CN105754586A - Fluorescein diacetate fluorescence probe containing aldehyde group structure and preparation method and application thereof - Google Patents

Fluorescein diacetate fluorescence probe containing aldehyde group structure and preparation method and application thereof Download PDF

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CN105754586A
CN105754586A CN201610187121.0A CN201610187121A CN105754586A CN 105754586 A CN105754586 A CN 105754586A CN 201610187121 A CN201610187121 A CN 201610187121A CN 105754586 A CN105754586 A CN 105754586A
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fluorescein
fluorescence probe
fda
preparation
probe
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于海波
李谷丽
宋有涛
方培菊
吕春娇
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Liaoning University
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    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract

The invention discloses a fluorescein diacetate fluorescence probe containing an aldehyde group structure and a preparation method and application thereof.The fluorescence probe is A-FDA, and the structural formula of A-FDA is shown in the formula (I).The A-FDA fluorescence probe is applied to indication for the activity of hydrolytic enzymes in living cells.By means of an aldehyde group in the probe, the lipophicity of the probe is improved, the specific selectivity of the probe is enhanced, reverse osmosis of hydrolyzed fluorescein generated after the probe enters the cells is relieved, and meanwhile good chemical stability, light stability, solubility and biocompatibility are achieved.A microimaging experiment shows that the probe has good cell targeting and permeability and has no toxic or side effect on the cells and organisms.The structural formula is shown in the description.

