Analysis of Trinitrophenylated Adenosine and Inosine by Capillary Electrophoresis and γ-Cyclodextrin-Enhanced Fluorescence Detection.

Monitoring molecules such as adenosine (Ado) and inosine (Ino) in the central nervous system has enabled the field of neuroscience to correlate molecular concentrations dynamics to neurological function, behavior, and disease. In vivo sampling techniques are commonly used to monitor these dynamics; however, many techniques are limited by the sensitivity and sample volume requirements of currently available detection methods. Here, we present a novel capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) method that analyzes Ado and Ino by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). These complexes exhibit ∼25-fold fluorescence enhancement upon the formation of inclusion complexes with γ-cyclodextrin (γ-CD). Association constants were determined as 4600 M(-1) for Ado and 1000 M(-1) for Ino by CE-LIF. The structure of the TNP-Ado:γ-CD complex was determined by 2D nuclear magnetic resonance (NMR) spectroscopy. Optimal trinitrophenylation reaction conditions and CE-LIF parameters were determined and resulted in the limit of detection of 1.6 µM for Ado and 4 µM for Ino. Ado and Ino were simultaneously quantified in homogenized rat forebrain samples to illustrate application of the technique. Simulated biological samples, desalted by ultrafiltration in the presence γ-CD, were concentrated on-capillary by large-volume sample stacking (LVSS) to achieve detection limits of 32 and 38 nM for TNP-Ado and TNP-Ino, respectively.

Solubilization of Hexafluorobenzene by the Micellar Aromatic Core Formed from Aggregation of Amphiphilic (2,3-O-Dibenzyl-6-O-sulfobutyl) Cyclodextrins.

Aggregation colloids that possess an aromatic pseudophase in an aqueous system could provide new avenues of research including micellar catalysis, aqueous remediation, and emulsion polymerization studies. The apparent aggregation of two macrocyclic surfactants, hexakis (2,3-O-dibenzyl-6-O-sulfobutyl) cyclomaltohexaose (DBSBA) and heptakis (2,3-O-dibenzyl-6-O-sulfobutyl) cyclomaltoheptaose (DBSBB), was investigated using diffusion ordered nuclear magnetic resonance (NMR) spectroscopy (DOSY), conductivity, and pyrene fluorescence techniques. These amphiphiles were found to possess near spherical symmetry at critical micelle concentrations of approximately 0.1 mM in all techniques used to study the phenomenon. Aggregation of both surfactants was found to be entropically driven at low temperatures but enthalpically driven at higher temperatures. The calculated compensation temperatures of DBSBA and DBSBB were determined to be 317 and 307 K, respectively. These surfactants contain a high percentage of aromatic moieties in their structures, which affects the thermodynamics of aggregation and their interior micellar environment. The proposed aromatic micellar core was tested using hexafluorobenzene (HFB) as a molecular probe in (19)F NMR experiments. (19)F NMR relaxation and chemical shift studies found the HFB quantitatively partitioned into the micellar interiors. Global regression analysis found that HFB interaction with DBSBA micelles possessed at least two association constants, differing by an order of magnitude, the largest being in excess of 8300 M(-1). DBSBB micellar interactions with HFB were found to be weaker, although in excess of 1100 M(-1), with a subsequent association constant of similar magnitude. Benzyl substituents of DBSBB are required for solubilization of HFB. Heteronuclear Overhauser effect spectroscopy (HOESY, (19)F-(1)H) of the DBSBB:HFB complex revealed strong interaction of HFB with benzyl substituents but not the cyclodextrin cavity.

Synthesis of aromatic sphingosine analogues by diastereoselective amination of enantioenriched trans-γ,δ-unsaturated ß-hydroxyesters.

An effective route to N-Boc-protected aromatic sphingosine analogues is accomplished. The strategy is based on the diastereoselective amination of enantioenriched trans-γ,δ-unsaturated ß-hydroxyesters to establish anti,N-Boc-α-hydrazino-ß-hydroxyesters. Nonreductive E1cB elimination is essential for the successful N-N bond cleavage of hydrazine while preserving the trans double bond. Either the (3R,2S) and (3S,2R) enantiomer of N-Boc-protected sphingosine analogues has been synthesized in five steps with excellent optical purity with ∼99% ee and >99% de.

Separation of sulfoalkylated cyclodextrins with hydrophilic interaction liquid chromatography.

