WO2019098756A2 - Colorant de marquage et kit le comprenant - Google Patents

Colorant de marquage et kit le comprenant Download PDF

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WO2019098756A2
WO2019098756A2 PCT/KR2018/014104 KR2018014104W WO2019098756A2 WO 2019098756 A2 WO2019098756 A2 WO 2019098756A2 KR 2018014104 W KR2018014104 W KR 2018014104W WO 2019098756 A2 WO2019098756 A2 WO 2019098756A2
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substituted
unsubstituted
labeling
ring
compound
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PCT/KR2018/014104
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Korean (ko)
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WO2019098756A3 (fr
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신봉기
김태영
송주만
양희정
제종태
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주식회사 에스에프씨
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Priority claimed from KR1020180135720A external-priority patent/KR102253839B1/ko
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Publication of WO2019098756A2 publication Critical patent/WO2019098756A2/fr
Publication of WO2019098756A3 publication Critical patent/WO2019098756A3/fr
Priority to US16/860,982 priority Critical patent/US11597842B2/en

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
    • C09B23/0033—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being bound through a sulfur atom
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0066—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/086—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines more than five >CH- groups
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/10—The polymethine chain containing an even number of >CH- groups
    • C09B23/102—The polymethine chain containing an even number of >CH- groups two heterocyclic rings linked carbon-to-carbon
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/10—The polymethine chain containing an even number of >CH- groups
    • C09B23/105—The polymethine chain containing an even number of >CH- groups two >CH- groups
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/10—The polymethine chain containing an even number of >CH- groups
    • C09B23/107—The polymethine chain containing an even number of >CH- groups four >CH- groups
    • 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
    • C09B57/00—Other synthetic dyes of known constitution
    • 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
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/02—Coumarine dyes

