Photocleavable protecting groups as nucleobase protections allowed the solid-phase synthesis of base-sensitive SATE-prooligonucleotides

Article abstract of DOI:10.1021/jo990479h

The first synthesis of oligodeoxynucleotide heteropolymers carrying base-sensitive S-pivaloylthioethyl (t-Bu-SATE) phosphotriester linkages has been performed. It is based on the use of 6-nitroveratryloxycarbonyl (NVOC) and 2,2'-bis(2-nitrophenyl)ethoxycarbonyl (diNPEOC) groups as nucleobase protections in combination with photolysis deprotection. The synthesis was realized using the phosphoramidite approach on solid support bearing a 1-(o- nitrophenyl)-1,3-propanediol linker. The removal of the protecting groups and the cleavage of the oligonucleotides from the solid support were accomplished in a single photolysis procedure upon UV irradiation at wavelengths > 300 nm. Faster deprotection rates were observed for diNPEOC-protected nucleosides and oligomers than with NVOC-protected ones. The synthesis of pentanucleoside t- Bu-SATE-phosphotriesters d((5')TpCpCpCpTp(3')), d((5')TpApApApAp(3')), and d((5')TpGpGpGpTp(3')) and of dodecanucleoside t-Bu-SATE-phosphotriesters and -phosphorothioate d((5')ApCpApCpCpCpApApTpTpCpTp(3')) and d((5')ApGpApApTpTpGpGpGpTpGpTp(3')) demonstrated the efficiency of the method.

Full text of DOI:10.1021/jo990479h

J . Org. Chem. 1999, 64, 6319-6328
6319
P h otoclea va ble P r otectin g Gr ou p s a s Nu cleoba se P r otection s
Allow ed th e Solid -P h a se Syn th esis of Ba se-Sen sitive
SATE-P r ooligon u cleotid es
Karine Alvarez, J ean-J acques Vasseur,* Thierry Beltran, and J ean-Louis Imbach
Laboratoire de Chimie Bio-Organique, UMR 5625 CNRS-UM II, Universite´ Montpellier II,
Place E. Bataillon, 34095 Montpellier Cedex 5, France
Received March 17, 1999
The first synthesis of oligodeoxynucleotide heteropolymers carrying base-sensitive S-pivaloyl-
thioethyl (t-Bu-SATE) phosphotriester linkages has been performed. It is based on the use of
6-nitroveratryloxycarbonyl (NVOC) and 2,2′-bis(2-nitrophenyl)ethoxycarbonyl (diNPEOC) groups
as nucleobase protections in combination with photolysis deprotection. The synthesis was realized
using the phosphoramidite approach on solid support bearing a 1-(o-nitrophenyl)-1,3-propanediol
linker. The removal of the protecting groups and the cleavage of the oligonucleotides from the solid
support were accomplished in a single photolysis procedure upon UV irradiation at wavelengths
>300 nm. Faster deprotection rates were observed for diNPEOC-protected nucleosides and oligomers
than with NVOC-protected ones. The synthesis of pentanucleoside t-Bu-SATE-phosphotriesters
d(5′TpCpCpCpTp3′), d(5′TpApApApAp3′), and d(5′TpGpGpGpTp3′) and of dodecanucleoside t-Bu-SATE-
phosphotriesters and -phosphorothioate d(5′ApCpApCpCpCpApApTpTpCpTp3′) and d(5′ApGpAp-
ApTpTpGpGpGpTpGpTp3′) demonstrated the efficiency of the method.
In tr od u ction
The natural affinity and the specificity of hybridization
of antisense oligonucleotides for nucleic acid targets have
opened perspectives of new therapies for the treatment
of various diseases.1,2 However, the effectiveness of
oligonucleotides is limited because of their instability in
serum, their inability to reach their target, their poor
cellular uptake, and adverse pharmacokinetics. These
limitations are mainly due to the anionic internucleo-
sidic linkages of phosphodiester oligonucleotides and of
their phosphorothioate analogues. To circumvent these
problems, we have proposed an approach wherein the
negative charges of oligonucleotides are temporarily
neutralized with enzymolabile protecting groups to form
neutral oligonucleotide prodrugs (prooligonucleotide) with
phosphotriester linkages.3 We have shown that thymidy-
late models with internucleoside phosphodiesters or
phosphorothioates protected with S-acylthioethyl (SATE)
groups are not degraded by nucleases,4 are stable in
human serum, and are selectively hydrolyzed to parent
phosphodiester or phosphorothioate oligonucleotides in
total cell extract, as a model for intracellular medium.4-6
The decomposition process is mediated by carboxy-
esterases (Figure 1).
F igu r e 1. Esterase-dependent decomposition of SATE-pro-
oligonucleotides. Upon incubation in total cell extract, thioester
bond breakage induced by carboxyesterases releases an un-
stable 2-mercaptoethyl phosphotriester which decomposes
spontaneously via intramolecular nucleophilic displacement
into the corresponding phosphodiester and ethylene sulfide.
nucleophiles and decompose during their solid-phase
synthesis under the standard basic conditions required
for removal of the common heterocyclic N-acyl protecting
groups7 and release of the oligonucleotide from the
regular succinyl-anchored solid support.8,9 Therefore, a
completely new strategy had to be set for the solid-phase
synthesis of these base-sensitive analogues. Require-
ments for such a synthesis are the achievement of the
appropriate S-acylthioethyl (SATE) phosphoramidite syn-
thons and the use of a solid support and of nucleobase
amino protecting groups which could be cleaved under
nonbasic and nonnucleophilic conditions. We recently
reported the solid-phase synthesis of dodecathymidine
SATE-phosphotriesters4 using a photolabile linker10 an-
Prooligonucleotides, being phosphotriester (or thiono-
phosphotriester) derivatives, are sensitive to bases and
(1) Tonkinson, J . L.; Stein, C. A. Cancer Invest. 1996, 14, 54-65.
(2) Crooke, S. T. In Antisense Research and Application; Crooke, S.
T., Ed.; Springer-Verlag: Berlin, Heidelberg, New York, 1998; Vol. 131,
pp 1-50.
(3) Barber, I.; Rayner, B.; Imbach, J .-L. Bioorg. Med. Chem. Lett.
1995, 5, 563-568.
(4) Tosquellas, G.; Alvarez, K.; Dell’Aquila, C.; Morvan, F.; Vasseur,
J .-J .; Imbach, J .-L.; Rayner, B. Nucleic Acids Res. 1998, 26, 2069-
2074.
(5) Barber, I.; Rayner, B.; Imbach, J .-L. Nucleosides Nucleotides
1995, 14, 1083-1085.
(6) Mignet, N.; Tosquellas, G.; Barber, I.; Morvan, F.; Rayner, B.;
Imbach, J . L. New J . Chem. 1997, 21, 73-79.
(7) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223-2311.
(8) Matteucci, M. D.; Caruthers, M. H. J . Am. Chem. Soc. 1981, 103,
3185-3191.
(9) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.;
Fisher, E. F.; McBride, L. J .; Matteucci, M.; Stabinsky, Z.; Tang, J . Y.
Methods Enzymol. 1987, 154, 287-313.
(10) Dell’Aquila, C.; Imbach, J . L.; Rayner, B. Tetrahedron Lett.
1997, 38, 5289-5292.
10.1021/jo990479h CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/03/1999

6320 J . Org. Chem., Vol. 64, No. 17, 1999
Alvarez et al.
chored to the solid support and thymidine phosphor-
amidite synthons having a SATE group in place of the
regular 2-cyanoethyl group. Our effort is now focused on
the solid-supported synthesis by the phosphoramidite
approach of SATE-phosphotriester heteropolymers con-
taining the four common nucleobases T, C, A, and G. For
this purpose, new N-protecting groups of the nucleobase
exocyclic amines are required.11
Photolabile protecting groups have been used in nucleic
acid chemistry for the protection of primary alcohols of
deoxynucleosides12,13 and of 2′-secondary alcohols of ri-
bonucleosides14,15 and phosphates.16,17 The efficiency of
photolabile solid-phase supports for the synthesis of
oligonucleotides has also been demonstrated.10,18 Surpris-
ingly, the use of photolabile groups for the protection of
exocyclic amino functions of the nucleobases C, G, and A
has never been reported.
be either isolated at this stage with a yield of 45% or
dimethoxytritylated by treatment of the crude dried
mixture with 4,4′-dimethoxytrityl chloride (1.2 equiv,
with respect to the nucleoside) to afford 5′-O-DMTr-4-
N-NVOC-2′-deoxycytidine (2) with a yield of 40% from
2′-deoxycytidine. Similarly, 4-N-diNPEOC-2′-deoxycyti-
dine (3) was isolated with a yield of 78% and then 5′-O-
tritylated to provide 5′-O-DMTr-4-N-diNPEOC-2′-deoxy-
cytidine (4) with a yield of 83%.
As part of our oligonucleotide prodrug program, we
decided to comparatively evaluate 6-nitroveratryloxycar-
bonyl (NVOC)19,20 and 2,2′-bis(2-nitrophenyl)ethoxycar-
bonyl (diNPEOC)21 as protecting groups of nucleobases.
We report herein their usefulness in the synthesis of
SATE-prooligonucleotides on solid support via the phos-
phoramidite approach.
