CN114085256A - 一种β-半乳糖苷酶响应的糖类衍生物及其应用 - Google Patents
一种β-半乳糖苷酶响应的糖类衍生物及其应用 Download PDFInfo
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- CN114085256A CN114085256A CN202111401720.5A CN202111401720A CN114085256A CN 114085256 A CN114085256 A CN 114085256A CN 202111401720 A CN202111401720 A CN 202111401720A CN 114085256 A CN114085256 A CN 114085256A
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Abstract
Description
技术领域
本发明涉及生物医药研究领域,具体涉及一种β-半乳糖苷酶响应的糖类衍生物及其应用。
背景技术
细胞表面的糖萼,主要是由蛋白多糖、糖蛋白和糖脂等组成,是哺乳动物细胞表面的一层致密的糖类化合物,在维持细胞功能方面起着重要的作用。癌细胞(肿瘤细胞)表面的糖萼的结构和性质与正常细胞的不一样,即异常的糖基化修饰。肿瘤糖萼的异常性,例如细胞表面聚糖最外层的唾液酸(sialic acid)与细菌感染、肿瘤发生和恶行肿瘤转移等密切相关。这些肿瘤相关异常糖基化修饰是肿瘤细胞标记、肿瘤诊断、肿瘤成像以及癌症治疗的重要靶点。因此,根据糖类化合物在细胞中代谢的特性,一系列的人工合成非天然糖衍生用于细胞的代谢标记。
利用人工合成非天然糖衍生物作为糖类前体,通过糖代谢途经标记肿瘤细胞已经成为了肿瘤标记、诊断和肿瘤治疗的一项重要的策略。例如,具有生物正交官能团(叠氮基团或炔基)的非天然唾液酸,被广泛用于细胞的标记中,并进一步用于聚糖分析、蛋白质组学分析、细胞标记、肿瘤治疗等中。然而,这种唾液酸代谢标记缺少细胞选择性,难以特异性标记肿瘤细胞,因此,如何提高糖代谢标记的细胞选择性对于更广泛应用这种策略非常重要。
β-半乳糖苷酶在许多恶性肿瘤中活性很强,因此,也是肿瘤的重要生物标记物,尤其是原发性卵巢癌和乳腺癌等癌症,针对β-半乳糖苷酶的肿瘤诊断和治疗策略的开发是一个热点方向。结合糖代谢工程策略,本发明提出了一种β-半乳糖苷酶响应的糖类衍生物的设计、制备及其在肿瘤标记、肿瘤成像、肿瘤治疗中的应用。通过对单糖、二糖进行β-半乳糖的修饰,使之仅能选择性地被β-半乳糖苷酶催化,释放出单糖、二糖前体,并且在β-半乳糖苷酶活性较高的肿瘤细胞中累积,从而增强肿瘤细胞的特异性标记,并可用于肿瘤的诊断和治疗中。
发明内容
本发明的目的是提供一种β-半乳糖苷酶响应的糖类衍生物及其在肿瘤诊断、标记中的应用,用于肿瘤细胞的糖代谢工程,含有高活性β-半乳糖苷酶的肿瘤细胞能够选择性地累积这些糖类衍生物,并表达在细胞表面,从而能够对肿瘤细胞进行选择性标记,并进一步用于肿瘤的成像和治疗。
为了解决现有技术存在的问题,本发明提供了如下技术方案:本发明的第一个目的是提供一种β-半乳糖苷酶响应的糖类衍生物,所述的β-半乳糖苷酶响应的糖类衍生物如式(I)所示的结构:
在糖上修饰一个β-半乳糖苷酶响应的化学结构,且在所述糖上修饰一个化学报告基团,所述的糖为单糖及单糖衍生物、二糖及二糖衍生物,所述的β-半乳糖苷酶响应的化学结构,含有一个β-糖苷键连接的半乳糖或半乳糖衍生物,含有一个自断裂的连接臂,在β-半乳糖苷酶的催化下,所述β-半乳糖苷酶响应的化学结构可以从糖类衍生物上断裂,裸露出单糖或二糖衍生物上的羟基。
进一步地,所述的β-半乳糖苷酶响应的糖类衍生物如式(II)所示的结构:
进一步地,所述的单糖及单糖衍生物为甘露糖、甘露糖胺、乙酰氨基甘露糖、唾液酸、葡萄糖、葡萄糖胺、半乳糖、半乳糖胺或岩藻糖,所述的二糖及二糖衍生物为海藻糖;所述的β-半乳糖苷酶为β-糖苷键连接的乙酰半乳糖。
进一步地,所述的化学报告基团,包括可用于点击化学反应的化学基团。
进一步地,所述的化学报告基团为含有叠氮-N3、炔基结构。
