Cypridina luciferin stabilization composition and method for storing cypridina luciferin

The invention is based on the Patent application title of invention is submitted by the 25 September 2006 " sea firefly fluorescein light-emitting substrate and manufacturing method thereof" of China Patent application 200680035410.3 applied for of.

Technical Field

The invention relates to a luciferase sea firefly reaction with fluorescein (light-emitting substrate) of the manufacturing method, model sea firefly fluorescein compound and its manufacturing technology established.

Furthermore, the invention relates to a light with natural sea firefly fluorescein derivatives of different wavelengths, a derivative of the background low, composition, method for luminescence assays.

Background Art

Luminescent crustacean sea firefly and its sibling species with secretory the luxAB (luciferase) and light-emitting substrate (luciferin), a luciferase sea fireflysea firefly fluorescein with the catalyst by oxidation reaction and of the maximum light-emitting wavelength is 460 nanometer blue optically.

Luciferase can be secreted to the outside has the characteristic of the cell, therefore, when the cloning of the cDNA is used as the reporter gene, in mammals, yeast cell protein synthesis in the cell can also be secreted. Therefore, does not destroy the cell because it can determine the luminous active of the luciferase sea firefly , therefore, for example, can be measured in cells of the mammalian in cell gene transcription activity (valeant literature 1-2, Patent literature 1-3). Furthermore, yeast cells can be used for detection of the report of the secreted luciferase gene (Patent literature 4).

In the utilization of the luxAB reports are as follows in examples: the secreted luxAB Image analysis and the secretion of proteins from cells visualized (valeant literature 3) example; by the use of the insert the growth hormone gene transcription activity report sea firefly a region of the gene into a mammalian cell, in continuously measuring biological cells of the examples of changes of the transcription activity of a (valeant literature 4). Furthermore, there are also the use of a luciferase sea firefly and fluorescent protein fusion body of active peptide from the protein quantitative example of processing (Patent document 5).

In the field of pharmacy, protein expression inhibitor and secretion inhibitor the development and exploration is important, to in cell of gene transcription activity of the target protein for the index of the change of the screening. Inhibitors of the transmission accompanied by the effect of changes of the transcription activity of a gene is the report of the task gene (protein). As a reporter protein, ON/OFF of the gene not only notice requirements, and can analyze the effect of inhibitors of secular change (time to decompose the can is high), or, the report requirements of the protein itself has no inhibiting effect or does not interfere with cell internal strength energy characteristics (there is no cytotoxicity). As realization of high time to decompose the report of can and gene has no cytotoxicity, or metabolic need of fast cells secretes a in makes of the report of the protein.

Luciferase sea firefly is secretome and can rapidly determine the change of the transcription activity of a cell outside, therefore, its wide use. Although the only useful method is, however, practical use of the luciferase sea firefly · in generalization also to the. This is because as the matrix of the fluorescein can not fully supply the relatively big problem.

Furthermore, as the problem of fluorescein sea firefly , can be enumerated: fluorescein it is difficult to stably supplied, such as the fluorescein and the albumin protein-light-emitting, light-emitting wavelength of the other light-emitting system as well.

To date, the report to the fluorescein sea firefly synthesis of fluorescein (etioluciferin) early as precursors to intermediates. For the purposes of synthesis of fluorescein at the beginning, the most short also need to 7 steps (valeant literature 5-7). As the beginning of the final step of the fluorescein sea firefly from fluorescein to poor yield of the steps, need a large number of preparation of raw material, therefore, the manufacturing cost is remarkably improve the fluorescein. According to the method described in the literature, using 3-methyl-2-oxo-valeric acid and the condensation reaction of fluorescein at the beginning of the optical activity of the synthetic yield fluorescein 3 stage is 2% (valeant literature 5, Figure 1). As mentioned above, due to the very low yield of the final step, therefore, the current status quo is, using organic synthesis of fluorescein sea firefly optical activity is difficult to be industrialized.

The racemic modification fluorescein (valeant literature 7) shows only a natural fluorescein about half of the activity, does not depend on the presence of luciferase fluorescein nonnatural luminous background, therefore, the optical activity of synthesizing fluorescein in the bioassay is very important issue.

On the other hand, the fluorescein and the sea sea firefly basic skeleton is kidney fluorescein imidazo Phrazinone skeleton, therefore, close to the maximum light-emitting wavelength, according to the structure of the luciferase is slightly different, are about 460 nanometer to about 480 nanometer, is difficult to be at the same time that the two light-emitting system.

Furthermore, despite the use of a luciferase sea firefly and fluorescent protein fusion the processing of peptides from the protein quantification of examples, but the light-emitting of the luciferase sea firefly maximum value (460 nanometer) the maximum value of the light-emitting fluorescent protein (525 nanometer) (stokes   shift) the difference is relatively small, for the 60 nanometer, therefore, can be seen the light-emitting of the luciferase sea firefly the fluorescent protein fluorescent interference. In the use of this optical interference processing quantification of fluorescent protein of a peptide in the form of a high background. Expect that developing maximum luminous wavelength different sea firefly fluorescein analogs.

Moreover, using fluorescein sea firefly white protein, and the like in place of the chemical light-emitting, light-emitting of the quantum yield is low. fluorescein enzyme quantity is not dependent on the self-light-emitting, the luciferase sea fireflyin cell of imaging or in the measurement of transcription activity as a background to observe. Background the development of desired sea firefly fluorescein analogs.

The prior synthesis of the fluorescein derivative sea firefly , and invention for superoxide anion such as the chemical luminescent reagent. However, there is no development a sea firefly derivatives of the substrate of the luciferase.

Patent literature 1 : WO90/01542

Patent literature 2: extra Kaiping 3-30678

Patent literature 3 : opens especially 2004-187652

Patent literature 4 : hopes especially 2005-169768

Patent literature 5:15828 PCT/JP03/

Patent literature 6: extra Kaiping -60697, the

Patent literature 7: extra Kaiping 5-286976, the

Valeant literature 1: Thompson, E.M. , Nagata, &   Tsuji S., F.I.Vargula hilgendorfii   luciferase:   monitoring   for a   secreted   reporter   gene expression   in   mammalian   enzyme   cells.Gene   96,257-62 (1990)

Valeant literature 2: Nakajima   Y, Kobayashi   K, Yamagishi   K,   T and   Y   Ohmiya Enomoto:   a cDNA   cloning   and   characterization   of   secreted   luciferase from   the   ostracod   Japanese   luminous, Cypridina   noctiluca.Biosci. Biotechnol.Biochem. 68,565-70, 2004

Valeant literature 3: Inouye, S. , Ohmiya, Y. &   Tsuji,   secretion   of luciferase   from F.Imaging   transformed   Chinese   hamster   ovary   A   Acad   S   Sci   cells.Proc Natl   U   89, 9584-7 (1992)

Valeant literature 4: Tanahashi, Y. , Ohmiya, Y. , Honma, S. , Katsuno, Y. , Ohta, H. , Nakamura, H. , Honma, K.Continuous   by   a   measurement   of   secreted   targeted promoter   reporter   activity   bioluminescence, Vargula hilgendorfii   luciferase.Anal   Biochem. 289,260-6 (2001)

Valeant literature 5: Kishi, Y. ; Goto, metric ton ; Inoue, S. ; Sugiura, S. ; Kishimoto, H.Tetrahedron   Lett. 1996.3445-3450

Valeant literature 6: Karpetsky, T.P. ; White, E.H.J.Am.Chem.Soc. 1971, 93, 2333-2334

Valeant literature 7: Nakamura, H.Aizawa, M.Takeuchi, D.Murai, A. Shimomura   O.Tetrahedron   lett. 2000, 41, 2185.

