Use of a 13C atom to differentiate two 15N-labeled nucleosides

Article abstract of DOI:10.1021/jo971206u

We report the first examples of the specifically ¹⁵N and ¹³C multilabeled nucleosides: [1,NH₂-¹⁵N₂]- and [2-¹³C-1,NH₂-¹⁵N₂-]- guanosine; [1,7,NH₂-¹⁵N₃]- and [2-¹³C-1,7,NH₂-¹⁵N₃]-2'- deoxyguanosine. In each set, the [¹³C] atom functions as a 'tag' that allows the N1 and N2 ¹⁵N atoms of two ¹⁵N-labeled guanines to be unambiguously differentiated in RNA and DNA fragments. The syntheses employ high-yield reactions in which protecting groups are not required and use relatively low cost sources of isotopes: [¹⁵N]-ammonium chloride and [¹⁵N]- or [¹³C,¹⁵N]-potassium cyanide.

Full text of DOI:10.1021/jo971206u

7
832
J . Org. Chem. 1997, 62, 7832-7835
Use of a 13C Atom To Differ en tia te Tw o N-La beled Nu cleosid es.
15
1
5
15
Syn th eses of [ NH
2
]-Ad en osin e, [1,NH
2
2
- N ]- a n d
1
3
15
15
[
2 2 2 3
2- C-1,NH - N ]-Gu a n osin e, a n d [1,7,NH - N ]- a n d
1
3
15
[
2 3
2- C-1,7,NH - N ]-2′-Deoxygu a n osin e
Hong Zhao, Alex R. Pagano, Weimin Wang, Anthony Shallop, Barbara L. Gaffney, and
Roger A. J ones*
Department of Chemistry, Rutgers, The State University of New J ersey, Piscataway, New J ersey 08855
Received J uly 2, 1997X
We report the first examples of the specifically 15N and 13C multilabeled nucleosides: [1,NH
15
2 2
- N ]-
1
3
15
15
13
15
2 2 2 3 2 3
and [2- C-1,NH - N -]-guanosine; [1,7,NH - N ]- and [2- C-1,7,NH - N ]-2′-deoxyguanosine. In
1
3
15
15
each set, the [ C] atom functions as a “tag” that allows the N1 and N2 N atoms of two N-
labeled guanines to be unambiguously differentiated in RNA and DNA fragments. The syntheses
employ high-yield reactions in which protecting groups are not required and use relatively low
1
5
15
13
15
cost sources of isotopes: [ N]-ammonium chloride and [ N]- or [ C, N]-potassium cyanide.
Specific 15N-labeling of DNA fragments with single
N] labels has demonstrated the value of these labels
singly labeled products. We have begun development of
routes designed to give multilabeled nucleosides, and the
[15
1
5
for NMR studies of nucleic acid structure and inter-
2 2
syntheses of [7,NH - N ]-adenosine and 2′-deoxyadenos-
actions.1
-11
To maximize the information available from
ine have been reported recently. Unfortunately, intro-
duction of more than one such multilabeled monomer into
a particular DNA or RNA fragment raises uncertainty
28
a single NMR experiment and a single synthesis, it would
be most useful to have in the molecule or complex as
many 15N labels as can be unambiguously distinguished.
15
about the identity of the N labels. The solution we have
1
5
13
The synthetic routes reported to date for N labeling of
devised is to add a C atom as a “tag” to one of a pair of
bases1
2-14
or nucleosides,
2,15-27
however, generally give
15
N-multilabeled nucleosides. Modern NMR spectrom-
eters are well suited to the use of molecules with both
X
13
15
13
Abstract published in Advance ACS Abstracts, October 15, 1997.
1) Gao, X.; J ones, R. A. J . Am. Chem. Soc. 1987, 109, 3169-3171.
2) Kupferschmitt, G.; Schmidt, J .; Schmidt, T.; Fera, B.; Buck, R.;
C and N labels. This C “tag” then offers a means
(
(
15
for differentiation of two otherwise identical N-labeled
residues. Because the most straightforward way to use
R u¨ 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.
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448-1450.
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Soc. 1991, 113, 5486-5488.
6) Gaffney, B. L.; Wang, C.; J ones, R. A. J . Am. Chem. Soc. 1992,
14, 4047-4050.
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15, 3832-3833.
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Soc. 1993, 115, 8742-8746.
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993, 115, 4921-4922.
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11) Gaffney, B. L.; Kung, P.-P.; Wang, C.; J ones, R. A. J . Am. Chem.
Soc. 1995, 117, 12281-12283.
