Use of 13C as an indirect tag in 15N specifically labeled nucleosides. Syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues.

Article abstract of DOI:10.1021/jo0345446

We have previously reported the use of a 13C tag at the C2 of 15N-multilabeled purine nucleosides to distinguish the adjacent-labeled 15N atoms from those in an untagged nucleoside. We now introduce the use of an indirect tag at the C8 of 15N7-labeled purine nucleosides. This tag allows unambiguous differentiation between a pair of 15N7-labeled purines in which only one is 13C8 labeled. Although the very small C8-N7 coupling (<1 Hz) precludes its direct detection in 1D 15N spectra, 2D 1H-15N NMR experiments display the large C8-H8 coupling (>200 Hz) because H8 is coupled to both N7 and C8. The 13C8 atom is introduced by means of a ring closure of the exocyclic amino groups of a pyrimidinone using [13C]sodium ethyl xanthate. Here, we present methods for the syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues.

Full text of DOI:10.1021/jo0345446

Use of 13C a s a n In d ir ect Ta g in 15N Sp ecifica lly La beled
Nu cleosid es. Syn th eses of [8-13C-1,7,NH2-15N3]Ad en osin e,
-Gu a n osin e, a n d Th eir Deoxy An a logu es
Anthony J . Shallop, Barbara L. Gaffney, and Roger A. J ones*
Department of Chemistry and Chemical Biology, Rutgers, The State University of New J ersey,
Piscataway, New J ersey 08854
jones@rutchem.rutgers.edu
Received April 28, 2003
We have previously reported the use of a 13C tag at the C2 of 15N-multilabeled purine nucleosides
to distinguish the adjacent-labeled 15N atoms from those in an untagged nucleoside. We now
introduce the use of an indirect tag at the C8 of 15N7-labeled purine nucleosides. This tag allows
unambiguous differentiation between a pair of 15N7-labeled purines in which only one is 13C8 labeled.
Although the very small C8-N7 coupling (<1 Hz) precludes its direct detection in 1D 15N spectra,
1
2D H-15N NMR experiments display the large C8-H8 coupling (>200 Hz) because H8 is coupled
to both N7 and C8. The 13C8 atom is introduced by means of a ring closure of the exocyclic amino
groups of a pyrimidinone using [13C]sodium ethyl xanthate. Here, we present methods for the
syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues.
In tr od u ction
To facilitate 15N NMR studies of specifically labeled
DNA and RNA, we have developed methods for efficient
synthesis of a variety of 15N-multilabeled purine nucleo-
sides.21-24 To permit spectral differentiation of two mul-
tilabeled nucleotides, we introduced the use of a 13C tag
at the purine C2 position to distinguish the neighboring
15N1, 15N3, and the guanine 15NH2 from those in an
untagged nucleotide based on the substantial 13C-15N
couplings (6-23 Hz).21,24 Use of this 13C2 tag allowed us
to probe local hydrogen bonding and stacking in a series
of specifically labeled RNA molecules with a minimum
of synthetic effort.10,25,26
15N NMR of labeled DNA and RNA fragments is a
valuable method for probing a variety of interactions at
specific nitrogen atoms since 15N chemical shifts are quite
sensitive to effects such as protonation,1,2 hydrogen
bonding,3-8 metal binding,9 and aromatic stacking.10
A
significant advantage of this use of 15N as a probe is that
it does not perturb the structure while giving straight-
forward spectra.11 In a related but different method,
uniformly 15N-labeled nucleic acid samples have proven
1
to be highly effective for structure determination by H
NMR,12 although nonhelical regions and larger molecules
can leave assignment ambiguities for which specific
labeling by chemical synthesis provides a clear resolu-
tion.13-15 Further, multilabeled nucleosides and nucleic
acids also provide useful mass tags for mass spectroscopic
work.16-20
We now extend this concept to the purine C8 position.
A preliminary account of this work with no synthetic
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D. J . J . Mol. Biol. 1998, 282, 619-636.
