CN113278001A - 一种近红外硒代半胱氨酸荧光探针及其制备方法与应用 - Google Patents
一种近红外硒代半胱氨酸荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种近红外硒代半胱氨酸荧光探针及其制备方法与应用。这种检测硒代半胱氨酸的近红外荧光探针的结构为(I),其具有反应速度快、选择性好、灵敏度高、检测限低的优点。在生理pH值下,荧光探针与不同浓度硒代半胱氨酸反应后荧光强度与浓度呈现良好的线性关系,适合定量检测硒代半胱氨酸,用于人肺癌细胞A549中硒代半胱氨酸的荧光成像,可定量检测细胞中硒代半胱氨酸。
Description
技术领域
本发明涉及荧光探针技术领域,尤其是一种近红外硒代半胱氨酸荧光探针及其制备方法与应用。
背景技术
硒是一种对人类健康至关重要的微量营养元素,硒摄入不足会引起克山病、大骨节病、糖尿病等疾病,但是摄入过量的硒会导致硒中毒,出现脱发、脱甲、肝损伤等。硒化合物在细胞内的主要存在形式是硒代半胱氨酸(selenocysteine,Sec),Sec在蛋白质合成中被称为第21种氨基酸,是构成硒蛋白(Selenoprotein,SeP)的重要成分。SeP都具有较高的酶催化活性,SeP酶参与人体中抗氧化防御、生育、肌肉的发育、甲状腺激素调节和免疫功能等生理功能的代谢。而Sec特异性地作为SeP的反应活性位点,Sec含量的高低与SeP 的高催化能力息息相关。由于硒代半胱氨酸在人体生理功能的重要作用,研究开发在生物体系中能快速可靠地检测硒代半胱氨酸的方法是非常有必要的。
由于荧光探针对生物活性物质检测具有灵敏度高、操作简便等优点,通过荧光探针方法检测生物样品中的硒代半胱氨酸吸引了研究者的广泛关注。也相继报到了一些选择性检测硒代半胱氨酸的荧光探针,但已报道的大多数硒代半胱氨酸荧光探针是在紫外-可见光区间的探针。紫外-可见光在组织内穿透性弱,且易被组织中血红蛋白及黑色素等物质吸收;另一方面,在可见光的激发下,生物体有荧光背景,这些因素严重影响了其检测分析的灵敏度和准确性,不利于活体及深层组织中成像。近红外光波长范围为650-900nm,组织在此区间内自发荧光较少,避免了生物背景对荧光信号的干扰;同时近红外光具有较强的穿透性,有利于深层组织成像;且具有较低的光损伤。因此,近红外荧光探针具有信噪比高、样本穿透性强和成像分辨率高的优势。
因此,开发高灵敏度的近红外硒代半胱氨酸荧光探针能为生物体内硒代半胱氨酸的生理检测与病理机制考查提供新的检测方法。
本发明公开了一种近红外硒代半胱氨酸荧光探针及其制备方法与应用。
发明内容
本发明的目的在于提供一种近红外硒代半胱氨酸荧光探针及其制备方法与应用。
本发明的近红外硒代半胱氨酸荧光探针YZ-M5,其特征在于具有如式(I)所示化学结构的化合物:
式(I)所述的近红外硒代半胱氨酸荧光探针的制备方法,其特征在于,其制备包括以下反应步骤:
上述所述的近红外硒代半胱氨酸荧光探针的制备方法,其特征在于,该方法包括以下步骤:
第一步:以浓硫酸为溶剂,4-(二乙氨基)水杨醛与环己酮进行反应,反应后倒入冰水中,再加入高氯酸形成沉淀制备化合物YZ-M3;
第二步:以醋酸为溶剂,化合物YZ-M3与对羟基苯甲醛进行反应,制备化合物 YZ-M4;
第三步:以二氯甲烷为溶剂,化合物YZ-M4与2,4-二硝基苯磺酰氯进行反应,制备化合物YZ-M5。
在第一步反应中,4-(二乙氨基)水杨醛与环己酮的摩尔比为1:1~3,反应的温度为80~120℃,反应时间为1~4h。
在第二步反应中,YZ-M3与对羟基苯甲醛的摩尔比为1:1~3,反应的温度为90~140℃,反应时间为1~4h。
在第三步中,YZ-M4与2,4-二硝基苯磺酰氯的摩尔比为1:1~3,化合物YZ-M4 与碳酸钾的摩尔比为1:0~4。
本发明还提供了上述近红外荧光探针作为检测硒代半胱氨酸的应用。
本发明尤其提供了上述近红外硒代半胱氨酸荧光探针在细胞及活体中检测硒代半胱氨酸的应用。
采用本发明的技术方案,优势如下:
本发明提供的检测硒代半胱氨酸的近红外荧光探针YZ-M5,具有反应速度快、选择性好、灵敏度高及检测限低(18.5nM)等优点。在生理pH值下,荧光探针与不同浓度硒代半胱氨酸反应后荧光强度与浓度呈现良好的线性关系,说明探针YZ-M5适合定量检测硒代半胱氨酸;探针YZ-M5实现了人肺癌细胞A549中硒代半胱氨酸的荧光成像;探针YZ-M5也实现了活体水平内源性硒代半胱氨酸的灵敏检测。由此可见,本发明制备的荧光探针YZ-M5是一种可定量检测细胞中硒代半胱氨酸的有效工具。
附图说明
图1为本发明实施例4中探针YZ-M5与硒代半胱氨酸反应前后的紫外可见光谱与荧光光谱。
图2为本发明实施例5中探针YZ-M5与硒代半胱氨酸(50μM)进行孵育时荧光强度与反应时间之间的关系。
