LC-ESI-MS及NMR技术应用于P_3m与氨基酸反应及合成d4T氢亚磷酸酯反应研究
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摘要
磷是组成生命体至关重要的元素,所有的生命活动过程都直接或间接有磷的参与。磷作为遗传物质DNA基本骨架存在于磷酸二酯键中;磷酸双酯(DNA和RNA)、磷酸酐(ATP)调控着整个生命过程,它们参与物质的代谢、能量的转化、遗传的表达等几乎所有的生化过程;蛋白质的磷酰化和去磷酰化在酶的活性调节、蛋白质的生物合成、开关血浆膜上某些特殊离子的通道、调节膜的渗透性等重要生理过程中都起着关键作用。正如诺贝尔奖获得者L.Todd所说的那样:“哪里有生命,哪里就有磷”。磷在生命活动中起着极为重要的作用,被誉为“生命活动的调控中心”。
电喷雾质谱(ESI-MS~n)和高效液相色谱—质谱联用技术(LC-MS)作为现代分析手段,广泛应用于复杂样品的分析和有机反应机理的研究。本论文以LC-MS/MS及ESI-MS为主要技术手段,结合~(31)p NMR和~(15)N-、D-同位素标记技术来研究动态的化学反应及其机理。
首先,本论文运用ESI-MS快速分析了单个氨基酸(α-、β-和γ-氨基酸)与偏三聚磷酸盐成肽反应产物,实验结果表明只有α-氨基酸,如甘氨酸、丙氨酸能成肽;β-和γ-氨基酸则很难,β-丙氨酸仅有少量的二肽生成,而γ-丁氨酸没有肽生成;运用高效液相色谱—质谱(HPLC-MS)联用技术对偏三聚磷酸盐和混合氨基酸的成肽反应进行分析。由于反应产物较为复杂,ESI-MS只能给出有限的信息,所以作者以全氟戊酸(NFPA)作为离子对试剂,采用反相离子对色谱技术对混合物进行了有效分离。甘氨酸与丙氨酸混合反应有2×2=4种二肽产生,甘氨酸与β-丙氨酸或γ-丁氨酸混合反应只有2×2~0=2种二肽产生,而β-丙氨酸与γ-丁氨酸混合反应没有肽的生成。这一实验结果进一步说明α-氨基酸在成肽反应中的重要性;通过~(15)N标记的磷酰化氨基酸模型化合物的合成,结合~(31)pNMR对该反应进行跟踪,研究了~(14)N的甘氨酸与~(15)N标记的甘氨酸与偏三聚磷酸盐反应的磷谱差异,研究结果表明反应过程中,化学位移在13.12 ppm的磷谱峰并不是文献所述的五元环混酐中间体,而可能是两个甘氨酸通过磷基团相连的一种磷酸盐所对应的磷谱峰,并推测它是由氨基酸的氨基在碱性条件下进攻五元环混酐的磷原子亲电中心而形成的。
其次,运用多级电喷雾质谱技术(ESI-MS~n)对一系列小肽的裂解规律做了研究。发现甘甘二肽[M+H-CO]~+峰还能失去一分子量为17 Da的中性碎片NH_3,表现出与其它二肽如丙丙二肽,亮亮二肽和甘丙二肽等小肽不同的质谱裂解规律。通过~(15)N标记技术,快速判断碎片离子及失去的中性分子中氮原子的个数;通过氘代实验判断碎片离子的活泼质子个数;高分辨多级质谱获得碎片离子的分子式。实验结果表明,[M+H-CO]~+峰并不是以前认为的CH_2=NH_2~+和H-Gly-OH通过氢键作用形成的络合物,而是经一重排反应形成自己的共价结构H_2NCH_2NHCH_2COOH;但这一重排反应只通用于甘甘二肽,对于其它侧链位阻较大的二肽则较难发生。
本论文还运用在线的高效液相色谱—多级电喷雾质谱(HPLC-MS~n)及NMR技术对PCl_3/醇体系一锅法合成d4T-5'-氢亚磷酸酯的反应机理进行了研究。首先利用高效液相色谱梯度洗脱对反应混合物中的各种组分进行了有效分离,得到9个组分的离子流峰,然后运用在线的多级质谱对各组分做离子流提取多级裂解研究,结合化学反应推测了反应各组分的可能化学结构,对其中的关键组分进行了标样合成表征;同时运用~(31)p NMR对反应进行了跟踪,提出反应机理,并比较使用单一醇解试剂和混合醇解试剂的优缺点。因此,本论文在合成d4T-5'-氢亚磷酸异丙酯时就没有使用混合醇解试剂,且产率较高。
最后,运用电喷雾多级质谱(ESI-MS~n)和多级高分辨质谱(HR-MS~n)对一系列高活性抗病毒d4T磷酰胺类衍生物的加钠离子作了深入研究。加钠离子和加钾离子的二级质谱相对来说较复杂,但二者裂解途径具有相似性。本文主要以加钠离子为例,作了较为细致地多级质谱研究,建立了这类化合物的多级质谱裂解数据信息,并发现了三个重排离子峰:m/z 259,m/z 240和m/z 160。对其中的两个主要重排峰m/z 240和m/z 160提出了五元环五配位磷中间体机理。加钾离子也有m/z 240和m/z 160这两个重排离子,但没有m/z 259。这些裂解规律及重排机理为此类化合物的合成鉴定和药物代谢等分析提供了较为有用的结构依据。
Phosphorus is a basic element for the life.All of the life processes are related to phosphorus.For example,the phosphorylation of protein;the linking group of the nucleosides of DNA and RNA are phosphates;ATP as energy currency has three phosphorus atoms,which participates in energy metabolism,the process of oxidation and phosphorylation,the translation and expression of the genetic information,the expression of protein and the biosynthesis and decomposition for cell.Nobel laureate L.Todd said:"Where there's life,there's phosphorus." Because of its importance, phosphorus has been considered the adjusting center for life activities.
Electrospray ionization mass spectrometry(ESI-MS) and high performance liquid chromatography(HPLC) coupled with ESI-MS as modern analytic methods are very powerful for the analysis of complicated mixtures and the investigation of the organic reaction mechanism.In this work,ESI-MS and LC-MS/MS combined with ~(31)P NMR, ~(15)N- and D- isotope techniques were used to investigate the mechanism of the reactions.
