WO1990002747A1 - Reactifs de phtalocyanine et tetrabenztriazaporphyrine decales vers le rouge - Google Patents

Reactifs de phtalocyanine et tetrabenztriazaporphyrine decales vers le rouge Download PDF

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Publication number
WO1990002747A1
WO1990002747A1 PCT/US1989/003807 US8903807W WO9002747A1 WO 1990002747 A1 WO1990002747 A1 WO 1990002747A1 US 8903807 W US8903807 W US 8903807W WO 9002747 A1 WO9002747 A1 WO 9002747A1
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WIPO (PCT)
Prior art keywords
aluminum
dna
reagent
group
phthalocyanine
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PCT/US1989/003807
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English (en)
Inventor
George E. Renzoni
Louis J. Theodore
Deborah C. Schindele
Clifford C. Leznoff
Barry V. Pepich
Karen L. Fearon
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Ultra Diagnostics Corporation
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Priority claimed from US07/398,433 external-priority patent/US5135717A/en
Application filed by Ultra Diagnostics Corporation filed Critical Ultra Diagnostics Corporation
Publication of WO1990002747A1 publication Critical patent/WO1990002747A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • This invention relates to phthalocyanine and tetrabenztriazaporphyrin reagents and their derivatives useful as fluorescent reporting groups, imaging agents, and also as therapeutic agents.
  • the fluorescent reagents are useful in nucleic acid sequence analysis, nucleic acid probe and hybridization assays, fluorescence microscopy, flow cytometry, immunoassay, and fluorescence imaging.
  • the reagents may also be useful as therapeutic agents in photodynamic applications.
  • Fluorescent compounds have been widely used in immunoassays, flow cytometry, fluorescence microscopy, and DNA sequencing. To date, the sensitivity of such assays has been limited by the spectral properties of available fluorophores.
  • Ideal fluorophores have five characteristics: a readily accessible excitation wavelength with a large molar absorptivity, a high fluorescence quantum yield, a large Stokes shift (> 50 nm), emission at long wavelengths (greater than 600 nm), and a sharp emission profile (full width at half maximum, FWHM ⁇ 40 nm).
  • Aluminum phthalocyanine (AlPc) has nearly ideal spectral properties. Excitation of aluminum phthalocyanine at 350 nm results in emission at 685 nm with fluorescence quantum yield ( ⁇ f ) of 0.58. Brandon, J.H., and D. Madge, J. Amer. Chem. Soc, 102:62-65, 1980.
  • Aluminum phthalocyanine is composed of a highly conjugated macrocycle and a trivalent aluminum atom.
  • the structure of the parent AlPc fluorophore is shown below.
  • L is a ligand such as OH when the AlPc is in water.
  • the trivalent aluminum atom provides axial ligation which serves to reduce aggregation and thereby increases fluorescence in solution.
  • phthalocyanine sulfonates have been determined to be effective in directed cell killing. Ben-Hur, E. and I. Rosenthal, Photochem. Photobiol. 42:129-133, 1985.
  • the advantage that phthalocyanines have over other photodynamic agents is their large molar absorptivity in the red region of the visible spectrum.
  • the large molar extinction coefficient coupled with the transparency of tissue at these red wavelengths provides for more efficient light penetration and subsequently more effective treatment of subcutaneous malignancies.
  • aluminum phthalocyanine derivatives red-shifted from the parent compound will provide an even greater depth of penetration and enable even more effective treatments. Derivatives attached to biological moieties such as probes or antibodies can be targeted to specific cell populations.
  • TBTAPs tetrabenztriazaporphyrins
  • the only structural difference is the replacement of the nitrogen at position twenty of the phthalocyanine with a substituted carbon.
  • No substituted derivatives of these compounds have been reported to date.
  • the spectral and luminescent properties of magnesium and palladium benzoporphyrins have been reported.
  • TTAP red-shifted, water-soluble phthalocyanine and tetrabenztriazaporphyrin
  • R 1 is independently selected from -XYW, -YW, -W, or -H.
  • X is CR 3 R 4 , where R 3 and R 4 are independently selected from hydrogen, alkyl (preferably C 1 -C 12 ), aryl (preferably C 8 -C 12 ), or aralkyl (preferably C 6 -C 12 ), or R 3 and R 4 together may be a carbonyl oxygen, or X is either phenyl or a heteroatom preferably selected from among oxygen, nitrogen, and sulfur.
  • Y is a linking group between X and W or between a benzo ring of the phthalocyanine or TBTAP macrocycle and W.
  • W is a water-soluble group.
  • R 2 comprises a biological entity such as an antibody, antigen, nucleot ⁇ de, nucleic acid, ol ⁇ gonucleotide, avidin, streptavidin, or a membrane probe, or R 2 is a reactive or activatable group suitable for conjugation to a biological entity.
  • Z is N or C-R, where R is H or an organic group such as alkyl (preferably C 1 -C 1 2 ), aryl (preferably C 8 -C 12 ), or aralkyl (preferably C 8 -C 12 ).
  • the R 1 and R 2 groups may be located on any of the four benzo rings of the TBTAP.
  • the biological entity is located on the meso carbon atom of the macrocycle rather than on a benzo ring.
  • the Unking group Y is preferably less than 4 atoms in length and may contain aliphatic, aromatic, polye ⁇ e, alkynyl, polyether, polyamide, peptide, amino acid, polyhydro ⁇ y, or sugar functionalities.
  • M is aluminum
  • each R 1 is -XYW
  • X is either an oxygen or sulfur atom
  • Y is a methylene group
  • W is a carboxylic acid
  • Z is nitrogen
  • R 2 is -X-CH 2 CO 2 H.
  • the substitution of R 1 occurs at the 1,8,15,22 positions (3 isomer) or at the 2,9,16,23 positions (4 isomer) of the macrocycle.
  • Se e FIGURE 1 for the phthalocyanine and tetrabenztriazaporphy ⁇ n ring numbering system.
  • M is aluminum
  • each R 1 is -XYW
  • X is either an oxygen or sulfur atom
  • Y is phenyl
  • W is sulfonate or sulfonyl chloride
  • Z is nitrogen
  • R 2 is -O-phenyl-sulfonate, -O-phenyl-sulfonyl chloride, -S- phenyl-sulfonate, or -5-phenyl-sulfonyl chloride.
  • the substitution of R 1 occurs at the 1,8,15,22 positions (3 isomer) or at the 2,9,16,23 positions (4 isomer) of th e macrocycle.
  • the preferred embodiments ar e as described above except that Z is a carbon substituted with either hydrogen or phenyl subst ⁇ tuents.
  • the phenyl may be unsubstituted or substituted by 1-5, preferably 1-2 substituents selected from among C 1 -C 6 alkyl, halogen (e.g. C1, Br, F, I), earboxy, nltro, or other subst ⁇ tutents that do not substantially interfere with the fluorescence or water solubility of the molecule.
  • the divalent metals (M), Cd, Mg, and Zn no axial ligand (L) is present.
  • Phosphorus (M) will bear either one or three axial ligands (L).
  • Reagent kits for detection of single analytes using a reagent described above are provided, as are kits and methods for sequencing DNA.
  • Reagent kits useful for simultaneous detection of a plurality of analytes in solution containing combinations of the subject reagents, each tethered to a different biological entity are also disclosed.
  • a second aspect of the present invention involves pyrazine porphyrazines, pyrazine tetrabenztriazaporphyrins, pyridine porphyrazines, and pyridine tetrabenztriazaporphyrins. These compounds have the same structure as formula I, with the exception that 1-4 of the benzo rings contain 1 nitrogen atom (pyridine derivatives) or 2 nitrogen atoms (pyrazine derivatives).
  • the benzo ring contains 1 nitrogen atom
  • both the 3 and 4 positional isomers are possible.
  • the 2 nitrogen atoms per ring in the pyrazine derivatives are generally oriented in a 1,4 arrangement in the benzo ring.
  • all 4 benzo rings will contain either 1 or 2 nitrogen atoms.
  • Mixed derivatives are also possible, in which 1-3 benzo rings contain one nitrogen atom (either isomer) and 3-1 benzo rings contain 2 nitrogen atoms.
  • the R 1 and R 2 groups may be attached to carbon atoms or nitrogen atoms in the benzo rings, but attachment to carbon atoms of the benzo rings is preferred.
  • -XYW When X is a heteroatom, -XYW will be attached to a carbon; when X is CR 3 R 4 or phenyl, -XYW may be attached to a carbon atom (preferred) or a nitrogen atom of the benzo ring.
  • Examples 12 and 13 herein Illustrate preferred compounds of the second aspect compounds. Additional preferred compounds are analogous to those identified for the compounds of the first aspect of the present invention.
  • the compounds of the second aspect of this invention may be used in the same applications as the phthalocyanine and tetrabenztriazaporphyrin compounds described above.
  • a third aspect of the present invention involves cation ⁇ c reagents having formula I above, except that R 2 is R 1 .
  • R 1 , X and Y are as described above.
  • W is -N + D 1 D 2 D 3 , wherein D 1 -D 3 are independently hydrogen, C 1 -C 12 alkyl, C 6 -C 12 aralkyl, or C 6 -C 12 aryl groups, or -N + D 1 D 2 D 3 forms a pyrid ⁇ nium ring.
  • the charged groups may be associated with any conventional counterion as long as it does not substantially interfere with fluorescence or synthesis of the reagent.
  • These reagents may be advantageously used to bind to (stain or label) oligo- and polynucleotides, especially DNA or RNA, for qualitative or quantitative determination.
  • the present invention provides intermediates for the synthesis of the compounds of formula I.
