ABSTRACT
The first method for solid support synthesis of all-
R
P-oligo(ribonucleoside phosphorothioate)s is presented as well as attempts
to increase the stereoselectivity of the key step in this approach. The synthetic strategy consists of (i) a solid support synthesis procedure, using 5
'
-
O
-(4-methoxytriphenylmethyl)-2
'
-
O
-
tert
-butyldimethylsilyl-ribonucleoside 3
'
-H- phosphonates, that due to stereoselectivity in the condensation step, gives oligomers with mostly
S
P-H- phosphonate diesters (72-89% under standard conditions), (ii) stereospecific sulfurization with S8 in
pyridine to produce oligo(ribonucleoside phophorothioate)s enriched with
internucleosidic linkages of
R
P configuration, (iii) treatment of the deprotected oligonucleotides with the enzyme Nuclease P1 from
Penicillium citrinum
, that specifically catalyses cleavage of
S
P-phosphorothioate diester linkages, which leaves a mixture of oligomers having all internucleosidic linkages as
R
P-phosphorothioates, and finally (iv) isolation and HPLC purification of the full length all-
R
P oligomer. Mixed sequences containing the four common nucleosidic residues up to the
chain length of a heptamer were synthesized. Change of
N
-4-protection on the cytidine building block from propionyl to
N
-methylpyrrolidin-2-ylidene gave a slightly improved diastereoselectivity in H-phosphonate diester formation. Increased selectivity up
to 99+ % was obtained with the guanosine building block when the amount of
pyridine in the coupling step was reduced.
Largely due to their usually higher stability towards enzymatic degradation (
1
), phosphorothioate internucleosidic linkages are currently the most common type
of phosphate modification in oligonucleotides evaluated for use as therapeutic
agents in treatment of various diseases (
2
,
3
). These oligo(nucleoside phosphorothioate)s usually have the sulfur in the
position of a non-bridging oxygen. The diastereotopicity of these oxygens gives rise to a
pair of diastereomeric phosphorothioates (denoted
R
P
and
S
P
) when one of them is replaced by sulfur. This chirality of phosphorothioates
makes them particularly suitable for analysis of the stereochemical course of
the phosphate transfer reaction. The use of nucleoside phosphorothioates as
tools in biological investigations, particularly for stereochemical analyses of
enzyme catalysed reactions, has been thoroughly reviewed (
4
-
7
). Examples where phosphorothioates are used to probe details of enzymatic
reaction mechanisms are also becoming more common (
8
-
11
).
The chirality of the phosphorothioates can be an asset but this also poses
difficulties in synthesis since most methods do not give stereochemically
homogeneous products. The most common method for synthesis of oligo(nucleoside
phosphorothioate)s (
1
) [based on the phosphoramidite (
12
,
13
) approach combined with sulfurization] gives stereoisomeric mixtures of
oligomers (
1
,
14
), although with a high degree of reproducibility (
15
) and with a slight preference for the
R
P
-isomer. Despite this type of oligonucleotide analog being one of the most
explored, it is only recently that it has been possible to obtain a high degree
of isomeric purity in chemically synthesized oligo(deoxyribonucleoside
phosphorothioate)s. The method by Stec
et al
. (
16
-
18
) is, to our knowledge, the only functional method for this purpose. In this
method, the oligonucleotidic chain is extended in a 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) catalysed reaction with 5'-
O
-dimethoxytritylnucleoside 3'-
O
-(2-thio-1,3,2-oxathiaphospholane)s, or the corresponding 2-thio-1,3,2-oxaselenaphospholanes (
19
) as a further possibility. For RNA fragments progress has been even slower and
before our present study only dimers and trimers that originated from
stereocontrolled chemical reactions were reported (
20
).
