CN102766346A - Cyanine fluorescent dye - Google Patents
Cyanine fluorescent dye Download PDFInfo
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- CN102766346A CN102766346A CN201210265837XA CN201210265837A CN102766346A CN 102766346 A CN102766346 A CN 102766346A CN 201210265837X A CN201210265837X A CN 201210265837XA CN 201210265837 A CN201210265837 A CN 201210265837A CN 102766346 A CN102766346 A CN 102766346A
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- thiazolinyl
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Abstract
The invention discloses a cyanine fluorescent dye used for biological fluorescence labelling. The fluorescent dye has a general formula shown in formula I and the characteristics of little synthetic steps, mild reaction conditions, simple purification processing mode, high yield and high light stability. Also, the fluorescent dye has a certain abilities to permeate cell membrane and the characteristics of low fluorescence background and specific recognizability to nucleic acid at the same time. When used as a nucleic acid stain in a flow cytometer, the fluorescent dye can effectively reduce the interference of the background fluorescence and increase the accuracy of the detection. The fluorescent dye provided by the invention can be used as leucocyte and reticulocyte stains in the blood.
Description
Technical field
The present invention relates to a kind of cyanine fluorochrome that can be used for biological fluorescent labelling, specifically, the present invention relates to a kind of optical dye that can under mild reaction conditions, prepare, its preparation method and the purposes in biological sample dyeing.
Background technology
Optical dye has been widely used in life science and clinical diagnose.In flow cytometry, optical dye during through laser irradiation area, sends the scattered light of fluorescence and multi-angle with after nucleic acid substances in the cell combines.Through detection, can obtain about the structure of cell and the bulk information of physical and chemical parameter to these fluorescence and scattered light signal.
In the clinical diagnose, be divided into lymphocyte, neutrophil leucocyte, monocyte, eosinophilic granulocyte, basophilic granulocyte etc. to the normal white corpuscle in the human peripheral blood usually.White corpuscle is under pathologic condition, and the nucleic acid component of its form, quantitative proportion and cell interior all can great changes will take place.In addition, the red corpuscle in the peripheral blood of human body is generally the sophisticated red corpuscle that does not contain RNA, under pathologic condition, the reticulocyte of the immature RNA of containing can occur.DNA/RNA nucleic acid through the optical dye pair cell carries out specific stain, utilize detection technique of fluorescence effectively examination go out these abnormal cellss.
The early stage dyestuff of using comprises new methylene blue (NMB), Brilliant cresyl blue (BCB), sends the peaceful Y of promise, ethidium bromide etc.They form mixture through embedding or electrostatic attraction and nucleic acid molecule, thereby can examine under a microscope the fluorescence after the enhancing.But self also can form dye composition these dyestuffs, can not obviously be different from the mixture that dyestuff and nucleic acid form, and causes high fluorescence background to disturb.Simultaneously, the fluorescence quantum efficiency of these dyestuffs is lower, has reduced fluorescence enhanced degree, influences the accuracy of detected result.
USP 3883247 discloses a kind of white corpuscle five sorting techniques, and it is to dye through SP 15 Lemon Yellow dialogue nucleus, and difference red through different white corpuscle subclass and the green fluorescence amount comes white corpuscle is classified.The SP 15 Lemon Yellow that this method adopts can form mixture with nucleic acid, and fluorescence is strengthened.This type dye molecule may cause optical quenching each other in the process of transmission ofenergy, make the dyestuff nucleic acid complexes not have fluorescence to send, and influences the detected result verity.In stream type cell analyzer, SP 15 Lemon Yellow and plastic piping have stronger effect, can increase background fluorescence intensity simultaneously.In order to remove the residual dyestuff in test back and to guarantee precision of test result, need instrument to carry out long pipeline and clean, this has just reduced the analysis efficiency of instrument.
USP 4882284 discloses a kind of white corpuscle five sorting techniques; It is that anhemolytic method is sharp to dye to the various cells in the hemocyte with the oxazine fluorochrome through adopting, and forward scatter light, side scattered light and the red fluorescence intensity through different cells comes white corpuscle is classified then.The fluorescence quantum efficiency of these dyestuffs is lower, has reduced fluorescence enhanced degree, influences the accuracy of detected result, owing to need to measure red corpuscle, white corpuscle and thrombocyte, then some abnormal erythrocytes such as macroerythrocyte etc. can influence the white corpuscle accurate counting simultaneously.
