AU2007353457A1

AU2007353457A1 - Novel hydrazone-based and oxime-based fluorescent and chromophoric/pro-fluorescent and pro-chromophoric reagents and linkers

Info

Publication number: AU2007353457A1
Application number: AU2007353457A
Authority: AU
Inventors: Leopoldo Mendoza; David A. Schwartz
Original assignee: SOLULINK Inc
Current assignee: SOLULINK Inc
Priority date: 2007-05-14
Filing date: 2007-05-14
Publication date: 2008-11-20
Also published as: EP2150858A1; WO2008140452A1; EP2150858A4

Description

WO 2008/140452 PCT/US2007/011529 TITLE OF THE INVENTION Novel Hydrazone-Based and Oxime-Based Fluorescent and Chromophoric/Pro-Fluorescent and Pro-Chromophoric Reagents and Linkers CROSS-REFERENCE TO RELATED APPLICATIONS None STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT None INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON COMPACT DISC None BACKGROUND OF THE INVENTION (1) Field of the Invention The present invention relates to compounds used to label biomolecules for diagnostic and therapeutic purposes. In particular, it relates to fluorescent, chromophoric, pro fluorescent and pro-chromophoric compounds that may be conjugated to biomolecules such as proteins and nucleic acids. Such compounds may be incorporated into linkers that may be used to link a ligand to a biomolecular probe allowing quantification of the ligand bound to that molecular probe. (2) Description of Related Art Methods to detect interactions between biomolecules continues to be an area of active research as new and more sensitive methods are required to increase sensitivity, reduce costs and enable new detection methods. One of the most widely used methods is to directly label a biomolecule with a fluorescent molecule that fluoresces at a desired frequency. For example, a fluorescent molecule is modified with a thiol- or amino-reactive moiety such as succinimidyl WO 2008/140452 PCT/US2007/011529 2 esters or maleimides that form a covalent bound - in the presence of a sulhydryl or amine group of a desired protein. The modified fluorescent molecule is isolated and reacted with the desired protein. The fluorescently labeled protein is then used to detect a desired target by monitoring the unique fluorescent 'frequency of the fluorophore. A variety of fluorophores have been modified with these moieties including fluorescein, rhodamine, Texas Red and cyanine dyes, Cy3 and Cy5. Unfortunately, the conjugation methods often cause quenching and photobleaching of the fluorophore and there can be interference with the observed signal if the unbound labeled biomolecule is not removed from the reaction mixture. Other biolmolecules such as nucleic acids for example DNA, RNA, polynucleotide and oligonucleotides have been labeled with fluorophores is commonly accomplished by incorporating a fluorophore on the base moiety of a nucleoside triphosphate. These fluorescently labeled triphosphates are added to the polymerase chain reaction (PCR) or reverse transcription reaction wherein the labeled nucleoside is incorporated in the amplicon yielding a fluorescently labeled polynucleotide. These fluorescently labeled polynucleotides are probed using oligonucleotide microarrays identifying sequences present in the target. Unfortunately, the fluorophores used for labeling these biomolecules are not often stable to these synthesis conditions. In addition, the long-term stability of conjugates are low -due to photobleaching, consequently, retention of the fluorescent signal is difficult when archiving microarrays. A variety of references cite the use of fluorescent hydrazides, thiosemicarbazides and hydrazides to react with aldehydes on biological molecules for the detection of the aldehydes. For example Ahn et al. (B. Ahn, S.G. Rhee and E.R. Stadtman, Anal. Biochem. 161:245 (1987) describe the use of fluorescein hydrazide and fluorescein WO 2008/140452 PCT/US2007/011529 3 thiosemicarbazide for the fluorometric determination of protein carbonyl groups and for the detection of oxidized proteins on polyacrylamide gels. Proudnikov and Mirzabekov (Nucl. Acids Res. 24-:4535, 1996)) describe labeling of DNA and RNA to identify acid-induced depurination that results in production of aldehyde moieties detected by reaction of fluorescent labels containing hydrazide groups in the presence of sodium cyanoborohydride. Others have labeled the reducing end of polysaccharides with fluorescent hydrazides. These methods are used to detect aliphatic aldehyde groups on biomolecules. In each of the references the fluorescent moiety is incorporated on the hydrazine or hydrazide that forms a hydrazone on reaction with the aldehyde present on the biomolecule. It has been documented that hydrazones formed between certain aromatic aldehydes and aromatic hydrazines and not aromatic hydrazides or aromatic thiosemicarbazides form fluorescent molecules (J. Wong and F. Bruscato, Tet. Lett. 4593, 1968). It has also been reported that hydrazones formed specifically from 2-substituted aldehyde heterocycles and 2-substituted hydrazine heterocycles become fluorescent on chelation to zinc (D.E. Ryan, F. Snape and M. Winpe, Anal. Chim. Acta 58:101, 1972) Schwartz et al. (U.S. 5,420,285; U.S. 5,753,520; U.S. 5,420,285; J. Nucl. Med. 31(12):2022, 1990 and Bioconjug. Chem. 2(5):333, 1991) describe the preparation of succinimidyl 6-hydraziniumnicotinate hydrochloride for the one-step modification of amino groups on proteins and other molecules to incorporate pyridylhydrazine moieties on proteins for the specific purpose of binding technetium-99m for in vivo diagnostic purpose. Subsequently Schwartz (U.S. 7,102,024) describe novel oligonucleotide aldehyde and hydrazine phosphoramidite reagents for incorporation of aldehydes and hydrazines on synthetic oligonucleotides including aromatic and heteroaromatic aldehydes and hydrazines. Triphosphates WO 2008/140452 PCT/US2007/011529 4 incorporating both aromatic hydrazine and aromatic aldehydes have been described by Schwartz and Hogrefe (U.S. 6,686,461). Cytidine and deoxycytidine moieties in polynucleotides can be transformed into 4-N-aminocytidine (4-hyd-C), an aromatic hydrazine, by treatment with hydrazine/bisulfite at neutral pH. Nitta et al. (Eur. J. Biochem. 157(2):427, 1986) has described crosslinking between 16S ribosomal RNA and protein S4 in E. coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate. Also Musso et al., - (U.S. 5,130,466) describe labeling of 4-N aminocytidine moieties on hydrazine/bisulfite treated DNA to yield a fluorescently labeled polynucleotide. Bittner et al. (U.S. 5,491,224) also describe the labeling of transaminated DNA with fluorescent moieties possessing moieties that react with the transaminated cytosine such as fluorophores possessing succinimidyl esters. In all of the aforementioned references the biomolecule is fluorescently labeled with a fluorescent molecule. Unfortunately, as previously stated, the processes or methods used to prepare the conjugate can often times cause quenching or photobleaching of the fluorophore. In addition, during use the unbound fluorescently labeled conjugate must be removed to obtain an accurate fluorescent signal. Therefore, there is a need in the field for a fluorescent label that is resistant to reaction conditions necessary for producing a labeled biomolecule and does not require removal of the unbound fluorescently labeled biomolecule from the detection reaction mixture to obtain a accurate and/or quantitative signal. There is also a need for fluorophores that may be formed under standard assay conditions from pro-fluorophores which, are stable under various laboratory conditions and by a reaction that is highly specific and efficient. To date the most commonly used method to link, immobilize and detect biomolecules is the biotin/streptavidin ligand/receptor couple. Biotin (Figure WO 2008/140452 PCT/US2007/011529 5 1) is a small molecule, MW 250, that binds to streptavidin with an association constant of 10". The extremely high binding constant and fast kinetics of binding and the stability of avidin under a variety of conditions make this an ideal ligand/receptor pair for these purposes. Biotin has been modified to include amino, thiol and carbohydrate reactive moieties, i.e. succinimidyl ester, maleimido and hydrazide respectively, to allow easy incorporation into a large variety of biomolecules. To accomplish detection of an analyte, biotin is conjugated to a probing biomolecule such as an antibody or an oligonucleotide. Following binding of the biotinylated biomolecule to its receptor or complement, an avidin/reporter conjugate such as an avidin/fluorophore conjugate or a avidin/reporter enzyme conjugate is added and allowed to bind to biotinylated probe and visualized by fluorescence detection or addition of a substrate that emits light or precipitates a colored insoluble product on enzymatic processing (Heitzmann H., Richards F.M., Proc. Natl. Acad. Sci. USA 71:3537-3541, 1974; Diamandis E.P., Christopoulos T.K., Clin. Chem. 37:625-636, 1991; Wilchek M. Methods Enzymol. Vol. 184, 1990; Savage, M.D. et al., 1992 Avidin-Biotin Chemistry: A Handbook. Rockford, IL: Pierce Chemical Co.). Following conjugation it is important to determine that the probe molecule has been biotinylated and to quantify the number of biotins now conjugated to the probe molecule. To this end two multi-step indirect assays have been developed. The first assay is' the HABA ([2-(4'-hydroxyazobenzene)] benzoic acid) assay developed by Green (Green, N.M. Biochem. J., 94, 23c-24, 1965). To quantify biotin label incorporation, a solution containing the biotinylated protein is added to a mixture of HABA and avidin. Because of its higher affinity for avidin, biotin displaces the HABA from its interaction' with avidin and the absorption at 500 nm decreases proportionately. By this method, an unknown amount of biotin present in a solution can be evaluated in a WO 2008/140452 PCT/US2007/011529 6 single cuvette by measuring' the absorbance of the HABA avidin solution before and after addition of the biotin containing sample. The change in absorbance relates to the amount of biotin in the sample. The second more sensitive fluorescence-based multi-step assay developed by Molecular Probes (recently acquired by Invitrogen Corporation, Carlsbad, CA) is the 'Fluoreporter Biotin Quantification Assay' that is based on fluorescence resonance energy transfer (FRET) quenching wherein an avidin molecule is labeled with a fluorophore and its binding sites are occupied with a fluorescent molecule that quenches the covalently linked fluorophore until the quencher in the binding site is displaced by a higher binding biotin molecule resulting in fluorescence of the covalently attached fluorophore. While this assay is sensitive to 50 100 pmol range it requires many processing steps and a fluorimeter or multi-well fluorimeter. It is also recommended to digest the biotinylated protein prior to the assay to expose any sterically encumbered biotins. Consequently there is a need in the field for a assay wherein the number of biotins covalently linked to a biomolecule could be determined by direct methods such as spectroscopic means. BRIEF SUMMARY OF THE INVENTION The present invention provides profluorescent/prochromophoric hydrazine and aldehyde reagent compounds for preparing novel- hydrazone-based fluorescent molecules. More specifically conjugationally extended profluorescent/prochromophoric hydrazine compounds of the formula (RR 2

