CN108129365A - 一种近红外检测半胱氨酸的荧光探针、其制备方法及应用 - Google Patents
一种近红外检测半胱氨酸的荧光探针、其制备方法及应用 Download PDFInfo
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- CN108129365A CN108129365A CN201810004650.1A CN201810004650A CN108129365A CN 108129365 A CN108129365 A CN 108129365A CN 201810004650 A CN201810004650 A CN 201810004650A CN 108129365 A CN108129365 A CN 108129365A
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- fluorescence
- cysteine
- piperazine
- probe
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
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- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 34
- 238000006243 chemical reaction Methods 0.000 claims description 32
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- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
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- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 4
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 4
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- 229910021380 Manganese Chloride Inorganic materials 0.000 description 4
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 4
- 229910001626 barium chloride Inorganic materials 0.000 description 4
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 4
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
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- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 4
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- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
- C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
- C07C311/21—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
- C07C303/36—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids
- C07C303/38—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of amides of sulfonic acids by reaction of ammonia or amines with sulfonic acids, or with esters, anhydrides, or halides thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/10—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by doubly bound oxygen or sulphur atoms
- C07D295/112—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by doubly bound oxygen or sulphur atoms with the ring nitrogen atoms and the doubly bound oxygen or sulfur atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D295/155—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- 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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- 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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- C—CHEMISTRY; METALLURGY
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- Chemical & Material Sciences (AREA)
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Abstract
本发明提供了一种具有背景干扰降低、样品穿透性强、选择性好、灵敏度高和成像分辨率好等优点的荧光“关‑开”型近红外检测半胱氨酸的荧光探针、其制备方法及应用。所述荧光探针以一种含多个双键的近红外荧光母核作为荧光团,以2,4‑二硝基苯磺酰胺部分作为猝灭单元使其荧光猝灭;实际检测中在半胱氨酸存在下,半胱氨酸的巯基对缺电子芳环发生亲核取代反应,磺酰胺键裂解,荧光猝灭剂解离,使得基于分子内电荷转移(ICT)过程的本身无荧光或弱荧光的探针分子释放出荧光母核,荧光增强或打开,产生选择性识别半胱氨酸荧光信号,从而达到对半胱氨酸(Cys)选择性识别和分析检测的目的。
Description
技术领域
本发明涉及化学分析检测技术领域,具体涉及一种荧光“关-开”型近红外检测半胱氨酸的荧光探针、其制备方法及应用。
背景技术
生物体内存在着多种重要的小分子巯基化合物,而半胱氨酸(Cys)是唯一含巯基(-SH)的天然必需氨基酸,是合成蛋白质的重要组分。半胱氨酸在生物体内的含量与许多生理疾病有着密切的联系,如阿尔兹海默病、心血管疾病等。因此,可以把半胱氨酸的含量高低作为这类疾病诊断的依据之一,同时,详尽认识半胱氨酸(Cys)的产生、分布及水平波动对细胞活性、机体生理和病理等研究有着重要的科学意义(吕建政.细胞内小分子巯基化合物检测的近红外荧光探针[D].山东师范大学,2014.)。
目前,应用于检测半胱氨酸的方法主要有高效液相色谱法、质谱法、毛细管电泳法、荧光检测法和电化学检测法等,荧光成像作为一种直观、原位的可视化观测技术在小分子检测方面得到了广泛应用,但是由于生物体及组织细胞中的某些成分,如胆红素、还原性烟酰胺腺嘌呤二核苷酸等会被激发产生自发荧光,与此同时生物样本的散射光强度大,严重干扰荧光检测及成像。由于细胞和组织的自发荧光在近红外波段最小,因此在检测复杂生物系统时,近红外荧光探针能提供更高的特异性和灵敏度,对细胞损伤小且具有更低的背景干扰。同时,由于光波在近红外区段的组织样品穿透性强,因此近红外荧光探针尤其适合体内荧光成像(活体成像)这一近几年迅速发展的新兴领域。目前对半胱氨酸(Cys)检测的探针多集中于可见光区或依赖灵敏度低的紫外-可见吸收法检测,甚至不能有效区分半胱氨酸和高半胱氨酸(如中国专利CN105693600A,CN105820810A,CN104447421A,Anal.Chem.,2016,88(14),pp 7178–7182),因其荧光发射和UV-vis吸收波长处在相对较短的波长区域,对生物成像应用有严重的限制,但是高灵敏度,特异性和专一性检测半胱氨酸(Cys)的近红外小分子荧光探针却鲜有报道。
发明内容
为了解决现有技术存在的问题,本发明提供了一种具有背景干扰降低、样品穿透性强、选择性好、灵敏度高和成像分辨率好等优点的荧光“关-开”型近红外检测半胱氨酸的荧光探针、其制备方法及应用。
本发明的目的是提供一种近红外检测半胱氨酸的荧光探针。
本发明的再一目的是提供所述荧光探针的制备方法。
本发明的再一目的是提供所述荧光探针的应用。
一种近红外检测半胱氨酸的荧光探针,所述荧光探针的结构为:
所述荧光探针以一种含多个双键的近红外荧光母核作为荧光团,以2,4-二硝基苯磺酰胺部分作为猝灭单元使其荧光猝灭;实际检测中在半胱氨酸存在下,半胱氨酸的巯基对缺电子芳环发生亲核取代反应,磺酰胺键裂解,荧光猝灭剂解离,使得基于分子内电荷转移(ICT)过程的本身无荧光或弱荧光的探针分子释放出荧光母核,荧光增强或打开,产生选择性识别半胱氨酸荧光信号,从而达到对半胱氨酸(Cys)选择性识别和分析检测的目的。
