CN117466798A - 近红外荧光染料及其在氧化应激检测中的应用 - Google Patents
近红外荧光染料及其在氧化应激检测中的应用 Download PDFInfo
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- CN117466798A CN117466798A CN202310954917.4A CN202310954917A CN117466798A CN 117466798 A CN117466798 A CN 117466798A CN 202310954917 A CN202310954917 A CN 202310954917A CN 117466798 A CN117466798 A CN 117466798A
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- 125000006017 1-propenyl group Chemical group 0.000 description 1
- 125000000530 1-propynyl group Chemical group [H]C([H])([H])C#C* 0.000 description 1
- WOBLHELYXSVBJE-UHFFFAOYSA-N 2,3,4-trimethyl-1h-benzo[g]indole Chemical class CC1=CC2=CC=CC=C2C2=C1C(C)=C(C)N2 WOBLHELYXSVBJE-UHFFFAOYSA-N 0.000 description 1
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- 125000000069 2-butynyl group Chemical group [H]C([H])([H])C#CC([H])([H])* 0.000 description 1
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- 125000000524 functional group Chemical group 0.000 description 1
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- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
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Abstract
本发明提供近红外荧光染料及其在氧化应激检测中的应用。具体地,本发明提供下式I或II所示的化合物,其中,X、Y、R1~R28如文中所定义。本发明的化合物可用作近红外增强型荧光染料探针,其波长均大于800nm。本发明提供的荧光染料能在过氧亚硝酸根等活性氧/活性氮作用下激活荧光信号,产生荧光信号的增强,可以实现氧化应激过程的特异性检测。
Description
技术领域
本发明属于荧光染料领域,具体地涉及一种近红外荧光染料及其在氧化应激检测中的应用。
背景技术
花菁染料是一类经典的近红外染料母核,可以通过调整推拉电子共轭链长度,或者改变推拉电子基团的结构,来将染料的最大吸收波长大幅红移。同时,花菁染料也在许多不同的生物应用场景中得到广泛应用,如细胞成像、活体成像、光热治疗、光动力治疗等,有良好的生物应用前景。值得注意的是,绝大部分花菁类分子探针的最大吸收波长仍然低于800nm。随着花菁染料波长进一步红移突破800nm后,染料的稳定性进一步降低,能级差变得更小,分子探针的构建变得尤为困难。因此,基于花菁类近红外荧光染料,尤其是近期领域中大量涌现的最大吸收波长大于800nm的花菁类荧光染料母核,开发通用型的分子探针构建策略,实现分子在近红外波段可激活的荧光增强,将能够基于此开发出的大量结构新颖的分子探针,用于疾病诊断、微环境检测和光热治疗等领域。
发明内容
本发明的目的是提供一种波长大、能够被活性氧/活性氮激活荧光信号、且可以对活体氧化应激过程进行特异性检测的荧光染料。
本发明的第一方面提供下式I或II所示的化合物:
式中:
X各自独立为卤素或-S-C1-4烷基-NHCO-R;
Y各自独立选自以下的阴离子:F-、Cl-、Br-、I-、OAc-、HSO4 -、H2PO4 -,ClO4 -、F3CCOO-、CH3SO3 -、CF3SO3 -、BF4 -、PF6 -、NO3 -和取代或未取代的四苯基硼酸根;
R为C1-6烷基、C2-6烯基或C2-6炔基;
R1、R2、R8、R9、R15、R16、R22和R23各组独立选自氢和C1-4烷基;
R7、R14、R21和R28各自独立选自任选被1-3个选自卤素、C1-4酰基和-SO3H的取代基取代的C1-20烷基、C2-20烯基或C2-20炔基;
R3~R6、R10~R13、R17~R20和R24~R27各自独立氢、卤素和C1-4烷基;或者,R2与R3、或R3与R4、或R4与R5、或R5与R6与它们所连接的碳一同形成苯环,和/或R9与R10、或R10与R11、或R11与R12、或R12与R13与它们所连接的碳一同形成苯环,和/或R16与R17、或R17与R18、或R18与R19、或R19与R20与它们所连接的碳一同形成苯环,和/或R23与R24、或R24与R25、或R25与R26、或R26与R27与它们所连接的碳一同形成苯环。
本发明第二方面提供一种近红外荧光染料,其含有本文任一实施方案所述的式I和/或式II所示的化合物。
本发明第三方面提供本文任一实施方案所述的式I和/或式II所示的化合物在制备用于检测氧化应激的试剂或试剂盒中的用途。
本发明第四方面提供一种体外检测ONOO-的方法,包括步骤:(1)在本文任一实施方案所述的式I和/或式II所示的化合物存在下,培养健康细胞,检测荧光;(2)在本文任一实施方案所述的式I和/或式II所示的化合物存在下,培养待测细胞,检测荧光;(3)比较步骤(2)和步骤(1)中的荧光强度,如果步骤(2)中的荧光增强,提示所述的待测细胞中含有ONOO-。
本文各方面更详细的描述如下文所述。
附图说明
图1是化合物Divac 800在CDCl3中的1H-NMR谱图。
图2是化合物Divac 800在CDCl3中的13C-NMR谱图。
图3是化合物Divac 800的HR-MS谱图。
图4是化合物Divac 800-SO3H在MeOD中的1H-NMR谱图。
图5是化合物Divac 800-SO3H在MeOD中的13C-NMR谱图。
图6是化合物Divac 800-SO3H的HR-MS谱图。
图7是化合物Divac 840在CDCl3中的1H-NMR谱图。
图8是化合物Divac 840在CDCl3中的13C-NMR谱图。
图9是化合物Divac 840的HR-MS谱图。
图10是化合物Divac 1040在DMSO-d6中的1H-NMR谱图。
图11是化合物Divac 1040的HR-MS谱图。
图12是化合物Divac 1040-B在CDCl3中的1H-NMR谱图。
图13是化合物Divac 1040-B在CDCl3中的13C-NMR谱图。
图14是化合物Divac 1040-B在CDCl3中的19F-NMR谱图。
图15是化合物Divac 1040-B的HR-MS谱图。
图16是化合物SC-1在CDCl3中的1H-NMR谱图。
图17是化合物SC-1在CDCl3中的13C-NMR谱图。
图18是化合物SC-1的HR-MS谱图。
图19是化合物SC-1的单晶结构示意图。
图20是化合物SC-2在CDCl3中的1H-NMR谱图。
