Electroanalytical performance of carbon films with near-atomic flatness.

Physicochemical and electrochemical characterization of carbon films obtained by pyrolyzing a commercially available photoresist has been performed. Photoresist spin-coated on to a silicon wafer was pyrolyzed at 1,000 degrees C in a reducing atmosphere (95% nitrogen and 5% hydrogen) to produce conducting carbon films. The pyrolyzed photoresist films (PPF) show unusual surface properties compared to other carbon electrodes. The surfaces are nearly atomically smooth with a root-mean-square roughness of <0.5 nm. PPF have a very low background current and oxygen/carbon atomic ratio compared to conventional glassy carbon and show relatively weak adsorption of methylene blue and anthraquinone-2,6-disulfonate. The low oxygen/carbon ratio and the relative stability of PPF indicate that surfaces may be partially hydrogen terminated. The pyrolyzed films were compared to glassy carbon (GC) heat treated under the same conditions as pyrolysis to evaluate the electroanalytical utility of PPF. Heterogeneous electron-transfer kinetics of various redox systems were evaluated. For Ru(NH3)6(3+/2+), Fe(CN)6(3-/4-), and chlorpromazine, fresh PPF surfaces show electron-transfer rates similar to those on GC, but for redox systems such as Fe3+/2+, ascorbic acid, dopamine, and oxygen, the kinetics on PPF are slower. Very weak interactions between the PPF surface and these redox systems lead to their slow electron-transfer kinetics. Electrochemical anodization results in a simultaneous increase in background current, adsorption, and electron-transfer kinetics. The PPF surfaces can be chemically modified via diazonium ion reduction to yield a covalently attached monolayer. Such a modification could help in the preparation of low-cost, high-volume analyte-specific electrodes for diverse electroanalytical applications. Overall, pyrolysis of the photoresist yields an electrode surface with properties similar to a very smooth version of glassy carbon, with some important differences in surface chemistry.

Noninvasive identification of materials inside USP vials with Raman spectroscopy and a Raman spectral library.

A commercial dispersive Raman spectrometer operating at 785 nm with a CCD detector was used to acquire spectra of USP reference materials inside amber USP vials. The laser and collection beams were directed through the bottom of the vials, resulting in a 60% loss of signal. The Raman shift was calibrated with a 4-acetamidophenol standard, and spectral response was corrected with a luminescent standard. After these corrections, the Raman spectra obtained inside the USP vial and on open powders differed by less than 5%. A spectral library of 309 reference materials was constructed, with spectral acquisition times ranging from 1 to 60 s. Of these, 8% had significant fluorescent background but observable Raman features, while 3% showed only fluorescence. A blind test of 26 unknowns revealed the accuracy of the library search to be 88-96%, depending on search algorithm, and 100% if operator discretion was permitted. The tolerance of the library search to degraded signal-to-noise ratio, resolution, and Raman shift accuracy were tested, and the search was very robust. The results demonstrate that Raman spectroscopy provides a rapid, noninvasive technique for compound identification.

Raman spectroscopic determination of the structure and orientation of organic monolayers chemisorbed on carbon electrode surfaces.

Azobenzene (AB) and 4-nitroazobenzene (NAB) were covalently bonded to carbon surfaces by electrochemical reduction of their diazonium derivatives. The N(1s) features of XPS spectra of modified surfaces had intensities expected for monolayer coverage. However, the Raman spectra were significantly more intense than expected, implying an increase in scattering cross section upon chemisorption. A likely explanation is resonance enhancement of the carbon/adsorbate chromophore analogous to that reported earlier for dinitrophenylhydrazine (DNPH) chemisorption. Vibrational assignments indicate that the C-C vibration between azobenzene and the carbon surface is in the 1240-1280 cm(-1) region, and this conclusion is supported by spectra obtained from [(13)C]graphite. Observation of depolarization ratios for 4-nitroazobenzene and DNPH on graphite edge plane indicate that NAB is able to rotate about the NAB/carbon C-C bond, while chemisorbed DNPH is not. The partial multiple bond character of the DNPH linkage to graphite is consistent with the observation that the DNPH π system remains parallel to the graphitic planes.

Raman spectroscopy of normal and diseased human breast tissues.

Raman spectra of histologically normal human breast biopsy samples were compared to those exhibiting infiltrating ductal carcinoma (IDC) or fibrocystic change. Experiments at 784 nm with CCD detectors reduced fluorescence interference and produced high SNR spectra with relatively low (10-200 mW) laser power. Sample to sample and patient to patient variation for normal specimens were less than 5% for the ratios of major Raman bands. The Raman spectra changed dramatically in diseased specimens, with much weaker lipid bands being evident. The spectrum of infiltrating ductal carcinoma samples is similar to that of human collagen. Differences between benign (fibrocystic) and malignant (IDC) lesions were smaller than those between normal and IDC specimens, but were still reproducible. Fiberoptic sampling through a hypodermic needle and with a remote probe were demonstrated. The possibility of rapid diagnosis with Raman spectroscopy is considered.

Characterization of human breast biopsy specimens with near-IR Raman spectroscopy.

