CN111675728B - 基于苯硒醚基团专一响应Cys的荧光探针、制备方法及应用 - Google Patents
基于苯硒醚基团专一响应Cys的荧光探针、制备方法及应用 Download PDFInfo
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Abstract
本发明提供了一种基于苯硒醚基团专一响应Cys的荧光探针、制备方法及应用。本探针选择BODIPY染料为荧光团，苯硒醚为识别团。检测机制是Cys同苯硒醚发生亲核取代‑重排反应，引起荧光信号变化。上述探针通过紫外和荧光光谱仪检测Cys且不受其他氨基酸和活性氧类的干扰，该检测过程简便、快速、灵敏，检测限为33 nM。更重要的是该探针可以检测细胞中Cys，在生物监测领域具有良好应用前景。
Description
技术领域
本发明涉及荧光探针，具体涉及一种基于苯硒醚基团专一响应Cys的荧光探针、制备方法及应用。
背景技术
生物硫化物对动植物和微观生命体代谢，食品加工等方面发挥着重要作用。小分子硫醇是生物硫化物的一部分其包括：半胱氨酸(Cys)，同型半胱氨酸(Hcy)和谷胱甘肽(GSH)。三种硫醇的浓度异常可能导致多种疾病，如肝脏损伤，心血管疾病，阿尔兹海默症，癌症等。在动物体内半胱氨酸(Cys)是一种蛋白质原性、半必需氨基酸，通过在折叠的蛋白质中提供二硫键来稳定蛋白质结构，增加蛋白质的刚性，调节蛋白质的功能。Cys合成的前体是Hcy，蛋氨酸转化为同型半胱氨酸，再与丝氨酸结合形成不对称的硫醚半胱氨酸Hcy结合丝氨酸在胱硫醚b-合酶(CBS)的作用下形成胱硫醚，之后在胱硫醚g-裂解酶(CSE)的作用下裂解成Cys。同时它还是GSH，牛磺酸和乙酰辅酶A合成的前体氨基酸。除此之外，Cys还可以从食物中获取，Cys存在于高蛋白食物中，包括肉类、鸡蛋、奶制品等。并且Cys已被粮农组织联合专家委员会(JECFA)指定为调味剂，因此，Cys的专一性检测非常重要。
但是，由于硫醇本身的结构相似性，都具有相同的活性位点巯基和胺基，特别是Cys和Hcy两者仅相差一个亚甲基的区别。所以实现三者区分仍是一个巨大的挑战。苯硒醚基团是易于离去的基团，容易受到亲核试剂的进攻，发生取代反应。而将苯硒醚作为检测团利用巯基的强亲核性对于活性硫化物报道非常少，特别是专一性识别Cys的探针还未曾报道。红色荧光探针因其长发射波长和强组织穿透性而众所周知。因此，开发基于苯硒醚为检测基团专一性响应动物细胞Cys的红色荧光探针具有重要意义。
发明内容
本发明提出了一种基于苯硒醚基团专一响应Cys的荧光探针、制备方法及应用，该探针制备方法操作方便，原料易得，能够实现细胞内Cys的专一性检测。
实现本发明的技术方案是：
一种基于苯硒醚基团专一响应Cys的荧光探针，探针的结构式为：
基于苯硒醚基团专一响应Cys的荧光探针的制备方法，步骤如下：
（1）N2保护冰水浴下，氢化钠加入到四氢呋喃溶剂中混合得到混合溶剂，将丙酮基对甲苯磺酸肟酯与对甲氧苯乙烯基甲基酮按照当量为1：2～3的比例溶于混合溶剂中，50℃反应4小时，冰水淬灭，二氯甲烷萃取，柱层析分离得到化合物1；
化合物1的结构式如下：
；
（2）步骤（1）制得的化合物1溶于四氢呋喃中，将N,N-二异丙基乙胺和三光气或硫光气按照当量为1：（1～1.5）的比例加入，25℃搅拌1-1.5h，然后减压去除溶剂，柱层析分离得到化合物2；
化合物2的结构式如下：
，
（3）将步骤（2）所得化合物2溶于二氯甲烷中，加入1.5当量的三氯氧磷反应过夜，之后加碱和三氟化硼乙醚按照当量为6：（8～10.5）加入上述体系中络合，25 ℃下络合4～6小时，柱层析分离得到化合物3；
化合物3的结构式如下：
，
（4）将二苯基二硒醚和四氢铝锂按照当量为1：2的比例溶于乙醇中还原，加入2～2.5当量的化合物3室温反应1～1.5小时，然后淬灭减压去除溶剂，残留物柱层析分离得到探针BDP-Se-3。
所述的制备方法制备的探针在专一检测Cys中的应用。