图21是化合物SC-2在CDCl3中的13C-NMR谱图。
图22是化合物SC-2的HR-MS谱图。
图23是化合物SC-3-B在CDCl3中的1H-NMR谱图。
图24是化合物SC-3-B在CDCl3中的13C-NMR谱图。
图25是化合物SC-3-B在CDCl3中的19F-NMR谱图。
图26是化合物SC-3-B的HR-MS谱图。
图27是化合物Divac 800-T1在CDCl3中的1H-NMR谱图。
图28是化合物Divac 800-T1在CDCl3中的13C-NMR谱图。
图29是化合物Divac 800-T1的HR-MS谱图。
图30是染料Divac 800在RAW264.7细胞外源性ONOO-的检测结果。
图31是染料Divac 800在RAW264.7细胞内源性ONOO-的检测结果。
图32是染料Divac1040对肝损伤小鼠的检测。a)探针检测肝损伤的原理;b)不同组别小鼠丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的含量;c)五组小鼠全身荧光信号随时间变化图;d)不同组别中小鼠肝组织切片的HE染色分析;e)不同组别小鼠肝脏部位荧光随时间变化曲线图。
图33是Divac 800、Cy800和ICG在808nm近红外光照射下的光热性能测试结果图。
图34是a)Divac 800、b)ICG、c)Cy 800在甲醇的单线态氧产率测试结果图。
图35是化合物Divac 800的单晶结构示意图。
具体实施方式
应理解,在本发明范围中,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,从而构成优选的技术方案。
术语
如本文所用,术语“烷基”本身或作为另一取代基的一部分,是指具有指定碳原子数的直链或支链烃基(例如C1-C20、C1-C10或C1-C6,其中C1-C20表示1-20个碳)。烷基的实例包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、仲丁基、正戊基、正己基、正庚基、正辛基等。
如本文所用,“烯基”指直链或支链含有2~20个碳原子(如2~10个碳原子、2~6个碳原子),除非碳链长度被另外限制,其中至少是链中的两个碳原子之间含有一个双键的基团。典型的链烯基包括乙烯基、1-丙烯基、2-丙烯基、2-甲基-1-丙烯基、1-丁烯基和2-丁烯基。
如本文所用,“炔基”指直链或支链含有2~20个碳原子(如2~10个碳原子、2~6个碳原子),除非碳链长度被另外限制,其中至少是链中的两个碳原子之间含有一个叁键的基团。典型的炔基包括乙炔基、1-丙炔基、1-甲基-2-丙炔基、2-丙炔基、1-丁炔基和2-丁炔基。
如本文所用,“卤素”或“卤原子”指F、Cl、Br、和I。“卤代的”是指被选自F、Cl、Br、和I的原子所取代。“卤素阴离子”是指卤素原子得到一个电子形成的阴离子,包括Cl—、Br—、I—、F—。
如本文所用,酰基指R-CHO基团,R为烷基。示例性的酰基包括甲酰基、乙酰基等。
化合物
本发明提供下式I或II所示的化合物:
式中:
X各自独立为卤素或-S-C1-4烷基-NHCO-R;
Y各自独立选自以下的阴离子:F-、Cl-、Br-、I-、OAc-、HSO4 -、H2PO4 -,ClO4 -、F3CCOO-、CH3SO3 -、CF3SO3 -、BF4 -、PF6 -、NO3 -和取代或未取代的四苯基硼酸根;
R为C1-6烷基、C2-6烯基或C2-6炔基;
R1、R2、R8、R9、R15、R16、R22和R23各组独立选自氢和C1-4烷基;
R7、R14、R21和R28各自独立选自任选被1-3个选自卤素、C1-4酰基和-SO3H的取代基取代的C1-20烷基、C2-20烯基或C2-20炔基;
R3~R6、R10~R13、R17~R20和R24~R27各自独立氢、卤素和C1-4烷基;或者,R2与R3、或R3与R4、或R4与R5、或R5与R6与它们所连接的碳一同形成苯环,和/或R9与R10、或R10与R11、或R11与R12、或R12与R13与它们所连接的碳一同形成苯环,和/或R16与R17、或R17与R18、或R18与R19、或R19与R20与它们所连接的碳一同形成苯环,和/或R23与R24、或R24与R25、或R25与R26、或R26与R27与它们所连接的碳一同形成苯环。
优选地,式I和II中,各X均为氯,或各X均为-S-C1-4烷基-NHCO-R;优选地,R为C2-6炔基。
优选地,式I和II中,各Y独立为:F-、Cl-、Br-、I-或取代或未取代的四苯基硼酸根;优选地,所述取代的四苯基硼酸根为四(卤代苯基)硼酸根,更优选为四(全氟代苯基)硼酸根,如:
优选地,式I和II中,R1、R2、R8、R9、R15、R16、R22和R23均为C1-4烷基,优选均为甲基或乙基;优选地,R1、R2、R8、R9、R15、R16、R22和R23相同。
优选地,式I和II中,R7、R14、R21和R28各自独立选自任选被1个选自-SO3H取代的C1-10烷基;优选地,R7、R14、R21和R28相同。
优选地,式I和II中,R3~R6、R10~R13、R17~R20和R24~R27均为氢;或R3与R4、R10与R11、R17与R18以及R24与R25分别与它们所连接的碳原子一起形成苯环,R5、R6、R12、R13、R19、R20、R26和R27为氢;或R2与R3、R9与R10、R16与R17以及R23与R24分别与它们所连接的碳原子一起形成苯环,R4~R6、R11~R13、R18~R20以及R25~R27均为氢。
优选地,式I和II中,被R1~R7取代的环、被R8~R14取代的环、被R15~R21取代的环以及被R22~R28取代的环各自独立选自:
其中,R1’和R2’各自独立选自C1-4烷基;R7’各自独立选自C1-12烷基;优选地,所述4个环相同。
优选地,所述式I化合物选自下组:
优选地,所述式II化合物选自下组:
化合物的合成路线
本发明利用已有的七甲川菁染料,通过端位取代基的改造,增加端基的供电子能力,实现染料吸收和发射波长的红移。发明人选择了Cy800、Cy840和Cy1040的端位取代基作为代表,用于菁染料螺环二聚体的构建。三个菁染料单体(Cy800/Cy840/Cy1040)的最大发射波长约为800/840/1040nm,能够满足不同生物应用情境下,对于染料波长的需求,实现通用型构建策略的目标。分别基于单键和螺环两种连接方式,构建得到单键连接菁染料二聚体SC-1/SC-2/和SC-3,以及螺环连接菁染料二聚体Divac 800/Divac 840/Divac 1040。
本文中,端位基团的合成可参照实施例2进行,中心连接基团的合成可参照实施例1进行。
氧化应激/过氧化亚硝酸盐(ONOO-)的检测和应用
氧化应激是指机体或细胞内氧自由基的产生与清除失衡,导致活性氧(ROS)和活性氮(RNS)在体内或细胞内蓄积而引起的氧化损伤过程。作为机体内的两大类氧化剂,ROS是指分子氧部分氧化还原形成比分子氧更具有活泼化学性质的氧的某些中间代谢产物或者是含氧衍生物,包括单线氧(1O2)、超氧阴离子(-O2-)、羟自由基(-OH)、过氧化氢(H2O2)、脂氧自由基LO-、脂过氧自由基LOO-及过氧化脂质LOOH等。RNS是以NO为中心的衍生物,包括一氧化氮(NO)、过氧化亚硝酸盐(ONOO-)等一系列含氮化合物。当产生氧化应激时,这些物质会导致不同组织细胞等遭受到不同程度的损伤与破坏,主要表现在脂质、氨基酸和蛋白质的氧化损伤、核酸和染色体等氧化损伤方面。
氧化应激可通过测量活性氧和活性氮来直接评估,或通过过度产生的活性氧和活性氮对脂质,蛋白质和核酸造成的相关损害来间接评估。由于活性氧和活性氮的瞬时特性,通常依赖于间接方法,但为了避免单独用某一种指标会检测不准确,一般应当综合考虑各种氧化代谢产物。
