AROMATIC TRIAMIDE-LANTHANIDE COMPLEXES

• US 20090036537A1
• Filed: 12/29/2004
• Published: 02/05/2009
• Est. Priority Date: 12/30/2003
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Petoud, Stephane, Xu, Jide, Raymond, Kenneth N.
• Granted Patent: US 8,551,453 B2
• Time in Patent Office: Days
• US Class Current: 514/616
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07C 235/60 having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
• CPC Class Codes: C07C 271/20 to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
• CPC Class Codes: C07D 277/16 Sulfur atoms
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present invention provides luminescent lanthanide metal chelates comprising a metal ion of the lanthanide series and a complexing agent comprising at least one phthalamidyl moiety. Also provided are probes incorporating the phthalamidyl ligands of the invention and methods utilizing the ligands of the invention and probes comprising the ligands of the invention.

Claims

1. A compound having the structure:

wherein

R¹, R², R³, R⁴, R⁵ and R⁶ are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocycloalkyl, wherein a member selected from R¹ and R²;

R³ and R⁴; and

R⁵ and R⁶, together with the nitrogen atom to which they are attached, optionally form a ring system selected from heteroaryl and heterocycloalkyl;

Y¹, Y² and Y³ are members independently selected from 0 and (H)₂;

Q is a member selected from H, a protecting group and a cleaveable group; and

a is 0 or 1.

2. The compound according to claim 1, wherein a member selected from R¹, R³ and R⁵ has the structure:

wherein

L¹ is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl; and

X¹ is a member selected from protected or unprotected reactive functional groups and non-covalent protein binding groups.

3. The compound according to claim 2, wherein a member selected from R¹, R³ and R⁵ is a member selected from:

X¹ is a member selected from;

in which R²¹ is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl;

v is an integer from 1 to 20; and

w is an integer from 1 to 1,000.

4. The compound according to claim 2, wherein said non-covalent protein binding group is sulfonate.

5. The compound according to claim 1, wherein a member selected from R¹, R³ and R⁵ has the structure:

wherein

L¹ is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

X² is a linking member adjoining L¹ to Z¹; and

Z¹ is a member selected from carrier molecules and detectable labels.

6. The compound according to claim 5, wherein said carrier molecule is a targeting agent.

7. The compound according to claim 2, having the structure:

wherein

X¹ is a member selected from NH₂, SH, COR⁷, O(CH₂)_mZ⁶, NHNH₂ and O(CH₂)₂(OCH₂CH₂)_sO(CH₂)₂Z⁶

wherein

R⁷ is a member selected from H, OR⁸, OCOR⁸, NR⁸R⁹,

wherein

R⁸ and R⁹ are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;

Z⁶ is a member selected from OR¹⁰, OCOR¹⁰, NR¹⁰R¹¹

wherein

R¹⁰ and R¹¹ are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;

m is an integer from 1 to 20; and

s is an integer from 1 to 1000.

8. The compound according to claim 1, having the structure:

wherein

L² is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl;

L³, L⁴, L⁵ and L⁶ are members independently selected from a single bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

Z², Z³, and Z⁴ are members independently selected from H, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

9. The compound according to claim 8, wherein Z², Z³, and Z⁴ are members independently selected from substituted or unsubstituted pyridyl, substituted or unsubstituted salicylamidyl, substituted or unsubstituted phthalamidyl, substituted or unsubstituted terephthalamidyl, substituted or unsubstituted catechol and

wherein

R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocycloalkyl, wherein a member selected from R⁷ and R⁸;

and R⁹ and R¹⁰, together with the nitrogen atom to which they are attached, form a ring system selected from heteroaryl and heterocycloalkyl;

Y⁴, Y⁵ and Y⁶ are members independently selected from 0 and (H)₂; and

Q is a member selected from H, a protecting group or a cleaveable group.

10. The compound according to claim 8, wherein L² is a substituted or unsubstituted C₁-C₆ alkyl group.

11. The compound according to claim 1, wherein at least one of R¹, R³ and R⁵ has the structure:

wherein,

Z⁵ is a member selected from H, OR¹⁷, SR¹⁷, NHR¹⁷, OCOR¹⁸, OC(O)NHR¹⁸, NHC(O)OR¹⁷, OS(O)₂OR¹⁷, and C(O)R¹⁸;

R¹⁷ is a member selected from H, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl;

R¹⁸ is a member selected from H, OR¹⁹, NR¹⁹NH₂, SH, C(O)R¹⁹, NR¹⁹H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R¹⁹ is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted alkyl;

X is a member selected from O, S and NR²⁰

wherein

R²⁰ is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

j an k are members independently selected from the group consisting of integers from 1 to 20.

12. The compound according to claim 1, having the structure:

in which p is an integer from 0 to 2.

