JPH04344464A - Fluorescent labeling pigment, biogenic material labeled by fluorescent labeling pigment and reagent coating them - Google Patents

Abstract

PURPOSE:To provide a reagent useful in analyzing various kinds of antigens and medicines by using a fluorescent labeling pigment represented by a specific formula, antigens and antibodies labelled by the fluorescent labeling pigment, or a reagent containing them. CONSTITUTION:A fluorescent labeling pigment expressed by a formula 1 (wherein M represents H2, AI or the like; aromatic circles respresented by A, B, C and D are thiophene circle, furan circle or the like; R<1>-R<4> represent -XQW, -QW, -W or hydrogen atom; X represents oxygen atom, nitrogen atom or the like; Q represents a coupled group of X and W; W represents -OH, -O or the like; K, I, (m) and (n) are integers of 0 to 4; Y represents halogen atom, -OR<13> or -NR<142>; and P is an integer f 0 to 2 representing the number of coupling of Y to M), and antigens and antibodies labeled by the fluorescent labeling pigment, or biogenic materials of nuclear acid, or a reagent containing those materials are used in analyzing various kinds of antigens and medicine in blood. An antigen/medicine analyzing reagent for measurement using a small, low- costed semiconductor laser having oscillation frequencies of not more than 660nm can thus be obtained.

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
No. 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]

[Problem to be Solved by the Invention] However, phthalocyanine has a Q(0,0) absorption band with a particularly large extinction coefficient of 665.
~680nm, and the absorption is relatively small in the wavelength range of 660nm or less, so when using a measurement device that uses an inexpensive and small semiconductor laser with an oscillation wavelength of 660nm or less, it is difficult to use as a detection drug. I cannot expect a good role from him.

The present invention provides a method for measuring using a small and inexpensive semiconductor laser having an oscillation wavelength of 660 nm or less.
The purpose of the present invention is to provide reagents or clinical test reagents useful for the analysis of various antigens, drugs, DNA base sequences, etc.

[0009]

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

[chemical 2
] [In chemical formula 2, M is H2, Al, Si, P, Ga, Ge
, Cd, Sc, Mg, Sn or Zn, A, B, C
The aromatic rings represented by , and D each independently represent a thiophene ring, a furan ring, a pyrrole ring, an imidazole ring, a thiazole ring, an oxazole ring, an isothiazole ring, or a pyrazole ring, and R1 , R2 , R3 and R4 are
Each independently represents -XQW, -QW, -W or a hydrogen atom; independently, a hydrogen atom,
It is an alkyl group, an aryl group or an aralkyl group, and R5
Carbonyl oxygen may be used as R6. ), or a phenylene group, Q represents a bonding group (linker) between X and W, and W represents -OH, -O-, -SH, -S-, -C
O2 H, -CO2 -, -OCH2 CO2 H,
-OCH2 CO2 - , -PO42 - , -PO
3-, -SO3-, -SO2-, -SO2C
l, -SO4 2 -, -NH2, -NHR7,
-NR8 R9 or -N(+)R10R11R
12 (However, R7 to R12 are each independently
They are a C1 to C10 alkyl group, a C6 to C12 aryl group, or a C6 to C12 aralkyl group. ), k, l, m and n each independently represent an integer of 0 to 4, Y is a halogen atom, -OR13
or -NR142 (However, R13 and R14 each independently represent a hydrogen atom, an alkyl group that may have a hydrophilic substituent, an acyl group that may have a hydrophilic substituent, a hydrophilic is a silyl group that may have a substituent or a phosphorus atom-containing group that may have a hydrophilic substituent), and p 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) above, 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, R1 to R
Specific examples of the alkyl groups for R5 and R6 in Examples of aryl groups include phenyl group, thienyl group, furyl group, pyrrolyl group, tolyl group, anisyl group, 4-aminophenyl group, and examples of aralkyl groups include benzyl group, 2-phenyl group, etc. Examples include ethyl group, 1-phenylethyl group, 3-phenylpropyl group, 2-phenylpropyl group, and 1-phenylpropyl group. Further, specific examples of the alkyl group which may have a hydrophilic substituent for R13 and R14 in Y include methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, Octyl group, nonyl group, decyl group, dodecyl group, tetradecyl group,
There are linear, branched, and alicyclic groups such as hexadecyl, octadecyl, eicosyl, and docosyl groups, and examples of acyl groups that may have hydrophilic substituents include formyl and acetyl groups. , propionyl group, butyryl group, 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, tripropylsilyl group, tributylsilyl group, triamylsilyl group, trihexylsilyl group, t-butyldimethylsilyl group, di(t-butyl)methylsilyl group,
Examples include dimethylphenylsilyl group, diphenylmethylsilyl group, and triphenylsilyl group.

