I would like to thank all who have answered my call for help. I
unfortunately have not had the time to digest everything nor to reply to
everyone; I want to do my homework before continuing the discussion any
further. Thanks again to the community; it will prove useful for our
in-house development.
Happy holidays to everyone,
Axel
As I was asked to post a summary, here are the answers I obtained in a
nut shell:
1----------------------------------------------------------------
[...] I took the advice of Dave Vander Velde (U Kansas) and went to
NOESY. Under normal circumstances, such molecules typically give little
nOe. However, using a solvent mixture of 70/30 d6-DMSO/D2O and reducing
the temp to about -15C, beautiful NOESY spectra were obtained.
See one of the following:
"A New Class of Nucleosides Possessing Unusual Physical Properties:
Syntheses, Hydration, and Structural Equilibria of
Uridine-6-carboxaldehydes" M.P. Groziak, A. Koohang, W.C. Stevens, and
P.D. Robinson, J. Org. Chem., 58, 4054-4060 (1993)
"Definitive Solution Structures for the 6-Formylated Versions of
1-(B-D-Ribofuranosyl)-, 1-(2'-Deoxy-B-D-Ribofuranisyl)-, and
1-B-D-Arabinofuranosyluracil, and of Thymidine," M.P. Groziak, R. Lin,
W.C. Stevens, L.L. Wotring, L.B. Townsend, J. Balzarini, M. Witvrouw,
and E. De Clercq, Nucleosides and Nucleotides, 15, 1041-1057 (1996)
"The Effect of a Chemical Modification on the Hydrated Adenosine
Intermediate Produced by Adenosine Deaminase and a Model Reaction for a
Potential Mechanism of Action of 5-Aminoimidazole Ribonucleotide
Carboxylase," M.P. Groziak, Z.-W. Huan, H. Ding, Z. Meng, W.C. Stevens
and P.D. Robinson, J. Med. Chem., 40, 3336-3345(1997)
Regards,
Bill
2----------------------------------------------------------------
[...] Roesy is better suited to small molecules such as this than noesy
because of the rapid tumbling rate in small molecules and the advantage
of being in the rotating frame that roesy offers. Negative roesy cross
peaks correspond to through space magnetization transfer while positive
cross peaks correspond to exchange peaks (i.e. an axial proton becoming
an equitorial proton due to conformer interconversion). Dispersive
peaks (negative and positive side by side) correspond to Tocsy (through
bond scalar coupling) bleed through. To minimize noise (heat stiples
that look like lines) it is better to run these experiments at lower
temps even though the lower temps cause line broadening due to shorter
T2's. Hope this is what you were seeking.
Bruce
3----------------------------------------------------------------
I guess the -ve crosspeaks in ROESY are due to exchange mechanism which
is operating in the similar time scales of ROESY.
regards-jagadeesh
4----------------------------------------------------------------
[...] but I think you should make both ROESY and NOESY and compare the
results. You do not mention anything about exchange between different
species or conformations. This could also lead to negative peaks in both
ROESY and NOESY.
Sincerely yours,
Svetlana
5----------------------------------------------------------------
A couple of suggestions:
- you can add a cosolvent to improve your compound solubility
- could you perhaps try at slightly higher temp. and see if the -ve
peaks go away. If they do then it's aggregation that's hurting you.
- I haven't had to do this, but perhaps a 13C T1 expt would say whether
all parts of the molecule are behaving
- are you sure the -ve peaks in the ROESY are real and not artifacts?
- might you have a 3-spin effect that is reported better in ROESY than
NOESY?
- could you do some 1D noe buildups to use as a "gold standard"?
You might want to look at the particular pulse sequences that you're
using and make sure you have the latest that use gradients, Z-filters,
adiabatic pulses, etc. as required. Best to contact the Varian app
lab.
Lotsa luck!
Mike
6----------------------------------------------------------------
I don't perform a lot of ROESY/NOESY work, but ROESY peaks should always
be positive -> perhaps you have some TOCSY peaks breaking through.
[...] Also, see "The Nuclear Overhauser Effect in Structural and
Conformational Analysis," Neuhas and Williamson authors, 1989 edition,
pg.327. Here they discuss a "False" transverse NOE transfer which could
produce negative crosspeaks.
Regards,
Bill (another bill ;o)
7----------------------------------------------------------------
I suggest you get your hands on a copy of 'The Nuclear Overhauser Effect
in Structural and Conformational Analysis' by Newhaus and Williamson. It
addresses all of your questions and more. I have a hard time keeping my
copy safe because everyone is always borrowing it.
Valerie
8----------------------------------------------------------------
For "small molecules", people tend to use ROESY other than NOESY. It has
to do with fast correlation time of small molecules.
Second, when you run ROESY, you should see the cross peaks and the
diagonal peaks have opposite signs, i.e., one is "-" and one is "+". It
is normal. But occasionally, you will see some cross peaks have
different signs. This may due to two things: 1), you have TOCSY
artifacts and 2), you have exchange cross peaks. In this case, you may
try a pulse sequence that will suppress these artifacts.
Good Luck,
Simon
9----------------------------------------------------------------
Both NOESY and ROESY spectra are phase sensitive. So, +ve and -ve are
dependent on how you phase your spectrum. If you phase your diagonal
peaks for +ve then "NOE cross peaks" will appear as -ve and vice versa.
But, "chemical exchange cross peaks" will have the SAME sign of diagonal
peaks. These "chemical exchange cross peaks" should not be included the
structure calculations.
Usually for medium sized molecules (mol. wt 700-1500) the NOE cross
peaks might be zero. So, NOESY will fail for those kind of molecules. In
that case you can ROESY.
What is mixing time you are using. This is very important. For small
molecules you can use around 900 msec. But, you should optimize it.
Good Luck.
Ilango.
10---------------------------------------------------------------
the 'intermolecular' crosspeaks due to aggregation are 'added' to
intramolecular dipolar coupling, so in noesy they can cancel out each
other.
Structure determination won't work then, I presume.
Safest is to compare spectra of different concentrations, e.g. 250uM,
500uM, 750uM, 1mM ...
which of course takes even more time than always measuring diluted
samples.
I hope that helps.
Greetings,
Robert
Received on Wed Dec 21 2005 - 09:12:57 MST