The antiviral defense mechanism in mammalian cells is induced
following virus infection by the action of interferon and subsequently
by the 2`,5`-oligoadenylate (2-5A) (
)synthetase/RNase
L/p68 kinase (PKR) system. This interferon-induced antiviral system is
also important in regulation of cell growth and differentiation,
pre-mRNA processing, and oncogene stability (Pestka, 1981; Lengyel,
1982; Dani et al., 1985; Wells and Mallucci, 1985, 1988; de
Maeyer and de Maeyer-Guignard, 1991; Hovanessian, 1991; Sperling et
al., 1991; McNair and Kerr, 1992). RNase L and PKR appear to
mediate the antiproliferative effects of interferon. Furthermore, PKR
has been reported to be a tumor suppressor gene (Koromilas et
al., 1992; Hassel et al., 1993). 2-5A synthetase is
an allosterically regulated enzyme, activated by dsRNA, which converts
ATP to 2-5A; 2-5A exerts its biological effects primarily
by binding to and activating its target enzyme, RNase L, an 80-kDa
2-5A-dependent endoribonuclease. Human and murine RNase L have
recently been cloned (Hassel et al., 1993; Zhou et
al., 1993). 2-5A and 2-5A derivatives also directly
inhibit HIV-1 reverse transcriptase (RT) by preventing HIV-1 RT/primer
complex formation (Montefiori et al., 1989;
Müller et al., 1991; Sobol et
al., 1993) and inhibit DNA topoisomerase I in HIV-1-infected cells
(Schröder et al., 1994).
Numerous
structurally and stereochemically modified 2-5A derivatives have
been synthesized to elucidate the biological role of the 2-5A
synthetase/RNase L system and the mechanism of activation of RNase L
(Johnston and Torrence, 1984; Hughes et al., 1985a, 1985b;
Kariko et al., 1987a, 1987b; Suhadolnik et al., 1987,
1988a, 1988b; Charachon et al., 1990; Kanou et al.,
1990, 1991; Charubala et al., 1989, 1991a, 1991b; Charubala
and Pfleiderer, 1992; Shimazu et al., 1993). Our laboratory
has examined the requirements for binding to and activation of RNase L
via stereochemical modification of the 2-5A molecule (Lee and
Suhadolnik, 1985; Kariko et al., 1987a, 1987b; Charachon et al., 1990; Charubala and Pfleiderer, 1992).
Phosphorothioate substitution provided the first 2-5A cores (i.e. 2-5A lacking 5`-phosphate moieties) able to
activate RNase L (Kariko et al., 1987b; Charachon et
al., 1990). The phosphorothioate trimer and tetramer
5`-monophosphate 2-5A derivatives activate RNase L at
concentrations equivalent to authentic 2-5A (Kariko et
al., 1987b; Charachon et al., 1990). Utilizing these
fully substituted phosphorothioate derivatives of 2-5A, we
established that changes in the stereochemical configuration of the
phosphodiester backbone of 2-5A have little or no effect on
binding affinity for RNase L; however, RNase L is a functionally stereoselective enzyme. 2-5A derivatives with S
chirality in all internucleotide linkages are
antagonists of RNase L; they bind to, but cannot activate, RNase L.
Indeed, the tetramer 5`-monophosphate 2-5A derivative,
pA(S
)A(S
)A(S
)A,
functions as an antagonist of RNase L when microinjected into vesicular
stomatitis virus-infected HeLa cells (Charachon et al., 1990).
The synthesis, characterization, and biological activities of
phosphorothioate/phosphodiester derivatives of 2-5A trimer and
tetramer cores and their 5`-monophosphates are described here. These
derivatives were designed as biological probes to examine the role of
phosphorothioate substitution in individual internucleotide linkages
with respect to stability, activation of RNase L, and inhibition of
HIV-1 replication. Enhanced uptake of the
phosphorothioate/phosphodiester 2-5A derivatives in
HIV-1-infected Sup T1 cells is demonstrated.
MATERIALS AND METHODS
Chemical Synthesis of Phosphorothioate/Phosphodiester
2-5A Cores
Precoated silica TLC sheets (F 1500 LS 254),
cellulose TLC sheets (F 1440), and PC paper chromatography sheets (58
60 cm) were from Schleicher & Schüll.
Silica Gel 60 (0.063-0.2 mesh) was obtained from Merck. DEAE
Sephadex A-25 was from Pharmacia Biotech Inc. Pyridine was dried by
distillation after refluxing with KOH, tosyl chloride, and
CaH
. CH
Cl
was distilled over
CaCl
and acetonitrile purified by refluxing with
CaH
, followed by distillation. Tetrazole was sublimed in
vacuum before use. Triethylammonium bicarbonate buffer (TEAB) (1 M) was prepared by passing CO
through a 1 M solution of triethylamine in water.HPLC was carried out with a
Merck-Hitachi D2000 chromatograph using a RP18 reverse-phase column (5
µm; 125
4 mm) with a flow rate of 1 ml/min. The column was
eluted with solvent 1 = 50 mM ammonium dihydrogen
phosphate (pH 7) or solvent 2 = MeOH/H
O (1/1, v/v)
with an isocratic gradient (t = 0 min, 30% B; t = 20 min, 30% B) followed by a linear gradient (t = 20 min 30% B; t = 50 min 40% B).
Purification of the 5`-monophosphates was carried out by HPLC
(Charachon et al., 1990). Medium pressure chromatography was
performed on a C-type column (400
39.5 mm) on silica gel
Lichoprep Si 60 (15-25 µm) at 2-5 bar.
P NMR spectroscopy (121.5 MHz) was performed using a
Bruker WH-300 (Shared Magnetic Resonance Facility, Temple University
School of Medicine) or using a 400-MHz Jeol spectrometer
(Universität Konstanz) with 85%
H
PO
as the standard.
H NMR was
performed using a Bruker WH-250, in ppm (s = singlet, d =
doublet, m = multiplet), with tetramethylsilane as the internal
standard.
H NMR spectra in D
O were determined
with a D
O signal as the standard (4.80 ppm).
Bis(diisopropylamino)-(
-cyanoethoxy)phosphane (3) (Kraszewski and Norris,
1987)
-Cyanoethanol (7 g; 0.1 mol) in absolute
CH
CN (40 ml) was added dropwise within 30 min to a solution
of freshly distilled PCl
(40 ml; 0.4 mol) at room
temperature and under nitrogen atmosphere. After stirring for 3.5 h,
the solvent and excess PCl
were removed in high vacuum, the
residue was dissolved in 450 ml of absolute ether and reacted at
-10 °C with N,N-diisopropylamine (127 ml;
0.9 mol) by dropwise addition within 1 h under nitrogen atmosphere. The
reaction mixture was stirred at -10 °C for 30 min and at room
temperature for 15 h. The precipitate was filtered under nitrogen, and
the solvent was removed in vacuo. The yellow crude product was
fractionally distilled over CaH
to give 14.7 g (49%) of
pure 3 of boiling point 114-118 °C. This reagent was
stored at -20 °C under nitrogen.
H NMR
(CDCl
), 3.75 (s, 2H, CH
), 3.52 (m, 4H, 4 N-CH),
2.60 (t, 2H,
-CH
), and 1.17 + 1.14 (2 d, 24H, 4
N-C(CH
)
);
P NMR
(CDCl
), 124.6 ppm.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenosine-2`-O-[(
-cyanoethyl)-N,N-diisopropylamino]phosphoramidite (4)
For Method A (Sinha et al.,
1984), compound 1 (Flockerzie et al., 1981) (3.79 g; 5
mmol) and diisopropylethylamine (3.5 ml) were dissolved in dry
CH
Cl
(20 ml) and
chloro-N,N-diisopropylaminocyanoethoxyphosphane (2.37 g; 10
mmol) was added. After 1.5 h of stirring under nitrogen at room
temperature, the reaction mixture was diluted with EtOAc (100 ml) and
the organic phase was washed with a saturated NaHCO
/NaCl
solution (2
80 ml). The organic layer was dried over
Na
SO
, filtered, and evaporated to dryness. The
residue was dissolved in CH
Cl
(10 ml) and added
dropwise to n-hexane (200 ml) at -60 °C. The product
was collected and evaporated to dryness in high vacuum for 8 h to give
4.3 g (89%) of a colorless amorphous solid. For Method B
(Kraszewski and Norris, 1987), compound 1 (3.79 g; 5 mmol) and
tetrazole (0.175 g; 2.5 mmol) were dissolved in dry
CH
Cl
(20 ml) and then
bis(diisopropylamino)-(
-cyanoethoxy)phosphane (3) (3 g; 10
mmol) was added. After stirring at room temperature under argon for 17
h, the reaction mixture was extracted with EtOAc (100 ml) and washed
with saturated NaHCO
/NaCl solution (80 ml). This was
repeated twice and work-up was performed analogous to Method A to give
4.49 g (94%) of a colorless amorphous
powder.

UV (MeOH), 
= 279 nm (4.33) or 229 nm
(4.43), R
on silica gel with toluol/EtOAc (1/1,
v/v): 0.64 and 0.61 (diastereomers).
P NMR
(CDCl
), 150.98, 151.34.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[O
-(2-cyanoethyl)-5`-]-N
-benzoyl-2`,3`-di-O-[(tertbutyl)dimethylsilyl]adenosine (6)
The phosphoramidite 4 (2.88 g; 3
mmol) and
N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine (5) (Flockerzie et al., 1981) (1.2 g; 2 mmol) were
dried at room temperature in high vacuum for 24 h and dissolved in dry
CH
Cl
(30 ml). Tetrazole (0.5 g; 8 mmol) was
added, and after 3 h of stirring at room temperature under argon, a
solution of I
(0.5 g of
H
O/pyridine/CH
Cl
(1/3/1, v/v/v))
was added dropwise until the brown color did not disappear. The mixture
was stirred for 15 min, then diluted with CHCl
(300 ml).
The organic phase was saturated with
Na
S
O
/NaCl (3
80 ml), dried
over Na
SO
, and evaporated to dryness. Final
coevaporation was done with toluene (3
20 ml). The crude
product was purified by silica gel column chromatography (15
2.5 cm) using CHCl
(100 ml), CHCl
/MeOH
(100/0.5, v/v; 1.5 liters), and CHCl
/MeOH: (100/1, v/v) to
elute the product. Product fractions were collected and evaporated to
dryness to give 2.33 g (79%) of dimer 6 as a solid
foam.

UV (MeOH): 
= 278 nm (4.62) and 230 nm
(4.62). R
on silica gel with CHCl
/MeOH
(95/5, v/v) = 0.56.
P NMR (CDCl
),
-0.74 and -1.07 ppm (diastereomers).
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[O
-(2-cyanoethyl)-5`]-N
-benzoyl-2`,3`-O-di-[(tert-butyl)dimethylsilyl]adenosine (7)
Compound 6 (2.22 g; 1.51 mmol)
was stirred with 2% p-TsOH in CH
Cl
/MeOH (4/1,
v/v; 30 ml) at room temperature for 30 min. The reaction mixture was
diluted with CH
Cl
(300 ml), washed with
phosphate buffer, pH 7.0 (2
100 ml), dried over
Na
SO
, and evaporated to dryness. The residue
was applied to a silica gel column (9
4.5 cm), washed with
CHCl
(0.7 liter) and CHCl
/MeOH (100/1, v/v; 300
ml). The product was eluted with CHCl
/MeOH (50/1, v/v, 300
ml; and 100/3, v/v, 300 ml). The combined product fractions were
evaporated to dryness in high vacuum to give 11.65 g (90%) of dimer 7 as an amorphous solid.