荧光探针的合成路线如下：
本发明基于苯硒醚作为响应基团用于专一检测Cys的荧光探针，该探针在溶液测试中，利用紫外和荧光光谱可判断出探针与Cys反应时间，浓度依赖性关系；在通过选择性和抗干扰能力测试观察出，探针可专一性检测Cys，不与活性氧化物反应且抗干扰能力强；并且探针的pH稳定性强，细胞毒性小。还可通过共聚焦荧光显微镜达到对HeLa细胞中的Cys检测的目的。
本发明的有益效果是：
（1）本发明一种利用苯硒醚作为响应基团专一性检测Cys的荧光探针，合成方法简单，操作方便；
（2）本发明的检测方法可以实现Cys专一性检测，而且不受其他氨基酸和活性氧化物类的干扰；
（3）本发明检测信号明显，为类近红外荧光增强型荧光探针。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为荧光探针BDP-Se-3的核磁氢谱图；
图2为荧光探针BDP-Se-3的核磁碳谱图；
图3为荧光探针BDP-Se-3与Cys作用的时间紫外变化；
图4为荧光探针BDP-Se-3与Cys作用的时间荧光变化；
图5为荧光探针BDP-Se-3测定Cys浓度滴定实验荧光变化；
图6为荧光最强发射波长610nm与Cys的浓度线性拟合图；
图7为常见氨基酸对探针BDP-Se-3检测Cys的荧光选择性；
图8为常见氨基酸对探针BDP-Se-3检测Cys的荧光干扰性；
图9为荧光探针BDP-Se-3和探针加Cys在不同pH缓冲溶液中最大荧光强度变化图；
图10为探针BDP-Se-3检测Cys的细胞毒性；
图11为荧光探针BDP-Se-3检测Cys HeLa细胞成像图。
具体实施方式
下面将结合本发明实施例，对本发明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付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。
实施例1
探针的制备步骤如下：
（1）化合物1的制备
N2保护冰水浴下，氢化钠加入到四氢呋喃溶剂中混合得到混合溶剂，将丙酮基对甲苯磺酸肟酯与对甲氧苯乙烯基甲基酮按照当量为1：2的比例溶于混合溶剂中，50℃反应4小时，冰水淬灭，二氯甲烷萃取，柱层析分离得到化合物1；
1H NMR (400 MHz, DMSO) δ 10.70 (s, 1H), 7.35 (d, J = 8.4 Hz, 2H),6.89 (d, J = 8.6 Hz, 2H), 6.76 (q, J = 16.5 Hz, 2H), 6.54 (s, 1H), 6.02 (s,1H), 3.75 (s, 3H), 2.01 (s, 3H)。
（2）化合物2的制备
将化合物1溶于四氢呋喃中，将N,N-二异丙基乙胺和三光气或硫光气按照当量为1：1的比例加入，25℃搅拌1h，然后减压去除溶剂，柱层析分离得到化合物2；
1H NMR (300 MHz, DMSO) δ 11.34 (s, 2H), 7.42 (d, J = 8.7 Hz, 5H),7.14 (d, J = 16.5 Hz, 3H), 7.00 – 6.89 (m, 7H), 6.34 (s, 2H), 3.77 (s, 6H),2.11 (s, 6H)。
（3）化合物3的制备
将化合物2溶于二氯甲烷，加入1.5当量的三氯氧磷反应过夜，之后加碱和三氟化硼乙醚按照当量为6：8加入上述体系中络合，25 ℃下络合4小时，柱层析分离得到化合物3；
1H NMR (400 MHz, CDCl3) δ 7.60 – 7.49 (m, 7H), 7.22 (s, 1H), 6.93 (d,J = 8.6 Hz, 4H), 6.71 (s, 2H), 3.85 (s, 6H), 2.52 (s, 6H)。
（4）BDP-Se-3的制备
将二苯基二硒醚和四氢铝锂按照当量为1：2的比例溶于乙醇中还原，加入2当量的化合物3室温反应1小时，然后淬灭减压去除溶剂，残留物柱层析分离得到探针BDP-Se-3。
