CN111748003A - N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物、制备方法与应用及肝靶向探针 - Google Patents
N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物、制备方法与应用及肝靶向探针 Download PDFInfo
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- CN111748003A CN111748003A CN202010588544.XA CN202010588544A CN111748003A CN 111748003 A CN111748003 A CN 111748003A CN 202010588544 A CN202010588544 A CN 202010588544A CN 111748003 A CN111748003 A CN 111748003A
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Abstract
本发明提供了一种N‑乙酰胺基半乳糖修饰的3‑硝基邻苯二甲酰亚胺衍生物、制备方法与应用及肝靶向探针。本发明荧光探针的制备方法简单，所得荧光探针以N‑乙酰胺基半乳糖为肝靶向基团，3‑硝基邻苯二甲酰亚胺为CO响应基团，可作为CO的检测试剂，选择性好、灵敏度高，能够靶向于肝细胞和肝组织，能够用于动态监测CO水平。
Description
技术领域
本发明涉及一种荧光探针，具体地说是涉及一种N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物、其制备方法与应用及肝靶向荧光探针。
背景技术
一氧化碳（CO）是一种无色无味的气体分子，可以和氧气竞争性结合血红蛋白，人类过量吸入会造成窒息死亡，被称为危害人类的“隐形杀手”，一直以来，被认为是一种气态环境污染物。近年来研究发现，CO是植物和动物体内一种内源性气体信号分子，其在维系动植物正常运行过程中扮演者重要的角色。动植物内源性CO均主要由血红素氧合酶（HO）催化分解亚铁血红素（heme）产生。植物内源性CO可以植物生长发育调节，如对逆境（如高盐、重金属、紫外线、活性氧等）响应、气孔运动、根系发育，以及与其他信号分子的相互作用等。动物内源性CO可以激活鸟苷酸环化酶活性、参与呼吸节律调节、缓解氧化应激、调节胰岛素释放和降低血压等。此外，相关研究发现内源性CO浓度异常与多种疾病的发生发展密切相关，包括神经退行性病变、心血管疾病、肺病、糖尿病、肥胖症、败血症和癌症等。因此，开发“可视化”内源CO的检测方法，是CO生物学功能和疾病诊断等相关研究发展迫切需要解决的问题之一。
CO的传统检测方法主要有电化学法、气相色谱法和比色法，这些都不适合活细胞和活体中实时、动态观测CO。相比之下，荧光探针法因其选择性好、灵敏度高、操作简便、实时检测，对生物样品无破坏性损伤以及适用于细胞和活体荧光成像等特点，已被广泛应用于活细胞和活体内多种生物分子的检测。CO的化学性质相对稳定，在生理条件下，能直接参与的化学反应较少。因此，与检测其他内源性活性分子的荧光探针相比，CO荧光探针的研究尚未成熟，仍有较大的发展空间。当前报道的CO荧光探针主要有三类：环钯金属配合物二聚体荧光探针、烯丙基醚/碳酸酯/碳酸酰胺二组分荧光探针和芳香硝基还原型荧光探针。其中，芳香硝基还原型荧光探针因其制备简单、使用方便受到广泛关注，但其仍存在选择性不高、灵敏度低以及对肝细胞和肝组织无靶向递送能力等问题。因此，发展选择性好、灵敏度高，靶向于肝细胞和肝组织以及能够动态监测CO水平的芳香硝基还原型荧光探针具有重要意义。
发明内容
本发明的目的之一是提供一种N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物。
本发明的目的之二是提供前述N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物的制备方法。
本发明的目的之三是提供前述N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物作为探针在检测一氧化碳，或荧光光谱检测，或细胞、活体成像中的应用。
本发明的目的之四是提供一种肝靶向荧光探针。
本发明的目的之五是提供一种一氧化碳的检测方法。
本发明的目的之一是这样实现的：
一种N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物，其化学分子式为C22H28N4O12，化学结构式如式I所示：
式I。
所述N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物的化学名称为：N-(2-(2-(((2R,3R,4R,5R,6R)-3-乙酰胺基-4,5-二羟基-6-(羟甲基)四氢化-2H-吡喃-2-烷基)氧杂)乙氧基)乙基)-2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酰胺；
