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ACS Chemical Neuroscience Jan 2021The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously...
The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of -azi-fropofol (AziF) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.
Topics: Anesthetics, General; Diazomethane; Propofol
PubMed: 33355437
DOI: 10.1021/acschemneuro.0c00667 -
ChemMedChem Oct 2022Identifying the protein targets of drugs is an important but tedious process. Existing proteomic approaches enable unbiased target identification but lack the throughput...
Identifying the protein targets of drugs is an important but tedious process. Existing proteomic approaches enable unbiased target identification but lack the throughput needed to screen larger compound libraries. Here, we present a compound interaction screen on a photoactivatable cellulose membrane (CISCM) that enables target identification of several drugs in parallel. To this end, we use diazirine-based undirected photoaffinity labeling (PAL) to immobilize compounds on cellulose membranes. Functionalized membranes are then incubated with protein extract and specific targets are identified via quantitative affinity purification and mass spectrometry. CISCM reliably identifies known targets of natural products in less than three hours of analysis time per compound. In summary, we show that combining undirected photoimmobilization of compounds on cellulose with quantitative interaction proteomics provides an efficient means to identify the targets of natural products.
Topics: Biological Products; Cellulose; Diazomethane; Mass Spectrometry; Proteins; Proteomics
PubMed: 35867055
DOI: 10.1002/cmdc.202200346 -
Philosophical Transactions of the Royal... Feb 2023Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic...
Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic gene clusters depends on complex regulations in bacteria. However, our current knowledge of NRPSs is based on enzymological studies using full NRPS systems and/or a single NRPS domain in heterologous hosts. Chemical and/or biochemical strategies to capture the endogenous activities of NRPSs facilitate studies on NRPS cell biology in bacterial cells. Here, we describe a chemical scaffold for the rapid and selective photoaffinity labelling of NRPSs in purified systems, crude biological samples and living bacterial cells. We synthesized photoaffinity labelling probes coupled with 5'--(phenylalanyl)sulfamoyladenosine with clickable alkyl diazirine or trifluoromethyl phenyl diazirine. We found that a trifluoromethyl phenyl diazirine-based probe cross-linked the Phe-activating domain of a GrsA-NRPS with high selectivity and sensitivity at shorter ultraviolet (UV) irradiation times (less than 5 min) relative to a prototypical benzophenone-based probe. Our results demonstrated that this quick labelling protocol can prevent damage to proteins and cells caused by long UV irradiation times, providing a mild photoaffinity labelling method for biological samples. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
Topics: Diazomethane; Bacteria; Peptide Synthases; Multigene Family
PubMed: 36633280
DOI: 10.1098/rstb.2022.0026 -
Chembiochem : a European Journal of... Jan 2020Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA-protein interactions in living...
Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA-protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA-protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA-protein interactions.
Topics: Cross-Linking Reagents; Diazomethane; Humans; Molecular Structure; RNA; RNA-Binding Proteins; Ultraviolet Rays
PubMed: 31658407
DOI: 10.1002/cbic.201900559 -
ACS Chemical Biology Aug 2021Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins....
Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.
Topics: Alkynes; Binding Sites; Cholesterol; Cyanobacteria; Diazomethane; Fluorescent Dyes; Ligand-Gated Ion Channels; Molecular Docking Simulation; Molecular Dynamics Simulation; Photoaffinity Labels; Protein Binding
PubMed: 34355883
DOI: 10.1021/acschembio.1c00364 -
Mercury-Free Synthesis of Pincer [C^N^C]Au Complexes by an Oxidative Addition/CH Activation Cascade.ChemSusChem Apr 2020Starting from the commercially available dimethyl sulfide-gold(I) chloride complex (DMSAuCl) and diazonium salts in the presence of 2,6-di-tert-butyl-4-methylpyridine as...
Starting from the commercially available dimethyl sulfide-gold(I) chloride complex (DMSAuCl) and diazonium salts in the presence of 2,6-di-tert-butyl-4-methylpyridine as base, symmetric and unsymmetric [C^N^C]Au Cl complexes were synthesized in a selective, photosensitizer-free, photochemical reaction using blue LED light. This new protocol provides the first mercury-free synthesis of these types of pincer-complexes in moderate-to-excellent yields, starting from a readily available gold(I) precursor. Owing to the extraordinary properties of the target compounds, like excellent luminescence and high anticancer activities, the synthesis of such complexes is a highly active field of research, which might make its way to an industrial application. Owing to the disadvantages of the known protocols, especially the toxicity and the selectivity issues in the case of unsymmetric complexes, avoiding the use of mercury, should further accelerate this ongoing development.
PubMed: 32134179
DOI: 10.1002/cssc.202000310 -
Analytical Chemistry Aug 2016Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid...
Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid instrument with improved fragmentation capacities in a systematic comparison to identify which fragmentation method would be best for the identification of cross-linked peptides. Specifically, we explored three fragmentation methods and two combinations: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human serum albumin (HSA) served as a test sample, yielding over all methods and in triplicate analysis in total 2602 matched PSMs and 1390 linked residue pairs at 5% false discovery rate, as confirmed by the crystal structure. HCD wins in number of matched peptide-spectrum-matches (958 PSMs) and identified links (446). CID is most complementary, increasing the number of identified links by 13% (58 links). HCD wins together with EThcD in cross-link site calling precision, with approximately 62% of sites having adjacent backbone cleavages that unambiguously locate the link in both peptides, without assuming any cross-linker preference for amino acids. Overall quality of spectra, as judged by sequence coverage of both peptides, is best for EThcD for the majority of peptides. Sequence coverage might be of particular importance for complex samples, for which we propose a data dependent decision tree, else HCD is the method of choice. The mass spectrometric raw data has been deposited in PRIDE (PXD003737).
Topics: Cross-Linking Reagents; Diazomethane; Humans; Peptides; Serum Albumin; Tandem Mass Spectrometry; Ultraviolet Rays
PubMed: 27454319
DOI: 10.1021/acs.analchem.6b02082 -
Organic Letters Mar 2021Aryl diazonium ions are important in synthesis and chemical biology, and the acid-labile triazabutadiene can protect this handle for future use. We report a Suzuki...
Aryl diazonium ions are important in synthesis and chemical biology, and the acid-labile triazabutadiene can protect this handle for future use. We report a Suzuki coupling strategy that is compatible with the triazabutadiene scaffold, expanding the scope of synthetically available triazabutadienes. Shown herein, the triazabutadiene scaffold remains intact and reactive after coupling, as demonstrated by releasing the aryl diazonium ion to label a tyrosine-rich model protein.
Topics: Diazonium Compounds; Ions; Molecular Structure; Proteins
PubMed: 33570414
DOI: 10.1021/acs.orglett.1c00257 -
Biochimica Et Biophysica Acta.... Oct 2022Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of...
Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of whether azide- or diazirine-modified lipids are suitable for such studies remains controversial. We have recently shown that diazirine-modified lipids can indeed form cross-links to membrane peptides after UV activation and that these cross-links can be precisely determined in their position by mass spectrometry (MS). However, we also observed an unexpected backfolding of the lipid's diazirine-containing stearoyl chain to the membrane interface challenging the potential application of this modified lipid for future cross-linking (XL)-MS studies of protein/lipid interactions. In this work, we compared an azide- (AzidoPC) and a diazirine-modified (DiazPC) membrane lipid regarding their self-assembly properties, their mixing behavior with saturated bilayer-forming phospholipids, and their reactivity upon UV activation using differential scanning calorimetry (DSC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and MS. Mixtures of both modified lipids with DMPC were further used for photo-chemically induced XL experiments with a transmembrane model peptide (KLAW23) to elucidate similarities and differences between the azide and the diazirine moiety. We showed that both photo-reactive lipids can be used to study lipid/peptide and lipid/protein interactions. The AzidoPC proved easier to handle, whereas the DiazPC had fewer degradation products and a higher cross-linking yield. However, the problem of backfolding occurs in both lipids; thus, it seems to be a general phenomenon.
Topics: Azides; Cross-Linking Reagents; Diazomethane; Mass Spectrometry; Membrane Lipids; Peptides; Scattering, Small Angle; X-Ray Diffraction
PubMed: 35841926
DOI: 10.1016/j.bbamem.2022.184004 -
Bioconjugate Chemistry Jan 2021Biological conjugation is an important tool employed for many basic research and clinical applications. While useful, common methods of biological conjugation suffer...
Biological conjugation is an important tool employed for many basic research and clinical applications. While useful, common methods of biological conjugation suffer from a variety of limitations, such as (a) requiring the presence of specific surface-exposed residues, such as lysines or cysteines, (b) reducing protein activity, and/or (c) reducing protein stability and solubility. Use of photoreactive moieties including diazirines, azides, and benzophenones provide an alternative, mild approach to conjugation. Upon irradiation with UV and visible light, these functionalities generate highly reactive carbenes, nitrenes, and radical intermediates. Many of these will couple to proteins in a non-amino-acid-specific manner. The main hurdle for photoactivated biological conjugation is very low yield. In this study, we developed a solid-state method to increase conjugation efficiency of diazirine-containing carbohydrates to proteins. Using this methodology, we produced multivalent carbohydrate-protein conjugates with unaltered protein charge and secondary structure. Compared to carbohydrate conjugates prepared with amide linkages to lysine residues using standard NHS conjugation, the photoreactive prepared conjugates displayed up to 100-fold improved binding to lectins and diminished immunogenicity in mice. These results indicate that photoreactive bioconjugation could be especially useful for applications, such as lectin targeting, where high binding affinity and low immunogenicity are desired.
Topics: Animals; Binding Sites; Carbohydrates; Diazomethane; Glycoconjugates; Light; Mice
PubMed: 33325683
DOI: 10.1021/acs.bioconjchem.0c00555