meso-1,2-bis(methylazo)-1,2-diphenylethane.

The title compound, meso-1,2-bis(methyldiazenyl)-1,2-diphenylethane, C16H18N4, is arranged in a disordered manner around an inversion point. The N-N atom distances in the azo group of 1.192 (8) and 1.195 (8) A, and the C-C atom distances in the ethylene moiety at 1.512 (8) and 1.503 (8) A in the two models [refined to 51.7 (6) and 48.3 (6)% occupancies] were not significantly different.

Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance.

Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.

Synthesis and characterization of photolabile derivatives of serotonin for chemical kinetic investigations of the serotonin 5-HT(3) receptor.

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.

Electron capture dissociation for structural characterization of multiply charged protein cations.

For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific.

A new photolabile precursor of glycine with improved properties: A tool for chemical kinetic investigations of the glycine receptor.

The synthesis and characterization of a new photolabile precursor of glycine (caged glycine) is described. The alpha-carboxyl group of glycine is covalently coupled to the alpha-carboxy-2-nitrobenzyl (alphaCNB) protecting group. Photolysis of the caged glycine with UV light produces free glycine. At 308 nm, the compound photolyzes with a quantum yield of 0.38. The absorption spectrum and the pH dependence of a transient absorption produced after laser-flash illumination are typical for aci-nitro intermediates of alphaCNB-protected compounds. The time constant for the major component of the aci-nitro intermediate decay ( approximately 84% of the total aci-nitro absorbance) was determined to be 7 micros at physiological pH. A minor component ( approximately 16%) decays with a rate constant of 170 micros. The compound does not activate or inhibit the alpha(1)-homomeric glycine receptor transiently expressed in HEK293 cells. After photolysis with a 10 ns pulse of 325 nm laser light, the glycine released from the caged compound activates glycine-mediated whole-cell currents in the same cells. The rise of these currents can be measured in a time-resolved fashion and occurs on a millisecond to sub-millisecond time scale. It can be described with a single-exponential function over >85% of the total current. The rate constant of the current rise is about 2 orders of magnitude slower than the rate constant of caged glycine photolysis. Thermal hydrolysis of the alphaCNB-caged glycine takes place with a half-life of 15.6 h at physiological pH. The new caged glycine is the first in a series of photoprotected glycine derivatives that has the required properties for use with chemical kinetic methods for investigation of glycine-activated cell surface receptors. Photolysis is rapid and efficient with respect to the receptor reactions to be studied; hydrolysis in aqueous solution is sufficiently slow, and the compound is biologically inert. It will, therefore, be a useful tool for investigation of the processes leading to channel opening of glycine receptor channels and the effects of mutations of the glycine receptor and of inhibitors on these processes.

Studies of structure and mechanism in acetonitrile chemical ionization tandem mass spectrometry of polyunsaturated fatty acid methyl esters.

Recently it has been shown that acetonitrile chemical ionization tandem mass spectrometry (CI-MS/MS) is a rapid, on-line means to determine double bond position in fatty acid methyl esters (FAME). The mechanism of this gas phase condensation reaction has been studied. Evidence of the (1-methyleneimino)-1-ethenylium ion (m/z 54), formed upon the reaction of acetonitrile with itself, adding across the double bond in a [2 + 2] cycloaddition reaction is observed. When this nascent complex undergoes collision-induced dissociation, two diagnostic ions emerge. One of these ions results from loss of the hydrocarbon end of the FAME, whereas the other ion results from loss of the methyl ester end, and when considered together, the diagnostic ions localize the positions of the double bonds in the FAME. Several labeling and MS/MS/MS experiments on the two diagnostic ions were performed to determine a plausible fragmentation mechanism of the stable (1-methyleneimino)-1-ethenylium-FAME complex. The first generation product ions, or diagnostic ions, appear to be formed though a charge-driven mechanism, whereas the second generation product ions are formed via charge-remote fragmentations. Plausible mechanisms for the formation and subsequent dissociation of the diagnostic ions are presented for the monounsaturated, diunsaturated, and polyunsaturated (3 or more double bonds) FAME.

Synthesis and characterization of a caged receptor ligand suitable for chemical kinetic investigations of the glycine receptor in the 3-microseconds time domain.

