15N-Multilabeled Adenine and Guanine Nucleosides. Syntheses of [1,3,NH2-15N3]- and [2-13C-1,3,NH2-15N3]-Labeled Adenosine, Guanosine, 2′-Deoxyadenosine, and 2′-Deoxyguanosine

Article abstract of DOI:10.1021/jo982372k

We report a high-yield route to the following specifically ¹⁵N- and ¹³C-multilabeled nucleosides: [1,3,NH₂-¹⁵N₃]- and [2-¹³C-1,3,NH₂-¹⁵N₃]-adenosine; [1,3,NH₂-¹⁵N₃]- and [2-¹³C-1,3,NH₂-¹⁵N₃]-guanosine; [1,3,NH₂-¹⁵N₃]- and [2-¹³C-1,3,NH₂-¹⁵N ₃]-2′-deoxyadenosine; [1,3,NH₂-¹⁵N₃]- and [2-¹³C-1,3,-NH₂-¹⁵N ₃]-2′-deoxyguanosine. In each set, the ¹³C2 atom functions as a "tag" that allows the ¹⁵N1 and ¹⁵N3 atoms to be unambiguously differentiated from the untagged versions in ¹⁵N NMR of RNA or DNA fragments. The key intermediate of this synthetic strategy for both the adenine and guanine nucleosides is [NH₂,CONH₂-¹⁵N ₂]-5-amino-4-iimdazolecarboxamide. The [2-¹³C]-label is added through a ring closure using [¹³C]-sodium ethyl xanthate (NaS¹³CSOEt). Enzymatic transglycosylation of either multilabeled 6-chloropurine or multilabeled 2-mercaptohypoxanthine and a final reaction with ¹⁵NH₃ give the adenine and guanine nucleosides. This is the first report of a [3-¹⁵N]-labeled guanine nucleoside.

Full text of DOI:10.1021/jo982372k

J . Org. Chem. 1999, 64, 6575-6582
6575
15N-Mu ltila beled Ad en in e a n d Gu a n in e Nu cleosid es. Syn th eses of
[1,3,NH2-15N3]- a n d [2-13C-1,3,NH2-15N3]-La beled Ad en osin e,
Gu a n osin e, 2′-Deoxya d en osin e, a n d 2′-Deoxygu a n osin e
J ose´-Luis Abad, Barbara L. Gaffney, and Roger A. J ones*,†
Department of Chemistry, Rutgers, The State University of New J ersey, Piscataway, New J ersey 08854
Received December 3, 1998
We report a high-yield route to the following specifically 15N- and 13C-multilabeled nucleosides:
[1,3,NH2-15N3]- and [2-13C-1,3,NH2-15N3]-adenosine; [1,3,NH2-15N3]- and [2-13C-1,3,NH2-15N3]-gua-
nosine; [1,3,NH2-15N3]- and [2-13C-1,3,NH2-15N3]-2′-deoxyadenosine; [1,3,NH2-15N3]- and [2-13C-1,3,-
NH2-15N3]-2′-deoxyguanosine. In each set, the 13C2 atom functions as a “tag” that allows the 15N1
and 15N3 atoms to be unambiguously differentiated from the untagged versions in 15N NMR of
RNA or DNA fragments. The key intermediate of this synthetic strategy for both the adenine and
guanine nucleosides is [NH2,CONH2-15N2]-5-amino-4-imidazolecarboxamide. The [2-13C]-label is
added through a ring closure using [13C]-sodium ethyl xanthate (NaS13CSOEt). Enzymatic
transglycosylation of either multilabeled 6-chloropurine or multilabeled 2-mercaptohypoxanthine
and a final reaction with 15NH3 give the adenine and guanine nucleosides. This is the first report
of a [3-15N]-labeled guanine nucleoside.
Specific 15N-labeling of DNA fragments with single 15
N
Here we report the syntheses of [1,3,NH2-15N3]- and
labels demonstrated its value for NMR studies of nucleic
acid structure and interactions.1-11 However, to maximize
the information available from a single NMR experiment
and a single synthesis, we have recently expanded these
studies to include in one nucleoside as many 15N labels
as can be unambiguously distinguished.12-14 Thus, we
have reported synthetic methods for triply labeled [1,7,-
NH2-15N3]-adenosine, [1,7,NH2-15N3]-guanosine, and their
deoxy analogues and a variety of doubly labeled versions.
Further, for the guanosine series, we have also reported
procedures that include a 2-13C label that functions as a
tag so that two otherwise identical 15N-multilabeled
nucleosides incorporated into DNA or RNA fragments can
be differentiated.13 We now extend this work to a new
family of multilabeled adenine and guanine nucleosides,
with and without 13C tags, that includes the N3 atom.
[2-13C-1,3,NH2-15N3]-labeled adenosine, guanosine, and
their deoxy analogues. While singly [3-15N]-labeled ad-
enine has been described previously,15-18 the work de-
scribed here is the first report of any [3-15N]-labeled
guanine nucleoside. Incorporation of these nucleosides
into DNA and RNA fragments will provide valuable
information, particularly regarding interactions with
minor groove binding agents.
Synthesis of singly [3-15N]-labeled adenine using nitra-
tion of 4-bromoimidazole has been reported by several
groups.15-17 Several years ago, we introduced an alterna-
tive method based on a milder azo coupling reaction to
introduce the 15N, which gave [NH2-15N]-5-amino-4-
imidazolecarboxamide (AICA).18 The syntheses reported
here begin with this same procedure but incorporate a
second 15N as part of the amide functionality. Thus, the
ethyl diester of the commercially available imidazole-4,5-
dicarboxylic acid is brominated with N-bromosuccinimide
(NBS) to afford 2 (see Scheme 1). Saponification of 2
followed by a diazocoupling with [â-15N]-4-bromoben-
zenediazonium ion, generated in situ by diazotization of
4-bromoaniline using Na15NO2, gives the singly labeled
azo acid 3 in 83% yield. Our previous route to the amide
using ethyl chloroformate18 worked well with excess NH3
but gives low yields when only a few equivalents of
15NH3 are employed (generated in situ with DBU/15NH4-
Cl in CH3CN). However, use of carbodiimides such as
dicyclohexylcarbodiimide (DCC) or 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (DEC) gives
the doubly labeled azoamide 4 in 93% yield with only 1.25
equiv of 15NH4Cl. Concomitant reduction of the bromo
† Phone: 732-445-4900. Fax: 732-445-5866. E-mail: jones@
rutchem.rutgers.edu.
(1) Gao, X.; J ones, R. A. J . Am. Chem. Soc. 1987, 109, 1275-1278.
(2) Kupferschmitt, G.; Schmidt, J .; Schmidt, T.; Fera, B.; Buck, R.;
Ru¨terjans, H. Nucleic Acids Res. 1987, 15, 6225-6241.
(3) Massefski, W., J r.; Redfield, A.; Sarma, U. D.; Bannerji, A.; Roy,
S. J . Am. Chem. Soc. 1990, 112, 5350-5351.
(4) Wang, C.; Gao, H.; Gaffney, B. L.; J ones, R. A. J . Am. Chem.
Soc. 1991, 113, 5486-5488.
(5) Wang, C.; Gao, X.; J ones, R. A. J . Am. Chem. Soc. 1991, 113,
1448-1450.
(6) Gaffney, B. L.; Marky, L. A.; J ones, R. A. Nucleic Acids Res. 1982,
10, 4351-4361.
(7) Goswami, B.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1993,
115, 3832-3833.
(8) Rhee, Y.; Wang, C.; Gaffney, B. L.; J ones, R. A. J . Am. Chem.
Soc. 1993, 115, 8742-8746.
(9) Macmillan, A. M.; Lee, R. J .; Verdine, G. L. J . Am. Chem. Soc.
1993, 115, 4921-4922.
(10) Gaffney, B. L.; Goswami, B.; J ones, R. A. J . Am. Chem. Soc.
1993, 115, 12607-12608.
(15) Barrio, M. D. C. G.; Scopes, D. I. C.; Holtwick, J . B.; Leonard,
N. J . Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 3986-3988.
(16) Sethi, S. K.; Gupta, S. P.; J enkins, E. E.; Whitehead, C. W.;
Townsend, L. B.; McCloskey, J . A. J . Am. Chem. Soc. 1982, 104, 3349-
3353.
(17) Baker, B. F.; Dervan, P. B. J . Am. Chem. Soc. 1989, 111, 2700-
2712.
(11) Gaffney, B. L.; Kung, P.-P.; Wang, C.; J ones, R. A. J . Am. Chem.
Soc. 1995, 117, 12281-12283.
(12) Pagano, A. R.; Lajewski, W. M.; J ones, R. A. J . Am. Chem. Soc.
1995, 117, 11669-11672.
(13) Zhao, H.; Pagano, A. R.; Wang, W.; Shallop, A.; Gaffney, B. L.;
J ones, R. A. J . Org. Chem. 1997, 62, 7832-7835.
(14) Pagano, A. R.; Zhao, H.; Shallop, A.; J ones, R. A. J . Org. Chem.
1998, 63, 3213-3217.
(18) Rhee, Y. S.; J ones, R. A. J . Am. Chem. Soc. 1990, 112, 8174-
8175.
10.1021/jo982372k CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/07/1999

6576 J . Org. Chem., Vol. 64, No. 18, 1999
Abad et al.
Sch em e 1
and azo groups to give the doubly labeled AICA, 5, is
accomplished using excess H2 with 10% Pd/C in methanol
saturated with NH3. Similar results are obtained using
Zn/HCl/Pd/10%C in 2-propanol, but the isolation and
purification of 5 by this procedure is more difficult.
guanosine affords the corresponding ribo- or 2′-deoxyri-
bonucleosides, 12a -d . In each case, 10% of the N7 isomer
was formed along with the N9 isomer.21 The regiochem-
istry observed for PNP couplings normally is exclusively
N9, but it has been reported that coupling of the
pyrimidopurine M1G by this method also gives 10% of
the N7 isomer.22 Oxidation of 12a -d to the corresponding
sulfoxides 13a -d with Oxone (2KHSO5-KHSO4-K2SO4)
in water followed by displacement of the methylsulfoxyl
group with [15N]-NH3 generated from 15NH4Cl in KHCO3
gives the guanine nucleosides 14a -d .
