CN106811192B - 耐碳青霉烯类抗生素病菌的荧光探针及其合成方法与应用 - Google Patents
耐碳青霉烯类抗生素病菌的荧光探针及其合成方法与应用 Download PDFInfo
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- CN106811192B CN106811192B CN201710027441.4A CN201710027441A CN106811192B CN 106811192 B CN106811192 B CN 106811192B CN 201710027441 A CN201710027441 A CN 201710027441A CN 106811192 B CN106811192 B CN 106811192B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D477/00—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring
- C07D477/10—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
- C07D477/12—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6
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Abstract
本发明耐碳青霉烯类抗生素病菌的荧光探针,其结构通式为:式中:X为碳原子或硫原子;当X为CH时,R1为甲基,能够为R或S构型;或者X为CH2或S;染料为氟硼二吡咯、萘酰亚胺、香豆素、荧光素或罗丹明的任意一种。所述荧光探针的合成方法包括以下步骤:⑴化合物3的制备;⑵化合物4的制备;⑶荧光探针CVB‑1的制备。将所述荧光探针制成试纸、试剂盒或者检测芯片可在检测碳青霉烯酶和含有碳青霉烯耐药菌中进行应用,通过荧光探针在荧光强度或颜色变化与否的现象来检测或区分碳青霉烯酶,进而快速检测有碳青霉烯酶表达的致病耐药菌,指导在治疗或临床上合理使用抗生素药物,对不用或少用抗生素药物具有重要的意义。
Description
技术领域
本发明涉及化合物制备技术领域,具体地说,是一种耐碳青霉烯类抗生素病菌的荧光探针及其合成方法以及所述荧光探针在检测碳青霉烯酶和含有碳青霉烯耐药菌中的应用。
背景技术
碳青霉烯类抗生素是一类具有特殊结构的β-内酰胺类抗生素,是由β-内酰胺环并五元环组成抗生素母核,其四元环上的取代基为反式结构。碳青霉烯类抗生素与其它类的β-内酰胺抗生素的构型相反,主要包括亚胺培南、美罗培南、多利培南及厄他培南等。因碳青霉烯类抗生素拥有广泛的抗菌活性且具有强烈抑制或杀灭不同类型细菌的能力(J.Antimicrob.Agents.1999,11,93;Antimicrob.Agents Chemother.2011,55,4943),使该类抗生素成为治疗细菌性严重感染的最后一道防线(Emerg.Infect.Dis.2011,17,1791-1798)。碳青霉烯类抗生素对革兰氏阴性与阳性菌引起的感染具有很好的疗效,对含有青霉素酶、头孢菌素酶的耐药菌亦具有很好的疗效。但是,随着此类抗生素的广泛使用,近年来在世界范围内逐渐出现了对之具有耐药性的细菌。研究发现,致病细菌产生耐药性的原因很多,如β-内酰胺抗生素的靶标青霉素结合蛋白发生了突变;另一个主要原因是细菌体内产生了一种叫β-内酰胺酶(β-lactamase)的抗生素灭活酶,这种酶能迅速水解常用的β-内酰胺类抗生素的β-内酰胺键,使抗生素失去药效。这类能使青霉烯类抗生素失效的β-内酰胺酶被称为碳青霉烯酶(carbapenemase)。所述碳青霉烯酶一般能水解碳青霉烯抗生素甚至几乎所有的β-内酰胺类抗生素,使之对碳青霉烯抗生素(亚胺培南、美罗培南等)的敏感性降低,治疗效果变差。
