| Abstract [eng] |
Nucleoside 5'-triphosphates [(d)NTPs] play an essential role in many biological processes. Natural (d)NTPs act as monomers for polymerase-mediated nucleic acid synthesis both in vivo and in vitro. Adenosine 5'-triphosphate (ATP) is the primary energy source for cellular functions. Cyclic mononucleotides such as cAMP and cGMP serve as intracellular signaling molecules. Dinucleotides such as nicotinamide adenine dinucleotide and flavin adenine dinucleotide are enzyme cofactors in many biochemical reactions. Due to the broad range of applications of (d)NTPs they are widely studied. Modified (d)NTPs are used in various cellular and molecular biology experiments. These compounds are utilized as substrates for polymerases, studied as inhibitors for enzymes, they are also often labelled with fluorescent or radioactive probes and are applied in the fields of therapeutics and diagnostics. Most often nucleobases are modified at the positions C5 of pyrimidines and C7 of 7-deazapurines. This ensures correct Watson-Crick base pairing and minimal enzymatic interference. Little is known about modifications at the positions N6 and O6 of purines and N4 and O4 of pyrimidines thus the aim of this study was to synthesize N4-amino acid modified nucleoside 5'-triphosphates and study them as substrates for DNA polymerases. To achieve this goal the following tasks have been formed: • To synthesize Boc-protected N4-amino acid modified 2'-deoxycytidine nucleosides. • To synthesize deprotected N4-amino acid modified 2'-deoxycytidine nucleosides. • To phosphorylate N4-modified 2'-deoxycytidine nucleosides into nucleoside 5'-triphosphates. • To investigate the synthesized nucleotides as substrates for enzymatic DNA synthesis. In total 15 new and uncharacterized compounds were synthesized, three of which were N4-acyl-Boc-2'-deoxycytidines (1-3) and four of which were N-(4-(2'-deoxycytidinyl))acylamides (5-8) (compound 4 (N4-phenylalaninoyl-Boc-2'-deoxycytidine) was synthesized by colleague T. Varnas). All of the compounds were phosphorylated into nucleoside 5'-triphosphates using Yoshikawa procedure and used as substrates for DNA polymerase-mediated deoxynucleic acid synthesis. Terminal deoxynucleotidyl transferase (TdT) incorporated all of the synthesized nucleotides (9-16) into DNA, the best substrates being N4-acyl-Boc-2'-deoxycytidine 5'-triphosphates. Likewise, DNA polymerase φ29 successfully utilized all of the compounds for primer extension reactions but to determine which of the substrates were the best additional experiments have to be made. |