英文名称为：
N-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-2-(4-nitro-1,3-dioxoisoindolin-2-yl)acetamide。
本发明的目的之二是这样实现的：
前述N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物的制备方法，包括如下步骤：
（a）以3-硝基邻苯二甲酸酐为原料，在冰醋酸回流的条件下，与甘氨酸反应，得到2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸；
（b）以D-(+)-半乳糖胺盐酸盐为原料，依次与乙酸酐发生乙酰化反应、与三氟甲磺酸三甲基硅酯和1,5-二氨基-3-氧杂戊烷发生糖苷化反应、与钯碳和氢气发生还原反应，得到端位氨基修饰的N-乙酰胺基半乳糖；
（c）2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸与端位氨基修饰的N-乙酰胺基半乳糖发生酰胺化反应，在三乙胺碱性条件下脱除乙酸酯基，得到N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物。
步骤（a）中，具体地，将3-硝基邻苯二甲酸酐、甘氨酸加入反应容器中，向其中加入冰醋酸，回流反应过夜，减压蒸除溶剂，甲醇重结晶，抽滤，干燥即得2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸。
步骤（b）中，发生乙酰化反应时，将D-(+)-半乳糖胺盐酸盐溶解于无水吡啶中，分别加无水三乙胺和DMAP，冰水下滴加乙酸酐，滴完后升至室温，搅拌过夜；将白色固体过滤，经甲苯洗、水洗，干燥得白色固体化合物2。
发生糖苷化反应时，将化合物2溶解于无水1,2-二氯乙烷中，滴加三氟甲磺酸三甲基硅酯，搅拌过夜，将NaHCO3溶解于冰水中，剧烈搅拌下将反应液倾入，继续搅拌30 min，依次经过二氯甲烷萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩得化合物3；将化合物3溶解于无水1,2-二氯乙烷中，加入1-羟基-5-叠氮-3-氧杂戊烷，滴加TMSOTf，室温搅拌4 h，将反应液缓慢倾入冷的NaHCO3水溶液，继续搅拌0.5 h，依次经二氯甲烷萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩得化合物4。
发生还原反应时，将化合物4溶解于无水四氢呋喃中，加入Pd/C，通入氢气（压力为20 bar），室温反应24 h；减压蒸除溶剂，得到端位氨基修饰的N-乙酰胺基半乳糖。
步骤（c）中，具体地，2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸与端位氨基修饰的N-乙酰胺基半乳糖发生酰胺化反应，在三乙胺碱性条件下脱除乙酸酯基，得到N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物。
将2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸和HATU溶于DMF中，室温搅拌15min；加入端位氨基修饰的N-乙酰胺基半乳糖和DIPEA，室温反应4 h；经二氯甲烷萃取，饱和食盐水洗，无水Na2SO4干燥，浓缩，得到白色固体化合物；将其溶于甲醇中，加入三乙胺，50℃反应24 h；减压蒸除溶剂，柱层析纯化，得到N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物。
本发明的目的之三是这样实现的：
前述在检测一氧化碳，或荧光光谱检测，或细胞、活体成像中的应用。
优选地，前述N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物作为探针在检测肝细胞或肝组织中的一氧化碳的应用，特别是采用荧光成像原位检测肝细胞或肝组织中的一氧化碳。
本发明的目的之四是这样实现的：
一种肝靶向荧光探针，其化学分子式为C22H28N4O12，其化学结构式如式I所示：
式I。
所示肝靶向荧光探针的化学名称为：N-(2-(2-(((2R,3R,4R,5R,6R)-3-乙酰胺基-4,5-二羟基-6-(羟甲基)四氢化-2H-吡喃-2-烷基)氧杂)乙氧基)乙基)-2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酰胺，
英文名称为：
N-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-2-(4-nitro-1,3-dioxoisoindolin-2-yl)acetamide。
所述肝靶向荧光探针可以实现对肝细胞或肝组织中CO的动态监测。
在CO浓度为10~100 μM时，荧光探针的荧光强度值y（F509nm）与CO浓度x（μM）的线性回归方程为y=125.39+2.90x。