12) Barrio, M. D. C. G.; Scopes, D. I. C.; Holtwick, J . B.; Leonard,
N. J . Proc. Natl. Acad. of Sci. U.S.A. 1981, 78, 3986-3988.
13) 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-
1
3
(
such a “tag” would be to have the C atom directly
15
connected to at least some of the N atoms involved, we
(
1
5
13
15
chose [1,NH
(8b ) and [1,7,NH
′-deoxyguanosine (8d ) as the first examples of this type
of multilabeled nucleoside pair.
2 2 2 2
- N ]- (8a ) and [2- C-1,NH - N ]-guanosine
1
1
5
13
15
(
2 3 2 3
- N ]- (8c) and [2- C-1,7,NH - N ]-
2
(
1
1
(
The syntheses of 8a and 8b are carried out by the same
route, which is shown in Scheme 1. In the first trans-
formation, inosine (1) is converted to the 6-chloro nucleo-
side (2) in 80% yield by the trifluoroacetic anhydride/
thionyl chloride/DMF procedure developed by Robins for
2′-deoxyinosine.29 Here, the trifluoroacetyl groups are
used for transient hydroxyl protection, rather than for
stabilization of the glycosidic bond. They are easily
removed by methanolysis after the chlorination. Am-
(
(
1
1
(
(
(
(
15
monolysis of 2 using [ N]-ammonium chloride and potas-
sium bicarbonate, as we reported recently,28 gives [ NH
15
]-
3
6
2
7
353.
2
(14) Griffey, R.; Poulter, C. D. Nucleic Acids Res. 1983, 11, 6497-
adenosine (3a ) in 90% yield. The subsequent steps
constitute an adenosine to guanosine transformation, in
which the adenosine amino group becomes the guanosine
N1, and the guanosine C2 and amino come from potas-
sium cyanide. It is based on a route we reported
504.
(
15) Wilson, M. H.; McCloskey, J . A. J . Org. Chem. 1973, 38, 2247-
249.
(
16) Poulter, C. D.; Livingston, C. L. Tetrahedron Lett. 1979, 9, 755-
58.
(
(
17) Gao, X.; J ones, R. A. J . Am. Chem. Soc. 1987, 109, 1275-1278.
1
5
22
18) Sarfati, S. R.; Kansal, V. K. Tetrahedron 1988, 44, 6367-
previously for singly [ N]-labeled 2′-deoxyguanosine,
6
1
1
8
6
372.
(19) Gaffney, B. L.; Kung, P.-P.; J ones, R. A. J . Am. Chem. Soc. 1990,
12, 6748-6749.
(25) Bleasdale, C.; Ellwood, S. B.; Golding, B. T.; Slaich, P. K.;
Taylor, O. J .; Watson, W. P. J . Chem. Soc., Perkin Trans. 1 1994, 2859-
2865.
(20) Adler, J .; Powell, W.; Wolfenden, R. J . Am. Chem. Soc. 1990,
12, 1247-1248.
21) Rhee, Y. S.; J ones, R. A. J . Am. Chem. Soc. 1990, 112, 8174-
175.
(
(26) Ariza, X.; Bou, V.; Vilarrasa, J . J . Am. Chem. Soc. 1995, 117,
3665-3673.
(
22) Goswami, B.; J ones, R. A. J . Am. Chem. Soc. 1991, 113, 644-
(27) Kamaike, K.; Takahashi, M.; Utsugi, K.; Tomizuka, K.; Ishido,
Y. Tetrahedron Lett. 1995, 36, 91-94.
47.
(
23) Acedo, M.; Fabrega, C.; Avi n˜ o, A.; Goodman, M.; Fagan, P.;
Wemmer, D.; Eritja, R. Nucleic Acids Res. 1994, 22, 2982-2989.
24) Sako, M.; Ishikura, H.; Hirota, K.; Maki, Y. Nucleosides
Nucleotides 1994, 13, 1239-1246.
(28) Pagano, A. R.; Lajewski, W. M.; J ones, R. A. J . Am. Chem. Soc.
1995, 117, 11669-11672.
(
(29) Robins, M. J .; Basom, G. L. Can. J . Chem. 1973, 51, 3161-
3169.