  • reactive or activatable intermediates in which R 2 in formula I is a group capable of being covalently attached to a biological entity are contemplated.
  • R 2 may be directly attached to the benzo ring or may be linked to the benzo ring by an XY or Y linkage.
  • Such R 2 groups include -SO 2 Cl; -CO 2 H; -COX', wherein X' is a leaving group such as N- hydroxy-succinim ⁇ de; maleimide; or isothiocyanate.
  • R 2 can also be a nucleophilic moiety, such as an amino group, for reaction with reactive groups on the biological entity.
  • a water soluble group ,W on the benzo rings may alternatively be conjugated to biological entities, in some embodiments.
  • the other variables in formula I are the same as defined herein. These compounds may be coupled to biological entities by standard coupling reactions. Once coupled, at least a portion of the reaetive group becomes a Y' group, as defined in connection with formula I.
  • FIGURE 1 shows the phthalocyanine and tetrabenztriazaporphyrin ring numbering system.
  • FIGURE 2 shows the absorbance and emission spectrum of aluminum phthalocyanine tetrasulf onate, 1, in water.
  • FIGURE 3 compares the absorbance spectra of the glycolic acid derivatives, 2 and 3, in water.
  • FIGURE 4 compares the emission spectra of the glycolic acid derivatives, 2 and 3, in water.
  • FIGURE 5 compares the emission of spectra of the two glycolic acid derivatives, 2 and 3, in aqueous cetyl trimethylammonium bromide (CTAB).
  • CTAB cetyl trimethylammonium bromide
  • FIGURE 6 compares the absorbance spectra of the oxygen substituted aluminum phthalocyanine sulfonates, 4 and 5, in water.
  • FIGURE 7 compares the emission spectra of the oxygen substituted aluminum phthalocyanine sulfonates, 4 and 5, in water.
  • FIGURE 8 compares the absorbance spectra of the sulfur substituted aluminum phthalocyanine sulfonates, 6 and 7, in water.
  • FIGURE 9 compares the emission spectra of the sulfur substituted aluminum phthalocyanine sulfonates, 6 and 7, in water.
  • FIGURE 10 compares the emission spectra of the oxygen substituted aluminum tetrabenztriazaporphyrin sulfonates, 8 and 9, in water.
  • FIGURE 11 compares the emission spectra of the sulfur substituted aluminum tetrabenztriazaporphyrin sulfonates, 10 and 11, in water.
  • FIGURE 12 shows the absorbance and emission spectra of aluminum 20-H tetrabenztriazaporphyrin sulf onate, 12, in water.
  • FIGURE 13 shows the absorbance and emission spectra of aluminum 20- phenyl tetrabenztriazaporphyrin sulfonate, 13, in water.
  • FIGURE 14 compares the absorbance spectra of the metal free phthalocyanine cationic f luorophore, 14a, in water with and without RNA.
  • FIGURE 15 compares the emission spectra of the metal free phthalocyanine cationic fluorophore, 14a, in water with and without RNA.
  • FIGURE 16 compares the absorbance spectra of the aluminum phthalocyanine cationic fluoropho ⁇ e, 14b, in water with and without RNA.
  • FIGURE 17 compares the emission spectra of the aluminum phthalocyanine cationic fluorophore, 14b, in water with and without RNA.
  • FIGURE 18 compares the emission spectra in water of four aluminum phthalocyanine sulfonates, 1, 4, 5 , and 7, suitable for DNA sequence analysis.
  • the invention in a first aspect, provides improved phthalocyanine and related reagents in 4he form of red-shifted, water-soluble, monomerically- tetherable derivatives according to formula I.
  • M represents either H 2 or is selected from among the following metals: aluminum, silicon, phosphorous, gallium, germanium, cadmium, scandium, magnesium, tin, and zinc.
  • Each R 1 is independently selected from -XYW, -YW, -W, or hydrogen.
  • X represents CR 3 R 4 , where R 3 and R 4 are independently selected from hydrogen, alkyl (preferably C 1 -C 1 2 ), aryl (preferably C 6 -C 12 ), or aralkyl (preferably C 6 -C 12 ), or R 3 and R 4 together may be a carbonyl oxygen, or X is phenyl, or X is a heteroatom selected from among oxygen, nitrogen, sulfur, phosphorus, silicon, and selenium.
  • Y represents a linking group; and W represents a water solubilizing group.
  • the substituent R 2 is selected from among -A, -Y'A, -XA, and -XY'A, where A denotes a biological entity such as an antibody, antibody fragment, antigen, oligonucleotide, nucleot ⁇ de, nucleic acid probe, avidin, streptavidin, or membrane probe.
  • R 2 may also be a reactive or activatable group which is directly attached to the benzo ring or attached by way of a linker, such as -X-alkylene- or -X-phenylene-, where X is defined above.
  • Z is N or C-R, where R is H, or an organic group such as alkyl (preferably C 1 -C 12 ), aryl (preferably C 6 -C 12 ), or aralkyl (preferably C 8 -C 12 ).
  • Y' is a linking group that tethers the biological entity (A) to the phthalocyanine or tetrabenztriazaporphyrin macrocycle.
  • the biological entities containing nucleotides or derivatives thereof are generally triphosphorylated, but mono- and di-phosphorylated compounds may also be employed.
  • Z is either a nitrogen atom or a carbon substituted with hydrogen, alkyl (preferably C 1 -C 12 ), aryl (preferably C 6 -C 12 ), or aralkyl (preferably, C 6 -C 12 ) groups.
  • Z is -CR 2 , in which case all of the variables on the benzo rings of the macrocycle will be R 1 groups; that is, the R 2 group may be attached to the meso carbon rather than to a benzo ring of the TBTAPs.
  • M is aluminum
  • each R 1 is -XYW
  • X is either oxygen or sulfur
  • Y is methylene
  • W is carboxylate
  • Z is nitrogen
  • R 2 is an activatable or reactive group attached to the benzo ring by way of an XY link.
  • R 2 may preferably be -XY-activatable group, where X is O, Y is methylene and the activatable group is -CO 2 H.
  • M is aluminum
  • each R 1 is -XYW
  • X is either oxygen or sulfur
  • Y is phenyl
  • W is sulfonate or sulfonyl chloride
  • R 2 is an activatable or reactive group attached to the benzo ring by way of an XY link
  • Z is nitrogen.
  • R 2 may preferably be -XY-reactive group, where X is O or S, Y is phenyl and the reactive group is -SO 2 Cl (located in the ortho, meta, or para positions of the phenyl ring).
  • a similar preferred embodiment is exactly as above except that Z is a carbon with either a hydrogen or phenyl substituent. These derivatives are referred to as tetrasubstituted tetrabenztriazaporphyrins.
  • some of the present compounds may occur as mixtures, particularly isomeric mixtures or mixtures of compounds with different numbers of water solubilizing groups. Such mixtures are within the scope of this invention.
  • phthalocyanines While the phthalocyanines all share a common absorbance wavelength in the ultraviolet near 350 nm, the visible absorbance is substituent dependent. A red shift of the visible absorbance maxima of phthalocyanines is attained by peripheral substitution with oxygen (ether) and sulfur (thioether) groups, X in formula I. Sulfur substitution results in a greater red shift of fluorescent emission than oxygen substitution, and substitution at the three positions (1,8,15,22 isomer) provides a greater shift than 4 substitution (2,9,16,23 isomer). The trend observed in the absorbance spectra is found in the fluorescence spectra. The trend is also observed in the absorbance and emission maxima of tetrabenztriazaporphyrins. In view of these observations, a preferred group of reagents are those in which at least one X is a heteroatom, although 2, 3 or 4 heteroatoms are also contemplated.
  • Substituent W is provided to impart water solubility to the reagent, preferably at 10 -6 M or lower concentrations.
  • the aqueous solubility should be maintained at temperatures ranging from about 4°C (e.g., for flow cytometric applications) to about 100°C (e.g., 67°C for gene probe applications).
  • W is chosen to provide maximum monomerism or, in other words, to minimize aggregation of the fluorophores in aqueous solution. Aggregation of the fluorophores results in the quenching of fluorescence and thus limits the sensitivity of the probe and therefore its utility in assay environments. Monomerism is discussed in greater detail hereinbelow. Since charge repulsion diminishes aggregation, W is preferably charged rather than neutral. However, W must not promote nonspecific binding. Thus, for nucleic acid sequencing, the W groups should be negatively charged (W is sulf onate, for example) in order to avoid ionic attraction to negatively charged DNA or RNA. Conversely, a positively charged phthalocyanine derivative (W is quaternary ammonium, for example) may be utilized to selectively stain DNA, RNA, and other negatively charged cellular constituents.
  • the water solubilizing groups W can be selected from among -OH, -poly-OH, -CO 2 H, -OCH 2 CO 2 H, -OCHD 1 CO 2 H, -OCD 1 D 2 CO 2 H, -PO 4 2- , -PO 3 -, -SO 3 -, -SO 2 -, -SO 4 2- , -SO 2 Cl, -N + H 3 /-NH 2 , -N + H 2 D/-NHD, -N + HD 1 D 2 /-ND 1 D 2 , and -N + D 1 D 2 D 3 with D-D3 being individually alkyl (preferably C 1 -C 12 ), aryl (preferably C 6 -C 12 ), or aralkyl (preferably C 6 -C 12 ), amino acids (such as one selected from the common 20 naturally occurring amino acids) or peptides (e.g.
  • sulfonate groups (preferably 2, 3 or 4) render the molecule water soluble over a wide range of pH (2-12).
