Benzocyanine compounds

Abstract

Compounds useful as labels with properties comparable to known fluorescent compounds. The compounds are conjugated to proteins and nucleic acids for biological imaging and analysis. Synthesis of the compounds, formation and use of the conjugated compounds, and specific non-limiting examples of each are provided.

Claims

1. A method of detecting at least one biomolecule, the method comprising combining at least one biomolecule with a composition comprising at least one excipient and a compound in an effective concentration to detect at least one biomolecule under conditions sufficient for binding the compound to the biomolecule, and detecting the biomolecule-bound compound, where the compound is selected from the group consisting of ##STR00264## ##STR00265## where each of R.sub.1, R.sub.2, R.sub.5, and R.sub.6 is the same or different and is independently selected from the group consisting of an aliphatic, heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO.sub.3, a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O).sub.s, where s is an integer from 3-6 inclusive, a sulfonamide group -L-SO.sub.2NHPZ, and a carboxamide group -LCONHPZ, where Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group; each of R.sub.7, R.sub.8, R.sub.11, R.sub.12, R.sub.13, and R.sub.14 is the same or different and is independently selected from the group consisting of H, SO.sub.3, a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O)s, where s is an integer from 3-6 inclusive, a sulfonamide-containing group -L-SO.sub.2NHPZ, and a carboxamide-containing group CONHPZ, where Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group; X is selected from OH, SH, NH.sub.2, NHNH.sub.2, F, Cl, Br, I, NHS (hydroxysuccinimidyl-/sulfosuccinimidyl), O-TFP (2,3,5,6-tetrafluorophenoxy), O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), O-benzotriazole, -benzotriazole, imidazole, azide, O-carbodiimide, NR-L-OH, NR-L-O-phosphoramidite, NR-L-SH, NR-L-NH.sub.2, NR-L-NHNH.sub.2, NR-L-CO.sub.2H, NR-L-CONHS, NR-L-CO-STP, NR-L-CO-TFP, NR-L-CO-benzotriazole, NR-L-CHO, NR-L-maleimide, NR-L-NHCOCH.sub.2I, or NR-LNHCOCH.sub.2Br wherein R is H or an aliphatic or heteroaliphatic group; L is selected from a divalent linear ((CH.sub.2).sub.t, t=0 to 15), crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom; Kat is a number of Na.sup.+, K.sup.+, Ca.sup.2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive; each of R.sub.3 and R.sub.4 is the same or different and is independently hydrogen, an aliphatic group, a heteroaliphatic group, or a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O).sub.s, where s is an integer from 3-6 inclusive, and Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group; or R.sub.3 and R.sub.4 together form a cyclic structure where R.sub.3 and R.sub.4 are joined using a divalent structural element selected from the group consisting of (CH.sub.2).sub.q, (CH.sub.2).sub.qO(CH.sub.2).sub.q, (CH.sub.2).sub.qS(CH.sub.2).sub.q, (CH.sub.2).sub.gCHCH, OCHCH where each of q and q is the same or different and is a integer from 2 to 6 inclusive; and Y is selected from the group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine, bromine, a substituted or unsubstituted aryl, phenylmercapto function, and a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O).sub.s, where s is an integer from 3-6 inclusive, and Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group.

2. The method of claim 1 wherein the biomolecule is selected from a protein, antibody, enzyme, nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid, carbohydrate, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, fluorescent proteins, organic polymer carrier material, inorganic polymeric carrier material, and combinations thereof.

3. The method of claim 1 wherein the at least one biomolecule is detected in an assay selected from fluorescence microscopy, flow cytometry, immunoassay, hybridization, chromatographic assay, electrophoretic assay, microwell plate based assay, fluorescence resonance energy transfer (FRET) system, high throughput screening, or microarray.

4. The method of claim 1 wherein the biomolecule is detected by in vivo imaging comprising providing the biomolecule-bound compound to at least one of a biological sample, tissue, or organism, and detecting the biomolecule within the at least one of a biological sample, tissue, or organism.

5. A method of labeling at least one biomolecule, the method comprising combining at least one biomolecule with a composition comprising at least one excipient and a compound in an effective concentration to label the at least one biomolecule under conditions sufficient for labeling the biomolecule with the compound, where the compound is selected from the group consisting of ##STR00266## ##STR00267## X is selected from OH, SH, NH.sub.2, NHNH.sub.2, F, Cl, Br, I, -NETS (hydroxysuccinimidyl-/sulfosuccinimidyl), O-TFP (2,3,5,6-tetrafluorophenoxy), O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), O-benzotriazole, -benzotriazole, imidazole, azide, O-carbodiimide, NR-L-OH, NR-L-O-phosphoramidite, NR-L-SH, NR-L-NH.sub.2, NR-L-NHNH.sub.2, NR-L-CO.sub.2H, NR-L-CONHS, NR-L-CO-STP, NR-L-CO-TFP, NR-L-CO-benzotriazole, NR-L-CHO, NR-L-maleimide, NR-L-NHCOCH.sub.2I, or NR-LNHCOCH.sub.2Br wherein R is H or an aliphatic or heteroaliphatic group; L is selected from a divalent linear ((CH.sub.2).sub.t, t=0 to 15), crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom; Kat is a number of Na.sup.+, K.sup.+, Ca.sup.2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive; each of R.sub.3 and R.sub.4 is the same or different and is independently hydrogen, an aliphatic group, a heteroaliphatic group, or a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O).sub.s, where s is an integer from 3-6 inclusive, and Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group; or R.sub.3 and R.sub.4 together form a cyclic structure where R.sub.3 and R.sub.4 are joined using a divalent structural element selected from the group consisting of (CH.sub.2).sub.q, (CH.sub.2).sub.qO(CH.sub.2).sub.q, (CH.sub.2).sub.qS(CH.sub.2).sub.q, (CH.sub.2).sub.gCHCH, OCHCH where each of q and q is the same or different and is an integer from 2 to 6 inclusive; and Y is selected from the group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine, bromine, a substituted or unsubstituted aryl, phenylmercapto function, and a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O).sub.s, where s is an integer from 3-6 inclusive, and Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group.

6. The method of claim 5 wherein the biomolecule is selected from the group consisting of a protein, antibody, enzyme, nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid, where each of R.sub.1, R.sub.2, R.sub.5, and R.sub.6 is the same or different and is independently selected from the group consisting of an aliphatic, heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO.sub.3, a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O).sub.s, where s is an integer from 3-6 inclusive, a sulfonamide group -L-SO.sub.2NHPZ, and a carboxamide group -LCONHPZ, where Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group; each of R.sub.7, R.sub.8, R.sub.11, R.sub.12, R.sub.13, and R.sub.14 is the same or different and is independently selected from the group consisting of H, SO.sub.3, a PEG group PZ where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH.sub.2CH.sub.2O).sub.s, where s is an integer from 3-6 inclusive, a sulfonamide-containing group -L-SO.sub.2NHPZ, and a carboxamide-containing group CONHPZ, where Z is selected from H, CH.sub.3, a CH.sub.3 group, an alkyl group, or a heteroalkyl group; carbohydrate, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, fluorescent proteins, organic polymer carrier material, inorganic polymeric carrier material, and combinations thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows absorption/emission profiles of some inventive compounds and commercial dyes.

(3) FIG. 2 graphs functional assay results with some commercial dyes and inventive compounds with one conjugate produced in one embodiment.

(4) FIG. 3 graphs functional assay results with some commercial dyes and inventive compounds with another conjugate produced in one embodiment.

(5) FIG. 4 graphs functional assay results with some commercial dyes and inventive compounds with another conjugate produced in one embodiment.

(6) FIG. 5 graphs functional assay results with some commercial dyes and inventive compounds with one conjugate produced in one embodiment.

(7) FIG. 6 graphs functional assay results with some commercial dyes and inventive compounds with one conjugate produced in one embodiment.

(8) FIG. 7 graphs functional assay results with some commercial dyes and inventive compounds with one conjugate produced in one embodiment.

