CN112209928B - 共价键标记丝氨酸类beta-内酰胺酶的试剂及制备和应用 - Google Patents
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Abstract
本发明公开了一种共价键标记丝氨酸类beta‑内酰胺酶的试剂及制备和应用。本发明的标记试剂,具有式I所示结构,各取代基的定义如说明书和权利要求书所述。通过蛋白及耐药细菌实验表明,本发明的标记试剂具有较强的对丝氨酸类beta‑内酰胺酶及表达丝氨酸类beta‑内酰胺酶的耐药病菌的靶向性。
Description
技术领域
本发明涉及生物标记及生物医药领域,具体地说,涉及一种共价键标记丝氨酸类beta-内酰胺酶的试剂及其制备方法与所述试剂在标记和靶向丝氨酸类beta-内酰胺酶及表达有丝氨酸类β-内酰胺酶的耐药菌中的应用。
背景技术
Beta-内酰胺类抗生素是目前应用于临床抗感染治疗最普遍的一类抗生素,而随着药物的广泛使用和致病菌的变迁,产生了病原菌对药物的耐药性问题,其中细菌产生beta-内酰胺酶(beta-lactamase)是病原菌耐药的主要原因。
Beta-内酰胺酶是一类能高效水解beta-内酰胺抗生素的水解酶。Beta-内酰胺酶按照以氨基酸序列同源性为基础的Ambler分类法将beta-内酰胺酶分为A、B、C、D四个类型,其中A、C、D三个类型为含丝氨酸的beta-内酰胺酶(serine-beta-lactamases,SBLs),B类为含金属锌离子的beta-内酰胺酶(metallo-beta-lactamases,MBLs)。丝氨酸类beta-内酰胺酶是目前最常见、最主要的beta-内酰胺酶。
Beta-内酰胺酶的产生是细菌对beta-内酰胺类抗生素产生耐药性的一个主要原因,但同时,beta-内酰胺酶也可以成为一类识别与鉴定耐药病菌的生物标记物,为快速检测耐药病菌提供可能。复杂的环境下通过直接的化学反应来标记耐药性有关的细菌耐药蛋白能够提供一个快速识别耐药病菌的机会,进而提供更为有效的诊疗手段。
丝氨酸类beta-内酰胺酶主要表达在耐药细菌表面,对酶进行标记可以实现对细菌的生物标记,并且beta-内酰胺酶在自然界中仅在部分细菌中出现,在真核细胞中并不存在,因此具有非常好的选择性。通过对耐药细菌的特异性标记,不仅可以高效地识别耐药细菌,也为后期对耐药细菌的靶向治疗提供了可能。
基于beta-内酰胺酶的高催化活性,这类蛋白分子在分子生物学中也有着广泛的应用。例如,诺贝尔化学奖获得者钱永健教授曾将丝氨酸beta-内酰胺酶(TEM-1)作为目标基因表达的报告基因,通过与丝氨酸beta-内酰胺酶相应的荧光底物作用,实现对目标基因表达情况的快速检测。此外,日本的Kikuchi教授曾报道了一种通过对丝氨酸beta-内酰胺酶的关键基因进行突变(E166N)从而使丝氨酸beta-内酰胺酶成为一个可作为对目标蛋白进行选择性标记的化学标签(Chemical tag)。这种生物大分子标记方式首先通过DNA编辑技术在细胞内的目标大分子中引入一段突变后的丝氨酸beta-内酰胺酶(E166NTEM-1),再通过后期加入可选择性识别beta-内酰胺酶的化学小分子配体来对目标大分子进行修饰与标记。这种方法易于通过后期引入多样性的荧光基团或其它功能的报告基团,具有较好的应用价值。
目前,本领域还需对丝氨酸类beta-内酰胺酶的标记进行研究。
发明内容
本发明的目的在于提供一种共价键标记丝氨酸类beta-内酰胺酶的试剂,具有高灵敏度、高选择性。
本发明的另一目的是,提供所述试剂的合成方法。
本发明的再一目的是,提供所述试剂在标记丝氨酸类beta-内酰胺酶和含有丝氨酸类beta-内酰胺酶抗生素耐药菌中的具体应用。
本发明的第一方面,提供一种式A所示化合物,
式中,X为H、Na、K、Ca或Mg。
本发明的第二方面,提供一种标记试剂,具有式I所示结构:
式中,X为H、Na、K、Ca或Mg;
L为连接基团;
