Oligo-2 ' -fluoro-2 ' -deoxynucleotide N3 ' -> P5 ' phosphoramidates: synthesis and properties
Ronald G.
Schultz
and
Sergei M.
Gryaznov*
Lynx Therapeutics Inc., 3832 Bay Center Place,
Hayward
, CA 94545,
USA
Received April 25, 1996;
Revised and Accepted June 7, 1996
ABSTRACT
Uniformly modified oligodeoxyribonucleotide N3
' ->
P5
'
phosphoramidates containing 2
'
-fluoro-2
'
-deoxypyrimidine nucleosides were synthesized using an efficient interphase
amidite transfer reaction. The 3
'
-amino group of solid phase-supported 2
'
-fluoro-2
'
- deoxynucleoside was used as an acceptor and 5
'
-diisopropylamino phosphoramidite as a donor of a phosphoramidite group in the tetrazole-catalyzed exchange reaction. Subsequent oxidation with aqueous iodine resulted in formation of an internucleoside phosphoramidate diester. The prepared oligo-2
'
-fluoro- nucleotide N3
' ->
P5
'
phosphoramidates form extremely stable duplexes with complementary nucleic acids: relative to isosequential
phosphodiester oligomers, the melting temperature
T
m of their duplexes with DNA or RNA was increased
~
4 or 5
o
C per modification respectively. Moreover, these compounds are highly resistant
to enzymatic hydrolysis by snake venom phosphodiesterase and they are 4-5 times more stable in acidic media (pH 2.2-5.3) than the parent oligo- 2
'
-deoxynucleotide N3
' ->
P5
'
phosphoramidates. The described properties of the oligo-2
'
-fluoronucleotide N3
' ->
P5' phosphoramidates suggest that they may have good potential for diagnostic and
antisense therapeutic applications.
INTRODUCTION
Synthetic oligonucleotides have become powerful tools in modern molecular
biology and nucleic acid-based diagnostics. They may become a new generation of rationally designed
therapeutic agents based upon specific interference with gene expression via
antisense or antigene modes of action (
1
,
2
). Additionally, use of natural and modified oligonucleotides as aptamers could offer an interesting approach to specific inactivation of proteins following high affinity binding (
3
,
4
). Several modifications have been introduced to improve binding properties of oligonucleotides and their resistance to enzymatic hydrolysis (
5
). Recently, we described a new class of oligonucleotide analogs, N3' -> P5' phosphoramidates, where the 3'-oxygen of each 2'-deoxyfuranose was substituted by
nitrogen (
6
-
8
). These compounds form unusually stable duplexes with complementary DNA and,
especially, RNA oligomers, as well as stable triplexes with polypurine-polypyrimidine double-stranded DNA targets. Thermal stabilization of the duplexes formed
by phosphoramidates with single-stranded RNA was enhanced by up to 2.7oC per modification and with single-stranded DNA by up to 0.7oC per modification. Additionally, these compounds form
very stable homoduplexes, whose melting temperatures
T
m
were 2.1-2.3oC per modification higher relative to their phosphodiester
counterparts. The nature of the unusual stability of N3' -> P5' phosphoramidate hybrids is not yet completely clear. One of
the factors contributing to the enhanced stability of the complex is a
preference for N-sugar puckering of the 2'-deoxyfuranose of the phosphoramidates over the favored S-sugar puckering of phosphodiesters (
7
,
9
). Another contributing factor may be increased hydration and rigidity of the
phosphoramidates relative to the parent phosphodiesters (
8
).
Oligonucleotide phosphoramidates are resistant to enzymatic hydrolysis by
phosphodiesterases. Chemically, these compounds are stable under neutral and
alkaline conditions and somewhat labile under acidic conditions. Acid-catalyzed hydrolysis of the phophoramidate presumably proceeds via protonation of 3'-nitrogen, followed by nucleophilic attack at phosphorus or by
metaphosphate formation. This leads to cleavage of the internucleoside N-P bond and formation of nucleotidic fragments with terminal 3'-amino or 5'-phosphate groups (
10
). One of the possible approaches to increase acid stability of oligonucleotide
phosphoramidates is to reduce the basicity of the 3'-nitrogen atom by placing a strong electron-withdrawing group nearby. An optimal candidate for this role
could be fluorine, which is both strongly electron withdrawing and sterically
undemanding. Phosphodiester and phosphorothioate oligonucleotides containing 2'-fluoro-2'-deoxynucleosides have been synthesized for
antisense (
11
,
12
) and ribozyme (
13
) applications and they appear to adopt A-form duplexes determined by 3'-
endo
or N-sugar, puckering (
14
,
15
).
