Glycoconjugates and methods for their use
10918732 ยท 2021-02-16
Assignee
Inventors
- Igor C. Almeida (El Paso, TX, US)
- Katja Michael (El Paso, TX, US)
- Nathaniel SCHOCKER (El Paso, TX, US)
- Susana Portillo (El Paso, TX, US)
- Rosa Maldonado (El Paso, TX, US)
Cpc classification
A61K47/643
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K47/646
HUMAN NECESSITIES
International classification
A61K47/64
HUMAN NECESSITIES
Abstract
Certain embodiments are directed to method for synthesizing and using glycoconjugates based on the immunodominant epitope Gal(1,3)Gal(1,4)GlcNAc (Gal3LN).
Claims
1. A neoglycoconjugate comprising a trisaccharide coupled to a carrier, wherein the trisaccharide is Gal(1,3)Gal(1,4)Glc, Gal(1,2)Gal(1,4)GlcNAc, Gal(1,2)Gal(1,4)Glc, or Gal(1,2)Gal(1,4)Glc.
2. The neoglycoconjugate of claim 1, further comprising a linker connecting the trisaccharide to the carrier.
3. The neoglycoconjugate of claim 1 wherein the carrier is a protein carrier.
4. The neoglycoconjugate of claim 3, wherein the protein carrier is bovine serum albumin.
5. The neoglycoconjugate of claim 1, wherein the carrier is peptide.
6. The neoglycoconjugate of claim 5, wherein the peptide is a T cell epitope.
7. The neoglycoconjugate of claim 1, wherein the trisaccharide comprises a terminal galactose residue.
8. A method of detecting a parasite comprising: (a) contacting a blood sample from a subject with the neoglycoconjugate of claim 1, wherein the neoglycoconjugate forms a complex with antibodies in the blood sample that bind a glycan having a terminal Gal; and (b) detecting the formation of an antibody-neoglycoconjugate complex.
9. The method of claim 8, wherein the subject is suspected of having Chagas disease, cutaneous and visceral leishmaniasis, or malaria.
10. A method for inducing an immune response against T. cruzi in a human comprising administering the neoglycoconjugate of claim 1, wherein an immune response is generated against a T. cruzi.
11. The method of claim 10, wherein the carrier is bovine serum albumin.
Description
DESCRIPTION OF THE DRAWINGS
(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.
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DESCRIPTION
(12) The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease, has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Gal(1,3)Gal(1,4)GlcNAc, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti--Gal antibodies in infected individuals. Embodiments described herein are directed to efficiently synthesizing the mercaptopropyl glycoside of a glycotope and conjugating the glycotope to a maleimide-derivatized carrier protein, such as but not limited to bovine serum albumin (BSA). Chemiluminescent-ELISA revealed that Gal(1,3)Gal(1,4)GlcNAc-BSA is recognized by purified anti--Gal antibodies from chronic Chagas disease patients 230-fold more strongly than by anti--Gal antibodies from sera of healthy individuals (NHS anti-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Gal(1,3)Gal(1,4)GlcNAc approximately 20-fold more strongly than pooled normal healthy serum (NHS). In contrast, the underlying disaccharide Gal(1,4)GlcNAc, and the monosaccharide GlcNAc or GlcNAc conjugated to BSA are poorly or not recognized by purified anti--Gal antibodies or sera from Chagasic patients or healthy individuals. These results highlight the importance of the terminal Gal moiety for recognition by Ch anti--Gal antibodies and the lack of antibodies against non-self Gal(1,4)GlcNAc and GlcNAc glycotopes.
(13) The substantial difference in binding of Ch vs. NHS anti--Gal antibodies to Gal(1,3)Gal(1,4)GlcNAc-BSA suggests that this neoglycoprotein is suitable for vaccine development. To this end, the Gal(1,3)Gal(1,4)GlcNAc-BSA neoglycoprotein was used to immunize 1,3-galactosyltransferase-knockout (1,3GalT-KO) mice, which produced antibody titers 40-fold higher as compared to pre-immunization titers. The synthetic Gal(1,3)Gal(1,4)GlcNAc and other linear or branched Gal-containing glycotopes (and embodiments thereof) coupled to a carrier protein or peptide could be used as diagnostic or prognostic (i.e., chemotherapy follow-up) biomarkers, or vaccine candidates for parasitic diseases, such as Chagas disease. These neoglycoconjugates can be employed for applications in visceral, cutaneous and mucocutaneous leishmaniasis; malaria; African trypanosomiasis; and hookworm and tapeworm infections.
(14) Glycoconjugates (glycolipids and glycoproteins) are major antigens on the surface of T. cruzi and contain highly immunogenic epitopes. The antigens remain largely unexplored as vaccine targets. Certain embodiments described herein are directed to glycoconjugate antigens from or mimicking T. cruzi glycoconjugate(s). The immunogenic epitopes contained in the glycoconjugate antigens of T. cruzi are reproduced and used as an immunogen. As described herein a mouse model that mimics a human response to T. cruzi infection or exposure was used to characterize the glycoconjugates described herein. In certain aspects, the glycoconjugate antigens and immunogenic epitopes can be administered as a vaccine. In certain aspects the vaccine is validated in a mouse model, as described herein. In still further aspects, a glycoconjugate-based vaccine can be used to induce a long-lasting, full-protection against T. cruzi.
(15) The immunodominant glycotope (glycan epitope), Gal(1,3)Gal(1,4)GlcNAc, is abundantly expressed in the mammal-dwelling T. cruzi trypomastigote stage (Almeida et al. 1994) and is not expressed on human cells, thus it is highly immunogenic to humans (Macher and Galili 2008, Travassos and Almeida 1993). The Gal(1,3)Gal(1,4)GlcNAc epitope contains a terminal, non-reducing Gal residue, which is highly conserved on trypomastigote-derived GPI-mucins (tGPI-mucins) of at least four major T. cruzi genotypes causing ChD in humans: TcI, TcII, TcV, and TcVI (Almeida et al. 1993, Izquierdo et al. 2013, Soares et al. 2012, Travassos and Almeida 1993).
(16) The Gal(1,3)Gal(1,4)GlcNAc glycotope contains the disaccharide Gal1,3Gal, which is strongly recognized by Chagasic (Ch) anti--Gal Abs and to a much lesser extent by the natural anti--Gal Abs from healthy individuals (NHS anti--Gal) (Almeida et al. 1994, Ashmus et al. 2013), which are produced mainly against Gram-negative enterobacteria of the human flora (Galili et al. 1999). These enterobacteria (e.g., E. coli, Enterobacter spp., Serratia spp., Salmonella spp., Shigella spp., Klebsiella spp., and Citrobacter spp.) have various types of non-reducing, terminal -Gal-linked glycans, mostly Gal1,2-R, Gal1,4-R, and Gal1,6-R (where R is the remaining side chain or core glycan) on the lipopolysaccharide (LPS) core oligosaccharides or O-antigens (Wilkinson 1996). The glycotope Gal(1,3)Gal(1,4)GlcNAc, so far not reported in enterobacteria, and most likely other as-yet unidentified T. cruzi-specific cell surface saccharides with terminal Gal moieties, induce the major lytic, protective antibodies (Ch anti--Gal Abs) produced during both the acute and chronic stages of ChD (Almeida et al. 1994; Almeida et al. 1991; Avila et al. 1989; Gazzinelli et al. 1991; Milani and Travassos 1988; Travassos and Almeida 1993). These studies strongly indicate that lytic Ch anti--Gal Abs could be one of the main immunological mechanisms for controlling the parasitemia in both stages of the disease in humans. Thus, Gal(1,3)Gal(1,4)GlcNAc offers a potential route toward a carbohydrate-based vaccine against Chagas disease. Glycoconjugates are still unexplored as vaccine targets in T. cruzi, although these molecules are the most abundant and immunogenic antigens on the plasma membrane of all T. cruzi developmental stages (Acosta-Serrano et al. 2007; Buscaglia et al. 2004; Frasch 2000).
(17) Embodiments described herein are directed to synthesizing glycosides of Gal(1,3)Gal(1,4)GlcNAc, and its truncated versions Gal(1,4)GlcNAc and GlcNAc, as well as its diastereomer GlcNAc, all equipped with a thiol functionality (glycosides 1-4,
I. GLYCOSIDE AND CONJUGATE SYNTHESIS
(18) The production of the trisaccharide Gal(1,3)Gal(1,4)GlcNAc and related analogs has been previously accomplished for a variety of uses, and mostly involves chemoenzymatic syntheses (Brinkmann et al. 2001; Fang et al. 1998; Qian et al. 1999; Vic et al. 1997), which are often efficient. However, some research groups prefer its chemical synthesis due to reagent availability, scalability, and derivatization options. For example, -Gal trisaccharides have been chemically synthesized and coupled to Sepharose (Dahmn et al. 2002), attached to a lipid for non-covalent association to target molecules (Litjens et al. 2005), or attached to linkers such as p-nitrophenol esters (Plaza-Alexander and Lowary 2013) and 3-aminopropyl groups (Hanessian et al. 2001; Wang et al. 2005) to allow for further functionalization.
