Optically active antiviral compounds having the formula ##STR1## wherein m is 0, 1, 2, or 3; n and q are selected from the group of 0 and 1, provided that n and q may not both be zero; and R, R.sub.1, and R.sub.2 are independently of each other selected from the group consisting of oxygen and sulfur, provided that all R, R.sub.1 and R.sub.2, may not be oxygen, and further provided that all R, R.sub.1, and R.sub.2 may not be sulfur. The compounds possess increased antiviral activity and/or metabolic stability.

FIELD OF THE INVENTION 
This invention relates to synthetic analogues of naturally occurring 
antiviral 2',5'-oligoadenylates wherein at least one of the 
internucleotide phosphodiester linkages is replaced with optically active 
phosphorothioate groups. The compounds with selected internucleotide 
phosphodiester linkages have antiviral activity and increased metabolic 
stability. 
BACKGROUND OF THE INVENTION 
The full nomenclature of the subject matter of the present invention 
involves lengthy terms. It is customary for those skilled in the art to 
abbreviate oligoadenylate analogues and related terms in a manner 
well-known to the art. These general and customary abbreviations are set 
forth herein below and may be utilized in the text of this specification. 
Abbreviations 
2-5A, 2',5'-oligoadenylate or p.sub.3 A.sub.n : Oligomer of adenylic acid 
with 2',5'-phosphodiester linkages and a 5'-terminal triphosphate group. 
A.sub.2, A.sub.3 and A.sub.4 : Dimer, trimer and tetramer of adenylic acid 
with 2',5'-phosphodiester linkages. 
pA.sub.3, ppA.sub.3 (or p.sub.2 A.sub.3), pppA.sub.3 (or p.sub.3 A.sub.3): 
5'-terminal mono-, di- and triphosphates of A.sub.3. 
pA.sub.4, ppA.sub.4 (or p.sub.2 A.sub.4), pppA.sub.4 (or p.sub.3 A.sub.4): 
5'-terminal mono-, di- and triphosphates of A.sub.4. 
ApA: Dimer of adenylic acid with 2'-5'-phosphodiester linkage. 
Ap*A: Dimer of adenylic acid with 2'-5'-phosphorothioate linkage. 
PR: The R stereoconfiguration about a chiral phosphorous atom in a 
phosphorothioate internucleotide linkage. 
PS: The S stereoconfiguration about a chiral phosphorous atom in a 
phosphorothioate internucleotide linkage. 
A.sub.Rp *ApA: (PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine. 
A.sub.Sp *ApA: (PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine. 
ApA.sub.Rp *A: Adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenosine. 
ApA.sub.Sp *A: Adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenosine. 
pA.sub.Rp *ApA, ppA.sub.Rp *ApA, pppA.sub.Rp *ApA, pA.sub.Sp *ApA, 
ppA.sub.Sp *ApA, pppA.sub.Sp *ApA, pApA.sub.Rp *A, ppApA.sub.Rp *A, 
pppApA.sub.Rp *A, pApA.sub.Sp *A, ppApA.sub.Sp *A, pppApA.sub.Sp *A: 
5'-mono-, di- and triphosphates of A.sub.Rp *ApA, A.sub.Sp *ApA, 
ApA.sub.Rp *A, and ApA.sub.Sp *A. 
A.sub.Rp *ApApA: 
(PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenylyl-(2',5')-adenosine. 
A.sub.Sp *ApApA: 
(PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenylyl-(2',5')-adenosine. 
Id.1 ApA.sub.Rp *ApA: Adenylyl-(2',5')-(PR) 
-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine. 
ApA.sub.Sp *ApA: 
Adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine. 
ApApA.sub.Rp *A: 
Adenylyl-(2',5')-(PR)-P-thioadenylyl-(2',5')-adenylyl-(2',5')-adenosine. 
ApApA.sub.Sp *A: 
Adenylyl-(2',5')-adenylyl-(2',5')-(PS)-P-thioadenylyl-(2',5')-adenosine. 
pA.sub.Rp *ApApA, ppA.sub.Rp *ApApA, pppA.sub.Rp *ApApA, pA.sub.Sp *ApApA, 
ppA.sub.Sp *ApApA, pppA.sub.Sp *ApApA, pApA.sub.Rp *ApA, ppApA.sub.Rp 
*ApA, pppApA.sub.Rp *ApA, pApA.sub.Sp *ApA, ppApA.sub.Sp *ApA, 
pppApA.sub.Sp *ApA, pApApA.sub.Rp *A, ppApApA.sub.Rp *A, pppApApA.sub.Rp 
*A, pApApA.sub.Sp *A, ppApApA.sub.Sp *A, pppApApA.sub.Sp *A: 5'-mono-, di- 
and triphosphates of the above tetramers. 
bz: benzoyl 
ce: cyanoethyl 
CFS: Chronic Fatigue Syndrome 
DEAE: 2-(diethylamino)ethyl 
DBU: 1.8 diazabicyclo5.3.0!undec-7-enc 
HIV: Human Immunodeficiency Virus 
MeOTr: monomethoxytrityl 
M.O.I: multiplicity of infection 
mRNA: Messenger RNA 
npe: 2-(4-niytophenyl)ethyl 
PBL: Peripheral blood lymphocytes 
pCp: Cytidine 3'-5'-bisphosphate 
(PS)-ATP-alpha-S: Adenosine 5'O--(PS)-(1-thiotriphosphate). 
RNase L: 2-5A-dependent endoribonuclease. 
rRNA: Ribosomal RNA 
RT: Reverse transcriptase 
SCP: Specific cleavage products 
tbds: (tert-butyl)dimethylsilyl 
Tris: tris(hydroxymethyl)aminomethane 
tRNA: Transfer RNA 
It is generally regarded that activation of RNase L by 2-5A is key to the 
antiviral defense mechanisms. Interferon induces transcription of the 
enzyme 2-5A synthetase which produces 2',5' linked oligoadenylates upon 
activation of double-stranded RNA. Previously, the only known biochemical 
effect of 2-5A is activation of RNase L. This enzyme hydrolyses mRNA and 
rRNA, thereby resulting in inhibition of protein synthesis. The activation 
of RNase L is transient unless 2-5A is continuously synthesized, since 
2-5A is rapidly degraded. RNase L activation thus plays a critical role in 
inhibiting replication, and therefore in defending against infection by 
viruses. 
A correlation has also been established between 2-5A metabolism and the 
growth cycle of HIV-1, i.e., high levels of 2-5A and activated RNase L 
correlate with failure of infected cells to release HIV-1, Schroder et 
al., J. Biol. Chem. 264: 5669-5673 (1989). Conversely, when the 
intracellular 2-5A pool decreases, RNase L can not be activated and HIV-1 
production increases. A role for 2-5A cores as inhibitors of HIV-1 
replication has been established with reports that 2-5A trimer and 
tetramer cores, 5'-monophosphates and 5'-triphosphates inhibit HIV-1 
reverse transcriptase/primer complex formation, Montefiori et al., Proc. 
Natl. Acad. Sci. USA 86: 7191-7194 (1989); Muller et al., Biochemistry 30: 
2027-2033 (1991); Sobol et al., Biochemistry 32: 1211-12118 (1993). 
The introduction of the phosphorothioate group in the 2',5'-internucleotide 
linkages of 2-5A, induces metabolic stability greater than authentic 2-5A 
and resulted in the first 2-5A cores (i.e. 2-5A lacking 5'-phosphate 
moieties) able to activate RNase L (Kariko et al. Biochemistry 26: 
7136-7142 (1987); Charachon et al. Biochemistry 29: 2550-2556 (1990)). 
Further, RNase L is a functionally stereoselective enzyme and 2-5A trimers 
and tetramers having at least one of the internucleotide phosphorothioate 
2',5'-linkages of the PS configuration have greatly enhanced metabolic 
stability. The chemical synthesis of the fully resolved 
2',5'-phosphorothioate adenylate trimer and tetramer cores has been 
reported, Suhadolnik et al., U.S. Pat. No. 4,924,624. Preparation of the 
stereoisomers via enzymatic synthesis is not possible due to the 
sterospecificity of 2-5A synthetase for the substrate (PS)-ATP-alpha-S, 
which yields trimer and tetramer products of the PR configuration 
exclusively. Further, while Lebleu et al., U.S. Pat. No. 4,981,957, 
discloses the enzymatic synthesis of a phosphorothioate-substituted 
derivative of 2',5' oligoadenylate, the compounds disclosed are not 
stereospecific. 
SUMMARY OF THE INVENTION 
Compounds of the present invention useful in inhibiting viral infections in 
plants and mammals have increased metabolic stability and/or antiviral 
activity. 
The compounds and the water-soluble salts thereof are of the formula 
##STR2## 
wherein m is zero, 1, 2 or 3; n and q are selected from the group of zero 
and 1, provided that n and q may not both be zero; and R, R.sub.1 and 
R.sub.2 are independently selected from the group of oxygen and sulfur, 
provided that all R, R.sub.1 and R.sub.2, may not be oxygen, and further 
provided that all R, R.sub.1 and R.sub.2 may not be sulfur. 
The invention also comprises a method of inhibiting viral infection in 
mammals or plants by administering an antivirally effective amount of a 
compound according to the above formula, or a water-soluble salt thereof, 
and antiviral compositions containing such compounds with a carrier. 
Compounds according to the formula wherein n is 1 and q is 1 may be 
utilized to form oligoadenylate conjugates with the macromolecular carrier 
poly(L-lysine) for intracellular transport. Such 
poly(L-lysine)/2',5'-phosphorothioate/phosphodiester oligoadenylate 
conjugates have the formula 
##STR3## 
wherein q is an integer from about 60 to about 70, and R is randomly R' or 
##STR4## 
From about five to about ten of the R groups comprise R'. R' has the 
following formula wherein m is 0, 1, 2 or 3; and where each R.sub.3, 
R.sub.4, or R.sub.5 are independently selected from the group of oxygen 
and sulfur; provided that all R.sub.3, R.sub.4 or R.sub.5 may not be 
oxygen; and further provided that all R.sub.3, R.sub.4 or R.sup.5, may not 
be sulfur. 
##STR5## 
Preferably, at least one of the internucleotide phosphorothioate groups 
##STR6## 
of the poly(L-lysine)/2',5'-phosphorothioate/phosphodiester oligoadenylate 
conjugates is of the PR configuration.

DETAILED DESCRIPTION OF THE INVENTION 
The individual nucleotide linkages of the trimer and tetramer derivatives 
of 2',5'-oligoadenylate (2-5A) were stereochemically modified via 
phosphorothiate substitution by phosphotriester and phosphoramidite 
chemical synthesis. The approach described herein utilizes fully protected 
monomeric building blocks (Schemes 1 and 2) below, which can be 
individually manipulated. The protecting groups remain in place during the 
chemical synthesis of the oligonucleotide chain and are removed at the end 
of the sequence by .beta.-elimination. 
The phosphorothioate/phosphodiester trimer cores, A.sub.Rp *ApA 10, 
A.sub.Sp *ApA 11, ApA.sub.Rp *A 23, and ApA.sub.Sp *A 24, were chemically 
synthesized and separated by preparative thin layer chromatography on 
silica gel, deblocked and purified by applying the residue on a DEAE 
Sephadex column. The four trimer cores are prepared from phosphoramidite 
intermediates 4 and 15. The synthesis relies on separation of fully 
resolved protected intermediates, 8, 9, 21 and 22 followed by removal of 
all blocking groups to yield the individually substituted 
2',5'-phosphorothioate/phosphodiester trimer adenylate cores. While not 
part of the invention, the preparation of the dimer core 6 is included for 
completeness. 
The selectively substituted tetramer cores, 38 and 39, 51 and 52 and 57 and 
58, were derived from the fully protected trimer cores, 30 and 31, and 
subsequently subjected to detrilylation and condensation to add the 
tetramer moiety. 
The compounds of the present invention comprise 2-5A derivatives that are 
(i) nuclease-resistant, (ii) non-toxic, (iii) able to activate or 
inactivate RNase L and (iv) able to inhibit HIV-1 replication. The 
inventive compounds are chemically synthesized 
phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives in 
which at least one 2',5'-phosphodiester bond has been selectively replaced 
with a 2',5'-phosphorothioate bond. The chemical synthesis of these 
phosphorothioate/phosphodiester derivatives utilizes the phosphotriester 
and phosphoramidite approach in which reactive functional groups are 
protected by blocking groups which can be individually manipulated. The 
phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives 
reveal heretofore unknown aspects of the stereochemical requirements for 
activation of RNase L, namely, that activation of RNase L requires PR 
chirality in the second internucleotide linkage from the 5'-terminus of 
the 2-5A molecule and that PS chirality in the second internucleotide 
linkage results in 2-5A derivatives that are antagonists of RNase L 
activation. Without wishing to be bound by any theory, it appears that PR 
chirality in the second internucleotide linkage from the 5'-terminus may 
serve to facilitate formation of a productive complex between RNase L, the 
allosteric activator (ApA.sub.Rp *A or ApA.sub.Rp *ApA) and the RNA 
substrate such that hydrolysis of HIV-1 RNA can occur. 
Phosphorothioate substitution of individual internucleotide linkages in the 
2-5A molecule has revealed that inhibition of HIV-1 replication is 
influenced by the location and stereoconfiguration of the chiral 
phosphorothioate group in the phosophorothioate/phosphodiester 
derivatives. Of the four phosphorothioate/phosphodiester trimer core 
derivatives, ApA.sub.Rp *A and ApA.sub.Sp *A were the most efficient 
inhibitors of HIV-1 induced syncytia formation (FIG. 4A). Of the six 
phosphorothioate/phosphodiester tetramer core derivatives, ApApA.sub.Rp *A 
and ApApA.sub.Sp *A were the most efficient inhibitors (FIG. 4B). A.sub.Rp 
*A, A.sub.Sp *A, 3',5'-A.sub.4, adenosine and adenine did not inhibit 
HIV-1 RT activity. Whereas ApA.sub.Rp *A and ApA.sub.Sp *A are both 
phosphodiesterase-resistant and inhibit HIV-1 RT, the ApA.sub.Rp *A 
enantiomer (but not the ApA.sub.Sp *A enantiomer) can also activate RNase 
L. 
In this regard it appears, again, without wishing to be bound by any 
theory, that the relative differences in the inhibition of HIV-1 
replication by the phosphorothioate/phospohodiester trimer and tetramer 
core derivatives may be explained by their resistance to hydrolysis by 
serum phosphodiesterases (see Table 1, infra.). In contrast to 
2',5'-phosphodiester bonds in authentic A.sub.2 and A.sub.3 which are 
totally hydrolyzed in serum-containing medium in 20 minutes, both PR and 
PS 2',5'-phosphorothioate bonds are more stable to hydrolysis by 
phosphodiesterases. The phosphorothioate/phosphodiester tetramer core 
derivatives which are stereochemically modified at the 5'-terminus 
(A.sub.Rp *ApApA and A.sub.Sp *ApApA) are rapidly hydrolyzed from the 
2',3'-terminus to their respective dimers, A.sub.Rp *A and A.sub.Sp *A. 
These dimers, although resistant to further hydrolysis, can neither 
activate RNase L nor inhibit HIV-1 replication. The remaining four 
phosphorothioate/phosphodiester tetramer core derivatives (ApA.sub.Rp 
*ApA, ApA.sub.Sp *ApA ApApA.sub.Rp *A and ApApA.sub.Sp *A) are hydrolyzed 
from the 5'-terminus to form their respective trimer cores, A.sub.Rp *ApA, 
A.sub.Sp *ApA, ApA.sub.Rp *A and ApA.sub.Sp *A, respectively. Because 
ApA.sub.Rp *A and ApA.sub.Sp *A are efficient inhibitors of HIV-1 
replication, this hydrolysis most likely accounts for the antiviral action 
of the tetramer derivatives, ApApA.sub.Rp *A and ApApA.sub.Sp *A. 