UV (MeOH): 
= 278 nm (4.60) and 232 nm
(4.42). R
on silica gel with CHCl
/MeOH
(95/5, v/v) = 0.36.
P NMR (CDCl
),
-0.77 and -1.30 ppm (diastereomers).
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-adenylyl-2`-[O
-(2-cyanoethyl)-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenylyl-2`-[O
-(2-cyanoethyl)-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine:
A(R
)ApA (8) and
A(S
)ApA
(9)
Phosphoramidite 4 (2.79 g;
2.92 mmol), 5`-hydroxy ApA dimer 7 (1.94 g; 1.62 mmol), and
tetrazole (0.567 g; 8.1 mmol) were dissolved in dry CH
CN
(8.1 ml) and stirred at room temperature under nitrogen. After 3 h,
S
(1.66; 6.48 mmol) and pyridine (7.8 ml) were added and
stirred further for 20 h at room temperature. The reaction mixture was
then diluted with CH
Cl
(300 ml), washed with
saturated NaCl (2
200 ml), dried over
Na
SO
, and evaporated to dryness. Final
coevaporation was with toluene (3
20 ml). The crude
diastereomeric mixture
A(R
,S
)ApA (8 and 9) was dissolved in CH
Cl
and applied to a silica gel column (21
3.5 cm). The
column was washed with CH
Cl
(450 ml) and
CH
Cl
/MeOH (99/1, v/v; 200 ml), and the product
was eluted with CH
Cl
/MeOH (97/3, v/v; 400 ml).
The product fractions were collected and evaporated to dryness to give
3.16 g (93%) of an isomeric mixture of A(R
)ApA (8) and A(S
)ApA (9). Separation into the pure diastereoisomers was
achieved by medium pressure chromatography as described above by
elution with CHCl
/MeOH (99/1, v/v; 800 ml; 20 ml/fraction;
fractions 1-40), followed by elution with CHCl
/MeOH
(95/5, v/v; 800 ml; 20 ml/fraction; fractions 41-80). Pure
A(R
)ApA isomer 8 (0.287 g) was
eluted in fractions 21-56 (20 ml/fraction). Fractions 57-61
gave the isomer mixture (0.07 g); pure A(S
)ApA
isomer 9 (0.132 g) was eluted in fractions 62-64.
Chromatographic separation was repeated with each 0.5 g of the crude
mixture to yield 1.62 g (51%) of A(R
)ApA 8 and 0.94 g (30%) of A(S
)ApA 9.

UV (MeOH): 
= 279 nm (4.76), 260 nm
(4.56), 236 nm (4.73). R
on silica gel with
CHCl
/MeOH (97/3, v/v) = 0.35.
P NMR
(CDCl
): 69.35 and -1.10
ppm.

UV (MeOH): 
= 279 nm (4.77), 260 nm
(4.57), and 236 nm (4.73).
P NMR (CDCl
), 68.33
and -0.84 ppm.
P-Thioadenylyl-2`-5`-adenylyl-2`-5`-adenosine:
A(R
)ApA isomer (10) and
A(S
)ApA isomer
(11)
The corresponding fully protected
trimers 8 and 9 were deblocked in the following manner;
the trimer (0.06 g; 0.029 mmol) was stirred with 2% p-TsOH in
CH
Cl
/MeOH (4/1, v/v; 1.2 ml) for 1.5 h at room
temperature. The reaction mixture was diluted with CHCl
(50
ml), washed with H
O (2
25 ml), dried, and
evaporated to dryness. The crude product was purified on preparative
silica gel plates (20
20
0.2 cm) in
CHCl
/MeOH (8/2, v/v). The product bands were eluted with
CHCl
/MeOH (4/1, v/v) and evaporated to a foam to give 0.04
g (84%) of A(R
)ApA 10 and 0.034 g (73%) of
A(S
)ApA 11. The 5`-hydroxy trimer (0.034 g;
0.08 mmol) was stirred with 0.5 M DBU in pyridine (5.0 ml),
and after stirring at room temperature for 20 h, the solution was
neutralized with 1 M acetic acid in dry pyridine (2.5 ml) and
evaporated to dryness. The residue was treated with methanolic ammonia
(5 ml), and after 48 h of stirring the solvents were removed in
vacuo. Desilylation was performed with 1 M tetrabutylammonium fluoride in THF (2 ml). After stirring for 48
h, the solvent was removed in vacuo, the residue was dissolved
in H
O (10 ml) and applied to a DEAE Sephadex A-25 column
(60
1 cm). The pure product was eluted with a linear gradient
of 0.14-0.17 M TEAB buffer, pH 7.5. After evaporation
and coevaporation with water several times, the trimer was applied to
paper sheets (35
50 cm) and developed in i-PrOH/concentrated
ammonia/H
O (6/1/3, v/v/v). The product band was cut out,
eluted with H
O, evaporated, and lyophilized to give 500
OD
units (79%) of A(R
)ApA 10 and 410 OD
units (65%) of
A(S
)ApA 11. UV 
in both
cases was 258 nm in H
O. A(R
)ApA (10): R
on cellulose in
i-PrOH/ammonia/H
O (6/1/3, v/v/v) = 0.33.
H NMR (D
O): 8.20; 8.19; 8.14 (3 s, 3H, H-C(8));
7.97 (1 s, 2H, 2 H-C(2)) and 7.76 (1 s, 1H, 1 H-C(2)); 6.08; 5.93; 5.82
(3 d, 3H, 3H-C(1`)). Retention time on reverse-phase HPLC was 5.60 min.
A(S
)ApA (11): R
on
cellulose in i-PrOH/ammonia/H
O (6/1/3, v/v/v) =
0.33.
H NMR (D
O): 8.14; 8.09; 8.02 (3 s, 3H,
H-C(8)); 7.94; 7.89: 7.80 (3 s, 3H, 2 H-C(2)); 6.03; 5.92; 5.80 (3 d,
3H, 3H-C(1`)). Retention time on reverse-phase HPLC was 6.51 min.
N,N-Diisopropyltrimethylsilylamine (Noth and Staudigl,
1982)
Methyl iodide (37.6 ml, 0.6 mol) in absolute ether (50 ml)
was added dropwise (over 90 min) to a suspension of 14.6 g (0.6 mol) of
magnesium and a few crystals of iodine in absolute ether (100 ml). The
reaction was then stirred for 30 min until all the magnesium dissolved.
Subsequently, N,N-diisopropylamine (78 ml, 0.55 mol) was added
within 10-15 min and the reaction was refluxed for 1 h. After
cooling to 0 °C, trimethylsilyl chloride (76 ml, 0.6 mol) was added
dropwise and the reaction mixture was again heated in an oil bath with
vigorous stirring to 80 °C for 20 h. The supernatant liquid was
decanted and the residue extracted with ether (4
50 ml). The
supernatant and ether extract were combined. The solvent, excess
trimethylsilyl chloride and unreacted N,N-diisoproylamine were
removed by distillation. The product was then isolated by distillation
under vacuum at an oil bath temperature of 60 °C to yield 73 g
(80%), boiling point at 18 mm Hg (Kp
) = 36-39
°C.
H NMR (CDCl
): 0.08 (s, 9H,
SiCH
); 1.04-1.07 (d, 12H, N-C-CH
), 3.2
(m, 2H, N-CH).
Chloro-N,N-diisopropylamino-2-(4-nitrophenyl)ethoxyphosphane (14)
p-Nitrophenylethanol (4.16 g; 25 mmol)
was added portion wise to a solution of freshly distilled PCl
(14 ml; 0.16 mol) in absolute ether (40 ml) at -30 °C
under a nitrogen atmosphere within 45 min. The reaction mixture was
stirred at room temperature for 1.5 h, and the solvent and excess
PCl
were removed in vacuo at 0 °C. The residue
was treated with N,N-diisopropyltrimethylsilylamine (4.33 g,
25 mmol) at 0 °C under a nitrogen atmosphere for 30 min and then at
room temperature for 20 h. The resulting trimethylsilyl chloride was
removed under high vacuum at room temperature to yield a syrupy pale
yellow product (7.1 g, 85%), which crystallized upon storage at
-20 °C. This material was then used for the subsequent
phosphitylation reactions.
H NMR (CDCl
):
8.1-8.2 (m, 2H, o to NO
); 7.39-7.43 (m, 2H, m
to NO
); 4.04-4.18 (m, 2H, P-O-CH
);
3.63-3.79 (m, 2H, N-CH); 3.07-3.13 (t, 2H,
P-O-C-CH
); 1.14-1.27 (2 d, 12H, N-C-CH
).
P NMR (CDCl
): 181.60 ppm.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenosine-2`-O-[(4-nitrophenyl)ethyl)-N,N-diisopropylamino]phosphoramidite (15)
For Method A, compound 1 (3.79
g, 5 mmol) and diisopropylethylamine (3.5 ml) were dissolved in dry
CH
Cl
(20 ml) and then
chloro-N,N-diisopropylamino-2-(4-nitrophenyl)ethoxyphosphane (14) (2.37 g, 10 mmol) was added dropwise under a nitrogen
atmosphere. After stirring at room temperature for 2 h, the reaction
mixture was diluted with EtOAc (200 ml) and the organic phase was
washed with a saturated NaHCO
/NaCl solution (3
80
ml), dried over Na
SO
, and evaporated to
dryness. The crude product was dissolved in toluene/EtOAc (7/3, v/v)
and chromatographed on a silica gel column (12
2 cm)
equilibrated with EtOAc/NEt
(95/5, v/v). The product
fractions were eluted with EtOAc/NEt
(95/5, v/v), collected
and evaporated to dryness, yielding 15 (5.28 g; 79%) as a
colorless solid foam.

UV (MeOH): 
= 277 nm (4.50), 229 nm
(4.48).
P NMR (CDCl
), 150.27, 150.01 ppm. R
on silica gel in toluene/EtOAc (1/1, v/v), 0.62
and 0.68 (diastereomers).
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine:
ApA(R
)A (21)
Triethylammonium
N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]5`-O-(monomethoxytrityl)adenosine-2`-[2-(4-nitrophenyl)ethyl]phosphate (20) 0.10 g; 0.1 mmol) (Charubala et al., 1981) and the
5`-hydroxy (R
) dimer 18 (0.066 g; 0.05
mmol) (Charubala and Pfleiderer, 1992) were coevaporated with dry
pyridine (3
5 ml) and dissolved in 1 ml of dry pyridine, and
(2,4,6-triisopropyl)benzenesulfonyl chloride (0.062 g; 0.2 mmol) and
3-nitro-1,2,4-triazole (0.068 g; 0.6 mmol) (Kroger and Mietchen, 1969;
Jones et al., 1980) were added. After stirring at room
temperature for 20 h, the reaction mixture was diluted with CHCl
(100 ml), washed with H
O (2
50 ml), dried,
and evaporated. Final evaporations were done with toluene (2
10
ml) to remove pyridine. The crude trimer 21 was purified by
silica gel column chromatography (15
2 cm) using first
CHCl
and then CHCl
/MeOH (100/1, v/v) as
eluants. The product fraction was collected and evaporated to a solid
foam, which was dried under high vacuum to give 0.08 g (70%) of 21.

UV (MeOH): 
= 276 nm (4.87) and 227 nm
(4.83). R
on silica gel in
CH
Cl
/EtOAc (1/1) = 0.63.
P
NMR (CDCl
), 69.88 and -1.0 ppm.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine:
ApA(S
)A (22)
Triethylammonium
N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenosine-2-[2-(4-nitrophenyl)ethyl]phosphate (20) 0.10 g; 0.1 mmol) (Charubala et al., 1981) and the
5`-hydroxy (S
) dimer 19 (0.066 g; 0.05
mmol) (Charubala and Pfleiderer, 1992) were coevaporated with dry
pyridine (3
5 ml) and dissolved in 1 ml of dry pyridine, and
(2,4,6-triisopropyl)benzenesulfonyl chloride (0.062 g; 0.2 mmol) and
3-nitro-1,2,4-triazole (0.068 g, 0.6 mmol) (Kroger and Mietchen, 1969;
Jones et al., 1980) were added. After stirring at room
temperature for 20 h, the reaction mixture was diluted with CHCl
(50 ml), washed with H
O (2
25 ml), dried, and
evaporated. Final evaporations were done with toluene (2
10 ml)
to remove pyridine. The crude trimer 22 was purified by silica
gel column chromatography (15
2 cm), using first CHCl
and then CHCl
/MeOH (100/1, v/v) as eluants. The
product fraction was collected and evaporated to a solid foam, which
was dried under high vacuum to give 0.088 g (77%) of 22.

