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Title:
L-RIBO-LNA ANALOGUES
Document Type and Number:
WIPO Patent Application WO/2000/066604
Kind Code:
A2
Abstract:
Nucleoside analogues wherein a 2'-4'-bridge locks the conformation of the nucleoside have been synthesised with an inverted stereochemistry at C-3' and C-4' to provide the L-ribo-configurated LNA nucleoside. The synthesis of L-ribo-LNA-nucleoside is applicable to all nucleobases including thymine, adenine, cytosine, guanine and uracil. These Locked Nucleic Acids (LNAs) with L-ribo-configuration have been utilised in the synthesis of 2'-O-4'-C-methylene-$g(a)-L-ribofuranosyl nucleotides as well as oligonucleotides with L-ribo-LNA nucleosides included therein. Methods of targeting complementary nucleic acids are greatly improved by use of these L-ribo-LNA modified oligonucleotides due to their high affinity for complementary nucleic acids.

Inventors:
WENGEL JESPER (DK)
Application Number:
PCT/DK2000/000225
Publication Date:
November 09, 2000
Filing Date:
May 04, 2000
Export Citation:
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Assignee:
EXIQON AS (DK)
WENGEL JESPER (DK)
International Classes:
C07H21/00; A61K31/7088; A61K48/00; A61P31/12; A61P35/00; C07H9/04; C07H19/06; C07H19/16; C07H21/02; C12N15/09; C12Q1/68; G01N33/53; G01N33/566; G01N33/58; (IPC1-7): C07H19/00
Domestic Patent References:
WO1999014226A21999-03-25
WO1998039352A11998-09-11
Other References:
KOSHKIN A ET AL: "LNA (Locked Nucleic Acids): Synthesis of the Adenine, Cytosine, Guanine, 5-Methylcytosine, Thymine and Uracil Bicyclonucleoside Monomers, Oligomerisation, and Unprecedented Nucleic Acid Recognition" TETRAHEDRON, vol. 54, 1998, pages 3607-3630, XP002901183
SINGH S K ET AL: "Synthesis of 2'-Amino-LNA: A Novel Conformationally Restricted High-Affinity Oligonucleotide Analogue with a Handle" J. ORG. CHEM., vol. 63, 1998, pages 10035-10039, XP002901184
OBIKA S ET AL: "Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2'-O,4'-C-methyleneribonucleosides" TETRAHEDRON LETTERS, vol. 39, 1998, pages 5401-5404, XP002901185
KOSHKIN A A ET AL: "LNA (Locked Nucleic Acid): An RNA Mimic Forming Exceedingly Stable LNA:LNA Duplexes" J. AM. CHEM. SOC. , vol. 120, 1998, pages 13252-13253, XP002901186
SINGH S K ET AL: "LNA (Locked Nucleic Acids): synthesis and high-affinity nucleic acid recognition" CHEM. COMMUN. , 1998, pages 455-456, XP002901187
OBIKA S ET AL: "Synthesis of 2'-O,4'-C-Methyleneuridine and -cytidine. Novel Bicyclic Nucleosides Having a Fixed C3,-endo Sugar Puckering" TETRAHEDRON LETTERS, vol. 38, no. 50, 1997, pages 8735-8738, XP002901188
SINGH S K ET AL: "Synthesis of Novel Bicyclo(2.2.1) Ribonucleosides: 2'-Amino- and 2'-Thio-LNA Monomeric Nucleosides" J. ORG. CHEM., vol. 63, 1998, pages 6078-6079, XP002901189
RAJWANSHI V K ET AL: "Synthesis and restricted furanose conformations of three novel bicyclic thymine nucleosides: a xylo-LNA nucleoside, a 3'-0,5'-C-methylene-linked nucleoside, and a 2'-0,5'-C-methylene-linked nucleoside" J. CHEM. SOC. PERKIN TRANS., vol. 1, 1999, pages 1407-1414, XP002901190
KOSHKIN A A ET AL: "Novel Convenient Syntheses of LNA (2.2.1) Bicyclo Nucleosides" TETRAHEDRON LETTERS, vol. 39, 1998, pages 4381-4384, XP002901191
KUMAR R ET AL: "The first analogues of LNA (Locked Nucleic Acids): phosphorothioate-LNA and 2'-Thio-LNA" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 8, 1998, pages 2219-2222, XP002901192
Attorney, Agent or Firm:
PLOUGMANN & VINGTOFT A/S (P.O. Box 3007, Copenhagen K, DK)
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Claims:
CLAIMS