Ring closure using diethoxymethyl acetate (DEMA), as
we have reported previously,12,18,19 gives the doubly 15N-
labeled hypoxanthine 7a , from which adenine nucleosides
10a ,c are obtained. To introduce the C2 label, and to
provide access to guanine nucleosides, we use an alterna-
tive ring closure reaction using sodium ethyl xanthate,
made from either [12C]- or [13C]-CS2, to give the mercap-
tohypoxanthines 6a ,b.20 The latter can be reduced to 7b
using Raney nickel in formic acid. Hypoxanthines 7a ,b
are then converted to the corresponding 6-chloropurines
8a ,b, which readily undergo enzymatic transglycosylation
using as sugar donors 7-methylguanosine or thymidine
to give 9a ,b and 9c,d , respectively. Finally, the third 15N
is introduced by chlorine displacement with 2 equiv of
15NH4Cl/KHCO3, to give the four multilabeled adenine
nucleosides, 10a -d .
These adenine nucleosides could be converted to the
corresponding guanine nucleosides by a five-step rear-
rangement,13 but the N1 label would be lost in the
process. The mercaptohypoxanthines 6a ,b offer an al-
ternate route which preserves this label. Methylation of
6a ,b with iodomethane gives the corresponding 2-meth-
ylthiohypoxanthines, 11a ,b. Enzymatic transglycosyla-
tion of 11a ,b with purine nucleoside phosphorylase (PNP)
in the presence of either 7-methylguanosine or 2′-deoxy-
Table 1 shows NMR chemical shifts and coupling
constants for the base protons and labeled atoms of the
final multilabeled nucleosides 10b,d and 14b,d . The
1
nucleosides without 13C labels had the same H, 13C, and
15N chemical shifts. In the adenine nucleosides 10b,d ,
the one-bond coupling between H2 and C2 is large (199
Hz) while the smaller two-bond couplings of H2 to N1
and N3 are similar (16 Hz), leading to doublets of
apparent triplets for H2. In the amino groups of all four
compounds, the one-bond coupling between hydrogen and
nitrogen is the normal ∼90 Hz. Small (3 Hz) three-bond
couplings between the amino hydrogens and the N1 are
apparent in the adenine nucleosides but not in the
guanine nucleosides.
(21) The side products we observed were found to return to the free
bases 11a ,b, upon treatment with acid, or isomerize to the correct
nucleosides 12a -d , upon treatment with PNP without a sugar donor.
To determine conclusively the identity of the minor products, we
prepared singly labeled [7-15N]-2-(methylthio)hypoxanthine by omitting
the RaNi desulfurization in a route we have reported previously.12 The
minor product formed upon transglycosylation using this [7-15N]-2-
(methylthio)hypoxanthine then was isolated. The N7 linkage was
confirmed by the observation of coupling (9 Hz) between the 15N7 and
the C1′.
(19) Gaffney, B. L.; Kung, P.-P.; J ones, R. A. J . Am. Chem. Soc. 1990,
112, 6748-6749.
(20) Yamazaki, A.; Kumashiro, I.; Takenishi, T. J . Org. Chem. 1967,
32, 1825-1828.
(22) Chapeau, M.-C.; Marnett, L. J . Chem. Res. Toxicol. 1991, 4,
636-638.

15N-Multilabeled Adenines
J . Org. Chem., Vol. 64, No. 18, 1999 6577
Ta ble 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 10b,d a n d 14b,d a
b
compd
H1
H2
8.12 (dt)
199: H2-C2
16: H2-N1,N3
8.12 (dt)
H8
NH2
C2b
N1b
N3b
223.3 (s)
NH2
10b
8.33 (s)
7.36 (dd)
90: H-N
3: H-N1
7.28 (dd)
90: H-N
3: H-N1
6.53 (d)
152.4 (dd)
236.7 (d)
5: N1-NH2
82.9 (d)
3: C2-N1/N3
2: C2-N1/N3
152.4 (br)
4: NH2-N1
10d
14b
8.32 (s)
7.91 (s)
236.7 (d)
5: N1-NH2
223.8 (s)
82.5 (d)
199: H2-C2
16: H2-N1,N3
4: NH2-N1
10.6 (br)
11.2 (br)
154.2 (ddd)
23: C2-NH2
13: C2-N1
7: C2-N3
155.0 (ddd)
23: C2-NH2
12: C2-N1
7: C2-N3
152.1 (d)
12: N1-C2
166.8 (t)
6: N3-C2,NH2
74.6 (dd)
23: NH2-C2
4: NH2-N3
90: H-N
14d
7.85 (s)
6.70 (d)
88: H-N
157.7 (d)
10: N1-C2
167.1 (t)
6: N3-C2,NH2
75.2 (ddd)
23: NH2-C2
6: NH2-N3
2: 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.
Ta ble 2. EI Ma ss Sp ectr a l Da ta for 10a -d a
compd
M
M - 30
M - 89
b + 30
b + 2
b + 1
b - 27/28
10a
10b
10c
10d
270 (3)
271 (4)
254 (2)
255 (3)
240 (10)
241 (11)
224 (4)
225 (7)
181 (28)
182 (33)
165 (22)
166 (36)
167 (74)
168 (84)
167 (10)
168 (11)
139 (27)
140 (67)
139 (18)
140 (26)
138 (100)
139 (100)
138 (100)
139 (100)
110 (10)
110 (13)
110 (17)
110 (11)
a
Entries for significant ions show mass followed by relative abundance in parentheses.
In the proton-decoupled 13C spectra, the one-bond C-N
couplings are significant in the guanines (23, 13, and 7
Hz) but are much smaller in adenosine (3 and 2 Hz) and
are not resolved in 2′-deoxyadenosine. This difference also
occurs in many of the intermediates.
The nitrogen chemical shifts are consistent with lit-
erature values.23 Some couplings among the C2 and the
three nitrogens are resolved in the proton-decoupled
spectra of these four compounds, while others are not.
For example, in adenosine, the largest coupling of N1 is
to the amino nitrogen, while, in guanosine, it is to C2.
We have previously noted the surprisingly large (23 Hz)
coupling between the amino nitrogen and C2 in the
guanosines.13 The 1H, 13C, and 15N NMR spectra for these
four compounds are included in the Supporting Informa-
tion.
spectra obtained for the guanine nucleosides have as
significant ions only the M + 1 and the B + 2 ions
(Experimental Section). The presence of three 15N atoms
in 14a ,c is seen in their molecular ions, M + 1 ) 287
and 271, respectively, which are three units larger than
unlabeled guanosine (283) and deoxyguanosine (267). The
additional 13C atom in 14b (M + 1 ) 288) and 14d (M +
1 ) 272) is also evident. Mass spectra for all eight final
products are included in the Supporting Information.
Exp er im en ta l Section
1
Gen er a l Meth od s. All H NMR spectra were acquired at
200 MHz, and 13C NMR spectra, at 50.3 MHz. 15N NMR
spectra were acquired at 40.5 MHz, and chemical shifts are
reported relative to NH3 using external 1 M [15N]-urea in
DMSO at 25 °C at 77.0 ppm as a reference.25 Analytical HPLC
was carried out with Waters C-18 Nova-Pak cartridges (8 ×
100 mm) using a gradient of 2-20% acetonitrile in 0.1 M
triethylammonium acetate (TEAA) at a flow rate of 2 mL/min.
Preparative reversed-phase HPLC was performed with three
Waters Delta-Pak PrepPak cartridges (40 × 100 mm, C18 300
Å, 15 µm) in series at a flow rate of 40 mL/min. UV data were
determined from multiwavelength HPLC using a diode array
detector and Millennium software.
Table 2 shows EI MS mass and relative abundance of
significant ions for adenine nucleosides 10a -d . The
results are consistent with known mass spectral data,
which are well-documented.1,24 The presence of three 15
N
atoms in 10a (m/z 270) and 10c (m/z 254) is seen in their
molecular ions, which are three units larger than unla-
beled adenosine (m/z 267) and deoxyadenosine (m/z 251),
respectively. The additional 13C atom in 10b (m/z 271)
and 10d (m/z 255) is also evident. Loss of the 5′ group as
CH2O results in ions 30 units smaller than the molecular
ions. Fragmentation of the sugar by loss of the 3′, 4′, and
5′ groups gives ions 89 units smaller than the molecular
ions, while loss of all but the 1′ group gives ions 30 units
larger than the base ions. The base + H ion is known to
fragment by successive losses of HCN units.24 Using
singly labeled [1-15N]-deoxyadenosine, we have previously
shown conclusively that the first HCN lost includes N1.1
Not surprisingly, the work reported here demonstrates
that this lost HCN also includes C2, since the resulting
ion (m/z 110) is the same in all four cases. The FAB
The [15N]-NH4Cl and [15N]-NaNO2 were obtained from Isotec
Inc., and the [13C]-CS2 was from Cambridge Isotope Labora-
tories. Thymidine phosphorylase was obtained from Sigma
Chemical Co., and purine nucleoside phosphorylase (PNP) was
a gift from Burroughs Wellcome Co. and is also available from
commercial sources. General reagents were obtained from
Aldrich Chemical Co.
Eth yl Im id a zole-4,5-d ica r boxyla te (1b).18 A mixture of
imidazole-4,5-dicarboxylic acid (1a ) (97% pure, 24 g, 149
mmol), 1.5 L of absolute ethanol, and 150 mL of H2SO4 was
refluxed for 2 days under N2 until a homogeneous solution was
obtained. The mixture was concentrated, neutralized with
saturated aqueous NaHCO3, and then continuously extracted
with CH2Cl2. The organic layer was washed twice with 0.1 M
NH4HCO3, and the combined aqueous layers were back-
washed with CH2Cl2. The combined organic layers were dried
(23) Levy, G. C. Nitrogen-15 nuclear magnetic resonance spectros-
copy; J ohn Wiley & Sons: New York, 1979.
