【作者】 王彬；

【导师】 吴玉清；

【作者基本信息】 吉林大学 ， 物理化学， 2011， 硕士

【摘要】 氨基酸具有较好的水溶性并且含有能与金属离子作用的活性基团,而形成多肽之后的结构更加丰富多彩。小肽具有更好的水溶性并且可以通过改变氨基酸残基的种类改变小肽的选择性和结合能力;小肽具有更多的结合点,能同时与不同的金属离子结合而具有设计成逻辑开关的潜力。本论文以丹磺酰基为荧光发色团,小肽做为识别基团,设计合成了几种丹酰拟肽类荧光探针。在水溶液中利用荧光滴定实验检测了它们对金属离子的识别特性,并利用多种表征方法对相关的识别机理做了初步研究。同时,构筑了拟肽类荧光探针/M²⁺复合物并初步尝试其在氨基酸检测中的应用。主要研究内容包括:1.设计、合成了丹酰甘酰色氨酸（DGT, 1）,丹酰甘酰甘酰色氨酸（DGGT, 2）,丹酰甘酰甘氨酸（DGG, 3）及丹酰甘酰谷氨酸（DGE, 4）四种丹酰肽类荧光离子探针,并检测了它们对金属离子的荧光响应。Hg²⁺能够诱导荧光探针分子1和2荧光蓝移而Cu²⁺却能猝灭它们的荧光;同时,Hg²⁺和Cu²⁺能够同时猝灭荧光分子3和4的荧光。根据上述荧光分子能够同时对Hg²⁺和Cu²⁺产生荧光响应的特性,我们将其应用到逻辑开关中。2.根据Cu²⁺能够猝灭丹酰拟肽类荧光探针荧光的现象,我们构筑了丹酰拟肽类荧光探针/Cu²⁺复合物并加以改进。利用氨基酸及蛋白质能够与Cu²⁺竞争结合的原理,成功地将“On-Off”型Cu²⁺荧光探针转化成“Off-On”型的氨基酸及蛋白质的检测试剂。同时,我们利用紫外-可见吸收光谱、荧光光谱、NMR、FT-IR、EPR、2D NOESY等表征方法对荧光探针分子对Hg²⁺和Cu²⁺的识别机理及结合模式进行了探究,并对其构效关系做了初步揭示,这些研究为设计多功能的荧光探针提供了条件,奠定了基础。更多还原