(19) Four features of the methods described herein for synthesis of an Gal(1,3)Gal(1,4)GlcNAc-containing NGP are: (i) predominant use of acyl protecting groups that can be easily installed and cleanly removed; (ii) utilization of 4,6-di-tertbutylsilyl protected galactosyl donor (Imamura et al. 2006) to ensure a stereoselective -galactosylation; (iii) utilization of easily accessible monosaccharide building blocks; and (iv) use of an allyl glycoside at the non-reducing end of the trisaccharide allowing for the installation of a thiol functional group, via a thiol-ene reaction, for covalent conjugation to a carrier protein. Implementing these features, the strategy involves the synthesis of an acyl-protected disaccharide (Gal1,3Gal), its conversion into a trichloroacetimidate donor, glycosylation of an appropriate allyl-glycoside GlcNAc acceptor to produce a Gal(1,3)Gal1,4)GlcNAc allyl glycoside, and further derivatization into a mercaptopropyl glycoside needed for protein conjugation.
(20) The neoglycoconjugates comprise a glycan attached to a carrier. The glycan can be attached via linker. In certain aspects the carrier can be a protein, peptide, or particle.
(21) In one example BSA was chosen for the generation of NGPs because of its large number of conjugation sites per BSA molecule, its solubility properties, and its suitability as a carrier protein (Makela and Seppala, 1986) and provider of T cell epitopes for the immunization of mice (Atassi et al., 1982), as well as its capability to attach non-covalently to wells of microtiter plates. Previously, it was discovered that Ch anti--Gal Abs recognize the disaccharide Gal(1,3)Gal, which comprises the two terminal sugars of the glycotope trisaccharide Gal(1,3)Gal(1,4)GlcNAc, much more strongly than Gal alone (Ashmus et al., 2013). In order to obtain information on the importance of Gal(1,4)GlcNAc or GlcNAc for antibody recognition, three additional BSA NPGs containing Gal1,4GlcNAc, GlcNAc, or GlcNAc were synthesized and tested by CL-ELISA.
(22) Other suitable carrier proteins include human serum albumin (HSA), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), tetanus toxoid (TT), recombinant proteins from T. cruzi containing CD4 and/or CD8 T cell epitopes, Neisseria meningitidis outer membrane protein complex, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens, protein D from Haemophilus influenzae, pneumolysin or its non-toxic derivatives, pneumococcal surface protein PspA, iron-uptake proteins, toxin A or B from Clostridium difficile, recombinant Pseudomonas aeruginosa exoprotein A (rEPA) and the like.
(23) In certain aspects the carrier can include one or more T-cell epitope. T cell epitopes, e.g., CD4+ T helper cell epitopes (Etlinger et al., 1990), are peptides that can induce T cell help and are known in the art. Epitopes that are useful in the present methods and compositions include those from diphtheria toxoid (DT), tetanus toxin (TI), Plasmodium falciparum circumsporozite, hepatitis B surface antigen, hepatitis B nuclear core protein, H. influenzae matrix protein, H. influenzae haemagglutinin, group B N. meningitidis outer membrane protein complex (OMPC), the pneumococcal toxin pneumolysin, and heat shock proteins, including those recombinantly produced and detoxified variants thereof.
(24) In certain aspects the T cell epitope may not include any lysine residues internally, but will be modified to include at least one lysine residue at an end, e.g., at the C terminus. In some embodiments, there is only one lysine residue at the C terminus or at the N terminus. In some embodiments, there will also be one or more amino acids between the carrier peptide sequence and the glycan component of the neoglycoconjugate, i.e., an amino acid spacer sequence. Such spacer sequences can be any amino acid, and will generally be flexible and have small R groups, to avoid steric hindrance and allow for optimal positioning of the linked carbohydrate for presentation to T cells and access of the peptide epitope to bind to an effector cell. Exemplary amino acids suitable for inclusion in the linker include glycine, alanine, and serine. In certain aspects the spacer does not contain lysine residues. In certain embodiments two or more carrier peptides are linked or cross-linked with two or more other carrier peptides.
(25) In other embodiments the carrier may be a nanoparticle carrier. The glycan or glycotope can be linked to biocompatible nanoparticles, with or without a linker or spacer between the glycan and the nanoparticle. The nanoparticles useful in the methods and compositions described herein are made of materials that are (i) biocompatible, i.e., do not cause a significant adverse reaction in a living animal when used in pharmaceutically relevant amounts; (ii) feature functional groups to which the binding moiety can be covalently attached, (iii) exhibit low non-specific binding of interactive moieties to the nanoparticle, and (iv) are stable in solution, i.e., the nanoparticles do not precipitate. The nanoparticles can be monodisperse (a single crystal of a material, e.g., a metal, per nanoparticle) or polydisperse (a plurality of crystals, e.g., 2, 3, or 4, per nanoparticle).
(26) A number of biocompatible nanoparticles are known in the art, e.g., organic or inorganic nanoparticles. Liposomes, dendrimers, carbon nanomaterials and polymeric micelles are examples of organic nanoparticles. Quantum dots can also be used. Inorganic nanoparticles include metallic nanoparticle, e.g., Au, Ni, Pt and TiO.sub.2 nanoparticles. Magnetic nanoparticles can also be used, e.g., spherical nanocrystals of 10-20 nm with a Fe.sup.2+ and/or Fe.sup.3+ core surrounded by dextran or PEG molecules. In some embodiments, colloidal gold nanoparticles are used, e.g., as described in U.S. Pat. Nos. 7,060,121; 7,232,474.
(27) The linkers or spacers can polymer or amino acid linkers. The linker or spacer will comprise a functional group that provide for attachment to the glycan and another functional group that provides for attachment to the carrier. A variety of linker molecules may be used, using conventional procedures. The linker can be any of a wide array of linking groups. Alternatively, the linker may be a single bond or a zero order linker.
(28) Said linker molecule is advantageously a homobifunctional or heterobifunctional molecule, for example adipic dihydrazide, ethylenediamine, cystamine, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-[4N-(2-iodoacetyl)]--alanyl propionate-propionate (SIAP), succinimidyl-4-(N-maleimido-methyl)cyclohexane-1 carboxylate (SMCC), 3,3-dithiodipropionic acid. In certain aspects the linker or spacer is a water-soluble polymer, and in one embodiment, the water-soluble polymer comprises poly(ethylene glycol).
II. IMMUNOGENIC COMPOSITIONS AND USES THEREOF
(29) One reason that a glycan-based vaccine against ChD has thus far been elusive is the lack of an adequate animal model closely mimicking the human anti-glycan immune response to T. cruzi. One example of a glycan-based epitope is Gal(1,3)Gal(1,4)GlcNAc (Gal3LN or Gal3LN). In certain aspect Gal3LN induces anti--Gal Abs. Except for humans and Old World monkeys, mice and all other mammals express this glycan on their cells and are, therefore, tolerant to the epitope. It is reasonable to assume then that the immune response observed in experimental vaccination with Gal-containing immunogens using any regular mouse model (e.g., BALB/c) would not account for the major protective humoral response against the parasite, i.e., Ch anti--Gal Abs. Accordingly, most if not all experimental vaccine studies using regular mouse models have been biased towards an almost exclusively CD8+ T cell-mediated protection. In part, this accounts for the fact that the overwhelming majority of experimental T. cruzi vaccines have employed CD8+ T cell epitopes. To circumvent this problem, 1,3GalT-KO mice were used that, like humans and Old World monkeys, do not express Gal1,3Gal epitopes on their cells due to the knock out of the enzyme alpha-1,3-galactosyltransferase. Therefore, the 1,3GalT-KO mouse model mimics humans in regard to the humoral (B cell-mediated) immune response against T. cruzi.
(30) An antigenic determinant is, unless otherwise indicated, a molecule that is able to elicit an immune response in a particular animal or species. Antigenic determinants include, for example, carbohydrate moieties, such as glycans. In certain aspects an antigenic determinant that is a carbohydrate can be referred to as a glycotope.
(31) Certain embodiments are directed to immunogenic compositions and methods comprising a Gal3LN conjugate. A Gal3LN conjugate is peptide or protein that has one or more Gal3LN moieties covalently attached, either directly or by a linker.
(32) As used herein, prophylactic and preventive vaccines are vaccines that are designed and administered to prevent infection, disease, and/or any related sequela(e) caused by or associated with a pathogenic organism, such as a trypanosome or other parasite.
(33) As used herein, therapeutic vaccines are vaccines that are designed and administered to patients already infected with a pathogenic organism. Therapeutic vaccines (e.g., therapeutic trypanosome vaccines) are used to prevent and/or treat the development of disease in these infected individuals.
(34) As used herein the phrase immune response or its equivalent immunological response refers to a humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secretion products) or both humoral and cellular response directed against an epitope of the invention in a subject or a donor subject. A donor subject is one in which an antibody is generated and isolated, the isolated antibody is then administered to a second subject. Treatment or therapy can be an active immune response induced by administration of immunogen or a passive therapy affected by administration of antibody, antibody-containing material, or vaccine-primed B and/or T cells.
(35) For purposes of this specification and the accompanying claims the terms epitope and antigenic determinant are used interchangeably to refer to a site on an antigen to which B and/or T cells respond or recognize.
(36) Embodiments described herein include methods for preventing or ameliorating parasite infections. As such, the invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared from immunogenic glycans and glycan conjugates.
(37) Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines.
