CN113292582B - 一种同时区分羟基自由基和过氧化氢的双功能荧光探针的合成与应用 - Google Patents
一种同时区分羟基自由基和过氧化氢的双功能荧光探针的合成与应用 Download PDFInfo
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Abstract
本发明公开了一种同时区分羟基自由基和过氧化氢的双功能荧光探针,该双功能荧光探针的化学结构式如下:
Description
技术领域
本发明属于分析化学技术领域,具体涉及一种同时区分羟基自由基和过氧化氢的双功能荧光探针的合成与应用。此类探针以不同的机理从各种活性氧物质中快速选择性检测羟基自由基和过氧化氢,青色荧光通道选择性检测羟基自由基,红色荧光选择性通道检测过氧化氢,具有大斯托克斯位移、高荧光量子产率,检测灵敏度高和可视化检测等优点。
背景技术
活性氧物种(ROS)是细胞正常代谢的天然副产物,在细胞信号转导和体内氧化还原平衡中起着至关重要的作用(Nature,2006,443,787–795)。诸如O2 ·-, H2O2,·OH,HOCl,1O2和ONOO-等多种活性氧通常在生命系统中共存,并且它们之间经常发生相互转化(Angew.Chem.Int.Ed.,2020,59,4216.)。在所有活性氧物质中,羟基自由基(·OH)被认为是最具反应活性和破坏力的ROS物种,它的寿命极短,并且可以与许多生物分子如DNA碱基,脂质和蛋白质反应。它的过量产生可导致细胞损伤,并与各种疾病有关。另一方面,在所有ROS中,过氧化氢(H2O2)由于其相对较长的半衰期和扩散范围,已成为氧化还原信号最重要的信使分子(Nat.Immunol.2002,3,1129–1134)。细胞过氧化氢在稳态浓度(0.1mM) 范围内可发挥信号分子作用,指示细胞生长和增殖,而更高的过氧化氢水平(例如100mM)则触发细胞生长停滞或凋亡(Cell Death Differ.,2015,22,377–388)。此外,细胞内通过芬顿反应,可使过氧化氢产生羟基自由基,它们之间存在着密切的相互关系。因此,监测细胞内羟基自由基和过氧化氢对于了解其生物学功能至关重要。
最近,已经报道了许多用于检测羟基自由基(·OH)/过氧化氢的荧光探针。其中,基于羟基自由基(·OH)特征反应的荧光探针,如芳香加成,自旋捕获和夺氢,对其他活性氧物质表现出相对较好的选择性。由于4-溴苯硼酸频哪醇酯良好的选择性,检测过氧化氢的荧光探针大都选择此基团作为识别位点。但是,到目前为止,还没有报道同时检测羟基自由基和过氧化氢的双功能荧光探针。说明双通道同时区分检测羟基自由基和过氧化氢仍然存在巨大挑战。
发明内容
鉴于上述情况,克服一些现有技术的不足,本发明的目的在于提供一种同时区分羟基自由基和过氧化氢的双功能荧光探针。该探针能在特定检测条件下,从各类活性氧化性物质中快速选择性荧光检测羟基自由基和过氧化氢。
本发明的目的还在于提供一种制备方法简单、高灵敏度、检测限低和成本较低的上述荧光分子探针的合成与应用方法。
本发明解决问题采取的具体技术方案为,一种同时区分羟基自由基和过氧化氢的双功能荧光探针的合成及其制备在环境中定量分析羟基自由基和过氧化氢,在活细胞中同时区分成像羟基自由基和过氧化氢的器件的应用,探针的化学结构式如下:
一种同时区分羟基自由基和过氧化氢的双功能荧光探针的合成,其特征在于,所述双功能荧光探针的制备方法包括以下步骤:
步骤1.合成3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯
a.将适量3-氟-4-硝基苯甲醚与L-脯氨酸甲酯盐酸盐加入无水乙腈中,再加入适量的三乙胺加热回流反应6小时,过滤除去三乙胺盐酸盐,旋干溶液,
b.将旋干后的溶液用甲醇溶解,缓慢加入适量的氯化铵和锌粉,室温下搅拌过夜,然后过滤除去锌粉,旋干溶剂,所得固体加入水中,过滤干燥得绿色固体,
c.将绿色固体用无水四氢呋喃溶解,加入适量硼氢化钠和三氟化硼乙醚,90℃下反应12小时,反应完全后,缓慢倒入水中,用氢氧化钠调节溶液pH至12,过滤,滤液用乙酸乙酯萃取,将有机层旋干,
d.将上述产品加入适量的丙烯酸甲酯和冰醋酸中回流反应6小时,过滤得到 3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯;
步骤2.合成3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯
I.在氮气保护条件下,将适量干燥重蒸的N,N-二甲基甲酰胺(DMF)缓慢加入等体积的三氯氧磷(POCl3)中,在20-50℃下搅拌30-60分钟,将3-(8-甲氧基-2,3,3a,4- 四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯溶于适量DMF中,逐滴加入到上述混合溶液中,混合物继续在氮气保护下60℃搅拌反应12小时;反应完全之后,将反应液倒入适量冰水中,用20%的NaOH溶液调节pH至5~6,然后用乙酸乙酯萃取,有机层旋干,得黄色液体产物,
