Oriol Pineda

Control of neurotransmitter release by an internal gel matrix in synaptic vesicles

Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

Relative Tendency of Carbonyl Compounds To Form Enamines

Equilibria between carbonyl compounds and their enamines (from O-TBDPS-derived prolinol) have been examined by NMR spectroscopy in DMSO-d(6). By comparing the exchange reactions between pairs (enamine A + carbonyl B → carbonyl A + enamine B), a quite general scale of the tendency of carbonyl groups to form enamines has been established. Aldehydes quickly give enamines that are relatively more stable than those of ketones, but there are exceptions to this expected rule; for example, 1,3-dihydroxyacetone acetals or 3,5-dioxacyclohexanones (2-phenyl-1,3-dioxan-5-one and 2,2-dimethyl-1,3-dioxan-5-one) show a greater tendency to afford enamines than many α-substituted aldehydes.

From vicinal azido alcohols to Boc-amino alcohols or oxazolidinones, with trimethylphosphine and Boc 2 O or CO 2

Abstract A practical solution to the problem of converting directly 1,2-azido alcohols to Boc-amino alcohols, without recourse to catalytic hydrogenation, involves the use of Me3P/Boc2O in THF (or CH2Cl2) and aqueous NaOH at rt (90–98% yields). The same azido alcohols can be converted in one-pot to the corresponding oxazolidinones with Boc2O/DMAP/Me3P or even better with CO2 and Me3P under basic catalysis (91–96% yields).

New chlorinated amphetamine-type-stimulants disinfection-by-products formed during drinking water treatment.

Previous studies have demonstrated high removal rates of amphetamine-type-stimulants (ATSs) through conventional drinking water treatments; however the behaviour of these compounds through disinfection steps and their transformation into disinfection-by-products (DBPs) is still unknown. In this work, for the first time, the reactivity of some ATSs such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) with chlorine has been investigated under simulated and real drinking water treatment conditions in order to evaluate their ability to give rise to transformation products. Two new DBPs from these illicit drugs have been found. A common chlorinated-by-product (3-chlorobenzo)-1,3-dioxole, was identified for both MDA and MDEA while for MDMA, 3-chlorocatechol was found. The presence of these DBPs in water samples collected through drinking water treatment was studied in order to evaluate their formation under real conditions. Both compounds were generated through treatment from raw river water samples containing ATSs at concentration levels ranging from 1 to 15 ng/L for MDA and from 2.3 to 78 ng/L for MDMA. One of them, (3-chlorobenzo)-1,3-dioxole, found after the first chlorination step, was eliminated after ozone and GAC treatment while the MDMA DBP mainly generated after the postchlorination step, showed to be recalcitrant and it was found in final treated waters at concentrations ranging from 0.5 to 5.8 ng/L.

Molecular recognition of peloruside A by microtubules. The C24 primary alcohol is essential for biological activity.

Peloruside is a microtubule‐stabilizing agent that targets the same site as laulimalide. It binds to microtubules with a 1:1 stoichiometry and with a binding affinity in the low‐μM range; thereby reducing the number of microtubular protofilaments in the same way as paclitaxel. Although the binding affinity of the compound is comparable to that of the low‐affinity stabilizing agent sarcodictyin, peloruside is more active in inducing microtubule assembly and is more cytotoxic to tumor cells; this suggests that the peloruside site is a more effective site for stabilizing microtubules. Acetylation of the C24 hydroxyl group results in inactive compounds. According to molecular modeling, this substitution at the C24 hydroxyl group presumably disrupts the interaction of the side chain with Arg320 in the putative binding site on α‐tubulin. The binding epitope of peloruside on microtubules has been studied by using NMR spectroscopic techniques, and is compatible with the same binding site.

Cyclostreptin derivatives specifically target cellular tubulin and further map the paclitaxel site.

Cyclostreptin is the first microtubule-stabilizing agent whose mechanism of action was discovered to involve formation of a covalent bond with tubulin. Treatment of cells with cyclostreptin irreversibly stabilizes their microtubules because cyclostreptin forms a covalent bond to β-tubulin at either the T220 or the N228 residue, located at the microtubule pore or luminal taxoid binding site, respectively. Because of its unique mechanism of action, cyclostreptin overcomes P-glycoprotein-mediated multidrug resistance in tumor cells. We used a series of reactive cyclostreptin analogues, 6-chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and [(14)C-acetyl]-8-acetyl-cyclostreptin, to characterize the cellular target of the compound and to map the binding site. The three analogues were cytotoxic and stabilized microtubules in both sensitive and multidrug resistant tumor cells. In both types of cells, we identified β-tubulin as the only or the predominantly labeled cellular protein, indicating that covalent binding to microtubules is sufficient to prevent drug efflux mediated by P-glycoprotein. 6-Chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and 8-acetyl-cyclostreptin labeled both microtubules and unassembled tubulin at a single residue of the same tryptic peptide of β-tubulin as was labeled by cyclostreptin (219-LTTPTYGDLNHLVSATMSGVTTCLR-243), but labeling with the analogues occurred at different positions of the peptide. 8-Acetyl-cyclostreptin reacted with either T220 or N228, as did the natural product, while 8-chloroacetyl-cyclostreptin formed a cross-link to C241. Finally, 6-chloroacetyl-cyclostreptin reacted with any of the three residues, thus labeling the pathway for cyclostreptin-like compounds, leading from the pore where these compounds enter the microtubule to the luminal binding pocket.

