PURINE BASE MODIFIED 2'-DEOXYRIBONUCLEOSIDES, USE IN TRIPLEX FORMING OLIGONUCLEOTIDES AND PROCESS FOR PREPARING THE SAME

Abstract: There are provided purine-base modified guanosine analogues, for example 2'-deoxy, and then ribonucleosides for incorporating into triplex-forming oligonucleotides. Also provided are triplex-forming oligonucleotides for binding into strands of DNA for treatment of disease and regulating genetic expression.

Description: PϋRINE BASE MODIFIED 2'-DEOXYRIBONUCLEOSIDES, USE IN TRIPLEX FORMING OLIGONUCLEOTIDES AND PROCESS FOR PREPARING THE SAME FIELD OF THE INVENTION This invention relates to the synthesis of novel purine base modified 2'-deoxyribonucleosides which are useful as constituents of oligonucleotides capable of forming a triple helix with a double stranded DNA. BACKGROUND Although the existence of triple helical structures is well documented, triplex formation is a relatively new concept as the basis for a program of pharmaceutical intervention. Recent work has made considerable progress towards the development of triplex forming oligonucleotides (TFO) reagents which bind to DNA in a site selective manner. This work has demonstrated that synthetic DNA oligonucleotides targeted to repeating A or G segments within a DNA gene can form stable triplexes at acidic pH. The formation of these triple helices is based upon hydrogen bonding of T to the AT bases in a DNA double helix or duplex and a protonated cytosine (C+) to a GC duplex. The requirement for protonation of cytosine while conceptually important is not of physiological significance since the pH of the C+GC triplex is almost 3. Moreover, this triplex is not stable at physiological pH. Attempts to solve this problem have included substituting cytosine with 5-methylcytosine and 5- bromouridine. These substitutions increased the apparent pH to allow triplex formation in the pH range between 6 and 7, however, this range is still not physiologic. Therefore, when considering the potential biological utility of TFOs, it is rather important to examine nucleoside modifications or other nucleoside congeners which would allow for sequence specific triplex formation at physiological pH (i.e., pH 7.4 to pH 7.8). During the past several years the prospect for oligonucleotide based therapy has been proposed * . Initial experiments were designed to determine the site selectivity for triplex formation along a sequence of DNA. The control region of the c-myc oncogene was used as a model. In these early experiments it was shown that, at physiological pH, a discrete 27 base long oligonucleotide had the capacity to form a stable triple helix with high selectivity. The synthetic oligonucleotide was designed to bind in the major groove to the purine rich strand of the DNA c-myc oncogene target and was stabilized by T binding to AT; A binding to AT; and G binding to GC. Subsequently it has been shown that GGC and TAT triplets result in stabilized triple helix formation in A or G rich sites within DNA. Additional refinements in triple helix forming oligonucleotide design has shown that significant stability enhancement can be achieved at most DNA target sites by forming triple helix compounds so that they bind antiparallel rather than parallel to the purine rich strand of the DNA. Thus, triple helix forming ligands can be designed which bind to purine-rich gene promoter segments at physiological pH, with dissociation constants in the lθ "9 - lθ "7 M range. The goal of TFO design is to develop molecules which can bind to any duplex DNA sequence, without regard for purine content or other symmetry consideration. H- bonding of the Hoogsteen or reverse Hoogsteen type occurs with purine bases in the major groove of an underlying duplex. Consequently, a polypurine/polypyrimidine duplex target presents an orderly array of bond formers positioned upon one side of the major helix groove. At sites of CG or TA inversion, the corresponding purine target base is placed upon the opposing half of the major groove and can be reached for the purposes of H-bonding only (a) upon extension of the TFO backbone, (b) by distension of the duplex, or (c) both. Indirect data suggests that duplex DNA assumes the A form upon triplex formation. In the A form, the major groove is deep and narrow, with dimensions well suited to the size of a bound third strand. Therefore, at sites of CG or TA inversion within a polypurine/polypyrimidine domain, at most 3-5A of transverse distortion is required to accommodate H-bonding at the "other side" of the major groove. That much lateral distortion can be partially accommodated by the conformational freedom available to the deoxyribose backbone or a duplex. Preliminary modelling suggests that, in order to form a standard TAT or GGC Hoogsteen or reverse Hoogsteen triplex at such sites, distortion of the duplex binding site might also be required. Evidence suggests that at sites of CG or TA inversion in an antiparallel triple helix, local triplet formation gives rise to substantial distortion of the duplex. The present invention provides a group of modified nucleoside analogues which form a strong triple helix at the sites of TA or CG inversion, without the requirement for binding site distortion. Because of the biostability, tissue distribution, cellular uptake and cost to manufacture, the exploration and synthesis of novel nucleoside surrogates is extremely important for the progression and eventual commercialization of these novel compounds. These novel co pounds should be very useful in treatment of many disorders. For example, since identification of a retrovirus HIV-1 as the etiological agent of AIDS, an intense effort has been made to identify drugs for the treatment and prevention of this debilitating, lethal disease. Although a variety of purine and pyrimidine nucleoside drugs have been shown to have in vitro anti-HIV activity, only one, AZT, has become widely available for treatment of AIDS and approved by the FDA for treatment of advanced AIDS cases. While clinically useful in many settings, AZT is associated with considerable toxicity, especially in myelosuppression, leading to serious hemotological side effects. Additionally, AZT-resistant HIV strains have been isolated from AIDS patents. Because of the toxicity of currently used drugs, new drugs must be discovered for chemical intervention of HIV infection. A radical novel therapeutic approach, commonly referred to "antisense" is now emerging which promises safe and effective treatments for a broad range of viral diseases, including HIV infection. This new approach entails the use of polymeric agents designed to specifically bind and inactivate selected viral genetic sequences, and thereby halt the disease process. The preliminary findings using antisense treatments, however, have been controversial. The present invention, however, is based upon the premise of a new method of treatment; triple helix formation. In the present invention, novel reagents are designed and synthesized with the potential to selectively inhibit HIV by direct binding to proviral DNA rather than messenger RNA, thereby forming a colinear triple helix. This approach can also be used in treatment of a variety of other diseases and in modulations of genetic expression. SUMMARY OF THE INVENTION A object of the present invention is the provision of novel analogues of guanosine. A further object of the present invention is the introduction of 2' -deoxyformycin A, thioguanosine and other guanosine analogues into TFOs. An additional object of the present invention is a method for treatment of AIDS. A further object of the present invention is to use these modified TFOs for treatment of disease. An additional object of the present invention is modified TFOs for regulation of gene expression. Thus, in accomplishing the ' foregoing objects, there is provided in accordance with one aspect of the present invention a series of guanosine analogues, compounds numbers 4, 10a, 10b, 14, 19 and 29 (Figures 5-9). In addition, there are also the specific compounds which were intermediates in the synthesis of these analogues. In the preferred embodiment, the 2' -deoxyformycin A and other guanosine analogues are incorporated into TFOs for use in the treatment of a variety of diseases and in regulation of proteins, hormones, and gene expression. Other and further objectives, features and advantages will be apparent and the invention more readily understood from a reading of the following specification and by reference to the accompanying drawings forming a part thereof, wherein the examples of the presently preferred embodiments of the invention are given for the purpose of disclosure. DESCRIPTIQN OF THE DRAWINGS Figure 1 is a model explaining triplet H-bonding at sites of CG and TA inversion. Figure 2 is a model showing the binding of f ormycin as a G homologue at sites of CG inversion. Figure 3 shows Cu-Phenanthroline hyper sensitivity at sites of CG and TA inversion in anti-parallel triple helices . Figure 4 is the band shift analysis showing the effect of 2 ' -deoxyformycin (dF) substitution. Figure 5 is a schematic of the synthesis of 7-N- benzoylamino-3- [ 3- 0_- (β-cyanoethyl) - N , N - diisopropylaminophosphoramidite-5-0-dimethoxytrityl-2- deoxy-β-D-eryti-ro-pentof uranosyl]pyrazolo [ 4 , 3-d.] - pyrimidine. Figure 6 is a schematic of the synthesis of 2- isobutyrylamino-9-[3 - _- (β-cyanoethyl) -N,N- diisopropylaminophosphoramidite-5-0-dimethoxytrityl-2- deoxy-α-D-erythro-pentofuranosyl]purin-6(lH)-one and 2- isobutyr lamino-9- [3- p_- (β-cyanoethyl) -N,N- diisopropylaminophosphoramidite-5-o-dimethoxytrityl-2- deoxy-α-D-eryt_hro-pentofuranosyl]purine-6 (lfi) -thione. Figure 7 is a schematic of the synthesis of 2- isobutyryl * amino-6-isobutyrylthio-9- [3-0- ( β-cyanoethyl) - N , N-diisopropylaminophosphoramidite-5-o-dimethoxytr ity 1- 2-deoxy-β-β-erythro-pentofuranosyl]purine. Figure 8 is a schematic of the synthesis of 2- isobutyrylamino-9- [ 3-0.