Paul Schuler

Determination of astaxanthin and astaxanthin esters in the microalgae Haematococcus pluvialis by LC-(APCI)MS and characterization of predominant carotenoid isomers by NMR spectroscopy

The oily product ZANTHIN® consists of natural astaxanthin, which is manufactured from the microalgae Haematococcus pluvialis by supercritical CO2 extraction. An HPLC method was developed to separate all of the components of the complex astaxanthin extract using a C30 column. The separation resulted in different isomers of astaxanthin accompanied by two other carotenoids. The main component consisted of astaxanthin singly esterified with several different fatty acids. C18:3, C18:2, C18:1 and C16:0 were identified as the most commonly occurring fatty acids. Doubly esterified astaxanthin was also found, although in lower concentrations compared to singly esterified astaxanthin. After performing a detailed fatty acid analysis by GC-MS, the peaks from the extract were assigned via HPLC-MS. A trans to cis transmutation of the all-trans compound was performed by thermal treatment in order to obtain an enrichment of cis isomers as the basis for unambiguous identification via NMR experiments. The all-trans as well as the 9- and 13-cis isomers of astaxanthin were characterized in detail by UV/Vis, 1H, and 1H,1H COSY NMR spectroscopy.

Online coupling of gas chromatography to nuclear magnetic resonance spectroscopy: method for the analysis of volatile stereoisomers.

The identification of volatile cis/trans-stereoisomers was accomplished by employing a hyphenated GC-NMR system. The chromatographic and spectroscopic conditions were optimized with respect to the (1)H NMR detection. A special processing technique was developed to handle the recorded NMR spectra in the gas phase with very low sample amounts. The processed stopped-flow (1)H NMR spectra of the investigated chromatographic peaks unequivocally revealed the structure of the corresponding compounds.

The ROS-induced cytotoxicity of ascorbate is attenuated by hypoxia and HIF-1alpha in the NCI60 cancer cell lines

Intravenous application of high‐dose ascorbate is used in complementary palliative medicine to treat cancer patients. Pharmacological doses of ascorbate in the mM range induce cytotoxicity in cancer cells mediated by reactive oxygen species (ROS), namely hydrogen peroxide and ascorbyl radicals. However, little is known about intrinsic or extrinsic factors modulating this ascorbate‐mediated cytotoxicity. Under normoxia and hypoxia, ascorbate IC50 values were determined on the NCI60 cancer cells. The cell cycle, the influence of cobalt chloride‐induced hypoxia‐inducible factor‐1α (HIF‐1α) and the glucose transporter 1 (GLUT‐1) expression (a pro‐survival HIF‐1α‐downstream‐target) were analysed after ascorbate exposure under normoxic and hypoxic conditions. The amount of ascorbyl radicals increased with rising serum concentrations. Hypoxia (0.1% O2) globally increased the IC50 of ascorbate in the 60 cancer cell lines from 4.5 ± 3.6 mM to 10.1 ± 5.9 mM (2.2‐fold increase, P < 0.001, Mann–Whitney t‐test), thus inducing cellular resistance towards ascorbate. This ascorbate resistance depended on HIF‐1α‐signalling, but did not correlate with cell line‐specific expression of the ascorbate transporter GLUT‐1. However, under normoxic and hypoxic conditions, ascorbate treatment at the individual IC50 reduced the expression of GLUT‐1 in the cancer cells. Our data show a ROS‐induced, HIF‐1α‐ and O2‐dependent cytotoxicity of ascorbate on 60 different cancer cells. This suggests that for clinical application, cancer patients should additionally be oxygenized to increase the cytotoxic efficacy of ascorbate.

Online coupling of enantioselective capillary gas chromatography with proton nuclear magnetic resonance spectroscopy

The hyphenation of enantioselective capillary gas chromatography and mass spectrometry is not always sufficient to distinguish between structural isomers, thus requiring peak identification by NMR spectroscopy. Here the first online coupling of enantioselective capillary gas chromatography with proton nuclear resonance spectroscopy is described for the unfunctionalized chiral alkane 2,4-dimethylhexane resolved on octakis(6-O-methyl-2,3-di-O-pentyl)-gamma-cyclodextrin at 60 degrees C. NMR allows constitutional and configurational isomers (diastereomers and enantiomers) to be distinguished. Enantiomers display identical spectra at different retention times, which enable an indirect identification of these unfunctionalized alkanes. The presented method is still at an early development stage, and will require instrumental optimization in the future.

Paramagnetische ionenpaare als vorstufen bei der synthese von zinn(IV)-sauerstoffheterocyclen

Triorganyltin hydroxides react with catechols in organic solvents in the presence of air oxygen at first to paramagnetic complexes which are investigated by ESR spectroscopy. The reaction has been run using 6 catechols and 5 different organometallic compounds in numerous solvents, so that its general application is proved. The ESR spectra of the solutions show typical hydrogen hyperfine structure and large splittings, which have to be assigned to magnetic coupling of the free electron with 117Sn and 119Sn nuclei. The tin splittings and the g-values significantly depend on the temperature and the solvent. These observations are interpreted in terms of ion pairing effects. A quantitative interpretation is given. The final products of these reactions are diamagnetic compounds which can be isolated in good yields. Based on the elemental analysis, 1H NMR and mass spectroscopy (electron impact, field desorption), the structures are assigned of partly dimerised o-phenylene dioxystannanes.

