RESUMEN
Peptoids belong to a class of sequence-controlled polymers comprising of N-alkylglycine. This study focuses on using tandem mass spectrometry techniques to characterize the fragmentation patterns of a set of singly and doubly protonated peptoids consisting of one basic residue placed at different positions. The singly protonated peptoids fragment by producing predominately high-abundant C-terminal ions called Y-ions and low-abundant N-terminal ions called B-ions. Computational studies suggest that the proton affinity (PA) of the C-terminal fragments is generally higher than that of the N-terminal fragments, and the PA of the former increases as the fragments are elongated. The B-ions are likely formed upon dissociating the proton-activated amide bonds via an oxazolone structure, and the Y-ions are produced subsequently by abstracting a proton from the newly formed B-ions, which is energetically favored. The doubly protonated peptoids prefer to fragment closest to either the N- or the C-terminus and produce corresponding B/Y-ion pairs. The basic residue seems to dictate the preferred fragmentation site, which may be the result of minimizing the repulsion between the two charges. Water and terminal neutral losses are a facile process accompanying the peptoid fragmentation in both charge states. The patterns appear to be highly influenced by the location of the basic residue.
Asunto(s)
Peptoides/química , Espectrometría de Masas en Tándem/métodos , Glicinas N-Sustituídas/química , Peptoides/síntesis química , ProtonesRESUMEN
There is a growing interest in new methods to generate bio-inspired, chemically diverse, sequence-defined synthetic polymers. Solid-phase submonomer approaches offer facile access to these types of materials, since they take advantage of readily available synthons. Submonomer approaches to date have been applied to peptidomimetics with oligo-amide backbones. Here we extend the approach to a phosphorous-containing backbone, where N-substituted phosphoramidate oligomers are constructed from a set of amine submonomers, diphenyl H-phosphonate, and cyclohexane diol. The key chemical steps in chain elongation are a chain extension reaction based on H-phosphonate (P III) chemistry, and a side chain attachment step based on the Atherton-Todd reaction. Cheap, stable chemical reagents are used without heating, all reaction times are 30 minutes or less and open to air, and no main-chain protecting groups are required. Phosphoramitoid tetramers and pentamers displaying a variety of side chain functionalities were synthesized by a three-step solid-phase submonomer method, typically with >85% crude purities.
Asunto(s)
Amidas/química , Ácidos Fosfóricos/química , Polímeros/química , Peptoides/síntesis química , Peptoides/química , Preparaciones Farmacéuticas/síntesis química , Preparaciones Farmacéuticas/química , Polímeros/síntesis química , Técnicas de Síntesis en Fase Sólida , EstereoisomerismoRESUMEN
A novel approach to sequentially degrade peptoid N-terminal N-(substituted)glycine residues on the solid-phase using very mild conditions is reported. This method relies on the treatment of resin-bound, bromoacetylated peptoids with silver perchlorate in THF, leading to an intramolecular cyclization reaction to liberate the terminal residue as a N-substituted morpholine-2,5-dione, resulting in a truncated peptoid upon hydrolysis and a silver bromide byproduct. Side-chain functional group tolerance is explored and reaction kinetics are determined. In a series of pentapeptoids possessing variable, non-nucleophilic side-chains at the second position (R(2) ), we demonstrate that sequential N-terminal degradation of the first two residues proceeds in 87% and 74% conversions on average, respectively. We further demonstrate that the degradation reaction is selective for peptoids, and represents substantial progress toward a mild, iterative sequencing method for peptoid oligomers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 726-736, 2016.
Asunto(s)
Peptoides , Proteolisis , Análisis de Secuencia de Proteína/métodos , Peptoides/química , Peptoides/genéticaRESUMEN
The relationship between the structure of sequence-defined peptoid polymers and their ability to assemble into well-defined nanostructures is important to the creation of new bioinspired platforms with sophisticated functionality. Here, the hydrophobic N-(2-phenylethyl)glycine (Npe) monomers of the standard nanosheet-forming peptoid sequence were modified in an effort to (1) produce nanosheets from relatively short peptoids, (2) inhibit the aggregation of peptoids in bulk solution, (3) increase nanosheet stability by promoting packing interactions within the hydrophobic core, and (4) produce nanosheets with a nonaromatic hydrophobic core. Fluorescence and optical microscopy of individual nanosheets reveal that certain modifications to the hydrophobic core were well tolerated, whereas others resulted in instability or aggregation or prevented assembly. Importantly, we demonstrate that substitution at the meta and para positions of the Npe aromatic ring are well tolerated, enabling significant opportunities to tune the functional properties of peptoid nanosheets. We also found that N-aryl glycine monomers inhibit nanosheet formation, whereas branched aliphatic monomers have the ability to form nanosheets. An analysis of the crystal structures of several N,N'-disubstituted diketopiperazines (DKPs), a simple model system, revealed that the preferred solid-state packing arrangement of the hydrophobic groups can directly inform the assembly of stable peptoid nanosheets.