First,the condensation reactions of single amino acid or each pair of amino acids such as glycine,L-alanine,β-alanine andγ-aminobutyric acid with sodium trimetaphosphate(P_(3m)) were reinvestigated by electrospray ion-trap mass spectrometry(ESI-MS) and high performance liquid chromatography.It was found that onlyα-amino acid could form peptide with inducement P_(3m).Withoutα-amino acid,theβ-amino acid orγ-amino acid could not form peptide either by themselves or with their mixtures under the same conditions.The reaction of ~(15)N labeled glycine with P_(3)m was traced by ~(31)P NMR.A possible mechanism of hetero- anhydride was proposed.
Second,the fragmentation pathway of protonated dipeptides was investigated by electrospray ionization tandem mass spectrometry.Protonated H-Gly-Gly-OH has a different dissociation pathway.On the contrary,other dipeptides,such as H-Ala-Ala-OH fragmented into form the[MH-CO]~+ by the a_1-y_1 pathway and did not show such rearrangement reaction.Additional mechanistic information was obtained by exchanging the active hydrogens of protonated H-Gly-Gly-OH for deuterium.In order to illustrate the mechanism,the ~(15)N labeled dipeptide ~(15)N-Gly-~(15)N-Gly was also investigated,and the structures of the fragment ions were confirmed by high-resolution electrospray ionization(ESI) tandem mass spectrometry.
Third,the mechanism of the reaction of d4T with PCl_3/alcohol was investigated by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry(HPLC-ESI-MS~n) and ~(31)P NMR.The reaction mixtures were separated by high performance liquid chromatography.And the sturctures of the mixture of the one-pot synthesis reaction,such as di-d4T hydrogen phosphonate and O-butyl H-phosphonate of d4T,were efficiently identified by on-line collision-induced dissociation(CID) tandem mass spectrometry.Furthermore,the reaction also was traced by ~(31)P NMR.According to the reaction byproducts,a possible reaction mechanism was proposed.It was found that tert-butanol was not necessary for the synthesis of O-isopropyl H-phosphonate of d4T.
Finally,a series of aryl and alkyl phosphoramidate derivatives of the anti-HIV drug 2',3'-dideoxy-2',3'-didehydrothymidine(d4T) were investigated by positive ion electrospray ionization mass spectrometry(ESI-MS).The fragmentation pathway of Na~+ and K~+ adducts were more complicated than proton adduct.In this work,the [M+Na]~+ ion was investigated in detail by tandem mass spectrometric(MS/MS) techniques.With positive ion mode,many abundant characteristic fragment ions were found,and several rearrangement ions such as m/z 259,m/z 240 and m/z 160 were observed.A rearrangement reaction mechanism was proposed,which involved a five-membered ring of five valent phosphorus intermediate.The results are very useful for the structural elucidation of aryl and alkyl phosphoramidate derivatives of d4T in organic synthesis and drug metabolism.At the same time,it was indicated that ESI-MS is a powerful tool for the structure determination of phosphoramidate derivatives of d4T.
