WO2020155743A1 - 基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途 - Google Patents

基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途 Download PDF

Info

Publication number
WO2020155743A1
WO2020155743A1 PCT/CN2019/117083 CN2019117083W WO2020155743A1 WO 2020155743 A1 WO2020155743 A1 WO 2020155743A1 CN 2019117083 W CN2019117083 W CN 2019117083W WO 2020155743 A1 WO2020155743 A1 WO 2020155743A1
Authority
WO
WIPO (PCT)
Prior art keywords
aggregation
cell membrane
staining reagent
purine skeleton
membrane targeted
Prior art date
Application number
PCT/CN2019/117083
Other languages
English (en)
French (fr)
Inventor
李坤
石磊
余孝其
刘艳红
于抗抗
Original Assignee
四川大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川大学 filed Critical 四川大学
Publication of WO2020155743A1 publication Critical patent/WO2020155743A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/14—Styryl dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to the field of biochemistry, in particular to the technical field of biofilm targeted staining, in particular to a purine skeleton-based no-washing aggregation-inducing cell membrane targeted staining reagent, and a preparation method and application thereof.
  • Cell membrane also called plasma membrane or cytoplasmic membrane
  • cytoplasmic membrane composed of a phospholipid bilayer and intercalated proteins
  • An important component of cells It has been shown to be involved in a variety of cellular processes and biological functions, such as cell migration, cell proliferation, phagocytosis, endocytosis, exocytosis, and selective penetration of substances.
  • Abnormal cell membrane is an important marker of extremely poor cell status and various diseases. Therefore, the development of highly selective and sensitive detection technology to accurately visualize cell membranes, especially in vivo visualization, is of great significance for exploring and solving basic problems in early medical diagnosis and research biology.
  • the methods for observing cell membranes mainly include ordinary optical microscope observation, fluorescent dye marking, transmission electron microscope, scanning electron microscope observation, atomic force microscope observation and other methods.
  • ordinary optical microscopes have low resolution and cannot observe the morphology of cell membranes in the inner tissue; scanning electron microscopes, transmission electron microscopes, atomic force microscopes, etc. usually need to fix cells to obtain dead cell samples, which is cumbersome and expensive.
  • fluorescent staining methods are widely used due to their easy operation, fast response, high sensitivity, and no damage to tissue cells.
  • the existing staining methods usually fall into the following two categories: 1Indirectly target cell membrane imaging by the protein on the target cell membrane; 2Stain and image the target phospholipid bilayer. Because different cells target cell membrane proteins in different expression quantities, and connecting specific recognition sites is time-consuming and laborious, and not efficient.
  • the existing phospholipid molecular layer targeting dyes although there are commercially available dyes such as DiO, DiI, CellMask, etc., due to their low specific targeting to cell membranes, most of the dyes still escape into cells, causing Signal interference, and multiple washings are required to remove background signals, which cannot meet the clinical needs of fast, accurate, simple and other requirements.
  • the washing process is incompatible with the continuous sensing or monitoring of biological processes, so it is particularly important to develop new and more advantageous no-wash cell membrane dyes.
  • the purpose of the present invention is to provide a purine skeleton-based no-clean aggregation-inducible cell membrane targeted staining reagent and its preparation method and application, so as to solve the operation of the existing cell membrane phospholipid molecular layer targeting dye due to multiple washings.
  • the process is complicated, time-consuming, low accuracy of imaging results, and inability to connect to sensing.
  • R 1 is C1-C20 alkyl
  • R 2 are each independently Wherein, R is each independently a C1-C10 alkyl chain or aromatic group, and Ar is an aromatic group;
  • R 3 is a C1-C20 alkyl group or a charged alkyl quaternary amine chain.
  • Ar is a benzene ring, furan or thiophene.
  • R 1 is a C1-C20 alkyl group
  • R 2 is R 3 is a C1 alkyl group or a charged alkyl quaternary amine chain.
  • a preparation method of a purine skeleton-based no-clean aggregation-inducible cell membrane targeted staining reagent comprising:
  • the first organic solvent is selected from one or a combination of DMSO and DMF
  • the first weak base is selected from sodium carbonate, One or more combinations of potassium carbonate, potassium phosphate and sodium phosphate;
  • the second organic solvent is one or a combination of dioxane and tetrahydrofuran
  • the second weak base is selected from n-butyl lithium, potassium tert-butoxide, sodium tert-butoxide , One or more combinations of potassium hydride, sodium hydride, potassium carbonate and sodium carbonate;
  • the catalyst is palladium tetrakistriphenylphosphorus
  • the mixed solvent is a mixed solvent of tetrahydrofuran and water or dioxane and water
  • the third weak base is selected from potassium carbonate, sodium carbonate, One or more combinations of potassium phosphate and sodium phosphate
  • the third organic solvent is selected from one or more combinations of dichloromethane, tetrahydrofuran, ethanol, methanol, N,N-dimethylformamide and acetic anhydride, and the fourth weak
  • the base is one or a combination of sodium acetate and piperidine.
  • the mixing ratio may be arbitrary.
  • the heating temperature in step (1) is 50-120°C
  • the heating temperature in step (2) is 50-100°C.
  • the invention effectively combines the characteristics of a purine-based aggregation-inducing compound with the lipophilic end of the alkyl chain and the hydrophilic end of the quaternary ammonium salt to design and synthesize an ultra-fast, wash-free, highly targeted, and highly stable cell membrane Staining reagents can be used for cell membrane staining of cultured cells and tissue cells in vitro.
  • the dyeing reagent prepared by the invention has a large Stokes shift (>150nm), which can effectively avoid the interference of background light, and the regulation of the lipophilic chain and the hydrophilic chain has strong guiding significance for the design and synthesis of cell membrane probes .
  • the staining reagent of the present invention has the characteristics of ultra-fast staining and no-wash imaging for cell membrane staining, can effectively reduce the interference of background fluorescence, and the no-wash process can solve the change of cell environment and cell loss caused by long-term staining and multiple washings Such problems have improved the accuracy of cell imaging results, reduced the complexity of surgery and made it possible to monitor biological processes over a long period of time.
  • the no-washing aggregation-inducible cell membrane targeted staining reagent for purine skeleton provided by the present invention has the advantages of small toxic and side effects, economical and easy-to-obtain raw materials, strong operability of the entire synthetic route, mild reaction conditions, and low overall cost.
  • Figure 1 is a synthetic route diagram of the preparation method of the present invention.
  • Figure 2(a) is a hydrogen spectrum of the dyeing reagent of Example 1.
  • Figure 2(b) is the carbon spectrum of the dyeing reagent of Example 1.
  • Figure 2(c) is a high resolution mass spectrum of the dyeing reagent of Example 1.
  • Figure 3(a) is a hydrogen spectrum of the staining reagent of Example 2.
  • Figure 3(b) is a carbon spectrum of the dyeing reagent of Example 2.
  • Figure 3(c) is a high-resolution mass spectrum of the staining reagent of Example 2.
  • Fig. 4(a) is a hydrogen spectrum of the staining reagent of Example 3.
  • Figure 4(b) is a carbon spectrum of the dyeing reagent of Example 3.
  • Figure 4(c) is a high-resolution mass spectrum of the staining reagent of Example 3.
  • Figure 5(a) is a hydrogen spectrum of the staining reagent of Example 4.
  • Figure 5(b) is the carbon spectrum of the dyeing reagent of Example 4.
  • Figure 5(c) is a high-resolution mass spectrum of the staining reagent of Example 4.
  • Fig. 6 is the ultraviolet absorption spectrum of the dyeing reagent of Example 1 in DMSO solution.
  • Figure 7 is the ultraviolet absorption spectrum of the dyeing reagent of Example 2 in DMSO solution.
  • Fig. 8 is the ultraviolet absorption spectrum of the dyeing reagent of Example 3 in DMSO solution.
  • Figure 9 is the ultraviolet absorption spectrum of the dyeing reagent of Example 4 in DMSO solution.
  • Fig. 10 shows the emission spectrum of the dyeing reagent of Example 1 in a DMSO/toluene mixed solution.
  • Figure 11 is the emission spectrum of the dyeing reagent of Example 2 in a DMSO/toluene mixed solution.
  • Figure 12 is the emission spectrum of the dyeing reagent of Example 3 in a DMSO/toluene mixed solution.
  • Figure 13 is the emission spectrum of the dyeing reagent of Example 4 in a DMSO/toluene mixed solution.
  • Figure 14 shows the ⁇ AIE values of the dyeing reagents of Examples 1, 2, 3, and 4 in a DMSO/toluene mixed solution.
  • Figure 15 shows the normalized fluorescence emission spectra of the staining reagents of Examples 1, 2, 3, and 4 in toluene.
  • Figure 16 shows the normalized fluorescence emission spectra of the dyeing reagents of Examples 1, 2, 3, and 4 in a solid state.
  • Figure 17 shows the MTS cytotoxicity experiment of the staining reagents of Examples 1, 2, 3, and 4.
  • Figure 18 is a laser confocal experiment of cell membrane staining in B16 cells with the staining reagents of Examples 1, 2, 3, and 4.
  • Figure S1 is a 3D reconstruction image of the laser confocal laser confocal experiment of the cell membrane staining of the staining reagent of Example 4 in the neuronal cells of mice.