UV (MeOH): 
= 277 nm (4.88) and 227 nm
(4.83). R
on silica gel in
CH
Cl
/EtOAc (1/1, v/v) = 0.55.
P NMR (CDCl
), 69.29 and -0.87 ppm.
Adenylyl-(2`-5`)-P-thioadenylyl-(2`-5`)-adenosine:
ApA(R
)A (23) and
ApA(S
)A (24)
The
fully protected trimers, ApA(R
)A (21) and
ApA(S
)A (22), were separately deblocked as
follows. The corresponding trimer (0.088 g; 0.037 mmol) was stirred
with 2% p-TsOH in CH
Cl
/MeOH (4/1, v/v; 0.8 ml).
After 30 min of stirring at room temperature, the reaction mixture was
diluted with CHCl
(50 ml) and washed with H
O (2
25 ml). The organic phase was dried over NaSO
and
evaporated to dryness. The crude product was purified on a silica gel
column (5
2 cm); the product was eluted with
CHCl
/MeOH (100/1, v/v), evaporated, and dried under high
vacuum to give 0.073 g (94%) of the 5`-hydroxy trimer
ApA(R
)A (21) and 0.061 g (84%) of the
5`-hydroxy trimer ApA(S
)A (22). The
resulting 5`-hydroxy trimer (0.04 g; 0.02 mmol) was then stirred with
10 ml of 0.5 M DBU in pyridine. After 24 h, the solution was
neutralized with 1 M acetic acid in pyridine (10 ml) and
evaporated to dryness. The residue was treated with saturated
methanolic ammonia (6 ml), and after stirring at room temperature for
48 h, the solvent was removed in vacuo and the residue was
desilylated with 1 M Bu
NF in THF (5 ml) for 48 h.
The solvent was then removed in vacuo, and the residue was
dissolved in water (10 ml) and applied onto a DEAE Sephadex A-25 column
(60
1 cm). The product was eluted with a linear gradient of
0.14-0.17 M TEAB buffer, pH 7.5. After evaporation and
coevaporation with water several times, the trimer was applied to paper
sheets (35
50 cm) and developed in i-PrOH/concentrated
ammonia/H
O (6/1/3, v/v/v). The product band was cut out,
eluted with H
O, evaporated, and lyophilized to give 354
OD
units (79%) of ApA(R
)A 23 and 410 OD
units (58%) of
ApA(S
)A 24. UV 
in both
cases was 258 nm in H
O. ApA(R
)A (23): R
on cellulose in
i-PrOH/ammonia/H
O (6/1/3, v/v/v) = 0.34.
H NMR (D
O): 8.17; 8.16; 8.09 (3 s, 3H, H-C(8));
7.90, 7.78 (2 s, 3H, 3
H-C(2)); 6.04; 5.96; 5.80 (3 d, 3H, 3
H-C(1`)). Retention time on reverse-phase HPLC was 5.98 min.
ApA(S
)A (24): R
on
cellulose in i-PrOH/ammonia/H
O (6/1/3, v/v/v) =
0.33.
H NMR (D
O): 8.17; 8.07; 8.04 (3 s, 3H, 3
H-C(8)); 8.01; 7.92: 7.72 (3 s, 3H, 2 3
H-C(2)); 6.04;
5.92; 5.82 (3 d, 3H, 3
H-C(1`)). Retention time on
reverse-phase HPLC was 7.23 min.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenosine-2`-O-[2-(4-nitrophenyl)ethyl]-N,N-diisopropylaminophosphoramidite (15)
Compound 15 was synthesized
using either
chloro-N,N-diisopropylamino-[2-(4-nitrophenyl)ethoxy]phosphane (14) (Method A) or
bis-(diisopropylamino)-[2-(4-nitrophenyl)ethoxy]phosphane (27) (Method B). To a solution of 1.52 g (2 mmol) of compound 1 in absolute CH
CN (10 ml),
bis(diisopropylamino)-[2-(4-nitrophenyl)ethoxy]phosphane (27) (1.59 g, 4 mmol) and tetrazole (0.07 g; 1 mmol) were added
under nitrogen atmosphere and the reaction mixture was stirred for 17 h
at room temperature. The reaction mixture was diluted with EtOAc (120
ml), washed twice with saturated NaHCO
/NaCl solution (60
ml), dried over Na
SO
, and evaporated to
dryness. The crude solid foam was applied onto a flash silica gel
column (20
2.5 cm) and chromatographed with toluene/EtOAc (1/1,
v/v; 250 ml). The product fraction (90 ml) was evaporated to give 15 (1.9 g, 90%) as a colorless solid foam.
Bis(diisopropylamino)-[2-(4-nitrophenyl)ethoxy]phosphane (27)
2-(4-Nitrophenyl)ethanol (8.35 g, 50
mmol) was added in small portions over 30 min to a solution of
distilled PCl
(28 ml; 280 mmol) in absolute ether (80 ml)
at -5 °C under a nitrogen atmosphere. After stirring for 15
min at -5 °C and 1.5 h at room temperature, the solvent and
excess PCl
were removed under high vacuum. The yellowish
syrupy residue was then dissolved in 200 ml of absolute ether and
reacted at -10 °C with N,N-diisopropylamine (64 ml,
450 mmol) by dropwise addition over 30 min under a nitrogen atmosphere.
The reaction mixture was stirred at -10 °C for 15 min and
room temperature for 16 h. The voluminous precipitate of N,N-diisopropylamine hydrochloride was filtered under
nitrogen, and the solvent was removed in vacuo. The yellowish
syrupy product (17.6 g; 89%), which crystallized on storage at
-20 °C, was pure enough to be used for phosphitylation
reactions.
H NMR (CDCl
): 8.10-8.13 (d,
2H, o to NO
); 7.36-7.40 (d, 2H, m to NO
);
3.75-3.82 (q, 2H, P-O-CH
); 3.36-3.51 (m, 2H,
N-CH); 2.95-3.00 (t, 2H, P-O-C-CH
); 1.05-1.12
(2d, 12H, N-C-CH
).
P NMR (CDCl
):
123.53 ppm.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-(monomethoxytrityl)adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine,
ApA (28and28a)
Phosphoramidite 15 (1.41 g;
1.34 mmol), N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine (5) (0.36 g, 0.93 mmol) and tetrazole (0.188 g, 2.68 mmol) were
stirred at room temperature in absolute CH
CN (9 ml) under a
nitrogen atmosphere. After 4 h, a solution of I
(0.5 g in
CH
Cl
/H
O/pyridine (1/1/3, v/v/v))
was added dropwise until the brown color did not disappear. The mixture
was stirred for 15 min, extracted with CH
Cl
(3
60 ml), and saturated
Na
S
O
/NaCl solution (2
60
ml). The CH
Cl
phase was collected, dried over
Na
SO
, evaporated, and coevaporated with toluene
(2
20 ml) to remove the pyridine. The crude dimer (1.85 g) was
dissolved in CH
Cl
, applied onto a flash silica
gel column (12
2.5 cm), and chromatographed using
CH
Cl
/1% MeOH (400 ml), 2% MeOH (200 ml), and 3%
MeOH (200 ml) to elute the product (600 ml). This fraction was
evaporated to dryness to give 1.45 g (quantitative yield) of dimer 28 as a colorless amorphous solid. The identity of the isolated
dimer 28 was proven by comparison with authentic material by
spectrophotometric methods. The authentic material was synthesized by
the phosphotriester method.

UV (MeOH): 
(log
) = 277 (4.69)
(260 (4.54); 231 (4.66)). R
on silica gel with
CHCl
/MeOH (49/1, v/v) = 0.37.
N
-Benzoyl-3`-O-(tert-butyl)dimethylsilyladenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine-5`-OH-ApA (29and29a)
The
crude dimer mixture 28 and 28a (2.24 g, 1.43 mmol) was
stirred with 2% p-TsOH in CH
Cl
/MeOH (4/1, v/v,
20 ml) at room temperature for 30 min. The reaction mixture was diluted
with CH
Cl
(200 ml), washed with H
O
(2
80 ml), dried over Na
SO
, and
evaporated to dryness. The colorless amorphous residue (2.0 g) was
applied onto a flash silica gel column (21
2.5 cm),
chromatographed with CH
Cl
(200 ml) and
CH
Cl
/2% MeOH (400 ml), and the product was
eluted with CH
Cl
/2% MeOH (500 ml). The product
fraction was evaporated and dried under high vacuum to give 1.2 g (75%
calculated to compound 5 over two steps) of 5`-OH dimer 29 as an amorphous solid. The identity of the isolated dimer 29 with authentic material was proven by chromatographic and
spectrophotometric comparison.

UV (MeOH): 
(log
) = 278 (4.68),
(259 (4.51), and 233 (4.46)). R
on silica gel with
toluene/EtOAc/MeOH (5:4:1) = 0.53.
P = NMR
(CDCl
: -0.36 and -0.73 ppm.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`-]N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine
A(R
)ApA (30) and
A(S
)ApA
(31)
Phosphoramidite 15 (0.59 g,
0.56 mmol), 5`-hydroxy ApA dimer 29 (0.52 g, 0.40 mmol) and
tetrazole (0.079 g; 1.12 mmol) were dissolved in dry CH
CN
(4 ml) and stirred at room temperature under a nitrogen atmosphere.
After 3 h, phosphoramidite 15 (0.464, 0.44 mmol) and tetrazole
(0.062 g, 0.88 mmol) were added again and the mixture was stirred for
another 3 h. Oxidation with S
(0.257 g, 1 mmol) and
pyridine (2.6 ml) was followed within 16 h at room temperature. The
reaction mixture was diluted with CH
Cl
(200
ml), washed with a saturated NaCl solution (2
80 ml), dried
over Na
SO
, and evaporated to dryness. Final
coevaporation was done with toluene (3
20 ml) to remove
pyridine. The crude diastereoisomeric mixture
A(R
,S
)ApA (30 and 31) was dissolved in CH
Cl
(20 ml),
applied onto a flash silica gel column (11
2.5 cm), and
chromatographed with CH
Cl
(400 ml),
CH
Cl
/0.5% MeOH (200 ml), 1% MeOH (200 ml). The
product was eluted with CH
Cl
/1.5% MeOH (200
ml). The product fraction was evaporated to dryness to give 0.713 g
(78%) of the isomeric mixture 30 and 31. Separation into
the pure diastereomers was achieved by application to preparative
silica gel plates (40
20
0.2 cm, eight plates) in
toluene/EtOAc (1/1, v/v, four developments). The isomeric product bands
were separately eluted with CH
Cl
/MeOH (4/1,
v/v) and evaporated to solid foams, which were dried under high vacuum
to give 0.311 g (34%) of A(R
)ApA 30 and
0.245 g (27%) of A(S
)ApA 31.

UV (MeOH): 
(log
) = 278 (4.87)
(260 (4.72); 231 (4.75)). R
on silica gel with
toluene/EtOAc (1/1, v/v, two developments) and toluene/EtOAc (1/2, v/v,
one development) = 0.37.

UV (MeOH): 
(log
) = 277 (4.86)
(260 (4.72); 231 (4.75)). R
on silica gel with
toluene/EtOAc (1:1, two developments) and toluene/EtOAc (1:2, one
development) = 0.27.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl])-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine
5`-hydroxy A(R
)ApA (32) and 5`-hydroxy
A(S
)ApA (33)
The
fully protected trimers 30 and 31 were separately
detritylated as follows: trimer (A(R
)ApA 30: 0.263 g, 0.115 mmol; A(S
)ApA 31:
0.21 g, 0.092 mmol) was stirred with 2% p-TsOH in
CH
Cl
/MeOH (4/1, v/v; for 30: 3.2 ml, for 31: 2.6 ml) for 75 min at room temperature. The reaction mixture
was diluted with CH
Cl
(120 ml), washed with
H
O (2
40 ml), dried over
Na
SO
, and evaporated to dryness. The crude
product was purified on preparative silica gel plates (40
20
0.2 cm) in toluene/EtOAc (3/7, v/v), the product bands were
eluted with CH
Cl
/MeOH (4/1, v/v) and evaporated
to a solid foam to give 0.2 g (86%) of 5`-hydroxy
A(R
)ApA 32 and 0.121 g (66%) of 5`-hydroxy
A(S
)ApA 33,
respectively.

UV (MeOH): 
(log
) = 278 (4.86)
(260 (4.71); 233 (4.64)). R
on silica gel with
toluene/EtOAc (3:7, two developments) = 0.35 (diastereomers).
P NMR, 69.60, 68.91, -0.36, and -0.56 ppm
(diastereomers).