Syn th esis of th e NVOC- a n d d iNP EOC-P r otected
2′-Deoxyr ib on u cleosid e 3′-t-Bu -SATE -p h osp h or -
a m id ites. The NVOC and the diNPEOC groups were
introduced on 2′-deoxycytidine through a transient-
protection protocol originally developed for the acylation
of exocyclic amino functions of deoxynucleosides.22 The
trimethylsilylation of the nucleoside in anhydrous pyri-
dine with trimethylsilyl chloride (5 equiv, with respect
to the nucleoside) was followed by the addition of either
the o-nitroveratryloxycarbonyl chloride (NVOC-Cl) or
the 2,2′-bis(2-nitrophenyl)ethyl chloroformate (diNPEOC-
Cl)21 (1.2 equiv, with respect to the nucleoside) and then
by the hydrolysis of trimethylsilyl groups with a methanol/
water solution. The 4-N-NVOC-2′-deoxycytidine (1) could
Nitroveratryloxycarbonylation of 5′,3′-di-O-TBDMSi-
2′-deoxyadenosine (5) was performed with in-situ-pre-
pared23 NVOC-tetrazolide (2 equiv, with respect to the
nucleoside) in hot THF, affording 5′,3′-di-O-TBDMSi-6-
N-NVOC-2′-deoxyadenosine (6) with a yield of 63%. This
compound was quantitatively desilylated with tetrabu-
tylammonium fluoride (TBAF), and the resulting 6-N-
NVOC nucleoside 7 was dimethoxytritylated to give 5′-
O-DMTr-6-N-NVOC-2′-deoxyadenosine (8) with a yield
of 53%. Similarly, the reaction of in-situ-prepared23
diNPEOC-tetrazolide with 5 afforded 5′,3′-di-O-TBDMSi-
6-N-diNPEOC-2′-deoxyadenosine (9) in 90% yield. After
desilylation, the resulting 5′,3′-diOH nucleoside 10 was
dimethoxytritylated to give 5′-O-DMTr-6-N-diNPEOC-2′-
deoxyadenosine (11). In comparison with this method, the
reaction of free 2′-deoxyadenosine with diNPEOC-Cl
following the transient-protection protocol22 resulted in
the concomitant formation of 10 (43% yield) and N,N-di-
(diNPEOC)-2′-deoxyadenosine (12, 25% yield).
(11) Alvarez, K.; Tworkowski, I.; Vasseur, J .-J .; Imbach, J .-L.;
Rayner, B. Nucleosides Nucleotides 1998, 17, 365-378.
(12) Pirrung, M. C.; Bradley, J . C. J . Org. Chem. 1995, 60, 6270-
6276.
(13) Pirrung, M. C.; Fallon, L.; McGall, G. J . Org. Chem. 1998, 63,
241-246.
(14) Hayes, J . A.; Brunden, M. J .; Gilham, P. T.; Gough, G. R.
Tetrahedron Lett. 1985, 26, 2407-2410.
(15) Tanaka, T.; Tamatsukuri; Ikehara, M. Nucleic Acids Res. 1987,
15, 7235-7248.
Similarly, the reaction of 2′-deoxyguanosine with di-
NPEOC-Cl following the same protocol afforded mono-
N-2-protected nucleoside 13 (15%) and N-2-diprotected
nucleoside 14 (20%). Simultaneous mono- and diprotec-
tions were reported for dansylethoxycarbonylation of 2′-
deoxyguanosine.24 Better results were obtained when the
3′,5′-di-O-silylated nucleoside 15 was reacted with di-
(16) Pirrung, M. C.; Shuey, S. W. J . Org. Chem. 1994, 59, 3890-
3897.
(17) Pirrung, M. C.; Fallon, L.; Lever, D. C.; Shuey, S. W. J . Org.
Chem. 1996, 61, 2129-2136.
(18) Venkatesan, H.; Greenberg, M. M. J . Org. Chem. 1996, 61, 525-
529.
(19) Pillai, V. N. R. Synthesis 1980, 1-26.
(20) Fodor, S. P. A.; Leigthon Read, J .; Pirrung, M. C.; Stryer, L.;
Lu, A. T.; Solas, D. Science 1991, 251, 767-773.
(21) Hasan, A.; Stengele, K.-P.; Giegrich, H.; Cornwell, P.; Isham,
K. R.; Sachleben, R. A.; Pfleiderer, W.; Foote, R. S. Tetrahedron 1997,
53, 4247-4264.
(23) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J . Am.
Chem. Soc. 1990, 112, 1691-1696.
(24) Wagner, T.; Pfleiderer, W. Helv. Chim. Acta 1997, 80, 200-
212.
(22) Ti, G. S.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1982,
104, 1316-1319.

Synthesis of Base-Sensitive SATE-Prooligonucleotides
J . Org. Chem., Vol. 64, No. 17, 1999 6321
Ta ble 1. P h otolysis Ra tes for N-P r otected Nu cleosid es 1,
3, 7, 10, 12, 13, a n d 14 in Dioxa n e/Wa ter , 3/2, v/v, 5%
CH3CO2H
NPEOC-Cl in the presence of tert-butylmagnesium
chloride23 to give 16 with an isolated yield of 50%.
Desilylation with TBAF produced 13, and subsequent
dimethoxytritylation afforded 5′-O-DMTr-2-N-diNPEOC-
2′-deoxyguanosine (17). The thymidine derivative 18 was
easily obtained from thymidine by reaction with dimeth-
oxytrityl chloride.
concn
no. nucleoside
N-protection
(mM)
tcompletion
1
3
dC
dC
dA
dA
NVOC
1
0.1
1
0.1
1
0.1
1
>4 h (t1/2 28 min)
40 min (t1/2 5 min)
20 min
diNPEOC
NVOC
The 3′-O-phosphoramidite monomer units bearing the
S-pivaloylthioethyl (t-Bu-SATE) instead of the standard
cyanoethyl were obtained by reaction of the 3′-OH
intermediates with the phosphitylating agent (S-piv-
aloylthioethyloxy)bis(diisopropylamino)phosphine25 (19,
1.5 equiv, with respect to the nucleoside), in the presence
of diisopropylammonium tetrazolide (0.5 equiv, with
respect to the parent nucleoside) in anhydrous dichlo-
romethane. This protocol is more convenient than the one
previously described4 for the synthesis of the 3′-phos-
phoramidite of thymidine in two steps by reaction with
(N,N-diisopropylamino)chlorophosphine in the presence
of N,N-diisopropylamine, giving rise to an unstable 3′-
phosphorodiamidite, which was then reacted in situ with
the SATE alcohols in the presence of tetrazole. Utilizing
the phosphitylating agent 19, the isolated yields of 20,
21, 22, 23, 24, and 25 after purification were 82, 84, 60,
78, 55, and 80%, respectively. In comparison the thymi-
dine phosphoramidite 25 was obtained with a yield of
55% using the two-step protocol.4
P h otod ep r otection Ra tes: Nu cleosid e Level. The
removal of photolabile protecting groups such as the
NVOC containing the o-nitrobenzyl moiety is generally
performed by irradiation at wavelengths >300 nm, which
do not damage nucleic acids.26 To avoid eventual side-
product formation between the released amino function
and the blocking group photoproduct (nitrosoaldehyde),
deprotection is carried out under slightly acidic condi-
tions.26 DiNPEOC deprotection does not require such
precautions as the photoproduct is not a nitrosoaldehyde
but an o-nitrostyrene derivative.21 However, to compare
deprotection rates of NVOC and diNPEOC derivatives,
the photolysis experiments were performed in the same
medium containing a small amount of acetic acid (1-
5%).
These experiments were run in a thermostated cuvette
(quartz) at room temperature using a high-pressure Hg
lamp. A Pyrex glass filter serves as a 300 nm cutoff filter.
Prior to photolysis, argon was bubbled through the
solutions (dioxane/water (3/2) + CH3CO2H) of the nucleo-
side samples to remove traces of O2. The rate of depro-
tection (Table 1) was determined by HPLC analysis.
Acetic acid percentages varying from 1 to 10% did not
affect the rate of NVOC removal; however, experiments
with less than 1% acetic acid showed a significant
decrease of the photolysis rate (data not shown).
N-4-NVOC-dC (1) and N-6-NVOC-dA (7) were depro-
tected by photolysis at approximately the same rate.
Similarly, no significant difference in the behavior of the
diNPEOC derivatives of dC 3 and dA 9 was noticed
(Table 1).
6 min
7
>4 h (t1/2 31 min)
40 min (t1/2 5 min)
20 min
10
diNPEOC
0.1
1
1
6 min
25 min
15 min
12
13
dA
dG
bis-(diNPEOC)
diNPEOC
0.1
1
5 min
15 min
14
dG
bis-(diNPEOC)
and 7 from 0.1 to 1 mM lowered the NVOC removal rate
by 6. In comparison, a 4-fold decrease for the removal of
diNPEOC protection was observed with the same in-
crease of concentration of the diNPEOC nucleosides 3 and
10.
The diNPEOC derivatives had photolysis rates ap-
proximately 5-fold greater than the corresponding NVOC
derivatives (Table 1). The effect of the protection on the
photolysis rates of N-protected nucleosides was consistent
with the results described for 5′-O-protected deoxythy-
midines.21 In this case, it was reported that the rate of
photololysis, replacing NVOC by diNPEOC, was en-
hanced by more than 1 order of magnitude (13×).
Finally, it was more difficult to remove the two
diNPEOC protecting groups in N,N′-6-bis(diNPEOC)-
deoxyadenosine (12) than in N,N′-2-bis(diNPEOC)-deoxy-
guanosine (14). One can notice that the deprotection of
both diNPEOC groups of the diprotected dG nucleoside
14 was as rapid as the removal of one diNPEOC in the
monoprotected dG derivative 13.
Syn th esis of th e SATE-P r ooligon u cleotid es on
Solid Su p p or t. Synthesis of t-Bu-SATE-phosphotriester
oligonucleotides was performed by use of phosphoramid-
ite methodology on an automated DNA-synthesizer.
To avoid nucleophilic and/or basic conditions used for
standard cleavage of oligonucleotides from solid support,
the regular succinyl linker was replaced with a photo-
labile linker bearing a 1-(o-nitrophenyl)-1,3-propanediol
moiety. This linker was successfully applied to the solid-
phase synthesis of oligododecathymidine methylphos-
photriesters10 as well as methyl or tert-butyl-SATE-
phosphotriesters and -phosphorothiotriesters.4 Because
of their poor solubility in acetonitrile, NVOC- and di-
NPEOC-protected nucleoside phosphoramidites were
solubilized in an acetonitrile/dichloromethane mixture (9/
1, v/v).
Despite their high purity, the coupling efficiency with
both NVOC- and diNPEOC-protected nucleoside phos-
phoramidites was low (less than 70% for diNPEOC)
compared with >98% for thymidine phosphoramidite 25
even when extended coupling times (one or two times 180
s instead of 30 s coupling for 25) were applied. These low
yields were explained by the presence of residual water
and were improved (90-95%) when solutions of the
diNPEOC-nucleoside phosphoramidites were dried over
4 Å molecular sieves for 24 h.27 The same result was
The NVOC removal was greatly dependent on the
concentration of the starting material. An increase of the
initial concentration in starting protected nucleosides 1
(25) Guzaev, A.; Boyode, B.; Balow, G.; Tivel, K. L.; Manoharan, M.