  • Figure S2 is a 2D image of a laser confocal experiment of cell membrane staining with the staining reagent of Example 4 in neuronal cells of mice.
  • Figure S3 is a confocal laser confocal experiment of cell membrane staining with the staining reagent of Example 4 in the red blood cells of mice.
  • Figure S4 is a 3D reconstruction image of the laser confocal experiment of cell membrane staining with the staining reagent of Example 4 in B16 cells.
  • Figure S5 is a 3D reconstruction image of a laser confocal experiment of cell membrane staining with commercially available cell membrane dyes in B16 cells.
  • Figure S6 is a laser confocal experiment of staining red blood cells in mouse brain with the staining reagent of Example 4.
  • Figure S7 is a confocal laser confocal experiment of dyeing living zebrafish epidermis with the staining reagent of Example 4.
  • 2,6-dichloropurine, indole, 4-formylphenylboronic acid, various solvents, catalysts, and bases were purchased from Inokia Technology Co., Ltd., and cell strains were purchased from ATCC (American Type Culture Collection). ), 10% fetal bovine serum (FBS) was purchased from Hyclone, and 1640 medium was purchased from Gibco, USA.
  • FBS fetal bovine serum
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • indole (1 g, 14 mmol) was added to a suspension of NaH (2 g, 21 mmol, 60% dispersed in mineral oil) in dry THF (500 mL). The resulting solution was stirred at 0°C for 1 hour, and then compound 1 (3.2 mL, 14 mmol, dissolved in 50 mL of dry THF) was slowly added. The mixture was heated to 70 degrees Celsius and stirred overnight. Then water was added to quench the reaction. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (30 mL ⁇ 3). The organic extracts were washed with brine, and dried over Na 2 SO 4.
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • Example 2 This example is basically the same as Example 1, except that the picoline salt in step (4) is replaced with 4-methyl-1-(3-(trimethylammonium)propyl)pyridine-1-ammonium bromide.
  • the synthetic route is as follows:
  • This example is basically the same as Example 1, except that the substituent of the third intermediate R 3 is changed and the picolinate involved in step (4) is different.
  • the synthetic route is as follows:
  • the aggregation-inducible cell membrane targeted staining reagents based on purine skeletons prepared in the foregoing Examples 1-4 were respectively formulated into 5mM DMSO mother liquor. Prepare 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 uL of DMSO solution respectively, scan the UV absorption value, and plot.
  • the ultraviolet absorption spectrum of the dyeing reagent of Example 1 is shown in FIG. 6, the ultraviolet absorption spectrum of the dyeing reagent of Example 2 is shown in FIG. 7, and the ultraviolet absorption spectrum of the dyeing reagent of Example 3 is shown in FIG.
  • the ultraviolet absorption spectrum of the dyeing reagent 4 is shown in Figure 9. As shown in the figure, each compound has two absorption peaks.
  • the staining reagents prepared in Examples 1, 2, 3, and 4 were prepared into 5mM DMSO mother liquor.
  • the mixed solution of DMSO, toluene (TL) and DMSO ⁇ TL was added to measure the fluorescence spectrum, and the fluorescence emission curve was obtained.
  • the maximum emission wavelength of the analyte has a significant red shift, and the fluorescence intensity gradually increases with the increase of the TL ratio.
  • the fluorescence intensity reaches the maximum when the TL ratio is 100%.
  • the fluorescence intensity change of the dyeing reagent of Example 1 is shown in Fig. 10
  • the fluorescence intensity change of the dyeing reagent of Example 2 is shown in Fig.
  • FIG. 11 the fluorescence intensity change of the dyeing reagent of Example 3 is shown in Fig. 12, and the dyeing reagent of Example 4 The change in fluorescence intensity is shown in Figure 13.
  • Figure 14 is a summary of the fluorescence emission spectra of the changes in the ⁇ AIE value of the staining reagents prepared in Examples 1, 2, 3, and 4 in TL
  • Figure 15 is the staining reagents prepared in Examples 1, 2, 3, and 4.
  • Figure 16 is a summary of the fluorescence emission spectra of the dyeing reagents prepared in Examples 1, 2, 3, and 4 in a solid state.
  • the present invention chooses dimethyl sulfoxide (DMSO) as its good solvent and toluene (TL) as its poor solvent.
  • DMSO dimethyl sulfoxide
  • TL toluene
  • Example 4 hardly emits fluorescence in DMSO, in DMSO/TL (>80% TL), Example 4 in an aggregated state emits strong fluorescence. This observed phenomenon may be due to the restricted intramolecular movement (RIM) process. Similar phenomena were also observed in Compound Example 1, Example 2, and Example 3.
  • RIM restricted intramolecular movement
  • Similar phenomena were also observed in Compound Example 1, Example 2, and Example 3.
  • the fluorescence intensity of Example 1, Example 2, Example 3, and Example 4 were 8.46, 14.6, 5.99, and 8.24 times higher than those in pure dimethyl sulfoxide, respectively.
  • B16 cells in the logarithmic growth phase were seeded in a 96-well culture plate, each well was seeded with 10,000 cells, with 10% fetal bovine serum (FBS), 1% double antibody (penicillin-streptomycin, 1000KU/L)
  • FBS fetal bovine serum
  • 1% double antibody penicillin-streptomycin, 1000KU/L
  • the DMEM(H) medium was cultured overnight at 37°C and 5% CO 2 . After the cells are completely attached to the wall, the staining reagents prepared in Examples 1, 2, 3, and 4 with different concentration gradients are added, and 3 multiple wells are set for each concentration, and a blank control group is set. After the drug was added, the culture was continued for 24 hours, and the inhibition rate of the cells was detected by the MTS method. The results are shown in Figure 17.
  • the compression of the long alkyl chain of DIO in the phospholipid bilayer membrane may interfere with physiological functions such as membrane fluidity, resulting in high toxicity.
  • the short alkyl chain of the probe has little effect on the phospholipid bilayer.
  • Test Example 4 Confocal laser imaging of cell membrane staining of B16 cells (mouse melanoma cells)
  • the imaging conditions were optimized. Due to the water solubility, electrostatic interaction and similar compatibility between the probe and the cell membrane, it is expected that the probe can be uniformly dispersed in the aqueous solution and can be quickly embedded in the plasma membrane. Therefore, the staining cycle of the probe was studied.
  • the primary neurons of the mice were inoculated in a 35 mm petri dish covered with polylysine in advance, and the inoculum was changed to neuron culture medium after 4 hours of sedimentation, and the medium was changed every two days. After culturing for 6 days, replace the culture medium in the petri dish with the neuron culture medium of Example 4, shake for 5-30 seconds, and use appropriate excitation and emission filters to treat neurons under a confocal laser microscope.
  • Figure S1 As can be seen from the figure, in the case of no-washing, the dye can image the cell membrane of neuronal cells well within 1 minute. In addition, in the 2D image S2, the tiny structures on the neuron cell membrane can be clearly presented.
  • red blood cells are separated. Disperse a small amount of red blood cells in the PBS solution containing the dye of Example 4, shake for 5-30 seconds, take 30 microliters and drop them onto the glass slide, and cover with a cover glass.
  • the dye can stain the cell membrane of red blood cells very well. In the enlarged image, you can also see a very obvious circular cell membrane. In addition, the red blood cells in the bright field image are dark, indicating that the heme inside is not released.
  • FIG. 1 is the 3D reconstruction image of the cell spheroid.
  • Figure A is the Z-axis slice of the cell spheroid. The distance between each layer is 5 microns. It can be seen that the dye almost stains the cell spheroid;
  • Figure B shows the dye in the cell spheroid.
  • Test Example 8 Deep-level imaging of mouse brain
  • mice The newborn mice (within 48 hours) were euthanized by carbon dioxide, and the brains were taken out. After clearing treatment, they were incubated with a 10 ⁇ M dye-containing PBS solution for 1 day under shaking and then placed in a confocal small dish.
  • ⁇ ex 405 nm
  • ⁇ em 470-600 nm.
  • Test Example 9 Imaging the zebrafish epidermis in vivo
  • the newborn zebrafish were incubated in a zebrafish culture medium containing 10 ⁇ M dye for 15 minutes, and then the zebrafish were placed in a zebrafish culture medium containing 0.003% tricaine methanesulfonate (anesthetic). After incubating for 20 seconds, the zebrafish were transferred to a double intaglio plate, fixed with an anesthetic solution containing 2.4% methylcellulose, and covered with a cover glass.
  • Figure S7C shows that the dye can stain zebrafish epidermis
  • Figures S7A and S7B show that the dye is highly specific to the cell membrane of living zebrafish epidermal cells, which illustrates the potential of the dye in this patent for application in in vivo imaging.
  • the present invention uses the purine skeleton as the basis of cell membrane dyes, and through reasonable regulation and design of the lipophilic end and hydrophilic end, a purine skeleton-based cell membrane targeted staining reagent is obtained.
  • This type of reagent can rapidly target staining at the same time , Can stay on the cell membrane for a long time, which is conducive to long-term monitoring.
  • the dyeing reagent has the characteristics of aggregation-inducing compound, it emits weak or no luminescence in a good solvent, and emits strong fluorescence in a poor solvent, so that this type of dye also has the specific performance of no-washing.
  • the preparation method of the invention has high yield, mild reaction conditions, and the prepared dyeing reagent has large Stokes shift and high targeting ability.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Materials Engineering (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