RESUMEN
N-Aryl glycines are a chemically diverse class of peptoid monomers that have strong structure-inducing propensities. Yet their use has been limited due to the sluggish reactivity of the weakly nucleophilic aniline submonomers. Here, we report up to a 76-fold rate acceleration of the displacement reaction using aniline submonomers in solid-phase peptoid synthesis. This is achieved by adding halophilic silver salts to the displacement reaction, facilitating bromide abstraction and AgBr precipitation. Mechanistic insight derived from analysis of a series of 15 substituted anilines reveals that the silver-mediated reaction proceeds through a transition state that has considerably less positive charge buildup on the incoming nucleophile and an enhanced leaving group. This straightforward enhancement to the submonomer method enables the rapid room temperature synthesis of a wide variety of N-aryl glycine-rich peptoid oligomers, possessing both electron-withdrawing and -donating substituents, in good yields.
Asunto(s)
Glicina/análogos & derivados , Glicina/química , Sustancias Macromoleculares/química , Peptoides/síntesis química , Modelos Moleculares , Peptoides/química , Técnicas de Síntesis en Fase Sólida/métodosRESUMEN
The ability to control and manipulate semiconductor/bio interfaces is essential to enable biological nanofabrication pathways and bioelectronic devices. Traditional surface functionalization methods, such as self-assembled monolayers (SAMs), provide limited customization for these interfaces. Polymer brushes offer a wider range of chemistries, but choices that maintain compatibility with both lithographic patterning and biological systems are scarce. Here, we developed a class of bioinspired, sequence-defined polymers, i.e., polypeptoids, as tailored polymer brushes for surface modification of semiconductor substrates. Polypeptoids featuring a terminal hydroxyl (-OH) group are designed and synthesized for efficient melt grafting onto the native oxide layer of Si substrates, forming ultrathin (â¼1 nm) monolayers. By programming monomer chemistry, our polypeptoid brush platform offers versatile surface modification, including adjustments to surface energy, passivation, preferential biomolecule attachment, and specific biomolecule binding. Importantly, the polypeptoid brush monolayers remain compatible with electron-beam lithographic patterning and retain their chemical characteristics even under harsh lithographic conditions. Electron-beam lithography is used over polypeptoid brushes to generate highly precise, binary nanoscale patterns with localized functionality for the selective immobilization (or passivation) of biomacromolecules, such as DNA origami or streptavidin, onto addressable arrays. This surface modification strategy with bioinspired, sequence-defined polypeptoid brushes enables monomer-level control over surface properties with a large parameter space of monomer chemistry and sequence and therefore is a highly versatile platform to precisely engineer semiconductor/bio interfaces for bioelectronics applications.
