Synthesis of mono- and dihydroxylated furanoses, pyranoses, and an oxepanose for the preparation of natural product analogue libraries

[structures: see text] Numerous biologically active natural products contain furanoses and pyranoses with mono- and dihydroxylated substituents. However, much of the structure-activity studies on such molecules is gathered on the aglycons without attention to the corresponding carbohydrate components. Consequently, there are few synthetic procedures that enable the rapid preparation of mono- and dihydroxyfuranoses and mono- and dihydroxypyranoses and no report for a 3,4-dihydroxyoxepanose. In this article we report the practical synthesis of orthogonally protected five-, six-, and seven-membered carbohydrate derivatives. The succinct manner in which these molecules were synthesized allows the rapid preparation of analogues aimed at discovering the role of ring size and individual hydroxyl moieties on the pyranose skeleton.     

Prediction of peptide binding to a major histocompatibility complex class I molecule based on docking simulation

Binding between major histocompatibility complex (MHC) class I molecules and immunogenic epitopes is one of the most important processes for cell-mediated immunity. Consequently, computational prediction of amino acid sequences of MHC class I binding peptides from a given sequence may lead to important biomedical advances. In this study, an efficient structure-based method for predicting peptide binding to MHC class I molecules was developed, in which the binding free energy of the peptide was evaluated by two individual docking simulations. An original penalty function and restriction of degrees of freedom were determined by analysis of 361 published X-ray structures of the complex and were then introduced into the docking simulations. To validate the method, calculations using a 50-amino acid sequence as a prediction target were performed. In 27 calculations, the binding free energy of the known peptide was within the top 5 of 166 peptides generated from the 50-amino acid sequence. Finally, demonstrative calculations using a whole sequence of a protein as a prediction target were performed. These data clearly demonstrate high potential of this method for predicting peptide binding to MHC class I molecules.     

Introduction of the CIITA gene into tumor cells produces exosomes with enhanced anti-tumor effects

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1- CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA- Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-α, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-γ cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.     

From the Laboratory to the Clinic: A Retrospective on Fully Synthetic Carbohydrate-Based Anticancer Vaccines Frequently used abbreviations are listed in the appendix

This review provides an account of our explorations into oligosaccharide and glycoconjugate construction for the creation and evaluation of vaccines based on carbohydrate-centered tumor antigens. Our starting point was the known tendency of transformed cells to express selective carbohydrate motifs in the form of glycoproteins or glycolipids. Anticancer vaccines derived from carbohydrate-based antigens could be effective targets for immune recognition and attack. Obtaining significant quantities of such structures from natural sources is, however, extremely difficult. With the total synthesis of tumor-associated carbohydrate antigens accomplished, we began to evaluate at the clinical level whether the human immune system can respond to such fully synthetic antigens in a focused and useful way. Toward this goal, we have merged the resources of chemistry and immunology in an attack on the problem. The synthesis and immunoconjugation of various tumor-associated carbohydrate antigens and the results of such constructs in mice vaccinations will be described. For fashioning an effective vaccine, conjugation to a suitable immunogenic carrier was necessary and conjugates of KLH (keyhole limpet cyanin) have consistently demonstrated the relevant immunogenicity. Preclinical and clinical studies with synthetic conjugate carbohydrate vaccines show induction of IgM- and IgG-antibody responses. Another approach to anticancer vaccines involves the use of clustered glycopeptides as targets for immune attack. Initial attention has been directed to mucin related O-linked glycopeptides. Synthetic trimeric clusters of glycoepitopes derived from the Tn-, TF- and Lewis(y)-antigens, appropriately bioconjugated, have been demonstrated to be immunogenic. The hope is that patients immunized in an adjuvant manner with synthetic carbohydrate vaccines would produce antibodies reactive with cancer cells and that the production of such antibodies would mitigate against tumor spread, thereby enabling a more favorable survival and "quality of life" prognosis.     

