CN113527347B - 近红外荧光标记脂肪酸及其制备方法 - Google Patents
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Abstract
本发明公开了近红外荧光标记脂肪酸及其制备方法,包括如下步骤:将甲基吡咯、苯甲醛、和三乙胺按照摩尔比2~2.5:1~1.3:0.01~0.3混合溶解到二氯甲烷中,室温,搅拌3h~24h,冰浴中缓慢滴加1~1.3当量的络合剂,搅拌10~30分钟。本发明采用近红外荧光探针与亚油酸(LA)或DHA通过酯化反应结合,其中近红外荧光特性有利于区别生物组织自身荧光信号,并对阿尔兹海默症早期生物标志物β‑淀粉样蛋白寡聚体有效识别结合呈现荧光,亚油酸和DHA是对人体非常重要的物质,参与脑细胞的形成和发育,维持神经细胞的正常生理活动。近红外荧光标记LA或DHA后协同作用使得该近红外荧光探针能跨越血脑屏障,到达中枢神经系统,更加有效达到阿尔兹默症早期诊断预防的目的。
Description
技术领域
本发明属于有机合成技术领域,具体涉及近红外荧光标记脂肪酸及其制备方法。
背景技术
由于我们国家年龄老龄化的趋势,平均每3秒就有一位阿尔兹海默症患者产生,日趋严重地危害着社会的稳定。目前,FDA批准的用于治疗阿尔兹海默病患者药物只有5种,它们对阿尔兹海默症几乎没有治疗的效果,只有延缓的作用。由于阿尔兹海默症(AD)早期准确诊断以及病况监测手段缺失,治疗药物研究进展更进一步受到阻碍。因此,对初期AD进行及时准确诊断是目前人类预防和治疗AD的关键所在。淀粉样蛋白(β-amyloid proteins,Aβ)增高是AD发病的主要原因之一,因此开发高效灵敏Aβ探针有利于AD的早期诊断和治疗。1959年,Vassar和Culli利用荧光染料硫黄素T(Thioflavin T,简称THT)首次成功标记了Aβ,为AD早期诊断奠定了基础。在此基础上,各种标记Aβ的探针相继问世,有半导体量子点、碳量子点、有机荧光染料(如THT衍生物)等。但由于重金属潜在的毒性和“blinking”现象,半导体量子点在生物应用中有一定局限性;低量子产率以及易聚集等问题导致碳量子点在AD早期诊断应用中存在较大困扰;THT衍生物尽管与Aβ具有很高的亲和力,但短波长信号易受生物体自身干扰,并且光稳定性不高,使其诊断的可靠性大大降低。近红外荧光能够有效穿透生物组织,避免生物自发光的干扰,在药物筛选以及疾病检测中呈现出巨大优势,因此,研究开发新型高量子产率、光稳定性好的近红外Aβ荧光探针对AD早期诊断具有重要的科学意义和应用价值。另外,由于血脑屏障的存在,荧光探针很难进入中枢神经系统完成成像功能,所以开发能穿越血脑屏障诊断剂对AD的诊断治疗具有重要意义。
发明内容
本发明的主要目的在于提供一种近红外长波长、高灵敏度、高选择性的阿尔兹海默症早期诊断荧光探针,可以有效解决背景技术中的问题。
为实现上述目的,本发明提供如下技术方案:
近红外荧光标记脂肪酸,其分子结构式为:
式中BDP的羟基与脂肪酸的羧基通过酯化反应结合在一起,所述脂肪酸为LA或DHA;
其反应路线如下式所示:
R-COOH:
所述R为
近红外荧光标记脂肪酸的制备方法,包括如下步骤:
(1)将甲基吡咯、苯甲醛、和三乙胺按照摩尔比2~2.5:1~1.3:0.01~0.3混合溶解到二氯甲烷中,室温,搅拌3h~24h,冰浴中缓慢滴加1~1.3当量的络合剂,搅拌10~30分钟,加入1~1.3当量的催化剂A,二氯甲烷萃取,无水Na2SO4干燥,25℃~60℃真空旋蒸除去溶剂,采用层析柱分离提纯,得到橙黄色固体产物X1;
(2)将X1、对-乙羟基苯甲醛、催化剂B按照1:1~1.3:0.01~0.3溶于甲苯和哌啶1:0.1~1(v/v)的混合溶液中,置于装有Dean-Stark装置的圆底烧瓶中,140℃-150℃加热回流,直到所有溶剂都被Dean-Stark装置收集,再向反应介质中加入1:0.1(v/v)甲苯和哌啶,重复2~4次,TLC跟踪至原料反应完全后,柱层析分离提纯,减压除去溶剂后,得到黑色固体产物BDP;