- (β-cyanoethyl) -N,N- diisopropylaminophosphoramidite-5-p.-dimethoxytrityl-2- deoxy-β-fi-ery t ro-pentof uranosyl ] pur ine-6 ( lfi) -thione . Figure 9 is a schematic of the synthesis of 2- isobutyrylamino-N 5 -isobutyry 1-7 - [ 3 -0- ( β-cyanoethyl ) -N , N- diisopropylaminophosphoramidite-5-0-dimethoxytrityl-2- deoxy-β-D-erytΛro-pentofuranosyl]pyrrolo[3,2-d]pyrimidin- 4(3H)-one. Figure 10 is a schematic of the synthesis of 7-N- benzoylamino-3-[2-0-methyl-3-0(β-cyanoethyl)-N,N-diiso- propylaminophosphoramidite-5-O-dimethoxytrityl-β-D- ribofuranosy1]pyrazolo-[4,3-d]pyrimidine. The drawings and figures are not necessarily to scale and certain features mentioned may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness. DETAILED DESCRIPTION OF THE INVENTION It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and the spirit of the invention. The term "TFO" or "triplex forming oligonucleotide" as used herein refers to the oligonucleotides of the present invention which are capable of forming a triplex by binding in the major groove with a duplex DNA structure. As used herein the term "major groove" refers to one of the grooves along the outer surface of the DNA helix which is formed because the sugar-phosphate backbone of the duplex DNA extends further from the axis than the bases do. The major groove is important for binding of regulator molecules to specific DNA sequences. The term "oligonucleotides" as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than 10. The exact size would depend on many factors including the specificity and binding affinity. In referring to "bases" herein the term includes both deoxyribonucleic acids and ribonucleic acids. The following abbreviations are used: "A" refers to adenine as well as its deoxyribose derivatives, "T" refers to thiamine as well as its deoxyribose derivatives, "G" refers to guanine as well as its deoxyribose derivative, "C" refers to cytosine as well as its deoxyribose derivative, "F" or "N" in sequences refers to guanosine analogues. Some examples of analogues are compounds 4, 10a, 10b, 14, 19, 29 and 35 (Figures 5-10). These compounds are called guanosine analogues because they can be used to replae guanosine in an oligonucleotide and will bind with C to form FCG Hoogsteen and reverse Hoogsteen triplets. One embodiment of the present invention are the compounds of Figure 5 which can be represented by the structure: wherein R, is H or -COC g H j ; R 2 is H or -COC 6 H 5 ; and R 3 is attached by a β linkage and is selected from the group consisting of wherein X is H or DMT and Y is H or These compounds of the present invention are synthesized by the process depicted in the schematic illustration represented in Figure 5. In the preferred embodiment compound 4 is synthesized. Another embodiment of the present invention are the compounds of Figure 6 which can be represented by the structure: wherein R 1 is 0 or S; R 2 is H or These compounds of the present invention are synthesized by the process depicted in the schematic illustration in Figure 6. Another embodiment of the present invention are the compounds of Figure 7 which can be represented by the structures: S-iBu wherein R, is TBDMS, DMT or H and R 2 is TBDMS, H or These compounds of the present invention are synthesized with the process depicted in the schematic illustration shown in Figure 7. Another alternative embodiment of the present invention are the compounds of Figure 8 which can be represented by the structure: These compounds of the present invention are synthesized by the process depicted in the schematic illustration of Figure 8. Another alternative embodiment of the present invention are the compounds of Figure 9 which can be represented by the structure: wherein , is H or (CH 3 ) 2 CH-CO-; R 2 is H or DMT; R 3 is H or These compounds of the present invention are synthesized by the process depicted in the schematic illustration of Figure 9. Another alternative embodiment of the present invention are compounds 23-26 (Figure 9) . A further embodiment of the present invention are the compounds of Figure 10 which can be represented by the structure: wherein R, is attached by a β linkage and is selected from the group consisting of: These compounds of the" present invention are synthesized by the process depicted in the schematic illustration represented in Figure 10. In the preferred embodiment, compound 35 is synthesized. In the preferred embodiment, there is a TFO comprising an oligonucleotide capable of forming a triple helix with a double-stranded DNA wherein at least one base of said oligonucleotide is replaced with at least one of the Compounds 4, 10a, 10b, 14, 19, 29 and 35 (Figures 5-10) . Additional embodiments of the present invention are the use of TFOs containing compounds of the present invention for the inhibition of HIV-1, and for altering the expression of genes selected from the group consisting of epidermal growth factor receptor, mouse insulin receptor, among others. A further embodiment of the present invention is a TFO containing a guanosine analogue. Specific embodiments of these TFOs are selected from the group consisting of SEQ. I.D. No. 1, SEQ. I.D. No. 2, SEQ. I.D. No. 3, SEQ. and I.D. No. 4. Another embodiment of the present invention is a TFO for treating AIDS comprising SEQ. I.D. No. 4. Another embodiment of the present invention is a triplex forming oligonucleotide for forming a colinear triplex with a target sequence in a duplex DNA. The TFO is comprised of a nucleotide sequence of at least about 20 nucleotides. The sequence includes G, T and guanosine analogues. In the TFO, G is used when the complementary location in the duplex DNA target possesses a G in the orienting (more purine rich) strand. Guanosine analogues are used instead of G when the duplex DNA target possesses a C in the orienting strand. T is used when the complementary location in the duplex DNA target is an AT base base. In general the TFO can be designed so as to form the above based triplets with either a net parallel or antiparallel TFO strand orientation with respect to the orienting strand of the duplex DNA target. However, the antiparallel TFO isomer is often favored for targets with a GC to AT base pair ratio substantially greater than one while the parallel isomer is often favored for targets with a GC to AT base pair ratio substantially less than one. Both TFO isomers can be equally favored for targets with a GC to AT base pair ratio near to one. In a preferred embodiment of the present invention there is a target sequence in a duplex DNA comprising a nucleotide sequence of at least about 20 nucleotides; said nucleotide sequence including G, T, and guanosine analogue, wherein G is used when the complementary location in the duplex DNA is a GC base pair, T is used en the complementary location in the duplex DNA is an _ιT base pair and guanosine analogue is used when there is a CG inversion within the duplex DNA target site; said sequence binding anti-parallel to the purine strand in the duplex DNA target site and said binding forming TAT, GGC and FCG Hoogsteen or reverse Hoogsteen triplets. The following examples are offered by way of illustration and are not intended to limit the invention in any manner. In examples, all percentages are by weight, if for solids and by volumes, if for liquids and all temperatures are in degrees Celsius unless otherwise noted. Example 1 Synthesis of 2'-deoxyformycin and its blocked DMT phosphoramidite derivative. A synthetic procedure to convert formycin to the corresponding 2•-deoxyribonucleoside (dF) , followed by activation to the corresponding blocked DMT- phosphoramidite was developed. Using commercially available formycin A(i) , 2 1 - deoxyformycin A can be prepared by a variety of known procedures. One method of synthesis of 2*-deoxyformycin A is described by T. C. Jain, et al., J. Org. Chem. 38:3179 (1973). This procedure involves reaction of 1 with acetoxyisobutyryl bromide, followed by ammonolysis and reductive dehalogenation. This approach produces a mixture of 2'-deoxyformycin A and the 3'-deoxy isomer, with the latter predominating by a ratio of more than 2:1. An alternative procedure is described by Rosowsky, et al., J. Med. Chem. 28:1096 (1985). This is a deoxygenation procedure and employs phenoxythiocarbonylation of the 2'-hydroxyl group of the corresponding 3*,5*-protected A. 3•,5*-Protection of 1. was accomplished by using l,3-dichloro-l,l,3,3- tetraisopropyldisiloxane, which in the presence of a proton acceptor gave the cyclic disiloxy derivative. Acylation of the 2"-hydroxyl of the disiloxy derivative with phenoxythiocarbonyl chloride, followed by reductive cleavage of the phenoxythiocarbonyl group with tri-n- butyltin hydride in the presence of a.a*- azobisisobutyronitrile gave the viable intermediate 7- amino-3-[3, 5- _- (1, 1, 3 , 3-tetraisopropyl-l, 3- disiloxanediyl-2-deoxy-β-D-ribof uranosyl) ] pyrazolo- [4,3- d]pyrimidine (2) . Example 2 Synthesis of 7-N.N'-Dibenzoylamino-3-r3.5-0- (1.1.3.3-tetraisopropyl-l.3-disiloxanediyl-2-deoxy-β-D- eryt ro-pentofuranosyl.1pyrazolor4.3-dipyrimidine (3.) To a cooled (0-5 e C) solution of 1.5 g of compound (2) (2.92 mmoles) in 25 ml anhydrous pyridine was added 1.36 ml