POTENTIAL INHIBITOR OF ACETYLCHOLINESTERASE
155
gradient reverse probe and a Silicon Graphics worksta-
tion. Chemical shifts were determined relative to
internal DSS (sodium 2,2-dimethyl-2-silapentane-5-
sulfonate).
of diffusion spin. Then, TrNOESY experiments were
performed and compared using an identical mixing
time (ꢆm ¼ 65 ms, which gave a reasonable experimental
signal-to-noise ratio) with various ratios R ¼ 223, 446
and 892. For each case, the intensities of the detected
transferred NOEs were measured. The results showed
that, for each cross peak, the relative intensities of
transferred NOEs determined with respect to the refer-
ence cross peak (H10, H20) were similar ( ꢃ 20%) in the
three experiments. Finally, a TrNOESY experiment from
the inhibitor–enzyme complex to the free inhibitor was
performed at 303 K under conditions that insured the best
signal-to-noise ratio and very limited spin diffusion. The
enzyme concentration was 2.24 mM and the [ligand]/
[AChE] ratio was 446. FIDs were acquired using a
mixing time of 65 ms. A total of 320 incremental values
of the evolution time were used with 320 scans, 2K
data block over 5000 Hz and a relaxation delay of 1.1 s.
Appropriate zero filling was carried out to yield a final
two-dimensional matrix of 2K ꢁ 1K real points. A 60 ꢅ
shifted squared sine-bell was applied in both dimensions.
The final matrix was baseline corrected separately on
each side of the water signal in the f2 dimension with a
third-order polynomial.
1
For 1D H NMR, FIDs were acquired over 5 kHz
spectral width with 32K data points; exponential apodi-
zation gave 1 Hz line broadening. Suppression of water
resonance was achieved by low-power irradiation during
the relaxation delay (2 s), with an attenuation of 60 and
55 dB in the absence and the presence of the enzyme,
respectively.
A 2D COSY23 experiment was acquired for 1 (1.2 mM)
at 298 K using 256 t1 increments with 2K data points and
128 scans each. Partial suppression of the residual HDO
signal was accomplished by presaturating during the 1.5 s
relaxation delay. The data were processed with a non-
shifted sine-bell window in both dimensions.
The phase-sensitive gradient selective HSQC24 and
gradient-selected HMBC25 (delay ¼ 70 ms corresponding
3
to J(C,H) ¼ 7 Hz) experiments were recorded at 298 K.
These experiments were acquired using 256 t1 increments
with 2K data points and 64 scans (HSQC) or 128 scans
(HMBC). Two zero-fillings were applied in the f1 dimen-
sion and a ꢄ/3 shifted squared sine-bell function was used
in both dimensions.
NOESY experiments were recorded using standard
techniques with the time-proportional phase incrementa-
tion mode (States-TPPI) with pulse sequence of Otting
et al.26 Suppression of the water resonance was achieved
by presaturating during the relaxation delay. The 2D
NOESY experiment for 1 (1 mM) in the absence of
AChE was acquired with a mixing time of 1 s and a
relaxation delay set to 1.1 s. The data matrix of 2K ꢁ 256
points was processed using a shifted sine-bell window
function (ꢄ/2) in both dimensions with zero filling in f1 to
2K ꢁ 1K.
Supplementary material
The Supplementary material available at the epoc website
in Wiley Interscience contains the NOESY spectrum of 1
(1 mM, pH* 7.8, 303 K) at 500.11 MHz, with a mixing
time of 1 s and the TrNOESY spectrum of 1 (1 mM, pH*
7.8, 303 K) in the presence of AChE (2.24 mM) at
500.11 MHz, with a mixing time of 65 ms.
From the technical point of view, attention was care-
fully paid to the spin-diffusion effect. The line broad-
ening of 1 was proportional to the amount of AChE. A
low enzymatic concentration (2.24 mM) with a large
concentration of ligand (1 mm) caused a half line broad-
ening of 1 signals. Under these conditions, (ratio
R ¼ [ligand]/[enzyme] ¼ 446), the TrNOE cross peaks
were well resolved. At higher concentrations of 1, the
resonances of H1, H8 and H3, H6 overlapped. On
increasing the concentration of AChE (ratio R ꢆ 66),
strong spin diffusion effects were observed for all protons
of bound ligand.
Initially, transferred NOEs were measured with a wide
range of NOE mixing time, ꢆm (50, 65, 80, 100, 150 and
300 ms), for a ratio R ¼ [ligand]/[enzyme] ¼ 223 (con-
centration of 1 ¼ 0.5 mM, concentration of enzyme ¼
2.24 mM) to determine the optimized value of ꢆm. For
ꢆm ¼ 50 ms, TrNOEs were scarcely detectable. Notable
spin diffusion effects appeared for mixing times >100 ms.
In consequence, it was not possible, in this study, to
obtain NOE build-up curves to evaluate the contribution
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Copyright # 2006 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2006; 19: 148–156