N⁶-Threonylcarbamoyladenosine (t⁶A) and its derivatives are universally conserved modified nucleosides found at position 37, 3΄ adjacent to the anticodon in tRNAs responsible for ANN codons. These modifications have pleiotropic functions of tRNAs in decoding and protein synthesis. In certain species of bacteria, fungi, plants and protists, t⁶A is further modified to the cyclic t⁶A (ct⁶A) via dehydration catalyzed by TcdA. This additional modification is involved in efficient decoding of tRNA^Lys. Previous work indicated that the chemical structure of ct⁶A is a cyclic active ester with an oxazolone ring. In this study, we solved the crystal structure of chemically synthesized ct⁶A nucleoside. Unexpectedly, we found that the ct⁶A adopted a hydantoin isoform rather than an oxazolone isoform, and further showed that the hydantoin isoform of ct⁶A was actually present in Escherichia coli tRNAs. In addition, we observed that hydantoin ct⁶A is susceptible to epimerization under mild alkaline conditions, warning us to avoid conventional deacylation of tRNAs. A hallmark structural feature of this isoform is the twisted arrangement of the hydantoin and adenine rings. Functional roles of ct⁶A37 in tRNAs should be reconsidered.

Along with the adoption of three-dimensional electron diffraction (3D ED/MicroED) as a mainstream tool for structure determination from sub-micron single crystals, questions about best practices regarding each step along the workflow, from data collection to structure solutions, arise. In this paper, we discuss three particular aspects of a 3D ED/MicroED experiment which, after hundreds of structures solved in Rigaku’s laboratories, we have found to be important to consider carefully. First, for a representative model system of a hydrated compound (trehalose dihydrate), we show that cryo-transfer of the sample into the diffractometer is an effective means to prevent dehydration, while cooling of the sample without cryo-transfer yields a marginal improvement only. Next, we demonstrate for a small (tyrosine) and a large (clarithromycin) organic compound, how a simplified and fast workflow for dynamical diffraction calculations can determine absolute crystal structures with high confidence. Finally, we discuss considerations and trade-offs for choosing an optimal effective crystal-to-detector distance; while a long distance is mandatory for a protein (thaumatin) example, even a small molecule with difficult diffraction behavior (cystine) yields superior results at longer distances than the one used by default.

The development of the unprecedented ortho-regioselective inverse-electron-demand hetero-Diels–Alder (IEDHDA) reaction is described. It has been demonstrated that by proper choice of reactants and reaction conditions the inverse-electron-demand hetero-Diels–Alder cycloaddition can be realized with unprecedented regioselectivity arising from the reaction between the terminal carbon atom of the dienophile and the heteroatom of the heterodiene.

The synthesis of α-, β- and γ-substituted α-phosphono-γ-lactones was accomplished using different ring closure and ring homologation strategies. It was found that the lactones could be selectively transformed into the corresponding ethyl cyclopropanecarboxylates by treatment with sodium ethoxide in boiling THF. The reported reaction provides an attractive alternative to the classical homologous Horner–Wadsworth–Emmons approach to the construction of cyclopropanes with electron-withdrawing functionalities.