影像科学与光化学  2018, Vol. 36 Issue (3): 236-244   PDF    
基于菁染料高选择性检测半胱氨酸的近红外荧光探针
马亚光, 张玉涛, 马一宇, 郭志前, 朱为宏     
华东理工大学 精细化工研究所, 上海 200237
摘要: 本文设计合成了以菁染料为荧光团,以4-(三氟甲基)苯硫基为半胱氨酸响应识别基团的近红外荧光探针(Cy-CF3)。利用探针分子Cy-CF3与半胱氨酸和谷胱甘肽反应发生的机理不同,实现了对半胱氨酸特异性识别。探针分子Cy-CF3与半胱氨酸发生芳香亲核取代反应生成巯基取代产物,进一步通过分子内重排反应生成氨基取代产物Cy-Cys。光谱研究结果表明,探针分子Cy-CF3与半胱氨酸作用后发生明显的吸收波长蓝移(160 nm),并且可观察到明显的颜色变化;荧光光谱中,随着半胱氨酸的加入,探针分子Cy-CF3在780 nm处的近红外荧光显著增强。Cy-CF3能高选择性检测半胱氨酸,并且不受其它氨基酸尤其是结构类似的谷胱甘肽干扰。探针分子Cy-CF3被成功地应用于活体细胞中检测半胱氨酸。
关键词: 近红外     半胱氨酸     荧光探针     菁染料    
Near-infrared Cyanine-based Fluorescent Probe for Cysteine with High Selectivity
MA Yaguang, ZHANG Yutao, MA Yiyu, GUO Zhiqian, ZHU Weihong     
Institute of Fine Chemicals, East China University of Science & Technology, Shanghai 200237, P. R. China
*Corresponding author: GUO Zhiqian, E-mail: guozq@ecust.edu.cn
Abstract: We developed a turn-on cyanine-based near-infrared (NIR) probe which can selectively detect cysteine. In this probe Cy-CF3, 4-(trifluoromethyl)thiophenol moiety is employed as both the recognition unit and fluorescence quenching group. With the titration of cysteine, a large blue shift (160 nm) is observed in the absorption spectra of Cy-CF3, accompanying with significant color changes. The probe Cy-CF3 shows obviously fluorescent enhancement at 780 nm in emission spectra. Over all, the probe Cy-CF3 exhibits high selectivity for cysteine over glutathione and other potential interferences. The reaction mechanism of Cy-CF3 with cysteine is verified by mass spectra, 1HNMR spectra and absorption spectra, indicating that 4-(trifluoromethyl)thiophenol in the probe Cy-CF3 could be replaced by cysteine, and then the PET process from 4-(trifluoromethyl)thiophenol to cyanine is blocked. The cell experiments also demonstrate that this probe could be successfully applied for the detection of cysteine in living cells.
Key words: near-infrared     cysteine     fluorescent probe     cyanine    

细胞内的生物小分子硫醇,如半胱氨酸(Cysteine,Cys)、高半胱氨酸(Homocysteine,Hcy)和还原性谷胱甘肽(Glutathione,GSH)在许多生理和病理过程中起着关键作用[1, 2]。研究表明,异常浓度水平的硫醇与发育迟缓、肝损伤以及皮肤病变等病症密切相关。半胱氨酸作为谷胱甘肽的前体氨基酸,可通过食物摄取或高半胱氨酸代谢途径产生,多种严重病症与半胱氨酸缺乏相关,例如发育迟缓、头发脱色、嗜眠和肝损伤等[3, 4]。尽管半胱氨酸和谷胱甘肽具有相类似的结构,但是鉴于它们在生命系统中扮演着各自重要的角色,其生理作用影响着不同的病症[5-7],因此,发展特异性检测生物样品中半胱氨酸的荧光探针,对于深入理解和研究半胱氨酸的生理功能,实现相关病症的早期诊断具有重要意义。

