Title:
Xylo-LNA analogues
Document Type and Number:
Kind Code:
A1

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,′-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.
Inventors:
Wengel, Jesper (Odense S, DK)
Application Number:
09/528110
Publication Date:
05/01/2003
Filing Date:
03/17/2000
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Referenced by:
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Primary Class:
435/6
Other Classes:
435/375, 514/44, 536/23.100
International Classes:
(IPC1-7): A61K048/00; C07H021/04; C12Q001/68
Attorney, Agent or Firm:
EDWARDS & ANGELL, LLP,Peter F Corless (P.O. Box 9169, Boston, MA, 02209, US)
Claims:
1. An oligomer comprising at least one nucleoside analogue of the general formula I 7embedded image wherein X is selected from —O—, —S—, —N(RN*), —C(R6R6*)—; B is selected from hydrogen, hydroxy, optionally substituted C1-4-alkoxy, optionally substituted C1-4-alkyl, optionally substituted C1-4-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 or equally applicable the substituent R5*; P* designates an internucleoside linkage to a preceding monomer, or a 3′-terminal group; R2* and R4* designate biradicals consisting of 1-4 groups/atoms selected from —C(RaRb)—, —C(Ra)═C(Ra)—, —C(Ra)═N—, —O—, Si(Ra)2—, —S—, —SO2—, —N(Ra)—, and <C═Z, wherein Z is selected from —O—, —S—, and —N(Ra)—, and Ra and Rb each is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino—C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino—C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-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 where two geminal substituents R* and Rb together may designate optionally substituted methylene olefin (═CH2); each of the substituents R1*, R2, R3*, R5, R5*, R6, and R6*which are present is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino—C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-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 where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NRN)— where RN is selected from hydrogen and C1-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and RN*, when present is selected from hydrogen and C1-4-alkyl; and basic salts and acid addition salts thereof.

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 at least 2, preferably at least 3, such as 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 2, wherein the oligonucleotide comprises at least 7, preferably at least 9, in particular at least 11, especially at least 13 successive Xylo-LNA monomers.

5. An oligomer according to claim 2, wherein all nucleoside monomers of an oligomer are Xylo-LNA.

6. An oligomer according to any of the claims 1-5, wherein the Xylo-LNA(s) has/have the following formula Ia 8embedded image wherein P, P*, B, X, R1*, R2, R2*, R3*, R4, R5* are as defined in claim 1.

7. An oligomer according to any of the claims 1-6, wherein X is selected from —(CR*R*), —O—, —S—, and —N(RN*), preferably —O—, —S—, and —N(RN*), in particular —O— .

8. An oligomer according to any of the claims 1-7, wherein the biradical constituted by R2* and R4* is selected from —(CR*R*)r—Y—(CR*R*)s—, —(CR*R*)r—Y—(CR*R*)s—Y—, —Y—(CR*R*)hd r+s—Y—, —Y—(CR*R*)r—Y—(CR*R*)s—, (CR*R*)r+s—, —Y—, —Y—Y—, wherein each Y is independently selected from —O—, —S—, —Si(R*)2, —N(R*)—, >C═O, —C(═O)—N(R*)—, and —N(R*)—C(═O)—, each R* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or di(C1-6-alkyl)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, and/or two adjacent (non-geminal) R* may together designate a double bond, and each of r and s is 0-4 with the proviso that the sum r+s is 1-4.

9. An oligomer according to claim 8, wherein the biradical is selected from —Y—, —(CR*R*)r+s—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—, wherein and each of r and s is 0-3 with the proviso that the sum r+s is 1-4.

10. An oligomer according to claim 9, wherein the biradical is selected from —O—, —S—, —N(R*)—, —(CR*R*)r+s+1—, —(CR*R*)r—O—(CR*R*)s—, —(CR*R*),—S—(CR*R*)s—, —(CR*R*),—N(R*)—(CR*R*)s—, —O—(CR*R*)r+s—O—, —S—(CR*R*)r+s—O—, —O—(CR*R*)r+s—S—, —N(R*)—(CR*R*)r+s—O—, —O—(CR*R*r+s—N(R*)—, —S—(CR*R*)r+s—S—, —N(R*)—(CR*R*)r+s—N(R*)—, —N(R*)—(CR*R*)r+s—S—, and —S— (CR*R*)r+s—N(R*)—, wherein each of r and s is 0-3 with the proviso that the sum r+s is 1-4, and where X is selected from —O—, —S—, and —N(RH)-where RH designates hydrogen or C1-4-alkyl.

11. An oligomer according to claim 10, wherein X is —O—, R2 is selected from hydrogen, hydroxy, and optionally substituted C1-6-alkoxy, and R1*, R3*, R5, and R5* designate hydrogen.

12. An oligomer according to claim 11, wherein the biradical is selected from —O—, —(CH2)0-1—O—(CH2)1-3—, —(CH2)0-1—S—(CH2)1-3—, and —(CH2)0-1—N(RN )—(CH2)1-3—.

13. An oligomer according to claim 12, wherein the biradical is selected from —O—CH2—, —S—CH2— and —N(RN)—CH2—.

14. An oligomer according to any of the claims 11-13, wherein B is selected from nucleobases.

15. An oligomer according to claim 14, 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.

16. An oligomer according to claim 8, wherein the biradical is —(CH2)2-4—.

17. An oligomer according to any of the claims 8-10, wherein one R* is selected from hydrogen, hydroxy, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and any remaining substituents R* are hydrogen.

18. An oligomer according to claim 17, wherein a group R* in the biradical of at least one Xylo-LNA is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

19. An oligomer according to any of the claims 1-18, wherein any internucleoside linkage of the Xylo-LNA(s) is selected from linkages consisting of 2 to 4, preferably 3, groups/atoms selected from —CH2—, —O—, —S—, —NRH—, >C═O, >C═NRH—, >C═S, —Si(R″)2— —SO—, —S(O)2—, —P(O)2—, —P(O,S)—, —P(S)2—, —PO(R″)—, —PO(OCH3)—, and —PO(NHRH)—, where RH is selected form hydrogen and C1-4-alkyl, and R* is selected from C1-6-alkyl and phenyl.