We have previously reported that condensation of a 2',3'-
O
-protected nucleoside with a 5'-
O
-protected 2'-
O
-
t
-butyldimethylsilyluridine 3'-H-phosphonate is a stereoselective reaction (
21
,
22
). About 85% of the reaction produces the
S
P
-diastereoisomer. Similar stereoselectivity has been reported for synthesis
of 2',5'-linkages, also with
t
-butyldimethylsilyl (TBDMS) protection for the secondary hydroxyl vicinal to the H-phosphonate function (
23
,
24
). We, and independently Seela and Kretschmer, have found that elemental sulfur converts dinucleoside H-phosphonates to the phosphorothioates in a stereospecific reaction (
21
,
22
,
25
,
26
). Taking advantage of this stereospecific sulfurization in combination with the stereoselective condensation we have started to explore the possibility to synthesize oligo(ribonucleoside phosphorothioate)s with homogeneous stereochemistry around the phosphorus centers. Using this
strategy we demonstrated synthesis of all-
R
P
-oligo(uridine phosphorothioate)s consisting of up to 12 nucleosidic
residues (
27
). We are herein presenting the first method for solid support synthesis of all-
R
P
-oligo(ribonucleoside phosphorothioate)s and attempt to increase the
stereoselectivity of the key step in this approach.
Our synthetic strategy is based on the H-phosphonate approach (
28
-
32
) and is shown in Scheme 1. (i) Using our method for H-phosphonate based RNA-synthesis (
33
-
36
) an oligo(nucleoside H-phosphonate), with a substantially higher content of
S
P
-linkages, is synthesized on solid support. (ii) The H-phosphonate linkages are, after completion of the elongation cycles,
stereospecifically sulfurized with S
8
in pyridine to produce an isomeric mixture of oligo(ribonucleoside
phosphorothioate)s enriched with linkages of
R
P
configuration (the production of isomeric mixtures of phophorothioates from oligo(ribonucleoside H-phosphonate)s has been reported previously (
37
) and the 2
n
stereoisomeric compounds formed after
n
number of condensations have sufficiently similar physical properties to makes their separation most difficult). (iii) The deprotected oligonucleotides are then subjected to the enzyme Nuclease P1 that specifically catalyses cleavage of
S
P
-phosphorothioate diester linkages (
38
) to leave a mixture of oligomers having all internucleosidic linkages as
R
P
-phosphorothioates. (iv) The full length all-
R
P
oligomer can then be easily separated from the shorter fragments by HPLC.
Pyridine (Labscan), acetonitrile (Merck pa) and triethylamine (Aldrich) were
dried by refluxing over CaH
2
, distilled and stored over molecular sieves or CaH
2
(in case of triethylamine). Dichloroethane (Merck pa) was stored over molecular
sieves. THF (Merck pa) was dried by refluxing over LAH and freshly distilled
prior to use. Pivaloyl chloride was distilled at atmospheric pressure and
stored at -20oC in sealed flasks. Trifluoroacetic acid was distilled at
atmospheric pressure and stored at room temperature in sealed flasks. Chloroform was passed through basic Al
2
O
3
prior to use. Uridine (Sigma), cytidine (Sigma), silver nitrate (Merck),
triethylamine trihydrofluoride (Aldrich),
tert
-butyldiphenylsilyl chloride (Aldrich), triisopropylsilyl chloride (Aldrich),
tert
-butyldimethylsilyl chloride (Aldrich), 4-methoxytriphenylmethyl chloride (Aldrich), sulfur (Aldrich),
phosphorus trichloride (Merck), ammonium acetate (Merck), potassium bromide
(Merck), lithium perchlorate (Merck), magnesium chloride (Merck), zinc sulfate
(Merck) and imidazole (Sigma) were all commercial grade. Concentrated (32%)
ammonia (Merck) and ethanol used for deprotection were kept in tightly closed
bottles at 8oC, mixed briefly before use and added cold to the solid support. The water
used was freshly doubly distilled and all glassware used were dried at 150oC overnight. Triethylammonium bicarbonate buffer (TEAB) (2 M, pH [approx]7) was prepared by passing carbon dioxide through an aqueous solution
containing the appropriate amount of triethylamine, and stored at 8oC. Column chromatography was performed in the flash mode using silica gel
(35-70 [mu]m) from Amicon Europe. All evaporations were carried out under
reduced pressure using rotatory evaporator.