Definitions

  • the present invention relates to dyes and kits for labeling which are excellent in fluorescence intensity and light stability.
  • the present invention corresponds to the results of a research project of the Advanced Technology Research Center (10076988), which was carried out with the support of the Ministry of Industry and Commerce.
  • Fluorescence is the most commonly used technique for tracking or analyzing biological molecules non-destructively in the life sciences.
  • Green fluorescent protein (GFP) and other self-emitting biomolecules are also present.
  • biomolecules such as biomolecules such as proteins, nucleic acids, and the like can be stained with fluorescent dyes, After labeling the dyes, we have obtained imaging data with various techniques, accompanied by optical equipment capable of detecting fluorescence signals.
  • a background art related to the present invention is Korean Patent Laid-Open Publication No. 10- 2017-0026245 (published on Mar. 23, 2017), which discloses a perylene compound, a method for producing the same, and a fluorescent dye containing the same All.
  • the object of the present invention is to provide a kit comprising the dye for the label.
  • Said linker comprising 1 to 150 non-hydrogen atoms, X is a reactive group,
  • Reference numeral 2 denotes a fluorescence terminal for generating a fluorescence signal
  • At least one of the above to is selected from the following formulas (3) to (5)
  • R to R and at least one of the functional group 7 is -LX.
  • a kit comprising the labeling dye.
  • the dye for labeling according to the present invention is excellent in optical properties such as fluorescence intensity and light stability and can be usefully used for a kit for labeling biomolecules.
  • FIGS. 1 and 2 show the standardized maximum absorption wavelengths and maximum fluorescence wavelengths of Antibody conjugates having similar D / P ratios
  • FIG. 3 is a graph comparing relative fluorescent intensities according to D / P ratios for Goat Anti-Mouse IgG labeled with [Compound 7], Alexa Fluor 647, and Cy 5, respectively.
  • FIG. 4 is a graph comparing relative fluorescence intensities according to D / P ratios for Goat anti-Rabbit IgG labeled with [Compound 7], Alexa Fluor 647, and Cy 5, respectively.
  • FIG. 5 is a graph comparing relative fluorescent intensities according to D / P rats for Goat anti-Human IgG labeled with [Compound 7], Alexa Fluor 647 and Cy 5, respectively.
  • Figure 6 is a graph comparing fluorescence emission for Alexa Fluor 647 GAM IgG, Compound 7 GAM IgG, background fluorescence.
  • Figure 7 compares relative fluorescence intensities according to D / P ratios using Goat Anti-Mouse IgG labeled with [Compound 7], Alexa Fluor 647 and Cy 5, using Mouse IgG and antigen antibody formation and FLISA It is a graph.
  • a dye for labeling represented by the following formula (1) or (2).
  • (1) is a ring-substituted polymethine optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or a substituent selected from nitrogen, oxygen and sulfur, Is also a ring-substituted polyene optionally containing one heteroatom,
  • Polymethine is a single bond and a double bond is an odd number of methine groups in combination alternately: the compounds consisting of a) (0161! 11, 1116 for), the polyene is a compound consisting of a methine group of the even number.
  • any carbon in the ring-substituted polymethine or polyene optionally containing a heteroatom may be substituted with at least one substituent, and adjacent substituents may be linked to each other to form a ring.
  • the following compounds may be specified by substituents present in the polymethine or polyene.
  • a substituent selected from the group consisting of a polymethine or a polyene and a substituent selected from the group consisting of [Formula 1] and [Formula 2] is connected to each other to form a ring, whereby [Compound 20], [Compound 21]
  • the labeling dye for display can be formed.
  • each of R 1 and R 2 is independently selected from the group consisting of hydrogen, deuterium, substituted or unsubstituted 01-010 alkyl, substituted or unsubstituted 01-010 heteroalkyl including at least one hetero atom, substituted Or a substituted or unsubstituted C2-10 alkenyl, a substituted or unsubstituted C2-10 alkynyl, a substituted or unsubstituted C2-10 alkynyl, a substituted or unsubstituted C2-10 alkynyl, a substituted or unsubstituted C2-10 alkynyl, a substituted or unsubstituted C2-10 alkynyl, , Halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, carbamate, 2019/098756 1 »(: 1 ⁇ ⁇ 2018/014104
  • the linker is a linker containing 1 to 150 non-hydrogen atoms
  • the V-reactive group is a fluorescent group that generates a fluorescence signal.
  • the O 3 -monophore means a functional group having 3 to I carbon atoms.
  • 3 - for alkyl is meant to & I) having two carbon atoms, saturated aliphatic groups, including straight-chain alkyl, such as alkyl and pulverized.
  • the alkyl is selected from the group consisting of methyl, ethyl, 11 -propyl, 11 -propyl, 11 -butyl, 5 -butyl, butyl, butyl, pent-1-yl, pent- Methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyl-1-yl, 11 -nucyl, II-heptyl and 11- octyl have .
  • alkoxy in the context of the present invention means both -O- (alkyl) group and -O- (unsubstituted cycloalkyl) group, and is a straight chain or branched hydrocarbon having at least one ether group and 1 to 10 carbon atoms.