  • Carboxyl ⁇ e acid groups are more sensitive to pH, thus limiting their versatility and performance in aqueous systems. Below pH 5, carboxylic acid groups are not ionized and therefore have limited solubility in water. Both sulfonic and phosphoric acids are ionized below pH 2. Quaternary ammonium groups are positively charged regardless of pH. Charged groups will be associated with a suitable counter ⁇ on.
  • the counterions are not necessarily limited and may be any known counterions that do not interfere with synthesis of the compounds or their desirable fluorescence characteristics.
  • Substituent Y is a group of atoms that links X with the water solubilizing group W or the reactive or activatable group R 2 .
  • Y is methylene (-CH 2 -); however, longer alkyl, aryl, or aralkyl chains are possible (preferably C 2 -C 12 ). Longer links may adversely impact water solubility and increase aggregation in solution leading to a diminution of fluorescence. Therefore, in a preferred embodiment Y has about 7 carbon atoms or less. Alternatively, the link Y may be hydrophilic or even charged to increase both water solubility and monomerism.
  • Suitable hydrophilic spacers include polyethers, polyamines, polyalcohols, and naturally occurring sugars, peptides, and nucleotides.
  • Y is phenyl with X at position one and W at position 4 (para substitution).
  • Y can be selected from among aliphatic, aromatic, mixed aliphatic/aromatic functionalities, polyene (eis or trans), mixed polyene and/or aliphatic and/or aromatic functionalities, alkynyl, mixed alkynyl and/or aliphatic and/or aromatic functionalities, polyether linked by aliphatic and/or aromatic and/or alkenyl and/or alkynyl functionalities, polyamides, peptides, amino acids, polyhydroxy functionalities, sugars, and nucleotides.
  • the precise nature of Y is unimportant, and practically any Y group will work as long as it does not interfere with water-solubility or fluorescence to an unacceptable degree and it is synthetically accessible.
  • Substituents R 1 are individually selected from among -XYW, -YW, -W, and hydrogen. In one preferred embodiment, all three R 1 groups are -XYW, -YW, or -W, especially -XYW. In another preferred embodiment, one R 1 is -XYW and the other two are -YW or -W.
  • Substituent R 2 may be a biological entity such as an antigen or an antibody attached to the macrocycle.
  • R 2 may also be an activatable group or a reactive group; as such, R 2 may be linked to the benzo ring by X or XY linkers, or may be directly attached to the benzo ring.
  • R 2 may be R 1 , in which case no biological entity is covalently bound to the fluorophore.
  • R 2 is attached to the meso carbon of a TBTAP or other derivatives of a triazaporphyrin described herein, and the remaining variables on the benzo rings of the macrocycle are each R 1 .
  • Representative biological entities (A) include natural or synthetic drugs (therapeutics and abused), drug metabolites, metabolites, hormones, peptides, nucleotides (e.g., ATP, CTP, GTP, TTP, UTP, dATP, dGTP, dCTP, dTTP, dUTP, ddATP, ddCTP, ddGTP, ddTTP, ddUTP, and derivatives thereof), neurotrarsmitters, enzyme substrates, DNA or RNA probes, DNA or RNA (oligo and polynucleotides), DNA/RNA hybrids, DNA/DNA hybrids, RNA/RNA hybrids, growth factors, antibody fragments (antigen binding fragments), antibodies (polyclonal or monoclonal), serum proteins, streptavidin, avidin, enzymes, intracellular organelles, cell surface antigens, receptors, ligand binding proteins or associated ligands, membrane probes etc.
  • nucleotides e.
  • the fluorescent moiety i.e., the macrocycle
  • R 2 monomerically to enhance fluorescence.
  • the particular nature of the biological entity is relatively unimportant. As long as the conjugation of the fluorophore to the biological entity does not destroy utility of the conjugate, it is contemplated to be within the scope of this invention.
  • membrane probe is meant a lipophilie organic moiety preferably having
  • the membrane probe is a long chain hydrocarbon group.
  • the hydrocarbon group is a saturated C 10 -C 30 alkyl group that may be straight chain, branched or may contain cyclic rings.
  • the membrane probe may be attached to a benzo ring or to the meso carbon of a TBTAP.
  • Preferred linkers Y' for connecting the biological entity to the phthalocyanine include sulfonam ⁇ de, amide, ether, thioether, ester, thioester, amine, and carbon-carbon bonds.
  • the biological entity should bear a terminal amino, carboxy, ⁇ , ⁇ -unsaturated earbonyl, thiol, sulfonyl chloride, or halide group for attachment to the phthalocyanine.
  • the phthalocyanine should bear a correspondingly reactive group, such as carboxy, amino, thiol, ⁇ , ⁇ -unsaturated earbonyl, sulfonyl chloride, or hydroxy.
  • the tether Y' to the biological entity, A, in R 2 is long enough for optimal recognition of A in typical biological assays.
  • Displacement of A from the phthalocyanine or tetrabenztriazaporphyrin can be further enhanced by the use of a rigid linker containing for example, alkene, acetylene, cyclic, aromatic or amide groups.
  • the water solubility of the phthalocyanine may also be enhanced by selection of hydrophilic or charged groups as part of the linker Y'.
  • Hydrophilic spacers include polyethers, polyamines, polyaleohols, and naturally occurring species such as sugars, peptides, and nucleotides. To reduce aggregation in aqueous solution long, hydrophobia tethers should be avoided.
  • the invention provides companion water soluble aluminum phthalocyanine derivatives.
  • the invention provides two aluminum tetraglycolylphthalocyanine isomers, 2 and 3, each having emission bands red-shifted relative to aluminum phthalocyanine trisulf onate (referred to as compound 1 herein).
  • the tetracarboxylic acid derivatives may be prepared as set forth in Example 1 herein. The only difference between the two phthalocyanines is the position of attachment of the glycolyl group (-OCH 2 CO 2 H) on the macrocycle. Substitution at the 2,9,16,23 positions provides 2, while 1,8,15,22 substitution gives 3.
  • the carboxylic acid groups present in these derivatives provides both water solubility and a reactive functionality for tethering compounds to biological entities.
  • exemplary biological entities for coupling to 2 and 3 are: antigens, antibodies or antibody fragments, receptors, intracellular organelles, proteins, such as avidin and streptavidin, enzyme substrates, membrane probes, nucleotides and derivatives thereof, nucleic acid probes, and nucleic acids.
  • the absorbance spectra of 2 and 3 in water are shown in FIGURE 3. Both exhibit a common excitation wavelength in the ultraviolet (350 nm) with molar absorptivities around 70,000. As shown in FIGURE 4, the emission maxima for the pair are distinguishable, with emission wavelengths of 704 nm for 2 and 727 nm and 3. The quantum yields of fluorescence are 0.55 and 0.43, for 2 and 3 respectively.
  • FIGURE 5 presents the emission spectra of 2 and 3 in aqueous cetyl trimethylammonium bromide (0.010 M CTAB). While the emission maximum of 3 remains essentially unchanged, a dramatic red shift to 716 nm occurs for 2.
  • the invention provides a family of four novel, water soluble, tetherable .aluminum phthalocyanine based fluorophores.
  • the family consists of two pairs of isomeric aluminum phthalocyanine derivatives. The emission of each fluorophore pair is unique and distinguishable from the other and all are red-shifted compared to 1.
  • the first pair of fluorophores are tetraphenoxy substituted aluminum phthalocyanines. Phthalocyanine formation from 4- phenoxyphthalonitrile yields a 2,9,16,23 phenoxy substituted phthalocyanine. Similar reaction with
  • 3-phenoxyphthalonitrile results in the formation of a 1,8,15,22 substituted phthalocyanine.
  • chlorosulfonic acid After the incorporation of aluminum, treatment of these derivatives with chlorosulfonic acid produces reactive sulfonyl chloride derivatives which may be coupled to biological entities, such as antigens, antibodies or antibody fragments, receptors, intracellular organelles, proteins, such as avidin and streptavidin, enzyme substrates, membrane probes, nucleotides and derivatives thereof, nucleic acid probes, and nucleic acids.
  • biological entities such as antigens, antibodies or antibody fragments, receptors, intracellular organelles, proteins, such as avidin and streptavidin, enzyme substrates, membrane probes, nucleotides and derivatives thereof, nucleic acid probes, and nucleic acids.
  • the second pair of fluorophores are tetrathiophenoxy substituted aluminum phthalocyanines.
  • 4-thiophenoxyphthalonitrile provides the 2,9,16,23 substituted phthalocyanine
  • 3-th ⁇ ophenoxyphthalonitrile gives the 1,8,15,22 substituted isomer.
  • chlorosulfonic acid After the incorporation of aluminum, treatment of these derivatives with chlorosulfonic acid yields a reactive form useful in coupling to biological entities. Hydrolysis produces sulfonates that are highly water soluble.
  • the absorbance spectra of the sulfonated tetrathiophenoxy aluminum phthalocyanines, (3 and 7, in water are shown in FIGURE 8.
  • the emission spectra are presented in FIGURE 9.
  • Example 2 The syntheses of 6 and 7 and a tabulation of their spectral properties are given in Example 2. These compounds may be attached to the above-mentioned reactive or activatable R 2 groups to yield conjugates that may be used for a variety of purposes, including sequencing of DNA.
  • a second family of four novel fluorophores derived from the tetrabenztriazaporphyrin (TBTAP) system is presented. These fluorophores differ from phthalocyanines 4 - 7 above only in position 20 of the ring system.
  • position 20 is a nitrogen atom
  • position 20 is a substituted carbon (in formula I, Z is N for phthalocyanines, Z is CR for the tetrabenztriaza- porphyrins).
  • the 20 carbon is phenyl substituted.
