Clemens Mügge

Prognostic implications of elevated whole blood choline levels in acute coronary syndromes.

Troponins I and T represent the current biomarker standard for diagnosis of myocardial infarction. Even small increases of cardiac troponins have prognostic implications, but not all patients at risk are correctly classified, particularly at admission. We identified elevated whole-blood choline as a promising marker and performed a prospective study of 327 patients with a suspected acute coronary syndrome that focused on the analysis of troponin-negative patients. Diagnostic classification of patients and the definition of troponin cutoffs were performed according to the new European Society of Cardiology/American College of Cardiology criteria. Blood was sampled serially and choline was measured using high-performance liquid chromatography mass spectrometry in whole blood. Patients were followed for 30 days. In patients with negative troponin I test results at admission (n = 250), choline was a predictor of cardiac death and nonfatal cardiac arrest (hazard ratio 6.0, p = 0.003), life-threatening arrhythmias (hazard ratio 3.75, p = 0.004), heart failure (hazard ratio 2.87, p = 0.002), and coronary angioplasty (hazard ratio 2.57, p = 0.001). In multivariate analysis of troponin-negative patients, choline was the strongest predictor of cardiac death or arrest (odds ratio 6.05, p = 0.01). Choline was not a marker for myocardial necrosis but indicated high-risk unstable angina in patients without acute myocardial infarction (sensitivity 86.4%, specificity 86.2%). Thus, an increased concentration of choline at hospital admission is a predictor of adverse cardiac events in patients with suspected acute coronary syndromes. Whole blood choline may be useful for early risk stratification of these patients, particularly if troponin results are negative on admission.

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: A monitor of intramolecular hydrogen bonds in helical peptides?

Summary2D 1H NMR spectroscopy of two α-helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from −10 to −6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the α-helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in α-helices in H2O/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in α-helices.

Quantitative Measurements in Continuous-Flow HPLC/NMR

Two methods for the quantitative determination of compounds in continuous-flow HPLC/NMR are described. The first method uses an internal standard (caffeine) of known concentration directly mixed into the mobile phase, while with the second method, a known amount of internal standard is injected onto the column during the chromatographic run. The latter method was validated using several nitroaromatic compounds and explosives. Deviations between the injected and calculated amounts of analytes are usually below 10% while the relative standard deviation ranges from 2% in the upper microgram range to 40% at the limit of detection.

Isolation and Structural Elucidation of Steroid Oligoglycosides from the StarfishAsteriasrubens by Means of Direct Online LC-NMR-MS Hyphenation and One- and Two-Dimensional NMR Investigations

LC-NMR-MS hyphenation has been applied in the screening of an asterosaponin subfraction obtained from the starfish Asterias rubens for unknown compounds. The suitability of this technique for characterizing compounds in the molecular weight range of 1200 to 1400 a.m.u. has been demonstrated. The additional information concerning the number of exchangeable protons provided by the LC-NMR-MS experiment proved to be particularly valuable for the structural elucidation. The LC-NMR-MS screening indicated the presence of hitherto unknown asterosaponins in the sample. Based on this information, four new compounds (ruberosides A−D; 2−4 and 6) have been isolated and their structures have been elucidated using one- and two-dimensional NMR techniques. A minor component has tentatively been assigned the structure 5.

Application of Continuous-Flow HPLC-Proton-Nuclear Magnetic Resonance Spectroscopy and HPLC-Thermospray-Mass Spectroscopy for the Structural Elucidation of Phototransformation Products of 2,4,6-Trinitrotoluene.

An aqueous solution of 2,4,6-trinitrotoluene (TNT) was irradiated by natural sunlight for a period of 1 month to generate phototransformation products of this compound. After solid-phase extraction on a poly(styrene-divinylbenzene) copolymer at pH 1, the structures of several acidic nitroaromatic compounds were identified by means of continuous-flow HPLC/(1)H NMR and HPLC/TSP-MS measurements of this extract. By interpretation of both NMR and MS spectra, it was even possible to characterize noncommercially available phototransformation products of TNT. The results obtained by continuous-flow HPLC/(1)H NMR were compared with those obtained by the investigation of a groundwater sample of a former ammunition site near Elsnig, Germany. The results show that several identified phototransformation products of TNT are also present in this groundwater sample.

Local THz Time Domain Spectroscopy of Duplex DNA via Fluorescence of an Embedded Probe

We demonstrate that THz vibrational activity of a biopolymer can be measured locally, on the effective length scale for polar solvation, with an embedded molecular probe. For this purpose, the polarity probe 2-hydroxy-7-nitrofluorene was linked into a 13mer DNA duplex opposite an abasic site. The NMR solution structure shows that the fluorene moiety occupies a well-defined position in place of a base pair but can flip around the long axis on a millisecond time scale. Femtosecond optical pump-probe experiments are used to measure the time-resolved Stokes shift of emission from the probe. The dynamic shifts for solution in H(2)O and D(2)O are quantified. Their difference is much larger than that expected for free water, implying that only bound water is observed. A weak 26 cm(-1) spectral oscillation of the emission band is observed, which is not present when the probe is free in solution and is therefore caused by the supramolecular structure (DNA and hydration water).