(38) Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Formulations can include such normally employed excipients and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
(39) The glycans and glycan-conjugates may be formulated into a vaccine as neutral or salt forms. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
(40) Typically, vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
(41) In certain instances, it will be desirable to have multiple administrations of the vaccine, e.g., 2, 3, 4, 5, 6 or more administrations. The vaccinations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, and 12 week intervals, including all ranges there between. Periodic boosters at intervals of 1-5 years will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies against the antigens.
(42) Carriers. A given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling an antigen to a carrier. An example of carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, human serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating an antigen to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde, and bis-biazotized benzidine.
(43) Adjuvants. The immunogenicity of a composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions. A number of adjuvants can be used to enhance an antibody response against an antigen. Adjuvants can (1) trap the antigen in the body to cause a slow release; (2) attract cells involved in the immune response to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) improve the spread of the antigen throughout the subject's body.
(44) Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP, BCG, aluminum salts, such as aluminum hydroxide or other aluminum compound, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion.
(45) Various methods of achieving adjuvant affect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70 to about 101 C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin; mixture with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of Gram-negative bacteria; emulsion in physiologically acceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed to produce an adjuvant effect.
(46) In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM) to enhance immune responses. BRMs have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ) and cytokines such as -interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.
III. EXAMPLES
(47) The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
Synthesis of Neoglycoproteins
(48) A. Results and Discussion
(49) The -Gal-containing disaccharide 11 was synthesized from the known allyl (-galactoside 5 (Stevenson and Furneaux, 1996), which was made from its peracetylated precursor following an optimized procedure (Khamsi et al., 2012). Disaccharide 11 was synthesized in a 55% overall yield, starting with p-methoxybenzylation of allyl glycoside 5 at position 3 via its tin acetal to give 6, followed by benzoylation of the three remaining hydroxyls to afford 7. Oxidative cleavage of the p-methoxybenzyl group with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone furnished the -Gal acceptor 8. This acceptor was glycosylated with the known di-tert-butylsilylidene equipped -Gal trichloroacetimidate donor 9 (Imamura et al., 2006), using trimethylsilyl trifluoromethanesulfonate catalysis to give disaccharide 10. The di-tert-butylsilylidene group was cleaved with a large excess of 70% hydrogen fluoride in pyridine in THF, followed by acetylation of the two hydroxyls to give the peracylated allyl disaccharide 11 (
(50) The -Gal-containing disaccharide 11 was then treated with palladium (II) chloride in methanol to give the hemiacetal, which was filtered immediately after consumption of the starting material to avoid the formation of a polar by-product that was observed after two hours of reaction, and converted into the trichloroacetimidate 12 with trichloroacetonitrile in the presence of 1,8-diazabicycloundec-7-ene (
(51) The Gal(1,4)GlcNAc disaccharide 2 was synthesized as shown in
(52) The mercaptopropyl glycosides oxidized to disulfides within hours-days of isolation, which could easily be reduced by tris(2-carboxyethyl)phosphine before their conjugation to BSA. The thiol groups on compounds 1-4 served as nucleophiles in the conjugate addition to commercially available maleimide-derivatized BSA in aqueous buffer at pH 7.2, as shown in
(53) The four NPGs Gal(1,3)Gal(1,4)GlcNAc-BSA, Gal(1,4)GlcNAc-BSA, GlcNAc-BSA, GlcNAc-BSA, and a BSA control conjugate in which the maleimide groups had been blocked with cysteine (Cys-BSA), were immobilized in 96-well polystyrene Nunc Maxisorp ELISA plates and antibody-binding responses were measured using CL-ELISA (Almeida et al. 1997), with pooled Chagasic human sera (ChHSP) and normal human sera (NHSP), as well as Ch anti--Gal Abs and NHS anti--Gal Abs, purified as described (Almeida et al. 1991). As shown in
(54) Next, the in vivo response to Gal(1,3)Gal(1,4)GlcNAc-BSA was evaluated in C57B1/6 1,3galactosyltransferase-knockout (1,3GalT-KO) mice. Akin to humans and in contrast to wild-type mice, these animals lack terminal Gal1,3-linked residues on glycoproteins, thus being able to produce high levels of anti--Gal antibodies (Tearle et al., 1996; Thall et al., 1996). Sera collected from immunized and control animals were pooled separately and analyzed by CL-ELISA (Ashmus et al., 2013). As shown in
(55) Based on the Gal(1,3)Gal(1,4)GlcNAc-BSA neoglycoprotein described above, two series of neoglycoconjugates can be produced, each composed of three components or modules (
(56) The mercaptopropyl glycoside of Gal(1,3)Gal(1,4)GlcNAc was efficiently synthesized in 12 steps from known monosaccharide building blocks. In contrast to the published chemical syntheses, this synthesis is the accessibility of the glycosyl acceptors, which are synthesized in 2-3 steps from commercially available starting materials. In addition, the synthesis utilizes common and inexpensive glycosylation catalysts. The two key steps in this synthesis are the stereoselective installation of the terminal Gal unit into disaccharide 10 in 92% yield, and the challenging glycosylation of the 2-deoxy-2-azido acceptor 14 to give the correct stereoisomer (trisaccharide 15) in 46% yield. With the exception of the p-methoxybenzyl group introduced into galactose derivative 6, the di-tert-butylsilylidene protecting group of the galactosyl donor 9, and the allyl group as a precursor of a hemiacetal in compound 11, easily installable and removable acetyl and benzoyl protecting groups were used throughout the synthesis. Utilizing anomeric allyl groups allowed for the convenient conversion into mercaptopropyl glycosides that were needed for the conjugation to maleimide-derivatized BSA. The mercaptopropyl group of these glycosides is highly versatile as it is suitable for the conjugation to a large variety of other biomolecules and surfaces by conjugate addition to maleimides, nucleophilic substitution, and thiol-ene reaction. Finally, the trisaccharide Gal(1,3)Gal(1,4)GlcNAc, which is an immunodominant glycotope in infective T. cruzi trypomastigotes, is highly immunogenic in the context of T. cruzi infection in both mice and humans. It is propose that the Gal(1,3)Gal(1,4)GlcNAc-BSA and its analogs containing different carrier proteins or peptides can be further used as diagnostic biomarkers or tools for the diagnosis and follow-up of chemotherapy of ChD, and as vaccine candidates for ChD in humans and nonhuman primates. In addition, these neoglycoconjugates can also be employed as vaccines, and diagnostic and prognostic (chemotherapy follow-up) biomarkers for other parasitic infections, including but not restricted to malaria, leishmaniasis, African trypanosomiasis, and hookworm and tapeworm infections.
(57) B. Materials and Methods
(58) Compound Isolation and characterization. Thin-layer chromatography was performed with silica gel on aluminum support, 8.0-12.0 m, Sigma-Aldrich, and visualized by UV light or with 2% H.sub.2SO.sub.4 in ethanol, followed by heating. Flash chromatography was performed with silica gel, grade A, 32-63 m, Dynamic Adsorbents. .sup.1H NMR spectra were recorded on a JEOL 600 MHz NMR spectrometer using tetramethylsilane or chloroform as an internal standard. .sup.13C NMR spectra were recorded on the same JEOL NMR spectrometer at 150 MHz. Optical rotations were recorded on an Atago AP300 automatic polarimeter. Mass spectra were recorded on a JEOL Accu TOF mass spectrometer using electrospray ionization, or on a Shimadzu Axima MALDI-TOF MS. Dichloromethane and pyridine were refluxed over calcium hydride and distilled, methanol was refluxed over magnesium and distilled. Reagents were purchased from Sigma-Aldrich, Acros Organics, Fisher Scientific, and Alfa Aesar. 96-well polystyrene Nunc MaxiSorp ELISA plates and CL-ELISA reagents were purchased from Thermo Scientific or Jackson ImmunoResearch, luminescence was recorded on a Luminoskan Ascent, Thermo Scientific.
(59) 3-thiopropyl -D-galactopyranosyl-(1.fwdarw.3)--D-galactopyranosyl-(1.fwdarw.4)-2-deoxy-2-acetamido--D-glucopyranoside (1). To a flask containing 17 (0.027 g, 0.018 mmol), 3 mL of 0.5M NaOMe was added under argon, and stirred at room temperature for 30 minutes. HRMS showed full removal of acyl protecting groups, and all material was present as a mixture of thiol and disulfide. Amberlyst-15 ion-exchange resin was added and stirred until the solution was neutral, followed by filtration through Celite and evaporation of the solvent. The remainder was dissolved in water and lyophilized to give 1 as a white powder (0.011 g, quant.). ESI-TOF HRMS [C.sub.23H.sub.41NO.sub.16S+Na].sup.+ calc. m/z=642.2044, found 642.1980.
(60) 3-thiopropyl -D-galactopyranosyl-(1.fwdarw.4)-2-deoxy-2-acetamido--D-glucopyranoside (2). To a flask containing 20 (0.045 g, 0.051 mmol), 4 mL of 0.5M NaOMe was added under argon, and stirred at room temperature for 30 minutes. HRMS showed full removal of acyl protecting groups, and all the material was present as a disulfide. Amberlyst-15 ion-exchange resin was added and stirred until the solution was neutral, followed by filtration through Celite and evaporation of the solvent. The remainder was dissolved in water and lyophilized to give 2 as a white powder (0.024 g, quant.). ESI-TOF HRMS [C.sub.34H.sub.60N.sub.2O.sub.22S.sub.2+H].sup.+ calc. m/z=913.3157, found 913.3046; [C.sub.34H.sub.60N.sub.2O.sub.22S.sub.2++Na].sup.+ calc. m/z=935.2977, found 935.2836.
