CN113801145B - 一种激活型近红外小分子荧光探针及其制备方法与应用 - Google Patents
一种激活型近红外小分子荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种激活型近红外小分子荧光探针及其制备方法与应用,将受体介导的结合以及滞留效应和酶响应的荧光激活策略相结合,设计并合成了一种PS靶向、Casp‑3激活的近红外荧光探针1‑DPA2。通过与PS结合,1‑DPA2可以有效地在受损的肾脏中停留从而避免了过快的清除;通过Casp‑3的激活作用,探针可以在受损的肾脏产生强的近红外荧光,从而实现对早期急性肾损伤更精准、更高信背比的成像检测分析。
Description
技术领域
本发明属于生物探针领域,具体涉及一种近红外激活型小分子荧光探针及其制备方法与应用。
背景技术
急性肾损伤是一种发病率和致死率很高的全球性疾病,主要表现为肾小球滤过作用的显著下降,导致肾功能紊乱。急性肾损伤有很多诱因,比如败血症、低血压、肾结石、器官衰竭以及药物的过度使用等等,在众多诱因中,药物过度使用引起的急性肾损伤最为普遍。传统诊断急性肾损伤的方法主要包括活肾穿刺活检或者测量血液中肌酐和血尿素氮的含量,但前者由于其侵入性的特点,可能会有潜在的其他器官感染的风险;后者由于检测的灵敏性不足,无法在急性肾损伤的早期阶段及时检出,这往往导致病人错过了最佳的治疗时机。因此,为了能对急性肾损伤患者做及时的干预,提高治疗的成功率,急需要开发高灵敏度的诊断工具,来实现对急性肾损伤早期、非侵入性检测。
分子荧光成像以其高的灵敏度、低成本、操作简易以及非侵袭性等优点,被广泛应用于对疾病的成像检测,在生物医学研究和临床诊断领域具有广阔的前景。近年来,科研工作者针对急性肾损伤,开发了许多能对其早期进行检测的光学成像探针。例如,来自美国德克萨斯大学的郑杰课题组开发了一种表面修饰了谷胱甘肽的修饰的金纳米颗粒,该纳米探针主要通过肾脏清除,来实现对不同阶段急性肾损伤的近红外成像。来自新加坡国立大学的浦侃裔课题组针对发生在早期急性肾损伤的不同分子事件,开发了一系列响应不同生物靶标的激活型荧光探针,实现对急性肾损伤小鼠的早期成像检测分析。尽管目前已有的荧光探针可以基本实现对急性肾损伤的早期检测,但是这些探针往往仅响应一个单独的分子事件且容易被清除,这会降低成像结果的准确性且难以监测急性肾损伤的发生和进程。因此,需要开发一种能够靶向多个生物靶标的激活型小分子荧光探针,使其靶向多个生物靶标,从而实现对急性肾损伤的早期高灵敏度的成像检测。
发明内容
发明目的:本发明所要解决的技术问题是针对现有技术的不足,提供一种激活型近红外荧光探针及其制备方法,从而实现对早期急性肾损伤更精准、更高信背比的成像检测分析。
为了实现上述目的,本发明采取的技术方案如下:
一种激活型近红外小分子荧光探针,它包括如下几个部分键合而成:
半胱天冬酶-3(Casp-3)特异性识别的多肽序列(G-DEVD-G):
特异性结合磷脂酰丝氨酸蛋白(PS)的二甲基吡啶胺-锌螯合物(DPA-Zn):
具有近红外荧光淬灭效应的淬灭剂(QC-1):
以及具有近红外荧光的花菁类荧光染料(IR-780dye):
具体地,它具有如下的分子结构:
进一步地,本发明还提供上述激活型近红外小分子荧光探针的制备方法,包括如下步骤:
(a)将化合物IR-780-Alkyne与化合物3进行点击化学反应,引入二甲基吡啶胺(DPA)基团;
(b)步骤(a)完成后,产物不纯化直接进行取代反应,引入叠氮(N3)基团;
(c)步骤(b)反应产物不纯化直接与G-DEVD-G-Alkyne进行点击化学反应,引入Casp-3识别多肽序列G-DEVD-G-Alkyne,得到化合物4;
(d)将化合物4进行取代反应,引入荧光猝灭基团QC-1;
(e)将步骤(d)反应产物不纯化直接进行配位反应,向二甲基吡啶胺(DPA)基团引入Zn2+,即得;
上述步骤反应式如下:
其中,步骤(a)中,所述的点击化学反应为将化合物IR-780-Alkyne、化合物3、无水硫酸铜(CuSO4)、抗坏血酸钠、三(3-羟丙基三唑甲基)胺(THPTA)溶于溶剂中,进行反应;其中,化合物IR-780-Alkyne、化合物3、抗坏血酸钠、无水硫酸铜、三(3-羟丙基三唑甲基)胺的摩尔比为5:(6-8):(5-8):(5-8):(5-8),优选为5:6:5:5;反应温度为20-30℃,优选为室温,搅拌状态下反应15-45min,优选为30min。所述溶剂包括但不限于二甲基亚砜,优选为二甲基亚砜。
步骤(b)中,将叠氮化钠(NaN3)溶于溶剂中,加入步骤(a)反应产物中进行取代反应;其中,化合物IR-780-Alkyne与叠氮化钠(NaN3)的摩尔比为5:(15-50),优选为5:25;反应温度为20-30℃,优选为室温,搅拌状态下反应10-30min,优选为15min。所述溶剂包括但不限于二次水,优选为二次水。
步骤(c)中,将化合物G-DEVDG-Alkyne溶于溶剂中,加入步骤(b)反应产物中进行反应;其中,化合物IR-7810-Alkyne与化合物G-DECDG-Alkyne的摩尔比为5:(5-10),优选为5:7.5;反应温度为20-30℃,优选为室温,搅拌状态下反应15-45min,优选为30min,得到含有化合物4的反应液,经纯化、冻干,得到化合物4;所述溶剂包括但不限于二甲基亚砜,优选为二甲基亚砜。
所述的化合物G-DEVDG-Alkyne结构为:
步骤(d)中,化合物4、QC-1活化酯和N,N-二异丙基乙胺溶于溶剂中进行反应;其中,化合物4、QC-1活化酯和N,N-二异丙基乙胺(DIPEA)的摩尔比为5:(5-8):(5-8),优选为5:6:8;反应温度为20-30℃,优选为室温,搅拌状态下反应0.5-1.5h,优选为1h;所述反应溶剂包括但不限于甲醇,优选为甲醇。
所述的QC-1活化酯结构为:
步骤(e)中,将氯化锌溶于溶剂,加入步骤(d)产物中进行配位反应;其中,化合物4与ZnCl2的摩尔比为5:(6-10),优选为5:10;反应温度为20-30℃,优选为室温,搅拌状态下反应0.5-1.5h,优选为1h;反应结束后将含有化合物的反应液纯化,冻干,即得。所述溶剂包括但不限于二次水,优选为二次水。
进一步地,本发明还要求保护上述激活型近红外小分子荧光探针用于在体外对Casp-3活性检测中的应用。
进一步地,本发明还要求保护上述激活型近红外小分子荧光探针用于对早期急性肾损伤成像检测中的应用。
本发明探针1-DPA2包含了能被Casp-3识别切断的GDEVDG多肽序列,具有PS蛋白靶向的DPA-Zn基团以及能淬灭近红外荧光的QC-1基团。由于QC-1与花菁类染料之间存在很强的荧光共振能量转移(FRET)效应,探针1-DPA2在刚开始时具有淬灭的近红外荧光。在Casp-3的作用下,QC-1连同着多肽序列GDEVD被切断,产生具有近红外荧光(808nm)的2-DPA2。当静脉注射1-DPA2后,1-DPA2由于良好的水溶性可以有效地通过肾脏清除。在健康的肾脏中,由于肾小管上皮细胞(REC)内部PS蛋白尚未外翻,Casp-3正处于未被激活的状态(Pro-Casp-3),探针1-DPA2无法停留在REC上且荧光无法被激活,这导致了1-DPA2在肾脏的快速清除,在肾脏部位表现出微弱的荧光。而在损伤的肾脏中,外翻的PS蛋白可以有效地结合1-DPA2,在激活的Casp-3作用下,探针1-DPA2的多肽序列GDEVD被切断,从而在受损的肾脏部位表现出强的近红外荧光。综上所述,探针1-DPA2通过同时响应两个早期凋亡的标志物(PS,Casp-3),从而实现了在早期对急性肾损伤无创、实时、高灵敏度、高信背比的近红外荧光成像检测。
有益效果:
本发明将受体介导的结合以及滞留效应和酶响应的荧光激活策略相结合,设计了合成了一种PS靶向、Casp-3激活的近红外荧光探针1-DPA2。通过与PS结合,1-DPA2可以有效地在受损的肾脏中停留从而避免了过快的清除;通过Casp-3的激活作用,探针可以在受损的肾脏产生强的近红外荧光,从而实现对早期急性肾损伤更精准、更高信背比的成像检测分析。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明,本发明的上述和/或其他方面的优点将会变得更加清楚。
图1为PS蛋白靶向、Casp-3激活的近红外荧光探针的设计及其作用机理。
图2为探针1-DPA2对Casp-3的响应。1-DPA2(1.0μM)与Casp-3(1.0μg/mL)或者Casp-3和Z-VAD-fmk(2.0μg/mL)在37℃下Casp-3酶切缓冲液中孵育2小时后的(a)HPLC分析以及(b)荧光光谱,荧光光谱激发波长为790nm。(c)1-DPA2(1.0μM)与不同浓度的Casp-3(0,0.01,0.05,0.1,0.2,0.5,1.0和2.0μg/mL)在37℃下于Casp-3酶切缓冲液中孵育2小时后的荧光光谱。(d)1-DPA2(1.0μM)与肾脏中的生物指标,比如Casp-3(1.0μg/mL),GGT(1.0μg/mL),GSH(5mM),H2O2,ClO-,ONOO-,OH.和O2 .-(150μM)在37℃下于Casp-3酶切缓冲液中孵育2小时后的荧光光谱。
图3为探针1-DPA2对肾损伤小鼠的活体成像检测。(a)向正常的小鼠(I)或肾损伤小鼠尾静脉注射5nmol 1-DPA2(II),Ctrl-QC(III),Ctrl-DPA2(V)或者提前0.5小时注射Z-VAD-fmk(2mg/kg)(IV)后,在0、1、2、4、8和12小时的荧光成像。在a的条件下对应的(b)肾脏(红箭头),(c)皮肤(黑圈)的荧光强度以及相应的(d)成像信背比。(e)在尾静脉注射1-DPA2后,对正常小鼠和肾损伤小鼠的离体器官荧光成像。(f)在尾静脉注射1-DPA2后,对正常小鼠和肾损伤小鼠的肾切片皮质区和髓质区的荧光成像,比例尺:40μm。在尾静脉注射1-DPA2后,收集小鼠24小时内的尿液,(g)荧光强度以及(h)HPLC分析。
图4为探针1-DPA2对肾损伤进程的监测。(a)对顺铂(20mg/kg,腹腔注射)处理的0,24,48,72和96小时的小鼠尾静脉注射1-DPA2(5nmol,200μL),在2小时的时间点采集荧光成像信号。(b)在a的条件下对应的肾(红箭头),皮肤(黑圈)的荧光以及(c)成像信背比。(d)顺铂(20mg/kg,腹腔注射)处理的0,24,48,72和96小时的小鼠的肾小球滤过率。(e)顺铂(20mg/kg,腹腔注射)处理的0,24,48,72和96小时的小鼠的肾切片的H&E染色,绿色箭头说明了铸型的形成,黄色箭头说明了肾小管细胞的坏死。比例尺:1mm。区域1和2分别为放大的皮质区和髓质区,比例尺:100μm。(f)顺铂(20mg/kg,腹腔注射)处理的0,24,48,72和96小时的小鼠的肾切片的Casp-3免疫荧光染色以及(g)其对应的荧光强度。比例尺:500mm。区域1和2分别为放大的皮质区和髓质区,比例尺:40μm。(h)1-DPA2对顺铂处理不同时间的小鼠的肾脏产生的荧光和相应肾脏Casp-3的荧光强度的相关性(皮尔森相关系数r=0.999)。
