CN115286619A - 一种特异性识别nqo-1的花菁类荧光探针及其制备方法和应用 - Google Patents
一种特异性识别nqo-1的花菁类荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种特异性识别NQO‑1的花菁类荧光探针及其制备方法和应用,本发明基于花菁衍生物作为探针的荧光母核,花菁衍生物的结构决定其荧光性质十分优异。通过修饰形成的探针本身荧光可以几乎完全掩蔽,与NQO‑1作用30分钟后,荧光强度迅速增强(荧光增强倍数在10倍左右)。检测限低至0.01μg/mL,灵敏度高,选择性和抗干扰性能好。
Description
技术领域
本发明涉及荧光探针技术领域,尤其是涉及一种特异性识别NQO-1的花菁类荧光探针及其制备方法和应用。
背景技术
胰腺癌是最具侵袭性的恶性肿瘤之一,5年生存率为9%,其发病率持续上升。由于胰腺癌患者预后不佳,且手术、化疗和放疗的效果有限,2020年胰腺癌死亡人数(466000人)与新发病例(496000人)相当,是男女癌症死亡的第七大原因。胰腺癌缺乏能够在早期发现肿瘤的主动筛查方法。因此,大多数患者被诊断为晚期转移或晚期,只有10-15%的患者能够通过手术切除。临床上,胰腺癌对化疗药物和放射治疗都产生了极强的细胞耐药性。这种耐药性是由与肿瘤微环境相关的细胞内、外因素产生的。因此,迫切需要针对胰腺癌的新疗法的出现。
光动力疗法(photodynamictherapy,PDT)是胰腺癌的一种临床辅助治疗方法,可诱导胰腺癌细胞凋亡和坏死,导致血管损伤,增强肿瘤组织的抗肿瘤免疫反应。胰腺癌对光动力疗法的抵抗力低于常规化疗和放疗,因此是治疗胰腺癌的一种有效手段。
NAD(P)H:醌氧化还原酶1(NQO1)是一种同型二聚体,利用普遍存在的黄素蛋白将醌还原为酚类,是胰腺癌治疗的特异靶点。NQO1在切除的胰腺癌样本中过表达大于85%,并且优先在胰腺癌中表达,而不是在非癌性邻近胰腺中表达。在胰腺上皮内瘤变中观察到NQO1表达增加,这是胰腺癌的前体病变。NQO1在原发性和恶性肿瘤细胞中进一步上调,与正常胰腺相比,胰腺癌组织中的基因表达增加了12倍。因此,NQO-1可以被认为是胰腺癌特异性识别的理想靶点。
此发明中,我们利用ICy-OH作为花菁类荧光母核,以对苯二醌衍生物为NQO-1识别基团设计并合成了一种新的NQO-1荧光探针(ICy-Q),用于选择性地识别和光动力杀伤NQO-1高表达的胰腺癌细胞,此外我们还发现探针ICy-Q和ICy-OH在无光照条件下能够选择性的诱导胰腺癌细胞死亡,实现对胰腺癌细胞的荧光识别与化疗杀伤。
发明内容
有鉴于现有技术的上述问题,本发明的目的是克服现有技术中的不足,提出一种特异性识别NQO-1的花菁类荧光探针及其制备方法和应用,并将该探针应用于胰腺癌细胞的化疗杀伤,荧光成像及光动力杀伤中研究。
本申请提供的一种特异性识别NQO-1的花菁类荧光探针,所述荧光探针分子式为C48H50IN2O5,其结构式为:
一种特异性识别NQO-1的花菁类荧光探针的制备方法,包括如下步骤:
S1、制备中间体ICy-OMe:将0.2mL哌啶、摩尔比为1:1的中间体3和中间体5溶于10mL乙酸酐中制备混合溶液的体积摩尔浓度为0.1-1.0mol/L,并在80℃条件下搅拌2小时,反应结束后将反应液倒入100g冰中,随后用二氯甲烷萃取,将萃取液用无水硫酸钠干燥,减压旋干脱除溶剂,柱层析进一步纯化,用二氯甲烷/甲醇(10:1至5:1)的洗脱剂洗脱后得到中间体ICy-OMe;
S2、制备中间体ICy-OH:将中间体ICy-OMe溶解于二氯甲烷中,然后缓慢滴入三溴化硼,在氩气保护下,反应混合物在50℃下搅拌4小时,随后让反应液冷却至室温,并缓慢加入甲醇淬灭反应,在真空下去除溶剂后,收集残渣,用水冲洗,并在真空中干燥,得到中间体ICy-OH;