S0022-3263(97)01206-1 CCC: $14.00 © 1997 American Chemical Society

Use of 13C To Differentiate Two 15N-Labeled Nucleosides
Sch em e 1
J . Org. Chem., Vol. 62, No. 22, 1997 7833
which had been derived from a set of transformations
3
0
first reported by Ueda. Oxidation of 3a to the N-oxide
a is followed by reaction with cyanogen bromide gener-
ated in situ from bromine and labeled KCN. The
4
1
5
synthesis of 8a uses [ N]-KCN, while synthesis of 8b
1
3
15
uses [ C, N]-KCN. The conversion of 4a to the 6-N-
methoxy derivatives 7a and 7b is carried out as a one-
flask set of reactions without purification of intermedi-
ates 5a , 5b, 6a , or 6b. Treatment of 5a or 5b with
triethylamine opens the oxazolidine ring so that the
N-oxide can be methylated with methyl iodide to give 6a
or 6b. Aqueous sodium hydroxide then opens the py-
rimidine ring of 6a or 6b. Neutralization of the mixture
followed by heating at 60 °C results in deformylation and
2
2,30
ring closure to give 7a or 7b.
7
After purification of
F igu r e 1. The 1H-decoupled 15N NMR resonances of (A) 15N1,
and (B) NH , of 8b at 40.543 MHz, in 10 mM sodium
2
a or 7b by preparative reversed-phase HPLC, enzymatic
deamination converts 7a or 7b to 8a or 8b to complete
the synthesis. Preparation of 8c and 8d , starting from
15
phosphate, pH 7, δ (ppm) 149.18 (d, J N-C ) 12.2 Hz), 71.95
(d, J N-C ) 24.4 Hz). Chemical shifts are reported relative to
1
5
28
15
[7,NH
2
- N
2
]-deoxyadenosine, 3b, uses the same se-
NH
3 3 2
, using external 1 M [ N]HNO in 90% D O at 25 °C at
3
75.8 ppm as a reference.
quence of reactions. During the oxidation to form 4b,
depurination of the deoxyadenosine by the m-chloroben-
zoic acid produced is minimal as long as the reaction time
is kept to 3 h.
the 13C2 and the 15N1 and 15NH
atoms in 8b and 8d . As
2
1
15
the H decoupled N NMR spectrum of 8b (Figure 1)
The key steps in the adenosine to guanosine transfor-
mation now have been optimized to a substantial extent.
Specifically, we have (1) improved the procedure for
preparation of the N-oxide to a 92% yield by using
m-chloroperoxybenzoic acid for the oxidation and pre-
parative reversed-phase HPLC for purification; (2) found
that it is important to avoid the presence of excess
bromine (orange color), in the reaction of the N-oxide with
the labeled cyanogen bromide; and (3) found that an 8:1
excess of methyl iodide in the methylation reaction is
preferable to the smaller excess we had used previously.
We have prepared 2-3 g quantities of 8a -d by these
procedures.
shows, these one-bond coupling constants are 12.2 Hz for
1
5
13
15
13
N1- C2 and 24.4 Hz for NH - C2 in water and
2
about the same in DMSO. The analogous coupling
constants for the deoxy compound 8d have the same
values, and they all are assumed to be negative. These
observed couplings are more than adequate to allow
1
5
differentiation of the N resonances of 8a and 8b in a
synthetic RNA fragment and all but the N7 15N resonance
of 8c and 8d in a DNA fragment.
When the Fermi contact term is the dominant spin-
1
spin coupling mechanism, most J C-N values are in the
range of -10 to -15 Hz and appear often to reflect the
percentage of s character (S) of the carbon and nitrogen
3
1-34
The use of these tagged nucleosides in NMR studies
of DNA and RNA fragments relies on coupling between
atoms (80J CN ) S
C
S
N
).
The 12.2 Hz coupling for the
1
5
N1 is in this range and is consistent with significant
(
30) Ueda, T.; Miura, K.; Kasai, T. Chem. Pharm. Bull. 1978, 26,
(31) Binsch, G.; Lambert, J . B.; Roberts, B. W.; Roberts, J . D. J .
Am. Chem. Soc. 1964, 86, 5564-5570.
2
122-2127.

7
834 J . Org. Chem., Vol. 62, No. 22, 1997
Zhao et al.
2
15
sp character. However, the 24 Hz coupling for the NH
2
Calcd for C10
H
11
O
4
N
4
Cl: C, 41.90; H, 3.87; N, 19.56. Found:
1
5
13
C, 42.00; H, 3.77; N, 19.59.
is significantly larger. In addition, the NH
2
- C6
1
5
coupling constant in 15NH
2
[ NH ]-Ad en osin e (3a ). A mixture of 2 (5.35 g, 18.7
2
-adenosine reported here is
1
5
mmol), [ N]-NH
6.1 mmol) in DMSO (28 mL) was sealed in a 100 mL bottle
which was kept in an oven at 80 °C for 2 days. The cooled (0
C) bottle was opened carefully (CO pressure), the contents
4 3
Cl (1.99 g, 37.2 mmol), and KHCO (5.62 g,
nearly as large, 20.6 Hz in DMSO. Although these
5
1
5
13
purine amino groups have unusually large N- C
coupling constants, their 15N- H coupling constants are
more typical (about 90 Hz). As we gain more information
about these 15N- C couplings within oligomers, we may
find that they provide additional structural information.