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10.1021/jo0345446 CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/08/2003
J . Org. Chem. 2003, 68, 8657-8661
8657

Shallop et al.
SCHEME 1
details was reported earlier as part of an overview of our
use of 13C tags with specifically 15N labeled nucleosides.27
Because the 13C8-15N7 coupling often is small (<1 Hz),
the 13C8 more conveniently serves as an indirect tag to
differentiate one of a pair of 15N7 labeled purines through
the combination of the 13C8-H8 coupling (>200 Hz) and
the H8-15N7 coupling (12 Hz). We present here proce-
dures for the syntheses of [8-13C-1,7,NH2-15N3]adenosine,
-guanosine, and their deoxy analogues.
xanthine has recently been reported,34 a procedure to
prepare the latter is not readily available.
Resu lts a n d Discu ssion
The synthetic focus of the work reported here is the
incorporation of 13C at the C8 position of adenosine and
guanosine. We have previously introduced 13C at the C2
position of purines either by a set of transformations
using [13C]cyanogen bromide24 or by closure of the py-
rimidine ring using [13C]sodium ethyl xanthate.21 The
latter procedure is directly applicable to closure of the
imidazole ring. The [13C]NaSCSOEt is easily synthesized
from [13C]CS2, and the resulting thiol can conveniently
be removed along with the thiol in the original starting
material.
A number of methods have previously been reported
for incorporation of 13C at the purine C8 position, either
as the only label,28-32 or along with some 15N, such as
[8-13C-NH2,9-15N2]adenosine.33 However, none of these
reports includes 15N at the biologically significant N7 in
the major groove. In addition, although the synthesis of
[8-13C-1,3,7,NH2-15N4]adenine from [8-13C-1,3,7,NH2-15N4]-
As shown in Scheme 1, the 15N7 can be incorporated
easily and in very high yield into the inexpensive pyrim-
idinone 1 by a nitrosation using [15N]NaNO2, followed by
reduction of the nitroso group to the 15N-labeled amino
group.22 The ring closure to incorporate the 13C8 occurs
in 90% yield by refluxing 2 in dimethylformamide (DMF)
with [13C]NaSCSOEt, which is preformed by reacting
[13C]CS2 with NaOH/EtOH. Both thiols in 3 are cleaved
with Raney nickel in 82% yield. Chlorination of 4 in
(27) Abad, J .-L.; Shallop, A. J .; Gaffney, B. L.; J ones, R. A. Biopoly-
mers (NAS) 1998, 48, 57-63.
(28) Kang, P.; Foote, C. S. J . Am. Chem. Soc. 2002, 124, 4865.
(29) LaFrancois, C. J .; Fujimoto, J .; Sowers, L. C. Nucleosides
Nucleotides 1999, 18, 23-37.
(30) Poe, M.; Germershausen, J . I.; MacCoss, M. Anal. Biochem.
1987, 164, 450-456.
(31) SantaLucia, J ., J r.; Shen, L. X.; Cai, Z.; Lewis, H.; Tinoco, I.,
J r. Nucleic Acids Res. 1995, 23, 4913-4921.
(32) Sharma, M.; Alderfer, J . L.; Box, H. C. J . Labelled Compd.
Radiopharm. 1983, 20, 1219-25.
(33) Orji, C. C.; Michalczyk, R.; Silks, L. A. P., III. J . Org. Chem.
1999, 64, 4685-4689.
(34) Chiriac, M.; Axente, D.; Palibroda, N.; Craescu, C. T. J . Labelled
Compd. Radiopharm. 1999, 42, 377-385.