(9) FIG. 8 shows functional assay results with some commercial dyes and inventive compounds in one embodiment.

(10) FIG. 9 graphs functional assay results with some commercial dyes and inventive compounds in one embodiment.

(11) FIG. 10 graphs functional assay results with some commercial dyes and inventive compounds in one embodiment.

(12) FIG. 11 tabulates functional assay results with some commercial dyes and inventive compounds in one embodiment.

(13) FIG. 12 graphs functional assay results with some some commercial dyes and inventive compounds in one embodiment.

(14) FIG. 13 tabulates functional assay results with some commercial dyes and inventive compounds in one embodiment.

(15) FIG. 14 is a histogram showing functional assay results with some commercial dyes and inventive compounds in one embodiment.

(16) FIGS. 15A-D show immunofluorescence data with some commercial dyes and inventive compounds forming a conjugate in one embodiment.

(17) FIGS. 16A-D show immunofluorescence data with some commercial dyes and inventive compounds forming a conjugate in one embodiment.

(18) FIGS. 17A-D show immunofluorescence data with some commercial dyes and inventive compounds in one embodiment.

(19) FIGS. 18A-D show immunofluorescence data with some commercial dyes and inventive compounds in one embodiment.

(20) FIGS. 19A-C show immunofluorescence data with commercial dyes and inventive compounds in one embodiment.

(21) FIG. 20A, column 1 A-E and column 2 A-E show immunofluorescence data with commercial dyes and inventive compounds in one embodiment.

(22) FIG. 20B, column 1 A-E and column 2 A-E show immunofluorescence data with commercial dyes and inventive compounds in one embodiment.

(23) FIG. 20C, column 1 A-E and column 2 A-E show immunofluorescence data with commercial dyes and inventive compounds in one embodiment.

(24) The following non-limiting examples further describe the compounds, methods, compositions, uses, and embodiments. They demonstrate that the inventive compounds exhibited desirable properties relative to commercially available fluorescent dyes. Signal to noise ratio (S/N) is the ratio between the desired signal and the mean of the blank, accounting for the standard deviation of the signal and the blank. Signal to background ratio (S/B) is the ratio between the desired average signal and the average blank.

EXAMPLE 1

Synthesis of 1,2-Dimethyl-1-(3-sulfopropyl)-1H-benzo[e]indole-6,8-disulfonic acid tri potassium salt

(25) ##STR00259##

(26) Five g (15.7 mmol) 6-hydrazino-naphthalene-1,3-disulfonic acid and 4.93 g (25 mmol) 4-methyl-5-oxohexane sulfonic acid were dissolved in 50 ml acetic acid. The solution was heated at 140 C. for four hours. The solvent was evaporated in vacuum. The oily residue was dissolved in 20 ml methanol, then 50 ml of a saturated solution of KOH in 2-propanol were added to yield a yellow precipitate. The solid was filtered off and dried in vacuum. Yield 4.1 g, MS (ESI): 158.2 [M].sup.3

EXAMPLE 2

Synthesis of 3-(2-Methoxy-ethyl)-1,2-dimethyl-6,8-disulfo-1-(3-sulfopropyl)-1H-benzo[e]indolium

(27) ##STR00260##

(28) A mixture of 7.56 g (12.8 mmol) 1,2-dimethyl-1-(3-sulfopropyl)-1H-benzo[e]indole-6,8-disulfonic acid tri potassium salt and 5.89 g (25.6 mmol) 2-methoxyethyl-p-toluene sulfonate was heated under argon for 24 h. The residue was purified by column chromatography (reversed phase silica, methanol/water, TFA). Yield 3.2 g, MS (ESI): 266.5 [M2H].sup.2

EXAMPLE 3

Synthesis of 3-(5-Carboxypentyl)-1,1,2-trimethyl-6,8-disulfo-1H-benzo[e]indolium

(29) ##STR00261##

(30) A mixture of 5.7 g (12.8 mmol) 1,1,2-trimethyl-1H-benzo[e]indole-6,8-disulfonic acid dipotassium salt and 5 g (25.6 mmol) 6-bromohexanoic acid was heated under argon for 24 h. The residue was purified by column chromatography (reversed phase silica, methanol/water, TFA). Yield 1.4 g, MS (ESI): 482.1 [M-H].sup.

EXAMPLE 4

Synthesis of 3-(5-Carboxypentyl)-1,1-dimethyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-6,8-disulfo-1H-benzo[e]indolium

(31) ##STR00262##

(32) 0.97 g (2 mmol) 3-(5-carboxypentyl)-1,1,2-trimethyl-6,8-disulfo-1H-benzo[e]indolium and 0.57 g (2.2 mmol) malonaldehyde-bisphenylimine-hydrochlorid were dissolved in 10 ml acetic acid and 10 ml acetic anhydride and stirred for four hours at 120 C. The solvent was removed under vacuum. The residue was washed carefully with ethyl acetate. A dark brown solid was obtained which was processed without further purification. MS (ESI): 611.2 [M-H].sup..

EXAMPLE 5

Synthesis of 679 Compound 1/1 (2-{(1E,3E)-5-[3-(5-Carboxypentyl)-1,1-dimethyl-6,8-disulfo-1,3-dihydro-benzo[e]indol-(2E)-ylidene]-penta-1,3-dienyl}-3-(2-methoxy-ethyl)-1-methyl-6,8-disulfo-1-(3-sulfopropyl)-1H-benzo[e]indolium)

(33) ##STR00263##

(34) 612 mg (1 mmol) 3-(5-carboxypentyl)-1,1-dimethyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-6,8-disulfo-1H-benzo[e]indolium and 533 mg (1 mmol) 3-(2-Methoxy-ethyl)-1,2-dimethyl-6,8-disulfo-1-(3-sulfopropyl)-1H-benzo[e]indolium were dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed by the addition of 200 mg of sodium acetate. The solution was stirred under reflux for 15 min. After cooling to room temperature, 20 ml diethylether was added. The resulting precipitate (mixture of the diastereomers 679 Compound 1/1 (isomer 1) and 679 Compound 1/1 (isomer 2)) was extracted by suction, washed with ether, and dried.

(35) The residue was purified by column chromatography (RP-18, acetonitrile/water and concentrated HCl) to separate the diastereomers from each other. The diastereomer that first eluted from the column was termed diastereomer 1 (679 Compound 1/1 (isomer 1)). The diastereomer that eluted second from the column was termed diastereomer 2 (679 Compound 1/1 (isomer 2)). The diastereomers were separated, followed by neutralization and evaporation. Purification of the single diastereomeric compound was completed on a RP-18 column, acetonitrile/water. The corresponding fractions were pooled and the solvent was removed by distillation. The two products (diastereomers 679 Compound 1/1 (isomer 1) and 679 Compound 1/1 (isomer 2)) were dried in high vacuum. 679 Compound 1/1 (isomer 1)

(36) yield: 10%

(37) UV-vis (PBS): .sub.max=679 nm .sub.em=698 nm

(38) MS (ESI) [M/z]: 262.5 [M].sup.4; 357.7 [M+Na].sup.3

(39) 679 Compound 1/1 (isomer 2)

(40) yield: 23%

(41) UV-vis (PBS): .sub.max=679 nm .sub.em=698 nm

(42) MS (ESI) [M/z]: 262.5 [M].sup.4; 357.7 [M+Na].sup.3

EXAMPLE 6

(43) The following properties of 679 Compound 1-NHS were compared with commercially available dyes.

(44) TABLE-US-00001 679 Company B Cy5.5 DyLight DyLight Compound Compound- Mono 680-NHS 680B-NHS 1-NHS NHS Ester MW (g/mol) 950 1196.16 1240.21 ~1150 1128.42 Ex (nm) 682 679 679 679 675 Em (nm) 715 698 698 702 694 140,000 180,000 180,000 184,000 250,000 (M 1 cm 1) (theoretical)

(45) Emission/excitation profiles for inventive and commercial compounds was determined by reconstituting the compounds in DMF at 10 mg/ml and then diluting in PBS buffer pH 7.2 to 10 g/ml. Absorbance spectra were collected on Cary UV spectrophotometer and emission spectra were collected on Tecan Safire, shown in FIG. 1 where solid lines represent absorbance spectra and dashed lines represent emission spectra, for DyLight 680-NHS (purple), DyLight 680B-NHS (blue), 679 Compound 1-NHS (red), and Company B Compound-NHS (black). The maximum absorbance and emission for each compound is shown below.