图5为探针1-DPA2用于监测NAC对急性肾损伤的疗效。(a)使用NAC对急性肾损伤小鼠治疗方案的示意图。通过腹腔注射顺铂(20mg/kg)来构建肾损伤模型。随后通过注射1次(1X,12小时),2次(2X,12小时和24小时),3次(3X,12小时,24小时和36小时)NAC(400mg/kg,腹腔注射)来治疗急性肾损伤。仅注射生理盐水的小鼠作为空白对照。(b)在顺铂处理的72小时后,向小鼠尾静脉注射5nmol 1-DPA2,在2小时的时间点采集荧光成像信号。在上述的条件下,小鼠(c)肾脏(红箭头),皮肤(黑圈)的荧光强度以及(d)相应的成像信背比。(e)在上述条件下,小鼠血清中肾损伤相关指标肌酐(CR),血尿素氮(BUN)以及胱抑素C(CysC)的测定。
具体实施方式
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
下述实施例中所述“%”,如无特殊说明,均为摩尔百分比。
试剂与仪器:所有化学试剂和溶剂均购自百灵威科技有限公司(中国上海),梯希爱(中国上海)化成工业发展有限公司和Sigma-Aldrich。分析溶剂与试剂为色谱纯,常规试剂均为分析纯且未经进一步纯化处理。荧光淬灭剂QC-1购自于LI-COR Biosciences(美国林肯)。
1H-NMR和13C-NMR光谱使用400MHz Bruker Avance III 400核磁仪获得,溶剂为DMSO-d6或Chloroform-d,化学位移(δ)以ppm为单位,单重峰,双重峰,三重峰,四重峰,dd(doublet of doublets)峰,多重峰以及宽峰分别以s,d,t,q,dd以及m表示;耦合常数(J)以Hz为单位;氢的个数用图谱的积分值确定,并标记为nH。高效液相色谱(HPLC)使用ThermoScientific Dionex Ultimate 3000,洗脱剂为CH3CN/H2O(1‰CF3COOH)。基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)分析使用AB SCIEX 4800Plus MALDI TOF/TOFTM质谱仪UV-Vis光谱使用Ocean Optics Maya 2000Pro光谱仪进行测量。荧光光谱使用HORIBAJobin Yvon Fluoromax-4荧光计测得。细胞和组织切片的荧光图像使用Olympus IX73荧光倒置显微镜获取。活体荧光图像用IVIS Lumina XR III系统获得,并使用Living image软件(PerkinElmer)测量圈取区域信号来量化荧光强度。
实施例1:
探针1-DPA2的设计、合成与表征
2-[2-(2-Azidoethoxy)ethoxy]-N,N-bis(2-pyridinylmethyl)ethanamine中间体化合物3(CAS:1613538-61-6)的合成路线如下:
反应条件:(a)NaN3,H2O,90℃,24h,96%;(b)TsCl,三乙胺,DCM,r.t.,4h,83%;(c)2,2-二甲基吡啶胺,三乙胺,CH3CN,80℃,24h,77%。
化合物1的合成:将2-氯乙氧基-2-乙氧基二乙醇(991mg,5.9mmol),NaN3(462mg,7.1mmol)溶于二次水(5mL)中,90℃下搅拌24小时。反应完成后,然后向反应混合物加入20mL乙酸乙酯萃取,一共萃取3次,有机层用无水Na2SO4干燥。过滤去除沉淀后旋蒸去除溶剂,得到化合物1为透明无色液体。产量:955mg(96%)1H NMR(400MHz,Chloroform-d)δ3.74(dd,J=8.1,3.8Hz,2H),3.69(d,J=2.8Hz,6H),3.62(dd,J=5.3,3.8Hz,2H),3.44–3.37(m,2H),2.79(s,1H).13C NMR(101MHz,CDCl3)δ72.57,70.63,70.35,70.01,61.68,50.64.MS:calcd.for C6H13N3O3Na+[(M+Na)+]:198.09;found ESI-MS:m/z 198.00
化合物2的合成:将化合物1(525mg,3mmol)溶解于10mL无水DCM中。随后将TsCl(684mg,3.6mmol),三乙胺(500μL,3.6mmol)分别加入反应溶液中,室温搅拌4小时。反应完成后,真空旋蒸除去DCM。残余物通过硅胶快速色谱法纯化,使用PE/EA(50:1至10:1v/v)的混合溶液作为洗脱剂,得到化合物2为无水液体。产量:436mg(83%)。1H NMR(400MHz,DMSO-d6)δ7.83–7.76(m,2H),7.53–7.44(m,2H),4.18–4.08(m,2H),3.71–3.62(m,1H),3.61–3.55(m,4H),3.49(m,4H),3.34(s,1H),2.43(s,3H).13C NMR(101MHz,DMSO)δ144.85,132.36,130.07,127.57,69.91,69.68,69.63,69.50,69.22,67.88,49.92,43.48,21.03.MS:calcd.for C13H19N3O5SNa+[(M+Na)+]:352.10;found ESI-MS:m/z 351.95.
