CN113234111A - 同时检测β-半乳糖苷酶和溶酶体pH的荧光探针及其制备方法和应用 - Google Patents
同时检测β-半乳糖苷酶和溶酶体pH的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了结构通式(I)所示化合物及其制备方法,该化合物可同时检测β‑半乳糖苷酶和溶酶体pH,是一种单分子比率型“二维”荧光探针,单分子比率型荧光探针在使用时可避免单功能的两个探针同时使用时产生的串扰,并通过两个发射带的自校准,实现更加准确的分析;通过对细胞内衰老相关的β‑半乳糖苷酶活性和衰老相关的溶酶体pH进行同时检测,实现了细胞衰老的精准检测。
Description
技术领域
本发明涉及一类同时检测β-半乳糖苷酶和溶酶体pH的荧光探针及其制备方法和应用,属于化学生物学传感器技术领域。
背景技术
细胞衰老是由DNA损伤、端粒缩短和功能障碍、致癌效应和其他类型的细胞应激引起的永久性细胞周期停滞。虽然该过程的主要作用是防止细胞异常增殖,但衰老细胞的逐渐积累会损害组织功能并加速器官老化,同时诱发多种衰老相关疾病。通过衰老生物标记物检测细胞衰老并进一步精确干预是缓解衰老和治疗衰老相关疾病的一种有前途的方法。目前被广泛使用的细胞衰老生物标志物包括衰老相关β-半乳糖苷酶、γ-H2AX、p21、p16、IL-6、MacroH2A、PPP1A、Smurf2、磷酸化的p38MAPK和PGM。其中,衰老相关β-半乳糖苷酶是一种内源性溶酶体酶,被认为是细胞衰老检测的金标准。然而,随着研究发现卵巢癌细胞和某些特定的细胞类型如破骨细胞和组织巨噬细胞中也会高表达内源性的β-半乳糖苷酶。这些非衰老相关的内源性β-半乳糖苷酶无论是在结构还是功能上都与衰老相关的内源性β-半乳糖苷酶相同,均由GLB1基因在溶酶体中编码、表达。因此,这些非衰老相关的内源性β-半乳糖苷酶对衰老相关β-半乳糖苷酶的检测产生的干扰问题已经成为衰老识别领域的瓶颈问题。
发明内容
本发明的目的之一是提供一种兼具检测β-半乳糖苷酶和溶酶体pH的荧光探针及其制备方法。
本发明的另一个目的是利用上述兼具溶酶体pH检测功能的β-半乳糖苷酶荧光探针,对细胞内衰老相关的β-半乳糖苷酶活性和衰老相关的溶酶体pH同时进行检测,以实现细胞衰老的精准检测。
本发明的实现过程如下:
结构通式(I)所示化合物,
结构通式(I)所示化合物的制备方法:化合物(A)与化合物(B)发生缩合反应得到化合物(I),
上述缩合反应中,使用醇类溶剂,反应温度为70-80 °C,所述醇类溶剂为甲醇、乙醇、丙醇。
结构通式(I)所示化合物在制备同时检测β-半乳糖苷酶和pH的荧光探针中的应用。
结构通式(I)所示化合物在制备同时检测细胞内内源性β-半乳糖苷酶和溶酶体pH的荧光探针中的应用。
结构通式(I)所示化合物在制备同时检测细胞内内源性衰老相关β-半乳糖苷酶和溶酶体pH的荧光探针中的应用。
结构通式(I)所示化合物在制备同时检测细胞内外源性β-半乳糖苷酶和细胞质pH的荧光探针中的应用。
结构通式(I)所示化合物在制备细胞成像和组织切片荧光成像试剂中的应用。
本发明首先以半花菁为荧光骨架,设计、合成了溶酶体pH敏感的荧光染料。该荧光染料通过在吲哚N上引入羧基来调控pKa值,使其pKa值与溶酶体的pH窗口匹配,从而实现对溶酶体pH的比率检测。然后,通过在上述溶酶体pH敏感的荧光染料的结构中引入β-D-半乳糖作为β-半乳糖苷酶受体和猝灭基团,合成了溶酶体pH敏感的衰老相关β-半乳糖苷酶“二维”荧光探针(I)。这些荧光探针自身无荧光,与β-半乳糖苷酶结合后其结构中的β-D-半乳糖苷键被切断,释放出溶酶体pH敏感的荧光染料。随后,这些溶酶体pH敏感的染料在较酸的溶酶体环境中发出绿色荧光,这是由于其主要分子内电荷转移效应较弱的酚羟基形式(“酸性形式”)存在。而在较碱的溶酶体环境中,溶酶体pH敏感的染料发出红色荧光,它们主要以分子内电荷转移效应较强的酚氧负离子形式(“碱性形式”)存在。本发明结合溶酶体内部pH随衰老逐渐升高的科学事实,设计、合成了兼具溶酶体pH识别功能的衰老相关β-半乳糖苷酶“二维”荧光探针,以实现细胞衰老的精准检测。
本发明的优点:
(1)首次开发了可同时检测β-半乳糖苷酶和溶酶体pH的单分子比率型“二维”荧光探针,单分子比率型荧光探针在使用时可避免单功能的两个探针同时使用时产生的串扰,并通过两个发射带的自校准,实现更加准确的分析;