R为具有荧光、放射或磁信号的报告基团或药物基团。
在另一优选例中,所述标记试剂为:
在另一优选例中,所述标记试剂为:
本发明的第三方面,提供式A所示化合物的制备方法,所述制备方法包括以下步骤:
式A1化合物与叠氮乙酸-N-琥珀酰亚胺酯反应获得式A所示化合物;
式中,X为H、Na、K、Ca或Mg。
本发明的第四方面,提供第二方面所述的标记试剂的制备方法,所述制备方法包括以下步骤:
式A所示化合物与具有荧光、放射或磁信号的报告分子或药物分子反应得到所述试剂。
本发明的第五方面,提供第二方面所述的标记试剂的用途,用于共价键标记或靶向丝氨酸类beta-内酰胺酶或其耐药菌。
本发明的第六方面,提供一种共价键标记丝氨酸类beta-内酰胺酶或其耐药菌的方法,包括将第二方面所述的标记试剂与丝氨酸类beta-内酰胺酶或其耐药菌共同孵育的步骤。
本发明的第七方面,提供巴坦类药物的应用,用于制备标记丝氨酸类beta-内酰胺酶或其耐药菌的试剂。
在另一优选例中,所述巴坦类药物选自:舒巴坦(Sulbactam)、他唑巴坦(Tazobactam)、阿维巴坦(Avibactam)、瑞来巴坦(Relebactam)。
本发明通过对阿维巴坦(Avibactam)或瑞来巴坦(Relebactam)进行结构修饰,制备得到了具有靶向丝氨酸类beta-内酰胺酶的分子,通过蛋白及耐药细菌实验表明,本发明的分子具有较强的对丝氨酸类beta-内酰胺酶及表达丝氨酸类beta-内酰胺酶的耐药病菌的靶向性。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。说明书中所揭示的各个特征,可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅,在此不再一一累述。
附图说明
图1示出标记原理。
图2为本发明实施例2制备的RLB-1荧光探针对TEM-1丝氨酸beta-内酰胺酶特异性标记图。
图3为本发明实施例2制备的RLB-1荧光探针与TEM-1丝氨酸beta-内酰胺酶共价成键后的质谱图。
图4为本发明实施例3制备的RLB-2荧光探针对TEM-1丝氨酸beta-内酰胺酶特异性标记图。
图5为本发明实施例3制备的RLB-2荧光探针对不同类型的beta-内酰胺酶特异性标记图。
图6为本发明实施例3制备的RLB-2荧光探针对丝氨酸beta-内酰胺酶的选择性荧光响应图。
图7为本发明实施例3制备的RLB-2荧光探针对耐药细菌选择性标记后的免洗荧光成像图。
具体实施方式
本申请的发明人经过广泛而深入地研究,首次研发出一种共价键标记丝氨酸类beta-内酰胺酶的试剂,通过对阿维巴坦(Avibactam)或瑞来巴坦(Relebactam)进行结构修饰,制备得到了具有靶向丝氨酸类beta-内酰胺酶的分子。所述试剂能够与丝氨酸类beta-内酰胺酶形成共价键连接从而实现对丝氨酸类beta-内酰胺酶或表达丝氨酸类beta-内酰胺酶的耐药病菌的标记在此基础上,完成了本发明。
标记试剂
本发明公开了一类具有共价键标记丝氨酸类beta-内酰胺酶的试剂,同时公开了这类试剂的合成与在丝氨酸类beta-内酰胺酶或表达丝氨酸类beta-内酰胺酶的耐药病菌中的应用。
本发明的试剂,具有式I所示结构:
式中H、Na、K、Ca或Mg;
L为连接基团;
R为具有荧光、放射或磁信号的报告基团或药物基团。
在另一优选例中,所述标记试剂结构为:
Reporter为具有荧光、放射或磁信号的报告基团。
在另一优选例中,所述标记试剂结构为:
Drug为具有治疗功能的药物。
在另一优选例中,所述标记试剂为:
所述试剂是通过连接臂与具有荧光的报告基团相连得到荧光探针,在丝氨酸类beta-内酰胺酶的作用下使活性位点开环,与酶之间形成共价键的结合,从而使所述荧光探针具有用于标记丝氨酸类beta-内酰胺酶及其耐药菌的性质,其标记原理如图1所示。
所述试剂能与生物标记物丝氨酸类beta-内酰胺酶选择性识别,从而起到定位丝氨酸类beta-内酰胺酶或标记抗生素耐药菌的作用。
