Nucleobase-modified nucleotides are widely used for a variety of purposes, where C5/C7 modified pyrimidines and purines are the main focus. However, a little attention has been paid to altering other positions of nucleobases such as C6/C8 or O4/N4. To increase the portfolio of accessible modified nucleotides not only the functional group should be the variable, but also the proper nucleobase position. Moreover, artificial nucleobases as well as nucleobase imitators bearing nucleotides should be explored more. Here, a set of novel modified pyrimidine nucleotides bearing N4, C5 or thio modifications as well as pyridone-based nucleobases was investigated. The application of these nucleotides for the template-independent DNA synthesis using terminal deoxynucleotidyl transferase was demonstrated. N4-acylated cytidine derivatives were also shown to be good substrates for the template-dependent DNA polymerases. N4-acyl-modified DNA was shown to be protected against the cleavage by the tested restriction endonucleases. In addition, N4-acyl-modified DNA can be demodified using an aqueous ethanolamine solution. Modified DNA bearing photoactive functional groups appeared to be suitable for the UV-induced covalent cross-linking with interacting proteins and a variety of solid supports. Therefore, novel modified pyrimidine nucleotides are proposed to expand the toolbox of nucleotide derivatives for the synthesis of functionalized DNA considerably