[6,6]‐Open and [6,6]‐Closed Isomers of C70(CF2): Synthesis, Electrochemical and Quantum Chemical Investigation

Novel difluoromethylenated [70]fullerene derivatives, C70(CF2 )n (n=1-3), were obtained by the reaction of C70 with sodium difluorochloroacetate. Two major products, isomeric C70(CF2 ) mono-adducts with [6,6]-open and [6,6]-closed configurations, were isolated and their homofullerene and methanofullerene structures were reliably determined by a variety of methods that included X-ray analysis and high-level spectroscopic techniques. The [6,6]-open isomer of C70(CF2 ) constitutes the first homofullerene example of a non-hetero [70]fullerene derivative in which functionalisation involves the most reactive bond in the polar region of the cage. Voltammetric estimation of the electron affinity of the C70(CF2 ) isomers showed that it is substantially higher for the [6,6]-open isomer (the 70-electron π-conjugated system is retained) than the [6,6]-closed form, the latter being similar to the electron affinity of pristine C70. In situ ESR spectroelectrochemical investigation of the C70(CF2 ) radical anions and DFT calculations of the hyperfine coupling constants provide evidence for the first example of an inter-conversion between the [6,6]-closed and [6,6]-open forms of a cage-modified fullerene driven by an electrochemical one-electron transfer. Thus, [6,6]-closed C70(CF2 ) constitutes an interesting example of a redox-switchable fullerene derivative.

Recognition of chirality in nitroxides using EPR and ENDOR spectroscopy

The question regarding chirality in nitroxides was rigorously investigated, namely, (a) are multiple EPR and ENDOR spectra expected from nitroxides with chiral centers, (b) which structures provide multiple structures and (c) under what conditions (temperature and solvent) are they obtained. It is shown that nitroxides with one chiral center produce only one EPR/ENDOR spectrum in toluene or ethanol (293–193 K); the nitroxyl function is not a chiral center under these conditions. Nitroxides with two chiral centers (α‐ and β‐position) give two EPR/ENDOR spectra consistent with a pair of diastereomeric configurations (see series II), but this observation was not apparent in every case (see series III). This anomaly was attributed to a stereoselective production of certain nitroxides which apparently do not produce diastereomeric mixtures in alcohols as solvent. Further examples of multiple EPR/ENDOR spectra were found when the position of chirality is in the α‐ and β‐position to a remote spin center such as the nitroxide function (see series IV and V). Finally, EPR/ENDOR spectra were obtained from nitroxide derivatives wherein the absolute configuration at the α‐carbon atom was known. Distinctly different EPR/ENDOR spectra were indeed obtained for every diastereomer, thus establishing this point unequivocally. Copyright

Chiral recognition by formation of paramagnetic diastereomeric complexes

The chiral 4-(α-hydroxy-benzyl)-2,6-di-tert. butyl-phenoxyl has been examined in its racemic form in toluene and carbontetrachloride. On adding of chiral auxiliaries (R)-resp. (S)-N,N-dimethyl-1-phenylethylamine or (S)-phenylethyl-amine two different couplings of the β-proton are recorded by ENDOR spectroscopy. The experimental results are interpreted by a ternary equilibrium between radical, solvent and auxiliary. A model for the suggested association processes is given and equilibrium constants and corresponding enthalpies are calculated.

Permutationsisomerisierung am pentakoordinierten Zinnatom. EPR- und ENDOR-Untersuchungen paramagnetischer Zinnkomplexe

Abstract Triphenyltin compounds and 9-tolyl-stannoles react with catechols or o -benzoquinones to give paramagnetic complexes. The structures were characterised by EPR and ENDOR spectroscopy. The spectra obtained were interpreted under the assumption that there is an equilibrium between two different paramagnetic species. Investigation of the temperature dependence leads to the kinetic parameters of these processes. From these values and the low temperature spectra a permutation isomerisation mechanism is proposed.

Synthesis and Spin Trapping Stereochemistry of the Chiral Spin Trap, 5,5-Dimethyl-3-phenylpyrroline-1-oxide

A chiral spin trap, 5,5‐dimethyl‐3‐phenylpyrroline‐1‐oxide (5), and several hydroxylamines were synthesized. The structures and conformations of these compounds were investigated mainly by 1H NMR spectroscopy. This spin trap was used to trap carbon‐ and oxygen‐centered radicals. The corresponding hydrogen spin adduct was prepared by oxidizing the hydroxylamine to the nitroxide radical. By the combination of 1H NMR and EPR results, it was shown that the conformation with the phenyl group of C‐3 at the equatorial position is exclusively populated and its lifetime is long compared with the EPR time‐scale. Addition of carbon‐centered radicals to 5 leads to trans adducts whereas oxygen‐centered radicals formed cis isomers. These could be confirmed by the investigation of the monovalent oxidation products of the hydroxylamines. The preference for these conformers is explained by the competition between steric and stereoelectronic effects. Despite the racemic nature of 5 and the formation of a new chiral center(s) in the spin adducts, the presence of different diastereomers would not be observed by EPR whereas 1H NMR studies of some of the nitrones showed clear evidence of diastereomeric mixtures. However, the spin adduct of sec‐hydroxybutyl radical showed some linewidth effects which could be attributed to the presence of two groups of diastereomers that were resolved by ENDOR spectroscopy. In general, the spin adducts of 5 are closely related to those of the well known DMPO, but the presence of a phenyl substituent at the 3‐position results in a variation of the β‐H coupling constants. In contrast to DMPO, 5 can scavenge short‐lived radicals in aqueous and non‐aqueous solutions.