benzoyl chlor^Je (11.7 mmoles) with stirring. After stirring the reaction mixture at room temperature for 2.5 h, it was partitioned between water and dichloromethane. The organic layer was separated, dried over anhydrous Na 2 S0 and then evaporated to dryness under reduced pressure. The residue was azeotroped with toluene (4 x 50 ml) to remove last traces of pyridine, and purified by chromatography over a silica gel column using a gradient of CH 2 C1 2 → MeOH (0-*l%) as the eluent. The homogeneous fractions were collected and evaporated under reduced pressure to yield 1.45 g (70.7%) of the title compound; mp 74-75 e C. IR (KBr) : υmax 1720 (C=0) , 3240 (NH) cm "1 ; UV (MeOH): λmax 230 nm (€24,000), 318 nm (€14,500); 1 H NMR (DMS0-d 6 ) : δ 0.98 {br s, 28 H, 2[(CH 3 ) 2 CH] 2 Si}, 2.40-2.90 (m, 2 H, C 2 ,H. and C 2 ,ϋ) , 3.7-4.0 (m, 3 H, C^.H and C 5 ,CH. 2 ) , 4.90 (q, 1 H, C 3 ,H) , 5.50 (dd, 1 H, C,,H) , 7.30-8.15 (m, 10 H, 2 COC^) , 9.07 (s, 1 H, C 5 H) and 12.0 (br s, 1 H, NH) . Anal. Calcd. for C^H^O^S^: C, 61.59; H, 6.75; N, 9.97. Found: C, 61.68; H, 6.81: N, 9.86. Example 3 Synthesis of 7-N-Benzoylamino-3-f2-deoxy-6-D- erythro-pentofuranosy1)pyrazolor4.3-d] yrimidine (6_) . To a solution of (3J (1.35 g, 1.96 mmoles) in anhydrous tetrahydrofuran (30 ml) was added tetra-n- butylammonium fluoride (8 ml, 1M solution in THF) at 0°C. After stirring the reaction mixture at room temperature for 2 h, the solvent was evaporated under reduced pressure and the residue was purified by chromatography over a silica gel column using a gradient of CH 2 C1 2 → MeOH (0→4%) as the eluent. The homogeneous fractions were collected and evaporated under reduced pressure to yield 0.46 g (68%) of analytically pure (j>) ; mp 250-251°C. IR (KBr) : umax 1690 (C=0) , 3100-3500 (OH, NH) cm "1 ; UV(MeOH) : λmax 248 nm (€14,500) , 320 nm (el3,700) ; 1 H NMR (DMS0-d 6 ) : S 2.00-2.20 (m, 1 H, C 2 ,B) , 2.60-2.80 (m, 1 H, C 2 „H) , 3.40-3.70 (m, 2 H, C 5 .CH 2 ) , 3.90 (m, 1 H, C 4 .fi) , 4.40 (d, 1 H, C j -E), 5.15 (br s, 2 H, 3' and 5 « OH), 5.50 (dd, 1 H, C^H), 7.50-8.15 (m, 5 H, COC^) , 8.75 (S, 1 H, C j H) , 11.80 (br s, 1 H, N,H) and 13.00 (br s, 1 H, N j fl) . Anal. Calcd. for C 17 H 17 N 5 0 4 • 1/4 H 2 0 : C, 56.74; H, 4.90; N, 19.46. Found: C, 56.93; H, 4.96; N, 19.35. Example 4 Synthesis of 7-N-Benzoylamino-3-f5-0- dimethoxvtritvl-2-deoxv-6-D-ervthro-pentofuranosvl.- pvrazolor4.3-dlpvrimidine (5_) . To a solution of (6 (0.4 g, 1.12 mmoles) in anhydrous pyridine (10 ml) was added 4,4•-dimethoxytrityl chloride (0.46 g, 1.35 mmoles) at room temperature with stirring. After stirring for 3 h, the reaction mixture was partitioned between CH 2 C1 2 and water (50 ml of each) . The organic layer was separated, dried over anhydrous Na 2 S0 4 and evaporated under reduced pressure. The residue was azeotroped with toluene (4 x 50 ml) to remove last -17- traces of pyridine, and purified by chromatography over a silica gel column using a gradient of CH 2 Cl 2 -»MeOH (0→2%) as the eluent. The homogeneous fractions were collected and evaporated under reduced pressure to yield 0.26 g (35.3%) of pure title compound; mp 107-108°C. IR (KBr) : υmax 1690 (C=0) , 3200-3400 (OH, NH) cm "1 ; UV (MeOH): λmax 238 nm (€31,700), 320 nm (el8,000); 1 H NMR (DMSO-d 6 ) : δ 2.05-2.30 and 2.75-2.95 (2m, 2 H, C 2 .H and C 2 „S) , 3.70 (S, 3 H, 0CH 3 ) , 3.71 (s, 3 H, 0CH. 3 ) , 3.98 (m, 2 H, C 5 ,CH. 2 ) , 4.40 (m, 1 H, C 4 ,H) , 5.20 (m, 1 H, C 3 .H) , 5.55 (dd, 1 H, C,,H), 6.70-8.30 (m, 18 H, COC^ and DMT), 8.65 (s, 1 H, Cg) , 11.85 (br s, 1 H, N,H) and 13.10 (br s, 1 H, N 7 H) . Example 5 Synthesis of 7-N-Benzoγlamino-3-f3-Q-.β-cyanoethyl)- N.N-diisopropylaminophosphoramidite-5-O-dimethoxytrityl- 2-deoxy-β-D-eryti * .ro-pentofuranosylJpyrazolof4.3- dlpvrimidine (4 . To a solution of (5_) (0.16 g, 0.2 mmoles) and N,N-diisopropylethylamine (0.14 ml, 0.83 mmoles) in anhydrous dichloromethane (3 ml) was added with stirring 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (56 μl, 0.25 mmoles) under an argon atmosphere. After 30 min an additional 45 μl of the phosphorylating agent was added. After stirring for an additional 15 min, the reaction mixture was diluted with 10% NEt 3 in EtOAc solution (50 ml) and washed with aqueous saturated NaHC0 3 solution (10 ml) . The aqueous layer was separated and reextracted with 10% NEt 3 -EtOAc. The combined organic layers were dried over anhydrous Na 2 S0 4 and evaporated to dryness under reduced pressure. A solution of the residue in 10% NEt 3 -EtOAc was passed through a silica gel column packed in CH 2 Cl 2 :EtOAc:NEt 3 (45:45:10) eluting with the same solvent system. The appropriate, homogeneous fractions were collected and evaporated under reduced pressure. The residue was dissolved in a small amount of CH 2 C1 2 (3 ml) and the product was precipitated by the addition to dry hexanes to yield 0.18 g (86.2%). 1 H NMR (CDC1 3 ) : δ 1.20 [m, 12 H, N(CMe 2 ) 2 ], 2.40-2.70 (m, 2 H, C 2 ,H and C 2 „fi) , 2.80-4.0 (m, 6 H, OCH 2 CH 2 CN, C 5 ,CH 2 ) , 3.75 *(s, 6 H, 2 OCfi 3 ), 4.35 (m, 1 H, C 4 .H), 4.75 (m, 1 H, C 3 ,fi) , 5.75 (q, 1 H, C,,H) , 6.50-8.20 (m, 18 H, COC^ and DMT), 8.64 and 8.65 (2 s, 1 H, C- j S of the isomers) , 12.20 (br s, l H, N 7 S); 31 P NMR (CDC1 3 ): δ 148.36. Anal. Calcd. for C 47 H 53 N 7 0 7 P. 1/2H 2 0: C, 65.11; H, 6.28; N, 11.31; P, 3.57. Found: C, 65.16, H, 6.32; N, 10.87; P, 3.70. Example 6 A New Synthesis of 2-Amino-9-.2-deoxy-α-D-ery thro- pentofuranosyl.purin-6.1H)-one (ji) . To a suspension of 2-amino-9-(2-deoxy-α-D-erytnro- pentofuranosyl)purine-6(lfi)-thione (2) [R. H. Iwamoto, E. M. Acton, and L. Goodman, J. Med. Chem. , 6, 684 (1963)] (2.0 g, 7.06 mmoles) in water (60 ml) were added NH 4 0H (30%, 20 ml), followed by H 2 0 2 (30%, 2.8 ml). After stirring at room temperature for 45 min, the reaction mixture was concentrated to half the original volume and allowed to stand at 25°C for an hour. The product that crystallized out from the aqueous solution was collected by filtration and the filtrate was neutralized with Dowex-50 (H + ) resin. The resin was removed by filtration, washed thoroughly with water (5 x 25 ml) and the combined filtrates were evaporated under reduced pressure to give a colorless solid. The solid was combined with the one obtained as above and crystallized from water to yield 1.62 g (86%) of the title compound, mp > 295 β C [Lit. mp > 300 β C, M. J. Robins and R. K. Robins, J. Org. Chem. , 34, 2160 (1969)]. IR (KBr) : υmax 1740 (C=0) , 3150, 3310, 3440 (OH, NH, NH 2 ) cm '1 ; UV: λmax (pHl) 254.5 n (€10,800); λmax (pH 7) 254 nm (el3,000); λmax (pH 11) 263 nm (€10,000); 1 H NMR (DMSO-d 6 ) : δ 2.20 (m, 1 H, C 2 ,H) 2.70 (m, 1 H, C 2 „ϋ) , 3.45 (t, 2 H, C 5 ,CH 2 ) , 4.05 (m, 1 H, C 4 .H) , 4.30 (m, 1 H, C 3 .fi) , 4.80 (t, 1 H, C 5 .OH) , 5.50 (d, 1 H, C 3 .OH) , 6.10 (dd, 1 H, J = 3.0 Hz, CH), 6.50 (s, 2 H, NH 2 ) , 8.0 (s, 1 H, CgH) , 10.64 (s, 1 H, N,H) • Anal. Calcd. for C 10 H 13 N 5 O 4 .l/3 H 2 0 : C, 43.95; H, 5.04; N, 25.63. Found: C, 43.97; H, 4.97; N, 25.58. Example 7 Synthesis of 2-Isobutyrylam.ino-9-.5-0- dimethqxytrityl- 2-deoxy-α-D-erytJ-ro-pentofuranosyl.- purin-6.lH.-one (la) . To a suspension of ≤. (1.8 g, 6.73 mmoles) in pyridine (30 ml) was added trimethylsilyl chloride (7.61 ml, 60 mmoles) at O'C. After stirring the reaction mixture for 2 h at room temperature, the reaction flask was cooled in an ice bath and isobutyryl chloride (0.85 ml, 8.07 mmoles) was added. The reaction was allowed to continue at room temperature for an additional 4 h, after which water (10 ml) was added, followed by CH 2 C1 2 (200 ml) . The organic layer was separated and evaporated under reduced pressure at 40°C. The resulting residue was azeotroped with toluene (5 x 25 ml) to remove the last traces of pyridine, and purified by chromatography over a silica gel column using MeOH:CH 2 Cl 2 (1:3, v/v) as the eluent. The homogeneous fractions were pooled and evaporated to yield 1.75 g (77%) of pure N 2 -isobutyry1-2'-deoxy-α-guanosine. A solution of the above N 2 -isobutyryl-2•-deoxy- α-guanosine (1.5 g, 4.45 mmoles) and dimethoxytrityl chloride (1.81 g, 5.34 mmoles) in pyridine (20 ml) was stirred at room temperature for 2.5 h. The reaction mixture was diluted with CH 2 C1 2 (200 ml) and the organic layer was washed with saturated aqueous sodium bicarbonate solution (2 x 50 ml) . The dried (over Na 2 S0 4 ) organic layer was evaporated under reduced pressure and the residue was purified on a silica gel column using a gradient of CH 2 C1 2 -+ MeOH (0-*4%) as the eluent. The homogeneous fractions were collected and evaporated under reduced pressure to yield 2.01 g (70.5%) of the pure title compound; mp 152-154°C (dec). IR (KBr) : υmax 1710 (C=0), 3100-3400 (OH, NH) cm *1 ; UV (MeOH): λmax 238 nm (€26,565), 262 nm (€14,600): H NMR (DMS0-d fi ) : δ 1.12 [d, 6 H, C(CH 3 ) 2 ], 2.30 - 2.45 (m, 1 H, C 2 ,E) , 2.80 (m, 1 H, C 2H 2) , 3.0-3.25 (m, 2 H, C^C^) " , 3.75 (s, 6 H, 2 OCH 3 ) , 4.31 (br S, 2 H, C^ϋ and C 4 .H) , 5.54 (d, 1 H, C^OH) , 6.27 (dd, 1 H, J = 4.0 HZ, C,,H), 6.89-7.41 (m, 13 H, DMT), 8.28 (s, 1 H, CgH), 11.67 (br S, 1 H, N,H) , 12.10 (br s, 1 H, NH) . Anal. Calcd. for C 35 B S7 N s 0 7 : C, 65.71; H, 5.83; N, 10.95. Found: C, 65.32; H, 5.94; N, 10.61. Example 8 Synthesis of 2-Isobutv * ι*-vlamino-9-r3-0-rB- cyanoethyl)- N.N-diisopropylaminophosphoramidite-5-O- dimethoxvtritvl-2-deoxv-α-D-ervt ro-pentofuranosvl1- p * urjn-6(lH)-one (lfia) . To a solution of Sa (0.75 g, 1.17 mmoles) and N,N-diisopropylethylamine (0.82 ml, 4.69 mmoles) in anhydrous dichloro ethane (4 ml) was added 2-cyanoethyl- N,N-diisopropylaminochlorophosphine (0.39 ml, 1.76 mmoles) under an argon atmosphere. After stirring for 30 min at room temperature, the reaction mixture was diluted with 10% NEt 3 in EtOAc solution (75 ml) . The organic phase was washed with saturated aqueous sodium bicarbonate solution (20 ml) and dried over anhydrous sodium sulfate. Evaporation of the solvent under reduced pressure and purification of the residue on a silica gel column using EtOAc:CH 2 C1 2 :NEt 3 (45:45:10) solution as the eluent gave 0.7lg (72.1%) of analytically pure product. 