Description

A kind of fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure and preparation method thereof And application
Technical field
The present invention relates to a kind of fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure and its preparation method and application.
Background technology
The fluorophor of fluorescein is the fluorescent derivatizing agent of modal covalent labeling albumen.Fluorescein(e) diacetate (FDA) hydrolysis is widely accepted as a method accurately and simply measuring total microbial activity.This method has Simplicity, quickly, sensitive advantage, and fluorescein(e) diacetate (FDA) hydrolysis be widely used in as weigh total microorganism live The instruction of property.For plant cell dyeing, measure the researchs such as the viability of pollen and isolated cells viability examination, therefore cell Fluorescent staining mark is widely used.Fluorescein(e) diacetate (FDA) is the diacetate esters of fluorescein, and lipophilicity is higher than fluorescence Element, is easily absorbed by cell through cell membrane.The fluorescence of fluorescein(e) diacetate (FDA) itself is more weak, but enters intracellular Fluorescein(e) diacetate (FDA) becomes to have the fluorescein of fluorescence through lipase hydrolysis, makes cell produce fluorescence, can be used for studying cell special Property and cell growing environment.Fluorescein(e) diacetate (FDA) can pass through cell membrane and accumulates in living cells as fluorescein.By Low compared with the hydrophily of BCECF or calcein in fluorescein.
Must possess as viable cell labelling probe: (1) probe can specifically be combined with intracellular specific substrates；(2) visit Pin mark note product signal is strong, can distinguish unlabeled cells；(3) probe is to target cell not damaged；(4) background fluorescence interference is little, I.e. fluorescence-causing substance seldom leaks out from intracellular.Fluorescein(e) diacetate, Aminofluorescein and isothiocyanates fluorescein etc. were once For marking living cells, but the leakage problems of fluorescence-causing substance is not well solved.Therefore it is badly in need of research and development and there is special selection The fluorescein(e) diacetate of property.
Summary of the invention
The present invention provides a kind of fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure and preparation method thereof and conduct To the application of hydrolytic enzyme activities instruction in living cells.This fluorescence probe not only improves its lipophilicity but also enhances specific selectivity, Reduce the counter-infiltration hydrolyzing fluorescein after entering cell, there is preferable chemistry, photostability, preferable dissolubility and life simultaneously Thing is compatible.Micro-imaging experiment shows that this kind of probe has the most cell targeted, permeability, to cell and organism without Toxic and side effect.
The technical solution used in the present invention is:
A kind of fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure, described fluorescence probe is A-FDA fluorescence probe, The structural formula of A-FDA fluorescence probe is as shown in (I):
The preparation method of the described fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure, comprise the steps: by Fluorescein list aldehyde and potassium carbonate join in ether, add dichloromethane so that dissolving；Drip appropriate acetic anhydride, under room temperature, magnetic Power stirring reaction 6h；Reactant is carried out suction filtration, takes off filtrate, be spin-dried for, purify through silica gel column chromatography, obtain white solid i.e. mesh Mark product A-FDA fluorescence probe.Described dichloromethane addition is that 1-6 drips.The addition of described acetic anhydride is 3-8ml.
Described fluorescein list aldehyde is 1:(1-3 with the mol ratio of potassium carbonate with ether): (2-4).
The synthesis of described fluorescein list aldehyde: take fluorescein in flask, adds chloroform and 15-crown-5 in flask, to Flask is slowly added dropwise NaOH solution, oil bath 55 DEG C, after reaction 7h, take product, carry out being acidified, suction filtration, be dried, cross post and purify To fluorescein list aldehyde.
Prepare reaction equation as follows:
A kind of fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure is as the application of fluorogenic hydrolase probe, mainly For detecting the instruction of living cells enzyme.The fluorescence probe of the present invention can be used for several samples, such as soil, compost sample microbial activity Analysis.
The method have the advantages that
Aldehyde radical in the fluorescent probe molecule of the present invention can improve its lipophilicity and specific selectivity, reduces entrance simultaneously Hydrolyze the counter-infiltration of fluorescein after cell, be applied to the detection of cytoactive, optimize analysis condition.It addition, this probe also has Preferably chemical stability, preferable dissolubility and bio-compatibility, not disturbing by species such as other common metal ions.
Accompanying drawing explanation
Fig. 1 is the A-FDA of the embodiment 1 preparation fluorescent microscopic imaging to living cells.
Fig. 2 is A-FDA in the cell fluorescence response of the time of staying.
Fig. 3 is A-FDA fluorescence response collection of illustrative plates of hydrolysis degree when different pH.
Fig. 4 is the collection of illustrative plates of the A-FDA mensuration Soil hydrolase of embodiment 2.Ab1: for the suction of the material that soil self produces Receive collection of illustrative plates；Ab2The absorption collection of illustrative plates hydrolyzed under the conditions of pH is 7.4 for A-FDA；Ac1In the presence of the while of A-FDA and soil Absorb collection of illustrative plates
Detailed description of the invention
The preparation of the embodiment 1 one kinds fluorescein(e) diacetate fluorescence probe (A-FDA fluorescence probe) containing aldehyde radical structure Method
1mol fluorescein list aldehyde and 2mol potassium carbonate being joined in 3mol ether, adding 3 dichloromethane so that dissolving, Dropping 5ml acetic anhydride, under room temperature, magnetic agitation reaction 6h, reactant is carried out suction filtration, takes off filtrate, be spin-dried for, through silica gel column layer Analysis purifies, and obtains white solid i.e. target product A-FDA fluorescence probe.
HRMS:444.0845.
Prepare reaction equation as follows:
Wherein, fluorescein list aldehyde synthetic method is: take 2g fluorescein in flask, add in flask 12ml chloroform, 0.4ml 15-crown-5, is being slowly added dropwise NaOH solution in flask, oil bath 55 DEG C, after reaction 7h, takes product, carries out being acidified, taking out Filter, be dried, cross post purify obtain fluorescein list aldehyde.
[1] A-FDA fluorescence response of hydrolysis degree when different pH
Take 0.0444g A-FDA in 5ml volumetric flask, add CHCl2 and dissolve, be settled to 5ml, obtain the A-of 2 × 10-5M FDA mother liquor.Take 0.05mlFDA, A-FDA mother liquor respectively in cuvette, add 3ml methyl alcohol, stir, wait 5min so that it is be fully hydrolyzed, tests fluorescence intensity.As seen from Figure 3, increase along with pH when pH is 1～3, fluorescence intensity It is held essentially constant.Have slight change when pH scope is 4-7, as pH > 9 time, fluorescence intensity is remarkably reinforced, and along with pH increases And constantly increase.
[2] fluorescent microscopic imaging
Carrying out contrast test, Hela cell line or nematode are dyeed by one group with A-FDA solution, and another group is blank test. To containing in the culture dish that living cells is Hela cell line or nematode, adding concentration is 5 × 10-5The dimethyl of the Rh-pH1 of M is sub- Sulfolane solution, mixes with cell culture fluid, after dyeing 5min, is carried out three times with the PBS of pH=7.4, Finally this culture dish is placed under Laser Scanning Confocal Microscope and observes.Experimental result find, be infected with A-FDA Hela cell line or Presenting obvious fluorescence in nematode, as it is shown in figure 1, test result indicate that, A-FDA has preferable cell-membrane permeable, energy Enough it is positioned in cell.Result display probe has good permeable membrane, can enter cell and always detect measurement in cell The instruction of microbial activity.
[3] time of staying measures
After cell is washed with deionized water 3 times after dyeing 10min, cell is added a certain amount of culture medium and continues to cultivate, so After be respectively separated 1,2,3,4,5h gather fluorescence picture, with the fluorescence intensity of fluorescence picture as ordinate, the time is that abscissa obtains The datagram gone out, as in figure 2 it is shown, this figure is longer in the intracellular time of staying after showing A-FDA dyeing.
Embodiment 2, A-FDA fluorescence probe measure Soil hydrolase
Take 2g soil in 125ml conical flask, in conical flask, add 15ml phosphate buffer solution (pH=7.4,60mM), Adding 0.2ml A-FDA solution (3mM) in conical flask, under the conditions of 30 DEG C, (volume ratio is 2:1 to cultivate 1h addition terminator Chloroform methanol mixed solution) take sample in 15ml centrifugal bottle, 2000 turns, centrifugal 3min, suction filtration supernatant, test, Result obtains Ac as shown in Figure 41；Set amount control group, one group is: take 2g soil in conical flask, adds 15ml phosphoric acid buffer Solution (pH=7.4,60mM), adds 0.2ml acetone in conical flask, and 30 DEG C of isothermal vibrations cultivate 1h, add terminator, centrifugal Obtaining supernatant, test, result obtains Ab as shown in Figure 41；Another group is: add equivalent phosphoric acid buffer in conical flask molten Liquid, 0.2mlA-FDA solution, cultivate 1h under the conditions of 30 DEG C, add terminator, add equivalent terminator, and equal conditions is centrifuged, Taking supernatant test, result obtains Ab as shown in Figure 42。
Soil activation is characterized as: formula: (Ac1-Ab1)/{ C (A-FDA) × Soil × time} obtains: 74 (M- 1Fluorescein g-1soil-1h).Visible, soil microbial activities can be detected by A-FDA.

