METHOD OF OBTAINING A DESIRED LOCALIZATION FOR CELLULAR IMAGING WITH THE USE OF PEPTIDOCONJUGATES

RELATED APPLICATIONS

This application claims priority from US Provisional Application No, 60/658,224, filed March 3, 2005 and entitled, "Method for Obtaining a Desired Localization for Cellular Imaging with the Use of Peptidoconjugates," which is hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT The invention was supported, in whole or in part, by a grant CHE-03349013 from

NSF. The Government has certain rights in the invention.

BACKGROUND

Photodynamic therapy, also referred to as photosensitization-based therapy (PDT), photoradiation therapy, phototherapy or photochemotherapy, is a medical treatment that employs a combination of light and a photosensitizing agent to generate a cytotoxic effect of cancerous or other unwanted tissues or organisms (See Dougherty, T. J., Photodynamic Therapy — New Approaches, Semin. Surg. Oncol. 5(1): 6-16 (1989); Liberman J. Light, Medicine of the Future, Santa Fe: Bear & Co. (1991); Hopper, C, Photodynamic Therapy: A Clinical Reality in the Treatment of Cancer, Lancet Oncol. 1: 212- 219 (2000)). The widely accepted PDT mechanism is that upon irradiation, the photosensitizer successively generates active oxygen species of high reactivity that interact with target molecules. Photodynamic therapy (PDT) consists of introducing a photoactive drug into a body and subsequent illumination of cells by visible or near infrared light. In the presence of oxygen, illumination activates the drug and in turn produces reactive oxygen species which are extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids, leading to cell destruction.

A large number of photosensitizing compounds have been developed for photodynamic therapy during the last ten years. For example, porphyrins and their derivatives absorb light strongly in the 690-880 nm region, and have been suggested for use as photosensitizers in photodynamic therapy. See U.S. Patent Nos. 5,268,371 and 5,272,142 and European Patent Nos. 213272 and 584552. See also Jori et al, "Controlled targeting of different subcellular sites by porphyrins in tumor-bearing mice", Br J Cancer 53:615-621 (1986).

Photodynamic therapy possesses high effectiveness and safety compared with the conventional chemotherapy because of its relative selectivity in most sites, its compatibility with other treatment, its repeatability, its ease of delivery, etc.

Photodynamic therapy has been effective in treating multiple types of cancer, including cancers of different tissues and organs, including benign and malignant tumors (See generally Oseroff, Photodynamic Therapy, Clinical Photomedicine, 387-402 (Marcel Dekker, Inc.) (1993)), early stage cancers of the lung, esophagus, stomach, cervix and cervical dysplasia, etc. See T. J. Dougherty, Photodynamic therapy: part II, Semin. Surg. Oncol, tl, 333-334 (1995). Moreover, numerous investigations demonstrate possible practical usefulness of photodynamic therapy in diverse disease conditions including dermatological diseases, atherosclerosis, infectious diseases, theumatoid arthritis, age-related macular degeneration, restenosis, AIDS, hematological diseases, etc. In Assignee's copending U.S. Patent Application No. 60/578,798 (filed on June 10,

2004), the entirety of which is incorporated herein by reference, the Assignee disclosed the use of novel peptidoconjugates which could produce therapeutic results via photodynamic therapy and methods of providing such therapy. However, a great improvement in the field of cellular imaging could be attained if the peptidoconjugates could be delivered to a desired cellular component (i.e., achieve a desired localization) and refrain from performing phototherapy, i.e. be available for cellular imaging without resulting in cell death. Further, a need exists for such a method to deliver peptidoconjugates to human cells for cellular imaging.

SUMMARY The present invention comprises a method of using a peptidoconjugate for cellular imaging. The method comprises providing a peptidoconjugate having an imaging agent and a peptide wherein a specific combination of a particular imaging agent and a particular peptide

allows for a desired localization within a human cell. Next, the method comprises delivering the peptidoconjugate to the human cell and allowing the peptidoconjugate to bind to a biomolecule within the cell. Further, the method comprises illuminating the human cell with a light energy wherein the light energy causes the imaging agent to deliver a measurable signal and further, observing the measurable signal. In one embodiment, the signal is a fluorescence.

In one aspect, provided herein are methods of cellular imaging, comprising providing a peptidoconjugate comprising an imaging agent and a peptide wherein peptidoconjugate specifically localizes within a cell, and delivering the peptidoconjugate to the cell. In one embodiment, the method further comprises allowing the peptidoconjugate to bind to a bio-molecule within the cell.

In one embodiment, the method further comprises detecting the peptidoconjugate to thereby determine its localization within the cell.

In one embodiment, the method further comprises obtaining the peptidoconjugate. In one embodiment, the method further comprises providing a therapeutic composition

In one aspect, provided herein are methods of using a peptidoconjugate for cellular imaging, comprising providing a peptidoconjugate having an imaging agent and a peptide wherein a specific combination of the imaging agent and the peptide allows for a desired localization within a cell; delivering the peptidoconjugate to the cell; allowing the peptidoconjugate to bind to a biomolecule within the cell; illuminating the cell with a light energy wherein the light energy causes the imaging agent to deliver a measurable signal; and observing the measurable signal.

In one embodiment, the cell is a HeLa cell. In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -F(d-R)FK.

In one embodiment, the peptidoconjugate specifically localizes to one or more mitochondria.

In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -F(d-R)FK.

In one embodiment, the peptidoconjugate specifically localizes to one or more mitochondria.

In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -F(d-R)FK.

In one embodiment, the peptidoconjugate specifically localizes to a nucleus. In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -d-(GRKKRRQRRR)(tat).

In one embodiment, the peptidoconjugate specifically localizes to one or more of a nucleus or a mitochondria.

In one aspect, provided herein are methods of using a peptidoconjugate for cellular imaging, comprising providing a peptidoconjugate having an imaging agent and a peptide wherein a specific combination of the imaging agent and the peptide allows for a desired localization within a human cell; delivering the peptidoconjugate to the human cell; allowing the peptidoconjugate to bind to a biomolecule within the cell; and observing a measurable signal. In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -F(d-R)FK.

In one embodiment, the peptidoconjugate specifically localizes to one or more mitochondria.

In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -F(d-R)FK.

In one embodiment, the peptidoconjugate specifically localizes to one or more mitochondria.

In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -F(d-R)FK.

In one embodiment, the peptidoconjugate specifically localizes a nucleus. In one embodiment, the imaging agent comprises,

and the peptide represented by X comprises -d-(GRE-KRRQRRR)(tat).

In one embodiment, the peptidoconjugate specifically localizes to one or more of a nucleus or a mitochondria.

In one aspect, provided herein are methods of using a peptidoconjugate for cellular imaging, comprising providing a plurality of peptidoconjugates wherein each peptidoconjugate includes an imaging agent and a peptide wherein a specific combination of the imaging agent and the peptide allows for a desired localization within a human cell; delivering each of the peptidoconjugates to the human cell; and allowing the peptidoconjugates to bind to a DNA molecule within the human cell.

In one embodiment, the method further comprises detecting the plurality of peptidoconjugates in the cell.

In one embodiment, the detecting comprises illuminating the human cell with a light energy wherein the light energy causes each imaging agent to deliver a fluorescence. 28. In one embodiment, the method further comprises observing the fluorescence of each imaging agent.

In one aspect, provided herein are imaging agents as described infa. Further aspects and embodiments are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present invention.

FIG. 1 shows several embodiments of dyes of the present invention. FIG. 2A and FIG. 2B show a desired localization of the present invention wherein the indicated localization is obtained by conjugating the indicated dye to the indicated peptide.

FIG. 3 A and FIG. 3B show an example of varying a desired localization by changing the dye of a peptidoconjugate.

FIG. 4A shows various embodiments of peptidoconjugates of the present invention wherein the dye is thiazole orange. FIG. 4B shows a photocleavage of pUC18 plasmid DNA by various peptidoconjugates;

FIG. 5A-FIG. 5C shows various confocal microscopy images of unfixed live HeLa cells.

FIG. 6A-FIG. 6F show localization profiles of several embodiments of peptidoconjugates of the present invention.

FIG. 7 shows a chart illustrating the phototoxicity of various peptidoconjugates.

While the above-identified drawings set forth preferred embodiments of the present invention, other embodiments of the present invention are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments of the present invention by way

of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the present invention.

DETAILED DESCRIPTION

Described herein are methods of cellular imaging. More specifically, described herein are uses of a peptidoconjugate having an imaging agent and a peptide wherein a specific combination of a particular imaging agent with a particular peptide allows for a desired localization within a human cell. The present invention comprises a method of using a peptidoconjugate for cellular imaging. The method comprises providing a peptidoconjugate having an imaging agent and a peptide wherein a combination of a particular imaging agent and a particular peptide allows for a desired localization within a human cell. As such, the method comprises delivering the peptidoconjugate to the human cell and allowing the peptidoconjugate to bind to a biomolecule within the cell. Further, the method comprises irradiating the human cell with a light energy wherein the light energy causes the imaging agent to deliver a measurable signal and allows for observing and measuring the measurable signal. In one embodiment, the measurable signal is a fluorescence.

