Oligonucleotide analogues

• US 7,034,133 B2
• Filed: 07/29/2002
• Issued: 04/25/2006
• 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:
  US10/208,650
• Publication Number:
  US 20030144231A1
• Time in Patent Office: 1,366 Days
• Field of Search: 536/23.1, 536/22.1, 536/24.3, 536/25.3, 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 with 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. A nucleoside analogue (hereinafter termed “

LNA”

) 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 a 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 or a 3′

-terminal group;

one pair of non-geminal substituents R^4*, and R^2*, designating a biradical selected from the following group;

(a) —

(CR*R*)_r—

O—

(CR*R*)_s- wherein r is 0 (zero) and s is greater than 1, or s is 0 (zero) and r is greater than 1,

and further wherein each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; 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, heteroarylcarbonyl, 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.

16. A kit for the isolation, purification, amplification, detection, identification, quantification, or capture of natural or synthetic nucleic acids, the kit comprising a reaction body and one or more LNAs as defined in claim 1.

17. A nucleic acid compound comprising the nucleoside analogue of claim 1.

24. The nucleoside analogue of claim 1, wherein the analogue is represented by the following structure:

B=adenine, guanine, thymine, 5-methyl-cytosine, cytosine, uracil, 2,6-diaminopurine.

25. The nucleoside analogue of claim 24, wherein the analogue is one of the following specific compounds:

a) (1R,5R,7R,8S)-8-hydroxy-5- (hydroxymethyl)-7- (adenin-9-yl)-2,6-dioxabicyclo[3.2.1]octane,

b) (1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(guanin-9-yl)-2,6-dioxabicyclo[3.2.1]octane,

c) (1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(thymin-1-yl)-2,6-dioxabicyclo[3.2.1]octane,

d) (1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(5-methyl-cytosin-1-yl)-2,6-dioxabicyclo[3.2.1]octane,

e) (1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(cytosin-1-yl)-2,6-dioxabicyclo[3.2.1]octane,

f) (1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(uracil-1-yl)-2,6-dioxabicyclo[3.2.1]octane; and

g) (1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(2,6-diaminopurin-9-yl)-2,6-dioxabicyclo[3.2.1]octane.

26. The nucleoside analogue of claim 1, wherein the analogue is represented by the following structure:

B=adenine, guanine, thymine, 5-methyl-cytosine, cytosine, until, 2,6-diaminopurine.

27. The nucleoside analogue of claim 1, wherein the analogue is one of the following specific compounds:

a) (1R,3R,4S,7R)-7-hydroxy-1-(hydroxymethyl)-3-(adenin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane,

b) (1R,3R,4S,7R)-7-hydroxy-1-(hydroxymethyl)-3-(guanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane,

c) (1R,3R,4S,7R)-7-hydroxy-1-(hydroxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,

d) (1R,3R,4S,7R)-7-hydroxy-1-(hydroxymethyl)-3-(5-methyl-cytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,

e) (1R,3R,4S,7R)-7-hydroxy-1-(hydroxymethyl)-3-(cytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,

f) (1R,3R,4S,7R)-7-hydroxy-1- (hydroxymethyl)-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane; and

g) (1R,3R,4S,7R)-7-hydroxy-1-(hydroxymethyl)-3-(2,6-diaminopurin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane.

29. A method of preparing an LNA modified oligonucleotide (an oligomer) comprising making the oligonucleotide with the LNA of claim 1.

30. The method of claim 29, wherein the LNA modified oligonucleotide comprises normal nucleosides.

2. A nucleoside analogue (hereinafter LNA) of the general formula II

wherein the substituent B is selected from nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

X is selected from —

O—

;

one of the substituents R², R³, and R^3* is a group Q*;

each of Q and Q* is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot—

O—

, Act-O—

, mercapto, Prot—

S—

, Act—

S—

, C_1-6-alkylthio, amino, ProtN(R^H)-, Act-N(R^H)-, mono- or di(C_1-6-alkyl)amino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkenyloxy, optionally substituted C_2-6-alkynyl, optionally substituted C_2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot—

O—

CH₂-, Act-O—

CH₂-, aminomethyl, Prot-N(R^H)—

CH₂-, Act-N(R^H)—

CH₂-, carboxymethyl, sulphonomethyl, where Prot is a protection group for —

OH, —

SH, and —

NH(R^H), respectively, Act is an activation group for —

OH, —

SH, and —

NH(R^H), respectively, and R^H is selected from hydrogen and C_1-6-alkyl;

wherein R^2* and R^4* together designate a biradical selected from the following group;