本发明提供了一种体外直接测量活性氧和活性氮的方法。在本发明的荧光染料分子存在下培养细胞,相比于健康细胞,如果细胞中含有活性氧或活性氮,荧光染料能够获得荧光开启,可以被仪器检测到荧光增强,进而提示细胞中含有活性氧或活性氮。细胞可以是人类或动物的离体细胞。在一些实施方案中,细胞可以是巨噬细胞。活性氧或活性氮可以是细胞内源的,也可以是细胞外源的。在一些实施方案中,本发明公开了一种体外检测ONOO-的方法,包括步骤:(1)在如本发明的荧光染料分子存在下,培养健康细胞,检测荧光;(2)在荧光染料分子存在下,培养待测细胞,检测荧光;(3)比较步骤(2)和步骤(1)中的荧光强度,如果步骤(2)中的荧光增强,提示所述的待测细胞中含有ONOO-。优选地,所述的细胞为巨噬细胞。优选地,所述的ONOO-为细胞外源性ONOO-和/或细胞内源性ONOO-。
因此,在一些实施方案中,本发明包括本文公开的式I和式II化合物在制备用于检测氧化应激的试剂或试剂盒中的应用。检测氧化应激包括检测活性氧和/或活性氮。活性氧和/或活性氮的种类如前所述。优选地,活性氧和/或活性氮选自:超氧化物、过氧化自由基、过氧化氢、羟自由基、过氧亚硝酸根。在一些实施方案中,试剂盒通过检测过氧亚硝酸根来检测氧化应激。
本发明的化合物的最大吸收波长在800nm以上,在近红外荧光成像、光热治疗、光动力治疗、太阳能光敏电池等方面有广泛的应用。因此,在一些实施方案中,本发明还包括本文公开的式I和式II化合物、其染料组合物在近红外荧光成像、光热治疗、光动力治疗或太阳能光敏电池中的应用,或在制备近红外荧光成像、光热治疗、光动力治疗或太阳能光敏电池中使用的试剂中的应用。
近红外荧光染料或染料组合物
本发明还提供近红外荧光染料或染料组合物,该组合物含有本发明的式I和/或式II化合物。该染料或组合物还可含有溶剂。
适用于本发明的溶剂包括但不限于MeOH、EtOH、PrOH、iPrOH、BuOH、丙酮、DMF、DMSO、吡啶、DCM、氯仿、二氯乙烷、MeCN、苯、甲苯、对二甲苯、氯苯、硝基苯、1,4-二噁烷、THF、乙酸乙酯和AcOH中的一种或多种的混合物。
试剂盒
本发明还提供了一种用于检测氧化应激的试剂盒,试剂盒中包含本文公开的式I和/或式II的化合物,或所述近红外荧光染料或染料组合物。
本发明的优点包括:
(1)本发明提供了一种新的近红外增强型探针的构建策略,并基于这一策略开发了一系列近红外增强型荧光染料探针。
(2)本发明提供的荧光染料具有较大的波长,均大于800nm。
(3)本发明提供的荧光染料能在过氧亚硝酸根等活性氧/活性氮作用下激活荧光信号,产生荧光信号的增强。
(4)本发明提供的荧光染料可以实现氧化应激过程的特异性检测,具有良好的生物应用价值。
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,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
实施例1:螺环连接菁染料二聚体的中心连接基团合成
具体合成步骤如下。
先将4-羟基-2-丁酮(10.0g,1.0eq 113.5mmol)溶于100mL甲苯,氩气保护下,加热至70℃,缓慢注射0.3mL浓硫酸,溶液变为棕黄色,继续升温至120℃回流并用分水器除水,反应1h。取3-环己烯-1-甲醛(12.5g,1.0eq,113.5mmol),缓慢注射入反应体系,反应2h。反应结束后,溶液呈棕色悬浊液,旋蒸除去溶剂,加适量水,加饱和碳酸氢钠溶液中和。用150mL的DCM萃取三次,有机相用无水Na2SO4干燥,旋干。粗产品用柱色谱分离,PE:EA=300:10(v/v),得到黄色液体6.4g,即为化合物1-1,产率35%。1HNMR(400MHz,CDCl3)δ6.79(d,J=10.2Hz,1H),5.91(d,J=10.2Hz,1H),5.81–5.72(m,1H),5.68–5.60(m,1H),2.53–2.39(m,2H),2.09(dd,J=4.2,1.7Hz,2H),2.06–2.00(m,2H),1.95(ddd,J=14.1,8.9,5.4Hz,2H),1.81(ddd,J=14.6,10.4,4.4Hz,2H),1.56(dt,J=13.0,6.6Hz,1H)。
将化合物1-1(5.0g,1.0eq 30.8mmol)用150mL无水甲醇溶解,加入氯化亚铜(2.9g,0.5eq,15.4mmol),冰浴下缓慢分批加入NaBH4固体(2.3g,2.0eq,61.6mmol)(第一次加入的量要稍大,使得反应液迅速变黑并放出气体,在确保安全的前提下,尽快把NaBH4加完),冰浴下敞口反应3h,TLC跟踪至原料完全反应完。缓慢滴加3M的盐酸淬灭未完全反应NaBH4,调节pH至弱酸性,旋干溶剂,加DCM/H2O萃取,有机相用无水Na2SO4干燥,旋蒸除去溶剂,柱层析分离(DCM),得到3.5g淡黄色液体,即为化合物1-2.产率68%。1H NMR(400MHz,CDCl3)δ5.74–5.53(m,2H),3.64(s,1H),1.98(dd,J=17.3,14.6Hz,3H),1.79–1.73(m,2H),1.55–1.34(m,7H),1.21–1.10(m,2H)。
将化合物1-2(3.0g,1.0eq,18.0mmol)溶解在100mL二氯甲烷中,冰浴下,缓慢加入mCPBA(6.2g,2.0eq,36.1mmol)。反应过夜,TLC点板跟踪。反应结束后,加过量亚硫酸钠固体淬灭mCPBA,反应2h,用淀粉碘化钾试纸检测mCPBA是否完全除去,抽滤,滤液加饱和碳酸钠溶液洗涤(除去生成的间氯苯甲酸),至水相呈中性。收集有机相,无水Na2SO4干燥,旋蒸除去溶剂。柱色谱分离,PE:EA=10:1(v/v),得到无色透明粘稠液体2.1g,即为化合物1-3,产率64%。1H NMR(400MHz,CDCl3)δ3.70–3.55(m,1H),3.18–3.06(m,2H),1.98(d,J=15.4Hz,1H),1.84–1.65(m,5H),1.54–1.42(m,3H),1.41–1.31(m,2H),1.22–1.02(m,3H)。
将化合物1-3(2.0g,1.0eq,11.0mmol,)溶解在100mL无水THF中,冰浴下,逐滴加入LiAlH4的THF溶液(2M,11.0mL,2.0eq,22.0mmol,),继续反应3h。TLC点板跟踪,至原料完全转化。先在反应体系中缓慢逐滴加入1mL水,然后加入1mL的15%的氢氧化钠水溶液,再加入3mL水,室温下搅拌40min,然后加入适量无水Na2SO4干燥,接着将反应液抽滤,DCM洗涤三遍,将有机相旋干,柱层析分离,DCM:MeOH=10:1,v/v。得到淡黄色粘稠液体1.4g,即为化合物1-4和1-5的混合物,总产率69%。1H NMR(400MHz,CDCl3)δ3.71(t,J=6.6Hz,2H),3.59(td,J=9.3,4.6Hz,3H),2.18(d,J=49.0Hz,6H),1.93(d,J=12.1Hz,2H),1.85–1.65(m,12H),1.38(dd,J=16.9,7.4Hz,6H),1.12(ddd,J=36.3,20.1,11.3Hz,6H)。
先配置Jones试剂,将2.4g CrO3、2.1mL浓硫酸、9.0mL水加入到25mL烧杯中搅匀。将化合物1-4(1.2g,1.0eq,6.5mmol)加入到50mL丙酮中,缓慢滴加5mL的Jones reagent,室温下搅拌3h。反应结束后,先加碳酸氢钠中和,然后用硅藻土抽滤,用30mL二氯甲烷洗三遍。用水萃取,有机相无水Na2SO4干燥,旋干,柱层析,DCM:MeOH=500:1(v/v),得到0.9g淡黄色液体,即为化合物螺[5.5]十一烷-3,9-二酮(1-6),产率80%。1H NMR(400MHz,CDCl3)δ2.39(t,J=6.8,8H)δ1.88(t,J=6.8,8H)。
