WO2011149778A1 - Synthetic oligosaccharides for neisseria meningitis vaccine - Google Patents
Synthetic oligosaccharides for neisseria meningitis vaccine Download PDFInfo
- Publication number
- WO2011149778A1 WO2011149778A1 PCT/US2011/037364 US2011037364W WO2011149778A1 WO 2011149778 A1 WO2011149778 A1 WO 2011149778A1 US 2011037364 W US2011037364 W US 2011037364W WO 2011149778 A1 WO2011149778 A1 WO 2011149778A1
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- WIPO (PCT)
- Prior art keywords
- acyl
- oligosaccharide
- antibody
- mmol
- meningitidis
- Prior art date
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- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
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- RZWZRACFZGVKFM-UHFFFAOYSA-N propanoyl chloride Chemical compound CCC(Cl)=O RZWZRACFZGVKFM-UHFFFAOYSA-N 0.000 description 1
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- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
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- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
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- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 1
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- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
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- 125000001544 thienyl group Chemical group 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
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- XETCRXVKJHBPMK-MJSODCSWSA-N trehalose 6,6'-dimycolate Chemical compound C([C@@H]1[C@H]([C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](COC(=O)C(CCCCCCCCCCC3C(C3)CCCCCCCCCCCCCCCCCC)C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)O2)O)O1)O)OC(=O)C(C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)CCCCCCCCCCC1CC1CCCCCCCCCCCCCCCCCC XETCRXVKJHBPMK-MJSODCSWSA-N 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 229940117013 triethanolamine oleate Drugs 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
- C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
- C07H5/06—Aminosugars
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/22—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Neisseriaceae (F), e.g. Acinetobacter
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Definitions
- the present invention relates to homogenous synthetic oligosaccharides, conjugates, and immunogenic and immunoprotective compositions derived therefrom.
- the present invention also relates to methods for making and using such composititions or antibodies thereto to prevent or treat diseases caused by Neisseria meningitidis bacteria, particularly group B (NmB) strains.
- NmB group B
- oligosaccharide derivatives and methods of their preparation and use for the prevention or treatment of diseases caused by Neisseria meningitidis and E. coli. They describe a de-N-acetylated oligosaccharide derivative in which one or more residues of the oligosaccharide has been modified by de-N-acetylation. Further, they describe de-N-acetylated oligosaccharide derivatives as well as conjugates in which the de-N-acetylated oligosaccharide derivative is linked to a carrier, e.g., a carrier protein, via its non-reducing terminus.
- a carrier e.g., a carrier protein
- the present invention provides a synthetic oligosaccharide 1a:
- R 1 and R 2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R 1 can be the same or different; and X is H or a protecting group.
- the oligosaccharides of the present invention have a specific number of monosaccharide units and a fixed, defined pattern of acylated and non-acylated residues.
- the present invention further provides antigens 1 b:
- R 1 and R 2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R 1 can be the same or different; X is a linker; and Y is H or a carrier.
- the present invention also includes compositions comprising an antigen 1 b and a pharmaceutically acceptable vehicle.
- the composition contains a single antigen or a known, defined mixture of antigens.
- the invention further provides vaccine compositions, including
- immunogenic and immunoprotective compositions comprising antigen 1 b and a pharmaceutically acceptable vehicle.
- These vaccine compositions can optionally include a pharmaceutically acceptable adjuvant.
- the vaccine is particularly preferred.
- compositions are endotoxin-free.
- the invention further provides a method for synthetically forming
- the invention further provides methods for diagnosing, treating, and preventing infections caused by N. meningitidis, particularly serotypes NmA, NmB, NmC and NmW-135 (See J Immunol 2009, 182:6610), particularly preferably NmB.
- FIG. 1 depicts the naturally occurring oligosaccharide associated with N. meningitidis.
- FIG. 2 depicts reaction scheme for forming building blocks as described in Example 1 .
- FIG. 3 depicts the assembly of exemplary protected oligosaccharides 1a of the present invention.
- FIG. 4 depicts the assembly of exemplary deprotected oligosaccharides 1a of the present invention.
- FIG. 5 depicts the conjugation of exemplary oligosaccharides 1a of the present invention to two carriers.
- oligosaccharide refers to a compound containing two or more monosaccharide units. Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the monosaccharide unit at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right.
- oligosaccharides described herein are described with the name or abbreviation for the non-reducing monosaccharide (e.g., Gal), preceded by the configuration of the glycosidic bond (a or ⁇ ), the ring bond, the ring position of the reducing monosaccharide involved in the bond, and then the name or
- the linkage between two sugars may be expressed, for example, as 2,3, 2 ⁇ 3, or 2-3.
- monosaccharide is a pyranose or furanose.
- monosaccharide or “monosaccharide unit” refers to a single sugar residue in an oligosaccharide, including derivatives therefrom. Within the context of an oligosaccharide, an individual monomer unit is a monosaccharide which is (or can be) bound through a hydroxyl group to another monosaccharide.
- endotoxin-free refers to an oligosaccharide that does not contain endotoxins or endotoxin components normally present in isolated bacterial carbohydrates and polysaccharides.
- synthetic refers to material with is assembled from monosaccharide building blocks that are chemically synthesized. These building blocks are coupled together to construct the oligosaccharide. Synthetic material is substantially or essentially free from components, such as endotoxins, glycolipids, oligosaccharides, etc., which normally accompany a compound when it is isolated. Typically, synthetic compounds are at least about 90% pure, usually at least about 95%, and preferably at least about 99% pure. Purity can be indicated by a number of means well known in the art. Preferably, purity is measured by HPLC. The identity of the synthetic material can be determined by mass spectroscopy and/or NMR spectroscopy.
- linker refers to either a bond or a moiety which at one end exhibits a grouping able to enter into a covalent bonding with a reactive functional group of the carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with a hydroxyl group or an amino group of an oligosaccharide according to the present invention.
- a biocompatible bridging molecule of suitable length, e.g.
- Linkers preferably include a substituted or unsubstituted (C1-C10) alkylene group or an substituted or unsubstituted (C2-C10) alkenylene group.
- protein carrier refers to a protein, peptide or fragment thereof, which is coupled or conjugated to an oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-protein carrier conjugate to a greater degree than the oligosaccharide alone.
- the protein carrier may serve as a T-dependent antigen which can activate and recruit T- cells and thereby augment T-cell dependent antibody production.
- conjugated refers to a chemical linkage, either covalent or non-covalent, that proximally associates an oligosaccharide with a carrier so that the oligosaccharide conjugate has increased immunogenicity relative to an unconjugated oligosaccharide.
- conjugate refers to an oligosaccharide chemically coupled to a carrier through a linker and/or a cross-linking agent.
- passive immunity refers to the administration of antibodies to a subject, whereby the antibodies are produced in a different subject (including subjects of the same and different species) such that the antibodies attach to the surface of the bacteria and cause the bacteria to be phagocytosed or killed.
- protection immunity means that a vaccine or
- immunization schedule that is administered to a animal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by a pathogen or diminishes or altogether eliminates the symptoms of the disease.
- Protective immunity may be predicted based on the ability of serum antibody to activate complement-mediated bactericidal activity or confer passive protection against a bacterial infection in a suitable animal challenge model.
- immunoprotective composition refers to a composition formulated to provide protective immunity in a host.
- Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunoassay
- antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab') 2 fragments, F(ab) molecules, Fv fragments, single chain fragment variable displayed on phage (scFv), single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule.
- monoclonal antibody refers to an antibody composition having a homogeneous antibody population.
- the term is not limited by the manner in which it is made.
- the term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab')2 fragments, Fv fragments, single chain fragment variable displayed on phage (scFv), and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule.
- telomere binding reaction which is based on and/or is probative of the presence of the antigen in a sample which may also include a heterogeneous population of other molecules.
- the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and does not bind in a significant amount to other molecules present in the sample.
- Specific binding to an antibody under such conditions may require an antibody or antiserum that is selected for its specificity for a particular antigen or antigens.
- antigen refers to any substance that may be specifically bound by an antibody molecule.
- immunogen and “immunogenic composition” refer to an antigenic composition capable of initiating lymphocyte activation resulting in an antigen-specific immune response.
- epitope refers to a site on an antigen to which specific B cells and/or T cells respond.
- the term is also used interchangeably with "antigenic determinant” or "antigenic determinant site.”
- B cell epitope sites on proteins, oligosaccharides, or other biopolymers may be composed of moieties from different parts of the macromolecule that have been brought together by folding. Epitopes of this kind are referred to as conformational or discontinuous epitopes, since the site is composed of segments the polymer that are discontinuous in the linear sequence but are continuous in the folded conformation(s). Epitopes that are composed of single segments of biopolymers or other molecules are termed continuous or linear epitopes.
- T cell epitopes are generally restricted to linear peptides. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
- Ac means acetyl (-C(O)CH 3 ).
- TBS means tert-butyldimethylsilyl
- Troc means 2,2,2-trichloroethoxycarbonyl.
- TCI means trichloroacetimidate
- Phth means phthaloyl
- TFA means trifluoroacetyl
- TCA means trichloroacetyl
- Cbz means benzyloxycarbonyl.
- Bz means benzoyl
- Bn means benzyl
- TES means triethylsilyl
- TBDPS means tert-butyldiphenylsilyl.
- MCA monochloracetyl
- Lev levulinoyl
- ADMB means 4-O-acetyl 2,2 dimethylbutanoyl
- Tr triphenylmethyl
- DMT dimethoxytrityl
- FMOC means 9-fluorenylmethoxycarbonyl.
- Alloc means Allyloxycarbonyl.
- Nap means naphthyl
- SEt means thioethyl
- SPh means thiophenyl
- STol means thiotolyl
- SAdm means thioadamantanyl.
- Synthetic oligosaccharides
- the present invention provides oligosaccharides 1a:
- R 1 and R 2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R 1 can be the same or different; and X is H or a protecting group.
- the present invention further provides antigens 1 b:
- R 1 and R 2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R 1 can be the same or different; L is a linker; and Y is H or a carrier.
- Preferred acyl groups are propionyl or acetyl. When more than one acyl group is present in the oligosaccharide, each can be the same or a different acyl group.
- n#, R1 i.e., n1 , R1 )
- n1 , R1 oligosaccharide of n units.
- the first unit is attached via a 2, 8 linkage to the monosaccharide bearing R 2 and the second, third, etc. units follow.
- n is 3
- the oligosaccharide is shown below:
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Abstract
The present invention provides synthetic oligosaccharides and conjugates thereof. The oligosaccharides may be synthesized by a chemical assembly methodology relying on a limited number of monosaccharide and disaccharide building blocks. The invention further provides immunogenic and immunoprotective compositions and antibodies derived therefrom for diagnosing, treating, and preventing infections caused by N. meningitidis.
Description
SYNTHETIC OLIGOSACCHARIDES FOR NEISSERIA MENINGITIS VACCINE
FIELD OF THE INVENTION
[0001] The present invention relates to homogenous synthetic oligosaccharides, conjugates, and immunogenic and immunoprotective compositions derived therefrom. The present invention also relates to methods for making and using such composititions or antibodies thereto to prevent or treat diseases caused by Neisseria meningitidis bacteria, particularly group B (NmB) strains.
BACKGROUND
[0002] Moe et al. (U.S. 7,595,307) describe compositions comprising
oligosaccharide derivatives, and methods of their preparation and use for the prevention or treatment of diseases caused by Neisseria meningitidis and E. coli. They describe a de-N-acetylated oligosaccharide derivative in which one or more residues of the oligosaccharide has been modified by de-N-acetylation. Further, they describe de-N-acetylated oligosaccharide derivatives as well as conjugates in which the de-N-acetylated oligosaccharide derivative is linked to a carrier, e.g., a carrier protein, via its non-reducing terminus.
SUMMARY
[0003] The present invention provides a synthetic oligosaccharide 1a:
where R1 and R2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R1 can be the same or different; and X is H or a protecting group.
[0004] The oligosaccharides of the present invention have a specific number of monosaccharide units and a fixed, defined pattern of acylated and non-acylated residues.
[0005] The present invention further provides antigens 1 b:
where R1 and R2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R1 can be the same or different; X is a linker; and Y is H or a carrier.
[0006] The present invention also includes compositions comprising an antigen 1 b and a pharmaceutically acceptable vehicle. Preferably the composition contains a single antigen or a known, defined mixture of antigens.
[0007] The invention further provides vaccine compositions, including
immunogenic and immunoprotective compositions, comprising antigen 1 b and a pharmaceutically acceptable vehicle. These vaccine compositions can optionally include a pharmaceutically acceptable adjuvant. Preferably, the vaccine
compositions are endotoxin-free.
[0008] The invention further provides a method for synthetically forming
oligosaccharides 1a and antigens 1 b.
[0009] The invention further provides methods for diagnosing, treating, and preventing infections caused by N. meningitidis, particularly serotypes NmA, NmB, NmC and NmW-135 (See J Immunol 2009, 182:6610), particularly preferably NmB.
BRI EF DESCRI PTION OF THE DRAWINGS
[0010] FIG. 1 depicts the naturally occurring oligosaccharide associated with N. meningitidis.
[0011] FIG. 2 depicts reaction scheme for forming building blocks as described in Example 1 .
[0012] FIG. 3 depicts the assembly of exemplary protected oligosaccharides 1a of the present invention.
[0013] FIG. 4 depicts the assembly of exemplary deprotected oligosaccharides 1a of the present invention.
[0014] FIG. 5 depicts the conjugation of exemplary oligosaccharides 1a of the present invention to two carriers.
DETAILED DESCRIPTION
[0015] Definitions
[0016] In order to provide a clear and consistent understanding of the
specification and claims, the following definitions are provided.
