN2B-Containing Receptors.

Gamma-L-glutamyl-L-glutamate (γ-Glu-Glu) was synthetized and further characterized for its activity on cultured neurons. We observed that γ-Glu-Glu elicited excitatory effects on neurons likely by activating mainly the N-methyl-D-aspartate (NMDA) receptors. These effects were dependent on the integrity of synaptic transmission as they were blocked by tetrodotoxin (TTX). We next evaluated its activity on NMDA receptors by testing it on cells expressing these receptors. We observed that γ-Glu-Glu partially activated NMDA receptors and exhibited better efficacy for NMDA receptors containing the GluN2B subunit. Moreover, at low concentration, γ-Glu-Glu potentiated the responses of glutamate on NMDA receptors. Finally, the endogenous production of γ-Glu-Glu was measured by LC-MS on the extracellular medium of C6 rat astroglioma cells. We found that extracellular γ-Glu-Glu concentration was, to some extent, directly linked to GSH metabolism as γ-Glu-Glu can be a by-product of glutathione (GSH) breakdown after γ-glutamyl transferase action. Therefore, γ-Glu-Glu could exert excitatory effects by activating neuronal NMDA receptors when GSH production is enhanced.

Ruxolitinib before allogeneic hematopoietic transplantation in patients with myelofibrosis on behalf SFGM-TC and FIM groups.

This multicenter prospective phase 2 trial analyzed disease-free survival (DFS) in myelofibrosis patients receiving ruxolitinib for 6 months before transplantation. Seventy-six patients were recruited. Age-adjusted dynamic international prognostic scoring system was intermediate-1, intermediate-2, and high in 27 (36%), 31 (41%), and 18 (24%) patients. All patients received ruxolitinib from inclusion to conditioning regimen (fludarabine-melphalan) or to progression. A donor was found in 64 patients: 18 HLA-matched sibling donor (MSD), 32 HLA-matched unrelated (UD10/10), and 14 HLA mismatched unrelated donor (UD9/10. Among 64 patients with a donor, 20 (31%) achieved a partial response before transplantation and 59 (92%) could be transplanted after ruxolitinib therapy (18/18 MSD, 30/21 UD10/10, 11/34 UD9/10), of whom 19 (32%) were splenectomized. Overall survival from inclusion was 68% at 12 months. One-year DFS after transplantation was 55%: 83%, 40%, and 34% after MSD, UD10/10 or UD9/10, respectively. Cumulative incidence of grade 2-4 acute graft-versus-host disease (GVHD) was 66% and non-relapse-mortality was 42% at 12 months. Short course of ruxolitinib before transplantation is followed by a high rate of transplantation. With the platform used in this protocol, outcome was much better in patients transplanted with HLA-matched sibling donor as compared to unrelated donor.

Characterization of l-Theanine Excitatory Actions on Hippocampal Neurons: Toward the Generation of Novel N-Methyl-d-aspartate Receptor Modulators Based on Its Backbone.

l-Theanine (or l-γ-N-ethyl-glutamine) is the major amino acid found in Camellia sinensis. It has received much attention because of its pleiotropic physiological and pharmacological activities leading to health benefits in humans, especially. We describe here a new, easy, efficient, and environmentally friendly chemical synthesis of l-theanine and l-γ-N-propyl-Gln and their corresponding d-isomers. l-Theanine, and its derivatives obtained so far, exhibited partial coagonistic action at N-methyl-d-aspartate (NMDA) receptors, with no detectable agonist effect at other glutamate receptors, on cultured hippocampal neurons. This activity was retained on NMDA receptors expressed in Xenopus oocytes. In addition, both GluN2A and GluN2B containing NMDA receptors were equally modulated by l-theanine. The stereochemical change from l-theanine to d-theanine along with the substitution of the ethyl for a propyl moiety in the γ-N position of l- and d-theanine significantly enhanced the biological efficacy, as measured on cultured hippocampal neurons. l-Theanine structure thus represents an interesting backbone to develop novel NMDA receptor modulators.

Body weight, body composition, and energy metabolism in lean and obese Zucker rats fed soybean oil or butter.

BACKGROUND: Dietary fat composition is thought to affect body weight regulation independent of the amount of fat ingested. OBJECTIVE: We analyzed the feeding behavior, body weight gain, body composition, and energy metabolism in lean and obese rats fed a diet in which fat was in the form of either butter or soybean oil. DESIGN: Ten lean (Fa/?) and 10 obese (fa/fa) adult Zucker rats were divided into 4 groups according to a 2 x 2 experimental design. They were fed a normally balanced diet over 11 wk in which 30% of energy was either soybean oil or butter. Food intake, body weight gain, and body composition were measured. Indirect calorimetry was used to study energy metabolism at rest and in relation to feeding and activity. RESULTS: Food intake increased similarly in lean and obese rats after butter feeding. Body weight gain increased in obese rats and decreased in lean rats after butter feeding. Body weight gain in obese rats was due mainly to an increase in the weight of lean tissues besides muscle, whereas adiposity and distribution of fat between the various pads did not change. Resting metabolic rates and postprandial lipid oxidation increased in butter-fed obese rats. Lipid oxidation during exercise was not significantly different between obese and lean rats. Fat oxidation increased in butter-fed lean rats during treadmill running at moderate intensity. CONCLUSIONS: In obese rats, basal metabolism and postprandial lipid oxidation increased during butter feeding, which appeared to prevent fat accumulation in the long term. In lean rats, butter feeding favored lipid utilization by working muscles, an observation that deserves further investigation in terms of endurance and performance.

Synthesis and characterization of a cyclooctapeptide analogue of ω-agatoxin IVB enhancing the activity of Cav2.1 calcium channels activity in cultured hippocampal neurons.

