A R T I C L E S
Luo et al.
previously.31,33 HsADA was purchased from Sigma or was a generous
gift of Dr. Kami Kim and Li-Min Ting (Department of Microbiology
and Immunology at Albert Einstein College of Medicine). PfADA was
prepared as described previously.22 All other reagents were obtained
from readily available commercial sources and used without further
purification.
[4- C]-, [4- N]-, and [4- C, 4- N]-5-Amino-1H-imidazolecar-
bonitrile (5a,b). Compound 4 (195 mg, 1 mmol) was dissolved in
TFA-MeOH (14 mL, 2:5), and the solution was stirred for 14 h.
Complete conversion from 4 to 5 was confirmed by TLC using 20%
13
15
13
15
3
MeOH-CHCl as the developing solvent. 3,4-Dihydro-2H-pyran, TFA,
and MeOH in the reaction mixture were removed by reduced pressure,
followed by overnight oil pump vacuum to give 5a + 5b quantitively.
The ratio of 5a,b is around 9:1, the latter of which was generated
through the migration of 3,4-dihydro-2H-pyran to 5-amino group of
5a under acidic conditions. However, this mixture was not subject to
further purification because the THP group was readily removed in
Synthesis. General Methods. All reagents and solvents were
purchased from commercially available sources and used without further
1
purification. Proton nuclear magnetic resonance ( H NMR), carbon
13
nuclear magnetic resonance ( C NMR), and nitrogen nuclear magnetic
1
5
3 6
resonance ( N NMR) were recorded in CD OD or DMSO-d using a
Bruker 300 MHz instrument. 1-D C and 15N NMR spectra were
collected using continuous proton-decoupling mode with at least 10 000
scans. Proton chemical shifts were referenced to internal 3-(trimeth-
1
3
the subsequent cyclization step. [4- C]-, [4- N]-, and [4- C, 4- N]-
13
15
13
15
1
labeled 5a: H NMR (300 Hz, CD
3
OD) δ ppm 8.04 (s, 1H, NdCH-
13
13 15
13
N). 4- C/ C N-labeled 5a: C NMR (75 Hz, CD OD) δ ppm 111.8
3
15
13
13
13 15
13
15
ylsilyl)propionate. N and C chemical shifts were referenced indirectly
from the proton chemical shift reference. Electrospray ionization (ESI)
(CN, singlet for C and doublet for C N). [4- C]-, [4- N]-, and
13
15
15
[4- C, 4- N]-labeled 5a: N NMR (75 Hz, CD OD) δ ppm 272
3
n
(singlet for N and doublet for 13C N); ESI MS (m/e) std C
15
15
mass spectra were determined on LCQ LC/MS (Finnigan Corp.) using
4
H
5
15
N
4
(M
13
either positive or negative modes.
+ 1) 109.13, found 110.13 for
C
1
C
3
15
H
5
N
4
, 110.13 for C
4
15
5 1 3
H N N ,
13
15
13
15
13
15
13
13
[4- C]-, [4- N]-, and [4- C, 4- N]-5-Nitro-1-(tetrahydropyran-
111.13 for
5b: 1H NMR (300 Hz, CD
(m, 1H, O-CH-N), 3.94 (m, 1H, O-CH
C
1
C
3
H
5
N N
1 3
. [4- C]-, [4- N]-, and [4- C, 4- N]-labeled
OD) δ ppm 7.73 (s, 1H, NdCH-N), 4.74
-C), 3.68 (m, 1H, O-CH
1
3
15
13
15
2
-yl)imidazolecarbonitriles 3. [4- C]-, [4- N]-, and [4- C, 4- N]-
3
34
labeled 3 were prepared according to the method described previously.
2
2
-
1
3
15
13
15
13
13 15
Briefly, [4- C]-, [4- N]-, and [4- C, 4- N]-5-nitroimidazolecarbo-
C), 1.7-2.3 (m, 6H, C-CH -C). [4- C]- and [ C N]-labeled 5b:
2
3
5
13
13
nitriles 2 (420 mg, 3 mmol), 3,4-dihydro-2H-pyran (0.563 mL, 6
mmol), and p-TsOH (15 mg) were added into 6 mL of EtOAc, and the
reaction mixture was stirred at room temperature for 2 h. Triethylamine
3
C NMR (75 Hz, CD OD) δ ppm 113.6 (CN, singlet for C and
doublet for 13C N). [4- N]- and [4- C, 4- N]-labeled 5b: N NMR
15
15
13
15
15
(75 Hz, CD
3
OD) δ ppm 266 (singlet for 15N and doublet for C N).
