基于异氟尔酮的L-精氨酸探针光谱性能研究

L-精氨酸（L-Arginine，L-Arg）是蛋白质的重要组成部分，也是人类某些疾病的重要诊断标准之一，L-Arg的浓度变化可能会导致许多疾病的问题，因此如何高效且灵敏地检测L-Arg极其重要。目前，对L-Arg的研究大多数是利用其作为NO（一氧化氮）的前体，来预防或缓解某些疾病的产生，对于L-Arg的定性检测并不多，而利用质子转移形成络合物/加成物的方式检测L-Arg更是少之又少。基于异氟尔酮设计并合成了一种含有丙二腈以及2,4-二羟基苯甲醛的比色探针(ISO-CN-OH)，该探针可以用于快速检测L-Arg。通过紫外吸收光谱研究发现，当向探针ISO-CN-OH溶液中加入L-Arg后，其紫外吸收光谱在669 nm处的吸收峰急剧增强，且溶液的颜色由橙黄色变为墨绿色，而其他氨基酸的加入不产生吸收峰以及溶液颜色的变化。通过对探针ISO-CN-OH进行抗干扰性实验，表明探针ISO-CN-OH对L-Arg具有专一选择性及良好的抗干扰能力。通过对L-Arg进行光谱滴定研究，实验结果表明在1.0～10.0×10-6 mol·L-1浓度范围内，具有良好的线性关系（R2=0.997），线性归方程式为y=0.020x+0.073，根据DL=3σ/K可计算出ISO-CN-OH的检出限为8.5×10-8 mol·L-1，这表明探针ISO-CN-OH具有高灵敏性。通过固定探针ISO-CN-OH与L-Arg的总浓度为100 μmol·L-1，改变L-Arg占总浓度的比例，根据其吸收光谱的Job’s plot滴定曲线分析可知，当L-Arg占总浓度比例为0.67时，探针ISO-CN-OH在669 nm处紫外吸收强度达到最大值，这表明探针ISO-CN-OH与L-Arg以1∶2的络合比进行配位络合。为了更进一步了解探针ISO-CN-OH与L-Arg的配位机理，进行了1H-NMR滴定实验，在探针ISO-CN-OH的DMSO-d₆溶液中分别加入0，0.5，1.0及2.0当量的L-Arg（d₂O），研究发现加入L-Arg后ISO-CN-OH的羟基峰消失，且其羟基周围氢质子共振峰的位置明显发生了偏移。该研究结果表明探针ISO-CN-OH通过将酸性的酚羟基质子转移到L-Arg碱性的胍基NH基团上，导致-OH基团附近形成负电荷，该负电荷与精氨酸的胍基部分络合，从而形成络合物/加成物，使得紫外可见光谱图在669 nm处出现新峰，且溶液颜色由橙黄色变为墨绿色。该研究为今后设计检测L-Arg探针分子奠定了基础。

Spectral Properties of L-Arginine Probe Based on Isoflurone

L-Arginine (L-Arg) is an important component of protein and one of the important diagnostic criteria for certain diseases in humans. The concentration change of L-Arg may cause many health problems. Therefore, it is very important to detect L-Arg efficiently and sensitively. At present, most research work based on L-Arg is used as the precursor of NO (Nitric Oxide) to prevent or alleviate some diseases. It are rarely reported for qualitative tests of L-Arg, and the detection of L-Arg by proton transfer to form complex/adduct is even less. In this paper, a colorimetric probe ISO-CN-OH based on isophorone, malononitrile and 2,4-dihydroxybenzaldehyde was designed and synthesized. The method based on proton transfer forming complex/adduct for detection of L-Arg rapidly were found. The UV-Vis spectra showed that absorption peak of the probe at 669 nm increased sharply when L-Arg was added into the ISO-CN-OH and the color of the solution changed from orange yellow to dark green. However, no color and absorption peak change was observed while other amino acids were added. Besides, ISO-CN-OH could detect L-Arg specifically without any interference by competition experiments. What’s more, the titration experiment of L-Arg, showed There is A good linear relationship (R2=0.997) between the relative absorption intensity (A-A₀) and the concentration of L-Arg within the concentration range of 1.0～10.0×10-6 mol·L-1. And the linear regression model wasy=0.020x+0.073. According toDL=3σ/K, the detection limit of ISO-CN-OH was 8.5×10-8 mol·L-1, which indicated that the probe had very high detection sensitivity. Fixing the total concentration of ISO-CN-OH and L-Arg was 100 μmol·L-1, and the ratio of L-Arg to the total concentration is changed to get the job’s plot titration curve. According to the job’s plot titration curve analysis, it is found that the UV absorption intensity of ISO-CN-OH reached the maximum at 669 nm when the ratio of L-Arg to the total concentration was 0.67, which indicated that ISO-CN-OH coordinated with L-Arg in the ratio of 1∶2. In order to further understand the coordination mechanism of ISO-CN-OH and L-Arg, 1H-NMR titration experiment was carried out. 0, 0.5, 1.0 and 2.0 equivalent of L-Arg (d₂O) were added into the DMSO-d₆ solution of ISO-CN-OH respectively. It was found that the hydroxyl peak of ISO-CN-OH disappeared and the hydrogen around the hydroxyl group shifted after adding L-Arg. The results showed that ISO-CN-OH causes the formation of negative charges near the -OH group by transferring acidic phenolic hydroxyl protons to l-ArG alkaline guanidine NH group. The forming negative charge complexed with the guanidine part of arginine to form a complex/admixture, which result in a new peak at 669 nm, and the color solution change. The study based on proton transfer forming complex/adduct for detection of L-Arg will provise certain guidelines for the design of L-Arg probe molecules in the future.