(2)首次通过对细胞内衰老相关的β-半乳糖苷酶活性和衰老相关的溶酶体pH进行同时检测,实现了细胞衰老的精准检测。
附图说明
图1为对溶酶体pH敏感的荧光染料KSAP1和KSAP2在pH值不同的缓冲溶液中的荧光光谱图;
图2为随米曲霉源β-半乳糖苷酶(A. oryzae β-gal)不断加入,荧光探针KSA01和KSA02在pH = 4.0的缓冲溶液中的紫外-可见吸收光谱和荧光光谱图;
图3为随大肠杆菌源β-半乳糖苷酶(E. coli β-gal)不断加入,荧光探针KSA01和KSA02在pH = 7.4的缓冲溶液中的紫外-可见吸收光谱和荧光光谱图;
图4为荧光探针KSA01和KSA02溶液中分别加入A. oryzae β-gal或者E. coli β-gal后荧光强度的时间动力学图;
图5为荧光探针KSA01和KSA02的选择性光谱图;图中的英文与中文对照如下:
Blank:空白;Trypsin:胰蛋白酶;Xanthine Oxidase:黄嘌呤氧化酶;Lysozyme:溶菌酶;Chymotrypsin:糜蛋白酶;Pepsin:胃蛋白酶;Hcy:高半胱氨酸;Cys:半胱氨酸;GSH:谷胱甘肽;H2O2:过氧化氢;E. coli β-gal:大肠杆菌源β-半乳糖苷酶;A. oryzae β-gal:米曲霉源β-半乳糖苷酶;
图6为荧光探针KSA01和KSA02分别在SKOV-3细胞,HepG2-Lacz (+)细胞和HepG2-Lacz (-)细胞中的荧光成像图;
图7为荧光探针KSA01在药物(XL413)诱导衰老的癌细胞中的荧光成像图;
图8为荧光探针KSA02在药物(XL413)诱导衰老的癌细胞中的荧光成像图。
具体实施方式
下面实施例中所使用的实验方法如无特殊说明,均为常规方法,所使用的材料、试剂等如无特殊说明,均可从商业途径得到。
实施例1 荧光探针KSA01的制备
化合物1的合成步骤:
准确称取化合物4−溴甲基苯甲酸1.18 g (5.5 mmol) 和2, 3, 3-三甲基吲哚803μL (5 mmol) 于50 mL的圆底烧瓶中,在瓶内加入20 mL乙腈,氩气保护下加热至80 °C回流24 h。24 h后停止反应,等反应液冷却到室温后,抽滤,然后用冷的丙酮溶液多次少量洗涤,得到的化合物1为白色固体 (1.24 g),产率为65%,熔点为239-241 °C。1H NMR (400 MHz,CD3OD) δ (ppm): 7.96 (d, J = 8 Hz, 2H), 7.88 (d, J = 7.2 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.63-7.52 (m, 4H), 5.92 (s, 2H), 2.97 (s, 3H), 1.61 (s, 6H).HRMS (ESI) exact mass calcd for C19H20NO2 ([M]+): 294.1489; found m/z:294.1485。
化合物2的合成步骤:
准确称量对羟基苯甲醛733 mg (6 mmol),无水硫酸钠2.13 g (15 mmol),四乙酰基-α-D-溴代半乳糖5.18 g (12.6 mmol) 和碳酸铯4.89 g (15 mmol) 于100 mL圆底烧瓶中,向瓶内加入40 mL乙腈,加热至80°C回流12 h, TLC监测。等反应完成后,将烧瓶内反应液冷却到室温,减压蒸馏将乙腈旋干,得褐色粗产品。粗品通过柱层析 (洗脱剂为: PE: EA= 1: 1, v: v) 进一步纯化,得到化合物2为白色固体 (2.28 g),产率84%,熔点为102-103°C。1H NMR (400 MHz, DMSO-d 6 ): δ (ppm): 9.92 (s, 1H), 7.92 (d, J = 8.6 Hz,2H), 7.18 (d, J = 8.6 Hz, 2H), 5.68 (d, J = 7.3 Hz, 1H), 5.38 (d, J = 2.4 Hz,1H), 5.30-5.24 (m, 2H), 4.50 (t, J = 6.1 Hz, 1H), 4.11 (d, J = 6.2 Hz, 2H),2.15 (s, 3H), 2.04 (d, J = 9.0 Hz, 6H), 1.96 (s, 3H); 13C NMR (100 MHz, DMSO-d6): 191.95, 170.41, 170.30, 169.99, 169.67, 161.32, 132.20, 131.81, 116.95,97.37, 71.11, 70.56, 68.68, 67.67, 61.77, 20.93, 20.86, 20.83, 20.78; HRMS(ESI) exact mass calcd for C21H24O11Na ( [M + Na]+): 475.1211; found m/z:475.1214. FT-IR (KBr, cm-1): 1739.72, 1593.13, 1382.90, 1220.89, 1070.45,935.44, 835.14。
