When a proton is saturated orinverted, spatially-close protons may experience an intensity enhancement, whichis termed the Nuclear Overhauser Effect (NOE). The NOE is unique among NMRmethods because it does not depend upon through-bond J couplings but dependsonly on the spatial proximity between protons. In other words, the strength of the NOE gives information onhow close two protons are. Forsmall molecules, an NOE may be observed between protons that are up to 4Āapart, while the upper limit for large molecules is about 5Ā.
There are many differentpossible NOE experiments (NOE or ROE, steady-state or transient, 1D or 2D,etc). The ones available on the400 and 500 are 1D selective-NOESY, 2D-NOESY, 2D-NOESY with zero-quantumsuppression, and 2D-ROESY. Some understanding of the theory of NOE is necessary for choosing thecorrect experiment and for interpreting it properly. Some of the important results from the theory are discussedbelow. For a full understanding,see the excellent text Neuhaus, David, and Williamson, Michael P., TheNuclear Overhauser Effect in Structural and Conformational Analysis,
Molecular Weight andMaximum NOE
The maximum possible NOEdepends on the molecular correlation time (or the inverse of the rate ofmolecular tumbling), which is in large part determined by the molecular weightand solvent viscosity. Largermolecular weights and higher viscosities lead to larger correlation times.
Time Dependence of NOE -Mixing Times
In transient experiments,such as NOESY and ROESY, the NOE dynamically builds up and then decays due torelaxation during the mixing time, as shown below in the plot of NOE versusmixing time. The NOE, thus, goes through a maximum as function of mixingtime. The location of the maximumNOE and rate of build-up depend on the correlation time, or its proxy, themolecular weight, and the distance between protons for a particular NOE.
There is only one mixing timespecified per NOE experiment, and it is the most important parameter for NOEexperiments. For small molecules, a mixing time that maximizes the NOE isdesirable, unless you intend to calculate an actual distance (see analysissection). Generally, one isinterested in a range of distances so the choice depends on molecular weightrather than a particular distance. For large molecules, the mixing time must be kept small so that the build-upobeys the linear approximation and spin diffusion is avoided (see analysissection). The following are guidelines:
1) small molecules 0.5 -1 sec. Start with 0.5 sec.
2) medium size molecules 0.1 -0.5 sec.
3) large molecules 0.05 - 0.2sec. Start with 0.1 sec.
1D versus 2D Methods
The choice between 2D (ROESYor NOESY) versus 1D (selective NOESY) depends on the amount of materialavailable and the amount of information needed. A single 2D experiment gives all NOE informationsimultaneously whereas 1D experiments provide NOEs one at atime. In general, Irecommend the 2D methods.
Spectral crowding will affectthe choice of experiment. Ifcritical peaks to be irradiated are very close (<30 Hz) to other peaks, thenthe selectivity of the 1D version will not be sufficient and the 2D versionwill be needed.
Artifacts and Their Suppression
Zero-quantum peaks are acommon artifact in all NOESY spectra. They occur between peaks that are J-coupled, such as ortho-protons on aring, as can be identified by their up-down DQF-COSY type of pattern.
There is a 2D NOESY sequencethat is designed to remove these zero-quantum peaks.
In ROESY spectra, a commonoccurrence is TOCSY transfer between protons that are J-coupled or symmetricwith respect to the center of the spectrum. This latter artifact can be removed by proper positioning ofo1p, the center of the spectrum. Finally, the cross-peak intensities have anoffset dependence. See analysis section for more detail.
If protons are undergoingchemical exchange, corresponding cross peaks occur in all NOE and ROEexperiments. In fact, chemicalexchange can be studied with these same NOE methods and are then termed EXESYexperiments.
In 1D selective NOESYexperiments, there are several types of possible artifacts: zero-quantum(up-down) peaks as well as the unsuppressed residual from very intensesinglets. Moreover, theexperiment uses selective pulses and their proper calibration is required foroptimal results and suppression of artifacts.
Choice of Experiment– a Prescription
Small molecules (MW < 600)
Theusual choice is 2D NOESY with zero-quantum suppression.
Medium sized molecules (700< MW < 1200)
Large Molecules (MW >1200)
Thechoice here is more complicated. The usual choice is 2D NOESY but 2D ROESY has advantages.
Dissolved oxygen or otherparamagnetic species such as Cu2+ can reduce or completely quenchthe NOE.
1) freeze the sample inliquid nitrogen or CO2/acetone.
2) evacuate the space abovethe solution.
3) turn off vacuum but keepsample isolated and allow to thaw. As it thaws, bubbling should be noticed.
4) repeat several times (3-4times).
5) backfill with N
When finished, the sampleshould, of course, be sealed in some manner. Tubes with attached stopcocks are available.
Sample size and tube options
When sample quantity is verylimited, it is advantageous to limit the amount of solvent in which it isdissolved. If a normal 5mm tube isused, however, this cannot be less than about 500
ANALYSIS – peakidentification
Relative Phase of Cross Peaks
Thephase of ROE, NOE and chemicalexchange cross peaks can be different and are summarized in the figure below.In this figure, it is assumed that protons A and B have an NOE or ROE whileprotons C and D are undergoing chemical exchange. Note that the phase behavior differs for large and smallmolecules. For small molecules, the diagonal peaks and NOE cross peaks haveopposite phase. If the diagonal is negative, then NOE cross peaks will bepositive. For large molecules, thediagonal and the NOE cross peaks have the same phase. The phase of the cross peaks, then, indicates whetherthe molecule is in the large or small molecule region, which has importantimplications for quantitation, as discussed below. Note that this phase behavior is due to thepositive/negative nature of NOE described at the beginning of this handout.
Crosspeaks due to chemical exchange, if it is occurring, have the same phase as thediagonal for both small and large molecules in both ROESY and NOESY.
InROESY, the diagonal peaks and ROE cross peaks have opposite phase for allmolecules since the ROE is always positive. TOCSY cross peaks are the major artifact in ROESYspectra. TOCSY peaks have the samephase as the diagonal, and are thus similar to exchange peaks.
When analyzing NOESY spectra,one must understand the consequences of spin diffusion.
ROESY spectra suffer muchless from spin diffusion; the phase of indirect contributions may be differentfrom direct contributions and allows their easy identification.
For organic molecules, it isgenerally sufficient to classify NOE peak intensities as strong, medium, andweak and make qualitative deductions about relative distances.
where aij is the NOE cross-peak volume and rijis the interproton distance of the the two protons i and j.
For this relation to bevalid, a strict experimental protocol must followed. First, the mixing time must be relatively short sothat the linear approximation is valid and spin diffusion is avoided. For smallmolecules, the mixing time must be less than several hundred milliseconds.
The purpose of running aNOESY is often to determine conformation by establishing a distance between twoprotons. When more than oneconformation is present, however, the NOE may give misleading distances.
the effective distance forNOE is less than the average distance, reff < rave.and the effective distance is weighted towards that of
ROESY – QuantitativeDistance Determination
In addition to the aboveconsiderations for NOESY, the ROESY has additional complications.
An additional complicationwith quantitation of ROESY spectra is that TOCSY transfer may occur and cancelor partially cancel ROESY cross peaks. This obviously has deleterious effects on distance determination.