S3、制备目标产物ICy-Q:将摩尔比为1:2的中间体ICy-OH、N,N二异丙基乙胺的混合物溶于无水乙腈中制备的混合溶液的体积摩尔浓度为0.1-1.0mol/L,氩气保护下,55℃搅拌0.5小时,随后加入中间体2,并继续在氩气保护下55℃搅拌5小时,反应结束后,减压旋干,柱层析进一步纯化,用二氯甲烷/甲醇(10:1至5:1)的洗脱剂洗脱后得到目标产物ICy-Q;
其中,中间体2的结构式为:
其中,中间体3的结构式为
其中,中间体5的结构式为
其中ICy-OMe的结构式为
其中ICy-OH的结构式为
其中ICy-Q的结构式为
进一步的,所述中间体2的制备方法包括如下步骤:
A1、制备中间体1:将摩尔比为1:1.4的4-甲氨基苯甲酸和CDI混合物溶于四氢呋喃中制备的混合溶液的体积摩尔浓度为0.1-1.0mol/L,25度搅拌1小时,随后向反应液加入水并搅拌,同时缓慢加入硼氢化钠,并继续搅拌2小时,反应结束后用稀盐酸中和反应液,并用乙酸乙酯萃取。将萃取液用无水硫酸钠干燥,减压旋干,柱层析进一步纯化,用石油醚/乙酸乙酯(2:1)的洗脱剂洗脱后得到中间体1。
A2、制备中间体2:将摩尔比为1:3的化合物Q、甲基吗啡啉溶于二氯甲烷中,将混合液冷却,并缓慢滴加氯甲酸异丁酯,并继续搅拌30分钟,随后添加含有中间体1的二氯甲烷溶液,并继续搅拌5小时,反应结束后将反应液减压去除溶剂,并重新用乙酸乙酯溶解,并依次用水,稀盐酸和碳酸氢钠饱和溶液洗涤,最后用无水硫酸钠干燥并减压去除溶剂;
将的到的产物(T-OH)和NBS混合物溶于5mL二氯甲烷中,再加入DMTU,室温搅拌2小时,反应完毕混合溶液用二氯甲烷稀释,然后用水洗涤三次,接着将二氯甲烷用无水硫酸钠干燥,最后将溶剂减压旋干,并使用石油醚/乙酸乙酯(3:1)作为洗脱液通过硅胶快速色谱纯化,以获得黄色的中间体2;
其中,结构式CDI的结构式为
其中,中间体1的结构式为
其中,化合物Q的结构式为
其中,结构式T-OH的结构式为
进一步的,所述中间体5的制备方法包括如下步骤:
将摩尔比为1:5的中间体4和碘甲烷溶于乙腈中以制备混合溶液,该混合溶液的体积摩尔浓度为0.1-1.5mol/L,将混合溶液在80℃条件下回流搅拌7小时,随后冷却至室温,通过过滤收集沉淀物,用乙醚洗涤,并在真空中干燥,得到中间体5;
其中,中间体4的结构式为
进一步的,所述步骤A2中,所述T-OH,NBS和DMTU混合物的摩尔比为1:1.5:0.45。
一种特异性识别NQO-1的花菁类荧光探针在制备检测、识别环境中或生物样品中NQO-1的检测试剂或者标记物中的应用。
进一步的,所述NQO-1的花菁类荧光探针用于制备正常胰腺细胞和胰腺癌细胞的NQO-1的检测试剂或者标记物中的应用。
进一步的,所述NQO-1的花菁类荧光探针测定NQO-1的检测方法如下:
通过荧光分光光度法,以650nm为激发波长,在710nm的波长处测定NQO-1的荧光强度,所述样品的检测限为0.01μg/mL。
通过采用上述检测方法:本发明的荧光探针其本身荧光微弱,在与NQO-1作用后迅速有很强的荧光发射,其最大吸收波长为650nm附近,与NQO-1反应后最大发射波长为710nm。
综上所述,本申请包括以下有益技术效果:
1、本发明的荧光探针对NQO-1有很好的选择性,在pH等于7.4的PBS缓冲溶液中,探针溶液荧光强度微弱,加入5μg/mL NQO-1,荧光强度逐渐增强,30分钟后增大到最初的10倍左右。而同样条件下分别加入其他可能干扰的离子和常见生理性亲核分子及常见酶,探针的荧光强度变化不明显;