Therefore, the decreased anti-HIV-1 activity observed with ApA.sub.Rp *ApA 
and ApA.sub.Sp *ApA (relative to ApApA.sub.Rp *A and ApApAp.sub.Sp *A) is 
likely due to hydrolysis from the 5'-terminus to form ApA.sub.Rp *A and 
ApA.sub.Sp *A, which are very efficient inhibitors of HIV-1 induced 
syncytia formation (compare FIGS. 4A and 4B). 
In preliminary experiments, all phosphorothioate/phosphodiester trimer and 
tetramer 2-5A core derivatives of the present invention have been shown to 
inhibit HIV-1 RT. Inhibition ranges from 22% to 70%. A.sub.Rp *A, A.sub.Sp 
*A, 3',5'-A.sub.4, adenosine and adenine did not inhibit HIV-1 RT 
activity. Whereas ApA.sub.Rp *A and ApA.sub.Sp *A are both 
phosphodiesterase-resistant and inhibit HIV-1 RT, the ApA.sub.Rp *A 
enantiomer (but not the ApA.sub.Sp *A enantiomer) can also activate RNase 
L. 
These three biological properties (i.e., resistance to hydrolysis by 
phospohodiesterases, inhibition of reverse transcriptase and activation of 
RNase L) may account for the 100% inhibition of HIV-1 replication observed 
with ApA.sub.Rp *A. 
The compounds of the invention are advantageously prepared as soluble salts 
of sodium, ammonium or potassium. The preparative scheme begins with 
6-N-benzoyl-3'-O-tert-butyldimethylsilyl-5'-O-monomethoxy-trityladenosine 
1, which is advantageously prepared from adenosine according to the 
procedure of Flockerzi et al., Liebig's Ann. Chem., 1568-1585 (1981). 
Preparation of the compounds of the present invention is illustrated in 
more detail by reference to the following non-limiting examples. 
3-Nitro-1,2,4-triazole and p-nitrophenylethanol used in the examples may be 
prepared advantageously from published procedures: Chattopadhyaya et al., 
Nucleic Acids Res. 8:2039-2053 (1980); Schwarz et al., Tetrahedron Lett., 
5513-5516 (1984); Uhlmann et al., Helv. Chim. Acta 64:1688-1703 (1981). 
These compounds are also available commercially in the United States. 
3-Nitro-1,2,4-triazole is available from Aldrich Chemical Co., P.O. Box 
355, Milwaukee, Wis. 53201 (1986-1987 cat. no. 24,179.2). 
p-Nitrophenylethanol is available from Fluka Chemical Corp. (cat. no. 
73,610). 
Pyridine and triethylamine used in the examples were purified by 
distillation over KOH, tosyl chloride and calcium hydride. Dichloromethane 
was distilled over calcium chloride and then passed through basic alumina. 
Pure acetonitrile was obtained by distillation over calcium hydride. 
Purification of the protected nucleotides was achieved by preparative 
column chromatography on silica gel 60 (0.063-0.2 mesh, Merck) and by 
preparative thick layer chromatography on silica gel 60 PF.sub.254 
(Merck). Thin layer chromatography ("TLC") was carried out on precoated 
thin layer sheets F 1500 LS 254 and cellulose thin layer sheets F 1440 
from Schleicher & Scheull. 
Scheme 1 is the reaction scheme for the preparation of the fully resolved 
trimers, having phosphorothioate substitution of the first internucleotide 
linkage A.sub.Rp *ApA 10 and A.sub.Sp *ApA 11, from the protected 
intermediates, 8 and 9, wherein "bz" denotes the benzoyl radical, "tbds" 
denotes the tert-butyldimethylsilyl radical, "ce" denotes the cyanoethyl 
radical, "npe" denotes the nitrophenylethoxy radical and "MeOTr" 
represents the monomethoxytrityl radical. The preparation of the trimer 
cores, 10 and 11 is set forth in Preparations 1 through 4 and Example 1. 
Preparation 4 illustrates the fully protected trimer core, while Example 1 
illustrates the removal of the blocking groups and the chemical 
purification of the fully resolved isomers. 
##STR7## 
PREATION 1 
a. Bis-(diisopropylamino)-(.beta.-cyanoethoxy)phosphane 3 
Preparation of the titled compound was in accord with the procedure of 
Kraszewski & Norris, Nucleic Acids Research Sump. Ser. 18: 177-80 (1987). 
.beta.-Cyanoethanol (7 g; 0.1 mole) in absolute CH.sub.3 CN (40 ml was 
added dropwise within 30 min to a solution of freshly distilled PCl.sub.3 
(40 ml; 0.4 mole) at room temperature ("r.t.") and under nitrogen 
atmosphere. After stirring for 3.5 h, the solvent and excess PCl.sub.3 
were removed in high vacuum, the residue was dissolved in 450 ml of 
absolute ether and at -10.degree. C. reacted with N,N-diisopropylamine 
(127 ml; 0.9 mole) by dropwise addition within 1 h under nitrogen 
atmosphere. The reaction mixture was stirred at -10.degree. C. for 30 min 
and at r.t. 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.sub.2 to give 14.7 g (49%) of pure 3 of b.p. 
114.degree.-118.degree. C. This reagent was stored at -20.degree. C. under 
nitrogen. .sup.1 H-NMR (CDCl.sub.3): 3.75 (s, 2H, CH.sub.2); 3.52 (m, 4H, 
4 N--CH); 2.60 (t, 2H, .beta.-CH.sub.2); 1.17+1.14 (2d, 24H, 4 
N--C(CH.sub.3).sub.2). .sup.31 P-NMR (CDCl.sub.3): 124.6 ppm. 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
osine-2'-O-(.beta.-cyanoethyl)-N,N-diisopropylamino!-phosphoramidite 4 
METHOD A 
The preparation of the titled compound was in accord with the procedures of 
Sinha et al., Nucleic Acids Res. 12: 4539-4557 (1984) wherein compound 1, 
Flockerzie et al. (1981), supra, (3.79 g; 5 mmole) and 
diisopropyl-ethylamine (3.5 ml) were dissolved in dry CH.sub.2 Cl.sub.2 
(20 ml) and chloro-N,N-diisopropylaminocyanoethoxy phosphane (2.37 g; 10 
mmole) was added. After 1.5 h stirring under nitrogen at r.t., the 
reaction mixture was diluted with EtOAc (100 ml) and the organic phase was 
washed with a saturated NaHCO.sub.3 /NaCl solution (2.times.80 ml). The 
organic layer was dried over Na.sub.2 SO.sub.4, filtered and evaporated to 
dryness. The residue was dissolved in CH.sub.2 Cl.sub.2 (10 ml) and added 
dropwise to n-hexane (200 ml) at -60.degree. 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. 
METHOD B 
Alternatively, the titled compound was prepared according to the procedure 
of Kraszewski and Norris (1987), supra. In this method compound 1 (3.79 g; 
5 mmole) and tetrazole (0.175 g; 2.5 mmole) were dissolved in dry CH.sub.2 
Cl.sub.2 (20 ml) and then 
bis-(diisopropylamino)-(.beta.-cyanoethoxy)phosphane 3 (3 g; 10 mmole) was 
added. After stirring at r.t. under argon for 17 h, the reaction mixture 
was extracted with EtOAc (100 ml) and washed with saturated NaHCO.sub.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. Anal. calc. for C.sub.52 H.sub.64 N.sub.7 O.sub.7 PSi.times.2 
H.sub.2 O (994.2): C 62.82, H 6.89, N 9.86. Found: C 62.52, H 7.08, N 
10.35. UV (MeOH): .lambda..sub.max (log.epsilon.) 279 nm (4.33); 229 nm 
(4.43). R.sub.f on silica gel with toluol/EtOAc (1/1, v/v): 0.64, 0.61 
(diastereomers). .sup.31 P-NMR (CDCl.sub.3): 150.98, 151.34. 
PREATION 2 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-adeny 
lyl-2'-O.sup.P 
-(2-cyanoethyl)-5'-!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-ad 
enosine 6 
The phosphoramidite 4 (2.88 g; 3 mmole) and 
6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!adenosine 5, Flockerzie 
et al. (1981), supra, (1.2 g; 2 mmole) were dried at r.t. in high vacuum 
for 24 h and dissolved in dry CH.sub.2 Cl.sub.2 (30 ml). Tetrazole (0.5 g; 
8 mmole) was added and after 3 h stirring at r.t. under argon, a solution 
of I.sub.2 0.5 g H.sub.2 O/pyridine/CH.sub.2 Cl.sub.2 (1/3/1, v/v/v)! was 
added dropwise until the brown color does not disappear. The mixture was 
stirred for 15 min, then diluted with CHCl.sub.3 (300 ml). The organic 
phase was saturated with Na.sub.2 S.sub.2 O.sub.3 /NaCl (3.times.80 ml), 
dried over Na.sub.2 SO.sub.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.sub.3 (100 ml), CHCl.sub.3 /MeOH (100/0.5, v/v; 1.5 L) and CHCl.sub.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 the dimer 6 in the form 
of a solid foam. Anal. calc. for C.sub.75 H.sub.94 N.sub.11 O.sub.13 
PSi.sub.3 (1490.9): C 60.42, H 6.49, N 10.33. Found: C 60.50, H 6.49, N 
10.22. UV (MeOH): .lambda..sub.max (log.epsilon.) 278 nm (4.62), 230 nm 
(4.62). R.sub.f on silica gel with CHCl.sub.3 /MeOH (95/5, v/v)=0.56. 
.sup.31 P-NMR (CDCl.sub.3): -0.74 and -1.07 ppm (diastereomers). 
PREATION 3 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'-O.sup.P 
-(2-cyanoethyl)-5'!-6-N-benzoyl-2',3'-O-di-(tert-butyl)dimethylsilyl!aden 
osine 7 
Compound 6 (2.22 g; 1.51 mmole) was stirred with 2% p-TsOH in CH.sub.2 
Cl.sub.2 /MeOH (4/1, v/v; 30 ml) at r.t. for 30 min. The reaction mixture 
was diluted with CH.sub.2 Cl.sub.2 (300 ml), washed with phosphate buffer, 
pH 7.0 (2.times.100 ml), dried over Na.sub.2 SO.sub.4 and evaporated to 
dryness. The residue was applied to a silica gel column (9.times.4.5 cm), 
washed with CHCl.sub.3 (0.7 L) and CHCl.sub.3 /MeOH (100/1, v/v; 300 ml). 
The product was eluted with CHCl.sub.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 5'-hydroxy dimer 7 as an amorphous 
solid. Anal. calc. for C.sub.55 H.sub.78 N.sub.11 O.sub.12 PSi.sub.3 
.times.H.sub.2 O (1218.5): C 54.21, H 6.62, N 12.64. Found: C 54.53, H 
6.58, N 12.62. UV (MeOH): .lambda..sub.max (log.epsilon.) 278 nm (4.60), 
232 nm (4.42.). R.sub.f on silica gel with CHCl.sub.3 /MeOH (95/5, 
v/v)=0.36. .sup.31 P-NMR (CDCl.sub.3): -0.77 and -1.30 ppm 
(diastereomers). 
PREATION 4 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR) 
-thioadenylyl-2'-O.sup.P 
-(2-cyanoethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl- 
2'-O.sup.P 
-(2-cyanoethyl)-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-ade 
nosine A.sub.Rp *ApA 8 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PS) 
-thioadenylyl-2'-O.sup.P 
-(2-cyanoethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl- 
2'-O.sup.P 
-(2-cyanoethyl)-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-ade 
nosine A.sub.Sp *ApA 9 
The phosphoramidite 4 (2.79 g; 2.92 mmole), the 5'-hydroxy dimer 7 (1.94 g; 
1.62 mmole) and tetrazole (0.567 g; 8.1 mmole) were dissolved in dry 
CH.sub.3 CN (8.1 ml) and stirred at r.t. under nitrogen. After 3 h, 
S.sub.8 (1.66; 6.48 mmole) and pyridine (7.8 ml) were added and stirred 
further for 20 h at r.t. The reaction mixture was then diluted with 
CH.sub.2 Cl.sub.2 (300 ml), washed with saturated NaCl (2.times.200 ml), 
dried over Na.sub.2 SO.sub.4 and evaporated to dryness. Final 
coevaporation was with toluene (3.times.20 ml). The crude diastereomeric 
mixture (A.sub.Rp *ApA 8+A.sub.Sp *ApA 9) was dissolved in CH.sub.2 
Cl.sub.2 and applied to a silica gel column (21.times.3.5 cm). The column 
was washed with CH.sub.2 Cl.sub.2 (450 ml) and CH.sub.2 Cl.sub.2 /MeOH 
(99/1, v/v; 200 ml) and the product was eluted with CH.sub.2 Cl.sub.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.sub.Rp *ApA 8 and A.sub.Sp *ApA 9. Separation into the pure 
diastereoisomers was achieved by medium pressure chromatography as 
described above by elution with CHCl.sub.3 /MeOH (99/1, v/v; 800 ml; 20 
ml/fraction; fractions 1-40) followed by elution with CHCl.sub.3 /MeOH 
(95/5, v/v; 800 ml; 20 ml/fraction; fractions 41-80). The fully protected 
A.sub.Rp *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) and the 
fully protected A.sub.Sp *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.sub.Rp *ApA 8 and 0.94 g (30%) of 
A.sub.Sp *ApA 9. Anal. calc. for A.sub.Rp *ApA-C.sub.101 H.sub.127 
N.sub.17 O.sub.19 P.sub.2 SSi.sub.4 (2089.6): C 58.05, H 6.13, H 11.40. 
Found: C 58.65, H 6.24, N 11.50. UV (MeOH): .lambda..sub.max 
(log.epsilon.) 279 nm (4.76), 260 nm (4.56), 236 nm (4.73). R.sub.f on 
silica gel with CHCl.sub.3 /MeOH (97/3, v/v)=0.35. .sup.31 P-NMR 
(CDCl.sub.3): 69.35 and -1.10 ppm. Anal. calc. for A.sub.Sp *ApA-C.sub.101 
H.sub.127 N.sub.17 O.sub.19 P.sub.2 SSi.sub.4 (2089.6): C 58.05, H 6.13, N 
11.25. Found: C 57.03, H 6.33, N 11.14. UV (MeOH): .lambda..sub.max 
(log.epsilon.) 279 nm (4.77), 260 nm (4.57), 236 nm (4.73). .sup.31 P-NMR 
(CDCl.sub.3): 68.33 and -0.84 ppm. 
EXAMPLE 1 
a. (PR)-P-Thioadenylyl-2'-5'-adenylyl-2'-5'-adenosine A.sub.Rp *ApA 10 
b. (PS)-P-Thioadenylyl-2'-5'-adenylyl-2'-5'-adenosine A.sub.Sp *ApA 11 
The corresponding fully protected trimers 8 and 9, respectively, were 
separately deblocked by stirring the trimer (0.06 g; 0.029 mmole) with 2% 
p-TsOH in CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v; 1.2 ml) for 1.5 h at r.t. The 
reaction mixture was diluted with CHCl.sub.3 (50 ml), washed with H.sub.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.sub.3 /MeOH (8/2, v/v). The product bands were eluted with CHCl.sub.3 
/MeOH (4/1, v/v) and evaporated to a foam to give 0.04 g (84%) of the 
A.sub.Rp *ApA isomer 10 and 0.034 g (73%) of the A.sub.Sp *ApA isomer 11. 
The 5'-hydroxy trimer (0.034 g; 0.08 mmole) was then stirred with 0.5M DBU 
in pyridine (5.0 ml) and after stirring at r.t. for 20 h, the solution was 
neutralized with 1M 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 stirring the solvents were removed in vacuo. Desilylation was 
performed with 1M tetrabutylammonium fluoride in THF (2 ml). After 48 h 
stirring, the solvent was removed in vacuo and the residue was dissolved 
in H.sub.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 
0.14-0.17M TEAB buffer, pH 7.5. After evaporation and coevaporation with 
water several times, the trimer was applied to four paper sheets 
(35.times.50 cm) and developed in i-PrOH/conc. ammonia/H.sub.2 O (6/1/3, 
v/v/v). The product band was cut out, eluted with H.sub.2 O, evaporated 
and lyophilized to give 500 O.D..sub.260 nm units (79%) of the A.sub.Rp 
*ApA isomer 10 and 410 O.D..sub.260 nm units (65%) of the A.sub.Sp *ApA 
isomer 11. UV .lambda..sub.max in both cases was 258 nm in H.sub.2 O. 