(46) TABLE-US-00002 Max Abs Max E (nm) nm) DyLight 680 677 715 DyLight 680B 680 704 679 Compound 1 679 704 Company B 676 706 Compound

(47) The following properties of 679 Compound 1-NHS, 679 Compound 4/4-NHS (V08-15173), and 679 Compound 4/4-NHS (V10-04152) were compared with commercially available dyes.

(48) TABLE-US-00003 Company B Company A DyLight 679 Compound V08-15173 V10-04152 Compound- Compound- 680B-NHS 1-NHS NHS NHS NHS NHS MW (g/mol) 1196.16 1240.21 1728.8 1524.75 ~1150 3241 Ex (nm) 679 679 684 689 679 681 Em (nm) 698 698 706 721 702 698 (M.sup.1cm.sup.1) 180,000 180,000 180,000 180,000 184,000 210,000 (theoretical) PEG (length/ 0 1/1 4/4 4/4 N/A ? # of chain) Sulfonate 5 5 4 2 3 ?

EXAMPLE 7

(49) Inventive and commercial compounds, each as the NHS ester, were conjugated to goat anti-mouse (GAM) antibodies, goat anti-rabbit (GAR) antibodies, and streptavidin (SA). GAM, GAR, and SA, at a concentration of 10 mg/ml in phosphate buffered saline (PBS), were dialyzed against 50 mM borate buffer, pH 8.5. The compounds were reconstituted in dimethylformamide (DMF) at 10 mg/ml and combined at 2, 4, 5, 7.5, or 10 molar excess with GAM, GAR, or SA for two hours at room temperature to label the antibodies or SA.

(50) The labeled compounds, also termed dyes or labels, were subjected to Pierce Dye Removal Resin (PDDR) to remove the unlabeled (free) compound; 100 l of the packed resin was used per mg of protein purified. The purified antibody-labeled dyes were then diluted 1:50 in PBS and scanned for absorbance from 700 nm to 230 nm to determine the protein concentration, and to determine the mole dye to mole protein ratio. Each conjugate was diluted 1:10 in 50% glycerol and heated in the presence of 10 mM dithiothreitol (DTT) for 5 min at 95 C., then separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE). The gels were scanned using the Typhoon 9400 Imager to verify removal of the unconjugated compound. Labeling efficiency was compared, with results showing degree of labeling below.

(51) TABLE-US-00004 DyLight DyLight 679 Company B 680 680B Compound 1 Compound Cy5.5 GAM-2X 0.9 1.9 2.0 1.6 GAM-3X 1.3 2.7 3.0 2.2 GAM-4X 2.0, 1.9 3.6, 3.6 3.6, 3.7 2.8 2.6 GAM-6X 2.3 5.0 5.1 3.9 GAM-8X 2.4 5.6 5.0 4.3 GAM-10X 3.0 6.5 6.8 5.4 GAM-12X 3.0 7.4 7.2 6.2 GAR-1X 0.5 0.9 1.0 0.7 GAR-2X 1.0 1.5 1.6 1.2 GAR-3X 1.3 2.1 2.4 1.7 GAR-4X 1.7, 1.8 2.6, 3.1 3.1, 3.1 2.3 2.2 GAR-6X 2.2 4.4 GAR-8X 2.4 5.5 5.6 4.2 GAR-10X 3.5 6.0 6.7 5.3 GAR-12X 3.4 6.6 6.7 6.2 SA-6X 2.9 4.1 4.0 4.1 SA-8X 3.4 4.7 5.0 4.8

(52) Labeling efficiency of GAM, GAR and SA was equivalent for 679 compound 1-NHS compared to DyLight 680B-NHS and higher compared to DyLight 680 and Company B compound. The antibodies were labeled, purified, and evaluated by SDS-PAGE as described above. Antibodies were also labeled with Company A compound, which was reconstituted in dimethylsulfoxide (DMSO) and combined at 2, 5, 7.5, 10, and 15 molar excess with GAM or GAR for sixty-five minutes at room temperature to label the antibodies.

(53) Inventive and commercial compounds, each as the NHS ester, were conjugated to goat anti-rat (GARat) antibodies. GARat, at a concentration of 10 mg/ml in phosphate buffered saline (PBS), was spiked with 10% v/v with 0.67 M borate buffer. The compounds were combined at 5 or 10 molar excess with GAR at for 65 minutes at room temperature to label the antibody. The antibodies were labeled, purified and evaluated by SDS-PAGE as described above.

(54) Labeling efficiency of GARat was slightly higher for 679 Compound 1-NHS compared to DyLight 680B-NHS, V08-15173-NHS and Company B compound-NHS.

(55) In another set of experiments, the labeling of GAM with the inventive and commercial compounds is shown below.

(56) TABLE-US-00005 Mole Dye/Mole Mole Dye/Mole Mole Dye/Mole Mole Dye/Mole Mole Dye/Mole Protein Ratio @ Protein Ratio @ Protein Ratio @ Protein Ratio @ Protein Ratio @ 2.5 X 5 X 7.5 X 10 X 15 X V08-15173 2.5 4.9 7.1 8.9 12.4 V10-04152 2.8 5.1 7.4 10.0 13.9 DY679P1 1.8 4.1 6.6 8.5 12.9 Company A 1.1 5.3 7.4 9.4 12.6 compound Company B 1.8 4.0 6.3 8.3 13.1 compound

(57) Labeling efficiency of GAM was similar for all the dyes at all molar excesses.

(58) In another set of experiments, the labeling of GAR with the inventive and commercial compounds is shown below, at a molar excess of 5, 15, and 25.

(59) TABLE-US-00006 Mole Dye/Mole Mole Dye/Mole Mole Dye/Mole Protein Ratio @ Protein Ratio @ Protein Ratio @ 5 X 15 X 25 X V08-15173 4.6 12.7 19.6 V10-04152 2.2 10.1 13.9 DY679P1 4.8 10.6 14.4 Company A 3.4 9.8 15.4 Compound Company B 4.1 15.2* 46.7* Compound

(60) At 5, the labeling efficiency of GAR for all the dyes was similar except for V10-04152. At 15 and 25, the labeling efficiency was similar except for Company B compound. *Company B compound conjugates precipitated in the Slide-A-Lyzer at molar excesses greater than 10, indicating this method of purification was not suitable for Company B dyes.

EXAMPLE 8

(61) Performance of the dye-GAM conjugates, dye-GAR conjugates, and dye-SA conjugates was evaluated in a functional assay. Wells of a 96 white opaque plate or black clear-bottom plate were coated with target proteins mouse IgG immunoglobulin, rabbit IgG immunoglobulin, or biotinylated bovine serum albumin (BBSA). One hundred l mouse or rabbit IgG, or BBSA at a concentration of 10 g/ml was applied to the corresponding wells in columns 1 and 2. The target proteins were serially diluted 1:1 from the wells in columns 2 to 11 using 100 l PBS. One hundred l of the samples from the wells in column 11 were discarded. One hundred l PBS was added to the wells in column 12. The plates were incubated overnight at 4 C. and then blocked 2200 l with Thermo Scientific SuperBlock Blocking Buffer. The coated plates were washed 2200 l with PBS-Tween and 1200 l with PBS. Based on the calculated concentrations, conjugates were diluted 1:250 in PBS, added to the corresponding plates (100 l/well) and then incubated for one hour in the dark. The plates were washed with 2200 l with PBS-Tween and 1200 l with PBS and filled with PBS buffer (100 l/well) prior to scanning the white opaque plates on Tecan Safire using 679 nm.sub.excitation/702 nm.sub.emission or scanning the black clear-bottom plates on LiCor Odyssey at 700 channel, to detect fluorescence intensity.