UV (MeOH): 
(log
) = 277 (4.85)
(260 (4.71); 233 (4.63)). R
silica gel with
toluene/EtOAc (3:7, two developments) = 0.42 (diastereomers).
P NMR, 69.28, 69.09, -0.33, and -0.56 ppm
(diastereomers).
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tertbutyl)dimethylsilyladenosine
ApA(R
)ApA (34) and
ApA(S
)ApA
(35)
Condensation to the fully
protected tetramers 34 and 35 was realized as follows.
Triethylammonium N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenosine-2`-[2-(4-nitrophenyl)ethyl]phosphate 20 (0.108 g, 0.1 mmol) and the 5`-hydroxy (R
) trimer 32 or (S
)
trimer 33 (0.1 g, 0.05 mmol), respectively, were coevaporated
with dry pyridine (3
2 ml) and dissolved in dry pyridine (0.5
ml), and then (2, 4, 6-triisopropyl)benzenesulfonyl chloride (0.061 g,
0.2 mmol) and 3-nitro-1,2,4-triazole (0.068 g, 0.6 mmol) were added.
The solution was stirred at room temperature for 21 h, extracted with
CH
Cl
(4
20 ml) and H
O (3
20 ml). The organic phase was collected, dried over
Na
SO
, evaporated, and coevaporated with toluene
(3
20 ml) to remove pyridine. The crude tetramers 34 and 35 were separately purified on preparative silica gel plates (40
20
0.2 cm) with toluene/EtOAc/MeOH (5/4/0.5, v/v/v),
and the product bands were eluted with
CH
Cl
/MeOH (4/1, v/v) and evaporated to solid
foams, which were dried under high vacuum to give 0.116 g (78%) of
ApA(R
)ApA 34 and 0.12 g (81%) of
ApA(S
)ApA 35.

UV (MeOH), 
(log
) = 277 (4.99)
(260 (4.85); 231 (4.85)). R
silica gel with
toluene/EtOAc/MeOH (5:4:0.5) = 0.63
(diastereomers).

UV (MeOH), 
(log
) = 277 (5.01)
(260 (4.88); 232 (4.89)). R
silica gel with
toluene/EtOAc/MeOH (5/4/0.5, v/v/v) = 0.62 (diastereomers).
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine
5`-OH-ApA(R
)ApA (36) and 5`-OH
ApA(S
)ApA (37)
The
fully protected tetramers (0.105 g; 0.035 mmol) 34 and 35 were separately detritylated by treatment with 2% p-TsOH in
CH
Cl
/MeOH (4/1, v/v, 1.2 ml) for 1 h at room
temperature. The reaction mixture was extracted with
CH
Cl
(3
40 ml) and washed with
H
O (3
30 ml). The organic phase was collected,
dried over Na
SO
, and evaporated to dryness. The
resulting residue was purified on preparative silica gel plates (20
20
0.2 cm) in toluene/EtOAc/MeOH (5/4/0.5, v/v/v). The
product bands were eluted with CH
Cl
/MeOH (4:1)
and evaporated to a solid foam, which was dried in high vacuum to give
0.064 g (67%) of the 5`-hydroxy ApA(R
)ApA 36 and 0.057 g (60%) of the corresponding (S
)
tetramer 37.

UV (MeOH), 
(log
) = 277 (5.00)
(260 (4.87); 235 (4.81)). R
silica gel with
toluene/EtOAc/MeOH (5:4:0.5) =
0.41.

UV (MeOH), 
(log
) = 277 (4.96)
(260 (4.82); 234 (4.74)). R
silica gel with
toluene/EtOAc/MeOH (5/4/0.5, v/v/v) = 0.39.
Adenylyl(2`-5`)-P-thioadenylyl-(2`-5`)-adenylyl-(2`-5`)-adenosine
ApA(R
)ApA (38) and
ApA(S
)ApA (39)
The
5`-hydroxy tetramers 36 and 37 were separately deblocked
in the following manner. Each 5`-hydroxy tetramer (0.056 g; 0.021 mmol)
was stirred with 0.5 M DBU in absolute CH
CN (2.5
ml) at room temperature; after 22 h, the solution was neutralized with
1 M AcOH in absolute CH
CN (1.25 ml) and evaporated
to dryness. The residual mixture was treated with methanolic ammonia
and after stirring at room temperature for 60 h, the solvent was
removed in vacuum. The residue was desilylated with 1 M Bu
NF in THF (5 ml) for 3 days. The solvent was
removed, and the residue was dissolved in H
O (10 ml),
applied onto a DEAE Sephadex A-25 column (30
2 cm), and
chromatographed first with H
O (200 ml) and then with a
linear gradient of 0-0.04 ml of TEAB buffer (pH 7.5) within 3000
ml (flow rate 2 ml/min). Under these conditions,
ApA(R
)ApA 38 was eluted with
0.23-0.28 M TEAB buffer and
ApA(S
)ApA 39 with 0.245-0.305 M TEAB buffer, respectively. The product fractions were collected,
evaporated, and coevaporated several times with MeOH. For further
purification, paper chromatography was performed using
i-PrOH/ammonia/H
O (55/10/35, v/v/v). The product band was
cut out, eluted with H
O, concentrated to a smaller volume,
and finally lyo-philized to give 728 OD
units (73%) of
ApA(R
)ApA 38 and 686 OD
units (69%) of ApA(S
)ApA 39,
respectively. ApA(R
)ApA (38): R
on cellulose in i-PrOH/ammonia/H
O
(55:10:35) = 0.36. UV (H
O): 
= 257 nm.
H NMR (D
O): 8.15, 8.07,
8.06, 7.93 (4 s, 4H, 4
H-C(8)); 7.92 (s, 2H, 2
H-C(2));
7.82, 7.79 (2 s, 2H, 2
H-C(2)); 6.03, 5.89, 5.86, 5.79 (4 d, 4
H-C(1`)). HPLC: on RP-18, A: 50 mM NH
H
PO
(pH 7.24). B:
MeOH/H
O (1/1, v/v); gradient: 0-1 min, 80% A, 20% B;
1-31 min, 30% A, 70% B; retention time: 9.55 min.
ApA(S
)ApA (39): R
on
cellulose in i-PrOH/ammonia/H
O (55/10/35, v/v/v) =
0.40. UV (H
O): 
= 257 nm. HPLC:
RP-18, A: 50 mM NH
H
PO
(pH
7.24). B: MeOH/H
O (1/1, v/v); gradient: 0-1 min, 80%
A, 20% B;1-31 min, 30% A, 70% B; retention time: 10.37 min.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`)-(monomethoxytrityl)P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenosine-2`-[2,5-dichlorophenyl-2-(4-nitrophenylethyl)phosphate]
A(R
,S
)Ap triester (43and44)
N
-Benzoyl-3`O-[(tert-butyl)dimethylsilyl]adenosine-2`-[2,5-dichlorophenyl,2-(4-nitrophenyl)ethylphosphate]
5`-OH-Ap triester 40 (0.52 g; 0.6 mmol) and phosphoramidite 15 (0.95 g; 0.9 mmol) were dissolved in absolute
CH
CN (6.5 ml) in the presence of tetrazole (0.126 g, 1.8
mmol) under nitrogen atmosphere. After stirring for 3 h at room
temperature, S
(0.39 g, 1.51 mmol) and absolute pyridine
(3.9 ml) were added and the reaction mixture was further stirred for 20
h, then extracted with CH
Cl
(2
80 ml)
and H
O (2
80 ml). The organic phase was collected,
dried over Na
SO
, and evaporated to dryness.
Final coevaporation was done with toluene (4
20 ml) to remove
pyridine. The crude diastereomeric mixture
A(R
,S
)Ap triester (43 and 44) was purified by flash silica gel column
chromatography (14
2.5 cm), using 200 ml of
CH
Cl
, CH
Cl
/1% MeOH, 2%
MeOH, 200 ml CH
Cl
/1% MeOH, 2% MeOH and finally
CH
Cl
/3% MeOH as eluants. The product fraction
(150 ml) was evaporated to a solid foam, which was dried in high vacuum
to give 0.975 g (88%) of 43 and 44 as a diastereomeric
mixture.

UV (MeOH): 
(log
) = 277 (4.75)
(228 (4.72)). R
on silica gel with
toluene/EtOAc/CHCl
(1/1/1, v/v/v) = 0.21.
P NMR (CDCl
) 69.87, 69.25, -6.89,
-7.22, and -7.31 ppm.
Triethylammonium
N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`)(monomethoxytrityl)-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenosine-2`-[2-(4-nitrophenylethyl)phosphate]
A(R
)Ap diester (45) and
A(S
)Ap diester
(46)
The solution of 0.558 g (3.36
mmol) of 4-nitrobenzaldehyde oxime in 15 ml of
H
O/dioxane/Et
N (1:1:1) was stirred for 30 min
at room temperature. Then, 0.62 g (0.336 mmol) of the diastereomeric
mixture of A(R
,S
)Ap triester (43 and 44) was added and stirred for 2.5 h at room
temperature. The mixture was evaporated and coevaporated with pyridine
(3
15 ml), toluene (3
15 ml), and finally with
CH
Cl
(3
15 ml). The residue was
dissolved in a small amount of CHCl
and chromatographed on
a flash silica gel column (15
2.5 cm) with CHCl
(150 ml), CHCl
/2% MeOH (200 ml), 4% MeOH (100 ml), 6%
MeOH (200 ml), CHCl
/6% MeOH/0.5% Et
N (300 ml),
and CHCl
/6% MeOH/2% Et
N (250 ml). The product
fraction (600 ml) was evaporated to a solid foam, which was dried under
high vacuum to give 0.55 g (91%) of the isomeric mixture 45 and 46. Separation into the pure diastereomers was achieved by
chromatography on preparative silica gel plates (seven plates, 40
20
0.2 cm) and three developments in
CHCl
/MeOH (9/1, v/v). The product bands were eluted with
CHCl
/MeOH (4/1, v/v) containing 1% Et
N and
evaporated to a solid foam to give 0.262 g (43%) of
A(R
)Ap diester 45, 0.144 (24%) of
A(S
)Ap diester 46, and 0.045 g (7%) of
A(R
,S
)Ap diester (45 and 46).

UV (MeOH): 
(log
) = 277 (4.69)
(260 (4.56); 231 (4.61)). R
on silica gel with
CHCl
/MeOH (9/1, v/v) = 0.37.
P NMR
(CDCl
), 69.66 and -0.09
ppm.

UV (MeOH): 
(log
) = 276 (4.60)
(260 (4.49); 232 (4.52)). R
on silica gel with
CHCl
/MeOH (9/1, v/v) = 0.28.
P NMR
(CDCl
), 68.96 and -0.06 ppm.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`)-(monomethoxytrityl)adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenosine-2`-[2,5-dichlorophenyl,2-(4-nitrophenyl)
ethylphosphate] ApAp triester (41and41a)
N
-Benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenosine-2`-[2,5-dichlorophenyl,2-(4-nitrophenyl)
ethylphosphate] 5`-OH-Ap triester 40 (0.43 g, 0.5 mmol) and
phosphoramidite 15 (0.735 g, 0.7 mmol) were dissolved in
absolute CH
CN (5 ml) in the presence of tetrazole (0.098 g,
1.5 mmol) under a nitrogen atmosphere. After stirring for 3.5 h at room
temperature, phosphoramidite (0.2 g, 0.19 mmol) and tetrazole (0.026 g,
0.37 mmol) were added again and the reaction mixture was stirred for
another 30 min. A solution of I
(0.5 g in
CH
Cl
/H
O/pyridine (1/1/3, v/v/v))
was added dropwise until the brown color did not disappear. The mixture
was stirred for another 10 min, diluted with CH
Cl
(20 ml), and washed with saturated
Na
S
O
/NaCl solution (2
80
ml). The organic phase was collected, dried over
Na
SO
, evaporated, and coevaporated with toluene
(3
30 ml) to remove the pyridine. The crude product was
purified by flash silica gel chromatography (15
2 cm) using
toluene/EtOAc (1/1, v/v), EtOAc, and EtOAc/2-4% MeOH as eluants.
The product fraction was evaporated to a solid foam, which was dried in
high vacuum at 30 °C to give 0.610 g (67%) of the ApAp triester.
The identity of the isolated compound with authentic material was
proved by spectrophotometric comparison. The authentic material was
synthesized from the Ap diester (20) (Charubala et al.,
1981) with the 5`-hydroxy P-triester (40) by the phosphotriester
method.