A.sub.Rp *ApA 10: R.sub.f on cellulose in i-PrOH/ammonia/H.sub.2 O (6/1/3, 
v/v/v)=0.33. .sup.1 H-NMR (D.sub.2 O): 8.20; 8.19; 8.14 (3s, 3H, H--C(8)); 
7.97 (1s, 2H, 2 H--C(2)) and 7.76 (1 s, 1H, 1 H--C(2)); 6.08; 5.93; 5.82 
(3d, 3H, 3 H--C(1')). Retention time on reverse-phase HPLC was 5.60 min. 
A.sub.Sp *ApA 11: R.sub.f on cellulose in i-PrOH/ammonia/H.sub.2 O (6/1/3, 
v/v/v)=0.33. .sup.1 H-NMR (D.sub.2 O): 8.14; 8.09; 8.02 (3s, 3H, H--C(8)); 
7.94; 7.89: 7.80 (3s, 3H, 2 H--C(2)); 6.03; 5.92; 5.80 (3d, 3H, 3 
H--C(1')). Retention time on reverse-phase HPLC was 6.51 min. 
Scheme 2 is the reaction scheme for the preparation of the remaining pair 
of trimer cores, ApA.sub.Rp *A 23 and ApA.sub.Sp *A 24, from the protected 
intermediates 21 and 22, and is outlined in detail in preparations 5 and 6 
and Example 2, below. 
##STR8## 
PREATION 5 
a. N,N-Diisopropyl-trimethylsilylamine 
The preparation of the titled compound was in accord with the procedure of 
Noth and Staudigl, Chem. Ber. 115: 3011-3024 (1982). Methyl iodide (37.6 
ml; 0.6 mole) in absolute ether (50 ml) was added dropwise (over 90 min) 
to a suspension of 14.6 g (0.6 mole) 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 was dissolved. Subsequently, 
N,N-diisopropylamine (78 ml; 0.55 mole) was added within 10-15 min and the 
reaction was refluxed for 1 h. After cooling to 0.degree. C., 
trimethylsilyl chloride (76 ml; 0.6 mole) was added dropwise and the 
reaction mixture was again heated in an oil-bath with vigorous stirring to 
80.degree. C. for 20 h. The supernatant liquid was decanted and the 
residue was extracted with ether (4.times.50 ml). The supernatant and the 
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.degree. C. to yield 73 g (80%), Kp.sub.18 
=36.degree.-39.degree. C. .sup.1 H-NMR (CDCl.sub.3): 0.08 (s, 9H, 
SiCH.sub.3); 1.04-1.07 (d, 12H, N--C--CH.sub.3), 3.2 (m, 2H, N--CH). 
b. Chloro-N,N-diisopropylamino-2-(4-nitrophenyl)ethoxy-phosphane 14 
p-Nitrophenylethanol (4.16 g; 25 mmole) was added portion wise to a 
solution of freshly distilled PCl.sub.3 (14 ml; 0.16 mole) in absolute 
ether (40 ml) at -30.degree. C. under a nitrogen atmosphere within 45 min. 
The reaction mixture was stirred at r.t. for 1.5 h and the solvent and 
excess PCl.sub.3 were then removed in vacuo at 0.degree. C. The residue 
was treated with N,N-diisopropyl-trimethylsilylamine (Preparation 5a) 
(4.33 g; 25 mmole) at 0.degree. C. under a nitrogen atmosphere for 30 min 
and then at r.t. for 20 h. The resulting trimethylsilyl chloride was 
removed under high vacuum at r.t. to yield a syrupy pale yellow product 
(7.1 g; 85%) which crystallized upon storage at -20.degree. C. This 
material was then used for the subsequent phosphitylation reactions. 
.sup.1 H-NMR (CDCl.sub.3): 8.1-8.2 (m, 2H, o to NO.sub.2); 7.39-7.43 (m, 
2H, m to NO.sub.2); 4.04-4.18 (m, 2H, P--O--CH.sub.2); 3.63-3.79 (m, 2H, 
N--CH); 3.07-3.13 (t, 2H, P--O--C--CH.sub.2); 1.14-1.27 (2d, 12H, 
N--C--CH.sub.3). .sup.31 P-NMR (CDCl.sub.3): 181.60 ppm. 
c. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
osine-2'-O-(4-nitrophenyl)ethyl)-N,N-diisopropylaimino!-phosphoramidite 15 
METHOD A 
Compound 1 (3.79 g; 5 mmole) and diisopropylethylamine (3.5 ml) were 
dissolved in dry CH.sub.2 Cl.sub.2 (20 ml) and then 
chloro-N,N-diisopropylamino-2-(4-nitrophenyl)-ethoxyphosphane 14 (2.37 g; 
10 mmole) was added dropwise under a nitrogen atmosphere. After stirring 
at r.t. for 2 h, the reaction mixture was diluted with EtOAc (200 ml), the 
organic phase was washed with a saturated NaHCO.sub.3 /NaCl solution 
(3.times.80 ml), dried over Na.sub.2 SO.sub.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.sub.3 (95/5, v/v). The product fractions were eluted with 
EtOAc/NEt.sub.3 (95/5, v/v), collected and evaporated to dryness, yielding 
15 (5.28 g; 79%) as a colorless solid foam. Anal. calc. for C.sub.57 
H.sub.68 N.sub.7 O.sub.9 PSi (1054.3): C 64.94, H 6.50, N 9.30. Found: C 
64.81, H 6.51, N 9.01. UV (MeOH): .lambda..sub.max (log.epsilon.) 277 nm 
(4.50), 229 nm (4.48). .sup.31 P-NMR (CDCl.sub.3): 150.27, 150.01 ppm. 
R.sub.f on silica gel in toluene/EtoAC (1/1, v/v): 0.62 and 0.68 
(diastereomers). 
METHOD B 
Alternatively, compound 15 was synthesized using 
bis-(diisopropylamino)-2-(4-nitrophenyl)ethoxy!-phosphane 27, infra. To a 
solution of 1.52 g (2 mmole) of compound 1 in absolute CH.sub.3 CN (10 
ml), bis-(diisopropylamino)-2-(4-nitrophenyl)ethoxy!-phosphane 27 (1.59 
g, 4 mmole) and tetrazole (0.07 g; 1 mmole) were added under nitrogen 
atmosphere and the reaction mixture was stirred for 17 h at r.t. The 
reaction mixture was diluted with EtOAc (120 ml) and washed twice with 
saturated NaHCO.sub.3 /NaCl solution (60 ml), dried over Na.sub.2 SO.sub.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. 
c. Bis-(diisopropylamino)-2-(4-nitrophenyl)ethoxy!-phosphane 27 
2-(4-Nitrophenyl)ethanol (8.35 g, 50 mmole) was added in small portions 
over 30 min to a solution of distilled PCl.sub.3 (28 ml; 280 mmole) in 
absolute ether (80 ml) at -5.degree. C. under a nitrogen atmosphere. After 
stirring for 15 min at -5.degree. C. and 1.5 h at r.t., the solvent and 
excess PCl.sub.3 were removed under high vacuum. Then, the yellowish 
syrupy residue was dissolved in 200 ml of absolute ether and reacted at 
-10.degree. C. with N,N-diisopropylamine (64 ml, 450 mmole) by dropwise 
addition over 30 min under a nitrogen atmosphere. The reaction mixture was 
stirred at -10.degree. C. for 15 min and r.t. for 16 h. The voluminous 
precipitate of N,N-diisopropyl-amine 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.degree. C. was 
pure enough to be used for phosphitylation reactions. .sup.1 -NMR 
(CDCl.sub.3): 8.10-8.13 (d, 2H, o to NO.sub.2); 7.36-7.40 (d, 2H, m to 
NO.sub.2); 3.75-3.82 (q, 2H, P--O--CH.sub.2); 3.36-3.51 (m, 2H, N--CH); 
2.95-3.00 (t, 2H, P--O--C--CH.sub.2); 1.05-1.12 (2d, 12H, N--C--CH.sub.3). 
.sup.3 P-NMR (CDCl.sub.3): 123.53 ppm. 
PREATION 6 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
ylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PR)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitro-phenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethyls 
ilyl!-adenosine ApA.sub.Rp *A 21 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
ylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PS)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitro-phenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethyls 
ilyl!-adenosine ApA.sub.Sp *A 22 
Triethylammonium 
6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)adeno 
sine-2'-2-(4-nitrophenyl)ethyl!-phosphate 20 (0.10 g; 0.1 mmole), 
Charubala et al., Liebig's Ann. Chem., 2392-2406 (1981), and the 
respective 5'-hydroxy dimers PR 18 and PS 19, (0.066 g; 0.05 mmole), 
Charubala and Pfleiderer (1992) supra, were coevaporated with dry pyridine 
(3.times.5 ml), dissolved in one ml dry pyridine and 
(2,4,6-triisopropyl)benzenesulfonyl chloride (0.062 g; 0.2 mmole) and 
3-nitro-1,2,4-triazole (0.068 g; 0.6 mmole), Kroger and Mietchen, Z. Chem. 
9: 378-379 (1969); Jones et al., Tetrahedron 36: 3075-3085 (1980), were 
added. After stirring at r.t. for 20 h, the reaction mixture was diluted 
with CHCl.sub.3 (100 ml), washed with H.sub.2 O (2.times.50 ml), dried and 
evaporated. Final evaporations were done with toluene (2.times.10 ml) to 
remove pyridine. The crude trimers 21 and 22, respectively, were purified 
by silica gel column chromatography (15.times.2 cm), using first 
CHCl.sub.3 and then CHCl.sub.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. Anal. calc. for C.sub.111 
H.sub.135 N.sub.17 O.sub.23 P.sub.2 SSi.sub.4 .times.2 H.sub.2 O (2317.8): 
C 57.52, H 5.95, N 10.27. Found: C 57.15, H 6.13, N 10.72. UV (MeOH): 
.lambda..sub.max (log.epsilon.) 276 nm (4.87), 227 nm (4.83). R.sub.f on 
silica gel in CH.sub.2 Cl.sub.2 /EtOAc (1/1)=0.63. .sup.31 P-NMR 
(CDCl.sub.3): 69.88 and -1.0 ppm. 
EXAMPLE 2 
a. Adenylyl-(2'-5')-(PR)-P-thioadenylyl-(2'-5')-adenosine ApA.sub.Rp *A 23 
b. Adenylyl-(2'-5')-(PS)-P-thioadenylyl-(2'-5')-adenosine and ApA.sub.Sp *A 
24 
The fully protected trimers, ApA.sub.Rp *A 21 and ApA.sub.Sp *A 22, were 
separately deblocked by stirring the corresponding trimer (0.088 g; 0.037 
mmole) with 2% p-TsOH in CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v; 0.8 ml. After 
30 min stirring at r.t., the reaction mixture was diluted with CHCl.sub.3 
(50 ml) and washed with H.sub.2 O (2.times.25 ml). The organic phase was 
dried over NaSO.sub.4 and evaporated to dryness. The crude product was 
purified or a silica gel column (5.times.2 cm) and the product eluted with 
CHCl.sub.3 /MeOH (100/1, v/v), evaporated and dried under high vacuum to 
give 0.073 g (94%) of the 5'-hydroxy trimer ApA.sub.Rp *A 21 and 0.061 g 
(84%) of the 5'-hydroxy trimer ApA.sub.Sp *A 22. The resulting 5'-hydroxy 
trimer (0.04 g; 0.02 mmole) was then stirred with 10 ml of 0.5M DBU in 
pyridine. After 24 h, the solution was neutralized with 1M acetic acid in 
pyridine (10 ml) and evaporated to dryness. The residue was treated with 
saturated methanolic ammonia (6 ml) and after stirring at r.t. for 48 h, 
the solvent was removed in vacuo and the residue was desilylated with 1M 
Bu.sub.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 0.14-0.17M TEAB buffer, pH 7.5. After evaporation and 
coevaporation with water several times, the trimer was applied to four 
paper sheets (35.times.50 cm) and developed in i-PrOH/conc. 
ammonia/H.sub.2 O (6/1/3, v/v/v). The product band was cut out, eluted 
with H.sub.2 O, evaporated and lyophilized to give 354 O.D..sub.260 nm 
units (79%) of the ApA.sub.Rp *A isomer 23 and 410 O.D..sub.260 nm units 
(58%) of the ApA.sub.Sp *A isomer 24. UV .lambda..sub.max in both cases 
was 258 nm in H.sub.2 O. ApA.sub.Rp *A 23: R.sub.f on cellulose in 
iPrOH/ammonia/H.sub.2 O (6/1/3, v/v/v) 0.34. .sup.1 H-NMR (D.sub.2 O): 
8.17; 8.16; 8.09 (3s, 3H, H--C(8)); 7.90, 7.78 (2 s, 3H, 3.times.H--C(2)); 
6.04; 5.96; 5.80 (3d, 3H, 3 H--C(1')). Retention time on reverse-phase 
HPLC was 5.98 min. ApA.sub.Sp *A (24): R.sub.f on cellulose in 
i-PrOH/ammonia/H.sub.2 O (6/1/3, v/v/v)=0.33. .sup.1 H-NMR (D.sub.2 O): 
8.17; 8.07; 8.04 (3s, 3H, 3.times.H--C(8)); 8.01; 7.92: 7.72 (3s, 3H, 2 
3.times.H--C(2)); 6.04; 5.92; 5.82 (3d, 3H, 3.times.H--C(1 )). Retention 
time on reverse-phase HPLC was 7.23 min. 
Preparations 7 and 8 begin the preparation for the fully resolved 
tetramers, ApA.sub.Rp *ApA 38 and ApA.sub.Sp *ApA 39, from their 
corresponding dimer 28 (Scheme 1). The reaction scheme continues with the 
addition of the trimer moiety in Preparations 9 and 10 (Scheme 2). 
PREATION 7 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-(monomethoxytrityl)-adenyly 
l-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine A.sub.(Rp,Sp) *A 28 
The phosphoramidite 15 (1.41 g; 1.34 mmole), 
6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsilyl!-adenosine 5 (0.36 g, 
0.93 mmole) and tetrazole (0.188 g, 2.68 mmole) were stirred at r.t. in 
absolute CH.sub.3 CN (9 ml) under a nitrogen atmosophere. After 4 h, a 
solution of I.sub.2 0.5 g in CH.sub.2 Cl.sub.2 /H.sub.2 O/pyridine 
(1/1/3, v/v/v)! was added dropwise until the brown color did not 
disappear. The mixture was stirred was stirred for another 15 min, then 
extracted with CH.sub.2 Cl.sub.2 (3.times.60 ml) and saturated Na.sub.2 
S.sub.2 O.sub.3 /NaCl solution (2.times.60 ml). The CH.sub.2 Cl.sub.2 
phase was collected, dried over Na.sub.2 SO.sub.4, evaporated and 
coevaporated with toluene (2.times.20 ml) to remove the pyridine. The 
crude dimer (1.85 g) was dissolved in CH.sub.2 Cl.sub.2 and applied onto a 
flash silica gel column (12.times.2.5 cm) and chromatographed using 
CH.sub.2 Cl.sub.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 (quant. yield) of the dimer 28 as a colorless amorphous solid. 
The identity of the isolated dimer 28 was proven by comparison with 
authentic material by spectrophotometric comparison. The authentic 
material was synthesized by the phosphotriester method: Anal. calc. for 
ApA 28=C.sub.80 H.sub.98 N.sub.11 O.sub.15 PSi.sub.3 (1569.0): C 61.24, H 
6.30, N 9.82. Found: C 61.24, H 6.24, N 9.65. UV (MeOH): .lambda..sub.max 
(log.epsilon.) 277 (4.69); 260 (4.54)!; 231 (4.66)!. R.sub.f on silica 
gel with CHCl.sub.3 /MeOH (49/1, v/v)=0.37. 