As used herein, a "peptide" refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. It also refers to either a full- length naturally-occurring amino acid sequence or a fragment thereof between about 2 and about 500 amino acids in length. Preferably, the peptide comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 , about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94,

about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500 amino acids. Additionally, unnatural amino acids, for example, β-alanine, phenyl glycine and homoarginine may be included. Commonly-encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L- optical isomer.

In addition to using D-amino acids, those of ordinary skill in the art are aware that modifications in the amino acid sequence of a peptide, polypeptide, or protein can result in equivalent, or possibly improved, second generation peptides that display equivalent or superior functional characteristics when compared to the original amino acid sequence. The present invention accordingly encompasses such modified amino acid sequences. Alterations can include amino acid insertions, deletions, substitutions, truncations, fusions, inversions, shuffling of subunit sequences, and the like, provided that the peptide sequences produced by such modifications have substantially the same functional properties as the naturally occurring counterpart sequences disclosed herein. Thus, for example, modified cell membrane-permeant peptides should possess substantially the same transmembrane translocation and internalization properties as the naturally occurring counterpart sequence. One factor that can be considered in making such changes is the hydropathic index of amino acids. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein has been discussed by Kyte and Doolittle (J. MoI. Biol., 157: 105-132, 1982). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resultant protein. This, in turn, affects the interaction of the protein with molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. As is known in the art, certain amino acids in a peptide or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide or protein having similar biological activity, i.e., which still retains biological functionality. In making such changes, it is preferable that amino acids having hydropathic indices within +/-2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within +/-1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within +/- 0.5.

Like amino acids can also be substituted on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 discloses that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0.+-.1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+/-1); alanine/histidine (-0.5); cysteine (- 1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Thus, one amino acid in a peptide, polypeptide, or protein can be substituted by another amino acid having a similar hydrophilicity score and still produce a resultant protein having similar biological activity, e.g., still retaining correct biological function. In making such changes, amino acids having hydropathic indices within +/-2 are preferably substituted for one another, those within +/-1 are more preferred, and those within +/-0.5 are most preferred.

As outlined above, amino acid substitutions in the peptides of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present peptides, etc., can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral non-polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. It should be noted that changes which are not expected to be advantageous can also be useful if these result in the production of functional sequences. Additionally, substitutions may be made based on sequence specific effects and the charge of particular amino acids. For example, it is of particular usefulness in the present invention to increase the cationic charge of the permeation peptide used in the conjugate to enhance cellular uptake. In addition, other substitutions may be made to increase or to

enhance cellular localization of the peptidoconjugate. One method of accomplishing this is the substitution of one or more positively charged amino acids for one or more negatively charged acids in the permeant peptide. For example, substitution of the positively charged amino acid Orn for the naturally occurring negatively charged amino acid at C-4 in the Tat basic peptide sequence increases the cellular uptake of a conjugate comprising such peptide. On the other hand, substituting at the same position with the negatively charged GIu, decreased cellular uptake.

Specifically localizes within a cell, as used herein includes one or having one or more specific localization, for example, a peptiodconjugate may specifically localize to both the mitochondria and the nucleus and be specific.

Binding to a bio-molecule within the cell may be direct or indirect binding.

Obtaining the peptidoconjugate refers to, for example, the purchase or manufacture of a peptidoconjugate.

Providing a therapeutic composition includes, upon correlation of the peptidoconjugate with a specific localization, a heath care provider may use the information for diagnosis and treatment.

As used herein, a "peptoid" refers to a polyamide of between 2 and 500 units having one or more substituent on the amide nitrogen atom. A peptoid is a synthetic analog of a peptide with the difference being that while a side-chain residue on a peptide is attached to a carbon atom α- to the carbonyl group, in a peptoid, the "side-chain residue" is attached to the amide nitrogen atom. Peptoids are synthetic polymers with controlled sequences and lengths, that can be made by automated solid-phase organic synthesis to include a wide variety of side-chains having different chemical functions. Preferably the peptoid comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83,

about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500

"side-chain residues." Peptoids have a number of notable structural features in comparison to peptides. For example, whereas the side-chain ("R") groups on biosynthetically produced peptides must be chosen from among the 20 naturally-occurring amino acids, peptoids can include a wide variety of different, non-natural side-chain residues because in peptoid synthesis the R group can be introduced as a part of an amine or by alkylation of the amine or the amide nitrogen. This is in contrast to synthetic peptides for which the incorporation of non-natural side-chain residues requires the use of non-natural α- protected amino acids. Peptoids can be synthesized in a sequence- specific fashion using an automated solid-phase protocol, e.g., the sub-monomer synthetic route. See, for example, Wallace et al., Adv. Amino Acid Mimetics Peptidomimetics, 1999, 2, 1-51 and references cited therein, all of which are incorporated herein in their entirety by this reference. Generally, when attached to a binding polymer, longer peptoids provide a higher ratio of charge/translational frictional drag (i.e., α value), than shorter peptoids. The synthesis of long peptoids can be achieved using the sub- monomer protocol. As used herein, a "subject" refers to any human or non-human organism.

As used herein, "imaging agent," includes, for example, agents that may be detected in vitro or in vivo, such as, for example, a fluorescent group, a phosphorescent group, a nucleic acid indicator, an ESR probe, a dye, a pH sensitive dye, a beacon probe, radioactive groups, or metals. The invention provides, for example, dyes that have one or more aromatic rings and one or more nonplanar substituents that project out of the plane of the aromatic ring. Certain dyes produce a signal when exposed to a change in environment, for example, a hydrophobicity, hydrogen bonding, polarity, or conformational change. Thus, a signal from certain dyes of the invention detectably changes upon exposure to a change in solvent, change in hydrogen bonding, change in the hydrophobicity of the environment, changed polarity or polarization, or change affecting the conformation of the dye. In one embodiment, the signal provided by the environmentally sensitive dye increases when the dye is exposed to an environment that is more hydrophobic. In another embodiment, the signal provided by the

environmentally sensitive dye increases when the dye is exposed to an environment where there is increased hydrogen binding between the dye and a component of the environment. Such an increase in hydrophobicity or an increase in hydrogen bonding can occur when a peptidoconjugate of the invention binds to a target protein or subcellular component. In other embodiments, the signal provided by the environmentally sensitive dye decreases when the dye is exposed to an environment that is more hydrophilic. In further embodiments, the signal provided by the dye decreases when the dye is exposed to an environment that has less hydrogen binding. Such an increase in hydrophiliciry or a decrease in hydrogen binding can occur when a peptidoconjugate of the invention is exposed to an aqueous environment or when such a peptidoconjugate becomes unbound from a target protein or subcellular component.

Aromatic rings useful as dyes include aromatic hydrocarbon radicals of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aromatic rings include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. Fused ring systems are also contemplated, including fused rings with heteroatoms such as nitrogen, sulfur or oxygen. Examples of aromatic rings that can be used include indole, indoline, benzothiophene, dihydrobenzothiophene and the like.

Dyes have many properties that make them particularly suitable for detection of targets and interactions in living cells. The dyes are, for example, bright, with long wavelengths outside of cellular autofluorescence background frequencies and that are less damaging to cells. Addition or deletion of parts of the aromatic system can shift excitation and/or emission wavelengths of the dyes so that more than one event can be monitored in a cell at the same time. The dyes of the invention can be designed to have enhanced water solubility, e.g., by attaching groups that sterically block aggregation without unduly increasing hydrophobicity. Dyes, for example, can be detected in cells by observing changes in intensity, a change in the shape or maxima of the excitation or emission peak, and/or dye lifetime, to permit ratio imaging and other techniques that can eliminate effects of uneven illumination, cell thickness etc. Linker Regions

Linker regions useful in linking the Tat or other peptides described herein and cargos such as drugs or diagnostic substances such as metal chelator moieties can comprise amino

acid residues or substituted or unsubstituted hydrocarbon chains. Useful linker regions include natural and unnatural biopolymers. Examples of natural linkers include oligonucleotides and L-oligopeptides, while examples of unnatural linkers are D- oligopeptides, lipid oligomers, liposaccharide oligomers, peptide nucleic acid oligomers, polylactate, polyethylene glycol, cyclodextrin, polymethacrylate, gelatin, and oligourea (Schilsky, et al., Eds., Principles of Antineoplastic Drug Development and Pharmacology, Marcel Dekker, Inc., New York, 1996, pp. 741). The linker region can be designed to be functional or non-functional.

