Post-Synthetic DNA-Functionalization Based on DNA-Templated Dynamic Chemistry
by Zeynep Kanlidere
Date of Examination:2015-04-15
Date of issue:2015-06-26
Advisor:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Lutz Ackermann
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Abstract
English
With the growing field of DNA technology there is an increasing demand for new modifications of DNA, which improve its versatility and promote the DNA as a fundamental scaffold for a wide field of applications like catalysis, encoding processes and supramolecular functionalized structures. The research described in this thesis includes the preparation of a modified oligonucleotide and is then focused on the functionalization of this modified oligonucleotide by a dynamic combinatorial chemistry approach. For the modification of the oligonucleotide, the ribose unit of the backbone in the native structure of DNA was replaced by D-threoninol. In order to achieve this purpose, in the first step D-threoninol was successfully converted to the phosphoramidite monomer 4 with an orthogonal protected amino group. The corresponding phosphoramidite monomer was necessary in order to make it suitable for automated DNA synthesis. It was successfully incorporated into the central position of a 13mer oligonucleotide by using automated solid phase synthesis. The advantage of the reactive amino group on the backbone of the modified oligonucleotide was that any functional molecule of interest functionalized as an aldehyde can be easily tethered into the oligonucleotide post-synthetically. In this work the natural nucleobases were synthesized with aldehyde groups to allow them to reversibly react with the free amino group on the modified oligonucleotide. This thesis is an example for the dynamic combinatorial chemistry approach applied to oligonucleotides demonstrating the selective attachment of aldehyde-modified nucleobase units to the abasic site of the double-stranded-oligonucleotide. This was achieved by allowing the modified oligonucleotide to react with four nucleobases through reversible imine formation in the presence of a complementary template strand. In the presence of four nucleobases, four possible imine products were obtained in the dynamic library but one of them was more dominant with respect to others. The correct nucleobase which paired with the nucleotide of the template strand according to Watson-Crick base pairing had the strongest binding to the template and was the dominant one in the library. However, the composition of the library did not necessarily consist of the singly most strongly bound product, but rather reflected a combination of products by arranging the whole system. It was interesting to see that when a new template was added, the library reorganized itself by amplifying the most stable product at the expense of others. The results demonstrated the adaptive and flexible nature of the dynamic library. The dynamic library was proven to be responsive to the external influences that alter the relative thermodynamic stabilities of the library members. The composition of the dynamic library was investigated by anion exchange-HPLC and subsequent ESI-MS analysis. The number of hydrogen bonds and the stacking interactions played an important role in the selectivity of the nucleobases. The higher selectivity was observed for the guanine and cytosine than adenine and thymine due to their higher number of hydrogen bonds. Furthermore, purine bases were incorporated with better yield and greater selectivity than pyrimidines (G>C and A>T). Not only nucleobases but also a metal ligand with π-surface was attached to the same modified oligonucleotide in order to gain a new function to the DNA. To achieve this purpose 1,10-phenantroline was functionalized as an aldehyde. Afterwards, the phenantroline ligand was successfully attached to the amine of the modified oligonucleotide in presence of the template strand. In future works, various phenantroline-based ligands with extended π-surfaces can be prepared in order to attach them to the backbone of the modified oligonucleotide. Dynamic combinatorial chemistry is a promising approach for the selection and screening of the best bound ligand. Furthermore, the phenantroline attached oligonucleotide was annealed with its complementary strand in the presence of copper ions in order to generate a DNA-based metal complex dsON1+P. Finally, this DNA-based metal complex was used as catalyst in an application for an enantioselective Diels-Alder reaction. In presence of Cu(II) catalyst (Cu(NO3)2), the Diels-Alder reaction between a diene and dienophile gave products in racemic fashion. In a second reaction, the DNA-based hybrid catalyst dsON1+P instead of free copper salts was used in order to obtain the products with enantiomeric stereoselectivity. However, the use of DNA-based hybrid catalyst dsON1+P did not show the predicted enantioselectivity in the reaction. The aim of designing an efficient enantioselective DNA-based catalyst was not completed due to time limitation, but it represents the further outlook of this work.
Keywords: Functional DNA; Dynamic Chemistry