寡聚酰胺-丝组二肽缀合物的设计合成、质谱解析及其切割DNA活性研究
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摘要
核酸切割剂在核酸化学研究领域中有至关重要的作用。在DNA突变、癌变以及细胞死亡等研究领域，核酸切割剂是一个很好的分子生物学工具，更是设计基因药物的模版。基于丝组二肽对DNA的切割活性和聚酰胺的特异性识别功能，本论文主要研究了寡聚酰胺-丝组二肽缀合物(NO_2-PyPyIm-SerHis)的设计合成、质谱解析及其切割DNA活性研究。
1．通过卤仿反应和DCC／HOBt偶合反应合成酰胺键，首次合成了寡聚酰胺-丝组二肽缀合物。该合成路线反应条件温和，产率适中。在目标分子的合成设计中，由寡聚酰胺作为该分子的DNA识别片断(DNA-BindingMotif，DBM)，同时，柔性链(Linker Motif，LM)与活性片断(Active Motif，AM)Ser-His的组氨酸残基C端相连，保证切割活性基团-丝氨酸残基上氨基和侧链羟基的状态和相对位置。生物活性实验验证此设计是合理的。
2．利用高分辨质谱、红外光谱、核磁共振谱(~1H NMR和~(13)C NMR)和电喷雾质谱(ESI-MS和ESI-MS／MS)分析鉴定化合物的结构，并分析总结了其质谱裂解规律。
3．在电喷雾质谱研究过程中，首次发现：质谱裂解过程中产生的某些酰胺类碎片离子和酯类化合物在离子阱中容易发生水解现象，并利用分子内的氨基催化分子离子水解反应的原理对此不常见的特征性裂解进行了详细解释。这可为同类酰胺或酯的质谱裂解规律和结构解析提供快捷、准确的依据。
4．论文首次提出在电喷雾质谱的裂解过程中，分子离子／碎片离子经过四元环、五元环和六元环过渡态发生氢重排，失去一个较为稳定的中性分子，趋于生成共轭体系的碎片离子。而当分子有羟基存在时容易发生酰胺键脱水现象。
5．利用紫外光谱和琼脂糖凝胶电泳两种方法测定了在B-R和Tri-HCl缓冲溶液中寡聚酰胺-丝组二肽缀合物与DNA的相互作用。发现Tri-HCl抑
摘要
制目标分子切割DNA。两种方法得到的实验结论是相吻合的。虽然我们
没有观察到寡聚酞胺一丝组二肤缀合物特异性地切割DNA，但琼脂糖凝
胶电泳图谱证明我们所合成的寡聚酞胺一丝组二肤缀合物
(N 02一PyPylm一serHis)切割DNA的活性明显高于丝组二肤。这可归因
于识别片断·(DBM)寡聚酞胺的存在增加了活性片断(AM)Se卜His的
局部浓度，相应地提高了活性片断(AM)丝组二肤切割DNA的活性。
寡聚酞胺一丝组二肤缀合物(N仇一PyPylm一serHis)是一个高效全新的DNA
切割剂，同时具有发展成为抗肿瘤前药的潜力。为设计合成更有效的特异性切割
DNA化合物，提供了理论和实践基础。
Artificial nucleic acid cleavage agents have attracted considerable attention on synthetic and biological part. As we all know, the amino acids histidine (His) and serine (Ser) function together as key catalytic amino acids in the active sites of such diverse enzymes as chymotrypsin, trypsin and uridine phosphorylase, and dipeptide seryl-histidine(Ser-His) is the shortest peptide ever reported to show DNA-cleavage activity, which mechanism and roles of functional group have been well documented. Besides, inspired by naturally occurring antibiotic distamycin A and its analogues, polyamide containing N-methylpyyrole/N-methylimidazolelt amino acids have been synthesized, and moreover, it was proved that these molecules recognized and binded in the minor groove of predetermined DNA sequence with high affinity and specificity.
To expand the possible efficient utility of Ser-His for wide range of biological and biomedical application that require more molecular recognition of DNA, we designed and synthesized the oligopolyamide-Seryl-Histidine conjugate, which is composed of DNA-recognizing polyamide chosen as DNA-binding motif (DBM), DNA-cleaving seryl-histidine acted as active motif (AM), and heptyldiamine (Linker Motif, LM) connected with carbon termini of histidine residues. Therefore, the hydroxyl functional group of N-terminal serine residue, and the imidazole function group of histidine residue can preserve their efficient, requisite function for DNA cleavage.
