Subhendu Sekhar Bag

T7 RNA polymerase (T7-RNAP) is a highly efficient transcriptional enzyme. However, it is prone to mutation at the functionally important Y571 residue, which strongly influences its functional properties. Here, we demonstrate a strategy to modulate the T7-RNAP enzyme by replacing the Y571 residue with a fluorescent triazolyl unnatural amino acid (FTUAA), TCNBAlaAc, via genetic code expansion (GCE) technology using two orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Thus, TCNBAlaAc-encoded variants, herein termed SSBT7-RNAP, were successfully expressed, purified, and characterized by SDS-PAGE, MALDI-TOF mass spectrometry, CD, UV–vis, and fluorescence spectroscopy. Incorporation of TCNBAlaAc introduced distinct and enhanced fluorescence signatures while largely preserving the overall secondary structures of the engineered enzymes. Notably, these novel TCNBAlaAc-encoded variants exhibited higher relative RNA yields than those of wild-type T7-RNAP under identical in vitro transcription conditions. Molecular docking further supported the structural compatibility of the modification with the promoter-bound enzyme. These findings establish GCE-based, site-specific incorporation of an FTUAA into a functionally relevant site of T7 RNA polymerase as a promising strategy for generating spectroscopically traceable, transcriptionally competent T7-RNAP variants for future enzyme engineering and mechanistic studies.

A series of triazole‐scaffolded enediyne‐amino acid hybrids featuring donor‐acceptor substituted aromatics at the two acetylenic termini has been rationally designed and synthesized. Their solid‐state thermal reactivity toward Bergman cyclization was investigated using differential scanning calorimetry (DSC), revealing the following reactivity trend: neutral–neutral > donor–acceptor (D–A) > donor–donor (D–D) > acceptor–acceptor (A–A). To rationalize this trend, both electronic perturbations and geometric distortions were considered and analyzed at the B3LYP/6‐31G(d,p) level of theory. Natural Bond Orbital (NBO) calculations highlight the critical role of through‐bond charge‐transfer‐delocalization in stabilizing the diradical transition state, particularly in D–A substituted enediynes. In contrast, A–A substituted enediynes exhibited destabilization due to increased dihedral distortion prohibiting through‐bond charge‐transfer‐delocalization, leading to coulombic repulsion at the C‐C bond‐forming termini in the TS, thereby hindering effective cyclization. Furthermore, the steric influence of the amino acid moiety linked to the triazole‐N of the hybrid was found to induce out‐of‐plane distortion in one of the enediyne arms, thereby playing a vital role in modulating thermal reactivity. UV–Vis spectroscopy, in conjunction with TD‐DFT calculations, further supports the influence of through‐bond charge‐transfer‐delocalization in tuning thermal reactivity. Overall, a notable enhancement in thermal Bergman cyclization reactivity via through‐bond charge‐transfer‐delocalization‐mediated stabilization of diradical TS is observed for donor–acceptor substituted enediynes.

The decoration of designer enediynes with donor-acceptor aromatics has been devised. The effect of electron donor-acceptor substituents on the two acetylenic termini of the enediynes on the thermal reactivity toward Bergman cyclization (BC) has been investigated. The thermal reactivity in terms of onset temperature from differential scanning calorimetry (DSC) among the various classes of enediynes follows the order: acceptor-acceptor > acceptor-donor > donor-donor. The reactivity variances among the designer enediynes were explained in terms of possible differences in through-bond charge transfer/withdrawal delocalization interactions, leading to stabilization of the TS of a possible cycloaromatization reaction. Further, the repulsion of the in-plane π-orbital is decreased while enediyne proceeds toward the TS in the case of acceptor-acceptor enediynes. This is in contrast to the effect of the presence of the donor-acceptor or donor-donor substituents. A UV-visible spectroscopic study and an NBO analysis support the charge delocalization stabilization of the activated complex of enediynes containing acceptor substituents in both arms. Overall, an electron-withdrawing substituent offers an accelerating effect on the thermal cyclization, resulting in the observed reactivity order.

Decorating biomolecular building blocks, such as amino acids, to afford desired and tuneable photophysical/biophysical properties would allow chemical biologists to use them for several biotechnological and biosensing applications. While many synthetic methodologies have been explored in this direction, advantages provided by click-derived triazole moieties are second to none. However, since their discovery, click-mediated triazoles have been majorly utilised as linkers for conjugating biomolecules, creating materials with novel properties, such as polymers or drug conjugates. Despite exploring their profound role as linkers, click-mediated triazoles as an integral part of biomolecular building blocks have not been addressed. 1,2,3-Triazole, a transamide mimic, exhibits high aromatic stacking propensity, high associability with biomolecules through H-bonding, and high stability against enzymatic hydrolysis. Furthermore, triazoles can be considered donors useable for installation/modulation of the photophysics of a fluorophore. Therefore, triazole with a chromophoric unit may rightly be utilised as an integral part of biomolecular building blocks to install microenvironment-sensitive solvofluorochromic properties suitable for biological sensing, studying inter-biomolecular interactions and introducing novel physicochemical properties in a biomolecule. This review mainly focuses on the facets of click-derived triazole in designing novel fluorescent amino acids and peptides with a particular emphasis on those wherein triazole acts as an integral part of amino acids, i.e. the side chain, generating a new class of fluorescent unnatural triazolyl amino acids. Thus, fluorescent triazolyl unnatural amino acids, peptidomimetics with such amino acids and aliphatic/aromatic triazolyl amino acids as scaffolds for peptidomimetics are the central part. However, to start with, a brief history, followed by a discussion on various other relevant facets of triazoles as linkers in various fields ranging from therapeutics, materials science, diagnostics, and bioconjugation to peptidomimetics, is cited. Additionally, the possible roles of CuAAC-mediated triazoles in shaping the future of bioorganic chemistry, medicinal chemistry, diagnostics, nucleoside chemistry and protein engineering are briefly discussed.

Chemical-capture-mediated sensing has had a great impact on proteomic research. Toward this end, we demonstrate the chemical trapping of BSA by the reactive formyl functionality of a newly developed fluorescent nucleoside probe, formylphenothiazine-labeled-2′-deoxyuridine. The probe is capable of trapping BSA via Schiff base formation leading to fluorescence ‘switch-on’ sensing with a large hypsochromic shift of ca. 100 nm. The α-amylase does not show any significant change in fluorescence response, demonstrating the efficiency of the probe in selective sensing of BSA. Docking studies suggest the preferential interaction of the phenothiazinylcarbaldehyde-labeled dU with the residual amino acids in site I of the BSA protein as compared to site II.