NOESY

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NOESY (Nuclear Overhauser Effect Spectroscopy) spectra provide information about
protons that are 5 Angstroms or less apart in space. The information is through space and
not through bond, like a COSY or TOCSY. The presence of a NOE peak is direct
evidence that two protons are within 5 Angstroms through space. The absence of a NOE
peak between protons does not necessarily mean that they are not within 5 Angstroms
since other factors can reduce a NOE peak even if the protons are close in space. A midsize
molecule (~1000-1500MW range) may have NOEs that are close to zero and a
ROESY may be required to see them. Large molecules generally give better NOEs at
higher field, but small molecules may actually give better NOEs at lower field. A 2-D
NOESY of a small molecule will have cross peaks of opposite phase to the diagonal. A
2-D NOESY of a large molecule will have cross peaks of the same phase as the diagonal.
Theoretically, these experiments should be symmetrical, but it is typical to see more
intense peaks on one side of the diagonal than the other.
Basic NOESY
NOESY - (all upper case) utilizes a probe file and gradients, no solvent suppression
If a probes file is not set up in the account, see documentation on setting up a probes file
or ask for help. This version allows as few as two transients allowing a faster acquisition
for concentrated samples. The macro is called by >NOESY. The top window should
have "seq:NOESY".
1. Make sure probe=’HCN’.
2. Find the 90-degree pulse width of the sample. Set pw=pw90. Collect a 1-D proton
and adjust the sweep width and tof. Users may also want to determine an approximate
T1 value so an optimal delay time between acquisitions can be set.
3. Call the macro by typing >NOESY in the command line. Use >dps to view the pulse
sequence.
4. Check that pw is set to the 90-degree pulse width, set d1 and np. Set the sw1=sw. Set
phase=1,2. Also, set nt and the gain using ni=1. The number of transients (nt) required
for this version can be as little as 2.
5. One parameter requiring forethought is the mix time. The mix is usually determined
by the size of the molecule under study. Small molecules require longer mix times, 0.5
to 1 second (suggested mix=0.5). Large molecules generally range from 0.05-0.3
seconds (suggested mix=0.15).
6. Set the ni and check >time. Adjust the delays, transients and/or increments to fit the
experiment into the time available. Use >go to start the experiment.
The all-caps versions of the NOESY includes linear prediction in the standard parameter
sets. To do simple processing of these spectra:
>setLP1
>gaussian
>wft2da
To turn off linear prediction set proc='ft' and proc1='ft'. Turning off linear prediction is
recommended for larger data sets because it can increase processing time dramatically.
Adding presaturation to the basic NOESY
Instructions for this can be found in the manual page for the NOESY pulse sequence
(man(‘NOESY’)). Set d1=0, set satflag=’ynnn’, satdly=1.5, satfrq=tof, satpwr=2.
The satfrq is normally equal the tof, but is not required. Values of satdly and satpwr
vary with the sample.
Figure 26: A simple NOESY spectrum of a small molecule should be relatively
symmetrical and gives proton-proton through space correlations.
Versions of NOESY
Multiple pulse programs exist for NOESY spectra in this facility. The other most
commonly used versions are included below.
a. noesy - (all lower case) no gradients, no water suppression
The macro is called by >noesy. The top window will have "seq:noesy". Parameters are
similar to the basic NOESY.
b. tnnoesy - (all lower case) no gradients, presaturation with transmitter
This macro is the same as "noesy" but includes the option of water suppression with the
transmitter. The macro is called by >tnnoesy.
"tnnoesy" is the same as "noesy" except includes set up for the presaturation parameters.
When acquiring the 1-D spectrum, move the transmitter onto the water peak (>nl
movetof). Also, be sure to determine the 90-degree pulse width and set the parameter
pw90=pw. Move the parameters the 1-D to a new experiment (i.e. mp(1,2)). Call the
macro >tnnoesy.