Asunto(s)
Polímeros , Polímeros/química , Adsorción , Propiedades de SuperficieRESUMEN
INTRODUCTION: Ventilator-associated pneumonia (VAP) occurs in up to 25% of mechanically ventilated patients, with an associated mortality up to 50%. Early diagnosis and appropriate empiric antibiotic coverage of VAP are crucial. Given the multitude of noninfectious clinical and radiographic anomalies within trauma patients, microbiology from bronchioalveolar lavage (BAL) is often needed. Empiric antibiotics are administered while awaiting BAL culture data. Little is known about the effects of these empiric antibiotics on patients with negative BAL microbiology if a subsequent VAP occurs during the same hospital course. METHODS: This is a retrospective chart review of intubated trauma patients undergoing BAL for suspected pneumonia over a 3-y period at a Level 1 trauma center. All patients with suspected VAP undergoing a BAL receive empiric antibiotics. If microbiology data are negative at 72 h, all antibiotics are stopped; however, if the BAL returns with ≥10(5) colony-forming units per milliliter, the diagnosis of VAP is confirmed. We divided patients into three groups. Group 1 consisted of patients in whom the initial BAL was positive for VAP. Group 2 consisted of patients with an initial negative BAL, who subsequently developed VAP at a later point in the hospital course. Group 3 consisted of patients with negative BAL who did not develop a subsequent VAP. RESULTS: We obtained 499 BAL specimens in 185 patients over the 3-y period. A total of 14 patients with 23 BAL specimens initially negative for VAP subsequently developed VAP later during the same hospital stay. These patients did not have an increase in the hospital length of stay, intensive care unit days, ventilator days, or mortality compared with those who had a positive culture on the first suspicion of VAP. There was a significant increase in the percentage of Enterobacter (21% versus 8%) and Morganella (8% versus 0%) as the causative organism in these 14 patients when the VAP occurred. Furthermore, the profile of the top two organisms in each group changed. Enterobacter (21%) and Pseudomonas (17%) were the principal organisms in the initial BAL-negative group, whereas the two predominant strains in the initial positive BAL group were methicillin-sensitive Staphylococcus aureus (21%) and Haemophilus influenza (11%). Interestingly, methicillin-resistant S. aureus remained the third most common organism in both groups. Empiric antibiotics also did not seem to induce the growth of multidrug-resistant organisms, and there was no increased rate of secondary infections such as Clostridium difficile. CONCLUSIONS: Ventilator-associated pneumonia remains a significant cause of morbidity and mortality in mechanically ventilated trauma patients. The diagnosis and treatment of VAP continue to be challenging. Once clinically suspected, empiric coverage decreases morbidity and mortality. Our data demonstrate that patients who receive empiric coverage exhibit a significantly different microbiologic profile compared with those who had an initial positive BAL culture. Initial empiric antibiotics in BAL-negative patients were not associated with an increase in multidrug-resistant organisms, hospital, or intensive care unit length of stay, ventilator days, and mortality or secondary infections.
Asunto(s)
Antibacterianos/uso terapéutico , Líquido del Lavado Bronquioalveolar/microbiología , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Bacterias Grampositivas/efectos de los fármacos , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Neumonía Asociada al Ventilador/tratamiento farmacológico , Acetamidas/farmacología , Acetamidas/uso terapéutico , Antibacterianos/farmacología , Lavado Broncoalveolar , Cefepima , Cefalosporinas/farmacología , Cefalosporinas/uso terapéutico , Farmacorresistencia Bacteriana/efectos de los fármacos , Quimioterapia Combinada , Femenino , Infecciones por Bacterias Gramnegativas/diagnóstico , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/mortalidad , Infecciones por Bacterias Grampositivas/diagnóstico , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/mortalidad , Humanos , Linezolid , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Oxazolidinonas/farmacología , Oxazolidinonas/uso terapéutico , Ácido Penicilánico/análogos & derivados , Ácido Penicilánico/farmacología , Ácido Penicilánico/uso terapéutico , Piperacilina/farmacología , Piperacilina/uso terapéutico , Neumonía Asociada al Ventilador/diagnóstico , Neumonía Asociada al Ventilador/microbiología , Neumonía Asociada al Ventilador/mortalidad , Estudios Retrospectivos , Tazobactam , Resultado del Tratamiento , Vancomicina/farmacología , Vancomicina/uso terapéutico , Heridas y Lesiones/terapiaRESUMEN
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Humanos , Liposomas , Nanopartículas , SARS-CoV-2/genéticaRESUMEN
Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.
Asunto(s)
Amiloide/química , Pliegue de Proteína , Deficiencias en la Proteostasis/diagnóstico , Amiloide/metabolismo , Diagnóstico Precoz , Humanos , Indicadores y Reactivos/química , Indicadores y Reactivos/metabolismo , Peso Molecular , Peptoides/química , Peptoides/metabolismo , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
The design and synthesis of protein-like polymers is a fundamental challenge in materials science. A biomimetic approach is to explore the impact of monomer sequence on non-natural polymer structure and function. We present the aqueous self-assembly of two peptoid polymers into extremely thin two-dimensional (2D) crystalline sheets directed by periodic amphiphilicity, electrostatic recognition and aromatic interactions. Peptoids are sequence-specific, oligo-N-substituted glycine polymers designed to mimic the structure and functionality of proteins. Mixing a 1:1 ratio of two oppositely charged peptoid 36mers of a specific sequence in aqueous solution results in the formation of giant, free-floating sheets with only 2.7 nm thickness. Direct visualization of aligned individual peptoid chains in the sheet structure was achieved using aberration-corrected transmission electron microscopy. Specific binding of a protein to ligand-functionalized sheets was also demonstrated. The synthetic flexibility and biocompatibility of peptoids provide a flexible and robust platform for integrating functionality into defined 2D nanostructures.
