CN109369632B - 一种水溶性聚集诱导发光探针及其制备方法与应用 - Google Patents
一种水溶性聚集诱导发光探针及其制备方法与应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及荧光探针领域,更具体地,涉及一种水溶性聚集诱导发光性质的荧光探针及其制备方法与应用。
背景技术
蛋白质在特定条件下,构象发生不规则变化,发生解折叠或者错误折叠最终使得可溶的蛋白质形成不可溶的规则或不规则的聚集体,其中所形成的富含折叠结构的纤维称为淀粉样纤维。淀粉样纤维被证明至少与多种严重的人类疾病即蛋白质的沉积疾病有关,这些疾病包括糖尿病、溶菌酶淀粉样变性病以及神经退化性变性病如阿尔兹海默症、帕金森疾病等。
由于荧光显微成像具有高灵敏度,高对比度,高分辨率,成像直观和成像速度快等优点,已被广泛应用于生物医学领域。生物学研究是在生理环境或水性介质中进行的,然而,目前所报道的Aβ斑块的荧光探针由于染色速度较慢,通常需要较长的染色时间(10min-30min)以确保探针与Aβ沉积物结合。此外,染色后的繁琐洗涤程序也是必要的,以便提高小鼠脑切片成像的信号背景比(SBR)。这两个缺点使得难以实现对整个小鼠脑中的Aβ斑块进行原位实时成像和3D成像。据我们所知,基于AIE活性的Aβ斑块的荧光探针是鲜有报道,并且目前还没有关于AIE活性的水溶性快速可免洗的Aβ探针的报道。开发基于AIE活性的快速可免洗的远红区或NIR发射的水溶性Aβ探针仍然是一项非常重要且具有挑战性的任务。
发明内容
本发明解决了现有技术中Aβ斑块的荧光探针无法实现在水溶性环境中检测,以及染色速度慢和染色后洗涤程序繁琐的技术问题提。
按照本发明的第一方面,提供了一种水溶性聚集诱导发光探针,所述荧光探针具有如式(一)或式(二)所示的结构通式:
所述式(一)和式(二)中,R1为叔胺基团,Ar1为苯环或者萘环,Ar2为含有吡啶或喹啉的鎓盐;所述式(二)中,Ar3为芳环或者芳杂环。
按照本发明的另一方面,提供了了一种水溶性聚集诱导发光探针的制备方法,所述制备方法为:
所述R1为叔胺基团,Ar1为苯环或者萘环,Ar2为含有吡啶或喹啉的鎓盐。
按照本发发明的另一方面,提供了一种水溶性聚集诱导发光探针的制备方法,所述制备方法为:
所述R1为叔胺基团,Ar1为苯环或者萘环,Ar2为含有吡啶或喹啉的鎓盐,Ar3为芳环或者芳杂环。
按照本发明的另一方面,提供了所述水溶性聚集诱导发光探针在β-淀粉样蛋白沉积斑块的荧光成像方面的应用。
按照本发明的另一方面,提供了所述水溶性聚集诱导发光探针用于定量检测纤维化蛋白的应用。
按照本发明的另一方面,提供了所述水溶性聚集诱导发光探针用于定量检测鸡卵清溶菌酶纤维的应用。
总体而言,通过本发明所构思的以上技术方案与现有技术相比,能够取得下列有益效果:
(1)本发明荧光探针电子供体部分和受体部分通过碳-碳双键桥接,在溶液状态下,电子供体部分和受体部分围绕碳碳双键自由旋转,发生非辐射衰减,表现出微弱的荧光。本发明所述荧光探针的哌啶基团能够识别Aβ,使得荧光探针进入Aβ的折叠结构中,且本发明所述荧光探针近平面线型结构更易于其插入折叠结构,使探针分子聚集,探针由于分子内运动受限制使非辐射衰减渠道被抑制,激发态荧光探针分子只能通过辐射衰减回到基态,而发出强烈的荧光。
(2)使用本发明所述荧光探针,能够直观地观察到Aβ沉积斑块,染色快速可免洗,且根据荧光强弱实现对鸡卵清溶菌酶纤维实现定量检测,丰富了聚集诱导分子的应用。
(3)本发明合成的荧光探针是具有良好水溶性的高灵敏性和特异性的Aβ荧光探针,由于其具有AIE活性,在水溶液中没有荧光只有在与Aβ沉积斑块结合后才发出很强的荧光,因此能够实现对AD鼠脑组织切片Aβ沉积斑块的免洗检测。此外,这种水溶性Aβ探针还具有快速染色的性质,1分钟内可对AD鼠脑组织切片Aβ沉积斑块实现快速染色。因此,此发明合成的荧光探针可对AD鼠脑组织切片Aβ沉积斑块进行快速免洗检测。
附图说明
