In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405 kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.     

Designing of precise vaccine construct against visceral leishmaniasis through predicted epitope ensemble: A contemporary approach

Visceral leishmaniasis (VL) caused by Leishmania donovani is a fatal parasitic disease affecting primarily the poor population in endemic countries. Increasing number of deaths as well as resistant to existing drugs necessitates the development of an effective vaccine for successful treatment of VL. The present study employed a combinatorial approach for designing monomer vaccine construct against L. donovani by applying forecasted B- and T- cell epitopes from 4 genome derived antigenic proteins having secretory signal peptides and glycophosphatidylinositol (GPI) anchors with ≤ 1 transmembrane helix. The forecasted population coverage of chosen T cell epitope ensemble (combined HLA class I and II) cover 99.14 % of world-wide human population. The predicted 3D structure of vaccine constructs (VC1/VC2) were modeled using homology modeling approach and docked to innate immune receptors TLR-2 and TLR-4 with respective docking energies −1231.4/−910.3 and −1119.4/−1476 kcal/mol. Overall, the aforementioned designed vaccine constructs were found appropriate for including in self-assembly protein nanoparticles (SAPN) for further study in developing cutting-edge precision vaccine against VL in short duration with cost-effective manner.     

Immunopeptidome screening to design An immunogenic construct against PRAME positive breast cancer; An in silico study

BackgroundMetastasis is the main cause of breast cancer (BC) lethality, especially in early stages, led to improvements in therapeutic procedures. Lately, by improvements in our perception of biological processes and immune system new classes of vaccines are emerged that grant us the opportunity of designing resolute constructs against desired antigens. In the current study, we used a variety of immunoinformatics tools to design a novel cancer vaccine against Preferentially Expressed Antigen of Melanoma (PRAME), which counts as a cancer testis antigen for various human cancers including BC. The PRAME up-regulation leads to strengthen BC stem cells maintenance, drug resistance, cell survival, adaptation, and apoptosis evading in cancerous cells.Methods and resultsThe PRAME co-expressed genes were mined and validated through BC RNA-sequencing of TCGA data. The immunodominant T-cell predicted epitopes were fused and engineered to form the vaccine. The safety, allergenicity, and immunogenic capabilities of the vaccine were confirmed by promising immunoinformatics tools. The vaccine’s structure was verified to be hydrophilic in most areas through Kyte and Doolittle hydrophobicity plotting. The interactions between the designed vaccine and immune receptors of TLR4 and IL1R were confirmed by protein-protein docking after modeling its tertiary structure. Finally, codon optimization and in silico cloning were performed to guarantee better in-vivo results.ConclusionIn conclusion, concerning in silico assessments’ results in this study, the designed vaccine can potentially boost immune responses against PRAME, therefore may decrease BC development and metastasis. According to the mined PRAME co-expressed genes and their functional annotation, cell cycle regulation is the prime mechanism opted by this construct and its adjacent regulatory genes along boosting immune reactions.     

Liposomal Antitumor Vaccines Targeting Mucin 1 Elicit a Lipid‐Dependent Immunodominant Response

The tumor‐associated antigen mucin 1 (MUC1) has been pursued as an attractive target for cancer immunotherapy, but the poor immunogenicity of the endogenous antigen hinders the development of vaccines capable of inducing effective anti‐MUC1 immunodominant responses. Herein, we prepared synthetic anti‐MUC1 vaccines in which the hydrophilic MUC1 antigen was N‐terminally conjugated to one or two palmitoyl lipid chains (to form amphiphilic Pam‐MUC1 or Pam₂‐MUC1). These amphiphilic lipid‐tailed MUC1 antigens were self‐assembled into liposomes containing the NKT cell agonist αGalCer as an adjuvant. The lipid‐conjugated antigens reshaped the physical and morphological properties of liposomal vaccines. Promising results showed that the anti‐MUC1 IgG antibody titers induced by the Pam₂‐MUC1 vaccine were more than 30‐ and 190‐fold higher than those induced by the Pam‐MUC1 vaccine and the MUC1 vaccine without lipid tails, respectively. Similarly, vaccines with the TLR1/2 agonist Pam₃CSK₄ as an adjuvant also induced conjugated lipid‐dependent immunological responses. Moreover, vaccines with the αGalCer adjuvant induced significantly higher titers of IgG antibodies than vaccines with the Pam₃CSK₄ adjuvant. Therefore, the non‐covalent assembly of the amphiphilic lipo‐MUC1 antigen and the NKT cell agonist αGalCer as a glycolipid adjuvant represent a synthetically simple but immunologically effective approach for the development of anti‐MUC1 cancer vaccines.     