电喷雾质谱(ESI-MS~n)和高效液相色谱—质谱联用技术(LC-MS)作为现代分析手段,广泛应用于复杂样品的分析和有机反应机理的研究。本论文以LC-MS/MS及ESI-MS为主要技术手段,结合~(31)p NMR和~(15)N-、D-同位素标记技术来研究动态的化学反应及其机理。
首先,本论文运用ESI-MS快速分析了单个氨基酸(α-、β-和γ-氨基酸)与偏三聚磷酸盐成肽反应产物,实验结果表明只有α-氨基酸,如甘氨酸、丙氨酸能成肽;β-和γ-氨基酸则很难,β-丙氨酸仅有少量的二肽生成,而γ-丁氨酸没有肽生成;运用高效液相色谱—质谱(HPLC-MS)联用技术对偏三聚磷酸盐和混合氨基酸的成肽反应进行分析。由于反应产物较为复杂,ESI-MS只能给出有限的信息,所以作者以全氟戊酸(NFPA)作为离子对试剂,采用反相离子对色谱技术对混合物进行了有效分离。甘氨酸与丙氨酸混合反应有2×2=4种二肽产生,甘氨酸与β-丙氨酸或γ-丁氨酸混合反应只有2×2~0=2种二肽产生,而β-丙氨酸与γ-丁氨酸混合反应没有肽的生成。这一实验结果进一步说明α-氨基酸在成肽反应中的重要性;通过~(15)N标记的磷酰化氨基酸模型化合物的合成,结合~(31)pNMR对该反应进行跟踪,研究了~(14)N的甘氨酸与~(15)N标记的甘氨酸与偏三聚磷酸盐反应的磷谱差异,研究结果表明反应过程中,化学位移在13.12 ppm的磷谱峰并不是文献所述的五元环混酐中间体,而可能是两个甘氨酸通过磷基团相连的一种磷酸盐所对应的磷谱峰,并推测它是由氨基酸的氨基在碱性条件下进攻五元环混酐的磷原子亲电中心而形成的。
其次,运用多级电喷雾质谱技术(ESI-MS~n)对一系列小肽的裂解规律做了研究。发现甘甘二肽[M+H-CO]~+峰还能失去一分子量为17 Da的中性碎片NH_3,表现出与其它二肽如丙丙二肽,亮亮二肽和甘丙二肽等小肽不同的质谱裂解规律。通过~(15)N标记技术,快速判断碎片离子及失去的中性分子中氮原子的个数;通过氘代实验判断碎片离子的活泼质子个数;高分辨多级质谱获得碎片离子的分子式。实验结果表明,[M+H-CO]~+峰并不是以前认为的CH_2=NH_2~+和H-Gly-OH通过氢键作用形成的络合物,而是经一重排反应形成自己的共价结构H_2NCH_2NHCH_2COOH;但这一重排反应只通用于甘甘二肽,对于其它侧链位阻较大的二肽则较难发生。
本论文还运用在线的高效液相色谱—多级电喷雾质谱(HPLC-MS~n)及NMR技术对PCl_3/醇体系一锅法合成d4T-5'-氢亚磷酸酯的反应机理进行了研究。首先利用高效液相色谱梯度洗脱对反应混合物中的各种组分进行了有效分离,得到9个组分的离子流峰,然后运用在线的多级质谱对各组分做离子流提取多级裂解研究,结合化学反应推测了反应各组分的可能化学结构,对其中的关键组分进行了标样合成表征;同时运用~(31)p NMR对反应进行了跟踪,提出反应机理,并比较使用单一醇解试剂和混合醇解试剂的优缺点。因此,本论文在合成d4T-5'-氢亚磷酸异丙酯时就没有使用混合醇解试剂,且产率较高。
最后,运用电喷雾多级质谱(ESI-MS~n)和多级高分辨质谱(HR-MS~n)对一系列高活性抗病毒d4T磷酰胺类衍生物的加钠离子作了深入研究。加钠离子和加钾离子的二级质谱相对来说较复杂,但二者裂解途径具有相似性。本文主要以加钠离子为例,作了较为细致地多级质谱研究,建立了这类化合物的多级质谱裂解数据信息,并发现了三个重排离子峰:m/z 259,m/z 240和m/z 160。对其中的两个主要重排峰m/z 240和m/z 160提出了五元环五配位磷中间体机理。加钾离子也有m/z 240和m/z 160这两个重排离子,但没有m/z 259。这些裂解规律及重排机理为此类化合物的合成鉴定和药物代谢等分析提供了较为有用的结构依据。
Phosphorus is a basic element for the life.All of the life processes are related to phosphorus.For example,the phosphorylation of protein;the linking group of the nucleosides of DNA and RNA are phosphates;ATP as energy currency has three phosphorus atoms,which participates in energy metabolism,the process of oxidation and phosphorylation,the translation and expression of the genetic information,the expression of protein and the biosynthesis and decomposition for cell.Nobel laureate L.Todd said:"Where there's life,there's phosphorus." Because of its importance, phosphorus has been considered the adjusting center for life activities.
