CN108147992A - 可标记深红荧光活性酯 - Google Patents
可标记深红荧光活性酯 Download PDFInfo
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- CN108147992A CN108147992A CN201611113245.0A CN201611113245A CN108147992A CN 108147992 A CN108147992 A CN 108147992A CN 201611113245 A CN201611113245 A CN 201611113245A CN 108147992 A CN108147992 A CN 108147992A
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- ir640b
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- fluorophor
- ester
- biomolecule
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- 150000002148 esters Chemical class 0.000 title claims abstract description 28
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- 238000002372 labelling Methods 0.000 claims abstract description 17
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- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims abstract description 16
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
-
- 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
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
-
- 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
- C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
-
- 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
- C09B23/12—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being branched "branched" means that the substituent on the polymethine chain forms a new conjugated system, e.g. most trinuclear cyanine 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
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Abstract
本发明属荧光成像试剂领域,涉及可对生物分子进行快速标记的活性深红荧光活性酯IR640B‑NHS及其前体IR640B,本发明中以2,3,3‑三甲基吲哚为母体,与丁磺酸内脂等原料反应,合成带磺酸基的花菁类荧光基团,再通过Suzuki‑Miyaura反应,将苯基羧酸通过碳‑碳键引入到荧光基团,通过对苯基羧酸修饰生成N‑羟基琥珀酰亚胺酯。所述活性酯在生理条件下与生物大分子中的伯胺基反应,将深红荧光基团通过酰胺键标记到目标分子上。本发明可实现对生物大分子如多肽、蛋白、抗体或聚合物分子进行快速、安全、有效、稳定的标记,可利用荧光显微镜、流式细胞仪等对目标生物大分子的受体靶向性、细胞内分布进行体外评价,和通过活体光学成像对目标分子无创监测和定量示踪。
Description
技术领域
本发明属荧光成像试剂领域,涉及可标记深红荧光活性酯,具体涉及一类可对目标生物分子进行快速标记的活性深红荧光活性酯及其前体。本发明提供了该类活性深红荧光活性酯及其前体的合成、表征、光学性质测定和在生物分子标记中的应用。
背景技术
