ABSTRACT
In recent years, the field of antibody drug conjugates (ADC) has seen a resurgence, largely driven by the clinical benefit observed in patients treated with ADCs incorporating camptothecin-based topoisomerase I inhibitor payloads. Herein, we present the development of a novel camptothecin ZD06519 (FD1), which has been specifically designed for its application as an ADC payload. A panel of camptothecin analogs with different substituents at the C-7 and C-10 positions of the camptothecin core was prepared and tested in vitro. Selected compounds spanning a range of potency and hydrophilicity were elaborated into drug-linkers, conjugated to trastuzumab, and evaluated in vitro and in vivo. ZD06519 was selected on the basis of its favorable properties as a free molecule and as an antibody conjugate, which include moderate free payload potency (â¼1 nmol/L), low hydrophobicity, strong bystander activity, robust plasma stability, and high-monomeric ADC content. When conjugated to different antibodies using a clinically validated MC-GGFG-based linker, ZD06519 demonstrated impressive efficacy in multiple cell line-derived xenograft models and noteworthy tolerability in healthy mice, rats, and non-human primates.
Subject(s)
Camptothecin , Immunoconjugates , Xenograft Model Antitumor Assays , Camptothecin/pharmacology , Camptothecin/chemistry , Immunoconjugates/pharmacology , Immunoconjugates/chemistry , Animals , Humans , Mice , Cell Line, Tumor , Drug Design , Female , RatsABSTRACT
Synthetic analogs based on the DNA bis-intercalating natural product peptides sandramycin and quinaldopeptin were investigated as antibody drug conjugate (ADC) payloads. Synthesis, biophysical characterization, and in vitro potency of 34 new analogs are described. Conjugation of an initial drug-linker derived from a novel bis-intercalating peptide produced an ADC that was hydrophobic and prone to aggregation. Two strategies were employed to improve ADC physiochemical properties: addition of a solubilizing group in the linker and the use of an enzymatically cleavable hydrophilic mask on the payload itself. All ADCs showed potent in vitro cytotoxicity in high antigen expressing cells; however, masked ADCs were less potent than payload matched unmasked ADCs in lower antigen expressing cell lines. Two pilot in vivo studies were conducted using stochastically conjugated DAR4 anti-FRα ADCs, which showed toxicity even at low doses, and site-specific conjugated (THIOMAB) DAR2 anti-cMet ADCs that were well tolerated and highly efficacious.
Subject(s)
Antineoplastic Agents , Immunoconjugates , Immunoconjugates/pharmacology , Immunoconjugates/chemistry , Peptides , Structure-Activity Relationship , Antigens , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistryABSTRACT
Despite a prevailing dogma wherein antibody drug conjugates (ADCs) increase the maximum tolerated dose of potent cytotoxin payloads while lowering the minimum effective dose, mounting clinical evidence argues that the tolerated doses of ADCs are not significantly different from those of related small molecules. Nonetheless, when dosed at or near the maximum tolerated dose, certain ADCs demonstrate improved efficacy. Understanding the challenges and opportunities for this class of biotherapeutics will help improve the design of next-generation ADCs.
Subject(s)
Antineoplastic Agents , Immunoconjugates , Humans , Immunoconjugates/therapeutic use , Antineoplastic Agents/therapeutic useABSTRACT
The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP-sialic acid sugar donors bearing fluorine atoms at the 7-position, starting from the corresponding 4-deoxy-4-fluoro-N-acetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7-fluorosialosides toward spontaneous hydrolysis (3- to 5-fold) and toward cleavage by GH33 sialidases (40- to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the N-glycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory half-lives of glycoproteins and may pave the way toward biobetters for therapeutic use.
