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Analytical Chemistry Feb 2021Mass-spectrometry-based chemoproteomics has enabled the rapid and proteome-wide discovery of functional and potentially 'druggable' hotspots in proteins. While numerous...
Mass-spectrometry-based chemoproteomics has enabled the rapid and proteome-wide discovery of functional and potentially 'druggable' hotspots in proteins. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or 'click' chemistry. The absence of bio-orthogonal chemistries that are functionally equivalent and complementary to CuAAC for chemoproteomic applications has hindered the development of multiplexed chemoproteomic platforms capable of assaying multiple amino acid side chains in parallel. Here, we identify and optimize Suzuki-Miyaura cross-coupling conditions for activity-based protein profiling and mass-spectrometry-based chemoproteomics, including for target deconvolution and labeling site identification. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual labeling. Multiplexed targeted deconvolution identified the protein targets of bifunctional cysteine- and lysine-reactive probes.
Topics: Alkynes; Azides; Catalysis; Click Chemistry; Copper; Cycloaddition Reaction; HEK293 Cells; Humans; Molecular Structure; Proteomics
PubMed: 33470097
DOI: 10.1021/acs.analchem.0c04726 -
Chemical Reviews Jun 2021The 1,4-conjugate addition reaction between activated alkynes or acetylenic Michael acceptors and nucleophiles (, the nucleophilic Michael reaction) is a historically... (Review)
Review
The 1,4-conjugate addition reaction between activated alkynes or acetylenic Michael acceptors and nucleophiles (, the nucleophilic Michael reaction) is a historically useful organic transformation. Despite its general utility, the efficiency and outcomes can vary widely and are often closely dependent upon specific reaction conditions. Nevertheless, with improvements in reaction design, including catalyst development and an expansion of the substrate scope to feature more electrophilic alkynes, many examples now present with features that are congruent with Click chemistry. Although several nucleophilic species can participate in these conjugate additions, ubiquitous nucleophiles such as thiols, amines, and alcohols are commonly employed and, consequently, among the most well developed. For many years, these conjugate additions were largely relegated to organic chemistry, but in the last few decades their use has expanded into other spheres such as bioorganic chemistry and polymer chemistry. Within these fields, they have been particularly useful for bioconjugation reactions and step-growth polymerizations, respectively, due to their excellent efficiency, orthogonality, and ambient reactivity. The reaction is expected to feature in increasingly divergent application settings as it continues to emerge as a Click reaction.
Topics: Alcohols; Alkynes; Amines; Biopolymers; Click Chemistry; Hydrogels; Sulfhydryl Compounds
PubMed: 33764739
DOI: 10.1021/acs.chemrev.0c01076 -
Biomacromolecules Jun 2023Oligonucleotides are powerful molecules for programming function and assembly. When arrayed on nanoparticle scaffolds in high density, the resulting molecules, spherical...
Oligonucleotides are powerful molecules for programming function and assembly. When arrayed on nanoparticle scaffolds in high density, the resulting molecules, spherical nucleic acids (SNAs), become imbued with unique properties. We used the copper-catalyzed azide-alkyne cycloaddition to graft oligonucleotides on Qβ virus-like particles to see if such structures also gain SNA-like behavior. Copper-binding ligands were shown to promote the click reaction without degrading oligonucleotide substrates. Reactions were first optimized with a small-molecule fluorogenic reporter and were then applied to the more challenging synthesis of polyvalent protein nanoparticle-oligonucleotide conjugates. The resulting particles exhibited the enhanced cellular uptake and protection from nuclease-mediated oligonucleotide cleavage characteristic of SNAs, had similar residence time in the liver relative to unmodified particles, and were somewhat shielded from immune recognition, resulting in nearly 10-fold lower antibody titers relative to unmodified particles. Oligonucleotide-functionalized virus-like particles thus provide an interesting option for protein nanoparticle-mediated delivery of functional molecules.
Topics: Oligonucleotides; Copper; Proteins; Nucleic Acids; Nanoparticles; Azides; Alkynes; Click Chemistry; Cycloaddition Reaction
PubMed: 37257068
DOI: 10.1021/acs.biomac.3c00178 -
Molecules (Basel, Switzerland) May 2023The subject of this investigation is a new method for the construction of sulfonylated heterocycles which overcomes the limitations of classical approaches using a cheap...
The subject of this investigation is a new method for the construction of sulfonylated heterocycles which overcomes the limitations of classical approaches using a cheap feedstock sulfonylating agent, especially under photocatalyst- and metal-free conditions.
Topics: Cyclization; Sodium; Alkynes
PubMed: 37298913
DOI: 10.3390/molecules28114436 -
The Journal of Physical Chemistry. B Jan 2023The C≡C stretching frequencies of terminal alkynes appear in the "clear" window of vibrational spectra, so they are attractive and increasingly popular as...
