CN114380862B - 一种比率型光学/光声双模式荧光探针dop-hno及其制备方法与应用 - Google Patents
一种比率型光学/光声双模式荧光探针dop-hno及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种比率型具有光学/光声双模式特性的硝酰基荧光探针DOP‑HNO及其制备方法和应用。本发明提供的双比率荧光/光声双模式探针DOP‑HNO发射光位于近红外区,有利于细胞或动物模型中硝酰基的检测,并且具有精细的组织成像深度和组织散射/自致荧光和探针浓度的低干扰,从而获得更高的成像分辨率和精准的信号检测结果。同时,本发明探针分子DOP‑HNO合成步骤简单,纯化方便,成像优异,具有广阔的应用前景。
Description
技术领域
本发明属于有机小分子荧光探针制备技术领域,具体涉及一种比率型光学/光声双模式荧光探针DOP-HNO及其制备方法与应用。
背景技术
硝酰基(HNO)是NO的单电子还原和质子化衍生物,在许多生理和病理过程中起着至关重要的作用。自它被发现以来,便引起了人们广泛关注,无数研究阐明了其在生物系统中的重要作用。例如,HNO已被证明是一种血管扩张剂,因此被认为是治疗心力衰竭的潜在治疗药物。作为醛脱氢酶的抑制剂,HNO在治疗中也显示出潜在的用途。尽管取得了这些令人兴奋的进展,但HNO的详细生物学功能及其内源性生成机制仍不清楚,这主要是由于缺乏有效的HNO检测方法。该分子具有高度反应活性,容易二聚和脱水成一氧化二氮,在生物样品中准确检测造成困难。因此,允许在生物环境中选择性和敏感地检测HNO是非常有意义的。
基于HNO重要的生物学意义,已经报道了多种检测HNO的荧光探针,荧光探针虽然具有灵敏度高和快速响应的优点,但它有限的组织穿透深度,使我们无法实现在动物模型中HNO的检测。光声(PA)成像技术结合了光学成像和声学成像的优点,具有光学成像的高对比度和超声成像的高空间分辨率。它可以克服传统光学成像的限制,在厘米级深度实现高分辨率成像,并能够在深层组织中实现高分辨率的可视化。此外,它还具有使用非电离辐射、无创辐射、对正常组织无影响的优点。同时,比值成像具有较强的自校正能力,可以消除探针在体内靶组织中的不均匀积累和光漂白所引起的干扰。
发明内容
针对现有技术的不足,本发明的目的在于提供一种比率型光学/光声双模式探针DOP-HNO及其制备方法,用于检测细胞和动物模型中的HNO。
为实现上述目的,本发明提供如下技术方案:
一种比率型光学/光声双模式荧光探针DOP-HNO,其化学结构式如下:
本发明还保护一种上述比率型光学/光声双模式荧光探针DOP-HNO的制备方法,合成路线如下:
优选的,具体包括以下步骤:
(1)在冰浴条件下,将三氯氧磷逐滴加入DMF和CH2Cl2的混合溶液中,再入加原料1,搅拌10min,撤去冰浴,加热进行反应,反应完成后分离提纯得到产物2;
(2)将原料3溶解在乙腈中,再加入碘乙烷和碳酸钾,加热反应,反应后分离提纯得到产物4;
(3)将步骤(1)中得到的产物2和步骤(2)中得到的产物4溶解在正丁醇和甲苯的混合溶液中,加热反应,反应结束后加入高氯酸搅拌,接着再分离提纯得到产物5;
(4)将步骤(3)中得到的产物5溶解在无水DMF中,加入间巯基苯酚,搅拌10min,再加入氢化钠,加热反应,反应后分离提纯得到产物6;
(5)将2-(二苯基膦基)苯甲酸溶解在二氯甲烷中,加入4-二甲氨基吡啶和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐,接着在15-30℃下搅拌10-20min,得到混合液;将步骤(4)中得到的产物6溶解于二氯甲烷中,加入上述混合液,在氮气氛下进行反应,反应完成后,进行分离提纯,即得到荧光探针DOP-HNO。
