RESUMEN
Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.
Asunto(s)
Conformación de Ácido Nucleico , División del ARN , ARN/química , Ribonucleasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Bioensayo/métodos , Catálisis , Cinética , Modelos Biológicos , Hibridación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Oligonucleótidos/aislamiento & purificación , Péptidos/síntesis química , Péptidos/química , Péptidos/aislamiento & purificación , Ribonucleasas/metabolismo , Relación Estructura-ActividadRESUMEN
Sequence-specific protein-based ribonucleases are not found in nature. Absolute sequence selectivity in RNA cleavage in vivo normally requires multi-component complexes that recruit a guide RNA or DNA for target recognition and a protein-RNA assembly for catalytic functioning (e.g. RNAi molecular machinery, RNase H). Recently discovered peptidyl-oligonucleotide synthetic ribonucleases selectively knock down pathogenic RNAs by irreversible cleavage to offer unprecedented opportunities for control of disease-relevant RNA. Understanding how to increase their potency, selectivity and catalytic turnover will open the translational pathway to successful therapeutics. Yet, very little is known about how these chemical ribonucleases bind, cleave and leave their target. Rational design awaits this understanding in order to control therapy, particularly how to overcome the trade-off between sequence specificity and potency through catalytic turnover. We illuminate this here by characterizing the interactions of these chemical RNases with both complementary and non-complementary RNAs using Tm profiles, fluorescence, UV-visible and NMR spectroscopies. Crucially, the level of counter cations, which are tightly-controlled within cellular compartments, also controlled these interactions. The oligonucleotide component dominated interaction between conjugates and complementary targets in the presence of physiological levels of counter cations (K+), sufficient to prevent repulsion between the complementary nucleic acid strands to allow Watson-Crick hydrogen bonding. In contrast, the positively-charged catalytic peptide interacted poorly with target RNA, when counter cations similarly screened the negatively-charged sugar-phosphate RNA backbones. The peptide only became the key player, when counter cations were insufficient for charge screening; moreover, only under such non-physiological conditions did conjugates form strong complexes with non-complementary RNAs.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Oligonucleótidos , ARN , Catálisis , ADN , Oligonucleótidos/genética , ARN/genética , RibonucleasasRESUMEN
Potent knockdown of pathogenic RNA in vivo is an urgent health need unmet by both small-molecule and biologic drugs. 'Smart' supramolecular assembly of catalysts offers precise recognition and potent destruction of targeted RNA, hitherto not found in nature. Peptidyl-oligonucleotide ribonucleases are here chemically engineered to create and attack bulge-loop regions upon hybridization to target RNA. Catalytic peptide was incorporated either via a centrally modified nucleotide (Type 1) or through an abasic sugar residue (Type 2) within the RNA-recognition motif to reveal striking differences in biological performance and strict structural demands of ribonuclease activity. None of the Type 1 conjugates were catalytically active, whereas all Type 2 conjugates cleaved RNA target in a sequence-specific manner, with up to 90% cleavage from 5-nt bulge-loops (BC5-α and BC5L-ß anomers) through multiple cuts, including in folds nearby. Molecular dynamics simulations provided structural explanation of accessibility of the RNA cleavage sites to the peptide with adoption of an 'in-line' attack conformation for catalysis. Hybridization assays and enzymatic probing with RNases illuminated how RNA binding specificity and dissociation after cleavage can be balanced to permit turnover of the catalytic reaction. This is an essential requirement for inactivation of multiple copies of disease-associated RNA and therapeutic efficacy.
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Oligonucleótidos/química , Péptidos/química , ARN/química , Ribonucleasas/química , Dominio Catalítico , Técnicas de Silenciamiento del Gen/métodos , Simulación de Dinámica Molecular , Péptidos/metabolismo , Ribonucleasas/metabolismoRESUMEN
Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.