In silico epitope identification of unique multidrug resistance proteins from Salmonella Typhi for vaccine development

Typhoid fever is a multisystemic illness caused by Salmonella enterica serovars Typhi and is resistant to most antibiotics and drugs. The resistance is conferred through multidrug resistance (MDR) proteins, which efflux most antibiotics and other drugs. We predicted potential candidate B-cell and T-cell epitopes using bio- and immune-informatics tools in the 11 MDR proteins - EmrA, EmrB, EmrD, MdtA, MdtB, MdtC, MdtG, MdtH, MdtK, MdtL and TolC. The antigenic potential of the MDR proteins was calculated using VaxiJen server. The B-cell and T-cell epitopes of the MDR proteins were predicted using BCPred and ProPredI and ProPred respectively. The binding affinities of the predicted T-cell epitopes were estimated using T-epitope designer and MHCPred tools. 10, 7, 5, 12, 14, 21, 26, 3, 3 and 3 B-cell epitopes were identified in EmrA, EmrB, EmrD, TolC, MdtA, MdtB, MdtC, MdtG, MdtH and MdtL respectively. We predicted 9 T-cell epitopes - YVSRRAVQP (EmrA), FGVANAISI (EmrB), MVNSQVKQA and YQGGMVNSQ (TolC), WDRTNSHKL (MdtA), FLRNIPTAI (MdtB), YVEQLGVTG (MdtG), VKWMYAIEA (MdtH) and LAHTNTVTL (MdtL) capable of eliciting both humoral and adaptive immune responses. These T-cell epitopes specifically bind to HLA alleles - DRB1*0101 and DRB1*0401. This is the first report of epitope prediction in the MDR proteins of S. Typhi. Taken together, these results indicate the MDR proteins – EmrA, MdtA and TolC are the most suitable vaccine candidates for S. Typhi. The findings of our study on the MDR proteins prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of typhoid fever.     

Chemical synthesis and immunological evaluation of new generation multivalent anticancer vaccines based on a Tn antigen analogue

Tumor associated carbohydrate antigens (TACAs), such as the Tn antigen, have emerged as key targets for the development of synthetic anticancer vaccines. However, the induction of potent and functional immune responses has been challenging and, in most cases, unsuccessful. Herein, we report the design, synthesis and immunological evaluation in mice of Tn-based vaccine candidates with multivalent presentation of the Tn antigen (up to 16 copies), both in its native serine-linked display (Tn-Ser) and as an oxime-linked Tn analogue (Tn-oxime). The high valent vaccine prototypes were synthesized through a late-stage convergent assembly (Tn-Ser construct) and a versatile divergent strategy (Tn-oxime analogue), using chemoselective click-type chemistry. The hexadecavalent Tn-oxime construct induced robust, Tn-specific humoral and CD4⁺/CD8⁺ cellular responses, with antibodies able to bind the Tn antigen on the MCF7 cancer cell surface. The superior synthetic accessibility and immunological properties of this fully-synthetic vaccine prototype makes it a compelling candidate for further advancement towards safe and effective synthetic anticancer vaccines.

A fully-synthetic anticancer vaccine candidate incorporating an hexadecavalent Tn antigen analogue display via oxime linkages induced tumor-specific IgG antibodies and cellular immune responses in mice coadministered with QS-21 as an adjuvant.     

