Cross-peaks

Muns ENDOR mvolves observation of the stimulated echo intensity as a fimction of the frequency of an RE Ti-pulse applied between tlie second and third MW pulse. In contrast to the Davies ENDOR experiment, the Mims-ENDOR sequence does not require selective MW pulses. For a detailed description of the polarization transfer in a Mims-type experiment the reader is referred to the literature [43]. Just as with three-pulse ESEEM, blind spots can occur in ENDOR spectra measured using Muns method. To avoid the possibility of missing lines it is therefore essential to repeat the experiment with different values of the pulse spacing Detection of the echo intensity as a fimction of the RE frequency and x yields a real two-dimensional experiment. An FT of the x-domain will yield cross-peaks in the 2D-FT-ENDOR spectrum which correlate different ENDOR transitions belonging to the same nucleus. One advantage of Mims ENDOR over Davies ENDOR is its larger echo intensity because more spins due to the nonselective excitation are involved in the fomiation of the echo. 

Each cross peak has x and y coordinates One coordinate corresponds to the chem real shift of a proton the other to the chemical shift to a proton to which it is coupled Because the diagonal splits the 2D spectrum m half each cross peak is duplicated on the other side of the other diagonal with the same coordinates except m reverse order This redundancy means that we really need to examine only half of the cross peaks To illustrate start with the lowest field signal (8 2 4) of 2 hexanone We assign fhis signal a friplef fo fhe protons af C 3 on fhe basis of ifs chemical shifl and fhe spin fmg evidenf m fhe ID speefrum... 

We look for cross peaks wifh fhe same x coordmafe by drawing a verfical line from 8 2 4 finding a cross peak wifh a y coordmafe of 8 1 6 This means that the protons responsi ble for the signal at 8 2 4 are coupled to the ones at 8 1 6 Therefore fhe chemical shifl of fhe C 4 prolons is 8 1 6... 

Now work from Ihese C 4 prolons Drawing a verfical line from 8 1 6 on fhe X axis finds Iwo cross peaks One cross peak simply confirms fhe coupling lo fhe pro tons al C 3 The olher has a y coordmafe of 8 1 3 and Iherefore musl correspond to fhe prolons al C 5... 

Finally a verfical line drawn from 8 2 1 mlersecls no cross peaks The smglel al 8 2 1 as expected is due to Ihe protons al C 1 which are nol coupled to any of Ihe olher protons m Ihe molecule... 

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

Now work from these C-4 protons. Drawing a vertical line from 8 1.6 on the x-axis finds two cross peaks. One cross peak simply confirms the coupling to the protons at C-3. The other has a y coordinate of 8 1.3 and, therefore, must correspond to the protons at C-5. 

Finally, a vertical line drawn from 8 2.1 intersects no cross peaks. The singlet at 8 2.1, as expected, is due to the protons at C-1, which are not coupled to any of the other protons in the molecule. 

Similarly, cross peak positions in RELAY spectra give valuable information about remote connectivities (spins coupled to common partner but not to each other) and are very useful in resolving chemical shift degeneracies. An AMX spin system with no coupling between A and X shows a cross peak at ... 

RELAY spectrum with appropriate delay 2r in contrast to the COSY cross peaks at and intensity of this peak depends on the magnitude of... 

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. 

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