化合物3的合成步骤:
准确称取2.28 g (1.82 mmol) 的化合物2于50 mL的梨形瓶中,氩气保护下加入25 mL的无水甲醇,在冰浴条件下向反应体系中加入甲醇钠的甲醇溶液4.8 mL,室温下搅拌2 h,TLC监测至反应结束。用阳离子交换树脂IR120 (钠型) 中和反应液,使溶液pH为6-7左右。抽滤,减压蒸馏除去甲醇,得粗产品。粗品经柱层析进一步纯化(洗脱剂: CH2Cl2: CH3OH= 10: 1, v/v),得化合物3为白色固体(1.12 g),产率78%,熔点为177-179 °C。1H NMR(400 MHz, DMSO-d 6 ) δ (ppm): 9.89 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.20 (d,J = 8.5 Hz, 2H), 5.23 (d, J = 5.0 Hz, 1H), 5.00 (d, J = 7.6 Hz, 1H), 4.90 (d,J = 5.3 Hz, 1H), 4.66 (t, J = 5.2 Hz, 1H), 4.54 (d, J = 4.4 Hz, 1H), 3.66-3.48 (m, 6H); 13C NMR (100 MHz, DMSO-d6): 191.92, 162.70, 132.07, 130.91,116.90, 100.82, 76.15, 73.69, 70.62, 68.56, 60.79; HRMS (ESI) exact masscalcd for C13H16O7Na ([M + Na]+): 307.0788; found m/z: 307.0802. FT-IR (KBr,cm-1): 3382.99, 1681.86, 1602.78, 1259.46, 1099.38, 842.86, 696.27, 628.77。
化合物KSA01的合成步骤:
准确称取化合物1 373 mg (1 mmol) 和化合物3 284 mg (1 mmol) 于50 mL的圆底烧瓶中,在烧瓶内加入20 mL的无水乙醇,氩气保护下加热回流12 h,TLC监测。待反应完成后,将反应体系冷却至室温,抽滤,用无水乙醇和无水***多次少量洗涤,得到化合物KSA01为黄色固体(294 mg),产率为46%,熔点为188-190 °C。1H NMR (400 MHz, DMSO-d6):8.59 (d, J = 16.0 Hz, 1H), 8.23 (d, J = 8.2 Hz, 2H), 7.96-7.91 (m, 3H), 7.5(d, J = 16.8 Hz, 2H), 7.60-7.52 (m, 2H), 7.47 (d, J = 7.7 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 6.11 (s, 2H), 5.26 (s, 1H), 5.04 (d, J = 7.3 Hz, 1H), 4.94 (s,1H ), 4.63 (d, J = 38.0 Hz, 2H), 3.67-3.43 (m, 6H), 1.88 (s, 6H); 13C NMR(100 MHz, DMSO-d6): 183.69, 167.21, 162.72, 155.93, 144.05, 141.22, 139.01,133.89, 131.20, 130.62, 129.70, 129.60, 128.61, 127.44, 123.68, 117.31,115.68, 110.83, 100.65, 76.20, 73.71, 70.59, 68.54, 60.77, 52.81, 49.53,26.52; HRMS (ESI) exact mass calcd for C32H34NO8 ([M]+): 560.2279; found m/z:560.2298. FT-IR (KBr, cm-1): 3390.71, 1706.93, 1575.77, 1523.70, 1247.89,1168.81, 1056.95, 759.92.)。