本发明公开了一种基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途，本发明以嘌呤骨架作为细胞膜染料的基础，通过合理的脂溶端亲水端的调控设计，得到了基于嘌呤骨架的细胞膜靶向染色试剂，该类试剂在快速靶向染色的同时，能够较长时间的停滞在细胞膜上，有利于长时的监测。此外，由于该染色试剂具有聚集诱导型化合物的特性，在良溶剂中发光很弱或不发光，在不良溶剂中发出强烈的荧光，使得该类染料还具有免洗的特异表现。本发明的制备方法收率高、反应条件温和，制得的染色试剂斯托克斯位移大、靶向性高。

Description

基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途 技术领域
本发明涉及生物化学领域，尤其涉及生物膜靶向染色技术领域，具体涉及一种基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途。
背景技术
细胞膜(也称为质膜或细胞质膜)，由磷脂双分子层与嵌插的蛋白组成，是将细胞的内部与外部环境分离的生物膜，它保护细胞不受其环境的影响，是生物体细胞的重要组成成分。它已被证明参与多种细胞过程和生物学功能，如细胞迁移、细胞扩散、吞噬、内吞、胞吐和物质的选择性渗透。细胞膜异常是细胞状态极差及多种疾病的重要标志物。因此，开发高选择性、高灵敏度的检测技术以准确对细胞膜进行可视化，尤其是活体可视化，对于探索与解决医学早期诊断和研究生物学中的基本问题具有重要意义。
目前，对于观测细胞膜的方法主要有普通光学显微镜观测、荧光染色标记、透射电镜、扫描电镜观测、原子力显微镜观测等方法。然而，普通光学显微镜分辨率不高，无法观测内层组织中的细胞膜形态；扫描电镜、透射电镜、原子力显微镜等通常需要对细胞进行固定得到死细胞样本，制样繁琐，设备昂贵。相比之下，荧光染色方法由于其易操作、快响应、高灵敏、对组织细胞无伤害等特点而被广泛采用。
现有的染色方法通常有以下两类：①通过标靶细胞膜上蛋白间接靶向细胞膜成像；②通过标靶磷脂双分子层进行染色成像。由于不同细胞靶向细胞膜蛋白的表达数量不同，且连接特异性识别位点费时费力，并不高效。现存的磷脂分子层靶向的染料中，虽然已有商售的DiO、DiI、CellMask等染料，但由于其对细胞膜的特异靶向性不高，仍有大部分染料逸散进细胞内，造成信号干扰，且需要多次的洗涤以去除背景信号，并不能满足临床上需要的快速、精准、简便等要求。此外，洗涤过程与生物过程的连续感测或监测是不相容的，因此开发新的更具优势的免洗细胞膜染料显得尤为重要。
发明内容
本发明的目的在于提供一种基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途，以解决现有细胞膜磷脂分子层靶向染料因需要多次洗涤而导致的操作过程复杂、耗时长、成像结果准确性低、不能连接感测的问题。
本发明解决上述技术问题的技术方案如下：
一种基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂，其结构如式(Ⅰ)所示：
Figure PCTCN2019117083-appb-000001
式(Ⅰ)中：
R 1为C1-C20烷基；
R 2各自独立地为
Figure PCTCN2019117083-appb-000002
其中，R各自独立地为C1-C10烷基链或芳香基团，Ar为芳香基团；
R 3为C1-C20烷基或带电荷的烷基季胺链。
进一步地，在本发明较佳的实施例中，Ar为苯环、呋喃或噻吩。
进一步地，在本发明较佳的实施例中，R 3为
Figure PCTCN2019117083-appb-000003
其中，n＝0-8。
进一步地，在本发明较佳的实施例中，R 1为C1-C20烷基，R 2为
Figure PCTCN2019117083-appb-000004
R 3为C1烷基或带电荷的烷基季胺链。
进一步地，在本发明较佳的实施例中，R 3为
Figure PCTCN2019117083-appb-000005
其中n＝0-3。
进一步地，在本发明较佳的实施例中，R 1为
Figure PCTCN2019117083-appb-000006
n＝1或3；R 2为
Figure PCTCN2019117083-appb-000007
R 3为
Figure PCTCN2019117083-appb-000008
Figure PCTCN2019117083-appb-000009
n＝0。
一种基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂的制备方法，所述制备方法包括：
(1)将2,6-二氯嘌呤和R 1的卤代物R 1-X溶于第一有机溶剂中，加入第一弱碱加热搅拌，得到第一中间体；其中，X为Cl，Br或I；
(2)将所述第一中间体加入至含有第二弱碱的第二有机溶剂中搅拌，然后加入R 2取代的
Figure PCTCN2019117083-appb-000010
加热搅拌，得到第二中间体；
(3)将2,4-甲酰基苯硼酸和催化剂溶于水和有机溶剂的混合溶剂中，加入第三弱碱，然后加入所述第二中间体混合，回流，得到第三中间体；
(4)将所述第三中间体与R 3取代的
Figure PCTCN2019117083-appb-000011
溶于第三有机溶剂，加入第四弱碱，搅拌，制得基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂。
进一步地，在本发明较佳的实施例中，步骤(1)中，所述第一有机溶剂选自DMSO和DMF中的一种或两种组合，所述第一弱碱选自碳酸钠、碳酸钾、磷酸钾和磷酸钠中的一种或多种组合；
步骤(2)中，所述第二有机溶剂为二氧六环和四氢呋喃中的一种或两种组合，所述第二弱碱选自正丁基锂、叔丁醇钾、叔丁醇钠、氢化钾、氢化钠、碳酸钾和碳酸钠中的一种或多种组合；
步骤(3)中，所述催化剂为四三苯基磷钯，所述混合溶剂为四氢呋喃和水或者二氧六环和水的混合溶剂，所述第三弱碱选自碳酸钾、碳酸钠、磷酸钾和磷酸钠中的一种或多种组合；
步骤(4)中，所述第三有机溶剂选自二氯甲烷、四氢呋喃、乙醇、甲醇、N,N-二甲基甲酰胺和 醋酸酐中的一种或多种组合，所述第四弱碱为醋酸钠和哌啶中的一种或两种组合。
需要说明的是，上述有机溶剂和碱为混合物时，其混合比例可以是任意的。
进一步地，在本发明较佳的实施例中，步骤(1)的加热温度为50-120℃，步骤(2)的加热温度为50-100℃。
基于本发明提供的嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂在生物体细胞膜上进行荧光成像中的应用。
本发明具有以下有益效果：
本发明将以嘌呤为基础的聚集诱导型化合物与由烷基链亲脂端和季铵盐亲水端特性有效结合，设计并合成了超快速、免洗、高靶向、高稳定性的细胞膜染色试剂，可用于体外培养细胞、组织细胞的细胞膜染色。本发明制得的染色试剂具有较大的斯托克斯位移(＞150nm)可以有效避免背景光的干扰，且亲脂链亲水链的调控对细胞膜探针的设计合成具有较强的指导意义。本发明的染色试剂对于细胞膜染色具有超快速染色、免洗成像的特性，能够有效降低背景荧光的干扰，且免洗的过程可解决长时染色、多次洗涤带来细胞环境的变化和细胞丢失等问题，提升了细胞成像结果的准确性，减小手术的复杂性并且使长时监测生物过程的成为可能。此外，本发明提供的嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂具有毒副作用小、原料经济易得、整条合成路线可操作性强、反应条件温和、总体成本较低等优势。
