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1.
Int J Nanomedicine ; 15: 7433-7450, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116480

RESUMO

Purpose: Salicyl (Sal) - among other oxygen functionalities - multi-walled carbon nanotubes (MWCNTs) and their nanohybrids are investigated as promising contrast agents (CA) in magnetic resonance imaging (MRI) or drug delivery platforms, due to their unique properties. The preliminary results and the literature reports were the motivation to endow high r2 relaxivities, excellent dispersibility in water, and biocompatibility to superparamagnetic MWCNTs nanohybrids. It was hypothesized that these goals could be achieved by, not described in the literature yet, two-stage oxygen functionalization of MWCNTs. Results: Two structurally different MWCNT materials differing in diameters (44 and 12 nm) and the iron content (4.7% and 0.5%) are studied toward the functionalization effect on the T2 relaxometric properties. MWCNT oxidation is typically the first step of functionalization resulting in "first generation" oxygen functional groups (OFGs) on the surface. Until now, the impact of OFGs on the relaxivity of MWCNT was not truly recognized, but this study sheds light on this issue. By follow-up functionalization of oxidized MWCNT with 4-azidosalicylic acid through [2+1] cycloaddition of the corresponding nitrene, "second generation" of oxygen functional groups is grafted onto the nanohybrid, ie, Sal functionality. Conclusion: The introduced OFGs are responsible for an almost 30% increase in the relaxivity, which leads to remarkable r2 relaxivity of 951 mM-1s-1 (419 (mg/mL)-1s-1), the unprecedented value reported to date for this class of CAs. Also, the resulting nanohybrids express low cytotoxicity and superb diffusion after subcutaneous injection to a mouse.


Assuntos
Meios de Contraste/química , Meios de Contraste/farmacologia , Imagem por Ressonância Magnética/métodos , Nanotubos de Carbono/química , Oxigênio/química , Animais , Azidas/química , Reação de Cicloadição , Camundongos Endogâmicos C57BL , Oxirredução , Prótons , Salicilatos/química , Água/química
2.
J Food Sci ; 85(10): 3509-3516, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32964461

RESUMO

Rapid and sensitive detection of live/infectious foodborne pathogens is urgently needed in order to prevent outbreaks and food recalls. This study aimed to (1) evaluate the incorporation of propidium monoazide (PMA) into PCR or LAMP assays to selectively detect viable Salmonella Enteritidis following sublethal heat or UV treatment, and autoclave sterilization; and (2) compare the detection of PMA-PCR and PMA-LAMP to DNA-based PCR and LAMP (without PMA), RNA-based RT-PCR and RT-LAMP, and culture-based methods. Nucleic acids (DNA or RNA) from 1-mL S. Enteritidis samples were used for PCR, RT-PCR, LAMP, and RT-LAMP assays. Serially diluted samples were plated on Xylose Lysine Tergitol-4 agar for cultural enumeration. Comparable detection of overnight cultured S. Enteritidis was obtained by PMA-PCR, PCR, and RT-PCR, though 1 to 2 log less sensitive than cultural assays. PMA-LAMP and RT-LAMP showed similar detection of overnight cultures, being 1 to 2 log less sensitive than the LAMP assay, and ∼4 log less than culture-based detection. Autoclaved S. Enteritidis did not test positive by RNA-based methods or PMA-PCR, but PMA-LAMP showed detection of 1 log CFU/mL. PMA-PCR and RT-PCR showed comparable detection of sublethal heat-treated cells to cultural assays, while PMA-LAMP showed 1 to 2 log less detection. Our results suggest that PMA-PCR and PMA-LAMP assays are not suitable for selective viable cell detection after UV treatment. While PMA-LAMP assay needs optimization, PMA-PCR shows promise for live/viable S. Enteritidis detection. PMA-PCR shows potential for routine testing in the food industry with results within 1-day, albeit depending on the inactivation method employed.