20. An oligomer according to claim 19, 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—, —NRH—CH2—CH2—, —CH2—CH2—NRH—, CH2NRH—CH2—, —CH2—CH2—NRH, —NRH—CO—O—, NRH—CO—NRH—, —NRH—CS—NRH—, —NRH—C(═NRH)—NRH—, —NRH—CO—CH2—N RH—, —O—CO—O—, —O—CO—CH2—O—, —O—CH2—CO—O—, —CH2—CO—NRH—, —O—CO—NRH—, —NRH—CO—CH2—, —O—CH2—CO—NR ,—O—CH 2—CH2—NRH—CH═N—O—, —CH2—NRH—O—, CH2—O—N═, —CH2—O—NRH—, —CO—NRH—CH2—, —CH2—NRH—O—, CH2—NRH—O—CO—, —NRH-CH2—, —O-NRH—, —O—CH2—S—, —S—CH2—O, —CH2—CH2—S—, —O—CH2CH2—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—NRH—, —NRH—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(NHRN)O, —O— P(O)2—NRH—, —NRH—P(O)2—O—, —O— P(O,NRH)—O—, and —O—Si(R″)2—O—.

21. An oligomer according to claim 20, wherein any internucleoside linkage of the Xylo-LNA(s) is selected from —CH2—CO—NRH—, —CH2—NRH—O—, S—CH2—O—, —O— P(O)2—O—, —O— P(O,S)—O—, —O— P(S)2—O—, —NRH—P(O)2—O—, —O— P(O, NRH)—O—, —O— PO(R″)—O—, —O— PO(CH3)—O—, and —O— PO(NHRN)—O—, where RH is selected form hydrogen and C1-4-alkyl, and R″ is selected from C1-6-alkyl and phenyl.

22. An oligomer according to any of the claims 1-21, wherein each of the substituents R1*, R2, R3*, R5, R5*, R6 and R6*of the Xylo-LNA(s), which are present, is independently selected from hydrogen, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, hydroxy, C1-6-alkoxy, C2-6-alkenyloxy, carboxy, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, formyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, C1-6-alkyl-carbonylamino, carbamido, azido, C1-6-alkanoyloxy, sulphono, sulphanyl, C1-6-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 RN*, when present and not involved in a biradical, is selected from hydrogen and C1-4-alkyl.

23. An oligomer according to any of the claims 1-22, wherein X is selected from —O—, —S—, and —NRN*, and each of the substituents R1*, R2, R3*, R5, R5*, R6 and R6* of the Xylo-LNA(s), which are present, designate hydrogen.

24. An oligomer according to any of the claims 1-23, wherein P is a 5′-terminal group selected from hydrogen, hydroxy, optionally substituted C1-6-alkyl, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkylcarbonyloxy, optionally substituted aryloxy, monophosphate, diphosphate, triphosphate, and —W—A′, wherein W is selected from —O—, —S—, and —N(RH)— where RH is selected from hydrogen and C1-6-alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

25. An oligomer according to any of the claims 1-24, wherein P* is a 3′-terminal group selected from hydrogen, hydroxy, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkylcarbonyloxy, optionally substituted aryloxy, and —W—A′, wherein W is selected from —O—, —S—, and —N(RH) where RH is selected from hydrogen and C1-6-alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

26. An oligomer according to any of the claims 1-25, having the following formula V: G—[Nu—L]n(0)—{[Xylo-LNA—L]m(q)—[Nu-L]n(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 I.

27. A nucleoside analogue (Xylo-LNA) of the general formula II 9embedded image 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—, —S—, —N(RN*), and —C(R6R6*)—; each of Q and Q* is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot—O—, Act—O—, mercapto, Prot—S—, Act—S—, C1-6-alkylthio, amino, Prot—N(RH), Act N(RH), mono- or di(C1-6-alkyl)amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkenyloxy, optionally substituted C2-6-alkynyl, optionally substituted C2-6-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(RH)—CH2—, Act—N(RH)—CH2—, carboxymethyl, sulphonomethyl, where Prot is a protection group for —OH, —SH, and —NH(RH), respectively, Act is an activation group for —OH, —SH, and —NH(RH), respectively, and RH is selected from hydrogen and C1-6-alkyl; and R2* and R4* together designate a biradical selected from —O—, —(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—, —S—(CR*R*)r+s—O—, —O—(CR*R*)r+s—N(R*)—(CR*R*)r+s—S—, and —S—(CR*R*)r+s—N(R*)—; N(R*)—(CR*R*)r+s—N(R*)—, —N(R*)—(CR*R*)s—S—, and —S—(CR*R*)r+s—N(R*)—; wherein each R* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or di(C1-6-alkyl)amino, optionally substituted C,16-alkoxy, optionally substituted C1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and/or two adjacent (non-geminal) R* may together designate a double bond, and each of r and s is 0-3 with the proviso that the sum r+s is 1-4; each of the present substituents R1*, R2, R3*, R5, R5*, R6, and R6* is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino—C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-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 where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NRN)— where RN is selected from hydrogen and C1-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and RN*, when present and not involved in a biradical, is selected from hydrogen and C1-4-alkyl; 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.

28. A nucleoside analogue according to claim 27, wherein the group B is selected from nucleobases and functional group protected nucleobases.

29. A nucleoside analogue according to any of the claims 27-28, wherein X is selected from —O—, —S—, and —N(RN*)—.

30. A nucleoside analogue according to any of the claims 27-29, wherein each of the substituents R1*, R2, R3*, R5, R5*, R6, and R6*, which are present, is independently selected from hydrogen, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, hydroxy, C1-6-alkoxy, C2-6-alkenyloxy, carboxy, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, formyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, C1-6-alkyl-carbonylamino, carbamido, azido, C1-6-alkanoyloxy, sulphono, sulphanyl, C1-6-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 RN*, when present and not involved in a biradical, is selected from hydrogen and C1-4-alkyl, with the proviso that any hydroxy, amino, mono(C1-6-alkyl)amino, sulfanyl, and carboxy is optionally protected.

31. A nucleotide analogue according to any of the claims 27-30, each of the substituents R1*, R2, R3*, R5, R5, R6 and R6*, which are present, designate hydrogen.

