RESUMO
The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts inâ situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells inâ vitro and for direct imaging of chemotherapy-induced apoptosis inâ vivo in mouse models of breast cancer.
Assuntos
Corantes FluorescentesRESUMO
The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts inâ situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells inâ vitro and for direct imaging of chemotherapy-induced apoptosis inâ vivo in mouse models of breast cancer.
RESUMO
Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics.
Assuntos
Apoptose , Imageamento Tridimensional , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Fagocitose/efeitos dos fármacos , Fosfatidilserinas/metabolismoRESUMO
Keratin 1 (KRT1) is overexpressed in squamous carcinomas and associated with aggressive pathologies in breast cancer. Herein we report the design and preparation of the first Trp-based red fluorogenic amino acid, which is synthetically accessible in a few steps and displays excellent photophysical properties, and its application in a minimally-disruptive labelling strategy to prepare a new fluorogenic cyclopeptide for imaging of KRT1+ cells in whole intact tumour tissues.
RESUMO
An activatable BODIPY probe for in vitro detection and fluorescence cell imaging of free Mg2+ without interference from Ca2+ is described. Fluorescence amplification of the probe is observed upon detection of physiological concentrations of Mg2+ due to reduced rotation of the fluorophore and effective chelation by a quinolizine-based core.
Assuntos
Corantes Fluorescentes/química , Magnésio/análise , Imagem Molecular/métodos , Animais , Quelantes , Humanos , Quinolizinas , RotaçãoRESUMO
Immunomodulatory agents represent one of the most promising strategies for enhancing tissue regeneration without the side effects of traditional drug-based therapies. Tissue repair depends largely on macrophages, making them ideal targets for proregenerative therapies. However, given the multiple roles of macrophages in tissue homeostasis, small molecule drugs must be only active in very specific subpopulations. In this work, we have developed the first prodrug-fluorophore conjugates able to discriminate closely related subpopulations of macrophages (i.e., proinflammatory M1 vs anti-inflammatory M2 macrophages), and employed them to deplete M1 macrophages in vivo without affecting other cell populations. Selective intracellular activation and drug release enabled simultaneous fluorescence cell tracking and ablation of M1 macrophages in vivo, with the concomitant rescue of a proregenerative phenotype. Ex vivo assays in human monocyte-derived macrophages validate the translational potential of this novel platform to develop chemical immunomodulatory agents as targeted therapies for immune-related diseases.
RESUMO
Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C2-BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).
Assuntos
Aspergillus fumigatus/citologia , Compostos de Boro/análise , Técnicas Microbiológicas/métodos , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Triptofano/análise , Compostos de Boro/síntese química , Triptofano/síntese químicaRESUMO
The rational design and synthesis of a Trp-BODIPY cyclic peptide for the fluorescent labelling of apoptotic bodies is described. Affinity assays, confocal microscopy and flow cytometry analysis confirmed the binding of the peptide to negatively-charged phospholipids associated with apoptosis, and its applicability for the detection and characterisation of subcellular structures released by apoptotic cells.
Assuntos
Compostos de Boro/química , Vesículas Extracelulares/química , Fluorescência , Corantes Fluorescentes/química , Peptídeos Cíclicos/química , Triptofano/química , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Coloração e RotulagemRESUMO
Stand-alone coupling reagents derived from bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride show efficient performance in solution and SPPS. In particular, the Oxyma Pure (Luxembourg Biotech., Tel Aviv, Israel) derivative shows the additional advantage of being highly soluble in DMF and even fairly soluble in CH3 CN, which can extend its use for the synthesis of complex peptides. These new stand-alone coupling reagents have the advantage of not bearing any counteranion such as PF6 or BH4 , whose presence can jeopardize the purification of final peptides prepared in solution.
Assuntos
Oxazolidinonas/síntese química , Peptídeos/síntese química , Ácidos Fosfínicos/síntese química , Sequência de Aminoácidos , Técnicas de Síntese em Fase Sólida , Solubilidade , SoluçõesRESUMO
The methodology for peptide bond formation is undergoing a continuous evolution where the main actors are being renewed. In recent years, coupling reagents based on the Oxyma scaffold, such as the uronium salt COMU, has been a groundbreaking contribution to the field. The advantages of COMU over classic benzotriazole-based reagents (HATU, HBTU, HCTU, TBTU) were proven in terms of solubility and coupling efficiency in bulky junctions in our groups and others. However, some aspects of the use of COMU need to be revised and improved, such as the stability of commercial samples in organic solvents, which hampers the compatibility with long synthesis in automated synthesizers. In this review, an overview of the main features and suggestions to improve the use of COMU are presented, along with a discussion on the best conditions for its use in microwave-assisted peptide robots.
Assuntos
Iminas/química , Morfolinas/química , Peptídeos/química , Triazóis/química , Acilação , Micro-OndasRESUMO
Peptide-bond formation is a pivotal process in the synthesis of peptide oligomers. Among the various coupling methodologies described, carbodiimides combine strong acylation potency and smooth reaction conditions, and they are commonly used in the presence of N-hydroxylamine additives. In recent years, acidic oxime templates, mainly ethyl 2-cyano-2-(hydroxyimino) acetate (Oxyma), have emerged as highly reactive alternatives to the classic and explosive-prone benzotriazolic additives, 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). However, to achieve certain biochemical targets, less reactive species, such as N-hydroxysuccinimide (HOSu) esters, are often required to obtain stability under aqueous conditions. In the present study, we report on a new family of water-soluble N-alkyl-cyanoacetamido oximes, most of which have proven useful in the construction of active carbonates for the introduction of fluorenylmethoxycarbonyl (Fmoc) with minimal impact of dipeptide impurities. We performed a direct comparison of these new N-alkyl-cyanoacetamido oximes with HOSu in order to evaluate their capacity to retain optical purity and their coupling efficiency in the assembly of bulky residues.
