WATER SOLUBLE FLUORESCENT COMPOUNDS

1 . A fluorescent compound comprising a structure according to Formula I:

wherein

the dotted lines encircling X and Y are each independently selected from atoms necessary for the formation of one ring to three fused rings having 4 to 7 atoms in each ring; and

at least one atom in the ring comprising C a is a nitrogen;

at least one atom in the ring comprising C b is a nitrogen;

w and z are integers independently selected from 0 to the number of atoms necessary for the formation of X or Y

wherein

w and z cannot both be 0;

each R 3 is a member independently selected from H, OR 30 , SR 30 , NR 30 R 30 , halogen, CN, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and a reactive functional group

wherein

two R 3 groups, together with the atoms to which they are attached, can be optionally joined to form a ring;

n is an integer selected from 0 to 4;

R 1 and R 2 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, reactive functional group, NO 2 , CN, P(O)(OR 4 )(OR 5 ), D 1 R 6 , NR 7 R 8 and C(D 2 )R 9

wherein

at least one of R 1 and R 2 is P(O)(OR 30 )(OR 30 );

D 1 is a member selected from O and S;

D 2 is a member selected from O, S and NH;

R 4 and R 5 are members independently selected from H, substituted or unsubstituted C 1 -C 4 alkyl, substituted or unsubstituted C 1 -C 4 heteroalkyl

R 6 , R 7 and R 8 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and a reactive functional group;

wherein

R 7 and R 8 , together with the nitrogen to which they are attached, can be optionally joined to form a member selected from a reactive functional group, —NHNH 2 , —N═N═N, —N═C═S and —N═C═O;

R 9 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a reactive functional group, NR 10 R 11 and OR 12

wherein

R 10 and R 11 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R 12 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R 13

wherein

R 13 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl

wherein each R 30 is independently selected from H, a negative charge, and a salt counterion.

2 . The fluorescent compound of claim 1 , wherein

has a structure according to

wherein

R 14 and R 15 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl; and

R 16 , R 17 , R 18 , R 19 and R 20 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, halogen, a reactive functional group, NO 2 , CN, P(O)(OR 4 )(OR 5 ), D 1 R 6 , NR 7 R 8 and C(D 2 )R 9

wherein

at least one of R 16 , R 17 , R 18 , R 19 and R 20 is P(O)(OR 30 )(OR 30 );

D 1 is a member selected from O and S;

D 2 is a member selected from O, S and NH;

R 4 and R 5 are members independently selected from H, substituted or unsubstituted C 1 -C 4 alkyl, substituted or unsubstituted C 1 -C 4 heteroalkyl

R 6 , R 7 and R 8 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and a reactive functional group;

wherein

R 7 and R 8 , together with the nitrogen to which they are attached, can be optionally joined to form a member selected from a reactive functional group, —NHNH 2 , —N═N═N, —N═C═S and —N═C═O;

R 9 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a reactive functional group, NR 10 R 11 and OR 12

wherein

R 10 and R 11 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R 12 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R 13

wherein

R 13 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl

wherein each R 30 is independently selected from H, a negative charge, and a salt counterion.

3 . The fluorescent compound of claim 2 , with the proviso that either a) R 1 and R 20 are independently selected from substituted or unsubstituted alkyl; or b) at least one of said R 1 and R 20 further comprises a member selected from a reactive functional group, solid support and carrier molecule.

4 . The fluorescent compound according to claim 1 , having a structure which is a member selected from

wherein

e is an integer selected from 1 to 2;

f is an integer selected from 1 to 4;

R 20 and R 21 are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl,

with the proviso that either a) R 20 and R 21 are independently selected from substituted or unsubstituted alkyl; or b) at least one of said R 20 and R 21 further comprises a member selected from a reactive functional group, solid support and carrier molecule;

R 22 and R 23 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, halogen, NO 2 , CN, P(O)(OR 4 )(OR 5 ), D 1 R 6 , NR 7 R 8 and C(D 2 )R 9

wherein

at least one of R 16 , R 17 , R 18 , R 19 , R 20 , R 22 and R 23 is P(O)(OR 30 )(OR 30 );

D 1 is a member selected from O and S;

D 2 is a member selected from O, S and NH;

R 4 and R 5 are members independently selected from H, substituted or unsubstituted C 1 -C 4 alkyl, substituted or unsubstituted C 1 -C 4 heteroalkyl

R 6 , R 7 and R 8 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and a reactive functional group;

wherein

R 7 and R 8 , together with the nitrogen to which they are attached, can be optionally joined to form a member selected from a reactive functional group, —NHNH 2 , —N═N═N, —N═C═S and —N═C═O;

R 9 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a reactive functional group, NR 10 R 11 and OR 12

wherein

R 10 and R 11 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R 12 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and C(O)R 13

wherein

R 13 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

5 . The fluorescent compound according to claim 4 , wherein R 17 is P(O)(OR 30 )(OR 30 ).