将10mL的DMF加入到烘干的双口烧瓶中,在氩气保护及冰浴下,缓慢注射10mL三氯氧磷,室温下反应1h,得到无色粘稠液体,即为Vilsmeier-Haack试剂。将化合物螺[5.5]十一烷-3,9-二酮(1.0g,5.6mmol)溶于10mL DCM和10mL DMF的混合溶剂中,氩气保护下,将4mL上述得到的Vilsmeier haack试剂加入到该溶液中,加热至80℃回流反应4h。反应结束后,将反应体系冷却至室温,然后将产物倒入200g冰水混合物中,在4℃冰箱中静置过夜。然后将上述液体用布氏漏斗抽滤,滤饼用石油醚洗涤5遍,干燥。得到1.3g黄色固体粉末,即为化合物1-7,其不稳定,容易被氧化,应除氧后存放在-20℃冰箱中。不经过进一步纯化,直接进行下一步反应。
实施例2:端基的合成
需要构建的端位基团共有5个,其中端基H1和H2的起始原料为2,3,3-三甲基吲哚,H3的起始原料为2,3,3-三甲基苯并吲哚,2-2和2-4的起始原料为1,8-萘内酰亚胺,这些起始原料均为商品化产品。
化合物H1、H2和H3的构建,都是利用氮原子的亲核性,与相应的亲电试剂通过一步亲核取代反应得到的,构建H1和H3的亲电试剂为碘乙烷,H2的为1,3-丙磺内酯。根据文献报道合成H1-H3。通用的合成策略是将不同的三甲基苯并吲哚与等当量的烷基化试剂在乙腈中回流进行予以制备。
端基2-2的构建需要先在1,8-萘内酰亚胺的氮原子上利用卤代烃进行烷基化,首先用碘乙烷进行烷基化,然后用甲基格式试剂处理,再经过一步脱水,得到所需的吲哚。
具体实验操作如下:
将1,8-萘内酰亚胺(2.0g,1.0eq,11.8mmol)溶于15mL无水THF,冰浴下分批缓慢加入NaH(0.9g,2.0eq,23.6mmol,60%in oil)加的过程中不断搅拌,加完后继续反应15分钟。然后缓慢加入碘乙烷(5.5g,3.0eq,35.5mmol),将反应升温至室温,继续反应3小时,TLC跟踪反应进程。反应结束后先加20mL氨水淬灭反应体系中未反应完的碘乙烷。用EA/H2O萃取三次,收集有机相,干燥,抽滤,旋蒸除去溶剂。柱色谱分离,PE:EA=50:1(v/v),得到黄色固体2.1g,即为化合物2-1,产率90%。1H NMR(400MHz,CDCl3)δ8.06(d,J=7.0Hz,1H),8.01(d,J=8.1Hz,1H),7.71(dd,J=8.1,7.1Hz,1H),7.53(d,J=8.4Hz,1H),7.47(dd,J=8.4,7.0Hz,1H),6.92(d,J=6.9Hz,1H),3.99(q,J=7.2Hz,2H),1.38(t,J=7.2Hz,3H)。
将化合物2-1(1.0g,1.0eq,5.1mmol)溶解于10mL无水THF中,氩气保护,冰浴下,逐滴加入甲基溴化镁(3.4mL,2.0eq,10.1mmol,3M in THF),逐渐升高至60℃反应1h,冷却至室温,逐滴加入6mL 3M的盐酸溶液,反应5min,观察到溶液从黄色变为绿色,加入5mL 1M碘化钾的甲醇溶液,搅拌5min,向滤液中加入20mL石油醚,使产物析出,过滤出产物,用石油醚洗涤3遍,干燥。得到0.8g黄色固体,即为化合物2-2,产率82%。.1H NMR(400MHz,DMSO)δ9.01(s,1H),8.82(s,1H),8.52(d,J=44.4Hz,2H),8.17(s,1H),8.01(s,1H),4.74(d,J=6.3Hz,2H),3.26(s,3H),1.56(t,J=7.0Hz,3H)。
同时,考虑到单体M1030的平面性非常好,极易发生堆积。因此,为了增加其溶解性,需在端基引入长链烷烃,这里选择引入辛烷。构建过程与引入乙基相似,具体操作如下:
先将1,8-萘内酰亚胺(5.0g,1.0eq,29.6mmol)溶于20mL无水THF中,冰浴下,缓慢加入NaH(2.4g,2.0eq,59.1mmol,60%in oil)反应20分钟后,加入1-碘辛烷(10.7g,1.5eq,44.3mmol),氩气保护下室温反应1小时。加入冰水淬灭NaH,旋蒸除去溶剂,柱层析分离,PE:DCM=5:1(v/v),得到6.0g黄色油状液体,即为化合物2-3,产率72.2%。1H NMR(400MHz,CDCl3)δ8.06(d,J=7.0Hz,1H),8.00(d,J=8.1Hz,1H),7.70(dd,J=8.0,7.1Hz,1H),7.52(d,J=8.3Hz,1H),7.49–7.42(m,1H),6.90(d,J=6.9Hz,1H),3.91(t,J=7.3Hz,2H),1.82–1.74(m,2H),1.43–1.23(m,10H),0.86(dd,J=8.4,4.6Hz,3H)。
将化合物2-3(2.0g,1.0eq,7.1mmol)溶于10mL无水THF中,氩气保护,冰浴下,逐滴加入甲基溴化镁(7.1mL,3.0eq,21.3mmol,3M in THF),逐渐升温至60℃反应1h,然后将其冷却至室温。逐滴加入10mL 3M的盐酸溶液,反应5min,观察到溶液从黄色变为绿色,加入5mL 1M碘化钾的甲醇溶液,搅拌5min,过滤除去无机盐,向滤液中加入20mL石油醚,使产物析出,过滤,用EA和PE各洗涤3遍,得到2.1g棕黄色固体,即为化合物2-4,产率73%。1H NMR(400MHz,CDCl3)δ8.94(d,J=7.3Hz,1H),8.60(d,J=8.1Hz,1H),8.35(d,J=7.4Hz,1H),8.27(d,J=8.2Hz,1H),8.06(t,J=7.7Hz,1H),7.90(t,J=7.8Hz,1H),4.87(t,J=7.5Hz,2H),3.49(s,3H),2.03(dt,J=15.3,7.7Hz,2H),1.54–1.45(m,2H),1.25(dd,J=11.9,6.0Hz,10H),0.83(d,J=7.1Hz,3H)。
实施例3:螺环菁染料二聚体的合成
将菁染料的端基和中心连接基团都合成完毕之后,需要进行最后一步构建菁染料的操作。选择通用的Cy7合成方法,即在弱碱(三乙胺或醋酸钠)条件下,吲哚和中心连接基团在乙酸和乙酸酐的混合溶剂中加热反应得到产物,其中酸酐充当脱水。
分子Divac 800的合成步骤和谱图表征如下:
在100mL烘干的双口烧瓶中依次加入化合物1-7(0.2g,1.0eq,0.6mmol)、醋酸钠(0.3g,6.0eq,3.7mmol)、化合物H1(0.7mg,6.0eq,3.7mmol),依次加入10mL乙酸和10mL乙酸酐,氮气除氧15min。在氩气保护下,加热至120℃回流,反应4h。反应结束后,将反应体系冷却至室温,旋蒸除去溶剂,粗产物用柱色谱分离,MeOH:DCM=30:1(v/v)。得到0.3g,有金属光泽的墨绿色固体,即为化合物Divac 800,产率46%。1H NMR(600MHz,CDCl3)δ8.34(d,J=14.1Hz,4H),7.37–7.31(m,8H),7.20(t,J=7.4Hz,4H),7.12(d,J=7.9Hz,4H),6.29(d,J=14.1Hz,4H),4.26(q,J=7.1Hz,8H),2.93(s,8H),1.64(s,24H),1.43(t,J=7.3Hz,12H).13CNMR(101MHz,CDCl3)δ171.97,148.67,145.08,141.66,141.10,128.85,125.41,122.33,110.71,101.44,53.48,49.26,41.33,40.20,36.61,31.57,29.04,28.09,27.64,22.60,20.42,18.75,14.30,14.10,12.78,11.41.HRMS(ESI),m/z,calcd.for C67H76Cl2N4 2+[M/2]:503.2718,found:503.2725。