Here we report synthesis and some physico-chemical properties of pyrimidine-containing oligo-2'-fluoro-3'-aminonucleotide N3' -> P5'
phosphoramidates. These oligo-2'-fluorophosphoramidates, as will be shown below, are more
stable to acid-catalyzed hydrolysis of the phosphoramidate backbone and form exceptionally stable duplexes with complementary DNA and RNA. Two different approaches
to the synthesis of these compounds were developed: one utilizing Atherton-Todd type oxidative phosphorylation coupling and another, more efficient
method utilizing a phosphoramidite transfer reaction.
RESULTS AND DISCUSSION
Preparation of monomers
Synthesis of the oligonucleotide N3' -> P5' phosphoramidates containing 2'-fluoro-2'-deoxynucleosides was
conducted using two types of monomers, the structures of which are shown in
Schemes 1-3.
Hydrolytic stability of oligonucleotide N3
' ->
P5
'
phosphoramidites
The stability of the oligo-2'-fluoro- in comparison with the oligo-2'-deoxynucleotide N3' -> P5'
phosphoramidates toward enzymatic hydrolysis was evaluated next. Thus, phosphoramidates
19
and
24
(Table
1
) were treated with snake venom phosphodiesterase and alkaline phosphatase (for
conditions see Materials and Methods) and analyzed at successive time points by
IE HPLC. Under the conditions used, oligo-2'-deoxyphosphoramidate
19
and oligo-2'-fluorophosphoramidate
24
were hydrolyzed progressively and at similar rates, with calculated half-lives of the full-length strands equal to 4.9 and 5.4 h respectively. In comparison, decathymidylic acid with natural phosphodiester linkages was completely digested to thymidine within 10 min under the same
conditions.
Acid stability of the oligonucleotide N3' -> P5' phosphoramidates
Experiment
Oligonucleotide
T
1/2
(h)
pH 2.2
pH 4.7
pH 5.3
1
U
np
U
np
U
np
U
np
U
np
U
np
U
np
U
np
U
np
T,
19
0.34
12.3
68
2
p
U
f
np
U
f
np
U
f
np
U
f
np
U
f
np
U
f
np
U
f
np
U
f
np
U
f
np
T,
24
1.0
66
309
Additionally, the stability of the phosphoramidates toward acid-catalyzed hydrolysis was studied. Oligonucleotides
19
and
24
were incubated at room temperature in 10% acetic acid, pH 2.2, or in 20 mM sodium acetate buffers, pH 4.7 and 5.3. The hydrolysis reactions
were monitored by IE HPLC and the data are summarized in Table
3
. The observed half-lives of full-length oligonucleotide
19
at pH 2.2, 4.7 and 5.3 were 21.5 min and 12.3 and 68 h respectively. The oligo-2'-fluorophosphoramidate
24
was noticeably more stable under these conditions, with respective half-lives of this full-length oligomer of 61 min and 66 and 309 h. These results
demonstrate a markedly reduced 3'-nitrogen basicity due to electron-withdrawing 2'-fluorine, with consequently greater acid stability of oligo-2'-fluorophosphoramidates
relative to the parent oligo-2'-deoxyphosphoramidates.
Notably, oligo-2'-fluoro-3'-aminouridine phosphoramidate
22
is not stable to prolonged heating in concentrated aqueous ammonia, with by-products becoming detectable by IE HPLC after heating at 55oC for more than 1 h. After incubation for 8 h, nearly 50% of
22
was transformed into closely eluting products, which were isolated and analyzed
by mass spectrometry. Two modifications were tentatively identified as arising
from O2 uracil-mediated elimination of 2'-fluorine: full-length oligonucleotides containing one
arabinonucleoside phosphoramidate or one 2,2'-anhydronucleoside phosphoramidate residue. Similar transformation
to arabinouridine has been reported for an oligomer containing 3'-terminal 2'-fluorouridine (
26
). A third type of modification was detected but not identified (M+15 a.m.u.). In contrast,
oligo-2'-deoxy-3'-aminouridine phosphoramidite
19
was stable to concentrated ammonia at 55oC even for 16 h.