(3)将LA或DHA、DCC/DMAP按照1:0.1~0.5(M:M)溶解到氯仿中,再加入BDP,25℃~90℃搅拌3~6h,25℃~60℃真空旋蒸除去溶剂,采用层析柱分离提纯,得到黑色固体产物BDP-R。
进一步地,所述步骤(1)中催化剂A为2,3-二甲基-5,6-二氰基苯醌作催化剂,所述络合剂为三氟化硼乙醚。
优选地,所述步骤(2)中催化剂B为对甲苯磺酰胺。
进一步地,所述步骤(3)中BDP和LA或BDP和DHA的摩尔比均为1:1~1.3。
与现有技术相比,本发明的有益效果是:
一、本发明采用近红外荧光探针与LA或DHA通过酯化反应结合,其中近红外荧光特性有利于区别生物组织自身荧光信号,并对阿尔兹海默症早期生物标志物β-淀粉样蛋白寡聚体有效识别结合呈现荧光,近红外荧光标记LA或DHA后协同作用使得该近红外荧光探针能跨越血脑屏障,到达中枢神经系统,更加有效达到阿尔兹默症早期诊断预防的目的;
二、亚油酸和DHA是对人体非常重要的物质,参与脑细胞的形成和发育,维持神经细胞的正常生理活动;LA和DHA是人体不能合成的必需的脂肪酸,有助于大脑细胞的修复生长,对于人体的修复起到积极作用;
三、化合物制备过程条件温和,步骤简单,有很好的潜在应用价值。
附图说明
图1是本发明得到的近红外荧光标记LA的核磁氢谱图。
图2是本发明得到的近红外荧光标记DHA的核磁氢谱图。
图3是本发明得到的近红外荧光标记LA与β-淀粉样蛋白共存160小时,β-淀粉样蛋白衍变的TEM形貌图。
图4是本发明得到的近红外荧光标记DHA监测β-淀粉样蛋白衍变过程荧光强度图(以ThT作为参照对比)。
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施方式,进一步阐述本发明。
以下实施例所用的X1制备方法如下:
将甲基吡咯(206mg,2.2mmol)、苯甲醛(106mg,1.0mmol)、和三乙胺(0.5ml)混合溶解到二氯甲烷中,室温搅拌过夜,0℃缓慢滴加三氟化硼乙醚(0.5ml),搅拌10分钟,加入2,3-二甲基-5,6-二氰基苯醌(227mg,1mmol),二氯甲烷萃取,无水Na2SO4干燥,25℃-60℃真空旋蒸除去溶剂,采用层析柱分离提纯,得到橙黄色固体产物X1。
实施例1
将X1(0.5mmol)、4-N,N-二苯基胺苯甲醛(1.0mmol)、对甲苯磺酰胺(0.01mmol)溶于甲苯(20mL)和哌啶(1mL)的混合溶液中,置于装有Dean-Stark装置的圆底烧瓶中,140℃加热回流,直到所有溶剂都被Dean-Stark装置收集,再向反应介质中加入甲苯(20mL)和哌啶(1mL),继续140℃加热回流直到所有溶剂都被Dean-Stark装置收集,重复加入甲苯(20mL)和哌啶(1mL)以及加热回流过程共3次,此过程通过TLC跟踪,原料反应完全后,柱层析,减压蒸馏除去溶剂后,得到黑色固体产物BDP,所述化合物通过核磁1H NMR谱图、质谱TOF MS EI+进行表征,1H NMR(300MHz,DMSO-D6):δ=7.67(d,J=8.8Hz,2H),7.33-7.34(m,5H),7.23(d,J=8.8Hz,2H),6.96(d,J=4.2Hz,2H),6.00(s,1H),6.02(s,1H),5.27(s,1H),4.61(s,2H),2.12(s,6H),2.07(s,3H);TOF MS EI+:442.2。
将LA(LA)(0.2mmol)、DCC(0.01mmol)、DMAP(0.01mmol)溶解到氯仿中,加入BDP(0.2mmol),70℃搅拌3h,60℃真空旋蒸除去溶剂,粗产品采用硅胶柱层析提纯,淋洗剂为二氯甲烷/乙酸乙酯(v/v,15/1),溶剂减压浓缩,得到黑色固体产物BDP-LA,所述化合物通过核磁1H NMR谱图、质谱TOF MS EI+进行表征,1H NMR(300MHz,DMSO-D6):δ=7.65(m,2H),7.31-7.35(m,5H),7.21(d,J=8.0Hz,2H),6.95(d,J=8.1Hz,2H),6.01(s,2H),5.48(m,2H),5.33(m,2H),5.22(s,2H),2.81(m,2H),2.33(m,2H),2.08-2.12(m,13H),1.68(m,2H),1.28-1.33(m,16H),0.88(m,3H);TOF MS EI+:718.5。