RESUMEN
DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide building blocks to provide sequence-specific, PCR-free fluorescent detection of unlabeled nucleic acid sequences. We discriminated at picomolar detection limits (<7 pM) "perfect-match" from mismatched sequences, down to a single nucleotide mutation, buried within longer lengths of the target. Detailed characterization by NMR, TEM, mass spectrometry, and rheology provided the structural understanding to design these hybrid peptide-oligonucleotide biomaterials with the desired sequence sensitivity and detection limit. We discuss the generic design, which is based on a highly predictable secondary structure of the oligonucleotide components, as a platform to detect genetic abnormalities and to screen for pathogenic conditions at the level of both DNA (e.g., SNPs) and RNA (messenger, micro, and viral genomic RNA).
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Hidrogeles/química , Ácidos Nucleicos/análisis , Reacción en Cadena de la Polimerasa/métodos , Disparidad de Par Base , Secuencia de Bases , Límite de Detección , Hibridación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismoRESUMEN
MicroRNAs (miRNAs) are active regulators in malignant growth and constitute potential targets for anticancer therapy. Consequently, considerable effort has focused on identifying effective ways to modulate aberrant miRNA expression. Here we introduce and assess a novel type of chemically engineered biomaterial capable of cleaving specific miRNA sequences, i.e. miRNA-specific artificial ribonucleases (hereafter 'miRNase'). The miRNase template presented here consists of the catalytic peptide Acetyl-[(LeuArg)2Gly]2 covalently attached to a miRNA-targeting oligonucleotide, which can be linear or hairpin. The peptide C-terminus is conjugated to an aminohexyl linker located at either the 3'- or 5'-end of the oligonucleotide. The cleavage efficacy, structural aspects of cleavage and biological relevance of a set of these designed miRNases was assayed with respect to highly oncogenic miR-21. Several miRNases demonstrated effective site-selective cleavage of miR-21 exclusively at G-X bonds. One of the most efficient miRNase was shown to specifically inhibit miR-21 in lymphosarcoma cells and lead to a reduction in their proliferative activity. This report provides the first experimental evidence that metallo-independent peptide-oligonucleotide chemical ribonucleases are able to effectively and selectively down-regulate oncogenic miRNA in tumour cells, thus suggesting their potential in development of novel therapeutics aimed at overcoming overexpression of disease-related miRNAs.
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Linfoma no Hodgkin/genética , Linfoma no Hodgkin/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Oligonucleótidos/administración & dosificación , Péptidos/administración & dosificación , Ribonucleasas/administración & dosificación , Animales , Línea Celular Tumoral , Ratones , Oligonucleótidos/química , Péptidos/química , Ribonucleasas/químicaRESUMEN
Traditional therapeutic interventions against abnormal gene expression in disease states at the level of expressed proteins are becoming increasingly difficult due to poor selectivity, off-target effects and associated toxicity. Upstream catalytic targeting of specific RNA sequences offers an alternative platform for drug discovery to achieve more potent and selective treatment through antisense interference with disease-relevant RNAs. We report a novel class of catalytic biomaterials, comprising amphipathic RNA-cleaving peptides placed between two RNA recognition motifs, here demonstrated to target the TΨC loop and 3'- acceptor stem of tRNAPhe. These unique peptidyl-oligonucleotide 'dual' conjugates (DCs) were created by phosphoramidate or thiol-maleimide conjugation chemistry of a TΨC-targeting oligonucleotide to the N-terminus of the amphipathic peptide sequence, followed by amide coupling of a 3'-acceptor stem-targeting oligonucleotide to the free C-terminal carboxylic acid functionality of the same peptide. Hybridization of the DCs bearing two spatially-separated recognition motifs with the target tRNAPhe placed the peptide adjacent to a single-stranded RNA region and promoted cleavage within the 'action radius' of the catalytic peptide. Up to 100% cleavage of the target tRNAPhe was achieved by the best candidate (i.e. DC6) within 4 h, when conformational flexibility was introduced into the linker regions between the peptide and oligonucleotide components. This study provides the strong position for future development of highly selective RNA-targeting agents that can potentially be used for disease-selective treatment at the level of messenger, micro, and genomic viral RNA.
