Oligothionucleotides

• US 5,639,873 A
• Filed: 08/04/1994
• Issued: 06/17/1997
• Est. Priority Date: 02/05/1992
• Status: Expired due to Term

• Current Assignee: Centre National De La Recherche Scientifique
• Original Assignee: Centre National De La Recherche Scientifique
• Inventors: Barascut, Jean-Louis, Imbach, Jean-Louis
• Primary Examiner(s): LeGuyader, John L.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US08/284,484
• Time in Patent Office: 1,048 Days
• Field of Search: 435/6, 435/91.1, 435/22.1, 536/23.1, 536/24.1, 536/24.3, 536/24.31, 536/24.5, 536/25.3, 536/25.31, 536/25.33, 536/25.34, 514/44
• US Class Current: 536/25.3
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/32 of the sugar
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/337 in alpha-anomeric form
• CPC Class Codes: C12N 2310/353 linked to the nucleic acid via an atom other than carbon
• CPC Class Codes: C12N 2310/3531 Hydrogen
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

Abstract

Chemical compounds consisting of an alpha or beta oligo-4'"'"'-thioribonucleotide or oligo-4'"'"'-thio-2'"'"'-deoxyribonucleotide characterized in that they comprise a concatenation of 4'"'"'-thioribonucleotides or 4'"'"'-thio-2'"'"'deoxyribonucleotides, respectively. The concatenation is optionally linked to an effector, in particular a radical corresponding to an intercalating agent or a photoactivatable or chemical radical, e.g. a radical carrying a function which reacts directly or indirectly with the nucleotide chains, or a radical whose presence enables easy and sensitive detection. Methods for preparing said compounds, and their uses in therapeutics, diagnostics and as laboratory reagents, are also described.

Claims

1. An oligomeric compound comprising a first oligothionucleotide, wherein said oligothionucleotide is an oligo-4'"'"'-thio-2'"'"'-deoxyribonucleotide which comprises 4'"'"'-thio-2'"'"'-deoxyribonucleotides linked by internucleotide linkages, or an oligo-4'"'"'-thioribonucleotide which comprises 4'"'"'-thioribonucleotides linked by internucleotide linkages.

2. The oligomeric compound of claim 1, having the formula:

##STR40## wherein;

the B radicals are, independently, nucleic acid bases and are attached to the glycoside ring according to a non-natural alpha anomeric configuration;

the X radicals are, independently, an oxoanion O^-, a thioanion S^-, an alkyl group, an alkoxy group, a thioalkyl group, an alkyl substituted by a nitrogen-containing heterocycle, an alkoxy radical substituted by a nitrogen-containing heterocycle, or a --Y--Z group;

R and R'"'"' are, independently, a hydrogen atom, a --Y--Z group, a Y'"'"'--Z'"'"' group, an RNA type oligonucleotide, or a DNA type oligonucleotide;

Y and Y'"'"' are, independently, a straight or branched alkylene radical --alk--, or a radical selected from the group consisting of ##STR41## wherein;

U is O, S, or N;

E is an oxoanion O^-, a thioanion S^-, an alkyl group, an alkoxy group, a thioalkyl group, an alkyl substituted by a nitrogen-containing heterocycle, or an alkoxy radical substituted by a nitrogen-containing heterocycle;

J is a hydrogen atom or a hydroxyl group;

Z and Z'"'"' are, independently, a hydroxyl group or an effector selected from the group consisting of polycyclic compounds having a planar configuration, acridine, proflavine, biotin, furocoumarin, daunomycin, anthracycline, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, porphyrins, 1,10-phenanthroline, phenanthridine, ellipticine, ellipticinium, dipyrido(1,2-a;

3'"'"',2'"'"'-d)imidazole, diazapyrene, 4-azidoacetophenone, ethylene-imine, beta-chloroethylamine, psoralen, methylpyrroporphyrin, and aromatic compounds absorbing near-ultraviolet or visible radiations;

n is an integer including 0;

L is an oxygen atom, a sulfur atom, or an --NH-- group.

3. The oligomeric compound of claim 2, wherein Y and Y'"'"' are independently ##STR42## wherein U is O, S, or N, and n'"'"' is an integer from 1 to 10.

4. The oligomeric compound of claim 2, wherein L is oxygen, R and R'"'"' are hydrogen atoms, and B is a natural nucleic acid base.

5. The oligomeric compound of claim 2, wherein Z and Z'"'"' are independently acridine, furocoumarin, daunomycin, 1,10-phenanthroline, phenanthridine, proflavine, porphyrins, dipyrido(1,2-a:

3'"'"'-d) imidazole, ellipticine, ellipticinium, or diazapyrene.

