CN108530483B - 基于香豆素骨架的Wittig试剂及其制备方法和用途 - Google Patents
基于香豆素骨架的Wittig试剂及其制备方法和用途 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
- C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
- C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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- Organic Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Plural Heterocyclic Compounds (AREA)
Abstract
Description
技术领域
本发明属于生物化学领域,具体涉及基于香豆素骨架的Wittig试剂及其制备方法和用途。
背景技术
DNA损伤在细胞内普遍存在[1]。5-醛基尿嘧啶(5fU)是一种与8-羟基脱氧鸟苷(8-oxodGuo)含量相当的DNA氧化损伤,由胸腺嘧啶(T)经系列氧化过程所得[2]。细胞内源性的活性氧(ROS),外源性的紫外辐射、离子辐射、芬顿试剂等均会使细胞内的5fU含量升高。与C-5位为甲基的胸腺嘧啶相比,C-5位为醛基的5-醛基尿嘧啶(5fU)其N-3位酸性明显增强,容易去质子以离子化形式存在,此离子化形式在DNA复制过程中易与G、C、T形成错配,造成基因突变;此外,活泼的醛基易与含氨基、巯基的化合物偶联引入新的碱基修饰损伤,进而干扰DNA的正常功能,甚至引发重大疾病[3]。
综上所述,开发高选择性、高灵敏度的检测技术以准确测定DNA中的5-醛基尿嘧啶(5fU)水平具有重要意义。
目前,色谱-质谱联用技[4]被广泛应用于检测DNA氧化损伤,但此法包含复杂繁琐的样品前处理过程,而且依赖于昂贵的大型分析仪器。相比之下,荧光传感技术具有操作简单、成本低廉、灵敏度高、选择性好等优势,受到了众多科研工作者的青睐。近年来,针对5-醛基尿嘧啶(5fU)的C-5位醛基,各种反应型小分子荧光探针相继被开发。他们大多为邻氨基苯硫酚、邻苯二胺、肼、吲哚等的衍生物,可选择性与醛基反应生成相应的苯丙噻唑、苯丙咪唑、C=N、C=C等共轭结构,实现对5-醛基尿嘧啶(5fU)的荧光识别[5]。尽管探针结构千变万化,但反应类型仅仅局限于席夫碱反应或类似Adol反应(羟醛缩合/醇醛缩合反应),因此,基于其他有机化学原理开发新的醛基化试剂以扩充5-醛基尿嘧啶(5fU)的荧光探针分子库是及其重要且必要的。
Wittig反应(叶立德反应)是德国化学家G.Wittig于1953年发现的,主要用于醛、酮直接合成烯烃。该方法具有收率高、反应条件温和、选择性好等优点,为此,G.Wittig获得了1979年的诺贝尔化学奖。香豆素是一种经济易得的化学原料,鉴于其优异的荧光性质及丰富的衍生手段,在化学生物学领域得到了广泛应用。截至目前,基于香豆素骨架改良传统的Wittig试剂构建比率型荧光探针,并将其应用于DNA中5-醛基尿嘧啶(5fU)的选择性识别与定量检测仍然是一个空白,而我们的设计拟将填补这个空白。
发明内容
本发明提供了一种基于香豆素骨架的Wittig试剂,用于DNA中5-醛基尿嘧啶(5fU)的检测。所述基于香豆素骨架的Wittig试剂的结构如式Ⅰ所示:
最优的,R为二乙基氨基。
本发明还提供了上述基于香豆素骨架的Wittig试剂的制备方法,其合成路线如下:
上述基于香豆素骨架的Wittig试剂的制备方法包括以下步骤:
a、将R取代的水杨醛和乙酰乙酸乙酯溶于有机溶剂,加入哌啶,回流4~12小时,制备得到中间体1;
b、将中间体1溶于AcOH(冰醋酸),加入液溴和氢溴酸,室温搅拌过夜,制备得到中间体2;
c、将中间体2,Ph3P(三苯基膦),KI(碘化钾)溶于有机溶剂,回流5~12小时,制备得到中间体3;
d、将中间体3溶于有机溶剂,加入碱水溶液,室温剧烈搅拌0.5~3小时,制备得到基于香豆素骨架的Wittig试剂。