Electrospray ionization mass spectrometry(ESI-MS) and high performance liquid chromatography(HPLC) coupled with ESI-MS as modern analytic methods are very powerful for the analysis of complicated mixtures and the investigation of the organic reaction mechanism.In this work,ESI-MS and LC-MS/MS combined with ~(31)P NMR, ~(15)N- and D- isotope techniques were used to investigate the mechanism of the reactions.
First,the condensation reactions of single amino acid or each pair of amino acids such as glycine,L-alanine,β-alanine andγ-aminobutyric acid with sodium trimetaphosphate(P_(3m)) were reinvestigated by electrospray ion-trap mass spectrometry(ESI-MS) and high performance liquid chromatography.It was found that onlyα-amino acid could form peptide with inducement P_(3m).Withoutα-amino acid,theβ-amino acid orγ-amino acid could not form peptide either by themselves or with their mixtures under the same conditions.The reaction of ~(15)N labeled glycine with P_(3)m was traced by ~(31)P NMR.A possible mechanism of hetero- anhydride was proposed.
Second,the fragmentation pathway of protonated dipeptides was investigated by electrospray ionization tandem mass spectrometry.Protonated H-Gly-Gly-OH has a different dissociation pathway.On the contrary,other dipeptides,such as H-Ala-Ala-OH fragmented into form the[MH-CO]~+ by the a_1-y_1 pathway and did not show such rearrangement reaction.Additional mechanistic information was obtained by exchanging the active hydrogens of protonated H-Gly-Gly-OH for deuterium.In order to illustrate the mechanism,the ~(15)N labeled dipeptide ~(15)N-Gly-~(15)N-Gly was also investigated,and the structures of the fragment ions were confirmed by high-resolution electrospray ionization(ESI) tandem mass spectrometry.
Third,the mechanism of the reaction of d4T with PCl_3/alcohol was investigated by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry(HPLC-ESI-MS~n) and ~(31)P NMR.The reaction mixtures were separated by high performance liquid chromatography.And the sturctures of the mixture of the one-pot synthesis reaction,such as di-d4T hydrogen phosphonate and O-butyl H-phosphonate of d4T,were efficiently identified by on-line collision-induced dissociation(CID) tandem mass spectrometry.Furthermore,the reaction also was traced by ~(31)P NMR.According to the reaction byproducts,a possible reaction mechanism was proposed.It was found that tert-butanol was not necessary for the synthesis of O-isopropyl H-phosphonate of d4T.
Finally,a series of aryl and alkyl phosphoramidate derivatives of the anti-HIV drug 2',3'-dideoxy-2',3'-didehydrothymidine(d4T) were investigated by positive ion electrospray ionization mass spectrometry(ESI-MS).The fragmentation pathway of Na~+ and K~+ adducts were more complicated than proton adduct.In this work,the [M+Na]~+ ion was investigated in detail by tandem mass spectrometric(MS/MS) techniques.With positive ion mode,many abundant characteristic fragment ions were found,and several rearrangement ions such as m/z 259,m/z 240 and m/z 160 were observed.A rearrangement reaction mechanism was proposed,which involved a five-membered ring of five valent phosphorus intermediate.The results are very useful for the structural elucidation of aryl and alkyl phosphoramidate derivatives of d4T in organic synthesis and drug metabolism.At the same time,it was indicated that ESI-MS is a powerful tool for the structure determination of phosphoramidate derivatives of d4T.
引文
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