CN108329366A - 一种用于检测β-半乳糖苷酶的荧光探针化合物及其制备方法 - Google Patents
一种用于检测β-半乳糖苷酶的荧光探针化合物及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测β‑半乳糖苷酶的荧光探针化合物及其制备方法。所述荧光探针为喹唑啉酮类衍生物,其结构式如下:先合成2‑(2‑羟基苯基)‑4(1H)‑喹唑啉酮,然后合成4(1H)‑喹唑啉酮,2‑[2‑[2,3,4,6‑四乙酰氧基‑(alpha‑D‑吡喃半乳糖基)氧基]苯基],最后合成即4(1H)‑喹唑啉酮,2‑[2‑(β‑D‑吡喃半乳糖基)氧基]苯基]。本发明合成的探针分子L可以通过荧光光谱法实现对β‑半乳糖苷酶的定性、定量的识别,而且在其他杂质,如无机盐、氨基酸、酶的存在的情况下依然有很好的抗干扰性、高选择性和高灵敏性。
Description
技术领域:
本发明涉及一种用于检测β-半乳糖苷酶的荧光探针化合物及其制备和应用,属于荧光探针技术领域。
技术背景:
相比其他众多的检测技术,如磁共振、正电子发射断层显像、单光子发射计算机断层显像、比色法、化学发光法等,荧光探针有高灵敏度、高选择性、便利性、稳定的生物成像性等优点,尤其是通过借助荧光共聚焦显微成像技术可实时检测活细胞内分子或离子浓度以及生物大分子结构的变化过程,在化学,生物,医学,环境等诸多领域受到了人们越来越多的关注,成为一大研究热点。
β-半乳糖苷酶是一种常用的检测转录和转染效率的报道基因,它同样是监测卵巢癌和细胞衰老的一个非常重要的生物标记物,其机理是在原发性卵巢癌中β-半乳糖苷酶会过度表达,因此可以作为卵巢癌可视化检测的分子靶点。相反,β-半乳糖苷酶在体内表达不足也会导致Morquio B综合症。近年来,β-半乳糖苷酶还被视为一种潜在治疗乳糖不耐症的药物,因此,研究出高选择性、高灵敏度检测β-半乳糖苷酶的传感器具有重要意义。
发明内容:
本发明的目的是为了改进现有技术的不足而提供一种用于检测β-半乳糖苷酶的荧光探针化合物,本发明的另一目的是提供上述化合物的制备方法,本发明还有一目的是提供上述化合物的应用,该探针不仅可以检测β-半乳糖苷酶,而且制备方法简单,适合放大和实际应用。
本发明的技术方案为:一种用于检测β-半乳糖苷酶的荧光探针化合物,其特征在于:所述荧光探针为喹唑啉酮类衍生物,其结构式如下:
本发明提供了制备如权利要求1所述的荧光探针化合物的方法,其具体步骤如下:
(1)化合物3即2-(2-羟基苯基)-4(1H)-喹唑啉酮的合成:在第一有机溶剂和催化剂的存在下,将水杨醛(化合物1)和2-氨基苯甲酰胺(化合物2)反应,回流反应结束后加入碱性溶液除去过量的催化剂,得到化合物3;
(2)化合物4即4(1H)-喹唑啉酮,2-[2-[2,3,4,6-四乙酰氧基-(alpha-D-吡喃半乳糖基)氧基]苯基]的合成:在第二有机溶剂和无机碱的存在下,将2-(2-羟基苯基)-4(1H)-喹唑啉酮和2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物反应,得到化合物4;
(3)探针分子L即4(1H)-喹唑啉酮,2-[2-(β-D-吡喃半乳糖基)氧基]苯基]的合成:在第三有机溶剂和无机碱的存在下,加入化合物4反应,后用酸中和至中性,得到探针分子L。
优选步骤(1)中所述水杨醛与2-氨基苯甲酰胺的摩尔比为1:(1~1.5);所述的催化剂与水杨醛的摩尔比为1:(1~1.2)。
优选步骤(1)中所述的第一有机溶剂为甲醇或乙醇等醇类;所述催化剂为碘;所述的碱性溶液为硫代硫酸钠水溶液。
优选步骤(2)中所述化合物3与2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物的摩尔比为1:(1.5~2.5);步骤(2)所述无机碱和化合物3的摩尔比为1:2.5~1:5。
优选步骤(2)中所述的第二有机溶剂为乙腈;所述无机碱为碳酸铯或碳酸钾等。
