RESUMO
The ionizable lipid ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), is a component of the lipid matrix of the prophylactic SARS-CoV-2 mRNA vaccine produced by Pfizer/BioNTech. This lipid ensures efficient vaccine assembly, protects the mRNA from premature degradation, and promotes the release of the nucleic acid into the cytoplasm for its further processing after endocytosis. The present work describes a simple and economical method for the synthesis of the ALC-0315 lipid, which can be taken advantage of in mRNA vaccine production.
RESUMO
The review considers liposomes as systems of substantial interest as adjuvant carriers in vaccinology due to their versatility and maximal biocompatibility. Research and development on the use of liposomes and lipid nanoparticles to create subunit vaccines for the prevention and treatment of infectious diseases has been going on for several decades. In recent years, the area has seen serious progress due to the improvement of the technology of industrial production of various high-grade lipids suitable for parenteral administration and the emergence of new technologies and equipment for the production of liposomal preparations. When developing vaccines, it is necessary to take into account how the body's immune system (innate and adaptive immunity) functions. The review briefly describes some of the fundamental mechanisms underlying the mobilization of immunity when encountering an antigen, as well as the influence of liposome carriers on the processes of internalization of antigens by immunocompetent cells and ways of immune response induction. The results of the studies on the interactions of liposomes with antigen-presenting cells in function of the liposome size, charge, and phase state of the bilayer, which depends on the lipid composition, are often contradictory and should be verified in each specific case. The introduction of immunostimulant components into the composition of liposomal vaccine complexes-ligands of the pathogen-associated molecular pattern receptors-permits modulation of the strength and type of the immune response. The review briefly discusses liposome-based vaccines approved for use in the clinic for the treatment and prevention of infectious diseases, including mRNA-loaded lipid nanoparticles. Examples of liposomal vaccines that undergo various stages of clinical trials are presented.
RESUMO
Previously, we showed that incorporation of methotrexate (MTX) in the form of a lipophilic prodrug (MTXDG) in 100-nm lipid bilayer liposomes of egg phosphatidylcholine can allow one to reduce toxicity and improve the antitumor efficiency of MTX in a mouse model of T-cell leukemic lymphoma. However, in our hemocompatibility tests in vitro, MTX liposomes caused complement (C) activation, obviously due to binding on the liposome surface and fragmentation of the C3 complement factor. In this work, we studied the interactions of MTX liposomes carrying stabilizing molecules phosphatidylinositol (PI), ganglioside GM1, or a lipid conjugate of N-carboxymethylated oligoglycine (CMG) in the bilayer with subpopulations of human blood leukocytes. Liposomes labeled with BODIPY-phosphatidylcholine were incubated with whole blood (30 min and 1 h, 37°C), blood cells were lysed with a hypotonic buffer, and the fluorescence of the liposomes bound but not internalized by the leukocytes was quenched by crystal violet. Cell suspensions were analyzed by flow cytometry. Incorporation of MTXDG dramatically enhanced the phagocytosis of liposomes of any composition by monocytes. Neutrophils consumed much less of the liposomes. Lymphocytes did not accumulate liposomes. The introduction of PI into MTX liposomes practically did not affect the specific consumption of liposomes by monocytes, while CMG was likely to increase the consumption rate regardless of the presence of MTXDG. The GM1 ganglioside presumably shielded MTX liposomes from phagocytosis by one of the monocyte populations and increased the efficiency of monocyte uptake by another population, probably one expressing C3b-binding receptors (C3b was detected on liposomes after incubation with blood plasma). MTX liposomes were shown to have different effects on TNF-α production by activated leukocytes, depending on the structure of the stabilizing molecule.
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The modern theory of lipid membrane structure incorporates the concept of lateral stress profile. The latter represents the forces that act on any solute inside the membrane. We used this concept to propose two lipid probes that introduce minimal distortions into the lipid bilayer packing: the surface pressure isotherms and volt-potentials of the pure and mixed (probe-containing) lipid monolayers are equal. The probes represent a FRET pair. They are applicable in lipid transfer and vesicle fusion experiments.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Lipossomos/química , Compostos de Boro/química , Fosfatidilcolinas/química , Teoria Quântica , Propriedades de SuperfícieRESUMO
We report that the action of the heterodimeric phospholipases A2 (PLA2s) from Vipera nikolskii, which comprises enzymatically active basic subunit and inactive acidic PLA2 homologue, on the lipid bilayer results in the aggregation and stacking of bilayers. These processes are demonstrated using two independent methods (fluorescence spectroscopy and electron microscopy). Aggregation of bilayers is possible because both subunits of the V. nikolskii heterodimer contain a membrane-binding site (also known as IBS). Thus, when the two IBSs bind to the membrane, the heterodimer acts as a connecting agent. Heterodimers induce aggregation of negatively charged bilayers composed of phosphatidylglycerol and do not induce aggregation of neutral bilayers composed of phosphatidylcholine.