Assuntos
Azidas/química , Propídio/análogos & derivados , Salmonella enteritidis/crescimento & desenvolvimento , Salmonella enteritidis/isolamento & purificação , Coloração e Rotulagem/métodos , Indústria de Processamento de Alimentos , Viabilidade Microbiana , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Propídio/química , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonella enteritidis/química , Salmonella enteritidis/genética
3.
Phys Chem Chem Phys ; 22(34): 19223-19229, 2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32812969

RESUMO

Azido stretch modes in a variety of azido-derivatized nonnatural amino acids and nucleotides have been used as a site-specific infrared (IR) probe for monitoring changes in their conformations and local electrostatic environments. The vibrational bands of azide probes are often accompanied by complex line shapes with shoulder peaks, which may arise either from incomplete background subtraction, Fermi resonance, or multiple conformers. The isotope substitution in the infrared probe has thus been introduced to remove Fermi resonances without causing a significant perturbation to the structure. Here, we synthesized and labeled the mid-N atoms of aliphatic azide derivatives with 15N to study the effects of isotope labelling on their vibrational properties. The FT-IR spectra of the aliphatic azide with asymmetric lineshape became a single symmetric band upon isotope substitution, which might be an indication of the removal of the hidden Fermi resonance from the system. We also noticed that the 2D-IR spectrum of unlabeled aliphatic azide has cross-peaks, even though it is not apparently identifiable. The 1D slice spectra obtained from the 2D-IR spectra reveal the existence of a hidden Fermi resonance peak. Furthermore, we show that this weak Fermi resonance does not produce discernible oscillatory beating patterns in the IR pump-probe spectrum, which has been used as evidence of the Fermi resonance. Therefore, we confirm that isotope labelling combined with 2D-IR spectroscopy is the most efficient and incisive way to identify the origin of small shoulder peaks in the linear and nonlinear vibrational spectra of various IR probe molecules.


Assuntos
Alanina/análogos & derivados , Azidas/química , Espectroscopia de Infravermelho com Transformada de Fourier , Alanina/química , Marcação por Isótopo , Modelos Químicos , Conformação Molecular , Isótopos de Nitrogênio/química , Eletricidade Estática , Vibração
4.
Phys Chem Chem Phys ; 22(24): 13358-13362, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32478770

RESUMO

Label-based functional studies of biomolecules in their native environment require labeling reactions inside living cells. In cell spin labeling using alkyne-azide click chemistry with a Gd3+-DOTAM-azide complex is shown to provide high spin label stability and narrow EPR lines for EPR spectroscopic detection of a spin labeled protein in living cells at ambient temperatures.


Assuntos
Escherichia coli/química , Gadolínio/química , Proteínas de Fluorescência Verde/análise , Marcadores de Spin , Acetamidas/química , Alquinos/química , Azidas/química , Química Click , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/citologia , Compostos Heterocíclicos com 1 Anel/química , Estrutura Molecular
5.
Nucleic Acids Res ; 48(13): 7356-7370, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32520335

RESUMO

To enable the optimal, biocompatible and non-destructive application of the highly useful copper (Cu+)-mediated alkyne-azide 'click' cycloaddition in water, we have isolated and characterized a 79-nucleotide DNA enzyme or DNAzyme, 'CLICK-17', that harnesses as low as sub-micromolar Cu+; or, surprisingly, Cu2+ (without added reductants such as ascorbate) to catalyze conjugation between a variety of alkyne and azide substrates, including small molecules, proteins and nucleic acids. CLICK-17's Cu+ catalysis is orders of magnitude faster than that of either Cu+ alone or of Cu+ complexed to PERMUT-17, a sequence-permuted DNA isomer of CLICK-17. With the less toxic Cu2+, CLICK-17 attains rates comparable to Cu+, under conditions where both Cu2+ alone and Cu2+ complexed with a classic accelerating ligand, THPTA, are wholly inactive. Cyclic voltammetry shows that CLICK-17, unlike PERMUT-17, powerfully perturbs the Cu(II)/Cu(I) redox potential. CLICK-17 thus provides a unique, DNA-derived ligand environment for catalytic copper within its active site. As a bona fide Cu2+-driven enzyme, with potential for being evolved to accept only designated substrates, CLICK-17 and future variants promise the fast, safe, and substrate-specific catalysis of 'click' bioconjugations, potentially on the surfaces of living cells.


