RESUMEN
OBJECTIVE: The development of an efficient, multifunctional drug delivery system overcoming different obstacles generally associated with drug formulations, including the poor accumulation of the active principle in the target site and its sustained release for prolonged time. METHODS: Our study proposes the development of a fluorinated poly(amidoamine) (PAMAM) carrier prodrug combining drug release boosted in alkaline environments with a possible implementation in 19F MRI applications. In particular, we functionalized the terminal primary amines of PAMAM G2 and G4 through an ad hoc designed fluorinated ibuprofen-arginine Michael acceptor to obtain multifunctional ibuprofen-PAMAM-Arg conjugates. RESULTS: These carriers demonstrated pH-dependent and sustained ibuprofen release for more than 5 days. This advantage was observed in both weak alkaline and physiological buffer solutions, allowing to overcome the limits associated to the burst release from similar fluorinated Arg-PAMAM dendrimers with ibuprofen physically encapsulated. CONCLUSION: These findings, coupled to the high biocompatibility of the system, suggest a potential synergistic biomedical application of our conjugates, serving as vehicles for drug delivery and as 19F magnetic resonance imaging contrast agents.
Asunto(s)
Arginina , Dendrímeros , Portadores de Fármacos , Liberación de Fármacos , Ibuprofeno , Profármacos , Ibuprofeno/administración & dosificación , Ibuprofeno/química , Dendrímeros/química , Concentración de Iones de Hidrógeno , Profármacos/química , Profármacos/administración & dosificación , Portadores de Fármacos/química , Arginina/química , Halogenación , Preparaciones de Acción Retardada/química , Sistemas de Liberación de Medicamentos/métodos , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacocinética , Humanos , Imagen por Resonancia Magnética/métodosRESUMEN
Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19F magnetic resonance imaging.
Asunto(s)
Dendrímeros , Transfección , Técnicas de Transferencia de Gen , Terapia GenéticaRESUMEN
A collection of peptidomimetics characterized by having an aspartic acid motif embedded in a rigid hydantoin heterocycle are synthesized through a sequential multicomponent domino process followed by standard regioselective deprotection/coupling reactions based on acid-base liquid/liquid purification protocols. 1H nuclear magnetic resonance experiments, molecular modeling, and X-ray analysis showed that the resulting hydantoin-based loops I (in particular) and II (to a lesser extent) can be considered novel ß-turn inducer motifs being able to project two peptide-like strands in a U-shaped conformation driven by the formation of intermolecular hydrogen bonds.
RESUMEN
The synthesis of a collection of enantiomerically pure, systematically substituted hydantoins as structural privileged universal mimetic scaffolds is presented. It relies on a chemoselective condensation/cyclization domino process between isocyanates of quaternary or unsubstituted α-amino esters and N-alkyl aspartic acid diesters followed by standard hydrolysis/coupling reactions with amines, using liquid-liquid acid/base extraction protocols for the purification of the intermediates. Besides the nature of the α carbon on the isocyanate moiety, either a quaternary carbon or a more flexible methylene group, conformational studies in silico (molecular modeling), in solution (NMR, circular dichroism (CD), Fourier transform infrared (FTIR)), and in solid state (X-ray) showed that the presented hydantoin-based peptidomimetics are able to project their substituents in positions superimposable to the side chains of common protein secondary structures such as α-helix and ß-turn, being the open α-helix conformation slightly favorable according to molecular modeling, while the closed ß-turn conformation preferred in solution and in solid state.
Asunto(s)
Hidantoínas , Peptidomiméticos , Hidantoínas/química , Conformación Molecular , Modelos Moleculares , Ciclización , Dicroismo Circular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Three model hydantoin-based universal peptidomimetics were designed and synthetized. Their preferred amphiphilic ß-turn conformation was assessed using molecular modeling and NMR experiments, and their antibacterial activity was tested against Gram-positive and Gram-negative bacteria strains, which demonstrated that these compounds could be a captivating class of antibiotics to fight emergent drug resistance.
