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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 -
Current Opinion in Chemical Biology Apr 2020Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful... (Review)
Review
Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.
Topics: Amino Acid Sequence; Amino Acids; Amino Acyl-tRNA Synthetases; Biocatalysis; Catalytic Domain; Diazonium Compounds; Enzymes; Genetic Code; Molecular Conformation; Oxidation-Reduction; Photochemical Processes; Protein Engineering; beta-Alanine
PubMed: 32163871
DOI: 10.1016/j.cbpa.2020.01.006 -
Chemical Reviews Mar 2016This review covers the addition of diazo compounds to ketones to afford homologated ketones, either in the presence or in the absence of promoters or catalysts.... (Review)
Review
This review covers the addition of diazo compounds to ketones to afford homologated ketones, either in the presence or in the absence of promoters or catalysts. Reactions with diazoalkanes, aryldiazomethanes, trimethylsilyldiazomethane, α-diazo esters, and disubstituted diazo compounds are covered, commenting on the complex regiochemistry of the reaction and the nature of the catalysts and promoters. The recent reports on the enantioselective version of ketone homologation reactions are gathered in one section, followed by reports on the use of cyclic ketones ring expansion in total synthesis. Although the first reports of this reaction appeared in the literature almost one century ago, the recent achievements, in particular, for the asymmetric version, forecast the development of new breakthroughs in the synthetically valuable field of diazo chemistry.
Topics: Chemistry Techniques, Synthetic; Diazonium Compounds; Heterocyclic Compounds; Hydrocarbons, Alicyclic; Ketones
PubMed: 26854865
DOI: 10.1021/acs.chemrev.5b00381 -
Chemical Society Reviews Mar 2021The interactions of biomolecules underpin all cellular processes, and the understanding of their dynamic interplay can lead to significant advances in the treatment of... (Review)
Review
The interactions of biomolecules underpin all cellular processes, and the understanding of their dynamic interplay can lead to significant advances in the treatment of disease through the identification of novel therapeutic strategies. Protein-protein interactions (PPIs) in particular play a vital role within this arena, providing the basis for the majority of cellular signalling pathways. Despite their great importance, the elucidation of weak or transient PPIs that cannot be identified by immunoprecipitation remains a significant challenge, particularly in a disease relevant cellular environment. Recent approaches towards this goal have utilized the in situ generation of high energy intermediates that cross-link with neighboring proteins, providing a snapshot of the biomolecular makeup of the local area or microenvironment, termed the interactome. In this tutorial review, we discuss these reactive intermediates, how they are generated, and the impact they have had on the discovery of new biology. Broadly, we believe this strategy has the potential to significantly accelerate our understanding of PPIs and how they affect cellular physiology.
Topics: Carbon-Nitrogen Ligases; Diazomethane; Escherichia coli; Escherichia coli Proteins; Free Radicals; Humans; Nucleic Acids; Protein Interaction Maps; Proteins; Repressor Proteins; Signal Transduction; Staining and Labeling
PubMed: 33458734
DOI: 10.1039/d0cs01366h -
Current Opinion in Chemical Biology Aug 2022Lipid-protein interactions serve as the basis for many of the diverse roles of lipids. However, these noncovalent binding events are often weak, transient, or dependent... (Review)
Review
Lipid-protein interactions serve as the basis for many of the diverse roles of lipids. However, these noncovalent binding events are often weak, transient, or dependent upon environmental cues. Photoaffinity labeling can preserve these interactions under native conditions, enabling their biochemical profiling. Typically, photoaffinity labeling probes contain a diazirine photocrosslinker and a click chemistry handle for enrichment and downstream analysis. In this review, we summarize recent advances in the understanding the mechanisms of diazirine photocrosslinking, and we provide an overview of recent applications of photoaffinity labeling to reveal the interactions of diverse types of lipids with specific members of the proteome.
Topics: Click Chemistry; Diazomethane; Lipids; Photoaffinity Labels
PubMed: 35724595
DOI: 10.1016/j.cbpa.2022.102173 -
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 -
Advances in Colloid and Interface... Nov 2015This paper reviews the surface modification strategies of a wide range of nanomaterials using aryldiazonium salts. After a brief history of diazonium salts since their... (Review)
Review
This paper reviews the surface modification strategies of a wide range of nanomaterials using aryldiazonium salts. After a brief history of diazonium salts since their discovery by Peter Griess in 1858, we will tackle the surface chemistry using these compounds since the first trials in the 1950s. We will then focus on the modern surface chemistry of aryldiazonium salts for the modification of materials, particularly metallic, semiconductors, metal oxide nanoparticles, carbon-based nanostructures, diamond and clays. The successful modification of sp(2) carbon materials and metals by aryldiazonium salts paved the way to innovative strategies for the attachment of aryl layers to metal oxide nanoparticles and nanodiamonds, and intercalation of clays. Interestingly, diazotized surfaces can easily trap nanoparticles and nanotubes while diazotized nanoparticles can be (electro)chemically reduced on electrode/materials surfaces as molecular compounds. Both strategies provided organized 2D surface assembled nanoparticles. In this review, aryldiazonium salts are highlighted as efficient coupling agents for many types of molecular, macromolecular and nanoparticulate species, therefore ensuring stability to colloids on the one hand, and the construction of composite materials and hybrid systems with robust and durable interfaces/interphases, on the other hand. The last section is dedicated to a selection of patents and industrial products based on aryldiazonium-modified nanomaterials. After nearly 160 years of organic chemistry, diazonium salts have entered a new, long and thriving era for the benefit of materials, colloids, and surface scientists. This tempts us to introduce the terminology of "diazonics" we define as the science and technology of aryldiazonium salt-derived materials.
PubMed: 26299313
DOI: 10.1016/j.cis.2015.07.011 -
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