传统检测生物小分子硫醇的荧光探针主要基于巯基的强亲核性,通过与荧光团之间特异性的化学反应,如迈克尔加成反应[8, 9]、硫硫键断裂反应[10, 11]、磺酰胺或磺酸酯键的裂解反应[12, 13]等,最终实现荧光变化检测小分子硫醇。近年来,基于上述化学反应已开发了大量检测生物小分子硫醇的荧光探针[14-28],但由于半胱氨酸、高半胱氨酸和谷胱甘肽具有相似的化学结构和反应活性,发展特异性检测区分半胱氨酸和谷光甘肽的荧光探针仍然具有很大的挑战性。特别是相对于细胞中含量丰富且结构相似的谷胱甘肽,半胱氨酸具有含量低、更难于检测的特点。最近,杨清正等[26]设计合成了基于氯代衍生物氟化硼二吡咯甲川(BODIPY)的比率型荧光传感器,基于芳香亲核取代-分子内重排检测机理,实现了对谷胱甘肽和半胱氨酸的选择性检测。赵春常等[29]设计合成了新型不对称BODIPY荧光探针CN-B-Cl,实现了选择性检测谷胱甘肽和半胱氨酸。但是,这些已报道的探针受制于其荧光发射波长较短,不利于生物活体成像应用。因此,发展高灵敏、特异性的近红外半胱氨酸荧光探针仍具有重要意义。

近红外荧光染料(发射波长650~900 nm)对生物组织损害小、组织穿透能力强、能有效消除组织荧光背景干扰,因而在生物荧光成像方面具有独特的优势[30, 31]。目前,文献已报道的生物小分子硫醇荧光探针主要基于荧光素[32, 33]、香豆素[34-36]、BODIPY[37, 38]等荧光团。本文选择菁染料IR-739作为荧光团母体构建近红外荧光探针,主要考虑七甲川菁染料作为典型的近红外发色团,光学性能优异[39, 40],并且其甲川链中位的氯原子容易被强亲核试剂取代,如酚羟基和硫醇等。设计合成了新型近红外荧光探针Cy-CF3,引入4-(三氟甲基)苯硫酚单元,使其与菁染料单元之间产生光致电子转移过程(PET),导致近红外荧光猝灭;含有强吸电子基团的苯硫酚单元在亲核试剂进攻时作为易离去基团。探针分子Cy-CF3实现了选择性区分检测Cys和GSH,其响应特点包括:显著的近红外荧光增强响应(780 nm)和大Stokes位移(100 nm)变化。

1 实验部分 1.1 试剂与仪器

核磁共振氢谱和碳谱(1HNMR和13CNMR)采用Brucker Avance 400超导傅里叶变换核磁共振波谱仪(400 MHz),选用CDCl3作为氘代溶剂进行测定(以TMS为内标,温度不加说明均为293 K);高分辨质谱(HRMS)采用Waters LCT Premier XE质谱仪,以MeOH或CH2Cl2作为溶剂进行测试;紫外-可见光吸收光谱采用Varian Cary 100紫外-可见分光光度计进行测定;荧光发射光谱采用Varian Cary Eclipse荧光光谱仪进行测定;共聚焦荧光图像采用Nikon A1R型共聚焦显微镜扫描测试。

合成过程中涉及的药品和国产分析纯级别试剂均为购买后直接使用未经进一步纯化,除非实验步骤中另有说明。紫外-可见吸收光谱和荧光发射光谱均采用V(DMSO):V(PBS)= 50:50的缓冲液(pH=7.4,10 mmol/L)作为测试体系, 在37 ℃下进行测试。

细胞培养及共聚焦成像:人宫颈癌细胞(HeLa)由华东理工大学生物工程学院提供。HeLa细胞在含有10%胎牛血清、50 unit/mL青霉素和50 μg/mL链霉素的DMEM培养液中,37 ℃和5% CO2条件下孵育生长。在进行细胞成像实验之前,将细胞随机分为两组,分别加入半胱氨酸和谷胱甘肽孵育10 min后,洗去细胞外游离的半胱氨酸和谷胱甘肽,再加入探针Cy-CF3继续共孵育1 h,用磷酸盐缓冲溶液(PBS)洗3遍。采用Nikon A1R/A1激光共聚焦显微镜(60倍油镜)对两组细胞分别进行二维扫描。激光器激发波长638 nm,荧光收集范围720~760 nm。

1.2 探针Cy-CF3的合成

图 1给出了荧光探针Cy-CF3的合成过程。首先通过3-乙基-1, 1, 2-三甲基-1H-苯并[e]吲哚碘盐与2-氯-1-甲酰基-3-羟甲基环己烯合成氯取代的七甲川菁染料IR-739,再通过IR-739与4-(三氟甲基)苯硫酚之间发生亲核反应生成目标化合物Cy-CF3