At this time, both 8a and 8b have been incorporated into
RNA fragments and were used successfully to help
1
°
2
were filtered and rinsed with DMSO, and the filtrate was
concentrated to ca. 10 mL. This solution was diluted with
water (20 mL) and the product purified by reversed phase
13
preparative chromatography (0-20% CH
combined product fractions were concentrated to dryness, and
the solid was dried in a vacuum desiccator over P for 24 h
to afford 4.27 g (84%) of 3a ‚0.25H O: mp 232-234 °C (lit.
) δ 8.34 (s, 1 H), 8.12 (s, 1
3
CN/water). The
2
O
5
determine the complete structure of an aptamer-AMP
4
0
2
complex3
5-37
and to characterize an intrahelical GU
1
2
33-234 °C); H NMR (DMSO-d
6
wobble pair.38 In addition, 8c and 8d have been incor-
porated into several different DNA molecules for NMR
studies which are under way.
H), 7.56 (d, 2 H, J ) 90.1 Hz), 5.86 (d, 1 H, J ) 6.2 Hz), 5.4
(m, 2 H), 5.17 (d, 1 H, J ) 4.4 Hz), 4.60 (m, 1 H), 4.14 (m, 1
1
3
H), 3.95 (m, 1 H), 3.8-3.4 (m, 2 H); C NMR (DMSO-d ) δ
6
1
7
1
56.4 (d, J C-15N ) 20.6), 152.7, 149.3, 140.2, 119.7, 88.2, 86.2,
+
3.7, 70.9, 62.0; MS (EI) m/z (relative intensity) 268 (M , 3),
Exp er im en ta l Section
+
15
36 ([b + H] , 100). Anal. Calcd for C10
H
13
O
4
N
4
N
1
2
‚0.25H O:
C, 44.04; H, 4.80; N, 25.68. Found: C, 43.78; H, 4.82; N, 25.69.
Gen er a l Meth od s. Melting points (mp) were determined
1
5
1
in soft glass capillary tubes and are uncorrected. The 15
N
[
2
NH ]-Ad en osin e N -Oxid e (4a ). To 2.55 g (9.19 mmol)
1
5
of [ NH
2
]-adenosine hydrate 3a dissolved in 300 mL of 30%
chemical shifts are reported relative to NH , using external 1
M [ N]HNO in 90% D O at 25 °C at 375.8 ppm as a reference.
3 2
3
1
5
aqueous dioxane was added 3.17 g (18.4 mmol) of 3-chloroper-
oxybenzoic acid (MCPBA). The mixture was allowed to stir
in the dark at room temperature for 3 h and was then
concentrated to about 50 mL. This solution was then washed
with 3 × 100 mL of ethyl ether, and the aqueous layer was
concentrated to a small volume and purified by preparative
Analytical HPLC was carried out with Waters C-18 Nova-Pak
cartridges (8 × 100 mm) using a gradient of 2-40% acetoni-
trile/0.1 M triethylammonium acetate (TEAA) at a flow rate
of 2 mL/min. Preparative HPLC was carried out 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.
The MCPBA from Aldrich (50-60% pure, along with 3-chlo-
robenzoic acid) was purified before use by dissolving in ether
and washing with three portions of 0.1 M aqueous potassium
phosphate (pH 7.5). Care should be taken while using this
reversed phase HPLC with a gradient of 0 to 12% CH
O in 30 min at a flow rate of 40 mL/min. Evaporation of
appropriate fractions gave pure 4a (2.31 g, 8.44 mmol, 92%):
mp 217 °C dec; UV (H O) λmax 235, 263 nm; UV (H O) λmin 254
nm; H NMR (200 MHz, DMSO-d ) δ (ppm) 8.62 (s, 1), 8.53 (s,
1), 8.30 (br, 2), 5.87 (d, 1, J ) 5.6 Hz), 5.54 (d, 1, J ) 6.0 Hz),
.22 (d, 1, J ) 5.0 Hz), 5.05 (t, 1, J ) 5.8 Hz), 4.52 (m, 1), 4.13
3
CN in
H
2
2
2
1
6
1
5
15
13
15
4
peroxy acid. The [ N]NH Cl, [ N]KCN, and [ C, N]KCN
5
(
were obtained from Isotec Inc. Adenosine deaminase (A-5773)
was obtained from Sigma Chemical Co. General reagents were
obtained from Aldrich Chemical Co.
m, 1), 3.93 (m, 1), 3.60 (m, 2); 13C NMR ( H decoupled, 50.3
MHz, DMSO-d ) δ (ppm) 148.6 (d, J ) 21.8 Hz), 143.6, 142.7,
42.2, 119.1, 87.7, 86.8, 74.1, 70.5, 61.5.
