Synthesis of N-Phosphonacetyl-L-isoasparagine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5937
Methods
under reduced pressure, and the residue obtained was triturated with
anhydrous diethyl ether. The precipitate was dried under nitrogen
to afford â-benzyl L-isoasparaginate (5) as the trifluoroacetic acid
Synthesis. All moisture-sensitive reactions were performed under
a nitrogen atmosphere with oven-dried glassware.
1
salt. Yield ) 505 mg (90%). H NMR (400 MHz, DMSO-d6) δ
Characterization. 1H and 13C NMR spectra were recorded on a
Varian 400 or 300 spectrometer. Proton chemical shifts are reported
in ppm (δ) relative to internal tetramethylsilane (TMS, δ 0.0) or
with the solvent reference relative to TMS employed as the internal
standard (CDCl3, δ 7.26 ppm; DMSO-d6, δ 2.50). Data reported
as follows: chemical shift (multiplicity [singlet (s), doublet (d),
triplet (t), and multiplet (m)], coupling constants [Hz], integration).
13C NMR spectra were recorded on a Varian 400 (100 MHz)
spectrometer with complete proton decoupling. The chemical shifts
were reported relative to CDCl3 (77.0 ppm) or DMSO-d6 (39.52
ppm) for solutions in CDCl3 and DMSO-d6, respectively. 31P NMR
spectra were recorded on a Varian 300 (121 MHz) spectrometer
using 85% H3PO4 as an external standard. Mass spectra were
obtained at the Mass Spectrometry Facilities of Boston College.
Fluorescence Measurements. Fluorescence emission spectra
were recorded at 20 ( 1 °C in 50 mM Tris acetate buffer, pH 8.3,
on a Jasco FP-6300 spectrofluorometer using pyrene-labeled
ATCase. The excitation wavelength was 338 nm; emission spectra
were collected between 360 and 560 nm; and the excitation and
emission bandwidths were set to 5 nm. To approximate the degree
of quaternary structure change for the enzyme, the intensity of the
fluorescence signal FT at 377 nm was measured as the population
of T-state enzyme.15 Saturation curves were determined by the
incremental addition of PALA or PALI to the pyrene-labeled
ATCase (1 mM enzyme) until there was no further change in
fluorescence. The data were normalized by dividing each data point
by FT in the absence of ligand.
2.89 (dd, J ) 7.8 Hz, 17.4 Hz, 1H), 3.0 (dd, J ) 4.4 Hz, 17.2 Hz,
1H), 4.05-4.08 (m, 1H), 5.12-5.19 (m, 2H), 7.34-7.40 (m, 5H),
7.65 (s, br, 1H), 7.87 (s, br, 1H), 8.2 (s, br, 3H); 13C NMR (100
MHz, DMSO-d6) δ 35.22, 48.76, 66.24, 127.90, 128.00, 128.27,
135.45, 168.77, 168.92; ESI-MS (m/z) 245.09 [M + Na]+
Preparation of â-Benzyl N-Chloroacetyl-L-isoasparaginate
(6). To a mixture of â-benzyl L-isoasparaginate trifluoroacetic acid
salt (5.12 g, 15 mmol) and pyridine (6.05 mL, 75 mmol) in 125
mL anhydrous dichloromethane, chloroacetic anhydride (5.65 g,
33 mmol) was added portion-wise, while keeping the temperature
at 0 °C. After the addition was complete, the reaction mixture was
stirred for 4 h at room temperature. Then the mixture was washed
with water (2 × 50 mL), saturated sodium bicarbonate (3 × 50
mL), and again with water (1 × 50 mL). The organic layer was
collected, dried over Na2SO4, and concentrated under reduced
pressure to afford the crude product. This was purified by repeated
precipitation using hexane from a saturated solution of crude
1
mixture in dichloromethane. Yield ) 3.6 g (80%). H NMR (400
MHz, CDCl3) δ 2.73 (dd, J ) 6.8 Hz, 17.2 Hz, 1H), 3.07 (dd, J )
4.0 Hz, 17.2 Hz, 1H), 4.02-4.10 (m, 2H), 4.81-4.86 (m, 1H),
5.17 (s, 2H), 5.51 (s, br, 1H), 6.45 (s, br, 1H), 7.33-7.40 (m, 5H),
7.85 (d, J ) 6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 35.87,
42.63, 49.40, 67.38, 128.43, 128.61, 128.72, 135.15, 166.34, 171.51,
171.64; ESI-MS (m/z) 320.9 [M + Na]+
Preparation of â-Benzyl N-(Diethoxyphosphinoyl)acetyl-L-
isoasparaginate (7). A mixture of â-benzyl N-chloroacetyl-L-
isoasparaginate (3.6 g, 12 mmol) and triethyl phosphite (20 mL)
were heated at 150 °C for 8 h and then excess triethyl phosphite
was removed by distillation under reduced pressure to give crude
product as a colorless viscous liquid. The pure product (7) was
obtained by silica column chromatography using a mixture of ethyl
Small-Angle X-ray Scattering. Small-angle X-ray scattering
curves were collected for the wild-type holoenzyme in the absence
of ligands and in the presence of PALA or PALI, according to
Stieglitz et al.32 Solution X-ray scattering experiments were
conducted at the Beam Line 4-2, at the Stanford Synchrotron
Radiation Laboratory, Menlo Park, CA.