N
-Methyl-2,2-dimethoxypyrrolidine, 5'-
O
-(4-methoxytriphenylmethyl)-4-
N
-(
N
-methylpyrrolidin-2-ylidene) cytidine, and 5'-
O
- (4-methoxytriphenylmethyl)-4-
N
-[2-(
tert
-butyldiphenyl-siloxy)-methyl]benzoyl]-2'-
O
-
tert
-butyldi-methyl- silyl-cytidine were prepared using published procedures (
51
,
52
). Silylation procedures for preparation of the different uridine derivatives were slight modifications of literature procedures (
46
,
58
,
59
). Ribonucleside H-phosphonates 1-5 were prepared using the PCl
3
/imidazole/triethylamine reagent system (
29
,
60
) with the exception that evaporation of a solution of the crude phosphonate in
pyridine-triethylamine (4:1) prior to chromatography was omitted. The solid
support, used for oligoribonucleotide synthesis was long chain alkylamine
controlled pore glass (LCAA-CPG, 500 Å pores) from Pierce. The solid support was functionalized with 5'-
O
-(4-methoxytriphenylmethyl)-6-
N
-butyryl-2'-
O
-
tert
-butyldimethylsilyladenosine 3'-
O
-succinate using a standard (
61
) (giving a loading of 25 [mu]mol/g) or a modified later procedure (
62
) giving a loading of 32 [mu]mol/g.
Machine assisted solid phase synthesis was carried out using a modified Gene
Assembler (Pharmacia). For syringe synthesis a gas-tight Hamilton syringe equipped with a sintered glass filter at the base
was used. Nuclease P1 (from
Penicillium citrinum
) and alkaline phosphatase (AP, from calf intestine 10 mg in 1 ml suspension pH
7 of 3.2 M ammonium sulfate, 1 mM magnesium chloride and 0.1 mM zinc chloride)
were purchased from Boehringer GmbH, and snake venom phosphodiesterase (SVPD,
from
Crotalus adamanteus
) was purchased from Sigma. All HPLC analyses and purifications were performed
with a Gilson HPLC-system detecting the eluate at 260 nm.
All the reactions were monitored using the following procedure. The appropriate
H-phosphonate
1-5
(50 [mu]mol) and 2',3'-di-
O
-benzoyluridine (27 mg, 60 [mu]mol) were rendered anhydrous by evaporation of added pyridine and dissolved in acetonitrile-pyridine (2 ml, the amount of pyridine in acetonitrile according to Tables
1
and
2
). Pivaloyl chloride (18 [mu]l, 150 [mu]mol) was added and the reaction was monitored by
31
P NMR in 10 mm tubes. The resonances corresponding to the produced H-phosphonate diesters were integrated in order to quantify each isomer
(error is within 3%). Variation of acquisition time, pulse delay,
1
H-decoupling power and comparison of coupled and
1
H-decoupled spectra did not give any substantial differences. It is assumed
that the major isomer is always the
S
P
isomer, which is also consistent with previous observations that the resonance
of this isomer is downfield of the
R
P
-isomer (
22
).
Oligoribonucleotide syntheses were performed using 0.5 [mu]mol support-bound nucleoside and included detritylation with 1% trifluoroacetic
acid in dichloroethane for 1 min and condensations during 1.5 min using an
alternating mode of segments (ratio 3:2) of H-phosphonate building blocks (50 mM) and pivaloyl chloride (225 mM) in
pyridine-acetonitrile (1:3 v/v). Washings using appropriate solvents were
performed between the chemical steps as follows: 2 min dichloroethane (DCE),
detritylation, 2 min DCE, 1 min MeCN, 1 min MeCN-pyridine (3:1), coupling, 1
min MeCN-pyridine (3:1), 1 min MeCN. After the desired number of elongations,
the solid support was dried under reduced pressure.