优选步骤(3)中所述化合物4与无机碱的摩尔比为1:4~1:5。
优选步骤(3)中所述的第三有机溶剂为乙醇或丙醇等醇类;所述无机碱为碳酸钾。
本发明还提供了上述的用于检测β-半乳糖苷酶的荧光探针化合物的应用,其特征在于:所述探针通过荧光光谱法实现对内源性和外源性β-半乳糖苷酶的识别。
本发明的荧光光谱测定:
将荧光探针分子配成浓度为10μM的磷酸盐缓冲溶液(pH=7.3),荧光光谱在荧光分光光度计上测量,激发波长333nm,每次转移3mL的探针磷酸盐缓冲溶液于石英比色皿中,用微量注射器分别加入不同体积的浓度为1mM的氯化铝、氯化钙、D-亮氨酸、L-半胱氨酸、谷胱甘肽的水溶液,和浓度为12.8U/mL的β-葡糖苷酸酶、溶菌酶、脂肪酶、酯酶和β-半乳糖苷酶的水溶液,用荧光光谱研究荧光探针与不同的无机盐、氨基酸、酶相互作用的光谱性质。
有益效果:
探针分子原料易得,合成路线简单,反应条件吻合,后处理简单方便,可以检测β-半乳糖苷酶。探针在四氢呋喃和水的混合溶液中溶解性较好,最大发射波长在495nm处,荧光信号较弱,随着β-半乳糖苷酶的加入,探针分子在波长495nm处出现较强的发射峰。因此可以用于检测β-半乳糖苷酶,此外这种探针能有效的检测活细胞中的β-半乳糖苷酶,可应用于生物医药领域当中。
附图说明:
图1为该荧光探针的1H NMR谱图。
图2为该荧光探针的13CNMR谱图。
图3为该荧光探针与β-半乳糖苷酶反应后的HR-MS谱图。
图4A为化合物4(10μmol/L)在四氢呋喃和水的混合溶液中不断增加水含量的荧光发射光谱图。
图4B为化合物4(10μmol/L)在四氢呋喃和水的混合溶液中不断增加水含量,在495nm处的荧光光谱图。
图5A为该荧光探针与β-半乳糖苷酶反应后,荧光发射光谱与反应时间的关系。图5B为该荧光探针与β-半乳糖苷酶反应后,体系在495nm处的荧光强度与反应时间的散点图。
图6A为该荧光探针与不同种类无机盐、氨基酸和酶结合后的荧光光谱图。
图6B为该荧光探针与不同种类无机盐、氨基酸和酶结合后,体系在495nm处的荧光光谱图。
图7A为该荧光探针与不同浓度的β-半乳糖苷酶作用后的荧光光谱图。
图7B该荧光探针与浓度为0-7U/mL的β-半乳糖苷酶用后,体系在495nm处的荧光强度与β-半乳糖苷酶浓度的折线图。
图8为荧光探针在OVCAR-3细胞的荧光共聚焦成像图。
图9为该荧光探针细胞毒性实验柱状图。
具体实施方式:
下面的实例将对本发明予以进一步的说明,但不会因此而限制本发明。
实施例1:检测β-半乳糖苷酶的荧光探针的制备,基本合成过程如下:
(1)化合物3的合成:向100mL反应瓶中加入10mmol水杨醛(化合物1)、10mmol 2-氨基苯甲酰胺(化合物2)和10mmol碘,向其中滴加60mL乙醇使其溶解,然后加热至回流反应5小时,溶液从浑浊至澄清,待反应结束后加入适量硫代硫酸钠水溶液以除去过量的碘。过滤得固体沉淀物,经水洗、甲醇洗后,有机相旋转蒸发,柱层析分离得到浅白色固体化合物3。洗脱剂为石油醚:二氯甲烷=1:8,产率为81%。所得化合物3的氢谱数据如下:1H NMR(400MHz,DMSO-d6)δ13.83(s,1H),12.48(s,1H),8.22(s,1H),8.16(s,1H),7.84(s,1H),7.75(s,1H),7.53(s,1H),7.45(s,1H),6.98(s,2H),所得化合物3的碳谱数据如下:13C NMR(400MHz,DMSO-d6)δ161.88(s),160.57(s),154.19(s),146.54(s),135.46(s),134.18(s),128.14(s),127.40(s),126.50(s),121.18(s),119.27(s),118.36(s),114.13(s),40.58(s),40.37(s),40.36–39.63(m),39.63–39.58(m),39.43(d,J=21.0Hz),39.30–38.68(m).