(24) Shaw, S. J .; Desiderio, D. M.; Tsuboyama, K.; McCloskey, J . A.
J . Am. Chem. Soc. 1970, 92, 2510-2522.
(25) Wishart, D. S.; Bigam, C. G.; Yao, J .; Abildgaard, F.; Dyson,
H. J .; Oldfield, E.; Markley, J . L.; Sykes, B. D. J . Biomol. NMR 1995,
6, 135-140.

6578 J . Org. Chem., Vol. 64, No. 18, 1999
Abad et al.
over anhydrous MgSO4, filtered, and concentrated to give 26
g (123 mmol, 83%) of pure 1b: IR 1705 cm-1; UV λmax 257 nm;
1H NMR (CDCl3) δ 12.10 (br s, 1H), 7.90 (s, 1H), 4.41 (q, J )
7 Hz, 4H), 1.38 (t, J ) 7 Hz, 6H); 13C NMR (CDCl3) δ 160.8,
137.7, 129.8, 61.4, 14.1.
OBr2‚0.25CH3OH: C, 32.14; H, 2.11; N, 18.29. Found: C,
32.26; H, 2.09; N, 18.01.
[NH 2,CONH 2-15N2]-5-Am in o-4-im id a zoleca r b oxa m id e
(AICA) (5). To a mixture of 4.4 g (12 mmol) of 4, 2.5 g of 10%
Pd/C, and 0.20 g of 1% Pt/C in a 1 L round-bottom flask was
cannulated under N2 0.95 L of a saturated solution of NH3(g)
in methanol. The resulting mixture was stirred for 10 min at
which point H2 was bubbled through the solution for 6 h from
balloons. The mixture was then filtered through a bed of Celite
to give a clear, pale yellow solution. After evaporation of the
solvent, the residue was dissolved in 40 mL of H2O, washed
with ether (3 × 20 mL), and then applied to a reversed phase
preparative column which was eluted with water. Formic acid
(2 mL/50 mL HPLC fraction) was added to fractions containing
the product. Evaporation of the solvent gave 1.8 g (10 mmol,
88%) of the formate salt of 5: mp 147-8 °C; UV λmax 230 nm
Eth yl 2-Br om oim id a zole-4,5-d ica r boxyla te (2).18 To a
mixture of 1b (26 g, 123 mmol) and 33 g (183 mmol) of
N-bromosuccinimide (NBS) was added 300 mL of dry aceto-
nitrile under N2. The resulting solution was stirred in the dark
for 24 h and concentrated to dryness. The residue was
dissolved in 1.2 L of ethyl acetate, and the solution was washed
four times with brine (250 mL), twice with saturated aqueous
Na2SO3 (100 mL), and twice again with brine (100 mL). The
organic layer was dried over MgSO4 and concentrated to
dryness. The residue was purified by flash chromatography
on silica gel using a gradient of 0-10% CH3OH in CH2Cl2 to
give 34 g (117 mmol, 95%) of pure 2: IR 1750 cm-1; UV λmax
1
(shoulder), 267 nm; H NMR (DMSO-d6) δ 8.14 (s, 1H), 7.19
286 ; H NMR (CDCl3) δ 11.00 (br s, 1H), 4.41 (q, J ) 7 Hz,
(s, 1H), 6.72 (d, J ) 88 Hz, 2H); 13C NMR (DMSO-d6) 164.7
(d, J ) 16 Hz), 163.3, 145.8 (d, J ) 16 Hz), 129.6, 107.5; 15N
NMR (DMSO-d6) 98, 50; HRMS m/z 128.0483 (calcd for C4H6-
ON215N2: 128.0482). Anal. Calcd for C4H6ON215N2‚H2O: C,
32.87; H, 5.52; N, 38.34. Found: C, 33.08; H, 5.55; N, 38.39.
1
4H), 1.37 (t, J ) 7 Hz, 6H); 13C NMR (CDCl3) δ 159.4, 131.8,
120.2, 61.7, 13.9.
[5-15N]-5-((4-Br om op h en yl)a zo)-2-b r om o-4-im id a zole-
ca r boxylic Acid (3).18 A suspension of 2 (25 g, 86 mmol) and
Na2CO3 (24 g) in 850 mL of water was stirred at 100 °C for 36
h and then cooled to 0 °C. To a cold solution of 4-bromoaniline
(97% pure, 13.4 g, 78 mmol) in 78 mL of 10% HCl and 350 mL
of water was added dropwise to a cold solution of Na15NO2
(5.7 g, 79 mmol) in 100 mL of water. After 20 min, when the
4-bromoaniline had reacted completely (HPLC), a cold solution
of Na2CO3 (11 g) in 125 mL of water was added slowly with
stirring. This cold mixture was then added slowly to the cold
aqueous solution of 2. After 5 min, the initially yellow solution
became red, and a thick precipitate formed. Stirring was
continued for 2 h, after which the solid was filtered out and
dissolved in 600 mL of aqueous Na2CO3 (10 g). A black residue
remained that contained no product. The aqueous solution was
washed with CH2Cl2 (5 × 100 mL) and then acidified with
concentrated HCl to afford a yellow precipitate. This precipi-
tate was collected by filtration, washed with cold water (3 ×
40 mL), and dried under vacuum over P2O5 to give 27 g (71
mmol, 83%) of pure 3: IR 1720 cm-1; UV λmax 235 nm, 357
nm, λmin 270 nm; 1H NMR (DMSO-d6) δ 13.50 (br s, 1H), 7.79
(s, 4H); 13C NMR (DMSO-d6) 160.3, 151.3 (d, J ) 6 Hz), 132.7,
125.2, 124.5 (br s), 123.1; 15N NMR (DMSO-d6) δ 486 (br s).
[5,CONH2-15N2]-5-((4-Br om op h en yl)a zo)-2-br om o-4-im -
id a zoleca r boxa m id e (4). To a mixture of 3 (9.5 g, 25 mmol),
hydroxybenzotriazole (3.5 g, 25 mmol), and 15NH4Cl (1.8 g, 32
mmol) in a 2 L flask was added 1.4 L of anhydrous CH3CN
under N2. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (DEC) (5.5 g, 29 mmol) was dissolved in 450 mL
of anhydrous CH3CN in a 500 mL round-bottom flask under
N2. Both flasks were kept for 2 h at -15 °C in a freezer after
which the DEC solution was cannulated to the 2 L flask kept
at -15 °C with stirring. During the transfer, 5.5 mL of DBU
(36 mmol) was added. Portions of water (3 × 20 mL) were
added after 4, 6, and 8 h, and stirring was continued at -10
°C for 36 h. The solvent was then evaporated, and the resulting
orange precipitate was suspended in 400 mL of H2O and
allowed to stand overnight at 4 °C. The orange precipitate was
collected by filtration. The filtrate was acidified with concen-
trated HCl until the pH was 5 and then extracted with CH2-
Cl2 (2 × 25 mL). The orange filter cake was dissolved in 3 L of
warm aqueous Na2CO3 (60 g), leaving a dark brown residue
that was discarded. The red solution was washed with CH2-
Cl2 (3 × 100 mL) and then neutralized with concentrated HCl.
The resulting yellow precipitate was filtered out and washed
with H2O (2 × 50 mL). Small amounts of additional product
were recovered from the combined CH2Cl2 layers by evapora-
tion and solubilization in warm aqueous Na2CO3 followed by
acidification. The combined products were dried under vacuum
over P2O5 to afford 8.8 g (24 mmol, 93%) of pure 4: IR 1663
cm-1; UV λmax 248 nm, 399 nm, λmin 275 nm; 1H NMR (DMSO-
d6) δ 8.2-7.2 (m); 13C NMR (DMSO-d6) 159.4 (d, J ) 18 Hz),
151.1 (d, J ) 5 Hz), 149.1, 132.5, 131.0, 125.0, 124.6, 123.1;
15N NMR (DMSO-d6) 470, 109; HRMS m/z 372.8957 (calcd for
[
13C]-Sod iu m Eth yl Xa n th a te (Na S13CSOEt). To a mix-
ture of 2.1 g of NaOH (53 mmol) dissolved by sonication in
200 mL of absolute ethanol was added 4 g (52 mmol) of [13C]-
CS2 (97-99%), and the pale yellow solution was stirred
overnight at room temperature. The solvent was evaporated,
and the white residue was dried under vacuum to afford 7.6 g
(52 mmol, 99%) of [13C]-sodium ethyl xanthate: UV λmax 300
nm; 1H NMR (DMSO-d6) δ 4.21 (dq, J 1 ) 4 Hz, J 2 ) 7 Hz,
2H), 1.16 (t, J ) 7 Hz, 3H); 13C NMR (DMSO-d6) δ 229.8, 66.0,
14.5.
[2-13C-1,3-15N2]-2-Mer ca p toh yp oxa n th in e (6b). A mix-
ture of 2.6 g of 5 (20 mmol) and 3.4 g (23 mmol) of [13C]-sodium
ethyl xanthate in 80 mL of anhydrous N,N-dimethylformamide
was stirred under N2 at room temperature for 20 min and then
refluxed for 4 h. The clear solution turned dark green, and a
white precipitate formed. The solution was allowed to cool, and
160 mL of CH3CN was added. The precipitate was filtered out
and washed with CH3CN (3 × 50 mL) to afford 4.6 g of crude
product. The filtrate was evaporated to dryness, and the
residue was purified by reversed phase preparative HPLC
using aqueous NH4HCO3 (pH ) 8) to afford an additional 0.3
g of product. The combined products were used without further
purification to make 7b or 11b. Pure analytical samples were
prepared by solubilization in water and precipitation with 10%
1
HCl: mp > 300 °C; UV λmax 280 nm; H NMR (DMSO-d6) δ
13.60 (br s, 1H), 13.23 (d, J ) 96 Hz, 1H), 12.16 (d, J ) 92 Hz,
1H), 8.05 (s, 1H); 13C NMR (DMSO-d6) 173.2 (C2), 154.1 (d,
J ) 8 Hz), 149.5 (d, J ) 16 Hz), 141.2, 111.3 (d, J ) 8 Hz); 15
N
NMR (DMSO-d6) 178 (d, J ) 11 Hz), 148 (d, J ) 14 Hz); HRMS
m/z 171.0087 (calcd for C413CH4ON215N2S: 171.0080). Anal.
