CA2488819A1 - Bis-transition-metal-chelate-probes - Google Patents

Abstract

A probe for labeling a target material is provided including two transition- metal chelates and detectable group. The probe has the general structural formula (I) wherein: (a) Y and Y' are each a transition metal, (b) R1~ and R 1~ are each independently CH(COO-), CH(COOH), or absent; (c)R2~ and R2 ~are linkers each having a length of from about 3.0 to about 20 .ANG.; and (d) X is a detectable group. The linkers may be linear or branched, may contain aromatic moeties, and may optionally be further substituted. Methods of use of the probe in detecting and analyzing target materials of interest also are provided.

Description

BIS-TRANSITION-METAL-CHELATE PROBES
This invention was made with Government support under Grant No. NIH RO1-GM41376, awarded by the National Institutes of Health. Therefore, the Government has certain rights in this invention.
FIELD OF THE INVENTION
This invention relates to compositions and methods for labeling molecules.
More particularly, the present invention relates to certain transition metal chelate probes capable of selectively associating with histidine- containing target sequences on compounds of interest and yielding a detectable signal.
BACKGROUND OF THE INVENTION
Characterization of proteins often requires the ability to incorporate detectable groups--e.g., fluorochromes, chromophores, spin labels, radioisotopes, paramagnetic atoms, heavy atoms, haptens, crosslinking agents, and cleavage agents--at specific, defined sites.
For proteins that do not contain pre-existing cysteine residues, site-specific labeling can be accomplished by use of site-directed mutagenesis to introduce a cysteine residue at the site of interest, followed by cysteine-specific chemical modification to incorporate the labeled probe. However, for proteins that contain pre-existing cysteine residues, site-specific labeling is difficult. Multiple strategies have been reported: (i) intein-mediated labeling ("expressed protein ligation"), (Muir, et al., Proc. Nat'l. Acad. .Sci. USA, 95:6705-6710 (1998)); (ii) transglutaminase-mediated labeling (Sato et al., Biochem.
35:13072-13080 (1996)); (iii) oxidation-mediated labeling (Geoghegan, et al., Bioconj. Chem., 3:138-146 (1992)); and (iv) trivalent-arsenic-mediated labeling (Griffin et al., Science 281:269-272, 1998) (U.S. Patent No. 6,008,378). Strategies (i)-(iii) do not permit in situ labeling (i.e., direct labeling of proteins in cuvettes, gels, blots, or biological samples--without the need for a subsequent purification step) or in vivo labeling (i.e., direct labeling of proteins in cells).
Strategy (iv) requires a structural scaffold presenting two trivalent-arsenic atoms in a precisely defined spatial relationship and therefore relates only to a limited number of detectable groups (such as those having a detectable xanthene, xanthanone, or phenoxazinestructural nucleus).
Transition-metal chelates consisting of a transition-metal ion, such as Ni2+, Co2+, Cu2+, or ZnZ+, in complex with a tridentate or tetradentate chelating ligand, such as iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA), exhibit high affinity for oligohistidine sequences, particularly hexahistidine sequences (Sulkowski, E., Trends Biotechnol., 3:1-7 (1985); Hochuli, et al., J. Chromat. 411:177-184 (1987); Hochuli, E. et al.
BioTechnol.
6:1321-1325 (1988). Figure 1 shows a proposed model for binding of neighboring hexahistidine residue to a Ni-NTA resin as disclosed in Crowe, J. et al., Methods Mol. Bdol., 31:371-387 (1994)).
The high affinity of interactions between transition-metal chelates and oligohistidine sequences, particularly hexahistidine sequences, has been verified using force microscopy experiments, which permit direct measurement of interaction forces on the single-molecule level and direct observation of molecular recognition of a single receptor-ligand pair (Kienberger, F. et al. Single Mol. 1:59-65 (2000); Schmitt, L. et al. Biophys.
J. 78: 3275-3285 (2000)).
The high affinity of interactions between transition-metal chelates and oligohistidine sequences, particularly hexahistidine sequences, has been used advantageously to purify biomolecules containing, or modified to contain, "oligohistidine tags,"
particularly "hexahistidine tags" (Hochuli, E. et al. BioTechnol. 6:1321-1325 (1988);
Crowe, J. et al., Methods Mol. Biol., 31:371-387 (1994)). In this application, termed "immobilized-metal-chelate affinity chromatography," a transition-metal chelate consisting of a transition-metal ion, such as Niz+, Co2+, Cu2+, or Zn2+, in complex with a tridentate or tetradentate chelating ligand, such as iminodiacetic acid (1DA) or nitrilotriacetic acid (NTA), is immobilized on a solid phase, such as chromatographic resin, and the resulting immobilized metal chelate is used to bind, and thereby purify from other components, tagged biomolecules.
The high affinity of interactions between transition-metal chelates and oligohistidine tags, particularly hexahistidine tags, also has been used advantageously in biosensor analysis of biomolecules containing, or modified to contain, oligohistidine tags, particularly hexahistidine tags (Gershon, et al. J. Immunol. Meths. 183:65-76 (1995);
Nieba, L. et al.
Anal. Biochem. 252:217-228 (1997)). Kienberger et al., Single Mol. 1; S9-65 (2000). In this application, a transition-metal chelate consisting of a transition-metal ion, such as Ni2+, Co2+
Cu2+, or Zn2+, in complex with a tridentate or tetradentate chelating ligand, such as iminodiacetic acid (mA) or nitrilotriacetic acid (NTA), is immobilized on a biosensor chip, such a surface-plasmon-resonance biosensor chip, and the resulting immobilized metal chelate is used to detect, quantify, and analyze tagged biomolecules.
It would be advantageous to be able to use the high affinity of interactions between transition-metal chelates and oligohistidine tags, particularly hexahistidine tags, in labeling and in situ detection of tagged biomolecules.
There is a need for improved methods and compositions for protein labeling. In particular, there is a need for methods and compositions that permit in situ labeling, that permit in vivo labeling, and that encompass a wide range of detectable groups with different properties.
SUMMARY OF THE INVENTION
The invention provides a molecule with two pendant metal-chelate moieties according to the general structural Formula (I), including tautomers, salts, and acids thereof:
R2/ X~ R2.
IRS, N N
~Y \O / .Y,. O
O O O O O O
(I) wherein: (a) Y and Y' are each a transition metal, (b) R' and R'' are each independently C(COO-), CH(COOH), or absent; (c) R2 and R2~ are linkers each having a length of from about 3.0 to about 20 A; and (d) X is a detectable group. The linkers may be linear or branched, may contain aromatic moieties, and optionally may be further substituted.
Additionally provided herein are methods of synthesis of compounds of the present invention involving coupling of:
(a) a synthon which includes a bis-activated-ester derivative of a detectable group; and (b) a synthon which includes an amine or hydrazide derivative of a chelator;
and then adding a transition metal.
Additionally provided herein are methods of synthesis of compounds of the present invention containing a non-sulfonated cyanine or squaraine detectable group, involving coupling of: (a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethylindole, mono-chelator-functionalized 2,3,3-trimethylbenzindole, mono-chelator-functionalized 2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole, mono-chelator-functionalized 2-methyl-napthothiazole, mono-chelator-functionalized 2-methyl-benzoxazole, and mono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthon, identical or nonidentical to the synthon in (a), selected from the group in (a); and (c) a synthon containing at least one carbon atom;
and then adding a transition metal.
Additionally provided herein are methods of synthesis of compounds of the present invention containing a disulfonated cyanine or squaraine detectable group, involving coupling of:
(a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized 2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized 2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized 2-methyl-5-sulfanato-benzothiazole, mono-chelator-functionalized 2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized 2-methyl-5-sulfanato-benzoxazole, and mono-chelator-functionalized 2-methyl-6-sulfanato-napthoxazole;

(b) a synthon, identical or nonidentical to the synthon in (a), selected from the group in (a);
and (c) a synthon containing at least one carbon atom; and then adding a transition metal.
Additionally provided herein are methods of synthesis of compounds of the present invention containing a monosulfonated cyanine or squaraine detectable group, involving coupling of: (a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethylindole, mono-chelator-functionalized 2,3,3-trimethylbenzindole, mono-chelator-functionalized 2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole, mono-chelator-functionalized 2-methyl-napthothiazole, mono-chelator-functionalized 2-methyl-benzoxazole, and mono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthon selected from mono-chelator-functionalized 2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized 2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized 2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized 2-methyl-6-sulfanato-benzothiazole, mono-chelator-functionalized 2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized 2-methyl-5-sulfanato-benzoxazole, and mono-chelator functionalized 2-methyl-6-sulfanato-napthoxazole; and (c) a synthon containing at least one carbon atom; and then adding a transition metal.
Additionally provided herein are methods of synthesis of compounds of the present invention containing a xanthene, xanthanone, or phenoxazine detectable group, involving reaction of a xanthene, xanthanone, or phenoxazine detectable group, a secondary-amine derivative of a chelator, and formaldehyde, according to the Mannich reaction (Mannich, C.
et al. Arch. Pharm. 250:647, 1912); followed by addition of a transition metal.
Additionally provided herein is a labeled target material including a target sequence of the form: (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6, and wherein the target sequence is bonded with a molecule according to Formula (I).
Also included is a detectable complex including a molecule according to Formula (I) and a target sequence, bonded thereto. The target sequence includes an amino acid sequence of the form: (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6.
The invention also includes a method for imparting fluorescent properties to a target material, including the step of reacting: (a) the target material having a target sequence of the form (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6, with (b) a molecule according to Formula (I), under conditions sufficient to permit metal-chelate moieties of said molecule according to Formula (I) to bond to the target sequence.
Furthermore, provided herein is a method for detecting a target material of interest, including the steps of: (a) providing a target material of interest having a target sequence of the form: (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6; (b) incubating the polypeptide with a molecule according to Formula (I), having a detectable group, for a time period sufficient to allow labeling of the target material; and (c) detecting the detectable group, thereby detecting the target material of interest.
Additionally, a method for imaging the localization, concentration or interactions of a target material of interest on or within cells, tissues, organs or organisms is provided, including the steps of: (a) providing a target material of interest having a target sequence of the form: (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6; (b) incubating the target material with a molecule according to Formula (I) for a time period sufficient to allow labeling of the polypeptide; and (c) detecting the detectable group of said molecule according to Formula (I), thereby imaging the localization, concentration or interactions of the target material of interest.
Furthermore, provided herein is an assay method for monitoring a binding process including the steps of: (a) reacting a first component of a specific binding pair with a second component of the pair, with the first component being labeled with a molecule according to Formula (I) having a detectable group; and (b) monitoring the reaction by monitoring a change in a signal of the detectable group.

