The subject invention relates to novel oligodeoxynucleotides 5' linked to covalently attached chemical groups via intermediate linked phosphoramidites, and their methods of production and use. More specifically, the subject invention involves the synthesis of an anthraquinone (or other) linked group to a phosphoramidite moiety outside the automatic synthesizer, followed by application of this compound as a terminator of the 5'-end of a phosphate-modified oligodeoxynucleotide in the automatic synthesizer. The compounds are used to attenuate or destroy mammalian gene expression or viral activity.

BACKGROUND OF THE INVENTION Recent developments in molecular genetics, such as the automated synthesis of oligodeoxynucleotides and the use of computer programs to calculate accessible RNA regions as likely targets for complementary base binding", provide the basis for new approaches to the control of gene expression and the potential for 'gene therapy' . One approach has been to prepare a linked product in which a chemotherapeutic agent is covalently attached to synthetic oligonucleotides with sequences directed toward complementary DNA or mRNA sequences. The standard method of preparing these linked products is to synthesize normal- phosphate oligonucleotides in the automatic synthesizer, followed by purification of the

oligonucleotides and covalent attachment of the che otherapeutic agent and/or linker group outside the automatic synthesizer. Helene et al constructed oligodeoxynucleotides with derivatized 3*-linked intercalating groups (acridines) using this standard method. Helene, C. , Montenay - Garestier, T., Saison, T. et al., Oligodeoxynucleotides covalently linked to intercalating agents: a new class of gene regulatory substances. Biochemie, 67:777-783, 1985.

Desired properties of these linked products include selective suppression of gene expression, stability against nucleases, and a balance between aqueous solubility and membrane transportability. - - Several examples of the covalent linking of active groups to oligodeoxynucleotides have been accomplished. For example, researchers have developed syntheses of iron EDTA chelating groups to normal phosphate oligodeoxynucleotides complementary to specific single stranded DNAs. These can give rise to reactive oxygen-containing radicals in the vicinity of the DNA. The compounds function by cleavage of the single stranded DNAs. Some problems of normal-phosphate oligodeoxynucleotides with linked EDTA chelating groups include: 1) not achieving complete specificity, 2) nonspecificity in that the Fe(ΣI) of the EDTA may not be tethered to a single nucleotide site, 3) autodegradation in that the highly efficient cleanage reagents themselves may destroy their own backbones, and 4) OH radicals may diffuse in many directions.

Researchers have also developed synthetic methods for linking alkylating groups to both the 3'-ends and 5'-ends of normal phosphate oligonucleotides. Knorre et al attached an

intercalating phenazine derivative to the 3'end of an oligodeoxynucleotide through an amine linker, and an alkylating group to the 5'end. Knorre et al. Reactive oligonucleotide derivatives and sequence - specific modification of nucleic acids. Biochemie, 67:785-789, 1985.

Phosphate modified oligodeoxynucleotides, such as phosphorothioate oligodeoxynucleotides in which one of the nonbriding oxygen atoms in each nucleotide is replaced by a sulfur atom, have the advantageous properties of good aqueous solubility, and automated synthesis via phosphoramidites, and have been noted for their antiviral effect. Phosphate-modified oligodeoxynucleotides with 5'- covalently attached chemical groups via intermediate linked phosphoramidites have not previously been obtained.

SPMMftRY PF THE INVENTIO Method of synthesis is provided for novel oligodeoxynucleotides 5' linked to covalently attached chemical groups via intermediate linked phosphoramidites. The synthesis of the covalently attached chemical group to a phosphoramidite moiety occurs outside the automatic synthesizer, followed by application of this complex as a terminator of the 5' end of a phosphate-modified oligodeoxynucleotide in the automatic synthesizer. The compounds are used to attenuate or destroy mammalian gene expression or viral activity. Actual or potential benefits of the present invention include anti-HIV activity in vitro, specificity by base sequence of the targeted cell's genes rather than by a protein, improved selectivity over

conventional drug therapy and decreased resistance, and stability against nucleases.

DOTATIED DESCRIPTION OF THE INVENTION

The present invention is directed to novel 5 oligodeoxynucleotides with 5'-linked chemical groups, via intermediate linked phosphoramidites and the synthesis and use thereof. These novel compounds are derived by synthesizing a phosphoramidite moiety to a covalently linked

10 chemical group outside an automatic synthesizer, followed by purification, and application of this phosphoramidite-covalently linked group complex as a terminator of the 5*-end of a normal or phosphate- modified oligodeoxynucleotide in an automatic

15 synthesizer.