Content of the invention

The purpose of this invention lies in, the optical activity of the fluorescein and of steps in the method for producing the problem of low yield, establishes at the same time light-emitting wavelength different fluorescein and fluorescein supply.

With the present invention provides the following novel fluorescein sea firefly luciferase reaction (light-emitting matrix) and its manufacturing method.

1. A kind of manufacturing general formula (2) method of a compound represented by the, characterized in that the general formula (1) compound represented by the reaction with the diazo methane compounds, and then the oxidizing agent and is selected from the group consisting of alcohols and glycols in the group consisting of at least 1 kind of reaction,

In the formula, X represents Cl or Br; R¹ and R² the same or different, represents a lower alkoxy, can be substituted aralkyloxy, or R¹ and R² with they are combined together with the carbon atom of said carbonyl, or R¹ and R² in expressing alkylene dioxy group.

2. General formula (2) of said compound,

In the formula, R¹ and R² the same or different, represents a lower alkoxy, can be substituted aralkyloxy, or R¹ and R² with they are combined together with the carbon atom of said carbonyl, or R¹ and R² in expressing alkylene dioxy group.

3. A kind of manufacturing general formula (4) of said sea firefly fluorescein or derivatives thereof, characterized in that the general formula (2) with a compound represented by the general formula (3) reaction of a compound represented by,

In the formula, R¹ and R² the same or different, represents a lower alkoxy, can be substituted aralkyloxy, or R¹ and R² with they are combined together with the carbon atom of said carbonyl, or R¹ and R² in expressing alkylene dioxy; R³ said aryl group which may be substituted or may be substituted heterocyclyl; Y¹ can be substituted preliminary Asia alkyl or lower alkenylene; Z¹ expressed amino, a low-grade alkyl amino, di-lower alkyl amino, guanidino or amidino, or by protecting group protected amino, guanidino or amidino.

4. A kind of manufacturing general formula (7) of said method for sea firefly fluorescein analogs, characterized in that the general formula (5) with a compound represented by the general formula (6) reaction of a compound represented by,

In the formula, R¹ and R² the same or different, represents a lower alkoxy, can be substituted aralkyloxy, or R¹ and R² with they are combined together with the carbon atom of said carbonyl, or R¹ and R² in expressing alkylene dioxy; R³ said aryl group which may be substituted or may be substituted heterocyclyl; R⁵ can be is substituted by fluorine atom of the lower alkyl, can be substituted with fluorine atom cycloalkyl, 5 or 6 of the $aromatic cyclic group or a heterocyclic group, aralkyl; Z¹ expressed amino, a low-grade alkyl amino, di-lower alkyl amino, guanidino or amidino, or by protecting group protected amino, guanidino or amidino.

5. General formula (7) of said compound,

In the formula, R³ said aryl group which may be substituted or may be substituted heterocyclyl; R⁵ can be is substituted by fluorine atom of the lower alkyl, can be substituted with fluorine atom cycloalkyl, 5 or 6 of the $aromatic cyclic group or a heterocyclic group, aralkyl; Z¹ expressed amino, a low-grade alkyl amino, di-lower alkyl amino, guanidino or amidino, or by protecting group protected amino, guanidino or amidino.

6. A kind of manufacturing general formula (8) of the fluorescein derivative sea firefly method, characterized in that the general formula (3) with a compound represented by the general formula (6) reaction of a compound represented by,

In the formula, R¹ and R² the same or different, represents a lower alkoxy, can be substituted aralkyloxy, or R¹ and R² with they are combined together with the carbon atom of said carbonyl, or R¹ and R² in expressing alkylene dioxy; R³ said aryl group which may be substituted or may be substituted heterocyclyl; R⁵ can be is substituted by fluorine atom of the lower alkyl, can be substituted with fluorine atom cycloalkyl, 5 or 6 of the $aromatic cyclic group or a heterocyclic group, aralkyl; Y¹ can be substituted preliminary Asia alkyl or lower alkenylene; Z¹ expressed amino, a low-grade alkyl amino, di-lower alkyl amino, guanidino or amidino, or by protecting group protected amino, guanidino or amidino.

7. General formula (8) the compound or its salt,

In the formula, R³ said aryl group which may be substituted or may be substituted heterocyclyl; R⁵ can be is substituted by fluorine atom of the lower alkyl, can be substituted with fluorine atom cycloalkyl, 5 or 6 of the $aromatic cyclic group or a heterocyclic group, aralkyl; Y¹ can be substituted preliminary Asia alkyl or lower alkenylene; Z¹ expressed amino, a low-grade alkyl amino, di-lower alkyl amino, guanidino or amidino, or by protecting group protected amino, guanidino or amidino; wherein, the R³ is indolyl or phenyl, Y¹ to 1, 2-propylidene, Z¹ under the condition of the guanidino, R⁵ is not sec-butyl.

8. If the above-mentioned 5 or 7 wherein the compound or its salt, it has than natural sea firefly the characteristic of fluorescein background low.

9. If the above-mentioned 5 or 7 wherein the compound or its salt, characterized in that the reaction of luciferase sea firefly the maximum light-emitting wavelength of the natural sea firefly fluorescein different.

10. The compound or its salt.

11. The compound or its salt.

12. The compound or its salt.

13. The compound or its salt.

14. The compound or its salt.

15. A stabilized composition fluorescein sea firefly , comprising sea firefly fluorescein or its analogues and antioxidant.

16. A save sea firefly method of fluorescein or analogues thereof, characterized in that the fluorescein or its analogue sea firefly the presence of the antioxidant to be stored.

17. A kind of kit, for the same biological sea firefly light-emitting system, wherein, comprising sea firefly fluorescein or its analogues and antioxidant.

18. At least 1 kind of antioxidant biological sea firefly for reducing the background application of the light-emitting system.

19. A measuring method for biological firefly class of the sea, characterized in that and the luciferase sea fireflysea firefly the fluorescein or its analogs in the bioluminescent determination system, the presence of the antioxidant determining biological light-emitting.

20. If the above-mentioned 15-20 the method, composition or kit, wherein, by the antioxidant is selected from ascorbic acid or its salt, isoascorbic acid or its salt, dithionous acid or its salt, bisulfite, sulfites, pyrosulfites, metabisulphites, cysteine, thioglycerols, butyl hydroxy anisole, polyphenol, thiophosphoro sulfuric acid or its salt, the group consisting of alkali metal borohydride.

Beneficial effects

According to the present invention, can be used for producing optically active simple and convenient method of fluorescein sea firefly. This is because, by the general formula (2) with a compound represented by the general formula (3) condensation of a compound, can be in the 1 stage of the reaction in order to obtain a high yield of the general formula (4) of the optical active body (Figure 1).

The invention of the general formula (7), (8) luciferase sea firefly compound can be used to make the light-emitting wavelength displacement.