ABSTRACT
BACKGROUND: Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting. OBJECTIVE: To investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy. DESIGN, SETTING, AND PARTICIPANTS: An ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC-treated MIBC patients. INTERVENTION: An rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Antineoplastic effects of targeting ofCS. RESULTS AND LIMITATIONS: In situ, ofCS was significantly overexpressed in residual tumors after NAC in two independent patient cohorts (p<0.02). Global gene-expression profiling and biochemical analysis of primary tumors and cell lines revealed syndican-1 and chondroitin sulfate proteoglycan 4 as ofCS-modified proteoglycans in MIBC. In vitro, ofCS was expressed on all MIBC cell lines tested, and VDC886 eliminated these cells in the low-nanomolar IC50 concentration range. In vivo, VDC886 effectively retarded growth of chemoresistant orthotopic bladder cancer xenografts and prolonged survival (p=0.005). The use of cisplatin only for the generation of chemoresistant xenografts are limitations of our animal model design. CONCLUSIONS: Targeting ofCS provides a promising second-line treatment strategy in cisplatin-resistant MIBC. PATIENT SUMMARY: Cisplatin-resistant bladder cancer overexpresses particular sugar chains compared with chemotherapy-naïve bladder cancer. Using a recombinant protein from the malaria parasite Plasmodium falciparum, we can target these sugar chains, and our results showed a significant antitumor effect in cisplatin-resistant bladder cancer. This novel treatment paradigm provides therapeutic access to bladder cancers not responding to cisplatin.
Subject(s)
Antigens, Protozoan/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/metabolism , Chondroitin Sulfates/metabolism , Cisplatin/therapeutic use , Drug Resistance, Neoplasm/drug effects , Oligopeptides/pharmacology , Urinary Bladder Neoplasms/drug therapy , Animals , Antigens, Protozoan/metabolism , Antineoplastic Agents/adverse effects , British Columbia , Cell Death/drug effects , Cell Line, Tumor , Cisplatin/adverse effects , Dose-Response Relationship, Drug , Europe , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitory Concentration 50 , Kaplan-Meier Estimate , Mice , Time Factors , Treatment Outcome , Tumor Burden/drug effects , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/mortality , Urinary Bladder Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.
Subject(s)
Antigens, Neoplasm/metabolism , Burkitt Lymphoma/metabolism , Chondroitin Sulfates/metabolism , Malaria, Falciparum/metabolism , Placenta/metabolism , Placenta/parasitology , Adolescent , Antibodies, Protozoan/blood , Antigens, Protozoan/immunology , Burkitt Lymphoma/parasitology , Cell Line, Tumor , Child , Child, Preschool , Erythrocytes/parasitology , Female , Humans , Immunoglobulin G/metabolism , Malaria, Falciparum/complications , Male , Plasmodium falciparum/immunology , Pregnancy , Proteoglycans/metabolism , Recombinant Proteins/metabolismABSTRACT
Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.
Subject(s)
Antigens, Protozoan/genetics , Chondroitin Sulfates/metabolism , Melanoma, Experimental/therapy , Placenta/metabolism , Recombinant Proteins/administration & dosage , Skin Neoplasms/therapy , Animals , Antigens, Protozoan/metabolism , Cell Line, Tumor , Chondroitin Sulfate Proteoglycans/metabolism , Female , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Hyaluronan Receptors/metabolism , Melanoma, Experimental/metabolism , Membrane Proteins/metabolism , Mice , Molecular Targeted Therapy , Oligopeptides/genetics , Oligopeptides/metabolism , Organ Specificity , Pregnancy , Recombinant Proteins/pharmacology , Skin Neoplasms/metabolismABSTRACT
Sphingolipid ceramide N-deacylase (SCDase) catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized. While SCDases show great value for the enzymatic synthesis of glycosphingolipids, they are relatively poorly characterized enzymes. In this work, the enzymatic properties of SCDase from Shewanella alga G8 (SA_SCD) were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp. TK4 (PS_SCD). The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5, respectively. Both activities were strongly inhibited by Zn(2+) and Cu(2+), while Fe(2+), Co(2+), Ni(2+), Mn(2+), Ca(2+), and Mg(2+) promoted the hydrolytic activity but inhibited the synthetic activity. SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis. Importantly, SA_SCD has a broader specificity for acyl donor acceptance than does PS_SCD, especially for unsaturated fatty acids and fatty acids with very short or long acyl chains. Further kinetic analysis revealed that the k cat/K M value for the hydrolytic activity of SA_SCD was 8.9-fold higher than that of PS_SCD for GM1a, while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1a (d18:1) than those of PS_SCD, respectively. The broad fatty acid specificity and high catalytic efficiency, together with the ease of expression of SA_SCD in Escherichia coli, make it a better biocatalyst than is PS_SCD for the synthesis and structural remodeling of glycosphingolipids.
