JPH04351961A - Fluorescent label-coloring matter, organism-derived material labeled tereby and reagent containing these - Google Patents

Abstract

PURPOSE:To provide a reagent or a clinical examination reagent useful for analyzing antigens, medicines, basic configurations of DNA and so on to be measured by means of a semiconductor laser having oscillation wavelength in 600-720nm by using a specific fluorescent label-coloring matter. CONSTITUTION:A fluorescent label-coloring matter as represented by a general equation and organism-derived materials such as antigens, antibodies, nucleotide and so on labeled by the fluorescent label-coloring matter or a reagent containing these are used for analyzing antigens/medicines in the blood or for analyzing basic configurations of DNA and so on. More specifically, M is H2, and R<1> is a 4-hydro carbonyl benzylthio group, and (n) is 2, and (m) is 0, and a compound without Y is exemplified. An absorption band Q(0, 0) of a compound as represented by a general equation moves not only to the same area as an absorption area of a phtalocyanine class but also to a short wavelength area, so that in the case of using a semiconductor laser having oscillation wavelength in 600-720nm, the compound can be used as a highly accurate reagent or clinical examination reagent useful for analyzing antigens/medicines or for analyzing the basic configurations of DNA and so on.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to fluorescent labeling dyes, biological substances labeled with fluorescent labeling dyes, and reagents containing them.

0002

BACKGROUND OF THE INVENTION Phthalocyanine pigments are organic pigments having four isoindole moieties linked by four nitrogen atoms constituting a cyclic conjugated chain. Compounds used in these pigments include phthalocyanine (blue-green), copper phthalocyanine (blue), chlorine-substituted copper phthalocyanine (green), and sulfonated copper phthalocyanine (green). Phthalocyanine pigments are commonly used in enamel, plastics, linoleum, inks, wallpaper, textiles, paper, rubber products, etc. On the other hand, it has been reported that free-based phthalocyanines, aluminum, cadmium, magnesium, silicon, tin and zinc phthalocyanines exhibit fluorescence (The Phthalocyanine).
nes 1:127, 1983).

[0003] It has also been reported that phthalocyanines can be used in various immunoassays (US Pat. No. 4
, 160,645, US Pat. No. 4,193,98
Publication No. 3, US Patent No. 4,220,450, US
Patent No. 4,233,402, US Patent No. 4,23
No. 5,869, US Patent No. 4,256,834, US Patent No. 4,277,437, US Patent No. 4,318,707, US Patent No. 4,483,9
No. 29, US Pat. No. 4,540,660, U.
S Patent No. 4,540,670, US Patent No. 4,5
60,534, US Patent No. 4,650,770, US Patent No. 4,656,129, US Patent No. 4,659,676).

Furthermore, phthalocyanines are used as catalysts in chemiluminescent immunoassay systems [Bull. Che
m. Soc. Jpn. Vol. 56, pp. 2965-2968 (1983), Vol. 56, pp. 2267-2271 (1983)
983), Vol. 57, pp. 587-588 (1984)
, Vol. 57, pp. 3009-3010 (1984), Vol. 58, pp. 1299-1303 (1985)]. Hara et al. use iron phthalocyanine as a catalyst for the chemiluminescent reaction between luminol and hydrogen peroxide, and quantify the analyte in the test sample from the amount of chemiluminescent signal. They investigated iron and cobalt phthalocyanines and porphyrin complexes of iron, palladium, platinum, manganese, and tin, and reported that iron phthalocyanine showed the best catalytic activity and high sensitivity.

Fluorescent substances are widely used in addition to colored substances in immunoassays, and fluorescent substances are also preferred over colored substances in enzyme immunoassays because they can increase sensitivity. It's starting to look like this. A well-known fluorophore-enzyme pair is alkaline phosphatase (al
kaline phosphatase) and 4-methylumbelliferyl phosphate (4-methyl
β-galactosidase and 4-methylumbelliferyl-D-galactopyranoside (4-methylumbelliferyl-D-g
horseradish peroxidase (alactopyranoside), horseradish peroxidase
roxidase) and p-hydroxyphenylacetic acid (p
-hydroxyphenyl acetic a
cid), etc., and the detection sensitivity of these systems is 10-15
It is M. However, even if we try to further increase the detection sensitivity, there are limits to the analytical properties of the produced fluorophore.