)N(H).(NH

2 )., wherein R is independently a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted WO 2008/140452 PCT/US2007/011529 7 conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 2 is independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1 10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; n is 0 when m is 2 and n is 1 when m is 1 may be combined with conjugationally extended profluorescent/prochromophoric carbonyl compounds of the formula O=C(RIR 2 ) wherein: R, is independently a substituted or.unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 2 is independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1 10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or WO 2008/140452 PCT/US2007/011529 8 more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; n is 0 when m is 2 and n is 1 when m is 1 to form fluorescent hydrazone compounds of the formula (RR 2

)NN=C(RR

2 ). In one embodiment the hydrazone compound has the formula: HN N' N R 1 N N R2 N

R

4 0

R

3

R

5 wherein R. is independently a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 2 is independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 .carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, WO 2008/140452 PCT/US2007/011529 9 sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R, is H or OH; R, is H or a nucleic acid moiety; and R 5 is PO or a nucleic acid moiety. In another embodiment these novel profluorophore hydrazine and carbonyl compounds may further comprise a linkable moiety at one of the R or R 2 positions wherein the linkable moiety is selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety. In yet another embodiment a biomolecule such as for example a nucleic acid, a nucleotide, a protein, an amino acid, a carbohydrate monomer or a polysaccharide is linked to the profluorescent/prochromophoric hydrazine and/or profluorescent/prochromophoric carbonyl by a linkable moiety. If the biomolecule is a nucleic acid it may be DNA, cDNA, RNA, or PNA and can comprise natural or unnatural bases or internucleotide linkages selected from the group consisting of phosphodiesters, phosphorothioates, phosphoramidites and peptide nucleic acids. In still another embodiment one or more of the profluorescent/prochromophoric hydrazine or carbonyl compounds may be bound to a polymer such as poly-lysine, poly-ornithine or polyethyleneglycol by one or more linkable moieties. In another aspect of the present invention methods of forming a hydrazone compound are provided by combining the conjugationally extended profluorescent/prochromophoric hydrazine of formula (RR 2

)N(H).(NH

2 ) with conjugationally extended profluorescent/prochromophoric carbonyl of the formula O=C(RR 2 ) for a time and under conditions that allow hydrazone formation. In one embodiment of this aspect of the invention the conjugationally extended profluorescent/prochromophoric hydrazine and/or the conjugationally extended WO 2008/140452 PCT/US2007/011529 10 profluorescent/prochromophoric carbonyl may further comprise a linkable moiety at either the R. or R 2 position. In yet another aspect of the invention a method for labeling a biomolecule with a fluorescent hydrazone compound is provided. In still another aspect the present invention provides oxyamine and aldehyde reagent compounds for preparing novel oxime-based fluorescent molecules. More specifically conjugationally extended profluorescent/prochromophoric oxyamine compound of formula: (R 1

R

2

)ONH

2 are provided wherein: R is a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; and R 2 is a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted- with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine. In one embodiment a profluorescent/prochromophoric oxyamine compound is provided wherein R, or R 2 further comprise a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive WO 2008/140452 PCT/US2007/011529 11 moiety, an ester moiety and a modified carbohydrate monomer moiety. In another embodiment a profluorescent/prochromophoric oxyamine compound is provided wherein the linker further comprises a biomolecule selected from the group consisting of a nucleic acid, a nucleotide, a protein an amino acid, a carbohydrate monomer and a polysaccharide. The nucleic acid may be selected from the group consisting of DNA, cDNA, RNA and PNA and may comprise natural or unnatural bases or internucleotide linkages selected from the group consisting of phosphodiesters, phosphorothioates, phosphoramidites and peptide nucleic acids. In another aspect of the invention a spectrophotometrically quantifiable linker is provided comprising of formula: A-B-C-D wherein A is an amino, thiol or carbohydrate reactive moiety; B is a chromophoric or fluorescent moiety; C is a flexible linker; and D is biotin or a receptor ligand. When A is an amino reactive moiety it may be selected from the group consisting of N hydroxysuccinimidyl, p-nitrophenyl, pentafluorophenyl and N hydroxybenzotriazolyl. When A is a thiol reactive moiety it may be selected from the group consisting of maleimido, a haloacetamido and pyridylsulfides. When A is a carbohydate reactive moiety it may be aminooxy. B may be a compound that fluoresces, emits light or precipitates a colored insoluble product on enzymatic processing. C is a flexible linker and may be a PEG flexible linker having no less than 8 carbon atoms and no more than 34 carbon atoms. D is a receptor ligand selected from the group consisting of receptor ligand pairs biotin/avidin, peptide S/ribonuclease, complimentary oligonucleotide pairs or antibody/ligand pairs, and digoxigenin/anti-digoxigenin antibody. In one embodiment of the present invention wherein the spectrophotometrically quantifiable linker is bound to a biomolecule via a .amino, thiol or carbohydrate reactive moiety and wherein the biomolecule is selected from the WO 2008/140452 PCT/US2007/011529 12 group consisting of a protein, a peptide, an oligonucleotide and a polynucleotide. Alternatively the spectrophotometrically quantifiable linker may be bound to a biomolecule via receptor ligand pairs such as biotin/avidin, peptide S/ribonuclease, digoxigenin/anti digoxigenin antibody complimentary oligonucleotide pairs or antibody/ligand pairs. Correspondingly, a first biomolecule may be bound via an amino, thiol or carbohydrate reactive moiety and a second biomolecule may be bound via a receptor ligand pair to . the spectrophotometrically quantifiable linker. In another aspect of the present invention a method of preparing a spectrophotometrically quantifiable linker is provided by the steps of preparing a first conjugate of a first biomolecule bound to one profluorescent/prochromophoric compound of a fluorescent pair via an amino, thiol or carbohydrate reactive moiety and preparing a second conjugate of a second biomolecule bound to a flexible linker via a biotin or- a receptor ligand and the other profluorescent/prochromophoric compound of a fluorescent pair and combining the first conjugate with the second conjugate for a time thereby forming a hydrazone bond between the profluorescent/prochromophoric compound pair forming a fluorescent moiety. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Figure 1: A diagrammatic representation of the chemistry for the formation of fluorescent hydrazones from conjugationally extended aldehydes and hydrazines; Figure 2: A diagrammatic representation of the tautomerization of bis- (2-heteroaromatic)hydrazone chelates; Figure 3: Hydrazine and aldehyde succinimidyl ester reagents, SANH and SFB respectively, developed for modification of amino moieties on biomolecules and a WO 2008/140452 PCT/US2007/011529 13 diagrammatic representation of the conjugation of a hydrazine-modified biomolecules with a benzaldehyde modified biomolecule; Figure 4: (A) PAGE gel of the results of the conjugation of a 5'-benzaldehyde-modified oligonucleotide to a hydrazine-modified antibody visualized by coomassie blue (CB) staining; (B) the same gel visualized by UV back shadowing to visualize the oligonucleotide conjugated to the protein; (C) nitrocellulose membrane of the blotted conjugate following hybridization of the fluorescein-labeled complementary oligonucleotide demonstrating retention of hybridization functionality of conjugated oligonucleotide; Figure 5: A diagrammatic representation of fluorescent hydrazone (3) formed from 6-hydrazinonicotinic acid (1; R = OH) and 4-dimethylaminocinnamaldehyde 2; Figure 6: (A) Chemical structure of benzaldehyde phosphoramidite used to incorporate benzaldehyde moieties on the 5'-terminus of oligonucleotides during their solid phase synthesis; (B) PAGE gel of purified oligonucleotide (Lane 1) and the product of the reaction of the oligonucleotide with trans-4 hydrazinostilbazole (1; Fluka Chemical Co, Milwaukee, WI); Figure 7: Absorbance and emission spectra of a 22mer oligonucleotide modified on the 5'-end with the hydrazone formed from the reaction of benzaldehyde and, trans-4 ' -Hydrazino-2-stilbazole; Figure 8: Chemical structure of bifunctional hydrazido amine modification reagent SHTH; Figure 9: A diagrammatic representation showing hydrazones prepared from conjugationally extended hydrazines and aldehydes form fluorescent species while hydrazones prepared from conjugationally extended hydrazides and WO 2008/140452 PCT/US2007/011529 14 aldehydes do not form substantially fluorescent species. 5'-(6-Hydrazinylpyridine)-modified oligonucleotide is reacted with 4 dimethylaminocinnamaldehyde (Reaction A) and naphthalene-1,2-dicarboxaldehyde (Reaction B) form fluorescent species. The hydrazone formed from the reaction of 5'- (6-hydrazidoterephalate) -modified oligonucleotide with 4-dimethylaminocinnamaldehyde is not fluorescent and the product with NDA forms a weakly fluorescent species based on the pyrollo-fused naphthalene product without conjugation through hydrazide moiety; Figure 10: A diagrammatic representation of the conversion of cytidine. to 4-N-aminocytidine with hydrazine/bisulfite; Figure 11: A diagrammatic representation of the incorporation of fluorescence into DNA wherein salmon sperm DNA was treated with hydrazine/bisulfite to convert cytidine moieties to 4-aminocytidine, an aromatic hydrazine. The modified DNA was treated with dimethylaminocinnamaldehyde (DAC; top reaction; Lane 2) or naphthalene-1,2-dicarboxladehyde (NDA; bottom reaction; Lane 4) and visualized following electrophoresis on an agarose gel (at left). Control reactions wherein untreated DNA was reacted with DAC and NDA were not fluorescent (Lanes 1 and 3 respectively); Figure 12: Chemical structure of commercially available aromatic hydrazines; Figure 13: Chemical structure of commercially available aldehydes; Figure 14: Chemical structure of cyanine dyes Cy3 and Cy5; Figure 15: Chemical -structure of cyanine pro-fluorophores and their parent fluorophores targeted for synthesis; Figure 16: A diagrammatic representation of the synthetic methods for the preparation of hydrazinoheterocyles; WO 2008/140452 PCT/US2007/011529 15 Figure 17: Chemical structure of benzimidazole pro fluorophores and synthesis schemes of their parent fluorophores targeted for synthesis; Figure 18: Chemical structure of biotin; Figure 19: A diagrammatic representation showing hydrazones prepared from conjugationally extended hydrazines and aldehydes that form fluorescent species while hydrazones prepared from conjugationally extended hydrazides and aldehydes do not form substantially fluorescent species. 5'-(6-Hydrazinylpyridine) modified oligonucleotide is reacted with 4-dimethyl aminocinnamaldehyde (Reaction A) and naphthalene-1, 2 dicarboxaldehyde (Reaction B) form fluorescent species. The hydrazone formed from the reaction of 5'-(6 hydrazidoterephalate) -modified oligonucleotide with 4 dimethylaminocinnamaldehyde is not fluorescent and the product with NDA forms a weakly fluorescent species based on the pyrollo-fused naphthalene product without conjugation through hydrazide moiety; Figure 20: A schematic representation of the synthesis of amino-reactive biotin/hydrazone chromophore 6; Figure 21: A graph showing amino-reactive biotin/hydrazone chromophore 6 and overlaid spectra of equivalent amounts (20 ug) native bIgG and bIgG modified with 5X, 1OX and 15X amino-reactive biotin/hydrazone chromophore 6 demonstrating the incorporation of chromophore/PEG4/biotin moiety by their absorbency at A354; Figure 22: Structure of a thiol-reactive chromophore linker of the present invention 7, aldehyde-reactive chromophore linker of the present invention 8 and an oxidized carbohydrate-reactive chromophore linker of the present invention 9; Figure 23: A schematic representation of the incorporation of a conjugationally extended aldehyde cytosine triphosphate 10 in a DNA amplicon (R=H) or RNA amplicon WO 2008/140452 PCT/US2007/011529 16 (R=OH) and labeling the modified amplicon with a linker of the present invention 11; Figure 24: Schematic representation of the synthesis of a linker of the present invention 11; Figure 25: Schematic representation of the synthesis of a linker of the present invention 1-8; Figure 26: Schematic representation of the synthesis of a linker of the present invention (2-10); Figure 27: Schematic representation of the synthesis of a linker of the present invention (3-4); Figure 28: Schematic representation of the synthesis of a linker of the present invention (4-2); Figure 29: Schematic representation of the synthesis of a linker of the present invention (6-2); Figure 30: Schematic representation of the synthesis of a linker of the present invention (7-8); and Figure 31: Schematic representation of the synthesis of a linker of the present invention (8-2). DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all terms used herein have the same meaning as are commonly understood by one of skill in the art to which this invention belongs. All patents, patent applications and publications referred to throughout the disclosure herein are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail. The term "biomolecule" as used herein refers to a compound of biological origin, or of biological activity, that may have, or may be modified to have, an amine group or carbonyl group that may be harnessed in the formation of a hydrazone bond with a novel carbonyl profluorophore or novel hydrazine pro-fluorophore of the present invention. Biomolecules include for example a nucleic acid, a nucleotide, a protein, an amino acid, a carbohydrate monomer WO 2008/140452 PCT/US2007/011529 17 and a polysaccharide. If the biomolecule is a nucleic acid it may be DNA, cDNA, RNA, or PNA and may comprise natural or unnatural bases or internucleotide linkages such as for example phosphodiesters, phosphorothioates, phosphoramidites or peptide nucleic acids. The term "profluorophore" as used herein refers to a compound that may, or may not fluoresce, but when joined with its corresponding profluorophore pair compound produces a fluorescent hydrazone/oxime compound that has - a peak emission wavelength substantially separate from the peak emission wavelength of either of the profluorophores that make up the fluorescent hydrazone/oxime compound. A profluorophore pair comprises a hydroxyamine-based profluorophore and a carbonyl-based profluorophore that when combined form a fluorescent hydrazone compound. The term "pro-chromophore" as used herein refers to a compound that may, or may not produce a visible color, but when joined with its corresponding pro-chromophoric pair compound produces a chromophoric compound that has a peak observable wavelength substantially separate from the peak observable wavelength of either of the prochromophores that make up the chromophoric hydrazone compound. A pro chromophoric pair comprises a hydroxyamine-based pro chromophore and a carbonyl-based pro-chromophore that when combined form a chromophoric hydrazone compound. The term "reactive linking moiety" as used herein refers to molecules used commercially for binding one molecule to another based on the presence of a particular chemical group on the molecule of interest. Some commercially sold molecules referred to herein as linking moieties include those that react with free amines on the target molecule, such as N-hydroxysuccinimidyl, p nitrophenyl, pentafluorophenyl and N-hydroxybenzotriazolyl ester and those that react with free sulfhydryls present on the target molecule such as maleimido, a-haloacetamido and pyridyldisulfides.