Here we report the development and characterization of a new photolabile protecting group for the carboxyl group of neurotransmitters, 2-methoxy-5-nitrophenyl. The synthesis and characterization of a photolabile derivative of beta-alanine, caged beta-alanine, are described. beta-Alanine can activate the glycine receptor, a major inhibitory receptor in the mammalian central nervous system; the 2-methoxy-5-nitrophenyl derivative of beta-alanine combined with a laser-pulse photolysis method makes it possible to investigate the chemical kinetic mechanism of the receptor in the 3-microseconds time domain. The derivative is photolyzed by a laser pulse to release free beta-alanine within 3 microseconds and with a product quantum yield of 0.2. In aqueous solution in the dark and at neutral pH, the compound is more stable, by a factor of approximately of 25, than the analogous derivative of glycine [Ramesh, D., Wieboldt, R., Niu, L., Carpenter, B. K., & Hess, G. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11074-11078]. 2-Methoxy-5-nitrophenyl-beta-alanine hydrolyzes in aqueous solution at neutral pH with a t1/2 of approximately 1.5 h. Neither the 2-methoxy-5-nitrophenyl-beta-alanine nor the 2-methoxy-5-nitrophenol photolysis side product activates, inhibits, or potentiates the response of glycine receptors in rat hippocampal neurons to glycine. Photolysis of 2-methoxy-5-nitrophenyl-beta-alanine by irradiation with a 600-ns laser pulse at 333 nm releases beta-alanine, which then activates glycine receptor-channels on neurons equilibrated with the caged compound, as detected by whole-cell current recording. Compared with the analogous derivative of glycine, in terms of quantum yield, photolysis rate, and stability, this new compound is not only a better candidate for use in chemical kinetic investigations of the glycine receptor, but can also be used in determining the location of glycine receptors in neuronal cells.

Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale.

Newly synthesized photolabile derivatives of glutamate, caged glutamate, that release free glutamate on a microsecond time scale after a pulse of UV laser light are described. 2-Nitrobenzyl derivatives were attached to the amino or carboxyl groups of glutamate. Substitution with a -CO2- group at the benzylic carbon accelerates the photolysis reaction when compared to -H and -CH3 substituents. gamma-O-(alpha-Carboxy-2-nitrobenzyl)glutamate is stable at neutral pH. In 100 mM phosphate buffer at pH 7.0, the compound is photolyzed at 308 nm with a quantum product yield of 0.14. The half-life of the major component of the photolytic reaction, as judged by the transient absorbance change at 430 nm, is 21 microseconds (approximately 90%); the half-life of a minor component (approximately 10%) is 0.2 ms. The amino-linked derivatives have half-lives in the millisecond region and a 4-fold lower quantum yield. The potential of the newly synthesized compound for use in rapid chemical kinetic investigations of glutamate receptors is demonstrated. (i) The caged glutamate at 1 mM concentration does not desensitize glutamate receptors in rat hippocampal neurons. (ii) Caged glutamate (1 mM) does not inhibit activation of the receptors by 50 microM glutamate. (iii) Photolysis of the compound induces rapid onset of transmembrane currents in rat hippocampal neurons.

Synthesis and photochemistry of photolabile derivatives of gamma-aminobutyric acid for chemical kinetic investigations of the gamma-aminobutyric acid receptor in the millisecond time region.

The gamma-aminobutyric acid (GABA) receptor is an abundant neuronal receptor in the mammalian and invertebrate nervous systems and is associated with an inhibitory chloride ion channel. GABA is the endogenous neurotransmitter for the receptor and can trigger both fast activation and a reversible desensitization of the receptor. A series of photolabile amine-linked o-nitrobenzyl derivatives of GABA were synthesized that photolyze rapidly to release free GABA. The photochemical properties of the GABA precursors were determined; the compounds undergo rapid photolysis, initiated with UV irradiation at 308 nm, and release free GABA on a millisecond time scale. The pH of the photolysis medium affects both the quantum yield and the rate of photolysis. For example, the quantum yield observed for N-(alpha-carboxy-2-nitrobenzyl)-gamma-aminobutyric acid increases from 0.06 at pH 5.0 to 0.1 at pH 10.5, and the half-life for the photolytic reaction decreases from 1.0 to 2.5 ms in the same pH range. Photolysis of the compounds induces rapid onset of transmembrane ion currents in mouse cortical neurons. The potential of the new compounds for use in rapid chemical kinetic investigations of the neuronal GABA receptor is demonstrated.

Photolysis of a protecting group for the carboxyl function of neurotransmitters within 3 microseconds and with product quantum yield of 0.2.

