©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of HIV-1 Replication and Activation of RNase L by Phosphorothioate/Phosphodiester 2`,5`-Oligoadenylate Derivatives (*)

(Received for publication, November 9, 1994; and in revised form, January 4, 1995)

Robert W. Sobol (1)(§) Earl E. Henderson (3) (2) Ning Kon (1) Jie Shao (1) Patricia Hitzges (1) Eli Mordechai (1) Nancy L. Reichenbach (1) Ramamurthy Charubala (4) Helga Schirmeister (4) Wolfgang Pfleiderer (4) Robert J. Suhadolnik (1) (3)(¶)

From the  (1)Departments of Biochemistry and (2)Microbiology and Immunology, and the (3)Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 and (4)Fakultät für Chemie, Universität Konstanz, Konstanz D-7750, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

2`,5`-Oligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/RNase L antiviral pathway. Stereochemical modification of individual internucleotide linkages of the 2-5A molecule was accomplished by phosphoramidite and phosphotriester chemical syntheses. Phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives revealed differences in the stereodynamics of activation of RNase L and inhibition of HIV-1 replication. The first and second internucleotide linkages are critical for activation of recombinant, human RNase L; A(R(p))ApA, A(S(p))ApA and ApA(R(p))A are agonists (IC = 2 times 10, 2 times 10, and 8 times 10M); ApA(S(p))A is an antagonist. The second and third internucleotide linkages are crucial for activation of murine RNase L; ApA(R(p))A, ApA(R(p))ApA, and ApApA(R(p))A are agonists (IC = 5 times 10M); ApA(S(p))A, ApA(S(p))ApA, and ApApA(S(p))A are antagonists. Inhibition of HIV-1-induced syncytia formation by the phosphorothioate/phosphodiester derivatives is specific for derivatives with substitution at the 2`,3`-terminus. ApA(R(p))A, ApA(S(p))A, ApApA(R(p))A, and ApApA(S(p))A are potent inhibitors of HIV-1-induced syncytia formation (80-, 10-, 40-, and 15-fold more inhibitory, respectively, than solvent control). HIV-1 infection results in enhanced uptake and accumulation of ApA(R(p))A and ApA(S(p))A (7- and 10-fold, respectively). These stereochemically modified 2-5A derivatives are taken up preferentially by HIV-1-infected cells and show promise in anti-HIV-1 chemotherapy.


INTRODUCTION

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) (^1)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(p) 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(p))A(S(p))A(S(p))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 times 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(2). CH(2)Cl(2) was distilled over CaCl(2) and acetonitrile purified by refluxing with CaH(2), followed by distillation. Tetrazole was sublimed in vacuum before use. Triethylammonium bicarbonate buffer (TEAB) (1 M) was prepared by passing CO(2) 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 times 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(2)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 times 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(3)PO(4) as the standard. ^1H NMR was performed using a Bruker WH-250, in ppm (s = singlet, d = doublet, m = multiplet), with tetramethylsilane as the internal standard. ^1H NMR spectra in D(2)O were determined with a D(2)O signal as the standard (4.80 ppm).

Bis(diisopropylamino)-(beta-cyanoethoxy)phosphane (3) (Kraszewski and Norris, 1987)

beta-Cyanoethanol (7 g; 0.1 mol) in absolute CH(3)CN (40 ml) was added dropwise within 30 min to a solution of freshly distilled PCl(3) (40 ml; 0.4 mol) at room temperature and under nitrogen atmosphere. After stirring for 3.5 h, the solvent and excess PCl(3) 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(2) to give 14.7 g (49%) of pure 3 of boiling point 114-118 °C. This reagent was stored at -20 °C under nitrogen. ^1H NMR (CDCl(3)), 3.75 (s, 2H, CH(2)), 3.52 (m, 4H, 4 N-CH), 2.60 (t, 2H, beta-CH(2)), and 1.17 + 1.14 (2 d, 24H, 4 N-C(CH(3))(2)); P NMR (CDCl(3)), 124.6 ppm.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenosine-2`-O-[(beta-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(2)Cl(2) (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(3)/NaCl solution (2 times 80 ml). The organic layer was dried over Na(2)SO(4), filtered, and evaporated to dryness. The residue was dissolved in CH(2)Cl(2) (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(2)Cl(2) (20 ml) and then bis(diisopropylamino)-(beta-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(3)/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), (max) = 279 nm (4.33) or 229 nm (4.43), R(F) on silica gel with toluol/EtOAc (1/1, v/v): 0.64 and 0.61 (diastereomers). P NMR (CDCl(3)), 150.98, 151.34.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[O^P-(2-cyanoethyl)-5`-]-N^6-benzoyl-2`,3`-di-O-[(tertbutyl)dimethylsilyl]adenosine (6)

The phosphoramidite 4 (2.88 g; 3 mmol) and N^6-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(2)Cl(2) (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(2) (0.5 g of H(2)O/pyridine/CH(2)Cl(2) (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(3) (300 ml). The organic phase was saturated with Na(2)S(2)O(3)/NaCl (3 times 80 ml), dried over Na(2)SO(4), and evaporated to dryness. Final coevaporation was done with toluene (3 times 20 ml). The crude product was purified by silica gel column chromatography (15 times 2.5 cm) using CHCl(3) (100 ml), CHCl(3)/MeOH (100/0.5, v/v; 1.5 liters), and CHCl(3)/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): (max) = 278 nm (4.62) and 230 nm (4.62). R(F) on silica gel with CHCl(3)/MeOH (95/5, v/v) = 0.56. P NMR (CDCl(3)), -0.74 and -1.07 ppm (diastereomers).

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[O^P-(2-cyanoethyl)-5`]-N^6-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(2)Cl(2)/MeOH (4/1, v/v; 30 ml) at room temperature for 30 min. The reaction mixture was diluted with CH(2)Cl(2) (300 ml), washed with phosphate buffer, pH 7.0 (2 times 100 ml), dried over Na(2)SO(4), and evaporated to dryness. The residue was applied to a silica gel column (9 times 4.5 cm), washed with CHCl(3) (0.7 liter) and CHCl(3)/MeOH (100/1, v/v; 300 ml). The product was eluted with CHCl(3)/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): (max) = 278 nm (4.60) and 232 nm (4.42). R(F) on silica gel with CHCl(3)/MeOH (95/5, v/v) = 0.36. P NMR (CDCl(3)), -0.77 and -1.30 ppm (diastereomers).

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-adenylyl-2`-[O^P-(2-cyanoethyl)-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenylyl-2`-[O^P-(2-cyanoethyl)-5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine: A(R(p))ApA (8) and A(S(p))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(3)CN (8.1 ml) and stirred at room temperature under nitrogen. After 3 h, S(8) (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(2)Cl(2) (300 ml), washed with saturated NaCl (2 times 200 ml), dried over Na(2)SO(4), and evaporated to dryness. Final coevaporation was with toluene (3 times 20 ml). The crude diastereomeric mixture A(R(p),S(p))ApA (8 and 9) was dissolved in CH(2)Cl(2) and applied to a silica gel column (21 times 3.5 cm). The column was washed with CH(2)Cl(2) (450 ml) and CH(2)Cl(2)/MeOH (99/1, v/v; 200 ml), and the product was eluted with CH(2)Cl(2)/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(p))ApA (8) and A(S(p))ApA (9). Separation into the pure diastereoisomers was achieved by medium pressure chromatography as described above by elution with CHCl(3)/MeOH (99/1, v/v; 800 ml; 20 ml/fraction; fractions 1-40), followed by elution with CHCl(3)/MeOH (95/5, v/v; 800 ml; 20 ml/fraction; fractions 41-80). Pure A(R(p))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(p))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(p))ApA 8 and 0.94 g (30%) of A(S(p))ApA 9.

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

UV (MeOH): (max) = 279 nm (4.77), 260 nm (4.57), and 236 nm (4.73). P NMR (CDCl(3)), 68.33 and -0.84 ppm.