[0017] Units, prefixes, and symbols may be denoted in their SI accepted form. Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. Unless otherwise noted, the terms "a" or "an" are to be construed as meaning "at least one of." The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
[0018] As used herein, "oligosaccharide" refers to a compound containing two or more monosaccharide units. Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the monosaccharide unit at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right. All oligosaccharides described herein are described with the name or abbreviation for the non-reducing monosaccharide (e.g., Gal), preceded by the configuration of the glycosidic bond (a or β), the ring bond, the ring position of the reducing monosaccharide involved in the bond, and then the name or
abbreviation of the reducing monosaccharide (e.g., GlcNAc). The linkage between two sugars may be expressed, for example, as 2,3, 2→3, or 2-3. Each
monosaccharide is a pyranose or furanose.
[0019] As used herein, "monosaccharide" or "monosaccharide unit" refers to a single sugar residue in an oligosaccharide, including derivatives therefrom. Within the context of an oligosaccharide, an individual monomer unit is a monosaccharide which is (or can be) bound through a hydroxyl group to another monosaccharide.
[0020] As used herein, "endotoxin-free" refers to an oligosaccharide that does not contain endotoxins or endotoxin components normally present in isolated bacterial carbohydrates and polysaccharides.
[0021] As used herein, "synthetic" refers to material with is assembled from monosaccharide building blocks that are chemically synthesized. These building blocks are coupled together to construct the oligosaccharide. Synthetic material is substantially or essentially free from components, such as endotoxins, glycolipids, oligosaccharides, etc., which normally accompany a compound when it is isolated. Typically, synthetic compounds are at least about 90% pure, usually at least about 95%, and preferably at least about 99% pure. Purity can be indicated by a number of means well known in the art. Preferably, purity is measured by HPLC. The identity of the synthetic material can be determined by mass spectroscopy and/or NMR spectroscopy.
[0022] As used herein the term "linker" refers to either a bond or a moiety which at one end exhibits a grouping able to enter into a covalent bonding with a reactive functional group of the carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with a hydroxyl group or an amino group of an oligosaccharide according to the present invention. Between the two functional groups of the linker molecule there is a biocompatible bridging molecule of suitable length, e.g. substituted or unsubstituted heteroalkylene, arylalkylene, alkylene, alkenylene, or (oligo)alkylene glycol groups. Linkers preferably include a substituted or unsubstituted (C1-C10) alkylene group or an substituted or unsubstituted (C2-C10) alkenylene group.
[0023] As used herein, the term "carrier" refers to a protein, peptide, lipid, polymer, dendrimer, virosome, virus-like particle (VLP), or combination thereof, which is coupled to the oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-carrier conjugate to a greater degree than the
oligosaccharide alone.
[0024] As used herein, "protein carrier" refers to a protein, peptide or fragment thereof, which is coupled or conjugated to an oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-protein carrier conjugate to a greater degree than the oligosaccharide alone. For example, when used as a carrier, the
protein carrier may serve as a T-dependent antigen which can activate and recruit T- cells and thereby augment T-cell dependent antibody production.
[0025] As used herein, "conjugated" refers to a chemical linkage, either covalent or non-covalent, that proximally associates an oligosaccharide with a carrier so that the oligosaccharide conjugate has increased immunogenicity relative to an unconjugated oligosaccharide.
[0026] As used herein, "conjugate" refers to an oligosaccharide chemically coupled to a carrier through a linker and/or a cross-linking agent.
[0027] As used herein, "passive immunity" refers to the administration of antibodies to a subject, whereby the antibodies are produced in a different subject (including subjects of the same and different species) such that the antibodies attach to the surface of the bacteria and cause the bacteria to be phagocytosed or killed.
[0028] As used herein, "protective immunity" means that a vaccine or
immunization schedule that is administered to a animal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by a pathogen or diminishes or altogether eliminates the symptoms of the disease. Protective immunity may be predicted based on the ability of serum antibody to activate complement-mediated bactericidal activity or confer passive protection against a bacterial infection in a suitable animal challenge model.
[0029] As used herein, "immunoprotective composition" refers to a composition formulated to provide protective immunity in a host.
[0030] As used herein, "in a sufficient amount to elicit an immune response" or "in an effective amount to stimulate an immune response" (e.g., to epitopes present in a preparation) means that there is a detectable difference between an immune response indicator measured before and after administration of a particular antigen preparation. Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunoassay
(ELISA), bactericidal assay (e.g., to detect serum bactericidal antibodies), flow cytometry, immunoprecipitation, Ouchter-Lowry immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays; cytotoxicity assays, and the like.
[0031] As used herein, "antibody" encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab')2 fragments, F(ab) molecules, Fv fragments, single chain fragment variable displayed on phage (scFv), single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule.
[0032] As used herein, "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited by the manner in which it is made. The term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab')2 fragments, Fv fragments, single chain fragment variable displayed on phage (scFv), and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule.
[0033] As used herein, "specifically binds to an antibody" or "specifically immunoreactive with", when referring to an oligosaccharide, protein or peptide, refers to a binding reaction which is based on and/or is probative of the presence of the antigen in a sample which may also include a heterogeneous population of other molecules. Thus, under designated immunoassay conditions, the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and does not bind in a significant amount to other molecules present in the sample. Specific binding to an antibody under such conditions may require an antibody or antiserum that is selected for its specificity for a particular antigen or antigens.
[0034] As used herein, "antigen" refers to any substance that may be specifically bound by an antibody molecule.
[0035] As used herein, "immunogen" and "immunogenic composition" refer to an antigenic composition capable of initiating lymphocyte activation resulting in an antigen-specific immune response.
[0036] As used herein, "epitope" refers to a site on an antigen to which specific B cells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." B cell epitope sites on proteins, oligosaccharides, or other biopolymers may be composed of moieties from different parts of the macromolecule that have been brought together by folding. Epitopes of this kind are referred to as conformational or discontinuous epitopes, since the site is
composed of segments the polymer that are discontinuous in the linear sequence but are continuous in the folded conformation(s). Epitopes that are composed of single segments of biopolymers or other molecules are termed continuous or linear epitopes. T cell epitopes are generally restricted to linear peptides. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
[0037] The term Ac means acetyl (-C(O)CH3).
[0038] The term TBS means tert-butyldimethylsilyl.
[0039] The term Troc means 2,2,2-trichloroethoxycarbonyl.
[0040] The term TCI means trichloroacetimidate.
[0041] The term Phth means phthaloyl.
[0042] The term TFA means trifluoroacetyl.
[0043] The term TCA means trichloroacetyl.
[0044] The term Cbz means benzyloxycarbonyl.
[0045] The term Bz means benzoyl.
[0046] The term Bn means benzyl.
[0047] The term TES means triethylsilyl.
[0048] The term TBDPS means tert-butyldiphenylsilyl.
[0049] The term MCA means monochloracetyl.
[0050] The term Lev means levulinoyl.
[0051] The term ADMB means 4-O-acetyl 2,2 dimethylbutanoyl
[0052] The term Tr means triphenylmethyl.
[0053] The term DMT means dimethoxytrityl.
[0054] The term FMOC means 9-fluorenylmethoxycarbonyl.
[0055] The term Alloc means Allyloxycarbonyl.
[0056] The term Nap means naphthyl.
[0057] The term SEt means thioethyl.
[0058] The term SPh means thiophenyl.
[0059] The term STol means thiotolyl.
[0060] The term SAdm means thioadamantanyl.
[0061] Synthetic oligosaccharides
[0062] In one aspect, the present invention provides oligosaccharides 1a:
[0063] where R1 and R2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R1 can be the same or different; and X is H or a protecting group.
[0064] The present invention further provides antigens 1 b:
[0065] where R1 and R2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R1 can be the same or different; L is a linker; and Y is H or a carrier.
[0066] Preferred acyl groups are propionyl or acetyl. When more than one acyl group is present in the oligosaccharide, each can be the same or a different acyl group.
[0067] Specific embodiments of the present invention are shown below and include disaccharides (Table 1 ), trisaccharides (Table 2), tetrasaccharides (Table 3), pentasaccharides (Table 4), hexasaccharides (Table 5), heptasaccharides (Table 6), octasaccharides (Table 7), nonasaccharides (Table 8), decasaccharides (Table 9).
[0068] In the following tables, the nomenclature "n#, R1 " (i.e., n1 , R1 )" identifies the monomeric unit in an oligosaccharide of n units. The first unit is attached via a 2, 8 linkage to the monosaccharide bearing R2 and the second, third, etc. units follow. For example, when n is 3, the oligosaccharide is shown below:
Table 1
Table 2
Table 3
compound R2 nl, Rl n2, Rl n3, Rl
1013. H H H H
1014. H H H acyl
1015. H H acyl H
1016. H H acyl acyl
1017. H acyl H H
1018. H acyl H acyl
1019. H acyl acyl H
1020. H acyl acyl acyl
1021. acyl H H H
compound 2 nl, Rl n2, Rl n3, Rl
1022. acyl H H acyl
1023. acyl H acyl H
1024. acyl H acyl acyl
1025. acyl acyl H H
1026. acyl acyl H acyl
1027. acyl acyl acyl H
1028. acyl acyl acyl acyl
Table 4
compound R2 nl, Rl n2, Rl n3, Rl n4, Rl
1029. H H H H H
1030. H H H H acyl
1031. H H H acyl H
1032. H H H acyl acyl
1033. H H acyl H H
1034. H H acyl H acyl
1035. H H acyl acyl H
1036. H H acyl acyl acyl
1037. H acyl H H H
1038. H acyl H H acyl
1039. H acyl H acyl H
1040. H acyl H acyl acyl
1041. H acyl acyl H H
1042. H acyl acyl H acyl
1043. H acyl acyl acyl H
1044. H acyl acyl acyl acyl
1045. acyl H H H H
1046. acyl H H H acyl
1047. acyl H H acyl H
1048. acyl H H acyl acyl
1049. acyl H acyl H H
1050. acyl H acyl H acyl
1051. acyl H acyl acyl H
1052. acyl H acyl acyl acyl
1053. acyl acyl H H H
1054. acyl acyl H H acyl
1055. acyl acyl H acyl H
1056. acyl acyl H acyl acyl
1057. acyl acyl acyl H H
1058. acyl acyl acyl H acyl
1059. acyl acyl acyl acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl
1060. acyl acyl acyl acyl acyl
Table 5
compound R2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl
1061. H H H H H H
1062. H H H H H acyl
1063. H H H H acyl H
1064. H H H H acyl acyl
1065. H H H acyl H H
1066. H H H acyl H acyl
1067. H H H acyl acyl H
1068. H H H acyl acyl acyl
1069. H H acyl H H H
1070. H H acyl H H acyl
1071. H H acyl H acyl H
1072. H H acyl H acyl acyl
1073. H H acyl acyl H H
1074. H H acyl acyl H acyl
1075. H H acyl acyl acyl H
1076. H H acyl acyl acyl acyl
1077. H acyl H H H H
1078. H acyl H H H acyl
1079. H acyl H H acyl H
1080. H acyl H H acyl acyl
1081. H acyl H acyl H H
1082. H acyl H acyl H acyl
1083. H acyl H acyl acyl H
1084. H acyl H acyl acyl acyl
1085. H acyl acyl H H H
1086. H acyl acyl H H acyl
1087. H acyl acyl H acyl H
1088. H acyl acyl H acyl acyl
1089. H acyl acyl acyl H H
1090. H acyl acyl acyl H acyl
1091. H acyl acyl acyl acyl H
1092. H acyl acyl acyl acyl acyl
1093. acyl H H H H H
1094. acyl H H H H acyl
1095. acyl H H H acyl H
1096. acyl H H H acyl acyl
1097. acyl H H acyl H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl
1098. acyl H H acyl H acyl
1099. acyl H H acyl acyl H
1100. acyl H H acyl acyl acyl
1101. acyl H acyl H H H
1102. acyl H acyl H H acyl
1103. acyl H acyl H acyl H
1104. acyl H acyl H acyl acyl
1105. acyl H acyl acyl H H
1106. acyl H acyl acyl H acyl
1107. acyl H acyl acyl acyl H
1108. acyl H acyl acyl acyl acyl
1109. acyl acyl H H H H
1110. acyl acyl H H H acyl
1111. acyl acyl H H acyl H
1112. acyl acyl H H acyl acyl
1113. acyl acyl H acyl H H
1114. acyl acyl H acyl H acyl
1115. acyl acyl H acyl acyl H
1116. acyl acyl H acyl acyl acyl
1117. acyl acyl acyl H H H
1118. acyl acyl acyl H H acyl
1119. acyl acyl acyl H acyl H
1120. acyl acyl acyl H acyl acyl
1121. acyl acyl acyl acyl H H
1122. acyl acyl acyl acyl H acyl
1123. acyl acyl acyl acyl acyl H
1124. acyl acyl acyl acyl acyl acyl
Table 6
compound R2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl
1125. H H H H H H H
1126. H H H H H H acyl
1127. H H H H H acyl H
1128. H H H H H acyl acyl
1129. H H H H acyl H H
1130. H H H H acyl H acyl
1131. H H H H acyl acyl H
1132. H H H H acyl acyl acyl
1133. H H H acyl H H H
1134. H H H acyl H H acyl
1135. H H H acyl H acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl
1136. H H H acyl H acyl acyl
1137. H H H acyl acyl H H
1138. H H H acyl acyl H acyl
1139. H H H acyl acyl acyl H
1140. H H H acyl acyl acyl acyl
1141. H H acyl H H H H
1142. H H acyl H H H acyl
1143. H H acyl H H acyl H
1144. H H acyl H H acyl acyl
1145. H H acyl H acyl H H
1146. H H acyl H acyl H acyl
1147. H H acyl H acyl acyl H
1148. H H acyl H acyl acyl acyl
1149. H H acyl acyl H H H
1150. H H acyl acyl H H acyl
1151. H H acyl acyl H acyl H
1152. H H acyl acyl H acyl acyl
1153. H H acyl acyl acyl H H
1154. H H acyl acyl acyl H acyl
1155. H H acyl acyl acyl acyl H
1156. H H acyl acyl acyl acyl acyl
1157. H acyl H H H H H
1158. H acyl H H H H acyl
1159. H acyl H H H acyl H
1160. H acyl H H H acyl acyl
1161. H acyl H H acyl H H
1162. H acyl H H acyl H acyl
1163. H acyl H H acyl acyl H
1164. H acyl H H acyl acyl acyl
1165. H acyl H acyl H H H
1166. H acyl H acyl H H acyl
1167. H acyl H acyl H acyl H
1168. H acyl H acyl H acyl acyl
1169. H acyl H acyl acyl H H
1170. H acyl H acyl acyl H acyl
1171. H acyl H acyl acyl acyl H
1172. H acyl H acyl acyl acyl acyl
1173. H acyl acyl H H H H
1174. H acyl acyl H H H acyl
1175. H acyl acyl H H acyl H
1176. H acyl acyl H H acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl
1177. H acyl acyl H acyl H H
1178. H acyl acyl H acyl H acyl
1179. H acyl acyl H acyl acyl H
1180. H acyl acyl H acyl acyl acyl
1181. H acyl acyl acyl H H H
1182. H acyl acyl acyl H H acyl
1183. H acyl acyl acyl H acyl H
1184. H acyl acyl acyl H acyl acyl
1185. H acyl acyl acyl acyl H H
1186. H acyl acyl acyl acyl H acyl
1187. H acyl acyl acyl acyl acyl H
1188. H acyl acyl acyl acyl acyl acyl
1189. acyl H H H H H H
1190. acyl H H H H H acyl
1191. acyl H H H H acyl H
1192. acyl H H H H acyl acyl
1193. acyl H H H acyl H H
1194. acyl H H H acyl H acyl
1195. acyl H H H acyl acyl H
1196. acyl H H H acyl acyl acyl
1197. acyl H H acyl H H H
1198. acyl H H acyl H H acyl
1199. acyl H H acyl H acyl H
1200. acyl H H acyl H acyl acyl
1201. acyl H H acyl acyl H H
1202. acyl H H acyl acyl H acyl
1203. acyl H H acyl acyl acyl H
1204. acyl H H acyl acyl acyl acyl
1205. acyl H acyl H H H H
1206. acyl H acyl H H H acyl
1207. acyl H acyl H H acyl H
1208. acyl H acyl H H acyl acyl
1209. acyl H acyl H acyl H H
1210. acyl H acyl H acyl H acyl
1211. acyl H acyl H acyl acyl H
1212. acyl H acyl H acyl acyl acyl
1213. acyl H acyl acyl H H H
1214. acyl H acyl acyl H H acyl
1215. acyl H acyl acyl H acyl H
1216. acyl H acyl acyl H acyl acyl
1217. acyl H acyl acyl acyl H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl
1218. acyl H acyl acyl acyl H acyl
1219. acyl H acyl acyl acyl acyl H
1220. acyl H acyl acyl acyl acyl acyl
1221. acyl acyl H H H H H
1222. acyl acyl H H H H acyl
1223. acyl acyl H H H acyl H
1224. acyl acyl H H H acyl acyl
1225. acyl acyl H H acyl H H
1226. acyl acyl H H acyl H acyl
1227. acyl acyl H H acyl acyl H
1228. acyl acyl H H acyl acyl acyl
1229. acyl acyl H acyl H H H
1230. acyl acyl H acyl H H acyl
1231. acyl acyl H acyl H acyl H
1232. acyl acyl H acyl H acyl acyl
1233. acyl acyl H acyl acyl H H
1234. acyl acyl H acyl acyl H acyl
1235. acyl acyl H acyl acyl acyl H
1236. acyl acyl H acyl acyl acyl acyl
1237. acyl acyl acyl H H H H
1238. acyl acyl acyl H H H acyl
1239. acyl acyl acyl H H acyl H
1240. acyl acyl acyl H H acyl acyl
1241. acyl acyl acyl H acyl H H
1242. acyl acyl acyl H acyl H acyl
1243. acyl acyl acyl H acyl acyl H
1244. acyl acyl acyl H acyl acyl acyl
1245. acyl acyl acyl acyl H H H
1246. acyl acyl acyl acyl H H acyl
1247. acyl acyl acyl acyl H acyl H
1248. acyl acyl acyl acyl H acyl acyl
1249. acyl acyl acyl acyl acyl H H
1250. acyl acyl acyl acyl acyl H acyl
1251. acyl acyl acyl acyl acyl acyl H
1252. acyl acyl acyl acyl acyl acyl acyl
Table 7
compound R2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl
1253. H H H H H H H H
1254. H H H H H H H acyl
1255. H H H H H H acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl
1256. H H H H H H acyl acyl
1257. H H H H H acyl H H
1258. H H H H H acyl H acyl
1259. H H H H H acyl acyl H
1260. H H H H H acyl acyl acyl
1261. H H H H acyl H H H
1262. H H H H acyl H H acyl
1263. H H H H acyl H acyl H
1264. H H H H acyl H acyl acyl
1265. H H H H acyl acyl H H
1266. H H H H acyl acyl H acyl
1267. H H H H acyl acyl acyl H
1268. H H H H acyl acyl acyl acyl
1269. H H H acyl H H H H
1270. H H H acyl H H H acyl
1271. H H H acyl H H acyl H
1272. H H H acyl H H acyl acyl
1273. H H H acyl H acyl H H
1274. H H H acyl H acyl H acyl
1275. H H H acyl H acyl acyl H
1276. H H H acyl H acyl acyl acyl
1277. H H H acyl acyl H H H
1278. H H H acyl acyl H H acyl
1279. H H H acyl acyl H acyl H
1280. H H H acyl acyl H acyl acyl
1281. H H H acyl acyl acyl H H
1282. H H H acyl acyl acyl H acyl
1283. H H H acyl acyl acyl acyl H
1284. H H H acyl acyl acyl acyl acyl
1285. H H acyl H H H H H
1286. H H acyl H H H H acyl
1287. H H acyl H H H acyl H
1288. H H acyl H H H acyl acyl
1289. H H acyl H H acyl H H
1290. H H acyl H H acyl H acyl
1291. H H acyl H H acyl acyl H
1292. H H acyl H H acyl acyl acyl
1293. H H acyl H acyl H H H
1294. H H acyl H acyl H H acyl
1295. H H acyl H acyl H acyl H
1296. H H acyl H acyl H acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl
1297. H H acyl H acyl acyl H H
1298. H H acyl H acyl acyl H acyl
1299. H H acyl H acyl acyl acyl H
1300. H H acyl H acyl acyl acyl acyl
1301. H H acyl acyl H H H H
1302. H H acyl acyl H H H acyl
1303. H H acyl acyl H H acyl H
1304. H H acyl acyl H H acyl acyl
1305. H H acyl acyl H acyl H H
1306. H H acyl acyl H acyl H acyl
1307. H H acyl acyl H acyl acyl H
1308. H H acyl acyl H acyl acyl acyl
1309. H H acyl acyl acyl H H H
1310. H H acyl acyl acyl H H acyl
1311. H H acyl acyl acyl H acyl H
1312. H H acyl acyl acyl H acyl acyl
1313. H H acyl acyl acyl acyl H H
1314. H H acyl acyl acyl acyl H acyl
1315. H H acyl acyl acyl acyl acyl H
1316. H H acyl acyl acyl acyl acyl acyl
1317. H acyl H H H H H H
1318. H acyl H H H H H acyl
1319. H acyl H H H H acyl H
1320. H acyl H H H H acyl acyl
1321. H acyl H H H acyl H H
1322. H acyl H H H acyl H acyl
1323. H acyl H H H acyl acyl H
1324. H acyl H H H acyl acyl acyl
1325. H acyl H H acyl H H H
1326. H acyl H H acyl H H acyl
1327. H acyl H H acyl H acyl H
1328. H acyl H H acyl H acyl acyl
1329. H acyl H H acyl acyl H H
1330. H acyl H H acyl acyl H acyl
1331. H acyl H H acyl acyl acyl H
1332. H acyl H H acyl acyl acyl acyl
1333. H acyl H acyl H H H H
1334. H acyl H acyl H H H acyl
1335. H acyl H acyl H H acyl H
1336. H acyl H acyl H H acyl acyl
1337. H acyl H acyl H acyl H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl
1338. H acyl H acyl H acyl H acyl
1339. H acyl H acyl H acyl acyl H
1340. H acyl H acyl H acyl acyl acyl
1341. H acyl H acyl acyl H H H
1342. H acyl H acyl acyl H H acyl
1343. H acyl H acyl acyl H acyl H
1344. H acyl H acyl acyl H acyl acyl
1345. H acyl H acyl acyl acyl H H
1346. H acyl H acyl acyl acyl H acyl
1347. H acyl H acyl acyl acyl acyl H