This synthetic method can provide a wide range of feasible compounds of the general formula R-L-ODN, in which ODN is oligodeoxynucleotide, L is a linker arm, and R is the covalently attached

20 group. An advantageous example of a compound synthesized by this method is anthraquinone 5'- 1inked to a phosphorothioate oligodeoxynucleotide

-÷5_- via a hydroxyethyl-piperazinyl linker arm, as seen in Example 1. Another advantageous example of a

25 compound synthesized by this method is phosphate- modified oligodeoxynucleotide with 5'-linked acridine unsubstituted except for the 9-amino position for linkage via a maleimide intermediate, as seen in Example 2.

30 The components of these compounds of novel oligodeoxynucleotides with 5'-linked chemical groups can be prepared as follows.

The linker arm is an intermediate linked phosphoramidite moiety. A preferred linker arm is a

piperazinyl derivative such as l-(2-hydroxyethyl) piperazine, although any linker arm which is convenient and appropriate for the chemical conditions employed in the synthesizer may be used. For example, acridine rings linked via the 9-amino group to oligos as prepared by Helene et al are subject to alkaline (ammonia) hydrolysis int he automatic synthesizer, although secondary amines are more stable than tertiary amines. In this situation, the preferred linker arm is a maleimide intermediate to 5* link 9-amino 6-chloro acridine onto an oligodeoxynucleotide. This synthesis of maleimide linked acridine oligodeoxynucleotide is in Example 2. It allows the synthesis of oligodeoxynucleotides with all bases unblocked by ammonia in the automatic synthesizer. Otherwise, without the use of ammonia one could obtain high yields of 6-thiophenol substituted acridine only with normal and phosphorothiate oliαo-dT's. which is the only unblocked base.

The covalently attached chemical group are preferably anthraquinone or acridine. Other covalently attached groups include intercalating groups, known drugs such as adriamycln, bleomycin, and hydrolytic groups such as imidazole. Another example would be anthraquinone attached via a polymethylene linker.

The covalently attached group 5'-linked 9- amino 6-chloro acridine was found to be subject to substitution at the 6 position by thiophenol used in the automatic synthesizer to de- ethylate the phosphotriester product. To overcome this problem, an acridine unsubstituted except for the 9 position for linkage was used. Also, a maleimide

intermediate linked the acridine onto the oligo-see - previous discussion and Example 2.

Oligodeoxynucleotides of 5-30 mer, preferably 12-28 mer, are the reasonable and necessary lengths for hybridization that may be used in the present invention. Longer oligodeoxynucleotides are more expensive. Also, the oligodeoxynucleotide may be a thiol-containing oligodeoxynucleotide.

The oligodeoxynucleotides may be of different base Sequences. Specific oligodeoxynucleotide base sequences may be synthesized to complement regions of a viral DNA, viral R A, mammalian DNA, or mammalian mRNA. The oligodeoxynucleotide is regarded as the means of delivering the covalently attached chemical group to the bidogical site in that it targets specific DNA or mRNA sequences. The oligodeoxynucleotide can be either a normal oligo or a phosphate-modified oligo such as phosphorothioate or a co-polymer combination of both. Phosphate-modified oligodeoxynucleotides are preferred. Covalently linked groups have not previously been attached to the 5' end of phosphate- modified oligonucleotides. λ series of oligonucleotides (of oligo base sequences dT _lr Td _; , dT ₈ , dT ₁₂ , dT ₁₅ , dc ₁₅ , antisense c- myc 15-mer, and antisense anti-rev HIV sequence) were constructed and 5'-linked to acridine or •anthraquinone via a hydroxyethyl-piperazinyl derivative, mien phosphorothioate oligodeoxynucleotides were substituted in place of normal oligodeoxynucleotides, the result was more effective inhibition of HIV expression in an in vitro T-eell assay. See Figure 1.

The 'following non-limiting examples illustrate the invention in more detail.