现有技术公开了所谓成像系为将一些无法用肉眼直接观察到的现象或者过程通过一定的途径转换为可视化的影像,从而方便被观察者对其进行观察和研究。据报道,德国物理学家伦琴在无意中首次发现X射线是能够穿透人体进行成像的射线,才有了之后的科学家对成像学的重视以及进一步的研究与应用,这即当代学术界炙手可热的成像学的起源。之后的事实证实了射线的发现为现代的生物医学领域打开了一扇大门,随之而来的是无数新的发现以及相关领域的发展,其中包括目前发展多变的影像学。
医学影像学是由生命科学和医学应用交叉组成的关键技术科学,其中,分子影像技术凭借其高靶向性及高灵敏度的独特优势,成为一门涉及影像学和现代分子生物学的新兴学科,在疾病早期诊断及治疗,实时的检测和监控等方面均有广泛应用;同时呈现出多极化发展的趋势,包括:光学成像、PET(正电子发射断层摄影术)、SPECT(单光子发射计算机断层摄影术)、X-CT(计算机X射线断层摄影术)、MRI(磁共振成像)等。研究实践显示,对比分子用影像学中的其他手段,光学成像以其组织穿透力强、灵敏度高、成像迅速的优点,被广泛用于肿瘤受体成像,心血管疾病成像等活体动态成像。
花菁类荧光染料是一种经典的荧光染料,因为其拥有消光系数大,荧光吸收和发射光谱易于调节,生物相容性好等优势,在生物荧光示踪技术中展现出巨大的潜力,其中,五甲川花菁类染料吸收和发射波长均处于600-700nm之间,接近近红外区域,被视为波长最短的近红外菁染料,在分子识别,蛋白标记,药物代谢分布研究,活体及细胞成像等领域展现出巨大的应用价值。
迄今,尚未上述荧光染料用于生物活性分子标记的深红荧光探针的报道。
基于现有技术的现状,本申请的发明人拟提供可标记深红荧光活性酯,本发明中采用花菁类深红荧光基团为母体,构建可以在生理中性条件下通过酰胺键标记到生物分子上的活性深红荧光基团及其前体。与现有的商品化深红荧光基团相比,本申请中的活性基团具有标记条件温和、吸光系数及量子产率高、荧光波长适合等优点。
发明内容
本发明的目的在于针对现有技术的现状,提供可标记深红荧光活性酯,具体涉及一类可对目标生物分子进行快速标记的活性深红荧光活性酯及其前体。
本发明中构建了一类活性深红荧光基团,此类基团可以在生理条件下通过酰胺键标记到生物分子上,从而在活体状态下实现对该类生物活性分子的无创示踪。
本发明基于光学成像中活体光学成像(In vivo optical imaging)的荧光技术(Fluorescence),通过共价键结合等手段,将荧光基团与待测生物分子或者靶细胞结合,之后通过光学成像仪器检测出的信号判断疾病情况、肿瘤转移有无、体内核酸变化等体内相关生物物质的变化。所述荧光基团具有标记率高、标记条件温和、荧光基团吸光系数和量子产率高、光化学性质稳定等特点,通过该类荧光基团的标记可实现目标生物分子在活体内分布的无创监测和定量。
具体的,本发明构建了一类可对生物分子进行快速标记的活性深红荧光活性酯(IR640B-NHS)及其前体(IR640B);
所述的可对生物分子进行快速标记的活性深红荧光活性酯及其前体,其通式如下:
IR640B和IR640B-NHS
其中IR640为花菁类深红荧光基团;B为荧光基团仲位通过碳-碳键引入的
芳香基团;NHS为N-羟基琥珀酰亚胺酯;
所述的对生物分子进行快速标记的活性深红荧光基团及其前体,其结构式如下:
其中,R1为H,卤素,烷基,芳香基,硝基,磺酸基,醛基或羧基;
R2为H,羧基,或者磺酸基;
n是1,2,3,4,5,6,7或8。
本发明中,卤素包括氯,溴或碘。
本发明中,烷基包括甲基,乙基,丙基,异丙基,丁基,异丁基。
本发明中,芳香基包括苯基,萘基,取代苯基。
本发明中,通过C-C键直接与对羧基苯环相连的方法修饰深红荧光染料。
本发明中,所述的活性深红荧光基团,以花菁类染料IR640类荧光基团为母体,利用Suzuki-Miyaura反应,也称作Suzuki偶联反应,在零价钯配合物催化下,通过羧基苯硼酸与IR640上的烯丙基溴反应将对苯基羧酸通过碳-碳键(C-C键)引入到荧光基团中;再通过对苯基羧酸修饰,生成N-羟基琥珀酰亚胺(NHS)活性酯;该活性酯可在生理中性条件下与生物分子中的伯氨基反应,将深红荧光基团通过生物稳定的酰胺键标记到目标分子生物活性分子上;
本发明中,使用一种参比荧光基团比较及衡量目标探针的各项光学性质,该参比基团为Suzuki偶联之前的IR640。
本发明提供了上述活性深红荧光基团及其前体的化学结构和制备方法,其包括,以2,3,3-三甲基吲哚为母体,与丁磺酸内脂等原料反应,合成带磺酸基的花菁类荧光基团;再通过Suzuki-Miyaura反应,将苯基羧酸通过碳-碳键(C-C键)引入到荧光基团中;再通过对苯基羧酸修饰,生成N-羟基琥珀酰亚胺酯(NHS ester);
其中包括步骤:
荧光基团骨架IR640的合成:
1.
2.
3.
目标深红荧光基团IR640B的合成:
4.
目标活性酯IR640B-NHS的合成:
5.
