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ACS Chemical Biology Dec 2016Diazo groups have broad and tunable reactivity. That and other attributes endow diazo compounds with the potential to be valuable reagents for chemical biologists. The... (Review)
Review
Diazo groups have broad and tunable reactivity. That and other attributes endow diazo compounds with the potential to be valuable reagents for chemical biologists. The presence of diazo groups in natural products underscores their metabolic stability and anticipates their utility in a biological context. The chemoselectivity of diazo groups, even in the presence of azido groups, presents many opportunities. Already, diazo compounds have served as chemical probes and elicited novel modifications of proteins and nucleic acids. Here, we review advances that have facilitated the chemical synthesis of diazo compounds, and we highlight applications of diazo compounds in the detection and modification of biomolecules.
Topics: Alkylation; Amino Acids; Animals; Biochemistry; Biological Products; Cycloaddition Reaction; Diazonium Compounds; Humans; Indicators and Reagents; Models, Molecular; Nucleic Acids; Proteins
PubMed: 27739661
DOI: 10.1021/acschembio.6b00810 -
Accounts of Chemical Research Oct 2023The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are...
The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are widespread in biology, occurring in all kingdoms of life and in all classes of RNA molecules. They regulate RNA structure, folding, and protein-RNA interactions, and have important roles in fundamental gene expression processes involving mRNA, tRNA, rRNA, and other types of RNA species. Our understanding of RNA modifications has advanced considerably; however, there are still many outstanding questions regarding the distribution of modifications across all RNA transcripts and their biological function. One of the major challenges in the study of RNA modifications is the lack of sequencing methods for the transcriptome-wide mapping of different RNA-modification structures. Furthermore, we lack general strategies to characterize RNA-modifying enzymes and RNA-modification reader proteins. Therefore, there is a need for new approaches to enable integrated studies of RNA-modification chemistry and biology.In this Account, we describe our development and application of chemoproteomic strategies for the study of RNA-modification-associated proteins. We present two orthogonal methods based on nucleoside and oligonucleotide chemical probes: 1) RNA-mediated activity-based protein profiling (RNABPP), a metabolic labeling strategy based on reactive modified nucleoside probes to profile RNA-modifying enzymes in cells and 2) photo-cross-linkable diazirine-containing synthetic oligonucleotide probes for identifying RNA-modification reader proteins.We use RNABPP with C5-modified cytidine and uridine nucleosides to capture diverse RNA-pyrimidine-modifying enzymes including methyltransferases, dihydrouridine synthases, and RNA dioxygenase enzymes. Metabolic labeling facilitates the mechanism-based cross-linking of RNA-modifying enzymes with their native RNA substrates in cells. Covalent RNA-protein complexes are then isolated by denaturing oligo(dT) pulldown, and cross-linked proteins are identified by quantitative proteomics. Once suitable modified nucleosides have been identified as mechanism-based proteomic probes, they can be further deployed in transcriptome-wide sequencing experiments to profile the substrates of RNA-modifying enzymes at nucleotide resolution. Using 5-fluorouridine-mediated RNA-protein cross-linking and sequencing, we analyzed the substrates of human dihydrouridine synthase DUS3L. 5-Ethynylcytidine-mediated cross-linking enabled the investigation of ALKBH1 substrates. We also characterized the functions of these RNA-modifying enzymes in human cells by using genetic knockouts and protein translation reporters.We profiled RNA readers for -methyladenosine (mA) and -methyladenosine (mA) using a comparative proteomic workflow based on diazirine-containing modified oligonucleotide probes. Our approach enables quantitative proteome-wide analysis of the preference of RNA-binding proteins for modified nucleotides across a range of affinities. Interestingly, we found that YTH-domain proteins YTHDF1/2 can bind to both mA and mA to mediate transcript destabilization. Furthermore, mA also inhibits stress granule proteins from binding to RNA.Taken together, we demonstrate the application of chemical probing strategies, together with proteomic and transcriptomic workflows, to reveal new insights into the biological roles of RNA modifications and their associated proteins.
Topics: Humans; Nucleosides; Adenosine; Proteomics; Diazomethane; Oligonucleotide Probes; RNA; AlkB Homolog 1, Histone H2a Dioxygenase
PubMed: 37733063
DOI: 10.1021/acs.accounts.3c00450 -
Biomaterials Nov 2020Driven by the clinical need for a strong tissue adhesive with elastomeric material properties, a departure from legacy crosslinking chemistries was sought as a...