制备方法
在另一优选例中,本发明的标记试剂的制备方法,包括以下步骤:
式A1化合物与叠氮乙酸-N-琥珀酰亚胺酯反应获得式A所示化合物;
式A所示化合物与具有荧光、放射或磁信号的报告分子或药物分子反应得到所述试剂。
在另一优选例中,所述制备方法包括以下步骤:
(1)化合物1的制备
将瑞来巴坦、叠氮乙酸-N-琥珀酰亚胺酯置于反应瓶中,加入N,N-二甲基甲酰胺(DMF)及N,N-二异丙基乙胺(DIPEA),将反应体系在室温下反应1小时,反应结束后,用反相C18制备柱纯化,冷冻干燥,得到白色的化合物,即所述化合物1;
(2)荧光探针(RLB-1)的制备
将化合物1、化合物2置于反应瓶中,加入二甲基亚砜(DMSO)、水、维生素C、硫酸铜及三(3-羟丙基三唑甲基)胺,将反应体系在室温下反应0.5小时,反应结束,用反相C18制备柱纯化,冷冻干燥,得到橙黄色的化合物,即所述荧光探针(RLB-1)。
(3)荧光探针(RLB-2)的制备
将化合物1、化合物3置于反应瓶中,加入二甲基亚砜(DMSO)、水、维生素C、硫酸铜及三(3-羟丙基三唑甲基)胺,将反应体系在室温下反应0.5小时,反应结束,用反相C18制备柱纯化,冷冻干燥,得到蓝色的化合物,即所述荧光探针(RLB-2)。
该合成路径的主要特征在于:将瑞来巴坦通过与叠氮乙酸-N-琥珀酰亚胺酯的缩合反应引入可后期修饰的叠氮基团,得到中间体,再通过点击化学反应实现与荧光报告基团的连接。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件(如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor LaboratoryPress,1989)中所述的条件)或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
在本发明的实施中,1H-NMR、13C-NMR用Bruker 400Mz型或Ascend 600Mz型仪器测定,测定溶剂为氘代水(D2O)、氘代氯仿(CDCl3)、氘代二甲基亚砜(DMSO-d6),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。
实施例1
共价键标记丝氨酸类beta-内酰胺酶的试剂(化合物1)的制备
将瑞来巴坦(0.144mmol(毫摩尔),50.0mg(毫克))、叠氮乙酸-N-琥珀酰亚胺酯(0.172mmol(毫摩尔),34.1mg(毫克))置于反应瓶中,加入N,N-二甲基甲酰胺300μL及N,N-二异丙基乙胺59μL。将反应体系在室温下反应1小时,反应结束后,用反相C18制备柱纯化,冷冻干燥,得到白色的化合物,即所述化合物1(38.0mg),产率为61%。
1H NMR(400MHz,D2O)δ4.41(d,J=13.2Hz,1H),4.36-4.22(m,3H),4.07(t,J=11.2Hz,2H),3.81(d,J=14.0Hz,1H),3.38(d,J=11.7Hz,1H),3.28(t,J=12.9Hz,1H),3.13(d,J=12.2Hz,1H),2.99(t,J=12.6Hz,1H),2.26(dd,J=15.0,5.1Hz,1H),2.14(dd,J=14.5,1.8Hz,1H),2.09-1.93(m,3H),1.93-1.81(m,1H),1.72-1.45(m,2H).13C NMR(150MHz,DMSO-d6)δ168.78,166.14,165.64,59.39,57.61,49.72,46.47,45.97,45.95,43.03,40.60,40.56,31.53,31.27,30.80,30.63,20.50,18.40.HRMS(ESI)m/z calcd forC14H21N7O7S[M-H]-430.1145,found 430.1146.