Support-bound nucleoside (10.0 [mu]mol) were treated sequentially by drawing into the syringe container
reagent solutions and washing solvents. Detritylation was performed with 1%
trifluoroacetic acid in dichloroethane (7 * 2 ml, 7 * 1 min). Condensations were performed by mixing solutions of H-phosphonate building blocks in pyridine (0.5 ml, 75 mM) and
a solution of pivaloyl chloride in acetonitrile (1.5 ml, 225 mM) and then
rapidly take up the mixture into the syringe with the solid support and to mix
the solution further by swirling throughout the coupling time. Washings using
appropriate solvents were performed between the chemical steps as follows: 5 * 2 ml dichloroethane (DCE), detritylation, 3 * 2 ml DCE, 5 * 2 ml MeCN-pyridine (3:1), coupling, 3 * 2 ml MeCN-pyridine (3:1), 4 * 2 ml MeCN. The solid support was dried under
reduced pressure.
The dried solid support with the assembled oligo(nucleoside H-phosphonate) was transferred to a 2 ml cryo vial (with screw cap) and
treated at room temperature with a solution of sulfur in pyridine (1 ml; 14 mg
sulfur is added to 1 ml pyridine whereupon most of the S
8
is dissolved, but a small fraction is not indicating that the solution is
saturated, concentration is ~0.055 M S
8
) for 18 h. The solid phase was filtered off and washed sequentially with pyridine, acetonitrile and dichloromethane and finally dried under reduced pressure.
After sulfurization, the solid support was transferred to a 2 ml cryo vial (with
screw cap) and treated at room temperature with 32% NH
3
(aq)
-EtOH (1.2 ml, 3:1, v/v) for 16 h. The support was filtered off and
lyophilization of the filtrate gave a white powder. Triethylammonium
trihydrofluoride (
41
,
42
) (0.3 ml) was added and the reaction was allowed to stand at room temperature
for 5 h. Water (0.5 ml) was added followed by extraction of the aqueous phase
by ethyl acetate (6 * 1 ml). Lyophilization of the aqueous phase gave the crude deprotected
oligo(ribonucleoside phosphorothioate) as a white powder.
The synthesized crude phosphorothioate oligomers were purified by reversed phase
HPLC (Supelcosilr LC 18 column, 15.0 cm * 4.6 mm) using a linear gradient of MeCN (0-10%) in NH
4
OAc (25 mM) under 90 min at a flow rate of 1 ml/min. All fractions containing
full length oligomers were collected, lyophilized and analysed as follows.
The crude oligomers were dissolved in (NH
4
)
2
SO
4
-buffer (150 [mu]l, 30 mM, pH 5.3) containing ZnSO
4
(0.44 mM) and treated with Nuclease P1 (0.05 mg, 15 U) at 37oC (
26
,
38
). Aliquots (5 [mu]l) were withdrawn at different times and filtered (ultrafreer-MC 10000 NMWL filter unit Millipore). The reaction was followed by
anion exchange HPLC (Watersr Gen Pak Fax) using a linear gradient of acetonitrile (0-27%) and lithium perchlorate (0-10 mM) in 20 mM sodium acetate buffer during 60 min and at a flow
rate of 1 ml/min. Crude and purified products were also analysed by reversed
phase HPLC (Supelcosilr LC 18 column, 15.0 cm * 4.6 mm) using a linear gradient of MeCN (0-10%) in NH
4
OAc (25 mM) under 90 min at a flow rate of 1 ml/min.
The crude deprotected oligomer was passed through a preparative reversed phase
HPLC column (Merck LiChrosorbr RP-18 7 [mu]m, 25.0 cm * 25 mm) using a linear gradient of acetonitrile (0-20%) in NH
4
OAc (25 mM) during 80 min at a flow rate of 7 ml/min. The appropriate fractions
(retention time 47-67 min for the heptamer) were collected and lyophilized.