For example, the compounds of the present invention 36 the maximum light-emitting wavelength can be displacement to 390 nanometer, the ultraviolet region of the light emitted. By making compound 36 analogs, can be easily synthesized the maximum light-emitting wavelength is about 380 nanometer-about 400 nanometer analogues. This kind of wavelength region of the light is absorbed by various compounds, thus can make the various chemical substance (such as an anti-cancer agent, such as rhodopsin) activation.

Also, fluorescein or derivatives thereof in the presence of the antioxidant, ascorbic acid or its salt, in particular, ISO-ascorbic acid or its salt is sea firefly class biological luminescence assays, background lower, than S/N is improved.

Luciferase in cells with contentsea fireflysea firefly (enzyme) or the expression of the luciferase gene in the system, in order to make the stability of the luciferase, such as with most of the albumin BSA, however, ascorbic acid, sulfite (for example Na₂ SO₃) antioxidant such as caused by the albumin can be suppressed in the background.

Description of drawings

Figure 1 representing natural of the prior synthesis of fluorescein sea firefly with the comparison of the synthesis method of the present invention.

Figure 2 that the fluorescein sea firefly of this invention the maximum light-emitting wavelength.

Figure 3 of the present invention said sea firefly fluorescein derivative application examples. Figure 3A sea fireflysea firefly and the luciferase expressed luciferase and fluorescent protein fusions light-emitting principle of (A1 expressed luciferase sea firefly and fluorescein analogs 36, A2 and the luciferase expressed sea firefly about half of the green fluorescent protein, fluorescein analogs 36. ), Chart 3B said double-report gene detection (determination 2 promoter sequence A, B of gene transcription activity of the gene constructs), chart 3C said gene detection of the double-report of the change of the light-emitting spectrum (I: the promoter of the spectrum of the active A (only expression promoter A), II: promoter activation B the spectrum before, III: promoter activation B the spectrum, ↑ : activation B as the promoter, relative to the light-emitting active (B/A) increase. ).

Mode of execution

In this specification, alkylene dioxy can enumerate: ethylidenedioxy, subpropyldioxy, ADEN dioxy, alkylidene asian fifth heavenly stem , carbon atoms, alkylidene asian oneself 2-6, preferably carbon atom number is 2-4 alkylene dioxy group.

Diazo methane compounds can be enumerated: diazomethane, trimethylsilyl diazomethane, and the like.

Lower alkyl may be enumerated: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl (R⁵ situation, preferably the optical active sec-butyl), tert-butyl, pentyl, hexyl, etc. carbon atom number is 1-6, preferably carbon atom number is 1-4 straight chain or a branched alkyl.

As by fluorine atom substituted lower alkyl, can enumerate contains fluorine atom, a hydrogen atom and carbon atom fluorinated lower alkyl and which contains fluorine atoms and carbon atoms of the perfluorinated lower alkyl group, a fluoro lower alkyl, can be listed in the above-mentioned lower alkyl group of 1 or more (not all) of the hydrogen atom is substituted by fluorine atom, a lower alkyl group. As the perfluoro lower alkyl group, in the above-mentioned lower alkyl group can be cited all of the hydrogen atom is substituted by fluorine atom, a lower alkyl group. As R⁵ the perfluoro lower alkyl, can enumerate: CF₃, CF₂ CF₃, CF₂ CF₂ CF₃, CF (CF₃)₂, CF₂ CF₂ CF₂ CF₃, CF₂ CF (CF₃)₂, CF (CF₃) CF₂ CF₃, C (CF₃)₃, CF₂ CF₂ CF₂ CF₂ CF₃, CF₂ CF (CF₃) CF₂ CF₃, CF (CF₂ CF₃)₂, CF (CF₃)₂ CF₂ CF₂ CF₂ CF₂ CF₂ CF₂ CF₂ CF₃, CF₂ CF (CF₃) CF₂ CF₂ CF₃, CF (CF₂ CF₃) (CF₂ CF₂ CF₃), C (CF₃)₂ CF₂ CF₂ CF₃, C (CF₃) (CF₂ CF₃)₂ carbon atoms such as 1-6 linear or branched perfluoro alkyl, preferably carbon atoms can be enumerated 3-6 with branched perfluoro alkyl.

Fluorocholine alkyl may be listed in the above-mentioned perfluoro lower alkyl group of 1 or more (not all) of the hydrogen atom is substituted by fluorine atom, a carbon atom number of 1-6 of the straight-chain or branched fluoroalkyl, preferably carbon atoms can be enumerated 3-6 with branched fluoro-alkyl.

Cycloalkyl can enumerate: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, carbon atoms such as link octyl 3-8, preferably carbon atom number is 5-6 cycloalkyl.

As atoms replace by fluorine a cycloalkyl, can enumerate contains fluorine atom, a hydrogen atom and carbon atom fluorinated cycloalkyl and a provided by fluorine atoms and carbon atoms of the perfluorinated cycloalkyl, as fluorocholine cycloalkyl, can enumerate the above-mentioned cycloalkyl of 1 a (not all) more than the hydrogen atom is substituted by fluorine atom of the cycloalkyl. As the full fluorocholine cycloalkyl, can be listed in the above-mentioned cycloalkyl group is substituted by fluorine atom all of hydrogen atoms of a cycloalkyl.

R⁵ of said 5 or 6 the aromatic ring or heterocyclic group can be enumerated: phenyl, pyridyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, Azoly, heteroploid Azoly, pyrimidinyl, pyrazinyl, thienyl, furyl, pyridazinyl.

Lower alkoxy can enumerate: methoxy, ethoxy, are propoxy, isopropoxy, positive butoxypolyethylene, isobuoxy, [...] , tert-butoxy, pentyloxy, hexyloxy, etc. carbon atom number is 1-6, preferably carbon atom number is 1-4 straight chain or a branched chain alkoxy.

A low-grade alkyl amino can enumerate: methylamino, ethylamino, is c amino, isopropylamino, is d amino, isobutyric amino, amino [...] , tert-butyl amino, fifth heavenly stem amino , caprolactam is the carbon atom number is amino 1-6, preferably carbon atom number is 1-4 straight chain or a branched chain of a substituted alkyl list amino.

The second low-level alkyl amino can enumerate: dimethylamino, diethylamino, di-n-c amino, diisopropyl amino, di-n-d amino, secondary amino-isobutene, [...] amino, amino two Uncle Ding , two fifth heavenly stems amino, the amino of dihexyl carbon atom number is 1-6, preferably carbon atom number is 1-4 straight chain or a branched chain alkyl group of the di-substituted amino.

R³ the heterocyclic radical can enumerate: 3-indolyl, 1-ISO-indolyl, 3H-Indol-2-yl, 1H-isoindol-3-yl, 3-isoquinolyl, 1-isoquinolyl, (2-, 3-or 4-) quinolyl, 1- [...] , 2- naphthyridine base , 2-quinazolinyl, 2-benzimidazolyl, (2-or 3-) benzofuranacetic, 1-ISO-benzofuran, (2-or 3-) benzo thiophene, 1- different benzene and thiophene, 1 a 6 aromatic ring of $(can be heterocyclic) and 1 a 5 or 6 great of $synthesis of the heterocyclic base.

