STEP-NOESY (and ROESY) Experiments
The advantages of selective 1D experiments over their 2D counterparts make them a very
attractive choice whenever they can be used. Generally the limitation is simply whether or not
the required selective excitation can be achieved. The most direct approach is simply to create a
frequency-selective pulse that excites just the resonance of interest. This can almost always be
done if the resonance of interest is separated from its nearest neighbor by roughly twice its own
width. For example, if one wishes to excite a multiplet 20 Hz wide, and that multiplet is
separated from its nearest neighbor by ~40 Hz or more, the experiment can be done. In cases in
which the peak separation is too small the technique called chemical shift selective filtering can
still often provided the needed selective excitation (by systematically cancelling the unavoidable
excitation of the neighboring resonance), but this experiment too eventually runs into limitations
(the separation between the desired and unwanted resonances must be >~ 5 Hz).
In this case there is a third experiment that can be used, called STEP-NOESY (or STEPROESY). The STEP acronym stands for Selective TOCSY-Edited Preparation, which describes
the basic idea of the experiment. If the desired resonance (the target resonance) cannot itself be
selectively excited, excitation of another resonance (the source resonance), for a spin coupled to
the target spin, followed by TOCSY transfer of magnetization from the source to the target
achieves the required selective excitation. It is in fact not even required that either the source or
the target resonance be completely selectively excited; this is because the TOCSY excitation is
phase cycled independently of the source, resulting in cancellation of unwanted signals from
either the source or the target excitation. (There is one exception: if unwanted signals excited by
both the source and target pulses are themselves for coupled spins the resulting spectrum will
show responses for both the desired target and its (unwanted) neighbor resonance.)
To set up a STEP-NOESY experiment simply select the source and target resonances. (This will
of course require some knowledge of resonance assignments, as is always true for selective
experiments.) It is best to select as the source resonance the one with the largest coupling to the
target, maximizing the efficiency of the TOCSY transfer. Enter the selected chemical shifts (in
ppm) as cnst21 (source) and cnst22 (target). Optimization of the TOCSY mixing time can also
be done (and probably should be, as it does not take long). To do this, include -DNO_NOE (or
-DNO_ROE, for ROESY) in the ZGOPTNS (-DCALC_SPOFFS must always be set; this is
included in the standard parameters for the experiment), and set ns to 4. Then use popt to
optimize the parameter d9 (TOCSY mixing time); a range of .02 to .05 in steps of .005 is
sufficient. The following figure shows the results (and the importance) of such an optimization