Calcd for C413CH4ON215N2S‚0.6H2O: C, 33.00; H, 2.88; N,
30.79. Found: C, 32.95; H, 2.78; N, 30.64.
[1,3-15N2]-Hyp oxa n th in e (7a ). To a mixture of 9.5 g (55
mmol) of the formate salt of 5 and 200 mL of anhydrous DMF
under N2 was added 9 mL of diethoxymethyl acetate (DEMA).
The resulting suspension was stirred for 15 min at room
temperature at which point 2.25 mL of formic acid was added
and the mixture was heated at 135 °C for 4 h. The suspension
was concentrated to a gray solid, resuspended in 100 mL of
refluxing acetonitrile, and then chilled. The solid was collected
by filtration and dried in a vacuum desiccator over P2O5 to
give 7.0 g (51 mmol, 92%) of pure 7a : mp > 300 °C; UV λmax
250 nm; 1H NMR (D2O/NaOD) δ 8.04 (t, J ) 13 Hz, 1H), 7.84
(s, 1H); 13C NMR (D2O/NaOD) 170.3 (t, J ) 4 Hz), 162.9 (d,
J ) 6 Hz), 153.9, 153.6 (d, J ) 2 Hz), 127.1; 15N NMR (D2O/
NaOD) 231, 220; HRMS m/z 138.0330 (calcd for C5H4-
ON215N2: 138.0326). Anal. Calcd for C5H4ON215N2‚0.125H2O:
C, 42.79; H, 3.05; N, 40.57. Found: C, 42.65; H, 2.88; N, 40.21.
[2-13C-1,3-15N2]-Hyp oxa n th in e (7b). A stirred mixture of
crude 6b (4.8 g) and 10 g of Raney nickel (50% water
suspension) in 250 mL of H2O was heated at 60 °C for 10 min,
and 3.5 mL of formic acid was added. The mixture was refluxed
for 30 min, 10 g of EDTA was added, and the mixture was
C
10H7ON315N2Br2: 372.8958). Anal. Calcd for C10H7N315N2-

15N-Multilabeled Adenines
J . Org. Chem., Vol. 64, No. 18, 1999 6579
refluxed for an additional 2 h. The hot reaction mixture was
filtered, and the Raney nickel filter cake was washed with
boiling water to give a blue solution which was combined with
the filtrate, concentrated, and purified by reversed phase
preparative HPLC using a gradient of 0-20% CH3CN in H2O.
Appropriate fractions of 7b were concentrated to dryness and
dried in a vacuum desiccator over P2O5 to afford 2.6 g (19
mmol, 95% from 5) of pure 7b: mp > 300 °C; UV λmax 250 nm;
1H NMR (D2O/NaOD) δ 8.04 (dt, J 1 ) 13 Hz, J 2 ) 195 Hz,
1H), 7.84 (s, 1H); 13C NMR (D2O/NaOD) 170.4, 163.3 (dd,
J 1 ) 2 Hz, J 2 ) 6 Hz), 154.3 (C2), 152.0, 127.5 (d, J ) 7 Hz);
15N NMR (D2O/NaOD) 228 (d, J ) 2 Hz), 220; HRMS m/z
139.0362 (calcd for C413CH4ON215N2: 139.0359). Anal. Calcd
for C413CH4ON215N2: C, 43.17; H, 2.90; N, 40.28. Found: C,
43.11; H, 2.80; N, 40.13.
CN in water. Appropriate fractions were combined and con-
centrated to a white solid, which was dried in a vacuum
desiccator over P2O5 to give 5.8 g (20.1 mmol, 92%) of pure
9a : mp 167-8 °C; UV λmax 267 nm; 1H NMR (DMSO-d6) δ
8.94 (s, 1H), 8.80 (t, J ) 15 Hz, 1H), 6.04 (d, J ) 5 Hz, 1H),
5.59 (d, J ) 5 Hz, 1H), 5.27 (d, J ) 5 Hz, 1H), 5.11 (t, J ) 5
Hz, 1H), 4.59 (q, J ) 5 Hz, 1H), 4.20 (q, J ) 4 Hz, 1H), 3.99
(q, J ) 4 Hz, 1H), 3.80-3.50 (m, 2 H); 13C NMR (DMSO-d6)
151.6 (dd, J 1 ) 2 Hz, J 2 ) 5 Hz), 149.3 (dd, J 1 ) 3 Hz, J 2 ) 5
Hz), 145.8 (t, J ) 9 Hz), 131.4 (t, J ) 2 Hz), 88.2, 85.7, 74.0,
70.1, 61.0; 15N NMR (DMSO-d6) 275, 251; HRMS m/z 288.0407
(calcd for C10H11O4N215N2Cl: 288.0410). Anal. Calcd for
C
10H11O4N215N2Cl‚0.125H2O: C, 41.27; H, 3.90; N, 19.94.
Found: C, 41.49; H, 3.76; N, 19.63.
[2-13C-1,3-15N2]-6-Ch lor o-9-(â-D-er yth r o-pen tofu r an osyl)-
p u r in e (9b). The same procedure used to prepare 9a , except
for starting with 8b rather than 8a , was used for 9b: mp 166-
[1,3-15N2]-6-Ch lor op u r in e (8a ). To a mixture of 7a (7.0 g,
51 mmol) and N,N-dimethylaniline (DMA) (18 mL, 0.14 mol)
under N2 was added POCl3 (175 mL, 1.9 mol). The resulting
mixture was heated at 130 °C for 30 min and monitored by
HPLC. The solution was then allowed to cool and was
concentrated to a black gum. The gum was dissolved in 150
mL of NH4OH (30%) with cooling (-20 °C). The aqueous
solution was filtered through a bed of Celite and washed once
with 100 mL of ethyl acetate and then twice with 50 mL of
ether. The NH3 was evaporated, and the residue was dissolved
in 80 mL of water, acidified to pH 2 with concentrated HCl
(with cooling in an ice bath), and then continuously extracted
with ether for 5 days. The ether layer was evaporated, and
the residue was dissolved in 20 mL of NH4OH (30%). This
aqueous solution was concentrated to 10 mL and applied to a
reversed phase preparative column. Elution with a gradient
of 0-10% CH3CN in water gave product fractions that were
concentrated to a white solid and dried in a vacuum desiccator
over P2O5 to give 7.0 g (45 mmol, 88%) of pure 8a : mp > 300
1
168 °C; UV λmax 267 nm; H NMR (DMSO-d6) δ 8.93 (s, 1H),
8.80 (dt, J 1 ) 15 Hz, J 2 ) 210 Hz, 1H), 6.04 (d, J ) 5 Hz, 1H),
5.56 (d, J ) 5 Hz, 1H), 5.24 (d, J ) 5 Hz, 1H), 5.08 (t, J ) 5
Hz, 1H), 4.58 (q, J ) 5 Hz, 1H), 4.20 (q, J ) 4 Hz, 1H), 4.00
(q, J ) 4 Hz, 1H), 3.80-3.50 (m, 2 H); 13C NMR (DMSO-d6)
151.7 (dd, J 1 ) 2 Hz, J 2 ) 4 Hz, C2), 149.3 (dd, J 1 ) 3 Hz,
J 2 ) 5 Hz), 145.8, 131.4 (d, J 1 ) 2 Hz), 88.3, 85.7, 74.1, 70.1,
61.0; 15N NMR (DMSO-d6) 275 (d, J ) 4 Hz), 251 (d, J ) 2
Hz); HRMS m/z 289.0432 (calcd for C913CH11O4N215N2Cl:
289.0443). Anal. Calcd for C913CH11O4N215N2Cl: C, 41.46; H,
3.83; N, 19.34. Found: C, 41.58; H, 3.70; N, 19.68.
[1,3-15N2]-6-Ch lor o-9-(2-d eoxy-â-d -er yth r o-p en tofu r a n o-
syl)p u r in e (9c). To a suspension of 8a (3.5 g, 22 mmol) and
thymidine (13 g, 44 mmol) in aqueous K2HPO4 (90 mL, 0.02
M) was added 6 N KOH until the pH was 7.0. To this mixture
was added purine nucleoside phosphorylase (400 µL, 2.1 units/
µL) and thymidine phosphorylase (dThd Pase, 800 units). The
mixture was kept at 43 °C with gentle agitation for 3 days.
Sodium chloride (15 g) was added, and the reaction mixture
was continuously extracted with CH2Cl2 for 48 h at which time
HPLC analysis indicated that all the 8a and 9c had been
extracted as well as small amounts of thymidine and thymine.
The organic phase (white suspension) was concentrated and
purified by reversed phase preparative chromatography using
a gradient of 0-15% CH3CN in water. The fractions containing
8a (0.26 g, 5% recovered yield) and 9c (5.1 g, 19 mmol, 83%)
were collected separately, concentrated, and then dried in a
vacuum desiccator over P2O5; mp > 147-8 °C; UV λmax 267
nm; 1H NMR (DMSO-d6) δ 8.90 (s, 1H), 8.78 (t, J ) 16 Hz,
1H), 6.47 (t, J ) 6 Hz, 1H), 5.38 (d, J ) 4 Hz, 1H), 4.98 (t,
J ) 5 Hz, 1H), 4.45 (m, 1H), 3.90 (q, J ) 3 Hz, 1H), 3.58 (m,
2H), 2.80-2.70 (m, 1H), 2.50-2.40 (m, 1H); 13C NMR (DMSO-
d6) 151.6 (t, J ) 4 Hz), 151.3 (dd, J 1 ) 2 Hz, J 2 ) 5 Hz), 149.2
(dd, J 1 ) 3 Hz, J 2 ) 4 Hz), 145.8, 131.4 (d, J ) 2 Hz), 88.1,
84.2, 70.4, 61.3; 15N NMR (DMSO-d6) 275, 251; HRMS m/z
272.0454 (calcd for C10H11O3N215N2Cl: 272.0460). Anal. Calcd
for C10H11O3N215N2Cl: C, 44.05; H, 4.07; N, 20.55. Found: C,
44.00; H, 4.04; N, 20.45.
