CN103342720A - 近红外bodipy类的羟基自由基探针及其合成方法和用途 - Google Patents
近红外bodipy类的羟基自由基探针及其合成方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一系列基于硼二吡咯亚甲基类近红外BODIPY化合物及其合成方法及在细胞成像中的应用。
背景技术
活性氧(ROS)包括超氧自由基(O2·-),羟基自由基(HO·),过氧化烷烃自由基(ROO·),过氧化氢(H2O2),单线态氧(1O2),次氯酸/次氯酸根(HOCl/-OCl)。活性氮(RNS),包括一氧化氮(NO),过氧化氮(ONOO-),二氧化氮自由基(NO2·)和硝酸盐(NO3 -)。[参见:(a)B.Halliwell and J.M.C.Gutteridge,Free Radicals in Biology andMedicine,Oxford University Press,Oxford,2007,pp.1-677.(b)L.L.Smith,Free RadicalBiol.Med.,2004,37,318-324.]ROS/RNS对人体健康和疾病非常重要,研究它们在生物学上的作用变得更加重要。但是,检测活体细胞中ROS/RNS的一个重要的障碍就是它们有很短的寿命并且细胞体内存在各种抗氧化剂。合成荧光探针使用荧光仪是一种最有效和最重要的检测ROS/RNS的手段,归功于该方法有一下几点优点,灵敏性高,操作简单,对于仪器的要求低。[参见:(a)S.-K.Ko,X.Chen,J.Yoon and J.Shin,Chem.Soc.Rev.,2011,40,2120.(b)X.Chen,Y.Zhou,X.Peng and J.Yoon,Chem.Soc.Rev.,2010,39,2120-2135.(c)K.Kikuchi,Chem.Soc.Rev.,2010,39,2048-2053.(d)J.S.Kim and D.T.Quang,Chem.Rev.,2007,107,3780-3799.(e)H.N.Kim,M.H.Lee,H.J.Kim,J.S.Kimand J.Yoon,Chem.Soc.Rev.,2008,37,1465-1472.]生物体的组织液及细胞内的DNA,蛋白质,酯质,通过生物体的降解可以产生羟基自由基。因此,羟基自由基在放射治疗癌症的过程中起到重要的作用。由于羟基自由基对人类身体的健康和疾病有很重要的作用,故而寻找一种检测羟基自由基的方法急需解决。当前通常用来检测羟基自由基的方法是利用电子顺磁共振(ESR)光谱检测,但是该方法灵敏性差,不能定量检测羟基自由基,另外不能用于检测生物细胞中的羟基自由基的存在。为了克服这些局限性,有文献报道合成了对羟基自由基敏感的荧光探针并且将它运用到生物样品中。Pou和他的课题组合成了一类基于硝基氧的探针,通过硝基氧被氧化后,荧光强度增强很大,但是没有选择性。[S.Pou,Y.-I.Huang,A.Bhan,V.S.Bhadti,R.S.Hosmane,S.Y.Wu,G.-L. Caoand G.M.Rosen,Anal.Biochem.,1993,212,85-90.]随后,许多课题组合成基于硝基氧的探针,通过连接不同的荧光团,例如萘、蒽、BODIPY化合物、二萘嵌苯、罗丹明。[(a)X.-F.Yang and X.-Q.Guo,Anal.Chim.Acta,2001,434,169-177.(b)X.-F.Yang and X.-Q.Guo,Analyst,2001,126,1800-1804.(c)P.Li,T.Xie,X.Duan,F.Yu,X.Wang and B.Tang,Chem.-Eur.J.,2010,16,1834-1840.(d)T.Maki,N.Soh,T.Fukaminato,H.Nak ajima,K.Nakano and T.Imato,Anal.Chim.Acta,2009,639,78-82.