(61) 3-thiopropyl 2-deoxy-2-acetamido--D-glucopyranoside (4). To a flask containing 22 (0.059 g, 0.127 mmol), 4 mL of 0.5M NaOMe was added under argon, and stirred at room temperature for 2 hours. HRMS showed full removal of acyl protecting groups, and most of the material was present as a disulfide. Amberlyst-15 ion-exchange resin was added and stirred until the solution was neutral, followed by filtration through Celite and evaporation of the solvent. The remainder was dissolved in water and lyophilized to give 4 as a white powder (0.037 g, quant.). ESI-TOF HRMS [C.sub.22H.sub.40N.sub.2O.sub.12S.sub.2+Na].sup.+ calc. m/z=611.1920, found 611.1707.
(62) Allyl 3-O-(4-methoxybenzyl)--D-galactopyranoside (6). A solution of 5 (Stevenson and Furneaux 1996) (0.409 g, 1.86 mmol) and Bu.sub.2SnO (0.693 g, 2.79 mmol) in 18 mL anhydrous MeOH was stirred and refluxed under argon for 8 h. The solution was then quickly concentrated, and resuspended in 18 mL benzene. Bu.sub.4NBr (0.30 g, 0.93 mmol) was added, followed by 4-methoxybenzyl chloride (0.378 mL, 2.79 mmol), and stirred at 80 C. for 12 h. The solution was concentrated, and purified by column chromatography on silica gel (CHCl.sub.3/MeOH 9:1) to give 6 as a white powder (0.430 g, 75%). Its .sup.1H and .sup.13C NMR spectra matched the ones previously described for this compound (Yoshida et al. 2001).
(63) Allyl 3-O-(4-methoxybenzyl)-2,4,6-tri-O-benzoyl--D-galactopyranoside (7). A solution of 6 (0.380 g, 1.22 mmol) in 5 mL anhydrous pyridine was cooled to 0 C. under argon. BzCl (0.854 mL, 7.35 mmol) was added dropwise, and stirred for 3 h. The solution was diluted with EtOAc, washed once with 1M HCl, once with a saturated NaHCO.sub.3 solution and once with brine, dried over MgSO.sub.4, filtered, concentrated, and purified by column chromatography on silica gel (hexanes/EtOAc 2:1) to give 7 as a white powder (0.658 g, 90%). [].sup.28.sub.D 72.4 (c=1 in CHCl.sub.3); R.sub.f=0.38 (MeOH/CHCl.sub.3 1:9); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.18; 8.05; 7.97; 7.56-7.61; 7.43-7.50 (5 m, 15H, 3Bz); 7.05; 6.59 (2 m, 4H, 4-OMe-benzyl); 5.90 (m, 1H, H-4); 5.77 (m, 1H, OCH.sub.2CHCH.sub.2); 5.55 (dd, 1H, .sup.3J.sub.H1/H2=8.9 Hz, .sup.3J.sub.H2/H3=8.9 Hz, H-2); 5.18 (m, 1H, OCH.sub.2CHCH.sub.2); 5.07 (m, 1H, OCH.sub.2CHCH.sub.2); 4.60-4.67 (m, 3H, H-1, H-6, CH.sub.2PhOMe); 4.41-4.47 (m, 2H, H-6, CH.sub.2PhOMe); 4.35 (m, 1H, OCH.sub.2CHCH.sub.2); 4.13 (m, 1H, OCH.sub.2CHCH.sub.2); 4.08 (m, 1H, H-5); 3.79 (dd, 1H, .sup.3J.sub.H3/H4=3.4 Hz, H-3); 3.70 (s, 3H, OCH.sub.3) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 166.3; 166.0; 165.3; 159.3; 133.7; 133.5; 133.4; 133.1; 130.3; 129.6-130.1; 129.5; 129.4; 128.5-128.7; 128.4; 117.7; 113.7; 100.2 (C-1); 75.8; 71.5; 71.3; 70.7; 70.1; 66.8; 62.8; 55.2 ppm. ESI-TOF HRMS [C.sub.38H.sub.36O.sub.10+Na].sup.+ calc. m/z=675.2206, found 675.2001; [C.sub.38H.sub.36O.sub.10+K].sup.+ calc. m/z=691.1946, found 691.2022.
(64) Allyl 2,4,6-tri-O-benzoyl--D-galactopyranoside (8). To a solution of 7 (0.633 g, 0.97 mmol) in 20 mL CH.sub.2Cl.sub.2 and 1.1 mL H.sub.2O, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (0.440 g, 1.94 mmol) was added in two portions, 30 minutes apart, and stirred vigorously for 12 h. The red and green solution was filtered through Celite, diluted with dichloromethane, and extracted with water (25 mL) and brine solution (25 mL), dried over MgSO.sub.4, filtered, concentrated, and purified by column chromatography on silica gel (EtOAc/hexanes 2:1) to give 8 as a white powder (0.504 g, 98%). Its .sup.1H and .sup.13C NMR spectra matched the ones previously described for this compound (Sherman et al. 2001).
(65) Allyl 4,6-di-O-tert-butylsilylene-2,3-di-O-benzoyl--D-galactopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl--D-galactopyranoside (10). A solution of acceptor 8 (0.175 g, 0.329 mmol) and 4,6-di-O-tertbutylsilyl-2,3-di-O-benzoyl--D-galactopyranosyl trichloroacetimidate donor 9 (Imamura et al. 2006) (0.266 g, 0.395 mmol) in anhydrous dichloromethane (6 mL) was added to a 10 mL round bottomed flask with freshly activated, crushed 4 molecular sieves and stirred under argon for 15 min. at 0 C. TMSOTf (0.010 mL, 0.059 mmol) was added dropwise, and the mixture was gradually brought to room temperature and stirred for 2 h. To quench the reaction, Et.sub.3N (0.010 mL, 0.072 mmol) was added and stirred. The solution was diluted with dichloromethane (50 mL) and extracted with water (225 mL) and brine solution (25 mL), dried over MgSO.sub.4, filtered, concentrated, and purified by column chromatography on silica gel (hexanes/EtOAc 3:1) to give 10 as a white powder (0.315 g, 92%). [].sup.28.sub.D 160.7 (c=1 in CHCl.sub.3); R.sub.f=0.55 (EtOAc/hexanes 1:2); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.09, 7.99, 7.84, 7.74, 7.60, 7.51, 7.41, 7.26, 7.13, 7.01 (10 m, 25H, 5Bz); 5.79-5.87 (m, 2H, OCH.sub.2CHCH.sub.2, GalH-4); 5.70-5.76 (m, 2H, GalH-2, GalH-2); 5.62 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GalH-1); 5.26 (m, 1H, OCH.sub.2CHCH.sub.2); 5.16 (m, 2H, OCH.sub.2CHCH.sub.2, GalH-3); 4.77 (d, 1H, .sup.3J.sub.H1/H2=8.3 Hz, GalH-1); 4.55 (dd, 1H, .sup.3J.sub.H5/H6=11.7 Hz, .sup.2J.sub.H6/H6=6.9 Hz, GalH-6); 4.40 (m, 1H, OCH.sub.2CHCH.sub.2); 4.22-4.30 (m, 3H, GalH-3, GalH-5, GalH-4); 4.19 (m, 1H, OCH.sub.2CHCH.sub.2); 4.03-4.14 (m, 2H, GalH-5, GalH-6); 3.64-3.71 (m, 2H, GalH-6, GalH-6); 1.02 (s, 9H, t-butyl); 0.79 (s, 9H, t-butyl) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 166.3, 166.1, 165.6, 165.4, 165.0, 133.6, 133.4; 133.4; 133.0; 132.9; 132.8; 129.5-129.9; 129.2; 128.8; 128.6; 128.3; 128.1; 118.0; 100.3 (C-1); 94.2 (C-1); 73.8; 71.5; 70.9; 70.7; 70.5; 70.2; 67.6; 67.1; 66.5; 65.9; 62.3; 27.4; 27.2; 25.4; 23.2; 20.7 ppm. ESI-TOF HRMS [C.sub.58H.sub.62O.sub.16Si+Na].sup.+ calc. m/z=1065.3705, found 1065.3587; [C.sub.58H.sub.62O.sub.16Si+K].sup.+ calc. m/z=1081.3444, found 1081.2728.
