4460 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Brief Articles
and exchanged into either an H2O or D2O solution (pH 6.5)
containing potassium phosphate (50 mM), sodium chloride
(100 mM), and dithiothreitol-d10 (5 mM). FKBP12/ligand
complexes (1/1) were prepared by incubating labeled FKBP12
with an excess amount of either unlabeled ascomycin or
unlabeled C24-deoxyascomycin for 24-48 h at room temper-
ature.
Krause, P. Brettheim, and J . Andrews for assistance in
collecting biological data.
Su p p or tin g In for m a tion Ava ila ble: Isotope-filtered 2D
TOCSY and NOESY spectra and structural statistics for C24-
deoxyascomycin bound to [U-13C,15N]FKBP; 13C(ω1), 1H(ω2)
planes from the 4D [13C,1H,15N,1H] NOESY spectrum of
[U-15N,13C]FKBP12 complexed with ascomycin or 24-deoxy-
ascomycin extracted at the 1HN,15N chemical shifts of the
individual residues Glu-54 and Gln-53, respectively; 13C(ω1),
1H(ω2) planes from the 4D [13C,1H,13C,1H] NOESY spectrum
of ligated [U-15N,13C]FKBP12 extracted at the 1H,13C chemical
shifts of Arg-57 Hδ2 and Val-55 Hγ1, respectively; chemistry
general methods. This information is available free of charge
NMR exp er im en ts: All NMR spectra of the FKBP/ligand
complexes were recorded on either a Bruker AMX500 (500
MHz) or an AMX600 (600 MHz) spectrometer. NMR spectra
were processed and analyzed using in-house written software
on Silicon Graphics computers. 2D isotope-filtered NOESY and
TOCSY spectra of the unlabeled ligand while bound to
[U-13C,15N]FKBP were recorded at 40 °C using pulse sequences
described earlier.18 Mixing times of 80 and 15 ms were used
for the NOESY and TOCSY experiments, respectively.
4D NOESY spectra of [U-13C,15N]FKBP while complexed
with unlabeled ligand were recorded at 30 °C. The 4D
[13C,1H,15N,1H]NOESY spectrum19 was acquired with eight
scans per experiment with 12 × 80 × 10 × 512 complex points
using sweep widths of 3289, 7463, 2128, and 10 000 Hz in
ω1(13C), ω2(1H), ω3(13C), and ω4(1H), respectively. Water sup-
pression was accomplished20 using two spin-lock pulses of 0.5-
and 2.5-ms duration. The 13C carrier frequency was set at 38
ppm. The regions from 70-49 and 27-5.5 ppm were folded
once. The 4D [13C,1H,13C,1H] NOESY spectrum21 was acquired
with eight scans per experiment with 12 × 64 × 12 × 512
complex points using sweep widths of 3289, 5208, 3289, and
10 000 Hz in ω1(13C), ω2(1H), ω3 (13C), and ω4 (1H), respectively.
The 13C spectral widths were folded in the same manner as in
the 4D [13C,1H,15N,1H] NOESY experiment. The 1H carrier was
centered at 3.7 ppm. In both 4D experiments, a 10-ms
homospoil was applied during the 50-ms NOESY mixing time
to suppress unwanted magnetization, and a series of randomly
spaced 90° pulses were used to saturate the 13C spins at the
beginning of the pulse sequence.21b
NOE-d er ived d ista n ce r estr a in ts a n d str u ctu r e ca l-
cu la tion s: The proton-proton distances used as restraints for
the structure calculations of the C24 deoxy derivative of
ascomycin were obtained by counting contours in the isotope-
filtered NOESY data set collected with a mixing time of 80
ms. NOEs were classified as either strong (1.8-2.8 Å), medium
(1.8-3.4 Å), or weak (1.8-4.4 Å). For distances involving
methyl groups, 1.0 Å was added to the upper bound to correct
for pseudoatom.22 A total of 126 NOE restraints were used
along with 5 lower bound restraints that were determined on
the basis of the lack of observable NOEs.
3D structures were calculated using a hybrid distance
geometry/dynamical simulated annealing protocol.14 Using the
XPLOR23 program and the NOE-derived distance restraints,
200 initial structures were generated and subjected to 200
steps of Powell restrained energy minimization to remove bad
van der Waals contacts. During this minimization, and
throughout the entire simulated annealing protocol, the NOE
force constant was maintained at 50 kcal‚mol-1‚Å-2, and
electrostatic terms were excluded. The minimization step was
followed by 7.5 ps of molecular dynamics (time-step of 3 fs) at
2000 K during which the van der Waals force constant was
decreased from its initial value of 20 kcal‚mol-1‚Å-2 to a value
of 0.003 kcal‚mol-1‚Å-2 while increasing all other force con-
stants (bond, angle, etc.). The structures were then cooled from
2000 to 100 K in steps of 50 K. Each step of the cooling process
consisted of 1.25 ps of restrained molecular dynamics (time-
step of 5 fs). The van der Waals force constant was increased
at each step by multiplying the previous value by 1.28 until a
final value of 4.0 kcal‚mol-1‚Å-2 was obtained. The van der
Waals radius was decreased stepwise to a final value of 0.8
times the value used in CHARM for Frepel.24 In the last stage
of the refinement, the structures were subjected to 1000 steps
of Powell restrained energy minimization.
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Ack n ow led gm en t. We thank the Analytical Re-
search Department of Abbott Laboratories for spectral
measurements. We also thank L. Miller, T. Fey, R.