Bioorg. Med. Chem. Lett. 1998, 8, 1123-1226.
(26) Robertson, S. A.; Ellman, J . A.; Schultz P. G. J . Am. Chem.
Soc. 1991, 113, 2722-2729.
(27) Reynolds, M. A.; Hogrefe, R. I.; J aeger, J . A.; Schwartz, D. A.;
Riley, T. A.; Marvin, W. B.; Daily, W. J .; Vaghefi, M. M.; Beck, T. A.;
Knowles, S. K.; Klem, R. E.; Arnold, L. J ., J r. Nucleic Acids Res. 1996,
24, 4584-4591.

6322 J . Org. Chem., Vol. 64, No. 17, 1999
Ta ble 2. P h otolysis Ra tes for N-P r otected Oligon u cleotid es
Alvarez et al.
calcd
found
concn
(mM)
no.
oligonucleotide
[M - H]- (m/z)
[M - H]- (m/z)
N-protector
tcompletion
26
d(5′TpCpTpTpTp3′)
2244.2
2243.6
NVOC
0.05
0.1
0.1
1
0.05
0.1
1
45 min
75 min
10 mina
15 min
45 min
10 mina
20 min
20 min
>5 h
diNPEOC
27
28
d(5′TpApTpTpTp3′)
2268.2
2284.2
2268.5
2283.3
NVOC
diNPEOC
d(5′TpGpTpTpTp3′)
diNPEOC
NVOC
1
d(5′TpCpCpApApTp3′)
0.05
0.005
2.5 hb
29
30
31
d(5′TpCpCpCpTp3′)
d(5′TpApApApTp3′)
d(5′TpGpGpGpTp3′)
2214.2
2286.2
2334.2
2214.0
2285.8
2334.3
diNPEOC
1
20 min
a
Time required for diNPEOC photolysis; a further 5-10 min photolysis time was then applied to totally cleave the oligomer from the
b
solid support. The oligonucleotide was not isolated and not characterized.
observed with extensive drying of the compounds as
powders under high vacuum over P2O5 and KOH. This
problem was not encountered with dT phosphoramidite
25 (coupling yield >98%). Even improved, the coupling
yields during solid-phase DNA synthesis were lower than
those obtained using standard N-acyl nucleoside phos-
phoramidites.
tert-Butyl hydroperoxide28,29 in anhydrous conditions
was used for the oxidation of phosphite triester interme-
diates to form t-Bu-SATE-phosphotriester linkages. This
oxidizer was preferred to the common iodine/water treat-
ment to prevent an eventual loss of the t-Bu-SATE
protection during aqueous oxidation.29,30
F igu r e 2. RP-HPLC profiles of the reaction mixture for NVOC
First, two pentanucleoside t-Bu-SATE-phosphotri-
esters, d(5′TpCpTpTpTp3′) 26 and d(5′TpApTpTpTp3′) 27,
bearing four t-Bu-SATE-phosphotriester internucleoside
linkages and one t-Bu-SATE-phosphodiester linkage at
their 3′-end were synthesized on a 1 µmol scale on a DNA
synthesizer using dT phosphoramidite 25 and NVOC-
phosphoramidites 20 and 22 as well as diNPEOC-
phosphoramidites 21 and 23 to evaluate the conditions
of the photolysis on oligonucleotides containing only one
adenine or one cytosine in their sequence.
The same protocol as the one described above for the
deprotection of the N-protected nucleosides was applied
to the supported oligonucleotides. Cleavage of these
compounds from the solid support was performed upon
exposure to the Pyrex-filter output of a high-pressure Hg
lamp at 25 °C. Prior to photolysis, argon was bubbled
through the suspensions (dioxane/water (3/2) + CH3-
CO2H) of the samples to remove traces of O2. At several
reaction times, aliquots were withdrawn and analyzed
by HPLC/ESI-MS (negative mode) to monitor the course
of the deprotection and characterize the compound.
After 5 min, the HPLC analysis (Figure 2) revealed
the presence of the undeprotected NVOC-dC oligonu-
cleotide (RT ) 31.2 min, m/z ) 2484) together with a
lower amount of the deprotected oligonucleotide 26
(RT ) 28.6 min, m/z ) 2243.6) and of a failure sequence,
5′-O-acetyl-d(5′TpTpTp3′) (RT ) 25.9 min, m/z ) 1301.7).
This truncated sequence was caused by the low coupling
yield of NVOC-dC phosphoramidite 20 (85%) during
oligonucleotide elongation. As observed with NVOC-
deprotection of the oligonucleotide d(TpCpTpTpTp) 26 (a) after
5 min of photolysis and (b) after 45 min of photolysis.
protected nucleosides, the removal of the protecting group
was dependent on the concentration of the starting
material (Table 2). Completion of the photolysis of the
NVOC-protected oligomer was observed after 45 and 75
min with 0.05 and 0.1 mM concentrations of oligonu-
cleotide, respectively. In comparison, the photolysis rate
of the NVOC-dC nucleoside was approximately 2-fold
greater than that of the NVOC-dC oligonucleotide. The
same result was observed with the NVOC-dA incorpo-
rated oligonucleotide. A 45 min photolysis reaction time
was necessary to ensure complete conversion of the
protected NVOC-dA oligonucleotide (RT ) 31.3 min) into
the deprotected oligomer 27 (RT ) 29.3 min, m/z )
2268.5).
Using the same experimental conditions as those
described for NVOC-oligonucleotides, much faster depro-
tection rates were observed for diNPEOC oligomers. After
10 min of photolysis of the corresponding solid-supported
oligonucleotides, the diNPEOC-protected intermediates
were not detected by HPLC/MS of the crude mixtures.
However, the photolysis was carried out for a further
5-10 min to ensure complete cleavage of the oligonu-
cleotides 26, 27, and 28 from the solid support.4,10 As
observed for the nucleoside derivatives, no significant
difference in the behavior toward photolysis of the
diNPEOC-oligonucleotides containing dC, dA, or dG was
noticed. More interestingly, the time of completion was
slightly influenced when the concentration was raised
from 0.1 to 1 mM.
(28) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett.
1986, 27, 4191-4194.
Use of NVOC protection was not pursued after an
attempt to obtain d(5′TpCpCpApApTp3′) from NVOC-
units assembly was unsuccessful. The deprotection of the
heterocyclic amino functions of this oligomer was not
(29) Alul, R. H.; Singman, C. N.; Zhang, G.; Letsinger, R. L. Nucleic
Acids Res. 1991, 19, 1527-1532.
(30) Nielsen, J .; Caruthers, M. H. J . Am. Chem. Soc. 1988, 110, 0,
6275-6276.

Synthesis of Base-Sensitive SATE-Prooligonucleotides
J . Org. Chem., Vol. 64, No. 17, 1999 6323
cycle incorporation (1 µmol scale). This indicates that
Ac2O capping treatment could not be applied in the
synthesis of dA-containing heteropolymers. Therefore,
this capping treatment was replaced by the less common
one using diethyl N,N-diisopropyl phosphoramidite in the
presence of tetrazole.31 Under such conditions, the syn-
thesis of the oligonucleotide 30 containing three adjacent
deoxyadenosines gave us the expected compound without
any detectable adduct as determined by ESI-MS analysis
(Figure 3). The synthesis of d(5′TpGpGpGpTp3′) 31 con-
taining three adjacent deoxyguanosines was carried out
using the same capping conditions as for oligomer 30.
Indeed, careful examination of the ESI spectrum of
isolated d(5′TpGpTpTpTp3′) 28 obtained with acetic an-
hydride as the capping reagent also revealed a small
amount of an acetylated oligonucleotide ([M - 2H +
CH3CO]-, m/z 2326.2; [M - 3H + CH3CO]2-, m/z 1162.1).
In addition, hydrolysis of the t-Bu-SATE oligomers 29,
30, and 31 with concentrated ammonia yielded the fully
unmasked phosphodiester oligonucleotides as the main
compounds.
Finally, the t-Bu-SATE-phosphotriester and -phospho-
rothionotriester analogues 32 and 33 of a dodecanucleo-
side, d(5′ApCpApCpCpCpApApTpTpCpTp3′), complemen-
tary to the splice acceptor site of mRNA coding for HIV-1
tat protein were prepared. Their complementary se-
quence analogue d(5′ApGpApApTpTpGpGpGpTpGpTp3′)
with phosphotriester linkages 34 was also synthesized.
tert-Butyl hydroperoxide28 was used as oxidizer for the
formation of phosphotriester linkages in 32 and 34.
Beaucage reagent32 was used for oxidation of phosphi-
tetriesters into thionophosphotriester internucleoside
linkages for oligonucleotide 33. Diethyl N,N-diisopropyl
phosphoramidite in the presence of tetrazole31 was used
as the capping reagent. After 40 min of photolysis, the
oligomers were purified by reversed-phase HPLC. In each
case, broad peaks were observed (32, Rt ) 21.2 min, 9.2
min from start to end of peak (Figure 4); 33, Rt ) 27.4
min over 5.4 min; 34, Rt ) 22.2 min over 8.6 min),
reflecting the presence of diastereomeric mixtures due
to Rp and Sp isomers at each internucleotide linkage.
The integrity of the oligonucleotides 32, 33, and 34 was
confirmed by ESI-MS analysis performed in positive
mode using solutions of the oligomers in CH3CN/H2O (7/
3, v/v) containing a small amount of formic acid (1%).33
The mass spectrum (Figure 4) for an aqueous solution of
oligomer 32 (1 OD260 nm in 500 µL of CH3CN/H2O (7/3)
containing 5 µL of HCO2H) generated charge states
[M + 4H]4+ at m/z 1340.6 (calcd 1341.1) and [M + 3H]3+
at m/z 1787.9 (calcd 1787.7). The measured mass of 5361
was consistent with the expected structure (calculated
mass 5360.2). Similarly, the mass spectrum of oligomer
33 showed [M + 5H]5+ at m/z 1111.7 (calcd 1111.6),
[M + 4H]4+ at m/z 1389.8 (calcd 1389.3), and [M + 3H]3+
at m/z 1851.9 (calcd 1852.0), and that of oligomer 34
showed [M + 4H]4+ at m/z 1839.2 (calcd 1388.8) and
[M + 3H]3+ at m/z 1851.1 (calcd 1851.4). Moreover,
hydrolysis of 32, 33, and 34 with concentrated aqueous
ammonia for 1 h at 55 °C afforded main compounds
Figu r e 3. Negative ion ESI mass spectra of d(5′TpApApApTp3′)
30, showing deprotonated monocharged ions: (A) use of Ac2O/
THF/2,6-lutidine (1/8/1, v:v:v) and 10% N-methylimidazole/
THF for the capping step during oligonucleotide elongation;
(B) use of diethyl N,N-diisopropyl phosphoramidite in the
presence of tetrazole as capping reagent.
complete even after 5 h of photolysis at a concentration
of 0.05 mM. Lowering the concentration from 50 to 5 µM
reduced the reaction time to 2.5 h. However, this reaction
time was too long, and HPLC analysis of the crude
mixture revealed numerous side products. Our attention
then focused on diNPEOC protection.