优选的,步骤(1)中DMF与CH2Cl2的体积比为1:1,原料1与三氯氧磷的摩尔比为1:1.5;反应条件为:在氮气保护气氛下回流进行,反应温度为80-100℃,反应时间为2-6h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醚混合溶剂。
优选的,步骤(2)中原料3、碘乙烷和碳酸钾的摩尔比为1-3:1-3:2-3;反应条件为:在氮气保护气氛下回流进行,反应温度为80-100℃,反应时间为12-18h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂。
优选的,步骤(3)中产物2和产物4的摩尔比为1:2,正丁醇和甲苯的体积比为7:3;反应条件为:在氮气保护气氛下回流进行,反应温度为100-120℃,反应时间为8-10h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂。
优选的,步骤(4)中产物5、间巯基苯酚和氢化钠的摩尔比为1:3:2;反应条件为:在氮气保护气氛下进行,反应温度为45-60℃,反应时间为8-16h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂。
优选的,步骤(5)中产物6、2-(二苯基膦基)苯甲酸、4-二甲氨基吡啶和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐的摩尔比为1-2:1-2:2-4:1-2;反应温度为20-30℃,反应时间为1-3h;提纯时硅胶柱层析所用洗脱剂为体积比为10:1的二氯甲烷/乙醇混合溶剂。
优选的,所述分离提纯具体步骤为将反应液通过减压蒸出溶剂,得到固体,将固体进行干燥,干燥后的固体通过硅胶柱层析进行提纯。
本发明还保护一种上述比率型光学/光声双模式荧光探针DOP-HNO在检测HNO中的应用。
优选的,该应用具体包括如下步骤:
(1)将制备得到的荧光探针DOP-HNO溶于二甲基亚砜溶剂,制成探针母液;
(2)将步骤(1)所得的探针母液加入到待测液或生物样品中;
(3)将不同当量HNO加入步骤(2)所得的探针母液的待测液中,用紫外分光光度计和荧光光谱仪观察荧光探针DOP-HNO的紫外吸收光谱和荧光发射光谱变化,或者将生物样品细胞与荧光探针共同孵育,然后在荧光显微镜下拍摄荧光图像,从而得到细胞荧光图像,或者将本探针用于活体中检测HNO,得到动物活体荧光成像图和光声成像图。
与现有技术相比,本发明具有如下的有益效果:
(1)本发明提供的技术合成路线基于分子内电荷转移(ICT)机理的首次实现了以含硫半花箐为荧光团,氯甲酸烯丙酯为响应基团的CO比率型光学/光声双模式检测。
(2)本发明提供的探针DOP-HNO合成步骤简单、纯化方便,成像优异;
(3)本发明提供的探针DOP-HNO生物相容性好,对细胞或动物的伤害小;
(4)本发明提供的探针DOP-HNO采用双比率荧光和光声检测,检测信号精准;
(5)本发明提供的探针DOP-HNO具有使用非电离辐射、无创辐射、对正常组织无影响的优点。
附图说明
图1是探针DOP-HNO的1H NMR图谱;
图2是探针DOP-HNO的13C NMR图谱;
图3是探针DOP-HNO对HNO的滴定紫外和荧光谱图变化的测试结果;
图4是探针DOP-HNO对HNO选择性的测试结果;
图5是探针DOP-HNO的细胞毒性测试结果;
图6是探针DOP-HNO共定位测试结果;
图7是探针DOP-HNO用于检测细胞外源性HNO测试结果;
图8是探针DOP-HNO用于检测小鼠肿瘤内HNO荧光成像实验结果;
图9是探针DOP-HNO用于检测小鼠肿瘤内HNO光声成像实验结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实施例中所用原料均可通过市售获得。
实施例1
一种比率型光学/光声双模式荧光探针DOP-HNO,其结构式如下:
本实施例中,所述比率型光学/光声双模式荧光探针DOP-HNO的具体合成步骤为:
(1)在冰浴条件下,将1.5mol三氯氧磷逐滴加入DMF和CH2Cl2的混合溶液(500mL)中,再入加1mol原料1,搅拌10min,撤去冰浴,在氮气保护下回流反应,反应温度为90℃,反应时间为4h,反应完成后分离提纯,提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醚混合溶剂,得到产物2;
(2)将1mol的原料3溶解在500mL乙腈中,再加入1mol碘乙烷和2mol碳酸钾,在80℃下反应14h,反应后分离提纯,提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂,得到产物4;
(3)将步骤(1)中得到的产物2(1mol)和步骤(2)中得到的产物4(2mol)溶解在体积比为7:3的正丁醇/甲苯混合溶液(500mL)中,在100℃下反应9h,反应结束后加入10mL高氯酸搅拌,接着再分离提纯,提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂,得到产物5;
(4)将步骤(3)中得到的产物5(1mol)溶解在无水DMF(500mL)中,加入3mol间巯基苯酚,搅拌10min,再加入2mol氢化钠,在45℃下反应10h,反应后分离提纯,提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂,得到产物6;
(5)将2-(二苯基膦基)苯甲酸(1mol)溶解在1000mL二氯甲烷中,加入DMAP(4-二甲氨基吡啶)(2mol)和EDC(1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐)(1mol),在15℃下搅拌10min,得到混合液;将步骤(4)中得到的产物6(1mol)溶解在500mL二氯甲烷中,加入上述混合液中,在氮气氛保护,20℃下反应2h,反应完毕后,将反应液减压蒸出溶剂,得到固体,将固体进行干燥,干燥后的固体通过硅胶柱层析,洗脱剂为二氯甲烷/乙醇(V/V=10:1),即得到荧光探针DOP-HNO。
如图1、图2所示,核磁及质谱表征:
1H NMR(600MHz,DMSO-d6)δ8.34(d,J=14.8Hz,1H),8.30(d,J=7.3Hz,1H),7.93(d,J=6.2Hz,1H),7.88(d,J=8.0Hz,1H),7.68(dd,J=20.6,7.3Hz,4H),7.62(d,J=7.5Hz,1H),7.50(d,J=5.1Hz,4H),7.46(d,J=5.3Hz,2H),7.35–7.25(m,6H),7.17(dd,J=10.2,2.1Hz,2H),6.98(d,J=9.3Hz,1H),4.61(q,J=7.2Hz,2H),2.84–2.80(m,2H),2.77(t,J=6.0Hz,2H),2.06(dq,J=20.2,6.9,6.4Hz,2H),1.85(s,6H),1.47(t,J=7.4Hz,3H).
13C NMR(151MHz,DMSO-d6)δ178.83,164.99,150.84,149.01,145.75,143.43,141.30,137.56,137.48,134.67,134.26,134.19(2C),134.05(2C),133.91,133.71,133.19,132.89,131.94,131.71,130.13,129.62(2C),129.44,129.41(2C),129.36(2C),129.28,128.73,128.13,127.58,123.57,122.01,118.61,114.56,109.20,51.59,40.50,35.58,30.28,27.59(2C),25.58,13.60.HRMS(ESI)m/z calcd for C46H41NO2PS+([M]+):702.2590;found 702.2551.