RESUMEN
Cationic lipids (CLs) have gained significant attention among nonviral gene delivery vectors due to their ease of synthesis and functionalization with multivalent moieties. In particular, there is an increasing request for multifunctional CLs having gene delivery capacity and antibacterial activity. Herein, we describe the design and synthesis of a novel class of aminoglycoside (AG)-based multifunctional vectors with high transfection efficiency and noticeable antibacterial properties. Specifically, cationic amphiphiles were built on a triazine scaffold, allowing for an easy derivatization with up to three potentially different substituents, such as neomycin (Neo) that serves as the polar head and one or two lipophilic tails, namely stearyl (ST) and oleyl (OL) alkyl chains and cholesteryl (Chol) tail. With the aim to shed more light on the effect of different types and numbers of lipophilic moieties on the ability of CLs to condense and transfect cells, the performance of Neo-triazine-based derivatives as gene delivery vectors was evaluated and compared. The ability of Neo-triazine-based derivatives to act as antimicrobial agents was evaluated as well. Neo-triazine-based CLs invariably exhibited excellent DNA condensation ability, even at a low charge ratio (CR, +/-). Besides, each derivative showed very good transfection performance at its optimal CR on two different cell lines, along with negligible cytotoxicity. CLs bearing symmetric two-tailed OL proved to be the most effective in transfection. Interestingly, Neo-triazine-based derivatives, used as either free lipids or lipoplexes, exhibited strong antibacterial activity against Gram-negative bacteria, especially in the case of CLs bearing one or two aliphatic chains. Altogether, these results highlight the potential of Neo-triazine-based derivatives as effective multifunctional nonviral gene delivery vectors.
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Antibacterianos/farmacología , Técnicas de Transferencia de Gen , Lípidos/química , Neomicina/química , Triazinas/química , Antibacterianos/química , CationesRESUMEN
We report the first synthesis of the complex amino acid labionin in a fully orthogonally protected and stereopure form. The structure-which incorporates five orthogonal protecting groups and three stereogenic centers-was assembled using two key synthetic steps: (1) a thia-Michael addition for installing the thioether bridge; (2) an electrophilic azidation for creating the central quaternary α-amino acid carbon in a stereochemically pure form. This work is expected to enable the solid phase synthesis of both natural and synthetic analogues labyrinthopeptins.
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Aminoácidos , Técnicas de Síntesis en Fase Sólida , SulfurosRESUMEN
The solid-phase synthesis of Gly-Ψ[CH(CF3)NH]-peptides is presented. In order to achieve this goal, the synthesis of Gly-Ψ[CH(CF3)NH]-dipeptides having the C-terminus unprotected, the N-terminus protected as Fmoc- or Teoc-, and possibly side chain functionalities protected with acid-labile protecting groups has been developed. A selected small library of six peptidomimetics, encompassing analogues of biological relevant peptides, have been obtained in high purity.
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Peptidomiméticos , Técnicas de Síntesis en Fase Sólida , Dipéptidos , PéptidosRESUMEN
Guanidinoglycosides are a class of non-cytotoxic molecular transporters capable of delivering high molecular weight bioactive cargos into cells at low nanomolar concentrations. Efficient bioconjugation with guanidinoglycosides has been previously demonstrated by utilizing a guanidinoneomycin decorated with a reactive but also unstable N-hydroxysuccinimmide ester-containing linker. Herein we report the synthesis, chemistry, and application of a new, stable guanidinoneomycin derivative armed with a highly specific maleimide moiety which allows for thiol-maleimide click chemistry, a highly popular bioconjugation strategy, widening the field of application of these intriguing and useful delivery vehicles.
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MaleimidasRESUMEN
We present the synthesis of polymeric amino- and guanidinoglycosides prepared by tethering neomycin and guanidinoneomycin to PAMAM dendrimers of generations 2 and 4. The ability of these conjugates to promote cellular uptake of high-molecular-weight cargo is discussed, together with their cytotoxicity and mechanisms of entry. We demonstrate that the presence of multiple guanidinoneomycin carriers on the PAMAM surface plays an important role in promoting cellular uptake of the dendrimers, maintaining the heparan sulfate specificity and negligible cytotoxicity typical of monomeric guanidinoglycoside molecular transporters.
