The nuclear Overhauser effect

We looked atthe stereoselectivity of Many occasions arise when even coupling constants do not help us in our quest for stereochemical 

The vicinal (3/) coupling constant between the two black Hs is 11 Hz. This is rather large and can be explained by a predominant conformation shown in the Newman projection, with the two large groups (PhCO and Ph) as far from each other as possible, the two medium groups (Br) as distant as possible, and the two black Hs in the places which are left. The dihedral angle between the black Hs is then 180° (they are anti-periplanar) and a large/is reasonable. 

But now see what happens when we react the dibromide with piperidine. A single diastereoisomer of an amine is formed, and there is good evidence that it has the opposite configuration from the di bromide in other words, replacement ofBrbyNhas occurred with inversion. 

A more serious situation arises when we treat this product with base. An unusual elimination product is formed, in which the amine group has moved next to the ketone. The reaction is interesting for this point alone, and one of the problems at the end of the chapter asks you to suggest a mechanism. But there is added interest, because the product is also formed as a single geometrical isomer, E or Z. But which one There is a hydrogen atom at one end of the alkene but not at the other so we can t use 3/ coupling constants to find out as there aren t any. 

What we need is a method that allows us to tell which groups are close to one another in space (though not necessarily through bonds) even when there are no coupling constants to help out. Very fortunately, an effect in NMR known as the nuclear Overhauser effect allows us to do this. 

Because biological macromolecules contain a large number of proton spins, we need to see how special pulse sequences can simplify the appearance of a carbon-13 spectrum and reveal such important information as the three-dimensional arrangement of the carbon backbones of proteins, nucleic acids, and lipids. 

Carbon-13 is a dilute-spin species in the sense that it is unlikely that more than one nucleus will be found in any given small molecule (provided the sample has not been enriched with that isotope the natural abundance of C is only 1.1 per cent). Even in large molecules, although more than one C nucleus may be present, it is unlikely that they will be close enough to give an observable splitting. Hence, it is not normally necessary to take into account spin- 

The technique described here is of considerable usefulness for the determination of the conformations of proteins and other biological macromolecules in their natural aqueous environments. 

Consider a very simple AX system in which the two spins interact by a magnetic dipole-dipole interaction. We expect two lines in the spectrum, one from A and the other from X. However, when we irradiate the system with radiofrequency radiation at the resonance frequency of X using such a high intensity that we saturate the transition (that is, we equalize the populations of the X levels), we find that the A resonance is modified. It may be enhanced, diminished, or even converted into an emission rather than an absorption. That modification of one resonance by saturation of another is called the nuclear Overhauser effect (NOE). 

To understand the effect, we need to think about the populations of the four levels of an AX system (Fig. 13.30). At thermal equilibrium, the population of the UaUx level is the greatest, and that of the PaPx level is the least the other two levels 

Correlations through space The nuclear Overhauser effect 

Which one of these two approaches is adopted in the laboratory may be dictated by the motional properties of the molecule(s) under study and more specifically the rates at which the molecules tumble in solution. Pre-empting what is to follow, it will be shown that the steady-state experiments are appropriate only for molecules that tumble rapidly in solution (we shall also see what defines rapidly in this context). Such measurements have traditionally been the home territory of small organic molecules in relatively non-viscous solutions. In contrast, very much larger molecules that tumble slowly in solution (or smaller molecules in very viscous solutions) can be meaningfully studied only with the transient NOE techniques, which may also be suitable for small-molecule studies. Between these two extremes of molecular tumbling rates, the conventional NOE can become weak and vanishingly small, a condition most likely to occur for those molecules with masses of around 1000-2000 daltons. It is here that rotating-frame NOE (ROE) measurements play a vital role, and these shall also be described. 

