本文選題：電子順磁共振 + 三苯甲基自由基PEG化　； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的:四硫取代三苯甲基自由基(trityl)是一類(lèi)新型的穩(wěn)定自由基,因其獨(dú)特的性質(zhì)在電子順磁共振(EPR)波譜及成像等領(lǐng)域具有廣泛的應(yīng)用前景。該類(lèi)穩(wěn)定自由基具有諸多優(yōu)點(diǎn),如高度的生物穩(wěn)定性、狹窄的EPR單線信號(hào)、高檢測(cè)靈敏度和長(zhǎng)弛豫時(shí)間(在25°C、無(wú)氧條件下的弛豫時(shí)間大于10μs)等。在過(guò)去十年,諸多努力已付諸于trityl自由基的性質(zhì)優(yōu)化和功能化。目前,通過(guò)對(duì)其分子結(jié)構(gòu)的優(yōu)化及修飾,trityl自由基實(shí)現(xiàn)了對(duì)細(xì)胞內(nèi)/外氧氣的位點(diǎn)特異性檢測(cè),對(duì)超氧、p H、生物體內(nèi)巰基化合物以及總體氧化還原態(tài)等生理參數(shù)的檢測(cè)。此外,該類(lèi)自由基及其衍生物在自旋標(biāo)記和高場(chǎng)動(dòng)態(tài)核極化領(lǐng)域也有超乎尋常的應(yīng)用。然而,trityl自由基與某些生物活性分子間的氧化還原反應(yīng)、較短的血液循環(huán)時(shí)間以及與白蛋白間較強(qiáng)的結(jié)合能力等方面仍存不足,大大限制了它們?cè)谏镝t(yī)學(xué)領(lǐng)域的應(yīng)用。同時(shí),trityl自由基及其衍生物對(duì)不同生理參數(shù)的檢測(cè)在靈敏性和選擇性方面遠(yuǎn)未實(shí)現(xiàn)最優(yōu)化。鑒于此,該論文旨在增強(qiáng)trityl自由基的生物相容性和提高trityl對(duì)生物巰基化合物的檢測(cè)靈敏性和特異性等方面開(kāi)展工作。方法:本論文研究包括兩部分:(1)有別于以前的“分散型”合成方法,本論文采用了“收斂型”方法高效合成了一系列具有不同PEG鏈長(zhǎng)和鏈數(shù)的新型PEG化trityl自由基。隨后,系統(tǒng)地研究了PEG化對(duì)trityl自由基的水溶性、自聚集濃度、氧氣敏感性及與白蛋白結(jié)合能力等理化性質(zhì)的影響,并研究了PEG化trityl自由基對(duì)對(duì)不同氧化還原物質(zhì)的穩(wěn)定性以及在小鼠總血和肝組織勻漿中的穩(wěn)定性。最后,采用L-band EPR研究了性質(zhì)優(yōu)良的d-TNP3(氘代樹(shù)枝狀PEG2000衍生化trityl自由基)在小鼠體內(nèi)的生物相容性、藥代動(dòng)力學(xué)以及EPR測(cè)氧方面的應(yīng)用;(2)通過(guò)四步反應(yīng)合成了硫甲磺酸酯trityl自由基-MTST,MTST與巰基化合物(主要是GSH和Cys)迅速反應(yīng)得到不同的產(chǎn)物MTST-GSH和MTST-Cys,它們具有顯著不同的EPR譜圖。對(duì)上述產(chǎn)物的EPR譜圖進(jìn)行模擬,獲得每種產(chǎn)物的組分以及每種組分相對(duì)應(yīng)的超精細(xì)裂分常數(shù)和化學(xué)交換速率等特征EPR參數(shù)。最后利用上述EPR參數(shù)模擬MTST在生物體內(nèi)的EPR譜圖,特異性檢測(cè)生物體內(nèi)不同巰基化合物的水平。結(jié)果:(1)PEG化明顯改善了trityl自由基的物理化學(xué)性質(zhì),相比于直鏈狀類(lèi)似物,樹(shù)枝狀PEG化trityl自由基具有更高的水溶性和自聚集濃度。氘代酯基化的衍生策略有效消除了酯基上氫原子的超精細(xì)裂分,賦予了trityl自由基狹窄的EPR單線信號(hào)和高的氧氣敏感性。此外,樹(shù)枝狀結(jié)構(gòu)的有效包裹使得PEG化trityl自由基對(duì)各類(lèi)氧化還原性物質(zhì)具有極高的穩(wěn)定性,在小鼠總血和肝勻漿中也有高度的生物穩(wěn)定性。小鼠活體研究證實(shí)d-TNP3在活體內(nèi)具有較好的生物相容性和較長(zhǎng)的動(dòng)力學(xué)時(shí)間。(2)MTST與GSH和Cys的反應(yīng)產(chǎn)物MTST-GSH和MTST-Cys具有顯著不同的EPR指紋圖譜,通過(guò)模擬上述EPR譜圖得到的特征EPR參數(shù)為特異性檢測(cè)生物體內(nèi)不同巰基化合物的水平提供了有效地理論依據(jù)。結(jié)論:本研究以trityl自由基為底物,對(duì)trityl自由基進(jìn)行了樹(shù)枝狀PEG化修飾,獲得了EPR性質(zhì)極佳、氧氣敏感性較高、生物相容性和穩(wěn)定性顯著提高的PEG化trityl自由基,實(shí)現(xiàn)了小鼠大腿肌肉中氧氣濃度的測(cè)定,在活體無(wú)創(chuàng)、監(jiān)測(cè)氧氣水平方面有較強(qiáng)的應(yīng)用前景;對(duì)trityl自由基進(jìn)行甲磺�；揎椀玫組TST,其與不同巰基化合物的反應(yīng)產(chǎn)物顯著不同EPR指紋圖譜,利用上述EPR譜圖所對(duì)應(yīng)的特征EPR參數(shù)實(shí)現(xiàn)了對(duì)巰基化合物的特異性檢測(cè),開(kāi)辟了生物體內(nèi)巰基化合物精準(zhǔn)檢測(cè)的EPR新方法。本論文的研究結(jié)果證實(shí)分子結(jié)構(gòu)修飾是增強(qiáng)trityl自由基的生物醫(yī)學(xué)應(yīng)用潛質(zhì),拓展其應(yīng)用領(lǐng)域的有效手段,有望解決EPR波譜和成像技術(shù)所面臨的諸多關(guān)鍵問(wèn)題。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R963;O652

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