(62) As shown in FIGS. 2-7, RFU and/or signal to background ratio (S/B) of the dyes were compared at various concentrations, using the indicated conjugation conditions.

(63) FIG. 2 shows Tecan Safire results of a functional assay using GAM conjugated with either 8 molar excess of the dyes (solid lines) or 10 molar excess of the dyes (dashed lines) of DyLight 680 (purple filled diamond/open square); DyLight 680B (blue filled triangle/X); 679 Compound 1 (red filled circle/asterisk); and Company B Compound (black filled square/+ sign). DyLight 680B-GAM (8) showed higher binding fluorescence compared to corresponding 679 Compound 1-GAM (8). At 10 molar excess, 679 Compound 1-GAM showed similar performance to DyLight 680B-GAM. Both DyLight 680 and Company B compound showed much lower intensity compared to DyLight 680B and 679 Compound 1 conjugates. Signal/Background (S/B) was higher for DyLight 680B-GAM conjugates than 679 Compound 1-GAM conjugates. Similar results were generally obtained for GAR conjugates.

(64) FIG. 3 shows Tecan Safire results of a functional assay using SA conjugated with either 6 molar excess of the dyes (solid lines) or 8 molar excess of the dyes (dashed lines) of DyLight 680 (purple filled diamond/open square); DyLight 680B (blue filled triangle/X); 679 Compound 1 (red filled circle/asterisk); and Company B Compound (black filled square/+ sign). 679 Compound 1-SA (6, 8) showed slightly lower binding fluorescence compared to corresponding DyLight 680B-SA (6, 8). There was no quenching trend with the conjugates at higher molar excesses. The S/B was slightly lower for 679 Compound 1-SA conjugates than for DyLight 680B-SA conjugates.

(65) FIG. 4 shows LiCor Odyssey results of a functional assay using GAM conjugated with either 2 molar excess of the dyes (solid lines) or 4 molar excess of the dyes (dashed lines) of DyLight 680 (purple diamond); DyLight 680B (blue triangle); 679 Compound 1 (red circle); and Cy5.5 (black asterisk). The following table compared S/B and raw intensity data.

(66) TABLE-US-00007 @ 1250 ng @ 39 ng @ 0 S/B coating coating (blank) DyLight 680-GAM-2X 25.3 9.8 1.0 DyLight 680-GAM-4X 6.4 2.3 1.0 DyLight 680B-GAM-2X 18.2 4.6 1.0 DyLight 680B-GAM-4X 31.9 8.1 1.0 679 Compound 1-GAM-2X 36.1 7.9 1.0 679 Compound 1-GAM-4X 65.2 15.9 1.0 Cy5.5-GAM-2X 62.3 17.7 1.0 Cy5.5-GAM-4X 19.6 6.1 1.0 @ 1250 ng @ 39 ng @ 0 Raw Intensity coating coating (blank) DyLight 680-GAM-2X 1941300 751514 76795 DyLight 680-GAM-4X 2596363 937396 403405 DyLight 680B-GAM-2X 5179777 1311798 284501 DyLight 680B-GAM-4X 8614438 2190343 270357 DY679P1-GAM-2X 5180336 1135001 143352 DY679P1-GAM-4X 7482510 1820225 114807 Cy5.5-GAM-2X 3957169 1124788 63513 Cy5.5-GAM-4X 4379901 1358039 223585

(67) FIG. 5 shows LiCor Odyssey results of a functional assay using GAR conjugated with either 2 molar excess of the dyes (solid lines) or 4 molar excess of the dyes (dashed lines) of DyLight 680 (purple diamond); DyLight 680B (blue triangle); 679 Compound 1 (red circle); and Cy5.5 (black asterisk). The following table compared S/B and raw intensity data.

(68) TABLE-US-00008 @ 1250 ng @ 39 ng @ 0 S/B coating coating (blank) DyLight 680-GAR-2X 66.7 19.8 1.0 DyLight 680-GAR-4X 62.2 17.4 1.0 DyLight 680B-GAR-2X 210.6 47.5 1.0 DyLight 680B-GAR-4X 167.2 39.3 1.0 DY679P1-GAR-2X 184.0 39.2 1.0 DY679P1-GAR-4X 141.9 33.4 1.0 Cy5.5-GAR-2X 57.3 16.1 1.0 Cy5.5-GAR-4X 26.7 9.0 1.0 @ 1250 ng @ 39 ng @ 0 Raw Intensity coating coating (blank) DyLight 680-GAR-2X 1868227 554324 28015 DyLight 680-GAR-4X 2140077 597726 34391 DyLight 680B-GAR-2X 4178812 943008 19839 DyLight 680B-GAR-4X 6285439 1476347 37592 DY679P1-GAR-2X 4039443 860737 21950 DY679P1-GAR-4X 6527381 1537020 46001 Cy5.5-GAR-2X 2408459 675886 42049 Cy5.5-GAR-4X 2929556 988970 109613

(69) FIG. 6 shows LiCor Odyssey results of a functional assay using GAM conjugated with either 8 molar excess of the dyes (solid lines) or 10 molar excess of the dyes (dashed lines) of DyLight 680 (purple diamond); DyLight 680B (blue square); 679 Compound 1 (red triangle); and Company B Compound (black X). The following table compared S/B data.

(70) TABLE-US-00009 2500 ng 39.1 ng mouse mouse IgG/well IgG/well DyLight 680-GAM-8X 173.6 48.9 DyLight 680-GAM-10X 137.2 37.6 DyLight 680B-GAM-8X 929.3 142.4 DyLight 680B-GAM-10X 914.5 165.6 DY679P1-GAM-8X 675.2 110.5 DY679P1-GAM-10X 871.5 183.5 Company B Compound-GAM-8X 459.9 112.4 Company B Compound-GAM-10X 374.0 96.1

(71) 679 Compound 1-GAM (10) showed equivalent bound fluorescence compared to corresponding DyLight 680B-GAM (10). S/B was lower for 679 Compound 1-GAM (8,10) compared to DyLight 680B-GAM (8,10). In general, similar results were obtained for GAR conjugates.

(72) FIG. 7 shows LiCor Odyssey results of a functional assay using SA conjugated with either 6 molar excess of the dyes (solid lines) or 8 molar excess of the dyes (dashed lines) of DyLight 680 (purple diamond); DyLight 680B (blue square); 679 Compound 1 (red triangle); and Company B Compound (black X). The following table compared S/B data.

(73) TABLE-US-00010 250 ng 3.9 ng BBSA/well BBSA/well DyLight 680-SA-6X 614.5 33.1 DyLight 680-SA-8X 436.3 25.1 DyLight 680B-SA-6X 2196.7 43.8 DyLight 680B-SA-8X 1969.5 75.6 DY679P1-SA-6X 1742.1 58.5 DY679P1-SA-8X 1406.0 54.7 Company B Compound-SA-6X 570.2 49.7 Company B Compound-SA-8X 427.7 36.1

(74) 679 Compound 1-SA (6, 8) showed lower bound fluorescence and S/B compared to corresponding DyLight 680B-SA (6, 8).

(75) 679 Compound 1 and Company Compound were conjugated to GAR at high molar excesses, and evaluated in a functional assay, as described above. FIG. 8 shows results expressed as RFU of a functional assay using GAR conjugated with 679 Compound 1 at either a 7.5 molar excess (blue diamond), 15 molar excess (red square), or 22.5 molar excess (green triangle), and Company A Compound at either 7.5 molar excess (purple X), 15 molar excess (turquoise asterisk), or 22.5 molar excess (orange circle).

(76) FIG. 9 shows VarioSkan Flash results with excitation and emission of 679 nm/702 nm, expressed as RFU of a functional assay using GAM conjugated with 5 molar excess of Company B compound (orange diamond), V08-15173 (blue triangle), and V10-04152 (red circle). Based on the data, V08-15173-GAM (5) showed higher binding fluorescence compared to Company B Compound-GAM (5).