化合物3的合成:在氮气保护下,将化合物2(395mg,1.2mmol)和2,2-二甲基吡啶胺(199mg,1.0mmol)溶于9mL无水乙腈中,然后将三乙胺(167μL,1.2mmol)溶于1mL无水乙腈中并滴加入反应液中,反应溶液在90℃回流并持续搅拌24小时。反应结束后,真空旋蒸除去乙腈,然后将反应混合物用50mL DCM稀释,并用饱和食盐水溶液萃取3次(每次20mL),有机层用无水Na2SO4干燥。过滤去除沉淀后旋蒸去除溶剂,残余物通过硅胶快速色谱法纯化,使用CH2Cl2/CH3OH(50:1至10:1v/v)的混合溶液作为洗脱剂,得到化合物3为金黄色液体。产量:274mg(77%)。1H NMR(400MHz,Chloroform-d)δ8.52(d,J=5.6Hz,2H),7.65(td,J=7.6,1.8Hz,2H),7.57(d,J=7.8Hz,2H),7.18–7.11(m,2H),3.91(s,4H),3.75–3.61(m,6H),3.57(dd,J=6.6,3.8Hz,2H),3.41–3.31(m,2H),2.84(t,J=5.9Hz,2H).13C NMR(101MHz,CDCl3)δ159.72,148.95,136.39,122.95,121.93,70.68,70.40,70.02,69.67,69.63,60.88,53.58,50.66.MS:calcd.for C18H25N6O2 +[(M+H)+]:357.20;found ESI-MS:m/z 357.10.
中间体化合物G-DEVD-G-Alkyne的合成路线如下:
反应条件:(a)HBTU,DIPEA,DMF,r.t.,2h,73%;(b)炔丙胺,HBTU,DIPEA,THF,r.t.,2h,81%;(c)95%TFA/DCM,r.t.,4h,74%。
化合物Boc-GDEVDG(OtBu)-OH的合成:通过标准固相肽合成(SPPS)程序(Nat.Protocol.,2007,2,3247),使用2-氯三苯甲基氯树脂,Fmoc保护的氨基酸和1,4,7,10-四氮杂环十二烷-1,4,7-乙酸三叔丁酯-10-乙酸进行多肽序列的合成。固相合成反应步骤结束后,使用1%TFA的DCM溶液从树脂上裂解产物。真空旋蒸除去溶剂后,加入冷的乙醚得到沉淀物,并将所得沉淀物离心,得到化合物Boc-GDEVDG(OtBu)-OH为淡黄色固体。产量:627mg(73%)。1H NMR(400MHz,DMSO-d6)δ8.27(d,J=8.1Hz,1H),8.09(d,J=8.2Hz,1H),8.00(t,J=5.8Hz,1H),7.89(d,J=7.9Hz,1H),7.78(d,J=8.5Hz,1H),7.03–6.90(m,1H),4.61(td,J=8.4,5.6Hz,2H),4.31(q,J=8.2Hz,1H),4.14(dd,J=8.4,6.6Hz,2H),3.83–3.63(m,2H),3.55(d,J=5.1Hz,2H),2.75–2.56(m,2H),2.49–2.42(m,2H),2.20(m,2H),2.00–1.81(m,2H),1.71(m,1H),1.40–1.34(m,36H),0.85–0.78(m,6H).13C NMR(101MHz,DMSO)δ171.72,170.83,170.76,170.56,170.38,170.20,169.26,169.07,155.74,80.18,80.11,79.54,78.09,57.51,51.88,49.29,49.21,43.26,40.73,37.39,37.32,31.31,30.59,28.13,27.70,27.61,27.31,19.12,17.92.MS:calcd.for C39H66N6O15Na+[(M+Na)+]:881.4586;found MALDI-MS:m/z 881.5767.
化合物Boc-GDEVDG(OtBu)-Alkyne的合成:将炔丙胺(17mg,0.3mmol),化合物Boc-GDEVDG(OtBu)-OH(172mg,0.2mmol),HBTU(114mg,0.3mmol)和DIPEA(70μL,0.4mmol)溶于无水THF(5mL)溶液中,室温搅拌2小时。反应完成后,真空旋蒸除去THF。残余物通过硅胶快速色谱法纯化,使用CH2Cl2/CH3OH(50:1至10:1v/v)的混合溶液作为洗脱剂,得到化合物2为白色固体。产量:150mg(84%)。1H NMR(400MHz,DMSO-d6)δ8.35(d,J=7.6Hz,1H),8.11(m,2H),8.03(t,J=5.8Hz,1H),7.90(d,J=7.9Hz,1H),7.79(d,J=8.3Hz,1H),6.97(t,J=5.6Hz,1H),4.57(m,2H),4.31(q,J=8.2Hz,1H),4.20–4.06(m,2H),3.93–3.79(m,2H),3.74–3.61(m,2H),3.55(d,J=5.2Hz,2H),3.09(t,J=2.5Hz,1H),2.74(d,J=5.6Hz,0H),2.70(d,J=5.5Hz,1H),2.62(dd,J=16.0,5.2Hz,1H),2.49–2.43(m,1H),2.20(m,2H),2.01–1.82(m,2H),1.71(m,1H),1.41–1.34(m,36H),0.82(t,J=6.1Hz,6H).13C NMR(101MHz,DMSO)δ171.71,170.90,170.85,170.34,170.24,169.32,169.28,168.26,155.74,80.81,80.19,79.55,78.09,72.96,57.61,51.88,49.68,49.23,43.27,42.06,37.37,37.01,31.32,30.51,28.13,27.84,27.70,27.63,27.26,19.10,18.02.MS:calcd.for C42H69N7O14Na+[(M+Na)+]:918.4902;found MALDI-MS:m/z 918.5758.