1
6
1
6
-Ch lor o-9-(â-D-r ibofu r a n osyl)p u r in e (2). A mixture of
inosine (1) (9.24 g, 34.4 mmol) and trifluoroacetic anhydride
55 mL) in CH Cl (180 mL) was stirred at rt for 16 h and
then concentrated to a white foam (high vacuum), dissolved
in CH Cl (920 mL), and cooled to 0 °C. To this solution was
added over 10 min a premixed solution of DMF (7 mL) and
thionyl chloride (14 mL) in CH Cl (370 mL), and the resulting
1
5
[
2 2
1,NH - N ]-2-Am in o-6-(m eth oxya m in o)-9-(â-D-r ibofu -
15
r a n osyl)p u r in e (7a ). To 1.00 g (15 mmol) of [ N]KCN
(
2
2
dissolved in 250 mL of anhydrous methanol and cooled to 0
°
C was added bromine (0.773 mL, 15 mmol). After 3 h of
stirring, 2.84 g (10 mmol) of 4a was added. After an additional
h, the reaction mixture was concentrated to dryness. The
residue was dissolved in a mixture of anhydrous DMF (45 mL)
and triethylamine (4 mL, 28.8 mmol) under N . The mixture
was stirred at room temperature for 40 min, after which 5 mL
of CH I (80.6 mmol) was added. Stirring was continued for a
2
2
3
2
2
cloudy suspension was refluxed for 16 h. This solution was
concentrated to ca. 300 mL and washed with saturated
NaHCO
with CH
were dried with Na
yellow oil was dissolved in MeOH (30 mL), and the mixture
was refluxed for 16 h. Ether (60 mL) was added to the cooled
0 °C) suspension, and the resulting precipitate was filtered
2
3
(200 mL). The aqueous phase was back extracted
Cl
(3 × 30 mL), and the combined organic phases
SO and concentrated. The resulting
3
2
2
further 4 h in darkness, whereupon the reaction mixture was
concentrated to dryness and the residue dissolved in 170 mL
of 0.25 N NaOH. After 1 h, the pH was adjusted to 7.4 with
2
4
1
N HCl. 95% Ethanol (180 mL) was then added, and the
(
3
9
mixture was heated at 60 °C for 4 h. The mixture was then
concentrated to a small volume and purified by preparative
reversed phase HPLC with a gradient of 2 to 14% acetonitrile
in 0.1 M ammonium bicarbonate in 40 min at a flow rate of
40 mL/min. Evaporation of appropriate fractions gave pure
and dried under vacuum (7.91 g, 80%): mp 160-163 °C (lit.
1
1
82-183.5 °C); H NMR (DMSO-d
6
) δ 8.97 (s, 1 H), 8.84 (s, 1
H), 6.06 (d, 1 H, J ) 5.1 Hz), 5.59 (d, 1 H, J ) 5.9 Hz), 5.28 (d,
H, J ) 5.1 Hz), 5.12 (t, 1 H, J ) 5.5 Hz), 4.58 (m, 1 H), 4.21
1
(
1
3
m, 1 H), 4.10 (m, 1 H), 3.5-3.8 (m, 2H); C NMR (DMSO-d
6
)
7
(H
(
1
a (2.25 g, 7.15 mmol, 72%): UV (H
2
2
O) λmax 214, 281 nm; UV
O) λmin 241 nm; H NMR (200 MHz, DMSO-d ) δ (ppm) 7.75
s, 1), 6.45 (d, 2, J ) 89.7 Hz), 5.62 (d, 1, J ) 5.8 Hz), 4.37 (m,
), 4.05 (m, 1), 3.84 (m, 1), 3.72 (s, 3), 3.54 (m, 2).
δ 151.8, 151.6, 149.4, 145.8, 131.4, 88.3, 85.8, 74.1, 70.1, 61.0;
1
+
37
+
+
35
+
6
MS (CI) 289 (M [ Cl] + H) , 287 (M [ Cl] + H) . Anal.
(
32) Christl, M.; Warren, J . P.; Hawkins, B. L.; Roberts, J . D. J .
Am. Chem. Soc. 1973, 95, 4392-4397.