The chapter is presented in two parts, the first covering the essential theory that underlies the NOE and the second addressing the practicalities of how one measures NOE enhancements, the experimental steps required to optimise such measurements and how to correctly 

NOE difference Establishing NOEs and hence spatial proximity between protons. Suitable only for small molecules (M, 1000), for 

NOESY(2D or ID) Establishing NOEs and hence spatial proximity between protons. Suitable for small M, 1000) and large molecules (My 2000) for which NOEs are positive and negative respectively, but may fail for mid-sized molecules (zero NOE). Observes transient NOEs generated from the inversion of a target. Estimates of intemuclear separations can be obtained in favourable cases. 

Many occasions arise when even coupling constants do not help us in our quest for stereochemical information. Consider this simple sequence. Bromination of the alkene gives as expected fratis addition and a single diastereoisomer of the dibromide. 

Dipole-dipole relaxation occurs when two nuclei are located close together and are moving at an appropriate relative rate (Section 5-1). Irradiation of one of these nuclei with a Bi field alters the Boltzmann population distribution of the other nucleus and therefore perturbs the inten.sity of its resonance. No J coupling need be present between the nuclei. The original phenomenon was discovered by Overhauser, but between nuclei and unpaired electrons. The nuclear Overhauser effect (when both spins are of nuclei) was observed first by Anet and Bourn and is of more interest to the chemist. It has great structural utility, because the dipole-dipole mechanism for relaxation depends on the distance between the two spins. (See eq. 5-1.) 

For small molecules (the extreme narrowing limit), the maximum increment in intensity Hmax Tirr/ Tobs SO that an initial intensity of unity (Iq = 1.0) increases up to (1 + irimax)- [In our example, A was irradiated ( irr ) and X observed ( obs ).] The maximum enhanced intensity, obtained by rearrangement of eq. 5-4, is given by 

The increase is almost always less than the maximum, because nondipolar relaxation mechanisms are present and because the observed nucleus is relaxed by nuclei other than the irradiated nucleus. 

Whenever the two nuclei are the same nuclide (e.g., both protons), the gyromagnetic ratios in eq. 5-5 cancel, becomes 0.5, and the maximum intensity enhancement (1 + max) is a factor of 1.5, or 50%. For the common case of broadband H irradiation with observation of C, [ C H)] tij x is 1.988, so the enhancement is a factor of up to 2.988, or about 200%. Other maximum Overhauser enhancement factors (1 + (]tr ax) include 2.24 

Certain nuclei have negative gyromagnetic ratios, so that becomes negative and a neg- 

---

Neuhaus D and Williamson M 1989 The Nuclear Overhauser Effect in Structural and Conformational Analysis (New York VCH)... 

Noggle J H and Schirmer R E 1971 The Nuclear Overhauser Effect (New York Academic)... 

Another technique often used to examine the stmcture of double-heUcal oligonucleotides is two-dimensional nmr spectroscopy (see AfAGNETiC SPIN resonance). This method rehes on measurement of the nuclear Overhauser effects (NOEs) through space to determine the distances between protons (6). The stmcture of an oligonucleotide may be determined theoretically from a set of iaterproton distances. As a result of the complexities of the experiment and data analysis, the quality of the stmctural information obtained is debated. However, nmr spectroscopy does provide information pertaining to the stmcture of DNA ia solution and can serve as a complement to the stmctural information provided by crystallographic analysis. 

Methods of disturbing the Boltzmann distribution of nuclear spin states were known long before the phenomenon of CIDNP was recognized. All of these involve multiple resonance techniques (e.g. INDOR, the Nuclear Overhauser Effect) and all depend on spin-lattice relaxation processes for the development of polarization. The effect is referred to as dynamic nuclear polarization (DNP) (for a review, see Hausser and Stehlik, 1968). The observed changes in the intensity of lines in the n.m.r. spectrum are small, however, reflecting the small changes induced in the Boltzmann distribution. 

The nuclear Overhauser effect resulting from the broad-band decoupling during the decoupled INEPT experiment also contributes to the signal enhancement of the C lines. 