Assuntos
Cobre/metabolismo , Reação de Cicloadição/métodos , DNA Catalítico/química , Alquinos/química , Azidas/química , Química Click/métodos , Oxirredução , Água/química
6.
Inorg Chem ; 59(12): 8468-8480, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32450042

RESUMO

The photochemical products of dinucleotides 5'-TpG/5'-GpT with a photoactivatable anticancer Pt(IV) complex (trans,trans,trans-[Pt(N3)2(OH)2(py)2], py = pyridine; 1) were characterized by electrospray ionization mass spectrometry. The primary MS showed the main products were monoplatinated and diplatinated adducts for both the dinucleotides accompanied by the formation of minor triplatinated dinucleotides, indicating that T-N3 and G-N1 may be platination sites additional to the well-known G-N7 site. Surprisingly, a series of minor platinated adducts with oxidation of guanine and/or thymine were observed. Although guanine is more sensitive to oxidation than thymine, thymine can compete with guanine for complex 1-induced oxidation, of which the oxidation adducts were identified as cis- and trans-diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine (cis,trans-ThdGly), 5-formyl-2'-deoxyuridine (5-FormdUrd), and 5-(hydroxymethyl)-2'-deoxyuridine (5-HMdUrd), respectively. While for guanine, apart from 8-hydroxyguanine (8-OH-G) and N-formylamidoiminohydantoin (RedSp), other guanine oxidized adducts such as spiroiminodihydantoin (Sp), dehydroguanidinohydantoin (DGh), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) were also identified. MS/MS analysis showed that unique fragments with a Pt moiety [Pt(N3)(py)] cross-linking the G and T bases were formed during the fragmentation of monoplatinated dinucleotides. Such binding mode to and oxidative damages on DNA bases imposed by the diazido Pt(IV) complex are apparently distinct from those of cisplatin, perhaps accounting for its unique mechanism of action.


Assuntos
Antineoplásicos/química , Complexos de Coordenação/química , Reagentes para Ligações Cruzadas/química , Guanina/química , Compostos Organoplatínicos/química , Timina/química , Azidas/química , Estrutura Molecular , Oxirredução , Processos Fotoquímicos
7.
Int J Food Microbiol ; 325: 108626, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32325345

RESUMO

AIMS: The aim of this study was to evaluate the performance of sampling methods [contact plates, sponges, and swabs] in the recovery of biofilm Listeria monocytogenes populations considering the seafood environment conditions (nature of conditioning, of materials and bacterial species). METHODS AND RESULTS: Different materials (stainless steel, polyvinyl chloride, polyurethane) were conditioned with two fish filtrates, the ready-to-eat the most consumed in Europe (smoked salmon, cod). After, we added the suspension of Listeria monocytogenes, alone or with Pseudomonas fluorescens or Carnobacterium strains, and incubated for 48 h at 8 °C. Then, the 48 h-biofilms were sampled with different methods (contact plates, sponges, and swabs). The cultivable bacterial populations were enumerated on agar, while the L. monocytogenes total and viable populations were quantified by qPCR and propidium monoazide-qPCR (PMA-qPCR), respectively. The amount of L. monocytogenes in biofilms was affected only by the nature of the conditioning with lowest adherent bacteria with cod versus with smoked salmon conditioning. Considering the amount of total population, the swab displayed the lowest values versus the sponges and the contact plates. An explanation was that the observations of the swab by microscopy showed the bacteria trapped within it. The recovery of cultivable bacterial populations was not significantly different with the three sampling methods. On the contrary, we showed that the VBNC populations were only detached by two of three methods (contact plates, sponges) while for the dead populations, those were contact plates and swabs. CONCLUSIONS: The nature of the conditioning influenced the amount of the bacteria in biofilms. And the performance of the recovery of the bacterial populations (dead, VBNC, cultivable) was dependent on the methods used. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the seafood environmental conditions influenced the biofilm formation and the assessment of the efficiency of cleaning and disinfectant operations could be significantly affected by the used sampling methods.