"Non-functional" as applied to linker regions means any non-reactive amino acid sequence, hydrocarbon chain, etc., that can bond covalently to Tat or other peptide residues on one end and a drug or chelating ligand, for example, on the other end. As used herein, the term "non-reactive" refers to a linker that is biologically inert and biologically stable when a complex containing the linker is contacted by cells or tissues. Upon characterization, the linker and conjugate can be shown to remain intact as the parent compound when analyzed by reverse phase HPLC or TLC. Non-functional linkers are desirable in the design and synthesis of complexes useful, for example, in non-specific labeling of white blood cells for imaging infections, in non-specific labeling of tissues for perfusion imaging, and in interaction with any intracellular receptor or other activity or site. Examples of nonfunctional linkers include, but are not limited to, amino hexanoic acid, glycine, alanine, or short peptide chains of nonpolar amino acids such as di- or tri-glycine or tri-alanine.

Hydrocarbon chain linkers can include both unsubstituted and substituted alkyl, aryl, or macrocyclic R groups, as disclosed in U.S. Pat. No. 5,403,574. R groups are found in the general formula -CR ₃ where R can be identical or different and includes the elements H, C, N, O, S, F, Cl, Br, and I. Representative examples include, but are not limited to, -CH ₃ , - CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C(CH ₃ ) ₃ , -C(CH ₃ ) ₂ , -OCH ₃ , -C(CH ₃ ) ₂ , -COOCH ₃ , -C(CH ₃ ) ₂ OCOCH ₃ , CONH ₂ , -C ₆ H ₅ , -CH ₂ (C ₆ H ₄ )OH, or any of their isomeric forms. "Alkyl" is intended to mean any straight, branched, saturated, unsaturated or cyclic Ci _-20 alkyl group. Typical Ci-C ₂₀ alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i- butyl, pentyl and hexyl groups. "Aryl" is intended to mean any aromatic cyclic hydrocarbon based on a six-membered ring. Typical aryl groups include, but are not limited to, phenyl, naphthyl, benzyl, phenethyl, phenanthryl, and anthracyl groups. The term "macrocycle" refers to R groups containing at least one ring containing more than seven carbon atoms. "Substituted" is intended to mean any alkyl, aryl or macrocyclic groups in which at least one

carbon atom is covalently bonded to any functional groups comprising the atoms H, C, N, O, S, F, Cl, Br or I.

"Functional" as applied to linker regions means, for example, amino acid residues, oligonucleotides, oligosaccharides, peptide nucleic acids, or substituted or unsubstituted hydrocarbon chains as discussed above that confer biological or physicochemical properties useful for the practice of this invention when incorporated into the linker component. Such properties include, for example, utility in medical imaging, radiotherapy, diagnosis, and pharmacological treatment of disease states by virtue of interaction of the functional linker region with intracellular components, which can be unique to, or highly characteristic of, cells in particular physiological or disease states. Such interaction can include, for example, binding or other reaction, for example cleavage, of the functional linker region due to interaction with intracellular components. However this interaction occurs, such interaction results in selective retention of the cargo molecule within particular cells due to the presence of a particular intracellular component(s) within such cells. The interaction of the functional linker with the intracellular component thereby confers target cell specificity to a peptide complex containing a particular functional linker moiety. Examples of functional linkers are peptide or protein binding motifs, protein kinase consensus sequences, protein phosphatase consensus sequences, or protease-reactive or protease-specific sequences. Additional examples include recognition motifs of exo- and endo-peptidases, extracellular metalloproteases, lysosomal proteases such as the cathepsins (cathepsin B), HIV proteases, as well as secretases, transferases, hydrolases, isomerases, ligases, oxidoreductases, esterases, glycosidases, phospholipases, endonucleases, ribonucleases and beta-lactamases.

Administration of Peptidoconjugates

Peptidoconjugates of the invention can be used in vitro and/or in vivo to detect target molecules of interest. In many cases, the peptidoconjugates can simply be added to test samples in a homogenous assay, not requiring addition of multiple reagents and/or wash steps before detection of the target.

Peptidoconjugates of the invention can typically contact target molecules in vitro by simple addition to a test sample containing the target molecules. Test samples for in vitro assays can be, e.g., molecular libraries, cell lysates, analyte eluates from chromatographic columns, and the like. The in vitro assay often takes place in a chamber, such as, e.g., a well of a multiwell plate, a test tube, an Eppendorf tube, a spectrophotometer cell, conduit of an

analytical system, channels of a microfluidic system, an open array, and the like. In an exemplary in vitro assay of the invention, an enzyme protein of interest is coated to the bottom of 96-well dishes also containing solutions representing a library of possible enzyme substrates. A peptidoconjugate of the invention with specific affinity for enzyme-substrate complex is added to each well. A multiwell scanning fluorometer is used to observe each well for fluorescence. Wells containing enzyme substrate can be identified as those in which fluorescent emissions at the wavelength of the peptidoconjugate dye. That is, in this example, the binding domain of the peptidoconjugate only binds to enzyme acting on substrate; the binding placing the dye into a binding pocket environment that significantly changes the emissions intensity of the dye.

Where peptidoconjugates of the invention are administered to living cells, binding can take place with targets on the cell surface, or the peptidoconjugate is transferred into the cell to make contact with an intracellular target molecule. In some cases, the peptidoconjugate can penetrate a cell suspected of containing a selected target passively by mere exposure of the cell to a medium containing the peptidoconjugate. In other embodiments, the peptidoconjugate is actively transferred into the cell by mechanisms known in the art, such as, e.g., poration, injection, transduction along with transfer peptides, and the like.

In some embodiments, one of skill in the art may choose to incorporate a translocation functionality on the peptidoconjugate in order to facilitate the translocation or internalization of that peptidoconjugate from the outside to inside the cell. As used herein, the term

"translocation functionality" refers to a chemical compound, group or moiety that increases the cell's ability to internalize another compound or material, for example, a peptidoconjugate. Examples of such translocation functionalities include peptide recognition/transport sequences, liposomal compositions, or the like. Alternative translocation methods and compositions are also utilized in accordance with the present invention to induce uptake of the second component, including, e.g., electroporation, cell permeating compositions containing, e.g. PEG, porins, saponins, streptolysin or the like.

Techniques useful for promoting uptake of peptidoconjugates include optoporation, for example, as described in Schneckenburger, H., Hendinger, A., Sailer, R., Strauss, W. S. & Schmitt, M. Laser-assisted optoporation of single cells. J Biomed Opt 7, 410-6 (2002); or Soughayer, J. S. et al., Characterization of Cellular Optoporation with Distance. Anal Chem 72, 1342-7 (2000). A variety of transduction peptides are also useful for promoting uptake of peptidoconjugates including those described in Zelphati, O. et al., Intracellular Delivery of

Proteins with a New Lipid-mediated delivery System. J Biol Chem 276, 35103-10 (2001); Yang, Y., Ma, J., Song, Z. & Wu, M., HTV-I TAT-Mediated Protein Transduction and Subcellular Localization Using Novel Expression Vectors. FEBS Lett 532, 36-44 (2002); and Torchilin, V. P. et al., Cell Transfection in Vitro and In Vivo with Nontoxic TAT Peptide- liposome-DNA Complexes. Proc Natl Acad Sci USA 100, 1972-7 (2003).

Additional techniques such as electroporation can also be used. Examples of electroporation procedures are provided in Glogauer, M. & McCulloch, C. A., Introduction of Large Molecules into Viable Fibroblasts by Electroporation: Optimization of Loading and Identification of Labeled Cellular Compartments. Exp Cell Res 200, 227-34 (1992); Teruel, M. N. & Meyer, T., Parallel Single-cell Monitoring of Receptor-triggered Membrane

Translocation of a Calcium-sensing Protein Module. Science 295, 1910-2 (2002); and Teruel, M. N., Blanpied, T. A., Shen, K., Augustine, G. J. & Meyer, T., A Versatile Microporation Technique for the Transfection of Cultured CNS Neurons. J Neurosci Methods 93, 37-48 (1999). Another procedure for introducing molecules such as peptidoconjugates into cells is the osmotic shock procedure. Examples of osmotic shock procedures include those described in Okada, C. Y. & Rechsteiner, M., Introduction of Macromolecules into Cultured Mammalian Cells by Osmotic Lysis of Pinocytic Vesicles. Cell 29, 33-41 (1982); and Park, R. D., Sullivan, P. C. & Storrie, B., Hypertonic Sucrose Inhibition of Endocytic Transport Suggests Multiple Early Endocytic Compartments. J Cell Physiol 135, 443-50 (1988).

One of skill in the art may also employ bead/syringe loading to introduce the peptidoconjugates of the invention into cells. Bead/syringe loading procedures are described in McNeil, P. L., Murphy, R. F., Lanni, F. & Taylor, D. L., A Method for Incorporating Macromolecules into Adherent Cells, J. Cell Biol. 98, 1556-1564 (1984); and McNeil, P. L. & Warder, E., Glass Beads Load Macromolecules into Living Cells. Journal of Cell Science 88, 669-678 (1987).