Asunto(s)
Biomimética , Glicina/análogos & derivados , Peptoides/química , Polímeros/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Tampones (Química) , Cristalización , Análisis de Fourier , Ligandos , Modelos Químicos , Nanoestructuras/química , Nanoestructuras/ultraestructura , Polímeros/síntesis química , Unión Proteica , Homología de Secuencia de Aminoácido , Electricidad Estática , Agua/químicaRESUMEN
BACKGROUND: Ethnic minorities and low income families tend to be in poorer health and have worse outcomes for a spectrum of diseases. Health care provider bias has been reported to potentially affect the distribution of care away from poorer communities, minorities, and patients with a history of substance abuse. Trauma is perceived as a disease of the poor and medically underserved. Minorities are overrepresented in low income populations and are also less likely to possess health insurance leading to a potential overlapping effect. Traumatic brain injury (TBI) is a predominant cause of mortality and long-term morbidity, which imposes a considerable social and financial burden. We therefore sought to determine the independent effect on outcome after TBI from race, insurance status, intoxication on presentation, and median income. METHODS: A 5-year retrospective chart review of admitted trauma patients aged 18 years and older to a Level I trauma center. Zip code of residency was a surrogate marker for socioeconomic status, because median income for each zip code is available from the US Census. Charts review included race, insurance status, mechanisms of trauma, and injuries sustained. Outcomes were placement of tracheostomy, hospital length of stay (HLOS), leaving Against Medical Advice (AMA), and discharge to home versus rehabilitation and mortality. RESULTS: A total of 3,101 TBI patients were included in the analyses. Multivariable logistic and proportional hazard regression analyses were undertaken adjusting for age, gender, Injury Severity Score, and mechanism. Rates of tracheostomy placement were unaffected by race, median income, or insurance status. Race and median income did not affect HLOS, but private insurance was associated with shorter HLOS and intoxication was associated with longer HLOS. Neither race nor intoxication affected rates of AMA, but higher income and private insurance was associated with lower rates of AMA. Non-Caucasian race and lack of insurance had significantly lower likelihood of placement in a rehabilitation center. Mortality was unaffected by race, increased in intoxicated patients, was variably affected by median income, and was lowest in patients with private insurance. CONCLUSIONS: An extremely complex interplay exists between socioethnic factors and outcomes after TBI. Few physicians would claim overt discrimination. Tracheostomy, the factor most directed by the surgeon, was unbiased by race, income, or insurance status. The likelihood of placement in a rehabilitation center was significantly impacted by both race and insurance status. Future prospective studies are needed to better address causation.
Asunto(s)
Lesiones Encefálicas/etnología , Lesiones Encefálicas/terapia , Etnicidad/estadística & datos numéricos , Clase Social , Adolescente , Adulto , Lesiones Encefálicas/mortalidad , Distribución de Chi-Cuadrado , Femenino , Humanos , Renta , Puntaje de Gravedad del Traumatismo , Seguro de Salud/estadística & datos numéricos , Tiempo de Internación/estadística & datos numéricos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Alta del Paciente/estadística & datos numéricos , Modelos de Riesgos Proporcionales , Sistema de Registros , Centros de Rehabilitación/estadística & datos numéricos , Estudios Retrospectivos , Factores de Riesgo , Factores Socioeconómicos , Traqueostomía/estadística & datos numéricos , Resultado del Tratamiento , Negativa del Paciente al Tratamiento/estadística & datos numéricosRESUMEN
BACKGROUND: In October 2008, Medicare and Medicaid stopped paying for care associated with catheter-related urinary tract infections (UTIs). Although most clinicians agree UTIs are detrimental, there are little data to support this belief. METHODS: This is a retrospective review of trauma registry data from a Level I trauma center between 2003 and 2008. Two proportional hazards regressions were used for analyses. The first predicted acquisition of UTI as a function of indwelling urinary catheter use, adjusting for age, diabetes, gender, and injury severity. The second predicted hospital mortality as a function of UTI, covarying for age, gender, chronic obstructive pulmonary disease, congestive heart failure, hypertension, diabetes, pneumonia, and injury severity. RESULTS: After excluding patients who stayed in the hospital <3 days and those with a UTI on arrival, 5,736 patients were included in the study. Of these patients, 680 (11.9%) met criteria for a UTI, with 487 (71.6%) indwelling urinary catheter-related infections. Predictors of UTI included the interaction between age and gender (p = 0.0018), Injury Severity Score (p = 0.0021), and indwelling urinary catheter use (p < 0.001). The development of a UTI predicted the risk of in-hospital death as a patient's age increased (p = 0.002). Similar results were seen when only catheter-associated UTIs are included in the analysis. CONCLUSIONS: Indwelling urinary catheter use is connected to the development of UTIs, and these infections are associated with a greater mortality as the age of a trauma patients increases.