6 . The fluorescent compound according to claim 4 , wherein R 14 and R 15 are members independently selected from substituted or unsubstituted C 1 -C 4 alkyl.

7 . The fluorescent compound according to claim 1 , wherein R 1 or R 2 is a member selected from

wherein

G is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted aryl;

R 25 is a member selected from H, a negative charge, a salt counterion, reactive functional group, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

8 . The fluorescent compound according to claim 7 , wherein said fluorescent compound is a member selected from

9 . The fluorescent compound according to claim 6 , wherein R 20 is substituted or unsubstituted C 1 -C 4 alkyl.

10 . The fluorescent compound according to claim 9 , wherein said fluorescent compound is a member selected from

11 . The fluorescent compound according to claim 1 , wherein —(C(R 3 )═C(R 3 )) n —C(R 3 )═ is a member selected from:

wherein

Z 4 is a member selected from H, OR 30 , SR 30 , NR 30 R 30 , halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

12 . The fluorescent compound according to claim 11 , wherein said fluorescent compound is a member selected from

13 . The fluorescent compound according to claim 1 , wherein said fluorescent compound is a member selected from

14 . The fluorescent compound according to claim 1 , wherein said fluorescent compound is a member selected from

15 . The fluorescent compound according to claim 1 , wherein at least one of said R 1 and R 20 further comprises —NR 35 R 36

wherein

R 35 and R 36 are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, and R 35 and R 36 , together with the nitrogen to which they are bound, are optionally joined to form a ring system.

16 . The fluorescent compound according to claim 1 , wherein said carrier molecule further comprises a quencher moiety.

17 . The fluorescent compound according to claim 15 , wherein said fluorescent compound, together with said quencher moiety, comprise a donor-acceptor energy transfer pair.

18 . The fluorescent compound according to claim 17 , wherein said quencher moiety has substantially no native fluorescence.

19 . The fluorescent compound according to claim 17 , wherein said quencher moiety comprises at least three residues

wherein

each of said residues is independently selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, and

at least two of said residues are covalently linked via an exocyclic diazo bond.

20 . The fluorescent compound according to claim 2 , wherein said fluorescent compound further comprises a nucleic acid at a position which is a member selected from the 3′-terminus, the 5′-terminus, a nucleobase, and a phosphorus-containing internucleotide bridge of said nucleic acid.

21 . The fluorescent compound according to claim 19 , wherein said nucleic acid further comprises a probe which is a member selected from a molecular beacon, scorpion probe, sunrise probe, conformationally assisted probe and TaqMan™ probe.

22 . The fluorescent compound according to claim 2 , wherein said carrier molecule is a peptide comprising a cleavage recognition site for an enzyme.

23 . The fluorescent compound according to claim 21 , wherein said enzyme is a member selected from protease, trypsin, enterokinase, HIV-1 protease, prohormone convertase, interleukin-1b-converting enzyme, adenovirus endopeptidase, cytomegalovirus assemblin, leishmanolysin, β-secretase for amyloid precursor protein, thrombin, renin, angiotensin-converting enzyme, cathepsin-D and a kininogenase.

24 . The fluorescent compound according to claim 1 , wherein said fluorescent compound further comprises a member selected from a protein, saccharide, nucleotide monophosphate, nucleotide diphosphate and nucleotide triphosphate.

25 . The fluorescent compound according to claim 24 , wherein said protein is an antibody.

26 . The fluorescent compound according to claim 24 , wherein said carbohydrate is dextran.

27 . The fluorescent compound according to claim 2 , in which a member selected from R 1 and R 20 has the formula:

wherein

L 1a is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

R 2a and R 3a are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl,

wherein

R 2a and R 3a , together with the nitrogen to which they are attached, are optionally joined to form a ring which is a member selected from substituted or unsubstituted 5-7 membered cycloalkyl and substituted or unsubstituted 5-7 membered heterocycloalkyl.

