Synthesis And DNA-Cleaving Activity Of Oligopolyaminde-Seryl-Histidine Conjugate

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 andESI-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 frominteracting with A, -DN...