T. Murase et al.
[13C2,15N2]Xanthine
J1 = 11.6 Hz, J2 = 7.6 Hz, J3 = 1.7 Hz, C2-H), 6.31 (1H, ddd,
J1 = 93.4 Hz, J2 = 7.6 Hz, J3 =0.9Hz, NH); MS (ESI) m/z 173 [M + H]+,
171 [M À H]À.
The hemisulfate [13C,15N2]2 (0.15 g, 0.773 mmol) was stirred with
[13C]formamide (0.25 g, 5.41 mmol) at 210 °C for 2 h. The
resulting mixture was dissolved in a 1 M NaOH aqueous
solution (2.3 mL) at 90 °C and filtered. The filtrate was
acidified with 1 M HCl (4.6 mL) at 4 °C, and the resulting
precipitate was collected and dried at 40 °C in vacuo to
[
13C2,15N3]5,6-Diaminopyrimidine-2,4(1H,3H)-dione hemisulfate
([13C2,15N3]2)
Sodium methoxide (781 mg, 14.5 mmol) was added to a
suspension of [13C,15N]1 (913 mg, 5.31 mmol) and [13C,15N2]urea
(304 mg, 4.83 mmol) in THF (9 mL), at room temperature. The
mixture was heated, EtOH (2.5 mL) was added, and it was
refluxed for 3 h. After cooling, the precipitate was filtered and
dried at 45 °C in vacuo to give a powder (1.41 g). The powder
was heated with 2 M H2SO4 (13 mL) and stirred for 10 min at
100 °C. After cooling in an ice bath, the resulting precipitate
was filtered, washed with water, and dried at 45 °C in vacuo to
give
[
13C2,15N2]xanthine (0.089 g, 74% yield): 1H NMR
(DMSO-d6) δ 7.93 (1H, d, J = 211 Hz, C8-H), 10.8 (1H, d,
J = 90.1Hz, NH), 11.5 (1H, d, J = 93.8Hz), 13.3 (1H, s, N7-H); HRMS
calculated for [C313C2H4N215N2O2 + H]+: m/z 157.0415; found:
157.0412 (À1.9 ppm).
Ethyl [13C,15N]2-acetamido-2-cyanoacetate ([13C,15N]1)
1
give [13C2,15N3]2 (172 mg, 18% yield): H NMR (DMSO-d6) δ 6.10
Potassium [13C]cyanide (1.00 g, 15.1 mmol) was added to a
solution of ethyl chloroacetate (2.03 g, 16.6 mmol) and 18-
crown-6 (200 mg, 0755 mmol) in acetonitrile (10 mL), in an ice
bath. The mixture was stirred at room temperature for two days
and filtered, and the solid washed using acetonitrile (10 mL). The
filtrate was concentrated under 70 Torr at 30–35 °C to give crude
(2H, s, NH2), 7.7 (3H, brs, NH2 and NH), 10.64 (1H, d, J = 91.4 Hz,
NH); MS (ESI) m/z 148 [M + H]+, 146 [M À H]À.
[13C3,15N3]UA
The hemisulfate [13C2,15N3]2 (0.170 g, 0.867 mmol) was mixed
with [13C]urea (0.0580 g, 0.954 mmol), pulverized in a mortar,
and stirred at 190 °C for 2 h. The resulting residue was dissolved
in 1 M NaOH (2.6 mL) at 90 °C and filtered. The filtrate was
acidified with 1 M HCl (5.3 mL) at 4 °C, and the resulting
precipitate was collected and dried at 40 °C in vacuo to give
[13C3,15N3]UA (0.12 g, 80% yield) as a pale gray powder: 1H
NMR (DMSO-d6) δ 10.5 (1H, d, J = 98.8 Hz, N7-H), 10.7 (1H, d,
J = 88.9 Hz, NH), 11.6 (2H, brs, NH × 2); HRMS calculated for
[C123C3H4N15N3O3 + H]+: m/z 175.0368; found: 175.0364 (À2.3 ppm).
[13C]3 (2.52 g) as an oil: H NMR (CDCl3) δ 1.32 (3H, t, J = 7.1 Hz,
CH2CH3), 3.49 (2H, d, J = 10.0 Hz, C2-CH2), 4.27 (2H, q, J = 7.1 Hz,
CH2CH3).