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Marcación de Gen/métodos , Nanoconjugados/química , Nanoconjugados/ultraestructura , Péptidos/química , ARN de Transferencia/química , ARN de Transferencia/genética , Sitios de Unión , Catálisis , Reactivos de Enlaces Cruzados/química , Diseño de Fármacos , Unión Proteica , Conformación Proteica , ARN de Transferencia/ultraestructura , Relación Estructura-ActividadRESUMEN
Artificial ribonucleases (aRNases) are small compounds catalysing RNA cleavage. Recently we demonstrated that aRNases readily inactivate various viruses in vitro. Here, for three series of aRNases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the aRNases display chaotropic-like and membranolytic activities. The levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation (enveloped, non-enveloped, RNA-, DNA-containing). We evaluated the impact of these activities on the efficiency of virus inactivation and found: i) the synergism between membranolytic and chaotropic-like activities is sufficient for the inactivation of enveloped viruses (influenza A, encephalitis, vaccinia viruses) for 1,4-diazabicyclo [2.2.2]octane based aRNases, ii) the inactivation of non-enveloped viruses (encephalomyocarditis, acute bee paralysis viruses) is totally dependent on the synergism of chaotropic-like and ribonuclease activities, iii) ribonuclease activity plays a leading role in the inactivation of RNA viruses by aRNases Dp12F6, Dtr12 and K-D-1, iv) peptide-like aRNases (L2-3, K-2) being effective virus killers have a more specific mode of action. Obtained results clearly demonstrate that aRNases represent a new class of broad-spectrum virus-inactivating agents.
Asunto(s)
Antivirales/farmacología , Ribonucleasas/farmacología , Inactivación de Virus/efectos de los fármacos , Virus/efectos de los fármacos , Animales , Antivirales/química , Línea Celular , Hemólisis/efectos de los fármacos , Humanos , Cinética , Estructura Molecular , Ribonucleasas/química , Virus Vaccinia/efectos de los fármacos , Virus/ultraestructuraRESUMEN
We report here the first experimental evidence of a self-assembling three-dimensional (3D) peptide hydrogel, with recognition motifs immobilized on the surface of fibres capable of sequence-specific oligonucleotide detection. These systems have the potential to be further developed into diagnostic and prognostic tools in human pathophysiology.
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Técnicas Biosensibles , Hidrogel de Polietilenoglicol-Dimetacrilato/síntesis química , Ácidos Nucleicos Inmovilizados/química , Péptidos/síntesis química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Péptidos/química , Propiedades de SuperficieRESUMEN
NRH:quinone oxidoreductase 2 enzyme (NQO2) is a potential therapeutic target in cancer and neurodegenerative diseases, with roles in either chemoprevention or chemotherapy. Here we report the design, synthesis and evaluation of non-symmetrical furan-amidines and their analogues as novel selective NQO2 inhibitors with reduced adverse off-target effects, such as binding to DNA. A pathway for the synthesis of the non-symmetrical furan-amidines was established from the corresponding 1,4-diketones. The synthesized non-symmetrical furan-amidines and their analogues showed potent NQO2 inhibition activity with nano-molar IC50 values. The most active compounds were non-symmetrical furan-amidines with meta- and para-nitro substitution on the aromatic ring, with IC50 values of 15 nM. In contrast to the symmetric furan-amidines, which showed potent intercalation in the minor grooves of DNA, the synthesized non-symmetrical furan-amidines showed no affinity towards DNA, as demonstrated by DNA melting temperature experiments. In addition, Plasmodium parasites, which possess their own quinone oxidoreductase PfNDH2, were inhibited by the non-symmetrical furan-amidines, the most active possessing a para-fluoro substituent (IC50 9.6 nM). The high NQO2 inhibition activity and nanomolar antimalarial effect of some of these analogues suggest the lead compounds are worthy of further development and optimization as potential drugs for novel anti-cancer and antimalarial strategies.