  • the family of four aluminum TBTAP fluorophores consists of two pairs of oxygen and sulfur positional isomers. Reaction of benzylmagnesium bromide with each of the four phthalonitriles, 4-phenoxyphthalonitrile, 3-phenoxyphthalonitrile, 4-thiophenoxyphthalonitrile, and 3-thiophenoxyphthalonitrile provides TBTAP ring systems which are metalated with aluminum and sulfonated to provide compounds 8 - 11, respectively.
  • the preparation of the aluminum 20-phenyl tetrabenztriazaporphyrins and the tabulation of their spectral properties are given in Example 3.
  • the emission spectra of the aluminum tetraphenoxy TBTAP derivatives are shown in FIGURE 10. Similarly, emission spectra of the tetrathiophenyl derivatives are shown in FIGURE 11. As with the aluminum phthalocyanines, the TBTAP sulfur analogs are red-shifted relative to the oxygen counterparts, and the 1,8,15,22 isomers are red-shifted compared to the 2,9,6,23 isomers.
  • Two novel, water soluble aluminum tetrabenztriazaporhphyrins are also described. These compounds are derived from phthalonitrile and are therefore unsubstituted. Reaction of methylmagnesium bromide with phthalonitrile and subsequent aluminum incorporation produced aluminum 20-H TBTAP. Similar reaction of phthalonitrile with benzylmagnesium bromide followed by the incorporation of aluminum gave aluminum 20-phenyl TBTAP. Both of these derivatives were rendered reactive to reactive groups on biological entities (e.g., -OH, -NH 2 , -SH) by treatment with chlorosulfonic acid.
  • biological entities e.g., -OH, -NH 2 , -SH
  • R 2 may be attached to a meso carbon, in which case the benzo rings of the macrocycle will each have an R 1 group attached thereto.
  • the red emission wavelength of 1 is one of the greatest advantages of this fluorophore. Since emission is shifted away from that of endogenous fluorescence (400-600 nm), background is reduced. Reduction of background leads to a higher signal-to- background ratio and greater sensitivity. This advantage may be realized regardless of where excitation is effected so long as there is absorbance at the excitation wavelength.
  • Excitation of 1 at 325 nm (helium cadmium laser), around 350 nm (Hg lamp source or argon ion laser), 633 nm (helium neon laser), 647 nm (krypton ion laser), or 670 nm (diode laser) leads to emission at 685 nm.
  • emission spectra of AlPc derivatives suffer less background interference. Interferences attributable to Rayleigh, Tyndall or Raman scatter can be reduced by more than 100 fold due to the large Stokes' shift (> about 300 nm) and long wavelength emission properties of phthalocyanines.
  • Aluminum phthalocyanines emit in the red (> 680 nm) at wavelengths beyond endogenous fluorescence (400-600 nm).
  • fluorescein and rhodamine derivatives currently marketed for nucleic acid sequence analysis, flow cytometry, immunoassay and nucleic acid probe assays have only 20-40 nm Stokes' shifts and emit at wavelengths less than 550 nm.
  • aluminum phthalocyanine based fluorophores have greater separation between emission wavelength maxima.
  • the range of emission maxima for known fluorescein families is only 21 nm with a typical separation of 6 nm between each dye.
  • the phthalocyanine family spans about 50 nm with an average separation between family members of greater than 15 nm.
  • phthalocyanine based fluorophores have sharp emission bands.
  • the full width at half maximum for fluorescein based dyes ranges from about 32-37 nm with significant red tailing.
  • fluorescence emission of phthalocyanines and TBTAPs may be enhanced by rendering the fluorophores monomerie rather than aggregated.
  • the degree of monomerism of a metallophthalocyanine or TBTAP in aqueous solution is a function of the metal. Divalent metals which cannot bear axial ligands tend to stack and exhibit reduced monomerism. Trivalent and greater metals are less prone toward aggregation due to axial ligation and are therefore more fluorescent in solution.
  • the most preferred metals for fluorescent reagents are therefore aluminum, gallium, scandium, silicon, germanium, and tin.
  • Metallophthalocyanines and TBTAPs suitable for magnetic resonance imaging applications would bear paramagnetic metals such as iron, manganese, and gadolinium. Here the metals are in the plus three oxidation state.
  • Metallophthalocyanines and TBTAPs suitable for radioactive imaging and therapeutic applications would bear radioisotopes of metals such as copper, cobalt, gallium, and technetium.
  • the radionuclides are gamma-emitters and are sensitive imaging probes.
  • the A(red)/A(blue) ratio of the subject conjugates should be > 1.75, and such conjugates are readily prepared by Method 2.
  • Phthalocyanine conjugates having A(red)/A(blue) ratios between about 1.5 and 1.75 While suitable for some purposes, have relatively limited sensitivity and so would not be useful.
  • Conjugates having A(red)/A(blue) ratios of less than 1 are considered to be not suitable for use as fluorescent markers.
  • the phthalocyanine and tetrabenztriazaporphyrin conjugates of this invention display similar tendencies in terms of their monomeric binding and its relation to the A(red)/A(blue) ratio.
  • some of the species disclosed in this invention have much stronger blue absorbances, e.g., compound 4, while others show diminished red absorbance, e.g., compound 12, as monomers in aqueous environments.
  • the most preferred methods of conjugation yield a range of A(red)/A(blue) from 1.4 to 2.0, depending on the fluorophore. Exemplary methods for preparing monomeric conjugates are provided below.
  • Example 6 describes the coupling of the reactive forms of the red-shifted aluminum phthalocyanines to streptavidin.
  • aluminum phthalocyanine may be coupled to a large molecule by a tether linker.
  • the tether linker may be any small bifunctional organic molecule.
  • the tether linker may be 2 to 12 atoms in length.
  • the tether linker is 7 to 12 atoms in length and sterically hindered.
  • a long sterically hindered tether ensures that aluminum phthalocyanine is displaced from the biological entity and that individual aluminum phthalocyanine moieties on the large molecule are displaced from one another.
  • the tether linker method may be utilized in conjunction with Methods 2 and 3.
  • Aluminum phthalocyanine may be coupled to large molecules with the use of an aqueous solvent containing a disaggregated organic such as DMF. Use of the disaggregant helps to ensure that aluminum phthalocyanine is bound in a monomeric rather than aggregated state.
  • Method 3 aluminum phthalocyanine may be coupled to large molecules by preincubation of the fluorophore in a disaggregating medium followed by coupling of the fluorophore to a large molecule in an aqueous solvent containing a disaggregating organic solvent such as DMF.
  • the preincubation is preferably performed by mixing a reactive derivative of aluminum phthalocyanine with dimethylformamide for one hour at 30°C prior to conjugation in a disaggregating medium.
  • the preincubation of fluorophore in a disaggregating organic solvent (e.g., DMF) prior to conjugation in a disaggregating medium is the first disclosure of such a method for generating monomeric conjugates with any fluorescent species including phthalocyanines and porphyrins.
  • a disaggregating organic solvent e.g., DMF
  • the counterion of these compounds may be any one that is stable and synthetically accessible, and that does not interfere with water solubility or desirable spectral properties.
  • Exemplary negative counterions are I-, Br-, Cl-, F-, borate etc.
  • Exemplary positive counterions are Ca +2 , Mg +2 , Na + , K + , quaternary ammonium, etc. These compounds may be used to detect DNA and RNA, generally by nonspecific binding to the DNA or RNA. Fluorescent detection of the compound bound to the DNA or RNA may then be carried out by standard fluorescent measurement components.
  • the reagents of the first and second aspects of the present invention may be used in combination with binding partners (or ligands) capable of specifically binding with a target substance, particularly an analyte.
  • binding partners or ligands
  • the reagent referred to as a reporter group in this context
  • the reporter group may be covalently or noncovalently bound to the binding partner and may be attached either prior to or after the analyte and binding partner are caused to interact and bind.
  • the reporter group is covalently linked to the binding partner before the binding partner and the analyte are caused to interact and bind.
  • the binding partner is caused to interact and bind with the analyte and after binding the reporter group is covalently or noncovalently attached to the binding partner.
  • the binding partner may be conjugated with biotin moieties and the reporter groups may be attached to avidin or streptavidin. Other specific binding pairs may also be used to join the binding partner and the reporter group.
  • binding partner/analyte pairs the following are representative, preferred embodiments:
  • nucleic acid probe or primer e.g., DNA or RNA having 5-10,000 nucleic acid bases
  • complementary target DNA or RNA e.g., DNA or RNA having 5-10,000 nucleic acid bases
  • the precise nature of the binding partner and analyte is relatively unimportant. All that is required is that the binding partner and analyte be capable of specific binding to each other and that a reagent as described herein be attachable to the binding partner, either before or after binding to the analyte and either covalently or via a second specific binding pair, e.g., a tightly binding pair such as avidin:biotin, strep tavidin:bio tin, and maltose binding protein:maltose. In another preferred embodiment, more than one analyte is determined simultaneously using a corresponding number of binding partners each attached to a different reagent according to the present invention for detection.
  • a second specific binding pair e.g., a tightly binding pair such as avidin:biotin, strep tavidin:bio tin, and maltose binding protein:maltose.
  • more than one analyte is determined simultaneously using a corresponding number of binding partners each attached
  • the different reagents are required to have substantially nonoverlapping emission spectra for separate detection.
  • the combinations of different reagents used in a particular assay may all be of the same general type (e.g. phthalocyanines or TBTAPs) or mixtures of reagent types (e.g. phthalocyanines and TBTAPs).
  • the fluorescence maxima must occur at different wavelengths, preferably separated by at least about 7 nm.