PREATION 8 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine-5'-OH-A.sub.(Rp,Sp) *A 29 
The crude dimer mixture 28 (2.24 g, 1.43 mmole) was stirred with 2% p-TsOH 
in CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v, 20 ml) at r.t. for 30 min. The 
reaction mixture was diluted with CH.sub.2 Cl.sub.2 (200 ml), washed with 
H.sub.2 O (2.times.80 ml), dried over Na.sub.2 SO.sub.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) and chromatographed with CH.sub.2 
Cl.sub.2 (200 ml), CH.sub.2 Cl.sub.2 /2% MeOH (400 ml) and the product was 
eluted with CH.sub.2 Cl.sub.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 2 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. Anal. calc. for 
5'-OH-ApA 29=C.sub.60 H.sub.82 N.sub.11 O.sub.14 PSi.sub.3 (1296.6): C 
55.58, H 6.37, N 11.88. Found: C 55.33, H 6.38, N 11.78. UV (MeOH): 
.lambda..sub.max (log.epsilon.) 278 (4.68); 259 (4.51)!; 233 (4.46). 
R.sub.f on silica gel with toluene/EtOAc/MeOH (5:4:1)=0.53. .sup.31 P=NMR 
(CDCl.sub.3 : -0.36 and -0.73 ppm. 
PREATION 9 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR) 
-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'-!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethyls 
ilyl!-adenosine A.sub.Rp *ApA 30 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PS) 
-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'-!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethyls 
ilyl!-adenosine A.sub.Sp *ApA 31 
The phosphoramidite 15 (0.59 g, 0.56 mmole), the 5'-hydroxy ApA dimer 29 
(0.52 g, 0.40 mmole) and tetrazole (0.079 g; 1.12 mmole) were dissolved in 
dry CH.sub.3 CN (4 ml) and stirred at r.t. under a nitrogen atmosphere. 
After 3 h, phosphoramidite 15 (0.464, 0.44 mmole) and tetrazole (0.062 g, 
0.88 mmole) were added again and the mixture was stirred for another 3 h. 
Then, oxidation with S.sub.8 (0.257 g, 1 mmole) and pyridine (2.6 ml) was 
followed within 16 h at r.t. The reaction mixture was diluted with 
CH.sub.2 Cl.sub.2 (200 ml) at r.t. The reaction mixture was diluted with 
CH.sub.2 Cl.sub.2 (200 ml), washed with a saturated NaCl solution 
(2.times.80 ml), dried over Na.sub.2 SO.sub.4 and evaporated to dryness. 
Final coevaporation was done with toluene (3.times.20 ml) to remove 
pyridine. The crude diastereoisomeric mixture A(.sub.Rp,Sp)*ApA 30+31 was 
dissolved in CH.sub.2 Cl.sub.2 (20 ml), applied onto a flash silica gel 
column (11.times.2.5 cm) and chromatographed with CH.sub.2 Cl.sub.2 (400 
ml), CH.sub.2 Cl.sub.2 /0.5% MeOH (200 ml), 1% MeOH (200 ml) and the 
product was eluted with CH.sub.2 Cl.sub.2 /1.5% MeOH (200 ml). The product 
fraction was evaporated to dryness to give 0.713 g (78%) of the isomeric 
mixture 30+31. Separation into the pure diastereomers was achieved by 
application to preparative silica gel plates (40.times.20.times.0.2 cm, 8 
plates) in toluene/EtOAc (1/1, v/v, 4 developments). The isomeric products 
bands were separately eluted with CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v) and 
evaporated to solid foams, which were dried under high vacuum to give 
0.311 g (34%) of the fully protected A.sub.Rp *ApA isomer 30 and 0.245 g 
(270%) of the fully protected A.sub.Sp *ApA isomer 31. Anal. calc. for 
A.sub.Rp *ApA 30=Cl.sub.111 H.sub.13-5 N.sub.17 O.sub.23 P.sub.2 SSi.sub.4 
.times.H.sub.2 O (2299.8): C 57.97, H 6.00, N 10.35. Found: C 57.63, H 
6.11, N 10.39. UV (MeOH): .lambda..sub.max (log.epsilon.) 278 (4.87); 260 
(4.72)!; 231. (4.75)!. R.sub.f on silica gel with toluene/EtOAc (1/1, 
v/v, 2 developments) and toluene/EtOAc (1/2, v/v, 1 development)=0.37. 
Anal. calc. for A.sub.Sp *ApA 31=C.sub.111 H.sub.135 N.sub.17 O.sub.23 
P.sub.2 SSi.sub.4 .times.2 H.sub.2 O (2317.8): C 57.52, H 6.05, N 10.27. 
Found: C 57.44, H 6.19, N 10.41. UV (MeOH): .lambda..sub.max 
(log.epsilon.) 277 (4.86); 260 (4.72)!; 231 (4.75)!. R.sub.f on silica 
gel with toluene/EtOAc (1:1, 2 developments) and toluene/EtOAc (1:2, 1 
development)=0.27. 
PREATION 10 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PR)-P-thioadenylyl-2'-O.sup 
.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine 5'-hydroxy A.sub.Rp *ApA 32 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-(PS)-P-thioadenylyl-2'-O.sup 
.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine 5'-hydroxy A.sub.Sp *ApA 33 
The corresponding fully protected trimers 30 and 31, respectively, were 
separately detritylated by stirring the trimer (A.sub.Rp *ApA 30: 0.263 g, 
0.115 mmole; A.sub.Sp *ApA 31: 0.21 g, 0.92 mmole) with 2% p-TsOH in 
CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v, for 30: 3.2 ml; for 31: 2.6 ml) for 75 
min at r.t. The reaction mixture was diluted with Ch.sub.2 Cl.sub.2 (120 
ml), washed with H.sub.2 O (2.times.40 ml), dried over Na.sub.2 SO.sub.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.sub.2C1 2/MeOH (4/1, v/v) and 
evaporated to a solid foam to give 0.2 g (86%) of the 5'-hydroxy A.sub.Rp 
*ApA 32 and 0.121 g (66%) of the 5'-hydroxy A.sub.Sp *ApA 33, 
respectively. Anal. Calc. for 5'-OH-A.sub.Rp *ApA 32=C.sub.91 H.sub.119 
N.sub.17 O.sub.22 SSI.sub.4 (2009.4: C 54.39, H 5.97, N 11.85. Found: C 
54.12, H 6.13, N 1174. UV (MeOH): .lambda..sub.max (log.epsilon.) 278 
(4.86); 260 (4.71)!; 233 (4.64!. R.sub.f on silica gel with 
toluene/EtOAc (3:7, 2 developments)=0.35 (diastereomers). .sup.31 P-NMR: 
69.60, 68.91, -0.36 and -0.56 ppm (diastereomers). Anal. calc. for 
5'-OH-A.sub.Sp *ApA 33=C.sub.91 H.sub.119 N.sub.17 O.sub.22 P.sub.2 
SSi.sub.4 (2009.4): C 54.39, H 5.97, N 11.85. Found: C 54.29, H 6.23, N 
11.51. UV (MeOH); .lambda..sub.max (log.epsilon.) 277 (4.85; 260 (4.71)!; 
233 (4.63)!. R.sub.f silica gel with toluene/EtOAc (3:7, 2 
developments=0.42 (diastereomers). .sup.31 P-NMR: 69.28, 69.09, -0.33 and 
-0.56 ppm (diastereomers). 
Scheme 3 is the reaction scheme for the preparation of the fully resolved 
tetramers, ApA.sub.Rp *ApA 38 and ApA.sub.Sp *ApA 39, from the protected 
intermediates, 34 and 35. The preparation of these compounds is outlined 
in Preparations 11 and 12 and Example 3. 
##STR9## 
PREATION 11 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
ylyl-2'-O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PR)-P-thioadenylyl-2'-O.sup.P 
-(2-(4-nitrophenyl)ethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl! 
-adenylyl-2'-O.sup.P 
-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethyls 
ilyl-adenosine ApA.sub.Rp *ApA 34 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
ylyl-2'-O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PS)-P-thioadenylyl-2'-O.sup.P 
-(2-(4-nitrophenyl)ethyl)-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl! 
-adenylyl-2'-O.sup.P 
-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl-adenosine ApA.sub.Sp *ApA 35 
The condensation to the fully protected tetramers 34 and 35, respectively, 
were separately realized by coevaporating 
triethylammonium-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monome 
thoxytrityl)adenosine-2'-2-(4-nitrophenyl)ethyl!-phosphate 20 (0.108 g, 
0.1 mmole) and the 5'-hydroxy PR trimer 32 or PS trimer 33 (0.1 g, 0.05 
mmole), respectively, with dry pyridine (3.times.2 ml), dissolved in dry 
pyridine (0.5 ml) and then (2,4,6-triisopropyl)benzenesulfonyl chloride 
(0.061 g, 0.2 mmole) and 3-nitro-1,2,4-triazole (0.068 g, 0.6 mmole) were 
added. The solution was stirred at r.t. for 21 h, then extracted with 
CH.sub.2 Cl.sub.2 (2.times.20 ml) and H.sub.2 O (3.times.20 ml). The 
organic phase was collected, dried over Na.sub.2 SO.sub.4, evaporated and 
coevaporated with toluene (3.times.20 ml) to remove pyridine. The crude 
tetramers 34 and 35, respectively, 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/), the product bands were eluted with CH.sub.2 Cl.sub.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.sub.Rp *ApA 34 and 0.12 g (8%) of 
the ApA.sub.Sp *ApA 35. Anal. calc. for ApA.sub.Rp *ApA 34=C.sub.142 
H.sub.172 N.sub.23 O.sub.32 P.sub.3 SSi.sub.5 .times.2 H.sub.2 O (3014.5): 
C 56.58, H 5.89, N 10.69; found: C 56.22, H 6.07, N 10.57. UV (MeOH): 
.lambda..sub.max (log.epsilon.) 277 (4.99): 260 (4.85)!; 231 (4.85)!. 
R.sub.f silica gel with toluene/EtOAc/MeOH (5:4:0.5)=0.63 (diastereomers). 
Anal. calc. for ApA.sub.Sp *ApA 35=C.sub.142 H.sub.172 N.sub.23 O.sub.32 
P.sub.3 SSi.sub.5 .times.H.sub.2 O (2996.5): C 56.92, H 5.85, N 10.75; 
found: C 56.40, H 5.89, N 10.61. UV (MeOH): .lambda..sub.max 
(log.epsilon.) 277 (5.01): 260 (4.88)!; 232 (4.89)!. R.sub.f silica gel 
with toluene/EtOAc/MeOH (5/4/0.5, v/v/v)=0.62 (diastereomers). 
PREATION 12 
a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PR)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethyls 
ilyl!-adenosine 5'-OH-ApA.sub.Rp *ApA 36 
b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PS)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethyls 
ilyl!-adenosine 5'-OH-ApA.sub.Sp *ApA 37 
The fully protected tetramers (0.105 g; 0.035 mmole) 34 and 35, 
respectively, were separately detritylated by treatment with 20% p-TsOH in 
CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v, 1.2 ml) for 1 h at r.t. The reaction 
mixture was extracted with CH.sub.2 Cl.sub.2 (3.times.40 ml) and washed 
with H.sub.2 O (3.times.30 ml). The organic phase was collected, dried 
over Na.sub.2 SO.sub.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.sub.2 Cl.sub.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.sub.Rp 
*ApA 36 and 0.057 g (60%) of the corresponding PS tetramer 37. Anal. calc. 
for 5'-OH-ApA.sub.Rp *ApA 36=C.sub.122 H.sub.156 N.sub.23 O.sub.31 P.sub.3 
SSi.sub.5 .times.H.sub.2 O (2724.1): C 53.79, H 5.85, N 11.83; found: C 
53.62, H 5.87, N 11.51. UV (MeOH): .lambda..sub.max (log.epsilon.) 277 
(5.00); 260 (4.87)!; 92.35 (4.81)!. R.sub.f silica gel with 
toluene/EtOAc/MeOH (5:4:0.5)=0.41. Anal. calc. for 5'-OH-ApA.sub.Sp *ApA 
37=C.sub.122 H.sub.156 N.sub.23 O.sub.31 P.sub.3 SSi.sub.5 .times.HO 
(2724.1): C 53.69, H 5.85, N 11.83; found: C 53.58, H 5,97, N 11.32. UV 
(MeOH): .lambda..sub.max (log.epsilon.) 277 (4.96): 260 (4.82)!; 234 
(4.74)!. R.sub.f silica gel with toluene/EtOAc/MeOH (5/4/0.5, v/v/v)=0.39. 
EXAMPLE 3 
a. Adenylyl (2'-5')-(PR)-P-thioadenylyl-(2'-5')-adenylyl-2(2'-5')-adenosine 
ApA.sub.Rp *ApA 38 
b. Adenylyl (2'-5')-(PS)-P-thioadenylyl-(2'-5')-adenylyl-2(2'-5')-adenosine 
ApA.sub.Sp *ApA 39 
The corresponding 5'-hydroxy tetramers 36 and 37, respectively, were 
separately deblocked by stirring each 5'-hydroxy tetramer (0.056 g; 0.021 
mmole) with 0.5M DBU in absolute CH.sub.3 CN (2.5 ml) at r.t. and after 22 
h, the solution was neutralized with 1M AcOH in absolute CH.sub.3 CN (1.25 
ml) and evaporated to dryness. The residual mixture was then treated with 
methanolic ammonia and after stirring at r.t. for 60 h, the solvent was 
removed in vacuum and finally the residue was disilylated with 1M Bu.sub.4 
NF in THF (5 ml) for three days. The solvent was then removed, the residue 
was dissolved in HO (10 ml), applied onto a DEAE Sephadex column A-25 
(30.times.2 cm) and chromatographed first with H.sub.2 O (200 ml) and then 
with a linear gradient of 0-0.04 ml TEAB buffer, pH 7.5, within 3000 ml 
(flow rate 2 ml/min). Under this condition, the ApA.sub.Rp *ApA tetramer 
38 was eluted with a 0.23-0.28M TEAB buffer and ApA.sub.Sp *ApA tetramer 
39 with 0.245-0.305M TEAB buffer, respectively. The product fractions were 
collected, evaporated and coevaporated several times with MeOH. For 
further purification, paper chromatography was performed using a system of 
i-PrOH/ammonia H.sub.2 O (55/10/35, v/v/v). The product band was cut out, 
eluted with H.sub.2 O, concentrated to a smaller volume and finally 
lyophilized to give 728 O.D..sub.260 nm units (73%) of the ApA.sub.Rp *ApA 
isomer 38 and 686 O.D..sub.260 nm units (69%) of the ApA.sub.Sp *ApA 
isomer 39, respectively. ApA.sub.Rp *ApA 38: R.sub.f on cellulose in 
i-PrOH/ammonia/H.sub.2 O (55:10:35)=0.36. UW (H.sub.2 O): .lambda..sub.max 
257 nm. .sup.1 H-NMR (D.sub.2 O): 8.15, 8.07, 8.06, 7.93 (4s, 4H, 
4.times.H--C(8)); 7.92 (s, 2H, 2.times.H--C(2)); 6.03, 5.89, 5.86, 5.79 
(4d, 4.times.H--C(1')). HPLC: on PR-18, A: 50 mM NH.sub.4 H.sub.2 PO.sub.4 
(pH 7.24). B: MeOH/H.sub.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.sub.Sp *ApA 39: 
R.sub.f on cellulose in i-PrOH/ammonia/H.sub.2 O (55/10/35, v/v/v)=0.40. 
UV (H.sub.2 O): .lambda..sub.max 257 nm. HPLC: PR-18, A: 50 mM NH.sub.4 
H.sub.2 PO.sub.4 (pH 7.24). B: MeOH/H.sub.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. 
Scheme 4 is the beginning of the reaction scheme for the preparation of the 
remaining tetramers from their intermediates, A.sub.Rp *Ap-diester 45 and 
the A.sub.Sp *Ap-diester 46, with the blocking groups in place. The 
preparation of the diesters is outlined in Preparations 13 and 14. 