荧光探针与CO反应后，溶液荧光由无色变为绿色，对CO具有高选择性。
荧光探针对CO的检测极限为19.2 nM。
荧光探针在pH为4~10的范围内，均可与CO呈现荧光响应。
荧光探针对于肝HepG2细胞内CO、APAP诱导斑马鱼肝脏内源性CO等的荧光成像检测。
本发明的目的之五是这样实现的：
利用I所示的N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物、肝靶向荧光探针检测CO浓度的方法，其采用荧光光谱法检测样品中CO的浓度。
可选地，采用荧光光谱法检测样品中CO的浓度，包括如下步骤：
（1）配制pH=7.4，浓度为10 mM的PBS缓冲液，备用；配制浓度为5 mM的式I所示肝靶向荧光探针的DMSO溶液，备用；配制浓度为10 mM CORM-2的DMSO溶液，备用。
（2）将2 mL PBS缓冲液、4 μL的式I所示肝靶向荧光探针的DMSO溶液和20 μL的CORM-2的DMSO溶液，依次加入到干净的荧光比色皿中，在荧光光谱仪上检测，在509 nm处有最大发射峰，随着待测样的加入，最大发射峰强度逐渐增强；
（3）将2 mL PBS缓冲液和4 μL的式I所示肝靶向荧光探针的DMSO溶液加入到荧光比色皿中，然后逐渐加入浓度为10 mM的CORM-2的DMSO溶液，当累积加入的CORM-2的DMSO溶液的体积分别为2 μL、4 μL、8 μL、12 μL、16 μL、20 μL时，荧光光谱仪上测定509 nm处对应的发射峰值分别为151.10、187.10、250.25、318.40、354.45、395.90；然后以比色皿中CO的浓度为横坐标，以509 nm处的荧光强度为纵坐标绘制图，得到CO浓度工作曲线；根据所述工作曲线拟合得到荧光强度值y（F509nm）与CO浓度x（μM）的线性回归方程为y=125.39+2.90x；
（4）将2 mL PBS缓冲液和4 μL的式I所示肝靶向荧光探针的DMSO溶液加入到荧光比色皿中，然后逐渐加入V μL的待测样品溶液，在荧光光谱仪上测定509 nm对应的发射峰值，然后将其代入步骤（3）所述的线性回归方程，将所得x值代入待测样品CO浓度计算公式C待测样=(2000+V) μL*x*10-6/V μL，可得待测样品中一氧化碳浓度C待测样的值。
本发明制备得到了一种N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物I，该化合物以3-硝基邻苯二甲酰亚胺为CO响应基团，以N-乙酰胺基半乳糖为肝靶向基团，可作为CO的检测试剂，对CO显示了极高的选择性和极低的检测限，能够靶向于肝细胞和肝组织，能够用于动态监测肝细胞和肝组织内源性CO变化。
附图说明
图1是肝靶向荧光探针与CO反应的荧光动力学测试结果，插图为荧光探针在509nm处的荧光发射峰强度与反应时间的拟合图。
图2是肝靶向荧光探针的荧光选择性测试结果。
图3是肝靶向荧光探针与CO反应溶液的荧光光谱测试结果，插图为荧光探针与CO反应溶液在509 nm处的荧光发射峰强度与10~100 μM浓度CO的线性关系结果。
图4是不同pH缓冲溶液中肝靶向荧光探针与CO反应前后509 nm处荧光强度结果。
图5是肝靶向荧光探针的细胞选择性荧光成像结果。
图6是肝靶向荧光探针用于肝细胞外源性CO的荧光成像结果。
图7是肝靶向荧光探针用于APAP诱导斑马鱼肝脏内源性CO的荧光成像结果。
具体实施方式
下面结合实施例对本发明做进一步的阐述，下述实施例仅作为说明，并不以任何方式限制本发明的保护范围。
在下述实施例中未详细描述的过程和方法是本领域公知的常规方法，实施例中所用试剂均为分析纯或化学纯，且均可市购或通过本领域普通技术人员熟知的方法制备。下述实施例均实现了本发明的目的。
实施例1
采用如下方式合成目标化合物，合成路线如下：
化合物2：
将2.15 g（10.0 mmol）化合物1盐即D-(+)-半乳糖胺盐酸盐溶解于20 mL无水吡啶中，分别加入1 mL无水三乙胺和122 mg（1.00 mmol）DMAP，冰水下滴加5.67 mL（60.0 mmol）乙酸酐，滴完后升至室温，搅拌过夜。将白色固体过滤，依次经过10 mL甲苯洗，50 mL水洗，干燥得3.35 g白色固体化合物2（产率86%）。
1 δ (ppm) 7.90 (d, J = 9.2 Hz, 1H), 5.63 (d, J = 8.8 Hz, 1H), 5.26(s, 1H), 5.06 (d, J = 9.6 Hz, 1H), 4.22 (s, 1H), 4.03-3.99 (m, 3H), 2.11 (s,3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.90 (s, 3H), 1.78 (s, 3H). 13C NMR (100 MHz,DMSO): δ (ppm) 170.4, 170.3, 170.1, 170.0, 169.4, 71.2, 70.4, 66.8, 61.8,48.5, 23.1, 20.9, 20.8.