WO 2008/140452 PCT/US2007/011529 18 The term "ligand/receptor couple" as used herein refers to a pair of molecules having a substantially high affinity of binding specifically to one another. One example of such a binding pair would be a receptor on a cell and the ligand that binds that receptor. Another example would be biotin and avidin, which are two molecules that have a strong affinity for binding each other having an association constant of around 10". Other pairs include Peptide S and ribonuclease A, digoxigenin and it receptor and complementary oligonucleotide pairs. " The term "greater than 400nm". as used herein refers to the wavelength at which the hydrazone and or oxime fluoresces. This term preferably includes wavelengths from about 400nm to about 900nm, more preferably from about 400nm to about 650nm and most preferably about 400nm to about 450nm wherein the Stoke's shift is about 100nm with respect to the fluorescence of the profluorophore molecules that form the hydrazone or oxime. To achieve the optimal signal from a fluorescent label it is important that the structural integrity of the fluorophore is retained throughout processing of the labeled reporter molecule. A disadvantage with commercially available fluorophores is their propensity to be hydrolytically unstable or photobleach. The ability to efficiently form fluorescent species in situ in biological media in contrast to present methods wherein a labile fluorescent species is present throughout all protocols would be extremely advantageous in yielding products with fully retained fluorescence for improved limits of detection. In one current example in DNA microarrays, fluorescently labeled triphosphates, e.g. Cy3 and Cy5 triphosphosphates (Amersham Biosciences, Piscataway, NJ), are incorporated during PCR or reverse transcriptase amplification however quenching of the fluorophores through photobleaching or hydrolysis occurs during the many manipulations required to isolate the desired fluorescently WO 2008/140452 PCT/US2007/011529 19 labeled polynucleotide. To overcome this problem a less than ideal two-step method has been developed wherein a 3 aminoallylcytidine triphosphate is incorporated during polynucleotide amplification with subsequent purification, labeling with fluorescent succinimidyl esters and final purification to remove excess unincorporated fluorescent molecules. This chemistry is based on an amino/succinimidyl ester reaction that requires large excess of succinimidyl ester due to its instability in water and steps to remove the excess hydrolyzed reagent.. This reaction proceeds over a small pH range, i.e. 7.2-8.0 and is concentration dependent. It would be advantageous to have a method wherein a stable non-fluorescent species is used to label a biomolecule that following all required processing in techniques such as PCi, 2-dimensional electrophoresis or immunohistochemistry can be reacted efficiently with a second non-fluorescent molecule to form a fluorescent species. The present invention describes a chemistry wherein a conjugationally extended hydrazine reacts with a conjugationally extended carbonyl in situ in aqueous media to form a fluorescent molecule (Figure 1). Both aldehydes and hydroylamines are stable in aqueous media and react efficiently to form stable hydrazones. The hydrazone formation is acid catalyzed and has an optimum pH of 4.7 but proceeds up to pH 8.0. This methodology could be extended to use with biosensors for biowarfare and pathogen detection, brand security and Near-IR products. These fluorophores may also be engineered for use in laser and photonics applications. D.E. Ryan, F. Snape and M. Winpe (Ligand Structure and Fluorescence of Metal chelates; N-Heterocyclic Hydrazones with Zinc, Anal. Chim. Acta 58:101, 1972) described a series of hydrazone chelates (Table 1) and that upon addition of Zn 2 the chelates complex the metal yielding a fluorescent WO 2008/140452 PCT/US2007/011529 20 metal chelate (Figure 2) . It was postulated how the non complexed chelate can exist in two different tautomers that have different fluorescent properties due to disrupted aromatic bonding. The addition of the zinc ion 'locks in' the tautomer with better conjugation and higher fluorescence. These authors further described the use of these chelates as analytical tools for determination of trace amounts, i.e. parts per million and parts per billion, of zinc. Full Name Abbreviated Relative Form excitation emission Fluorescence* Pyridine-2-aldehyde-2-pyridyl hydrazone PAPH 455 515 1 Quinoline-2-aldehyde-2-pyridylhydrazone QAPH 490 540 2 Phenanthridine-2-aldehyde-2-pyridylhydrazone PDAPH 490 545 7 Pyridine-2-aldehyde-2-quinolylhydrazone PAQH 470 535 660 Quinoline-2-aldehyde-2-quinolylhydrazone QAQH 495 595 30 Phenanthridine-2-aldehyde-2- PDAQH 525 610 16 quinolylhydrazone Pyridine-2-aldehyde-2- PAPDH 450 540 100 phenanthrdinylhydrazone Quinoline-2-aldehyde-2- QAPDH 510 600 110 phenanthrdinylhydrazone Phenanthridine-2-aldehyde-2- PDAPDH 580 620 230 phenanthrdinylhydrazone Benzimidazole-2-aldehyde-2- pyridylhydrazone BAPH 440 510 140 470B550enzimidazole-2-aldehyde-2- BAQH 470 520 2000 quinolylhydrazone Benzimidazole-2-aldehyde-2- BAPDH 480 530 440 phenanthrdinylhydrazone Phenyl-2-pyridylketone-2-pyridylhydrazone PPKPH 420 470 8 Phenyl-2-pyridylketone-2-quinolylhydrazone PPKQH 470 550 450 Phenyl-2-pyridylketone-2- PPKPDH 490 575 1520 phenanthrdinylhydrazone Table 1 lists the bis-(2-heteroaromatic)hydrazones prepared by Ryan et al., supra. and including their excitation and emission wavelengths and relative fluorescence properties. D.E. Ryan, F. Snape and M. Winpe (Anal. Chim. Acta 58, 1972) describe a series of hydrazone chelates that upon addition of Zn2" complex the metal yielding a fluorescent WO 2008/140452 PCT/US2007/011529 21 metal chelate (Figure 2). Linkable hydrazone and oxime chelates are provided for covalent attachment to any other molecule including biomolecules, polymers, dendrimer, metals or surfaces of the following structure VII: X' X" 1

R

3 N R4 N Y nX2 n. yT

R

1

X

2 = NR 2 orO VII wherein X' and X'' are N; X 2 is NR 2 or O; n' and n'' are either 0 or 1 carbon atom or hetero-atom; Y or Y' is an alcohol, amino or a thiol reactive moiety; R 1 is a single or plurality of substitutents of any combination of one or more alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic or combination of polyaromatic and heteroaromatic moieties that are contiguously conjugated. Further R, can be unsubstituted or substituted with one or more substituents that extend the conjugation of R. comprising groups such as but not limited to hydroxy, alkoxy, alkenes, alkynes, unsubstituted amines and substituted primary, secondary, tertiary and quaternary amines, nitro, carboxy and sulfo. When X 2 is N, R 2 is H or straight chain, branched or cyclic aliphatic moiety of 1-10 carbon atoms or plurality of substituents of any combination of one or more alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic or combination of polyaromatic and heteroaromatic moieties that are contiguously conjugated. Further R2 can be unsubstituted or substituted with one or more substituents that extend the conjugation of RI comprising groups such as but not limited to hydroxy, WO 2008/140452 PCT/US2007/011529 22 alkoxy, alkenes, alkynes, unsubstituted amines and substituted primary, secondary, tertiary and quaternary amines, nitro, carboxy and sulfo.