实施例2
探针DOP-HNO与HNO反应的紫外吸收和荧光发射实验。
配置实施例1中制备探针DOP-HNO的DMSO母液,浓度为5mM;然后分别在紫外和荧光皿中加入1998μL液体(PBS:DMSO=7:3)和4μL探针母液,随后在荧光皿中加入HNO,将上述溶液用紫外-可见分光光度计和荧光光谱仪测试其吸收光谱和荧光发射光谱,测试结果见图3。
从图3a中可以观察到紫外光谱峰在610nm下降,740nm上升,从图3b中可以观察到荧光光谱峰在720nm下降,783nm上升,呈现一个比色比值的变化。
实施例3
探针DOP-HNO的选择性实验。
将实例2中配置探针DOP-HNO的方法同样适用于选择性的测试。已检测的相关生物成分包括生物硫醇,盐,活性氧等,具体为Cys,GSH,Hcy,GSNO,CaCl2,NaS2O3,ZnCl2,NaOCl,NaCl,H2O2,KO2,Na2S,NaNO2,NaNO3,ONOO,·OH,NO和HNO。测试结果见图4。
如图4所示,当只有HNO存在时,荧光比值才显著增强,说明与其它组分相比,探针DOP-HNO对HNO有极好的选择性,可以在复杂的生物环境中特异性检测HNO。
实施例4
按照前述制备方法制备得到探针DOP-HNO,并将其用于细胞毒性测试。
测试方法如下:分别将消化好的HeLa,4T1细胞悬浮液以每孔1×105个细胞180μL-1的密度接种于96孔培养板中,放入细胞培养箱培养24小时。在显微镜观察细胞密度为80~90%时向每个孔中分别加入20μL探针(0、2、4、6、8和10μM)后继续培养24小时后加入MTT试剂(10μL,5mg/mL MTT)孵育6小时。除去细胞上清液,每孔加入100mL DMSO,放置于摇床上低速震荡15分钟以充分溶解紫色的甲瓒结晶物。通过酶标仪测试甲瓒在570nm处的吸光度并计算细胞的存活率;细胞存活率表示为实验组平均值与空白组平均值的百分比。测试结果如图5所示。
从图5中可以看到,探针DOP-HNO在细胞内的细胞毒性较低说明探针的生物相容性较好。
实施例5
将实施例1制备得到的探针DOP-HNO进行共定位实验。
测试方法如下:将4T1细胞贴壁培养于内含10%胎牛血清的低糖培养液中,在37℃,5%CO2的饱和湿度孵箱中培养,每隔3天更换培养液,并进行传代培养和将细胞移入共聚焦皿中进行培养。取出1个共聚焦皿将里面培养液换成1mL新鲜培养液,将5mM的探针母液,取2μL于共聚焦皿中与商业探针LYDG(200nM)孵育15分钟;再取出1个共聚焦皿将里面培养液换成1mL新鲜培养液,将5mM的探针母液,取2μL于共聚焦皿中与商业探针MTDG(200nM)孵育15分钟;将共同染色的4T1细胞进行共聚焦显微镜成像。其中探针的绿色通道激发波长为488nm,荧光收集波长为505-560nm;红色通道激发波长为638nm,荧光收集波长为650-750nm;测试结果如图6所示。
从图6中可以看到,探针DOP-HNO定位于线粒体。
实施例6
实施例1制备得到的探针DOP-HNO在细胞中外源性响应HNO共聚焦荧光成像实验。
将适当密度的4T1细胞接种到共聚焦皿(35mm),培养24h后,用吸管吸除瓶内的培养基,加入1mL新鲜培养基。探针在细胞中HNO的外源性响应实验设置空白组和实验组共4组。空白组加入10μM的探针孵育15min,移除培养基,用PBS润洗2遍,进行成像。实验组先加10μM的探针孵育15min,移除培养基,加入1mL新鲜培养基,再分别加5eq,10eq,15eq的HNO孵育30min,用吸管吸除皿内的培养基,用PBS润洗2遍,进行成像。荧光成像实验过程中,激发源选择638nm,收集波段分别为650nm-720nm和730nm-795nm。实验结果如图7所示。
从图7中可以看出,空白组不加入HNO时通道1发出绿色荧光,而通道2没有荧光,当加入不同当量的HNO时,通道2的红色荧光不断增强,以剂量依赖的方式,同时,通道1的绿色荧光不断减弱,说明探针DOP-HNO具有比值荧光成像能力,且能在细胞环境中很好的响应HNO。
实施例7
实施例1制备得到的探针DOP-HNO用于检测小鼠肿瘤HNO荧光成像实验。
为了在体内验证这种HNO反应的比值荧光,我们采用了一些异种移植乳腺癌模型。在肿瘤平均体积增加到200mm3后,对小鼠肿瘤进行瘤内注射探针DOP-HNO和HNO进行成像。实验结果如图8所示。
从图8中可以看出,通道1(λex=630nm滤片700nm)的图像显示,肿瘤区域荧光在下降,而通道2(λex=720nm滤片790nm)的图像显示,肿瘤区域明显的荧光增强功能。
