藉由光氧化反應快速合成含有犬尿胺酸和 N-甲醯犬尿胺酸的胜肽

Rapid synthesis of peptides containing kynurenine and N- formylkynurenine residues by dye-sensitized photooxidation

陳韻嬛(Yun-Hsuan Chen)

指導教授 ： 戴桓青

國立臺灣大學/理學院/化學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802049

摘要

細胞呼吸時會不斷產生對細胞成分造成氧化損傷的活性氧化物質。活性氧化物質造成的蛋白質氧化與老化及神經退化疾病相關。在不同的活性氧化物質中，雖然單線態氧的反應性較溫和但可優先在胺基酸側鏈中氧化色胺酸。氧化性的後轉譯修飾可將含色胺酸的殘基轉變成 N-甲醯犬尿胺酸和犬尿胺酸。儘管這種後轉譯修飾在細菌和哺乳動物細胞中已經被報導過，但它們的生理和病理作用尚未詳細研究。缺乏有效的化學合成方法一直是該研究領域的主要障礙。
在文獻中含犬尿胺酸多肽的合成路徑，使用了犬尿胺酸單體進行固相胜肽合成，或在樹脂上進行臭氧分解以將色胺酸轉化為犬尿胺酸。然而，這些程序太繁瑣且需要化學合成方面的技術。在此我們闡發了一個簡單的一鍋化反應，利用孟加拉玫瑰紅為光敏劑產生單線態氧，在胜肽鏈上將色胺酸殘基轉化為犬尿胺酸和 N-甲醯犬尿胺酸殘基。我們測試了三種模型多肽，並且都觀測到色胺酸首先轉化為 N-甲醯犬尿胺酸。接著 N-甲醯犬尿胺酸可以通過鹼化或酸化的處理條件轉化為犬尿胺酸。我們以毫克的規模獲得了含有 100%犬尿胺酸和 N-甲醯犬尿胺酸修飾的胜肽。其可作為電噴灑串聯式質譜的標準品，分子標記物或免疫試劑。
該合成方法仍受到胜肽的結構和其他胺基酸的組成的限制，但對於某些胜肽序列其仍然是有效的方法。我們的目標進一步研究犬尿胺酸和 N-甲醯犬尿胺酸在哺乳動物細胞中的作用，並分析蛋白質機制的降解。

關鍵字

N-甲醯犬尿胺酸殘基 ； 犬尿胺酸 ； 活性氧化物質 ； 光敏感劑 ； 單線態氧

並列摘要

Cellular respiration constantly generates reactive oxygen species (ROS), which cause oxidative damages to cellular components. Protein oxidation by ROS is associated with aging and age-related disorders. Among different ROS, singlet oxygen is only moderately reactive, but it preferentially oxidizes tryptophan (Trp) among amino acid side chains. This oxidative post-translational modification (PTM) transforms Trp residues into N-formylkynurenine (NFK) or kynurenine (Kyn). Although such PTMs have been reported in bacteria and mammalian cells, their physiological and pathological roles have not been studied in detail. The lack of efficient chemical synthesis strategies has been a primary obstacle in this research field.
Previous synthetic routes to obtain Kyn-containing peptides have used solid phase peptide synthesis with Kyn building blocks, or on-resin ozonolysis to convert Trp to Kyn. However, these procedures were cumbersome and required expertise in chemical synthesis. Here we describe a simple, one-pot reaction to transform a Trp residue into an NFK or Kyn residue on a peptide chain, utilizing rose bengal as photosensitizer to generate singlet oxygen. Three model peptides were tested and, in all cases, Trp was first transformed into NFK. NFK could be converted to Kyn by either basic or acidic workup conditions, depending on peptide sequence. We could therefore obtain peptides containing 100% NFK modification or 100% Kyn modification at milligram scales that may serve as LC-MS/MS standards, molecular probes, or immunization agents. This will allow many laboratories interested in NFK and Kyn modification to create their own peptide reagents.

並列關鍵字

N-formylkynurenine ； kynurenine ； reactive oxygen species ； photosensitizer ； singlet oxygen

參考文獻

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