33) Schulman, J . M.; Venanzi, T. J . Am. Chem. Soc. 1976, 98,
1
5
[
2 2
1,NH - N ]-Gu a n osin e (8a ). To 2.25 g (7.15 mmol) of 7a
(
dissolved in 150 mL of 0.1 M phosphate buffer (pH 7.4) was
added adenosine deaminase (430 units). The mixture was
shaken in an oven at 37 °C for 2 days, during which time the
product crystallized. The mixture was then cooled to 0 °C and
filtered, and the crude product was purified by recrystallization
4
701-4705.
(
(
34) Wasylishen, R. D. Can. J . Chem. 1976, 54, 833-839.
35) J iang, F.; Kumar, R. A.; J ones, R. A.; Patel, D. J . Nature
(
London) 1996, 382, 183-186.
36) J iang, F.; Fiala, R.; Live, D.; Kumar, R. A.; Patel, D. J .
Biochemistry 1996, 35, 13250-13266.
37) J iang, F.; Patel, D. J .; Zhang, X.; Zhao, H.; J ones, R. A. J .
Biomol. NMR 1997, 9, 55-62.
38) Zhang, X.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1997,
19, 6432-6433.
39) Zemlicka, J .; Owens, J . In Nucleic Acid Chemistry; Townsend,
(
from water to give 1.78 g of 8a (5.01 mmol, 70%): mp 239 °C
1
(
dec; H NMR (200 MHz, DMSO-d
6
) δ (ppm) 10.61 (d, 1, J )
88.5 Hz), 7.92 (s, 1), 6.44 (d, 2, J ) 89.5 Hz), 5.68 (d, 1, J )
(
5
.8 Hz), 5.37 (d, 1, J ) 5.9 Hz), 5.09 (d, 1, J ) 4.3 Hz), 5.02 (t,
1
(
L. B., Tipson, R. S., Eds.; Wiley-Interscience: New York, 1978; Vol. 2,
pp 611-614.
(40) Sober, H. A. Handbook of Biochemistry; 2nd ed.; The Chemical
Rubber Co.: Cleveland, Ohio, 1970.

Use of 13C To Differentiate Two 15N-Labeled Nucleosides
J . Org. Chem., Vol. 62, No. 22, 1997 7835
1
5
1
, J ) 5.0 Hz), 4.36 (m, 1), 4.07 (m, 1), 3.85 (m, 1), 3.56 (m, 2);
2 3
[1,7,NH - N ]-2-Am in o-6-(m eth oxya m in o)-9-(2-d eoxy-
1
3
1
C NMR ( H decoupled, 50.3 MHz, DMSO-d
d, J ) 10.7 Hz), 153.7 (d of d, J ) 23 Hz, J ) 13 Hz), 151.4,
35.6, 116.3 (d, J ) 7.9 Hz), 86.3, 85.2, 73.7, 70.4, 61.4; 15
) δ (ppm) 149.98 (d, J ) 70.2 Hz),
5.87 (t, J ) 88.5 Hz); EI MS m/z 285, 153, 135, 109.
6
) δ (ppm) 156.8
â-D-er yth r o-p en tofu r a n osyl)p u r in e (7c). The procedure for
7c was analogous to that for 7a , using 0.73 g (11.0 mmol) of
[ N]KCN, dissolved in 250 mL anhydrous methanol, 0.55 mL,
(10.6 mmol) of bromine, 2.11 g (7.85 mmol) of 4b, 40 mL of
(
1
15
N
NMR (40.543 MHz, DMSO-d
7
6
DMF, 4 mL of triethylamine, and 4 mL of CH
3
I (64.5 mmol),
to give 7c (1.77 g, 5.91 mmol, 75%): H NMR (200 MHz,
DMSO-d
1
3
15
1
[
2 2
2- C-1,NH - N ]-Gu a n osin e (8b). To 2.03 g (30 mmol)
1
5
13
of [ N, C]-KCN dissolved in 450 mL of anhydrous methanol
and cooled to 0 °C was added bromine (1.55 mL, 30 mmol).
After 3 h of stirring, 5.67 g (20.0 mmol) 4a was added. After
an additional 3 h, the reaction mixture was concentrated to
dryness. The residue was dissolved in a mixture of anhydrous
DMF (80 mL) and triethylamine (8 mL, 57.6 mmol) under N
The mixture was stirred at room temperature for 40 min, after
which 8 mL of CH I (129 mmol) was added. Stirring was
continued for a further 4 h in darkness, whereupon the
reaction mixture was concentrated to dryness and the residue
dissolved in 340 mL of 0.25 N NaOH. After 1 h, the pH was
adjusted to 7.4 with 1 N HCl. 95% Ethanol (400 mL) was then
added, and the mixture was heated at 60 °C for 4 h. The
mixture was then concentrated to a small volume and purified
by preparative reversed phase HPLC with a gradient of 2 to
6
) δ (ppm) 7.74 (d, 1, J N-H ) 12.0 Hz), 6.45 (d, 2, J N-H
) 89.2 Hz), 6.05 (t, 1, J ) 6.6 Hz), 5.20 (m, 2); 4.30 (m, 1),
3.77 (m, 1), 3.72 (s, 3), 3.50 (m, 2); 2.50 and 2.16 (m and m, 2).