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Amidinas/farmacología , Antimaláricos/farmacología , Antineoplásicos/farmacología , Furanos/farmacología , Malaria/tratamiento farmacológico , Plasmodium/efectos de los fármacos , Amidinas/síntesis química , Amidinas/química , Antimaláricos/síntesis química , Antimaláricos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Furanos/síntesis química , Furanos/química , Humanos , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-ActividadRESUMEN
Described here is a new class of peptidyl-oligonucleotide conjugates (POCs) which show efficient cleavage of a target RNA in a sequence-specific manner. Through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to amphiphilic peptide sequences containing leucine, arginine, and glycine, zero-linker conjugates are created which exhibit targeted phosphodiester cleavage under physiological conditions. tRNA(Phe) from brewer's yeast was used as a model target sequence in order to probe different structural variants of POCs in terms of selective TΨC-arm directed cleavage. Almost quantitative (97-100%) sequence-specific tRNA cleavage is observed for several POCs over a 24 h period with a reaction half-life of less than 1 h. Nontargeted cleavage of tRNA(Phe) or HIV-1 RNA is absent. Structure-activity relationships reveal that removal of the peptide's central glycine residue significantly decreases tRNA cleavage activity; however, this can be entirely restored through replacement of the peptide's C-terminal carboxylic acid group with the carboxamide functionality. Truncation of the catalytic peptide also has a detrimental effect on POC activity. Based on the encouraging results presented, POCs could be further developed with the aim of creating useful tools for molecular biology or novel therapeutics targeting specific messenger, miRNA, and genomic viral RNA sequences.
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Oligonucleótidos/química , Oligonucleótidos/metabolismo , Péptidos/química , Péptidos/metabolismo , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Hongos/química , ARN de Transferencia/química , Saccharomyces cerevisiae/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Docking-based virtual screening is an established component of structure-based drug discovery. Nevertheless, scoring and ranking of computationally docked ligand libraries still suffer from many false positives. Identifying optimal docking parameters for a target protein prior to virtual screening can improve experimental hit rates. Here, we examine protocols for virtual screening against the important but challenging class of drug target, protein tyrosine phosphatases. In this study, common interaction features were identified from analysis of protein-ligand binding geometries of more than 50 complexed phosphatase crystal structures. It was found that two interactions were consistently formed across all phosphatase inhibitors: (1) a polar contact with the conserved arginine residue, and (2) at least one interaction with the P-loop backbone amide. In order to investigate the significance of these features on phosphatase-ligand binding, a series of seeded virtual screening experiments were conducted on three phosphatase enzymes, PTP1B, Cdc25b and IF2. It was observed that when the conserved arginine and P-loop amide interactions were used as pharmacophoric constraints during docking, enrichment of the virtual screen significantly increased in the three studied phosphatases, by up to a factor of two in some cases. Additionally, the use of such pharmacophoric constraints considerably improved the ability of docking to predict the inhibitor's bound pose, decreasing RMSD to the crystallographic geometry by 43% on average. Constrained docking improved enrichment of screens against both open and closed conformations of PTP1B. Incorporation of an ordered water molecule in PTP1B screening was also found to generally improve enrichment. The knowledge-based computational strategies explored here can potentially inform structure-based design of new phosphatase inhibitors using docking-based virtual screening.
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Evaluación Preclínica de Medicamentos , Proteínas Tirosina Fosfatasas/química , Interfaz Usuario-Computador , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Agua/química , Rayos XRESUMEN
Using biomimetic chemical reduction or Clostridium perfringens cell extract containing azoreductase, the dimer-fluorescent probe 2,4-O-bisdansyl-6,7-diazabicyclooct-6-ene, which possesses a conformationally constrained cis-azo bridge, is reduced to the tetra-equatorial 2,4-O-bisdansyl-cyclohexyl-3,5-bisammonium salt which exhibits fluorescence indicative of a dansyl monomer.
RESUMEN
Design of site-selective artificial ribonucleases (aRNases) is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containing multiple imidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.