II.将适量的铝和碘加入无水乙腈中,反应10分钟,将上述黄色液体溶入适量无水乙腈中,滴加至铝和碘的乙腈溶液中,回流反应6小时,反应完全后,反应液加入冰水中,过滤,滤液用乙酸乙酯萃取,旋干,柱层析纯化得3-(7-甲酰基-8- 羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯;
步骤3.合成双功能荧光探针甲基3-(7-(2,2-二氰基乙烯基)-8-((4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苄基)氧基)-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉 -5(1H)-基)丙酸酯
i.将3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯和 4-溴苯硼酸频哪醇酯加入适量乙腈中,再加入适量碳酸钾,回流过夜,反应完毕后柱层析纯化得黄色固体,
ii.将步骤i中黄色固体和丙二腈加入适量乙醇中,再加入适量哌啶,室温下搅拌反应过夜,过滤得到所述的荧光分子探针3-(7-(2,2-二氰基乙烯基)-8-((4- (4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苄基)氧基)-2,3,3a,4-四氢吡咯并[1,2-a] 喹喔啉-5(1H)-基)丙酸酯。
本发明的一种同时区分羟基自由基和过氧化氢的双功能荧光探针的使用方法:无特殊说明,通常在室温下将探针分子溶解在有机相和水相体积比为2:8的环境下用于分析检测,有机相为乙醇(EtOH),水相为pH=7.4的磷酸盐缓冲溶液(PBS) 和分析物的水溶液。
本发明的双功能荧光探针同时区分羟基自由基和过氧化氢的具体特征如下:该双功能荧光探针用二甲基亚砜(DMSO)溶解,探针分子溶解在有机相和水相(2:8,v/v) 溶液中,与过氧化氢室温反应30分钟后,在470nm激发波长下发射600nm的红色荧光;与羟基自由基室温反应15分钟后,在430nm激发波长下发射490nm 的强绿色荧光。因此实现特定激发与荧光发射信号检测特定分析物,当两种活性氧均存在时,利用不同的激发和荧光发射信号也能很好地将两者区分。上述荧光分子探针实现了在同一检测条件下同时区分检测羟基自由基和过氧化氢,对其他活性氧、活性硫、常见的氨基酸、金属离子和活性氮均无明显响应,对过氧化氢和羟基自由基两者的检测限分别低至18.6nM和4.9nM。因此,本发明公开的双功能荧光探针能够实现对羟基自由基和过氧化氢的高灵敏区分定量检测。
附图说明
图1本发明所述的双功能荧光探针的核磁共振氢谱图。
图2本发明所述的双功能荧光探针响应羟基自由基和过氧化氢的光谱图。
图3本发明所述的双功能荧光探针同时成像Raw 264.7细胞中的羟基自由基和过氧化氢。
具体实施方式
下面结合附图对本发明做进一步的说明。
本发明所述的双功能荧光分子探针的合成路线如下所示:
实施例1.合成3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯
a.将30g(175.31mmol)3-氟-4-硝基苯甲醚与37.5g(227.9mmol)L-脯氨酸甲酯盐酸盐加入200mL无水乙腈中,再加入73mL(525.9mmol)三乙胺加热回流反应6小时,过滤除去三乙胺盐酸盐,旋干溶液,
b.将旋干后的溶液用300mL甲醇溶解,缓慢加入46.76g(874.13mmol)氯化铵和57.15g(874.13mmol)锌粉,室温下搅拌过夜,然后过滤除去锌粉,旋干溶剂,固体加入水中,过滤干燥得绿色固体,
c.将绿色固体用400mL无水四氢呋喃溶解,加入36.4g(962.17mmol)硼氢化钠和122mL(962.17mmol)三氟化硼乙醚,90℃下反应12小时,反应完全后,缓慢倒入水中,用氢氧化钠调节pH至12,过滤,滤液用乙酸乙酯萃取,将有机层旋干,
d.将上述产品加入适量的丙烯酸甲酯中回流反应12小时,过滤得到3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯26.4g,产率为64.27%。
实施例2.合成3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基) 丙酸甲酯
I.在氮气保护条件下,将适量干燥重蒸的10mL N,N-二甲基甲酰胺(DMF)缓慢加入10mL(61.99mmol)三氯氧磷(POCl3)中,在20-50℃下搅拌30-60分钟,得黄色溶液,将10g(34.44mmol)3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉 -5(1H)-基)丙酸甲酯溶于40mL N,N-二甲基甲酰胺(DMF)中,逐滴加入到黄色的混合溶液中,混合物继续在氮气保护下60℃搅拌反应12小时;反应完全之后,将反应液倒入适量冰水中,用20%的NaOH溶液调节pH至5~6,然后用乙酸乙酯萃取,有机层旋干,得黄色液体产物。