28 . A method for determining whether a sample contains an enzyme, said method comprising:

(a) contacting said sample with a peptide construct comprising

i) a fluorescent compound according to claim 1 ;

ii) a quencher; and

iii) a cleavage recognition site for said enzyme,

wherein said peptide is in a conformation allowing donor-acceptor energy transfer between said fluorophore and said quencher when said fluorophore is excited;

(b) exciting said fluorescent compound; and

(c) determining a fluorescence property of said sample, wherein the presence of said enzyme in said sample results in a change in said fluorescence property.

29 . A method for determining whether a compound alters an activity of an enzyme, said method comprising:

(a) contacting a sample comprising said enzyme and said compound with a peptide construct comprising

i) a fluorescent compound according to claim 1 ;

ii) a quencher; and

iii) a cleavage recognition site for said enzyme,

wherein said peptide is in a conformation allowing donor-acceptor energy transfer between said fluorescent compound and said quencher when said fluorescent compound is excited;

(b) exciting said fluorescent compound; and

(c) determining a fluorescence property of said sample, wherein said activity of said enzyme in said sample results in a change in said fluorescence property.

30 . A method for detecting a nucleic acid target sequence, said method comprising:

(a) contacting said target sequence with a detector oligonucleotide comprising a target binding sequence, said detector oligonucleotide having linked thereto,

i) a fluorescent compound according to claim 1 ; and

ii) a quencher,

wherein said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorescent compound and said quencher when said fluorescent compound is excited;

(b) hybridizing said target binding sequence to said single-stranded target sequence, thereby altering said conformation of said detector oligonucleotide, causing a change in a fluorescence parameter; and

(c) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.

31 . The method according to claim 30 , wherein said complementary strand is synthesized in a target amplification reaction.

32 . The method according to claim 31 , wherein said complementary strand is synthesized by extension of the target sequence using said detector oligonucleotide as a template.

33 . The method according to claim 32 , wherein said fluorescence parameter is detected in-real time.

34 . A method for detecting amplification of a target sequence comprising, in an amplification reaction:

(a) hybridizing to said target sequence a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly associated secondary structure 5′ to said target binding sequence, wherein at least a portion of said detector sequence is a single stranded tail which is available for hybridization to said target sequence, said detector oligonucleotide having linked thereto,

i) a fluorescent compound according to claim 1 ; and

ii) a quencher,

wherein said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorescent compound and said quencher when said fluorescent compound is excited;

(b) extending said hybridized detector oligonucleotide on said target sequence with a polymerase to produce a detector oligonucleotide extension product and separating said detector oligonucleotide extension product from said target sequence;

(c) hybridizing a primer to said detector oligonucleotide extension product and extending the primer with said polymerase, thereby linearizing said intramolecularly associated secondary structure and producing a change in a fluorescence parameter; and

(d) detecting said change in said fluorescence parameter, thereby detecting said target sequence.

35 . The method according to claim 34 , wherein said target sequence is amplified by a method selected from Strand Displacement Amplification, Polymerase Chain reaction, Self Sustained Sequence Replication, Transcription Mediated Amplification, and Nucleic Acid Sequence Based Amplification.

36 . The method according to claim 34 , wherein said secondary structure further comprises a partially or entirely single-stranded restriction endonuclease site.

37 . The method according to claim 34 , wherein a change in fluorescence intensity is detected.

38 . The method according to claim 37 , wherein said change in fluorescence intensity is detected in real-time.

39 . The method according to claim 34 , wherein said intramolecularly associated secondary structure comprises a portion of said target binding sequence.

40 . A fluorescent compound having the formula:

in which ring systems A and B are members independently selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;

wherein

at least one of ring systems A and B comprise a substituent, and said substituent is PO 3 ;

R 1 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and COOR 20

wherein

R 20 is a member selected from substituted or unsubstituted alkyl

R 12 is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

Q is a member selected from substituted or unsubstituted unsaturated alkyl, substituted or unsubstituted unsaturated cycloalkyl, substituted or unsubstituted unsaturated heteroalkyl, and substituted or unsubstituted unsaturated heterocycloalkyl.