2、本发明的荧光探针对NQO-1的检测表现出较高的灵敏度。探针溶液的荧光强度,随着增加NQO-1浓度而递增,大约在加入2μg/mL NQO-1时达到峰值。在0到2μg/mL倍量NQO-1的区间内,探针溶液荧光强度与NQO-1浓度有很好的线性关系;
3、在本发明中,我们开发了一种花菁结构的NQO-1荧光增强探针,对NQO-1具有高度的选择性。探针能够选择性的对NQO-1高表达的胰腺癌细胞进行荧光成像和光动力杀伤,同时还能选择性诱导胰腺癌细胞焦亡;
4、本发明的花菁衍生物作为探针的荧光母核,花菁衍生物的结构决定其荧光性质十分优异。通过修饰形成的探针本身荧光可以几乎完全掩蔽,与NQO-1作用30分钟后,荧光强度迅速增强(荧光增强倍数在10倍左右)。检测限低至0.01μg/mL,灵敏度高,选择性和抗干扰性能好。
综上所述,本发明的荧光探针是一种便捷,灵敏,适用于体外和活细胞内部检测NQO-1的工具,在化学分析检测领域具有广阔的应用前景。
附图说明
图1为本发明荧光探针制备过程示意图;
图2本发明中间体化合物1的质谱谱图;
图3本发明中间体化合物2的质谱谱图;
图4本发明中间体化合物5的核磁氢谱谱图;
图5本发明中间体化合物5的核磁碳谱谱图;
图6本发明中间体化合物5的质谱谱图;
图7本发明中间体化合ICy-OMe核磁氢谱谱图;
图8本发明中间体化合ICy-OMe核磁碳谱谱图;
图9本发明中间体化合ICy-OMe的质谱谱图;
图10本发明中间体化合ICy-OH核磁氢谱谱图;
图11本发明中间体化合ICy-OH核磁碳谱谱图;
图12本发明中间体化合ICy-OH的质谱谱图;
图13本发明中间体化合ICy-Q核磁氢谱谱图;
图14本发明中间体化合ICy-Q核磁碳谱谱图;
图15本发明中间体化合ICy-Q的质谱谱图;
图16为本发明的荧光探针ICy-Q(5μM)和ICy-OH(5μM)在PBS缓冲溶液(pH=7.4)中的吸收光谱图和荧光光谱图;
图17为本发明的荧光探针ICy-Q(5μM)在PBS缓冲溶液(pH=7.4)中,加入NQO-1(5μg/mL),反应60分钟的荧光光谱图和荧光强度变化图;
图18为本发明的荧光探针与NQO-1反应机理图;
图19为本发明的荧光探针(5μM)在PBS缓冲溶液(pH=7.4)中,加入递增浓度NQO-1(0-5μg/mL),反应60分钟后的荧光强度变化曲线。其插图为本发明的荧光探针(5μM)在PBS缓冲溶液(pH=7.4)中,加入递增浓度NQO-1(0-2μg/mL)的线性关系图;
图20为本发明荧光探针的各种不同离子和分子选择性的柱状图。其中a图,从序号1到8彩色柱分别代表常见金属离子(100μM)与探针(5μM)作用后的荧光强度:空白组,氯化钠,硝酸钾,硫酸镁,硫酸亚铁,氯化铁,硫酸铜,NQO-1;其中b图,从序号1到8彩色柱分别代表生理性亲核分子和常见酶类(100μg/mL)与探针(5μM)作用后的荧光强度:酪氨酸酶,谷氨酰胺转移酶,白蛋白,半乳糖苷酶,NADH,半胱氨酸,还原型谷胱甘肽,NQO-1;
图21为本发明的荧光探针ICy-Q和探针ICy-OH对正常胰腺细胞的CCK-8毒性实验,探针浓度梯度(最终浓度):4μM、3.5μM、3.0μM,2.5μM、2.0μM、1.5μM、1.5μM、0.5μM、0μM(对照),细胞存活率大于70%,基本无毒性;
图22为本发明的荧光探针ICy-Q和探针ICy-OH对胰腺癌细(MIA-PaCa-2)的CCK-8毒性实验,探针浓度梯度(最终浓度):4μM、3.5μM、3.0μM,2.5μM、2.0μM、1.5μM、1.5μM、0.5μM、0μM(对照),无光照条件下细胞存活率低于50%,近红外光(650nm)照条件下,毒性进一步增强;