Nucleic acids encoding binding domains of the invention can optionally be introduced into cells in expression plasmids, e.g., by transduction or other forms of transformation. Once inside the living cells, the binding domain can be translated from the nucleic acid to a functional peptide. Peptidoconjugates of the invention can enter the cell, e.g., by injection of diffusion to become linked to the expressed binding domain to generate a peptidoconjugate in situ. According to one aspect the invention reporter moieties are polypeptides that act as

signaling entities. The reporter moieties may be any polypeptides that show fluorescence at known wavelengths upon exposure to external light. Examples of reporter moieties include cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP), which are mutants of the green fluorescent protein (GFP). Detection of Target-Peptidoconjugate Binding Reactions

A wide variety of binding reactions can be detected and monitored using the present peptidoconjugates, for example, protein-protein interactions, receptor-ligand interactions, nucleic acid interactions, protein-nucleic acid interactions, and the like. Detection of a target molecule can provide identification of the target in a specified state, quantification of the target, and/or localization of the target. Multiple measurements can allow determination of kinetics. The ability to monitor multiple targets can permit monitoring of the balance between different signaling activities. In the intracellular environment, many of these reaction types are involved in the multiplicity of steps of signal transduction within cells. For example, activation of a particular cellular event is often triggered by the interaction between a cell surface receptor and its ligand. The signal from the receptor is often transmitted along via the binding of enzymes to other proteins, for example, kinases, which then pass the signal on through the cell until the ultimate cell system response is achieved. In many cases, the signal or ultimate response can be detected using peptidoconjugates of the invention. For example, signal transduction often involves phosphorylation of system molecules that can be detected directly with the phosphate involved in the binding site, or indirectly through conformational changes induced by the phosphorylation.

In one embodiment, the invention provides methods for identifying the activation status of endogenous proteins in living cells. Peptidoconjugates of the invention can permit identification, quantification, and resolution of the spatial, temporal and compartmental regulation of receptor phosphorylation and activation during various processes, for example, endocytosis. In another embodiment, the peptidoconjugates and methods of the invention can permit observation of epidermal growth factor receptor (EGFR) effects on the development and progression of breast cancer. In a further embodiment, complex formation between HIV gpl20 and CD4 cell receptors can be monitored. In accordance with the present invention, binding interactions can occur between a peptidoconjugate and one or more target molecules or components of the cell. A "target molecule of interest" is a molecule that is known by one of skill in the art and is selected for

interaction with a peptidoconjugate of the invention. A target molecule often comprises an endogenous unlabeled and/or untagged component of a test solution or cell. Endogenous components can be, e.g., expressed by the cell naturally, or present as a result of introduction of an appropriate genetic construct within the cell. For example, nucleic acid or protein target molecules can be expressed in the cell, either naturally (e.g., constitutively) or by induction of an appropriate genetic construct introduced into the cell line.

As used herein, the term "sample" as used in its broadest sense, refers to any plant, animal or viral material containing DNA or RNA, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. The sample containing nucleic acids can be drawn from any source and can be natural or synthetic. The sample containing nucleic acids may contain deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or copolymers of deoxyribonucleic acids and ribonucleic acids or combinations thereof. Alternatively, the sample may have been subject to purification (e.g. extraction) or other treatment. The term "sample" can also refer to "a biological sample."

As used herein, the term "a biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "A biological sample" further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, and organs. Most often, the sample has been removed from an animal, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, i.e., without removal from animal. Typically, a "biological sample" will contain cells from the animal, but the term can also refer to non- cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure the cancer-associated polynucleotide or polypeptides levels. "A biological sample" further refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.

As used herein, a "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.

As used herein, a "therapeutic composition" refers to a composition that upon delivery into a cell or a subject, acts upon the cell or subject to correct or compensate for an underlying molecular deficit, or counteract a disease state or syndrome of the cell.

As used herein, the singular forms "a," "an," and "the" used in the specification and claims include both singular and plural referents unless the content clearly dictates otherwise.

The present invention is a method comprising providing a peptidoconjugate wherein the peptidoconjugate has an imaging agent conjugated to a peptide. In one embodiment, a specific combination of a particular imaging agent with a particular peptide allows for a desired localization within a cell. In one embodiment, the method comprises allowing the peptidoconjugate to bind to a biomolecule within the cell. In one embodiment, the biomolecule is a DNA molecule. In one embodiment, the biomolecule is an RNA molecule. Those skilled in the art will recognize that various biomolecules are within the spirit and scope of the present invention.

In one embodiment, the method comprises irradiating the cell with an energy source. In one embodiment, the energy source is a light source. In one embodiment, the imaging agent of the peptidoconjugate is activated by the energy source. In one embodiment, the imaging agent of the peptidoconjugate gives off a measurable signal when it is illuminated by the energy source. In one embodiment of the invention, the imaging agent gives off a measurable fluorescence in response to the energy source.

In one embodiment of the invention, the fluorescence given off by the imaging agent in response to the light source may be observed and measured. In one embodiment of the invention, the fluorescence is observed and measured with a confocal microscope. Those skilled in the art will recognize that various devices used to observe and measure fluorescence are within the spirit and scope of the present invention.

Imaging Agents

As stated, the method of the present invention comprises providing a peptidoconjugate wherein a particular imaging agent is conjugated to a particular peptide. The following are a summary of the preferred imaging agents of the present invention. FIG. 1 shows the structure of four preferred dyes. FIG. 1 also shows an absorbance of each dye at

various wavelengths. Those skilled in the art will recognize that similar dyes and derivatives of those dyes shown in FIG. 1 and described below are included within the spirit and scope of the present invention.

In one embodiment, the dye of the peptidoconjugate is Victoria Blue ("BO")- The structure of BO may be represented as follows:

In the above-identified structure, "X" represents a peptide conjugated to the dye.

In one embodiment, the dye of the peptidoconjugate is thiazole orange ("TO"). The structure of TO may be represented as follows:

In the above-identified structure, "X" represents a peptide conjugated to the dye.

In one embodiment, the dye of the peptidoconjugate is Victoria Blue-3 (BO-3). The structure of BO-3 may be represented as follows:

In the above-identified structure, "X" represents a peptide conjugated to the dye.

In one embodiment, the dye of the peptidoconjugate is thiazole orange-3 ("TO-3"). The structure of TO-3 may be represented as:

In the above-identified structure, "X" represents a peptide conjugated to the dye. Peptide and Peptoid

In one embodiment of the invention, the dye of the peptidoconjugate is conjugated to either a peptide or a peptoid to form a peptidoconjugate. In general, the peptide or peptoid is covalently conjugated to a dye via the amide group on the amino acid backbone. Generally, the peptide or peptoid comprises between about 1 and about 500 amino acid units in length, and can be the natural L-enantiomer, or the unnatural D-enantiomer, or a D- and L- enantiomer mixture. In one embodiment, the peptide or peptoid length is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,

about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500 amino acid units in length. Preferably, the length is between about 1 and about 25 amino acid units. Synthesis of the Peptidoconjugate

Another aspect of the present invention provides methods of synthesizing the peptidoconjugate compounds of the present invention. In general, the peptidoconjugate compounds can be formed by a general process of synthesizing the dye part and the peptide or peptoid parts, followed by conjugating the dye parts to the peptide or peptoid parts. Different approaches including those well known in the art, can be used to synthesize the dye parts. Examples of synthesizing a dye include but are not limited to teachings and disclosures in Neckers, D. C; Paczkowski, J. Tetrahedron, 42, 4671 (1986), Svanvik, N.; Westman, G.; Wang, D.; Kubista, M. Anal. Biochem., 281, 26 (2000), and Benson, S. C; Singh, P.; Glazer, A. Nucl. Acids Res., 21, 5727 (1993), the disclosures are herein incorporated by reference.

Peptides can be synthesized by different methods well known in the art. For example, including ribosomally-directed fermentation methods, as well as non- ribosomal strategies and chemical synthesis methods. Peptides containing the 20 natural amino acids and those greater than about 30 residues can be prepared via recombinant expression systems that utilize the ribosomally directed peptide synthesis machinery of a host organism, e.g., E. coli. Smaller peptides (less than 30 residues) and peptides which contain unnatural or non- proteninogenic amino acids or modified amino acid side chains often prepared through a

more general solution-phase chemical synthesis of peptides (e.g., using N-Boc protection and the activated ester route). Protocols for sequence solution-phase chemical synthesis of peptides have been described in Andersson et al., Biopolymers 55:227-250 (2000). One current method used for generating peptides is solution-phase chemical synthesis, which employs a N-tert-butoxy (N-Boc) protected amino acid and a C-protected amino acid

(Andersson et al., Biopolymers 55: 227-250 (2000)). An alternative solution-phase method for chemically-catalyzed peptide synthesis employs pre-activated esters as the carboxyl component for coupling (Andersson et al., Biopolymers 55: 227-250 (2000)). In addition, en2yme-mediated solid-phase peptide synthesis has also been employed. Solid-phase peptide synthesis (SPPS) uses insoluble resin supports, and has simplified and accelerated peptide synthesis and facilitated purification (Merrifield, R.B., J. Am. Chem. Soc. 85: 2149-2154 (1963)). Since the growing peptide is anchored on an insoluble resin, unreacted soluble reagents can be removed by simple filtration or washing without manipulative losses. Solid phase peptide synthesis can be performed using automation. Those skilled in the art will recognize that various peptides are within the spirit and scope of the present invention.