  • halogen is used herein to denote fluoro (-10, Means bromo (-) or iodo (-1), and haloalkyl means alkyl substituted with the above-mentioned halogen.
  • halomethyl may be substituted with at least one of the hydrogens of methyl by halogen - 3 ⁇ 4 or 3 ⁇ 4).
  • aralkyl is a functional group in which aryl is substituted with a carbon atom of alkyl, and is a generic name of - (big 2 ) 11 .
  • aralkyls include benzyl ( 2 : 2 (: 6 3 ⁇ 4) or phenethyl (- 2 (: 3 ⁇ 4 ( 6 3 ⁇ 4)).
  • the polyalkylene oxide may be additionally substituted as necessary to the extent that the properties of the polymer are maintained.
  • the substitution may be a chemical bond to increase or decrease the chemical or biological stability of the polymer.
  • a polyalkylene oxide Any carbon or terminal carbon may be substituted with hydroxy, alkyl ether (methyl ether, ethyl ether, propyl ether, etc.), carboxyl methyl ether, carboxyethyl ether, benzyl ether, dibenzyl methylene ether or dimethylamine.
  • the polyalkylene oxide may be a polyalkylene oxide (mPEG) terminated with an alkyl ether, for example, represented by the formula - (CH 2 CH 2 O) n CH 3 . Further, the polyalkylene oxide may be a polyalkylene oxide (mPEG) terminated with hydrogen, and may be represented by, for example, a formula of _ (CH 2 CH 2 O) n H.
  • the size of mPEG may vary depending on the size of n corresponding to the number of ethylene glycol repeating units.
  • L is a linker containing 1 to 150 non-hydrogen atoms
  • X is carboxyl, succinimidyl ester, sulfo-succinimidyl ester, succinimidyl ester, isothiocyanate, maleimide, haloacetamide, hydrazide, vinyl sulphone, dichlorotriazine, A phosphoramidite, an alkyl halide, an acyl halide, a carbohydrazide, a hydroxylamine, a ketone, an alkyne, ), Azides, aliphatic and aromatic amines, sulfotetrafluorophenyl esters, sul fodi chlorophenyl esters, carbonyl azides (such as, for example, car boryl chloride, sul fonyl fluoride, boronic acid, isocyanate, halogen-substituted triazine
  • L is the same linker as L of the -L-X functional group, which is a fluorophore generating a fluorescence signal.
  • Z is selected from the group consisting of coumarins, cyanine, bodipy, fuluoresceins, rhodamines, pyrenes, carbopyronin, oxazine, , Xanthenes, thioxanthene and acridines, or a fluorescent moiety selected from the structures represented by the formulas (1) and (2) Can be single. 2019/098756 1 »(: 1/10/06 018/014104
  • 11 may be unsubstituted fullerene.
  • a condensation ring means an annular structure in which two or more rings share two or more atoms.
  • the aliphatic condensed ring include cyclic nucleic acid, cyclopentane, cycloheptane, cyclooctane, and cyclic dicarbonate.
  • the aromatic condensed rings include naphthalene, anthracene, phenanthryl, triphenylen, fluoranthene, Perylene, klysene, acenaphthalene, fluorene, or tetracene.
  • Examples of the aliphatic heterocyclic ring containing one or more of the above-mentioned 0 and atomic atoms include piperidine, tetrahydrothiopyran, tetrahydropyran, dioxane, pyrrolidine, tetrahydrofuran and tetrahydrothiophene
  • aromatic heterocyclic rings containing one or more of the 10 atoms and the aromatic heterocyclic rings include quinoline, cinnoline, quinazoline, quinoxaline, phthalazine, naphthyridine, phenanthroline, acridine, Benzoquinoline, indole, benzooxazole, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, benzoyl, be
  • the condensed ring may be further substituted with one or more substituents such as the following formula (6).
  • substituents such as the following formula (6).
  • To [Chemical Formula 6] [Chemical Formula 1] 3 ⁇ 4 to about 5 is required at least two are connected to each other is selected from a condensed ring type property, and the substituents of: and 1? 18 are included.
  • lipids include fatty acids, phospholipids, lipopolysaccharides and the like.
  • carbohydrate include monosaccharides, disaccharides, polysaccharides (e.g., dextran) .
  • the biomolecule is a functional group for reacting with any functional group of the labeling dye represented by the formula (1) or (2) or a reactive group bonded to the labeling dye, and includes amino, sulphydryl, , Hydroxyl, carboxyl, phosphate, and thiophosphate, or may have a derivative form thereof.
  • the biomolecule may be an oxy or dioxane having at least one or a derivative form selected from amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate, 2019/098756 1 »(: 1 ⁇ ⁇ 2018/014104
  • a dye for labeling represented by the [formula 1] or [formula 2] of the present invention drill amino, seolpeu high in addition to the above-described biomolecule (1 1 ⁇ 7 (1 1 ), carbonyl, hydroxyl, carboxyl, phosphate (Including a receptor ligand), a water-soluble vitamin, a fat-soluble vitamin, an inorganic salt, a receptor, an enzyme or an enzyme substrate, a cell, a cell membrane, a toxin, a microorganism or a nano- (Such as polystyrene microspheres) and the like.
  • the water-soluble vitamins include vitamin 8 group, vitamin 0, pantothenic acid, niacin, folic acid, biotin and inositol
  • fat-soluble vitamins may include vitamins vitamin 0 and vitamins 1 and the like.
  • the inorganic salts may include, but are not limited to, calcium, calcium monophosphate, potassium phosphate dibasic, potassium chloride, magnesium phosphate, sodium hydrogen carbonate, zinc oxide, ferric pyrophosphate, manganese sulfate and potassium iodide. It is not.