按Ambler分类法,所述β-内酰胺酶(碳青霉烯酶)可分为A、B、C、D四个类型(PhilosTrans R Soc Lond B Biol Sci 1980;289:321–31),其中A、C、D三个类型为含有丝氨酸的β-内酰胺酶;B类为含有金属锌离子的β-内酰胺酶。所述A类β-内酰胺酶包括KPC、GES类型的β-内酰胺酶等,能够在许多致病菌里检测到,例如肠杆菌属、粘质沙雷氏菌、克雷伯菌属等等。早在1996年,美国就从北卡罗来纳州一临床病例中分离出了含KPC酶的克雷伯菌,研究发现,这一菌株能够耐所有抗生素药。之后,在新德里的医疗机构和生态环境中发现了属于B类β-内酰胺酶的新德里含金属β-内酰胺酶(NDM),这些酶的活性中心锌离子能与酰胺键结合,使抗生素水解失去药效。所述新德里含金属β-内酰胺酶的致病菌又被称为“超级细菌”,这类“超级细菌”在被发现后曾很快通过病人和游客向世界各地传播,引起世界范围内的担忧。在中国,2005~2014年间对碳青霉烯抗生素具有耐药性的克雷伯肺炎菌的比例由2.4%上升为13.4%(Clin Microbiol Infect 2016;22:S9–S14)。
目前,检测有碳青霉烯酶表达的细菌的方法主要有三种:表型检测法、基因检测法和基于碳青霉烯酶的检测法。所述表型检测法通常是通过测定细菌对碳青霉烯抗生素的敏感性来检测的,它又包括琼脂扩散测试法、最小抑菌浓度测定(MIC)法、双纸片协同测试法(DDST)、修饰的霍奇检测法(MHT)等。美国临床与实验室标准研究所基于这些方法制定了标准的抗生素药敏试验来检测致病菌的药敏性,它在一定程度上反映了细菌的耐药性,也给细菌性严重感染病人的治疗带来极大的帮助。但是,这些方法缺乏良好的专一性和灵敏性,且检测耗时长,通常需要24~48小时,此外,这类方法无法及时提供选择抗生素的必要信息。所述基因检测法主要是通过聚合酶链反应(PCR)来进行检测,通过实时定量逆转录酶-PCR(rt qRT-PCR)能检测碳青霉烯酶编码基因的mRNA表达。所述基因检测法具有很高的精确性和灵敏度,但是,它使用的仪器昂贵、花费高且不能检测到新的碳青霉烯酶基因。所述基于碳青霉烯酶的检测法,它通过检测碳青霉烯酶的活性能提供细菌抗生素耐力的重要信息。通过比色原理用头孢硝基噻吩(Nitrocefin)能成功地检测碳青霉烯酶,其检测原理为:头孢硝基噻吩的β-内酰胺环能被β-内酰胺酶快速水解而开环,由于底物分子共轭结构的变化从而使其颜色由黄色变成红色,通过颜色的变化来检测其耐药菌,操作简单,使用方便,但是,由于颜色变化比较缓慢、灵敏度不高等原因在很大程度制约了该检测方法的实际应用。
近年来,荧光探针受到了人们广泛的关注。所述荧光探针具有背景信号低、灵敏度高、仪器成本低等诸多优点,使其在生物医药领域的应用广受关注,目前,已有较多的荧光探针化合物应用于高灵敏度和实时对β-内酰胺酶的检测。1998年,诺贝尔奖获得者钱永健教授在《科学》杂志上撰文(Science 1998,279,84-88),首次报道了基于β-内酰胺结构的分子荧光探针,利用荧光能量转移(FRET)机理,用于监测β-内酰胺酶的活性。之后,陆陆续续报道了一些检测β-内酰胺酶活性的荧光探针化合物。但是,这些荧光探针化合物是基于头孢菌素母核设计的,它们不能区分普通β-内酰胺酶和碳青烯酶。国际专利申请WO2012/003955A1公开了一种“基于碳青霉烯母核结构设计的具有一定荧光性能的碳青霉烯酶荧光探针”,但该专利申请所述的荧光探针没有说明具体的光学性质,没有引入典型的荧光报告基团,故而其检测性能是不清楚的。