本发明还提供了所述荧光探针的制备方法,所述制备方法包括以下步骤:
步骤一、化合物4-哌嗪-1-苯甲醛的合成
a.在反应瓶中加入哌嗪、水、乙二醇单甲醚,将对氟苯甲醛溶于乙二醇单甲醚并置于漏斗中,室温搅拌下缓慢加入反应瓶中,滴加完毕后,回流搅拌反应3-5小时;
b.将步骤a中的反应液冷却至室温,再将反应液倒入水中,过滤,然后将滤饼中加入水中,加入盐酸溶液,过滤除去不溶物,滤液用氢氧化钠溶液调节pH至10,使用二氯甲烷(DCM)萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压旋干,石油醚重结晶,过滤,真空干燥得4-哌嗪-1-苯甲醛;
步骤二、化合物(E)-2-(5,5-二甲基-3-(4-(哌嗪-1-基)苯乙烯基)环己-2-烯-1-亚基)丙二腈的合成
Ⅰ.向反应瓶中加入4-哌嗪-1-苯甲醛、2-(3,5,5-三甲基环己-2-烯亚基)丙二腈、乙醇和催化量的哌啶,搅拌回流反应至完全;
Ⅱ.将步骤Ⅰ中的反应液冷却至室温,过滤,滤饼用乙醇洗涤,石油醚洗涤,真空干燥;
步骤三、探针(E)-2-(3-(4-(4-((2,4-二硝基苯基)磺酰基)哌嗪-1-基)苯乙烯基)-5,5-二甲基环己-2-烯-1-亚基)丙二腈的合成
A.惰性气体保护下,向反应瓶中加入(E)-2-(5,5-二甲基-3-(4-(哌嗪-1-基)苯乙烯基)环己-2-烯-1-亚基)丙二腈,加入无水二氯甲烷,冷至0℃,再加入2,4-二硝基苯磺酰氯和干燥重蒸的三乙胺,搅拌反应,并自然升温至室温;
B.将步骤A中的反应液减压浓缩除去溶剂二氯甲烷,柱分离得目标探针分子。
优选的,步骤b中,盐酸溶液的浓度为10%,氢氧化钠溶液的浓度为20%。
优选的,所述反应瓶为圆底烧瓶。
优选的,步骤a中哌嗪和对氟苯甲醛摩尔比为3-4:1。
优选的,步骤Ⅰ中4-哌嗪-1-苯甲醛和2-(3,5,5-三甲基环己-2-烯亚基)丙二腈的摩尔比为1:1-1.05。
优选的,步骤A中2-(3,5,5-三甲基环己-2-烯亚基)丙二腈和2,4-二硝基苯磺酰氯的摩尔比为1:1-1.05。
本发明还提供了所述荧光探针检测半胱氨酸的用途。
进一步的,所述荧光探针在细胞内半胱氨酸荧光成像中的用途。
利用所述荧光探针,使用裸眼或荧光检半胱氨酸。
所述荧光探针的具体使用方式为:将所述荧光探针用二甲基亚砜(DMSO)溶解,探针分子在磷酸缓冲液(PBS,10mM,pH=8)中时其最大紫外吸收波长在450nm,加入半胱氨酸(Cys)后在37–C反应2小时,反应液在450nm处的吸收峰明显增强,随着半胱氨酸(Cys)浓度的增高,吸收峰逐渐上升;而且,探针分子在磷酸缓冲液(PBS,10mM,pH=8)中时荧光很弱,当加入半胱氨酸(Cys)后在37–C反应2小时,反应液在658nm处的荧光明显增强(约100倍),说明其对半胱氨酸的响应效果好,荧光发射波长为近红外区域,背景干扰小,成像分辨率高,非常有利于生物细胞成像分析。
同时该分子探针不受其他离子和氨基酸的干扰,如:FeCl3,MgCl2,CdCl2,CoCl2,BaCl2,CuCl2,AgNO3,ZnCl2,MnCl2,Hg(NO3)2,NaBr,NaF,N2H4,GSH,L-谷氨酸,L-脯氨酸,L-天冬氨酸,L-酪氨酸,DL-组氨酸,2-氨基丙酸等,荧光发射波长处于近红外波段,背景干扰低、样品穿透性强、灵敏度和成像分辨率好。
本发明的有益效果为:
1、本发明提供了一种具有背景干扰降低、样品穿透性强、选择性好、灵敏度高和成像分辨率好等优点的荧光“关-开”型近红外检测半胱氨酸的荧光探针,所述荧光探针的荧光强度与检测物半胱氨酸(Cys)浓度呈线性变化,可对半胱氨酸的含量进行定量检测,检测限低至32nM,使得该荧光探针在生物化学领域具有潜在的应用价值。
2、本发明还提供了所述荧光探针的制备方法,所述制备方法能够制备出所述选择性好、灵敏度高的近红外检测半胱氨酸的荧光探针。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为纯探针(25μM)和探针加入Cys(50μM)的荧光谱图,λex=430nm,λem=658nm。图1表示:所述荧光探针为“关-开”型探针。
图2为探针(25μM)中加入不同浓度Cys的荧光谱图,自下而上Cys浓度依次为0,10,20,25,50,125,150,375,500,750μM。图2表示:所述荧光探针的荧光强度与检测物(Cys)的浓度呈线性关系,可对半胱氨酸的含量进行定量检测。图中,由于下方几条线有部分重合,特作出说明:最下方有两条线有部分重合,其上方有三条线部分重合。
图3为探针(25μM)和探针(25μM)加入50μM不同干扰物的荧光谱图(λex=430nm,λem=658nm),从1到22所加的物质分别为空白(纯探针),Cys,FeCl3,MgCl2,CdCl2,CoCl2,BaCl2,CuCl2,AgNO3,ZnCl2,MnCl2,Hg(NO3)2,NaBr,NaF,N2H4,GSH,L-谷氨酸,L-脯氨酸,L-天冬氨酸,L-酪氨酸,DL-组氨酸,2-氨基丙酸。图3表示:所述荧光探针的选择性很好,只对半胱氨酸有响应。