Assuntos
Biofilmes/crescimento & desenvolvimento , Microbiologia de Alimentos/métodos , Listeria monocytogenes/isolamento & purificação , Alimentos Marinhos/microbiologia , Ágar , Animais , Azidas/química , Carnobacterium/isolamento & purificação , Desinfetantes/farmacologia , Europa (Continente) , Peixes/microbiologia , Propídio/análogos & derivados , Propídio/química , Pseudomonas fluorescens/isolamento & purificação , Aço Inoxidável/análise
8.
Molecules ; 25(7)2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32290240

RESUMO

The reaction between organic azides and alkyne derivatives via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient strategy to combine phthalocyanines and analogues with different materials. As examples of such materials, it can be considered the following ones: graphene oxide, carbon nanotubes, silica nanoparticles, gold nanoparticles, and quantum dots. This approach is also being relevant to conjugate phthalocyanines with carbohydrates and to obtain new sophisticated molecules; in such way, new systems with significant potential applications become available. This review highlights recent developments on the synthesis of phthalocyanine, subphthalocyanine, and porphyrazine derivatives where CuAAC reactions are the key synthetic step.


Assuntos
Azidas/química , Indóis/química , Azidas/síntese química , Catálise , Técnicas de Química Sintética , Química Click , Eletrodos , Indóis/síntese química , Estrutura Molecular , Polímeros/química
9.
J Vis Exp ; (157)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32176211

RESUMO

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Corantes Fluorescentes , Antibacterianos/química , Azidas/química , Bactérias/efeitos dos fármacos , Química Click , Reação de Cicloadição , Corantes Fluorescentes/química , Testes de Sensibilidade Microbiana , Microscopia Confocal
10.
Org Biomol Chem ; 18(12): 2252-2263, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32149317

RESUMO

Glycoconjugates, due to their diverse functions, are widely regarded as biologically important molecules. Artemisinic acid 1 occurs naturally in the plant Artemisia annua and is considered to be the biogenetic precursor of the antimalarial drug, artemisinin 2. We report herein the design and synthesis of diverse artemisinic acid derived glycoconjugates. We have synthesized 12-O-artemisinic acid-glycoconjugates (7a-k) and 12-N-artemisinic acid-glycoconjugates (8a-k) by utilizing Cu(i)-catalyzed azide-alkyne cycloaddition reactions (Click chemistry) with various synthesized sugar azides (6a-k) in good to excellent yields along with two fluorescently labeled compounds, 12-O-artemisinic acid-glycoconjugate 11 and 12-N-artemisinic acid-glycoconjugate 12, to investigate the mode of action of these compounds in biological systems. All the synthesized artemisinic acid glycoconjugates were assayed for their efficacy against the MCF7 cell line. Our anticancer studies indicated that all the synthesized compounds inhibited the growth of MCF7 cells in a dose dependent manner, barring compounds 4 and 7d. However, these compounds exhibit moderate cytotoxicity, as is evident from their IC50 values.


Assuntos
Antineoplásicos/síntese química , Artemisininas/química , Glicoconjugados/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Azidas/química , Química Click , Reação de Cicloadição , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Glicoconjugados/farmacologia , Humanos , Concentração Inibidora 50 , Células MCF-7 , Sesquiterpenos/química
11.
Chem Asian J ; 15(9): 1420-1429, 2020 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-32144862

RESUMO

The development of advanced bioorthogonal reactions for detection and labeling of biomolecules is significant in chemical biology. Recently, researchers have found that multifluorinated aryl azides hold great potential for the development of improved bioorthogonal reactions. The fluorine atom can be a perfect substituent group because of its properties of excellent electronegativity and small steric hindrance. In this Minireview, we discuss recent developments of improved hydrogen sulfide (H2 S) fluorescence probes, fast strain-promoted azide-alkyne cycloaddition (SPAAC) and nonhydrolysis Staudinger reactions based on the use of multifluorinated aryl azides. Additionally, kinetic studies and biological applications of these reactions are also presented.