P-Thioadenylyl-2`-5`-adenylyl-2`-5`-adenosine: A(R(p))ApA isomer (10) and A(S(p))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(2)Cl(2)/MeOH (4/1, v/v; 1.2 ml) for 1.5 h at room temperature. The reaction mixture was diluted with CHCl(3) (50 ml), washed with H(2)O (2 times 25 ml), dried, and evaporated to dryness. The crude product was purified on preparative silica gel plates (20 times 20 times 0.2 cm) in CHCl(3)/MeOH (8/2, v/v). The product bands were eluted with CHCl(3)/MeOH (4/1, v/v) and evaporated to a foam to give 0.04 g (84%) of A(R(p))ApA 10 and 0.034 g (73%) of A(S(p))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(2)O (10 ml) and applied to a DEAE Sephadex A-25 column (60 times 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 times 50 cm) and developed in i-PrOH/concentrated ammonia/H(2)O (6/1/3, v/v/v). The product band was cut out, eluted with H(2)O, evaporated, and lyophilized to give 500 OD units (79%) of A(R(p))ApA 10 and 410 OD units (65%) of A(S(p))ApA 11. UV (max) in both cases was 258 nm in H(2)O. A(R(p))ApA (10): R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.33. ^1H NMR (D(2)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(p))ApA (11): R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.33. ^1H NMR (D(2)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 times 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(18)) = 36-39 °C. ^1H NMR (CDCl(3)): 0.08 (s, 9H, SiCH(3)); 1.04-1.07 (d, 12H, N-C-CH(3)), 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(3) (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(3) 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. ^1H NMR (CDCl(3)): 8.1-8.2 (m, 2H, o to NO(2)); 7.39-7.43 (m, 2H, m to NO(2)); 4.04-4.18 (m, 2H, P-O-CH(2)); 3.63-3.79 (m, 2H, N-CH); 3.07-3.13 (t, 2H, P-O-C-CH(2)); 1.14-1.27 (2 d, 12H, N-C-CH(3)). P NMR (CDCl(3)): 181.60 ppm.

N^6-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(2)Cl(2) (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(3)/NaCl solution (3 times 80 ml), dried over Na(2)SO(4), and evaporated to dryness. The crude product was dissolved in toluene/EtOAc (7/3, v/v) and chromatographed on a silica gel column (12 times 2 cm) equilibrated with EtOAc/NEt(3) (95/5, v/v). The product fractions were eluted with EtOAc/NEt(3) (95/5, v/v), collected and evaporated to dryness, yielding 15 (5.28 g; 79%) as a colorless solid foam.

UV (MeOH): (max) = 277 nm (4.50), 229 nm (4.48). P NMR (CDCl(3)), 150.27, 150.01 ppm. R(F) on silica gel in toluene/EtOAc (1/1, v/v), 0.62 and 0.68 (diastereomers).

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine: ApA(R(p))A (21)

Triethylammonium N^6-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(p)) dimer 18 (0.066 g; 0.05 mmol) (Charubala and Pfleiderer, 1992) were coevaporated with dry pyridine (3 times 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(3) (100 ml), washed with H(2)O (2 times 50 ml), dried, and evaporated. Final evaporations were done with toluene (2 times 10 ml) to remove pyridine. The crude trimer 21 was purified by silica gel column chromatography (15 times 2 cm) using first CHCl(3) and then CHCl(3)/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): (max) = 276 nm (4.87) and 227 nm (4.83). R(F) on silica gel in CH(2)Cl(2)/EtOAc (1/1) = 0.63. P NMR (CDCl(3)), 69.88 and -1.0 ppm.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine: ApA(S(p))A (22)

Triethylammonium N^6-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(p)) dimer 19 (0.066 g; 0.05 mmol) (Charubala and Pfleiderer, 1992) were coevaporated with dry pyridine (3 times 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(3) (50 ml), washed with H(2)O (2 times 25 ml), dried, and evaporated. Final evaporations were done with toluene (2 times 10 ml) to remove pyridine. The crude trimer 22 was purified by silica gel column chromatography (15 times 2 cm), using first CHCl(3) and then CHCl(3)/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): (max) = 277 nm (4.88) and 227 nm (4.83). R(F) on silica gel in CH(2)Cl(2)/EtOAc (1/1, v/v) = 0.55. P NMR (CDCl(3)), 69.29 and -0.87 ppm.

Adenylyl-(2`-5`)-P-thioadenylyl-(2`-5`)-adenosine: ApA(R(p))A (23) and ApA(S(p))A (24)

The fully protected trimers, ApA(R(p))A (21) and ApA(S(p))A (22), were separately deblocked as follows. The corresponding trimer (0.088 g; 0.037 mmol) was stirred with 2% p-TsOH in CH(2)Cl(2)/MeOH (4/1, v/v; 0.8 ml). After 30 min of stirring at room temperature, the reaction mixture was diluted with CHCl(3) (50 ml) and washed with H(2)O (2 times 25 ml). The organic phase was dried over NaSO(4) and evaporated to dryness. The crude product was purified on a silica gel column (5 times 2 cm); the product was eluted with CHCl(3)/MeOH (100/1, v/v), evaporated, and dried under high vacuum to give 0.073 g (94%) of the 5`-hydroxy trimer ApA(R(p))A (21) and 0.061 g (84%) of the 5`-hydroxy trimer ApA(S(p))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(4)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 times 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 times 50 cm) and developed in i-PrOH/concentrated ammonia/H(2)O (6/1/3, v/v/v). The product band was cut out, eluted with H(2)O, evaporated, and lyophilized to give 354 OD units (79%) of ApA(R(p))A 23 and 410 OD units (58%) of ApA(S(p))A 24. UV (max) in both cases was 258 nm in H(2)O. ApA(R(p))A (23): R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.34. ^1H NMR (D(2)O): 8.17; 8.16; 8.09 (3 s, 3H, H-C(8)); 7.90, 7.78 (2 s, 3H, 3 times 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(p))A (24): R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.33. ^1H NMR (D(2)O): 8.17; 8.07; 8.04 (3 s, 3H, 3 times H-C(8)); 8.01; 7.92: 7.72 (3 s, 3H, 2 3 times H-C(2)); 6.04; 5.92; 5.82 (3 d, 3H, 3 times H-C(1`)). Retention time on reverse-phase HPLC was 7.23 min.

N^6-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(3)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(3)/NaCl solution (60 ml), dried over Na(2)SO(4), and evaporated to dryness. The crude solid foam was applied onto a flash silica gel column (20 times 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(3) (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(3) 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. ^1H NMR (CDCl(3)): 8.10-8.13 (d, 2H, o to NO(2)); 7.36-7.40 (d, 2H, m to NO(2)); 3.75-3.82 (q, 2H, P-O-CH(2)); 3.36-3.51 (m, 2H, N-CH); 2.95-3.00 (t, 2H, P-O-C-CH(2)); 1.05-1.12 (2d, 12H, N-C-CH(3)). P NMR (CDCl(3)): 123.53 ppm.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-(monomethoxytrityl)adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine, ApA (28and28a)

Phosphoramidite 15 (1.41 g; 1.34 mmol), N^6-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(3)CN (9 ml) under a nitrogen atmosphere. After 4 h, a solution of I(2) (0.5 g in CH(2)Cl(2)/H(2)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(2)Cl(2) (3 times 60 ml), and saturated Na(2)S(2)O(3)/NaCl solution (2 times 60 ml). The CH(2)Cl(2) phase was collected, dried over Na(2)SO(4), evaporated, and coevaporated with toluene (2 times 20 ml) to remove the pyridine. The crude dimer (1.85 g) was dissolved in CH(2)Cl(2), applied onto a flash silica gel column (12 times 2.5 cm), and chromatographed using CH(2)Cl(2)/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): (max) (log) = 277 (4.69) (260 (4.54); 231 (4.66)). R(F) on silica gel with CHCl(3)/MeOH (49/1, v/v) = 0.37.