图23为本发明的荧光探针ICy-Q(2.5μM)在不同条件下细胞中成像结果。(a)加入ICy-Q探针处理的MIA-PaCa-2胰腺癌细胞中明场成像(BL)、荧光成像(FL)以及Merge图;(b)加入ICy-Q探针和NQO-1抑制剂处理的MIA-PaCa-2胰腺癌细胞中明场成像、荧光成像以及Merge图;(c)加入ICy-Q探针处理的HPNE-1正常胰腺细胞中明场成像、荧光成像以及Merge图。使用640nm激发,比例尺:30微米;
图24为二维码,通过手持终端扫码可获取图1-23的彩色原图。
具体实施方式
以下结合附图1-24对本申请作进一步详细说明。
本申请实施例公开一种特异性识别NQO-1的花菁类荧光探针的制备方法。
本申请的中间体3(Org.Biomol.Chem.2015,13(30),8169-8172.)和中间体4(Chem.Science.2019,10,10586.)按照相应文献方法合成,化合物Q从上海毕得医药科技有限公司购买。
实施例1:
该荧光探针的合成路线如图1所示,在室温下,将N,N-羰基二咪唑(即CDI,456mg,2.81mmol)添加到4-(甲氨基)苯甲酸(302mg,2.0mmol)THF(四氢呋喃,5mL)溶液中。在室温下(25℃)搅拌溶液1小时,并添加水(1mL)搅拌。当缓慢添加(20分钟内添加完毕)NaBH4(142mg,3.75mmol)时,以400转每分钟速度搅拌该混合物2h,待反应结束后用稀盐酸(1毫升)淬灭反应(中和反应液)。搅拌30分钟后,用乙酸乙酯(3X 10mL)提取(萃取)。将合并的萃取液用无水硫酸钠干燥,过滤,减压旋干浓缩以得到灰白色的粘稠物。粗产物通过层析柱柱(SiO2)纯化,用洗脱剂洗脱后得到192mg(70%产率)无色粘稠状中间体化合物1。其质谱图,如图2所示。
HRMS(ESI)calculated for C8H12NO+,[M+H]+,138.0913,found,138.0914.
化合物Q(250mg,1.0mmol),甲基吗啡啉(300mg,3.0mmol)溶于5mL二氯甲烷中,将混合液冷却至-55℃,并缓慢滴加氯甲酸异丁酯(164mg,1.2mmol),并继续搅拌30分钟。随后缓慢滴加含有中间体1(137mg,1.0mmol)的二氯甲烷溶液,并继续搅拌5小时。反应结束后将反应液减压去除溶剂,并重新用乙酸乙酯溶解,并依次用水,稀盐酸和碳酸氢钠饱和溶液洗涤,最后用无水硫酸钠干燥并减压去除溶剂。将得到的产物(T-OH)和NBS(265mg,1.5mmol,1.5eq.)混合物溶于5mL二氯甲烷中,再加入DMTU(47mg,0.45mmol,0.45eq.),室温搅拌2小时。反应完毕混合溶液用二氯甲烷稀释,然后用水洗涤三次,接着将二氯甲烷用无水硫酸钠干燥,最后将溶剂减压旋干,并使用石油醚/乙酸乙酯(5:1至2:1)作为洗脱液通过硅胶快速色谱纯化,以获得黄色的中间体2(267mg,产率62%)。其质谱图,如图3所示。
将中间体4(2.85g,10mmol)溶解于乙腈(50mL)中,然后添加碘甲烷(7.05g,50.0mmol)。将反应混合物回流加热至80℃条件下搅拌7h,冷却至室温,通过过滤收集沉淀物,用乙醚洗涤,并在真空中干燥。中间体化合物5为粉红色晶体(2.9g,产率为70%)。其核磁图谱和质谱图,如图4-6所示。
1H NMR(400MHz,DMSO-d6):δ=1.52(s,6H),2.74(s,3H),3.94(s,3H),7.73(d,J=8.38Hz,1H),8.01(d,J=8.50Hz,1H),8.30(s,1H).13C NMR(DMSO-d6,101MHz):δ=196.6,144.2,142.4,137.9,132.7,117.6,96.5,54.5,35.2,21.9,14.6.HRMS(ESI)calculatedfor C12H15IN+,[M]+,300.0244,found,300.0245.