Thermal stability of the phosphoramidite duplexes
We evaluated the ability of the oligo-2'-fluoronucleoside phosphoramidates to hybridize with
complementary DNA and RNA. Melting temperatures were determined for duplexes
formed under close to physiological salt concentrations and the results are
summarized in Table
2
. Substitution of one 2'-deoxynucleoside by one 2'-fluoronucleoside in a phosphoramidate decamer led to
an increase in
T
m
by ~2oC for either DNA or RNA hybrids (experiments 2 and 3, Table
2
). Accordingly, replacement of two 2'-deoxynucleosides with 2'-fluoronucleosides in the same phosphoramidate decamer
led to an increase in duplex
T
m
of 3.5-6.4oC. In contrast, others have reported that substitution with two
central 2'-fluoronucleosides destabilizes the duplexes of phosphodiester
oligomers (
27
).
Substitution of all 2'-fluoronucleosides for 2'-deoxynucleosides in the decamer phosphoramidate
resulted in significant enhancement of duplex thermal stability:
T
m
values were increased by 16-25oC (compare experiments 2 and 5, 6 and 7 Table
2
). The data show that further increases in the proportion of N-sugar ring pucker in the N3' -> P5' phosphoramidates as well as additional negative polarization of 3'-amino groups (by 2'-fluorine) substantially stabilized
oligonucleotide duplexes. It is noteworthy that
T
m
values of the duplexes formed by 2'-fluoroamidates were 38-44oC higher than those of isosequential phosphodiester compounds, with 4-5oC per modification increases in melting
temperatures (compare experiments 1, 5 and 6, Table
2
).
The mixing curves obtained for the oligonucleotides from experiment 6 (Table
2
) in melting buffers containing 10 mM magnesium chloride demonstrate 1:1
stoichiometry of the complex formed by phosphoramidate
23
and the complementary polypurine strand. Moreover, thermal dissociation
experiments conducted within the 15-80oC temperature range resulted in single transition melting curves
with highest hypochromicity (~25%) for the 1:1 mixtures of purine and pyrimidine strands, but not for the
1:2 mixtures (15%). These data indicate that duplexes, not triplexes, are
likely formed by the tested oligonucleotide phosphoramidates under the
hybridization conditions used.
The same trend in duplex thermal stability was observed for mixed base 11mer
27
(Table
2
), which formed more stable hybrids with complementary DNA and RNA than did the analogous oligo- 2'-deoxynucleoside phosphoramidate
26
(compare experiments 9 and 10, Table
2
).
In conclusion, the oligonucleotide N3' -> P5' phosphoramidates containing 2'-fluoro-3'-aminonucleosides were
synthesized using two different approaches, with the phosphoramidite transfer reaction shown as
an efficient method for assembly of uniformly modified oligomers. The 2'-fluoro-modified phosphoramidates form extremely stable duplexes with
complementary DNA and RNA under close to physiological conditions, where
T
m
values were increased by 4-5oC per modification relative to natural phosphodiesters. In addition,
these compounds were more stable in acidic media than 2'-deoxynucleoside phosphoramidates and are comparably resistant to
enzymatic digestion by snake venom phosphodiesterase. The described properties of the oligo-2'-fluoro- 3'-aminonucleotide phosphoramidates indicate
that they have good potential as diagnostic and possible antisense agents.
MATERIALS AND METHODS
General methods
Phosphodiester oligodeoxyribonucleotides and oligoribonucleotides were prepared
on an ABI 380B DNA synthesizer using standard protocol via the phosphoramidite
method (
28
). Oligonucleotide N3' -> P5' phosphoramidates, containing 2'-deoxyribonucleosides and one or two 2'-fluoronucleosides were synthesized
using the oxidative phosphorylation method on a ABI 394 synthesizer as
previously described (
8
). Uniformly modified oligo-2'-fluoronucleotide N3' -> P5' phosphoramidates were prepared by the
amidite transfer reaction on an ABI 380B synthesizer using the following
protocol: (i) detritylation, 5% dichloroacetic acid in dichloromethane, 1 min; (ii) coupling, 0.1 M phosphoramidite
2
and 0.45 M tetrazole in acetonitrile, 3 min; (iii) oxidation, 0.1 M iodine in tetrahydrofuran/pyridine/water, 10/10/1 v/v/v,
1 min; (iv) capping, acetylation of unreacted 3'-amino groups by standard ABI capping solutions, 30 s.