Three other pentanucleoside t-Bu-SATE-phosphotri-
esters, i.e., d(5′TpCpCpCpTp3′) 29, d(5′TpApApApTp3′) 30,
and d(5′TpGpGpGpTp3′) 31, were prepared on a 1 µmol
scale using diNPEOC-phosphoramidites 21, 23, and 24,
respectively, to study the photolysis on oligonucleotides
containing three diNPEOC-protected nucleosides in their
sequence. At 1 mM concentration in oligonucleotide,
photolysis was carried out for 20 min. Longer reaction
times did not modify the HPLC profile of the crude
mixtures. The oligomers were then purified by reversed-
phase HPLC and characterized by ESI-MS analysis
(negative mode). Surprisingly, while the ESI-MS analysis
mass of the isolated oligomer 29 was in accord with its
calculated mass (m/z calcd 2214.2, found 2214.0), the
analysis of the isolated oligomer 30 revealed a mixture
of the expected oligomer (m/z 2285.2) with adducts at m/z
2327.8 and 2369.5.0 (Figure 3). One possible explanation
might be partial acetylation (+42 and +2 × 42) of the
oligomer under the capping step conditions used during
the oligomer elongation. Standard capping is generally
performed with a mixture of Ac2O/THF/2,6-lutidine (1/
8/1, v/v/v) and 10% N-methylimidazole/THF for 20 s per
(31) Yu, D.; Tang, J .; Iyer, R. P.; Agrawal, S. Tetrahedron Lett. 1994,
35, 8565-8568.
(32) Iyer, R. P.; Egan, W.; Regan, J . B.; Beaucage, S. L. J . Am. Chem.
Soc. 1990, 112, 1253-1254.
(33) Baker, T. R.; Keough, T.; Dobson, R. L. M.; Riley, T. A.;
Hasselfield, J . A.; Hesselberth, P. E. Rapid Commun. Mass Spectrom.
1993, 7, 190-194.

6324 J . Org. Chem., Vol. 64, No. 17, 1999
Alvarez et al.
gel plates (Kieselgel 60 F254, Merck), and column flash chro-
matography was run on silica gel Kieselgel Merck 60, 40-63
µm. Elution for TLC was performed with methanol/dichlo-
romethane mixtures (2/98 to 20/80, v/v) except where otherwise
stated. 1H NMR spectra were recorded at 200, 250, or 400
MHz. Tetramethylsilane, CHD2CN, CHCl3, and DMSO-d5 were
used as internal standards. 31P NMR spectra were recorded
at room temperature at 80 or 100 MHz relative to phosphoric
acid as an external standard. Glycerol, glycerol/thioglycerol
(1/1, v/v, GT), 3-nitrobenzylic alcohol (NOBA),and poly-
(ethylene glycol) (PEG) were used as the sample matrix for
fast-atom-bombardment mass spectra (FAB-MS). Electrospray
mass spectra (ESI-MS) were recorded on a quadrupole mass
spectrometer. Oligonucleotides with molecular weight below
2500 were analyzed in negative mode. Crude materials were
analyzed by HPLC/ESI-MS. Purified samples (1 OD260 nm) were
dissolved in acetonitrile/water (500 µL, v/v, 1/1). Longer
oligonucleotides were analyzed by ESI-MS in positive mode,
after HPLC purification. Samples (1 OD260 nm) were dissolved
in acetonitrile/water (500 µL, v/v, 7/3) containing formic acid
(5 µL, 1%) and introduced into the mass spectrometer at a 10
µL/min flow rate with a Harvard Apparatus 22 pump model.
HPLC analyses were performed on an instrument equipped
with a photodiode array UV detector. Reversed-phase HPLC
analysis of reaction mixtures and purified compounds as well
as HPLC purification of the oligonucleotides were performed
on a C18 (5 µm) Nucleosil column (150 × 4.6 mm, Macherey-
Nagel, Germany) at a flow rate of 1 mL/min. Solid-phase
syntheses of the prooligonucleotides were performed using a
photolabile CPG solid support13,14 (63.6 µmol/g) on a 1 µmol
scale (15-16 mg of solid support) using a DNA synthesizer.
Photolyses were conducted upon exposition of the samples in
dioxane/water in a 1 cm path length quartz cell to the Pyrex-
filtered output of a high-pressure Hg lamp (HPK 125, Philips,
Nederlands).
F igu r e 4. (A) RP-HPLC profile of the purified oligonucleotide
32 d(ApCpApCpCpCpApApTpTpCpTp) obtained after 40 min
of photolysis. (B) Positive ion ESI mass spectrum of d(ApCp-
ApCpCpCpApApTpTpCpTp) 32 showing protonated ions with
charges of 3 and 4. The measured mass was 5361, and the
averaged calculated mass is 5360.2.
4-N-(6-Nitr over a tr yloxyca r bon yl)-2′-d eoxycytid in e (1).
Prior to use, the 2′-deoxycytidine hydrochloride (300 mg, 1.13
mmol) was dried three times by coevaporation of anhydrous
pyridine and then suspended in anhydrous pyridine (10 mL).
To that suspension was added under argon trimethylchlorosi-
lane (0.72 mL, 5.67 mmol). After the mixture was stirred for
30 min to 1 h, NVOC-Cl (376 mg, 1.36 mmol) was added, and
the reaction mixture was stirred at room temperature over-
night. Water/methanol (1/1, v/v, 5 mL) was added and the
reaction mixture was stirred for 30 min and then concentrated
under reduced pressure. Dichloromethane (100 mL) was
added, and the resulting mixture was washed with saturated
aqueous NaHCO3 (80 mL) and then with brine (80 mL). The
organic layer was dried (Na2SO4), filtered, and evaporated to
dryness. Residual pyridine was removed by coevaporations
with toluene (3×). The residue was purified by flash column
chromatography (silica gel, gradient 0-10% MeOH/CH2Cl2).
The appropriate fractions were combined and evaporated to
dryness. The resulting foam was dissolved in dioxane and
lyophilized to give 1 (237 mg, 45%) as a colorless powder: 1H
NMR (pyridine-d5) δ 8.86 (d, J ) 7.5 Hz, 1 H, H-6), 7.77 (s, 1
H, H ortho to NO2 of NVOC), 7.54 (s, 1 H, H meta to NO2 of
NVOC), 7.45 (d, J ) 7.5 Hz, 1 H, H-5), 7.26 (d, 1 H, OH-3′),
7.01 (t, 1 H, OH-5′), 6.93 (m, 1 H, H-1′), 5.86 (s, 2 H, CO2CH2),
5.00 (m, 1 H, H-3′), 4.52 (m, 1 H, H-4′), 4.22-4.18 (m, 2 H,
H-5′, H-5′′), 3.84 and 3.79 (2 s, 6 H, 2 OCH3), 2.95 and 2.62 (2
m, 2 H, H-2′, H-2′′); FABMS (positive mode) m/z 467 (MH)+,
351 (4-N-NVOC-Cyt + H)+, 228 (M - NVOC + 2H)+ ) (dC +
H)+; HRMS calcd for C19H23N4O10 467.1414, found 467.1489;
FABMS (negative mode) m/z 465 (M - H)-, 226 (M -
NVOC)- ) (dC - H)-.
which comigrated on reversed-phase HPLC with authen-
tic samples of dodecanucleoside phosphodiesters d(Ap-
CpApCpCpCpApApTpTpCpTp), its phosphorothioate ana-
logue, and d(ApGpApApTpTpGpGpGpTpGpTp), respec-
tively.
Con clu sion
For the first time, photocleavable protecting groups for
nucleobases combined with an anchored photolabile solid
support allowed the synthesis of base-sensitive SATE-
phosphotriester oligonucleotides of various sequences.
The diNPEOC protecting groups are removed, and the
oligonucleotides are cleaved from the solid support in a
single deprotection step by UV irradiation at wavelengths
>300 nm without affecting the phosphotriester inter-
nucleoside linkages.
Exp er im en ta l Section
Gen er a l Met h od s. Commercial chemical reagents
were reagent grade and were used without purification except
where otherwise stated. o-Nitroveratryloxycarbonyl chloride
(NVOC-Cl), N,N-bis(diisopropylamino)chlorophosphine, tet-
razole (>99%), and redistilled N,N-diisopropylethylamine were
obtained from Aldrich (St. Quentin Fallavier, France). Meth-
ylene chloride (Aldrich) was dried over P2O5 followed by
distillation. DNA synthesis reagents, except oxidizers, were
from Perseptive Biosystems Ltd. (Voisins le Bretonneux,
France). Anhydrous tert-butyl hydroperoxide (3 M in toluene)
was from Fluka and was diluted with anhydrous methylene
chloride. 3H-1,2-Benzodithiol-3-one-1,1-dioxide (Beaucage re-
agent) was a gift from Isis Pharmaceuticals (Carlsbad, CA).