图 1 荧光探针Cy-CF3的合成 Fig.1 Synthesis of fluorescent sensor Cy-CF3
1.2.1 化合物IR-739的合成

在100 mL单口瓶中加入3-乙基-1, 1, 2-三甲基-1H-苯并[e]吲哚碘盐(5.30 g, 14.5 mmol)、2-氯-1-甲酰基-3-羟甲基环己烯(1.34 g, 7.2 mmol)和无水乙酸钠(1.19 g, 14.5 mmol),溶于乙酸酐(30 mL)中。油浴100 ℃,氩气保护下回流反应1.5 h,反应过程中固体逐渐溶解,溶液逐渐变为绿色,利用薄板层析监测判断反应结束。随后减压蒸馏除去乙酸酐,得红色固体产物,再加入5 mL二氯甲烷溶解,并将二氯甲烷溶液滴入1000 mL石油醚中,析出固体,抽滤得到红色固体,用无水甲醇进行重结晶,得到橘红色晶体4.6 g,产率85.0%。

1HNMR (400 MHz, CDCl3): 1.53 (t, J=7.2 Hz, 6H, —CH2CH3), 2.03(s, 12H, —CH3), 2.13~2.07 (m, 2H, —CH2—), 2.79 (t, J= 6.0 Hz, 4H, —CH2—), 4.46~4.33 (m, 4H, —CH2CH3), 6.28 (d, J=14.0 Hz, 2H, alkene-H), 7.53~7.43 (m, 4H, Ph-H), 7.60 (t, J=7.6 Hz, 2H, Ph-H), 8.00~7.91 (m, 4H, Ph-H), 8.13 (d, J=8.4 Hz, 2H, Ph-H), 8.45 (d, J=14.0 Hz, 2H, alkene-H)。13CNMR (100 MHz, CDCl3):12.80, 20.77, 26.82, 27.61, 40.32, 51.14, 100.59, 122.06, 125.13, 127.32, 127.78, 128.16, 130.19, 130.89, 131.96, 133.95, 139.22, 143.45, 149.90, 173.19。HRMS(ESI-MS):calcd for [C42H44ClN2]+ 611.3188, found 611.3179。

1.2.2 化合物Cy-CF3的合成

在50 mL单口瓶中,将IR-739(50 mg, 0.07 mmol)溶于10 mL DMF中,并在冰浴下冷却搅拌。再将4-(三氟甲基)苯硫酚(15.0 mg, 0.08 mmol)溶于5 mL N, N-二甲基甲酰胺(DMF)中,并用恒压滴液漏斗缓慢滴加到上述反应溶液中,待溶液滴加完毕后油浴加热至70 ℃,反应12 h。直接减压蒸馏除去反应溶剂DMF,并加入3 mL二氯甲烷溶解,滴入剧烈搅拌的正己烷(500 mL)中,观察到有絮状沉淀立即析出,再抽滤干燥得绿色固体40.1 mg,产率65.0%。

1HNMR (400 MHz, CDCl3):1.51 (t, J= 7.2 Hz, 6H, —CH2CH3), 1.76 (s, 12H, —CH3), 2.15~2.07 (m, 2H, —CH2—), 2.90 (t, J=6.8 Hz, 4H, —CH2—), 4.43~4.40 (m, 4H, —CH2CH3), 6.40 (d, J=14.0 Hz, 2H, alkene-H), 7.49 (d, J= 8.0 Hz, 2H, Ph-H), 7.48~7.41 (m, 4H, Ph-H), 7.93 (d, J=8.8 Hz, 4H, Ph-H), 8.04 (d, J= 8.8 Hz, 2H, Ph-H), 8.69 (d, J= 14.0 Hz, 2H, alkene-H)。13CNMR (100 MHz, CDCl3):12.84, 26.83, 27.29, 40.18, 50.95, 101.38, 110.61, 122.03, 125.03, 125.10, 126.27, 127.68, 128.10, 130.16, 130.82, 131.94, 134.08, 134.16, 139.20, 144.45, 173.12。19FNMR(376 MHz, CDCl3):―62.51。HRMS(ESI-MS):calcd for [C49H48F3N2S]+ 735.3485, found 735.3485。

