Nuclear Overhauser Enhancement Measurements

For the measurement of homonuclear NOE it is normal to switch off the decoupler just before measurement, in order to reduce artifacts. Small NOE s on sensitive nuclei such as H are conveniently identified by computer subtraction of the enhanced and unenhanced spectra, i.e., by NOE difference spectroscopy. As this subtraction is rarely perfect, it is wise to check for the presence of an overall peak integral to confirm that an apparent residual resonance is not merely a subtraction artifact. The percentage NOE of a peak is readily calculated by comparison of its area with the (negative) area of the large peak which will always arise from saturation at the position of irradiation. Irradiation sufficient for such spectroscopy can be specific within a typical range of +40 Hz. 

In some cases it is possible to obtain even greater sensitivity enhancements by using other polarization transfer techniques, some of which are described at the end of the next section. 

Although Ti may often be readily obtained from the linewidth IjuTi, there are also frequent cases where this linewidth is obscured by overlapping or, in the case of 

One limitation on the spin-echo measurement of T2 is that if the 180° pulse inverts the resonances of spins coupled to each other, then this is equivalent to symmetrical switching of the Larmor precession frequencies of each multiplet component. Thus although each component, say of a doublet, will refocus adequately, it will do so with a phase error of tJ, where J is the doublet separation. The echo train will thus be modulated according to J. 

Fourier transformation of the echo permits one to inspect the time evolution of such individual resonances. Indeed, their evolution may be treated as a new type of FID, and therefore Fourier-transformed in a second dimension. This is the basis of /-resolved two-dimensional spectroscopy, which is described in a more general context in Section 8. 

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Pulsed n.m.r. methods have been used to probe the conformations adopted by 2 -amino-2 -deoxyadenosine, 3 -amino-3 -deoxyadenosine, and puromycin. Longitudinal proton relaxation time and nuclear Overhauser enhancement measurements were carried out to characterize orientation of the base relative to ribose. The latter two compounds have a preferred N-anti-g+ conformation, while 2 -amino-2 -deoxyadenosine adopts the S-syn-g jt family of conformations. These findings corroborate the previous proposal relating the N state of ribose with a/t//-orientation of the base and the S state with syn orientation. Nuclear Overhauser enhancements have also been used to determine the site of glycosylation in nucleosides of -triazole the n.O.e. of H-r due to 3-Me or 5-Me showed that the structure of the major product of fusion of l-0-acetyl-2,3,5-tri-0-benzoyl-j8-D-ribofuranose with 3,5-dimethyl-1,2,4-triazole was (9) and that of the minor was (10). High resolution... 

Nuclear Overhauser enhancement (NOE) spectroscopy has been used to measure the through-space interaction between protons at and the protons associated with the substituents at N (20). The method is also useful for distinguishing between isomers with different groups at and C. Reference 21 contains the chemical shifts and coupling constants of a considerable number of pyrazoles with substituents at N and C. NOE difference spectroscopy ( H) has been employed to differentiate between the two regioisomers [153076 5-0] (14) and [153076 6-1] (15) (22). N-nmr spectroscopy also has some utility in the field of pyrazoles and derivatives. 

N-protonation the absolute magnitude of the Ad values is larger than for Af-methylation <770MR(9)53>. Nuclear relaxation rates of and have been measured as a function of temperature for neat liquid pyridazine, and nuclear Overhauser enhancement has been used to separate the dipolar and spin rotational contributions to relaxation. Dipolar relaxation rates have been combined with quadrupole relaxation rates to determine rotational correlation times for motion about each principal molecular axis (78MI21200). NMR analysis has been used to determine the structure of phenyllithium-pyridazine adducts and of the corresponding dihydropyridazines obtained by hydrolysis of the adducts <78RTC116>. 

More recent studies on the folded toxin structure by Norton and colleagues have utilized h- and C-NMR techniques (19,20). By using 2D-FT-NMR, it was possible to localize a four stranded, antiparallel )5-pleated sheet "backbone structure in As II, Ax I, and Sh I (21,22), In addition, Wemmer et al. (23) have observed an identical )5-pleated structure in Hp II. No a-helix was observed in these four variants. In the near future, calculated solution conformations of these toxins, utilizing distance measurements from extracted Nuclear Overhauser Enhancement (NOE) effects should greatly stimulate structure-activity investigations. 

The 50.31 MHz 13C NMR spectra of the chlorinated alkanes were recorded on a Varian XL-200 NMR spectrometer. The temperature for all measurements was 50 ° C. It was necessary to record 10 scans at each sampling point as the reduction proceeded. A delay of 30 s was employed between each scan. In order to verify the quantitative nature of the NMR data, carbon-13 Tj data were recorded for all materials using the standard 1800 - r -90 ° inversion-recovery sequence. Relaxation data were obtained on (n-Bu)3SnH, (n-Bu)3SnCl, DCP, TCH, pentane, and heptane under the same solvent and temperature conditions used in the reduction experiments. In addition, relaxation measurements were carried out on partially reduced (70%) samples of DCP and TCH in order to obtain T data on 2-chloropentane, 2,4-dichloroheptane, 2,6-dichloroheptane, 4-chloroheptane, and 2-chloroheptane. The results of these measurements are presented in Table II. In the NMR analysis of the chloroalkane reductions, we measured the intensity of carbon nuclei with T values such that a delay time of 30 s represents at least 3 Tj. The only exception to this is heptane where the shortest T[ is 12.3 s (delay = 2.5 ). However, the error generated would be less than 10%, and, in addition, heptane concentration can also be obtained by product difference measurements in the TCH reduction. Measurements of the nuclear Overhauser enhancement (NOE) for carbon nuclei in the model compounds indicate uniform and full enhancements for those nuclei used in the quantitative measurements. Table II also contains the chemical... 