Diethyl N,N-diisopropyl phosphoramidite was from Chem-
Genes (Waltham, MA). 2,2′-Bis(2-nitrophenyl)ethyl chlorofor-
mate (diNPEOC-Cl) was prepared according to a published
procedure.31 Analytical TLC was performed on precoated silica
4-N-(2,2′-Bis(2-n itr oph en yl)eth yloxycar bon yl)-2′-deoxy-
cytid in e (3). 2′-Deoxycytidine hydrochloride (204 mg, 0.77
mmol) was treated with the same protocol as for preparation
of 1, except the NVOC-Cl was replaced with diNPEOC-Cl
(330.5 mg, 0.94 mmol). After purification by flash column
chromatography (silica gel, gradient 2-6% MeOH/CH2Cl2), the
appropriate fractions were combined and evaporated to dry-
ness. The resulting foam was dissolved in dioxane and lyoph-
ilized to give 3 (325 mg, 78%) as a colorless powder: 1H NMR

Synthesis of Base-Sensitive SATE-Prooligonucleotides
J . Org. Chem., Vol. 64, No. 17, 1999 6325
(CD3CN) δ 8.44 (s, 1 H, NH), 8.27 (d, J ) 7.52 Hz, 1 H, H-6),
7.97 (dd, J ortho ) 8.04 Hz, J meta ) 1.06 Hz, 2 H, H ortho to NO2
of diNPEOC), 7.69-7.51 (m, 6 H, H meta and para to NO2 of
diNPEOC), 7.08 (d, J ) 7.50 Hz, 1 H, H-5), 6.10 (m, 1 H, H-1′),
5.55 (t, J ) 6.17 Hz, 1 H, CO2CH2CH), 4.90 (d, J ) 6.14 Hz, 2
H, CO2CH2CH), 4.30 (m, 1 H, H-3′), 3.96 (m, 1 H, H-4′), 3.70
(m, 2 H, H-5′, H-5′′), 3.40 (d, 1 H, OH-3′), 3.26 (t, 1 H, OH-5′),
2.39 and 2.14 (2 m, 2 H, H-2′, H-2′′); FABMS (positive mode)
m/z 542 (MH)+, 426 (4-N-diNPEOC-Cyt + H)+; HRMS calcd
for C24H24N5O10 542.1523, found 542.1520; FABMS (negative
mode) m/z 540 (M - H)-, 226 (M - diNPEOC)- ) (dC - H)-.
(MH)+, 780 (2-N-bis-diNPEOC-Gua + H)+; 582 (2-N-diNPEOC-
dG + H)+, 466 (2-N-diNPEOC-Gua + H)+; HRMS calcd for
C
40H34N9O16 896.2124, found 896.2155; FABMS (negative
mode) m/z 894 (M - H)-, 778 (2-N-bis-diNPEOC-Gua - H)-,
580 (2-N-diNPEOC-dG - H)-, 464 (2-N-diNPEOC-Gua - H)-.
5′,3′-Bis(O-ter t-b u t yld im et h ylsilyl)-6-N-(6-n it r over a -
tr yloxyca r bon yl)-2′-d eoxya d en osin e (6). A THF solution
of 6-nitroveratryloxycarbonyltetrazolide was prepared by add-
ing NVOC-Cl (500 mg, 1.81 mmol) to a mixture of 1H-
tetrazole (113, 1.6 mmol) and Et3N (242 µL, 1.73 mmol) in
anhydrous THF (20 mL) at 0 °C. The resulting mixture was
stirred at 0 °C for 10 min and then at room temperature for
30 min. The precipitate was filtered off through a pad of Celite
and washed with THF. The THF solution of NVOC-tetrazole
was concentrated and added to 5′,3′-bis(O-tert-butyldimethyl-
silyl)-2′-deoxyadenosine (5) (385 mg, 0.8 mmol) in anhydrous
THF (15 mL). The resulting mixture was heated to 70 °C for
3 h and then kept at room temperature overnight. After
concentration under reduced pressure, the oil residue was
diluted with ethyl acetate (100 mL) and washed with saturated
aqueous NaHCO3 (80 mL) and then with brine (80 mL). The
organic layer was dried (Na2SO4) and then evaporated to
dryness. The residue was purified by flash column chroma-
tography (silica gel, gradient 70/30 to 100/0, v/v, of CH2Cl2/
cyclohexane), and the appropriate fractions were combined and
evaporated to dryness. The resulting foam was dissolved in
dioxane and lyophilized to afford 6 (362 mg, 63%): 1H NMR
(CDCl3) δ 8.77 (s, 1 H, H-8), 8.36 (s, 1 H, H-2), 7.76 (s, 1 H, H
ortho to NO2 of NVOC), 7.27 (s, 1 H, H meta to NO2 of NVOC),
6.52 (m, 1 H, H-1′), 5.75 (s, 2 H, CO2CH2), 4.63 (m, 1 H, H-3′),
4.06 (m, 1 H, H-4′), 4.02 and 3.99 (2 s, 6H, 2 OCH3), 3.99-
3.76 (m, 2 H, H-5′, H-5′′), 2.66 and 2.50 (2 m, 2 H, H-2′, H-2′′),
0.93 (m, 18 H, 2 C(CH3)3), 0.12 (m, 12H, 2 Si(CH3)2); FABMS
(positive mode) m/z 719 (MH)+; HRMS calcd for C32H51N6O9-
Si2 719.3252, found 719.3346; FABMS (negative mode) m/z 717
(M - H)-.
5′,3′-Bis(O-ter t-b u t yld im et h ylsilyl)-6-N-(2,2′-b is(2-n i-
tr oph en yl)eth yloxyca r bon yl)-2′-d eoxya d en osin e (9). 5′,3′-
Bis(O-tert-butyldimethylsilyl)-2′-deoxyadenosine (5) (1.7 g, 3.5
mmol) was treated with the same protocol as for the prepara-
tion of 6, except 2,2′-bis(2-nitrophenyl)ethyloxycarbonyl-
tetrazolide prepared as above from diNPEOC-chloride (1.6
g, 4.56 mmol) replaced 6-nitroveratryloxycarbonyltetrazolide.
Compound 9 was in with 90% yield (2.51 g): 1H NMR (CDCl3)
δ 8.75 (s, 1 H, H-8), 8.40 (sl, 1 H, NH), 8.30 (s, 1 H, H-2), 8.00
(dd, J ortho ) 8.06 Hz, J meta ) 1.22 Hz, 2 H, H ortho to NO2 of
diNPEOC), 7.60-7.42 (m, 6 H, H meta and para to NO2 of
diNPEOC), 6.50 (m, 1 H, H-1′), 5.81 (t, J ) 6.81 Hz, 1 H, CO2-
CH2CH), 4.99 (d, J ) 6.84 Hz, 2 H, CO2CH2CH), 4.62 (m, 1 H,
H-3′), 4.04 (m, 1 H, H-4′), 3.94-3.75 (m, 2 H, H-5′, H-5′′), 2.63
and 2.51 (2 m, 2 H, H-2′, H-2′′), 0.93 (m, 18 H, 2 C(CH3)3),
0.12 (m, 12H, 2 Si(CH3)2); FABMS (positive mode) m/z 794
(MH)+; HRMS calcd for C37H52N7O9Si2 794.3365, found
794.3455; FABMS (negative mode) m/z 792 (M - H)-, 478
(M - diNPEOC - H)-.
6-N-(2,2′-Bis(2-n itr oph en yl)eth yloxycar bon yl)-2′-deoxy-
a d en osin e (10) a n d 6-N-Bis(2,2′-bis(2-n itr op h en yl)eth yl-
oxyca r bon yl)-2′-d eoxya d en osin e (12). 2′-Deoxyadenosine
(200 mg, 0.79 mmol) was treated as 2′-deoxycytidine for the
preparation of 3. After purification by flash column chroma-
tography (silica gel, gradient 0-5% MeOH/CH2Cl2), the ap-
propriate fractions were combined and evaporated to dryness.
The resulting foams were dissolved in dioxane and lyophilized
to afford 10 (192 mg, 43%) and 12 (173 mg, 25%) as colorless
powders. Compound 10: 1H NMR (CD3CN) δ 8.90 (s, 1 H, NH),
8.57 (s, 1 H, H-8), 8.25 (s, 1 H, H-2), 7.93 (dd, J ortho ) 8.11 Hz,
J meta ) 1.07 Hz, 2 H, H ortho to NO2 of diNPEOC), 7.66-7.57
(m, 6 H, H meta and para to NO2 of diNPEOC), 6.40 (m, 1 H,
H-1′), 5.60 (t, J ) 6.32 Hz, 1 H, CO2CH2CH), 4.95 (d, J ) 6.25
Hz, 2 H, CO2CH2CH), 4.56 (m, 2 H, H-3′, OH-3′), 4.04 (m, 1
H, H-4′), 3.74 (m, 2 H, H-5′, H-5′′), 3.47 (m, 1 H, OH-5′), 2.79
and 2.39 (2 m, 2 H, H-2′, H-2′′); FABMS (positive mode) m/z
566 (MH)+, 450 (6-N-diNPEOC-Ade + H)+; HRMS calcd for
C25H24N7O9 566.1636, found 566.1611; FABMS (negative mode)
m/z 564 (M - H)-, 448 (6-N-diNPEOC-Ade - H)-. Compound
12: 1H NMR (CD3CN) δ, 8.52 (s, 1 H, H-8), 8.13 (s, 1 H, H-2),
7.88 (m, 4 H, H ortho to NO2 of diNPEOC), 7.49-7.22 (m, 12
H, H meta and para to NO2 of diNPEOC), 6.42 (m, 1 H, H-1′),
5.47 (t, 2 H, 2 CO2CH2CH), 5.05-4.85 (m, 6 H, 2 CO2CH2CH,
H-3′, OH-5′), 4.29 (m, 1 H, H-4′), 3.96 (m, 2 H, H-5′, H-5′′),
2.99 and 2.37 (2 m, 2 H, H-2′, H-2′′); FABMS (positive mode)
m/z 880 (MH)+, 764 (6-N-bis-diNPEOC-Ade + H)+, 566 (6-N-
diNPEOC-dA + H)+, 450 (6-N-diNPEOC-Ade + H)+; HRMS
calcd for C40H34N9O15 880.2174, found 880.2140; FABMS
(negative mode) m/z 878 (M - H)-, 762 (6-N-bis-diNPEOC-
Ade - H)-, 564 (6-N-diNPEOC-dA - H)-, 448 (6-N-diNPEOC-
Ade - H)-.