Determination of the charged state distributions of partially- and fully-substituted sulfoalkylated cyclodextrins was achieved using hydrophilic interaction liquid chromatography (HILIC). HILIC analysis of a spiked sample of the partially sulfopropylated cyclodextrins was achieved using a gradient to baseline resolve the charged states from -1 to -14. The fully-substituted CDs yielded a major peak with some trace impurities and the partially-substituted sulfopropylated cyclodextrins showed a wide range of charge states present in the mixture. Small changes in the structure of the cyclodextrins have a significant impact on the retention times of the various types of cyclodextrins investigated.

Fluorescence enhancement of a Meisenheimer complex of adenosine by gamma-cyclodextrin: a thermodynamic and kinetic investigation.

The fluorescent properties of a trinitrophenylated Meisenheimer complex of adenosine (TNP-Ado) in water were examined in the presence of alpha-, beta-, and gamma-cyclodextrins (CDs). The TNP-Ado complex exhibits minimal fluorescence in water, whereas addition of 10 mM alpha-CD, beta-CD, and gamma-CD enhances fluorescence by factors of 2, 7, and 110, respectively. The large enhancement by gamma-CD is attributed to its larger hydrophobic cavity, which is able to accommodate the TNP moiety of TNP-Ado. (1)H NMR spectra demonstrate 1:1 stoichiometry of the complex, which undergoes slow exchange on the NMR time scale. (1)H NMR and 2D ROESY spectra reveal substantial interaction of the TNP hydrogens with gamma-CD. Equilibrium constants were determined by fluorimetry from 10 to 20 degrees C by nonlinear curve fitting. Fluorescence is temperature dependent, with maximum fluorescence increasing with decreasing temperature. Complexation is exothermic with large negative entropy, consistent with formation of a tight complex between TNP-Ado and gamma-CD. Rate constants and activation parameters for both complexation and dissociation were determined by a combination of fluorimetry and 2D NMR exchange spectroscopy (EXSY).

Microbial degradation of usnic acid in the reindeer rumen.

Reindeer (Rangifer tarandus) eat and utilize lichens as an important source of energy and nutrients in winter. Lichens synthesize and accumulate a wide variety of phenolic secondary compounds, such as usnic acid, as a defense against herbivores and to protect against damage by UV-light in solar radiation. We have examined where and to what extent these phenolic compounds are degraded in the digestive tract of the reindeer, with particular focus on usnic acid. Three male reindeer were given ad libitum access to a control diet containing no usnic acid for three weeks and then fed lichens ad libitum (primarily Cladonia stellaris) containing 9.1 mg/g DM usnic acid for 4 weeks. Usnic acid intake in reindeer on the lichen diet was 91-117 mg/kg BM/day. In spite of this, no trace of usnic acid or conjugates of usnic acid was found either in fresh rumen fluid, urine, or feces. This suggests that usnic acid is rapidly degraded by rumen microbes, and that it consequently is not absorbed by the animal. This apparent ability to detoxify lichen phenolic compounds may gain increased importance with future enhanced UV-B radiation expected to cause increased protective usnic acid/phenol production in lichens.

Separation and sensitive detection of D-amino acids in biological matrices.

The increase in our understanding of D-amino acid function and distribution in mammals is in many ways a result of the initial development of sensitive enantioselective separation strategies that allow for quantification in real biological samples. This article reviews progress on the development of chiral selective separation and detection of D-amino acids including enzyme-based microbiosensors, GC/MS, HPLC/fluorescence, HPLC/MS-MS, cEKC/fluorescence, and MEKC/fluorescence. Only methods capable of analyzing D-isomers in biological matrices are given here and significant effort is made to highlight approaches that offer speed, resolution, high sensitivity, and versatility.

Simultaneous efflux of endogenous D-ser and L-glu from single acute hippocampus slices during oxygen glucose deprivation.

D-serine and L-glutamate play crucial roles in excitotoxicity through N-methyl-D-aspartate receptor coactivation, but little is known about the temporal profile of efflux during cerebral ischemia. We utilized a newly designed brain slice microperfusion device coupled offline to capillary electrophoresis laser-induced fluorescence to monitor dynamic efflux of endogenous D-ser and L-glu in response to oxygen glucose deprivation (OGD) in single acute hippocampus slices. Efflux profiles with 2-min temporal resolution in response to 24-min OGD show that efflux of D-ser slightly precedes efflux of L-glu by one 2-min sampling interval. Thus both coagonists are available to activate NMDA receptors by the time when glu is released. The magnitude of D-ser efflux relative to baseline values is, however, less than that for L-glu. Peak efflux during OGD, expressed as pre-OGD baseline values, was as follows: D-ser 254% +/- 24%, L-glu 1,675% +/- 259%, L-asp 519% +/- 128%, and L-thr 313% +/- 33%. L-glutamine efflux was shown to decrease significantly in response to OGD. The microperfusion/CE-LIF approach shows several promising attributes for studying endogenous chemical efflux from single, acute brain slices.