8658 J . Org. Chem., Vol. 68, No. 22, 2003

13C as an Indirect Tag in 15N-Labeled Nucleosides
TABLE 1. NMR Ch em ica l Sh ifts a n d Cou p lin g Con sta n ts for Ba se P r oton s a n d La beled Atom s for 11a a n d b a n d 15a
a n d ba
b
compd
13C8b
15N1b
15N7b
15NH2
75 (s)
H1
H2
H8
15NH2
11a
135.3 (s)
148 (s)
244 (s)
10.62 (d)
7.92 (dd)
6.44 (d)
<1:C8-N7
89:H1-N1
214:H8-C8
12:H8-N7
7.92 (dd)
213:H8-C8
12:H8-N7
8.34 (dd)
213:H8-C8
12:H8-N7
8.31 (dd)
90:NH 2-NH2
11b
15a
15b
135.2 (s)
<1:C8-N7
148 (s)
249 (s)
242 (s)
242 (s)
74 (s)
10.63 (d)
89:H1-N1
6.44 (d)
90:NH 2-NH2
139.9 (s)
<1:C8-N7
237 (d)
5:N1-NH2
83 (d)
5:NH2-N1
8.13 (d)
16:H2-N1
7.34 (dd)
90:NH2-NH2
3:NH2-N1
7.28 (dd)
139.5 (s)
237 (d)
82 (d)
8.12 (d)
<1:C8-N7
5:N1-NH2
5:NH2-N1
16:H2-N1
213:H8-C8
12:H8-N7
90:NH2-NH2
3:NH2-N1
a
Each entry shows chemical shift (δ) and splitting pattern in parentheses, followed by coupling constants (Hz) and designation of
b
coupled atoms. Proton decoupled.
refluxing POCl3 to give 5 works well,22 but hydrolysis
back to 4 during workup of this reaction can be a
problem. We have found that care in removing excess
POCl3 and having a pH > 10 avoid this hydrolysis.
Enzymatic transglycosylation to give 6a and b occurs
in high yield22 with purine nucleoside phosphorylase and
either 7-methylguanosine for the ribonucleoside or thy-
midine and thymidine phosphorylase for the deoxy-
nucleoside. The second 15N label is then readily incorpo-
rated in 85-90% yield by ammination of 6a and b to give
the adenosine products 7a and b.22 Their oxidation gives
the N1-oxides 8a and b in 92% yield.24
the 13C8-H8 coupling constants are very large (>200 Hz)
and the 15N7-H8 constants are moderate (12 Hz) but the
13C-15N constants are small (<1 Hz). Thus, while use of
the 13C8 as a direct tag for the adjacent 15N7 atom may
be difficult, it serves most conveniently as an indirect tag,
via the larger H8 coupling to both the 13C8 (>200 Hz)
1
and the 15N7 (12 Hz). A heteronuclear H-15N 2D NMR
spectrum displays a H8, N7 cross-peak that is split by
1
>200 Hz along the H dimension (see Supporting Infor-
mation). With this approach, a tagged 15N7 resonance
from a DNA or RNA fragment can be distinguished
readily from an untagged one.
We have further optimized our previously reported24
adenosine to guanosine transformation. In the first step,
the N1-oxides 8a and b are reacted with cyanogen
bromide, generated in situ by addition of [15N]KCN to
Br2, to give 9a and b. In our previous procedure, the
reverse order of addition (Br2 to KCN) may have gener-
ated some cyanogen, (CN)2, resulting in the loss of the
15N label. In the next steps, treatment of 9a and b with
Et3N opens the oxazolidine ring so that the N1-oxide can
be methylated with CH3I. Neutralization followed by
heating results in deformylation and ring closure to give
10a and b. The time required for some of these steps has
been significantly reduced, resulting in cleaner reaction
mixtures and higher yields of 10a and b (∼80%). Enzy-
matic deamination completes the transformation to give
11a and b in 89-95% yield.
The triply labeled adenosine N1-oxides 8a and b can
also be converted to quadruply labeled adenosine 15a and
b by our reported methods.23 After an N1-alkoxy-medi-
ated Dimroth rearrangement transposes the labeled
amino to the N1 position in 12a and b, the resulting N6-
methoxy group is removed with Raney Ni to give 13a and
b in an overall yield of 81% from 8a and b. The final
label is introduced by converting the 6-NH2 to a 6-triazole
group in 14a and b, based on a high-yield procedure
developed by Robins.35,36 The displacement of the 6-tria-
zole with [15N]NH3 gives 15a and b in 81-83% yield.