化合物3：
将1.95 g（5.00 mmol）化合物2溶解于20 mL无水1,2-二氯乙烷中，滴加1.08 mL（6.00mmol）三氟甲磺酸三甲基硅酯（TMSOTf），滴完后，搅拌过夜。将840 mg（10.0 mmol）NaHCO3溶解于50 mL冰水中，剧烈搅拌下将反应液倾入，继续搅拌30 min。依次经过二氯甲烷萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩得化合物3淡黄色粘稠液体1.65 g，定量产率。未分离直接进行下一步。
1 3 δ (ppm) 5.90 (d, J = 6.8 Hz, 1H), 5.36 (s, 1H), 4.80 (s, 1H), 4.15-4.02 (m, 3H), 3.90 (s, 1H), 2.11 (s, 3H), 2.03 (s, 3H), 1.97 (s, 9H). 13C NMR(100 MHz, CDCl3): δ (ppm) 170.2, 170.0, 169.6, 166.1, 101.2, 71.5, 69.2,65.0, 63.3, 61.3, 20.5, 20.4, 20.3, 14.2.
化合物4：
将1.65 g（5.00 mmol）化合物3溶解于20 mL无水1,2-二氯乙烷中，加入786 mg（6.00mmol）2-(2-叠氮乙氧基)乙醇，滴加181 μL（1.00 mmol）TMSOTf。室温搅拌4 h。将反应液缓慢倾入冷的NaHCO3水溶液，继续搅拌0.5 h。依次经过二氯甲烷萃取，饱和食盐水洗，无水硫酸钠干燥，浓缩得化合物4淡黄色粘稠液体1.84 g，两步产率80%。
1 3 δ (ppm) 5.82 (d, J = 8.8 Hz, 1H), 5.33 (d, J = 2.8 Hz, 1H), 5.18(dd, J = 3.2 Hz, J = 11.2 Hz, 1H), 4.74 (d, J = 8.4 Hz, 1H), 4.17-4.02 (m,3H), 3.96-3.88 (m, 2H), 3.79-3.74 (m, 1H), 3.64-3.60 (m, 4H), 3.45-3.35 (m,2H), 2.13 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.95 (s, 3H).
化合物5：
将460 mg化合物4（1.00 mmol）溶解于10 mL无水四氢呋喃中，加入40 mg Pd/C，通入氢气（压力为20 bar），室温反应24 h。然后减压蒸除溶剂，得到黄色固体5即端位氨基修饰的N-乙酰胺基半乳糖，无需纯化，可直接用于后续反应。
化合物7：
将1.93 g 3-硝基邻苯二甲酸酐（10 mmol）和0.826 g甘氨酸（11 mmol）加入50 mL圆底烧瓶中，加入20 mL冰醋酸，回流反应过夜。然后减压蒸除溶剂，甲醇重结晶，抽滤，干燥，得2.28 g白色固体7即2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸（91%）。
1 d 6：δ (ppm): 13.38 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 7.6Hz, 1H), 8.11 (t, J = 8.0 Hz, 1H), 4.35 (s, 2H).
式I目标化合物HEP-CO:
将250 mg化合物7（1 mmol）和HATU（570 mg, 1.5 mmol）溶于8 mL DMF中，室温搅拌15min。依次加入434 mg化合物5（1 mmol）和523 μL DIPEA（3 mmol），室温反应4 h。依次经二氯甲烷萃取，饱和食盐水洗，无水Na2SO4干燥，浓缩，得到白色固体化合物。将其溶于5 mL甲醇中，加入33 μL三乙胺（1 mmol），50 ℃反应24 h。减压蒸除溶剂，柱层析（v(二氯甲烷/甲醇) = 5/1）纯化，得55.1 mg白色固体荧光探针HEP-CO即N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物（20%）。
1 d 6 δ (ppm): 8.31 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 7.2 Hz, 1H), 8.09(t, J = 7.6 Hz, 1H), 4.27 (d, J = 8.8 Hz, 1H), 4.22 (s, 2H), 3.81 - 3.76 (m,1H), 3.73 - 3.66 (m, 1H), 3.63 (d, J = 3.2 Hz, 1H), 3.50 - 3.46 (m, 4H), 3.43- 3.38 (m, 3H), 3.31 (t, J = 6.0 Hz, 1H), 3.25 - 3.18 (m, 2H), 1.80 (s, 3H);13C NMR (101 MHz, DMSO-d 6):δ (ppm): 170.4, 170.3, 166.3, 166.3, 166.1, 163.5,144.8, 137.0, 134.0 129.0, 127.6, 123.5, 101.8, 75.7, 71.7, 69.9, 69.3, 68.0,67.9, 60.9, 52.4, 46.2, 41.0, 23.5, 23.4. HRMS (C22H29N4O12): Cald for541.1776, found 541.1768.