图1、2和3分别是化合物Divac 800在CDCl3中的1H-NMR谱图、在CDCl3中的13C-NMR谱图和HR-MS谱图。从Divac 800单晶结构(图35)的主视图可以清晰的看到,两个Cy7染料是呈现倒V字型交叠的。
分子Divac 800-SO3H的合成步骤和谱图表征如下:
在100mL烘干的双口烧瓶中依次加入化合物1-7(0.3g,1.0eq,0.9mmol)、醋酸钠(0.4g,5.0eq,4.6mmol)、化合物H2(1.3g,5.0eq,4.6mmol),加入30mL乙酸和30mL乙酸酐,氮气除氧15min。在氩气保护下,加热至120℃回流,反应4h。反应结束后,将反应体系冷却至室温,旋蒸除去溶剂,粗产物用柱色谱分离,MeOH:DCM:CH3COOH=100:400:10(v/v/v)。得到0.5g有金属光泽的墨绿色固体,即为化合物Divac 800-SO3H,产率40%。1H NMR(400MHz,MeOD)δ8.48(d,J=14.1Hz,4H),7.45(d,J=7.1Hz,8H),7.39(t,J=7.5Hz,4H),7.24(t,J=7.4Hz,4H),6.41(d,J=14.1Hz,4H),4.47–4.31(m,8H),2.98(t,J=6.5Hz,8H),2.81(s,8H),2.30–2.16(m,8H),1.67(s,24H).13C NMR(151MHz,MeOD)δ174.65,145.11,142.05,128.57,125.18(d,J=2.6Hz),122.03,111.08,101.28,49.22,48.04,47.89,47.82–47.54,47.47,47.33,47.18,42.75,35.60,27.04,22.80,19.92.HRMS(ESI)m/z calcd forC71H80Cl2N4O12S4 2-[M+3H]+:1381.4262;found:1381.4832。
图4~6分别是化合物Divac 800-SO3H在MeOD中的1H-NMR谱图、在MeOD中的13C-NMR谱图、以及HR-MS谱图。
分子Divac 840的合成步骤和谱图表征如下:
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在100mL烘干的双口烧瓶中依次加入化合物1-7(0.2g,1.0eq,0.6mmol)、醋酸钠(0.3g,6.0eq,3.7mmol)、化合物H3(0.8g,6.0eq,3.7mmol),加入10mL乙酸和10mL乙酸酐,氮气除氧15min。在氩气保护下,加热至120℃回流,反应10h。反应结束后,将反应体系冷却至室温,旋蒸除去溶剂,粗产物用柱色谱分离,MeOH:DCM=20:1(v/v)。得到0.3g有金属光泽的墨绿色固体,即为化合物Divac 840,产率40%。1H NMR(600MHz,CDCl3)δ8.49(d,J=14.1Hz,4H),8.07(d,J=8.5Hz,4H),7.92–7.89(m,8H),7.60–7.57(m,4H),7.45(d,J=7.8Hz,4H),7.42–7.39(m,4H),6.38(d,J=14.2Hz,4H),4.43(dd,J=14.4,7.1Hz,8H),3.06–2.99(m,8H),1.96(s,24H),1.53(t,J=7.3Hz,12H).,13C NMR(151MHz,CDCl3)δ173.28,148.03,144.35,139.17,133.96,131.96,130.87,130.14,128.14,127.77,125.47,125.11,122.05,110.58,101.09,51.03,40.48,36.74,31.41,27.68,13.14.HRMS(ESI)m/z calcd.forC67H76Cl2N4 2+,[(M+2H)/2]:604.3109,found:604.3039。
图7~9分别是化合物Divac 840在CDCl3中的1H-NMR谱图、在CDCl3中的13C-NMR谱图、以及HR-MS谱图。
分子Divac 1040的合成步骤和谱图表征如下:
将化合物1-7(150.0mg,1.0eq,0.5mmol)和化合物2-4(883.6mg,6.0eq,2.7mmol)溶解于5mL乙酸,缓慢加入三乙胺(276.7mg,6.0eq,2.7mmol)和5mL乙酸酐,氩气保护下,加热至90℃反应3小时。冷却至室温,加入20mL乙酸乙酯,抽滤,滤饼用乙酸乙酯和石油醚洗涤四次,然后用乙醇重结晶(度不高于60℃),得到黑色固体,柱色谱分离,DCM:MeOH=10:1(v/v),得到黑色固体280.0mg,即为化合物Divac 1040,产率62%。化合物溶解性有所改善,但仍然不够理想,没有取得理想的碳谱数据,只有氢谱和质谱的数据。1H NMR(600MHz,DMSO-d6)δ8.71(d,J=13.8Hz,4H),8.23(d,J=7.5Hz,4H),8.01(d,J=8.0Hz,4H),7.71(dd,J=15.9,7.8Hz,8H),7.64–7.61(m,4H),7.54(d,J=7.2Hz,4H),6.80(d,J=14.0Hz,4H),4.33(s,8H),2.95(s,8H),1.85–1.78(m,8H),1.41–1.37(m,8H),1.29–1.26(m,8H),1.13(s,8H),1.07–1.05(m,10H),0.67(t,J=6.8Hz,12H).HRMS(ESI)m/z calcd.for C95H108Cl2N4 2+[(M+2H)/2]:688.3943,found:688.3984。
图10~11分别是化合物Divac 1040在DMSO-d6中的1H-NMR谱图及其HR-MS谱图。
同时,发明人通过加大位阻阴离子的方式,提高化合物Divac 1040的溶解性,具体合成步骤如下:
将Divac 1040(178.0mg,1.0eq,109.0M)溶于10mL丙酮,将四(全氟取代苯基)硼酸钠(191.0mg,2.5eq,272.4M)溶于去离子水,配置成10%的水溶液。向Divac 1040的丙酮溶液中逐滴加入上述水溶液,室温下,氩气保护,搅拌反应3h。反应结束后用无水Na2SO4干燥,抽滤,柱色谱分离(纯DCM),旋蒸除去溶剂,得到棕褐色固体270.0mg,即为染料Divac 1040-B,产率91%。1H NMR(600MHz,CDCl3)δ8.80(d,J=13.9Hz,4H),8.17(d,J=7.5Hz,4H),7.84(d,J=8.0Hz,4H),7.56(dd,J=15.4,7.9Hz,8H),7.51–7.47(m,4H),7.05(d,J=7.2Hz,4H),6.45(d,J=13.9Hz,4H),4.01(t,J=7.1Hz,8H),2.76(s,8H),1.80(dt,J=14.7,7.4Hz,8H),1.34(t,J=7.3Hz,10H),1.26(t,J=7.5Hz,10H),1.17–1.12(m,20H),0.75(t,J=7.0Hz,12H).13C NMR(151MHz,CDCl3)δ154.33,148.97,148.17,147.37,143.71,140.74,138.93,137.01,135.41,131.67,129.80,129.56,129.50,129.30,129.27,128.28,127.63,124.71,124.00,110.01,106.63,48.50,44.35,40.70,36.70,35.89,32.63,31.64,31.45,30.21,29.18,29.08,29.03,28.38,26.98,22.51,13.90.19F NMR(377MHz,CDCl3)δ-132.43(s),-163.03(t,J=20.7Hz),-166.72(t,J=18.3Hz).HRMS(ESI)m/z calcd.forC95H108Cl2N4 2+[(M+2H)/2]:688.3943,found:688.3984。
