Oligonucleotide analogues

• US 6,794,499 B2
• Filed: 09/11/1998
• Issued: 09/21/2004
• Est. Priority Date: 09/12/1997
• Status: Expired due to Term

• Current Assignee: Exiqon A/S (Qiagen NV)
• Original Assignee: Exiqon A/S (Qiagen NV)
• Inventors: Nielsen, Poul, Wengel, Jesper
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/152,059
• Publication Number:
  US 20020068708A1
• Time in Patent Office: 2,202 Days
• Field of Search: 536/22.1, 536/23.1, 536/24.3, 536/25.3, 514/1, 514/44
• US Class Current: 536/23.1
• 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

Abstract

Novel oligomers, and synthesis thereof, comprising one or more bi-, tri, or polycyclic nucleoside analogues are disclosed herein. The nucleoside analogues have a “locked” structure, termed Locked Nucleoside Analogues (LNA). LNA'"'"'s exhibit highly desirable and useful properties. LNA'"'"'s are capable of forming nucleobase specific duplexes and triplexes will single and double stranded nucleic acids. These complexes exhibit higher thermostability than the corresponding complexes formed with normal nucleic acids. The properties of LNA'"'"'s allow for a wide range of uses such as diagnostic agents and therapeutic agents in a mammal suffering from or susceptible to, various diseases.

Claims

1. An oligomer comprising at least one LNA nucleoside of the general formula I

wherein

X is selected from —

O—

;

B is selected from hydrogen, hydroxy, optionally substituted C_1-4-alkoxy, optionally substituted C_1-4-alkyl, optionally substituted C_1-4-acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

P designates the radical position for an internucleoside linkage to a succeeding monomer, or a 5′

-terminal group, such internucleoside linkage or 5′

-terminal group optionally including the substituent R⁵;

one of the substituents R², R^2*, R³, and R^3* is a group P* which designates an internucleoside linkage to a preceding monomer, or a 3′

-terminal group,

one pair of non-geminal substituents R^4*, and R^2*, designating a biradical consisting of 2-5 groups/atoms selected from —

(CR*R*)r—

Y—

(CR*R*)s—

, —

(CR*R*)r—

Y—

(CR*R*)s—

Y—

, —

Y—

(CR*R*)r+s—

Y—

, —

Y—

(CR*R*)r—

Y—

(CR*R*)s—

, —

(CR*R*)r+s—

, each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, Y is —

O—

, —

S—

, 0 (zero) or —

N(RN)—

, and each of r and s is 0-4 with the proviso that the sum r+s is 1-4, and provided that when the biradical is —

(CR*R*)r—

Y—

(CR*R*)s—

, then Y is —

S—

or —

N(R^N′

)—

; and

each of the substituents R^1*, R², R³, R⁵, and R^5*, which are present and not involved in P, P* is independently selected from hydrogen, optionally substituted C_1-12-alkyl, optionally substituted C_2-12-alkenyl, optionally substituted C_2-12-alkynyl, hydroxy, C_1-12-alkoxy, C_2-12-alkenyloxy, carboxy, C_1-12-alkoxycarbonyl, C_1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarycarbonyl, amino, mono- and di(C_1-6-alkyl)amino, carbamoyl, mono- and di(C_1-6-alkyl)-amino-carbonyl, amino-C_1-6-alkyl-aminocarbonyl, mono- and di(C_1-6-alkyl)amino-C_1-6-alkyl-aminocarbonyl, C_1-6-alkyl-carbonylamino, carbamido, C_1-6-alkanoyloxy, sulphono, C_1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C_1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted;

and basic salts and acid addition salts thereof.

2. An oligomer of claim 1 wherein the oligomer comprises 1 to 10000 nucleosides of the formula I and 0-10000 nucleosides selected from naturally occurring nucleosides and nucleoside analogues, with the proviso that the sum of the number of nucleosides and the number of LNA(s) is at least 2.

3. An oligomer of claim 2 wherein at least one LNA nucleoside comprises a nucleobase as the substituent B.

4. An oligomer of claim 1 wherein one of the substituents R³ and R^3* designates P*.

5. An oligomer of 1 wherein one or more nucleosides have the following formula Ia.

wherein P, P*, B, X, R^1*,R², R^2*, R¹, R^4*, R⁵ and R^5* are as defined in claim 1.