(77) FIG. 10 shows LiCOR Odyssey results, using the 700 channel, expressed as Raw Fluorescence Intensity of a functional assay using GAM conjugated with 5 molar excess of 679 Compound 1/1 (green triangle), Company A Compound (purple circle), and V08-15173 (blue square). FIG. 11 shows LiCOR Odyssey results expressed as S/B of a functional assay using GAM conjugated at 2.5, 5, 10, or 15 molar excess of 679 Compound 1/1, Company A Compound, and V08-15173. V08-15173-GAM (5) showed similar binding fluorescence to Company A Compound-GAM (5). 679 Compound 1/1-GAM (5) binding fluorescence was lower compared to the other two conjugates.

(78) FIG. 12 shows LiCOR Odyssey results, using the 700 channel, expressed as Raw Fluorescence Intensity of a functional assay using GAR conjugated with 5 and 25 molar excess of 679 Compound 1/1 (green 5, diamond, solid line; green 25, diamond, dashed line), Company A Compound (purple 5, triangle, solid line; purple 25, triangle, dashed line), V08-15173 (light blue 5, square, solid line; dark blue 25, square, dashed line), and V10-04152 (yellow 5, circle, solid line; red 25, circle, dashed line). FIG. 13 shows LiCOR Odyssey results expressed as S/B of a functional assay using GAR conjugated at 5, 15, or 25 molar excess of 679 Compound 1/1, Company A Compound, V08-15173, and V10-04152. Based on the Raw Fluorescence Intensity data, there was no apparent quenching for the V08-15173-GAR at 25 molar excess. 679 Compound 1/1, V10-04152, and Company A Compound were saturating at 25. FIG. 14 shows summary Raw Fluorescence Intensity data, and apparent quenching at 1000 ng/well at 25 for 679 Compound 1/1 (679 1), V10-04152, and Company A Compound, but no apparent quenching at 25 for V08-15173.

EXAMPLE 9

(79) The inventive compounds were evaluated for immunofluorescence in cell based assays using the following protocol. Frozen A549 cell plates stored at 20 C. were placed for 30 min 50 C. Storage buffer (PBS) was removed and the cells were permeabilized for 15 min (100 l/well) with 0.1% Triton-X100 in 1PBS buffer. Plates were blocked for 30 min in 2% BSA in 1PBS-0.1% Trion-X100. Primary antibodies diluted in 2% BSA in 1PBS-0.1% Trion-X100 were added to the plates (column 1-11; column 12 included only blocker) and incubated three hours at room temperature. Mouse anti-lamin A was added at 1 g/ml and rabbit anti-lamin B1 was added at 3 g/ml. After overnight incubation, the antibody solution was removed from the plates and the plates were washed with PBS-0.5% Tween-20 (2100 l/well). GAM and GAR secondary antibodies labeled with DyLight 680-NHS, DyLight 680B-NHS, 679 Compound 1-NHS and Company B Compound-NHS were diluted to 4 g/ml in PBS, added and incubated for one hour at room temperature. The plates were then washed three times with 100 l/well PBS, and Hoechst stain diluted to 0.1 g/ml in PBS was added to each well (100 l/well). The plates were scanned on ArrayScan Plate Reader for imaging and quantitation.

(80) FIG. 15 shows results of an immunofluorescence assay using mouse anti-lamin A as a primary antibody, and DyLight 680-GAM (FIG. 15A), DyLight 680B-GAM (FIG. 15B), 679 Compound 1-GAM (FIG. 15C), or Company B Compound-GAM (FIG. 15D) as secondary antibody, where the compound was conjugated to GAM (secondary antibody) at 4 molar excess (column 1), 6 molar excess (column 2), 8 molar excess (column 3), 10 molar excess (column 4), or 12 molar excess (column 5). 679 Compound 1 conjugated to GAM showed very similar performance to corresponding DyLight 680B conjugates.

(81) The inventive compounds and commercial dye were evaluated for immunofluorescence in a second cell based assay using the following protocol. Frozen U20S cell plates stored at 20 C. were placed overnight at 4 C. Storage buffer (PBS) was removed and the cells were permeabilized for 15 min (100 l/well) with 0.1% Triton-X100 in 1PBS buffer. Plates were blocked for 30 min in 2% BSA in 1PBS-0.1% Trion-X100. Primary antibodies diluted in 2% BSA in 1PBS-0.1% Trion-X100 were added to the plates (column 1-11; column 12 included only blocker) and incubated five hours at room temperature. Mouse anti-lamin A was added at 2 g/ml and rabbit anti-lamin B1 was added at 4 g/ml. After incubation, the antibody solution was removed from the plates and the plates were washed PBS-0.5% Tween-20 (2100 l/well). Next, GAM and GAR secondary antibodies labeled with DyLight 680-NHS, DyLight 680B-NHS, 679 Compound 1-NHS, and Cy5.5 Mono Ester were diluted to 4 g/ml in PBS and incubated with the cells for one hour at room temperature. GAM and GAR conjugated to DyLight 680-NHS, DyLight 680B-NHS, 679 Compound 1-NHS, and Company B Compound at 10 molar excess were diluted to 4 g/ml in PBS and then serially diluted 1:1 in the plate to the following concentrations: 2 g/ml, 1 g/ml, 0.5 g/ml, 0.25 g/ml, 0.125 g/ml and/or 0.0625 g/ml. The plates were washed 3 with 100 l/well PBS, and Hoechst stain diluted to 0.1 g/ml in PBS was added to each well (100 l/well). The plates were scanned on ArrayScan Plate Reader for imaging and quantitation.

(82) FIG. 16 shows results of an immunofluorescence assay using mouse anti-lamin A as a primary antibody, and either DyLight 680-GAM (FIG. 16A), DyLight 680B-GAM (FIG. 16B), 679 Compound 1-GAM (FIG. 16C), or Cy5.5-GAM (FIG. 16D) as secondary antibody, where the compound was conjugated to GAM (secondary antibody) at 2 molar excess (column 1), 3 molar excess (column 2), or 4 molar excess (column 3). Performance in immunofluorescence of 679 Compound 1 conjugate was similar to the performance of corresponding DyLight 680B conjugates. Quantitative analysis of FIG. 16 data, expressed as Mean Total Intensity, which is the average total intensity of all pixels within a defined area or defined primary object such as a nucleus, is shown below.

(83) TABLE-US-00011 Mean Total Intensity 2X 3X 4X DyLight 680 91899 122829 144792 DyLight 680B 150542 228678 305700 DY679P1 232482 263580 330865 Cy5.5 211945 342208 451111

(84) Fluorescence signal intensity for DyLight 680B and 679 Compound 1 GAM conjugates was 2-4 times higher, depending on the molar excess, compared to DyLight 680 or Cy5.5 GAM conjugates, and S/B for DyLight 680B & 679 Compound 1 GAM conjugates at the low molar excesses was comparable to each other and to DyLight 680. Overall fluorescence signal intensity for DyLight 680B and 679 Compound 1 GAR conjugates was about two times higher compared to DyLight 680. The S/B for DyLight 680B and 679 Compound 1 GAR labeled at 2, 3 or 4 molar excess were comparable to each other and to DyLight 680 conjugates.

(85) In the indicated experiments, the inventive compounds were evaluated for immunofluorescence in cell based assays using the following protocol. Frozen A549 cell plates stored at 20 C were placed for 30 min 50 C. Storage buffer (PBS) was removed and the cells were permeabilized for 15 min (100 l/well) with 0.1% Triton-X100 in 1PBS buffer. Plates were blocked for 30 min in 2% BSA in 1PBS-0.1% Trion-X100. Primary antibodies diluted in 2% BSA in 1PBS-0.1% Trion-X100 were added to the plates (column 1-11; column 12 included only blocker) and incubated three hours at room temperature. Mouse anti-lamin A was added at 1 g/ml and rabbit anti-lamin B1 was added at 3 g/ml. After overnight incubation, the antibody solution was removed from the plates and the plates were washed with PBS-0.5% Tween-20 (2100 l/well). GAM and GAR secondary antibodies labeled with DyLight 680-NHS, DyLight 680B-NHS, 679 Compound 1-NHS and Company B Compound-NHS were diluted to 4 g/ml in PBS, added and incubated for one hour at room temperature. The plates were then washed three times with 100 l/well PBS, and Hoechst stain diluted to 0.1 g/ml in PBS was added to each well (100 l/well). The plates were scanned on ArrayScan Plate Reader for imaging and quantitation.

