[6-15NH2]-2′,3′,5′-Tr i-O-a cetyl-N6-ben zoyla d en osin e (2a *).
The above general procedure was followed using 6-bromo-9-
(2′,3′,5′-tri-O-acetyl-â-D-ribofuranosyl)-9H-purine13 (1a , 55 mg,
0.12 mmol) and [15N]-benzamide.14 After 3 h at 80 °C, the
purification of the crude product by flash chromatography gave
5 mg (9%) of starting material 1a and 44 mg (74%) of [6-15NH2]-
performed by reaction of 4* with NBS in the presence of
HMPT and LiBr.13 Pd-catalyzed addition of 1.1 equiv of
labeled benzamide14 to the labeled 6-bromopurine 1a *
afforded the double-labeled adenosine 2a ** in 74% yield
which was deacetylated to the deprotected double-labeled
N-benzoyladenosine 5**. The spectroscopic data of 5**
showed a doublet at δ -101.9 (2J NH ) 15 Hz) and a
doublet at δ -230.8 (1J NH ) 85 Hz) in the proton-coupled
15N NMR spectrum;16 the splitting of C6 (dd, 1J CN ) 18.4
2′,3′,5′-tri-O-acetyl-N6-benzoyladenosine (2a *) as
a colorless
oil: 1H NMR (CDCl3, 400 MHz) δ 9.08 (d, J ) 88.4 Hz, 1H),
8.80 (s, 1H), 8.19 (s, 1H), 8.03 (d, J ) 7.2 Hz, 2H), 7.63-7.51
(m, 3H), 6.27 (d, J ) 5.5 Hz, 1H), 5.96 (t, J ) 5.5 Hz, 1H), 5.68
(dd, J ) 5.5, 4.3 Hz, 1H), 4.50-4.38 (m, 3H), 2.16 (s, 3H), 2.13
(s, 3H), 2.09 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 170.3, 169.5,
169.3, 164.6 (d, J ) 13.0 Hz), 152.9 (d, J ) 2.3 Hz), 151.7, 149.7
(d, J ) 19.9 Hz), 141.2, 133.4 (d, J ) 9.2 Hz), 132.8, 128.8, 127.9,
123.6 (d, J ) 2.3 Hz), 86.3, 80.5, 73.1, 70.6, 63.0, 20.7, 20.5, 20.3;
15N NMR (CDCl3, 30 MHz) δ -236.2 (d, J ) 88 Hz); HRMS
(FAB) calcd for C23H24N415NO8 (M + H)+ 499.1595, found
499.1579.
1
Hz, J CN ) 4.6 Hz) in the 13C NMR spectrum confirmed
the incorporation of the second label.
In conclusion, we have developed a simple procedure
for the introduction of amides in position 6 of purines.
The overall process can be used to prepare directly 15N-
labeled adenosines protected as amides.
[1-15N,6-15NH 2]-2′,3′,5′-Tr i-O-a ce t yl-N 6-b e n zoyla d e n o-
sin e (2a **). The above general procedure was followed using
[1-15N]-6-bromo-(2′,3′,5′-tri-O-acetyl-â-D-ribofuranosyl)-9H-pu-
rine (1a *, 56 mg, 0.122 mmol) and [15N]benzamide.14 After 3 h
at 80 °C, the purification of the crude product by flash chroma-
tography gave 45 mg (74%) of [1-15N,6-15NH2]-2′,3′,5′-tri-O-acetyl-
N6-benzoyladenosine (2a **) as a colorless oil: 1H NMR (CDCl3,
400 MHz) δ 9.09 (dd, J ) 88.8, 2.0 Hz, 1H), 8.80 (d, J ) 15.8
Hz, 1H), 8.19 (s, 1H), 8.03 (d, J ) 7.2 Hz, 2H), 7.63-7.51 (m,
3H), 6.26 (d, J ) 5.5 Hz, 1H), 5.96 (t, J ) 5.5 Hz, 1H), 5.68 (dd,
J ) 5.5, 4.3 Hz, 1H), 4.49-4.37 (m, 3H), 2.17 (s, 3H), 2.13 (s,
3H), 2.09 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 170.3, 169.5,
169.3, 164.6 (d, J ) 13.0 Hz), 152.9 (dd, J ) 2.7, 2.3 Hz), 151.7
(dd, J ) 2.3, 1.2 Hz), 149.7 (dd, J ) 19.9, 5.2 Hz), 141.2, 133.4
(d, J ) 9.2 Hz), 132.8, 128.8, 127.9, 123.6 (dd, J ) 3.1, 2.3 Hz),
86.3, 80.5, 73.1, 70.6, 63.0, 20.7, 20.5, 20.3; 15N NMR (CDCl3,
30 MHz) δ -236.1 (dd, J ) 89, 4 Hz), -114.0 (dd, J ) 16, 4 Hz);
HRMS (FAB) calcd for C23H24N315N2O8 (M + H)+ 500.1566, found
500.1556.