(2)化合物4的合成:将步骤(1)中得到的2mmol化合物3溶于50mL乙腈中,再向其中加入5mmol碳酸铯和3mmol 2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物,室温下反应20小时,反应结束后,过滤溶液,旋转蒸发,柱层析分离得到白色固体化合物4。洗脱剂为石油醚:乙酸乙酯=1:3,产率为57.2%。所得化合物4的氢谱数据如下:1H NMR(400MHz,DMSO-d6)δ8.10(d,J=8.1Hz,1H),8.03(q,J=8.2Hz,2H),7.81–7.72(m,2H),7.55(t,J=7.8Hz,1H),7.31(d,J=8.3Hz,1H),7.25(t,J=7.4Hz,1H),6.63(d,J=7.9Hz,1H),5.54(d,J=3.0Hz,1H),5.50(d,J=8.1Hz,1H),5.43(d,J=8.7Hz,2H),5.35(d,J=2.5Hz,1H),5.22(dd,J=10.3,3.1Hz,1H),5.08(s,1H),4.55(s,1H),4.49(s,1H),4.26–4.19(m,1H),4.14(d,J=4.9Hz,1H),4.13–4.10(m,1H),4.09(s,1H),4.07–4.02(m,1H),2.21(s,4H),2.13(d,J=8.0Hz,4H).
(3)分子探针L的合成:将步骤(2)中得到的1mmol化合物4溶于10mL甲醇中,向其中滴加4mmol碳酸钾的甲醇溶液,滴加完毕后,室温反应2小时。反应结束后用1N的盐酸溶液中和至中性,后过滤、旋转蒸发,柱层析分离得到白色固体喹唑啉酮衍生物,即探针分子L。洗脱剂为二氯甲烷:甲醇=20:1,产率为41%。所得探针分子L的氢谱如图1所示,具体数据如下:1H NMR(400MHz,DMSO-d6)δ8.12(d,J=8.5Hz,1H),7.86(d,J=7.6Hz,1H),7.70(s,1H),7.61(d,J=9.9Hz,1H),7.52(s,2H),7.37(d,J=8.2Hz,1H),7.21(dd,J=17.7,10.0Hz,1H),5.32(t,J=4.7Hz,1H),3.71(s,1H),3.69–3.62(m,2H),3.57(s,2H),3.51(s,1H),2.00(dd,J=15.1,7.1Hz,4H),所得探针分子L的碳谱数据如图2所示,具体数据如下:13C NMR(400MHz,DMSO-d6)δ169.80(s),169.49(s),169.29(d,J=4.7Hz),167.94(s),164.05(s),159.38(s),154.67(s),151.38(s),129.78(s),113.56(s),98.52(s),93.49(s),61.28(s),40.01(s),39.84(s),39.67(s),39.54(s),39.42(d,J=20.9Hz),39.17(s),39.01(s),20.50–19.94(m),19.38(s).ESI-MS:[M]=400.13;found[M+H]+:401.1349.
实施例2:检测β-半乳糖苷酶的荧光探针的制备,基本合成过程如下:
(1)化合物3的合成:在反应瓶中加入50mmol水杨醛(化合物1)、75mmol 2-氨基苯甲酰胺(化合物2)和60mmol碘,向其中滴加150mL乙醇使其溶解,然后加热至回流反应9小时,待反应结束后加入适量硫代硫酸钠水溶液以除去过量的碘。过滤得固体沉淀物,经水洗、甲醇洗后,有机相旋转蒸发,柱层析分离得到浅白色固体化合物3。洗脱剂为石油醚:二氯甲烷=1:8,产率为83%。所得化合物3的氢谱数据如下:1H NMR(400MHz,DMSO-d6)δ13.83(s,1H),12.48(s,1H),8.22(s,1H),8.16(s,1H),7.84(s,1H),7.75(s,1H),7.53(s,1H),7.45(s,1H),6.98(s,2H),所得化合物3的碳谱数据如下:13C NMR(400MHz,DMSO-d6)δ161.88(s),160.57(s),154.19(s),146.54(s),135.46(s),134.18(s),128.14(s),127.40(s),126.50(s),121.18(s),119.27(s),118.36(s),114.13(s),40.58(s),40.37(s),40.36–39.63(m),39.63–39.58(m),39.43(d,J=21.0Hz),39.30–38.68(m).