Mechanism of action of the cytotoxic macrolides amphidinolide X and J.

Microtubules and actin filaments play important biological roles in mitosis, cytokinesis, cell signaling, intracellular transport, and cell motility of eukaryotic cells. 2] Molecules that target these cytoskeleton proteins are potential antitumor or anti-HIV agents. In fact, there are several clinical drugs that target the stabilization (paclitaxel-like behavior) or destabilization (vinca-like or colchicine-like behavior) of microtubules, specifically their heterodimeric component, a,b-tubulin. On the other hand, no actin-targeting drug has yet entered clinical studies. Amphidinolides are a series of structurally dissimilar cytotoxic macrolides isolated from dinoflagellates (Amphidinium sp.). Their mechanisms of action are unknown, except that for one that has one of the largest rings, the 26-membered macrolide amphidinolide H (Amp-H, MW = 562.73) ; this shows cytotoxicity in the nanomolar range against several carcinoma cell lines. Amp-H drastically and irreversibly deformed actin fibers; the actin fibers completely disappeared, and only a few disorganized aggregates remained in the cells. Amp-H induced multinucleated cells by disrupting actin organization (polyploid cells). In vitro assays on purified actin indicated that Amp-H stimulates actin polymerization, and stabilizes the actin filaments (F-actin). 11] In contrast, most of the smallest amphidinolides are cytotoxic in the micromolar range. For example, amphidinolide X (1, MW = 448.59) [12] and amphidinolide J (4, MW = 390.56) [13] have IC50 values of 1.3 and 6.9 mm, respectively, against the lymphocytic leukemia cell line L1210, although their mechanisms of action have not been reported. As these small amphidinolides are easier to synthesize than the larger molecules, 16] it would be desirable to identify their binding sites. Appropriate chemical modifications of these natural products might afford leads with activities below 0.1 mm that might eventually give rise to new antitumor agents. We report here biological studies of 1, the structurally related synthetic diolides 2 and 3, and 4 (Scheme 1). We examined their effect on the proliferation of A2780 (human ovarian carcinoma) and of LoVo (human colon carcinoma) cell lines, as well as on the cytoskeleton proteins tubulin, actin, and intermediate filaments in A549 (lung carcinoma) and PtK2 cells. Their effects on actin polymerization was then studied in vitro.

Nucleophile-Catalyzed Additions to Activated Triple Bonds. Protection of Lactams, Imides, and Nucleosides with MocVinyl and Related Groups

Additions of lactams, imides, (S)-4-benzyl-1,3-oxazolidin-2-one, 2-pyridone, pyrimidine-2,4-diones (AZT derivatives), or inosines to the electron-deficient triple bonds of methyl propynoate, tert-butyl propynoate, 3-butyn-2-one, N-propynoylmorpholine, or N-methoxy-N-methylpropynamide in the presence of many potential catalysts were examined. DABCO and, second, DMAP appeared to be the best (highest reaction rates and E/Z ratios), while RuCl3, RuClCp*(PPh3)2, AuCl, AuCl(PPh3), CuI, and Cu2(OTf)2 were incapable of catalyzing such additions. The groups incorporated (for example, the 2-(methoxycarbonyl)ethenyl group that we name MocVinyl) serve as protecting groups for the above-mentioned heterocyclic CONH or CONHCO moieties. Deprotections were accomplished via exchange with good nucleophiles: the 1-dodecanethiolate anion turned out to be the most general and efficient reagent, but in some particular cases other nucleophiles also worked (e.g., MocVinyl-inosines can be cleaved with succinimide anion). Some structural and mechanistic details have been accounted for with the help of DFT and MP2 calculations.

Further Insight into the Interactions of the Cytotoxic Macrolides Laulimalide and Peloruside A with Their Common Binding Site

The binding site of the macrolides laulimalide and peloruside A, which is different from that of the clinically useful drugs paclitaxel/taxol and ixabepilone (tax site), is known to be between two adjacent β-tubulin units (ext site). Here, we report our study of the binding of these molecules to an α1β1/α2β2-tubulin “tetramer” model. AutoDock 4.2.6//AutoDock Vina dockings predicted that the affinities of laulimalide and peloruside A for the tax site are quite similar to those for the ext site. However, molecular dynamics (MD) simulations indicated that only when these two ligands are located at the ext site, there are contacts that help stabilize the system, favoring the β1/β2 interactions. The binding affinity of laulimalide for this site is stronger than that of peloruside A, but this is compensated for by additional β1/β2 contacts that are induced by peloruside A. MD studies also suggested that epothilones at the tax site and either laulimalide or peloruside A at the ext site cause similar stabilizing effects (mainly linking the M-loop of β1 and loop H1–B2 of β2). In a “hexamer” model (3 units of αβ-tubulin), the effects are confirmed. Metadynamics simulations of laulimalide and peloruside A, which are reported for the first time, suggest that peloruside A produces a stronger change in the M-loop, which explains the stabilization of the β1/β2 interaction.