32. A nucleoside analogue according to any of the claims 27-31, wherein Q is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot—O—, mercapto, Prot—S—, C1-6-alkylthio, amino, Prot—N(RH)—, mono- or di(C1-6-alkyl)amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkenyloxy, optionally substituted C2-6-alkynyl, optionally substituted C2-6-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(RH)—CH2—, carboxymethyl, sulphonomethyl, where Prot is a protection group for —OH, —SH, and —NH(RH), respectively, and RH is selected from hydrogen and C1-6-alkyl; and Q* is selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Act—O—, mercapto, Act—S—, C1-6-alkylthio, amino, Act—N(RH)—, mono- or di(C1-6-alkyl)amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkenyloxy, optionally substituted C2-6-alkynyl, optionally substituted C2-6-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(RH), respectively, and RH is selected from hydrogen and C1-6-alkyl.

33. A nucleotide analogue according to any of the claims 27-32, where B is in the “β-configuration”

34. A nucleoside analogue according to claim 33, wherein X is —O—, R2 selected from hydrogen, hydroxy, and optionally substituted C1-6-alkoxy, and R1*, R3, R5, and R5* designate hydrogen.

35. A nucleoside analogue according to claims 37-34, wherein the biradical is selected from —O—, —(CH2)0-1—O—(CH2)1-3—, —(CH2)0-1-N(RN)—(—CH2)1-3.

36. A nucleoside analogue according to claim 35, wherein the biradical is selected from —O—CH2—, —S—CH2— and —N(RN)—CH2—.

37. A nucleoside analogue according to any of the claims 34-36, wherein B is selected from nucleobases.

38. A nucleoside analogue according to claim 37, wherein B is selected from adenine, guanine, thymine, cytosine and uracil.

39. A nucleoside analogue according to claim 38, wherein the biradical is —(CH2)2-4—, preferably —(CH2)2—.

40. A nucleoside analogue according to any of the claims 34-39, wherein one R* is selected from hydrogen, hydroxy, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and any remaining substituents R* are hydrogen.

41. The use of a Xylo-LNA as defined in any of the claims 27-40 for the preparation of an Xylo-LNA modified oligonucleotide (an oligomer) according to any of the claims 1-34.

42. The use according to claim 41, wherein the incorporation of Xylo-LNA modulates the ability of the oligonucleotide to act as a substrate for nucleic acid active enzymes.

43. The use of a Xylo-LNA as defined in any of the claims 27-40 for the preparation of a conjugate of an Xylo-LNA modified oligonucleotide and a compound selected from proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, and PNA.

44. A conjugate of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 and a compound selected from proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, and PNA.

45. The use of a Xylo-LNA as defined in any of the claims 27-40 as a substrate for enzymes active on nucleic acids.

46. The use according to claim 45, wherein the substituent Q in the formula II in claim 31 designates a triphosphate,

47. The use according to claim 45, wherein the Xylo-LNA is used as a substrate for DNA and RNA polymerases.

48. The use of a Xylo-LNA as defined in any of the claims 27-40 as a therapeutic agent.

49. The use of a Xylo-LNA as defined in any of the claims 27-40 for diagnostic purposes.

50. The use of one or more Xylo-LNA as defined in any of the claims 27-40 in the construction of solid surface onto which LNA modified oligonucleotides of different sequences are attached.

51. The use of Xylo-LNA modified oligomers (ribozymes) as defined in any of the claims 1-26 in the sequence specific cleavage of target nucleic acids.

52. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 in therapy, e.g. as an antisense, antigene or gene activating therapeutic.

53. The use according to claim 52, wherein the LNA modified oligonucleotide recruits RNAseH.

54. The use of complexes of more than one Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 in therapy, e.g. as an antisense, antigene or gene activating therapeutic.

55. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 as an aptamer in therapeutic applications.

56. The use of an Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 in diagnostics, e.g. for the isolation, purification, amplification, detection, identification, quantification, or capture of natural or synthetic nucleic acids.

57. The use according to claim 56, wherein the oligonucleotide comprises a photochemically active group, a thermochemically active group, a chelating group, a reporter group, or a ligand that facilitates the direct or indirect detection of the oligonucleotide or the immobilisation of the oligonucleotide onto a solid support.

58. The use according to claim 57, wherein the photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand includes a spacer (K), said spacer comprising a chemically cleavable group.

59. The use according to claim 58, wherein the photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand is attached via the biradical (i.e. as R*) of at least one of the LNA(s) of the oligonucleotide.

60. The use according to claim 58 for capture and detection of naturally occurring or synthetic double stranded or single stranded nucleic acids such as RNA or DNA.

61. The use according to claim 57 for purification of naturally occurring double stranded or single stranded nucleic acids such as RNA or DNA.

62. The use according to claim 57 as a probe in in-situ hybridisation, in Southern hydridisation, Dot blot hybridisation, reverse Dot blot hybridisation, or in Northern hybridisation.

63. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 as an aptamer in molecular diagnostics.

64. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 as an aptamer in RNA mediated catalytic processes.

65. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 as an aptamer in specific binding of antibiotics, drugs, amino acids, peptides, structural proteins, protein receptors, protein enzymes, saccharides, polysaccharides, biological cofactors, nucleic acids, or triphosphates.

66. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 as an aptamer in the separation of enantiomers from racemic mixtures by stereospecific binding.

67. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 for the labelling of cells.

68. The use according to claim 67, wherein the label allows the cells to be separated from unlabelled cells.

69. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 to hybridise to non-protein coding cellular RNAs, such as tRNA, rRNA, snRNA and scRNA, in vivo or in-vitro.

70. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 in the construction of an oligonucleotide comprising a fluorophor and a quencher, positioned in such a way that the hybridised state of the oligonucleotide can be distinguished from the unbound state of the oligonucleotide by an increase in the fluorescent signal from the probe .

71. The use of a Xylo-LNA modified oligonucleotide (an oligomer) as defined in any of the claims 1-26 in the construction of Taqman probes or Molecular Beacons.

72. 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 (oligomer) as defined in any of the claims 1-26.

73. A kit according to claim 72, wherein the Xylo-LNA modified oligonucleotides are immobilised onto said reaction body.

74. 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 any of the claims 27-40.

75. A kit according to claim 74, wherein the Xylo-LNAs are immobilised onto said reactions body.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to the field of xylo-configurated bicyclic nucleoside analogues and to the synthesis of such nucleoside analogues which are useful in the formation of synthetic oligonucleotides capable of forming nucleobase specific duplexes with complementary single stranded and double stranded nucleic acids. The invention also relates to the field of xylo-configurated bicyclic nucleoside analogues which may be used as therapeutic drugs and which may be incorporated in oligonucleotides.