(86) In the indicated experiments, the inventive compounds and commercial dye were evaluated for immunofluorescence in a cell based assay using the following protocol. Frozen U20S cell plates which were stored at 80 C. were thawed for 45 minutes at 50 C. Storage buffer (PBS) was removed and the cells were permeabilized for 15 minutes with 0.1% Triton-X100 in 1PBS buffer (100 l/well). The cell plate was blocked for 60 minutes in 2% BSA/PBS-0.1% Triton-X100. Primary antibody, either rat anti-Grp94 (5 g/ml), mouse anti-lamin A (10 g/ml), or rabbit anti-lamin B1 (10 g/ml), diluted in 2% BSA/PBS-0.1% Triton-X100 was added to the plate and incubated for 1 hour at room temperature. Control wells contained only 2% BSA/PBS-0.1% Triton-X100 blocker. After incubation, the antibody solution was removed from the plate and the plate was washed three times with 100 l/well of PBS-0.5% Tween-20 and one time with 100 l/well PBS. GARat, GAM, or GAR secondary antibodies labeled with various molar excess of the inventive or commercial compound were diluted to 4 g/ml in PBS and incubated for 1 hour at room temperature. The plates were washed three times with 100 l/well of PBST and once with 100 l/well PBS, and Hoechst (diluted to 0.1 g/ml in PBS) was added to each well (100 l/well). The plates were scanned on ArrayScan Plate Reader or ToxInsight Instrument.

(87) FIG. 17 shows detection of Grp94 in U20S cells (column 1) with 679 Compound 1-GARat (FIG. 17A), DyLight 680B-GARat (FIG. 17B), V08-15173-GARat (FIG. 17C), and Company B Compound-GARat (FIG. 17D) conjugated at a 5 molar excess; and associated controls (column 2).

(88) FIG. 18 shows detection of Grp94 in U20S cells (column 1) with 679 Compound 1-GARat (FIG. 18A), DyLight 680B-GARat (FIG. 18B), V08-15173-GARat (FIG. 18C), and Company B Compound-GARat (FIG. 18D) conjugated at a 10 molar excess; and associated controls (column 2).

(89) As shown in FIGS. 17-18, no non-specific binding was observed with V08-15173-GARat and Compound B Compound-GARat conjugates but there was with DY679P1 and DyLight 680B-GARat conjugates.

(90) Quantitative analysis of the data of FIGS. 17-18, expressed as Mean Total Intensity, which is the average total intensity of all pixels within a defined area or defined primary object such as a nucleus, is shown below.

(91) TABLE-US-00012 Negative Controls 5X 10X 5X 10X S/B (5X) S/B (10X) 679 Compound 1 - 81221 97162 36744 70585 2.2 1.4 GARat DyLight 680B-GARat 77855 91720 34773 61355 2.2 1.5 V08-15173-GARat 76825 70881 28190 26341 2.7 2.7 Company B Compound- 64881 56119 26762 30379 2.4 1.8 GARat
S/B was slightly better for V08-15173-GARat conjugates (5, 10) compared to the corresponding 679 Compound 1-GARat, DyLight 680B-GARat, and Company B Compound-GARat conjugates.

(92) 679 Compound 1-GAM, Company A Compound-GAM, and Company A Compound R-GAM were evaluated for immunofluorescence in a cell based assay using detection of PDI in cells with a mouse anti-PDI antibody. FIG. 19 shows results of 679 Compound 1-GAM at a 15 molar excess (FIG. 19A), Company A Compound-GAM at a 15 molar excess (FIG. 19B), and Company A Compound R-GAM at a 15 molar excess (FIG. 19C). As FIG. 19 shows, 679 Compound 1-GAM exhibited proper staining of PDI in the ER while Company A Compound-GAM and Company A Compound R-GAM exhibited non-specific staining with staining found throughout the cell. Similar results were obtained for 679 Compound 1-GAM at 7.5 and 22.5 molar excesses, as well as Company A Compound-GAM and Company A Compound R-GAM at 7.5 molar excess (data not shown).

(93) FIG. 20A-C shows detection of lamin A in U20S cells (column 1) with V08-15173-GAM, V10-04152-GAM, 679 Compound 1/1-GAM, Company A Compound-GAM, and Company B Compound-GAM conjugates (4 g/ml) at 5 molar excess (FIG. 20A), 10 molar excess (FIG. 20B), and 15 molar excess (FIG. 20C) with V08-15173-GAM (row A FIGS. 20A, 20B, 20C), V10-04152-GAM (row B FIGS. 20A, 20B, 20C), 679 Compound 1/1-GAM (row C FIGS. 20A, 20B, and 20C), Company A Compound-GAM (row D FIGS. 20A, 20B, 20C), Company B Compound-GAM (row E FIGS. 20A, 20B, 20C), and associated negative controls (column 2). There was very little non-specific binding observed with V08-15173-GAM and 679 Compound 1/1-GAM. Company A Compound-GAM and Company B Compound-GAM conjugates showed high non-specific binding starting from 5 molar excess. At low molar excess, Company A Compound-GAM exhibited staining of the nucleus, which was greater than the other dyes. V10-04152-GAM exhibited good specificity but was not very bright. Staining of the nucleus with Company B Compound-GAM was greatly improved at 15 molar excess, but there was an increase in non-specific binding.

(94) The following table shows quantitative analysis of the FIGS. 20A-C data expressed as Mean Total Intensity, which is the average total intensity of all pixels within a defined area or defined primary object such as a nucleus, and S/B ratios.

(95) TABLE-US-00013 679 Compound Company A Company B V08-15173 V10-04152 1/1 Compound Compound Negative Negative Negative Negative Negative Average control control control control control 2.5 X 17366 5966 11946 6085 29229 5628 54496 9107 7092 6329 5.0 X 32820 6643 12788 7357 49279 5859 72289 20303 11922 8360 7.5 X 42876 7100 12513 6503 28782 152502 51104 18894 13351 10 X 47756 9690 21147 6266 30098 6115 188505 136044 25020 18330 15 X 49060 10779 15942 6746 48071 7277 679693 665370 67705 24350 679 Compound Company A Company B S/B V08-15173 V10-04152 1/1P1 Compound Compound 2.5 X 2.9 2.0 5.2 6.0 1.1 5.0 X 4.9 1.7 8.4 3.6 1.4 7.5 X 6.0 1.9 4.9 3.0 1.4 10 X 4.9 3.4 4.9 1.4 1.4 15 X 4.6 2.4 6.6 1.0 2.8

(96) Lamin A, a nuclear protein, should show staining specific to the nucleus. Any lamin A staining outside the nucleus is non-specific staining. In addition, negative control conditions that lack a primary antibody are also used to determine the antibody staining specificity. Company A and Company B Compounds showed no non-specific binding. V08-15173 and 679 Compound 1/1 exhibited a minimal amount of non-specific binding. Due to the strong non-specific binding at higher molar excesses, Company A and Company B Compounds exhibited decreased signal to background (S/B) levels.

(97) Quantitative analysis of a repeat experiment of FIG. 20A-C are shown below.

(98) TABLE-US-00014 Company A 679 Compound V08-15173 Compound 1/1 Negative Negative Negative Average control control control 2.5 X 118440 12053 198471 24758 77195 8711 5 X 183078 9441 351666 84270 140772 8674 10 X 391473 13094 638569 330337 159948 20481 15 X 211270 17260 560119 632936 199626 16277 Company A 679 Compound S/B V08-15173 Compound 1/1 2.5 X 9.8 8.0 8.9 5 X 19.4 4.2 16.2 10 X 29.9 1.9 7.8 15 X 12.2 0.9 12.3

(99) Company A Compound showed much higher non-specific binding compared to V08-15173 and 679 Compound 1/1, with the non specific binding appearing at the 2.5 condition for Company A Compound-GAM.

