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US 2003/0198982 A1Published application
Nucleic acid binding compounds containing pyrazolo[3,4-d]pyrimidine analogues of purin-2,6-diamine and their uses
AssigneeRoche Molecular Systems, Inc.
InventorsFrank Seela, Frank Bergmann, Herbert Von Der Eltz, Dieter Heindl, Christoph Seidel, Georg Becher
Life of this patent
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Abstract
The present invention is in the field of nucleic acid binding compounds comprising 7-substituted 7-deaza-8aza-2,6-diamino-purine bases, compounds useful for the preparation of such compounds, various uses thereof and methods for the determination of nucleic acids using said compounds in the field of diagnostics.
Claims92
A nucleic acid binding compound comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a group of the general formula I
- 52
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of
- (1) —F, —Cl, —Br or —I,
- (2) Nitro
- (3) Cyano
- (4) —COO−
- (5) —(C1-C10)-alkyl substituted according to (10)
- (6) —(C2-C10)-alkenyl substituted according to (10)
- (7) —(C2-C10)-alkynyl substituted according to (10)
- (8) —(C6-C22)-aryl substituted according to (10)
- (9) —W—(C1-C10)-alkyl, —W—(C2-C10)-alkenyl, —W—(C2-C10)-alkynyl, —W—(C6-C22)-aryl or W—H, wherein W≡S—, —O—, —NH—, —S—S—, —CO—, —COO—, —CO—NH—, —NH—CO—, —NH—CO—NH—, —NH—CS—NH—, —(CH2)n-[O—(CH2)r]s—, where r and s are, independently of each other, an integer between 1 to 18 and n is 0 or 1 independently from r and s,
- (10) substituents (5) to (9) wherein any alkyl, alkenyl, alkynyl or aryl can be substituted by one or more moieties selected from the group consisting of -halogen, —SH, —NO2, —CN, —S—(C1-C6)-alkyl, —NR5R6, —N+R5R6R12, —OR -COR11, —NH—CO—NR5R6, —NH—CS—NR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- wherein R11 is selected from the group consisting of —NHR12, OR12, and —SR12
- wherein R5, R6 and R12 are selected independently from the group consisting of —H, —(C1-C10)-alkyl, —(C2-C10)-alkenyl, —(C2-C10)-alkinyl, —(C6-C22)-aryl and a reporter group, a group which facilitates intracellular uptake or a group that, when the nucleic acid binding compound hybridizes to its complementary nucleic acid, attacks the latter while binding, cross-linking or cleaving,
- said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n-[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R2, R3 is independent from X, R1, R2 and R3 and is selected from the group of,
- (1)—H
- (2)(C1-C10)-alkyl,
- (3)(C2-C10)-alkenyl,
- (4) (C2-C10)-alkynyl,
- (5)(C6-C22)-aryl,
- (6) -Z-(C1-C10)-alkyl, -Z-(C2-C10)-alkenyl, -Z-(C2-C10)-alkynyl, -Z-(C6-C22)-aryl or Z-H, wherein Z=—CO—, —CO—NH—, —CS—NH—, —(CH2)n—[O—(CH2)r]s—, where r and s are, independently of each other, an integer between 1 to 18 and n is 1 or 2 independently from r and s,
- (7)substituents (2) to (6)
- wherein any alkyl, alkenyl, alkynyl or aryl can be substituted by one or more moieties selected from the group consisting of -halogen, —NO2, —OR12, —CN, —SH, —S—(C1-C6)-alkyl, —NR5R6, —N+R5R6R12, COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- wherein R11 is selected from the group consisting of —NHR12 and OR12,
- wherein R5, R6 and R12 are selected independently from the group consisting of —H, —(C1-C10)-alkyl, —(C2-C10)-alkenyl, —(C2-C10)-alkynyl, —(C6-C22)-aryl and a reporter group,
- said alkyl, alkenyl, alkynyl or aryl in substituents (2) to (7) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s;
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH;
- and
- D is the position of attachment of the group to the rest of the nucleic acid binding compound
- or any salts thereof.