The cyanine fluorochrome that uses at present is as having unsymmetrical cyanine dye TOTO (thiazole orange dimer), the YOYO of high affinity by the exploitations such as Glazer of the U.S. to dsDNA! oxazole Huang dimer), TOTAB, TOTIN (thiazole orange heterodimer), and [K.M. Sovenyhazy such as asymmetric single methylene radical cyanine dyes TO-PRO-3, PO-PRO-2 and BO-PRO-2 that derive on this basis; J.A. Bordelon, J.T. Petty. Nucleic Acids Res, 2003; 31; 2561], their absorption and emission are all in the near-infrared region (670-1000 nm), but the molecular structure of this type dyestuff is complicated; Synthetic route is longer; Purification process length consuming time and yield are lower, thereby expensive, have limited its use range.
Therefore need the new cyanine fluorochrome of exploitation; This fluorochrome has following characteristics: synthesis step is few, and synthesis condition is gentle, and the purification process mode is simple; Product yield is high; Light stability is high, has the penetrating ability of certain cytolemma, has low fluorescence background simultaneously and to the specific recognition capability of nucleic acid.
Summary of the invention
The object of the invention: a kind of cyanine fluorochrome of the present invention is the deficiency that exists to prior art, on its basis, repeatedly improve and test, and that a type of obtaining has a synthesis step is few; Synthesis condition is gentle; The purification process mode is simple, and product yield is high, and light stability is high; Have the penetrating ability of certain cytolemma, have low fluorescence background simultaneously and the cyanine fluorochrome of the specific recognition capability of nucleic acid.
Content of the present invention: the present invention has announced a kind of cyanine fluorochrome with structure general formula I:
Wherein
X is C (CH
3)
2, O or S;
R1 is selected from H, OH, C1-18 alkyl, phenyl;
R2 is selected from H, OH, NHR5, N (R5) 2;
R3 is selected from benzyl, C3-18 thiazolinyl, C3-18 alkynyl, and wherein following substituting group is optional to be replaced benzyl: halogen, hydroxyl, sulfydryl, cyanic acid, nitro, alkyl, aryl, alkoxyl group, heterocyclic radical, haloalkyl, amino, alkylamino, amido, carboxyl by being selected from;
R4 is selected from C3-18 thiazolinyl, C3-18 alkynyl;
R5 is selected from C1-18 alkyl, C3-18 thiazolinyl;
Y-is a negative ion.
Technical scheme of the present invention: one aspect of the present invention provides a kind of cyanine fluorochrome, and said optical dye can obtain through following step:
The compound dissolution of compound and general formula III that will have general formula I I is in the mixing solutions of pyridine/acetic anhydride, and stirring reaction is 2 ~ 10 hours under the normal temperature, promptly obtains cyanine fluorochrome.
The inventor finds in research process; Can increase its reactive behavior through substituted radical among adjustment general formula compound II and the III; In pyridine/acetic anhydride solution, can react smoothly under the room temperature; And greatly reduce the formation of impurity, simplified the difficulty of purification process, as long as through the repeatedly operation of washing and recrystallization.
The present invention also provides a kind of conjugate, said conjugate to comprise above-mentioned formula I compound on the one hand.
Another aspect of the present invention also is provided for the compsn of biological sample dyeing, and said compsn comprises above-mentioned formula I compound or its conjugate.
Further aspect of the present invention also provides formula I compound of the present invention or its conjugate or the purposes of compsn in biological sample is dyeed.
Compound of the present invention, conjugate or compsn have good dyeing to biological sample such as nucleic acid, white corpuscle, reticulocyte etc.; The mixture emission wavelength of its formation is in the near-infrared region; Avoided the fluorescence background of biology self to disturb; Help to improve the tolerance range of detected result, can on stream type cell analyzer, be used as the staining agent of various biological samples simultaneously.