Also provided herein is an assay method for monitoring a binding process including the steps of: (a) reacting a first component of a specific binding pair with a second component of the pair, with the first component being labeled with a molecule according to Formula (I) having a detectable group; and (b) monitoring the reaction by monitoring fluorescence emission intensity, fluorescence lifetime, fluorescence polarization, fluorescence anisotropy, or fluorescence correlation of the detectable group.
Additionally provided herein is an assay method for monitoring a binding process, including the steps of: (a) reacting a first component of a specific binding pair with a second component of the pair, with the first component being labeled with a molecule according to Formula (I) wherein X of Formula (I) is a fluorochrome, and with the second component containing Y, wherein Y is selected from the group including a fluorochrome and chromophore, Y being capable of participating in fluorescence energy transfer, fluorescence quenching, or exciton formation with X; and (b) monitoring the reaction by monitoring fluorescence of X.
The invention also provides an assay method for monitoring a binding process, including the steps of: (a) reacting a first component of a specific binding pair with a second component of the pair, with the first component being labeled with a molecule according to Formula (I) wherein X of Formula (I) is selected from the group consisting of a fluorochrome and a chromophore, and with the second component containing Y, wherein Y is a fluorochrome able to participate in fluorescence energy transfer, fluorescence quenching, or exciton formation with X; and (b) monitoring the reaction by monitoring fluorescence of Y.
The invention further provides an assay method for monitoring a reaction, including the steps of: (a) reacting a first participant in a reaction with a second participant in the reaction, the first participant being labeled with a molecule according to Formula (I); and (b) monitoring the reaction by monitoring a change in a detectable property of the detectable group.
Furthermore, provided herein is a method for isolating a target material of interest including the steps of: (a) contacting molecules according to Formula (I) immobilized on a solid support, with a solution containing a polypeptide of interest, the polypeptide including a target sequence of the form: (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6, under conditions that allow binding of the target material to immobilized molecules of Formula (I); and (b) eluting the target material of interest with a low-molecular weight monothiol or low-molecular-weight dithiol.
The invention also includes a method for immobilizing a target material of interest including the steps of: (a) contacting molecules according to Formula (I) immobilized on a solid support, with a solution containing a target material, the target material containing a target sequence of the form (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6, under conditions that allow binding of the target material to immobilized molecules according to Formula (I).
Additionally provided herein is a kit including: (a) a molecule according to Formula (I); and (b) a molecule containing a target sequence including an amino acid sequence of the form: (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6.
Further provided herein is a kit including: (a) a molecule according to Formula (I);
and (b) a reagent that promotes the formation of a complex between the molecule according to Formula (I) and a peptide having a target sequence of the form: (H);, wherein H is histidine, and i is 4 to 12, preferably 4 to 8, and most preferably 6.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a prior-art model for the binding of neighboring hexahistidine residues to a NTA:Ni2+ resin.
FIGS. 2 and 3 show results of fluorescence anisotropy experiments verifying specific interactions between bis-transition-metal-chelate probes according to the invention with a hexahistidine-tagged protein.
FIG. 4 is a model structure of a DNAF-CAP-His6 complex showing the position of the fluorescein of DNAF (circle), the position of the hexahistidine tag of each CAP-His6 promotor (diamond), the distance between fluorescein and the hexahistidine tag of the proximal CAP-His6 promotor (~55 A), and the distance between fluorescein and the hexahistidine tag of the distal CAP-Hiss promotor (~80 A).
FIGS. 5 and 6 show results of FRET experiments verifying high-affinity, specificinteractions of bis-transition-metal-chelate probes according to the present invention with a hexahistidinetagged protein.
FIGS. 7 and 8 show results of FRET experiments verifying stoichiometric interactions of nickel containing probes according to the present invention with the hexahistidine tag.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The inventors have found, as set forth herein, that a molecule having two transition-metal chelates and a detectable group binds with high affinity and high specificity to oligohistidine target sequences, particularly hexahistidine target sequences.
Furthermore, the inventors have found that a molecule having two transition-metal chelates and a detectable group binds with much higher affinity (more than 10 times higher affinity) and much higher specificity (more than 10 times higher specificity) to oligohistidine target sequences, particularly hexahistidine target sequences, than does a molecule having only a single transition-metal chelate and a detectable group.
Furthermore, the inventors have found that a molecule having two transition-metal chelates and a detectable group can be used to label, detect, and analyze target materials containing, or derivatized to contain, oligohistidine target sequences, particularly hexahistidine target sequences.
Furthermore, the inventors have found that a molecule having two transition-metal chelates and a detectable group can be used in in situ labeling, detection, and analysis of target materials containing, or derivatized to contain, oligohistidine target sequences, particularly hexahistidine target sequences (i.e., direct labeling, detection, and analysis of said target materials--without the need for a subsequent purification step).
Compositions of the Invention The present invention provides a probe for detecting a target material of interest. The probe includes two transition-metal chelates and a detectable group, according to the following general structural Formula (I), and tautomers, salts, and acids thereof:
R2/ X~ R2, Rt R1, N N
O OYO \O O OY~O O
(I) wherein: (a) Y and Y' are each a transition metal, (b) R' and R'~ are each independently CH(COO-), CH(COOH), or absent, (c) RZ and R2~ are linkers each having a length of about 3.0 to 20 ~, and preferably about 3.0 to 15 ~, and (d) X is a detectable group.
The linkers may be linear or branched, may contain aromatic moieties, and may optionally be further substituted.

"Y" in Formula (I) is a transition metal. Y can be any transition metal capable of specific interaction with a oligohistidine tag. Transition metals are those metals having incompletely filled d-orbitals and variable oxidation states. Examples of suitable transition metals include:
nickel, cobalt, copper, and zinc. In a preferred embodiment, Y is a divalent transition-metal ion. In a particularly preferred embodiment, Y is selected from the group consisting of Ni2+, Co2+, Cu2+, and Zn2+.
When R' in Formula (I) is absent, the chelator is iminodiacetic acid (IDA).
When R' is CH(COO-) or CH(COOH), the chelator is nitrilotriacteic acid (NTA).
Similarly, when R'~ in Formula (I) is absent, the chelator is iminodiacetic acid (IDA).
When R'' is CH(COO-) or CH(COOH), the chelator is nitrilotriacetic acid (NTA).
R2 and RZ' in Formula (I) are linkers. The structures of R2 and R2' should permit the two pendant transition-metal chelates to be separated by a distance comparable to the dimensions of a oligohistidine target sequence, particularly a hexahistidine target sequence.
Thus, the structures of RZ and RZ' should permit the two pendant transition-metal chelates to be separated by about 2.5 to 25 A, and preferably by about 5 to 20 A
(distances measured metal-to-metal). RZ and R2' may be linear or branched, may optionally contain cyclic groups, ZO and may optionally be further substituted. RZ and R2' may be the same or different.
Preferably, R2 and R2' are the same. RZ and R2' may be connected to different atoms of X
(preferably two atoms on the same edge or face of X). Alternatively, RZ and RZ' may be connected to the same atom of X. Alternatively, RZ and RZ' may be connected to a single atom, which in turn is connected, directly or through a linker of maximal length 4 t~, to X.
X in Formula (I) is a detectable group. "Detectable group" as used herein refers to any chemical moiety that can be detected. Examples of detectable groups include fluorescent moieties, phosphorescent moieties, luminescent moieties, absorbent moieties, photosensitizers, spin labels, radioisotopes, isotopes detectable by nuclear magnetic resonance, paramagnetic atoms, heavy atoms, haptens, crosslinking agents, cleavage agents, and combinations thereof.

In one embodiment, X is detected by monitoring a signal. Some signals which may be monitored due to the presence of a detectable group include, for example, fluorescence (fluorescence emission intensity, fluorescence lifetime, fluorescence polarization, fluorescence anisotropy, or fluorescence correlation), luminescence, phosphorescence, absorbance, singlet-oxygen production, electron spin resonance, radioactivity, nuclear magnetic resonance, and X-ray scattering.
In another embodiment, X is detected by receptor-binding, protein-protein or protein-nucleic acid crosslinking, or protein or nucleic acid cleavage.
Preferred detectable groups include fluorescent moieties. In one preferred embodiment, cyanine fluorescent moieties are used. These include, but are not limited to:
Cy3: 1-R-2-[3-[1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propeny 1]-3,3-dimethyl-5-sulfo-3H-indolium, CyS: 1-R-2-[5-[1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-penta dienyl]-3,3-dimethyl-5-sulfo-3H-indolium, Cy7: 1-R-2-[7-[1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrienyl]-3,3-dimethyl-5-sulfo-3H-indolium, indocyanine green and IRDye (1-R-2-[2-[2-R'-3-[(1-R-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene) ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-5-sulfo-3H-indolium), and mono- and non-sulfonated derivatives thereof. In another ?0 preferred embodiment, squaraine fluorescent moieties are used. In another preferred embodiment, xanthene, xanthanone, and phenoxazine fluorescent moieties are used.
Examples of cyanine, squaraine, xanthene, xanthanone, and phenoxazine detectable ?5 groups fluorescent moieties are described, inter alia, in Southwick et al., 1990, Cytometry 11:418-430; Mujumdar et al., 1993, Bioconjugate Chemistry 4:105-111; Waggoner and Ernst, Fluorescent Regents for Flow Cytometry, Part 1: Principles of Clinical Flow Cytometry (1993) and Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Inc. 6'h edition (1996) and Berling and Reiser, Methoden der 30 Organischer Chemie, p 231-299 (1972), Oswald et al., Analytical Biochemistry 280: 272-277 (2000), Oswald et al. Photochemistry and Photobiology 74(2): 237-245 (2001), Oswald et al.