  • the fluorophores For simultaneous use of fluorescent reagents, the fluorophores must be readily distinguishable for quantitation or quantifiable by ratioing methods.
  • a sequencing primer is modified with an amino group at the 5' terminus or each of the four dideoxynucleotides is labeled with one of each of four fluorescent reagents.
  • cell surface antigens expressed by certain subsets of cells may be labeled either directly or indirectly with a fluorescent reagent and antibody or antibody fragment.
  • the number of cell subsets that may be labeled and quantitated is determined by the number of unique fluorescent labels employed.
  • each of any number of fluorescent reagents may be attached to a different antigen, antibody, or antibody fragment.
  • a simultaneous thyroid immunoassay test panel may be performed by labeling triiodothyronine (T3) with one fluorescent reagent, thyroxine (T4) with a second fluorescent reagent, and anti-thyroid stimulating hormone (anti-TSH) with a third fluorescent reagent.
  • T3 triiodothyronine
  • T4 thyroxine
  • anti-TSH anti-thyroid stimulating hormone
  • any number of fluorescent reagents may be attached to a different nucleic acid probe to perform simultaneous probe analysis.
  • the number of probes that may be detected as the result of a single hybridization step is determined by the number of fluorescent reagents utilized.
  • the reagents of the first or second aspects are used to sequence nucleic acid molecules or fragments.
  • the most common approach for DNA sequence analysis is the Sanger dideoxynucleotide sequencing method.
  • a family of four aluminum phthalocyanine derivatives is required. The derivatives may be used to label either sequencing primers or each of the four dideoxynucleotides (ddNTP's).
  • ddNTP's dideoxynucleotides
  • the present compounds may be effectively used to sequence DNA without degradation.
  • a single primer is labeled with each of four different fluorescent labels.
  • labeled chain terminators such as dideoxynucleotides rather than labeled primers.
  • all four of the sequencing reactions may be performed in a single vessel and then loaded onto a single lane of the sequencing gel.
  • the macrocycles involved in the present reagents are larger than the corresponding fluorescein or rhodamine reagents previously used for sequencing and are relatively more planar. As a result, it was unpredictable whether the fluorophore labled primer of this invention would be compatible with the sequencing enzyme.
  • researchers at corporations that develop sequencing fluorophores predicted trouble with both sequencing enzyme compatibility and electrophoretic mobility of the sequencing primer and fragments.
  • the fluorophore labeled primer was found to be compatible with the sequencing enzyme and the electrophoretic mobility of the dye labeled primer and sequencing fragments is not significantly different from that of the amino modified primer or sequencing fragments.
  • the kits will generally contain one or more containers of reagents of the present invention, and may contain other chemicals, controls, etc., as may be necessary or desirable.
  • the labeled chain terminating dideoxynucleotides are selected so that their fluorescence emission spectra are distinguishable, i.e. substantially non-overlapping.
  • substantially non-overlapping is meant that the emission spectra have wavelengths of maximum emission that are separated by at least about 7 nm, preferably at least about 10-20 nm.
  • An alternative DNA sequencing kit may have a container of fluorophorelabeled primer (a reagent of the present invention), containers of deoxynucleotides, e.g., dATP, dTTP, dGTP, dCTP; containers of chain terminators, e.g., ddATP, ddTTP, ddGTP, ddCTP, ddUTP; and a container of a DNA polymerase.
  • a container of fluorophorelabeled primer a reagent of the present invention
  • containers of deoxynucleotides e.g., dATP, dTTP, dGTP, dCTP
  • chain terminators e.g., ddATP, ddTTP, ddGTP, ddCTP, ddUTP
  • a container of a DNA polymerase e.g., a DNA polymerase.
  • two or more reagents with maximum spectral resolution are required.
  • Use of at least two different fluorophores having nonoverlapping emission maxima allows the user to perform two color analyses.
  • Two or more color analyses are generally effected by labeling subsets of cells using antibodies specific for each cell type, either indirectly (e.g., via intervening biotin:avidin binding) or directly (i.e., covalently) attached to a fluorescent reagent or dye as disclosed herein.
  • AIDS testing may be performed by simultaneous analysis of two T cell subsets within a sample of peripheral blood containing lymphocytes. A ratio of
  • T-Helper cells one color
  • T-Suppressor cells the second color
  • Multicomponent immunoassay allows for the simultaneous detection of more than one analyte. Cost and time considerations make this a preferred method for many clinical applications.
  • a single patient sample may be used for detection of a panel of therapeutic drugs, abused drugs, infectious disease agents, hormones or any combination thereof if each of the analytes or antibodies specific for each of the analytes is labeled with a different fluorescent dye.
  • Multicomponent probe assays enable detection of infectious disease agents, cancers and genetic abnormalities. Since there are probe libraries available for detection of many agents and abnormalities, one would like to have as many fluorophores that may be excited with common wavelengths as possible. In this application, each probe specific for regions of chromosomes associated with disease agents, cancers, or genetic abnormalities (leading to birth defects or genetic diseases) is labeled with a different fluorophore.
  • the cancers treatable or detectable by the present reagents are not necessarily limited and any one for which a therapeutic or diagnostic agent has been developed may potentially be treated or diagnosed using the appropriate fluorophores described herein.
  • the reagents disclosed herein, particularly those of the first and second aspects, may also be used for photodynamic therapy employing standard methods. See Example 15.
  • Tetrasubstituted phthalocyanines derived from monosubstituted phthalonitriles are necessarily an inseparable mixture of four isomeric products.
  • the product phthalocyanines arise from the differences in orientation of the phthalonitrile during the cyclization process. Cyclization of a 4-substituted phthalonitrile leads to the formation of 2,9,16,23-tetrasubstituted phthalocyanine, as well as three other tetrasubstituted isomers, namely, 2,9,16,24; 2,10,16,24; and 2,9,17,24.
  • cyclization of a 3-substituted phthalonitrile provides the corresponding 1,8,15,22-tetrasubst ⁇ tuted phthalocyanine along with three other tetrasubstituted derivatives, 1,8,15,25; 1,11,15,25; 1,8,18,25. Recognizing this, we have for simplicity designated tetrasubstituted phthalocyanines derived from 3- substituted phthalonitriles as 1,8,15,22 and phthalocyanines derived from 4- substituted phthalonitriles as 2,9,16,23. See FIGURE 1 for macrocycle position numbering.
  • Tetrasubstituted aluminum phthalocyanines may be prepared from monosubstituted phthalonitriles. Nitro displacement from either 3- or 4-nitrophthalonitrile with oxygen or sulfur nucleophiles provide the corresponding phthalonitriles in good yield. The oxygen or sulfur reagent used in the nitro displacement may impart to the phthalocyanine water solubility and tetherability, or may be further elaborated to provide these required properties. Reagents such as hydroxyacetic acid and thioacetic acid may provide appropriately funetionalized phthalonitriles directly (X is O or S, Y is CH 2 , and W Is CO 2 H).
  • tetraoxy or tetrathio substituted phthalocyanines may be treated with an alkylating agent such as methyl bromoacetate to provide the fully funetionalized phthalocyanine.
  • Cleavage of the neopentyl group is accomplished upon reaction with boron tribromide in benzene as generally disclosed by Leznoff, C.C. et al., Photochem. Photobiol. 46:959-963, 1987.
  • the cleavage product, aluminum hydroxy 2,9,16,23-tetrahydroxyphthalocyanine, is a versatile intermediate which may be treated with a variety of alkylating agents to provide a family of tetraalkoxy substituted phthalocyanines.
  • alkylation of the tetrahydroxy derivative with methyl bromoacetate and potassium carbonate (forty molar equivalents of each) in refluxing methanol affords the tetra methyl ester derivative.
  • the alkylated product may be directly hydrolyzed to the tetracarboxylic acid by heating in a solution of 0.5 M methanolic potassium hydroxide.
  • Aluminum hydroxy 2,9,16,23- tetraglycolylphthalocyanine was isolated by precipitation, 2, from an aqueous acid solution.
  • FIGURES 3 and 4 The absorbance and emission spectra in water are shown in FIGURES 3 and 4, respectively.
  • CTAB cetyl trimethlyammonium bromide
  • Tetrasubstituted oxygen and sulfur substituted aluminum phthalocyanine sulfonates are described in Example 2.
  • the four tetrasubstituted reagents of Example 2 are prepared from monosubstituted phthalonitriles.
  • the following is a detailed description of the preparation of a family of four aluminum phthalocyanine based reagents. The presentation is organized into sections which detail phthalocyanine preparation, phthalocyanine metalation, reactive phthalocyanine formation, and water soluble phthalocyanine formation. Within each section a detailed procedure is given for one member of the family of four reagents followed by a comment on the procedures for the other three reagents. Any differences in procedure are highlighted.
  • 3-thiophenylphthalonitrile produced 1,8,15,22-tetrathiophenylphthalocyanine in 87% yield.
  • the product was isolated by precipitation from methylene chloride without the addition of methanol. The spectral properties are tabulated below.
  • Aluminum Acetylacetonate Tetraphenoxyphthalocyanine Sulfonates Tetraphenoxyphthalocyanine Sulfonates.
  • Water soluble aluminum phthalocyanine sulfonates were prepared from aluminum acetylacetonate 2,9,16,23- and 1,8,15,22-tetraphenoxyphthalocyanines as described above for the corresponding axial hydroxy compounds.
  • the absorbance and emission wavelengths as well as quantum yields in water are tabulated below. Phthalocyanine Absorbance Emission Quantum Yield
  • the spectral data summarized above for the acetylacetonate ligated aluminum phthalocyanine sulfonates contrasts significantly with the data for the corresponding hydroxylated derivatives.
