磷酰化二羟基蒽醌的合成及其与牛血清白蛋白的弱相互作用研究
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摘要
羟基(9，10-)蒽醌具有抗癌、抗肿瘤、抑制生物体中由于过氧化反应导致的多种疾病的作用；而许多药物分子在磷酰化后具备了更多独特的生物活性和药理特性。因此磷酰化羟基蒽醌的合成和从分子水平上研究其与生物大分子之间的相互作用，对药物开发与研制具有重要意义。
本文利用经改造并较简化的Atheron-Todd合成法，分别以亚磷酸二乙酯和亚磷酸二异丙酯为磷酰化试剂，成功对1，2-二羟基蒽醌、2，6-二羟基蒽醌、1，8-二羟基蒽醌和1，4-二羟基蒽醌进行了磷酰化改造；合成了九个未见文献报道的羟基蒽醌类的磷酰酯类衍生物。其合成路线如下图：
所得化合物结构均经核磁共振、红外和电喷雾质谱确证。对四个二羟基蒽醌中不同取代位置的羟基的反应活性进行对比，发现β-羟基的反应活性高于α-羟基的反应活性，量子化学计算其电荷分布也得到了同样的结论。
本文通过荧光分析法研究了磷酰化羟基蒽醌与牛血清白蛋白(BSA：Bovine serum albumin)之间的弱相互作用，并用荧光猝灭原理计算了它们之间的结合常数，作用力类型。结果证明，磷酰化羟基蒽醌与牛血清白蛋白发生了弱相互作用，它们之间的结合常数均为10~5，有1个结合位点，作用力类型属于氢键或范德华力。
9,10- anthraquinones multi hydroxy derivatives have anticancer function and can inhibit multifarious diseases in organism caused by oxidation reactions. Many medical molecules have many unique activities and characteristics after being phosphorylated. It is very important to synthesis new phosphoryl dihydroxy-anthraquinones and research interaction detween them and biological macromoleculer for exploration of new drug.
In this thesis,by Atheron-Todd reaction ,using diethyl phosphate and diisopropyl phosphate as phosphorylate reagents,we successfully synthesized a series of new dihydroxy-anthraquinones phosphate derivatives. The synthesis route was shown as following scheme. Their structures were confirmed by NMR, IR and ESI-MS. It was found that the reaction activities ofβ-hydroxyl multi- anthraquinones is much higher than those ofα-hydroxyl, which have been further confirmed by charge distributing analysis.
The interactions of phosphoryly dihydroxy anthraquinone and BSA was confirmed by Fluorescence Analysis, which coubld be further used as a a powerful tool for screening numerous small molecule-protein complexes, due to its rapidity of data acquisition and the relative simplicity of interpretation. The results show that the binding constants between them are all about 10~5, and the number of bind site is 1 respectively, and the type of applied force between them is Van-der Waals Force and hydrogen bond.
本文利用经改造并较简化的Atheron-Todd合成法，分别以亚磷酸二乙酯和亚磷酸二异丙酯为磷酰化试剂，成功对1，2-二羟基蒽醌、2，6-二羟基蒽醌、1，8-二羟基蒽醌和1，4-二羟基蒽醌进行了磷酰化改造；合成了九个未见文献报道的羟基蒽醌类的磷酰酯类衍生物。其合成路线如下图：
所得化合物结构均经核磁共振、红外和电喷雾质谱确证。对四个二羟基蒽醌中不同取代位置的羟基的反应活性进行对比，发现β-羟基的反应活性高于α-羟基的反应活性，量子化学计算其电荷分布也得到了同样的结论。
本文通过荧光分析法研究了磷酰化羟基蒽醌与牛血清白蛋白(BSA：Bovine serum albumin)之间的弱相互作用，并用荧光猝灭原理计算了它们之间的结合常数，作用力类型。结果证明，磷酰化羟基蒽醌与牛血清白蛋白发生了弱相互作用，它们之间的结合常数均为10~5，有1个结合位点，作用力类型属于氢键或范德华力。
9,10- anthraquinones multi hydroxy derivatives have anticancer function and can inhibit multifarious diseases in organism caused by oxidation reactions. Many medical molecules have many unique activities and characteristics after being phosphorylated. It is very important to synthesis new phosphoryl dihydroxy-anthraquinones and research interaction detween them and biological macromoleculer for exploration of new drug.