13. A polymer comprising a subunit having said structure according to claim 1.

14. The polymer according to claim 13, wherein said polymer is a biomolecule.

15. The polymer according to 1, having the structure:

wherein

L⁷ is a member selected from a single bond, substituted or unsubstituted alkyl and substituted or unsubstituted aryl; and

X³ is linking member joining L⁷ to A;

A is a carrier molecule.

16. The polymer according to claim 15 wherein A is a member selected from biopolymers, poly(amino acids), polyethers, polyimines, polysaccharides, dendrimers, cyclodextrins, pharmaceutical agents.

17. The polymer according to claim 16, wherein said biopolymer is a member selected from polypeptides, nucleic acids and saccharides.

18. The polymer according to claim 17, wherein said protein is a member selected from antibodies, enzymes, and serum proteins

19. A chelate of a metal ion comprising an organic ligand having said structure according to claim 1.

20. The chelate according to claim 19, wherein said metal ion is a lanthanide ion.

21. The chelate according to claim 20, wherein said chelate is luminescent.

22. The chelate according to claim 19, wherein said chelate is covalently attached to a carrier molecule.

23. A method for detecting enzyme in a sample, said method comprising:

(a) contacting said sample with a peptide construct comprising;

i) a peptide sequence, said sequence comprising a cleavage site for said enzyme;

ii) a complex according to claim 19 covalently bound to said peptide; and

iii) a quencher of light energy covalently bound to said peptide sequence, said quencher having an absorbance band overlapping an emission band of said complex,

wherein said peptide sequence conformation allows light energy transfer between said complex and said quencher when said complex is excited;

(b) exciting said complex;

(c) determining a fluorescence property of said sample; and

(d) comparing said fluorescence property from step (c) with a reference fluorescence property for said peptide construct, wherein said activity of said enzyme in said sample alters said light energy transfer, resulting in a change in said fluorescence property.

24. A method of determining the effect of a compound on enzyme activity, said method comprising:

(a) contacting a sample comprising said enzyme with a peptide construct comprising;

iii) a peptide sequence, said sequence comprising a cleavage site for said enzyme;

iv) a complex according to claim 19 covalently bound to said peptide sequence; and

iii) a quencher of light energy covalently bound to said peptide sequence, said quencher having an absorbance band overlapping an emission band of said complex,

wherein said peptide sequence conformation allows light energy transfer between said complex and said quencher when said complex is excited;

(b) exciting said complex;

(c) determining a fluorescence property of said sample; and

(d) comparing said fluorescence property from step (c) with a reference fluorescence property for said peptide construct, wherein said activity of said enzyme in said sample alters said light energy transfer, resulting in a change in said fluorescence property.

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

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

i) a complex according to claim 19;

ii) a quencher of light energy having an absorbance band overlapping an emission band of said complex,

wherein said detector nucleic acid conformation allows fluorescence energy transfer between said complex and said quencher when said complex is excited;

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

(c) determining a fluorescence property of said sample; and

(d) comparing said fluorescence property from step (c) with a reference fluorescence property for said peptide construct, wherein said activity of said enzyme in said sample alters said light energy transfer, resulting in a change in said fluorescence property.

26. The method according to claim 25, wherein said detector oligonucleotide has a format selected from molecular beacons, scorpion probes, sunrise probes, light up probes and TaqMan™

probes.

27. The method according to claim 23, 24 or 25, wherein said fluorescence property is detected in-real time.

28. The method according to claim 23, 24 or 25, wherein said change and said fluorescence property measured is a change in fluorescence intensity.

29. A microarray comprising a complex according to claim 19, wherein said complex is conjugated to a solid support or to a carrier molecule attached to said solid support.

30. The microarray according to claim 29, wherein said carrier molecule is a member selected from a nucleic acid, a peptide, a peptide nucleic acid, a pharmaceutical agent and combinations thereof.

31. The microarray according to claim 29, wherein said solid support is divided into a first region and a second region, said first region having attached thereto a first complex, and said second region having attached thereto a second.

32. A method of providing radiation therapy to a subject requiring such therapy, said method comprising:

administering to said subject a complex according to claim 19, said complex having radiosensitization properties; and

administering ionizing radiation to said subject, thereby providing radiation therapy to said subject.

33. A method for photodynamic therapy of a lesion or of a lesion beneath melanodermic tissue of a subject, said method comprising:

(a) administering a complex according to claim 19 to said subject; and

(b) photoirradiating said lesion.

34. The method according to claim 33, wherein said photoirradiating is with light having a wavelength range of about 610 to about 1150 nanometers.

35. The method of claim 34 wherein the photoirradiating is with light having a wavelength range of about 730 to about 770 nanometers.

36. The complex according to claim 19, wherein said complex comprises a component of an ink or a dye.

37. The complex according to claim 19, wherein said complex comprises a component of a substrate for the transmission and amplification of light.

38. The complex according to claim 37, wherein said substrate comprises a member selected from glass, organic polymers, inorganic polymers and combinations thereof.

39. A method for amplifying light transmitted by a substrate, said method comprising transmitting light through a substrate according to claim 37, thereby amplifying said light.