- MeSH
- interpretace obrazu počítačem metody přístrojové vybavení MeSH
- laboratorní automatizace metody přístrojové vybavení MeSH
- mikroskopie klasifikace metody MeSH
- objevování léků * metody MeSH
- rychlé screeningové testy metody MeSH
- techniky kombinatorické chemie metody MeSH
- vyvíjení léků metody přístrojové vybavení MeSH
- Publikační typ
- přehledy MeSH
Bioorthogonal chemistry provides one of the possibilities to modify various biomolecules in their native environment. The combination of Click chemistry with the BONCAT method (bioorthogonal non-canonical amino acid tagging) is widely used for tagging and analysis of newly synthesized proteins, which are clearly distinguishable from the pre-existing protein pool. However, the commonly used procedure results in low quality 2D electrophoretic profiles. We put a lot of effort into obtaining clear results using a standard Click protocol, with a negligible effect. Here we describe a Click-on-membrane approach which we successfully used not only to monitor de novo protein synthesis but also to detect newly synthesized RNA.
Nucleic acid aptamers are single-stranded (ss)DNA or RNA oligonucleotides that can take various conformations and bind specifically and with high affinity to selected targets. While the introduction of SELEX (systematic evolution of ligands by exponential enrichment) revolutionized the production of the aptamers, this procedure is impeded by the formation of undesirable by-products reflecting hybridization among complementary oligonucleotides in the ssDNA libraries during asymmetric PCR. To reduce nonspecific amplification we tested cellulose-derived compounds and found that sodium carboxymethylcellulose (CMC) at a concentration 0.05%-0.2% efficiently suppressed production of undesirable large DNA amplicons during asymmetric PCR in the course of SELEX. Formation of the PCR by-products was reduced by CMCs of low and medium viscosity more than by CMCs of high viscosity, and all of them bound to DNA oligonucleotides as determined by electrophoresis in agarose gels. In contrast to CMC, methylcellulose did not reduce the formation of the PCR by-products and did not bind to DNA. DNA aptamers selected in the presence of CMC could be used directly in enzyme-linked immunosorbent-like assay. The combined data suggest that CMC binds weekly to DNA oligonucleotides through hydroxyl groups and in this way inhibits low-affinity DNA-DNA hybridization and enhances the production of specific amplicons in asymmetric PCR.
- MeSH
- aptamerová technika SELEX metody MeSH
- aptamery nukleotidové chemie MeSH
- ELISA metody MeSH
- jednovláknová DNA chemie MeSH
- methylcelulosa chemie MeSH
- polymerázová řetězová reakce metody MeSH
- sodná sůl karboxymethylcelulosy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA-encoded chemical libraries (DECLs) are powerful tools for modern drug discovery. A DECL is a pooled mixture of small molecule compounds, each of which is tagged with a unique DNA sequence which functions as a barcode. After incubation with a drug target and washing to remove non-binders, the bound molecules are eluted and submitted for DNA sequencing to determine which molecules are binding the target. While the DECL technology itself is ultra-high throughput, the following re-synthesis of identified compounds for orthogonal validation experiments remains the bottleneck. Using existing DNA-small molecule conjugates directly for affinity measurements, as opposed to complete compound resynthesis, could accelerate the discovery process. To this end, we have tested various geometries of fluorescently-labelled DNA constructs for fluorescence anisotropy (FA) experiments. Minimizing the distance between the fluorescent moiety and ligand can maximize the correlation between ligand-protein interaction and corresponding change in fluorophore rotational freedom, thus leading to larger, easier to interpret changes in FA values. However, close proximity can also cause artifacts due to potentially promiscuous interactions between fluorophore and protein. By balancing these two opposite effects, we have identified applicable fluorescently labelled DNA constructs displaying either a single ligand or pairs of fragments for affinity measurement using a FA assay.