6. An oligomer of claim 5 wherein R^3* designates P*.

7. An oligomer of claim 6 wherein the oligomer comprises one biradical constituted by two non-geminal substituents.

8. An oligomer of claim 1 wherein R^3* designates P*.

9. An oligomer of claim 8 wherein, R² is selected from hydrogen, hydroxy, and optionally substituted C_1-4-alkoxy, and R^3*, R³, R⁵, and R^5* designate hydrogen.

10. An oligomer of claim 9 wherein B is selected from nucleobases.

11. An oligomer of claim 10 wherein the oligomer comprises at least one LNA nucleoside wherein B is selected from adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴, N⁶-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 2,6-diaminopyrimidino,2,6-diaminopyrazine,1-methyl-pyrazolo[4,3-d]pyrimidino-5,7(4H,6H)-dione, 1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, and inosine.

12. An oligomer according to claim 1 wherein any internucleoside linkage of the one or more LNA nucleosides is selected from linkages consisting of 2 to 4 groups/atoms selected from —

CH₂—

, —

O—

, —

S—

, —

NR^H—

, >

C═

O, >

NR^H, >

C═

S, —

Si(R″

)₂—

—

SO—

, —

S(O)₂—

, —

P(O)₂—

, —

P(O,S)—

, —

P(S)₂—

, —

PO(R″

)—

, —

PO(OCH₂)—

, and —

PO(NHR^H)—

, where R^H is selected form hydrogen and C_1-4-alkyl, and R″

is selected from C_1-6-alkyl and phenyl.

13. An oligomer of claim 12 wherein any internucleoside linkage of the one or more LNA nucleosides is selected from —

CH₂—

CH₂—

CH₂—

, —

CH₂—

CO—

CH₂—

CH₂—

CHOH—

CH₂—

, —

O—

CH₂—

O—

, —

O—

CH₂—

CH₂—

, —

O—

CH₂—

CH═

, —

CH₂—

CH₂—

O—

, —

NR^H—

CH₂—

, —

CH₂—

CH₂—

NR^H—

, —

CH₂—

NR^H—

CH₂—

, —

O—

CH₂—

CH₂NR^H—

, —

NR^H—

CO—

O—

, —

NR^H—

CO—

NR_H—

, —

NR^H—

CS—

NR^H—

, —

NR^H—

C(═

NR^H)—

NR^H—

, —

NR^H—

CO—

CH₂—

NR^H—

, —

O—

CO—

O—

, —

O—

CO—

CH₂—

O—

, —

O—

CH₂—

CO—

O—

, —

CH₂—

CO—

NR^H—

, —

O—

CO—

NR^H—

, —

NR^H—

CO—

CH₂—

, —

O—

CH₂—

CO—

NR^H—

, —

O—

CH₂—

CH₂—

NR^H—

, —

CH═

N—

O—

, —

CH₂—

NR^H—

O—

, —

CH₂—

O—

N═

, —

CH₂—

O—

NR^H—

, —

CO—

NR^H—

CH₂—

, —

CH₂—

NR^H—

O—

, —

CH₂—

NR^H—

CO—

, —

O—

NR^H—

CH₂—

, —

O—

NR^H—

, —

O—

CH₂—

S—

, —

S—

CH₂—

O—

, —

CH₂—

CH₂—

S—

, —

O—

CH₂—

CH₂—

S—

, —

S—

CH₂—

CH═

, —

S—

CH₂—

CH₂—

, —

CH₂—

CH₂—

O—

, —

S—

CH₂—

CH₂—

S—

, —

CH₂—

S—

CH₂—

, —

CH₂—

SO—

CH₂—

, —

CH₂—

SO₂—

CH₂—

, —

O—

SO—

O—

, —

S(O)₂—

O—

, —

O—

S(O)₂—

CH₂—

, —

O—

S(O)₂—

NR^H—

, —

NR^H—

S(O)₂—

CH₂—

, —

O—

S(O)₂—

CH₂—

, —

O—

P(O)₂—

O—

, —

O—

P(O,S)—

O—

, —

O—

P(S)₂—

O—

, —

S—

P(O)₂—

O—

, —

S—

P(O,S)—

O—

, —

S—

P(S)₂—

O—

, —

O—

P(O)₂—

S—

, —

O—

P(O,S)—

S—

, —

O—

P(S)₂—

S—

, —

S—

P(O)₂—

S—

, —

S—

P(O,S)—

S—

, —

S—

P(S)₂—

S—

, —

O—

PO(R″

)—

O—

, —

O—

PO(OCH₃)—

O—

, —

O—

PO(BH₃)—

O—

, —

O—

PO(NHR^H)—

O—

, —

O—

P(O)₂—

NR^H—

, —

NR^H—

P(O)₂—

O—

, —

O—

P(O,NR^H)—

O—

, and —

O—

Si(R″

)₂—

O—

.