Functional Characterization of the Recombinant N-Methyltransferase Domain from the Multienzyme Enniatin Synthetase

A 51 kDa fusion protein incorporating the N‐methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S‐adenosyl methionine (AdoMet) as demonstrated by cross‐linking experiments with 14C‐methyl‐AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H2 and H8 of the adenine moiety, H1’ of D‐ribose, and SCH3 group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl‐N‐acetylcysteamine thioesters (aminoacyl‐SNACs) of L‐Val, L‐Ile, and L‐Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4′‐phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl‐SNAC and 14C‐methyl‐AdoMet for various lengths of time, for up to 30 min. N‐[14C‐Methyl]‐aminoacyl‐SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N‐[14C‐methyl] amino acids. Further proof for the formation of N‐14C‐methyl‐aminoacyl‐SNACs came from MALDI‐TOF mass spectrometry which yielded 23 212 Da for N‐methyl‐valyl‐SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L‐Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D‐Val was not accepted as a substrate; this indicates selectivity for the L isomer.

Novel disulfide-constrained pentapeptides as models for β-VIa turns in proteins

The conformational behavior of cyclic peptides of the amino acid sequence Cys‐Phe/Ala‐Pro‐Ala‐Cys has been investigated through the combined use of molecular simulation methods and NMR experiments to find models for β‐VIa turns of proteins. Both oxidized (cyclic) peptides and reduced (linear) forms were investigated. At least 95% of the cyclic peptides show a cis conformation of the Xaa‐Pro bond in solution in DMSO or water, whereas all other peptide bonds are trans. Furthermore, we observed a hydrogen bond between the NH group of residue Ala4 and the C = O group of residue Cys1. Both properties are indicative of β‐VIa turns. After reduction of the disulfide bridge, the all‐trans form of the peptide bonds predominates.

Structure–reactivity relationship of Amadori rearrangement products compared to related ketoses

Structure-reactivity relationships of Amadori rearrangement products compared to their related ketoses were derived from multiple NMR spectroscopic techniques. Besides structure elucidation of six Amadori rearrangement products derived from d-glucose and d-galactose with l-alanine, l-phenylalanine and l-proline, especially quantitative (13)C selective saturation transfer NMR spectroscopy was applied to deduce information on isomeric systems. It could be shown exemplarily that the Amadori compound N-(1-deoxy-d-fructos-1-yl)-l-proline exhibits much higher isomerisation rates than d-fructose, which can be explained by C-1 substituent mediated intramolecular catalysis. In combination with a reduced carbonyl activity of Amadori compounds compared to their related ketoses which results in an increased acyclic keto isomer concentration, the results on isomerisation dynamics lead to a highly significant increased reactivity of Amadori compounds. This can be clearly seen, comparing approximated carbohydrate milieu stability time constants (ACuSTiC) which is 1 s for N-(1-deoxy-d-fructos-1-yl)-l-proline and 10 s for d-fructose at pD 4.20 ± 0.05 at 350 K. In addition, first NMR spectroscopic data are provided, which prove that α-pyranose of (amino acid substituted) d-fructose adopts both, (2)C5 and (5)C2 conformation.

Comparison of proline and N-methylnorleucine induced conformational equilibria in cyclic pentapeptides

The cyclic, imido acid containing pentapeptides cyclo(Asp‐Trp‐(NMe)Nle‐Asp‐Phe) (cpp[NMeNle3]) and cyclo(Asp‐Trp‐Pro‐Asp‐Phe) (cpp[Pro3]) have been investigated by 1H‐NMR spectroscopy in DMSO and by restrained molecular dynamics methods. The spectra indicate the existence of at least four cis/trans isomers for cpp[NMeNle3] and two cis/trans isomers for cpp[Pro3]. In addition to the imido peptide bonds, cpp[NMeNle3] shows cis/trans isomerization of the Asp4‐Phe5 and Phe5‐Asp1 peptide bonds whereas only the Phe5‐Asp1 peptide bond isomerizes in the Pro‐containing peptide. In cpp[Pro3] all cis bonds are centred in βVIb turns. Also, cpp[NMeNle3] prefers backbone angles around the cis bonds which are rather similar to the angles of a βVIb turn. The higher number of cis/trans isomers and slight deviations in the backbone angles of comparable isomers of both peptides are caused by an enhanced flexibility of cpp[NMeNle3] due to the possibility of the φ‐(NMe)Nle rotation.