2014年,Rao等人报道了用于检测对碳青霉烯具有耐药性的肠科杆菌的荧光探针(Angew.Chem.Int.Ed.2014,53,8113–8116),所述荧光探针是基于常见的头孢菌素母核设计的,它通过结构调整,引入香豆素作为荧光报告基团后,将6,7-位的构象由顺式转变为反式构象,合成一系列的荧光探针化合物,从而实现对β-碳青霉烯酶及表达有碳青霉烯酶细菌的选择性检测,但是,其不同的荧光探针对不同的碳青霉烯酶的选择性和检测灵敏度有很大的不同。此外,华东理工大学在2016年的一项专利申请也提出了一种耐碳青霉烯类抗生素病菌的荧光探针,所述荧光探针以碳青霉烯抗生素的母核结构为酶特异性识别基团,在碳青霉烯抗生素母核的3'-位引入具有离去功能的染料,在碳青霉烯酶的作用下使β-内酰胺环开环,从而产生荧光信号,实现对碳青霉烯酶的选择性检测。但是,这一荧光探针主要是基于香豆素为报告基团(fluorophore)的,其激发光位于紫外区域,发色光位于蓝色光区域,易于被病菌样本可能存在的自身荧光信号所干扰,从而降低荧光探针检测的灵敏度。
发明内容
本发明的目的在于克服现有技术的不足,提供一类耐碳青霉烯类抗生素病菌的荧光探针,它们是基于青霉烯类抗生素设计的用于检测产生碳青霉烯酶致病菌的荧光探针,具有高灵敏度、高选择性、且使用费用低。本发明的第二目的是,提供所述荧光探针的合成方法。本发明的第三目的是,提供所述荧光探针在检测碳青霉烯酶和含有碳青霉烯耐药菌中的具体应用方法。
为实现上述目的,本发明采取了以下技术方案。
一类耐碳青霉烯类抗生素病菌的荧光探针,其特征在于,所述荧光探针的结构通式为:
式中:
X为碳原子或硫原子;当X为CH时,R1为甲基,能够为R或S构型;或者X为CH2或S;
染料(dye)为氟硼二吡咯(BODIPY)、萘酰亚胺、香豆素、荧光素或罗丹明的任意一种。
进一步,所述氟硼二吡咯(BODIPY)的通式结构为:
式中:
R2、R3、R4、R5、R6独自选自氢、甲基、乙基;
R7、R8、R9、R10和R11独自选自氢、甲基、乙基、卤素、烷氧基、羟基、羧基、磺酸基、氰基、醛基、酯基或聚乙二醇基,其中的含酸基团(如羧基、磺酸基),包括其锂、钠、钾、镁、钙盐类。
进一步,含有氟硼二吡咯(BODIPY)通式结构中荧光探针的优先结构(CVB-1)为:
进一步,所述优先荧光探针能与生物标记物碳青霉烯酶选择性识别,从而起到检测碳青霉烯酶的作用。
进一步,所述萘酰亚胺的通式结构为:
式中:
R选自1至6碳的烷基、饱和羧基、饱和酯基或聚乙二醇基。
一类耐碳青霉烯类抗生素病菌的荧光探针,其特征在于,所述荧光探针是在培南类(penem)抗生素母核的3-位引入双键后,再与BODIPY(氟硼二吡咯)的2-位相连得到荧光探针,在碳青霉烯酶(carbapenemase)的作用下使β-内酰胺环开环,使整个荧光探针发生一定的结构变化,从而产生光学性质的变化,使所述荧光探针具有用于检测青霉烯酶及其耐药菌的性质,其检测原理表示为:
为实现上述第二目的,本发明采取了以下技术方案。
一种耐碳青霉烯类抗生素病菌的荧光探针的合成方法,其特征在于,包括以下步骤:
(1)化合物3的制备
将将化合物1、化合物2置于反应瓶中,加入N,N-二甲基甲酰胺(DMF)及三乙胺(TEA),将所得混合物经冷冻-真空-溶解三个循环脱气后,加入醋酸钯(PdOAc2)和三(邻甲基苯基)膦(P(o-Tol)3,再脱气两次;
将反应体系在氮气氛围下80℃反应16小时,冷却后,加入乙酸乙酯稀释反应体系,将乙酸乙酯相用水洗,再用饱和食盐水洗,用无水硫酸钠干燥、浓缩;
将所得混合物用石油醚与乙酸乙酯作为流动相经硅胶柱柱层析纯化后得到化合物3;
(2)化合物4的制备
