CN107739375B - 一种新型喹嗪类pH荧光分子探针的制备及用途 - Google Patents
一种新型喹嗪类pH荧光分子探针的制备及用途 Download PDFInfo
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Abstract
本发明公开了一种新型pH响应荧光分子探针，具有全新作用机制，如下所示：
其中，骨架结构如式II化合物所示的4H‑喹嗪‑4‑亚胺，在碱性条件下，脒键中C‑N键断开，并发生相应的荧光减弱，以及发射波长或吸收波长的红移或蓝移。在酸性条件下脒键中C‑N键重建，并发生荧光减弱/增强，以及发射波长或吸收波长的红移或蓝移，可用作比率探针检测pH值。该新型荧光探针具有较大的Stokes shift和较强的量子效率，并在细胞成像中表现出特异性标记细胞溶酶体，该pH荧光探针为基于喹嗪环的开环闭环实现荧光开启和关闭，丰富了pH响应荧光分子探针的种类，具有十分广泛的工业化应用前景，也为科学研究提供了重要的参考价值。
Description
技术领域
本发明属于荧光分子探针领域，尤其涉及一种分子内开环/闭环调节荧光的pH响应荧光分子探针及其应用。
背景技术
pH值是影响物质理化性质及反应性能的一个重要参数，同时细胞内环境的pH值是一项重要的生理参数, 在细胞、酶和很多人体组织活动中起到重要指示作用, 如细胞增殖、细胞凋亡、抗药性、离子传输、肌肉收缩等等。因此监测生命环境的pH值的变化对于生命细胞行为研究和神经系统研究具有重要意义。一般情况下, 生命细胞环境存在两种pH范围, 一种是pH=6.8~7.4, 如细胞质；另一种是pH=4.5~6.0, 即酸性的细胞器, 如溶酶体。正常范围的细胞pH环境有利于细胞生长, 而生命pH环境异常往往与癌症、阿尔茨海默病等病症相关联。因此, 对生命环境的pH值的测量在生命科学领域具有十分重要的意义。
目前最广泛使用的pH测量方法就是pH比色指示剂和玻璃电极。玻璃电极虽然灵敏，但是用于较小的细胞检测时会有电流干扰以及机械损伤，因此玻璃电极同样不适用于细胞内pH检测和细胞成像，且在极端pH值检测时误差较明显。自发现石蕊试液具有随着pH值变化发生颜色变化的特性依赖，各种酸碱指示剂得到了广泛的应用，但是该方法也存在缺陷，即无法用于生命体内进行活体细胞造影，而且比色法的灵敏度较低，无法检测到细胞中0.10-0.20个单位的pH变化。相比以上两种检测方法，基于荧光探针的光学检测方法则不具有上述缺陷，其不仅便于荧光显微学研究，而且可实时监测pH值的动态分布和区域变化。因此，开发新型用于pH准确测量的荧光探针成为当代分子荧光识别领域的一个研究热点。
现在商用的pH荧光探针主要是基于荧光素发展得到，如BCECF-AM, BCFL-AM和BCFL-SE等，荧光素骨架响应pH的机制为螺环在碱性条件下，受到OH-的影响发生开环，从而发出强的荧光，在酸性条件下，受到H+的影响，螺环发生闭环，荧光消失，所以在不同pH值情况下，荧光强度不同，强度随pH变化发生变化，可实现细胞内的pH值监测。
荧光素骨架响应pH值的机制为螺环的开闭。但是荧光素骨架存在一些缺陷，如Stokes shift较小，溶解度较差，细胞摄取较差，作为荧光团分子量较大。为了克服这些缺陷，我们设计了一类新的喹嗪类分子，是一种分子内开关环响应pH值的荧光团。
发明内容
为了克服上述诸多缺陷，本发明人进行了大量的调研与创造性工作，从而开发了一类新型分子内开闭环响应pH值的荧光探针、制备方法及用途。
具体而言，本发明的技术方案和内容涉及以下三个方面。
第一方面，本发明的技术方案和内容涉及一类化学通式如式IV所示的化合物，
其中，R1为芳香基团或取代芳香基团，如R1为
