5-羥甲基胞嘧啶pKa值的理論研究

靳玲俠，閔鎖田,王志銀,趙夢(mèng)婷，任宏江

(1.陜西理工學(xué)院 化學(xué)與環(huán)境科學(xué)學(xué)院,陜西 漢中 723001; 2.西安文理學(xué)院 化學(xué)工程學(xué)院,西安 710065)

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5-羥甲基胞嘧啶pKa值的理論研究

靳玲俠1，閔鎖田1,王志銀1,趙夢(mèng)婷1，任宏江2

(1.陜西理工學(xué)院 化學(xué)與環(huán)境科學(xué)學(xué)院,陜西 漢中 723001; 2.西安文理學(xué)院 化學(xué)工程學(xué)院,西安 710065)

采用兩種不同動(dòng)力學(xué)循環(huán)方案，在B3LYP/6-311++G(d,p)+ZPE和B3LYP/aug-cc-pVTZ//B3LYP/6-311++G(d,p)+ZPE水平上，同時(shí)考慮PCM和CPCM模型對(duì)5-羥甲基胞嘧啶(5-HMeCyt)中N3、N4、O2不同位點(diǎn)質(zhì)子化的pKa值進(jìn)行計(jì)算研究.結(jié)果表明：采用經(jīng)驗(yàn)值法，5-HMeCyt中N3位置質(zhì)子化的pKa值最大；采用質(zhì)子交換法，以N3質(zhì)子化的胞嘧啶為參考酸，發(fā)現(xiàn)CPCM模型計(jì)算結(jié)果對(duì)計(jì)算方法的依賴性較大.

B3LYP；5-甲基胞嘧啶；胞嘧啶；5-羥甲基胞嘧啶

5-羥甲基胞嘧啶(5-HMeCyt)可能是一個(gè)重要的表觀遺傳學(xué)標(biāo)記，也可能是5-甲基胞嘧啶(5-MeCyt)去甲基向胞嘧啶(Cyt)轉(zhuǎn)化的中間產(chǎn)物.已有文獻(xiàn)報(bào)道，pKa值對(duì)氫原子轉(zhuǎn)移、質(zhì)子交換、電子轉(zhuǎn)移等反應(yīng)機(jī)制的闡明尤為重要[1-4].Arnott等[5]實(shí)驗(yàn)發(fā)現(xiàn)在一定pH值下，胞嘧啶的N3位質(zhì)子可轉(zhuǎn)移至鳥嘌呤.同時(shí)，胞嘧啶的堿基序列對(duì)pH值會(huì)產(chǎn)生一定影響.因此，可通過pKa值控制堿基配對(duì)的最佳條件.另外，在酸性介質(zhì)中，Cyt存在3個(gè)可能的質(zhì)子化位置，分別為N3、O2和N4.綜合文獻(xiàn)報(bào)道[6-7]，Cyt在酸性條件下形成6個(gè)質(zhì)子化異構(gòu)體，用滴定方法較難準(zhǔn)確測(cè)量有機(jī)物具體位點(diǎn)的pKa值.因此采用理論與計(jì)算化學(xué)估算嘧啶堿基的pKa值對(duì)于理解以質(zhì)子轉(zhuǎn)移為基礎(chǔ)的化學(xué)物質(zhì)和生物進(jìn)程具有非常重要的意義.

在實(shí)驗(yàn)方面，F(xiàn)loria′n等人在研究胞嘧啶質(zhì)子化異構(gòu)體紅外光譜時(shí)發(fā)現(xiàn)，胞嘧啶的N3，N4，O2位置上均可發(fā)生質(zhì)子化反應(yīng)，同時(shí)發(fā)現(xiàn)N3和O2質(zhì)子化異構(gòu)體較其它異構(gòu)體穩(wěn)定[8].Saenger等人通過實(shí)驗(yàn)檢測(cè)到胞嘧啶的pKa值為4.5[9-10].由于胞嘧啶質(zhì)子化位置的復(fù)雜性且部分異構(gòu)體的不穩(wěn)定性，導(dǎo)致實(shí)驗(yàn)難于準(zhǔn)確檢測(cè)不穩(wěn)定異構(gòu)體的pKa值.在理論研究方面，Moser等人[11]采用密度泛函理論方法得出胞嘧啶的pKa值為6.29～6.98，并預(yù)測(cè)甲基化對(duì)胞嘧啶pKa值的影響，而George等采用Poisson-Boltzmann方法對(duì)胞嘧啶在第三螺旋結(jié)構(gòu)的N3位置pKa值進(jìn)了計(jì)算，得出N3質(zhì)子化胞嘧啶的pKa值為4.3[12]，與實(shí)驗(yàn)值極為接近(4.5)，5-HMeCyt是一種重要的表觀遺傳修飾，可能與去甲基化過程有關(guān)，但其作用機(jī)制仍不明確.鑒于此，本文將系統(tǒng)考察5-HMeCyt不同位點(diǎn)質(zhì)子化的pKa值(圖1)，同時(shí)探討PCM和CPCM對(duì)pKa值的影響，解決質(zhì)子化位置是否起顯著催化作用.

