Diaziridinyl-aryl and bis-[di(chloroethyl)amino]-aryl oligonucleotide conjugates and reagents for making the same

Claim 2

An oligonucleotide having the structure shown by formula (1) or formula (2) ##STR15## where X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group represents normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons; t is an integer having the values 1-3; ODN represents an oligonucleotide sequence that is complementary to a target sequence in nucleic acid, said ODN optionally having a tail moiety attached at either of the 5' or 3' ends, or both, and optionally having a reporter group, intercalator group, minor groove binder moiety, chelating group or lipophilic group attached to it, and LINKER is a group having the length of 1 to 20 atoms, and which covalently connects the ODN to the Q ring.

Claim 3

The oligonucleotide in accordance with claim 1, having the structure of formula (1) wherein X is selected from Cl, Br and I.

Claim 4

The oligonucleotide in accordance with claim 1 wherein Q is selected from a phenyl group optionally substituted with one or more R 1 groups, a 1,4-quinone group optionally substituted with one or more R 1 groups and 1,4-dihydroxyphenyl group optionally substituted with one or more R 1 groups.

Claim 5

The oligonucleotide in accordance with claim 1 having approximately 6 to 500 nucleotide units.

Claim 6

The oligonucleotide in accordance with claim 4 having approximately 6 to 200 nucleotide units.

Claim 7

The oligonucleotide in accordance with claim 1 that is triplex forming and wherein the sequence ODH is complementary in the triplex forming sense to a target sequence in double stranded nucleic acid.

Claim 8

An oligonucleotide in accordance with claim 1 where t is 1.

Claim 9

An oligonucleotide having the formula ##STR16## wherein B is a heterocyclic base; R 1 independently is H, O--C 1-6 alkyl, OC 2-6 alkenyl, or F; R 8 independently is H or C 1-6 alkyl; w is an integer between approximately 5 to approximately 199; X 1 is independently O or S; X 2 is independently O or NH; t 1 is 0 or 1; t 2 is 0 or 1; t 3 is an integer between 0 and 3; t 4 is 0 or 1, wherein the sum of t 1 , t 2 , t 3 and t 4 is at least one and does not exceed 3; W 1 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety; W 2 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety, and Z represents the groups shown by formula (a) or by formula (b) ##STR17## where X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons, and LINKER is a group having the length of 1 to 20 atoms, and which covalently connects the ODN to the Q ring, said oligonucleotide having a substantially continuous sequence of at least 6 nucleotides that is complementary to a target sequence in nucleic acid, and said oligonucleotide optionally having one or more covalently attached lipophilic group, a minor groove binder group, a reporter group, or chelating group.

Claim 10

An oligonucleotide having the formula ##STR16## wherein B is a heterocyclic base; R 1 independently is H, O--C 1-6 alkyl, OC 2-6 alkenyl, or F; R 8 independently is H or C 1-6 alkyl; w is an integer between approximately 5 to approximately 199; X 1 is independently O or S; X 2 is independently O or NH; t 1 is 0 or 1; t 2 is 0 or 1; t 3 is an integer between 0 and 3; t 4 is 0 or 1, wherein the sum of t 1 , t 2 , t 3 and t 4 is at least one and does not exceed 3; W 1 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety; W 2 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety, and Z represents the groups shown by formula (a) or by formula (b) ##STR17## where X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons, and LINKER is a group having the length of 1 to 20 atoms, and which covalently connects the ODN to the Q ring, said oligonucleotide having a substantially continuous sequence of at least 6 nucleotides that is complementary to a target sequence in nucleic acid, and said oligonucleotide optionally having one or more covalently attached lipophilic group, a minor groove binder group, a reporter group, or chelating group.

Claim 11

The oligonucleotide in accordance with claim 8 that is triplex forming.

Claim 12

The oligonucleotide in accordance with claim 8 wherein R 7 is H.

Claim 13

The oligonucleotide in accordance with claim 8 wherein X 1 and X 2 are O.

Claim 14

The oligonucleotide in accordance with claim 8 wherein t 3 and t 4 each is 0.

Claim 15

The oligonucleotide in accordance with claim 8 wherein Q is selected from a phenyl group optionally substituted with one or more R 1 groups, a 1,4-quinone group optionally substituted with one or more R 1 groups and 1,4-dihydroxyphenyl group optionally substituted with one or more R 1 groups.

Claim 16

The oligonucleotide in accordance with claim 13 wherein Z is the group shown by formula (a) and X is Cl.

Claim 17

A triplex forming oligonucleotide that includes a substantially continuous sequence of at least 6 nucleotides, which sequence is complementary in the triplex forming sense to a target sequence in duplex nucleic acid, the oligonucleotide having the formula ##STR18## wherein B is a heterocyclic base; w is an integer between approximately 5 to approximately 199; t 1 is 0 or 1; t 2 is 0 or 1 wherein at least one of t 1 and t 2 is 1; W 1 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety; W 2 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety, and Z represents the groups shown by formula (a) or by formula (b) ##STR19## where X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons, and LINKER is a group having the length of 1 to 20 atoms, and which covalently connects the ODN to the Q ring.