N^6-Benzoyl-3`-O-(tert-butyl)dimethylsilyladenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-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(2)Cl(2)/MeOH (4/1, v/v, 20 ml) at room temperature for 30 min. The reaction mixture was diluted with CH(2)Cl(2) (200 ml), washed with H(2)O (2 times 80 ml), dried over Na(2)SO(4), and evaporated to dryness. The colorless amorphous residue (2.0 g) was applied onto a flash silica gel column (21 times 2.5 cm), chromatographed with CH(2)Cl(2) (200 ml) and CH(2)Cl(2)/2% MeOH (400 ml), and the product was eluted with CH(2)Cl(2)/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): (max) (log) = 278 (4.68), (259 (4.51), and 233 (4.46)). R(F) on silica gel with toluene/EtOAc/MeOH (5:4:1) = 0.53. P = NMR (CDCl(3): -0.36 and -0.73 ppm.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`-]N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine A(R(p))ApA (30) and A(S(p))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(3)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(8) (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(2)Cl(2) (200 ml), washed with a saturated NaCl solution (2 times 80 ml), dried over Na(2)SO(4), and evaporated to dryness. Final coevaporation was done with toluene (3 times 20 ml) to remove pyridine. The crude diastereoisomeric mixture A(R(p),S(p))ApA (30 and 31) was dissolved in CH(2)Cl(2) (20 ml), applied onto a flash silica gel column (11 times 2.5 cm), and chromatographed with CH(2)Cl(2) (400 ml), CH(2)Cl(2)/0.5% MeOH (200 ml), 1% MeOH (200 ml). The product was eluted with CH(2)Cl(2)/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 times 20 times 0.2 cm, eight plates) in toluene/EtOAc (1/1, v/v, four developments). The isomeric product bands were separately eluted with CH(2)Cl(2)/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(p))ApA 30 and 0.245 g (27%) of A(S(p))ApA 31.

UV (MeOH): (max) (log) = 278 (4.87) (260 (4.72); 231 (4.75)). R(F) 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): (max) (log) = 277 (4.86) (260 (4.72); 231 (4.75)). R(F) on silica gel with toluene/EtOAc (1:1, two developments) and toluene/EtOAc (1:2, one development) = 0.27.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl])-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine 5`-hydroxy A(R(p))ApA (32) and 5`-hydroxy A(S(p))ApA (33)

The fully protected trimers 30 and 31 were separately detritylated as follows: trimer (A(R(p))ApA 30: 0.263 g, 0.115 mmol; A(S(p))ApA 31: 0.21 g, 0.092 mmol) was stirred with 2% p-TsOH in CH(2)Cl(2)/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(2)Cl(2) (120 ml), washed with H(2)O (2 times 40 ml), dried over Na(2)SO(4), and evaporated to dryness. The crude product was purified on preparative silica gel plates (40 times 20 times 0.2 cm) in toluene/EtOAc (3/7, v/v), the product bands were eluted with CH(2)Cl(2)/MeOH (4/1, v/v) and evaporated to a solid foam to give 0.2 g (86%) of 5`-hydroxy A(R(p))ApA 32 and 0.121 g (66%) of 5`-hydroxy A(S(p))ApA 33, respectively.

UV (MeOH): (max) (log) = 278 (4.86) (260 (4.71); 233 (4.64)). R(F) 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): (max) (log) = 277 (4.85) (260 (4.71); 233 (4.63)). R(F) 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^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`-di-O-[(tertbutyl)dimethylsilyladenosine ApA(R(p))ApA (34) and ApA(S(p))ApA (35)

Condensation to the fully protected tetramers 34 and 35 was realized as follows. Triethylammonium N^6-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(p)) trimer 32 or (S(p)) trimer 33 (0.1 g, 0.05 mmol), respectively, were coevaporated with dry pyridine (3 times 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(2)Cl(2) (4 times 20 ml) and H(2)O (3 times 20 ml). The organic phase was collected, dried over Na(2)SO(4), evaporated, and coevaporated with toluene (3 times 20 ml) to remove pyridine. The crude tetramers 34 and 35 were separately purified on preparative silica gel plates (40 times 20 times 0.2 cm) with toluene/EtOAc/MeOH (5/4/0.5, v/v/v), and the product bands were eluted with CH(2)Cl(2)/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(p))ApA 34 and 0.12 g (81%) of ApA(S(p))ApA 35.

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

UV (MeOH), (max) (log) = 277 (5.01) (260 (4.88); 232 (4.89)). R(F) silica gel with toluene/EtOAc/MeOH (5/4/0.5, v/v/v) = 0.62 (diastereomers).

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine 5`-OH-ApA(R(p))ApA (36) and 5`-OH ApA(S(p))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(2)Cl(2)/MeOH (4/1, v/v, 1.2 ml) for 1 h at room temperature. The reaction mixture was extracted with CH(2)Cl(2) (3 times 40 ml) and washed with H(2)O (3 times 30 ml). The organic phase was collected, dried over Na(2)SO(4), and evaporated to dryness. The resulting residue was purified on preparative silica gel plates (20 times 20 times 0.2 cm) in toluene/EtOAc/MeOH (5/4/0.5, v/v/v). The product bands were eluted with CH(2)Cl(2)/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(p))ApA 36 and 0.057 g (60%) of the corresponding (S(p)) tetramer 37.

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

UV (MeOH), (max) (log) = 277 (4.96) (260 (4.82); 234 (4.74)). R(F) 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(p))ApA (38) and ApA(S(p))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(3)CN (2.5 ml) at room temperature; after 22 h, the solution was neutralized with 1 M AcOH in absolute CH(3)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(4)NF in THF (5 ml) for 3 days. The solvent was removed, and the residue was dissolved in H(2)O (10 ml), applied onto a DEAE Sephadex A-25 column (30 times 2 cm), and chromatographed first with H(2)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(p))ApA 38 was eluted with 0.23-0.28 M TEAB buffer and ApA(S(p))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(2)O (55/10/35, v/v/v). The product band was cut out, eluted with H(2)O, concentrated to a smaller volume, and finally lyo-philized to give 728 OD units (73%) of ApA(R(p))ApA 38 and 686 OD units (69%) of ApA(S(p))ApA 39, respectively. ApA(R(p))ApA (38): R(F) on cellulose in i-PrOH/ammonia/H(2)O (55:10:35) = 0.36. UV (H(2)O): (max) = 257 nm. ^1H NMR (D(2)O): 8.15, 8.07, 8.06, 7.93 (4 s, 4H, 4 times H-C(8)); 7.92 (s, 2H, 2 times H-C(2)); 7.82, 7.79 (2 s, 2H, 2 times H-C(2)); 6.03, 5.89, 5.86, 5.79 (4 d, 4 times H-C(1`)). HPLC: on RP-18, A: 50 mM NH(4)H(2)PO(4) (pH 7.24). B: MeOH/H(2)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(p))ApA (39): R(F) on cellulose in i-PrOH/ammonia/H(2)O (55/10/35, v/v/v) = 0.40. UV (H(2)O): (max) = 257 nm. HPLC: RP-18, A: 50 mM NH(4)H(2)PO(4) (pH 7.24). B: MeOH/H(2)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^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`)-(monomethoxytrityl)P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenosine-2`-[2,5-dichlorophenyl-2-(4-nitrophenylethyl)phosphate] A(R(p),S(p))Ap triester (43and44)

N^6-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(3)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(8) (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(2)Cl(2) (2 times 80 ml) and H(2)O (2 times 80 ml). The organic phase was collected, dried over Na(2)SO(4), and evaporated to dryness. Final coevaporation was done with toluene (4 times 20 ml) to remove pyridine. The crude diastereomeric mixture A(R(p),S(p))Ap triester (43 and 44) was purified by flash silica gel column chromatography (14 times 2.5 cm), using 200 ml of CH(2)Cl(2), CH(2)Cl(2)/1% MeOH, 2% MeOH, 200 ml CH(2)Cl(2)/1% MeOH, 2% MeOH and finally CH(2)Cl(2)/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): (max) (log) = 277 (4.75) (228 (4.72)). R(F) on silica gel with toluene/EtOAc/CHCl(3) (1/1/1, v/v/v) = 0.21. P NMR (CDCl(3)) 69.87, 69.25, -6.89, -7.22, and -7.31 ppm.