A peptoid can be synthesized by a similar method to the synthesis of a peptide described above. For example, synthesis of a peptoid can be carried out by methods described in Murphy, J. E.; Uno, T.; Hamer, J. D.; Dwarki, V. Zuckermann, R. N., Proc. Natl. Acad. ScL USA, 95, 1517 (1998), Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.; Proc. Natl. Acad. Sd. USA, 89, 9367 (1992), and Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am. Chem. Soc, 114, 10646 (1992), all are incorporated herein by reference. Those skilled in the art will recognize that various methods of preparing peptoids are within the spirit and scope of the present invention. Peptidoconjugates can be synthesized with a method known in the art. Typically, the dye part bearing an electrophilic moiety reacts with a nucleophilic group, i.e., amino terminus on a peptide or peptoid. For example the method can use commercially available Rink amide resin on a solid support for coupling the dye to the peptide or peptoid. Yield from coupling reactions can be assessed by spectroscopy. For example, couplings can be performed using 4 equivalents of Fmoc protected amino acid, 4 equivalents of HBTU and 8 equivalents of Hϋnig's base in DMF for 3 hours. Deprotection of the Fmoc group can be achieved using 20% piperidine in DMF for 30 minutes (to minimize diketopiperazine formation, dipeptides were deprotected using 50% piperidine in DMF for 5 min). The dye moiety is attached to a

resin-bound peptide as described below. The dye-peptide conjugates are simultaneously deprotected and cleaved from the resin with a 95:5 TFA : TIPS solution. The solution is then concentrated in vacuo and purified via RP-HPLC (H ₂ O/CH ₃ CN in 0.1% TFA). The resulting products can be isolated by lyophilization and characterized by MALDI-TOF mass spectrometry. Those skilled in the art will recognize that various methods of preparing a peptidoconjugate are within the spirit and scope of the present invention.

Localization

In one embodiment, the method comprising administering the peptidoconjugate to a cell and achieving a desired localization. In one embodiment of the invention, the method comprising administering the peptidoconjugate to a human cell and achieving a desired localization. In one embodiment, the method comprising administering the peptidoconjugate to a HeLa cell and achieving a desired localization. In one embodiment, a plurality of peptidoconjugates are delivered to a cell and each peptidoconjugate achieves a desired localization. A desired localization refers to a peptidoconjugate being specifically sequestered in a desired cellular component. Those skilled in the art will recognize that any number of peptidoconjugates of the present invention may be delivered to a cell and the method remains within the spirit and scope of the present invention. In addition, those skilled in the art will recognize that cellular imaging various types of cells from various types of sources are within the spirit and scope of the present invention. In one embodiment of the invention, the desired localization is achieved by conjugating a specific dye to a specific peptide. FIG. 2A and FIG. 2B provide an overview of the specific localization of particular peptidoconjugates of the present invention.

In one embodiment, a BO dye is conjugated to the following peptide: -F(d-R)FK (SEQ. ID 1). As such, In one embodiment, the peptidoconjugate is: BO-F(d-R)FK. In one embodiment, the peptidoconjugate of BO-F(d-R)FK allows for mitochondrial localization. As such, the desired localization of this embodiment is mitochondrial localization.

In one embodiment, a TO dye is conjugated to the following peptide: -F(d-R)FK. As such, In one embodiment, the peptidoconjugate is: TO-F(d-R)FK. In one embodiment, the peptidoconjugate of TO-F(d-R)FK allows for mitochondrial localization. As such, the desired localization of this embodiment is mitochondrial localization.

In one embodiment, a TO-3 dye is conjugated to the following peptide: -F(d-R)FK. As such, In one embodiment, the peptidoconjugate is: TO-3-F(d-R)FK. In one embodiment,

the peptidoconjugate of TO-3-F(d-R)FK allows for nuclear localization. As such, the desired localization of this embodiment is nuclear localization.

In one embodiment, a TO dye is conjugated to the following peptide: -d- (GRKKRRQRRR)(tat) (SEQ. ID 2). As such, In one embodiment, the peptidoconjugate is: TO-d-(GRKKRRQRRR)(tat). In one embodiment, the peptidoconjugate of TO-d- (GRKKRRQRRR)(tat) allows for either nuclear or mitochondrial localization. As such, the desired localization of this embodiment is nuclear localization or mitochondrial localization.

Those skilled in the art will recognize that various derivatives of the dye and/or the peptides of the above embodiments are within the spirit and scope of the present invention.

FIG. 3 A and FIG. 3B further illustrate the dependence of localization on the identify of the dye in the peptidoconjugate. In one embodiment shown in FIG. 3B, the dye of the peptidoconjugate is Rose Bengal ("RB") and the peptide is TAT (structure shown in FIG. 3A). The peptidoconjugate may be represented as:

In one embodiment of FIG. 3B, the dye of the peptidoconjugate is fluorescein and the peptide is TAT. The peptidoconjugate may be represented as:

Next, FIG 3B shows the dye of the peptidoconjugate is TO and once again the peptide is TAT, represented as:

The cells shown in FIG. 3B shows the differences in localization created by changing the dye of the peptidoconjugate. More specifically, FIG. 3B shows that different dyes conjugated to the same peptide can alter the localization of the peptidoconjugate. As shown in the cell images of FIG. 3B, a TOTat peptidoconjugate is localized in the nucleus of the cell. Next, a RB-Tat peptidoconjugate is excluded from the nucleus of the cell. Finally, a FL-Tat peptidoconjugate shows no specific localization.

Pharmaceutical Composition and Administration

Another aspect of the present invention provides a pharmaceutical composition that comprises a peptidoconjugate compound described herein and a pharmaceutically acceptable carrier. Either solid or liquid pharmaceutically acceptable carriers can be employed. Solid carriers include but are not limited to, starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Liquid carriers include but are not limited to, syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like. Similarly, the carrier or diluent may include any prolonged release material. When liquid carriers are used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension. A summary of such pharmaceutical compositions may be found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton Pa. (Gennaro 18th ed. 1990).

The pharmaceutical composition can be used in its solid form or dissolved in an appropriate solvent for addition to the carrier (solid or liquefied) or dissolved in an appropriate solvent. Preferred mixtures should be in appropriate solvents for dissolving both medicament and carrier, and at the desired degree of medicament purity. It is preferred that upon hydration, at the appropriate pH for the pharmaceutical composition, the peptidoconjugate and the carrier form a complex which facilitates delivery of the

peptidoconjugate to a target. Other additives and pharmaceutical excipients can also be added, during or after formulation, to improve the ease of formulation, formulation stability, speed of reconstitution and delivery of the formulation. These include, but are not limited to, penetration enhancers, targeting aids, anti-oxidants, preservatives, buffers, stabilizers and solid support materials. The composition may include osmoregulators if required, such as but not limited to, physiologically buffered saline (PBS), carbohydrate solution such as lactose, trehalose, higher polysaccharides, or other injectable material. A wide variety of excipients and stabilizers are known in the art and their use will depend on formulation type and application requirements. The function of stabilizers is to provide increased storage stability in cases where the peptidoconjugate or carrier is labile to heat, cold, light or oxidants or other physical or chemical agents. Other purposes for stabilizer can be for maintaining peptidoconjugate and/or carrier in a form appropriate for transport to and uptake at the target site. Depending on the solubility, the excipients or stabilizers can be added either prior to the deposition step or after the hydration step. Moreover, the pharmaceutical composition can be foπnulated into dosage forms such as capsules, impregnated wafers, ointments, lotions, inhalers, nebulizers, tablets, or injectable preparations.

The pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. One of ordinary skill in the art will appreciate that the route and/or mode of administration will vary depending on the conditions of target organisms and the desired results. For example, the pharmaceutical composition can be administered by methods including but being not limited to, oral, topical, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

Effective amounts or doses of the composition for cellular imaging may be determined using recognized in vitro systems or in vivo animal models. One of the factors that determine the dosage is the irradiation time. If it is desired to irradiate only for short time, the concentration of the composition can be increased. Dosage regimens are adjusted to provide the optimum desired response (e.g., cellular imaging). For example, a single dosage can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the situation.

Irradiation

Methods for irradiation include, but are not limited to, the administration of laser, nonlaser, or broad band light. Irradiation can be produced by extracorporeal or intraarticular generation of light of the appropriate wavelength. Light used in the invention can be administered using any device capable of delivering the requisite power of light including, but not limited to, fiber optic instruments, arthroscopic instruments, or instruments that provide transillumination. Delivery of the light to a recessed, or otherwise inaccessible physiological location can be facilitated by flexible fiber optics (implicit in this statement is the idea that one can irradiate either a broad field, such as the lung or a lobe of the lung, or a narrow field where bacterial cells may have localized). Those skilled in the art will recognize that various devices and methods for irradiation are within the spirit and scope of the present invention. Further, those skilled in the art will recognize that various amounts and/or types of energy may be delivered to the cells and remain within the spirit and scope of the present invention.

