Yamamoto Junpei, Tanaka Yoshiyuki, Iwai Shigenori
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
Org Biomol Chem. 2009 Jan 7;7(1):161-6. doi: 10.1039/b815458a. Epub 2008 Nov 10.
We synthesized a dinucleoside monophosphate of the (15)N-labeled (6-4) photoproduct, which is one of the major UV-induced lesions in DNA, to investigate the (6-4) photolyase repair mechanism, and characterized its protonation state by measuring (15)N NMR spectra as a function of pH. We expected that chemical-shift changes of the pyrimidone (15)N3, due to protonation, would be observed at pH 3, as observed for the (15)N-labeled 5-methylpyrimidin-2-one nucleoside. Interestingly, however, the changes were observed only in alkaline solutions. In UV absorption spectroscopy and HPLC analyses under acidic conditions, a change in the maximum absorption wavelength, due to the protonation-induced hydrolysis, was observed at and below pH 1, but not at pH 2, whereas the protonation of 5-methylpyrimidin-2-one occurred at pH values between 2 and 3. These results indicated that the pK(a) value for this N3 is remarkably lower than that of a normal pyrimidone ring, and strongly suggest that an intramolecular hydrogen bond is formed between the N3 of the 3' base and the 5-OH of the 5' base under physiological conditions. The results of this study have implications not only for the recognition and reaction mechanisms of (6-4) photolyase, but also for the chemical nature of the (6-4) photoproduct.
我们合成了一种（15）N标记的（6-4）光产物的二核苷单磷酸，（6-4）光产物是DNA中主要的紫外线诱导损伤之一，以研究（6-4）光解酶的修复机制，并通过测量作为pH函数的（15）N NMR光谱来表征其质子化状态。我们预期，由于质子化，在pH 3时会观察到嘧啶酮（15）N3的化学位移变化，这与（15）N标记的5-甲基嘧啶-2-酮核苷的情况相同。然而，有趣的是，这种变化仅在碱性溶液中观察到。在酸性条件下的紫外吸收光谱和HPLC分析中，在pH 1及以下观察到由于质子化诱导的水解导致的最大吸收波长变化，但在pH 2时未观察到，而5-甲基嘧啶-2-酮的质子化发生在pH值2至3之间。这些结果表明，该N3的pK（a）值明显低于正常嘧啶酮环的pK（a）值，并强烈表明在生理条件下，3'碱基的N3与5'碱基的5-OH之间形成了分子内氢键。本研究结果不仅对（6-4）光解酶的识别和反应机制有影响，而且对（6-4）光产物的化学性质也有影响。