Beneficial effect of the present invention: the synthesis step of cyanine fluorochrome described in the present invention is few, and synthesis condition is gentle, and the purification process mode is simple; Product yield is high; Light stability is high, has the penetrating ability of certain cytolemma, has low fluorescence background simultaneously and to the specific recognition capability of nucleic acid.
These feature and advantage of the present invention and other feature and advantage will become obvious after with reference to following accompanying drawing and embodiment of the present invention.
Description of drawings
Fig. 1 is to use one embodiment of the invention I as Arneth's count reagent, the scatter diagram that the lateral scattering light intensity and the fluorescence intensity signals of mensuration blood sample generated.
Fig. 2 is to use one embodiment of the invention II as Arneth's count reagent, the scatter diagram that the lateral scattering light intensity and the fluorescence intensity signals of mensuration blood sample generated.
Fig. 3 is to use one embodiment of the invention III as Arneth's count reagent, the scatter diagram that the lateral scattering light intensity and the fluorescence intensity signals of mensuration blood sample generated.
Fig. 4 is to use one embodiment of the invention IV conduct as reticulocyte determination reagent, the scatter diagram that the lateral scattering light intensity and the fluorescence intensity signals of mensuration blood sample generated.
Embodiment
Definition
Unless otherwise indicated, the term that uses among this paper has following implication.
No matter the term that uses among this paper " alkyl " is to use separately or be used in combination with other groups, refers to comprise 1-18, preferred 1-12, more preferably 1-8, the most preferably straight chained alkyl and the branched-chain alkyl of 1-6 carbon atom.As mention single alkyl like " n-propyl ", and then only refer in particular to straight chained alkyl, as mention single branched-chain alkyl like " sec.-propyl ", then only refer in particular to branched-chain alkyl.For example, " C1-6 alkyl " comprises C1-4 alkyl, C1-3 alkyl, methyl, ethyl, n-propyl, sec.-propyl and the tertiary butyl.Similarly rule also is applicable to other group that uses in this specification sheets, such as the definition for thiazolinyl and alkynyl.
The term that uses among this paper " halogen " comprises fluorine, chlorine, bromine and iodine.
The term that uses among this paper " biological sample " includes but not limited to white corpuscle, reticulocyte in nucleic acid, the blood etc.
Compsn of the present invention
The present invention also provides the compsn that comprises above-mentioned formula I compound or its conjugate, and said compsn is used for the dyeing of biological sample.
Compsn of the present invention also can comprise needed other component of biological sample dyeing, for example solvent, osmotic pressure regulator, pH regulator agent, tensio-active agent etc. except that comprising formula I compound or its conjugate.Compsn of the present invention can be used as aqueous solution form and exists, and perhaps can be used as to face other suitable form existence that is formulated as solution with suitable solvents with preceding.
Preparation and the purifying of embodiment 1 cyanine fluorochrome I
3-benzyl-5-hydroxyl-2-(2-(N-phenylacetylamino)-vinyl) the benzothiazole bromine salt and 4.1mmol 7-Propylamino-4-methyl isophthalic acid-(propenyl) quinoline bromine salt that in the 50mL of dried and clean there-necked flask, add 4.0mmol; To wherein adding 10ml pyridine and 2ml acetic anhydride; Magnetic agitation reaction at room temperature stopped after 4.5 hours, obtained the bullion of reaction product.This crude product poured in the anhydrous diethyl ether stir, product is separated out.With washing with 3 * 25ml ETHYLE ACETATE after the filtration of crude product, then with obtaining compound shown in the title after an amount of recrystallizing methanol.Product yield 72%.
Maximum absorption band: 630nm (methyl alcohol/terepthaloyl moietie)
MS?(EI)?C
32H
32BrN
3OS m/z:?506.6?[M-Br]
?+。
Preparation and the purifying of embodiment 2 cyanine fluorochrome II
3-(hexene-2-yl)-5-hydroxyl-2-(2-(N-phenylacetylamino)-vinyl) benzothiazole bromine salt and 4.1mmol 7-butylamine base-4-methyl isophthalic acid-(hexene-2-yl) quinoline bromine salt of in the 50mL of dried and clean there-necked flask, adding 4.0mmol; To wherein adding 10ml pyridine and 3ml acetic anhydride; Magnetic agitation reaction at room temperature stopped after 5 hours, obtained the bullion of reaction product.This crude product poured in the anhydrous diethyl ether stir, product is separated out.With washing with 3 * 25ml ETHYLE ACETATE after the filtration of crude product, then with obtaining compound shown in the title after an amount of recrystallizing methanol.Product yield 65%.
