CN114957388B - 一种短肽荧光探针及其制备方法和应用 - Google Patents
一种短肽荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种短肽荧光探针及其制备方法和应用，所述短肽荧光探针的结构如式I所示。本发明提供的短肽荧光探针能够有效靶向乏氧微环境肿瘤区域，可以实现乏氧条件下的成像，可用于肿瘤细胞的荧光成像，对肿瘤的检测和治疗提供有利帮助，且该材料对细胞无毒性，安全性高。
Description
技术领域
本发明属于化学成像材料领域，具体涉及一种短肽荧光探针及其制备方法和应用，尤其涉及一种靶向效果好的短肽荧光探针及其制备方法和应用。
背景技术
荧光成像是非常重要的成像方式，由于其简单、快速和灵敏度高的优点被广泛应用于生物医药领域。4-氯-7硝基苯并-2-氧杂-1,3-二唑(NBD)是一种用途广泛的荧光分子，常用于作为蛋白、带有氨基官能团的纳米颗粒的标记物，但NBD本身也具有一些局限性，例如不具备靶向性，因此开发具备靶向性的NBD材料在本领域研究中具备实际意义。
CN112745303B公开了一种乏氧荧光探针，具有荧光团和乏氧识别基团，所述乏氧识别基团为1-5个吸电子基团取代的苯基偶氮，所述吸电子基团选自三氟甲基、氟基、硝基、氰基、磺酸基、磺酸酯基、羧基、脂基、酰氨基、烷酰基中的一种或几种。本发明所述的乏氧荧光探针能有效靶向线粒体，可用于细胞器乏氧循环检测，特别是用于细胞/组织/活体乏氧成像、生物标记或传感领域。
CN113527416A公开了一种硝基还原酶响应氨基酸及肿瘤乏氧荧光探针的制备方法。基于光延反应，合成了具有硝基还原酶(NTR)响应性的2-硝基咪唑修饰的丙氨酸(Fmoc-A(2NI))。在该非天然氨基酸的基础上合成了具有还原响应形貌转变的多肽序列Fmoc-A(2NI)VE，以及荧光分子IR780共价修饰的多肽衍生物，通过共组装的方式制备了超分子纳米探针。该发明的优点是:开发了一种硝基还原酶响应的可用于调控多肽组装和荧光探针制备的非天然氨基酸，构筑而成的纳米探针具有硝基还原酶作用下荧光显著增强的特点，以及乏氧响应性组装形貌转变，具有良好的肿瘤深度乏氧区域近红外荧光成像的性质。制备方法简单，易于工业化生产，应用领域范围广阔。
由于NBD是一种优秀的荧光分子，但其缺乏靶向性使其使用情况受到严重影响。因此，如何提供一种靶向的基于NBD的材料，成为了亟待解决的问题。
发明内容
针对现有技术的不足，本发明的目的在于提供一种短肽荧光探针及其制备方法和应用，尤其提供一种靶向效果好的短肽荧光探针及其制备方法和应用。本发明提供的短肽荧光探针能够有效靶向乏氧微环境肿瘤区域，可以实现乏氧条件下的成像，可用于肿瘤细胞的荧光成像，对肿瘤的检测和治疗提供有利帮助，且该材料对细胞无毒性，安全性高。
为达到此发明目的，本发明采用以下技术方案：
第一方面，本发明提供了一种短肽荧光探针，所述短肽荧光探针的结构如式I所示：
本发明提供的短肽荧光探针包含硝基咪唑基团和半胱氨酸中的巯基结构，使其可在乏氧条件下发生缩合，从而大量聚集，能够有效靶向乏氧微环境肿瘤区域，可以实现乏氧条件下的成像，可用于肿瘤细胞的荧光成像，对肿瘤的检测和治疗提供有利帮助，且该材料对细胞无毒性，安全性高。
第二方面，本发明提供了一种根据如上所述的短肽荧光探针的制备方法，所述制备方法包括以下步骤：
(1)将树脂活化，之后与Fmoc-Cys(StBu)-OH(N-芴甲氧羰基-S-叔丁硫基-L-半胱氨酸)、缩合剂、有机碱混合反应，之后进行活性位点灭除、脱保护，得到第一肽链；