步骤1:在乙醇中,苯胺与粘溴酸发生加成反应;
步骤2:若R2为磺酸基,则2,3,3-三甲基吡啶(包含5位被R1取代的和未取代的)与1,4-丁磺酸內酯在邻二氯苯中发生取代反应,若R2为烷基,羟基或者羧基,则2,3,3-三甲基吡啶(包含5位被R1取代的和未取代的)与碘代的羧酸或者醇以及碘代的烷烃在乙腈中发生取代反应;
步骤3:步骤1制得的产物和步骤2制得的产物在醋酸酐中于60-90℃下,由醋酸钠催化发生加成反应制得IR640骨架;
步骤4:步骤3制得的IR640骨架在水中由四三苯基磷钯催化与对羧基苯硼酸发生suzuki反应制得IR640B;
步骤5:步骤4制得的IR640B在无水DMF中,在EDC活化下与NHS发生酯化反应,制得IR640B-NHS。
本发明提供了上述两类化合物的1)化学结构,2)制备方法,以及下述实施方式中3)生物分子标记的应用。该发明提供了简单易行的IR640B,IR640B-NHS的合成策略,通过此法合成的IR640B以及其活化酯IR640B-NHS具有量子产率高(是ICG的大约19倍),荧光强度好,光稳定性优良等特点;另外因为其吸收/发射波长分别为:640/664nm,可直接应用于当前广泛使用的商品化的流式细胞仪,激光共聚焦显微镜,荧光显微镜,小动物活体荧光成像仪等科研仪器,使其在生物荧光示踪等领域具有突出的价值。除此之外,本发明的实施例中还提供了活性酯IR640B-NHS应用于生物标记的实例和方法,该活性酯可在生理条件下与生物大分子中的伯胺基反应,将深红荧光基团通过生理稳定的酰胺键标记到目标分子上;多个实验结果证实IR640B-NHS可在室温下对包含伯氨基的生物分子进行高效稳定的标记。结果说明IR640B-NHS在进行蛋白,抗体,生物高分子等的标记和荧光示踪上具有极大的应用潜力。
本发明的优点有,
可实现对生物大分子如多肽、蛋白、抗体或聚合物分子进行快速、安全、有效、稳定的标记,不但可以利用荧光显微镜、流式细胞仪等对目标生物大分子的受体靶向性、细胞内分布进行体外评价,而且还能够通过活体光学成像对目标分子进行无创监测和定量示踪。
附图说明
图1:IR640B(红色线条)和IR640(黑色线条)的吸收(左)光谱和发射(右)光谱,分子浓度为1μM。
图2:目标荧光基团IR640B(A)和参比荧光基团IR640(B)以及ICG(C)在溶液中的荧光强度与吸光度比值计算量子产率,(D)线性拟合数据分析和量子产率计算结果。
图3:(A)目标荧光基团IR640B和参比荧光基团IR640在pH 7.4缓冲溶液中的光化学稳定性研究。(B)IR640B,IR640的荧光强度随时间变化。
图4:IR640B-NHS标记树枝状分子[NH2(CH2)2NH2]前后的吸收(左)和发射光谱(右)。
图5:IR640B-NHS标记牛血清白蛋白前后的吸收(左)和发射光谱(右)。
图6:肿瘤细胞摄取[NH2(CH2)2NH]-CONHSIR640后荧光成像结果。
图7:细胞未摄取[NH2(CH2)2NH]-CONHSIR640和已摄取[NH2(CH2)2NH]-CONHSIR640流式细胞数据分析结果。
具体实施方式
通过下述实施例将有助于进一步理解本发明,但并不能以这些实例限制本发明的内容。
实施例1
化合物1的合成:
将粘溴酸(5.94g,23.8mM)溶解于40mL乙醇,将苯胺(4.29g,4.2mL,46.1mM)用20mL乙醇稀释,之后将稀释好的苯胺溶液逐滴加入粘溴酸的乙醇溶液中,大概用时10min。反应容器需在剧烈搅拌以及40℃的条件下进行反应。苯胺加完后,会有二氧化碳产生,直到其产生完全后,才表明反应结束。接着加入冰乙醚50mL,有亮黄色固体析出。过滤,并用冰乙醚洗涤,干燥,即得产物。
实施例2
化合物2的合成:
将2.3.3-三甲基吲哚(2.00g,12.6mM)和丁横酸内酯(5.60g,41.2mM)溶于5.0mL邻二氯苯中,120℃油浴,搅拌,回流反应12h完毕,冷却至室温后,将其滴入450mL乙醚中沉淀,过滤得粗产物。经水溶解后以氯仿萃取3次得水层溶液,冻干得纯品磺酸基吲哚。
实施例3
化合物3的合成:
将化合物2(0.413g,1.40mM)和化合物1(0.267g,0.700mM)及无水乙酸钠(0.116g,1.41mM)溶于13mL乙酸酐中,70℃油浴加热3h。反应完毕冷却后,乙醚沉淀得固体产物。产物以柱层析桂胶(100-120目)纯化,甲醇:二氯甲烷(0-20%)梯度洗脱收集产物。
实施例4
化合物4的合成:
化合物3(0.281g,0.400mM),碳酸钾(0.119g,0.860mM)和对羧基苯硼酸(0.0986g,0.720mM)加入到2mL水中,反应温度调至95摄氏度后,加入[Pd(PPh3)4](27.0mg,0.0217mM)搅拌,反应两小时后,薄层色谱板显示有大极性的产物产生,产物经柱层析纯化(CH2Cl2:CH3OH=10:4),冻干后得宝蓝色固体产物。
实施例5
化合物5的合成:
将化合物4(0.149mg,0.200mM)溶解在3mL无水DMF中,加入NHS(28.0mg,0.243mM),在1-(3-二甲氨基丙基)-3-乙基碳二亚胺(37.0mg,0.194mM)的作用下脱水缩合,于黑暗环境中反应12h,得蓝色溶液。将反应所得的蓝色液体逐滴加入50mL冰乙醚中,抽滤后得到墨蓝色沉淀。将该沉淀用无水乙腈反复快速冲洗2-3次,充分除掉过量的缩合剂。最后,用适量不超过5mL的冰水,将pH值控制在4-6之间,将漏斗上的沉淀迅速冲洗溶解,得到的液体冷冻干燥,得到宝蓝色固体。
实施例6
目标荧光基团IR640B和参比荧光基团IR640的吸收和发射光谱:
首先用pH 7.4的PBS磷酸缓冲溶液将目标荧光基团IR640B和参比荧光基团IR640分别配成1μM的PBS溶液。于室温平衡。取3mL样品加入石英比色皿中(10×10mm),用SHIMADZU UV-2550紫外分光度计测其吸收谱(实验参数为:扫描速度为0.5nm/s,狭缝宽度为5.0nm,测量波长为400-900nm);用SHIMAZDU RF-5301PC荧光分度计测其发射谱(实验参数为:扫描速度设置Medium,激发波长为640nm(IR640)和745nm(IR640B),测量波长为500-900nm,激发/发射狭缝宽度为5/5nm,灵敏度为Low);图1显示了IR640B和IR640的吸收光谱(左)和发射光谱(右),吸收光谱信号收集范围400-900nm,发射光谱激发波长为640nm;其中,左图显示芳香环的引入使吸光度有所提高,右图发射光谱显示芳环的引入大大提高了荧光强度。
实施例7
目标荧光基团IR640和参比荧光基团IR640B的摩尔吸收系数:
根据实施例6荧光基团的单光子吸收光谱和荧光光谱,得到目标荧光基团IR640B和参比荧光基团IR640的最大吸收和最大发射波长,同时根据朗伯—比尔定律计算其摩尔吸光系数;
表1.IR640B和IR640在PBS溶液中的光学参数
结果显示,目标荧光基团IR640B其荧光强度较参比荧光基团IR640有显著增强,荧光强度增加约3倍。
实施例8
目标荧光基团IR640B和参比荧光基团IR640在水溶液中的量子产率:
以ICG(一种深红荧光基团,已被美国FDA批准临床使用)在PBS缓冲溶液中的量子产率计算目标荧光基团IR640B和参比荧光基团IR640在水溶液中的量子产率,每种化合物均稀释到0.25-1.5μM测量,并在其最大吸收波长处激发,荧光强度以曲线下面积计算,以荧光强度对最大波长处吸光度拟合直线(如图2所示);量子产率根据如下等式计算:
在等式中,s和r分别对应参比样品和实验样品。φ代表量子产率。Grad拟合直线斜率。η代表溶剂的折光率,q代表激发波长的校正因子。由于所有的光谱在非常接近的激发波长处测量,因此我们假设qs/qr为1。该方法测量所得量子产率误差在10%以内;