Assuntos
Alquinos/química , Azidas/química , Corantes Fluorescentes/química , Sulfeto de Hidrogênio/análise , Azidas/síntese química , Reação de Cicloadição , Corantes Fluorescentes/síntese química , Halogenação , Estrutura Molecular , Imagem Óptica
12.
Biomater Sci ; 8(9): 2507-2513, 2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32211707

RESUMO

The design of multiple stimuli-responsive, stable polymeric drug carriers is key for efficient drug release against solid tumors. Herein, core-crosslinked micelles were readily prepared from a pair of redox/pH-sensitive clickable copolymers. The two copolymers comprised the same poly(ethylene glycol) (PEG)-poly(ε-benzyloxycarbonyl-l-lysine) (PZLL) block but with either disulfide-linked azadibenzocyclooctyne (DBCO) or azide (AZ) group-tagged branched polyethylenimine (BPEI, 1.8 kDa). The data showed that an equivalent of the two copolymers could self-assemble into nanosized micelles with the crosslinked core via the DBCO-AZ click chemistry. The click-crosslinked micelles showed excellent size stability under multiple dilutions but destabilization in an acidic or reductive environment. Besides, they could load doxorubicin (DOX), an anticancer drug, and mediate slow drug release in a neutral environment but sufficient drug unloading under acidic plus reductive conditions. In vitro, DOX-loaded crosslinked micelles led to higher DOX accumulation in the cellular nucleus in comparison with non-crosslinked micelles from the PEG-PZLL-BPEI copolymer (PP), thus causing more marked cytotoxicity in SKOV-3 cells. In vivo, DOX-loaded crosslinked micelles caused significant growth inhibition of SKOV-3 tumors xenografted in BALB/c nude mice, and showed superior anticancer efficacy to non-crosslinked PP micelles. Chemotherapy with core-crosslinked micelles had no adverse side effects on the health (serum levels and body weight) of the mice. This study highlights the design of clickable block copolymers to easily construct core-crosslinked and multiple stimuli-responsive micelles for enhanced anticancer therapy.


Assuntos
Antineoplásicos/administração & dosagem , Compostos Aza/administração & dosagem , Azidas/administração & dosagem , Ciclo-Octanos/administração & dosagem , Doxorrubicina/administração & dosagem , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Compostos Aza/química , Compostos Aza/farmacocinética , Azidas/química , Azidas/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Octanos/química , Ciclo-Octanos/farmacocinética , Doxorrubicina/química , Doxorrubicina/farmacocinética , Liberação Controlada de Fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Micelas , Neoplasias/tratamento farmacológico , Polímeros/administração & dosagem , Polímeros/química , Polímeros/farmacocinética , Distribuição Tecidual
13.
Molecules ; 25(3)2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32046110

RESUMO

In this report, we describe the synthetic elaboration of the easily available enantiomerically pure ß-amino alcohols. Attempted direct substitution of the hydroxyl group by azido-functionality in the Mitsunobu reaction with hydrazoic acid was inefficient or led to a diastereomeric mixture. These outcomes resulted from the participation of aziridines. Intentionally performed internal Mitsunobu reaction of ß-amino alcohols gave eight chiral aziridines in 45-82% yield. The structural and configuration identity of products was confirmed by NMR data compared to the DFT calculated GIAO values. For 1,2,3-trisubstituted aziridines slow configurational inversion at the endocyclic nitrogen atom was observed by NMR at room temperature. Moreover, when aziridine was titrated with Zn(OAc)2 under NMR control, only one of two N-epimers directly participated in complexation. The aziridines underwent ring opening with HN3 to form the corresponding azido amines as single regio- and diastereomers in 90-97% yield. Different results were obtained for 1,2-disubstituted and 1,2,3-trisubstituted aziridines. For the later aziridines ring closure and ring opening occurred at different carbon stereocenters, thus yielding products with two inverted configurations, compared to the starting amino alcohol. The 1,2-disubstituted aziridines produced azido amines of the same configuration as the starting ß-amino alcohols. To obtain a complete series of diastereomeric vic-diamines, we converted the amino alcohols into cyclic sulfamidates, which reacted with sodium azide in SN2 reaction (25-58% overall yield). The azides obtained either way underwent the Staudinger reduction, giving a series of six new chiral vic-diamines of defined stereochemistries.