BACKGROUND OF THE INVENTION

[0002] Synthetic oligonucleotides are widely used compounds in disparate fields such as molecular biology and DNA-based diagnostics and therapeutics.

[0003] General Considerations

[0004] To be useful in the extensive range of the different applications outlined above oligonucleotides have to satisfy a large number of different requirements. As therapeutics, for instance, a useful oligonucleotide must be able to penetrate the cell membrane, have good resistance to extra- and intracellular nucleases and preferably have the ability to recruit endogenous enzymes like RNAseH. In DNA-based diagnostics and molecular biology other properties are important such as, e.g., the ability of oligonucleotides to act as efficient substrates for a wide range of different enzymes evolved to act on natural nucleic acids, such as e.g. polymerases, kinases, ligases and phosphatases. The fundamental property of oligonucleotides, however, which underlies all uses is their ability to recognise and hybridise sequence specifically to complementary single stranded nucleic acids employing either Watson-Crick hydrogen bonding (A-T and G-C) or other hydrogen bonding schemes such as the Hoogsteen mode. The two important terms, affinity and specificity, are commonly used to characterise the hybridisation properties of a given oligonucleotide. Affinity is a measure of the binding strength of the oligonucleotide to its complementary target sequence (expressed as the thermostability (T m ) of the duplex). Each nucleobase pair in the duplex adds to the thermostability and thus affinity increases with increasing size (number of nucleobases) of the oligonucleotide. Specificity is a measure of the ability of the oligonucleotide to discriminate between a fully complementary and a mismatched target sequence. In other words, specificity is a measure of the loss of affinity associated with mismatched nucleobase pairs in the target.

[0005] At constant oligonucleotide size, the specificity increases with increasing number of mismatches between the oligonucleotide and its targets (i.e. the percentage of mismatches increases). Conversely, specificity decreases when the size of the oligonucleotide is increased at a constant number of mismatches (i.e. the percentage of mismatches decreases). Stated another way, an increase in the affinity of an oligonucleotide occurs at the expense of specificity and vice-versa.

[0006] Given the shortcomings of natural oligonucleotides, new approaches for enhancing specificity and affinity are highly desirable for DNA-based therapeutics, diagnostics and for molecular biology techniques in general.

[0007] Conformationally Restricted Nucleosides

[0008] It is known that oligonucleotides undergo a conformational transition in the course of hybridising to a target sequence, from the relatively random coil structure of the single stranded state to the ordered structure of the duplex state.

[0009] Thus, conformational restriction has in recent years been applied to oligonucleotides in the search for analogues displaying improved hybridisation properties compared to the unmodified (2′-deoxy)oligonucleotides. For example bicyclo[3.3.0]nucleosides with an additional C-3′,C-5′-ethano-bridge (M. Tarköy, M. Bolli, B. Schweizer and C. Leumann, Helv. Chem. Acta, 1993, 76, 481; Tarköy and C. Leumann, Angew. Chem., Int. Ed. Engl., 1993, 32, 1432; M. Egli, P. Lubini, M. Dobler and C. Leumann, J. Am. Chem. Soc., 1993, 115, 5855; M. Tarköy, M. Bolli and C. Leumann, Helv. Chem. Acta, 1994, 77, 716; M. Bolli 30 and C. Leumann, Angew. Chem., Int. Ed. Engl., 1995, 34, 694; M. Bolli, P. Lubini and C. Leumann, Helv. Chem. Acta, 1995, 78, 2077; J. C. Litten, C. Epple and C. Leumann, Bioorg. Med. Chem. Lett., 1995, 5, 1231; J. C. Litten and C. Leumann, Helv. Chem. Acta, 1996, 79, 1129; M. Bolli, J. C. Litten, R. Schultz and C. Leumann, Chem. Biol., 1996, 3, 197; M. Bolli, H. U. Trafelet and C. Leumann, Nucleic Acids Res., 1996, 24, 4660), bicarbocyclo[3.1.0]nucleosides with an additional C-1′,C-6′- or C-6′,C-4′-methano-bridge (K. -H. Altmann, R. Kesselring, E. Francotte and G. Rihs, Tetrahedron Lett., 1994, 35, 2331; K. -H. Altmann, R. Imwinkelried, R. Kesselring and G. Rihs, Tetrahedron Lett., 1994, 35, 7625; V. E. Marquez, M. A. Siddiqui, A. Ezzitouni, P. Russ, J. Wang, R. W. Wagner and M. D. Matteucci, J. Med. Chem., 1996, 39, 3739; A. Ezzitouni and V. E. Marquez, J. Chem. Soc., Perkin Trans. 1, 1997, 1073), bicyclo[3.3.0]- and [4.3.0]nucleosides containing an additional C-2′,C-3′-dioxalane ring synthesised as a dimer with an unmodified nucleoside where the additional ring is part of the internucleoside linkage replacing a natural phosphodiester linkage (R. J. Jones, S. Swaminathan, J. F. Millagan, S. Wadwani, B. S. Froehler and M. Matteucci, J. Am. Chem. Soc., 1993, 115, 9816; J. Wang and M. D. Matteucci, Bioorg. Med. Chem. Lett., 1997, 7, 229), dimers containing a bicyclo[3.1.0]nucleoside with a C-2′,C-3′-methano bridge as part of amide- and sulfonamide-type internucleoside linkages (C. G. Yannopoulus, W. Q. Zhou, P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378), bicyclo[3.3.0] glucose-derived nucleoside analogue incorporated in the middle of a trimer through formacetal internucleoside linkages (C. G. Yannopoulus, W. Q. Zhou, P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378) and bicyclic[4.3.0]- and [3.3.0]nucleosides with additional C-2′,C-3′-connected six- and five-membered ring (P. Nielsen, H. M. Pfundheller, J. Wengel, Chem. Commun., 1997, 826; P. Nielsen, H. M. Pfundheller, J. Wengel, XII International Roundtable: Nucleosides, Nucleotides and Their Biological Applications; La Jolla, Calif., Sep. 15-19, 1996; Poster PPI 43) have been synthesised and incorporated into oligodeoxynucleotides. Unfortunately, oligonucleotides comprising these analogues form, in most cases, less stable duplexes with complementary nucleic acids compared to the unmodified oligonucleotides. In cases where a moderate improvement in duplex stability is observed, this relates only to either a DNA or an RNA target, or it relates to fully but not partly modified oligonucleotides or vice versa.