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 R7 to R12 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.

In addition, the substituents containing a phosphorus atom represented by R13 and R14 include -P(=O)R15R16, -P(=O)(NR1
7R18)2, -PR192 (R15 to R19 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; ) may have various substituents.

In formula 2, Y is -OR13 or -NR142
(R13 and R14 represent an alkyl group, an acyl group, and a silyl group), and the compound in which p 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. The compound in which Y is -OH or -NH2 and p is 1 or 2 in Chemical Formula 2, Y is a halogen atom in Chemical Formula 2,
It can be obtained by hydrolyzing or ammonolyzing a compound in which p is 1 or 2. During 2nd year,
Compounds in which Y is a halogen atom and p is 1 or 2, and compounds in which p is zero and do not have Y can be synthesized by the following two routes.

The first route is described in the literature (J. Chem.
oc. Referring to the method described on page 911 (1937), chemical formula 3

[Formula 3] (wherein, M is H2, Al, Si, P, Ga, Ge, C
d, Sc, Mg, Sn or Zn, Y represents a halogen atom, p represents an integer of 0 to 2 representing the number of bonds of Y to M, and aromatic represented by A, B, C, and D. The rings each independently represent a thiophene ring, a furan ring, a pyrrole ring, an imidazole ring, a thiazole ring, an oxazole ring, an isothiazole ring, or a pyrazole ring. ) is obtained, and then, in chemical formula 2, substituents R1 and R2 are added to this compound.
, R2, R3 which can form R3 or R4
Alternatively, the compound represented by the formula 2 is obtained by reacting a chemical species having an R4 site.

The second route is chemical compound 4

[C4] Or 5

embedded image (In Chemical Formulas 4 and 5, the aromatic rings represented by A each independently represent a thiophene ring, a furan ring, a pyrrole ring, an imidazole ring, a thiazole ring, an oxazole ring, an isothiazole ring, or a pyrazole ring, R1 and k have the same meanings as in Chemical Formula 2) can be obtained by reacting a compound represented by the formula (R1 and k have the same meanings as in Chemical Formula 2) with a corresponding metal or metal salt. On the other hand, when M in Chemical Formula 2 is H2, it can be obtained by reacting the compounds represented by Chemical Formulas 4 and 5 with Na or Li alkoxide, and then hydrolyzing. The compound represented by Chemical Formula 5 can be obtained by reacting the compound represented by Chemical Formula 4 with ammonia in methanol in the presence of sodium methoxide.

[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.

The compound represented by chemical formula 2 has a large absorption maximum based on the Q(0,0) band in a short wavelength region (660 nm or less) compared to phthalocyanine, so it has a large absorption maximum at 660 nm.
Analysis of various antigens, drugs, or DN
It can be used as a highly accurate reagent useful for analysis of the base sequence of A or as a clinical test reagent.

[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 Zn, R1 to R4 are all hydroxycarbonyl groups, A, B, C and D are all thiophene rings, k, l, m and n are all 1, and Synthesis literature of compounds with p=0 and no Y [J. Chem. Soc. , 911 pages (19
37)] With reference to the method described, in chemical formula 2, M is Zn.
, R1 to R4 are all hydroxycarbonyl groups, A,
A compound in which B, C, and D are all thiophene rings, k, l, m, and n are all 1, p is 0, and Y is absent was synthesized.