14. An oligomer of claim 13 wherein any internucleoside linkage of the one or more LNA nucleosides is selected from —

CH₂—

CO—

NRH—

, —

CH₂—

NR^H—

O—

, —

S—

CH₂—

O—

, —

O—

P(O)₂—

, —

O—

P(O,S)—

O—

, —

O—

P(S)₂—

O—

, —

NR^H—

P(O)₂—

O—

, —

O—

P(O,NR^H)—

O—

, —

O—

PO(R″

)—

O—

, —

O—

PO(CH₂)—

O—

, and —

O—

PO(NHR^N)—

O—

, where R^H is selected form hydrogen and C_1-4-alkyl, and R″

is selected from C_1-6-alkyl and phenyl.

15. An oligomer of claim 1 wherein each of the substituents R^1*, R², R³, R^3*, R⁵, and R^5*, of the one or more LNA nucleosides, which are present and not involved in P, P* is independently selected from hydrogen, optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl), hydroxy, C_1-6-alkoxy, C_2-6-alkenyloxy, carboxy, C_1-6-alkoxycarbonyl, C_1-6-alkylcarbonyl, formyl, amino, mono- and di(C_1-6-alkyl)amino, carbamoyl, mono- and di(C_1-6-alkyl)-amino-carbonyl, C_1-6-alkyl-carbonylamino, cabamido, azido, C_1-6-alkanoyloxy, sulphono, sulphanyl, C_1-6-alkylthio, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and halogen, where two geminal substituents together may designate oxo, and where R^N*, when present and not involved in a biradical, is selected from hydrogen and C_1-4-alkyl.

16. An oligomer of claim 1 wherein each of the substituents R^1*, R², R¹, R^3*, R⁵, and R^5*, of the LNA(s), which are present and not involved in P, P* designate hydrogen.

17. An oligomer of claim 1 wherein P is a 5′

-terminal group selected from hydrogen, hydroxy, optionally substituted C_1-6-alkyl, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkylcarbonyloxy, optionally substituted aryloxy, monophosphate, diphosphate, triphosphate, and —

W—

A′

, wherein W is selected from —

O—

, —

S—

, and —

N(R^H)—

where R^H is selected from hydrogen and C_1-6-alkyl, and where A′

is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

18. An oligomer of claim 1 wherein P* is a 3′

-terminal group selected from hydrogen, hydroxy, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkylcarbonyloxy, optionally substituted aryloxy, and —

W—

A′

, wherein W is selected from —

O—

, —

S—

, and —

N(R^H)—

where R^H is selected from hydrogen and C_1-6-alkyl, and where A′

is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

19. An oligomer of claim 1 wherein the oligomer corresponds to the following formula V:

20. An oligomer of claim 1 further comprising a PNA mono- or oligomer segment of the formula

wherein B is a defined above for the formula I, AASC designates hydrogen or an amino acid side chain, t is b 1-5, and w is 1-50.

21. An oligomer of claim 1 which has an increased specificity towards complementary ssRNA or ssDNA compared to a corresponding reference oligonucleotide which does not contain any LNA units.

22. An oligomer of claim 1 which has an increased affinity towards complementary ssRNA or ssDNA compared to a corresponding reference oligonucleotide which does not contain any LNA units.

23. An oligomer of claim 1 which is capable of binding to a target sequence in a dsDNA or dsRNA molecule by of strand displacement or by triple helix formation.

24. An oligomer of claim 1 which is more resistant to nucleases than a corresponding reference oligonucleotide which does not contain any LNA units.

25. An oligomer according to claim 1 which has nucleic acid catalytic activity.

26. A diagnostic or analysis kit comprising an oligonucleotide of claim 1.

27. A kit of claim 26 wherein the oligonucleotide is immobilized on a solid support.

28. A diagnostic or analysis kit comprising a reaction body and one or more oligonucleotides of claim 1.

29. The kit of claim 28 wherein the one or more oligonucleotides are immobilized on the reaction body.

30. A diagnostic or analysis kit comprising a reaction body and one or more oligonucleotides of claim 1.

31. The kit of claim 30 wherein the one or more oligonucleotides are immobilized on the reaction body.

32. A diagnostic or analysis kit comprising a reaction body and one or more oligonucleotides of claim 9.

33. The kit of claim 32 wherein the one or more oligonucleotides are immobilized on the reaction body.