附图说明
图1为本发明制备方法的合成路线图。
图2(a)为实施例1的染色试剂的氢谱。
图2(b)为实施例1的染色试剂的碳谱。
图2(c)为实施例1的染色试剂的高分辨质谱。
图3(a)为实施例2的染色试剂的氢谱。
图3(b)为实施例2的染色试剂的碳谱。
图3(c)为实施例2的染色试剂的高分辨质谱。
图4(a)为实施例3的染色试剂的氢谱。
图4(b)为实施例3的染色试剂的碳谱。
图4(c)为实施例3的染色试剂的高分辨质谱。
图5(a)为实施例4的染色试剂的氢谱。
图5(b)为实施例4的染色试剂的碳谱。
图5(c)为实施例4的染色试剂的高分辨质谱。
图6为实施例1的染色试剂在DMSO溶液中的紫外吸收光谱。
图7为实施例2的染色试剂在DMSO溶液中的紫外吸收光谱。
图8为实施例3的染色试剂在DMSO溶液中的紫外吸收光谱。
图9为实施例4的染色试剂在DMSO溶液中的紫外吸收光谱。
图10为实施例1的染色试剂在DMSO/甲苯混合溶液中的发射光谱。
图11为实施例2的染色试剂在DMSO/甲苯混合溶液中的发射光谱。
图12为实施例3的染色试剂在DMSO/甲苯混合溶液中的发射光谱。
图13为实施例4的染色试剂在DMSO/甲苯混合溶液中的发射光谱。
图14为实施例1、2、3、4的染色试剂在DMSO/甲苯混合溶液中的αAIE值。
图15为实施例1、2、3、4的染色试剂在甲苯中的归一化荧光发射光谱。
图16为实施例1、2、3、4的染色试剂在固体状态下的归一化荧光发射光谱。
图17为实施例1、2、3、4的染色试剂的MTS细胞毒性实验。
图18为实施例1、2、3、4的染色试剂在B16细胞中的细胞膜染色激光共聚焦实验。
图S1为实施例4的染色试剂在小鼠的神经元细胞中的细胞膜染色激光共聚焦实验的3D重构化图。
图S2为实施例4的染色试剂在小鼠的神经元细胞中的细胞膜染色激光共聚焦实验的2D图。
图S3为实施例4的染色试剂在小鼠的血红细胞中的细胞膜染色激光共聚焦实验。
图S4为实施例4的染色试剂在B16细胞中的细胞膜染色激光共聚焦实验的3D重构化图。
图S5为商购细胞膜染料在B16细胞中的细胞膜染色激光共聚焦实验的3D重构化图。
图S6为实施例4的染色试剂在小鼠脑部红细胞染色激光共聚焦实验。
图S7为实施例4的染色试剂在对活体斑马鱼表皮染色激光共聚焦实验。
具体实施方式
以下结合附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例中，2,6-二氯嘌呤、吲哚、4-甲酰基苯硼酸、各类溶剂、催化剂、碱购于伊诺凯科技有限公司，细胞株购于ATCC(American Type Culture Collection)，10％胎牛血清(FBS)购于Hyclone，1640培养基购于美国Gibco。
本发明实施例的合成路线如图1所示，过程包括：
(1)将2,6-二氯嘌呤和R 1的卤代物R 1-X溶于第一有机溶剂中，加入第一弱碱加热搅拌，得到第一中间体；其中，X为Cl，Br或I；
(2)将所述第一中间体加入至含有第二弱碱的第二有机溶剂中搅拌，然后加入R 2取代的
Figure PCTCN2019117083-appb-000012
加热搅拌，得到第二中间体；
(3)将2,4-甲酰基苯硼酸和催化剂溶于水和有机溶剂的混合溶剂中，加入第三弱碱，然后加入所述第二中间体混合，回流，得到第三中间体；
(4)将所述第三中间体与R 3取代的
Figure PCTCN2019117083-appb-000013
溶于第三有机溶剂，加入第四弱碱，搅拌，制得基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂。
下面结合实施例对本发明进一步说明。
实施例1
本实施例的基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂的制备方法，包括以下步骤：
(1)合成第一中间体：2,6-二氯-9-正丙基-9-氢-嘌呤
合成路线如下：
Figure PCTCN2019117083-appb-000014
将2,6-二氯嘌呤(1.0mmol)、1-溴丙烷(1.5mol)和碳酸钾(3.0mmol)在DMSO(5mL)中混合搅拌6小时，随后加入100mL水。分离有机层，用乙酸乙酯(30mL×3)萃取水层。将有机萃取物用盐水洗涤，并使用Na 2SO 4干燥。溶剂除减压蒸馏后，用200-300目硅胶柱层析纯化。用石油醚/乙酸乙酯(3:2)洗脱，得到第一中间体为白色固体，产率为57％。洗脱剂为乙酸乙酯/石油醚＝2:3(V/V)。最终得白色固体，产率61％。
1H NMR(400MHz,CDCl 3)δ8.11-8.08(s,1H),4.22-4.16(t,2H),1.95-1.85(m,2H),0.94-0.88(t,3H)。
(2)合成第二中间体：2-氯-6-(1-氢-吲哚)-9-正丙基-9-氢-嘌呤
合成路线如下：
Figure PCTCN2019117083-appb-000015
在氮气保护下，将吲哚(1g，14mmol)加入到NaH(2g，21mmol，60％分散在矿物油中)在干燥THF(500mL)中的悬浮液中。将得到的溶液在0℃搅拌1小时，然后缓慢加入化合物1(3.2mL，14mmol，在50mL干THF中溶解)。将混合物加热至70摄氏度并搅拌过夜。随后加入水使反应猝灭。分离有机层，用乙酸乙酯(30mL×3)萃取水层。将有机萃取物用盐水洗涤，并使用Na 2SO 4干燥。溶剂除减压蒸馏后，用200-300目硅胶柱层析纯化。用石油醚/乙酸乙酯(3:1)洗脱，得到第二中间体为白色固体，产率为57％。
1H NMR(400MHz,CDCl 3)δ89.14-9.11(d,1H),8.97-8.93(d,1H),7.96-7.94(s,1H),7.64-7.58(d,1H),7.42-7.36(t,1H),7.31-7.26(t,1H),6.81-6.78(d,1H),4.23-4.17(t,2H),2.00-1.90(m,2H),1.01-0.95(t,3H)。
(3)合成第三中间体：4-(6-(1-氢-吲哚)-9-正丙基-9氢-嘌呤)-2-苯甲醛
合成路线如下：
Figure PCTCN2019117083-appb-000016
于氮气保护下，将化合物2(342mg，1.1mmol)、4-甲酰基苯硼酸(1.5eq)、四三苯基磷钯(0.05eq)和2.0mL碳酸钠水溶液(2M)加入10.0mL二氧六环中，回流8小时，薄层色谱监测反应完全后，将反应混合物倒入100mL水中，用二氯甲烷萃取。有机层用盐水、水洗涤，并使用Na 2SO 4干燥。溶剂除减压蒸馏后，用200-300目硅胶柱层析纯化。用二氯甲烷洗脱，得到第三中间体为白色固体，产率为88％。
1H NMR(400MHz,CDCl 3)δ10.12-10.09(s,1H),9.22-9.19(d,1H),9.03-8.99(d,1H),8.71-8.65(t,3H),8.14-8.09(d,2H),7.72-7.68(d,1H),7.46-7.41(t,1H),7.32-7.27(t,1H),6.95-6.91(d,1H),4.37-4.30(t,2H),2.00-1.91(m,2H),0.94-0.88(t,3H)。