##STR10## 
PREATION 13 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5')-(monomethoxytrityl)-adeny 
lyl -2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denosine-2'-2,5-dichlorophenyl,2-(4-nitrophenyl)-ethylphosphate!ApAp-tries 
ter 41 and 41a 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2,5-dichlorophen 
yl,2-(4-nitrophenyl)ethylphosphate!5'-OH-Ap-triester 40 (0.43 g, 0.5 mmole) 
and the phosphoramidite 15 (0.735 g, 0.7 mmole) were dissolved in absolute 
CH.sub.3 CN (5 ml) in the presence of tetrazole (0.098 g, 1.5 mmole) under 
a nitrogen atmosphere. After stirring for 3.5 h at r.t., phosphoramidite 
(0.2 g, 0.19 mmole) and tetrazole (0.026 g, 0.37 mmole) were added again 
and the reaction mixture was stirred for another 30 min. A solution of 
I.sub.2 0.5 g in Ch.sub.2 Cl.sub.2 /H.sub.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.sub.2 Cl.sub.2 (20 ml) and 
washed with saturated Na.sub.2 S.sub.2 O.sub.3 NaCl solution (2.times.80 
ml). The organic phase was collected, dried over Na.sub.2 SO.sub.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.degree. C. to give 0.610 g 
(67%) of the ApAp-triester 41 and 41a. 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. Anal. calc. for ApAp-triester=C.sub.88 H.sub.94 
N.sub.12 O.sub.20 Cl.sub.2 P.sub.2 SSi.sub.2 (1828.8): C 57.80, H 5.18, N 
9.9. Found: C 57.77, H 5.20, N 9.02. UV (MeOH): .lambda..sub.max 
(log.epsilon.): 277 (4.75); 260 (4.62)!; 228 (4.72)!. R.sub.f on silica 
gel with toluene/EtOAc/MeOH (5/4/1, v/v/v)=0.78. 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5')-(monomethoxytrityl)-(PR,P 
S)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denosine-2'-2,5-dichlorophenyl-2-(4-nitrophenylethyl)-phosphate! 
A(.sub.Rp,Sp)*Ap-triester 43+44 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2,5-dichlorophen 
yl,2-(4-nitrohenyl)-ethylphosphate!5'-OH-Ap-triester 40 (0.52 g; 0.6 mmole) 
and the phosphoramidite 15 (0.95 g; 0.9 mmole) were dissolved in absolute 
CH.sub.3 CN (6.5 ml) in the presence of tetrazole (0.126 g, 1.8 mmole) and 
under nitrogen atmosphere. After stirring for 3 h at r.t., S.sub.8 (0.39 
g, 1.51 mmole) and absolute pyridine (3.9 ml) were added and the reaction 
mixture was further stirred for 20 h, then extracted with CH.sub.2 
Cl.sub.2 (2.times.80 ml) and H.sub.2 O (2.times.80 ml). The organic phase 
was collected, dried over Na.sub.2 SO.sub.4 and evaporated to dryness. 
Final coevaporation was done with toluene (4.times.20 ml) to remove the 
pyridine. The crude diastereomeric mixture A(.sub.Rp,Sp)*Ap-triester 43+44 
was purified by flash silica gel column chromatography (14.times.2.5 cm), 
using 200 ml CH.sub.2 Cl.sub.2, CH.sub.2 Cl.sub.2 /1% MeOH, 2% MeOH and 
finally 200 ml CH.sub.2 Cl.sub.2 /1% MeOH, 2% MeOH and finally CH.sub.2 
Cl.sub.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. Anal. calc. for 
A(.sub.Rp,Sp)*Ap-triester 43+44=C.sub.88 H.sub.94 N.sub.12 O.sub.19 
Cl.sub.2 P.sub.2 SSi.sub.2 (1844.9): C 57.29, H 5.14, N 9.11. Found: C 
56.96, H 5.16, N 9.09. UV (MeOH): .lambda..sub.max (log.epsilon.): 277 
(4.75); 228 (4.72)!. R.sub.f on silica gel with toluene/EtOAc/CHCl.sub.3 
(1/1/1, v/v/v)=0.21. .sup.31 P-NMR (CDCl.sub.3) 69.87, 69.25, -6.89, -7.22 
and -7.31 ppm. 
PREATION 14 
a. 
Triethylammonium-N-6-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5')-(monome 
thoxytrityl)-(PR)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)-ethyl-5'!-N-6-benzoyl-3'-O-(tert-butyl)dimethylsilyl!- 
adenosine-2'-2-(4-nitrophenylethyl)-phosphate! A.sub.Rp *Ap-diester 45 
b. 
Triethylammonium-N-6-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5')-(monome 
thoxytrityl)-(PS)-P-thioadenylyl-2'-(O.sup.P -2-(4-nitrophenyl)-ethyl 
-5'!-N-6-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenosine-2'-2-(4-nitro 
phenylethyl)-phosphate! A.sub.Sp *Ap-diester 46 
The solution of 0.558 g (3.36 mmole) of 4-nitrobenzaldehyde oxime in 15 ml 
of H.sub.2 O/dioxane/Et.sub.3 N (1:1:1) was stirred for 30 min at r.t. 
Then, 0.62 g (0.336 mmole) of the diastereomeric mixture of the 
A(.sub.Rp,Sp)*Ap-triester 43+44 was added and stirred for 2.5 h at r.t. 
The mixture was evaporated, then coevaporated with pyridine (3.times.15 
ml), toluene (3.times.15 ml) and finally with CH.sub.2 Cl.sub.2 
(3.times.15 ml). The residue was dissolved in a small amount of CHCl.sub.3 
and chromatographed on a flash silica gel column (15.times.2.5 cm) with 
CHCl.sub.3 (150 ml), CHCl.sub.3 /2% MeOH (200 ml), 4% MeOH (100 ml), 6% 
MeOH (200 ml), CHCl.sub.3 /6% MeOH/0.5% Et.sub.3 N (300 ml) and CHCl.sub.3 
/6% MeOH/2% Et.sub.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+46. Separation into the pure 
diastereomers was achieved by chromatography on preparative silica gel 
plates (7 plates, 40.times.20.times.0.2 cm) and three developments in 
CHCl.sub.3 /MeOH (9/1, v/v). The product bands were eluted with CHCl.sub.3 
/MeOH (4/1, v/v) containing 1% Et.sub.3 N and evaporated to a solid foam 
to give 0.262 g (43%) of A.sub.Rp *Ap-diester 45, 0.144 (24%) of A.sub.Sp 
*Ap-diester 46 and 0.045 g (7%) of A(.sub.Rp,Sp)*Ap-diester 45+46. Anal. 
calc. for A.sub.Rp *Ap-diester 45=C.sub.88 H.sub.107 N.sub.13 O.sub.19 
P.sub.2 SSi.sub.2 .times.2 H.sub.2 O (183.7.1) C 57.53, H 6.09, N 9.91. 
Found: C 57.30, H 6.70, N 9.75. UV (MeOH): .lambda..sub.max 
(log.epsilon.); 277 (4.69); 260 (4.56)!; 231 (4.61)!. R.sub.f on silica 
gel with CHCl.sub.3 /MeOH (9/1, v/v)=0.37. .sup.31 P-NMR (CDCl.sub.3): 
69.66 and -0.09 ppm. Anal. calc. for A.sub.Sp *Ap-diester 46=C.sub.88 
H.sub.107 N.sub.13 O.sub.19 P.sub.2 SSi.sub.2.times.2 H.sub.2 O (1837.1): 
C 57.53, H 6.09, N 9.91. Found: C 56.44, H 7.15, N 8.61. UV (MeOH): 
.lambda..sub.max (log.epsilon.); 276 (4.60); 260 (4.49)!; 232 (4.52)!. 
R.sub.f on silica gel with CHCl.sub.3 /MeOH (9/1, i/v)=0.28. .sup.31 P-NMR 
(CDCl.sub.3): 68.96 and -0.06 ppm. 
Scheme 5 is the reaction scheme for the preparation of the remaining fully 
resolved tetramers, ApApA.sub.Rp *A 51, ApApA.sub.Sp *A 52, A.sub.Rp 
*ApApA 57, and A.sub.Sp *ApApA 58, from their respective protected 
intermediates, 47, 48, 53 and 54. The corresponding preparations are 
outlined in Preparation 15, 16, and 17 and Examples 4 and 5. 
##STR11## 
PREATION 15 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
ylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!- 
adenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PR)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)-dimethyls 
ilyl!-adenosine ApApA.sub.Rp *A 47 
Triethylammonium 
6-N-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5'-O-(monomethoxytrityl)-ade 
nylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denosine-2'-2-(4-nitrophenyl)ethyl-phosphate! 42 (0.14 g; 0.078 mmole) and 
the 5'-hydroxy PR dimer 18 (0.08 g, 0.06 mole) 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 mmole) and 
3-nitro-1,2,4-triazole (0.053 mg, 0.47 mmole) were added. The solution was 
stirred at r.t. for 22 h, extracted with CH.sub.2 Cl.sub.2 (2.times.30 
ml), washed with H.sub.2 O (2.times.20 ml), dried over Na.sub.2 SO.sub.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.sub.2 Cl.sub.2 (50 ml), then with CH.sub.2 Cl.sub.2 /1% MeOH (100 ml), 
2% MeOH (50 ml) and finally with CH.sub.2 Cl.sub.2 /3% MeOH (100 ml). The 
product fraction (80 ml) was evaporated to dryness to give 0.11 g (62%) of 
the fully protected tetramer ApApA.sub.Rp *A 47 as a colorless foam after 
drying under high vacuum at 350.degree. C. Anal. calc. for C.sub.142 
H.sub.172 N.sub.23 O.sub.32 P.sub.3 SSi.sub.5 .times.H.sub.2 O (2996.5): C 
56.92, H 5.85, N 10.75. Found: C 56.51, H 5.91, N 10.37. UV (MeOH): 
.lambda..sub.max (log.epsilon.) 277 (4.99); 259 (4.84)!; 233 (4.85)!. 
R.sub.f on silica gel with CHCl.sub.3 /MeOH (19/1, v/v)=0.46. 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-aden 
ylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)-ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!- 
(PS)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)-dimethyls 
ilyl!-adenosine ApApA.sub.Sp *A 48 
ApAp-diester 41 (0.14 g, 0.078 mmole) and the 5'-hydroxy PS dimer 19 (0.08 
g, 0.06 mmole) 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 mmole) and 
3-nitro-1,2,4-triazole (0.053 mg, 0.46 mmole) were added. After stirring 
for 4.5 h at r.t., (2,4,6-triisopropyl)benzenesulfonyl chloride (0.024 g, 
0.078 mmole) and 3-nitro-1,2,4-triazole (0.027 g, 0.234 mmole) were added 
again. The solution was stirred at r.t. for 16.5 h, then extracted with 
CH.sub.2 Cl.sub.2 (4.times.20 ml) and added with H.sub.2 O (3.times.20 
ml), dried over Na.sub.2 SO.sub.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.sub.2 Cl.sub.2 
and CH.sub.2 Cl.sub.2 /1-3% MeOH to give 0.107 g (60%) of the fully 
protected tetramer ApApA.sub.Sp *A 48 as a colorless foam after drying 
under high vacuum at 35.degree. C. Anal. calc. for C.sub.142 H.sub.172 
N.sub.23 O.sub.32 P.sub.3 SSi.sub.5 .times.H.sub.2 O (2996.5): C 56.92, H 
5.85, N 10.75. Found: C 56.51, H 5.91, N 10.85. UV (MeOH): 
.lambda..sub.max (log.epsilon.) 277 (4.99); 259 (4.85)!; 231 (4.86)!. 
R.sub.f on silica gel with CHCl.sub.3 /MeOH (19/1, v/v)=0.46. 
PREATION 16 
a. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PR)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine 5'-OH-ApApA.sub.Rp *A 49 
b. 6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-adenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-( 
PS)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine 5'-OH-ApApA.sub.Sp *A 50 
The fully protected tetramer ApApA.sub.Rp *A 47 (0.104 g, 0.035 mmole) was 
stirred with 2% p-TsOH in CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v, 1.4 ml) at 
r.t. After 1.5 h, the reaction mixture was extracted with CH.sub.2 
Cl.sub.2 (3.times.40 ml) and H.sub.2 O (2.times.40 ml). The combined 
organic phase was dried over Na.sub.2 SO.sub.4 and evaporated to dryness. 
The crude product was purified on a flash silica gel column (11.times.1 
cm) and the product eluted with 20 ml CH.sub.2 Cl.sub.2 and 50 ml CH.sub.2 
Cl.sub.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 the hydroxy tetramer 
ApApA.sub.Rp *A 49. Anal. calc. for C.sub.122 H.sub.156 N.sub.23 O.sub.31 
P.sub.3 SSi.sub.5 .times.H.sub.2 O (2706.5): C 54.15, H 5.81, N 11.90. 
Found: C 53.97, H 6.02, N 11.65. UV (MeOH): .lambda..sub.max 
(log.epsilon.) 277 (5.00); 260 (4.85)!; 234 (4.77)!. R.sub.f on silica 
gel with CHCl.sub.3 /MeOH (19/1, v/v)=0.43. 
The fully protected tetramer ApApA.sub.Sp *A 48 was treated in an analogous 
manner through the purification stage. The crude product 50 was purified 
on two preparative silica gel plates (20.times.20.times.0.2 cm) in 
CHCl.sub.3 /MeOH (19/1, v/v), the product band was eluted with CH.sub.2 
Cl.sub.2 /MeOH (4/1, v/v) and evaporated to a solid foam to give 0.068 g 
(72%) of the 5'-hydroxy tetramer ApApA.sub.Sp *A 50. 
EXAMPLE 4 
a. Adenylyl-(2'-5')-adenylyl-(2'-5')-(PR)-P-thioadenylyl-(2'-5')-adenosine 
ApApA.sub.Rp *A 51 
b. Adenylyl-(2'-5')-adenylyl-(2'-5')-(PS)-P-thioadenylyl-(2'-5')-adenosine 
ApApA.sub.Sp *A 52 
The corresponding 5'-hydroxy tetramers 49 and 50, respectively, were 
deblocked separately by stirring the 5'-hydroxy tetramer (0.067 g, 0.025 
mmole) with 0.5M DBU in absolute CH.sub.3 CN (3 ml) at r.t. for 20 h, the 
solution was neutralized with 1M AcOH in absolute CH.sub.3 CN (1.5 ml) and 
evaporated to dryness. R.sub.f on silica gel with 
{EtOAc/i-PrOH/ammonia/H.sub.2 O, 7/1/2, v/v/v) 7/3, v/v: ApApA.sub.Rp 
*A=0.58; ApApA.sub.Sp *A=0.66. The residue was then treated with 
methanolic ammonia (10 ml) and after 3 days reaction time, the solvent was 
removed under vacuum. R.sub.f on silica gel with 
EtOAc/i-PrOH/ammonia/H.sub.2 O, 7/1/2, v/v/v) 1/1, v/v}: ApApA.sub.Rp 
*A=0.38; ApApA.sub.Sp *A=0.36!. Desilylation was done with 1M Bu.sub.4 NF 
in THF (5 ml). The reaction mixture was stirred at r.t. for 48 h and then 
the solvent was evaporated in vacuum. The residue was taken up in H.sub.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.sub.Rp *A was eluted with 
0.15-0.20M TEAB buffer, pH 7.5, and in the case of the tetramer 
ApApA.sub.Sp *A with 0.24-0.32M 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.sub.2 O 
(6/1/3, v/v/v). The product band was cut out, eluted with H.sub.2 O, 
concentrated to a smaller volume and finally lyophilized to give 675 
O.D..sub.260 nm units (57%) of ApApA.sub.Rp *A isomer 51 and 753 
O.D..sub.260 nm (65%) of ApApA.sub.Sp *A isomer 52. ApApA.sub.Rp *A 51: 
R.sub.f on cellulose in i-PrOH/ammonia/H.sub.2 O (6/1/3, v/v/v)=0.33. UV 
(H.sub.2 O): .lambda..sub.max 258 nm. HPLC: PR-18, A: 50 mM NH.sub.4 
H.sub.2 PO.sub.4, pH 7.2. B: MeOH/H.sub.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.sub.Sp *A 52: R.sub.f on cellulose in i-PrOH/ammonia/H.sub.2 O 
(6/1/3, v/v/v)=0.21. UV (H.sub.2 O): .lambda..sub.max 258 nm. HPLC: PR-18, 
A: 50 mM NH.sub.4 H.sub.2 PO.sub.4, pH 7.2. B: MeOH/H.sub.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. 
