CN114394924B - 一种用于粘度检测的有机双态荧光分子探针及其制备方法与应用 - Google Patents
一种用于粘度检测的有机双态荧光分子探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种用于粘度检测的有机双态荧光分子探针及其制备方法与应用，涉及小分子荧光探针领域。通过外延蒽基，设计合成了吲哚方酸菁荧光分子探针(Z)‑2‑((4‑(蒽‑1‑基)‑2‑(甲氧羰基)苯基)氨基)‑3‑氧代‑4‑((1,3,3‑三甲基‑3H‑吲哚‑1‑鎓‑2‑基)亚甲基)环丁烯‑1‑醇酸盐。该分子探针在不同粘度的环境变化中呈现出不一样的荧光响应/显色，可实现不同溶液体系中粘度的快速定量测定/可视化监测，方便快捷，适用于制备检测粘度的试剂盒/试纸条。并且分子探针可实现双态发光，在稀溶液态和聚集态均表现出了明亮的红色荧光，感光度极好，稳定发光不猝灭，同时也适用于生物成像。
Description
技术领域
本发明涉及小分子荧光探针领域，具体地，涉及一种用于粘度检测的有机双态荧光分子探针及其制备方法与应用。
背景技术
生物环境中的粘度水平对整个生命体系起着至关重要的作用。研究发现，粘度的异常与多种疾病相关，如动脉粥样硬化、糖尿病、阿耳茨海默氏病、恶性肿瘤等。高粘度很容易导致肿瘤患者出现血栓，直接危及患者生命。因此，急需开发有效检测粘度的工具，通过检测粘度能够及时发现并早期诊断疾病，这对促进疾病预防和开展临床诊断具有重要作用。
随着有机双态荧光材料的优势逐渐在实际应用中突显出来，目前已经开发出许多性能优良的有机双态荧光分子探针。对于生物成像应用而言，有机双态荧光分子探针无论是在稀溶液态还是在聚集态都表现出强荧光性能，感光度极好，稳定发光不猝灭。特别是发射在红/近红外区可以极大减少背景荧光干扰和光损伤。因此，有机双态荧光分子探针以其高灵敏度，无创实时检测，快速无损分析等优点，可以实现非常理想的可视化和监测癌症模型中的粘度变化，在生物成像领域有着极大的发展潜力和应用前景。
发明内容
本发明旨在提供一种用于粘度检测的有机双态荧光分子探针及其制备方法与应用。通过外延蒽基，设计合成了吲哚方酸菁荧光分子探针(Z)-2-((4-(蒽-1-基)-2-(甲氧羰基)苯基)氨基)-3-氧代-4-((1,3,3-三甲基-3H-吲哚-1-鎓-2-基)亚甲基)环丁烯-1-醇酸盐(SQ-1)，其化学结构式是：
本发明提供的用于粘度检测的有机双态荧光分子探针的制备方法，包括以下步骤：
(1)将2-氨基-5-溴苯甲酸甲酯和苯硼酸频那醇酯倒入烧瓶中，加入四(三苯基膦)钯、碳酸铯、二氧六环和水，在氮气保护下加热反应。反应结束冷却至室温后，再加入1-溴蒽，在氮气保护下加热反应，得到2-氨基-5-(蒽-1-基)苯甲酸甲酯(1-an)。
(2)将3-羟基-4-((1,3,3-三甲基吲哚-2-亚基)甲基)-3-环丁烯-1,2-二酮和1-an倒入烧瓶中，加入无水乙醇溶解，在氮气保护下加热反应，自然冷却后旋干。通过柱层析纯化得到目标产物(Z)-2-((4-(蒽-1-基)-2-(甲氧羰基)苯基)氨基)-3-氧代-4-((1,3,3-三甲基-3H-吲哚-1-鎓-2-基)亚甲基)环丁烯-1-醇酸盐(SQ-1)。
本发明所述荧光探针SQ-1的制备中，优选的，步骤(1)中，2-氨基-5-溴苯甲酸甲酯与苯硼酸频那醇酯的摩尔比优选为1:1，反应时间优选为12h，加样顺序优选为先加2-氨基-5-溴苯甲酸甲酯和苯硼酸频那醇酯，再按顺序加入四(三苯基膦)钯、碳酸铯、二氧六环和水；步骤(2)中，3-羟基-4-((1,3,3-三甲基吲哚-2-亚基)甲基)-3-环丁烯-1,2-二酮和1-an的摩尔比优选为1:1，反应温度优选为90℃，反应时间优选为3h，洗脱液优选为二氯甲烷:甲醇＝100:1(体积比)。