The C≡C stretching frequencies of terminal alkynes appear in the "clear" window of vibrational spectra, so they are attractive and increasingly popular as site-specific probes in complicated biological systems like proteins, cells, and tissues. In this work, we collected infrared (IR) absorption and Raman scattering spectra of model compounds, artificial amino acids, and model proteins that contain terminal alkyne groups, and we used our results to draw conclusions about the signal strength and sensitivity to the local environment of both aliphatic and aromatic terminal alkyne C≡C stretching bands. While the IR bands of alkynyl model compounds displayed surprisingly broad solvatochromism, their absorptions were weak enough that alkynes can be ruled out as effective IR probes. The same solvatochromism was observed in model compounds' Raman spectra, and comparisons to published empirical solvent scales (including a linear regression against four meta-aggregated solvent parameters) suggested that the alkyne C≡C stretching frequency mainly reports on local electronic interactions (i.e., short-range electron donor-acceptor interactions) with solvent molecules and neighboring functional groups. The strong solvatochromism observed here for alkyne stretching bands introduces an important consideration for Raman imaging studies based on these signals. Raman signals for alkynes (especially those that are π-conjugated) can be exceptionally strong and should permit alkynyl Raman signals to function as probes at very low concentrations, as compared to other widely used vibrational probe groups like azides and nitriles. We incorporated homopropargyl glycine into a transmembrane helical peptide via peptide synthesis, and we installed -ethynylphenylalanine into the interior of the fatty acid acyl carrier protein using a genetic code expansion technique. The Raman spectra from each of these test systems indicate that alkynyl C≡C bands can act as effective and unique probes of their local biomolecular environments. We provide guidance for the best possible future uses of alkynes as solvatochromic Raman probes, and while empirical explanations of the alkyne solvatochromism are offered, open questions about its physical basis are enunciated.
Topics: Alkynes; Spectrum Analysis, Raman; Solvents
PubMed: 36538691
DOI: 10.1021/acs.jpcb.2c06176 -
South African Medical Journal =... Dec 2021Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor, has been a component of first-line antiretroviral therapy (ART) in the South African HIV/AIDS...
BACKGROUND
Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor, has been a component of first-line antiretroviral therapy (ART) in the South African HIV/AIDS programme since 2004. It is extensively used in ART programmes in other low- and middle-income countries. The natural history of the previously recognised EFV drug-induced liver injury (DILI) is not known.
OBJECTIVES
To define and establish a causality assessment for EFV DILI and document its natural history by detailing a patient cohort. All relevant features characterising the patterns of clinical and histological injury, the duration of clinical and biochemical recovery and the associated mortality rate were documented. Factors associated with specific histological patterns of liver injury were analysed.
METHODS
Patients were prospectively included after meeting causality and inclusion criteria for EFV DILI. Clinical, demographic and liver histological features (where possible) were documented from the time of presentation and throughout follow-up. Prednisone at 0.25 - 0.5 mg/kg was initiated at the discretion of the treating hepatologist.
RESULTS
Fifty patients were prospectively included in the analysis. The median age was 34 (interquartile range (IQR) 29 - 39) years, males being older than females (p=0.014). Most (92%) were female, and 86% were of black African ethnicity. The median duration of ART at presentation was 6 months, with half of the women having initiated ART during pregnancy, at a median gestation of 24 (IQR 11 - 36) weeks. The median CD4 nadir at ART treatment initiation was 517 cells/µL, with no significant difference in CD4 nadir between those who were pregnant and those who were not (p=0.6). The median RUCAM (Roussel Uclaf Causality Assessment Method) score was 7, and among the 75% of patients who had liver biopsies, three histological patterns were identified: submassive necrosis (60%), nonspecific hepatitis (35%), and mixed cholestatic hepatitis (5%). On multivariate analysis, predictors for the development of submassive necrosis included younger age (<30 years; p=0.045), ART initiation in pregnancy (p=0.02), and a baseline CD4 count >350 cells/µL (p=0.018). For the nonspecific hepatitis group, pregnancy was also an associated factor (p=0.04). The mortality rate was 14%, with a median time from admission to death of 15 days. The median (IQR) time to initial hospital discharge was a lengthy 33 (24 - 52) days. Biochemical recovery was prolonged, necessitating a follow-up period of more than a year at an outpatient specialist clinic, with 86% of patients initiating a protease inhibitor-based ART regimen successfully.
CONCLUSIONS
EFV DILI is a severe drug complication of ART with appreciable mortality and significant inpatient morbidity, requiring prolonged hospitalisation and follow-up.
Topics: Adult; Alkynes; Benzoxazines; Chemical and Drug Induced Liver Injury; Cyclopropanes; Female; HIV Infections; Humans; Male; Prospective Studies; Reverse Transcriptase Inhibitors; South Africa
PubMed: 34949306
DOI: 10.7196/SAMJ.2021.v111i12.14584 -
Journal of Medicinal Chemistry Aug 2022High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that...
High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter.
Topics: Alkynes; Azides; Cycloaddition Reaction; High-Throughput Screening Assays; Mass Spectrometry
PubMed: 35912476
DOI: 10.1021/acs.jmedchem.1c02206 -
MBio Aug 2021Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources and possess a broad a range of bioactivities. In...
Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources and possess a broad a range of bioactivities. In bacteria, the basic biosynthesis of polyynes is known, but their biosynthetic gene cluster (BGC) distribution and evolutionary relationship to alkyne biosynthesis have not been addressed. Through comprehensive genomic and phylogenetic analyses, the distribution of alkyne biosynthesis gene cassettes throughout bacteria was explored, revealing evidence of multiple horizontal gene transfer events. After investigation of the evolutionary connection between alkyne and polyyne biosynthesis, a monophyletic clade was identified that possessed a conserved seven-gene cassette for polyyne biosynthesis that built upon the conserved three-gene cassette for alkyne biosynthesis. Further diversity mapping of the conserved polyyne gene cassette revealed a phylogenetic subclade for an uncharacterized polyyne BGC present in several Pseudomonas species, designated . Pathway mutagenesis and high-resolution analytical chemistry showed the Pseudomonas protegens BGC directed the biosynthesis of a novel polyyne, protegencin. Exploration of the biosynthetic logic behind polyyne production, through BGC mutagenesis and analytical chemistry, highlighted the essentiality of a triad of desaturase proteins and a thioesterase in both the P. protegens and Trinickia caryophylli (formerly Burkholderia caryophylli) caryoynencin pathways. We have unified and expanded knowledge of polyyne diversity and uniquely demonstrated that alkyne and polyyne biosynthetic gene clusters are evolutionarily related and widely distributed within bacteria. The systematic mapping of conserved biosynthetic genes across the available bacterial genomic diversity proved to be a fruitful method for discovering new natural products and better understanding polyyne biosynthesis. Natural products bearing alkyne (triple carbon bond) or polyyne (multiple alternating single and triple carbon bonds) moieties exhibit a broad range of important biological activities. Polyyne metabolites have been implicated in important ecological roles such as cepacin mediating biological control of plant pathogens and caryoynencin protecting Lagriinae beetle eggs against pathogenic fungi. After further phylogenetic exploration of polyyne diversity, we identified a novel gene cluster in Pseudomonas bacteria with known biological control abilities and proved it was responsible for synthesizing a new polyyne metabolite, protegencin. The evolutionary analysis of polyyne pathways showed that multiple biosynthetic genes were conserved, and using mutagenesis, their essentiality was demonstrated. Our research provides a foundation for the future modification of polyyne metabolites and has identified a novel polyyne, protegencin, with potential bioactive roles of ecological and agricultural importance.
Topics: Biosynthetic Pathways; Evolution, Molecular; Genome, Bacterial; Genomics; Multigene Family; Phylogeny; Polyynes; Pseudomonas
PubMed: 34340549
DOI: 10.1128/mBio.00715-21 -
Nature Communications Mar 2022As one of the typical bioorthogonal reactions, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction holds great potential in organic synthesis,...
As one of the typical bioorthogonal reactions, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction holds great potential in organic synthesis, bioconjugation, and surface functionalization. However, the toxicity of Cu(I), inefficient catalytic activity, and the lack of cell specific targeting of the existing catalysts hampered their practical applications in living systems. Herein, we design and construct a DNA-based platform as a biocompatible, highly efficient, and precisely targeted bioorthogonal nanocatalyst. The nanocatalyst presents excellent catalytic efficiency in vitro, which is one order of magnitude higher than the commonly used catalyst CuSO/sodium ascorbate. The theoretical calculation further supports the contribution of DNA structure and its interaction with substrates to the superior catalytic activity. More importantly, the system can achieve efficient prodrug activation in cancer cells through cell type-specific recognition and produce a 40-fold enhancement of transformation compared to the non-targeting nanocatalyst, resulting in enhanced antitumor efficacy and reduced adverse effects. In vivo tumor therapy demonstrates the safety and efficacy of the system in mammals.
Topics: Alkynes; Animals; Azides; Catalysis; Click Chemistry; Copper; Cycloaddition Reaction; DNA; Mammals
PubMed: 35304487
DOI: 10.1038/s41467-022-29167-x -
Journal of Labelled Compounds &... Apr 2022Base catalysed exchange with sodium hydroxide, calcium oxide or N,N,N,N-tetramethylguanidine in deuterium oxide is a viable procedure for the preparation of terminally...
Base catalysed exchange with sodium hydroxide, calcium oxide or N,N,N,N-tetramethylguanidine in deuterium oxide is a viable procedure for the preparation of terminally deuterated alkynes for those alkynes stable to strong base. The use of silver perchlorate as a catalyst is an alternative practical option when labelling alkynes which are sensitive to base or contain functionalities which would lead to labelling elsewhere in the molecule. Labelling with this catalyst takes place smoothly at ambient temperature in a mixture of N,N-dimethylformamide and deuterium oxide.
Topics: Alkynes; Catalysis; Deuterium
PubMed: 35067956
DOI: 10.1002/jlcr.3963