1
°C; UV λmax 267 nm; H NMR (DMSO-d6) δ 8.76 (dd, J 1 ) 15
Hz, J 2 ) 16 Hz, 1H), 8.71 (s, 1H); 13C NMR (DMSO-d6) 154.1
(dd, J 1 ) 2 Hz, J 2 ) 4 Hz), 151.5 (br s), 147.7 (dd, J 1 ) 3 Hz,
J 2 ) 5 Hz), 146.3, 129.3; 15N NMR (DMSO-d6) 273, 256; HRMS
m/z 155.9980 (calcd for C5H3N215N2Cl: 155.9987). Anal. Calcd
for C5H3N215N2Cl‚0.125H2O: C, 37.81; H, 2.06; N, 35.28.
Found: C, 37.76; H, 1.87; N, 35.70.
[2-13C-1,3-15N2]-6-Ch lor op u r in e (8b). The same procedure
used to prepare 8a , except for starting with 7b instead of 7a ,
was used for 8b: mp > 300 °C; UV λmax 267 nm; 1H NMR
(DMSO-d6) δ 8.73 (dt, J 1 ) 15 Hz, J 2 ) 209 Hz, 1H), 8.68 (s,
1H); 13C NMR (DMSO-d6) 154.1, 151.4 (dd, J 1 ) 3 Hz, J 2 ) 4
Hz, C2), 147.7 (dd, J 1 ) 3 Hz, J 2 ) 5 Hz), 146.2, 129.3 (dd,
J 1 ) 3 Hz, J 2 ) 12 Hz); 15N NMR (DMSO-d6) 273 (d, J ) 4
Hz), 256; HRMS m/z 157.0015 (calcd for C413CH3N215N2Cl:
157.0020). Anal. Calcd for C413CH3N215N2Cl‚0.25H2O: C, 37.06;
H, 2.18; N, 34.58. Found: C, 37.30; H, 1.84; N, 34.79.
7-Meth ylgu a n osin e. A suspension of guanosine (41 g, 140
mmol) and dimethyl sulfate (28 mL, 296 mmol) in N,N-
dimethylacetamide (350 mL) was stirred at room temperature
for 6 h. The pH of the homogeneous solution was adjusted to
10 with NH4OH (30%), and the solution was poured into 900
mL of chilled acetone (0 °C). The white precipitate was filtered
out, suspended in absolute ethanol (400 mL), refiltered,
suspended in dry ether (400 mL), and finally filtered again.
The white product was dried in a vacuum desiccator over P2O5
to give 40 g (130 mmol, 93%) of 7-methylguanosine, which was
used without further purification.
[2-13C-1,3-15N2]-6-Ch lor o-9-(2-d eoxy-â-D-er yth r o-p en to-
fu r a n osyl)p u r in e (9d ). The same procedure used to prepare
9c, except for starting with 8b rather than 8a , was used for
1
9d : mp 146-148 °C; UV λmax 267 nm; H NMR (DMSO-d6) δ
8.88 (s, 1H), 8.78 (dt, J 1 ) 15 Hz, J 2 ) 210 Hz, 1H), 6.46 (t,
J ) 6 Hz, 1H), 5.36 (d, J ) 4 Hz, 1H), 4.95 (t, J ) 5 Hz, 1H),
4.44 (m, 1H), 3.89 (q, J ) 3 Hz, 1H), 3.58 (m, 2H), 2.80-2.70
(m, 1H), 2.50-2.40 (m, 1H); 13C NMR (DMSO-d6) 151.5 (t,
J ) 3 Hz, C2), 149.1 (d, J ) 3 Hz), 145.7, 131.3 (d, J ) 11 Hz),
88.1, 84.2, 70.4, 61.3; 15N NMR (DMSO-d6) 275 (d, J ) 4 Hz),
[1,3-15N2]-6-Ch lor o-9-(â-D-er yth r o-p en t ofu r a n osyl)p u -
r in e (9a ). To a suspension of 8a (3.4 g, 22 mmol) and
7-methylguanosine (13 g, 44 mmol) in aqueous K2HPO4 (70
mL, 0.02 M) was added 6 N KOH until the pH was 7.4. To
this mixture was added purine nucleoside phosphorylase (500
µL, 2.1 units/µL). The mixture was kept at 43 °C with gentle
agitation for 7 days. The reaction mixture was then filtered
and the filter cake extracted using sonication with DMF (2 ×
80 mL) and then H2O (100 mL). All solutions were combined
and concentrated under vacuum just until a white precipitate
started to appear (60 mL). Water was added (60 mL), and 40
mL of the warm solution was filtered directly onto a reversed
phase preparative column which was eluted with 0-10% CH3-
251 (d, J ) 2 Hz); HRMS m/z 273.0483 (calcd for C913CH11
-
O3N215N2Cl: 273.0494). Anal. Calcd for C913CH11O3N215N2Cl:
C, 43.89; H, 4.05; N, 20.47. Found: C, 43.74; H, 3.98; N, 20.50.
[1,3,NH2-15N3]-Ad en osin e (10a ). A mixture of 9a (5.5 g,
19 mmol), [15N]-NH4Cl (2.2 g, 40 mmol), and KHCO3 (6.1 g,
60 mmol) in DMSO (14 mL) was sealed in a 100 mL flask and
kept in an oven at 80 °C for 7 days. The cooled (0 °C) reaction
vial was opened carefully, the contents were diluted with 70
mL of water, and the pH was adjusted to 7 with glacial acetic
acid. The product was purified by reversed phase preparative

6580 J . Org. Chem., Vol. 64, No. 18, 1999
Abad et al.
chromatography using a gradient of 0-20% CH3CN in water.
Appropriate fractions were concentrated to dryness, and the
solid was dried in a vacuum desiccator over P2O5 to afford 4.6
g (17 mmol, 89%) of pure 10a : mp 233-4 °C; UV λmax 260
nm; 1H NMR (DMSO-d6) δ 8.33 (s, 1H), 8.12 (t, J ) 15 Hz,
1H), 7.34 (dd, J 1 ) 2 Hz, J 2 ) 90 Hz, 2H), 5.86 (d, J ) 5 Hz,
1H), 5.43 (2H), 5.18 (d, J ) 4 Hz, 1H), 4.60 (q, J ) 5 Hz, 1H),
4.12 (m, 1H), 3.95 (m, 1H), 3.78-3.40 (2H); 13C NMR (DMSO-
d6) 156.2 (dt, J 1 ) 2 Hz, J 2 ) 16 Hz), 152.4 (br s), 149.1 (dd,
J 1 ) 2 Hz, J 2 ) 5 Hz), 140.0, 119.4, 88.0, 86.0, 73.5, 70.7, 61.8;
15N NMR (DMSO-d6) 237, 223, 83; HRMS m/z 270.0873 (calcd
for C10H13O4N215N3: 270.0879). Anal. Calcd for C10H13O4N2-
15N3: C, 44.45; H, 4.85; N, 25.92. Found: C, 44.57; H, 4.91; N,
26.18.
filtration and was washed with CH3CN (3 × 50 mL) to afford
4.6 g of crude product. The filtrate was evaporated to dryness,
and the residue was purified by reversed phase preparative
HPLC using aqueous NH4HCO3 (pH ) 8) to afford an ad-
ditional 0.3 g of product. The combined products were used
without further purification to make 11a . Pure analytical
samples of 6a were prepared by solubilization in water and
precipitation with 10% HCl: mp > 300 °C; UV λmax 280 nm;
1H NMR (DMSO-d6) δ (ppm) 13.54 (br s, 1H), 13.10 (br s, 1H),
12.19 (d, J ) 92 Hz, 1H), 8.06 (s, 1H); 13C NMR (DMSO-d6)
173.4 (dd, J 1 ) 11 Hz, J 2 ) 15 Hz), 153.5 (d, J ) 9 Hz), 149.1
(d, J ) 19 Hz), 141.6, 110.4 (dd, J 1 ) 3 Hz, J 2 ) 9 Hz); 15N
NMR (DMSO-d6) 178, 148; HRMS m/z 170.0040 (calcd for
C5H4ON215N2S: 170.0047). Anal. Calcd for C5H4ON215N2S‚
0.66H2O: C, 32.96; H, 2.95; N, 30.76. Found: C, 33.32; H, 2.62;
N, 30.76.
[2-13C-1,3,NH2-15N3]-Ad en osin e (10b). The same proce-
dure used to prepare 10a , except for starting with 9b rather
than 9a , was used for 10b: mp 233-234 °C; UV λmax 260 nm;
1H NMR (DMSO-d6) δ 8.33 (s, 1H), 8.12 (dt, J 1 ) 16 Hz,
J 2 ) 199 Hz, 1H), 7.36 (dd, J 1 ) 3 Hz, J 2 ) 90 Hz, 2H), 5.87
(d, J ) 6 Hz, 1H), 5.40 (2H), 5.18 (1H), 4.60 (t, J ) 5 Hz, 1H),
4.13 (dd, J 1 ) 3 Hz, J 2 ) 4 Hz, 1H), 3.96 (q, J ) 3 Hz, 1H),
3.78-3.40 (m, 2H); 13C NMR (DMSO-d6) 156.1 (dt, J 1 ) 3 Hz,
J 2 ) 19 Hz), 152.4 (br s, C2), 149.1 (dd, J 1 ) 2 Hz, J 2 ) 5 Hz),
140.0, 119.4, 88.0, 86.0, 73.5, 70.7, 61.8; 15N NMR (DMSO-d6)
237 (d, J ) 5 Hz), 223, 83 (d, J ) 4 Hz); HRMS m/z 271.0906
(calcd for C913CH13O4N215N3: 271.0912). Anal. Calcd for
C913CH13O4N215N3‚0.25 H2O: C, 43.56; H, 4.94; N, 25.40.