R

3 and RA are H or straight chain, branched or cyclic aliphatic moiety of 1-10 carbon atoms or plurality of substitutents of any combination of one or more alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic or combination of polyaromatic and heteroaromatic moieties that are contiguously conjugated. Further R, and R 4 can be unsubstituted or substituted with one or more substituents that extend the conjugation comprising groups such as but not limited to hydroxy, alkoxy, alkenes, alkynes, unsubstituted amines and substituted primary, secondary, tertiary and quaternary amines, nitro, carboxy and sulfo. Y or Y' is linkable moiety chosen from amino reactive moieties including succinimidyl esters, tetrafluorophenol esters and N-hydroxybenzotriazolyl esters or thiol reactive moieties including maleimides and a-bromoacetamides or monomers used in the solution phase or solid phase syntheses of biomolecules including amidites used for the preparation of oligonucleotides, nucleoside triphosphates for the transferase-mediated syntheses of RNA and DNA polynucleotides acid or ester used in the preparation of peptides and modified carbohydrate monomers used in the syntheses of oligosaccharides. The hydrazones of structure VII are prepared from aromatic or heteroaromatic hydrazines or hydroxylamines of structure VIII WO 2008/140452 PCT/US2007/011529 23 H2N I XR X2n' y'

X

2 = NR 2 or O Vi/ and aromatic or heteroaromatic carbonyls of structure IX R3 I I 0 Y "o R1 Ix Upon addition of Zn 2 + or any other metal that chelates to the hydrazone a fluorescent moiety results. For Example, such linkable chelates include: 0 0 N-O N O O N O N 0- N H H 0 0 0 0 N 01 N H _ r4- 0

HH

WO 2008/140452 PCT/US2007/011529 24 Bifunctional hydrazine and carbonyl reagents to modify biomolecules have been prepared. Figure 3 outlines this chemistry. The hydrazine/carbonyl bioconjugation couple has significant advantages over currently used maleimido/thiol couple in that both the aldehyde and hydrazine moieties are stable following incorporation on biomolecules, simple addition of an aldehyde-modified biomolecule to a hydrazine modified biomolecule yields a stable hydrazone without the requirement of a reduction reaction to stabilize the bond, the stability of the functional groups allows conjugations to be performed at low concentrations, i.e. <100 microgram/mL and the chemistry has been engineered to prepare conjugates from all biomolecules. Figure 4 shows the conjugation of a 5'-[4 formylbenzamidel-modified oligonucleotide to a hydrazine modified antibody. The results demonstrate complete conversion of modified protein to conjugate by the simple addition of the stable 5'-[4-formylbenzamide]-modified oligonucleotide to the modified-hydrazine modified protein forming a stable hydrazone mediated conjugate. The linkers have been prepared as reagents for the solid phase syntheses of peptides (6-hydrazinonicotinamide carboxylic acids) and oligonucleotides (4-formylbenzamide phosphoramidites). Aldehyde-modified deoxy- and ribo triphosphates have also been prepared and demonstrated to be incorporated into polynucleotide amplicons. In the initial demonstration of the fluorescence of conjugationally extended hydrazones, 6-hydrazinonicotinic acid I (Solulink Biosciences, San Diego, CA) was reacted with 4-dimethylcinnamaldehyde 2 (Aldrich Chemical Co., Milwaukee, WI) to yield fluorescent hydrazone 3 (Figure 5). Hydrazone 3 absorbed at 397 nm and emitted at 508 nm a Stokes shift of 109 nm. Other hydrazones prepared from commercially available conjugationally extended hydrazines and aldehydes were prepared and their respective excitation WO 2008/140452 PCT/US2007/011529 25 and emission wavelengths are presented in Table 2 below. It should be noted that the Stoke's shifts for hydrazones 2, 3 and 4 all are 100 nm or greater. absorbance omission 0 nm nm ' 385 407 H H 355 472 H H 2 0 397 508 H 3 450 550 H H 4 Table 2 shows the fluorescent hydrazones and their absorbance and emission maxima. In another demonstration 4-formylbenzamide phosphoramidite has been prepared that is used to incorporate benzaldehyde moieties directly on the 5'-end of oligonucleotides during solid phase oligonucleotide synthesis. The incorporation of this moiety is accomplished with identical procedures and yields as incorporation of DMT-amino modified phosphoramidites. Reaction of an oligonucleotide with trans-4 '-hydrazino-2-stilbazole dihydrochloride quantitatively yields a fluorescent oligonucleotide (Figure 6). The emission and absorbance spectra of hydrazone 4 (see Table 2 above) linked to a 22mer oligonucleotide are presented in Figure 7. Methods have been developed to prepare both hydrazino and hydrazido-modified oligonucleotides. Hydrazinopyridine modified oligonucleotides can be prepared by the reaction of amino-modified oligonucleotides with SANH and hydrazido modified oligonucleotides can be prepared using SHTH (Figure 8). To demonstrate that hydrazones prepared from conjugationally extended hydrazines but not. conjugationally WO 2008/140452 PCT/US2007/011529 26 extended hydrazides both oligonucleotides were reacted with 4-dimethylaminocinnamaldehyde (Figure 9, reactions A and C) but only the hydrazine derived hydrazone was fluorescent. In another demonstration both hydrazino- and hydrazido modified oligonucleotides were reacted with 1,2-naphthalene dicarboxaldehyde (NDA; reactions B and D). It is known that amines react with NDA yield a fluorescent species. The products from the reaction of these oligonucleotides were both fluorescent however the hydrazine derived product absorbed and emitted qualitatively more intensely and at longer wavelengths than the hydrazido-modified oligonucleotide. In another demonstration salmon sperm DNA was treated with hydrazine/bisulfite to convert cytidine moieties to 4 N-aminocytidine, an aromatic hydrazine (Figure 10; Negishi, K., Harada, C., Ohara, Y., Oohara, K., Nitta, N. and Hayatsu, H., 4-N-aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity, Nucleic Acids Res. 1983, 11, 5223-33)). The reaction of the modified DNA with both 4-dimethylaminocinnamaldehyde and naphthalene-1, 2 dicarboxaldehyde (NDA) yielded fluorescent DNA. (Figure 11). It should be noted that the hydrazine-modified cytidine is a component of the fluorophore and not solely a linkage point. It is anticipated that conjugationally extended aldehydes that yield hydrazones with more intensely fluorescent properties can be developed to convert reverse transcribed DNA to fluorescent species thereby using all natural triphosphates in the reverse transcription reaction and not substituted triphosphates whose incorporation is random and not quantitatively reproducible batch to batch. A library of hydrazone fluorophores may be prepared from commercially available aromatic hydrazines and aldehydes using the methods described. Figure 12 below presents structures of commercially available hydrazines that may be purchased and reacted to form hydrazone fluorophores.