6. The oligomeric compound of claim 2, wherein the nucleic acid bases are thymine, adenine, 2-aminoadenine, cytosine, guanine, uracil, 5-bromouracil, 8-azidoadenine, 7-deazaadenine, 7-deazaguanine, 4-azidocytosine, or 4-azidothymine.

7. The oligomeric compound of claim 2, wherein J is OH.

8. The oligomeric compound of claim 1 further comprising an RNA type oligonucleotide or a DNA type oligonucleotide linked by an internucleotide linkage to said compound of claim 1, wherein oxygen is the heteroatom of the furanose ring of the nucleotides of said DNA type oligonucleotide or RNA type oligonucleotide.

9. The oligomeric compound of claim 1 further comprising an RNA type oligonucleotide or a DNA type oligonucleotide linked by internucleotide linkages at one or each of its ends to said compound of claim 1, wherein oxygen is the heteroatom of the furanose ring of the nucleotides of said DNA type oligonucleotide or RNA type oligonucleotide.

10. The oligomeric compound of claim 2 further comprising an RNA type oligonucleotide or a DNA type oligonucleotide linked by an internucleotide linkage to said compound of claim 2, wherein oxygen is the heteroatom of the furanose ring of the nucleotides of said DNA type oligonucleotide or RNA type oligonucleotide.

11. The oligomeric compound of claim 2 further comprising an RNA type oligonucleotide or a DNA type oligonucleotide linked by internucleotide linkages at one or each of its ends to said compound of claim 2, wherein oxygen is the heteroatom of the furanose ring of the nucleotides of said DNA type oligonucleotide or RNA type oligonucleotide.

12. The oligomeric compound of claim 8, further comprising a second oligothionucleotide linked to said DNA type oligonucleotide, wherein said second oligothionucleotide comprises 4'"'"'-thlo-2'"'"'-deoxyribonucleotides or 4'"'"'-thioribonucleotides linked by internucleotide linkages.

13. The oligomeric compound of claim 10, further comprising a second oligothionucleotide linked to said DNA type oligonucleotide, wherein said second oligothionucleotide comprises 4'"'"'-thio-2'"'"'-deoxyribonucleotides or 4'"'"'-thioribonucleotides linked by internucleotide linkages.

14. The oligomeric compound of claim 12, wherein said first and second oligothionucleotides are oligo-4'"'"'-thioribonucleotides.

15. The oligomeric compound of claim 13, wherein said first and second oligothionucleotides are oligo-4'"'"'-thioribonucleotides.

16. A process for preparing the oligomeric compound of claim 1, comprising the steps:

(a) synthesizing an oligothionucleotide using 4'"'"'-thionucleotides, wherein the internucleotide linkages of said oligothionucleotide are selected from the group consisting of phosphodiester, phosphotriester, phosphoramidite, and hydrogen phosphonate, and wherein said 4'"'"'-thionucleotides are completely protected; and

(b) removing the protecting groups to obtain said oligomeric compound.

17. A process for preparing the oligomeric compound of claim 2, wherein both Z and Z'"'"' are OH, by a phosphoramidite method comprising the steps:

(a) protecting the 5'"'"' position of a 4'"'"'-thionucleotide or an oligo-4'"'"'-thionucleotide with dimethoxytrityl and protecting the 3'"'"'position of a 4'"'"'-thionucleotide or an oligo-4'"'"'-thionucleotide with methyl diisopropylaminophosphoramidite;

(b) functionalizing a solid support having an amino group by incorporating a 4'"'"'-thionucleoside derivative having a succinyl linkage between the 3'"'"'-hydroxyl group of the 4'"'"'-thionucleoside derivative and the amino group of the solid support;

(c) elongating the oligothionucleotide chain in a synthesizing reactor; and

(d) detaching the oligothionucleotide from the solid support, deprotecting the 5'"'"' and 3'"'"' positions, and purifying said oligomeric compound.

18. The oligomeric compound of claim 1 further comprising a linked effector, which is an intercalating agent or a chemical or photoactivable radical, wherein said effector is a polycyclic compound having a planar configuration, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, porphyrins, 1,10-phenanthroline, 4-azidoacetophenone, ethylene-imine, beta-chloroethylamine, psoralen, acridine, proflavine, biotin, furocoumarin, daunomycin, anthracycline, phenanthridine, ellipticine, ellipticinium, methylpyrroporphyrin, dipyrido(1,2-a:

3'"'"',2'"'"'-d) imidazole, diazapyrene, or an aromatic compound absorbing near-ultraviolet or visible radiations.