RESUMO
Small angle neutron scattering is a powerful complementary technique in structural biology. It generally requires, or benefits from, deuteration to achieve its unique potentials. Molecular deuteration has become a mature expertise, with deuteration facilities located worldwide to support access to the technique for a wide breadth of structural biology and life sciences. The sorts of problems well answered by small angle scattering and deuteration involve large (>10Å) scale flexible movements, and this approach is best used where high-resolution methods (crystallography, NMR, cryo-EM) leave questions unanswered. This chapter introduces deuteration, reviewing biological deuteration of proteins, lipids and sterols, and then steps through the ever-expanding range of deuterated molecules being produced by chemical synthesis and enabling sophisticated experiments using physiologically relevant lipids. Case studies of recent successful use of deuteration may provide illustrative examples for strategies for future experiments. We discuss issues of nomenclature for synthesised molecules of novel labeling and make recommendations for their naming. We reflect on our experiences, with cost associated with achieving an arbitrary deuteration level, and on the benefits of experimental co-design by user scientist, deuteration scientist, and neutron scattering scientist working together. Although methods for biological and chemical deuteration are published in the public domain, we recommend that the best method to deuterate is to engage with a deuteration facility.
Assuntos
Biologia Molecular , Nêutrons , Espalhamento a Baixo Ângulo , Cristalografia , LipídeosRESUMO
The deuterium kinetic isotope effect has been known for a period of 40 years, but it is only relatively recently that new drug entities (NDEs) incorporating deuterium demonstrating beneficial pharmacokinetics, pharmacodynamics, and toxicology have arrived to market. Determination of the precise location to deuterate and subsequently any evaluation for a kinetic isotope effect (KIE) is challenging. Typically, such an evaluation would be performed in an in vitro metabolic assay (e.g. liver microsomes) in separate reaction media for both the deuterated and non-deuterated analogues. Here, we have devised an approach whereby we incubate a 1:1 ratio of both the deuterated and protio-form of an imaging agent together in the same liver microsomal assay and determine the relative rate of consumption of both moieties, based upon specific MS-MS transitions unique to both molecules without the need for liquid chromatography-mass spectrometry (LC-MS) separation and quantification. Any deviation of the ratio of the MS transitions from the initial starting point indicated an observable KIE. A site specific deuteration of PBR111, a neuroinflammation imaging agent, was chosen for a proof-of-concept study. Based upon prior mechanistic knowledge of PBR111, two locations were selected for deuteration; an active and inactive site, to corroborate that there was no significant KIE for the inactive site and confirm the efficacy of the developed methodology.
Assuntos
Deutério/química , Corantes Fluorescentes/química , Microssomos Hepáticos/química , Cromatografia Líquida , Humanos , Cinética , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Espectrometria de Massas em TandemRESUMO
Phytantriol is an interfacially-active lipid that is chemically robust, non-digestible and forms particles with internal bicontinuous cubic phase structures (cubosomes) when dispersed with non-ionic surfactants at ambient and physiological temperatures. The liquid crystalline internal structure of phytantriol-based cubosomes can be changed to alter the interfacial contact area/topology with the aqueous dispersant to trigger bioactive payload release or to alter the local membrane curvature around bound or embedded proteins. To enable the study of payload distribution, structure and transformation kinetics within phytantriol particles by neutron scattering techniques it is desirable to have access to a deuterated version of this molecule but to date a synthetic route has not been available. The first successful synthesis of phytantriol-d39 is presented here alongside a preliminary physical characterisation of related particle structures when phytantriol-d39 is dispersed using two non-ionic surfactants, Tween® 80 and Pluronic® F127. Synchrotron small angle X-ray scattering (SAXS) was used to confirm that phytantriol-d39-based nanoparticles in D2O form similar liquid crystalline structures to those of their natural isotopic abundance (phytantriol/H2O) counterparts as a function of temperature. Finally, small angle neutron scattering (SANS) with solvent contrast to match out the phytantriol-d39 structuring was used to show that the spatial correlations between the Tween® and Pluronic® non-ionic surfactant molecules are different within dispersed phytantriol-d39 particles with different liquid crystalline structures in D2O. The surfactant molecules in phytantriol-d39/Tween® 80 particles with Im3m cubic structures were found to follow a self-avoiding walk, whereas in phytantriol-d39/Pluronic® F127 particles with Pn3m cubic structures they were found to follow a more rod-like packing arrangement.