1H NMR (400 MHz, CDCl3): δ7.62 (s, 1H), 7.59 (d, J = 8.6 Hz, 5H), 7.29(d, J = 7.5 Hz, 3H), 7.23 (s, 3H), 7.18 (d, J = 7.0 Hz, 1H), 6.94 (d, J = 8.7Hz, 14H), 6.71(s, 2H), 3.86 (s, 6H), 2.50 (s, 6H)。13C NMR (101 MHz, CDCl3) δ160.61, 152.70, 143.72, 136.78, 133.81, 129.82, 129.57, 129.22, 128.22,126.38, 119.07, 117.18, 114.34, 55.40, 18.20。
实施例2
探针的制备步骤如下：
（1）化合物1的制备
N2保护冰水浴下，氢化钠加入到四氢呋喃溶剂中混合得到混合溶剂，将丙酮基对甲苯磺酸肟酯与对甲氧苯乙烯基甲基酮按照当量为1：2.5的比例溶于混合溶剂中，50℃反应4小时，冰水淬灭，二氯甲烷萃取，柱层析分离得到化合物1；
（2）化合物2的制备
将化合物1溶于四氢呋喃中，将N,N-二异丙基乙胺和三光气或硫光气按照当量为1：1.3的比例加入，25℃搅拌1-1.5h，然后减压去除溶剂，柱层析分离得到化合物2；
（3）化合物3的制备
将化合物2溶于二氯甲烷，加入1.5当量的三氯氧磷反应过夜，之后加碱和三氟化硼乙醚按照当量为6：9加入上述体系中络合，25 ℃下络合5小时，柱层析分离得到化合物3；
（4）BDP-Se-3的制备
将二苯基二硒醚和四氢铝锂按照当量为1：2的比例溶于乙醇中还原，加入2.3当量的化合物3室温反应1.3小时，然后淬灭减压去除溶剂，残留物柱层析分离得到探针BDP-Se-3。
实施例3
探针的制备步骤如下：
（1）化合物1的制备
N2保护冰水浴下，氢化钠加入到四氢呋喃溶剂中混合得到混合溶剂，将丙酮基对甲苯磺酸肟酯与对甲氧苯乙烯基甲基酮按照当量为1：3的比例溶于混合溶剂中，50℃反应4小时，冰水淬灭，二氯甲烷萃取，柱层析分离得到化合物1；
（2）化合物2的制备
将化合物1溶于四氢呋喃中，将N,N-二异丙基乙胺和三光气或硫光气按照当量为1：1.5的比例加入，25℃搅拌1.5h，然后减压去除溶剂，柱层析分离得到化合物2；
（3）化合物3的制备
将化合物2溶于二氯甲烷，加入1.5当量的三氯氧磷反应过夜，之后加碱和三氟化硼乙醚按照当量为6：10.5加入上述体系中络合，25 ℃下络合6小时，柱层析分离得到化合物3；
（4）BDP-Se-3的制备
将二苯基二硒醚和四氢铝锂按照当量为1：2的比例溶于乙醇中还原，加入2.5当量的化合物3室温反应1.5小时，然后淬灭减压去除溶剂，残留物柱层析分离得到探针BDP-Se-3。
实施例4
溶液中检测Cys的荧光探针BDP-Se-3的反应时间测试：
用二甲基亚砜(DMSO)配制1 mM BDP-Se-3的荧光探针储备液；探针BDP-Se-3（10 µM）与Cys（100 µM）在溶液体系为CH3CN/PBS缓冲液(v/v＝1/1，pH＝7.4)中进行反应，如图3所示，从紫外吸收图谱观察BDP-Se-3在675 nm处的吸收峰伴随着时间的推移逐渐下降，并在10 min内下降到最低；而新生成的545 nm处紫外吸收峰逐渐增大至最高值，反应前后的吸收峰发生了130 nm的蓝移。同时，在荧光图谱上可观察到，在用525 nm的激发下，613 nm处出现一个新的荧光发射峰并增强约80倍(图4所示)。
实施例5
溶液中检测Cys的荧光探针BDP-Se-3的浓度滴定测试以及浓度线性关系：
在浓度滴定实验中，发现随着Cys的浓度逐渐增加，613 nm处的荧光峰也逐渐增强，其荧光强度在Cys浓度至100 µM时达到最。(见图5所示)。
以Cys浓度为横坐标，探针BDP-Se-3在613 nm处的荧光强度为纵坐标，绘制图并进行线性拟合，得到该探针的线性回归方程为：Y = 14.4 X + 3.89，线性相关系数R2 =0.999并计算出检测限为33 nM。(见图6所示)。