[0006]Recently, reagents using phthalocyanines that have a high fluorescence quantum yield and high solubility in water have been proposed (WO Patent No. 88/04777, WO Patent No. 90/02747, JP-A-1-233222).

[0007]

[Problems to be Solved by the Invention] However, phthalocyanines have a Q(0,0) absorption band with a particularly large extinction coefficient of 65
In the wavelength range of 5 to 700 nm, absorption is relatively small in the wavelength range of 650 nm or less. Therefore, 600-65
If a measuring device equipped with a semiconductor laser with an oscillation wavelength of 0 nm is used, an excellent role as a detection drug cannot be expected.

The present invention applies not only to semiconductor lasers with an oscillation wavelength of 650 to 700 nm, but also to semiconductor lasers with an oscillation wavelength of 600 to 650 nm.
The purpose of this invention is to provide reagents or clinical test reagents that are useful for the analysis of various antigens and drugs, or the analysis of DNA base sequences, etc., which can be measured using a small and inexpensive semiconductor laser with an oscillation wavelength in the vicinity of m. shall be.

[0009]

[Means for Solving the Problems] The present invention provides the following (1) to (
9). That is, (1) chemical formula 2

[Chemical formula 2] [In chemical formula 2, M is H2, Al, Si, P, Ga, Ge
, Cd, Sc, Mg, Sn or Zn, and R1 is -
XQW, -QW, -W or a hydrogen atom; X is an oxygen atom, a nitrogen atom, a sulfur atom, a phosphorus atom, a silicon atom,
Selenium atom, CR2 R3 (However, R2 and R3
are each independently a hydrogen atom, an alkyl group, an aryl group or an aralkyl group, and R2 R3 may be carbonyl oxygen. ), or a phenylene group, Q
indicates a bonding group (linker) between X and W, W is -OH,
-O-, -SH, -S-, -CO2 H, -CO2
-, -OCH2 CO2 H, -OCH2 CO2
- , -PO42 - , -PO3- , -SO3-
, -SO2- , -SO2 Cl, -SO4
2-, -NH2, -NHR4, -NR5
R6 or -N(+)R7R8R9 (however, R4
~R9 are each independently a C1 to C10 alkyl group, a C6 to C12 aryl group, or a C6 to C12 aryl group
12 aralkyl groups. ), and n is
Indicates an integer of 0 to 2, Y is a halogen atom, -OR10
or -NR112 (However, R10 and R11 each independently represent a hydrogen atom, an alkyl group which may have a hydrophilic substituent, an acyl group which may have a hydrophilic substituent, a hydrophilic is a silyl group which may have a substituent or a phosphorus atom-containing group which may have a hydrophilic substituent), m is Y
An integer from 0 to 2 representing the number of bonds to M. ] Fluorescent labeling dye. (2) A reagent containing the fluorescent labeling dye of (1) above. (3) A biological substance labeled with the fluorescent labeling dye of (1) above. (4) A reagent containing the labeled biological substance of (3) above. (5) The reagent according to (3) or (4) above, wherein the biological substance is an antigen, antibody, or nucleotide. (6) The reagent according to (5) above, wherein the antigen is a drug. (7) The reagent according to (5) above, wherein the antibody is a monoclonal antibody. (8) The reagent according to (5), wherein the nucleotide is an oligonucleotide or a polynucleotide. (9) Nucleotides are ATP, CTP, GTP, TTP
, UTP, dATP, dCTP, dGTP, dTTP,
dUTP, ddATP, ddCTP, ddGTP, dd
The above (5) which is TTP, ddUTP or a derivative thereof
) reagent.

In the compound of formula 2 of the present invention, specific examples of the alkyl groups of R2 and R3 in X of R1 include methyl group, ethyl group, propyl group, sec-propyl group, n-butyl group, iso- butyl group, t-butyl group,
Examples of aryl groups include phenyl group, thienyl group, furyl group, pyrrolyl group, tolyl group, anisyl group, and 4-aminophenyl group; examples of aralkyl groups include benzyl group. , 2-phenylethyl group, 1-phenylethyl group, 3-phenylpropyl group, 2-phenylpropyl group, 1-phenylpropyl group, etc. Further, specific examples of the alkyl group which may have a hydrophilic substituent for R10 and R11 in Y include a methyl group, an ethyl group, a propyl group, a butyl group,
There are straight chain, branched and alicyclic groups such as pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, eicosyl group, docosyl group, etc. Examples of the acyl group which may have a hydrophilic substituent include formyl group, acetyl group, propionyl group, butyryl group,
Examples include valeryl group, pivaloyl group, hexanoyl group, octanoyl group, lauryl group, palmityl group, stearyl group, etc. Silyl groups that may have hydrophilic substituents include trimethylsilyl group, triethylsilyl group, tripropyl group, etc. Silyl group, tributylsilyl group, triamylsilyl group, trihexylsilyl group, t-butyldimethylsilyl group, di(t-butyl)methylsilyl group, dimethylphenylsilyl group, diphenylmethylsilyl group, triphenylsilyl group, etc. .