NMR chemical shifts and coupling constants for the
15N/13C atoms and selected base protons in the final four
nucleosides are shown in Table 1, and their full spectra
are included in the Supporting Information. As expected,
Exp er im en ta l Section
[8-13C-7-15N]-2,8-Dith ioxoh yp oxa n th in e (3). Dry 222 (11.3
g, 71 mmol) and [13C]-NaSCSOEt21 (11.3 g, 78 mmol) were
suspended in anhydrous DMF (200 mL) and refluxed for 2.5
h. The reaction mixture was cooled in an ice bath, and the
product was precipitated by addition of cold CH3CN. The crude
product was filtered, washed with cold CH3CN, and dried to
give 10.5 g. The filtrate was concentrated and purified by
reversed phase C18 chromatography using water to give an
additional 5.69 g. The crude products were combined and used
without further purification.
[8-13C-7-15N]Hyp oxa n th in e (4). To a suspension of 3 (4.00
g, 17.8 mmol) in 250 mL of water was added 50% aqueous
Raney Ni (16.0 g) and 96% formic acid (7.0 mL). The resulting
suspension was refluxed for 15 min, ethylenediaminetetraace-
tic acid and dipotassium salt (EDTA) (6.30 g, 15.6 mmol) was
added, and refluxing was continued for 2 h. The hot reaction
mixture was filtered, and the solid was washed with boiling
water. The filtrate was concentrated and the residue purified
on a C18 column using a gradient of water to 40% CH3CN in
water over 30 min to give 2.23 g (16.1 mmol, 92% from 2) of 4.
[8-13C-7-15N]-6-Ch lor op u r in e (5). A mixture of 4 (4.00 g,
29.0 mmol), POCl3 (100 mL, 1.07 mol), and N,N-dimethyl-
aniline (9.0 mL) was refluxed for 45 min until a homogeneous
black solution was obtained. The solution was cooled and
concentrated to a black gum to remove all traces of POCl3.
The gum was dissolved with cooling in 45 mL of 30% aqueous
NH3 and washed first with ethyl acetate and then ether, taking
care to keep the pH above 10. The aqueous layer was then
concentrated to dryness, and the residue was dissolved in
water (100 mL) and acidified with cooling to pH 2 using 1 M
HCl. This solution was then extracted with ether for 4 days
using a liquid/liquid continuous extractor. The ether was
concentrated to dryness, and the residue was dissolved in
water and purified on a C18 column using a gradient of water
to 20% CH3CN in water over 30 min to give 3.98 g (25.4 mmol,
88%) of 5.
(35) Samano, V.; Miles, R. W.; Robins, M. J . J . Am. Chem. Soc. 1994,
116, 9331-9332.
(36) Miles, R. W.; Samano, V.; Robins, M. J . J . Am. Chem. Soc. 1995,
117, 5951-5957.
J . Org. Chem, Vol. 68, No. 22, 2003 8659

Shallop et al.
[8-13C-7-15N]-6-Ch lor o-9-(â-D-r ibofu r a n osyl)p u r in e (6a ).
A suspension of 5 (4.75 g, 30.3 mmol) and 7-methylguanosine
(13.7 g, 46.0 mmol) in aqueous K2HPO4 (120 mL, 0.02 M) was
adjusted to pH 7.4 with 6 N aqueous NaOH. To this mixture
was added purine nucleoside phosphorylase (1500 units). The
resulting mixture was kept at 30 °C with gentle agitation for
2 days. DMF (50 mL) was then added, and the mixture was
stirred at room temperature for 5 h. This mixture was filtered
to remove most of the 7-methylguanine. The filtrate was
concentrated to a small volume and the residue purified on a
C18 column using a gradient of water to 20% CH3CN in water
over 40 min to give 8.17 g (28.3 mmol, 93%) of 6a .