  • the wavelengths of fluorescence emission of the acetylacetonates are roughly 10 nm blue shifted relative to their hydroxy analogs. The blue shift limits their utility in multicomponent analysis when used in conjunction with the parent, aluminum phthalocyanine sulfonate, which emits at 684 nm.
  • the ideal family of fluorophores for multicomponent analysis will have spectrally resolved emission bands.
  • aluminum phthalocyanine sulfonates employs axial hydroxy rather than acetylacetonate ligands.
  • Tetrasubstituted oxygen and sulfur substituted aluminum tetrabenztriazaporphyrins are described in Example 3.
  • the four tetrasubstituted reagents of Example 3 are prepared from monosubstituted phthalonitriles.
  • the following is a detailed description of the preparation of a family of four aluminum tetrabenztriazaporphyrin based reagents.
  • the presentation is organized into sections which detail tetrabenztriazaporphyrin preparation, metalation, reactive derivative formation, and water-soluble derivative formation. Within each section a detailed procedure is given followed by a comment on the procedures for the other three reagents. Any differences in procedure are highlighted.
  • the reaction mixture was diluted with 200 mL methylene chloride and washed first with 3-200 mL portions saturated aqueous sodium bicarbonate and then with 200 mL 5% v/v aqueous hydrochloric acid. The organic phase was dried over sodium sulfate, filtered and concentrated.
  • the crude reaction product was chromatographed on silica gel eluting with chloroform. The fractions containing the desired product were combined, concentrated and twice more chromatographed on silica gel, eluting with 85% chloroform in hexane to afford 204 mg (19%) 20-phenyl 2,9,16,23- tetraphenoxytetrabenztriazaporphyrin as a deep blue-green solid.
  • Silica thin layer chromatography eluting with 65% methylene chloride in hexane gave a homogeneous product with an R f of 0.54. Spectral data are tabulated below.
  • the sulfonyl chloride derivatives of the four tetrasubstituted aluminum hydroxy 20-phenyl tetrabenztriazaporphyrins were prepared by treatment with chlorosulfonic acid as described previously for the corresponding aluminum phthalocyanines in Example 2.
  • Aluminum tetrabenztriazaporphyrins sulfonates substituted at position twenty with either hydrogen, 12, or phenyl, 13, are described in Example 4. These water solution and reactive derivatives have performance characteristics similar to the aluminum phthalocyanines sulfonates and possess the optical properties of the aluminum tetrabenztriazaporphyrins. The following is a detailed description of the preparation of these compounds. The presentation is organized into sections which detail tetrabenztriazaporphyrin preparation, metalation, reactive TBTAP preparation, and water soluble TBTAP preparation. Within each section a detailed procedure is given for the 20-hydrogen derivative followed by a comment on the procedure for the 20-phenyl derivative.
  • magnesium 20-phenyl tetrabenztriazaporphyrin was prepared.
  • the product was isolated by dilution of the quinoline reaction mixture with 500 mL distilled water.
  • the crude product was collected by filtration and dried in vacuo.
  • the product was purified by chromatography on silica eluting with hexane: tetrahydrofuran (1:1). Spectral data are tabulated below.
  • Example 5 Exemplary cationic phthalocyanines are presented in Example 5.
  • the derivatives in Example 5 satisfy formula I where M is either H 2 or aluminum, each R 1 is -XYW, X is oxygen, Y is ethylene (-CH 2 CH 2 -), W is trimethylammonium iodide, Z is nitrogen, and R 2 is -XYW, -YW, or -W.
  • the positively charged tetrasubstituted phthalocyanines are prepared from monosubstituted phthalonitriles.
  • the phthalocyanine precursor 4-dimethylaminoethanoxyphthalon ⁇ trile
  • the phthalocyanine precursor was prepared by displacement of nitro from 4-nitrophthalonitrile with 2- d ⁇ methylaminoethanol. Formation of the diim ⁇ noisoindol ⁇ ne and subsequent cyclization resulted in the metal free tetrasubstituted phthalocyanine.
  • the amino groups were quaternized with methyl iodide.
  • Aluminum was incorporated by treatment with aluminum tr ⁇ acetylacetonate.
  • the aluminum phthalocyanine was rendered water soluble by alkylat ⁇ on with methyl iodide to provide the tetraquaternary ammonium compound 14b.
  • the absorbance spectrum of 14a in water presented in FIGURE 14 shows nearly complete aggregation.
  • the fluorescence quantum yield is less than 0.01.
  • RNA Torquea yeast
  • the absorbance spectrum of 14a in the presence of RNA, FIGURE 14, is indicative of a monomeric phthalocyanine.
  • the emission spectra for the two solutions are compared in FIGURE 15.
  • the fluorescence enhancement of 14a upon RNA binding is 450-fold.
  • the oorresponding absorbance and emission spectra for aluminum derivative 14b are shown in FIGURES 16 and 17, respectively.
  • the fluorescence enhancement upon RNA binding is 340. No fluorescence enhancement was observed for either 14a or 14b in the presence of bovine serum albumin.
  • Tabulated below are the spectral data for the metal free and aluminum phthalocyanine derivatives prepared as described above.
  • the emission wavelength and fluorescence enhancement of the fluorophores in the presence of RNA are presented.
  • the absorbance data was recorded with a fluorophore concentration of 5 X 10 -6 M and an RNA (Torula Yeast) concentration of 1.0 mg/mL. The fluorescence data was obtained for these solutions at 100-fold dilution.
  • the counterion is iodide.
  • the reaction was quenched by the addition of 250 ⁇ L of a 10 mg/mL solution of lysine in 0.2 M sodium bicarbonate in phosphate buffered saline containing 0.02% sodium azide as a preservative.
  • the conjugate was purified by size exclusion chromatography on Sephadex G-50 in phosphate buffered saline containing 0.02% sodium azide. Spectral data for the conjugate is tabulated below. Direct Coupling of Aluminum Hydroxy 1,8,15,22-Tetraphenoxyphthalocyanine Sulfonyl Chloride to Streptavidin.
  • the 20-substituted tetrabenztriazaporphyrins (TBTAP) described above, like the phthalocyanines, are useful as reagents for fluorescence analysis.
  • TBTAP tetrabenztriazaporphyrins
  • One unique property of the TBTAP system is the position 20 substituent.
  • Grignard reagent used in the preparation of the TBTAP (see Examples 3 and 4)
  • a reactive 20-substituent may be synthesized.
  • the Grignard reagent may either contain the functional group of choice or be capable of further elaboration to the group of choice.
  • the resulting 20-substituted TBTAP is then monofunctionally reactive.
  • Particularly useful reactive groups as R 2 enable efficient coupling to biological entities.
  • Preferred reactive groups would include sulfonyl chloride, carboxylic acid and derivatives, amino, isothiocyanate, maleimide, and im ⁇ date among others.
  • TBTAP reagent An example of a useful monofunctionally reactive TBTAP reagent would be one with an isothiocyanate or N-hydroxysuccinimide ester moiety at position 20. These reagents may be useful in various applications such as immunoassays, nucleic acid sequencing, nucleic acid probe assays, flow cytometry or for selective functionalization. As an example of selective functionalization, the isothiocyanate derivative could serve as a fluorescent reagent in protein sequence analysis utilizing the Edman degradation process. The isothiocyanate portion of the fluorophore couples to the N terminus of the peptide to be sequenced which is im mobilized (C terminus) on a solid phase.
  • Degradation of the peptide follows with the fluorophore labeled terminal amino acid being cleaved from the peptide.
  • the fluorophore labeled amino acid is then removed from the immobilized peptide and the amino acid is identified.
  • the new N terminus of the remaining peptide, now one amino acid residue shorter, is ready for the next cycle. Repetition of the process results in the sequential identification of the amino acid residues of the peptide of interest.
  • Highly fluorescent reagents such as phthalocyanines and TBTAPs, would improve the detection limits of protein sequence analysis and enable the sequencing of smaller quantities of protein.
  • the advantage of highly sensitive fluorophores is particularly relevant when only trace quantities of rare proteins are available.
  • the 20-subst ⁇ tuent of the TBTAP ring system may be designed to create the desired optical properties of the TBTAP.
  • the wavelengths of absorbance and fluorescent emission may be manipulated by the choice of substituent at position 20.
  • Electron donating groups are expected to red shift both absorbance and fluorescence wavelengths while a blue shift is anticipated for electron withdrawing groups.
  • TBTAP derivatives such as trifluoro methyl (CF 3 ) and perfluorophenyl (C 6 F 5 ) may be prepared from commercially available 1,1,1- trifluoro-2-bromoethane and 2,3,4,5,6-pentafluorobenzyl bromide, respectively. These TBTAP bearing electron withdrawing substituents are predicted to absorb and emit light at wavelengths blue of the parent.
  • tetrasubstituted phthalocyanines and tetrabenztriazaporphyrins described in Examples 2 and 3 are derived from unsubst ⁇ tuted phenoxy or thiophenylphthalonitriles. Substituted phenoxy or thiophenylphthalonitriles may also be prepared and cyclized to the corresponding phthalocyanines or tetrabenztriazaporphyrin systems. These modified derivatives may serve to fine tune the optical properties of the parent tetrasubstituted material.
  • 3-(4-fluorophenoxy)phthalonitrile may be prepared by treatment of 4-fluorophenol with 3-nitrophthalonitrile in a procedure analogous to that which results in the production of 3-phenoxyphthalonitrile.
  • Cyclization of the fluoro substituted phthalonitrile to the phthalocyanine or TBTAP will result in the formation of a species slightly different from its nonfluorinated parent.