30 more elements
A composition for analyzing interactions between nucleic acid binding compounds comprising an array of a plurality of nucleic acid binding compounds having different sequences, wherein said plurality of nucleic acid binding compounds are coupled to a solid substrate at known locations and are selected to bind to complementary nucleic acid binding compounds
- characterized in that
- only the nucleic acid binding compounds or the nucleic acid binding compounds and the complementary nucleic acid binding compounds together are nucleic acid binding compounds comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases, wherein a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof.
A compound of formula VI
- 59
- wherein
- A is selected from the group consisting of O, S, CH2 and N-(C1-C6)-alkyl,
- R14 is selected from the group consisting of —H, —OH, —(C1-C10)-alkoxy, O-protecting group, S-protecting group, NH-protecting group, —(C2-C10)-alkenyloxy, -halogen, -azido, —SH, —(C1-C6)-alkylmercapto, O-reporter group and —NH2,
- R15 and R16 are independently selected from the group consisting of —H, —(C1-C8)-alkyl, —(C2-C18)-alkenyl, —(C2-C18)-alkynyl, —(C2-C18)-alkyl-carbonyl, —(C3-C19)-alkenyl-carbonyl, —(C3-C19)-alkynyl-carbonyl, —(C6-C14)-aryl-(C1-C8)-alkyl, a protecting group or a compound of formula IV, with the proviso that R or R is a group of formula IV,
- 60
- wherein
- T is selected from the group consisting of oxo, thioxo and selenoxo,
- U is selected from the group consisting of —OH, —SH, —SeH, —(C1-C10)-alkoxy, —(C1-C10)-alkyl, —(C6-C22)-aryl, —(C6-C14)-aryl-(C1-C10)-alkyl, —NR23R24, and —O—(C1-C10)-alkyl-O—(C1-C10)-alkyl-R25, or wherein NR23R24 can together with N be a 5-6-membered heterocyclic ring,
- R23 and R24 are independently selected from the group consisting of —(C1-C10)-alkyl, —(C1-C20)-aryl, —(C6-C14)-aryl-(C1-C10)-alkyl, —(C1-C6)-alkyl-[NH(CH2)c]d—NR26R27,
- R25 is selected from the group consisting of —H, —OH, -halogen, amino, —(C1-C18)-alkylamino, —COOH, —CONH2 and COO(C1-C4)-alkyl,
- R26 and R27 are independently selected from the group consisting from —H, —(C1-C6)-alkyl, and —(C1-C4)-alkoxy-(C1-C6)-alkyl,
- R29 is selected from the group consisting of —OR30 and —SR30,
- R30 is selected from the group consisting of —H, —(C1-C10)-alkyl, —(C2-C10)-alkenyl, —(C6-C22)-aryl, a protecting group, a phosphate, a diphosphate and a reporter group, and
- M and M′ are independently selected from the group consisting of oxy, sulfanediyl, —NR22, —(C1-C10)-alkyl, or —O—(C1-C10)-alkyl-O—, and —S—(C1-C10)-alkyl-O— and —NR22—(C1-C6)-alkyl-O—,
- R22 is selected from the group of —H and —(C1-C10)-alkyl, and
- c is an integer from 2 to 6,
- d is an integer from 0 to 6, and
- B is a moiety of formula I,
- 61
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of substituents (1) to (10) for R1 in formula I in claim 1 and
- 62
- wherein Nuc is the link to formula I and n is any integer from 1 to 18 said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R1, R3 is independent from X, R1, R2 and R3 and is selected from the group of substituents (1) to (7) for R2 and R3 in formula I in claim 1,
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH
- D is the position of attachment of the group to the rest of the nucleic acid binding compound.