Triethylammonium N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`)(monomethoxytrityl)-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenosine-2`-[2-(4-nitrophenylethyl)phosphate] A(R(p))Ap diester (45) and A(S(p))Ap diester (46)

The solution of 0.558 g (3.36 mmol) of 4-nitrobenzaldehyde oxime in 15 ml of H(2)O/dioxane/Et(3)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(p),S(p))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 times 15 ml), toluene (3 times 15 ml), and finally with CH(2)Cl(2) (3 times 15 ml). The residue was dissolved in a small amount of CHCl(3) and chromatographed on a flash silica gel column (15 times 2.5 cm) with CHCl(3) (150 ml), CHCl(3)/2% MeOH (200 ml), 4% MeOH (100 ml), 6% MeOH (200 ml), CHCl(3)/6% MeOH/0.5% Et(3)N (300 ml), and CHCl(3)/6% MeOH/2% Et(3)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 times 20 times 0.2 cm) and three developments in CHCl(3)/MeOH (9/1, v/v). The product bands were eluted with CHCl(3)/MeOH (4/1, v/v) containing 1% Et(3)N and evaporated to a solid foam to give 0.262 g (43%) of A(R(p))Ap diester 45, 0.144 (24%) of A(S(p))Ap diester 46, and 0.045 g (7%) of A(R(p),S(p))Ap diester (45 and 46).

UV (MeOH): (max) (log) = 277 (4.69) (260 (4.56); 231 (4.61)). R(F) on silica gel with CHCl(3)/MeOH (9/1, v/v) = 0.37. P NMR (CDCl(3)), 69.66 and -0.09 ppm.

UV (MeOH): (max) (log) = 276 (4.60) (260 (4.49); 232 (4.52)). R(F) on silica gel with CHCl(3)/MeOH (9/1, v/v) = 0.28. P NMR (CDCl(3)), 68.96 and -0.06 ppm.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`)-(monomethoxytrityl)adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenosine-2`-[2,5-dichlorophenyl,2-(4-nitrophenyl) ethylphosphate] ApAp triester (41and41a)

N^6-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(3)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(2) (0.5 g in CH(2)Cl(2)/H(2)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(2)Cl(2) (20 ml), and washed with saturated Na(2)S(2)O(3)/NaCl solution (2 times 80 ml). The organic phase was collected, dried over Na(2)SO(4), evaporated, and coevaporated with toluene (3 times 30 ml) to remove the pyridine. The crude product was purified by flash silica gel chromatography (15 times 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): (max) (log) = 277 (4.75) (260 (4.62); 228 (4.72)). R(F) on silica gel with toluene/EtOAc/MeOH (5/4/1, v/v/v) = 0.78.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O^P-2- (4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenylyl-2`-[(O^P-2- (4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P- thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6- benzoyl- 2`,3`-di-O-[(tertbutyl)dimethylsilyl]adenosine ApApA(R(p))A (47)

Triethylammonium N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`- O-(monomethoxytrityl)adenylyl2`-[(O^P-2- (4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O- [(tert-butyl)dimethylsilyl]adenosine-2`-[2-(4-nitrophenyl)ethylphosphate] 42 (0.14 g; 0.078 mmol) and the 5`-hydroxy (R(p)) dimer (18) (0.08 g, 0.06 mol) were coevaporated with dry pyridine (4 times 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(2)Cl(2) (2 times 30 ml), washed with H(2)O (2 times 20 ml), dried over Na(2)SO(4), and evaporated to dryness. Pyridine was removed by coevaporation with toluene (3 times 20 ml). The crude tetramer 47 was purified by flash silica gel column chromatography (15 times 1 cm) and eluted first with CH(2)Cl(2) (50 ml), then with CH(2)Cl(2)/1% MeOH (100 ml), 2% MeOH (50 ml), and finally with CH(2)Cl(2)/3% MeOH (100 ml). The product fraction (80 ml) was evaporated to dryness to give 0.11 g (62%) of ApApA(R(p))A 47 as a colorless foam after drying under high vacuum at 35 °C.

UV (MeOH): (max) (log) = 277 (4.99) (259 (4.84); 233 (4.85)). R(F) on silica gel with CHCl(3)/MeOH (19/1, v/v) = 0.46.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O^P-2-(4nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine ApApA(S(p))A (48)

ApAp diester 41 (0.14 g, 0.078 mmol) and the 5`-hydroxy (S(p)) dimer 19 (0.08 g, 0.06 mmol) were coevaporated with dry pyridine (4 times 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(2)Cl(2) (4 times 20 ml) and with H(2)O (3 times 20 ml), dried over Na(2)SO(4), and evaporated. Final coevaporations were done with toluene (4 times 15 ml) to remove pyridine. The crude tetramer 48 was purified by flash silica gel column chromatography (15 times 1 cm) and eluted analogous to tetramer 47 with CH(2)Cl(2) and CH(2)Cl(2)/1-3% MeOH to give 0.107 g (60%) of the tetramer ApApA(S(p))A 48 as a colorless foam after drying under high vacuum at 35 °C.

UV (MeOH): (max) (log) = 277 (4.99) (259 (4.85); 231 (4.86)). R(F) on silica gel with CHCl(3)/MeOH (19/1, v/v) = 0.46.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`di-O-[(tert-butyl)dimethylsilyl]adenosine 5`-OH-ApApA(R(p))A (49)

The fully protected tetramer ApApA(R(p))A 47 (0.104 g, 0.035 mmol) was stirred with 2% p-TsOH in CH(2)Cl(2)/MeOH (4/1, v/v, 1.4 ml) at room temperature. After 1 h, the reaction mixture was extracted with CH(2)Cl(2) (3 times 40 ml) and H(2)O (2 times 40 ml). The combined organic phase was dried over Na(2)SO(4) and evaporated to dryness. The crude product was purified on a flash silica gel column (11 times 1 cm); the product was eluted with 20 ml of CH(2)Cl(2) and 50 ml of CH(2)Cl(2)/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(p))A 49.

UV (MeOH): (max) (log) = 277 (5.00); 260 (4.85); 234 (4.77)). R(F) on silica gel with CHCl(3)/MeOH (19/1, v/v) = 0.43.

Adenylyl-(2`-5`)-adenylyl-(2`-5`)-P-thioadenylyl-(2`-5`)-adenosine ApApA(R(p))A (51) and ApApA(S(p))A (52)

The fully protected tetramer ApApA(S(p))A (48) (0.104 g 0.35 mmol) was stirred with 2% p-TsOH in CH(2)Cl(2)/MeOH (4/1, v/v, 1.4 ml) at room temperature. After 1.5 h, the reaction mixture was extracted with CH(2)Cl(2) (3 times 40 ml) and H(2)O (2 times 40 ml). The organic phase was collected, dried over Na(2)SO(4), and evaporated to dryness. The crude product was purified on two preparative silica gel plates (20 times 20 times 0.2 cm) in CHCl(3)/MeOH (19/1, v/v), the product band was eluted with CH(2)Cl(2)/MeOH (4/1, v/v) and evaporated to a solid foam to give 0.068 g (72%) of the 5`-hydroxy tetramer ApApA(S(p))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(3)CN (3 ml) at room temperature for 20 h, the solution was neutralized with 1 M AcOH in absolute CH(3)CN (1.5 ml), and evaporated to dryness. (R(F) on silica gel with EtOAc/i-PrOH/ammonia/H(2)O (7/1/2, v/v/v): ApApA(R(p))A = 0.58; ApApA(S(p))A = 0.66.) The residue was then treated with methanolic ammonia (10 ml), and after 3 days, the solvent was removed under vacuum. (R(F) on silica gel with EtOAc/i-PrOH/ammonia/H(2)O (7/1/2, v/v/v); ApApA(R(p))A = 0.38; ApApA(S(p))A = 0.36). Desilylation was done with 1 M Bu(4)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(2)O (10 ml) and applied to a DEAE Sephadex A-25 column (30 times 2 cm). With flow rates of 2 ml/min, the pure tetramer ApApA(R(p))A was eluted with 0.15-0.20 M TEAB buffer (pH 7.5) and in the case of the tetramer ApApA(S(p))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 times 50 cm) and developed in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v). The product band was cut out, eluted with H(2)O, concentrated to a smaller volume, and finally lyophilized to give 675 OD units (57%) of ApApA(R(p))A 51 and 753 OD (65%) of ApApA(S(p))A 52. ApApA(R(p))A (51): R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.33. UV (H(2)O): (max) = 258 nm. HPLC: RP-18, A: 50 mM NH(4)H(2)PO(4), pH 7.2. B: MeOH/H(2)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(p))A (52): R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.21. UV (H(2)O): (max) = 258 nm. HPLC: RP-18, A: 50 mM NH(4)H(2)PO(4), pH 7.2. B: MeOH/H(2)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^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-thioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6benzoyl-3`-O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4nitrophenyl)ethyl-5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl) dimethylsilyl]adenosine Ap(R(p))ApApA (53)