Microfluidic biofunctionalisation protocols to form multi‐valent interactions for cell rolling and phenotype modification investigations

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro‐fluidic protocols. Many available processes either require expensive and time‐consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi‐valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin–biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC‐I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC‐I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC‐I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC‐I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.     

Transmembrane Signaling of T Lymphocytes by Ligand-Induced Receptor Complex Assembly

T lymphocytes (T cells) are the central cell type initiating all immune responses. They are able to recognize other cells in the body that have been invaded by foreign living or nonliving matter. In such cells, foreign peptides generated by intracellular breakdown are complexed with molecules of the major histocompatibility complex (MHC) specially designed for peptide binding. Peptide-loaded MHC molecules appear on the surface of these cells and alert the immune system. The molecular complex which T cells use for recognition of peptide-loaded MHC molecules is among the most sophisticated and versatile receptor systems in biology. It consists of specific and nonspecific transmembrane components which assemble to a functional signal transduction unit as the result of ligand binding. Correct assembly leads to activation and relocation of enzymes including membrane-associated, tyrosin-specific protein kinases and phosphatases. Transmembrane signaling in T cells depends on the correct assembly and cooperation among multiple molecular components. This may be related to a multitude of different cellular responses of T cells at different stages of differentiation, all elicited through the T cell receptor complex.     

基于环境的编码方法在预测HLA-A*0201结合多肽中的应用

T淋巴细胞对抗原的识别是产生与调节有效免疫应答的关键, T细胞只识别主要组织相容性复合物(MHC)呈递上来的抗原, 因此MHC与抗原多肽的结合就成为一系列免疫应答过程中基础的一环. 为了辅助疫苗设计, 多种机器学习技术已被普遍应用于MHC结合多肽的预报领域中. 本文以支持向量机(SVM)为手段, 以HLA-A*0201的实验数据集为对象, 对多种肽段编码方法形成的模型进行评价, 得到的AUC值的范围在0.932~0.936之间. 提出一种新的利用抗原多肽结合环境的编码方法, 使预报的AUC值提高到0.953. 对独立数据集进行建模预报, 同样证明环境编码模型的预报准确率高于传统编码方法的准确率.     

Synthesis and biological evaluation of two chemically modified peptide epitopes for the class I MHC protein HLA-B*2705

The T-cell receptor of a CD8(+) T-cell recognises peptide epitopes bound by class I major histocompatibility complex (MHC) glycoproteins presented in a groove on their upper surface. Within the groove of the MHC molecule are 6 pockets, two of which mostly display a high degree of specificity for binding amino acids capable of making conserved and energetically favourable contacts with the MHC. One type of MHC molecule, HLA-B*2705, preferentially binds peptides containing an arginine at position 2. In an effort to increase the affinity of peptides for HLA-B*2705, potentially leading to better immune responses to such a peptide, we synthesised two modified epitopes where the amino acid at position 2 involved in anchoring the peptide to the class I molecule was replaced with the alpha-methylated beta,gamma-unsaturated arginine analogue 2-(S)-amino-5-guanidino-2-methyl-pent-3-enoic acid. The latter was prepared via a multi-step synthetic sequence, starting from alpha-methyl serine, and incorporated into dipeptides which were fragment-coupled to resin-bound heptameric peptides yielding the target nonameric sequences. Biological characterisation indicated that the modified peptides were poorer than the native peptides at stabilising empty class I MHC complexes, and cells sensitised with these peptides were not recognised as well by cognate CD8(+) T-cells, where available, compared to those sensitised with the native peptide. We suggest that the modifications made to the peptide have decreased its ability to bind to the peptide binding groove of HLA-B*2705 molecules which may explain the decrease in recognition by cytotoxic T-cells when compared to the native peptide.     

Synthesis of a Monophosphoryl Derivative of Escherichia coli Lipid A and Its Efficient Coupling to a Tumor‐Associated Carbohydrate Antigen

Monophosphoryl lipid A is a safe and potent immunostimulant and vaccine adjuvant, which is potentially useful for the development of effective carbohydrate‐based conjugate vaccines. This paper presents a convergent and efficient synthesis of a monophosphoryl derivative of E. coli lipid A that has an alkyne functionality at the reducing end, which is suitable for coupling with various molecules. The coupling of this derivative to an N‐modified analogue of tumor‐associated antigen GM3 through click chemistry is also presented.     