2 结果与讨论 2.1 设计与合成

本文设计的两步反应操作简单(见图 1),合成产率较高(分别为85%和65%)。中间体IR-739和目标化合物Cy-CF3均通过1HNMR、13CNMR、HRMS表征,证明其化学结构的正确性。值得注意的是, 该类菁染料衍生物IR-739和Cy-CF3中甲川链的双键均为反式结构,其烯氢的耦合常数均为14.0 Hz,由于受连接基团以及电子共轭体系的影响,甲川链上两组烯氢之间的化学位移差异较大。

2.2 探针Cy-CF3对半胱氨酸的紫外-可见和荧光滴定光谱

图 2A为探针Cy-CF3在DMSO:PBS=50:50 (V:V, 10 mmol/L, pH = 7.4, 37 ℃)的缓冲溶液中,加入不同当量的Cys进行紫外吸收光谱滴定实验。在未加入Cys时,探针Cy-CF3的最大吸收峰在845 nm,随着加入半胱氨酸的浓度增加,其初始845 nm处的吸收强度不断降低并在685 nm处出现一个新的吸收峰(图 2A),最大吸收波长蓝移约160 nm。如图 2C,在685 nm波长激发下,未加入Cys时,探针Cy-CF3初始荧光强度很弱,这是由于苯硫酚与菁染料单元之间发生PET过程猝灭了荧光。当加入Cys后,探针Cy-CF3在780 nm处的近红外荧光显著增强,表明Cys的巯基通过硫酯交换取代,有效阻断了4-(三氟甲基)苯硫酚诱导的PET过程(图 2C)。图 2B2D是荧光探针Cy-CF3在加入Cys(10 μmol/L)后吸收光谱和荧光谱图随时间变化图。可以看出,Cy-CF3在827 nm处的吸光强度随着时间延长逐渐降低,但685 nm处的吸光强度随时间延长逐渐增强,并在反应30 min后达到平衡,其后保持不变。相似的,在荧光光谱中,探针Cy-CF3在780 nm处荧光强度随着时间延长明显增强,并在30 min后基本保持不变。

图 2 探针Cy-CF3(10 μmol/L)滴定不同浓度的半胱氨酸(0、2、4、8、12、16、24、32、40、80、120、160、200 μmol/L) 20 min后紫外吸收光谱(A)和荧光光谱(C)。加入半胱氨酸(10 μmol/L)后,探针Cy-CF3(10 μmol/L)吸收光谱 在685 nm和827 nm处吸光度(B)和荧光光谱在780 nm处荧光强度(D)时间变化曲线图(激发波长685 nm)溶液体系为DMSO:PBS=50:50 (V:V, 10 mmol/L, pH = 7.4, 37 ℃) Fig.2 Absorption (A) and fluorescence (C) spectra of Cy-CF3 (10 μmol/L) with the titration of Cys (0, 0, 2, 4, 8, 12, 16, 24, 32, 40, 80, 120, 160, 200 μmol/L) for 20 min. Time dependence of absorbance at 685 and 827 nm (B) and fluorescent intensity at 780 nm (D) of CY-CF3 in the presence of Cys (10 μmol/L) All mixed solutions are DMSO:PBS=50:50 (V:V, 10 mmol/L, pH = 7.4, 37 ℃) and λex=685 nm
2.3 探针Cy-CF3的识别机理研究

根据上述吸收光谱和荧光光谱的测试数据,并结合之前已有的相关研究工作,我们提出了Cy-CF3与Cys的反应机理(图 3)。当pH=7.4时,由于Cys中巯基具有强的亲核性,其与探针Cy-CF3中含强吸电子基团的氟代苯硫酚单元之间发生亲核取代反应,首先生成中间体化合物1,进一步通过五元环过渡态发生分子内取代反应,生成氨基取代化合物Cy-Cys。相比之下,GSH由于分子构型较大,其位阻效应严重阻碍了亲核硫取代反应发生,使进一步发生分子取代反应更加困难。

图 3 探针Cy-CF3与半胱氨酸的响应机理示意图 Fig.3 Proposed mechanism of Cy-CF3 with cysteine

利用质谱分析Cy-CF3与Cys的反应机理:图 4A是未加Cys的Cy-CF3质谱图,荷质比(m/z)为735.35的峰对应Cy-CF3的分子离子峰[C49H48F3N2S]+,而在Cy-CF3与Cys反应的质谱图(图 4B)中,除了观察到Cy-CF3相应的分子离子峰,m/z=696.36的峰与亲核取代反应后的产物Cy-Cys分子离子峰[C45H50N3O2S]+对应。质谱结果分析进一步证实了Cy-CF3选择性检测半胱氨酸的机理。