(4)合成基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂：(E)-4-(4-(6-(1-氢-吲哚)-9-正丙基-9-氢-嘌呤)-2-苯乙烯基)-1-甲基吡啶-1-六氟磷酸化物
合成路线如下：
Figure PCTCN2019117083-appb-000017
在乙醇(10mL)中加入化合物3(381mg，1mmol)和1,4-二甲基吡啶-1-碘化物(235mg，1mmol)后，将哌啶(0.05mL)滴入搅拌液中。然后将混合物在室温下搅拌约12小时，以薄层色谱监控反应完全后，旋蒸除去溶剂，然后用饱和的六氟磷酸钾丙酮溶液(10mL)溶解。在室温下搅拌2小时后，减压蒸馏去丙酮，然后过滤得到粗产物。粗品经中性氧化铝柱层析纯化。用甲醇/二氯甲烷＝20/1(V:V)洗脱，得到深黄色固体，产率为25％。
1H NMR(400MHz,CDCl 3)δ8.60(s,1H),7.73(dd,J＝12.5,7.4Hz,6H),7.54(t,J＝6.9Hz,3H),7.47(dt,J＝7.1,3.5Hz,6H),7.31(d,J＝8.7Hz,1H),6.54(dd,J＝8.8,2.3Hz,1H),6.48(d,J＝2.1Hz,1H),5.55(d,J＝28.6Hz,1H),3.40(q,J＝7.1Hz,4H),1.20(t,J＝7.1Hz,6H)。 13C NMR(101MHz,CDCl 3)δ177.7,161.9,157.2,151.1,144.0,133.3,133.2,131.9,130.3,128.9,128.8,127.3,126.4,108.9,96.5,56.4,55.4,44.8,12.5。HRMS(ESI)C 33H 30NO 3P[M+H] +520.2040。
本实施例制得的基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂的氢谱、碳谱和高分辨质谱分别如图2(a)-图2(c)所示。
实施例2
本实施例与实施例1基本相同，区别在于改变第三中间体R 3的取代基，其合成路线如下：
Figure PCTCN2019117083-appb-000018
在乙醇(10mL)中加入第三中间体(381mg，1mmol)和1,4-二甲基吡啶-1-碘化物(235mg，1mmol)后，将哌啶(0.05mL)滴入搅拌液中。然后将混合物在室温下搅拌约12小时，以薄层色谱监控反应完全后，旋蒸除去溶剂，然后用饱和的六氟磷酸钾丙酮溶液(10mL)溶解。在室温下搅拌2小时后，减压蒸馏去丙酮，然后过滤得到粗产物。粗品经中性氧化铝柱层析纯化。用甲醇/二氯甲烷＝20/1(V:V)洗脱，得到黄色固体，产率为37％。
1H NMR(400MHz,DMSO-d 6)δ9.22-9.20(d,1H),9.06-9.03(d,1H),8.88-8.84(d,2H),8.67-8.65(s,1H),8.59-8.52(d,2H),8.25-8.21(d,2H),8.09-8.02(d,1H),7.96-7.91(d,2H),7.73-7.69(d,1H),7.64-7.57(d,1H),7.47-7.41(t,1H),7.33-7.28(t,1H),6.94-6.92(d,1H),4.36-4.30(t,2H),4.27-4.24(s,3H),1.99-1.90(t,2H),1.38-1.27(m,4H),0.89-0.83(t,3H). 13C NMR(101MHz,DMSO-d 6)δ157.04,154.01,152.62,149.00,146.10,145.63,140.20,139.57,137.38,135.66,130.67,129.00,128.80,124.80,124.34,124.14,123.23,121.53,121.20,116.73,108.56,47.44,43.74,29.28,28.62,22.00,14.26.HRMS(ESI):m/z:Calcd for C 32H 31N 6 +:499.2605；[M-PF 6] +Found:499.2605.
本实施例制得的基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂的氢谱、碳谱和高分辨质谱分别如图3(a)-图3(c)所示。
实施例3
本实施例与实施例1基本相同，区别在于将步骤(4)中的甲基吡啶盐换为4-甲基-1-(3-(三甲铵)丙基)吡啶-1-溴化铵，其合成路线如下：
Figure PCTCN2019117083-appb-000019
得到深褐色固体，产率33％。 1H NMR(400MHz,DMSO-d 6)δ9.23-9.20(d,1H),9.09-9.03(m,3H),8.71-8.69(s,1H),8.62-8.57(d,2H),8.37-8.33(d,2H),8.20-8.14(d,1H),8.02-7.98(d,2H),7.74-7.67(m,2H),7.47-7.42(t,1H),7.34-7.28(t,1H),6.95-6.93(d,1H),4.65-4.60(t,2H),4.40-4.34(t,2H),3.11-3.7(s,9H),1.99-1.92(m,2H),0.89-0.83(t,3H). 13C NMR(101MHz,DMSO-d 6)δ157.08,154.10,153.50,149.06,146.25,145.04,140.83,139.78,137.37,135.68,130.69,129.15,129.00,128.93,124.83,124.58,124.36,123.26,121.57,121.29,116.71,109.99,108.61,62.25,57.24,52.91,45.50,24.47,23.06,11.51.HRMS(ESI):m/z:Calcd for C 35H 39F 6N 7P +:702.2903；[M-PF 6] +Found:702.2902.
本实施例制得的基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂的氢谱、碳谱和高分辨质谱分别 如图4(a)-图4(c)所示。
实施例4
本实施例与实施例1基本相同，区别在于改变第三中间体R 3的取代基以及参与步骤(4)的甲基吡啶盐不同，其合成路线如下：
Figure PCTCN2019117083-appb-000020
得到深褐色固体，产率37％。 1H NMR(400MHz,DMSO-d 6)δ9.24-9.20(d,1H),9.07-9.03(d,1H),9.01-8.87(d,2H),8.70-8.68(s,1H),8.62-8.58(d,2H),8.36-8.31(d,2H),8.18-8.12(d,1H),8.02-7.97(d,2H),7.74-7.65(m,2H),7.45-7.42(t,1H),7.34-7.29(t,1H),6.95-6.93(d,1H),4.61-4.45(t,2H),4.40-4.34(t,2H),3.11-3.7(s,9H),1.99-1.93(m,2H),1.41-29(m,4H),0.89-0.84(t,3H).1.61-1.58(s,2H),1.40-1.27(m,4H),0.89-0.83(t,3H). 13C NMR(101MHz,DMSO-d 6)δ157.04,154.05,153.48,149.04,146.17,145.03,140.79,139.74,137.37,135.67,130.68,129.13,128.98,128.87,124.83,124.56,124.36,123.26,121.55,121.24,116.72,108.59,62.25,57.24,52.90,43.76,29.28,28.61,24.49,22.00,14.26.HRMS(ESI):m/z:Calcd for C 37H 43F 6N 6P +:730.3216；[M-PF 6] +Found:730.3217.