1
acetate/methanol (9.5:0.5). Yield ) 4.6 g (95%). H NMR (400
MHz, CDCl3) δ 1.33 (t, J ) 7.0 Hz, 6H), 2.76-3.02 (m, 3H), 3.11
(dd, J ) 5.4 Hz, 17.4 Hz, 1H), 4.09-4.19 (m, 4H), 4.93-4.98 (m,
1H), 5.07-5.14 (m, 2H), 6.21 (s, br, 1H), 7.28-7.32 (m, 5H), 7.50
(s, br, 1H), 7.62 (d, J ) 9.2 Hz, 1H); 13C NMR (100 MHz, CDCl3)
δ 16.63, 35.74, 36.22 (d, JCP ) 127 Hz), 49.30, 62.97, 63.75, 66.98,
128.26, 128.37, 128.61, 135.48, 164.22, 171.59, 173.22; 31P NMR
(121 MHz, CDCl3) δ 22.77; ESI-MS (m/z) 423.13 [M + Na]+.
Anal. (C17H25N2O7P), C, H, N.
Crystallization and Freezing of Crystals. The purified enzyme
was crystallized by microdialysis, using 50 µL wells. The enzyme
solution, at 18 mg/mL, was dialyzed against the crystallization
buffer, which consisted of 50 mM maleic acid, 1 mM PALI, and
3 mM sodium azide at pH 5.7. Crystals formed within 3 days, with
average dimensions of 0.3 × 0.3 × 0.2 mm. Crystals were soaked
in a solution of 30% 2-methyl-2,4-pentanediol in crystallization
buffer for approximately 1 min prior to freezing in liquid nitrogen.
X-ray Data Collection and Processing. The diffraction data
were collected using a Rigaku/MSC R-axis IV++ detector while
X-rays were generated using a Rigaku/MSC RU-200 rotating-anode
generator operating at 50 kV and 100 mA at the Boston College
Crystallographic Facility. The diffraction data, collected to 2.3 Å,
were integrated, scaled, and averaged using the program d*TREK
(Rigaku/MSC).33
Structural Refinement. The data were refined using the
coordinates of wild-type E. coli ATCase complexed with PALA
(PDB entry 1DO9), as the initial model.17 Prior to refinement, all
waters and ligands were removed. The structure was refined using
CNS,34 and refinement statistics are summarized in Table 1. Initially
rigid body refinement was carried out followed by simulated
annealing. After rigid body, simulated annealing, energy minimiza-
tion, and B-factor refinement initial maps were analyzed. Trouble
areas of the structure, the N-termini of the regulatory chains
(residues 1-11), the 50s loop of the regulatory chains, and residues
65-72 of the R6 regulatory chain were manually rebuilt using
XtalView.35 The N-termini of the regulatory chains were highly
disordered even after several rounds of rebuilding, therefore,
residues 1-9 of the regulatory chains were omitted from the
structure. After rebuilding was complete, PALI was fit into the Fo-
Fc electron density. Placement of the PALI was verified using
simulated annealing omit maps.
Preparation of â-Benzyl N-Phosphonacetyl-L-isoasparaginate
(8). To a solution of â-benzyl N-(diethoxyphosphinoyl)acetyl-L-
isoasparaginate (400 mg, 1 mmol) in anhydrous acetonitrile (10
mL), trimethylsilyl bromide was added (560 mL, 5.6 mmol) at 0
°C and then the mixture was stirred overnight at room temperature.
The solvents were evaporated under vacuum and 5 mL of water
was added. The mixture was stirred for 1 h at room temperature,
washed with dichloromethane, and lyophilized to give pure product
1
8 in quantitative yield. H NMR (400 MHz, D2O) δ 2.62-2.78
(m, 2H), 2.85 (dd, J ) 8.2 Hz, 16.6 Hz, 1H), 2.95 (dd, J ) 5.1 Hz,
16.4 Hz, 1H), 4.72 (m, 1H), 5.15-5.37 (m, 2H), 7.36-7.40 (m,
5H); 31P NMR (121 MHz, D2O) δ 15.98; ESI-MS (m/z) 367.06
[M + Na]+
Preparation of N-Phosphonacetyl-L-isoasparagine (1). To a
solution of â-benzyl N-phosphonacetyl-L-isoasparaginate (275 mg,
0.8 mmol) in THF/ethanol (25 mL/15 mL) was added 10% Pd/C
(132 mg), and the mixture was stirred overnight under a hydrogen
atmosphere. After the completion of the reaction, Pd was removed
by filtration and the filtrate was concentrated under reduced pressure
to give a viscous colorless liquid. This was dissolved in 10 mL
water and washed with dichloromethane, and the aqueous phase
was lyophilized to give pure product 1 as a hygroscopic solid. Yield
) 186 mg (92%). 1H NMR (400 MHz, D2O) δ 2.78-2.99 (m, 4H),
4.72-4.79 (m, 1H); 31P NMR (121 MHz, D2O) δ 17.95;
ESI-MS (m/z) 253.02 [M - H]-. Anal. (C6H11N2O7P‚H2O‚
0.3EtOH), C, H, N.
Waters were added to the structure using CNS and XtalView on
the basis of the Fo-Fc electron density maps at or above the 2.8 σ