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；R2如氰基，
炔基，烯丙基，炔丙基，甲酰基，乙酰基，丙酰基，丁酰基；
进一步的，式IV所示化合物限定为式V所示化合物，
其中，R1为
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第二方面，本发明的技术方案和内容涉及上述式II的衍生物的制备方法，所述方法为：
a，将甲酸乙酯加入到新鲜配制的乙醇钠乙醇溶液中，搅拌5分钟，加入芳香取代乙腈，室温反应过夜，次日用5%盐酸溶液调节反应液的pH至中性左右，用二氯甲烷萃取，有机层合并，干燥，柱层析纯化得到化合物1；
b, 化合物1与溴乙烷溶于DMF中，在碱的作用下，加热至60℃反应过夜，反应液倒入水中，二氯甲烷萃取后，有机层干燥，蒸除溶剂，柱层析纯化得到化合物2；
c, 化合物3加入到新鲜配制的乙醇钠乙醇溶液中，搅拌5分钟，加入化合物2，室温反应过夜，次日用5%盐酸溶液调节反应液的pH至中性左右，用二氯甲烷萃取，有机层合并，干燥，柱层析纯化得到目标化合物IMQU。
本发明所述化合物IMQU具有以下所示的pH响应机制，
该类荧光探针在pH小于化合物pK a的环境时，主要以A形态存在，发出强的荧光；在pH大于化合物pK a的环境时，主要以C形态存在，荧光强度减弱数倍；
进一步的，本发明中式A、式B和式C中R2基团可为氰基或吡啶-2-基乙腈基等；
本发明所述A-C或C-A过程，其发射波长存在红移或蓝移，同时荧光强度发生变化，吸收波长蓝移。
第三方面，本发明的技术方案和内容涉及所述的式II化合物用于荧光分析或生物pH检测领域的用途。所述式II化合物可用作荧光探针分子，可实现对不同pH 下对H+或OH-的识别，从而丰富了pH响应荧光分子探针的种类，开拓了脒键官能团的新用途，为有机分析和光化学提供了新型的探针分子。
与现有技术相比，本发明的有益效果为：
1、首次实现以脒键为pH响应的基团，具有pK a值可控的特点；
2、本发明的荧光团具有光漂白性低的特点，适合在生物成像中的长时间观测；
3、本发明荧光探针的制备方法简单，具有较大的Stokes shift。
附图说明
图1为本发明实施例25采集到的典型pH-荧光响应谱图。
图2为本发明实施例26采集到的IMQU-8在A549细胞中的荧光成像图。
图3为IMQU-8分子荧光探针监测氨水对A549细胞中pH影响的荧光成像图。
具体实施方式
以下结合实施例对本发明作进一步的阐述。
实施例仅用于说明本发明，而不是以任何方式来限制本发明。
实施例1：3-氧-2-苯基丙腈（1a）的制备.
准确称取金属钠（236 mg, 1.3 eq.），并在0℃下加入到绝对乙醇（10 mL）中，搅拌至金属钠完全转化，然后先后加入甲酸乙酯（825 μL, 1.1 eq.）和苯乙腈（1.0 g, 1.0eq.），升温至40℃继续搅拌4-8小时，反应转化完全后，用1 N 盐酸溶液将反应液的pH值调节至6-8，用二氯甲烷萃取三次，有机层用饱和食盐水洗涤一次，二氯甲烷层干燥后，真空蒸除溶剂，所得固体用柱层析纯化，得到白色固体902 mg，产率78.7%。1H NMR (400 MHz,DMSO-d 6) δ 8.63 (s, 1H), 7.74 (d, J = 7.2 Hz, 2H), 7.07 (t, J = 7.2 Hz, 3H),6.73 (t, J = 7.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d 6) δ 174.66, 139.54, 129.33,128.62, 127.91, 122.90, 120.73, 120.48, 77.71。
实施例2：2-(4-氯苯基)-3-氧丙腈（1b）的制备.