Assuntos
2,2'-Dipiridil/química , Amino Álcoois/química , Diaminas/química , Aminas/química , Azidas/química , Aziridinas/química , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Estereoisomerismo
14.
Carbohydr Res ; 489: 107948, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32062397

RESUMO

Rh/Al2O3 can be used as an effective chemo-selective reductive catalyst that combines the mild conditions of catalytic hydrogenation with high selectivity for azide moieties in the presence of other hydrogenolysis labile groups such as benzyl and benzyloxycarbonyl functionalities. The practicality of this strategy is exemplified with a range of azide-containing carbohydrate and amino acid derivatives.


Assuntos
Aminas/síntese química , Azidas/química , Ródio/química , Aminas/química , Configuração de Carboidratos , Catálise , Hidrogenação
15.
Phys Chem Chem Phys ; 22(9): 4875-4879, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32072999

RESUMO

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E. coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Alquinos/química , Azidas/química , Catálise , Cobre/química , Reação de Cicloadição , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Óxidos de Nitrogênio/química , Marcadores de Spin
16.
Nat Methods ; 17(3): 311-318, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015544

RESUMO

Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-type-specific profiling of gene expression. Current metabolic labeling methods rely on exogenous pyrimidine analogs that are only incorporated into RNA in cells expressing an exogenous enzyme. This approach assumes that off-target cells cannot incorporate these analogs. We disprove this assumption and identify and characterize the enzymatic pathways responsible for high background incorporation. We demonstrate that mammalian cells can incorporate uracil analogs and characterize the enzymatic pathways responsible for high background incorporation. To overcome these limitations, we developed a new small molecule-enzyme pair consisting of uridine/cytidine kinase 2 and 2'-azidouridine. We demonstrate that 2'-azidouridine is only incorporated in cells expressing uridine/cytidine kinase 2 and characterize selectivity mechanisms using molecular dynamics and X-ray crystallography. Furthermore, this pair can be used to purify and track RNA from specific cellular populations, making it ideal for high-resolution cell-specific RNA labeling. Overall, these results reveal new aspects of mammalian salvage pathways and serve as a new benchmark for designing, characterizing and evaluating methodologies for cell-specific labeling of biomolecules.


Assuntos
RNA/química , Uracila/química , Animais , Azidas/química , Biotinilação , Domínio Catalítico , Técnicas de Cocultura , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Células HEK293 , Células HeLa , Humanos , Cinética , Camundongos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Núcleosídeo-Fosfato Quinase/metabolismo , Domínios Proteicos , RNA Interferente Pequeno/genética , Uridina/química , Uridina Quinase/metabolismo
17.
Molecules ; 25(3)2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-32012736

RESUMO

To enhance the versatility of organic azides in organic synthesis, a better understanding of their photochemistry is required. Herein, the photoreactivity of azidoisoxazole 1 was characterized in cryogenic matrices with IR and UV-Vis absorption spectroscopy. The irradiation (λ = 254 nm) of azidoisoxazole 1 in an argon matrix at 13 K and in glassy 2-methyltetrahydrofuran (mTHF) at 77 K yielded nitrosoalkene 3. Density functional theory (DFT) and complete active space self-consistent field (CASSCF) calculations were used to aid the characterization of nitrosoalkene 3 and to support the proposed mechanism for its formation. It is likely that nitrosoalkene 3 is formed from the singlet excited state of azidoisoxazole 1 via a concerted mechanism or from cleavage of an intermediate singlet nitrene that does not undergo efficient intersystem crossing to its triplet configuration.