Two highly similar LAEDDTNAQKT and LTDKIGTEI epitopes in G glycoprotein may be useful for effective epitope based vaccine design against pathogenic Henipavirus

Nipah virus and Hendra virus, two members of the genus Henipavirus, are newly emerging zoonotic pathogens which cause acute respiratory illness and severe encephalitis in human. Lack of the effective antiviral therapy endorses the urgency for the development of vaccine against these deadly viruses. In this study, we employed various computational approaches to identify epitopes which has the potential for vaccine development. By analyzing the immune parameters of the conserved sequences of G glycoprotein using various databases and bioinformatics tools, we identified two potential epitopes which may be used as peptide vaccines. Using different B cell epitope prediction servers, four highly similar B cell epitopes were identified. Immunoinformatics analyses revealed that LAEDDTNAQKT is a highly flexible and accessible B-cell epitope to antibody. Highly similar putative CTL epitopes were analyzed for their binding with the HLA-C 12*03 molecule. Docking simulation assay revealed that LTDKIGTEI has significantly lower binding energy, which bolstered its potential as epitope-based vaccine design. Finally, cytotoxicity analysis has also justified their potential as promising epitope-based vaccine candidate. In sum, our computational analysis indicates that either LAEDDTNAQKT or LTDKIGTEI epitope holds a promise for the development of universal vaccine against all kinds of pathogenic Henipavirus. Further in vivo and in vitro studies are necessary to validate the obtained findings.     

Fully synthetic Mincle-dependent self-adjuvanting cancer vaccines elicit robust humoral and T cell-dependent immune responses and protect mice from tumor development

A new strategy based on a macrophage-inducible C-type lectin (Mincle) agonist was established to construct synthetic cancer vaccines. Using sialyl-Tn (STn) as a model antigen, four conjugates with the Mincle agonist as a built-in adjuvant were designed and synthesized through a facile and efficient method. All conjugates could induce BMDMs to produce inflammatory cytokines in a Mincle-dependent manner and were found to elicit robust humoral and T cell-dependent immune responses alone in mice. The corresponding antibodies could recognize, bind and exhibit complement-dependent cytotoxicity to STn-positive cancer cells, leading to tumor cell lysis. Moreover, all conjugates could effectively inhibit tumor growth and prolong the mice survival time in vivo, with therapeutic effects better than STn-CRM197/Al. Notably, compared to conventional glycoprotein conjugate vaccines, these fully synthetic conjugate vaccines do not cause “epitope suppression.” Mincle ligands thus hold great potential as a platform for the development of new vaccine carriers with self-adjuvanting properties for cancer treatment. Preliminary structure–activity relationship analysis shows that a vaccine containing one STn antigen carried by vizantin exhibits the best efficacy, providing support for further optimization and additional investigation into Mincle agonists as the carrier of self-adjuvanting cancer vaccines.

A new strategy based on a Macrophage-inducible C-type lectin (Mincle) agonist was established to construct synthetic cancer vaccines.     

Synthesis and immunological study of a wall teichoic acid-based vaccine against E. faecium U0317

ABSTRACT

A repeat unit of cell wall teichoic acids (WTA) isolated from E. faecium U0317 was chemically synthesized efficiently by a stepwise strategy. It was derivatized with a 5-aminopentanyl linker to facilitate conjugation with carrier proteins KLH and HSA. Immunological studies of the KLH conjugate 1 demonstrated that it could provoke robust immune responses and high titers of IgG antibodies, which could successfully recognize the synthesized WTA repeat unit 3. This result suggested that synthetic glycoconjugate 1 could be a promising vaccine candidate against E. faecium for further studies.     

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam₃CSK₄, α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.     