本实施例制得的基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂的氢谱、碳谱和高分辨质谱分别如图5(a)-图5(c)所示。
试验例1紫外吸收光谱
将上述实施例1-4制得的基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂分别配制成5mM的DMSO母液。分别配为0.5、1、2、3、4、5、6、7、8、9、10uL的DMSO溶液，扫描紫外吸收值，绘图。实施例1的染色试剂的紫外吸收光谱如图6所示，实施例2的染色试剂的紫外吸收光谱如图7所示，实施例3的染色试剂的紫外吸收光谱如图8所示，实施例4的染色试剂的紫外吸收光谱如图9所示。如图所示，每个化合物有两个吸收峰。其中一个在λ＝330nm附近，这归因于π-π*跃迁，另一个在λ＝375nm附近出现，这表示分子内电荷转移(ICT)跃迁。另外，无论是延长给电子烷基链还是增加带正电荷的吸电子亲水端，都会略微调整给电子供体-受体(D-A)的性质，导致吸收峰红移。
试验例2聚集诱导特性表征
将实施例1、2、3、4制得的染色试剂配为5mM的DMSO母液。分别加入DMSO、甲苯(TL)和DMSO\TL的混合溶液测定其荧光光谱，得到荧光发射曲线。与DMSO溶液相比，在DMSO\TL的混合溶液中，待测物的最大发射波长发生明显红移，荧光强度随TL比例的增加逐渐增强，含TL比例为100％时荧光强度达到最大。其中，实施例1的染色试剂的荧光强度变化如图10，实施例2的染色试剂的荧光强度变化如图11，实施例3的染色试剂的荧光强度变化如图12，实施例4的染色试剂的荧光强度变化如图13。此外，图14为实施例1、2、3、4制得的染色试剂的α AIE值变化在TL中的荧光发射光谱汇总，图15为实施例1、2、3、4制得的染色试剂在TL中的荧光发射光谱汇总， 图16为实施例1、2、3、4制得的染色试剂在固体状态下的荧光发射光谱汇总。由于这四种化合物都属于有机盐，本发明选择二甲基亚砜(DMSO)作为其良溶剂，甲苯(TL)作为不良溶剂。例如，虽然在DMSO中，实施例4几乎不发射荧光，但在DMSO/TL(>80％TL)中，聚集态的实施例4发射出强烈的荧光。这种观察到的现象可能归因于分子内运动受限(RIM)过程。在化合物实施例1、实施例2、实施例3中也观察到类似现象。此外，当TL含量增加到99.9％时，实施例1、实施例2、实施例3和实施例4的荧光强度分别比它们在纯二甲基亚砜中高8.46、14.6、5.99和8.24倍。
试验例3 MTS细胞毒性实验
处于对数生长期的B16细胞接种于96孔培养板中，每孔接种10000个细胞，用含10％胎牛血清(FBS)、1％双抗的(青霉素-链霉素，1000KU/L)的DMEM(H)培养基在37℃，5％CO 2条件下培养过夜。待细胞完全贴壁，加入不同浓度梯度的实施例1、2、3、4制得的染色试剂，每个浓度设3个复孔，同时设空白对照组。加药后继续培养24小时，MTS法检测细胞的抑制率，结果如图17所示。在20μm的高浓度下，无论探针或DIO对B16细胞都几乎没有细胞毒性。然而，当浓度上升到40μm，探针的细胞通过率在68％左右时，DIO染料的细胞活性降低了31％。这种现象可能归因于，在高浓度时，长烷基链对细胞膜生理活性有比较大的影响，这意味着DIO的毒性在较大浓度时比探针高许多。当在较低浓度时，烷基链插入磷脂双分子层后，质膜的生理功能仍能比较好的保持。然而，在较高的DIO浓度下，DIO在磷脂双层膜中的长烷基链的压缩可能会干扰膜的流动性等生理功能，导致高毒性。与DIO相比，探针的短烷基链对磷脂双层的影响很小。
试验例4对B16细胞(小鼠黑色素瘤细胞)细胞膜染色的激光共聚焦成像
将B16细胞在35毫米培养皿中培养一夜。在一定浓度下染一定时间后(在DMSO<0.1vol％的1mL培养基中加入1μL 5mM的化合物母液)，震荡摇晃5秒-30秒钟，在激光共聚焦显微镜下用适当的激发和发射滤光片对染料进行成像：λ ex＝405nm，λ em＝470-600nm。结果如图18所示。首先对其成像条件进行了优化。由于探针具有水溶性以及和细胞膜之间的静电相互作用和相似相容性，预计探针可以均匀地分散在水溶液中，并且可以快速嵌入到质膜中。因此，对探针的染色周期进行了研究。值得注意的是，这些探针在进行细胞染色后不用清洗直接用于成像。在小鼠黑色素瘤细胞(小鼠癌细胞B16)中可以清楚地观察到它们的细胞膜。染色周期实验的结果表明，当染色时间减少到1分钟、30秒、10秒甚至5秒时，各探针的荧光成像质量没有明显变化，表明其可用于超快成像。
试验例5对小鼠原代神经元成像
将小鼠的原代神经元接种在提前铺有多聚赖氨酸的35毫米培养皿中，沉降4小时后将接种液更换为神经元培养基，每两天换液一次。经培养6天后，将培养皿中的培养基更换为含有实施例4的神经元培养基，震荡摇晃5-30秒钟，在激光共聚焦显微镜下用适当的激发和发射滤光片对神经元细胞进行逐层扫描：λ ex＝405nm，λ em＝470-600nm，层间距0.6微米。经过3D重构化后，结果如图S1所示。由图中可以看到，在免洗的情况下，染料可以在1分钟内对神经元细胞的细胞膜进行良好的成像。此外，在2D图S2中，神经元细胞膜上的微小结构都可以被清晰的呈现。
试验例6对小鼠血红细胞成像
将小鼠新鲜提取的血液经至少三次稀释离心后，分离得到纯净的血红细胞。将少量血红细胞分散在含有实施例4染料的PBS溶液中，震荡摇晃5-30秒，取30微升滴加到载玻片上，盖上盖玻片。在 激光共聚焦显微镜下用适当的激发和发射滤光片对血红细胞进行成像：λ ex＝405nm，λ em＝470-600nm。结果如图S3所示。染料可以很好的对血红细胞的细胞膜染色，在放大图中也可以看到很明显的圆形细胞膜，此外，明场图中的红细胞成深色，说明其内部的血红素并未释放，细胞膜结构完整，表明染料的低毒性。相对的，使用商售细胞膜染料DiO进行对照，可以明显的发现红细胞的血红素外渗，且染料进入细胞内部，不能清晰的显示出细胞膜。这组对比，说明了本发明中细胞膜染料的高细胞膜靶向性和高生物相容性。
试验例7对3D B16细胞球成像
将细胞以含有0.24％甲基纤维素的无菌DMEM培养液稀释为1×10 6个每毫升的细胞悬液，取40微升滴加到10毫升培养皿的皿盖上，倒置培养24小时候，转入预铺有1％琼脂糖的96孔板中继续培养48小时。将待用3D细胞球转入到玻底96孔板中，将普通培养基置换为含有10μM染料的培养基溶液，摇晃10-20秒，于37摄氏度下孵育1.5小时后，在激光共聚焦显微镜下用适当的激发和发射滤光片对血红细胞进行成像：λ ex＝405nm，λ em＝470-600nm。图S4为细胞球的3D重构化图，图A为细胞球的Z轴层切图，每层间距5微米，可以看到染料几乎将细胞球染透；图B为染料在细胞球中x-y、y-z、x-z三个方向的分布状态，可以看到染料在细胞球中染色均匀；图C为细胞球的整体3D重构图，可以看出细胞球的大体形貌；图D为细胞球的整体/局部放大图，在图d5中可以清晰地看到蜂窝状的细胞膜结构。此外，在与商售细胞膜染料DiO进行共定位染色时发现，商售试剂几乎无法在复杂的致密体系中分辨细胞膜(图S5)，这说明本发明中，染料的高灵敏性与高特异性。