制备方法同实施例1，收率为74.0 %。此化合物以烯醇式和酮式1:1比例的混合物形式存在。1H NMR (400 MHz, DMSO-d 6) δ 12.37 (ketone form) (brs, 1H), 8.09(ketone form) (s, 1H), 7.72 (enol form) (s, 1H), 7.69 (ketone form) (d, J =8.8 Hz, 2H), 7.43 (ketone and enol form) (m, 7H). 13C NMR (100 MHz, DMSO-d 6) δ160.80 (ketone form), 159.22 (enol form), 131.70 (ketone form), 131.47(ketone form), 131.38 (enol form), 131.06 (enol form), 130.44 (enol form),129.35 (ketone form), 129.28 (ketone form), 129.02 (enol form), 128.54(ketone form, 2C), 126.17 (enol form, 2C), 120.34 (enol form), 117.01 (ketoneform), 89.25 (ketone form), 88.99 (enol form)。
实施例3：2-(2,4-二氟苯基)-3-氧丙腈 (1c)的制备.
制备方法同实施例1，收率为77.2%。此化合物以烯醇式和酮式0.6:1比例的混合物形式存在。1H NMR (400 MHz, DMSO) δ 12.19 (ketone form) (s, 1H), 7.78 (ketoneform) (s, 1H), 7.72 (enol form) (s, 0.6 H), 7.54 (enol form) (dd, J = 15.6,8.8 Hz, 0.6 H), 7.45 (ketone form) (dd, J = 15.6, 9.2 Hz, 1H), 7.30 (ketoneform and enol form) (m, 1 + 0.6 H), 7.12 (ketone form and enol form) (t, J =8.4 Hz, 1 + 0.6 H). 13C NMR (100 MHz, DMSO) δ 162.91 (enol form), 160.60(ketone form, 2C), 160.45 (enol form), 132.74 (enol form), 130.83 (ketoneform), 119.94 (enol form), 117.10 (ketone form), 116.67 (ketone form), 115.53(enol form), 112.67 (ketone form), 112.26 (enol form), 105.30 (ketone form),105.04 (ketone form), 104.79 (enol form), 104.53 (enol form), 83.37 (ketoneform), 82.58 (enol form)。
实施例4：2-(4-乙氧基-2,3,5,6-四氟苯基)-3-氧丙腈(1d)的制备.
制备方法同实施例1， 收率为65.9 %。1H NMR (400 MHz, DMSO-d 6) δ 7.94(ketone form) (s, 1 H), 7.78 (s, 0.7 H), 4.32 (ketone form, enol form) (m, 2H + 1.4 H), 1.34 (ketone form, enol form) (m, 3 H + 2.1 H). 13C NMR (100 MHz,DMSO-d 6) δ 166.44 (ketone form), 164.00 (enol form), 145.78 (enol form),143.31 (ketone form), 142.64 (enol form), 140.19 (ketone form), 137.33 (enolform), 136.81 – 135.85 (ketone form), 118.55 (enol form), 115.66 (ketoneform), 106.19 (ketone form), 104.37 (enol form), 74.83 (enol form), 74.18(ketone form), 71.49 (enol form + ketone form), 15.69 (enol form), 15.64(ketone form)。
实施例5：2-(4-氟苯基)-3-氧丙腈(1e)的制备.
制备方法同实施例1，收率68.2 %。此化合物以烯醇式和酮式1:1比例的混合物形式存在。1H NMR (400 MHz, DMSO-d 6) δ 12.16 (ketone form) (brs, 1H), 7.99 (ketoneform) (s, 1H), 7.70 (enol form) (dd, J = 8.8, 5.6 Hz, 2H), 7.66 (enol form)(s, 1H), (ketone form) 7.45 (dd, J = 8.8, 6.8 Hz, 2H), 7.28 – 7.14 (ketoneform and enol form) (m, 4H). 13C NMR (100 MHz, DMSO-d 6) δ 162.65 (enol form),162.26 (ketone form), 160.23 (ketone form), 160.05 (enol form), 159.83 (enolform), 158.19 (ketone form), 129.06 (ketone form), 128.95 (ketone form),128.50 (enol form), 126.59 (enol form), 120.57 (enol form), 117.25 (ketoneform), 116.21 (ketone form), 115.91 (enol form), 89.30 (enol form), 89.09(ketone form)。
实施例6：2-(4-溴苯基)-3-氧丙腈(1f)的制备.
