Synthesis of [1Ј,2Ј,5Ј,2-13C4]-2Ј-Deoxy-D-adenosine
39.6 Hz, C-4Ј), 119.2 (C-5), 140.5 (C-8), 148.6 (C-3), 152.6 (strong oven (100 °C) overnight afforded 1.60 g (11.1 mmol, 86%) of the
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signal, C-2), 155.8 (C-6) ppm. HRMS (ESI): m/z calcd. for
title compound. H NMR (300 MHz, [D6]DMSO): δ ϭ 4.75 (s, 1
H, 5-H), 7Ϫ5 (br. s, 4 H, NH and SH) ppm. 13C NMR (75 MHz,
[D6]DMSO): δ ϭ 78.6 (C-5), 155.2 (C-6), 162.7 (C-4), 174.8 (strong
signal, C-2) ppm. MS (ESI): m/z ϭ 144 [M ϩ H].
C613C4H13O4N5 255.1153; found 255.1168.
[2-13C]-Adenine (2a): Compound 11a (100 mg, 0.64 mmol) was dis-
solved in ethanol (3 mL), containing concentrated aqueous NH3
(1.4 mmol NH3). The solution was transferred into a Carius tube
and frozen by application of liquid nitrogen. The tube was sealed
and heated at 140 °C, and left to cool overnight. The sealed tube
was frozen with liquid nitrogen and opened. Evaporation of the
solvent afforded a white residue, which contained, in addition to
[2-13C]-adenine, ammonium chloride (135 mg, quantitative). This
solid was used without further purification. 1H NMR (300 MHz,
D2O): δ ϭ 7.90 (s, 1 H, 8-H), 8.26 (s, 1 H, 8-H), 8.26 (s, 1 H, 8-H)
ppm. 13C NMR (75 MHz, D2O): 138.9 (C-8), 150.4 (strong signal,
C-2) ppm. HRMS (ESI): m/z calcd. for C413CH6N5 137.0720;
found 137.0757.
[2-13C]-6-Amino-5-nitroso-2-thioxo-1,2-dihydro-4(3H)-pyrimidinone
(7a): Compound 6a (1.55 g, 10.7 mmol) was suspended in HCl (1
, 40 mL) and cooled in ice. To this mixture was added a solution
of sodium nitrite (785 mg, 11.3 mmol) in water (10 mL). Stirring
and cooling was continued for 7 h, after which time the red suspen-
sion was filtered and washed with water and ethanol (10 mL). Dry-
ing in a vacuum desiccator (100 °C) yielded 1.71 g (9.9 mmol, 92%)
of a red solid matter. 1H NMR (300 MHz, [D6]DMSO): δ ϭ 7.7
(br. s), 11.2, 12.6 ppm. 13C NMR (75 MHz, [D6]DMSO): 148.7 (C-
5), 159.4 (C-6), 174.6 (C-4), 176.4 (strong signal, C-2) ppm. MS
(ESI): m/z ϭ 174 [M ϩ H], 196 [M ϩ Na].
[2-13C]-5,6-Diamino-2-thioxo-1,2-dihydro-4(3H)-pyrimidinone (8a):
Compound 7a (1.6 g, 9.3 mmol) was suspended in saturated
NaHCO3 (38 mL) and cooled in ice. Sodium dithionite (4.25 g,
24.3 mmol) was added in four portions and the reaction mixture
was stirred for 6 h. To the now yellow suspension was (slowly)
added acetic acid, and the solid was collected by filtration. Washing
with water and ethanol and drying in a vacuum desiccator afforded
1.5 g (9.3 mmol, 94%) of a slightly yellow solid. 1H NMR
(200 MHz, [D6]DMSO): δ ϭ 8Ϫ5 (br. s) ppm. 13C NMR (50 MHz,
[D6]DMSO): δ ϭ 102.4 (C-5), 140.8 (C-6), 157.9 (C-4), 167.6
(strong signal, C-2) ppm. MS (ESI): m/z ϭ 160 [M ϩ H], 182 [M
ϩ Na].
Ethyl Cyanoacetate (5): Cyanoacetic acid (3.41 g, 40 mmol) was
dissolved in dichloromethane (150 mL), after which ethanol
(12 mL, 200 mmol) and H2SO4 (65 µL, 1.2 mmol) were added. This
solution was heated under reflux for 18 h, using a reversed
DeanϪStark apparatus to trap the liberated water. After the reac-
tion period, the solution was poured into 100 mL of 10% NaHCO3
solution, and after the separation of the ensuing layers the aqueous
layer was extracted twice with dichloromethane (100 mL). The so-
lution was dried with MgSO4, followed by filtration and removal
of the solvent in vacuo. The residual colourless oil weighed 4.3 g
(38 mmol, 95%) and consisted of ethyl cyanoacetate. 1H NMR
(300 MHz, CDCl3): 1.32 (t, 3JHϪH ϭ 7.2 Hz, 3 H, Et), 3.49 (s, 2 H,
2-H), 4.27 (q, 3JHϪH ϭ 7.2 Hz, 2 H, Et) ppm. 13C NMR (75 MHz,
CDCl3): 13.8 (Et), 24.6 (C-2), 62.8 (Et), 113.1 (C-3),162.9 (C-1)
ppm. MS (ESI): m/z ϭ 114 [M ϩ H], 136 [M ϩ Na].