  • the present invention provides a labeling kit comprising the labeling dye.
  • the labeling kit may further include an enzyme for reaction with target molecules, a solvent (buffer solution, etc.), and other reagents.
  • a solvent buffer solution, etc.
  • the solvent a buffer selected from the group consisting of phosphate buffer, carbonate buffer and Tris buffer, organic solvent selected from dimethylsulfoxide, dimethylformamide, dichloromethane, methanol, ethanol and acetonitrile, or water
  • the solubility can be controlled by introducing various functional groups into the dye for the display depending on the kind of the solvent. Labeling of biomolecules using labeling dyes (1 3 61 )
  • a single-chain probe nucleic acid having a complementary base sequence to the target nucleic acid is prepared, and the single-chain-modified target nucleic acid and the probe nucleic acid are hybridized to each other through the reaction (that is, the dye is intercalated into the target nucleic acid) After hybridization on the plate, the fluorescent signal can be generated by allowing the fluorescent dye to be intercalated into the target nucleic acid.
  • a library of cDNA such as cDNA, a library of the genome, or all the genomes are used as templates (bell type) Can be used.
  • the method of immobilizing the probe nucleic acid on the substrate can be appropriately selected depending on the kind of the nucleic acid and the type of the substrate. For example, a method of electrostatic bonding to a substrate surface-treated with a cation such as polylysine using the charge of DNA may be used.
  • the probe complementary to the base sequence of the target nucleic acid to be labeled is labeled with a dye, and the target nucleic acid is reacted with the target nucleic acid before or after the amplification of the target nucleic acid to measure the fluorescence of the target nucleic acid.
  • the elongation reaction of the target nucleic acid is carried out by an enzyme (DNA polymerase, RNA polymerase).
  • an enzyme DNA polymerase, RNA polymerase.
  • the double-stranded nucleic acid sequence formed by the primer consisting of the target nucleic acid and the oligonucleotide is recognized by the enzyme, An elongation reaction takes place from one position, and only the desired gene region is amplified.
  • the synthesis reaction is carried out using the nucleotide (dNTP, NTP) as a raw material.
  • nucleotides having dyes are added to an ordinary nucleotide (dNTP, NTP) at an arbitrary ratio, a nucleic acid into which the dye has been introduced can be synthesized.
  • a nucleotide having an amino group at an arbitrary ratio may be introduced by PCR, followed by binding of a labeling dye to synthesize a nucleic acid into which a labeling dye has been introduced.
  • Enzyme makin the synthesis reaction consists of the nucleotide to the composite as a raw material, the right light used for changing ereul H of the 3 'nucleotide of this time, the longer without being the extension reaction of the nucleic acid achieved, the reaction was terminated at that point, do.
  • a terminator is introduced at a certain probability to terminate the reaction, so that nucleic acids of various lengths are synthesized.
  • the DNA is lined up in sequence of lengths.
  • each kind of terminator is labeled with a different labeling dye, the tendency to depend on each base is observed at the end point (3 'end) of the synthesis reaction, and the dye for labeling, which is labeled on the terminator, By reading the information, nucleotide sequence information of the target nucleic acid can be obtained.
  • a primer labeled with a labeling dye in advance may be used in place of the terminator to hybridize with the target nucleic acid.
  • PNA peptide nucleic acid
  • PNA is a substitution of a pentane / phosphate skeleton, which is a basic skeleton structure of a nucleic acid, with a polyamide skeleton of a glycine unit. It has a three-dimensional structure resembling a nucleic acid and has a highly specific And is strongly coupled. It can be used as a reagent for telomere studies by applying it to telomere PNA probes as well as conventional DNA analysis methods such as I SH (inverse hybridization) method .
  • I SH inverse hybridization
  • double-stranded DNA is hybridized on a label by contacting it with PNA labeled with a labeling dye having a base sequence complementary to all or a part of the DNA base sequence, and heating the mixture to produce single-stranded DNA , And the mixture is slowly cooled to room temperature to prepare a PNA-DNA complex and measure the fluorescence thereof.
  • the method of measuring the fluorescence intensity of the product by amplifying the target nucleic acid by the method has been described.
  • the size of the product is confirmed by electrophoresis, and then the fluorescence intensity is measured, .
  • the amount of product can be measured in real time using a probe designed to generate fluorescence by using an energy transfer of a fluorescent dye and hybridizing it to the product of the PCR method.
  • DNA labeled donor and acceptor can be used.
  • Specific labeling methods include the molecular beacon method, the TaqMan-PCR method, and the cycling probe method, which confirm the presence of a nucleic acid of a specific sequence.