The crude oligomer (0.3-0.4 [mu]mol) was dissolved in: (NH
4
)
2
SO
4
buffer (150 [mu]l, 30 mM, pH 5.3) containing ZnSO
4
(0.44 mM) and Nuclease P1 (0.05 mg, 15 U) (
26
,
38
). The mixture was kept at 37oC for 18 h [too long treatment leads to somewhat lower yields since the enzyme also catalyses cleavage of the
R
P
-linkages, albeit at a considerably lower rate (
63
). For example, the peak arising from the all-
R
P
heptamer is still the tallest one after 48 h digestion]. The enzyme was then
filtered off (ultrafreer-MC 10000 NMWL filter unit Millipore) and the resulting solution was passed through a strong anion-exchange HPLC column (Shandonr 5 [mu]m Hypersil WP SAX column, 25 cm * 7 mm) using a linear gradient of KBr (0-75 mM) in a buffer of NH
4
OAc in H
2
O:MeCN (50 mM, 95:5, v/v) during 60 min at a flow rate of 2.5 ml/min. The last
eluated peak with retention time 23 min was collected and lyophilized. The
product was purified by reversed phase HPLC (Supelcosilr LC 18 column, 15.0 cm * 4.6 mm) using a linear gradient of MeCN (0-10%) in NH
4
OAc (25 mM) under 90 min at a flow rate of 1 ml/min. The large peak with
retention time 56 min corresponding to the all-
R
P
7mer was collected and lyophilized. The overall isolated yield of 7mer was in
average 4-5% (calculated from the loading on support as determined by trityl
assay). The theoretical yield is ~30% total based on coupling efficiency and stereoselectivity. Analysis of
the all-
R
P
-5'-A
P(S)
G
P(S)
G
P(S)
U
P(S)
U
P(S)
C
P(S)
A-3' by negative ion MALDI-TOF mass spectrometry gave the expected molecular ion (96 m/z
units higher than the nonthioate 7mer used as standard i.e., [M-H]
-
m/z = 2299.5).
The purified all-
R
P
7mer (0.2 [mu]mol) was dissolved in Tris-HCl buffer (50 [mu]l, 0.1 M, pH 8.7) and MgCl
2
(0.3 mM) and a solution (50 [mu]l) of snake venom phosphodiesterase (0.14 mg, 0.05 U) in Tris-HCl (50 mM, pH 8.0) and MgCl
2
(10 mM) was added followed by alkaline phosphatase (20 [mu]l) (
45
,
64
). The digestion was performed at 37oC and aliquots were taken out after 5 and 18 h filtered (ultrafreer-MC 10000 NMWL filter unit Millipore) and analysed by reversed phase
HPLC (Supelcosilr LC 18 column, 15.0 cm * 4.6 mm) and using a linear gradient of MeCN (0-10%) in NH
4
OAc (25 mM) under 90 min at a flow rate of 1 ml/min.
1
H and
31
P NMR spectra were recorded on a Jeol GSX-270 FT spectrometer. Chemical shifts are given in p.p.m. relative to
tetramethylsilane (
1
H, CDCl
3
, 25oC) or 2% H
3
PO
4
in D
2
O as external reference (
31
P, 25oC). Assignments are based on chemical shifts and the observed coupling
pattern, specifically making sure in the H-phosphonates that coupling with phosphorus is to the 3'-hydrogen. High resolution FAB mass spectra were recorded Jeol
SX-102 instrument. MALDI-TOF MS was recorded on a Finnigan-MAT instrument.
5'-
O
-(4-methoxytriphenylmethyl)uridine (2.52 g, 4.90 mmol) was dissolved in
pyridine (30 ml) and silver nitrate (1.00 g, 5.86 mmol) was added, followed by
tert
-butyldiphenylsilyl chloride (1.55 ml, 5.86 mmol). The reaction mixture was
stirred at room temperature with exclusion of light for 18 h, by which time TLC
(chloroform-methanol, 9:1 v/v) indicated complete reaction. THF (100 ml) was
added, the reaction was filtered through Celite and the solution was
evaporated. The residue was partitioned between saturated NaHCO
3
(100 ml) and toluene (2 * 100 ml) the combined organic extracts were dried over Na
2
SO
4
and evaporated. Silica gel column chromatography using a stepwise gradient of
ethyl acetate (14-25%) in toluene as eluent afforded the title compound as a white foam.
Yield 1.313 g (36%). The poor yield was due to formation of a large amount (52%) of the 3'-
O
-
tert
-butyldiphenylsilyl isomer.