[0010] An appraisal of most of the reported analogues is further complicated by the lack of data on analogues with G, A and C nucleobases and lack of data indicating the specificity and mode of hybridisation. In many cases, synthesis of the reported monomer analogues is very complex while in other cases the synthesis of fully modified oligonucleotides is incompatible with the widely used standard phosphoramidite chemistry.

[0011] Recently, oligomers comprising Locked Nucleic Acids (LNA) have been reported (Nielsen, P., Pfundheller, H. M., Olsen, C. E. and Wengel, J., J. Chem. Soc., Perkin Trans. 1, 1997, 3423; Nielsen, P., Pfundheller, H. M., Wengel, J., Chem. Commun., 1997, 9, 825; Christensen, N. K., Petersen, M., Nielsen, P., Jacobsen, J. P. and Wengel, J., J. Am. Chem. Soc., 1998, 120, 5458; Koshkin, A. A. and Wengel, J., J. Org. Chem., 1998, 63, 2778; Obika, S., Morio, K. -I., Hari, Y. and Imanishi, T., Bioorg. Med. Chem. Lett., 1999, 515). Interestingly, incorporation of LNA monomers containing a 2′-O,4′-C-methylene bridge into an oligonucleotide sequence led to unprecedented improvement in the hybridisation ability of the modified oligonucleotide (Singh, S. K., Nielsen, P., Koshkin, A. A., Olsen, C. E. and Wengel, J., Chem. Commun., 1998, 455; Koshkin, A. K., Singh, S. K., Nielsen, P., Rajwanshi, V. K., Kumar, R., Meldgaard, M., Olsen, C. E., and Wengel, J., Tetrahedron, 1998, 54, 3607; Koshkin, A. A. Rajwanshi, V. K., and Wengel, J., Tetrahedron Lett., 1998, 39, 4381; Singh, Sanjay K. and Wengel, J., Chem. Commun., 1998, 1247; Kumar, R., Singh, S. K., Koshkin, A. A., Rajwanshi, V. K., Meldgaard, M., and Wengel, J., Bioorg. Med. Chem. Lett., 1998, 8, 2219; Obika, S. et al. Tetrahedron Lett., 1997, 38, 8735; Obika, S. et al. Tetrahedron Lett., 1998, 39, 5401; Singh, S. K., Kumar, R., and Wengel, J., J. Org. Chem., 1998, 63, 6078; Koshkin, A. A., Nielsen, P., Meldgaard, M., Rajwanshi, V. K., Singh, S. K., and Wengel, J., J. Am. Chem. Soc., 1998, 120, 13252; Singh, S. K., Kumar, R., and Wengel, J., J. Org. Chem., 1998, 63, 10035). Oligonucleotides comprising these LNA monomers and the corresponding 2′-thio-LNA analogue form duplexes with complementary DNA and RNA with thermal stabilities not previously observed for bi- or tricyclic nucleosides modified oligonucleotides (ΔT m /modification=+3 to +11° C.) and show improved selectivity. In a series of papers, Seela et al. have studied xylo-DNA ( FIG. 1 , Base=adenin-9-yl, cytosin-1-yl, guanin-9-yl or thymin-1-yl) comprising one or more 2′-deoxy-β-D-xylofuranosyl nucleotide monomers (Rosemeyer, H.; Seela, F. Helv. Chem. Acta 1991, 74, 748; Rosemeyer, H.; Krecmerova, M.; Seela, F. Helv. Chem. Acta 1991 74, 2054; Seela, F.; Wörner, Rosemeyer, H. Helv. Chem. Acta 1994, 77, 883; Seela, F.; Heckel, M.; Rosemeyer, H. Helv. Chem. Acta 1996, 79, 1451; Rosemeyer, H.; Seela, F. Nucleosides Nucleotides, 1995, 14, 1041; Schoeppe, A.; Hinz, H. -J.; Rosemeyer, H.; Seela, F. Eur. J. Biochem. 1996, 239, 33). Compared with the corresponding natural 2′-deoxy-β-D-ribofuranosyl oligonucleotides, xylo-DNA generally display a mirror-image-like secondary structure, entropically favourable duplex formation, increased stability towards exonucleases, and, for oligonucleotides comprising a small number of 2′-deoxy-β-D-xylofuranosyl monomers, decreased thermal affinity towards complementary DNA (Rosemeyer, H.; Seela, F. Helv. Chem. Acta 1991, 74, 748; Rosemeyer, H.; Krecmerova, M.; Seela, F. Helv. Chem. Acta 1991, 74, 2054; Seela, F.; W{umlaut over (r)}ner, Rosemeyer, H. Helv. Chem. Acta 1994 , 77, 883; Seela, F.; Heckel, M.; Rosemeyer, H. Helv. Chem. Acta 1996, 79, 1451).

SUMMARY OF THE INVENTION

[0012] 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.; Wörner, 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.

[0013] Thus, the present inventors have now provided novel LNA nucleoside analogues (Xylo-LNAs) and oligonucleotides having Xylo-LNA nucleoside analogues included therein. The novel Xylo-LNA nucleoside analogues have been synthesised with thymine as the nucleobase but can easily be synthesised with the other four nucleobases thereby providing a full set of nucleoside analogues for incorporation in oligonucleotides.

[0014] The present invention relates to oligomers comprising at least one nucleoside analogue (hereinafter termed “Xylo-LNA”) of the general formula I 1 embedded image

[0015] wherein X is selected from —O—, —S—, —N(R N* )—, —C(R 6 R 6* )—;

[0016] 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;

[0017] 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 R 5 or equally applicable the substituent R 5 ′;

[0018] P* designates an internucleoside linkage to a preceding monomer, or a 3′-terminal group;

[0019] R 2* and R 4* designate biradicals consisting of 1-4 groups/atoms selected from —C(R a R b )—, C(R a )═C(R a )—, —C(R a )═N—, —O—, —Si(R a ) 2 —, —S—, —SO 2 —, —N(R a )—, and >C═Z,

[0020] wherein Z is selected from —O—, —S—, and —N(R a )—, and R a and R b each 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 where two geminal substituents R a and R b together may designate optionally substituted methylene olefin (═CH 2 );

[0021] each of the substituents R 1* , R 2 , R 3* , R 5 , R 5* , R 6 , and R 6* which are present 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 where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NR N ) where R N is selected from hydrogen and C 1-4 -alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and R N* , when present, is selected from hydrogen and C 1-4 -alkyl;

[0022] and basic salts and acid addition salts thereof.