化合物G-DEVD-G-Alkyne的合成:将化合物Boc-GDEVDG(OtBu)-Alkyne(90mg,0.1mmol)溶于95%TFA/DCM(10mL)中室温搅拌4小时。反应结束后旋蒸去除溶剂,加入冷的乙醚(20mL)得到沉淀物,并将所得沉淀物离心,得到化合物G-DEVD-G-Alkyne为白色固体。产量:63mg(81%)。1H NMR(400MHz,DMSO-d6)δ12.38(s,2H),8.69(d,J=7.8Hz,1H),8.39(d,J=7.1Hz,1H),8.22(d,J=7.8Hz,1H),8.12(t,J=5.6Hz,1H),8.06(t,J=5.7Hz,3H),7.72(d,J=8.3Hz,1H),4.67(td,J=8.3,4.5Hz,1H),4.50(q,J=7.3Hz,1H),4.29(td,J=8.4,5.4Hz,1H),4.12(dd,J=8.1,6.6Hz,1H),3.92–3.79(m,2H),3.72–3.64(m,2H),3.58(s,2H),3.46(s,3H),3.08(t,J=2.5Hz,1H),2.80–2.62(m,2H),2.60–2.53(m,1H),2.35–2.16(m,2H),2.03–1.86(m,2H),1.81–1.68(m,1H),0.82(t,J=7.3Hz,6H).13C NMR(101MHz,DMSO)δ174.06,171.79,171.36,171.06,170.65,170.29,168.38,165.87,80.87,72.93,57.53,52.13,49.73,49.47,42.10,36.27,35.65,30.53,30.19,27.85,26.98,19.09,17.91.MS:calcd.for C25H37N7O12Na+[(M+Na)+]:650.2500;found MALDI-MS:m/z 650.2961.
探针1-DPA2的合成路线如下:
反应条件:(a)化合物3,CuSO4,抗坏血酸钠,THPTA,r.t.,DMSO/H2O,30min;(b)NaN3,r.t.,H2O,15min;(c)G-DEVD-G-Alkyne,CuSO4,抗坏血酸钠,THPTA,r.t.,DMSO/H2O,30min,74%;(d)QC-1,DIPEA,r.t.,DMSO,30min;(e)ZnCl2,r.t.,CH3OH/H2O,1h,76%。
化合物4的合成:首先,根据已报道的方法(J.Am.Chem.Soc.2020,142,2787-2794.)合成了近红外荧光染料IR-780-Alkyne。(a)将化合物3(16mg,0.044mmol)和IR-780-Alkyne(26mg,0.02mmol)溶于DMSO中,将CuSO4(2.5mg,0.01mmol),抗坏血酸钠(2.0mg,0.01mmol)和THPTA(4.3mg,0.01mmol)溶解于二次水中,加入到DMSO中,室温搅拌30分钟引入DPA基团。(b)随后,直接向反应液中加入溶于二次水中的NaN3(6.5mg,0.1mmol),室温搅拌15分钟引入N3基团。(c)最后将化合物G-DEVD-G-Alkyne(19mg,0.03mmol)溶解于DMSO中,将其加入到反应液中,并补加溶于二次水(200μL)的CuSO4(2.5mg,0.01mmol),抗坏血酸钠(2.0mg,0.01mmol)和THPTA(4.3mg,0.01mmol),室温下继续搅拌30分钟引入多肽序列G-DEVD-G-Alkyne。反应完成后,通过制备HPLC纯化残余物,通过冻干得到化合物4为墨绿色固体。产量:29mg(74%)。MS:calcd.for C101H129N24O16 +[M+]:1934.0013;found MALDI-MS:m/z1934.0253.
化合物1-DPA2的合成:(d)将QC-1活化酯(1.1mg,0.001mmol),化合物4(2.3mg,0.0012mmol)和DIPEA(0.26μL,0.0015mmol)溶于二甲基亚砜(200μL)溶液中,室温搅拌0.5小时引入荧光猝灭基团QC-1。(e)随后,将氯化锌(0.34mg,0.0025mmol)溶解在50%的CH3OH/H2O的混合液中,加入反应液继续在室温下搅拌1小时,将Zn2+引入DPA基团。反应结束后通过制备HPLC纯化残余物,通过冻干获得化合物1-DPA2为墨绿色固体。产量:2.4mg(76%)。MS:calcd.for C150H194ClN27O33S4Zn2 2+[M2+]:3192.1476;found ESI-MS:m/z1596.0705.
