Bilha Fischer

Acetyl Chloride-Methanol as a Convenient Reagent for: A) Quantitative Formation of Amine Hydrochlorides B) Carboxylate Ester Formation C) Mild Removal of N-t-Boc-Protective Group.

Abstract Hydrogen chloride qualitatively generated in situ by the addition of acetyl chloride to alcoholic solutions is a useful reagent for carboxylic acid esterification, N-t-Boc deprotection and phosphoramide solvolysis reactions.

Aβ40, either Soluble or Aggregated, Is a Remarkably Potent Antioxidant in Cell-Free Oxidative Systems

The brains of individuals diagnosed with Alzheimers disease (AD) are characterized by amyloid plaques, of which the major component is Abeta peptide. Excessive Cu and Fe ions binding to Abeta were suggested to have a deleterious effect on promoting both the aggregation of Abeta and the generation of reactive oxygen species (ROS). Other studies suggested that Abeta plays a protective role by acting as an antioxidant at nanomolar concentrations. The apparent confusion regarding the antioxidant and pro-oxidant properties of Abeta(40) encouraged us to explore the modulatory role of Abeta(40) at the molecular level under oxidative stress conditions. Here, we focused on Abeta(40) in the simplest oxidative system, namely, Cu(I)/Cu(II)/Fe(II)-H(2)O(2). Using ESR, we monitored the production of OH radicals in the above-mentioned systems in the presence of Abeta(40). We found that Abeta(40), either in its soluble or in its aggregated form, functioned as a remarkably potent antioxidant in Cu(I)/Fe(II)-catalyzed radical-producing systems and slightly less potently in the presence of Cu(II) with IC(50) values of 13-62 muM. Abeta(40) proved to be 3.8-6.5 and 15-42 times more potent than the soluble Abeta(28) and the potent antioxidant Trolox, respectively, in the Cu(I)/Fe(II)-H(2)O(2) systems. Time-dependent enhancement of ROS production by Abeta(40) occurs only at low concentrations of aggregated Abeta(40) and in the presence of Cu(II). On the basis of the extremely low IC(50) values of Abeta(40) and the extensive oxidative damage caused to Abeta(40) in Cu(I)/Fe(II)-H(2)O(2) systems, we propose that radical scavenging is the major mechanism of antioxidant activity of Abeta(40) in addition to metal ion chelation. In summary, Abeta(40), either soluble or aggregated, at either nanomolar or micromolar concentrations is a highly potent antioxidant in cell-free oxidative systems, acting mainly as a radical scavenger. Therefore, we propose that it is not the Abeta(40)-Cu(I)/Fe(II) complex per se that is responsible for the oxidative damage in AD.

Comparative studies on the affinities of ATP derivatives for P2X‐purinoceptors in rat urinary bladder

1 Radioligand binding assays have been used to determine the affinities of a series of ATP derivatives with modifications of the polyphosphate chain, adenine and ribose moieties of the ATP molecule for [3H]‐α,β‐methylene ATP ([3H]‐α,β‐MeATP) binding sites in rat urinary bladder. 2 The replacement of the bridging oxygen in the triphosphate chain of ATP (pIC50 = 5.58) with a methylene or imido group markedly increased the affinity (691 fold in IC50 values for β,γ‐imidoATP, 15 fold for β,γ‐methylene ATP), and the replacement of an ionized oxygen on the γ‐phosphate with a sulphur (ATPγS) also led to increased affinity (5623 fold in IC50 values). 3 Modifications at N6, N1, and C‐8 positions on the purine base usually reduced the affinity of ATP (a decrease of 2.8 fold in IC50 values for N6‐methylATP and 8.9 fold for 8‐bromo ATP), while the attachment of an alkylthio group to the C‐2 position greatly increased the affinity for P2X‐purinoceptors (from 3.5 to 98 fold increase in IC50 values). 4 Replacement of the 3′‐hydroxyl group on the ribose with substituted amino or acylamino groups produced more potent P2X‐purinoceptor agonists (an increase of 447 fold in IC50 values for 3′‐deoxy‐3′‐benzylamino ATP and 28 fold for 3′‐deoxy‐3′‐(4‐hydroxyphenylpropionyl)amino ATP. 5 Diadenosine polyphosphates (Ap[n]A) were also shown to displace the [3H]‐α,β‐MeATP binding. The rank order of potency was Ap6A > Ap5A > Ap4A >> Ap3A >> Ap2A. 6 Suramin, PPADS, and reactive blue 2 could competitively displace the binding of [3H]‐α,β‐MeATP to P2X‐purinoceptors, with pIC50 values of 6.26, 5.35, and 6.22, respectively.

Identification of potent P2Y-purinoceptor agonists that are derivatives of adenosine 5'-monophosphate.

1 A series of chain‐extended 2‐thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C‐linked P2Y‐purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase‐linked P2Y‐purinoceptor of C6 rat glioma cells, and the cloned human P2U‐receptor stably expressed in 1321N1 human astrocytoma cells. 2 Although adenosine monophosphate itself was not an agonist in the two P2Y‐purinoceptor test systems, eleven different 2‐thioether‐substituted adenosine monophosphate analogues were full agonists. The most potent of these agonists, 2‐hexylthio AMP, exhibited an EC50 value of 0.2 nM for activation of the C6 cell receptor. This potency was 16,000 fold greater than that of ATP and was only 10 fold less than the potency of 2‐hexylthio ATP in the same system. 2‐hexylthio adenosine was inactive. 3 Monophosphate analogues that were the most potent activators of the C6 cell P2Y‐purinoceptor were also the most potent activators of the turkey erythrocyte P2Y‐purinoceptor. However, agonists were in general more potent at the C6 cell receptor, and potency differences varied between 10 fold and 300 fold between the two receptors. 4 Although 2‐thioether derivatives of adenosine monophosphate were potent P2Y‐purinoceptor agonists no effect of these analogues on the human P2U‐purinoceptor were observed. 5 These results support the view that a single monophosphate is sufficient and necessary for full agonist activity at P2Y‐purinoceptors, and provide insight for strategies for development of novel P2Y‐purinoceptor agonists of high potency and selectivity.