实施例6
干扰性和抗干扰性离子实验：
在不同的荧光比色皿中，分别加入4 mL CH3CN/PBS缓冲液(v/v＝1/1，pH＝7.4)和40μL荧光探针储备液，如图7所示，当探针BDP-Se-3分别加入上述选择的氨基酸，活性硫化物以及活性氧化物分析物种（100 µM）后（1: Cys; 2: Hcy; 3: GSH; 4: Ala; 5: Gln; 6:Ile; 7: Met; 8: Arg; 9: His; 10: Tyr; 11: Asp; 12: Glu; 13: Leu; 14: Gly; 15:Phe; 16: Lys; 17:Thr; 18: Ser; 19: Pro; 20: Try; 21: SO3 2ˉ; 22: S2O3 2ˉ; 23:S2O4 2ˉ; 24: S2O5 2ˉ; 25: HSO3ˉ; 26: NaHS; 27: NO·; 28: TBHP; 29: HOCl; 30:H2O2 ; 31: 1O2 ; 32: ONOO−），探针BDP-Se-3可以对Cys进行专一性识别，并在613 nm处出现明亮的红色荧光，而跟Hcy/GSH和其它种类分析物反应后没有荧光出现，因此，探针可以实现专一性响应Cys。当BDP-Se-3（10 µM）在分别加入上述分析物（0: blank; 1: Hcy; 2:GSH; 3: Ala; 4: Gln; 5: Ile; 6: Met; 7: Arg; 8: His; 9: Tyr; 10: Asp; 11:Glu; 12: Leu; 13: Gly; 14: Phe; 15: Lys; 16:Thr; 17: Ser; 18: Pro; 19: Try;20: SO3 2ˉ; 21: S2O3 2ˉ; 22: S2O4 2ˉ; 23: S2O5 2ˉ; 24: HSO3ˉ; 25: NaHS; 26: NO·; 27:TBHP; 28: H2O2 ; 29: 1O2）后再加入100 µM Cys反应10分钟后，可以看出Cys在与各种分析物共存情况下，BDP-Se-3在复杂的溶液体系内依然能够专一性检测到Cys，且荧光程度较强。实验证明，BDP-Se-3能够在响应Cys时不受其它物质的干扰(见图8所示)。
实施例7
pH值的响应性实验：
将探针BDP-Se-3溶于二甲基亚砜中得到10mM探针母液，配置pH为6.0、6.4、6.8、7.2、7.6、8.0的磷酸二氢钾和氢氧化钠缓冲溶液，对探针以及探针和Cys反应后荧光强度的变化进行了研究。
结果如图9所示，探针荧光强度在pH值为6.0‒8.0的溶液中基本保持不变；在加入Cys后，随着pH值的不断升高，其位于613 nm处荧光强度不断增强，且在生理范围7.2‒8.0的pH值下，荧光强度最强。实验证明探针BDP-Se-3能够适应生物体内的pH环境。
实施例8
MTT细胞毒性实验：
利用探针BDP-Se-3对HeLa细胞的MTT细胞毒性实验，结果如图10所示。HeLa细胞用含有不同浓度的探针（1.25, 2.5, 5.0, 10, 20 μM）培养液孵育后，计算细胞的存活百分比。结果如图9所示，低浓度下细胞存活率可高达90%，探针几乎没有细胞毒性。
实施例9
检测半胱氨酸的荧光探针对HeLa细胞内Cys的检测性能测试；
检测Cys的荧光探针BDP-Se-3对人体宫颈癌（HeLa）细胞中Cys的荧光共聚焦成像。结果如图11所示，(a)明场和(b)BDP-Se-3（10 μM）和HeLa细胞一起孵育30分钟后，再用PBS缓冲液洗涤三次后的显微镜下荧光成像；(d)明场和(e)HeLa细胞先用5 mM的NEM孵育40分钟，用PBS缓冲液洗涤三次，BDP-Se-3（10 μM）再与细胞共孵育30分钟；(c)和(f)为共聚焦荧光与明场的重叠。通过共聚焦红色通道观察，检测Cys的荧光探针在HeLa细胞中产生明亮的红色荧光；对照组实验中，将细胞先与NEM(N-乙基马来酰亚胺，生物硫醇掩蔽剂)共同孵育然后再与BDP-Se-3共同孵育，由成像实验结果可以观察红色通道荧光响应微弱。由此说明探针可以检测细胞内源性Cys。该结果也表明，本发明的荧光探针具有检测HeLa细胞内Cys的良好应用前景。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种基于苯硒醚基团专一响应Cys的荧光探针的制备方法，其特征在于，探针的结构式为：