准确称取化合物1 820 mg (2.19 mmol) 对羟基苯甲醛320 mg (2.63 mmol) 于50 mL的圆底烧瓶中,在烧瓶内加入20 mL的无水乙醇,氩气保护下加热回流8 h,TLC监测。待反应完成后,将反应体系冷却至室温,旋干溶剂后获得粗产品,粗品经柱层析进一步纯化(洗脱剂: CH2Cl2: CH3OH = 10: 1, v/v),用无水乙醇和无水***多次少量洗涤,得到化合物KSAP1为红色固体(732 mg),产率为70%,熔点为252-253 °C。. 1H NMR (400 MHz, DMSO-d6): δ (ppm): 8.46 (d, J = 25.6 Hz, 1H), 8.08 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 8.1 Hz, 2H), 7.85 (d, J = 4.3 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.52-7.44(m, 5H), 6.87 (d, J = 8.5 Hz, 2H), 6.00 (s, 2H), 1.85 (s, 6H); HRMS (ESI)exact mass calcd for C26H24NO3 ([M]+): 398.1751; found m/z: 398.1765. FT-IR(KBr, cm-1): 3417.71, 2974.11, 2366.56, 1714.63, 1571.92, 1523.70, 1271.03,1163.03, 752.20。
由1和2制备,具体合成步骤同KSA01。
实施例2 荧光探针KSA02的制备
化合物4的合成步骤:
准确称取化合物8−溴辛酸 446 mg (2 mmol) 和2, 3, 3-三甲基吲哚321 μL (2mmol) 于甲苯 25 mL的圆底烧瓶中,氩气保护下加热至100 °C回流20 h。20 h后停止反应,等反应液冷却到室温后,抽滤,然后用冷的***溶液多次少量洗涤,得到的化合物4紫粉色固体 (309 mg),产率为40%,熔点为140-142 °C。1H NMR (600 MHz, DMSO-d6): δ (ppm):11.87 (s, 1H), 8.05 (d, J = 4.8 Hz, 1H), 7.88 (d, J = 4.1 Hz, 1H), 7.54 (t, J = 3.3 Hz, 2H), 4.51 (d, J = 6.5 Hz, 2H), 2.94 (s, 3H), 2.12 (t, J = 7.1 Hz,2H), 1.82 (s, 2H), 1.53 (s, 6H), 1.40 (d, J = 6.5 Hz, 4H) 1.25-1.16 (m, 6H).HRMS (ESI) exact mass calcd for C25H29N2O ([M]+): 302.2115; found m/z:302.2099。
化合物KSA02的合成步骤:
准确称取134 mg (0.35 mmol)化合物4 和100 mg (0.35 mmol) 化合物3 于25mL的圆底烧瓶中,在烧瓶内加入5 mL的无水乙醇,氩气保护下加热回流12 h,TLC监测。待反应完成后,将反应体系冷却至室温,抽滤,用无水乙醇和无水***多次少量洗涤,得到化合物KSA02为黄色固体(102 mg),产率为45%,熔点为140-142 °C。1H NMR (600 MHz, DMSO-d6): δ(ppm): 11.99 (s, 1H), 8.47 (d, J = 16.1 Hz, 1H), 8.26 (s, 2H), 7.92 (d,J = 7.0 Hz, 1H), 7.89 (d, J = 6.6 Hz, 1H), 7.61(s, 3H), 7.22 (d, J = 8.6 Hz,2H), 5.28 (s, 1H), 5.05 (d, J = 7.6 Hz, 1H), 4.99-4.97 (m, 1H), 4.79-4.64 m,4H), 3.75 (s, 1H), 3.68 (t, J = 6.2 Hz, 1H), 3.65-3.61 (m, 1H), 3.57-3.55 (m,1H), 3.47 (s, 2H), 2.17 (t, J = 7.3 Hz, 2H), 1.80 (s, 6H), 1.48-1.40 (m, 4H),1.32-1.24 (m, 6H). 