【Abstract】 The design and synthesis of optical probes with high selectivity and sensitivity to heavy transition-metal ions （HTMs）, particularly mercury, lead, and copper ions, have attracted special attention due to the toxicity of these ions, both in physiological systems and in the environment. In the previous studies, the concept that introducing amino acids as binding site of probe have been proved successful. Highly sensitive fluorescence probes derived from pyrene butyric acid for Pb²⁺ and dansyl moiety for Hg²⁺have been achieved, which displayed rapid and specific optical responses to target metal ion in buffered aqueous solution. In the present study, instead of using single amino acid, the motif of peptide will be introduced to design fluorescence probe for metal ions. By tuning the number and sequence of participant amino acids several attractive features of probe will be achieved as: （i） felicitous hydrophilicity and hydrophobicity, in keeping the balance between water-solubitily and cell permeability; （ii） easily optimized high selectivity and sensitivity to different metal ions; and essentially （iii） posessing more binding site in comparison to a single amino acid, which supply a possibility to be used as a logic gate or molecular switch to different kinds of metal ions. Although some probes based on peptide motif had been developed for metal ions, they could not response to different metal ions simultaneously. In the present study, a a series of dansyl labeled peptides, were synthesized, in possessing of multiple binding sites that would confer the potential to be used as a logic gate or molecular switch for different kinds of metal ions.Firstly, dansly-Gly-Trp （DGT, 1）, dansly-Gly-Gly-Trp （DGGT, 2）, dansyl-Gly-Gly （DGG, 3）, dansyl-Gly-Glu （DGE, 4） were synthesized as efficient fluorescence probe for metal ions. The probe 1 could respond to Hg²⁺ with enhanced and blue-shifted fluorescence emission but to Cu²⁺ with obvious fluorescence quenching. The combination of these intrinsic properties with the selective responses to different chemical inputs allows this system to be implemented as an ionic switch. The underlying mechanisms of the probe to different kind of metal ion were explored successfully by using either 1H NMR, NOESY, electron paramagnetic resonance （EPR） or FT-IR spectra. In addition, the chemical shifts of H in indole ring demonstrated that the indole ring may have a cooperative effect on Hg²⁺ complexation. The NOESY spectrum showed that the indole ring and methylene were spatially close to naphthalene. Electron paramagnetic resonance （EPR） spectra indicated a strong interaction between CuCl2 and probe 1. A multitude of obvious spectral features (hyperfine splittings of the 14N nuclear spins of the peptide with the unpaired electron spin of Cu²⁺) appeared at 325-350 mT, proving that a complex formed between Cu²⁺ and 1. In addition, to investigate the binding model of 1/Hg²⁺ and 1/Cu²⁺, simulations were also performed by using density functional theory （DFT） and reasonable binding configurations were achieved for these two complexes.In the previous work, we have developed a tetrafunctional probe based on peptide and dansyl motif, which responded to Hg²⁺ and Cu²⁺ in distinct modes with distinguishing optical properties. After the fluorescence was quenched by Cu²⁺, further addition of histidine or other amino acids could almost fully recover the fluorescence of probe. The results demonstrated that the general amino acid can snatch the Cu²⁺ from the reported probe/Cu²⁺ complex, which supplied a potential application of ligand in the detection of amino acid. Although we have improved the water-solubility of probe at some extend, the selectivity to amino acids was still poor. Almost all the amino acids can snatch Cu²⁺ in the presence of probe because the stronger binding affinity between Cu²⁺ and amino acids. Therefore, further efforts would be paid to improve the binding force between Cu²⁺ and probe by optimizing the structure. As the ATCUN sequence has been proved to be the most strong Cu²⁺ binding structure, the tryptophan was firstly employed to simulate the Cu²⁺ coordination structure. The previous dipeptide sensor, DGT, containing the indole ring which was expected to form loop structure with Cu²⁺ through sulfonamide, amide and the nitrogen of indole ring, although actually the indole ring did not. In fact, generally the pyrrole-type nitrogen of indole could not chelate Cu²⁺ so efficiently as the pyridine-type nitrogen in imidazole. Such limitation inspired us to design a new fluorescence probe, Dansyl-Gly-Py （5）, for Cu²⁺ by introducing the amino methyl pyridine by mimicing the ATCUN structure more closely. To do so, we hoped the binding affinity of the new peptide probe to Cu²⁺ could be improved, and finally the selectivity of probe/Cu²⁺ complex to amino acids could be improved significantly. the fluorescence emission of 5/Cu²⁺ could be released strongly toward the addition of histidine or cysteine, but no such extensive response to other amino acids. For the fluorescence recovery induced by histidine, we proposed that the stronger binding affinity bewteen 5 and Cu²⁺ exceeded the binding between 5 and most of amino acids, which resulted in only histidine could snatch Cu²⁺ from the 5/Cu²⁺ complex while the other amino acids were faint. Such an obvious fluorescence response of 5/Cu²⁺ to histidine illustrated the high selectivity of it to different amino acids. 5/Cu²⁺ could be further used to distinguish histidine-rich biomolecules, such as histidine-tagged proteins.In conclusion, we synthesized a series of probe based on a dipeptide motif and a dansyl moiety, which responded to Hg²⁺ and Cu²⁺. The concepts presented here will contribute to the future development of miniaturized molecular probes with multi-functions. In additon, we have developed a new luminesce switch-on probe/Cu²⁺ complex, which could be used to distinguishing histidine and cysteine from other amino acids, and further to detecting proteins with and without His-tags either in buffer solution or in standard foetal bovine serum solution, indicating that probe/Cu²⁺ had potential application in real sample.更多还原