NOESY

There are two basic 2-D NMR experiments that make use of the NOE the NOESY and the ROESY [1] experiments. NOESY stands for nuclear Overhauser effect spectroscopy and ROESY stands for rotational Overhauser effect spectroscopy. The ROESY experiment is also referred to in some of the literature as the CAMELSPIN experiment. The principal difference between the NOESY and ROESY experiments lies in the time scale associated with the dipolar relaxation mechanism. 

Rotational Overhauser effect spectroscopy, ROESY. Syn. CAMELSPIN experiment A 2-D NMR experiment similar to the 2-D NOESY experiment except that the ROESY experiment employs a spin-lock using the B, field of the applied RE, thus skirting the problem of the cancellation of the NOE cross peak when correlation times become long enough to reduce the rate constant for the dipolar doubl quantum spin flip. 

A large number of ID nOe experiments may have to be performed if the spatial relationships among many protons in a molecule are to be determined. In such cases, instead of employing multipulse ID nOe experiments, we can opt for the nOe spectroscopy (NOESY) experiment. If many protons have close chemical shifts, then NOESY may be particularly advanta- 

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NOE-difFerence spectroscopy is particularly valuable for distinguishing stereoisomers, for it relies solely on intemuclear distances, and thus avoids any problems of ambiguity or absence associated with couplings. With smallish molecules, it is best carried out in the above 1D maimer, because 2 s are necessary for tire transmission of the NOE. The transmission process becomes more efficient with large molecules and is almost optimal for proteins. However, problems can occur with molecules of intemiediate size [3f]. A 2D version of the NOE-difference experiment exists, called NOESY. 

For large molecules, such as proteins, the main method in use is a 2D technique, called NOESY (nuclear Overhauser effect spectroscopy). The basic experiment [33, 34] consists of tluee 90° pulses. The first pulse converts die longitudinal magnetizations for all protons, present at equilibrium, into transverse magnetizations which evolve diirhig the subsequent evolution time In this way, the transverse magnetization components for different protons become labelled by their resonance frequencies. The second 90° pulse rotates the magnetizations to the -z-direction. 

Figure Bl.13.6. The basic elements of a NOESY spectrum. (Reproduced by penuission of Wiley from Williamson M P 1996 Encyclopedia of Nuclear Magnetic Resonance ed D M Grant and R K Harris (Chichester Wiley) pp 3262-71).

The tenn slow in this case means that the exchange rate is much smaller than the frequency differences in the spectrum, so the lines in the spectrum are not significantly broadened. Flowever, the exchange rate is still comparable with the spin-lattice relaxation times in the system. Exchange, which has many mathematical similarities to dipolar relaxation, can be observed in a NOESY-type experiment (sometimes called EXSY). The rates are measured from a series of EXSY spectra, or by perfonning modified spin-lattice relaxation experiments, such as those pioneered by Floflfman and Eorsen [20]. 

The 2-D nuclear Overhauser effect spectroscopy (2-D-NOESY) experiment resembles the COSY however, the cross-peaks arise from... 

Generally, the most powerful method for stmctural elucidation of steroids is nuclear magnetic resonance (nmr) spectroscopy. There are several classical reviews on the one-dimensional (1-D) proton H-nmr spectroscopy of steroids (267). C-nmr, a technique used to observe individual carbons, is used for stmcture elucidation of steroids. In addition, C-nmr is used for biosynthesis experiments with C-enriched precursors (268). The availability of higher magnetic field instmments coupled with the arrival of 1-D and two-dimensional (2-D) techniques such as DEPT, COSY, NOESY, 2-D J-resolved, HOHAHA, etc, have provided powerful new tools for the stmctural elucidation of complex natural products including steroids (269). 

Figure 2.21. HFI NOE difference spectra (b, c) and FIFI NOESY diagram (d) of a-pinene (1) with /-/ NMR spectrum (a) for comparison [(CD3)2CO, 10% v/v, 25 °C, 200 MHz, section from <5 = 0.85 to 2.34 ]. Vertical arrows in (b) and (c) indicate the irradiation frequencies in the HH NOESY plot (d), cross-signals linked by a dotted line show the NOE detected in (c)...

A comparison of the methods of proton-proton NOE detection has shown that two-dimensional NOE detection such as NOESY and ROESY are better suited to the investigation of the stereochemistry of biopolymers whereas for small- to medium-sized molecules (up to 30 C atoms) NOE difference spectroscopy is less time consuming, more selective and thus more conclusive. 

NOESY Nuclear Overhauser effect spectroscopy, detection of NOE in the HH COSY square format, traces out closely spaced protons in larger molecules... 

All these experiments involve at least three distinct time periods preparation (tp), evolution (tl), and detection (t2) these periods are usually separate by rf pulses. Some experiments (e.g., NOESY, RELAY) further contain an additional "Mixing period, tm, between the evolution and detection periods. 

Figure 1. Pulse sequences of some typical 2D-NMR experiments. COSY = correlation SpectroscopY, DQFCOSY = Double Quantum Filtered COSY, RELAY = RELAYed Magnetization Spectroscopy, and NOESY = Nuclear Overhauser Effect SpectroscopY.

A related experiment TOCSY (Total Correlation Spectroscopy) gives similar information and is relatively more sensitive than the REIAY. On the other hand, intensity of cross peak in a NOESY spectrum with a short mixing time is a measure of internuclear distance (less than 4A). It depends on the correlation time and varies as . It is positive for small molecules with short correlation time (o r <<1) and is negative for macromolecules with long correlation time (wr >>l) and goes through zero for molecules with 1 Relaxation effects should be taken into consideration for quantitative interpretation of NOE intensities, however. 

It was straightforward to identify the spin systems of four valines, five threonines, four alanines, three glycines, two glutamates, and AMX type residues (Asp, Cys, Phe, Tyr, Trp, and Ser) of this protein from the COSY, RELAY, and NOESY spectra in D O solution. The RELAY spectrum was particularly useful in identification of Val, Ala, Thr, and Glu spin systems. 

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