Heterocyclyl, aralkyl, aralkyloxy, aryl substituent can be enumerated: a halogen atom (in particular chlorine atom or a fluorine atom), methyl, ethyl, acetyl, methoxy, OH, SH, COOH, amino, methylamino, dimethylamino, nitro, cyano, trifluoromethyl, etc., can be 1-3 of these substituent substituted.

R³ the aryl group can be enumerated: naphthyl, phenyl, biphenyl, anthryl, Fikki, fluorenyl, indenyl, that non-alkenyl (Phenalenyl), [...] , Pyren, and the like.

Aralkyl can enumerate: benzyl, 1-phenethyl, 2-phenethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, menaphthyl carbon atoms such as 7-12 aralkyl.

Aralkyloxy can enumerate: benzyloxy, 1-phenethyl propoxycyclohexyl, 2-phenethyl propoxycyclohexyl, 1-phenylpropyl propoxycyclohexyl, 2-phenylpropyl propoxycyclohexyl, 3-phenylpropyl propoxycyclohexyl, menaphthyl propoxycyclohexyl and other carbon atom number is 7-12 the aralkyloxy.

Z¹ expressed amino, guanidino or amidino the protecting group can be widely instantiated: uncle Ding Yangtang (BOC) group, animal pen oxygen carbo- (Cbz) group, the Fmoc protecting group is usually such.

Y¹preliminary Asia alkyl may be cited of:CH₂, CH (CH₃), CH₂ CH₂, CH₂ CH (CH₃), CH (CH₃) CH₂, CH₂ CH₂ CH₂, CH₂ CH (CH₃) CH₂, CH₂ CH₂ CH₂ CH₂ carbon atoms such as 1-6, preferably carbon atom number is 1-4 alkylene, lower alkenylene can enumerate: CH=CH, CH=C (CH₃), C (CH₃) =CH, CH=CHCH₂, CH=C (CH₃) CH₂, CH₂ CH=CH, CH=CHCH₂ CH₂ carbon atoms such as 1-6, preferably carbon atom number is 1-4 the alkenylene.

Next, the route 1 of compound 2 a description of the manufacturing method.

< line 1>

(In the formula, R¹ and R² such as the above-mentioned definition. )

The manufacturing method of the Arndt-Eistert reaction set as the main reaction of the three steps. First of all, with respect to the (+)-2-methyl butyrate (1A) 1 mol, the use of 1 molar left and right to the excess of thionyl chloride, to enable its reaction at room temperature 1-24 hours, thereby obtaining the acid chlorides. Furthermore, when using 2 equivalents of trimethyl silyl diazo methane or diazomethane in the acyl chloride 0-4 the reaction under [...] 1-4 hours, diazo ketone compound obtained. The purposes of a diazo ketone compound, the use of 1 equivalent of the oxidizing agent (in particular tert-butyl hypochlorite), or the equivalent in the presence of an excess of the alcohol or diol, the 0-4 the reaction under [...] 1-2 hours, therefore can obtain a novel optically active compound (2). Furthermore, R¹ and R² of the benzyloxy, the amount of the catalyst can be the presence of platinum oxide , by a hydrogenation decomposition represented by the general formula (2) compound (aldehyde body).

An oxidizing agent such as hypochlorous acid T-butyl ester can be cited.

The R¹ and R² speaking, and with an oxidizing agent for the alcohol (methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, secondary butyl alccohol, tertiary butyl alcohol, the carbon atom number is 1-6 of the straight-chain or branched lower alcohol, or benzyl alcohol, and the like can have a substituent of the alkyl alcohol) or glycol (ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, 1, 2-butanediol, 1, 3-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2, 3-butanediol, etc. carbon atom number is 2-6 diol) and is introduced into the, for example, when the use of methanol, R¹ and R² are methoxy, is the use of benzyl alcohol, R¹ and R² is benzyloxy. Furthermore, if and for methanol and ethanol, can be obtained the R¹ and R² are methoxy and ethoxy compound.

In the present invention 1 in one embodiment, as a substituent aralkyloxy, can be cited such as:is hydroxy, lower alkoxy, cyano, nitro, amino, a low-grade alkyl amino, di-lower alkyl amino, halogen atom (F, Cl, Br, I), lower alkyl, SH, alkylthio (S-lower alkyl), ethylenedioxy substituent such as for 1-3 a substituted aralkyloxy.

< line 2>

(In the formula, R¹, R², R³, Y¹ and Z¹ such as the above-mentioned definition. )

The purposes of the reaction, with respect to the general formula (3) compound 1 mole, using the formula (2) compound about 1 mole to about 4 mole, or in the presence of an acid in the presence of not, at about 65 the [...] -about 100 the temperature of [...] to enable its reaction 1-2 hours to advantageously carry out.

General formula (3) for the compounds of the method of manufacturing the line 3 to explain the.

< line 3>

(In the formula, R³ and Z¹ such as the above-mentioned definition. Y^1a expressed CH₂ CH₂ CH₂. R⁴ represents a halogen atom, especially Cl or Br. )

Routeings 3 expressed by a general formula (5) or the general formula (3) of a description of process for the production of compounds.

Z¹ expressed amino, a low-grade alkyl amino, di-lower alkyl amino, guanidino or amidino, or by protecting group protected amino, guanidino or amidino, in the above-mentioned intermediates in the manufacturing method, is preferably protected amino, guanidino or amidino.

General formula (5) or the general formula (3) method of manufacturing set as the Suzuki coupling reaction main reaction. When the with respect to the known 2-amino -3, 5-di-halogenated amino pyrazine (9A) 1 mole, using 1 mole of vinyl boronic acid reagent (9B) 1 mole, and a palladium catalyst in the presence of alkali to enable its reaction 1-2 hours, olefin compounds can be obtained (the general formula (9)). By the general formula (9) by using the compounds of R³ B (OH)₂ can be obtained by the Suzuki coupling reaction formula (5) compound.

On the other hand, relative to the olefin compound (9) 1 mol, in 10% molar of the presence of platinum oxide , by hydrogenating after reduction of the alkene, can likewise by using R³ B (OH)₂ represented by the general formula of the Suzuki coupling reaction (3a) of the compound.

Using the following line 4 to the general formula (7) of a description of process for the production of compounds.

< line 4>

(In the formula, R¹, R², R³, R⁵ and Z¹ such as the above-mentioned definition. )

With respect to the general formula (5) the compound of 1 molar using the formula (6) compound about 1 mole to about 4 mole, or in the presence of an acid in the presence of no, in the 65-100 [...] the temperature of the reaction to make the 1-2 hours, can be obtained by the general formula (7) compound.

< line 5>

(In the formula, R¹, R², R³, R⁵, Y¹ and Z¹ such as the above-mentioned definition. )

With respect to the general formula (3) compound 1 molar using the formula (6) compound about 1 mole to about 4 mole, or in the presence of an acid in the presence of no, in the 65-100 [...] the temperature of the reaction to make the 1-2 hours, can be obtained by the general formula (8) compounds.

The compounds of the present invention is useful as a fluorescein sea firefly.