结果显示,相比于商品化的ICG花菁染料,IR640和IR640B的量子产率分别是它的8.4倍和18.9倍,实验证明IR640和IR640B均具有较高的量子产率,在荧光示踪上有很大的应用潜力。
实施例9
目标荧光基团IR640B和参比荧光基团IR640的光化学稳定性研究
将IR640和IR640B配成1μM的PBS(pH=7.4)溶液,取150微升加入96孔板中,于0h,1h,2h,4h,8h,12h,24h在小动物活体荧光成像仪(Maestro2)中进行拍摄,记录荧光强度,用origin软件进行分析,分别得目标荧光基团IR640B和参比荧光基团IR640在pH 7.4缓冲溶液中的光化学稳定性结果,激发波长为633nm,收集波长为680nm,以及IR640B,IR640的荧光强度随时间变化,激发波长:640nm,发射波长:664nm(如图3所示);
通过origin软件定量分析后表明,经Suzuki反应生成IR640B以及IR640的光学稳定性均十分良好,但前者荧光强度约为IR640的3倍,因此,此类碳-碳键修饰的荧光基团更适合于标记目标生物活性分子,并对其在活体内分布进行无创监测。
实施例10
IR640B-NHS标记树枝状分子(PAMAM)后的吸收与发射测量
取分子量为28000的树枝状分子(PAMAM G5)[NH2(CH2)2NH2]20mg溶于2-3mL pH为7.4的PBS缓冲液中;另外取2mg目标分子IR640B-NHS溶于200μL的DMF中,最后将此溶液,逐滴滴加至前述已配好的含PAMAM的PBS溶液中,置于摇床上过夜反应;反应完后,透析24h之后进行冻干,取少量固体进行吸收与发射测量(实验参数见实施例8),实验结果如图4所示,标记后的Dendrimer分子式为[NH2(CH2)2NH]-CONHSIR640;
与标记前相比,标记后的PAMAM分子显示出特征的IR640B的最大吸收峰和发射峰,证明PAMAM已被IR640B-NHS成功标记,PAMAM在药剂学的研究中被广泛用作纳米药物递释材料,而本实验证明IR640B-NHS在室温下能高效标记到树枝状高分子,胶束,脂质体等纳米载体上,显示了其在研究此类纳米递释材料在生物体内的代谢分布的可行性。
实施例11
IR640B-NHS标记牛血清白蛋白的吸收与发射测量
取牛血清白蛋白溶于PBS磷酸盐缓冲溶液中,调节pH为7.4。将IR640B-NHS溶于100μL的DMF中,逐滴加至反应溶液中,至于摇床,反应2h;反应结束后,反应液加入截流分子量为10000的超滤管中进行超滤(4000rpm,30×3min)取超滤管上层溶液冻干得标记IR640B的牛血清白蛋白固体,取少量固体进行吸收与发射测量(实验参数见实施例8),实验结果如图5所示,
与标记前相比,标记后的BSA分子显示出特征的IR640B的最大吸收峰和发射峰,证明BSA已被IR640B-NHS成功标记;鉴于抗体,多肽以及其他多种蛋白在生命科学研究领域用途广泛,本实验结果证明IR640B-NHS可以高效的标记到蛋白分子上,对于细胞成像,动物病灶显像等多种用途均有很大的潜力。
实施例12肿瘤细胞荧光染色
取标记的树枝状分子PAMAM-G5[NH2(CH2)2NH]-CONHSIR640取2mg溶于pH为7.4的500μL的PBS中,将此溶液加入DMEM培养基(10%FBS,1%双抗),之后,用此培养基孵育C6细胞(大鼠脑胶质瘤细胞)过夜,用PBS清洗,再置于激光共聚焦活细胞成像系统中(型号:CarlZeiss LSM710活细胞成像工作站,),设定激发波长为633nm,发射波长为660nm。收集成像结果(如图6所示),成像结果显示标记上IR640B的PAMAM分子被肿瘤细胞大量摄取,最终在共聚焦显微镜下呈现出较强的荧光亮度,结果证明,IR640B可用于细胞荧光成像,在研究病理毒理机制,药物摄取机制等均有应用价值。
实施例13
