WO2008140452A1 - Nouveaux réactifs et lieurs fluorescents et chromophores/pro-fluorescents et pro-chromophores a base hydrazone et d'oximes - Google Patents

Nouveaux réactifs et lieurs fluorescents et chromophores/pro-fluorescents et pro-chromophores a base hydrazone et d'oximes Download PDF

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WO2008140452A1
WO2008140452A1 PCT/US2007/011529 US2007011529W WO2008140452A1 WO 2008140452 A1 WO2008140452 A1 WO 2008140452A1 US 2007011529 W US2007011529 W US 2007011529W WO 2008140452 A1 WO2008140452 A1 WO 2008140452A1
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moiety
substituted
conjugationally
extended
unsubstituted
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David A. Schwartz
Leopoldo Mendoza
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Solulink, Inc.
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Priority to AU2007353457A priority Critical patent/AU2007353457A1/en
Priority to EP07794844A priority patent/EP2150858A4/fr
Priority to PCT/US2007/011529 priority patent/WO2008140452A1/fr
Publication of WO2008140452A1 publication Critical patent/WO2008140452A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B26/00—Hydrazone dyes; Triazene dyes
    • C09B26/02—Hydrazone dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to compounds used to label biomolecules for diagnostic and therapeutic purposes.
  • 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 .
  • a fluorescent molecule that fluoresces at a desired frequency.
  • a fluorescent molecule is modified with a thiol- or amino-reactive moiety such as succinimidyl 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 .
  • fluorophores have been modified with these moieties including fluorescein, rhodamine, Texas Red and cyanine dyes, Cy3 and Cy5.
  • fluorescein, rhodamine, Texas Red and cyanine dyes Cy3 and Cy5.
  • 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.
  • 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.
  • 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.
  • 4-N-aminocytidine (4-hyd-C)
  • Nitta et al . (Eur. J. Biochem. 157(2) :427, 1986) has described crosslinking between 16S ribosomal RWA and protein S4 in E. coli ribosomal 3OS subunits effected by treatment with bisulfite/hydrazine and bromopyruvate .
  • Musso et al. " (U.S.
  • the biomolecule is fluorescently labeled with a fluorescent molecule.
  • the processes or methods used to prepare the conjugate can often times cause quenching or photobleaching of the fluorophore.
  • the unbound fluorescently labeled conjugate must be removed to obtain an accurate fluorescent signal .
  • Biotin (Figure 1) is a small molecule, MW 250, that binds to streptavidin with an association constant of 10 15 .
  • 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 .
  • biotin is conjugated to a probing biomolecule such as an antibody or an oligonucleotide.
  • a probing biomolecule such as an antibody or an oligonucleotide.
  • 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.
  • the first assay is ' the HABA ( [2- (4 '-hydroxyazobenzene) ] benzoic acid) assay developed by Green (Green, N.M. Biochem. J., 94, 23c-24, 1965).
  • HABA [2- (4 '-hydroxyazobenzene) ] benzoic acid
  • Green Green, N.M. Biochem. J., 94, 23c-24, 1965.
  • 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 ran decreases proportionately.
  • an unknown amount of biotin present in a solution can be evaluated in a 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 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-
  • FRET fluorescence resonance energy transfer
  • 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) n (NH 2 ) n , 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
  • 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;
  • R 2 is independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10
  • R 1 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 alken
  • 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.
  • 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.
  • 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.
  • 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.
  • conjugationally extended profluorescent/prochromophoric hydrazine and/or the conjugationally extended profluorescent/prochromophoric carbonyl may further comprise a linkable moiety at either the R 1 or R 2 position.
  • 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 1 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
  • a profluorescent/prochromophoric oxyamine compound 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.
  • a profluorescent/prochromophoric oxyamine compound 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 intemucleotide linkages selected from the group consisting of phosphodiesters, phosphorothioates, phosphoramidites and peptide nucleic acids.
  • 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.
  • A is an amino reactive moiety it may be selected from the group consisting of N- hydroxysuccinimidyl, p-nitrophenyl, pentafluorophenyl and N- hydroxybenzotriazolyl .
  • A 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.
  • 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 group consisting of a protein, a peptide, an oligonucleotide and a polynucleotide.
  • 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.
  • 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 .
  • a method of preparing a spectrophotometrically quantifiable linker 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.
  • 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 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 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 aldehydes do not form substantially fluorescent species .
  • 5 ' - ( 6-Hydrazinylpyridine) -modified oligonucleotide is reacted with 4- dimethylaminocinnamaldehyde (Reaction A) and na ⁇ hthalene-l,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) .