其中,上述基于香豆素骨架的Wittig试剂的制备方法中,步骤a所述的乙酰乙酸乙酯的摩尔量为R取代水杨醛的1.5~2.5倍;所述哌啶的摩尔量为R取代水杨醛的0.1~1.6倍;所述的有机溶剂为无水乙醇、异丙醇、乙腈或二氯甲烷中的任意一种。
其中,上述基于香豆素骨架的Wittig试剂的制备方法中,步骤b所述的液溴的摩尔量为中间体1的1.5~2.0倍,所述氢溴酸的摩尔量为中间体1的3~5倍。
其中,上述基于香豆素骨架的Wittig试剂的制备方法中,步骤c所述的三苯基膦的摩尔量为中间体2的1.2~2.5倍;所述碘化钾的摩尔量为中间体2的0.1~0.5倍;所述的有机溶剂为二氯甲烷、甲苯、四氢呋喃、乙腈、甲醇或N,N-二甲基甲酰胺中的任意一种。
其中,上述基于香豆素骨架的Wittig试剂的制备方法中,步骤d所述的有机溶剂为二氯甲烷、甲苯、四氢呋喃、乙腈、甲醇或N,N-二甲基甲酰胺中的任意一种;所述碱水溶液的体积为有机溶剂体积的20%~80%(V/V);所述碱水溶液中的碱为碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾或三乙胺中任意一种,其摩尔量为中间体3的2~10倍。
本发明还提供了上述基于香豆素骨架的Wittig试剂对5-醛基尿嘧啶(5fU)进行荧光识别的用途。
本发明有效结合荧光传感技术和有机人名反应,将香豆素荧光骨架引入传统的Wittig试剂,设计并合成了首例比率型小分子荧光探针用于DNA中5-醛基尿嘧啶(5fU)的选择性识别与定量检测。该探针可以在较温和的条件下与5-醛基尿嘧啶(5fU)的C-5位醛基发生反应生成C=C双键,此C=C双键及其紧邻的嘧啶环扩大了香豆素分子的共轭平面,使反应后的荧光发射波长发生红移,从而达到比率荧光识别的目的。该系列化合物对5-醛基尿嘧啶(5fU)具有优良的选择性及灵敏性,与现有的“off-on”型荧光探针相比,本发明设计的比率型荧光探针有效降低了背景荧光的干扰,使检测的准确性大大提高。此外,本发明提供的基于香豆素骨架的Wittig试剂具有毒副作用小、原料经济易得、整条合成路线可操作性强、反应条件温和、总体成本较低等优势。
附图说明
图1化合物3的氢谱、碳谱、高分辨质谱。
图2化合物4的氢谱、碳谱、高分辨质谱。
图3化合物4对5fU核苷的荧光选择性。
图4化合物4对ODN-5fU的荧光选择性。
图5化合物4标记的ODN-5fU的凝胶电泳图。
图6化合物4的MTT细胞毒性实验。
图7 10μM化合物4与乙醇固定化的HeLa细胞孵化16h后的共聚焦荧光成像图。其中,a-c图的细胞辐射过γ射线但未与化合物4共孵化;d-f图的细胞未辐射过γ射线但与化合物4共孵化;g-i图的细胞既辐射过γ射线又与化合物4共孵化;j图为d、g两图的平均荧光强度(488nm激发,500-600nm收集)。
具体实施方式
基于香豆素骨架的Wittig试剂的制备方法包括以下步骤:
a、将R取代的水杨醛和乙酰乙酸乙酯溶于有机溶剂,加入哌啶,回流4~12小时,制备得到中间体1;所述的乙酰乙酸乙酯的摩尔量为R取代水杨醛的1.5~2.5倍,哌啶的摩尔量为R取代水杨醛的0.1~1.6倍;所述的有机溶剂为无水乙醇、异丙醇、乙腈或二氯甲烷中的任意一种。
b、将中间体1溶于冰醋酸,加入液溴和氢溴酸,室温搅拌过夜,制备得到中间体2;所述的液溴的摩尔量为中间体1的1.5~2.0倍,所述氢溴酸的摩尔量为中间体1的3~5倍。
c、将中间体2,Ph3P(三苯基膦),KI(碘化钾)溶于有机溶剂,回流5~12小时,制备得到中间体3;所述的三苯基膦的摩尔量为中间体2的1.2~2.5倍,所述碘化钾的摩尔量为中间体2的0.1~0.5倍;所述的有机溶剂为二氯甲烷、甲苯、四氢呋喃、乙腈、甲醇或N,N-二甲基甲酰胺中的任意一种。
d、将中间体3溶于有机溶剂,加入碱的水溶液,室温剧烈搅拌0.5~3小时,制备得到基于香豆素骨架的Wittig试剂;所述的有机溶剂为二氯甲烷、甲苯、四氢呋喃、乙腈、甲醇或N,N-二甲基甲酰胺中的任意一种;所述碱水溶液的体积为有机溶剂体积的20%~80%(V/V);所述碱水溶液中的碱为碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾或三乙胺中任意一种,其摩尔量为中间体3的2~10倍。