1. An oligomer comprising at least one nucleoside analogue of the general formula I wherein X is selected fromO,S,N (R'),C(RR); B is selected from hydrogen, hydroxy, optionally substituted C, 4alkoxy, optionally substituted C, 4alkyl, optionally substituted C, 4acyloxy, 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 R 5*; P* designates an internucleoside linkage to a preceding monomer, or a 3'terminal group; R2 and R4 designate biradicals consisting of 14 groups/atoms selected from C (RaRb),C (Ra) =C (Ra),C (Ra) =N,0,Si (Ra) 2,S,SO2,N (Ra), and >C=Z, wherein Z is selected fromO,S, andN (Ra), and Ra and Rb each is independently selected from hydrogen, optionally substituted C,, 2alkyl, optionally substituted C2, 2alkenyl, optionally substituted C212alkynyl, hydroxy, C,, 2alkoxy, C112alkoxycarbonyl,C112alkylcarbonyl,formyl,aryl,C212alkenyloxy,carboxy, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, monoand di (C16alkyl) amino, carbamoyl, monoand di (C, 6alkyl)aminocarbonyl, aminoC, 6alkyl aminocarbonyl, monoand di (C, 6alkyl) aminoC, 6alkylaminocarbonyl, C, 6alkyl carbonylamino, carbamido, C16alkylsulphonyloxy,sulphono, nitro, azido, sulphanyl, C1 6alkylthio, 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 Ra and Rbtogether may designate optionally substituted methylene olefin (=CH2); each of the substituents R*, R2, R3, R5, R'*, R6, and R6* which are present is independently selected from hydrogen, optionally substituted C,, 2alkyl, optionally substituted C2, 2alkenyl, optionally substituted C2, 2alkynyl, hydroxy, C,, 2alkoxy, C2, 2alkenyloxy, carboxy, C,, 2alkoxycarbonyl, C,, 2alkylcarbonyl, formyl, aryl, aryloxy carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, monoand di (C, 6alkyl) amino, carbamoyl, monoand di (C, 6alkyl)aminocarbonyl, aminoC, 6alkylaminocarbonyl, monoand di (C, 6 alkyl) aminoC, 6alkylaminocarbonyl, C, 6alkylcarbonyiamino, carbamido, C, 6 alkanoyloxy, sulphono, C, 6alkylsulphonyloxy, nitro, azido, sulphanyl, C, _6alkylthio, 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 15 carbon atom (s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected fromO,S, and(NRN)where RN is selected from hydrogen and C, 4alkyl, and where two adjacent (nongeminal) substituents may designate an additional bond resulting in a double bond; and RN, when present is selected from hydrogen and C, 4alkyl; and basic salts and acid addition salts thereof.
2. An oligomer according to claim 1, comprising 110000 LriboLNA (s) of the general formula I and 010000 nucleosides selected from naturally occurring nucleosides and nucleoside analogues, with the proviso that the sum of the number of nucleosides and the number of LriboLNA (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 LriboLNA 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 LriboLNA monomers.
5. An oligomer according to claim 2, wherein all nucleoside monomers of an oligomer are LriboLNA.
6. An oligomer according to any of the claims 15, wherein the LriboLNA (s) has/have the following formula la wherein P, P*, B, X, R', R2, R2, R3, R4, R5, and Rus are as defined in claim 1.
7. An oligomer according to any of the claims 16, wherein X is selected from (CR6R6),O,S,(CR6R6),O,S, andN (RN), preferablyO,S, andN (RN), in particularO.
8. An oligomer according to any of the claims 17, wherein the biradical constituted by R2* and R'issetectedfrom(CR'R')rY(CR'R\,(CR'R')rY(CR'R')sY,Y(CR*R*)r.sY, Y (CR*R*) rY (CR*R') s, (CR*R*) r+s,Y,YY, wherein each Y is independently selected fromO,S,Si (R) 2,N (R'), >C=O,C (=O)N (R*), andN (R')C (=O), each R* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, monoor di (C,6alkyl) amino, optionally substituted C, 6alkoxy, optionally substituted C, 6alkyl, 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 04 with the proviso that the sum r+s is 14.