Chemical steps within the cycle were followed by acetonitrile washing and flushing with dry argon for 0.2-0.4 min. After cleavage from the solid support and deprotection with concentrated aqueous ammonia (1-1.5 h, 55oC) oligonucleotides were analyzed and purified by IE HPLC. Oligonucleotides were desalted on
Pharmacia NAP-5 or NAP-10 gel filtration columns immediately after purification and stored
frozen or lyophilized at -18oC.
Preparation of the 5'-phosphorylated oligonucleotides was upon sulfone-derivatized CPG (
29
) and 5'-hydroxyl oligomers were synthesized upon oligonucleotide-succinyl CPG.
Oligonucleotide thermal dissociation experiments were carried out as previously
described (
7
), with melting buffers as listed in Table
2
.
Acid hydrolysis of 0.17 OD
260
of the dimer dU
f
np
T was in 25 [mu]l 64% acetic acid (2 h at 55oC) and the reaction mixture was analyzed by RP HPLC. Approximately 83%
of the dimer, retention time (Rt) 15.0 min, was hydrolyzed to mainly 5'-thymidylic acid, Rt 10.6 min, and 2'-fluoro-3'-aminouridine, Rt 11.2 min, as identified by co-injection with authentic
standards. Also, ~7.5% of thymidine, Rt 12.1 min, was found in the reaction mixture.
Acid hydrolysis of the oligonucleotide phosphoramidates (Table
3
), 1-3 OD
260
of each, was carried out at room temperature in 0.1-0.2 ml 10% acetic acid, pH 2.2, or in 20 mM sodium acetate buffers, pH
4.7 and 5.3. For enzymatic digestion, 0.2 OD
260
of oligonucleotides
19
and
22
(Table
2
) were treated with 0.02 U snake venom phosphodiesterase and 0.8 U alkaline
phosphatase (Sigma, St Louis, MO) in 0.2 ml 10 mM Tris-HCl buffer, pH 8.9, at room temperature. Aliquots from the reaction
mixtures were taken at multiple time points and analyzed by IE HPLC.
5
'
-
O
-DMT-3
'
-azido-3
'
-deoxyarabinouridine (5)
To 13.8 g (26 mmol)
4
in 500 ml acetone was added 200 ml H
2
O and 12.0 g (185 mmol) NaN
3
. The mixture was refluxed overnight, then concentrated
in vacuo
to remove the acetone. The resultant slurry was extracted with 600 ml CH
2
Cl
2
, which in turn was washed with water (3 * 250 ml). Concentration of the CH
2
Cl
2
layer and flash chromatography of the crude product provided 6.0 g (40%) of a
pale yellow solid. Mass spectrometry, FAB
+
, M+H
+
, calculated, 572.2145; observed, 572.2147.
1
H NMR [delta] 9.4 (br s, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.4-7.3 (mm), 6.89 (d, J = 7.9 Hz, 4H), 6.10 (d, J = 5.5 Hz, 1H), 5.46
(d, J = 8.1 Hz, 1H), 4.55 (m, 1H), 4.19 (dd, J = 7.2, 7.7 Hz, 1H), 3.84 (m,
1H), 3.83 (s, 6H), 3.64 (dd J = 2.6, 11.3 Hz, 1H), 3.43 (dd, J = 2.6, 11.4 Hz,
1H).
To 2.9 g (5.1 mmol)
6
in 75 ml 95% ethanol was added 0.5 g 10% palladium on carbon. The mixture was
hydrogenated at 40 p.s.i. overnight and then the catalyst removed by
filtration. The solvent was removed
in vacuo
and the resultant solid purified by flash chromatography to give 1.2 g (43%) of
product as a white powder. Mass spectrometry, FAB
+
, M+H
+
, calculated, 548.2197; observed, 548.2206.