Found: C, 43.37; H, 4.83; N, 25.57.
[1,3-15N2]-2-(Meth ylth io)h yp oxa n th in e (11a ). To a solu-
tion of 4 g of crude 6a in 150 mL of water was added 1.4 mL
(22 mmol) of iodomethane. The mixture was stirred vigorously
in darkness at room temperature for 24 h. A white precipitate
formed, and the starting material disappeared. The mixture
was concentrated to 50 mL, and the precipitate was collected
by filtration. Additional product was obtained from the filtrate
by preparative reversed phase HPLC using a gradient of
0-20% CH3CN in H2O, giving a total of 3.2 g (17 mmol, 85%
from 5) of pure 11a : mp 290-1 °C (dec); UV λmax 262 nm; 1H
NMR (D2O/NaOD) δ (ppm) 7.71 (s, 1H), 2.52 (s, 3H); 13C NMR
(D2O) 169.9 (d, J ) 4 Hz), 164.2 (m), 152.9, 124.5, 16.4; 15N
NMR (D2O/NaOD) 227, 213; HRMS m/z 184.0210 (calcd for
C6H6ON215N2S: 184.0203). Anal. Calcd for C6H6ON215N2S‚
0.66H2O: C, 36.73; H, 3.77; N, 28.56. Found: C, 36.93; H, 3.53;
N, 28.30.
[1,3,NH2-15N3]-2′-Deoxya d en osin e (10c). A mixture of 9c
(4.8 g, 18 mmol), [15N]-NH4Cl (1.93 g, 36 mmol), and KHCO3
(5.5 g, 54 mmol) in DMSO (12 mL) was sealed in a 100 mL
flask and kept in an oven at 80 °C for 7 days. The cooled (0
°C) reaction vial was opened carefully, the contents were
diluted with 66 mL of water, and the pH was adjusted to 7
with glacial acetic acid. The product was purified by reversed
phase preparative chromatography using a gradient of 0-20%
CH3CN in water. Appropriate fractions were concentrated to
dryness, and the solid was dried in a vacuum desiccator over
P2O5 to afford 4.3 g (17 mmol, 94%) of pure 10c: mp 190-1
°C (dec); UV λmax 260 nm; 1H NMR (DMSO-d6) δ 8.33 (s, 1H),
8.13 (t, J ) 16 Hz, 1H), 7.33 (dd, J 1 ) 3 Hz, J 2 ) 90 Hz, 2H),
6.34 (t, J ) 7 Hz, 1H), 5.31 (d, J ) 4 Hz, 1H), 5.26 (t, J ) 5
Hz, 1H), 4.40 (m, 1H), 3.88 (m, 1H), 3.72-3.38 (m, 2H), 2.73
[2-13C-1,3-15N2]-2-(Meth ylth io)h yp oxa n th in e (11b). The
same procedure used to prepare 11a , except for starting with
6b rather than 6a , was used to prepare 11b: 1H NMR (D2O/
NaOD) δ (ppm) 7.72 (s, 1H), 2.53 (d, J ) 4 Hz, 3H); 13C NMR
(D2O) 169.8 (d, J ) 4 Hz), 168.5, 164.4 (C2), 152.3 (dd, J 1 ) 3
Hz, J 2 ) 29 Hz, 124.2 (d, J ) 5 Hz), 16.3 (d, J ) 9 Hz); 15N
NMR (D2O/NaOD) 227, 213; HRMS m/z 185.0243 (calcd for
C513CH6ON215N2S: 185.0237). Anal. Calcd for C513CH6ON215N2S‚
0.75H2O: C, 36.26; H, 3.81; N, 28.20. Found: C, 36.40; H, 3.63;
N, 27.96.
[1,3-15N2]-2-(Meth ylth io)in osin e (12a ). To a suspension
of 11a (3.3 g, 18 mmol) and 7-methylguanosine (12 g, 41 mmol)
in aqueous K2HPO4 (70 mL, 0.02 M) was added 6 N potassium
hydroxide until the pH was 7.4. To this mixture was added
purine nucleoside phosphorylase (500 µL, 2.1 units/µL). The
mixture was kept at 43 °C with gentle agitation for 7 days.
The reaction mixture was then filtered and the filter cake
extracted by sonication with DMF (2 × 80 mL) and then H2O
(100 mL). All solutions were combined and concentrated under
vacuum until a white precipitate first appeared (∼60 mL).
Then 60 mL of water was added, and 40 mL portions of the
warm solution were filtered directly onto the reversed phase
preparative column and eluted with 0-10% CH3CN in water.
Appropriate fractions were combined, concentrated, and dried
in a vacuum desiccator over P2O5 to give 4.8 g (15 mmol, 83%)
(m, 1H), 2.25 (ddd, J 1 ) 3 Hz, J 2 ) 6 Hz, J 3 ) 11 Hz, 1H); 13
C
NMR (DMSO-d6) 156.1 (dt, J 1 ) 4 Hz, J 2 ) 21 Hz), 152.4, 148.9
(dd, J 1 ) 2 Hz, J 2 ) 5 Hz), 139.6, 119.3, 88.1, 84.0, 71.1, 62.0;
15N NMR (DMSO-d6) 237, 224, 82; HRMS m/z 254.0925 (calcd
for C10H13O3N215N3: 254.0929). Anal. Calcd for C10H13O3N215N3‚
0.875H2O: C, 44.48; H, 5.51; N, 25.94. Found: C, 44.33; H,
5.31; N, 26.19.
[2-13C-1,3,NH2-15N3]-2′-Deoxya d en osin e (10d ). The same
procedure used to prepare 10c, except for starting with 9d
rather than 9c, was used for 10d : mp 191-192 °C; UV λmax
1
260 nm; H NMR (DMSO-d6) δ 8.32 (s, 1H), 8.12 (dt, J 1 ) 16
Hz, J 2 ) 199 Hz, 1H), 7.28 (dd, J 1 ) 3 Hz, J 2 ) 90 Hz, 2H),
6.33 (t, J ) 7 Hz, 1H), 5.29 (d, J ) 4 Hz, 1H), 5.28 (t, J ) 5
Hz, 1H), 4.40 (m, 1H), 3.87 (m, 1H), 3.72-3.38 (2H), 2.73 (m,
1H), 2.25 (ddd, J 1 ) 3 Hz, J 2 ) 6 Hz, J 3 ) 11 Hz, 1H); 13C
NMR (DMSO-d6) 156.1 (d, J ) 20 Hz), 152.4 (C2), 148.9 (dd,
1
of pure 12a : mp 245-6 °C (dec); UV λmax 263 nm; H NMR
(DMSO-d6) δ (ppm) 12.6 (br d, J ) 83 Hz, 1H), 8.20 (s, 1H),
5.83 (d, J ) 6 Hz, 1H), 5.46 (d, J ) 6 Hz, 1H), 5.20 (d, J ) 4
Hz, 1H), 4.98 (t, J ) 5 Hz, 1H), 4.52 (q, J ) 5 Hz, 1H), 4.12 (q,
J ) 4 Hz, 1H), 3.90 (q, J ) 4 Hz, 1H), 3.57 (m, 2H), 2.55 (s,
3H); 13C NMR (DMSO-d6) 157.7 (d, J ) 7 Hz), 156.8 (d, J ) 9
Hz), 148.5 (d, J ) 6 Hz), 138.1 (m), 121.2 (d, J ) 6 Hz), 87.3,
85.5, 73.9, 70.3, 61.4, 13.2; 15N NMR (DMSO-d6) 206,
J 1 ) 2 Hz, J 2 ) 5 Hz), 139.6, 119.3, 88.1, 84.0, 71.1, 62.0; 15
N
NMR (DMSO-d6) 237 (d, J ) 5 Hz), 224, 83 (d, J ) 4 Hz);
HRMS m/z 255.0952 (calcd for C913CH13O3N215N3: 255.0963).
Anal. Calcd for C913CH13O3N215N3‚0.5H2O: C, 45.45; H, 5.34
N, 26.51. Found: C, 45.06; H, 5.24; N, 26.46.
Sod iu m Eth yl Xa n th a te (Na SCSOEt). The same proce-
dure described above was used except unlabeled CS2 was
employed.
171; HRMS (FAB) m/z 317.0706 (calcd for
C11H15O5-
N215N2S: 317.0704). Anal. Calcd for C11H14O5N215N2S‚H2O: C,
39.51; H, 4.82; N, 16.76. Found: C, 39.78; H, 4.77; N, 16.97.
[2-13C-1,3-15N2]-2-(Meth ylth io)in osin e (12b). The same
procedure used to prepare 12a , except for starting with 11b
rather than 11a , was used to prepare 12b: 1H NMR (DMSO-
d6) δ (ppm) 12.6 (br s, 1H), 8.20 (s, 1H), 5.82 (d, J ) 5 Hz,
1H), 5.44 (br s, 1H), 5.21 (br s, 1H), 4.99 (br s, 1H), 4.53 (t,
J ) 5 Hz, 1H), 4.12 (t, J ) 4 Hz, 1H), 3.90 (q, J ) 3 Hz, 1H)
3.57 (m, 2H), 2.54 (d, J ) 5 Hz, 3H); 13C NMR (DMSO-d6) 157.9
[1,3-15N2]-2-Mer ca p t oh yp oxa n t h in e (6a ). A mixture of
2.6 g (20 mmol) of [NH2,CONH2-15N2]-5-amino-4-imidazole-
carboxamide (5) and 3.4 g (24 mmol) of sodium ethyl xanthate
in 80 mL of anhydrous N,N-dimethylformamide was stirred
under N2 at room temperature for 20 min and then refluxed
for 4 h. The clear solution turned dark green, and a white
precipitate formed. The solution was allowed to cool, and 160
mL of CH3CN was added. The precipitate was collected by

15N-Multilabeled Adenines
J . Org. Chem., Vol. 64, No. 18, 1999 6581
(dd, J 1 ) 2 Hz, J 2 ) 10 Hz), 148.5 (d, J ) 5 Hz), 138.1, 121.2
(m), 87.3, 85.5, 73.9, 70.4, 61.4, 13.3; 15N NMR (DMSO-d6) 206,
173); HRMS (FAB) m/z 318.0753 (calcd for C1013CH15O5-
N215N2S: 318.0737). Anal. Calcd for C1013CH14O5N215N2S‚
H2O: C, 39.40; H, 4.81; N, 16.71. Found: C, 39.64; H, 4.65, N,
17.01.