Neuhaus, D., and Williamson, M. (1989). The nuclear Overhauser effect in structural and confirma-tional analysis. VCH Pubs., New York, p. 278. 

J.H. Noggle and R.E. Schirmer, The Nuclear Overhauser Effect, Chemical Applications, Academic Press, New York, NY (1971). 

Whereas spin decoupling, COSY and TOCSY techniques are used to establish connectivities between protons through bonds, techniques that make use of the nuclear Overhauser effect (NOE), such as 1-D NOE and NOESY, 1- and 2-D GOESY, 1- and 2-D ROESY, can establish connectivities through space. Before looking at these techniques in detail, it s worth spending a little time considering the NOE phenomenon itself - in a nonmathematical manner, of course ... 

At the beginning of this section, we listed the various experiments that are available which make use of the Nuclear Overhauser Effect but as yet, we have made no attempt to indicate the pros and cons of each of these and under what circumstances one may be preferable over another. It is virtually impossible to give cast iron advice regarding the selection of one NOE experiment over another as the decision has to be based on a huge number of considerations, and on the instrumentation and software available to you. Having said that, we shall now attempt to establish some broad guidelines. 

Convincing evidence was found that the majority of acyclic aldo-nitrones exist in the Z-form, by investigating the ASIS-effect (aromatic solvent induced shift effect) (399). However, in some cases, specified by structural factors and solvent, the presence of both isomers has been revealed. Thus, in C -acyl-nitrones the existence of Z -and -isomers was detected. Their ratio appears to be heavily dependant on the solvent polar solvents stabilize Z-isomers and nonpolar, E-isomers (399). A similar situation was observed in a- methoxy-A-tert-butylnitrones. In acetone, the more polar Z-isomer was observed, whereas in chloroform, the less polar E-isomer prevailed. The isomer assignments were made on the basis of the Nuclear Overhauser Effect (NOE) (398). /Z-Isomerization of acylnitrones can occur upon treatment with Lewis acids, such as, MgBr2 (397). Another reason for isomerization is free rotation with respect to the C-N bond in adduct (218) resulting from the reversible addition of MeOH to the C=N bond (Scheme 2.74). The increase of the electron acceptor character of the substituent contributes to the process (135). 

Bell, R. A., Some Chemical Applications of the Nuclear Overhauser Effect, 7, 1. 

As with the COSY experiment, the sequence starts with a pulse followed by an evolution period, but now the mechanism that couples the two spins (which must be in close proximity, typically <6 A) is the Nuclear Overhauser Effect (NOE). The second pulse converts magnetization into population disturbances, and cross-relaxation is allowed during the mixing time. Finally, the third pulse transfers the spins back to the x-y-plane, where detection takes place. The spectrum will resemble a COSY spectrum, but the off-diagonal peaks now indicate through-space rather than through-bond interactions. 

The observed polarization is primarily associated with the former parahydrogen protons. However, other protons may also experience a drastic signal enhancement due to nuclear spin polarization transferred to these nuclei via the nuclear Overhauser effect (NOE) or similar processes, both in the final reaction products as well as in their precursor intermediates. 

When one resonance in an NMR spectrum is perturbed by saturation or inversion, the net intensities of other resonances in the spectrum may change. This phenomenon is called the nuclear Overhauser effect (NOE). The change in resonance intensities is caused by spins close in space to those directly affected by the perturbation. In an ideal NOE experiment, the target resonance is completely saturated by selected irradiation, while all other signals are completely unaffected. An NOE study of a rigid molecule or molecular residue often gives both structural and conformational information, whereas for highly flexible molecules or residues NOE studies are less useful. 

The principle source of experimental conformational data in an NMR structure determination is constraints on short interatomic distances between hydrogen atoms obtained from NMR measurements of the nuclear Overhauser effect (NOE). NOEs result from cross-relaxation mediated by the dipole-dipole interaction between spatially proximate nu-... 

---