1
5
2 3
[1,7,NH - N ]-2′-Deoxygu a n osin e (8c). To 1.77 g (5.91
mmol) of 7c dissolved in 120 mL of 0.1 M TEAA (pH 7.4) was
added adenosine deaminase (355 units). The mixture was
shaken in an oven at 37 °C for 5 days and then directly purified
by preparative reversed phase HPLC with a gradient of 0 to
12% acetonitrile in 0.1 M ammonium bicarbonate in 40 min
at a flow rate of 40 mL/min. Evaporation of appropriate
fractions gave 1.51 g of 8c (5.59 mmol, 95%): mp 245 °C dec;
H NMR (200 MHz, DMSO-d ) δ (ppm) 10.59 (d, 1, J N-H )
88.3 Hz), 7.90 (d, 1, J N-H ) 12.0 Hz), 6.43 (d, 2, J N-H ) 89.5
Hz), 6.10 (d, 1, J ) 6.2 Hz), 5.24 (d, 1, J ) 4.0 Hz) 4.93 (t, 1,
J ) 5.5 Hz), 4.32 (m, 1), 3.79 (m, 1), 3.85 (m, 1), 3.50 (m, 2),
2.49 and 2.20 (m and m, 2); 13C NMR ( H decoupled, 50.3 MHz,
2
.
3
1
6
1
1
4% acetonitrile in 0.1 M ammonium bicarbonate in 35 min
at a flow rate of 40 mL/min. Evaporation of appropriate
DMSO-d ) δ (ppm) 156.9 (d of d, J ) 10.8 Hz, J ) 6.3 Hz),
6
1
3
fractions gave 4.04 g (12.8 mmol, 64%) of pure [2- C-1,NH
2
-
153.7 (d of d, J ) 22.8 Hz, J ) 13.3 Hz), 150.9, 135.3, 116.7
1
5
15
N
2
]-2-amino-6-(methoxyamino)-9-(â-D-ribofuranosyl)purine
which was dissolved in 270 mL of 0.1 M phosphate buffer (pH
.4). Adenosine deaminase (770 units) was then added. The
(d, J ) 7.7 Hz), 87.6, 82.6, 70.8, 61.8; N NMR (40.543 MHz,
DMSO-d ) δ (ppm) 250. 81 (d, J N-H ) 12.2 Hz), 149.71 (s), 75.42
6
7
(t, J ) 91.6 Hz); EI MS m/z 270, 154, 136, 110.
13 15
mixture was shaken in an oven at 37 °C for 2 days, during
which time the product crystallized. The mixture was then
cooled to 0 °C and filtered. The crude product was purified
2 3
[2- C-1,7,NH - N ]-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 (7d ). The pro-
cedure for 7d was analogous to that for 7a , using 0.73 g (11.0
1
3
15
by recrystallization from water to give 2.63 g of 8b (9.13 mmol,
mmol) of [ C, N]-KCN, dissolved in 250 mL of anhydrous
methanol, 0.55 mL (10.6 mmol) of bromine, 2.11 g (7.85 mmol)
1
7
1
5
4
1%): mp 239 °C dec; H NMR (200 MHz, DMSO-d
6
) δ (ppm)
0.61 (d, 1, J ) 89.0 Hz), 7.92 (s, 1), 6.44 (d, 2, J ) 89.5 Hz),
3
of 4b, 40 mL of DMF, 4 mL of triethylamine, and 4 mL of CH I
1
.68 (d, 1, J ) 5.9 Hz), 5.38 (d, 1, J ) 6.2 Hz), 5.11 (d, 1, J )
(64.5 mmol), to give 7d (1.75 g, 5.83 mmol, 74%): H NMR
(200 MHz, DMSO-d ) δ (ppm) 7.71 (d, 1, J N-H ) 12.1 Hz), 6.49
.7 Hz), 5.02 (t, 1, J ) 5.0 Hz), 4.38 (m, 1), 4.07 (m, 1), 3.84
6
1
(
m, 1), 3.56 (m, 2); 13C NMR ( H decoupled, 50.3 MHz, DMSO-
(d, 2, J N-H ) 89.3 Hz), 6.04 (t, 1, J ) 6.2 Hz), 5.22 (m, 1), 4.97
(m, 1), 4.30 (m, 1), 3.76 (m, 1), 3.72 (s, 3), 3.50 (m, 2), 2.50 and
2.16 (m and m, 2).
