4498
D. Zielinska et al. / Tetrahedron Letters 47 (2006) 4495–4499
6. Phosphoramidites 1a,g,c,t were thoroughly characterized
by an appropriate NMR spectroscopy methods, mass
spectrometry and RP HPLC analysis; for 1t: ESI MS,
M+H+ 684.54; 31P NMR, ppm, (acetonitrile-d3), 149.39,
149.26, Rp/Sp-isomers; for 1a: ESI MS, M+H+ 797.55;
31P NMR, ppm, (acetonitrile-d3), 149.03, 148.64, Rp/Sp-
isomers; for 1g: ESI MS, M+H+ 779.33; 31P NMR, ppm,
(acetonitrile-d3),149.30,148.60, Rp/Sp-isomers; for 1c: ESI
MS, M+H+ 773.56; 31P NMR, ppm, (acetonitrile-d3),
149.48, 149.17, Rp/Sp-isomers.
yield via crystallization from dichloromethane/aceto-
nitrile. Moreover, it appears that the quality of iBu–Cl
plays an important role in achieving good yield of the
product. The presence of products of hydrolysis of
iBu–Cl in the reaction mixture during the N2-protection
reaction led to formation of significant amounts (up to
50%) of
a
major by-product: 30-NH-iBu-N2-Tr–
guanosine.10
7. Representative 30-NH-tritylation and base-protecting
procedures. 30-NH-Tr-30-dT: 30-amino-30-deoxythymidine
(1.3 g, 5.4 mmol) was co-evaporated and then dissolved in
30 ml of anhydrous pyridine and 4.6 ml of diisopropyl-
ethylamine. After 10 min of stirring Tr–Cl (1.5 g,
5.4 mmol) was added and the reaction mixture was left
overnight. Following the disappearance of the starting
material (TLC control), the reaction was quenched with
methanol and concentrated in vacuo. The obtained oil was
dissolved in methylene chloride, washed with satd sodium
bicarbonate, dried over sodium sulfate, concentrated in
vacuo, and precipitated from methylene chloride-hexane
as a white powder product (2.2 g , 85% yield). 30-NH-Tr-
20,30-ddG:30-amino-20,30-ddG, 5 g (18.8 mmol), was co-
evaporated (3·) with dry pyridine and suspended in 300 ml
of anhydrous DMF; 2.6 ml (18.8 mmol) of TEA was
added and the reaction mixture was heating at 50ꢁC in an
oil bath for 30 min while stirring. Then 5.24 g (18.8 mmol)
of Tr–Cl was added and the reaction mixture was stirred
for 1 h at rt. After the disappearance of the starting 30-
amino nucleoside (by TLC analysis; more Tr–Cl can be
added if the starting material is still present) the reaction
mixture was poured into ꢀ300 ml of water and placed into
the freezer (ꢁ18ꢁC) for 2–3 h. The desired product was
filtered-off as a white solid. This solid was re-crystallized
from hot pyridine and acetonitrile (7:3, v/v,) to obtain
essentially pure 30-NH-Tr-20,30-ddG (93% yield). N2-iBu-
30-NH-Tr-20,30-ddG via N,O-peracylation: 5 g (9.8 mmol)
30-NH-Tr-20,30-ddG was co-evaporated (2·) with pyri-
dine, suspended in 200 ml of pyridine and cooled in an ice
bath; 1.13 ml (10.8 mmol) iBu–Cl was added to 8 ml of
pyridine and the reaction mixture was stirred for 2 h (TLC
control). After the disappearance of the starting material
the reaction mixture was poured into 100 ml of ice cold aq
Na-bicarbonate, and extracted with a 200 ml portion (2·)
of ethyl acetate. The organic layer was washed with water
and concentrated in vacuo to a yellow gum. The gum was
dissolved in 50–100 ml of ethanol on an ice bath and an
equal volume of ice-cold 1 M sodium hydroxide in water/
ethanol, 1:1 (v/v) was added. The reaction mixture was
stirred for 20–30 min and 1.2 equiv of ammonium chloride
was added. The mixture was poured into 200 ml of aq
sodium bicarbonate and extracted (2·) with 200 ml of
ethyl acetate. The combined organic layers were dried over