Asunto(s)
Infecciones Relacionadas con Catéteres/epidemiología , Mortalidad Hospitalaria/tendencias , Cateterismo Urinario/efectos adversos , Infecciones Urinarias/epidemiología , Heridas y Lesiones/epidemiología , Adulto , Infecciones Relacionadas con Catéteres/diagnóstico , Infecciones Relacionadas con Catéteres/terapia , Causas de Muerte , Comorbilidad , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/diagnóstico , Traumatismo Múltiple/epidemiología , Traumatismo Múltiple/terapia , Sistema de Registros , Estudios Retrospectivos , Medición de Riesgo , Análisis de Supervivencia , Centros Traumatológicos , Resultado del Tratamiento , Infecciones Urinarias/diagnóstico , Infecciones Urinarias/terapia , Heridas y Lesiones/diagnóstico , Heridas y Lesiones/terapiaRESUMEN
Peptoids are a diverse family of sequence-defined oligomers of N-substituted glycine monomers, that can be readily accessed by the solid-phase submonomer synthesis method. Due to the versatility and efficiency of this chemistry, and the easy access to hundreds of potential monomers, there is an enormous potential sequence space that can be explored. This has enabled researchers from many different fields to custom-design peptoid sequences tailored to a wide variety of problems in biomedicine, nanoscience and polymer science. Here we provide detailed protocols for the synthesis of peptoids, using optimized protocols that can be performed by non-chemists. The submonomer method is fully compatible with Fmoc-peptide synthesis conditions, so the method is readily automated on existing automated peptide synthesizers using protocols provided here. Although the submonomer synthesis for peptoids is well established, there are special considerations required in order to access many of the most useful and desirable sidechains. Here we provide methods to include most of the amino-acid-like side chains, some of the most important non-natural monomer classes, as well as the creation of peptoid conjugates and peptide-peptoid hybrids.
Asunto(s)
Peptoides , Glicina , Sustancias Macromoleculares , Péptidos , Técnicas de Síntesis en Fase SólidaRESUMEN
BACKGROUND: Normal vital signs are typically associated with improved outcomes in trauma patients. Whether this association is true for geriatric patients is unclear. METHODS: A Level 1 trauma center retrospective chart review of vital signs on presentation (heart rate [HR] and blood pressure) in young (aged 17-35 years) and geriatric (aged 65 years or older) blunt trauma victims from September 2003 to September 2008 was preformed. Generalized nonlinear using piecewise regression for the linear portion of standard logistic models was used to model risk of mortality as a function of HR and blood pressure. Independent models were selected for elderly and young trauma patients based on blood pressure and HR. Models of the same complexity were then fit within each gender and age. RESULTS: There were 2,194 geriatric and 2,081 young patients. Two hundred fifty-one (11.4%) geriatric and 49 (2.4%) young patients died. At all points of "normality," the mortality of the geriatric patients was higher than the young group. Mortality increases considerably in the elderly patients for HRs >90 beats per minute (bpm), an association not seen until HR of 130 bpm in the young group. Mortality significantly increases with systolic blood pressure (SBP) <110 mm Hg in the geriatric patients but not until a SBP of 95 mm Hg in the young patients. HR and mortality association was most variable in the male geriatric patients. CONCLUSIONS: Vital signs on presentation are less predictive of mortality in geriatric blunt trauma victims. Geriatric blunt trauma patients warrant increased vigilance despite normal vital signs on presentation. New trauma triage set points of HR >90 or SBP <110 mm Hg should be considered in the geriatric blunt trauma patients.