图1为本发明实施例1-实施例3制备的荧光探针的合成路线示意图。
图2是本发明实施例1和实施例2制备的荧光探针水溶液的紫外吸收光谱和聚集态的荧光发射光谱图;图2(a)是本发明实施例1和实施例2制备的荧光探针在水溶液中的紫外吸收光谱;图2(b)是本发明实施例1和实施例2制备的荧光探针在聚集态时的荧光发射光谱图。
图3是本发明实施例1和实施例2制备的荧光探针在不同四氢呋喃-水纳米分散液中的发射光谱。图3(a)为实施例1制备的PD-BZ-OH在不同四氢呋喃含量的THF-水纳米分散液中的荧光发射光谱。图3(b)为实施例2制备的PD-NA-OH在不同四氢呋喃含量的THF-水纳米分散液中的荧光发射光谱。
图4(a)是本发明实施例1的制备的荧光探针PD-BZ-OH在具有不同HEWL纤维含量的PBS缓冲液中的荧光发射光谱;图4(b)是本发明实施例2的制备的荧光探针PD-NA-OH在具有不同HEWL纤维含量的PBS缓冲液中的荧光发射光谱;图4(c)分别为PD-BZ-OH在605nm和PD-NA-OH在656nm处发射峰相对强度(I/I0)与HEWL纤维浓度的拟合直线。
图5是使用本发明实施例1和实施例2的制备的荧光探针PD-BZ-OH和PD-NA-OH与文献报道的Aβ染料PD-NA-TEG染色转基因小鼠脑组织切片中Aβ斑块的荧光成像图片。其中图5(a)和5(d)为文献报道的Aβ染料PD-NA-TEG对转基因小鼠的脑组织切片中的Aβ斑块染色后的荧光成像图片;图5(b)和图5(e)分别为实施例1和实施例2的制备的荧光探针PD-BZ-OH和PD-NA-OH对转基因小鼠的脑组织切片中的Aβ斑块染色后的荧光成像图片;图5(c)为图5(a)和图5(b)的叠加图片;图5(f)为图5(d)和图5(e)的叠加图片;图5(g)和图5(h)分别为文献报道的Aβ染料PD-NA-TEG和实施例1制备的荧光探针PD-BZ-OH对Tg小鼠的全脑切片的荧光成像图。
图6是本发明实施例1制备的荧光探针PD-BZ-OH和文献报道的Aβ染料PD-NA-TEG对Tg小鼠脑切片Aβ斑块染色后漂洗和免洗的荧光成像;其中图6(a)为文献报道的Aβ染料PD-NA-TEG对Tg小鼠脑切片Aβ斑块染色后免洗的荧光成像;图6(b)为实施例1制备的荧光探针PD-BZ-OH对Tg小鼠脑切片Aβ斑块染色后免洗的荧光成像;图6(c)为实施例2制备的荧光探针PD-NA-OH对Tg小鼠脑切片Aβ斑块染色后免洗的荧光成像;图6(d)为文献报道的Aβ染料PD-NA-TEG对Tg小鼠脑切片Aβ斑块染色后漂洗的荧光成像;图6(e)为实施例1制备的荧光探针PD-BZ-OH对Tg小鼠脑切片Aβ斑块染色后漂洗的荧光成像;图6(f)为实施例2制备的荧光探针PD-NA-OH对Tg小鼠脑切片Aβ斑块染色后漂洗的荧光成像;图6(g)为PD-NA-TEG染色的脑片成像图(a)和(d)中直线区域的背景比(SBR);图6(h)为实施例1制备的荧光探针PD-BZ-OH染色的脑片成像图(b)和(e)中直线区域的背景比(SBR);图6(i)为实施例2制备的荧光探针PD-NA-OH染色的脑片成像图(c)和(f)中直线区域的背景比(SBR)。
图7是本发明实施例1制备的荧光探针PD-BZ-OH对Tg小鼠脑切片染色0分钟到15分钟时的荧光成像图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
实施例1
(1)化合物1的合成:将4-甲基吡啶(5g,53.76mmol,1eq.)和2-溴乙醇(6.67g,53.76mmol,1eq.)溶于CH3CN(100mL)中90℃下搅拌16小时。真空浓缩,得到化合物1(11.82g,粗品),为黄色油状物,不经进一步纯化用于下一步反应。1H NMR(600MHz,DMSO-d6)δ8.94(d,J=6.6Hz,2H),8.02(d,J=6.6Hz,2H),5.17(brs,1H),4.67(t,J=4.8Hz,2H),3.85(t,J=4.8Hz,2H),2.63(s,3H).
