Negatively charged minor groove binders

1. An oligonucleotide-negatively charged minor groove binder conjugate comprising:

a negatively charged minor groove binder moiety comprising:

at least one aryl moiety, and

at least one acidic moiety capable of ionizing under physiological conditions, wherein said acidic moiety is covalently attached to at least one of said aryl moiety and optionally comprises an acidic moiety linker;

an oligonucleotide moiety which is covalently attached to said negatively charged minor groove binder moiety, wherein said negatively charged minor

groove binder moiety is covalently attached to 3′-position, 5′-position or an internal sugar moiety of said oligonucleotide; and

wherein the conjugate retains said at least one acidic moiety of the negatively charged minor groove binder moiety.

2. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 , wherein said negatively charged minor groove binder moiety is covalently attached to a heterocyclic base portion of said oligonucleotide moiety.

3. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 , wherein said negatively charged minor groove binder moiety comprises a plurality of said acidic moieties.

4. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 3 , wherein said negatively charged minor groove binder moiety comprises at least three of said acidic moieties.

5. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 , wherein said oligonucleotide comprises from about 3 to about 100 nucleotide units.

6. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 , wherein at least one of said acidic moiety is covalently attached to at least one of said aryl moiety through said acidic moiety linker.

7. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 6 , wherein said acidic moiety linker comprises from 1 to about 30 atoms selected from the group consisting of C, N, O, S, P and a combination thereof.

8. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 7 , wherein the combination of said acidic moiety and said acidic moiety linker is of the formula:

—(X 1 ) a —[C(═O)] b —(R 1 ) c —X 2

wherein

each of a, b and c is independently 0 or 1;

each X 1 is independently selected from the group consisting of:

(i) O,

(ii) NR 2 , where each R 2 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl and a nitrogen protecting group, and

(iii) alkylene;

each R 1 is independently selected from the group consisting of alkylene, cycloalkylene, arylene and a combination thereof; and

each X 2 is independently said acid moiety.

9. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 , wherein said acidic moiety has pKa of about 6 or less.

10. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 9 , wherein each of said acidic moiety is independently selected from the group consisting of:

(i) —(O) d S(O) e OH, wherein d 0 or 1 and e is 1 or 2, and

(ii) —(O) f P(O) g (OR a1 ) h (OH) i , wherein each R a1 is independently selected from the group consisting of alkyl, aralkyl and aryl; f is 0 or 1; each of g and h is independently 0, 1, or 2; and i is 1, 2 or 3, provided the sum of g+h+i is 2 or 3;

(iii) —CO 2 H; and

(iv) salts thereof.

11. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 , wherein said acidic moiety is covalently attached to a phenyl moiety or a heteroatom of a heteroaryl portion of said aryl moiety, optionally through said acidic moiety linker.

12. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 11 , wherein said aryl moiety of negatively charged minor groove binder is selected from the group consisting of phenyl, a heteroaryl, a fused phenyl-heteroaryl, a fused heteroaryl-phenyl-heterocyclyl and a combination thereof.

13. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 further comprising a first linker moiety, wherein said negatively charged minor groove binder moiety is covalently attached to said oligonucleotide moiety through said first linker moiety.

14. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 13 further comprising a quencher moiety, wherein said quencher moiety is covalently attached to said first linker moiety.

15. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 14 , wherein said quencher moiety has an absorbance maximum of from about 400 nm to about 1000 nm.

16. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 further comprising a fluorophore moiety covalently attached to said oligonucleotide moiety, wherein said fluorophore moiety is covalently attached to said oligonucleotide moiety through an optional second linker moiety.

17. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 16 , wherein the emission wavelength of said fluorophore moiety is from about 400 to about 1000 nm.

18. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 1 of the formula:

wherein

NMGB is said negatively charged minor groove binder;

ODN is said oligonucleotide;

FL is a fluorophore;

Q is a quencher;

L 1 is a first linker comprising a chain of from 3 to about 100 atoms selected from the group consisting of C, N, 0, S, P and combinations thereof;

L 2 is a second linker comprising a chain of from 1 to about 30 atoms selected from the group consisting of C, N, O, S, P and combinations thereof; and

each of a 1 , b 1 , c 1 and d 1 is independently 0 or 1.

19. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 18 , wherein NMGB moiety is of the formula:

wherein

n is an integer from 2 to 10;

R 3 is selected from the group consisting of:

(a) alkoxy,

(b) aryloxy,

(c) R a —O-L 3 —N(R b )—, where L 3 is a third linker comprising a chain of from 3 to 20 atoms selected from the group consisting of C, N, O, S, P and combinations thereof; and R a is hydrogen, a hydroxyl protecting group or attached to L 1 ; and R b is hydrogen, alkyl, cycloalkyl or a nitrogen protecting group,

(d) a moiety of the formula:

where

each of X 3 is independently selected from the group consisting of hydrogen, alkyl, alkoxy, halide, cyano, nitro, the acidic moiety optionally comprising an acidic linker and —NR b1 —C(═O)R c , where each R b1 is hydrogen, alkyl, cycloalkyl or a nitrogen protecting group, each R c is independently selected from the group consisting of hydrogen, alkyl, and cycloalkyl;

each of R 4 is independently selected from the group consisting of:

(a) hydrogen,

(b) alkyl,

(c) the acidic moiety optionally comprising an acidic moiety linker,

(d) —C(═O)R 71 , where R 71 is hydrogen, alkyl, hydroxy or alkoxy,

(e) —NR′R″, where each of R′ and R″ is independently hydrogen or alkyl, and

(f) -alkylene)-OR 72 , where R 72 is hydrogen or alkyl,

each R 5 is independently selected from the group consisting of:

(a) hydrogen,

(b) alkyl,

(c) alkoxy,

(d) cycloalkyl,

(e) halide,

(f) cyano,

(g) nitro,

(h) —[X 4 ] m1 —C(═O)—[O] m2 —R 8 , where each m1 and m2 is independently 0 or 1, X 4 is O, NR b1 , where R b1 is hydrogen, alkyl, cycloalkyl or a nitrogen protecting group and R 8 is hydrogen, alkyl or cycloalkyl, provided when m2 is 1, R 8 is alkyl or cycloalkyl, and

(i) —C(═O)—NR e R f , where each of R e and R f is independently hydrogen, alkyl, cycloalkyl and a nitrogen protecting group,

(j) the acidic moiety optionally comprising an acidic moiety linker,

(k) —NR′R″, where each of R′ and R″ is independently hydrogen or alkyl, and

(l) -(alkylene)-OR 72 , where R 72 is hydrogen or alkyl;

each of R 6 and R 7 is selected from the group consisting of:

(a) hydrogen,

(b) alkyl,

(c) cycloalkyl,

(d) -L x -Z x , where L x is a linker comprising from 3 to 20 atoms selected from the group consisting of C, N, O, S, P and combinations thereof; Z x is hydrogen, a protecting group, a solid support or a point of attachment to said first linker L 1 ,

(e) the acidic moiety optionally comprising an acidic moiety linker, and

(f) a moiety of the formula -(Z 1 ) j -C(═O)—(R 10 ) k —[C(═O)] l —R 11 ,

where

each of j, k and l is independently 0 or 1;

each Z 1 is independently selected from the group consisting of O, NR 12 and alkylene;

each R 10 is independently selected from the group consisting of alkylene and cycloalkylene;

each R 11 is independently selected from the group consisting of alkyl, alkoxy, aryloxy, —NR 13 R 14 , —NR 15 —NR 16 R 17 , hydroxyalkyl and thioalkyl; and

each of R 12 , R 13 , R 14 , R 15 , R 16 , and R 17 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl and a nitrogen protecting group;

(g) —NR′R″, where each of R′ and R″ is independently hydrogen or alkyl;

(h) -(alkylene)-OR 72 , where R 72 is hydrogen or alkyl; and

(i) —CHO

provided at least one of X 3 , R 4 , R 6 , or R 7 comprises said acidic moiety optionally comprising said acidic moiety linker, and provided that one of R 3 , R 6 , R 7 and R a is a point of attachment to said first linker L 1 .

20. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 18 , wherein NMGB moiety is of the formula:

wherein

W 1 is N or CR x30 , where R x30 is hydrogen, alkyl, or hydroxy;

W 2 is NR 19 , S or O;

p is an integer from 2 to 12;

each R 19 is independently selected from the group consisting of:

(a) hydrogen,

(b) alkyl,

(c) a nitrogen protecting group, and

(b) said acidic moiety optionally comprising an acidic moiety linker;

each of R 18 and R 20 is independently selected from the group consisting of:

(a) hydrogen,

(b) alkyl,

(c) cycloalkyl,

(d) said acidic moiety; and

(e) -(Z 1 ) j -C(═O)—(R 10 ) k —[C(═O)] l —R 11 ,

where

each of j, k and l is independently 0 or 1;

each Z 1 is independently selected from the group consisting of O, NR 12 and alkylene;

each R 10 is independently selected from the group consisting of alkylene and cycloalkylene;

each R 11 is independently selected from the group consisting of alkyl, alkoxy, aryloxy, —NR 13 R 14 , —NR 15 —NR 16 R 17 , hydroxyalkyl and thioalkyl; and

each of R 12 , R 13 , R 14 , R 15 , R 16 , and R 17 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl and a nitrogen protecting group;

provided at least one of R 18 , R 19 or R 20 is said acidic moiety, optionally comprising said acidic moiety linker, and provided that one of R 18 and R 20 is a point of attachment to said first linker L 1 .

21. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 20 , wherein NMGB moiety is of the formula:

wherein

p, R 18 , R 19 and R 20 are those defined in claim 20 .

22. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 18 , wherein NMGB moiety is of the formula:

wherein

R 21 is an optionally substituted aryl-heterocyclyl;

each of R 22 and R 24 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl and a nitrogen protecting group;

Ar 1 is optionally substituted aryl moiety; and

R 23 is selected from the group consisting of hydrogen and said acidic moiety optionally comprising said acidic moiety linker,

provided when R 23 is hydrogen at least one of Ar 1 or R 21 is substituted with said acidic moiety optionally comprising said acidic moiety linker, and provided that one of R 21 , R 22 , R 23 and R 24 is a point of attachment to said first linker L 1 .

23. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 18 , wherein said quencher moiety, Q, is of the formula:

wherein

Y is selected from the group consisting of substituted phenyldiazenyl, nitro and —NR 50 R 51 , where each of R 50 and R 51 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl and a nitrogen protecting group;

each of z and w is independently an integer from 0 to 4;

each R z is independently selected from the group consisting of hydrogen, nitro, cyano, halide and —S(O) aa NR 52 R 53 , where aa is 0, 1 or 2 and each of R 52 and R 53 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl and a nitrogen protecting group, or two adjacent R z 's and carbon atom to which they are attached to forms a five- or six-membered ring having from zero to three heteroatoms as ring members; and

each R w is independently selected from the group consisting of alkoxy, halide and —NR 54 —C(═O)R 55 , where R 54 is selected from the group consisting of hydrogen, alkyl and a nitrogen protecting group, and R 55 is selected from the group consisting of hydrogen, alkyl and cycloalkyl, or two adjacent R w 's and carbon atom to which they are attached to forms a five- or six-membered ring having from zero to three heteroatoms as ring members.

24. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 23 , wherein said quencher moiety is of the formula:

wherein

w, z, R w and R z are those defined in claim 24 ; and

Y is selected from the group consisting of nitro and —N(CH 3 ) 2 .

25. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 23 , wherein said quencher moiety is of the formula:

wherein

Y is selected from the group consisting of nitro and —NR 50 R 51 ; and

w, z, R w , R z , R 50 and R 51 are those defined in claim 23 .

26. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 18 , wherein said fluorophore has emission wavelength of from about 400 to about 1000 nm.

27. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 26 , wherein said fluorophore, FL, is selected from the group consisting of a derivative of fluoresceine, cyanine, resorufin and coumarin.

28. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 27 , wherein said fluorophore, FL, is a coumarin derivative selected from the group consisting of:

wherein

each of R 61 , R 62 , R 63 , R 64 , R 65 , R 66 and R 67 is independently selected from the group consisting of hydrogen, halide, nitro, cyano, —SO 3 R 70 , —SO 2 N(R 70 ) 2 , —C(═O)OR 70 , —C(═O)N(R 70 ) 2 , —CNS, —OR 70 , —C(═O)R 70 , —SR 70 , —NHC(O)R 70 , —CF 3 , —N(R 70 ) 2 , wherein each R 70 is independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, aryl and a protecting group compatible with oligonucleotide synthesis, or

two adjacent groups of R 61 through R 66 together with the carbon atoms to which they are attached form a five- or six-membered ring having from zero to three heteroatoms as ring member; and

Z is O or S;

provided that at least one of R 61 through R 67 is a point of attachment to said second linker L 2 or to said oligonucleotide ODN.

29. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 18 , wherein L 1 comprises a moiety selected from the group consisting of:

wherein a 2 and b 2 are independently an integer from 2 to 10;

wherein a 2 , b 2 and c 2 are independently an integer from 2 to 10, X is O, CH 2 or NR i and each of R g , R h and R i is independently hydrogen, alkyl, cycloalkyl or a nitrogen protecting group; and

wherein a 2 , b 2 and c 2 are independently an integer from 2 to 10, X is O, CH 2 or NR i and each of R g and R i is independently hydrogen, alkyl, cycloalkyl or a nitrogen protecting group.

30. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 29 of the formula:

wherein

c 1 , d 1 , L 2 , Q, ODN, NMGB and FL are those defined in claim 18 ;

each of a 3 , b 3 and c 3 is 0 or 1; and

each of L a , L b and L c is independently a linker comprising an acyclic chain of from 1 to about 10 atoms, wherein each atom of the acyclic chain is independently selected from the group consisting of C, N, O, S and P.

31. The oligonucleotide-negatively charged minor groove binder conjugate according to claim 29 of the formula:

wherein

c 1 , d 1 , L 2 , FL, Q, ODN and NMGB are those defined in claim 18 ; and

a 2 and b 2 are those defined in claim 29 .

32. A pharmaceutical composition comprising an oligonucleotide-negatively charged minor groove binder conjugate of claim 1 .