Peptidoconjugate Kits

The invention further provides a packaged composition such as a kit or other container for detecting, monitoring or otherwise observing a target molecule. The kit or container can hold a peptidoconjugate of the invention and instructions for using the peptidoconjugate for detecting, monitoring or otherwise observing a target molecule. The peptidoconjugate includes at least one binding domain and a dye.

The kits of the invention can also comprise containers with solutions or tools useful for manipulating or using the peptidoconjugates of the invention. Such tools include buffers, reaction tubes, reagents and the like. The kit can also contain a container of buffer at roughly neutral pH (e.g. sodium phosphate buffer, pH 7.5). The following examples are illustrative of the present invention, but are not limiting.

Numerous variations and modifications on the invention as set forth can be effected without departing from the spirit and scope of the present invention.

EXAMPLES

The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, pending patent applications and published patents, cited through this application, are hereby expressly incorporated by reference.

Example 1. Phototoxity of Peptidoconiugates Modulated Bv A Single Amino Acid:

Oxidative stress resulting from the intracellular release of chemical oxidants or free radicals is known to exert deleterious effects on biological function. In cells, exposure to light of sensitizers can result in the formation of singlet oxygen ( ¹ O ₂ ), a highly reactive, mutagenic, and genotoxic species that induces oxidative stress.'- ^1"3 -' These properties have been harnessed in photodynamic chemotherapy, an anticancer treatment that involves the photosensitization of ¹ O ₂ to promote cell death within solid tumors. ^[4] Chemical modification of essential cellular components likely underlies the detrimental effects of ¹ O ₂ , as direct damage to DNA, proteins, and lipids is observed.'- ¹ ' ³ ' ^5"8 -' Given that the reaction of ¹ O ₂ with DNA and amino acids generates transient species that are highly reactive (e.g. endoperoxides and peroxyl radicals), ^[8"10] cross-reactions between nucleic acids and bound proteins are probable. Understanding and exploiting biomolecular crossreactions that occur when ¹ O ₂ is generated intracellularly may lead to new photodynamic therapeutics with enhanced photodynamic therapeutics with enhanced potencies. Applicants have developed a strategy towards studying oxidative cross reactions between amino acids and DNA promoted by ¹ O ₂ that relies on a family of DNA-binding peptidoconjugates containing a photoactive intercalator, thiazole orange ("TO") J ¹² ' ¹³ ^ TO generates ¹ O ₂ upon photoexcitation, serving as an oxidant source in addition to providing a DNA-binding anchor. In assignee's co-pending application, U.S. Provisional Application No. 60/578,798, the entirety of which is incorporated herein, TO-dipeptide conjugates were identified which exhibited DNA photocleavage activity that depended upon the composition of the peptide. ¹ - ¹⁴¹ Conjugates containing certain aromatic amino acids, in particular tryptophan ("W") and tyrosine ("Y"), photocleave DNA. However, it was discovered that conjugates containing glycine ("G") or phenylalanine ("F") do not promote strand scission. It has been established that a subset of protein residues (including tryptophan, cysteine, histidine, and tyrosine) reacts with ¹ O ₂ to form peroxides.'- ¹ ' ^8"10 -' Peroxyl radicals are reasonable candidates as the active DNA cleaving species, as precedent exists for strand scission via hydrogen abstraction from the DNA backbone by thermally-generated peroxides. ^ ⁵ ^ Indeed, studies of TO-peptidoconjugates displaying strand scission activity revealed that amino acid-based peroxides are the active species inducing DNA cleavage.'- ¹⁴ -'

This experimental section describes TO-peptidoconjugates that access human cells and exhibit amino acid dependent phototoxity. The TO-conjugates feature a portion (residues 49-57) of HIV-I transactivator of transcription ("Tat") peptide sequence previously used by

other laboratories to deliver appended cargoes into cellsJ ¹⁶ - ¹ Based on previous findings, Applicants wanted to investigate whether cell-permeable TO-peptidoconjugates were toxic to human cells upon photoexcitation and to determine if toxicity would be triggered by the presence of specific amino acids as previously demonstrated when the DNA strand-scission activity was initially characterized. ¹¹⁴³ Indeed, a TOTat peptidoconjugate containing tryptophan cleaves DNA in vitro and exhibits appreciable phototoxicity, while a glycine analogue does not significantly cleave DNA or cause cell death. These results represent an example of novel peptide-containing photodynamic agents with activities that can be tuned through the manipulation of sequence composition. Further, these results provide a novel method for cellular imaging of human cells .

Preparation ofpeptidoconjugates

TO D-peptide conjugates were prepared on a Rink amide support and the N-terminus was capped with a TO derivative ¹ - ¹⁴³ using standard solid-phase Fmoc chemistry. Subsequent cleavage from the resin and purification by HPLC afforded TO-peptidoconjugates (see structure Ia in FIG. 4A). The unnatural D-peptide structure was used to impart resistance to protease degradation J ¹⁷ - ¹ Two versions of the Tat peptide were prepared with a glycine or tryptophan residue incorporated proximal to the dye to evaluate if selective DNA cleavage and phototoxicity would be observed. TO-dipeptide conjugates containing Z)-amino acids are analogous to those previously reported containing Z-amino acids were also synthesized to provide benchmark controls for DNA cleavage.

The photocleavage properties of the TO-Tat peptidoconjugates were investigated using a plasmid nicking assay. Upon irradiation with visible light, the tryptophan-containing conjugate (see structure Ib in FIG. 4A), caused high levels of strand scission. The observation of a higher yield of DNA cleavage for the tryptophan containing conjugate (inducing direct DNA cleavage because of the W-peroxide formed upon the production of

¹ O ₂ ) relative to non-tryptophan conjugates (see structure Ia of FIG. 4A)(exhibitmg only a low level cleavage because of direct ¹ O ₂ -PrOmOtCd damage) indicates that these TO-conjugates display analogous photoreactivity to those described previously. ^ Interestingly a control dipeptide conjugate made from d-amino acids (see structure 2b of FIG. 4A), cleaved supercoiled plasmid DNA more efficiently than the tryptophan-containing conjugate, demonstrating that tryptophan exhibits greater reactivity when presented to DNA within the context of a dipeptide rather than a Tat decapeptide. ^[18] This effect may arise from different conformations of tryptophan that are induced by the two peptides. Nonetheless, these results

indicated that the amino acid dependent DNA cleavage previously discovered and characterized ¹ - ¹⁴¹ in dipeptide conjugates could be extrapolated to a more complex peptide structure. Furthermore, the TO-W-Tat peptidoconjugate, which should have good cellular uptake properties, is a suitable probe for studies inside cells. Confocal fluorescence microscopy was employed to conform cellular uptake of the

TO-Tat peptidoconjugates. As TO undergoes a dramatic increase in its fluorescent quantum yield when bound to DNA or RNA, ^ ¹³ - ¹ it can be used as an intrinsic probe for the location of the conjugates within cells. Both TO-Tat peptidoconjugates, structures Ia and Ib of FIG. 4A, exhibited identical localization patterns indicating that they were efficiently imported into live unfixed HeLa cells. Less than five percent of cells were stained when incubated with propidium iodide (which detects dead cells), reflecting that the conditions used to evaluate uptake patterns of the peptidoconjugates did not induce cell death.

To further investigate the location of the TO-Tat peptidoconjugates within human cells, co-localization experiments were performed utilizing two dyes as controls. Syto-85 binds to nucleic acids and provides a marker for nuclear uptake, while Mito Tracker Deep Red-663 selectively stains mitochondria. Interestingly, the TO-Tat peptidoconjugates were found to enter both the mitochondria and the nucleoli of live cells. ^ ⁹ - ¹ This finding is in stark contrast to the prior work of Ross et al. who found that the Tat sequence did not enter the mitochondria, ^ ⁰ - ¹ however the peptide was conjugated to a different dye (Oregon Green); different cargoes can markedly change uptake and localization patterns. ^[16] For the TO- conjugates described here, it is also noteworthy that these probes are most emissive when bound to DNA. Thus, while it is clear that the compounds have penetrated the nucleus, there may also be appreciable concentrations of the compounds in regions of the cell that appear dark in the images shown. As the TO-Tat peptidoconjugates were shown to enter human cells efficiently, these probes presented an appropriate system for the analysis of phototoxicity. When incubated with HeLa cells and irradiated, the TO-W-Tat peptidoconjugate, structure Ib of FIG. 4A, was significantly phototoxic to cells as a function of irradiation time, while cells exposed to the G-containing conjugate, structure Ia of FIG. 4A, were unaffected (see FIG. 4B). After irradiation, the cells were incubated with fresh media for 24 hours before viability was analyzed to allow the effects of the compounds to be assessed. Cell death was not observed if cells containing either conjugate were kept in the dark. ^[21] Interestingly, we observed that the

Tat peptide effectively abolished the dark toxicity of unmodified TO, as the parent compound (unmodified TO) causes quantitative cell death even in the absence of light.