Imidazole (1.3 g, 19 mmol), dried by evaporation of added acetonitrile (50 ml) was dissolved in acetonitrile (15 ml). The reaction mixture was cooled and stirred on an ice-salt bath, and phosphorus trichloride (0.52 ml, 6.0 mmol) followed by triethylamine (2.6 ml, 19 mmol) were added. To this mixture was a solution of 5'-
O
-(4-methoxytriphenylmethyl)-2'-
O
-
tert
-butyldiphenylsilyluridine
(1.3 g, 1.7 mmol, dried by evaporation of added acetonitrile) in acetonitrile
(15 ml) added dropwise over 30 min. The cooling bath was removed and the
reaction was allowed to stand for an additional 15 min. Water (5 ml) was added,
the solvent was evaporated and the residue was partitioned between chloroform
and 1 M triethylammonium bicarbonate (
aq
) whereafter the chloroform part was collected and evaporated under reduced
pressure. Silica gel column chromatography using a stepwise gradient of MeOH (1-25%) in CHCl
3
-Et
3
N (99.9:0.1, v/v) as eluent and subsequent evaporation of collected fractions
afforded the title compound as a white foam. Yield 1.3 g (83%).
31
P NMR (in pyridine): [delta] = 3.5;
1
H NMR (in CDCl
3
) [delta] = 8.18 (s, 1 H, NH), 7.76 (dd,
3
J = 6.6 Hz, 2 H, aromatic protons), 7.55 (dd,
3
J = 7.0 Hz, 2 H, aromatic protons), 7.37-7.49 (m, 3 H, aromatic protons), 7.33 (d,
3
J = 8.1 Hz, 1 H, 6-H), 7.03-7.24 (m, 16 H, aromatic protons), 7.02 (d,
1
J = 624 Hz, P-H), 6.67 (d,
3
J = 8.8 Hz, 2 H, aromatic protons), 6.27 (d,
3
J = 7.0 Hz, 1 H, 1'-H), 4.86 (d,
3
J = 8.1 Hz, 1 H, 5-H), 4.63 (m, 1 H, 2'-H, 3'-H), 4.48 (s, 1 H, 4'-H), 3.76 (s, 3 H, CH
3
O), 3.22 and 3.48 (2 dd, 2 H, 5'-H), 3.02 (q, 6 H, CH
2
N), 1.33 (t, 9 H, CH
3
CH
2
N), 1.09 (s, 9 H,
t
-Bu). HRMS Found: M-, 817.2706 C
45
H
46
O
9
N
2
SiP requires
M
, 817.2710.
This compound was prepared as described for 5'-
O
-(4-methoxytriphenylmethyl)-2'-
O
-
tert
-butyldiphenylsilyluridine, using triisopropylsilyl chloride (1.25 ml, 5.86
mmol). Yield 2.6 g (80%). Attempts to perform the silylation in THF proved to
be too slow whereas the DMF/imidazole procedure (
45
) afforded acceptable regioselectivity, 68%.
This compound was prepared as described for compound
1b
, using 5'-
O
-(4-methoxytriphenylmethyl)-2'-
O
-triisopropylsilyl-uridine (4.0 g, 6.0 mmol) Yield 4.2 g (83%).
31
P NMR (in pyridine): [delta] = 3.2;
1
H NMR (in CDCl
3
) [delta] = 7.94 (d,
3
J = 8.1 Hz, 1H, 6-H), 6.98 (d,
1
J = 622 Hz, 1 H P-H), 7.38-7.23 (m, 13 H, aromatic protons), 6.83 (d,
3
J = 8.8 Hz, 2 H, aromatic protons), 6.07 (d,
3
J = 5.5 Hz, 1 H, 1'-H), 5.14 (d,
3
J = 8.1 Hz, 1 H, 5-H), 4.82 (m, 1 H, 3'-H), 4.64 (t, 1 H, 2'-H), 4.48 (d, 1 H, 4'-H), 3.79 (s, 3 H, CH
3
O), 3.46 and 3.58 (2 dd, 2 H, 5'-H), 2.98 (q, 6 H, CH
2
N), 1.30 (t, 9 H, CH
3
CH
2
N), 1.10 (m, 18 H, CH
3
C). HRMS Found: M-, 735.2818 C
38
H
48
O
9
N
2
SiP requires
M
, 735.2867.