WO 2008/140452 PCT/US2007/011529 27 Figure 13 presents structures of commercially available aldehydes that will be purchased to be reacted to form hydrazone fluorophores. The initial pro-fluorophore structures targeted for syntheses in this program are based on cyanine dyes. These dyes are extremely sensitive and have been developed for a variety of commercial uses including life sciences applications as well as photographic uses (A. Mishra, R.K. Behera, P.K. Behera, .K. Mishra and G.B. Behera, Cyanines during the 1990's: A Review, Chem. Rev., 100:1973, 2000). Figure 14 below presents the structures of the most used cyanine dyes, Cy3 and Cy5, for life science applications. These dyes are routinely used as reporter molecules in both gene and protein microarrays. Figure 15 presents aldehyde and hydrazine cyanine-based profluorophores and their parent fluorophores targeted for synthesis. Two methods have been developed for the preparation of hydrazino-substituted aromatic compounds (Figure 16). The classical method for the synthesis of 2 hydrazinoheteroaromatic compounds is direct nucleophilic aromatic substitution of 2-chloro-heterocycles with hydrazine. Arterburn et al. (J.B. Arterburn, K. V. Rao, R. Ramdaa and B.R. Dible, Org. Lett. 2001, 3, 1351 and J.B. Arterburn, B.D. Bryant and D. Chen, Chem. Comm. 2003, 1890) have developed palladium-catalyzed protocols to convert 2 substituted bromo, chloro and trifloro substituted pyridines to 2-hydrazinylpyridines. Aromatic aldehydes can be prepared by a variety of methods including direct oxidation of methyl-substituted aromatic moieties and reduction of aromatic nitriles. Aromatic aldehydes can be conjugationally extended using the Mannich reaction. Due to the fluorescence of benzimidazole-quinoline hydrazone 5 a variety of pro-fluorophores based on this parent core structure have been investigated. Figure 17 WO 2008/140452 PCT/US2007/011529 28 presents target pro-fluorophores and their respective parent fluorophores. Diverse libraries with varying fluorescent properties can be readily prepared as any carbonyl and any hydrazine prepared or commercially available can be combined to yield a fluorescent hydrazone. The excitation and emission characteristics desired can be tailored by incorporation of substituents such as dimethylamino, alkoxy and nitro groups. The photophysical characteristics of the fluorophores may be observed using a QM-2 Spectrofluorimeter (Photon Technologies International, Inc, Trenton, NJ), with a nitrogen-dye laser/second harmonic generator excitation source. A Xe arc lamp may be utilized having excitation that allows for the collection of steady state excitation and emission spectra, the characterization of quantum yield, photo-bleaching, and an degradation of fluorescence from these species. The response of this instrument may be characterized by fluorescence quantum yield standards (i.e. quinine sulfate) to determine the quantum yield of the various fluorophores. The laser system with the laser-strobe detection attachment allows for the collection of sub nanosecond time-decays. The time decay curves may be analyzed to determine the excited-state lifetimes of these fluorophores. In addition a Nd:YAG laser pumped OPO system, will allow for tunable excitation between 400 nm and 3000 nm. The detection system includes a Jobin-Yvon 0.5m monochromator with both PMT and CCD detection. The CCD camera is sensitive in the visible and Near Infrared regions of the electromagnetic spectrum. This system may be used for the characterization of fluorophores in the far-red region of the visible spectrum and in the NIR region. The tunable excitation will provide a means to excite fluorophores, regardless of their absorption spectra in the visible/NIR regions WO 2008/140452 PCT/US2007/011529 29 The stability of the commercially available fluorophores has limited the full range of development of a variety of applications. The advantageous characteristics of this technology includes: elimination of the need to remove the excess second moiety from the in situ formed fluorescent species as it is either not fluorescent or has completely different fluorescent properties that do not interfere with detection of the new fluorescent species; increased efficiency of the formation of the fluorescent species >90%, in buffered aqueous media, pH 5.0-8.0; the ability to prepare a wide variety of fluorophores of different absorbance and emission wavelengths by varying the structures of the two moieties of the final fluorescent molecule; utilizing a linker moiety that may be incorporated on either of the pro-fluorescent species for covalent linking to a biomolecule or a surface; significant reduction in photobleaching or increased hydrolytic stability of the initial pro-fluorophore as has it will be in a lower energy state than fully conjugated fluorophores currently employed; and the development of fluorescent species having well separate spectral absorbance and emission properties, i.e. a Stoke's shift >100 nm. United States patent appl-ication serial no.: 60/546,104 to Schwartz incorporated herein in its entirety has described the in situ preparation of hydrazone fluorophores by the reaction of a conjugationally extended aldehyde with a conjugationally extended hydrazine one of which is linked to biomolecular probe such as an antibody or an oligonucleotide. Figure 19 presents the reaction scheme for the reaction of a conjugationally extended hydrazine with a conjugationally extended aldehyde linked to an oligonucleotide forming an oligonucleotide linked fluorescent hydrazone. The scheme also presents results that demonstrated that the reaction i-s specific for a conjugationally extended hydrazine and not a hydrazide. In contrast to forming chromophore/fluorophores in situ the WO 2008/140452 PCT/US2007/011529 30 present invention incorporates a pre-formed chromophoric/fluorescent hydrazone into the linker comprising the ligand for direct spectrophotometric quantification of the level of incorporation of the ligand when bound to a biomolecule such as a protein or nucleic acid. Figure 20 presents the construction of an amino reactive biotin moiety that has incorporated in its chain a chromophoric/fluorescent hydrazone for spectrophotometric quantitiation and a short PEG linker that is required to retain the binding affinity of biotin to streptavidin. This tri-functional molecule can be readily quantified spectrophotometrically following conjugation to a biomolecule because of its unique molar extinction coefficient (generally >20000) and its unique absorbance or fluorescence (generally at wavelengths greater than 300 nm and at frequencies having no, or only minimal, observable signals prior to conjugation). It is anticipated that more highly conjugated systems than presented in Figure 20 will absorb at longer wavelengths with greater extinction coefficients or fluorescence allowing even greater sensitivity. Figure 21 presents constructions of thiol and oxidized carbohydrate-reactive linkers of the present invention. The incorporation of labels into nucleic acids such as cDNA or cRNA using polymerases and reverse transcriptases respectively for gene expression analysis by microarrays is a multi-step procedure that requires high levels of reproducibility so results can be reliably compared between experiments. One current method for labeling cDNA or cRNA is the use of a nucleoside modified to incorporate a biotin molecule on the minor groove side. One of the most commonly used methods to label and detect labeled cDNA and cRNA is using a biotinylated nucleoside triphosphate (NTP). As there are only labor-intensive methods to quantitate the level of biotin incorporation in the amplicon, the biotin-modified WO 2008/140452 PCT/US2007/011529 31 amplicon is used directly without quantification. It would be extremely advantageous to be able to directly quantitate the level of biotin incorporated into cDNA or cRNA. Figure 22 is a schematic diagram of the synthesis of a nucleoside triphosphate modified with a conjugationally extended aldehyde such as a benzaldehyde moiety and to label the amplicon after elongation by reaction with a biotinylated conjugationally extended hydrazine. United States patent 6,686,461 to ' D. Schwartz and R. Hogrefe which is incorporated herein by reference in its entirety more fully discloses this synthesis. The chemistry described herein is advantageous in that the formation of the hydrazone is high yielding at near stoichiometric amounts, a chromophore is formed that will allow batch-to-batch quantification of levels of incorporation of biotin and a short polyethylene linker is incorporated is necessary to retain the affinity of the biotin to its cognate receptor avidin. In another protocol the amplicon -may be hybridized prior to reaction with the biotin hydrazinonicotinamide and subsequently detected with a fluorescently labeled avidin or anti-biotin antibody. The benzaldehyde-labeled amplicon can be quantified by removing an aliquot and treating it with a hydrazide pro-fluorophore to form a fluorescent hydrazone and spectrophotometrically quantifying the level of aldehyde incorporation. This may be advantageous as the hybridization reaction will have minimal modification resulting in less sterically encumbered hybridization. In use the linker moiety reacts with a biomolecule such as an antibody under appropriate reaction conditions. The conjugate is then purified and the protein concentration determined. The number of biotin molecules/protein molecule is determined by observing the absorbance of a known concentration of the conjugate in solution at a wavelength >300 nm. The concentration of the chromophore and therefore the biotin is determined by dividing the absorbance reading by the extinction coefficient of the chromophore WO 2008/140452 PCT/US2007/011529 32 incorporated in the chain. This concentration is divided by the mM concentration of the protein and the number of biotin molecules per conjugated is determined. EXAMPLES Example 1 Synthesis of Biotin/PEG/hydrazone succinimidyl ester 6, (Figure 20) PMR spectra were obtained on a Bruker 500 MHz NMR at NuMega Laboratories (San Diego, CA) and electrospray mass spectral data was obtained at HT Laboratories (San Diego, CA). 1. Synthesis of Mono-Boc-1, 13-diamino-4,7,10 trioxatetradecane (1; (3-{2-[2-(3-Amino-propoxy) ethoxyJ-ethoxy}-propyl)-carbamic acid tert-butyl ester), Amine 1. To a solution of 4,7,10-trioxa-1,13-tridecanediamine (Figure 20) (30 g; mmol) in dichloromethane (1000 mL) was added a solution of di-t-butyl dicarbonate (10 g; mmol; Aldrich Chemical Co., Mi-lwaukee, WI) in dichloromethane (200 mL) over 2 h. The reaction mixture was stirred at room temperature for 4 hours. Thin layer chromatography (TLC, silica gel) using dichloromethane/methanol/triethylamine (90/10/1);ninhydrin development) indicated the presence of two new spots, a minor spot at Rf 0.8 ascribed to the bis BOC product and a major spot at Rf (0.2) for the desired product. The reaction mixture was washed with water (4 X 500 mL) to remove the excess diamine and the organic phase was dried over magnesium sulfate, filtered and concentrated to give a viscous oil that was purified by flash chromatography over silica gel using DCM/MeOH/TEA (95/5/1) to give 10.5 g of desire Amine 1 as an oil.

WO 2008/140452 PCT/US2007/011529 33 2. Synthesis of ((3-{2-[2-(3-{[6-(N'-Isopropylidene hydrazino) -pyridine-3-carbonyl] -amino} -propoxy) -. ethoxy] -ethoxy}propyl) -carbamic acid tert-butyl ester), Hydrazone 2. To a solution of Amine 1 (1.05 g; 3.28 mmol) in DCM (20 mL) was added a solution of succinimidyl 6 hydrazinonicotiniate acetone hydrazone (0.951 g; 3.28 mmol; Solulink Biosciences, Inc., San Diego, CA) in DCM (10 mL). The reaction mixture was stirred at room temperature for 6 hours. Subsequently the reaction mixture was washed with water and brine. The organic phase was dried (magnesium sulfate), filtered and concentrated to give 1.2 g of Hydrazone 2 as colorless thick oil. 3. Synthesis of (4-{[5-(3-{2-[2-(3-tert-Butoxy carbonylamino-propoxy) -ethoxy] -ethoxy} propylcarbamoyl) -pyridin-2-yl ] -hydrazonomethyl} benzoic acid), Hydrazone 3. To Hydrazone 2 (0.405 g: 0.81 mmol) in MeOH (5 mL) and 100 mM MES, 150 mM NaCl (5 mL) was added a solution of 4 carboxybenzaldehyde (0.121; 0.81 mmol) in MeOH (3 mL) . The reaction mixture is allowed to stir at room temperature overnight. Copious precipitate formed. The reaction mixture was centrifuged and the solids were washed with a 1/1 solution of MeOH/MES. The solids were dried under vacuum to yield 0.42 g of Hydrazone 3 as a pale yellow solid and used directly in the next step. 4. Synthesis of (4-{[5-(3-{2-[2-(3-Amino-propoxy) ethoxy] -ethoxy} -propylcarbamoyl) -pyridin-2-yl] hydrazonomethyll-benzoic acid hydrochloride salt), chromophore Hydrazone 4.