实施例2
将X1(0.3mmol)、4-N,N-二苯基胺苯甲醛(0.61mmol)、对甲苯磺酰胺(0.01mmol)溶于甲苯(2mL)和哌啶(0.5mL)的混合溶液中,置于装有Dean-Stark装置的圆底烧瓶中,145℃加热回流,直到所有溶剂都被Dean-Stark装置收集,再向反应介质中加入甲苯(2mL)和哌啶(0.5mL),继续145℃加热回流直到所有溶剂都被Dean-Stark装置收集,重复加入甲苯(2mL)和哌啶(0.5mL)以及加热回流过程共3次,此过程通过TLC跟踪,原料反应完全后,柱层析,减压蒸馏除去溶剂后,得到黑色固体产物BDP,所述化合物通过核磁1H NMR谱图、质谱TOF MSEI+进行表征。
将LA(0.2mmol)、DCC(0.003mmol)、DMAP(0.003mmol)溶解到氯仿中,加入BDP(0.2mmol),70℃搅拌3h,60℃真空旋蒸除去溶剂,粗产品采用硅胶柱层析提纯,淋洗剂为二氯甲烷/乙酸乙酯(v/v,15/1),溶剂减压浓缩,得到黑色固体产物BDP-LA,所述化合物通过核磁1H NMR谱图、质谱TOF MS EI+进行表征。
实施例3
将X1(0.54mmol)、4-N,N-二苯基胺苯甲醛(1.08mmol)、对甲苯磺酰胺(0.01mmol)溶于甲苯(25mL)和哌啶(1mL)的混合溶液中,置于装有Dean-Stark装置的圆底烧瓶中,143℃加热回流,直到所有溶剂都被Dean-Stark装置收集,再向反应介质中加入甲苯(25mL)和哌啶(1mL),继续143℃加热回流直到所有溶剂都被Dean-Stark装置收集,重复加入甲苯(25mL)和哌啶(1mL)以及加热回流过程共4次,此过程通过TLC跟踪,原料反应完全后,柱层析,减压蒸馏除去溶剂后,得到黑色固体产物BDP,所述化合物通过核磁1H NMR谱图、质谱TOF MS EI+进行表征,1H NMR(300MHz,DMSO-D6):δ=7.54(d,J=8.8Hz,2H),7.18(d,J=8.8Hz,2H),6.75-6.79(m,5H),6.02(s,1H),5.65-5.67(m,2H),3.11(s,12H),2.12(s,9H);TOF MS EI+:498.3。
将DHA(0.5mmol)、DCC(0.01mmol)、DMAP(0.01mmol)溶解到氯仿中,加入BDP(0.5mmol),70℃搅拌3h,60℃真空旋蒸除去溶剂,粗产品采用硅胶柱层析提纯,淋洗剂为二氯甲烷/乙酸乙酯(v/v,15/1),溶剂减压浓缩,得到黑色固体产物BDP-DHA,所述化合物通过核磁1H NMR谱图、质谱TOF MS EI+进行表征,1H NMR(300MHz,DMSO-D6):δ=7.67(m,2H),7.33-7.37(m,5H),7.23(d,J=8.2Hz,2H),6.95(d,J=8.1Hz,2H),6.02(s,2H),5.35-5.49(m,12H),5.20(s,2H),2.78-2.82(m,10H),2.35-2.38(m,4H),2.12(s,6H),2.07(s,3H),2.01(m,2H),0.79(m,3H);TOF MS EI+:752.4。
实施例4
将X1(1.0mmol)、4-N,N-二苯基胺苯甲醛(2.3mmol)、对甲苯磺酰胺(0.03mmol)溶于甲苯(5mL)和哌啶(1mL)的混合溶液中,置于装有Dean-Stark装置的圆底烧瓶中,142℃加热回流,直到所有溶剂都被Dean-Stark装置收集,再向反应介质中加入甲苯(5mL)和哌啶(1mL),继续142℃加热回流直到所有溶剂都被Dean-Stark装置收集,重复加入甲苯(5mL)和哌啶(1mL)以及加热回流过程共3次,此过程通过TLC跟踪,原料反应完全后,柱层析,减压蒸馏除去溶剂后,得到黑色固体产物BDP,所述化合物通过核磁1H NMR谱图、质谱TOF MS EI+进行表征。