13C NMR (150 MHz, DMSO-d6): 182.15, 174.92, 162.68,154.31, 144.12, 140.94, 133.71, 129.48, 128.54, 123.54, 117.05, 115.49,111.08, 100.71, 76.17, 73.82, 70.98, 68.45, 60.73, 52.56, 46.82, 34.03,31.49, 29.00,26.39, 26.17, 24.84, 22.51, 14.42. HRMS (ESI) exact mass calcdfor C32H42NO8 ([M]+): 568.2905; found m/z: 568.2905. FT-IR (KBr, cm-1): 3427.71,2935.11, 1740.87, 1586.73, 1530.25, 1474.65, 1068.54, 836.16。
准确称取308 mg化合物4 (0.81 mmol)和118 mg对羟基苯甲醛(0.97 mmol) 于25mL的圆底烧瓶中,在烧瓶内加入6 mL的无水乙醇,氩气保护下加热回流12 h,TLC监测。待反应完成后,将反应体系冷却至室温,溶剂旋干得粗产品,粗品经柱层析进一步纯化(洗脱剂:CH2Cl2: CH3OH = 10: 1, v/v),得到化合物KSAP2为红色固体(205 mg),产率为52%,熔点高于300 °C。. 1H NMR (600 MHz, DMSO-d6): δ (ppm): 11.90 (s, 1H), 8.37 (d, J =15.9 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 7.83 (t, J = 7.7 Hz, 2H), 7.58 (t, J = 7.8 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.40 (d, J = 16.0 Hz, 1H), 6.93 (d,J = 7.9 Hz, 2H), 4.59 (t, J = 7.3 Hz, 2H), 2.16 (t, J = 6.3 Hz, 2H), 1.77 (s,6H), 1.49-1.45 (m, 2H), 1.43-1.39 (m, 2H), 1.32-1.21 (m, 6H); 13C NMR (125MHz, DMSO-d6): δ (ppm): 181.10, 174.85, 165.09, 154.64, 143.58, 141.16,134.40, 129.33, 128.86, 125.93, 123.30, 117.27, 114.85, 108.29, 99.81, 51.94,33.91, 28.70, 28.66, 28.36, 26.47, 26.02, 24.69; HRMS (ESI) exact mass calcdfor C26H32NO3 ([M]+): 406.2377; found m/z: 406.2378. FT-IR (KBr, cm-1): 3391.64,2928.34, 1719.06, 1569.40, 1519.77, 1466.37, 1164.05, 834.66。
由4和2制备,具体合成步骤同KSA02。
实施例3
实施例1和2制备得到的KSAP1和KSAP2在不同pH下的荧光性质实验。
取荧光染料KSAP1和KSAP2溶于二甲基亚砜(DMSO)中,配置成1 mmol/L储备液。从储备液中取50 μL加入到10 mL比色管中,分别用不同pH值(3.0~10.0)的PBS缓冲溶液(100mmol)稀释定容至5 mL,测量荧光团在pH值不同的缓冲溶液中的荧光性质。如图1所示,当pH从3.0增加到10.0时,两个荧光团的短波长(KSAP1和KSAP2分别为536 nm和534 nm) 的荧光强度逐渐降低,而长波长(KSAP1和KSAP2分别为569 nm和562 nm)的荧光强度逐渐增大,实验结果表明荧光染料KSAP1和KSAP2能实现pH的比率型检测。
实施例4