(e)N.B.Yapici,S.Jockusch,A.Moscatelli,S.R.Mandalapu,Y.Itagaki,D.K.Bates,S.Wiseman,K.M.Gibson,N.J.Turro,L.Bi,Org.Lett.2012,14,50-53.]同时,还出现许多其它类型的探针,其中基于FRET类型的探针,利用DNA基团连接两种荧光团。当有羟基自由基存在时,阻止FRET能量转移,导致荧光的变化。[(a)N.Soh,K.Makihara,E.Sakoda and T.Imato,Chem.Commun.,2004,496-497.(b)B.Tang,N.Zhang,Z.Chen,K.Xu,L.Zhuo,L.An and G.Yang,Chem.-Eur.J.,2008,14,522-528.]基于苯酚的探针,利用苯酚易被氧化为苯醌的原理,来检测自由基。[(a)X.Qu,L.J.Kirschenbaum and E.T.Borish,Photochem.Photobiol.,2000,71,307-313.(b)G.M.Makrigiorgos,J.Baranowska-Kortylewicz,E.Bump,S.K.Sahu,R.M.Berman and A.I.Kassis,Int.J.Radiat.Biol.,1993,63,445-458.(c)N.Soh,K.Makihara,T.Ariyoshi,D.Seto,T.Maki,H.Nak ajima,K.Nakano and T.Imato,Anal.Sci.,2008,24,293-296.]探针运用到细胞成像需要满足以下几点要素,1)为了避免激发射波长干扰发射波长,需要探针具有大的斯托克斯位移;2)荧光的最大发射波长应该处于红外或者近红外区。[(a)R.Bandichhor,A.D.Petrescu,A.Vespa,A.B.Kier,F.Schroeder,K.Burgess,J.Am.Chem.Soc.,2006,128,10688.(b)P.W.Du,S.J.Lippard,Inorg.Chem.,2010,49,10753.]
2003年stalke和他的课题组合成化合物,基于二苯基磷连接到蒽基团的中间位置,当三价磷被氧化为五价磷后,化合物有很强的荧光产生。表明二苯基磷可以捕捉到自由基,将该基团引入发光团中可以作为自由基的探针。[Z.Fei,N.Kocher,C.J.Mohrschladt,H.Ihmels,D.Stalke,Angew.Chem.Int.Ed.,2003,42,783-787.]本文选择硼-二吡咯亚甲基(BODIPY)作为荧光团,基于以下几点因素:1),BODIPY刚性结构使它具有优异的光物理性质和光化学特性,紫外吸收光谱的摩尔消光系数大,荧光量子产率高、尖锐、稳定性好,易修饰的化学结构;2)鉴于经典的简单BODIPY紫外吸收和荧光发射光谱位于500nm左右,为了获得近红外BODIPY,取代简单BODIPY的吡咯环上的3-,5-,1-,7-的甲基,从而得到了理想的近红外荧光探针。[(a)Z.Shen,H.K.Rurack,H.Uno,M.Spieles,B.Schulz,G.Reck,N.Ono,Chem.-Eur.J.,2004,10,4853.(b)A.Loudet,K.Burgess,Chem.Rev.,2007,107,4891.(c)J.Han,K.Burgess,Chem.Rev.,2010,110,2709.(d)G.Ulrich,R.Ziessel,A.Harriman,Angew.Chem.,Int.Ed.,2008,47,1184.(e)K.M.Kadish,K.M.Smith,R.Guiland,the handbook of porphyrin science,8,1.(f)T.Bura,P.Retailleau,G.Ulrich,R.Ziessel,J.Org.Chem.,2011,76,1109.]