制备方法同实施例1，收率70.3%。此化合物以烯醇式和酮式1:1比例的混合物形式存在。1H NMR (400 MHz, DMSO-d 6) δ 12.22 (ketone form) (brs, 1H), 7.81 (ketoneform) (s, 1H), 7.75 (enol form) (s, 1H), 7.61 – 7.51 (ketone form) (m, 1H),7.47 (enol form) (m, 1H), 7.29 (enol form) (m, 2H), 7.14 (ketone form) (m,2H). 13C NMR (100 MHz, DMSO-d 6) δ 160.71 (ketone form), 159.23 (enol form),132.13 (ketone form, 2C), 132.09 (ketone form), 131.88 (enol form, 2C),131.43 (enol form), 128.82 (ketone form, 2C), 126.45 (enol form, 2C), 120.28(enol form), 119.96 (ketone form), 119.74 (enol form), 116.95 (ketone form),89.39 (ketone form), 89.15 (s, 1H)。
实施例7：3-氧-2-(吡啶-3-基)丙腈（1g）的制备.
制备方法同实施例1，收率35.2%。1H NMR (400 MHz, DMSO-d 6) δ 9.07 (s, 1H),8.64 (s, 1H), 8.42 (s, 1H), 8.34 (s, 1H), 8.24 (s, 1H), 8.18 (s, 1H), 8.10(d, J = 8.2 Hz, 1H), 7.83 (s, 1H), 7.54 (s, 1H), 7.38 (s, 1H). 13C NMR (100MHz, DMSO-d 6) δ 167.28 (enol form), 162.09 (ketone form), 147.41 (ketoneform), 145.12 (ketone form), 142.21 (enol form), 141.87 (enol form), 134.73(ketone form), 132.70 (enol form), 132.18 (ketone form), 129.32 (enol form),125.22 (ketone form), 124.34 (ketone form), 121.91 (enol form), 116.77 (enolform), 86.65 (ketone form), 82.13 (enol form)。
实施例8：3-氧-2-(吡啶-2-基)丙腈（1h）的制备.
制备方法同实施例1，收率74.1%。1H NMR (400 MHz, DMSO-d 6) δ 15.20 (brs,1H), 9.07 (s, 1H), 8.31 (d, J = 6.0 Hz, 1H), 8.09 (t, J = 8.0 Hz, 1H), 7.29(d, J = 8.8 Hz, 1H), 7.22 (t, J = 6.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d 6) δ182.67, 152.69, 143.41, 137.97, 121.02, 119.52, 117.62, 77.21。
实施例9：(Z/E)-3-乙氧基-2-苯基丙烯腈（2a）的制备.
准确称取化合物1a（500 mg, 1.0 eq.）和K2CO3（713 mg, 1.5 eq.）于圆底烧瓶中，加入DMF（6 mL），室温搅拌5分钟后，加入溴乙烷（336 μL, 1.3 eq.），升温至50℃，反应过夜。次日，真空抽滤除去无机盐，加入三倍于滤液体积的水，用二氯甲烷萃取三次，饱和食盐水洗涤有机层一次，二氯甲烷层干燥后蒸干，粗品用柱层析纯化，得到白色固体484 mg，产率81.1%。1H NMR (400 MHz, DMSO-d 6) δ 7.66 (m, 3H), 7.43 (t, J = 7.8 Hz, 2H),7.30 (t, J = 7.4 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H).13C NMR (100 MHz, DMSO-d 6) δ 161.00, 131.25, 129.14 (2C), 127.89, 127.17 (2C),119.78, 91.39, 72.28, 15.65。
实施例10：(Z/E)-2-(4-氯苯基)-3-乙氧丙烯腈(2b)的制备.