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Aminoglicósidos/química , Dendrímeros/química , Portadores de Fármacos/química , Animales , Biotina/química , Células CHO , Carbocianinas/química , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Portadores de Fármacos/farmacología , Portadores de Fármacos/toxicidad , Endocitosis/efectos de los fármacosRESUMEN
In the present study, an attempt was made to achieve separation of enantiomers within a minute in nano-LC and CEC. In order to achieve this goal several parameters were optimized from the viewpoint of the property of chiral analytes, concentration of the chiral selector in the packing material, capillary dimensions, and separation mode. The enantiomers of several of the applied chiral sulfoxides could be resolved with the analysis time <1 min. Some instrumental obstacles hindering further reduction of analysis time are also highlighted.
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Electrocromatografía Capilar/métodos , Cromatografía Liquida/métodos , Nanotecnología/métodos , Modelos Químicos , Estereoisomerismo , Sulfóxidos/análisis , Sulfóxidos/química , Sulfóxidos/aislamiento & purificación , Factores de TiempoRESUMEN
Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.
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Cationes/química , ADN/química , Nanopartículas/química , Compuestos Orgánicos/química , Polímeros/química , Benzotiazoles , Supervivencia Celular , Quitosano/análogos & derivados , Quitosano/química , Diaminas , Células HeLa , Heparina/química , Humanos , Polietileneimina/análogos & derivados , Polietileneimina/química , Quinolinas , Espectrometría de Fluorescencia , TransfecciónRESUMEN
The development of multifunctional vectors for efficient and safe gene delivery is one of the major challenges for scientists working in the gene therapy field. In this context, we have designed a novel type of aminoglycoside-rich dendrimers with a defined structure based on polyamidoamine (PAMAM) in order to develop efficient, nontoxic gene delivery vehicles. Three different conjugates, i.e., PAMAM G4-neamine, -paromomycin, and -neomycin, were synthesized and characterized by nuclear magnetic resonance (NMR) and MALDI analysis. The conjugates were found to self-assemble electrostatically with plasmid DNA, and unlike neamine conjugate, each at its optimum showed increased gene delivery potency compared to PAMAM G4 dendrimer in three different cell lines, along with negligible cytotoxicity. These results all disclosed aminoglycosides as suitable functionalities for tailoring safe and efficient multifunctional gene delivery vectors.
Asunto(s)
Aminoglicósidos/química , Dendrímeros/química , Vectores Genéticos/química , Vectores Genéticos/genética , Nylons/química , Transfección/métodos , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Conformación Molecular , Plásmidos/química , Plásmidos/genética , Células Tumorales CultivadasRESUMEN
Recent years have witnessed a growing interest in the development of new methods for linking sugars to peptides or proteins because natural glycopeptides or neoglycoconjugates with well defined chemical structures are very important tools to study diverse biological phenomena. Herein we report a novel, one-pot, three-component process for the synthesis of peptide-urea conjugates incorporating a hexafluorovaline or an aspartic acid alkyl ester residue under very mild conditions and high yields. The reaction has been exploited for the synthesis of a wide array of structurally diverse peptide-sugar conjugates through a regiospecific four-component, one-pot sequential domino process, by generating the reacting sugar-carbodiimides in situ from readily accessible starting materials.
