CN109053822B - 含糖基萘酰亚胺类荧光探针及其应用 - Google Patents
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Abstract
本发明公开了一种含糖基萘酰亚胺类荧光探针及其在糖苷酶抑制剂分子的筛选和细胞成像中的应用。本发明所述的含糖基萘酰亚胺类荧光探针的结构式如式I所示，其中糖基部分为β‑葡萄糖、β‑N‑乙酰氨基葡萄糖、β‑半乳糖、β‑N‑乙酰氨基半乳糖,n＝1‑6。本发明设计并合成的含糖基萘酰亚胺类荧光探针具有优良的水溶性和荧光性质，可以应用于糖苷酶抑制剂分子筛选中，并且可以通过肉眼快速筛选抑制率大于70％的抑制剂分子；该探针可进一步用于活细胞内糖苷酶的荧光成像。
Description
技术领域
本发明涉及一种新颖的糖基萘酰亚胺类衍生物，以及其制备方法与作为荧光探针在糖苷酶抑制剂分子的筛选和细胞成像中的应用。
背景技术
糖苷酶是一种能够水解糖元之间或糖与非糖之间的糖苷键的糖基水解酶。它是维持生物体基本功能的重要酶类之一，在自然界广有分布，几乎各种类型的动、植物和微生物中，都有其存在。与此同时，糖苷酶在生物技术、人类疾病、真菌和昆虫的控制等方面都有着重要作用，是一个很好的靶酶。它的小分子抑制剂不仅可以作为糖尿病治疗剂和杀虫剂，而且也是一种有用的生物学工具。
糖苷酶的检测常用糖基化的对硝基苯酚底物和4-甲基伞形酮底物,然而两种底物都有一定的缺点。例如糖基化的对硝基苯酚底物，利用紫外吸收法测定，灵敏度弱,检测区间窄(0-0.5)，底物使用浓度高(500μM)，化合物颜色对反应测定存在干扰，室温易分解；糖基化的4-甲基伞形酮底物酶解后，溶解性低，需要加饱和碳酸钠放大荧光信号，其测量结果易受环境影响，而且必须依靠仪器检测，不能肉眼直观评估酶活，这导致了它们的应用有一定的局限性。鉴于以上方面的缺点，目前极需要更好的糖苷酶底物实现酶抑制剂的快速检测。当前荧光传感器由于其安全、方便、快速，可以实现体外检测和体内成像研究等优点，在生物学和医学上有着广泛的应用。因此，本文设计了新型的水溶性好的含糖基萘酰亚胺结构的分子，实现其在中性条件下作为荧光探针在糖苷酶抑制剂分子的筛选的应用；另外它能实现活细胞内糖苷酶荧光成像，可用于检测细胞内是否存在相应的糖苷酶以及实现在活细胞内检测相应的糖苷酶的抑制剂分子。
发明内容
本发明涉及一种新颖的糖基萘酰亚胺类衍生物，其制备方法与作为荧光探针在糖苷酶抑制剂分子的筛选和细胞成像中的应用。
本发明提供的一种新颖的糖基萘酰亚胺类荧光探针，其结构式如式I所示，
式I中，R1＝H,R2＝OH,R3＝H,R4＝OH，即糖基部分为葡萄糖；R1＝H,R2＝NHAc,R3＝H,R4＝OH,即糖基部分为N-乙酰氨基葡萄糖；R1＝H,R2＝OH,R3＝OH,R4＝H,即糖基部分为半乳糖；R1＝H,R2＝NHAc,R3＝OH,R4＝H,即糖基部分为N-乙酰氨基半乳糖；n为1-6。
上述式I所示化合物的制备方法，包括如下步骤：
(1)式II与式III在三光气作用下得到含保护基的糖基萘酰亚胺衍生物式IV；
式II中，R5＝H,R6＝OAc或OBz,R7＝H,R8＝OAc或OBz，即糖基部分为含保护基的葡萄糖；R5＝H,R6＝NHAc,R7＝H,R8＝OAc或OBz，即糖基部分为含保护基的N-乙酰氨基葡萄糖；R5＝H,R6＝OAc或OBz,R7＝OAc或OBz,R8＝H，即糖基部分为含保护基的半乳糖；R5＝H,R6＝NHAc,R7＝OAc或OBz,R8＝H，即糖基部分为含保护基的N-乙酰氨基半乳糖；R9为Ac或Bz；n＝1-6。
(2)式IV在碱性条件下脱除保护基，即得到式I所示化合物。
式I中，R1＝H,R2＝OH,R3＝H,R4＝OH，即糖基部分为葡萄糖；R1＝H,R2＝NHAc,R3＝H,R4＝OH,即糖基部分为N-乙酰氨基葡萄糖；R1＝H,R2＝OH,R3＝OH,R4＝H,即糖基部分为半乳糖；R1＝H,R2＝NHAc,R3＝OH,R4＝H,即糖基部分为N-乙酰氨基半乳糖；n为1-6。
步骤(2)所述碱性条件采用的试剂为甲醇钠、甲醇氨、甲醇甲氨中的至少一种；所述反应的温度可为室温，时间可为10～50h，具体可为12h、12～24h、24～48h或10～35h。