将步骤(1)得到的化合物3溶解于N-甲基吡咯烷酮(NMP)与N,N-二甲基甲酰胺的混合液中,N-甲基吡咯烷酮与N,N-二甲基甲酰胺的体积比为1:3;
加入二氟化氢铵(NH4·HF2),在室温下反应;
反应结束后加入乙酸乙酯稀释反应体系,将乙酸乙酯相用水洗,再用饱和食盐水洗,用无水硫酸钠干燥、浓缩;
将所得混合物用石油醚与乙酸乙酯作为流动相经硅胶柱柱层析纯化后得到化合物4;
(3)荧光探针(CVB-1)的制备
将步骤(2)得到的化合物4溶解于四氢呋喃(THF)中,加入pH为6.0的0.35M磷酸盐缓冲液(PB)以及活化锌粉(Zn),在20℃下反应1小时;
将反应液过滤,用色谱乙腈洗涤,用反相C18制备柱纯化,冷冻干燥,得到黑紫色的化合物,即所述的荧光探针(CVB-1);
所述荧光探针(CVB-1)的合成路径为:
(该合成路径的主要特征在于:将化合物1通过与碘、溴或三氟甲磺酸酯取代的染料〈以碘取代的氟硼二吡咯2为例〉通过Heck偶联反应,实现母核与荧光报告基团的共轭偶联;再经过两步脱保护,得到优选荧光探针CVB-1)。
进一步,所述化合物1为关键中间体,以此为衍生物能够合成其他的荧光探针化合物。
为实现上述第三目的,本发明采取了以下技术方案。
将所述耐碳青霉烯类抗生素病菌的荧光探针制成试纸、试剂盒或者检测芯片在检测碳青霉烯酶和含有碳青霉烯耐药菌中的应用。
进一步,所述应用包括以下步骤:
(1)将耐碳青霉烯类抗生素病菌的荧光探针与待测样品在一定条件下混合,形成具有光学性质的化合物;
(2)测量具有光学性质的化合物的光学信号变化,包括荧光、紫外吸收或颜色的变化,从而确定待测样品中碳青霉烯酶或者含有碳青霉烯耐药菌的含量或浓度。
本发明的积极效果:
(1)本发明对原有的碳青霉烯酶荧光探针重新进行了设计,利用自己研究的荧光增强机理,克服了原先研究的荧光探针的缺点,发展了新的、检测灵敏度更高、检测范围更广的荧光探针。
(2)基于碳青霉烯抗生素为母核,保证了荧光探针对碳青霉烯酶的专一性响应,对非碳青霉烯酶不产生响应。
(3)本发明的荧光探针(CVB-1)是在3-位引入与母核共轭的氟硼二吡咯作为荧光报告基团,在碳青霉烯酶水解触发后,经自动结构变化后产生荧光响应。
(4)以氟硼二吡咯作为荧光报告基团,其激发波长为503nm,最大发射波长在512nm,激发发射光波长且在绿色光范围内,能大大提高对碳青霉烯酶、耐药致病菌或包含有前两者的血样、尿样的检测的灵敏度。
(5)荧光探针(CVB-1)在碳青霉烯酶的作用下不仅能产生荧光变化,还能发生颜色变化,因此,可通过颜色变化来检测样品,不需要借助仪器,使其应用更简单方便。
(6)本发明提供的荧光探针可应用于临床耐药菌检测,可快速检测有碳青霉烯酶表达的细菌及其耐药菌并具有高选择性、高精确性和高灵敏度,且使用费用低、简便易行,对在医疗中不用或者少用抗生素药物具有非常重要的意义。
附图说明
图1为本发明耐碳青霉烯类抗生素病菌的荧光探针的合成方法的流程框图。
图2为应用实施例1荧光探针在碳青霉烯酶IMP-1下吸收光谱变化图。
图3为应用实施例1荧光探针(100μM)在有碳青霉烯酶IMP-1(100nM)与无碳青霉烯酶IMP-1(0nM)情况下的颜色变化。
图4为应用实施例1荧光探针在一定浓度的碳青霉烯酶下随时间的荧光光谱变化图。
图5为应用实施例2不同β-内酰胺酶与荧光探针混合培养1小时的荧光变化图。
图6为应用实施例3含不同重组β-内酰胺酶大肠杆菌与荧光探针混合培养2小时的荧光变化图。
图7为应用实施例3含不同临床耐药菌及野生大肠杆菌酶与荧光探针混合培养2小时的相对荧光强度图。
具体实施方式
以下结合附图具体介绍本发明的具体实施方式和相关检测效果。但是需要指出,本发明的实施不限于以下的实施方式,实施例中未注明具体条件的实验方法,通常是按照常规条件或者是按照制造厂商所建议的条件进行。