[8-13C-7-15N]-6-Ch lor o-9-(2′-d eoxy-â-D-er yth r o-p en tofu -
r a n osyl)p u r in e (6b). A suspension of 5 (3.32 g, 21.2 mmol)
and thymidine (15.4 g, 63.7 mmol) in aqueous K2HPO4 (100
mL, 0.02 M) was adjusted to pH 7 with 6 N aqueous NaOH.
To this mixture was added purine nucleoside phosphorylase
(716 units) and thymidine phosphorylase (714 units). The
resulting mixture was kept at 37 °C with gentle agitation for
2 days and then extracted with CH2Cl2 for 2 days using a
liquid/liquid continuous extractor. The organic layer was
concentrated to dryness, and the residue was dissolved in
water and purified as for 6a to give 4.77 g (17.5 mmol, 83%)
of 6b.
[8-13C-1,7,NH 2-15N3]-2-Am in o-6-(m et h oxya m in o)-9-(2′-
d eoxy-â-D-er yth r o-p en tofu r a n osyl)p u r in e (10b). To bro-
mine (1.05 g, 6.6 mmol) in water (2.0 mL) cooled to 0 °C was
slowly added [15N]KCN (0.45 g, 6.8 mmol) in water (10 mL).
After 30 min of stirring, this solution was added to 8b (1.30 g,
4.8 mmol) in 45 mL of water and the mixture was treated as
for 10a , using 2.5 mL (40.2 mmol) of CH3I and 82 mL of 0.25
M NaOH. The product was purified on a C18 column using a
gradient of 0.1 M NH4HCO3 (pH 7) to 10% CH3CN in 0.1 M
NH4HCO3 over 1 h to give 1.16 g (3.9 mmol, 81%) of 10b.
[8-13C-1,7,NH2-15N3]Gu a n osin e (11a ). To 10a (2.87 g, 9.1
mmol) dissolved in 190 mL of 0.1 M aqueous K2HPO4 (pH 7.4)
was added adenosine deaminase (546 units). The solution was
kept at 37 °C with gentle agitation for 5 days during which
time the product crystallized. The mixture was cooled to 0 °C
and filtered, and the product was recrystallized from water to
give 2.33 g (8.10 mmol, 89%) of 11a .
[8-13C-1,7,NH2-15N3]-2′-Deoxygu a n osin e (11b). To 10b
(0.92 g, 3.1 mmol) dissolved in 60 mL of 0.1 M triethylammo-
nium acetate (TEAA) (pH 7.4) was added adenosine deaminase
(211 units). The mixture was kept at 37 °C with gentle
agitation for 6 days. The solution was purified on a C18 column
using a gradient of water to 12% CH3CN in water over 40 min
to give 0.75 g (2.6 mmol, 85%) of pure 11b.
[8-13C-7,NH2-15N2]Ad en osin e (7a ). A mixture of 6a (3.05
g, 10.6 mmol), [15N]NH4Cl (1.16 g, 21.3 mmol), and KHCO3
(3.25 g, 32.5 mmol) in dimethyl sulfoxide (DMSO) (16 mL) was
sealed in a bomb and kept at 80 °C for 3 days. The cooled bomb
(-20 °C) was carefully opened, and the contents were diluted
with water (25 mL) and adjusted to pH 7 with acetic acid. The
product was purified on a C18 column using a gradient of
water to 20% CH3CN in water over 30 min to give 2.61 g (9.3
mmol, 88%) of 7a .
[8-13C-7,NH2-15N2]-2′-Deoxya d en osin e (7b). A mixture of
6b (4.00 g, 14.7 mmol), [15N]NH4Cl (1.73 g, 31.8 mmol), and
KHCO3 (4.72 g, 47.1 mmol) in DMSO (24 mL) was sealed in a
bomb and kept at 80 °C for 4 days. The product was treated
as for 7a to give 3.64 g (13.4 mmol, 91%) of 7b.
[8-13C-7,NH2-15N2]Ad en osin e N1-Oxid e (8a ). A mixture of
7a (3.00 g, 10.7 mmol) and m-chloroperoxybenzoic acid (MCP-
BA) (3.70 g, 21.4 mmol) in 50% aqueous CH3OH (90 mL) was
stirred at room temperature for 18 h. The mixture was diluted
with water and then washed with ether. The aqueous layer
was concentrated to a small volume and the residue purified
on a C18 column using a gradient of water to 20% CH3CN in
water over 30 min to give 2.97 g (10.4 mmol, 97%) of 8a .