Subject(s)
Amidohydrolases/metabolism , Fatty Acids/metabolism , Glycosphingolipids/metabolism , Pseudomonas/enzymology , Shewanella/enzymology , Amidohydrolases/chemistry , Amidohydrolases/genetics , Cations, Divalent/metabolism , Enzyme Activators/metabolism , Enzyme Inhibitors/metabolism , Enzyme Stability , Escherichia coli/genetics , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Metals/metabolism , Protein Binding , Shewanella/genetics , Substrate SpecificityABSTRACT
Glycans bearing modified hydroxyl groups are common in biology but because these modifications are added after assembly, enzymes are not available for the transfer and coupling of hydroxyl-modified monosaccharide units. Access to such enzymes could be valuable, particularly if they can also introduce 'bio-orthogonal tags'. Glycosynthases, mutant glycosidases that synthesize glycosides using glycosyl fluoride donors, are a promising starting point for creation of such enzymes through directed evolution. Inspection of the active site of a homology model of the GH1 Agrobacterium sp. ß-glycosidase, which has both glucosidase and galactosidase activity, identified Q24, H125, W126, W404, E411 and W412 as amino acids that constrain binding around the 3-OH group, suggesting these residues as targets for mutation to generate an enzyme capable of handling 3-O-methylated sugars. Site-directed saturation mutagenesis at these positions within the wild-type ß-glycosidase gene and screening via an on-plate assay yielded two mutants (Q24S/W404L and Q24N/W404N) with an improved ability to hydrolyze 4-nitrophenyl 3-O-methyl-ß-D-galactopyranoside (3-MeOGal-pNP). Translation of these mutations into the evolved glycosynthase derived from the same glucosidase (2F6) yielded glycosynthases (AbgSL-T and AbgNN-T, where T denotes transferase) capable of forming 3-O-methylated glucosides on multi-milligram scales at rates approximately 5 and 40 times greater, respectively, than the parent glycosynthase.
Subject(s)
3-O-Methylglucose/metabolism , Agrobacterium/enzymology , Directed Molecular Evolution/methods , Mutagenesis, Site-Directed/methods , beta-Glucosidase/genetics , beta-Glucosidase/metabolism , Agrobacterium/chemistry , Agrobacterium/genetics , Agrobacterium/metabolism , Catalytic Domain , Models, Molecular , Mutation , Substrate Specificity , beta-Glucosidase/chemistryABSTRACT
An LLG-3 oligosaccharide-fluoride can be assembled chemoenzymatically and readily coupled with various sphingosines by an engineered endoglycoceramidase glycosynthase. The lyso-ganglioside products are acylated to generate the individual isomers identified in the heterogeneous natural isolates, as well as modified glycosphingolipids.
Subject(s)
Gangliosides/chemical synthesis , Glycoside Hydrolases/chemistry , Neuroprotective Agents/chemical synthesis , Animals , Carbohydrate Sequence , Gangliosides/chemistry , Gangliosides/pharmacology , Glycoside Hydrolases/metabolism , Molecular Sequence Data , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacology , Sialic Acids/chemistry , Starfish , StereoisomerismABSTRACT
Glycosynthase mutants of Rhodococcus sp. endo-glycoceramidase II efficiently synthesize complex glycosphingolipids. Glycosyl fluoride donors may be assembled via sequential glycosyltransferase-catalysed glycosylation of lactosyl fluoride. Alternatively, lactosyl fluoride may be coupled to sphingosine prior to subsequent glycosylation steps.
Subject(s)
Glycoside Hydrolases/metabolism , Glycosphingolipids/biosynthesis , Glycosyltransferases/metabolism , Glycoside Hydrolases/genetics , Glycosphingolipids/chemistry , Glycosylation , Glycosyltransferases/genetics , Lactose/analogs & derivatives , Lactose/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/enzymologyABSTRACT
Sialic acids play important roles in various biological processes and typically terminate the oligosaccharide chains on the cell surfaces of a wide range of organisms, including mammals and bacteria. Their attachment is catalyzed by a set of sialyltransferases with defined specificities both for their acceptor sugars and the position of attachment. However, little is known of how this specificity is encoded. The structure of the bifunctional sialyltransferase Cst-II of the human pathogen Campylobacter jejuni in complex with CMP and the terminal trisaccharide of its natural acceptor (Neu5Ac-α-2,3-Gal-ß-1,3-GalNAc) has been solved at 1.95 Å resolution, and its kinetic mechanism was shown to be iso-ordered Bi Bi, consistent with its dual acceptor substrate specificity. The trisaccharide acceptor is seen to bind to the active site of Cst-II through interactions primarily mediated by Asn-51, Tyr-81, and Arg-129. Kinetic and structural analyses of mutants modified at these positions indicate that these residues are critical for acceptor binding and catalysis, thereby providing significant new insight into the kinetic and catalytic mechanism, and acceptor specificity of this pathogen-encoded bifunctional GT-42 sialyltransferase.