II.将621.8mg(23.05mmol)的铝和5.85g(23.05mmol)的碘加入15mL无水乙腈中,反应10分钟,将上述黄色溶液溶入30mL无水乙腈中,将两混合液混合回流反应6小时,反应完全后,反应液加入冰水中,过滤,滤液用乙酸乙酯萃取,旋干,柱层析纯化得黄色固体3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯1.2g,产率11.45%。
实施例3.合成甲基3-(7-(2,2-二氰基乙烯基)-8-((4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苄基)氧基)-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基) 丙酸酯
i.将3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯 200mg(658.15μmol)和4-溴苯硼酸频那醇酯390.30mg(1.30mmol)加入8mL乙腈中,再加入碳酸钾136.16mg(985.70μmol),回流过夜,反应完毕后柱层析纯化得黄色固体120.0mg,产率35.09%。
ii.将步骤i中黄色固体120mg(230.58μmol)和丙二腈18.3mg(276.69μmol)加入6mL乙醇中,再加入一滴哌啶,室温下反应过夜,过滤得到所述的双功能荧光探针3-(7-(2,2-二氰基乙烯基)-8-((4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2- 基)苄基)氧基)-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸酯94mg,产率为71.71%。
实施例4.双功能荧光探针在体外环境中区分检测羟基自由基和过氧化氢本发明所述的双功能荧光探针光谱性质实验:将探针溶解在二甲基亚砜(DMSO) 中配置成浓度为1mM的探针溶液,分别配置浓度为10mM的羟基自由基和过氧化氢水溶液。具体测试方式为:取20μL 1mM的探针溶液,再加入所需量的分析物溶液,最后加入的380μL的分析纯EtOH和所需量的PBS,所有测试均保持有机相和水相的体积比为2:8(每一个测试样品总体积为2mL)。例如当要求测试过氧化氢浓度为20μM时探针与过氧化氢反应后的荧光强度,配制样品情况为:取20μL的1mM的探针溶液,40μL的1mM的过氧化氢水溶液,再加入380μL的分析纯EtOH和1560μL的PBS缓冲溶液于2mL的样品管中,室温下震荡摇匀30分钟后即可用470nm的激发波长测量其荧光发射强度,其他测试操作与上述步骤类似。该双功能探针分子实现了用不同的激发波长和荧光发射信号区分检测羟基自由基和过氧化氢这两种活性氧,具有很高的灵敏度,检测限分别低至18.6nM和4.9nM,非常适合活细胞内源性羟基自由基和过氧化氢的成像 /定量分析。
实施例5.RAW246.7(巨噬细胞)内源性羟基自由基和过氧化氢双通道荧光成像分析将RAW246.7细胞传代至共聚焦皿细胞培养基中,在标准生长条件下培养24小时后,加入适量探针(5μM)继续在标准生长条件下培养30分钟,然后在共聚焦荧光显微镜下照相,分别用绿色、红色荧光通道进行荧光成像RAW246.7细胞内源性羟基自由基和过氧化氢,从图3可以看出,本发明的双功能荧光探针成功实现了细胞中内源性的羟基自由基和过氧化氢的双通道荧光成像分析。
本发明提供的一种同时区分羟基自由基和过氧化氢的双功能荧光探针,利用芳香加成性检测羟基自由基,反应后在430nm激发波长下发射490nm的绿光,通过过氧化氢氧化性,高选择性识别过氧化氢,在470nm激发波长下发射600nm的红光,荧光现象明显。且反应后产物水溶性较好,荧光量子产率高,斯托克斯位移大等优点。在生物化学、分析检测等领域都具有较大的实际应用价值。尽管本发明的内容已经通过上述优选实施例作了详细的介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,具有本文所述技术特征的双功能荧光探针及其同时检测羟基自由基和过氧化氢的应用,均落入本专利的保护范围。
Claims (4)
2.如权利要求1所述的双功能荧光探针的合成方法 ,其特征在于,所述双功能荧光探针的合成方法包括以下步骤:
步骤1.合成3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯
a.将适量3-氟-4-硝基苯甲醚与L-脯氨酸甲酯盐酸盐加入无水乙腈中,再加入适量的三乙胺加热回流反应6小时,过滤除去三乙胺盐酸盐,旋干溶液,
b.将旋干后的溶液用甲醇溶解,缓慢加入适量的氯化铵和锌粉,室温下搅拌过夜,然后过滤除去锌粉,旋干溶剂,所得固体加入水中,过滤干燥得绿色固体,
c.将绿色固体用无水四氢呋喃溶解,加入适量硼氢化钠和三氟化硼乙醚,90℃下反应12小时,反应完全后,缓慢倒入水中,用氢氧化钠调节溶液pH至12,过滤,滤液用乙酸乙酯萃取,将有机层旋干,
d.将上述产品加入适量的丙烯酸甲酯和冰醋酸中回流反应6小时,过滤得到3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯;