Bioconjugate Chemistry 10: 925-931 (1999), U.S. Patent No: 6,086,737. The structures in these publications are all incorporated herein by reference.
In a preferred embodiment, X may be selected from the following cyanine detectable groups:

R ~ ~ R3, U V
N \ \ ~N+
n~
(II) R3, / U\ \ V
n~
(III) R3, - \

(IV) R3 R3.
U
N \ ~ ~N+
n~
(V) R3, ~I
N \ ~ ~N+
(VI) wherein U and V are each independently C(R4)Z, NH, O, S, or (CH)Z; R3 and R3' are each independently H or sulfonate; R4 is H, CH3, CHZCH3, or (CH2)ZCH3; and n is 0 or an integer of from 1 to 6.
In another preferred embodiment, X may be selected from the following squaraine detectable groups:

R3 ~ I ~ R3, U O V
N \ \ ~N+
Jn~~ Jn l (VII) R3, I
U O V
N \ \ ~N+
n. n' (VIII) R3, Rs I
\
N N+
(IX) R3 Rs R3, \
N+
..
(X) R3, 3 ' R
N N+
.. U ..
(XI) R3 R3, Rs I
U O V
N \ \ ~N+
n, O n.
(XII) Rs R3' U O V
I~
....
(XIII) R3' R3 Rs N N+
(XIV) N N
N N

R3 R3, Rs I
N N+
(XV) wherein U and V are each independently C(R4)Z, NH, O, S, or (CH)2; R3 and R3~
are each independently H or sulfonate; R4 is H, CH3, CHZCH3, or (CHZ)2CH3; RS is absent or is selected from the group consisting of H, an alkyl group, and an aryl group;
and n' is 0 or an integer of from 1 to 3.
In another preferred embodiment, X may be selected from the following xanthene, xanthanone, and phenoxazine detectable groups:
R6, OH
(XVI) N N

R6, OH
(XVII) O
R6, H OH
(XVIII) R6, OH
(XIX) 6 6' R / ~N / R
O ~ O ~ OH
(XX) R~ R6, OH
(XXI) wherein R6, RG', R6", R6"', R6~~", and R6"'~~ are each independently hydrogen, halogen, hydroxyl, or alkoxyl; and R7, when present, is hydrogen, carboxyl, carboxylate or sulfonate.
One preferred molecule of the present invention includes two pendant transition-metal chelates and a cyanine detectable group according to the following general structural formula:

R3, U V
N \ \ ~N+
R2. n IR2, R1 R1, I
N N
O OYO O O Oy0 p (XXII) wherein Y, Y', R', R'~, R2, and RZ' are as defined previously; wherein U and V
are each independently C(R4)2, NH, O, S, or (CH)2; R3 and R3' are each independently H
or sulfonate;
R4 is H, CH3, CHZCH3, or (CH2)ZCH3; and n is 0 or an integer of from 1 to 6.
Particularly preferred embodiments include the aforementioned structure where n is 1, 2 or 3. In an even more preferred embodiment, n is 1, 2, or 3; and R2 and RZ' are identical n 6" wT D 6", and are about 3.0 to 15 A in length. In an especially preferred embodiment, n is l, 2, or 3; R2 and R2' are identical and about 3.0 to 15 ~ in length; and Y and Y' are each Niz+.
One preferred molecule of the present invention includes two pendant transition-metal chelates and a cyanine detectable group according to the following general structural formula:

R3, U V
N+
O
NH HN
O O
1~
O~ N ~i N ~ i O- \Y~ ~y~ -O.
O O
(XXIII) wherein Y and Y' are as defined previously; U and V are each independently C(R4)Z, NH, O, S, or (CH)Z; R3 and R3' are each independently H or sulfonate; R4 is H, CH3, CHZCH3, or (CHZ)zCH3; and n is 0 or an integer of from 1 to 6. In a particularly preferred embodiment, n is 1, 2, or 3; and Y and Y' are each Ni2+.
Furthermore, provided herein is a molecule with two pendant transition-metal chelates and a detectable group according to the following general structural formula:

O O
NH HN
O O
~O O
O
N N i O- \Y~ ~ ~ ~y~ ~O_ O
O O
(XXIV) wherein Y and Y' are as defined previously; R3 and R3~ are each independently H or sulfonate;
and n is 1, 2, 3, or 4. In a particularly preferred embodiment, n is 1, 2, or 3; and Y and Y' are each Ni2+
There are no particular limitations to the detectable groups of the compounds of the present invention, so long as the ability of the bis-transition-metal-chelate moieties to bind to a target sequence is maintained. The points) of attachment between the bis-transition-metal-chelate moieties and the detectable group may vary.
Modifying groups that aid in the use of the bis-transition-metal-chelate derivative may also be incorporated. For example, the bis-transition-metal-chelate derivative may be substituted at one or more positions to add a solid-phase binding group or a crosslinking group.
For applications involving labeling of target materials within cells, the bis-transition-metal-chelate derivative preferably is capable of traversing a biological membrane. Smaller molecules are generally able to traverse a biological membrane better than larger derivatives.

Bis-transition-metal-chelate derivatives of less than 2000 Daltons are preferable for membrane traversal.
The polarity of the bis-transition-metal-chelate derivative can also determine the ability of the bis-transition-metal-chelate derivative to traverse a biological membrane.
Generally, a hydrophobic bis-transition-metal-chelate derivative is more likely to traverse a biological membrane. The presence of polar groups can reduce the likelihood of a molecule to traverse a biological membrane. A bis-transition-metal-chelate derivative that is unable to traverse a biological membrane may be further derivatized by addition of groups that enable or enhance the ability of the molecule to traverse a biological membrane.
Preferably, such derivatization does not significantly alter the ability of the bis-transition-metal-chelate derivative to react subsequently with a target sequence. The bis-transition-metal-chelate derivative may also be derivatized transiently. In such instances, after traversing the membrane, the derivatizing group is eliminated to regenerate the original bis-transition-metal-chelate derivative. Examples of derivatization methods that increase membrane traversability include ether formation with acyloxyalkyl groups. For example, an acetoxymethyl ether is readily cleaved by endogenous mammalian intracellular esterases.
Jansen, A. and Russell, T.J., J. Chem. Soc., 2127-2132 (1965). Also, pivaloyl ester is useful in this regard. Madhu et al., J. Occul. Pharmaco. Ther., 14:389-399 (1998).
Methods of Synthesis of Compositions of the Invention The invention provides methods of synthesis of compounds of the present invention which include coupling of: (a) a synthon which includes a bis-activated-ester derivative of a detectable group; and (b) a synthon which includes an amine or hydrazide derivative of a chelator; and then adding a transition metal.
The invention also provides methods of synthesis of non-sulfonated cyanine or squaraine compounds of the present invention which include coupling of: (a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethylindole, mono-chelator-functionalized 2,3,3-trimethylbenzindole, mono-chelator-functionalized 2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole, mono-chelator-functionalized 2-methyl-napthothiazole, mono-chelator-functionalized 2-methyl-benzoxazole, and mono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthon, identical or nonidentical to the synthon in (a), selected from the group in (a); and (c) a synthon containing at least one carbon atom; and then adding a transition metal.
The invention also provides methods of synthesis of disulfonated cyanine or squaraine compounds of the present invention which include coupling of: (a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized 2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized 2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized 2-methyl-5-sulfanato-benzothiazole, mono-chelator-functionalized 2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized 2-methyl-5-sulfanato-benzoxazole, and mono-chelator-functionalized 2-methyl-6-sulfanato-napthoxazole; (b) a synthon, identical or nonidentical to the synthon in (a), selected from the group in (a); and (c) a synthon containing at least one carbon atom; and then adding a transition metal.
The invention also provides methods of synthesis of monosulfonated cyanine or squaraine compounds of the present invention which include coupling of: (a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethylindole, mono-chelator-functionalized 2,3,3-trimethylbenzindole, mono-chelator-functionalized 2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole, mono-chelator-functionalized 2-methyl-napthothiazole, mono-chelator-functionalized 2-methyl-benzoxazole, and mono-chelator-functionalized 2-methyl-napthoxazole; (b) a synthon selected from mono-chelator-functionalized 2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized 2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized 2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized 2-methyl-6-sulfanato-benzothiazole, mono-chelator-functionalized 2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized 2-methyl-5-sulfanato-benzoxazole, and mono-chelator-functionalized 2-methyl-6-sulfanato-napthoxazole; and (c) a synthon containing at least one carbon atom; and then adding a transition metal.
Coupling of the synthons referred to herein can be accomplished in a single step, or in two steps. For example, for symmetric compounds (i.e., where (a) and (b) are identical), coupling of the reactants (a), (b), and (c) desirably is carned out in a single step. For asymmetric compounds (i.e., where (a) and (b) are non-identical), coupling of the reactants (a), (b), and (c) desirably is carned out in two steps: i.e., reaction of (a) with (c), followed by reaction of the resultant product with (c); or, alternatively, reaction of (b) with (c), followed by reaction of the resultant product with (a).
Coupling of the synthons referred to herein can be performed in solution, or with one or more synthons attached to a solid support.
Coupling of the synthons referred to herein can be performed with the chelator in an unprotected form, or with the chelator in a protected form initially and deprotected thereafter.
The invention also provides methods of synthesis of xanthene, xanthanone, or phenoxazine compounds of the present invention which include reaction of a xanthene, xanthanone, or phenoxazine detectable group, a secondary-amine derivative of a chelator, and formaldehyde, according to the Mannich reaction (Mannich, C. et al. Arch.
Pharm. 250:647, 1912); followed by addition of a transition metal.
The Mannich reaction referred to herein can be performed with the chelator in an unprotected form, or with the chelator in a protected form initially and deprotected thereafter.
Target Materials and Target Seguences of the Invention The invention provides detectable complexes of molecules according to Formula (I) with target sequences. Detectable complexes as used herein refer to the association between target amino acid sequences and bis-transition-metal-chelate derivatives according to the invention.
Suitable target materials include, but are not limited to, polypeptides, and polypeptide mimetics (such as peptide nucleic acid). Preferably, the target material is a polypeptide.
As used herein, "polypeptide" refers to both short chains, commonly referred to as "peptides, "oligopeptides," or "oligomers," and to longer chains, generally referred to as "proteins." Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides may include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well-known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in research literature. Thus "polypeptide" includes peptides, oligopeptides, polypeptides and proteins, all of which terms are used interchangeably herein.
The target material contains, or is modified to contain, at least one copy of an oligohistidine target sequence, herein referred to interchangeably as the "target sequence" or "tag." The target sequence is generally of the form: (H);, wherein H is histidine and i is an integer of from 4 to 12 (i.e., SEQ B7 NOS. 1-9), preferably 4 to 8, and most preferably 6.
The target sequence may be incorporated at any desired site, or set of sites, within a target material, but preferably is incorporated at a site that is (a) accessible and (b) not essential for structure and function of the target material.
For example, when the target material is a protein, the target sequence preferably is incorporated at the N-terminal region, at the C-terminal region, at an internal loop region, at a surface-exposed non-essential loop, at an internal linker region, or at combinations thereof.
The specific site, or set of sites, can be chosen to accommodate the functional requirements of a protein. For example, it is known that N-terminal modification of chemokines can affect their activity; therefore, in applications with chemokines, either C-terminal modification or internal modification would be preferable. Since labeling is performed at defined, user-selected sites, effects on the activity of target material can be avoided.
When it is important to preserve the activity of the tagged target material, specific activity testing of the tagged vs.
the untagged tareget material may be conducted to verify activity. See, for example, Mas et al,. Science, 233:788-790 (1986).
Target-sequence-containing polypeptides may be generated by total synthesis, partial synthesis, in vitro translation, or in vivo bacterial, archaeal, or eukaryotic production.

