Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
J Am Chem Soc. 2012 Oct 10;134(40):16773-80. doi: 10.1021/ja307215z. Epub 2012 Sep 28.
Tyrosine-derived hydroperoxides are formed in peptides and proteins exposed to enzymatic or cellular sources of superoxide and oxidizing species as a result of the nearly diffusion-limited reaction between tyrosyl radical and superoxide. However, the structure of these products, which informs their reactivity in biology, has not been unequivocally established. We report here the complete characterization of the products formed in the addition of superoxide, generated from xanthine oxidase, to several peptide-derived tyrosyl radicals, formed from horseradish peroxidase. RP-HPLC, LC-MS, and NMR experiments indicate that the primary stable products of superoxide addition to tyrosyl radical are para-hydroperoxide derivatives (para relative to the position of the OH in tyrosine) that can be reduced to the corresponding para-alcohol. In the case of glycyl-tyrosine, a stable 3-(1-hydroperoxy-4-oxocyclohexa-2,5-dien-1-yl)-L-alanine was formed. In tyrosyl-glycine and Leu-enkephalin, which have N-terminal tyrosines, bicyclic indolic para-hydroperoxide derivatives were formed ((2S,3aR,7aR)-3a-hydroperoxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid) by the conjugate addition of the free amine to the cyclohexadienone. It was also found that significant amounts of the para-OH derivative were generated from the hydroxyl radical, formed on exposure of tyrosine-containing peptides to Fenton conditions. The para-OOH and para-OH derivatives are much more reactive than other tyrosine oxidation products and may play important roles in physiology and disease.
酪氨酸衍生的过氧化物是在暴露于超氧化物和氧化物质的酶或细胞来源的肽和蛋白质中形成的，这是由于酪氨酸自由基和超氧化物之间的近乎扩散限制反应。然而，这些产物的结构，它们在生物学中的反应性信息，尚未得到明确的建立。我们在这里报告了超氧化物（由黄嘌呤氧化酶产生）与几种肽衍生的酪氨酸自由基加成形成的产物的完全特征，这些自由基是由辣根过氧化物酶形成的。反相高效液相色谱、液质联用和 NMR 实验表明，超氧化物加成到酪氨酸自由基的主要稳定产物是对位过氧化物衍生物（相对于酪氨酸中 OH 的位置），可以还原为相应的对位醇。在甘氨酰酪氨酸的情况下，形成了稳定的 3-(1-过氧-4-氧代环己-2,5-二烯-1-基）-L-丙氨酸。在具有 N-末端酪氨酸的酪氨酸-甘氨酸和亮氨酸脑啡肽中，通过环己二烯酮的游离胺的共轭加成，形成了双环吲哚对位过氧化物衍生物（（2S,3aR,7aR）-3a-过氧-6-氧代-2,3,3a,6,7,7a-六氢-1H-吲哚-2-羧酸）。还发现，在含酪氨酸的肽暴露于芬顿条件下形成羟基自由基时，会产生大量的对位-OH 衍生物。对位-OOH 和对位-OH 衍生物比其他酪氨酸氧化产物更具反应性，可能在生理学和疾病中发挥重要作用。