The synthesis of a photosensitive blocking group for the carboxyl function of neurotransmitters, in this case glycine, is reported. The compound, 2-methoxy-5-nitrophenyl glycine ester (caged glycine), is photolyzed by a laser pulse at 308 or 337 nm within 3 microseconds and with a product quantum yield of 0.2. The compound is hydrolyzed in water with a time constant tau of 6.1 min at pH 7.1 and 3 hr at pH 4.0. Mouse cerebral cortical neurons containing glycine receptors were used in biological assays. A cell-flow device, in which solutions of caged glycine at pH 4.0 were mixed with buffer to give a final pH of 7.1, was used to equilibrate the compound with receptors on the cell surface. Neither the caged compound nor the 2-methoxy-5-nitrophenol photolysis product affected the glycine receptors or modified their response to glycine. When cells equilibrated with caged glycine are irradiated by a laser pulse at 337 nm, glycine receptor channels are opened, as detected in whole-cell current recordings. The approach described may be used in the synthesis and characterization of photolabile precursors of neurotransmitters and other compounds that contain carboxyl groups and for kinetic investigations of neurotransmitter receptors in central nervous system cells in the microsecond time domain.

Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.

4-halo-3-hydroxyanthranilic acids: potent competitive inhibitors of 3-hydroxy-anthranilic acid oxygenase in vitro.

The mechanism of action of three potent inhibitors of 3-hydroxyanthranilic acid oxygenase (3HAO), the enzyme responsible for the production of the endogenous excitotoxin quinolinic acid, was examined in vitro. Using either liver homogenate or purified 3HAO, and following the rapid synthesis of the immediate enzymatic product alpha-amino-beta-carboxymuconic acid omega-semialdehyde spectrophotometrically, 4-halogenated (F, Cl, Br) 3-hydroxyanthranilic acids were found to inhibit enzymatic activity in a reversible fashion. Because of the very tight binding of the drugs to 3HAO, reversibility was detected only after warming the protein-inhibitor complexes at 37 degrees. Further studies showed that enzyme inhibition was competitive in nature (apparent Ki values: 190, 6 and 4 nM for the F-, Cl- and Br-compounds, respectively), and suggested that the drugs are metabolized by the enzyme. Specific, reversible, and tightly binding 3HAO inhibitors can be expected to become valuable tools for the study of quinolinate neurobiology. The drugs could also be of interest for the diagnostics and therapeutics of brain diseases which have been speculatively linked to a pathological overabundance of quinolinic acid.

Preparation of 4-halo-3-hydroxyanthranilates and demonstration of their inhibition of 3-hydroxyanthranilate oxygenase activity in rat and human brain tissue.

The chemical syntheses of 4-fluoro-, 4-chloro-, and 4-bromo-3-hydroxyanthranilic acids are described. Their actions as inhibitors of 3-hydroxyanthranilate oxygenase in cell-free homogenates from rat and human brain and in rat brain slices are presented.

Synthesis, photochemistry, and biological activity of a caged photolabile acetylcholine receptor ligand.

A biologically inert photolabile precursor of carbamoylcholine has been synthesized; it is photolyzed to carbamoylcholine, a well-characterized acetylcholine analogue, with a half-time of 40 microseconds at pH 7.0 and a quantum yield of 0.8. The compound, N-(alpha-carboxy-2-nitrobenzyl)carbamoylcholine, was synthesized from (2-nitrophenyl)glycine. The photolysis rates (of five compounds) and the biological activity (of two compounds) were determined, and both properties were found to depend on the nature of the substituents on the photolabile protecting group. Laser pulse photolysis at wavelengths between 308 and 355 nm was used to investigate the wavelength dependence, quantum yield, and rate of the photolysis reaction. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis and by their ability to activate the nicotinic acetylcholine receptor. BC3H1 muscle cells containing those receptors and a cell-flow method were used in the biological assays. The approach described may be useful in the preparation and characterization of other photolabile precursors of neurotransmitters that contain amino groups. The importance of these rapidly photolyzed, inert precursors of neurotransmitters is in chemical kinetic investigations of the reactions involving diverse neuronal receptors; such studies have been hampered because the available techniques have an insufficient time resolution.

Identification and quantification of kynurenic acid in human brain tissue.

Serial ion-exchange and high-performance liquid chromatography separations were employed for the tissue extraction and purification of kynurenic acid (KYNA). Subsequently, the compound isolated from postmortem human brain tissue was unequivocally identified as KYNA by nuclear magnetic resonance and mass spectrometric analyses. Regional distribution analyses revealed the highest concentration of KYNA (1.58 +/- 0.43 pmol/mg tissue) in the caudate nucleus with lower levels in the thalamus, globus pallidus, hippocampus, parietal cortex and frontal cortex. Of the brain structures examined, the lowest concentration of KYNA (0.14 +/- 0.02 pmol/mg tissue) was found in the cerebellum.