将0.2mL哌啶,中间体3(224mg,1.0mmol)和中间体5(427mg,1.0mmol)溶于10mL乙酸酐中,80℃条件下搅拌2小时。反应结束后将反应液倒入100g冰中,随后用二氯甲烷萃取。将萃取后的二氯甲烷用无水硫酸镁或用无水硫酸钠干燥,随后减压旋干脱除溶剂,然后用柱层析纯化的到蓝色固体ICy-OMe 400mg,产率61%。其核磁图谱和质谱图,如图7-9所示。
将化合物ICy-OMe(651mg,1.0mmol)溶解于DCM(二氯甲烷,10mL)中,然后缓慢滴入BBr3(2.5g,10.0mmol)。在氩气保护下,反应混合物在50℃温度下搅拌4小时,并通过TLC(约2~4小时)进行监测。当起始材料消失时,将反应液冷却至室温,通过添加甲醇(10mL)并搅拌20min水解反应混合物(淬灭反应)。在真空下去除溶剂后,收集残渣,用水冲洗,并在真空中干燥。以蓝色固体形式获得化合物ICy-OH(585mg,产率为95%)。其核磁图谱和质谱图,如图10-12所示。
1H NMR(400MHz,DMSO-d6):δ=8.55(d,J=14.8Hz,4H),8.17(d,J=1.3Hz,4H),7.88(dd,J=1.2,8.3Hz,1H),7.61(s,4H),7.52(d,J=8.4Hz,4H),7.46(d,J=8.4Hz,3H),6.96(s,4H),6.89(dd,J=2.1,8.4Hz,4H),6.45(d,J=14.8Hz,3H),3.81(s,3H),2.75(br.s.,2H),2.68(t,J=5.3Hz,2H),1.92-1.81(m,2H),1.77-1.71(m,6H).13C NMR(101MHz,DMSO-d6):δ=176.7,162.3,161.9,154.7,144.8,144.4,142.8,137.8,135.5,131.7,129.8,126.4,115.3,115.3,115.0,114.7,104.1,102.5,92.1,50.5,32.9,28.8,27.7,24.1,20.5.HRMS(ESI)calculated for C26H24INO2 +,[M]+,510.0924,found,510.0924.
将ICy-OH(32mg,0.05mmol)和N,N-二异丙基乙胺(DIPEA,17.5μL,0.10mmol)的混合物溶解在无水乙腈中(混合溶液的体积摩尔浓度为0.1-1.0mol/L),在Ar气氛下,55℃搅拌30分钟后。将中间体2(43mg,0.10mmol)逐滴添加到混合物中,并且反应混合物在Ar气氛下在55℃下继续搅拌5h。完成后,在减压下浓缩所得混合物,并使用CH2Cl2/CH3OH(10:1至5:1)作为洗脱液通过硅胶快速色谱纯化,以获得蓝色固体形式的化合物ICy-Q(39mg,产率45%)。其核磁图谱和质谱图,如图13-15所示。
1H NMR(400MHz,DMSO-d6):δ=8.56(d,J=14.7Hz,1H),8.33(s,1H),8.17(s,1H),7.90(d,J=8.1Hz,1H),7.68-7.58(m,2H),7.56(s,1H),7.48(d,J=8.4Hz,1H),7.38(br.s.,1H),7.25(s,1H),7.14(d,J=8.8Hz,1H),6.51(d,J=15.0Hz,1H),5.35(br.,2H),3.84(s,3H),3.07(br.,2H),2.74(br.s.,2H),2.68(br.,5H),2.02(s,5H),1.91(br.,5H),1.76(s,6H),1.23(br.,6H).13C NMR(75MHz,CDCl3-d):δ=191.3,187.7,176.3,172.0,162.5,162.3,154.8,154.6,145.7,143.9,143.6,143.3,142.2,138.2,137.8,136.2,135.5,135.0,131.4,129.4,129.2,127.7,127.5,116.3,115.7,114.5,114.0,103.7,102.2,91.2,70.5,50.3,47.7,38.1,37.1,34.1,29.2,28.5,28.1,24.5,20.3,14.1,12.8,12.1.HRMS(ESI)calculated for C48H50IN2O5 +,[M]+,861.2759,found,861.2758.
