Hetero Diels-Alder (HDA) reactions with the participation of E-2-aryl-1-cyano-1-nitroethenes and methylenecyclopentane were evaluated on the basis of experimental as well as quantumchemical data. It was found that contrary to most known HDA reactions, title processes are realised under non-catalytic conditions and with full regiocontrol. The DFT study shows, without any doubt, the polar but single-step reaction mechanism. Deeper exploration using Bonding Evolution Theory (BET) techniques gives a clear image of the sequences of electron density reorganisation along the reaction coordinate. The first C4-C5 bond is created in phase VII by merging two monosynaptic basins, while the second O1-C6 bond is created in the last phase by a donation of the nonbonding electron density of O1 to C6. Based on the research, we can conclude that the analysed reaction proceeds according to a two-stage one-step mechanism.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch of the histidine triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the structure of the human HINT1-adenosine 5'-monophosphate (AMP) complex at 1.38 Å resolution obtained from a new monoclinic crystal form is reported. The final structure has R _cryst = 0.1207 (R _free = 0.1615) and the model exhibits good stereochemical quality. Detailed analysis of the high-resolution data allowed the details of the protein structure to be updated in comparison to the previously published data.

A 52-nucleotide DNA/2′-OMe-RNA oligomer mimicking 10–23 DNAzyme in the complex with its substrate was synthesized, purified and crystallized by the hanging-drop method using 0.8 M sodium potassium tartrate as a precipitant. A data set to 1.21 Å resolution was collected from a monocrystal at 100 K using synchrotron radiation on a beamline BL14.1 at BESSY. The crystal belonged to the P2₁ group with unit-cell a = 49.42, b = 24.69, c = 50.23, β = 118.48.

Oxathiaphospholane derivatives of 2′-OMe-ribonucleosides and 2′-O-TBDMS-ribonucleosides (^MN-OTP and ^TN-OTP, respectively; nucleobase protected) were synthesized and separated into pure P-diastereomers. X-ray analysis showed the R_P absolute configuration of the phosphorus atom in the fast-eluting diastereomer of ^TA-OTP. The fast- and slow-eluting P-diastereomers of ^MN-OTP and ^TN-OTP were used in the solid-phase synthesis of phosphorothioate dinucleotides (^MN_PST and N_PST, respectively), which were subsequently hydrolyzed with R_P-selective phosphodiesterase svPDE and S_P-selective nuclease P1 to determine the absolute configuration of the phosphorus atoms. P-Stereodefined phosphorothioate ([PS]) 10-mer chimeric oligomers [PS]-{DNA:(2′-OMe)-RNA} and isosequential [PS]-{DNA:RNA} containing two ^MN_PS or N_PS units were synthesized. Melting experiments performed for their complexes with Watson–Crick paired DNA matrix showed that ^MN_PS or N_PS units decrease the thermal stability of the duplexes (ΔT_m = −0.5 ÷ −5.5 °C per modification) regardless of the absolute configuration of the P-atoms. When the (2′-OMe)-RNA matrix was used an increase in T_m was noted in all cases (ΔT_m = +1 ÷ +7 °C per modification). The changes in thermal stability of the duplexes formed by [PS]-chimeras with DNA and (2′-OMe)-RNA matrices do not correlate with the absolute configuration of the phosphorus atoms.

Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet. An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2'-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution. HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2'-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar K_d and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors. Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2. Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.