Triethylammonium N^6benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-Pthioadenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`-O-[(tertbutyl)dimethylsilyl]adenosine-2`-[(O^P-2-(4-nitrophenyl)ethylphosphate] A(R(p))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 times 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(2)Cl(2) (60 ml) and washed with H(2)O (2 times 30 ml), dried over Na(2)SO(4), and evaporated to dryness. Pyridine was removed by coevaporation with toluene (3 times 20 ml). The crude tetramer 53 was purified by flash silica gel column chromatography (11 times 1 cm) and eluted first with CH(2)Cl(2) (50 ml), then with CH(2)Cl(2)/1% MeOH (100 ml), 2% MeOH (200 ml), and 3% MeOH (50 ml), and finally with CH(2)Cl(2)/5% MeOH (50 ml). The product fraction (200 ml) was evaporated to dryness. The residue was chromatographed again on preparative silica gel plates (20 times 20 times 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(2)Cl(2)/MeOH (4/1, v/v) and evaporated to a solid foam to give 0.053 g (30%) of A(R(p))ApApA 53 after drying in high vacuum at 35 °C.

UV (MeOH): (max) (log) = 277 (4.96) (260 (4.82); 232 (4.82)). R(F) on silica gel with toluene/EtOAc/MeOH (5:4:1) = 0.78.

N^6-Benzoyl-3`-O-[(tert-butyl)dimethylsilyl]-5`-O-(monomethoxytrityl)-P-thioadenylyl-2`- [(O^P-2-(4-nitrophenyl)ethyl-5`]-N^6-benzoyl-3`O-[(tert-butyl)dimethylsilyl]adenylyl- 2`-[(O^P-2-(4-nitrophenyl)ethyl5`]N^6-benzoyl-3`- O-[(tert-butyl)dimethylsilyl]adenylyl-2`-[(O^P-2-(4-nitrophenyl)ethyl- 5`]-N^6-benzoyl-2`,3`-di-O-[(tert-butyl)dimethylsilyl]adenosine A(S(p))ApApA (54)

A(S(p))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 times 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(2)Cl(2) (60 ml), washed with H(2)O (2 times 30 ml), dried over Na(2)SO(4), and evaporated to dryness. Further work-up was performed analogous to that described for 53 to give 43 mg (24%) of A(S(p))ApApA 54 in the form of a solid foam.

UV (MeOH): (max) (log) = 277 (4.98) (260 (4.84); 232 (4.85)). R(F) 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(p))ApApA (57) and A(S(p))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(p)) tetramer 53 ((S(p)) tetramer 54: 0.032 g, 0.012 mmol) in 2% p-TsOH in CH(2)Cl(2)/MeOH (4/1, v/v; for (R(p)): 0.5 ml; for (S(p)): 0.38 ml) was stirred for 1 h at room temperature. The reaction mixture was diluted with CH(2)Cl(2) (60 ml), washed with H(2)O (2 times 30 ml), dried over Na(2)SO(4), and evaporated to dryness. The crude products were purified on preparative silica gel plates (20 times 20 times 0.2 cm) in CHCl(3)/MeOH (19/1, v/v), and the product bands were eluted with CH(2)Cl(2)/MeOH (4/1, v/v) and evaporated to solid foams to give 0.034 g (80%) of the 5`-hydroxy A(R(p))ApApA isomer 55 and 0.02 g (68%) of the 5`-hydroxy A(S(p))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(3)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(4)NF in THF (2.5 ml). After stirring at room temperature for 60 h, the solvent was removed under vacuum. Some H(2)O (10 ml) was added to the resulting residue and applied to a DEAE Sephadex A-25 column (32 times 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(2)O, 55/10/35, v/v/v) gave, after lyophilization, 347 OD units (58%) of A(R(p))ApApA (57) and 111 OD units (31%) of A(S(p))ApApA (58), respectively. A(R(p))ApApA (57): UV (H(2)O) = 257 nm. R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.21. HPLC: RP18, A: 50 mM NH(4)H(2)PO(4) (pH 7.2). B: MeOH/H(2)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(p))ApApA (58): UV (H(2)O) = 257 nm. R(F) on cellulose in i-PrOH/ammonia/H(2)O (6/1/3, v/v/v) = 0.32. HPLC: RP18, A: 50 mM NH(4)H(2)PO(4), pH 7.2. B: MeOH/H(2)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(2) in air at 37 °C. Aliquots (30 µl) were removed at t(0) 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 DH5alpha (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 times 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(2), 0.1 mM ATP, and 14 mM beta-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(3)A(4)[P]pCp was synthesized by ligation of [P]pCp (specific activity 3000 Ci/mmol) to p(3)A(4) 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(2) 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 times 10^2 to 2 times 10^6 cells/well) was seeded into multiple wells of a 96-well microtiter plate. Immediately, 2 times 10^5 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(p))A and ApA(S(p))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(p))A or ApA(S(p))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(p))A and ApA(S(p))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(18) 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(2)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(p) and S(p) 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 beta-elimination.