- whereby optionally at least one protecting group substitutes one or two hydrogen atoms of a —OH, —SH, NH2, NH-alkyl, —NH-alkenylene, —NH-alkynylene, or a —NH-aryl group,
- and any salts thereof.
27 more elements
A compound of formula VII
- 63
- wherein
- A is selected from the group consisting of O, S, CH2 and N-(C1-C6)-alkyl,
- M and M′ are independently selected from the group consisting of oxy, sulfanediyl, —NR22, —(C1-C10)-alkyl, or —O—(C1-C10)-alkyl-O—, and —S—(C1-C10)-alkyl-O— and —NR22—(C1-C6)-alkyl-O—,
- R22 is selected from the group of —H and —(C1-C10)-alkyl,
- R14 is selected from the group consisting of —H, —OR31, —(C1-C10)-alkoxy, —(C2-C10)-alkenyloxy, —(C2-C10)-alkynyloxy, -halogen, -azido, NHR31, SR31,
- R31 is a protecting group or a reporter group,
- R32 and R17 are independently selected from the group consisting of —H, —(C1-C10)-alkyl, —(C2-C10)-alkenyl, —(C6-C22)-aryl, or wherein NR32R17 can form together with N a 5-6-membered heterocyclic ring,
- R18 is selected from the group consisting of —(C2-C6)-alkenyloxy, substituted or unsubstituted —(C1-C6)-alkyl, unsubstituted —(C1-C6)-alkoxy or —(C1-C6)-alkoxy substituted one or more times by a group selected from the group consisting of -halogen, p-nitroaryloxy and -cyano, and
- B is a group of formula I
- 64
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of substituents (1) to (10) for R1 in formula I in claim 1 and
- 65
- wherein Nuc is the link to formula I and n is any integer from 1 to 18 said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R2, R3 is independent from X, R1, R2 and R3 and is selected from the group of substituents (1) to (7) for R2 and R3 in formula I in claim 1;
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH
- D is the position of attachment of the group to the rest of the nucleic acid binding compound.
- with the proviso that one or two hydrogen atoms of any —OH, —SH, —NH2, —NH-alkyl, —NH-alkenylene, —NH-alkynylene, or —NH-aryl group are substituted by a protecting group,
- and any salts thereof.
18 more elements
A compound of general formula IX
- 66
- wherein
- A is selected from the group consisting of O, S, CH2 and N-(C1-C6)-alkyl,
- M and M′ are independently selected from the group consisting of oxy, sulfanediyl, —NR22, —(C1-C10)-alkyl, or —O—(C1-C10)-alkyl-O—, and —S—(C1-C10)-alkyl-O— and —NR22-(C1-C6)-alkyl-O—,
- R22 is selected from the group of —H and —(C1-C10)-alkyl,
- R14 is selected from the group consisting of —H, —OR31, —(C1-C10)-alkoxy, —(C2-C10)-alkenyloxy, —(C2-C10)-alkynyloxy, -halogen, -azido, NHR31, SR31, or O-reporter group,
- R31 is a protecting group or a reporter group,
- R32 and R17 are independently selected from the group consisting of —H, —(C1-C10)-alkyl, —(C2-C10)-alkenyl, —(C6-C22)-aryl, or wherein NR32R17 can form together with N a 5-6-membered heterocyclic ring,
- R18 is selected from the group consisting of —(C2-C6)-alkenyloxy, substituted or unsubstituted —(C1-C6)-alkyl, unsubstituted —(C1-C6)-alkoxy or —(C1-C6)-alkoxy substituted one or more times by a group selected from the group consisting of -halogen, p-nitroaryloxy and -cyano, and
- B is a group of formula I
- 67
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of substituents (1) to (10) for R1 in formula I in claim 1 and
- 68
- wherein Nuc is the link to formula I and n is any integer from 1 to 18 said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R2, R3 is independent from X, R1, R2 and R3 and is selected from the group of substituents (1) to (7) for R2 and R3 in formula I in claim 1
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH
- D is the position of attachment of the group to the rest of the nucleic acid binding compound.
