during the capping process. Synthesis (1 µmol) was carried
out on the photolabile linker previously reported by us on
polystyrene support (16) using automated synthesis cycles
similar to those reported by Scaringe in his paper introducing
2′-ACE technology for the synthesis of RNA.3,10 The
oligonucleotide was deprotected in three steps. First, dem-
ethylation of the phosphate triesters was accomplished using
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihy-
drate (0.2 M, 15 min, 25 °C).3,11 The resin was washed
following filtration. The washings were combined with the
excess reagents, evaporated to dryness, and treated with
concentrated aqueous ammonia. Following demethylation,
the resin was treated with Et3N in DMF (1:1 by volume, 16
h, 55 °C, eight or more half-lives established in studies on
the monomers; see Table 1) in order to cleave the exocyclic
amine-protecting groups. Again, the excess reagents and
solvent obtained from washing of the resin were combined
and evaporated to dryness. Finally, the fully deprotected,
resin-bound oligonucleotide was cleaved from the solid-phase
support via photolysis using a transilluminator (365 nm, 3
h).10 Following collection of the solution and washing of
the resin, this concentrated sample, as well as the washings
from the previous two steps, was purified by denaturing
polyacrylamide gel electrophoresis. Trace amounts of DNA
were observed in the concentrated washings of the dem-
ethylation reaction, and none was detected in the respective
washings obtained following cleavage of the exocyclic
amines. The remainder of the oligonucleotide (17) was
obtained from the sample subjected to all three deprotection
steps, indicating that the carbonate linkage was stable to the
exocyclic amine and phosphate-deprotection steps. Com-
parison of the yield of DNA obtained from photolysis to an
equivalent sample, which was cleaved from the resin using
concentrated aqueous ammonia, indicated that the photolysis
proceeded in 67% yield. Analysis of the two samples of DNA
by ESI-MS indicated that the photolytically cleaved material,
which migrated as a single band in the gel, was a mixture
of the desired product and modifications of this biopolymer
containing between one and four acetyl groups (data not
shown). The acetyl groups were assumed to result from the
capping process, as observed above for the reaction of the
carbamate protected monomers.
Scheme 3a
a Key: (a) sec-BuLi, DMPU, phenylacetyl chloride; (b) TFA,
H2O, THF; (c) Et3N‚3HF, THF.
analogue) were tested separately for their stability to the
desilylation (anhydrous Et3N‚3HF, DMF), oxidation (t-
BuOOH), and capping reagents. Exposure of the silyl
protected â-cyano-R,R-dimethyl-ethyl of deoxyadenosine to
acetic anhydride, 2,6-lutidine, and N-methylimidazole (stan-
dard capping reagents employed in oligonucleotide synthesis)
resulted in a slow transformation into the respective N-acetyl
derivative (65% in 15 h). Removal of N-methylimidazole
from the reaction mixture eliminated this decomposition
reaction.
Given that the substitution of the acetyl group for the
carbamate was significantly slower than the capping proce-
dure during automated synthesis, we attempted to synthesize
an oligonucleotide (17, Figure 2) utilizing N-methylimidazole
Subsequently, synthesis of 18 was carried out omitting
N-methylimidazole in the capping process. The three-step
deprotection procedure was carried out as described above.
Less efficient capping was evident in the purification gel,
which revealed the presence of greater amounts of deletion
products close in size to the full-length product. However,
the product isolated from this gel was pure, as determined
by ESI-MS analysis, and did not exhibit any evidence of
N-acetylation (Figure 3). The suitability of the method was
further demonstrated by the syntheses of 19, which also was
obtained as a single product (Figure 4). No evidence for
nucleophilic trapping of acrylonitrile or the more hindered
(10) Venkatesan, H.; Greenberg, M. M. J. Org. Chem. 1996, 61, 525.
The alkylamine-derivatized polystyrene support was manufactured by
Amersham Pharmacia Biotech.
(11) Dahl, B. J.; Bjergarde, K.; Henriksen, L.; Dahl, O. Acta Chem.
Scand. 1990, 44, 639.
Figure 2. Solid-phase support and oligonucleotides prepared in
this study.
Org. Lett., Vol. 2, No. 23, 2000
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