Development of Biocompatible Ene-Ligation Enabled by Prenyl-Based β-Caryophyllene with Triazoline/Selectfluor under Physiological Conditions

et al. 2022

Preprint

Self Cite

Chemical modifications in RNAs play critical roles in structural diversification and functional regulation of many vital biochemical processes. Several hydrophobic prenyl-modifications have been discovered in a variety of RNA species since the 1990s. Prenyl groups are the feedstocks of terpene and many other biological molecules and the processes of prenylation in different macromolecules have been widely studied. We present here a new chemical biology technique to identify and label i6A, a prenyl-modified RNA residue, based on the unique reactivity of the prenyl group. We also found that iodine-mediated cycloaddition reactions of the prenyl group occurs in a superfast manner, and converts i6A from a hydrogen-bond acceptor into a donor. Based on this reactivity, we developed an iodine-mediated oxidation and reverse transcription (IMORT) method to profile cellular i6A residues with a single-base resolution, allowing for the transcriptome-wide detection and analysis of various i6A-containing RNA species.

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Bioorthogonal in-cell Labeling and Profiling of N⁶-isopentenyladenosine (i⁶A) Modified RNA

Wang

et al. 2022

Preprint

Self Cite

“…Next, the dual fluorescent labeling of the target protein lysozyme with citronellol, as well as HRAS and KRAS, has also been evaluated via NEL (SI Figures S17−19) and SACR approaches (SI Figures S20 −21). With respect to citronellolmodified lysozymes, the dual fluorescent labelings with the 1-NO pyrene (2t, 50 μM) targeting lysine residue as well as the PTAD-DBCO-Cy5 (50 μM) 31 targeting tyrosine residue have been carried out simultaneously. It turned out that the in-gel blue fluorescence, red fluorescence, as well as merged pink fluorescence have been determined.…”

Section: Explorations Of the Reaction On Proteinsmentioning

confidence: 99%

Deciphering Regulatory Proteins of Prenylated Protein via the FRET Technique Using Nitroso-Based Ene-Ligation and Sequential Azidation and Click Reaction

Gan

et al. 2022

Org. Lett.

Self Cite

We report here the selective incorporations of nitroso species into a wide range of proteins targeting lysine residue(s). The corresponding azo functionalities were formed in a highly selective manner with excellent yields, displaying rather good stability under physiological conditions. Furthermore, the azodation proceeded smoothly in high yields on targeted peptides. Fluorescent and/or dual fluorescent labeling of varied proteins following this protocol have been determined efficiently and selectively. With this established protocol, we aim to determine its usage in the evaluation of the interaction of prenylated proteins with their interacted enzyme(s) via FRET assays. Delightedly, chemically modified proteins with a 1-pyrenyl fluorophore through 254 nm UV irradiation and the sequential azodation and click reaction of protein prenyl functionality, which enable the incorporation of naphthene, indeed increase the fluorescence energy transferred since we observed significantly enhanced absorption located at 218 nm in lysed HEK293T cells and a clearly strengthened greenish fluorescence in living HEK293T cells.

“…For instance, the use of IEDDA reaction has been limited in living cells due to the fact that thiol groups in cysteines of many proteins may also react with the coupling reagent trans -cyclooctene (TCO) . Our research group has been interested in lipid modifications on nucleic acids in the past decade. − Toward the labeling, sequencing, and manipulation of these unique and important natural RNA modifications, we have developed the ene-ligation method by taking advantage of the reactivity between naturally occurring and strain-promoted β-caryophyllene (βCP) and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) . The reaction displays a great potential in biocompatible ligation with high efficiency and selectivity, showing a kinetic of 1.0 mol –1 ·s –1 , which advances over the classic strain-promoted azide–alkyne click chemistry reaction.…”

Section: Introductionmentioning

confidence: 99%

Development of Nitroso-Based Probes for Labeling and Regulation of RAS Proteins in Cancer Cells via Sequential Ene-Ligation and Oxime Condensation

Liu

et al. 2023

J. Org. Chem.

Self Cite

Prenyl functionalities have been widely discovered in natural products, nucleic acids, and proteins with significant biological roles in both healthy and diseased cells. In this work, we develop a series of new nitroso-based probes for the labeling, enrichment, and regulation of prenylated RAS protein, which is highly associated with ∼20% of human cancers and used to be regarded as an “undruggable” target via a sequential ene-ligation and oxime condensation (SELOC) process. We found that these nitroso species can rapidly react with prenyl-containing molecules through ene-ligation and install a molecular tag for functional applications under physiological conditions. We first investigated this ligation process on two peptide models and demonstrated its labeling efficiency on various proteins such as myoglobin, lysozyme, RNase A, BSA, and HSP40. We further coupled this reactive platform with proteolysis-targeting chimera technology targeting to increase its efficiency and accuracy, as well as to expand its application range. Using the prenylated RAS protein as the model, we demonstrated that RAS could be efficiently decorated with our nitroso probes, which further condensate with oxime and rapidly react with a pomalidomide-containing hydroxylamine probe for protein degradation. As a result, the RAS protein in both HeLa and A549 cell lines has been determined to be efficiently degraded both in vitro and in vivo. This is the first case targeting post-translational modification other than ligand–protein interaction to degrade and regulate RAS proteins. We envision that our SELOC strategy will have great potential in studying the fundamental structures and functions of prenylated biomolecules and developing new drugs based on these unique cellular pathways.