In this thesis,by Atheron-Todd reaction ,using diethyl phosphate and diisopropyl phosphate as phosphorylate reagents,we successfully synthesized a series of new dihydroxy-anthraquinones phosphate derivatives. The synthesis route was shown as following scheme. Their structures were confirmed by NMR, IR and ESI-MS. It was found that the reaction activities ofβ-hydroxyl multi- anthraquinones is much higher than those ofα-hydroxyl, which have been further confirmed by charge distributing analysis.
The interactions of phosphoryly dihydroxy anthraquinone and BSA was confirmed by Fluorescence Analysis, which coubld be further used as a a powerful tool for screening numerous small molecule-protein complexes, due to its rapidity of data acquisition and the relative simplicity of interpretation. The results show that the binding constants between them are all about 10~5, and the number of bind site is 1 respectively, and the type of applied force between them is Van-der Waals Force and hydrogen bond.
引文
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11. F. R. Atherton, H. T. Openshaw, A. R. Todd. Phosphorylation. Ⅱ. Reaction of dialkyl phosphites with polyhalogen compounds in presence of bases. New method for the phosphorylation of amines [J], J. Chem Soc., 1945, 382.
12. F. R. Atherton, H. T Howard. A. R. Todd. Phosphorylation. Ⅳ. Further studies on the use of dibenzyl chlorophosphonate and the examination of certain alternative phosphorylation methods[J], J Chem Soc, 1948, 1106.
13．R．Y Chen (陈茹玉)，L．Z．Liu (刘纶祖)．Study on Organophosphorus Chemistry(有机磷化学研究)[M]．Beijing：High Education Press，2001，49
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28. Fujii, N. Induction of topoisomerase Ⅱ-mediated DNA cleavage by the plant naphthoquinones plumbagin and shikonin[J]. Antimicrob. Agents Chemother. 1992, 36, 2589-2594.
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2. L. Todd, "Where there's Life, There's Phosphorus" [M], ( K. Makoto, N, Keiko, O. Tairo, eds). Tokyo, Japan, Sci. Soc. Press, 1981, 275-279.
3. L. Todd, "Science and Scientists"[M], (Kagegama, M. eds), Scientific Soc. Press, Tokyo, Japan 1981, 275.
4. O. M. Rosen, E. G. Krebs. Protein Phosphorylation, Papers from a conference[M], Cold Spring Harbor Laboratory, Cold Spring Harbor, 1981, New York.
5. G. Thomas, E. J. Podesta, J. E. Gordon. Protein Phosphorylation and Bio-regulation[M], 1980, S. Karyer. Baei.
6. Hunter T., Tyrosine phosphorylation: past, present and future [J], Biochem. Soc. Trans. 1996, 24, 307-327
7. Fischer E. H., Cell signaling by protein tyrosine phosphorylation[J] Advances in Enzyme Regulation, 1999, 39 (1), 359-369.
8. Clemens M. J., Protein phosphorylation in cell growth regulation[M], published by harwood academic publishers GmbH, 1996, pp1-13.
9．陈晓岚，博士毕业论文[C]，郑州大学化学系，河南郑州，2005．05．10．
10. Athertion FR, Dpenshaw HT, Todd AR. Studies on phosphorylation, Part Ⅰ dibenzyl chlorophosponate as a phosphorlating agent [J]. Chem Soc, 1945: 382-385.
11. F. R. Atherton, H. T. Openshaw, A. R. Todd. Phosphorylation. Ⅱ. Reaction of dialkyl phosphites with polyhalogen compounds in presence of bases. New method for the phosphorylation of amines [J], J. Chem Soc., 1945, 382.
12. F. R. Atherton, H. T Howard. A. R. Todd. Phosphorylation. Ⅳ. Further studies on the use of dibenzyl chlorophosphonate and the examination of certain alternative phosphorylation methods[J], J Chem Soc, 1948, 1106.