实施例1和2制备得到的KSA01和KSA02的米曲霉源β-半乳糖苷酶(A. oryzae β-gal)滴定光谱实验。
取荧光探针KSA01和KSA02溶于二甲基亚砜(DMSO)中,配置成1 mmol/L储备液。从储备液中取50 μL加入到10 mL比色管中,用pH = 4的PBS缓冲溶液(100 mmol/L, A. oryzae β-gal测试使用pH = 4的PBS缓冲液)稀释,再加入不同单位浓度的A. oryzae β-gal标准溶液(终浓度为0−1 U/mL),最后用pH = 4的PBS缓冲溶液定容至5 mL。上述样品在37 ℃孵育30 min后测量其溶液的吸光度和荧光强度。如图2a和2b所示,探针KSA01和KSA02溶液的吸收光谱的最大吸收波长分别为405 nm和385 nm,随着A. oryzae β-gal不断加入,其最大吸收波长分别移动到435 nm和423 nm处。如图2c和2d所示,探针KSA01和KSA02溶液几乎无荧光,随着A. oryzae β-gal的不断加入,其分别在536 nm和534 nm处发射绿色荧光且荧光强度逐渐增强。在一定A. oryzae β-gal浓度范围内,探针最大发射波长的荧光强度与酶浓度呈现良好的线性关系,且根据检测限公式LOD = 3σ/k(σ是11次空白对照组的标准偏差,k是线性方程的斜率)可以计算出探针KSA01和KSA02对A. oryzae β-gal浓度的最低检出范围分别为8.3×10-3 U/mL 和 6.8×10-3 U/mL。以上结果表明探针KSA01和KSA02在pH = 4.0的缓冲溶液中对A. oryzae β-gal响应良好。
实施例5
实施例1和2制备得到的KSA01和KSA02的大肠杆菌源β-半乳糖苷酶(E. coli β-gal)滴定光谱实验。
取荧光探针KSA01和KSA02溶于二甲基亚砜(DMSO)中,配置成1 mmol/L储备液。从储备液中取50 μL加入到10 mL比色管中,用pH = 7.4的PBS缓冲溶液(100 mmol/L, E. coli β-gal测试使用pH = 7.4的PBS缓冲液)稀释,再加入不同单位浓度的E. coli β-gal标准溶液(终浓度为0−1.1 U/mL不等),最后用pH = 7.4的PBS缓冲溶液定容至5 mL。上述样品在37 ℃孵育30 min后测量其溶液的荧光强度。如图3a和3b所示,随着E. coli β-gal的不断加入,KSA01的最大吸收峰从425 nm移动到530 nm,KSA02的最大吸收峰从409 nm移动到523 nm。如图3c和3d所示,探针KSA01和KSA02溶液几乎无荧光,随着E. coli β-gal的不断加入,其分别在569 nm和562 nm处发射红色荧光且荧光强度逐渐增强。在一定E. coliβ-gal浓度范围内,探针最大发射波长的荧光强度与酶浓度呈现良好的线性关系,探针KSA01和KSA02对E. coli β-gal浓度的最低检出范围分别为4.6×10-3 U/mL和3.2×10-3U/mL。以上结果表明探针KSA01和KSA02对E. coli β-gal响应良好,而且在E. coli β-gal最适pH为7.4时发射红色荧光,这与探针对A. oryzae β-gal的响应不同。
实施例6
实施例1和2制备得到的KSA01和KSA02的酶动力学实验。
在10 mL的比色管中加入50 μL荧光探针KSA01和KSA02储备液(1 mmol/L储备液),然后加入1mL 的pH = 4.0或pH = 7.4 的PBS缓冲溶液后分别加A. oryzae β-gal或E. coli β-gal(均1 U/mL)最终用pH = 4.0或pH = 7.4 的PBS缓冲溶液稀释至 5 mL,测定上述样品的最大发射波长荧光强度随时间的变化。探针分子的时间动力学曲线如图4a和4c所示,在pH = 4.0的PBS缓冲溶液中加入A. oryzae β-gal后,绿色荧光强度逐渐升高,并分别在13 min和9 min达到最大值并稳定。如图4b和4d所示,在pH = 7.4的PBS缓冲溶液中加入E. coli β-gal后,红色荧光强度逐渐升高,并分别在14 min和13 min达到最大值并稳定。
实施例7
实施例1和2制备得到的KSA01和KSA02对β-半乳糖苷酶的选择性实验。