(66) Allyl 2,3-di-O-benzoyl-4,6-di-O-acetyl--D-galactopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl--D-galactopyranoside (11). A solution of 10 (0.464 g, 0.444 mmol) in anhydrous THF (7 mL) was added to a 50 mL plastic conical tube and stirred under argon at rt. A solution of HF-pyridine (70% HF, 30% pyridine) (0.223 mL, 8.88 mmol) was added to the reaction mixture and stirred for 3 h, then quenched with 0.5 mL saturated NaHCO.sub.3. The solution was diluted with EtOAc and extracted with water and brine, dried over MgSO.sub.4, and concentrated. The compound was then added to a 25 mL round bottom flask in 5 mL anhydrous pyridine, and Ac.sub.2O was added (0.252 mL; 2.66 mmol) and stirred for 12 h. The solvent was then co-evaporated with toluene, and the remainder was purified by column chromatography on silica gel (hexanes/EtOAc 2:1) to give 11 as a white powder (0.389 g, 89% in 2 steps). [].sup.28.sub.D 163.0 (c=1 in CHCl.sub.3); R.sub.f=0.20 (EtOAc/hexanes 1:2); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.15; 7.99; 7.70; 7.61; 7.51; 7.44; 7.39; 7.28; 7.24; 7.10; 7.01 (11 m, 25H, 5Bz); 5.80-5.86 (m, 2H, OCH.sub.2CHCH.sub.2, GalH-4); 5.76 (dd, 1H, .sup.3J.sub.H2/H3=9.6 Hz, GalH-2); 5.67 (d, 1H, .sup.3J.sub.H1/H2=4.1 Hz, GalH-1); 5.61 (dd, 1H, .sup.3J.sub.H2/H3=11.0 Hz, GalH-2); 5.41 (dd, 1H, .sup.3J.sub.H3/H4=3.4 Hz, GalH-3); 5.26 (m, 1H, OCH.sub.2CHCH.sub.2); 5.09-5.17 (m, 2H, OCH.sub.2CHCH.sub.2, GalH-4); 4.78 (d, 1H, .sup.3J.sub.H1/H2=7.6 Hz, GalH-1); 4.57 (dd, 1H, .sup.3J.sub.H5/H6=11.0 Hz, .sup.2J.sub.H6/H6=6.2 Hz, GalH-6); 4.41 (m, 1H, OCH.sub.2CHCH.sub.2); 4.27-4.32 (m, 2H, GalH-3, GalH-5); 4.19 (m, 2H, OCH.sub.2CHCH.sub.2, GalH-5); 4.11 (dd, 1H, .sup.3J.sub.H5/H6=6.6 Hz, GalH-6); 3.96 (m, 2H, GalH6, GalH6); 1.97-2.03 (m, 6H, 2Ac) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 170.1; 169.8; 166.2; 166.0; 165.4; 165.1; 164.9; 133.6; 133.5; 133.4; 133.1; 133.0; 129.8; 129.8; 129.7; 129.5; 129.5; 129.3; 128.8; 128.6; 128.4; 128.3; 128.3; 128.2; 118.1; 100.3 (C-1); 93.4 (C-1); 73.4; 71.5; 70.6; 70.2; 67.9; 67.6; 66.8; 65.5; 62.3; 61.5; 20.8; 20.6 ppm. ESI-TOF HRMS [C.sub.54H.sub.50O.sub.18+NH.sub.4].sup.+ calc. m/z=1004.3341, found 1004.3070.
(67) Trichloroacetimidate 2,3-di-O-benzoyl-4,6-di-O-acetyl--D-galactopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl--D-galactopyranoside (12). To a solution of 11 (0.369 g, 0.374 mmol) in MeOH (6 mL), PdCl.sub.2 (0.0398 g, 0.225 mmol) was added and stirred for 2 h at room temperature until consumption of most of the starting material. After 2 h, a degradation product can be observed. The solution was filtered through Celite, concentrated, and purified by column chromatography on silica gel (EtOAc/hexanes 2:3) to give the and anomers (0.308 g, 87%). A recovered compound assumed to be remaining starting material was actually the vinyl glycoside. The anomeric product mixture was then placed into a round-bottomed flask, 10 mL anhydrous CH.sub.2Cl.sub.2 was added under argon, and the solution was cooled to 0 C. CCl.sub.3CN (0.325 mL, 3.24 mmol) was added, followed by dropwise addition of DBU (0.015 mL, 0.097 mmol) and the mixture was brought to rt over 3 h. The solution was concentrated and purified by column chromatography on silica gel (EtOAc/hexanes 1:2) to give 12 as a white powder (0.295 g, 84%). [].sup.27.sub.D 186.2 (c=1 in CHCl.sub.3); R.sub.f=0.65 (acetone/hexanes 1:1); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.64 (s, 1H, NH); 8.10; 7.94; 7.67-7.71; 7.53-7.63; 7.47; 7.36-7.41; 7.23-7.30; 7.12; 7.02 (9 m, 25H, 5Bz); 6.90 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GalH-1) 5.99 (d, 1H, .sup.3J.sub.H4/H5=2.8 Hz, GalH-4); 5.94 (dd, 1H, .sup.3J.sub.H2/H3=10.3 Hz, GalH-2); 5.76 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GalH-1); 5.65 (dd, 1H, .sup.3J.sub.H2/H3=10.3 Hz, GalH-2); 5.49 (dd, 1H, .sup.3J.sub.H3/H4=3.4 Hz, GalH-3); 5.28 (m, 1H, GalH-4); 4.76 (dd, 1H, .sup.3J.sub.H3/H4=3.4 Hz, GalH-3); 4.65 (m, 1H, GalH-5); 4.44 (dd, 1H, 7.6 Hz, 11.7 Hz, GalH-6); 4.41 (dd, 1H, .sup.3J.sub.H5/H6=11.7 Hz, GalH-5); 4.31 (dd, 1H, 5.5 Hz, 11.7 Hz, GalH-6); 4.09-4.14 (m, 1H, GalH-6); 4.02 (dd, 1H, .sup.2J.sub.H6/H6=6.2 Hz, GalH-6); 1.98-2.06 (m, 6H, 2Ac) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 170.2; 169.8; 166.0; 165.6; 165.3; 165.0; 160.5; 133.9; 133.0-133.3; 129.6-129.9; 129.4; 128.8; 128.5; 128.3; 128.2; 93.8 (GalC-1); 93.2 (GalC-1); 90.9 (CCl.sub.3); 70.1; 69.5; 68.7; 67.8; 66.7; 66.0; 62.5; 60.9; 20.9; 20.6 ppm. ESI-TOF HRMS did not show a molecular ion peak for [C.sub.53H.sub.46Cl.sub.3NO.sub.18].sup.+.
(68) Allyl 2-deoxy-2-acetamido-3,6-di-O-benzoyl--D-glucopyranoside (13). To a solution of allyl 2-deoxy-2-acetamido--D-glucopyranoside (Gavard et al. 2003) (3.98 g, 15.24 mmol) in 80 mL anhydrous AcCN, 1-benzoylimidazole (5.46 mL, 36.56 mmol) was added via a plastic syringe, and was heated at 80 C. for 12 h. After evaporation of the solvent the remainder was dissolved in EtOAc and extracted twice with water and once with brine solution, dried over MgSO.sub.4, filtered, concentrated, and purified by column chromatography on silica gel (toluene/EtOAc 2:1) to give 13 as a white powder (4.79 g, 67%). Its .sup.1H and .sup.13C NMR spectra matched the ones previously described for this compound (Danac et al. 2007). A minor byproduct with a higher R.sub.f value was identified as the tri-O-benzoylated compound.
(69) Allyl 2-deoxy-2-azido-3,6-di-O-benzoyl--D-glucopyranoside (14). Compound 14 was prepared similarly to a published synthesis with slight variations in the solvent and the time period over which BzCl was added (Danac et al. 2007): A solution of allyl 2-deoxy-2-azido--D-glucopyranoside (Gavard et al. 2003) (0.30 g, 1.223 mmol) in 10 mL anhydrous pyridine was cooled to 20 C., and BzCl (0.350 mL, 3.01 mmol) was added dropwise in 3 portions of 0.117 mL each over 1 h, and stirred for an additional 1 h. The solution was diluted with EtOAc and extracted twice with water and once with brine solution, dried over MgSO.sub.4, filtered, concentrated, and purified by column chromatography on silica gel (hexanes/EtOAc 5:2) to give 14 as a white powder (0.364 g, 66%). [].sup.25.sub.D 161.0 (c=1 in CHCl.sub.3) R.sub.f=0.48 (EtOAc/hexanes 1:2); NMR (600 MHz, CDCl.sub.3, 300K) 8.05-8.11; 7.58; 7.43-7.48 (m, 10H, 2Bz); 5.95 (m, 1H, OCH.sub.2CHCH.sub.2); 5.64 (dd, 1H, .sup.3J.sub.H3/H4=9.6 Hz, H-3); 5.36 (m, 1H, OCH.sub.2CHCH.sub.2); 5.25 (m, 1H, OCH.sub.2CHCH.sub.2); 5.09 (d, 1H, .sup.3J.sub.H1/H2=4.1 Hz, H-1); 4.73 (dd, 1H, .sup.3J.sub.H5/H6=4.8 Hz, .sup.2J.sub.H6/H6=12.4 Hz, H-6); 4.60 (dd, 1H, .sup.3J.sub.H5/H6=2.1 Hz, H-6); 4.29 (m, 1H, CH.sub.2CHCH.sub.2); 4.12 (m, 2H, H-5, CH.sub.2CHCH.sub.2); 3.77 (dd, 1H, .sup.3J.sub.H4/H5=9.6 Hz, H-4); 3.47-3.54 (broad, 1H, 4-OH); 3.44 (dd, 1H, .sup.3J.sub.H2/H3=11.0 Hz, H-2) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 167.2; 167.0; 133.7; 133.4; 133.0; 130.1; 129.7-130.0; 129.2; 128.6; 128.6; 118.5; 97.0 (C-1); 74.0; 70.7; 70.0; 69.0; 63.5; 61.2 ppm. ESI-TOF HRMS [C.sub.23H.sub.23N.sub.3O.sub.7+H].sup.+ calc. m/z=454.1614, found 454.1912. A minor byproduct of this reaction was identified as the tri-O-benzoylated compound.