图 4 探针Cy-CF3未加入半胱氨酸(A)和加入半胱氨酸后(B)的质谱图 Fig.4 Mass spectra of Cy-CF3 in the absence (A) and (B) presence of Cys
2.4 荧光探针Cy-CF3的选择性

以生物体内常见的氨基酸作为干扰物,考察探针Cy-CF3的抗干扰能力。在Cy-CF3(10 μmol/L)的DMSO/PBS(pH=7.4, 50/50, V/V,10 mmol/L)缓冲液中,分别加入50 μmol/L的精氨酸(Arg)、丙氨酸(Ala)、天冬氨酸(Asp)、天冬酰胺(Asn)、谷氨酸(Glu)、谷胱甘肽(GSH)、异亮氨酸(Ile)、亮氨酸(Leu)、苯丙氨酸(Phe)、脯氨酸(Pro)、丝氨酸(Ser)、苏氨酸(Thr)、酪氨酸(Tyr)。选择性测试结果如图 5所示,探针Cy-CF3只有在与Cys作用时表现出明显的荧光增强,而在相同测试条件下,其它被检测的氨基酸包括GSH对探针Cy-CF3的荧光光谱基本无影响。由此证明探针Cy-CF3对半胱氨酸具有专一的识别能力。

图 5 探针Cy-CF3(10 μmol/L)加入5当量(50 μmol/L)各种氨基酸的荧光强度比值(780 nm),溶液体系为DMSO:PBS=50:50 (V:V, 10 mmol/L, pH=7.4, 37 ℃) Fig.5 Fluorescence responses (780 nm) of Cy-CF3 (10 μmol/L) toward various amino acids (50 μmol/L) in DMSO:PBS=50:50 (V:V, 10 mmol/L, pH=7.4, 37 ℃) solution
2.5 细胞成像实验

通过细胞成像实验进一步验证荧光探针Cy-CF3可以在细胞内高选择性检测半胱氨酸。采用HeLa细胞作为模型,首先分别加入半胱氨酸(10 μmol/L)和谷胱甘肽(10 μmol/L)孵育10 min,在洗去细胞外游离的半胱氨酸和谷胱甘肽后,再加入探针Cy-CF3(10 μmol/L)继续共孵育1 h。荧光成像图如图 6所示,加入半胱氨酸孵育的HeLa细胞观察到明显的荧光信号(图 6A),加入谷胱甘肽的HeLa细胞并未观察到荧光信号(图 6B),说明探针Cy-CF3在细胞中的荧光变化与光谱测试结果一致,表现出对半胱氨酸专一性的识别响应。

图 6 HeLa细胞加入半胱氨酸(10 μmol/L)(A1~A3)和谷胱甘肽(10 μmol/L)(B1~B3)孵育10 min后,再加入探针Cy-CF3(10 μmol/L)共孵育1 h的细胞共聚焦荧光成像图 Fig.6 Fluorescence images of HeLa cells incubated with Cys (10 μmol/L) (A1-A3) and GSH (10 μmol/L) (B1-B3) for 10 min and then Cy-CF3 (10 μmol/L) for 1 h, respectively
3 结论

本文设计并合成了基于七甲川菁染料的高选择性检测半胱氨酸的近红外荧光探针Cy-CF3。探针分子Cy-CF3中的4-(三氟甲基)苯硫酚基团与半胱氨酸发生芳香亲核取代反应,先生成巯基取代产物中间体,进一步通过分子内重排反应生成氨基取代产物Cy-Cys。由于取代反应发生过程中PET被阻断,从而使其荧光显著增强。探针Cy-CF3检测半胱氨酸过程中表现出专一性、高灵敏度、抗干扰的优点。通过实验进一步验证了Cy-CF3可用于细胞中实现对Cys的特异性检测。此外,探针Cy-CF3在加入半胱氨酸前后表现出从绿色到红色的显著颜色变化,可直接用肉眼观察到,亦十分值得关注。总之,本文发展的荧光探针分子有望应用于活体组织内选择性测试半胱氨酸。

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