Driven by the clinical need for a strong tissue adhesive with elastomeric material properties, a departure from legacy crosslinking chemistries was sought as a multipurpose platform for tissue mending. A fresh approach to bonding wet substrates has yielded a synthetic biomaterial that overcomes the drawbacks of free-radical and nature-inspired bioadhesives. A food-grade liquid polycaprolactone grafted with carbene precursors yields CaproGlu. The first-of-its-kind low-viscosity prepolymer is VOC-free and requires no photoinitiators. Grafted diazirine end-groups form carbene diradicals upon low energy UVA (365 nm) activation that immediately crosslink tissue surfaces; no pre-heating or animal-derived components are required. The hydrophobic polymeric environment enables metastable functional groups not possible in formulations requiring solvents or water. Activated diazirine within CaproGlu is uniquely capable of crosslinking all amino acids, even on wet tissue substrates. CaproGlu undergoes rapid liquid-to-biorubber transition within seconds of UVA exposure-features not found in any other bioadhesive. The exceptional shelf stability of CaproGlu allows gamma sterilization with no change in material properties. CaproGlu wet adhesiveness is challenged against current unmet clinical needs: anastomosis of spliced blood vessels, anesthetic muscle patches, and human platelet-mediating coatings. The versatility of CaproGlu enables both organic and inorganic composites for future bioadhesive platforms.
Topics: Adhesiveness; Animals; Biocompatible Materials; Diazomethane; Humans; Tissue Adhesives; Viscosity
PubMed: 32891870
DOI: 10.1016/j.biomaterials.2020.120215 -
Analytical Methods : Advancing Methods... Nov 2021For electrochemical immunosensors, inexpensive electrodes with fast redox kinetics, and simple stable methods of electrode functionalization are vital. However, many...
For electrochemical immunosensors, inexpensive electrodes with fast redox kinetics, and simple stable methods of electrode functionalization are vital. However, many inexpensive and easy to fabricate electrodes suffer from poor redox kinetics, and functionalization can often be difficult and/or unstable. Diazonium tosylates are particularly stable soluble salts that can be useful for electrode functionalization. Recently developed thermoplastic electrodes (TPEs) have been inexpensive, moldable, and highly electroactive carbon composite materials. Herein, the synthesis and grafting of diazonium tosylate salts were optimized for modification of TPEs and used to develop the first TPE immunosensors. With diazonium tosylates, TPEs were amine functionalized either directly through grafting of -aminophenyl diazonium salt or indirectly through grafting -nitrophenyl diazonium salt followed by electrochemical reduction to an amine. Diazonium tosylates were synthesized as a paste in 6 min. Once the reaction paste was spread over the electrodes, near monolayer coverage (1.0 ± 0.2 nmol cm) was achieved for -nitrophenyl diazonium salt within 5 min. Amine functionalized electrodes were conjugated to C-reactive protein (CRP) antibodies. Antibody-modified TPEs were applied for the sensitive detection of CRP, a biomarker of cardiovascular disease using electrochemical enzyme-linked immunosorbent assays (ELISA). LODs were determined to be 2 ng mL in buffer, with high selectivity against interfering species for both functionalization methods. The direct -aminophenyl modification method had the highest sensitivity to CRP and was further tested in spiked serum with an LOD of 10 ng mL. This low-cost and robust TPE immunosensor platform can be easily adapted for other analytes and multiplexed detection.
Topics: Biosensing Techniques; Diazonium Compounds; Electrochemical Techniques; Electrodes; Immunoassay
PubMed: 34651620
DOI: 10.1039/d1ay00965f -
Journal of the American Chemical Society May 2022Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the...
Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. To overcome this restriction, we have engineered variants of protoglobin (Pgb) that use diazirines as carbene precursors. While the enhanced stability of diazirines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered Pgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an Pgb variant, determined by microcrystal electron diffraction (MicroED), reveals that evolution has enhanced access to the heme active site to facilitate this new-to-nature catalysis. Using readily prepared aryl diazirines as model substrates, we demonstrate the application of these highly stable carbene precursors in biocatalytic cyclopropanation, N-H insertion, and Si-H insertion reactions.
Topics: Azo Compounds; Biocatalysis; Catalysis; Diazomethane; Methane
PubMed: 35561334
DOI: 10.1021/jacs.2c02723 -
The Journal of Organic Chemistry Dec 2021We recently reported the incorporation of diazirine photo-cross-linkers onto the -GlcNAc posttranslational modification in mammalian cells, enabling the identification...