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Metabolismo de los Hidratos de Carbono , Péptidos/química , Péptidos/metabolismo , Ácido Aspártico/química , Ácidos Carboxílicos/química , Técnicas de Química Sintética , Hidrólisis , Especificidad por Sustrato , Urea/química , Valina/análogos & derivados , Valina/químicaRESUMEN
Diabetic peripheral neuropathy (DPN) is characterized by myelin abnormalities; however, the molecular mechanisms underlying such deficits remain obscure. To uncover the effects of diabetes on myelin alterations, we have analyzed myelin composition. In a streptozotocin-treated rat model of diabetic neuropathy, analysis of sciatic nerve myelin lipids revealed that diabetes alters myelin's phospholipid, FA, and cholesterol content in a pattern that can modify membrane fluidity. Reduced expression of relevant genes in the FA biosynthetic pathway and decreased levels of the transcriptionally active form of the lipogenic factor sterol-regulatory element binding factor-1c (SREBF-1c) were found in diabetic sciatic nerve. Expression of myelin's major protein, myelin protein zero (P0), was also suppressed by diabetes. In addition, we confirmed that diabetes induces sciatic nerve myelin abnormalities, primarily infoldings that have previously been associated with altered membrane fluidity. In a diabetic setting, synthetic activator of the nuclear receptor liver X receptor (LXR) increased SREBF-1c function and restored myelin lipid species and P0 expression levels to normal. These LXR-modulated improvements were associated with restored myelin structure in sciatic nerve and enhanced performance in functional tests such as thermal nociceptive threshold and nerve conduction velocity. These findings demonstrate an important role for the LXR-SREBF-1c axis in protection from diabetes-induced myelin abnormalities.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Vaina de Mielina/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Animales , Colesterol/metabolismo , Diabetes Mellitus Experimental/genética , Regulación de la Expresión Génica , Lípidos/química , Receptores X del Hígado , Masculino , Proteína P0 de la Mielina/metabolismo , Vaina de Mielina/química , Fosfolípidos/metabolismo , Proteínas Quinasas/genética , Ratas , Ratas Sprague-Dawley , Nervio Ciático/patología , Estearoil-CoA Desaturasa/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , EstreptozocinaRESUMEN
The development of new practical and green methods for the synthesis of small heterocycles is an attractive area of research due to the well-known potential of heterocyclic small molecule scaffolds in the drug discovery process. Herein we report a one-pot, three-component sequential procedure for the synthesis of diversely 1,3,5- and 1,3,5,5-substituted hydantoins, in high yields and very mild conditions, using readily accessible starting materials such as azides, iso(thio)cyanates and substituted α-halo-acetic carboxylic acids. This methodology is especially convenient for the synthesis of spiro-hydantoins, which are particularly interesting bioactive compounds in medicinal chemistry.
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Técnicas de Química Sintética/métodos , Descubrimiento de Drogas/métodos , Hidantoínas/síntesis química , Azidas/química , Ácidos Carboxílicos/química , Química Farmacéutica , Hidantoínas/química , Hidrocarburos Halogenados/química , Isocianatos/química , Estructura Molecular , Tiocianatos/químicaRESUMEN
Sulfur as a stereogenic center can be found in synthetic compounds and natural products. The current study evaluated the enantioseparation of 16 chiral (benzylsulfinyl)benzamide compounds by capillary electrophoresis using charged cyclodextrins (CDs) as chiral selectors in 50 mM sodium acetate buffer, pH 5.5. The sulfoxides varied in the type and position of the substituent of the benzyl moiety as well as the position and methylation of the amide group. Typically, randomly substituted CDs separated the majority of the model analytes in contrast to single isomer CDs. In case of random substitution, γ-CD derivatives displayed higher resolution ability toward the set of model compounds followed by ß-CD and α-CD derivatives. Except for a few examples, the (+)-enantiomer of the analytes migrated before the (-)-isomer irrespective of the type of the CD so that the chiral recognition appeared to be also mostly independent on the structure of the sulfoxides. Evaluation of complexation constants and complex mobilities of selected CD-analyte pairs revealed that the separations were based on the stereoselective complexation by the CD expressed as complexation constants but examples for complex mobilities as the determining factor for the enantiomer migration order were also found. In case of 2-(4-bromobenzylsulfinyl)-N-methyl benzamide in the presence of heptakis(2,3-di-O-methyl-6-O-sulfo)-α-CD reversal of the enantiomer migration order as a function of the CD concentration was observed. Using neutral CD derivatives in the presence of sodium dodecyl sulfate-based micelles at pH 9.0 only few sulfoxides could be enantioseparated.