J ) 4 Hz, 1H), 3.94 (q, J ) 3 Hz, 1H) 3.60 (m, 2H), 2.96 (d,
J ) 5 Hz, 3H); 13C NMR (DMSO-d6) 160.9 (C2), 157.0 (d, J )
8 Hz), 147.6, 139.9, 124.2, 87.4 (d, J ) 4 Hz), 85.8, 74.0 (d,
J ) 8 Hz), 70.4 (d, J ) 2 Hz), 61.3; 15N NMR (DMSO-d6) 213,
176 (br s); HRMS (FAB) m/z 334.0688 (calcd for C1013CH15O6-
N215N2S: 334.0687). Anal. Calcd for C1013CH14O6N215N2S‚
H2O: C, 37.60; H, 4.59; N, 15.95. Found: C, 37.63; H, 4.46; N,
15.86.
[1,3-15N2]-2-(Meth ylth io)-2′-d eoxyin osin e (12c). To a
suspension of 11a (2.5 g, 14 mmol) and 2′-deoxyguanosine (4.3
g, 15 mmol) in aqueous K2HPO4 (70 mL, 0.02 M) was added 6
N potassium hydroxide until the pH was 8.4. To this mixture
was added purine nucleoside phosphorylase (400 µL, 2.1 units/
µL). The flask was sealed and the mixture kept at 43 °C with
gentle agitation for 2 days. The reaction mixture was then
filtered and the filter cake was extracted by sonication and
heating with 2% NH4OH (4 × 50 mL). All solutions were
combined and concentrated under vacuum until a white
precipitate first formed. The mixture was heated and the warm
solution was filtered directly onto the reversed phase prepara-
tive column which was then eluted with 0-5% CH3CN in 0.25
M NH4HCO3. Unreacted 11a eluted with 2′-deoxyguanosine,
so fractions containing both products were combined, evapo-
rated, heated again with PNP, and then purified. Final
fractions containing pure product were combined and concen-
trated to a white solid, which was dried in a vacuum desiccator
over P2O5 to give 3.1 g (10 mmol, 71%) of pure 12c: mp 245-6
°C (dec); UV λmax nm; 1H NMR (DMSO-d6) δ (ppm) 8.13 (s,
1H), 6.26 (t, J ) 7 Hz, 1H), 5.32 (br s, 1H), 4.95 (br s, 1H),
4.38 (m, 1H), 3.83 (m, 1H), 3.52 (m, 2H), 2.67 (m, 1H), 2.53 (s,
3H), 2.26 (ddd, J 1 ) 2 Hz, J 2 ) 6 Hz, J 3 ) 12 Hz, 1H); 13C
NMR (DMSO-d6) 158.2 (dd, J 1 ) 2 Hz, J 2 ) 9 Hz), 157.9 (d,
J ) 8 Hz), 148.3 (d, J ) 5 Hz), 137.5, 121.2 (dd, J 1 ) 2 Hz,
J 2 ) 7 Hz), 87.8, 83.4, 70.8, 61.7, 13.2 (d, J ) 4 Hz); 15N NMR
(DMSO-d6) 206, 171; HRMS (FAB) m/z 301.0762 (calcd for
C11H15O4N215N2S: 301.0754). Anal. Calcd for C11H14O4N215N2S‚
0.75H2O: C, 42.10; H, 4.98; N, 17.85. Found: C, 41.92; H, 4.92;
N, 18.04.
[1,3-15N2]-2-(Meth ylsu lfoxyl)-2′-d eoxyin osin e (13c). To
a cold stirred mixture of 3.5 g (12 mmol) of 12c in 650 mL of
H2O was added dropwise a cold solution of 4.1 g (6.6 mmol,
1.15 equiv) of Oxone (2KHSO5-KHSO4-K2SO4) in 100 mL of
H2O over 2 h. The mixture was stirred vigorously for an
additional 2 h at 0 °C. The mixture became a clear solution,
and Na2SO3 (0.26 g, 2 mmol) was added. The solution was
stirred for 10 min, and the pH was adjusted to 6.7 with
NaHCO3. The resulting solution was concentrated, and the
diastereoisomeric mixture was purified by reversed phase
preparative HPLC using a gradient of 0-20% CH3CN in H2O
to obtain 3.3 g of nearly pure 13c, which was used without
1
further purification: UV λmax 258 nm; H NMR (DMSO-d6) δ
(ppm) 7.99 (s, 1H), 6.25 (dd, J 1 ) 6 Hz, J 2 ) 8, 1H), 5.32 (d,
J ) 4 Hz, 1H), 5.04 (br m, 1H), 4.37 (br m, 1H), 3.83 (br m,
1H), 3.52 (br m, 2H), 2.75-2.55 (br m, 1H), 2.68 (s, 3H), 2.19
(ddd, J 1 ) 2 Hz, J 2 ) 6 Hz, J 3 ) 13 Hz, 1H); 13C NMR (DMSO-
d6) 166.6, 166.0, 149.2 (d, J ) 5 Hz), 137.0, 124.7, 87.8, 83.5,
71.0, 62.0, 48.6, 39.7; 15N NMR (DMSO-d6) 242 (d, 15 Hz), 204
(d, 13 Hz).
[2-13C-1,3-15N2]-2-(Meth ylsu lfoxyl)-2′-deoxyin osin e (13d).
The same procedure used to prepare 13c, except for starting
with 12d rather than 12c, was used to prepare 13d : 1H NMR
(DMSO-d6) δ (ppm) 7.99 (s, 1H), 6.25 (dd, J 1 ) 6 Hz, J 2 ) 8
Hz, 1H), 5.33 (br s, 1H), 5.06 (br m, 1H), 4.37 (br m, 1H), 3.83
(br m, 1H) 3.52 (br m, 2H), 3.35 (s, 1H), 2.75-2.55 (br m, 1H),
2.68 (d, J ) 4 Hz, 3H), 2.19 (ddd, J 1 ) 2 Hz, J 2 ) 6 Hz, J 3
)
13 Hz, 1H); 13C NMR (DMSO-d6) 166.7, 166.0 (C2), 149.2 (d,
J ) 5 Hz), 137.0, 124.7, 87.8, 83.5, 71.1, 62.0, 39.7; 15N NMR
(DMSO-d6) 242 (d, 15 Hz), 204 (d, 13 Hz).
[2-13C-1,3-15N2]-2-(Meth ylth io)-2′-deoxyin osin e (12d). The
same procedure used to prepare 12c, except for starting with
11b rather than 11a , was used to prepare 12d : 1H NMR
(DMSO-d6) δ (ppm) 12.5 (br s, 1H), 8.17 (s, 1H), 6.27 (t, J ) 7
Hz, 1H), 5.32 (br s, 1H), 4.91 (br s, 1H), 4.38 (br m, 1H), 3.83
(m, 1H), 3.52 (m, 2H), 2.67 (m, 1H), 2.54 (d, J ) 5 Hz, 3H),
2.26 (ddd, J 1 ) 2 Hz, J 2 ) 6 Hz, J 3 ) 12 Hz, 1H); 13C NMR
(DMSO-d6) 157.6 (dd, J 1 ) 2 Hz, J 2 ) 10 Hz, C2), 156.9 (d,
J ) 8 Hz), 148.2 (d, J ) 5 Hz), 137.9, 121.2 (dd, J 1 ) 2 Hz,
J 2 ) 7 Hz), 87.8, 83.3, 70.7, 61.7, 13.2 (d, J ) 4 Hz); 15N NMR
(DMSO-d6) 206, 171; HRMS (FAB) m/z 302.0793 (calcd for
[1,2,3-15N3]-Gu a n osin e (14a ). A mixture of 13a (3.3 g, 9.9
mmol), [15N]-NH4Cl (1.7 g, 30 mmol), and KHCO3 (2 g, 20
mmol) in anhydrous DMSO (32 mL) was sealed in a 100 mL
vial and was kept at 78 °C for 14 days. The cooled (0 °C)
reaction vial was opened carefully, and the contents were
concentrated under vacuum to 10 mL, diluted with 90 mL of
hot water, and purified in two parts by reversed phase
preparative chromatography using a gradient of 0-10% CH3-
CN in water. The combined product fractions were concen-
trated to dryness, and the solid was dried in a vacuum
desiccator over P2O5 to give 2.3 g (8.0 mmol, 81%) of pure
C1013CH15O4N215N2S: 302.0788). Anal. Calcd for C1013CH14
-
O4N215N2S‚H2O: C, 41.37; H, 5.05; N, 17.55. Found: C, 41.21;
H, 4.67; N, 17.85.
1
[1,3-15N2]-2-(Meth ylsu lfoxyl)in osin e (13a ). To a cold
stirred mixture of 4.5 g (14 mmol) of 12a in 700 mL of H2O
was added dropwise a solution of 3.8 g (7.5 mmol, 1.2 eq) of
Oxone (2KHSO5-KHSO4-K2SO4) in 100 mL H2O over 2 h.