图3为本发明实施例6中荧光探针YZ-M5在pH=7.4的PBS中与不同浓度硒代半胱氨酸反应后的荧光光谱,激发波长为580nm,发射波长为660nm,从下往上硒代半胱氨酸浓度依次增加。
图4为本发明实施例6中荧光探针YZ-M5在pH=7.4的PBS中与不同浓度硒代半胱氨酸反应后的荧光强度与Sec(0-40μM)浓度的线性关系。
图5为本发明实施例7中荧光探针YZ-M5与不同生物活性因子反应5分钟时的荧光强度,图例:1、空白;2、硒代半胱氨酸;3、高半胱氨酸;4、半胱氨酸;5、谷胱甘肽;6、谷氨酸;7、天冬氨酸;8、缬氨酸;9、苯丙氨酸;10、脯氨酸;11、苏氨酸;12、精氨酸;13、异亮氨酸;14、亮氨酸;15、组氨酸;16、赖氨酸;17、色氨酸;18;丝氨酸;19、Na2SeO3;20、Na2Se。
图6为本发明实施例8中荧光探针YZ-M5在有无硒代半胱氨酸情况下与A549 细胞共同孵育15min和30min后,用580nm激发得到的荧光显微镜成像图。
具体实施方式
下面以实施例对本发明进一步详细说明,但本发明的保护范围不仅限于这些实施例。
实施例1
化合物YZ-M3的制备
在一个50mL圆底烧瓶中,加入15mL浓H2SO4,然后在浓H2SO4中滴加环己酮(2.94g,0.03mol),冷却到室温后,加入4-(二乙氨基)水杨醛(3.86g,0.02mol),90℃剧烈搅拌反应2.0h。反应完全后,冷却并倒入200mL冰水中,然后加入70%高氯酸1.5mL,静置过夜,析出红色沉淀,过滤,用冷水(50mL×3)洗涤,干燥。得到化合物YZ-M3红色固体5.05g,产率71%。
实施例2
化合物YZ-M4的制备
化合物YZ-M3(711mg,2mmol)和对羟基苯甲醛(366mg,3mmol)溶于醋酸(20 mL)中。将反应混合物加热至110℃反应2h。反应完全后,减压蒸馏除去溶剂。粗产物通过使用硅胶柱色谱纯化(DCM:MeOH=20:1,v/v),获得黑色固体598mg,产率65%。1H NMR(400MHz,d6-DMSO)δ10.33(s,1H),8.48(s,1H),8.03(s,1H),7.87(d,J=9.4Hz,1H), 7.58(d,J=8.6Hz,2H),7.43(dd,J=9.4,1.9Hz,1H),7.27(s,1H),6.93(d,J=8.6Hz,2H), 3.70(q,J=7Hz,4H),2.87(dd,J=18.3,13.0Hz,4H),1.89-1.77(m,2H),1.26(t,J=7Hz, 6H).13C NMR(100MHz,d6-DMSO)δ191.42,163.49,160.15,158.85,156.04,148.68,137.56, 133.97,132.55,132.29,126.90,125.49,123.43,118.80,118.45,116.43,116.29,95.85,45.95,27.23,21.48,13.01.HRMS(C24H26NO2)+:360.1956,calcd for(C24H26NO2)+:360.1958.
实施例3
荧光探针YZ-M5的制备
将化合物YZ-M4(230mg,0.5mmol)和无水碳酸钾(138mg,1mmol)加入DCM(10 mL)溶液中,降温到0℃,然后将2,4-二硝基苯磺酰氯(192mg,0.75mmol)溶于10mL无水 DCM中,并滴加至上述反应液中。反应完全后,减压蒸馏除去溶剂。粗品通过硅胶柱层析 (DCM:MeOH=30:1,v/v)纯化得到荧光探针YZ-M5黑色固体234mg,产率68%。1H NMR (400MHz,d6-DMSO)δ9.14(d,J=2Hz,1H),8.64(dd,J=9,2Hz,1H),8.58(s,1H),8.32(d,J =9Hz,1H),8.03(s,1H),7.94(d,J=9Hz,1H),7.70(d,J=9Hz,2H),7.53(dd,J=9,2Hz,1H), 7.35(d,J=9Hz,2H),7.30(d,J=2Hz,1H),3.73(q,J=7Hz,4H),2.89(t,J=6Hz,4H),1.89– 1.80(m,2H),1.26(t,J=7Hz,6H).13C NMR(100MHz,d6-DMSO)δ161.23,159.18,156.80, 152.05,149.38,148.89,148.60,135.68,134.04,133.46,132.96,132.74,131.20,130.26,128.01,124.10,122.84,121.58,120.32,119.59,95.86,55.38,46.26,27.13,26.85,21.56. HRMS(C28H26NO4)+:590.1588,calcd for(C28H26NO4)+:590.1592.