In the 1 preferred embodiment, the compound of this invention, the reaction with the same luciferase sea firefly , its maximum light-emitting wavelength of the natural sea firefly compared with fluorescein, to the long wavelength side or the short wavelength side displacement 15 nanometer the above, therefore, can be with the sea assaying the fluorescein of the kidney.

And, the 1 preferred embodiment, the compounds of this invention are stable compound, and the characteristic of having a low background. Specifically, the compounds of the present invention compared with the natural luciferin, background is reduced by about 5% or more.

In the present invention, for through and sea firefly fluorescein or its analogues and antioxidant, can make the stabilized sea firefly fluorescein or analogs thereof. Moreover, the presence of the antioxidant comprising the luciferase sea firefly and sea fireflysea firefly class fluorescein or the like, the measuring, the luminescent signal can be enhanced, and reduce the luminous background, than S/N can be greatly improved.

Antioxidant can enumerate: ascorbic acid or its salt, isoascorbic acid or its salt, dithionous acid or its salt, bisulfite, sulfites, pyrosulfites, metabisulphites, cysteine, thioglycerols, butyl hydroxy anisole, polyphenol, thiophosphoro sulfuric acid or its salt, such as alkali metal borohydride, the antioxidant can be used independently 1 or combined use of 2 or more kinds. Preferably can enumerate ascorbic acid, isoascorbic acid or its salt. The combined 2 when more antioxidant, preferably, the combined ascorbic acid and at least 1 kind of other antioxidant.

As a salt of ascorbic acid, isoascorbic acid salt, bisulfite, sulfites, pyrosulfites, metabisulphites, thiosulfate, can be enumerated: sodium, potassium, lithium, an alkali metal salt such as nuclide strontium, ammonium salt, calcium, magnesium and other alkali earth metal salt, and the like. Furthermore, an alkali metal borohydride can be enumerated: nabh (NaBH₄), boron potassium hydride, li-borohydride , such as hydroboration cesium.

The ascorbic acid is added in the light-emitting system when biological sea firefly , the adding concentration is preferably about 0.05M-about 1 meter

Fluorescein or its analogue comprising sea firefly and antioxidant compositions, in the fluorescein or its analogue sea firefly (solution or powder, particles, solid component crystallization, and the like) stabilization, preferably. In the composition, per 1 part by weight of fluorescein or its analogue sea firefly , mixed antioxidant about 40000-about 800000 parts by weight.

sea firefly of the present invention is adding fluorescein or its analogs in the system containing sea firefly fluorescein, in in such a system, mostly in about 0.1% to about 1% of albumin (HAS or BSA, in particular BSA) in the presence of a luciferase detection. The white protein to determine the background significantly increase in the system, but the presence of ascorbic acid in the assay system, when the reducing agent such as sodium sulfite, can remarkably inhibit the background caused by the rising of the albumin.

Therefore, containing sea firefly fluorescein or its analogues and antioxidant (in particular ascorbic acid, sulfite) composition, not only the fluorescein or its analogue sea firefly has a storage stability at room temperature, but also in determining the background caused by the rise in the albumin, therefore, is particularly preferred.

Embodiment

Next, based on the embodiment, more specifically represents invention.

Embodiment 1: formula (2A) compound (optically active 1, 1-diethoxy-3-methylglutaryl-2-one) of manufacturing

In the optical active S-2-methyl butyric acid (1g) the CH₂ Cl₂ solution, instillment thionyl chloride (1.2 milliliter). Directly on the stir at room temperature 24 hours. Concentrating the reaction liquid, the production of acid chlorides. For the 1 [...] 1 THF-acetonitrile of (15 milliliter) the acid chlorides dissolved, in 0 the add [...] 2M the TMSCHN₂ (10 milliliter), one hour after concentrating the solution. Using ethanol (10 milliliter) make this solution dissolving, in 0 the dropping under [...] tert-BuOCl (1 milliliter). In 0 the stirring the reaction liquid under [...] 1 hour. Concentrating the reaction liquid, with silica gel column to purify, get S-1, 1-diethoxy-3-methylglutaryl-2-one (0.6g, 39%).¹ H   NMR (500MHz, CD₃ Cl) 0.86 (3H, t, J=7Hz), 1.08 (3H, d, J=7Hz), 1.24 (6H, t, J=7Hz), 1.31-1.73 (2H, m), 2.93 (1H, q, J=7Hz), 3.51-3.71 (4H, m), 4.63 (1H, s); IR (KBr) 2955, 2871, 1727, 1486, 1367, 1120, 1063 centimeter^-1,_D²⁴ = + 22 (c   1.0 dichloromethane).

Embodiment 2: using the formula (2A) and formula (10) a compound of general formula (11) the compound of (fluorescein sea firefly optical activity)

In the eggplant-shaped flask (5 milliliter) compound is added in (10) (10 milligram), water (0.1 milliliter), 1, 1-diethoxy-3-methylglutaryl-2-one (0.02 milliliter), ethanol (0.1 milliliter), in 80 the heating [...] 30 minutes. Adding 48% HBr (0.02 milliliter), further heating 10 minutes, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (11) (7.1 milligram, 50%).¹ H   NMR (500MHz, CD₃ OD) 0.96 (3H, t, J=7Hz), 1.45 (3H, d, J=7Hz), 1.81-1.99 (2H, m), 2.33 (2H, quintet, J=7Hz), 3.17 (1H, sextet, J=7Hz), 3.41 (2H, q, J=7Hz), 3.44 (2H, q, J=7Hz), 7.18-7.21 (2H, m), 7.50 (1H, d, J=7Hz), 8.01 (1H, s), 8.07 (1H, d, J=7Hz), 8.44 (1H, s),_D²⁴ = + 22 (0.046, acetonitrile-water -10% TFA: 39-60-1), the obtained compound (11) is the same as the activity of the natural luciferin (test example 1).

Embodiment 3: using formula (12) and the formula (10) a compound of general formula (11) the compound of (fluorescein sea firefly optical activity)

In the eggplant-shaped flask (5 milliliter) compound is added in (10) (11 milligram), water (0.2 milliliter), glyoxal-secondary butyl (+) (0.01 milliliter), methanol (0.1 milliliter), the 65 the lower heating [...] 1 hour. After the return to the reaction solution at room temperature, adding a drop of 48% HBr of, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain 4 milligram compound (11) (31%).¹ H   NMR (500MHz, CD₃ OD) 0.96 (3H, t, J=7Hz), 1.45 (3H, d, J=7Hz), 1.81-1.99 (2H, m), 2.33 (2H, quintet, J=7Hz), 3.17 (1H, sextet, J=7Hz), 3.41 (2H, q, J=7Hz), 3.44 (2H, q, J=7Hz), 7.18-7.21 (2H, m), 7.50 (1H, d, J=7Hz), 8.01 (1H, s), 8.07 (1H, d, J=7Hz), 8.44 (1H, s).

Embodiment 4: general formula (13)-(16) of the compound

Compound (13) of synthetic.

