The 5' cap is a hallmark of eukaryotic mRNA involved in the initiation of translation. Its modification with a single photo-cleavable group can bring translation of mRNA under the control of light. However, UV irradiation causes cell stress and downregulation of translation. Furthermore, complex processes often involve timed expression of more than one gene. The approach would thus greatly benefit from the ability to photo-cleave by blue light and to control more than one mRNA at a time. We report the synthesis of a 5' cap modified with a 7-(diethylamino)coumarin (CouCap) and adapted conditions for in vitro transcription. Translation of the resulting CouCap-mRNA is muted in vitro and in mammalian cells, and can be initiated by irradiation with 450 nm. The native cap is restored and no non-natural residues nor sequence alterations remain in the mRNA. Multiplexing for two different mRNAs was achieved by combining cap analogs with coumarinand ortho-nitrobenzyl-based photo-cleavable groups.

Conspectus The central dogma of molecular biology hinges on messenger RNA (mRNA), which presents a blueprint of the genetic information encoded in the DNA and serves as a template for translation into proteins. In addition to its fundamental importance in basic research, this class of biomolecules has recently become the first approved Covid vaccine, underscoring its utility in medical applications. Eukaryotic mRNA is heavily processed, including the 5′ cap as the primary hallmark. This 5′ cap protects mRNA from degradation by exoribonucleases but also interacts specifically with several proteins and enzymes to ensure mRNA turnover and processing, like splicing, export from the nucleus to the cytoplasm, and initiation of translation. The absence of a 5′ cap leads to a strong immune response, and the methylation status contributes to distinguishing self from non-self RNA. Non-natural modifications of the 5′ cap provide an avenue to label mRNAs and make them accessible to analyses, which is important to study their cellular localization, trafficking, and binding partners. They bear potential to engineer mRNAs, e.g., more stable or immunogenic mRNAs that are still translated, by impacting select interactions in a distinct manner. The modification of the 5′ cap itself is powerful as it can be applied to make long mRNAs (∼1000 nt, not directly accessible by solid-phase synthesis) by in vitro transcription. This Account describes our contribution to the field of chemo-enzymatic modification of mRNA at the 5′ cap. Our approach relies on RNA methyltransferases (MTases) with promiscuous activity on analogues of their natural cosubstrate S-adenosyl-L-methionine (AdoMet). We will describe how RNA MTases in combination with non-natural cosubstrates provide access to site-specific modification of different positions of the 5′ cap, namely, the N2 and N7 position of guanosine and the N6 position of adenosine as the transcription start nucleotide (TSN) and exemplify strategies to make long mRNAs with modified 5′ caps. We will compare the chemical and enzymatic synthesis of the AdoMet analogues used for this purpose. We could overcome previous limitations in methionine adenosyltransferase (MAT) substrate scope by engineering variants (termed PC-MATs) with the ability to convert methionine analogues with benzylic and photocaging groups at the sulfonium ion. The final part of this Account will highlight applications of the modified mRNAs. Like in many chemo-enzymatic approaches, a versatile strategy is to install small functional groups enzymatically and use them as handles in subsequent bioorthogonal reactions. We showed fluorescent labeling of mRNAs via different types of click chemistry in vitro and in cells. In a second line of applications, we used the handles to make mRNAs amenable for analyses, most notably next-generation sequencing. In the case of extremely promiscuous enzymes, the direct installation of photo-cross-linking groups was successful also and provided a way to covalently bind protein-interaction partners. Fina...

Tropolone (2‐hydroxycyclohepta‐2,4,6‐triene‐1‐one and tautomer) is a non‐benzenoid bioactive natural chromophore with pH‐dependent fluorescence character and extraordinary metal binding affinities, especially with transition‐metal ions Cu2+/Zn2+/Ni2+. This report describes the syntheses and biophysical studies of a new tropolonyl thymidine [(4(5)‐hydroxy‐5(4)‐oxo‐5(4)H‐cyclohepta‐1,3,6‐trienyl)thymidine] (tr‐T) nucleoside and of corresponding tropolone‐conjugated DNA oligonucleotides that form B‐form DNA duplex structures with a complementary DNA strand, although their duplex structures are less stable than that of the control. Furthermore, the stabilities of those DNA duplex structures are lowered by the presence of increasing numbers of tr‐T residue or by decreasing pH of their environments. Most importantly, these duplex structures are made fluorescent because of the presence of the tropolone moieties conjugated to the thymidine residues. The fluorescence behavior of those duplex structures exhibits pH dependence, with stronger fluorescence at lower pH and weaker fluorescence at high pH. Importantly, the fluorescence characters of tr‐DNA oligonucleotides are significantly enhanced by nearly threefold after duplex structure formation with their complementary control DNA oligonucleotide. Further, the fluorescence behavior of these tr‐DNA duplex structures is also dependent on the pH conditions. Hence, tropolonyl‐conjugated DNA represents a class of new fluorescent analogues that might be be employed for sensing DNA duplex formation and provide opportunities to improve fluorescence properties further.

Pyrazole, pyrazolone, and aminopyrazolone derived molecules are bioactive molecules and considered as potential therapeutic drug candidates because of their unique structural properties. These molecules have abilities to interact with several bio‐macromolecules via non‐covalent interactions such as hydrogen bonding and π–π interactions. In structural organization of dipeptides, pyrazole containing aromatic amino acid/dipeptides have been explored and considered as potential amino acid residue. In repertoire of unnatural aromatic amino acids, this report describes the synthesis of 4‐aminopyrazolonyl containing amino acids and their crystal structures. The incorporation of 4‐aminopyrazolonyl at N‐terminal of native amino acid/dipeptides influences the conformational changes of respective peptide which induces the formation of distinctive supramolecular self‐assembly structures such as β‐sheet and α‐helices in their solid‐state crystal. The structural conformation of those peptides, here, are also demonstrated in solution phase by 1H‐NMR (1D/2D) and [D6]DMSO titration methods which support the formation of inter‐/intramolecular hydrogen bonding in solution. Hence, these unnatural amino acid analogues can tune the secondary structure of natural amino acid/peptides by introducing at N‐terminal via amide bond.