(2)将步骤(1)得到的第一肽链依次与Fmoc-Phe-OH((N-芴甲氧羰基-L-苯丙氨酸))、Fmoc-Lys(Boc)-OH(N-芴甲氧羰基-N’-叔丁氧羰基-L-赖氨酸)在缩合剂和有机碱条件下反应，得到第二肽链；
(3)将步骤(2)得到的第二肽链与硝基咪唑乙酸、缩合剂、有机碱混合反应，得到NIKFC(StBu)，之后将树脂切割，得到NIKFC-S肽段；
(4)将步骤(3)得到的NIKFC-S肽段与4-氯-7硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)反应，之后脱保护，得到所述短肽荧光探针；
反应路线如下：
上述方法通过采用固相合成方法能够有效合成所述短肽荧光探针。
优选地，步骤(1)所述树脂包括2-氯三苯甲基氯树脂或王树脂中任意一种。
优选地，步骤(1)、步骤(2)、步骤(3)所述反应的时间独立地为0.8-1.2h，例如0.8h、0.9h、1h、1.1h或1.2h等，但不限于以上所列举的数值，上述数值范围内其他未列举的数值同样适用。
优选地，步骤(3)所述切割包括以下步骤：将NIKFC(StBu)与三氟乙酸混合处理，之后抽滤、收集滤液，得到NIKFC-S肽段。
优选地，步骤(4)所述NIKFC-S肽段与4-氯-7硝基苯并-2-氧杂-1,3-二唑的摩尔比为(0.9-1.1):(0.9-1.1)。
优选地，步骤(4)所述反应的时间为12-24h。
其中，NIKFC-S肽段与4-氯-7硝基苯并-2-氧杂-1,3-二唑的摩尔比中，NIKFC-S肽段的份数可以是0.9、1或1.1等，4-氯-7硝基苯并-2-氧杂-1,3-二唑的份数可以是0.9、1或1.1等，但不限于以上所列举的数值，上述数值范围内其他未列举的数值同样适用。
优选地，步骤(3)所述切割后、步骤(4)所述反应后、步骤(4)所述脱保护后独立地还进行纯化。
优选地，所述缩合剂包括HBTU(苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐)、HOBT(1-羟基苯并三唑)或TBTU(2-(1H-苯并三偶氮L-1-基)-1,1,3,3-四甲基脲四氟硼酸酯)中任意一种。
优选地，所述有机碱包括DIPEA(N,N-二异丙基乙胺)或DIC(N,N'-二异丙基碳二亚胺)。
第三方面，本发明还提供了如上所述的短肽荧光探针在制备细胞荧光成像材料中的应用。
相对于现有技术，本发明具有以下有益效果：
本发明提供了一种短肽荧光探针，其能够有效靶向乏氧微环境肿瘤区域，可以实现乏氧条件下的成像，可用于肿瘤细胞的荧光成像，对肿瘤的检测和治疗提供有利帮助，且该材料对细胞无毒性，安全性高。
附图说明
图1是实施例1中短肽荧光探针的核磁氢谱图；
图2是实施例1中短肽荧光探针的高分辨电喷雾质谱图；
图3是短肽荧光探针对乏氧组Hela细胞成像时细胞的状态图；
图4是短肽荧光探针对常氧组Hela细胞成像的结果图；
图5是短肽荧光探针对乏氧组Hela细胞成像的结果图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1
本实施例提供了一种短肽荧光探针的制备方法，具体步骤如下：
(1)树脂活化：将0.4g 2-氯三甲基氯树脂放入固相合成管中，用5mL二氯甲烷(DCM)溶解。然后用氮气搅拌溶液10分钟。之后，用真空泵抽去树脂上的溶剂。然后再次添加5mL二氯甲烷(DCM)，并用氮气搅拌10分钟。随后，抽去液体，并用3mL N,N-二甲基甲酰胺(DMF)清洗(用氮气搅拌30s，然后真空泵抽去溶剂)，重复五次。
(2)Cys-树脂的合成：将0.8mmol的Fmoc-Cys(StBu)-OH溶解在3mL DMF中，然后向溶液中添加0.4mL的DIPEA。之后，将溶液添加到固相合成管中，并用氮气搅拌1h。反应后，抽去树酯上的溶剂并用3mL DMF洗涤5次。