(70) Allyl 2,3-di-O-benzoyl-4,6-di-O-acetyl--D-galactopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl--D-galactopyranosyl-(1.fwdarw.4)-2-deoxy-2-azido-3,6-di-O-benzoyl--D-glucopyranoside (15). A solution of acceptor 14 (0.126 g, 0.279 mmol) and donor 12 (0.304 g, 0.279 mmol) in anhydrous CH.sub.2Cl.sub.2 (6 mL) was placed in a 10 mL round bottomed flask with freshly activated, crushed 4 molecular sieves and stirred under argon for 15 min at 0 C. TMS-OTf (0.015 mL, 0.0835 mmol) was added dropwise to the reaction mixture, which was gradually brought to room temperature and stirred for 2 h. The reaction was quenched with Et.sub.3N (0.02 mL, 0.143 mmol), filtered through Celite, concentrated and purified by column chromatography on silica gel (hexanes/EtOAc 2:1) to give 15 as a slightly yellow powder (0.175 g, 46%). [].sup.26.sub.D 106.1 (c=1 in CHCl.sub.3) R.sub.f=0.53 (EtOAc/hexanes 1:1); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.20; 8.02; 7.97; 7.68; 7.55-7.64; 7.36-7.52; 7.31; 7.23; 7.11; 6.97 (10 m, 35H, 7Bz); 5.93 (m, 1H, OCH.sub.2CHCH.sub.2); 5.87 (dd, 1H, .sup.3J.sub.H2/H3=9.5 Hz, .sup.3J.sub.H3/H4=9.5 Hz, GlcH-3); 5.66 (dd, 1H, .sup.3J.sub.H1/H2=9.5 Hz, GalH-2); 5.56 (m, 1H, GalH-4); 5.53 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GalH-1); 5.34 (m, 1H, OCH.sub.2CHCH.sub.2); 5.30 (dd, 1H, .sup.3J.sub.H2/H3=10.3 Hz, GalH-2); 5.24 (m, 1H, OCH.sub.2CHCH.sub.2); 5.06 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GlcH-1); 4.95 (m, 1H, GalH-3); 4.80 (d, 1H, .sup.3J.sub.H1/H2=7.9 Hz, GalH-1); 4.53-4.60 (m, 2H, GalH-6, GalH-6); 4.26 (m, 1H, OCH.sub.2CHCH.sub.2); 4.05-4.18 (m, 5H, OCH.sub.2CHCH.sub.2, GalH-3, GalH-5, GlcH-4, GalH-4); 4.01 (m, 1H, GalH-5); 3.87 (dd, 1H, .sup.3J.sub.H5/H6=11.0 Hz, .sup.2J.sub.H6/H6=6.9 Hz, GalH-6); 3.82 (dd, 1H, .sup.3J.sub.H5/H6=11.7 Hz, GalH-6); 3.73-3.78 (m, 2H, GlcH-5, GlcH-6); 3.40-3.49 (m, 2H, GlcH-2, GlcH-6); 1.95-1.99 (m, 6H, 2Ac) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 170.3; 169.7; 166.0; 165.9; 165.7; 165.2; 164.8; 164.5; 133.8; 133.5; 133.2; 133.0-133.1; 132.9; 129.2-130.0; 128.5-128.9; 128.0-128.4; 118.7; 101.3 (GalC-1); 96.9 (GlcC-1); 92.9 (GalC-1); 76.4; 73.2; 71.3; 70.8; 70.6; 69.1; 69.0; 67.9; 67.8; 67.3; 66.8; 64.6; 62.5; 61.8; 61.4; 61.1; 20.7; 20.5 ppm. ESI-TOF HRMS [C.sub.74H.sub.67N.sub.3O.sub.24+Na].sup.+ calc. m/z=1399.4458, found 1399.4391; [C.sub.74H.sub.67N.sub.3O.sub.24+K].sup.+ calc. m/z=1420.3752, found 1420.3016.
(71) Allyl 2,3-di-O-benzoyl-4,6-di-O-acetyl--D-galactopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl--D-galactopyranosyl-(1.fwdarw.4)-2-deoxy-2-acetamido-3,6-di-O-benzoyl--D-glucopyranoside (16). To a flask containing 15 (0.125 g, 0.0904 mmol), was added 8 mL of thioacetic acid, and was stirred for 24 h at 40 C. The solution was concentrated by two co-evaporations with toluene, and purified by column chromatography on silica gel (EtOAc/hexanes 1:1.fwdarw.3:1) to give 16 as a white powder (0.097 g, 77%). [].sup.26.sub.D 94.3 (c=1 in CHCl.sub.3); R.sub.f=0.15 (hexanes/EtOAc 1:1); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.20; 8.02; 7.96; 7.68; 7.55-7.63; 7.47-7.53; 7.36-7.45; 7.28-7.35; 7.22; 7.15; 7.03; 6.95 (10 m, 35H, 7Bz); 5.84-5.92 (m, 2H, OCH.sub.2CHCH.sub.2, NH); 5.52-5.67 (m, 5H, GalH-2, GalH-4, GalH-1, GlcNAcH-3); 5.26-5.31 (m, 2H, GalH-2, OCH.sub.2CHCH.sub.2); 5.23 (m, 1H, OCH.sub.2CHCH.sub.2); 4.90-4.94 (m, 2H, GlcNAcH-1, GalH-3); 4.80 (d, 1H, .sup.3J.sub.H1/H2=7.6 Hz, GalH-1); 4.50-4.59 (m, 3H, GlcNAcH-2); 4.06-4.22 (m, 4H, GalH-3, OCH.sub.2CHCH.sub.2); 4.00 (m, 2H, OCH.sub.2CHCH.sub.2); 3.88 (dd, 1H); 3.78-3.84 (m, 2H); 3.66-3.72 (m, 2H); 1.96-2.00 (m, 6H, 2Ac); 1.86 (s, 3H, NHAc) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 170.3; 170.2; 169.7; 166.6; 166.1; 165.9; 165.7; 165.2; 164.8; 164.6; 133.9; 133.5; 133.3; 133.1; 133.1; 132.9; 130.0; 129.5-129.8; 129.4; 129.2; 128.9; 128.6-128.8; 128.0-128.4; 118.6; 101.3 ((3Ga1C-1); 96.4 (aGlcNAcC-1); 92.9 (aGa1C-1); 75.9; 73.2; 71.7; 71.4; 70.8; 69.0; 68.8; 67.9; 67.8; 67.3; 66.8; 64.6; 62.5; 61.8; 61.1; 52.1; 29.8; 23.3; 20.8; 20.5 ppm. ESI-TOF HRMS [C.sub.76H.sub.71NO.sub.25+H].sup.+ calc. m/z=1398.4393, found 1398.4308; [C.sub.76H.sub.71NO.sub.25+Na].sup.+ calc. m/z=1420.4213, found 1420.4487; [C.sub.76H.sub.71NO.sub.25+K].sup.+ calc. m/z=1436.3935, found 1436.3893.
(72) 3-(acetylthio)propyl 2,3-di-O-benzoyl-4,6-di-O-acetyl--D-galactopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl--D-galactopyranosyl-(1.fwdarw.4)-2-deoxy-2-acetamido-3,6-di-O-benzoyl--D-glucopyranoside (17). To a solution of 16 (0.030 g, 0.022 mmol) and AIBN (0.004 g, 0.022 mmol) in anhydrous THF (3 mL), thioacetic acid (0.016 mL, 0.222 mmol) was added and stirred under argon for 5 min. The solution was then placed in a Rayonet UV reactor (350 nm) and stirred for 12 h under water cooling (rt). The solution was concentrated by two co-evaporations with toluene, and purified by column chromatography on silica gel (EtOAc/Hex 2:1) to give 17 as a white powder (0.028 g, 89%). [].sup.26.sub.D 94.2 (c=0.5 in CHCl.sub.3); R.sub.f=0.48 (EtOAc/hexanes 2:1); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.20; 8.01; 7.96; 7.68; 7.55-7.63; 7.47-7.53; 7.20-7.45; 7.13; 7.06; 6.95 (10 m, 35H, 7Bz); 6.19 (d, 1H, .sup.3J.sub.H1/H2=9.3 Hz, NH); 5.52-5.67 (m, 4H, GalH-2, GalH-2); 5.52 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GalH-1); 5.28 (dd, 1H, .sup.3J.sub.H2/H3=10.3 Hz, .sup.3J.sub.H3/H4=10.3 Hz, GalH-3); 4.92 (m, 1H, GalH-4); 4.83 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GlcNAcH-1); 4.79 (d, 1H, .sup.3J.sub.H1/H2=8.3 Hz, GalH-1); 4.52-4.59 (m, 3H, GlcNAcH-2); 4.09-4.16 (m, 2H); 4.04 (m, 1H); 4.00 (m, 1H, GalH-5); 3.87 (dd, 1H, .sup.3J.sub.H5/H6=11.7 Hz, .sup.2J.sub.H6/H6=6.9 Hz, GalH-6); 3.81 (dd, 1H, .sup.3J.sub.H5/H6=11.7 Hz, GalH-6); 3.68-3.78 (m, 3H, OCH.sub.2CH.sub.2CH.sub.2); 3.62 (dd, 1H); 3.44 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 3.09 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 2.95 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 2.32 (s, 3H, SAc); 1.97-1.99 (m, 6H, 2Ac); 1.85-1.92 (m, 5H, NHAc, OCH.sub.2CH.sub.2, OCH.sub.2CH.sub.2) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 195.8; 170.5; 170.3; 169.7; 166.5; 165.9; 165.7; 165.1; 164.6; 133.9; 133.5; 133.2; 133.1; 133.1; 133.0; 132.8; 129.3-130.0; 128.6-129.2; 128.0-128.4; 101.3 (GalC-1); 97.4 (GlcNAcC-1); 92.9 (Ga1C-1); 76.1; 73.2; 71.8; 71.4; 70.8; 69.1; 67.9; 67.8; 67.3; 66.8; 66.0; 64.6; 62.5; 61.8; 61.1; 51.9; 30.7; 29.8; 29.3; 25.5; 23.2; 20.8; 20.5 ppm. ESI-TOF HRMS [C.sub.78H.sub.75NO.sub.26S+H].sup.+ calc. m/z=1474.4376, found 1474.4222.
