一种含聚乙二醇非离子亲水基团的新型水溶性荧光染料的合成及细胞成像

doi:10.11944/j.issn.1000-0518.2016.01.150252

摘要/Abstract

摘要：

通过向吲哚环“N”位上引入一个含聚乙二醇(PEG)醚链的非离子亲水基团,合成了一种新型水溶性不对称五甲川吲哚菁染料。用核磁(¹H NMR)和高分辨质谱(HRMS)表征染料的结构,测试了染料的光谱性能,标记牛血清白蛋白(BSA),并对固定细胞和活细胞分别染色。结果表明,该染料在水中的最大紫外吸收波长和荧光发射波长分别为648和668 nm,斯托克斯(stokes)位移为20 nm,荧光量子产率(Ф)为0.13,以碘钨灯为光源光照8 h后,染料光降解率为5.8%。用染料的NHS活性酯标记牛血清白蛋白,标示率(D/P)为1.16。对固定细胞染色发现,染料可对细胞整体着色,对细胞核染色最明显,能清晰看到核仁。对活细胞染色发现,有少量染料跨膜进入细胞内部,对细胞质和细胞核有微弱染色,但在活细胞膜上聚集明显。

关键词: 聚乙二醇醚链, 非离子亲水基团, 五甲川菁染料, 荧光, 细胞成像

Abstract:

In this work, a novel asymmetric pentamethine cyanine(Cy5) dye containing one nonionic hydrophilic group with two polyethylene glycol(PEG) ether chains on the indoline “N” was synthesized and characterized by ¹H nuclear magnetic resonance(¹H NMR) and high resolution mass spectrometer(HRMS). The spectra character and light stability of this Cy5 were investigated. The results show that the λ_ab and λ_em of the dye in water are 648 nm and 668 nm, respectively, and Stokes shifts reach 20 nm. The fluorescence quantum yield(Ф) in water is 0.13. After irradiated for 8 h, the photo-degradation of the aqueous soluble dyestuff of 5.8% happens. Albumin Bovine Serum(BSA) is labeled with this Cy5, and the dye/protein molar ratio(D/P) of fluorescent labeling reachs 1.16. By comparing fixed cells staining and viable cells staining, it is found that fixed cells can be overall stained by the new Cy5, the nucleoli of which are the most obvious and can be seen clearly, and that a small amount of the new Cy5 across the cell membrane of viable cells into the cell can stain cytoplasm and cell nucleus weakly, but a lot of the dye clumps together in the viable cell membrane.

Key words: polyethylene glycol ether chain, nonionic hydrophilic group, pentamethine cyanine, fluorescence, cell imaging

王升, 邱娜, 张鹏超, 汤昆, 张付利. 一种含聚乙二醇非离子亲水基团的新型水溶性荧光染料的合成及细胞成像[J]. 应用化学, 2016, 33(1): 32-38.

WANG Sheng, QIU Na, ZHANG Pengchao, TANG Kun, ZHANG Fuli. Synthesis and Cell markers of Novel Water-soluble Asymmetric Fluorescent Dyes with Nonionic Hydrophilic Group[J]. Chinese Journal of Applied Chemistry, 2016, 33(1): 32-38.

