5436 J. Am. Chem. Soc., Vol. 119, No. 23, 1997
Kahn et al.
Scheme 1
accomplished using literature procedures as follows. Bromoacetic acid
for several hours. The 13C NMR spectra were proton decoupled and
were collected with a sweep width of 49682 Hz at 17 °C with a Bruker
AVANCE DRX500 instrument; 128 scans were sufficient to detect the
signal due to the primary oxidation product. Higher spectral resolution
(0.2 Hz versus 0.6 Hz), lower temperature (5 °C), and longer acquisition
1
3
6
was converted to methyl cyanoacetate and condensed with urea to
form aminouracil, which was isolated from the chilled reaction mixture
by filtration. The aminouracil was converted to 5,6-diaminouracil by
14
nitrosation and reduction; the crude product was recovered by filtration
and was washed with H O and acetone. The crude bisulfite salt was
1
8
2
times (1024 scans) were used in experiments conducted with H
2
O
suspended in concentrated HCl and warmed to 80 °C under nitrogen,
which resulted in the precipitation of the HCl salt from the suspension.
The diaminouracil hydrochloride was recovered by filtration and washed
(>99 atom % 18O, purchased from Isotec). The spectra in H 18O were
2
resolution enhanced with a Gaussian window function. 1,4-Dioxane
(66.5 ppm) served as an internal chemical shift standard.
with H
2
O and acetone. This material was immediately used for the
Computational Methods. An ab initio quantum mechanical
approach was used to study uric acid, urate mono- and dianions, and
the oxidation products. All calculations were performed with Gaussian
next step in the synthesis, the condensation with urea, following the
published procedure. The crude uric acid was suspended in H O and
2
6
17a
17b
slowly dissolved by the gradual addition of 1 N NaOH. The pH of
the solution was not allowed to exceed 9.5, since urate is susceptible
to decomposition at high pH. After all of the material was dissolved,
the uric acid was precipitated from solution by the addition of HCl.
The product was further purified by repeated precipitations from an
alkaline solution or by precipitation from a solution of concentrated
sulfuric acid. The crystalline product was slightly yellow, with UV-
vis and C NMR spectra matching the spectra of authentic uric acid.
Typical yields for the overall synthesis of uric acid from bromoacetic
acid and KCN were 30%. Incorporation of C into C2 of urate was
92 and Gaussian 94 programs. Uric acid, the four possible urate
monoanions, and the six possible urate dianions were optimized at the
HF/6-31+G(d,p) level, and frequency analysis was performed to verify
the minima. The structures were kept planar, and energy was
minimized with respect to all geometrical parameters unless frequency
analysis indicated that the planar structure was not the minimum energy
structure. In the latter cases, the nonplanar structures were optimized
without constraints. The stability of the restricted Hartree-Fock (RHF)
wave function was tested for uric acid and for the urate monoanions;
the wave functions showed real stability. A value of 0.92 was used as
1
3
15
1
3
1
3
18
accomplished by condensing [ C]urea with methyl cyanoacetate;
a correction factor to scale the HF/6-31+G(d,p) zero-point energies.
13
13
13
[
4- C]urate was synthesized using K CN; [5- C]urate was synthesized
Urate monoanions were also optimized at the MP2(Full)/6-31+G(d)
level and in the presence of a solvent reaction field at the HF/6-31+G(d)
1
3
13
using [2- C]bromoacetic acid; [6- C]urate was synthesized using
13
13
19
[
1- C]bromoacetic acid; and [8- C]urate was synthesized by condens-
level using the Onsager self-consistent reaction field model. Two
1
3
ing diaminouracil with [ C]urea.
tautomers of 5-hydroxyisouric acid and their corresponding monoanions
NMR Experiments. Unlabeled allantoin (Sigma Chemical Co.) and
were optimized at the HF/6-31+G(d,p) level.