细胞摄取标记后的PAMAM分子后流式分析测定
取标记的PAMAM分子[NH2(CH2)2NH]-CONHSIR640适量,加入已长满80%肿瘤细胞(C6,大鼠脑胶质瘤细胞)含培养基10mL的培养皿中,使其终浓度为5μM,孵育细胞12h,置于流式细胞仪(BD FACSAria II流式细胞分选仪)下进行分析测定(激发波长:633nm,发射波长:660nm),用Flowjo软件处理得图7所示结果,显示用标记的PAMAM分子[NH2(CH2)2NH]-CONHSIR640孵育细胞后,相比于孵育之前,99%以上的细胞均呈现出较强的荧光,证明细胞对探针的高摄取率以及IR640B的高量子产率和荧光强度,表明IR640B对应用于细胞荧光成像及流式细胞筛分具有极大的应用价值。
Claims (9)
1.可对生物分子进行快速标记的活性深红荧光活性酯IR640B-NHS及其前体IR640B,其通式如下:
IR640B和IR640B-NHS
其中,IR640为花菁类深红荧光基团;B为荧光基团仲位通过碳-碳键引入的芳香基团;NHS为N-羟基琥珀酰亚胺酯。
2.如权利要求1所述的可对生物分子进行快速标记的活性深红荧光活性酯IR640B-NHS及其前体IR640B,,其结构式如下:
其中,R1为H,卤素,烷基,芳香基,硝基,磺酸基,醛基或羧基;
R2为H,羧基,或者磺酸基;
n是1,2,3,4,5,6,7或8。
3.如权利要求2所述的可对生物分子进行快速标记的活性深红荧光活性酯IR640B-NHS及其前体IR640B,其特征在于,所述的卤素选自氯或溴或碘。
4.如权利要求2所述的可对生物分子进行快速标记的活性深红荧光活性酯IR640B-NHS及其前体IR640B,其特征在于,所述的烷基选自甲基,乙基,丙基,异丙基,丁基或异丁基。
5.如权利要求2所述的可对生物分子进行快速标记的活性深红荧光活性酯IR640B-NHS及其前体IR640B,其特征在于,所述的芳香基选自苯基,萘基或取代苯基。
6.如权利要求1或2所述的可对生物分子进行快速标记的活性深红荧光活性酯IR640B-NHS及其前体IR640B的制备方法,其特征在于,其包括,以2,3,3-三甲基吲哚为母体,与丁磺酸内脂等原料反应,合成带磺酸基的花菁类荧光基团;再通过Suzuki-Miyaura反应,将苯基羧酸通过碳-碳键(C-C键)引入到荧光基团中;再通过对苯基羧酸修饰,生成N-羟基琥珀酰亚胺酯(NHSester)。
7.按权利要求6的方法,其特征在于,其包括步骤:
荧光基团骨架IR640的合成:
1).
2).
3).
目标深红荧光基团IR640B的合成:
4).
目标活性酯IR640B-NHS的合成:
5).
步骤1:在乙醇中,苯胺与粘溴酸发生加成反应;
步骤2:若R2为磺酸基,则2,3,3-三甲基吡啶与1,4-丁磺酸內酯在邻二氯苯中发生取代反应,若R2为烷基,羟基或者羧基,则2,3,3-三甲基吡啶与碘代的羧酸或者醇以及碘代的烷烃在乙腈中发生取代反应;
步骤3:步骤1制得的产物和步骤2制得的产物在醋酸酐中于60-90℃下,由醋酸钠催化发生加成反应制得IR640骨架;
步骤4:步骤3制得的IR640骨架在水中由四三苯基磷钯催化与对羧基苯硼酸发生suzuki反应制得IR640B;
步骤5:步骤4制得的IR640B在无水DMF中,在EDC活化下与NHS发生酯化反应,制得IR640B-NHS。
8.按权利要求7的方法,其特征在于,所述步骤2中:2,3,3-三甲基吡啶包含5位被R1取代的和未取代的。
9.如权利要求1所述的所述的可对生物分子进行快速标记的活性深红荧光活性酯IR640B-NHS及其前体IR640B,其特征是所述的活性酯在生理中性条件下与目标分子中的伯氨基反应,将深红荧光基团通过生物稳定的酰胺键标记到目标分子上。
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