We recently reported the incorporation of diazirine photo-cross-linkers onto the -GlcNAc posttranslational modification in mammalian cells, enabling the identification of binding partners of -GlcNAcylated proteins. Unfortunately, the syntheses of the diazirine-functionalized substrates have exhibited inconsistent yields. We report a robust and stereoselective synthesis of cell-permeable GlcNAc-1-phosphate esters based on the use of commercially available bis(diisopropylamino)chlorophosphine. We demonstrate this approach for two diazirine-containing GlcNAc analogues, and we report the cellular incorporation of these compounds into glycoconjugates to support photo-cross-linking applications.
Topics: Acetylglucosamine; Animals; Diazomethane; Glycoconjugates; Phosphates; Proteins
PubMed: 34618463
DOI: 10.1021/acs.joc.1c01781 -
Proceedings of the National Academy of... Jan 2021Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame...
Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
Topics: Amino Acid Sequence; Animals; Cattle; Chromatin; Diazomethane; Gene Expression Regulation; Genetic Loci; HEK293 Cells; HeLa Cells; Histones; Humans; K562 Cells; Lysine; Mice; Open Reading Frames; Pan troglodytes; Peptides; Protein Binding; Protein Interaction Mapping; Rats; Sequence Alignment; Sequence Homology, Amino Acid; Transcription, Genetic; Transgenes; Ultraviolet Rays
PubMed: 33468658
DOI: 10.1073/pnas.2021943118 -
Molecules (Basel, Switzerland) Feb 2023In materials (polymer) science and medicinal chemistry, heteroaromatic derivatives play the role of the central skeleton in development of novel devices and discovery of... (Review)
Review
In materials (polymer) science and medicinal chemistry, heteroaromatic derivatives play the role of the central skeleton in development of novel devices and discovery of new drugs. On the other hand, (3-trifluoromethyl)phenyldiazirine (TPD) is a crucial chemical method for understanding biological processes such as ligand-receptor, nucleic acid-protein, lipid-protein, and protein-protein interactions. In particular, use of TPD has increased in recent materials science to create novel electric and polymer devices with comparative ease and reduced costs. Therefore, a combination of heteroaromatics and (3-trifluoromethyl)diazirine is a promising option for creating better materials and elucidating the unknown mechanisms of action of bioactive heteroaromatic compounds. In this review, a comprehensive synthesis of (3-trifluoromethyl)diazirine-substituted heteroaromatics is described.
Topics: Photoaffinity Labels; Diazomethane; Chemistry, Pharmaceutical; Proteins; Nucleic Acids
PubMed: 36771073
DOI: 10.3390/molecules28031408 -
Journal of Flow Chemistry 2024Azo compounds find use in many areas of science, displaying crucial properties for important applications as photoconductive organic pigments, fluorescent quenchers,... (Review)
Review
Azo compounds find use in many areas of science, displaying crucial properties for important applications as photoconductive organic pigments, fluorescent quenchers, paints, cosmetics, inks, and in the large and valuable dye industry. Due to the unstable intermediates, and the exothermic and fast reactions used in their synthesis, high value azo compounds are excellent candidates for continuous flow manufacturing. This comprehensive review covers the progress made to date on developing continuous flow systems for azo synthesis and reflects on the main challenges still to be addressed, including scale up, conversion, product purity, and environmental impact. The further development of integrated continuous flow processes has the potential to help tackle these challenges and deliver improved methods for azo compound generation.
PubMed: 38882391
DOI: 10.1007/s41981-024-00307-2 -
Current Protocols Jul 2021This protocol enables identification of the interaction partners of O-GlcNAcylated proteins. The method involves the introduction of the diazirine photocrosslinker onto...
This protocol enables identification of the interaction partners of O-GlcNAcylated proteins. The method involves the introduction of the diazirine photocrosslinker onto the O-GlcNAc modification within living cells. The photocrosslinker is activated by UV light to yield covalent crosslinking between O-GlcNAcylated proteins and neighboring molecules. The binding partners can be further characterized by immunoblot or proteomics mass spectrometry methods. The benefits of using the photocrosslinker include the capacity to trap low-affinity binding interactions and the ability to selectively target the interaction partners of the O-GlcNAcylated form of the protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: In-cell production and crosslinking of O-GlcNDAzylated proteins Basic Protocol 2: Immunoblot analysis to assess O-GlcNDAz crosslinking Support Protocol: Detection of UDP-GlcNDAz from cell lysates.
Topics: Acetylglucosamine; Diazomethane; Mass Spectrometry; Protein Processing, Post-Translational; Proteins
PubMed: 34288588
DOI: 10.1002/cpz1.201