UV (MeOH): 
(log
) = 277 (4.75)
(260 (4.62); 228 (4.72)). R
on silica gel with
toluene/EtOAc/MeOH (5/4/1, v/v/v) = 0.78.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O
-2-
(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenylyl-2`-[(O
-2-
(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-
thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-
benzoyl- 2`,3`-di-O-[(tertbutyl)dimethylsilyl]adenosine
ApApA(R
)A (47)
Triethylammonium
N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`- O-(monomethoxytrityl)adenylyl2`-[(O
-2-
(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-
[(tert-butyl)dimethylsilyl]adenosine-2`-[2-(4-nitrophenyl)ethylphosphate] 42 (0.14 g; 0.078 mmol) and the 5`-hydroxy (R
) dimer (18) (0.08 g, 0.06 mol) were
coevaporated with dry pyridine (4
0.5 ml), dissolved in dry
pyridine (0.6 ml), and then (2, 4, 6-triisopropyl)benzenesulfonyl
chloride (0.047 mg, 0.156 mmol) and 3-nitro-1,2,4-triazole (0.053 mg,
0.47 mmol) were added. The solution was stirred at room temperature.
for 22 h, extracted with CH
Cl
(2
30
ml), washed with H
O (2
20 ml), dried over
Na
SO
, and evaporated to dryness. Pyridine was
removed by coevaporation with toluene (3
20 ml). The crude
tetramer 47 was purified by flash silica gel column
chromatography (15
1 cm) and eluted first with
CH
Cl
(50 ml), then with
CH
Cl
/1% MeOH (100 ml), 2% MeOH (50 ml), and
finally with CH
Cl
/3% MeOH (100 ml). The product
fraction (80 ml) was evaporated to dryness to give 0.11 g (62%) of
ApApA(R
)A 47 as a colorless foam after
drying under high vacuum at 35 °C.

UV (MeOH): 
(log
) = 277 (4.99)
(259 (4.84); 233 (4.85)). R
on silica gel with
CHCl
/MeOH (19/1, v/v) = 0.46.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O
-2-(4nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine
ApApA(S
)A (48)
ApAp diester 41 (0.14 g, 0.078 mmol) and the 5`-hydroxy (S
) dimer 19 (0.08 g, 0.06 mmol) were
coevaporated with dry pyridine (4
0.5 ml), dissolved in dry
pyridine (0.6 ml), and then (2, 4, 6-triisopropyl)benzenesulfonyl
chloride (0.47 mg, 0.156 mmol) and 3-nitro-1,2,4-triazole (0.053 mg,
0.46 mmol) were added. After stirring for 4.5 h at room temperature,
(2,4,6-triisopropyl)benzenesulfonyl chloride (0.024 g, 0.078 mmol) and
3-nitro-1,2,4-triazole (0.027 g, 0.234 mmol) were added again. The
solution was stirred at room temperature for 16.5 h, extracted with
CH
Cl
(4
20 ml) and with H
O
(3
20 ml), dried over Na
SO
, and
evaporated. Final coevaporations were done with toluene (4
15
ml) to remove pyridine. The crude tetramer 48 was purified by
flash silica gel column chromatography (15
1 cm) and eluted
analogous to tetramer 47 with CH
Cl
and
CH
Cl
/1-3% MeOH to give 0.107 g (60%) of
the tetramer ApApA(S
)A 48 as a colorless
foam after drying under high vacuum at 35
°C.

UV (MeOH): 
(log
) = 277 (4.99)
(259 (4.85); 231 (4.86)). R
on silica gel with
CHCl
/MeOH (19/1, v/v) = 0.46.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`di-O-[(tert-butyl)dimethylsilyl]adenosine
5`-OH-ApApA(R
)A (49)
The fully
protected tetramer ApApA(R
)A 47 (0.104 g,
0.035 mmol) was stirred with 2% p-TsOH in
CH
Cl
/MeOH (4/1, v/v, 1.4 ml) at room
temperature. After 1 h, the reaction mixture was extracted with
CH
Cl
(3
40 ml) and H
O (2
40 ml). The combined organic phase was dried over
Na
SO
and evaporated to dryness. The crude
product was purified on a flash silica gel column (11
1 cm);
the product was eluted with 20 ml of CH
Cl
and
50 ml of CH
Cl
/1% MeOH to 5% MeOH. The product
fraction (100 ml) was evaporated and dried under high vacuum to give
0.075 g (80%) of ApApA(R
)A 49.

UV (MeOH): 
(log
) = 277 (5.00);
260 (4.85); 234 (4.77)). R
on silica gel with
CHCl
/MeOH (19/1, v/v) = 0.43.
Adenylyl-(2`-5`)-adenylyl-(2`-5`)-P-thioadenylyl-(2`-5`)-adenosine
ApApA(R
)A (51) and
ApApA(S
)A (52)
The
fully protected tetramer ApApA(S
)A (48)
(0.104 g 0.35 mmol) was stirred with 2% p-TsOH in
CH
Cl
/MeOH (4/1, v/v, 1.4 ml) at room
temperature. After 1.5 h, the reaction mixture was extracted with
CH
Cl
(3
40 ml) and H
O (2
40 ml). The organic phase was collected, dried over
Na
SO
, and evaporated to dryness. The crude
product was purified on two preparative silica gel plates (20
20
0.2 cm) in CHCl
/MeOH (19/1, v/v), the product
band was eluted with CH
Cl
/MeOH (4/1, v/v) and
evaporated to a solid foam to give 0.068 g (72%) of the 5`-hydroxy
tetramer ApApA(S
)A 50. The corresponding
5`-hydroxy tetramers 49 and 50 were deblocked as follows.
The 5`-hydroxy tetramer (0.067 g, 0.025 mmol) was stirred with 0.5 M DBU in absolute CH
CN (3 ml) at room temperature
for 20 h, the solution was neutralized with 1 M AcOH in
absolute CH
CN (1.5 ml), and evaporated to dryness. (R
on silica gel with
EtOAc/i-PrOH/ammonia/H
O (7/1/2, v/v/v):
ApApA(R
)A = 0.58;
ApApA(S
)A = 0.66.) The residue was then
treated with methanolic ammonia (10 ml), and after 3 days, the solvent
was removed under vacuum. (R
on silica gel with
EtOAc/i-PrOH/ammonia/H
O (7/1/2, v/v/v);
ApApA(R
)A = 0.38;
ApApA(S
)A = 0.36). Desilylation was done
with 1 M Bu
NF in THF (5 ml). The reaction mixture
was stirred at room temperature for 48 h, and the solvent was
evaporated in vacuo. The residue was taken up in
H
O (10 ml) and applied to a DEAE Sephadex A-25 column (30
2 cm). With flow rates of 2 ml/min, the pure tetramer
ApApA(R
)A was eluted with 0.15-0.20 M TEAB buffer (pH 7.5) and in the case of the tetramer
ApApA(S
)A with 0.24-0.32 M TEAB
buffer (pH 7.5). After evaporation and coevaporation with MeOH several
times, the tetramer was applied onto eight paper sheets (25
50
cm) and developed in i-PrOH/ammonia/H
O (6/1/3, v/v/v). The
product band was cut out, eluted with H
O, concentrated to a
smaller volume, and finally lyophilized to give 675 OD
units (57%) of ApApA(R
)A 51 and 753
OD
(65%) of ApApA(S
)A 52.
ApApA(R
)A (51): R
on
cellulose in i-PrOH/ammonia/H
O (6/1/3, v/v/v) =
0.33. UV (H
O): 
= 258 nm. HPLC:
RP-18, A: 50 mM NH
H
PO
, pH
7.2. B: MeOH/H
O (1/1, v/v), gradient: 0-1 min, 80% A,
20% B; 1-31 min, 30% A, 70% B; retention time: 9.70 min.
ApApA(S
)A (52): R
on
cellulose in i-PrOH/ammonia/H
O (6/1/3, v/v/v) =
0.21. UV (H
O): 
= 258 nm. HPLC:
RP-18, A: 50 mM NH
H
PO
, pH
7.2. B: MeOH/H
O (1/1, v/v), gradient: 0-1 min, 80% A,
20% B; 1-31 min, 30% A, 70% B; retention time: 13.49 min.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-thioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4nitrophenyl)ethyl-5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)
dimethylsilyl]adenosine Ap(R
)ApApA (53)
Triethylammonium N
benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-Pthioadenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenosine-2`-[(O
-2-(4-nitrophenyl)ethylphosphate]
A(R
)Ap diester 45 (0.141 g, 0.078 mmol) and
the 5`-hydroxy dimer 29 (0.078 g, 0.06 mmol) were coevaporated
with dry pyridine (4
0.5 ml) and finally dissolved in dry
pyridine (0.6 ml). Then (2,4,6-triisopropyl)benzenesulfonyl chloride
(0.047 mg, 0.156 mmol) and 3-nitro-1,2,4-triazole (0.053 mg, 0.47 mmol)
were added and stirred at room temperature for 21 h. The reaction
mixture was diluted with CH
Cl
(60 ml) and
washed with H
O (2
30 ml), dried over
Na
SO
, and evaporated to dryness. Pyridine was
removed by coevaporation with toluene (3
20 ml). The crude
tetramer 53 was purified by flash silica gel column
chromatography (11
1 cm) and eluted first with
CH
Cl
(50 ml), then with
CH
Cl
/1% MeOH (100 ml), 2% MeOH (200 ml), and 3%
MeOH (50 ml), and finally with CH
Cl
/5% MeOH (50
ml). The product fraction (200 ml) was evaporated to dryness. The
residue was chromatographed again on preparative silica gel plates (20
20
0.2 cm) in toluene/EtOAc/MeOH (5/4/0.5, v/v/v) to
remove a small amount of 5`-hydroxy dimer. The tetramer product band
was eluted with CH
Cl
/MeOH (4/1, v/v) and
evaporated to a solid foam to give 0.053 g (30%) of
A(R
)ApApA 53 after drying in high vacuum at
35 °C.

UV (MeOH): 
(log
) = 277 (4.96)
(260 (4.82); 232 (4.82)). R
on silica gel with
toluene/EtOAc/MeOH (5:4:1) = 0.78.
N
-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-thioadenylyl-2`-
[(O
-2-(4-nitrophenyl)ethyl-5`]-N
-benzoyl-3`O-[(tert-butyl)dimethylsilyl]adenylyl-
2`-[(O
-2-(4-nitrophenyl)ethyl5`]N
-benzoyl-3`-
O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O
-2-(4-nitrophenyl)ethyl-
5`]-N
-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine
A(S
)ApApA (54)
A(S
)Ap diester 46 (0.141 g, 0.078 mmol) and the 5`-hydroxy dimer 29 (0.078 g, 0.06 mmol) were coevaporated with dry pyridine (4
0.5 ml) and dissolved in dry pyridine (0.6 ml).
(2,4,6-Triisopropyl)benzenesulfonyl chloride (0.047 mg, 0.156 mmol) and
3-nitro-1,2,4-triazole (0.053 mg, 0.47 mmol) were added, and the
mixture was stirred at room temperature. After 21 h,
(2,4,6-triisopropyl)benzenesulfonyl chloride (0.024 mg, 0.079 mmol) and
3-nitro-1,2,4-triazole (0.027 mg, 0.24 mmol) were added again. The
reaction mixture was stirred for 1 h, then diluted with
CH
Cl
(60 ml), washed with H
O (2
30 ml), dried over Na
SO
, and evaporated
to dryness. Further work-up was performed analogous to that described
for 53 to give 43 mg (24%) of A(S
)ApApA 54 in the form of a solid foam.