Little difference was noted when peak heights were used. The error in the T data is less than + 10%. Nuclear Overhauser enhancement factors (q) were obtained by measuring the integrated intensity of peaks in a difference spectrum from one with enhancement minus one with no enhancement and dividing that value by the integral from the one with no enhancement i.e. n ( nOe no nOe / (I nOe" Accuracy should be 10% or better. Linewidtns were measured at half heights, and chemical shifts are relative to TMS. 

The latter, in contrast to nuclear Overhauser enhancement and exchange spectroscopy (NOESY), always feature positive NOEs (negative cross-peaks with respect to diagonal), eliminating known problems of NOEs vanishing or spin diffusion, depending on correlation time, when high field spectrometers are used for measurements of medium-size compounds. 

Spin-lattice relaxation times were measured by the fast inversion-recovery method (24) with subsequent data analysis by a non-linear three parameter least squares fitting routine. (25) Nuclear Overhauser enhancement factors were measured using a gated decoupling technique with the period between the end of the data acquisition and the next 90° pulse equal to eibout four times the value. Most of the data used a delay of eibout ten times the Ti value. (26)... 

The layout of this chapter is as follows. The aspects of relaxation theory of interest for this article are summarized very briefly in Section 2. Section 3 deals with general aspects of relaxation measurements, including polarization transfer techniques for improving the sensitivity. Sections 4, 5 and 6 cover measurements of T, T2 and the nuclear Overhauser enhancement, respectively. 

The stereochemistry of the double bond in 4-(a-arylethylidene)-2-phenyl-5(4//)-oxazolones can be determined by measurements of long-range heteronuclear selective carbon-13 proton nuclear Overhauser enhancements. In the (Z)-isomers 774, large nuclear Overhauser enhancements are observed for the carbonyl carbon atom upon presaturation of the methyl group (Fig. 7.65). These effects are much smaller for the ( ) isomers. ... 

Although crosslinked polymers and polymer gels are not soluble, the spectra of swollen, low crosslink density networks exhibit reasonably narrow C-13 NMR line widths, sufficiently resolved to reveal details of microstructure 13S). Thus, recording the spectra under scalar low power decoupling yields characterization information and some dynamic measurements, concerning T, T2 (line widths) and nuclear Overhauser enhancement (NOE) for lightly crosslinked polymers. 

Notably, two isomeric products can be generated. The usual infrared (IR) and mass spectra as well as H and 13C NMR chemical shifts could not define which isomer was formed. The authors used different NMR techniques, such as 2-D heteronuclear multiple bond correlation (HMBC) experiments and phase-sensitive nuclear overhauser enhancement spectroscopy (NOESY) measurements to elucidate the product s structure. 

A third type of information available from NMR comes from the nuclear Overhauser enhancement or NOE. This is a direct through-space interaction of two nuclei. Irradiation of one nucleus with a weak radio frequency signal at its resonant frequency will equalize the populations in its two energy levels. This perturbation of population levels disturbs the populations of nearby nuclei so as to enhance the intensity of absorbance at the resonant frequency of the nearby nuclei. This effect depends only on the distance between the two nuclei, even if they are far apart in the bonding network, and varies in intensity as the inverse sixth power of the distance. Generally the NOE can only be detected between protons (XH nuclei) that are separated by 5 A or less in distance. These measured distances are used to determine accurate three-dimensional structures of proteins and nucleic acids. 

Even before the common use of Fourier transform NMR, it was known that the homonuclear nuclear Overhauser enhancement (NOE) could be used to identify pairs of protons separated by a few angstroms in space.2 It was also clear, however, that many such distances and accurate assignments to specific protons would be needed to define a solution structure fully. The advent of two-dimensional NMR was a major step toward providing the necessary data,3-4 but the introduction of the two-dimensional NOE measurement (NOESY)5-6 truly revolutionized the ability both to assign spectral peaks and to acquire large quantities of distance information. 

We described the basic aspects of NOESY in Section 10.1 as an introductory example of a 2D experiment. NOESY is very widely used in measuring macro-molecular conformation, as we see in Chapter 13. However, as shown in Fig. 8.4, the H— H nuclear Overhauser enhancement 17 varies from its value of +0.5 in small molecules to a limiting value of — 1 in large polymers with very long Tc, and at intermediate values of rc the NOE may vanish. An alternative is to use the NOE measured in the rotating frame, as this quantity is always positive. By analogy to NOESY, this technique has the acronym ROESY (rotating frame Overhauser enhancement spectroscopy),... 

After completion of the spectral assignment, NMR-derived short- and long-range distances can be determined by measurement of nuclear Overhauser enhancements (or NOEs). An NOE is an interaction between a pair of atoms <5.0 A apart, and the... 

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