Xylo-LNA analogues

• US 7,084,125 B2
• Filed: 03/17/2000
• Issued: 08/01/2006
• Est. Priority Date: 03/18/1999
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Exiqon A/S (Qiagen NV)
• Inventors: Wengel, Jesper
• Primary Examiner(s): Epps Ford, Janet
• Application Number:
  US09/528,110
• Publication Number:
  US 20030082807A1
• Time in Patent Office: 2,328 Days
• Field of Search: 536/22.1, 536/24.5, 536/23.1, 536/25.34, 514/44, 435/91.5
• US Class Current: 514/44.00A
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 9/04 Cyclic acetals

Abstract

Based on the above and on the remarkable properties of the 2′-O,4′-C-methylene bridged LNA monomers it was decided to synthesise oligonucleotides comprising one or more 2′-O,4′-C-methylene-β-D-xylofuranosyl nucleotide monomer(s) as the first stereoisomer of LNA modified oligonucleotides. Modelling clearly indicated the xylo-LNA monomers to be locked in an N-type furanose conformation. Whereas the parent 2′-deoxy-β-D-xylofuranosyl nucleosides were shown to adopt mainly an N-type furanose conformation, the furanose ring of the 2′-deoxy-β-D-xylofuranosyl monomers present in xylo-DNA were shown by conformational analysis and computer modelling to prefer an S-type conformation thereby minimising steric repulsion between the nucleobase and the 3′-O-phopshate group (Seela, F.; Wömer, Rosemeyer, H. Helv. Chem. Acta 1994, 77, 883). As no report on the hybridisation properties and binding mode of xylo-configurated oligonucleotides in an RNA context was believed to exist, it was the aim to synthesise 2′-O,4′-C-methylene-β-D-xylofuranosyl nucleotide monomer and to study the thermal stability of oligonucleotides comprising this monomer. The results showed that fully modified or almost fully modified Xylo-LNA is useful for high-affinity targeting of complementary nucleic acids. When taking into consideration the inverted stereochemistry at C-3′ this is a surprising fact. It is likely that Xylo-LNA monomers, in a sequence context of Xylo-DNA monomers, should have an affinity-increasing effect.

Claims

1. An oligomer comprising at least one nucleoside analogue (Xylo-LNA) of the general formula I

2. An oligomer according to claim 1, comprising 1–

10000 Xylo-LNA(s) of the general 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 Xylo-LNA(s) is in the range of 2–

15000.

3. An oligomer according to claim 2, wherein at least one Xylo-LNA comprises a nucleobase as the substituent B.

4. An oligomer according to claim 1, wherein the Xylo-LNA(s) has/have the following formula 1a

5. An oligomer according to claim 1, wherein the biradical is selected from —

(CR*R*)_r+s+1—

, —

(CR*R*)_r—

O—

(CR*R*)_s—

, —

(CR*R*)_r—

S—

(CR*R*)_s—

, —

(CR*R*)_r—

N(R*)—

(CR*R*)_s—

, —

O—

(CR*R*)_r+s—

O—

, wherein each of r and s is 0–

3 with the proviso that the sum r+s is 1–

4.

6. An oligomer according to claim 5, wherein the biradical is —

O—

CH₂—

, —

S—

CH₂—

or —

N(R^N)—

CH₂—

.

7. An oligomer according to claim 1, wherein the oligomer comprises at least one Xylo-LNA wherein B is selected from adenine and guanine and at least one Xylo-LNA wherein B is selected from thymine, cytosine and uracil.

8. An oligomer according to claim 5, wherein the biradical is —

(CH₂)_2-4—

.

9. An oligomer according to any of the claims 6–

8, wherein one R* is selected from hydrogen, hydroxy, optionally substituted C_1-6-alkoxy, optionally substituted C_1-6-alkyl, and any remaining substituents R* are hydrogen.

10. An oligomer according to claim 1, wherein any internucleoside linkage of the Xylo-LNA(s) is selected from linkages consisting of 3, groups/atoms selected from —

CH₂—

, —

O—

, —

S—

, —

NR^H—

, >

C═

O, >

C═

NR^H, >

C═

S, —

Si(R″

)₂—

, —

SO—

, —

S(O)₂—

, —

P(O)₂—

, —

P(O,S)—

, —

P(S)₂—

, —

PO(R″

)—

, —

PO(OCH₃)—

, and —

PO(NHR^H)—

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

is selected from C_1-6-alkyl and phenyl.