Assuntos
Bicamadas Lipídicas/química , Fosfolipases A2/química , Venenos de Víboras/química , Viperidae , Animais , Dimerização , Lipossomos/química , Fosfatidilcolinas/química , Fosfatidilgliceróis/químicaRESUMO
Magnetic fluid-loaded liposomes (MFLs) were fabricated using magnetite nanoparticles (MNPs) and natural phospholipids via the thin film hydration method followed by extrusion. The size distribution and composition of MFLs were studied using dynamic light scattering and spectrophotometry. The effective ranges of magnetite concentration in MNPs hydrosol and MFLs for contrasting at both T2 and T1 relaxation were determined. On T2 weighted images, the MFLs effectively increased the contrast if compared with MNPs hydrosol, while on T1 weighted images, MNPs hydrosol contrasting was more efficient than that of MFLs. In vivo magnetic resonance imaging (MRI) contrasting properties of MFLs and their effects on tumor and normal tissues morphology, were investigated in rats with transplanted renal cell carcinoma upon intratumoral administration of MFLs. No significant morphological changes in rat internal organs upon intratumoral injection of MFLs were detected, suggesting that the liposomes are relatively safe and can be used as the potential contrasting agents for MRI.
Assuntos
Meios de Contraste/química , Lipossomos/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Animais , Carcinoma de Células Renais/patologia , Interações Hidrofóbicas e Hidrofílicas , Rim/patologia , Neoplasias Renais/patologia , Masculino , Transplante de Neoplasias , Tamanho da Partícula , Ratos , Ratos WistarRESUMO
Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.
Assuntos
Análise Química do Sangue/métodos , Corantes Fluorescentes , Fosfolipases A2 Secretórias/sangue , Biomarcadores/sangue , Análise Química do Sangue/estatística & dados numéricos , Corantes Fluorescentes/química , Humanos , Hidrólise , Mediadores da Inflamação/sangue , Cinética , Micelas , Modelos Biológicos , Fosfolipídeos/química , Sensibilidade e Especificidade , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Immediately upon contact with blood, nanosized drug delivery systems become coated with a so-called protein corona. The quantitative and qualitative composition of the corona defines not only the behavior of the nanocarrier in the circulation but, ultimately, the pharmacokinetics and biodistribution of the encapsulated drug as well. In turn, the composition of the protein corona depends on the surface properties of the nanoparticles, such as size and distribution of charge and functional groups on the particle surface. Liposomes belong to the most bio- and hemocompatible drug delivery systems feasible for intravenous route of administration required in chemotherapy of metastasizing tumors. However, knowledge on the interactions of liposomes of various compositions with blood plasma proteins remains fragmentary. Moreover, all nanosized drug delivery systems are potential targets for the innate immunity system, primarily the complement (C) system, which underlies frequent cases of hypersensitivity reactions. Recently, in a panel of in vitro hemocompatibility tests, we demonstrated that liposomes built of natural phospholipids - egg phosphatidylcholine and phosphatidylinositol from Saccharomyces cerevisiae - and loaded with diglyceride conjugates of anticancer drugs melphalan and methotrexate, did not affect the morphology and numbers of the main blood cell types. While preparations with melphalan prodrug were also inert in coagulation and C activation tests, methotrexate-loaded liposomes caused impaired coagulation and C activation. The aim of this work was to study the interactions of liposomes carrying prodrugs of melphalan and methotrexate with blood plasma proteins in vitro. Data on protein binding capacity of liposomes obtained with classical gel permeation chromatography techniques allowed for prediction of rather rapid elimination of the liposomes from circulation. A number of differences revealed through immunoblotting of the liposome-bound proteins agree with the previously obtained data on C activation. The possible mechanism of C activation by methotrexate-containing liposomes is discussed.
