In recent publications, oligonucleotides joined by 2',5'-linkages were found to bind to complementary single-stranded RNA but to bind weakly, or not at all, to single-stranded DNA [e.g., P. A. Giannaris and M. J. Damha (1993) Nucleic Acids Res. 21, 4742-4749]. In this work, the biochemical and physicochemical properties of 2',5'-linked oligoribonucleotides containing mixed sequences of the four nucleobases (A, G, C, and U) were evaluated. CD spectra of RNA:2', 5'-RNA duplexes were compared with the spectra of DNA:DNA, RNA:RNA, and DNA:RNA duplexes of the same base sequence. The CD results indicated that the RNA:2',5'-RNA duplex structure more closely resembles the structure of the RNA:DNA hybrid, being more A-form than B-form in character. The melting temperature (Tm) values of the backbone-modified duplexes were compared with the Tm values of the unmodified duplexes. The order of thermal stability was RNA:RNA > DNA:DNA approximately RNA:DNA approximately DNA:RNA > RNA:2',5'-RNA > 2',5'-RNA:2',5'-RNA >> DNA:2',5'-RNA (undetected). RNA:2',5'-RNA duplexes are not substrates of the enzyme RNase H (Escherichia coli, or HIV-1 reverse transcriptase), but they can inhibit the RNase H-mediated cleavage of a natural DNA:RNA substrate. Structural models that are consistent with the selective association properties of 2',5'-linked oligonucleotides are discussed.

The antivirally active 3'-deoxyadenylyl-(2'-5')-3'-deoxyadenylyl-(2'-5')-3'-deoxyadenosine (cordycepin trimer core) was modified at the 2'-or 5'-terminus, by attachment of cholesterol via a carbonate bond (+ 15) or a succinate linker (-+ 16 and 27) to improve cell permeability. The corresponding monomeric conjugates 4,7, and 21 of cordycepin were prepared as model substances to study the applicability of the anticipated protecting groups the monomethoxytrityl (MeOTr), the (rert -butyl)dimethylsilyl (tbds), and the / J ' -eliminating 2-(4-nitrophenyl)ethyl (npe) and 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) groups ~ for the final deblocking steps without harming the ester bonds of the conjugate trimers. The syntheses were performed in solution using phosphoramidite chemistry. The fully protected trimer conjugates 13, 14, and 26 as well as all intermediates were characterized by elemental analyses, UV and 'H-NMR spectra. The deblocked conjugates 15, 16, and 27 were pure according to HPLC and showed the correct compositions by mass spectra. Comparative biological studies indicated that cordycepincholesterol conjugate trimers 16 and 27 were 333-and 1000-fold, respectively, more potent inhibitors of HIV-I-induced syncytia formation than cordycepin trimer core.

The syntheses of biodegradable 2 -and 5'-ester and 2 -and 5'-carbonate conjugates of the antivirally active 3'-deoxyadenylyl-(2'5')-3'-deoxyadenylyl-(2'-5')-3'-deoxyadenosine (cordycepin-trimer core) with the vitamins, E, D,, and A and the lipids 1,2-di-0 -palmitoylglycerol and 1,2-di-0 -hexadecylglycerol were achieved first by preparation of the trimeric educts 19-21 (Scheme 1). Secondly, these substances were condensed with the lipophilic residues via a succinate or carbonate linker and then deprotected byp -elimination of the npeoc and npe protecting groups and acid treatment for detritylation without harming the ester and carbonate functions, respectively (Scheme 2). Metabolically stable cordycepin-trimer-vitamin and -lipid conjugates are a new class of bioconjugates that inhibit HIV-1-induced syncytia formation with IC,, values of 7, 18, and 24 p~ for 39,29, and 42, respectively, and inhibit HIV-1 reverse transcriptase (RT) activity from 14 to 96% (see Table). Of the nine conjugates tested, inhibition of HIV-1 replication by 28,29,32,40, and 42 may be attributed in part to the activation of the RNase LjPKR antiviral pathways. Trimer conjugate 42 showed the greatest inhibition of HIV-1 replication, i.e., a 120-fold decrease in HIV-I-induced syncytia formation and an 88 % inhibition of HIV-1 reverse transcriptase (RT). This inhibition of replication of HIV-1 by 42 can be attributed in part to the activation of recombinant, human RNase L. The inhibition of HIV-1 replication by the cordycepin-trimer-vitamin and -lipid conjugates is significantly greater than that observed for the (2-5') A-trimer core or cordycepin-trimer core.

To improve cell permeability, monomeric 3'-deoxyadenosine (cordycepin) and antivirally active trimeric 3'-deoxyadenylyl-(2'-5')-3'-deoxyadenylyI-(2' -5')-3'-deoxyadenosine (2'-5')d3'(A-A-A) ; cordycepin-trimer core) were modified at the 2'-0-or 5'-0-position by myristic, cholic, and folic acid = tetradecanoic, 3a,7a,12~(-trihydroxy-5p-cholan-24-oic, and N-{4-{ [(2-amino-3,4-dihydro-4-oxopteridin-6-yl)methyl]amino}be~oyl}-~-glutamic acid, resp., linked by a 6-aminohexanoyl spacer. Syntheses of the trimeric educts 21, 27, and 28 were performed by phosphoramidite chemistry, using P-eliminating 2-(4-nitrophenyl)ethyl (npe), 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) and (9H-fluoren-9-y1)methoxycarbonyl (fmoc) groups which allow a deprotection by DBU in an aprotic solvent without harming the ester-bounded conjugates, to give the products 24-26 and 31-33. The metabollically stable (2'-5')A derivatives 26 and 33, covalently linked to folic acid either at the 2'-terminus or at the 5'-terminus of (2'-5')d3'(A-A-A), respectively, are a new class of the anti-HIV-1 compounds. Inhibition of HIV-1 reverse transcriptase (RT) activity by 26 and 33 was 45 and 81 %, respectively. Only 33 activated recombinant, human RNase L by 35%.

Monomeric 3'-deoxyadenosine (cordycepin) was modified at the 2-0 -(13-18) and S'-O-position (25-29) by the vitamins E, D,, and A and by the two lipids 1,2-di-O-palmitoylglycerol and 1,2-di-O-hexadecylglycerol via succinate or carbonate linkages. These base-labile conjugates afforded protection groups like the 2-(4-nitropheny1)ethoxycarbonyl (npeoc) and monomethoxytrityl group (MeOTr) that are cleavable without harming the ester and carbonate bonds, respectively. Monomeric conjugates of cordycepin and vitamin E, vitamin D,, 1,2-di-0-palmitoylglycerol, and 1,2-di-O-hexadecylglycerol (see 13, 14, 17, 18, 25, 26, 28, and 29) inhibited HIV-l-induced syncytia formation 1.7 to 6.2 fold compared to 1.5-fold for cordycepin (see Table); ICsO values for 25 and 28 were 257 and 267 p~, respectively. In addition, the monomeric cordycepin-vitamin and -lipid conjugates inhibited HIV-1 RT activity 2 8 4 9 % which compares with a 13% inhibition of HIV-1 RT observed for cordycepin. The minimal inhibition of HIV-I-induced syncytia formation and HIV-1 RT activity did not proceed by the activation of RNase L. The monomeric conjugates tested (13, 14) increased PKR expression.