UV (MeOH): 
(log
) = 277 (4.98)
(260 (4.84); 232 (4.85)). R
on silica gel with
toluene/EtOAc/MeOH (5/4/1, v/v/v) = 0.78.
P-Thioadenylyl-(2`-5`)-adenylyl-(2`-5`)-adenylyl-(2`-5`)-adenosine
A(R
)ApApA (57) and
A(S
)ApApA (58)
The
corresponding fully protected tetramers 53 and 54 were
deblocked in the following manner. A solution of 0.047 g (0.016 mmol)
of (R
) tetramer 53 ((S
) tetramer 54: 0.032 g, 0.012 mmol)
in 2% p-TsOH in CH
Cl
/MeOH (4/1, v/v; for (R
): 0.5 ml; for (S
): 0.38
ml) was stirred for 1 h at room temperature. The reaction mixture was
diluted with CH
Cl
(60 ml), washed with
H
O (2
30 ml), dried over
Na
SO
, and evaporated to dryness. The crude
products were purified on preparative silica gel plates (20
20
0.2 cm) in CHCl
/MeOH (19/1, v/v), and the product
bands were eluted with CH
Cl
/MeOH (4/1, v/v) and
evaporated to solid foams to give 0.034 g (80%) of the 5`-hydroxy
A(R
)ApApA isomer 55 and 0.02 g (68%) of the
5`-hydroxy A(S
)ApApA isomer 56. Solutions
of the 5`-hydroxy tetramers 55 (0.034 g, 0.013 mmol) and 56 (0.02 g, 0.007 mmol) were separately stirred with 0.5 M DBU in absolute CH
CN (55: 1.5 ml; 56:
0.9 ml) for 18 h at room temperature, neutralized by addition of 1 M AcOH (55: 0.75 ml; 56: 0.45 ml), and
evaporated. The residue was treated with 10 ml of saturated methanolic
ammonia, and the solution, after stirring at room temperature for 60 h,
was evaporated to dryness. Desilylation was done by treatment with 1 M Bu
NF in THF (2.5 ml). After stirring at room
temperature for 60 h, the solvent was removed under vacuum. Some
H
O (10 ml) was added to the resulting residue and applied
to a DEAE Sephadex A-25 column (32
2 cm) and eluted with
0-0.5 M TEAB buffer, pH 7.5. The fractions of the main
peak were collected, evaporated and coevaporated several times with
MeOH. Further purification by paper chromatography
(i-PrOH/ammonia/H
O, 55/10/35, v/v/v) gave, after
lyophilization, 347 OD
units (58%) of
A(R
)ApApA (57) and 111 OD
units (31%) of A(S
)ApApA (58),
respectively. A(R
)ApApA (57): UV
(H
O) = 257 nm. R
on cellulose
in i-PrOH/ammonia/H
O (6/1/3, v/v/v) = 0.21. HPLC:
RP18, A: 50 mM NH
H
PO
(pH
7.2). B: MeOH/H
O (1/1, v/v), gradient: 0-1 min, 80%
A, 20% B; 1-31 min, 30% A, 70% B; retention time: 7.47 min.
A(S
)ApApA (58): UV (H
O)
= 257 nm. R
on cellulose in
i-PrOH/ammonia/H
O (6/1/3, v/v/v) = 0.32. HPLC: RP18,
A: 50 mM NH
H
PO
, pH 7.2. B:
MeOH/H
O (1/1, v/v), gradient: 0-1 min, 80% A, 20% B;
1-31 min, 30% A, 70% B; retention time: 9.84 min.
5`-Monophosphorylation of Phosphorothioate/Phosphodiester
Derivatives of 2-5A Trimer and Tetramer Cores
The
phosphorothioate/phosphodiester 2-5A trimer and tetramer cores
were 5`-phosphorylated by T4 polynucleotide kinase in the presence of
ATP and purified by HPLC as described (Kariko et al., 1987b;
Charachon et al., 1990). Yields of phosphorylation ranged from
15-68%.
Stability of the Phosphorothioate/Phosphodiester
2-5A Trimer and Tetramer Core Derivatives to Serum
Phosphodiesterase
2-5A and phosphorothioate/phosphodiester
core derivatives (300 µM) were incubated in 200 µl of
RPMI 1640 medium supplemented with 10% FBS in 5% CO
in air
at 37 °C. Aliquots (30 µl) were removed at t
and t
. Hydrolysis products were identified
by HPLC (Kariko et al., 1987a; solvent system 2).
Cell Culture and HIV-1
L929 cells were maintained
in monolayer culture in Dulbecco's minimum essential medium
(Eagle's) supplemented with 5% calf serum (Biofluids, Inc.).
Extracts were prepared from confluent monolayer cultures as described
(Kariko et al., 1987a). Sup T1 cells and HIV-1 IIIB in Molt4
cells were obtained from the AIDS Reference and Reagent Repository. Sup
T1 cells were maintained in RPMI 1640 medium supplemented with 10% FBS
as described (Henderson et al., 1991). HIV-1 IIIB was prepared
as described previously (Henderson et al., 1991).
Escherichia coli Expression of Recombinant, Human RNase
L
Recombinant, human RNase L was expressed as the fusion protein
glutathione S-transferase (GST)-RNase L in E. coli.
Cloning followed standard methods. PCR reactions used as a template
pZC5-1, which contains the full-length human RNase L cDNA (generously
provided by Dr. R. H. Silverman). Reactions contained the 5` primer,
5`-ATCGGGATCCATATGGAGAGCAGGGATCATAAC-3`, and the 3` primer,
5`-ACCGGAATTCACCAAGTGACTGTTCTCTC-3`, to amplify a 1.2-kb DNA fragment
containing a NdeI restriction site at the ATG translation
start codon. The PCR fragment (1.2 kb) was digested with BamHI
and EcoRI, purified by agarose gel electrophoresis, and cloned
into the BamHI and EcoRI sites of pRSET-C
(Invitrogen). The sequence of the PCR fragment was confirmed by
dideoxynucleotide sequencing (U. S. Biochemical Corp.). The resulting
plasmid was digested with PstI and HindIII. The
fragment containing pRSET-C and the 5`-fragment of RNase L cDNA (NdeI-PstI) was purified by agarose gel
electrophoresis and ligated to the 3`-fragment of RNase L cDNA (PstI-HindIII) from pZC5-1 to regenerate the entire
RNase L cDNA (pNK11). The complete RNase L cDNA (2.2 kb) contained in
pNK11 was digested with NdeI, filled in with Klenow fragment,
and ligated into the SmaI site of pGEX2T to make pNK12.
Orientation and correct reading frame were verified by restriction
endonuclease mapping. pNK12 was transformed into E. coli DH5
(Life Technologies, Inc.) for expression.Cell growth
and expression of recombinant, human GST-RNase L were performed as
described (Smith and Johnson, 1988). Cell extracts were prepared by
passage through a French pressure cell (5000 p.s.i., twice). The
homogenate was clarified by centrifugation (20,000
g,
60 min, 4 °C). The supernatant was passed through a 2-ml
GSH-Sepharose column (1 ml/min). After washing with 40 ml of
phosphate-buffered saline, the GST-RNase L fusion protein was eluted
from the column with 20 mM glutathione in 50 mM Tris-HCl, pH 8.0.
Further purification of GST-RNase L was
accomplished with a fast protein liquid chromatography system
(Pharmacia Biotech Inc.) (Dong et al., 1994). GST-RNase L (1
mg) was loaded onto a Superdex 200 (Hiload 16/60) column (Pharmacia
Biotech Inc.), equilibrated with 25 mM Tris-HCl (pH 7.4), 50
mM KCl, 1 mM EDTA, 5 mM MgCl
,
0.1 mM ATP, and 14 mM
-mercaptoethanol.
GST-RNase L was eluted from the column (110 kDa) at 1 ml/min and stored
at -80 °C.
Radiobinding Assays
Assays were performed as
described (Wreschner et al., 1981) with L929 cell extracts as
the source of RNase L.
p
A
[
P]pCp was synthesized
by ligation of [
P]pCp (specific activity 3000
Ci/mmol) to p
A
with T4 RNA ligase.
RNase L Assays
For Method 1, core-cellulose assays
for activation of murine RNase L were performed as described
(Silverman, 1985; Kariko et al., 1987a). Activation of RNase L
was measured by conversion of poly(U)[
P]pCp to
acid-soluble fragments. For Method 2, ribosomal RNA cleavage
assays were performed as described (Kariko et al., 1987a). For Method 3, activity of recombinant, human GST-RNase L was
determined by hydrolysis of poly(U)[
P]pCp in the
presence of 2-5A or 2-5A derivatives according to Dong et al.(1994). Reaction mixtures (15 µl) containing 20 ng
of GST-RNase L, 2000-5000 cpm
poly(U)[
P]pCp and 2-5A or derivative were
incubated at 30 °C for 15 min. The remaining RNA was precipitated
with 1 ml of 5% trichloroacetic acid (0 °C) in 0.2% pyrophosphate
with 0.1 ml of 5 mg/ml yeast RNA as carrier. Precipitated RNA was
filtered onto glass-fiber filters and quantitated by scintillation
spectrometry (counting efficiency >99%).
Assay of HIV-1 (IIIB)-induced Syncytia
Formation
The infected-centers assay was used to quantify the
effects of the phosphorothioate/phosphodiester 2-5A derivatives
on the ability of HIV-1 to replicate in human cells (Henderson et
al., 1991). Briefly, freshly isolated peripheral blood lymphocytes
(PBL) were treated with 2-5A or derivatives for 2 h and infected
with partially purified HIV-1 strain IIIB (m.o.i. of 0.1). The infected
PBL were maintained in RPMI 1640 medium supplemented with 10% fetal
bovine serum at 37 °C in a humidified 5% CO
atmosphere.
After 48 h, the cells were washed twice in Hank's balanced salt
solution and serially diluted, and a known number of infected cells
(from 1
10
to 2
10
cells/well)
was seeded into multiple wells of a 96-well microtiter plate.
Immediately, 2
10
exponentially growing Sup T1
cells were added to each well. The wells were visually examined daily
for syncytia; final results were read at 72 h. Each syncytium was
counted as a single infected cell. The number of infected cells
required for syncytia formation in 50% of the wells was calculated
using the Reed-Muench formula. Statistical significance was determined
using the Student's t test.
Assay of Cell Survival
Cell viability was
determined by trypan blue exclusion. Post-treatment colony forming
ability was determined by growth in microtiter wells (Kraemer et
al., 1980).
Uptake Assays
Accumulation of
ApA(R
)A and ApA(S
)A in
control and HIV-1-infected Sup T1 cells was determined as follows. Sup
T1 cells were infected with HIV-1 (m.o.i. of 0.1). After 48 h (at which
time syncytia were evident), ApA(R
)A or
ApA(S
)A (300 µM) was added and
incubated for 4 h at 37 °C. The cells were harvested, washed, and
extracted with Nonidet P-40. 2-5A derivatives were extracted with
trichloroacetic acid:freon (Silverman, 1985). The acid-salt elution
method (Haigler et al., 1980) was also used to distinguish
between membrane-associated and cytoplasmic 2-5A derivatives.
Quantitation of Intracellular ApA(R
)A and
ApA(S
)A
Intracellular uptake and quantitation of the
2-5A derivatives was determined by HPLC as described (Suhadolnik et al., 1983; Kariko et al., 1987a). Reverse phase
HPLC analyses of the trichloroacetic acid:freon extracts were performed
with a Waters C
µBondapak analytical column (flow rate
1 ml/min). The column was eluted with solvent A = 50 mM ammonium phosphate, pH 7.0, and solvent B =
methanol/H
O (1:1, v/v) with a linear gradient (beginning at
1 min) (t = 1 min, 10% B; t = 31 min,
40% B) followed by maintenance at 40% B for 20 min.
RESULTS
Chemical Synthesis of the
Phosphorothioate/Phosphodiester 2-5A Derivatives
The
chemical synthesis and resolution of phosphorothioate/phosphodiester
trimer and tetramer derivatives of 2-5A are described using
phosphotriester and phosphoramidite approaches to introduce R
and S
chirality into
individual internucleotide linkages. The success of this approach
depends on the choice of appropriate protecting groups in the monomeric
building blocks as illustrated in Fig. 1and 2. Reactive
functional groups are protected by transient and/or permanent blocking
groups, which can be individually manipulated. Chemical syntheses of
2-5A derivatives with various phosphorothioate configurations
have been reported (Nelson et al., 1984; Charubala and
Pfleiderer, 1987; Charubala et al., 1991a; Battistini et
al., 1992; Shimazu et al., 1993). A new synthetic
approach is described here in which relatively base-stable blocking
groups are introduced that can be deprotected by
-elimination.
Figure 1:
Synthesis of the
phosphorothioate/phosphodiester 2-5A trimer cores:
A(R
)ApA (compound 10),
A(S
)ApA (compound 11),
ApA(R
)A (compound 23), and
ApA(S
)A (compound
24).
Stability of the Phosphorothioate/Phosphodiester
2-5A Core Derivatives to Serum Phosphodiesterase