本发明的另一个目的在于，提供了一种新型的靶向糖苷酶的荧光探针(式I所示化合物)，其含有聚乙二醇修饰的萘酰亚胺荧光团、氨基甲酸酯连接臂和糖苷酶识别基团(β-葡萄糖或β-N-乙酰氨基葡萄糖或β-半乳糖或β-N-乙酰氨基半乳糖)。它们具有优良的光学和物理性质，与β-葡萄糖苷酶或β-N-乙酰己糖胺酶或β-半乳糖苷酶或β-N-乙酰半乳糖苷酶反应后，在540nm附近产生了新的荧光发射，不仅可以用于糖苷酶抑制剂分子的高效筛选，也可以用于活细胞内糖苷酶荧光成像。
本发明的有益效果为：本发明以多种糖苷酶为研究对象，合成出结构新颖的糖苷酶的荧光探针。本发明所述的糖基萘酰亚胺化合物具有优良的光学和物理性质，与糖苷酶反应后，在540nm附近产生了新的荧光发射，从而应用于糖苷酶抑制剂分子筛选中，并且可以通过肉眼快速筛选抑制率大于70％的抑制剂分子；该探针可进一步用于活细胞内糖苷酶的荧光成像。本发明所述探针分子具有很高的医药及农药应用研究价值。
附图说明
图1为本发明所合成的含糖基的萘酰亚胺类化合物的结构式。
图2为实施例1中探针的氢谱图。
图3为实施例2中有无β-N-乙酰己糖胺酶存在下探针溶液的光学性质。
图4为实施例4中探针分子与β-N-乙酰己糖胺酶反应的动力学曲线。
具体实施方式
下面结合附图及实施例对本发明作进一步描述。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法，均可从商业途径得到；所实施例中化合物I如无特殊说明，均选用R1＝H,R2＝NHAc,R3＝H,R4＝OH,n＝4,即以含β-N-乙酰氨基葡萄糖结构的荧光分子为实例说明。
实施例1，式I中R1＝H,R2＝NHAc,R3＝H,R4＝OH,n＝4的化合物制备，具体步骤如下：
(1)在250mL的圆底烧瓶中加入N-乙酰氨基葡萄糖(10g,40.3mmol)、乙酰氯(40ml,186.8mmol)，于室温下反应48h，TLC监测反应完全。将反应液加入到500mL冰水中，用200mLDCM萃取得到有机层，再用饱和NaHCO3水溶液(100mL)、水(100mL)洗涤，无水Na2SO4干燥，浓缩后得到2-乙酰氨基-3,4,6-三-O-乙酰基-2-脱氧-α-D-吡喃葡萄糖苷氯化物，无需进一步纯化直接进行下一步反应。
在500mL的反应瓶中加入4-羟基苯甲醛(2.5g,20.5mmol)，Na2CO3(2.2g，20.5mmol)，四丁基溴化铵(6.6g，20.5mmol)，100mL DCM和150mL H2O，在35℃下滴加2-乙酰氨基-3,4,6-三-O-乙酰基-2-脱氧-α-D-吡喃葡萄糖苷氯化物(5.0g，13.7mmol)的DCM(50mL)溶液。在35℃下搅拌12h，TLC确定反应完成。分液，有机相用水(2×100mL)洗涤，无水Na2SO4干燥，浓缩，甲醇重结晶，得4.5g白色固体化合物，产率72.6％。
结构确证数据如下：[α]D 25-16.3(c＝1.0,CHCl3)；1H NMR(300MHz,DMSO-d6)δ9.92(s,1H,CHO),8.08(d,J＝9.0Hz,1H,NH),7.90(d,J＝8.7Hz,2H,ArH),7.22(d,J＝8.7Hz,2H,ArH),5.54(d,J＝8.4Hz,1H,H-1),5.25(t,J＝9.3Hz,1H,H-3),4.96(t,J＝9.3Hz,1H,H-4),4.26-4.18(m,2H,H-2,H-6b),4.14–4.01(m,2H,H-6a,H-5),2.01(s,6H,2CH3),1.96(s,3H,CH3),1.79(s,3H,CH3)；13C NMR(75MHz,DMSO-d6)δ191.59,170.07,169.79,169.66,169.42,161.36,131.75,131.27,116.67,97.12,72.43,71.18,68.45,61.71,53.20,22.75,20.57,20.52,20.43。
(2)取(1)中所得产物(4.5g，10.0mmol)溶于120mL DCM和40mL H2O中，在室温下分批加入NaBH4(0.57g，15.0mmol)，继续室温搅拌3h，TLC确定反应完成。分液，有机相依次用0.5M HCl(50mL)、水(2×50mL)洗涤，无水Na2SO4干燥，浓缩，得到白色固体化合物4.2g，产率93.3％。