Computer-Aided Discovery of Potent Broad-Spectrum Vaccine Adjuvants

Adjuvants stimulate the immune system to vigorously respond to a vaccine. While current adjuvants such as aluminum salts and oil-in-water emulsions have been used for decades, they do not generate broad and long-lasting responses in many vaccines. Consequently, more potent adjuvants are needed. Here, using computer-aided molecule design and machine learning, we discovered 2 new, broad-spectrum adjuvants that can boost vaccine responses. Our library containing 46 toll-like receptor (TLR)-targeting agonist ligands were assembled on Au nanoparticles. Comprehensive in vitro, ex vivo and in vivo studies showed both leads promoted dendritic cell activation via multiple TLRs and enhanced antigen presentation to T cells. When used together with tumor-specific antigens to immunize mice against B16-OVA melanoma and 4T1-PD1 breast cancer, both adjuvants unleashed strong immune responses that suppressed tumor growth and lung metastases. Our results show computer-aided design and screening can rapidly uncover potent adjuvants for tackling waning immunity in current vaccines.     

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with ��2-adrenergic agonist

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that β₂-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since β₂-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced T_h2-biased immunity against HSV antigen, as evidenced by IgG isotypes and T_h1/T_h2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.     

Study of intact virus‐like particles of human papillomavirus by capillary electrophoresis

Virus‐like particles of human papillomavirus (HPV‐VLP), resulting from the self‐assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil^® and Cervarix^®, are based on HPV‐VLP L1. Analytical techniques currently used to characterize HPV‐VLP, such as SDS‐PAGE, Western blot, ELISA, are time‐consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16‐VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV‐VLP analysis in CE was achieved using poly(ethylene oxide)‐coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV‐VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV‐VLP peak was confirmed by comparison with a sample obtained from a wild‐type baculovirus and with VLP‐based vaccine, Gardasil^®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP‐based vaccines, demonstrating that CE could be successfully used for vaccine quality control.     

Using random forest to classify T-cell epitopes based on amino acid properties and molecular features

T-lymphocyte (T-cell) is a very important component in human immune system. T-cell epitopes can be used for the accurately monitoring the immune responses which activation by major histocompatibility complex (MHC), and rationally designing vaccines. Therefore, accurate prediction of T-cell epitopes is crucial for vaccine development and clinical immunology. In current study, two types peptide features, i.e., amino acid properties and chemical molecular features were used for the T-cell epitopes peptide representation. Based on these features, random forest (RF) algorithm, a powerful machine learning algorithm, was used to classify T-cell epitopes and non-T-cell epitopes. The classification accuracy, sensitivity, specificity, Matthews correlation coefficient (MCC), and area under the curve (AUC) values for proposed method are 97.54%, 97.22%, 97.60%, 0.9193, and 0.9868, respectively. These results indicate that current method based on the combined features and RF is effective for T-cell epitopes prediction.     

Injectable Biodegradable Chitosan‐Alginate 3D Porous Gel Scaffold for mRNA Vaccine Delivery

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide‐based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low‐transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan‐alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold‐mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex‐loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN‐γ secretion are seen with in vivo scaffold‐mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold‐mediated mRNA lipoplex delivery, while protein‐based immunization did not elicit IgG production until 2 weeks post‐injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.     

Novel multi-epitope protein containing conserved epitopes from different Leishmania species as potential vaccine candidate: Integrated immunoinformatics and molecular dynamics approach

Leishmaniosis, caused by intracellular parasites of the genus Leishmania, has become a serious public health problem around the world, and for which there are currently extensive limitations. In this work, a theoretical model was proposed for the development of a multi-epitope vaccine. The protein GP63 of the parasite was selected for epitopes prediction, due to its important biological role for the infection process and abundance. IEDB tools were used to determine epitopes B and T in Leishmania braziliensis; besides, other conserved epitopes in three species were selected. To improve immunogenicity, 50S ribosomal protein L7 / L12 (ID: P9WHE3) was used as a domain of adjuvant in the assembly process. The folding arrangement of the vaccine was obtained through homologous modeling multi-template with MODELLER v9.21, and a Ramachandran plot analysis was done. Furthermore, physicochemical properties were described with the ProtParam tool and secondary structure prediction combining GOR-IV and SOPMA tools. Finally, a molecular dynamics simulation (50 ns) was performed to establish flexibility and conformational changes. The analysis of the results indicates high conservancy in the epitopes predicted among the four species. Moreover, Ramachandran plot, physicochemical parameters, and secondary structure prediction suggest a stable conformation of the vaccine, after a minimum conformational change that was evaluated with the free energy landscape. The conformational change does not drive any substantial change for epitope exposition on the surface. The vaccine proposed could be tested experimentally to guide new approaches in the development of pan-vaccines; vaccines with regions conserved in multiple species.     