In one preferred embodiment, the target sequences and/or target-sequence-containing polypeptides used in the invention are prepared using solid-phase synthesis (see, e.g., Merrifield et al. J. Am. Chem. Soc., 85:2149, (1962) Steward and Young, Solid Phase Pe tides Synthesis, Freeman, San Francisco, (1969), and Chan and White, Fmoc Solid Phase Peptide Synthesis - A Practical Approach, Oxford Press (2000)).
In another preferred embodiment, the target sequences and/or target-sequence-containing polypeptides used in the invention are prepared using native chemical ligation (Dawson et al., Science, 266, 1994).
In an especially preferred embodiment, the target sequences and/or target-sequence-containing polypeptides are generated by in vivo bacterial, archaeal, or eukaryotic expression of a recombinant nucleic acid sequence encoding the target-sequence-containing polypeptide.
Methods for the construction of recombinant nucleic acid sequences encoding a tag-containing polypeptide are well known in the art (Sambrook and Russel, Molecular Cloning A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, New York (2001), the entirety of which is herein incorporated by reference. In addition, techniques for transient or stable introduction of recombinant nucleic acid sequences into cells (see, for example, Ausubel et al., Current Protocols In Molecular Biolo~y, John Wiley & Sons, Inc. (1995)), for replacement of native nucleic acid sequences by recombinant nucleic acid sequences in cells (see, for example, Ausubel et al., Current Protocols In Molecular Biolo~y, John Wiley &
Sons, Inc. (1995)), and for expression of recombinant nucleic acid sequences in cells (see e.g., Lee and Arthans, H.J. Biol. Chem., 263:3521, (1988); Rosenberg, et al., Gene, 56:125 (1987)), are well known in the art.
The bis-transition-metal-chelate moieties of the molecules according to Formula (I) bind to the oligohistidine target sequence. The transition metals of the bis-transition-metal-chelate moieties bind to imidazole groups of histidines of the oligohistidine target sequence.
Although not intending to be limited to such interpretation, it is believed that the affinity of the bis-transition-metal-chelate probe for oligohistidine target sequences relates to the presence of two tridentate (where R' or R'~ is absent) or tetradentate (where R' or R'~ is CH(COO-) or CH(COOH)) transition-metal chelates, each having a transition metal with at least two coordination sites available for interaction with electron-donor groups.
Oligohistidine target sequences comprising 4 to 12 histidine residues have appropriate electron-donor functionality, size, and flexibility to interact with available coordination sites of the bis-transition-metal-chelate probe, creating a stable linkage therewith.
An example of a transition-metal-chelate probe of the invention in association with a oligohistidine target sequence, in this case a hexahistidine target sequence, is depicted as follows:
~3$ $~7 / ~ ~ \
N ~ N+
n O O
NH HN
O O
O O
O O N~/
i N
w ~ ~O.
~o% v . o , N
N \N N \N N
NI1 ' NH NH \ NH NH
R- N N N N N N R°
H H H H H H
O O O O O O
Labeling is accomplished by contacting a bis-transition-metal-chelate molecule according to Formula (I) with a target-sequence-containing target material.
The bis-transition-metal-chelate molecule may be contacted with a target-sequence-containing target material located in, for example, a test tube, a microtiter-plate well, a cuvette, a flow cell, or a capillary, or immobilized on, for example a surface or other solid support.
Alternatively, the bis-transition-metal-chelate molecule may be contacted with a target-sequence-containing target material located within a cell, tissue, organ, or organism (in which embodiment, the bis-transition-metal-chelate derivative preferably is capable of traversing an intact biological membrane).
In one embodiment, the bis-transition-metal-chelate molecules according to Formula (I) are used to label target-sequence-containing molecules within cells. The bis-transition-metal-chelate molecules of this invention may be introduced into cells by diffusion (for bis-transition-metal-chelate derivatives capable of traversing biological membranes) or by microinjection, electroporation, or vesicle fusion (for any bis-transition-metal-chelate derivative). The target-sequence-containing molecules may be introduced into cells by microinjection, electroporation, or vesicle fusion, or by expression of recombinant genes in situ.
In one preferred embodiment, a target-sequence-containing protein produced by expression of a recombinant gene within cells is contacted with a probe of this invention by incubating cells in medium containing the probe. Following labeling, and optionally following further manipulations, cells are imaged using an epi-illumination, confocal, or total-internal-reflection optical microscope with an optical detector, such as a CCD camera, an intensified CCD camera, a photodiode, or a photomultiplier tube, and fluorescence signals are analyzed.
Uses of the Compositions of the Invention It is contemplated that bis-transition-metal-chelate molecules of the invention may be used in a variety of in vitro and in vivo applications.
The bis-transition-metal-chelate molecules of the invention may be used in numerous standard assay formats, as are well known in the art. Some examples of assay formats include fluorescence emission intensity, fluorescence polarization (FP), fluorescence anisotropy (FA), fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), fluorescence-activated cell--or particle--sorting (FACS), x/y-fluorescence scanning (FluorImaging), epi-illumination optical microscopy, confocal optical microscopy, total-internal-reflection optical microscopy, absorbance spectroscopy, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), scintillation proximity assay (SPA), autoradiography, and assays formats that involve use of biotin or other hapten incorporation to provide a recognition event for binding or immobilization of one or more components.
Some examples, which are intended to be illustrative and not limiting of possible assay formats and applications that could use site specific bis-transition-metal-chelate-labeled target materials, are set forth below.
For example, the bis-transition-metal-chelate derivatives of the present invention may be used to detect and/or quantify a polypeptide of interest containing, or derivatized to contain, a target sequence. The target-sequence-containing polypeptide is incubated with a molecule according to Formula (I) for a time period sufficient to allow labeling thereof.
Labeled target-sequence-containing polypeptide optionally may be separated from unbound material before the detection step using any method known in the art, and the detectable group X is detected, thereby detecting the polypeptide of interest. The target-sequence-containing polypeptide may be included in any material, including, but not limited to, cuvettes, microtiter plates, capillaries, flow cells, test tubes, gels, blots, and biological samples.
The invention also provides an assay method for monitoring a binding process.
In this method, a first component of a specific reaction pair is labeled with a molecule according to Formula (I) and is reacted with a second component of the pair. The reaction can be monitored by monitoring a change in a signal of the detectable group X.
Examples of specific reaction pairs include, but are not restricted to, antibodies/antigens, hormone/receptor, enzyme/substrate, and protein/analyte.
In a fluorescence-emission-intensity assay, the sample is exposed to light of a first wavelength (able to be absorbed by a fluorescent moiety), and fluorescence-emission intensity is monitored at a second wavelength (emitted by said fluorescent moiety).

Fluorescence-emission intensity is dependent on the quantity of the fluorescent moiety and on the local environment of the fluorescent moiety.
A fluorescence-emission-intensity assay to detect and quantify binding between two molecules, molecule 1 and molecule 2, may be configured as follows: A reaction mixture is prepared by combining molecule 1 labeled with fluorescent moiety X according to the current invention and molecule 2. Complex formation results, directly or indirectly, from a change in the local environment of X, and, correspondingly, in a change in the fluorescence emission intensity of X. The progress of the reaction is monitored by observing the change in fluorescence emission intensity of X. Equilibrium association and dissociation constants may be extracted from the concentration-dependence of the reaction.
In a fluorescence-polarization (FP) or fluorescence-anisotropy (FA) assay, a sample is exposed to polarized light of a first wavelength (able to be absorbed by a fluorescent moiety), and fluorescence-emission polarization or anisotropy is monitored at a second wavelength (emitted by said fluorescent moiety). Fluorescence-emission polarization or anisotropy is inversely related to the rotational dynamics, and thus to the size, of said fluorescent moiety (or, if said fluorescent moiety is attached to a molecule or complex, to the rotational dynamics, and thus to the size, of the molecule or complex). FP or FA assays permit detection of reactions that result in changes in size of molecules or complexes, including especially, macromolecule-association and macromolecule-dissociation reactions.
An FP or FA assay to detect and quantify binding between two molecules, molecule 1 and molecule 2, may be configured as follows: A reaction mixture is prepared by combining ZS molecule 1 labeled with fluorochrome X according to the current invention and molecule 2.
Complex formation results in formation of a higher-molecular-weight, higher-FP, higher-FA
species. The progress of the reaction is monitored by observing the decrease in FP or FA.
Equilibrium association and dissociation constants are extracted from the concentration-dependence of the reaction.
A further FP or FA assay may be used to detect and quantify proteolytic activity and may be configured as follows: A reaction mixture is prepared by combining a substrate molecule labeled with fluorochrome X according to the present invention and a sample containing a proteolytic enzyme. Cleavage of the substrate molecule by the proteolytic enzyme results in the production of lower-molecular-weight, lower-FP, lower-FA
fragments.
The progress of the reaction is monitored by observing the decrease in FP or FA.
Fluorescence resonance energy transfer (FRET) is a physical phenomenon that permits measurement of distance). FRET occurs in a system having a fluorescent probe serving as a donor and a second fluorescent probe serving as an acceptor, where the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. In such a system, upon excitation of the donor with light of the donor excitation wavelength, energy can be transferred from the donor to the acceptor, resulting in excitation of the acceptor and emission at the acceptor emission wavelength. FRET readily can be detected--and the efficiency of FRET readily can be quantified--by exciting with light of the donor excitation wavelength and monitoring emission of the donor, emission of the acceptor, or both. The efficiency of energy transfer, E, is a function of the Forster parameter, R~, and of the distance between the donor and the acceptor, R:
E = [1 + (R/R~)6]n wherein the Forster parameter (in angstroms, A), is:
Ro (in ~) _ (0.211 X lO~s)(n-4QDK2.~1/6 wherein n is the refractive index of the medium, QD is the donor quantum yield in the absence of the acceptor, Kz is the orientation factor relating the donor acceptor transition dipoles, and J is the spectral overlap integral of the donor emission spectrum and the acceptor excitation spectrum.
If one performs FRET experiments under conditions where Ro is constant, measured changes in E permit detection of changes in R, and, if one performs experiments under conditions where Ro is constant and known, the measured absolute magnitude of E permits determination of the absolute magnitude of R.