WO 2008/140452 PCT/US2007/011529 34 A solution of Hydrazone 3 (0.388 g; 0.66 mmol) in dioxane (15 mL) was prepared with heating. The solution was cooled to room temperature and 4 N HC1 in dioxane (4 mL; Aldrich Chemical Co., Milwaukee, WI) was added succinimidyl and the reaction was stirred at room temperature for 16 h. A precipitate formed on stirring. The reaction mixture was centrifuged and the solids were washed with dioxane (3 X 10 mL). The solids were resuspended in dioxane and concentrated under vacuum to yield 240 mg of amino/PEG4/Hydrazone 4 as a pale yellow solid.. Electrospray mass spec: expected m/e 487; found positive mode 488 (M+H), negative mode 486 (M-H) and 522 (M+Cl~) 5. Synthesis of Biotin/PEG4/chromophore succinimidyl ester 6, (5-(N'-{4-[2-(2,5-Dioxo-pyrrolidin-1-yl) 2-oxo-acetyl] -benzylidene} -hydrazino) -pyridine-2 carboxylic acid {3-[2-(2-{3-(5-(2-oxo-hexahydro thieno[3,4-d]imidazol-4-yl)-pentanoylaminol propoxy} -ethoxy) -ethoxyl -propyl} -amide). To a solution of amino/PEG4/hydrazone 4 (0.780 g; 1.60 mmol) in DMF (25 mL) was added biotin succinimidyl ester (0.546 g; 1.60 mmol) followed by the addition of triethylamine (0.726 mL; 4.80 mmol). The solution was stirred at room temperature until complete as determined by silica gel TLC using DCM/MeOH/TEA (90/10/1) as eluant (developed by UV to visualize the pyridine chromophore and dimethylaminocinnamaldehyde/sulfuric acid/ethanol spray followed by heating to visualize the biotin moiety). To the reaction mixture N-hydroxysuccinimide (0.184 g; 1.60 mmol) and DCC (0.330 g; 1.60 mmol) were added and stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness and partitioned between DCM and water. The organic phase was further washed with brine, dried (magnesium sulfate), filtered and concentrated to give WO 2008/140452 PCT/US2007/011529 35 a yellow sticky solid. The solids were triturated with ethyl acetate.. The solids were isolated by filtration to give 830 mg of a yellow solid. TLC (DCM/MeOH/TEA (90/10/1) indicated one major spot (visualized by UV and dimethylaminocinnamaldehyde/sulfuric acid/ethanol solution) and HPLC analysis (YMC C-18, 150 -X 4.6 cm; 5 .m; 120A; gradient mobile phase A: water/acetonitrile/trifluoroacetic acid (20/80/0.1), mobile phase B: 0.1% TFA in water; gradient 10%A/90%B to 100%A-over.20 min; retention time 8.8 min, detection at A254 and A350. PMR (DMSO-d) .-: 11. 64, s (1 H), 8.65, d, (1 H), 8.37 t, (1 H) NH, 8.12 dd (1 H), 7.95 and 8.11 ab system (4 H), 7.73 t (1 H) NH, 7.36 d (1 H), 6.41 s (1 H), 6.35 s (1 H), 5.57 d (1 H), 4.29 br. t (1 H), 4.11 br. t (1 H), 3.3 - 3.55 m (12 H), 3.08 m (4 H), 2.90 s (4 H), 2.88 dd (1 H), 2.57 d (1 H), 2.03 t (2 H), 1.75 m (2 H), 1.59 m (2 H), 1.2-1.5 m (8 H). The extinction coefficient of Biotin/PEG4/chromophore succinimidyl ester 6 was determined by dissolving Biotin/PEG4/chromophore succinimidyl ester 6 (1.0 mg) in DMF (1 mL) and diluting into PBS. The absorbance maximum was A354 and the molar extinction coefficient was determined to be 23,250. 6. Fluorescence Data of Biotin/PEG4/chromophore succinimidyl ester 6. To a solution of Chromalink Biotin 354S (23.6 mg) in DMF (1.0 mL) was added ethanolamine (1.77 eL). The reaction was incubated overnight at room temperature. C18-RP HPLC indicated complete conversion to product. The solvent was removed under reduced pressure and the product was used directly in the determination of its fluorescence. Fluorescence was measured on a Quanta Master-2-2005 Sensitivity Enhanced instrument (QM-4-2005 SE: Photon Technology International, Birmingham, NJ). On excitation at 395 nm the molecule fluoresced at 420 nm. The excitation bandpass was 4 mm and the emission bandpass was 4 mm. The WO 2008/140452 PCT/US2007/011529 36 lifetime of fluorescence was <10 microseconds- shorter than can be measured quantitatively. Example 2 Protein labeling with Biotin/PEG4/chromophore/succinimidyl ester 6. Bovine immunoglobulin (bIgG; Sigma Chemical Co., St. Louis, MO) was dissolved in modification buffer (100 mM phosphate, 150. mM NaCl, pH 7.2) to prepare a 5 mg/mL solution. A solution of Biotin/ PEG4/chromophore/ succinimidyl ester 6 (1 mg) dissolved in DMF (100 mL) was prepared. Three separate reactions were performed wherein 5 mole equiv., 10 mol equiv. and 15 mol equiv. of Biotin/ PEG4/chromophore/succinimidyl ester 6 (1.3, 2.6 and 3.9 uL,) respectively were added to 0.5 mg bIgG solution. The reaction was allowed to incubate at room temperature for 2 hours. The reaction mixtures were desalted into PBS using Biomax diafiltration apparatuses (Millipore, Inc., Bedford, MA). Protein concentrations of all the modified proteins were determined using the BCA assay (Pierce Chemical Co., Rockford, IL). Spectral analyses of each product were performed by diluting 20 mg of modified protein to 100 mL in PBS. The number of moles of chromophore incorporated was calculated by determining the absorbance of the protein at A354 dividing by the molar extinction coefficient, i.e. 29000, of the chromophore. The overlaid spectra of the products as well as unmodified IgG are present in Figure 21A. The number of incorporated biotins in the modified proteins was further analyzed by the HABA assay (Pierce Chemical Co., Rockford, IL). The results, both tabular and graphically, from both the UV spectral assay and the HABA assay are presented below. IgG/HABA IgG/A354 WO 2008/140452 PCT/US2007/011529 37 5X 1.03 2.45 loX 1.60 4.71 15X 2.22 6.25 A further experiment to demonstrate retention of binding activity of the chromophore/biotinylated bIgG the modified proteins were incubated with streptavidin and the reaction products were analyzed by PAGE gel electrophoresis. Figure 21B presents the results. Example 3 Synthesis of Biotin/PEG/hydrazone 10, (Figure 24). 1. Synthesis of ({3-[2-(2-{3-[5-(2-Oxo-hexahydro thieno(3,4-d]imidazol-4-yl)-pentanoylamino) propoxy} -ethoxy) -ethoxy] -propyl} -carbamic acid tert-butyl ester),1-biotinamido/PEG/BOC-amino 14. To a solution of Amine 1 (0.544 g; 1.70 mmol) in DMF (15 mL) was added a solution of biotin succinimidyl ester (0.580 g; 1.70 mmol) in DMF followed by the addition of TEA (0.75 mL; 5.09 mmol). The reaction mixture was stirred at room temperature for 16 h. The solvent was removed on the rotavap and the residue was partitioned between DCM and water. The organic phase was further washed with brine, dried (magnesium sulfate), filtered and concentrated to give 415 mg of 1-biotinamido/PEG/BOC-amino 14 as an amorphous solid. The product was a single spot by TLC (DCM/MeOH/TEA (90/10/1); developed by dimethylcinnamaldehyde/ethanol/ sulfuric acid/heat to visualize the biotin moiety). The product was used directly in the next step. 2.. Synthesis of (5- (2-Oxo-hexahydro-thieno[3,4 dlimidazol-4-yl)-pentanoic acid (3-{2-[2-(3-amino- WO 2008/140452 PCT/US2007/011529 38 propoxy) -ethoxy] -ethoxy} -propyl) -amide), 1 biotinamido/PEG/amino 15. To a solution of 1-biotinamido/PEG/BOC- amino 14 (400 mg; 0.73 mmol) was dissolved in dioxane (20 mL) with mild heating. The solution was cooled to room temperature and a solution of 4 N HCl in dioxane (10 mL; Aldrich Chemical Co., Milwaukee, WI) was added. The reaction was stirred for 14 h. The solvent was removed on the rotavap and the residue was co-evaporated twice from dry dioxane. The product, 1 biotinamido/PEG/amino 15, was used directly without purification. 3. Synthesis of (5- (N' -Methylene-hydrazino) -pyridine 2-carboxylic acid {3-[2-(2-{3-[5-(2-oxo hexahydro-thieno[3, 4- djimidazol-4-yl) pentanoylamino -propoxy} -ethoxy) -ethoxy propyl}-amide), 1-biotinamido/PEG/amido-6 hydrazino-4-nicotinamide 11. To a solution of 1-biotinamido/PEG/amino 15 (0.375 g; 0.78 mmol) in DMF (25 mL) was added a solution of SANH (0.225 g; 0.78 mmol) and triethylamine (0.645 mL; 4.66 mmol). The reaction mixture was stirred at room temperature for 16 h. The solvent was removed on the rotavap and the residue was partitioned between DCM and water. The organic phase was further washed with brine, dried (magnesium sulfate), filtered and concentrated to give 290 mg of 1 biotinamido/PEG/amido-6-hydrazino-4-nicotinamide 11 as an amorphous solid. The product was a single spot, Rf 0.33, by TLC (DCM/MeOH/TEA (90/10/1) developed by dimethylcinnamaldehyde/ethanol/sulfuric acid/heat to visualize the biotin moiety). Mass spectral data: exptd m/e 621; pos mod exptd m/e 622 (M+H) ; found 622 and exptd 644 (M+Na); found 644; neg mode exptd m/e (M-H) 620; found 620 and (M+Cl~) 656; found 656.

WO 2008/140452 PCT/US2007/011529 39 Example 4 Synthesis of compound 1-8, (Figure 25). 1. Synthesis of 1- (Boc-amido)-13-(N biotinamido)4,7,10-trioxa-1,13-tridecane 1-2. A solution of BOCNH/PEG3/NH 2 1-1 (3.40 g; 10.6 mmol) in DCM (50 mL) was added TEA (4.43 mL) followed by the dropwise addition of a solution of biotin succinimidyl ester (3.44 g; 10.6 mmol) . The reaction mixture was allowed to stir at room temperature. The progress of the reaction was followed by TLC (DCM/MeOH/TEA (90/10/1); visualized using an ethanol/sulfuric acid dimethylcinnamaldehyde solution and heat) . Following completion of the reaction the solvent was removed under reduced pressure and the product was purified by silica chromatography using a DCM/MeOH/TEA from 95/5/1 to 90/10/1 as eluant. Isolated 4.29 g; 74% yield. 2. Synthesis of 1- (Amino) -13- (N-biotinamido) -4, 7, 10 trioxa-1, 13-tridecane 1-3. To a solution of BOCNH/PEG3/Biotin (679.6mg, 1.24mmol) in dry dioxane (10.OmL) was added 4 N HCl/dioxane (5.0 mL; Aldrich Chemical Co. St. Louis, MO). The reaction mixture was allowed to stir at room temperature for 16 h. TLC of the product indicated complete conversion to product and the precipitated product was isolated by decanting solvent and washed with dry dioxane (2x 10ml). The product was used in the subsequent step without purification (isolated yield 524mg, 94.0%). 3. Synthesis of 4-{[5-(2,5-Dioxo-cyclopent-3-enyl) pyridin-2-yl]-hydrazonomethyl}-benzoic acid 1-6.