将DHA(1.0mmol)、DCC(0.03mmol)、DMAP(0.03mmol)溶解到氯仿中,加入BDP(1.0mmol),70℃搅拌3h,60℃真空旋蒸除去溶剂,粗产品采用硅胶柱层析提纯,淋洗剂为二氯甲烷/乙酸乙酯(v/v,15/1),溶剂减压浓缩,得到黑色固体产物BDP-DHA,所述化合物通过核磁1H NMR谱图、质谱TOF MS EI+进行表征。
实施例1所制备物质的应用一
近红外荧光标记LA与β-淀粉样蛋白共存160小时,β-淀粉样蛋白衍变的TEM形貌图(以ThT作为参照对比):
20℃下,加入2μm纯化的Aβ单体,用100mM Tris-HCl缓冲液(pH值7.4)孵化,每30分钟搅拌1分钟(500rpm),160小时后,用TEM观察Aβ的形貌,发现无BDP-LA存在下Aβ衍变成斑块,BDP-LA存在下Aβ保持了自身的有序结构,说明BDP-LA对Aβ的聚集有一定的抑制作用。
实施例3所制备物质的应用二
近红外荧光标记DHA监测β-淀粉样蛋白衍变过程荧光强度图(以ThT作为参照对比):
Aβ衍变聚集过程中24h时会有Aβoligomers形成,72h时会有Aβfibrils形成,ThT检测Aβfibrils,用ThT参比对照,用BDP-DHA监测Aβ单体反应聚集过程,反应开始时在96孔板中加入BDP-DHA和ThT,20℃下,加入2μm纯化的Aβ单体,用100mM Tris-HCl缓冲液(pH值7.4)孵化,每30分钟搅拌1分钟(500rpm),然后使用荧光光谱仪测量一次BDP-DHA荧光,并使用玻尔兹曼方程进行拟合,发现BDP-DHA对Aβ的聚集有一定的抑制作用。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (5)
1.近红外荧光标记脂肪酸,其特征在于,其分子结构式为:式中BDP的羟基与脂肪酸的羧基通过酯化反应结合在一起,所述脂肪酸为LA或DHA;
其反应路线如下式所示:
所述R为或/>。
2.如权利要求1所述的近红外荧光标记脂肪酸的制备方法,其特征在于,包括如下步骤:
(1)将甲基吡咯、苯甲醛和三乙胺按照摩尔比2~2.5:1~1.3:0.01~0.3混合溶解到二氯甲烷中,室温,搅拌3h~24h,冰浴中缓慢滴加1~1.3当量的络合剂,搅拌 10~30 分钟,加入1~1.3当量的催化剂A,二氯甲烷萃取,无水 Na2SO4干燥,25℃~60℃真空旋蒸除去溶剂,采用层析柱分离提纯,得到橙黄色固体产物X1;
(2)将X1、对羟甲基苯甲醛、催化剂B按照1:1~1.3:0.01~0.3溶于甲苯和哌啶体积比为1:0.1~1的混合溶液中,置于装有Dean-Stark装置的圆底烧瓶中,140℃-150℃加热回流,直到所有溶剂都被Dean-Stark装置收集,再向反应介质中加入体积比为1:0.1的甲苯和哌啶,重复2-4次,TLC跟踪至原料反应完全后,柱层析分离提纯,减压除去溶剂后,得到黑色固体产物BDP;
(3)将LA或DHA、DCC/DMAP按照摩尔比1:0.1~0.5溶解到氯仿中,再加入BDP,25℃~90℃搅拌3~6h,25℃~60℃真空旋蒸除去溶剂,采用层析柱分离提纯,得到黑色固体产物BDP-R。
3.权利要求2所述的近红外荧光标记脂肪酸的制备方法,其特征在于:所述步骤(1)中催化剂A为2,3-二甲基-5,6-二氰基苯醌,所述络合剂为三氟化硼乙醚。
4.权利要求2所述的近红外荧光标记脂肪酸的制备方法,其特征在于:所述步骤(2)中催化剂B为对甲苯磺酰胺。
5.权利要求2所述的近红外荧光标记脂肪酸的制备方法,其特征在于:所述步骤(3)中BDP和LA或BDP和DHA的摩尔比均为1:1~1.3。
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