Maximum absorption band: 632nm (methyl alcohol/terepthaloyl moietie)
MS?(EI)?C
33H
44BrN
3OS m/z:?539.3?[M-Br]
?+。
Preparation and the purifying of embodiment 3 cyanine fluorochrome III
The 6-hydroxyl-3 that in the 50mL of dried and clean there-necked flask, adds 4.0mmol; 3-dimethyl--2-(2-(N-phenylacetylamino)-vinyl)-1-(propine-2-yl)-benzothiazole salt compounded of iodine and 4.1mmol 7-N; TMSDEA N diethylamine base-4-methyl isophthalic acid-benzyl-quinoline salt compounded of iodine; To wherein adding 10ml pyridine and 0.8ml acetic anhydride, magnetic agitation reaction at room temperature stopped after 8 hours, obtained the bullion of reaction product.This crude product poured in the anhydrous diethyl ether stir, product is separated out.With washing with 3 * 25ml ETHYLE ACETATE after the filtration of crude product, then with obtaining compound shown in the title after an amount of recrystallizing methanol.Product yield 78%.
Maximum absorption band: 635nm (methyl alcohol/terepthaloyl moietie)
MS?(EI)?C
36H
38IN
3O m/z:?528.7?[M-I]
?+。
Preparation and the purifying of embodiment 4 cyanine fluorochrome IV
3-benzyl-5-ethyl-the 2-(2-(N-phenylacetylamino)-vinyl) benzoxazole bromine salt and 4.1mmol 7-hydroxy-4-methyl-1-(heptenyl) quinoline bromine salt that in the 50mL of dried and clean there-necked flask, add 4.0mmol; To wherein adding 10ml pyridine and 1ml acetic anhydride; Magnetic agitation reaction at room temperature stopped after 4 hours, obtained the bullion of reaction product.This crude product poured in the anhydrous diethyl ether stir, product is separated out.With washing with 3 * 25ml ETHYLE ACETATE after the filtration of crude product, then with obtaining compound shown in the title after an amount of recrystallizing methanol.Product yield 73%.
Maximum absorption band: 627nm (methyl alcohol/terepthaloyl moietie)
MS?(EI)?C
35H
37BrN
2O
2 m/z:?517.6?[M-Br]
?+。
Measure the blood that adds 20uL process EDTA.2K anti-freezing processing in the reagent at the 1mL leukocyte differential count; Add the optical dye I of 20uL 20ppm then, be modulated into specimen, lucifuge was hatched 10 minutes; Suck stream type cell analyzer through appropriate reconstruction; Collect 5000 leukocytic red fluorescences and side scattered light intensity signal, the result is as shown in Figure 1, and white corpuscle is divided into four types of lymphocyte, monocyte, neutrophil leucocyte and eosinophilic granulocytes.
Embodiment 6 cyanine fluorochrome II are as the leukocyte differential count test agent
Measure the blood that adds 20uL process EDTA.2K anti-freezing processing in the reagent at the 1mL leukocyte differential count; Add the optical dye II of 20uL 20ppm then, be modulated into specimen, lucifuge was hatched 10 minutes; Suck stream type cell analyzer through appropriate reconstruction; Collect 5000 leukocytic red fluorescences and side scattered light intensity signal, the result is as shown in Figure 2, and white corpuscle is divided into four types of lymphocyte, monocyte, neutrophil leucocyte and eosinophilic granulocytes.
Embodiment 7 cyanine fluorochrome III test test agent as leukocyte differential count
Measure the blood that adds 20uL process EDTA.2K anti-freezing processing in the reagent at the 1mL leukocyte differential count; The optical dye III that adds 20uL 20ppm then; Be modulated into specimen, suck stream type cell analyzer, collect 5000 leukocytic red fluorescences and side scattered light intensity signal through appropriate reconstruction; The result is as shown in Figure 3, and white corpuscle is divided into four types of lymphocyte, monocyte, neutrophil leucocyte and eosinophilic granulocytes.