实施例4
探针YZ-M5与硒代半胱氨酸反应的紫外可见光谱与荧光光谱
探针YZ-M5与硒代半胱氨酸反应前后的紫外可见光谱与荧光光谱如图1如示。在荧光光谱中,探针YZ-M5自身几乎无荧光,与硒代半胱氨酸(20μM)反应后,荧光强度显著增加,其最大发射波长660nm,与YZ-M4的荧光光谱一致。在紫外可见光谱中,探针YZ-M5最大吸收峰在380和560nm,与硒代半胱氨酸(20μM)反应后的最大吸收峰在425和580nm,这与荧光母核YZ-M4的吸收峰一致。
实施例5
探针YZ-M5与硒代半胱氨酸反应的时间
将探针YZ-M5与硒代半胱氨酸(50μM)孵育,荧光强度与反应时间之间的关系如图2。随着孵育时间的增加,在660nm处产生的荧光强度逐渐增强,5min时,荧光强度达到峰值,反应趋于完全,说明探针YZ-M5与硒代半胱氨酸的反应速度快。
实施例6
荧光探针YZ-M5与硒代半胱氨酸反应的线性关系及检测限
图3为荧光探针YZ-M5在pH=7.4的PBS中与不同浓度硒代半胱氨酸反应后的荧光光谱,激发波长为580nm,发射波长为660nm处的荧光强度随硒代半胱氨酸浓度增加依次增强。图4为荧光强度与Sec(0-40μM)浓度范围内呈现良好的线性关系,线性方程为y=5.44824x+39.94063(R2=0.98685),YZ-M5检测硒代半胱氨酸的检测限为18.5nM,说明YZ-M5的检测灵敏度高。
实施例7
探针YZ-M5对生物活性因子的选择性
图5为荧光探针YZ-M5与不同生物活性因子反应5分钟时的荧光强度变化,其中:1、空白;2、硒代半胱氨酸;3、高半胱氨酸;4、半胱氨酸;5、谷胱甘肽;6、谷氨酸;7、天冬氨酸;8、缬氨酸;9、苯丙氨酸;10、脯氨酸;11、苏氨酸;12、精氨酸;13、异亮氨酸;14、亮氨酸;15、组氨酸;16、赖氨酸;17、色氨酸;18;丝氨酸;19、Na2SeO3; 20、Na2Se。可知与硒代半胱氨酸反应后的荧光强度增强,而与其他生物硫醇、氨基酸和硒化物反应后荧光强度增加不明显,说明荧光探针YZ-M5对硒代半胱氨酸选择性好。
实施例8
探针YZ-M5在A549细胞中的荧光成像应用
图6为荧光探针YZ-M5在有无硒代半胱氨酸情况下与A549细胞共同孵育15min 和30min后,用580nm激发得到的荧光显微镜成像图,在细胞内荧光探针YZ-M5与硒代半胱氨酸反应后,显示红色荧光,说明荧光探针YZ-M5可用于细胞中的荧光成像。
Claims (8)
3.根据权利要求2所述的近红外硒代半胱氨酸荧光探针YZ-M5的制备方法,其特征在于,该方法包括以下步骤:
第一步:以浓硫酸为溶剂,4-(二乙氨基)水杨醛与环己酮进行反应,反应后倒入冰水中,再加入高氯酸形成沉淀制备化合物YZ-M3;
第二步:以醋酸为溶剂,化合物YZ-M3与对羟基苯甲醛进行反应,制备化合物YZ-M4;
第三步:以二氯甲烷为溶剂,化合物YZ-M4与2,4-二硝基苯磺酰氯进行反应,制备化合物YZ-M5。
4.根据权利要求3所述的近红外硒代半胱氨酸荧光探针YZ-M5的制备方法,其特征在于,在第一步反应中,4-(二乙氨基)水杨醛与环己酮的摩尔比为1:1~3,反应的温度为80~120℃,反应时间为1~4h。
5.根据权利要求3所述的近红外硒代半胱氨酸荧光探针YZ-M5的制备方法,其特征在于,在第二步反应中,YZ-M3与对羟基苯甲醛的摩尔比为1:1~3,反应的温度为90~140℃,反应时间为1~4h。
6.根据权利要求3所述的近红外硒代半胱氨酸荧光探针YZ-M5的制备方法,其特征在于,在第三步反应中,YZ-M4与2,4-二硝基苯磺酰氯的摩尔比为1:1~3,化合物YZ-M4与碳酸钾的摩尔比为1:0~4。
7.一种权利要求1所述的近红外荧光探针YZ-M5用作检测硒代半胱氨酸的应用。
8.根据权利要求7所述的应用,其特征在于:所述的近红外硒代半胱氨酸荧光探针在细胞中检测硒代半胱氨酸的应用。
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