Exp er im en ta l Section
For general methods, see ref 15. Compound 1a ′ was prepared
according to ref 17. Coupling constants (J ) are given in Hz. 15N
NMR chemical shifts are referred to external concentrated H15
-
NO3 (negative values upfield). HRMS were registered in the FAB
positive mode.
[1-15N]-6-Br om o-9-(2′,3′,5′-tr i-O-a cetyl-â-D-r ibofu r a n osyl)-
9H-p u r in e (1a *). To a solution of [1-15N]-2′,3′,5′-tri-O-acetyli-
nosine15 (4*, 125 mg, 0.32 mmol) and N-bromosuccinimide (169
mg, 0.95 mmol) in CH3CN (5 mL) at -20 °C was added dropwise
hexamethylphosphorous triamide (HMPT, (Me2N)3P) (144 µL,
0.79 mmol). The reaction mixture was stirred at room temper-
ature for 0.5 h. Afterward, LiBr (137 mg, 1.58 mmol) was added,
and the reaction mixture was heated at 70 °C for 5 h. The brown
mixture was cooled to room temperature, and the volatile
materials were removed by rotatory evaporation. Purification
of the crude product by flash chromatography (CH2Cl2-MeOH
98:2) afforded 114 mg (79%) of [1-15N]-6-bromo-9-(2′,3′,5′-tri-O-
acetyl-â-D-ribofuranosyl)-9H-purine (1a *) as
a pale yellow
[1-15N,6-15NH2]-N6-Ben zoyla d en osin e (5**). To a solution
of [1-15N,6-15NH2]-2′,3′,5′-tri-O-acetyl-N6-benzoyladenosine (2a **,
36 mg, 0.072 mmol) in ethanol-pyridine 1:1 (v/v, 439 µL), a
mixture of 2 N NaOH and ethanol (299 µL + 299 µL) was added.
After the mixture was stirred for 6 min at room temperature,
Amberlite IR-120 was added to neutralize the base. The resin
was filtered and washed with ethanol (1 mL) and pyridine (1
mL). Combined filtrates were evaporated, and the crude product
was purified by flash chromatography (CH2Cl2-MeOH 80:20)
to give 27 mg (99%) of [1-15N,6-15NH2]-N6-benzoyladenosine (5**):
1H NMR (DMSO-d6, 400 MHz) δ 11.17 (d, J ) 84.6 Hz, 1H), 8.76
(d, J ) 15.4 Hz, 1H), 8.72 (s, 1H), 8.05 (d, J ) 7.6 Hz, 2H), 7.68-
7.53 (m, 3H), 6.05 (d, J ) 6.0 Hz, 1H), 5.55 (d, J ) 5.7 Hz, 1H),
5.25 (d, J ) 3.9 Hz, 1H), 5.13 (t, J ) 5.7 Hz, 1H), 4.66 (m, 1H),
4.20 (m, 1H), 3.99 (m, 1H), 3.74-3.55 (m, 2H); 13C NMR (DMSO-
d6, 100 MHz) δ 165.7 (d, J ) 13.0 Hz), 152.2 (dd, J ) 2.7, 1.2
Hz), 151.6, 150.4 (dd, J ) 18.4, 4.6 Hz), 143.1, 133.4 (d, J ) 9.2
Hz), 132.4, 128.5, 125.9 (dd, J ) 3.1, 1.5 Hz), 87.6, 85.7, 73.7,
70.4, 61.3; 15N NMR (DMSO-d6, 30 MHz) δ -230.8 (broad d, J
) 85 Hz),16 -101.9 (broad d, J ) 15 Hz); HRMS (FAB) calcd for
foam: 1H NMR (CDCl3, 300 MHz) δ 8.73 (d, J ) 15.9 Hz, 1H),
8.32 (s, 1H), 6.23 (d, J ) 5.1 Hz, 1H), 5.96 (dd, J ) 5.2, 5.1 Hz,