本发明的荧光分子探针SQ-1是一个典型的分子转子结构，其中平面蒽环部分的转动可以促使SQ-1在短波区域的发射，方酸部分则增强SQ-1的长波发射。随着体系环境中粘度的上升，蒽环部分的转动逐渐受阻，短波发射逐渐减弱，而方酸部分导致的长波发射增强，实现了从短波发射到长波发射的过渡，因而SQ-1可以实现从蓝光到白光，再从白光到红光的显色变化。本发明的荧光分子探针响应灵敏，对粘度的响应时间在数秒内，可实现不同溶液体系中粘度的快速定量/可视化检测，方便快捷，尤其适用于制备检测粘度的试剂盒/试纸条。
本发明的有益效果是：
本发明的荧光分子探针SQ-1是有机双态分子探针，不需要任何繁杂的分子修饰和封装，不会受到探针浓度的限制，无论是在稀溶液态还是在聚集态都表现出强荧光性能，感光度极好，稳定发光不猝灭。且SQ-1的发射是在红/近红外区可以极大减少背景荧光干扰和光损伤，同时适用于生物成像。
附图说明
图1是本发明实施例1的分子探针的合成路线图。
图2是本发明实施例2的分子探针在不同溶剂中的紫外-可见吸收光谱图。
图3是本发明实施例2的分子探针在不同溶剂中的荧光光谱图。
图4是本发明实施例2的分子探针的荧光显微镜图。
图5是本发明实施例2的分子探针的晶体态的荧光光谱图。
图6是本发明实施例3的分子探针在不同甘油比例的粘度中的荧光光谱图。
图7是本发明实施例3的分子探针的荧光强度与体系粘度的相关曲线图。
图8是本发明实施例3的分子探针在不同粘度中的CIE色度坐标图。
图9是本发明实施例4的分子探针在细胞成像中的应用(左上：商用染料对细胞染色，左下为SQ-1对细胞染色，右上为细胞在明场未被染色，右下为叠加图)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步阐述，本发明并不限于此；
实施例1
本发明通过外延蒽基，设计合成了吲哚方酸菁荧光分子探针(Z)-2-((4-(蒽-1-基)-2-(甲氧羰基)苯基)氨基)-3-氧代-4-((1,3,3-三甲基-3H-吲哚-1-鎓-2-基)亚甲基)环丁烯-1-醇酸盐(SQ-1)，其化学结构式是：
探针制备方法包括以下步骤，合成路线如图1所示：
(1)将2-氨基-5-溴苯甲酸甲酯和苯硼酸频那醇酯(摩尔比1:1)倒入烧瓶中，按顺序加入四(三苯基膦)钯、碳酸铯、二氧六环和水，在氮气保护下加热反应12h。反应结束冷却至室温后，再加入1-溴蒽，在氮气保护下加热反应12h，得到2-氨基-5-(蒽-1-基)苯甲酸甲酯(1-an)。
(2)将3-羟基-4-((1,3,3-三甲基吲哚-2-亚基)甲基)-3-环丁烯-1,2-二酮和1-an(摩尔比1:1)倒入烧瓶中，加入无水乙醇溶解，在氮气保护下加热90℃反应3h，自然冷却后旋干。通过柱层析(洗脱液为二氯甲烷:甲醇＝100:1(体积比))纯化得到目标产物(Z)-2-((4-(蒽-1-基)-2-(甲氧羰基)苯基)氨基)-3-氧代-4-((1,3,3-三甲基-3H-吲哚-1-鎓-2-基)亚甲基)环丁烯-1-醇酸盐(SQ-1)。
1HNMR(400MHz,CDCl3,ppm)：δ＝11.62(s,1H),8.86(d,J＝8.69Hz,1H),8.37(dd,J＝15.31,11.61Hz,3H),8.13(s,1H),8.06-7.97(m,2H),7.97-7.89(m,2H),7.67(dd,J＝8.82,1.69Hz,1H),5.90(s,1H),3.94(d,J＝15.97Hz,3H),1.81-1.59(m,7H),3.53(s,3H),7.43-7.37(m,2H),7.29(ddd,J＝12.17,9.46,4.13Hz,2H),7.20-7.09(m,2H),6.99(d,J＝7.87Hz,1H).13CNMR(101MHz,CDCl3,ppm)：δ＝128.51,128.06,127.17,126.96,125.68,125.06,124.61,123.66,123.44,121.30,76.26,76.00,75.68,51.74,28.67,26.00.MS(m/z)：579.2272.