试验例8对小鼠脑部的深层次成像
将新生小鼠(48小时内)二氧化碳安乐死，取脑，经透明化处理后，以含有10μM染料的PBS溶液在震荡摇晃的情况下孵育1天，然后将脑置于共聚焦小皿中，在激光共聚焦显微镜下用适当的激发和发射滤光片对血红细胞进行成像：λ ex＝405nm，λ em＝470-600nm。其中，对小鼠脑部毛细血管中红细胞的成像(图S6A)与神经元细胞的横向3D重构图(图S6B)与纵向3D重构图(图S6C)均表明染料对细胞膜的特异性染色，以及在生物体样本中的应用潜力。
试验例9对活体斑马鱼表皮成像
将新生的斑马鱼置于含有10μM染料的斑马鱼培养液中孵育15分钟，然后将斑马鱼置于含有0.003％三卡因甲基磺酸盐(麻醉剂)的斑马鱼培养液中。孵育20秒后，将斑马鱼转移到双凹版上，以含有2.4％甲基纤维素的麻醉液固定后，盖上盖玻片。在激光共聚焦显微镜下用适当的激发和发射滤光片对血红细胞进行成像：λ ex＝405nm，λ em＝470-600nm。图S7C表明染料可以对斑马鱼表皮进行染色，而图S7A与图S7B的放大图则表明染料对于活体斑马鱼表皮细胞细胞膜的高度特异性，说明了本专利中染料在活体成像中应用的潜力。
综上所述，本发明以嘌呤骨架作为细胞膜染料的基础，通过合理的脂溶端亲水端的调控设计，得到了基于嘌呤骨架的细胞膜靶向染色试剂，该类试剂在快速靶向染色的同时，能够较长时间的停滞在细胞膜上，有利于长时的监测。此外，由于该染色试剂具有聚集诱导型化合物的特性，在良溶剂中发光很弱或不发光，在不良溶剂中发出强烈的荧光，使得该类染料还具有免洗的特异表现。本发明的制备方法收率高、反应条件温和，制得的染色试剂斯托克斯位移大、靶向性高。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂，其特征在于，其结构如式(Ⅰ)所示：
    Figure PCTCN2019117083-appb-100001
    式(Ⅰ)中：
    R 1为C1-C20烷基；
    R 2各自独立地为
    Figure PCTCN2019117083-appb-100002
    其中，R各自独立地为C1-C10烷基链或芳香基团，Ar为芳香基团；
    R 3为C1-C20烷基或带电荷的烷基季铵链。
  2. 根据权利要求1所述的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂，其特征在于，Ar为苯环、呋喃或噻吩。
  3. 根据权利要求1所述的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂，其特征在于，R 3为
    Figure PCTCN2019117083-appb-100003
    其中，n＝0-8。
  4. 根据权利要求1所述的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂，其特征在于，R 1为C1-C20烷基，R 2为
    Figure PCTCN2019117083-appb-100004
    R 3为C1烷基或带电荷的烷基季胺链。
  5. 根据权利要求4所述的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂，其特征在于，R 3为
    Figure PCTCN2019117083-appb-100005
    其中n＝0-3。
  6. 根据权利要求1所述的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂，其特 征在于，R 1为
    Figure PCTCN2019117083-appb-100006
    n＝1或3；R 2为
    Figure PCTCN2019117083-appb-100007
    R 3为
    Figure PCTCN2019117083-appb-100008
    n＝0。
  7. 权利要求1-6中任意一项所述的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂的制备方法，其特征在于，所述制备方法包括：
    (1)将2,6-二氯嘌呤和R 1的卤代物R 1-X溶于第一有机溶剂中，加入第一弱碱加热搅拌，得到第一中间体；其中，X为Cl，Br或I；
    (2)将所述第一中间体加入至含有第二弱碱的第二有机溶剂中搅拌，然后加入R 2取代的
    Figure PCTCN2019117083-appb-100009
    加热搅拌，得到第二中间体；
    (3)将2,4-甲酰基苯硼酸和催化剂溶于水和有机溶剂的混合溶剂中，加入第三弱碱，然后加入所述第二中间体混合，回流，得到第三中间体；
    (4)将所述第三中间体与R 3取代的
    Figure PCTCN2019117083-appb-100010
    溶于第三有机溶剂，加入第四弱碱，搅拌，制得基于嘌呤骨架的聚集诱导型细胞膜靶向染色试剂。
  8. 根据权利要求7所述的制备方法，其特征在于，
    步骤(1)中，所述第一有机溶剂选自DMSO和DMF中的一种或两种组合，所述第一弱碱选自碳酸钠、碳酸钾、磷酸钾和磷酸钠中的一种或多种组合；
    步骤(2)中，所述第二有机溶剂为二氧六环和四氢呋喃中的一种或两种组合，所述第二弱碱选自正丁基锂、叔丁醇钾、叔丁醇钠、氢化钾、氢化钠、碳酸钾和碳酸钠中的一种或多种组合；
    步骤(3)中，所述催化剂为四三苯基磷钯，所述混合溶剂为四氢呋喃和水或者二氧六环和水的混合溶剂，所述第三弱碱选自碳酸钾、碳酸钠、磷酸钾和磷酸钠中的一种或多种组合；
    步骤(4)中，所述第三有机溶剂选自二氯甲烷、四氢呋喃、乙醇、甲醇、N,N-二甲基甲酰胺和醋酸酐中的一种或多种组合，所述第四弱碱为醋酸钠和哌啶中的一种或两种组合。
  9. 根据权利要求7所述的制备方法，其特征在于，步骤(1)的加热温度为50-120℃，步骤(2)的加热温度为50-100℃。
  10. 权利要求1-6中任意一项所述的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂或者由权利要求7-9中任意一项所述的制备方法制备的基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂在生物体细胞膜上进行荧光成像中的应用。
PCT/CN2019/117083 2019-01-28 2019-11-11 基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途 WO2020155743A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910079970.8A CN109722059B (zh) 2019-01-28 2019-01-28 基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途
CN201910079970.8 2019-01-28