With fluorochromes and chromophores known in the art, FRET is useful over distances of about 1 nm to about 15 nm, which are comparable to the dimensions of biological macromolecules and macromolecule complexes. Thus, FRET is a useful technique for investigating a variety of biological phenomena~that produce changes in molecular proximity. When FRET is used as a detection mechanism, colocalization of proteins and other molecules can be imaged with spatial resolution beyond the limits of conventional optical microscopy.
A FRET assay to detect and quantify binding between two molecules, molecule 1 and molecule 2, may be configured as follows: A reaction mixture is prepared by combining molecule 1 labeled with a molecule according to Formula (I) where detectable group X is a fluorescent moiety and molecule 2 is labeled with a fluorescent moiety Y or a chrompohore Y, wherein X and Y are able to participate in FRET. Complex formation results in increased proximity between X and Y, and, correspondingly, in increased FRET. The progress of the reaction is monitored by observing the increase in FRET. Equilibrium association and dissociation constants may be extracted from the concentration-dependence of the reaction.
A FRET assay to detect and quantify proteolytic activity may be configured as follows: A reaction mixture is prepared by combining a) a substrate molecule labeled at site 1 with Formula (I) wherein detectable group X is a fluorescent moiety and labeled at site 2 with fluorochrome Y, wherein sites 1 and 2 are on opposite sides of the proteolytic-cleavage site, and wherein X and Y are able to participate in FRET, and b) a sample containing a proteolytic enzyme. Cleavage of the substrate molecule by the proteolytic enzyme results in decreased proximity between X and Y and, correspondingly, in decreased FRET.
The progress of the reaction is monitored by observing the decrease in FRET.
A FRET assay to detect conformation change within molecule 1 induced upon interaction with molecule 2, may be configured as follows: A reaction mixture is prepared by combining (a) molecule 1 labeled at one site with fluorochrome X according to the current invention and labeled at another site with fluorochrome Y, wherein X and Y are able to participate in FRET, and (b) molecule 2. Conformation change within molecule 1 induced upon interaction with molecule 2 results in a change in proximity between X
and Y, and, correspondingly, a change in FRET. The progress of the reaction is monitored by observing the change in FRET.
A FRET assay to measure the distance between two sites, 1 and 2, within a molecule of interest, may be configured as follows: the molecule of interest is labeled at site 1 with fluorochrome X according to the current invention and is labeled at site 2 with fluorochrome Y, wherein X and Y are able to participate in FRET; fluorescence excitation and emission spectra are collected for X and Y; and the distance, R, is calculated as described supra.
Fluorescence emission intensity, lifetime, polarization, aniosotropy and FRET
are further described in the following references: Brand, L. and Johnson, M.L., Eds., Fluorescence Spectroscopy (Methods in Enzymology, Volume 278), Academic Press (1997), Cantor, C.R. and Schimmel, P.R., Biophysical Chemistry Part 2, W.H. Freeman (1980) pp.
433-465. Dewey, T.G., Ed., Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, Plenum Publishing (1991). Guilbault, G.G., Ed., Practical Fluorescence, Second Edition, Marcel Dekker (1990). Lakowicz, J.R., Ed., Topics in Fluorescence Spectroscopy: Techniques (Volume l , 1991 ); Principles (Volume 2, 1991 );
Biochemical Applications (Volume 3, 1992); Probe Design and Chemical Sensing (Volume 4, 1994);
Nonlinear and Two-Photon Induced Fluorescence (Volume 5, 1997); Protein Fluorescence (Volume 6, 2000), Plenum Publishing.
Fluorescence imaging using epi-illumination, confocal, or total-internal-reflection optical microscopy permits characterization of the quantities, locations, and interactions of fluorochrome-labeled target materials within cells. All fluorescence observables that can be analyzed in vitro--emission intensity, emission lifetime, fluorescence correlation, FP/FA, and FRET--also can be analyzed in cells (See Nakanishi et al. Anal. Chem. 73:2920-2928 (2001);
Maiti, S. et al. Proc. Natl. Acad. Sci. USA 94: 11753-11757 (1997); Eigen and Rigler, Proc.
Natl. Acad. Sci. USA 91:5740-5747 (1994) for example of uses of fluorescence in cells).
The bis-transition-metal-chelate derivatives of this invention may be used to label target-sequence-containing molecules within cells. The bis-transition-metal-chelate derivatives of this invention may be introduced into cells by diffusion (for bis-transition-metal-chelate derivatives capable of traversing biological membranes) or by microinjection, electroporation, or vesicle fusion (for any bis-transition-metal-chelate derivative). The target-sequence-containing molecules may be introduced into cells by microinjection, electroporation, or vesicle fusion, or by expression of recombinant genes in situ.
In one embodiment, a target-sequence-containing protein produced by expression of a recombinant gene within cells is contacted with a bis-transition-metal-chelate derivative of this invention by incubating cells in medium containing the bis-transition-metal-chelate derivative. Following labeling, and optionally following further manipulations, the cells are imaged using an epi-illumination, confocal, or total-internal-reflection optical microscope with an optical detector, such as a CCD camera, an intensified CCD camera, a photodiode, or a photomultiplier tube, and fluorescence signals are analyzed.
The fluorescent molecules of the present invention also can be used, in vitro or in vivo, in single-molecule fluorescence assays with single-molecule detection, wherein fluorescence emission intensity, fluorescence correlation, FPlFA, or FRET is analyzed from individual single molecules.
The fluorescent molecules of the present invention also can be used, in vitro or in vivo, in fluorescence assays with "multiplex" detection, wherein a plurality of different fluorescent molecules are attached to a plurality of different primary molecules, molecule la, lb, ...ln, with each primary molecule being specific for a different secondary component, 2a, 2b, ...2n, in order to monitor a plurality of reactions between primary molecules and secondary molecules in a single reaction mixture. According to this method of use, each of the primary molecules is separately labeled with a fluorochrome having a different, distinguishable excitation and/or emission wavelength. The primary molecules are then reacted, as a group, with the secondary molecules, as a group, and fluorescence is monitored at each of different, distinguishable excitation and/or emission wavelengths.
The fact that the present invention is compatible with fluorochromes having different, distinguishable excitation and emission wavelengths (see, e.g., Table 1 for excitation maxima and emission maxima of derivatives of Cy3, CyS, and Cy7 in Examples), makes the invention particularly important for applications involving multiplex detection.
Most or all of the assays above, in vitro or in vivo, can be adapted for high-throughput screening, using formats, equipment, and procedures apparent to persons skilled in the art.
Examples of fluorochromes and chromophores suitable for use in assays above, in conjunction with the molecules of the invention, are presented in Haugland R.
P. Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, sixth edition (1996), ISBN
0-9652240-0-7 (Spence, MTZ, ed). Said fluorochromes and chromophores can be incorporated into polypeptides and other molecules of interest by any suitable method, many of which are well known in the art, including, but not limited to, chemical synthesis, enzymatic synthesis, ribosomal synthesis, chemical ligation, chemical modification, and hapten binding (see Haugland R. P. Handbook of Fluorescent Probes and Research Chemicals, supra). Alternatively, fusions of autofluorescent proteins, such as green fluorescent protein, to a polypeptide of interest can be encoded as nucleic-acid fusion constructs, produced in cells, and analyzed in cells or in vitro.
The methods of the invention may be used in many areas of biology and biological research including drug screening, diagnostics and academic research.
It further is contemplated that the bis-transition-metal-chelate molecules of the invention may be used for immobilization andlor affinity-purification of target-sequence-containing molecules.
Immobilization may be accomplished by: (a) covalently attaching a bis-transition-metal-chelate derivative to a surface or other solid support (via detectable group X or via a linker); (b) contacting the resulting bis-transition-metal-chelate-derivative-containing surface or other solid support with a solution containing a target-sequence-containing target material;
and (c) optionally washing the surface or the solid support to remove unbound material.

Affinity purification may be accomplished by: (a) covalently attaching a bis-transition-metal-chelate derivative to a surface or other solid support, (b) contacting the resulting bis-transition-metal-chelate-derivative-containing surface or other solid support with a solution containing a target-sequence-containing molecule, (c) optionally washing the surface or other solid support to remove unbound material, and (d) eluting the target-sequence-containing molecule with a low-molecular-weight monothiol (e.g., (3-mercaptoethanol) or, preferably, a low-molecular-weight dithiol (e.g., dithiothreitol or ethanedithiol).
The invention also provides a kit including a molecule according to Formula (I) and a target material including a target sequence of the form: (H);, wherein H is histidine and i is an integer of from 4 to 12 (i.e., SEQ )D NOS. 1-9), preferably 4 to 8, and most preferably 6.
The invention also provides a kit. The kit includes a molecule according to Formula (I) and a reagent the promotes the formation of a complex between the molecule of Formula (I) and a target sequence of the invention.
It will be apparent that the present invention has been described herein with reference to certain preferred or exemplary embodiments. The preferred or exemplary embodiments described herein may be modified, changed, added to, or deviated from without departing from the intent, spirit and scope of the present invention, and it is intended that all such additions, modifications, amendments and/or deviations be included within the scope of the following claims.

EXAMPLES
FX A MPT .F. 1 Synthesis of (Ni2+-NTA)~-Cy3 A. Swnthesis of (NTA)~-Cy3 N-(5-amino-1-carboxypentyl)iminodiacetic acid (Dojindo; 26 mg, 80 p,mol) was dissolved in 1.6 ml O.1M sodium carbonate and was added to Cy3 bis-succinimidyl-ester ("Cy3 Reactive Dye" from Amersham-Pharmacia Biotech). Following reaction for 1 hour (with vortexing at 15-min intervals) at 25°C in the dark, products were purified from excess N-(5-amino-1-carboxypentyl)iminodiacetic acid using a Sep-Pak C18 cartridge ((Millipore;
pre-washed with 10 ml of acetonitrile and 10 ml water; washed with 20 ml water; eluted with 1 ml 60% methanol), dried, re-dissolved in 200 ~.l methanol, and purified by preparative TLC
[1000A silica gel (Analtech); NH40H:ethanol:water 55:35:10 v/v/v]. Three bands were resolved, corresponding to (NTA)2-Cy3 (rf=0.2), (NTA),-Cy3 mono acid (r,=0.5), and (NTA)2-Cy3 bis acid (rt=0.8). (NTA)Z-Cy3 was eluted using 60% methanol, dried, re-dissolved in 2 ml water and quantified spectrophotometrically (esso-150,OOOM-'crri'). The yield was 64 nmol, 8%. ES-MS: mle 1197.0 (calculated 1197.4).
B. Synthesis of (Niz+-NTA)z-Cy3 NiCl2 (Aldrich; 350 nmol of NiCl2 in 3 p,l of O.O1N HCl) was added to (NTA)2-Cy3 (70 nmol in 2 ml water), and the solution was brought to pH 7 by addition of 0.8 ml 50 mM
sodium acetate (pH 7), 200 mM NaCI. Following reaction for 30 min. at 25°C in the dark, the product was purified using a Sep-Pak C18 cartridge ((Millipore; procedure as above) and dried. ES-MS: mle 1316.8 (calculated 1315.7). Niz+ content [determined by performing analogous reaction with ~3NiC12 (New England Nuclear) and quantifying reactivity in product by scintillation counting in Scintiverse II (Fischer)]: 1.4 mol Ni2+ per mol.
Spectroscopic properties are reported in Table 1.

o~
NH HN
O O
-o 0 N N
o l ' / ' ~~O_ _ O--Ni ~ ~ IYi O
~O O O
XXV

S ectrosco is Pro erties of (Ni2+-NTA)2-C
3 and (Ni2+-NTA)2-C
3a fluorochrome ~ max exc ~ max em (nm)uantum yield (nm) (Q) (Ni'-NTA)Z- 552 565 0.04 (Ni'-NTA)2- 650 668 0.05 Cy5 a Ni'+-free analogues exhibit identical ~, n,ax, eXC
and ~, max,em and 3.8-fold higher Q (with the higher Q presumably reflecting the unavailability of nonradiative decay involving Ni2+ unoccu ied d orbitals).