如图1所示,PS蛋白靶向、Casp-3激活的近红外荧光探针的设计及其作用机理:(a)1-DPA2的化学结构及其在Casp-3的作用下转化为具有近红外荧光的2-DPA2。(b)探针1-DPA2对急性肾损伤的成像检测作用机制。当静脉注射探针1-DPA2后,探针可以通过肾脏清除进入到肾脏。在正常的肾中,由于PS蛋白没有外翻到细胞膜上以及Casp-3尚未激活,因此1-DPA2的荧光保持淬灭且快速被清除。而在损伤的肾中,探针可以和外翻的PS结合并产生一定的停留效应,同时具有活性的Casp-3可以切断1-DPA2上相应的多肽序列(QC-GDEVD)使得1-DPA2的近红外荧光激活,从而实现对急性肾损伤的早期检测。
对比例:
对照探针Ctrl-DPA2、Ctrl-QC的设计、合成与表征
对照探针Ctrl-DPA2的合成路线如下:
反应条件:(a)ZnCl2,r.t.,CH3OH/H2O,1h,79%。
化合物Ctrl-DPA2的合成:将化合物4(2.3mg,0.0012mmol)溶解于甲醇(500μL)溶液中。随后,将溶解于二次水(500μL)的氯化锌(0.34mg,0.0025mmol)加入其中丙在室温下搅拌1小时。反应结束后通过制备HPLC纯化残余物,通过冻干获得化合物Ctrl-DPA2为墨绿色固体。产量:2mg(79%)。MS:calcd.for C101H135N24O20Zn2 3+[M3+]:2131.8851;found ESI-MS:m/z 711.2940.
对照探针Ctrl-QC的合成路线如下:
反应条件:(a)化合物1,CuSO4,抗坏血酸钠,THPTA,r.t.,DMSO/H2O,30min;(b)NaN3,r.t.,H2O,15min;(c)G-DEVD-G-Alkyne,CuSO4,抗坏血酸钠,THPTA,r.t.,DMSO/H2O,30min,73%;(d)QC-1,DIPEA,r.t.,DMSO,30min,89%。
化合物5的合成:(a)将化合物1(7.7mg,0.044mmol)和IR-780-Alkyne(26mg,0.02mmol)溶于二甲基亚砜(1mL)中,将CuSO4(2.5mg,0.01mmol),抗坏血酸钠(2.0mg,0.01mmol)和THPTA(4.3mg,0.01mmol)溶解于二次水(200μL)中,随后加入到反应液中室温搅拌30分钟引入PEG链。(b)随后,直接向反应液中加入溶于二次水的NaN3(mg,0.1mmol),室温搅拌15分钟引入N3基团。(c)最后将化合物G-DEVD-G-Alkyne(19mg,0.03mmol)溶解于DMSO中,将其加入到反应液中,并补加溶于二次水(200μL)的CuSO4(2.5mg,0.01mmol),抗坏血酸钠(2.0mg,0.01mmol)和THPTA(4.3mg,0.01mmol),室温下继续搅拌30分钟引入多肽序列G-DEVD-G-Alkyne。反应完成后,通过制备HPLC纯化得到化合物5为墨绿色固体。产量:23mg(73%)。MS:calcd.for C77H107N18O18 +[M+]:1571.8005;found MALDI-MS:m/z1571.1277.
化合物Ctrl-QC的合成:(d)将QC-1活化酯(1.1mg,0.001mmol),化合物5(1.9mg,0.0012mmol)和DIPEA(0.26μL,0.0015mmol)溶于二甲基亚砜(200μL)溶液中,室温搅拌0.5小时引入荧光猝灭基团QC-1。反应结束后通过制备HPLC纯化残余物,通过冻干获得化合物Ctrl-QC为墨绿色固体。产量:2.3mg(89%)。MS:calcd.for C126H167ClN21O31S4 +[M+]:2633.0703;found ESI-MS:m/z 2633.0705.
探针性能测试
1、探针对Casp-3的酶切响应
在Casp-3的酶切缓冲液(含有50mM HEPES缓冲液,100mM NaCl,1mM EDTA,10mMTCEP,40mM NaHCO3,10wt%丙三醇and 0.1wt%CHAPS)中,研究探针1-DPA2对Casp-3的响应能力。如图2a所示,在探针1-DPA2(1μM)与Casp-3(1.0μg/mL)在37℃下孵育2h后,通过高效液相色谱(HPLC)分析,结果显示1-DPA2(12.5min)能够在Casp-3的作用下完全转化为2-DPA2(12.7min),1-DPA2的荧光也被Casp-3显著激活,其荧光打开的倍数约为10倍(图2b)。此外,由于Z-VAD-fmk(2.0μg/mL)对Casp-3的活性的抑制,导致探针1-DPA2和Casp-3,Z-VAD-fmk共孵育2h后,1-DPA2并没有转化为2-DPA2(图2a),其荧光也没有被激活(图2b)。
2、探针对Casp-3的检测灵敏性
取1-DPA2(2μM,100μL)与100μL不同浓度的Casp-3酶切缓冲溶液混合,探针最终的工作浓度为1μM,Casp-3最终的浓度分别为0、0.01、0.05、0.1、0.2、0.5、1.0以及2.0μg/mL,每组试验重复三次。每个浓度组三个平行试验,将上述溶液于37℃下孵育25分钟,然后使用HORIBA Jobin Yvon Fluoromax-4荧光仪获得反应溶液的荧光光谱(λex=760nm,λem=808nm)对Casp-3浓度做图,得到各自拟合直线的斜率k。检测限LOD=3δ/k。其中,δ为测量11个空白探针孔溶液的强度值的标准方差。