As
reported previously from this laboratory (Kariko et al.,
1987), A(R
)A and A(S
)A were
not hydrolyzed in 6 h (Table 1). In addition, no hydrolysis of
the phosphorothioate/phosphodiester trimer core derivatives was
observed. However, the phosphorothioate/phosphodiester tetramer core
derivatives were hydrolyzed from the 5`- or 2`,3`-terminus depending on
the location of the phosphorothioate-substituted linkage. Under these
conditions, authentic A
and A
were completely
hydrolyzed to inosine and hypoxanthine in 20 min.
E. coli Expression of Recombinant, Human
2-5A-dependent Endoribonuclease
The study of the
stereodynamics of the activation of RNase L by 2-5A and the
phosphorothioate/phosphodiester 2-5A derivatives was extended
using the E. coli expressed fusion protein, GST-RNase L. The
fusion protein was induced following IPTG treatment. The GST-RNase L
was soluble. The GST-RNase L specifically bound 2-5A as measured
by photolabeling with [
P]pApAp-8-azidoA; in
nontransformed E. coli cell extracts, no protein was
photolabeled. (
)These data were in agreement with
photolabeling of RNase L in L929 cell extracts (Charubala et
al., 1989).
Purification of GST-RNase L
Purification of
GST-RNase L was performed using GSH-Sepharose affinity chromatography.
One liter of E. coli culture yielded about 1 mg of GST-RNase
L. Cleavage of the GST-RNase L by thrombin revealed a
thrombin-susceptible site in RNase L, which resulted in partial
cleavage of RNase L to 42- and 36-kDa peptide fragments. 2-5A
binding activity was mainly retained by the 42-kDa protein as
determined by [
P]pApAp-8-azidoA photolabeling
(data not shown). Therefore, GST-RNase L was used in the
poly(U)[
P]pCp hydrolysis assays. The GST domain
of the GST-RNase L fusion protein did not affect the activation of
RNase L by 2-5A or phosphorothioate/phosphodiester 2-5A
derivatives. GST expressed during induction co-purified with GST-RNase
L by GSH Sepharose. Therefore, GST-RNase L was further purified by gel
filtration with Superdex 200 and FPLC. The final GST-RNase L fusion
protein preparation was homogeneously pure as determined by
SDS-polyacrylamide gel electrophoresis (not shown).
Binding of Phosphorothioate/Phosphodiester 2-5A
Derivatives to Partially Purified Murine RNase L
A
,
pA
, and p
A
bound to RNase L with
IC
values of 1
10
M, 1
10
M, and 1
10
M, respectively. Binding of the
phosphorothioate/phosphodiester trimer and tetramer core and
5`-monophosphate 2-5A derivatives to murine RNase L was
equivalent to or slightly better than the corresponding authentic
2-5A cores and 5`-monophosphates (IC
s from 8
10
M to 8
10
M for the cores and from 1
10
M to 1
10
M for the
5`-monophosphates). The trimer and tetramer 2-5A core derivatives
with phosphorothioate substitution in the first internucleotide linkage
from the 5`-terminus exhibited lower affinity compared to those with
substitution in the second or third internucleotide linkage. These
results were consistent with the binding of fully substituted
phosphorothioate 2-5A trimer and tetramer derivatives reported
from this laboratory (Kariko et al., 1987b; Charachon et
al., 1990).
Hydrolysis of Poly(U)[
P]pCp by
Partially Purified Murine RNase L
In core-cellulose assays with
murine RNase L, p
A
, p
A
,
pA
, and pA
had IC
values of 5
10
M, 5
10
M, 2
10
M, and 2
10
M, respectively.
ApA(R
)A was the only
phosphorothioate/phosphodiester trimer core able to activate partially
purified murine RNase L (IC
of 5
10
M) (Fig. 3A,
). Three of the
phosphorothioate/phosphodiester trimer 5`-monophosphate derivatives
activated the partially purified murine RNase L;
pApA(R
)A was the most potent activator (IC
of 1
10
M) (Fig. 3B,
). pA
(
),
pA(R
)ApA (
), and
pA(S
)ApA (
) were 100-fold less potent, and
pApA(S
)A (
) was completely inactive.
ApA(R
)ApA (
) and
ApApA(R
)A (
) were the only tetramer core
derivatives able to activate murine RNase L (IC
s of 5
10
M) (Fig. 3C).
Five of the six phosphorothioate/phosphodiester tetramer
5`-monophosphate derivatives (pA(R
)ApApA,
pA(S
)ApApA, pApA(R
)ApA,
pApApA(R
)A, and pApApA(S
)A)
activated murine RNase L (IC
s >6
10
M to 8
10
M), whereas the pApA(S
)ApA
enantiomer did not activate murine RNase L at concentrations as high as
1
10
M (Fig. 3D,
).
Figure 3:
Hydrolysis of
poly(U)[
P]pCp by partially purified murine RNase
L with phosphorothioate/phosphodiester trimer core (A), trimer
5`-monophosphate (B), tetramer core (C), and tetramer
5`-monophosphate (D) 2-5A derivatives. PanelsA and B, p
A
(
),
pA
(
), A
(
),
A(R
)ApA (
), A(S
)ApA
(
), ApA(R
)A (
),
ApA(S
)A (
), pA(R
)ApA
(
), pA(S
)ApA (
),
pApA(R
)A (
), and
pApA(S
)A (
). PanelsC and D, p
A
(
), pA
(
), A(R
)ApApA (
),
A(S
)ApApA (
),
ApA(R
)ApA (
),
ApA(S
)ApA (
), ApApA(R
)A
(
), ApApA(S
)A (
),
pA(R
)ApApA (
),
pA(S
)ApApA (
),
pApA(R
)ApA (
),
pApA(S
)ApA (
),
pApApA(R
)A (
),
pApApA(S
)A (
). 100% represents 25,000 dpm
of poly(U)[
P]pCp bound to glass fiber
filters.
Hydrolysis of Ribosomal RNA by Partially Purified Murine
RNase L
Consistent with results from core-cellulose assays (Fig. 3A), ApA(R
)A (1
10
M) was the only trimer core able to
activate murine RNase L to cleave rRNA giving rise to the specific
cleavage products characteristic of RNase L (Fig. 4A, lane5). A(R
)ApA,
A(S
),ApA and ApA(S
)A, as well
as authentic A
, were unable to activate the murine RNase L
at concentrations as high as 1
10
M (Fig. 4A, lanes3, 4, 6, and 7). Authentic p
A
activated the murine RNase L at 1
10
M (Fig. 4A, lane2).
pA(R
)ApA, pA(S
)ApA, and
pApA(R
)A activated murine RNase L at 1
10
M, 1
10
M, and 2
10
M,
respectively (Fig. 4B, lanes4-6), compared with pA
, which activates
RNase L at 1
10
M (lane3). However, pApA(S
)A did not
activate the murine RNase L at 1
10
M (lane7); no activity was observed at
concentrations as high as 5
10
M (data not shown). ApA(R
)ApA and
ApApA(R
)A were the only tetramer core derivatives
able to activate RNase L (1
10
M) (Fig. 5A, lanes6 and 8). As
observed in the core-cellulose assays (Fig. 3D), five
of the six phosphorothioate/phosphodiester tetramer 5`-monophosphates
could activate the partially purified murine RNase L
(pA(R
)ApApA, pA(S
)ApApA,
pApA(R
)ApA, pApApA(R
)A, and
pApApA(S
)A) (Fig. 5B, lanes
4-6, 8, and 9, respectively). The most
potent activator was pApA(R
)ApA (1
10
M) (Fig. 5B, lane6). pApA(S
)ApA was unable to
activate RNase L at concentrations as high 1
10
M (Fig. 5B, lane7).
Figure 4:
Ribosomal RNA cleavage assay with
partially purified murine RNase L and phosphorothioate/phosphodiester
trimer core (A) and trimer 5`-monophosphate (B) 2-5A
derivatives. Panel A, L929 cell extracts were incubated in the
absence (lane 1) or presence of p
A
(10
M) (lane 2),
A(R
)ApA (10
M) (lane 3), A(S
)ApA (10
M) (lane 4), ApA(R
)A
(10
M) (lane 5),
ApA(S
)A (10
M) (lane 6), or A
(10
M) (lane 7). Panel B, L929 cell extracts were incubated
in the absence (lane 1) or presence of p
A
(2
10
M) (lane 2),
pA
(10
M) (lane 3),
pA(R
)ApA (10
M) (lane 4), pA(S
)ApA (10
M) (lane 5), pApA(R
)A (2
10
M) (lane 6) or
pApA(S
)A (10
M) (lane 7). Arrows indicate positions of specific
cleavage products (SCP) of RNase L.
Figure 5:
Ribosomal RNA cleavage assay with
partially purified murine RNase L and phosphorothioate/phosphodiester
tetramer core (A) and tetramer 5`-monophosphate (B)
2-5A derivatives. Panel A, L929 cell extracts were
incubated in the absence (lane1) or presence of
p
A
(10
M) (lane2), A
(10
M) (lane3), A(R
)ApApA
(10
M) (lane4),
A(S
)ApApA (10
M) (lane5), ApA(R
)ApA
(10
M) (lane6),
ApA(S
)ApA (10
M) (lane7), ApApA(R
)A
(10
M) (lane8), or
ApApA(S
)A (10
M) (lane9). PanelB, L929 cell
extracts were incubated in the absence (lane1) or
presence of p
A
(10
M) (lane2), pA
(10
M) (lane3),
pA(R
)ApApA (10
M) (lane4), pA(S
)ApApA
(10
M) (lane5),
pApA(R
)ApA (10
M) (lane6), pApA(S
)ApA
(10
M) (lane7),
pApApA(R
)A (10
M) (lane8), or pApApA(S
)A
(10
M) (lane9).
Degradation of Poly(U)[
P]pCp by
Recombinant, Human GST-RNase L
Purified recombinant, human
GST-RNase L was activated to hydrolyze
poly(U)[
P]pCp by p
A
,
pA
, and A
at IC
values of 5
10
M, 5
10
M, and 5
10
M,
respectively (Fig. 6). The IC
values for
A(R
)ApA and A(S
)ApA were 2
10
M and 2
10
M, respectively, whereas the IC
for
ApA(R
)A was 8
10
M. ApA(S
)A did not activate the
recombinant, human GST-RNase L at concentrations as high as 1
10
M. Whereas A
and
A(S
)A dimer cores did not activate recombinant,
human GST-RNase L at 1
10
M,
A(R
)A (1
10
M)
did activate the human recombinant GST-RNase L. Recombinant, human
GST-RNase L was not activated by 3-5A
or
3-5A(R
)A(R
)A. GST-RNase
L did not hydrolyze poly(C)[
P]pCp with or
without 2-5A (not shown).
Figure 6:
Hydrolysis of
poly(U)[
P]pCp by recombinant, human GST-RNase L
with phosphorothioate/phosphodiester trimer core 2-5A
derivatives. p
A
(
), pA
(
), A
(
), A(R
)ApA
(
), A(S
)ApA (
),
ApA(R
)A (
), ApA(S
)A
(
). 100% represents 2,000 dpm of
poly(U)[
P]pCp bound to glass fiber
filters.
Inhibition of Murine RNase L Activation by
pApA(S
)A
The binding affinity of
pApA(S
)A (1
10
M) for murine RNase L and the observation that
pApA(S
)A does not activate RNase L suggested that
pApA(S
)A might be a stereospecific inhibitor of
RNase L. Indeed, pApA(S
)A inhibited activation of
RNase L by p
A
or pApA(R
)A (Fig. 7A). Authentic p
A
activated RNase L at 10
M or
10
M (lanes1 and 3). However, addition of pApA(S
)A
(10
M) resulted in the inhibition of RNase
L-catalyzed hydrolysis of rRNA (lanes2 and 4). Similarly, whereas pApA(R
)A activated
murine RNase L at 10
M or 10
M (lanes5 and 7), addition
of pApA(S
)A (10
M)
inhibited activation (lanes6 and 8). The
inhibitory activity of pApA(S
)A was also observed
with partially purified murine RNase L (Fig. 7B).
p
A
activated RNase L (IC
of 5
10
M (
)); however, upon
addition of pApA(S
)A (1
10
M), the observed IC
value shifted to 1
10
M (
), demonstrating
specific inhibition of p
A
-mediated activation
of murine RNase L by pApA(S
)A.
Figure 7:
Inhibition of activation of partially
purified murine RNase L by pApA(S
)A in L929 cell
extracts (A) and by partially-purified RNase L (B). A, incubations were in the presence of p
A
(10
M) (lanes1 and 2), p
A
(10
M) (lanes3 and 4),
pApA(R
)A (10
M) (lanes5 and 6),
pApA(R
)A (10
M) (lanes7 and 8), and
pApA(S
)A (10
M) (lanes2, 4, 6, and 8). B, activation of RNase L partially purified from L929
cell extracts as determined by conversion of
poly(U)[
P]pCp to acid-soluble fragments.
p
A
(
), p
A
, and
pApA(S
)A at 10
M (
). 100% represents 25,000 dpm of
poly(U)[
P]pCp bound to glass fiber
filters.
Inhibition of Cell Growth by
Phosphorothioate/Phosphodiester 2-5A Core
Derivatives
Little or no decrease in survival was observed with
any trimer or tetramer phosphorothioate/phosphodiester 2-5A core
derivative (Fig. 8). On the basis of the lack of cytotoxicity
and calculated 1% uptake of [
H]cordycepin trimer
core by lymphocytes (Suhadolnik et al., 1983), 3
10
M was chosen as the concentration for
screening the phosphorothioate/phosphodiester derivatives for anti-HIV
activity.
Figure 8:
Inhibition of HIV-1 (IIIB)-induced
syncytia formation and cell growth by phosphorothioate/phosphodiester
trimer (A) and tetramer (B) 2-5A derivatives.
PBL (2
10
cells/ml) were treated with
A(R
)ApA, A(S
)ApA,
ApA(R
)A, ApA(S
)A,
A(R
)ApApA, A(S