2-N-(2,2′-Bis(2-n it r op h en yl)et h yloxyca r b on yl)-2′-d e-
oxygu a n osin e (13) a n d 2-N-Bis(2,2′-bis(2-n itr op h en yl)-
eth yloxyca r bon yl)-2′-d eoxygu a n osin e (14). The procedure
applied to 2′-deoxyadenosine in the preparation of 10 and 12
was modified for 2′-deoxyguanosine (342 mg, 1.28 mmol). After
standing overnight with diNPEOC-Cl (583 mg, 1.66 mmol),
the mixture was treated for 15 min with 10% aqueous
ammonia (1.5 mL) at room temperature and then filtered. The
filtrate was evaporated, redissolved in CH2Cl2/MeOH (95/5,
v/v), and purified by flash column chromatography (silica gel,
gradient 5-10% MeOH/1% Et3N/CH2Cl2), and the appropriate
fractions were combined and evaporated to dryness. The
resulting foams were dissolved in dioxane and lyophilized to
afford 13 (103 mg, 14%) and 14 (209 mg, 18%) as colorless
powders. Compound 13: 1H NMR (DMSO-d6) δ 8.12 (s, 1 H,
H-8), 7.99 (dd, J ortho ) 7.71 Hz, J meta ) 0.97 Hz, 2 H, H ortho
to NO2 of diNPEOC), 7.76-7.53 (m, 6 H, H meta and para to
NO2 of diNPEOC), 6.15 (m, 1 H, H-1′), 5.40 (t, J ) 6.69 Hz, 1
H, CO2CH2CH), 5.31 (m, 1 H, OH-3′), 5.04 (m, 1 H, OH-5′),
4.86 (d, J ) 6.50 Hz, 2 H, CO2CH2CH), 4.35 (m, 1 H, H-3′),
3.82 (m, 1 H, H-4′), 3.57-3.47 (m, 2 H, H-5′, H-5′′), 2.21 (m, 2
H, H-2′, H-2′′); FABMS (positive mode) m/z 582 (MH)+, 466
(2-N-diNPEOC-Gua + H)+; HRMS calcd for C25H24N7O10
582.1585, found 582.1583; FABMS (negative mode) m/z 580
(M - H)-, 464 (2-N-diNPEOC-Gua - H)-. Compound 14: 1H
NMR (DMSO-d6) δ 8.21 (s, 1 H, H-8), 7.90 (m, 4 H, H ortho to
NO2 of diNPEOC), 7.46-7.18 (m, 12 H, H meta and para to
NO2 of diNPEOC), 6.08 (m, 1 H, H-1′), 5.33 (m, 3 H, 2 CO2-
CH2CH, OH-5′), 4.86 (m, 5H, 2 CO2CH2CH, OH-3′), 4.23 (m, 1
H, H-3′), 3.86 (m, 1 H, H-4′), 3.55-3.42 (m, 2 H, H-5′, H-5′′),
2.09 (m, 2 H, H-2′, H-2′′); FABMS (positive mode) m/z 896
6-N-(6-Nit r over a t r yloxyca r bon yl)-2′-d eoxya d en osin e
(7). To a solution of 6 (362 mg, 0.5 mmol) in anhydrous THF
(15 mL) was added a 1 M solution of TBAF in THF (1.51 mL,
1.51 mmol), and the resulting mixture was stirred at room
temperature for 1.5 h. After evaporation, the residue was
purified by flash column chromatography (silica gel, gradient
0-10% MeOH/CH2Cl2), and the appropriate fractions were
combined and evaporated to dryness. The resulting foam was
dissolved in dioxane and lyophilized to afford 7 in quantitative
1
yield. H NMR (pyridine-d5) δ 9.15 (s, 1 H, H-8), 9.02 (s, 1 H,
H-2), 7.77 (s, 1 H, H ortho to NO2 of NVOC), 7.54 (s, 1 H, H
meta to NO2 of NVOC), 7.32 (d, 1 H, OH-3′), 6.97 (t, 1 H, OH-
5′), 6.77 (m, 1 H, H-1′), 6.01 (s, 2 H, CO2CH2), 5.19 (m, 1 H,
H-3′), 4.59 (m, 1 H, H-4′), 4.23-4.14 (m, 2 H, H-5′, H-5′′), 3.76
and 3.74 (2 s, 6H, 2 OCH3), 3.15 and 2.77 (2 m, 2 H, H-2′,
H-2′′); FABMS (positive mode) m/z 491 (MH)+; 252 (M -
NVOC + 2H)+ ) (dA + H)+; HRMS calcd for C20H23N6O9
491.1527, found 491.1572; FABMS (negative mode) m/z 489
(M - H)-, 250 (M - NVOC)- ) (dC - H)-.

6326 J . Org. Chem., Vol. 64, No. 17, 1999
Alvarez et al.
6-N-(2,2′-Bis(2-n itr oph en yl)eth yloxycar bon yl)-2′-deoxy-
a d en osin e (10). The TBAF treatment described above for the
synthesis of 7 was performed with 9 (2.5 g, 3.16 mmol) and
afforded 10 in quantitative yield.
5′-O-(4,4′-Dim eth oxytr ityl)-4-N-(2,2′-bis(2-n itr op h en yl)-
eth yloxyca r bon yl)-2′-d eoxycytid in e (4): 1H NMR (CDCl3)
δ 8.23 (d, J ) 7.55 Hz, 1 H, H-6), 7.99 (dd, J ortho ) 8.00 Hz,
J meta ) 1.34 Hz, 2 H, H ortho to NO2 of diNPEOC), 7.60-7.26
(m, 15 H, 6 H meta and para to NO2 of diNPEOC, 9 H DMTr),
6.87 (m, 5 H, 4 H DMTr, H-5), 6.25 (m, 1 H, H-1′), 5.81 (t, J )
6.32 Hz, 1 H, CO2CH2CH), 4.93 (d, J ) 6.33 Hz, 2 H, CO2CH2-
CH), 4.50 (m, 1 H, H-3′), 4.12 (m, 1 H, H-4′), 3.82 (s, 6 H, 2
OCH3), 3.48 (m, 2 H, H-5′, H-5′′), 2.70 and 2.25 (2 m, 2 H,
H-2′, H-2′′); FABMS (positive mode) m/z 844 (MH)+, 426 (4-
N-diNPEOC-Cyt + H)+, 303 (DMTr)+; HRMS calcd for
C45H42N5O12 844.2830, found 844.2729; FABMS (negative
mode) m/z 842 (M - H)-, 528 (M - DMTr)-.
5′-O-(4,4′-Dim eth oxytr ityl)-6-N-(6-n itr over a tr yloxyca r -
bon yl)-2′-d eoxya d en osin e (8): 1H NMR (CDCl3) δ 8.70 (s, 1
H, H-8), 8.15 (s, 1 H, H-2), 7.79 (s, 1 H, H ortho to NO2 of
NVOC), 7.43-7.24 (m, 10 H, 9 aromatic H of DMTr, H meta
to NO2 of NVOC), 6.81 (m, 4 H, 4 aromatic H of DMTr), 6.49
(m, 1 H, H-1′), 5.75 (s, 2 H, CO2CH2), 4.74 (m, 1 H, H-3′), 4.18
(m, 1 H, H-4′), 4.01 and 3.99 (2 s, 6 H, 2 OCH3 of NVOC), 3.80
(s, 6 H, 2 OCH3 of DMTr), 3.44 (m, 2 H, H-5′, H-5′′), 2.89 and
2.61 (2 m, 2 H, H-2′, H-2′′).
5′,3′-Bis(O-ter t-b u t yld im et h ylsilyl)-6-N-(2,2′-b is(2-n i-
tr op h en yl)eth yloxyca r bon yl)-2′-d eoxygu a n osin e (16). To
a solution of 5′,3′-bis(O-tert-butyldimethylsilyl)-2′-deoxygua-
nosine (15) (1 g, 4 mmol) in THF (20 mL) and HMPA (3 mL)
was added dropwise a 1 M solution of tert-butylmagnesium
chloride in THF (5 mL), and the mixture was stirred at 25 °C
for 1 h. To this solution was added diNPEOC-Cl (1.5 g, 8
mmol), and the resulting mixture was stirred for 4 h. Methanol
(5 mL) was added and the resulting mixture evaporated to an
oil, which was dissolved in ether (100 mL). This solution was
washed with an aqueous solution of 0.15 M EDTA (80 mL)
and then with saturated aqueous NaHCO3 (50 mL) and brine
(50 mL). The crude residue was subjected to silica gel column
chromatography (silica gel, gradient 0-2% MeOH/CH2Cl2), and
the appropriate fractions were combined and evaporated to
dryness. The resulting foam was dissolved in dioxane and
lyophilized to afford 16 in 50% yield: 1H NMR (CDCl3) δ 11.14
(sl, 1 H, NH), 8.02 (dd, J ortho ) 8.00 Hz, J meta ) 1.41 Hz, 2 H,
H ortho to NO2 of diNPEOC), 7.97 (s, 1 H, H-8), 7.91 (sl, 1 H,
NH), 7.66-7.39 (m, 6 H, H meta and para to NO2 of diNPEOC),
6.20 (m, 1 H, H-1′), 5.81 (t, J ) 7.17 Hz, 1 H, CO2CH2CH),
4.96 (d, J ) 7.16 Hz, 2 H, CO2CH2CH), 4.57 (m, 1 H, H-3′),
3.99 (m, 1 H, H-4′), 3.78 (m, 2 H, H-5′, H-5′′), 2.39 (2 m, 2 H,
H-2′, H-2′′), 0.92 (m, 18 H, 2 C(CH3)3), 0.11 (m, 12H, 2
Si(CH3)2); FABMS (positive mode) m/z 810 (MH)+, 496 (M -
diNPEOC + 2H)+; HRMS calcd for C37H52N7O10Si2 810.3314,
found 810.3352; FABMS (negative mode) m/z 808 (M - H)-,
494 (M - diNPEOC - H)-.
6-N-(2,2′-Bis(2-n it r op h en yl)et h yloxyca r b on yl)-2′-d e-
oxygu a n osin e (13). To a solution of 16 (1.5 g, 1.8 mmol) in
anhydrous THF (15 mL) was added a 1 M solution of TBAF
in THF (5.66 mL, 5.66 mmol), and the resulting mixture was
stirred at room temperature for 1.5 h. After concentration, the
mixture was extracted with an 8:2 mixture of dichloromethane
and pyridine, washed with saturated aqueous NaHCO3 (50
mL) and brine (50 mL), and then dried and concentrated. The
crude product was subjected to silica gel column chromatog-
raphy (silica gel, gradient 0-10% MeOH/CH2Cl2), and the
appropriate fractions were combined and evaporated to dry-
ness. The resulting foam was dissolved in dioxane and lyoph-
ilized to afford 13 in quantitative yield.