图4为探针(25μM)加入Cys(50μM)后再加入不同干扰物的荧光谱图(λex=430nm,λem=658nm),从1到22所加的物质分别为空白,Cys,FeCl3,MgCl2,CdCl2,CoCl2,BaCl2,CuCl2,AgNO3,ZnCl2,MnCl2,Hg(NO3)2,NaBr,NaF,N2H4,GSH,L-谷氨酸,L-脯氨酸,L-天冬氨酸,L-酪氨酸,DL-组氨酸,2-氨基丙酸,且4-12干扰物的浓度为1mM,4-22干扰物的浓度为10mM。图4表示:所述荧光探针与半胱氨酸反应后的生成物也很稳定,不受其他物质干扰。
图5为本发明的荧光探针分子的核磁共振氢谱图。图5表示:所述荧光探针分子的结构。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
实施例1
一种荧光探针的制备方法,所述制备方法包括以下步骤:
具体实验操作如下:
4-哌嗪-1-苯甲醛的合成:
在100mL两口圆底烧瓶中加入15g(174.14mmol)哌嗪,然后加入18mL水和25mL乙二醇甲醚;用量筒量取5mL(46.61mmol)对氟苯甲醛,然后加入恒压滴液漏斗中,加入5mL乙二醇甲醚与之混合,搅拌下缓慢滴加入上述反应瓶中;室温滴加完毕后加热回流反应4小时;待反应液冷却至室温,将反应液倒入200mL水中,过滤。将滤饼加入150mL10%盐酸溶液中搅拌10分钟,过滤除去不溶物,用20%的氢氧化钠溶液将溶液pH调至10,DCM萃取(80mL×3),饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压旋干;石油醚重结晶得黄色固体6g,产率为60%。1H NMR(500MHz,Chloroform-d)δ9.76(s,1H),7.747-7.730(d,J=8.5Hz,2H),6.910-6.892(d,J=9.0Hz,2H),3.357-3.338(t,4H),3.016-2.996(t,4H);
(E)-2-(5,5-二甲基-3-(4-(哌嗪-1-基)苯乙烯基)环己-2-烯-1-亚基)丙二腈的合成:
向50mL圆底烧瓶中加入0.5g(2.63mmol)4-(哌嗪-1-基)苯甲醛,0.5g(2.68mmol)2-(3,5,5-三甲基环戊-2-烯-1-亚基)丙二腈,乙醇20mL,搅拌回流反应至完全;反应完毕后将反应液冷却至室温,过滤,滤饼用冷乙醇洗涤3次,石油醚洗涤3次,真空干燥得红色固体0.82g,产率为87%。1H NMR(500MHz,Chloroform-d)δ7.427-7.409(d,J=9.0Hz,2H),7.020-6.988(d,J=16.0Hz,1H),6.891-6.852(t,3H),6.820-6.788(d,J=16.0Hz,1H),3.281 3.261(t,4H),3.043 3.023(t,4H),2.570(s,2H),2.446(s,2H),1.063(s,6H);
探针(E)-2-(3-(4-(4-((2,4-二硝基苯基)磺酰基)哌嗪-1-基)苯乙烯基)-5,5-二甲基环己-2-烯-1-亚基)丙二腈((E)-2-(3-(4-(4-((2,4-dinitrophenyl)sulfonyl)piperazin-1-yl)styryl)-5,5-dimethylcycl ohex-2-en-1-ylidene)malononitrile)的合成:
氩气保护下,向25mL两口烧瓶中加入0.36g(1mmol)(E)-2-(5,5-二甲基-3-(4-(哌嗪-1-基)苯乙烯基)环己-2-烯-1-亚基)丙二腈,使用注射器加入15mL无水二氯甲烷并冷至0℃左右,迅速称取0.27g(1.01mmol)2,4-二硝基苯磺酰氯,加入反应瓶中并向其中滴加3滴已重蒸的三乙胺,搅拌反应并自然升温至室温;待反应完毕后减压浓缩除去溶剂二氯甲烷,柱层析分离得红色固体267mg,产率为45%。1H NMR(500MHz,Chloroform-d)δ8.5(dd,J=8.6,2.3Hz,1H),8.50(d,J=2.2Hz,1H),8.27(d,J=8.6Hz,1H),7.45(dd,J=9.2,2.6Hz,2H),7.01(d,J=15.9Hz,1H),6.93-6.84(m,3H),6.81(s,1H),3.63-3.35(m,8H),2.60(s,2H),2.47(s,2H),1.09(s,6H)。
实施例2
本实施例和实施例1的流程基本相同,其中区别点为:
哌嗪和对氟苯甲醛摩尔比为3:1(139.83mmol:46.61mmol);
4-哌嗪-1-苯甲醛和2-(3,5,5-三甲基环己-2-烯亚基)丙二腈的摩尔比为1:1.05(2.63mmol:2.76mmol);
2-(3,5,5-三甲基环己-2-烯亚基)丙二腈和2,4-二硝基苯磺酰氯的摩尔比为1:1.05(1mmol:1.05mmol)。
本实施例中,最终的结果和实施例1中的实验产物相同,说明在上述配比下,也能够顺利合成所述荧光探针。