)ApApA,
ApA(R
)ApA, ApA(S
)ApA,
ApApA(R
)A, ApApA(S
)A,
A
, or A
(300 µM) or adenosine (900
µM) for 2 h and infected with HIV-1 (strain IIIB). Cell
survival was determined by colony formation of uninfected Sup T1 cells
in three independent experiments. The number of cells required for
syncytia formation in 50% of the wells was
determined.
Inhibition of HIV-1-induced Syncytia Formation by
Phosphorothioate/Phosphodiester 2-5A Derivatives
The
infected centers assay was used to measure the ability of the
phosphorothioate/phosphodiester 2-5A derivatives to inhibit
HIV-1-induced syncytia formation, an indicator of HIV-1 replication in
T cells. ApA(R
)A was a highly efficient inhibitor
of syncytia formation, with 8
10
cells required for
the formation of infected centers in 50% of the wells compared to
untreated cells, an 80-fold reduction in syncytia formation (p < 0.059) (Fig. 8A). The
ApA(S
)A enantiomer was also an effective inhibitor
of syncytia formation, with 1
10
cells required for
formation of infected centers in 50% of the wells, a 10-fold reduction
in syncytia formation (p = 0.07).
A(R
)ApA and A(S
)ApA had
little or no effect on HIV-1-induced syncytia formation compared to
untreated cells (p = 0.6 and 0.6). Authentic A
and A
(3
10
M)
inhibited syncytia formation slightly; adenosine (9
10
M) was not inhibitory. The numbers of
infected cells required for infected centers in 50% of the wells in the
presence of ApApA(R
)A and
ApApA(S
)A were 8
10
and 3
10
cells, respectively, which represent 40-and
15-fold reductions in syncytia formation (Fig. 8B). The
other tetramer diastereomers (A(R
)ApApA,
A(S
)ApApA, ApA(R
)ApA, and
ApA(S
)ApA) and A
were not effective
inhibitors. A(R
)A, A(S
)A,
A
dimer cores (3
10
M),
adenine (9
10
M), or 3`,5`-A
(3
10
M) were not inhibitory
(data not shown).
Enhanced Uptake and Accumulation of ApA(R
)A
and ApA(S
)A in HIV-1-infected Sup T1 Cells
To
determine whether HIV-1 infection affects uptake of
ApA(R
)A and ApA(S
)A, Sup T1
cells were incubated with 300 µM ApA(R
)A or ApA(S
)A.
Trichloroacetic acid:freon extracts were prepared and analyzed by HPLC.
ApA(R
)A and ApA(S
)A were
eluted at 31 and 33 min, respectively, identical to authentic
ApA(R
)A and ApA(S
)A (Fig. 9). The identity of ApA(R
)A and
ApA(S
)A in the trichloroacetic acid:freon extracts
was further confirmed by HPLC analyses in which authentic
ApA(R
)A or ApA(S
)A was
co-injected with the trichloroacetic acid:freon extracts; only one peak
was observed in each case. Quantitation revealed a 7- and 10-fold
enhancement of uptake of ApA(R
)A and
ApA(S
)A by HIV-1-infected cells compared to
uninfected cells (Fig. 10). At 4 h post-treatment, the
accumulation of intracellular ApA(R
)A and
ApA(S
)A was 126 and 255 pmol/10
cells,
respectively, in HIV-1-infected cells. In uninfected cells, the uptake
was 20 and 28 pmol/10
cells for
ApA(R
)A and ApA(S
)A,
respectively. No metabolic degradation products were detected by HPLC.
Figure 9:
HPLC analyses of
ApA(R
)A (A) and ApA(S
)A (B) treated Sup T1 cells (uninfected or HIV-1-infected). Sup
T1 cells were infected with HIV-1 at a m.o.i. of 0.1. After 48 h,
ApA(R
)A or ApA(S
)A (300
µM) was added and incubated for 4 h at 37 °C. Cell
extract preparation, 2-5A derivative extraction, and quantitation
by HPLC were as described under ``Materials and
Methods.''
Figure 10:
Enhancement of
ApA(R
)A (A) and
ApA(S
)A (B) uptake and accumulation in
HIV-1-infected Sup T1 cells. Experimental conditions were as described
for Fig. 9.
DISCUSSION
It has been established that the growth cycle of HIV-1 and
2-5A metabolism, i.e. high levels of 2-5A and
activated RNase L, correlate with failure of infected cells to release
HIV-1 (Schröder et al., 1989, 1994).
Stimulation of 2-5A synthetase by HIV-1 trans-activation
response element (TAR) has been reported (Schröder et al., 1989, 1990; SenGupta et al., 1989; Maitra et al., 1994). Conversely, with depletion of the intracellular
2-5A pool, RNase L cannot be activated and HIV-1 production
increases. Our approach to circumvent the HIV-1 Tat-induced blockade of
the 2-5A synthetase/RNase L antiviral pathway has been to augment
the depleted intracellular 2-5A pool with 2-5A derivatives
that are nuclease-resistant, are non-toxic, activate RNase L, and
inhibit HIV-1 replication (Montefiori et al., 1989;
Müller et al., 1991; Sobol et
al., 1993; Schröder et al., 1994). In
this study, we have employed chemically synthesized
2`,5`-phosphorothioate/phosphodiester derivatives as biological probes
to examine (i) the stereochemical requirements for binding to and
activation of RNase L (recombinant, human GST-RNase L and partially
purified murine RNase L) and (ii) the inhibition of HIV-1 induced
syncytia formation. Enhanced uptake of ApA(R
)A and
ApA(S
)A in HIV-1-infected Sup T1 cells has been
demonstrated.
Marked differences were observed in the stereochemical
requirements for activation of partially purified murine RNase L and
recombinant, human GST-RNase L. The recombinant, human GST-RNase L
revealed a stereochemical preference for the R
configuration at the internucleotide linkage closest to the
5`-terminus. A(R
)ApA activates human RNase L 10
times more effectively than does A(S
)ApA.
Stereochemical discrimination was even more evident at the
2`,3`-terminal linkage. ApA(R
)A activates the
recombinant, human GST-RNase L, whereas ApA(S
)A is
an antagonist of RNase L activation. With respect to the dimer cores,
A(R
)A, but not A
or
A(S
)A, activates the human GST-RNase L. Murine
RNase L, unlike human RNase L, was not activated by trimer core
2-5A derivatives with phosphorothioate substitution at the
5`-terminal linkage (A(R
)ApA and
A(S
)ApA). Murine and recombinant, human RNase L
show the same activation pattern with trimer core 2-5A
derivatives modified at the 2`,3`-terminal linkage, i.e. ApA(R
)A, but not
ApA(S
)A, activates murine RNase L. Furthermore,
dimer cores A(R
)A, A(S
)A, and
A
do not activate murine RNase L. With the tetramer core
derivatives, only those with the R
configuration
at the second or third internucleotide linkage from the 5`-terminus
activated murine RNase L (ApA(R
)ApA and
ApApA(R
)A). Neither the corresponding S
tetramer cores (ApA(S
)ApA
and ApApA(S
)A) nor the 5`-terminal substituted
tetramer core derivatives (A(R
)ApApA and
A(S
)ApApA) could activate murine RNase L.
With
the enantiomeric pairs,
pApA(R
)A/pApA(S
)A and
pApA(R
)ApA/pApA(S
)ApA, only
pApA(R
)A and pApA(R
)ApA
positively contribute to the allosteric activation of murine RNase L.
Five of the six tetramer 5`-monophosphate 2-5A derivatives
activate murine RNase L, whereas pApA(S
)ApA was an
antagonist of RNase L activation. Therefore, the R
configuration in the second internucleotide linkage of the
phosphorothioate/phosphodiester tetramer 5`-monophosphate derivatives (i.e. pApA(R
)ApA) is crucial for
formation of a productive ribonucleoprotein complex between RNase L,
its allosteric activator, and substrate RNA. The
2`,5`-phosphorothioate/phosphodiester cores with S
chirality bind to but do not form a productive ribonucleoprotein
complex, indicating that the R
configuration at
the 2`,3`-terminus of the trimer cores is important for allosteric
interaction with RNase L. With the tetramer cores, the R
configuration at the second and third internucleotide linkage
from the 5`-terminus is important for allosteric activation of RNase L.
In support of this hypothesis, the 2-5A binding domain of human
RNase L (which contains the basic amino acid residues, histidine 223,
lysine 286, and arginine 295) has been shown to be critical for binding
to and interaction with the second internucleotide linkage from the
5`-terminus of 2-5A (Zhou et al., 1993).
The
phosphorothioate/phosphodiester trimer derivatives have also revealed
new information with respect to the stereodynamics of inhibition of
HIV-1-induced syncytia formation. The most effective inhibitor of
HIV-1-induced syncytia formation was ApA(R
)A
(80-fold decrease in syncytia formation compared to vehicle alone) (Fig. 8A). ApA(S
)A inhibited
syncytia formation 10-fold. Derivatives with phosphorothioate
substitution at the 5`-terminus did not effectively inhibit
HIV-1-induced syncytia formation. With the
phosphorothioate/phosphodiester tetramer cores, the most potent
inhibitors of syncytia formation were derivatives with stereochemical
substitution at the 2`,3`-terminus (ApApA(R
)A and
ApApA(S
)A). Phosphorothioate substitution at the
5`-terminus or second internucleotide linkage offered little or no
protection against HIV-1-induced syncytia formation.
The enhanced
inhibition of HIV-1 replication by ApA(R
)A and
ApA(S
)A trimer cores may be explained in part by
their resistance to hydrolysis by serum phosphodiesterases (see Table 1). In contrast, authentic A
and A
(which do not significantly inhibit HIV-1-induced syncytia
formation) are hydrolyzed in serum-containing medium within 20 min
(Kariko et al., 1987). Earlier reports from this laboratory
demonstrated that the 2`,5`-phosphorothioates inhibit HIV-1 replication
via the inhibition of HIV-1 RT/primer complex formation (Sobol et
al., 1993). Therefore, the nearly total inhibition of HIV-1
replication observed with ApA(R
)A may be explained
by several activities, including nuclease resistance, activation of
RNase L, inhibition of HIV-1 RT and inhibition of DNA topoisomerase I.
The decreased inhibition of HIV-1 replication by
ApA(S
)A relative to ApA(R
)A
may be due to the inability of ApA(S
)A to activate
RNase L.
HIV-1 infection was found to enhance uptake of
ApA(R
)A and ApA(S
)A in
infected Sup T1 cells (7- and 10-fold increases, respectively).
Enhanced uptake of ApA(R
)A and
ApA(S
)A in HIV-1-infected cells was also observed
using the acid wash method of Pastan and co-workers (Haigler et
al., 1980), indicating that the uptake observed was due to
internalized, and not membrane-associated, oligonucleotide. Similarly,
uptake of S-(dC)
was enhanced in HSV-2-infected cells
compared to uninfected cells (Gao et al., 1990). Silverman and
co-workers (Maran et al., 1994) have reported on the unaided
uptake of 2-5A linked to antisense oligonucleotides into HeLa
cells. One explanation for the enhanced uptake of
ApA(R
)A and ApA(S
)A in
HIV-1-infected cells may be binding of ApA(S
)A and
ApA(R
)A to highly phosphorylated and glycosylated
HIV-1 surface proteins and/or HIV-1 RT and subsequent uptake into the
cell. Support for this suggestion is our finding that there is no
difference in the uptake of ApA(S
)A by PBL 4 h
after HIV-1 infection (about 9 pmol/10
cells). This
observation is in marked contrast to the enhanced uptake observed 48 h
after infection ( Fig. 9and Fig. 10). These observations
suggest that the enhanced uptake observed in HIV-1-infected cells is
related to expression of viral function late in infection. The
molecular basis underlying the differential uptake of
ApA(R
)A and ApA(S
)A in
HIV-1-infected cells compared to uninfected cells is under study. The
findings described contribute to our understanding of the
stereochemical requirements for allosteric activation of RNase L and
inhibition of HIV-1 replication. 2-5A derivatives, which act
distal to the HIV-1 Tat-induced blockade in the 2-5A
synthetase/RNase L antiviral pathway, may have potential as anti-HIV-1
agents.