圖1 5-HMeCyt及其N3、O2、N4位質(zhì)子化結(jié)構(gòu)示意圖

1 計(jì)算方法

為比較計(jì)算方法對(duì)計(jì)算結(jié)果的影響，本文選用B3LYP/6-311++G(d,p)+ZPE和B3LYP/aug-cc-pVTZ//B3LYP/6-31G(d,p)+ZPE兩種計(jì)算方法[13-14]，并且使用兩種循環(huán)方案分別對(duì)5-HMeCyt不同位點(diǎn)質(zhì)子化的pKa值進(jìn)行計(jì)算.以氣相分子作為研究對(duì)象，應(yīng)用Gaussian03程序包[15]，采用密度泛函理論B3LYP方法對(duì)5-HMeCyt及其質(zhì)子化異構(gòu)體的初始幾何構(gòu)型進(jìn)行優(yōu)化和能量計(jì)算，并采用頻率進(jìn)行驗(yàn)證，以確定其最穩(wěn)定的異構(gòu)體結(jié)構(gòu).同時(shí)考察CPM和CPCM模型對(duì)5-HMeCyt的質(zhì)子化異構(gòu)體pKa值的影響，對(duì)各異構(gòu)體的溶劑化自由能進(jìn)行了計(jì)算[16].

1.1 經(jīng)驗(yàn)值法[17]

Scheme1 經(jīng)驗(yàn)值法:

↑-ΔGs(C5H8N3O+)↓ΔGs(C5H7N3O2)↓ΔGs(H+)

Scheme2 質(zhì)子交換法:

↑-ΔGs(C5H8N3O+)↑-ΔGs(C4H5N3O)↓ΔGs(C5H7N3O2)↓ΔGs(C4H6N3O+)

Scheme3采用經(jīng)驗(yàn)值法計(jì)算5-HMeCyt質(zhì)子化異構(gòu)體的pKa值:

(1)

pKa=ΔG(aq)/(2.303RT)

(2)

Scheme4采用質(zhì)子交換法計(jì)算5-HMeCyt質(zhì)子化異構(gòu)體的pKa值:

(3)

pKa=ΔG(aq)/(2.303RT)+pKa(C4H6N3O+)

(4)

1.2 質(zhì)子交換法[18]

表1 氣相自由能Gg、吉布斯自由能校正值GZEP、自由能在水溶液中變化值ΔGaq(a.u.)及pKa值

注:aCPCM and PCM,B3LYP/6-311++G(d,p)+ZPE；SP-CPCM and SP-PCM,B3LYP/aug-cc-pVTZ//B3LYP/6-31G(d,p)+ZPE.

2 結(jié)果與討論

本文利用上述兩種熱力學(xué)循環(huán)方法計(jì)算出質(zhì)子化5-HMeCyt在水溶液中的pKa值.

比較經(jīng)驗(yàn)值法采用不同模型計(jì)算出5-HMeCyt不同位點(diǎn)質(zhì)子化異構(gòu)體的pKa值，發(fā)現(xiàn)不同位點(diǎn)質(zhì)子化異構(gòu)體的pKa值由N4、O2、N3依次增大，說明5-HMeCyt不同位點(diǎn)質(zhì)子化異構(gòu)體的pKa值存在顯著差別.在PCM模型下，5-HMeCyt對(duì)計(jì)算方法的依賴較小.在CPCM模型下采用B3LYP/6-311++G(d,p)+ZPE與B3LYP/aug-cc-pVTZ方法計(jì)算的結(jié)果偏差較大.

采用質(zhì)子交換法運(yùn)用不同計(jì)算方法對(duì)5-HMeCyt的N3位質(zhì)子化異構(gòu)體pKa值的計(jì)算結(jié)果表明，在相同計(jì)算方法下采用CPCM和PCM模型計(jì)算結(jié)果存在較大偏差.

3 結(jié)論

本文通過密度泛函理論、溶劑化模型和兩種熱力學(xué)循環(huán)方案對(duì)5-HMeCyt不同位點(diǎn)的pKa值進(jìn)行了詳細(xì)計(jì)算研究，得出以下結(jié)論：(1)5-HMeCyt不同位點(diǎn)的pKa值具有顯著差別；(2)在PCM模型下5-HMeCyt不同位點(diǎn)pKa值對(duì)計(jì)算方法的依賴性較小，而CPCM模型對(duì)計(jì)算方法依賴性較大.

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[責(zé)任編輯 馬云彤]

Theoretical Research on pKa Value of the 5-Hydroxymethylcytosine

JIN Ling-xia1, MIN Suo-tian1, WANG Zhi-yin1, ZHAO Meng-ting1, REN Hong-jiang2

(1. College of Chemistry and Environmental Science, Shaanxi University of Technology,Hanzhong 723001, China; 2. School of Chemical Engineering, Xi’an University, Xi’an 710065, China)

The protonated pKa values in different positions of O2, N3, N4 for 5-hydroxymethylcytosine (5-HMeCyt) are calculated by adopting two different dynamic cycle schemes at the B3LYP/6-311++G(d, p)+ZPE and B3LYP/aug-cc-pVTZ//B3LYP/6-311++G(d, p)+ZPE level, which consider the effect of the PCM and CPCM models. The results show that the protonated pKa value is the largest in the N3 position of the 5-HMeCyt by the empirical value method. The results of CPCM model greatly depend on the calculation method when taking the N3 position of cytosine as reference by the proton exchange method.

B3LYP; the 5-methylcytocine; cytosine; the 5-hydroxymethylcytosine

1008-5564(2016)04-0053-04

2016-03-24

陜西理工學(xué)院科研基金資助項(xiàng)目(SLGQD14-10)

靳玲俠(1981—)，女，陜西大荔人，陜西理工學(xué)院化學(xué)與環(huán)境科學(xué)學(xué)院講師，博士，主要從事生物小分子反應(yīng)機(jī)理的理論研究.

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