5-HOMedC-containing oligodeoxynucleotides (ODNs) is need-
ed. The building block currently used for the synthesis of
5-HOMedC containing DNA strands necessitates a rather tedious
chemical synthesis via an unstable bromothymidine inter-
mediate.8,9 In addition, deprotection of the embedded
5-HOMedC unit in our hands required heating of the synthesized
oligonucleotides for 60 h at 60 °C with conc. ammonia,9
which prohibits any derivatization of the oligonucleotides
with fluorescence or biotin labels typically needed for many
biochemical experiments.
Scheme 1
.
Synthesis of the Phosphoramidite Building Block 6
(C* ) 5-HOMedC)
Here we report the development of a novel 5-HOMedC
phosphoramidite building block, available in just seven steps
from the stable and commercially available 5-iodo-deoxy-
cytidine 1. We found that the building block enables syn-
thesis of 5-HOMedC containing DNA strands using standard
phosphoramidite chemistry with excellent coupling yield.
Furthermore, deprotection of the new building block was
achieved with just dilute NaOH solution over 12 h at room
temperature.
We chose to protect the amino and hydroxy groups of the
5-HOMedC base as a cyclic carbamate. This group elegantly
inactivates both nucleophilic groups of 5-HOMedC and is one
of the smallest possible protective groups, therefore allowing
efficient coupling in the DNA synthesizer. Furthermore, it
can be easily deprotected simultaneously with cleavage of
the DNA strand from the resin by simple base treatment in
one step.
The synthesis is depicted in Scheme 1. The starting point
is 5-iododeoxycytidine 1,10 which was reacted with TBS-Cl
to protect the hydroxyl groups. The further synthesis can
alternatively be carried out without OH protection; however,
the yields of the following reactions turned out be lower,
and the purification is more tedious. To insert the hydroxy-
methylene group, we chose to utilize a Pd-catalyzed formy-
lation reaction with CO. This reaction turned out to be
extremely efficient even in the presence of the unprotected
exocyclic amino group, providing 2 in yields of above 95%.
We next reduced the obtained formyl group at C5 with
NaBH4 to obtain compound 3. For this step application of
Luche conditions is absolutely crucial.11 Without the addition
of CeCl3 the hydride presumably adds to the extremely
electrophilic C6 position of the base resulting in decomposi-
tion of the starting material 2. To introduce the cyclic
carbamate, compound 3 was treated with 4-nitrophenylchlo-
roformate12 to give the protected compound 4 in very good
yield. Subsequent deprotection of the silyl groups was
achieved with HF in pyridine. In ethylacetate as solvent, the
diol 5 precipitates after completion of the deprotection
allowing its isolation by simple centrifugation. We finally
converted 5 into the 5-HOMedC phosphoramidite building block
6 using standard procedures.13
DNA synthesis was performed using standard procedures.
To develop the appropriate deprotection method, we prepared
ODN1 (Scheme 1) using the phosphoramidite 6. Coupling
times with 6 were doubled to allow efficient incorporation
into the oligonucleotide chain. Initial attempts to deprotect
the strands with a standard protocol (conc. ammonia at room
temperature overnight) furnished oligonucleotides containing
5-HOMedC. However, the urea derivative 7 and the aminom-
ethyl-dC nucleobase 8 were formed as major byproducts
(Figure 2). To prevent these undesired side reactions, we
Figure 2. Nucleosides 7 and 8 were obtained after deprotection of
the oligonucleotides using standard NH3-based conditions. Depro-
tection with NaOH, however, yields exclusively 5-HOMedC.
(8) Shiau, G. T.; Schinazi, R. F.; Chen, M. S.; Prusoff, W. H. J. Med.
Chem. 1980, 23, 127–133
(9) Tardy-Planechaud, S.; Fujimoto, J.; Lin, S. S.; Sowers, L. C. Nucleic
Acids Res. 1997, 25, 553–558
.
.
(10) Hwang, C. H.; Park, J. S.; Won, J. H.; Kim, J. N.; Ryu, E. K.
Arch. Pharm. Res. 1992, 15, 69–72.
used 0.4 M NaOH in MeOH/H2O 4:1 as the deprotection
solution at room temperature overnight. Cleavage of the
DNA strand from the solid support and deprotection of all
(11) Luche, J. L. J. Am. Chem. Soc. 1978, 100, 2226–2227.
(12) Sammet, B. Synlett 2009, 3050–3051.
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Org. Lett., Vol. 12, No. 24, 2010