制备方法同实施例9，收率69.4%。1H NMR (400 MHz, DMSO-d 6) δ 7.71 (s, 1H),7.64 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 4.32 (q, J = 7.2 Hz, 2H),1.34 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d 6) δ 161.72, 132.22, 130.20,129.22 (2C), 128.80 (2C), 119.44, 90.30, 72.58, 15.67。
实施例11：(Z/E)-2-(2,4-二氟苯基)-3-乙氧丙烯腈(2c)的制备.
制备方法同实施例9，收率70.1%。1H NMR (400 MHz, DMSO-d 6) δ 7.79 (Z) (s, 1H), 7.75 (E) (s, 1.3 H), 7.51 (Z + E) (m, 2.3 H), 7.39 – 7.28 (Z + E) (m,2.3H), 7.22 – 7.11 (Z + E) (m, 2.3 H), 4.30 (Z) (q, J = 7.6 Hz, 2H), 4.23 (E)(q, J = 7.2 Hz, 2.6 H), 1.33 (Z) (t, J = 6.8 Hz, 3H), 1.25 (E) (t, J = 7.2Hz, 3.9 H). 13C NMR (100 MHz, DMSO-d 6) δ 165.63 (Z), 163.27 (E), 132.54 (E),130.93 (Z), 119.01 (E), 116.34 (Z), 115.99 (Z), 115.00 (E), 112.65 (Z),112.36 (E), 105.38 (Z), 105.21 (E), 105.12 (Z), 104.95 (E), 104.86 (Z),104.69 (E), 84.36 (Z), 83.94 (E), 71.84 (E), 71.63 (Z), 15.60 (Z), 15.54 (E)。
实施例12：(Z/E)-3-乙氧基-2-(4-乙氧基-2,3,5,6-四氟苯基)丙烯腈(2d)的制备.
制备方法同实施例9，收率42.6%。1H NMR (400 MHz, DMSO-d 6) δ 7.99 (s, 1H),7.83 (s, 1H), 4.38 – 4.23 (m, 8H), 1.40 – 1.29 (m, 9H), 1.24 (t, J = 7.1 Hz,4H)。
实施例13：(Z/E)-3-乙氧基-2-(4-氟苯基)丙烯腈(2e)的制备.
制备方法同实施例9，收率81.5%。1H NMR (400 MHz, DMSO) δ 8.01 (Z) (s,0.17 H), 7.66 (E) (m, 2.4 H), 7.47 (m, 0.34 H), 7.25 (Z + E) (m, 0.34 + 1.6H), 4.28 (Z + E) (m, 0.34 + 1.6 H), 1.32 (Z + E) (m, 0.68 + 2.4 H). 13C NMR(100 MHz, DMSO) δ 163.07 (Z), 162.64 (Z + E), 160.86 (E), 160.20 (Z + E),129.27 (E, 2C), 127.69 (Z), 126.86 (Z, 2C), 119.68 (E), 116.39 (Z, 2C),116.15 (E, 2C), 90.36 (E), 90.11 (Z), 72.34 (E), 71.35 (Z), 15.67 (E + Z)。
实施例14：(Z/E)-2-(4-溴苯基)-3-乙氧丙烯腈(2f)的制备.
制备方法同实施例9，收率76.5%。1H NMR (400 MHz, DMSO-d 6) δ 8.13 (Z) (s,1H), 7.72 (E) (s, 1.6 H), 7.58 (E + Z) (m, 6.4 + 2H), 7.41 (Z) (d, J = 8.4Hz, 2H), 4.36 – 4.26 (Z + E) (m, 2 + 3.2H), 1.34 (Z + E) (m, 3 + 4.8H). 13CNMR (100 MHz, DMSO-d 6) δ 163.71 (Z), 161.76 (E), 132.31 (Z, 2C), 132.12 (E,2C), 131.30 (Z), 130.57 (E), 129.06 (E, 2C), 126.68 (Z, 2C), 120.75 (E),120.38 (Z), 119.38 (E), 116.27 (Z), 90.38 (E), 90.15 (Z), 72.60 (E), 71.59(Z), 15.67 (E + Z)。
实施例15：(Z/E)-3-乙氧基-2-(吡啶-3-基)丙烯腈（2g）的制备.