9. An oligomer according to claim 8, wherein the biradical is selected from Y, (CR*R*)r+s , (CR*R*)rY(CR*R*)s, and Y(CR*R*)r+sY, wherein each of r and s is 03 with the proviso that the sum r+s is 14.
10. An oligomer according to claim 9, wherein the biradical is selected fromO,S, N(R*),(CR*R*)ri (CR*R*)r0(CR'R')s,(CR*R')rS(CR*R*)s,(CR'R')rN(R') (CR'R')s,0(CR*R*)r.s0,S(CR'R')r.s0,0(CR'R')r.sS,N(R*)(CR'R')r.s0,0 (CR'R') r.sN(R'),S(CR*R')S,N(R')(CR'R')r.sN(R'),N(R')(CR*R')r.sS, andS (CR*R')r+sN(R), wherein each of r and s is 03 with the proviso that the sum r+s is 14, and where X is selected fromO,S, andN (R") where R"designates hydrogen or C14 alkyl.
11. An oligomer according to claim 10, wherein X is O, R2 is selected from hydrogen, hydroxy, and optionally substituted C, 6alkoxy, and R', R', R5, and R designate hydrogen.
12. An oligomer according to claim 11, wherein the biradical is selected from0, (CH2) o and(CH2)01N(RN)(CH2)13,suchasOCH2,1O(CH2)13,(CH2)01S(CH2)13, SCH2andN (RN)CH2.
13. An oligomer according to any of the claims 1112, wherein B is selected from nucleobases.
14. An oligomer according to claim 8, wherein the biradical is (CH2) 24.
15. An oligomer according to any of the claims 810, wherein one e R* is selected from hydrogen, hydroxy, optionally substituted C, 6alkoxy, optionally substituted C, 6alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and any remaining substituents R'are hydrogen.
16. An oligomer according to any of the claims 115, wherein any internucleoside linkage of the LriboLNA (s) is selected from linkages consisting of 2 to 4, preferably 3, groups/atoms selected fromCH2,O,S,NR", >C=O, >C=NR", >C=S,Si (R") 2 SO, S (O) 2,P (O) 2,P (O, S),P (S) 2,PO (R"),PO (OCH3), andPO (NHRH), where RH is selected form hydrogen and C, 4alkyl, and R"is selected from C, 6alkyl and phenyl.
17. An oligomer according to claim 16, wherein any internucleoside linkage of the Lribo LNA (s) is selected fromCH2CH2CH2,CH2COCH2,CH2CHOHCH2,OCH2O, CH2CH2O,NRHCH2CH2,CH2CH2NRH,CH2NRH OCH2CH2,OCH2CH=, <BR> <BR> <BR> CH2,OCH2CH2NR",NR"COO,NR"CONR",NR"CSNR",<BR> <BR> <BR> <BR> <BR> NRHC (=NR")NR",NR"COCH2NR",OCOO,OCOCH2O,OCH2COO,<BR> <BR> <BR> <BR> CH2CONR",OCONR",NRHCOCH2,OCH2CONR",OCH2CH2NR", _ CH2ON=,CH2ONRH,CONRHCH2,CH2NRHO,CH2CH=NO,CH2NRHO, ONRH,OCH2S,SCH2O,CH2CH2S,OCH2CH2S,NRHCO,ONRHCH2, SCH2CH=,SCH2CH2,SCH2CH20,SCH2CH2S,CH2SCH2,CH2SOCH2, CH2SO2CH2,OSOO,OS (0) 2O,OS (0) 2CH2,OS (0) 2NR",NR"S (0) 2CH2, OP(O,S)O,OP(S)2O,SP(O)2O,SP(O,S)O,S OS(O)2CH2,OP(O)2O, P (S) 2O, OP (0) 2S, OP (O, S)S,OP (S) 2S,SP (0) 2S,SP (O, S)S,SP (S) 2S, OPO(BH3)O,OPO(NHRN)O,OP(O)2NRH,NRH OPO(R")O,OPO(OCH3)O, P (0) 2O OP (O, NR")O, andOSi (R") 2O.