  • 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 fluorophofes targeted for synthesis
  • Figure 16 A diagrammatic representation of the synthetic methods for the preparation of hydrazinoheterocyles
  • Figure 17 Chemical structure of benzimidazole pro- fluorophores and synthesis schemes of their parent fluorophores targeted for synthesis
  • FIG. 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 blgG and blgG 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 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);
  • Figure 31 Schematic representation of the synthesis of a linker of
  • biomolecule 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 and a polysaccharide.
  • the biomolecule is a nucleic acid it may be DNA, cDNA, KNA, or PNA and may comprise natural or unnatural bases or internucleotide linkages such as for example phosphodiesters, phosphorothioates, phosphoramidites or peptide nucleic acids.
  • profluorophore 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.
  • pro-chromophore 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.
  • linking moieties 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 .
  • ligand/receptor couple refers to a pair of molecules having a substantially high affinity of binding specifically to one another.
  • 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 15 .
  • 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 lOOnm with respect to the fluorescence of the profluorophore molecules that form the hydrazone or oxime.
  • fluorescently labeled triphosphates e.g.
  • Cy3 and Cy5 triphosphosphates 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 labeled polynucleotide.
  • 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 .
  • 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) .
  • 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 .
  • Table 1 lists the bis- (2-heteroaromatic)hydrazones prepared by Ryan et a.1. , supra. and including their excitation and emission wavelengths and relative fluorescence properties.
  • X' and X' ' are N;
  • X 2 is NR 2 or 0;
  • 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.
  • R 1 can be unsubstituted or substituted with one or more substituents that extend the conjugation of R 1 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.
  • 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.
  • R 2 can be unsubstituted or substituted with one or more substituents that extend the conjugation of R 1 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.
  • R 3 and R 4 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.
  • R 3 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.
  • 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 biomolecule
  • the hydrazones of structure VII are prepared from aromatic or heteroaromatic hydrazines or hydroxylamines of structure VIII
  • X 2 NR 2 or O
  • linkable chelates include:
  • 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- formylbenzamide] -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.
  • 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 .
  • Table 2 shows the fluorescent hydrazones and their absorbance and emission maxima.
  • 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) .
  • SHTH SHTH
  • oligonucleotides were reacted with 4-dimethylaminocinnamaldehyde ( Figure 9, reactions A and C) but only the hydrazine derived hydrazone was fluorescent .
  • 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.
  • 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., 100:1913, 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 .
  • 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.
  • Figure 17 presents target pro-fluorophores and their respective parent fluorophores .
  • 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 .
  • a Nd:YAG laser pumped OPO system will allow for tunable excitation between 400 nm and 3000 run.
  • 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
  • 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 abs ⁇ rbance 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.
  • the present invention incorporates a pre-formed chromophoric/fluorescent hydrazone into the linker comprising the ligand for direct spectrophotometry 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) .
  • Figure 21 presents constructions of thiol and oxidized carbohydrate-reactive linkers of the present invention.
  • NTP biotinylated nucleoside triphosphate
  • 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.
  • a conjugationally extended aldehyde such as a benzaldehyde moiety
  • U. 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.
  • 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.
  • 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 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.
  • Electrospray mass spec expected m/e 487; found positive mode 488 (M+H) , negative mode 486 (M-H) and 522 (M+Cl " ) .
  • 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 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 dimethyla ⁇ dnocinnamaldehyde/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.
  • the extinction coefficient of Biotin/PEG4/chromophore succinimidyl ester 6 was determined by dissolving Biotin/PEG4/chromo ⁇ hore 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.
  • Bovine immunoglobulin (blgG; 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.
  • modification buffer 100 mM phosphate, 150. mM NaCl, pH 7.2
  • 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 blgG 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.
  • 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.
  • 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 1 H-NMR, HPLC and mass spectroscopy.
  • 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 iriM 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 DEAE-sephadex using a gradient from water to 0.6 M LiCl as the eluting buffers. The product is characterized by 1 H-NMR, HPLC and mass spectroscopy.