31 P NMR (CDC1 3 ) : δ 149.20 and 150.28; 1 H NMR (CDC1 3 ) : δ 1.0 - 1.40 [m, 21 H, N(CHCH 3 ) 2 and COCH(Cβ 3 ) 2 ], 3.80 (s, 6 H, 2 OCfl 3 ) , 6.30 (m, 1 H, C,.H), 8.12, 8.16 (2s, CgH of the isomers) . Anal. Calcd. for C 44 H 54 N 7 0 8 P. H 2 0 : C, 61.60; H, 6.58; N, 11.43; P, 3.61. Found: C, 61.26; H, 6.69; N, 11.47; P, 3.76. Example 9 Synthesis of 2-Isobutyrγlamino-9- 5-Q- dimethpxytrityl- 2-deoxy-α-D-eryt ro-pentofuranosγl.- purine-6 (1H)-thione (9b) . To a suspension of 2 (4.0 g, 14.12 mmoles) in pyridine (40 ml) was added trimethylsilyl chloride (17.1 ml, 140 mmoles) at 0 β C. After stirring the reaction mixture for 2 h at room temperature, the reaction flask was cooled in an ice bath and isobutyryl chloride (1.78 ml, 16.95 mmoles) was added. The reaction was allowed to continue at room temperature for an additional 3 h, after which water (40 ml) was added, followed by dichloromethane (200 ml) . The organic phase was separated, dried over Na 2 S0 4 and evaporated under reduced pressure at 40°C. The resulting residue was redissolved in methanol (50 ml) and evaporated to dryness under reduced pressure. The residue was dissolved in CH 2 C1 2 , filtered and the filtrate was used directly in the next step without further purification. Evaporation of the solvent gave a residue, which was dried by co-evaporation with anhydrous pyridine (2 x 20 ml) , redissolved the resulting residue in pyridine (40 ml) to which dimethoxytrityl chloride (4.88 g, 14 mmoles) was added. After stirring the reaction mixture at room temperature for 3.5 h, the mixture was diluted with water (50 ml) . The aqueous mixture was extracted with CH 2 C1 2 (3 x 100 ml) and the combined organic phase was dried over anhydrous Na 2 S0 4 . Evaporation of the solvent at reduced pressure gave a residue, which was purified on a silica gel column using a gradient of CH 2 C1 2 → MeOH (0 → 5%) as the eluent. The homogeneous fractions were collected and evaporated under reduced pressure to yield 6.02 g (65%, for 2 steps) of pure title compound; mp 139-141°C (dec.) . IR (KBr) : umax 1690 (C=0) , 3100-3400 (OH, NH) cm "1 ; UV (MeOH): λmax 234 nm (€22,300), 332 nm (e20,300); 1 H NMR (DMSO-d 6 ) : δ 1.13 [d, 6 H, C(Cfi 3 ) 2 ], 2.30 - 2.45 (m, 1 H, C 2 ,H) , 2.80 (m, 2 H, C^fi and CO-CH<) , 3.0 - 3.30 (m, 2 H, C j .CHg), 3.74 (s, 6 H, 2 0CH 3 ) , 4.33 (br s, 2 H, C^.H and C 4 .H) , 5.53 (d, 1 H, C j .OH) , 6.29 (dd, 1 H, J - 3.8 Hz, C,,H), 6.89-7.37 (m, 13 H, DMT), 8.42 (s, 1 H, CgH), 12.14 (s, 1 H, N,H) r 12.64 (br s, 1 H, NH.) . Anal. Calcd. for C 35 H 37 N 5 0 6 S. 1/3 CH 3 0H : C, 63.68; H, 5.79; N, 10.51, S, 4.81. Found: C, 63.34; H, 5.59; N, 10.36; S, 4.64 Example 10 Synthesis of 2-Isobutvrvlamino-9-r3-0-f β- cvanoethvl . - N . N-diisopropvlaminophosphoramidite-5-O- dimethoxvtritvl-2-deoxv-α-D-ervthro-pentof uranosvl 1 - wine-e . iH. -thipne (10b,) . To a solution of 9b (0.65 g, 1 mmole) and N,N-diisopropylethylamine (0.7 ml, 4 mmoles) in anhydrous dichloromethane (8 ml) was added 2-cyanoethyl- N,N-diisopropylaminochlorophosphine (0.29 l, 1.3 mmoles) under an argon atmosphere. After stirring for 20 min at room temperature an additional 0.29 ml of phosphorylating agent was added, stirred for further 30 min and the reaction was terminated by the addition of anhydrous methanol (20 μl) . After stirring for 5 min, the reaction mixture was partitioned between 10% NEt 3 in EtOAc and saturated aqueous NaHC0 3 solution (50 ml) . The organic phase was separated, dried over anhydrous Na 2 S0 4 and evaporated to dryness under reduced pressure. The resulting residue was purified on a silica gel column using EtOAc:CH 2 C1 2 :NEt 3 (45:45:10) solution as the eluent to give 0.678 g (79.5%) of the title compound as colorless amorphous solid. P NMR (CDC1 3 ) : δ 149.15 and 150.6; 1 H NMR (CDC1 3 ) : δ 0.9 - 1.4 [m, 12 H, N(CHCH 3 ) 2 and COCH(CH 3 ) 2 ]. 3.80 (s, 6 H, 20CH 3 ) , 6.25 (m, 1 H, C V H) , 8.23 and 8.26 (2s, CgH of the isomers) . Anal. Calcd. for C^H^NyO f PS : C, 61.74; H, 6.36; N, 11.45; P, 3.62. Found: C, 61.50; H, 6.59; N, 11.12; P, 3.71. Example 11 Synthesis of 3' .5'-Di-O-(t-butyl.dimethylsilyl-2'- deoxy-6-thioσuanosine {12,) • To a suspension of 2*-deoxy-6-thioguanosine (11) [R. H. Iwa oto, E. M. Acton and L. Goodman, J. Med. Chem. , 6, 684 (1963)] (1.8 g, 6.3 mmoles) in anhydrous N,N-dimethyl-formamide (25 ml) was added t-butyldimethylsilyl chloride (3.35 g, 22 mmoles), followed by dry imidazole (2.98 g, 41.9 mmoles). The reaction mixture was protected from moisture and stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure at 40°C to give a white solid. The solid was purified by chromatography over a silica gel column using a gradient of CH 2 C1 2 → MeOH (0 → 5%) as the eluent. The appropriate homogeneous fractions were pooled and evaporated under reduced pressure to yield 2.4 g (73.6%) of pure title compound; mp > 300°C (dec). IR (KBr) : υmax 1257 (C=S) , 3100-3400 (NH, NH 2 ) cm "1 ; UV (MeOH) : λmax 226 nm (€5,500), 270 nm (€3,500), 344 nm (€11,800); 1 H NMR (DMSO-d 6 ) : δ 0.07 [d, 6 H, t-Bu-Si(Cg 3 ) 2 ], 0.88 (d, 18 H, t-Bu-SiMe 2 ) , 2.20-2.73 (m, 2 H, C 2 .H and C 2 „H) , 3.60 - 3.85 (m, 3 H, C 4 .H and C 5 .CH 2 ) , 4.48 (d, 1 H, C 3I H) , 6.10 (t, 1 H, C,,H) , 6.83 (s, 2 H, NH 2 ) , 8.0 (S, 1 H, CgH) and 11.97 (S, 1 H, N,H) . Anal. Calcd. for C 22 H 41 N 5 0 3 SSi 2 : C, 51.61; H, 8.07; N, 13.68; S, 6.26; Si, 10.97. Found: C, 51.36; H, 8.05; N, 13.65; S, 6.27; Si, 10.99. Example 12 Synthesis of 2-Isobutyrylamino-6-isobutyrylthio-9- f3.5-di-0- (t-butyl)dimethγlsilyl-2-deoxy-β-D-eryt ro- pentofuranosyl1purine (H) . To an ice-cold solution of 12. (2.2 g, 4.2 mmoles) in anhydrous pyridine (100 ml) was added isobutyryl chloride (1.37 ml, 12.8 mmoles) . The reaction mixture was stirred at room temperature for 3 h before it was poured into saturated aqueous sodium bicarbonate solution (300 ml) containing crushed ice (100 g) and ethyl acetate (400 ml) . The aqueous mixture was stirred for 15 min. The organic phase was separated, washed with water (2 x 50 ml) and dried over anhydrous sodium sulfate. Evaporation of the solvent under reduced pressure gave a residue, which was co-evaporated with toluene (3 x 20 ml) to remove last traces of pyridine. The residual pale yellow oil was purified by chromatography over silica gel using 2% MeOH in CH 2 C1 2 as the eluent to give 2.1 g (75%) of pure title compound; mp 85°C. IR (KBr) : υmax 1690 (C=0) , 3100-3400 (NH)cm '1 ; UV (MeOH): λmax 226 n (€9,700), 332 nm (€11,500); 'H NMR (DMSO-d 6 ) : δ 0.08 [d, 6 H, * fc-Bu-Si(CH 3 ) 2 ], 0.88 (d, 18 H, £-Bu-SiMe 2 ) , 1.14 [d, 12 H, 2CO-CH(CH 3 ) 2 ], 2.28 - 2.89 (m, 3 H, C 2 .H, C 2 „H and CHMe 2 ) , 3.61 - 3.89 (m, 3 H, C 4 .H and CJ.CH-J) , 4.52 (s, 1 H, C^H), 6.20 (t, 1 H, C j .II) , 8.38 (s, l H, C g H) and 11.93 (br s, 1 H, NH). Exa ple 13 Synthesis of 2-Isobutyrylamino-6-isobutyrylthio- 9-.2-deoxy-β-D-er thro-pentofuranosyl)purine (16) . To a solution of 13 (2.1 g, 3.6 mmoles) in anhydrous tetrahydrofuran (25 ml) was added tetra-n-butylammonium fluoride (17.5 ml, 17.5 mmoles, 1M solution in THF) at 0°C. After stirring at room temperature for 3 h, the solvent was evaporated under reduced pressure and the residual brown colored solid was purified by chromatography over a silica gel column using a gradient of CH 2 C1 2 → MeOH (0→10%) as the eluent. The homogeneous fractions were collected and evaporated under reduced pressure to give 1.15 g (84.2%) of the title compound; mp > 300°C. IR (KBr) : Umax 1700 (C=0) , 3100-3600 (NH, OH) cm "1 ; UV (MeOH): λmax 232 nm (€8,600), 246 nm (ell,500), 294 nm (€7,400); 1 H NMR (DMSO-d 6 ) : δ 1.12 [d, 12 H, 2COCH(CH 3 ) 2 ], 2.28 - 2.96 (m, 3 H, C 2 ,fi, C 2 „H and CHMe 2 ) , 3.53 - 3.86 (m, 3 H, C 4 .H and C 5 ,CH 2 ) , 4.43 (s, 1 H, C 3 ,fi) , 4.94 (t, 1 H, C 5 ,Ofl) , 5.36 (d, 1 H, C j .OH.) , 6.35 (t, 1 H, C H), 8.61 (s, 1 H, CgH) and 10.63 (s, 1 H, NH) . Example 14 Synthesis of 2-Isobutyrylamino-6-isobutyrylthio-9- .5-0-dimethoxytrityl-2-deoxy-β-D-erythro-pentofuranosyl)- purine (1.) . Compound 16. (1.0 g, 2.36 mmoles) was dried by repeated co-evaporation with anhydrous pyridine (5 x 20 ml) , and redissolved in dry pyridine (10 ml) , to which was added 4,4•-dimethoxytrityl chloride (1.15 g, 3.4 mmoles). After stirring for 2.5 h at room temperature, the reaction mixture was diluted with dichloromethane (200 ml) . The organic phase was washed sequentially with water (100 ml) , saturated NaHC0 3 solution (100 ml) and then with water (100 ml) , before it was dried over anhydrous Na 2 S0 4 . The solvent was evaporated under reduced pressure and the residue was purified by chromatography over a silica gel column using 2% MeOH in CH 2 C1 2 as the eluent. The homogeneous fractions were pooled and evaporated to dryness to give a white solid. The solid was dissolved in minimum volume of CH 2 C1 2 (- 5 ml) and reprecipitated by the addition to pentane (200 ml) to yield 0.15 g (85%) of the pure title compound, mp 108 β C (dec). IR (KBr) : υmax 1700 (C=0) , 3200-3600 (NH, OH) cm "1 ; UV (MeOH): λmax 236 nm (€15,700), 296 nm (€6,400); 1 H NMR (DMSO-d 6 ) : δ 1.09 [q, 12 H, 2 COCH(CH 3 ) 2 ], 2.30 - 2.90 (m, 4 H,. C 2 .H, C 2M fi, 2 CHMe 2 ) , 3.70 (S, 6 H, 2 OCH 3 ) , 3.98 (d, 1 H, C 4 ,H) , 4.55 (m, 1 H, C j .H) , 5.33 (d, 1 H, C^.C-H) , 6.40 (t, 1 H, .H) , 6.70 - 7.28 (m, 13 H, DMT), 8.49 (s, 1 H, C g H) and 10.56 (s, 1 H, NH) • Example 15 Synthesis of 2-Isobutyrylamino-6-isobutyrylthio-9- r3-0- (6-cvanoethvl.