18. An oligomer according to any of the claims 117, wherein each of the substituents R1*, R2, R3*, R5, R5*, R6 and R6 of the LriboLNA (s), which are present, is independently selected from hydrogen, optionally substituted C, 6alkyl, optionally substituted C2 6 alkenyl, hydroxy, C, 6alkoxy, C2 6alkenyloxy, carboxy, C16alkoxycarbonyl, C16 alkylcarbonyl, formyl, amino, monoand di (C, 6alkyl) amino, carbamoyl, monoand di (C1 carbamido,azido,C16alkanoyloxy,6alkyl)aminocarbonyl,C16alkylcarbonylamino, sulphono, sulphanyl, C16alkylthio, 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 C14alkyl.
19. An oligomer according to any of the claims 118, wherein X is selected fromO,S, andNRN^, and each of the substituents R1*, R2, R3*, R',R*,R"andR'of the Lribo LNA (s), which are present, designate hydrogen.
20. An oligomer according to any of the claims 119, wherein P is a 5'terminal group selected from hydrogen, hydroxy, optionally substituted C, 6alkyl, optionally substituted C, 6alkoxy, optionally substituted C, 6alkylcarbonyloxy, optionally substituted aryloxy, monophosphate, diphosphate, triphosphate, andWA', wherein W is selected fromO, S, andN (RH)where RH is selected from hydrogen and C, 6alkyl, and where A'is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
21. An oligomer according to any of the claims 120, wherein P* is a 3'terminal group selected from hydrogen, hydroxy, optionally substituted C, 6alkoxy, optionally substituted C, 6alkylcarbonyloxy, optionally substituted aryloxy, andWA', wherein W is selected fromO,S, andN (R") where R"is selected from hydrogen and C, 6alkyl, and where A'is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
22. An oligomer according to any of the claims 121, having the following formula III: G[NuL]n(o){[(LriboLNA)L]m(q)[NuL]n(q)}qG*III wherein q is 150; each of n (0),.., n (q) is independently 010000; each of m (1),.., m (q) is independently 110000; with the proviso that the sum of n (0),.., n (q) and m (1),.., m (q) is 215000; G designates a 5'terminal group; each Nu independently designates a nucleoside selected from naturally occurring nucleosides and nucleoside analogues; each LriboLNA independently designates a nucleoside analogue; each L independently designates an internucleoside linkage between two groups selected from Nu and LriboLNA, or L together with G* designates a 3'terminal group; and each LriboLNAL independently designates a nucleoside analogue of the general formula 1.
23. A nucleoside analogue (LriboLNA) of the general formula 11 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 fromO,S,N (RN*), andC (RR) ; each of Q and Q' is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, ProtO, ActO, mercapto, ProtS, ActS, C16alkylthio, amino, ProtN (RH), ActN (R"), monoor di (C, 6alkyl) amino, optionally substituted C, 6alkoxy, optionally substituted C, 6alkyl, optionally substituted C2 6alkenyl, optionally substituted C26 alkenyloxy, optionally substituted C26alkynyl, optionally substituted C26alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, ProtOCH2, ActOCH2, aminomethyl, ProtN (RH) CH2, ActN (R")CH2, carboxymethyl, sulphonomethyl, where Prot is a protection group forOH,SH, andNH (R"), respectively, Act is an activation group forOH,SH, and NH (RH), respectively, and R"is selected from hydrogen and C, 6alkyl; and R2 and R4* together designate a biradical selected from0,(CR'R')rr,(CR*R*)r0 <BR> <BR> <BR> (CR'R') s,(CR'R')rS(CR'R'),,(CR'R')rN(R')(CR'R'),,0(CR'R')r.s0,S(CR'R')r.s<BR> <BR> <BR> <BR> 0,0(CR'R')r.sS,N(R')(CR'R')r.s0,0(CR'R')r.sN(R'),S(CR'R')r.sS, andS(CR*R*)r+sN(R*);N(R*)(CR*R*)r+sN(R*),N(R*)(CR*R*)r+sS, wherein each R* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, monoor di (C, 6alkyl) amino, optionally substituted C, 6alkoxy, optionally substituted C16alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and/or two adjacent (nongeminal) R may together designate a double bond, and each of r and s is 03 with the proviso that the sum r+s is 14; each of the present substituents R1*, R2, R3, R5, Rs, R6, and R6* is independently selected from hydrogen, optionally substituted substitutedC212alkenyl,optionally optionally substituted C112alkoxy,C212alkenyloxycarboyx,C112hydroxy, alkoxycarbonyl, C,, 2alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, monoand di (C, 6alkyl) amino, carbamoyl, monoand di (C, 6alkyl)aminocarbonyl, aminoC, 6alkyl aminocarbonyl, monoand di (C, 6alkyl) aminoC, 6alkylaminocarbonyl, C, 6alkyl carbonylamino, carbamido, C, _6alkanoyloxy, sulphono, C,6alkylsulphonyloxy, nitro, azido, sulphanyl, C, 6alkylthio, 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 15 carbon atom (s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected fromO,S, and (NRN)where RN is selected from hydrogen and C, 4alkyl, and where two adjacent (nongeminal) 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 C, 4alkyl; 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.
