CN111333619A - 一类488nm激发的高稳定性超分辨荧光染料及其合成和应用 - Google Patents
一类488nm激发的高稳定性超分辨荧光染料及其合成和应用 Download PDFInfo
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- CN111333619A CN111333619A CN201811554278.8A CN201811554278A CN111333619A CN 111333619 A CN111333619 A CN 111333619A CN 201811554278 A CN201811554278 A CN 201811554278A CN 111333619 A CN111333619 A CN 111333619A
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- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
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- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明提供了一类488nm激发的高稳定性超分辨荧光染料及其合成和应用,该荧光染料的结构式如(1)所示。其氮杂环丁烷、四氢吡咯等具有刚性结构的取代基在萘酰亚胺4,5‑位的引入抑制了分子内扭转,提升了分子的稳定性及量子产率(水中最高达0.80以上)。同时,对称的双供电基结构使此类染料紫外吸收与荧光发射峰半峰宽变窄(<40nm),避免了荧光多色成像中的串色现象。此外,此类染料对极性、温度、黏度均不敏感,不同微环境下荧光发射波长、峰型、荧光强度没有明显变化,最大限度保持了荧光信号的稳定性。由于光稳定性的提升此类染料能够用于多种超分辨荧光成像,在生物成像、识别等领域中具有巨大的潜在应用价值。
Description
技术领域
本发明属于荧光染料领域,具体涉及一类488nm激发的高稳定性超分辨荧光染料及其合成和应用。
背景技术
超分辨荧光成像技术能够借助荧光染料克服光学衍射极限,将光学显微镜的分辨率提升至20nm,这使科学家在生物学、材料学、医学等领域观测到更为精细的结构。但在分辨率提升的同时对荧光染料的稳定性、荧光亮度提出了更高的要求。正如超分辨技术的创始人Stefan.W.Hell所说:“在Ernst Abbe的时代,成像质量是由物镜决定的;而今天,成像质量则由荧光团决定。”即荧光染料的稳定性、亮度是目前制约超分辨荧光成像技术的重要因素。
常用的488nm激发下的荧光染料为FITC与Alexa488,其中FITC由于氧负离子的存在变得极为不稳定,在高激光强度下很容易被氧氧化而发生淬灭。而Alexa488为罗丹明类染料,虽然其稳定性相比FITC有很大提升,但是染料不易改造、功能化,且Alexa488所携带的正电荷容易导致本身在线粒体聚集,非特异性标记严重。因此,488nm激发下的荧光染料仍然匮乏,缺少用于超分辨的高稳定性且适用性强的荧光染料,这一波段荧光染料的开发会极大促进超分辨技术的进一步发展。
发明内容
本发明的目的之一是提供一类488nm激发的高稳定性超分辨荧光染料,该类染料水中荧光量子产率最高可达0.80,稳定性提升,能够用于SIM(结构光照明显微镜)、STED(受激辐射损耗)等多种超分辨技术中。
本发明的另一目的是提供一类488nm激发的高稳定性超分辨荧光染料的合成方法,该方法步骤简单、易于提纯、易功能化等优点。
本发明提供一类488nm激发的高稳定性超分辨荧光染料,通过萘酰亚胺4,5-位刚性环的引入实现了荧光稳定性、亮度的大幅度提升。此类染料对pH、黏度、温度等微环境均不敏感,能够最大限度保持荧光信号的准确性。
本发明提供一类功能化488nm激发的高稳定性超分辨荧光染料,基于高稳定性荧光染料,通过不同靶向基团的连接(如:吗啡啉、苄基鸟嘌呤、三苯基膦、紫杉醇等)实现了对不同目标的荧光标记及超分辨荧光成像。
本发明提供一类具有反应活性的488nm激发的高稳定性超分辨荧光染料,基于以上荧光染料,连接NHS、叠氮、炔基、四唑分子等活性基团使该类染料能够经多种方式与目标物实现共价结合。
一类488nm激发的高稳定性超分辨荧光染料,该荧光染料通过氮杂环丁烷、四氢吡咯、乙二胺类衍生物及其他刚性结构的引入使该类染料吸收达到480nm左右,荧光半峰宽变窄,荧光量子产率最高可达0.80以上;该类染料包含N-丁基-4,5-取代萘酰亚胺类染料、线粒体荧光染料、SNAP-tag荧光染料、Halo-tag荧光染料、活性酯荧光染料或药物靶向荧光染料。
所述N-丁基-4,5-取代萘酰亚胺类染料,吸收波长在460-495nm,适合488nm激光进行激发,其结构式如下:
R1,R2分别独立则为中的一种,若R1,R2独立则为以整体结构存在,R3,R4各自独立为H、C1-4烷基、(CH2CH2O)nH、(CH2)mSO3M;若R3不为H时,R4必为非H取代基;m、n为0-4整数。
一类488nm激发的高稳定性超分辨荧光染料的合成方法,所述N-丁基-4,5-取代萘酰亚胺类染料合成路线,如下:
具体合成步骤如下:
(1)染料N-丁基-4,5-二脂肪胺基-1,8-萘酰亚胺的合成:
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺溶于乙二醇甲醚中,并向其中加入脂肪环胺;将反应液缓慢升温至100-140℃,并在氮气保护下反应10-24h;减压除去溶剂,硅胶柱分离,以体积比为400-30:1的二氯甲烷和甲醇为洗脱剂,除去溶剂,得棕黄色固体N-丁基-4,5-二脂肪胺基-1,8-萘酰亚胺。
其中,N-丁基-4-溴-5-硝基-1,8-萘酰亚胺与脂肪环胺的质量比为1:1-3;N-丁基-4-溴-5-硝基-1,8-萘酰亚胺的质量与乙二醇甲醚的体积比为1:50-200g/mL;所述脂肪环胺为氮杂环丁烷、四氢吡咯、乙二胺衍生物及环己二胺衍生物。
所述线粒体荧光染料,能够特异性标记细胞内线粒体,其结构式如下:
R1,R2分别独立则为中的一种,若R1,R2独立则为以整体结构存在,R3,R4各自独立为H、C1-4烷基、(CH2CH2O)nH、(CH2)mSO3M;若R3不为H时,R4必为非H取代基;m、n为0-4整数。一类488nm激发的高稳定性超分辨荧光染料的合成方法,线粒体荧光染料合成路线:
具体合成步骤如下:
(1)中间体N-溴烷基-4,5-二取代-1,8-萘酐的合成:
将N-羟烷基-4,5-二取代-1,8-萘酐于乙酸乙酯,向其中滴加三溴化磷,缓慢升温至60-80℃搅拌4-12h,反应结束后减压除去溶剂,硅胶色谱柱分离得到N-溴烷基-4,5-二取代-1,8-萘酐;
其中,N-羟烷基-4,5-二取代-1,8-萘酐与三溴化磷的质量比为1:1.7-5;N-羟烷基-4,5-二取代-1,8-萘酐的质量与乙酸乙酯的体积比为20-30:1mg/mL。
(2)中间体N-三苯基膦基烷基-4,5-二取代-1,8-萘酐的合成:
将N-溴烷基-4,5-二取代-1,8-萘酐和三苯基膦溶于乙腈中,升温至120-140℃,反应18-30h结束后减压除去溶剂,硅胶色谱柱分离得到N-三苯基膦基烷基-4,5-二取代-1,8-萘酐;
其中,N-溴烷基-4,5-二取代-1,8-萘酐与三苯基膦的质量比为:1:2.7-8;N-溴烷基-4,5-二取代-1,8-萘酐的质量与乙腈的体积比为15-30:1mg/mL。
(3)线粒体探针的合成:
将N-三苯基膦基烷基-4,5-二取代-1,8-萘酐溶于乙二醇甲醚,向其中滴加脂肪胺,升温至100-140℃搅拌,反应10-15h后减压除去溶剂,硅胶色谱柱分离得到线粒体探针。
其中,N-三苯基膦基烷基-4,5-二取代-1,8-萘酐与脂肪胺的质量比为:1.6-2.4:1;N-三苯基膦基烷基-4,5-二取代-1,8-萘酐的质量与乙二醇甲醚的体积比为5.3-24:1;所述脂肪环胺为氮杂环丁烷、四氢吡咯、乙二胺衍生物及环己二胺衍生物。
SNAP-tag荧光染料,能够特异性识别SNAP-tag蛋白,实现活细胞免洗标记,其结构式如下:
R1,R2分别独立则为中的一种,若R1,R2独立则为以整体结构存在,R3,R4各自独立为H、C1-4烷基、(CH2CH2O)nH、(CH2)mSO3M;若R3不为H时,R4必为非H取代基;m、n为0-4整数。
一类488nm激发的高稳定性超分辨荧光染料的合成方法,所述SNAP-tag荧光染料的合成路线:
具体合成步骤如下:
(1)SNAP-tag探针的合成
将N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾和2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤置于史莱克瓶中,氮气置换2-5次后加入干燥的N,N-二甲基甲酰胺;室温反应3-10h后,加压出去溶剂,硅胶柱分离,以体积比为100-10:1的二氯甲烷和甲醇为洗脱剂,除去溶剂得靶向SNAP-tag蛋白的荧光探针。
其中,N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾、2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤的质量比为1:1-5:1-5;N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺的体积比为1:80-200g/mL。