(100) 679 Compound 1/1-NHS and V08-15173-NHS showed on average a 20% lower intensity compared to Company A Compound-NHS. V10-04152 intensity was about 50% lower than V08-15173, and about 65% lower than Company A Compound. GAM labeling efficiency was similar for all dyes at all molar excesses. At 5 molar excess, the GAR labeling efficiency for all dyes was similar, except for V10-04152. At 15 and 25 molar excesses, the GAR labeling efficiency similar for all compounds except Company B Compound. In immunofluorescence studies, Company A Compound, 679 Compound 1/1, and Company B Compound GAM conjugates showed high non-specific binding. V08-15173 and V10-04152 GAM conjugates showed little non-specific binding. Performance of conjugates in immunofluorescence appeared to be highly dependent on the performance of the primary antibody.

EXAMPLE 10

(101) The inventive compounds are used for in vivo imaging to obtain information about biological tissues that are primarily accessible. The compounds are responsive to light in the near infrared region of the spectrum, a part of the spectrum that has minimal interference from the absorbance of biological materials. In one embodiment, the compounds are used for fluorescent imaging of targets within animals. For example, in vivo imaging information can be obtained using methods such as X-ray, magnetic resonance imaging, positron emission tomography, ultrasound imaging and probing, and other non-invasive methods used for diagnosing and treating disease. Light in the near infrared range (NIR), from about 650 nm to about 1000 nm wavelength, can permeate through several centimeters of tissue and thus can be used for in vivo imaging. Fluorescent dyes, such as the inventive compounds that are responsive to light in these longer wavelengths, can be used as conjugates with targeting molecules such as antibodies to bind and accumulate in, e.g., diseased tissue such as tumors, and may be used to distinguish healthy from diseased tissue. In some methods, the inventive compound may be attached to a biomolecule, such as a protein, peptide, or a drug, which is localized or retained in the desired tissue environment. Fluorescent in vivo imaging using NIR dyes such as the inventive compounds are diagnostic agents to discretely target disease tissue directly within animals or humans.

(102) For in-vivo imaging, the compound, an isomer of the compound, or a conjugate of the compound or isomer with a targeting agent, is administered to a tissue (e.g., intravenously), permitted to accumulate with excess compound removed by the circulatory system, then the tissue is irradiated with light at an appropriate wavelength. The NIR fluorescent light is recorded and/or an image is generated from the data obtained to specifically detect and visualize the targeted cells or tissues. The dose of compound administered can differ and would be known by one skilled in the art depending upon the specific tissue, application, etc., as long as the method achieves a detectable concentration of the compound in the tissue to be assessed.

EXAMPLE 11

In Vivo Imaging Using an Inventive Compound Conjugated to Anti-HER2 Antibody

(103) 779 Compound 1-NHS is conjugated to a rabbit anti-HER2 antibody (Genscript USA, Piscataway N.J.) by reconstituting the compound in dimethylformamide (DMF) at 10 mg/ml, then incubating at 10 molar excess with rabbit anti-HER2 antibody (0.1 mg) for one hour at room temperature to result in 779 Compound 1-anti-HER2 conjugate. The conjugation reaction is then subjected to PDDR to remove unlabeled (free) 779 Compound 1. Ten microgram of conjugate is injected intravenously to athymic mice bearing BT474 tumors. The animals are imaged overtime at 1, 24, 48, 72, 96, and 120 hours post-injection using Pearl Imager, LI-COR Biosciences (LI-COR Instruments, Lincoln Nebr.).

(104) Upon whole body imaging, fluorescence intensity is observed to be distributed over the whole animal during the first hour imagining and diminishes significantly at 72 hours. After 96 hours, the signal is localized and specific to the tumor.

(105) 679 Compound 4/4-NHS is conjugated to a rabbit anti-HER2 antibody (Genscript USA, Piscataway N.J.) by reconstituting the compound in dimethylformamide (DMF) at 10 mg/ml, then incubating at 10 molar excess with rabbit anti-HER2 antibody (0.1 mg) for one hour at room temperature to result in 679 Compound 4/4-anti-HER2 conjugate. The conjugation reaction is then subjected to PDDR to remove unlabeled (free) 679 Compound 4/4. Ten microgram of conjugate is injected intravenously to athymic mice bearing BT474 tumors. The animals are imaged over time at 1, 24, 48, 72, 96, and 120 hours post-injection using Pearl Imager, LI-COR Biosciences (LI-COR Instruments, Lincoln Nebr.).

(106) Upon whole body imaging, fluorescence intensity is observed to be distributed over the whole animal during the first hour imagining and diminishes significantly at 72 hours. After 96 hours, the signal is localized and specific to the tumor.

EXAMPLE 12

In Vivo Imaging Using Either Monosulfonated or Disulfonated Inventive Compound

(107) The compound may be rendered less hydrophilic, i.e., more hydrophobic, by altering the number of sulfonate groups. The fewer sulfonates, the more hydrophobic the compound becomes. In this embodiment, the compound may be more readily retained in a desired tissue or location if the appropriate number of sulfonates is determined; e.g., compound penetration into cells is more efficient if fewer sulfonates are present on the molecule. The compound may contain one, two, three, or four sulfonate groups. Hydrophobic compounds are also known to more efficiently cross the cell membrane, and thus are more desirable when the target of interest is located within the cell.

(108) Alendronate, a compound that binds to, and is retained in, LNCap prostate cancer cells, is conjugated with disulfonated or monosulfonated benzo 779 Compound 1 by incubating a solution containing 1 mM disulfonated or monosulfonated 779 Compound 1-NHS in 1 ml of PBS and 0.5 ml tetrahydrofuran (THF) with 0.1 mM alendronate and 0.2 mM diisopropylethylamine at room temperature overnight. The conjugate is purified using reverse phase HPLC with 0-50% methanol against a 0.1 M ammonium acetate buffer, and is then lyophilized.

(109) LNCap cells are grown orthotopically in nude mice. 779 Compound 1-alendronate (5 nmole) is injected into the tumor. Control mice are injected with free 779 Compound 1 containing a carboxylic acid residue instead of the reactive NHS ester. X-ray and near infra-red fluorescence images are captured.

(110) Upon imaging the whole mouse, 779 Compound 1-alendroneate conjugate is retained in mouse tissue greater than the unconjugated compound; the conjugate is retained in the LNCap cell-induced tumor for at least 18 hrs.

(111) Alendronate, a compound that binds to, and is retained in, LNCap prostate cancer cells, is conjugated with disulfonated or monosulfonated 679 Compound 4/4 by incubating a solution containing 1 mM disulfonated or monosulfonated 679 Compound 4/4-NHS in 1 ml of PBS and 0.5 ml tetrahydrofuran (THF) with 0.1 mM alendronate and 0.2 mM diisopropylethylamine at room temperature overnight. The conjugate is purified using reverse phase HPLC with 0-50% methanol against a 0.1 M ammonium acetate buffer, and is then lyophilized.

(112) LNCap cells are grown orthotopically in nude mice. 679 Compound 4/4-alendronate (5 nmole) is injected into the tumor. Control mice are injected with free 679 Compound 4/4 containing a carboxylic acid residue instead of the reactive NHS ester. X-ray and near infra-red fluorescence images are captured.

(113) Upon imaging the whole mouse, 679 Compound 4/4-alendroneate conjugate is retained in mouse tissue greater than the unconjugated compound; the conjugate is retained in the LNCap cell-induced tumor for at least 18 hrs.