-N.N-diisopropvlaminophosphorami- dite-5-O-dimethoxvtritvl-2-deoxv-B-D-er hro-pento- uranosvl1purine (1£) . To a solution of 15 (0.30 g, 0.42 mmoles) and N,N-diisopropylethylamine (0.29 ml, 1.68 mmoles) in anhydrous dichloromethane (5 ml) was added with stirring 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.19 ml, 0.84 mmoles) under an argon atmosphere. The reaction mixture was stirred at room temperature for 1 h, after which ethyl acetate (45 ml) and NEt 3 (5 ml) were added. The mixture was partitioned between cold (0 - 5°C) saturated aqueous NaHC0 3 solution (50 ml) . The organic phase was dried over anhydrous Na 2 S0 4 and evaporated under reduced pressure. The residual oil was purified by chromatography over a silica gel column using EtOAc:CH 2 Cl 2 :NEt 3 (45:45:10, v/v) as the eluent. The homogeneous fractions were combined and evaporated under reduced pressure to yield 0.18 g (47%) of the title compound as amorphous white solid. 1 H NMR (DMSO-d 6 ) : «5 1.34 [m, 24 H, 2COCH(CH 3 ) 2 and NCH(CH 3 ) 2 ], 2.5 - 4.0 (m, 8 H, C 2 .fl, C 2 „H, OCH 2 CH 2 CN, 2COCfi) , 3.71 (s, 6 H, 2 OCH 3 ) , 3.88 - 4.12 (m, 3 H, C 4 .H and C 5 .CH 2 ) , 4.83 (m, 1 H, C 3 .H) , 6.43 (t, 1 H, C,,H) , 6.69 - 7.29 (m, 13 H, DMT), 8.52 (s, 1 H, C g H) and 10.5 (br s, 1 H, NH) ; 31 P NMR (DMSO-d 6 ) : δ 134 and 144. Example 16 Synthesis of 2-Isobutyrylamino-9-.2-deoxy-β-D- e tftro-pentofuranosyl)purjne-6(IB)-th-pne (13.) • To a suspension of 2*-deoxy-6-thioguanosine (12) [N. B. Hanna, K. Ramasa y, R. K. Robins, and G. R. Revankar, J. Heterocycl . Chem. , 25, 1899 (1988)] (2.0 g, 7.1 mmoles) in anhydrous pyridine (25 ml) was added trimethylsilyl chloride (10.8 ml, 71 mmoles) at 0°C. After stirring the reaction mixture for 2 h at room temperature, the reaction flask was cooled in an ice bath and isobutyryl chloride (0.91 ml, 8.6 mmoles) was added. The reaction was allowed to continue at 0°C for 30 min and for 3 h at room temperature. Water (40 ml) was added to the reaction mixture, followed by dichloromethane (200 ml) . After shaking for 5 min, the organic layer was separated and evaporated under reduced pressure at 40°C. The resulting residue was azeotroped with toluene (5 x 25 ml) to remove last traces of pyridine, and purified by chromatography over a silica gel column using 5% MeOH in CH 2 C1 2 as the eluent. The homogeneous fractions were combined and evaporated to yield 1.6 g (63%) of the title compound, mp > 240°C (dec). IR (KBr) : υmax 1290 (C=S) , 1690 (C=0) , 3150-3250 (NH, OH) cm *1 ; UV (MeOH): λmax 232 nm (€8,100), 330 nm (€10,700); 1 H NMR (DMSO-d 6 ) : δ 1.14 [d, 6 H, CH(CH 3 ) 2 ], 2.24-2.86 (m, 3 H, C 2 .H, C 2H H, CH<) , 3.53 (m, 2 H, C 5 .CH 2 ) , 3.85 (m, 1 H, C 4 .H) , 4.38 (s, 1 H, C J .H) , 4.98 (t, 1 H, C 5 .OH) , 5.33 (t, 1 H, C 3 ,OH) , 6.21 (t, 1 H, C,,H) , 8.42 (s, 1 H, C g H), 12.0 (br s, 1 H, N,H) and 13.30 (br s, 1 H, NHCO) . Anal. Calcd. for C 14 H 19 N 5 0 4 S.H 2 0: C,45.27; H,5.69; N,18.85; S,8.63. Found: C,45.46; H,5.41; N,18.54; S,8.97. Example 17 Synthesis of 2-Isobutyrylamino-9-.5-0- dimethoxytrityl-2-deoxy-β-D-erytJιro-pentofuranosyl.- purine-6f1H.-thione (20) . A solution of 18. (1.5 g, 4.2 mmoles) in anhydrous pyridine was evaporated several times (4 x 25 ml) under diminished pressure to obtain a dry residue, which was redissolved in dry pyridine (20 ml) . To the pyridine solution was added 4,4•-dimethoxytrityl chloride (1.53 g, 4.5 mmoles) and stirred at room temperature for 4 h. The reaction mixture was partitioned between CH 2 C1 2 (200 ml) and water (200 ml) . The organic layer was separated, dried over anhydrous Na 2 S0 4 and evaporated under reduced pressure. The residue was azeotroped with toluene (4 x 50 ml) to remove last traces of pyridine, and purified by chromatography over a silica gel column using a gradient of CH 2 C1 2 → MeOH (0 → 4%) as the eluent. The homogeneous fractions were pooled and evaporated under reduced pressure to give a solid. The white solid was dissolved in minimum volume of CH 2 C1 2 (3 ml) and reprecipitated by the addition to pentane (-100 ml) to yield 1.12 g (67%) of the title compound; mp 146 e C (dec). IR (KBr) : υmax 1250 (C=S) , 1690 (C=0) , 3100-3600 (NH, OH) ; UV (MeOH) : λmax 236 nm (€17,700), 286 nm (€5,200), 332 nm (€14,300); 1 H NMR (DMSO-d 6 ) : δ 1.14 [d, 6 H, CH(CH 3 ) 2 ], 2.15 - 2.86 (m, 3 H, C 2 ,H, C 2 „H and CH) , 3.72 (s, 6 H, 2 OCH 3 ) , 3 .96 (m, 1 H, C 4 .H) , 4.43 (t, 1 H, C 3 .H) , 5.37 (d, 1 H, C 3 .OH) , 6.27 (t, 1 H, C V H) , 6.76 - 7. 30 (m, 13 H, DMT) , 8.90 (s , 1 H, C g H) , 11.93 (s, 1 H, NH) and 13.43 (s, 1 H, NHCO-) . Anal . Calcd. for C 35 H 37 N 5 0 6 S . 1/2 CH 3 OH: C, 63 .47 ; H, 5.85 ; N, 10.42 ; S, 4.77. Found: C, 63.20; H, 5.69 ; N, 10.33 ; S , 4 .78. Example 18 Synthesis of 2-Isobutyrylamino-9-r3-Q-(β- cyanoethyl)-N.N-diisopropylaminophosphoramidite-5-O- dimethoxytritγl-2-deoxy-β-D-eryti.ro-pentofuranosyl]- purine-6 iH)-thione fl9.. To a solution of 2J> (0.35 g, 0.53 mmoles) and N,N-diisopropylethylamine (0.37 ml, 2.1 mmoles) in anhydrous dichloromethane (5 ml) was added with stirring 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.25 ml, 1.06 mmoles) under an argon atmosphere. The reaction mixture was stirred at room temperature for 50 min. Ethyl acetate (45 ml) and triethylamine (5 ml) were added to the reaction mixture and the resulting solution was partitioned between ice-cold saturated aqueous NaHC0 3 solution (50 ml) . The organic phase was separated, dried over anhydrous Na 2 S0 4 and evaporated under reduced pressure to give an oil. The residual oil was dissolved in CH 2 C1 2 (3 ml) and purified by chromatography on a silica gel column using EtOAc:CH 2 C1 2 :NEt 3 (45:45:10, v/v) as the eluent. The appropriate homogeneous fractions were pooled and evaporated to give 0.43 g (94%) of 19. as amorphous white solid. 31 P NMR(CDC1 3 ) : δ 148.01 and 149.08; 1 H NMR (CDC1 3 ) : δ 0.84-1.35 [m, 18 H, CH(CH 3 ) 2 and 2 NCH(Cfi 3 ) 2 ] , 2.5-4.0 (m, 8 H, C 2 .H, C 2 „H, 2CH- and OCH 2 CH 2 CN) , 3.72 (d, 6 H, 20CH 3) , 4.1-4.85 (m, 3 H, C 4 .H and C 51 CH 2 ) , 6 « 18 (t, 1 H, C,,H) , 6.73-7.46 (m, 13 H, DMT) , 7.90 (d, 1 H, C g H), 8.13 (d, 1 H, NH) and 8.55 (s, 1 H, NH) • Anal. Calcd. for C 44 H 54 N 7 0 7 SP. 1.5 H 2 0: C,59.85; H,6.50; N,11.10; S,3.36; P,3.50. Found: C,60.01; H,6.79; N,11.23; S,3.26, P,4.01. Example 19 Synthesis of 2-Isobutyrylamino-7-β-D-ribofuranosyl- pvrrolor3.2-dlpvrimidin-4(3H.5H.-one (21) . To a suspension of 2-amino-7-β-D-ribofuranosyl- pyrrolo-[3,2-d]pyrimidin-4(3H,5H)-one (21) [N. S. Girgis, M. A. Michael, D. F. Smee, H. A. Alaghamandan, R. K. Robins and H. B. Cotta , J. Med. Chem. , 33, 2750 (1990)] (2.0 g, 7.1 mmoles) in anhydrous pyridine (20 ml) was added trimethylsilyl chloride (9.0 ml, 71 mmoles) at 0°C. After stirring the reaction mixture for 2 h at room temperature, the reaction flask was cooled in the ice-bath and isobutyryl chloride (0.90 ml, 8.5 mmoles) was added. The reaction was allowed to continue at room temperature for an additional 4 h, after which cold water (20 ml) was added, followed by CH 2 C1 2 (150 ml) . The organic phase was separated and evaporated under reduced pressure at 40°C. The resulting residue was azeotroped with toluene (4 x 25 ml) to remove last traces of pyridine, and triturated with dry CH 2 C1 2 . The colorless solid that separated was collected by filtration and dried. The filtrate which contains a small amount of the product was chromatographed on a silica gel column using 5% MeOH in CH 2 C1 2 as the eluent. The homogeneous fractions were collected, evaporated to dryness in vac o and combined with the above product to yield 1.90 g (76%) of the title compound; mp 222-224 e C. IR (KBr) : υmax 1680 (C=0), 3050-3500 (OH, NH) cm *1 ; UV (MeOH): λmax 246 nm (€15,400), 274 nm (el0,700); 1 H NMR (DMS0-d 6 ) : δ 1.10 [d, 6 H, C(CH 3 ) 2 ], 2.75 (m, 1 H, COCH) , 3.30 - 3.80 (m, 3 H, C 4 .H, C 5 ,CH 2 ), 3.90 (t, 1 H, C 3 .H), 4.09 (t, 1 H, C 2 .H) , 4.81 (d, ' l H, J = 6.0 Hz, CpH), 7.42 (d, 1 H, J = 2.8 Hz, Cg) , 11.35 (br s, 1 H, N 5 H) and 11.98 (br s, 2 H, N j H and NH-iBu). Anal. Calcd. for C 15 H 20 N 4 O 6 . 1/2 MeOH : C, 50.54; H, 6.02; N, 15.21. Found: C, 50.81; H, 5.70; N, 14.83. Example 20 Synthesis of 2-Isobutvrvlamino-7-r3.5-O-fl.l.3.3- tetraisopropvl-1.3-disiloxanedivl)-B-D-ribofuranosyll- pyrrolor .2-d ~ pyrimidin-4f3H.5H)-one (23) . To a solution of 22 (2.68 g, 7.61 mmoles) in anhydrous pyridine (30 ml) * was added 1,3-dichloro-l,1,3,3,-tetraisopropyldisiloxane (2.91 ml, 9.13 mmoles). After stirring the reaction mixture for 2.5 h at room temperature, it was partitioned between dichloromethane (100 ml) and water (100 ml). The organic phase was separated, dried over anhydrous Na 2 S0 4 and evaporated under reduced pressure. The residue was purified by chromatography over a silica gel column using 2% MeOH in dichloromethane as the eluent. The appropriate homogeneous fractions were pooled, solvent evaporated under reduced pressure to yield 3.07 g (67.8%) of the title compound, mp 148-150 β C. IR (KBr) : 1680 (C=0) , 3000 - 3500 (OH, NH) cm '1 ; UV (MeOH): λmax 244 nm (€30,400), 276 nm (€12,600); 1H NMR (DMSO-d 6 ) : δ 0.90 - 1.20 [m, 34 H, COCH(CH 3 ) 2 , and 0-Si(CHMe 2 ) 2 -0-Si(CHMe 2 ) 2 ], 2.75 (m, 1 H, COCHMe 2 ), 3.75-4.30 (m, 5 H, C 2 .H, C^H, C 4 ,H and C 5 ,CH 2 ), 4.92 (d, 1 H, J - 2.6 Hz, C H) , 7.28 (s, 1 H, &) , 11.40 (s, 1 H, N 5 H and 12.00 (br s, 2 H, N j H and NH-iBu) . Anal. Calcd. for C 2r iA V i 0 J 8i 2 . 