- with the proviso that one or two hydrogen atoms of any —OH, —SH, —NH2, —NH-alkyl, —NH-alkenylene, —NH— alkynylene, or —NH-aryl group are substituted by a protecting group, and any salts thereof.
17 more elements
A compound of general formula X
- 69
- wherein
- M and M′ are independently selected from the group consisting of oxy, sulfanediyl, —NR22, —(C1-C10)-alkyl, or —O—(C1-C10)-alkyl-O—, and —S—(C1-C10)-alkyl-O— and —NR22—(C1-C6)-alkyl-O—,
- R22 is selected from the group of —H and —(C1-C10)-alkyl,
- R14 is selected from the group consisting of —H, —OR31, —(C1-C10)-alkoxy, -(C2-C10)-alkenyloxy, —(C2-C10)-alkynyloxy, -halogen, -azido, NHR31, SR31, or O-reporter,
- R31 is a protecting group or a reporter group,
- B is a group of formula I
- 70
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of substituents (1) to (10) for R1 in formula I in claim 1 and
- 71
- wherein Nuc is the link to formula I and n is any integer from 1 to 18 said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R2, R3 is independent from X, R1, R2 and R3 and is selected from the group of substituents (1) to (7) for R2 and R3 in formula I in claim 1
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH
- D is the position of attachment of the group to the rest of the nucleic acid binding compound.
- with the proviso that one or two hydrogen atoms of any —OH, —SH, —NH2, —NH-alkyl, —NH-alkenylene, —NH-alkynylene, or —NH-aryl group are substituted by a protecting group,
- and any salts therof.
15 more elements
A precursor for the synthesis of a nucleic acid binding compound comprising a backbone , wherein the backbone comprises a moiety of the general formula VI
- 72
- wherein
- A is selected from the group consisting of O, S, CH2 and N-(C1-C6)-alkyl,
- M and M′ are independently selected from the group consisting of oxy, sulfanediyl, —NR22—, —(C1-C10)-alkyl-, or —O—(C1-C10)-alkyl-O—, and —S—(C1-C10)-alkyl-O— and —NR22—(C1-C6)-alkyl-O—,
- R22 is selected from the group of —H, —(C1-C10)-alkyl, a protecting group and a reporter group,
- R14 is selected from the group consisting of —H, —OR31, —(C1-C10)-alkoxy, O-protecting group, S-protecting group, NH2-protecting group, —(C2-C10)-alkenyloxy, —(C2-C10)-alkynyloxy, -halogen, -azido, SH, —(C1-C10)-alkylmercapto, and —O-solid phase,
- R15 and R16 are independently selected from the group consisting of —H, —(C1-C6)-alkyl, —(C2-C10)-alkenyl, —(C2-C10)-alkynyl, —(C2-C10)-alkyl-carbonyl, —(C3-C19)— alkenyl-carbonyl, —(C3-C19)-alkynyl-carbonyl, —(C6-C14)-aryl-(C1-C10)-alkyl, protecting group and a solid phase
- B is the link to a moiety of formula I,
- 73
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of substituents (1) to (10) for R1 in formula I in claim 1 and
- 74
- wherein Nuc is the link to formula I and n is any integer from 1 to 18 said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R2, R3 is independent from X, R1, R2 and R3 and is selected from the group of substituents (1) to (7) for R2 and R3 in formula I in claim 1
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH
- D is the position of attachment of the group to the rest of the nucleic acid binding compound.
- with the proviso that one or two hydrogen atoms of any —OH, —SH, —NH2, —NH-alkyl, —NH-alkenylene, —NH-alkynylene, or —NH-aryl group are substituted by a protecting group,
- and any salts thereof.
