Koo Kyung A, Waisbourd-Zinman Orith, Wells Rebecca G, Pack Michael, Porter John R
Department of Biological Sciences, University of the Sciences , Philadelphia, Pennsylvania 19104, United States.
Division of Pediatric Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia , Philadelphia, Pennsylvania 19104, United States.
Chem Res Toxicol. 2016 Feb 15;29(2):142-9. doi: 10.1021/acs.chemrestox.5b00308. Epub 2016 Jan 13.
In our previous work, we identified a natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis, endemic plants associated with outbreaks of biliary atresia in Australian neonatal livestock. Biliatresone is a very rare isoflavonoid with an α-methylene ketone between two phenyls, 1,2-diaryl-2-propenone, along with methylenedioxy, dimethoxyl, and hydroxyl functional groups, that causes extrahepatic biliary toxicity in zebrafish. The toxic core of biliatresone is a methylene in the α-position relative to the ketone of 1,2-diaryl-2-propenone that serves as an electrophilic Michael acceptor. The α-methylene of biliatresone spontaneously conjugated with water and methanol (MeOH), respectively, via Michael addition in a reverse phase high-performance liquid chromatography (RP-HPLC) analysis. We here report the reactivity of biliatresone toward glutathione (GSH), several amino acids, and other thiol- or imidazole-containing biomolecules. LC-MS and HPLC analysis of the conjugation reaction showed the reactivity of biliatresone to be in the order histidine > N-acetyl-d-cysteine (D-NAC) = N-acetyl-l-cysteine (L-NAC) > histamine > glutathione ≥ cysteine ≫ glycine > glutamate > phenylalanine, while serine and adenine had no reactivity due to intramolecular hydrogen bonding in the protic solvents. The reactivity of ethyl vinyl ketone (EVK, 1-penten-3-one), an example of a highly reactive α,ß-unsaturated ketone, toward GSH gave a 6.7-fold lower reaction rate constant than that of biliatresone. The reaction rate constant of synthetic 1,2-diaryl-2-propen-1-one (DP), a core structure of the toxic molecule, was 10-fold and 1.5-fold weaker in potency compared to the reaction rate constants of biliatresone and EVK, respectively. These results demostrated that the methylenedioxy, dimethoxyl, and hydroxyl functional groups of biliatresone contribute to the stronger reactivity of the Michael acceptor α-methylene ketone toward nucleophiles compared to that of DP and EVK.
在我们之前的工作中，我们从澳大利亚新生家畜胆汁闭锁疫情相关的本土植物团集扁芒菊和滨海扁芒菊中鉴定出一种天然毒素——双硫双氢愈创木酸。双硫双氢愈创木酸是一种非常罕见的异黄酮，在两个苯基之间有一个α-亚甲基酮（1,2-二芳基-2-丙烯酮），还带有亚甲二氧基、二甲氧基和羟基官能团，它会在斑马鱼中引发肝外胆管毒性。双硫双氢愈创木酸的毒性核心是相对于1,2-二芳基-2-丙烯酮的酮基在α位的一个亚甲基，它作为亲电迈克尔受体。在反相高效液相色谱（RP-HPLC）分析中，双硫双氢愈创木酸的α-亚甲基分别通过迈克尔加成与水和甲醇（MeOH）自发共轭。我们在此报告双硫双氢愈创木酸与谷胱甘肽（GSH）、几种氨基酸以及其他含硫醇或咪唑的生物分子的反应活性。共轭反应的液相色谱-质谱（LC-MS）和高效液相色谱（HPLC）分析表明，双硫双氢愈创木酸的反应活性顺序为：组氨酸＞N-乙酰-D-半胱氨酸（D-NAC）=N-乙酰-L-半胱氨酸（L-NAC）＞组胺＞谷胱甘肽≥半胱氨酸≫甘氨酸＞谷氨酸＞苯丙氨酸，而丝氨酸和腺嘌呤由于在质子溶剂中的分子内氢键作用而没有反应活性。乙烯基乙基酮（EVK，1-戊烯-3-酮）是一种高反应活性的α,β-不饱和酮，它与谷胱甘肽的反应速率常数比双硫双氢愈创木酸低6.7倍。有毒分子的核心结构合成1,2-二芳基-2-丙烯-1-酮（DP）的反应速率常数分别比双硫双氢愈创木酸和EVK的反应速率常数弱10倍和1.5倍。这些结果表明，双硫双氢愈创木酸的亚甲二氧基、二甲氧基和羟基官能团使得迈克尔受体α-亚甲基酮相对于DP和EVK对亲核试剂具有更强的反应活性。
