th  - 1987 - 51st ENC Conference

MK7
IMPROVEMENTS OF THE 2D TRANSFERRED NOE EXPERIMENT AND APPLICATION IN THE
CONFORMATIONAL ANALYSIS OF INHIBITORS BOUND TO CMP-KDO SYNTHETASE
Stephen W. Fesik* , Edward T • Olejniczak • and William E • Kohlbrenner •
Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, IL 60064.
Transferred nuclear Overhauser effects (TRNOE) provide a useful means of
determining the conformation of enzyme-bound ligands. However, in the 2D
version of the experiment certain precautions must be taken to alleviate some
of the problems associated with the experiment• One of the problems is the
presence of large protein signals which, in many cases, obscures the
resonances of the ligand. In order to suppress these unwanted signals, we
have taken advantage of the shorter spin-spin relaxation times (T 2) of the
protein signals compared to the averaged signals of the ligand. This was
accomplished by inserting a Carr-Purcell-Meiboo m-Gill pulse sequence before
the evolution time (t 1) of the conventional 2D NOE experiment, markedly
improving the quality of the spectra. Another problem, the presence of large
zero quantum peaks, were eliminated by standard means, maintaining a constant
1
mixing time.
The modified 2D TRNOE experiment described above was applied in the study
of the bound conformations of CMP-KDO synthetase inhibitors. These studies
were aimed at providing conformational information towards the design of more
potent inhibitors of lipopolysaccharide biosynthesis and the development of a
new class of antibiotics• Based on 2D TRNOE experiments using several
inhibitors, it was found that their bound conformations depended on the
hydrophobicity of their side chains. The bound conformations were used to
propose the location of a hydrophobic and hydrophilic binding site.
1. S. Macura, K. Wuthrich, and R.R. Ernst, J.Magn. Reson., 4__7_7, 351 (1982).