No abstract

H and 13 C NMR spectra were recorded on a Bruker DRX 500 instrument (BBO probe) at 500.13 and 125.76 MHz, respectively, in steps of 10 K between 210 and 280 K (15 mg of the base form in 600 L CDCl 3 ), between 180 and 280 K (10 mg of the hydrochloride in 600 L [d4]methanol), and 298 K. Temperature calibration was done with a methanol reference sample. 1 H NMR spectra were acquired using a spectral width of 12 kHz, an acquisition time of 5.5 s and 16 scans, zerofilled to 128 k datapoints (0.09 Hz per point) and processed with a 1 Hz exponential decay. 13 C NMR spectra were acquired using a spectral width of 30 kHz, an acquisition time of 1.1 s and 2048 scans in about 1h, zerofilled to 128 k datapoints (0.23 Hz per point) and also processed without apodization. Peaks were fitted to a Lorentzian lineshape using MestreNova 8.0.0 (MestreLab Research S.L.) To increase the signal-to-noise ratio for peaks broadened beyond 20 Hz, the 13 C NMR spectra were reprocessed with a 5 Hz exponential decay. 1 H-EXSY spectra were recorded on a TBI inverse probe at 210 K (both samples), 200 and 190 K (hydrochloride in methanol-d 4 ). The spectra were acquired using a spectral width of 4 kHz, 1024 x 512 complex time domain datapoints, a mixing period of 50 ms (additionally 200 ms at 190 K) and 8 scans in about 4.5 h. The spectra were zerofilled to 2048 x 2048 datapoints and processed with a shifted square sine bell apodization in both dimensions. Populations and exchange rates were obtained from diagonal-and crosspeak integrals using EXSYCalc (MestreLab Research S.L.).

Sirtuins are nicotinamide dinucleotide-dependent class III histone deacetylases catalyzing various physiological processes involved in cell proliferation, differentiation, apoptosis, and ageing. This makes them attractive targets in drug research. In order to simplify sirtuin substrates for assay development, two N(ɛ)-acetyllysine derivatives, N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine amide, and N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine methyl ester were synthesized and evaluated as substrates for human SIRT1 in a capillary electrophoresis-based enzyme assay. Substrate, deacetylated product, and the coproduct nicotinamide were separated in a 200 mM phosphate/Tris buffer at pH 2.85. Field-amplified sample injection was employed to achieve sufficient assay sensitivity. While the ester derivative was not recognized by the enzyme, the amide substrate was effectively converted to the deacetylated product. The assay was subsequently validated with respect to range, linearity, limit of detection, and limit of quantification. Michaelis-Menten kinetic parameters, K(m) = 83 μM and V(max) = 6.8 μM/min were determined. The applicability of the assay for inhibitor screening was demonstrated using the known inhibitors sirtinol and the suramin derivate NF258. Resveratrol did not increase the deacetylation rate at concentrations of up to 200 μM. Docking experiments revealed the necessity of an amide function at the C-terminus of nonpeptide substrates while more structural freedom is tolerated at the N-terminus of N(ɛ) -acetyllysine.

The SPE of leukotrienes and eicosatetraenoic acids using anion exchange materials was compared to the classical extraction with C18 columns. A silica-based strong anion exchanger, a polymer-based weak anion exchanger, and a polymer-based mixed-mode strong anion exchanger were studied. All anion exchange materials displayed a higher recovery of the analytes with values between 70 and 90% when extracting standard solutions and analyzing by HPLC. The effect was less pronounced for the analysis of the compounds in incubations of polymorphonuclear leukocytes. Using MEKC with head-column field-amplified sample stacking for analyte quantification, much lower values of the peak areas were observed compared to the determination of the recovery of the analytes by HPLC. Using MEKC analysis, the highest values were found for the polymer-based weak anion exchange material, while values below 10% were found for the polymer-based mixed mode strong anion exchanger. This could be attributed to the presence of electrolytes in the eluates that compromised the stacking efficiency. The extent of residual electrolytes depended on the SPE protocol, resulting in large differences of the amount of analyte determined by MEKC when applying head-column field-amplified sample stacking for online analyte concentration.

Structural aspects such as chemical exchange, dimerization, solvent association, nitrogen inversion and protonation status of strychnine were investigated using experimental and calculated data. The information was mainly interpreted in view of a successful determination of the absolute configuration (AC) with strychnine (base and salt) as test molecule due to its importance in chemistry. By geometry optimization a stable isomer of protonated strychnine was found with an inverted nitrogen, however, 25 kcal/mol higher in energy. It is shown that solvent association can be assumed in protic solvents such as methanol and dimerization to a small extent in polar/protic solvents. However, the monomeric structural model neglecting explicit solvent molecules still allows the correct prediction of the AC of base and hydrochloride using optical rotation and ECD data.