本发明的荧光探针对NQO-1的检测机理详情如下所述:探针ICy-Q的酚羟基被醚化,醚键削弱了酚羟基的分子内电荷转移效应导致荧光被掩蔽掉。探针ICy-Q的醌基团被NQO-1还原成酚,酚的亲核作用导致分子内酰胺键发生断裂露出胺基,胺基通过消除反应引发醚键断裂,导致酚羟基暴露,发射很强的荧光。探针ICy-OH和ICy-Q的吸收和荧光光谱如图16所示,从荧光光谱图中可知,同等浓度的ICy-Q的荧光强度明显弱于ICy-OH。探针ICy-Q与NQO-1作用后的荧光光谱和荧光强度变化如图17所示,探针ICy-Q与NQO-1作用后的荧光强度随时间增加而增强,大约30分钟左右达到峰值。探针ICy-Q响应机理如图18所示。
实验分析:
一、荧光强度与NQO-1浓度的关系
取荧光探针(5μM)加入PBS缓冲溶液(pH=7.4)中,接着加入递增浓度NQO-1(0-5μg/mL),分别有0μg、0.1μg/mL、0.5μg/mL、1.0μg/mL、2.0μg/mL、3.0μg/mL、5μg/mL、反应60分钟后,其荧光光谱图如图19a所示,其荧光强度与NQO-1的浓度的线性关系图如图19b所示,计算得到该分子的检测限可以达到0.01μg/mL,适合微量检测。可见本发明的荧光探针具有良好用前景。
二、荧光探针抗干扰能力检测
取探针分子溶液ICy-Q(5μM)于在PBS缓冲溶液(pH=7.4)中配成待测溶液,再分别加入各种干扰物分子,氯化钠,硝酸钾,硫酸镁,硫酸亚铁,氯化铁,硫酸铜,酪氨酸酶,谷氨酰胺转移酶,白蛋白,半乳糖苷酶,NADH,还原型谷胱甘肽,反应60分钟后测量,其结果如图20所示,溶液的荧光几乎没有明显变化,而只加入NQO-1的探针溶液荧光大幅度增强(约10倍),可见该荧光探针能够对NQO-1实现专属识别。本发明的荧光探针对NQO-1的检测具有强的抗干扰能力。
三、荧光探针ICy-Q和探针ICy-OH对正常胰腺细胞的CCK-8毒性实验
HPNE-1细胞接种于96孔培养皿(0.8×103个/孔),放置于细胞培养箱中,使之完全贴壁。之后更换新鲜培养液,加入200μL不同浓度4.0μM、3.5μM、3.0μM、2.5μM,2.0μM、1.5μM、1.0μM、0.5μM、0μM的荧光探针(ICy-Q和ICy-OH)分散液,培养24小时后,在酶标仪上将波长设定为530nm,测定96孔板每孔溶液的吸光度(OD值),按下列公式计算细胞生存率:细胞存活率=(OD待测组-OD空白组)/(OD细胞组-OD空白组)×100%。从图21中看出,细胞存活率大于70%,基本无毒性。
四、荧光探针ICy-Q和探针ICy-OH对胰腺癌细(MIA-PaCa-2)的CCK-8毒性实验
MIA-PaCa-2细胞接种于96孔培养皿(0.8×103个/孔),放置于细胞培养箱中,使之完全贴壁。之后更换新鲜培养液,加入200μL不同浓度4.0μM、3.5μM、3.0μM、2.5μM,2.0μM、1.5μM、1.0μM、0.5μM、0μM的荧光探针(ICy-Q和ICy-OH)分散液继续培养6小时,随后用功率为15mW/cm2的660nm LED灯照射20分钟,然后继续培养18小时。暗毒性组则不经过光照,加入探针后继续培养24小时,其余与LED灯照射组相同。最后,在酶标仪上将波长设定为530nm,测定96孔板每孔溶液的吸光度(OD值),按下列公式计算细胞生存率:细胞存活率=(OD待测组-OD空白组)/(OD细胞组-OD空白组)×100%。从图22左中看出,探针ICy-Q对胰腺癌细胞毒性较大,IC50低于2.5μM,光照后毒性进一步增强IC50低于1.5μM。从图22右中看出,探针ICy-OH对胰腺癌细胞毒性也较大,IC50低于2.5μM,光照后毒性进一步增强IC50低于1.5μM。