制备方法同实施例9，收率76.8%。1H NMR (400 MHz, DMSO-d 6) δ 8.67 (d, J =2.4 Hz, 1H), 8.47 (dd, J = 4.8 Hz, J = 1.2 Hz, 1H), 8.17 (s, 1H), 7.83 (dt, J= 2.0 Hz, J = 8.0 Hz, 1H), 7.42 (dd, J = 8.0 Hz, J = 4.8 Hz, 1H), 4.31 (q, J= 6.8 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d 6) δ 164.37,148.51, 145.76, 132.28, 128.19, 124.31, 115.98, 87.87, 71.77, 15.68。
实施例16：(Z/E)-3-乙氧基-2-(吡啶-2-基)丙烯腈（2h）的制备.
制备方法同实施例9，收率62.3%。1H NMR (400 MHz, DMSO-d 6) δ 8.50 (d, J =4.7 Hz, 1H), 8.39 (s, 1H), 7.81 (t, J = 8.6 Hz, 1H), 7.45 (d, J = 8.0 Hz,1H), 7.29 – 7.23 (m, 1H), 4.37 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 6.8 Hz, 3H).13C NMR (100 MHz, DMSO-d 6) δ 166.02, 151.00, 149.74, 137.87, 122.45, 119.01,116.03, 92.13, 72.20, 15.68。
实施例17：4-亚氨基-3-苯基-4H-喹嗪-1-腈（IMQU-1）的制备.
准确称取金属钠（52 mg, 1.3 eq.），并在0℃下加入到绝对乙醇（10 mL）中，搅拌至金属钠完全转化，然后加入2-吡啶乙腈（225.1 mg, 1.1 eq.），室温搅拌5分钟后，加入中间体2a（300 mg, 1.0 eq），室温继续搅拌过夜，反应转化完全后，用1 N 盐酸溶液将反应液的pH值调节至6-8，用二氯甲烷萃取三次，二氯甲烷层用饱和食盐水洗涤一次，干燥后，减压蒸除溶剂，所得固体用柱层析纯化，得到橘黄色固体278 mg，产率65.1%。1H NMR (400 MHz,DMSO-d 6) δ 9.38 (d, J = 7.2 Hz, 1H), 7.81 (t, J = 8.8 Hz, 1H), 7.74 (brs,1H), 7.70 (d, J = 8.8 Hz, 2H), 7.53 -7.41 (m, 6H), 7.26 (t, J = 6.4 Hz, 1H).13C NMR (100 MHz, DMSO-d 6) δ 155.95, 145.69, 137.38, 135.67, 132.67, 130.47,129.73 (2C), 128.97 (2C), 128.65, 122.62, 121.16, 118.82, 116.55, 79.67. HRMS(Q-TOF): calculated for [M+H]+ 245.0953, found 246.1027。
实施例18：3-(4-氯苯基)-4-亚氨基-4H-喹嗪-1-腈(IMQU-2)的制备.
制备方法同实施例17，收率为62.3%。1H NMR (400 MHz, DMSO-d 6) δ 9.39 (d, J= 7.6 Hz, 1H), 7.84 (m, 2H), 7.74 (d, J = 9.2 Hz, 1H), 7.53 (q, J = 8.6 Hz,4H), 7.48 (s, 1H), 7.29 (t, J = 7.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d 6) δ155.47, 145.85, 136.25, 135.96, 133.15, 130.95 (2C), 130.58, 129.70 (2C),122.67, 119.79, 118.76, 116.74, 99.99, 79.74. HRMS (Q-TOF): calculated for [M+H]+ 279.0563, found 280.0643。
实施例19：3-(2,4-二氟苯基)-4-亚氨基-4H-喹嗪-1-腈(IMQU-3)的制备.