Synthesis of (Ni2+-NTA)z-CySA. Synthesis of (NTA)?-Cy5 N-(5-amino-1-carboxypentyl)iminodiacetic acid (Dojindo; 40 mg; 125 ~.mol) was dissolved in 0.8 ml O.1M sodium carbonate and was added to Cy5 bis-succinimidyl-ester ("Cy5 Reactive Dye" Amersham-Pharmacia Biotech; 800 nmol). Following reaction for 1 h (virtexed at 15 minute intervals) at 25°C in the dark, products were purified from excess N-(5-amino-1-carboxypentyl)iminodiacetic acid using a Sep-Pak C18 cartridge ((Millipore;
procedure as above), dried, re-dissolved in 200 ~.1 methanol, and purified in 100 ~,m portions by preparative TLC [silica gel, 1000 A (Analtech); NH~OH:ethanol:water in a 55:35:10 v/v/v.
Three bands were resolved, corresponding to (NTA)2-Cy5 (rt=0.2), (NTA)~-Cy5 mono acid (rf=0.6), and (NTA)2-Cy5 bis acid (rt=0.8). The (NTA)Z-Cy5 was eluted with 60%
methanol, dried, re-dissolved in 2 ml water and quantified spectrophotometrically (e5so = 250,OOOM-lcm-~). Yield: 60 nmol; 7.5%.
B. Synthesis of (Ni2+-NTA)~-Cy5 NiCl2 (Aldrich; 90 nmol in 1 p,l of 0.01 N HCl) was added to (NTA)Z-Cy5 (30 mmol in 1 ml water), and the solution was bought to pH 7 by addition of 0.5 ml 50 mM sodium acetate (pH 7), 70 mM NaCI. Following reaction for 30 min. at 25°C in the dark, the product was purified using a Sep-Pak C18 cartridge ((Millipore; procedure as above) and dried. ES-MS: mle 1341.0 (calculated 1341.7). Spectroscopic properties are reported in Table 1.
'nc O
mn ~ N ~~
N ~ i o- it ~ ~ ~ ;_o_ '~o~0 0 0 (XXVI) Preparation of a C-terminall~hexahistidine tagged derivative of the transcriptional activator CAP (CAP-Hiss) A. Preparation of CAPHis6 Plasmid pAKCRP-Hiss encodes CAP-His6 under the control of bacteriophage T7 gene promotor. Plasmid AKCRP-His6 was constructed from plasmid pAKCRP (as described in Kapanidis, A. et al., J. Mol. Biol. 312:453-468 (2001) by using site-directed mutagenesis (as 10 described in Kukel, et al., J. Meths. Enzymol., 204:125-138 (1991)) to insert six His codons (CAC-CAC-CAC-CAC-CAC-CAC) after codon 209 of the crp gene.
To prepare CAP-His6, a culture of E. coli strain BL21(DE3) (Novagen) transformed with pAKCRP-Hiss was shaken at 37°C in 1 L LB (as described in Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1972)) containing 200 mg/ml ampicillin until OD600 = 0.5, induced by addition of isopropyl-thio-(3-D-galactoside to 1 mM, and shaken an additional 3 h at 37°C. The culture was harvested by centrifugation (4,500 x g; 15 min. at 4°C), the cell pellet was re-suspended in 15 ml buffer A
[20 mM Tris-HCl (pH 7.9), 500 mM NaCI, 5 mM imidazole], cells were lysed by sonication, ZO and the lysate was cleared by centrifugation (30,000 x g; 30 min. at 4°C). The sample was adjusted to 15 ml with buffer A, adsorbed onto 2 ml Ni2+-NTA agarose (Qiagen) in buffer A, washed with 12 ml buffer A containing 20 mM imidazole, and eluted with 6 x 1 ml buffer A
containing 200 mM imidazole.
Fractions containing CAP-Hiss were pooled, desalted twice into buffer B [40 mM
Tris-HCl (pH 8), 100 mM NaCI, 1 mM dithiothreitol, 5% glycerol] by gel-filtration chromatography on NAP-10 (Amersham-Pharmacia Biotech), quantified spectrophotometrically (s27g, protomer = 20,000 M-' cm'), and stored in aliquots at -80°C. Yield 20 mg/L culture. Purity > 99%.

Verification of Affinit a~~ecificity of Association of (Ni2+-NTA)2Cy3 and (Ni2+-NTA)~CyS with Target Material Affinity and specificity of association of the probe with target material were evaluated using fluorescence anisotropy assays (methods as in Jameson and Dwyer, Methods Enzymol., 246:283-300 (1995)). Formation of a complex of the probe with a tagged protein was detected as an increase in fluorescence anisotropy, A, arising from the increase in molecular size and corresponding decrease in rotational dynamics.
A. Titration of (Ni2+-NTA)~Cy3 and (Ni2+-NTA)~CyS with CAP-His6 Reaction mixtures [200 p,l, in 100 ~.l quartz micro-cuvettes (Starna)]
contained 50 nM
of (Ni2+-NTA)Z-Cy3 or (Ni2+-NTA)2-Cy5 in buffer C [40 mM Tris-HCl (pH 8), 100 mM
NaCI, 1 mM dithiothreitol, 0.5 mM imidazole, 0.2 mM cAMP, 100 ~.g/ml bovine serum albumin, and 5% glycerol]. Reaction mixtures were titrated with 0-3 ~,M CAP-His6 (or CAP) by successive addition of 0.5-4 ~.l aliquots of 2-4 ~.M CAP-Hiss (or CAP) in the same buffer.
Fluorescence anisotropy was determined at the start of the titration and 5 min after each successive addition in the titration. All solutions were maintained at 25°C.
B. Detection of Fluorescence Anisotropy Fluorescence measurements were performed using a commercial steady-state fluorescence instrument (QM-1, PTI) equipped with T-format Glan-Thompson polarizers (PTI). Excitation wavelengths were 530 nm for (Ni2+-NTA)2-Cy3 and 630 nm for (Ni2+-NTA)Z-CyS; emission wavelengths were 570 nm for Ni2+-NTA)Z-Cy3 and 670 nm for Ni2+-NTA)z-CyS. Slit widths were lOmn. Fluorescence emission intensities were corrected for background by subtraction of fluorescence emissions intensities for control reactions containing identical concentrations of CAP-His6 or CAP but not containing probe.
Fluorescence anisotropy, A, was calculated using: A = (Ivy-GIvH)/(Ivv + 2GvH) where Ivy and IvH are the fluorescent intensities with the excitation polarizer at a vertical position and the emission polarizers at vertical and horizontal positions, respectively, and G is the grating correction factor. Data were plotted as: (A-A~/Ao) where A is the fluorescence anisotropy in the presence of the indicated concentration of CAP-His6 or CAP, and Ao is the fluorescence anisotropy in the absence of CAP-His6 or CAP. Equilibrium dissociation constants were calculated using linear regression.
Referring now to FIG. 2, a graphical representation of results of titration of (Ni2~-NTA)2-Cy3 with His6-CAP is shown (filled circles). Specific interaction between (Ni2+-NTA)2-Cy3 and CAP-His6 is evidenced by a large, saturable increase in fluorescence anisotropy. High affinity of interaction is evidenced by a low equilibrium dissociation constant (KD = 1.0 p,M). Specificity of interaction is evidenced by the absence of a significant increase in fluorescence anisotropy in a control titration with CAP (open circles;
>95% specificity). ).
Referring now to FIG. 3, a graphical representation is shown of titration of (NTA)Z-Cy5 with CAP-His6 is shown (filled circles). Specific interaction between (Ni2+-NTA)2-Cy5 and Hiss-CAP is evidenced by a large, saturable increase in fluorescence anisotropy. High affinity of interaction is evidenced by a low equilibrium dissociation constant (KD = 0.4 pM).
Specificity of interaction is evidenced by the absence of a significant increase in fluorescence anisotropy in a control titration with CAP (open circles; (>95% specificity).
ZO

Verification of Affinit ~,~Specificity, and Stoichiometry of Association of (Ni2+-NTA)?
and (Ni2+-NTA)aCyS with Target Material Using_FRET
ZS The affinity, specificity, and stoichiometry of interactions between probes according to the invention and the His6 tag also were verified using FRET assays. A His6-tagged protein-DNA complex, (CAP-His6)-DNAF, was prepared. FRET assays using the probes according to the invention then were performed to verify interactions, to detect a target material, and to measure an intermolecular distance.
A. Preparation of DNAF

DNAF, 53 base pair fluorescein-labelled DNA fragment containing the consensus DNA site for CAP (fluorescein incorporated at position -9 relative to the consensus DNA site for CAP) was prepared as described in Ebright, R. et al., J. Mol. Biol.
312:453-468 (2001).
B. FRET Assays--Standard TitrationsReaction mixtures [200 p.l, in 50 p.l quartz micro-cuvettes (Starna)] contained 5 nM DNAF and 50 nM CAP-His6 (or CAP) in buffer C.
Reaction mixtures were titrated with 0-3.2 p.M 2a or 2b by successive addition of 0.3-1.2 gl aliquots of 30-300 pM of (Niz+-NTA)z-Cy3 or (Niz+-NTA)z-Cy5 in the same buffer.
Fluorescence anisotropy was determined at the start of the titration and 5 min after each successive addition in the titration. All solutions were maintained at 25°C.
Fluorescent emission intensities, F, were measured using a commercial steady-state fluorescence instrument (QM-1, PTI) equipped with T-format Glan-Thompson polarizers (PTI) set at 54.7° ("magic angle"). Excitation wavelength was 480 nm;
emission wavelength range were 500-600 nm (titrations with (Niz+-NTA)z-Cy3) or 500-700 (titrations with (Niz+:NTA)z-CyS; excitation slit width was 10 nm; emission slit width was 15 nm.
Fluorescence emission intensities were corrected for background (by subtraction of fluorescence emission intensities for control reaction mixtures containing identical concentrations of (Niz+-NTA)z-Cy3 or (Niz+-NTA)z-CyS, but not containing CAP-His6 or CAP) and for dilution.
Efficiencies of FRET, E, were calculated as: E = 1 -(Fszo,aso~szoiago°) where Fszo,4ao is the fluorescence emission intensity of the fluorescein label at the indicated concentration of (Niz+-NTA)z-Cy3 or (Niz+-NTA)z-Cy5 and Fszoiaso° is the fluorescence emission intensity of the fluorescein label at 0 ~.M of (Niz+-NTA)z-Cy3 or (Niz+-NTA)z-CyS. Data were plotted as E vs. titrant concentration, and binding curves and equilibrium dissociation constants were calculated using non-linear regression (as described in Gunasekera, A. et al., J. Biol. Chem., 267:14,713-14,720 (1992)).
Referring now to FIG. 5, a graphical representation of results of titration of the (CAP-His6)-DNAF complex with (Niz~-NTA)z-Cy3 is shown (filled circles). Specific interaction between the (CAP-His6)-DNAFCOmplex and N( iz+-NTA)z-Cy3 is evidenced by a large, saturable increase in FRET. High affinity of interaction is evidenced by a low equilibrium dissociation constant (Kp = 0.9 pM). Specificity of interaction is evidenced by the absence of a significant increase in fluorescence anisotropy in a control titration with the CAP-DNAF
complex (open circles; (>95% specificity).
Refernng now to FIG. 6, a graphical representation of results of titration of the (CAP-His6)-DNAF complex with (Ni2~-NTA)Z-Cy5 is shown (filled circles). Specific interaction between the (CAP-His6)-DNAF complex and (Ni2+-NTA)2-Cy5 is evidenced by a large, saturable increase in FRET. High affinity of interaction is evidenced by a low equilibrium dissociation constant (Kp = 0.3 pM). Specificity of interaction is evidenced by the absence of a significant increase in fluorescence anisotropy in a control titration with the CAP-DNAF
complex (open circles; (>95% specificity).
C. FRET Assays--Stoichiometric Titrations Stoichiometric titrations were performed analogously to standard titrations (as described in Example 5B), using reaction mixtures containing 0.6-2.6 p.M (CAP-His6)-DNAF
[prepared by equilibration of DNAF with excess CAP-His6 for 20 min. at 25°C, followed by removal of unbound CAP-His6 by filtration through Bio-Rex 70 (Bio-Rad) (according to methods described in Kapanidis, A.N., et al., J. Mol. Biol. 312:453-468 (2001)], and titrating with 0-12 ~.M of (Ni2+-NTA)z-Cy3 or (Ni2+-NTA)2-Cy5 by successive addition of 0.3-1.2 ~.l aliquots of p,M (Ni2+-NTA)2-Cy3 or (Ni2+-NTA)z-CyS. Fluorescence emission intensities were corrected for dilution and background, and values of E were corrected for non-specific interactions (by subtraction of values of E for control reaction mixtures omitting CAP-His6).
Corrected values of E were plotted as E/Esac vs. titrant concentration where Esat is the E at saturating titrant concentrations).
Referring now to FIG. 7, a graphical representation of results of stoichiometric titration of the (CAP-Hiss)-DNAF complex with (Niz~-NTA)2-Cy3 is shown (filled circles).
The interaction between with (Ni2~-NTA)2-Cy3 and His6 has a stoichiometry is 1:1, as evidenced inflection of the titration curve at a ratio of 1 mole (Ni2~-NTA)2-Cy3 to 1 mole CAP-His6 protomer.