所述Halo-tag荧光染料,能够特异性识别Halo-tag蛋白,实现活细胞免洗标记,其结构式如下:
R1,R2分别独立则为中的一种,若R1,R2独立则为以整体结构存在,R3,R4各自独立为H、C1-4烷基、(CH2CH2O)nH、(CH2)mSO3M;若R3不为H时,R4必为非H取代基;m、n为0-4整数。
一类488nm激发的高稳定性超分辨荧光染料的合成方法,所述Halo-tag荧光染料合成路线:
具体合成步骤如下:
(1)Halo-tag探针的合成
将N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH置于史莱克瓶中,并氮气置换2-5次;将1-碘-6-氯己烷溶于干燥的N,N-二甲基甲酰胺后,加入反应液中;室温下搅拌1-5h后,减压除去溶剂,硅胶柱分离,以体积比为100~400:1的二氯甲烷和甲醇为洗脱剂,除去溶剂得到靶向Halo-tag蛋白的荧光探针。
其中,N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH的质量比为5-10:1;N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与1-碘-6-氯己烷体积比为0.5-1mg/μL;N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺体积比为5-20:1mg/mL。
所述活性酯荧光染料,能够用于与氨基的缩合实现对目标的共价连接,其结构式如下:
R1,R2分别独立则为中的一种,若R1,R2独立则为以整体结构存在,R3,R4各自独立为H、C1-4烷基、(CH2CH2O)nH、(CH2)mSO3M;若R3不为H时,R4必为非H取代基;m、n为0-4整数。
一类488nm激发的高稳定性超分辨荧光染料的合成方法,活性酯荧光染料:
具体合成步骤如下:
(1)中间体N-1-(羧基)烷基-4,5-二脂肪氨基-1,8-萘酰亚胺COOH-DF系列化合物
COEt-DF系列化合物溶于甲醇中,并向反应液中滴加2M氢氧化钠溶液。室温下反应1-3h后,减压蒸馏除去甲醇,过滤并用水洗涤滤饼干燥后得COOH-DF系列化合物;
其中,COEt-DF系列化合物的质量与甲醇的体积比为10-20:1mg/mL;COEt-DF系列化合物的质量与2M氢氧化钠溶液的体积比为10-20:1mg/mL;COEt-DF系列化合物的质量与水的体积比为10-20:1mg/mL。
(2)带有NHS活性基团的荧光染料合成
将COOH-DF系列化合物,DCC溶于干燥的N,N-二甲基甲酰胺后,室温搅拌10-40min。N-羟基琥珀酰亚胺溶于1mL干燥的N,N-二甲基甲酰胺并加入反应液中;2-5h后减压除去溶剂,硅胶柱分离,以体积比20:1-4:1的二氯甲烷和乙酸乙酯为洗脱剂,除去溶剂后得NHS活性基团的荧光染料染料。
其中,COOH-DF系列化合物、DCC、NHS质量比为1:1-5:1-10;COOH-DF系列化合物的质量与N,N-二甲基甲酰胺的体积比为10-20:1mg/mL。
所述药物靶向荧光染料,可用于488nm激光进行激发用于检测与成像,其结构式如下:
R1,R2分别独立则为中的一种,若R1,R2独立则为以整体结构存在,R3,R4各自独立为H、C1-4烷基、(CH2CH2O)nH、(CH2)mSO3M;若R3不为H时,R4必为非H取代基;m、n为0-4整数。
一类488nm激发的高稳定性超分辨荧光染料的合成方法,药物靶向荧光染料合成路线:
具体合成步骤如下:
(1)含药物性的荧光染料的合成
10-30mg带有NHS活性基团的系列染料与带有活性氨基的药物分子置于史莱克瓶中,并用氮气置换2-5次;2-20μL二异丙基乙基胺溶于0.5-2mL干燥的二甲基亚砜中并加入反应瓶中;室温下搅拌3-10h后,水洗并用二氯甲烷萃取得有机相,硅胶柱分离得药物分子为靶向基团的荧光染料;
其中,NHS活性基团的系列染料与药物分子的质量比为1:0.5-1;NHS活性基团的系列染料的质量与二异丙基乙基胺的体积比为2-5:1mg/μL;二异丙基乙基胺与二甲基亚砜的体积比为1:100-300。
药物分子包括紫杉醇、秋水仙素、磺胺、生物素或叶酸。
上述一类488nm激发的高稳定性超分辨荧光染料易于改造、功能化,荧光发射峰窄(可达30nm)、量子产率高(水中最高可达0.80),能够在SIM,STED等超分辨领域得到广泛应用。
一类488nm激发的高稳定性超分辨荧光染料在细胞、组织及活体内的荧光成像领域的应用。
一类488nm激发的高稳定性超分辨荧光染料用于SNAP-tag蛋白的识别与检测。
一类488nm激发的高稳定性超分辨荧光染料用于Halo-tag蛋白的识别与检测。
一类488nm激发的高稳定性超分辨荧光染料在单分子检测中的应用。
一类488nm激发的高稳定性超分辨荧光染料在超分辨成像技术中的应用。
本发明具有以下特征:
该类488nm激发的高稳定性超分辨荧光染料拥有合成原料低价、方法简单通用、易于修饰功能化、便于批量生产等优点。
该类488nm激发的高稳定性超分辨荧光染料稳定性高于同波段的FITC\Alexa488,水中荧光量子产率可达0.80,半峰宽最窄可达30nm。
该类488nm激发的高稳定性超分辨荧光染料对于极性、黏度、pH、温度等微环境均不敏感,能够保持荧光信号的稳定性。
该类488nm激发的高稳定性超分辨荧光染料由于稳定性及亮度的提升能够应用于多种超分辨荧光成像。
该类488nm激发的高稳定性超分辨荧光染料的功能化分子具有较好定位及识别效果,细胞渗透性高。
附图说明
图1实施例1制备的BuAN-DAze的核磁谱图氢谱。
图2实施例3制备的BuAN-AzeAzo的核磁谱图氢谱。
图3实施例5制备的BuAN-DAC的核磁谱图氢谱。
图4实施例7制备的Halo-DAze的核磁谱图氢谱。
图5实施例9制备的SNAP-DAze的核磁谱图氢谱。
图6实施例15制备的Mito-DAze的核磁谱图氢谱。
图7实施例16制备的COOH-DAze的核磁谱图氢谱。
图8实施例19制备的Col-DAC的高分辨质谱。
图9实施例20制备的DTX-DAC的高分辨质谱。
图10实施例1制备的BuAN-DAze在乙醇中的归一化的荧光激发与发射谱图,横坐标为波长,纵坐标为归一化强度,荧光染料的浓度为10μM。
图11实施例2制备的BuAN-DAzo在不同溶剂中的归一化的紫外吸收谱图,横坐标为波长,纵坐标为归一化吸收强度,荧光染料的浓度为10μM。
图12实施例2制备的BuAN-DAzo在不同溶剂中的归一化的荧光发射谱图,横坐标为波长,纵坐标为归一化荧光强度,荧光染料的浓度为10μM。
图13实施例11制备的SNAP-DMEDA在PBS中与1μM SNAP-tag蛋白结合的动力学曲线图,横坐标为时间,纵坐标为荧光强度,荧光探针的浓度为1μM。
图14实施例7制备的Halo-DAze在转染的pHALOf-H2B的HeLa细胞荧光共聚焦成像图,荧光探针的浓度为1μM。
图15实施例9制备的SNAP-DAze在转染的pSNAPf-H2B的HEK293细胞荧光共聚焦成像图,荧光探针的浓度为1μM。
图16实施例10制备的SNAP-DAC在转染的pSNAPf-Cox8A的HEK293细胞荧光共聚焦成像图,荧光探针的浓度为1μM。
图17实施例15制备的Mito-DAze在RWPE细胞中荧光共聚焦成像图,荧光探针的浓度为1μM。
图18实施例15制备的Mito-DAze在HT29细胞中荧光共聚焦成像图,荧光探针的浓度为1μM。
图19实施例14制备的Mito-DAC在HeLa细胞荧中光共聚焦成像图,荧光探针的浓度为1μM。
图20实施例15制备的Mito-DAze在RWPE细胞中结构光照明显微成像图,荧光探针的浓度为1μM。
图21实施例14制备的Mito-DAC在MCF细胞中结构光照明显微成像图,荧光探针的浓度为1μM。
图22实施例12制备的SNAP-DAC在转染的pSNAPf-H2B的HeLa细胞中STED超分辨荧光成像图,荧光探针的浓度为1μM。
具体实施方式
实施例1
N-丁基-4,5-二氮杂环丁基-1,8萘酰亚胺(BuAN-DAze)的合成
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺(100mg,0.26mmol)溶于20mL乙二醇甲醚中,并向其中加入氮杂环丁烷(300mg,5.26mmol)。将反应液缓慢加热至120℃,并反应24h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=150:1,V/V),得黄色固体80mg,产率51%。实施例1制备的BuAN-DAze的核磁谱图氢谱如图1所示,氢谱与碳谱具体数据为:
1H NMR(400MHz,CDCl3)δ8.38(d,J=8.5Hz,2H),6.38(d,J=8.5Hz,2H),4.21–4.12(m,2H),4.05(s,2H),2.42(s,8H),1.69(dt,J=15.2,7.6Hz,2H),1.43(dq,J=14.8,7.4Hz,2H),0.95(t,J=7.3Hz,3H).13C NMR(101MHz,CDCl3)δ164.44,155.52,133.05,132.82,110.29,108.05,106.30,54.79,39.68,30.41,20.49,16.90,13.93.
其高分辨质谱数据如下:高分辨质谱理论值calcd for C22H26N3O2[M+H]+364.2025,实测值364.2035.
经检测,其结构如上式BuAN-DAze所示,其荧光性能如下:
将BuAN-DAze溶解于DMSO溶液中,配制成2mM母液,根据需要配制成不同浓度测试溶液,以检测其荧光光谱与激发光谱。
每次取20μL染料母液,分别加入4mL乙醇中,配制成10μM的荧光探针测试液,并进行荧光激发与发射光谱测试。
BuAN-DAze荧光激发与发射光谱如图10所示:BuAN-DAze在乙醇中激发波长在480nm,荧光发射波长在488nm,荧光发射半峰宽只有32nm。这说明BuAN-Daze能够适用于488nm激发的荧光成像与检测。
实施例2
N-丁基-4,5-二(氮杂环戊)基-1,8萘酰亚胺(BuAN-DAzo)的合成
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺(50mg,0.13mmol)溶于5mL乙二醇甲醚中,并向其中加入四氢吡咯200mg。将反应液缓慢加热至120℃,并反应10h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=100:1,V/V),得黄色固体38mg,产率75%。实施例9制备的BuAN-DAzo的核磁谱图氢谱与碳谱具体数据为:
1H NMR(400MHz,CDCl3)δ8.32(d,J=8.7Hz,2H),6.65(d,J=8.7Hz,2H),4.18(t,J=7.0Hz,2H),3.60(s,2H),3.37(d,J=4.9Hz,1H),3.28(d,J=4.2Hz,2H),2.67(d,J=8.0Hz,2H),2.19(s,2H),1.97(s,4H),1.70(dt,J=15.2,7.6Hz,2H),1.57(s,2H),1.45(dt,J=15.1,7.4Hz,2H),0.96(t,J=7.3Hz,3H).13C NMR(101MHz,CDCl3)δ164.33,154.20,133.79,132.69,109.52,108.97,106.15,52.27,49.49,39.53,30.45,25.89,25.54,20.51,13.97.
其高分辨质谱数据如下:分辨质谱理论值C24H30N3O2[M+H]+392.2338,实测值392.2343.
经检测,其结构如上式BuAN-DAzo所示,其荧光性能如下:
将BuAN-DAzo溶解于DMSO溶液中,配制成2mM母液,根据需要配制成不同浓度测试溶液,以检测其荧光光谱与激发光谱。
BuAN-DAzo在乙腈、氯仿、二甲基亚砜、乙醇、水中紫外吸收与荧光发射光谱测试。每次取20μL BuAN-DAzo母液加入4mL乙腈、氯仿、二甲基亚砜、乙醇、水中,配制成10μM的荧光染料测试液,进行紫外吸收与荧光发射光谱的测试。
BuAN-DAzo在乙腈、氯仿、二甲基亚砜、乙醇、水的吸收谱图如图11所示:BuAN-DAzo在乙腈、氯仿、二甲基亚砜、乙醇、水吸收波长在488nm左右,其中乙醇为485nm。
BuAN-DAzo在不同溶剂中荧光发射谱图如图12所示:BuAN-DAzo在不同溶剂中荧光发射波长在496nm左右,该染料适用作488nm激发的荧光染料。此外,该染料的荧光发射波长及紫外吸收波长随着极性的变化没有明显变化,对极性环境变化不敏感。
实施例3
N-丁基-4-氮杂环戊基-5-氮杂环丁基-1,8萘酰亚胺(BuAN-AzeAzo)的合成
N-丁基-4-溴-5-氮杂环丁基-1,8萘酰亚胺(BuAN-BrAze)的合成
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺(100mg,0.26mmol)溶于8毫升乙二醇甲醚中,并向其中加入氮杂环丁烷40mg。反应液在50℃下搅拌1h后,减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=200:1,V/V),得棕色固体75mg,产率72%。
N-丁基-4-氮杂环戊基-5-氮杂环丁基-1,8萘酰亚胺(BuAN-AzeAzo)的合成
将BuAN-BrAze(80mg,0.21mmol)溶于10mL乙二醇甲醚中并向反应液中加入200mg四氢吡咯,而后将反应液缓慢加热至120℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=100:1,V/V),得深黄色固体52mg,产率67%。实施例3制备的BuAN-AzeAzo的核磁谱图氢谱如图2所示,具体数据为:
1H NMR(400MHz,CDCl3)δ8.28(dd,J=10.1,8.7Hz,2H),6.61(d,J=8.7Hz,1H),6.27(d,J=8.5Hz,1H),4.16–3.90(m,4H),3.68–3.49(m,4H),2.95(s,2H),2.45–2.23(m,2H),2.09–1.89(m,2H),1.87(s,2H),1.62(dt,J=15.2,7.6Hz,2H),1.36(dq,J=14.7,7.4Hz,2H),0.88(t,J=7.3Hz,3H).13C NMR(101MHz,CDCl3)δ163.50,163.32,155.24,152.31,132.38,131.85,131.67,109.05,108.50,107.97,105.48,104.86,54.66,52.05,50.07,38.62,29.41,28.68,24.70,19.49,15.78,12.92.
其高分辨质谱数据如下:高分辨质谱理论值C23H28N3O2[M+H]+378.2182,实测值378.2093.
经检测,其结构如上式BuAN-AzeAzo所示,其荧光性能如下:水中其荧光发射波长为493nm,吸收波长达到481nm,适用于488nm激光进行激发。
实施例4
N-丁基-4,5-乙二胺基-1,8萘酰亚胺(BuAN-EDA)的合成
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺(100mg,0.27mmol)溶于30mL乙二醇甲醚中,并向其中加入乙二胺150mg。将反应液缓慢加热至70℃,并反应24h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=70:1,V/V),得黄色固体71mg,产率87%。其核磁谱图氢谱与碳谱具体数据为:
1H NMR(400MHz,DMSO-d6)δ8.29(s,2H),8.03(d,J=8.6Hz,2H),6.67(d,J=8.7Hz,2H),4.01–3.92(m,2H),3.51(s,4H),1.54(dt,J=14.9,7.5Hz,2H),1.31(dt,J=14.8,7.4Hz,2H),0.90(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO-d6)δ163.36,155.59,135.28,133.35,110.27,107.36,105.79,46.73,38.97,30.40,20.35.
经检测,其结构如上式BuAN-EDA所示,其荧光性能如下:水中其荧光发射波长为487nm,吸收波长达到481nm,适用于488nm激光进行激发。
实施例5
N-丁基-4,5-(1,2-环己二胺)基-1,8萘酰亚胺(BuAN-DAC)的合成
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺(100mg,0.27mmol)溶于10mL乙二醇甲醚中,并向其中加入环己二胺350mg。将反应液缓慢加热至120℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=70:1,V/V),得黄色固体31mg,产率32%。实施例5制备的BuAN-DAC的核磁谱图氢谱如图3所示,具体数据为:
1H NMR(400MHz,DMSO-d6)δ8.04(d,J=8.6Hz,2H),7.50(s,2H),6.83(d,J=8.7Hz,2H),4.04–3.83(m,2H),3.16(t,J=7.0Hz,2H),2.19(d,J=11.2Hz,2H),1.73(d,J=8.1Hz,2H),1.54(dt,J=14.9,7.6Hz,2H),1.30(dq,J=14.3,7.2Hz,6H),0.90(t,J=7.3Hz,3H).13C NMR(101MHz,DMSO-d6)δ163.43,154.52,134.70,133.32,110.56,107.86,106.52,59.52,55.38,32.09,30.40,23.64,20.35,14.28.
高分辨质谱理论值C22H26N3O2[M+H]+364.2025,实测值364.2029.
经检测,其结构如上式BuAN-DAC所示,其荧光性能如下:水中其荧光发射波长为488nm,吸收波长达到481nm,适用于488nm激光进行激发。
实施例6
N-丁基-4,5-(1,2-环己二胺)基-1,8萘酰亚胺(BuAN-DMC)的合成
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺(100mg,0.27mmol)溶于10mL乙二醇甲醚中,并向其中加入N,N’-二甲基环己二胺350mg。将反应液缓慢加热至120℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=100:1,V/V),得黄色固体31mg,产率30%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,CDCl3)δ8.40(d,J=8.2Hz,1H),6.81(d,J=8.4Hz,1H),4.35–3.94(m,1H),3.10(s,2H),2.22(s,1H),1.81(d,J=8.3Hz,1H),1.70(dt,J=15.2,7.5Hz,1H),1.44(dq,J=14.8,7.4Hz,1H),1.20(s,1H),0.96(t,J=7.3Hz,2H).
经检测,其结构如上式BuAN-DMC所示,其荧光性能如下:水中其荧光发射波长为515nm,吸收波长达到460nm。
实施例7
Halo-DAze的合成
将Halo-OH(30mg,0.08mmol)与NaH(6mg,0.25mmol)置于10mL史莱克瓶中,用氮气置换三次。将15μL 1-碘-6-氯己烷溶于6mL干燥的DMF后,并加入反应液。室温下搅拌5h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=200:1,V/V),得棕色固体20mg,产率50%。实施例7制得的Halo-DAze核磁谱图氢谱如图4所示,氢谱与碳谱具体数据如下:
1H NMR(400MHz,CDCl3)δ8.37(d,J=8.5Hz,2H),6.38(d,J=8.5Hz,2H),4.41(t,J=6.5Hz,2H),4.07(s,8H),3.78(t,J=6.5Hz,2H),3.71–3.65(m,2H),3.60–3.54(m,2H),3.43(t,J=6.6Hz,2H),2.43(s,4H),2.02(dd,J=14.1,7.1Hz,2H),1.80–1.70(m,2H),1.54(dd,J=13.8,6.9Hz,2H),1.41(dd,J=15.2,7.8Hz,2H).13C NMR(101MHz,CDCl3)δ164.41,155.61,133.22,132.94,110.11,108.02,107.86,106.32,77.22,71.21,70.13,68.21,54.55,38.61,33.56,29.70,26.74,25.42,25.38.
其高分辨质谱数据如下:高分辨质谱理论值C28H37ClN3O4[M+H]+514.2473,实测值514.2477.
经检测,其结构如上式Halo-DAze所示,在水中的紫外吸收波长为484nm,荧光发射波长为493nm,能够用于Halo-tag的荧光标记。
实施例8
Halo-DAC的合成
将OAN-DAC(50mg,0.13mmol)与NaH(10mg,0.42mmol)置于10mL史莱克瓶中,用氮气置换三次。将50μL 1-碘-6-氯己烷溶于5mL干燥的DMF后,并加入反应液。室温下搅拌1h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=100:1,V/V),得棕色固体36mg,产率56%。其核磁谱图氢谱与碳谱具体数据如下:
1H NMR(400MHz,CDCl3)δ8.25(d,J=8.3Hz,2H),6.52(d,J=8.3Hz,2H),5.00(s,2H),4.39(t,J=6.2Hz,2H),3.81(t,J=6.2Hz,2H),3.70(s,2H),3.58(d,J=4.4Hz,2H),3.41(dd,J=11.1,6.3Hz,2H),3.21(d,J=7.7Hz,2H),2.13(d,J=11.4Hz,2H),1.86(d,J=7.5Hz,2H),1.79–1.64(m,2H),1.56–1.27(m,10H).13C NMR(101MHz,CDCl3)δ164.33,152.36,133.81,114.40,110.73,110.57,107.77,71.20,70.18,70.12,68.15,59.46,45.18,38.69,33.58,32.67,32.56,29.51,26.75,25.41,23.61.
其高分辨质谱数据如下:高分辨质谱理论值C28H37ClN3O4[M+H]+514.2473,实测值514.2477.
经检测,其结构如上式Halo-DAC所示,其荧光性能如下:Halo-DAC在水光发射波长在490nm左右,激发波长在480nm,荧光半峰宽只有40nm。
实施例9
SNAP-DAze的合成:
将BA-DAze(40mg,0.09mmol)、BG+(40mg,0.16mmol)、叔丁醇钾(40mg,0.36mmol)置于10mL史莱克瓶中,用氮气置换三次并加入5mL干燥DMF。室温下搅拌6h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=20:1,V/V),得棕色固体24mg,产率45%。实施例9制备的核磁谱图氢谱如图5所示,核磁谱图氢谱与碳谱具体数据如下:
1H NMR(400MHz,DMSO-d6)δ12.40(s,1H),8.17(d,J=8.4Hz,2H),7.79(s,1H),7.41(d,J=7.4Hz,2H),7.28(d,J=7.6Hz,2H),6.48(d,J=8.5Hz,2H),6.26(s,2H),5.42(s,2H),5.22(s,2H),4.14(s,8H),2.38(s,4H).13C NMR(101MHz,DMSO-d6)δ163.47,160.31,160.09,156.01,155.65,138.90,138.22,135.60,133.37,132.87,128.87,127.74,113.97,108.21,106.94,106.73,56.50,54.63,42.37,19.02.
其高分辨质谱数据如下:高分辨质谱理论值C31H29N8O3[M+H]+561.2363,实际值561.2380.
经检测,其结构如上式SNAP-DAze所示,其荧光性能如下:SNAP-DAze在乙腈、氯仿、二甲基亚砜、乙醇、水光发射波长在490nm左右,且随着极性的变化荧光发射波长及荧光峰型均没有明显变化。
实施例10
SNAP-DAzo的合成
将BA-DAzo(30mg,0.07mmol)、BG+(150mg,0.63mmol)、叔丁醇钾(150mg,0.91mmol)置于10mL史莱克瓶中,用氮气置换四次并加入6mL干燥DMF。室温下搅拌6h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=25:1,V/V),得棕色固体23mg,产率60%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.60(s,1H),8.27(d,J=8.4Hz,2H),7.78(s,1H),7.41(d,J=7.4Hz,2H),7.28(d,J=7.6Hz,2H),6.48(d,J=8.5Hz,2H),6.26(s,2H),5.42(s,2H),5.22(s,2H),4.62(d,J=5.8Hz,2H),3.71(s,2H),3.45(s,2H),3.39(s,2H),2.71(s,2H),2.21(s,2H),1.98(dt,J=15.9,7.8Hz,4H),1.62(s,2H).
经检测,其结构如上式SNAP-DAzo所示,其在水中的荧光发射波长为495nm,吸收波长为485nm左右,能有用于488nm激光激发。
实施例11
SNAP-DMEDA的合成
将BA-DMDEA(50mg,0.12mmol)、BG+(95mg,0.37mmol)、叔丁醇钾(100mg,0.89mmol)置于10mL史莱克瓶中,用氮气置换四次并加入4mL干燥DMF。室温下搅拌3h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=25:1,V/V),得棕色固体27mg,产率40%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.40(s,1H),8.25(d,J=8.6Hz,2H),7.79(s,1H),7.42(d,J=7.8Hz,2H),7.32(d,J=8.1Hz,2H),6.86(d,J=8.7Hz,2H),6.27(s,2H),5.42(s,2H),5.22(s,2H),3.63(s,4H),3.12(s,6H).
经检测,其结构如上式SNAP-DMEDA所示,其在水中的荧光发射波长为510nm左右,吸收波长为458nm左右,能快速特异性识别SNAP-tag。
实施例12
SNAP-DAC的合成
将BA-DAC(40mg,0.09mmol)、BG+(95mg,0.37mmol)、叔丁醇钾(84mg,0.75mmol)置于10mL史莱克瓶中,用氮气置换四次并加入3mL干燥DMF。室温下搅拌10h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=25:1,V/V),得棕色固体28mg,产率53%。其核磁谱图氢谱与碳谱具体数据如下:
1H NMR(400MHz,DMSO-d6)δ12.39(s,1H),8.05(d,J=8.6Hz,2H),7.79(s,1H),7.56(s,2H),7.40(d,J=8.0Hz,2H),7.30(d,J=8.0Hz,2H),6.84(d,J=8.7Hz,2H),6.27(s,2H),5.41(s,2H),5.17(s,2H),3.16(d,J=8.5Hz,2H),2.19(d,J=11.3Hz,2H),1.73(d,J=6.6Hz,2H),1.40–1.25(m,4H).13C NMR(101MHz,DMSO-d6)δ163.39,160.30,160.09,155.65,154.76,138.94,138.22,135.61,134.96,133.53,128.86,127.99,113.94,110.71,107.56,106.40,99.99,66.98,59.47,42.38,32.06,23.62.
其高分辨质谱数据如下:高分辨质谱理论值C31H29N8O3[M+H]+561.2363,实际值561.2380.
经检测,其结构如上式SNAP-DAC所示,其在水中的荧光发射波长为485nm,吸收波长为479nm左右,能够对SNAP-tag进行免洗标记。
实施例13
SNAP-AzeAzo的合成
将BA-AzeAzo(20mg,0.05mmol)、BG+(36mg,0.14mmol)、叔丁醇钾(51mg,0.45mmol)置于10mL史莱克瓶中,用氮气置换三次并加入4mL干燥DMF。室温下搅拌8h后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=20:1,V/V),得棕色固体14mg,产率52%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.45(s,1H),8.13(dd,J=15.7,8.0Hz,2H),7.80(s,1H),7.41(d,J=5.9Hz,2H),7.29(d,J=7.2Hz,2H),6.76(dd,J=18.6,9.6Hz,1H),6.42(t,J=8.4Hz,1H),6.25(s,2H),5.43(s,2H),5.23(s,2H),3.63(s,2H),3.19(s,2H),2.90(s,2H),2.07(m,6H).13C NMR(101MHz,DMSO-d6)δ163.51,163.36,160.09,156.69,155.65,154.68,153.83,138.19,133.73,132.76,132.66,132.59,128.82,127.77,113.99,107.87,107.39,107.15,106.84,106.24,67.01,52.46,49.75,42.31,25.93,25.63,25.30,16.69.
经检测,其结构如上式SNAP-AzeAzo所示,其在水中的荧光发射波长为494nm,吸收波长为485nm左右,能够对SNAP-tag进行免洗标记。
实施例14
荧光探针Mito-DAC的合成。
中间体N-(6-羟基己基)-4-溴-5-硝基-1,8-萘酐的合成
4-溴-5-硝基-1,8-萘酰亚胺(1.30g,3.11mmol)溶于50mL乙醇中,并向其中滴加6-氨基-1-己醇(363mg,3.11mmol)。70℃下1h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚:二氯甲烷=2:1,V/V)分离得米白色固体620mg,产率53%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,CDCl3)δ8.71(d,J=7.8Hz,1H),8.51(d,J=7.9Hz,1H),8.22(d,J=7.9Hz,1H),7.93(d,J=7.8Hz,1H),4.25–4.07(m,2H),3.65(t,J=6.5Hz,2H),1.75(dt,J=14.4,7.0Hz,2H),1.59(dd,J=13.2,6.5Hz,2H),1.48–1.43(m,4H).13C NMR(101MHz,CDCl3)δ162.83,162.06,151.31,135.98,132.36,131.24,130.55,125.74,124.15,123.55,122.45,121.23,62.77,40.76,32.55,27.86,26.68,25.29.
高分辨质谱数据如下:C18H18BrN2O5[M+H]+计算值:421.0399,实验值:421.0396.
经验证,上述结构为N-(6-羟基己基)-4-溴-5-硝基-1,8-萘酐。
中间体N-(6-溴己基)-4-溴-5-硝基-1,8-萘酐的合成
将N-(6-羟基己基)-4-溴-5-硝基-1,8-萘酐(500mg,1.19mmol)溶于二氯甲烷中,并向其中滴加三溴化磷(1.61g,5.95mmol)。70℃下搅拌6h后,用饱和碳酸钠溶液洗涤有机相。所得有机相用无水硫酸钠干燥后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:石油醚=1:1,V/V),得白色固体230mg,产率40%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,CDCl3)δ8.71(d,J=7.8Hz,1H),8.52(d,J=7.9Hz,1H),8.22(d,J=7.9Hz,1H),7.93(d,J=7.8Hz,1H),4.22–4.11(m,2H),3.41(t,J=6.8Hz,2H),1.94–1.83(m,2H),1.75(dt,J=15.0,7.6Hz,2H),1.58–1.49(m,2H),1.44(dd,J=14.8,5.8Hz,2H).
高分辨质谱数据如下:C18H16Br2N2O4[M+H]+计算值:481.9477,实验值:481.9482.
经验证,上述结构为N-(6-溴己基)-4-溴-5-硝基-1,8-萘酐。
中间体N-(6-三苯基膦己基)-4-溴-5-硝基-1,8-萘酐的合成
将N-(6-溴己基)-4-溴-5-硝基-1,8-萘酐(200mg,0.41mmol)与三苯基膦(1.08g,4.13mmol)溶于10mL无水乙腈中,并置于密封管中。140℃下反应24h后,减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=400:1,V/V),得白色固体485mg,产率60%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,CDCl3)δ8.66(d,J=7.3Hz,1H),8.47(d,J=8.0Hz,1H),8.20(d,J=7.3Hz,1H),8.01–7.40(m,16H),4.11(t,J=6.8Hz,2H),3.72(s,2H),1.80–1.33(m,8H).13C NMR(101MHz,CDCl3)δ162.73,161.96,151.21,135.98,135.13,133.77,133.67,132.32,132.13,132.03,131.96,131.25,130.64,130.52,128.56,128.44,125.68,124.05,123.59,122.40,121.16,118.57,117.71,53.46,40.58,30.11,29.95,27.43,26.55.
高分辨质谱数据如下:C36H31N2O4P+[M]+计算值:665.1205,实验值:665.1208.
经验证,上述结构为N-(6-三苯基膦己基)-4-溴-5-硝基-1,8-萘酐。
荧光探针Mito-DAC的合成
将N-(6-三苯基膦己基)-4-溴-5-硝基-1,8-萘酐(100mg,0.13mmol)溶于10毫升乙二醇甲醚中,并向其中加入1,2-二氨基环己二胺(60mg,0.52mmol)。将反应液缓慢加热至120℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=200:1,V/V),得黄色固体40mg,产率89%.其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,CDCl3)δ8.04(d,J=8.5Hz,2H),7.83(t,J=6.8Hz,3H),7.68(dd,J=13.9,6.4Hz,12H),6.83(d,J=8.5Hz,2H),5.86(s,2H),4.02(t,J=6.5Hz,2H),3.42–3.31(m,2H),3.18(d,J=9.7Hz,2H),2.33(d,J=12.5Hz,2H),1.80(d,J=8.2Hz,2H),1.63(s,4H),1.48(d,J=9.7Hz,2H).13C NMR(101MHz,CDCl3)δ164.31,153.34,135.46,134.31,133.53,133.43,130.75,130.63,118.30,117.44,111.04,109.26,107.18,59.65,38.94,32.67,29.71,27.28,25.53,23.65.
高分辨质谱数据如下:C42H43N10O21P+[M]+计算值:652.3087,实验值:652.3128.
经检测,该化合物结构如Mito-DAC所示,适用于多种生理状态下的活细胞线粒体成像且光性能不受微环境影响,亮度高稳定性强可以满足超分辨成像对线粒体的长时间动态追踪,荧光发射波长在481nm左右。
实施例15
荧光探针Mito-DAze的合成。
中间体N-(6-羟基己基)-4-溴-5-硝基-1,8-萘酐的合成
4-溴-5-硝基-1,8-萘酰亚胺(1.30g,3.11mmol)溶于50mL乙醇中,并向其中滴加6-氨基-1-己醇(363mg,3.11mmol)。70℃下1h后,减压蒸馏除去溶剂,残余物经硅胶柱(石油醚:二氯甲烷=2:1,V/V)分离得米白色固体620mg,产率53%。核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,CDCl3)δ8.71(d,J=7.8Hz,1H),8.51(d,J=7.9Hz,1H),8.22(d,J=7.9Hz,1H),7.93(d,J=7.8Hz,1H),4.25–4.07(m,2H),3.65(t,J=6.5Hz,2H),1.75(dt,J=14.4,7.0Hz,2H),1.59(dd,J=13.2,6.5Hz,2H),1.48–1.43(m,4H).13C NMR(101MHz,CDCl3)δ162.83,162.06,151.31,135.98,132.36,131.24,130.55,125.74,124.15,123.55,122.45,121.23,62.77,40.76,32.55,27.86,26.68,25.29.
高分辨质谱数据如下:C18H18BrN2O5[M+H]+计算值:421.0399,实验值:421.0396.
经验证,上述结构为N-(6-羟基己基)-4-溴-5-硝基-1,8-萘酐。
中间体N-(6-溴己基)-4-溴-5-硝基-1,8-萘酐的合成
将化合物N-(6-羟基己基)-4-溴-5-硝基-1,8-萘酐(500mg,1.19mmol)溶于二氯甲烷中,并向其中滴加三溴化磷(1.61g,5.95mmol),于70℃下搅拌6h后,用饱和碳酸钠溶液洗涤有机相。所得有机相用无水硫酸钠干燥后减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:石油醚=1:1,V/V),得白色固体230mg,产率40%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,CDCl3)δ8.71(d,J=7.8Hz,1H),8.52(d,J=7.9Hz,1H),8.22(d,J=7.9Hz,1H),7.93(d,J=7.8Hz,1H),4.22–4.11(m,2H),3.41(t,J=6.8Hz,2H),1.94–1.83(m,2H),1.75(dt,J=15.0,7.6Hz,2H),1.58–1.49(m,2H),1.44(dd,J=14.8,5.8Hz,2H).
高分辨质谱数据如下:C18H16Br2N2O4[M+H]+计算值:481.9477,实验值:481.9482.
经验证,上述结构为N-(6-溴己基)-4-溴-5-硝基-1,8-萘酐。
中间体N-(6-三苯基膦己基)-4-溴-5-硝基-1,8-萘酐的合成
将化合物N-(6-溴己基)-4-溴-5-硝基-1,8-萘酐(200mg,0.41mmol)与三苯基膦(1.08g,4.13mmol)溶于10mL无水乙腈中,并置于密封管中。140℃下反应24h后,减压除去溶剂,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=400:1,V/V),得白色固体485mg,产率60%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,CDCl3)δ8.66(d,J=7.3Hz,1H),8.47(d,J=8.0Hz,1H),8.20(d,J=7.3Hz,1H),8.01–7.40(m,16H),4.11(t,J=6.8Hz,2H),3.72(s,2H),1.80–1.33(m,8H).13C NMR(101MHz,CDCl3)δ162.73,161.96,151.21,135.98,135.13,133.77,133.67,132.32,132.13,132.03,131.96,131.25,130.64,130.52,128.56,128.44,125.68,124.05,123.59,122.40,121.16,118.57,117.71,53.46,40.58,30.11,29.95,27.43,26.55.
高分辨质谱数据如下:C36H31N2O4P+[M]+计算值:665.1205,实验值:665.1208.
经验证,上述结构为N-(6-三苯基膦己基)-4-溴-5-硝基-1,8-萘酐所示。
荧光探针Mito-DAze的合成
将化合物N-(6-三苯基膦己基)-4-溴-5-硝基-1,8-萘酐(100mg,0.13mmol)溶于10mL乙二醇甲醚中,并向其中加入氮杂环丁烷(30mg,0.52mmol)。将反应液缓慢加热至120℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=200:1,V/V),得黄色固体40mg,产率89%。
实施例15制备的Mito-DAze核磁谱图氢谱如图6所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ8.31(d,J=8.4Hz,2H),7.76(dd,J=21.9,9.4Hz,15H),6.38(d,J=8.4Hz,2H),4.22–3.83(m,10H),3.50(s,2H),2.43(s,4H),1.66(s,4H),1.38(s,4H).13C NMR(101MHz,CDCl3)δ155.69,135.22,133.65,133.55,132.86,130.68,130.56,118.51,109.73,107.73,106.34,55.05,39.31,29.67,27.53,26.15,22.51,16.92.
高分辨质谱数据如下:C42H43N3O2P+[M]+计算值:652.3088,实验值:652.3109.
经检测,上述产物结构为Mito-DAze,该化合物在活细胞成像实验中能快速准确定位于线粒体,亮度高、稳定性强。
实施例16
NHSM-DAze的合成
COOH-DAze的合成
(1)COMe-DAze的合成
将COMe-NBr(200mg,0.49mmol)溶于10mL乙二醇甲醚中,并向其中加入氮杂环丁烷400mg。将反应液缓慢加热至120℃,并反应10h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=100:1,V/V),得深黄色固体60mg,产率31%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,CDCl3)δ8.74(d,J=7.9Hz,1H),8.54(d,J=7.7Hz,1H),8.24(d,J=7.9Hz,1H),7.94(d,J=7.8Hz,1H),4.93(s,2H),4.23(q,J=7.2Hz,2H),4.19–3.90(m,8H),2.43(s,4H),1.32(t,J=7.2Hz,3H).
(2)COOH-DAze的合成
COMe-DAze(40mg,0.10mmol)溶于4mL甲醇中,并向反应液中缓慢滴加2M氢氧化钠溶液4mL。滴加完毕后,反应液在室温下反应1h后,减压蒸馏除去甲醇,浑浊液过滤并用4mL水洗涤滤饼干燥后得COOH-DAze 32mg,产率86%。实施例16制备的COOH-DAze核磁谱图氢谱如图7所示,具体数据如下:
1H NMR(400MHz,DMSO-d6)δ8.15(d,J=8.3Hz,2H),6.48(d,J=8.3Hz,2H),4.49(s,2H),4.06(s,8H),2.39(s,4H).13C NMR(101MHz,DMSO-d6)δ163.49,155.72,133.21,132.50,109.02,107.32,106.59,54.80,43.24,16.81.
其高分辨质谱数据如下:高分辨质谱理论值C20H20N3O4[M+H]+366.1454,实测值366.1440.
NHSM-DAze的合成
COOH-DAze(30mg,0.08mmol)与二环己基碳亚(DCC)(30mg,0.15mmol)溶于1mL N,N-二甲基甲酰胺中,并在室温下搅拌20min。N-羟基琥珀酰亚胺(100mg,0.87mmol)溶于3mLN,N-二甲基甲酰胺后,滴加至反应液。2h后减压除去溶剂,硅胶柱分离,以二氯甲烷:乙酸乙酯=5:1为洗脱剂,除去溶剂得土黄色固体32mg,产率85%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ8.18(d,J=8.2Hz,2H),6.38(d,J=8.4Hz,2H),4.51(s,2H),4.06(b,8H),2.87(s,4H),2.39(s,4H).
经检测,其结构如上式NHSM-DAze所示,其在水中荧光发射波长为493nm,能够与活性氨基进行室温缩合。
实施例17
NHSB-DAC的合成
中间体COOH-DAC的合成
(1)COMe-DAC的合成
将COMe-NBr(200mg,0.49mmol)溶于20mL乙二醇甲醚中,并向其中加入1,2-环己二胺400mg。将反应液缓慢加热至100℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=80:1,V/V),得深黄色固体124mg,产率64%。
其高分辨质谱数据如下:高分辨质谱理论值C22H24N3O4[M+H]+394.1767,实测值394.1788.
(2)COOH-DAC的合成
COMe-DAC(60mg,0.10mmol)溶于3mL甲醇中,并向反应液中缓慢滴加2M氢氧化钠溶液3mL。滴加完毕后,反应液在室温下反应3h后,减压蒸馏除去甲醇,浑浊液过滤并用3mL水洗涤滤饼干燥后得COOH-DAC46mg,产率83%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.72(s,1H),8.03(d,J=8.6Hz,2H),7.59(s,2H),6.84(d,J=8.7Hz,2H),4.62(s,2H),3.16(d,J=5.9Hz,2H),2.20(d,J=11.7Hz,2H),1.73(d,J=6.9Hz,2H),1.31(dt,J=31.3,16.1Hz,4H).13C NMR(101MHz,DMSO-d6)δ170.62,163.06,154.85,135.08,133.45,110.71,107.28,106.37,59.46,41.02,32.06,23.62.
其高分辨质谱数据如下:高分辨质谱理论值C20H20N3O4[M+H]+366.1454,实测值652.3109.
经检测,其结构如上式COOH-DAC所示。
NHSM-DAC的合成
COOH-DAC(20mg,0.05mmol)与二环己基碳亚(DCC)(100mg,0.50mmol)溶于1mL N,N-二甲基甲酰胺中,并在室温下搅拌30min。N-羟基琥珀酰亚胺(200mg,1.74mmol)溶于2mLN,N-二甲基甲酰胺后,滴加至反应液。5h后减压除去溶剂,硅胶柱分离,以二氯甲烷:乙酸乙酯=6:1为洗脱剂,除去溶剂得土黄色固体22mg,产率87%。其核磁谱图氢谱数据如下:
1H NMR(400MHz,DMSO-d6)δ8.10–7.83(m,2H),7.56(s,2H),6.84(d,J=8.7Hz,2H),4.25(s,2H),3.18(d,J=9.1Hz,2H),2.82(s,4H),2.19(d,J=11.4Hz,2H),1.73(d,J=7.2Hz,2H),1.33(dt,J=27.8,15.1Hz,4H).
经检测,其结构如上式NHSM-DAC所示,其在水中荧光发射波长为487nm,能够与活性氨基进行室温缩合。
实施例18
BCOOH-DAC的合成
(1)BCOMe-DAC的合成
将BCOMe-NBr(200mg,0.46mmol)溶于10mL乙二醇甲醚中,并向其中加入1,2-环己二胺600mg。将反应液缓慢加热至100℃,并反应12h。减压除去乙二醇甲醚,残余物经硅胶柱分离残余物(二氯甲烷:甲醇=80:1,V/V),得深黄色固体103mg,产率53%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,DMSO-d6)δ8.04(d,J=8.6Hz,2H),7.51(s,2H),6.82(d,J=8.7Hz,2H),4.00(dt,J=14.1,5.3Hz,4H),3.14(d,J=8.8Hz,2H),2.30(t,J=7.5Hz,2H),2.19(d,J=11.7Hz,2H),1.89–1.80(m,2H),1.73(d,J=6.8Hz,2H),1.31(dt,J=30.1,15.8Hz,4H),1.14(t,J=7.1Hz,3H).13C NMR(101MHz,DMSO-d6)δ172.88,163.49,154.56,134.79,133.35,110.58,107.74,106.44,60.18,59.48,38.55,32.07,31.80,23.75,23.63,14.53.
其高分辨质谱数据如下:高分辨质谱理论值C24H28N3O4[M+H]+422.2080,实测值422.2108.
(2)BCOOH-DAC的合成
BCOMe-DAC(80mg,0.19mmol)溶于5mL甲醇中,并向反应液中缓慢滴加2M氢氧化钠溶液8mL。滴加完毕后,反应液在室温下反应1h后,减压蒸馏除去甲醇,浑浊液过滤并用5mL水洗涤滤饼干燥后得BCOOH-DAC 65mg,产率87%。其核磁谱图氢谱与碳谱数据如下:
1H NMR(400MHz,DMSO-d6)δ12.01(s,1H),8.04(d,J=8.6Hz,2H),7.51(s,2H),6.82(d,J=8.7Hz,2H),3.99(dd,J=9.2,4.6Hz,2H),3.15(d,J=9.1Hz,2H),2.21(dd,J=16.7,9.3Hz,4H),1.88–1.76(m,2H),1.72(d,J=8.0Hz,2H),1.42–1.19(m,4H).13C NMR(101MHz,DMSO-d6)δ174.48,163.50,154.57,134.79,133.36,110.58,107.76,106.47,59.50,47.97,33.82,32.08,31.90,25.79,24.93,23.86,23.63.
其高分辨质谱数据如下:高分辨质谱理论值C22H24N3O4[M+H]+394.1767,实测值394.1824.
经检测,其结构如上式BOOH-DAC所示。
NHSB-DAC的合成
BCOOH-DAC(50mg,0.12mmol)与二环己基碳亚(DCC)(112mg,0.54mmol)溶于2mL N,N-二甲基甲酰胺中,并在室温下搅拌20min。N-羟基琥珀酰亚胺(200mg,1.74mmol)溶于2mLN,N-二甲基甲酰胺后,滴加至反应液。3h后减压除去溶剂,硅胶柱分离,以二氯甲烷:乙酸乙酯=5:1为洗脱剂,除去溶剂得土黄色固体55mg,产率89%。其核磁谱图氢谱与碳谱具体数据如下:
1H NMR(400MHz,DMSO-d6)δ8.19–7.93(m,2H),7.53(s,2H),6.83(d,J=8.7Hz,2H),4.05(t,J=6.5Hz,2H),3.15(d,J=9.2Hz,2H),2.80(s,4H),2.72(t,J=7.7Hz,2H),2.19(d,J=11.4Hz,2H),1.97–1.88(m,2H),1.73(d,J=7.2Hz,2H),1.31(dt,J=28.8,15.2Hz,4H).13C NMR(101MHz,DMSO-d6)δ170.66,169.11,163.47,154.65,134.87,133.42,110.63,107.66,106.43,59.48,38.35,32.07,28.69,25.90,23.73,23.63.
其高分辨质谱数据如下:高分辨质谱理论值C26H27N4O6[M+H]+491.1931,实测值491.1981.
经检测,其结构如上式NHSB-DAC所示,其在水中荧光发射波长为487nm,能够与活性氨基进行室温缩合。
实施例19
Col-DAC的合成
NHSB-DAC(20mg,0.04mmol)与氨基秋水仙素(15mg,0.04mmol)置于5mL史莱克瓶中,并用氮气置换3次。将5μL二异丙基乙基胺(DIPEA)溶于2mL二甲基亚砜(DMSO)中,而后将混合液加入反应瓶。室温下搅拌3h后,将反应液倒入10mL水中,并用50mL二氯甲烷萃取得有机相,无水硫酸钠干燥后经硅胶柱分离得(二氯甲烷:甲醇=80:1,V/V)棕黄色固体23mg,产率77%。
实施例19制备的Col-DAC的高分辨质谱如图8所示,质谱数据为:高分辨质谱理论值C42H45N4O8[M+H]+733.3237,实测值733.3220.
经检测,其结构如上式Col-DAC所示,其在水中荧光发射波长为489nm,吸收为481nm。
实施例20
DTX-DAC的合成
NHSB-DAC(10mg,0.02mmol)与氨基紫杉醇(14mg,0.02mmol)置于5mL史莱克瓶中,并用氮气置换3次。将3μL二异丙基乙基胺(DIPEA)溶于1mL二甲基亚砜(DMSO)中,而后将混合液加入反应瓶。室温下搅拌2h后,将反应液倒入10mL水中,并用50mL二氯甲烷萃取得有机相,无水硫酸钠干燥后经硅胶柱分离(二氯甲烷:甲醇=20:1,V/V)得棕黄色固体16mg,产率72%。
实施例20制备的DTX-DAC的高分辨质谱如图9所示,质谱数据为:高分辨质谱理论值C60H67N4O15[M+H]+1083.4603,实测值1083.4603.
经检测,其结构如上式DTX-DAC所示,其在水中荧光发射波长为488nm,吸收为481nm。
将该类染料分别溶解于DMSO溶液中,配制成不同染料的2mM母液,根据需要配制成不同浓度测试溶液,以检测其荧光光谱测试及细胞内荧光成像。
实施例21
SNAP-DMEDA在PBS中与1μM SNAP-tag蛋白结合的动力学曲线测试。取0.5μL SNAP-DMEDA母液溶于1mL PBS中,而后加入等浓度蛋白后检测500nm处荧光强度,激发波长为450nm。
SNAP-DMEDA与SNAP-tag蛋白结合的动力学曲线如图13所示:SNAP-DMEDA在加入SNAP-tag后迅速与蛋白发生特异性结合,荧光恢复,荧光强度在1分钟内达到稳定。SNAP-DMEDA与SNAP-tag反应常数大于15000M-1S-1,t1/2=6s。此类染料的引入不影响靶向基团的靶向性,能够广泛应用于不同的靶向分子设计中。
实施例22
探针Halo-DAze、SNAP-DAze、SNAP-DAC在转染细胞中荧光共聚焦成像。将细胞分别接种在共聚焦培养皿中,采用1mL含有10%胎牛血清的DMED高糖培养基进行培养。在37℃和5%二氧化碳条件下孵育48小时后,用PBS缓冲液轻柔洗涤细胞2次后更换新鲜不含有血清的培养液。然后向其分别加入含有Halo-tag或SNAP-tag相应质粒(NEB)的Lipo 2000(Invitrogen)转染工作液,并置于培养箱中进行培养,4小时后,更换为含有血清的新鲜培养基。24小时后取0.5μL以上所述染料母液溶于1mL细胞培养液中,而后置于37℃下孵育30分钟后进行荧光共聚焦成像,无需洗去培养液。
Halo-DAze在转染的Halo-H2B的HeLa细胞的荧光共聚焦成像图如图14所示:Halo-DAze能够在活细胞内对目标蛋白达到免洗荧光成像,具有好的细胞相容性。
SNAP-DAze在转染有pSNAPf-H2B的HEK293细胞的荧光共聚焦成像图如图15所示:SNAP-DAze能够在活细胞内对目标蛋白达到免洗荧光成像,细胞核轮廓清晰。
SNAP-DAC在转染有pSNAPf-Cox8A的HEK293细胞的荧光共聚焦成像图如图16所示:SNAP-DAC能够在活细胞内对融合有SNAP-tag的线粒体相关蛋白(Cox8A)进行免洗荧光成像,线粒体形态及轮廓清晰。
实施例23
Mito-DAze、Mito-DAC在活细胞内(RWPE、HeLa、HT29等)荧光共聚焦成像及结构光照明显微成像。取0.5μL以上探针母液溶于1mL细胞培养液中,而后置于37℃下对细胞进行孵育10-30分钟后进行荧光成像。
Mito-DAze对RWPE细胞中线粒体成像图如图17所示:(a)为Mito-DAze在RWPE细胞中能够对线粒体成像图;(b)为商业的深红色线粒体染料在RWPE细胞中能够对线粒体成像图;(c)为前两个信号通道叠加图。这说明Mito-DAze能够对RWPE细胞内进行精准定位,无需洗去培养基。
Mito-DAze对HT29细胞中线粒体成像图如图18所示:Mito-DAze在HT29细胞中能够对线粒体实现精准定位,达到免洗荧光成像,HT29细胞线粒体特有的粗短形态清晰可见。
Mito-DAC对HeLa细胞中线粒体成像图如图19所示:Mito-DAC在HeLa细胞荧中能够对线粒体实现免洗荧光成像,HeLa细胞中线形线粒体清晰可见。
Mito-DAze在RWPE细胞中对线粒体结构照明显微成像图如图20所示:通过Mito-DAze能够看到RWPE线粒体更为精细的结构,部分结构可以看到染料在线粒体膜上的分布。
Mito-DAC在MCF细胞中对线粒体结构照明显微成像图如图21所示:通过Mito-DAC能够得到MCF线粒体更高分辨率的荧光成像图,部分结构可以看到染料在线粒体膜上的分布。
实施例24