1/3 MeOH : C, 54.22; H, 7.88; N, 9.25. Found: c, 54.62; H, 7.93; N, 8.83. Example 21 Synthesis of 2-Isobutyrylamino-7-r3.5-Q-f1.1.3.3- tetraisopropyl-1.3-disiloxanediyl)-2-phenoxythio- carbonyl-β-D-ribofuranosyl pyrrolor3.2-d'pγrimidin- 4(3H)-one (26) . To a suspension of 22 (0.80 g, 1.34 mmoles) and 4-dimethylaminopyridine (0.33 g, 2.7 mmoles) in anhydrous acetonitrile (10 ml) was added phenoxythiocarbonyl chloride (0.20 ml, 1.47 mmoles) under an argon atmosphere. After stirring at room temperature for 6 h, the reaction mixture was diluted with CH 2 C1 2 (75 ml) . The organic phase was separated, washed with water (2 x 25 ml) and dried over anhydrous Na 2 S0 4 . The solvent was evaporated under reduced pressure. The residue thus obtained was purified by chromatography over silica gel column using 1.5% MeOH in CH 2 C1 2 as the eluent. The appropriate homogeneous fractions were pooled and evaporated to yield 0.71 g (72.5%) of the title compound, mp 98-99 e C. UV (MeOH): λmax 262 nm (€26,000), 332 nm (€12,500); 1 H NMR (DMSO-d 6 ) : 0.80 - 1.30 [m, 34 H, COCH(CH 3 ) 2 and 0-Si(CHMe 2 ) 2 -0-Si(CHMe) 2 ], 2.80 (m, 1 H, COCHMe.,) . 3.80 - 4.20 (m, 4 H, C^.H, C 4 ,H and C 5 ,CH 2 ) , 4.92 (br S, 1 H, C 2 .H), 5.21 (d, 1 H, J = 3.8 Hz, C,,H) , 7.10 - 7.60 (m, 5 H, OC^), 8.03 (s, 1 H, CgH), 11.79 (br s, 1 H, N-sH) and 12.24 (br s, 2 H, N^ and NH-iBu). Example 22 Synthesis of 2-Isobutvrvlamino-7-r3.5-0-(1.1.3.3- tetraisopropvl-1.3-disiloxanedivl)-2-deoxv-β-D-ervtJιro- pentofuranosvllPvrrolor3.2-d.pvrimidin-4(3H.5H.-one (25) . To a solution of 6 (0.65 g, 0.845 mmoles) and α,α,-azobisisobutyronitrile (50 mg) in anhydrous toluene (25 ml) was added tri-n-butyltin hydride (1.36 ml, 5.1 mmoles) under an argon atmosphere. The mixture was heated with stirring at 65°C for 2.5 h, after which the solvent was evaporated to dryness under reduced pressure. The residue thus obtained was purified by chromatography over a silica gel column using 2% MeOH in CH 2 C1 2 as the eluent. The appropriate homogeneous fractions were collected and evaporated to yield 0.30 g (61.2%) of the ' pure title compound, mp 93-95°C. IR (KBr) : υmax 1680 (C=0), 3100-3500 (NH) cm "1 ; UV (MeOH): λmax 248 nm (€24,200), 282 nm (€10,600); 1 H NMR (DMSO-d 6 ) : δ 0.80 - 1.20 [m, 34 H, COCH(Cfi 3 ) 2 and 0-Si(CHMe 2 ) 2 -o-Si(CHMe 2 ) 2 ] , 2.75 - 3.10 (m, 3 H, C0CHMe 2 , C 2 .H and C 2 „H) , 3.75 - 4.30 (m, 4 H, Cj.H, C 4 .H and C 5 ,CH 2 ) , 4.92 (dd, 1 H, C,,H) , 7.24 (S, 1 H, C^) , 11.41 (s, 1 H, N- j H) and 11.93 (s, 1 H, NH-iBu). Anal. Calcd. for C^^N^Si^MeOH : C, 55.06; H, 8.24; N, 9.16. Found: C, 55.36; H, 8.06; N, 8.46. Selective alkylation of a solution of 2J5 in dry acetonitrile with isobutyryl chloride in the presence of NaH will give the N-5 isobutyrylated derivative (24.) . When the diisobutyryl derivative (24) is treated with a molar solution of tetra-n-butylammonium flouride in THF, deprotection of the 3',5•-hydroxyl groups will occur to give 2-isobutyrylamino-N 5 -isobutyryl-7-β-fi-erythro- pentofuranosyl)pyrrolo[3,2-d]pyrimidin-4(3H)-one (22) . Selective protection of the 5'-hydroxyl group of 22 with 4,4•-dimethoxytrityl ether will follow the conventional protocol to obtain the 5'-ODMT derivative (2S) . The 3'-hydroxyl group of 28 will be converted into the phosphoramidite (22) by the reaction with 2-cyanoethyl- N,N-diisopropylchlorophosphoramidite in the presence of diisopropylethylamine in anhydrous dichloromethane. Example 23 Stressed triolet formation of sites of TA and GC inversion. In normal anti-parallel triplex formation the TFO binds to duplex DNA by means of a GGC or TAT triplet base within the major helix groove. In this class of triple helix structure the third strand selectively binds anti¬ parallel with respect to the more purine duplex strand. However, occasionally there are sites within these triplexes where the purine rich strand is interrupted by pyrimidine. Such a disruption constitutes the formation of an altered binding site and is related to the polypurine reference isomer by- inversion about a local base pair diad. Preliminary molecular modeling suggested that T and G bases in the third strand can form a TAT and GGC triplet at sites of TA and CG inversion but only upon substantial distortion of the duplex and TFO backbone. In that instance duplex sites of TA and CG inversion develop a locally altered duplex structure of a kind which could be recognized by a structured specific probe. To measure this altered duplex structure mapping of the chemical cleavage which results from binding of copper phenanthroline, (Cu(0P) 2 ) a duplex DNA intercalator was used as a structural probe. Copper phenanthroline has the capacity to cleave DNA near its binding site. Mapping analysis of triplexes which form at two sites within the EGFR promoter region were examined. As seen in Figure 3 upon binding of the corresponding anti-parallel TFO, the duplex develops high Cu(OP) 2 sensitivity relative to uncomplexed DNA only at sites of TA or CG inversion. The Cu(OP) 2 is generally excluded from binding elsewhere within the triple helix as would be expected from simple competition between the -35- two classes of bound ligand. The EGFR sites are shown in Table 1. Table 1 shows a detailed map of the 3'A-EG36ap and 3'A-EG4ap complexes. Controlled TFOs 3'A-HIV38p and 3'AEG31ap are also shown. This is a detailed map of the EGFR promoter region near -352 from the start codon. The low resolution structure of the 3'AEG4ap and 3'AEG36ap triplex is presented. Bracketed numbers refer to distance from the radio-labeled Hind III terminus shown in Figure 3. Controlled TFOs 3'-AHIV38ap and 3'A-EG31ap do not bind. Sites are underlined where a polypurine/polypyrimidine stretch is interrupted by TA or CG inversions within the triplex binding site. Note that in both complexes Cu(0P) 2 hypersensitivity is induced at the inversion sites by site selective triplex formation. This effect was not detected in the uncomplexed duplex or upon adding control oligonucleotides. Although this mapping data cannot define the details of distortion which is detected, it does provide evidence of hypersensitivity and suggests that the bound TFO may not lay passively as a spacer over such sites. Instead, the inversion site forms triplet H bonds which require substantial compensatory distortion of the duplex binding site. Local narrowing of the major groove necessarily SUBSTITUTESHEET -35a- SUBSTITUTE SHEET -36- accompanied by expansion of the minor groove presents a specific structural possibility. Example 24 Model To Explain Triplet Formation At Sites of TA and CG Inversion. One structural hypothesis to explain the -stretched triplet formation at sites of TA and GC inversion is presented in Figure 1 which is based upon a reverse Hoogsteen/anti(RH/a) model for a stable triplet formation within adjoining polypurine runs. In Figure 1 the triplet schemes have been presented in a schematic form to emphasize symmetry and H bonding relationships. . In the process of standard GGC triplet formation, a third strand guanosine donates two H-bonds to G of the underlying duplex. Similarly, T donates one and accepts one H-bond from A in the underlying duplex. In both instances, G and T possess a pseudo twofold axis of symmetry and constantly can engage in both Hoogsteen and reverse Hoogsteen type triplets. The top half of Figure 1 is a schematic representation of the reverse Hoogsteen triplets. The thick bar associated with the third strand G or T represents the position assumed by the furanose plane (end-on and is projection) in an anti- confirmation. Such a reverse Hoogsteen anti(RH/a) configuration places the third strand anti-parallel with respect to the purine rich reference strand of the duplex target as required by low resolution structure determination. The referenced strand of the underlying duplex is positioned to the left in Figure 1. The symmetry among the three strands which comprise such triplets is presented graphically with the arrows. The bottom half of Figure 1 depicts the corresponding situation resulting from duplex sites with CG (left) or TA (right) inversion about the base diad. -37- This produces a dislocation within a polypurine/polypyrimidine target site. If a base triplet is to be formed at such a defect, continuity must be maintained with flanking regions of the triplex. To satisfy that connectivity requirement, the TFO strand at the site of CG or TA inversion must retain an overall antiparallel orientation with respect to the purine rich reference strand of the duplex target. This is equivalent to the requirement that triplets must switch to a parallel strand orientation relative to the purine containing strand at the site of TA or CG inversion. Such a change of symmetry is depicted by arrows in the lower portion of Figure 1. For an unmodified base, there are two degrees of freedom which can be exercised to achieve such local reversal of triplet symmetry: 1. conversion from Hoogsteen to reverse Hoogsteen H-bonding, or 2. anti/syn glycosidic rotation. We have displayed the Hoogsteen to reverse Hoogsteen degree of freedom in the lower portion of Figure 1. In this configuration the glycosidic bond is kept anti to produce H/a triplets at the sites of inversion. Because of the pseudo two fold symmetry of G and T, there is little difference in bond strength for the Hoogsteen and reverse Hoogsteen triplets. Therefore, in evaluating the possibility