实施例2
将实施例1所得目标化合物进行光谱性能测试。
1、探针母液的配制
准确称量5.4048 mg探针固体，用2 mL无水DMSO溶解，配制成浓度为5 mM的探针母液；以CORM-2 (CAS: 22549-69-0)作为CO释放分子，准确称量5.1201 mg CORM-2固体，用无水DMSO溶解，配制成浓度为5 mM的CO母液；测试溶液为磷酸盐缓冲体系(10 mM, pH = 7.4)。
2、探针与CO反应动力学测试
向2 mL PBS中，依次加入探针分子HEP-CO (10 μM)和CO (100 μM)，37度下检测反应时间0-40 min内探针溶液的荧光发射光谱，其中，激发波长为420 nm，发射波长收集范围为440-700 nm，激发和发射狭缝宽度均为10 nm。所得结果如图1所示。从图中可以看出，加入CO后，探针反应迅速。
3、探针对CO选择性光谱检测
选取阴阳离子（Fe3+、Mg2+、Cu2+、NO3 -、NO2 -、SO3 2-）活性硫物种（H2S、Cys、Hcy、GSH），活性氮物种（NO），活性氧物种（ONOO-、H2O2、NaClO、KO2、t-BuOOH），其它。
选择性测试条件：向2 mL PBS中，依次加入探针分子HEP-CO (10 μM)、不同干扰分子或CO (100 μM)。其中，Cys、Hcy浓度为1 mM，GSH浓度为5 mM，其余干扰分子浓度均为100μM，37度下反应60 min。所得结果如图2所示。从图中可以看出，探针对CO具有高选择性。
4、探针对CO的检测范围与检测极限测试
向2 mL PBS中，依次加入探针HEP-CO (10 μM)和不同浓度的CO (10, 20, 40, 60,80, 100 μM)，37度下反应30 min。分别检测上述探针溶液的荧光发射光谱，所得结果如图3所示，在CO浓度为10~100 μM时，荧光探针的荧光强度值y（F509nm）与CO浓度x（μM）的线性回归方程为y=125.39+2.90x。
向2 mL PBS中，依次加入探针HEP-CO (10 μM)和不同浓度的CO (0, 2, 4, 6, 8,10 μM)，37度下反应30 min，分别检测上述探针溶液的荧光发射光谱。单独测试12次仅加入探针HEP-CO (10 μM)的荧光发射光谱，计算509 nm处的荧光强度值的标准偏差（σ）；对低浓度CO（0-10 μM）与探针反应的荧光发射光谱中509 nm处的荧光强度值与CO浓度进行线性拟合，得到直线斜率k。通过公式计算检测极限LOD = 3*σ/k。探针对CO的检测极限为19.2 nM。
5、探针对CO在不同pH下的光谱检测
配制不同pH（4.0、5.0、6.0、7.0、8.0、9.0、10.0）的PBS (10 mM)。向不同pH的PBS中，依次加入探针HEP-CO (10 μM)和CO (100 μM)，37度下作用30 min。然后分别检测不同pH下的探针与CO反应前后溶液的荧光发射光谱，所得结果如图4所示。在pH 4-10范围内，探针与CO反应后，均呈现较大的荧光响应，具有较宽的pH适用范围。
实施例3
细胞选择性实验
选用人肝癌HepG2、人宫颈癌Hela、人非小细胞肺癌A549和人胃粘膜上皮细胞癌GES-1四种细胞作为研究对象。将四种细胞分别培养至贴壁，第一组实验中，分别向各种细胞中加入探针HEP-CO (10 μM, 2 mL)，与细胞共孵育1 h；第二组实验中，分别向各种细胞中加入探针HEP-CO (10 μM, 2 mL)，与细胞共孵育1 h后，用生理PBS清洗三次，以除去过量的探针，再加入CO (100 μM, 2 mL)，继续孵育30 min。利用激光共聚焦显微镜，对上述细胞进行荧光成像。选择405 nm激光器，绿色荧光通道收集475 - 575 nm范围内的信号。所得结果如图5所示，则探针可以选择性靶向肝HepG2细胞，用于HepG2细胞内CO的荧光成像检测。
实施例4
细胞外源性CO的荧光成像