本发明的耐碳青霉烯类抗生素病菌的荧光探针,是在培南类(penem)抗生素母核的3-位引入双键后再与氟硼二吡咯的2-位相连得到荧光探针,在碳青霉烯酶(carbapenemase)的作用下使β-内酰胺环开环,使整个荧光探针发生一定的结构变化,从而产生光学性质的变化,使所述荧光探针具有用于检测青霉烯酶及其耐药菌的性质,其结构通式的表示为:
式中:
X为碳原子或硫原子;当X为CH时,R1为甲基,能够为R或S构型;或者X为CH2或S;
R2、R3、R4、R5、R6独自选自氢、甲基、乙基,其中,R2-5优选为甲基,R6优选为氢;
R7、R8、R9、R10和R11独自选自氢、甲基、乙基、卤素、烷氧基、羟基、羧基、磺酸基、氰基、醛基、酯基或聚乙二醇基,其中,R7-11优选为氢;其中的含酸基团(如羧基、磺酸基)包括其锂、钠、钾、镁、钙盐类。
以下介绍本发明耐碳青霉烯类抗生素病菌的荧光探针的合成方法的具体实施方式。说明:在本发明的实施中,1H-NMR用Bruker 400Mz型仪器测定,测定溶剂为氘代三氯甲烷(CDCl3),内标为四甲基硅烷(TMS);所有溶剂均为色谱纯、分析纯或化学纯,所使用的无水溶剂均是按标准方法干燥处理获得的。产品的纯化除另有说明以外均使用硅胶柱色谱法,所使用的硅胶为200~300目。
实施例1(参见图1)
一种耐碳青霉烯类抗生素病菌的荧光探针的合成方法,其化合物3的制备方法为:
将化合物1〔0.39mmol(毫摩尔),189mg(毫克)〕、化合物2(0.3mmol,135mg)置于反应瓶中,加入N,N-二甲基甲酰胺1.2mL以及三乙胺0.3mL;将所得混合物经过冷冻-真-溶解三个循环脱气后,加入醋酸钯0.03mmol(6.7mg)和三(邻甲基苯基)膦0.045mmol(13.7mg),再脱气两次。
将反应体系在氮气氛围下80℃反应16h,冷却后加入乙酸乙酯稀释反应体系,将乙酸乙酯相用水洗,再用饱和食盐水洗,用无水硫酸钠干燥、浓缩。
将所得混合物用石油醚与乙酸乙酯作为流动相经硅胶柱柱层析纯化后得到化合物3(57mg)产率为24%。
其合成路径及结构通式为:
谱图特征为:1H NMR(400MHz,CDCl3)δ8.20(d,J=8.7Hz,2H),7.66(d,J=8.7Hz,2H),7.49(m,4H),7.30–7.27(m,2H),6.71(d,J=16.7Hz,1H),6.04(s,1H),5.44(d,J=14.0Hz,1H),5.25(d,J=14.0Hz,1H),4.31–4.24(m,1H),4.22(dd,J=9.2,2.5Hz,1H),3.54–3.44(m,H),3.24(dd,J=5.5,2.6Hz,1H),2.68(s,3H),2.58(s,3H),1.46(s,3H),1.38(s,3H),1.27(d,J=3.3Hz,3H),1.26(d,J=3.2Hz,3H),0.86(s,9H),0.09(s,3H),0.08(s,3H).13C NMR(101MHz,CDCl3)δ172.75,161.20,157.60,154.57,150.50,147.64,144.75,143.16,142.07,138.71,134.92,132.47,131.08,129.38,129.29,128.13,128.11,127.67,127.34,124.27,123.79,122.39,121.89,66.02,65.14,59.10,56.23,38.93,25.80,22.58,18.04,17.20,14.86,14.71,13.87,13.17,-4.13,-4.85.HRMS(ESI)m/z calcd forC44H51BF2N4NaO6Si(M+Na)+831.3537,found 831.3547.