Referring now to FIG. 8, a graphical representation of results of stoichiometric titration of the (CAP-His6)-DNAF complex with (Ni2~-NTA)2-Cy5 is shown (filled circles).
The interaction between with (Ni2~-NTA)Z-Cy5 and His6 has a stoichiometry is 1:1, as evidenced inflection of the titration curve at a ratio of 1 mole (Ni2~-NTA)2-Cy5 to 1 mole CAP-His6 protomer.
D. FRET Assays--Distance Determinations Donor-acceptor distances, R, were determined using the measured efficiencies of FRET at saturation, Esa~ (0.45 for titration with (Ni2~-NTA)2-CyS; 0.25 for titration (Ni2~-NTA)2-CyS; see FIGS. 5, 6), and the measured Forster parameters, Ro:
E = Ro6/(Ro6 + R~) Ro (in A) _ (0.2 11 X lO-5)(n-4QDKZJ)1~6 wherein n is the refractive index of the medium (1.4 for dilute protein solutionsg), QD is the donor quantum yield in the absence of acceptor [0.4; measured using quinine sulfate in 0.1 N
N2S04 as standard (QQs = 0.51)], KZ 1S the orientation factor relating the donor emission dipole and acceptor dipole [approximated as 2/3 due to the low fluorescent anisotropy of the donor], and J is the spectral overlap integral of the donor emission spectrum and the acceptor excitation spectrum:
J = [JFD(~)~a,(~)~4d~]/[JFu(~)d~]
wherein FD(7~) is the normalized corrected emission spectrum of donor, EA(~,) is the molar extinction coefficient of acceptor, and ~, is the wavelength.
The analysis above yields a donor-acceptor distance of 56(~4) ~. This distance is in excellent agreement with the distance of about 55 A expected based on structural information as illustrated in FIG. 3 (corresponding to the distance between the fluorescein on DNA and the Hisb of the proximal CAP-His6 protomer).

It will be apparent that the present invention has been described herein with reference to certain preferred or exemplary embodiments. The preferred or exemplary embodiments described herein may be modified, changed, added to, or deviated from without departing from the intent, spirit and scope of the present invention, and it is intended that all such additions, modifications, amendments and/or deviations be included within the scope of the following claims.

SEQUENCE LISTING
<110> Ebright, Richard H.
Ebright, Yon W.
<120> Transition Metal Containing Chelete Probes <130> 744-37P
<150> 60/410,267 <151> 2002-09-13 <150> 60/367,775 <151> 2002-03-28 <160> 9 <170> PatentIn version 3.1 <210> 1 <211> 4 <212> PRT
<213> Artificial Sequence <220>
<223> Target Sequence <400> 1 His His His His <210> 2 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<223> Target Sequence <400> 2 His His His His His <210> 3 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Target sequence <400> 3 His His His His His His <210> 4 <211> 7 <212> PRT
<213> Artificial Sequence <220>
<223> Target sequence <400> 4 His His His His His His His <210> 5 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> Target sequence <400> 5 His His His His His His His His <210> 6 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Target sequence <400> 6 His His His His His His His His His <210> 7 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Target Sequence <400> 7 His His His His His His His His His His <210> 8 <211> 11 <212> PRT
<213> Artificial Sequence <220>
<223> Target sequence <400> 8 His His His His His His His His His His His <210> 9 <211> 12 <212> PRT
<213> Artificial Sequence <220>
<223> Target sequence <400> 9 His His His His His His His His His His His His

Claims (76)

WE CLAIM:

1. A probe for labeling a target material, comprising:
a conjugate of a transition metal compound with a detectable group, said conjugate having the general structural formula (I), and tautomers, salts, and acids thereof:
wherein (a) Y and Y' are each a transition metal; (b) R1 and R1' are each independently CH(COO-), CH(COOH), or absent; (c) R2 and R2' are linear or branched, group.
optionally substituted, linkers of from about 3.0 to about 20 .ANG. long; and (d) X is a detectable group.

2. The molecule according to claim 1, wherein (R1 + R2) and (R1' + R2') are each independently linkers of from about 3.0 .ANG. to about 15 .ANG. long, with the proviso that the difference in length between (R1 + R2) and (R1' + R2') is less than or equal to about 6 .ANG..

3. The molecule according to claim 2, wherein the length of (R1 + R2) is equal to the length of (R1' + R2').

4. The molecule according to claim 1, wherein Y and Y' are each independently selected from the group consisting of Ni2+, Co2+, Cu2+, and Zn2+.

5. The molecule according to claim 4, wherein Y and Y' are each Ni2+.

6. The molecule according to claim 1, wherein the detectable group is selected from the group consisting of a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, an absorbent moiety, a photosensitizer, a spin label, a radioisotope, an isotope detectable by nuclear magnetic resonance, a paramagnetic atom, a heavy atom, a hapten, a crosslinking agent, a cleavage agent, and combinations thereof.

7. The molecule according to claim 1, wherein X is a fluorescent moiety.

8. The molecule according to claim 1, wherein X is derived from a cyanine dye.

9. The molecule according to claim 1, wherein X is derived from a squaraine dye.

10. The molecule according to claim 1, where X is selected from the group consisting of:
wherein (a) U and V are each independently C(R4)2, NH, O, S, or (CH)2; (b) R3 and R3' are each independently H or sulfonate; (c) R4 is H, CH3, CH2CH3, or (CH2)2CH3; and (d) n is 0 or an integer of from 1 to 6.

11. The molecule according to claim 10, wherein n is 1, 2 or 3.

12. The molecule according to claim 1, where X is selected from the group consisting of:
wherein (a) U and V are each independently C(R4)2, NH, O, S, or (CH)2; (b) R3 and R3 are each independently H or sulfonate; (c) R4 is H, CH3, CH2CH3, or (CH2)2CH3; (d) R5 is absent or is selected from the group consisting of H, an alkyl group, and an aryl group; and (e) n' is 0 or an integer of from 1 to 3

13. The molecule according to claim 12, wherein n is 0, 1, or 2.

14. The molecule according to claim 1, where X is selected from the group consisting of:
wherein (a) R6, R6~, R6~~, R6~~~, R6~~~~, and R6~~~~~ are each independently hydrogen, halogen, hydroxyl, or alkoxyl; and (b) R7, when present, is hydrogen, carboxyl, carboxylate or sulfonate.

15. The molecule according to claim 1, wherein said molecule is capable of traversing a biological membrane.

16. A molecule having two pendant transition-metal-chelate moieties according to the general structural formula:
wherein (a) Y and Y' are each a transition metal; (b) U and V are each independently C(R4)2, NH, O, S, or (CH)2; (c) R1 and R1~ are each independently CH(COO-), CH(COOH), or absent; (d) R2 and R2~ are each independently linear or branched, optionally substituted, linkers of from about 3.0 to about 20 .ANG. long; (e) R3 and R3' are each independently H or sulfonate; (f) R4 is H, CH3, CH2CH3, or (CH2)2CH3; and (g) n is 0 or an integer of from 1 to 6.

17. The molecule according to claim 16, wherein (R1 + R2) and (R1' + R2') are each independently linkers of from about 3.0 .ANG. to about 15 .ANG. long, with the proviso that the difference in length between (R1 + R2) and (R1' + R2') is less than or equal to about 6 .ANG..

18. The molecule according to claim 16, wherein Y and Y' are each independently selected from the group consisting of Ni2+, Co2+, Cu2+, and Zn2+.

19. The molecule according to claim 16, wherein Y and Y' are each Ni2+.

20. The molecule according to claim 16, wherein n is 1, 2, or 3.

21. A molecule with two pendant transition-metal-chelate moieties according to general structural formula:
wherein (a) Y and Y' are each a transition metal; (b) U and V are each independently C(R4)2, NH, O, S, or (CH)2; n is 0 or an integer of from 1 to 6; (c) R3 and R3~ are each independently H or sulfonate; (e) R4 is H, CH3, CH2CH3, or (CH2)2CH3; and (f) n is 0 or an integer of from 1 to 6.

22. The molecule according to claim 21, wherein Y and Y' are each independently selected from the group consisting of Ni2+, Co2+, Cuz+, and Zn2+.

23. The molecule according to claim 21, wherein Y and Y' are each Ni2+

24. The molecule according to claim 21, wherein n is 1, 2, or 3.

25. A molecule with two pendant transition-metal-chelate moieties according to general structural formula:
wherein Y and Y' are each a transition metal; R3 and R3~ are each independently H or sulfonate; and n is 1, 2, 3, or 4.