结果如图2c所示,探针的荧光强度随着Casp-3浓度的增大而增强。荧光强度与Casp-3浓度(0-0.2μg/mL)成线性关系。探针对Casp-3的检测限(LOD)经计算为1.35ng/mL,表明探针可用于对Casp-3的高灵敏检测。
3、探针对Casp-3的检测特异性
取1-DPA2(2μM,100μL)与100μL含有不同酶,活性氧簇的Casp-3酶切缓冲溶液混合,探针最终工作浓度为1μM,不同酶,活性氧簇分别为GGT(1.0μg/mL)、Casp-3(1.0μg/mL)、GSH(5mM)、H2O2(150μM)、ClO-(150μM)、ONOO-(150μM)、OH.(150μM)以及O2 .-(150μM)。每组试验重复3次,将上述溶液于37℃下孵育2h,然后使用HORIBA Jobin Yvon Fluoromax-4荧光仪获得反应溶液在808nm处的荧光强度,激发波长为790nm。
结果如图2d所示,只有加入Casp-3才可以激活探针1-DPA2,并产生显著的荧光增强,以上结果说明探针1-DPA2对Casp-3具有很强的特异性。
4、探针在活体水平对顺铂诱导的急性肾损伤的成像检测
取15只6-8周龄的雌性BALB/C裸鼠,任选其中的12只,通过腹腔注射顺铂(20mg/kg)的方式,构建急性肾损伤模型,剩余的3只小鼠仅注射生理盐水作为健康小鼠对照。在48h后,将含有1-DPA2,Ctrl-QC,Ctrl-DPA2(25μM,200μL)的生理盐水通过尾静脉注射进入小鼠体内。在注射后0h,1h,2h,4h,8h和12h的时间点使用IVIS Lumina XR III成像系统采集全身荧光图像。
如图3a所示,在尾静脉注射1-DPA2后,在肾脏的部位可以观察到明亮的近红外荧光,其荧光强度在2h的时间点达到了最大。1-DPA2在急性肾损伤小鼠的肾脏产生的更强的荧光,其荧光强度约为正常小鼠的2.1倍(图3b)。当急性肾损伤的小鼠提前0.5h尾静脉注射Z-VAD-fmk(2mg/kg),其肾部的荧光明显被减弱。对于尾静脉注射了Ctrl-QC的急性肾损伤小鼠,其肾部最大的荧光强度约为注射了1-DPA2的75%。当尾静脉注射了常亮探针Ctrl-DPA2后,其肾脏部位的荧光在1小时的时候达到最大。但由于该探针是常亮型探针,Ctrl-DPA2在小鼠上产生的背景荧光要高于1-DPA2以及Ctrl-QC(图3c),这导致了其在2h时的荧光信背比(SBR)仅为1.8±0.2,仅为1-DPA2产生的荧光信背比(~5.7±0.3)的32%(图3d)。
尾静脉注射1-DPA2后2h,正常小鼠和肾损伤小鼠的主要器官被取出并进行离体的荧光成像。从图3e中我们可以看出,相比于其他的器官,注射了1-DPA2的小鼠肾脏部位表现出最强的荧光,且其在肾损伤小鼠上的荧光强度约为正常小鼠的3.5倍。此外,肾脏组织切片的荧光成像也证明(图3f),来自肾损伤小鼠的肾切片的近红外荧光明显强于正常小鼠的肾切片,且其荧光主要分布在肾皮质层的肾小管上皮细胞上。这证明了在对于顺铂引起的早期急性肾损伤,其损伤主要发生在了肾皮质层区域的肾小管上皮细胞上。
收集1-DPA2处理后小鼠在24h内的尿液,通过IVIS Lumina XR III采集荧光(图3g)以及HPLC分析(图3h),我们可以看出探针1-DPA2可以在肾损伤小鼠的肾脏被激活且通过尿液排出,其尿液的荧光强度约为正常小鼠的2.1倍。
从以上的结果可以看出,探针1-DPA2可以通过肾脏清除,且可以在肾损伤小鼠的肾脏部位被激活、停留,并产生明显的近红外荧光,适合用于对药物诱导的早期急性肾损伤的非侵入性近红外荧光成像检测。
5、探针在活体水平对顺铂诱导的不同程度急性肾损伤的成像检测
任选15只6-8周龄的雌性BALB/C裸鼠,用顺铂(20mg/kg,腹腔注射)处理不同的时长(0,24,48,72和96h),构建不同程度的急性肾损伤模型。通过尾静脉注射的方式将含有1-DPA2(25μM,200μL)的生理盐水注射进入小鼠体内。在注射后2h的时间点使用IVIS LuminaXR III成像系统采集全身荧光图像。
如图4a所示,在尾静脉注射1-DPA2后,随着顺铂处理时间延长,其肾脏部位产生的近红外荧光也不断增加,在顺铂处理72h后,其肾脏部位的荧光强度最高(图4b),约为没有经过顺铂处理小鼠肾脏的2.5倍。此外,其荧光成像信背比同样在顺铂处理72h的小鼠中达到6.3(图4c),远高于顺铂处理0h(~2.1),24h(~3.6),48h(~5.6)以及96h(~5.9)所产生的成像信背比。
测定顺铂诱导的急性肾损伤小鼠的肾小球滤过率(GFR),可以看到在注射了顺铂0,24,48h后,小鼠的肾小球滤过率还没有明显下降(图4d);但在注射顺铂72h后,可以看到小鼠的肾小球滤过率显著下降,约为5.15μL min-1g-1。随后,取顺铂处理不同时间的小鼠肾脏切片做H&E染色,如图4e所示,只有在经过顺铂处理72h后的肾切片上才能看到明显的铸型形成以及细胞核的坏死。同时,做肾切片Casp-3免疫荧光染色,如图4f所示,随着顺铂处理时间的延长,肾脏的Casp-3含量也显著提高,在顺铂处理后72h达到了最大,其荧光强度约为不经过顺铂处理的46倍(图4g)。不仅如此,探针1-DPA2在肾脏部位的荧光强度和肾脏部位的Casp-3的荧光强度之间的还存在很好的相关性(皮尔森相关系数r=0.999,图4h)。以上结果说明随着顺铂处理时间的增加,肾脏损伤的程度也在不断加剧,探针1-DPA2在肾脏产生的荧光可以很好的反映出肾脏的损伤程度,适用于对药物引起的急性肾损伤的成像监测。