We schemed out the synthesis route for the oligopolyamide-Seryl-Histidine conjugate. By the use of haloform and DCC/HOBt couple reaction, the building blocks prepared such as NO2-Py-COOH and NO2-PyPy-COOH and other materials Boc-L-His-OH, Boc-L-Ser(Bn)-OH were effectively connected to construct the target molecule. The synthetic condition is mild and the yield is moderate. The chemical structure was identified by the use of HRFAB-MS, IR, 1H NMR, 13C NMR and
ESI-MS, ESI-MSn.
The structures of those compounds were positively identified by electrospray ionization tandem mass sepectrometry (ESI-MSn). During the identification of monoimidazole/polyamine amides, we observed an odd fragmentation pathway in which the characteristic fragment ions m/z 183 or 197 always produced the fragment ion at m/z 172 by the apparent expulsion of 'impossible' neutral fragments of mass 11 or 25 Da. In order to clarify this phenomenon, we synthesized additional relevant compounds and specifically deuterated molecules. A proposed mechanism led to a good understanding of the fragmentation pathway in which amides and esters hydrolyzed, presumably in the ion trap itself, and were catalyzed by the intramolecular amino group.
Since successful application of ESI-MS in the areas such as drug metabolism, degradant identification and pharmacokinetics depends on a thorough understanding of ESI-MS and ESI-MS/MS data of the compounds. The dissertation reveals some unique features of ESI-MS/MS spectra of this important class of oligopolyamide-Seryl-Histidine conjugate. The analysis of the ESI-MS/MS data showed protonated compounds undergoes complicated gas-phase rearrangements in addition to simple bond cleavages. Rearrangement can be followed by elimination of a neutral fragment, which occurs with the migration of a positive center via four, five, six-center. The target molecule easily eliminated water contributing to the hydroxyl group of serine residue.
It was found that there is hyperchromic effect and red-shift on UV absorption spectrom as -DNA was interacted with the small molecule oligopolyamide-Seryl-Histidine conjugate in B-R buffer. In addition, as the mixture was incubated in B-R buffer at 50C for 48hours, agarose gel analysis showed that -DNA was cleaved into heterogeneous smear. However, in the same condition, when the Tri-HCl buffer was chosen as incubation condition instead of B-R buffer, there was no cleavage for double-stranded -DNA, which was consistent with the UV results. Then it could be proved that Tri-HCl inhibited the target molecule from
interacting with A, -DN
1．通过卤仿反应和DCC／HOBt偶合反应合成酰胺键，首次合成了寡聚酰胺-丝组二肽缀合物。该合成路线反应条件温和，产率适中。在目标分子的合成设计中，由寡聚酰胺作为该分子的DNA识别片断(DNA-BindingMotif，DBM)，同时，柔性链(Linker Motif，LM)与活性片断(Active Motif，AM)Ser-His的组氨酸残基C端相连，保证切割活性基团-丝氨酸残基上氨基和侧链羟基的状态和相对位置。生物活性实验验证此设计是合理的。
2．利用高分辨质谱、红外光谱、核磁共振谱(~1H NMR和~(13)C NMR)和电喷雾质谱(ESI-MS和ESI-MS／MS)分析鉴定化合物的结构，并分析总结了其质谱裂解规律。
3．在电喷雾质谱研究过程中，首次发现：质谱裂解过程中产生的某些酰胺类碎片离子和酯类化合物在离子阱中容易发生水解现象，并利用分子内的氨基催化分子离子水解反应的原理对此不常见的特征性裂解进行了详细解释。这可为同类酰胺或酯的质谱裂解规律和结构解析提供快捷、准确的依据。
4．论文首次提出在电喷雾质谱的裂解过程中，分子离子／碎片离子经过四元环、五元环和六元环过渡态发生氢重排，失去一个较为稳定的中性分子，趋于生成共轭体系的碎片离子。而当分子有羟基存在时容易发生酰胺键脱水现象。
5．利用紫外光谱和琼脂糖凝胶电泳两种方法测定了在B-R和Tri-HCl缓冲溶液中寡聚酰胺-丝组二肽缀合物与DNA的相互作用。发现Tri-HCl抑
摘要
制目标分子切割DNA。两种方法得到的实验结论是相吻合的。虽然我们
没有观察到寡聚酞胺一丝组二肤缀合物特异性地切割DNA，但琼脂糖凝
胶电泳图谱证明我们所合成的寡聚酞胺一丝组二肤缀合物
(N 02一PyPylm一serHis)切割DNA的活性明显高于丝组二肤。这可归因
于识别片断·(DBM)寡聚酞胺的存在增加了活性片断(AM)Se卜His的
局部浓度，相应地提高了活性片断(AM)丝组二肤切割DNA的活性。
寡聚酞胺一丝组二肤缀合物(N仇一PyPylm一serHis)是一个高效全新的DNA
切割剂，同时具有发展成为抗肿瘤前药的潜力。为设计合成更有效的特异性切割
DNA化合物，提供了理论和实践基础。
