Trityl自由基的PEG化效应及其功能化为选择性的巯基探针

发布时间：2018-05-01 01:33

本文选题：电子顺磁共振 + 三苯甲基自由基PEG化　；参考：《天津医科大学》2017年博士论文

【摘要】：目的:四硫取代三苯甲基自由基(trityl)是一类新型的稳定自由基,因其独特的性质在电子顺磁共振(EPR)波谱及成像等领域具有广泛的应用前景。该类稳定自由基具有诸多优点,如高度的生物稳定性、狭窄的EPR单线信号、高检测灵敏度和长弛豫时间(在25°C、无氧条件下的弛豫时间大于10μs)等。在过去十年,诸多努力已付诸于trityl自由基的性质优化和功能化。目前,通过对其分子结构的优化及修饰,trityl自由基实现了对细胞内/外氧气的位点特异性检测,对超氧、p H、生物体内巯基化合物以及总体氧化还原态等生理参数的检测。此外,该类自由基及其衍生物在自旋标记和高场动态核极化领域也有超乎寻常的应用。然而,trityl自由基与某些生物活性分子间的氧化还原反应、较短的血液循环时间以及与白蛋白间较强的结合能力等方面仍存不足,大大限制了它们在生物医学领域的应用。同时,trityl自由基及其衍生物对不同生理参数的检测在灵敏性和选择性方面远未实现最优化。鉴于此,该论文旨在增强trityl自由基的生物相容性和提高trityl对生物巯基化合物的检测灵敏性和特异性等方面开展工作。方法:本论文研究包括两部分:(1)有别于以前的“分散型”合成方法,本论文采用了“收敛型”方法高效合成了一系列具有不同PEG链长和链数的新型PEG化trityl自由基。随后,系统地研究了PEG化对trityl自由基的水溶性、自聚集浓度、氧气敏感性及与白蛋白结合能力等理化性质的影响,并研究了PEG化trityl自由基对对不同氧化还原物质的稳定性以及在小鼠总血和肝组织匀浆中的稳定性。最后,采用L-band EPR研究了性质优良的d-TNP3(氘代树枝状PEG2000衍生化trityl自由基)在小鼠体内的生物相容性、药代动力学以及EPR测氧方面的应用;(2)通过四步反应合成了硫甲磺酸酯trityl自由基-MTST,MTST与巯基化合物(主要是GSH和Cys)迅速反应得到不同的产物MTST-GSH和MTST-Cys,它们具有显著不同的EPR谱图。对上述产物的EPR谱图进行模拟,获得每种产物的组分以及每种组分相对应的超精细裂分常数和化学交换速率等特征EPR参数。最后利用上述EPR参数模拟MTST在生物体内的EPR谱图,特异性检测生物体内不同巯基化合物的水平。结果:(1)PEG化明显改善了trityl自由基的物理化学性质,相比于直链状类似物,树枝状PEG化trityl自由基具有更高的水溶性和自聚集浓度。氘代酯基化的衍生策略有效消除了酯基上氢原子的超精细裂分,赋予了trityl自由基狭窄的EPR单线信号和高的氧气敏感性。此外,树枝状结构的有效包裹使得PEG化trityl自由基对各类氧化还原性物质具有极高的稳定性,在小鼠总血和肝匀浆中也有高度的生物稳定性。小鼠活体研究证实d-TNP3在活体内具有较好的生物相容性和较长的动力学时间。(2)MTST与GSH和Cys的反应产物MTST-GSH和MTST-Cys具有显著不同的EPR指纹图谱,通过模拟上述EPR谱图得到的特征EPR参数为特异性检测生物体内不同巯基化合物的水平提供了有效地理论依据。结论:本研究以trityl自由基为底物,对trityl自由基进行了树枝状PEG化修饰,获得了EPR性质极佳、氧气敏感性较高、生物相容性和稳定性显著提高的PEG化trityl自由基,实现了小鼠大腿肌肉中氧气浓度的测定,在活体无创、监测氧气水平方面有较强的应用前景;对trityl自由基进行甲磺酰基修饰得到MTST,其与不同巯基化合物的反应产物显著不同EPR指纹图谱,利用上述EPR谱图所对应的特征EPR参数实现了对巯基化合物的特异性检测,开辟了生物体内巯基化合物精准检测的EPR新方法。本论文的研究结果证实分子结构修饰是增强trityl自由基的生物医学应用潜质,拓展其应用领域的有效手段,有望解决EPR波谱和成像技术所面临的诸多关键问题。
[Abstract]:Objective: four sulfur substituted three benzyl free radical (trityl) is a new class of stable free radicals. Because of its unique properties, it has a wide range of applications in the fields of electronic paramagnetic resonance (EPR) spectroscopy and imaging. This kind of stable free radical has many advantages, such as high biological stability, narrow EPR single line signal, high detection sensitivity and long length. Relaxation time (at 25 C, relaxation time under anaerobic conditions is greater than 10 s). In the past ten years, many efforts have been put into the optimization and functionalization of the properties of trityl radicals. At present, by optimizing and modifying their molecular structures, trityl free radicals have realized the specific detection of intracellular / external oxygen, and in SUPEROXYGEN, P H, in vivo The detection of physiological parameters such as sulfhydryl compounds and the overall redox state. In addition, the free radicals and their derivatives also have extraordinary applications in the field of spin labeling and high field dynamic nuclear polarization. However, the redox reaction between trityl radicals and some bioactive molecules, short blood circulation time and with albumin The strong binding ability is still inadequate, which greatly limits their application in the field of biomedicine. At the same time, the trityl free radical and its derivatives are far from optimizing the sensitivity and selectivity of different physiological parameters. In view of this, this paper aims to enhance the biocompatibility of trityl free radicals and improve the trityl reproduction. Work on the sensitivity and specificity of the detection of sulfhydryl compounds. Methods: this thesis consists of two parts: (1) different from the previous "dispersive" synthesis methods. This paper uses a "convergent" method to efficiently synthesize a series