CN112745340B - 基于bodipy染料靶向溶酶体选择性检测h2s的荧光探针、制备及应用 - Google Patents
基于bodipy染料靶向溶酶体选择性检测h2s的荧光探针、制备及应用 Download PDFInfo
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Abstract
本发明提供了一种基于BODIPY荧光染料靶向溶酶体选择性响应H2S的荧光探针、制备及应用,本探针选择BODIPY染料为荧光团,2,4‑二硝基苯磺酰基为识别团。传感机制是基于H2S诱导探针中2,4‑二硝基苯磺酰基裂解,从而抑制光诱导电子转移(PET)过程,导致探针荧光恢复。上述探针通过紫外和荧光光谱仪检测H2S且不受其他氨基酸、活性硫化物和代表性阴离子等的干扰,该检测过程简便、快速、灵敏,检测限为4.75 nM。更重要的是该探针可以靶向溶酶体并同时检测细胞中的H2S,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领域,具体涉及一种基于BODIPY荧光染料靶向溶酶体选择性响应H2S的荧光探针、制备及应用。
背景技术
硫化氢(H2S)因被认为是生命***中继一氧化碳(CO)与一氧化氮(NO)后的第三种重要气体信号分子,而受到研究者们极大的关注。近年来,越来越多的研究结果表明,内源性产生的硫化氢在广泛的生理和病理过程中起着重要作用。另一方面,H2S含量的异常与许多严重疾病密切相关,如阿尔茨海默病、唐氏综合症、糖尿病和肝硬化等,尽管有这些发现,但是H2S的潜在分子属性仍然不清楚。细胞溶酶体是一种重要的动态性亚细胞器,通过参与细胞内吞作用、分泌作用、自噬过程、及吞噬膜转运过程,承担着细胞内接受或降解诸如蛋白质、DNA、RNA等大分子的职能。溶酶体参与许多生理过程,如骨组织重塑、质膜修复和胆固醇体内平衡,以及细胞死亡和细胞信号传导等。因此,开发能够用于检测活细胞溶酶体中H2S的有效分子工具,可以帮助我们更好地理解H2S的生物学作用。
在发展用于检测H2S的各种方法中,荧光探针法具有很大的优越性,包括高灵敏度、可实现实时生物成像和操作简便性等。过去几年中,已报道过多种识别H2S的荧光探针,虽然取得了很大进展,但大多数已开发的探针或多或少存在一些局限性,例如检测荧光波长短(<500 nm),和响应时间长(>20 min)等。此外,许多探针受到生物硫醇干扰的影响,通常来自半胱氨酸(Cys),同型半胱氨酸(Hcy)和谷胱甘肽(GSH),因为这些生物硫醇不仅表现出与H2S相似的化学性质(例如,它们都可以通过巯基进行亲核反应),且它们也与H2S共存,还在生命***中含量更丰富。为了克服这些局限,非常需要开发一种能够快速响应,高选择性和高灵敏度的荧光探针用于H2S的检测。
发明内容
本发明提出了一种基于BODIPY荧光染料靶向溶酶体选择性响应H2S的荧光探针、制备及应用,该探针制备方法操作方便,原料易得,能够实现细胞内外源性和内源性H2S的检测,并且探针具有溶酶体定位作用。
实现本发明的技术方案是:
所述的荧光探针的制备方法,步骤如下:
(1)N2保护冰水浴下,将1~1.5当量三苯基膦加入到四氢呋喃溶剂中得到混合溶剂,将2-吗啉乙醇与对羟基苯甲醛按照当量为1:(1~1.5)的比例溶于混合溶剂中,再加入1~1.5当量的偶氮二甲酸二异丙酯,室温反应12~18 h,减压除去溶剂,柱层析分离得到化合物1;
(2)步骤(1)制得的化合物1溶于四氢呋喃中,加入(2~3)当量的2,4-二甲基吡咯,加入0.1~0.2 mL三氟乙酸,室温反应10~15 h,然后再加入(1~1.5)当量的四氯苯醌,室温反应0.5~1 h,三乙胺和三氟化硼***按照当量(5~7):(8~10)的比例添加到上述混合溶液,室温反应6~12 h,二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,柱层析分离得到化合物2;