进一步地,所述β-半乳糖苷酶响应的化学结构修饰在单糖的6位羟基上,或二糖的6位羟基上。
进一步地,所述化学报告基团修饰在单糖的2位氨基或羟基上,或二糖的2位羟基上。
本发明的另一个目的是提供一种β-半乳糖苷酶响应的糖类衍生物在制备肿瘤诊断、标记中的应用。
本发明的另一个目的是提供一种β-半乳糖苷酶响应的糖类衍生物在肿瘤成像中的应用。
有益效果:本发明的糖类衍生物上修饰有化学报告基团,所述化学报告基团用于标记或治疗。所述β-半乳糖苷酶响应的糖类衍生物能够被肿瘤细胞大量摄取,并通过糖代谢途经,将非天然的糖表达在肿瘤细胞表面,从而用于肿瘤的标记和肿瘤的治疗。
与传统的具有化学报告基团的糖类衍生物前体相比,本发明具有如下优点:(1)本发明的β-半乳糖苷酶响应的糖类衍生物,能够通过β-半乳糖苷酶的催化,选择性地对肿瘤细胞进行标记,且能够在肿瘤细胞中大量积累,避免对正常细胞的副作用,促进肿瘤标记的效率和肿瘤治疗的效率。这一策略提供了一种简单而有效的方法来增强肿瘤细胞的选择性标记,且可以进一步用于肿瘤成像和肿瘤治疗,能够广泛应用于肿瘤诊断和治疗领域。
(2)本发明所述的糖前体,因其具有β-半乳糖苷酶响应的特性,能够提高肿瘤细胞标记的选择性,能够在β-半乳糖苷酶活性较高的肿瘤细胞中大量积累,从而促进肿瘤标记的效率和肿瘤治疗的效率。提供了一种具有肿瘤细胞选择性的糖代谢标记前体化合物,是一种B-半乳糖苷酶响应的糖前体,对现有的糖代谢工程进行了合理的改造,且所发明的糖前体衍生物具有很高的标记效率,降低了正常细胞的标记,从而降低副作用。该发明可以用于肿瘤的标记、成像、诊断及肿瘤治疗中。
附图说明
图1为本发明的β-半乳糖苷酶响应的乙酰甘露糖胺(Gal-AAM)及其合成路线图。
反应条件:(a)(i)MeOH,MeONa;(ii)TEA,氯乙酸酐,r.t,12h;(b)DMF,NaN3,12h;(c)TBDPSCl,Py,DMAP,50℃,24h;(d)Py,AC2O,r.t,12h;(e)TBAF,THF,r.t,12h;(f)Py,AC2O,r.t,12h;(g)DCM,HBr/AcOH,0℃4h;(h)Ag2O,CH3CN,4-羟基-3-硝基苯甲醛,r.t,3h;(i)NaBH4,iPrOH/CHCl3,r.t,3h;(j)4-硝基苯基羰基氯酸盐,DCM,TEA,r.t,3h;(k)DMAP,Py,50℃,18h;
图2为本发明的Gal-AAM的核磁共振氢谱图。
图3为本发明的Gal-AAM的核磁工程碳谱图。
图4为本发明的共聚焦显微镜观察经DAPI、DBCO-Cy5标记的Ac4ManNAc、AC3MANAz或Gal-AAM处理的MDA-MB-231细胞和HEK293细胞图。
图5为本发明的流式细胞检测经DBCO-Cy5标记的Ac4ManNAc、AC3MANAz或Gal-AAM处理的细胞的相应平均荧光强度图,****:P<0.0001。
具体实施方式
下面结合附图和实施例对本发明作更进一步的说明。
根据下述实施例,可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
本发明的第一个目的是提供一种β-半乳糖苷酶响应的糖类衍生物,所述的β-半乳糖苷酶响应的糖类衍生物如式(I)所示的结构:
在糖上修饰一个β-半乳糖苷酶响应的化学结构,且在所述糖上修饰一个化学报告基团,所述的糖为单糖及单糖衍生物、二糖及二糖衍生物,所述的β-半乳糖苷酶响应的化学结构,含有一个β-糖苷键连接的半乳糖或半乳糖衍生物,含有一个自断裂的连接臂,在β-半乳糖苷酶的催化下,所述β-半乳糖苷酶响应的化学结构可以从糖类衍生物上断裂,裸露出单糖或二糖衍生物上的羟基。
所述的单糖及单糖衍生物为甘露糖、甘露糖胺、乙酰氨基甘露糖、唾液酸、葡萄糖、葡萄糖胺、半乳糖、半乳糖胺或岩藻糖,所述的二糖及二糖衍生物为海藻糖;所述的β-半乳糖苷酶为β-糖苷键连接的乙酰半乳糖。
所述的化学报告基团,包括可用于点击化学反应的化学基团。
所述的化学报告基团为含有叠氮-N3、炔基结构。
所述β-半乳糖苷酶响应的化学结构修饰在单糖的6位羟基上,或二糖的6位羟基上。
所述化学报告基团修饰在单糖的2位氨基或羟基上,或二糖的2位羟基上。