(a) —

(CR*R*)_r—

O—

(CR*R*)_s-wherein r is 0 (zero) and s is greater than 1, or s is 0 (zero) and r is greater than 1,

and further wherein each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; and

each of the substituents R^1*, R², R³, R⁵, and R^5*, which are not involved in Q, Q*, 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, heteroarylcarbonyl, amino, mono- and di(C_1-6-alkyl)amino, carbamoyl, mono- and di(C_1-6-alkyl)-amino-carbonyl, amino-C_1-6-alkylaminocarbonyl, mono- and di(C_1-6-alkyl)amino- C_1-6-alkyl-aminocarbonyl, C_1-6 alkylcarbonylamino, 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;

with the proviso that,

any chemical group (including any nucleobase), which is reactive under the conditions prevailing in oligonucleotide synthesis, is optionally functional group protected.

3. A nucleoside analogue according to claim 2, wherein the group B is selected from nucleobases and functional group protected nucleobases.

4. A nucleoside analogue according to any of the claims 2-3, wherein each of the substituents R^1*, R², R³, R^3*, R⁵, and R^5*, which are present and not involved in Q, Q*, 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-alkylcarbonylamino, carbamido, azido, C_1-6-alkanoyloxy, sulphono, sulphanyl, C_1-6-alkylthio, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, and halogen, and where R^N*, when present and not involved in a biradical, is selected from hydrogen and C_1-4-alkyl, with the proviso that any hydroxy, amino, mono(C_1-6-alkyl)amino, sulfanyl, and carboxy is optionally protected.

5. A nucleoside analogue according to any of the claim 2 or 3, each of the substituents R^1*, R², R³, R^3*,and R⁵, R^5*, which are present and not involved in Q* designate hydrogen.

6. A nucleoside analogue according to any of the claim 2 or 3, wherein R^3* designates P*.

7. A nucleoside analogue according to claim 2, wherein Q is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot—

O—

, mercapto, Prot—

S—

, C_1-6-alkylthio, amino, Prot-N(R^H)-, mono- or di(C_1-6 alkyl)amino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6 alkenyloxy, optionally substituted C_2-6-alkynyl, optionally substituted C_2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot—

O—

CH₂-, aminomethyl, Prot-N(R^H)—

CH₂-, carboxymethyl, sulphonomethyl, where Prot is a protection group for —

OH, —

SH, and —

NH(R^H), respectively, and R^H is selected from hydrogen and C_1-6-alkyl; and

Q* is selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Act-O—

, mercapto, Act—

S—

, C_1-6-alkylthio, amino, Act-N(R^H)-, mono- or di(C_1-6-alkyl amino, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6-alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkenyloxy, optionally substituted C_2-6 alkynyl, optionally substituted C_2-6 alkynyloxy, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, where Act is an activation group for —

OH, —

SH, and —

NH(R^H), respectively, and R^H is selected from hydrogen and C_1-6-alkyl.

8. A nucleoside analogue according to claim 2, having the general formula IIa

wherein the substituents Q, B, R^1*, R², R^2*, R³, R^3*, R^4*, R⁵, and R^5* are as defined in claim 2 provided the nucleoside analogue has a configuration other than α

-L.

9. A nucleoside analogue according to claim 8, wherein R^3* designates P*.

10. A nucleoside analogue according to claim 9, wherein R² selected from hydrogen, hydroxy, and optionally substituted C_1-6-alkoxy, and R^1*, R³, R⁵, and R^5*, designate hydrogen.

11. A nucleoside analogue according to any of the claim 9 or 10, wherein B is selected from nucleobases.

12. A nucleoside analogue according to claim 11, wherein B is selected from adenine and guanine thymine, cytosine uracil purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine, 5- (C₃-C₆)-alkynyl-cytosine, 2,6-diaminopyrimidine,2,6-diaminopyrazine,1-methyl-pyrazolo[4,3-d]pyrimidine-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.