(3)将步骤(2)所得化合物2溶于甲苯中,加入1~2当量的对羟基苯甲醛,再加入5~6当量的哌啶、8~10当量的乙酸,110 °C回流6~12 h,柱层析分离得到化合物3;
(4)将化合物3溶于二氯甲烷中,然后加入2~4当量的2,4-二硝基苯磺酰氯和6~10当量的三乙胺,45 ℃回流2~3 h,柱层析分离得到探针BDP-DNBS。
荧光探针的合成路线如下:
本发明基于BODIPY染料靶向溶酶体选择性检测H2S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H2S反应时间及浓度依赖性关系;在通过选择性和抗干扰能力测试观察出,探针可选择性检测H2S,不受其他氨基酸干扰,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细胞中溶酶体靶向并进行H2S检测的目的。
本发明的有益效果是:
(1)本发明一种利用2,4-二硝基苯磺酰基作为响应基团选择性检测H2S,吗啉基进行溶酶体定位的荧光探针,合成方法简单,操作方便;
(2)本发明的检测方法可以实现H2S选择性性检测,而且不受其他氨基酸的干扰,该检测过程简便、快速、灵敏,检测限为4.75 nM;
(3)本发明检测信号明显,可以靶向溶酶体并同时检测细胞中的H2S,为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-DNBS的核磁氢谱图;
图2为荧光探针BDP-DNBS的核磁碳谱图;
图3为荧光探针BDP-DNBS与H2S作用的时间紫外变化;
图4为荧光探针BDP-DNBS与H2S作用的时间荧光变化;
图5为荧光探针BDP-DNBS测定H2S浓度滴定实验荧光变化;
图6为荧光最强发射波长587 nm与H2S的浓度线性拟合图;
图7为常见氨基酸对探针BDP-DNBS检测H2S的荧光选择性;
图8为常见氨基酸对探针BDP-DNBS检测H2S的荧光抗干扰性;
图9为荧光探针BDP-DNBS和探针加H2S在不同pH缓冲溶液中最大荧光强度变化图;
图10为探针BDP-DNBS检测H2S的细胞毒性;
图11为荧光探针BDP-DNBS对HeLa细胞内H2S检测的细胞成像图;
图12为荧光探针BDP-DNBS 对HeLa细胞溶酶体定位图。
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,将1.1当量的三苯基膦加入到四氢呋喃溶剂中得到混合溶剂,将2-吗啉乙醇与对羟基苯甲醛按照当量为1:1.2的比例溶于混合溶剂中,再加入1.1当量的偶氮二甲酸二异丙酯,室温反应15 h,减压除去溶剂,柱层析分离得到化合物1;
1H NMR (400 MHz, CDCl3) δ 9.77 (s, 1H), 7.72 (d, J = 8.7 Hz, 2H), 6.92(d, J = 8.7 Hz, 2H), 4.10 (t, J = 5.7 Hz, 2H), 3.69 – 3.54 (m, 4H), 2.73 (t,J = 5.7 Hz, 2H), 2.54 – 2.40 (m, 4H).
(2)化合物2的制备
将化合物1溶于四氢呋喃中,加入2.2当量的2,4-二甲基吡咯,加入0.2 mL三氟乙酸,室温反应12 h,然后再加入1.1当量的四氯苯醌,室温反应1 h,按照当量为6:8比例添加三乙胺和三氟化硼***,室温反应6 h,二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,柱层析分离得到化合物2;
1H NMR (400 MHz, CDCl3) δ 7.16 (d, J = 8.6 Hz, 1H), 7.01 (d, J = 8.6Hz, 1H), 5.97 (s, 1H), 4.16 (t, J = 5.7 Hz, 1H), 3.80 – 3.72 (m, 2H), 2.85(t, J = 5.7 Hz, 1H), 2.66 – 2.58 (m, 2H), 2.55 (s, 3H), 1.42 (s, 3H).