本发明实施例中,细胞株购于ATCC(American Type Culture Collection),10%胎牛血清(FBS)购于Hyclone,DMEM(H)培养基购于美国Gibco,10000×Gel-Red(核酸染料)购于Biosharp。
本发明实施例中,ODN-5fC(含有5-醛基胞嘧啶的DNA)、ODN-5fU(含有5-醛基尿嘧啶的DNA)、ODN-AP(含有脱碱基位点的DNA)购于Takara Biotechnology,ODN-C、ODN-T购于生工生物工程(上海)股份有限公司。各个ODN的序列参见表1。
表1本发明实施例中使用的各个ODN的核苷酸序列
实施例1 3-乙酰基-7-(二乙氨基)香豆素(化合物1)的合成:
将4-(二乙氨基)水杨醛(1.93g,10mmol)、乙酰乙酸乙酯(1.85g,15mmol)、哌啶(1mL,39.5mmol)溶于200mL无水乙醇,80℃下回流。反应完全后冷却至室温,抽滤,冰乙醇洗涤,真空干燥得到光泽黄色固体2.15g(8.3mmol),产率为83%。
1H NMR(400MHz,CDCl3)δ8.43(s,1H),7.39(d,J=9.0Hz,1H),6.61(dd,J=9.0,2.4Hz,1H),6.46(d,J=2.3Hz,1H),3.45(q,J=7.1Hz,4H),2.67(s,3H),1.24(t,J=7.1Hz,6H)。
实施例2 3-(2-溴乙酰基)-7-(二乙氨基)香豆素(化合物2)的合成:
将化合物1(1.5g,5.78mmol)、50%氢溴酸水溶液(2.8g,17.3mmol)溶于150mL冰醋酸中,搅拌条件下逐滴滴加液溴(1.48g,9.25mmol),滴毕,室温搅拌过夜。70℃减压除去溶剂,加入100ml水,用饱和碳酸氢钠溶液调节pH=8-9,二氯甲烷萃取三次,合并有机相,无水硫酸钠干燥,过滤,旋干。三氯甲烷重结晶得橙色固体1.29g(3.81mmol),产率为66%。
1H NMR(400MHz,CDCl3)δ8.53(s,1H),7.43(d,J=9.0Hz,1H),6.64(dd,J=9.0,2.5Hz,1H),6.48(d,J=2.4Hz,1H),4.77(s,2H),3.48(q,J=7.2Hz,4H),1.25(t,J=7.1Hz,6H)。
实施例3 3-(2-(溴化三苯基膦盐)乙酰基)-7-(二乙氨基)香豆素(化合物3)的合成:
将化合物2(1.29g,3.81mmol)、三苯基膦(1.12g,4.57mmol)、碘化钾(0.063mg,0.38mmol)溶于二氯甲烷,40℃回流8小时。冷却至室温,减压蒸馏除去有机溶剂,粗产品用200-300目硅胶柱分离,洗脱剂为甲醇/二氯甲烷=1:30(V/V)。最终得棕红色固体1.65g(3.16mmol),产率83%。
1H NMR(400MHz,CDCl3)δ8.87(s,1H),7.80(m,16H),6.63(d,J=7.3Hz,1H),6.43(s,1H),5.83(s,2H),3.45(dd,J=12.8,5.8Hz,4H),1.23(t,J=7.1Hz,6H)。13C NMR(101MHz,CDCl3)δ177.7,161.9,157.2,151.1,144.0,133.2,133.1,132.0,130.3,128.9,128.8,127.3,126.4,108.9,96.5,56.4,55.3,44.8,12.5。HRMS(ESI)C33H31NO3P+[M]+520.2038。
实施例4 3-(2-(三苯基膦)乙酰基)-7-(二乙氨基)香豆素(化合物4)的合成:
将化合物3(0.3g,0.58mmol)溶于10mL二氯甲烷中,加入20mL饱和碳酸钾水溶液,室温剧烈搅拌3小时,分液,二氯甲烷萃取,合并有机相,无水硫酸钠干燥,过滤,旋干。粗产品用200-300目硅胶柱分离,洗脱剂为甲醇/二氯甲烷=1:50(V/V)。最终得棕红色固体0.286g(0.55mmol),产率95%。