图12~15分别是化合物Divac 1040-B在CDCl3中的1H-NMR谱图、在CDCl3中的13C-NMR谱图、在CDCl3中的19F-NMR谱图及其HR-MS谱图。
实施例4:单键连接菁染料二聚体的合成
SC-1化合物的合成路线如下所示:
具体的合成步骤如下所示:
先在干燥的两口烧瓶中加入2.8mL的DMF(36.0mmol,7.0eq)氩气保护,冰浴下,加入2.9mL的POCl3(30.9mmol,6.0eq),搅拌反应40min。用10mL的1,2-二氯乙烷将4,4'-二氧代联环己烷(1.0g,1.0eq,5.2mmol)溶解,然后加入到上述混合液中,加热至80℃反应3h。反应结束后,冷却至室温,将上述反应液倒入200mL冰水混合物中,在4℃冰箱中静置过夜。次日,会有大量黄色固体析出,抽滤,滤饼用石油醚洗涤三遍,干燥,得到淡黄色固体1.5g,即为化合物3-1,产率85%。不经过进一步纯化,直接进行下一步反应。
将化合3-1(1.0g,1.0eq,2.9mmol)、无水醋酸钠(1.4g,6.0eq 17.5mmol)、化合物H1(3.2g,6.0eq,17.5mmol)溶于5mL乙酸中,加入5mL乙酸酐,氩气保护下,加热至125℃反应4h。反应结束后,过滤除去无机盐,旋蒸除去溶剂,柱层析分离(DCM:MeOH=15:1),得到绿色带有红色金属光泽的固体1.3g,即为染料SC-1,产率44%。1H NMR(400MHz,CDCl3)δ8.40(d,J=14.1Hz,4H),7.36(d,J=7.4Hz,8H),7.24(d,J=7.3Hz,4H),7.12(d,J=7.9Hz,4H),6.51(d,J=14.1Hz,4H),4.51–4.31(m,8H),3.11(d,J=12.9Hz,4H),2.95–2.79(m,4H),2.29(s,2H),1.73(d,J=3.9Hz,24H),1.44(dd,J=9.3,4.9Hz,12H).13CNMR(151MHz,CDCl3)δ171.89,150.31,144.74,141.99,141.39,128.71,127.52,125.01,122.18,110.64,102.14,49.28,40.47,36.44,30.17,29.70,28.15,28.12,12.74.HRMS(ESI)m/z calcd.forC68H78Cl2N4 2+[M/2]:510.2786,found:510.2806。
图16~19是化合物SC-1在CDCl3中的1H-NMR谱图、在CDCl3中的13C-NMR谱图、HR-MS谱图和单晶结构示意图。
从SC-1的单晶结构(图19)的主视图和侧视图可以清晰的看到,在菁染料H聚集体中,两个Cy800染料呈现出平行交叠的特点,且均保持了良好的平面性,这与最初的设想是完全一致的。
接下来继续进行另外两个H聚集体的合成,操作步骤如下:
将化合物3-1(150.0mg,1.0eq,0.4mmol)、三乙胺(265.4mg,6.0eq,2.6mmol)、化合物H3(957.8mg,6.0eq,2.6mmol)溶于5mL乙酸中,加入5mL乙酸酐,氩气保护下,加热至125℃反应4h。反应结束后,过滤除去无机盐,旋蒸除去溶剂,柱层析分离(DCM:MeOH=15:1),得到墨绿色带有红色金属光泽的固体220.0mg,即为染料SC-2,产率44%。1H NMR(600MHz,CDCl3)δ8.52(d,J=14.1Hz,4H),8.13(d,J=8.5Hz,4H),7.92(dd,J=8.4,4.5Hz,8H),7.63–7.58(m,4H),7.46(dd,J=11.2,3.9Hz,4H),7.39(d,J=8.8Hz,4H),6.56(d,J=14.2Hz,4H),4.55(dd,J=14.5,7.3Hz,8H),3.27(dd,J=7.2,5.7Hz,2H),3.17(d,J=12.3Hz,4H),2.98–2.89(m,4H),2.04(d,J=5.1Hz,24H),1.50(t,J=7.2Hz,12H).13C NMR(151MHz,CDCl3)δ173.27,170.01,149.60,143.73,139.39,134.07,131.86,130.69,130.15,128.19,127.65,127.30,124.94,122.09,110.70,101.64,53.45,51.08,40.62,36.42,34.45,30.19,29.70,29.32,27.72,27.61,23.40,22.54,14.87,14.13,13.05.HRMS(ESI)m/z calcd.forC84H86Cl2N4 2+[(M+2H)/2]:611.3109,found:611.3119。
图20~22分别是化合物SC-2在CDCl3中的1H-NMR谱图、在CDCl3中的13C-NMR谱图、HR-MS谱图。
将化合物3-1(150.0mg,1.0eq,0.4mmol)、三乙胺(265.4mg,6.0eq,2.6mmol)、化合物2-4(1.1g,6.0eq,2.6mmol)溶于5mL乙酸中,加入5mL乙酸酐,氩气保护下,加热至120℃反应3h。反应结束后,冷却至室温,加入40mL的乙酸乙酯,有固体析出,抽滤,用正己烷洗涤滤饼三次,干燥,得到黑绿色固体400.0mg,即为化合物SC-3,产率56%。
将SC-3(177.4mg,1.0eq,107.9M)溶于10mL丙酮,将四(全氟取代苯基)硼酸钠(189.3mg,2.5eq,269.6M)溶于去离子水,配置成10%的水溶液。向SC-3的丙酮溶液中逐滴加入上述水溶液,室温下,氩气保护,搅拌反应3h。反应结束后用无水硫酸钠干燥,抽滤,柱色谱分离(纯DCM),旋蒸除去溶剂,得到棕褐色固体255.0mg,即为染料SC-3-B,产率86%。1HNMR(600MHz,CDCl3)δ8.92(d,J=10.9Hz,4H),8.41(d,J=7.2Hz,4H),8.10(d,J=8.0Hz,4H),7.82(t,J=7.7Hz,4H),7.71(d,J=3.4Hz,4H),7.59(t,J=7.7Hz,4H),7.13(dd,J=6.0,2.4Hz,4H),6.56(s,4H),4.01(d,J=22.0Hz,8H),2.93(s,4H),2.71(s,4H),2.28–2.23(m,2H),1.78(dt,J=15.0,7.4Hz,8H),1.32–1.26(m,16H),1.21–1.17(m,8H),1.14–1.10(m,17H),0.73(t,J=7.1Hz,12H).13CNMR(151MHz,CDCl3)δ147.86,146.27,142.21,140.01,138.37,137.86,135.93,134.30,130.77,128.94,128.83,127.18,125.04,124.13,122.84,120.56,113.04,108.89,105.60,102.22,43.21,30.59,30.41,29.29,29.17,28.67,28.15,28.00,27.89,25.89,21.45,12.85.19F NMR(565MHz,CDCl3)δ-132.25(s),-162.59(s),-166.50(s).HRMS(ESI)m/z calcd.for C96H110Cl2N4 2+[(M+2H)/2]:695.9143,found:695.4060。