13．R．Y Chen (陈茹玉)，L．Z．Liu (刘纶祖)．Study on Organophosphorus Chemistry(有机磷化学研究)[M]．Beijing：High Education Press，2001，49
14．Z．H．Li (李正化)．Pharmaceutical Chemistry (药物化学)．[M]．Beijing：People's Health Press，1993，152．
15．尹应武．博士毕业论文，清华大学，1994
16. B. Oussaid, M. Soufiaoui, B. Garrigues. The Atherton-Todd reactions under sonochemical activation[J], Synthetic. Commun., 1995, 25(6), 871.
17. Moret Ed, E., M., Hilbers, H. W. in Vivo Activity and Hydrophobicity of Cytostatic Aziridinylquinones[J]. J. Med. Chem. 1996. 39 (3): 720
18．李伟，醋酸镁比色法测定首乌丸中1，8-二羟基蒽醌的含量[J]，广西中医学院学报，2003，6(4)：49-50．
19．丁玉玲．大黄蒽醌类的研究概况[J]，时珍国医国药，2005，3：11-11．
20．马永涛，玄延花，曹东铉．大黄醇提液抗柯萨奇B3病毒的初步研究[J]，中国中医药科技，2001，8(5)：308-309．
21．刘冠贤，叶任高，谭志明．大黄素对狼疮性肾炎成纤维细胞生物学行为的影响[J]．中国中西医结合杂志，2000，20(3)：196-198．
22．陈高翔，屈遂林，方勤．大黄素对人成纤维细胞产生纤溶酶原激活物抑制剂的影响[J]．交通医学，2000，14(6)：576-580．
23．江苏新医学院．中药大辞典[M]．上海：上海人民出版社，1977．
24. Moore, H. W. Bioactivation as a model for drug design bioreductive alkylation[J], Science 1977, 197, 527-532.
25. Johnson, M. G.; Kiyokawa, H. Bioorg. Antitumor agents-CLXVII. Synthesis and structure-activity correlations of the cytotoxic anthraquinone 1, 4-bis-(2, 3-epoxypropylamino)-9, 10-anthracenedione, and of related compounds[J], Med. Chem. 1997, 58, 1469-1479.
26. Miller, M. G. Mechanisms of toxicity of naphthoquinones to isolated hepatocytes[J], Biochem. Pharmacol. 1986, 35, 1177-1184.
27. Moore, H. W. Science 1977, 197, 527.
28. Fujii, N. Induction of topoisomerase Ⅱ-mediated DNA cleavage by the plant naphthoquinones plumbagin and shikonin[J]. Antimicrob. Agents Chemother. 1992, 36, 2589-2594.
29．李秀才，大黄的研究进展[J]，中国药学杂志，1998，33(10)：581-584
30．大蒲谚吉 大黄的药理药效 现代东洋医学，1991；12：87
31．王素贤，苏秀玲，乔亚芳，等．茜草蒽醌类成分的研究[J]．药学学报，1992，27(10)：734．
32．张海容，郭祀远，李琳等．荧光法研究帕沙星与白蛋白的作用[J]．光谱学与光谱分析[J]2001，21 (6)：829
33. Mcmenamy R H. Albumin Structure, Function and Uses[M]. Oxford; Dergamon Press, 1977: 143
34．徐景达，药物结构与活性的关系，北京，人民卫生出版社，1987：174．
35. Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy[M], Plenum Press, New York. 1983.
36．陈新谦，金有豫，新编药物学，北京，人民药物出版社，第十四版，1997
37. E. Eisenstein, G L. Gilliland, O. Herzberg, J. Moult, J. Orban, R. J. Poljak, L. Banerjei, D. Richardson, A. J. Howard. Biological function made crystal clearannotation of hypothetical proteins via structural genomics[J], Curr. Opin. Biotechnol. 2000, 11, 25-30.
38. A. Pandey, M. Mann. Proteomics to study genes and genomes Nature, 2000, 405, 837-846.
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