The 2-amino -3, 5-dibromo-amino pyrazine (1g, 4mmol) and vinyl boronic acid reagent (0.8g) of the Alkanes 72 milliliter-saturated Na₂ CO₃ (24 milliliter) solution by adding Pd (PPh₃)₄ (50 milligram). In 80 the heating the mixture under [...] 1 hour. After cooling, the reaction mixture is diluted acetic acid ethyl ester, used for the washing of salt water, the sodium sulfate for the drying. Concentration is carried out after filtering, to obtain the crude extract. Purification of crude extract by chromatography, to obtain the desired product (450 milligram, 34%).¹ H   NMR (500MHz, CDCl₃), 1.44 (9H, s), 3.96 (2H, br), 4.69 (2H, br), 4.82 (1H, br), 6.40 (1H, d, J=15Hz), 6.87 (1H, br), 7.95 (1H, s).

Compound (14) synthesis of

The compound (13) (0.4g, 1mmol) and 1-(benzenesulfonyl)-3-indole boronic acid (0.73g, 2mmol) of the Alkanes 50 milliliter-saturated Na₂ CO₃ (24 milliliter) solution by adding Pd (PPh₃)₄ (30 milligram). In 80 the heating the mixture under [...] 1 hour. After cooling, the reaction mixture is diluted acetic acid ethyl ester, used for the washing of salt water, the sodium sulfate for the drying. Concentration is carried out after filtering, to obtain the crude extract. Purification of crude extract by chromatography, to obtain the desired product. For the resultant of the desired 8 milliliter methanol-b Dissolving alkane solution , adding 5N sodium hydroxide (2 milliliter). Stirred for a night at room temperature. After post-processing, purification of the mixture by chromatography, to obtain compound (14) (170 milligram, 46%).¹ H   NMR (500MHz, CDCl₃), 1.45 (9H, s), 4.04 (2H, br), 4.55 (2H, br), 4.80 (1H, br), 6.61 (1H, d, J=15Hz), 6.94 (1H, br), 7.21-7.27 (2H, m), 7.41 (1H, d, J=7Hz), 7.66 (1H, d, J=2.5Hz), 8.41 (1H, br), 8.35 (1H, s).

Compound (15) synthesis of

TFA (trifluoroacetic acid) solution for the compound (14) is dissolved, with 1 hour stirring and concentrated. Purification by chromatography of a mixed solution of concentrated, to obtain compound (15) (170 milligram).

¹ HNMR (500MHz, CD₃ OD) 3.85 (2H, d, J=7Hz), 6.98-7.17 (4H, m), 7.41 (1H, d, J=7Hz), 7.77 (1H, s), 8.28 (1H, d, J=7Hz), 8.34 (1H, s).

Compound (16) synthesis of

Compound (15) (150 milligram, 0 . 6mmol), pyrazole-1-methyl-amidine (175 milligram), DIEA (303 milligram) DMF solution of stirred for a night at room temperature. Adding ether to, get precipitation. Dissolving the precipitated with methanol, by recrystallization to obtain compound (16) (95 milligram, 53%).¹ H   NMR (500MHz, CD₃ OD) 4.20 (2H, d, J=7Hz), 7.05-7.17 (4H, m), 7.43 (1H, d, J=7Hz), 7.87 (1H, s), 8.12 (1H, d, J=7Hz), 8.08 (1H, s).

Embodiment 5: the general formula (16) with the formula (12) and the reaction of a compound of general formula (17) compounds

Compound (17) synthesis of

The compound is added in the eggplant-shaped flask (16) (7 milligram, 22mmol), water (0.1 milliliter), compound (12) (0.02 milliliter), ethanol (0.05 milliliter), in 100 the heating [...] 30 minutes. Adding 48% HBr (0.02 milliliter), further heating 10 minutes, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (17) (3.5 milligram, 39%).¹ H   NMR (500MHz, CD₃ OD) 0.96 (3H, t, J=7Hz), 1.44 (3H, d, J=7Hz), 1.81-1.99 (2H, m), 4.20 (2H, d, J=7Hz), 7.18-7.21 (4H, m), 7.42 (1H, d, J=7Hz), 7.94 (1H, s), 8.07 (1H, d, J=7Hz), 8.11 (1H, s).

Embodiment 6: general formula (10) the synthesis of the compounds of

Compound (18) synthesis of

The compound (13) (1g, 0.3mmol) dissolved in ethanol (50 milliliter) in, adding platinum oxide (100 milligram). Stirring vigorously under a hydrogen environment 24 hours. A diatomaceous earth pad to the reaction solution to filter and a glass filter. Concentrating the filtrate, and column chromatographying, get compound (18). (800 milligram, 79%)compound (18) the bussei:¹ H   NMR (500MHz, CDCl₃) 1.44 (9H, s), 2.03 (2H, d, J=7Hz), 2.80 (2H, d, J=7Hz), 3.28 (2H, d, J=7Hz), 7.95 (1H, s).

Compound (10) with the embodiment of for 4 said compound of (16) the method for manufacturing method for manufacturing the same. Compound (10) the bussei:¹ H   NMR (500MHz, CDCl₃) 2.03 (2H, d, J=7Hz), 2.80 (2H, d, J=7Hz), 3.28 (2H, d, J=7Hz), 7.08-7.16 (2H, m), 7.41 (1H, d, J=7Hz), 7.90 (1H, s), 8.30 (1H, s).

Embodiment 7: represented by the general formula (10) with a compound of formula (6) of the compound (19), (20), (21), (22), (23) reaction, are respectively synthesis of compound (24), (25), (26), (27), (28).

Compound (24) of synthetic: compound is added in the eggplant-shaped flask (10) (30 milligram), water (0.4 milliliter), 19 (0.030 milliliter), ethanol (0.2 milliliter), 49% HBr (0.03 milliliter), in 100 the heating [...] 1 hour. After return to the reaction solution at room temperature, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (24) (15 milligram, 38%).¹ H   NMR (500MHz, CD₃ OD) 2.33 (2H, quintet, J=7Hz), 2.54 (3H, s), 3.31 (2H, t, J=7Hz), 3.43 (2H, t, J=7Hz), 7.17-7.22 (2H, m), 7.47 (1H, d, J=7Hz), 7.98 (1H, s), 8.01 (1H, d, J=7Hz), 8.31 (1H, s); 1R (KBr) 3376, 3175, 1661, 1550, 1454 centimeter^-1.

Compound (25) can be used with compound (24) a method of manufacturing method of the same. Compound (25) the bussei:¹ H   NMR (500MHz, CD₃ OD) 1.41 (3H, t, J=7Hz), 2.33 (2H, quintet, J=7Hz), 2.95 (2H, q, J=7Hz), 3.35 (2H, t, J=7Hz), 3.43 (2H, t, J=7Hz), 7.16-7.22 (2H, m), 7.47 (1H, d, J=7Hz), 7.99 (1H, s), 8.02 (1H, d, J=7Hz), 8.35 (1H, s); IR (KBr) 3376, 3175, 1661, 1550, 1454 centimeter^-1.

Compound (26) can be used with compound (24) a method of manufacturing method of the same. Compound (26) the bussei:¹ H   NMR (500MHz, CD₃ OD) 1.50 (6H, t, J=7Hz), 2.29 (2H, quintet, J=7Hz), 3.32 (1H, sextet, J=7Hz), 3.37 (2H, q, J=7Hz), 3.43 (2H, q, J=7Hz), 7.11-7.14 (2H, m), 7.40 (1H, d, J=7Hz), 7.96 (1H, s), 7.94 (1H, d, J=7Hz), 8.22 (1H, s); IR (KBr) 3376, 3175, 1655, 1548, 1452 centimeter^-1.