Subject(s)
Bacterial Proteins/chemistry , Campylobacter jejuni/enzymology , Cytidine Monophosphate/chemistry , Sialyltransferases/chemistry , Trisaccharides/chemistry , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Campylobacter jejuni/genetics , Catalysis , Crystallography, X-Ray , Cytidine Monophosphate/genetics , Cytidine Monophosphate/metabolism , Humans , Kinetics , Mutation, Missense , Protein Structure, Tertiary , Sialyltransferases/genetics , Sialyltransferases/metabolism , Structure-Activity Relationship , Trisaccharides/genetics , Trisaccharides/metabolismABSTRACT
Glycosyltransferases (GTs) offer very attractive approaches to the synthesis of complex oligosaccharides. However, the limited number of available GTs, together with their instability and strict substrate specificity, have severely hampered the broad application of these enzymes. Previous attempts to broaden the range of substrate scope and to increase the activity of GTs via protein engineering have met with limited success, partially because of the lack of effective high-throughput screening methods. Recently, we reported an ultra-high-throughput screening method for sialyltransferases based on fluorescence-activated cell sorting (Aharoni et al. Nat. Methods 2006, 3, 609-614). Here, we considerably improve this method via the introduction of a two-color screening protocol to minimize the probability of false positive mutants and demonstrate its generality through directed evolution of a neutral sugar transferase, beta-1,3-galactosyltransferase CgtB. A variant with broader substrate tolerance than the wild-type enzyme and 300-fold higher activity was identified rapidly from a library of >10(7) CgtB mutants. Importantly, the variant effected much more efficient synthesis of G(M1a) and asialo G(M1) oligosaccharides, the building blocks of important therapeutic glycosphingolipids, than did the parent enzyme. This work not only establishes a new methodology for the directed evolution of galactosyltransferases, but also suggests a powerful strategy for the screening of almost all GT activities, thereby facilitating the engineering of glycosyltransferases.
Subject(s)
Campylobacter jejuni/enzymology , Flow Cytometry/methods , Glycosyltransferases/genetics , High-Throughput Screening Assays/methods , Mutation , Escherichia coli/genetics , Gene Expression , Glycosyltransferases/isolation & purification , Glycosyltransferases/metabolismABSTRACT
Mammalian cell surfaces are modified by complex arrays of glycoproteins, glycolipids and polysaccharides, many of which terminate in sialic acid and have central roles in essential processes including cell recognition, adhesion and immunogenicity. Sialylation of glycoconjugates is performed by a set of sequence-related enzymes known as sialyltransferases (STs). Here we present the crystal structure of a mammalian ST, porcine ST3Gal-I, providing a structural basis for understanding the mechanism and specificity of these enzymes and for the design of selective inhibitors.
Subject(s)
Sialyltransferases/chemistry , Animals , Catalytic Domain , Crystallography, X-Ray , Humans , Models, Molecular , N-Acetylneuraminic Acid/metabolism , Protein Structure, Secondary , Sialyltransferases/genetics , Sialyltransferases/metabolism , Substrate Specificity , Swine , beta-Galactoside alpha-2,3-SialyltransferaseABSTRACT
Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immune diseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in their synthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screening strategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, and we identified several mutants with enhanced activities. These new catalysts, which are capable of producing a broad range of homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids and demonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.
Subject(s)
Directed Molecular Evolution , Glycoside Hydrolases/chemistry , Glycosphingolipids/chemical synthesis , Catalysis , Enzyme-Linked Immunosorbent Assay , Glycoside Hydrolases/genetics , Glycosphingolipids/chemistry , Point Mutation , Rhodococcus/enzymology , Substrate SpecificityABSTRACT
Most circulating human proteins exist as heterogeneously glycosylated variants (glycoforms) of an otherwise homogeneous polypeptide. Though glycan heterogeneity is most likely important to glycoprotein function, the preparation of homogeneous glycoforms is important both for the study of the consequences of glycosylation and for therapeutic purposes. This review details selected approaches to the production of homogeneous human N- and O-linked glycoproteins with human-type glycans. Particular emphasis is placed on recent developments in the engineering of glycosylation pathways within yeast and bacteria for in vivo production, and on the in vitro remodeling of glycoproteins by enzymatic means. The future of this field is very exciting.