Structure and ligand-binding site characteristics of the human P2Y11 nucleotide receptor deduced from computational modelling and mutational analysis

The P2Y11-R (P2Y11 receptor) is a less explored drug target. We computed an hP2Y11-R (human P2Y11) homology model with two templates, bovine-rhodopsin (2.6 A resolution; 1 A=0.1 nm) and a hP2Y1-ATP complex model. The hP2Y11-R model was refined using molecular dynamics calculations and validated by virtual screening methods, with an enrichment factor of 5. Furthermore, mutational analyses of Arg106, Glu186, Arg268, Arg307 and Ala313 confirmed the adequacy of our hP2Y11-R model and the computed ligand recognition mode. The E186A and R268A mutants reduced the potency of ATP by one and three orders of magnitude respectively. The R106A and R307A mutants were functionally inactive. We propose that residues Arg106, Arg268, Arg307 and Glu186 are involved in ionic interactions with the phosphate moiety of ATP. Arg307 is possibly also H-bonded to N6 of ATP via the backbone carbonyl. Activity of ATP at the F109I mutant revealed that the proposed p-stacking of Phe109 with the adenine ring is a minor interaction. The mutation A313N, which is part of a hydrophobic pocket in the vicinity of the ATP C-2 position, partially explains the high activity of 2-MeS-ATP at P2Y1-R as compared with the negligible activity at the P2Y11-R. Inactivity of ATP at the Y261A mutant implies that Tyr261 acts as a molecular switch, as in other G-protein-coupled receptors. Moreover, analysis of cAMP responses seen with the mutants showed that the efficacy of coupling of the P2Y11-R with Gs is more variable than coupling with Gq. Our model also indicates that Ser206 forms an H-bond with Pgamma (the gamma-phosphate of the triphosphate chain of ATP) and Met310 interacts with the adenine moiety.

Soluble Amyloid β1-28-Copper(I)/Copper(II)/Iron(II) Complexes Are Potent Antioxidants in Cell-Free Systems

Amyloid beta (Abeta) is a central characteristic of Alzheimers disease (AD). Currently, there is a long-standing dispute regarding the role of Abeta-metal ion (Zn, Cu, and Fe) complexes in AD pathogenesis. Here, we aim to decipher the connection between oxidative damage implicated in AD and Abeta-metal ion complexes. For this purpose we study, using ESR, the modulation of Cu/Fe-induced H 2O 2 decomposition by Abeta 1-28 (Abeta 28), a soluble model of Abeta 40/42. The addition of H 2O 2 to 0.6 nM-360 microM Abeta 28 solutions containing 100 microM Cu(II)/Cu(I)/Fe(II) at pH 6.6 results in a concentration-dependent sigmoidal decay of [*OH] with IC 50 values of 61, 59, and 84 microM, respectively. Furthermore, Abeta 28 reduces 90% of *OH production rate in the Cu(I)-H 2O 2 system in 5 min. Unlike soluble Abeta 28, Abeta 28-Cu aggregates exhibit poor antioxidant activity. The mode of antioxidant activity of soluble Abeta 28 is twofold. The primary (rapid) mechanism involves metal chelation, whereas the secondary (slow) mechanism involves (*)OH scavenging and oxidation of Cu(Fe)-coordinating ligands. On the basis of our findings, we propose that soluble Abeta may play a protective role in the early stages of AD, but not in healthy individuals, where Abetas concentration is nanomolar. Yet, when Abeta-metal ion complexes undergo aggregation, they significantly lose their protective function and allow oxidative damage to occur.

Do oxazoliks undergo Diels-Alder reactions with heterodienophiles?☆

Abstract 5-Alkoxy and 2-alkoxyoxazoles were shown to react with NN, CN or CO dienophiles to form products that in most cases can be explained to result from a Diels-Alder addition, or by nucleophilic attack of the oxazole on the dienophile, followed by rearrangement. The products are triazolines, imidazolines or oxazolines respectively. Relative reactivitles were established and mechanistic pathways discussed.

Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5′‐O‐(1‐boranotriphosphate) derivatives

P2Y‐nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P‐boronated ATP derivatives (ATP‐α‐B) were synthesized by substitution of a nonbridging O at P with a BH3 group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl‐ or methylthio‐). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y1 and rat P2Y2 receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y1‐GFP, rP2Y2‐GFP). We investigated agonist‐induced receptor endocytosis, [Ca2+]i rise and arachidonic acid (AA) release. Agonist‐induced endocytosis of rP2Y1‐GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP‐α‐B analogs. Both 2‐MeS‐substituted diastereoisomers induced a greater degree of agonist‐induced receptor endocytosis as compared to the 2‐Cl‐substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca2+ release by these compounds in HEK 293 cells stably transfected with rP2Y1. In case of rP2Y2‐GFP, the borano‐nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca2+ release, AA release and in inducing receptor endocytosis. The different ATP‐α‐B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y1 receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes.

An Improved One-Pot Synthesis of Nucleoside 5′-Triphosphate Analogues

Nucleoside 5′-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5′-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to −15°C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using 31P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.

Opposite diastereoselective activation of P2Y1 and P2Y11 nucleotide receptors by adenosine 5′-O-(α-boranotriphosphate) analogues

We explored the stereoselective activation of the P2Y11 receptor, stably expressed and tagged with GFP, in 1321N1 cells, in comparison to its closest homologue, the P2Y1 receptor.