WO 2008/140452 PCT/US2007/011529 40 To a solution of MHPH (1-4; 208.49 mg, 0.66 mmol; Solulink Biosciences, San Diego, CA) in MeOH (5.OmL) was added a solution of 4-carboxybenzaldehyde (1-5; 100.0 mg; 0.66 mmol) in MeOH (5.0 mL). The reaction mixture was stirred at room temperature and the progress of the reaction was followed by TLC (DCM/MeOH/acetic acid (90/10/1)). On completion the product was isolated by suction filtration and dried under reduced pressure. Isolated 133.0 mg; 56.0% yield. 4. Synthesis of Pentafluorophenyl 4-{[5-(2,5-Dioxo cyclopent-3-enyl) -pyridin-2-yll -hydrazonomethyl} benzoate 1-7. To a solution of maleimido/ hydrazone 1-6 (133.0 mg; 0.37 mmol) in DMF (1.0 mL) was added pentafluorophenol (83.3 mg; 0.45 mmol) followed by the dropwise added of EDC (86.84 g; 0.45 mmol) in DMF (1.0 mL) . The reaction mixture was stirred at room temperature and monitored by TLC (DCM /MeOH/TEA (90/10/1)). Upon completion the solvent was removed on the rotavap under reduced pressure and the crude product was partitioned between EA and water. The organic phase was further washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give 194 mg, 99.4% yield. 5. Synthesis of Maleimido Chromalink" 1-8. To a solution of biotin/PEG3/NH 2 (154 mg; 0.345 mmol) in DMF (1 mL) was added a solution of maleimido/hydrazone pentafluorophenol ester 1-7 (178 mg; 0.345 mmol) in DMF (1 mL) followed by the addition of TEA (48 uL). The reaction mixture was allowed to stir at room temperature and was monitored by TLC (DCM /MeOH/TEA (90/10/1)). Following completion of the reaction the solvent was removed on the rotavap under reduced pressure and the crude product was WO 2008/140452 PCT/US2007/011529 41 dissolved in DCM and purified by silica gel chromatography by initially eluting with ethyl acetate to remove non polar impurities followed by DCM/isopropanol (80/20) to isolate the product (100 mg, 38%). Example 5 Synthesis of compound 2-10, (Figure 26). 1. Synthesis of 1-(Boc-amino)-13- (4-formylbenzamido) 4,7,10-trioxa-tridecane 2-2. To a solution of 1-1 (5.22g, 16.3mmol) in DCM and TEA (4.54 ml, 32.6 mmol) was added a solution of succinimidyl 4 formylbenzoate (2-1; 3.62 g, 14.7 mmol; Solulink Biosciences, San Diego, CA). The homogenous solution was allowed to stir at room temp, monitored by TLC (running solvent: 90:10:01 (DCM:MeOH:TEA). After completion (product rf:0.6) solvent was removed on the rotavap under reduced pressure. The product was purified using silica gel chromatography with DCM/MeOH/TEA (97/3/1) as eluant. 2. Synthesis of 1-(Boc-amino)-13-(4-1-(Boc-amino)-13 (6- [N' - (4-carbamoyl-benzylidene) -hydrazino) nicotinamido)- -4,7,10-trioxa-tridecane 2-4. To a solution of 2-2 (1 equiv) in MeOH is added 6 hydrazinonicotinic acid 2-3 (1 equiv; Solulink Biosciences, San Diego, CA) and the reaction mixture is stirred at room temperature for 16 h. The reaction mixture is concentrated and the product is purified using silica gel chromatography. 3. Synthesis of 1-(Amino)-13-(6-[N'-(4-carbamoyl benzylidene) -hydrazinol -nicotinamido) - 4,7,10 trioxa-tridecane 2-5.

WO 2008/140452 PCT/US2007/011529 42 To a solution of 2-4 in dioxane is added a equal volume of 4 N HCl/dioxane (Aldrich Chemical Co., St. Louis, MO) and the reaction mixture is stirred at room temperature. Following complete conversion of starting material to product as determined by TLC the solvent is removed on the rotavap under reduced pressure. The residue was co evaporated from xylenes and the product was used directly in the following step. 4. . Synthesis of 3-(12-0-Acetyl-digoxigeninyl) chloroformate.2-7. To a solution of 12-0-Acetyl-digoxigenin (2-6; 1 equiv) and TEA (2 equiv) in DCM or -other non-hydroxylic solvent is added phosgene (1.2 equiv as a 20% solution in toluene; Aldrich Chemical Co., St. Louis, MO) . The reaction mixture is stirred at room temperature until complete as determined by TLC. The reaction mixture is diluted into DCM and washed with 5% sodium bicarbonate solution and brine. The organic phase is dried over anhydrous sodium sulfate, filtered and concentrated. The product is used in the next step without further purification. 5. Synthesis of compound 2-8. To. a solution of 2-5 (1 equiv) . in DMF is added a solution of 2-7 (1 equiv) in DMF and triethylamine (3 equiv) . The reaction mixture is stirred at room temperature and monitored by TLC and RP-HPLC. On completion of the reaction the solvent is removed on the rotavap under reduced pressure and the product is isolated by silica gel chromatography. 6. Synthesis of compound 2-9.

WO 2008/140452 PCT/US2007/011529 43 To a solution of 2-8 (1 equiv) in DMF is a added an equal volume of 5% sodium bicarbonate solution and the pH of the reaction mixture is adjusted to pH 8.5 and stirred at room temperature until complete as determined by TLC. The reaction mixture is diluted with DCM and the organic phase is further washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The product is purified by silica gel chromatography. 7. Synthesis of compound 2-10. To a solution of 2-9 (1 equiv) in DMF is added NHS (1.05 equiv) and EDC (1.1 equiv) . The reaction mixture is stirred at room temperature until >90% conversion to product as determined by C18-RP HPLC. The solvent is removed on the rotavap and the residue is partitioned between DCM and water. The organic phase is further washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The product is purified by silica gel chromatography. Example 7 Synthesis of compound 3-4, (Figure 27). 1. Synthesis of compound 3-2. To a solution of 1-1 in DMF is added a solution of 1-7 (1 equiv; 1-(amino)-13-(6-[N'-(4-carbamoyl-benzylidene) hydrazino -nicotinamido) -4,7, 10-trioxa-tridecane); 1 equiv) in DMF. The progress of the reaction is followed by RP-HPLC and TLC. Upon completion of the reaction the solvent is removed under reduced pressure and the product 3-2 is isolated by silica gel chromatography. 2. Synthesis of compound 3-3.

WO 2008/140452 PCT/US2007/011529 44 To a solution of 3-2 (1 equiv) in dioxane is added an equal volume of 4N HCl/dioxane and the reaction mixture is stirred at room temperature overnight.' The progress of the reaction is followed by RP-HPLC and TLC. Upon completion of the reaction the solvent is removed under reduced pressure and the solvent is removed and co-evaporated from dry dioxane and xylenes. The product 3-3 is used directly in the next step. 3. Synthesis of compound 3-4. To a solution of 3-3 (1 equiv) in DMF is added a. solution of 2-6 (1 equiv) in DMF. To the stirred reaction mixture is added TEA (4 equiv). The pH of the reaction mixture is checked by spotting 0.5 uL of reaction mixture on wet pH paper. If the pH is <7.4 more TEA is added until pH >8.0. The progress of the reaction is followed by RP-HPLC and TLC. Upon completion of the reaction the solvent is removed under reduced pressure and the residue is partitioned between DCM and water. The organic phase is further washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The product 3-4 is purified by silica gel chromatography. Example 8 Synthesis of compound 4-2, (Figure 28). To a solution of compound 4-1 (200.0 mg, 0.28 mmol) in DMA (5 mL) were added sulfo NHS (73.0 mg 0.56 mmol) and DCC (63.59 mg, 0.308 mmol). The solution was stirred at 55 0 C for 48 hours. Progress of the-reacti6n was monitored by C18-RP HPLC. At completion the solution was filtered to remove unreacted sulfo NHS and precipitated DCU. The filtrate was condensed under reduced pressure via the rotavap. Remaining residue was dissolved in hot isopropanol. The solution was WO 2008/140452 PCT/US2007/011529 45 allowed to cool and product crystallized. The product, compound 4-2, was isolated by filtration and dried under reduced pressure. Example 9 Synthesis of compound 6-3, (Figure 29). 1. Synthesis of ChromaLink' Biotin sulfo-NHS ester 6-2. A mixture of 50 mg/mL solution of acid compound 4-1 (12.9 mg; 0.018 mmol) in DMSO (0.256 mL) is prepared. To the solution is added sulfo-NHS (4.7 mg; 0.22 mmol; 1.2 equiv) and EDC (4.2 mg; 0.22 mmol; 1.2 equiv) and stirred overnight at room temperature. The reaction was followed by C18 RP-HPLC that indicated >90% conversion to product. The solution was used directly in the next step. 2. Synthesis of ChromaLink" Biotin/PEG3/dUTP 6-3. A solution of 5-aminoallyl dUTP (1.0 mg; 1.93 uL of a, 75 mg/mL solution in water; Trilink Biotechnologies, San Diego, CA) is dissolved in 100 mM borate, pH 8.0). 6.1 (0.22 mg; 5.5 uL; 1.5 equiv of a 50 mg/mL solution in DMSO) was added and the reaction mixture is incubated at room temperature. The progress of the reaction is followed by C 18 RP-HPLC. The product is isolated by ion-exchange chromatography using DEAE-Sephadex using a gradient from water to 0.6 M LiCl as the eluting buffers. The product is characterized by 'H-NMR, HPLC and mass spectroscopy. Example 10 Synthesis of compound 7-8, (Figure 30). 1. Synthesis of compound 7-3.