发明内容
本发明内容是设计并提供了一系列近红外荧光探针,基于3,5-(二苯基磷苯乙烯基)硼-二吡咯亚甲基(BODIPY)及其制备方法和用途。
本发明的技术方案如下:
一类基于硼二吡咯亚甲基类近红外BODIPY化合物,它有如下结构:取代基R对应化合物如下
一种制备上述的基于近红外硼二吡咯亚甲基(BODIPY)化合物B5-B8的方法,它可以按如下反应制备,
它包括下列步骤:
在100mL的两颈烧瓶中,加入(0.16mmol)的简单BODIPY化合物B1-B4中的一种,(0.32mmol)2-(二苯基基磷)苯甲醛,0.4mL无水哌啶,0.4mL冰醋酸和50mL无水乙腈,氩气保护下,使用分水器,加热到90℃,反应过程用TLC监测,至B1-B4原料点全部消失。反应结束后,冷却至室温,反应后有大量的固体析出,直接抽滤,得到的固体为相应的目标化合物B5-B8。
上述的制备基于近红外硼二吡咯亚甲基(BODIPY)化合物B5-B8的方法,所述的分离出固体产品后的滤液中存在的产品,处理方法为,分别用水,饱和食盐水萃取,无水硫酸钠干燥,减压得到粗产物,柱层析,得到目标化合物B5-B8。
一种制备上述的基于近红外硼二吡咯亚甲基(BODIPY)化合物B9-B10的方法,它包括下列步骤:
在100mL的圆底烧瓶中,加入(0.02mmol)的化合物B5或B6,溶解到四氢呋喃中,然后加入(0.2mmol)的双氧水,再加入(0.12mmol)的硫酸亚铁,反应30分钟,反应过程用TLC监测,至原料点全部消失,滤液分别用水,饱和食盐水萃取,无水硫酸钠干燥,减压得到产品,即为纯产品B9或B10。
经二氯甲烷和正己烷混合溶剂重结晶后得化合物B9或B10。
用1H-NMR、UV-Vis、fluorescence spectral、MALDI-TOF MASS及晶体结构表征并证实了该BODIPY羟基自由基探针的结构(见附图和附表)。检测所用仪器为:BrukerARX500型核磁共振仪(TMS为内标,氘代CDCl3为溶剂),岛津UV-4500型紫外-可见分光光度计(扫描范围350~900nm,光路狭缝2nm),日立F-4600美国ThermoELECTRON CORPORATION质谱工作站。
本发明的有益效果
本发明与现有技术相比,其显著优点是:首次运用磷的价态变化,达到检测检测羟基自由基并且不受其RON/ROS的干扰,通过引入2-(二苯基磷)苯乙烯基检测羟基自由基的官能团到BODIPY的3-,5-位,一方面达到高效检测羟基自由基的效果,另一方面,增大共轭性达到红移的结果,使得探针B5-B8均红移到近红外区,处于生物窗口的范围内(650-900nm)。探针B8分子内存在能量转移并且具有天线效应,除了二甲基亚砜溶液中,在其它溶液中的能量转移效率为150%和334nm的假斯托克斯位移。四个探针在二甲基亚砜溶液中表现较弱的荧光量子产率,在其它溶剂中均表现出较高的荧光量子产率。四个探针中重点测试了B5和B6对羟基自由基的响应和选择性,结果表明均能高效,快速检测羟基自由基,并且B6能够成功应用到生物成像中,这对以后荧光探针的研究具有重要的意义。
附图说明
图1-图6为探针B5-B10的核磁图谱;
图7为探针B9的晶体结构;
图8随着增加·OH的不断滴加,探针B5在DMSO溶液中的荧光光谱变化图。(右上角)在580nm激发波长下,探针B5在650nm处荧光强度随·OH浓度变化的动力学曲线。
图9随着增加·OH的不断滴加,探针B6在DMSO溶液中的荧光光谱变化图。(右上角)在580nm激发波长下,探针B6在650nm处荧光强度随·OH浓度变化的动力学曲线。