Subject(s)
Genetic Engineering , Glycoproteins/chemical synthesis , Glycoproteins/metabolism , Bacteria/genetics , Gene Expression Regulation , Glycoproteins/genetics , Humans , Saccharomyces cerevisiae/geneticsABSTRACT
Analogues of the tumor-associated gangliosides GM(3) and GM(2) containing terminal S-linked neuraminic acid residues and an amino terminated, truncated ceramide homologue have been synthesized and conjugated to a protein. The synthesis involved coupling of a S-linked sialyl alpha(2-->3) galactose disaccharide with a glucosyl sphingosine analogue, followed by elaboration and deprotection to give amino-terminated glycosyl ceramide 1. Glycosyltransferase-catalyzed extension of the trisaccharide 1 provided access to the modified GM(2) tetrasaccharide 2 or sulphur-containing GD(3) analogue 30. Owing to their potentially enhanced resistance to endogenous exo-glycoside hydrolases and their inherent non-self character, carbohydrate antigens containing non-reducing terminal thioglycosidic linkages may be more immunogenic than O-linked antigens and may stimulate the production of antibodies capable of recognizing naturally occurring oligosaccharides. Our initial results suggest that in fact these antigens are viable immunogens and furthermore, that immune sera cross reacts with O-gangliosides in the context of a heterologous glycoprotein conjugate.
Subject(s)
Antigens, Neoplasm/chemistry , Carbohydrates/chemistry , G(M3) Ganglioside/chemistry , Glycosyltransferases/chemistry , Neuraminic Acids/chemistry , Vaccines, Synthetic/biosynthesis , Animals , Mice , Mice, Inbred BALB C , Proteins/chemistry , Vaccines, Synthetic/chemistryABSTRACT
Analogues of GM3 and GM2 gangliosides were chemoenzymatically synthesized on a multifunctional ceramide-type tether designed to facilitate diverse strategies for glycoconjugate synthesis. The truncated ceramide aglycon maintains the stereogenic centres of natural ceramide while avoiding extensive hydrophobicity that can hamper synthesis and purification of the glycolipids. Tetanus toxoid and BSA glycoconjugates of these two gangliosides were prepared for immunization of mice, and for solid phase assays to screen for ganglioside-specific antibodies. Inhibition experiments showed that antibodies generated by tetanus toxoid conjugates of GM3 and GM2 exhibited specificity for the carbohydrate epitope and the stereogenic centres of the ceramide.
Subject(s)
Ceramides/chemistry , G(M2) Ganglioside/chemical synthesis , G(M3) Ganglioside/chemical synthesis , Glycoconjugates/chemical synthesis , Animals , Antibodies/analysis , Ceramides/immunology , Combinatorial Chemistry Techniques , Epitopes , Glycoconjugates/immunology , Immunization , Mice , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/immunology , Serum Albumin, Bovine/therapeutic use , Tetanus Toxoid/chemistry , Tetanus Toxoid/immunology , Tetanus Toxoid/therapeutic useSubject(s)
Antibody Specificity , Antigens, Bacterial/immunology , Antigens, Neoplasm/immunology , Oligosaccharides/immunology , Sulfur Compounds/immunology , Vaccines, Conjugate/immunology , Antigens, Bacterial/chemistry , Antigens, Fungal/chemistry , Antigens, Fungal/immunology , Antigens, Neoplasm/chemistry , Carbohydrate Sequence , Molecular Sequence Data , Oligosaccharides/chemistry , Sulfur Compounds/chemistry , Vaccines, Conjugate/chemistryABSTRACT
A disaccharide portion of the A-side chain of the rhamnogalacturonan II oligosaccharide has been prepared. Glycosylation of methyl (methyl 3,4-O-isopropylidene-alpha-D-galactopyranosid)uronate with p-tolyl 2,3-di-O-acetyl-3-C-(benzyloxymethyl)-1-thio-alpha/beta-D-erythrofuranoside was carried out using N-iodosuccinimide as promoter and silver trifluoromethanesulfonate as catalyst. Removal of the protecting groups gave the beta-d-Apif-(1-->2)-alpha-D-GalpA-OMe disaccharide.