Figure 1: Synthesis of the phosphorothioate/phosphodiester 2-5A trimer cores: A(R(p))ApA (compound 10), A(S(p))ApA (compound 11), ApA(R(p))A (compound 23), and ApA(S(p))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(p))A and A(S(p))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(2) and A(3) 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. (^2)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(3), pA(3), and p(3)A(3) bound to RNase L with IC values of 1 times 10M, 1 times 10M, and 1 times 10M, 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 (ICs from 8 times 10M to 8 times 10M for the cores and from 1 times 10M to 1 times 10M 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(3)A(3), p(3)A(4), pA(3), and pA(4) had IC values of 5 times 10M, 5 times 10M, 2 times 10M, and 2 times 10M, respectively. ApA(R(p))A was the only phosphorothioate/phosphodiester trimer core able to activate partially purified murine RNase L (IC of 5 times 10M) (Fig. 3A, up triangle). Three of the phosphorothioate/phosphodiester trimer 5`-monophosphate derivatives activated the partially purified murine RNase L; pApA(R(p))A was the most potent activator (IC of 1 times 10M) (Fig. 3B, up triangle). pA(3) (circle), pA(R(p))ApA (box), and pA(S(p))ApA () were 100-fold less potent, and pApA(S(p))A () was completely inactive. ApA(R(p))ApA (box) and ApApA(R(p))A (up triangle) were the only tetramer core derivatives able to activate murine RNase L (ICs of 5 times 10M) (Fig. 3C). Five of the six phosphorothioate/phosphodiester tetramer 5`-monophosphate derivatives (pA(R(p))ApApA, pA(S(p))ApApA, pApA(R(p))ApA, pApApA(R(p))A, and pApApA(S(p))A) activated murine RNase L (ICs >6 times 10M to 8 times 10M), whereas the pApA(S(p))ApA enantiomer did not activate murine RNase L at concentrations as high as 1 times 10M (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(3)A(3) (bullet), pA(3) (circle), A(3) (), A(R(p))ApA (box), A(S(p))ApA (), ApA(R(p))A (up triangle), ApA(S(p))A (), pA(R(p))ApA (box), pA(S(p))ApA (), pApA(R(p))A (up triangle), and pApA(S(p))A (). PanelsC and D, p(3)A(4) (bullet), pA(4) (circle), A(R(p))ApApA (down triangle), A(S(p))ApApA (), ApA(R(p))ApA (box), ApA(S(p))ApA (), ApApA(R(p))A (up triangle), ApApA(S(p))A (), pA(R(p))ApApA (down triangle), pA(S(p))ApApA (), pApA(R(p))ApA (box), pApA(S(p))ApA (), pApApA(R(p))A (up triangle), pApApA(S(p))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(p))A (1 times 10M) 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(p))ApA, A(S(p)),ApA and ApA(S(p))A, as well as authentic A(3), were unable to activate the murine RNase L at concentrations as high as 1 times 10M (Fig. 4A, lanes3, 4, 6, and 7). Authentic p(3)A(3) activated the murine RNase L at 1 times 10M (Fig. 4A, lane2). pA(R(p))ApA, pA(S(p))ApA, and pApA(R(p))A activated murine RNase L at 1 times 10M, 1 times 10M, and 2 times 10M, respectively (Fig. 4B, lanes4-6), compared with pA(3), which activates RNase L at 1 times 10M (lane3). However, pApA(S(p))A did not activate the murine RNase L at 1 times 10M (lane7); no activity was observed at concentrations as high as 5 times 10M (data not shown). ApA(R(p))ApA and ApApA(R(p))A were the only tetramer core derivatives able to activate RNase L (1 times 10M) (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(p))ApApA, pA(S(p))ApApA, pApA(R(p))ApA, pApApA(R(p))A, and pApApA(S(p))A) (Fig. 5B, lanes 4-6, 8, and 9, respectively). The most potent activator was pApA(R(p))ApA (1 times 10M) (Fig. 5B, lane6). pApA(S(p))ApA was unable to activate RNase L at concentrations as high 1 times 10M (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(3)A(3) (10M) (lane 2), A(R(p))ApA (10M) (lane 3), A(S(p))ApA (10M) (lane 4), ApA(R(p))A (10M) (lane 5), ApA(S(p))A (10M) (lane 6), or A(3) (10M) (lane 7). Panel B, L929 cell extracts were incubated in the absence (lane 1) or presence of p(3)A(3) (2 times 10M) (lane 2), pA(3) (10M) (lane 3), pA(R(p))ApA (10M) (lane 4), pA(S(p))ApA (10M) (lane 5), pApA(R(p))A (2 times 10M) (lane 6) or pApA(S(p))A (10M) (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(3)A(4) (10M) (lane2), A(4) (10M) (lane3), A(R(p))ApApA (10M) (lane4), A(S(p))ApApA (10M) (lane5), ApA(R(p))ApA (10M) (lane6), ApA(S(p))ApA (10M) (lane7), ApApA(R(p))A (10M) (lane8), or ApApA(S(p))A (10M) (lane9). PanelB, L929 cell extracts were incubated in the absence (lane1) or presence of p(3)A(4) (10M) (lane2), pA(4) (10M) (lane3), pA(R(p))ApApA (10M) (lane4), pA(S(p))ApApA (10M) (lane5), pApA(R(p))ApA (10M) (lane6), pApA(S(p))ApA (10M) (lane7), pApApA(R(p))A (10M) (lane8), or pApApA(S(p))A (10M) (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(3)A(3), pA(3), and A(3) at IC values of 5 times 10M, 5 times 10M, and 5 times 10M, respectively (Fig. 6). The IC values for A(R(p))ApA and A(S(p))ApA were 2 times 10M and 2 times 10M, respectively, whereas the IC for ApA(R(p))A was 8 times 10M. ApA(S(p))A did not activate the recombinant, human GST-RNase L at concentrations as high as 1 times 10M. Whereas A(2) and A(S(p))A dimer cores did not activate recombinant, human GST-RNase L at 1 times 10M, A(R(p))A (1 times 10M) did activate the human recombinant GST-RNase L. Recombinant, human GST-RNase L was not activated by 3-5A(3) or 3-5A(R(p))A(R(p))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(3)A(3) (bullet), pA(3) (circle), A(3) (), A(R(p))ApA (box), A(S(p))ApA (), ApA(R(p))A (up triangle), ApA(S(p))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(p))A

The binding affinity of pApA(S(p))A (1 times 10M) for murine RNase L and the observation that pApA(S(p))A does not activate RNase L suggested that pApA(S(p))A might be a stereospecific inhibitor of RNase L. Indeed, pApA(S(p))A inhibited activation of RNase L by p(3)A(3) or pApA(R(p))A (Fig. 7A). Authentic p(3)A(3) activated RNase L at 10M or 10M (lanes1 and 3). However, addition of pApA(S(p))A (10M) resulted in the inhibition of RNase L-catalyzed hydrolysis of rRNA (lanes2 and 4). Similarly, whereas pApA(R(p))A activated murine RNase L at 10M or 10M (lanes5 and 7), addition of pApA(S(p))A (10M) inhibited activation (lanes6 and 8). The inhibitory activity of pApA(S(p))A was also observed with partially purified murine RNase L (Fig. 7B). p(3)A(3) activated RNase L (IC of 5 times 10M (bullet)); however, upon addition of pApA(S(p))A (1 times 10M), the observed IC value shifted to 1 times 10M (circle), demonstrating specific inhibition of p(3)A(3)-mediated activation of murine RNase L by pApA(S(p))A.


Figure 7: Inhibition of activation of partially purified murine RNase L by pApA(S(p))A in L929 cell extracts (A) and by partially-purified RNase L (B). A, incubations were in the presence of p(3)A(3) (10M) (lanes1 and 2), p(3)A(3) (10M) (lanes3 and 4), pApA(R(p))A (10M) (lanes5 and 6), pApA(R(p))A (10M) (lanes7 and 8), and pApA(S(p))A (10M) (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(3)A(3) (bullet), p(3)A(3), and pApA(S(p))A at 10M (circle). 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 [^3H]cordycepin trimer core by lymphocytes (Suhadolnik et al., 1983), 3 times 10M 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 times 10^5 cells/ml) were treated with A(R(p))ApA, A(S(p))ApA, ApA(R(p))A, ApA(S(p))A, A(R(p))ApApA, A(S(p))ApApA, ApA(R(p))ApA, ApA(S(p))ApA, ApApA(R(p))A, ApApA(S(p))A, A(3), or A(4) (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(p))A was a highly efficient inhibitor of syncytia formation, with 8 times 10^5 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(p))A enantiomer was also an effective inhibitor of syncytia formation, with 1 times 10^5 cells required for formation of infected centers in 50% of the wells, a 10-fold reduction in syncytia formation (p = 0.07). A(R(p))ApA and A(S(p))ApA had little or no effect on HIV-1-induced syncytia formation compared to untreated cells (p = 0.6 and 0.6). Authentic A(3) and A(4) (3 times 10M) inhibited syncytia formation slightly; adenosine (9 times 10M) was not inhibitory. The numbers of infected cells required for infected centers in 50% of the wells in the presence of ApApA(R(p))A and ApApA(S(p))A were 8 times 10^5 and 3 times 10^5 cells, respectively, which represent 40-and 15-fold reductions in syncytia formation (Fig. 8B). The other tetramer diastereomers (A(R(p))ApApA, A(S(p))ApApA, ApA(R(p))ApA, and ApA(S(p))ApA) and A(4) were not effective inhibitors. A(R(p))A, A(S(p))A, A(2) dimer cores (3 times 10M), adenine (9 times 10M), or 3`,5`-A(4) (3 times 10M) were not inhibitory (data not shown).

Enhanced Uptake and Accumulation of ApA(R(p))A and ApA(S(p))A in HIV-1-infected Sup T1 Cells

To determine whether HIV-1 infection affects uptake of ApA(R(p))A and ApA(S(p))A, Sup T1 cells were incubated with 300 µM ApA(R(p))A or ApA(S(p))A. Trichloroacetic acid:freon extracts were prepared and analyzed by HPLC. ApA(R(p))A and ApA(S(p))A were eluted at 31 and 33 min, respectively, identical to authentic ApA(R(p))A and ApA(S(p))A (Fig. 9). The identity of ApA(R(p))A and ApA(S(p))A in the trichloroacetic acid:freon extracts was further confirmed by HPLC analyses in which authentic ApA(R(p))A or ApA(S(p))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(p))A and ApA(S(p))A by HIV-1-infected cells compared to uninfected cells (Fig. 10). At 4 h post-treatment, the accumulation of intracellular ApA(R(p))A and ApA(S(p))A was 126 and 255 pmol/10^6 cells, respectively, in HIV-1-infected cells. In uninfected cells, the uptake was 20 and 28 pmol/10^6 cells for ApA(R(p))A and ApA(S(p))A, respectively. No metabolic degradation products were detected by HPLC.