Predicting sequences and structures of MHC-binding peptides: a computational combinatorial approach

Peptides bound to MHC molecules on the surface of cells convey critical information about the cellular milieu to immune system T cells. Predicting which peptides can bind an MHC molecule, and understanding their modes of binding, are important in order to design better diagnostic and therapeutic agents for infectious and autoimmune diseases. Due to the difficulty of obtaining sufficient experimental binding data for each human MHC molecule, computational modeling of MHC peptide-binding properties is necessary. This paper describes a computational combinatorial design approach to the prediction of peptides that bind an MHC molecule of known X-ray crystallographic or NMR-determined structure. The procedure uses chemical fragments as models for amino acid residues and produces a set of sequences for peptides predicted to bind in the MHC peptide-binding groove. The probabilities for specific amino acids occurring at each position of the peptide are calculated based on these sequences, and these probabilities show a good agreement with amino acid distributions derived from a MHC-binding peptide database. The method also enables prediction of the three-dimensional structure of MHC-peptide complexes. Docking, linking, and optimization procedures were performed with the XPLOR program [1].     

Divergent cycloaddition and ring-closing metathesis approaches to indolizidine and pyrrolo[1,2-a]azepine skeletons from a chiral precursor: an expeditious route to (-)-8-epi-swainsonine triacetate

[reaction: see text] A divergent strategy for the synthesis of diverse azabicyclic ring systems has been developed in which a chiral N-allylpyrrolidine derivative, obtained from a carbohydrate precursor was converted to (-)-8-epi-swainsonine triacetate by RCM and to a pyrrolo[1,2-a]azepine derivative and a 3-hydroxymethyl-substituted indolizidine by N-allylcarbohydrate nitrone and nitrile oxide cycloadditions.     

Computational investigation of peptide binding stabilities of HLA-B*27 and HLA-B*44 alleles

Major Histocompatibility Complex (MHC) is a cell surface glycoprotein that binds to foreign antigens and presents them to T lymphocyte cells on the surface of Antigen Presenting Cells (APCs) for appropriate immune recognition. Recently, studies focusing on peptide-based vaccine design have allowed a better understanding of peptide immunogenicity mechanisms, which is defined as the ability of a peptide to stimulate CTL-mediated immune response. Peptide immunogenicity is also known to be related to the stability of peptide-loaded MHC (pMHC) complex. In this study, ENCoM server was used for structure-based estimation of the impact of single point mutations on pMHC complex stabilities. For this purpose, two human MHC molecules from the HLA-B*27 group (HLA-B*27:05 and HLA-B*27:09) in complex with four different peptides (GRFAAAIAK, RRKWRRWHL, RRRWRRLTV and IRAAPPPLF) and three HLA-B*44 molecules (HLA-B*44:02, HLA-B*44:03 and HLA-B*44:05) in complex with two different peptides (EEYLQAFTY and EEYLKAWTF) were analyzed. We found that the stability of pMHC complexes is dependent on both peptide sequence and MHC allele. Furthermore, we demonstrate that allele-specific peptide-binding preferences can be accurately revealed using structure-based computational methods predicting the effect of mutations on protein stability.     