选用人肝癌HepG2细胞作为研究对象。将四种细胞分别培养至贴壁，对照组，仅对细胞成像；实验组，分别向细胞中加入探针HEP-CO (10 μM, 2 mL)，与细胞共孵育1 h后，用生理PBS清洗三次，以除去过量的探针，再分别加入不同浓度CO (50 μM、200 μM)的细胞培养液(2 mL)，继续孵育30 min。利用激光共聚焦显微镜，对上述细胞进行荧光成像。选择405 nm激光器，绿色荧光通道收集475 – 575 nm范围内的信号。所得结果如图6所示，则探针可用于HepG2细胞内外源性CO的荧光成像检测。
实施例5
对乙酰氨基酚(APAP)诱导斑马鱼肝脏内源性CO的荧光成像：
斑马鱼受精卵在E3培养液中，28℃下控光（光照14 h/黑暗10 h）培育至5-7日龄，进行荧光成像实验。第一组实验中，向培养液中加入探针HEP-CO (10 μM)，培育1 h；第二组实验中，向培养液中加入APAP (2 mM)，用培养液清洗斑马鱼三次以除去过量APAP，再加入探针HEP-CO (10 μM)，培育1 h，用E3培养液清洗斑马鱼三次除去过量探针。利用激光共聚焦显微镜，对上述细胞进行荧光成像。选择405 nm激光器，绿色荧光通道收集475 -575 nm范围内的信号。所得结果如图7所示，则探针可以用于APAP诱导斑马鱼肝脏内源性CO的荧光成像检测。
Claims (9)
1.一种N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物，其特征在于，化学分子式为C22H28N4O12，化学结构式如式I所示：
式I。
2.权利要求1所述的N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物的制备方法，其特征在于，包括如下步骤：
（a）以3-硝基邻苯二甲酸酐为原料，在冰醋酸回流的条件下，与甘氨酸反应，得到2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸；
（b）以D-(+)-半乳糖胺盐酸盐为原料，依次与乙酸酐发生乙酰化反应、与三氟甲磺酸三甲基硅酯和1,5-二氨基-3-氧杂戊烷发生糖苷化反应、与钯碳和氢气发生还原反应，得到端位氨基修饰的N-乙酰胺基半乳糖；
（c）2-(4-硝基-1,3-二氧代异吲哚啉-2-基)乙酸与端位氨基修饰的N-乙酰胺基半乳糖发生酰胺化反应，在三乙胺碱性条件下脱除乙酸酯基，得到N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物。
3.权利要求1所述的N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物在非诊断检测一氧化碳，或荧光光谱检测，或细胞、组织、活体成像中的应用。
4.根据权利要求3所述的N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物，其特征在于，N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物作为探针在检测肝细胞或肝组织中的一氧化碳的应用。
5.一种肝靶向荧光探针，其特征在于，化学分子式为C22H28N4O12，化学结构式如式I所示：
式I。
6.权利要求5所述的肝靶向荧光探针在检测肝细胞或肝组织中的一氧化碳的应用。
7.根据权利要求6所述的肝靶向荧光探针在检测肝细胞或肝组织中的一氧化碳的应用，其特征在于，所述肝靶向荧光探针对CO的检测极限为19.2 nM。
8.根据权利要求6所述的肝靶向荧光探针在检测肝细胞或肝组织中的一氧化碳的应用，其特征在于，所述肝靶向荧光探针在pH为4~10的范围内与CO呈现荧光响应。
9.一种检测一氧化碳浓度的方法，其特征在于，采用权利要求1所述的N-乙酰胺基半乳糖修饰的3-硝基邻苯二甲酰亚胺衍生物作为探针，采用荧光光谱法检测样品中CO的浓度。
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