N^1-甲基鸟嘌呤阳离子脱质子动力学的研究

Deprotonation Kinetics of 1-Methylguanine After One-Electron Oxidation

在所有DNA碱基中,鸟嘌呤碱基G具有最低的氧化电位,导致其最容易被氧化.G碱基被单电子氧化成为G正离子自由基(G+?),G+?存在两个脱质子位点,其中脱嘧啶环上亚氨基质子N1-H比脱环外氨基质子N2-H更有利,因而在普通G碱基中研究脱N2-H的过程无法排除脱N1-H过程的干扰,使得其脱N2-H的动力学迄今尚不明确.在本文中,通过将G碱基上的N1-H用CH3取代(即mG),采用纳秒时间分辨瞬态紫外可见吸收光谱方法研究了mG碱基单电子氧化后脱质子N2-H的动力学.根据瞬态紫外可见吸收光谱,确定了mG+?脱质子的产物是mG(N2-H)?,即脱质子的位点是N2-H.进一步通过测量mG(N2-H)?的生成速率常数与mG的浓度依赖关系,得到室温下SO4-?单电子氧化mG生成mG+?的速率常数为(3.7±0.1)×109 L?mol-1?s-1以及mG+?脱N2-H的速率常数为(7.1±0.2)×106 s-1.并通过检测不同温度下mG+?脱N2-H的速率常数,利用阿仑尼乌斯方程得出脱质子N2-H的活化能为19.9±1.0 kJ?mol-1.这些结果可为DNA碱基的氧化损伤过程提供更为丰富的动力学信息.

Among the four natural DNA bases, guanine(G) is the most sensitive to oxidation due to its lowest oxidation potential. When G base is oxidized to guanine cation radical(G+?), it will deprotonate from both the imino proton N1-H and the amino proton N2-H. According to the pKa values for N1-H and N2-H deprotonation, the main deprotonation site in G base is N1-H which would interfere with the N2-H deprotonation, making the kinetics of N2-H deprotonation difficult to be measured. Herein, the N2-H deprotonation kinetics is investigated using 1-methylguanosine(mG), where N1-H is substituted by methyl group to avoid the N1-H deprotonation and N9-H is substituted by ribose to ensure enough solubility of methylguanine in water, by nanosecond transient absorption(ns-TA) spectroscopy. By 355 nm photolysis of Na2S2O8, the highly oxidizing radical SO4-? is generated, which will oxidize mG to mG+? instantaneously. The time-resolved absorption spectra obtained for reaction of mG with SO4-? exhibits transient absorptions for mG(N2-H)? featured by absorption band at 600 nm, indicating that the mG+? deprotonation product is mG(N2-H)? and the deprotonation site is therefore validated to be N2-H. The mG concentration dependence of mG(N2-H)? formation rate constant is assessed through changing the mG concentration from 0.25 mmol?L-1 to 5 mmol?L-1. The concentration dependence experiment reveals that the rate-limiting step to form mG(N2-H)? is the bimolecular reaction of mG with SO4-? when mG concentration is lower than 2 mmol?L-1 and the bimolecular reaction rate constant to form mG+? is(3.7±0.1)×109 L?mol-1?s-1; when mG concentration is above 2 mmol?L-1, the rate-limiting step to form mG(N2-H)? is the first-order mG+? deprotonation and the N2-H deprotonation rate constant is(7.1±0.2)×106 s-1. Furthermore, the N2-H deprotonation rate constant is measured at different temperatures varying from 278 K to 298 K. According to Arrhenius equation, the activation energy barrier for the N2-H deprotonation is determined to be 19.9±1.0 kJ? mol-1. These results can provide valuable kinetic information on the oxidative damage of DNA.