- MeSH
- DNA chemická syntéza chemie MeSH
- fluorescenční barviva chemická syntéza chemie MeSH
- fluorescenční polarizace MeSH
- knihovny malých molekul chemická syntéza chemie farmakologie MeSH
- ligandy MeSH
- objevování léků MeSH
- preklinické hodnocení léčiv MeSH
- techniky kombinatorické chemie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Herein, we report a stereoselective formation of tetrahydro-6H-benzo[e][1,4]oxazino[4,3-a][1,4]diazepine-6,12(11H)-diones. Their preparation consisted in solid-phase synthesis of linear intermediates starting from polymer-supported Ser(tBu)-OH. Using various 2-nitrobenzoic acids and bromoketones, the key intermediates were obtained in five steps and subjected to trifluoroacetic acid-mediated cleavage from the resin, followed by stereoselective reduction with triethylsilane. Subsequent catalytic hydrogenation of the nitro group and cyclization yielded the target compounds with full retention of the C12astereocenter configuration.
Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.
- MeSH
- aminocukry chemická syntéza chemie metabolismus MeSH
- galektin 3 antagonisté a inhibitory metabolismus MeSH
- glykoproteiny chemická syntéza chemie metabolismus farmakologie MeSH
- glykosylace MeSH
- lidé MeSH
- ligandy MeSH
- oligosacharidy chemická syntéza chemie metabolismus MeSH
- sérový albumin hovězí chemická syntéza chemie metabolismus farmakologie MeSH
- skot MeSH
- techniky kombinatorické chemie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We designed a combinatorial library of trifunctional scaffold-derived compounds, which were derivatized with 30 different in-house-made azides. The compounds were proposed to mimic insulin receptor (IR)-binding epitopes in the insulin molecule and bind to and activate this receptor. This work has enabled us to test our synthetic and biological methodology and to prove its robustness and reliability for the solid-phase synthesis and testing of combinatorial libraries of the trifunctional scaffold-derived compounds. Our effort resulted in the discovery of two compounds, which were able to weakly induce the autophosphorylation of IR and weakly bind to this receptor at a 0.1 mM concentration. Despite these modest biological results, which well document the well-known difficulty in modulating protein-protein interactions, this study represents a unique example of targeting the IR with a set of nonpeptide compounds that were specifically designed and synthesized for this purpose. We believe that this work can open new perspectives for the development of next-generation insulin mimetics based on the scaffold structure.
- MeSH
- azidy chemická syntéza chemie MeSH
- inzulin analogy a deriváty chemie metabolismus MeSH
- knihovny malých molekul chemická syntéza chemie metabolismus farmakologie MeSH
- měď analýza MeSH
- molekulární struktura MeSH
- receptor inzulinu chemie metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- techniky kombinatorické chemie * MeSH
- techniky syntézy na pevné fázi MeSH
- vazba proteinů MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
An efficient and high-yielding solid phase synthesis of a small library of imidazolidin-2-ones and imidazol-2-ones was carried out employing a high chemo- and regioselective gold-catalyzed cycloisomerization as a key step. Polymer-supported amino acids derivatized with several alkyne functionalities combined with tosyl- and phenylureas have been subjected to gold-catalysis exhibiting exclusively C-N bond formation. The present work proves the potential of solid phase synthesis and homogeneous gold catalysis as an efficient and powerful synthetic tool for the generation of drug-like heterocycles.
This Review summarizes all of the currently described strategies applicable for the solid-phase synthesis of purine derivatives. The individual approaches are classified according to the immobilization procedure used resulting in a linkage of the final scaffold at various positions.
Synthesis of 2,3-dihydrobenzo[f][1,2,5]thiadiazepin-4(5H)-one 1,1-dioxides from polymer-supported α-amino acids is described herein. Different α-amino acids immobilized on Wang resin were sulfonylated with various 2-nitrobenzenesulfonyl chlorides. The resulting 2-nitrobenzenesulfonamides were alkylated with alcohols according to the Fukuyama-Mitsunobu procedure. After reduction of the nitro group and cleavage from the polymer support, the final intermediates were reacted with thionyl chloride, and target compounds of good crude purity and acceptable overall yields were obtained. The chiral HPLC studies revealed the impact of the cyclization step on the resulting stereochemistry. The developed strategy allows for simple production of desired compounds with the application of parallel/combinatorial solid-phase synthesis using commercially available building blocks.
