Andrew S. Zektzer

Response-intensity modulation in long-range heteronuclear two-dimensional NMR chemical-shift correlation

Two-dimensional NMR spectroscopy has had a very significant impact on the way in which structure elucidation and spectral assignment studies are performed. Investigators have begun to exploit long-range heteronuclear coupling constants by utilizing delays in the conventional 2D NMR heteronuclear chemical-shift correlation experiment optimized for long-range magnetization transfer through two, three, and/or four bonds (‘Jcu, 3JcH, and “J,,) (1-16). Alternatively, several new experiments specifically for the observation of long-range heteronuclear spin-coupling information (14, 15, 2 7-22) have been reported. Despite the now numerous applications of longrange heteronuclear chemical-shift correlation contained in the literature, factors affecting the magnetization transfer step in these experiments have not been carefully investigated. Most long-range experiments are optimized for a 10 Hz magnetization transfer based upon the work of Reynolds and co-workers (2) or that of Wemly and Lauterwein (9), although other optimizations have also been used with success (I, 4, 10). As a consequence of the absence of responses in some of our own work despite optimization of the experiment at or near the magnitude of the coupling in question we now wish to communicate the results of our preliminary investigation into factors affecting response intensity in long-range heteronuclear chemical-shift correlation. Schenker and von Philipsborn (23) have reported a systematic investigation of the optimization of the INEPT and DEPT experiments in weakly coupled spin-f systems. They have shown that when magnetization is transferred via small heteronuclear couplings, that the transfer may be modulated by other homoand/or heteronuclear spin couplings which are experienced by those nuclei involved in the polarization transfer. Population transfer involved in the long-range heteronuclear chemical-shift correlation experiments may also be modulated in similar fashion, as recently suggested by Freeman (18) and Bax (21).

Trans-4-methoxypipecolic acid, an amino acid from Inga paterno

Abstract A new trans -4-methoxy derivative of pipecolic acid was isolated from the tropical legume, Inga paterno . Structure elucidation and conformational analysis were ascertained using a variety of NMR techniques.

The lanthanide-induced-shift-assisted determination of proton spin-lattice relaxation times

Abstract A new method is proposed for the determination of NMR relaxation times in second-order (strongly coupled) spectra. The method exploits the shifting abilities of the lanthanide shift reagent, Eu(fod)3. It is shown that, in favorable cases, the spectral dispersion afforded by a shift reagent can allow one to determine T2s and then extrapolate them back to zero LIS (absence of shift reagent) to get the relaxation times for the protons in the strongly coupled spectra. It is shown that complications due to cross-relaxation are absent. The technique is illustrated for the case of adamantanone interacting with Eu(fod)3 in CDCI3 as solvent. Data derived from 13C NMR spin-lattice relaxation on the same molecule can be used to determine what percentage of the proton relaxation is caused by the dipolar relaxation mechanism.

Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes

Hepatic microsomal membranes, prepared under various conditions that yield either intact or disrupted microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of N-ethylmaleimide. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the W/S ratio. For latent microsomes, the value of this parameter was determined to be 0.65 +/- 0.02 and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The W/S ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The W/S ratio was observed to be dependent upon the method of microsome preparation yielding values of 1.02 +/- 0.02 for hypertonically disrupted vesicles and 1.28 +/- 0.02 for mechanically disrupted vesicles. Microsomal marker enzymes such as cytochrome P-450 and FAD-containing monooxygenase retained significant levels of functionality following nitroxide incorporation.