Publications (1)

Publication Number Publication Date
WO2020155743A1 true WO2020155743A1 (zh) 2020-08-06

Family

ID=66301224

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/117083 WO2020155743A1 (zh) 2019-01-28 2019-11-11 基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途

Country Status (2)

Country Link
CN (1) CN109722059B (zh)
WO (1) WO2020155743A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112341463B (zh) * 2020-03-17 2022-01-28 江苏科技大学 一种基于嘌呤母体的荧光探针化合物及其制备方法和应用
CN111533730B (zh) * 2020-04-28 2023-02-10 皖南医学院 一种免洗型细胞膜靶向荧光探针及其制备方法和应用
CN111393441B (zh) * 2020-04-28 2021-05-04 四川大学 基于嘌呤骨架的聚集诱导型脂滴靶向染色试剂及其制备方法和应用
CN113214673B (zh) * 2021-04-13 2022-04-05 大连理工大学 基于吡唑啉酮的糖蛋白结合型膜染料、其制备方法及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105367566A (zh) * 2015-11-30 2016-03-02 四川大学 取代的香豆素-噻唑橙衍生物及其制备方法和用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101148448B (zh) * 2006-09-20 2010-08-04 浙江医药股份有限公司新昌制药厂 N2-喹啉取代的嘌呤衍生物的制备方法
WO2010005558A2 (en) * 2008-07-07 2010-01-14 Xcovery, Inc. Pi3k isoform selective inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105367566A (zh) * 2015-11-30 2016-03-02 四川大学 取代的香豆素-噻唑橙衍生物及其制备方法和用途

Also Published As

Publication number Publication date
CN109722059B (zh) 2020-02-07
CN109722059A (zh) 2019-05-07

Similar Documents

Publication Publication Date Title
WO2020155743A1 (zh) 基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途
CN108484590A (zh) 一种基于咔唑的双光子粘度荧光探针及其制备方法和用途
CN111393441B (zh) 基于嘌呤骨架的聚集诱导型脂滴靶向染色试剂及其制备方法和应用
CN110981842B (zh) 一种区分正常细胞和癌细胞的特异性检测脂滴的荧光探针及应用
EP3798221A1 (en) Fluorescent probe and preparation method and use thereof
KR101715543B1 (ko) 메가스토크스 아미노-트리아졸릴-bodipy 화합물 및 살아있는 뉴런 염색 및 인간 혈청 알부민 fa1 약물 부위 프로빙에 대한 적용
EP2778161B1 (en) Two-photon fluorescent probe using naphthalene as matrix and preparation method and use thereof
CN108299438B (zh) pH响应性近红外荧光探针化合物及其制备方法和应用
CN107746367A (zh) β‑石竹烯衍生物及其制备方法与应用
CN105367566B (zh) 取代的香豆素-噻唑橙衍生物及其制备方法和用途
CN106634964A (zh) 噁嗪类化合物在制备近红外荧光探针中的应用
CN109503550B (zh) 2-氮杂芳基-6-取代氨基喹唑啉酮化合物及其制备方法和应用
CN109369569B (zh) 一类检测丙酮醛的荧光探针及其制备方法和应用
CN115160327B (zh) 一种靶向μ阿片受体的小分子荧光探针及其制备和应用
CN110357896A (zh) 一类化合物及制备与其在检测二价铜离子和强酸pH中的应用
CN109280017A (zh) 一种双光子荧光高尔基体定位剂及其制备方法和应用
CN114230494A (zh) 大斯托克斯位移近红外荧光探针的合成及其在检测硫化氢中的应用
CN111961072B (zh) 一种溶酶体靶向的红外二窗发射荧光染料及其制备方法和应用
CN114805262A (zh) 一种粘度和极性响应型平台荧光探针、硫化氢检测荧光探针及其合成工艺与应用
CN108440386B (zh) 一种双光子荧光pH探针的制备方法及其在细胞成像中的应用
CN112341453A (zh) 一种基于香豆素的荧光探针及其制备方法和应用
JP2016193897A (ja) ｐＨ依存性蛍光化合物
EP3000848B1 (en) Compound for generating second harmonic of light, dye composition for generating second harmonic of light, and cell examination method
CN112574240B (zh) Egfr特异识别的荧光化合物，其制备方法和用途
WO2020246616A1 (ja) Ｅｎｐｐ活性検出用蛍光プローブ

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19913844

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19913844

Country of ref document: EP

Kind code of ref document: A1