Gen er a l P r oced u r e for th e 5′-O-(4,4′-Dim eth oxy)tr ityl-
a tion of N-P r otected 2′-Deoxyn u cleosid es. Prior to use,
the N-protected 2′-deoxynucleosides 1, 3, 7, 10, and 13 (0.5
mmol) were dried three times by coevaporation of anhydrous
pyridine and then dissolved in anhydrous pyridine (10 mL).
4,4′-Dimethoxytrityl chloride (200 mg, 0.6 mmol) and the
resulting mixture were stirred at room temperature overnight.
Methanol (10 mL) was added, and the mixture was stirred for
a further 10 min, concentrated under reduced pressure, then
diluted with dichloromethane (100 mL), and washed with
saturated aqueous NaHCO3 (80 mL) and then with brine (80
mL). The organic layer was dried (Na2SO4), filtered, and
evaporated to dryness. Residual pyridine was removed by
coevaporations with toluene (3×). The residues were purified
by flash column chromatography (silica gel, gradient 0-2%
MeOH/CH2Cl2 containing 1% Et3N). The appropriate fractions
were combined and evaporated to dryness. The resulting foams
were dissolved in dioxane and lyophilized to give 2 (40% yield),
4 (83% yield), 8 (53% yield), 11 (60% yield), and 17 (60% yield),
respectively.
5′-O-(4,4′-Dim eth oxytr ityl)-4-N-(6-n itr over a tr yloxyca r -
bon yl)-2′-d eoxycyt id in e (2): 1H NMR (CDCl3) δ 8.24 (d,
J ) 7.35 Hz, 1 H, H-6), 7.72 (s, 1 H, H ortho to NO2 of NVOC),
7.28 (m, 9H, aromatic H of DMTr), 7.08 (s, 1 H, H meta to
NO2 of NVOC),6.93-6.69 (m, 5 H, 4 aromatic H, H-5), 6.26
(m, 1 H, H-1′), 5.60 (s, 2 H, CO2CH2), 4.49 (m, 1 H, H-3′), 4.12
(m, 1 H, H-4′), 3.96 and 3.94 (2 s, 6 H, 2 OCH3), 3.77 and 3.68
(2 s, 6 H, 2 OCH3), 3.44 (m, 2 H, H-5′, H-5′′), 2.24 and 1.44 (2
m, 2 H, H-2′, H-2′′).
5′-O-(4,4′-Dim eth oxytr ityl)-6-N-(2,2′-bis(2-n itr op h en yl)-
eth yloxycar bon yl)-2′-deoxyaden osin e (11): 1H NMR (CDCl3)
δ 8.68 (s, 1 H, H-8), 8.10 (s, 1 H, H-2), 8.01 (dd, J ortho ) 7.98
Hz, J meta ) 1.36 Hz, 2 H, H ortho to NO2 of diNPEOC), 7.60-
7.24 (m, 15 H, 6 H meta and para to NO2 of diNPEOC, 9 H of
DMTr), 6.81 (m, 4 H, 4 H of DMTr), 6.48 (m, 1 H, H-1′), 5.82
(t, J ) 6.74 Hz, 1 H, CO2CH2CH), 4.99 (d, J ) 6.79 Hz, 2 H,
CO2CH2CH), 4.72 (m, 1 H, H-3′), 4.16 (m, 1 H, H-4′), 3.80 (s,
6 H, 2 OCH3), 3.43 (m, 2 H, H-5′, H-5′′), 2.87 and 2.60 (2 m, 2
H, H-2′, H-2′′); FABMS (positive mode) m/z 868 (MH)+, 450
(4-N-diNPEOC-Ade + H)+, 303 (DMTr)+; HRMS calcd for
C
46H42N7O11 868.2942, found 868.2943; FABMS (negative
mode) m/z 866 (M - H)-, 552 (M - DMTr)-.
5′-O-(4,4′-Dim eth oxytr ityl)-2-N-(2,2′-bis(2-n itr op h en yl)-
eth yloxycar bon yl)-2′-deoxygu an osin e (17): 1H NMR (CDCl3)
δ 8.39 (s, 1 H, NH), 7.92 (dd, J ortho ) 8.08 Hz, J meta ) 1.39 Hz,
2 H, H ortho to NO2 of diNPEOC), 7.76 (s, 1 H, H-8), 7.54-
7.18 (m, 15 H, 6 H meta and para to NO2 of diNPEOC, 9 H
DMTr), 6.74 (m, 4 H, DMTr), 6.25 (m, 1 H, H-1′), 5.74 (t, J )
6.78 Hz, 1 H, CO2CH2CH), 4.90 (d, J ) 6.76 Hz, 2 H, CO2CH2-
CH), 4.76 (m, 1 H, H-3′), 4.16 (m, 1 H, H-4′), 3.76 (s, 6 H, 2
OCH3), 3.27 (m, 2 H, H-5′, H-5′′), 2.59 (m, 2 H, H-2′, H-2′′);
FABMS (positive mode) m/z 884 (MH)+, 303 (DMTr)+; HRMS
calcd for C46H42N7O12 884.2891, found 884.2979; FABMS
(negative mode) m/z 882 (M - H)-, 568 (M - DMTr)-.
(S-P iva loyl-2-t h ioet h yl)-N,N-b is(d iisop r op yla m in o)-
p h osp h in e (19). To a solution at 0 °C of N,N-bis(diisopropyl-
amino)chlorophosphine (5 g, 18.7 mmol) in diethyl ether (40
mL) was added dropwise a mixture of S-pivaloyl-2-thioethanol
(3 g, 18.7 mmol) and Et3N (2.9 mL, 20.6 mmol). The resulting
mixture was stirred at room temperature for 3 h and then
filtered; the glassware was washed with cyclohexane, and the
combined filtrates were concentrated until a viscous oil was
obtained. The oil was purified by flash column chromatography
(silica gel, elution with cyclohexanes/ethyl acetate/triethyl-
amine, 89/10/1, v/v/v). The appropriate fractions were com-
bined and evaporated to dryness to give 17 as an oil (6.3 g,
86%): 31P NMR (CDCl3) δ 126.01; 1H NMR (CDCl3) δ 3.66 (m,
2 H, OCH2CH2S), 3.54 (m, 4 H, CH(CH3)2), 3.10 (t, J ) 6.4
Hz, 2 H, OCH2CH2S), 1.25 (s, 9 H, COC(CH3)3), 1.19 (m, 24 H,
CH(CH3)2); FABMS (positive mode) m/z 393 (MH)+.
Gen er a l P r oced u r e for t h e P h osp h it yla t ion of 5′-O-
(4,4′-Dim eth oxytr ityl) N-P r otected 2′-Deoxyn u cleosid es.
Prior to use, the 5′-O-DMTr N-protected 2′-deoxynucleosides
2, 4, 8, 11, 17, and 18 (0.5 mmol) and diisopropylammonium
tetrazolide (43 mg, 0.25 mmol) were separately dried three
times by coevaporation of anhydrous acetonitrile and then
mixed and dissolved in anhydrous dichloromethane (14 mL).
A solution of (S-pivaloyl-2-thioethyl)-N,N-bis(diisopropylami-
no)phosphine (19; 235 mg, 0.6 mmol) in dichloromethane (3
mL) was added under argon. The resulting mixture was stirred
overnight at room temperature and then diluted with ethyl

Synthesis of Base-Sensitive SATE-Prooligonucleotides
J . Org. Chem., Vol. 64, No. 17, 1999 6327
acetate (60 mL) and washed with saturated aqueous NaHCO3
(50 mL) and then with brine (50 mL). The organic layer was
dried (Na2SO4), filtered, and evaporated to dryness. The
residues were purified by flash column chromatography (silica
gel, gradient 30-100% ethyl acetate/1% Et3N/cyclohexane).
The appropriate fractions were combined, evaporated to dry-
ness, and dissolved in acetonitrile (20 mL). The resulting
solution were filtered on a Millex filter, evaporated to dryness,
redissolved in benzene, and lyophilized to, respectively, afford
the resulting phosphororamidites 20 (82%), 21 (84%), 22 (60%),
23 (78%), 24 (55%), and 25 (80%) as colorless powders.
5′-O-(4,4′-Dim eth oxytr ityl)-4-N-(6-n itr over a tr yloxyca r -
bon yl)-2′-deoxycytidin e 3′-O-(S-P ivaloyl-2-th ioeth yl) N,N-
Diisop r op ylp h osp h or a m id ite (20): 31P NMR (CDCl3) δ
148.95, 148.35; 1H NMR (CDCl3) δ 8.35 (m, 1 H, H-6), 7.76 (s,
1 H, H ortho to NO2 of NVOC), 7.43-7.25 (m, 9H, H of DMTr),
7.04 (s, 1 H, H meta to NO2 of NVOC), 6.85 (m, 5 H, 4 H of
DMTr, H-5), 6.28 (m, 1 H, H-1′), 5.64 (s, 2 H, CO2CH2), 4.66
(m, 1 H, H-3′), 4.23 (m, 1 H, H-4′), 4.01 and 3.99 (2 s, 6 H, 2
OCH3 of NVOC), 3.81 (s, 6 H, 2 OCH3 of DMTr), 3.75-3.43
(m, 6 H, CH(CH3)2, OCH2CH2S, H-5′, H-5′′), 3.10-2.94 (m, 2
H, OCH2CH2S), 2.76 and 2.32 (2 m, 2 H, H-2′, H-2′′), 1.27-
1.06 (m, 21 H, CH(CH3)2, CH(CH3)3); FABMS (positive mode,
PEG) m/z 1060 (MH)+, 303 (DMTr)+.
(m, 15 H, 6 H meta and para to NO2 of diNPEOC, 9 H DMTr),
6.78 (m, 4 H, H DMTr), 6.23 (m, 1 H, H-1′), 5.78 (m, 1 H, CO2-
CH2CH), 4.93 (m, 2 H, CO2CH2CH), 4.67 (m, 1 H, H-3′), 4.24
(m, 1 H, H-4′), 3.79 (s, 6 H, 2 OCH3), 3.75-3.51 (m, 4 H, 2
CH(CH3)2, OCH2CH2S), 3.30 (m, 2 H, H-5′, H-5′′), 3.03 (m, 2
H, OCH2CH2S), 2.58 (2 m, 1 H, H-2′, H-2′′), 1.28-1.09 (m, 21
H, CH(CH3)2, CH(CH3)3); FABMS (positive mode, PEG) m/z
1175 (MH)+, 303 (DMTr)+; HRMS (NOBA) calcd for C59H68
-
N8O14PS 1175.4313, found 1175.4338; FABMS (negative mode,
PEG) m/z 1173 (M - H)-, 1189, (M + O - H)-.