实施例2(参见图1)
一种耐碳青霉烯类抗生素病菌的荧光探针的合成方法,其化合物4的制备方法为:
将实施例1制备的化合物3共57mg(0.07mmol)溶解于0.7毫升的N-甲基吡咯烷酮/N,N-二甲基甲酰胺中,N-甲基吡咯烷酮与N,N-二甲基甲酰胺的体积比为1:3;
之后加入二氟化氢铵15.9mg(0.28mmol),在室温(25℃)下反应36h;
反应结束后加入乙酸乙酯稀释反应体系,将乙酸乙酯相分别用水和饱和食盐水洗,再用无水硫酸钠干燥后浓缩;
将得到的混合物用石油醚与乙酸乙酯作为流动相经硅胶柱柱层析纯化后得到化合物4共39.6毫克,产率为79%。
其合成路径及结构通式为:
谱图特征为:1H NMR(400MHz,CDCl3)δ8.22(d,J=8.8Hz,2H),7.66(d,J=8.8Hz,2H),7.54–7.48(m,3H),7.45(d,J=16.8Hz,1H),7.29–7.26(m,2H),6.72(d,J=16.7Hz,1H),6.05(s,1H),5.49(d,J=13.8Hz,1H),5.23(d,J=13.9Hz,1H),4.31–4.25(m,1H),4.23(dd,J=9.0,2.5Hz,1H),3.61–3.51(m,1H),3.28(dd,J=6.7,2.5Hz,1H),2.66(s,3H),2.58(s,3H),1.44(s,3H),1.38(s,3H),1.28(s,3H),1.27(s,3H).
实施例3(参见图1)
一种耐碳青霉烯类抗生素病菌的荧光探针的合成方法,其荧光探针(CVB-1)的制备方法为:
将实施例2得到的化合物4共13.9mg(0.02mmol)溶解于0.5毫升的四氢呋喃中,加入pH为6.0的0.35M磷酸盐缓冲液(PB)0.3mL以及活化锌粉26.2mg(0.02mmol),在20℃下反应1h;
将反应液过滤,用色谱乙腈洗涤,用反相C18制备柱纯化,冷冻干燥,得到黑紫色的化合物,即所述的荧光探针(CVB-1)。
所述荧光探针(CVB-1)的合成路径及结构通式为:
谱图特征为:1H NMR(400MHz,CDCl3)δ7.63(d,J=16.9Hz,1H),7.50–7.44(m,2H),7.32–7.26(m,2H),6.46(d,J=16.9Hz,1H),6.00(s,1H),4.30–4.19(m,1H),4.15(d,J=8.9Hz,1H),3.47–3.37(m,1H),3.20(d,J=4.7Hz,1H),2.70(s,3H),2.56(s,3H),1.42(s,3H),1.37(s,3H),1.33(d,J=6.0Hz,3H),1.22(d,J=7.1Hz,3H).HRMS(ESI)m/z calcd forC31H31BF2N3O4(M-1)-558.2376,found 558.2377.
