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S. Cortial et al. / Journal of Molecular Catalysis B: Enzymatic 76 (2012) 1–8
2. Experimental
under reduced pressure. The product was purified by preparative
thin layer silica gel chromatography to offer 26 mg (62% yield). 1
H
2.1. Reagents
NMR (300 MHz, CD3OD): ı = 6.61 (s, 1H), 3.34 (s, 3H), 3.30 (s, 3H).
13C NMR (62.5 MHz, CD3OD): ı = 161.2, 150.4, 145.2, 121.5, 36.8,
28.4. m/z = 155 [MH+].
All chemicals were of the highest grade commercially available
and purchased from Sigma–Aldrich, Fluka, or Merck. Para-
banic acid is from TCI-Europe N.V., Belgium. Analytical grade
solvents were from SDS (Peypin, France). Formic acid is HPLC-
grade suprapur (Merck). (14C)-methyl iodide was purchased from
Amersham-Pharmacia-Biotech (Saclay, France). Superoxide dismu-
tase (SOD, 4400 U/mg solid) and catalase (15,300 U/mg) were from
Sigma–Aldrich.
2.5. Reduction of dim-NU double bond
500 mg (2.7 mmol) of dim-NU were dissolved in 50 mL of methy-
lene chloride and 1 mL MeOH. 450 mg of NaBH4 were added
dropwise in ice and the reaction monitored by TLC. The reaction
is stopped by the addition of water, and the compound extracted
in dichloromethane. 370 mg of the ketone 2 were obtained from
flash chromatography (75% yield). 2: 1H NMR (300 MHz, CD3OD):
ı = 5·24 (t, J = 4.7 Hz, 1H), 3.95 (q, J = 13.9 Hz, 1H), 3.94 (q, J = 13.9 Hz,
1H), 3.22 (s, 3H), 3.05 (s, 3H). 13C NMR (62.5 MHz, CD3OD): ı = 160.1,
152.1, 81.3, 46.9, 36.2, 28.7. MS (ES): m/z = 187.
Ketone 2 was stirred in distilled water overnight. 12 mg of the
enol 3 were obtained by preparative HPLC. 3: 1H NMR (300 MHz,
CD3OD): ı = 4.15 (q, J = 14.4 Hz, 1H), 3.20 (s, 3H), 3.08 (s, 3H). 13C
NMR (62.5 MHz, CD3OD): ı = 164.8, 156.0, 83.0, 48.4, 38.1, 30.8. MS
(ES): m/z = 187.
2.2. N-methylation general procedure
This reaction was applied to uracil, 5-nitrouracil, 5-fluorouracil,
parabanic acid and dialuric acid [24]. The desired quantity of the
starting compound is solubilized in dimethylformamide DMF, and
the solution stirred in ice under nitrogen atmosphere. 6 equiv. of
NaH were added dropwise and allowed to react for 4 h. 2.5 equiv.
of methyl iodide were then added and temperature rised to room
temperature under stirring. The reaction is monitored by TLC or
HPLC and stopped after total consumption of the starting material
(3 h to overnight). DMF is evaporated under vacuum and the residue
purified by flash chromatography on silica gel. Yields range from 65
to 90%.
2.6. Synthesis of 5-hydroxy-N,N-dimethyluracil
N-N-dimethyl-5-nitrouracil 1: 1H-NMR (300 MHz, CDCl3):
ı = 8.72 (s, 1H), 3.59 (s, 3H), 3.38 (s, 3H). 13C-NMR (62.5 MHz,
CDCl3): ı = 154.5, 150.0, 147.2, 125.2, 38.7, 29.0, MS (ES): m/z = 185.
N-N-dimethyl-parabanic acid: 1H NMR (300 MHz, CDCl3):
ı = 3.13 (s, 6H). 13C NMR (62.5 MHz, CDCl3): ı = 160.4, 157.5, 28.43.
MS (ES): m/z = 142.
Methylation of 5-hydroxyuracil according to the procedure
reported above lead to the trimethylated derivative 5-methoxy-
N,N-dimethyluracil. The methoxy group was selectively demethy-
lated as follows: 132 mg of 5-methoxy-N,N-dimethyluracil were
dissolved in 3 mL of methylene chloride and mixed with 15.5 mL
of 1 M solution of BBr3 in methylene chloride. The mixture was
allowed to react at 4 ◦C until total disappearance of the starting
material in TLC, then the pH is adjusted to 9 with NH4OH and the
solvent evaporated. The mixture is recovered in 100 mL water and
extracted with 6 × 50 mL of methylene chloride. 5-hydroxy-N,N-
dimethyluracil was purified by flash chromatography in 75% yield.