杯[4]芳烃硫脲衍生物的合成及其对阴离子的络合性能

杯[4]芳烃四丁醚经过对位硝化反应后在水合肼作用下被还原成氨基衍生物， 并进一步与异硫氰酸苯酯反应得到单取代与1，3－二取代的杯[4]芳烃硫脲衍生物 ，总收率为50％。它们对系列阴离子的络合实验表明氢键的形成在络合过程中有重 要作用，其中杯[4]双硫脲衍生物5b对H2PO4^-显现出良好的选择性络合能力。     

Recovery of known T-cell epitopes by computational scanning of a viral genome

A new computational method (EpiDock) is proposed for predicting peptide binding to class I MHC proteins, from the amino acid sequence of any protein of immunological interest. Starting from the primary structure of the target protein, individual three-dimensional structures of all possible MHC-peptide (8-, 9- and 10-mers) complexes are obtained by homology modelling. A free energy scoring function (Fresno) is then used to predict the absolute binding free energy of all possible peptides to the class I MHC restriction protein. Assuming that immunodominant epitopes are usually found among the top MHC binders, the method can thus be applied to predict the location of immunogenic peptides on the sequence of the protein target. When applied to the prediction of HLA-A^*0201-restricted T-cell epitopes from the Hepatitis B virus, EpiDock was able to recover 92% of known high affinity binders and 80% of known epitopes within a filtered subset of all possible nonapeptides corresponding to about one tenth of the full theoretical list.The proposed method is fully automated and fast enough to scan a viral genome in less than an hour on a parallel computing architecture. As it requires very few starting experimental data, EpiDock can be used: (i) to predict potential T-cell epitopes from viral genomes (ii) to roughly predict still unknown peptide binding motifs for novel class I MHC alleles.     

Hexaazamacrocyclic nickel and copper complexes and their reactivity with tetracyanoquinodimethane

The hexaazamacrocycle 1,4,7,10,13,16-hexaazacyclooctadecane, [18]ane-N6, forms mono- and dinuclear derivatives with copper chloride depending on the reaction stoichiometries and times. The mononuclear derivative, [Cu([18]ane-N6)]Cl2.H2O, presents the macrocycle wrapped around the metal atom in a distorted octahedral coordinative environment, while the dinuclear derivative, [Cu2([18]ane-N6)Cl2]Cl2.4H2O, is formed by a central Cu2Cl2 core surrounded by an almost planar macrocycle. The crystal structure of both derivatives is stabilized by a network of hydrogen bonds involving the amine macrocyclic groups, the chloride anions, and the crystallization water molecules. The copper atoms in the dinuclear derivative show a strong antiferromagnetic coupling, as expected for the crystal structure parameters. A mononuclear nickel derivative has also been obtained from nickel nitrate by following the same synthetic procedure. These compounds react with TCNQ salts with formation of two types of derivatives, [M([18]ane-N6)](TCNQ)2 and [M([18]ane-N6)](TCNQ)4, depending on the use of radical-anionic or mixed-valence TCNQ salts in the reaction. The crystal structures of the nickel derivatives show that the former derivatives are built up by macrocyclic metal cations surrounded by dimeric dianions (TCNQ)22-, either isolated or stacked along the crystal. The derivative with four TCNQ units/formula consists of alternated chains of metallomacrocyclic cations and stacked TCNQ anions. The crystal parameters suggest that every TCNQ holds approximately 0.5 electrons and overlaps with a neighboring unit to form dimeric monoanions, (TCNQ)2-.     

Resolving Multiple Protein–Peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease

Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE–FL) can resolve multiple MHC–peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE–FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.     

Interaction of a lithiated nitronyl nitroxide with polyfluorinated 1,4-naphthoquinones

A 4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl lithium derivative was found to react with 2-methoxypentafluoro-1,4-naphthoquinone to form a product of addition at the carbonyl function: radical 2-(3,5,6,7,8-pentafluoro-1-hydroxy-2-methoxy-4-oxo-1,4-dihydronaphthalen-1-yl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl. The yield of the addition product increased with temperature and reached 84% at 0?°C. The reaction of the lithium derivative with hexafluoro-1,4-naphthoquinone gave rise to a product of addition at both carbonyl groups, namely, nitronyl nitroxide diradical 2,3,5,6,7,8-hexafluoro-1,4-bis(4,4,5,5-tetramethyl-3-oxide-1-oxyl-4,5-dihydro-1H-imidazole-2-yl)-1,4-dihydronaphthalene-1,4-diol in a 16% yield. The structures of both mono- and diradical were solved by X-ray diffraction analysis, which revealed formation of an intramolecular H-bond between the OH group and nitroxide oxygen. According to electron paramagnetic resonance (EPR) spectroscopy, the obtained mono- and dinitroxide are prone to spontaneous deoxygenation in a toluene solution to give corresponding iminonitroxides. In water, they are much more stable.     