图23~26是化合物SC-3-B在CDCl3中的1H-NMR谱图、在CDCl3中的13C-NMR谱图、在CDCl3中的19F-NMR谱图、HR-MS谱图。
实施例5:螺环连接菁染料二聚体的结构修饰
得到菁染料二聚体之后,为了便于进行之后的生物应用,需要在染料分子上进行功能化基团的修饰,目前使用较多的是生物正交的方法(Bioorthogonality),即叠氮化合物与末端带炔基的化合物进行的1,3-偶极环加成反应,产物为三氮唑。该反应在体外发生时需要一价铜离子的催化,在室温下即可发生,非常高效。发明人在染料上修饰炔基,以偶联其他叠氮修饰的生物标志物或生物大分子。引入炔基,一般需要用亲核试剂将染料中心氯原子进行亲核取代。N取代的胺化菁染料会导致波长变短,不利于探针的应用,而氧和硫取代则对染料的波长影响很小,是合适的修饰方法。发明人构建了端位分别暴露炔基和巯基的连接臂,具体制备过程如下所示:
将4-戊炔酸(5.0g,1.0eq,51.0mmol)、N-羟基琥珀酰亚胺(7.0g,1.2eq,61.2mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)(11.7g,1.2eq,61.2mmol)、三乙胺(12.9g,2.5eq,127.4mmol)溶解在150mL的DCM中,氩气保护,室温下反应30小时。TLC点板跟踪反应进程,发现原料完全反应,旋蒸除去溶剂,以EA/H2O萃取三次,收集有机相,无水硫酸钠干燥,旋蒸除去溶剂后用柱色谱分离,PE:EA=20:1(v/v),得到浅黄色固体7.5g,即为化合物T1-1,产率为75%。不经过进一步纯化,直接进行下一步反应。
将化合物T1-1(5.0g,2.0eq,25.6mmol)、化合物胱胺(2.0g,1.0eq,12.8mmol)和三乙胺(5.2g,4.0eq,51.2mmol)溶解于150mL的DMF中(超声助溶,可适当加热促进溶解),氩气保护下,室温反应20小时,TLC点板跟踪反应进程,观察到原料完全消耗。旋蒸除去溶剂,用EA/H2O萃取三遍,收集有机相,无水Na2SO4干燥,抽滤,旋蒸除去溶剂后得到2.9g乳白色固体,即为化合物T1-2,产率73%。1H NMR(400MHz,DMSO-d6)δ8.13(t,J=5.5Hz,2H),3.36–3.30(m,4H),2.79–2.73(m,6H),2.37–2.32(m,4H),2.27(dd,J=11.3,4.2Hz,4H).13C NMR(101MHz,DMSO)δ170.90,84.15,71.85,38.43,37.60,34.58,14.65.HRMS(ESI)m/z.Calcd.for C14H20N2O2S2[M+H]+=313.1039,found:313.1032。
将化合物Divac 800(250.0mg,1.0eq,198.4mmol)、T1-2(124.0mg,2.0eq,396.7mmol)、三(2-羧乙基)膦(148.9mg,3.0eq,0.6mmol)、三乙胺(100.4mg,5.0eq,1.0mmol)溶于10mL无水DMF,氮气除氧20min,氩气保护下,室温反应2h,TLC点板跟踪反应进程,(反应结束后溶液呈现棕黄色,说明有大量染料在碱性条件下被破坏)发现原料完全消耗,旋蒸除去溶剂,直接用柱层析分离,DCM:MeOH=20:1(v/v),得到墨绿色固体85.0mg,即为化合物Divac 800-T1,产率35%。1H NMR(600MHz,CDCl3)δ8.83(d,J=14.1Hz,4H),8.66(s,2H),7.33(dd,J=13.6,7.1Hz,8H),7.19(t,J=7.5Hz,4H),7.03(d,J=7.9Hz,4H),6.19(d,J=14.1Hz,4H),4.10(dd,J=13.8,6.6Hz,8H),3.50–3.40(m,4H),3.08–3.01(m,4H),2.71(s,8H),2.48–2.40(m,9H),1.80(d,J=2.4Hz,2H),1.68(s,24H),1.37(t,J=7.1Hz,12H).13C NMR(151MHz,CDCl3)δ175.92,172.15,171.89,157.03,146.91,141.78,141.05,131.34,128.67,125.04,122.35,110.17,100.98,83.42,68.58,49.22,45.74,40.02,39.47,37.01,35.93,34.71,31.93,30.91,29.70,29.32,27.96,22.70,20.99,14.72,14.13,12.37,8.60,1.02.HRMS(ESI)m/zcalcd.for C81H96S2N6O2 2+[M/2]:624.3513;found,624.3517。
图27~29分别是化合物Divac 800-T1在CDCl3中的1H-NMR谱图、在CDCl3中的13C-NMR谱图、HR-MS谱图。
实施例6:化合物Divac 800的细胞成像图
该项实验分为两个部分,第一部分是探究分子能否对细胞外源性的ONOO-进行检测,第二部分是探究分子能否对细胞内源性的ONOO-进行检测。本实验所选用的细胞系为小鼠巨噬细胞系RAW264.7细胞,将其作为生物实验模型,是因为巨噬细胞在脂多糖(LPS)、干扰素γ(IFN-γ)和佛波酯(PMA)的刺激下,能够产生活性氧(ROS)和活性氮(RNS)。
首先想要探究的是,分子能否对细胞外源性的ONOO-进行检测,实验中选用的外源性ONOO-供体是林西多明,商品名为SIN-1。ONOO-的淬灭剂为米诺环素。实验所用的成像设备是发明人自行搭建的近红外共聚焦荧光显微镜,选择的激发波长为808nm,接收波段为850nm-890nm。
结果如图30所示。从图30的数据可知,分子Divac 800能够很容易的进入细胞(只需孵育10分钟),进入细胞后分子本身会呈现出一定的荧光(control组)。当加入SIN-1孵育1h之后,荧光有明显增强。文献中报道,释放ONOO-的最大浓度约为加入初始SIN-1浓度的1.2%-3.6%,并且,不同的细胞以及不同的培养环境都会对释放速率产生影响。于是,继续增加SIN-1的用量,从0.1mM提高到1mM,观察到荧光相较于0.1mM有一定的增强,但并不显著,便没有继续增加SIN-1的浓度。该实验结果证明,分子Divac 800能够对细胞外源性的ONOO-进行Turn-ON型检测,当加入SIN-1的浓度为1mM时,荧光增强2.3倍。
进一步地,探究分子Divac 800能否对细胞内源性的ONOO-进行检测。根据文献报道的实验条件进行实验,得到如图31所示的实验结果。
从上述实验可知,在LPS等物质诱导细胞产生内源性ONOO-之后,探针分子的荧光相较于对照组有明显增强,荧光增强约2.2倍。当用一氧化氮合酶抑制剂氨基胍(AG)预处理细胞时,受LPS等物质刺激的细胞中仅能检测到较弱的荧光,表明AG很好地阻碍了细胞生成ONOO-。以上实验结果表明,探针Divac 800能够对细胞内源性的ONOO-进行Turn-ON型检测。
实施例7:化合物Divac 1040的活体成像图
过量摄入对乙酰氨基酚(APAP)能诱导小鼠产生肝损伤,这一损伤过程是剂量依赖的,一般超过300mg/kg的APAP就能造成小鼠肝损伤。这一损伤过程会造成肝脏部位过表达ONOO-等ROS/RNS,这些物质可以激活Divac系列化合物。此处选择波长最长的Divac 1040进行近红外荧光成像的实验。