WO 2008/140452 PCT/US2007/011529 46 To a solution of BOC-EDA (381 mg; 2.38 mmol) in THF (7 mL) was added a heterogeneous mixture of S-HyNic (SANH; 690 mg; 2.38 mmol; Solulink Biosciences, San Diego, CA) and TEA (660 uL; 4.76 mmol). Following stirring for 1 h the reaction mixture became homogeneous and was complete .as determined by TLC (DCM/MeOH/TEA (90/10/1)). The solvent was removed on the rotavap and the residue was partitioned between DCM and water. The organic phase was further washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to give 950 mg of pale yellow oil. The oil solidified on standing and was suspended in hexanes/ethyl acetate (1/1) and isolated by filtration to give 350 mg of a pale yellow solid that was a single spot by TLC. 2. Synthesis of compound 7-4. 7-3 (332 mg; 0.99 mmol) was dissolved MeOH (7 mL) with mild heating. The solution was cooled to room temperature. 100 mM MES, 0.9% NaCl, pH 4.7 (7 mL) was added followed by the addition of 4-carboxybenzaldehyde (150 mg; 0.99 mmol) in MeOH (7mL). Following stirring for 30 min a precipitate formed. The reaction mixture was stirred for 8 h and the solids were isolated by filtration and dried at 50 0C under high vacuum to give 320 mg of a pale yellow solid. 3. Synthesis of compound 7-5. To a mixture of 7-5 (150 mg; 0.35 mmol) in dry dioxane (3 mL) was added 4 N HCl/dioxane (3 mL) and the reaction mixture was stirred overnight at room temperature. The reaction mixture never became homogeneous. The reaction mixture was transferred to a test tube and centrifuged. The supernatant was discarded and the solids were washed with dry dioxane (10 mL) . The washing procedure was repeated three times. The solids were dried under vacuum to give 125 WO 2008/140452 PCT/US2007/011529 47 mg of a pale yellow solid. Mass spectral analysis: expected (neg mode): 326; found 326. The solids were used directly in the synthesis of 7-6. 4. Synthesis of compound 7-6. To a solution of 7-5 (111.0 mg; 0.31 mmol) in DMF (4 mL) was added biotin succinimidyl ester (94 mg; 0.28 mmol) and TEA (340 uL; 2.4 mmol). The reaction mixture was allowed to stand at room temperature for 48 h. Solids that precipitated were isolated by filtration to give 124 mg of a pale yellow solid. TLC indicated at single spot Rf 0.2 (DCM/MeOH/TEA (80/20/1); developed with dimethylcinnamaldehyde solution in ethanol/2% sulfuric acid followed by heating). Mass spectral analysis: expected (neg mode): 553; found 552 and 588 (+Cl~). 5. Synthesis of compound 7-7. A mixture of 20 mg/mL solution of acid 7-6 (22.6 mg; 41 umol) in DMA (dimethylacetamide; 1.12 mL) was prepared. To the solution was added sulfo-NHS (10.6 umol; 1.2 equiv) and EDC (9.4 mg; 1.2 equiv) and stirred overnight at room temperature. The reaction was followed by C18 RP-HPLC that indicated >90% conversion to product. The solution was used directly in the next step. 6. Synthesis of compound 7-8. A solution of 5-aminoallyl dUTP (0.10 mg; 1.3 uL of a 75 mg/mL solution in water; Trilink Biotechnologies, San Diego, CA) was dissolved in 100 mM borate, pH 8.0). 7-7 (0.167 mg of a 20 mg/mL solution in DMA) was added and the reaction mixture was incubated at room temperature. The progress of the reaction was followed by C-18 RP-HPLC. The product is isolated by ion-exchange chromatography using WO 2008/140452 PCT/US2007/011529 48 DEAE-sephadex using a gradient from water to 0.6 M LiC1 as the eluting buffers. The product is characterized by 'H-NMR, HPLC and mass spectroscopy. Example 11 Synthesis of ChromaLink" Dige dUTP (8-2, Figure 31). 1. Synthesis of ChromaLink" Dige sulfo-NHS ester 8-1. A mixture of 20 mg/mL solution of acid 2-9 (1 equiv) in DMA (dimethylacetamide; 1.12 mL) is prepared. To the solution is added sulfo-NHS (1.2 equiv) and EDC (1.2 equiv) and stirred overnight at room temperature. The reaction was followed by C18 RP-HPLC that indicated >90% conversion to product. The solution was used directly in the next step. 2. Synthesis of ChromaLink" Dige dUTP 8-2. A solution of 5-aminoallyl dUTP (1 equiv of a 75 mg/mL solution in water; Trilink Biotechnologies, San Diego, CA) is dissolved in 100 mM borate, pH 8.0). 8-1 (1.5 equiv of a 20 mg/mL solution in DMA) is added and the reaction mixture is incubated at room temperature. The progress of the reaction is followed by C-18 RP-HPLC. The product is isolated by ion-exchange chromatography using DEAE-sephadex using a gradient from water to 0.6 M LiCl as the eluting buffers. The product is characterized by 'H-NMR, HPLC and mass spectroscopy.

Claims

1. A fluorescent hydrazone compound of formula I, (R,R 2 ) NN=C (RR 2 } wherein: Ri are independently a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 2 are independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the - unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine and wherein said composition has an emission frequency equal to or greater than 400nm.

2. A fluorescent hydrazone compound according to claim 1 wherein one of R, one of R or one of R 2 further WO 2008/140452 PCT/US2007/011529 50 comprises a linker moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety. .

3. A fluorescent hydrazone compound according to claim 2 wherein said linker further comprises a biomolecule.

4. A fluorescent composition of the formula III, H *%N IN YR1 N R2 N 0 R 4 0 R3 R 5 III wherein; R, is a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 2 is a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1 10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an WO 2008/140452 PCT/US2007/011529 51 alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 3 is H or OH; R 4 is H or a nucleic acid moiety; and R, is PO, or a nucleic acid moiety.

5. A profluorescent hydrazine compound of formula IV, (RR2) N(H), (NH2),, IV wherein: R is a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 2 is a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1 10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of WO 2008/140452 PCT/US2007/011529 52 one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; n is 0 when m is 2 and n is 1 when m is 1.

6. A profluorescent hydrazine compound according to claim 5 wherein RI or R 2 further comprise a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety..

7. A profluorescent hydrazine compound according to claim 6 wherein said linker further comprises a biomolecule.

8. A composition comprising a polymer having one or more profluorescent hydrazine compounds according to claim 5 bound to said polymer by one or more linker moieties.

9. A polymer according to claim 8 wherein said polymer is poly-lysine, poly-ornithine, amino dextran or polyethyleneglycol.

10. A polymer according to claim 8 wherein the polymer is a dendrimer or a panam dedrimer.

11. A profluorescent oxyamine compound of formula VI, (R 1 R 2 ) ONH 2 VI wherein: R2 is a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted WO 2008/140452 PCT/US2007/011529 53 primary, secondary, tertiary and quaternary amine; and R 2 is a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1 10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine.

12. A profluorescent oxyamine compound according to claim 11 wherein R 1 or R 2 further comprise a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety.

13. A profluorescent oxyamine compound according to claim 11 wherein said linker further comprises a biomolecule.

14. A composition comprising a polymer having one or more profluorescent oxyamine compounds according to claim 11 bound to said polymer by one or more linker moieties.

15. A polymer according to claim 14 wherein said polymer is poly-lysine, poly-ornithine, amino dextran or polyethyleneglycol.

16. A polymer according to claim 14 wherein the polymer is a dendrimer or a panam dedrimer.

17. A method of preparing a fluorescent hydrazone according to claim 1 by combining a hydrazine of formula IV, (RR 2 ) N (H),(NH2) IV WO 2008/140452 PCT/US2007/011529 54 with a carbonyl of formula V: O=C (RR 2 ) V wherein: R, are independently a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; R 2 are independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; n is 0 when m is 2 and n is 1 when m is 1 for a time and under conditions that allow hydrazone formation. WO 2008/140452 PCT/US2007/011529 55

18. A method according to claim 17 wherein R2 or R 2 of formula IV further comprises a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety.

19. A method according to claim 17 wherein R, or R 2 of formula V further comprises a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety.

20. A spectrophotometrically quantifiable linker comprising of formula: A-B-C-D wherein; A is an amino thiol or carbohydrate reactive moiety; B is a chromophoric or fluorescent moiety; C is a flexible linker; and D is biotin or a receptor ligand.

21. A spectrophotometrically quantifiable linker according to claim 20 wherein B is a compound that fluoresces emits light or produces a colored product on enzymatic processing.

22. A spectrophotometrically quantifiable linker according to claim 20 bound to a biomolecule via a amino, thiol or carbohydrate reactive moiety.

23. A spectrophotometrically quantifiable linker according to claim 22 wherein the biomolecule is selected from the group consisting of protein, peptide, and oligonucleotide, polynucleotide.

24. A spectrophotometrically quantifiable linker according to claim 20 bound to a biomolecule via receptor ligand pairs; biotin/avidin, peptide S/ribonuclease, digoxigenin/anti-digoxigenin antibody, complimentary oligonucleotide pairs or antibody/ligand pairs. WO 2008/140452 PCT/US2007/011529 56

25. A spectrophotometrically quantifiable linker according to claim 20 wherein a first biomolecule is bound via an amino, thiol or carbohydrate reactive moiety and a second biomolecule is bound via a receptor ligand pair.

26. A compound of the formula: 0 0 N0 0 H44m H N N ,Cf 0 HN NH H H Y

27. A compound of the formula: 0 HNH HN / N H N H 0 o 0

28. A compound of the formula: 0 O N O i CH 0 NH H H HH WO 2008/140452 PCT/US2007/011529 57

29. A compound of the formula: 0 - N HHN NH HN N H NH / O ON H HN H N ON SO 0 0

30. A compound of the formula: 0 0 N H HN NHN N 7H H H Na H N O ON O 0 0

31. A compound of the formula: 0 NHN HHNNHHN /\NO OH H 0 0

32. A compound of the formula: 0 0 N HNI HN / N H H H HN s N N N 0 0 WO 2008/140452 PCT/US2007/011529 58

33. A compound of the formula: 0 0 0 -% 0 I N H O NN 0 HN NH H H 0 \\ /%-0 P- 01 I\ 0 ' wherein R = H or OH.

34. A compound of the formula: 0 HNA 0 H NH H .N O 0 N N HH H H 00 o O 0 I\ P t% P- 0 I J a 4 Na OH *Na-O Oa O-Na* a* O ONa~ wherein R = H or OH.

35. A compound of the formula: 0 OH HC \ HH N N H OH OO R HO -0 -P +0 *0 0 -O 0 wherein R = H or OH. WO 2008/140452 PCT/US2007/011529 59

36. A method of preparing a spectrophotometrically quantifiable linker comprising the steps: a. preparing a first conjugate of a first biomolecule bound to one profluorescent compound of a fluorescent pair via a n amino, thiol or carbohydrate reactive moiety; b. preparing a second conjugate of a second biomolecule bound to a flexible linker via a biotin or a receptor ligand and the other profluorescent compound of a fluorescent pair and c. ' combining the first conjugate with the second conjugate for a time thereby forming a hydrazone bond between the profluorescent compound pair forming a fluorescent moiety.