Q is a group that connects X and W, and is a C1 to C8 saturated or unsaturated linear, branched or alicyclic bonding group;
For example, in addition to methylene group, ethylene group, trimethylene group, tetramethylene group, propylene group, vinylene group, propenylene group, cyclopropylene group, cyclopentylene group, cyclohexylene group, etc., groups such as polyether, polyamine, polyalcohol, etc. There is.

In W, the C1 to C10 alkyl groups of R4 to R9 include methyl group, ethyl group, propyl group,
There are linear, branched, and cyclic groups such as butyl group, amyl group, hexyl group, heptyl group, nonyl group, and decyl group, and C
Examples of the 6-C12 aryl group include phenyl group, tolyl group, anisyl group, naphthyl group, biphenyl group, etc.
C6 to C12 aralkyl groups include benzyl group, 2
-phenylethyl group, 1-phenylethyl group, 3-phenylpropyl group, 2-phenylpropyl group, 1-phenylpropyl group, etc.

[0013] Furthermore, the substituents containing a phosphorus atom represented by R10 and R11 include -P(=O)R12R13,
-P(=O)(NR14R15)2, -PR
162 (R12 to R16 each independently represent an alkyl group, an aryl group, an acyl group, a cycloalkyl group, an alkoxyl group, an aryloxyl group, a polyether group, a hydroxyl group, or a halogen atom, and they can be substituted with various substituents. ).

In formula 2, Y is -OR10 or -NR112
(R10 and R11 represent an alkyl group, an acyl group, and a silyl group), and the compound in which m represents 1 or 2 is:
A compound in which Y is -OH or -NH2 in Chemical Formula 2 can be synthesized by reacting with a corresponding alcohol, acyl chloride, silanol, chlorosilane, chlorophosphine, chlorophosphite, phosphoryl chloride, or the like.

[0015] The compound in which Y is -OH or -NH2 and m is 1 or 2 in formula 2 is a compound in which Y is a halogen atom and m is 1 or 2 in formula 2. It can be obtained by decomposition or ammoniolysis. Compounds in which Y is a halogen atom and m is 1 or 2 in Chemical Formula 2, and compounds in which m is zero and do not have Y are described in the literature [Inorg. Chem. , Volume 19, 383
Page (1980); J. Am. Chem. Soc.
．． , Vol. 112, p. 7408 (1980); Zh.
Obshch. Khim. , Volume 49, 1408
Page (1979); vol. 47, p. 2143 (197
7); Vol. 43, p. 2025 (1973)], it can be synthesized by the following route.

That is, chemical formula 3

A compound represented by the following formula (in formula 3, R1 and n have the same meanings as in formula 2) can be obtained by reacting with a corresponding metal or metal salt. On the other hand, when M in Chemical Formula 2 is H2, it can be obtained by decomposing a compound in which M in Chemical Formula 2 is Mg or Zn with an acid.