(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 Zn, R1 to R4 are all hydroxycarbonyl groups, A, B, C and D are all thiophene rings, k, l, m and n are all 1,
Synthesis of an oligonucleotide primer labeled with a compound with p of 0 and no Y. The compound synthesized in (1) above and the oligonucleotide synthesized in (2) above with a linker attached to the phosphoric acid moiety of the 5'-terminal guanosine. - Primer in 0.2M sodium carbonate buffer (pH 9.3)
) and kept warm at 25°C overnight in a dark place.
By purifying with PLC, in chemical formula 2, M is Zn, R1 to R4 are all hydroxycarbonyl groups, A, B, C and D are all thiophene rings, k,
A primer was obtained in which l, m, and n were all 1, p was 0, and a compound was bound without Y.

(4) Analysis of DNA base sequence DNA with a known base sequence is used as a sample, and in the formula 2, M is Zn, R1 to R4 are all hydroxycarbonyl groups, A, B, C, and D is a thiophene ring, k,
Using a primer bound to a compound in which l, m, and n are all 1, p is 0, and there is no Y, a Sanger reaction is performed with each of the four types of bases, and then electrophoretic separation is performed in separate lanes. The analysis was performed using a DNA sequencer equipped with a semiconductor laser with an oscillation wavelength of 650 nm. As a result, up to 340 bases of DNA could be determined with 99% accuracy.

Example 2 Reference [J. Chem. Soc. , 911 pages (19
37)] With reference to the method described, in chemical formula 2, M is H2
, R1 to R4 are all sulfonic acid groups, A, B, C, and D are all thiophene rings, k, l, m, and n are all 1, p is 0, and a compound without Y was synthesized. 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, up to 380 bases of the DNA were 99% The decision was made with precision.

Example 3 Literature [J. Chem. Soc. , 911 pages (19
37)] With reference to the method described, in chemical formula 2, M is Al.
, R1 to R4 are all 5-carboxypropionyl groups, A, B, C and D are all thiophene rings, k, l,
A compound in which m and n are both 1, p is 0, and Y is absent was synthesized. 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.
Up to 280 bases of A could be determined with 99% accuracy.

Example 4 Literature [J. Chem. Soc. , 911 pages (19
37)] With reference to the method described, in chemical formula 2, M is Zn.
, R1 to R4 are all 5-carboxyvaleryl groups, A
, B, C, and D are all thiol rings, k, l, m, and n are all 1, p is 0, and a compound without Y was synthesized. Hereinafter, in the same manner as in Example 1, a primer to which this compound was bound via a linker was obtained, and as a result of analyzing DNA with a known base sequence using this primer, 3
Up to 20 bases could be determined with 99% accuracy.

[0027]

Effects of the Invention The present invention provides reagents or clinical test reagents useful for analyzing various antigens, drugs, DNA base sequences, etc., for measurement using a semiconductor laser having an oscillation wavelength in the wavelength range of 660 nm or less. We were able to provide

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, A, B, C
The aromatic rings represented by , and D each independently represent a thiophene ring, a furan ring, a pyrrole ring, an imidazole ring, a thiazole ring, an oxazole ring, an isothiazole ring, or a pyrazole ring, and R1 , R2 , R3 and R4 are
Each independently represents -XQW, -QW, -W or a hydrogen atom; independently, a hydrogen atom,
It is an alkyl group, an aryl group or an aralkyl group, and R5
Carbonyl oxygen may be used as R6. ), or a phenylene group, Q represents a bonding group (linker) between X and W, W is -OH, -O-, -SH, -S-, -C
O2 H, -CO2 -, -OCH2 CO2 H,
-OCH2 CO2 - , -PO42 - , -PO
3-, -SO3-, -SO2-, -SO2
Cl, -SO4 2 -, -NH2, -NHR7
, -NR8 R9 or -N(+)R10R1
1R12 (However, R7 to R12 are each independently a C1 to C10 alkyl group, a C6 to C12
aryl group or C6 to C12 aralkyl group. ), k, l, m and n each independently represent an integer of 0 to 4, Y is a halogen atom, -OR
13 or -NR142 (However, R13 and R14 each independently represent a hydrogen atom, an alkyl group that may have a hydrophilic substituent, an acyl group that may have a hydrophilic substituent, or a hydrophilic substituent. is a silyl group that may have a hydrophilic substituent or a phosphorus atom-containing group that may have a hydrophilic substituent), and p represents the number of bonds of Y to M. Indicates an integer between ~2. ] 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.