24. A nucleoside analogue according to claim 23, wherein the group B is selected from nucleobases and functional group protected nucleobases.
25. A nucleoside analogue according to any of the claims 2324, wherein X is selected fromO,S, andN (RN).
26. A nucleoside analogue according to any of the claims 2325, wherein each of the substituents R', R2, R', R5, R5*, R6, and R6, which are present, is independently selected from hydrogen, optionally substituted C, 6alkyl, optionally substituted C26alkenyl, hydroxy, C, 6alkoxy, C2 6alkenyloxy, carboxy, C, 6alkoxycarbonyl, C, 6alkylcarbonyl, formyl, amino, monoand di (C, 6alkyl) amino, carbamoyl, monoand di (C, 6alkyl)amino carbonyl, C, 6alkylcarbonylamino, carbamido, azido, C, 6alkanoyloxy, sulphono, sulphanyl, C, 6alkylthio, 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 C, 4alkyl, with the proviso that any hydroxy, amino, mono (C, 6alkyl) amino, sulfanyl, and carboxy is optionally protected.
27. A nucleoside analogue according to any of the claims 2326, each of the substituents R1*, R2, R3, R5, R', R6 and R6, which are present, designate hydrogen.
28. A nucleoside analogue according to any of the claims 2327, wherein Q is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, ProtO, mercapto, ProtS, C16alkylthio, amino, ProtN (RH), monoor di (C, 6alkyl) amino, optionally substituted C, 6alkoxy, optionally substituted C16alkyl, optionally substituted C26alkenyl, optionally substituted C26alkenyloxy, optionally substituted C26alkynyl, optionally substituted C26alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, ProtOCH2, aminomethyl, ProtN (R")CH2, carboxymethyl, sulphonomethyl, where Prot is a protection group forOH,SH, andNH (R"), respectively, and RH is selected from hydrogen and C16alkyl ; and Q* is selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, ActO, mercapto, ActS, C16alkylthio, amino, ActN (R"), monoor di (C, 6alkyl) amino, optionally substituted C, 6alkoxy, optionally substituted C16alkyl, optionally substituted C26alkenyl, optionally substituted C26alkenyloxy, optionally substituted C26alkynyl, optionally substituted C26alkynyloxy, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, where Act is an activation group forOH,SH, andNH (R"), respectively, and RH is selected from hydrogen and C16alkyl.
29. A nucleoside analogue according to any of the claims 2328, wherein X is O, R2 selected from hydrogen, hydroxy, and optionally substituted C, 6alkoxy, and R1*, R3 R5, and R'designate hydrogen.
30. A nucleoside analogue according to claims 2329, wherein the biradical is selected (CH2)01O(CH2)13,(CH2)01S(CH2)13,and(CH2)01N(RN)(CH2)13.fromO,.
31. A nucleoside analogue according to claim 30, wherein the biradical is selected from andN(RN)CH2.OCH2,SCH2.
32. A nucleoside analogue according to claim 2331, wherein the biradical is (CH2) 24, preferably (CH2) 2.
33. A conjugate of a LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 126 and a compound selected from proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, and PNA.
34. The use of an LriboLNA as defined in any of the claims 2332 for the preparation of an LriboLNA modified oligonucleotide (an oligomer) according to any of the claims 134.
35. The use according to claim 33, wherein the incorporation of LriboLNA modulates the ability of the oligonucleotide to act as a substrate for nucleic acid active enzymes.