Calculations of NMR spectra were performed by the GIAO method
in Gaussian 94 using a density functional theory approach. The most
satisfactory results were obtained using Becke’s exchange functional
1
3
specifically C-labeled uric acids in 100 mM phosphate (D
2
O) were
20
used to study the pH dependence of the carbon chemical shifts. To
generate and study the product of the urate oxidase reaction, 5 mM
21
1
3
solutions of [ C]urate were prepared in 100 mM phosphate, pD 7.6 in
00% D O. The solution was degassed and then saturated with oxygen
22
with the Lee, Yang, and Parr (LYP) correlation functional. Two basis
1
2
sets, D95+(2d,p) and AUG-cc-pVDZ, were chosen for NMR calcula-
tions on the basis of the results of preliminary calculations of acetone
and uric acid shieldings. The geometries used for NMR calculations
were optimized at the B3LYP/6-31++G(2d,p) level. Calculated
shieldings were converted to chemical shift values by the formula δ )
in a small round-bottom flask using a Firestone valve. In a typical
experiment, 0.7 units of purified soybean root nodule urate oxidase,
5
either native enzyme or recombinant enzyme expressed in Escherichia
1
6
coli as a thioredoxin fusion protein, was added to the oxygenated
urate solution. After 5 min, catalase (400 units) was added to remove
hydrogen peroxide. Control experiments showed that the same
oxidation products formed in the absence as in the presence of catalase.
The reaction mixture was gently bubbled with oxygen for 15 min, and
the progress of the reaction was monitored spectrophotometrically (urate
has an absorption maximum at 292 nm while the product has increased
absorbance relative to urate between 302 and 306 nm). The solution
was then transferred to a 5 mm NMR tube and spectra were acquired
(17) (a) Frisch, M. J.; Trucks, G. W.; Head-Gordon, M.; Gill, P. M. W.;
Wong, M. W.; Foresman, J. B.; Johnson, B. G.; Schlegel, H. B.; Robb, M.
A.; Replogle, E. S.; Gomperts, R.; Andres, J. L.; Raghavachari, K.; Binkley,
J. S.; Gonzalez, C.; Martin, R. L.; Fox, D. J.; Defrees, D. J.; Baker, J.;
Stewart, J. P.; Pople, J. A. Gaussian 92; Gaussian, Inc.: Pittsburgh, PA,
1
992. (b) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.;
Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.; Keith, T.; Petersson, G.
A.; Montgomery, J. A.; Raghavachari, K.; Al-Laham, M. A.; Zakrzewski,
V. G.; Ortiz, J. V.; Foresman, F. B.; Cioslowski, J.; Stefanov, B. B.;
Nanayakkara, F.; Challacombe, M.; Peng, C. Y.; Ayala, P. Y.; Chen, W.;
Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gomperts, R.; Martin, R. F.;
Fox, D. J.; Binkley, J. S.; Defrees, D. J.; Baker, J.; Stewart, J. P.; Head-
Gordon, M.; Gonzalez, C.; Pople, J. A. Gaussian 94, Rev. C.3; Gaussian,
Inc.: Pittsburgh, PA, 1995.
(
13) Lazarus, R. A.; Sulewski, M. A.; Benkovic, S. J. J. Labelled Cmpd.
Radiopharm. 1982, 19, 1189-1195.
14) Sherman, W. R.; Taylor, E. C. Organic Syntheses; Wiley: New
York, 1963; Collect. Vol. 4, pp 247-249.
15) Coxon, B.; Fatiadi, A. J.; Sniegoski, L. T.; Hertz, H. S.; Schaffer,
R. J. Org. Chem. 1977, 42, 3132-3140.
(
(
(18) Scott, A. P.; Radom, L. J. Phys. Chem. 1996, 100, 16502-16513.
(19) Wong, M. W.; Frisch, M. J.; Wiberg, K. B. J. Am. Chem. Soc. 1991,
113, 4776-4782.
(
16) The urate oxidase-thioredoxin fusion protein was created by splicing
5
the gene encoding soybean root nodule urate oxidase in-frame to the
pThioHis A vector (Invitrogen). This protein was expressed and purified
in the same manner as the recombinant protein; no significant differences
(20) Wolinski, K.; Hilton, J. F.; Pulay, P. J. Am. Chem. Soc. 1990, 112,
8251-8260.
5
in kinetic behavior were noted between the fusion protein, the recombinant
protein, and native enzyme isolated from soybean root nodules.
(21) Becke, A. D. Phys. ReV. A 1988, 38, 3098-3100.
(22) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV B 1988, 37, 785-789.