图10随着增加·OH的不断滴加,探针B8在DMSO溶液中的荧光光谱变化图。(左上角)在334nm激发波长下,探针B8在650nm处荧光强度随·OH浓度变化的动力学曲线。
图11为探针B5在580nm激发波长下加入ROS/RON的相对荧光强度;加入羟基自由基的相对荧光强度。
图12为探针B6在580nm激发波长下加入下述ROS/RON的相对荧光强度;加入羟基自由基的相对荧光强度。
图13为探针B8在334nm激发波长下加入下述ROS/RON的相对荧光强度;加入羟基自由基的相对荧光强度。
图14探针B6的细胞成像图,a图为细胞与10μM的探针B6孵育30分钟后的细胞成像图,b图为a图的明场细胞成像图,c图为a图和b图的叠加图,d图为细胞与探针B6孵育2小时,然后加入PMA到细胞中继续孵育2小时候的细胞成像图,e图为d图的明场细胞成像图,f图为d图和e图的叠加图。
图15探针B8的生物成像图,a图为细胞与10μM的探针B8孵育30分钟后的细胞成像图,b图为a图的明场细胞成像图,c图为a图和b图的叠加图,d图为细胞与探针B8孵育2小时,然后加入PMA到细胞中继续孵育2小时候的细胞成像图,e图为d图的明场细胞成像图,f图为d图和e图的叠加图。
具体实施方式
实施例1.化合物B1-B4的合成:
250mL的圆底烧瓶中,加入(2mmol)相对应的醛,(4mmol)2,4-二甲基吡咯,0.01mL的三氟乙酸和100mL无水二氯甲烷,室温搅拌过夜。然后加入(2mmol)DDQ氧化,反应1h,依次加入3mL的三乙胺和三氟化硼乙醚溶液,反应5h,加水淬灭反应。待反应结束后,用二氯甲烷萃取,减压旋干,柱层析,用二氯甲烷比石油醚为1∶2的展开剂过柱,得到相应的BODIPY产物。
B1C19H19BF2N2(324.16)yellow solid,38.6%yield,1H NMR(CDCl3,500MHz):7.45(t,J=5Hz,3H),7.27(s,2H),6.95(s,2H),2.53(s,6H),1.34(s,6H).
B2C51H83BF2N2O2(804.65)red solid,62.2%yield,1H NMR(CDCl3,500MHz):6.95(t,J=10Hz,13H),6.78(m,2H),5.98(s,2H),4.04(t,2H),3.95(t,2H),3.18(m,2H),2.96(m,9H),2.55(s,6H),1.48(s,6H),1.84(m,4H),1.26(m,82H),0.88(m,9H).
B3C21H21BF2N2(350.18)yellow solid,22.6%yield,1H NMR(CDCl3,500MHz):7.54(d,2H),7.23(s,1H),6.77(q,1H),5.98(s,2H),5.86(d,1H),5.36(d,1H),2.56(s,6H),1.42(s,6H).
B4C26H22BF2N3S(457.16)red solid;65%yield;m.p.>250℃;1H NMR(CDCl3,500MHz):8.19(d,1H,J=10Hz),8.08(d,1H,J=10.0Hz),7.81(m,2H),7.76(d,1H,J=3.5Hz),7.71(t,1H,J=5.0Hz),7.51(t,1H,J=5.0Hz),7.04(d,1H,J=5.0Hz),6.02(s,2H),2.57(s,6H),1.76(s,6H).