EXAMPLE 1 λ mixture of 1-chloroanthraquinone (l.g) and l-(2-hydroxyethyl) piperazine (5g) is heated at 150* for 30 minutes. After cooling to room temperature, water is added. The mixture is filtered. l-[l-(2- hydroxyethyljpiperazinyl]anthraquinone, m.p. 168* (after recrystallization from CHC1 ₃ ) is obtained. 1-[1-(2-hydroxyethyl) piperazinyl] anthraquinone (336 mg, 1 mmol) is dissolved in CH ₂ C1 ₂ (2 ml) and N-ethyl-diisopropylamine (760 p, 4 mmol) is added. Then, N-N-diisopropyl-methyl- phosphoramidic chloride (194 ft, 4 mmol) is added to the solution. After 30 minutes, pour the solution into ethylacetate (5 ml, previously washed with 5% NaHCO-} and extract with 2 x 5 il of 5. NaHC0 ₃ and 2 x 5 ml of saturated NaCl. The ethyl acetate phase was dried over Na ₂ S0 ₄ and evaporated to an oil. A diisopropyl phosphamidite (di-isopropylamino phosphate) 0-methyl ester is obtained. The TLC on silica gel (ethylacetate:triethylamine » 9:1) showed complete reaction. Rf of the starting material-O.3, Rf of the product-0.7. ³¹ PNMR spectroscopy peak of 148. Without further purification, the oil was dissolved in the appropriate volume of acetonitrile and used directly in oligσnucleotide preparation. The yield was about 65%.

Synthesis of phosphorothioate oligodeoxynucleotide with the anti-sense anti rev HIV base sequence d(5'-TCG TCG CTG TCT CCG CTT CTT

CCT GCC A) was carried out using standard phosphoramidite techniques. In an automatic synthesizer (an ABI 380B) , attach the diisopropyl phosphamidite (diisopropyla ino phosphate) 0-methyl

ester to the 5' end of the phosphorothioate oligodeoxynucleotide by means of the standard nucleotide cycle. There is thus obtained 5'- anthraquinone oligodeoxynucleotide, which is then deblocked and.purified by HPLC.

EXAMPLE 2 ■Synthesis of Maleimide Linked Acridine piiσodeoxynucleotide S-Trityl-3-mercaptopropanol(l)ol (334 mg, 1 mmol) were dissolved in methylene chloride (2 ml) . N-ethyldiisopropylamine (760 k, 4 mmol) was added, and methoxydiisopropylchloriophosphite (180?., 1.2 mmol) was slowly added at 0C. The mixtures were left for a further 15 minutes at 0C. The solutions were poured into ethylacetate (5 ml) (contained with triethyla ine 0.5 ml) and washed with 2x5ml of 5% NaHCO _j and 2x5ml of saturated NaCl. The organic phases were dried over Na ₂ S0 _; and evaporated to oil. ³¹ P NMR spectrum showed a single peak, -146 and TLC on silica gel (petroleum ether 9, triethylamine 1; Rf of starting material 0.3, Rf of product 0.45). Without further purification, the clear oil was dissolved in dry acetonitrile for 100 M solution. Typical yield was about 40%. Oligodeoxynucleotide synthesis was carried out using standard solid-phase phosphoramidite techniques whereupon an oligodeoxynucleotide containing an s-triphenylmethyl group attached to the 5' phosphate group via a carbon chain was obtained. The S-triphenylmethyl group is removed with silver according to Connolly, B., 13 Nucleic Acids Research 4485 (1985) . Thiol-containing oligodeoxynucleotides are obtained.

The thiol-containing oligodeoxynucleotides (1 jiol scale synthesis) was dissolved in 1% NaHC0 ₃ (2ml), and N-(9-acridinyl) maleimide was added. N- (9-acridinyl) maleimide had been synthesized according to Nara, Y. and Tuzimura, K., 42 Agric. Biol. Chem. 793, 1978. The mixture was kept at 4C for 16 hours, and then extracted with 3x2 ml of ethylacetate. There is thus obtained maleimide linked acridine oligodeoxynucleotides. The mixture (aqueous phase) was concentrated with flashed N ₂ gas. The products were purified by HPLC. Yields of 35- 40% were obtained from lmol scale synthesis.

While preferred embodiments of the invention have been described herein, it will be evident to those skilled in the art from a reading of the foregoing disclosure of specific oligodeoxynucleotide base sequences synthesized with 5'-linked anthraquinone or other chemical groups, that oligodeoxynucleotides of different base sequences can be used. Various changes and modifications, especially pertaining to the covalently attached chemical groups, may be made without departing from the spirit of the present invention.