Cytidine (1.46 g, 6.0 mmol) was dried by evaporation of added pyridine (3 * 20 ml).
N
-Methyl-2,2-dimethoxypyrrolidine (
52
) (1.96 ml, 12 mmol) and methanol (10 ml) were added. After 90 min the solution
was quenched with H
2
O (0.1 ml), concentrated, and the residue dried by evaporation of added pyridine
(3 * 30 ml). The residue was dissolved in pyridine (50 ml) and the resulting
solution cooled on an ice-bath. 4-Methoxytriphenylmethyl chloride (2.24 g, 7.2 mmol) was then added
and the stirred reaction mixture was allowed to slowly reach room temperature.
After 18 h TLC using chloroform-methanol (9:1, v/v) indicated complete reaction
and excess reagent was quenched by the addition of methanol (50 [mu]l). The solution was concentrated and partitioned between saturated NaHCO
3
(100 ml) and chloroform (2 * 100 ml). The combined organic phases were dried over Na
2
SO
4
and evaporated. Silica gel column chromatography using a stepwise gradient of
MeOH (1-8%) in CHCl
3
as eluent afforded the title compound as a foam. Yield 1.8 g (51%).
1
H NMR (in CDCl
3
) [delta] = 7.80 (d,
3
J = 7.3 Hz, 1H, 6-H), 7.33-7.15 (m, 12 H, aromatic protons), 6.84 (d,
3
J = 8.8 Hz, 2 H, aromatic protons), 5.70 (d,
3
J = 2.6 Hz, 1 H, 1'-H), 5.51 (d,
3
J = 7.3 Hz, 1 H, 5-H), 4.02 (m, 1 H, 3'-H), 3.89 (m, 2 H, 2'-H, 4'-H), 3.66 (s, 3 H, CH
3
O), 3.37 (m, 2 H, 5'-H, 5''-H), 3.29 (m, 2 H, CH
2
N), 2.87 (m, 5 H, CH
2
C, CH
3
N), 1.87 (m, 2H, CH
2
CH
2
N).
This compound was prepared as described for 5'-
O
-(4-methoxytriphenylmethyl)-2'-
O
-
tert
-butyldiphenylsilyluridine, using 5'-
O
-(4- methoxytriphenylmethyl)-4-
N
-(
N
-methylpyrrolidin-2-ylidene)- cytidine
(0.85 g, 1.4 mmol),
tert
-butyldimethylsilyl chloride (0.21 mL, 1.4 mmol) and
pyridine-THF (1:1, v/v) (20 ml) as solvent. Yield 0.69 g (69%).
1
H NMR (in CDCl
3
) [delta] = 8.03 (d,
3
J = 7.3 Hz, 1H, 6-H), 7.45 (d,
3
J = 7.0 Hz, 4 H, aromatic protons), 7.21-7.31 (m,10 H, aromatic protons), 6.91 (d, 1J = 618 Hz, 1 H P-H), 6.85 (d,
3
J = 9.8 Hz, 2 H, aromatic protons), 5.93 (d, 1 H, 1'-H), 5.75 (d, 1 H, 5-H), 4.30 (m, 2 H, 2'-H, 3'-H), 4.07 (m, 1 H, 4'-H), 3.80 (s, 3 H, CH
3
O), 3.48 (m, 4 H, 5'-H, 5''-H, CH
2
N), 3.07 (m, 5 H, CH
2
C, CH
3
N), 2.08 (m, 2 H, CH
2
CH
2
N), 0.92 (s, 9 H, CH
3
C),0.31 (s, 3 H, CH
3
Si); 0.19 (s, 3 H, CH
3
Si).
