1,2,4-Triazines have recently been identified as versatile dienes participating in the inverse electron-demand Diels-Alder reaction with strained dienophiles. However, their widespread utility in bioconjugation reactions is still limited. Herein, we report a systematic study on the reactivity of various 1,2,4-triazines with trans-cyclooctenes showing that the structure of both the triazine and the dienophile significantly affect the reaction rate. Our kinetic study led to the discovery of novel cationic 1,2,4-triazines with superior properties for bioconjugation reactions. We have developed an efficient method that enables their late-stage functionalization and allows for easy access to various useful heterobifunctional scaffolds. In addition, these charged dienes form unprecedented fluorescent products upon reaction with trans-cyclooctenes and can be used for fluorogenic labeling of subcellular compartments in live cells.

• Publikační typ
• časopisecké články MeSH

• Publikační typ
• časopisecké články MeSH

Electrophoretic mobility shift assay (EMSA) is a well-established technique to monitor interactions between biomolecules particularly DNA and proteins. Even though numerous variations of this method have been presented, challenges in the form of detection sensitivity and/or variations in the stability of the formed complex still remain. With advances in the area of nanomaterials improvements in EMSA have been also suggested. Recently, Zhang and Wang (Electrophoresis 2015, 36, 1011-1015) presented electrophoretic mobility shift method for determination of number of DNA molecules conjugated to quantum dots (QDs), which was further utilized for calculation of enzymatic activity, sequence specific DNA detection, and neutral molecule quantification.

• Klíčová slova
• DNA, Electrophoretic mobility shift assay, Protein, Quantum dots,
• MeSH
• DNA analýza   chemie   MeSH
• kvantové tečky analýza   chemie   MeSH
• nanokonjugáty analýza   chemie   MeSH
• retardační test metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• nanokonjugáty MeSH

Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC reaction of ABNOH-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable tricyclic fused hexahydropyrrolo-indole conjugates. Similarly modified ABNOH-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNOH-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.

• Klíčová slova
• Bioconjugations, N-oxyl radicals, Nucleotides, Oligonucleotides, Peptides,
• MeSH
• DNA-dependentní DNA-polymerasy metabolismus   chemie   MeSH
• DNA * chemie   MeSH
• nukleotidy * chemie   MeSH
• peptidy * chemie   MeSH
• proteiny chemie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• tryptofan * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• DNA * MeSH
• nukleotidy * MeSH
• peptidy * MeSH
• proteiny MeSH
• reagencia zkříženě vázaná MeSH
• tryptofan * MeSH

Lanthanide-doped upconversion nanoparticles (UCNPs) display highly beneficial photophysical features for background-free bioimaging and bioanalysis; however, they are instable in high ionic strength buffers, have no functional groups, and are nonspecifically interacting. Here, we have prepared NIR-excitable UCNPs that are long-term colloidally stable in buffered media and possess functional groups. Heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide were attached to UCNPs via a ligand exchange. Streptavidin (SA)-conjugates were prepared by click reaction of UCNP@PEG-alkyne and SA-azide. Antihuman serum albumin pAbF antibody was modified with azide groups and conjugated to UCNP@PEG-alkyne via click reaction; alternatively, the antibody, after mild reduction of its disulfide bonds, was conjugated to UCNP@PEG-maleimide. We employed these nanoconjugates as labels for an upconversion-linked immunosorbent assay. SA-based labels achieved the lowest LOD of 0.17 ng/mL for the target albumin, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL).

• MeSH
• nanočástice * MeSH
• polyethylenglykoly MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• polyethylenglykoly MeSH

A bioorthogonal 'catch and photorelease' strategy, which combines alkyne-azide cycloaddition between p-hydroxyphenacyl azide and alkyne derivatives to form a 1,2,3-triazole adduct and subsequent photochemical release of the triazole moiety via a photo-Favorskii rearrangement, is introduced. The first step can also involve photorelease of a strained alkyne and its Cu-free click reaction with azide.

• Publikační typ
• časopisecké články MeSH

In this study, biotin-conjugated glutathione was synthesized using peptide bonding of the biotin carboxy group and amino group of the γ-glutamic acid to prepare an alternative coating for CdTe quantum dots (QDs). This type of coating combines the functionality of the biotin with the fluorescent properties of the QDs to create a specific, high-affinity fluorescent probe able to react with avidin, streptavidin and/or neutravidin. Biotin-functionalized glutathione-coated CdTe QDs were prepared by a simple one-step method using Na₂ TeO₃ and CdCl₂. Obtained QDs were separated from the excess of the biotin-conjugated glutathione by CE employing 300 mM borate buffer with pH 7.8 as a background electrolyte. The detection of sample components was performed by the photometric detection at 214 nm and LIF employing Ar⁺ ion laser (488 nm).