[0023] The present invention furthermore relates to nucleoside analogues (Xylo-LNAs) of the general formula II 2 embedded image

[0024] wherein the substituent B is selected from nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

[0025] X is selected from —O—, —S—, —N(R N* )—, and —C(R 6 R 6* )—;

[0026] 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(RH)—, 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—CH 2 —, Act—O—CH 2 —, aminomethyl, Prot—N(R H )— CH 2 —, Act—N(R H )—CH 2 —, 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; and

[0027] R 2* and R 4* together designate a biradical selected from —O—, —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—, —S—(CR*R*) r+s — O—, —O—(CR*R*) r+s —S—, —N(R*)—(CR*R*) r+s —O—, —O—(CR*R*) r+s —N(R*)—, —S—(CR*R*) r+s —N(R*)—(CR*R*) r+s —N(R*)—, —N(R*)—(CR*R*) r+s —S— and —S—(CR*R*) r+s —N(R*)—;

[0028] wherein each R* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or di(C 1-6 -alkyl)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/or two adjacent (non-geminal) R* may together designate a double bond, and each of r and s is 0-3 with the proviso that the sum r+s is 1-4;

[0029] each of the present substituents R 1* , R 2 , R 3* , R 5 , R 5* , R 6 , and R 6* 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 where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NR N )— where R N is selected from hydrogen and C 1-4 -alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and R N* , when present and not involved in a biradical, is selected from hydrogen and C 1-4 -alkyl;

[0030] and basic salts and acid addition salts thereof;

[0031] 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.

[0032] The present invention also relates to the use of the nucleoside analogues (Xylo-LNAs) for the preparation of oligomers, and the use of the oligomers as well as the nucleoside analogues (Xylo-LNAs) in diagnostics, molecular biology research, and in therapy.

DETAILED DESCRIPTION OF THE INVENTION

[0033] When used herein, the term “Xylo-LNA” (X/o-configurated Locked Nucleoside Analogues) refers to xylo-configurated bicyclic nucleoside analogues, either incorporated in the oligomer of the invention (general formula 1) or as discrete chemical species (general formula 11). The term “monomeric Xylo-LNA” specifically refers to the latter case.

[0034] Oligomers and Nucleoside Analogues

[0035] As mentioned above, the present invention i.a. relates to novel oligomers (oligonucleotides) comprising one or more xylo-configurated bicyclic nucleoside analogues. The xylo-configurated bicyclic nucleoside analogues are hereinafter referred to as “Xylo-LNA”.

[0036] Each of the possible Xylo-LNAs incorporated in an oligomer (oligonucleotide) has the general formula I 3 embedded image

[0037] wherein X is selected from —O— (the xylofuranose motif), —S—, —N(R N* )—, C(R 6 R 6* )—, where R 6 , R 6* , and R N* are as defined further below. Thus, the Xylo-LNAs incorporated in the oligomer comprise a 5-membered ring as an essential part of the bicyclic structure.

[0038] Among the possible 5-membered rings, the situations where X designates —O—, —S—, and —N(R N* ) seem especially interesting, and the situation where X is —O— appears to be particularly interesting.

[0039] The substituent B may designate a group which, when the oligomer is complexing with DNA or RNA, is able to interact (e.g. by hydrogen bonding or covalent bonding or electronic interaction) with DNA or RNA, especially nucleobases of DNA or RNA. Alternatively, the substituent B may designate a group which acts as a label or a reporter, or the substituent B may designate a group (e.g. hydrogen) which is expected to have little or no interactions with DNA or RNA. Thus, the substituent B is preferably 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.

[0040] In the present context, the terms “nucleobase” covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered “non-naturally occurring” have subsequently been found in nature. Thus, “nucleobase” includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Illustrative examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 , N 4 -ethanocytosine, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5—(C 3 -C 6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoiso-cytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine and the “non-naturally occurring” nucleobases described in Benner et al., U.S. Pat. No. 5,432,272. The term “nucleobase” is intended to cover all of these examples as well as analogues and tautomers thereof. Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.

[0041] When used herein, the term “DNA intercalator” means a group that can intercalate into a DNA or RNA helix, duplex or triplex. Examples of functional parts of DNA intercalators are acridines, anthracenes, quinones such as anthraquinone, indole, quinoline, isoquinoline, dihydroquinones, anthracyclines, tetracyclines, methylene blue, anthracyclinone, psoralens, coumarins, ethidium-halides, dynemicin, metal complexes such as 1,10-phenanthroline-copper, tris(4,7-diphenyl-1,10-phenanthroline), ruthenium-cobalt-enediynes such as calcheamicin, porphyrins, distamycin, netropcin, viologen, daunomycin. Especially interesting examples are acridines, quinones such as anthraquinone, methylene blue, psoralens, coumarins, and ethidium-halides.

[0042] In the present context, the term “photochemically active groups” covers compounds which are able to undergo chemical reactions upon irradiation with light. Illustrative examples of functional groups hereof are quinones, especially 6-methyl-1,4-naphthoquinone, anthraquinone, naphthoquinone, and 1,4-dimethyl-anthraquinone, diazirines, aromatic azides, benzophenones, psoralens, diazo compounds, and diazirino compounds.

[0043] In the present context “thermochemically reactive group” is defined as a functional group which is able to undergo thermochemically-induced covalent bond formation with other groups. Illustrative examples of functional parts thermochemically reactive groups are carboxylic acids, carboxylic acid esters such as activated esters, carboxylic acid halides such as acid fluorides, acid chlorides, acid bromide, and acid iodides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives.