Breast biopsy samples were examined with Raman spectroscopy with laser wavelengths ranging from 406 to 830 nm. A combination of a single-stage spectrograph, band reject filter, and CCD detector permitted low laser powers and minimal risk of sample radiation damage. Spectra of formalin-fixed human tissue revealed Raman features for lipids and carotenoids. The best defined lipid features were observed for 782- and 830-nm laser excitation, while carotenoid features were strongest in the 488-515-nm range due to resonance enhancement. Comparison of the spectra with those of fatty acid esters revealed that the major lipid component is a derivative of oleic acid. Lipid and carotenoid Raman bands were superimposed on a luminescent background which was less prominent at longer laser wavelengths. A compact, portable, diode laser spectrometer was tested in a clinical setting with fiber optic sampling. The results indicate that substantial biochemical information is available from near-IR Raman spectroscopy and the technique may have clinical applications.

Synthetic aci-reductones: 3,4-dihydroxy-2H-1-benzopyran-2-ones and their cis- and trans-4a,5,6,7,8,8a-hexahydro diastereomers. Antiaggregatory, antilipidemic, and redox properties compared to those of the 4-substituted 2-hydroxytetronic acids.

Synthetic procedures for the elaboration of aci-reductones belonging to the 6- or 7-mono- or bis-substituted-3,4-dihydroxy-2H-1-benzopyran-2-ones (6-10) and their cis- and trans-4a,5,6,7,8,8a-hexahydro diastereomers (11, 12) are described. hexahydrobenzopyranone aci-reductones were conveniently prepared by using Meldrum's synthon (2,2-dimethyl-1,3-dioxane-4,6-dione, 49). Certain of these substances were evaluated for antilipidemic activity in the cholesterol-fed rat model, and all analogues were studied for their ability to inhibit aggregation of human platelets. Results are compared to aci-reductones belonging to the 4-aryl- and 4-spiroalkyl-2-hydroxytetronic acid systems (4,5a,b). Redox potentials for all aci-reductones were determined with cyclic voltammetry. It would appear that the 4-aryl-2-hydroxytetronic acids represent leads for further study as antiatherosclerotic drugs owing to their favorable antilipidemic and antiaggregatory properties whereas the benzopyranones are of most interest as probes for platelet antiaggregatory mechanism studies.

Side-chain effects on phenothiazine cation radical reactions.

The cation radical of each of the phenothiazine tranquilizers is a likely intermediate in the metabolism of the drugs to at least two of the three major metabolic classes, the sulfoxides and the hydroxylated derivatives. Previous work has shown that the reactions of the radical are highly dependent on the environment, particularly the presence of nucleophiles. The present report discusses the effect of cation radical structure on the formation of sulfoxide and hydroxylated metabolites in vitro. Cyclic voltammetry, spectrophotometry, and liquid chromatography were used to examine reactions of various phenothiazine radicals in aqueous buffers. A radical with a three-carbon aliphatic side chain (e.g., chlorpromazine) forms solely sulfoxide and parent unless amine nucleophiles are present, in which case hydroxylation occurs. A shorter side chain (e.g., promethazine) causes radical dimerization and pronounced hydroxylation, regardless of external nucleophiles. A piperazine side chain (e.g., fluphenazine) promotes hydroxylation, with some sulfoxide observed. The results indicate that a deprotonated amine is necessary for hydroxylation and that the amine may be present in the original drug rather than an external nucleophile. In addition to information about cation radical reactions, the redox properties of several different phenothiazines are presented.

Effect of structure on phenothiazine cation radical reactions in aqueous buffers.

The reactions of the cation radicals of 11 phenothiazine tranquilizers were examined in mildly acidic aqueous buffers. Of the 11, those having an aminopropyl side chain in the 10 position reacted to form 0.5 mol of sulfoxide and 0.5 mol of parent drug per mole of initial radical. Cation radicals with different side chains react to form additional products, which remain to be identified but probably result from hydroxylation of the phenothiazine ring. The decay kinetics of three of the cation radicals undergoing reactions with known stoichiometry, namely, chloropromazine, promazine, and triflupromazine, were studied in detail, and it was concluded that they all react via the same mechanism. The mechanism involves attack of the cation radical by a nucleophile, and radicals with electron-withdrawing groups in the 2 position react more quickly. Since the cation radicals with faster reaction rates with nucleophiles are more pharmacologically active, it is hypothesized that the cation radical-nucleophile interaction may be responsible for binding of phenothiazines to receptor proteins.

Electrochemical oxidation of hydroxylated phenothiazine and imipramine derivatives.

The electrochemical oxidations of several hydroxylated derivatives of promazine, chlorpromazine, imipramine, and 3-chloroimipramine are examined and compared. Oxidation of the monohydroxyphenothiazine derivatives leads to both dihydroxy species and substituted benzoquinones, while oxidation of hydroxylated imipramines leads to only the corresponding benzoquinones. The oxidation potentials of 17 tricyclic psychoactive drugs and metabolites are tabulated and compared. The potential importance of these results to drug activity and side effects is discussed.