Claim 18

A triplex forming oligonucleotide that includes a substantially continuous sequence of at least 6 nucleotides, which sequence is complementary in the triplex forming sense to a target sequence in duplex nucleic acid, the oligonucleotide having the formula ##STR18## wherein B is a heterocyclic base; w is an integer between approximately 5 to approximately 199; t 1 is 0 or 1; t 2 is 0 or 1 wherein at least one of t 1 and t 2 is 1; W 1 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety; W 2 is H, a phosphate or thiophosphate group, a tail moiety or a tail moiety attached through a phosphate moiety, and Z represents the groups shown by formula (a) or by formula (b) ##STR19## where X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons, and LINKER is a group having the length of 1 to 20 atoms, and which covalently connects the ODN to the Q ring.

Claim 19

The oligonucleotide in accordance with claim 15 wherein Z represents the group of formula (a).

Claim 20

The oligonucleotide in accordance with claim 16 wherein X is Cl.

Claim 21

The oligonucleotide in accordance with claim 15 wherein Z represents the group of formula (b).

Claim 22

The oligonucleotide in accordance with claim 17 wherein Q is selected from a phenyl group optionally substituted with one or more R 1 groups, a 1,4-quinone group optionally substituted with one or more R 1 groups and 1,4-dihydroxyphenyl group optionally substituted with one or more R 1 groups.

Claim 23

The oligonucleotide in accordance with claim 18 wherein Q is selected from a phenyl group optionally substituted with one or more R 1 groups, a 1,4-quinone group optionally substituted with one or more R 1 groups and 1,4-dihydroxyphenyl group optionally substituted with one or more R 1 groups.

Claim 24

A reagent including a cross-linking function and a functional group suitable for reacting with a nucleophilic amino group attached to an oligonucleotide while substantially retaining intact the cross-linking function, the reagent having the formula ##STR20## or the formula ##STR21## wherein X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons; SPACER is a group having the length of 1 to 20 atoms, and which covalently connects the R 4 group to the Q ring, said SPACER terminating in the carbonyl (C═O) groups shown in the formula, and R 4 is a leaving group, said R 4 leaving group forming an active ester with said carbonyl group.

Claim 25

A reagent including a cross-linking function and a functional group suitable for reacting with a nucleophilic amino group attached to an oligonucleotide while substantially retaining intact the cross-linking function, the reagent having the formula ##STR20## or the formula ##STR21## wherein X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons; SPACER is a group having the length of 1 to 20 atoms, and which covalently connects the R 4 group to the Q ring, said SPACER terminating in the carbonyl (C═O) groups shown in the formula, and R 4 is a leaving group, said R 4 leaving group forming an active ester with said carbonyl group.

Claim 26

The reagent of claim 21 wherein the R 4 group is 2,3,5,6-tetrafluorophenyloxy (TFP) or para-nitrophenyloxy (PNP).

Claim 27

Diaziridinyl-aryl and bis-[di(chloroethyl)amino]-aryl DNA cross-linking agents covalently bonded to DNA targeting agents, having the formula ##STR22## or the formula ##STR23## wherein X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons; T.A. represents a DNA targeting agent selected from the group consisting of intercalators, minor groove binders, peptide nucleic acids, polyamines and synthetic polyamides. LINKER* represents a group having the length of 1 to 60 atoms which covalently connect the DNA targeting agent to the Q ring.

Claim 28

Diaziridinyl-aryl and bis-[di(chloroethyl)amino]-aryl DNA cross-linking agents covalently bonded to DNA targeting agents, having the formula ##STR22## or the formula ##STR23## wherein X is a leaving group; Q is a 5 or 6 membered aromatic or quinone ring containing 0 to 3 heteroatoms independently selected from N, O and S, the Q ring being unsubstituted or substituted with one or more R 1 groups where R 1 is F, Cl, Br, I, alkyl, Oalkyl, Salkyl, Oalkenyl, Salkenyl, CO-alkyl, OH, O═, OCOalkyl, N(R 3 ) 2 , NHCOalkyl, SO 2 alkyl, COOH, COOalkyl, CN, CF 3 , NO 2 , tetrazol or aryl where R 3 is H or alkyl, the alkyl represents normal alkyl of 1 to 10 carbons, branch-chained alkyl of 3 to 10 carbons and cycloalkyl of 3 to 10 carbons, alkenyl group normal alkenyl of 2 to 10 carbons, branch-chained alkenyl and cycloalkenyl of 3 to 10 carbons; T.A. represents a DNA targeting agent selected from the group consisting of intercalators, minor groove binders, peptide nucleic acids, polyamines and synthetic polyamides. LINKER* represents a group having the length of 1 to 60 atoms which covalently connect the DNA targeting agent to the Q ring.

Claim 29

The DNA cross-linking agents in accordance with claim 23 wherein Q is selected from a phenyl group optionally substituted with one or more R 1 groups, a 1,4-quinone group optionally substituted with one or more R 1 groups and 1,4-dihydroxyphenyl group optionally substituted with one or more R 1 groups.