实施例8
实施例1制备得到的探针DOP-HNO用于检测小鼠肿瘤HNO光声成像实验。
通过体内比值PA成像进一步证实了探针DOP-HNO与HNO的反应。将探针DOP-HNO和HNO注射到肿瘤区域后,在PA670和PA735通道中均观察到明显的光声信号。实验结果如图9所示。
从图9中可以看出,通道PA670的图像显示,肿瘤区域光声信号在下降,而通道PA735的图像显示,肿瘤区域光声信号增强功能。说明探针可能适合作为肿瘤中定量检测HNO的PA探针。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (10)
3.根据权利要求2所述的制备方法,其特征在于,具体包括以下步骤:
(1)在冰浴条件下,将三氯氧磷逐滴加入DMF和CH2Cl2的混合溶液中,再加入原料1,搅拌10min,撤去冰浴,加热进行反应,反应完成后分离提纯得到产物2;
(2)将原料3溶解在乙腈中,再加入碘乙烷和碳酸钾,加热反应,反应后分离提纯得到产物4;
(3)将步骤(1)中得到的产物2和步骤(2)中得到的产物4溶解在正丁醇和甲苯的混合溶液中,加热反应,反应结束后加入高氯酸搅拌,接着再分离提纯得到产物5;
(4)将步骤(3)中得到的产物5溶解在无水DMF中,加入间巯基苯酚,搅拌10min,再加入氢化钠,加热反应,反应后分离提纯得到产物6;
(5)将2-(二苯基膦基)苯甲酸溶解在二氯甲烷中,加入4-二甲氨基吡啶和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐,接着在15-30℃下搅拌10-20min,得到混合液;将步骤(4)中得到的产物6溶解于二氯甲烷中,加入上述混合液,在氮气氛下进行反应,反应完成后,进行分离提纯,即得到荧光探针DOP-HNO。
4.根据权利要求3所述的制备方法,其特征在于,步骤(1)中DMF与CH2Cl2的体积比为1:1,原料1与三氯氧磷的摩尔比为1:1.5;反应条件为:在氮气保护气氛下回流进行,反应温度为80-100℃,反应时间为2-6h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醚混合溶剂。
5.根据权利要求3所述的制备方法,其特征在于,步骤(2)中原料3、碘乙烷和碳酸钾的摩尔比为1-3:1-3:2-3;反应条件为:在氮气保护气氛下回流进行,反应温度为80-100℃,反应时间为12-18h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂。
6.根据权利要求3所述的制备方法,其特征在于,步骤(3)中产物2和产物4的摩尔比为1:2,正丁醇和甲苯的体积比为7:3;反应条件为:在氮气保护气氛下回流进行,反应温度为100-120℃,反应时间为8-10h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂。
7.根据权利要求3所述的制备方法,其特征在于,步骤(4)中产物5、间巯基苯酚和氢化钠的摩尔比为1:3:2;反应条件为:在氮气保护气氛下进行,反应温度为45-60℃,反应时间为8-16h;提纯时柱层析所用洗脱剂为体积比10:1的二氯甲烷/乙醇混合溶剂。
8.根据权利要求3所述的制备方法,其特征在于,步骤(5)中
产物6、2-(二苯基膦基)苯甲酸、4-二甲氨基吡啶和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐的摩尔比为1-2:1-2:2-4:1-2;反应温度为20-30℃,反应时间为1-3h;提纯时硅胶柱层析所用洗脱剂为体积比为10:1的二氯甲烷/乙醇混合溶剂。
9.一种如权利要求1所述的比率型光学/光声双模式荧光探针DOP-HNO在检测硝酰基中的应用。
10.根据权利要求9所述的应用,其特征在于,包括如下步骤:
(1)将制备得到的荧光探针DOP-HNO溶于二甲基亚砜溶剂,制成探针母液;
(2)将步骤(1)所得的探针母液加入到待测液或生物样品中;
(3)将不同当量硝酰基加入步骤(2)所得的探针母液的待测液中,用紫外分光光度计和荧光光谱仪观察荧光探针DOP-HNO的紫外吸收光谱和荧光发射光谱变化,或者将生物样品细胞与荧光探针共同孵育,然后在荧光显微镜下拍摄荧光图像,从而得到细胞荧光图像,或者将本探针用于活体中检测硝酰基,得到动物活体荧光成像图和光声成像图。
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