Reactions of chloropromazine cation radical with physiologically occurring nucleophiles.

The reactions between chlorpromazine cation radical and a variety of physiologically occurring nucleophiles, which involve formation of a covalent, yet reversible bond, have been examined. As reported earlier, this reaction does not involve disproportionation of the radical but, rather, direct reaction between radical and nucleophile. The resulting adduct further reacts to form chlorpromazine sulfoxide or hydroxylated derivatives, and the original nucleophile is regenerated. The products and kinetics of the reaction depend strongly on the identity of the nucleophile, with the sulfhydryl group being the fastest and water being the slowest of the nucleophiles studied. The likely involvement of these reactions in the metabolism of chlorpromazine is discussed. In addition, it is proposed that the radical/nucleophile interaction is a reasonable model reaction for the effects of chlorpromazine radical on neuronal enzymes and receptor sites.

Chemical and electrochemical oxidation of 7-hydroxychlorpromazine.

The oxidation of 7-hydroxychlorpromazine, a process associated with several side effects of chlorpromazine therapy, was examined in vitro by electrochemistry and rapid-scanning spectrophotometry. At pH 2, the oxidation results in a quantitative yield of 7,8-dioxochlorpromazine, but several intermediates are observable during the course of the reaction. These include a quinone imine with a half-life of 0.1 s, a monosubstituted benzoquinone with a half-life of approximately 50 s, and a disubstituted benzoquinone with a half-life of approximately 5 min. The concentrations of each intermediate were determined quantitatively as a function of time, and a complete oxidation mechanism is proposed. At pH 7, the yield of 7,8-dioxochlorpromazine is less than at pH 2, and an additional reaction pathway involving direct hydroxylation of the quinone imine is observed. The relationship of these reactions to the pharmacology of the hydroxylated chlorpromazine metabolites is discussed.

Oxidative reactions of hydroxylated chlorpromazine metabolites.

The oxidation pathways of two hydroxylated chlorpromazine metabolites were investigated using modern electrochemical techniques. Upon oxidation, the 7-hydroxy derivative of chlorpromazine rapidly reacts to form the 7,8-dihydroxy derivative and a substituted quinone. The oxidation potentials for both compounds were determined in the pH 3-8 range. The importance of these redox reactions and potentials to the pharmacology of the materials is discussed.

A kinetic analysis of a catechol-specific binding site in the microsomal fraction from the rabbit aorta.

(-)-3/-Norepinephrine (3H-NE) binding to the microsomal fraction of the rabbit aorta has been studied. Binding appears to increase linearly with time up to at least 30 min, shows no evidence of stereoselectivity and may be inhibited only by compounds possessing the catechol or 3-methoxy-4hydroxyphenyl moieties, with the latter being 100-fold less effective. 3H-NE binding is saturable with a Km of 8.5 X 10(-8) M and V max of 28 pmoles/mg protein. A Hill plot indicates that binding is noncooperative whereas a Scatchard plot suggests that two sites may be present. Binding does not appear to require physiological concentrations of Ca2+ or Mg2+ and is inhibited significantly by EDTA and sodium metabisulfite. In addition, binding is markedly enhanced by low and high pH values. This binding is also inhibited by sodium metabisulfite which suggests that an oxidized form of the catecholamine is the active binding species. Experiments with several group specific reagents indicate that binding may require a free sulfhydryl group but not a carboxyl function. The binding process requires an energy of activation of 14.8 kcal/mole whose magnitude may be partly explained, with the aid of optical rotatory dispersion spectra, by a non-stereoslective conformational change in protein structure induced by the amine. The characteristics of the 3H-NE binding sites observed in the microsomal fractional of the rabbit aorta appear to be different from those expected if binding were to the adrenoreceptors. A possible mechanism for catecholamine binding to free sulfhydryl groups on protein is presented.

Intracyclization rates of 6-hydroxydopamine and 6-aminodopamine analogs under physiological conditions.

It is agreed that the neurotoxic action of 6-hydroxydopamine and 6-aminodopamine is related to their ease of oxidation. The initial oxidation products, the p-quinone and p-quinone imine, readily undergo 1,2-intracyclization. These reactions could represent an important loss of active neurotoxic agent available uptake. A variety of substituted 6-aminodopamine analogs was prepared and their formal potentials and cyclization rates were measured accurately. The effect of the balance of ease of oxidation vs. rate of cyclization on their neurotoxicity was examined. The results are in general accord with in vivo lifetimes for 6-hydroxydopamine and 6-aminodopamine in rat caudate nucleus.

Potential oxidative pathways of brain catecholamines.

The possibility that catecholamines can be oxidized via aberrant pathways in vivo is open to question, but in vitro oxidation via aerobic manipulations is established. Assuming oxidation does occur, we have examined quantitatively the fast chemical reactions of the initial oxidation products, the o-quinones. The nature and rates of these reactions were studies under the conditions simulating closely those which presumably exist in mammalian brain. The results are in close accord with existing literature and especially support oxidation pathways recently reported in [3H]-norepinephrine binding to particulate cell fractions.