36. The use of a LriboLNA as defined in any of the claims 2332 for the preparation of a conjugate of an LriboLNA modified oligonucleotide and a compound selected from proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, and PNA.
37. The use of a LriboLNA as defined in any of the claims 2332 as a substrate for enzymes active on nucleic acids.
38. The use according to claim 37, wherein the LriboLNA is used as a substrate for DNA and RNA polymerases.
39. The use of an LriboLNA as defined in any of the claims 2332 as a therapeutic agent.
40. 49 The use of an LriboLNA as defined in any of the claims 2332 for diagnostic purposes.
41. The use of one or more LriboLNA as defined in any of the claims 2332 in the construction of solid surface onto which LNA modified oligonucleotides of different sequences are attached.
42. The use of LriboLNA modified oligomers (ribozymes) as defined in any of the claims 122 in the sequence specific cleavage of target nucleic acids.
43. The use of a LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 122 in therapy, such as an antisense, an antigene or a gene activating therapeutic.
44. The use according to claim 42, wherein the LNA modified oligonucleotide recruits RNAseH.
45. The use of complexes of more than one LriboLNA modified oligonucleotide (an oligomer) said oligonucleotides being defined according to any of the claims 122, in therapy, such as as an antisense, an antigene or gene activating therapeutic.
46. The use of an aLriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 622 in therapy, wherein the aLriboLNA modified oligonucleotide specifically interact with RNA selected from the group consisting of tRNAs, rRNAs, snRNAs and scRNAs thereby inhibiting any of the following cellular processes selected from the group consisting of translation, RNA splicing, RNA processing, and other essential cellular processes.
47. The use of an LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 622 in diagnostics suc as for the isolation, purification, amplification, detection, identification, quantification, or capture of natural or synthetic nucleic acids.
48. The use of an aLriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 622 in diagnostics such as for the isolation, purification, amplification, detection, identification, quantification, or capture of natural or synthetic nucleic acids, said (xLriboLNA modified oligonucleotide being able to discriminate between RNA and DNA thereby selectively hybridizing to the target RNA.
49. The use according to any of claims 46 or 47, 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.
50. The use according to claim 48, 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.
51. The use according to claim 49, 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.
52. The use according to claim 50 for capture and detection of naturally occurring or synthetic double stranded or single stranded nucleic acids such as RNA or DNA.
53. The use according to claim 47 for purification of naturally occurring double stranded or single stranded nucleic acids such as RNA or DNA.
54. The use of an LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 122 as an aptamer in molecular diagnostics.
55. The use of an LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 122 as an aptamer in RNA mediated catalytic processes.
56. The use of an LriboLNA modified oligonucleotide (an ohgomer) as defined in any of the claims 122 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.
57. The use of an LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 122 as an aptamer in the separation of enantiomers from racemic mixtures by stereospecific binding.
58. The use of a LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 122 for the labelling of cells.
59. The use of an LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 122 to hybridise to nonprotein coding cellular RNAs, such as tRNA, rRNA, snRNA and scRNA, in vivo or invitro.
60. The use of a LriboLNA modified oligonucleotide (an oligomer) as defined in any of the claims 122 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.
61. 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 LriboLNA modified oligonucleotides (oligomer) as defined in any of the claims 1 22.
62. 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 LriboLNAs as defined in any of the claims 2332.