1
H NMR [delta] 8.04 (d, J = 8.0 Hz, 1H), 7.9 (br m, 1H), 7.2-7.4 (mm), 6.85 (d, J = 8.4 Hz, 4H), 6.00 (d, J = 16.2 Hz, 1H),
5.32 (d, J = 8.7 Hz, 1H), 4.83 (dd, J = 3.9, 52.2 Hz, 1H), 3.88 (br d, J = 11
Hz, 1H), 3.8 (s, 6H), 3.8-3.7 (mm, 2H), 3.52 (dd, J = 2.6, 11.1 Hz, 1H);
19
F NMR [delta] -200.1 (ddd, J = 16.4, 27.5, 52.1 Hz).
3
'
-
O
-Methanesulfonyl-5
'
-
O
-benzoyl-2,2
'
-anhydroarabinouridine (7)
This was prepared in two steps from
3
according to the procedure of Codington
et al
. (
17
) in 69-77% overall yields.
5
'
-
O
-benzoyl-2
'
,3
'
-anhydrolyxouridine (8)
This was prepared in two steps from
7
according to the procedure of Codington
et al.
(18) in 63-77% overall yields.
3
'
-Azido-5
'
-
O
-benzoyl-3
'
-deoxyarabinouridine (9)
This was prepared from
8
and anhydrous NH
4
N
3
(
30
), according to the procedure of Reichman
et al.
(
19
), but without successful recrystallization. Mass yields were 98% or greater,
but NMR suggested 25-35% of the regioisomer, 2'-azido-5'-
O
-benzoyl-2'- deoxyxylouridine
9i
, which co-eluted with the desired product on silica gel TLC.
1
H NMR, major component
9
[delta] 10.8 (br s, 1H), 8.11 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 8.1 Hz, 1H),
7.62 (d, J = 7.3 Hz, 1H), 7.5 (m, 2H), 6.19 (d, J = 3.6 Hz, 1H), 5.40, (d, J =
8.0 Hz, 1H), 4.84 (m, 1H), 4.73 (d, J = 5.7 Hz, 1H), 4.63 (br d, J = 4.2 Hz,
1H), 4.2 (mm, 2H); minor component
9i
[delta] 10.6 (br s, 1H), 8.11 (d, J = 7.5 Hz, 2H), 7.81 (d, J = 8.1 Hz, 1H),
7.64 (d, J = 7.5 Hz, 1H), 7.5 (m, 2H), 5.85 (s, 1H), 5.47, (d, J = 8.1 Hz, 1H),
4.86 (m, 1H), 4.76 (d, J = 5.4 Hz, 1H), 4.62 (br d, J = 4.0 Hz, 1H), 4.3-4.2 (mm, 2H).
To 1.0 g (2.9 mmol)
11
in 50 ml anhydrous pyridine was added 1.0 g (3.2 mmol) 4-methoxytrityl chloride. The mixture was stirred overnight, 5 ml saturated
aqueous NaHCO
3
was added and the mixture concentrated
in vacuo
to an oil. The oil was dissolved in 125 ml ethyl acetate, which was washed with water (3 * 100 ml) and reconcentrated
in vacuo
to 2.05 g of foam. The foam was dissolved in a mixture of 40 ml methanol, 40 ml dioxane and 10 ml water. NaOH (1 g, 25 mmol) was added and the mixture stirred overnight. The
solution was concentrated
in vacuo
to a syrup, which was dissolved in 100 ml ethyl acetate and washed with water
(3 * 100 ml). Concentration
in vacuo
of the organic layer gave 1.11 g of a foam, which upon flash chromatography
gave 1.05 g (76%) of a white solid. Mass spectrometry, FAB
+
, M+H
+
, calculated, 518.2091; observed, 518.2076.
1
H NMR [delta] 8.64 (br d J = 4.2 Hz, 1H), 8.14 (br s, 1H), 7.57 (mm, 5H), 7.48 (d J =
8.7 Hz, 1H), 7.3 (mm, 8H), 6.83 (d J = 8.8 Hz, 2H), 5.67 (d, J = 17.7 Hz, 1H),
5.62 (d, J = 8.1 Hz, 1H), 4.23 (m, 2H), 4.03 (br d, J = 10.2 Hz, 1H), 3.80 (s,
3H), 3.31 (dddd, J = 3.6, 10.3, 10.9, 25.8 Hz, 1H), 2.80 (dd, J = 3.6, 50.9 Hz,
1H), 2.51 (dd, J = 3.0, 11.2 Hz, 1H);
19
F NMR [delta] -192.5 (dddd, J = 2.9, 17.7, 26.1, 50.9 Hz).