实施例2
荧光探针(RLB-1)的制备
将化合物1(0.012mmol,5.0mg)、化合物2(0.013mmol,5.3mg)置于反应瓶中,加入二甲基亚砜50μL、水50μL、维生素C(0.048mmol,8.5mg)、硫酸铜(0.001mmol,0.2mg)及三(3-羟丙基三唑甲基)胺(0.001mmol,0.4mg)。将反应体系在室温下反应0.5小时,反应结束,用反相C18制备柱纯化,冷冻干燥,得到橙黄色的化合物(4.3mg),即所述荧光探针(RLB-1)。
1H NMR(600MHz,DMSO-d6)δ9.43(t,J=5.7Hz,1H),8.50(s,1H),8.28(d,J=8.1Hz,1H),8.04(d,J=5.8Hz,1H),7.90(s,1H),7.38(d,J=8.0Hz,1H),6.68(d,J=2.3Hz,2H),6.61-6.51(m,4H),5.53-5.35(m,2H),4.58(d,J=5.6Hz,2H),4.21(d,J=12.7Hz,1H),3.99(s,1H),3.87(d,J=12.4Hz,2H),3.72(d,J=6.4Hz,1H),3.17(t,J=12.7Hz,1H),3.03-2.93(m,2H),2.83-2.70(m,1H),2.09-1.98(m,1H),1.91-1.61(m,5H),1.60-1.48(m,1H),1.45-1.32(m,1H).13C NMR(150MHz,DMSO-d6)δ168.90,168.20,166.24,164.65,164.12,159.70,154.79,151.89,144.51,135.97,134.77,129.24,126.61,124.75,124.36,123.52,112.77,109.13,102.32,59.44,57.67,50.62,46.50,46.04,46.01,43.23,40.74,40.69,35.08,31.59,31.34,30.80,30.64,20.54,18.45.HRMS(ESI)m/z calcd for C38H35N8O13S[M-H]-843.2044,found 843.2043.
实施例3
荧光探针(RLB-2)的制备
将化合物1(0.012mmol,5.0mg)、化合物3(0.013mmol,6.2mg)置于反应瓶中,加入二甲基亚砜50μL、水50μL、维生素C(0.048mmol,8.5mg)、硫酸铜(0.001mmol,0.2mg)及三(3-羟丙基三唑甲基)胺(0.001mmol,0.4mg)。将反应体系在室温下反应0.5小时,反应结束,用反相C18制备柱纯化,冷冻干燥,得到蓝色的化合物(3.7mg),即所述荧光探针(RLB-2)。
1H NMR(600MHz,DMSO-d6)δ8.04(dd,J=7.8,2.7Hz,1H),7.79-7.66(m,4H),7.59(d,J=7.4Hz,1H),7.45(t,J=7.5Hz,2H),7.39-7.24(m,5H),7.22-7.15(m,2H),7.08(s,1H),7.00(s,1H),5.81(d,J=14.0Hz,1H),5.50-5.33(m,2H),4.22(d,J=12.7Hz,1H),3.99(s,1H),3.94-3.78(m,4H),3.72(d,J=6.3Hz,1H),3.17(t,J=12.9Hz,1H),3.02-2.92(m,2H),2.82-2.72(m,1H),2.60-2.53(m,2H),2.11-1.57(m,14H),1.57-1.49(m,1H),1.43-1.34(m,1H).
13C NMR(150MHz,DMSO-d6)δ190.94,174.36,172.00,168.86,166.19,164.13,148.82,145.88,145.20,142.28,141.10,141.04,133.67,129.68,129.32,128.59,128.42,128.36,126.99,124.25,123.74,122.46,121.25,117.49,110.50,99.73,59.40,57.63,50.59,48.72,46.48,46.01,45.98,43.20,42.89,40.72,40.67,39.52,31.60,31.36,30.78,30.62,26.56,26.25,22.43,20.52,18.42.
HRMS(ESI)m/z calcd for C48H50N8O9S[M-H]-913.3343,found 913.3345.