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Signos Vitales , Heridas no Penetrantes/diagnóstico , Adolescente , Adulto , Factores de Edad , Anciano , Presión Sanguínea/fisiología , Femenino , Frecuencia Cardíaca/fisiología , Mortalidad Hospitalaria , Humanos , Modelos Logísticos , Masculino , Dinámicas no Lineales , Pronóstico , Estudios Retrospectivos , Riesgo , Factores Sexuales , Análisis de Supervivencia , Centros Traumatológicos , Heridas no Penetrantes/mortalidad , Adulto JovenRESUMEN
The ability of antibodies to bind a wide variety of analytes with high specificity and high affinity makes them ideal candidates for therapeutic and diagnostic applications. However, the poor stability and high production cost of antibodies have prompted exploration of a variety of synthetic materials capable of specific molecular recognition. Unfortunately, it remains a fundamental challenge to create a chemically diverse population of protein-like, folded synthetic nanostructures with defined molecular conformations in water. Here we report the synthesis and screening of combinatorial libraries of sequence-defined peptoid polymers engineered to fold into ordered, supramolecular nanosheets displaying a high spatial density of diverse, conformationally constrained peptoid loops on their surface. These polyvalent, loop-functionalized nanosheets were screened using a homogeneous Förster resonance energy transfer (FRET) assay for binding to a variety of protein targets. Peptoid sequences were identified that bound to the heptameric protein, anthrax protective antigen, with high avidity and selectivity. These nanosheets were shown to be resistant to proteolytic degradation, and the binding was shown to be dependent on the loop display density. This work demonstrates that key aspects of antibody structure and function-the creation of multivalent, combinatorial chemical diversity within a well-defined folded structure-can be realized with completely synthetic materials. This approach enables the rapid discovery of biomimetic affinity reagents that combine the durability of synthetic materials with the specificity of biomolecular materials.
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Anticuerpos/química , Técnicas Químicas Combinatorias , Descubrimiento de Drogas , Nanoestructuras/química , Peptoides/química , Transferencia Resonante de Energía de Fluorescencia , Estructura Molecular , Tamaño de la Partícula , Peptoides/síntesis química , Ingeniería de Proteínas , Propiedades de SuperficieRESUMEN
A method for the rapid sequence determination of peptoids [oligo(N-substituted glycines)] and peptide-peptoid hybrids selected from one-bead-one-compound combinatorial libraries has been developed. In this method, beads carrying unique peptoid (or peptide-peptoid) sequences were subjected to multiple cycles of partial Edman degradation (PED) by treatment with a 1:3 (mol/mol) mixture of phenyl isothiocyanate (PITC) and 9-fluorenylmethyl chloroformate (Fmoc-Cl) to generate a series of N-terminal truncation products for each resin-bound peptoid. After PED, the Fmoc group was removed from the N-terminus and any reacted side chains via piperidine treatment. The resulting mixture of the full-length peptoid and its truncation products was analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, to reveal the sequence of the full-length peptoid. With a slight modification, the method was also effective in the sequence determination of peptide-peptoid hybrids. This rapid, high-throughput, sensitive, and inexpensive sequencing method should greatly expand the utility of combinatorial peptoid libraries in biomedical and materials research.
Asunto(s)
Péptidos/química , Peptoides/química , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Biblioteca de Péptidos , Análisis de Secuencia de Proteína/economía , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/economíaRESUMEN
The anti-granulocyte receptor-1 (Gr-1) mAb, RB6-8C5, has been used extensively to deplete neutrophils in mice and to investigate the role of these cells in host defense. RB6-8C5 binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes. It is thus likely that in vivo administration of RB6-8C5 may deplete not only neutrophils but also other Gr-l+ (Ly6C+) cells. This study describes the use of an Ly6G-specific mAb, 1A8, as an alternative means to deplete neutrophils. In vivo administration of RB6-8C5 reduced blood neutrophils and Gr-1+ monocytes, whereas administration of 1A8 reduced blood neutrophils but not Gr-1+ monocytes. Plasma TNF-alpha in endotoxemia was increased 20-fold by RB6-8C5 pretreatment and fourfold by 1A8 pretreatment. In a wound model, pretreatment with either antibody decreased wound neutrophils and macrophages. TNF-alpha staining in brefeldin-treated wound leukocytes was increased by pretreatment with RB6-8C5, but not 1A8. Neutrophil depletion with 1A8 offers advantages over the use of RB6-8C5, as it preserves non-neutrophil Gr-1+ cells depleted by the anti-Gr-1 antibody. The loss of non-neutrophil Gr-1+ populations in RB6-8C5-treated animals is associated with increased TNF-alpha responses, suggesting these cells may function to suppress TNF-alpha production.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos Ly/inmunología , Neutrófilos/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Modelos Animales de Enfermedad , Endotoxemia/inmunología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/inmunología , Neutrófilos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunología , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/inmunologíaRESUMEN
