Three-Dimensional Control of DNA Hybridization by Orthogonal Two-Color Two-Photon Uncaging.

We successfully introduced two-photon-sensitive photolabile groups ([7-(diethylamino)coumarin-4-yl]methyl and p-dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three-dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double-strand displacement in a hydrogel and in neurons. Orthogonal two-photon uncaging of the two cages is possible, thus enabling complex scenarios of three-dimensional control of hybridization with light.

Characterization of one- and two-photon photochemical uncaging efficiency.

The idea of using light to unleash biologically active compounds from inert precursors (uncaging) was introduced over 30 years ago. Recent efforts prompted the development of photoremovable protecting groups that have increased photochemical efficiencies for one- and two-photon excitation to allow more sophisticated applications. This requires characterization of one- and two-photon photochemical efficiencies of the uncaging processes.

A step toward the reactivation of aged cholinesterases--crystal structure of ligands binding to aged human butyrylcholinesterase.

Organophosphorus nerve agents irreversibly inhibit cholinesterases. Phosphylation of the catalytic serine can be reversed by the mean of powerful nucleophiles like oximes. But the phosphyl adduct can undergo a rapid spontaneous reaction leading to an aged enzyme, i.

Influence of GABA(A) receptor α subunit isoforms on the benzodiazepine binding site.

Classical benzodiazepines, such as diazepam, interact with α(x)β(2)γ(2) GABA(A) receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands.

The donor-acceptor biphenyl platform: a versatile chromophore for the engineering of highly efficient two-photon sensitive photoremovable protecting groups.

Different photoremovable protecting groups in the o-nitrobenzyl, phenacyl, and 2-(o-nitrophenyl)propyl series with a donor-acceptor biphenyl backbone, known to display excellent two-photon absorption cross-sections, were investigated in order to develop efficient two-photon sensitive photoremovable protecting groups. The 2-(o-nitrophenyl)propyl series was a more versatile platform to increase the two-photon sensitivity of photoremovable protecting groups, leading to the p-alkoxy and p-bisalkylamino-4-nitro-[1,1'-biphenyl]-3-yl)propyl derivatives: PENB and EANBP respectively. Those two photoremovable protecting groups are to date the best caging groups for two-photon excitation at 800 and 740 nm respectively, offering attracting perspectives in chemical biology.

Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents.

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs.

Retrochalcone derivatives are positive allosteric modulators at synaptic and extrasynaptic GABA(A) receptors in vitro.

Background And Purpose: Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA(A) receptors (GABA(A)Rs). We previously reported that trans-6,4'-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA(A)Rs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4'-methoxyretrochalcone (Rc-Br) and 4,3'-dimethoxychalcone (Ch-OMe) on GABA(A)Rs.

Binding modes of noncompetitive GABA-channel blockers revisited using engineered affinity-labeling reactions combined with new docking studies.

The binding modes of noncompetitive GABA(A)-channel blockers were re-examined taking into account the recent description of the 3D structure of prokaryotic pentameric ligand-gated ion channels, which provided access to new mammalian or insect GABA receptor models, emphasizing their transmembrane portion. Two putative binding modes were deciphered for this class of compounds, including the insecticide fipronil, located nearby either the intra- or the extracellular part of the membrane, respectively. These results are in full agreement with previously described affinity-labeling reactions performed with GABA(A) noncompetitive blockers (Perret et al.

Small photoactivatable molecules for controlled fluorescence activation in living cells.

The search for chemical probes which allow a controlled fluorescence activation in living cells represent a major challenge in chemical biology. To be useful, such probes have to be specifically targeted to cellular proteins allowing thereof the analysis of dynamic aspects of this protein in its cellular environment. The present paper describes different methods which have been developed to control cellular fluorescence activation emphasizing the photochemical activation methods known to be orthogonal to most cellular components and, in addition, allowing a spatio-temporal controlled triggering of the fluorescent signal.

Live-cell one- and two-photon uncaging of a far-red emitting acridinone fluorophore.

Total synthesis and photophysical properties of PENB-DDAO, a photoactivatable 1,3-dichloro-9,9-dimethyl-9H-acridin-2(7)-one (DDAO) derivative of a far-red emitting fluorophore, are described. The photoremovable group of the DDAO phenolic function comprises a donor/acceptor biphenyl platform which allows an efficient (> or = 95%) and rapid (< 15 micros time-range) release of the fluorescent signal and displays remarkable two-photon uncaging cross sections (delta(a) x Phi(u) = 3.7 GM at 740 nm).

Photochemical tools to study dynamic biological processes.

Light-responsive biologically active compounds offer the possibility to study the dynamics of biological processes. Phototriggers and photoswitches have been designed, providing the capability to rapidly cause the initiation of wide range of dynamic biological phenomena. We will discuss, in this article, recent developments in the field of light-triggered chemical tools, specially how two-photon excitation, "caged" fluorophores, and the photoregulation of protein activities in combination with time-resolved x-ray techniques should break new grounds in the understanding of dynamic biological processes.

Relative positioning of diazepam in the benzodiazepine-binding-pocket of GABA receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket.

Flavylium salts as in vitro precursors of potent ligands to brain GABA-A receptors.

The synthesis of a series of derivatized flavylium cations was undertaken and the affinity to the benzodiazepine binding site of the GABA-A receptor evaluated. The observed high affinity for some derivatives (sub-muM range) was explained by an in vitro transformation of the flavylium cations into the corresponding trans-retrochalcones, components which are proposed to be the active species in this series.

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs.

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2).

Reactive derivatives for affinity labeling in the ifenprodil site of NMDA receptors.

To prepare thiol-reactive ifenprodil derivatives designed as potential probes for cysteine-substituted NR2B containing NMDA receptors, electrophilic centers were introduced in different areas of the ifenprodil structure. Intermediates and final compounds were evaluated by binding studies and by electrophysiology to determine the structural requirements for their selectivity. The reactive compounds were further tested for their stability and for their reactivity in model reactions; some were found suitable as structural probes to investigate the binding site and the docking mode of ifenprodil in the NR2B subunit.

Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor.

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker.

Photolabile glutamate protecting group with high one- and two-photon uncaging efficiencies.

A pi-extended [2-(2-nitrophenyl)propoxy]carbonyl (NPPOC) derivative has been prepared as an efficient UV and near-IR photolabile protecting group for glutamate. This glutamate cage compound exhibits efficient photorelease upon one-photon excitation (epsilonPhi=990 M(-1) cm(-1) at 315 nm). In addition, it also shows efficient photorelease in activation of glutamate receptors in electrophysiological recordings.

Synthesis and photochemical properties of a light-activated fluorophore to label His-tagged proteins.

Rapid and efficient light-induced fluorescence enhancement is demonstrated on a DMNPB-"caged" coumarin derivative carrying a His-tag recognition motif.

Use of a 'caged' analogue to study the traffic of choline within acetylcholinesterase by kinetic crystallography.

Acetylcholinesterase plays a crucial role in nerve-impulse transmission at cholinergic synapses. The apparent paradox that it displays high turnover despite its active site being buried raises cogent questions as to how the traffic of substrates and products to and from the active site can occur so rapidly in such circumstances. Here, a kinetic crystallography strategy aimed at structurally addressing the issue of product traffic in acetylcholinesterase is presented, in which UV-laser-induced cleavage of a photolabile precursor of the enzymatic product analogue arsenocholine, 'caged' arsenocholine, is performed in a temperature-controlled X-ray crystallography regime.

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors.

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101.

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators.

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists.