CN111333644B - 一类近红外脂滴荧光染料及其的合成方法和应用 - Google Patents
一类近红外脂滴荧光染料及其的合成方法和应用 Download PDFInfo
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Abstract
本发明提供了一类近红外脂滴荧光染料及其合成方法和应用，该荧光染料是基于苝酰亚胺，在供电基一端引入环己二胺设计合成的一类高亮度近红外荧光染料，其结构式如(1)所示，环己二胺的强供电性大幅度提升了染料的荧光激发与发射波长，这导致这类染料在乙醇中的激发波长达到了710nm以上，荧光发射达到750nm。此外，环己二胺在增加分子刚性结构的同时使分子对称性得到提升，这使得染料对溶剂极性极不敏感，且半峰宽只有45nm。这类染料在不同溶剂的荧光量子产率均达到0.4以上，摩尔消光系数达到50000M‑1cm‑1左右。本项目设计的高亮度近红外染料对细胞毒性小，组织穿透深，能够广泛应用于组织及活体成像。
Description
技术领域
本发明属于用于脂滴标记的荧光染料领域，具体涉及一类近红外脂滴荧光染料及其合成方法和应用。
背景技术
脂滴是由磷脂单分子层及中性脂(三酰基甘油、甾醇酯等)构成的疏水核，是机体内各组织细胞脂肪存储的主要形式，在膜的运输、蛋白降解、病毒识别等过程中起着重要作用。然而在不同组织与细胞中，脂滴具有特异性，其数目、大小、分布并不相同，从100nm到100μm大小不一；同一细胞内随其生理环境条件变化其脂滴的数目与尺寸变化也很快。这就需要脂滴的监测需要高的时间及空间分辨率，同时需要在细胞、组织甚至活体中实现原位分析。
近几年有机小分子荧光染料凭借其高时空分辨率及原位分析的优势被广泛应用于脂滴相关研究中，用于脂滴的示踪、定量分析等。然而，目前商业脂滴染料的荧光发射很难进入到近红外区，这也导致组织及或体内对脂滴的研究受到极大限制。例如：应用最广泛的商业脂滴染料尼罗红与BODIPY515，二者合适的激发均在488nm，发射在500-580nm，这一波段活细胞内存在较高荧光背景，且这一波长很难穿透500μm以上样本。此外，商业脂滴染料多以氟硼吡咯为荧光母体，光稳定较差。因此，近红外区的尤其700nm以上脂滴染料存在巨大空白，亟待借助这类染料实现脂滴在组织及活体层次的相关研究。
发明内容
本发明的目的是提供一类近红外脂滴荧光染料的合成及应用。
本发明提供一类近红外脂滴荧光染料，以苝酰亚胺为荧光基团，在其9,10- 位引入环己二胺结构，使得荧光激发波长及荧光发射波长红移至近红外区。该荧光染料对多种细胞进行染色后能特定的标记细胞内脂滴，具有波长长、染色浓度低、快速染色、生物相容性好等特点，能够用于活细胞、活体成像。
一类近红外脂滴荧光染料，该荧光染料具有如下结构：
其中R1为
一类近红外脂滴荧光染料，该染料荧光发射波长达到750nm，荧光亮子产率达到0.4，能够广泛应用于组织及活体成像。
一类近红外脂滴荧光染料的合成方法，用于脂滴标记的荧光染料合成路线，如下：
具体合成步骤如下：
(1)中间体N-烷基-9,10-二溴-1,6,7,12-四氯苝酰亚胺的合成：
将9,10-二溴-1,6,7,12-四氯苝酰亚胺与二甘醇胺或丁胺溶于N-甲基吡咯烷酮与冰醋酸混合液中；将反应液加热至100-140℃，搅拌1-10h；将反应液泠却至室温后倒入冰水中抽滤得黑色固体，真空干燥，经200-300目二氧化硅硅胶柱分离，以体积比1:0.25～6的二氯甲烷和石油醚为洗脱剂，减压除去溶剂得深红色固体N-烷基-9,10-二溴-1,6，7,12-四氯苝酰亚胺；
其中，9,10-二溴-1,6,7,12-四氯苝酰亚胺与二甘醇胺或丁胺的质量比为 1-10:1；9,10-二溴-1,6,7,12-四氯苝酰亚胺的质量与N-甲基吡咯烷酮与醋酸混合液的体积比为1:20-120(g：mL)；N-甲基吡咯烷酮与冰醋酸的体积比为1-3:3-4。
(2)探针N-烷基-9,10-环己二胺基-1,6,7,12-四氯苝酰亚胺的合成：
将N-烷基-9,10-二溴-1,6，7,12-四氯苝酰亚胺，溶于乙二醇甲醚中，并向其中加入环己二胺；而后将反应液缓慢升温至90-130℃，并在氮气保护下反应 10-24h；减压除去溶剂，硅胶柱(200-300目二氧化硅)分离，以体积比1:0～1 的二氯甲烷和石油醚为洗脱剂，减压除去溶剂，得蓝色固体探针N-烷基-9,10- 二脂肪胺基-1,6,7,12-四氯苝酰亚胺。
其中，N-烷基-9,10-二溴-1,6，7,12-四氯苝酰亚胺与反式环己二胺的质量比为1-2:1-6；反式环己二胺与乙二醇甲醚质量与体积比为5-120:1(mg：mL)。
一种近红外脂滴荧光染料的合成方法，该方法具有操作方便、原料廉价、提纯简单等优点。
一种近红外脂滴荧光染料在细胞与活体内脂滴标记、脂滴的荧光成像、脂滴定量分析及脂滴的实时监测领域的应用。
上述用于脂滴标记的一类荧光染料在活细胞及活体内能够对脂滴进行特异性标记并实现荧光成像。
本发明具有以下特点：
本发明的类染料拥有合成原料价廉、方法简单且易于拓展等优点。
本发明的染料在在乙醇中荧光激发波长能够达到710nm以上，荧光量子产率>0.40；荧光发射波长达到750nm左右。此类染料荧光发射波长及激发波长均达到了近红外区，组织穿透能力强，对细胞损伤小，更有利于活细胞、组织及活体成像。
此类染料在活细胞中能够对脂滴进行精准定位；同时能够对斑马鱼活体进行实时荧光成像。
附图说明
图1实施例3制备的N-(2-(2-羟基)-乙氧基)乙基-9,10-环己二胺基-1,6,7,12-四氯苝酰亚胺(OLD-710)的核磁谱图氢谱。
图2为实施例3制备的脂滴染料OLD-710在不同溶剂中的荧光谱图，横坐标为波长，纵坐标为荧光强度，荧光探针的浓度为10μM。
图3为实施例3制备的脂滴染料OLD-710在不同溶剂中的紫外吸收谱图，横坐标为波长，纵坐标为吸收强度，荧光探针的浓度为10μM。
图4中实施例3制备的脂滴染料OLD-710的MCF活细胞荧光成像图。
图5中实施例3制备的脂滴染料OLD-710对斑马鱼染色后的荧光成像图，左图为斑马鱼的宽场成像图，右图为斑马鱼荧光成像图。
具体实施方式
实施例1
近红外染料BuLD-710的合成方法。
中间体N-丁基-9,10-二溴-1,6,7,12-四氯苝酰亚胺的合成：
将1,6,7,12-四氯-9,10-二溴-3,4-苝酐(1.2g，1.96mmol)溶于乙酸与N-甲基吡咯烷酮混合液60mL(4:1,V/V),而后向其中滴加正丁胺(428mg,5.86mmol)。 120℃反应6h后，将反应液倒入200mL冰水中，沉降并过滤得黑色固体。黑色固体经硅胶柱分离(石油醚：二氯甲烷＝1:1，V/V)得红色固体600mg，产率46％。其核磁谱图氢谱数据如下：
1H NMR(400MHz,CDCl3)δ8.59(s,2H),8.14(s,2H),4.38–4.11(m,2H), 1.94–1.66(m,2H),1.56–1.38(m,2H),0.99(t,J＝7.1Hz,3H).
染料N-丁基-9,10-环己二胺基-1,6,7,12-四氯苝酰亚胺的合成：
将N-丁基-1,6,7,12-四氯-9,10-二溴-3,4-苝酰亚胺(200mg，0.30mmol)与反式环己二胺(228mg，2.00mmol)溶于10mL乙二醇甲醚，并将其加热至90℃。 14h后减压除去溶剂，残余物经硅胶柱分离(石油醚：二氯甲烷＝1:4，V/V) 得蓝绿色固体56mg，产率30％。其核磁谱图氢谱数据如下：
1H NMR(400MHz,DMSO-d6)δ8.24(d,J＝3.7Hz,2H),8.11(s,1H),7.96(s, 1H),7.17(d,J＝10.8Hz,2H),4.28(t,J＝6.5Hz,2H),3.21(d,J＝10.0Hz,2H), 2.26(d,J＝10.9Hz,2H),1.94-1.68(m,2H),1.77(s,2H),1.56-1.38(m,2H),1.41(s, 2H),1.25(s,2H),0.99(t,J＝7.1Hz,3H).
经检测，其结构如上式BuLD-710所示，其乙醇中荧光发射波长为750nm 左右，已达到近红外区，且能够标记活细胞内脂滴。
实施例2
脂滴荧光染料OLD-710的合成方法。
中间体N-(2-(2-羟基)-乙氧基)乙基-9,10-二溴-1,6,7,12-四氯苝酰亚胺的合成：
将1,6,7,12-四氯-9,10-二溴-3,4-苝酐(0.6g，0.98mmol)溶于乙酸与N-甲基吡咯烷酮混合液72mL(1:1,V/V),而后向其中滴加二甘醇胺(600mg,17.4 mmol)。140℃反应1h后，将反应液倒入250mL冰水中，沉降并过滤得黑色固体。黑色固体经硅胶柱分离(石油醚：二氯甲烷＝1:1-1:4，V/V)得红色固体128mg，产率37％。其核磁谱图氢谱数据如下：
1H NMR(400MHz,CDCl3)δ8.60(s,2H),8.13(s,2H),4.67–4.41(m,2H), 3.88(d,J＝5.3Hz,2H),3.71(d,J＝4.5Hz,2H),3.67(d,J＝3.6Hz,2H),2.38(s, 1H).
染料N-(2-(2-羟基)-乙氧基)乙基-9,10-环己二胺基-1,6,7,12-四氯苝酰亚胺(OLD-710)的合成：
将N-(2-(2-羟基)-乙氧基)乙基-9,10-二溴-1,6,7,12-四氯苝酰亚胺(100mg，0.14mmol)与反式环己二胺(600mg，5.26mmol)溶于5mL乙二醇甲醚，并将其加热至130℃。18h后减压除去溶剂，残余物经硅胶柱分离(展开剂：二氯甲烷)得蓝绿色固体48mg，产率52％。其核磁谱图氢谱数据如下：
1H NMR(400MHz,DMSO-d6)δ8.25(d,J＝3.8Hz,2H),8.11(s,1H),7.94(s, 1H),7.187(d,J＝10.7Hz,2H),4.60(s,1H),4.26(t,J＝6.5Hz,2H),3.65(t,J＝6.6 Hz,2H),3.48(s,4H),3.22(d,J＝10.1Hz,2H),2.26(d,J＝10.8Hz,2H),1.76(s, 2H),1.43(s,2H),1.27(s,2H).
经检测，其结构如上式OLD-710所示，其在乙醇中荧光发射波长为750nm 左右，已达到近红外区，且能够标记活细胞内脂滴。
实施例3
脂滴荧光染料OLD-710的合成方法。
中间体N-(2-(2-羟基)-乙氧基)乙基-9,10-二溴-1,6,7,12-四氯苝酰亚胺的合成：
将1,6,7,12-四氯-9,10-二溴-3,4-苝酐(1.2g，1.96mmol)溶于乙酸与N-甲基吡咯烷酮混合液24mL(3:1,V/V),而后向其中滴加二甘醇胺(120mg,3.48 mmol)。100℃反应6h后，将反应液倒入250mL冰水中，沉降并过滤得黑色固体。黑色固体经硅胶柱分离(石油醚：二氯甲烷＝1:1-1:4，V/V)得红色固体380mg，产率55％。其核磁谱图氢谱数据如下：
1H NMR(400MHz,CDCl3)δ8.60(s,2H),8.13(s,2H),4.67–4.41(m,2H), 3.88(d,J＝5.3Hz,2H),3.71(d,J＝4.5Hz,2H),3.67(d,J＝3.6Hz,2H),2.38(s, 1H).
染料N-(2-(2-羟基)-乙氧基)乙基-9,10-环己二胺基-1,6,7,12-四氯苝酰亚胺(OLD-710)的合成：
将N-(2-(2-羟基)-乙氧基)乙基-9,10-二溴-1,6,7,12-四氯苝酰亚胺(200mg，0.28mmol)与反式环己二胺(100mg，0.88mmol)溶于20mL乙二醇甲醚，并将其加热至120℃。24h后减压除去溶剂，残余物经硅胶柱分离(展开剂：二氯甲烷)得蓝绿色固体80mg，产率43％。实施例3制备的N-(2-(2-羟基)- 乙氧基)乙基-9,10-二-氮杂环丁基-1,6,7,12-四氯苝酰亚胺(OLD-710)的核磁谱图氢谱如图1所示，具体数据如下：
1H NMR(400MHz,DMSO-d6)δ8.24(d,J＝3.7Hz,2H),8.10(s,1H),7.94(s, 1H),7.17(d,J＝10.8Hz,2H),4.60(s,1H),4.26(t,J＝6.5Hz,2H),3.65(t,J＝6.6 Hz,2H),3.48(s,4H),3.21(d,J＝10.0Hz,2H),2.26(d,J＝10.9Hz,2H),1.76(s, 2H),1.40(s,2H),1.22(s,2H).
经检测，其结构如上式OLD-710所示，其乙醇中荧光发射波长为750nm 左右，已达到近红外区，且能够标记活细胞内脂滴。
将该染料分别溶解于DMSO溶液中，配制成不同染料的2mM母液，根据需要制配成不同浓度测试溶液，检测其荧光光谱变化及细胞、活体内脂滴荧光成像。
OLD-710在乙腈、氯仿、乙醇、二甲基亚砜等溶剂中的荧光发射光谱测试。每次取20μL OLD-710母液，分别加入4mL乙腈、氯仿、乙醇、二甲基亚砜，配制成10μM的荧光探针测试液，并进行荧光光谱的测试。
OLD-710在乙腈、氯仿、乙醇、二甲基亚砜中的归一化的荧光谱图如图2 所示：荧光染料浓度为10μM，OLD-710在乙腈、氯仿、乙醇、二甲基亚砜中的荧光发射波长均大于720nm，乙醇及DMSO中波长为750nm左右，达到了近红外发射波长。
OLD-710在乙腈、氯仿、乙醇、二甲基亚砜等溶剂中归一化的紫外吸收。每次取20μL荧光染料母液，分别加入4mL乙腈、氯仿、乙醇、二甲基亚砜，配制成10μM的荧光染料测试液，并进行紫外吸收光谱的测试。
OLD-710在乙腈、氯仿、乙醇、二甲基亚砜中的归一化的紫外吸收谱图如图3所示：荧光染料浓度为10μM。OLD-710在乙腈、氯仿、乙醇、二甲基亚砜中的紫外吸收波长均大于650nm，乙醇及二甲基亚砜中达到了710nm以上。
实施例4
OLD-710对活细胞染色后荧光成像检测。取0.5μL OLD-710母液溶于1mL 细胞培养液中，37℃，5％CO2下孵育15分钟后分别进行荧光共聚焦成像。
OLD-710终浓度为1μM的细胞培养液孵育乳腺癌细胞(MCF)15分钟后共聚焦荧光成像图如图4所示：MCF细胞内圆形脂滴清晰可见，可观察到不同脂滴的大小及分布。
实施例5
OLD-710对斑马鱼染色后荧光成像检测。取1μL OLD-710母液溶于1mL 斑马鱼幼鱼培养液中，并在室温孵育30分钟后进行共聚焦荧光成像。
OLD-710终浓度为2μM的斑马鱼培养液孵育斑马鱼幼鱼30分钟后共聚焦荧光成像图如图5中右图所示：OLD-710能够对斑马鱼体内脂类代谢中心肝脏及腹部进行特异性标记。
Claims (3)
1.一类近红外脂滴荧光染料的合成方法，其特征在于该荧光染料的结构如下：
其中R1为
近红外脂滴荧光染料合成方法包含步骤如下：
(1)中间体N-烷基-9,10-二溴-1,6,7,12-四氯苝酰亚胺的合成：
将1,6,7,12-四氯-9,10-二溴-3,4-苝酐与二甘醇胺或丁胺溶于N-甲基吡咯烷酮与冰醋酸混合液中；将反应液加热至100-140℃，搅拌1-10h；将反应液冷却至室温后倒入冰水中抽滤得黑色固体，真空干燥，经200-300目二氧化硅硅胶柱分离，以体积比1:0.25～6的二氯甲烷和石油醚为洗脱剂，减压除去溶剂得深红色固体N-烷基-9,10-二溴-1,6，7,12-四氯苝酰亚胺；
(2)荧光染料N-烷基-9,10-环己二胺基-1,6,7,12-四氯苝酰亚胺的合成：
将N-烷基-9,10-二溴-1,6，7,12-四氯苝酰亚胺，溶于乙二醇甲醚中，并向其中加入环己二胺；而后将反应液缓慢升温至90-130℃，并在氮气保护下反应10-24h；减压除去溶剂，经200-300目二氧化硅硅胶柱分离，以体积比1:0～1的二氯甲烷和石油醚为洗脱剂，减压除去溶剂，得到蓝色固体荧光染料N-烷基-9,10-环己二胺基-1,6,7,12-四氯苝酰亚胺。
2.根据权利要求1所述的一类近红外脂滴荧光染料的合成方法，其特征在于所述步骤(1)中，1,6,7,12-四氯-9,10-二溴-3,4-苝酐与二甘醇胺或丁胺的质量比为1-10:1；
1,6,7,12-四氯-9,10-二溴-3,4-苝酐的质量与N-甲基吡咯烷酮与冰醋酸混合液的体积比为1:20-120g/mL；
N-甲基吡咯烷酮与冰醋酸的体积比为1-3:3-4。
3.根据权利要求1所述的一类近红外脂滴荧光染料的合成方法，其特征在于所述步骤(2)中，N-烷基-9,10-二溴-1,6，7,12-四氯苝酰亚胺与环己二胺的质量比为1-2:1-6；
环己二胺的质量与乙二醇甲醚的体积比为5-120:1g/mL。
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