Set d1=0. Do not use a delay between acquisition and presaturation that would allow the
water signal to relax back. Set satmode='ynn', satdly=1.5, satfrq=tof, satpwr=2. Start
with the satdly at 1.5 sec and the satpwr at 2. Adjust these values but do not use a
satpwr > 20 ever and typical values are 10 or below. Be sure to increase the gain after
adjusting the water suppression.
c. gnoesywg - gradient NOESY with watergate suppression
This macro can be used with the macro "autowatergatenoesy". The parameters can be
loaded from the parameter set in /vnmr/parlib.
Run a 1-D spectrum and move the transmitter onto the water peak (nl movetof). Find the
90-deg pulse width and set pw90=pw. Two options exist for setting up at this point:
First, use the "autowatergatenoesy" macro. After setting the above parameters, type
>autowatergatenoesy. The macro will then set up a series of 1-D arrayed experiments
to find the best watergate parameters and run one increment of the final parameters.
Second, if a user has already run gwatergate 1-D, load the parameters for gnoesywg from
/vnmr/parlib. The parameters are called watergatenoesy.par. Then enter the watergate
parameters from the 1-D into the 2-D parameter set. These parameters are: p180, p1,
p1lvl, phincr1. Run one increment to check to see that the water suppression is working
and increase the receiver gain (i.e.gain=40).
Set the mix time, d1, at, sw =sw1, nt, ni, and phase=1,2. Check the amount of time the
experiment will take. Adjust the delay, nt or ni accordingly to fit the time allowed for
the experiment.
d. wgnoesy - gradient NOESY with watergate suppression
This version is from the BioPack pulse sequences and requires BioPack activated in the
account before using it. This version can be autocalibrated from the setup menu. Be sure
to check the linear prediction settings in this version.
This version of watergate noesy is supplied through the Varian package, BioPack. If
BioPack is not in the account, it must be first activated. Activating BioPack takes a few
minutes – see section on BioPack activation. BioPack will do an autocalibration for the
experiment. Make sure probe=’HCN’. Use the Menu Bar and select: [Setup] [Water]
[Auto Calibration] [Watergates] then [3-9-19 NOESY]
A user will be prompted to use the values from the standard parameter sets. Answer 'n'
and put in the values for the tpwr and pw90. The operator will also be asked for a d1
time, mix time, and for a flippw time (typically between 1-2 ms).
The macro will then run a series of arrayed 1-D experiments finding the optimal values
for the water suppression including an optimized tof value. The last thing it will do is run
one increment at the optimized parameters. Set the sw, sw1, nt, ni, and at. Be sure to
turn the receiver gain up before starting the experiment. Check the amount of time the
experiment will take using the macro >time.
e. WET NOESY
WET NOESY can be set up manually for multiple solvent suppression, please refer to the
section on Water Suppression, under WET. To run WET NOESY for water suppression
only, the experiment can be set up with auto calibration in BioPack by:
[SetUp] [Water] [AutoCalibration] [WET NOESY]
Answer the questions, use a calibrated pw instead of the standard parameter set and be
ready to supply a mixing time in sec.
Processing a NOESY
Most NOESY spectra can be processed through the menu system as follows:
[ProcMenu]
[Phase F2]
The first increment will appear (will possibly have a 180-degree phase shift). Phase the
baseline as a 1-D spectrum.
[Adjust Weighting]
Use any weighting function desired, but a common one for a NOESY is a gaussian (gf).
[Return] [Transform F2]
A spectrum will appear. Place the cursor on a FID (not solvent) that has a reasonable
signal and [Adjust Weighting]. Again use a gaussian function or other function.
[Return] [Transform F1]
The spectrum should appear on the screen. If an error about being outside of range
occurs, use >f full dconi. To further correct phase on the spectrum and/or do a drift
correct. For drift correct:
>dc('f1') or dc('f2')
or
>abc
Alternatively, process the spectrum as follows:
>wft(1) Phase the spectrum. >wti
Adjust the weighting function for the FID and use >wft1da.
Select a trace and use >wti. Apply the second weighting function and use >wft2da.

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[此贴子已经被作者于2006-11-14 13:33:25编辑过]