(2)PD-BZ-OH的合成:醛2(1eq.),化合物1(1eq.)和哌啶(催化量)的乙醇溶液,80℃下搅拌16小时。浓缩,粗品用乙醇和乙酸乙酯重结晶,得到目标化合物,为红棕色固体。1HNMR(600MHz,DMSO-d6)δ8.73(d,J=6.0Hz,2H),8.10(d,J=6.6Hz,2H),7.92(d,J=16.2Hz,1H),7.60(d,J=8.4Hz,2H),7.24(d,J=16.2Hz,1H),7.00(d,J=8.4Hz,2H),5.24(t,J=5.4Hz,1H),4.53–4.45(m,2H),3.83(q,J=4.8Hz,2H),3.38–3.35(m,4H),1.59(s,6H).13CNMR(151MHz,DMSO-d6)δ154.18,152.94,144.55,142.08,130.58,124.33,122.73,118.52,114.75,62.09,60.52,48.46,25.41,24.44.MS(ESI,m/z):309(M-Br)+.
实施例2
PD-NA-OH的合成:醛3(1eq.),化合物1(1eq.)和哌啶(催化量)的乙醇溶液,80℃下搅拌16小时。浓缩,粗品用乙醇和乙酸乙酯重结晶,得到目标化合物,为红棕色固体。1H NMR(600MHz,DMSO-d6)δ8.86(d,J=6.6Hz,2H),8.24(d,J=6.6Hz,2H),8.13(d,J=16.2Hz,1H),8.03(s,1H),7.83–7.79(m,2H),7.77(d,J=9.0Hz,1H),7.54(d,J=16.2Hz,1H),7.42(dd,J=9.0,2.4Hz,2H),7.20(s,1H),5.28(t,J=5.3Hz,1H),4.56(t,J=5.4Hz,2H),3.86(q,J=5.4Hz,2H),3.35–3.33(m,4H),1.69–1.64(m,4H),1.63–1.59(m,2H).13C NMR(151MHz,DMSO-d6)δ153.13,150.31,144.37,141.41,135.68,129.77,129.33,129.21,127.10,126.54,123.79,122.89,121.23,119.28,108.42,61.79,59.94,48.85,24.99,23.87.MS(ESI,m/z):359(M-Br)+.
实施例3
(1)化合物6的合成:将4-哌啶基溴苯(500mg,2.08mmol,1eq.)5-醛基-2-噻吩硼酸(488mg,3.12mmol,1.5eq.),K2CO3(574mg,4.16mmol,2eq.)和Pd(PPh3)4(240mg,0.208mmol,0.1eq.)和甲苯/水(20mL/6mL)的混合物,在90℃在N2保护下搅拌16小时。向其中加入EtOAc(50mL),有机相用水和盐水洗涤,用Na2SO4干燥并真空浓缩。粗品通过硅胶色谱法分离提纯(石油醚:DCM=20-70%),得到化合物6,淡黄色固体(189mg)。1H NMR(600MHz,Chloroform-d)δ9.84(s,1H),7.69(d,J=3.9Hz,1H),7.57(s,2H),6.94(d,J=15.2Hz,2H),3.28(s,4H),1.86–1.58(m,6H).