Structure‐based redesign of proteins for minimal T‐cell epitope content

The protein universe displays a wealth of therapeutically relevant activities, but T‐cell driven immune responses to non‐“self” biological agents present a major impediment to harnessing the full diversity of these molecular functions. Mutagenic T‐cell epitope deletion seeks to mitigate the immune response, but can typically address only a small number of epitopes. Here, we pursue a “bottom‐up” approach that redesigns an entire protein to remain native‐like but contain few if any immunogenic epitopes. We do so by extending the Rosetta flexible‐backbone protein design software with an epitope scoring mechanism and appropriate constraints. The method is benchmarked with a diverse panel of proteins and applied to three targets of therapeutic interest. We show that the deimmunized designs indeed have minimal predicted epitope content and are native‐like in terms of various quality measures, and moreover that they display levels of native sequence recovery comparable to those of non‐deimmunized designs. © 2013 Wiley Periodicals, Inc.     

Synthesis of LeX and LeY Oligosaccharides with Azido-Type Spacer-Arms. Comparison of 3- and 4-Methoxybenzyl Groups as Key Temporary Protective Groups

Abstract

5-Azido-3-oxa-l-pentanol was prepared from 2-(2-chloroethoxy)ethanol and used as a spacer in the chemical synthesis of the trisaccharide β-D-Gal-(1→4)-[α-L-Fuc-(1→3)]-GlcNAc and the tetrasaccharide α-L-Fuc-α-(1→2)-β-D-Gal-(1→4)-[α-L-Fuc-(1→3)]-GlcNAc that represent the epitopes defining the human blood groups Le^x and Le^y. The classical 4-methoxybenzyl group and the remarably acid-stable 3-methoxybenzyl group were compared as temporary protective groups for position 3 at the glucosamine unit to circumvent the problems associated with the simultaneous presence of allyl and azido groups. The resulting oligosaccharides were coupled to proteins with high efficiency.

     

Synthesis and Characterisation of Self‐Assembled and Self‐Adjuvanting Asymmetric Multi‐Epitope Lipopeptides of Ovalbumin

Designing a lipopeptide (LP) vaccine with a specific asymmetric arrangement of epitopes may result in an improved display of antigens, increasing host‐cell recognition and immunogenicity. This study aimed to synthesise and characterise the physicochemical properties of a library of asymmetric LP‐based vaccine candidates that contained multiple CD4⁺ and CD8⁺ T‐cell epitopes from the model protein antigen, ovalbumin. These fully synthetic vaccine candidates were prepared by microwave‐assisted solid phase peptide synthesis. The C12 or C16 lipoamino acids were coupled to the N or C terminus of the OVA CD4 peptide epitope. The OVA CD4 LPs and OVA CD8 peptide constructs were then conjugated using azide–alkyne Huisgen cycloaddition to give multivalent synthetic vaccines. Physiochemical characterisation of these vaccines showed a tendency to self‐assemble in aqueous media. Changes in lipid length and position induced self‐assembly with significant changes to their morphology and secondary structure as shown by transmission electron microscopy and circular dichroism.     

An Economical Synthesis of Lewis X,Sialyl Lewis X and Their α-Galactosyl Analogues

Abstract

Economical syntheses of the Lewis X trisaccharide 8 and sialyl Lewis X tetrasaccharide 18 epitopes and the syntheses of the α-galactosyl epimers 9 and 20 of these structures are described. Thioglycosides 2, 5, 11 and 15 were used as glycosyl donors to construct the desired compounds in a stepwise manner in dimethyl(methylthio)sulphonium triflate promoted couplings. Benzyl 3-O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-2-acetamido-6-O-benzyl-2-deoxy-α-D-glucopyranoside (4) was a key structure in these syntheses, and was synthesised in multi-gram scale.