[2-13C]-5,6-Diamino-4(3H)-pyrimidinone (9a): Compound 8a (1.5 g,
9.3 mmol) was dissolved in NH3 (5%, 40 mL). Raney nickel (5 mL
of a 50% slurry in water) was added to this solution, and the reac-
tion mixture was heated under reflux for 90 min. The black mixture
was filtered hot through two fibreglass filter papers, which were
washed with boiling water (2 ϫ 15 mL). The filtrate was concen-
trated in vacuo to afford 1.10 g of a yellow solid (8.7 mmol, 92%).
1H NMR (200 MHz, [D6]DMSO): δ ϭ 5.4 (br. s, 5 H), 7.63 (d,
1JCϪH ϭ 200.0 Hz, 1 H, 2-H) ppm. 13C NMR (200 MHz,
[D6]DMSO): δ ϭ 110.5 (C-5), 138.8 (strong signal, C-2), 147.7 (C-
6), 156.7 (C-4) ppm. MS (ESI): m/z ϭ 128 [M ϩ H].
6-Amino-2-thioxo-1,2-dihydro-4(3H)-pyrimidinone (6): Compound
13 (400 mg, 1.72 mmol) was placed in a 100-mL three-necked flask,
containing a propylene (black) magnetic stirring bar. The flask and
contents were cooled to Ϫ70 °C and liquid NH3 (50 mL) was intro-
duced. Na(s) (43 mg, 1.89 mmol) was added to this colourless solu-
tion in small portions. Once the reaction mixture remained blue,
indicating the presence of solvated electrons in NH3, NH4Cl (1 g)
was added to quench the reaction. While stirring, the flask was
opened and the NH3 was allowed to evaporate. The white residue
was dissolved in water (4 mL) with the aid of the minimum amount
of 1 NaOH required for complete dissolution and precipitated
with a small amount of acetic acid. Filtration and washing with
water, ethanol and acetone (3 mL of each) afforded, after drying,
[2-13C]-Hypoxanthine (10a): Compound 9a (800 mg, 6.3 mmol) was
suspended in water (2 mL) to which were added H2SO4 (617 mg,
6.3 mmol) and formic acid (290 mg, 6.3 mmol). The mixture was
heated under reflux overnight and was subsequently allowed to
cool to room temperature. Additional water (1 mL) was added to
keep the mixture fluid and the mixture was neutralised with ammo-
nium hydroxide (25% in water) and acetic acid. The precipitate was
cooled on ice, filtered and washed with water (twice, 4 mL), ethanol
(96%, twice, 4 mL), and acetone (twice, 4 mL). The solid was dried
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200 mg (1.4 mmol, 81%) of a white powder. H NMR (300 MHz,
[D6]DMSO): δ ϭ 4.75 (s, 1 H, 5-H), 7Ϫ5 (br. s, 4 H, NH and SH)
ppm. 13C NMR (75 MHz, [D6]DMSO): δ ϭ 78.6 (C-5), 155.2 (C-
6), 162.7 (C-4), 174.8 (C-2) ppm. MS (ESI): m/z ϭ 143 [M ϩ H].
in vacuo to give 802 mg (5.9 mmol, 94%) of a slightly coloured
[2-13C]-6-Amino-2-thioxo-1,2-dihydro-4(3H)-pyrimidinone
(6a):
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solid. 1H NMR (200 MHz, [D6]DMSO): δ ϭ 8.0 (d, JCϪH
ϭ
204.5 Hz, 1 H, 2-H), 8.10 (s, 8-H, 1 H) ppm. 13C NMR (50 MHz,
[D6]DMSO): δ ϭ 119.2 (C-5), 140.2 (C-8), 144.7 (strong signal,
C-2), 153.3 (C-4), 155.4 (C-6) ppm. HRMS (ESI): m/z calcd. for
C413CH4N4O 138.04969; found 138.04973.
Ethyl cyanoacetate (1.47 g, 13.0 mmol) was added to a solution of
sodium ethoxide, prepared by dissolving sodium (298 mg,
13.0 mmol) in dry ethanol (10 mL). After about 1 min, a white
suspension developed and [13C]-thiourea (1.0 g, 12.96 mmol) was
added. This suspension was heated under reflux for 2 h, during
which the suspension became a clear, yellowish solution and then
a white suspension again. The ethanol was removed in vacuo and
the solid residue was dissolved in water (15 mL). After precipitation
with acetic acid, the solid was collected by filtration and washed
with water, ethanol and acetone (10 mL each). Drying in a vacuum
[2-13C]-6-Chloropurine (11a): POCl3 (22 mL) was added to N,N-di-
methylaniline (1.8 mL), followed by 10a (800 mg, 5.9 mmol). The
mixture was heated under reflux for 30 min, during which time it
slowly turned brown. Excess POCl3 was removed with a water as-
pirator and the last traces were removed with an oil pump. The
Eur. J. Org. Chem. 2002, 2356Ϫ2362
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