  • Other labeling methods include the molecular beacon method, the TaqMan-PCR method, and the cycling probe method, which confirm the presence of a nucleic acid of a specific sequence.
  • the labeling dye of the present invention can be used to observe the phenomenon of intracellular signal transduction.
  • a variety of enzymes are involved in internal signal transduction or subsequent cellular responses.
  • a representative signal In the transfer phenomenon it is known that a specific protein kinase is activated, thereby initiating signal transduction by inducing protein phosphorylation.
  • Binding and hydrolysis of nucleotides play a critical role in their activity, and by introducing a labeling dye into a nucleotide derivative, intracellular signal transduction can be observed with high sensitivity have.
  • RNAi RNA interference
  • dsRNA double-stranded RNA
  • reaction was carried out using the same method as that described in the above 2, and the reaction was carried out using reversed phase chromatography and anion exchange resin, and the compound 4 0 2012/027623 show 2) (12, 11.9%).
  • Compound 3 was prepared by using 2-methyl-1,3-benzoxazole-6-sulfonic acid instead of 2,3,3-trimethylindole-5-sulfonic acid as a raw material in the method for producing Compound 4 (15 ⁇ , 15.4 %).
  • Compound 11 was prepared using ion exchange resin after reaction in the same manner as in Journal of Photochemistry and Photobiology A: Chemistry 168 (2004) 53-57 using 18 prepared by the same method as in the above 2 (60 mg, 27.5%).
  • Actin filaments in group 41 are effectively stained.
  • Compound 43 was added to the 70000 aminodextran solution to obtain a dye / dextran of about 12. After 6 hours, the conjugate was purified to pH (-50) using water as the eluent.
  • Goat Ant i -Mouse IgG was diluted with 0.1 M sodium bicarbonate buffer to prepare 1 mg / ml protein solution, and 125 1 protein solution was added to each tube.
  • Dye solution (5 mg / ml) containing compound 7 was prepared in 1, 2, 3, 4, 5 yl of prepared tube and immediately vortexed. Then, the reaction solution was added to the Ami con centrifuge filter after shaking the locker for 30 minutes at room temperature. Filtered with PBS (14000 rpm, 10 min, 5 times) until free dye was removed with Centrifuge.
  • the filtrate was centrifuged (lOOOrpm, 2 min), diluted with 1 ml of PBS, and immediately visualized by UV, PL and microplate reader.
  • the ratio of the dye to the protein was determined using the following equation.
  • a dye Absorbance at the maximum absorption wavelength of the dye after labeling
  • a 280 absorbance at 280 nm
  • E prot molar extinction coefficient of protein at 280 nm (210,000 for IgG)
  • E dye molar extinction coefficient of dye at maximum absorption wavelength
  • the maximum absorption wavelength is similar to [conjugate labeled with a compound group (Antibody conjugate) labeled with Alexa Fluor 647 and Cy 5].
  • the degree of agglutination is relatively smaller than that of conjugate labeled with Alexa Fluor 647 and Cy 5 from the H band.
  • the fluorescence wavelength is similar to that of Conjugate labeled with Alexa Fluor 647 and Cy 5 (Antibody conjugate labeled with compound group).
  • FIG. 3 is a graph comparing relative fluorescence intensities according to D / P ratios of Goat Anti-Mouse IgG against [Compound 7], Alexa Fluor 647 and Cy 5, respectively.
  • an antibody conjugate labeled with a compound group (Antibody
  • the fluorescence intensity is at its maximum at a D / P ratio of about 5.
  • the fluorescence intensity is at its maximum at a D / P ratio of about 5.
  • the Antibody Conjugate labeled with Alexa Fluor 647 and Cy 5 from the D / P ratio of 3 or more [antibody conjugate labeled with compound group
  • Rabbit IgG was used for the experiment.
  • FIG. 4 is a graph showing the results of the comparison of [Compound 7], Alexa Fluor 647 and Cy 5 labeled
  • fluorescence intensities are maximum at a D / P ratio of about 4.
  • Human IgG was used in the same manner.
  • FIG. 5 is a graph showing the results of a comparison between [Compound 7], Alexa Fluor 647, and Cy 5
  • an antibody conjugate labeled with a compound group (Antibody
  • fluorescence intensities are maximum at a D / P ratio of about 8.
  • FIG. 6 is a graph comparing the fluorescence emission of Alexa Fluor 647 GAM IgG, Compound 7 GAM IgG, and background fluorescence, and fluorescence was measured by the method according to Experimental Example 2).
  • FIG. 7 shows relative fluorescence intensities according to D / P ratios, using FLISA after mouse IgG and antigen antibody formation for Goat Ant i -Mouse IgG labeled with [Compound 7], Alexa Fluor 647 and Cy 5 respectively This is a comparative graph.
  • Lymphocytes were obtained from a spleen of naive C57BL / 6 mice (5 weeks old). The spleen was placed on a metal screen, and the spleen was mush up with a plunger to dissolve the red blood cells with 0.83% ammonium chloride for 2 minutes. After that, 1% fetal bovine serum, 1% penicillin / streptomycin
  • SA compound 7 in SA-compound 7 was 10 times brighter than SA-Cy5 and brighter than SA-Alexa fluor 647 and SA-compound 7 was the brightest Experimental Example 5
  • Fluorescence wavelength 1 ⁇ _
  • molar topcoverage £ Solvent. Measurements were made using the following parameters: A 0, ethanol (A), methylene chloride (A), water (B) and And the measurement results are shown in Table 3 below.

