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Methods in Molecular Biology (Clifton,... 2021Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of...
Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.
Topics: Animals; Diazomethane; High-Throughput Screening Assays; Humans; Isomerism; Lipidomics; Lysophospholipids; Methylation; Mice; Monoglycerides; Phosphatidylglycerols; Phosphatidylinositol Phosphates; Spectrometry, Mass, Electrospray Ionization; Trimethylsilyl Compounds
PubMed: 33954941
DOI: 10.1007/978-1-0716-1410-5_6 -
Chemistry (Weinheim An Der Bergstrasse,... Jun 2021Starting from commercially available DMSAuCl and diazonium salts, cationic [ ]Au complexes were synthesized in a selective, photosensitizer-free, photochemical reaction...
Starting from commercially available DMSAuCl and diazonium salts, cationic [ ]Au complexes were synthesized in a selective, photosensitizer-free, photochemical reaction by irradiation with blue LED light. This new protocol represents the first easy synthesis of these types of pincer complexes in moderate to excellent yield starting from a readily available gold(I) precursor with nitrogen as the only by-product. Owing to the disadvantages of known protocols, especially the toxicity in the case of a transmetalation with mercury or the necessity for a mostly twofold excess of a gold precursor, this method offers an attractive alternative towards this kind of gold(III) complexes. In addition, the first arylated [ ]Au(III) pincer complex was synthesized by using this technology.
PubMed: 33929076
DOI: 10.1002/chem.202100035 -
Molecular & Cellular Proteomics : MCP 2021Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While...
Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MS. The MS-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.
Topics: Chromatography, Liquid; Cross-Linking Reagents; Diazomethane; Fungal Proteins; Mass Spectrometry; Photochemical Processes; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Serum Albumin, Bovine
PubMed: 33915260
DOI: 10.1016/j.mcpro.2021.100084 -
Communications Chemistry Apr 2021The development of miniaturized flow platforms would enable efficient and selective synthesis of drug and lead molecules by rapidly exploring synthetic methodologies and...
The development of miniaturized flow platforms would enable efficient and selective synthesis of drug and lead molecules by rapidly exploring synthetic methodologies and screening for optimal conditions, progress in which could be transformative for the field. In spite of tremendous advances made in continuous flow technology, these reported flow platforms are not devised to conduct many different reactions simultaneously. Herein, we report a metal-based flow parallel synthesizer that enables multiplex synthesis of libraries of compounds and efficient screening of parameters. This miniaturized synthesizer, equipped with a unique built-in flow distributor and n number of microreactors, can execute multiple types of reactions in parallel under diverse conditions, including photochemistry. Diazonium-based reactions are explored as a test case by distributing the reagent to 16 (n = 16) capillaries to which various building blocks are supplied for the chemistry library synthesis at the optimal conditions obtained by multiplex screening of 96 different reaction variables in reaction time, concentration, and product type. The proficiency of the flow parallel synthesizer is showcased by multiplex formation of various C-C, C-N, C-X, and C-S bonds, leading to optimization of 24 different aryl diazonium chemistries.
PubMed: 36697557
DOI: 10.1038/s42004-021-00490-6 -
Toxicity Report Series Mar 2021Trimethylsilyldiazomethane (TMSD) is a methylating reagent widely used in organic chemistry. TMSD is structurally related to the compound diazomethane, which is a known... (Comparative Study)
Comparative Study
Trimethylsilyldiazomethane (TMSD) is a methylating reagent widely used in organic chemistry. TMSD is structurally related to the compound diazomethane, which is a known lethal respiratory toxicant in humans and in animal models. TMSD is less reactive (with lower explosive potential) than diazomethane and is considered a safer, less toxic alternative. Few toxicity data are available to support this claim, however, and TMSD is readily available commercially from chemical suppliers. Concern over the inhalation toxicity of TMSD originates from reports of the death of two chemists resulting from lung injury and acute respiratory distress syndrome following exposure to TMSD in the workplace. Other concerns include the known inhalation toxicity of diazomethane and the absence of inhalation toxicity data for TMSD. The National Toxicology Program (NTP) conducted this study to evaluate the acute inhalation toxicity of TMSD in vivo.(Abstract Abridged).
Topics: Administration, Inhalation; Animals; Carcinogenicity Tests; Carcinogens; Diazomethane; Dose-Response Relationship, Drug; Male; Mice; Mice, Inbred Strains; Rats; Rats, Sprague-Dawley; Survival Rate; Symptom Assessment; Trimethylsilyl Compounds
PubMed: 33819212
DOI: 10.22427/NTP-TOX-101 -
Journal of the American Chemical Society Mar 2021Here we report the first example of alkyne trifunctionalization through simultaneous construction of C-C, C-O, and C-N bonds via gold catalysis. With the assistance of a...
Here we report the first example of alkyne trifunctionalization through simultaneous construction of C-C, C-O, and C-N bonds via gold catalysis. With the assistance of a γ-keto directing group, sequential gold-catalyzed alkyne hydration, vinyl-gold nucleophilic addition, and gold(III) reductive elimination were achieved in one pot. Diazonium salts were identified as both electrophiles (N source) and oxidants (C source). Vinyl-gold(III) intermediates were revealed as effective nucleophiles toward diazonium, facilitating nucleophilic addition and reductive elimination with high efficiency. The rather comprehensive reaction sequence was achieved with excellent yields (up to 95%) and broad scope (>50 examples) under mild conditions (room temperature or 40 °C).
Topics: Alkynes; Catalysis; Diazonium Compounds; Gold; Oxidation-Reduction; Quantum Theory; Temperature; Vinyl Compounds
PubMed: 33661619
DOI: 10.1021/jacs.1c01811 -
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 -
ACS Chemical Biology Feb 2021Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It...
Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It participates in a variety of biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD enables photo-cross-linking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.
Topics: Azides; Click Chemistry; Cross-Linking Reagents; Diazomethane; HEK293 Cells; Humans; NAD; Poly (ADP-Ribose) Polymerase-1; Poly ADP Ribosylation; Protein Processing, Post-Translational; Proteome; Proteomics; Ultraviolet Rays
PubMed: 33524253
DOI: 10.1021/acschembio.0c00937 -
Molecules (Basel, Switzerland) Jan 2021The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first...
The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.
Topics: Allosteric Site; Aspartate Kinase; Binding Sites; Chorismate Mutase; Diazomethane; Enzyme Inhibitors; Humans; Mass Spectrometry; Molecular Structure; Phosphoglycerate Dehydrogenase; Protein Domains; Structure-Activity Relationship
PubMed: 33477510
DOI: 10.3390/molecules26020477 -
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