实施例2
从图2和图3可以明显看出，SQ-1在不同极性溶剂条件下的紫外-可见吸收光谱(450-600nm)和荧光发射光谱(525-675nm)呈现出镜像对称关系，并伴有明显双峰。其中最大吸收峰位于530-550nm，最大发射峰集中在550-570nm。且随着溶剂从正己烷、二氯甲烷、四氢呋喃到二甲基亚砜，溶剂极性的增强，SQ-1紫外吸收和荧光发射峰的位置逐渐红移，推测可能是电荷转移效应所致。因为SQ-1中的扭曲构象可以很好抑制共轭分子间的π-π堆积作用，减少非辐射能量的损耗，表现出较大的Stokes位移和较高的量子产率。
为了研究SQ-1在聚集态下的发射行为，通过液相自组装的方式在二氯甲烷/正己烷的混合溶剂中制备了相应的微晶分子，图4是SQ-1的微晶在荧光显微镜下的图片，为细长的针状结构，呈现出了明亮的红色荧光。这个结果与SQ-1晶体态的荧光光谱图(图5)相符合，其最大发射波长位于670nm。
实施例3
在占比不同的甘油/甲醇溶剂中进一步探究了SQ-1的发光行为(图6)，SQ-1是一个典型的分子转子结构，对不同粘度的环境变化呈现出不一样的荧光响应。随着甘油比例的不断上升，SQ-1在400-530nm处的短波峰逐渐减弱，在530-650nm处的长波峰逐渐增强。荧光强度(570nm)与粘度的相关曲线如图7所示，在粘度为3.4-627.5cP范围内，荧光强度的对数与粘度的对数呈线性正相关，据此可以检测粘度值。因而探针能够高灵敏定量检测粘度的变化。
鉴于SQ-1在不同粘度环境下的荧光性质有所差异，结合SQ-1的发射行为绘制出了相应的CIE色度坐标图(图8)。根据光的互补色原理，随着环境粘度的增强，SQ-1可以实现从蓝光到白光，再从白光到红光的显色变化，因此，探针可实现不同溶液体系中粘度的快速可视化检测，方便快捷，尤其适用于制备检测粘度的试剂盒/试纸条。
实施例4
为了进一步探究SQ-1在生物成像领域的应用，在激光扫描共聚焦显微镜下使用SQ-1对人脐静脉内皮细胞进行成像实验(图9)，经过SQ-1的共孵育染色后，细胞形态并未发生明显改变，说明SQ-1具有良好的生物相容性。与商用荧光染料共定位的结果表明SQ-1用于生物细胞成像的准确性和可靠性，除此之外进入细胞质的SQ-1无论在溶液态还是聚集态都呈现出了明亮的红色荧光，这也更强有力说明了SQ-1不需要任何繁杂的分子修饰和封装，即可直接实现活细胞的荧光成像，并且表现出稳定的长波区域发射。鉴于良好的生物相容性、结果的准确可靠性以及优异的红光发射等优势，SQ-1在生物成像领域有着极大的发展潜力和应用前景。
应当说明的是，本发明的上述所述之技术内容仅为使本领域技术人员能够获知本发明技术实质而进行的解释与阐明，故所述之技术内容并非用以限制本发明的实质保护范围。本发明的实质保护范围应以权利要求书所述之为准。本领域技术人员应当知晓，凡基于本发明的实质精神所作出的任何修改、等同替换和改进等，均应在本发明的实质保护范围之内。
Claims (4)
1.一种用于粘度检测的有机双态荧光分子探针，其特征在于：通过外延蒽基，设计合成了吲哚方酸菁荧光分子探针(Z)-2-((4-(蒽-1-基)-2-(甲氧羰基)苯基)氨基)-3-氧代-4-((1,3,3-三甲基-3H-吲哚-1-鎓-2-基)亚甲基)环丁烯-1-醇酸盐，其化学结构式是：
2.一种如权利要求1所述的用于粘度检测的有机双态荧光分子探针的制备方法，其特征在于：包括以下步骤：
(1)将2-氨基-5-溴苯甲酸甲酯和苯硼酸频那醇酯倒入烧瓶中，加入四(三苯基膦)钯、碳酸铯、二氧六环和水，在氮气保护下加热反应；反应结束冷却至室温后，再加入1-溴蒽，在氮气保护下加热反应，得到2-氨基-5-(蒽-1-基)苯甲酸甲酯1-an；
(2)将3-羟基-4-((1,3,3-三甲基吲哚-2-亚基)甲基)-3-环丁烯-1,2-二酮和1-an倒入烧瓶中，加入无水乙醇溶解，在氮气保护下加热反应，自然冷却后旋干；通过柱层析纯化得到目标产物(Z)-2-((4-(蒽-1-基)-2-(甲氧羰基)苯基)氨基)-3-氧代-4-((1,3,3-三甲基-3H-吲哚-1-鎓-2-基)亚甲基)环丁烯-1-醇酸盐。
3.根据权利要求2所述的用于粘度检测的有机双态荧光分子探针的制备方法，其特征在于：所述步骤(1)中，2-氨基-5-溴苯甲酸甲酯与苯硼酸频那醇酯的摩尔比为1:1，反应时间为12h，加样顺序为先加2-氨基-5-溴苯甲酸甲酯和苯硼酸频那醇酯，再按顺序加入四(三苯基膦)钯、碳酸铯、二氧六环和水。
4.根据权利要求2所述的用于粘度检测的有机双态荧光分子探针的制备方法，其特征在于：所述步骤(2)中，3-羟基-4-((1,3,3-三甲基吲哚-2-亚基)甲基)-3-环丁烯-1,2-二酮和1-an的摩尔比为1:1，反应温度为90℃，反应时间为3h，洗脱液体积比为二氯甲烷:甲醇＝100:1。
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