NOE difference measurements

The nOe difference measurements not only help in stereochemical assignments but also provide connectivity information. A large nOe at 8 6.91 (G-8H), resulting from the irradiation of the methyl singlet of the methoxy group (8 3.94), confirms their proximity in space. This nOe result is consistent with structure A for the coumarin. 

Nuclear Overhauser effect (NOE) difference measurements were used to assign structure 79 for the product of reaction of diphenylnitrile imine with 5-ethylsulfonyl-2-methyl(27/)pyridazinone. Thus in the H NMR spectrum the ot/, o-protons of the arylhydrazino moiety (which were identified by two-dimensional heteronuclear multiple quantum correlation (2-D HMQC) spectroscopy) were shown in differential NOE (DNOE) experiment to be significantly enhanced on irradiation of pyridazine hydrogen H-7, proving their steric proximity <2000JST13>. 

In Chapter 5 we observed NOE interactions by ID NOE difference, measuring the steady-state NOE resulting from a long (several seconds), low-power continuous-wave irradiation of one nucleus. The modern selective (DPFGSE) ID NOE experiment... 

The clear advantages of the gradient-selected NOE experiment over the conventional steady-state NOE difference means this is becoming a popular tool in small molecule structural studies. However, there are fundamental differences between the data presented by the two experimental protocols, with steady-state experiments observing equilibrium NOEs and transient experiments observing kinetic NOEs. As a consequence, ID NOESY experiments demand a somewhat different approach to data interpretation over that currently adopted for steady-state NOE difference measurements, some of the key considerations include ... 

The structure of another closely related alkaloid, valachine, has also been established using NOE difference measurements. The NOE effects (% increases) observed between protons lying in close proximity to one another are shown in Figure 3.23. 

NOE difference spectroscopy is used to identify short, throughspace interactions and measure these interactions to an upper limit of about... 

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. 

The nOe difference spectrum has the advantage that it allows measurements of small nOe effects, even 1% or below. The experiment involves switching on the decoupler to allow the buildup of nOe. It is then switched off, and a w/2 pulse is applied before acquisition. The nOe is not affected much by the decoupler s being off during acquisition, since the nOes do not disappear instantaneously (the system takes several Ti seconds to return to its equilibrium state). 

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... 

Fig. 12a—c NOE experiments carried out at 200 MHz on compound 3. a Normal spectrum, with expansion of methine doublet b selective NOE spectrum, total time required 18 min c NOE difference spectrum, total time required (preparation, measurement) 42 min... 

There are two types of NOE experiments that can be performed. These are referred to as the steady-state NOE and the transient NOE. The steady-state NOE experiment is exemplified by the classic NOE difference experiment [15]. Steady-state NOE experiments allow one to quantitate relative atomic distances. However, there are many issues that can complicate their measurement, and a qualitative interpretation is more reliable [16]. Spectral artifacts can be observed from imperfect subtraction of spectra. In addition, this experiment is extremely susceptible to inhomogeneity issues and temperature fluctuations. 

Temperature consistency between measurements performed on different spectrometers is particularly critical for accurate interpretation of the data (see Refs. [19, 20] for post-acquisition temperature consistency tests). However, temperature control and equalization are also important for the combined analysis of T1, T2, and NOE data measured on the same spectrometer, because of the possible temperature differences between these measurements. Fig. 12.1 illustrates the sensitivity of relaxation parameters to temperature variations. Accurate measurement of protein dynamics requires that all experiments be done at the same temperature. To improve temperature consistency between Tlr T2, and... 

X-ray crystallography, docking modes can be validated by various NMR techniques NOEs may be observed between the ligand and the receptor protein by heteronuclear-filtered NOE spectroscopy [51], chemical shift changes of protein resonances upon binding can be analyzed by simulation of shifts caused by ring currents and electrostatic effects [52], and saturation transfer difference measurements indicate which part of the ligand is in direct contact with the protein [52]. 

The simplest and most popular experimental method is the well known one-dimensional (ID) NOE difference procedure [3], which is very easily implemented in any spectrometer and which can be routinely set up even by novice spectrometer operators. However, this difference method is based on subtraction of the unperturbed spectrum from the NOE-containing one, both separately recorded, and therefore the required difference information contributes only a small part of the recorded signal. Furthermore, the difference spectrum is very sensitive to subtraction errors, as well as pulse imperfections or missettings, or other spectrometer instabilities, all of which often result in prominent phase distortions or other subtraction artifacts which prevent the accurate measurement of the desired NOE values. Therefore the reliable measurement (or even detection) of enhancements below 1 % is not generally available using this difference method. 

Figure 2 Average NOE difference spectra measurements for 3-methyl-1,2,4-trioxolane.

A large enhancement of H2 from H3 would not have been expected for conformation a because H2 is much closer to Hy. So there must also be a large population of conformation b. We cannot easily quantify the relative populations from this NOE difference spectrum because H2 and H4 are in such different proton environments ( 6.5.3). Equilibration between the two conformations is obviously fast on the chemical shift timescale. The appropriate experiment here is separate irradiation of H2 and H4 and measurement of the relative enhancements at H3 and H . This experiment is illustrated below, and the resulting spectra are shown on the next page. 

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