1H NMR (300 MHz, CDCl3): ı = 6.85 (s, 1H), 3.39 (s, 3H), 3.36 (s,
3H). 13C NMR (62.5 MHz, CDCl3): ı = 161.4, 150.5, 131.8, 121.5, 37.2,
28.7. MS (ES): m/z = 185.
2.3. Synthesis of 5-nitro-N,N-(14C)-dimethyluracil
[
14C] methyl iodide (15 mCi, 55 mCi/mmole, from Amersham)
was gauged into a vacuum ramp to afford 0.22 mmol of radioac-
tive methyl iodide, i.e. 12.1 mCi. Non-labeled (0.56 mmol) methyl
iodide in the gaseous form was mixed into the vacuum ramp with
the radioactive methyl iodide to obtain an isotopic dilution to
15.4 mCi/mmol (0.78 mmol–12.1 mCi).
The [14C] methyl iodide (12.1 mCi–15.4 mCi/mmol–0.78 mmol)
was transferred under vacuum on a mixture of 53 mg (80% purity
– 1.8 mmol) of sodium hydride, 45 mg (0.28 mmol) of nitrouracil in
5 mL of anhydrous dimethylformamide. The vacuum was broken
under dry gaseous nitrogen and the mixture was allowed to react
under stirring at room temperature during 5 h. The solution was
evaporated and the residue purified.
2.7. Reduction of N,N-dimethyl-parabanic acid to
N,N-dimethyl-hydroxyhydantoin
140 mg (1 mmol) of N,N-dimethyl-parabanic acid were dis-
solved in 20 mL of methanol and the solution cooled in ice. 40 mg
of NaBH4 (1 equiv.) were added dropwise and the reaction closely
monitored by HPLC to avoid any formation of the diol. The reaction
is stopped by the addition of water, and the mixture evaporated.
52 mg of pure N,N-dimethyl-hydroxyhydantoin were obtained
through flash chromatography.
Chromatography on silica gel offered 4.7 mCi of pure [14C]
dimethyl nitrouracil (radioactive yield: 39%). Chemical (98.7%) and
radiochemical (96.1%) purities were determined by HPLC. Structure
was assigned by NMR and mass spectrometry.
N-N-dimethyl-hydroxyhydantoïn 4: 1H NMR (300 MHz, CDCl3):
ı = 5.25 (s, 1H), 3.02 (s, 3H), 2.99 (s, 3H). 13C NMR (62.5 MHz, CDCl3):
ı = 173.3, 157.2, 79.6, 26.4, 24.5. MS (ES): m/z = 144.
Specific activity was 27 mCi/mmol as measured by mass
spectrometry and UV spectrophotometry. Stock solutions were
prepared at 0.1 mCi/mL methanol (MeOH). Radiochemical purity
was determined by HPLC. The original procedure is available in
the lab book CEA/DSV/390 and the original spectra in the analysis
report of CMM-2150.
2.8. Synthesis of N,N-dimethylhydantoin
N,N-dimethylhydantoin was synthesized according to the liter-
ature [16]. 176 mg (2 mmol) of dimethyl-urea and 200 mg of glyoxal
(2 mmol) were mixed in pH 4 aqueous solution and held to react
at 50 ◦C for 4 h. The solution is evaporated by vacuo, recovered in
20% NaHCO3 and heated to 60 ◦C for 4 h. The mixture was evap-
orated by vacuo and extracted by ether. Ether was removed and
the residue purified by flash chromatography to offer 115 mg of
N,N-dimethyl-hydantoin.
2.4. Nitro-reduction of dim-NU
55 mg (0.3 mmol) of dim-NU were dissolved in 10 mL of MeOH
then 15 mg of palladium on carbon catalyst (Pd/C) were added in a
vessel equipped with a septum. Hydrogen was allowed to bubble
into the mixture via a needle 2 h at atmospheric pressure and room
temperature. The mixture is then filtrated and the solvent removed