SNAP-DAC在转染的pSNAPf-H2B的HeLa细胞中STED超分辨荧光成像实验。取0.5μLSNAP-DAC母液溶于1mL细胞培养液中,37℃,5%CO2下孵育30分钟后,通过4%甲醛溶液对细胞进行固定后置于1mL PBS缓冲液中,用于STED超分辨荧光成像。
SNAP-DAC在转染的pSNAPf-H2B的HeLa细胞中STED超分辨荧光成像图如图22所示:SNAP-DAC能够对HeLa细胞内细胞核进行特异性标记。由于光稳定性的提升,SNAP-DAC能够在GW/cm2级高强度激光下进行多次成像、重构得到更高分辨率图像。
Claims (18)
1.一类488nm激发的高稳定性超分辨荧光染料,其特征在于:该荧光染料通过氮杂环丁烷、四氢吡咯、乙二胺类衍生物及其他刚性结构的引入使该类染料吸收达到480nm左右,荧光半峰宽变窄,荧光量子产率最高可达0.80以上;该类染料包含N-丁基-4,5-取代萘酰亚胺类染料、线粒体荧光染料、SNAP-tag荧光染料、Halo-tag荧光染料、活性酯荧光染料或药物靶向荧光染料。
8.一种如权利要求2所述的488nm激发的高稳定性超分辨荧光染料的合成方法,其特征包在于含步骤如下:
(1)染料N-丁基-4,5-二脂肪胺基-1,8-萘酰亚胺的合成:
将N-丁基-4-溴-5-硝基-1,8-萘酰亚胺溶于乙二醇甲醚中,并向其中加入脂肪环胺;将反应液缓慢升温至100-140℃,并在氮气保护下反应10-24h;减压除去溶剂,硅胶柱分离,以体积比为400-30:1的二氯甲烷和甲醇为洗脱剂,除去溶剂,得棕黄色固体N-丁基-4,5-二脂肪胺基-1,8-萘酰亚胺。
其中,N-丁基-4-溴-5-硝基-1,8-萘酰亚胺与脂肪环胺的质量比为1:1-3;N-丁基-4-溴-5-硝基-1,8-萘酰亚胺的质量与乙二醇甲醚的体积比为1:50-200g/mL;所述脂肪环胺为氮杂环丁烷、四氢吡咯、乙二胺衍生物及环己二胺衍生物。
9.一种如权利要求3所述的488nm激发的高稳定性超分辨荧光染料的合成方法,其特征在于包含以下步骤:
(1)中间体N-溴烷基-4,5-二取代-1,8-萘酐的合成:
将N-羟烷基-4,5-二取代-1,8-萘酐于乙酸乙酯,向其中滴加三溴化磷,缓慢升温至60-80℃搅拌4-12h,反应结束后减压除去溶剂,硅胶色谱柱分离得到N-溴烷基-4,5-二取代-1,8-萘酐;
其中,N-羟烷基-4,5-二取代-1,8-萘酐与三溴化磷的质量比为1:1.7-5;N-羟烷基-4,5-二取代-1,8-萘酐的质量与乙酸乙酯的体积比为20-30:1mg/mL;
(2)中间体N-三苯基膦基烷基-4,5-二取代-1,8-萘酐的合成:
将N-溴烷基-4,5-二取代-1,8-萘酐和三苯基膦溶于乙腈中,升温至120-140℃,反应18-30h结束后减压除去溶剂,硅胶色谱柱分离得到N-三苯基膦基烷基-4,5-二取代-1,8-萘酐;
其中,N-溴烷基-4,5-二取代-1,8-萘酐与三苯基膦的质量比为:1:2.7-8;N-溴烷基-4,5-二取代-1,8-萘酐的质量与乙腈的体积比为15-30:1mg/mL;
(3)线粒体探针的合成:
将N-三苯基膦基烷基-4,5-二取代-1,8-萘酐溶于乙二醇甲醚,向其中滴加脂肪胺,升温至100-140℃搅拌,反应10-15h后减压除去溶剂,硅胶色谱柱分离得到线粒体探针;
其中,N-三苯基膦基烷基-4,5-二取代-1,8-萘酐与脂肪胺的质量比为:1.6-2.4:1;N-三苯基膦基烷基-4,5-二取代-1,8-萘酐的质量与乙二醇甲醚的体积比为5.3-24:1;所述脂肪环胺为氮杂环丁烷、四氢吡咯、乙二胺衍生物及环己二胺衍生物。
10.一种如权利要求4所述的488nm激发的高稳定性超分辨荧光染料的合成方法,其特征在于包含步骤如下:
(1)SNAP-tag探针的合成
将N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾和2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤置于史莱克瓶中,氮气置换2-5次后加入干燥的N,N-二甲基甲酰胺;室温反应3-10h后,加压出去溶剂,硅胶柱分离,以体积比为100-10:1的二氯甲烷和甲醇为洗脱剂,除去溶剂得靶向SNAP-tag蛋白的荧光探针。
其中,N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺、叔丁醇钾、2-氨基-6-(N-甲基)四氢吡咯基鸟嘌呤的质量比为1:1-5:1-5;N-(4-羟甲基)苄基-4,5-脂肪胺基-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺的体积比为1:80-200g/mL。
11.一种如权利要求5所述的一类488nm激发的高稳定性超分辨荧光染料的合成方法,其特征在于包含步骤如下:
(1)Halo-tag探针的合成
将N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH置于史莱克瓶中,并氮气置换2-5次;将1-碘-6-氯己烷溶于干燥的N,N-二甲基甲酰胺后,加入反应液中;室温下搅拌1-5h后,减压除去溶剂,硅胶柱分离,以体积比为100~400:1的二氯甲烷和甲醇为洗脱剂,除去溶剂得到靶向Halo-tag蛋白的荧光探针;
其中,N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺与NaH的质量比为5-10:1;N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与1-碘-6-氯己烷体积比为0.5-1mg/μL;N-(2-(2-羟基)-乙氧基)乙基-4,5-取代-1,8-萘酰亚胺的质量与N,N-二甲基甲酰胺体积比为5-20:1mg/mL。
12.一种如权利要求6所述的488nm激发的高稳定性超分辨荧光染料的合成方法,其特征在于包含步骤如下:
(1)中间体N-1-(羧基)烷基-4,5-二脂肪氨基-1,8-萘酰亚胺COOH-DF系列化合物
COEt-DF系列化合物溶于甲醇中,并向反应液中滴加2M氢氧化钠溶液。室温下反应1-3h后,减压蒸馏除去甲醇,过滤并用水洗涤滤饼干燥后得COOH-DF系列化合物;
其中,COEt-DF系列化合物的质量与甲醇的体积比为10-20:1mg/mL;COEt-DF系列化合物的质量与2M氢氧化钠溶液的体积比为10-20:1mg/mL;COEt-DF系列化合物的质量与水的体积比为10-20:1mg/mL;
(2)带有NHS活性基团的荧光染料合成
将COOH-DF系列化合物,DCC溶于干燥的N,N-二甲基甲酰胺后,室温搅拌10-40min;N-羟基琥珀酰亚胺溶于1mL干燥的N,N-二甲基甲酰胺并加入反应液中;2-5h后减压除去溶剂,硅胶柱分离,以体积比4~20:1的二氯甲烷和乙酸乙酯为洗脱剂,除去溶剂后得NHS活性基团的荧光染料染料。
其中,COOH-DF系列化合物、DCC、NHS质量比为1:1-5:1-10;COOH-DF系列化合物的质量与N,N-二甲基甲酰胺的体积比为10-20:1mg/mL。
13.一种如权利要求7所述的488nm激发的高稳定性超分辨荧光染料的合成方法,其特征在于包含步骤如下:
(1)含药物性的荧光染料的合成
10-30mg带有NHS活性基团的系列染料与带有活性氨基的药物分子置于史莱克瓶中,并用氮气置换2-5次;2-20μL二异丙基乙基胺溶于0.5-2mL干燥的二甲基亚砜中并加入反应瓶中;室温下搅拌3-10h后,水洗并用二氯甲烷萃取得有机相,硅胶柱分离得药物分子为靶向基团的荧光染料;
其中,NHS活性基团的系列染料与药物分子的质量比为1:0.5-1;NHS活性基团的系列染料的质量与二异丙基乙基胺的体积比为2-5:1mg/μL;二异丙基乙基胺与二甲基亚砜的体积比为1:100-300。
药物分子包括紫杉醇、秋水仙素、磺胺、生物素或叶酸。
14.如权利要求1所述的一类488nm激发的高稳定性超分辨荧光染料在细胞、组织及活体内的荧光成像领域的应用。
15.如权利要求1所述的一类488nm激发的高稳定性超分辨荧光染料用于SNAP-tag蛋白的识别与检测。
16.如权利要求1所述的一类488nm激发的高稳定性超分辨荧光染料用于Halo-tag蛋白的识别与检测。
17.如权利要求1所述的一类488nm激发的高稳定性超分辨荧光染料在单分子检测中的应用。
18.如权利要求1所述的一类488nm激发的高稳定性超分辨荧光染料在超分辨成像技术中的应用。
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