The structure of the toxin ω-agatoxin IVB, extracted from the venom of funnel-web spider Agelenopsis aperta, is an important lead structure when considering the design of modulators of synaptic transmission which largely involves P/Q-type (CaV2.1) voltage gated calcium channels (VGCC) at central synapses. Focusing on the loop 2 of the ω-agatoxin IVB that seems to be the most preeminent interacting domain of the toxin with the CaV2.1 VGCC, cyclooctapeptides mimicking this loop were synthesized. While (14)Trp is essential for the binding of the neurotoxin to the CaV2.1 VGCC, the substitution of the (12)Cys for a glycidyl residue led to a cyclooctapeptide named EP14 able to enhance CaV2.1 VGCC-associated currents measured with patch-clamp recordings and to evoke ω-agatoxin IVA-sensitive intracellular Ca(2+) increase as measured by fura-2 spectrofluoroimaging. Furthermore, this cyclooctapeptide was able to potentiate spontaneous excitatory synaptic transmission in a network of cultured hippocampal neurons, consistent with the activation of presynaptic VGCC by EP14. In addition, this peptide did not affect cell survival measured with the MTT assay. Therefore, such new cyclopeptidic structures are potential good candidates for synthesis of new agents aimed at the restoration deficient excitatory synaptic transmission.

Peptide neurotoxins that affect voltage-gated calcium channels: a close-up on ω-agatoxins.

Peptide neurotoxins found in animal venoms have gained great interest in the field of neurotransmission. As they are high affinity ligands for calcium, potassium and sodium channels, they have become useful tools for studying channel structure and activity. Peptide neurotoxins represent the clinical potential of ion-channel modulators across several therapeutic fields, especially in developing new strategies for treatment of ion channel-related diseases. The aim of this review is to overview the latest updates in the domain of peptide neurotoxins that affect voltage-gated calcium channels, with a special focus on ω-agatoxins.

Synthesis of cyclooctapeptides: constraints analogues of the peptidic neurotoxin, omega-agatoxine IVB-an experimental point of view.

omega-AGA IVB is an important lead structure when considering the design of effectors of glutamate release inducting P/Q-type calcium channels. The best route to achieve the analogues possessing the three-dimensional arrangement corresponding to the native binding loop was the introduction of constraint by ring formation via side chain to side chain lactamization for suitably protected Lys and Glu residues. Since tryptophane residue located at position 14 of this neuropeptide has been suggested as essential for binding, analogues in which this amino acid was replaced by aza-tryptophane and alanine were synthesized. The synthesis was carried out on various acid-labile resins (BARLOS chlorotrityl, Rink amide, PEG-based or Wang resins), by Fmoc strategy. In this paper, we describe optimization of the peptide cyclization with various protecting groups, and on resin or in solution cyclization experimental parameters.

An intermediate in a new synthesis approach to beta-substituted beta-hydroxyaspartame.

The crystal and molecular structure of 1-tert-butyl 4-ethyl (2'R,3'R,5'R,2S,3S)-3-bromomethyl-3-hydroxy-2-[(2'-hydroxy-2',6',6'-trimethylbicyclo[3.1.1]hept-3'-ylidene)amino]succinate, C(21)H(34)BrNO(6), is presented. This compound is an intermediate in the new synthetic route to beta-substituted beta-hydroxyaspartates, which are blockers of glutamate transport.

Vitamins and minerals: a model for safe addition to foods.

BACKGROUND: Significant subgroups in most European populations have intakes below nationally recommended levels for several vitamins, minerals and trace elements, placing individuals at risk of suboptimal intake of important vitamins and minerals. The voluntary addition of micronutrients to the appropriate foods may help address the risks associated with low micronutrient intakes. However, concerns need to be addressed regarding the potential for unacceptably high intakes, particularly for those people consuming very large amounts of food. AIM OF THE STUDY: To develop a model to estimate the level of each micronutrient that can be added safely to foods. METHODS: A theoretical model was developed based on the critical factors which determine the risk of unacceptably high intake for each micronutrient at high levels of food/energy intakes. These included 1) Tolerable Upper Intake Levels (UL), 2) high micronutrient intakes in Europe at the 95(th) percentile intake for each nutrient, 3) the proportion of fortified foods in the diets of individuals at the 95(th) percentile for energy intakes, 4) the proportion of foods to which micronutrients could practically be added, and 5) a range of estimates for the fractions of foods which might be actually fortified for each nutrient. A maximum level was set up for each micronutrient per typical serving or 100 kcal portion. The outputs of the model were then compared against a recent model developed by AFSSA, based on the food intake data in France. RESULTS: Three categories of micronutrients were identified, in which micronutrients could be added safely to foods at levels (per serving, e. g., 100 kcal) 1) greater than 1 European Commission Recommended Daily Intake (EC RDA): vitamin B12, vitamin C, vitamin E, riboflavin, panthothenic acid, niacin and thiamine; 2) between 50 and 100 % of the EC RDA: vitamin B6, vitamin D, folic acid, biotin, copper, iodine and selenium; 3) between 10 and 40 % of the EC RDA: iron, zinc, calcium, phosphorus and magnesium. A fourth category consisting of retinol, for which high end intake levels are close to UL for some population subgroups in Europe and thus requires further consideration. CONCLUSIONS: A wide range of vitamins and minerals can be added safely to foods at nutritionally important levels in the current diets of Europeans.

Optically pure beta-substituted beta-hydroxy aspartates as glutamate transporter blockers.

A short asymmetric synthesis of optically pure beta-substituted beta-hydroxy aspartates is described. The key step is an aldol reaction between a glycine enolate derived from an oxazinone intermediate used as chiral auxiliary and various alpha-keto esters. Excellent diastereomeric excesses are obtained.