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.

Methylation and alkylation are important techniques used for the synthesis and derivatisation of small molecules and natural products. Application of S‐adenosylmethionine (SAM)‐dependent methyltransferases (MTs) in biotechnological hosts such as Escherichia coli lowers the environmental impact of alkylation compared to chemical synthesis and facilitates regio‐ and chemoselective alkyl chain transfer. Here, we address the limiting factor for SAM synthesis, methionine supply, to accelerate in vivo methylation activity. Introduction of the direct sulfurylation pathway, consisting of O‐acetylhomoserine sulfhydrolase (ScOAHS) and O‐acetyltransferase (ScMET2), from S. cerevisiae into E. coli and supplementation with methanethiol or the corresponding disulfide improves atom‐economic methylation activity in three different MT reactions. Up to 17‐fold increase of conversion compared to the sole expression of the MT and incorporation of up to 79 % of the thiol compound added were achieved. Promiscuity of ScOAHS allowed in vivo production of methionine analogues from organic thiols. Further co‐overproduction of a methionine adenosyltransferase yielded SAM analogues which were further transferred by MTs onto different substrates. For methylation of non‐physiological substrates, conversion rates up to 73 % were achieved, with an isolated yield of 41 % for N‐methyl‐2,5‐aminonitrophenol. The here described technique enables E. coli to become a biotechnological host for improved methylation and selective alkylation reactions.

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 (Φ_F ≈ 10⁻²). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (Φ_R ≈ 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 K_a ≈ 10⁵–10⁶ M⁻¹, as well as with bovine serum albumin (BSA) K_a ≈ 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.

Methylation and alkylation are important techniques used for the synthesis and derivatisation of small molecules and natural products. Application of S‐adenosylmethionine (SAM)‐dependent methyltransferases (MTs) in biotechnological hosts such as Escherichia coli lowers the environmental impact of alkylation compared to chemical synthesis and facilitates regio‐ and chemoselective alkyl chain transfer. Here, we address the limiting factor for SAM synthesis, methionine supply, to accelerate in vivo methylation activity. Introduction of the direct sulfurylation pathway, consisting of O‐acetylhomoserine sulfhydrolase (ScOAHS) and O‐acetyltransferase (ScMET2), from S. cerevisiae into E. coli and supplementation with methanethiol or the corresponding disulfide improves atom‐economic methylation activity in three different MT reactions. Up to 17‐fold increase of conversion compared to the sole expression of the MT and incorporation of up to 79 % of the thiol compound added were achieved. Promiscuity of ScOAHS allowed in vivo production of methionine analogues from organic thiols. Further co‐overproduction of a methionine adenosyltransferase yielded SAM analogues which were further transferred by MTs onto different substrates. For methylation of non‐physiological substrates, conversion rates up to 73 % were achieved, with an isolated yield of 41 % for N‐methyl‐2,5‐aminonitrophenol. The here described technique enables E. coli to become a biotechnological host for improved methylation and selective alkylation reactions.