  • the optical properties will also vary slightly from the parent.
  • Octasubstituted phthalocyanines and tetrabenztriazaporphyrins may be prepared from disubstituted phthalonitriles in procedures analogous to those described in Examples 2 and 3 for the preparation of tetrasubstituted phthalocyanines and TBTAPs from monosubstituted phthalonitriles.
  • the octasubstituted derivatives may be broadly categorized based on the position of the substitution.
  • Symmetrical phthalocyanines and TBTAPs are derived from 3,6- and 4,5-disubstituted phthalonitriles. Less symmetrical and more difficult to prepare are 3,4- and 3,5-disubstituted phthalonitriles.
  • the 3,6-methoxy derivative exhibits a significant red shift. However, the fluorescence quantum yield is low. The 4,5-methoxy derivative is actually blue shifted and retains more of a fluorescence emission. Both of these derivatives may be further elaborated to water soluble and reactive reagents by a reaction sequence completely analogous to that described for the isomeric aluminum tetraneopentoxyphthaloeyanines described in Example 1.
  • Octasubstituted derivatives composed of four sulfur substituents and four oxygen substituents may also be prepared as described in the Examples above. These derivatives may be prepared from phthalonitriles substituted with both an oxygen and a sulfur substituent, for example, 3-thiophenyl-5- phenoxyphthalonitrile.
  • the phenyl groups in the example may be other than phenyl and the position of the substituents may also vary.
  • the optical properties of these mixed derivatives is expected to be intermediate between the octaoxy and the octathio analogs.
  • 4,5-Octasubstituted carbon derivatives may also be prepared.
  • the system is known as a naphthalocyanine.
  • These highly conjugated derivatives are approximately 100 nm red shifted relative to their phthalocyanine counterparts.
  • Tabulated below are the spectral characteristics of aluminum phthalocyanine and naphthalocyanine chlorides in dimethylformamide.
  • phthalocyanines Closely related in structure to phthalocyanines are pyrazine porphyrazines.
  • Phthalocyanines bear four benzo rings appended to the macrocycle while pyrazine porphyrazines have four pyrazine (1,4-diazabenzene) rings.
  • These derivatives may be prepared from either 2,3-dicyanopyridine or 3,4- dicyanopyridine. Cyclization of 2,3-dicyanopyridine gives 3-pyridine porphyrazine while 3,4-dicyanopyridine produces 4-pyridine porphyrazine. Like pyrazine porphyrazines, the pyridine porphyrazines absorb at wavelengths blue-shifted relative to phthalocyanines, with the 3-pyridine isomer blue-shifted relative to the 4-pyridine porphyrazine. Metalation with aluminum chloride in quinoline provided the aluminum derivatives.
  • the reagents of this invention are organometallic compounds and as such many different metals may be bound.
  • the macroeyclic ring systems disclosed are eapable of efficient chelation of a variety of metals useful in image analysis and therapeutic applications, such as magnetic resonance imaging, radionuelide imaging, and as radiopharmaeeuticals. Active metals for these applications may be, incorporated into the macrocycle and directed to the site of interest.
  • the targeting of the metal bearing reagent may be a naturally selective uptake of the reagent by the site of interest, an antibody directed against an antigen present at the site of interest to which the reagent is conjugated, a complementary fragment of DNA to which the reagent is coupled, a membrane probe to which the reagent is coupled or some other delivery mechanism.
  • Paramagnetic metals useful for magnetic resonance imaging contrast agents include gadolinium, manganese, and iron.
  • Radioactive metal complexes of copper 67, technetium 99, cobalt 57, and gallium 67 have been used as radiopharmaeeuticals in both diagnostic and therapeutic applications.
  • the reagents of this invention may be useful in the applications described above by virtue of their metal binding capabilities. Also, the biological conjugates of this invention will serve to act as targeting agents for the applications described above.
  • Representative malignancies that can be treated by the radionuclides are: leukemia, ovarian cancer, lymphoma, breast cancer, myeloma, kidney, liver, and colorectal cancer, and the like.
  • PDT agents photosensitizers
  • the activated photosensitizers effectively kill cells in their immediate vicinity presumably by the generation of singlet oxygen.
  • the reagents of this invention offer two improvements over the existing technology. The first advantage lies in the deep red absorbance of the disclosed reagents and the second in the targeting of these reagents made possible by their biological binding conjugates. Phthalocyanines and TBTAPs which absorb in the deep red with large molar absorptivities will enable treatment of more tissue.
  • PDT reagents are limited by their relatively blue abosrbance profiles with respect to depth of penetration of activating light. Since human tissue is nearly transparent in the near infrared, PDT agents which absorb in this region will be most effective. The utilization of red-shifted phthalocyanines and TBTAPs will enable access to tissues which would be unaffected by currently employed blue absorbing sensitizers.
  • the targeting of the photosensitizer is a critical aspect in PDT.
  • the reagents of this invention by virtue of their conjugation to biological
  • oligonucleotides can seek out and bind to sites requiring photodynamic treatment.
  • the conjugation of these deep red absorbing phthalocyanines and TBTAPs to antibodies (or antigen binding antibody fragments) directed against cancerous tissue or cancer-associated antigens enables efficient delivery of the photoactivatable agents to the cancer.
  • the coupling of red absorbing phthalocyanines and TBTAPs to a complementary fragment of DNA enables the use of anti-sense oligonucleotides or DNA probes as targeting agents.
  • Another targeting method involves covalently attaching the reagent to a membrane probe, as defined above.
  • malignancies that could be treated by PDT using the present reagents are: bladder cancer, skin cancer (melanoma), esophogeal cancer, brain tumors, other solid tumors, and the like.
  • a representative example of a two color system for AIDS testing that employs the phthalocyanine based fluorophores is as follows.
  • R 1 and R 2 group are located at the 1, 8, 15,
  • each subset of cells may be quantitated simultaneously using a flow cytometer equipped with optical filters that allow for discrimination of the two different fluorophores.

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Abstract

L'invention concerne des dérivés à fixation monomère, fluorescents, solubles dans l'eau, décalés vers le rouge, de formule (I), dans laquelle M représente soit H2 soit est choisi parmi les métaux suivants: aluminium, silicium, phosphore, gallium, germanium cadmium, scandium, magnésium, étain, et zinc. Chaque R1 est choisi indépendamment entre -XYW, -YW, et -W. Y représente soit un carbone, soit un hétérocarbone choisi parmi oxygène, azote, soufre, phosphore, silicium et sélénium; Y représente un groupe de liaison; et W représente un groupe de solubilisation dans l'eau. Le substituant R2 est choisi parmi-A, -Y'A, -XA et XY'A, A indiquant une entité biologique telle qu'un anticorps, un fragment d'anticorps, un nucléotide, une sonde d'acide nucléique, un antigène, un oligonucléotide, un désoxynucléotide, un didésoxynucléotide, une avidine, une streptavidine ou une sonde de membrane, ou R2 est un groupe réactif ou activable adapté pour une conjugaison avec une entité biologique. Y' est un groupe de liaison fixant l'entité au macrocycle de phtalocyanine ou de tétrabenztriazaporphyrine. Z représente soit un atome d'azote soit un carbone à substitution hydrogène, alkyle, aryle ou groupes aralkyle. Z peut aussi être fixé à R2. L'invention concerne aussi des dérivés des composés de formule (I) dans lesquels 1 à 4 des cycles benzo contiennent 1 ou 2 atomes N. L'invention concerne également des procédés de séquençage d'ADN et de détection d'analytes, comprenant des cellules, à l'aide de ces dérivés, ainsi que des kits pour procéder à des analyses des analytes et de cytométrie d'écoulement. L'invention concerne en outre des procédés de détection d'ADN à l'aide de composés cationiques de formule (I) dans laquelle R2 = R1 et W = -N+D1D2D3. On peut, par ailleurs, utiliser des composés contenant Tc, Gd, etc. comme métal dans la formule (I) pour l'imagerie ou la thérapie.