(3)活性位点封端：向固相合成管中加入5mL封端试剂(体积比DCM:MeOH:DIPEA＝16:3:1)，用氮气搅拌10min，抽去溶剂。然后重复上述步骤一次。完成后，用3mL DMF清洗树脂5次。
(4)Cys的Fmoc脱保护：向固相合成管中加入5mL脱保护溶剂(DMF中体积分数30％哌啶)，并与氮气搅拌30min。反应后，用3mL DMF洗涤树酯5次。
(5)Lys-Phe-Cys-树脂的合成：先将0.8mmol的Fmoc-Phe-OH和0.88mmol的HBTU溶解在3mL DMF中，然后添加0.4mL的DIPEA。之后，将溶液添加到固相合成管中，并用氮气搅拌1h。反应后，抽去溶剂并用3mL DMF洗涤5次。之后将0.8mmol的Fmoc-Lys(Boc)-OH和0.88mmol的HBTU溶解在3mL DMF中，然后添加0.4mL的DIPEA。之后，将溶液添加到固相合成管中，并用氮气搅拌1h。反应后，抽去溶剂并用3mL DMF洗涤5次。Fmoc对Lys的脱保护步骤与Cys相同。
(6)NI-Lys-Phe-Cys树脂的合成：将1.2mmol的硝基咪唑乙酸和1.32mmol的HBTU溶解在3mL DMF中，然后添加0.4mL的DIPEA。将溶液加入固相合成管中，并与氮气搅拌1h。反应后，抽去溶剂，并分别用3mL DMF、DCM、MeOH和正己烷(每种溶剂五次)清洗。
(7)多肽切割：向固相合成管中加入10mL三氟乙酸(TFA)进行切割。首先在0℃下切割树脂30分钟，然后在20℃下切割30分钟。切割完成后，抽去并收集溶液，然后用氮气浓缩过滤。
(8)沉淀：向无水乙醚中缓慢滴加浓缩过滤后的TFA溶液得到沉淀，然后过滤沉淀，得到浅黄色粉末状固体，这是反应的粗产物。粗产物经高效液相色谱纯化(以水:乙腈＝70:30体积比为初始梯度，0:100体积比为终止梯度进行梯度淋洗)，最终得到白色粉末状固体NIKFC-S肽段。
(9)连接NBD：将NIKFC-S肽段(63.7mg，0.1mmol)溶于甲醇(1.2mL)和水(1mL)的混合溶剂中，加入Na2CO3(27.8mg，0.2mmol)调节溶液的pH至8，搅拌溶液至其变为澄清。在此之后，将NBD-Cl(20mg，0.1mmol)溶于0.4mL的甲醇之中，并将NBD-Cl的甲醇溶液缓慢滴加入之前混合好的NIKFC-S肽段的溶液中，在20℃下搅拌反应12小时。反应结束后，通过旋转蒸发的方法去除体系内的甲醇，随后，用HCl溶液将溶液的pH调节至3，再通过离心(7500rpm×5min)沉淀、过滤去除水，得到粗产物，其为红褐色固体。将粗产物通过HPLC分离纯化(以水:乙腈＝50:50体积比为初始梯度，0:100体积比为终止梯度进行梯度淋洗)后，得到终产物，其为红褐色固体。
(10)对上述红褐色固体的水溶液使用10倍当量还原性谷胱甘肽(GSH)脱去巯基的保护，随后进行纯化，得到式I的短肽荧光探针，表征数据如下：1H NMR(400MHz,DMSO)δ8.50(d,J＝4.4Hz,1H),8.48(d,J＝3.2Hz,1H),8.23(d,J＝8.0Hz,1H),8.10(d,J＝8.4Hz,1H),7.60(d,J＝0.8Hz,1H),7.23(m,5H),7.18(d,J＝0.8Hz,1H),5.11(s,2H),4.56-4.54(m,1H),4.51-4.47(m,1H)4.38-4.33(m,1H),3.13-3.09(dd,J＝5.2Hz,3.2Hz,1H),3.08-3.03(dd,J＝3.2Hz,4.0Hz,1H),3.00-2.95(dd,J＝3.6Hz,4.4Hz,1H),2.81-2.75(dd,J＝5.6Hz,6.4Hz,1H),2.69-2.66(m,2H)observed ESI-MS:m/z 712.24。核磁氢谱如图1所示，高分辨电喷雾质谱如图2所示。
