CN109369636A - 一种区分不同粘度的双光子荧光探针及其制备方法和应用 - Google Patents
一种区分不同粘度的双光子荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种可区分不同粘度的双光子荧光探针,化学名称为3‑(2‑苯并噻唑乙腈)‑9‑乙基咔唑。所述双光子粘度探针可以通过3‑苯甲醛‑9‑乙烷‑9H‑咔唑与2‑苯并噻唑乙腈反应获得。所述双光子荧光探针可用于检测溶液和细胞中的粘度。本发明提供的区分不同粘度的双光子荧光探针,具有较高的灵敏度,良好的光学稳定性以及对粘度特异性响应;并且实现了在细胞以及斑马鱼活体内粘度的检测。本发明提供的该探针的合成方法,步骤简单、纯化方便、收率高。
Description
技术领域
本发明属于有机小分子荧光探针领域,具体涉及一种区分不同粘度的双光子荧光探针及其制备方法和应用。
背景技术
粘度是衡量一种浓稠流体的流动性和扩散性的主要因素,同时是流体扩散速率的主要参考指标。微环境的粘度在病理学研究中起到非常重要的作用,因为粘度的变化往往会影响细胞微环境中各种新陈代谢的进行。荧光成像技术由于具有实时监测,背景信号低,灵敏度高等优点而成为检测生物分子以及生物微环境重要的一种手段。
荧光成像技术由于具有实时监测,背景信号低,灵敏度高等优点而成为检测生物分子以及生物微环境重要的一种手段。双光子性质具有高穿透度,对生物样品损伤少等优点而被广泛应用到生物成像中。所以,发展一种新的双光子荧光探针对于检测细胞内的粘度具有重要意义。
发明内容
针对现有技术中存在的问题题,本发明提供一种可区分不同粘度的双光子荧光探针,该探针可用于细胞及斑马鱼活体成像,且选择性好、灵敏度高。
本发明的另一目的是提供一种上述荧光探针的合成方法,原料易得、合成步骤简单、收率高。
为实现上述目的,本发明采用如下技术方案。
一种可区分不同粘度的双光子荧光探针,化学名称为3-(2-苯并噻唑乙腈)-9-乙基咔唑,简称为CB,结构式如式(I):
式(I)。
一种上述双光子粘度探针的合成方法,包括以下步骤:
(1)3-溴咔唑的氢氧化钠溶液与溴乙烷于四氢呋喃中加热反应,分离纯化得到白色固体,即3-溴-9-乙烷-9H-咔唑:
;
(2)保护气氛中,3-溴-9-乙烷-9H-咔唑与4-甲酰基苯硼酸在碳酸钾和四(三苯基膦)钯存在下于四氢呋喃中加热回流,分离纯化得3-苯甲醛-9-乙烷-9H-咔唑:
;
(3)2-氨基硫酚与丙二腈在冰乙酸存在下于乙醇中室温搅拌反应,抽滤得2-苯并噻唑乙腈:
;
(4)保护气氛中,2-苯并噻唑乙腈与3-苯甲醛-9-乙烷-9H-咔唑在哌啶存在下于乙醇中加热回流,抽滤分离得3-(2-苯并噻唑乙腈)-9-乙基咔唑:
。
步骤(1)中,所述3-溴咔唑:氢氧化钠:溴乙烷的摩尔比为1:2:6。
步骤(1)中,所述分离纯化步骤为:将反应后的体系减压蒸馏,旋干溶剂得到粗产物后,经硅胶色谱柱分离得到提纯产物;所述色谱柱的流动相优选为体积比为1:30的乙酸乙酯与石油醚。
步骤(1)和(2)中,所述反应温度为55℃,反应时间为12h。
步骤(2)中,所述3-溴-9-乙烷-9H-咔唑:碳酸钾:4-甲酰基苯硼酸:四(三苯基膦)钯的摩尔比为1:3:1.2:0.03。
步骤(2)中,所述分离纯化步骤为:将反应后的体系减压蒸馏,旋干溶剂得到粗产物后,经硅胶色谱柱分离得到提纯产物;所述硅胶色谱柱分离的流动相优选为体积比为1:10的乙酸乙酯与石油醚。
步骤(3)中,所述2-氨基硫酚:丙二腈:冰乙酸的摩尔比为1:1:0.01。
步骤(3)中,所述反应时间为10h。
步骤(4)中,所述2-苯并噻唑乙腈:3-苯甲醛-9-乙烷-9H-咔唑:哌啶的摩尔比为2:1:0.002。
步骤(4)中,所述反应温度为80℃,反应时间为7h。
一种上述双光子荧光探针在检测溶液和细胞粘度中的应用。
本发明荧光探针的识别机理如下:
由于9-乙烷-9H-咔唑与另外一部分通过单键相连,所以在低粘度的情况下,咔唑可以通过单键自由旋转,使整个探针分子不共平面,探针几乎没有荧光即荧光处于“关闭”状态;当在粘度比较大的体系中时,由于苯甲醛的自由旋转受到限制,使得整个探针分子共平面,探针就会发出很强的荧光,即荧光开关被打开。所以,通过这种“开关型”荧光探针可以检测不同的粘度。
本发明的有益效果为:
本发明提供的区分不同粘度的双光子荧光探针,具有较高的灵敏度,良好的光学稳定性以及对粘度特异性响应;并且实现了在细胞以及小鼠活体内粘度的检测。本发明提供的该探针的合成方法,步骤简单、纯化方便、收率高。
附图说明
图1是探针的1H NMR谱;
图2是探针的13C NMR谱;
图3是探针在不同粘度体系中的发射光谱;
图4是探针的细胞成像应用;
图5是探针在斑马鱼中的成像应用。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 荧光探针CB的合成
(1)将2.46 g 3-溴咔唑(10 mmol)和0.8 g氢氧化钠 (20 mmol)加入装有20 mL四氢呋喃的高压管中,搅拌均匀后,放入55℃油浴锅中恒温反应,1.5h后加入6.56 g溴乙烷(60mmol),反应约12h,反应完成后,将反应后的体系减压蒸馏,旋干溶剂得到粗产物后,以体积比为1:30的乙酸乙酯与石油醚为流动相进行柱色谱分离提纯得3-溴-9-乙烷-9H-咔唑;
(2)称量0.274 g3-溴-9-乙烷-9H-咔唑(1 mmol),和0.18 g4-甲酰基苯硼酸(1.2mmol)、37 mg四-(三苯基膦钯)(0.03 mmol)、0.445g碳酸钾(3.23 mmol)于四氢呋喃溶剂中混合反应,除氧,升温至55 ℃回流,在N2氛围环境下反应12 h后,进行减压蒸馏,旋干溶剂,然后以体积比为1:30的乙酸乙酯与石油醚为流动相进行柱色谱分离提纯得3-苯甲醛-9-乙烷-9H-咔唑;
(3)称量0.125 g2-氨基硫酚(0.01 mmol),和0.66 g丙二腈(0.01 mmol)、0.54 ml冰乙酸(0.0001 mmol)于乙醇溶剂中混合反应,室温搅拌反应10h后,出现沉淀,抽滤后,可得纯产物2-苯并噻唑乙腈;
(4)称量0.299 g3-苯甲醛-9-乙烷-9H-咔唑(0.1 mmol),和0.348 g2-苯并噻唑乙腈(0.2 mmol)、0.25 ml哌啶(0.002 mmol)于乙醇溶剂中混合反应,除氧,升温至80 ℃回流,在N2氛围环境下反应7 h后,反应后体系直接抽滤,可得纯净物3-(2-苯并噻唑乙腈)-9-乙基咔唑,即探针CB;
探针的1H NMR谱如图1:1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.65 (d, J= 1.6 Hz, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.03 (q, J = 8.8 Hz, 4H), 7.90 (dd,J1=8.4 Hz, J2= 2, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H),7.49 (m, 1H), 7.24 (t, J = 7.4 Hz, 1H), 4.48 (q, J = 7.0 Hz, 2H), 1.33 (t, J= 7.0 Hz, 3H);
探针的13C NMR谱如图2:13C NMR (101 MHz, DMSO-d6) δ 192.34, 146.71, 139.85,139.52, 134.00 , 129.96, 129.18, 126.79, 125.91, 124.73, 122.72, 122.14,120.50, 118.97, 118.88, 109.47, 109.14, 36.88, 13.49。
实施例2 荧光探针CB在不同粘度体系中的荧光光谱
配制浓度为1 mM实施例1所得荧光探针的二甲基亚砜(DMSO)的测试母液待用。
测试液中,分别取3ml不同比例甘油与甲醇的溶剂(甘油:甲醇=0:10,1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,9:1,10:0),然后加入探针母液(终浓度为10 μM),进行荧光扫描(激发波长425nm,检测波段450-850nm),测得各体系中相对荧光强度,如图3所示。由图3可知,随着溶剂粘度的增加,相对荧光强度变强。
实施例3 荧光探针CB在细胞中的成像
配制浓度为1 mM实施例1所得荧光探针的二甲基亚砜(DMSO)的测试母液待用。