2
'
-Fluoro-3
'
-(4-methoxytrityl)amino-2
'
,3
'
-dideoxyuridine 5
'
-(2-cyanoethyl
N
,
N
-diisopropyl)phosphoramidite (2u)
Mass spectrometry, FAB
+
, M+H
+
, calculated, 718.3170; observed; 718.3194.
19
F NMR [delta] -190.9 (ddd, J = 21.7, 21.8, 51.3 Hz), -193.4 (ddd);
31
P NMR [delta] 150.5, 149.4.
N
4,5
'
-
O
-Dibenzoyl-2
'
-fluoro-3
'
-amino-2
'
,3
'
-dideoxycytidine (13)
To 6.9 g (18.4 mmol) crude
10
(containing 35%
10i
) in 50 ml anhydrous CH
3
CN was added an ice-cold solution of 11.7 g (169 mmol) 1,2,4-triazole and 3.35 ml (36.1 mmol) POCl
3
in 90 ml anhydrous CH
3
CN. The mixture was cooled in an ice bath and anhydrous triethylamine (23 ml,
165 mmol) was added, then the reaction allowed to warm to room temperature with
stirring. After 90 min, 15 ml (108 mmol) triethylamine and 4 ml water were
added and the mixture stirred for 10 min. The solvent was removed
in vacuo
, then 250 ml ethyl acetate was added and the solution was washed with saturated aqueous NaHCO
3
(2 * 250 ml) and with 250 ml water. TLC indicated a fluorescent intermediate with the
same mobility as the starting material. The mixture was concentrated
in vacuo
to 6.7 g of a foam. Dioxane (100 ml) and 20 ml concentrated aqueous ammonia
were added and, after 3 h, the mixture was concentrated
in vacuo
to a yellow gel. The gel was dissolved in 100 ml ethyl acetate and washed with water (3 * 200 ml). Concentration
in vacuo
and vacuum desiccation over P
2
O
5
yielded 5.4 g of a solid which gave only one spot on silica gel TLC. Only two
significant signals were observed by
19
F NMR, major component [delta] -192.8 (ddd, J = 22.8, 22.8, 53.1 Hz); minor component [delta] -200.7 (ddd, J = 13.6, 19.9, 53.4 Hz).
Anhydrous pyridine (100 ml) was added and the solution cooled to 4oC. Benzoyl chloride (11.7 ml 100 mmol) was added with stirring and the
mixture allowed to warm to room temperature. After 2 h, 5 ml water was added
and the solvent removed
in vacuo
, giving a brown oil, which was dissolved in 200 ml ethyl acetate, washed with water (3 * 200 ml) and then reconcentrated
in vacuo
to an oily foam. Ethanol (150 ml) and 2 g 10% palladium on activated carbon were added and the mixture was hydrogenated
at 40 p.s.i. H
2
overnight. TLC indicated formation of two slower, closely migrating compounds.
The catalyst was removed by filtration and the filtrate concentrated
in vacuo
to an oily yellow foam. Silica gel flash chromatography (500 ml silica, eluted
with 0-3% CH
3
OH in CH
2
Cl
2
) provided 1.85 g of semi-pure product, which was dissolved in 10 ml CH
2
Cl
2
. A solid quickly precipitated, which was collected by filtration and washed
with fresh CH
2
Cl
2
. Vacuum desiccation yielded 1.5 g of product
13
(11% yield from
9
and
9i
) as fine white crystals. Mass spectrometry, FAB
+
, M+H
+
, calculated, 453.1574; observed, 453.1574.
1
H NMR [delta] 8.21 (d, J = 7.5 Hz, 1H), 8.08-8.13 (mm, 3H), 7.94 (d, J = 7.4 Hz, 2H), 7.46-7.7 (mm, 8H), 6.04 (d, J = 16.9 Hz, 1H), 5.08 (dd, J = 3.6,
51.5 Hz, 1H), 4.85 (dd, J = 3.3, 12.8 Hz, 1H), 4.80 (dd, J = 2.1, 12.8 Hz, 1H),
4.26 (m, 1H), 3.48 (dm, J = 27 Hz, 1H);
19
F NMR [delta] -200.1 (m).