Embodiment 8 cyanine fluorochrome IV knit red test agent as net
In 3mL reticulocyte determination reagent, add the blood that 5uL handles through antithrombotics; The optical dye IV that adds 30uL 50ppm then; Be modulated into specimen, lucifuge was hatched 10 minutes, sucked the stream type cell analyzer through appropriate reconstruction; Collect the red fluorescence and the side scattered light intensity signal of 100,000 cells, the result is as shown in Figure 4.According to different fluorescence intensities, can distinguish the reticulocyte of high fluorescent and the mature erythrocyte of low fluorescence intensity.
Through above-mentioned each embodiment and specific embodiment the present invention is made an explanation, but skilled person in the art will appreciate that under the situation that does not depart from aim of the present invention and scope, can make various modifications, change and replacement the present invention.
Claims (13)
1. cyanine fluorochrome, its general structure is shown in I:
Wherein
X is C (CH
3)
2, O or S;
R1 is selected from H, OH, C1-18 alkyl, phenyl;
R2 is selected from H, OH, NHR5, N (R5) 2;
R3 is selected from benzyl, C3-18 thiazolinyl, C3-18 alkynyl, and wherein following substituting group is optional to be replaced benzyl: halogen, hydroxyl, sulfydryl, cyanic acid, nitro, alkyl, aryl, alkoxyl group, heterocyclic radical, haloalkyl, amino, alkylamino, amido, carboxyl by being selected from;
R4 is selected from C3-18 thiazolinyl, C3-18 alkynyl;
R5 is selected from C1-18 alkyl, C3-18 thiazolinyl;
Y-is a negative ion.
2. the compound of claim 1, wherein X is C (CH
3)
2, O or S.
3. each compound in the claim 1 ~ 2, wherein R1 is selected from H, OH, C1-18 alkyl, phenyl.
4. each compound in the claim 1 ~ 3, wherein R2 is selected from H, OH, NHR5, N (R5) 2.
5. each compound in the claim 1 ~ 4; Wherein R3 is selected from benzyl, C3-18 thiazolinyl, C3-18 alkynyl, and wherein following substituting group is optional to be replaced benzyl: halogen, hydroxyl, sulfydryl, cyanic acid, nitro, alkyl, aryl, alkoxyl group, heterocyclic radical, haloalkyl, amino, alkylamino, amido, carboxyl by being selected from.
6. each compound in the claim 1 ~ 5, wherein R4 is selected from C3-18 thiazolinyl, C3-18 alkynyl.
7. each compound in the claim 1 ~ 6, wherein R5 is selected from C1-18 alkyl, C3-18 thiazolinyl.
8. each compound in the claim 1 ~ 7, wherein Y-is halide-ions, ClO
4 -, PF
6 -, CF
3SO
3 -, BF
4 -, acetate moiety or tosic acid negative ion.
9. each compound among the claim 1-8, wherein said compound is:
10. compsn that is used for biological sample dyeing, wherein said compsn comprise among the claim 1-9 each compound.
11. the compsn of claim 10, wherein said biological sample are selected from the white corpuscle in nucleic acid, the blood, the reticulocyte in the blood.
12. leukocytic method in the analyzing blood, this method comprises:
1) blood sample to be measured is dyeed with each compsn among the claim 1-9;
2) white corpuscle in the blood sample of analysis dyeing back on stream type cell analyzer.
13. the method for reticulocyte in the analyzing blood, this method comprises:
1) with blood sample to be measured with among the claim 1-9 each compsn dyeing;
2) reticulocyte in the blood sample of analysis dyeing back on stream type cell analyzer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105026928A (en) * | 2013-02-28 | 2015-11-04 | 希森美康株式会社 | Urine sample analysis device and urine sample analysis method |
| CN109030156A (en) * | 2018-10-19 | 2018-12-18 | 武汉百合龙腾生物科技有限责任公司 | A kind of flow cytometer showed dyeing liquor |
| CN114437057A (en) * | 2020-10-30 | 2022-05-06 | 深圳市瑞图生物技术有限公司 | Fluorescent dye and preparation method and application thereof |
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