实施例2
在本实施例中，通过以下方法进行NIKFC-NBD(SH)的细胞成像，具体包括以下步骤：
将HeLa细胞分两组，按分组接种于两个不同的六孔板的14mm爬片中，其中第一组放入普通培养箱37℃孵育24h，另一组放入厌氧培养箱37℃孵育24h。在此之后，分别对两组细胞加入终浓度为80μM的实施例1中的短肽荧光探针孵育4h，然后使用Olmpus倒置荧光显微镜成像，激光器波长488nm。
图3为短肽荧光探针对乏氧组Hela细胞成像时细胞的状态图，可以发现图中细胞状态良好，反映了本发明提供的探针具有对细胞无毒性，安全性高的优点。
短肽荧光探针对Hela细胞成像的结果如图4-5所示，可以发现在乏氧组(图5)中，细胞内出现明显的荧光信号，而常氧组(图4)荧光信号不明显，充分证明了本发明提供的短肽荧光探针具有较好的细胞成像能力和靶向效果，能够实现乏氧环境下的成像。
申请人声明，本发明通过上述实施例来说明本发明的短肽荧光探针及其制备方法和应用，但本发明并不局限于上述实施例，即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
Claims (10)
1.一种短肽荧光探针，其特征在于，所述短肽荧光探针的结构如式I所示：
2.一种根据权利要求1所述的短肽荧光探针的制备方法，其特征在于，所述制备方法包括以下步骤：
(1)将树脂活化，之后与Fmoc-Cys(StBu)-OH、缩合剂、有机碱混合反应，之后加入封端试剂进行活性位点封端、脱保护，得到第一肽链；
(2)将步骤(1)得到的第一肽链依次与Fmoc-Phe-OH、Fmoc-Lys(Boc)-OH在缩合剂和有机碱条件下反应，得到第二肽链；
(3)将步骤(2)得到的第二肽链与硝基咪唑乙酸、缩合剂、有机碱混合反应，得到NIKFC(StBu)，之后将树脂切割，得到NIKFC-S肽段；
(4)将步骤(3)得到的NIKFC-S肽段与4-氯-7硝基苯并-2-氧杂-1,3-二唑反应，之后脱保护，得到所述短肽荧光探针；
所述有机碱包括DIPEA或DIC；
反应路线如下：
3.根据权利要求2所述的短肽荧光探针的制备方法，其特征在于，步骤(1)所述树脂包括2-氯三苯甲基氯树脂或王树脂中任意一种。
4.根据权利要求2或3所述的短肽荧光探针的制备方法，其特征在于，步骤(1)、步骤(2)、步骤(3)所述反应的时间独立地为0.8-1.2h。
5.根据权利要求2所述的短肽荧光探针的制备方法，其特征在于，步骤(3)所述切割包括以下步骤：将NIKFC(StBu)与三氟乙酸混合处理，之后抽滤、收集滤液，得到NIKFC-S肽段。
6.根据权利要求2所述的短肽荧光探针的制备方法，其特征在于，步骤(4)所述NIKFC-S肽段与4-氯-7硝基苯并-2-氧杂-1,3-二唑的摩尔比为(0.9-1.1):(0.9-1.1)。
7.根据权利要求2所述的短肽荧光探针的制备方法，其特征在于，步骤(4)所述反应的时间为12-24h。
8.根据权利要求2所述的短肽荧光探针的制备方法，其特征在于，步骤(3)所述切割后、步骤(4)所述反应后、步骤(4)所述脱保护后独立地还进行纯化。
9.根据权利要求2所述的短肽荧光探针的制备方法，其特征在于，所述缩合剂包括HBTU、HOBT或TBTU中任意一种。
10.一种根据权利要求1所述的短肽荧光探针在制备细胞荧光成像材料中的应用。
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