EXAMPLE 13

In Vivo Imaging Using Either Monosulfonated or Disulfonated Inventive Compound

(114) A drug delivery nanoparticle system conjugated with disulfonated or monosulfonated 779 Compound 1 is prepared as follows. A solution containing 1 mM disulfonated or monosulfonated 779 Compound 1-NHS in 1 ml of PBS is incubated overnight at room temperature with 0.1 mM of an anti-cancer drug conjugated with transferrin in the form of a nanoparticle. The resulting 779 Compound 1-nanoparticle conjugate is purified by centrifugation and then lyophilized.

(115) The 779 Compound 1 (isomer 1)-nanoparticle conjugate (1 nmole) is injected intravenously into a mouse tail vein. Control mice are injected with free 779 Compound 1 dye. X-ray and near infra-red fluorescence images of mouse brain are captured.

(116) 779 Compound 1-nanoparticle conjugate localizes in the mouse brain for greater than about 24 hours after injection. Tumor size progressively decreases after injection of 779 Compound 1-nanoparticle conjugate, compared to 779 Compound 1-nanoparticle without the anti-cancer drug.

(117) A drug delivery nanoparticle system conjugated with disulfonated or monosulfonated 679 Compound 4/4 is prepared as follows. A solution containing 1 mM disulfonated or monosulfonated 679 Compound 4/4-NHS in 1 ml of PBS is incubated overnight at room temperature with 0.1 mM of an anti-cancer drug conjugated with transferrin in the form of a nanoparticle. The resulting 679 Compound 4/4-nanoparticle conjugate is purified by centrifugation and then lyophilized.

(118) The 679 Compound 4/4 (isomer 1)-nanoparticle conjugate (1 nmole) is injected intravenously into a mouse tail vein. Control mice are injected with free 679 Compound 4/4 dye. X-ray and near infra-red fluorescence images of mouse brain are captured.

(119) 679 Compound 4/4-nanoparticle conjugate localizes in the mouse brain for greater than about 24 hours after injection. Tumor size progressively decreases after injection of 679 Compound 4/4-nanoparticle conjugate, compared to 679 Compound 4/4-nanoparticle without the anti-cancer drug.

EXAMPLE 14

(120) The mono-sulfonated derivative is on any one of eight possible positions on the 579, 679, or 779 compound, accounting for the stereochemistry around the carbon positions on the rings as well as the non-symmetrical nature of the two ends of each dye. Similarly, the di- and tri-substituted sulfonates can be on multiple possible positions on the inventive compounds.

EXAMPLE 15

(121) The inventive compounds are used for in vivo imaging as described in J. Gastrointest Surg (2008) 12:1938-1950. Briefly, human pancreatic cell lines are maintained in media supplemented with penicillin/streptomycin at 37 C. with 5% CO.sub.2. Mouse anti-CEA antibody and Control Mouse IgG (in PBS with 0.20% sodium azide) are conjugated to V08-15173. The dye is reconstituted at 10 mg/ml in DMF and then added to the antibody at a 10 molar excess. The reaction is carried out for one hour at room temperature. The samples are then dialyzed against 32 L of PBS. The cell lines are plated in 96-well plates at 510.sup.4 cells per well. After 48 hours culture in appropriate media, the cells are incubated with 1 g of V08-15173 labeled anti-CEA antibody or V08-15173-labeled control mouse IgG for four hours at 37 C. The cells are then washed three times with PBS and then imaged with an inverted Nikon De-485 microscope and Spot camera RD.

(122) Surgical procedures and intravital imaging are performed with the animals anesthesized by intramuscular injection of 0.02 ml of 50% ketamine, 38% xylazine and 12 acepromazine maleate. Human pancreatic and colorectal cancer cell lines are harvested by trypsinization and washed twice with serum free medium and washed twice with serum-free medium. Cells (110.sup.6 in 100 l of serum-free media) are injected subcutaneously within 30 minutes of harvesting over the right flank in female nu/nu mice between 4-6 weeks of age. Subcutaneous tumors are allowed to grow for 7-14 days until they reached diameter of 1-2 mm prior to the delivery of conjugated antibody. For ASPC-1 implants, the cells are harvested by trypsinization and washed 3 in serum-free media. The cells are resuspended in serum-free media. The cells are resuspended in serum-free media at 510.sup.6/ml. A volume of 200 l of the cell suspension is then injected directly into the peritoneal cavity within 30 minutes of harvesting.

(123) For antibody delivery, one to two weeks after subcutaneous, orthotopic, or intraperitoneal tumor implantation, animals are given intravenous (i.v.) injection of either conjugated anti-CEA or conjugated control IgG antibody diluted in PBS to a final volume of 100 l. All i.v. injections are done via the tail vein. For the dose-response experiment, the antibody dose is 75 g. For the in vivo time course, the animals are anesthesized and imaged at 30 min, 1, 2, 6, 24 hours and 8 and 15 days after systemic antibody delivery.

(124) Predicted in vivo and ex vivo analysis results are that post-experiment surgical exposure reveals accumulation of dye in the liver, bladder, and a region of inflammation in the subcutaneous tissue. Ex vivo analysis of the vital organs confirms the presence of dye predominantly in the liver with some signal detected in the spleen intestines and lungs.

EXAMPLE 16

(125) The inventive compounds are used for in vivo imaging as described in J. Gastrointest Surg (2008) 12:1938-1950. Briefly, human pancreatic cell lines are maintained in media supplemented with penicillin/streptomycin at 37 C. with 5% CO.sub.2. Mouse anti-CEA antibody and Control Mouse IgG (in PBS with 0.20% sodium azide) are conjugated to 779 Compound 1. The dye is reconstituted at 10 mg/ml in DMF and then added to the antibody at a 10 molar excess. The reaction is carried out for one hour at room temperature. The samples are then dialyzed against 32 L of PBS. The cell lines are plated in 96-well plates at 510.sup.4 cells per well. After 48 hours culture in appropriate media, the cells are incubated with 1 g of V08-15173 labeled anti-CEA antibody or V08-15173-labeled control mouse IgG for four hours at 37 C. The cells are then washed three times with PBS and then imaged with an inverted Nikon De-485 microscope and Spot camera RD.

(126) Surgical procedures and intravital imaging are performed with the animals anesthesized by intramuscular injection of 0.02 ml of 50% ketamine, 38% xylazine and 12 acepromazine maleate. Human pancreatic and colorectal cancer cell lines are harvested by trypsinization and washed twice with serum free medium and washed twice with serum-free medium. Cells (110.sup.6 in 100 l of serum-free media) are injected subcutaneously within 30 minutes of harvesting over the right flank in female nu/nu mice between 4-6 weeks of age. Subcutaneous tumors are allowed to grow for 7-14 days until they reached diameter of 1-2 mm prior to the delivery of conjugated antibody. For ASPC-1 implants, the cells are harvested by trypsinization and washed 3 in serum-free media. The cells are resuspended in serum-free media. The cells are resuspended in serum-free media at 510.sup.6/ml. A volume of 200 l of the cell suspension is then injected directly into the peritoneal cavity within 30 minutes of harvesting.

(127) For antibody delivery, one to two weeks after subcutaneous, orthotopic or intraperitoneal tumor implantation, animals are given intravenous (i.v.) injection of either conjugated anti-CEA or conjugated control IgG antibody diluted in PBS to a final volume of 100 l. All i.v. injections are done by tail vein. For the dose-response experiment, the antibody dose is 75 g. For the in vivo time course, the animals are anesthesized and imaged at 30 min, 1, 2, 6, 24 hours and 8 and 15 days after systemic antibody delivery.

(128) Predicted in vivo and ex vivo analysis results are that post-experiment surgical exposure reveals accumulation of dye in the liver, bladder, and a region of inflammation in the subcutaneous tissue. Ex vivo analysis of the vital organs confirms the presence of dye predominantly in the liver with some signal detected in the spleen intestines and lungs.

(129) The embodiments shown and described in the specification are only specific embodiments of inventors who are skilled in the art and are not limiting in any way. Therefore, various changes, modifications, or alterations to those embodiments may be made without departing from the spirit of the invention or the scope of the following claims. The references cited are expressly incorporated by reference herein in their entirety.