Claims (7)

1. the fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure, it is characterised in that described fluorescence probe is A-FDA Fluorescence probe, the structural formula of A-FDA fluorescence probe is as shown in (I):
。
2. the preparation method of the fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure as claimed in claim 1, its feature Being, comprise the steps: to join in ether by fluorescein list aldehyde and potassium carbonate, adding dichloromethane so that dissolving；Dropping Appropriate acetic anhydride, under room temperature, magnetic agitation reaction 6h；Reactant is carried out suction filtration, takes off filtrate, be spin-dried for, through silica gel column chromatography Purify, obtain white solid i.e. target product A-FDA fluorescence probe.
3. preparation method as claimed in claim 2, it is characterised in that described fluorescein list aldehyde and potassium carbonate and ether mole Ratio is 1:(1-3): (2-4).
4. as claimed in claim 2 or claim 3 preparation method, it is characterised in that the synthesis of described fluorescein list aldehyde: take fluorescein in In flask, in flask, add chloroform and 15-crown-5, in flask, be slowly added dropwise NaOH solution, oil bath 55 DEG C, react 7h After, take product, carry out being acidified, suction filtration, be dried, cross post and purify and obtain fluorescein list aldehyde.
5. preparation method as claimed in claim 2, it is characterised in that described dichloromethane addition is that 1-6 drips.
6. preparation method as claimed in claim 2, it is characterised in that the addition of described acetic anhydride is 3-8ml.
7. the fluorescein(e) diacetate fluorescence probe containing aldehyde radical structure as claimed in claim 1 is in cell enzyme activity indicates Application.
CN201610187121.0A 2016-03-26 2016-03-26 Fluorescein diacetate fluorescence probe containing aldehyde group structure and preparation method and application thereof Pending CN105754586A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106632215A (en) * 2016-11-17 2017-05-10 陕西师范大学 Fluorescent protein dyeing agent as well as preparation method and application of fluorescent protein dyeing agent
CN108918495A (en) * 2018-08-21 2018-11-30 辽宁大学 The method of spectrophotometry quantitative detection cyanide ion based on 2- aldehyde radical rhodamine derivative

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
冯志明等: "氢化荧光素二乙酸酯的合成及其在活细胞染色中的应用", 《湖南师范大学学报(医学版)》 *
李卓: "基于罗丹明B及荧光素的新型荧光分子探针的设计与合成", 《中国优秀硕士学位论文全文数据库》 *
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王流芳等: "四种荧光素酯的化学物理性质", 《有机化学》 *
葛凤燕: "荧光素衍生物的合成及表征", 《中国博士学位论文全文数据库》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106632215A (en) * 2016-11-17 2017-05-10 陕西师范大学 Fluorescent protein dyeing agent as well as preparation method and application of fluorescent protein dyeing agent
CN108918495A (en) * 2018-08-21 2018-11-30 辽宁大学 The method of spectrophotometry quantitative detection cyanide ion based on 2- aldehyde radical rhodamine derivative
CN108918495B (en) * 2018-08-21 2020-10-09 辽宁大学 Method for quantitatively detecting cyanide ions based on spectrophotometry of 2-aldehyde rhodamine derivatives

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