Compound (27) can be used with compound (24) a method of manufacturing method of the same. Compound (27) the bussei:¹ H   NMR (500MHz, CD₃ OD) 1.03 (6H, d, J=7Hz), 2.18 (1H, m), 2.33 (2H, quintet, J=7Hz), 2.78 (2H, d, J=7Hz), 3.35 (2H, t, J=7Hz), 3.43 (2H, t, J=7Hz), 7.16-7.22 (2H, m), 7.47 (1H, d, J=7Hz), 7.99 (1H, s), 8.04 (1H, d, J=7Hz), 8.36 (1H, s); 1R (KBr) 3376, 3175, 1661, 1550, 1454 centimeter^-1.

Compound (28) can be used with compound (24) a method of manufacturing method of the same. Compound (28) the bussei:¹ H   NMR (500MHz, CD₃ OD) 2.27 (2H, quintet, J=7Hz), 2.99 (2H, t, J=7Hz), 3.41 (2H, t, J=7Hz), 7.14-7.22 (2H, m), 7.43 (1H, d, J=7Hz), 7.87 (1H, s), 8.02 (1H, s), 8.06 (1H, d, J=7Hz).

Embodiment 8

Compound (30) synthesis of: compound is added in the eggplant-shaped flask (10) (10 milligram), water (0.1 milliliter), 29 (0.020 milliliter), ethanol (0.1 milliliter), 49% HBr (0.01 milliliter), in 100 the heating [...] 1 hour. After return to the reaction solution at room temperature, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (30) (6 milligram, 42%).¹ H   NMR (500MHz, CD₃ OD) 2.24 (2H, quintet, J=7Hz), 3.25 (2H, t, J=7Hz), 3.39 (2H, t, J=7Hz), 4.27 (2H, s), 7.10 (2H, dd, J=7Hz), 7.24 (1H, t, J=7Hz), 7.30-7.45 (5H, m), 7.93 (1H, dd, J=7Hz), 7.96 (1H, s), 8.27 (1H, s).

Compound (32) synthetic: compound is added in the eggplant-shaped flask (10) (10 milligram), water (0.1 milliliter), 31 (0.020 milliliter), ethanol (0.1 milliliter), 49% HBr (0.01 milliliter), in 100 the heating [...] 1 hour. After return to the reaction solution at room temperature, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (32) (5.0 milligram, 34%).¹ H   NMR (500MHz, CD₃ OD) 1.84 (3H, d, J=7Hz), 2.29 (2H, quintet, J=7Hz), 3.38 (2H, t, J=7Hz), 3.42 (2H, t, J=7Hz), 4.60 (1H, q, J=7Hz), 7.16 (2H, dd, J=7Hz), 7.24 (1H, t, J=7Hz), 7.32 (2H, t, J=7Hz), 7.40 (2H, d, J=7Hz), 7.43 (1H, dd, J=7Hz), 7.97 (1H, dd, J=7Hz), 7.98 (1H, s), 8.27 (1H, s).

Compound (34) synthetic: compound is added in the eggplant-shaped flask (10) (10 milligram), water (0.1 milliliter), 33 (0.020 milliliter), ethanol (0.1 milliliter), 49% HBr (0.01 milliliter), in 100 the heating [...] 1 hour. After return to the reaction solution at room temperature, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (32) (5.2 milligram, 35%).¹ H   NMR (500MHz, CD₃ OD) 0.99 (3H, t, J=7Hz), 1.84 (3H, d, J=7Hz), 2.32 (4H, m), 3.41 (2H, t, J=7Hz), 3.44 (2H, q, J=7Hz), 4.29 (1H, t, J=7Hz), 7.16-7.23 (3H, m), 7.30 (2H, t, J=7Hz), 7.44 (2H, d, J=7Hz), 7.47 (1H, dd, J=7Hz), 7.95 (1H, dd, J=7Hz), 7.97 (1H, s), 8.34 (1H, s).

Compound (35) synthetic: the compound (13) (0.2g, 0 . 6mmol) and 2-naphthyl boronic acid (103 milligram, 0 . 6mmol) of the Alkanes 50 milliliter-saturated Na₂ CO₃ (24 milliliter) solution by adding Pd (PPh₃)₄ (40 milligram). In 80 the heating the mixture under [...] 1 hour. After cooling, the reaction mixture is diluted acetic acid ethyl ester, used for the washing of salt water, the sodium sulfate for the drying. Concentration is carried out after filtering, to obtain the crude extract. Purification of crude extract by chromatography, to obtain the desired product. With 1 milliliter TFA (trifluoroacetic acid) solution of the obtained compound is dissolved, the stirring 1 hour and concentrated. Further by 3.5 milliliter DMF the trifluoroacetic acid salt of compound is dissolved, add pyrazole-1-methyl-amidine (280 milligram), DIEA (301 milligram), stirred for a night at room temperature. Adding ether to, get precipitation. Dissolving the precipitated with methanol, and recrystallization, get compound (35) (135 milligram, 70%).¹ H   NMR (500MHz, CD₃ OD) 2.21 (2H, quintet, J=7Hz), 2.90 (2H, t, J=7Hz), 3.37 (2H, t, J=7Hz), 7.46 (2H, d, J=7Hz), 7.50 (1H, d, J=7Hz), 7.86 (2H, d, J=7Hz), 8.01 (1H, dd, J=7Hz), 8.30 (1H, s), 8.37 (1H, s).

Compound (36) synthesis of: compound is added in the eggplant-shaped flask (35) (10 milligram), water (0.1 milliliter), compound (2A) (0.020 milliliter), ethanol (0.1 milliliter), 49% HBr (0.01 milliliter), in 100 the heating [...] 1 hour. After return to the reaction solution at room temperature, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (36) (3.5 milligram, 27%).¹ H NMR (500MHz, CD₃ OD) 0.96 (3H, t, J=7Hz), 1.47 (3H, d, J=7Hz), 1.86 (2H, m), 2.35 (2H, quintet, J=7Hz), 3.19 (1H, m), 3.43 (2H, t, J=7Hz), 3.48 (2H, t, J=7Hz), 7.55 (2H, d, J=7Hz), 7.90 (1H, d, J=7Hz), 8.01 (2H, d, J=7Hz), 8.14 (1H, d, J=7Hz), 8.60 (1H, s), 8.78 (1H, s).

Compound (37) synthetic: compound is added in the eggplant-shaped flask (16) (10 milligram), water (0.1 milliliter), compound 33 (0.020 milliliter), ethanol (0.1 milliliter), 49% HBr (0.01 milliliter), in 100 the heating [...] 1 hour. After return to the reaction solution at room temperature, reducing concentrated. Dissolve the residue with ethanol, using LH-20 column to carry out the purification, to obtain compound (37) (4.4 milligram, 30%).¹ H NMR (500MHz, CD₃ OD) 1.83 (3H, d, J=7Hz), 4.20 (2H, d, J=7Hz), 4.60 (1H, q, J=7Hz), 7.18-7.32 (8H, m), 7.42 (1H, d, J=7Hz), 7.94 (1H, s), 8.07 (1H, d, J=7Hz), 8.11 (1H, s).