PREATION 17 
a. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR) 
-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine A.sub.Rp *ApApA 53 
Triethylammonium 
6-N-benzoyl-3'-O-(tert-butyl)-dimethylsilyl!-5'-O-(monomethoxytrityl)-(PR 
)-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denosine-2'-(O.sup.P -2-(4-nitrophenyl)ethyl-phosphate! A.sub.Rp 
*Ap-diester 45 (0.141 g, 0.078 mmole) and the 5'-hydroxy dimer 29 (0.078 
g, 0.06 mmole) 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 mmole) and 
3-nitro-1,2,4-triazole (0.053 mg, 0.47 mmole) were added and stirred at 
r.t. for 21 h. The reaction mixture was diluted with CH.sub.2 Cl.sub.2 (60 
ml) and washed with H.sub.2 O (2.times.30 ml), dried over Na.sub.2 O.sub.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 chromatograpy (11.times.1 cm) and eluted first with 
CH.sub.2 Cl.sub.2 (50 ml), then with CH.sub.2 Cl.sub.2 /1% MeOH (100 ml), 
2% MeOH (200 ml), 3% MeOH (50 ml) and finally with CH.sub.2 Cl.sub.2 /5% 
MeOH (50 ml). The product fraction (200 ml) was evaporated to dryness. The 
residue was chromatographed again on two preparative silica gel plates 
(20.times.20.times.0.2 cm) in toluene/EtOAc/MeOH (5/4/0.5, v/v/v) to 
remove small amount of 5'-hydroxy dimer. The tetramer product band was 
eluted with CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v) and evaporated to a solid 
foam to give 0.053 g (30%) of the tetramer A.sub.Rp *ApApA 53 after drying 
in high vacuum at 35.degree. C. Anal. calc. for C.sub.142 H.sub.172 
N.sub.23 O.sub.32 P.sub.3 SSi.sub.5 .times.3 H.sub.2 O (3032.5): C 56.24, 
H 5.92, N 10.62. Found: C 55.75, H. 5.71, N 9.83. UV (MeOH): 
.lambda..sub.max (log.epsilon.) 277 (4.96); 260 (4.82)!; 232 (4.82)!. 
R.sub.f on silica gel with toluene/EtOAc/MeOH (5:4:1)=0.78. 
b. 
6-N-Benzoyl-3'-O-(tert-butyl)dimethylsilyl!-5'-O-(monomethoxytrityl)-(PS) 
-P-thioadenylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-3'-O-(tert-butyl)dimethylsilyl!-a 
denylyl-2'-(O.sup.P 
-2-(4-nitrophenyl)ethyl-5'!-6-N-benzoyl-2',3'-di-O-(tert-butyl)dimethylsi 
lyl!-adenosine A.sub.Sp *ApApA 54 
A.sub.Sp *Ap-diester 46 (0.141 g, 0.078 mmole) and the 5'-hydroxy dimer 29 
(0.078 g, 0.06 mmole) 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 mmole) and 
3-nitro-1,2,4-triazole (0.053 mg, 0.47 mmole) were added and the mixture 
was stirred at r.t. After 21 h, (2,4,6-triisopropyl)benzenesulfonyl 
chloride (0.024 mg, 0.079 mmole) and 3-nitro-1,2,4-triazole (0.027 mg, 
0.24 mmole) were added again. The reaction mixture was stirred for another 
hour, then diluted with CH.sub.2 Cl.sub.2 (60 ml) and washed with H.sub.2 
O (2.times.30 ml), dried over Na.sub.2 SO.sub.4 and evaporated to dryness. 
Further work-up was performed analagous to that described for tetramer 53 
to give 43 mg (24%) of A.sub.Sp *ApApA 54 in the form of a solid foam. 
Anal. calc. for C.sub.142 H.sub.172 N.sub.23 O.sub.32 P.sub.3 SSi.sub.5 
.times.H.sub.2 O (2996.5): C 56.92, H 5.85, N 10.75. Found: C 56.63, H 
6.08, N 10.18. UV (MeOH): .lambda..sub.max (log.epsilon.) 277 (4.98); 260 
(4.84)!; 232 (4.85)!. R.sub.f on silica gel with toluene/EtOAc/MeOH 
(5/4/1, v/v/v)=0.78. 
EXAMPLE 5 
a. (PR)-P-Thioadenylyl-(2'-5')-adenylyl-(2'-5')-adenylyl-(2'-5')-adenosine 
A.sub.Rp *ApApA 57 
b. (PS)-P-Thioadenylyl-(2'-5')-adenylyl-(2'-5')-adenylyl-(2'-5')-adenosine 
A.sub.Sp *ApApA 58 
The corresponding fully protected tetramers 53 and 54 were deblocked by 
stirring a solution of 0.047 g (0.016 mmole) of PR tetramer 53 (PS 
tetramer 54: 0.032 g, 0.012 mmole) in 2% p-TsOH in CH.sub.2 Cl.sub.2 /MeOH 
(4/1; v/v; for PR: 0.5 ml; for PS: 0.38 ml) for 1 h at r.t. The reaction 
mixture was diluted with CH.sub.2 Cl.sub.2 (60 ml), washed with H.sub.2 O 
(2.times.30 ml), dried over Na.sub.2 SO.sub.4 and evaporated to dryness. 
The crude products were purified on preparative silica gel plates 
(20.times.20.times.0.2 cm) in CHCl.sub.3 /MeOH (19/1, v/v), the product 
bands were eluted with CH.sub.2 Cl.sub.2 /MeOH (4/1, v/v) and evaporated 
to solid foams to give 0.034 g (80%) of the 5'-hydroxy A.sub.Rp *ApApA 
isomer 55 and 0.02 g (68%) of the 5'-hydroxy A.sub.Rp *ApApA isomer 56. 
The solution of the 5'-hydroxy tetramers 55 (0.034 g, 0.013 mmole) and 56 
(0.02 g, 0.007 mmole), respectively, were separately stirred with 0.5M DBU 
in absolute CH.sub.3 CN (55: 1.5 ml; 56: 0.9 ml)! for 18 h at r.t., then 
neutralized by addition of 1M 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 r.t. for 60 h, was evaporated 
to dryness. Desilylation was done by treatment with 1M Bu.sub.4 NF in THF 
(2.5 ml). After stirring at r.t. for 60 h, the solvent was removed under 
vacuum. Some H.sub.2 O (10 ml) was added to the resulting residue and 
applied to a DEAE Sephadex column A-25 (32.times.2 cm) and eluted with 
0-0.5M 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.sub.2 O, 55/10/35, v/v/v) gave, 
after lyophilization, 347 O.D..sub.260 nm units (58%) of A.sub.Rp *ApApA 
57 and 111 O.D..sub.260 nm units (31%) of A.sub.Sp *ApApA 58, 
respectively. A.sub.Rp *ApApA 57: UV (H.sub.2 O)=257 nm. R.sub.f on 
cellulose in i-PrOH/ammonia/H.sub.2 O (6/1/3, v/v/v)=0.21. HPLC: PR-18, A: 
50 mM NH.sub.4 H.sub.2 PO.sub.4 (pH 7.2). B: MeOH/H.sub.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.sub.Sp *pApA 58: UV (H.sub.2 O)=257 nm. R.sub.f on cellulose 
in iPrOH/ammonia/H.sub.2 O (6/1/3, v/v/v)=0.32. HPLC: PR-18, A: 50 mM 
NH.sub.4 H.sub.2 PO.sub.4, pH 7.2. B: MeOH/H.sub.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. 
Preparation of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylate 
5'-Monophosphates 
The phosphorothioate/phosphodiester trimer and tetramer cores were 
synthesized as described above in Examples 1, 2, 3, 4, and 5. The trimer 
and tetramer 5'-monophosphates were enzymatically synthesized according to 
the procedure of Sambrook et al., Molecular Cloning--A Laboratory Manual, 
2 ed., Cold Spring Harbor Laboratory Press, pp. 5.68-5.71 (1989) from 
their corresponding cores and ATP with T4 polynucleotide kinase. 
5'-Monophosphorylation was determined by reverse-phase HPLC analysis and 
confirmed by the subsequent hydrolysis of each 5'-monophosphate derivative 
by 5'-nucleotidase (data not shown). Yields of phosphorylation ranged from 
15% to 68%. 
Preparation of 2',5'-Phosphorothioate/phosphodiester Oligoadenylate 
5'-Diphosphate and 5'-Triphosphate 
The 5'-diphosphate and 5-triphosphate of the 
2',5'-phosphorothioate/phosphodiester oligoadenylates may be prepared from 
the 5'-monophosphate by following the procedure of Example 6. 
EXAMPLE 6 
All reactions are performed in glassware oven-dried at 125.degree. C. for 
18-24 hr. A 2',5'-phosphorothioate/phosphodiester oligoadenylate 
stereoisomer (trimer or tetramer, 400 OD units at 260 nm) is dissolved in 
500 microliters of dry dimethylformamide ("DMF") and dried in vacuo in a 
10 ml conical flask at 35.degree. C. This process is repeated three times. 
To the dry residue, 50 micromoles of triphenylphosphine, 100 micromoles of 
imidazole and 50 micromoles of dipyridinyl disulfide are added. The 
mixture is dissolved in 500 microliters dry DMF plus 50 microliters of dry 
dimethylsulfoxide. The solution is stirred with a stirring bar for 2 hr at 
room temperature. After 2 hr the solution is homogeneous (after 30 
minutes, the solution begins to change to yellow). The solution is 
transferred dropwise to 10 ml of a 1% NaI/dry acetone (w/v) solution. The 
clear colorless precipitate which forms is the sodium salt of the 
5'-phosphoroimidazolidate. The precipitate is centrifuged at room 
temperature, the supernatant is decanted, and the precipitate is washed 
three times with 10 ml dry acetone. The centrifuging is repeated. The 
precitipate is dried over P.sub.2 O.sub.5 in vacuo for 2 hr. The 
precipitate is dissolved in 200 microliters of freshly prepared 0.5M 
tributylammonium pyrophosphate in dry DMF. The solution is maintained at 
room temperature for 18 hr after which time the DMF is removed in vacuo. 
The residue is dissolved in 0.25M triethylammonium bicarbonate buffer 
("TEAB") (pH 7.5). The 5'-di and 5'-triphosphate products are separated 
using a DEAE-Sephadex A25 column (HCO.sub.3 -form; 1.times.20 cm) with a 
linear gradient of 0.25M to 0.75N TEAB. Fractions (10 ml) are collected. 
The product is observed by ultraviolet spectroscopy at 254 nm. The 
fractions containing the 5'-di and 5'-triphosphates are separately pooled 
and dried in vacuo. by lyophilization. The yield of the 5'-diphosphate is 
about 5%; the yield of the 5'-triphosphate is about 60%. 
Stability of the Phosphorothioate/Phosphodiester Trimer and Tetramer Core 
Derivatives to Serum Phosphodiesterase 
The stability of authentic 2-5A and phosphorothioate/phosphodiester trimer 
and tetramer core derivatives (300 .mu.M) was determined by incubation in 
200 .mu.L of RPMI-1640 medium supplemented with 10% fetal calf serum in 5% 
CO.sub.2 -in-air at 37.degree. C. Aliquots (30 .mu.L) were removed at time 
zero and 6 hours. The hydrolysis products were identified by HPLC as 
described in Kariko et al. , Biochemistry 26: 7127-7135 (1987). Under the 
conditions described therein, authentic A.sub.2 and A.sub.3 were 
completely hydrolyzed to inosine and hypoxanthine in 20 min (Table 1), 
while A.sub.Rp *A and A.sub.Sp *A were not hydrolyzed. 
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 
internucleotide linkage. For example, A.sub.Rp *ApApA and A.sub.Sp *ApApA 
were 50% degraded to their respective dimer cores, A.sub.Rp *A and 
A.sub.Sp *A, whereas ApA.sub.Rp *ApA and ApA.sub.Sp *ApA are degraded from 
the 2',3'-terminus to form the trimer cores, ApA.sub.Rp *A and ApA.sub.Sp 
*A. ApApA.sub.Rp *A and ApApA.sub.Sp *A are degraded from the 5'-terminus 
to yield ApA.sub.Rp *A and ApA.sub.Sp *A, respectively. 
TABLE 1 
______________________________________ 
Hydrolysis of Phosphorothioate/Phosphodiester Trimer and Tetramer 
2-5A Core Derivatives by Serum Phosphodiesterases 
2-5A or 
Derivative 
% Hydrolysis.sup.a 
Hydrolysis Products.sup.b 
______________________________________ 
A.sub.2 100 (20 min) inosine, hypoxanthine 
A.sub.3 100 (20 min) inosine, hypoxanthine 
A.sub.Rp *A 
0.sup.b not hydrolyzed 
A.sub.Sp *A 
0.sup.b not hydrolyzed 
A.sub.Rp *ApA 
0 not hydrolyzed 
A.sub.Sp *ApA 
0 not hydrolyzed 
ApA.sub.Rp *A 
0 not hydrolyzed 
ApA.sub.Sp *A 
0 not hydrolyzed 
A.sub.Rp *ApApA 
100 inosine, hypoxanthine, 
A.sub.Rp *A 
A.sub.Sp *ApApA 
100 inosine, hypoxanthine, 
A.sub.Sp *A 
ApA.sub.Rp *ApA 
50 inosine, hypoxanthine, 
A.sub.Rp *ApA 
ApA.sub.Sp *ApA 
50 inosine, hypoxanthine, 
A.sub.Sp *ApA 
ApApA.sub.Rp *A 
30 inosine, hypoxanthine, 
ApA.sub.Rp *A 
ApApA.sub.Sp *A 
33 inosine, hypoxanthine, 
ApA.sub.Sp *A 
______________________________________ 
.sup.a Incubations were for 6 h as described in text. Number in 
parentheses indicates the time at which 100% hydrolysis was observed. 
.sup.b Identified as described by Kariko et al., Biochemistry 26: 7127713 
(1987). 
Binding of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylates to RNase L 
The affinity of the phosphorothioate/phosphodiester 2-5A derivatives for 
RNase L was determined in radiobinding assays according to the method of 
Knight et al., Meth. Enzymol 79: 216-227 (1981). Authentic A.sub.3, 
pA.sub.3 and p.sub.3 A.sub.3 bind to RNase L with IC.sub.50, values of 
1.times.10.sup.-6 M, 1.times.10.sup.-9 M and 1.times.10.sup.-9 M, 
respectively. According to the invention, the binding of the 
phosphorothioate/phosphodiester core and their 5'1-monophosphates to RNase 
L was equivalent to or slightly better than the corresponding authentic 
2-5A cores and 5'-monophosphates, with IC.sub.50 values from 
8.times.10.sup.-7 M to 8.times.10.sup.-6 M for the cores and from 
1.times.10.sup.-8 M to 1.times.10.sup.-9 M for the 5'-monophosphates. The 
trimer and tetramer 2-5A core derivatives with phosphorothioate 
substitution in the first internucleotide linkage from the 5'-terminus 
exhibited lower affinity compared to those with phosphorothioate 
substitution in the second or third internucleotide linkage. 
Activation of RNase L by Phosphorothioate/Phosphodiester Derivatives of 
2-5A 
Correlation of biological properties with absolute configuration has only 
been possible with the preparation of the fully resolved 
2',5'-phosphorothioate/phosphodiester adenylate trimer cores. However, the 
trimer core compounds have been found to bind and/or activate RNase L only 
modestly. RNase L activation by the 2',5'-phosphorothioate core molecules 
is significantly enhanced by 5'-phosphorylation. 