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Abstract

La présente invention concerne : un colorant de marquage ayant une excellente intensité de fluorescence et une excellente photostabilité; et un kit.
PCT/KR2018/014104 2017-11-16 2018-11-16 Colorant de marquage et kit le comprenant WO2019098756A2 (fr)

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US16/860,982 US11597842B2 (en) 2017-11-16 2020-04-28 Labeling dye and kit including same

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KR10-2017-0153074 2017-11-16
KR20170153074 2017-11-16
KR10-2018-0135720 2018-11-07
KR1020180135720A KR102253839B1 (ko) 2017-11-16 2018-11-07 표지용 염료 및 이를 포함하는 키트

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WO2019098756A3 WO2019098756A3 (fr) 2019-07-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4047057A4 (fr) * 2019-11-19 2024-01-17 Sfc Co Ltd Extincteur et utilisations associées

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KR101149535B1 (ko) * 2009-04-17 2012-05-25 한국과학기술연구원 생체 분자 표지를 위한 신규 시아닌 화합물 및 그 제조방법
WO2017010852A1 (fr) * 2015-07-16 2017-01-19 에스에프씨 주식회사 Composé colorant
KR20170101360A (ko) * 2016-02-26 2017-09-06 에스에프씨 주식회사 시아닌계 화합물, 이를 포함하는 생체분자 표지용 염료, 키트 및 조영제 조성물
KR20170105662A (ko) * 2016-03-09 2017-09-20 에스에프씨 주식회사 시아닌계 화합물, 이를 포함하는 생체분자 표지용 염료, 키트 및 조영제 조성물

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4047057A4 (fr) * 2019-11-19 2024-01-17 Sfc Co Ltd Extincteur et utilisations associées

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