15
13
15
(TEA, 0.02 mL) and hexane (9 mL) were added, and the supernatant
13
13
15
[
6- C], [6- N], and [6- C, 6- N]Adenines 6. TEA (1.3 mL, 10
was subject to a short silica column. The impurities were eluted with
mmol) was added into 10 mL of 2-ethoxyethanol containing 9:1 5a,b
120 mg, 1 mmol) and formamidine acetate (440 mg, 4 mmol). All
solids were dissolved when the reaction was heated to 90 °C. The
reaction mixture was then stirred under these conditions for another
2 h, and volatile residues were removed under reduced pressure,
followed by oil pump vacuum for 24 h. The solid residues were subject
to recrystallization in hot water to give pure [6- C], [6- N], and [6- C,
- N]adenines (125-135 mg, 90-98%). [6- C]-labeled adenine 6:
H NMR (300 Hz, CD OD) δ ppm 8.19 (d, 1H, 2 position), 8.10 (s,
1
3
15
4
[
0% EtOAc/hexane, followed by the elution of [4- C]-, [4- N]-, and
(
1
3
15
4- C, 4- N]-labeled 3 with 80% EtOAc/hexane. The solvents were
1
3
15
13 15
removed in vacuo to give 0.63-0.65 g of C/ N/ C N-labeled
1
3
15
13
15
products with yield 96-98%. [4- C]-, [4- N]-, and [4- C, 4- N]-
1
1
labeled 3: H NMR (300 Hz, CD
3
OD) δ ppm 8.18 (s, 1H, NdCH-
-C), 3.81 (m, 1H,
-C); C NMR (75 Hz, CD
N), 5.56 (dd, 1H, O-CH-N), 4.12 (m, 1H, O-CH
2
13
15
13
1
3
O-CH
2
-C), 1.7-2.3 (m, 6H, C-CH
2
3
-
15
13
6
1
1
3
15
OD) δ ppm 153.2, 139.7, 109.2 (CN, singlet for C and N and doublet
3
for C N), 102.5 (singlet for N and doublet for 13C and 13C N),
1
3
15
15
15
13
13
1
H, 8 position); C NMR (75 Hz, CD
ESI MS (m/e) std C (M - 1) 136.13, found 137.13 for
3
OD) δ ppm 156.4 (s, CN);
1
5
13
15
15
8
8.2, 70.1, 32.3, 26.6, 23.5. [4- N]- and [4- C, 4- N]-labeled 3:
N
5 8 5
H N
1
5
NMR (75 Hz, CD
3
OD) δ ppm 284 (singlet for N and doublet for
13
15
1
1 4 8 5 3
C C H N . [6- N]-labeled adenine 6: H NMR (300 Hz, CD OD) δ
13
15
C N).
[
ppm 8.19 (s, 1H, 2-H), 8.10 (s, 1H, 8-H); 15N NMR (75 Hz, DMSO-
1
3
15
13
15
4- C]-, [4- N]-, and [4- C,4- N]-5-Amino-1-(tetrahydropyran-
15
d
6
) δ ppm 82 (s, C N); ESI MS (m/e) std C
found 137.13 for C
Hz, CD
C NMR (75 Hz, CD OD) δ ppm 156.4 (d, C N); N NMR (75
Hz, DMSO-d
) 136.13, found 138.13 for 13C
1 4 8 1 4
C H N N .
H
5 8
N
5
(M - 1) 136.13,
1
3
15
2
[
(
-yl)imidazolecarbonitriles 4. A mixture of [4- C]-, [4- N]-, and
15
13
15
1
5
H
8
N
1
N
4
. [6- C, 6- N]adenine 6: H NMR (300
13
15
4- C, 4- N]-5-nitro-1-(tetrahydropyran-2-yl)imidazole-carbonitriles 3
446 mg, 2 mmol) and 160 mg of Raney nickel in 20 mL of EtOH-
3
OD) δ ppm 8.19 (d, 1H, 2 position), 8.10 (s, 1H, 8 position);
13
13 15
15
3
H
2
O (95:5) was stirred under 1 atm H
2
at room temperature for 16 h.