(3)化合物3的制备
将化合物2溶于甲苯中,加入1.2当量的对羟基苯甲醛,再加入5当量的哌啶、8当量的乙酸,110 °C回流6 h,柱层析分离得到化合物3;
1H NMR (300 MHz, CDCl3) δ 7.49 (dd, J = 18.1, 12.4 Hz, 3H), 7.24 –7.11 (m, 3H), 6.99 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 6.57 (s,1H), 5.99 (s, 1H), 4.18 (t, J = 5.6 Hz, 2H), 3.90 – 3.71 (m, 4H), 2.87 (t, J= 5.5 Hz, 2H), 2.64 (dd, J = 15.5, 11.1 Hz, 7H), 1.45 (d, J = 10.4 Hz, 6H).
(4)探针BDP-DNBS的制备
将化合物3溶于二氯甲烷中,然后加入2.5当量的2,4-二硝基苯磺酰氯和6当量的三乙胺,45 ℃回流2~3 h,柱层析分离得到探针BDP-DNBS。
1H NMR (300 MHz, CDCl3) δ 10.04 (s, 1H), 8.71 (dd, J = 5.5, 2.1 Hz,1H), 8.60 – 8.48 (m, 1H), 8.23 (dd, J = 27.0, 8.6 Hz, 1H), 7.98 (d, J = 8.6Hz, 1H), 7.71 – 7.51 (m, 3H), 7.46 (d, J = 8.6 Hz, 1H), 7.24 – 7.16 (m, 3H),7.10 (d, J = 8.5 Hz, 2H), 6.60 (s, 1H), 6.10 (s, 1H), 4.26 (t, J = 5.6 Hz,2H), 3.95 – 3.74 (m, 4H), 2.93 (t, J = 5.5 Hz, 2H), 2.68 (dd, J = 15.8, 11.3Hz, 7H), 1.53 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 159.46, 156.85, 148.91,148.54, 144.27, 142.12, 141.21, 136.79, 135.68, 134.15, 133.85, 133.28,132.85, 131.75, 129.34, 128.83, 126.97, 126.58, 122.83, 122.32, 121.99,120.34, 117.35, 115.32, 66.92, 57.71, 54.24, 14.78, 14.19.
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,将1当量的三苯基膦加入到四氢呋喃溶剂中得到混合溶剂,将2-吗啉乙醇与对羟基苯甲醛按照当量为1:1的比例溶于混合溶剂中,再加入1.5当量的偶氮二甲酸二异丙酯,室温反应12 h,减压除去溶剂,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,加入2当量的2,4-二甲基吡咯,加入0.1 mL三氟乙酸,室温反应15 h,然后再加入1.5当量的四氯苯醌,室温反应0.5 h,按照当量5:8比例添加三乙胺和三氟化硼***,室温反应10 h,二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,柱层析分离得到化合物2;
(3)化合物3的制备
将化合物2溶于甲苯中,加入1当量的对羟基苯甲醛,再加入5.5当量的哌啶、9当量的乙酸,110 °C回流12 h,柱层析分离得到化合物3;
(4)探针BDP-DNBS的制备
将化合物3溶于二氯甲烷中,然后加入4当量的2,4-二硝基苯磺酰氯和8当量的三乙胺,45 ℃回流3 h,柱层析分离得到探针BDP-DNBS。
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,将1.5当量的三苯基膦加入到四氢呋喃溶剂中得到混合溶剂,将2-吗啉乙醇与对羟基苯甲醛按照当量为1:1.5的比例溶于混合溶剂中,再加入1当量的偶氮二甲酸二异丙酯,室温反应18 h,减压除去溶剂,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,加入3当量的2,4-二甲基吡咯,加入0.2 mL三氟乙酸,室温反应12 h,然后再加入1.1当量的四氯苯醌,室温反应0.8 h,按照7:9比例添加三乙胺和三氟化硼***,室温反应12 h,二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,柱层析分离得到化合物2;
(3)化合物3的制备