1H NMR(400MHz,CDCl3)δ8.60(s,1H),7.73(dd,J=12.5,7.4Hz,6H),7.54(t,J=6.9Hz,3H),7.47(dt,J=7.1,3.5Hz,6H),7.31(d,J=8.7Hz,1H),6.54(dd,J=8.8,2.3Hz,1H),6.48(d,J=2.1Hz,1H),5.55(d,J=28.6Hz,1H),3.40(q,J=7.1Hz,4H),1.20(t,J=7.1Hz,6H)。13C NMR(101MHz,CDCl3)δ177.7,161.9,157.2,151.1,144.0,133.3,133.2,131.9,130.3,128.9,128.8,127.3,126.4,108.9,96.5,56.4,55.4,44.8,12.5。HRMS(ESI)C33H30NO3P[M+H]+520.2040。
实施例5化合物4对5fU核苷的选择性实验:
将化合物4(10mM溶于乙醇,1μL)、核苷(10mM溶于水,50μL)、500μL乙醇以及450μL水加入1.5mL离心管中,37℃反应48小时。反应结束后,扫描荧光光谱(Ex=460nm)。
如图3所示,5fU与化合物4共孵后,化合物4在495nm处的发射峰下降,伴随着555nm处的新峰出现,而A、G、C、T、5hmC、5hmU、5fC与化合物4共孵后,其荧光发射光谱与对照组基本重叠,说明化合物4对5fU具有优异的比率荧光选择性。
实施例6化合物4对ODN-5fU的选择性实验:
将化合物4(10mM溶于乙醇,25μL)、ODN(100μM溶于水,50μL)、以及50μL乙醇加入1.5mL离心管中,37℃反应48小时。反应结束后,蒸干乙醇,加入500μL水,二氯甲烷洗去过量的化合物4(500μl×3),然后扫描水相的荧光光谱(Ex=495nm)。
如图4所示,与ODN-5fC、ODN-T、ODN-C、ODN-AP相比,ODN-5fU与化合物4共孵后,在555nm处出现明显的荧光增强,说明化合物4对ODN-5fU具有优异的荧光选择性。
实施例7化合物4与ODN共孵后的凝胶电泳实验:
将化合物4(0.1mM溶于乙醇,4μL)、ODN(0.1mM溶于水,65μL)、以及31μL乙醇加入1.5mL离心管中,37℃反应48小时。反应结束后,涡旋混匀,取3μL,再加入6μL去离子甲酰胺、1μL loading buffer(上样缓冲液)制成上样溶液。
电泳所用凝胶为20%的变性聚丙烯酰胺,包含1×TBE(89mM硼酸、2mM EDTA(乙二胺四乙酸)、89mM Tris碱(三羟甲基氨基甲烷))、7M尿素等组分。电泳条带的迁移在1×TBE缓冲液中进行,室温下150V恒电压电泳60min。电泳结束后,将凝胶置于凝胶成像系统中对化合物4标记的ODN-5fU成像,然后将凝胶用3×Gel-Red浸染45min,再将其置于凝胶成像系统中对不能被化合物4标记的ODN-5fC、ODN-T、ODN-C、ODN-AP成像。
如5图所示,Gel-Red染色前,在凝胶成像系统中仅ODN-5fU对应的跑道呈现绿色荧光,此绿色荧光来源于标记在ODN-5fU上的化合物4,而其他DNA(ODN-5fC、ODN-T、ODN-C、ODN-AP)序列中不含有5-醛基尿嘧啶(5fU)或其他能与化合物4发生反应的位点,所以不能被化合物4标记并在对应的跑道上呈现化合物4的绿色荧光;Gel-Red染色后,所有跑道均呈现出绿色荧光,此绿色荧光来源于核酸染色剂Gel-Red。此实验再一次验证了化合物4对ODN-5fU的高度特异性与灵敏性。
实施例8化合物4对HeLa细胞(子宫颈癌细胞)中的5-醛基尿嘧啶(5fU)的共聚焦成像:
首先,将HeLa细胞置于含有10%胎牛血清(FBS)、1%双抗的(青霉素-链霉素,1000KU/L)的DMEM(H)培养基中,通5%CO2,37℃培育24小时。然后将细胞暴露在60Coγ射线源下以18Gy/min的速率辐射60min,辐射结束后,弃去培养基,用PBS洗3次,加入80%乙醇固定20min,再换成70%乙醇于4℃冰箱中保存。最后向其中加入1μL化合物4的乙醇母液(10mM),37℃孵化16h,孵化结束后,取出培养皿,用PBS(磷酸盐缓冲溶液)洗3次,并将其置于荧光共聚焦显微镜上拍照得成像图7。其中,a-c图的细胞辐射过γ射线但未与化合物4共孵化;d-f图的细胞未辐射过γ射线但与化合物4共孵化;g-i图的细胞既辐射过γ射线又与化合物4共孵化。