11. An oligomer according to claim 10, wherein any internucleoside linkage of the Xylo-LNA(s) 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—

CH₂—

NR^H—

, —

CH₂—

NR^H—

CH₂—

, —

O—

CH₂—

CH₂—

NR^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—

, —

S—

CH₂—

CH₂—

O, —

S—

CH₂—

CH₂—

S—

, —

CH₂—

S—

CH₂—

, —

CH₂—

SO—

CH₂—

, —

CH₂—

SO₂—

CH₂—

, —

O—

SO—

O—

, —

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^N)—

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—

.

12. An oligomer according to claim 1, wherein each of the substituents R^1*, R², R^3*, R⁵, R^5*, of the Xylo-LNA(s), which are present, designate hydrogen.

13. An oligomer according to claim 1, having the following formula V:

G-[Nu-L]_n(0){[Xylo-LNA-L]_m(q)[NU-L]_n(q)}_q-G* 

 

V

wherein

q is 1–

50;

each of n(0), . . . , n(q) is independently 0–

10000;

each of m(1), . . . , m(q) is independently 1–

10000;

with the proviso that the sum of n(0), . . . , n(q) and m(1), . . . , m(q) is 2–

15000;

G designates a 5′

-terminal group;

each Nu independently designates a nucleoside selected from naturally occurring nucleosides and nucleoside analogues;

each Xylo-LNA independently designates a nucleoside analogue;

each L independently designates an internucleoside linkage between two groups selected from Nu and Xylo-LNA, or L together with G* designates a 3′

-terminal group; and

each Xylo-LNA-L independently designates a nucleoside analogue of the general formula 1.

21. The Xylo-LNA modified oligonucleotide as defined in claim 1 conjugated with a compound selected from the group consisting of proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, and PNA.

22. 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 Xylo-LNA modified oligonucleotides as defined in claim 1.

23. 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 Xylo-LNA modified oligonucleotides as defined in claim 1.

24. A kit according to claim 22, wherein the Xylo-LNA modified oligonucleotides are immobilised onto said reaction body.

27. A composition comprising one or more Xylo-LNA modified oligonucleotides of claim 1.

28. The Xylo-LNA modified oligonucleotide as defined in claim 1 further comprising a pharmaceutically acceptable carrier.

29. The oligomer of claim 1, wherein R1* is in a β

configuration.

14. A nucleoside analogue (Xylo-LNA) of the general formula II

15. A nucleoside analogue according to claim 14, wherein the group B is selected from nucleobases and functional group protected nucleobases.

16. A nucleoside analogue according to claim 14, wherein each of the substituents R^1*, R², R^3*, R⁵, R^5*, which are present, 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, 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, with the proviso that any hydroxy, amino, mono(C_1-6-alkyl)amino, sulfanyl, and carboxy is optionally protected.

17. A nucleoside analogue according to claim 14, wherein each of the substituents R^1*, R², R^3*, R⁵, R^5*, which are present, designate hydrogen.

18. A nucleoside analogue according to claim 14, 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.

19. A nucleoside analogue according to claim 14, wherein the biradical is —

O—

, and —

(CH₂)_0-1—

O—

(CH₂)_1-3—

.

20. A nucleoside analogue according to claim 14, wherein B is selected from adenine, guanine, thymine, cytosine and uracil.

25. 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 Xylo-LNAs as defined in claim 14.

26. A kit according to claim 25, wherein the Xylo-LNAs are immobilised onto said reaction body.

30. The nucleoside analogue of claim 14, wherein R1* is in a β

configuration.

32. The nucleoside analogue (Xylo-LNA) of claim 14, wherein Q* represents an activation group for —

OH, —

SH, and —

NH(R^H).

33. The nucleoside analogue (Xylo-LNA) of claim 32, wherein said activation group is an optionally substituted O-phosphoramidite.

34. The nucleoside analogue (Xylo-LNA) of claim 14, wherein the nucleoside analogue is a 3′

-phosphoramidite derivative.

35. The nucleoside analogue (Xylo-LNA) of claim 33, wherein said O-phosphoramidite is a N,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.

31. An oligomer comprising at least one nucleoside analogue (Xylo-LNA) of the general formula I

36. A nucleoside analogue (Xylo-LNA) having the following general formula:

37. The nucleoside analogue (Xylo-LNA) of claim 36, wherein R₁ is dimethoxytrityl (DMT) and R₂ is —

P(N(iPr)₂)(O(CH₂)₂CN.

38. An oligomer comprising at least one nucleoside analogue (Xylo-LNA) having the following general formula:

39. 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 Xylo-LNA modified oligonucleotides as defined in claim 38.

40. A composition comprising one or more Xylo-LNA modified oligonucleotides of claim 38.

41. The Xylo-LNA modified oligonucleotide as defined in claim 38 further comprising a pharmaceutically acceptable carrier.