Assuntos
Proteínas Sanguíneas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Lipossomos/química , Lipossomos/metabolismo , Melfalan/química , Metotrexato/química , Complemento C3/metabolismo , Humanos , Pró-Fármacos/química , Especificidade por SubstratoRESUMO
A fluorescent analog of the lipophilic prodrug of antitumor agent methotrexate has been synthesized. The conjugate consists of a residue of rac-1-[13-(Me4-BODIPY-8)tridecanoyl]-2-oleoylglycerol connected to methotrexate by ester bond via ß-Ala-N-carbonylmethylene linker (Me4-BODIPY-8,4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl). The probe is designed for incorporation in the membrane of liposomal vehicle to study a mechanism of interaction with tumor cells and an intracellular traffic.
Assuntos
Técnicas de Química Sintética , Metotrexato/química , Pró-Fármacos/química , Compostos de Boro/química , Corantes Fluorescentes/síntese química , Glicerídeos/química , Lipossomos/química , Metotrexato/síntese química , Pró-Fármacos/síntese químicaRESUMO
Colchicine site binders--blockers of tubulin polymerization--are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable formulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine--yeast phosphatidylinositol--palmitic or oleic prodrug, 8 : 1: 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.
Assuntos
Colchicina/administração & dosagem , Neoplasias/tratamento farmacológico , Pró-Fármacos/administração & dosagem , Triazóis/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colchicina/síntese química , Colchicina/química , Ácidos Graxos/síntese química , Ácidos Graxos/química , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Neoplasias/patologia , Polimerização/efeitos dos fármacos , Pró-Fármacos/síntese química , Pró-Fármacos/química , Triazóis/síntese química , Triazóis/química , Tubulina (Proteína)/efeitos dos fármacosRESUMO
The antimitotic agent combretastatin A4 (CA-4) has been suggested as an antivascular agent for anticancer therapy relatively recently. To reduce systemic toxicity by means of administration in liposome formulations, in this study new lipophilic prodrugs, oleic derivatives of CA-4 and its 4-arylcoumarin analog (CA4-Ole and ArC-Ole, respectively), have been synthesized: Liposomes of 100 nm mean diameter prepared on the basis of egg phosphatidylcholine and phosphatidylinositol from bakers yeast have been shown to include completely up to 10 mol. % of CA4-Ole, or 7 mol. % of ArC-Ole. Also, prodrug bearing liposomes decorated with tetrasaccharide selectin ligand Sialyl Lewis X (SiaLe(x)) have been constructed to achieve targeting to endothelium under neovascularization. The antitumor activity in vivo was studied in the model of slowly growing mouse breast cancer. Under the used dose (22 mg/kg) as well as the regimen of treatment (four injections, one per a week, starting from the appearance of palpable tumors) cytostatic CA-4 did not reveal any anticancer effect, and oppositely even stimulated tumor growth. Liposome formulations of CA4-Ole did not show such stimulation. However, to achieve pronounced antitumor effect, number of injections of liposomes should be apparently elevated. New antimitotic agent ArC revealed cytotoxic activity of only one tenth value obtained for CA-4 in vitro in the culture of human breast carcinoma cells. Nevertheless, in vivo in the mouse model of breast cancer this compound showed antitumor effect under double CA-4 equivalent dose. The results demonstrate availability of SiaLe(x)-liposomes loaded with ArC-Ole: this preparation began to inhibit tumor growth already after the second injection. It is necessary further to choose doses and regimens of administration both for ArC and liposome formulations bearing ArC-Ole.