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Abstract

L'invention porte: sur des compositions d'hydrazines étendues par conjugaison de formule (RR2)N(H)n(NH2)n, sur des compositions d'hydrazones fluorescentes de formule (RR2)NN=C (R1R2), sur des méthodes de formation d'hydrazones par réaction d'hydrazines étendues par conjugaison avec des carbonyles étendus par conjugaison, et sur leurs méthodes d'utilisation dans des systèmes de bioessais. L'invention porte également sur l'utilisation de ces compositions d'hydrazines étendues par conjugaison et d'oximes dans des bioessais directs de colorimétrie et de fluorométrie impliquant l'incorporation d'un chromophore ou d'un fluorophore dans un lieur placé entre un fragment réactif de liaison et une molécule de biotine. Plus spécifiquement le lieur comprend une molécule de paire de liaison à haute affinité telle que par exemple la biotine de la paire de liaison biotine/avidine à haute affinité reliée à une molécule espaceur telle que par exemple une longueur de polyéthylèneglycol suivi d'un fragment pro-chromophore, chromophore, pro-fluorescent ou fluorescent relié à un fragment réactif amino-, thiol d'hydrate de carbone tel que par exemple un groupe succinimidyle, maléimido ou aminoxy, pouvant se fixer par covalence à une biomolécule.
PCT/US2007/011529 2007-05-14 2007-05-14 Nouveaux réactifs et lieurs fluorescents et chromophores/pro-fluorescents et pro-chromophores a base hydrazone et d'oximes WO2008140452A1 (fr)

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AU2007353457A AU2007353457A1 (en) 2007-05-14 2007-05-14 Novel hydrazone-based and oxime-based fluorescent and chromophoric/pro-fluorescent and pro-chromophoric reagents and linkers
EP07794844A EP2150858A4 (fr) 2007-05-14 2007-05-14 Nouveaux réactifs et lieurs fluorescents et chromophores/pro-fluorescents et pro-chromophores a base hydrazone et d'oximes
PCT/US2007/011529 WO2008140452A1 (fr) 2007-05-14 2007-05-14 Nouveaux réactifs et lieurs fluorescents et chromophores/pro-fluorescents et pro-chromophores a base hydrazone et d'oximes

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US20120258880A1 (en) * 2010-11-22 2012-10-11 The University Of Chicago Methods and/or Use of Oligonucleotide Conjugates for Assays and Flow Cytometry Detections
US8309306B2 (en) 2008-11-12 2012-11-13 Nodality, Inc. Detection composition
US9222936B2 (en) 2007-04-18 2015-12-29 Solulink, Inc. Methods and/or use of oligonucleotide conjugates for suppressing background due to cross-hybridization
US9575067B2 (en) 2007-05-23 2017-02-21 Ventana Medical Systems, Inc. Polymeric carriers for immunohistochemistry and in situ hybridization

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US5512400A (en) * 1993-12-15 1996-04-30 Hodogaya Chemical Co., Ltd. Hydrazone compound and electrophotographic photoreceptor and organic electroluminescent element both containing the same
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US9222936B2 (en) 2007-04-18 2015-12-29 Solulink, Inc. Methods and/or use of oligonucleotide conjugates for suppressing background due to cross-hybridization
US9575067B2 (en) 2007-05-23 2017-02-21 Ventana Medical Systems, Inc. Polymeric carriers for immunohistochemistry and in situ hybridization
EP2167963B1 (fr) * 2007-05-23 2019-04-17 Ventana Medical Systems, Inc. Supports polymères pour immunohistochimie et hybridation in situ
EP3561513A1 (fr) * 2007-05-23 2019-10-30 Ventana Medical Systems, Inc. Supports polymères pour immunohistochimie et hybridation in situ
US8309306B2 (en) 2008-11-12 2012-11-13 Nodality, Inc. Detection composition
US20120258880A1 (en) * 2010-11-22 2012-10-11 The University Of Chicago Methods and/or Use of Oligonucleotide Conjugates for Assays and Flow Cytometry Detections
US20120258881A1 (en) * 2010-11-22 2012-10-11 The University Of Chicago Methods and/or Use of Oligonucleotide Conjugates for Assays and Microscopy/Imaging Detections
US20130123121A1 (en) * 2010-11-22 2013-05-16 The University Of Chicago Methods and/or Use of Oligonucleotide-Bead Conjugates for Assays and Detections

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