N
4,5
'
-
O
-Dibenzoyl-2
'
-fluoro-3
'
-(4-methoxytrityl)amino-2
'
,3
'
-dideoxycytidine (14)
To 0.9 g (2.0 mmol)
13
in 25 ml anhydrous pyridine was added 0.86 g (2.8 mmol) 4-methoxytrityl chloride and the mixture stirred overnight. The reaction was
quenched with 0.5 ml H
2
O and concentrated
in vacuo
. CH
2
Cl
2
(50 ml) was added and washed with 50 ml saturated aqueous NaHCO
3
and with water (2 * 50 ml). The solvent was removed
in vacuo
, replaced with 10 ml CH
2
Cl
2
and pipetted into 80 ml rapidly stirred 1/1 hexane/ether. After further
stirring for 2 h, the product was collected by filtration and dried under
vacuum, giving 1.3 g (88% yield) of product as a white powder. Mass
spectrometry, FAB
+
, M+H
+
, calculated, 725.2775; observed, 725.2761.
1
H NMR [delta] 8.59 (br s, 1H), 8.07 (br d, J = 5.7 Hz, 1H), 7.89 (br d, J = 7 Hz, 2H),
7.83 (dd, J = 1.3, 6.7 Hz, 2H), 7.68 (dd, J = 7.4, 7.4 Hz, 2H), 7.5-7.6 (m, 8H), 7.43 (dd, J = 2.1, 6.9 Hz, 2H), 7.1-7.3 (mm, 7H), 6.71 (d, J = 8.9 Hz, 2H), 5.80 (d, J = 15.4 Hz, 1H),
5.03 (dd, J = 2.0, 13.0 Hz, 1H), 4.98 (dd, J = 2.3;, 13.1 Hz, 1H), 4.41 (br d,
J = 10.5 Hz, 1H), 3.63 (s, 3H), 3.36 (dddd, J = 3.1, 11.1, 11.1, 25.7 Hz, 1H),
2.84 (dd, J = 3.1, 49.9 Hz, 1H), 2.52 (dd, J = 2.7, 11.5 Hz, 1H);
19
F NMR [delta] -196.3 (m).
To 1.3 g (1.75 mmol)
14
in 20 ml 65/30/5 pyridine/methanol/water, cooled in an ice bath, was added 10
ml cold 2 M NaOH in 65/30/5 pyridine/methanol/water. The mixture was stirred
cold for 20 min, then neutralized with pyridinium-H
+
form BioRad AG 50W-X8 cation exchange resin. After 5 min, the resin was removed by filtration
and washed with methanol. The combined filtrate and wash were concentrated
in vacuo
to an oil, which was dissolved in 100 ml ethyl acetate. The mixture was washed
with 100 ml saturated aqueous NaHCO
3
and with water (2 * 100 ml). After concentration
in vacuo
to a foam, the product was dissolved in 10 ml CH
2
Cl
2
and pipetted into 75 ml rapidly stirred hexane/ether, 2/1. The product was
collected by filtration and dried under vacuum, giving 1.13 g (~100%) of product as a white powder. Mass spectrometry, FAB
+
, M+Cs
+
, calculated, 753.1489; observed, 753.1499.
1
H NMR [delta] 8.30 (br d, J = 6.8 Hz, 1H), 7.89 (br d, J = 6.7 Hz, 2H), 7.64 (dd, J =
7.4, 7.4 Hz, 1H), 7.44-7.56 (mm, 9H), 7.22-7.32 (mm, 9H), 6.82 (d, J = 8.8 Hz, 2H), 5.80 (d, J = 15.7 Hz,
1H), 4.26 (mm, 2H), 4.13 (d, J = 10.2 Hz, 1H), 3.81 (s, 3H), 3.26 (dddd, J =
3.4, 10.7, 10.8, 26.5 Hz, 1H), 2.93 (dd, J = 3.3, 50.5 Hz, 1H), 2.50 (dd, J =
2.8, 11.0 Hz, 1H);
19
F NMR [delta] -195.3 (m).
N
4-Benzoyl-2
'
-fluoro-3
'
-(4-methoxytrityl)amino-2
'
,3
'
- dideoxycytidine 5
'
-(2-cyanoethyl
N
,
N
-diisopropyl) phosphoramidite (2c)
Mass spectrometry, FAB
+
, M+Cs
+
, calculated, 953.2568; observed, 953.2531.
19
F NMR [delta] -193.6 (m);
31
P NMR [delta] 150.4, 149.4.