13. A nucleoside analogue according to claim 2 of the general formula IIa

wherein X is —

O—

;

B is selected from nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

R^3* is a group Q*;

each of Q and Q* is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot—

O—

, Act-O—

, mercapto, Prot—

S—

, Act—

S—

, C,_1-6-alkylthio, amino, Prot-N(R^H)-, Act-N(R^H)-, mono- or di(C_1-6-alkyl)amino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkenyloxy, optionally substituted C_2-6-alkynyl, optionally substituted C_2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot—

O—

CH₂-, Act-O—

CH₂-, aminomethyl, Prot-N(R^H)—

CH₂-, Act-N(R^H)—

CH₂-, carboxymethyl, sulphonomethyl, where Prot is a protection group for —

OH, —

SH, and —

NH(R^H), respectively, Act is an activation group for —

OH, —

SH, and —

NH(R^H), respectively, and R^H is selected from hydrogen and C_1-6-alkyl;

R^2*and R^4* together designate a biradical selected from the following group;

(a) —

(CR*R*)_t—

O—

(CR*R*)_s-wherein r is 0 (zero) and s is greater than 1, or s is 0 (zero) and r is greater than 1,

and further wherein each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

each of the substituents R^1*, R², R³, R⁵, and R^5* 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, carbamido, 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; and

basic salts and acid addition salts thereof; and

with the proviso that any chemical group (including any nucleobase), which is reactive under the conditions prevailing in oligonucleotide synthesis, is optionally functional group protected.

14. A nucleoside analogue according to any of the claim 13, wherein B is selected from nucleobases.

15. A nucleoside analogue according to claim 14, wherein B is selected from adenine and guanine thymine, cytosine uracil purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C³-C⁶)-alkynyl-cytosine, 2,6-diaminopyrimidine, 2,6-diaminopyrazine, 1-methyl-pyrazolo[4,3-d]pyrimidine-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.

18. The nucleoside analogue of claim 13, wherein Q* represents an activation group for —

OH, —

SH, and —

NH(R^H).

19. The nucleoside analogue of claim 13, wherein said activation group is an optionally substituted phosphoramidite.

20. The nucleoside analogue of claim 13, wherein the nucleoside analogue is a 3′

-phosphoramidite derivative.

21. The nucleoside analogue of claim 20, wherein the nucleoside analogue is an O-phosphoramidite.

22. The nucleoside analogue of claim 21, wherein the O-phosphoramidite is N,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.

28. A nucleoside analogue according to claim 2, having the general formula IIa

wherein the substituents Q, B, R^1*, R², R^2*, R³, R^3*, R^4*, R⁵, and R^5* are as defined in claim 2 provided the nucleoside analogue has a configuration other than β

-D.

31. A nucleoside analogue according to claim 4, wherein each of the substituents R^1*, R²,R³, R^3*, and R⁵, R^5*,which are present and not involved in Q* designate hydrogen.

32. A nucleoside analogue according to claim 4, wherein R^3* designates P*.

33. A nucleoside analogue according to claim 5, wherein R^3* designates P*.

23. A nucleoside analogue of the general formula IIa

wherein X is —

O—

;

B is selected from nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

R^3* is a group Q*;

each of Q and Q* is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot—

O—

, Act-O—

, mercapto, Prot—

S—

, Act—

S—

, C_1-6-alkylthio, amino, Prot-N(R^H)-, Act-N(R^H)-, mono- or di(C_1-6-alkylamino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkynyl, optionally substituted C_2-6-alkenyloxy, optionally substituted C_2-6-alkynyl, optionally substituted C_2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot—

O—

CH₂-, Act-O—

CH₂- , aminomethyl, Prot-N(R^H)—

CH2-, Act-N(R^H)—

CH₂-, carboxymethyl, sulphonomethyl, where Prot is a protection group for —

OH, —

SH, and —

NH(R^H), respectively, Act is an activation group for —

OH, —

SH, and —

NH(R^H), respectively, and R^H is selected from hydrogen and C_1-6-alkyl;

R^2* and R^4* together designate a biradical selected from the following group;

(a) —

(CR*R*)_r—

O—

(CR*R*)_s- wherein r is 0 (zero) and s is greater than 1, or s is 0 (zero) and r is greater than 1,

and further wherein each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

each of the substituents R^1*, R², R³, R⁵, and R^5* 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, carbamido, 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;

and basic salts and acid addition salts thereof;

and with the proviso that any chemical group (including any nucleobase), which is reactive under the conditions prevailing in oligonucleotide synthesis, is optionally functional group protected.