63. A kit according to claim 60 or 61, wherein the LriboLNAs are immobilised onto said reactions body.
Description:
INTERNATIONAL SEARCH REPORT Int tional Application No PCT/DK 00/00225 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X SINGH S K ET AL :"Synthesis of 1-62 2'-Amino-LNA : A Novel Conformationally Restricted High-Affinity Oligonucleotide Analogue with a Handle" J. ORG. CHEM., vol. 63,1998, pages 10035-10039, XP002901184 the whole document X OBIKA S ET AL:"Stability and structural 1-62 features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2'-0, 4'-C-methyleneribonucleosides" TETRAHEDRON LETTERS, vol. 39,1998, pages 5401-5404, XP002901185 the whole document X KOSHKIN A A ET AL :"LNA (Locked Nucleic 1-62 Acid): An RNA Mimic Forming Exceedingly Stable LNA: LNA Duplexes" J. AM. CHEM. SOC., vol. 120,1998, pages 13252-13253, XP002901186 the whole document X SINGH S K ET AL :"LNA (Locked Nucleic 1-62 Acids): synthesis and high-affinity nucleic acid recognition" CHEM. COMMUN., 1998, pages 455-456, XP002901187 the whole document X WO 98 39352 A (IMANISHI TAKESHI) 1-34 11 September 1998 (1998-09-11) the whole document X OBIKA S ET AL :"Synthesis of 23-32 2'-0, 4'-C-Methyleneuridine and-cytidine. Novel Bicyclic Nucleosides Having a Fixed C3,-endo Sugar Puckering" TETRAHEDRON LETTERS, vol. 38, no. 50,1997, pages 8735-8738, XP002901188 the whole document X SINGH S K ET AL :"Synthesis of Novel 2-22, Bicyclo (2.2.1) Ribonucleosides : 2'-Amino-33-62 and 2'-Thio-LNA Monomeric Nucleosides" J. ORG. CHEM., vol. 63, 1998, pages 6078-6079, XP002901189 claims 1,23-32 3 3 INTERNATIONAL SEARCH REPORT Im.. ational Application No PCT/DK 00/00225 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X RAJWANSHI V K ET AL :"Synthesis and 2-22, restricted furanose conformations of three 33-62 novel bicyclic thymine nucleosides : a xylo-LNA nucleoside, a 3'-0,5'-C-methylene-linked nucleoside, and a 2'-0, 5'-C-methylene-linked nucleoside" J. CHEM. SOC. PERKIN TRANS., vol. 1,1999, pages 1407-1414, XP002901190 claims 1,23-32 X KOSHKIN A A ET AL :"Novel Convenient 1,23-32 Syntheses of LNA (2.2.1) Bicyclo Nucleosides" TETRAHEDRON LETTERS, vol. 39,1998, pages 4381-4384, XP002901191 the whole document A 2-22, 33-62 X KUMAR R ET AL :"The first analogues of 2-22, LNA (Locked Nucleic Acids): 33-62 phosphorothioate-LNA and 2'-Thio-LNA" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 8,1998, pages 2219-2222, XP002901192 claims 1,23-32 3 International application No. INTERNATIONAL SEARCH REPORT PCT/DK 00/00225 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This International Search Report has not been established in respect of certain claims under Article 17 (2) (a) for the following reasons: 1. Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: 2. Claims Nos. : 1-62 because they relate to parts of the international Application that do not comply with the prescribed requirements to such an extent that no meaningful International Search can be carried out, specifically: see FURTHER INFORMATION sheet PCT/ISA/210 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4 (a). Box li Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: 1. As a ! i required additional search fees were timely paid by the applicant, this International Search Report covers all searchable claims. 2. As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this International Search Report covers only those claims for which fees were paid, specifically claims Nos.: 4. No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest g The additional search fees were accompanied by the applicant's protest. u No protest accompanied the payment of additional search fees. u FURTHER INFORMATION CONTINUED FROM PCT/ISA/210 Continuation of Box 1. 2 Claims Nos.: 1-62 Present claims 1-62 relate to an extremely large number of possible compounds. In fact, the claim contains so many options, variables, possible permutations and provisos that a lack of clarity I concisenness within the meaning of Article 6 PCT arises to such an extent as to render a meaningful search of the claims impossible.

Consequently, the search has been carried out for those parts of the application which appear to be clear and concise, namely the i recited in the examples.

The initial phase of the search revealed a very large number of documents relevant to the issue of novelty. So many documents were retrieved that it is impossible to determine which parts of the claim (s) may be said to define subject-matter for which protection might legitimately be sought (Article 6 PCT). For these reasons, a meaningful search over the whole breadth of the claim (s) is impossible. Consequently, the search has been restricted INTERNATIONAL SEARCH REPORT | ím.. ational Application No Information on patent family members Patent document Publication Patent family Publication cited in search report date member (s) date WO 9914226 A 25-03-1999 AU 9063398 A 05-04-1999 EP 1015469 A 05-07-2000 WO 9839352 A 11-09-1998 AU 720472 B 01-06-2000 AU 6120998 A 22-09-1998 EP 1013661 A 28-06-2000 JP 10304889 A 17-11-1998