Assuntos
Cumarínicos/síntese química , Lipossomos/administração & dosagem , Pró-Fármacos/síntese química , Estilbenos/síntese química , Animais , Antimitóticos/administração & dosagem , Antimitóticos/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cumarínicos/administração & dosagem , Cumarínicos/farmacologia , Esquema de Medicação , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oligossacarídeos/química , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Antígeno Sialil Lewis X , Estilbenos/administração & dosagem , Estilbenos/farmacologiaRESUMO
In the current study a technique for microencapsulation of human breast adenocarcinoma cells MCF-7 in alginate-chitosan microcapsules is used. Microencapsulation is proposed to generate multicellular tumor spheroids (MTS) based on these cells and to test them further as an in vitro model for anti-tumor drug screening. Cytotoxicity of methotrexate (MTX) was studied on the obtained MTS. A set of MTS with mean size of 150, 200 and 300 m was prepared in function of a cultivation time. After incubation of MTS in cultivation medium containing MTX at concentrations of 1, 2, 10, 50 and 100 nM for 48 hs cell viability was evaluated. MTS were shown to be more resistant to MTX than the monolayer culture, and the resistance to MTX was increased with enhancing a spheroid size. At MTX concentration of 100 nM a number of viable cells in MTS with the size of 300 m was 2.5-fold bigger than that one in monolayer culture. It is suggested that the cells in microencapsulated MTS can better mimic cell behavior in a small size solid tumor than the cells in a monolayer culture. In future microencapsulated MTS can be proposed as a novel in vitro model for anticancer drug screening.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Esferoides Celulares , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Imobilizadas , Feminino , Humanos , Metotrexato/farmacologia , Modelos BiológicosRESUMO
The efficiency of the chemotherapeutic agent methotrexate (MTX) in tumor cells is limited by the frequent development of the drug resistance of tumor cells. We had previously shown in vitro using human acute leukemia cells with various sensitivity to MTX (T-lymphoblastic CCRF-CEM line and resistant CEM/MTX subline) that MTX incorporation into liposomes as a lipophilic prodrug, diglyceride conjugate (MTX-DG), allows for the overcoming of cell resistance due to the impaired active transmembrane transport. In this work, we have studied the profile of binding with carbohydrates of the cell lines mentioned using carbohydrate fluorescent probes (poly(acryl amide) conjugates). Lipophilic conjugates of tetrasaccharide SiaLe(x), 6'-HSO3LacNAc, and also inactive pentaol for incorporation into liposomes, have been synthesized. The cytotoxicity of MTX-DG liposomes equipped with the SiaLe(x) ligand toward the sensitive CCRF-CEM cell culture was demonstrated to be 3.5 times higher than that of MTX-DG liposomes bearing the control inactive pentaol. The activity of MTX liposomes bearing 6'-HSO3LacNAc toward resistant CEM/MTX was 1.6-fold increased. The use of carbohydrate ligands as molecular addresses for drug-carrying liposomes as a potential method of treating heterogeneous tumor tissue is discussed.
Assuntos
Resinas Acrílicas/química , Antimetabólitos Antineoplásicos/administração & dosagem , Diglicerídeos/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Metotrexato/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Ligantes , Lipídeos/química , Lipossomos , Metotrexato/farmacologiaRESUMO
It has recently been shown that the influenza virus can specifically bind the residue of a nonsialylated sulfated oligosaccharide Gal(6SO(3)H)beta1-4GlcNAcbeta (6'SLacNAc). To identify by photoaffinity labeling the virion component that binds 6'SLacNAc, we synthesized a carbohydrate probe containing a (125)I labeled diazocyclopentadien-2-yl carbonyl group as an aglycone. According to the electrophoretic data, the labeled areas corresponded to a large hemagglutinin subunit, a nucleocapsid protein, and neuraminidase (NA). Probing in the presence of an excess of 6'SLacNAcbeta-OCH(2)CH(2)NHAc glycoside resulted in redistribution of the labeling intensity, with the maximum inhibition being observed for NA. The data obtained indicate that NA is a viral 6'SLacNAc-binding protein.