that the H/a triplet scheme can occur at a TA or CG dislocation, the principal concern is not H-bond strength or strand orientation per se, but is instead the substantial transverse dislocation of the third strand backbone required to accommodate the defect. That dislocation is identified by horizontal arrows at the top of Figure l and amounts to approximately a 3-5A displacement. Alteration of local -38- base stacking interaction is also associated with such a dislocation, which could also contribute a local change in triplex stability. The triple helix structure and thermodynamics can be evaluated in the context of the model in Figure 1. The 3-δA dislocation and attendant stacking change might be difficult to accommodate into an orderly triplex, thereby- resulting in local reduction of base stacking and triplet H-bonding energy, relative to the stable interactions within polypurine/polypyrimidine runs. The destabilizing effect of such defects upon triplex stability is therefore consistent with the model. The model in Figure 1 suggests that, although the triplets are imperfect, they can form at sites of CG and TA inversion and may have a net stabilizing influence on triple helix formation. This conclusion from the model is consitent with the Cu(OP) 2 hypersensitivity assay that the duplex becomes deformed at these sites in the triplex. This deformation may help facilitate formation of the stressed triplets. Example 25 2"-Deoxyformycin A model to improve triplet formation at sites of CG and TA inversion. If triplet formation is made stable at sites of TA and CG inversion, oligonucleotides can be designed against any duplex site, rather than just at purine rich targets. Based upon the model in Figure 2, one solution to the binding problem at CG sites is to alter the position of the two hydrogen bond donors of guanosine, relative to the site of glycosidic bond attachment to the furanose. This solution is displayed in Figure 2. As can be seen the result is a reduction of the transverse dislocation required to accommodate triplet H/a bonding at sites of CG inversion. -39- Figure 2 displays an approximation to that proposed alteration, employing 2'-deoxyformycin as the corresponding G homologue. Formycin (1, Figure 5) is a naturally occurring nucleoside antibiotic, with structural similarity to adenosine. However, due to its unusual C-C glycosidic bond, the aglycon moiety becomes more aromatic (abs. max shifts to 310nM) and its state of " protonation is altered relative to A. At neutral pH, the formycin ring is protonated at sites t and N 2 (Figure 5) . As such, the ring is capable of donating 2 H-bonds to an acceptable donor and is therefore homologous to G, with respect to triplet formation. Thus, by substituting 2*-deoxyformycin for G, the third strand backbone becomes less distended at sites of CG inversion. Note also, the improved similarity of furanose placement, relative to a GGC triplet at sites of CG inversion (lower part of Figure 2) . Example 26 Introduction of dF into a progesterone response element A duplex DNA target is presented in Table 2 which corresponds to the binding domain for progesterone response elements in a progesterone response promoter. The two protein binding sites are underlined. The TFO specific for this site is presented in the preferred antiparallel orientation. The dF-DMT-phosphoramidite (1) was introduced into the TFO 3'A-PRE2ap. As an unmodified TFO, the progesterone response element PRE2ap binds selectively to the purine rich target site described in Table 2. TABLE 2 d∑ CONTAINING TFO: 3'A-PRE2Fap (SEQ. ID. No. 1). 5 ' -G&'TCTCTACAL'l'L'l'U'l^ 3 • TARGET 3 ' -40- 36BP site, 31/36=87% purine, 60% GC, 3 sites of dF substitution. The corresponding dF derivative (3Α-PRE2Fap) was synthesized by the standard solid phase method. The TFOs are prepared employing solid-phase synthesis using the CPG method and β-cyanoethylphosphoromidite chemistry on a Milligen 8700 synthesizer. The TFOs are modified at the 3'-terminus as primary amine, i.e. 3'-OCH 2 CHOHCH 2 N + H 3 , which is introduced as part of the link to the CPG support: "3'-amino modifier" provided by Glenn Research. In all instances, TFOs are purified by C 18 HPLC, followed by detritylation, organic extraction, then rechromatography over a G25 molecular sieve. The purity of the resulting material is analyzed by high resolution electrophoresis and by HPLC. Example 27 Comparison of Standard TFO with dF TFO The binding of 3•-A-PRE2ap and its 2•-deoxyformycin homologue were monitored at 37°C by the band shift method. The standard binding buffer: lOmM Tris/HCl, lOmM MgCl 2* , pH 7.8 was used. In this experiment, the radiolabelled duplex target fragment of Table 2 is titrated with increasing concentration of unlabeled TFO. Binding is then detected by the characteristic electrophoretic mobility difference between duplex and triplex on a native 10% acrylamide gel. In this assay the 2'-deoxyformycin containing TFO displayed approximately 10-fold increase in affinity as compared to its unmodified counterpart (Figure 4) . There is an apparent titration midpoint decrease from 10 "7 M to 10 * ^ in TFO equivalents. This significant -41- eπhancement of bidning affinity resulting from dF substitution provides a method to accommodate sites of CG (and TA) inversion within target sites for antiparallel triplex formation. Example 28 Introduction of dF into an insulin receptor site TFO. A purine rich domain within the mouse insulin receptor gene has been identified (Table 3). Its TFO compelment 3'A-IR2ap is presented bound in an antiparallel orientation. 3'A-IR2ap is presented in the orientation which would result if it were to bind so as to form GGC and TAT triplets in a antiparallel orientation. Numbering refers to the principal capping site described by Sibley, et al. iProc. Natl . Acad. Sci . USA 86:9732 (1989)]. TABLE 3 THE IR, SITE 2 UPSTREAM REGION WITH TC REPEATS, IN HUMAN AND MOUSE (IR2Fap; SEQ. ID. No. 2) -138 -99 38BP site, 84% purine, 76% CG, 5 sites of dF. substitution. Example 29 Introduction of dF into the EGF promoter. A purine rich domain within the EGFR promoter has been identified, along with its antiparallel TFO complement -42- 3'A-EG36ap (Table 4). Numbering refers to the position relative to the start codon. TABLE 4 THE EGFR,EG36 SITE TARGET FUNCTION: Spl BINDING SITE #4 (EGFR36Fap; SEQ. ID. No. 3). -352 -317 5 ' -TTCTCCTCCTOΓTCΓGCTCCTCXX:GATC^CTCCTCC- 3 ' TARGET 3 ' -AAGAGGAGGAGGAGAGGAGGAGGGCTAGGGAGGAGG-5 ' SITE 5'-TTGTGGTGGTGGTGTGGTGGTGGGGTTGGGTGGTGG-3' EGFR36ap 5'-TTGTGGTG3TGGTGTFGTGGTGGGFTTGGGTGGTGG-3' EGFR36Fap 36BP site, 92% purine, 67% GC, 2 sites of £F substitution Example 30 Introduction of dF into HIV-1 site. A target site within the HIV-l LTR is presented, corresponding to a series of adjacent Spl binding sites. Numbering refers to position relative to the viral mRNA start site. TABLE 5 PROPOSED Spl TRANSCRIPTION ACTIVATOR BINDING SITE 4 (HIV38Fp; SEQ. ID. No. 4) . -80 -51 9476 9501 5 , -AGGGAG CσTGGCCTGσσCGGGACTGGGGAGTGGα3AG-3 , TARGET 5'-TGGGTGGGGTGGGGTG GGGGGTGTGGGGTGTGGGGTG-3' HIV38p 5'-TGGGTGGFGTGGFFTGGGFGGGTFTGGGGTGTGGFGTG-3' HIV38Fp 38BP site, 77% GC, 74% purine, 6 sites of dF substitution -43- Example 31 Synthesis of 7-N-benzoylamino-3-r2-Q-methyl-3-0-(β- cyanoethyl)-N.N-diisopropylaminophosphoramidite-5-O- dimethoxytrityl-β-D-ribofuranosvπpyrazolor4.3.dl- pvrimidine (35) . The synthesis of compound 3_5 follows the reaction sequence shown in Figure 10. Treatment of a suspension of formycin A (1) in anhydrous pyridine with trimethylsilyl chloride at 0°C for 2 h, followed by the addition of 1.1 molar equivalent of benzoyl chloride and subsequent hydrolysis of the trimethylsilyl protecting groups with water yields N-7-benzoylformycin A (3.0) . Protection of the 3' ,5' -hydroxyl groups of 3) was accomplished by the reaction with 1,3-dichloro-l,1,3,3- tetraisopropyldisiloxane to afford 7-N-benzoylamino-3- [3,5-0-(l,1,3,3-tetraisopropyl-l,3-disiloxanediyl-β-D- ribofuranosyl]pyrazolo[4,3-d]pyrimidine (21). Selective methylation of the 2' -hydroxyl group of 21 with either diazomethane in the presence of 1 mM stannous chloride [M.J. Robins, S.R. Naik and A.S.K. Lee, J. Org. Chem. , 39: 1891 (1974)] or with methyl iodide in the presence of freshly precipitated silver oxide [E. Sochacka and A. Malkiewicz, Nucleosides Nucleotides, 9: 793 (1990)] yields 7-N-benzoylamino-3-[3,5-0-(1,1,3,3-tetraisopropy1- l , 3 -disiloxanediyl-2- 0_- methyl-β- D_- ribofuranosyl]pyrazolo[4,3-d]pyrimidine (24.)