Core-cellulose assays were performed according to the method of Silverman, 
Anal. Biochem. 144: 450-460 (1985) and Kariko et al. (1987), supra, in 
which RNase L was partially purified from L929 cell extracts by 
immobilization on 2-5A.sub.4 core-cellulose. Activation of RNase L was 
measured by the conversion of poly(U) .sup.32 p!pCp to acid soluble 
fragments. The results indicate that authentic p.sub.3 A.sub.3, p.sub.3 
A.sub.4, pA.sub.3 and pA.sub.4 have IC.sub.50 values of 5.times.10.sup.-10 
M, 5.times.10.sup.-10 M, 2.times.10.sup.-7 M and 2.times.10.sup.-8 M, 
respectively, while surprisingly, of the phosphorothioate/phosphodiester 
trimer core derivatives, only ApA.sub.Rp *A can activate RNase L 
(IC.sub.50 of 5.times.10.sup.-7 M) (FIG. 1A, .DELTA.). Three of the 
phosphorothioate/phosphodiester trimer 5'-monophosphates can activate 
RNase L, with pApA.sub.Rp *A being the most potent activator of RNase L 
(IC.sub.50 of 1.times.10.sup.-9 M) (FIG. 1B, .DELTA.). pA.sub.Rp *ApA 
(.quadrature.) and pA.sub.Sp *ApA (.box-solid.) are 100-fold less potent 
activators of RNase L. Of the six phosphorothioate/phosphodiester tetramer 
core derivatives, only ApA.sub.Rp *ApA (.quadrature.) and ApApA.sub.Rp *A 
(.DELTA.) can activate RNase L (IC.sub.50 of 5.times.10.sup.-7 M and 
5.times.10.sup.-7 M, respectively) (FIG. 1C). Five of the six 
phosphorothioate/phosphodiester tetramer 5'-monophosphates activate RNase 
L (IC.sub.50 values &gt;6.times.10.sup.-7 M to 8.times.10.sup.-10 M). The 
pApA.sub.Sp *ApA enantiomer did not activate RNase L, even at 
concentrations as high as 1.times.10.sup.-5 M (FIG. 1D, .box-solid.). 
Activation of RNase L by the phosphorothioate/phosphodiester trimer and 
tetramer 2-5A derivatives was also measured in a rRNA cleavage assay using 
L929 cell extracts according to the method of Kariko et al. (1987), supra, 
in which extracts of L929 cells were incubated for 1 h at 30.degree. C. in 
the presence or absence of 2-5A or 2-5A derivative. Consistent with the 
results from the core-cellulose assays (FIG. 1A), ApA.sub.Rp *A 
(1.times.10.sup.-6 M) was the only trimer core able to activate RNase L to 
cleave rRNA to the well-characterized specific cleavage products (SCP) of 
RNase L (FIG. 2A, lane 5). A.sub.Rp *ApA, A.sub.Sp *ApA and ApA.sub.Sp *A, 
as well as authentic A.sub.3, did not activate RNase L at concentrations 
as high as 1.times.10.sup.-6 M (FIG. 2A, lanes 3, 4, 6, 7). Authentic 
p.sub.3 A.sub.3 was active at 1.times.10.sup.-8 M (FIG. 2A, lane 2). The 
corresponding 5'-monophosphates, pA.sub.Rp *ApA, pA.sub.Sp *ApA and 
pApA.sub.Rp *A, activated at 1.times.10.sup.-7 M, 1.times.10.sup.-7 M, and 
2.times.10.sup.-9 M, respectively (FIG. 2B, lanes 4-6), as compared with 
pA.sub.3 which was active at 1.times.10.sup.-6 M (lane 3). Incubation with 
pApA.sub.Sp *A, even at concentrations as high as 5.times.10.sup.-6 M, did 
not result in detectable rRNA degradation (data not shown). 
Comparable degradation of rRNA was observed with two of the six 
phosphorothioate/phosphodiester tetramer core derivatives relative to the 
authentic p.sub.3 A.sub.4 control. ApA.sub.Rp *ApA and ApApA.sub.Rp *A 
activated RNase L at 1.times.10.sup.-5 M (FIG. 3A, lanes 6 and 8). As was 
observed in the core-cellulose assays (FIG. 1D), five of the six 
phosphorothioate/phosphodiester tetramer 5'-monophosphates were able to 
activate RNase L (pA.sub.Rp *ApApA, pA.sub.Sp *ApApA, pApA.sub.Rp *ApA, 
pApApA.sub.Rp *A, and pApApA.sub.Sp *A) (FIG. 3B, lanes 4, 5, 6, 8, 9, 
respectively). The most efficient activator or RNase L was pApA.sub.Rp 
*ApA (1.times.10.sup.-8 M) (FIG. 3B, lane 6), while pApA.sub.Sp *ApA was 
an antagonist of RNase L activation and was unable to activate RNase L 
even at concentrations as high as 1.times.10.sup.-5 M (FIG. 3B, lane 7). 
Inhibition of RNase L Activation by pApA.sub.Sp *A 
The high affinity of pApA.sub.Sp *A for RNase L and the observation that 
pApA.sub.Sp *A does not activate RNase L, suggests that it might be a 
specific inhibitor of RNase L. Indeed, pApA.sub.Sp *A inhibits the 
activation of RNase L by p.sub.3 A.sub.3 or pApA.sub.Rp *A (FIG. 4A). 
Authentic p.sub.3 A.sub.3 activates RNase L to hydrolyze 28S and 18S rRNA 
to SCP at 10.sup.-9 M or 10.sup.-8 M (lanes 1 and 3). However, addition of 
pApA.sub.Sp *A (10.sup.-6 M ) results in the inhibition of RNase 
L-catalyzed hydrolysis of rRNA (lanes 2 and 4). Similarly, whereas 
pApA.sub.Rp *A activates RNase L at 10.sup.-9 M or 10.sup.-8 M (lanes 5 
and 7), the addition of pApA.sub.Sp *A (10.sup.-6 M ) inhibits this 
activation (lanes 6 and 8). The inhibitory activity of pApA.sub.Sp *A was 
also observed with partially-purified RNase L (FIG. 4B). p.sub.3 A.sub.3 
activates RNase L with an IC.sub.50 value of 5.times.10.sup.-10 M 
(.circle-solid.); however, upon addition of pApA.sub.Sp *A 
(1.times.10.sup.-6 M ), the observed IC.sub.50 value shifts to 
1.times.10.sup.-8 M (.smallcircle.), demonstrating specific inhibition of 
p.sub.3 A.sub.3 -mediated activation of RNase L by pApA.sub.Sp *A. 
Notwithstanding, pApA.sub.Sp *A is useful as a probe in the evaluation of 
the role of RNase L in the interferon-induced biological cascade. Most 
importantly, pApA.sub.Sp *A selectively inhibits activation of RNase L at 
physiological concentrations, and is metabolically stable to specific and 
non-specific phosphodiesterases. The molecule provides the means to 
selectively inhibit RNase L activation. 
Moreover, it is expected that pApA.sub.Sp *A has therapeutic activity. 
Individuals afflicted with chronic myelogenous leukemia ("CML") display a 
highly elevated RNase L activity, as evidenced by novel rRNA CML-specific 
cleavage products. Thus, pApA.sub.Sp *A, which is a metabolically stable 
inhibitor of RNase L, has potential utility in treating chronic 
myelogenous leukemia. 
Additionally, individuals afflicted with chronic fatigue syndrome ("CFS") 
(also known as myalgic encephalomyelitis (ME) or low natural killer ("NK") 
cell disease) and other HHV-6 related disorders also display a highly 
elevated RNase L activity compared to controls (mean basal level=466.+-.23 
compared to 123.+-.12 in controls; p&lt;0.0001), Suhadolnik et al, Clinical 
Infectious Disease 18 (SUPPL. 1): 96-104 (1994). In experiments performed 
using extracts of peripheral blood mononuclear cells ("PBMC") from 
individuals with CFS before and during therapy with a biological response 
modifier, poly (I)-poly (C.sub.12 U) (mismatched dsRNS, Ampligen.RTM.), as 
compared to healthy individuals, the mean basal latent 2-5A synthetase 
level in PMBC extracts was significantly decreased following therapy 
(610.+-.220 picomoles 2-5A/mg protein/hour) compared to controls 
(2035.+-.325 picomoles 2-5A/mg protein/hour, P&lt;0.001). Id. Further, all 
pretherapy PBMC extracts tested were positive for human herpes virus-6 
(HHV-6) replication. Therapy resulted in a significant decrease in HHV-6 
activity (p&lt;0.01) and down regulation of the 2-5A synthetase/RNase L 
pathway in temporal association with clinical and neuropsychological 
improvement. Without wishing to be bound by any theory, it appears that 
the upregulated 2-5A pathway observed in CFS pretherapy is consistent with 
a hypothesis that an activated immune state and persistent viral infection 
may play a pathogenesis of CFS. Thus, pApA.sub.Sp *A, which, as stated 
above, is a metabolically stable inhibitor of RNase L, also has potential 
utility in treating CFS. 
Inhibition of Cell Growth by Phosphorothioate/Phosphodiester 2-5A Core 
Derivatives 
Cell viability was determined by Trypan blue exclusion. Post-treatment 
colony forming ability was determined by growth in microtiter wells as 
outlined in the procedure of Kraemer et al. Mutation Res. 72: 285-292 
(1980). The results indicate that no decrease in survival or inhibition of 
Sup T1 cell growth in microtiter plates was observed with any of the 
dimer, trimer or tetramer phosphorothioate/phosphodiester 2-5A core 
derivatives. On the basis of the lack of cytotoxicity and estimated uptake 
of 1% on a previous report with the cordycepin derivative of 2-5A, 
Suhadolnik et al., Nucleosides and Nucleotides 2: 351-366 (1983), 
3.times.10.sup.-4 M was chosen as the concentration at which to screen the 
phosphorothioate/phosphodiester derivatives for anti-HIV-1 activity. 
Effect of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylates on 
Inhibition of HIV-1-Induced Syncytia Formation 
The infected centers assay as described by Henderson et al., Virology 182: 
186-198 (1991), was used to measure the ability of the 
phosphorothioate/phosphodiester derivatives of 2-5A trimer and tetramer 
cores to inhibit HIV-1 induced syncytia formation, an indicator of HIV-1 
replication in T cells. Freshly isolated peripheral blood lymphocytes 
(PBL) were treated with 2-5A or derivatives for 2 h and infected with 
HIV-1 strain IIIB at a m.o.i. of approximately 0.1. The infected PEL were 
maintained in RPMI-1640 medium supplemented with 10% (v/v) 
heat-inactivated fetal calf serum at 37.degree. C. in a humidified 5% 
CO.sub.2 in air atmosphere. After 48 h, the cells were washed twice in 
Hank's balaned salt solution, serially diluted and seeded into multiple 
wells of a 96-well microtiter plate. Immediately, 2.times.10.sup.5 
exponentially growing Sup T1 cells were added to each well; Sup T1 cells 
readily form a syncytium with a cell which is productively infected with 
HIV-1. The wells were examined daily for the presence of syncytia, using a 
tissue culture microscope. The first signs of syncytia formation can be 
seen in 12 h, with some complete syncytia developing by 24 h. Final 
results were read at 72 h. Each syncytium was counted as a single infected 
cell. The number of syncytia per seeded cell is determined and expressed 
as an infected center per infected cell. In the control (no 2-5A 
derivative added), 100% syncytia formation was equivalent to 12.+-.3 
syncytia per 200 HIV-1 infected cells. 
The data is shown in FIGS. 5A and 5B. As shown in FIG. 5A, ApA.sub.Rp *A 
was a highly efficient inhibitor of syncytia formation, with 100% 
inhibition observed at 3.times.10.sup.-4 M. Its PS enantiomer, ApA.sub.Sp 
*A inhibited syncytia formation 78%. A.sub.Rp *ApA and A.sub.Sp *ApA 
inhibited syncytia formation only 10% and 15%, respectively. Authentic 
A.sub.3 and A.sub.4 (3.times.10.sup.-4 M) inhibited syncytia formation 21% 
and 15%, respectively while adenosine (9.times.10.sup.-4 M) did not 
inhibit syncytia formation. Of the six phosphorothioate/phosphodiester 
tetramer core derivatives, ApApA.sub.Rp *A and ApApA.sub.Sp *A were the 
most inhibitory (90% and 76% inhibition, respectively) (FIG. 5B). 
ApA.sub.Rp *ApA, ApA.sub.Sp *ApA, A.sub.Rp *ApApA and A.sub.Sp *ApApA 
inhibited syncytia formation 26%, 32%, 16% and 18%, respectively. 
Adenosine and A.sub.4 (FIG. 5B), as well as the A.sub.Rp *A and A.sub.Sp 
*A dimers, adenine or 3',5'-A.sub.4 (data not shown), were not able to 
inhibit syncytia formation. 
Effect of 2',5'-Phosphorothioate/Phosphodiester Oligoadenylates on HIV-1 
Reverse Transcriptase Activity 
Sup T1 cells were treated with 2-5A or a phosphorothioate/phosphodiester 
derivative at 300 .mu.M for 6 hours and then infected with HIV-1 at a 
multiplicity of infection (M.O.I) of approximately 0.1. Adenosine and 
adenine were tested at 900 .mu.M. At 96 hours post-infection, culture 
supernatant was removed and HIV-1 RT activity was assayed in triplicate as 
described by Henderson et al., Virology 182:186-198 (1991). Briefly in 
this method, 25 .mu.l of culture supernatant was added to a 50 .mu.l 
cocktail containing 50 mM Tris (pH 8.0), 20 mM dithiothreitol, 10 mM 
MgCl.sub.2, 60 mM NaCl, 0.05 Nonidet p-40, 5 .mu.g/ml oligodeoxythymidylic 
acid, 10 .mu.g/ml polyriboadenylic acid, 10 .mu.M deoxythymidine 
triphosphate and 1 mCi .sup.32 p!thymidine 5'-triphosphate. The mixture 
was incubated at 37.degree. C. for 2 hours. Fifty microliters of the 
cocktail were then spotted onto diethylaminoethyl (DEAE) paper, dried, 
washed with 2.times. SSC solution (three times for 10 minutes each time) 
and 95% ethanol (two times for 5 minutes each time), dried and exposed to 
radiographic film for 18 to 24 hours at -80.degree. C. The filters were 
cut and final quantitation was determined by scintillation spectrometry. 
The data for the HIV-1 RT activity is shown in Table 2. As indicated, the 
trimer ApA.sub.Sp *A was the most efficient inhibitor of HIV-1 RT activity 
(78%). On the contrary, its PR enantiomer, ApA.sub.Rp *A inhibited HIV-1 
reverse transcription by 31%. Similarly, the tetramer with the PS 
phosphorothioate/phosphodiester linkage adjacent to the 2' terminal 
linkage, ApApA.sub.Sp *A, was able to suppress RT activity 62% while its 
PR counterpart was only 38% effective in inhibiting this activity. 
TABLE 2 
______________________________________ 
Inhibition of HIV-1 Reverse Transcriptase Activity 
by Phosphorothioate/Phosphodiester 2-5A 
2-5A or Percent Inhibition of HIV-1 
Derivative Reverse Transcriptase.sup.1 
______________________________________ 
2',5'-A.sub.Rp *ApA 
32 
2',5'-A.sub.Sp *ApA 
56 
2',5'-ApA.sub.Rp *A 
31 
2',5'-ApA.sub.Sp *A 
78 
2',5'-A.sub.Rp *ApApA 
57 
2',5'-A.sub.Sp *ApApA 
54 
2',5'-ApA.sub.Rp *ApA 
52 
2',5'-ApA.sub.Sp *ApA 
42 
2',5'-ApApA.sub.Rp *A 
38 
2',5'-ApApA.sub.Sp *A 
62 
2',5'-A.sub.4 26 
A.sub.4 8 
Adenosine 0 
Adenine 4 
______________________________________ 
.sup.1 Average of triplicate determinations. Intraassay variation for 
replicates was &lt;10%. 
The compounds of the present invention may be combined with appropriate 
pharmaceutical or agricultural carriers to form an antiviral composition. 
For pharmaceutical use, the compounds of the invention may be taken up in 
pharmaceutically acceptable carriers, such as, solutions, suspensions, 
tablets, capsules, ointments, elixirs and injectable composition and the 
like. They are administered to subjects suffering from viral infection. 