• MeSH
• biotin chemie   metabolismus   MeSH
• elektroforéza kapilární MeSH
• fluorescence MeSH
• glutathion chemie   metabolismus   MeSH
• kvantové tečky * MeSH
• kyselina glutamová chemie   MeSH
• sloučeniny kadmia chemie   MeSH
• streptavidin chemie   MeSH
• telur chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biotin MeSH
• cadmium telluride MeSH Prohlížeč
• glutathion MeSH
• kyselina glutamová MeSH
• sloučeniny kadmia MeSH
• sodium tellurate(IV) MeSH Prohlížeč
• streptavidin MeSH
• telur MeSH

Peptide-peptide interactions are crucial in the living cell as they lead to the formation of the numerous types of complexes. In this study, synthetic peptides containing 11 of cysteines (α-domain of metallothionein (MT)) and sialic acid binding region (130-loop of hemagglutinin (HA)) were employed. The aim of the experiment was studying the interactions between MT and HA-derived peptides. For this purpose, fragments were tagged with cysteines at C-terminal part to serve as ligand sites for PbS and CuS quantum dots (QDs), and therefore these conjugates can be traced and quantified during wide spectrum of methods. As a platform for interaction, γ-Fe2O3 paramagnetic particles modified with tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane (hydrodynamic diameter 30-40 nm) were utilized and MT/HA interactions were examined using multi-instrumental approach including electrochemistry, electrophoretic methods, and MALDI-TOF/TOF mass spectrometry. It was found that peptides enter mutual creation of complexes, which are based on some of nonbonded interactions. The higher willingness to interact was observed in MT-derived peptides toward immobilized HA. Finally, we designed and manufactured flow-through electrochemical 3D printed device (reservoir volume 150 μL) and utilized it for automated analysis of the HA/MT metal labels. Under the optimal conditions, (deposition time and flow rate 80 s and 1.6 mL/min for CuS and 120 s and 1.6 mL/min PbS, respectively), the results of peptide-conjugated QDs were comparable with atomic absorption spectrometry.

• Klíčová slova
• 3D printing, Advanced materials, Influenza, Lab-On-a-Chip, Magnetic particle,
• MeSH
• 3D tisk * MeSH
• magnetické nanočástice chemie   MeSH
• mikrofluidní analytické techniky přístrojové vybavení   MeSH
• peptidy analýza   chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• magnetické nanočástice MeSH
• peptidy MeSH

A series of new hypervalent iodine reagents based on the 1,3-dihydro-3,3-dimethyl-1,2-benziodoxole and 1,2-benziodoxol-3-(1H)-one scaffolds, which contain a functionalized tetrafluoroethyl group, have been prepared, characterized, and used in synthetic applications. Their corresponding electrophilic fluoroalkylation reactions with various sulfur, oxygen, phosphorus, and carbon-centered nucleophiles afford products that feature a tetrafluoroethylene unit, which connects two functional moieties. A related λ(3) -iodane that contains a fluorophore was shown to react with a cysteine derivative under mild conditions to give a thiol-tagged product that is stable in the presence of excess thiol. Therefore, these new reagents show a significant potential for applications in chemical biology as tools for fast, irreversible, and selective thiol bioconjugation.

• Klíčová slova
• bioconjugation, fluorine, fluoroalkylation, hypervalent compounds, iodine, tetrafluoroethylene,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fluoroalkylation reagents based on hypervalent iodine are widely used to transfer fluoroalkyl moieties to various nucleophiles. However, the transferred groups have so far been limited to simple structural motifs. We herein report a reagent featuring a secondary amine that can be converted to amide, sulfonamide, and tertiary amine derivatives in one step. The resulting reagents bear manifold functional groups, many of which would not be compatible with the original synthetic pathway. Exploiting this structural versatility and the known high reactivity toward thiols, the new-generation reagents were used in bioconjugation with an artificial retro-aldolase, containing an exposed cysteine and a reactive catalytic lysine. Whereas commercial reagents based on maleimide and iodoacetamide labeled both sites, the iodanes exclusively modified the cysteine residue. The study thus demonstrates that modular fluoroalkylation reagents can be used as tools for cysteine-selective bioconjugation.

• Klíčová slova
• bioconjugation, cysteine, enzymes, fluorine, fluoroalkylation, hypervalent iodine,
• MeSH
• aminy chemie   MeSH
• cystein chemie   MeSH
• jod chemie   MeSH
• karbodiimidy chemie   MeSH
• lysin chemie   MeSH
• proteiny chemie   metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• sulfhydrylové sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 1-ethyl-3-(3-(diethylamino)propyl)carbodiimide MeSH Prohlížeč
• aminy MeSH
• cystein MeSH
• jod MeSH
• karbodiimidy MeSH
• lysin MeSH
• proteiny MeSH
• sulfhydrylové sloučeniny MeSH