26. The molecule according to claim 25, wherein Y and Y' are each independently selected from the group consisting of Ni2+, Co2+, Cu2+, and Zn2+.

27. The molecule according to claim 25, wherein Y and Y' are each Ni2+

28. The molecule according to claim 25, wherein n is 1, 2, or 3.

29. A method for imparting detectable properties to at least one target material, the method comprising the step of reacting:
(a) a target material having a target sequence comprising an amino acid sequence of the form: (H); wherein H is histidine, and i is an integer of from 4 to 12;
and (b) a molecule according to Formula (I) under conditions sufficient to permit transition-metal-chelate moieties of said molecule to associate with said target sequence.

30. The method according to claim 29, wherein said target material is a polypeptide.

31. The method according to claim 29, wherein said target sequence is SEQ ID
NO. 3.

32. A method for detecting at least one target material of interest, said method comprising:
(a) providing a target material containing a target sequence, said target sequence comprising an amino acid sequence of the form: (H); wherein H is histidine, and i is an integer of from 4 to 12;
(b) incubating said target material with a molecule according to Formula (I) having a detectable group, for a time period sufficient to allow labeling of said target material; and (c) detecting said detectable group, thereby detecting said target material.

33. The method according to claim 32, wherein said target material is located within a material selected from the group consisting of a cuvette, a microtiter plate, a capillary, a flow cell, a test tube, a gel, a blot and a biological sample.

34. The method according to claim 32, wherein said target material is a polypeptide.

35. The method according to claim 32, wherein step (b) is performed in a gel matrix.

36. The method according to claim 32, wherein step (b) is performed in a complex mixture of components.

37. The method according to claim 32, wherein labeled target material is separated from other components following step (b).

38. The method according to claim 32, wherein labeled target material is not separated from other components following step (b).

39. The method according to claim 32, wherein said detectable group is a fluorescent moiety.

40. The method according to claim 32, wherein said detecting step includes detecting a fluorescence property.

41. The method according to claim 40, wherein said fluorescence property is at least one of a fluorescence-emission intensity, a fluorescence lifetime, a fluorescence anisotropy, a fluorescence polarization, and a fluorescence correlation.

42. A method for determining the localization, concentration, or interactions of at least one target material of interest on or within a cell, tissue, organ, or organism, comprising the steps of:

(a) providing a a cell, tissue, organ, or organism containing a target material containing a target sequence, said target sequence comprising an amino acid sequence of the form: (H); wherein H is histidine, and i is an integer of from 4 to 12;
(b) incubating said cell, tissue, organ, or organism with a molecule according to Formula (I) having a detectable group, for a time period sufficient to allow labeling of said target material; and (c) detecting said detectable group, thereby determining the localization, concentration, or interactions of said target material.

43. The method according to claim 42, wherein said target material is a polypeptide.

44. The method according to claim 42, wherein said detectable group is a fluorescent moiety.

45. The method according to claim 42, wherein said detecting step includes detecting a fluorescence property.

46. The method according to claim 45, wherein said fluorescence property is at least one of a fluorescence-emission intensity, a fluorescence lifetime, a fluorescence anisotropy, a fluorescence polarization, and a fluorescence correlation.

47. An assay method for monitoring a binding process comprising the steps of:
(a) reacting a first component of a specific binding pair with a second component of said pair, with said first component being labeled with a molecule according to Formula (I) having a detectable group; and (b) monitoring said reaction by monitoring a change in a signal of said detectable group.

48. An assay method for monitoring a binding process, comprising the steps of:
(a) reacting a first component of a specific binding pair with a second component of said pair, with said first component being labeled with a molecule according to Formula (I) having a detectable group; and (b) monitoring said reaction by monitoring at least one of a fluorescence-emission intensity, a fluorescence lifetime, a fluorescence anisotropy, a fluorescence polarization, and a fluorescence correlation of said detectable group.

49. An assay method for monitoring a binding process comprising the steps of:
(a) reacting a first component of a specific binding pair with a second component of said pair, with said first component being labeled with a molecule according to Formula (I) wherein X of Formula (I) is a fluorochrome, and said second component including Z, wherein Z is capable of participating in fluorescence energy transfer, fluorescence quenching or exciton formation with X and is selected from the group including a fluorochrome and chromophore; and (b) monitoring said reaction by monitoring fluorescence of X.

50. An assay method for monitoring a binding process comprising the steps of:
(a) reacting a first component of a specific binding pair with a second component of said pair, with said first component being labeled with a molecule according to Formula (I) wherein X of Formula (I) is selected from the group consisting of a fluorochrome and a chromophore, and said second component including Z, wherein Z is a fluorochrome able to participate in fluorescence energy transfer, fluorescence quenching, or exciton formation with X; and (b) monitoring the reaction by monitoring fluorescence of Z.

51. An assay method for monitoring a reaction, comprising the steps of:
(a) reacting a first analyte with a second analyte, said first analyte being labeled with a molecule according to formula (I) having a detectable group; and (b) monitoring said reaction by monitoring a charge in a detectable property of said detectable group.

52. The method according to claim 51, wherein said reaction is selected from the group consisting of a protein-protein binding event, a protein-self-association event, a protein-protein cleavage event, and a conformational charge of a protein.

53. A method for isolating at least one target material of interest comprising:
(a) contacting at least one molecule according to Formula (I) immobilized on a solid support, with a solution containing a target material having a target sequence of the form: (H)i wherein H is histidine, and i is an integer of from 4 to 12, under conditions that allow binding of said polypeptide to said immobilized molecule of Formula (I);
and (b) eluting said target material with a low-molecular weight monothiol or low-molecular-weight dithiol.

54. The method according to claim 53, further comprising the step of washing said solid support to remove unbound material before eluting said target material.

55. The method according to claim 53, wherein said solid support is selected from the group consisting of a surface, a bead, a gel, and a chromatographic matrix.

56. A method for immobilizing at least one target material of interest including:
(a) contacting at least one molecule according to Formula (I) immobilized on a solid support with a solution containing a target material having a target sequence of the form: (H)i wherein H is histidine, and i is an integer of from 4 to 12, under conditions that allow binding of said target material to said immobilized molecule of Formula (I).

57. The method of claim 56, further comprising the step of washing said solid support to remove unbound material.

58. The method according to claim 56, wherein said solid support is selected from the group consisting of a surface, a bead, a gel, and a chromatographic matrix.

59. A kit, comprising:
(a) a molecule according to Formula (I); and (b) a molecule including a target sequence, said target sequence comprising an amino acid sequence of the form: (H)i wherein H is histidine, and i is an integer of from 4 to 12.

60. A kit comprising:

(a) a molecule according to formula (I); and (b) a reagent that promotes the formation of a complex between the molecule according to formula (I) and a target sequence, said target sequence comprising an amino acid sequence of the form: (H)i wherein H is histidine, and i is an integer of from 4 to 12.

61. The method of synthesis of a compound of claim 1 by coupling:
(a) a synthon consisting of a bis-activated-ester derivative of a detectable group; and (b) a synthon consisting of an amine or hydrazide derivative of a chelator;
and then adding a transition metal.

62. The method of claim 61, wherein said chelator is protected during said coupling and deprotected thereafter.

63. The method of synthesis of a compound of claim 1 by coupling:
(a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethylindole, mono-chelator-functionalized 2,3,3-trimethylbenzindole, mono-chelator-functionalized 2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole, mono-chelator-functionalized 2-methyl-napthothiazole, mono-chelator-functionalized 2-methyl-benzoxazole, and mono-chelator-functionalized 2-methyl-napthoxazole;
(b) a synthon, identical or nonidentical to the synthon in (a), selected from the group in (a); and (c) a synthon containing at least one carbon atom;
and then adding a transition metal.

64. The method of claim 63, wherein said coupling is performed as a single reaction step.

65. The method of claim 63, wherein said coupling comprises: either (i) first reacting (a) and (c) to form a product, followed by further reacting the product with (b);
or (ii) first reacting (b) and (c) to form a product, followed by further reacting the product with (a).

66. The method of claim 63, wherein said chelator is protected during said coupling and deprotected thereafter.

67. The method of synthesis of a compound of claim 1 by coupling:
(a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized 2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized 2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized 2-methyl-5-sulfanato-benzothiazole, mono-chelator-functionalized 2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized 2-methyl-5-sulfanato-benzoxazole, and mono-chelator-6-sulfanato-functionalized 2-methyl-napthoxazole;
(b) a synthon, identical or nonidentical to the synthon in (a), selected from the group in (a); and (c) a synthon containing at least one carbon atom;
and then adding a transition metal.

68. The method of claim 67, wherein said coupling is performed as a single reaction step.

69. The method of claim 67, wherein said coupling comprises: either (i) first reacting (a) and (c) to form a product, followed by further reacting the product with (b);
or (ii) first reacting (b) and (c) to form a product, followed by further reacting the product with (a).

70. The method of claim 67, wherein said chelator is protected during said coupling and deprotected thereafter.

71. The method of synthesis a compound of claim 1 by coupling:
(a) a synthon selected from mono-chelator-functionalized 2,3,3-trimethylindole, mono-chelator-functionalized 2,3,3-trimethylbenzindole, mono-chelator-functionalized 2-methyl-pyridine, mono-chelator-functionalized 2-methyl-benzothiazole, mono-chelator-functionalized 2-methyl-napthothiazole, mono-chelator-functionalized 2-methyl-benzoxazole, and mono-chelator-functionalized 2-methyl-napthoxazole;
(b) a synthon selected from mono-chelator-functionalized 2,3,3-trimethyl-5-sulfanato-indole, mono-chelator-functionalized 2,3,3-trimethyl-6-sulfanato-benzindole, mono-chelator-functionalized 2-methyl-5-sulfanato-pyridine, mono-chelator-functionalized 2-methyl-6-sulfanato-benzothiazole, mono-chelator-functionalized 2-methyl-6-sulfanato-napthothiazole, mono-chelator-functionalized 2-methyl-5-sulfanato-benzoxazole, and mono-chelator-functionalized 2-methyl-6-sulfanato-napthoxazole; and (c) a synthon containing at least one carbon atom;
and then adding a transition metal.

72. The method of claim 71, wherein said coupling is performed as a single reaction step.

73. The method of claim 71, wherein said coupling comprises: either (i) first reacting (a) and (c) to form a product, followed by further reacting the product with (b);
or (ii) first reacting (b) and (c) to form a product, followed by further reacting the product with (a).

74. The method of claim 71, wherein said chelator is protected during said coupling and deprotected thereafter.

75. The method of synthesis a compound of claim 1 by performing a Mannich reaction involving a xanthene, xanthanone, or phenoxazine detectable group, a secondary-amine derivative of a chelator, and formaldehyde; and then adding a transition metal.

76. The method of claim 75, wherein said chelator is protected during said coupling and deprotected thereafter.