16 more elements
A precursor or intermediate of a nucleic acid binding compound, wherein the backbone comprises a moiety of the general formula III
- 75
- wherein
- A is selected from the group consisting of O, S, CH2 and N-(C1-C6)-alkyl,
- M is selected from the group consisting of oxy, sulfanediyl, —NR22—, —(C1-C10)-alkyl-, or —O—(C1-C10)-alkyl-O—, and —S—(C1-C10)-alkyl-O—, O—CO—, —NR22—(C1-C6)-alkyl-O—,
- R22 is selected from the group of —H, —(C1-C10)-alkyl, a protecting group and a reporter group,
- R14 is selected from the group consisting of —H, —OH, —(C1-C10)-alkoxy, —(C2-C10)-alkenyloxy, —(C2-C10)-alkynyloxy, -halogen, -azido, SH, —(C1-C10)-alkylmercapto, O-reporter group, O-solid phase and —NH2 linked to a protecting group,
- R15 is selected from the group consisting of —H, —(C1-C6)-alkyl, —(C2-C10)-alkenyl, —(C2-C10)-alkynyl, —(C2-C10)-alkyl-carbonyl, —(C3-C19)-alkenyl-carbonyl, —(C3-C19)-alkynyl-carbonyl, —(C6-C14)-aryl-(C1-C10)-alkyl and a solid phase,
- B is the link to a moiety of formula I,
- 76
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of substituents (1) to (10) for R1 in formula I in claim 1 and
- 77
- wherein Nuc is the link to formula I and n is any integer from 1 to 18 said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R2, R3 is independent from X, R1, R2 and R3 and is selected from the group of substituents (1) to (7) for R2 and R3 in formula I in claim 1,
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH
- D is the position of attachment of the group to the rest of the nucleic acid binding compound.
- wherein R14 is O-solid phase or R15 is solid phase,
- with the proviso that one or two hydrogen atoms of any —OH, —SH, —NH2, —NH-alkyl, —NH-alkenylene, —NH-alkynylene, or —NH-aryl group are substituted by a protecting group,
- and any salts thereof.
17 more elements
A precursor or intermediate compound comprising a backbone, said backbone having attached heterocyclic groups characterized in that a heterocyclic group is a group of the general formula I
- 78
- wherein
- R1 is independent from X, R2 or R3 and is selected from the group consisting of substituents (1) to (10) for R1 in formula I in claim 1 and
- 79
- wherein Nuc is the position of attachment of the group to the rest of the group of formula I and n is any integer from 1 to 18 said alkyl, alkenyl, alkynyl or aryl in substituents (5) to (10) being unsubstituted or substituted by one or more moieties selected from the group consisting of -halogen, —SH, —S—(C1-C6)-alkyl, —(C1-C6)-alkoxy, —OH, —NR5R6, —COR11, —NH—CONR5R6, —NH—CSNR5R6 and —(CH2)n—[O—(CH2)r]s—NR5R6, r and s are independently of each other an integer of from 1 to 18 and n is 0 or 1 independently from r and s,
- with the proviso that R5, R6 or R12 is not a reporter group if the radicals (5) to (7) are substituted by —NR5R6, NHR12, OR12, or SR12;
- R2, R3 is independent from X, R1, R2 and R3 and is selected from the group of substituents (1) to (7) for R2 and R3 in formula I in claim 1,
- X is independent from R1, R2 or R3 and is selected from the group consisting of N and CH
- D is the position of attachment of the group to the rest of the nucleic acid binding compound.
- wherein said backbone is solid phase bound,
- with the proviso that one or two hydrogen atoms of any —OH, —SH, —NH2, —NH-alkyl, —NH-alkenylene, —NH-alkynylene, or —NH-aryl group are substituted by a protecting group,
- and any salts thereof.
9 more elements
Use of a first nucleic acid binding compound in a hybridization reaction to form a parallel or antiparallel duplex with a second nucleic acid binding compound wherein the first nucleic acid binding compound and/or the second nucleic acid binding compound comprise a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof.
Use of a nucleic acid binding compound having a backbone as a capture probe, whereby the backbone has attached thereto heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof.
Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound to increase the melting temperature of a parallel or antiparallel duplex with a second nucleic acid binding compound whereby the increase in melting temperature is compared to the melting temperature of a duplex of the first nucleic acid binding compound with a second nucleic acid binding compound wherein the heterocyclic group in the first nucleic acid binding compound is complementary to a heterocyclic group in the second nucleic acid binding compound.
Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound used as probe in an amplification reaction, to increase the melting temperature of a duplex with a second nucleic acid binding compound in comparison to the melting temperature of a primer used in the amplification reaction, whereby the increase in melting temperature is compared to the melting temperature of a duplex of the first nucleic acid binding compound with the second nucleic acid binding compound wherein the heterocyclic group in the first nucleic acid binding compound is complementary to a heterocyclic group in the second nucleic acid binding compound.
Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound to harmonize the contribution of each base pair to the melting temperature of a parallel or antiparallel duplex with a second nucleic acid binding compound.
Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a first nucleic acid binding compound for enhanced detection of sequences in a second nucleic acid binding compound having mismatches in a duplex with the first nucleic acid binding compound.
Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a nucleic acid binding compound to increase the melting temperature of an intramolecular duplex of the nucleic acid binding compound whereby the increase in melting temperature is compared to the melting temperature of the intramolecular duplex of the nucleic acid binding compound wherein the heterocyclic group in the nucleic acid binding compound is complementary to a heterocyclic group in the hybridizing part of the nucleic acid binding compound.
Use of a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof in place of a heterocyclic group in a nucleic acid binding compound for enhanced detection of subtypes in a target nucleic acid.
A method for the determination of the presence, absence or amount of a nucleic acid comprising the steps
- providing a sample suspected to contain the nucleic acid,
- providing a nucleic acid binding compound comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, which is essentially complementary to a part or all of the nucleic acid,
- contacting said sample with the nucleic acid binding compound under conditions for binding the nucleic acid binding compound to the nucleic acid to form a duplex,
- determining the binding product or the degree of hybridization between the nucleic acid and the nucleic acid binding compound as a measure of the presence, absence or amount of the nucleic acid.
1 more element
A method for distinguishing related nucleotide sequences in a nucleic acid, the method comprising the following steps:
- a) providing a nucleic acid binding compound having a defined sequence, wherein the nucleic acid binding compound comprises a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof;
- b) providing a nucleic acid with two related nucleotide sequences, each of which comprises a target sequence, wherein one of the nucleotide sequence is a target sequence that is perfectly complementary to the nucleic acid binding compound and one other of the segments is a related target sequence;
- c) incubating the nucleic acid with the nucleic acid binding compound under hybridization conditions to form a duplex;
- and
- d) determining the degree of hybridization between the nucleic acid binding compound and each of the segments.
2 more elements
A method for detecting the presence of a target sequence in a nucleic acid, the method comprising the following steps:
- a) providing a nucleic acid which is to be tested for the presence of the target sequence;
- b) providing a nucleic acid binding compound comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, wherein the nucleic acid binding compound has a sequence that is substantially complementary to the target sequence;
- c) incubating the nucleic acid and the nucleic acid binding compound under hybridization conditions;
- and
- d) identifying hybridized nucleic acids;
2 more elements
A method for primer extension, the method comprising the following steps:
- a) providing a nucleic acid containing a target sequence,
- b) providing one or more nucleic acid binding compound complementary to the target sequence wherein the nucleic acid binding compound comprises a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof,
- c) providing a polymerizing enzyme and nucleotide substrates, and
- d) incubating the nucleic acid, the nucleic acid binding compounds, the enzyme and the substrates under conditions favorable for polymerization.
1 more element
A method for determining the nucleotide sequence of a nucleic acid, the method comprising the following steps:
- a) providing an array of nucleic acid binding compounds having different known sequences, wherein the nucleic acid binding compound comprises a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, with the proviso that the nucleic acid binding compounds do not contain a reporter group;
- b) incubating the nucleic acid with the array under hybridization conditions, and
- c) determining to which of the nucleic acid binding compounds in the array the nucleic acid hybridizes.