在10 mL的比色管中加入50 μL荧光探针KSA01和KSA02储备液,然后加入1mL 的pH= 4.0或pH = 7.4 的PBS缓冲溶液后分别加A. oryzae β-gal、E. coli β-gal、Trypin、Xanthine、Oxidase、Lysozyme、Chymotrypsin、Pepsin等生物酶(均1 U/mL),及Hcy、Cys、GSH、H2O2等小分子(10 μM),最终用pH = 4.0或pH = 7.4的PBS缓冲溶液稀释至5 mL后孵育30 min, 然后测试上述样品的荧光光谱。结果如图5a和5c所示,显示探针KSA01和KSA02在pH = 4.0的缓冲溶液中选择性响应A. oryzae β-gal,发射绿色荧光,且不受其它分析物的干扰。如图5b和5d所示,在pH = 7.4的缓冲溶液中选择性响应E. coli β-gal,发射红色荧光,同样不受其它分析物的干扰。
实施例8
实施例1和2制备得到的KSA01和KSA02在各种细胞模型中的荧光成像实验。
分别向SKOV-3细胞(人卵巢癌细胞),HepG2-Lacz(+)细胞和HepG2-Lacz(-)细胞中加入探针KSA01或KSA02(300 μM)孵育30 min,PBS清洗三次细胞后,采用激光共聚焦拍摄荧光成像图片(激发波长为 405 nm和488 nm,收集波长范围500-550 nm和570-620 nm),得到荧光成像图6。富含内源性卵巢癌相关β-半乳糖苷酶的SKOV-3细胞的红、绿通道均显示荧光信号,且绿色通道的荧光强度高于红色通道,这是由于内源性卵巢癌相关的β-半乳糖苷酶位于pH较低的溶酶体环境中。然而,高表达E. coli β-gal的Lacz基因转染的HepG2细胞的红、绿通道同样显示强的荧光信号,不同的是红通道荧光强度高于绿通道的,这是由于这类细菌β-gal在pH较高的细胞质环境中表达。这些结果表明,我们的探针不但能够检测活细胞中内源性或外源性β-半乳糖苷酶,而且还能区分检测他们所处的微环境(溶酶体或细胞质)的pH。
实施例9
实施例1和2制备得到的KSA01和KSA02在衰老细胞模型中的荧光成像实验。
经XL413诱导不同时间的HepG2细胞作为药物诱导衰老癌细胞模型。诱导不同时间的HepG2细胞(人肝癌细胞)用探针KSA01和KSA02(300 μM)孵育30 min,PBS清洗三次细胞后,采用激光共聚焦拍摄荧光成像图片(激发波长为 405 nm和488 nm,收集波长范围500-550 nm和570-620 nm),得到荧光成像图7和图8。XL413通过抑制DNA复制激酶CDC7诱导肝癌细胞衰老。随着XL413诱导时间增加,HepG2细胞逐渐衰老,其溶酶体内衰老相关的β-半乳糖苷酶逐渐累积。由图7和图8可以看出,随着XL413诱导时间增加,两个探针在HepG2细胞的红、绿通道荧光强度均逐渐增加,且红通道荧光强度的增加程度均高于绿色荧光通道,表明随着衰老,癌细胞内的衰老相关的β-半乳糖苷酶逐渐累积且其所在溶酶体pH逐渐升高。KU-60019被报道可作为潜在的抗衰老药物。经XL413诱导7天后的衰老HepG2细胞近一步用KU-60019处理两天后,用探针KSA02(300 μM)孵育30 min,PBS清洗三次细胞后,进行共聚焦成像。从图8中可以看出,与探针在XL413诱导7天的HepG2细胞中的成像结果相比,探针在进一步经KU-60019处理的HepG2细胞中的红、绿通道的荧光强度显著降低,且红通道与绿通道的荧光强度比也显著降低,表明β-半乳糖苷酶水平和溶酶体pH均下降,成功可视化了KU-60019的抗衰药效。该探针有望成为此类抗衰老药物的快速、准确可视化评价工具。
Claims (9)
3.根据权利要求2所述制备方法,其特征在于:缩合反应使用醇类溶剂,反应温度为70-80 °C。
4.根据权利要求3所述制备方法,其特征在于:所述醇类溶剂为甲醇、乙醇、丙醇。
5.权利要求1所示化合物在制备同时检测β-半乳糖苷酶和pH的荧光探针中的应用。
6.权利要求1所示化合物在制备同时检测细胞内内源性β-半乳糖苷酶和溶酶体pH的荧光探针中的应用。
7.权利要求1所示化合物在制备同时检测细胞内内源性衰老相关β-半乳糖苷酶和溶酶体pH的荧光探针中的应用。
8.权利要求1所示化合物在制备同时检测细胞内外源性β-半乳糖苷酶和细胞质pH的荧光探针中的应用。
9.权利要求1所示化合物在制备细胞成像和组织切片荧光成像试剂中的应用。
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