6、探针用于在活体水平NAC对顺铂诱导的急性肾损伤的疗效评估
取15只6-8周龄的雌性BALB/C裸鼠,任选其中12只腹腔顺铂(20mg/kg)构建急性肾损伤模型。剩余3只注射生理盐水作为空白对照。随后通过腹腔注射NAC(400mg/kg)的方式对小鼠肾损伤进行1次(1X,12h)、2次(2X,12和24h)以及3次(3X,12,24和36h)的治疗(图5a)。在顺铂处理后的72h,将含有1-DPA2(25μM,200μL)的生理盐水尾静脉注射进入小鼠体内。在注射后2h的时间点使用IVIS Lumina XR III成像系统采集全身荧光图像。
如图5b所示,在尾静脉注射1-DPA2后,随着治疗次数的增加,小鼠肾脏部位产生的近红外荧光逐渐下降。对于仅经过顺铂处理的小鼠,其肾脏的荧光的强度以及信背比分别为健康小鼠的2.3、3.1倍(图5c,5d)。而对于接受了3次NAC治疗的肾损伤小鼠,其肾脏的荧光强度和信背比与健康小鼠相比没有显著性差异。随后,分析小鼠血清中的肌酐(CR),血尿素氮(BUN)和胱抑素C(CysC)的含量(图5e),可以看出随着NAC治疗次数的增加,小鼠血液中肌酐,血尿素氮以及胱抑素C的含量也逐渐趋于正常水平,证明了NAC对急性肾损伤具有较好的疗效。以上结果也说明了探针1-DPA2适用于药物对急性肾损伤疗效的成像评估。
本发明提供了一种激活型近红外小分子荧光探针及其制备方法与应用的思路及方法,具体实现该技术方案的方法和途径很多,以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (9)
2.权利要求1所述的激活型近红外小分子荧光探针的制备方法,其特征在于,包括如下步骤:
(a)将化合物IR-780-Alkyne与化合物3进行点击化学反应,引入二甲基吡啶胺基团;
(b)步骤(a)完成后,产物不纯化直接进行取代反应,引入叠氮基团;
(c)步骤(b)反应产物不纯化直接与G-DEVD-G-Alkyne进行点击化学反应,引入半胱天冬酶-3识别多肽序列G-DEVD-G-Alkyne,得到化合物4;
其中,所述G-DEVD-G-Alkyne结构为:
(d)将化合物4进行取代反应,引入荧光猝灭基团QC-1;
其中,所述QC-1结构为:
(e)将步骤(d)反应产物不纯化直接进行配位反应,向二甲基吡啶胺基团引入Zn2+,即得;
上述步骤反应式如下:
3.根据权利要求2所述的激活型近红外小分子荧光探针的制备方法,其特征在于,步骤(a)中,所述的点击化学反应为将化合物IR-780-Alkyne、化合物3、无水硫酸铜、抗坏血酸钠、三(3-羟丙基三唑甲基)胺溶于溶剂中,进行反应;其中,化合物IR-780-Alkyne、化合物3、抗坏血酸钠、无水硫酸铜、三(3-羟丙基三唑甲基)胺的摩尔比为5:(6-8):(5-8):(5-8):(5-8),反应温度为20-30℃,搅拌状态下反应15-45min。
4.根据权利要求2所述的激活型近红外小分子荧光探针的制备方法,其特征在于,步骤(b)中,将叠氮化钠溶于溶剂中,加入步骤(a)反应产物中进行取代反应;其中,化合物IR-780-Alkyne与叠氮化钠的摩尔比为5:(15-50);反应温度为20-30℃,搅拌状态下反应10-30min。
5.根据权利要求2所述的激活型近红外小分子荧光探针的制备方法,其特征在于,步骤(c)中,将化合物G-DEVDG-Alkyne溶于溶剂中,加入步骤(b)反应产物中进行反应;其中,化合物IR-7810-Alkyne与化合物G-DECDG-Alkyne的摩尔比为5:(5-10);反应温度为20-30℃,搅拌状态下反应15-45min,得到含有化合物4的反应液,经纯化、冻干,得到化合物4。
6.根据权利要求2所述的激活型近红外小分子荧光探针的制备方法,其特征在于,步骤(d)中,化合物4、QC-1活化酯和N,N-二异丙基乙胺溶于溶剂中进行反应;其中,化合物4、QC-1活化酯和N,N-二异丙基乙胺的摩尔比为5:(5-8):(5-8);反应温度为20-30℃,搅拌状态下反应0.5-1.5h。
7.根据权利要求2所述的激活型近红外小分子荧光探针的制备方法,其特征在于,步骤(e)中,将氯化锌溶于溶剂,加入步骤(d)产物中进行配位反应;其中,化合物4与ZnCl2的摩尔比为5:(6-10);反应温度为20-30℃,搅拌状态下反应0.5-1.5h;反应结束后将含有化合物的反应液纯化,冻干,即得。
8.权利要求1所述的激活型近红外小分子荧光探针用于在体外对半胱天冬酶-3活性检测中的应用。
9.权利要求1所述的激活型近红外小分子荧光探针在制备成像检测早期急性肾损伤试剂中的应用。
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