- Upon subsequent deprotection of the 3' ,5' -O-protected silyl gorups by the treatment with tetra-n-butylammonium fluoride (1M solution in THF) at 0-5 e C for 2 h, the desired key intermediate 2' -O-methyl-N-7-benzoylformycin A (22) is produced. Coupling of 22 with 1.1 molar equivalent of 4,4' -dimethoxytrityl chloride in anhydrous pyridine at ambient temperature for 4 h, yields 7-N- benzoylamino-3-[2-0-methyl-5-0-dimethoxytrityl-β-D- -44- ribofuranosyl]pyrazolo[4,3-d]pyrimidine (22). With 2' - and 5' -hydroxyl groups protected, the 3' -hydroxyl of 22. is now activated to the phosphoramidite by the treatment with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of N,N-diisopropylethylamine to afford the target compound 25. The product 3_5 can be purified on a silca gel column. All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. One skilled in the art would readily appreciate the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compounds, methods, procedures and techniques described herein are presently representative of the preferred embodiments and are intended to be exemplary and not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention or defined by the scope of the appended claims. -45- SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Revan ar, Ganapathi R. Hogan, Michael Ξ. Rao, Takkellapati S. Schroff, Hitesh N. (ii) TITLE OF INVENTION: 2'-DEOXYRIBONUCLEOSIDES, USE IN TRIPLEX FORMING OLIGONUCLEOTIDES AND PROCESS FOR PREPARING THE SAME (ϋi) NUMBER OF SEQUENCES: 15 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Thomas D. Paul (B) STREET: 1301 McKinney, Suite 5100 (C) CITY. Houston (D) STATE: Texas (E) COUNTRY: U.S.A. (F) ZIP: 77010-3095 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Paul, Thomas D. (B) REGISTRATION NUMBER: 32,714 (C) REFERENCE/DOCKET NUMBER: D-5348-PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 713/651-5151 (B) TELEFAX: 713/651-5246 (C) TELEX: 762829 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES SUBSTITUTE SHEET -46- (xi) SEQUENCE DESCRIPTION: SEQ ID N :l: GTGTNTTGTG GGGGGTGGGG GTNTTGTGTG TNTTGT 36 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ϋi) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGTGGGGNTN GTGTNGTNNG TTTGTGGGGG TGGGGGGGG 39 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTGTGGTGGT GGTGTNGTGG TGGGNTTGGG TGGTGG 36 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TGGGTGGNGT GGNNTGGGNG GGTNTGGGGT GTGGNGTG 38 (2) INFORMATION FOR SEQ ID NO:5: SUBSTITUTE SHEET -47- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGTCCCCGC TGCTGGTTCT CCTCCTCCTC CTCGCATTCT CCTCCTCCTC TGCTCCTCCC 60 GATCCCTCCT CCGCCGCCTG GTCCC 85 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TTGTGGTGGT GGTGTGGTGG TGGGGTTGGG TGGTGG 36 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGGGTGGGGT GGGGTGGGGG GGTGTGGGGT GTGGGGTG 38 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET -47a- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTGTGGTGGT GGTGGTGGGT TTGTGGTGGT GGTGT 35 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TGGGTGGTGG TGGGGGGGTG GGTGGG 26 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CCTCCCCGAG CTCTGCTGGC TTTCTCCCCC TCCCCCCCC 39 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO s UBSTITUTE SHEET -47b- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGTGGGGGTG GTGTGGTGGG TTTGTGGGGG TGGGGGGGG 39 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GATCTGTACA CTCTGTTCTC CCCCCACCCC CTGTACACTC TGTTCTG 47 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TTCTCCTCCT CCTCTGCTCC TCCCGATCCC TCCTCC 36 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GTGTGTTGTG GGGGGTGGGG GTGTTGTGTG TGTTGT 36 SUBSTITUTESHEET -47c- (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: AGGGAGGCGT GGCCTGGGCG GGACTGGGGA GTGGCGAG 38 SUBSTITUTE SHEET

Claims: -48- CLAIMS What is Claimed is: 1. A compound of the structure: wherein R 1 is H or -COC^; R 2 is H or -COC g H j ; and R 3 is attached by a β linkage and is selected from the group consisting of wherein X is H or DMT; and ¥ is H or NC(CH 2 ) 2 0 -49- 2. The compound of claim 1, wherein R, and R 2 are -COC 6 H 5 , R 3 is 3. The compound of claim 1 , wherein R, is H; R 2 is -COC 6 H5 and R 3 is 4. The compound of claim 3, wherein R 3 is 5. The compound of claim 3, wherein R 3 is -50- 6. A triplex forming oligonucleotide comprising an oligonucleotide capable of forming a triple helix with a double stranded DNA, wherein at least one base of said oligonucleotide is replaced with the compound of claim 5. 7. A compound of the structure: -51- 8. A compound of the structure: wherein R, is 0 or S; R 2 is H or 9. The compound of claim 8, wherein , is 0 and R 2 is 10. A triplex forming oligonucleotide comprising an oligonucleotide capable of forming a triple helix with a double stranded DNA, wherein at least one base of said oligonucleotide is replaced with the compound of claim 9. 11. The compound of claim 8, wherein R 1 is S and R 2 is NC(CH 2 ) 2 0-' I N , -52- 12. A triplex forming oligonucleotide comprising an oligonucleotide capable of forming a triple helix with a double stranded DNA, wherein at least one base of said oligonucleotide is replaced with the compound of claim 11. 13. A compound of the structure: 14. A compound of the structure: -53- wherein R, is TBDMS, DMT or H; and R 2 is TBDMS, H or 15. The compound of claim 14, wherein R, is DMT and 16. A triplex forming oligonucleotide comprising an oligonucleotide capable of forming a triple helix with a double stranded DNA, wherein at least one base of said oligonucleotide is replaced with the compound of claim 15. 17. A compound of the structure: -54- 18. The compound of claim 17, wherein R, is DMT and R 2 is 19. Atriplex forming oligonucleotide comprising an oligonucleotide capable of forming a triple helix with a double stranded DNA, wherein at least one base of said oligonucleotide is replaced with the compound of claim 18. 20. A compound of the structure: -55- wherein R 1 is H or -CO-CH (CH 3 ) 2 ; R 2 is H or DMT; R 3 is H or R 4 is OH or H. 21. The compound of claim 20, wherein R 1 is -CO-CH( and R 4 is H. 22. A triplex forming oligonucleotide comprising an oligonucleotide capable of forming a triple helix with a double stranded DNA, wherein at least one base of said oligonucleotide is replaced with the compound of claim 21. 23. A compound of the structure: -56- wherein R, is H or -OC-CH(CH 3 ) 2 ; and R 2 is OH, H or S I -O-C-OPh 24. A compound of the structure wherein R t is attached by a β linkage and is selected from the group consisting of wherein X is H or CH 3 ; Y is H or and Z is H or DMT. 25. The compound of claim 24, wherein R^s attached by a β linkage and is -57- 26. A triplex forming oligonucleotide comprising an oligonucleotide capable of forming a triple helix with a double stranded DNA, wherein at least one base of said oligonucleotide is replaced with the compound of claim 25. 27. A triplex forming oligonucleotide, wherein at least one base of said oligonucleotide is replaced with a guanosine analogue. 28. The triplex forming oligonucleotide of claim 27, wherein the guanosine analogue is selected from the group consisting of compounds 4, 10a, 10b, 14, 19, 29 and 35. 29. The triplex forming oligonucleotide of claim 27 wherein said oligonucleotide is selected from the group Oconsisting of SEQ. I.D. No. 1, SEQ. I.D. No. 2, SEQ. I.D. No. 3 and SEQ. I.D. No. 4. 30. The triplex forming oligonucleotide of claim 27 for treating AIDS comprising SEQ. I.D. No. 4. 31. A triplex forming oligonucleotide (TFO) for forming a colinear triplex with a target sequence in a duplex DNA comprising a nucleotide sequence of at least about 20 nucleotides; said nucleotide sequence including G, T, and guanosine analogue, wherein G is used when the complementary location in the duplex DNA is a GC base pair, T is used when the complementary location in the duplex DNA is an AT base pair and guanosine analogue is used when there is a CG inversion within the duplex DNA target site; said sequence binding to a orienting strand in the duplex DNA target site and said binding forming -58- TAT, GGC and NCG Hoogsteen or reverse Hoogsteen triplets, wherein N is a guanosine analogue. 32. The TFO of claim 31, wherein the guanosine analogue is selected from the group consisting of compounds 4, 10a, 10b, 14, 19, 29 and 35. 33. The TFO of claim 31, wherein the TFO binds anti-parallel to the orienting strand and the target GC to AT base pair ratio is substantially greater than one. 34. The TFO of claim 31, wherein the TFO binds parallel to the orienting strand and the target GC to AT base pair ratio is substantially less than one. 35. The TFO of claim 31, wherein the TFO is selected from the group consisting of SEQ. I.D. No. 1, SEQ. I.D. No. 2, SEQ. I.D. No. 3 and SEQ. I.D. No. 4. 36. The TFO of claim 31, for treating HIV-1 infection wherein said TFO is SEQ. I.D. No. 4. 37. A triplex forming oligonucleotide (TFO) for forming a colinear triplex with a target sequence in a duplex DNA comprising a nucleotide sequence of at least about 20 nucleotides; said nucleotide sequence including, G, T, and guanosine analogue, wherein G is used when the complementary location in the duplex DNA target possesses a G in the orienting strand, guanosine analogue is used when the duplex DNA target possesses a C in the orienting strand and T is used when the complementary location in the duplex DNA is an AT base pair; said sequence binding parallel or anti-parallel with respect to the orienting strand. 38. The oligonucleotide of claim 37, wherein the target sequnece has a GC to AT base pair ratio substantially greater than one and the TFO binds anti¬ parallel to the orienting strand. 39. The oligonucleotide of claim 37, wherein the target sequence has a GC to AT base pair ratio -59- substantially less than one and the TFO binds parallel to the orienting strand. 40. The TFO of claim 37, wherein the guanosine analogue is selected from the group consisting of compounds 4, 10a, 10b, 14, 19, 29 and 35. 41. The TFO of claim 37, wherein the TFO is selected from the group consisting of SEQ. I.D. No. 1, SEQ. I.D. No. 2, SEQ. I.D. No. 3 and SEQ. I.D. No. 4. 42. The TFO of claim 37, for treating HIV-1 infection wherein said TFO is SEQ. I.D. No. 4.

Inventors: HOGAN MICHAEL EDWARD, RAO TAKKELLAPATI SUDHAKAR, REVANKAR GANAPATHI RAMAKRISHNA, SHROFF HITESH NAVINCHANDRA, HOGAN, MICHAEL, EDWARD, RAO, TAKKELLAPATI, SUDHAKAR, REVANKAR, GANAPATHI, RAMAKRISHNA, SHROFF, HITESH, NAVINCHANDRA
Applicants: BAYLOR COLLEGE MEDICINE, TRIPLEX PHARMA CORP, BAYLOR COLLEGE OF MEDICINE, TRIPLEX PHARMACEUTICAL CORPORATION
Application year: 1992
Main classification: C07H19
IPC codes: A61K31, A61P31, C07F7, C07H19, C07H21, C07H23, C12N15, C12Q1