Figure 9: HPLC analyses of ApA(R(p))A (A) and ApA(S(p))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(p))A or ApA(S(p))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(p))A (A) and ApA(S(p))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(p))A and ApA(S(p))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(p) configuration at the internucleotide linkage closest to the 5`-terminus. A(R(p))ApA activates human RNase L 10 times more effectively than does A(S(p))ApA. Stereochemical discrimination was even more evident at the 2`,3`-terminal linkage. ApA(R(p))A activates the recombinant, human GST-RNase L, whereas ApA(S(p))A is an antagonist of RNase L activation. With respect to the dimer cores, A(R(p))A, but not A(2) or A(S(p))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(p))ApA and A(S(p))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(p))A, but not ApA(S(p))A, activates murine RNase L. Furthermore, dimer cores A(R(p))A, A(S(p))A, and A(2) do not activate murine RNase L. With the tetramer core derivatives, only those with the R(p) configuration at the second or third internucleotide linkage from the 5`-terminus activated murine RNase L (ApA(R(p))ApA and ApApA(R(p))A). Neither the corresponding S(p) tetramer cores (ApA(S(p))ApA and ApApA(S(p))A) nor the 5`-terminal substituted tetramer core derivatives (A(R(p))ApApA and A(S(p))ApApA) could activate murine RNase L.

With the enantiomeric pairs, pApA(R(p))A/pApA(S(p))A and pApA(R(p))ApA/pApA(S(p))ApA, only pApA(R(p))A and pApA(R(p))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(p))ApA was an antagonist of RNase L activation. Therefore, the R(p) configuration in the second internucleotide linkage of the phosphorothioate/phosphodiester tetramer 5`-monophosphate derivatives (i.e. pApA(R(p))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(p) chirality bind to but do not form a productive ribonucleoprotein complex, indicating that the R(p) configuration at the 2`,3`-terminus of the trimer cores is important for allosteric interaction with RNase L. With the tetramer cores, the R(p) 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(p))A (80-fold decrease in syncytia formation compared to vehicle alone) (Fig. 8A). ApA(S(p))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(p))A and ApApA(S(p))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(p))A and ApA(S(p))A trimer cores may be explained in part by their resistance to hydrolysis by serum phosphodiesterases (see Table 1). In contrast, authentic A(3) and A(4) (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(p))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(p))A relative to ApA(R(p))A may be due to the inability of ApA(S(p))A to activate RNase L.

HIV-1 infection was found to enhance uptake of ApA(R(p))A and ApA(S(p))A in infected Sup T1 cells (7- and 10-fold increases, respectively). Enhanced uptake of ApA(R(p))A and ApA(S(p))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(p))A and ApA(S(p))A in HIV-1-infected cells may be binding of ApA(S(p))A and ApA(R(p))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(p))A by PBL 4 h after HIV-1 infection (about 9 pmol/10^6 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(p))A and ApA(S(p))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.


FOOTNOTES

*
This work was supported by U.S. Public Health Service Grant P30-CA12227, National Science Foundation Grant DMB-900139 (to R. J. S.), and by Federal Work Study funds (to R. W. S. and E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, TX 77555.

To whom correspondence should be addressed. Tel.: 215-707-4607; Fax: 215-707-3515.

(^1)
The abbreviations used are: 2-5A, 2`,5`-oligoadenylates (p(3)A); p(3)A(3) and p(3)A(4): trimer and tetramer of adenylic acid with 2`,5`-phosphodiester linkages and a 5`-triphosphate; pA(3) and pA(4): trimer and tetramer of adenylic acid with 2`,5`-phosphodiester linkages and a 5`-monophosphate; A(2), A(3), and A(4), 5`-dephosphorylated p(3)A(2), p(3)A(3) and p(3)A(4); A(R(p))A, A(S(p))A, A(R(p))ApA, A(S(p))ApA, ApA(R(p))A, ApA(S(p))A, A(R(p))ApApA, A(S(p))ApApA, ApA(R(p))ApA, ApA(S(p))ApA, ApApA(R(p))A, and ApApA(S(p)),: phosphorothioate/phosphodiester derivatives of A(2), A(3), and A(4) with R(p) or S(p) stereoconfiguration in one chiral center (assignment of configuration from the 5` to the 2` terminus); pA(R(p))ApA, pA(S(p))ApA, pApA(R(p))A, pApA(S(p))A, pA(R(p))ApApA, pA(S(p))ApApA, pApA(R(p))ApA, pApA(S(p))ApA, pApApA(R(p))A, and pApApA(S(p))A: phosphorothioate/phosphodiester derivatives of pA(3) and pA(4) with R(p) or S(p) stereoconfiguration in one chiral center; pApAp-8-azidoA, adenylyl(2`-5`)-adenylyl-(2`-5`)-8-azidoadenosine 5`-monophosphate; DBU, 1,8-diazabicyclo(5.4.0)undec-7-ene; dsRNA, double-stranded RNA; GST, glutathione S-transferase; HIV-1, human immunodeficiency virus type 1; HPLC, high performance liquid chromatography; IFN, interferon; IPTG, isopropyl-beta-thiogalactopyranoside; PBL, peripheral blood lymphocytes; pCp, cytidine 3`,5`-bisphosphate; PKR, dsRNA-dependent p68 kinase; RNase L, 2-5A-dependent endoribonuclease; RT, reverse transcriptase; TEAB, triethylammonium bicarbonate buffer; TLC, thin layer chromatography.

(^2)
N. Kon, Z. Yu, and R. J. Suhadolnik, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Charles Grubmeyer and Yiming Xu, Department of Biochemistry, Temple University School of Medicine, for expert technical assistance in the use of FPLC for the purification of human GST-RNase L, and Somchai Pornbanlualap and Frank Sullivan, Department of Biochemistry, Temple University School of Medicine, for expert technical assistance with HPLC analyses.


