Commercially available trans-4-hydroxy-L-proline has been used as a starting material for the synthesis of prolinol-based nucleotide analogues with N-phosphonomethyl moiety attached to the nitrogen atom of prolinol ring. The synthetic methodology based on the inversion of configuration at both 1- and 4- positions led, in result, to all diastereoisomeric O-protected 4-mesyloxyprolinol-N-methylphosphonates. Alkylation of nucleobases using the synthons afforded the nucleotide analogues corresponding to alpha- and beta-nucleotides in both L- and D-series. The NMR-based conformational study of alpha- and beta-nucleotides in aqueous solution performed at two different pH values securing either N-fully protonated or deprotonated forms revealed in both cases occurrence of the same mostly populated conformer. All final prolinol-based nucleoside phosphonic acids were tested for cytotoxic and antiviral properties, but no significant activity was found.