RESUMO
Non-typeable Haemophilus influenzae (NTHi) is the most common pathogen in primary ciliary dyskinesia (PCD) patients. We hypothesised that abnormal ciliary motility and low airway nitric oxide (NO) levels on airway epithelial cells from PCD patients might be permissive for NTHi colonisation and biofilm development.We used a primary epithelial cell co-culture model to investigate NTHi infection. Primary airway epithelial cells from PCD and non-PCD patients were differentiated to ciliation using an air-liquid interface culture and then co-cultured with NTHi.NTHi adherence was greater on PCD epithelial cells compared to non-PCD cells (p<0.05) and the distribution of NTHi on PCD epithelium showed more aggregated NTHi in biofilms (p<0.001). Apart from defective ciliary motility, PCD cells did not significantly differ from non-PCD epithelial cells in the degree of ciliation and epithelial integrity or in cytokine, LL-37 and NO production. Treatment of PCD epithelia using exogenous NO and antibiotic significantly reduced NTHi viability in biofilms compared with antibiotic treatment alone.Impaired ciliary function was the primary defect in PCD airway epithelium underlying susceptibility to NTHi biofilm development compared with non-PCD epithelium. Although NO responses were similar, use of targeted NO with antibiotics enhanced killing of NTHi in biofilms, suggesting a novel therapeutic approach.
Assuntos
Células Epiteliais/microbiologia , Infecções por Haemophilus/fisiopatologia , Síndrome de Kartagener/microbiologia , Óxido Nítrico/farmacologia , Adolescente , Adulto , Antibacterianos/farmacologia , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Estudos de Casos e Controles , Criança , Pré-Escolar , Citocinas/metabolismo , Feminino , Haemophilus influenzae/patogenicidade , Haemophilus influenzae/fisiologia , Humanos , Síndrome de Kartagener/fisiopatologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Adulto JovemRESUMO
Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above-zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic α-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into α-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3°C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein α-helix.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Água/metabolismo , Fosfatidilcolinas/metabolismo , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Propriedades de SuperfícieRESUMO
Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3'-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with ß-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following ß-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (ß-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks ß-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of ß-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the ß-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Compostos Azo/farmacologia , Biofilmes/efeitos dos fármacos , Cefalosporinas/farmacologia , Doadores de Óxido Nítrico/farmacologia , Pró-Fármacos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Amoxicilina/farmacologia , Anti-Inflamatórios não Esteroides/química , Azitromicina/farmacologia , Compostos Azo/química , Cefalosporinas/química , Óxido Nítrico/análise , Doadores de Óxido Nítrico/química , Penicilinase/química , Plâncton/microbiologia , Pró-Fármacos/químicaRESUMO
PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking ß-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.
Assuntos
Compostos Azo/farmacologia , Biofilmes/efeitos dos fármacos , Cefalosporinas/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Pró-Fármacos/farmacologia , Antibacterianos/farmacologia , Azitromicina/farmacologia , Cromatografia Líquida , Farmacorresistência Bacteriana , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Óxidos de Nitrogênio/metabolismo , Proteômica , beta-Lactamases/metabolismoRESUMO
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), an unsaturated acyl chain containing lipid, is often the predominant lipid in eukaryotic cell membranes in which it is crucial for the fluidity of membranes under physiological conditions. Commercially available, partially deuterated [D31 ]1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D31 ]POPC) does not provide sufficient isotopic contrast for detailed structural studies of multicomponent membranes through neutron techniques. Herein, a relatively straightforward and generic chemical deuteration method is discussed for the asymmetric synthesis of perdeuterated [D31 ]1-palmitoyl-[D33 ]2-oleoyl-sn-[D5 ]glycero-[D13 ]3-phosphocholine ([D82 ]POPC) that also allows selective deuteration of any of its constituent groups. Neutron reflectivity of a [D82 ]POPC-supported bilayer was used to experimentally determine the neutron scattering length density profile of the lipid. The acyl chains of [D82 ]POPC are closely contrast-matched to heavy water, whereas the very high scattering length density of the deuterated glycerophosphocholine head groups provides good contrast to membrane-binding agents in both deuterated and non-deuterated solvent environments.
RESUMO
Broadband phase-sensitive vibrational sum frequency generation (SFG) spectroscopy was utilized to study the molecular orientation of molecules adsorbed on dielectric solid substrates. A gold thin film was employed to generate a SFG signal as a local oscillator (LO). To simplify the phase measurement, a self-assembled monolayer (SAM) of octadecyltrichlorosilane (OTS) was used as a standard sample for phase correction of the phase-sensitive SFG measurements on the solid/air interface. It was demonstrated that the absolute orientation of molecules in the LB films on a fused quartz surface can be clearly distinguished by phase-sensitive SFG measurement. In addition, the observation on the SAM of d35-OTS reveals that the two C-H stretching modes for α-CH2 group are in opposite phase. Furthermore, by using the present phase-sensitive SFG setup, the orientation flipping of water molecules on positively and negatively charged solid/liquid interface can be distinguished.