Solid -P h a se Elon ga tion of P r ooligon u cleotid es Usin g
NVOC-P r otected Nu cleosid e P h osp h or a m id ites 20 a n d
22. Nucleoside phosphoramidites 20 and 22 solutions (0.1 M)
in anhydrous acetonitrile/dichloromethane (9/1, v/v) were used.
dT phosphoramidite 25 was dissolved in anhydrous acetoni-
trile. During each coupling step, a 20-fold molar excess of 20,
22, and 25 was introduced into the column bearing the
photolabile CPG solid support.13,14 A modified “1 µmol” cycle
was applied. Modifications included extended coupling times
(180 s “wait”) and an extended capping time (300 s “wait”) for
the first capping step (8 s “wait” for the next capping steps).
A solution of tert-butyl hydroperoxide (1.1 M) in toluene/
dichloromethane (obtained from commercial 3 M solution in
toluene) was then used as the oxidizer for the formation of
phosphotriester internucleoside linkages (60 s “wait”). The
coupling efficiency was evaluated by measuring the release of
the 4,4′-dimethoxytrityl carbocation by spectrophotometry (λ
498 nm) at the end of each incorporation cycle. Yields for 20
and 22 couplings were between 87 and 93% whereas the
coupling efficiency of 25 was >98%.
Solid -P h a se Elon ga tion of P r ooligon u cleotid es Usin g
d iNP EOC-P r otected Nu cleosid e P h osp h or a m id ites 21,
23, a n d 24. Nucleoside amidite 21, 23, and 24 solutions (0.06
M) in acetonitrile/dichloromethane (9/1, v/v) were dried over
4 Å molecular sieves for 24 h and then filtered off on a 0.45
µm Millex filter. A 15-fold molar excess of the amidites was
introduced into the column bearing the photolabile CPG solid
support.13,14 Modifications of the standard “1 µmol” cycle were
extended coupling times (360 s “wait”), replacement of the
common capping step by a treatment with diethyl N,N-
diisopropyl phosphoramidite (0.1 M in acetonitrile) in the
presence of 0.5 M tetrazole solution in acetonitrile31 (capping
time of 3 × 120 s “wait”). The intermediate phosphite triesters
were oxidized by a 60 s treatment with a 1.1 M solution of
tert-butyl hydroperoxide in toluene/dichloromethane or by a
30 s treatment with a solution of 3-H-1,2-benzodithiol-3-one
1,1-dioxide (0.05 M) to afford phosphate triester and thiono-
phosphate triester internucleoside linkages, respectively. The
coupling yield of nucleoside amidites 21, 23, and 24 was
between 90 and 96%.
5′-O-(4,4′-Dim eth oxytr ityl)-4-N-(2,2′-bis(2-n itr op h en yl)-
eth yloxyca r bon yl)-2′-d eoxycytid in e 3′-O-(S-P iva loyl-2-
th ioeth yl) N,N-Diisopr opylph osph or am idite (21): 31P NMR
1
(CD3CN) δ 148.96, 148.65; H NMR (CD3CN) δ 8.42 (s, 1 H,
NH), 8.21 and 8.15 (2 d, 1 H, H-6), 7.95 (dd, 2 H, H ortho to
NO2 of diNPEOC), 7.69-7.30 (m, 15 H, 6 H meta and para to
NO2 of diNPEOC, 9 H DMTr), 6.89 (m, 4 H, H DMTr), 6.81
(d, 1 H, H-5), 6.09 (m, 1 H, H-1′), 5.57 (t, 1 H, CO2CH2CH),
4.90 (d, J ) 6.27 Hz, 2 H, CO2CH2CH), 4.60 (m, 1 H, H-3′),
4.11 (m, 1 H, H-4′), 3.79 (s, 6 H, 2 OCH3), 3.67-3.54 (m, 4 H,
2 CH(CH3)2, OCH2CH2S), 3.41 (m, 2 H, H-5′, H-5′′), 3.02 (m, 2
H, OCH2CH2S), 2.53 and 2.32 (2 m, 2 H, H-2′, H-2′′), 1.29-
1.05 (m, 21 H, CH(CH3)2, CH(CH3)3); FABMS (positive mode,
PEG) m/z 1135 (MH)+, 826 (M - DMTr + 2H)+, 303 (DMTr)+;
HRMS (NOBA) calcd for C58H67N6O14PSNa 1157.4071, found
1157.4034; FABMS (negative mode, PEG) m/z 1133 (M - H)-,
1149, (M + O - H)-.
5′-O-(4,4′-Dim eth oxytr ityl)-6-N-(6-n itr over a tr yloxyca r -
bon yl)-2′-d eoxya d en osin e 3′-O-(S-P iva loyl-2-th ioeth yl)
N,N-Diisop r op ylp h osp h or a m id ite (22): 31P NMR (CDCl3)
1
δ 149.07, 149.04; H NMR (CDCl3) δ 8.70 (s, 1 H, H-8), 8.22
and 8.20 (2 s, 1 H, H-2), 7.76 (s, 1 H, H ortho to NO2 of NVOC),
7.43-7.20 (m, 10 H, 9 H of DMTr, H meta to NO2 of NVOC),
6.79 (m, 4 H, H of DMTr), 6.52 (m, 1 H, H-1′), 5.75 (s, 2 H,
CO2CH2), 4.79 (m, 1 H, H-3′), 4.35 (m, 1 H, H-4′), 4.01 and
3.99 (2 s, 6 H, 2 OCH3 of NVOC), 3.79 and 3.76 (2 s, 6 H, 2
OCH3 of DMTr), 3.72-3.44 (m, 6 H, CH(CH3)2, OCH2CH2S,
H-5′, H-5′′), 3.20-2.87 (m, 4 H, OCH2CH2S, H-2′, H-2′′), 1.29-
1.11 (m, 21 H, CH(CH3)2, CH(CH3)3); FABMS (positive mode,
PEG) m/z 1084 (MH)+, 303 (DMTr)+.
Dep r otection a n d Relea se of th e P r ooligon u cleotid es
fr om th e Solid Su p p or t. When the required number of cycles
were completed, the columns were flushed with argon and then
disassembled and the supported 5′-detritylated prooligonu-
cleotides (∼15 mg) were divided into portions (4 mg, ∼0.25
µmol). Each portion was suspended within a 1 cm path length
quartz cell in the appropriate volume of dioxane/water (3/2)
containing acetic acid (5%). To obtain a desired ∼0.1 mM
theoretical prooligo concentration (Ctheor), 2.5 mL of solution
was used, and 250 µL of solution was used for Ctheor ≈ 1 mM.
Argon was bubbled through the suspensions, and then the
magnetically stirred suspensions were exposed at 20 °C to the
light of a high-pressure Hg lamp filtered with a Pyrex glass
(2 mm thick). Aliquots of the solutions were withdrawn and
analyzed by reversed-phase HPLC. After completion of the
photolyses (table), the glass beads were filtered off on a 0.45
µm Millex filter and washed with the same solvent mixture
(2 × 0.5 mL). The prooligonucleotides were purified by
reversed-phase HPLC using linear gradients of acetonitrile (0-
90%) in 0.05 M aqueous triethylammonium acetate (pH 7). The
appropriate fractions were concentrated under reduced pres-
sure. Residues were dissolved in dioxane/water (1/1 to 3/2, v/v)
and lyophilized to afford colorless powders.
5′-O-(4,4′-Dim eth oxytr ityl)-6-N-(2,2′-bis(2-n itr op h en yl)-
eth yloxyca r bon yl)-2′-d eoxya d en osin e 3′-O-(S-P iva loyl-2-
th ioeth yl) N,N-Diisopr opylph osph or am idite (23): 31P NMR
1
(CD3CN) δ 148.79, 148.64; H NMR (CD3CN) δ 8.73 (s, 1 H,
NH), 8.51 (s,1 H, H-8), 8.20(s, 1 H, H-2), 7.93 (dd, 2 H, H ortho
to NO2 of diNPEOC), 7.66-7.42 (m, 6 H, 6 H meta and para
to NO2 of diNPEOC), 7.41-7.18 (m, 9 H, H DMTr), 6.77 (m, 4
H, H DMTr), 6.41 (m, 1 H, H-1′), 5.58 (t, J ) 6.21 Hz, 1 H,
CO2CH2CH), 4.94 (d, J ) 6.16 Hz, 2 H, CO2CH2CH), 4.88 (m,
1 H, H-3′), 4.22 (m, 1 H, H-4′), 3.74 (s, 6 H, 2 OCH3), 3.61 (m,
4 H, 2 CH(CH3)2, OCH2CH2S), 3.29 (m, 2 H, H-5′, H-5′′), 3.03
(m, 3 H, OCH2CH2S, H-2′), 2.59 (2 m, 1 H, H-2′′), 1.20-1.09
(m, 21 H, CH(CH3)2, CH(CH3)3); FABMS (positive mode, PEG)
m/z 1159 (MH)+, 1175 (M + O + H)+, 850 (M - DMTr + 2H)+,
303 (DMTr)+; HRMS (NOBA) calcd for C59H68N8O13PS
1159.4364, found 1159.4333.
5′-O-(4,4′-Dim eth oxytr ityl)-2-N-(2,2′-bis(2-n itr op h en yl)-
et h yloxyca r bon yl)-2′-d eoxygu a n osin e 3′-O-(S-P iva loyl-
2-th ioeth yl) N,N-Diisop r op ylp h osp h or a m id ite (24): 31P
NMR (CDCl3) δ 148.94, 148.65; 1H NMR (CDCl3) δ 8.03 (dd, 2
H, H ortho to NO2 of diNPEOC), 7.82 (s,1 H, H-8), 7.68-7.21

6328 J . Org. Chem., Vol. 64, No. 17, 1999
Alvarez et al.
Su p p or tin g In for m a tion Ava ila ble: Copies of the 1H and
31P NMR spectra of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
Ack n ow led gm en t. We thank the Association pour
la Recherche sur le Cancer (ARC, France) and the
Agence Nationale de Recherche sur le SIDA (ANRS,
France) for financial support.
J O990479H