将化合物2溶于甲苯中,加入2当量的对羟基苯甲醛,再加入5当量的哌啶、10当量的乙酸,110 °C回流6 h,柱层析分离得到化合物3;
(4)探针BDP-DNBS的制备
将化合物3溶于二氯甲烷中,然后加入3当量的2,4-二硝基苯磺酰氯和10当量的三乙胺,45 ℃回流3 h,柱层析分离得到探针BDP-DNBS。
实施例4
溶液中检测H2S的荧光探针BDP-DNBS的反应时间测试:
用色谱乙腈(CH3CN)配制0.5 mM BDP-DNBS的荧光探针储备液;探针BDP-DNBS(5 µM)与H2S(50 µM)在溶液体系为CH3CN/PBS缓冲液(v/v=4/6,pH=7.4)中进行反应,如图3所示,从紫外吸收图谱观察BDP-DNBS在555 nm处的吸收峰伴随着时间的推移逐渐下降右移到566 nm,1 min内反应完全。同时,在荧光图谱上可观察到,在用465 nm的激发下,587 nm处出现一个新的荧光发射峰并增强约42倍(图4所示)。
实施例5
溶液中检测H2S的荧光探针BDP-DNBS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H2S的浓度逐渐增加,587 nm处的荧光峰也逐渐增强,其荧光强度在H2S浓度至30 µM时达到最大,30-50 µM荧光强度不再增加。(见图5所示)。
以H2S浓度为横坐标,探针BDP-DNBS在587 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 = 11.8 X – 10.7,线性相关系数R2 =0.994并计算出检测限为4.75 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=4/6,pH=7.4)和40 μL荧光探针储备液,如图7所示,当探针BDP-DNBS分别加入上述选择的氨基酸、活性硫分析物种(50 µM)后(1. H2S; 2. Cys; 3. Hcy; 4. GSH; 5. Glu; 6. Leu; 7. Gly; 8.Ile; 9. Phe; 10. Ala; 11. Thr; 12. Gln; 13. Asn; 14. Met; 15. Ser; 16. Pro;17. Lys; 18. TrP; 19. Arg; 20. His; 21. Tyr; 22. S2O3 2−; 23. HSO3 −; 24. SO3 2−;25. S2O4 2−; 26. S2O5 2−; 27 Cl−; 28. Br−; 29. I−; 30. NO2 −; 31. CO3 2−; 32. HCO3 −;33. CH3COO−.),探针BDP-DNBS可以对H2S进行选择性识别,并在587 nm处出现明亮的红色荧光,而跟其它种类分析物反应后的荧光基本可以忽略,因此,探针可以实现选择性响应H2S。当BDP-DNBS(5 µM)在分别加入上述分析物(0. blank; 1. Cys; 2. Hcy; 3. GSH; 4.Glu; 5. Leu; 6. Gly; 7. Ile; 8. Phe; 9. Ala; 10. Thr; 11. Gln; 12. Asn; 13.Met; 14. Ser; 15. Pro; 16. Lys; 17. TrP; 18. Arg; 19. His; 20. Tyr; 21. S2O3 2 −; 22. HSO3 −; 23. SO3 2−; 24. S2O4 2−; 25. S2O5 2−; 26. Cl−; 27. Br−; 28. I−; 29 NO2 −;30. CO3 2−; 31. HCO3 −; 32. CH3COO−)后再加入50 µM H2S反应60s后,可以看出H2S在与各种分析物共存情况下,BDP-DNBS在复杂的溶液体系内依然能够选择性检测到H2S,且荧光程度较强。实验证明,BDP-DNBS能够在响应H2S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-DNBS溶于乙腈中得到10 mM探针母液,配置pH为3.0、4.0、5.0、6.0、7.0、8.0、9.0的柠檬酸和磷酸氢二钠缓冲溶液,对探针以及探针和H2S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3.0‒9.0的溶液中基本保持不变;在加入H2S后,其位于587 nm处荧光强度明显增强,在pH范围4.0‒9.0的pH值下,具有良好的荧光强度。实验证明探针BDP-DNBS能够适应于活细胞中溶酶体定位并进行硫化氢检测。
实施例8
CKK-8细胞毒性实验:
利用探针BDP-DNBS对HeLa细胞的CKK-8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(0, 2.0, 5.0, 10, 20, 50 μM)培养液孵育后,计算细胞的存活百分比。结果如图10所示,在50 μM浓度下细胞存活率大于80%,探针具有低细胞毒性。