A method for determining the nucleotide sequence of a target sequence in a nucleic acid, the method comprising the following steps:
- a) providing a nucleic acid comprising the target sequence;
- b) providing at least two nucleic acid binding compounds comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic group is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof with a known sequence and wherein one of the at least two nucleic acid binding compounds has a sequence that is perfectly complementary to the target sequence and at least one other of the nucleic acid binding compounds has a related target sequence;
- c) incubating the at least two nucleic acid binding compounds with the nucleic acid under hybridization conditions;
- and
- d) determining the degree of hybridization between each of the nucleic acid binding compounds and the nucleic acid.
2 more elements
A method for examining gene expression in a cell, the method comprising the following steps:
- a) providing a population of nucleic acids representative of the genes expressed in the cell,
- b) providing an array of nucleic acid binding compounds having different sequences wherein the nucleic acid binding compounds comprise a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, and with the proviso that the nucleic acid binding compounds do not contain a reporter group;
- c) incubating the population of nucleic acids with the array under hybridization conditions, and
- d) determining which of the nucleic acid binding compounds in the array become hybridized to nucleic acids.
1 more element
A method for identifying a mutation in a target sequence of a gene of interest, the method comprising the following steps:
- a) providing a nucleic acid that comprises the target sequence.
- b) providing an array of nucleic acid binding compounds comprising a backbone, said backbone having attached heterocyclic groups capable of base pairing to nucleobases characterized in that a heterocyclic groups is a substituted pyrazolo[3,4-d]pyrimidine or an analogue thereof, wherein the nucleic acid binding compounds have different sequences, wherein the different sequences include the wild-type target sequence and different mutant target sequences, with the proviso that the nucleic acid binding compounds do not contain a reporter group;
- c) incubating the nucleic acid with the array under hybridization conditions, and
- d) determining which of the nucleic acid binding compounds in the array become hybridized to the nucleic acid.
1 more element
A building block for the synthesis of an oligonucleotide
- comprising the nucleosides adenosine or desoxyadenosine, guanosine or desoxyguanosine, isoguanosine or desoxyisoguanosine, cytidine or deoxycytidine, uridine or desoxyuridine, thymidine or desoxythymidine, or analogues of these nucleosides
- wherein a substituent is attached to the base moiety selected from the group of phthalimidoalkyl, phthalimidoalkenyl, or phthalimidoalkynyl groups with the following formulas
- 80
- wherein Nuc is the position of attachment of the substituent to the base moiety and n is any integer from 1 to 18,
- with the proviso that the phthalimidoalkynyl or phthalimidoalkyl group is not attached to the C5-atom of deoxyuridine and the proviso that the phthalimidoalkynyl group is not attached to the C7-atom of 7-deaza-deoxyguanosine.
2 more elements
Use of a phthaloyl group as a protecting group in a method for the synthesis of an oligonucleotide from building blocks comprising the nucleosides adenosine or desoxyadenosine, guanosine or desoxyguanosine, isoguanosine or desoxyisoguanosine, cytidine or deoxycytidine, uridine or desoxyuridine, thymidine or desoxythymidine, or analogues of these nucleosides
- wherein the amino groups of —(CH2)n—NH2, —CH═CH—(CH2)n—NH2, or —C≡C—(CH2)n—NH2 attached to the base moiety are derivatized with the phthaloyl group and n is any integer from 1 to 18,
- with the proviso that the —(CH2)n—NH2 or —C≡C—(CH2)n—NH2 group is not attached to the C5-atom of deoxyuridine and the proviso that the —C≡C—(CH2)n—NH2 group is not attached to the C7-atom of 7-deaza-deoxyguanosine.
Dependencies are inferred from the claim text.