1348. H acyl H acyl acyl acyl acyl acyl
1349. H acyl acyl H H H H H
1350. H acyl acyl H H H H acyl
1351. H acyl acyl H H H acyl H
1352. H acyl acyl H H H acyl acyl
1353. H acyl acyl H H acyl H H
1354. H acyl acyl H H acyl H acyl
1355. H acyl acyl H H acyl acyl H
1356. H acyl acyl H H acyl acyl acyl
1357. H acyl acyl H acyl H H H
1358. H acyl acyl H acyl H H acyl
1359. H acyl acyl H acyl H acyl H
1360. H acyl acyl H acyl H acyl acyl
1361. H acyl acyl H acyl acyl H H
1362. H acyl acyl H acyl acyl H acyl
1363. H acyl acyl H acyl acyl acyl H
1364. H acyl acyl H acyl acyl acyl acyl
1365. H acyl acyl acyl H H H H
1366. H acyl acyl acyl H H H acyl
1367. H acyl acyl acyl H H acyl H
1368. H acyl acyl acyl H H acyl acyl
1369. H acyl acyl acyl H acyl H H
1370. H acyl acyl acyl H acyl H acyl
1371. H acyl acyl acyl H acyl acyl H
1372. H acyl acyl acyl H acyl acyl acyl
1373. H acyl acyl acyl acyl H H H
1374. H acyl acyl acyl acyl H H acyl
1375. H acyl acyl acyl acyl H acyl H
1376. H acyl acyl acyl acyl H acyl acyl
1377. H acyl acyl acyl acyl acyl H H
1378. H acyl acyl acyl acyl acyl H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl
1379. H acyl acyl acyl acyl acyl acyl H
1380. H acyl acyl acyl acyl acyl acyl acyl
1381. acyl H H H H H H H
1382. acyl H H H H H H acyl
1383. acyl H H H H H acyl H
1384. acyl H H H H H acyl acyl
1385. acyl H H H H acyl H H
1386. acyl H H H H acyl H acyl
1387. acyl H H H H acyl acyl H
1388. acyl H H H H acyl acyl acyl
1389. acyl H H H acyl H H H
1390. acyl H H H acyl H H acyl
1391. acyl H H H acyl H acyl H
1392. acyl H H H acyl H acyl acyl
1393. acyl H H H acyl acyl H H
1394. acyl H H H acyl acyl H acyl
1395. acyl H H H acyl acyl acyl H
1396. acyl H H H acyl acyl acyl acyl
1397. acyl H H acyl H H H H
1398. acyl H H acyl H H H acyl
1399. acyl H H acyl H H acyl H
1400. acyl H H acyl H H acyl acyl
1401. acyl H H acyl H acyl H H
1402. acyl H H acyl H acyl H acyl
1403. acyl H H acyl H acyl acyl H
1404. acyl H H acyl H acyl acyl acyl
1405. acyl H H acyl acyl H H H
1406. acyl H H acyl acyl H H acyl
1407. acyl H H acyl acyl H acyl H
1408. acyl H H acyl acyl H acyl acyl
1409. acyl H H acyl acyl acyl H H
1410. acyl H H acyl acyl acyl H acyl
1411. acyl H H acyl acyl acyl acyl H
1412. acyl H H acyl acyl acyl acyl acyl
1413. acyl H acyl H H H H H
1414. acyl H acyl H H H H acyl
1415. acyl H acyl H H H acyl H
1416. acyl H acyl H H H acyl acyl
1417. acyl H acyl H H acyl H H
1418. acyl H acyl H H acyl H acyl
1419. acyl H acyl H H acyl acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl
1420. acyl H acyl H H acyl acyl acyl
1421. acyl H acyl H acyl H H H
1422. acyl H acyl H acyl H H acyl
1423. acyl H acyl H acyl H acyl H
1424. acyl H acyl H acyl H acyl acyl
1425. acyl H acyl H acyl acyl H H
1426. acyl H acyl H acyl acyl H acyl
1427. acyl H acyl H acyl acyl acyl H
1428. acyl H acyl H acyl acyl acyl acyl
1429. acyl H acyl acyl H H H H
1430. acyl H acyl acyl H H H acyl
1431. acyl H acyl acyl H H acyl H
1432. acyl H acyl acyl H H acyl acyl
1433. acyl H acyl acyl H acyl H H
1434. acyl H acyl acyl H acyl H acyl
1435. acyl H acyl acyl H acyl acyl H
1436. acyl H acyl acyl H acyl acyl acyl
1437. acyl H acyl acyl acyl H H H
1438. acyl H acyl acyl acyl H H acyl
1439. acyl H acyl acyl acyl H acyl H
1440. acyl H acyl acyl acyl H acyl acyl
1441. acyl H acyl acyl acyl acyl H H
1442. acyl H acyl acyl acyl acyl H acyl
1443. acyl H acyl acyl acyl acyl acyl H
1444. acyl H acyl acyl acyl acyl acyl acyl
1445. acyl acyl H H H H H H
1446. acyl acyl H H H H H acyl
1447. acyl acyl H H H H acyl H
1448. acyl acyl H H H H acyl acyl
1449. acyl acyl H H H acyl H H
1450. acyl acyl H H H acyl H acyl
1451. acyl acyl H H H acyl acyl H
1452. acyl acyl H H H acyl acyl acyl
1453. acyl acyl H H acyl H H H
1454. acyl acyl H H acyl H H acyl
1455. acyl acyl H H acyl H acyl H
1456. acyl acyl H H acyl H acyl acyl
1457. acyl acyl H H acyl acyl H H
1458. acyl acyl H H acyl acyl H acyl
1459. acyl acyl H H acyl acyl acyl H
1460. acyl acyl H H acyl acyl acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl
1461. acyl acyl H acyl H H H H
1462. acyl acyl H acyl H H H acyl
1463. acyl acyl H acyl H H acyl H
1464. acyl acyl H acyl H H acyl acyl
1465. acyl acyl H acyl H acyl H H
1466. acyl acyl H acyl H acyl H acyl
1467. acyl acyl H acyl H acyl acyl H
1468. acyl acyl H acyl H acyl acyl acyl
1469. acyl acyl H acyl acyl H H H
1470. acyl acyl H acyl acyl H H acyl
1471. acyl acyl H acyl acyl H acyl H
1472. acyl acyl H acyl acyl H acyl acyl
1473. acyl acyl H acyl acyl acyl H H
1474. acyl acyl H acyl acyl acyl H acyl
1475. acyl acyl H acyl acyl acyl acyl H
1476. acyl acyl H acyl acyl acyl acyl acyl
1477. acyl acyl acyl H H H H H
1478. acyl acyl acyl H H H H acyl
1479. acyl acyl acyl H H H acyl H
1480. acyl acyl acyl H H H acyl acyl
1481. acyl acyl acyl H H acyl H H
1482. acyl acyl acyl H H acyl H acyl
1483. acyl acyl acyl H H acyl acyl H
1484. acyl acyl acyl H H acyl acyl acyl
1485. acyl acyl acyl H acyl H H H
1486. acyl acyl acyl H acyl H H acyl
1487. acyl acyl acyl H acyl H acyl H
1488. acyl acyl acyl H acyl H acyl acyl
1489. acyl acyl acyl H acyl acyl H H
1490. acyl acyl acyl H acyl acyl H acyl
1491. acyl acyl acyl H acyl acyl acyl H
1492. acyl acyl acyl H acyl acyl acyl acyl
1493. acyl acyl acyl acyl H H H H
1494. acyl acyl acyl acyl H H H acyl
1495. acyl acyl acyl acyl H H acyl H
1496. acyl acyl acyl acyl H H acyl acyl
1497. acyl acyl acyl acyl H acyl H H
1498. acyl acyl acyl acyl H acyl H acyl
1499. acyl acyl acyl acyl H acyl acyl H
1500. acyl acyl acyl acyl H acyl acyl acyl
1501. acyl acyl acyl acyl acyl H H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl nl, Rl
1502. acyl acyl acyl acyl acyl H H acyl
1503. acyl acyl acyl acyl acyl H acyl H
1504. acyl acyl acyl acyl acyl H acyl acyl
1505. acyl acyl acyl acyl acyl acyl H H
1506. acyl acyl acyl acyl acyl acyl H acyl
1507. acyl acyl acyl acyl acyl acyl acyl H
1508. acyl acyl acyl acyl acyl acyl acyl acyl
Table 8
compound R2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1509. H H H H H H H H H
1510. H H H H H H H H acyl
1511. H H H H H H H acyl H
1512. H H H H H H H acyl acyl
1513. H H H H H H acyl H H
1514. H H H H H H acyl H acyl
1515. H H H H H H acyl acyl H
1516. H H H H H H acyl acyl acyl
1517. H H H H H acyl H H H
1518. H H H H H acyl H H acyl
1519. H H H H H acyl H acyl H
1520. H H H H H acyl H acyl acyl
1521. H H H H H acyl acyl H H
1522. H H H H H acyl acyl H acyl
1523. H H H H H acyl acyl acyl H
1524. H H H H H acyl acyl acyl acyl
1525. H H H H acyl H H H H
1526. H H H H acyl H H H acyl
1527. H H H H acyl H H acyl H
1528. H H H H acyl H H acyl acyl
1529. H H H H acyl H acyl H H
1530. H H H H acyl H acyl H acyl
1531. H H H H acyl H acyl acyl H
1532. H H H H acyl H acyl acyl acyl
1533. H H H H acyl acyl H H H
1534. H H H H acyl acyl H H acyl
1535. H H H H acyl acyl H acyl H
1536. H H H H acyl acyl H acyl acyl
1537. H H H H acyl acyl acyl H H
1538. H H H H acyl acyl acyl H acyl
1539. H H H H acyl acyl acyl acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1540. H H H H acyl acyl acyl acyl acyl
1541. H H H acyl H H H H H
1542. H H H acyl H H H H acyl
1543. H H H acyl H H H acyl H
1544. H H H acyl H H H acyl acyl
1545. H H H acyl H H acyl H H
1546. H H H acyl H H acyl H acyl
1547. H H H acyl H H acyl acyl H
1548. H H H acyl H H acyl acyl acyl
1549. H H H acyl H acyl H H H
1550. H H H acyl H acyl H H acyl
1551. H H H acyl H acyl H acyl H
1552. H H H acyl H acyl H acyl acyl
1553. H H H acyl H acyl acyl H H
1554. H H H acyl H acyl acyl H acyl
1555. H H H acyl H acyl acyl acyl H
1556. H H H acyl H acyl acyl acyl acyl
1557. H H H acyl acyl H H H H
1558. H H H acyl acyl H H H acyl
1559. H H H acyl acyl H H acyl H
1560. H H H acyl acyl H H acyl acyl
1561. H H H acyl acyl H acyl H H
1562. H H H acyl acyl H acyl H acyl
1563. H H H acyl acyl H acyl acyl H
1564. H H H acyl acyl H acyl acyl acyl
1565. H H H acyl acyl acyl H H H
1566. H H H acyl acyl acyl H H acyl
1567. H H H acyl acyl acyl H acyl H
1568. H H H acyl acyl acyl H acyl acyl
1569. H H H acyl acyl acyl acyl H H
1570. H H H acyl acyl acyl acyl H acyl
1571. H H H acyl acyl acyl acyl acyl H
1572. H H H acyl acyl acyl acyl acyl acyl
1573. H H acyl H H H H H H
1574. H H acyl H H H H H acyl
1575. H H acyl H H H H acyl H
1576. H H acyl H H H H acyl acyl
1577. H H acyl H H H acyl H H
1578. H H acyl H H H acyl H acyl
1579. H H acyl H H H acyl acyl H
1580. H H acyl H H H acyl acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1581. H H acyl H H acyl H H H
1582. H H acyl H H acyl H H acyl
1583. H H acyl H H acyl H acyl H
1584. H H acyl H H acyl H acyl acyl
1585. H H acyl H H acyl acyl H H
1586. H H acyl H H acyl acyl H acyl
1587. H H acyl H H acyl acyl acyl H
1588. H H acyl H H acyl acyl acyl acyl
1589. H H acyl H acyl H H H H
1590. H H acyl H acyl H H H acyl
1591. H H acyl H acyl H H acyl H
1592. H H acyl H acyl H H acyl acyl
1593. H H acyl H acyl H acyl H H
1594. H H acyl H acyl H acyl H acyl
1595. H H acyl H acyl H acyl acyl H
1596. H H acyl H acyl H acyl acyl acyl
1597. H H acyl H acyl acyl H H H
1598. H H acyl H acyl acyl H H acyl
1599. H H acyl H acyl acyl H acyl H
1600. H H acyl H acyl acyl H acyl acyl
1601. H H acyl H acyl acyl acyl H H
1602. H H acyl H acyl acyl acyl H acyl
1603. H H acyl H acyl acyl acyl acyl H
1604. H H acyl H acyl acyl acyl acyl acyl
1605. H H acyl acyl H H H H H
1606. H H acyl acyl H H H H acyl
1607. H H acyl acyl H H H acyl H
1608. H H acyl acyl H H H acyl acyl
1609. H H acyl acyl H H acyl H H
1610. H H acyl acyl H H acyl H acyl
1611. H H acyl acyl H H acyl acyl H
1612. H H acyl acyl H H acyl acyl acyl
1613. H H acyl acyl H acyl H H H
1614. H H acyl acyl H acyl H H acyl
1615. H H acyl acyl H acyl H acyl H
1616. H H acyl acyl H acyl H acyl acyl
1617. H H acyl acyl H acyl acyl H H
1618. H H acyl acyl H acyl acyl H acyl
1619. H H acyl acyl H acyl acyl acyl H
1620. H H acyl acyl H acyl acyl acyl acyl
1621. H H acyl acyl acyl H H H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1622. H H acyl acyl acyl H H H acyl
1623. H H acyl acyl acyl H H acyl H
1624. H H acyl acyl acyl H H acyl acyl
1625. H H acyl acyl acyl H acyl H H
1626. H H acyl acyl acyl H acyl H acyl
1627. H H acyl acyl acyl H acyl acyl H
1628. H H acyl acyl acyl H acyl acyl acyl
1629. H H acyl acyl acyl acyl H H H
1630. H H acyl acyl acyl acyl H H acyl
1631. H H acyl acyl acyl acyl H acyl H
1632. H H acyl acyl acyl acyl H acyl acyl
1633. H H acyl acyl acyl acyl acyl H H