(2)化合物4的合成:将步骤(1)中得到的10mmol化合物3溶于120mL乙腈中,再向其中加入50mmol碳酸铯和25mmol 2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物,室温下反应30小时,反应结束后,过滤溶液,旋转蒸发,柱层析分离得到白色固体化合物4。洗脱剂为石油醚:乙酸乙酯=1:3,产率为58%。所得化合物4的氢谱数据如下:1H NMR(400MHz,DMSO-d6)δ8.10(d,J=8.1Hz,1H),8.03(q,J=8.2Hz,2H),7.81–7.72(m,2H),7.55(t,J=7.8Hz,1H),7.31(d,J=8.3Hz,1H),7.25(t,J=7.4Hz,1H),6.63(d,J=7.9Hz,1H),5.54(d,J=3.0Hz,1H),5.50(d,J=8.1Hz,1H),5.43(d,J=8.7Hz,2H),5.35(d,J=2.5Hz,1H),5.22(dd,J=10.3,3.1Hz,1H),5.08(s,1H),4.55(s,1H),4.49(s,1H),4.26–4.19(m,1H),4.14(d,J=4.9Hz,1H),4.13–4.10(m,1H),4.09(s,1H),4.07–4.02(m,1H),2.21(s,4H),2.13(d,J=8.0Hz,4H).
(3)分子探针L的合成:将步骤(2)中得到的5mmol化合物4溶于30mL甲醇中,向其中缓慢滴加25mmol碳酸钾的甲醇溶液,滴加完毕后,室温反应4小时。反应结束后用1N的盐酸溶液中和至中性,后过滤、旋转蒸发,柱层析分离得到白色固体喹唑啉酮衍生物,即探针分子L。洗脱剂为二氯甲烷:甲醇=20:1,产率为41.6%。所得探针分子L的氢谱数据如下:1HNMR(400MHz,DMSO-d6)δ8.12(d,J=8.5Hz,1H),7.86(d,J=7.6Hz,1H),7.70(s,1H),7.61(d,J=9.9Hz,1H),7.52(s,2H),7.37(d,J=8.2Hz,1H),7.21(dd,J=17.7,10.0Hz,1H),5.32(t,J=4.7Hz,1H),3.71(s,1H),3.69–3.62(m,2H),3.57(s,2H),3.51(s,1H),2.00(dd,J=15.1,7.1Hz,4H),所得探针分子L的碳谱数据如下:13C NMR(400MHz,DMSO-d6)δ169.80(s),169.49(s),169.29(d,J=4.7Hz),167.94(s),164.05(s),159.38(s),154.67(s),151.38(s),129.78(s),113.56(s),98.52(s),93.49(s),61.28(s),40.01(s),39.84(s),39.67(s),39.54(s),39.42(d,J=20.9Hz),39.17(s),39.01(s),20.50–19.94(m),19.38(s).ESI-MS:[M]=400.13;found[M+H]+:401.1349.
实施例3:聚集诱导效应的检测
喹唑啉酮类荧光探针具有聚集诱导特性,它们溶解在纯有机溶剂中时,无荧光产生,但当它们聚集时就会发出强烈荧光。探针L与β-半乳糖苷酶反应后,体系之所以产生荧光是由于生成了具有聚集诱导效应的化合物3。故这里首先测定化合物3在四氢呋喃和水混合溶液中的聚集诱导特性。
探针L与β-半乳糖苷酶反应后的HR-MS如图3所示,结果表明探针L与β-半乳糖苷酶反应后的产物为化合物3。ESI-MS:[M]=238.07;found[M-H]+:237.0664
如图4A所示,化合物3在纯四氢呋喃溶液中几乎无荧光发射。在四氢呋喃和水混合溶液中,随着水馏分的增加,化合物3的溶解度降低,化合物3得以聚集。虽然化合物3在水含量为0-90%时,荧光强度较低,随着水含量从90%增加到99%时,体系荧光强度迅速增加(如图4B),这一现象表明,化合物3具有聚集诱导效应。
实施例4:探针分子的动力学测试
在磷酸盐缓冲溶液(pH=7.3)中,将10μM的探针L与12.8U/mL的β-半乳糖苷酶的水溶液进行动力学试验,每2.5分钟记录一次加入β-半乳糖苷酶后体系在495nm处的荧光强度。结果如图5A所示,在探针L的溶液中加入β-半乳糖苷酶后,荧光强度迅速增加,如图5B所示,在20分钟处达到了稳定状态,延长时间,荧光强度并没有明显增加。之后的检测可以将时间定为20分钟。