Artificial nucleic acid cleavage agents have attracted considerable attention on synthetic and biological part. As we all know, the amino acids histidine (His) and serine (Ser) function together as key catalytic amino acids in the active sites of such diverse enzymes as chymotrypsin, trypsin and uridine phosphorylase, and dipeptide seryl-histidine(Ser-His) is the shortest peptide ever reported to show DNA-cleavage activity, which mechanism and roles of functional group have been well documented. Besides, inspired by naturally occurring antibiotic distamycin A and its analogues, polyamide containing N-methylpyyrole/N-methylimidazolelt amino acids have been synthesized, and moreover, it was proved that these molecules recognized and binded in the minor groove of predetermined DNA sequence with high affinity and specificity.
To expand the possible efficient utility of Ser-His for wide range of biological and biomedical application that require more molecular recognition of DNA, we designed and synthesized the oligopolyamide-Seryl-Histidine conjugate, which is composed of DNA-recognizing polyamide chosen as DNA-binding motif (DBM), DNA-cleaving seryl-histidine acted as active motif (AM), and heptyldiamine (Linker Motif, LM) connected with carbon termini of histidine residues. Therefore, the hydroxyl functional group of N-terminal serine residue, and the imidazole function group of histidine residue can preserve their efficient, requisite function for DNA cleavage.
We schemed out the synthesis route for the oligopolyamide-Seryl-Histidine conjugate. By the use of haloform and DCC/HOBt couple reaction, the building blocks prepared such as NO2-Py-COOH and NO2-PyPy-COOH and other materials Boc-L-His-OH, Boc-L-Ser(Bn)-OH were effectively connected to construct the target molecule. The synthetic condition is mild and the yield is moderate. The chemical structure was identified by the use of HRFAB-MS, IR, 1H NMR, 13C NMR and
ESI-MS, ESI-MSn.
The structures of those compounds were positively identified by electrospray ionization tandem mass sepectrometry (ESI-MSn). During the identification of monoimidazole/polyamine amides, we observed an odd fragmentation pathway in which the characteristic fragment ions m/z 183 or 197 always produced the fragment ion at m/z 172 by the apparent expulsion of 'impossible' neutral fragments of mass 11 or 25 Da. In order to clarify this phenomenon, we synthesized additional relevant compounds and specifically deuterated molecules. A proposed mechanism led to a good understanding of the fragmentation pathway in which amides and esters hydrolyzed, presumably in the ion trap itself, and were catalyzed by the intramolecular amino group.
Since successful application of ESI-MS in the areas such as drug metabolism, degradant identification and pharmacokinetics depends on a thorough understanding of ESI-MS and ESI-MS/MS data of the compounds. The dissertation reveals some unique features of ESI-MS/MS spectra of this important class of oligopolyamide-Seryl-Histidine conjugate. The analysis of the ESI-MS/MS data showed protonated compounds undergoes complicated gas-phase rearrangements in addition to simple bond cleavages. Rearrangement can be followed by elimination of a neutral fragment, which occurs with the migration of a positive center via four, five, six-center. The target molecule easily eliminated water contributing to the hydroxyl group of serine residue.
It was found that there is hyperchromic effect and red-shift on UV absorption spectrom as -DNA was interacted with the small molecule oligopolyamide-Seryl-Histidine conjugate in B-R buffer. In addition, as the mixture was incubated in B-R buffer at 50C for 48hours, agarose gel analysis showed that -DNA was cleaved into heterogeneous smear. However, in the same condition, when the Tri-HCl buffer was chosen as incubation condition instead of B-R buffer, there was no cleavage for double-stranded -DNA, which was consistent with the UV results. Then it could be proved that Tri-HCl inhibited the target molecule from
interacting with A, -DN
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