Communications
J . Org. Chem., Vol. 62, No. 20, 1997 6713
to thymidylyl-(3′f5′)-thymidine (TpT) 12 which was
identical to TpT prepared by conventional 2-cyanoethyl
phosphoramidite chemistry. The deprotection kinetics
of the (N-trifluoroacetylamino)butyl phosphate protecting
The (N-trifluoroacetylamino)butyl and (N-trifluoro-
acetylamino)pentyl phosphate protecting groups reported
herein can serve as alternatives to the traditional 2-cya-
noethyl phosphate protecting group in the solid-phase
synthesis of oligonucleotides and may be superior in
specific circumstances. Since the (N-trifluoroacetylami-
3
1
group from the dimer 6 has been evaluated by P-NMR
spectroscopy.6 The NMR data are consistent with an
initial rate-limiting cleavage of the N-trifluoroacetyl
group, followed by a rapid cyclodeesterification of the
intermediate 8 to produce pyrrolidine and the dinucleo-
side phosphodiester 11. The cyclodeesterification step
proceeded so readily that only a very small accumulation
of 8 was detected during the first 30 min of the depro-
tection reaction. Complete conversion of 6 to the dinucle-
otide 11 occurred within 2 h at 25 °C.6 Interestingly,
deprotection of the dimer 7 under the conditions used for
4
no)butyl group can be removed by concd NH OH at
ambient temperature, this phosphate protecting group
could be as effective as the 2-cyanoethyl group in the
synthesis of oligonucleotides carrying base-sensitive nu-
cleobases. In addition, the (N-trifluoroacetylamino)butyl
or the (N-trifluoroacetylamino)pentyl phosphate protect-
ing group may be useful in the synthesis of oligoribo-
nucleotides when the 5′-hydroxy function is protected
with groups such as the (fluoren-9-ylmethoxy)carbonyl
(Fmoc)11 or the (2-dansylethoxy)carbonyl (Dnseoc) group.
The stepwise removal of these groups is typically carried
out under non-nucleophilic basic conditions (0.1 M DBU
in MeCN) which are more compatible with the (N-
trifluoroacetylamino)alkyl phosphate protecting groups
than the 2-cyanoethyl group. Another potential limita-
tion of the 2-cyanoethyl group for phosphate protection
is the release of acrylonitrile during its deprotection.
Depending on the base used, addition of acrylonitrile to
12
5
gave the dithymidine (5-aminopentyl) phosphate tri-
ester 10 as a major product (ca. 90%). Complete depro-
tection of the dinucleotide 7 to TpT required treatment
o
with concd NH
4
OH at 56 C for 2 h. No evidence of
internucleotidic phosphodiester cleavage was detected
under the conditions used for the deprotection of either
5
or 7. Phosphoramidites 2a , 2b, and 3 have also been
applied to the solid phase synthesis of octadecathymidylic
dT18) and octadecadeoxycytidylic (dC18) acids using
(
1
3
standard protocols. These polynucleotides were cleaved
from their respective CPG support upon treatment with
thymine/uracil at N-3 has been well-documented. By
comparison, the release of pyrrolidine or piperidine
during the deprotection of the (N-trifluoroacetylami-
no)butyl or (N-trifluoroacetylamino)pentyl phosphate
protecting group is quite innocuous.
concd NH
was further treated with concd NH
4
OH for 2 h at 25 °C. The oligodeoxycytidylate
OH at 55 °C (10 h)
4
to ensure complete nucleobase deprotection. The overall
synthetic quality and recovery of dT18 and dC18 were
The strategy we have outlined for the protection of
phosphodiesters is not limited to the use of the trifluo-
roacetyl group for masking the amino function of the
comparable to that obtained by the use of 2-cyanoethyl
phosphoramidites.6 These crude oligonucleotides were
4
-aminobutyl and 5-aminopentyl groups. Other groups
completely hydrolyzed by snake venom phosphodi-
esterase (SVP) and bacterial alkaline phosphatase (BAP)
to their corresponding nucleosides. Neither thymine or
cytosine modification nor incomplete deprotection of
requiring very unique deprotection conditions may con-
1
4
ceivably be employed. In this context, the Fmoc and
7
the (benzyloxy)carbonyl groups have been applied to the
protection of an aminoethyl phosphotriester function.
Thus, the application of N-protected 4-aminobutyl or
N-protected 5-aminopentyl groups to phosphate protec-
tion in oligonucleotide synthesis may potentially provide
a much greater versatility in deprotection conditions than
currently possible with the conventional 2-cyanoethyl
group.
phosphotriester functions were detected by RP-HPLC
analysis of the hydrolysates.6 These results also suggest
that conventional nucleobase protecting groups do not
interfere with deprotection of the (N-trifluoroacetylami-
no)butyl phosphotriester function.
In an effort to detect the formation of pyrrolidine or
piperidine during the deprotection of (N-trifluoroacety-
lamino)butyl- or (N-trifluoroacetylamino)pentyl phos-
phate protecting groups, the non-nucleosidic phosphot-
riesters 13 and 14 were prepared, and each treated with
deuterium oxide saturated with ammonia until complete
cyclodeesterification to O,O-diethyl phosphoric acid di-
ester was observed by 31P-NMR spectroscopy. The gen-
eration of pyrrolidine and piperidine during the depro-
tection of 13 and 14, respectively, was unambiguously
confirmed by 1H-NMR spectroscopy.6 These findings
strongly support the cyclodeesterification mechanism
depicted in Scheme 1 and are consistent with related
literature data.7
The synthetic approach to the phosphoramidites 2a ,
2
b, and 3 is being extended to the other nucleobases, and
N-protecting groups other than trifluoroacetyl for the
-aminobutyl function will be tested in the solid phase
4
synthesis of oligonucleotides. Results of these findings
will be reported in due course.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of
1
-3, 13, and 14; mechanistic study of phosphate deprotection
using 13 and 14 as models; comparative polyacrylamide gel
electrophoresis of dT18 that has been prepared from 2a , 3, and
the parent 2-cyanoethyl phosphoramidite; and HPLC analysis
of SVP and BAP hydrolysates of unpurified dT18 and dC18 (13
pages).
-10
J O970962X
(
9) Murahashi, S.-I.; Kondo, K.; Hakata, T. Tetrahedron Lett. 1982,
2
3, 229-232.
(
10) Hendrickson, J . B.; Hussoin, M. S. Synlett 1990, 423-424.
(11) Lehmann, C.; Xu, Y.-Z.; Christodoulou, C.; Tan, Z.-K.; Gait, M.
J . Nucl. Acids Res. 1989, 17, 2379-2390.
12) Bergmann, F.; Pfleiderer, W. Helv. Chim. Acta 1994, 77, 203-
15.
(
2
(
(
(
6) Data shown as Supporting Information.
7) Brown, D. M.; Osborne, G. O. J . Chem. Soc. 1957, 2590-2593.
8) Denney, D. B.; Powell, R. L.; Taft, A.; Twitchell, D. Phosphorus
(13) Chambers, R. W. Biochemistry 1965, 4, 219-226; see also
Ogilvie, K. K.; Beaucage, S. L. Nucl. Acids Res. 1979, 7, 805-823.
(14) Seliger, H.; Krist, B.; Berner, S. Nucleosides Nucleotides 1991,
10, 303-306.
1
971, 1, 151.