PCT/US1989/003807 1988-09-08 1989-08-31 Reactifs de phtalocyanine et tetrabenztriazaporphyrine decales vers le rouge WO1990002747A1 (fr)

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US24160888A 1988-09-08 1988-09-08
US241,608 1988-09-08
US398,433 1989-08-29
US07/398,433 US5135717A (en) 1986-12-24 1989-08-29 Tetrabenztriazaporphyrin reagents and kits containing the same

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Cited By (25)

* Cited by examiner, † Cited by third party
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WO1991014456A2 (fr) * 1990-03-22 1991-10-03 Quadra Logic Technologies Inc. Compositions de therapie photodynamique ameliorees
EP0502723A2 (fr) * 1991-03-05 1992-09-09 Hitachi Chemical Co., Ltd. Tétraazaporphine soluble dans l'eau et fluorochrome pour marquer
US5238940A (en) * 1990-03-22 1993-08-24 Quadra Logic Technologies Inc. Compositions for photodynamic therapy
EP0597389A1 (fr) * 1992-11-10 1994-05-18 Hitachi Chemical Company, Ltd. Tétraazaporphines solubles à l'eau et fluorochromes pour marquage
EP0609894A2 (fr) * 1993-02-05 1994-08-10 Canon Kabushiki Kaisha Complexe marqué et méthode d'analyse utilisant le-même
WO1996009315A1 (fr) * 1994-09-21 1996-03-28 Board Of Regents, The University Of Texas System Photodecoupage d'adn a l'aide de texaphyrines
WO1996011937A1 (fr) * 1994-10-14 1996-04-25 Stratagene Marquage de polynucleotides par porphyrines
US5565552A (en) * 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5567687A (en) * 1989-03-06 1996-10-22 University Of Texas Texaphyrins and uses thereof
WO1996038461A1 (fr) * 1995-06-02 1996-12-05 Pharmacyclics, Inc. Clivage d'arn au moyen de complexes metalliques
US5595726A (en) * 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5633354A (en) * 1994-09-21 1997-05-27 Pharmacyclics, Inc. Phosphoramidite derivatives of texaphyrins
US5714328A (en) * 1995-06-07 1998-02-03 Board Of Regents, The University Of Texas System RNA photocleavage using texaphyrins
US5837866A (en) * 1994-09-21 1998-11-17 Board Of Regents, The University Of Texas Phosphoramidite derivatives of macrocycles
US5880287A (en) * 1990-05-15 1999-03-09 Hyperion, Inc. Polyoxyhydrocarbyl related products and methods for fluorescence assays
US5919922A (en) * 1990-05-15 1999-07-06 Hyperion, Inc. Fluorescent dyes free of aggregation and serum binding
US6022959A (en) * 1996-08-20 2000-02-08 Pharmacyclics, Inc. Nucleic acids internally-derivatized with a texaphyrin metal complex and uses thereof
US6060598A (en) * 1990-05-15 2000-05-09 Hyperion, Inc. Fluorescence immunoassays using fluorescent dyes free of aggregation and serum binding
WO2000031187A1 (fr) * 1998-11-25 2000-06-02 Hyperion, Inc. Colorants fluorescents solubles dans l'eau sans agregation et sans liaison au serum, produits et procedes associes
WO2001047719A1 (fr) * 1999-12-28 2001-07-05 Mitsui Chemicals, Incorporated Support d'enregistrement optique et nouveaux composes azaporphyrine
WO2005024065A2 (fr) * 2003-09-05 2005-03-17 Ebara Corporation Compose colorant cationique permettant de detecter un acide nucleique double brin et procede et appareil utilisant ce compose
WO2007017602A2 (fr) * 2005-08-11 2007-02-15 Laboratoires Synth-Innove Marqueurs, leur procede de fabrication et leurs applications
EP1758917A1 (fr) * 2004-06-14 2007-03-07 Hereth, Hannjorg Azaporphyrines substituees en tant que marqueurs de fluorescence
US8524891B2 (en) 2005-07-14 2013-09-03 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Tetraazaporphyrin-based compounds and their uses
CN105820170A (zh) * 2016-05-05 2016-08-03 厦门大学 以三正辛基膦为溶剂合成磺基金属酞菁荧光化合物的方法

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CA2056431A1 (fr) * 1989-06-07 1990-12-08 David Dolphin Photosensibilisation de derives de la porphyrine obtenus par additions diels-alder
GB9317881D0 (en) * 1993-08-27 1993-10-13 Secr Defence Photosensitizers
DE19539409C2 (de) * 1995-10-11 1999-02-18 Diagnostikforschung Inst Kontrastmittel für die Nahinfrarot-Diagnostik
US6573258B2 (en) * 2000-09-27 2003-06-03 Frontier Scientific, Inc. Photodynamic porphyrin antimicrobial agents
ATE298341T1 (de) * 2001-03-21 2005-07-15 Molteni & C Metallsubstituierte, nicht zentrosymmetrische phthalocyanin-analoga, deren herstellung und verwendung zur photodynamischen therapie, und als in-vivo-diagnostikum
JP4648637B2 (ja) * 2004-02-04 2011-03-09 株式会社日本触媒 カルボキシル基および／またはスルホン酸基を有するフタロシアニン化合物ならびにその製造方法

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EP0252683A2 (fr) * 1986-07-02 1988-01-13 E.I. Du Pont De Nemours And Company Procédé et réactifs pour la détermination de séquences ADN
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Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5567687A (en) * 1989-03-06 1996-10-22 University Of Texas Texaphyrins and uses thereof
US5238940A (en) * 1990-03-22 1993-08-24 Quadra Logic Technologies Inc. Compositions for photodynamic therapy
WO1991014456A3 (fr) * 1990-03-22 1991-12-12 Quadra Logic Tech Inc Compositions de therapie photodynamique ameliorees
WO1991014456A2 (fr) * 1990-03-22 1991-10-03 Quadra Logic Technologies Inc. Compositions de therapie photodynamique ameliorees
US5919922A (en) * 1990-05-15 1999-07-06 Hyperion, Inc. Fluorescent dyes free of aggregation and serum binding
US6060598A (en) * 1990-05-15 2000-05-09 Hyperion, Inc. Fluorescence immunoassays using fluorescent dyes free of aggregation and serum binding
US5880287A (en) * 1990-05-15 1999-03-09 Hyperion, Inc. Polyoxyhydrocarbyl related products and methods for fluorescence assays
US5438135A (en) * 1991-03-05 1995-08-01 Hitachi Chemical Company Water-soluble tetraazaporphins and fluorochrome for labeling
EP0502723A3 (en) * 1991-03-05 1993-01-27 Hitachi Chemical Co., Ltd. Water-soluble tetraazaporphins and fluorochrome for labeling
EP0502723A2 (fr) * 1991-03-05 1992-09-09 Hitachi Chemical Co., Ltd. Tétraazaporphine soluble dans l'eau et fluorochrome pour marquer
US5595726A (en) * 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5565552A (en) * 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5607924A (en) * 1992-01-21 1997-03-04 Pharmacyclics, Inc. DNA photocleavage using texaphyrins
EP0597389A1 (fr) * 1992-11-10 1994-05-18 Hitachi Chemical Company, Ltd. Tétraazaporphines solubles à l'eau et fluorochromes pour marquage
US5627028A (en) * 1992-11-10 1997-05-06 Hitachi Chemical Company, Ltd. Water-soluble tetraazaporphins and fluorochrome for labeling
EP0609894A3 (fr) * 1993-02-05 1998-01-07 Canon Kabushiki Kaisha Complexe marqué et méthode d'analyse utilisant le-même
EP0609894A2 (fr) * 1993-02-05 1994-08-10 Canon Kabushiki Kaisha Complexe marqué et méthode d'analyse utilisant le-même
US5798491A (en) * 1993-06-09 1998-08-25 Board Of Regents, The University Of Texas System Multi-mechanistic chemical cleavage using certain metal complexes
US5633354A (en) * 1994-09-21 1997-05-27 Pharmacyclics, Inc. Phosphoramidite derivatives of texaphyrins
US5837866A (en) * 1994-09-21 1998-11-17 Board Of Regents, The University Of Texas Phosphoramidite derivatives of macrocycles
WO1996009315A1 (fr) * 1994-09-21 1996-03-28 Board Of Regents, The University Of Texas System Photodecoupage d'adn a l'aide de texaphyrines
WO1996011937A1 (fr) * 1994-10-14 1996-04-25 Stratagene Marquage de polynucleotides par porphyrines
WO1996038461A1 (fr) * 1995-06-02 1996-12-05 Pharmacyclics, Inc. Clivage d'arn au moyen de complexes metalliques
US5714328A (en) * 1995-06-07 1998-02-03 Board Of Regents, The University Of Texas System RNA photocleavage using texaphyrins
US6022959A (en) * 1996-08-20 2000-02-08 Pharmacyclics, Inc. Nucleic acids internally-derivatized with a texaphyrin metal complex and uses thereof
WO2000031187A1 (fr) * 1998-11-25 2000-06-02 Hyperion, Inc. Colorants fluorescents solubles dans l'eau sans agregation et sans liaison au serum, produits et procedes associes
WO2001047719A1 (fr) * 1999-12-28 2001-07-05 Mitsui Chemicals, Incorporated Support d'enregistrement optique et nouveaux composes azaporphyrine
US6969764B2 (en) 1999-12-28 2005-11-29 Mitsui Chemicals, Inc. Optical recording medium and novel azaporphyrin compounds
WO2005024065A2 (fr) * 2003-09-05 2005-03-17 Ebara Corporation Compose colorant cationique permettant de detecter un acide nucleique double brin et procede et appareil utilisant ce compose
WO2005024065A3 (fr) * 2003-09-05 2005-08-18 Ebara Corp Compose colorant cationique permettant de detecter un acide nucleique double brin et procede et appareil utilisant ce compose
EP1758917A4 (fr) * 2004-06-14 2009-11-11 Hereth Hannjorg Azaporphyrines substituees en tant que marqueurs de fluorescence
EP1758917A1 (fr) * 2004-06-14 2007-03-07 Hereth, Hannjorg Azaporphyrines substituees en tant que marqueurs de fluorescence
US8524891B2 (en) 2005-07-14 2013-09-03 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Tetraazaporphyrin-based compounds and their uses
WO2007017602A3 (fr) * 2005-08-11 2007-08-02 Synth Innove Lab Marqueurs, leur procede de fabrication et leurs applications
WO2007017602A2 (fr) * 2005-08-11 2007-02-15 Laboratoires Synth-Innove Marqueurs, leur procede de fabrication et leurs applications
US8034626B2 (en) 2005-08-11 2011-10-11 Laboratoires Synth-Innove Labels, their production process and their uses
CN105820170A (zh) * 2016-05-05 2016-08-03 厦门大学 以三正辛基膦为溶剂合成磺基金属酞菁荧光化合物的方法
CN105820170B (zh) * 2016-05-05 2019-11-19 厦门大学 以三正辛基膦为溶剂合成磺基金属酞菁荧光化合物的方法

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EP0434727A1 (fr) 1991-07-03
JPH04500516A (ja) 1992-01-30
CA1337754C (fr) 1995-12-19

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