d
6
) δ (ppm) 156.8 (d, J ) 10.7 Hz), 153.6 (d of d, J ) 22.6 Hz,
J ) 13.1 Hz), 151.4, 135.6, 116.3 (d, J ) 7.9 Hz), 86.3, 85.2,
1
5
13
15
7
1
J
3.7, 70.4, 61.4; N NMR (40.543 MHz, DMSO-d
6
) δ (ppm)
2 3
[2- C-1,7,NH - N ]-2′-Deoxygu a n osin e (8d ). The pro-
50.01 (d of d, J N-H ) 88.5 Hz, J N-C ) 12.2 Hz), 75.79 (t of d,
N-H ) 88.5 Hz, J N-C ) 24.4 Hz); EI MS m/z 286, 154, 136,
cedure for 8d was analogous to that for 8c using 1.75 g (5.83
mmol) of 7d dissolved in 120 mL of 0.1 M TEAA (pH 7.4), and
1
10.
350 units of adenosine deaminase to give 1.58 g of 8d (5.82
1
5
1
1
[
7,NH
2
- N
2
]-2′-Deoxya d en osin e N -Oxid e (4b). To 2.72
mmol, 99%): mp 245 °C dec; H NMR (200 MHz, DMSO-d
6
) δ
1
5
28
g (10.0 mmol) of [7,NH
2
- N
2
]-2′-deoxyadenosine hydrate, 3b,
(ppm) 10.59 (d, 1, J N-H ) 88.9 Hz), 7.90 (d, 1, J N-H ) 12.0
Hz), 6.43 (d, 2, J N-H ) 89.7 Hz), 6.10 (d, 1, J ) 6.2 Hz), 5.24
(d, 1, J ) 3.8 Hz), 4.93 (t, 1, J ) 5.5 Hz), 4.31 (m, 1), 3.79 (m,
dissolved in 400 mL of 50% aqueous methanol was added 6.67
g (38.6 mmol) of 3-chloroperoxybenzoic acid (MCPBA). The
mixture was stirred in the dark at room temperature for 3 h
and was then concentrated to give about 150 mL of a cloudy
mixture. It was washed with 2 × 200 mL of ethyl ether, and
the aqueous layer was immediately neutralized to pH 7 with
1
3
1), 3.85 (m, 1), 3.51 (m, 2); 2.49 and 2.20 (m and m, 2);
C
1
NMR ( H decoupled, 50.3 MHz, DMSO-d ) δ (ppm) 156.8 (d of
6
d, J ) 10.6 Hz and J ) 6.4 Hz), 153.6 (d of d, J ) 22.8 Hz and
J ) 13.3 Hz), 150.9, 135.3, 116.7 (d, J ) 5.5 Hz), 87.6, 82.6,
70.8, 61.8; 15N NMR (40.543 MHz, DMSO-d
) δ (ppm) 250.81
0
.25 M NaOH. The solution was then concentrated to a small
6
volume and purified by preparative reversed phase HPLC with
a gradient of 0 to 12% acetonitrile in water in 30 min at a
flow rate of 40 mL/min. Evaporation of appropriate fractions
(d, J N-H ) 12.2 Hz), 149.64 (d, J N-C ) 12.2 Hz), 75.42 (t of d,
N-H ) 88.5 Hz, J N-C ) 24.4 Hz); EI MS m/z 271, 155, 137,
111.
J
gave pure 4b (2.35 g, 8.74 mmol, 87%): mp 212 °C dec; UV
1
(
H
2
O) λmax 232, 262 nm; λmin 251 nm; H NMR (200 MHz,
DMSO-d
(
1
2
d
1
6
) δ (ppm) 8.62 (s, 1), 8.50 (d, 1, J N-H ) 12.1 Hz), 8.20
Ack n ow led gm en t. This work was supported by
grants from the National Institutes of Health (GM48802
and GM31483).
br, 2), 6.31 (t, 1, J ) 6.6 Hz), 5.34 (d, 1, J ) 4.0 Hz), 4.95 (t,
, J ) 5.2 Hz), 4.39 (m, 1), 3.85 (m, 1), 3.55 (m, 2), 2.69 and
3 1
.32 (m and m, 2); 1 C NMR ( H decoupled, 50.3 MHz, DMSO-
) δ (ppm) 148.3 (d of d, J ) 21.9 Hz, J ) 6.0 Hz), 143.3,
42.3, 141.6, 118.8, 88.0, 83.6, 70.6, 61.6.
6
J O971206U