结构确证数据:[α]D 25+12.1(c＝1.0,CHCl3)；1H NMR(300MHz,CDCl3)δ7.26(d,J＝8.4Hz,2H,ArH),6.95(d,J＝8.6Hz,2H,ArH),5.99(d,J＝8.8Hz,1H,OH),5.46–5.34(t,J＝9.6Hz,1H,H-3),5.24(d,J＝8.3Hz,1H,H-1),5.12(t,J＝9.6Hz,1H,H-4),4.62(s,2H,CH2),4.27(dd,J＝12.2,5.4Hz,1H,H-6b),4.20–4.08(m,2H,H-6a,H-2),3.94–3.80(m,1H,H-5),2.07(s,3H,OAc),2.06(s,3H,OAc),2.05(s,3H,OAc),1.93(s,3H,NAc)；HRMS(ESI)calcdfor C21H28NO10(M+H+)454.1713,found454.1725。
(3)在-10℃下，先将含四乙基乙二醇单甲醚结构的萘酰亚胺(4.2g，10mmol)溶于干燥的DCM(40mL)再加入Et3N(1.5g，15mmol)混合，然后氮气保护下，将溶有三光气(3.3g，11mmol)的干燥DCM(25mL)逐滴加入到上述发应液中。在室温下继续搅拌3h，TLC检测反应完成。减压蒸馏除去溶剂，将剩余物溶于60mL干燥DCM中，并在0℃下滴加步骤(2)的产物(4.5g，10mmol)，反应6h，TLC确定反应完成。反应液中加入100mL水淬灭，用DCM(3×50mL)萃取。有机相用无水Na2SO4干燥，浓缩，硅胶柱层析纯化得到乙酰基保护的探针6.5g，产率73.8％。
结构表征：[α]D 25+99.8(c＝1.0,DMSO)；1H NMR(300MHz,DMSO-d6)δ10.35(s,1H,NHCO),8.70(d,J＝8.8Hz,1H,ArH),8.48(t,J＝7.8Hz,2H,ArH),8.20(d,J＝8.3Hz,1H,ArH),8.11(d,J＝9.1Hz,1H,NHAc),7.86–7.76(m,1H,ArH),7.48(d,J＝8.7Hz,2H,ArH),7.07(d,J＝8.7Hz,2H,ArH),5.37(d,J＝8.5Hz,1H,H-1),5.28–5.18(m,3H,H-3,PhCH2 ),4.94(t,J＝9.6Hz,1H,H-4),4.28–4.18(m,3H,H-6b,CH2),4.18–3.96(m,3H,H-6a,H-2,H-5),3.66(t,J＝6.3Hz,2H,CH2),3.59–3.52(m,2H,CH2),3.51–3.46(m,2H,CH2),3.46–3.31(m,8H,4CH2),3.20(s,3H,CH3),2.01(s,3H,OAc),2.01(s,3H,OAc),1.96(s,3H,OAc),1.79(s,3H,NAc)；13C NMR(75MHz,DMSO-d6)δ170.08,169.75,169.61,169.44,163.58,163.01,156.88,154.06,140.93,131.83,131.04,130.53,130.12,129.49,128.44,126.43,123.88,122.20,118.14,117.00,116.67,98.03,72.60,71.33,71.01,69.89,69.80,69.76,69.63,68.59,67.07,66.33,61.79,58.11,53.35,22.78,20.60,20.54,20.47；HRMS(ESI)calcdfor C43H52N3O17(M+H+)882.3297,found 882.3311。
乙酰基保护的探针(0.88g，1.0mmol)溶解在10mL无水的甲醇溶液中，然后向反应液中逐滴滴加饱和甲醇氨溶液至反应液的pH值达到9.5。室温下搅拌50h，TLC确定反应完成，浓缩，用乙醚重结晶，得到0.63g淡黄色固体，产率83.3％。