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Ciclodextrinas , Benzamidas , Ciclodextrinas/química , Electroforesis Capilar/métodos , Estereoisomerismo , SulfóxidosRESUMEN
Neuroactive steroids act in the peripheral nervous system as physiological regulators and as protective agents for acquired or inherited peripheral neuropathy. In recent years, modulation of neuroactive steroids levels has been studied as a potential therapeutic approach to protect peripheral nerves from damage induced by diabetes. Nuclear receptors of the liver X receptor (LXR) family regulate adrenal steroidogenesis via their ability to control cholesterol homeostasis. Here we show that rat sciatic nerve expresses both LRXα and ß isoforms and that these receptors are functional. Activation of liver X receptors using a synthetic ligand results in increased levels of neurosteroids and protection of the sciatic nerve from neuropathy induced by diabetes. LXR ligand treatment of streptozotocin-treated rats increases expression in the sciatic nerve of steroidogenic acute regulatory protein (a molecule involved in the transfer of cholesterol into mitochondria), of the enzyme P450scc (responsible for conversion of cholesterol into pregnenolone), of 5α-reductase (an enzyme involved in the generation of neuroactive steroids) and of classical LXR targets involved in cholesterol efflux, such as ABCA1 and ABCG1. These effects were associated with increased levels of neuroactive steroids (e.g., pregnenolone, progesterone, dihydroprogesterone and 3α-diol) in the sciatic nerve, and with neuroprotective effects on thermal nociceptive activity, nerve conduction velocity, and Na(+), K(+)-ATPase activity. These results suggest that LXR activation may represent a new pharmacological avenue to increase local neuroactive steroid levels that exert neuroprotective effects in diabetic neuropathy.
Asunto(s)
Neuropatías Diabéticas/metabolismo , Neuropatías Diabéticas/prevención & control , Receptores Nucleares Huérfanos/metabolismo , Esteroides/metabolismo , Esteroides/uso terapéutico , Análisis de Varianza , Animales , Benzoatos/farmacología , Bencilaminas/farmacología , Peso Corporal/efectos de los fármacos , Cromatografía Liquida/métodos , Diabetes Mellitus Experimental/complicaciones , Neuropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/etiología , Ligandos , Receptores X del Hígado , Masculino , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Conducción Nerviosa/fisiología , Umbral del Dolor , Ratas , Ratas Sprague-Dawley , Nervio Ciático/metabolismo , Nervio Ciático/microbiología , Nervio Ciático/patología , Nervio Ciático/fisiopatología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
The mechanism of the reaction between carbodiimides and activated α,ß-unsaturated carboxylic acids yielding fully substituted hydantoins and variable amounts of N-acyl urea by-products was studied using density functional theory calculations. Two alternative pathways featuring N-acyl ureas and imino-oxazolidinones as intermediates for the formation of the hydantoin product were taken into account. The results obtained using two different computational models indicate that the overall barriers are similar for the two pathways considered. In all cases, inclusion of a second molecule of carboxylic acid was required to mediate tautomerizations and rearrangement steps. The calculations successfully reproduce the experimentally observed regioselectivity with respect to both N-acyl urea and hydantoin products.
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Hidantoínas/síntesis química , Teoría Cuántica , Hidantoínas/química , Estructura Molecular , EstereoisomerismoRESUMEN
The development of new methods for linking sugars to heterocycles and peptides is an attractive area of research because glyco-conjugates play important roles in biology and medicine and are indispensable tools for probing several processes. Herein we report a one-pot, three-component sequential procedure for the synthesis of a novel class of glyco-conjugates, i.e. glyco-hydantoin conjugates, in high yields and very mild conditions, using readily accessible starting materials. We also demonstrated that some of the glyco-hydantoin conjugates obtained are synthons for the preparation of a novel class of glyco-pseudopeptides in which the amino acid is tethered to the sugar through the hydantoin ring.