Prions cause transmissible infectious diseases in humans and animals and have been found to be transmissible by blood transfusion even in the presymptomatic stage. However, the concentration of prions in body fluids such as blood and urine is extremely low; therefore, direct diagnostic tests on such specimens often yield false-negative results. Quantitative preanalytical prion enrichment may significantly improve the sensitivity of prion assays by concentrating trace amounts of prions from large volumes of body fluids. Here, we show that beads conjugated to positively charged peptoids not only captured PrP aggregates from plasma of prion-infected hamsters, but also adsorbed prion infectivity in both the symptomatic and preclinical stages of the disease. Bead absorbed prion infectivity efficiently transmitted disease to transgenic indicator mice. We found that the readout of the peptoid-based misfolded protein assay (MPA) correlates closely with prion infectivity in vivo, thereby validating the MPA as a simple, quantitative, and sensitive surrogate indicator of the presence of prions. The reliable and sensitive detection of prions in plasma will enable a wide variety of applications in basic prion research and diagnostics.
Asunto(s)
Bioensayo/métodos , Microesferas , Peptoides , Enfermedades por Prión/sangre , Enfermedades por Prión/diagnóstico , Priones/sangre , Animales , Biomarcadores , Corteza Cerebral/metabolismo , Cricetinae , Modelos Animales de Enfermedad , Ratones , Peptoides/química , Enfermedades por Prión/mortalidad , Sensibilidad y EspecificidadRESUMEN
Mass spectrometric techniques and more particularly collision-induced dissociation (CID) experiments represent a powerful method for the determination of the primary sequence of (bio)molecules. However, the knowledge of the ion fragmentation patterns say the dissociation reaction mechanisms is a prerequisite to reconstitute the sequence based on fragment ions. Previous papers proposed that protonated peptoids dissociate following an oxazolone-ring mechanism starting from the O-protonation species and leading to high mass Y sequence ions. Here we revisit this backbone cleavage mechanism by performing CID and ion mobility experiments, together with computational chemistry, on tailor-made peptoids. We demonstrated that the B/Y cleavages of collisionally activated O-protonated peptoids must involve the amide nitrogen protonated structures as the dissociating species, mimicking the CID behavior of protonated peptides. Upon the nucleophilic attack of the oxygen atom of the N-terminal adjacent carbonyl group on the carbonyl carbon atom of the protonated amide, the peptoid ions directly dissociate to form an ion-neutral complex associating an oxazolone ion to the neutral truncated peptoid residue. Dissociation of the ion/neutral complex predominantly produces Y ions due to the high proton affinity of the secondary amide function characteristic of truncated peptoids. Whereas the production of Yx ions from acetylated peptoids also involves the B/Y pathway, the observation of abundant Yx ions from non-acetylated peptoid ions is shown in the present study to arise from an A1-Yx mechanism. The consecutive and competitive characters of the A1-Yx and the B/Y mechanisms are also investigated by drift time-aligned CID experiments.
Asunto(s)
Peptoides/química , Protones , Espectrometría de Masa por Ionización de Electrospray/métodos , Iones/químicaRESUMEN
Peptoid nanosheets are supramolecular protein-mimetic materials that form from amphiphilic polypeptoids with aromatic and ionic side chains. Nanosheets have been studied at the nanometer scale, but the molecular structure has been difficult to probe. We report the use of 13C-13C dipolar recoupling solid-state NMR measurements to reveal the configuration of backbone amide bonds selected by 13C isotopic labeling of adjacent α-carbons. Measurements on the same molecules in the amorphous state and in nanosheets revealed that amide bonds in the center of the amino block of peptoid (NaeNpe)7-(NceNpe)7 (B28) favor the trans configuration in the amorphous state and the cis configuration in the nanosheet. This unexpected result contrasts with previous NMR and theoretical studies of short solvated peptoids. Furthermore, examination of the amide bond at the junction of the two charged blocks within B28 revealed a mixture of both cis and trans configurational states, consistent with the previously predicted brickwork-like intermolecular organization.