制备方法同实施例17，收率为49.7%。1H NMR (400 MHz, DMSO-d 6) δ 9.39 (d, J= 6.4 Hz, 1H), 7.86 (t, J = 7.6 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.53 –7.48 (m, 4H), 7.30 (t, J = 6.0 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H). 13C NMR (100MHz, DMSO-d 6) δ 154.78, 146.26, 136.32, 134.53, 133.70, 130.50, 122.69,118.68, 116.81, 114.60, 113.01, 112.82, 105.53, 105.28, 105.02, 79.01. HRMS(Q-TOF): calculated for [M+H]+ 281.0765, found 282.0838。
实施例20：3-(4-乙氧基-2,3,5,6-四氟苯基)-4-亚氨基-4H-喹嗪-1-腈(IMQU-4)的制备.
制备方法同实施例17，收率为60.8%。1H NMR (400 MHz, DMSO-d 6) δ 9.44 (d, J= 7.2 Hz, 1H), 7.98 (s, 1H), 7.96 – 7.88 (m, 1H), 7.76 (d, J = 8.9 Hz, 1H),7.62 (s, 1H), 7.34 (t, J = 7.0 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 1.39 (t, J= 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d 6) δ 153.26, 146.87, 137.24 (2C),136.53 (2C), 130.71 (2C), 122.79 (2C), 118.46, 117.23 (2C), 109.60, 105.94,78.61, 71.45, 15.73. HRMS (Q-TOF): calculated for [M+H]+ 361.0838, found362.0874。
实施例21：3-(4-氟苯基)-4-亚氨基-4H-喹嗪-1-腈(IMQU-5)的制备.
制备方法同实施例17，收率为54.6%。1H NMR (400 MHz, DMSO-d 6) δ 9.38 (d, J= 7.2 Hz, 1H), 7.82 (t, J = 7.6 Hz, 2H), 7.72 – 7.67 (m, 3H), 7.47 – 7.43 (m,3H), 7.28 (t, J = 7.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d 6) δ 155.41, 145.86,136.66, 135.95, 133.01, 132.61 (2C), 131.20 (2C), 130.57, 122.64, 121.71,119.79, 118.78, 116.70, 79.62. HRMS (Q-TOF): calculated for [M+H]+ 263.0859,found 264.0939。
实施例22：3-(4-溴苯基)-4-亚氨基-4H-喹嗪-1-腈(IMQU-6)的制备.
制备方法同实施例17，收率为57.2%。1H NMR (400 MHz, DMSO-d 6) δ 9.38 (d, J= 7.2 Hz, 1H), 7.80 (t, J = 8.8 Hz, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.52 (dd,J = 3.2 Hz, 5.6 Hz, 2H), 7.42 (d, J = 0.8 Hz, 1H), 7.33 (t, J = 8.8 Hz, 2H),7.26 (t, J = 6.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d 6) δ 163.47, 161.03, 155.91,145.81, 135.73, 133.74, 132.73, 131.32, 131.23, 130.50, 122.59, 120.20,118.84, 116.70, 116.49, 79.35. HRMS (Q-TOF): calculated for [M+H]+ 323.0058,325.0038, found 324.0131, 326.0112。
实施例23：4-亚氨基-3-(吡啶-3-基)-4H-喹嗪-1-腈（IMQU-7）的制备.
制备方法同实施例17，收率70.5%。1H NMR (400 MHz, DMSO-d 6) δ 9.40 (d, J =7.2 Hz, 1H), 8.67 (d, J = 1.6 Hz, 1H), 8.62 (dd, J = 4.8, 1.6 Hz, 1H), 7.92(dt, J = 8.0, 2.0 Hz, 1H), 7.86 (t, J = 8.8 Hz, 1H), 7.75 (d, J = 8.8 Hz,1H), 7.57 (s, 1H), 7.52 (q, J = 4.8 Hz,, 1H), 7.31 (t, J = 7.2 Hz, 1H). HRMS(Q-TOF): calculated for [M+H]+ 246.0905, found 247.0982。
实施例24：4-亚氨基-3-(吡啶-2-基)-4H-喹嗪-1-腈（IMQU-8）的制备.