将适当密度的Hela细胞、A549细胞和HeG2细胞分别接种到灭菌的35 mm成像培养皿中,在CO2培养箱(温度为37 ℃,5 % CO2)中培养,待细胞贴壁后,第一组加入10μM粘度荧光探针CB孵育半小时后进行生物成像(单光子成像:激发波长:561nm,发射波长:600-620nm;双光子成像:激发波长780nm,发射波长:600-620nm);第二组先加入10μM粘度刺激物莫能菌素(Monensin),40分钟后再加入10μM粘度荧光探针CB,孵育半小时后进行生物成像;第三组先加入10μM粘度刺激物制霉菌素(Nystatin),40分钟后加入10μM粘度荧光探针CB,孵育半小时后进行生物成像;成像结果如图4所示。通过分析比较可以看出无论在单光子还是在双光子条件下,在只有探针的情况下细胞只发微弱的光;在加有探针以及粘度刺激物(莫能菌素,制霉菌素)的情况下细胞发出强烈的红光;因此不同粘度的细胞可以通过本发明合成的探针进行细胞成像来区分。
实施例4 荧光探针CB在斑马鱼中的成像
配制浓度为1 mM实施例1所得荧光探针的二甲基亚砜(DMSO)的测试母液待用。
配制浓度为1 mM实施例1所得荧光探针的二甲基亚砜(DMSO)的测试母液待用。
将斑马鱼放入不同组成像盘中,分成四组,第一组仅加入PBS=7.4的缓冲溶液,半小时后进行双光子生物成像;第二组加入10μM粘度荧光探针CB孵育半小时后进行双光子生物成像;第三组先加入10μM粘度刺激物莫能菌素(Monensin),40分钟后再加入10μM粘度荧光探针CB,孵育半小时后进行双光子生物成像(单光子成像:激发波长:561nm,发射波长:600-620nm;双光子成像:激发波长780nm,发射波长:600-620nm);第四组先10μM粘度刺激物制霉菌素(Nystatin),40分钟后加入10μM粘度荧光探针CB,孵育半小时后进行生物成像;成像结果如图5所示。通过分析比较可以看出,无论在单光子还是在双光子条件下,在只有PBS的条件下斑马鱼没有光;在只有探针的情况下斑马鱼只发微弱的光;在加有探针以及粘度刺激物(莫能菌素,制霉菌素)的情况下斑马鱼发出强烈的红光;因此不同粘度的斑马鱼可以通过本发明合成的探针进行斑马鱼成像来区分。
Claims (6)
1.一种可区分不同粘度的双光子荧光探针,化学名称为3-(2-苯并噻唑乙腈)-9-乙基咔唑,其结构式如式(I):
式(I)。
2.一种如权利要求1所述的双光子粘度探针的合成方法,其特征在于,包括以下步骤:
(1)3-溴咔唑的氢氧化钠溶液与溴乙烷于四氢呋喃中加热反应,分离纯化得到白色固体,即3-溴-9-乙烷-9H-咔唑;
(2)保护气氛中,3-溴-9-乙烷-9H-咔唑与4-甲酰基苯硼酸在碳酸钾和四(三苯基膦)钯存在下于四氢呋喃中加热回流,分离纯化得3-苯甲醛-9-乙烷-9H-咔唑;
(3)2-氨基硫酚与丙二腈在冰乙酸存在下于乙醇中室温搅拌反应,抽滤得2-苯并噻唑乙腈;
(4)保护气氛中,2-苯并噻唑乙腈与3-苯甲醛-9-乙烷-9H-咔唑在哌啶存在下于乙醇中加热回流,抽滤分离得3-(2-苯并噻唑乙腈)-9-乙基咔唑。
3.根据权利要求2所述的合成方法,其特征在于,步骤(1)中,所述3-溴咔唑:氢氧化钠:溴乙烷的摩尔比为1:2:6;
步骤(2)中,所述3-溴-9-乙烷-9H-咔唑:碳酸钾:4-甲酰基苯硼酸:四(三苯基膦)钯的摩尔比为1:3:1.2:0.03;
步骤(3)中,所述2-氨基硫酚:丙二腈:冰乙酸的摩尔比为1:1:0.01;
步骤(4)中,所述2-苯并噻唑乙腈:3-苯甲醛-9-乙烷-9H-咔唑:哌啶的摩尔比为2:1:0.002。
4.根据权利要求2所述的合成方法,其特征在于,步骤(1)和(2)中,所述反应温度为55℃,反应时间为12h;
步骤(3)中,所述反应时间为10h;
步骤(4)中,所述反应温度为80℃,反应时间为7h。
5.根据权利要求2所述的合成方法,其特征在于,步骤(1)中,所述分离纯化步骤为:将反应后的体系减压蒸馏,旋干溶剂得到粗产物后,经硅胶色谱柱分离得到提纯产物;所述色谱柱的流动相优选为体积比为1:30的乙酸乙酯与石油醚;
步骤(2)中,所述分离纯化步骤为:将反应后的体系减压蒸馏,旋干溶剂得到粗产物后,经硅胶色谱柱分离得到提纯产物;所述硅胶色谱柱分离的流动相优选为体积比为1:10的乙酸乙酯与石油醚。
6.一种如权利要求1所述的双光子荧光探针在检测溶液和细胞粘度中的应用。
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AD01 | Patent right deemed abandoned |