[0044] In the present context, the term “chelating group” means a molecule that comprises more than one binding site and frequently binds to another molecule, atom or ion through more than one binding site at the same time. Examples of functional parts of chelating groups are iminodiacetic acid, nitrilotriacetic acid, ethylenediamine tetraacetic acid (EDTA), aminophosphonic acid, etc.

[0045] In the present context, the term “reporter group” means a group that is detectable either by itself or as a part of a detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g. light or X-rays, of a certain wavelength, and which subsequently re-emits the energy absorbed as radiation of longer wavelength; illustrative examples are dansyl (5-dimethylamino)-1-naphthalenesulfonyl), DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines, coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems, Inc.), erytrosine, coumaric acid, umbelliferone, Texas Red, rhodamine, tetramethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene, fluorescein, europium, ruthenium, samarium, and other rare earth metals, radioisotopic labels, chemiluminescence labels (labels that are detectable via the emission of light during a chemical reaction), spin labels (a free radical (e.g. substituted organic nitroxides) or other paramagnetic probes (e.g. Cu 2+ , Mg 2+ ) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy), enzymes (such as peroxidases, alkaline phosphatases, β-galactosidases, and glucose oxidases), antigens, antibodies, haptens (groups which are able to combine with an antibody, but which cannot initiate an immune response by themselves, such as peptides and steroid hormones), carrier systems for cell membrane penetration such as: fatty acid residues, steroid moieties (cholesteryl), vitamin A, vitamin D, vitamin E, folic acid peptides for specific receptors, groups for mediating endocytose, epidermal growth factor (EGF), bradykinin, and platelet derived growth factor (PDGF). Especially interesting examples are biotin, fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin, ruthenium, europium, Cy5 and Cy3.

[0046] In the present context, the term “ligand” means something which binds. Ligands can comprise functional groups such as: aromatic groups (such as benzene, pyridine, naphtalene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, C 1-20 alkyl groups optionally interrupted or terminated with one or more heteroatoms such as oxygen atoms, nitrogen atoms, and/or sulphur atoms, optionally comprising aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-β-alanine, polyglycine, polylysine, peptides, oligo/polysaccharides, oligo/polyphosphates, toxins, antibiotics, cell poisons, and steroids, and also “affinity ligands”, i.e. functional groups or biomolecules that have a specific affinity for sites on particular proteins, antibodies, poly- and oligosaccharides, and other biomolecules.

[0047] It will be clear for the person skilled in the art that the above-mentioned specific examples under DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands correspond to the “active/functional” part of the groups in question. For the person skilled in the art it is furthermore clear that DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands are typically represented in the form M-K— where M is the “active/functional” part of the group in question and where K is a spacer through which the “active/functional” part is attached to the 5-membered ring. Thus, it should be understood that the group B, in the case where B is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, has the form M-K—, where M is the “active/functional” part of the DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group, and ligand, respectively, and where K is an optional spacer comprising 1-50 atoms, preferably 1-30 atoms, in particular 1-15 atoms, between the 5-membered ring and the “active/functional” part.

[0048] In the present context, the term “spacer” means a thermochemically and photochemically non-active distance-making group and is used to join two or more different moieties of the types defined above. Spacers are selected on the basis of a variety of characteristics including their hydrophobicity, hydrophilicity, molecular flexibility and length (e.g. see Hermanson et. al., “Immobilized Affinity Ligand Techniques”, Academic Press, San Diego, Calif. (1992), p. 137-ff). Generally, the length of the spacers is less than or about 400 Å, in some applications preferably less than 100 Å. The spacer, thus, comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulphur atoms. Thus, the spacer K may comprise one or more amide, ester, amino, ether, and/or thioether functionalities, and optionally aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-p-alanine, polyglycine, polylysine, and peptides in general, oligosaccharides, oligo/polyphosphates. Moreover the spacer may consist of combined units thereof. The length of the spacer may vary, taking into consideration the desired or necessary positioning and spatial orientation of the “active/functional” part of the group in question in relation to the 5-membered ring. In particularly interesting embodiments, the spacer includes a chemically cleavable group. Examples of such chemically cleavable groups include disulphide groups cleavable under reductive conditions, peptide fragments cleavable by peptidases, etc.

[0049] In one embodiment of the present invention, K designates a single bond so that the “active/functional” part of the group in question is attached directly to the 5-membered ring.

[0050] In a preferred embodiment, the substituent B in the general formulae I and II is preferably selected from nucleobases, in particular from adenine, guanine, thymine, cytosine and uracil.

[0051] In the oligomers of the present invention (formula I), P designates the radical position for an internucleoside linkage to a succeeding monomer, or to a 5′-terminal group. The former possibility applies when the Xylo-LNA in question is not the 5′-terminal “monomer”, whereas the latter possibility applies when the Xylo-LNA in question is the 5′-terminal “monomer”. It should be understood (which also will be clear from the definition of internucleoside linkage and 5′-terminal group further below) that such an internucleoside linkage or 5′-terminal group may include the substituent R 5 (or equally applicable: the substituent R 5* ) thereby forming a double bond to the group P (5′-Terminal refers to the position corresponding to the 5′ carbon atom of a ribose moiety in a nucleoside)

[0052] On the other hand, P designates the radical position for an internucleoside linkage to a preceding monomer or a 3′-terminal group. Analogously, the former possibility applies when the Xylo-LNA in question is not the 3′-terminal “monomer”, whereas the latter possibility applies when the Xylo-LNA in question is the 3′-terminal “monomer” (3′-terminal refers to the position corresponding to the 3′-carbon atom of a ribose moiety in a nucleoside.)

[0053] In the present context, the term “monomer” relates to naturally occurring nucleosides, non-naturally occurring nucleosides, PNAs, LNAs etc. as well as Xylo-LNAs. Thus, the term “succeeding monomer” relates to the neighbouring monomer in the 5′-terminal direction and the “preceding monomer” relates to the neighbouring monomer in the 3′-terminal direction. Such succeeding and preceding monomers, seen from the position of an Xylo-LNA monomer, may be naturally occurring nucleosides or non-naturally occurring nucleosides, or even further Xylo-LNA monomers.

[0054] Consequently, in the present context (as can be derived from the definitions above), the term “oligomer” means an oligonucleotide modified by the incorporation of one or more Xylo-LNA(s). Furthermore, the term “oligomer” means an oligonucleotide modified by the incorporation of one or more Xylo-LNA(s) and one or more “monomers” as defined supra.