实施例4
RLB-1荧光探针性能检测
实验分组:
1号孔:5μM TEM-1+10μM RLB-1;
2号孔:5μM TEM-1+10μM RLB-1+500μM avibactam;
3号孔:bacteria lysate+5μM TEM-1+10μM RLB-1;
4号孔:bacteria lysate+10μM RLB-1。
实验条件为:将上述样品在37℃水浴条件下孵育2h,150V跑胶,再分别对其进行荧光成像(右,激发波长为365nm,发射波长为535nm)及考马斯蓝染色(左)。
结果如图1所示,实验结果表明RLB-1能够选择性地对TEM-1beta-内酰胺酶进行标记。
将TEM-1beta-内酰胺酶溶液和TEM-1beta-内酰胺酶+RLB-1溶液在37℃水浴条件下孵育1h后,进行生物质谱检测。RLB-1荧光探针与TEM-1丝氨酸beta-内酰胺酶共价成键后的质谱图如图2所示。
由结果可得,TEM-1和TEM-1+RLB-1的分子量分别为29983.8和30828.0,两者的差值844.2与RLB-1的分子量完全吻合,证明RLB-1能与TEM-1进行很好的结合。
实施例5
RLB-2荧光探针性能检测
(1)RLB-2荧光探针对TEM-1丝氨酸beta-内酰胺酶特异性标记
实验分组:
1号孔:5μM TEM-1+10μM RLB-2
2号孔:5μM TEM-1+10μM RLB-2+500μM avibactam
3号孔:bacteria lysate+5μM TEM-1+10μM RLB-2
4号孔:bacteria lysate+10μM RLB-2。
实验条件为:将上述样品在37℃水浴条件下孵育2h,150V跑胶,再分别对其进行荧光成像(右,激发波长为685nm,发射波长为720nm)及考马斯蓝染色(左)。
结果如图3所示,实验结果表明RLB-2能够选择性地对TEM-1beta-内酰胺酶进行标记。
(2)RLB-2荧光探针对不同类型的beta-内酰胺酶特异性标记
实验分组:
1号孔:10μM RLB-2+5μM TEM-1
2号孔:10μM RLB-2+5μM AmpC
3号孔:10μM RLB-2+5μM KPC-2
4号孔:10μM RLB-2+5μM OXA-1
5号孔:10μM RLB-2+5μM NDM-1
6号孔:10μM RLB-2+5μM IMP-1
7号孔:10μM RLB-2+5μM VIM-27
8号孔:10μM RLB-2+5μM CphA。
实验条件为:将上述样品在37℃水浴条件下孵育2h,150V跑胶,再分别对其进行荧光成像(右,激发波长为685nm,发射波长为720nm)及考马斯蓝染色(左)。
结果如图4所示,实验结果表明RLB-2能够选择性地对A、C、D类beta-内酰胺酶(TEM-1、AmpC、KPC-2、OXA-1)进行标记,而无法对B类beta-内酰胺酶(NDM-1、IMP-1、VIM-2、7CphA)进行标记。
(3)RLB-2荧光探针对丝氨酸beta-内酰胺酶的选择性荧光响应
实验分组
1号:1mM RLB-2+1mM TEM-1
2号:1mM RLB-2+1mM TEM-1+50mM avibactam
3号:1mM RLB-2+1mM NDM-1
4号:1mM RLB-2+1mM IMP-1
5号:1mM RLB-2+1mM VIM-27
6号:1mM RLB-2+1mM CphA
7号:1mM RLB-2+1mM BSA
8号:1mM RLB-2+1x108cfu/ml E.coli。
实验条件为:37℃水浴条件下孵育3h,利用荧光仪光谱仪检测655nm处荧光强度(激发波长为629nm)。
结果如图5所示,实验结果表明RLB-2探针能够对TEM-1beta-内酰胺酶实现选择性荧光增强效果。
(4)RLB-2荧光探针对耐药细菌选择性标记后的免洗荧光成像
ATCC BAA 1143为表达AmpC beta-内酰胺酶的临床细菌,E.coli为不表达beta-内酰胺酶的常见细菌,对其进行细菌标记实验。
实验操作为:在与抑制剂avibactam进行1h的预孵育之后,将1μM RLB-2的ATCCBAA 1143、E.coli菌液和1μM RLB-2+50μM avibactam的ATCC BAA 1143菌液在37℃条件下孵育3h,之后进行共聚焦荧光成像,激发波长为633nm。
结果如图6所示,实验结果表明对于表达beta-内酰胺酶的细菌,RLB-2有很好的标记作用(上),在抑制剂的作用下,RLB-2的标记效果消失(中),对不表达beta-内酰胺酶的细菌,没有标记作用(下)。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (6)
5.如权利要求2所述的标记试剂的用途,其特征在于,用于制备共价键标记或靶向丝氨酸类beta-内酰胺酶或其耐药菌的试剂。
6.一种巴坦类药物的应用,用于制备如权利要求2所述的标记试剂,所述巴坦类药物为阿维巴坦或瑞来巴坦。
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