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This article presents an overview of new emerging approaches for nucleic acid detection via hybridization techniques that can potentially be applied to genomic analysis and SNP identification in clinical diagnostics. Despite the availability of a diverse variety of SNP genotyping technologies on the diagnostic market, none has truly succeeded in dominating its competitors thus far. Having been designed for specific diagnostic purposes or clinical applications, each of the existing bio-assay systems (briefly outlined here) is usually limited to a relatively narrow aspect or format of nucleic acid detection, and thus cannot entirely satisfy all the varieties of commercial requirements and clinical demands. This drives the diagnostic sector to pursue novel, cost-effective approaches to ensure rapid and reliable identification of pathogenic or hereditary human diseases. Hence, the purpose of this review is to highlight some new strategic directions in DNA detection technologies in order to inspire development of novel molecular diagnostic tools and bio-assay systems with superior reliability, reproducibility, robustness, accuracy and sensitivity at lower assay cost. One approach to improving the sensitivity of an assay to confidently discriminate between single point mutations is based on the use of target assembled, split-probe systems, which constitutes the main focus of this review.
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ADN/análisis , Genoma Humano/genética , Hibridación de Ácido Nucleico/métodos , Polimorfismo de Nucleótido Simple , ADN/genética , Sondas de ADN/genética , Genotipo , Humanos , Reproducibilidad de los ResultadosRESUMEN
Molecular dynamics investigations into active site plasticity of Trypanosoma cruzi trans-sialidase, a protein implicated in Chagas disease, suggest that movement of the Trp(312) loop plays an important role in the enzyme's sialic acid transfer mechanism. The observed Trp(312) flexibility equates to a molecular shovel action, which leads to the expulsion of the donor aglycone leaving group from the catalytic site. These computational simulations provide detailed structural insights into sialyl transfer by the trans-sialidase and may aid the design of inhibitors effective against this neglected tropical disease.
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Glicoproteínas/química , Glicoproteínas/metabolismo , Neuraminidasa/química , Neuraminidasa/metabolismo , Trypanosoma cruzi/enzimología , Triptófano/metabolismo , Biocatálisis , Dominio Catalítico , Glicosilación , Simulación de Dinámica Molecular , Ácido N-Acetilneuramínico/metabolismoRESUMEN
myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P(3)], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P(3) is considered to be important to complex Fe(3+) in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P(4)], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe(3+), evident from excimer fluorescence induced by pi-pi stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca(2+) cations exceeding a 1 : 1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe(3+) have been determined. The complexes formed between Fe(3+) and 4,6-bispyrenoyl Ins(1,2,3,5)P(4) display similar stability to those formed with Ins(1,2,3)P(3), indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P(4), which closely resemble those obtained for Ins(1,2,3)P(3). The data presented confirms that Fe(3+) binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif.
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Colorantes Fluorescentes/química , Fosfatos de Inositol/química , Quelantes del Hierro/química , Pirenos/química , Inositol 1,4,5-Trifosfato/química , Relación Estructura-ActividadRESUMEN
Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe(3+) in the unstable penta-axial conformation.
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Colorantes Fluorescentes/síntesis química , Inositol 1,4,5-Trifosfato/química , Hierro/química , Oligoelementos/química , Colorantes Fluorescentes/química , Modelos Moleculares , Conformación Molecular , Difracción de Rayos XRESUMEN
2-O-tert-Butyldimethylsilyl-4,6-bis-O-pyrenoyl-myo-inositol-1,3,5-orthoformate (6) and 2-O-tert-butyldimethylsilyl-4-O-[4-(dimethylamino)benzoyl]-6-O-pyrenoyl-myo-inositol-1,3,5-orthoacetate (10) adopt conformationally restricted unstable chairs with five axial substituents. In the symmetrical diester 6, the two pi-stacked pyrenoyl groups are electron acceptor-donor partners, giving a strong intramolecular excimer emission. In the mixed ester 10, the pyrenoyl group is the electron acceptor and the 4-(dimethylamino)benzoyl ester is the electron donor, giving a strong intramolecular exciplex emission. The conformation of the mixed ester 10 was assessed using 1H NMR spectroscopy (1H-NOESY) and computational studies. which showed the minimum inter-centroid distance between the two aromatic systems to be approximately 3.9 A. Upon addition of acid, the orthoformate/orthoacetate trigger in 6 and 10 was cleaved, which caused a switch of the conformation of the myo-inositol ring to the more stable penta-equatorial chair, leading to separation of the aromatic ester groups and loss of excimer and exciplex fluorescence, respectively. This study provides proof of principle for the development of novel fluorescent molecular probes.