结构表征：[α]D 25+100.7(c＝1.0,DMSO)；1H NMR(300MHz,DMSO-d6)δ8.70(d,J＝8.6Hz,1H,ArH),8.52–8.42(m,2H,ArH),8.21(d,J＝8.3Hz,1H,ArH),7.89–7.74(m,2H,ArH,NHAc),7.45(d,J＝8.6Hz,2H,ArH),7.03(d,J＝8.5Hz,2H,ArH),5.22(s,2H,PhCH2 ),5.14(s,1H,OH),5.01(d,J＝8.5Hz,1H,H-1),4.23(t,J＝6.2Hz,2H,CH2),3.80–3.67(m,2H,H-3,H-4),3.66–3.61(m,2H,CH2),3.58–3.44(m,6H,3CH2,),3.44–3.38(m,6H,3CH2),3.38–3.34(m,3H,H-2,H-5,H-6b),3.30(dd,J＝5.5,1.2Hz,1H,H-6a),3.20(s,3H,OCH3),1.82(s,3H,CH3)；13C NMR(75MHz,DMSO-d6)δ169.38,163.60,163.03,157.68,154.07,140.95,131.87,131.05,130.16,129.88,129.45,128.45,126.44,123.86,122.23,118.09,116.98,116.57,99.32,77.42,74.14,71.33,70.49,69.89,69.80,69.76,69.63,67.07,66.45,60.89,58.11,55.67,48.72,23.20；HRMS(ESI)calcd for C37H46N3O14(M+H+)756.2980,found756.2963。
实施例2实施例1中制备的化合物I(R1＝H,R2＝NHAc,R3＝H,R4＝OH,n＝4)在识别β-N-乙酰己糖胺酶(Hex)中荧光光谱的变化。
其具体操作方法及结果如下：
向1cm×1cm×4cm的比色皿中依次加入160μL 200μM化合物I,1440μL PBS缓冲溶液，制备浓度为20μM化合物I溶液，测定其荧光光谱。另外一组则向比色皿中依次加入160μL200μM化合物I，1440μL含有Hex的PBS缓冲溶液，温浴30分钟,测定其荧光光谱。从图3可以看出探针与酶反应后，探针发射波长发生了红移，而且探针与酶反应前后溶液由无色变为了黄色，易于肉眼可视。
实施例3化合物I和Hex的作用与时间的关系
在实施例2的基础上，我们研究了化合物I和Hex的作用与时间的关系。向比色皿中依次加入160μL 200μM化合物I，1440μL含有Hex的PBS缓冲溶液，室温下测定反应在0-80分钟的荧光光谱变化。我们发现前10分钟波长为438nm的荧光强度一直在降低，而在540nm新增了一个荧光发射峰，后70分540nm的荧光发射峰快速的增强，远远大于438nm处的荧光强度。说明随着时间的变化，探针与酶有着很好的响应。
实施例4化合物I与Hex的动力学参数
进一步地，我们探究了化合物I与Hex的动力学参数。首先在比色皿中配置一系列浓度的化合物I溶液，测定其荧光强度与浓度的关系，得到y＝166.95x-1.5388(R2＝0.9994)的线性曲线，然后配置含有Hex的不同浓度的化合物I溶液，37℃温浴10分钟，测量其荧光值。得到线性曲线y＝16.18x+0.40677(R2＝0.9995),进而得到Km＝39.76nm(图4),表明化合物I与酶有很好的亲和力。
实施例5化合物I在筛选抑制剂分子中的应用
使用20μM化合物I作为荧光探针测试了14个文献报道的β-N-乙酰己糖胺酶(Hex)抑制剂分子在100μM浓度下的抑制率，结果表明，抑制率的测试结果与以伞形酮(4-MU-GlcNAc)为底物测试的抑制剂结果相符。
表1：部分抑制剂分子抑制率的测试结果
进一步按照0-10％，10-20％,30-40％,40-50％,50-60％,60-70％,70-80％,80-90％,90-100％的抑制率对这些抑制剂分子进行了归类。然后向2mL透明螺纹液相色谱进样瓶中依次加入200μL 1mM各个抑制范围的典型抑制剂分子，200μL 200μM化合物I，100μLHex和1500μL PBS缓冲溶液，37℃下温浴20分钟。从测试结果可以看出，化合物I的溶液颜色随着抑制剂分子抑制率的增大，深黄色在逐渐变淡；当抑制率大于70％，其溶液则由深黄色变为近乎无色。另外，在抑制率约为50％时，化合物I的溶液颜色发生了“突变”，由无色变为了浅黄色。说明探针I可以实现肉眼识别高效糖苷酶抑制剂分子，从而达到肉眼筛选抑制率大于70％的抑制剂分子的目的。