REFERENCES

  1. Battistini, C., Brasca, M. G. & Fustinoni, S. (1992) Tetrahedron 48, 3209-3226 [CrossRef]
  2. Charachon, G., Sobol, R. W., Bisbal, C., Salehzada, T., Silhol, M., Charubala, R., Pfleiderer, W., Lebleu, B. & Suhadolnik, R. J. (1990) Biochemistry 29, 2550-2556 [Medline] [Order article via Infotrieve]
  3. Charubala, R. & Pfleiderer, A. (1987) Nucleosides & Nucleotides 6, 2314-2317
  4. Charubala, R. & Pfleiderer, W. (1992) Helv. Chim. Acta 75, 471-479
  5. Charubala, R., Uhlmann, E. & Pfleiderer, W. (1981) Liebig's Ann. Chem. , 2392-2406
  6. Charubala, R., Pfleiderer W., Sobol, R. W., Li, S. W. & Suhadolnik, R. J. (1989) Helv. Chim. Acta 72, 1354-1361
  7. Charubala, R., Sobol, R. W., Suhadolnik, R. J. & Pfleiderer, W. (1991a) Nucleosides & Nucleotides 10, 383-388
  8. Charubala, R., Sobol, R. W., Kon, N., Suhadolnik, R. J. & Pfleiderer, W. (1991b) Helv. Chim. Acta 74, 892-898
  9. Dani, C., Mechti, N., Piechaczyk, M., Lebleu, B., Jeanteur, P. & Blanchard, J. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4895-4899
  10. de Maeyer, E. & de Maeyer-Guignard, J. (1991) in The Cytokine Handbook (Thomson, A. W., ed) pp. 215-239, Academic Press, New York
  11. Dong, B., Xu, L., Zhou, A., Hassel, B. A., Lee, X., Torrence, P. F. & Silverman, R. H. (1994) J. Biol. Chem. 269, 14153-14158 [Abstract/Free Full Text]
  12. Flockerzie, F., Silber, G., Charubala, R., Schlosser, W., Varma, R., Creegan, F. & Pfleiderer, W. (1981) Liebig's Ann. Chem. 1568-1585
  13. Gao, W. Y., Jaroszewski, J. Y., Cohen, J. S. & Cheng, Y. C. (1990) J. Biol. Chem. 265, 20172-20178 [Abstract/Free Full Text]
  14. Haigler, H. T., Maxfield, F. R., Willingham, M. C. & Pastan, I. (1980) J. Biol. Chem. 255, 1239-1241 [Abstract/Free Full Text]
  15. Hassel, B. A., Zhou, A., Sotomayor, C., Maran, A. & Silverman, R. H. (1993) EMBO J. 12, 3297-3304 [Abstract]
  16. Henderson, E. E., Yang, J.-Y., Zhang, R.-D. & Bealer, M. (1991) Virology 182, 186-198 [Medline] [Order article via Infotrieve]
  17. Hovanessian, A. G. (1991) J. Interferon Res. 11, 199-205 [Medline] [Order article via Infotrieve]
  18. Hughes, B. G., Srivastava, P. C., Muse, D. D. & Robins, R. K. (1983) Biochemistry 22, 2116-2126 [Medline] [Order article via Infotrieve]
  19. Hughes, B. G. & Robins, R. K. (1983) Biochemistry 22, 2127-2135 [Medline] [Order article via Infotrieve]
  20. Jones, S. S, Rayner, B., Reese, C. B., Ubasawa, A. & Ubasawa, M. (1980) Tetrahedron 36, 3075-3085 [CrossRef]
  21. Johnston, M. I. & Torrence, P. F. (1984) Interferon: Mechanisms of Production and Action (Friedman, R. M., ed) Vol. 3, pp. 189-298, Elsevier Science Publishers, New York
  22. Kanou, M., Ohomori, H., Takaku, H., Yokoyama, S., Kawai, G., Suhadolnik, R. J. & Sobol, R. (1990) Nucleic Acids Res. 18, 4439-4446 [Abstract]
  23. Kanou, M., Ohomori, H., Nagai, K., Yokoyama, S., Suhadolnik, R. J., Sobol, R. & Takaku, H. (1991) Biochem. Biophys. Res. Commun. 176, 769-774 [Medline] [Order article via Infotrieve]
  24. Kariko, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R. & Pfleiderer, W. (1987a) Biochemistry 26, 7127-7135 [Medline] [Order article via Infotrieve]
  25. Kariko, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R. & Pfleiderer, W. (1987b) Biochemistry 26, 7136-7142 [Medline] [Order article via Infotrieve]
  26. Koromilas, A. E., Roy, S., Barber, G. N., Katze, M. G. & Sonenberg, N. (1992) Science 257, 1685-1689 [Medline] [Order article via Infotrieve]
  27. Kraemer, K. H., Waters, H. L. & Buchanan, J. K. (1980) Mutat. Res. 72, 285-292 [Medline] [Order article via Infotrieve]
  28. Kraszewski, A. & Norris, K. E. (1987) Nucleic Acids Research Symp. Ser. 18, 177-180
  29. Kroger, C. F. & Mietchen, R. (1969) Z. Chem. 9, 378-379
  30. Lee, C. & Suhadolnik, R. J. (1985) Biochemistry 24, 551-555 [Medline] [Order article via Infotrieve]
  31. Lengyel, P. (1982) Annu. Rev. Biochem. 51, 251-282 [CrossRef][Medline] [Order article via Infotrieve]
  32. Maitra, R. K., McMillan, N. A., Desai, S., McSwiggen, J., Hovanessian, A. G., Sen, G., Williams, B. R. G. & Silverman, R. H. (1994) Virology 204, 823-827 [CrossRef][Medline] [Order article via Infotrieve]
  33. Maran, A., Maitra, R. K., Kumar, A., Dong, B., Xiao, W., Li, G., Williams, B. R. G., Torrence, P. F. & Silverman, R. H. (1994) Science 265, 789-792 [Medline] [Order article via Infotrieve]
  34. McNair, A. N. B. & Kerr, I. M. (1992) Pharmacol. Ther. 56, 79-95 [Medline] [Order article via Infotrieve]
  35. Montefiori, D. C., Sobol, R. W., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., Pfleiderer, W., Modliszewski, A., Robinson, W. E., Jr. & Mitchell, W. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7191-7194 [Abstract]
  36. Müller, W. E. G., Weiler, B. E., Charubala, R., Pfleiderer, W., Leserman, L., Sobol, R. W., Suhadolnik, R. J. & Schröder, H. C. (1991) Biochemistry 30, 2027-2033 [Medline] [Order article via Infotrieve]
  37. Nelson, P. S., Bach, C. T. & Verheyden, J. P. H. (1984) J. Org. Chem. 49, 2314-2317
  38. Noth, H. & Staudigl, R. (1982) Chem. Ber. 115, 3011-3024
  39. Pestka, S. (1981) Methods Enzymol. 78, 79Schröder, H. C., Wenger, R., Kuchino, Y. &Müller, W. E. G. (1989) J. Biol. Chem.264, 5669-5673
  40. Schröder, H. C., Ugarkovic, D., Wenger, R., Okamoto, T. & Müller, W. E. G. (1990) AIDS Res. Hum. Retrovir. 6, 659-672 [Medline] [Order article via Infotrieve]
  41. Schröder, H. C., Kelve, M., Schäcke, H, Pfleiderer, W., Charubala, R., Suhadolnik, R. J. & Müller, W. E. G. (1994) Chem.-Biol. Interact. 90, 169-183 [Medline] [Order article via Infotrieve]
  42. SenGupta, D. N. & Silverman, R. H. (1989) Nucleic Acids Res. 17, 969-978 [Abstract]
  43. Shimazu, M., Shinozuka, K. & Sawai, K. (1993) Angew. Chem. Int. Ed. Engl. 32, 870-872
  44. Silverman, R. H. (1985) Anal. Biochem. 144, 450-460 [Medline] [Order article via Infotrieve]
  45. Sinha, N. D., Biernat, J., McManus, J. & Köster, H. (1984) Nucleic Acids Res. 12, 4539-4557 [Abstract]
  46. Smith, D. B. & Johnson, K. S. (1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  47. Sobol, R. W., Fisher, W. L., Reichenbach, N. L., Kumar, A., Beard, W. A., Wilson, S. H., Charubala, R., Pfleiderer, W. & Suhadolnik, R. J. (1993) Biochemistry 32, 12112-12118 [Medline] [Order article via Infotrieve]
  48. Sperling, J., Chebath, J., Arad-Dann, H., Offen, D., Spann, P., Lehrer, R., Goldblatt, D., Jolles, B. & Sperling, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10377-10381 [Abstract]
  49. Suhadolnik, R. J., Doetsch, P. W., Devash, Y., Henderson, E. E., Charubala, R. & Pfleiderer, W. (1983) Nucleosides & Nucleotides 2, 351-366
  50. Suhadolnik, R. J., Lee, C., Kariko, K. & Li, S. W. (1987) Biochemistry 26, 7143-7149 [Medline] [Order article via Infotrieve]
  51. Suhadolnik, R. J., Kariko, K., Sobol, R. W., Jr., Li, S. W., Reichenbach, N. L. & Haley, B. E. (1988a) Biochemistry 27, 8840-8846 [Medline] [Order article via Infotrieve]
  52. Suhadolnik, R. J., Li, S. W., Sobol, R. W., Jr. & Haley, B. (1988b) Biochemistry 27, 8846-8851 [Medline] [Order article via Infotrieve]
  53. Wells, V. & Mallucci, L. (1985) Exp. Cell Res. 159, 27-36 [Medline] [Order article via Infotrieve]
  54. Wells, V. & Mallucci, L. (1988) J. Interferon Res. 8, 793-802 [Medline] [Order article via Infotrieve]
  55. Wreschner, D. H., McCauley, J. W., Skehel, J. J. & Kerr, I. M. (1981) Nature 289, 414-417 [Medline] [Order article via Infotrieve]
  56. Zhou, A., Hassel, B. A. & Silverman, R. H. (1993) Cell 72, 753-765 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.