[0017] The biological substances used in the present invention include proteins, peptides, nucleotides, etc. obtained from living organisms such as animals, plants, and microorganisms (including viruses).
There are sugars, lipids, hormones, vitamins, alkaloids, antibiotics, and complexes thereof, and these may be extracted from nature, completely synthetically synthesized, or semi-synthesized artificially. You can. Specific examples of proteins/peptides include serum albumin, IgG
- Immunoglobulins such as IgA, IgM, IgD, and IgE, monoclonal antibodies against various proteins and leukocyte membrane antigens, enzymes such as peroxidase, glucose oxidase, and alkaline phosphatase, etc., and specific examples of nucleotides include DNA and RNA. , synthetic oligonucleotide, synthetic polynucleotide, ATP, CT
P, GTP, TTP, UTP, dATP, dCTP, d
GTP, dTTP, dUTP, ddATP, ddCTP
, ddGTP, ddTTP, ddUTP, or derivatives thereof. Specific examples of saccharides include polysaccharides such as glycogen, starch, and mannan, as well as monosaccharides such as oligosaccharides, glucose, and mannose; Examples include phosphatidylcholine, phosphatidylethanolamine, fats, fatty acids, etc. Hormones include insulin, growth hormone, oxytocin, vasopressin, secretin, epidermal growth factor, gastrin, glucagon, peptide hormones such as calcitonin, androgens, estrogens, Examples include steroid hormones such as hydrocortisone, catecholamines such as adrenaline and noradrenaline, and various vitamins such as vitamin A, vitamin B1, B2, B6, B12, biotin, folic acid, vitamin C, vitamin D, and vitamin E. Examples of alkaloids include opium alkaloids such as morphine, tropane alkaloids such as atropine and scopolamine, indole alkaloids such as vinblastine and vincristine, and isoquinoline alkaloids such as ouren. Examples of antibiotics include penicillin, cephalosporin, Examples include kanamycin, erythromycin, chloramphenicol, and the like.

[0018] In order to bond a fluorescent labeling dye to a biological substance, the functional groups such as amino groups and hydroxyl groups in the biological substance and the carboxyl group and sulfone group in the fluorescent labeling dye are used. directly, ionic or covalently bonded, or after chemical modification such as adding a binding group (linker) to a part of the biological material so that the fluorescent labeling dye can react with it. , just react. A biological substance labeled with a fluorescent labeling dye can be purified by conventional separation means such as chromatography and recrystallization.

By changing the substituent represented by R1, the Q(0,0) absorption band of the compound represented by formula 2 is not only in the same region as the absorption region of phthalocyanines, but also has a wavelength shorter than that. Using this fact, the compound represented by chemical formula 2 can be used to analyze various antigens and drugs using any semiconductor laser with an oscillation wavelength of 600 to 720 nm. It can also be used as a highly accurate reagent or clinical test reagent useful for DNA base sequence analysis.

[0020]

EXAMPLES The present invention will now be explained in more detail with reference to Examples. Example 1 (1) In chemical formula 2, M is H2, R1 is a 4-hydroxycarbonylbenzylthio group, n is 2, m is 0, and there is no Y, synthesis literature [J. Am. Chem. Soc. , 11th
2, p. 7408 (1990)], synthesized a compound in which M is H2, R1 is a 4-hydroxycarbonylbenzylthio group, n is 2, m is 0, and there is no Y. did.

(2) Synthesis of linker-bound oligonucleotide primer The primer (5'-GTTTCCCAGT
CACGAC-3') was synthesized. Phosphorylation of the synthesized primers was carried out using 50mM Tris-HCl (pH 7.6),
The reaction was carried out in a 100 μl reaction solution containing 10 mM magnesium chloride, 10 mM dithiothreitol, 3 mM ATP, and T4-nucleotide kinase at 37° C. for 1 hour. The phosphorylated primer was separated by high performance liquid chromatography (HPLC) using a gel filtration column, the peak of the phosphorylated primer was collected, and the solvent was removed by lyophilization. Next, this was mixed with 250mM of 1,
2-diaminoethane (pH 6.0), 200 mM ethyl-3(3-diethylaminopropyl)carbodiimide and 100 mM N-methylimidazole (pH 6.0).
) in 100 μl of a reaction solution containing
' A linker [NH2-(
CH2)2-NH-] was combined.

(3) In formula 2, M is H2, R1 is a 4-hydroxycarbonylbenzylthio group, n is 2, m is 0, and Y
Oligonucleotides labeled with compounds without
Synthesis of primers The compound synthesized in (1) above and 5 synthesized in (2) above
'An oligonucleotide primer with a linker attached to the phosphate moiety of the terminal guanosine was mixed in 0.2M sodium carbonate buffer (pH 9.3), incubated overnight at 25°C in the dark, and then analyzed by HPLC. By purification, a primer was obtained in which a compound of formula 2 in which M is H2, R1 is a 4-hydroxycarbonylbenzylthio group, n is 2, m is 0, and there is no Y is bound via a linker.

(4) DNA base sequence analysis A primer to which a compound of formula 2, where M is H2, R1 is a 4-hydroxycarbonylbenzylthio group, n is 2, m is 0, and there is no Y, is bound via a linker. DN of the known base sequence using
Using A as a sample, each sample was subjected to Sanger reaction with four types of bases, separated by electrophoresis in separate lanes, and analyzed using a DNA sequencer equipped with a semiconductor laser with an oscillation wavelength of 670 nm. As a result, up to 310 bases of DNA could be determined with 99% accuracy.

