Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure.

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.

Photochemical Reactivity of Naphthol-Naphthalimide Conjugates and Their Biological Activity.

Quinone methide precursors 1a-e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10-2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10-5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105-106 M-1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M-1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a-e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.

Substituted adamantylphthalimides: Synthesis, antiviral and antiproliferative activity.

In this study, three groups of adamantylphthalimides, bearing different substituents at the phthalimide moiety, N-(4'-R2 )phthalimidoadamantanes (1-7), 3-[N-(4'-R2 )phthalimido]-1-adamantanols (8-10), and 3-[N-(4'-R2 )phthalimido]adamantane-1-carboxylic acids (11-15), were synthesized and screened against tumor cells and viruses. The most potent compounds are not substituted at the adamantane and bear an OH or NH2 substituent at the phthalimide (compounds 3 and 5). The antiproliferative activities of compounds 3 and 5 are in the micromolar range, much higher than the one of thalidomide. A minor antiviral activity against cytomegalovirus and varicella-zoster virus was found for compounds 3 and 5, but these compounds lacked selectivity. The results presented are important for the rational design of the next-generation compounds with anticancer and antiviral activities.