Quantitative determination of secondary metabolites in Cladina stellaris and other lichens by micellar electrokinetic chromatography.

The quantitative determination of usnic acid (UA), perlatolic acid (PA), and atranorin (AT) in Alaska lichens by micellar electrokinetic chromatography (MEKC) is reported. The background electrolyte (BGE) included sodium docecyl sulfate (SDS), and beta-cyclodextrin (beta-CD) in a high-pH borate buffer. The presence of beta-CD in the buffer significantly decreases peak width, especially for UA, as it decreases migration time for both UA and PA. Linear calibration curves for UA, PA, and AT were established using an internal standard of benzoic acid (BA). Concentration limits of detection (cLODs) are 2.5, 2.2 and 2.0microg/mL (S/N 3) for UA, PA, and AT, respectively. Dry samples of lichen were extracted at room temperature with acetone for 24h in the presence of BA as internal standard. Recoveries of UA from spiked samples ranged from 92 to 98%. Amounts of UA and PA in the lichen samples ranged from 0.28 to 1.7% dry weight and 0.02 to 0.23%, respectively.

Enantioseparation and stacking of Cyanobenz[f]isoindole-amino acids by reverse polarity capillary electrophoresis and sulfated beta-cyclodextrin.

A capillary electrophoresis method with laser-induced fluorescence detection for the chiral separation of cyanobenz[f]isoindole (CBI) derivatives of amino acids was developed and optimized. The enantioseparations are accomplished with sulfated beta-CD (S-beta-CD) as chiral selector at low pH and reverse polarity. BGE conditions were optimized for CBI-serine and then applied to other CBI-amino acids. Baseline resolution of 13 CBI-amino acids was achieved using a single BGE formulation of 2 wt % S-beta-CD in 25 mM phosphate buffer at pH 2.00 and a voltage of -30 kV. pH is the most critical BGE parameter affecting resolution. At 2 wt % S-beta-CD, CBI-serine enantiomers are baseline-resolved at pH 2.00 but no resolution is obtained at pH 3.00. l-Glutamate, l-aspartate and d-serine are simultaneously quantified in the microdialysate of an arctic ground squirrel to illustrate the application to biological samples. Dilute solutions of the CBI-amino acids in water can be stacked by hydrodynamic injection with a 100-fold improvement in signal-to-noise ratio without loss of chiral resolution. The stacking is proposed to consist of field-amplified migration, pH-mediated stacking, and sweeping by S-beta-CD. The limit of detections for CBI-dl-serine and CBI-dl-glutamate are determined as 0.20 and 0.30 nM, respectively. The stacking method was not applicable to the high ionic strength microdialysates.

Nonaqueous synthesis of a selectively modified, highly anionic sulfopropyl ether derivative of cyclomaltoheptaose (beta-cyclodextrin) in the presence of 18-crown-6.

A highly anionic cyclomaltooligosaccharide (cyclodextrin, CD) derivative containing sulfopropyl functional groups on the primary face of the CD was synthesized. Heptakis(2,3-di-O-methyl)cyclomaltoheptaose [heptakis(2,3-di-O-methyl)-beta-cyclodextrin] was reacted with 1,3-propane sultone and potassium hydride (KH) in anhydrous tetrahydrofuran in the presence of 18-crown-6 to yield highly substituted potassium heptakis(2,3-di-O-methyl-6-O-sulfopropyl)cyclomaltoheptaose [heptakis(KSPDM)-beta-CD] with an average degree of substitution (DSCE) of 6.9 as determined by inverse detection capillary electrophoresis (CE). The principal species in the product is the fully substituted heptakis(KSPDM)-beta-CD. Complete NMR assignments of the hydrogen and carbon atoms are made using a combination of gCOSY and gHSQC. In the absence of 18-crown-6, the reaction generates a mixture of multiply charged derivatives with average DSCE of 4.1. The possible roles of the crown ether in the reaction are discussed. The ROESY NMR spectrum of the inclusion complex that forms between heptakis(KSPDM)-beta-CD and 2-naphthoic acid in D2O reveals that 2-naphthoic acid inserts with the carboxyl group toward the derivatized primary rim of the cyclodextrin.