Test example 1: fluorescent sea firefly determining spectrum in other words, a light-emitting

In 0.1 milliliter of 50 mm (pH7.5) buffer   Tris-HCl in, the luciferase sea firefly (final concentration 100ng/ml) and sea firefly with olefin is fluorescein or fluorescein analogs sea firefly (compound (17)) mixed, measuring and accumulating 30 seconds of the light-emitting spectrum, of the results of the, compound (17) the light-emitting of the largest natural fluorescein compared with the wavelength, the long wavelength side displacement about 15 nanometer.

Test example 2: fluorescence sea firefly in other words, the determination of the light-emitting spectrum

In 0.1 milliliter of 50 mm (pH7.5) buffer   Tris-HCl in, the luciferase sea firefly (final concentration 100ng/ml) and model sea firefly fluorescein analogs (compounds (30), (32), (34), (36), (37)) mixed, measuring luminous spectrum (Figure 2).

Results can be known, the fluorescein sea firefly 2 with benzyl on the compound (30) and 2 has on the [...] 6 has on the naphthyl compound (36) is, biological light-emitting wavelength of the natural fluorescein (11) compared, to the short wavelength displacement; 2 with branched chain containing on the benzyl compound (32), (34) with the natural fluorescein (11) compared, to long wavelength displacement. Control can not be realized in the past can be controlled by the light-emitting wavelength of the luciferase sea firefly. Especially compound (36) of the light-emitting, the maximum wavelength of ultraviolet region to prominently (390 nanometer) displacement, and can display the different light-emitting system of the light-emitting color. In the light-emitting the largest wavelength 390 nanometer is, almost not by the sea kidney fluorescein (blue) or firefly fluorescein enzyme (from yellow-green to red) of the light-emitting of the light interference, therefore, not to use filter light-emitting can be measured. Furthermore, because this compound (36) with the largest wavelength of the green fluorescent protein, yellow fluorescent protein fluorescence maximum wavelength (stokes   shift) the difference is large, therefore, if using the substrate, the light-emitting of the luciferase sea firefly the fluorescent protein to the fluorescence maximum wavelength of the light interference is reduced, therefore, is suitable for utilizing sea firefly luciferase and fluorescent protein from protein fusion body to carry out quantitative treatment of the peptide.

Embodiment 9

In containing or not containing 0.3M sodium ascorbate 0.1 milliliter of the 100 mm buffer   Tris-HCl (pH7.5) or 0.1 milliliter of the 100 mm buffer   Tris-HCl (pH7.5)/ 0.3M in the NaCl, mixins sea firefly fluorescein (final concentrations of the 1  M), measuring 10 seconds of optimizing the (background), then adding sea firefly (final concentration 5ng/ml) luciferase, measuring 10 seconds of optimizing the generated by luciferase.

The results are shown in table 1.

Moreover, the room temperature measured 24 hours after the activity of the fluorescein. The results are shown in table 2.

As shown in table 1 illustrated, the buffer system containing ascorbic acid sodium salt, at least, to improve S/N 16 times.

Furthermore, the table 2 is shown, containing the luciferin 0.3M ascorbic acid sodium salt of 0.1 milliliter of 100 mm (pH7.5) buffer   Tris-HCl in, even at the room temperature to be stable.

[Table 1] after just adding fluorescein sea firefly the measured value of

[Table 2]

Adding sea firefly fluorescein 24 hours after the measured value of

Embodiment 10: more than 24 hours at room temperature of the preservation of sea firefly fluorescein

In containing 0.3M sodium ascorbate and 0.02M of sodium sulfite is 100 mm (pH7.5) buffer   Tris-HCl in, mixins sea firefly fluorescein (final concentrations of the 1  M) and sea firefly (final concentration 5ng/ml) luciferase, measuring 10 seconds of optimizing the generated by luciferase. Moreover, the measuring of the fluorescein in at room temperature 24 hours after the biological of the light-emitting active. The results are shown in table 3.

[Table 3]

The table 3 is shown, containing fluorescein, 0.3M ascorbic acid sodium salt and 0.02M the Na₂ SO₃ of 0.1 milliliter of the 100 mm buffer   Tris-HCl (pH7.5) and the separate 0.3M ascorbic acid sodium salt (embodiment 9 of the 24 hours later, table 2) compared with the, further improved the stability of the room temperature.

Embodiment 11: the use of ascorbic acid as an antioxidant BSA sodium and sodium sulfite reducing the background

Under the condition of without antioxidant, containing the 0.3M sodium ascorbate, 0.3M sodium ascorbate and 0.02M the Na₂ SO₃ of 100 mm   Tris-HCl buffer/1% BSA (p H7.5) in, mixins sea firefly fluorescein (final concentrations of the 1  M), measuring 10 seconds of optimizing the (background), then adding sea firefly (final concentration 5ng/ml) luciferase, measuring 10 seconds of optimizing the generated by luciferase. The results are shown in table 4.

[Table 4]

From table 4 can be known is shown, containing fluorescein, 0.3M ascorbic acid sodium salt and 0.02M the Na₂ SO₃ of 0.1 milliliter of the 100 mm buffer   Tris-HCl (pH7.5), caused by the BSA the luminous background is reduced by approximately 10 times, improving the ratio of S/N 14 times.

Industrial practicability

Fluorescein sea firefly of the present invention light-emitting matrix, and can be applied to the use of a luciferase sea fireflysea firefly fluorescein analogs (different light-emitting wavelength), such as double-report gene detection method. Specifically:

1. The use of light-emitting the maximum wavelength is 390-620nm, a determination condition does not depend on the light-emitting wavelength of the light 2 and the luciferase sea fireflysea firefly a luciferase and fluorescent protein gene fusions 2 gene transcription activity, for extracellular proteins are secreted into the measuring system.

2. Using sea fireflysea firefly fluorescein analogs and the luciferase reaction to reach the light-emitting, the maximum wavelength 390-520nm light-emitting reaction. If the fluorescein analogs sea firefly 36, the maximum light-emitting wavelength of about 390 nanometer (Figure 3A).

3. Luciferase and fluorescent protein using sea firefly and about half of the fluorescein analogs sea firefly the largest wavelength and the light-emitting reaction reaches 500-620nm light-emitting reaction. For example, if the luciferase sea firefly and GFP, is about 510 nanometer (Figure 3A).

4.2, 3 combined preferably with the maximum light-emitting wavelength difference of 100 nanometer or more, but if it is the 30 nanometer, can be color segmentation, double the report for gene detection.

5. sea firefly luciferase gene in the promoter upstream insertion A, the luciferase sea firefly and fluorescent protein promoter upstream insertion fusant B (Figure 3B), the 2 gene constructs in transfected in cell , cultured for certain time after the transfection, then to the recovery of the culture or culture plate in sea firefly fluorescein analogs directly added, color light filter used for separating, determining the amount of emitted light of the two kinds, and to determine the promoter A, the active amount B (because does not need to carry on cytoclastically, therefore can be directly movable cell measurement of gene transcription activity) (Figure 3C). A the active paste only promoter, can be observed only a maximum luminous wavelength 390 nanometer light-emitting, but with the activation of the promoter B, in 510 nanometer near peak can be observed, it increases. The light-emitting spectrum can be easily separated through the filter.