WO1990012802A1 - Novel oligodeoxynucleotides with 5'-linked chemical groups, method of production thereof and use thereof - Google Patents
Novel oligodeoxynucleotides with 5'-linked chemical groups, method of production thereof and use thereof Download PDFInfo
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- WO1990012802A1 WO1990012802A1 PCT/US1990/001501 US9001501W WO9012802A1 WO 1990012802 A1 WO1990012802 A1 WO 1990012802A1 US 9001501 W US9001501 W US 9001501W WO 9012802 A1 WO9012802 A1 WO 9012802A1
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- oligodeoxynucleotide
- covalently attached
- attached group
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- linker arm
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Definitions
- 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.
- 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., Huawei, T. et al.
- 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( ⁇ I) 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.
- 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.
- 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
- 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
- An advantageous example of a compound synthesized by this method is anthraquinone 5'- 1inked to a phosphorothioate oligodeoxynucleotide
- 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.
- 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.
- 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.
- 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 oli ⁇ o-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.
- ⁇ 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.
- EXAMPLE 1 ⁇ 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.
- 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.
- the oil was dissolved in the appropriate volume of acetonitrile and used directly in olig ⁇ nucleotide preparation. The yield was about 65%.
- CCT GCC A was carried out using standard phosphoramidite techniques.
- an automatic synthesizer an ABI 380B
- 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.
Abstract
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.
Description
NOVEL OLIGODEOXYNUCLEOTIDES WITH 5'-LINKED CHEMICAL GROUPS, METHOD OF PRODUCTION THEREOF AND USE THEREOF
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 dTlr Td;, dT8, dT12, dT15, dc15, 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 CHC13) is obtained. 1-[1-(2-hydroxyethyl) piperazinyl] anthraquinone (336 mg, 1 mmol) is dissolved in CH2C12 (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. NaHC03 and 2 x 5 ml of saturated NaCl. The ethyl acetate phase was dried over Na2S04 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. 31PNMR 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% NaHCOj and 2x5ml of saturated NaCl. The organic phases were dried over Na2S0; and evaporated to oil. 31P 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% NaHC03 (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 N2 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.
Claims
1. λ compound comprising an oligodeoxynucleotide linked at its 5' end with a linker arm to a covalently attached group, wherein a) said oligodeoxynucleotide is a phosphate- modified analog; b) said linker arm is part of an intermediate linked phosphoramidite moiety; and c) said covalently attached group confers biological activity upon said compound.
2. The compound according to claim 1, wherein the oligodeoxynucleotide is phosphorothioate.
3. The compound according to claim 1, wherein the oligodeoxynucleotide comprises a eopolymer combination of a normal oligodeoxynucleotide and a phosphate-modified oligodeoxynucleotide.
4. The compound according to claim 1, wherein the linker arm is a piperazinyl derivative.
5. The compound according to claim 1, wherein the covalently attached group is an intercalating group.
6. The compound according to claim 1, wherein the covalently attached group is anthraquinone.
7. The compound according to claim 1, wherein the covalently attached group is selected from the group consisting of adriamycin, bleomycin, i idazole, and hydrolytic compounds.
8. The compound according to claim 1, wherein the oligodeoxynucleotide is a thiol- containing oligodeoxynucleotide, the covalently attached group is acridine unsubstituted except for the 9-amino position for linkage, and the linker arm is a maleimide intermediate.
9. λ method of producing a compound of claim 1, wherein said covalently attached group is synthesized linked to said linker arm to form a linker arm-attached group complex outside an automatic synthesizer, followed by attachment of said linker arm-attached group complex to the 5' end of said oligodeoxynucleotide in an automatic synthesizer, wherein said linker arm is appropriate for the chemical conditions employed in said automatic synthesizer.
10. λ method of producing a compound of claim 1, wherein said oligodeoxynucleotide is synthesized in an automatic synthesizer, followed by covalent attachment of the linker arm and/or the covalently attached group outside an automatic synthesizer.
11. λ method of producing a compound of claim 8, wherein said thiol-containing oligodeoxynucleotide is synthesized in an automatic synthesizer, followed by covalent attachment of the acridine and the maleimide intermediate outside an automatic synthesizer.
12. The compound according to claim 1, wherein the oligodeoxynucleotide is linked at its 3' end with a linker arm to a covalently attached group.
13. The compound according to claim 1, wherein the oligodeoxynucleotide is linked at intermediate points within the oligodeoxynucleotide with a linker arm to a covalently attached group.
14. A method of destroying or attenuating viral activity, said method comprising administering an effective amount of a compound as set forth in claim 1 to a mammal infected with said virus, wherein said oligodeoxynucleotide's base sequence complements regions of the viral DNA or viral RNA and thereby delivers the covalently attached group to the targeted biological site.
15. λ method of destroying or attenuating mammalian gene expression, said method comprising administering an effective amount of a compound as set forth in claim 1 to a mammal that contains said gene, wherein said oligodeoxynucleotide's base sequence complements regions of said mammalian gene's DNA or mammalian mRNA and thereby delivers the covalently attached group to the targeted biological site.