1
Sodium [15N]nitrite (1.00 g, 14.3 mmol) and, subsequently, a
solution of AcOH (948 mg, 15.8 mmol) in water (0.55 mL) was
added to the crude [13C]3 (2.52 g) in water (0.51 mL), in an
ice bath. The mixture was stirred at room temperature
overnight. After ethyl acetate (20 mL) and 5% sodium
bicarbonate aqueous solution (15 mL) were added to the
reaction mixture in the ice bath, the organic layer was
separated, and the aqueous layer was extracted using ethyl
acetate (20 mL × 2). The organic layers were combined, dried
over sodium sulfate, concentrated, and dried in vacuo at 40 °
C to give crude ethyl [13C,15N]2-cyano-2-hydroxyiminoacetate
([13C,15N]6) (1.46 g).
Preparation of enzyme extracts from mouse plasma
All animal experiments were approved by the Committee on
Animal Care of Sanwa Kagaku Kenkyusho Co., Ltd. Retired ICR
mice obtained from Charles River Laboratories (Yokohama,
Japan) were sacrificed under 2.5% isoflurane anesthesia. Blood
was collected in heparinized tubes and immediately centrifuged
at 2000 × g for 10 min at 4 °C to obtain plasma.
A suspension of the crude [13C,15N]6 in AcOH (2.0 mL) was
added slowly to a suspension of zinc powder (3.22 g) in a solvent
of acetic anhydride (3.0 mL) and AcOH (3.0 mL), in an ice bath at
7–15 °C under nitrogen. Additional AcOH (1 mL × 2) was used to
wash the vessel of the crude [13C,15N]6. The mixture was stirred
overnight at room temperature. The resulting mixture was
filtered, and the insolubles were washed with AcOH (4 mL × 2).
Thereafter, the insolubles were suspended in a mixed solvent
of AcOH (2mL) and chloroform (5mL), and filtered; this washing
was repeated, and the filtrates were combined and
concentrated. The residue was solved in chloroform (20 mL),
and NaOH aqueous solution 5% (25 mL) was added at 5–10 °C.
The organic layer was separated, and the aqueous layer was
extracted with chloroform (30 mL × 2). The organic layers were
combined, dried over anhydrous sodium sulfate, and
concentrated to give a white solid (1.62 g). The solid was
refluxed in ethyl acetate (5 mL) and left to stand first at 20 °C
for 2 h, and subsequently, at 6 °C for three days. The obtained
crystals were filtered, dried in vacuo at 40 °C for 3 h to give
Calibration curve of [13C2, 15N2]UA with human plasma
To prepare calibration standard samples, each 100-μL pooled
human plasma sample spiked with 20–4000 nM [13C2,15N2]UA
was mixed with 2 μM [13C3,15N3]UA as ISTD and the total volume
was adjusted to 250 μL using a Tris-buffer (pH 8.5); 500 μL MeOH
was added, and the mixture was centrifuged at 2000 × g for
15 min at 4 °C, the supernatant was evaporated and reconstituted
with 150 μL Tris-buffer. Each solution was filtered through an
ultrafiltration membrane, and the amount of [13C2,15N2]UA was
measured using LC/TQMS. The calibration curve was constructed
by plotting the peak area ratio of [13C2,15N2]UA (Analyte) to
[13C3,15N3]UA (ISTD) versus the concentrations of [13C2,15N2]UA.
Confirmation of sensitivity: XOR activity assay of diluted
mouse plasma
[
13C,15N]1 (755 mg) as colorless crystals. The filtrate was
concentrated, and subjected to silica gel column The XOR activity was measured with the serially diluted mouse
chromatography (n-hexane:ethyl acetate = 2:1) to give plasma as a model reaction of a less active biological sample to
[
13C,15N]1 (185 mg). The total amount of [13C,15N]1 was 940 mg confirm the sensitivity of the assay utilizing [13C2,15N2]xanthine. A
obtained in 36% yield from potassium [13C]cyanide: 1H NMR 100-μL mouse plasma sample diluted 1, 4, 16, 64, 256, or 1024 times
(CDCl3) δ 1.37 (3H, t, J = 7.1 Hz, CH2CH3), 2.12 (3H, d, J = 1.5 Hz, was mixed with 16 μM [13C2,15N2]xanthine, 40 μM NAD+, 7.98μM
CH3CO), 4.37 (2H, q, J = 7.1 Hz, CH2CH3), 5.53 (1H, ddd, oxonate, and 2 μM [13C3,15N3]UA, and the total volume was adjusted
Copyright © 2016 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2016, 59 214–220