；
制备步骤如下：
（1）N2保护冰水浴下，将丙酮基对甲苯磺酸肟酯与对甲氧苯乙烯基甲基酮溶于混合溶剂中，50℃反应4小时，冰水淬灭，二氯甲烷萃取，柱层析分离得到化合物1；
化合物1的结构式如下：
；
（2）步骤（1）制得的化合物1溶于四氢呋喃中，加入N,N-二异丙基乙胺和三光气，室温搅拌，然后减压去除溶剂，柱层析分离得到化合物2；
化合物2的结构式如下：
；
（3）将步骤（2）所得化合物2溶于二氯甲烷中，加入三氯氧磷反应过夜，之后加碱和三氟化硼乙醚络合，柱层析分离得到化合物3；
化合物3的结构式如下：
；
（4）将二苯基二硒醚和四氢铝锂溶于乙醇中还原，加入化合物3室温反应，然后淬灭减压去除溶剂，残留物柱层析分离得到探针BDP-Se-3。
2.根据权利要求1所述的基于苯硒醚基团专一响应Cys的荧光探针的制备方法，其特征在于：所述步骤（1）中混合溶剂为氢化钠加入到四氢呋喃溶剂中混合得到，丙酮基对甲苯磺酸肟酯与对甲氧苯乙烯基甲基酮按照当量为1：（2-3）的比例溶于混合溶剂中。
3.根据权利要求1所述的基于苯硒醚基团专一响应Cys的荧光探针的制备方法，其特征在于：所述步骤（2）中N,N-二异丙基乙胺和三光气按照当量为1：（1～1.5）比例添加，室温搅拌1-1.5h。
4.根据权利要求1所述的基于苯硒醚基团专一响应Cys的荧光探针的制备方法，其特征在于：所述步骤（3）中碱为N,N-二异丙基乙胺或三乙胺，碱与三氟化硼乙醚按照当量为6：（8～10.5）添加，室温络合4-6h。
5.根据权利要求1所述的基于苯硒醚基团专一响应Cys的荧光探针的制备方法，其特征在于：所述步骤（4）中二苯基二硒醚和四氢铝锂按照当量为1:2的比例混合溶解，加入2-2.5当量化合物3室温反应1-1.5h。
6.权利要求1-5任一项所述的制备方法制备的探针在制备专一检测Cys荧光探针中的应用。
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| A novel near-infrared styryl-BODIPY fluorescent probe for discrimination ofGSH and its application in living cells;Chengjun Wang et al.;《Dyes and Pigments》;20180203;第152卷;85-92 * |
| Development of Lysosome-Targeted Fluorescent Probes for Cys by Regulating the Boron-dipyrromethene (BODIPY) Molecular Structure;Jinhua Gao et al.;《Chemistry A European Journal》;20191231;第25卷;11246-11256 * |
| Meso-heteroaryl BODIPY dyes as dual-responsive fluorescent probes probesfor discrimination of Cys from Hcy and GSH;Jian Zhang et al.;《Sensors and Actuators B: Chemical》;20180103;第260卷;861-869 * |
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