Example 2 Reference [J. Am. Chem. Soc. , 11th
2, page 7408 (1990)], synthesized a compound in which M is Al, R1 is a 4-sulfobenzylthio group, n is 2, m is 0, and there is no Y. did. Hereinafter, in the same manner as in Example 1, a primer to which this compound was bound via a linker was obtained, and DNA with a known base sequence was analyzed using this primer. As a result, 370
Even bases could be determined with 99% accuracy.

Example 3 Literature [Zh. Obshch. Khim. , Vol. 43, p. 2025 (1973)], a compound in which M is Zn, R1 is a 4-hydroxycarbonylphenyl group, n is 2, m is 0, and there is no Y is prepared. After the synthesis, the same procedure as in Example 1 was carried out to obtain a primer to which this compound was bound via a linker. Next, using this, we analyzed DNA with known base sequences using a laser beam with an oscillation wavelength of 610 nm (based on a combination of N2 laser and dye laser). As a result, up to 270 bases of DNA were analyzed with 99% accuracy. I was able to decide.

Example 4 Literature [Zh. Obshch. Khim. , Vol. 43, p. 2025 (1973)], a compound in which M is Al, R1 is a sulfophenyl group, n is 2, m is 0, and there is no Y was synthesized. . Hereinafter, in the same manner as in Example 3, a primer to which this compound was bound via a linker was obtained, and DNA with a known base sequence was analyzed using this primer. As a result, up to 350 bases of DNA were
It was determined with an accuracy of 9%.

[0027]

Effects of the Invention The present invention provides reagents or clinical test reagents useful for analyzing various antigens, drugs, DNA base sequences, etc., which are measured using a semiconductor laser with an oscillation wavelength of 600 to 720 nm. did it.

Claims (9)

[Claims] Claim 1: Chemical formula 1 [Chemical formula 1] [In chemical formula 1, M is H2, Al, Si, P, Ga, Ge
, Cd, Sc, Mg, Sn or Zn, and R1 is -
XQW, -QW, -W or a hydrogen atom; X is an oxygen atom, a nitrogen atom, a sulfur atom, a phosphorus atom, a silicon atom,
Selenium atom, CR2 R3 (However, R2 and R3
are each independently a hydrogen atom, an alkyl group, an aryl group or an aralkyl group, and R2 R3 may be carbonyl oxygen. ), or a phenylene group, Q
indicates a bonding group (linker) between X and W, W is -OH,
-O-, -SH, -S-, -CO2 H, -CO2
-, -OCH2 CO2 H, -OCH2 CO2
- , -PO42 - , -PO3- , -SO3-
, -SO2- , -SO2 Cl, -SO4
2-, -NH2, -NHR4, -NR5
R6 or -N(+)R7R8R9 (however, R4
~R9 are each independently a C1 to C10 alkyl group, a C6 to C12 aryl group, or a C6 to C12 aryl group
12 aralkyl groups. ), and n is
Indicates an integer of 0 to 2, Y is a halogen atom, -OR10
or -NR112 (However, R10 and R11 each independently represent a hydrogen atom, an alkyl group which may have a hydrophilic substituent, an acyl group which may have a hydrophilic substituent, a hydrophilic is a silyl group which may have a substituent or a phosphorus atom-containing group which may have a hydrophilic substituent), m is Y
An integer from 0 to 2 representing the number of bonds to M. ] Fluorescent labeling dye. 2. A reagent containing the fluorescent labeling dye according to claim 1. 3. A biological substance labeled with the fluorescent labeling dye according to claim 1. 4. A reagent containing the labeled biological substance according to claim 3. 5. The reagent according to claim 3 or 4, wherein the biological substance is an antigen, an antibody, or a nucleotide. 6. The reagent according to claim 5, wherein the antigen is a drug. Claim 7: Claim 5 wherein the antibody is a monoclonal antibody.
Reagents listed. 8. The reagent according to claim 5, wherein the nucleotide is an oligonucleotide or a polynucleotide. Claim 9: The nucleotide is ATP, CTP, GTP,
TTP, UTP, dATP, dCTP, dGTP, dT
TP, dUTP, ddATP, ddCTP, ddGTP
, ddTTP, ddUTP or a derivative thereof.