实施例2.化合物B5-B8的合成:
100mL的两颈烧瓶中,加入(0.16mmol)的简单BODIPY化合物B1-B4,(0.32mmol)2-(二苯基基磷)苯甲醛,0.4mL无水哌啶,0.4mL冰醋酸和50mL无水乙腈,氩气保护下,使用分水器,加热到90℃,反应过程用TLC监测,至B1-B4原料点全部消失。反应结束后,冷却至室温,反应后有大量的固体析出,直接抽滤,得到固体为产品。滤液分别用水,饱和食盐水萃取,无水硫酸钠干燥,减压得到粗产物,柱层析,得到目标化合物B5-B8。核磁图谱见附图1-图4。
实施例3.化合物B9和B10的合成:
100mL的圆底烧瓶中,加入(0.02mmol)的化合物B5或B6,溶解到四氢呋喃中,然后加入(0.2mmol)的双氧水,再加入(0.12mmol)的醋酸钴,反应30分钟,反应过程用TLC监测,至原料点全部消失。滤液分别用水,饱和食盐水萃取,无水硫酸钠干燥,减压除去溶剂,得到产品,即为纯产品。核磁图谱见附图5和图6。探针B9的晶体结构件见附图7,探针B9单晶的测试数据见表1。
表1探针B9的单晶数据
实施例4.探针B5-B8探针作为羟基自由基探针
探针B5-B8可以选择性的识别羟基自由基,将探针溶解到DMSO溶液中,浓度为10μM,逐渐滴加不同浓度的羟基自由基(Co(OAc)2和H2O2的Fenton试剂)到该溶液中,然后得到相应的荧光发射光谱,发现随着羟基自由基浓度的增加,探针的荧光强度在逐渐增强(见图8,图9和图10)。
将探针B5-B8溶解到DMSO溶液中,浓度为10μM,分别将ROS/RON加入探针的溶液中,ROS/RON分别是探针摩尔量的100倍,H2O2,-OCl,·O2,TBHP,NO,NO2 -,Vc,Fe3+,GSH,结果发现探针的荧光发射光谱基本没有变化(见图11,图12和图13)。
实施例5.探针B6或B8探针在生物成像中应用。
探针B6或B8可以成功应用到细胞成像并检测细胞中羟基自由基的存在。温度控制在37℃,将Hela细胞和浓度为10μM探针放在一起孵育30分钟,激发测得很弱的红色荧光。接着,控制温度在37℃,补加50μM PMA到该细胞中孵育1小时,激发测得很强的红色荧光。明视场的实验测试·OH和探针,进一步证实细胞成像的可视性。同时得到了明视场和图像叠加谱图,进一步证实探针B6或B8可以检测羟基自由基(见图14和图15)。
Claims (5)
1.一类基于硼二吡咯亚甲基类近红外BODIPY化合物,其特征是它有如下结构:取代基R对应化合物如下
2.一种制备权利要求1所述的基于近红外硼二吡咯亚甲基(BODIPY)化合物B5-B8的方法,其特征是它包括下列步骤:
在两颈烧瓶中,加入0.16mmol的BODIPY化合物B1-B4中的一种,0.32mmol的2-(二苯基基磷)苯甲醛,0.4mL的无水哌啶,0.4mL的冰醋酸和50mL的无水乙腈,氩气保护下,使用分水器,加热到90℃,反应过程用TLC监测,至B1-B4原料点全部消失,反应结束后,冷却至室温,反应后有大量的固体析出,直接抽滤,得到的固体为相应的目标化合物B5-B8。
3.根据权利要求2所述的制备基于近红外硼二吡咯亚甲基(BODIPY)化合物B5-B8的方法,其特征是:所述的分离出固体产品后的滤液中存在的产品,用如下方法处理:滤液分别用水,饱和食盐水萃取,无水硫酸钠干燥,减压得到粗产物,柱层析,得到目标化合物B5-B8。
4.一种制备权利要求1所述的基于近红外硼二吡咯亚甲基(BODIPY)化合物B9-B10的方法,其特征是它包括下列步骤:
在圆底烧瓶中,加入0.02mmol的化合物B5或B6,溶解到四氢呋喃中,然后加入0.2mmol的双氧水,再加入0.12mmol的硫酸亚铁,反应30分钟,反应过程用TLC监测,至原料点全部消失,滤液分别用水,饱和食盐水萃取,无水硫酸钠干燥,减压除去溶剂得到产品,即为B9或B10,经二氯甲烷和正己烷混合溶剂重结晶后得化合物B9或B10。
5.权利要求1所述的基于硼二吡咯亚甲基化合物在制备检测羟基自由基和生物成像探针中的应用。
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