Assuntos
Corantes Fluorescentes/química , Vírus da Influenza A Subtipo H1N1/química , Oligossacarídeos/química , Proteínas Virais/química , Sítios de Ligação , Corantes Fluorescentes/síntese química , Hemaglutininas Virais/química , Neuraminidase/química , Proteínas do Nucleocapsídeo/química , Ligação Proteica , Vírion/químicaAssuntos
Gangliosídeo G(M1)/análogos & derivados , Interleucina-4/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Gangliosídeo G(M1)/metabolismo , Interleucina-4/química , Modelos Moleculares , Marcadores de Fotoafinidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We have recently synthesized a lipid conjugate of the anticancer agent methotrexate (MTX-DG) and showed that the conjugate is quantitatively included in the lipid bilayer of liposomes prepared by a standard extrusion technique from an 8 : 1 : 1 (mol) egg phosphatidylcholine-yeast phosphatidylinositol-MTX-DG mixture. Both the size of liposomes (126 +/- 30 nm) and the MTX-DG concentration (4.4 mM) are relevant for systemic injections in mammals. The liposomal formulation of MTX-DG was shown to overcome the resistance of tumor cells in vitro to methotrexate: the cytotoxic activities (IC50) of MTX in cultures of the human T-lymphoblastic leukemia cell line CEM-CCRF and the MTX-resistant subline CEM/MTX were 0.075 +/- 0.005 and 16.4 +/- 4.9 microM, respectively, while, in the case of liposomes loaded with MTX-DG, the IC50 values were much closer: 0.77 +/- 0.06 and 3.8 +/- 1.9 microM.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Diglicerídeos/química , Portadores de Fármacos/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Metotrexato/farmacologia , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Concentração Inibidora 50 , Leucemia/patologia , Lipossomos , Metotrexato/administração & dosagem , Metotrexato/química , Tamanho da PartículaRESUMO
This review contains a brief consideration of some theoretical aspects of photoaffinity (photoreactive) labeling (PAL), and the most widely used photoreactive groups, such as arylazide, benzophenone, and 3-(trifluoromethyl)-3-phenyldiazirine, are characterized in comparison. Experimental methodology is described, including modern approaches of mass spectrometry for analysis of cross-linking products between the photoreactive probes and biomolecules. Examples of PAL application in diverse fields of structural biology during the last five-ten years are presented. Potential drug targets, transport processes, stereochemistry of interaction of G-protein-coupled receptors with ligands, as well as structural changes in nicotinic acetylcholine receptor are considered. Applications of photoaffinity ganglioside and phospholipid probes for studying biological membranes and of nucleotide probes in investigations of replicative and transcriptional complexes, as well as photoaffinity glycoconjugates for detecting carbohydrate-binding proteins are covered. In combination with modern techniques of instrumental analysis and computer-aided modeling, PAL remains the most important approach in studies on the organization of biological systems.
Assuntos
Estrutura Molecular , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/síntese química , Coloração e Rotulagem/métodos , Azidas/síntese química , Azidas/química , Azirinas/síntese química , Azirinas/química , Benzofenonas/síntese química , Benzofenonas/química , Glicoconjugados/química , Ligantes , Lipídeos/química , Membranas/química , Membranas/metabolismo , Modelos Moleculares , Nucleotídeos/químicaRESUMO
A lipophilic methotrexate prodrug capable of incorporation into membranes of carrier liposomes was synthesized. The conjugate consists of a lipophilic rac-1,2-dioleoylglycerol anchor connected to methotrexate through beta(Ala)-N-carbonylmethylene linker, which should be located in the polar region of the lipid bilayer. The ester bond between the hydrophilic linker and the antitumor agent can be hydrolyzed by intracellular esterases. The liposomal formulation of the prodrug exhibited a cytotoxic activity in vitro. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
Assuntos
Diglicerídeos/síntese química , Metotrexato/análogos & derivados , Metotrexato/síntese química , Animais , Sobrevivência Celular/efeitos dos fármacos , Diglicerídeos/farmacologia , Humanos , Lipossomos , Metotrexato/farmacologia , Ressonância Magnética Nuclear Biomolecular , Células Tumorais CultivadasRESUMO
A method for the synthesis of photoaffinity neoglycolipid probes with a highly efficient carbene-generating diazocyclopentadien-2-ylcarbonyl (Dcp) label, which can be radioiodinated under standard oxidation conditions, was developed. The probes are intended for incorporation into the lipid bilayer. They are lipophilic glycoconjugates on the basis of an amphiphilic aglycone built up from a diacylglycerol and a polyethylene glycol spacer (with a polymerization degree of 9-16) bearing the Dcp label at the terminal unit. The location of the label in the aglycone provides the possibility of one-step preparation of a wide range of probes using various carbohydrate synthons. We have synthesized photoaffinity neoglycoconjugates containing the oligosaccharides: sialyl LewisX tetrasaccharide and A trisaccharide, which is specific to some tumor cells. A probe containing an inactive pentaol (aminodeoxyglucitol) was also synthesized to detect nonspecific binding. The Dcp label is bound to the probe molecule by ester bond; its lability under alkaline conditions facilitates the analysis of cross-linked products after photoaffinity labeling. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.
Assuntos
Glicolipídeos/síntese química , Lectinas/química , Proteínas de Membrana/química , Sondas Moleculares , Marcadores de Afinidade , Ressonância Magnética Nuclear Biomolecular , FotoquímicaRESUMO
The subunits of the F0 membrane sector of bovine heart mitochondrial H(+)-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H(+)-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn- glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn- glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero- 3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The cross-linked products were identified by SDS-PAGE and MALDI mass spectrometry.