The dosage administered depends upon the nature and severity of the 
infection, the disease stage, and, when administered systematically, the 
size and weight of the infected subject. 
The compounds are generally administered in the form of water-soluble 
salts. Pharmaceutically acceptable water soluble salts include, for 
example, the sodium, potassium or ammonium salts of the active compounds. 
They are readily dissolved in water or saline solution. Thus, the 
preferred formulation for pharmacological use comprises a saline solution 
of the desired compound in salt form. The formulation may further contain 
an agent, such as a sugar or protein, to maintain osmotic balance. The 
salt form of the compound is preferred owing to the relatively high 
acidity (about pH 3) of the acid form of the compounds. 
The compounds of the invention may be used as a treatment or 
prophylactically for humans and animals from viral infectives such as 
Herpes simplex, rhinovirus, hepatitis and other infections of the 
hepatitis virus family, Epstein Barr virus, measles virus, multiple 
sclerosis (which may be caused by a viral agent) and the various Human 
T-Lymphotropic Viruses ("HTLV"), such as HTLV-1, which causes cutaneous T 
cell lymphoma, HTLV-2, which causes Sezary lymphoma, and HTLV-3, which is 
responsible for Acquired Immune Deficiency Syndrome ("AIDS"). The 
compounds of the invention inhibit the HIV-1 Induced Syncytia formation. 
The compounds may be applied topically to treat skin cancers caused by 
radiation, carcinogens or viral agents. Such skin cancers include 
cutaneous T-cell lymphoma, Sezary lymphoma, Xeroderma pigmentosium, ataxia 
telangiectasia and Bloom's syndrome. A sufficient amount of a preparation 
containing a compound of the invention is applied to cover the lesion or 
affected area. An effective concentration of active agent is between about 
10.sup.-3 M and 10.sup.-5 M, with 10.sup.-4 M being preferred. 
The compounds of the present invention may also be used to treat 
plant-infecting virus, particularly tobacco mosaic virus, and other 
viruses which cause necrosis in turnips, cucumber, orchids and in other 
plants. Such viruses include, but are not limited to, tobacco vein 
mottling virus, vesicular stomatitis virus, vaccinia virus, turnip 
necrosis virus, and cymbidium orchid virus. 
The compounds may be administered effectively to plants by topical 
application by abrasion of the leaf surface, aerosol spray, treatment of 
the soil, spraying, or dusting. 
An effective antiviral composition may be formed by combining one or more 
of the compounds of the invention with a carrier material suitable for 
agricultural use. While the individual stereoisomers are preferred for 
pharmaceutical use, mixtures of one or more of stereoisomers may be 
employed in agricultural applications. The active compound may also be 
administered by spraying insect vectors such as aphids, thrips and 
whiteflies which carry virus to plants. The dosage administered depends 
upon the severity of the infection. 
The compounds of the invention may be applied to plant seeds prior to 
germination to control viruses contained in the germ plasm. The seeds may 
be soaked in a solution of polyethylene glycol ("PEG") containing one or 
more of the compounds. PEG brings the seeds to physiological activity and 
arrest. The relative concentration of active compound to PEG depends upon 
the type of seed under treatment. 
Plants may be effectively treated with an aqueous formulation containing 
from about 10.sup.-1 to about 10.sup.-2 M concentration of active 
ingredient. The compounds of the invention may be applied at very low 
concentrations. An effective amount of active ingredient on the plant 
surface is from about 10.sup.-8 to about 10.sup.-12 mole per cm.sup.2 of 
being preferred. For the typical tobacco plant of 1,000 cm.sup.2, 
10.sup.-5 M of compound is effective. At this rate, one pound of active 
ingredient is sufficient to treat 2.times.10.sup.8 tobacco plants. 
For agricultural application, the compounds are advantageously administered 
in the form of water-soluble salts, e.g. ammonium or potassium salts. 
Sodium salts are generally avoided in treating edible plants. 
The compounds of the invention are readily dissolved in water, particularly 
at such low concentrations. Aqueous formulations for agricultural use may 
optionally contain a sticker and/or a UV-stabilizer. Such agents are 
well-known to those skilled in the art. Fatty acids (1%) are useful as 
spreader sticker agents. Effective UV-stabilizers include, for example, 
p-aminobenzoic acid. 
For antiviral use in mammals, the compounds of the invention are 
administered parenterally, such as intravenously, intraarterially, 
intramuscularly, subcutaneously or when administered as an anti-cancer 
agent, intratumorally. The preferred route of administration for antiviral 
therapy is intravenous injection. The compounds of the invention may be 
administered to mammals at very low concentrations. The actual dosage 
administered may take into account the size and weight of the patient, 
whether the nature of the treatment is prophylactic or therapeutic in 
nature, the age, health and sex of the patient, the route of 
administration, the nature and stage of the affliction, and other factors. 
An effective daily dosage of active ingredient, based upon in vivo studies 
involving other 2-5A analogues, is from about 0.25 g per 70 kg of body 
weight (approximately 152 lbs) to about 2.5 g per 70 kg of body weight. 
The preferred daily dosage is about 0.5 g per 70 kg of body weight. Those 
skilled in the art should readily be able to derive appropriate dosages 
and schedules of aministration to suit the specific circumstance and needs 
of the patient. 
It is expected that an effective treatment regimen includes administration 
of the daily dosage for two days. Treatment is continued at least until 
the disease condition is substantially abated. 
Preferably, the therapeutic end point is determined by testing for the 
continued presence of viral DNA. Such testing can be done by polymerase 
chain reaction (PCR) in which the presence of viral DNA is assayed 
according to convential PCR. PCR primers of appropriate nucleotide 
sequences for amplification of viral DNA can be prepared from known viral 
nucleotide sequences. To obtain DNA for testing, patient peripheral blood 
mononuclear cells are lysed with an appropriate lysing agent, such as 
NP-40. 
Alternatively, testing for the continued presence of the virus can be 
performed by an antigen-antibody assay using any of the known monoclonal 
or polyclonal antisera against a protein antigen of the target virus' 
protein coat. For example, an antigen-antibody assay may be employed to 
detect any of the protein antigen in the antigens HIV protein coat, for 
example, the gp120, p17 or p24. Moreover, the target antigen is not 
limited merely to coat protein antigens. Antisera can be targeted against 
a suitable non-coat protein antigen, such as the HIV reverse transcriptase 
(RT) molecule. Monoclonal antibodies to HIV RT are known. Sobol et al., 
Biochemistry 30: 10623-10631 (1991). 
Additionally, testing for the presence of the infecting virus during or 
post-treatment could be accomplished by an assay which assesses the viral 
load in the patient's blood stream. This can be done by determining the 
level of syncytia formation, i.e., by measuring the formation of viral 
particles. See procedure outlined in Henderson et al., Virology 182: 
186-198 (1994). 
In addition to administration with conventional carriers, the compounds of 
the present invention may be administered by a variety of specialized 
oligonucleotide or nucleic acid delivery techniques. 2-5A and its 
analogues have been successfully encapsulated in various encapsulating 
materials, such as in unilamellar liposomes and delivered with the aid of 
monoclonal antibodies to cells, Bayard et al., Eur. J. Biochem. 
151:319-325 (1985). Reconstituted Sendai virus envelopes have been 
successfully used to deliver RNA and DNA to cells, Arad et al., Biochem. 
Biophys. Acta. 859: 88-94 (1986). Moreover, the virus envelope is not 
limited to Sendai virus, but could include encapsulation in any retroviral 
amphotrophic particle. For example, an HIV envelope could be formed from 
any part or all of the outer protein coat of a non-infectious HIV 
particle. Such particles as gp 120 can be cloned by known recombinant 
techniques. These techniques may be utilized for introduction of the 
present 2',5'-phosphorothioate/phosphodiester oligoadenylates into cells. 
It is further contemplated that the compounds of the invention may be 
administered in the form of prodrugs in which lipophilic groups are 
attached to, for example, the 5'-terminal hydroxyl group of the core 
compound. 
Conjugation of 2',5'-Phosphorothioate Tetramer Adenylates 
The 2',5'-phosphorothioate/phosphodiester tetramers of the invention may be 
conjugated with the carrier (poly)L-lysine. (Poly)L-lysine has been shown 
to be an effective vector for introducing 2',5'-oligoadenylates and 
analogues into intact cells. Bayard et al., Biochemistry 25: 3730-3736 
(1986) Poly(L-lysine) conjugation to trimer molecules is not feasible, 
owing to the destruction of the 2 -terminal ribosyl moiety and subsequent 
inactivation of the molecule. Conjugation to poly(L-lysine) permits 
efficient intracellular transport of the 
2',5'-phosphorothioate/phosphodiester oligoadenylates of the invention, 
while preserving intact within the conjugate the trimer moiety believed 
necessary for good biological activity. 
The conjugates are formed by introducing two aldehyde functions at the 2' 
end of the tetramer by periodate oxidation of the alpha-glycol group of 
the ribose residue. The resulting aldehyde groups are then randomly 
coupled to the epsilon-amino groups of lysine residues of poly(L-lysine) 
by Schiff base formation, and then reduced with sodium cyanoborohydride at 
pH 8.0. This procedure converts the 2',3'-terminal ribose ring into a 
morpholine structure. The poly(L-lysine) peptide preferably contains from 
about 60 to about 70 lysine residues. From about five to about ten of the 
lysine residues are coupled in this manner to tetramer moieties. The 
resulting 2',5'-phosphorothioate/phosphodiester/(poly)L-lysine conjugates 
may then be isolated by gel filtration chromatography on a Sephadex G-50 
column. 
The 2',5'-phosphorothioate/phosphodiester oligoadenylate poly(L-lysine) 
conjugates have the formula: 
##STR12## 
wherein q is an integer from about 60 to about 70 and each R is 
independently R' or 
##STR13## 
From about five to about ten of the R groups comprise R'. The R' group has 
the following formula: 
##STR14## 
wherein m is zero, 1, 2 or 3; and R.sub.3, R.sub.4 and R.sub.5 are 
independently selected from the group of oxygen and sulfur, provided that 
all R.sub.3, R.sub.4 and R.sub.5 may not be oxygen, and further provided 
that all R.sub.3, R.sub.4 and R.sub.5 may not be sulfur. 
The conjugates may be advantageously prepared by the procedure of Bayard et 
al., Biochemistry 25: 3730-3736 (1986): 
EXAMPLE 7 
Preparation of Poly(L-lysine)/2',5'-Phosphorothioate/Phosphodiester 
Oligoadenylate Conjugates 
A 4-microliter aliquot of sodium metaperiodate (0.6 micromole in 0.1M 
sodium acetate buffer, pH 4.75) is added to an ice-cold solution of 
2',5'-phosphorothioate/phosphodiester tetramer adenylate in 400 microliter 
of distiller water. The reaction mixture is stirred on ice for 30 min; 400 
microliter of poly(L-lysine) (0.14 micromole in 0.2M phosphate buffer, pH 
8.0) and 200 microliter of sodium cyanoborohydride (20 micromole in 0.2M 
phosphate buffer, pH 8.0) are added. The mixture is incubated for 2 h at 
room temperature and then loaded on a Sephadex G-50 column equilibrated 
with 0.1M sodium acetate buffer, pH 4.75. Each fraction is assayed for its 
phosphorothioate/phosphodiester oligoadenylate/poly(L-lysine) content by 
the method described by Lowry et al., J. Biol. Chem. 193:265-275 (1951), 
and by absorbance at 260 nm. 
Conjugation of the 2',5'-phosphothioate/phosphodiester tetramer to 
poly(L-lysine) leaves the remaining three 2',5'-linked 
phosphorothioate/phosphodiester adenylic residues intact for optimal RNase 
L binding and activation. 
Liposome Encapsulation of 2',5'Phosphorothioate/phosphodiester 
Oligoadenylates 
Encapsulation of the compounds of the present invention comprises another 
attractive non-disruptive technique for introduction into cells. Liposome 
encapsulation may be advantageously accomplished according to the 
technique described by Kondorosi et al., FEBS Lett. 120:37-40 (1980). 
EXAMPLE 8 
Preparation of Large Unilamellar Vesicles (Liposomes) Loaded with 
2',5'-Phosphorothioate/Phosphodiester Oligoadenylates 
Briefly, a phospholipid mixture from bovine brain (Sigma Chemical Co., 
Folch fraction III composed of 80-85% phosphatidylserine with the 
remaining 15% composed of other brain lipids; 35 mg) is suspended in 5 ml 
of buffer A 0.1M NaCl, 2 mM histidine, 2 mM 
N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acit ("TES"), 0.4 mM 
EDTA (pH 7.4) by vortexing. The suspension is sonicated under nitrogen for 
10 minutes at 0.degree. C. The suspension is further incubated for 1 hr at 
37.degree. C. after adjusting the final concentration of Ca.sup.++ to 20 
mM by the addition of 125 microliters of 800 mM CaCl.sub.2. The resulting 
precipitate is sedimented by centrifugation (2500.times.g, 10 min), 
vortexing and mixing with 100 microliters of 1.times.10.sup.-4 M 
2',5'-phosphorothioate/phosphodiester oligoadenylate, which is dissovled 
in phosphate-buffered saline. The final concentration of EDTA is then 
adjusted to 120 mM by the addition of 400 microliters of buffer B 150 mM 
EDTA, pH 7.4, 0.1M NaCl, 2 mM histidine, 2 mM TES!. Liposomes are formed 
after incubation of this mixture for 30 minutes at 37.degree. C. The 
excess of EDTA and non-encapsulated components are removed by passing the 
liposomes through a Sephadex G-25 column which is equilibrated with 
phosphate-buffered saline. About 10% of the 
2',5'-phosphorothioate/phosphodiester oligoadenylate is encapsulated into 
liposomes by this procedure. The liposome suspension is stable at 
4.degree. C. for one week following preparation. 
Preparation of Reconstituted Sendai Virus Envelopes Containing 
2',5'-Phosphorothioate/Phosphodiester Oligoadenylates 
Reconstituted Sendai virus envelopes may be used as efficient vehicles for 
the introduction of polynucleotides into cells. Arad et al., Biochimica et 
Biophysica Acta 859: 88-94 (1986), discloses introduction of poly(I) 
.circle-solid.poly(C) into cultured cells by the use of reconstituted 
Sendai virus envelopes. Fusion of the aforesaid reconstituted Sendai virus 
envelopes leads to introduction of the enclosed macromolecules into the 
recipient cell cytoplasm. Reconstituted Sendai virus envelopes may be 
obtained by detergent solubilization of intact Sendai virus particles. The 
reconstituted envelopes are fusogenic vesicles consisting of the viral 
envelope phospholids and their glycoproteins, devoid of the viral genomic 
RNA. 
Incorporation of the compounds of the present invention into reconstituted 
Sendai virus envelopes for fusion-mediated micro-injection may be 
accomplished by following the procedure of Arad et al., Biochimica et 
Biophysica Acta 859: 88-94 (1986). Briefly, a pellet of Sendai virus 
particles (1.5 mg protein) is dissolved in 30 microliters of a solution 
containing 10% Triton X-100, 100 mM NaCl, 50 mM Tris-HCl (pH 7.4) and 0.1 
mM phenylmethylsulfonyl fluoride (Triton X-100:protein ratio, 2:1, w/w). 
To the clear supernatant obtained after centrifugation, 
2',5'-phosphorothioate/phosphodiester oligoadenylate dissolved in a 
solution A (160 mM NaCl, 20 mM Tris-HCl (pH 7.4)) is added to give a final 
concentration of active ingredient of 5-20 mg/ml and a final volume of 150 
microliters. Triton X-100 is removed from the supernatant by direct 
addition of 40 mg of SM-2 Bio-Beads. The turbid suspension obtained 
(containing reconstituted Sendai virus envelopes) is centrifuged at 
100,000.times.g for 1 h. The pellet, containing about 10% of the original 
viral protein, is then suspended in solution A to give a final protein 
concentration of 25 micrograms/ml. 
The present invention may be embodied in other specific forms without 
departing from the spirit or essential attributes thereof and, 
accordingly, reference should be made to the appended claims, rather than 
to the foregoing specification, as indicating the scope of the invention.