The correlation of phototoxicity with the DNA photocleavage activity of the TO- peptidoconjugates described here strongly suggests that the W-based peroxides formed on TO-conjugates containing this amino acid are responsible for this selectivity. While we cannot unequivocally prove that the decreased cell viability results from DNA cleavage, the location of the conjugates within the cellular compartments that contain genomic DNA present the possibility that strand scission may underlie the toxicity described. This represents the first study that utilizes designed TAT-peptide sequences to induce selective DNA damage and to produce phototoxic agents.

Synthesis of dye-peptide conjugates

Dye-peptide conjugates were synthesized on solid support using Rink amide resin (NovaBiochem). Couplings were performed using 4 equivalents of Fmoc-Z>-protected amino acid (Advanced ChemTech), 4 equivalents of HBTU (Advanced ChemTech) and 8 equivalents of N,N-diisopropylethylamine (Acros) in DMF for 3 hours. Deprotection of the Fmoc group was achieved using 20% piperidine in DMF for 30 minutes (to minimize diketopippiperazine formation, dipeptides were deprotected using 50% piperidine in DMF for 5 minutes). The deprotected N-terminus was capped with 4 equivalents of TO-COOH ¹ - ¹⁴ -' under standard coupling conditions as described above. To minimize byproducts resulting from the Rink amide resin under high concentrations of TFA, a 2-step procedure for detachment/deprotection of the resin was performed as described. ¹ - ²²¹ The dye peptide conjugates were detached from the resin by slurrying in 10% TFA:DCM [v/v] and transferred to a glass funnel with a fine stinter. The solvent was allowed to drip slowly through the resin bed and was washed with 5% TFA:DCM [v/v] and concentrated in vacuo. Deprotection was achieved by stirring the residue in 95:5 TFA:TIS solution at room temperature for 0.5-2h. The solution was concentrated in vacuo and ET ₂ O was added to precipitate the peptide. The resulting red solid was dissolved in 0.1% TFA:H ₂ O and purified via RP-HPLC (H ₂ O)/CH ₃ CN in 0.1% TFA). The products were isolated by lyophilization and characterized by MALDI-TOF mass spectrometry. The purity of the peptides was >95% as determined by RP-HPLC (H ₂ O)/CH ₃ CN in 0.1% TFA). A molar extinction coefficient (E)-OS ₉ OOOM ^-1 Cm ^'1 in H ₂ O) at 500 nm was used to quantitate TO-peptide conjugates' ¹ - ¹³¹

DNA photocleavage

lμM TO-peptide conjugate was added to 75 μM (bp) pUC18 in 1OmM sodium cacodylate (pH 7) in the dark. Irradiation was performed for 30 minutes at 501nm with an Oriel Instruments Spectral Luminator tunable light source. Lamp intensity was 1.36 mW/cm ² . Cleavage efficiencies were evaluated using 1% agarose gel electrophoresis visualized by ethidium bromide staining. Minimal cleavage was observed when identical samples were incubated in the dark or when DNA samples were irradiated alone.

Cell culture

HeLa 229 cells (ATCC) were cultured as subconfluent monolayers on 25 or 75 cm ² cell culture plates with vent caps (Corning) in 1 X minimum essential medium α medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (ATCC) in a humidified incubator at 37 ⁰ C containing 5%CO ₂ (gas).

Confocal microscopy

HeLa cells that had been grown to subconfulence were dissociated from the surface with 2mL of 0.05% Trypsin/0.53 mM EDTA (Cellgro) for 15 minutes at 37 ⁰ C. Aliquots of IXlO ⁵ cells were plated in four-well Lab-Tek glass bottom coverslips (Nalge Nunc Inc.) and cultured overnight to allow cell adherence. The culture media was removed and the cells were rinsed in Ix Ca ²⁺ and Mg ²⁺ -free PBS, pH 7.4 (Cellgro). HeLa cells were incubated for 1.5 hours at 37 ⁰ C with 500 mL of media containing lOμM of structures Ia or Ib of FIG. 4A. For co-localization studies, cells were incubated with lOμM Ia and 50OnM Syto-85 (Molecular Probes) or 3μM Mito Tracker Deep Red-633 (Molecular Probes). Cells were washed 3 times for 5 minutes with 1 mL of PBS. After washing, PBS (500 μL) was added and the cells were placed on ice. Images were taken with an inverted Leica TCS SP2 scanning confocal microscope with a 4Ox oil immersion lens. The images were analyzed with the Leica confocal software program. Cells incubated with Syto-85, Mito Tracker Deep Red-633, or 1 a were used to identify appropriate emission collection parameters and to minimize bleed-through of the co- localized fluorophore. The excitation wavelength for both structures Ia and Ib of FIG. 4A was 488 ran and emission was collected from 500-571 nm. Syto-85 (λ _{ex max} =567nm) and Mito Tracker Deep Red-633 (λ _e χ _ma χ ⁼ 644nm) were excited at 543 nm and collected from 556-648 nm and 600-750 nm respectively. These parameters were used in all experiments. Cells were exposed to propidium iodide (MP Biomedicals) to determine the extent of cell death (<5%). Propidium iodide was excited at 488nm and collected from 550-

700nm. Cells that brightly fluoresced in subsequent experiments were assumed to be dead and were not used in the evaluation of conjugates.

Phototoxicity

HeLa cells were split as described above and 100 μL aliquots were seeded (1 x 10 cells) into 96 well clear flat bottom microplates (Costar). After overnight incubation, the media was replaced with 100 μL of new media. Freshly prepared solutions of structures Ia and Ib of FIG. 4A (3μM) were added to each well. Cells were incubated for 30 minutes in the dark at 37 ⁰ C, and then irradiated at 501 ran for 2, 5, 10, 15, and 20 minutes (UVA doses of 0.804, 2.01, 4.02, 6.03, and 8.04 J cm ^'2 respectively) in triplicate, the media was replaced. For the dark controls, fresh media was also added.

Cells were analyzed with the Cell Counting Kit-8 (CCK-8, Dojindo) to determine cell viability. After an overnight incubation following irradiation, the media was removed and fresh media (90 μL) containing 10 μL of CCK-8 was introduced. After a 1 hour incubation at 37 ⁰ C, the absorbance of each sample was measured at 470nni on a ThermaMax plate reader (Molecular Devices). Samples that contained only CCK-8 and media were subtracted from all samples. Wells without conjugates were used as controls to determine the extent of cell death. FIG. 4 represents an average of three separate trials.

FIG. 4A shows structures of various peptidoconjugates wherein the dye is TO. In FIG. 4A, structure Ia is TO-G-Tat; structure Ib is TO-W-Tat; structure 2a is TO-GK; and structure 2b is TO-WK.

FIG. 4B shows a photocleavage of pUC18 plasmid DNA by TO-peptidoconjugates analyzed by agarose gel electrophoresis. Solutions contained 75 μM (bp) pUC18, 1OmM sodium cacodylate (pH 7) and 1 μM TO-peptdiconjugate. Samples were irradiated for 30 minutes at 501 nm as indicated and the conversion of supercoiled to nicked circular plasmid was monitored to evaluate DNA cleavage.

FIG. 5A-5C show confocal microscopy images of unfixed live HeLa cells incubated for 1.5 hours at 37 ⁰ C with lOμM of structures Ia of FIG. 4A and structure Ib of FIG. 4A. FIG. 5A shows a transmission image of HeLa cells incubated with structure Ib of FIG. 4A. FIG. 5B shows a red fluorescence (λ _ex =488 nm, λ _em =500-571 nm) of the same cells illustrating both cytoplasmic and nuclear uptake for structure Ib of FIG. 4A. FIG. 5C shows a peptidoconjugate of structure Ia of FIG. 4A having an identical internalization pattern to structure Ib of FIG. 4A.