实施例5:探针分子对β-半乳糖苷酶的选择性和抗干扰性
将荧光探针分子配成浓度为10μM的磷酸盐缓冲溶液(pH=7.3),荧光光谱在荧光分光光度计上测量,激发波长333nm,每次转移3mL的探针磷酸盐缓冲溶液于石英比色皿中,用微量注射器分别加入不同体积的浓度为1mM的氯化铝、氯化钙、D-亮氨酸、L-半胱氨酸、谷胱甘肽的水溶液,和浓度为12.8U/mL的β-葡糖苷酸酶、溶菌酶、脂肪酶、酯酶和β-半乳糖苷酶的水溶液,结果如图6A和图6B所示,表明了加入其他无机盐、氨基酸、酶对探针分子的荧光强度几乎没有影响,这表明该探针分子对β-半乳糖苷酶具有较高的选择性和较好的抗干扰能力。
实施例6:探针分子对β-半乳糖苷酶的灵敏度实验
取实例1中3mL浓度为10μM的探针磷酸盐缓冲溶液(pH=7.3)于石英比色皿中,,向其中加入不同浓度β-半乳糖苷酶,随β-半乳糖苷酶浓度依次增加,其荧光光谱发生明显红移增强的变化,如图7A所示,表明探针与β-半乳糖苷酶结合以后发生了变化。通过酶浓度与荧光强度的关系曲线可知,在0-7.0U/ml的范围内,β-半乳糖苷酶浓度与荧光强度呈现良好的线性关系(R2=0.99),其线性方程为:y=13.709x+9.6642。如图7B所示。
实施例7:荧光探针在细胞内检测β-半乳糖苷酶
向已经培养好的OVCAR-3细胞中加入10μM的探针,培养30分钟,用吸管吸掉培养基,用PBS缓冲液洗涤细胞3次,加入2mL PBS缓冲液进行成像实验,结果如图8(A)-(C)所示,然后在向其中加入外源性β-半乳糖苷酶,进行成像实验结果如图8(D)-(F)所示,细胞荧光强度明显增强,证明改探针可以实现在细胞内检测内源性和外源性的β-半乳糖苷酶。
实施例8:荧光探针的细胞毒性实验
在OVCAR-3细胞中分别加入浓度分别为10,、20、50、100μM的探针L溶液进行MTT细胞毒性试验。
结果如图9所示,在37℃下,即使用浓度为100μM的探针L处理OVCAR-3细胞24h,OVCAR-3细胞仍能保持较高细胞活力(≥85.92%),这表明探针L细胞毒性低,可安全用于OVCAR-3细胞的生物成像。
Claims (9)
1.一种用于检测β-半乳糖苷酶的荧光探针化合物,其特征在于:所述荧光探针为喹唑啉酮类衍生物,其结构式如下:
2.一种制备如权利要求1所述的荧光探针化合物的方法,其具体步骤如下:
(1)化合物3即2-(2-羟基苯基)-4(1H)-喹唑啉酮的合成:在第一有机溶剂和催化剂的存在下,将水杨醛和2-氨基苯甲酰胺反应,回流反应结束后加入碱性溶液除去过量的催化剂,得到化合物3;
(2)化合物4即4(1H)-喹唑啉酮,2-[2-[2,3,4,6-四乙酰氧基-(alpha-D-吡喃半乳糖基)氧基]苯基]的合成:在第二有机溶剂和无机碱的存在下,将2-(2-羟基苯基)-4(1H)-喹唑啉酮和2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物反应,得到化合物4;
(3)探针分子L即4(1H)-喹唑啉酮,2-[2-(β-D-吡喃半乳糖基)氧基]苯基]的合成:在第三有机溶剂和无机碱的存在下,加入化合物4反应,后用酸中和至中性,得到探针分子L。
3.如权利要求2所述的方法,其特征在于步骤(1)中所述水杨醛与2-氨基苯甲酰胺的摩尔比为1:(1~1.5);所述的催化剂与水杨醛的摩尔比为1:(1~1.2)。
4.如权利要求2所述的方法,其特征在于步骤(1)中所述的第一有机溶剂为甲醇或乙醇;所述催化剂为碘;所述的碱性溶液为硫代硫酸钠水溶液。
5.如权利要求2所述的方法,其特征在于步骤(2)中所述化合物3与2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物的摩尔比为1:(1.5~2.5);步骤(2)所述无机碱和化合物3的摩尔比为1:2.5~1:5。
6.如权利要求2所述的方法,其特征在于步骤(2)中所述的第二有机溶剂为乙腈;所述无机碱为碳酸铯或碳酸钾。
7.如权利要求2所述的方法,其特征在于步骤(3)中所述化合物4与无机碱的摩尔比为1:4~1:5。
8.如权利要求2所述的方法,其特征在于步骤(3)中所述的第三有机溶剂为乙醇或丙醇;所述无机碱为碳酸钾。
9.一种如权利要求1所述的用于检测β-半乳糖苷酶的荧光探针化合物的应用,其特征在于:所述探针通过荧光光谱法实现对内源性和外源性β-半乳糖苷酶的识别。
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