) δ ppm 82 (d, 13C N); ESI MS (m/e) std C
15
6
5
H N
8 5
(M -
The disappearance of 3 and formation of 4 were confirmed by thin
layer chromatography (TLC) using 80% EtOAc-hexane and 10%
15
1
13
15
Synthesis of Isotopically Labeled Adenosines. [6- C]-, [6- N]-,
6- C, 6- N]-, [1- N]-, [1′- H]-, and [1′- C]-labeled adenosines were
prepared from radiolabeled riboses 7 and isotopically labeled adenines
through two one-pot enzymatic reactions. Specifically labeled ATPs
MeOH-CHCl
3
as developing solvents, respectively. The reaction
13
15
15
3
14
[
mixture was then passed through a 3-cm self-packed Celite 521 column.
The volatile residues of the filtrate were removed under reduced
pressure, followed by oil pump vacuum for overnight to give 4 (375-
33
were synthesized as described previously with some modification.
1
3
15
13
15
3
4
(
80 mg, 97-98% yield). [4- C]-, [4- N]-, and [4- C, 4- N]-labeled
Here ribose was used as a starting material instead of glucose.
Radiolabeled ribose 5-phosphate was generated by ribokinase (RK).
Briefly, 2 mM adenine and 1 mM ribose (final concentrations) were
added into a solution containing 20 mM phosphoenolpyruvate, 0.1 mM
ATP, 100 mM phosphate, 50 mM glycylglycine, 50 mM KCl, 20 mM
1
:
H NMR (300 Hz, CD
3
OD) δ ppm 7.60 (s, 1H, NdCH-N), 5.25
-C), 3.69 (m, 1H, O-CH
-C); C NMR (75 Hz, CD OD) δ ppm
dd, 1H, O-CH-N), 4.07 (m, 1H, O-CH
2
2
-
1
3
C), 1.7-2.3 (m, 6H, C-CH
1
2
3
1
3
13 15
58.8, 137.6, 113.4 (CN, singlet for C and doublet for C N
and N), 84.5, 83.2 (singlet for N and doublet for C and C N),
8.8, 32.3, 26.6, 23.5. [4- N]- and [4- C, 4- N]-labeled 4: N NMR
75 Hz, CD
OD) δ ppm 284 (singlet for 15N and doublet for
C N); ESI MS (m/e) std C O (M + 1) 192.80, found
1
5
15
13
13 15
MgCl
the addition of an enzyme stock containing 0.01 unit of ribokinase,
.01 unit of adenine phosphoribosyltransferase, 0.01 unit of phospho-
2
, and 2 mM DTT (pH ) 7.4). The reaction was initialized by
15
13
15
15
6
(
3
0
1
3
15
9
14 4
H N
D-ribosyl-1-pyrophosphate synthase, 1 unit of adenylate kinase, and 1
unit of pyruvate kinase. The reaction mixture was incubated at 30 °C
for 12 h to generate radiolabeled ATPs and then heated to 95 °C for 3
min. Into this solution 5 mM glucose, 4 units of hexokinase, 5 units of
adenylate kinase, and 10 units of alkaline phosphatase were added.
The reaction mixture was incubated at 30 °C for 10 h to allow the
conversion of ATP to adenosine. The conversion from ATP to adenosine
is slow in the presence of only alkaline phosphatase, probably due to
1
3
15
1
93.80 for
1 8 13 4 9 13 1 3
C C H N O, 193.73 for C H N N O, 194.89 for
13
1 8 1 3
C C H N O.
1315N
(
(
(
33) Parkin, D. W.; Leung, H. B.; Schramm, V. L. J. Biol. Chem. 1984, 259,
411-9417.
34) Wanner, M. J.; Koomen, G. J. J. Chem. Soc., Perkin Trans. 1 2002, 1877-
880.
35) Suwinski, J.; Swierczek, K. J. Labelled Compd. Radiopharm. 2002, 45,
95-801.
9
1
7
8010 J. AM. CHEM. SOC.
9
VOL. 129, NO. 25, 2007