用本发明的合成方法制备的荧光探针制成试纸、试剂盒或者检测芯片可在生物、预防保健、临床诊断各领域检测碳青霉烯酶和含有碳青霉烯耐药菌中的进行应用。以下提供3个应用实施例以对本发明所述的应用进行说明。
应用实施例1
本发明制备的荧光探针在检测碳青霉烯酶和含有碳青霉烯耐药菌中的应用,包括以下步骤:
(1)将本发明的耐碳青霉烯类抗生素病菌的荧光探针与待测样品在一定条件下混合,形成具有光学性质或颜色变化的化合物(参见图2、3、4)。
从图2可知:荧光探针CVB-1在IMP-1的作用下吸收发生明显变化,最终产生典型的氟硼二吡咯的吸收谱图。
从图3可知:荧光探针CVB-1在IMP-1的作用下吸收发生明显颜色变化,由紫色变为粉色。
从图4可知:荧光探针CVB-1在碳青霉烯酶作用下吸收荧光显著增强。
(2)测量具有光学性质的化合物的光学信号变化或观察荧光探针的颜色变化,从而确定待测样品中碳青霉烯酶或者含有碳青霉烯耐药菌的含量或浓度。
应用实施例2
测试碳青霉烯酶为A类重组表达的碳青霉烯酶KPC-3;含有锌离子的B类重组表达的碳青霉烯酶VIM-27、IMP-1、NDM-1;D类碳青霉烯酶OXA-48以及非碳青霉烯酶TEM-1、TEM-3、CTX-M-9。
测定荧光探针在β-内酰胺酶作用下的荧光变化:
将检测样品(β-内酰胺酶酶或耐药菌)混在磷酸盐缓冲液{1X PBS,pH=7.4,0.1%表面活性剂〔CHAPS,3-[3-(胆酰胺丙基)二甲基铵]-1-丙磺酸内盐〕}中置于酶标微板内,在室温(25℃)下通过酶标仪测量荧光强度的变化,激发波长为500nm,发射波长为535nm,监测1h左右。
测定结果参见图5,不同β-内酰胺酶(碳青霉烯酶VIM-27、IMP-1、KPC-3、NDM-1、OXA-48及非碳青霉烯酶TEM-1、TEM-3、CTX-M-9及没有酶)与荧光探针混合培养1小时的荧光变化。从图5可以看出:荧光探针CVB-1在低浓度的碳青霉烯酶(IM-27、IMP-1、KPC-3、NDM-1、OXA-48)的作用下即可发生明显的荧光强度的变化,而在没有酶或高浓度非碳青霉烯酶的作用下,荧光探针CVB-1的荧光强度并不发生明显的变化;这一荧光强度变化与否的现象说明:本发明制备的荧光探针CVB-1能用于检测或区分碳青霉烯酶。
应用实施例2只是检测样品的通用条件。需要指出的是:应用实施例2中使用的检测样品(酶样、菌样、血液样品、尿液样品等)、各种试剂(缓冲体系、酶稳定剂等)、检测条件(酸碱度、温度等)并不局限于上述待检测样品和通用条件。
应用实施例3
本发明制备的荧光探针在检测表达有重组碳青霉烯酶的大肠埃希菌和临床菌中的应用
将表达有不同β-内酰胺酶的临床大肠杆菌在LB培养基中37℃培养过夜,通过测量600nm处的吸光度得到相应的细菌数量并用CFU/mL(每毫升的菌落形成单位)进行表示。对于每个用于研究的菌株按照1︰10的四个梯度进行一系列的稀释;将细菌检测实验在黑色的384孔板上进行(总体积为15μL)。在所述384孔板上的每个孔内同时加入5μL的梯度稀释的细菌,然后加入10μL的7.5μM荧光探针CVB-1,将得到的待检测样品在25℃室温下通过酶标仪监测荧光强度的变化。
检测结果见图6——含不同重组β-内酰胺酶大肠杆菌与荧光探针CVB-1混合培养2小时的荧光变化图。图7——含不同临床耐药菌(VIM-27、IMP-1、KPC-3、TEM-1)及野生大肠杆菌酶与荧光探针混合培养2小时的相对荧光强度图。
在图7中:
NDM-1-Kp为NDM-1克雷伯肺炎菌(ATCC BAA2146)。
VIM-1-Kp为VIM-1克雷伯肺炎菌(NCTC 13440)。
MDR-Ab为多耐药鲍氏不动杆菌(ATCC BAA1605)。