(2)PD-BZTh-OH的合成:醛6(1eq.),化合物1(1eq.)和哌啶(催化量)的乙醇溶液,80℃下搅拌16小时。浓缩,粗品用乙醇和乙酸乙酯重结晶,得到目标化合物,为红棕色固体。1HNMR(600MHz,DMSO-d6)δ8.80(d,J=6.3Hz,2H),8.21(d,J=15.8,2.4Hz,1H),8.17(d,J=6.4Hz,2H),7.56(d,J=8.4Hz,2H),7.46(dd,2H),7.09(d,J=15.9Hz,1H),6.99(d,J=8.5Hz,2H),5.25(t,J=5.3Hz,1H),4.51(d,J=5.1Hz,2H),3.85(q,J=5.1Hz,2H),3.26(t,J=5.1Hz,4H),1.72–1.43(m,6H).13C NMR(151MHz,DMSO-d6)δ153.26,151.89,148.98,144.80,138.03,134.63,134.53,127.16,123.40,123.18,122.67,121.00,115.65,62.32,60.50,49.02,25.42,24.40.MS(ESI,m/z):391(M-Br)+.
结果分析:
图2是本发明实施例1和实施例2制备的荧光探针水溶液的紫外吸收光谱和聚集态的荧光发射光谱图。图2(a)是本发明实施例1和实施例2制备的荧光探针在水溶液中的紫外吸收光谱,可以得出,PD-BZ-OH和PD-NA-OH两种探针在水中的最大紫外吸收峰位于410nm,图2(b)是本发明实施例1和实施例2制备的荧光探针在聚集态时的荧光发射光谱图,可以得出,聚集态时发射出红色荧光,峰值波长为595和660nm。
图3是本发明实施例1和实施例2制备的荧光探针在不同四氢呋喃-水纳米分散液中的发射光谱。图3(a)为实施例1制备的PD-BZ-OH在不同四氢呋喃含量的THF-水纳米分散液中的荧光发射光谱。当四氢呋喃含量从0%增加到90%时,荧光强度逐渐增强,但增强幅度较小。然而,随着四氢呋喃含量从80%提高到90%,荧光强度显着增强。图3(b)为实施例2制备的PD-NA-OH在不同四氢呋喃含量的THF-水纳米分散液中的荧光发射光谱。当四氢呋喃含量从0%增加到90%时,荧光强度逐渐增强,但增强幅度较小。然而,随着四氢呋喃含量从80%提高到90%,荧光强度显着增强。从图3可以得出,本发明制备的水溶性荧光探针,随着含THF含量的升高,探针分子溶解度降低,发生聚集,荧光显著增强,由于分子内运动受限(RIM)机制,两种分子表现出AIE特性。
图4(a)是本发明实施例1的制备的荧光探针PD-BZ-OH在具有不同HEWL纤维含量的PBS缓冲液中的荧光发射光谱;图4(b)是本发明实施例2的制备的荧光探针PD-NA-OH在具有不同HEWL纤维含量的PBS缓冲液中的荧光发射光谱;图4(c)分别为PD-BZ-OH在605nm和PD-NA-OH在656nm处发射峰相对强度(I/I0)与HEWL纤维浓度的拟合直线。从图4可以得出,在荧光探针的PBS缓冲液中添加少量HEWL纤维时,其荧光开启,并随着HEWL纤维的逐渐增加荧光逐渐增强,两种荧光分子都可检测纤维化的HEWL,并可通过拟合的直线方程对淀粉样纤维进行定量分析。计算所得PD-BZ-OH和PD-NA-OH对HEWL纤维的检测限分别为0.8017μM和0.6579μM。