1634. H H acyl acyl acyl acyl acyl H acyl
1635. H H acyl acyl acyl acyl acyl acyl H
1636. H H acyl acyl acyl acyl acyl acyl acyl
1637. H acyl H H H H H H H
1638. H acyl H H H H H H acyl
1639. H acyl H H H H H acyl H
1640. H acyl H H H H H acyl acyl
1641. H acyl H H H H acyl H H
1642. H acyl H H H H acyl H acyl
1643. H acyl H H H H acyl acyl H
1644. H acyl H H H H acyl acyl acyl
1645. H acyl H H H acyl H H H
1646. H acyl H H H acyl H H acyl
1647. H acyl H H H acyl H acyl H
1648. H acyl H H H acyl H acyl acyl
1649. H acyl H H H acyl acyl H H
1650. H acyl H H H acyl acyl H acyl
1651. H acyl H H H acyl acyl acyl H
1652. H acyl H H H acyl acyl acyl acyl
1653. H acyl H H acyl H H H H
1654. H acyl H H acyl H H H acyl
1655. H acyl H H acyl H H acyl H
1656. H acyl H H acyl H H acyl acyl
1657. H acyl H H acyl H acyl H H
1658. H acyl H H acyl H acyl H acyl
1659. H acyl H H acyl H acyl acyl H
1660. H acyl H H acyl H acyl acyl acyl
1661. H acyl H H acyl acyl H H H
1662. H acyl H H acyl acyl H H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1663. H acyl H H acyl acyl H acyl H
1664. H acyl H H acyl acyl H acyl acyl
1665. H acyl H H acyl acyl acyl H H
1666. H acyl H H acyl acyl acyl H acyl
1667. H acyl H H acyl acyl acyl acyl H
1668. H acyl H H acyl acyl acyl acyl acyl
1669. H acyl H acyl H H H H H
1670. H acyl H acyl H H H H acyl
1671. H acyl H acyl H H H acyl H
1672. H acyl H acyl H H H acyl acyl
1673. H acyl H acyl H H acyl H H
1674. H acyl H acyl H H acyl H acyl
1675. H acyl H acyl H H acyl acyl H
1676. H acyl H acyl H H acyl acyl acyl
1677. H acyl H acyl H acyl H H H
1678. H acyl H acyl H acyl H H acyl
1679. H acyl H acyl H acyl H acyl H
1680. H acyl H acyl H acyl H acyl acyl
1681. H acyl H acyl H acyl acyl H H
1682. H acyl H acyl H acyl acyl H acyl
1683. H acyl H acyl H acyl acyl acyl H
1684. H acyl H acyl H acyl acyl acyl acyl
1685. H acyl H acyl acyl H H H H
1686. H acyl H acyl acyl H H H acyl
1687. H acyl H acyl acyl H H acyl H
1688. H acyl H acyl acyl H H acyl acyl
1689. H acyl H acyl acyl H acyl H H
1690. H acyl H acyl acyl H acyl H acyl
1691. H acyl H acyl acyl H acyl acyl H
1692. H acyl H acyl acyl H acyl acyl acyl
1693. H acyl H acyl acyl acyl H H H
1694. H acyl H acyl acyl acyl H H acyl
1695. H acyl H acyl acyl acyl H acyl H
1696. H acyl H acyl acyl acyl H acyl acyl
1697. H acyl H acyl acyl acyl acyl H H
1698. H acyl H acyl acyl acyl acyl H acyl
1699. H acyl H acyl acyl acyl acyl acyl H
1700. H acyl H acyl acyl acyl acyl acyl acyl
1701. H acyl acyl H H H H H H
1702. H acyl acyl H H H H H acyl
1703. H acyl acyl H H H H acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1704. H acyl acyl H H H H acyl acyl
1705. H acyl acyl H H H acyl H H
1706. H acyl acyl H H H acyl H acyl
1707. H acyl acyl H H H acyl acyl H
1708. H acyl acyl H H H acyl acyl acyl
1709. H acyl acyl H H acyl H H H
1710. H acyl acyl H H acyl H H acyl
1711. H acyl acyl H H acyl H acyl H
1712. H acyl acyl H H acyl H acyl acyl
1713. H acyl acyl H H acyl acyl H H
1714. H acyl acyl H H acyl acyl H acyl
1715. H acyl acyl H H acyl acyl acyl H
1716. H acyl acyl H H acyl acyl acyl acyl
1717. H acyl acyl H acyl H H H H
1718. H acyl acyl H acyl H H H acyl
1719. H acyl acyl H acyl H H acyl H
1720. H acyl acyl H acyl H H acyl acyl
1721. H acyl acyl H acyl H acyl H H
1722. H acyl acyl H acyl H acyl H acyl
1723. H acyl acyl H acyl H acyl acyl H
1724. H acyl acyl H acyl H acyl acyl acyl
1725. H acyl acyl H acyl acyl H H H
1726. H acyl acyl H acyl acyl H H acyl
1727. H acyl acyl H acyl acyl H acyl H
1728. H acyl acyl H acyl acyl H acyl acyl
1729. H acyl acyl H acyl acyl acyl H H
1730. H acyl acyl H acyl acyl acyl H acyl
1731. H acyl acyl H acyl acyl acyl acyl H
1732. H acyl acyl H acyl acyl acyl acyl acyl
1733. H acyl acyl acyl H H H H H
1734. H acyl acyl acyl H H H H acyl
1735. H acyl acyl acyl H H H acyl H
1736. H acyl acyl acyl H H H acyl acyl
1737. H acyl acyl acyl H H acyl H H
1738. H acyl acyl acyl H H acyl H acyl
1739. H acyl acyl acyl H H acyl acyl H
1740. H acyl acyl acyl H H acyl acyl acyl
1741. H acyl acyl acyl H acyl H H H
1742. H acyl acyl acyl H acyl H H acyl
1743. H acyl acyl acyl H acyl H acyl H
1744. H acyl acyl acyl H acyl H acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1745. H acyl acyl acyl H acyl acyl H H
1746. H acyl acyl acyl H acyl acyl H acyl
1747. H acyl acyl acyl H acyl acyl acyl H
1748. H acyl acyl acyl H acyl acyl acyl acyl
1749. H acyl acyl acyl acyl H H H H
1750. H acyl acyl acyl acyl H H H acyl
1751. H acyl acyl acyl acyl H H acyl H
1752. H acyl acyl acyl acyl H H acyl acyl
1753. H acyl acyl acyl acyl H acyl H H
1754. H acyl acyl acyl acyl H acyl H acyl
1755. H acyl acyl acyl acyl H acyl acyl H
1756. H acyl acyl acyl acyl H acyl acyl acyl
1757. H acyl acyl acyl acyl acyl H H H
1758. H acyl acyl acyl acyl acyl H H acyl
1759. H acyl acyl acyl acyl acyl H acyl H
1760. H acyl acyl acyl acyl acyl H acyl acyl
1761. H acyl acyl acyl acyl acyl acyl H H
1762. H acyl acyl acyl acyl acyl acyl H acyl
1763. H acyl acyl acyl acyl acyl acyl acyl H
1764. H acyl acyl acyl acyl acyl acyl acyl acyl
1765. acyl H H H H H H H H
1766. acyl H H H H H H H acyl
1767. acyl H H H H H H acyl H
1768. acyl H H H H H H acyl acyl
1769. acyl H H H H H acyl H H
1770. acyl H H H H H acyl H acyl
1771. acyl H H H H H acyl acyl H
1772. acyl H H H H H acyl acyl acyl
1773. acyl H H H H acyl H H H
1774. acyl H H H H acyl H H acyl
1775. acyl H H H H acyl H acyl H
1776. acyl H H H H acyl H acyl acyl
1777. acyl H H H H acyl acyl H H
1778. acyl H H H H acyl acyl H acyl
1779. acyl H H H H acyl acyl acyl H
1780. acyl H H H H acyl acyl acyl acyl
1781. acyl H H H acyl H H H H
1782. acyl H H H acyl H H H acyl
1783. acyl H H H acyl H H acyl H
1784. acyl H H H acyl H H acyl acyl
1785. acyl H H H acyl H acyl H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1786. acyl H H H acyl H acyl H acyl
1787. acyl H H H acyl H acyl acyl H
1788. acyl H H H acyl H acyl acyl acyl
1789. acyl H H H acyl acyl H H H
1790. acyl H H H acyl acyl H H acyl
1791. acyl H H H acyl acyl H acyl H
1792. acyl H H H acyl acyl H acyl acyl
1793. acyl H H H acyl acyl acyl H H
1794. acyl H H H acyl acyl acyl H acyl
1795. acyl H H H acyl acyl acyl acyl H
1796. acyl H H H acyl acyl acyl acyl acyl
1797. acyl H H acyl H H H H H
1798. acyl H H acyl H H H H acyl
1799. acyl H H acyl H H H acyl H
1800. acyl H H acyl H H H acyl acyl
1801. acyl H H acyl H H acyl H H
1802. acyl H H acyl H H acyl H acyl
1803. acyl H H acyl H H acyl acyl H
1804. acyl H H acyl H H acyl acyl acyl
1805. acyl H H acyl H acyl H H H
1806. acyl H H acyl H acyl H H acyl
1807. acyl H H acyl H acyl H acyl H
1808. acyl H H acyl H acyl H acyl acyl
1809. acyl H H acyl H acyl acyl H H
1810. acyl H H acyl H acyl acyl H acyl
1811. acyl H H acyl H acyl acyl acyl H
1812. acyl H H acyl H acyl acyl acyl acyl
1813. acyl H H acyl acyl H H H H
1814. acyl H H acyl acyl H H H acyl
1815. acyl H H acyl acyl H H acyl H
1816. acyl H H acyl acyl H H acyl acyl
1817. acyl H H acyl acyl H acyl H H
1818. acyl H H acyl acyl H acyl H acyl
1819. acyl H H acyl acyl H acyl acyl H
1820. acyl H H acyl acyl H acyl acyl acyl
1821. acyl H H acyl acyl acyl H H H
1822. acyl H H acyl acyl acyl H H acyl
1823. acyl H H acyl acyl acyl H acyl H
1824. acyl H H acyl acyl acyl H acyl acyl
1825. acyl H H acyl acyl acyl acyl H H
1826. acyl H H acyl acyl acyl acyl H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1827. acyl H H acyl acyl acyl acyl acyl H
1828. acyl H H acyl acyl acyl acyl acyl acyl
1829. acyl H acyl H H H H H H
1830. acyl H acyl H H H H H acyl
1831. acyl H acyl H H H H acyl H
1832. acyl H acyl H H H H acyl acyl
1833. acyl H acyl H H H acyl H H
1834. acyl H acyl H H H acyl H acyl
1835. acyl H acyl H H H acyl acyl H
1836. acyl H acyl H H H acyl acyl acyl
1837. acyl H acyl H H acyl H H H
1838. acyl H acyl H H acyl H H acyl
1839. acyl H acyl H H acyl H acyl H
1840. acyl H acyl H H acyl H acyl acyl
1841. acyl H acyl H H acyl acyl H H
1842. acyl H acyl H H acyl acyl H acyl
1843. acyl H acyl H H acyl acyl acyl H
1844. acyl H acyl H H acyl acyl acyl acyl
1845. acyl H acyl H acyl H H H H
1846. acyl H acyl H acyl H H H acyl
1847. acyl H acyl H acyl H H acyl H
1848. acyl H acyl H acyl H H acyl acyl
1849. acyl H acyl H acyl H acyl H H
1850. acyl H acyl H acyl H acyl H acyl
1851. acyl H acyl H acyl H acyl acyl H
1852. acyl H acyl H acyl H acyl acyl acyl
1853. acyl H acyl H acyl acyl H H H
1854. acyl H acyl H acyl acyl H H acyl
1855. acyl H acyl H acyl acyl H acyl H
1856. acyl H acyl H acyl acyl H acyl acyl
1857. acyl H acyl H acyl acyl acyl H H
1858. acyl H acyl H acyl acyl acyl H acyl
1859. acyl H acyl H acyl acyl acyl acyl H
1860. acyl H acyl H acyl acyl acyl acyl acyl
1861. acyl H acyl acyl H H H H H
1862. acyl H acyl acyl H H H H acyl
1863. acyl H acyl acyl H H H acyl H
1864. acyl H acyl acyl H H H acyl acyl
1865. acyl H acyl acyl H H acyl H H
1866. acyl H acyl acyl H H acyl H acyl
1867. acyl H acyl acyl H H acyl acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1868. acyl H acyl acyl H H acyl acyl acyl
1869. acyl H acyl acyl H acyl H H H
1870. acyl H acyl acyl H acyl H H acyl
1871. acyl H acyl acyl H acyl H acyl H
1872. acyl H acyl acyl H acyl H acyl acyl
1873. acyl H acyl acyl H acyl acyl H H
1874. acyl H acyl acyl H acyl acyl H acyl
1875. acyl H acyl acyl H acyl acyl acyl H
1876. acyl H acyl acyl H acyl acyl acyl acyl
1877. acyl H acyl acyl acyl H H H H
1878. acyl H acyl acyl acyl H H H acyl
1879. acyl H acyl acyl acyl H H acyl H
1880. acyl H acyl acyl acyl H H acyl acyl
1881. acyl H acyl acyl acyl H acyl H H
1882. acyl H acyl acyl acyl H acyl H acyl
1883. acyl H acyl acyl acyl H acyl acyl H
1884. acyl H acyl acyl acyl H acyl acyl acyl
1885. acyl H acyl acyl acyl acyl H H H
1886. acyl H acyl acyl acyl acyl H H acyl
1887. acyl H acyl acyl acyl acyl H acyl H
1888. acyl H acyl acyl acyl acyl H acyl acyl
1889. acyl H acyl acyl acyl acyl acyl H H
1890. acyl H acyl acyl acyl acyl acyl H acyl
1891. acyl H acyl acyl acyl acyl acyl acyl H
1892. acyl H acyl acyl acyl acyl acyl acyl acyl
1893. acyl acyl H H H H H H H
1894. acyl acyl H H H H H H acyl
1895. acyl acyl H H H H H acyl H
1896. acyl acyl H H H H H acyl acyl
1897. acyl acyl H H H H acyl H H
1898. acyl acyl H H H H acyl H acyl
1899. acyl acyl H H H H acyl acyl H
1900. acyl acyl H H H H acyl acyl acyl
1901. acyl acyl H H H acyl H H H
1902. acyl acyl H H H acyl H H acyl
1903. acyl acyl H H H acyl H acyl H
1904. acyl acyl H H H acyl H acyl acyl
1905. acyl acyl H H H acyl acyl H H