FIG. 6A-FIG. 6F show localization profiles of various TO-Tat peptidoconjugates. HeLa cells were incubated with 10 μM of structure 1 of FIG. 4A and 50OnM Syto-85 or 3 μM Mito Tracker Red-633 for 1.5 hours at 37 ⁰ C. FIG. 6A and FIG. 6D show red fluorescent image of cells stained with structure Ia of FIG. 4 A. FIG. 6B shows a visualization of Mito Tracker Deep Red-633 staining of the mitochondria. FIG. 6C shows a merged image of red and blue fluorescence images illustrating co-localization of structure Ia of FIG. 4 A with Mito Tracker Deep Red-633. FIG. 6E shows a green fluorescent image illustrating nucleolar staining by Syto-85. FIG. 6F shows a merged image showing that structure Ia of FIG. FIG. 4A co-localizes with Syto-85 in the nucleoli of these cells. Red fluorescence: λ _ex =488 nm, λ _em ⁼ 500-571 nm; Green fluorescence: λ _ex ^: =543 nm, λ _em ⁼ 556-648 nm; Blue fluorescence: λ _ex =543 nm, λ _em =600-750 nm. ^[19]

FIG. 7 shows a chart related to the phototoxicity of TO-Tat peptidoconjugates. Toxicity was evaluated 12 hours after incubation and irradiation at 501nm with 3 μM of structure Ia of FIG. 4A and structure Ib of FIG. 4A by CCK-8 assay. Data shown in FIG. 7 represent mean values and error bars are standard deviation values. All assays were performed in triplicate and three independent trials were conducted. Dark controls were performed to confirm that cell viability was maintained in the absence of photoexcitation. In addition, light controls were performed to confirm that the irradiation conditions did not harm the cells. Example 2. Synthesis of a peptoid

Peptoids were synthesized according to the method of Zuckerman. The Fmoc-Rink amide resin (1.0 equiv.) was treated with 20% piperidine in DMF for 30 minutes. The free resin-bound amine was then treated with a solution of bromoacetic acid (10 equiv.) and diisopropylcarbodiimide (10 equiv.) in DMF for 30 minutes. This procedure was repeated. The resin was then treated with a solution of primary amine (40 equiv.) in DMF for 12 hours. These two steps were repeated until an oligomer of desired length was obtained. The resin was then treated with Fmoc-protected amino acid (4.0 equiv.), HBTU (4.0 equiv.), and DIPEA (8.0 equiv.) in DMF for 3 hours. The dye moiety was attached to the resin as described earlier. The dye-peptoid conjugates were simultaneously deprotected and cleaved from the resin with a 95:5 TFA : TIPS solution. The solution was concentrated in vacuo and purified via RP-HPLC (H ₂ O/CH ₃ CN in 0.1% TFA). The products were isolated by lyophilization and characterized by MALDI-TOF mass spectrometry. The purity of the peptides was > 95% as determined by analytical RP-HPLC (H ₂ O/CH ₃ CN in 0.1% TFA).

Example 3. DNA-binding characterization of Thiazole Orange ( ^" TOVPeptide Conjugates

Solvents were purchased from Fisher and reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Acros Organics (Morris Plains, NJ). Amino acids were purchased from Advanced ChemTech (Louiville, KY). Calf thymus DNA (CT DNA) was purchased from Sigma (St. Louis, MO). All solvents and reagents were used without further purification. HPLC grade acetonitrile and Millipore water were used for HPLC analysis. The buffer used in all experiments was 50 mM sodium phosphate, 10 mM sodium chloride (pH 7).

Reversed-phase HPLC was performed using a HP 1100 system with a Varian 250 x 4.6 mm stainless steel column packed with Microsorb-MV 300 Cl 8 (5 μM). A flow rate of 1.0 mL/min. was used with an aqueous solution buffered with 50 mM ammonium acetate and a linear gradient from 20 to 100% acetonitrile over 80 min. 'H NMR spectra were recorded on a Varian 400 and 500 MHz spectrometer. Proton chemical shifts are reported in ppm (δ) relative to the solvent reference relative to tetramethylsilane (TMS) (dβ-DMSO, δ 2.50; CD3OD, δ 3.30). Data are reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m)], coupling constants [Hz], integration). Carbon NMR spectra were recorded on a Varian 500 (125 MHz) spectrometer with complete proton decoupling. Carbon chemical shifts were reported in ppm (δ) relative to TMS with the respective solvent resonance as the internal standard (DMSO, 39.5; CD3OD, 49.2). Mass spectral analysis was performed by the Boston College Mass Spectrometry Facility. Samples were analyzed by accurate mass electrospray mass spectrometry (ES-MS) operating in positive mode on a Micromass LCT mass spectrometer. UV analysis was performed on a Hewlett Packard 8452A Diode Array Spectrophotometer. Steady state fluorescence measurements were performed on a Jobin Yvon Horiba Fluorolog®-3. For all steady state measurements the 470 nm and the 490- 650 nm. Dissociation constants were measured using a Perkin Elmer Wallac Victor Fluorescence reader fitted with a 450-490 nm excitation filter and a 515 nm long pass emission filter.

Hypochromicitv measurements

Absorbance values for 4 μM solution of TO-conjugates were measured before and after the addition of 45 μM (bp) CT DNA. Percent hypochromicity was calculated as the percent change in absorbance at λmax.

Quantum yield measurements

Quantum yields are reported relative to a 50 nM fluorescein standard in 0.1 M NaOH (φ= 0.93). See Weber, G.; Teale, F. W. J. Trans. Faraday Soc. 53, 646 (1957). Absorbance values at the excitation wavelength (470 nm) were measured for solutions containing 13.5 μM TO-conjugate and 405 uM CT DNA bp. The sample was then diluted to a final TO- conjugate concentration of 1.5 μM and CT DNA concentration of 45 μM (bp) to ensure that the absorbance of the sample was less than 0.06 for measurement of emission spectra. The integral of the emission spectrum was corrected for variations in absorbance and reported relative to the fluorescein standard. Measurement of dissociation constants

Dissociation constants were determined with fluorescence titrations performed in a 384 well plate with a total volume of 40 μl in each well. The concentration of Toconjugates was kept constant at 50 nM and the concentration of CT DNA was increased until fluorescence signals plateaued. Each sample was run in triplicate and the values of each concentration point were averaged. Scatchard analysis was used to obtain Kd values.

Diffusional quenching experiments

Samples containing 1.5 μM TO-conjugate and 45 μM CT DNA bp were titrated with hexaamineruthenium(III) chloride and the fluorescence was measured after each addition. The data was plotted according to the Stern- Volmer equation (Eq. 1) and the slope of the best-fit line was used to determine the value for kq. Lifetimes are relative to the reported lifetime for thiazole orange and were based on the ratio of the quantum yield of each sample to thiazole orange (φ= 0.11). See Netzel, T. L.; Nafisi, K.; Zhao, M.; Lenhard, J. R.; Johnson, I. J. Phys. Chem., 99, 17936 (1995), Nygren, T. L.; Svanvik, N.; Kubista, M. Biopolymers, 46, 39 (1998). Eq. 1 lot I = k _q τ[ QJ + 1

Displacement of distamycin A

The methods used for the displacement assay are analogous to those described by Boger. See Boger, D. L.; Tse, W. C; Bioorg. Med. Chem., 9, 2511 (2001), Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C; Hedrick, M. P. J. Am. Chem. Soc, 123, 5878 (2001). Fluorescence of a solution containing 2.0 μM TO-conjugate and 12 μM CT DNA bp was measured. Distamycin A was added to produce a final concentration of 2.0 μM and the

fluorescence was remeasured once again. Percent decrease in fluorescence was calculated as the percent change in the integral of the emission spectrum upon addition of distamycin A.

References

[I] M. J. Davies, Biochem. Biophys. Res. Cornmun. 2003, 305, 761-770. [2] B. Epe, Chem. Biol. Interact. 1991, 80, 239-260.

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[4] D. E. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 3, 380-387; b) M. R. Detty, S. L. Gibson, S. J. Wagner, J. Med. Chem. 2004, 47, 3897-3915; c) N. L. Oleinick, R. L. Morris, I. Belichenko, Photochem. Photobiol. ScL 2002, 1, 1-2 1. [5] H. Sies, Mutat. Res. 1993, 299, 183-191.

[6] A. Michaeli, J. Feitelson, Photochem. Photobiol. 1994, 59, 284-289. [7] A. W. Girotti, W. Korytowski, Methods Enzymol. 2000, 319, 85-100.

[8] A. Wright, W. A. Bubb, C. L. Hawkins, M. J. Davies, Photochem. Photobiol. 2002, 76, 35-46.

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[15] T. A. Dix, K. M. Hess, M. A. Medina, R. W. Sullivan, S. L. Tilly, T. L. Webb, Biochemistry 1996, 35, 4578-4583. [16] A. Joliot, A. Prochiantz, Nat. Cell. Biol. 2004, 6, 189-196. [17] P. M. Fischer, Curr. Protein Pept. ScL 2003, 4, 339-356.

[18] The DνA cleavage observed with Ib was comparable to that obtained with the TO- WK /-peptidoconjugate previously described. ¹⁴

[19] Fluorescent images were assigned colors to maximize the ability to visualize the overlap between different fluorophores using the Leica confocal software. They do not reflect the emission properties of the fluorophore.

[20] M. F. Ross, A. Filipovska, R. A. Smith, M. J. Gait, M. P. Murphy, Biochem. J. 2004, 383, 457-468.

[21] Dark toxicities were negligible at 3 μM, however as the concentrations of the conjugates approached 7 μM, significant dark toxicity was observed, therefore IC ₅₀ values could not be determined.

[22] Novabiochem Catalog, 2004/5, 3.15-3.16 (Method 3-19).

All patents, patent applications, and published references cited herein are hereby incorporated herein by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.