16. A method of inhibiting HIV expression in in vitro T-cells, said method comprising administering an effective amount of a compound as set forth in claim 1, wherein said oligodeoxynucleotide complements regions of the viral DNA or viral RNA of said HIV, and said covalently attached group is anthraquinone or acridine.
17. λ method of inhibiting HIV expression in in vitro T-cells, said method comprising administering an effective amount of a compound as set forth in dais 1, wherein said oligodeoxynucleotides' base sequence is the antisense oligomer against rev and said covalently attached group is anthraquinone or acridine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US34007389A | 1989-04-18 | 1989-04-18 | |
| US340,073 | 1989-04-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1990012802A1 true WO1990012802A1 (en) | 1990-11-01 |
Family
ID=23331752
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1990/001501 WO1990012802A1 (en) | 1989-04-18 | 1990-03-23 | Novel oligodeoxynucleotides with 5'-linked chemical groups, method of production thereof and use thereof |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0469042A4 (en) |
| JP (1) | JPH04500679A (en) |
| AU (1) | AU633911B2 (en) |
| CA (1) | CA2049361A1 (en) |
| WO (1) | WO1990012802A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0490434A1 (en) * | 1990-12-10 | 1992-06-17 | Akzo Nobel N.V. | Labelled, modified oligonucleotides |
| US5733523A (en) * | 1990-12-10 | 1998-03-31 | Akzo Nobel N.V. | Targeted delivery of a therapeutic entity using complementary oligonucleotides |
| WO2001004129A1 (en) * | 1999-07-07 | 2001-01-18 | Exiqon A/S | Synthesis of stable quinone and photoreactive ketone phosphoramidite reagents for solid phase synthesis of photoreactive-oligomer conjugates |
| US6531591B1 (en) | 1999-07-07 | 2003-03-11 | Exiqon A/S | Synthesis of stable quinone and photoreactive ketone phosphoramidite reagents for solid phase synthesis of photoreactive-oligomer conjugates |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3687808A (en) * | 1969-08-14 | 1972-08-29 | Univ Leland Stanford Junior | Synthetic polynucleotides |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1440626A (en) * | 1973-05-02 | 1976-06-23 | Farmaceutici Italia | |
| IL91673A0 (en) * | 1988-09-20 | 1990-04-29 | Us Health | Method for tagging an oligodeoxynucleotide |
| AU5418290A (en) * | 1989-03-31 | 1990-11-05 | University Patents Inc. | Polynucleotide phosphorodithioates as therapeutic agents for retroviral infections |
| AU6603690A (en) * | 1989-10-05 | 1991-04-28 | University Patents Inc. | Nucleoside and polynucleotide thiophosphoramidite and phosphorodithioate compounds and processes |
-
1990
- 1990-03-23 JP JP50641790A patent/JPH04500679A/en active Pending
- 1990-03-23 AU AU55263/90A patent/AU633911B2/en not_active Ceased
- 1990-03-23 WO PCT/US1990/001501 patent/WO1990012802A1/en not_active Application Discontinuation
- 1990-03-23 CA CA 2049361 patent/CA2049361A1/en not_active Abandoned
- 1990-03-23 EP EP19900906714 patent/EP0469042A4/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3687808A (en) * | 1969-08-14 | 1972-08-29 | Univ Leland Stanford Junior | Synthetic polynucleotides |
Non-Patent Citations (4)
| Title |
|---|
| Gene, Vol. 72, 28 February 1988, C.A. STEIN et al., "Phosphorothioate and normal oligodeoxyribonucleatides with 5'-linked acridine: characterization and preliminary kinetics of cellular uptake", pages 333-341. * |
| Nucleic Acid Research, Vol. 14, No. 15, 11 August 1986 SUDHIR AGRAWAL et al., "Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides", see pages 6227-6245. * |
| Proc. Natl. Acad. Sci. USA, Vol. 84, 30 November 1987, MAKOTO MATSUKURA et al., "Phosphorothioate analogs of oligodeoxynucleotides: Inhibitors of replication and cytopathic effects of human immunodeficiency virus", pages 7706-7710. * |
| See also references of EP0469042A4 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0490434A1 (en) * | 1990-12-10 | 1992-06-17 | Akzo Nobel N.V. | Labelled, modified oligonucleotides |
| US5733523A (en) * | 1990-12-10 | 1998-03-31 | Akzo Nobel N.V. | Targeted delivery of a therapeutic entity using complementary oligonucleotides |
| WO2001004129A1 (en) * | 1999-07-07 | 2001-01-18 | Exiqon A/S | Synthesis of stable quinone and photoreactive ketone phosphoramidite reagents for solid phase synthesis of photoreactive-oligomer conjugates |
| US6531591B1 (en) | 1999-07-07 | 2003-03-11 | Exiqon A/S | Synthesis of stable quinone and photoreactive ketone phosphoramidite reagents for solid phase synthesis of photoreactive-oligomer conjugates |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0469042A1 (en) | 1992-02-05 |
| CA2049361A1 (en) | 1990-10-19 |
| AU633911B2 (en) | 1993-02-11 |
| EP0469042A4 (en) | 1992-07-01 |
| JPH04500679A (en) | 1992-02-06 |
| AU5526390A (en) | 1990-11-16 |
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