本实验的实验条件如下:Divac 1040的剂量:1mg/mL,100μL;激发光:808nm;长通滤光片:1200nm;曝光时间:1000ms;激光功率:180mW/cm2。
实验结果如图32所示。
实施例8:化合物Divac 800的光热效果测试
为了研究Divac 800的光热转换效果,对其光热性能进行了测试。在含5%DMSO的PBS(pH=7.4,10mM)中,以1W/cm2功率密度的808nm激光照射,进行Divac 800、Cy800和ICG在近红外光照射下的光热性能测试(染料浓度均为20μM)。对每个时间点的温度进行记录,得到图33所示的曲线图。从图中可知,在激光照射下,三个测试体系温度均逐渐升高。其中,在前50秒,分子Divac 800升温速率最快,温差也最大。280秒之后,三个测试体系的温度几乎保持平稳,不再增加。
对温度数据进行整理,得到表1的数据。从表1中可知,在三个分子中,分子Divac800温度上升的最多(21.3℃),终温也最高(45.6℃),其次是单体(Cy 800,升温18.1℃,终温41.9℃),最后是ICG(升温15.3℃,终温38.5℃)。这说明相较于ICG和Cy 800,分子Divac800有着更好的光热效果。
表1:Divac 800、Cy800和ICG在808nm近红外光照射下的温度变化
染料名称 | ICG | Cy800 | Divac 800 |
温度差/℃ | 15.3 | 18.1 | 21.3 |
终温/℃ | 38.5 | 41.9 | 45.6 |
实施例9:化合物Divac 800的光动力效果测试
测试三种菁染料(Divac 800、Cy 800和ICG)的单线态氧产率,从而了解分子Divac800做光动力治疗的潜力。在甲醇中,Divac 800的波长与ICG的波长相当,因此选择ICG作为单线态氧产率测定的参照。根据文献报道,ICG在甲醇中的单线态氧产率为0.008。实验条件如下:各染料的浓度为1μM;DPBF的浓度为100μM;采用0.33W/cm2功率密度的808nm激光照射。
如图34所示,在808nm激光照射下,三类菁染料均可产生单线态氧(1O2),这部分产生的1O2由1O2捕捉剂1,3-二苯基异苯并呋喃(DPBF)捕获,从而破坏其结构,于是便观察到在410nm处(DPBF的最大吸收处)的吸收值下降。在测试1O2时,需保证DPBF在410nm处的吸光度值为1左右。计算1O2产率时,将410nm处的吸光度与激光照射时间进行线性拟合,通过公式1-1可以对1O2产率进行计算。
公式中Φ表示单线态氧产率,k代表在410nm处,紫外吸收值随时间延长而减小的斜率,F是吸收校正因子,F=1-10-OD其中OD代表的是最大吸收处的吸光度值,f代表保准品,而s代表待测品。
通过上述公式可以计算得到分子Divac 800的单线态氧产率为0.002,而Cy 800的1O2产率为0.012。从计算数据可知,分子Divac 800的1O2产率比Cy800和ICG均要低,结合该分子的摩尔消光系数和荧光量子产率数据推测,分子Divac 800可能将吸收的光子能量主要转化为热能或者分子运动的动能,而较少地进入三线态产生1O2。
Claims (10)
1.下式I或II所示的化合物:
式中:
X各自独立为卤素或-S-C1-4烷基-NHCO-R;
Y各自独立选自以下的阴离子:F-、Cl-、Br-、I-、OAc-、HSO4 -、H2PO4 -,ClO4 -、F3CCOO-、CH3SO3 -、CF3SO3 -、BF4 -、PF6 -、NO3 -和取代或未取代的四苯基硼酸根;
R为C1-6烷基、C2-6烯基或C2-6炔基;
R1、R2、R8、R9、R15、R16、R22和R23各组独立选自氢和C1-4烷基;
R7、R14、R21和R28各自独立选自任选被1-3个选自卤素、C1-4酰基和-SO3H的取代基取代的C1-20烷基、C2-20烯基或C2-20炔基;
R3~R6、R10~R13、R17~R20和R24~R27各自独立为氢、卤素和C1-4烷基;或者,R2与R3、或R3与R4、或R4与R5、或R5与R6与它们所连接的碳一同形成苯环,和/或R9与R10、或R10与R11、或R11与R12、或R12与R13与它们所连接的碳一同形成苯环,和/或R16与R17、或R17与R18、或R18与R19、或R19与R20与它们所连接的碳一同形成苯环,和/或R23与R24、或R24与R25、或R25与R26、或R26与R27与它们所连接的碳一同形成苯环。
2.如权利要求1所述的化合物,其特征在于;
各X均为氯,或各X均为-S-C1-4烷基-NHCO-R;优选地,R为C2-6炔基;和/或
各Y独立为:F-、Cl-、Br-、I-或取代或未取代的四苯基硼酸根;优选地,所述取代的四苯基硼酸根为四(卤代苯基)硼酸根,更优选为四(全氟代苯基)硼酸根。
3.如权利要求1所述的化合物,其特征在于,R1、R2、R8、R9、R15、R16、R22和R23均为C1-4烷基,优选均为甲基或乙基;优选地,R1、R2、R8、R9、R15、R16、R22和R23相同。
4.如权利要求1所述的化合物,其特征在于,R7、R14、R21和R28各自独立选自任选被1个选自-SO3H取代的C1-10烷基;优选地,R7、R14、R21和R28相同。
5.如权利要求1所述的化合物,其特征在于:
R3~R6、R10~R13、R17~R20和R24~R27均为氢;或
R3与R4、R10与R11、R17与R18以及R24与R25分别与它们所连接的碳原子一起形成苯环,R5、R6、R12、R13、R19、R20、R26和R27为氢;或
R2与R3、R9与R10、R16与R17以及R23与R24分别与它们所连接的碳原子一起形成苯环,R4~R6、R11~R13、R18~R20以及R25~R27均为氢;
优选地,被R1~R7取代的环、被R8~R14取代的环、被R15~R21取代的环以及被R22~R28取代的环各自独立选自:
其中,R1’和R2’各自独立选自C1-4烷基;R7’各自独立选自C1-12烷基;优选地,所述4个环相同。
6.如权利要求1所述的化合物,其特征在于,所述的式I化合物选自下组:
所述的式II化合物选自下组:
7.一种近红外荧光染料或染料组合物或试剂盒,其含有权利要求1~6中任一项所述的化合物和任选的溶剂;优选地,所述溶剂选自MeOH、EtOH、PrOH、iPrOH、BuOH、丙酮、DMF、DMSO、吡啶、DCM、氯仿、二氯乙烷、MeCN、苯、甲苯、对二甲苯、氯苯、硝基苯、1,4-二噁烷、THF、乙酸乙酯和AcOH中的一种或多种的混合物。
8.权利要求1~6中任一项所述的化合物在制备用于检测氧化应激的试剂或试剂盒中的用途,或在制备近红外荧光成像、光热治疗、光动力治疗或太阳能光敏电池中使用的试剂中的用途。
9.如权利要求8所述的用途,其特征在于,所述氧化应激的检测包括检测活性氧和/或活性氮;优选地,所述的活性氧和/或活性氮选自下组:超氧化物、过氧化自由基、过氧化氢、羟自由基、过氧亚硝酸根;更优地,所述的活性氧和/或活性氮为过氧亚硝酸根。
10.一种体外检测ONOO-的方法,其特张在于,包括步骤:(1)在如权利要求1-6中任一项所述的化合物存在下,培养健康细胞,检测荧光;(2)在如权利要求1-6中任一项所述的化合物存在下,培养待测细胞,检测荧光;(3)比较步骤(2)和步骤(1)中的荧光强度,如果步骤(2)中的荧光增强,提示所述的待测细胞中含有ONOO-;优选地,所述的细胞为巨噬细胞;优选地,所述的ONOO-为细胞外源性ONOO-和/或细胞内源性ONOO-。
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