图5是使用本发明实施例1和实施例2的制备的荧光探针PD-BZ-OH和PD-NA-OH与文献报道的Aβ染料PD-NA-TEG染色转基因小鼠脑组织切片中Aβ斑块的荧光成像图片。其中图5(a)和5(d)为文献报道的Aβ染料PD-NA-TEG对转基因小鼠的脑组织切片中的Aβ斑块染色后的荧光成像图片;图5(b)和图5(e)分别为实施例1和实施例2的制备的荧光探针PD-BZ-OH和PD-NA-OH对转基因小鼠的脑组织切片中的Aβ斑块染色后的荧光成像图片;图5(c)为图5(a)和图5(b)的叠加图片;图5(f)为图5(d)和图5(e)的叠加图片;图5(g)和图5(h)分别为文献报道的Aβ染料PD-NA-TEG和实施例1制备的荧光探针PD-BZ-OH对Tg小鼠的全脑切片的荧光成像图。从图5可以得出,共定位的荧光成像表明,在PD-BZ-OH和PD-NA-TEG染色的脑切片中观察到许多荧光点。叠加图像表明,PD-BZ-OH的红色斑点和PD-NA-TEG的绿色斑点基本完全重合。经过计算,PD-BZ-OH和PD-NA-TEG的皮尔逊相关系数(PCC)为0.823。通过图像强度相关性分析,PD-BZ-OH和PD-NA-TEG的共定位程度大于87%,表现出很高的相关性。PD-NA-OH表现出与PD-BZ-OH相似的特性,表明两种AIE荧光探针,是优异的小鼠脑中的Aβ斑块的检测探针。
图6是本发明实施例1制备的荧光探针PD-BZ-OH和文献报道的Aβ染料PD-NA-TEG对Tg小鼠脑切片Aβ斑块染色后漂洗和免洗的荧光成像;其中图6(a)为文献报道的Aβ染料PD-NA-TEG对Tg小鼠脑切片Aβ斑块染色后免洗的荧光成像;图6(b)为实施例1制备的荧光探针PD-BZ-OH对Tg小鼠脑切片Aβ斑块染色后免洗的荧光成像;图6(c)为实施例2制备的荧光探针PD-NA-OH对Tg小鼠脑切片Aβ斑块染色后免洗的荧光成像;图6(d)为文献报道的Aβ染料PD-NA-TEG对Tg小鼠脑切片Aβ斑块染色后漂洗的荧光成像;图6(e)为实施例1制备的荧光探针PD-BZ-OH对Tg小鼠脑切片Aβ斑块染色后漂洗的荧光成像;图6(f)为实施例2制备的荧光探针PD-NA-OH对Tg小鼠脑切片Aβ斑块染色后漂洗的荧光成像;图6(g)为PD-NA-TEG染色的脑片成像图(a)和(d)中直线区域的背景比(SBR);图6(h)为实施例1制备的荧光探针PD-BZ-OH染色的脑片成像图(b)和(e)中直线区域的背景比(SBR);图6(i)为实施例2制备的荧光探针PD-NA-OH染色的脑片成像图(c)和(f)中直线区域的背景比(SBR)。可以看到文献报道的脂溶性探针PD-NA-TEG,未经漂洗背景荧光很强,斑块成像不明显,必须经过漂洗才能降低背景干扰,观察到明显的斑块。而实施例1和实施例2制备的水溶性荧光探针PD-BZ-OH和PD-NA-OH,染色后,未经漂和经过漂洗的小鼠脑切片,背景干扰很弱,都可以观察到明显的斑块。这说明我们合成的水溶性荧光探针,在成像时可以免洗,这为断层扫描实时成像奠定了基础。
图7是本发明实施例1制备的荧光探针PD-BZ-OH对Tg小鼠脑切片染色0分钟到15分钟时的荧光成像图。可以看到在染色1分钟后就可以观测到明显的斑块,延迟染色时间没有明显变化,这说明所合成的荧光探针具有快速染色的能力。
本发明所合成的荧光探针能够检测纤维化淀粉样蛋白。作为基于AIE活性的新型水溶性荧光探针,实验表明,该系列AIE活性的荧光分子是小鼠脑内Aβ斑块结合的优良荧光探针。能够实现对Aβ斑块快速免洗检测,利用其免洗和快速染色的特点,借助于荧光断层扫描显微成像(fMOST)系统,可实现对鼠脑中Aβ斑块分布的三维立体成像。
本领域的技术人员容易理解,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (5)
3.如权利要求1所述水溶性聚集诱导发光探针在非疾病的诊断与治疗目的的β-淀粉样蛋白沉积斑块的荧光成像方面的应用。
4.如权利要求1所述水溶性聚集诱导发光探针用于非疾病的诊断与治疗目的定量检测纤维化蛋白的应用。
5.如权利要求1所述水溶性聚集诱导发光探针用于非疾病的诊断与治疗目的的定量检测鸡卵清溶菌酶纤维的应用。
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