实施例3
本实施例和实施例的流程基本相同,其中区别点为:
哌嗪和对氟苯甲醛摩尔比为4:1(186.44mmol:46.61mmol);
4-哌嗪-1-苯甲醛和2-(3,5,5-三甲基环己-2-烯亚基)丙二腈的摩尔比为1:1(2.63mmol:2.63mmol);
2-(3,5,5-三甲基环己-2-烯亚基)丙二腈和2,4-二硝基苯磺酰氯的摩尔比为1:1.05(1mmol:1mmol)。
本实施例中,最终的结果和实施例1中的实验产物相同,说明在上述配比下,也能够顺利合成所述荧光探针。
实施例4
本实施例为所述荧光探针的应用举例:
实验结果如图1-5所述。
将所述荧光探针用二甲基亚砜(DMSO)溶解,配制1mM的探针溶液,取探针溶液50μL,加入相应的磷酸缓冲液(PBS,10mM,pH=8)配成探针浓度为25μM的溶液,当需要加入检测物时,保持探针的浓度为25μM和每个样品溶液体积为2mL,调整加入检测物和PBS缓冲液的体积;测试其紫外吸收光谱和荧光发射光谱变化情况。纯探针溶液的最大紫外吸收波长在450nm,加入半胱氨酸(Cys)后在37–C反应2小时,反应液在450nm处的吸收峰明显增强,随着半胱氨酸(Cys)浓度的增高,吸收峰逐渐上升;而且,纯探针分子在磷酸缓冲液(PBS,10mM,pH=8)中时荧光很弱,当加入半胱氨酸(Cys)后在37–C反应2小时,反应液在658nm处的荧光明显增强(λex=430nm),说明其对半胱氨酸的响应性极好,荧光发射波长为近红外区域,背景干扰小,成像分辨率高,非常有利于生物细胞成像分析。同时该分子探针不受其他离子和氨基酸的干扰,如:FeCl3,MgCl2,CdCl2,CoCl2,BaCl2,CuCl2,AgNO3,ZnCl2,MnCl2,Hg(NO3)2,NaBr,NaF,N2H4,GSH,L-谷氨酸,L-脯氨酸,L-天冬氨酸,L-酪氨酸,DL-组氨酸,2-氨基丙酸等。同时,半胱氨酸的荧光探针的荧光强度与检测物半胱氨酸(Cys)浓度呈线性变化,可对半胱氨酸的含量进行定量检测,检测限低至32nM,使得该荧光探针在生物化学领域极具应用价值。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.一种近红外检测半胱氨酸的荧光探针,其特征在于,所述荧光探针的结构为:
。
2.权利要求1所述荧光探针的制备方法,其特征在于,所述制备方法包括以下步骤:
步骤一、化合物4-哌嗪-1-苯甲醛的合成
a.在反应瓶中加入哌嗪、水、乙二醇单甲醚,将对氟苯甲醛溶于乙二醇单甲醚并置于漏斗中,室温搅拌下缓慢加入反应瓶中,滴加完毕后,回流搅拌反应3-5小时;
b.将步骤a中的反应液冷却至室温,再将反应液倒入水中,过滤,然后将滤饼中加入水中,加入盐酸溶液,过滤除去不溶物,滤液用氢氧化钠溶液调节pH至10,使用二氯甲烷(DCM)萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,过滤,减压旋干,石油醚重结晶,过滤,真空干燥得4-哌嗪-1-苯甲醛;
步骤二、化合物(E)-2-(5,5-二甲基-3-(4-(哌嗪-1-基)苯乙烯基)环己-2-烯-1-亚基)丙二腈的合成
Ⅰ.向反应瓶中加入4-哌嗪-1-苯甲醛、2-(3,5,5-三甲基环己-2-烯亚基)丙二腈、乙醇和催化量的哌啶,搅拌回流反应至完全;
Ⅱ.将步骤Ⅰ中的反应液冷却至室温,过滤,滤饼用乙醇洗涤,石油醚洗涤,真空干燥;
步骤三、探针(E)-2-(3-(4-(4-((2,4-二硝基苯基)磺酰基)哌嗪-1-基)苯乙烯基)-5,5-二甲基环己-2-烯-1-亚基)丙二腈的合成
A.惰性气体保护下,向反应瓶中加入(E)-2-(5,5-二甲基-3-(4-(哌嗪-1-基)苯乙烯基)环己-2-烯-1-亚基)丙二腈,加入无水二氯甲烷,冷至0℃,再加入2,4-二硝基苯磺酰氯和干燥重蒸的三乙胺,搅拌反应,并自然升温至室温;
B.将步骤A中的反应液减压浓缩除去溶剂二氯甲烷,柱分离得目标探针分子。
3.根据权利要求2所述的制备方法,其特征在于,步骤b中,盐酸溶液的浓度为10%,氢氧化钠溶液的浓度为20%。
4.根据权利要求2所述的制备方法,其特征在于,所述反应瓶为圆底烧瓶。
5.根据权利要求2所述的制备方法,其特征在于,步骤a中哌嗪和对氟苯甲醛摩尔比为3-4:1。
6.根据权利要求2所述的制备方法,其特征在于,步骤Ⅰ中4-哌嗪-1-苯甲醛和2-(3,5,5-三甲基环己-2-烯亚基)丙二腈的摩尔比为1:1-1.05。
7.根据权利要求2所述的制备方法,其特征在于,步骤A中2-(3,5,5-三甲基环己-2-烯亚基)丙二腈和2,4-二硝基苯磺酰氯的摩尔比为1:1-1.05。
8.权利要求1所述的荧光探针检测半胱氨酸的用途。
9.根据权利要求8所述的用途,其特征在于,所述荧光探针在细胞内半胱氨酸荧光成像中的用途。
10.根据权利要求8所述的用途,其特征在于,利用所述荧光探针,使用裸眼或荧光检半胱氨酸。
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