五、荧光探针ICy-Q(2.5μM)在不同条件下细胞中成像结果
为了探究探针的生物应用性,我们利用探针ICy-Q检测细胞水平的NQO-1含量。将HPNE-1细胞和MIA-PaCa-2细胞分别在Dulbecco’s modified Eagle’s medium(DMEM)中培养。荧光成像前,将HPNE-1细胞和MIA-PaCa-2分别细胞接种于共聚焦培养皿(1.3×104个),培养1天。第一组MIA-PaCa-2细胞用含有ICy-Q(2.5μM)的培养基中处理3小时,第二组MIA-PaCa-2细胞用含双香豆素(NQO-1抑制剂)和ICy-Q(2.5μM)的培养基中处理3小时,第三组HPNE-1细胞用含ICy-Q(2.5μM)的培养基中处理3小时。共聚焦成像前,将培养皿中用过的培养基废弃,用磷酸盐缓冲液清洗3次。最后所有组细胞用激光显微镜成像,其中激发波长为640nm,收集波段为680-740nm。第一组在Cy5通道中观察到强烈的荧光(图23a)。第二组加入了双香豆素处理的胰腺癌细胞,荧光较第一组明显弱(图23b)。第三组NQO-1低表达的正常胰腺细胞,荧光强度也明显弱于第一组(图23c),这一结果表明ICy-Q可以检测到活细胞中NQO-1含量的差异。
以上结果验证了ICy-Q可以作为一种良好的荧光探针选择性检测活细胞中的NQO-1。
为便于对图1-23更直观的观察,特附上图1-23的彩色原图的二维码。具体的,通过手持终端对图24的二维码进行扫描。此外,若说明书附图中存在不清楚的地方,亦可参照二维码的彩图加以分辨和理解。
以上均为本申请的较佳实施例,并非依此限制本申请的保护范围,故:凡依本申请的结构、形状、原理所做的等效变化,均应涵盖于本申请的保护范围之内。
Claims (9)
2.根据权利要求1所述的一种特异性识别NQO-1的花菁类荧光探针的制备方法,其特征在于:包括如下步骤:
S1、制备中间体ICy-OMe:将0.2mL哌啶、摩尔比为1:1的中间体3和中间体5溶于10mL乙酸酐中制备混合溶液的体积摩尔浓度为0.1-1.0mol/L,并在80℃条件下搅拌2小时,反应结束后将反应液倒入100g冰中,随后用二氯甲烷萃取,将萃取液用无水硫酸钠干燥,减压旋干脱除溶剂,柱层析进一步纯化,用洗脱剂洗脱后得到中间体ICy-OMe;
S2、制备中间体ICy-OH:将中间体ICy-OMe溶解于二氯甲烷中,然后缓慢滴入三溴化硼,在氩气保护下,反应混合物在50℃下搅拌4小时,随后让反应液冷却至室温,并缓慢加入甲醇淬灭反应,在真空下去除溶剂后,收集残渣,用水冲洗,并在真空中干燥,得到中间体ICy-OH;
S3、制备目标产物ICy-Q:将摩尔比为1:2的中间体ICy-OH、N,N二异丙基乙胺的混合物溶于无水乙腈中制备的混合溶液的体积摩尔浓度为0.1-1.0mol/L,氩气保护下,55℃搅拌0.5小时,随后加入中间体2,并继续在氩气保护下55℃搅拌5小时,反应结束后,减压旋干,柱层析进一步纯化,用洗脱剂洗脱后得到目标产物ICy-Q;
其中,中间体2的结构式为:
其中,中间体3的结构式为
其中,中间体5的结构式为
其中ICy-OMe的结构式为
其中ICy-OH的结构式为
其中ICy-Q的结构式为
3.根据权利要求2所述的一种特异性识别NQO-1的花菁类荧光探针的制备方法,其特征在于:所述中间体2的制备方法包括如下步骤:
A1、制备中间体1:将摩尔比为1:1.4的4-甲氨基苯甲酸和CDI混合物溶于四氢呋喃中制备的混合溶液的体积摩尔浓度为0.1-1.0mol/L,25度搅拌1小时,随后向反应液加入水并搅拌,同时缓慢加入硼氢化钠,并继续搅拌2小时,反应结束后用稀盐酸中和反应液,并用乙酸乙酯萃取。将萃取液用无水硫酸钠干燥,减压旋干,柱层析进一步纯化,用洗脱剂洗脱后得到中间体1。
A2、制备中间体2:将摩尔比为1:3的化合物Q、甲基吗啡啉溶于二氯甲烷中,将混合液冷却,并缓慢滴加氯甲酸异丁酯,并继续搅拌30分钟,随后添加含有中间体1的二氯甲烷溶液,并继续搅拌5小时,反应结束后将反应液减压去除溶剂,并重新用乙酸乙酯溶解,并依次用水,稀盐酸和碳酸氢钠饱和溶液洗涤,最后用无水硫酸钠干燥并减压去除溶剂;
将的到的产物(T-OH)和NBS混合物溶于5mL二氯甲烷中,再加入DMTU,室温搅拌2小时,反应完毕混合溶液用二氯甲烷稀释,然后用水洗涤三次,接着将二氯甲烷用无水硫酸钠干燥,最后将溶剂减压旋干,并使用洗脱液通过硅胶快速色谱纯化,以获得黄色的中间体2;
其中,结构式CDI的结构式为
其中,中间体1的结构式为
其中,化合物Q的结构式为
其中,结构式T-OH的结构式为
5.根据权利要求4所述的一种特异性识别NQO-1的花菁类荧光探针的制备方法,其特征在于:所述步骤A2中,所述T-OH,NBS和DMTU混合物的摩尔比为1:1.5:0.45。
6.根据权利要求5所述的一种特异性识别NQO-1的花菁类荧光探针的制备方法,其特征在于:所述步骤S1和S3中的洗脱剂均为二氯甲烷/甲醇(10:1至5:1);
所述步骤A1中的洗脱剂为石油醚/乙酸乙酯(2:1);所述步骤A2中的洗脱剂为石油醚/乙酸乙酯(3:1)。
7.根据权利要求1所述的一种特异性识别NQO-1的花菁类荧光探针在制备检测、识别环境中或生物样品中NQO-1的检测试剂或者标记物中的应用。
8.根据权利要求7所述的一种特异性识别NQO-1的花菁类荧光探针的应用,其特征在于:所述NQO-1的花菁类荧光探针用于制备胰腺癌细胞的NQO-1的检测试剂或者标记物中的应用。
9.根据权利要求8所述的一种特异性识别NQO-1的花菁类荧光探针的应用,其特征在于:所述NQO-1的花菁类荧光探针测定NQO-1的检测方法如下:
通过荧光分光光度法,以650nm为激发波长,在710nm的波长处测定NQO-1的荧光强度,所述样品的检测限为0.01μg/mL。
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CN116891479A (zh) * | 2023-07-13 | 2023-10-17 | 中国药科大学 | 一种靶向mor的响应性半花菁荧光探针、其合成方法及应用 |
CN116891479B (zh) * | 2023-07-13 | 2024-04-19 | 中国药科大学 | 一种靶向mor的响应性半花菁荧光探针、其合成方法及应用 |
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