(73) Allyl 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(1.fwdarw.4)-2-deoxy-2-acetamido-3,6-di-O-benzoyl--D-glucopyranoside (19). To a solution of acceptor 13 (Danac et al. 2007) (3.60 g, 7.63 mmol) and donor 18 (Schmidt and Michel 1980) (14.0 g, 28.42 mmol) in 60 mL anhydrous CH.sub.2Cl.sub.2, BF.sub.3-OEt.sub.2 (1.93 mL, 15.25 mmol) was added and immediately brought to 35-40 C. After 3 h, Et.sub.3N (2.35 mL, 16.87 mmol) was added. The solution was washed one time with a saturated NaHCO.sub.3 solution, and the aqueous layer was extracted with CH.sub.2Cl.sub.2. The organic phases were combined, dried over MgSO.sub.4, filtered, concentrated, and purified by column chromatography on silica gel (EtOAc/Hex 2.3:1) to give 19 as a white powder (5.10 g, 83%). [].sup.22.sub.D 56.6 (c=1 in CHCl.sub.3); R.sub.f=0.30 (EtOAc/hexanes 2:1); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.07; 7.61; 7.52; 7.47 (4 m, 10H, 2Bz); 5.91 (m, 1H, OCH.sub.2CHCH.sub.2); 5.85 (d, 1H, .sup.3J.sub.H1/H2=9.6 Hz, NH); 5.62 (dd, 1H, .sup.3J.sub.H2/H3=11.0 Hz, .sup.3J.sub.H3/H4=8.3 Hz, GlcNAcH-3); 5.30 (m, 1H, OCH.sub.2CHCH.sub.2); 5.25 (m, 1H, OCH.sub.2CHCH.sub.2); 5.13 (m, 1H, GalH-4); 5.10 (dd, 1H, .sup.3J.sub.H2/H3=10.3 Hz, GalH-2); 4.91 (d, 1H, .sup.3J.sub.H1/H2=3.4 Hz, GlcNAcH-1); 4.82 (dd, 1H, .sup.3J.sub.H3/H4=3.4 Hz, GalH-3); 4.69 (m, 1H, GlcNAcH-6); 4.61 (d, 1H, .sup.3J.sub.H1/H2=8.3 Hz, GalH-1); 4.47 (m, 1H, GlcNAcH-2); 4.41 (dd, 1H, .sup.2J.sub.H6/H6=4.1 Hz, .sup.3J.sub.H5/H6=11.7 Hz, GlcNAcH-6); 4.22 (m, 1H, OCH.sub.2CHCH.sub.2); 4.07-4.15 (m, 2H, GlcNAcH-4, GlcNAcH-5); 4.03 (m, 1H, OCH.sub.2CHCH.sub.2); 3.64 (dd, 1H, .sup.3J.sub.H5/H6=8.3 Hz, .sup.2J.sub.H6/H6=11.0 Hz, GalH-6); 3.48 (dd, 1H, .sup.3J.sub.H5/H6=5.5 Hz, GalH-6); 3.36 (dd, 1H, 6.2 Hz, 8.3 Hz, GalH-5); 1.80-2.10 (m, 15H, 4Ac, NHAc) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 170.2; 169.9; 169.4; 166.4; 166.1; 133.6; 133.5; 133.2; 129.8; 129.7; 128.7; 128.6; 118.6; 101.1 (GalC-1); 96.5 (GlcNAcC-1); 76.4; 72.2; 71.0; 70.6; 69.4; 68.9; 68.8; 66.3; 62.6; 60.0; 52.2; 23.2; 20.5-20.8 ppm. ESI-TOF HRMS [C.sub.39H.sub.45NO.sub.17+H].sup.+ calc. m/z=800.2766, found 800.2864; [C.sub.39H.sub.45NO.sub.17+Na].sup.+ calc. m/z=822.2585, found 822.2022; [C.sub.39H.sub.45NO.sub.17+K].sup.+ calc. m/z=838.2325, found 838.1110.
(74) 3-(acetylthio)propyl 2,3,4,6-tetra-O-acetyl--D-galactopyranosyl-(1.fwdarw.4)-2-deoxy-2-acetamido-3,6-di-O-benzoyl--D-glucopyranoside (20). To a solution of 19 (0.050 g, 0.063 mmol) and AIBN (0.010 g, 0.063 mmol) in anhydrous THF (3 mL), thioacetic acid (0.045 mL, 0.63 mmol) was added and stirred under argon for 5 min. The solution was then placed in a Rayonet UV reactor (350 nm) stirred for 12 h under water cooling (rt). The solution was concentrated by two co-evaporations with toluene, and purified by column chromatography on silica gel (EtOAc/Hex 2:1) to give 20 as a white powder (0.046 g, 84%). [].sup.22.sub.D 45.6 (c=0.9 in CHCl.sub.3); R.sub.f=0.25 (EtOAc/hexanes 2:1); .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 8.08; 7.61; 7.52; 7.47 (4 m, 10H, 2Bz); 6.15 (d, 1H, .sup.3J.sub.H1/H2=9.6 Hz, NH); 5.58 (dd, 1H, .sup.3J.sub.H2/H3=10.3 Hz, .sup.3J.sub.H3/H4=8.3 Hz, GlcNAcH-3); 5.13 (m, 1H, GalH-4); 5.10 (dd, 1H, .sup.3J.sub.H2/H3=10.3 Hz, GalH-2); 4.80-4.84 (m, 2H, GlcNAcH-1, GalH-3); 4.70 (m, 1H, GlcNAcH-6); 4.60 (d, 1H, .sup.3J.sub.H1/H2=8.3 Hz, GalH-1); 4.48 (m, 1H, GlcNAcH-2); 4.40 (dd, 1H, .sup.2J.sub.H6/H6=4.1 Hz, .sup.3J.sub.H5/H6=11.7 Hz, GlcNAcH-6); 4.09 (m, 2H, GlcNAcH-4, GlcNAcH-5); 3.80 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 3.61 (dd, 1H, .sup.3J.sub.H5/H6=8.3 Hz, .sup.2J.sub.H6/H6=11.0 Hz, GalH-6); 3.47 (m, 2H, OCH.sub.2CH.sub.2CH.sub.2, GalH-6); 3.37 (m, 1H, GalH-5); 3.10 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 2.96 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 2.35 (s, 3H, SAc); 1.85-2.05 (m, 17H, NHAc, 4Ac, OCH.sub.2CH.sub.2CH.sub.2) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 195.7; 170.5; 170.1; 169.9; 169.4; 166.3; 166.1; 133.5; 129.8; 129.7; 128.7; 128.6; 101.1 (GalC-1); 97.4 (GlcNAcC-1); 76.4; 72.2; 71.0; 70.6; 69.4; 68.9; 66.3; 66.1; 62.6; 60.0; 52.1; 30.7; 29.8; 29.3; 25.6; 23.1; 20.5-20.8 ppm. ESI-TOF HRMS [C.sub.41H.sub.49NO.sub.18S+H].sup.+ calc. m/z=876.2749, found 876.3192; [C.sub.41H.sub.49NO.sub.18S+Na].sup.+ calc. m/z=898.2568, found 898.2413.
