CN111333622B - 含活性酯的高亮度、高稳定性荧光染料及其合成和应用 - Google Patents
含活性酯的高亮度、高稳定性荧光染料及其合成和应用 Download PDFInfo
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Abstract
本发明提供一类含活性酯的高亮度、高稳定性荧光染料及其合成和应用,该荧光染料通过萘酰亚胺4,5‑位刚性环的引入达到了荧光稳定性、亮度得到大幅度提升,其结构式如(1)所示,该类染料在水中荧光量子产率最高可达0.80以上,荧光发射波长在495nm左右,半峰宽小于35nm,能有效避免串色现象。在不同微环境下该类染料能够保持荧光信号的稳定性,对温度、极性等均不敏感。此外,由于活性酯的存在该类染料能够用于标记抗体、多肽等生物分子,实现对目标物的监测与荧光成像。
Description
技术领域
本发明属于荧光标记领域,具体涉及一类含活性酯的高亮度、高稳定性荧光染料及其合成和应用。
背景技术
由于灵敏度高、原位实时响应、检测周期短的优势,荧光分析法在各研究领域(如:荧光PCR检测、抗体标记等)已经被广泛应用于识别与标记,其能够通过荧光信号将微观的信息变化以宏观的形式显示出来。而荧光分析法应用的同时也催动着荧光分子向更高亮度、更高稳定性发展,其中有机小分子荧光染料凭借易修饰、可选择性多、颜色丰富等特点备受研究工作者的青睐。借助活性酯基团即可实现有机小分子荧光染料与目标物氨基基团的共价连接,达到稳定的荧光标记。
目前,用于荧光分析的有机小分子荧光染料主要为共振(resonant)染料,拥有窄的吸收与发射峰、溶剂不敏感、斯托克斯位移小、高摩尔消光系数及高量子产率等特点,如罗丹明、花菁染料、荧光素等。但是此类染料分子本身会带有电荷,严重影响分子的标记性能、稳定性、相容性,如荧光素分子负离子容易被氧化为淬灭,不易穿透细胞膜;花菁染料的聚甲炔链极易被单线态氧进攻,烯烃的扭转导致量子产率降低等。尤其在超分辨与单分子检测技术蓬勃发展的时代,有机小分子荧光染料的稳定性、亮度仍需进一步提高,亟需高亮度、高稳定、相容性好的中性荧光小分子的出现。
发明内容
本发明的目的之一是提供一类含活性酯的高亮度、高稳定性荧光染料,该类染料为不带电荷中性分子,在水中量子产率最高可达0.80以上,可用于对目标物氨基的修饰。
本发明的另一目的是提供一类含活性酯的高亮度、高稳定性荧光染料的合成方法,该方法步骤简单、易于提纯、通用性好等优点。
本发明提供一类含活性酯的高亮度、高稳定性荧光染料,通过萘酰亚胺4,5- 位刚性环引入达到荧光稳定性、亮度得到大幅度提升。该类染料呈现出共振染料亮度高、半峰宽窄等优势的同时保持自身不带电荷的特性,对多种微环境变化均不敏感,保持了荧光信号的准确性。
一类含活性酯的高亮度、高稳定性荧光染料,该系列荧光探针具有如下结:
其中n为0-2整数。
一类含活性酯的高亮度、高稳定性荧光染料的合成方法,此系列荧光探针合成路线,如下:
具体合成步骤如下:
(1)中间体N-(酯基)烷基-4-溴-5-硝基-1,8-萘酰亚胺COEt-NBr的合成:
将4-溴-5-硝基-1,8-萘酐,1-氨基脂肪酸酸乙酯盐酸盐,三乙胺溶于无水乙醇中。将反应液加热至40-90℃,搅拌1-24h。将反应液泠却至室温后,减压除去溶剂后,硅胶柱分离,以体积比为1:0.25~6的二氯甲烷和石油醚或体积比为 1:0~0.01的二氯甲烷和甲醇为洗脱剂,减压除去溶剂得米白色固体N-(酯基) 烷基-4-溴-5-硝基-1,8-萘酰亚胺COEt-NBr;
(2)中间体N-(酯基)烷基-4,5-二脂肪氨基-1,8-萘酰亚胺COEt-DF的合成:将COEt-NBr系列化合物,溶于乙二醇甲醚中,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50~400:1的二氯甲烷和甲醇为洗脱剂,除去溶剂,得棕黄色固体N-(酯基)烷基-4,5-二脂肪氨基-1,8-萘酰亚胺COEt-DF;
(3)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;
(4)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;
步骤(1)中,4-溴-5-硝基-1,8-萘酐、1-氨基脂肪酸酸乙酯盐酸盐、三乙胺的质量比为1:1-3:1-3;4-溴-5-硝基-1,8-萘酐的质量与乙醇的体积比为1:20-80 g/mL;
步骤(2)中,N-(酯基)烷基-4-溴-5-硝基-1,8-萘酰亚胺与脂肪环胺的质量比为1:0.5-3;N-烷基-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 10-20:1mg/mL;
所述脂肪环胺为氮丙啶、氮杂环丁烷、四氢吡咯、环己二胺;
步骤(3)中,COEt-DF系列化合物的质量与甲醇的体积比为10-20:1 mg/mL;COEt-DF系列化合物的质量与2M氢氧化钠溶液的体积比为10-20:1 mg/mL;COEt-DF系列化合物的质量与水的体积比为10-20:1mg/mL;
步骤(4)中,COOH-DF系列化合物、DCC、NHS质量比为1:1-5:1-10; BCOOH-DAC的质量与N,N-二甲基甲酰胺的体积比为10-20:1mg/mL。
一类含活性酯的高亮度、高稳定性荧光染料在抗体的荧光标记领域的应用。
一类含活性酯的高亮度、高稳定性荧光染料在荧光成像领域的应用。
上述一类含活性酯的高亮度、高稳定性荧光染料能够用于对含有氨基等亲核基团的目标物进行荧光标记,荧光亮度高、稳定性强,在荧光成像、标记领域有较大应用前景。本发明具有以下特点:
本发明涉及的荧光染料拥有合成原料低价、方法简单通用等优点。
本发明涉及的荧光染料4,5-位刚性结构的引入使荧光量子产率大幅升,水中量子产率在0.80以上,摩尔消光系数达到40000M-1/cm-1以上。
本发明涉及的荧光染料能够通过活性酯与生物大分子氨基残基进行缩合达到对生物大分子的稳定荧光标记。
本发明涉及的荧光染料对于温度、黏度、极性等微环境均不敏感,能够最大限度保持荧光信号的准确性。
附图说明
图1实施例1制备的COOH-DAze的核磁谱图氢谱。
图2实施例2制备的COOH-DAC的核磁谱图氢谱。
图3实施例3制备的NHSB-DAC的核磁谱图氢谱。
图4实施例1制备的探针COOH-DAze在水中的归一化紫外吸收与荧光发射光谱图,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5实施例2制备的探针COOH-DAC在氯仿、水中的归一化荧光发射光谱图,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6实施例3制备的探针NHSB-DAC标记的细胞内骨架结构的荧光共聚焦成像图。
具体实施方式
实施例1
NHSM-DAze的合成
中间体2-(N-(4-溴-5-硝基-1,8萘酰亚胺))氨基乙酸乙酯(COMe-NBr) 的合成
4-溴-5-硝基-1,8-萘酰亚胺(1.00g,3.11mmol)溶于80mL乙醇中,并向其中加入甘氨酸乙酯盐酸盐(1000mg,7.17mmol)与1.00g三乙胺。90℃下反应24h后,减压蒸馏除去溶剂,残余物经硅胶柱(二氯甲烷:石油醚=3:1, V/V)分离得白色固体962mg,产率76%。其核磁谱图数据如下:
1H NMR(400MHz,CDCl3)δ8.73(d,J=7.8Hz,1H),8.53(d,J=7.9Hz,1H), 8.24(d,J=7.9Hz,1H),7.94(d,J=7.8Hz,1H),4.92(s,2H),4.26(q,J=7.1Hz, 2H),1.31(t,J=7.1Hz,3H).13C NMR(101MHz,CDCl3)δ167.36,162.48,161.75, 151.56,136.06,132.66,131.64,130.74,125.26,124.71,123.60,122.00,121.35, 61.94,41.64,14.16.
其高分辨质谱数据如下:高分辨质谱理论值C16H12BrN2O6[M+H]+406.9879, 实测值406.9888.
中间体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1制备的COOH-DAze核磁谱图氢谱如图1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)δ 163.49,155.72,133.21,132.50,109.02,107.32,106.59,54.80,43.24,16.81.
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.
经检测,其结构如上式COOH-DAze所示,其荧光性能如下:
将COOH-DAze溶解于DMSO溶液中,配制成mM母液,根据需要配制成不同浓度测试溶液,以检测其荧光光谱及紫外吸收光谱。
COOH-DAze在水中紫外吸收与荧光发射光谱测试。每次取20μL COOH-DAze母液加入4mL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。
COOH-DAze在水中归一化紫外吸收与荧光发射光谱如图4所示: COOH-DAze在水光发射波长在489nm,吸收波长在480nm,荧光发射半峰宽只有35nm。
NHSM-DAze的合成
COOH-DAze(30mg,0.08mmol)与二环己基碳亚(DCC)(30mg,0.15 mmol)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493 nm,能够与活性氨基进行室温缩合。
实施例2
NHSM-DAC的合成
中间体2-(N-(4-溴-5-硝基-1,8萘酰亚胺))氨基乙酸乙酯(COMe-NBr) 的合成
4-溴-5-硝基-1,8-萘酰亚胺(1.00g,3.11mmol)溶于20mL乙醇中,并向其中加入甘氨酸乙酯盐酸盐(3000mg,21.5mmol)与3.00g三乙胺。40℃下反应1h后,减压蒸馏除去溶剂,残余物经硅胶柱(二氯甲烷:石油醚=3:1,V/V) 分离得白色固体418mg,产率33%。
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-DAC 46mg,产率83%。实施例2制备的COOH-DAC核磁谱图氢谱如图2所示,核磁谱图氢谱与碳谱具体数据如下:
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=11.7 Hz,2H),1.73(d,J=6.9Hz,2H),1.31(dt,J=31.3,16.1Hz,4H).13C NMR(101 MHz,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,其荧光性能如下:
将COOH-DAC溶解于DMSO溶液中,配制成mM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。
COOH-DAC在氯仿、水中荧光发射光谱测试。每次取20μL COOH-DAC 母液加入4mL氯仿、水中,配制成10μM的荧光染料测试液,进行荧光发射光谱的测试。
COOH-DAC在氯仿、水中归一化荧光发射光谱如图5所示:COOH-DAC 在氯仿、水光发射波长分别在482nm与478nm,即COOH-DAC染料受环境中极性影响较小,荧光发射波长与峰型几乎不变,能够保持荧光信号的准确性。
NHSM-DAC的合成
COOH-DAC(20mg,0.05mmol)与二环己基碳亚(DCC)(100mg,0.50 mmol)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3
NHSB-DAC的合成
中间体6-(N-(4-溴-5-硝基-1,8萘酰亚胺))氨基丁酸乙酯(BCOMe-NBr) 的合成
4-溴-5-硝基-1,8-萘酰亚胺(1.00g,3.11mmol)溶于80mL乙醇中,并向其中加入4-氨基丁酸乙酯盐酸盐(1.00g,6.21mmol)与3.00g三乙胺。80℃下反应10h后,减压蒸馏除去溶剂,残余物经硅胶柱(二氯甲烷:石油醚=3:1, V/V)分离得白色固体608mg,产率45%。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),8.52(d,J=7.9Hz,1H), 8.22(d,J=7.9Hz,1H),7.93(d,J=7.8Hz,1H),4.25(t,J=7.1Hz,2H),4.10(q,J =7.1Hz,2H),2.44(t,J=7.4Hz,2H),2.09(p,J=7.3Hz,2H),1.24(t,J=7.1Hz, 3H).13C NMR(101MHz,CDCl3)δ172.72,162.85,162.09,151.33,136.00,132.40, 131.30,130.57,125.65,124.24,123.56,122.36,121.24,60.53,40.11,31.82,23.20, 14.23.
其高分辨质谱数据如下:高分辨质谱理论值C18H16BrN2O6[M+H]+435.0192, 实测值435.0193.
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 65 mg,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.
NHSB-DAC的合成
BCOOH-DAC(50mg,0.12mmol)与二环己基碳亚(DCC)(112mg,0.54 mmol)溶于2mLN,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2mL N,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%。实施例3制备的NHSB-DAC核磁谱图氢谱如图3所示,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。
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,以用于蛋白标记及细胞内荧光成像。
实施例4
实施例3制备的荧光探针NHSB-DAC标记多克隆抗体并纯化。
NHSB-DAC母液10μL加入到含有多克隆抗体羊抗小鼠IgG(0.5mg/mL) 的100μL溶液中,室温下静置1h,过葡聚糖凝胶柱G-25除去多余的荧光小分子。
实施例5
实施例4制备的NHSB-DAC标记多克隆抗体标记微管蛋白。将NHSB-DAC 标记多克隆抗体溶于水溶液中配制成0.5mg/mL的母液备用。将Hela细胞(增殖表皮癌细胞)铺在培养皿中,皿中含有10%胎牛血清的DMED高糖培养基1 mL,在37℃和5%二氧化碳条件下培养至细胞密度约为70%,用PBS缓冲液轻柔洗涤细胞2次后,用4%多聚甲醛固定30min,弃掉固定液用PBS洗3次,然后用0.2%的TritonX-100透化20min后用PBS洗3次,每次5min,然后用 5%的BSA封闭液封闭20分钟后再用PBS洗3次。加入含有抗α-微管蛋白的单克隆抗体(约10μg/mL)的200μLPBS溶液,4℃孵育过夜。第二天用PBS洗 3遍后加入含NHSB-DAC标记的多克隆抗体(约10μg/mL)的200μLPBS溶液, 37℃孵育3小时。最后用PBS清洗3遍后在荧光共聚焦显微镜下成像。
NHSB-DAC标记多克隆抗体对细胞内细胞骨架共聚焦荧光成像如图6所示:NHSB-DAC标记的细胞骨架结构清晰,荧光背景较低。
Claims (8)
2.如权利要求1所述的一类含活性酯的高亮度、高稳定性荧光染料的合成方法,其特征包含步骤如下:
将4-溴-5-硝基-1,8-萘酐,1-氨基脂肪酸乙酯盐酸盐,三乙胺溶于无水乙醇中;将反应液加热至40-90℃,搅拌1-24h;将反应液冷却至室温后,减压除去溶剂后,硅胶柱分离,以体积比为1:0.25~6的二氯甲烷和石油醚或体积比为1:0~0.01的二氯甲烷和甲醇为洗脱剂,减压除去溶剂得米白色固体
将溶于乙二醇甲醚中,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50~400:1的二氯甲烷和甲醇为洗脱剂,除去溶剂,得棕黄色固体所述脂肪环胺为氮丙啶、氮杂环丁烷、四氢吡咯、环己二胺;
(4)含活性酯的高亮度、高稳定性荧光染料的合成
3.根据权利要求2所述的一类含活性酯的高亮度、高稳定性荧光染料的合成方法,其特征在于步骤(1)中,4-溴-5-硝基-1,8-萘酐、1-氨基脂肪酸乙酯盐酸盐、三乙胺的质量比为1:1-3:1-3;4-溴-5-硝基-1,8-萘酐的质量与乙醇的体积比为1:20-80g/mL。
7.一类如权利要求1所述的含活性酯的高亮度、高稳定性荧光染料在抗体的荧光标记领域的应用。
8.一类如权利要求1所述的含活性酯的高亮度、高稳定性荧光染料在荧光成像领域的应用。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011011605A1 (en) * | 2009-07-22 | 2011-01-27 | Opti Medical Systems | Hydrogen peroxide sensors based upon photo-induced electron transfer |
WO2012116949A1 (en) * | 2011-02-28 | 2012-09-07 | F. Hoffmann-La Roche Ag | Application of quantum dots for nuclear staining |
CN107603269A (zh) * | 2016-07-11 | 2018-01-19 | 华东理工大学 | 一类基于萘酰亚胺的荧光染料、其制备方法及应用 |
CN108069902A (zh) * | 2016-11-14 | 2018-05-25 | 中国科学院大连化学物理研究所 | 一类标记和/或检测细胞中脂滴的荧光探针及其制备和应用 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011011605A1 (en) * | 2009-07-22 | 2011-01-27 | Opti Medical Systems | Hydrogen peroxide sensors based upon photo-induced electron transfer |
WO2012116949A1 (en) * | 2011-02-28 | 2012-09-07 | F. Hoffmann-La Roche Ag | Application of quantum dots for nuclear staining |
CN107603269A (zh) * | 2016-07-11 | 2018-01-19 | 华东理工大学 | 一类基于萘酰亚胺的荧光染料、其制备方法及应用 |
CN108069902A (zh) * | 2016-11-14 | 2018-05-25 | 中国科学院大连化学物理研究所 | 一类标记和/或检测细胞中脂滴的荧光探针及其制备和应用 |
Non-Patent Citations (2)
Title |
---|
Aziridinyl Fluorophores Demonstrate Bright Fluorescence and Superior Photostability by Effectively Inhibiting Twisted Intramolecular Charge Transfer;Xiaogang Liu et al;《 J. Am. Chem. Soc.》;20160520;第138卷;6960-6963 * |
含N-羟基琥珀酰亚胺酯的三枝BODIPY染料的合成;甄文;《工程科技Ⅰ辑》;20180331;第1.5、1.5.1节 * |
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