Recently, a new type of nucleic acid analogues with modified phosphate group, namely, phosphoryl guanidine oligonucleotides, has been described. In the present work, we assess the difference between diastereomers of a mono-substituted phosphoryl guanidine oligonucleotide and analyze their resistance to nuclease digestion. Individual diastereomers ('fast' and 'slow') of a trideoxynucleotide d (TpCp*A) were isolated by reverse-phase HPLC. Snake venom phosphodiesterase digestion showed that the native trideoxynucleotide was fully degraded after 30 min, whereas both 'fast' and 'slow' diastereomers of d (TpCp*A) were not completely digested even after 7 days. UV and CD spectra revealed similarities in the structure of the diastereomers. Structural analysis by 1D and 2D NMR spectroscopy also uncovered significant similarity in the properties of Rp and Sp diastereomers. Structural analysis of nuclear Over-hauser effect spectroscopy (NOESY) data and restrained molecular dynamics methods showed very flexible single-stranded oligonucleotide structures. Detailed computational analysis of restraint penalty energies via restrained molecular dynamics simulations with the 2D NMR interproton distance data allowed us to conclude that most likely, the 'fast' isomer is the Sp diastereomer, and the 'slow' isomer is the Rp diastereomer. (C) 2019 Elsevier Inc. All rights reserved.