[0055] The crucial part of the present invention is the xylo-configuration of the 5-membered ring combined with the provision that R 2* and R 4* together designate a biradical forming a fused ring onto the 5-membered ring.

[0056] In the groups constituting the biradical(s), Z is selected from —O—, —S—, and —N(R a ) r and R a and R b each 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 the latter groups may include a spacer as defined for the substituent B), where aryl and heteroaryl may be optionally substituted. Moreover, two geminal substituents R a and R b together may designate optionally substituted methylene (═CH 2 optionally substituted one or two times with substituents as defined as optional substituents for aryl).

[0057] It is believed that biradicals which are bound to the ring atoms of the 5-membered rings 5 are preferred in that inclusion of the substituents R 5 and R 5* may cause an undesired sterical interaction with internucleoside linkage. Thus, it is preferred that the one or two pairs of non-geminal substituents, which are constituting one or two biradical(s), respectively, are selected from the present substituents of R 1* , R 6 , R 6 , R N* , R 2 , and R 3* .

[0058] In the present context, i.e. in the present description and claims, the orientation of the biradicals are so that the left-hand side represents the substituent with the lowest number and the right-hand side represents the substituent with the highest number. Thus, when R 2* and R 4 together designate a biradical “—O—CH 2 —”, it is understood that the oxygen atom represents R 2* and the methylene group represents R 4* .

[0059] Considering the interesting possibilities for the structure of the biradical(s) in Xylo-LNA(s) incorporated in oligomers according to the invention, it is believed that the biradical(s) constituted by pair(s) of non-geminal substituents preferably is/are selected from —(CR*R*) r —Y—(CR*R*) s —, —(CR*R*) r —Y—(CR*R*) s —Y—, —Y—(CR*R*) r+s , —Y—(CR*R*) r+s Y—(CR*R*) s —, —(CR*R*) r+s —, —Y—, —Y—Y—, wherein each Y is independently selected from —O—, —S—, —Si(R*) 2 —, —N(R*)—, >C═O, —C(═O)—N(R*)—, and —N(R*)—C(═O)—, each R* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or di(C 1-6 -alkyl)amino, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating 25 groups, reporter groups, and ligands, and/or two adjacent (non-geminal) R* may together designate a double bond; and each of r and s is 0-4 with the proviso that the sum r+s is 1-4. Particularly interesting situations are those wherein each biradical is independently selected from —Y—, —(CR*R*) r+s —, —(CR*R*) r —Y—(CR*R*) s , and —Y—(CR*R*) r+s —Y—, wherein and each of r and s is 0-3 with the proviso that the sum r+s is 1-4.

[0060] Particularly interesting oligomers are those wherein the following criteria applies for the Xylo-LNA(s) in the oligomers: R 2* and R 4* together designate a biradical selected from —O—, —S—, —N(R*)—, —(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*) r —, —O—(CR*R*) r+s —, —S—(CR*R*) r+s —O—(CR*R*) r —S—N(R*)—(CR*R*) r+s —O—, —O—(CR*R*) r+s —N(R*)—, —S—(CR*R*) r+s —N(R*)—(CR*R*) r+s —N(R*)—, —N(R*)—(CR*R*) r+s —S—, and —S—(CR*R*) r+s —N(R*)—; wherein each of r and s is 0-3 with the proviso that the sum r+s is 1-4, and where R* is selected from hydrogen, hydroxy, 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 any remaining substituents R* are hydrogen.

[0061] In one preferred embodiment, one group R in the biradical of at least one LNA is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands (where the latter groups may include a spacer as defined for the substituent B).

[0062] In another preferred embodiment, one group R* in the biradical of at least one LNA is selected from hydrogen, hydroxy, 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 any remaining substituents R* are hydrogen.

[0063] With respect to the substituents R 1* , R 2 , R 3 , R 5 , R 5* , R 6 , and R 6* which are present, are 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 the latter groups may include a spacer as defined for the substituent B), where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NR N )— where R N is selected from hydrogen and C 1-4 -alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and R N* , when present, is selected from hydrogen and C 1-4 -alkyl.

[0064] Preferably, each of the substituents R 1* , R 2 , R 3* , R 5 , R 5* , R 6 , and R 6* of the Xylo-LNA(s), 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, and ligands, and halogen, where two geminal substituents together may designate oxo, and where R N* , when present, is selected from hydrogen and C 1-4 -alkyl.

[0065] In a preferred embodiment of the present invention, X is selected from —O—, —S—, and —NR N ,in particular —O—, and each of the substituents R 1* , R 2 , R 3 , R 5 , R 5* , R 6 , and R 6* of the Xylo-LNA(s), which are present, designate hydrogen.

[0066] In an even more preferred embodiment of the present invention, X is —O—, the substituents R 1* , R 2 , R 3 , R 5 , and R 5* designate hydrogen, and R 2 and R* of an Xylo-LNA incorporated into an oligomer together designate a biradical, selected from —O—, —(CH 2 ) 0-1 —O—(CH 2 ) 1-3 —, —(CH 2 ) 0-1 —S—(CH 2 ) 1-3 —, —(CH 2 ) 0-1 —N(R N )—(CH 2 ) 1-3 —, and —(CH 2 ) 2-4 —, in particular from —O—CH 2 —, —S—CH 2 —, and —NR H —CH 2 —. Generally, with due regard to the results obtained so far, it is preferred that the biradical constituting R 2* and R 4* forms a two atom bridge, i.e. the biradical forms a five membered ring with the furanose ring (X═O).

[0067] In one embodiment of the present invention the biradical is —(CH 2 ) 2-4 —.

[0068] For these interesting embodiments, it is preferred that the Xylo-LNA(s) has/have the following general formula Ia. 4 embedded image

[0069] Also interesting as a separate aspect of the present invention is the variant of formula Ia where B is in the “α-configuration”.

[0070] The oligomers according to the invention typically comprise 1-10000 Xylo-LNA(s) of the general formula I (or of the more detailed general formula Ia) and 0-10000 nucleosides selected from naturally occurring nucleosides and nucleoside analogues. The sum of the number of nucleosides and the number of Xylo-LNA(s) (n) is at least 2, preferably at least 3, in particular at least 5, especially at least 7,