实施例9
荧光探针对HeLa细胞内H2S的检测性能测试;
检测H2S的荧光探针BDP-DNBS对人体***(HeLa)细胞中H2S的荧光共聚焦成像。结果如图11所示,(a) 为BDP-DNBS(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;(b) 为HeLa细胞先用100 μM H2S孵育30分钟,用PBS缓冲液洗涤三次,BDP-DNBS(10 μM)再与细胞共孵育30分钟;(c) 为HeLa细胞先用100 μM Cys孵育30分钟,用PBS缓冲液洗涤三次,BDP-DNBS(10 μM)再与细胞共孵育30分钟;(d) 为HeLa细胞先用200 μM NEM孵育60分钟,用PBS缓冲液洗涤三次,BDP-DNBS(10 μM)再与细胞共孵育30分钟;(e-h)为(a-d)对应的细胞核成像,HeLa细胞用Hoechst 33342染色;(i-l) 分别为探针红色通道和蓝色通道的重叠。通过共聚焦红色通道观察,检测H2S的荧光探针在HeLa细胞中产生微弱的红色荧光;对照组实验中,细胞中加入外源性H2S再与BDP-DNBS共同孵育,红色通道产生强烈荧光;细胞中加入Cys(能刺激细胞产生H2S)再与BDP-DNBS共同孵育,红色通道产生显著荧光;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-DNBS共同孵育,红色通道几乎检测不到荧光。由此说明探针对活细胞内源性和外源性H2S具有良好的检测活性。该结果也表明,本发明的荧光探针具有检测HeLa细胞内源性和外源性H2S的良好应用前景。
实施例10
荧光探针对HeLa细胞内溶酶体定位的性能测试;
探针BDP-DNBS在溶酶体内的靶向定位能力测试。如图12所示,HeLa细胞与10 μMBDP-DNBS和100 nM Mito-Tracker Green、200 nM Lyso-Tracker Green DND-26细胞器追踪器共孵育30 min,洗涤3次后,使用共聚焦显微镜成像。结果表明,来自探针的红色荧光信号与LysoTracker Green DND−26的绿色荧光能够很好地重叠,描述两个通道之间强度分布相关性的皮尔森共定位系数计算为0.67。此外两者的ROI(线性感兴趣区域)强度曲线具有高度重叠。相比之下,BDP-DNBS与Mito-Tracker Green并没有很好的重叠(Pearson相关系数为0.33)。该测试结果证明探针BDP-DNBS可以实现对溶酶体的定位,并能够对溶酶体内的H2S实现靶向定位检测。
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)
2.权利要求1所述的荧光探针的制备方法,其特征在于,步骤如下:
(2)步骤(1)制得的化合物1溶于四氢呋喃中,依次加入2,4-二甲基吡咯和三氟乙酸,一次室温反应,然后再加入四氯苯醌二次室温反应,接着再加入三乙胺和三氟化硼***,三次室温反应,二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,柱层析分离得到化合物2,所述化合物2的结构式如下:;
(4)将化合物3溶于二氯甲烷中,然后加入三乙胺和2,4-二硝基苯磺酰氯,回流,柱层析分离得到探针BDP-DNBS。
3.根据权利要求2所述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1~1.5当量的三苯基膦加入到四氢呋喃溶剂中混合得到;2-吗啉乙醇与对羟基苯甲醛按照当量为1:(1~1.5)的比例溶于混合溶剂中,偶氮二甲酸二异丙酯按照1~1.5当量添加到上述溶液中,室温反应12~18 h。
4.根据权利要求2所述荧光探针的制备方法,其特征在于:所述步骤(2)中化合物1、2,4-二甲基吡咯、四氯苯醌、三乙胺和三氟化硼***按照当量为1:(2~3):(1~1.5):(5~7):(8~10)比例添加,三氟乙酸加入量为0.1~0.2 mL,一次室温反应10~15 h,二次室温反应0.5~1h,三次室温搅拌6~12 h。
5.根据权利要求2所述荧光探针的制备方法,其特征在于:所述步骤(3)中化合物1、对羟基苯甲醛、哌啶和乙酸按照当量为1:(1~2):(5~6):(8-10)比例添加,110 ℃回流6~12 h。
6.根据权利要求2所述荧光探针的制备方法,其特征在于:所述步骤(4)中化合物3、2,4-二硝基苯磺酰氯和三乙胺按照当量为1:(2~4):(6~10)的比例添加,45 ℃回流2~3 h。
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