To a stirred solution of imidazole (0.75 g, 11 mmol) and triethylamine (1.6 ml,
12 mmol) in CH
2
Cl
2
(40 ml) was added phosphorous trichloride (0.32 ml, 3.6 mmol). After 30 min,
the reaction mixture was cooled to -78oC, and 5'-
O
-(4-methoxytriphenylmethyl)-4-
N
-(
N
-methylpyrrolidin-2-ylidene)-2'-
O
-
tert
-butyldimethylsilylcytidine (0.74 g, 1.0 mmol), previously dried by
evaporation of added pyridine (20 ml), dissolved in CH
2
Cl
2
(15 ml) was added dropwise over 15 min. The ice bath was then removed and the
reaction was allowed to stand for an additional 15 min. The reaction was poured
into triethylammonium bicarbonate buffer (1 M, 75 ml) with stirring. The
mixture was transferred to a separatory funnel, the phases separated, the
aqueous phase extracted with CH
2
Cl
2
(50 ml), and the combined organic phases dried over Na
2
SO
4
and evaporated. Silica gel column chromatography using a stepwise gradient of
MeOH (1-25%) in CHCl
3
-Et
3
N (99.9:0.1, v/v) as eluent afforded the title compound. Yield 0.74 g (82%).
31
P NMR (in pyridine-MeCN, 1-3): [delta] = 1.8;
1
H NMR (in CDCl
3
) [delta] = 7.95 (d,
3
J = 7.3 Hz, 1H, 6-H), 7.43 (d,
3
J = 7.3 Hz, 4 H, aromatic protons), 7.21-7.31 (m,10 H, aromatic protons), 6.91 (d,
1
J = 618 Hz, 1 H P-H), 6.83 (d,
3
J = 8.8 Hz, 2 H, aromatic protons), 6.00 (d, 1 H, 1'-H), 5.60 (d,
3
J = 8.1 Hz, 1 H, 5-H), 4.67 (m, 1 H, 3'-H), 4.47 (t, 1 H, 2'-H), 4.41 (m, 1 H, 4'-H), 3.78 (s, 3 H, CH
3
O), 3.52 (m, 2 H, 5'-H, 5''-H), 3.45 (t, 2 H, CH
2
N), 3.18 (t, 2 H, CH
2
C), 3.00 (m, 9 H, CH
3
N, CH
3
N), 2.03 (m, 2 H, CH
2
CH
2
N), 1.28 (t, 9 H, CH
3
CH
2
N), 0.89 (s, 9 H, CH
3
C), 0.17 (d, 6 H, CH
3
Si); HRMS Found: M-, 773.3170 C
40
H
50
O
8
N
4
SiP requires
M
, 773.3136.
This compound was prepared as described for compound
4c
, using 5'-
O
-(4-methoxytriphenylmethyl)-4-
N
-[2-(
tert
-butyldiphenylsiloxy)-methyl]bensoyl]-2'-
O
-(
tert
-butyldimethylsilyl)-cytidine (1.0 g, 1.0 mmol). Yield 0.93 g (80%).
1
H NMR (in CDCl
3
) [delta] = 8.51 (d, 1H, 6-H), 7.67-7.14 (m, 27 H, aromatic protons, 5-H), 6.82 (d,
1
J = 652 Hz, 1 H P-H), 6.81 (d,
3
J = 9.0 Hz, 2 H, aromatic protons), 5.71 (d, 1 H, 1'-H), 5.00 (d,
3
J = 2.6 Hz, 2 H, O-CH2), 4.56 (m, 1 H, 3'-H), 4.39 (d, 1 H, 2'-H), 4.30 (d,
3
J = 8.2 Hz, 1 H, 4'-H), 3.97 (m, 2 H, 5'-H, 5''-H), 3.80 (s, 3 H, CH
3
O), 3.05 (m, 6 H, CH
3
N), 1.32 (t, 9 H, CH
3
CH
2
N), 1.07 (s, 9 H, CH
3
C), 0.93 (s, 9 H, CH
3
C), 0.20 (s, 3 H, CH
3
Si), 0.14 (s, 3 H, CH
3
Si). HRMS Found: M-, 1064.4103 C
59
H
67
O
10
N
3
Si
2
P requires
M
, 1064.4102.
We thank Dr Jan Johansson at the Karolinska Institute for help with MALDI-TOF MS analysis, the Swedish Natural Science Research Council and
Procordia Research Funds for financial support.
*To whom correspondence should be addressed at present address: Laboratory of
Organic and Bioorganic Chemistry, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden
REFERENCES
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