Assuntos
Alcenos/química , Azidas/química , Temperatura Baixa , Isoxazóis/química , Compostos Nitrosos/química , Fotólise , Alcenos/análise , Azidas/efeitos da radiação , Isoxazóis/efeitos da radiação , Compostos Nitrosos/análise , Teoria Quântica
18.
Org Biomol Chem ; 18(10): 1957-1967, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32101244

RESUMO

1,4- and 1,5-Disubstituted triazole amino acid monomers have gained increasing interest among peptidic foldamers, as they are easily prepared via Cu- and Ru-catalyzed click reactions, with the potential for side chain variation. While the latter is key to their applicability, the synthesis and structural properties of the chiral mono- or disubstituted triazole amino acids have only been partially addressed. We here present the synthesis of all eight possible chiral derivatives of a triazole monomer prepared via a ruthenium-catalyzed azide alkyne cycloaddition (RuAAC). To evaluate the conformational properties of the individual building units, a systematic quantum chemical study was performed on all monomers, indicating their capacity to form several low energy conformers. This feature may be used to effect structural diversity when the monomers are inserted into various peptide sequences. We envisage that these results will facilitate new applications for these artificial oligomeric compounds in diverse areas, ranging from pharmaceutics to biotechnology.


Assuntos
Peptidomiméticos/síntese química , Triazóis/síntese química , Alquinos/química , Azidas/química , Química Click , Reação de Cicloadição , Modelos Moleculares , Polimerização , Polímeros/síntese química , Teoria Quântica , Estereoisomerismo , Termodinâmica
19.
Molecules ; 25(4)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32102403

RESUMO

The exceptional reactivity of the azide group makes organic azides a highly versatile family of compounds in chemistry and the material sciences. One of the most prominent reactions employing organic azides is the regioselective copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition with alkynes yielding 1,2,3-triazoles. Other named reactions include the Staudinger reduction, the aza-Wittig reaction, and the Curtius rearrangement. The popularity of organic azides in material sciences is mostly based on their propensity to release nitrogen by thermal activation or photolysis. On the one hand, this scission reaction is accompanied with a considerable output of energy, making them interesting as highly energetic materials. On the other hand, it produces highly reactive nitrenes that show extraordinary efficiency in polymer crosslinking, a process used to alter the physical properties of polymers and to boost efficiencies of polymer-based devices such as membrane fuel cells, organic solar cells (OSCs), light-emitting diodes (LEDs), and organic field-effect transistors (OFETs). Thermosets are also suitable application areas. In most cases, organic azides with multiple azide functions are employed which can either be small molecules or oligo- and polymers. This review focuses on nitrene-based applications of multivalent organic azides in the material and life sciences.


Assuntos
Alquinos/química , Azidas/química , Reagentes para Ligações Cruzadas/química , Nitrogênio/química , Triazóis/síntese química , Catálise , Reação de Cicloadição , Humanos , Ciência dos Materiais/métodos , Estrutura Molecular , Processos Fotoquímicos , Fotólise
20.
Bioelectrochemistry ; 133: 107484, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32087557

RESUMO

Electro-click methodology was employed to prepare an electrochemical immunosensor for the cytokine interleukin 1ß (IL-1ß). The strategy involved binding of ethynylated IgG to azide-MWCNTs modified electrodes by Cu(I) catalyzed-cycloaddition reaction where the catalyst was electrochemically synthesized. This electro-click protocol is significantly faster and greener than the methods for catalyst generation through chemical reduction. The oriented immobilization of the capture antibody onto IgG-MWCNTs conjugates allowed the preparation of a sandwich-type immunosensor using biotinylated anti-IL-1ß as detector antibody labeled with alkaline phosphatase-streptavidin (AP-strept). Differential pulse voltammetric transduction through the 1-naphthylphosphate/1-naphthol system was carried out. The analytical characteristics achieved with the electrochemical immunosensor showed a calibration curve exhibiting two linear ranges between 10 and 200 pg mL-1 (r2 = 0.998), and from 200 to 1200 pg mL-1 (r2 = 0.998), and a LOD value of 5.2 pg mL-1, an improvement compared with those claimed for commercial ELISA kits. In addition, the assay time was at least one hour shorter. Excellent performance was observed in the determination of IL-1ß in saliva with no need for sample treatment, and by simple interpolation using a calibration plot constructed with standard solutions of the target cytokine.


Assuntos
Anticorpos Imobilizados/química , Técnicas Eletroquímicas/instrumentação , Interleucina-1beta/análise , Saliva/química , Azidas/química , Técnicas Biossensoriais/instrumentação , Química Click , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Imunoglobulina G/química , Limite de Detecção , Nanotubos de Carbono/química
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