1H), 5.65 (dd, J ) 5.2, 4.4 Hz, 1H), 4.50-4.37 (m, 3H), 2.16 (s,
3H), 2.12 (s, 3H), 2.09 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 170.2,
169.5, 169.3, 152.2 (d, J ) 4.6 Hz), 149.9 (d, J ) 2.7 Hz), 143.7
(d, J ) 2.1 Hz), 143.4, 134.9 (d, J ) 2.7 Hz), 86.9, 80.5, 73.1,
70.4, 62.8, 20.7, 20.5, 20.3; 15N NMR (CDCl3, 30 MHz) δ -61.1
(d, J ) 16 Hz); HRMS (FAB) calcd for C16H18BrN315NO7 (M +
H)+ 458.0329, 460.0309, found 458.0325, 460.0319.
Gen er a l P r oced u r e for th e Rea ction of 6-Br om op u r in e
Nu cleosid es w ith Ben za m id e. An oven-dried vial was charged
with Pd2dba3‚CHCl3 (0.05 mmol, 10 mol % Pd) and 1,1′-bis-
(diphenylphosphino)ferrocene (dppf, 0.15 mmol, 1.5 equiv/Pd).
The vial was capped with a rubber septum, and a solution of
nucleoside (1.00 mmol) in toluene (3.0 mL) was added via
cannula. Then, benzamide (1.10 mmol) and Cs2CO3 (1.40 mmol)
were added as solids, and the resulting yellow mixture was
stirred at 80 °C. The reaction was monitored by thin-layer
chromatography. After complete consumption of the starting
nucleoside, the resulting brown suspension was allowed to cool
to room temperature and concentrated in vacuo. The crude
material was adsorbed onto silica gel and purified by flash
chromatography (hexanes-EtOAc 25:75 and later CH2Cl2-
MeOH from 99:1 to 98:2).
C
17H17N315N2NaO5 (M + Na)+ 396.1068, found 396.1065.
Ack n ow led gm en t. This work was supported by
funds from Spanish MCYT (BQU2000-0647) and Gen-
eralitat de Catalunya (2000SGR021, 2001SGR51).
(15) The key intermediate is 2′,3′,5′-triacetyl-1-nitroinosine: (a)
Ariza, X.; Bou, V.; Vilarrasa, J . J . Am. Chem. Soc. 1995, 117, 3665-
3673. For the use of N-nitronucleobases in 15N-labeling, see also: (b)
Ariza, X.; Bou, V.; Vilarrasa, J .; Tereshko, V.; Campos, J . L. Angew.
Chem., Int. Ed. Engl. 1994, 33, 2454-2455. (c) Ariza, X.; Farra`s, J .;
Serra, C.; Vilarrasa, J . J . Org. Chem. 1997, 62, 1547-1549. (d) Ariza,
X.; Vilarrasa, J . J . Org. Chem. 2000, 65, 2827-2829.
(16) This last splitting was only observed in dilute samples. In
concentrated samples, fast proton exchanges prevented to see this
splitting.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization data of compounds 2b, 2c, 3a ,
1
and 5*. H, 13C and 15N NMR spectra for all compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Zemlicka, J .; Owens, J . Nucl. Acid Chem. 1978, 2, 611-614.
J O049490U
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