(75) 3-(acetylthio)propyl 2-deoxy-2-acetamido-3,4,6-tri-O-acetyl--D-glucopyranoside (22). To a solution of 21 (Kiso and Anderson 1979) (0.081 g, 0.209 mmol) and AIBN (0.034 g, 0.209 mmol) in anhydrous THF (5 mL), thioacetic acid (0.149 mL, 2.09 mmol) was added and stirred under argon for 5 min. The solution was then placed in a Rayonet UV reactor (350 nm) and stirred for 12 h under water cooling (rt). The solution was concentrated by two co-evaporations with toluene, and purified by column chromatography on silica gel (CHCl.sub.3/MeOH 25:1) to give 22 as a white powder (0.082 g, 85%). [].sup.26.sub.D 11.9 (c=1 in CHCl.sub.3); R.sub.f=0.30 (MeOH/CHCl.sub.3 1:9) .sup.1H NMR (600 MHz, CDCl.sub.3, 300K) 6.20 (d, 1H, .sup.3J.sub.H1/H2=8.9 Hz, NH); 5.17 (dd, 1H, .sup.3J.sub.H2/H3=8.9 Hz, .sup.3J.sub.H3/H4=10.3 Hz, H-3); 5.02 (dd, 1H, .sup.3J.sub.H4/H5=9.62 Hz, H-4); 4.50 (d, 1H, .sup.3J.sub.H1/H2=8.3 Hz, H-1); 4.20 (dd, 1H, .sup.3J.sub.H5/H6=12.4 Hz, .sup.2J.sub.H6/H6=4.8 Hz, H-6); 4.06 (dd, 1H, .sup.3J.sub.H5/H6=12.4 Hz, H-6); 3.92 (m, 1H, H-2); 3.85 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 3.64 (m, 1H, H-5); 3.42 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 3.00 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 2.75 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2); 2.28 (s, 3H, SAc); 1.80-2.10 (m, 13H, NHAc, 3Ac, OCH.sub.2CH.sub.2CH.sub.2); 1.69 (m, 1H, OCH.sub.2CH.sub.2CH.sub.2) ppm. .sup.13C NMR (150 MHz, CDCl.sub.3, 300K): 196.7; 171.0; 170.8; 170.6; 169.5; 100.8 (C-1); 72.9; 71.8; 68.7; 67.5; 62.2; 54.4; 30.7; 29.4; 25.4; 23.3; 20.6-20.9 ppm. ESI-TOF HRMS [C.sub.19H.sub.29NO.sub.10S+H].sup.+ calc. m/z=464.1590, found 464.1340; [C.sub.19H.sub.29NO.sub.10S+Na].sup.+ calc. m/z=486.1410, found 486.1100; [C.sub.19H.sub.29NO.sub.10S+K].sup.+ calc. m/z=502.1149, found 502.0768.
(76) Immunization Protocol. Groups of five female C57B1/6 1,3GalT-KO mice (Tearle et al. 1996; Thall et al. 1996) were immunized subcutaneously with 20 g Gal(1,3)Gal(1,4)GlcNAc-BSA in 200 l PBS/dose/immunization or 20 g BSA alone in 200 l PBS. All animals were immunized four times at 7-day intervals and sacrificed 14 days after the last immunization. Blood was collected by cardiac puncture and serum was separated through centrifugation for analysis by CL-ELISA. All animal procedures were performed according to the vertebrate animal protocols A-201211-1 and A-201411-1, approved by the University of Texas at El Paso's Institutional Animal Care and Use Committee.
(77) Protocol for the conjugation of thiols to BSA. For the conjugation of mercaptopropyl glycosides to maleimide-activated BSA a conjugation kit Imject Maleimide Activated Carrier Protein Spin Kit from Thermo Scientific, product #77667, was used, and the protocol provided by the manufacturer was followed.
(78) Tris(2-carboxyethyl)phosphine (TCEP, 0.8 mg, 2.79 mol) was dissolved in 250 L of Imject Maleimide Conjugation Buffer (83 mM sodium phosphate buffer, 0.1 M EDTA, 0.9 M sodium chloride, 0.02% sodium azide, pH 7.2) and added to microcentrifuge tubes containing sugar-disulfide (2.40 mol), and stirred. After 1 hour, 10 L was removed to determine the initial thiol concentration. Vials of maleimide-activated BSA (2 mg, 15-25 moles of maleimide/mole of BSA) were reconstituted by adding 200 L of ultrapure water. The remaining 240 L of sugar-conjugation buffer solution was added to each vial. Vials were flushed with argon, sealed with parafilm, and mixed for 3 hours on a shaker. Reaction Buffer was prepared (0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA) and a solution of Ellman's Reagent [5,5-dithiobis-(2-nitrobenzoic acid)=DTNB] (4 mg DTNB in 1 mL of Reaction Buffer). After 3 hours, 18.3 L was removed from each conjugation solution to determine the thiol concentration after the conjugation. Each sample to be tested was diluted to 250 L with Reaction Buffer and added to a test tube containing 50 L of Ellman's Reagent Solution and 2.5 mL of Reaction Buffer, and mixed at room temperature for 15 minutes. With a spectrophotometer set to 412 nm, the absorbance of each sample was measured. Using the molar extinction coefficient of 2-nitro-5-thiobenzoic acid (TNB, =14,150 M.sup.1 cm.sup.1), the concentration of sulfhydryls in each sample and the amount of sugar loaded (average: 2.0 mol) was determined.
(79) Conjugates were then diluted to 1 mL and added to Amicon Ultra 3K Centrifugal Filter Devices for desalting. Filters were centrifuged for 20 minutes at 4000g, then 1 mL of ultrapure water was added to the filter, and centrifuging was continued for 20 minutes at 4000g. The filtrate tube was then removed, and filters were inverted and centrifuged at 1000g for 2 minutes to collect in the concentrate tube. The collected material was lyophilized, and stock solutions of the protein were prepared. The protein concentrations were determined using a Pierce BCA (bicinchoninic acid) Protein Assay Reagent kit using a spectrophotometer at a detection wavelength of 562 nm.
(80) CL-ELISA protocol. 12 ng of each NGP were diluted in 0.2 M carbonate-bicarbonate buffer (pH 9.6), immobilized on 96-well MaxiSorp microplates (NUNC, Thermo Fisher Scientific) and incubated overnight at 4 C. Free binding sites were blocked with 200 l/well of 1% bovine serum albumin (BSA) in 1 phosphate buffered saline (PBS, pH 7.4). 50 l of Chagasic and normal human sera ( 1/800) or purified antibodies (1 g/ml) were added as primary antibodies diluted in 1% BSA-PBS with 0.05% Tween 20 (Promega). 50 l of goat anti-human IgG (H+L) biotin conjugated (Thermo Fisher Scientific) ( 1/10,000) diluted in 1% BSA-PBS with 0.05% Tween 20 was added as secondary antibody. 50 l of High Sensitivity NeutrAvidin-HRP (Thermo Fisher Scientific) ( 1/5,000) diluted in 1% BSA-PBS with 0.05% Tween 20 was then added. Finally, microplate was developed by adding 50 L of SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific), diluted in 0.2 M carbonate-bicarbonate buffer, pH 9.6 with 0.1% BSA. Relative luminescent units (RLU) were obtained using a Luminoskan luminometer (Labsystems, Thermo). All incubations between steps were carried out for 1 hour at 37 C. Microplates were washed three times with PBS-0.05% Tween 20 between all steps except before blocking.
Example 2
Assessment of Immune Response in A1,3Gal T-KO Mouse Model
(81) The immune response of 1,3GalT-KO mice (C57BL/6 background) was evaluated against the immunodominant Gal3LN epitope, covalently linked to bovine serum albumin (BSA) as carrier protein. The Gal3LN-BSA-vaccinated group showed significantly lower initial parasitemia than the control groups (BSA or PBS) (
(82) It is established that CD4+ T cells play a critical role in the control of ChD. Here, a protocol was developed to evaluate the role of CD4+ T cells in 1,3GalT-KO mice immunized with Gal3LN-BSA and challenged with mammalian cell tissue culture-derived Trypanosoma cruzi trypomastigotes (TCTs). Mice were depleted of CD4.sup.+ T cells by a single injection of 500 g anti-CD4 mAb (clone gK1.5, BD). A day later, the depletion efficiency (98%) was assessed in the total blood by flow cytometry. Two control groups without any immunization, in which mice were depleted then infected or infected then depleted, were also evaluated. All mice infected before or after the depletion died within 30 days, thus validating the crucial role of CD4.sup.+ T cells in the 1,3GalT-KO mouse model of ChD (
(83) Abbreviations use throughout this specification include: 1,3-GalT-KO, 1,3-galactosyltransferase-knockout; Abs, antibodies; AcSH, thioacetic acid; AIBN, azobisisobutyronitrile; BF.sub.3-Et.sub.2O, boron trifluoride etherate; BSA, bovine serum albumin; Ch anti--Gal, anti--Gal antibodies purified from sera of patients with chronic Chagas disease; ChD, Chagas disease; CL-ELISA, chemiluminescent ELISA; ChHSP, pooled sera of chronic Chagas disease patients; DBU, 1,8-diazabicycloundec-7-ene; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DTBS, di-tertbutylsilyl; DTB S(OTf).sub.2, di-tertbutylsilyl bis(trifluoromethanesulfonate); FPLC, fast protein liquid chromatography; HF-pyr, hydrogen fluoride in pyridine; MALDI-TOF, Matrix-assisted laser desorption ionization Time-of-Flight; NGP, neoglycoprotein; NHSP, normal human serum pool; NHS anti--Gal, anti--Gal antibodies from sera of healthy individuals; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; PMB, para-methoxybenzyl; RLU, relative luminescence units; TCEP, tris(2-carboxyethyl)phosphine; tGPI-mucins, trypomastigote-derived GPI-mucins; TMSOTf, trimethylsilyl trifluoromethanesulfonate
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