[8-13C-7,NH2-15N2]-2′-Deoxya d en osin e N1-Oxid e (8b). A
mixture of 7b (1.59 g, 5.8 mmol) and MCPBA (3.89 g, 22.5
mmol) in 50% aqueous CH3OH (200 mL) was stirred at room
temperature for 3 h. The reaction mixture was concentrated,
giving a suspended white solid, and washed with ether. The
aqueous layer was concentrated and the residue purified as
for 8a to give 1.50 g (5.6 mmol, 95%) of 8b.
[8-13C-1,7,NH2-15N3]-2-Am in o-6-(m eth oxya m in o)-9-(â-D-
r ibofu r a n osyl)p u r in e (10a ). To bromine (2.09 g, 13.1 mmol)
in water (3.8 mL) cooled to 0 °C was slowly added [15N]KCN
(0.83 g, 12.6 mmol) in water (35 mL). After 30 min of stirring,
this solution was added to 8a (2.56 g, 8.9 mmol) dissolved in
water (90 mL). After 1.5 h, the reaction mixture was concen-
trated to a small volume from 50% DMF/CH3CN three times.
DMF (67 mL) and Et3N (4.7 mL, 34 mmol) were added under
nitrogen. The mixture was stirred at room temperature for
45 min, after which CH3I (4.7 mL, 75.5 mmol) was slowly
added. Stirring was continued in darkness for 2 h, and the
reaction mixture was then concentrated to a yellow oil. This
oil was dissolved in 0.25 M NaOH (150 mL) and stirred at
room temperature for 10 min. The pH was adjusted to 7.4 with
1 M HCl, and ethanol (160 mL) was added. The mixture was
heated at 60 °C for 4 h and then concentrated to 50 mL. The
product was purified on a C18 column using a gradient of
water to 20% CH3CN in water over 30 min to give 2.20 g (7.0
mmol, 78%) of 10a .
[8-13C-1,7-15N2]-6-(Meth oxyam in o)-9-(â-D-r ibofu r an osyl)-
p u r in e (12a ). A mixture of 8a (1.50 g, 5.2 mmol) and CH3I
(1.0 mL, 16.1 mmol) in 25 mL of N,N-dimethylacetamide was
stirred at room temperature for 2.5 h. The reaction mixture
was chilled in an ice bath, 2 M dimethylamine in CH3OH (14.5
mL) was added slowly, and stirring was continued at room
temperature for 1 h. The mixture was concentrated, dissolved
in 20 mL of CH3OH, and refluxed for 48 h. The mixture was
again concentrated to give crude 12a , which was used without
further purification.
[8-13C-1,7-15N2]-6-(Meth oxya m in o)-9-(2′-d eoxy-â-D-er yth -
r o-p en tofu r a n osyl)p u r in e (12b). A mixture of 8b (1.50 g,
5.6 mmol) and CH3I (1.1 mL, 16.9 mmol) in 24 mL of N,N-
dimethylacetamide was stirred at room temperature for 24 h
and then treated as for 12a to give crude 12b.
[8-13C-1,7-15N2]Ad en osin e (13a ). Crude 12a was dissolved
in 35 mL of 30% aqueous NH3 and heated to 95 °C, whereupon
50% aqueous Raney Ni (8.00 g) was added slowly, and the
suspension was maintained at 95 °C for 2.5 h. The hot mixture
was filtered, the Raney Ni was washed with boiling water, the
filtrate was concentrated to a small volume, and the residue
was purified on a C18 column using a gradient of water to
20% CH3CN in water over 30 min to give 1.23 g (4.4 mmol,
84% from 8a ) of 13a .