制备方法同实施例17，收率72.4%。1H NMR (400 MHz, DMSO-d 6) δ 10.81 (s,1H), 9.57 (d, J = 7.6 Hz, 1H), 8.67 (d, J = 4.8 Hz, 1H), 8.16 (s, 1H), 8.02(d, J = 8.0 Hz, 1H), 7.91 (t, J = 7.6 Hz, 2H), 7.76 (d, J = 8.8 Hz, 1H), 7.36(q, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d 6) δ 155.84, 153.31, 148.03,146.12, 137.73, 137.09, 134.15, 130.82, 122.60, 122.30 (2C), 118.80, 117.09,113.54, 79.34. HRMS (Q-TOF): calculated for [M+H]+ 246.0905, found 247.0979。
实施例25：IMQU-8在不同pH值的水溶液中的荧光谱图采集.
取2.46mg化合物IMQU-8，溶于1mL二甲亚砜，制得探针母液，浓度为10-3M。分别取100微升探针母液，加入到10mL不同pH的二甲亚砜的水溶液中（二甲亚砜：水=2:8）测其荧光发射强度变化发现：pH从12变化至6的过程中，528 nm处荧光强度明显增强，pH从6升至3的过程中，荧光强度无明显变化，如图1所示。
实施例26：IMQU-7在不同pH值的水溶液中的荧光谱图采集.
取2.46mg化合物IMQU-7，溶于1mL二甲亚砜，制得探针母液，浓度为10-3M。分别取100微升探针母液，加入到10mL不同pH的二甲亚砜的水溶液中（二甲亚砜：水=2:8）测其荧光发射强度变化发现：pH从12变化至6的过程中，528 nm处荧光强度明显增强，pH从6升至3的过程中，荧光强度无明显变化。
实施例27：IMQU-8在肿瘤细胞A549中的荧光成像.
将A549细胞接种于培养瓶中，加入5 mL 含有10%胎牛血清（FBS）、100 μg/mL青链霉素混合液、DMEM的混合培养基，在37 °C、5%二氧化碳的加湿细胞培养箱中培养。每天给细胞换培养液液一次，隔两天传代一次，传代时用酶消化法传代（用胰蛋白酶-EDTA消化液），细胞传至3~4代后，将其接种至直径35 mm 的玻璃底培养皿继续培养，过夜后将细胞用磷酸盐缓冲液（PBS）洗涤两次，用少量二甲基亚砜溶解探针IMQU-8，加入到上述混合培养基中，配置成终浓度为10 μM的工作液，用移液枪移取3 mL 加入培养皿中，将其放入培养箱中培养30 min后，用PBS 洗涤三次，再加入1 mL 空白混合培养基，准备进行荧光共聚焦成像。激发波长为405 nm，荧光采集波段为510 nm-570 nm。
Claims (5)
1.一种式II所示喹嗪类pH荧光探针，其特征在于，所述pH荧光探针包含如下式II所示核心结构单元，该结构单元在H+/OH-介导下发生脒键的C-N键断裂或重建，相应发生荧光减弱/增强，以及发射波长或吸收波长的红移或蓝移，如下图所示：
其中，R为：
2.权利要求1所述式II的pH荧光探针，其特征在于，pKa的范围为6.0～9.0之间。
3.权利要求1所述式II的pH荧光探针，其特征在于，可用于肿瘤细胞和正常细胞内pH变化的监测。
4.权利要求1所述pH荧光探针在pH响应荧光检测领域的应用，其特征在于，采用荧光法，pH荧光探针在溶液中对pH有荧光强度响应。
5.权利要求4所述pH荧光探针在pH响应荧光检测领域的应用，其特征在于，所述溶液的溶剂选自水、甲醇、乙醇、乙腈、丙酮、N,N-二甲基甲酰胺、二甲基亚砜中的一种或几种。
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| CN103242327A (zh) * | 2013-04-11 | 2013-08-14 | 天津师范大学 | 对N-甲基环戊醛基罗丹明6GpH荧光分子探针及其制备方法与用途 |
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