Assuntos
Silanos/química , Ar , Eletrólitos/química , Ouro/química , Espectrofotometria Infravermelho , Propriedades de Superfície , Água/químicaRESUMO
Deuterated arylamines demonstrate great potential for use in optoelectronic devices, but their widespread utility requires a method for large-scale synthesis. The incorporation of these deuterated materials into optoelectronic devices also provides the opportunity for studies of the functioning device using neutron reflectometry based on the difference in the scattering length density between protonated and deuterated compounds. Here we report mild deuteration conditions utilising standard laboratory glassware for the deuteration of: diphenylamine, N-phenylnaphthylamine, N-phenyl-o-phenylenediamine and 1-naphthylamine (via H/D exchange in D2O at 80 °C, catalysed by Pt/C and Pd/C). These conditions were not successful in the deuteration of triphenylamine or N,N-dimethylaniline, suggesting that these mild conditions are not suitable for the deuteration of tertiary arylamines, but are likely to be applicable for the deuteration of other primary and secondary arylamines. The deuterated arylamines can then be used for synthesis of larger organic molecules or polymers with optoelectronic applications.
Assuntos
Deutério/química , Difenilamina/síntese química , Fenilenodiaminas/síntese química , Aminas , Difenilamina/química , Fenilenodiaminas/químicaRESUMO
We describe a straightforward method, for synthesis of large scale (gram quantities) of highly deuterated phytanic acid from commercially available phytol while preserving the stereochemistry around the chiral centres. The subsequent synthesis of tail-deuterated analogues of the archeabacterial membrane lipids 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC) from perdeuterated phytanic acid is also described. Both lipids were employed in construction of two different model membranes, namely Langmuir monolayers and a tethered bilayer membrane (TBM) on a solid substrate, characterised by pressure area isotherm and neutron reflectometry techniques. At 10 mN/m pressure the head-group thickness of both monolayers was similar while the thickness of the tail region was significantly larger for tail-deuterated DPhyPC, which was evident from a smaller area per molecule. At 20 mN/m the thickness of the head and tail regions in both lipids was comparable, yet the area per molecule of tail-deuterated DPhyPC was 10% smaller than tail-deuterated DPEPC. In the TBM bilayer model membrane, the thickness of the lipid tails in both inner and outer leaflets was 8.2 Å, giving a total of 16.4 Å. Deuteration enabled unambiguous determination of the relative proportion of the hydrogenous tether, phospholipid and subphase.
Assuntos
Deutério/química , Bicamadas Lipídicas/síntese química , Difração de Nêutrons/métodos , Fosfolipídeos/síntese química , Ácido Fitânico/síntese química , Fitol/química , Fluidez de Membrana , Conformação MolecularRESUMO
Oleic acid and its phospholipid derivatives are fundamental to the structure and function of cellular membranes. As a result, there has been increasing interest in the availability of their deuterated forms for many nuclear magnetic resonance, infrared, mass spectroscopy and neutron scattering studies. Here, we present for the first time a straightforward, large-scale (gram quantities) synthesis of highly deuterated [D32 ]oleic acid by using multiple, yet simple and high yielding reactions. The precursors for the synthesis of [D32 ]oleic acid are [D14 ]azelaic acid and [D17 ]nonanoic acid, which were obtained by complete deuteration (>98% D) of their (1) H forms by using metal catalysed hydrothermal H/D exchange reactions. The oleic acid was produced with ca. 94% D isotopic purity and with no contamination by the trans-isomer (elaidic acid). The subsequent synthesis of [D64 ]dioleoyl-sn-glycero-3-phosphocholine from [D32 ]oleic acid is also described.
Assuntos
Deutério/química , Glicerilfosforilcolina/análogos & derivados , Ácido Oleico/química , Ácido Oleico/síntese química , Técnicas de Química Sintética , Ácidos Dicarboxílicos/química , Ácidos Graxos/química , Glicerilfosforilcolina/síntese química , Glicerilfosforilcolina/química , Metilação , FosfatidilcolinasRESUMO
Just say NO to biofilms: NO-donors are used to disperse a bacterial biofilm so that co-administered antibiotics will kill the more susceptible unattached cells. The chemically stable cephalosporin-3'-diazeniumdiolate NO-donor prodrug is activated by bacterial ß-lactamases and facilitates this two-step biofilm erradication.