The mixture was stirred vigorously for an additional 2 h at 0
°C. The mixture became a clear solution, and Na2SO3 (0.26 g,
2 mmol) was added. The solution was concentrated, and the
diastereoisomeric mixture was purified by reversed phase
preparative HPLC using 0-20% CH3CN in H2O to give 4.5 g
(13.5 mmol, 96%) of pure 13a : UV λmax 258 nm; 1H NMR
(DMSO-d6) δ (ppm) 8.43 (s, 1H), 5.88 (d, J ) 6 Hz, 1H), 5.48
(br s, 1H), 5.21 (br s, 1H), 4.97 (br s, 1H), 4.52 (q, J ) 3 Hz,
1H), 4.14 (t, J ) 4 Hz, 1H), 3.93 (q, J ) 4 Hz, 1H) 3.57 (m,
2H), 2.95 (s, 3H); 13C NMR (DMSO-d6) 160.8 (d, J ) 8 Hz),
156.9 (d, J ) 8 Hz), 147.6 (d, J ) 5 Hz), 140.1, 124.2 (d, J )
7 Hz), 87.4, 85.7 (d, J ) 15 Hz), 74.1, 70.3 (m), 61.3; 15N NMR
(DMSO-d6) 213, 174 (br s); HRMS (FAB) m/z 333.0638 (calcd
for C11H15O6N215N2S: 333.0653). Anal. Calcd for C11H14O6N2-
15N2S‚0.5H2O: C, 38.71; H, 4.43; N, 16.42. Found: C, 38.81;
H, 4.49; N, 16.32.
14a : mp 248-9 °C (dec); UV λmax 253 nm; H NMR (DMSO-
d6) δ (ppm) 10.65 (br s, 1H), 7.93 (s, 1H), 6.45 (d, J ) 89 Hz,
2H), 5.69 (d, J ) 5 Hz, 1H), 5.38 (br s, 1H), 5.11 (br s, 1H),
5.03 (br s, 1H), 4.38 (br s, 1H), 4.07 (br s, 1H), 3.86 (q, J ) 3
Hz, 1H), 3.56 (m, 2H); 13C NMR (DMSO-d6) 156.9 (d, J ) 11
Hz), 153.7 (ddd, J 1 ) 7 Hz, J 2 ) 13 Hz, J 3 ) 23 Hz), 151.4 (dd,
J 1 ) 3 Hz, J 2 ) 7 Hz), 135.7 (m), 116.7 (dd, J 1 ) 2 Hz, J 2 ) 8
Hz), 86.4, 85.3, 73.7, 70.4, 61.5; 15N NMR (DMSO-d6) 166 (d,
J ) 5 Hz), 148, 74 (d, J ) 5 Hz); MS (FAB) m/z 287 (61%,
M + 1); 155 (100%, b + 2); HRMS (FAB) m/z 287.0903 (calcd
for C10H14O5N215N3: 287.0906). Anal. Calcd for C10H13O5N2-
15N3‚0.25H2O: C, 41.31; H, 4.68; N, 24.09. Found: C, 41.00;
H, 4.90; N, 24.21.
[2-13C-1,2,3-15N3]-Gu a n osin e (14b). The same procedure
used to prepare 14a , except for starting with 13b rather than
1
13a , was used to prepare 14b: H NMR (DMSO-d6) δ (ppm)
10.6 (br s, 1H), 7.91 (s, 1H), 6.53 (d, J ) 90 Hz, 2H), 5.68 (d,
J ) 6 Hz, 1H), 5.5-4.8 (br m, 3H), 4.40 (t, J ) 5 Hz, 1H), 4.08
(dd, J 1 ) 3 Hz, J 2 ) 5 Hz, 1H), 3.86 (q, J ) 4 Hz, 1H), 3.57 (m,
2H); 13C NMR (DMSO-d6) 157.7 (d, J ) 11 Hz), 154.2 (ddd,
J 1 ) 7 Hz, J 2 ) 13 Hz, J 3 ) 23 Hz, C2), 151.4 (dd, J 1 ) 3 Hz,
J 2 ) 7 Hz), 135.6, 116.8 (dd, J 1 ) 2 Hz, J 2 ) 7 Hz), 86.5, 85.3,
73.7, 70.5, 61.5; 15N NMR (DMSO-d6) 167 (t, J ) 6 Hz), 152
(d, J ) 12 Hz), 75 (dd, J 1 ) 4 Hz, J 2 ) 23 Hz); MS (FAB) m/z
288 (100%, M + 1); 156 (99%, b + 2); HRMS (FAB) m/z
[2-13C-1,3-15N2]-2-(Meth ylsu lfoxyl)in osin e (13b). The same
procedure used to prepare 13a , except for starting with 12b
rather than 12a , was used to prepare 13b: 1H NMR (DMSO-
d6) δ (ppm) 8.44 (s, 1H), 5.88 (d, J ) 6 Hz, 1H), 5.53 (br s,
1H), 5.26 (br s, 1H), 5.02 (br s, 1H), 4.53 (br s, 1H), 4.14 (t,

6582 J . Org. Chem., Vol. 64, No. 18, 1999
Abad et al.
288.0941 (calcd for C913CH14O5N215N3: 288.0940). Anal. Calcd
for C913CH13O5N215N3‚H2O: C, 39.35; H, 4.95; N, 22.95.
Found: C, 39.56; H, 4.91; N, 23.15.
[2-13C-1,2,3-15N3]-2′-Deoxygu a n osin e (14d ). The same
procedure used to prepare 14c, except for starting with 13d
rather than 13c, was used to prepare 14d : H NMR (DMSO-
1
[1,2,3-15N3]-2′-Deoxygu a n osin e (14c). A mixture of nearly
pure 13c (3.2 g), [15N]-NH4Cl (1.7 g, 30 mmol), and KHCO3 (2
g, 20 mmol) in anhydrous DMSO (32 mL) was sealed in a 100
mL vial and was kept at 78 °C for 14 days. The cooled (0 °C)
reaction vial was opened carefully, and the contents were
concentrated under vacuum to 10 mL, diluted with 90 mL of
hot water, and purified in two parts by reversed phase
preparative chromatography using a gradient of 0-10% CH3-
CN in water. The combined product fractions were concen-
trated to dryness, and the solid was dried in a vacuum
desiccator over P2O5 to give 2.3 g (8.5 mmol, 71% from 12c) of
pure 14c: mp > 300 °C (dec); UV λmax 253 nm; 1H NMR
(DMSO-d6) δ (ppm) 10.6 (br d, J ) 68 Hz, 1H), 7.91 (s, 1H),
6.44 (d, J ) 89 Hz, 2H), 6.11 (dd, J 1 ) 6 Hz, J 2 ) 7 Hz, 1H),
5.24 (d, J ) 4 Hz, 1H), 4.93 (t, J ) 5 Hz, 1H), 4.32 (br m, 1H),
3.79 (br m, 1H), 3.51 (br m, 2H), 2.6-2.4 (br m, 1H), 2.18 (ddd,
J 1 ) 3 Hz, J 2 ) 6 Hz, J 3 ) 13 Hz, 1H); 13C NMR (DMSO-d6)
d6) δ (ppm) 11.2 (br), 7.85 (s, 1H), 6.70 (d, J ) 88 Hz, 2H),
6.11 (dd, J 1 ) 6 Hz, J 2 ) 8 Hz, 1H), 5.7-4.8 (br s, 2H), 4.33
(br m, 1H), 3.80 (br m, 1H), 3.52 (br m, 2H), 2.6-2.4 (m, 1H),
2.17 (ddd, J 1 ) 3 Hz, J 2 ) 6 Hz, J 3 ) 13 Hz, 1H); 13C NMR
(DMSO-d6) 158.9 (d, J ) 11 Hz), 155.0 (ddd, J 1 ) 7 Hz, J 2
)
12 Hz, J 3 ) 23 Hz, C2), 151.0 (dd, J 1 ) 3 Hz, J 2 ) 7 Hz), 135.1,
116.8 (dd, J 1 ) 2 Hz, J 2 ) 8 Hz), 87.7, 82.8, 70.9, 61.9, 39.6;
15N NMR (DMSO-d6) 167 (t, J ) 6 Hz), 158 (d, J ) 10 Hz), 75
(ddd, J 1 ) 2 Hz, J 2 ) 6 Hz, J 3 ) 23 Hz); MS (FAB) m/z 272
(37%, M + 1); 156 (100%, b + 2); HRMS (FAB) m/z 272.0983
(calcd for C913CH14O4N215N3: 272.0990). Anal. Calcd for C9-
13CH13O4N215N3‚H2O: C, 41.52; H, 5.23; N, 24.22. Found: C,
41.36; H, 5.15; N, 24.22.
Ack n ow led gm en t. This work was supported by
grants from the National Institutes of Health (GM48802
and GM31483). A postdoctoral fellowship from CIRIT-
Generalitat de Catalunya to J .-L.A. is also gratefully
acknowledged.
156.9 (d, J ) 11 Hz), 153.7 (ddd, J 1 ) 8 Hz, J 2 ) 13 Hz, J 3
)
23 Hz), 150.9 (dd, J 1 ) 3 Hz, J 2 ) 8 Hz), 135.4, 116.7 (dd,
J 1 ) 2 Hz, J 2 ) 8 Hz), 87.6, 82.7, 70.8, 61.8, 39.6; 15N NMR
(DMSO-d6) 167 (d, J ) 6 Hz), 148, 74 (d, J ) 5 Hz); MS (FAB)
m/z 271 (37%, M + 1); 155 (100%, b + 2); HRMS (FAB) m/z
271.0945 (calcd for C10H14O4N215N3: 271.0957). Anal. Calcd for
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 15N
NMR spectra for 10b,d and 14b,d , as well as mass spectra
for 10a -d and 14a -d . This material is available free of charge
via the Internet at http://pubs.acs.org.
C
10H13O4N215N3‚0.5H2O: C, 43.01; H, 5.05; N, 25.08. Found:
C, 43.31; H, 5.02; N, 25.29.
J O982372K