Unique rodlike surface morphologies in trisilanolcyclohexyl polyhedral oligomeric silsesquioxane films

A trisilanol derivative of polyhedral oligomeric silsesquioxane (POSS), trisilanolisobutyl-POSS, has recently been reported to form stable monolayers at the air/water interface. This paper explores the mono- and multilayer properties of another POSS derivative, trisilanolcyclohexyl-POSS, with pi-A isotherm and Brewster angle microscopy measurements. Results show that with continuously increasing surface concentration via symmetrical compression, trisilanolcyclohexyl-POSS amphiphiles at the air/water interface undergo a series of phase transitions from traditional Langmuir monolayers (one-POSS-molecule thick) to unique rodlike hydrophobic aggregates in multilayer films (approximately eight-POSS-molecules thick) that are dramatically different from "collapsed" morphologies seen in other systems. Stable and hydrophobic rodlike structure formation on water is presumably due to trisilanolcyclohexyl-POSS' unique molecular structure and strong tendency to form intermolecular hydrogen bonds in the solid state. This result is consistent with existing POSS/polymer composite research, which shows that POSS molecules tend to aggregate and crystallize into lamellar nanocrystals.     

In silico designing breast cancer peptide vaccine for binding to MHC class I and II: A molecular docking study

Antigenic peptides or cancer peptide vaccines can be directly delivered to cancer patients to produce immunologic responses against cancer cells. Specifically, designed peptides can associate with Major Histocompatibility Complex (MHC) class I or II molecules on the cell surface of antigen presenting cells activating anti-tumor effector mechanisms by triggering helper T cell (Th) or cytotoxic T cells (CTL). In general, high binding to MHCs approximately correlates with in vivo immunogenicity. Consequently, a molecular docking technique was run on a library of novel discontinuous peptides predicted by PEPOP from Human epidermal growth factor receptor 2 (HER2 ECD) subdomain III. This technique is expected to improve the prediction accuracy in order to identify the best MHC class I and II binder peptides. Molecular docking analysis through GOLD identified the peptide 1412 as the best MHC binder peptide to both MHC class I and II molecules used in the study. The GOLD results predicted HLA-DR4, HLA-DP2 and TCR as the most often targeted receptors by the peptide 1412. These findings, based on bioinformatics analyses, can be exploited in further experimental analyses in vaccine design and cancer therapy to find possible proper approaches providing beneficial effects.     

Selective Capture and Collection of Live Target Cells Using a Photoreactive Silicon Wafer Device Modified with Antibodies via a Photocleavable Linker

A device for the capture and recollection of live target cells is described. The platform was a silicon (Si) wafer modified with an anti-HEL antibody (anti-HEL-IgG, HEL = hen egg lysozyme) through a photocleavable 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker. The modification processes of the Si wafer surface were monitored by Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and fast-scanning atomic force microscopy (FS-AFM). The attachment of IgG and its release reaction on the Si surface via the photochemical cleavage of the ANP linker were observed directly by FS-AFM. The results of an enzyme-linked immunosorbent assay (ELISA) indicated that the photorelease of the complex of anti-HEL-IgG with the secondary antibody-alkaline phosphatase hybrid (secondary IgG-AP) from the Si surface occurs with minimum damage. Furthermore, it was possible to collect SP2/O cells selectively that express HEL on their cell membranes (SP2/O-HEL) on the Si wafer device. Photochemical cleavage of the ANP linker facilitated the effective release of living SP2/O cells whose viability was verified by staining experiments using tripan blue. Moreover, it was possible to reculture the recovered cells. This methodology represents an effective strategy for isolating intact target cells in the biological and medicinal sciences and related fields.