其中，这14个文献报道的β-N-乙酰己糖胺酶(Hex)抑制剂分子具体结构参见文献(a)Kong,H.；Chen,W.；Lu,H.；Yang,Q.；Dong,Y.；Wang,D.；Zhang,J.Carbohydr.Res.2015,413,135-144.(b)Kong,H.；Chen,W.；Liu,T.；Lu,H.；Yang,Q.；Dong,Y.；Liang,X.；Jin,S.；Zhang,J.Carbohydr.Res.2016,429,54-61.(c)Shen,S.；Chen,W.；Dong,L.；Yang,Q.；Lu,H.；Zhang,J.J.Enzyme Inhib.Med.Chem.2018,33,445-452.(d)Chen,W.；Shen,S.；Dong,L.；Zhang,J.；Yang,Q.Bioorg.Med.Chem.2018,26,394-400。
实施例6化合物I在细胞内β-N-乙酰己糖胺酶(Hex)成像中的应用
用10μM化合物I分别加到含有活的过表达Hex酶的SW480细胞和不表达Hex酶的MDKB细胞的PBS缓冲液中，37℃下温浴1小时。对照组则预先将56μM PUGNAc加入到SW480细胞，37℃下温浴1小时后再去除剩余的PUGNAc，之后加入10μM的化合物I进一步孵育1h，用共聚焦荧光显微镜Carlzeiss lsm710捕捉荧光图像。结果可以看出，化合物I可以很好的响应Hex酶，从而产生绿色荧光，而当酶被抑制或细胞不表达Hex酶则产生蓝色荧光。
Claims (2)
1.一种含糖基的萘酰亚胺类化合物，所述糖基萘酰亚胺化合物的结构如式I所示：
式I中，R1＝H,R2＝NHAc,R3＝H,R4＝OH,即糖基部分为N-乙酰氨基葡萄糖；n为4。
2.权利要求1所述的含糖基的萘酰亚胺类化合物的制备方法，包含以下步骤：
(1)式II与式III在三光气作用下得到含保护基的糖基萘酰亚胺衍生物式IV；
式II中，R5＝H,R6＝NHAc,R7＝H,R8＝OAc或OBz，即糖基部分为含保护基的N-乙酰氨基葡萄糖；R9为乙酰基或苯甲酰基的一种；n＝4；
(2)式IV在碱性条件下脱除保护基，即得到式I所示化合物；
式I中，R1＝H,R2＝NHAc,R3＝H,R4＝OH,即糖基部分为N-乙酰氨基葡萄糖；n＝4。
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| CN104946242A (zh) * | 2015-06-09 | 2015-09-30 | 华南理工大学 | 用于检测β-半乳糖苷酶的荧光探针及制备方法和应用 |
| CN107383120A (zh) * | 2017-07-31 | 2017-11-24 | 中国农业大学 | 一种糖基萘酰亚胺衍生物及其制备方法与应用 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946242A (zh) * | 2015-06-09 | 2015-09-30 | 华南理工大学 | 用于检测β-半乳糖苷酶的荧光探针及制备方法和应用 |
| CN107383120A (zh) * | 2017-07-31 | 2017-11-24 | 中国农业大学 | 一种糖基萘酰亚胺衍生物及其制备方法与应用 |
Non-Patent Citations (2)
| Title |
|---|
| A Practical and High-Affinity Fluorescent Probe for Uridine Diphosphate Glucuronosyltransferase 1A1: A Good Surrogate for Bilirubin;Xia Lv，等;《Journal of Medicinal Chemistry》;20171110;第60卷;第9664-9675页 * |
| Diamagnetic Imaging Agents with a Modular Chemical Design for Quantitative Detection of β‑Galactosidase and β‑Glucuronidase Activities with CatalyCEST MRI;Gabriela Fernández-Cuervo，等;《Bioconjugate Chem.》;20160922;第27卷;第2549-2557页 * |
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