步骤2.合成3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯
I.在氮气保护条件下,将适量干燥重蒸的N,N-二甲基甲酰胺(DMF)缓慢加入等体积的三氯氧磷(POCl3)中,在20-50℃下搅拌30-60分钟,将3-(8-甲氧基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯溶于适量DMF中,逐滴加入到上述混合溶液中,混合物继续在氮气保护下60℃搅拌反应12小时;反应完全之后,将反应液倒入适量冰水中,用20%的NaOH溶液调节pH至5~6,然后用乙酸乙酯萃取,有机层旋干,得黄色液体产物,
II.将适量的铝和碘加入无水乙腈中,反应10分钟,将上述黄色液体溶入适量无水乙腈中,滴加至铝和碘的乙腈溶液中,回流反应6小时,反应完全后,反应液加入冰水中,过滤,滤液用乙酸乙酯萃取,旋干,柱层析纯化得3-(7-甲酰基-8- 羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯;
步骤3.合成双功能荧光探针甲基3-(7-(2,2-二氰基乙烯基)-8-((4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苄基)氧基)-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸酯
i.将3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯和4-溴苯硼酸频哪醇酯加入适量乙腈中,再加入适量碳酸钾,回流过夜,反应完毕后柱层析纯化得黄色固体,
ii.将步骤i中黄色固体和丙二腈加入适量乙醇中,再加入适量哌啶,室温下搅拌反应过夜,过滤得到所述的双功能荧光探针3-(7-(2,2-二氰基乙烯基)-8-((4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苄基)氧基)-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸酯。
3.如权利要求2所述的双功能荧光 探针的合成方法,其特征在于,所述步骤i的3-(7-甲酰基-8-羟基-2,3,3a,4-四氢吡咯并[1,2-a]喹喔啉-5(1H)-基)丙酸甲酯和4-溴苯硼酸频哪醇酯摩尔比为1:2。
4.如权利要求1所述的双功能荧光探针的应用,其特征在于,所述的双功能荧光探针制备在环境中定量分析羟基自由基和过氧化氢,在活细胞中同时区分荧光成像羟基自由基和过氧化氢的器件的应用。
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CN106220663A (zh) * | 2016-07-22 | 2016-12-14 | 济南大学 | 一种过氧化氢荧光探针化合物的制备与应用 |
CN106928263A (zh) * | 2017-03-31 | 2017-07-07 | 济南大学 | 一种用于快速检测过氧化氢的荧光探针的制备与应用 |
CN107286927A (zh) * | 2016-04-11 | 2017-10-24 | 中国科学院理化技术研究所 | 一种检测细胞内过氧亚硝酸根离子的荧光探针及其合成方法 |
CN111072699A (zh) * | 2019-12-30 | 2020-04-28 | 广州中医药大学(广州中医药研究院) | 一种羟基自由基比率式荧光探针及其制备方法和应用 |
-
2021
- 2021-05-11 CN CN202110511420.6A patent/CN113292582B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107286927A (zh) * | 2016-04-11 | 2017-10-24 | 中国科学院理化技术研究所 | 一种检测细胞内过氧亚硝酸根离子的荧光探针及其合成方法 |
CN106220663A (zh) * | 2016-07-22 | 2016-12-14 | 济南大学 | 一种过氧化氢荧光探针化合物的制备与应用 |
CN106928263A (zh) * | 2017-03-31 | 2017-07-07 | 济南大学 | 一种用于快速检测过氧化氢的荧光探针的制备与应用 |
CN111072699A (zh) * | 2019-12-30 | 2020-04-28 | 广州中医药大学(广州中医药研究院) | 一种羟基自由基比率式荧光探针及其制备方法和应用 |
Non-Patent Citations (1)
Title |
---|
A novel fluorescent probe for selective imaging of cellular cysteine with large Stokes shift and high quantum yield;Yabing Gan 等;《Talanta》;20191207;第210卷;120612 * |
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