1906. acyl acyl H H H acyl acyl H acyl
1907. acyl acyl H H H acyl acyl acyl H
1908. acyl acyl H H H acyl acyl acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1909. acyl acyl H H acyl H H H H
1910. acyl acyl H H acyl H H H acyl
1911. acyl acyl H H acyl H H acyl H
1912. acyl acyl H H acyl H H acyl acyl
1913. acyl acyl H H acyl H acyl H H
1914. acyl acyl H H acyl H acyl H acyl
1915. acyl acyl H H acyl H acyl acyl H
1916. acyl acyl H H acyl H acyl acyl acyl
1917. acyl acyl H H acyl acyl H H H
1918. acyl acyl H H acyl acyl H H acyl
1919. acyl acyl H H acyl acyl H acyl H
1920. acyl acyl H H acyl acyl H acyl acyl
1921. acyl acyl H H acyl acyl acyl H H
1922. acyl acyl H H acyl acyl acyl H acyl
1923. acyl acyl H H acyl acyl acyl acyl H
1924. acyl acyl H H acyl acyl acyl acyl acyl
1925. acyl acyl H acyl H H H H H
1926. acyl acyl H acyl H H H H acyl
1927. acyl acyl H acyl H H H acyl H
1928. acyl acyl H acyl H H H acyl acyl
1929. acyl acyl H acyl H H acyl H H
1930. acyl acyl H acyl H H acyl H acyl
1931. acyl acyl H acyl H H acyl acyl H
1932. acyl acyl H acyl H H acyl acyl acyl
1933. acyl acyl H acyl H acyl H H H
1934. acyl acyl H acyl H acyl H H acyl
1935. acyl acyl H acyl H acyl H acyl H
1936. acyl acyl H acyl H acyl H acyl acyl
1937. acyl acyl H acyl H acyl acyl H H
1938. acyl acyl H acyl H acyl acyl H acyl
1939. acyl acyl H acyl H acyl acyl acyl H
1940. acyl acyl H acyl H acyl acyl acyl acyl
1941. acyl acyl H acyl acyl H H H H
1942. acyl acyl H acyl acyl H H H acyl
1943. acyl acyl H acyl acyl H H acyl H
1944. acyl acyl H acyl acyl H H acyl acyl
1945. acyl acyl H acyl acyl H acyl H H
1946. acyl acyl H acyl acyl H acyl H acyl
1947. acyl acyl H acyl acyl H acyl acyl H
1948. acyl acyl H acyl acyl H acyl acyl acyl
1949. acyl acyl H acyl acyl acyl H H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1950. acyl acyl H acyl acyl acyl H H acyl
1951. acyl acyl H acyl acyl acyl H acyl H
1952. acyl acyl H acyl acyl acyl H acyl acyl
1953. acyl acyl H acyl acyl acyl acyl H H
1954. acyl acyl H acyl acyl acyl acyl H acyl
1955. acyl acyl H acyl acyl acyl acyl acyl H
1956. acyl acyl H acyl acyl acyl acyl acyl acyl
1957. acyl acyl acyl H H H H H H
1958. acyl acyl acyl H H H H H acyl
1959. acyl acyl acyl H H H H acyl H
1960. acyl acyl acyl H H H H acyl acyl
1961. acyl acyl acyl H H H acyl H H
1962. acyl acyl acyl H H H acyl H acyl
1963. acyl acyl acyl H H H acyl acyl H
1964. acyl acyl acyl H H H acyl acyl acyl
1965. acyl acyl acyl H H acyl H H H
1966. acyl acyl acyl H H acyl H H acyl
1967. acyl acyl acyl H H acyl H acyl H
1968. acyl acyl acyl H H acyl H acyl acyl
1969. acyl acyl acyl H H acyl acyl H H
1970. acyl acyl acyl H H acyl acyl H acyl
1971. acyl acyl acyl H H acyl acyl acyl H
1972. acyl acyl acyl H H acyl acyl acyl acyl
1973. acyl acyl acyl H acyl H H H H
1974. acyl acyl acyl H acyl H H H acyl
1975. acyl acyl acyl H acyl H H acyl H
1976. acyl acyl acyl H acyl H H acyl acyl
1977. acyl acyl acyl H acyl H acyl H H
1978. acyl acyl acyl H acyl H acyl H acyl
1979. acyl acyl acyl H acyl H acyl acyl H
1980. acyl acyl acyl H acyl H acyl acyl acyl
1981. acyl acyl acyl H acyl acyl H H H
1982. acyl acyl acyl H acyl acyl H H acyl
1983. acyl acyl acyl H acyl acyl H acyl H
1984. acyl acyl acyl H acyl acyl H acyl acyl
1985. acyl acyl acyl H acyl acyl acyl H H
1986. acyl acyl acyl H acyl acyl acyl H acyl
1987. acyl acyl acyl H acyl acyl acyl acyl H
1988. acyl acyl acyl H acyl acyl acyl acyl acyl
1989. acyl acyl acyl acyl H H H H H
1990. acyl acyl acyl acyl H H H H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl
1991. acyl acyl acyl acyl H H H acyl H
1992. acyl acyl acyl acyl H H H acyl acyl
1993. acyl acyl acyl acyl H H acyl H H
1994. acyl acyl acyl acyl H H acyl H acyl
1995. acyl acyl acyl acyl H H acyl acyl H
1996. acyl acyl acyl acyl H H acyl acyl acyl
1997. acyl acyl acyl acyl H acyl H H H
1998. acyl acyl acyl acyl H acyl H H acyl
1999. acyl acyl acyl acyl H acyl H acyl H
2000. acyl acyl acyl acyl H acyl H acyl acyl
2001. acyl acyl acyl acyl H acyl acyl H H
2002. acyl acyl acyl acyl H acyl acyl H acyl
2003. acyl acyl acyl acyl H acyl acyl acyl H
2004. acyl acyl acyl acyl H acyl acyl acyl acyl
2005. acyl acyl acyl acyl acyl H H H H
2006. acyl acyl acyl acyl acyl H H H acyl
2007. acyl acyl acyl acyl acyl H H acyl H
2008. acyl acyl acyl acyl acyl H H acyl acyl
2009. acyl acyl acyl acyl acyl H acyl H H
2010. acyl acyl acyl acyl acyl H acyl H acyl
2011. acyl acyl acyl acyl acyl H acyl acyl H
2012. acyl acyl acyl acyl acyl H acyl acyl acyl
2013. acyl acyl acyl acyl acyl acyl H H H
2014. acyl acyl acyl acyl acyl acyl H H acyl
2015. acyl acyl acyl acyl acyl acyl H acyl H
2016. acyl acyl acyl acyl acyl acyl H acyl acyl
2017. acyl acyl acyl acyl acyl acyl acyl H H
2018. acyl acyl acyl acyl acyl acyl acyl H acyl
2019. acyl acyl acyl acyl acyl acyl acyl acyl H
2020. acyl acyl acyl acyl acyl acyl acyl acyl acyl
Table 9
compound R2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2021. H H H H H H H H H H
2022. H H H H H H H H H acyl
2023. H H H H H H H H acyl H
2024. H H H H H H H H acyl acyl
2025. H H H H H H H acyl H H
2026. H H H H H H H acyl H acyl
2027. H H H H H H H acyl acyl H
2028. H H H H H H H acyl acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2029. H H H H H H acyl H H H
2030. H H H H H H acyl H H acyl
2031. H H H H H H acyl H acyl H
2032. H H H H H H acyl H acyl acyl
2033. H H H H H H acyl acyl H H
2034. H H H H H H acyl acyl H acyl
2035. H H H H H H acyl acyl acyl H
2036. H H H H H H acyl acyl acyl acyl
2037. H H H H H acyl H H H H
2038. H H H H H acyl H H H acyl
2039. H H H H H acyl H H acyl H
2040. H H H H H acyl H H acyl acyl
2041. H H H H H acyl H acyl H H
2042. H H H H H acyl H acyl H acyl
2043. H H H H H acyl H acyl acyl H
2044. H H H H H acyl H acyl acyl acyl
2045. H H H H H acyl acyl H H H
2046. H H H H H acyl acyl H H acyl
2047. H H H H H acyl acyl H acyl H
2048. H H H H H acyl acyl H acyl acyl
2049. H H H H H acyl acyl acyl H H
2050. H H H H H acyl acyl acyl H acyl
2051. H H H H H acyl acyl acyl acyl H
2052. H H H H H acyl acyl acyl acyl acyl
2053. H H H H acyl H H H H H
2054. H H H H acyl H H H H acyl
2055. H H H H acyl H H H acyl H
2056. H H H H acyl H H H acyl acyl
2057. H H H H acyl H H acyl H H
2058. H H H H acyl H H acyl H acyl
2059. H H H H acyl H H acyl acyl H
2060. H H H H acyl H H acyl acyl acyl
2061. H H H H acyl H acyl H H H
2062. H H H H acyl H acyl H H acyl
2063. H H H H acyl H acyl H acyl H
2064. H H H H acyl H acyl H acyl acyl
2065. H H H H acyl H acyl acyl H H
2066. H H H H acyl H acyl acyl H acyl
2067. H H H H acyl H acyl acyl acyl H
2068. H H H H acyl H acyl acyl acyl acyl
2069. H H H H acyl acyl H H H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2070. H H H H acyl acyl H H H acyl
2071. H H H H acyl acyl H H acyl H
2072. H H H H acyl acyl H H acyl acyl
2073. H H H H acyl acyl H acyl H H
2074. H H H H acyl acyl H acyl H acyl
2075. H H H H acyl acyl H acyl acyl H
2076. H H H H acyl acyl H acyl acyl acyl
2077. H H H H acyl acyl acyl H H H
2078. H H H H acyl acyl acyl H H acyl
2079. H H H H acyl acyl acyl H acyl H
2080. H H H H acyl acyl acyl H acyl acyl
2081. H H H H acyl acyl acyl acyl H H
2082. H H H H acyl acyl acyl acyl H acyl
2083. H H H H acyl acyl acyl acyl acyl H
2084. H H H H acyl acyl acyl acyl acyl acyl
2085. H H H acyl H H H H H H
2086. H H H acyl H H H H H acyl
2087. H H H acyl H H H H acyl H
2088. H H H acyl H H H H acyl acyl
2089. H H H acyl H H H acyl H H
2090. H H H acyl H H H acyl H acyl
2091. H H H acyl H H H acyl acyl H
2092. H H H acyl H H H acyl acyl acyl
2093. H H H acyl H H acyl H H H
2094. H H H acyl H H acyl H H acyl
2095. H H H acyl H H acyl H acyl H
2096. H H H acyl H H acyl H acyl acyl
2097. H H H acyl H H acyl acyl H H
2098. H H H acyl H H acyl acyl H acyl
2099. H H H acyl H H acyl acyl acyl H
2100. H H H acyl H H acyl acyl acyl acyl
2101. H H H acyl H acyl H H H H
2102. H H H acyl H acyl H H H acyl
2103. H H H acyl H acyl H H acyl H
2104. H H H acyl H acyl H H acyl acyl
2105. H H H acyl H acyl H acyl H H
2106. H H H acyl H acyl H acyl H acyl
2107. H H H acyl H acyl H acyl acyl H
2108. H H H acyl H acyl H acyl acyl acyl
2109. H H H acyl H acyl acyl H H H
2110. H H H acyl H acyl acyl H H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2111. H H H acyl H acyl acyl H acyl H
2112. H H H acyl H acyl acyl H acyl acyl
2113. H H H acyl H acyl acyl acyl H H
2114. H H H acyl H acyl acyl acyl H acyl
2115. H H H acyl H acyl acyl acyl acyl H
2116. H H H acyl H acyl acyl acyl acyl acyl
2117. H H H acyl acyl H H H H H
2118. H H H acyl acyl H H H H acyl
2119. H H H acyl acyl H H H acyl H
2120. H H H acyl acyl H H H acyl acyl
2121. H H H acyl acyl H H acyl H H
2122. H H H acyl acyl H H acyl H acyl
2123. H H H acyl acyl H H acyl acyl H
2124. H H H acyl acyl H H acyl acyl acyl
2125. H H H acyl acyl H acyl H H H
2126. H H H acyl acyl H acyl H H acyl
2127. H H H acyl acyl H acyl H acyl H
2128. H H H acyl acyl H acyl H acyl acyl
2129. H H H acyl acyl H acyl acyl H H
2130. H H H acyl acyl H acyl acyl H acyl
2131. H H H acyl acyl H acyl acyl acyl H
2132. H H H acyl acyl H acyl acyl acyl acyl
2133. H H H acyl acyl acyl H H H H
2134. H H H acyl acyl acyl H H H acyl
2135. H H H acyl acyl acyl H H acyl H
2136. H H H acyl acyl acyl H H acyl acyl
2137. H H H acyl acyl acyl H acyl H H
2138. H H H acyl acyl acyl H acyl H acyl
2139. H H H acyl acyl acyl H acyl acyl H
2140. H H H acyl acyl acyl H acyl acyl acyl
2141. H H H acyl acyl acyl acyl H H H
2142. H H H acyl acyl acyl acyl H H acyl
2143. H H H acyl acyl acyl acyl H acyl H
2144. H H H acyl acyl acyl acyl H acyl acyl
2145. H H H acyl acyl acyl acyl acyl H H
2146. H H H acyl acyl acyl acyl acyl H acyl
2147. H H H acyl acyl acyl acyl acyl acyl H
2148. H H H acyl acyl acyl acyl acyl acyl acyl
2149. H H acyl H H H H H H H
2150. H H acyl H H H H H H acyl
2151. H H acyl H H H H H acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2152. H H acyl H H H H H acyl acyl
2153. H H acyl H H H H acyl H H
2154. H H acyl H H H H acyl H acyl
2155. H H acyl H H H H acyl acyl H
2156. H H acyl H H H H acyl acyl acyl
2157. H H acyl H H H acyl H H H
2158. H H acyl H H H acyl H H acyl
2159. H H acyl H H H acyl H acyl H
2160. H H acyl H H H acyl H acyl acyl
2161. H H acyl H H H acyl acyl H H
2162. H H acyl H H H acyl acyl H acyl
2163. H H acyl H H H acyl acyl acyl H
2164. H H acyl H H H acyl acyl acyl acyl
2165. H H acyl H H acyl H H H H
2166. H H acyl H H acyl H H H acyl