sodium sulfate and evaporated in vacuo to a yellow solid,
which was then crystallized from hot acetonitrile to obtain
a white solid product (3 g, 53% yield). N2-iBu-30-NH–Tr-
20,30-ddG via TMS-based transient protection: 5 g
(9.8 mmol) 30-NH-Tr-20,30-ddG was co-evaporated (2·)
with pyridine, suspended in 200 ml of pyridine and cooled
in an ice bath. 6.3 ml (49 mmol) of TMS–Cl was slowly
added to the reaction mixture. After 30 min, 5.1 ml
(49 mmol) of iBu–Cl in pyridine was added and the
reaction mixture was removed from the ice bath and
stirred for 2 h (TLC control). The mixture was chilled in
the ice bath and then 20 ml of cold water followed, in
15 min, by the addition of 20 ml of concentrated aqueous
ammonia were added, and stirred for an additional
30 min. The reaction mixture was concentrated to oil
Preparation of phosphoramidite 1c was conducted in a
similar fashion (Scheme 4). First, 30-amino group of
30-amino-20, 30-dideoxycytidine was regio-selectively pro-
tected with trityl chloride in pyridine and DMF mixture,
1:4 (v/v), in presence of triethylamine (4 M equiv).
Second, we sought to utilize the high nucleophilicity of
the exocyclic N4-amino group of 30-amino cytidine,
relative to the Ade and Gua counterparts. Hence, N4-
amino group was protected with benzoic anhydride in a
mixture of acetonitrile and methanol, 9:1 (v/v), at 50 ꢁC.
This reaction results in the formation of N4-Bz-30-NH-
Tr-50-OH–cytidine precursor as the predominant prod-
uct, with an isolated yield of ꢀ70%, based on the starting
30-amino nucleoside. Importantly, no significant 50-O-
benzoylation (est. <5%) was observed under the reaction
conditions used. Finally, standard 50-O-phosphitylation
produced the desirable phosphoramidite 1c.6,7 This new
30-amino nucleoside-based procedure for preparation of
1c reduces the total number of chemical steps (from seven
to three) with significant increase in the process efficiency
and overall yield of final product (16% cf. 60%).11
In conclusion, we report an efficient and simple method
for preparation of 30-aminonucleoside-50-phosphor-
amidites, the key building blocks used for assembly of
oligonucleotide N30 ! P50 phosphoramidates and thio-
phosphoramidates, which are currently under clinical
development as potential therapeutic agents.
References and notes
1. Herbert, B.-S.; Pongracz, K.; Shay, J. W.; Gryaznov, S.
M. Oncogene 2002, 21, 638–642.
2. Ford, L. P.; Zou, Y.; Pongracz, K.; Gryaznov, S. M.;
Shay, J. W.; Wright, W. E. J. Biol. Chem. 2001, 276,
32198–32203; Gryaznov, S. M. Biochem. Biophys. Acta
1999, 1489, 131–140.
3. Nelson, J. S.; Fearon, K.; Nguyen, M. Q.; McCurdy, S.
N.; Frediani, J. F.; Foy, M. F.; Hirschbein, B. L. J. Org.
Chem. 1997, 62, 7278–7287.
4. The 30-amino purine nucleosides: 30-amino-20, 30-dideoxy-
adenosine and 30-amino-20, 30-dideoxyguanosine were
acquired from Metkinen Oy (Finland). These compounds
were prepared by an enzymatic trans-glycosylation process
starting from 30-amino-30-deoxythymidine, which was
obtained via reduction of readily available 30-azido-30-
deoxythymidine (AZT). 30-Amino-20,30-dideoxycytidine
was prepared as described: Glinski, R. P.; Khan, S. M.;
Kalamas, R. L.; Stevens, C. L. J. Chem. Soc. D 1970, 915–
916.
5. N- versus O-selective tritylation of aliphatic non-nucleo-
side-based amino alcohols was previously reported; for
example: Buckus, P.; Saboniene, R.; Lemesiene, D. Zh.
Org. Khimi (Russian) 1970, 6, 1984–1987.