OXA-48-Kp为OXA-48克雷伯肺炎菌(NCTC 13442)。
TEM-3-E.coli为TEM-3大肠杆菌(NCTC 13351)。
CTX-M-9-Ec为CTXM-9阴沟肠杆菌(NCTC 13464)。
SHV-18-Kp为SHV-18克雷伯肺炎菌(ATCC 700603)。
TEM-1-E.coli为EM-1大肠杆菌(ATCC 35218)。
E.coli为不含内酰胺酶的大肠杆菌(LMG194)。
从图6、图7可以看到:在直接荧光成像后,只有表达有碳青霉烯酶的临床耐药菌能对荧光探针CVB-1产生响应,而且产生的荧光信号明显。而对照组非碳青霉烯酶的临床耐药菌以及野生型大肠杆菌都不能对荧光探针CVB-1产生响应,它们几乎没有荧光信号产生。
应用实施例1~3的结果证明:
本发明的耐碳青霉烯类抗生素病菌的荧光探针能够应用于对碳青霉烯酶和含有碳青霉烯耐药菌的检测,可通过所述荧光探针在荧光强度或颜色变化与否的现象来检测或者区分碳青霉烯酶,进而可快速检测有碳青霉烯酶表达的致病耐药菌,指导在医疗临床上合理使用抗生素药物。此外,本发明的耐碳青霉烯类抗生素病菌的荧光探针在生物、预防保健、临床诊断各领域也具有潜在的、积极的应用价值。
Claims (5)
1.一类耐碳青霉烯类抗生素病菌的荧光探针,其特征在于,所述荧光探针的结构通式为:
式中:
X为碳原子或硫原子;当X为CH时,R1为甲基,能够为R或S构型;或者X为CH2或S;
染料为氟硼二吡咯。
2.根据权利要求1所述的耐碳青霉烯类抗生素病菌的荧光探针,其特征在于,含有氟硼二吡咯通式结构中荧光探针的优先结构为:
所述优先结构的荧光探针能与生物标记物碳青霉烯酶选择性识别,从而起到检测碳青霉烯酶的作用。
3.一类耐碳青霉烯类抗生素病菌的荧光探针,其特征在于,所述荧光探针是在培南类抗生素母核的3-位引入双键后,再与氟硼二吡咯的2-位相连得到荧光探针,在碳青霉烯酶的作用下使β-内酰胺环开环,使整个荧光探针发生一定的结构变化,从而产生光学性质的变化,使所述荧光探针具有用于检测青霉烯酶及其耐药菌的性质,其检测原理表示为:
4.一种耐碳青霉烯类抗生素病菌的荧光探针的合成方法,其特征在于,包括以下步骤:
(1)化合物3的制备
将将化合物1、化合物2置于反应瓶中,加入N,N-二甲基甲酰胺及三乙胺,将所得混合物经冷冻-真空-溶解三个循环脱气后,加入醋酸钯和三(邻甲基苯基)膦,再脱气两次;
将反应体系在氮气氛围下80℃反应16小时,冷却后,加入乙酸乙酯稀释反应体系,将乙酸乙酯相用水洗,再用饱和食盐水洗,用无水硫酸钠干燥、浓缩;
将所得混合物用石油醚与乙酸乙酯作为流动相经硅胶柱柱层析纯化后得到化合物3;
(2)化合物4的制备
将步骤(1)得到的化合物3溶解于N-甲基吡咯烷酮与N,N-二甲基甲酰胺的混合液中,N-甲基吡咯烷酮与N,N-二甲基甲酰胺的体积比为1:3;
加入二氟化氢铵,在室温下反应;
反应结束后加入乙酸乙酯稀释反应体系,将乙酸乙酯相用水洗,再用饱和食盐水洗,用无水硫酸钠干燥、浓缩;
将所得混合物用石油醚与乙酸乙酯作为流动相经硅胶柱柱层析纯化后得到化合物4;
(3)荧光探针CVB-1的制备
将步骤(2)得到的化合物4溶解于四氢呋喃中,加入pH为6.0的0.35M磷酸盐缓冲液以及活化锌粉,在20℃下反应1小时;
将反应液过滤,用色谱乙腈洗涤,用反相C18制备柱纯化,冷冻干燥,得到黑紫色的化合物,即所述的荧光探针CVB-1;
所述荧光探针CVB-1的合成路径为:
5.根据权利要求4所述的合成方法,其特征在于,所述化合物1为关键中间体,以此为衍生物能够合成其他的荧光探针化合物。
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