2167. H H acyl H H acyl H H acyl H
2168. H H acyl H H acyl H H acyl acyl
2169. H H acyl H H acyl H acyl H H
2170. H H acyl H H acyl H acyl H acyl
2171. H H acyl H H acyl H acyl acyl H
2172. H H acyl H H acyl H acyl acyl acyl
2173. H H acyl H H acyl acyl H H H
2174. H H acyl H H acyl acyl H H acyl
2175. H H acyl H H acyl acyl H acyl H
2176. H H acyl H H acyl acyl H acyl acyl
2177. H H acyl H H acyl acyl acyl H H
2178. H H acyl H H acyl acyl acyl H acyl
2179. H H acyl H H acyl acyl acyl acyl H
2180. H H acyl H H acyl acyl acyl acyl acyl
2181. H H acyl H acyl H H H H H
2182. H H acyl H acyl H H H H acyl
2183. H H acyl H acyl H H H acyl H
2184. H H acyl H acyl H H H acyl acyl
2185. H H acyl H acyl H H acyl H H
2186. H H acyl H acyl H H acyl H acyl
2187. H H acyl H acyl H H acyl acyl H
2188. H H acyl H acyl H H acyl acyl acyl
2189. H H acyl H acyl H acyl H H H
2190. H H acyl H acyl H acyl H H acyl
2191. H H acyl H acyl H acyl H acyl H
2192. H H acyl H acyl H acyl H acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2193. H H acyl H acyl H acyl acyl H H
2194. H H acyl H acyl H acyl acyl H acyl
2195. H H acyl H acyl H acyl acyl acyl H
2196. H H acyl H acyl H acyl acyl acyl acyl
2197. H H acyl H acyl acyl H H H H
2198. H H acyl H acyl acyl H H H acyl
2199. H H acyl H acyl acyl H H acyl H
2200. H H acyl H acyl acyl H H acyl acyl
2201. H H acyl H acyl acyl H acyl H H
2202. H H acyl H acyl acyl H acyl H acyl
2203. H H acyl H acyl acyl H acyl acyl H
2204. H H acyl H acyl acyl H acyl acyl acyl
2205. H H acyl H acyl acyl acyl H H H
2206. H H acyl H acyl acyl acyl H H acyl
2207. H H acyl H acyl acyl acyl H acyl H
2208. H H acyl H acyl acyl acyl H acyl acyl
2209. H H acyl H acyl acyl acyl acyl H H
2210. H H acyl H acyl acyl acyl acyl H acyl
2211. H H acyl H acyl acyl acyl acyl acyl H
2212. H H acyl H acyl acyl acyl acyl acyl acyl
2213. H H acyl acyl H H H H H H
2214. H H acyl acyl H H H H H acyl
2215. H H acyl acyl H H H H acyl H
2216. H H acyl acyl H H H H acyl acyl
2217. H H acyl acyl H H H acyl H H
2218. H H acyl acyl H H H acyl H acyl
2219. H H acyl acyl H H H acyl acyl H
2220. H H acyl acyl H H H acyl acyl acyl
2221. H H acyl acyl H H acyl H H H
2222. H H acyl acyl H H acyl H H acyl
2223. H H acyl acyl H H acyl H acyl H
2224. H H acyl acyl H H acyl H acyl acyl
2225. H H acyl acyl H H acyl acyl H H
2226. H H acyl acyl H H acyl acyl H acyl
2227. H H acyl acyl H H acyl acyl acyl H
2228. H H acyl acyl H H acyl acyl acyl acyl
2229. H H acyl acyl H acyl H H H H
2230. H H acyl acyl H acyl H H H acyl
2231. H H acyl acyl H acyl H H acyl H
2232. H H acyl acyl H acyl H H acyl acyl
2233. H H acyl acyl H acyl H acyl H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2234. H H acyl acyl H acyl H acyl H acyl
2235. H H acyl acyl H acyl H acyl acyl H
2236. H H acyl acyl H acyl H acyl acyl acyl
2237. H H acyl acyl H acyl acyl H H H
2238. H H acyl acyl H acyl acyl H H acyl
2239. H H acyl acyl H acyl acyl H acyl H
2240. H H acyl acyl H acyl acyl H acyl acyl
2241. H H acyl acyl H acyl acyl acyl H H
2242. H H acyl acyl H acyl acyl acyl H acyl
2243. H H acyl acyl H acyl acyl acyl acyl H
2244. H H acyl acyl H acyl acyl acyl acyl acyl
2245. H H acyl acyl acyl H H H H H
2246. H H acyl acyl acyl H H H H acyl
2247. H H acyl acyl acyl H H H acyl H
2248. H H acyl acyl acyl H H H acyl acyl
2249. H H acyl acyl acyl H H acyl H H
2250. H H acyl acyl acyl H H acyl H acyl
2251. H H acyl acyl acyl H H acyl acyl H
2252. H H acyl acyl acyl H H acyl acyl acyl
2253. H H acyl acyl acyl H acyl H H H
2254. H H acyl acyl acyl H acyl H H acyl
2255. H H acyl acyl acyl H acyl H acyl H
2256. H H acyl acyl acyl H acyl H acyl acyl
2257. H H acyl acyl acyl H acyl acyl H H
2258. H H acyl acyl acyl H acyl acyl H acyl
2259. H H acyl acyl acyl H acyl acyl acyl H
2260. H H acyl acyl acyl H acyl acyl acyl acyl
2261. H H acyl acyl acyl acyl H H H H
2262. H H acyl acyl acyl acyl H H H acyl
2263. H H acyl acyl acyl acyl H H acyl H
2264. H H acyl acyl acyl acyl H H acyl acyl
2265. H H acyl acyl acyl acyl H acyl H H
2266. H H acyl acyl acyl acyl H acyl H acyl
2267. H H acyl acyl acyl acyl H acyl acyl H
2268. H H acyl acyl acyl acyl H acyl acyl acyl
2269. H H acyl acyl acyl acyl acyl H H H
2270. H H acyl acyl acyl acyl acyl H H acyl
2271. H H acyl acyl acyl acyl acyl H acyl H
2272. H H acyl acyl acyl acyl acyl H acyl acyl
2273. H H acyl acyl acyl acyl acyl acyl H H
2274. H H acyl acyl acyl acyl acyl acyl H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2275. H H acyl acyl acyl acyl acyl acyl acyl H
2276. H H acyl acyl acyl acyl acyl acyl acyl acyl
2277. H acyl H H H H H H H H
2278. H acyl H H H H H H H acyl
2279. H acyl H H H H H H acyl H
2280. H acyl H H H H H H acyl acyl
2281. H acyl H H H H H acyl H H
2282. H acyl H H H H H acyl H acyl
2283. H acyl H H H H H acyl acyl H
2284. H acyl H H H H H acyl acyl acyl
2285. H acyl H H H H acyl H H H
2286. H acyl H H H H acyl H H acyl
2287. H acyl H H H H acyl H acyl H
2288. H acyl H H H H acyl H acyl acyl
2289. H acyl H H H H acyl acyl H H
2290. H acyl H H H H acyl acyl H acyl
2291. H acyl H H H H acyl acyl acyl H
2292. H acyl H H H H acyl acyl acyl acyl
2293. H acyl H H H acyl H H H H
2294. H acyl H H H acyl H H H acyl
2295. H acyl H H H acyl H H acyl H
2296. H acyl H H H acyl H H acyl acyl
2297. H acyl H H H acyl H acyl H H
2298. H acyl H H H acyl H acyl H acyl
2299. H acyl H H H acyl H acyl acyl H
2300. H acyl H H H acyl H acyl acyl acyl
2301. H acyl H H H acyl acyl H H H
2302. H acyl H H H acyl acyl H H acyl
2303. H acyl H H H acyl acyl H acyl H
2304. H acyl H H H acyl acyl H acyl acyl
2305. H acyl H H H acyl acyl acyl H H
2306. H acyl H H H acyl acyl acyl H acyl
2307. H acyl H H H acyl acyl acyl acyl H
2308. H acyl H H H acyl acyl acyl acyl acyl
2309. H acyl H H acyl H H H H H
2310. H acyl H H acyl H H H H acyl
2311. H acyl H H acyl H H H acyl H
2312. H acyl H H acyl H H H acyl acyl
2313. H acyl H H acyl H H acyl H H
2314. H acyl H H acyl H H acyl H acyl
2315. H acyl H H acyl H H acyl acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2316. H acyl H H acyl H H acyl acyl acyl
2317. H acyl H H acyl H acyl H H H
2318. H acyl H H acyl H acyl H H acyl
2319. H acyl H H acyl H acyl H acyl H
2320. H acyl H H acyl H acyl H acyl acyl
2321. H acyl H H acyl H acyl acyl H H
2322. H acyl H H acyl H acyl acyl H acyl
2323. H acyl H H acyl H acyl acyl acyl H
2324. H acyl H H acyl H acyl acyl acyl acyl
2325. H acyl H H acyl acyl H H H H
2326. H acyl H H acyl acyl H H H acyl
2327. H acyl H H acyl acyl H H acyl H
2328. H acyl H H acyl acyl H H acyl acyl
2329. H acyl H H acyl acyl H acyl H H
2330. H acyl H H acyl acyl H acyl H acyl
2331. H acyl H H acyl acyl H acyl acyl H
2332. H acyl H H acyl acyl H acyl acyl acyl
2333. H acyl H H acyl acyl acyl H H H
2334. H acyl H H acyl acyl acyl H H acyl
2335. H acyl H H acyl acyl acyl H acyl H
2336. H acyl H H acyl acyl acyl H acyl acyl
2337. H acyl H H acyl acyl acyl acyl H H
2338. H acyl H H acyl acyl acyl acyl H acyl
2339. H acyl H H acyl acyl acyl acyl acyl H
2340. H acyl H H acyl acyl acyl acyl acyl acyl
2341. H acyl H acyl H H H H H H
2342. H acyl H acyl H H H H H acyl
2343. H acyl H acyl H H H H acyl H
2344. H acyl H acyl H H H H acyl acyl
2345. H acyl H acyl H H H acyl H H
2346. H acyl H acyl H H H acyl H acyl
2347. H acyl H acyl H H H acyl acyl H
2348. H acyl H acyl H H H acyl acyl acyl
2349. H acyl H acyl H H acyl H H H
2350. H acyl H acyl H H acyl H H acyl
2351. H acyl H acyl H H acyl H acyl H
2352. H acyl H acyl H H acyl H acyl acyl
2353. H acyl H acyl H H acyl acyl H H
2354. H acyl H acyl H H acyl acyl H acyl
2355. H acyl H acyl H H acyl acyl acyl H
2356. H acyl H acyl H H acyl acyl acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2357. H acyl H acyl H acyl H H H H
2358. H acyl H acyl H acyl H H H acyl
2359. H acyl H acyl H acyl H H acyl H
2360. H acyl H acyl H acyl H H acyl acyl
2361. H acyl H acyl H acyl H acyl H H
2362. H acyl H acyl H acyl H acyl H acyl
2363. H acyl H acyl H acyl H acyl acyl H
2364. H acyl H acyl H acyl H acyl acyl acyl
2365. H acyl H acyl H acyl acyl H H H
2366. H acyl H acyl H acyl acyl H H acyl
2367. H acyl H acyl H acyl acyl H acyl H
2368. H acyl H acyl H acyl acyl H acyl acyl
2369. H acyl H acyl H acyl acyl acyl H H
2370. H acyl H acyl H acyl acyl acyl H acyl
2371. H acyl H acyl H acyl acyl acyl acyl H
2372. H acyl H acyl H acyl acyl acyl acyl acyl
2373. H acyl H acyl acyl H H H H H
2374. H acyl H acyl acyl H H H H acyl
2375. H acyl H acyl acyl H H H acyl H
2376. H acyl H acyl acyl H H H acyl acyl
2377. H acyl H acyl acyl H H acyl H H
2378. H acyl H acyl acyl H H acyl H acyl
2379. H acyl H acyl acyl H H acyl acyl H
2380. H acyl H acyl acyl H H acyl acyl acyl
2381. H acyl H acyl acyl H acyl H H H
2382. H acyl H acyl acyl H acyl H H acyl
2383. H acyl H acyl acyl H acyl H acyl H
2384. H acyl H acyl acyl H acyl H acyl acyl
2385. H acyl H acyl acyl H acyl acyl H H
2386. H acyl H acyl acyl H acyl acyl H acyl
2387. H acyl H acyl acyl H acyl acyl acyl H
2388. H acyl H acyl acyl H acyl acyl acyl acyl
2389. H acyl H acyl acyl acyl H H H H
2390. H acyl H acyl acyl acyl H H H acyl
2391. H acyl H acyl acyl acyl H H acyl H
2392. H acyl H acyl acyl acyl H H acyl acyl
2393. H acyl H acyl acyl acyl H acyl H H
2394. H acyl H acyl acyl acyl H acyl H acyl
2395. H acyl H acyl acyl acyl H acyl acyl H
2396. H acyl H acyl acyl acyl H acyl acyl acyl
2397. H acyl H acyl acyl acyl acyl H H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2398. H acyl H acyl acyl acyl acyl H H acyl
2399. H acyl H acyl acyl acyl acyl H acyl H
2400. H acyl H acyl acyl acyl acyl H acyl acyl
2401. H acyl H acyl acyl acyl acyl acyl H H
2402. H acyl H acyl acyl acyl acyl acyl H acyl
2403. H acyl H acyl acyl acyl acyl acyl acyl H
2404. H acyl H acyl acyl acyl acyl acyl acyl acyl
2405. H acyl acyl H H H H H H H
2406. H acyl acyl H H H H H H acyl
2407. H acyl acyl H H H H H acyl H
2408. H acyl acyl H H H H H acyl acyl
2409. H acyl acyl H H H H acyl H H
2410. H acyl acyl H H H H acyl H acyl
2411. H acyl acyl H H H H acyl acyl H
2412. H acyl acyl H H H H acyl acyl acyl
2413. H acyl acyl H H H acyl H H H
2414. H acyl acyl H H H acyl H H acyl
2415. H acyl acyl H H H acyl H acyl H
2416. H acyl acyl H H H acyl H acyl acyl
2417. H acyl acyl H H H acyl acyl H H
2418. H acyl acyl H H H acyl acyl H acyl
2419. H acyl acyl H H H acyl acyl acyl H
2420. H acyl acyl H H H acyl acyl acyl acyl
2421. H acyl acyl H H acyl H H H H
2422. H acyl acyl H H acyl H H H acyl