参考文献

[1]	Mujumbdar S R,Mujumbdar R B,Grant C M,et al.Cyanine-Labeling Reagents: Sulfobenzin-docyanine Succinimidylesters[J]. Bioconjugate Chem,1996,7(3):356-362.
[2]	Andrea L,Stadler J O,Santos D,et al.Fluorescent DNA Nanotags Featuring Covalently Attached Intercalating Dyes:Synthesis, Antibody Conjugation, and Intracellular Imaging[J]. Bioconjugate Chem,2011,22(8):1491-1502.
[3]	Dan L,Liu S L,Liu R,et al.EphB4-Targeted Imaging with Antibody h131, h131-F(ab)2 and h131-Fab[J]. Mol Pharm,2013,10(12):4527-4533.
[4]	XIANG Decheng,LIU Heng,MENG Qinghua,et al.Synthesis of a Carbocyanine Type Anti-photobleaching pH Fluorescence Probe and the Application in Cell Imaging[J]. Acta Chim Sin,2013,71(10):1435-1440 (in Chinese). 向德成,刘恒,孟庆华,等. 一种耐光漂白的碳菁型pH荧光探针的合成与细胞成像[J]. 化学学报,2013,71(10):1435-1440.
[5]	Li D,Liu S L,Liu R,et al.Targeting the EphB4 Recept or for Cancer Diagnosis and Therapy Monitoring[J]. Mol Pharm,2013,10(1):329-336.
[6]	Choi K Y,Liu G,Lee S,et al.Theranostic Nanoplatforms for Simultaneous Cancer Imaging and Therapy:Current Approaches and Future Perspectives[J]. Nanoscale,2012,4(2):330-342.
[7]	Pauli J,Kai L C,Janis Berkemeyer,et al.New Fluorescent Labels with Tunable Hydrophilicity for the Rational Design of Bright Optical Probes for Molecular Imaging[J]. Bioconjugate Chem,2013,24(7):1174-1185.
[8]	Sano K,Nakajima T,Miyazaki K,et al.Short PEG-Linkers Improve the Performance of Targeted, Activatable Monoclonal Antibody-Indocyanine Green Optical Imaging Probes[J]. Bioconjugate Chem,2013,24(5):811-816.
[9]	Mujumdar R B,Ernst L A,Mujumdar S R,et al.Cyanine Dye Labeling Reagents:Sulfoindocyanine Succinimidyl Esters[J]. Bioconjugate Chem,1993,4(2):105-111.
[10]	Wang L,Peng X,Zhang R,et al.Syntheses and Spectral Properties of Fluorescent Trimethine Sulfo-3H-indocyanine Dyes[J]. Dyes Pigm,2002,54(2):107-111.
[11]	WANG Li,DAI Bin,PENG Xiaojun,et al.Novel Water-soluble Asymmetric Cy5 Dyes:Synthesis, Photo-stabilies and Fluorescent Labeling Protein[J]. Chem J Chinese Univ,2010,31(7):1381-1385(in Chinense). 王丽,戴彬,彭孝军,等. 新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记[J]. 高等学校化学学报,2010,31(7):1381-1385.
[12]	TANG Kun,QIU Na,WU Pin,et al.Novel Water-soluble Asymmetric Pentamethine Cyanine Dyes:Synthesis, Fluorescent Properties and Fluorescent Labeling[J]. Chinese J Appl Chem,2014,31(11):1256-1260(in Chinese). 汤昆,邱娜,吴品,等. 新型水溶性不对称五甲川吲哚菁染料的合成、荧光性能及荧光标记[J]. 应用化学,2014,31(11):1256-1260.
[13]	LI Chunlan,WANG Lanying,SUN Guofeng,et al.Advances in Study of the Indocyanine Dyes[J]. Chinese J Org Chem,2006,4(26):442-453(in Chinese). 李春兰,王兰英,孙国峰,等. 吲哚菁染料的研究进展[J]. 有机化学,2006,4(26):442-453.
[14]	Toutchkine A,Nalbant P,Hahn K M. Facile Synthesis of Thiol-Reactive Cy3 and Cy5 Derivatives with Enhanced Water Solubility[J]. Bioconjugate Chem,2002,13(3):387-391.
[15]	Patonay G,Salon J,Sowell J,Strekowski L. Noncovalent Labeling of Biomolecules with Red and Near-infrared Dyes[J]. Molecules,2004,9(3):40-49.
[16]	Strekowski L,Lipowska M,Patonay G. Facile Derivatizations of Heptamethine Cyanine Dyes[J]. Synth Commun,1992,22(17):259-2598.
[17]	Zaheer A,Lenkinskl R E,Mahmood A,et al.In Vivo Near-infrared Fluorescence Imaging of Osteoblastic Activity[J]. Nat Biotechnol,2001,19(2):1148-1154.
[18]	SHEN Beifen,CHEN Zhinan,LIU Minpei. Recombinant Antibody[M]. Beijing:Science Press,2005:440-441(in Chinese). 沈倍奋,陈志南,刘民培. 重组抗体[M]. 北京:科学出版社. 2005:440-441.