图7中激发光为488nm,收集500-600nm波段。从图7可以看出,未经γ射线辐射的细胞,细胞内的5fU含量极低,与化合物4共孵后仅呈现出很微弱的绿色荧光;而经γ射线辐射后的细胞,细胞内的5fU含量大大提高[6],与化合物4共孵后,绿色荧光明显增强,进一步对d、g两图中的荧光强度进行测量,发现g图的平均荧光强度约为d图的2倍,说明化合物4能够检测细胞内的5-醛基尿嘧啶(5fU)。
实施例9化合物4的MTT细胞毒性实验:
将处于对数生长期的Hela细胞接种于96孔培养板中,每孔接种10000个细胞,用含10%胎牛血清(FBS)、1%双抗的(青霉素-链霉素,1000KU/L)的DMEM(H)培养基在37℃,5%CO2条件下培养过夜。待细胞完全贴壁,加入不同浓度梯度的化合物4,每个浓度设3个复孔,同时设空白对照组。加药后继续培养24小时,MTT法检测细胞的抑制率。
如图6所示,在浓度范围1.25~20μM范围内,化合物4的细胞毒性非常小。
本发明有效结合荧光传感技术和有机人名反应,将香豆素荧光骨架引入传统的Wittig试剂,设计并合成了首例比率型小分子荧光探针用于DNA中5-醛基尿嘧啶(5fU)的选择性识别与定量检测,该试剂具有选择性好、灵敏度高、毒副作用小、原料经济易得、整条合成路线可操作性强、反应条件温和、总体成本较低等优势,为反应型5-醛基尿嘧啶(5fU)荧光探针的设计提供了新思路。
参考文献:
1、Bauer,N.C.;Corbett,A.H.;Doetsch,P.W.,Nucleic Acids Res.2015,43,10083-10101。
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序列表
<110> 四川大学
<120> 基于香豆素骨架的Wittig试剂的制备方法及其用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gactcaacag ccgta 15
Claims (11)
2.根据权利要求1所述的基于香豆素骨架的Wittig试剂,其特征在于:R1~R3独立地为C1~C4烷基。
5.根据权利要求4所述的基于香豆素骨架的Wittig试剂,其特征在于:R为二乙基氨基。
6.权利要求1~5任意一项所述的基于香豆素骨架的Wittig试剂的制备方法,包括以下步骤:
a、将R取代的水杨醛和乙酰乙酸乙酯溶于有机溶剂,加入哌啶,回流4~12小时,制备得到中间体1;
b、将中间体1溶于冰醋酸,加入液溴和氢溴酸,室温搅拌过夜,制备得到中间体2;
c、将中间体2,三苯基膦,碘化钾溶于有机溶剂,回流5~12小时,制备得到中间体3;
d、将中间体3溶于有机溶剂,加入碱水溶液,室温剧烈搅拌0.5~3小时,制备得到基于香豆素骨架的Wittig试剂。
7.根据权利要求6所述的基于香豆素骨架的Wittig试剂的制备方法,其特征在于:步骤a所述的乙酰乙酸乙酯的摩尔量为R取代水杨醛的1.5~2.5倍;所述哌啶的摩尔量为R取代水杨醛的0.1~1.6倍;所述的有机溶剂为无水乙醇、异丙醇、乙腈或二氯甲烷中的任意一种。
8.根据权利要求6所述的基于香豆素骨架的Wittig试剂的制备方法,其特征在于:步骤b所述的液溴的摩尔量为中间体1的1.5~2.0倍,所述氢溴酸的摩尔量为中间体1的3~5倍。
9.根据权利要求6所述的基于香豆素骨架的Wittig试剂的制备方法,其特征在于:步骤c所述的三苯基膦的摩尔量为中间体2的1.2~2.5倍;所述碘化钾的摩尔量为中间体2的0.1~0.5倍;所述的有机溶剂为二氯甲烷、甲苯、四氢呋喃、乙腈、甲醇或N,N-二甲基甲酰胺中的任意一种。
10.根据权利要求6所述的基于香豆素骨架的Wittig试剂的制备方法,其特征在于:步骤d所述的有机溶剂为二氯甲烷、甲苯、四氢呋喃、乙腈、甲醇或N,N-二甲基甲酰胺中的任意一种;所述碱水溶液的体积为有机溶剂体积的20%~80%;所述碱水溶液中的碱为碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾或三乙胺中任意一种,其摩尔量为中间体3的2~10倍。
11.权利要求1~5任意一项所述的基于香豆素骨架的Wittig试剂在对5-醛基尿嘧啶进行荧光识别中的用途。
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