[8-13C-1,7-15N2]-2′-Deoxya d en osin e (13b). Crude 12b was
dissolved in 60 mL of 30% aqueous NH3, heated to 95 °C,
treated with Raney Ni, and purified as for 13a to give 1.24 g
(4.6 mmol, 82%) of 13b.
[8-13C-1,7-15N2]-9-(â-D-Ribofu r a n osyl)-6-(1,2,4-tr ia zol-4-
yl)-p u r in e (14a ). A mixture of 13a (0.97 g, 3.5 mmol) and
N,N-dimethylformamide azine dihydrochloride (2.69 g, 12.5
mmol) was dried by evaporation from pyridine three times to
a final volume of 10 mL. Trimethylsilyl chloride (TMSCl) (1.75
mL, 13.8 mmol) was added to the solution, which was then
heated at 100 °C for 22 h. The solution was concentrated and
the residue dissolved in cold CH2Cl2 (35 mL). This solution
was washed twice with cold brine/saturated NaHCO3 and then
twice with cold brine/2 M HCl. The organic layer was concen-
trated and the residue dissolved in CH3OH (27 mL). After 3 h
with stirring, the suspension was cooled (0 °C) and filtered.
The solid was resuspended in cold ether, filtered again, and
dried to give 0.87 g of 14a without further purification.
[8-13C-1,7-15N2]-9-(2′-Deoxy-â-D-er yth r o-pen tofu r an osyl)-
6-(1,2,4-tr ia zol-4-yl)-p u r in e (14b). A mixture of 13b (1.93
g, 7.1 mmol) and N,N-dimethylformamide azine (4.39 g, 30.9
mmol) was dried by evaporation from pyridine three times to
a final volume of 30 mL. TMSCl (2.0 mL, 16.6 mmol) was
added to the solution, which was treated as for 14a , except
8660 J . Org. Chem., Vol. 68, No. 22, 2003

13C as an Indirect Tag in 15N-Labeled Nucleosides
that the final product was suspended in cold CH2Cl2 rather
than ether, to give 1.61 g of 14b without further purification.
[8-13C-1,7,NH2-15N3]Ad en osin e (15a ). A mixture of [15N]-
NH4Cl (1.49 g, 27.4 mmol) and K2CO3 (3.73 g, 27.0 mmol) in
24 mL of DMSO was sealed in a bomb and kept at 85 °C for
30 min. The cooled bomb (-20 °C) was carefully opened, and
crude 14a (0.85 g) was quickly added. The resealed bomb was
heated at 85 °C for 5 days. The cooled bomb was carefully
opened and the contents diluted in 40 mL of water and
adjusted to pH 7 with acetic acid. The product was purified
on a C18 column using a gradient of water to 20% CH3CN in
water over 30 min to give 0.60 g (2.1 mmol, 81%) of 15a .
[8-13C-1,7,NH2-15N3]-2′-Deoxya d en osin e (15b). A mixture
of [15N]NH4Cl (0.28 g, 5.1 mmol) and potassium tert-butoxide
(0.52 g, 4.6 mmol) in 7.0 mL of DMSO was sealed in a bomb
and kept at 85 °C for 30 min. The cooled bomb (-20 °C) was
carefully opened, and 14b (0.70 g, 2.3 mmol) was quickly
added. The resealed bomb was heated at 85 °C for 2 days. The
cooled bomb was carefully opened, and the contents were
diluted with 20 mL of water. The product was purified as for
15a to give 0.47 g (1.9 mmol, 83%) of 15b.
Ack n ow led gm en t. We thank Timothy J . Hyde for
assistance with the 2D NMR. This work was supported
by grants from the National Institutes of Health
(GM31483, GM48802, and GM60485).
Su p p or tin g In for m a tion Ava ila ble: General methods,
characterization of compounds (UV, mp, 1H, 13C, 15N NMR
chemical shifts and coupling constants, HRMS, and elemental
analysis), 1H, 13C, 15N 1D NMR spectra for selected intermedi-
ates and final compounds, and 2D HSQC 1H-15N NMR for final
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O0345446
J . Org. Chem, Vol. 68, No. 22, 2003 8661