Chemical Reasearch On Selectivity Of Mechanism Of Nucleic Acid Base Hydroxylation Catalyzed By TET1 Protein

ObjectiveAs a demethylases,TET1 proteins can catalyze 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hmC),which is related to various disease.Meanwhile,thymine(T)can also be transformed to 5-hydroxymethyluracil(5hmU)under the reaction with TET1 protein.The function of TET1 protein have sparked great interest,whereas their mechanism still remains controversial.Meanwhile,researches show that active ingredients of Chinese herbs can inhibit its effection.The aim of this study is to find reaction mechanism of TET1 protein and its selectivity from the viewpoint of chemistry,and further to summarize general rule of this kind of reaction so as to provide the basis for biological study.MethodsIn this study,TET1 protein was used as a research object.In the presence of Fe(II)and a-KG,TET1 was reacted with some representative compounds(such as cyclohexane,hexahydrotoluene,ethylbenzene,cumene,methyl salicylate,ethyl salicylate,2-Amino-4,6-dimethylpyrimidine,6-methyl purine,methionine,theophylline and 5-ethyluracil)in vitro.Then the reaction products were analyzed using GC-MS or UPLC-MS method.The position of the substituted H atom and the chemical selectivity were deduced based on the results of mass spectrograms.Results1)5hmU and uracil(demethylation products of T)were detected in the reaction solution of T that catalyzed by TET1 protein,and the conversion rate was 9.2%in vitro.2)Hydroxides of cyclohexane,methylcyclohexane weren't present in the reaction solution.3)Ethylbenzene,cumene,2-amino 4,6-dimethylpyrimidine and 6-methylpurine were did hydroxylated by TET1 protein,and the-CH3 of which H atom was replaced by OH was connected to a double bond.4)In the reaction solution of methyl salicylate,ethyl salicylate(whose-CH3 is connected to an O atom),methionine(whose-CH3 is conjuncted with a S atom)and theophylline(-CH3 is conjuncted with a N atom),their hydroxides were found.5)Hydroxide was found in the reaction of 5-ethyluracil,but uracil(deethylation products of 5-ethyluracil)was not.ConclusionT can be hydrolated by TETI protein in the presence of a-KG and Fe(?)to generate 5hmU.TET1 protein can catalyzed the hydrolation of-CH3 which is connected to a double bond possibly due to the p-? conjugate after losing H atom.Meanwhile,the H atom in-CH3 which conjunct to O atom,S atom,N atom,because of the electron attracting effect(-1).The four kinds of C-H bound dissociation energy is relativiely low.This suggest that TET1 protein enables catalyze the hydroxylation of the methyl group in the DNA.But the H atom which is connected to terminal carbon in the ethyl group in the DNA fails to be hydrolated by TET1 protein.Therefore,TET1 protein exhibits demethylation rather than dealkylation.