本发明的另一个目的是提供一种β-半乳糖苷酶响应的糖类衍生物在制备肿瘤诊断、标记中的应用。
本发明的另一个目的是提供一种β-半乳糖苷酶响应的糖类衍生物在肿瘤成像中的应用。
实施例2
实施例2与实施例1的区别在于:所述的β-半乳糖苷酶响应的糖类衍生物如式(II)所示的结构:
实施例3
β-半乳糖苷酶响应的糖类衍生物的合成
本发明设计了一种β-半乳糖苷酶响应的乙酰基化-N-叠氮乙酰基-甘露糖胺,通过自断裂连接臂在其C6位置上修饰乙酰化β-半乳糖,即化合物Gal-AAM,式(II)所示的结构:
其合成路线如图1所示。
以D-半乳糖和D-甘露糖胺盐酸盐为原料,合成了半乳糖胺。化合物6(Ac3ManNAz),根据文献报道,从D-甘露糖胺盐酸盐中仅需五步即可获得。然后根据文献报告,合成带有可切割连接臂的半乳糖衍生物(图1中标注的化合物11),并用4-硝基苯基氯甲酸酯活化以生成碳酸盐化合物12。在DMAP、吡啶存在下,通过将化合物6偶联到化合物12,制备前体Gal-AAM,反应后通过硅胶柱层析纯化,产率为42%。
对Gal-AAM进行结构鉴定,其核磁共振氢谱和碳谱,如图2和图3所示,核磁共振谱图证明所合成的化合物正确,且乙酰半乳糖的连接方式为β-半乳糖苷键。
试验例1
Gal-AAM在不同细胞系中的代谢标记
具有高β-半乳糖活性的乳腺癌细胞系MDA-MB-231和低β-半乳糖活性的HEK293细胞,分别与100μM GalAAM孵育24小时,并在37℃下用DBCO-Cy5(50μM)标记1小时,同时分别使用Ac4ManAc和Ac3ManAz作为阴性和阳性对照。通过激光扫描共聚焦显微镜收集细胞图像。如图4所示,MDA-MB-231细胞和HEK293细胞经过Ac4ManNAc、AC3MANAz或Gal-AAM处理,并通过DAPI、DBCO-Cy5标记,其中,MDA-MB-231能够成功被Gal-AAM代谢标记,显示出较强的荧光信号。而对于低β-半乳糖苷酶活性的HEK293细胞,Gal-AAM则无法标记。与AC3MANAz相比,Gal-AAM对肿瘤细胞具有更好的选择性,且具有相似的代谢效率。图5流式细胞的结果进一步验证可知,Gal-AAM能够选择性地对高β-半乳糖苷酶活性的肿瘤细胞进行标记。
与传统的用于肿瘤细胞标记的化合物Ac4ManAz或Ac3ManAz等相比,本发明的化合物Gal-AAM,能够在β-半乳糖活性较高的肿瘤细胞MDA-MB-231中高效代谢,其代谢效率较高,且在正常细胞中不能代谢,因此,本发明的Gal-AAM化合物既保留了高效的代谢效率,又具有肿瘤细胞的选择性,从而更有效地用于肿瘤细胞的诊断和标记中,并用于肿瘤诊断试剂等相关的开发和应用中。
以上所述仅是本发明的优选实施方式,应对指出: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。
Claims (9)
3.根据权利要求1所述的一种β-半乳糖苷酶响应的糖类衍生物,其特征在于:所述的单糖及单糖衍生物为甘露糖、甘露糖胺、乙酰氨基甘露糖、唾液酸、葡萄糖、葡萄糖胺、半乳糖、半乳糖胺或岩藻糖,所述的二糖及二糖衍生物为海藻糖;所述的β-半乳糖苷酶为β-糖苷键连接的乙酰半乳糖。
4.根据权利要求1所述的β-半乳糖苷酶响应的糖类衍生物,其特征在于:所述的化学报告基团,包括可用于点击化学反应的化学基团。
5.根据权利要求3所述的β-半乳糖苷酶响应的糖类衍生物,其特征在于:所述的化学报告基团为含有叠氮-N3、炔基结构。
6.根据权利要求1所述的β-半乳糖苷酶响应的糖类衍生物,其特征在于:所述β-半乳糖苷酶响应的化学结构修饰在单糖的6位羟基上,或二糖的6位羟基上。
7.根据权利要求1所述的β-半乳糖苷酶响应的糖类衍生物,其特征在于:所述化学报告基团修饰在单糖的2位氨基或羟基上,或二糖的2位羟基上。
8.权利要求1至7任一项所述的β-半乳糖苷酶响应的糖类衍生物在制备肿瘤标记物的试剂中的应用。
9.权利要求1至7任一项所述的β-半乳糖苷酶响应的糖类衍生物在肿瘤成像中的应用。
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