Graphene as adsorbent for highly efficient extraction of modified nucleosides in urine prior to liquid chromatography-tandem mass spectrometry analysis

RNA modifications have been involved in numerous biological processes, and aberrations of these modifications are tightly associated with various diseases including cancer. Herein, we developed graphenebased solid-phase extraction and robust ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) combined with stable isotope-dilution for simultaneous enrichment and accurate determination of 17 modified nucleosides in human urine. We found graphene could effectively adsorb various modified nucleosides in human urine samples. With this method, we identified and quantified these modified nucleosides in urine samples collected from lung cancer patients and healthy controls.We revealed that the levels of 12 modified nucleosides were all diminished in urine from lung cancer patients, compared with healthy controls. It is worth noting that we demonstrated, for the first time, the presence of 5,2-O-dimethyluridine(m~5U_m) in human urine. Together, we established a robust analytical method for simultaneous determinations of 17 modified nucleosides in human urine, and our results revealed a close correlation between the concentrations of urinary modified nucleosides and the occurrence of lung cancer, implying the potential applications of these modified nucleosides as noninvasive biomarkers for the early detection of lung cancer. Moreover, this study will stimulate future investigations on the regulatory roles of RNA modifications in the initiation and progression of lung cancer.

Metabolic Labeling and Imaging of Cellular RNA via Bioorthogonal Cyclopropene−Tetrazine Ligation

RNA labeling is vital for the study of an RNA structure,cellular distribution,localization,and metabolism.Herein,we report N6 cyclopropane-modified adenosine(cpA)as a new analog for metabolic RNA labeling.We successfully applied inverse electrondemand Diels–Alder(iEDDA)chemistry to label cellular RNA with cpA.This labeling technique is practical and provides a new platform to study RNA roles in cells in a metal-free manner.This simple and robust assay represents a significant advancement in the profiling methods of the nascent transcriptome using chemical approaches.

Synthesis and cytotoxic evaluation of substituted 3-(3'-indolyl-/3'-pyridyl)-isoxazolidines and bis-indoles

Regio-and stereoselective 1,3-dipolar cycloadditions of C-(3-indolyl)-N-phenylnitrone(10)were carried out with different mono-substituted,disubstituted and cyclic dipolarophiles under mono-mode microwave irradiation to obtain substituted 3-(indol-30-yl)-N-phenyl-isoxazolidines(16–22).Reactions of nitrone(10)with allenic esters under similar conditions afforded,via a domino process,bis-indole derivatives(23a–c)along with compounds 24 and 25.Similarly,reactions of C-(3-pyridyl)-N-phenylnitrone(26)with mono-substituted,disubstituted and cyclic dipolarophiles were carried out in refluxing dry toluene to obtain substituted 3-(30-pyridyl)-Nphenylisoxazolidines(27–34).Some of the compounds(16f,18b,23a,23c,27c and 29f)display significant cytotoxicity against a number of human cancer cell lines.

Systematic investigation of bioorthogonal cellular DNA metabolic labeling in a photo-controlled manner

Bioorthogonal cleavage and ligation reactions together form one more integrated system about the repertoire of bioorthogonal chemistry,capacitating an array of thrilling new biological applications.The bond-cleavage type and position of biomolecular remain a great challenge,which determines the metabolic pathway of the targets in living systems.Herein we designed two linkages of methylene and carbonyl group attached the N-3 position of the 5-ethynyl-2’-deoxyuridine(EdU)base or the oxygen atom at deoxyribose 3’position to a photocaging group,which would be cleaved by irradiation with 365 nm ultraviolet light.EdU derivatives linked by methylene at the N-3 position had better photodecage efficiency and stability in the absence of light.This paper provides a strategy for studying the nucleoside metabolic pathways in cells,which can easily and conveniently evaluate the effect of the position and type of the linkages.The developed strategy affords a reference for controlling spatial and temporal metabolism of small-molecule drugs,allowing direct manipulation of intact cells under physiological conditions.