of new PEG trityl free radicals with different PEG chain length and chain number. The effects of PEG on the water solubility, self aggregation, oxygen sensitivity and binding ability to albumin of trityl radicals were studied. The stability of PEG trityl radicals to different redox substances and the stability in the total blood and liver homogenate of mice were studied. Finally, the properties of L-band EPR were studied. The biocompatibility of d-TNP3 (deuterated dendrite PEG2000 derivatization trityl radical) in mice, pharmacokinetics and the application of EPR in oxygen measurement; (2) the trityl free radical -MTST of thiorulfate was synthesized by the four step reaction, and MTST and sulfhydryl compounds (mainly GSH and Cys) were quickly reacted to obtain different products MTST-GSH and MTST -Cys, they have significant different EPR spectra. The EPR spectra of the above products are simulated, the components of each product and the EPR parameters of the hyperfine splitting constant and chemical exchange rate corresponding to each component are obtained. Finally, the EPR parameters of the above EPR parameters are used to simulate the EPR spectrum of MTST in the organism, and the specificity is used to detect the differences in the organism. Results: (1) the physicochemical properties of the trityl radical are obviously improved by PEG, and the dendritic PEG trityl free radical has higher water solubility and self aggregation than the direct chain like analogues. The deuterated derivative strategy effectively eliminates the hyperfine splitting of the hydrogen atom on the ester group and gives trityl freedom. Based on the narrow EPR single line signal and high oxygen sensitivity, the effective encapsulation of the dendritic structure makes the PEG trityl free radical highly stable to all kinds of redox substances, and also has high biological stability in the total blood and liver homogenate of mice. The study of mouse in vivo has proved that d-TNP3 has better biological phase in living body. Capacitive and long kinetic time. (2) the reaction products of MTST and GSH and Cys, MTST-GSH and MTST-Cys, have significant different EPR fingerprints. The characteristic EPR parameters obtained by simulating the above EPR spectra provide an effective theoretical basis for the specific detection of different levels of sulfhydryl compounds in organisms. Conclusion: This study is based on trityl free freedom. Based on the substrate, the trityl free radical was modified by dendritic PEG. The PEG trityl free radical with excellent EPR properties, high oxygen sensitivity and significant improvement in biocompatibility and stability was obtained. The determination of oxygen concentration in the mouse thigh muscles was achieved. It has a strong application prospect in living body noninvasive and monitoring oxygen level; trityl The free radical was modified by methsulfonyl group to obtain MTST, and its reaction products with different sulfhydryl compounds significantly different EPR fingerprints. The specific detection of sulfhydryl compounds was realized by the characteristic EPR parameters corresponding to the EPR spectra above, and a new method for the accurate detection of sulfhydryl compounds in living organisms was opened up. The results of this paper have been proved in this paper. Real molecular structure modification is an effective means to enhance the biomedical applications of trityl free radicals and expand its application field. It is expected to solve many key problems in EPR spectroscopy and imaging technology.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R963;O652

【相似文献】