RESUMO
The presence of low pKa N-hydroxylamines is beneficial in peptide chemistry as they reduce some base-mediated side reactions. Here we evaluated the applicability and buffering capacity of Ethyl 2-cyano-2-(hydroxyimino) acetate (Oxyma) in the prevention of aspartimide/piperidide formation and Pro-based overcoupling and compared it with the performance of HOBt and HOAt. In addition, the compatibility of these additives with the highly acid-labile 2-chlorotrityl chloride resin is examined.
Assuntos
Hidroxilaminas/química , Peptídeos/química , Peptídeos/síntese química , Pregnadienos/química , Compostos de Tritil/química , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Concentração de Íons de Hidrogênio , Estrutura MolecularRESUMO
4-(4,6-Di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate (DFET/NMM/BF(4)) was prepared and used as a reagent for coupling sterically hindered substrates. The formation of the appropriate triazine "superactive" ester in a reaction of DFET/NMM/BF(4) with carboxylic acids was confirmed. The efficiency of the reagent has been studied in the synthesis of Leu-enkephaline pentapeptide carried out on a Fmoc-RinkAmide-AM-PS resin, by systematically modifying the -Gly-Gly- fragment for N-methyl or α,α-disubstituted residues and compared with the efficiency of classic aminium salt 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) under a variety of reaction conditions. In syntheses of Aib-Aib (Aib: α-aminoisobutyric acid), MeVal-MeVal, and MeLeu-MeLeu, the considerably superior performance of enkephaline analogues was obtained for DFET/NMM/BF(4) relative to TBTU, regardless of reaction conditions. Analysis of the couplings involving triazine reagent suggests that factors controlling efficiency of coupling sterically hindered substrates are the structure of the leaving group permitting formation of the cyclic intermediate or cyclic transition state and the absence of strongly solvating solvents. It has to be considered as highly probable that the absence of strongly solvating milieu favors cyclic intermediates or the cyclic transition state. Arrangement of both components into the cyclic intermediate or cyclic transition state by accumulation of the geminal (vicinal) substituents effect (known as the Thorpe-Ingold effect) would compensate retardation of the coupling process caused by steric hindrance.
Assuntos
Boratos/química , Morfolinas/química , Triazinas/química , Alquilação , Aminoácidos/química , Indicadores e Reagentes/química , Nitrogênio/química , Oligopeptídeos/síntese química , Oligopeptídeos/químicaRESUMO
Recent studies described the great impact of a non-benzotriazolic family of coupling reagents based on ethyl 2-cyano-2-(hydroxyimino)acetate, Oxyma, as a powerful coupling methodology for peptide synthesis. Here we present the synthesis and evaluation of the derived phosphonium salts O-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyOxP) and tetrafluoroborate (PyOxB). Both coupling reagents exhibited higher capacity to suppress racemization in various peptide models and enhanced solubility in DMF and DCM than benzotriazole-based reagents. In addition, the hexafluorophosphate analog PyOxP, combined excellent stability with outstanding efficiency in the assembly of demanding penta and decapeptides that include consecutive Aib residues. Cyclization models revealed the advantages of PyOxP, which rendered a higher percentage of cyclic material than other known potent phosphonium salts.
Assuntos
Compostos Organofosforados/química , Pregnadienos/química , Ciclização , Hidrólise , Estrutura Molecular , Solubilidade , EstereoisomerismoRESUMO
We describe a new family of uronium-type coupling reagents that differ in their iminium moieties and leaving groups. The presence of the morpholino group in conjunction with an oxime derivative--especially ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma)--had a marked influence on the solubilities, stabilities, and reactivities of the reagents. Finally, the new uronium salt derived from Oxyma (COMU) performed extremely well in the presence of only 1 equiv of base, thereby confirming the effect of the hydrogen bond acceptor in the reaction. COMU also showed a less hazardous safety profile than the benzotriazole-based HDMA and HDMB, which exhibited unpredictable autocatalytic decompositions. Furthermore, the Oxyma moiety contained in COMU suggests a lower risk of explosion than in the case of the benzotriazole derivatives.
Assuntos
Morfolinas/química , Compostos Organofosforados/química , Peptídeos/síntese química , Triazóis/química , Compostos Aza/química , Cristalografia por Raios X , Digoxigenina/análogos & derivados , Digoxigenina/química , Conformação Molecular , Peptídeos/química , Pregnadienos/química , Succinimidas/químicaRESUMO
Oxyma [ethyl 2-cyano-2-(hydroxyimino)acetate] has been tested as an additive for use in the carbodiimide approach for formation of peptide bonds. Its performance in relation to those of HOBt and HOAt, which have recently been reported to exhibit explosive properties, is reported. Oxyma displayed a remarkable capacity to inhibit racemization, together with impressive coupling efficiency in both automated and manual synthesis, superior to those of HOBt and at least comparable to those of HOAt, and surpassing the latter coupling agent in the more demanding peptide models. Stability assays showed that there was no risk of capping the resin under standard coupling conditions. Finally, calorimetry assays (DSC and ARC) showed decomposition profiles for benzotriazole-based additives that were consistent with their reported explosivities and suggested a lower risk of explosion in the case of Oxyma.