2423. H acyl acyl H H acyl H H acyl H
2424. H acyl acyl H H acyl H H acyl acyl
2425. H acyl acyl H H acyl H acyl H H
2426. H acyl acyl H H acyl H acyl H acyl
2427. H acyl acyl H H acyl H acyl acyl H
2428. H acyl acyl H H acyl H acyl acyl acyl
2429. H acyl acyl H H acyl acyl H H H
2430. H acyl acyl H H acyl acyl H H acyl
2431. H acyl acyl H H acyl acyl H acyl H
2432. H acyl acyl H H acyl acyl H acyl acyl
2433. H acyl acyl H H acyl acyl acyl H H
2434. H acyl acyl H H acyl acyl acyl H acyl
2435. H acyl acyl H H acyl acyl acyl acyl H
2436. H acyl acyl H H acyl acyl acyl acyl acyl
2437. H acyl acyl H acyl H H H H H
2438. H acyl acyl H acyl H H H H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2439. H acyl acyl H acyl H H H acyl H
2440. H acyl acyl H acyl H H H acyl acyl
2441. H acyl acyl H acyl H H acyl H H
2442. H acyl acyl H acyl H H acyl H acyl
2443. H acyl acyl H acyl H H acyl acyl H
2444. H acyl acyl H acyl H H acyl acyl acyl
2445. H acyl acyl H acyl H acyl H H H
2446. H acyl acyl H acyl H acyl H H acyl
2447. H acyl acyl H acyl H acyl H acyl H
2448. H acyl acyl H acyl H acyl H acyl acyl
2449. H acyl acyl H acyl H acyl acyl H H
2450. H acyl acyl H acyl H acyl acyl H acyl
2451. H acyl acyl H acyl H acyl acyl acyl H
2452. H acyl acyl H acyl H acyl acyl acyl acyl
2453. H acyl acyl H acyl acyl H H H H
2454. H acyl acyl H acyl acyl H H H acyl
2455. H acyl acyl H acyl acyl H H acyl H
2456. H acyl acyl H acyl acyl H H acyl acyl
2457. H acyl acyl H acyl acyl H acyl H H
2458. H acyl acyl H acyl acyl H acyl H acyl
2459. H acyl acyl H acyl acyl H acyl acyl H
2460. H acyl acyl H acyl acyl H acyl acyl acyl
2461. H acyl acyl H acyl acyl acyl H H H
2462. H acyl acyl H acyl acyl acyl H H acyl
2463. H acyl acyl H acyl acyl acyl H acyl H
2464. H acyl acyl H acyl acyl acyl H acyl acyl
2465. H acyl acyl H acyl acyl acyl acyl H H
2466. H acyl acyl H acyl acyl acyl acyl H acyl
2467. H acyl acyl H acyl acyl acyl acyl acyl H
2468. H acyl acyl H acyl acyl acyl acyl acyl acyl
2469. H acyl acyl acyl H H H H H H
2470. H acyl acyl acyl H H H H H acyl
2471. H acyl acyl acyl H H H H acyl H
2472. H acyl acyl acyl H H H H acyl acyl
2473. H acyl acyl acyl H H H acyl H H
2474. H acyl acyl acyl H H H acyl H acyl
2475. H acyl acyl acyl H H H acyl acyl H
2476. H acyl acyl acyl H H H acyl acyl acyl
2477. H acyl acyl acyl H H acyl H H H
2478. H acyl acyl acyl H H acyl H H acyl
2479. H acyl acyl acyl H H acyl H acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2480. H acyl acyl acyl H H acyl H acyl acyl
2481. H acyl acyl acyl H H acyl acyl H H
2482. H acyl acyl acyl H H acyl acyl H acyl
2483. H acyl acyl acyl H H acyl acyl acyl H
2484. H acyl acyl acyl H H acyl acyl acyl acyl
2485. H acyl acyl acyl H acyl H H H H
2486. H acyl acyl acyl H acyl H H H acyl
2487. H acyl acyl acyl H acyl H H acyl H
2488. H acyl acyl acyl H acyl H H acyl acyl
2489. H acyl acyl acyl H acyl H acyl H H
2490. H acyl acyl acyl H acyl H acyl H acyl
2491. H acyl acyl acyl H acyl H acyl acyl H
2492. H acyl acyl acyl H acyl H acyl acyl acyl
2493. H acyl acyl acyl H acyl acyl H H H
2494. H acyl acyl acyl H acyl acyl H H acyl
2495. H acyl acyl acyl H acyl acyl H acyl H
2496. H acyl acyl acyl H acyl acyl H acyl acyl
2497. H acyl acyl acyl H acyl acyl acyl H H
2498. H acyl acyl acyl H acyl acyl acyl H acyl
2499. H acyl acyl acyl H acyl acyl acyl acyl H
2500. H acyl acyl acyl H acyl acyl acyl acyl acyl
2501. H acyl acyl acyl acyl H H H H H
2502. H acyl acyl acyl acyl H H H H acyl
2503. H acyl acyl acyl acyl H H H acyl H
2504. H acyl acyl acyl acyl H H H acyl acyl
2505. H acyl acyl acyl acyl H H acyl H H
2506. H acyl acyl acyl acyl H H acyl H acyl
2507. H acyl acyl acyl acyl H H acyl acyl H
2508. H acyl acyl acyl acyl H H acyl acyl acyl
2509. H acyl acyl acyl acyl H acyl H H H
2510. H acyl acyl acyl acyl H acyl H H acyl
2511. H acyl acyl acyl acyl H acyl H acyl H
2512. H acyl acyl acyl acyl H acyl H acyl acyl
2513. H acyl acyl acyl acyl H acyl acyl H H
2514. H acyl acyl acyl acyl H acyl acyl H acyl
2515. H acyl acyl acyl acyl H acyl acyl acyl H
2516. H acyl acyl acyl acyl H acyl acyl acyl acyl
2517. H acyl acyl acyl acyl acyl H H H H
2518. H acyl acyl acyl acyl acyl H H H acyl
2519. H acyl acyl acyl acyl acyl H H acyl H
2520. H acyl acyl acyl acyl acyl H H acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2521. H acyl acyl acyl acyl acyl H acyl H H
2522. H acyl acyl acyl acyl acyl H acyl H acyl
2523. H acyl acyl acyl acyl acyl H acyl acyl H
2524. H acyl acyl acyl acyl acyl H acyl acyl acyl
2525. H acyl acyl acyl acyl acyl acyl H H H
2526. H acyl acyl acyl acyl acyl acyl H H acyl
2527. H acyl acyl acyl acyl acyl acyl H acyl H
2528. H acyl acyl acyl acyl acyl acyl H acyl acyl
2529. H acyl acyl acyl acyl acyl acyl acyl H H
2530. H acyl acyl acyl acyl acyl acyl acyl H acyl
2531. H acyl acyl acyl acyl acyl acyl acyl acyl H
2532. H acyl acyl acyl acyl acyl acyl acyl acyl acyl
2533. acyl H H H H H H H H H
2534. acyl H H H H H H H H acyl
2535. acyl H H H H H H H acyl H
2536. acyl H H H H H H H acyl acyl
2537. acyl H H H H H H acyl H H
2538. acyl H H H H H H acyl H acyl
2539. acyl H H H H H H acyl acyl H
2540. acyl H H H H H H acyl acyl acyl
2541. acyl H H H H H acyl H H H
2542. acyl H H H H H acyl H H acyl
2543. acyl H H H H H acyl H acyl H
2544. acyl H H H H H acyl H acyl acyl
2545. acyl H H H H H acyl acyl H H
2546. acyl H H H H H acyl acyl H acyl
2547. acyl H H H H H acyl acyl acyl H
2548. acyl H H H H H acyl acyl acyl acyl
2549. acyl H H H H acyl H H H H
2550. acyl H H H H acyl H H H acyl
2551. acyl H H H H acyl H H acyl H
2552. acyl H H H H acyl H H acyl acyl
2553. acyl H H H H acyl H acyl H H
2554. acyl H H H H acyl H acyl H acyl
2555. acyl H H H H acyl H acyl acyl H
2556. acyl H H H H acyl H acyl acyl acyl
2557. acyl H H H H acyl acyl H H H
2558. acyl H H H H acyl acyl H H acyl
2559. acyl H H H H acyl acyl H acyl H
2560. acyl H H H H acyl acyl H acyl acyl
2561. acyl H H H H acyl acyl acyl H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2562. acyl H H H H acyl acyl acyl H acyl
2563. acyl H H H H acyl acyl acyl acyl H
2564. acyl H H H H acyl acyl acyl acyl acyl
2565. acyl H H H acyl H H H H H
2566. acyl H H H acyl H H H H acyl
2567. acyl H H H acyl H H H acyl H
2568. acyl H H H acyl H H H acyl acyl
2569. acyl H H H acyl H H acyl H H
2570. acyl H H H acyl H H acyl H acyl
2571. acyl H H H acyl H H acyl acyl H
2572. acyl H H H acyl H H acyl acyl acyl
2573. acyl H H H acyl H acyl H H H
2574. acyl H H H acyl H acyl H H acyl
2575. acyl H H H acyl H acyl H acyl H
2576. acyl H H H acyl H acyl H acyl acyl
2577. acyl H H H acyl H acyl acyl H H
2578. acyl H H H acyl H acyl acyl H acyl
2579. acyl H H H acyl H acyl acyl acyl H
2580. acyl H H H acyl H acyl acyl acyl acyl
2581. acyl H H H acyl acyl H H H H
2582. acyl H H H acyl acyl H H H acyl
2583. acyl H H H acyl acyl H H acyl H
2584. acyl H H H acyl acyl H H acyl acyl
2585. acyl H H H acyl acyl H acyl H H
2586. acyl H H H acyl acyl H acyl H acyl
2587. acyl H H H acyl acyl H acyl acyl H
2588. acyl H H H acyl acyl H acyl acyl acyl
2589. acyl H H H acyl acyl acyl H H H
2590. acyl H H H acyl acyl acyl H H acyl
2591. acyl H H H acyl acyl acyl H acyl H
2592. acyl H H H acyl acyl acyl H acyl acyl
2593. acyl H H H acyl acyl acyl acyl H H
2594. acyl H H H acyl acyl acyl acyl H acyl
2595. acyl H H H acyl acyl acyl acyl acyl H
2596. acyl H H H acyl acyl acyl acyl acyl acyl
2597. acyl H H acyl H H H H H H
2598. acyl H H acyl H H H H H acyl
2599. acyl H H acyl H H H H acyl H
2600. acyl H H acyl H H H H acyl acyl
2601. acyl H H acyl H H H acyl H H
2602. acyl H H acyl H H H acyl H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2603. acyl H H acyl H H H acyl acyl H
2604. acyl H H acyl H H H acyl acyl acyl
2605. acyl H H acyl H H acyl H H H
2606. acyl H H acyl H H acyl H H acyl
2607. acyl H H acyl H H acyl H acyl H
2608. acyl H H acyl H H acyl H acyl acyl
2609. acyl H H acyl H H acyl acyl H H
2610. acyl H H acyl H H acyl acyl H acyl
2611. acyl H H acyl H H acyl acyl acyl H
2612. acyl H H acyl H H acyl acyl acyl acyl
2613. acyl H H acyl H acyl H H H H
2614. acyl H H acyl H acyl H H H acyl
2615. acyl H H acyl H acyl H H acyl H
2616. acyl H H acyl H acyl H H acyl acyl
2617. acyl H H acyl H acyl H acyl H H
2618. acyl H H acyl H acyl H acyl H acyl
2619. acyl H H acyl H acyl H acyl acyl H
2620. acyl H H acyl H acyl H acyl acyl acyl
2621. acyl H H acyl H acyl acyl H H H
2622. acyl H H acyl H acyl acyl H H acyl
2623. acyl H H acyl H acyl acyl H acyl H
2624. acyl H H acyl H acyl acyl H acyl acyl
2625. acyl H H acyl H acyl acyl acyl H H
2626. acyl H H acyl H acyl acyl acyl H acyl
2627. acyl H H acyl H acyl acyl acyl acyl H
2628. acyl H H acyl H acyl acyl acyl acyl acyl
2629. acyl H H acyl acyl H H H H H
2630. acyl H H acyl acyl H H H H acyl
2631. acyl H H acyl acyl H H H acyl H
2632. acyl H H acyl acyl H H H acyl acyl
2633. acyl H H acyl acyl H H acyl H H
2634. acyl H H acyl acyl H H acyl H acyl
2635. acyl H H acyl acyl H H acyl acyl H
2636. acyl H H acyl acyl H H acyl acyl acyl
2637. acyl H H acyl acyl H acyl H H H
2638. acyl H H acyl acyl H acyl H H acyl
2639. acyl H H acyl acyl H acyl H acyl H
2640. acyl H H acyl acyl H acyl H acyl acyl
2641. acyl H H acyl acyl H acyl acyl H H
2642. acyl H H acyl acyl H acyl acyl H acyl
2643. acyl H H acyl acyl H acyl acyl acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2644. acyl H H acyl acyl H acyl acyl acyl acyl
2645. acyl H H acyl acyl acyl H H H H
2646. acyl H H acyl acyl acyl H H H acyl
2647. acyl H H acyl acyl acyl H H acyl H
2648. acyl H H acyl acyl acyl H H acyl acyl
2649. acyl H H acyl acyl acyl H acyl H H
2650. acyl H H acyl acyl acyl H acyl H acyl
2651. acyl H H acyl acyl acyl H acyl acyl H
2652. acyl H H acyl acyl acyl H acyl acyl acyl
2653. acyl H H acyl acyl acyl acyl H H H
2654. acyl H H acyl acyl acyl acyl H H acyl
2655. acyl H H acyl acyl acyl acyl H acyl H
2656. acyl H H acyl acyl acyl acyl H acyl acyl
2657. acyl H H acyl acyl acyl acyl acyl H H
2658. acyl H H acyl acyl acyl acyl acyl H acyl
2659. acyl H H acyl acyl acyl acyl acyl acyl H
2660. acyl H H acyl acyl acyl acyl acyl acyl acyl
2661. acyl H acyl H H H H H H H
2662. acyl H acyl H H H H H H acyl
2663. acyl H acyl H H H H H acyl H
2664. acyl H acyl H H H H H acyl acyl
2665. acyl H acyl H H H H acyl H H
2666. acyl H acyl H H H H acyl H acyl
2667. acyl H acyl H H H H acyl acyl H
2668. acyl H acyl H H H H acyl acyl acyl
2669. acyl H acyl H H H acyl H H H
2670. acyl H acyl H H H acyl H H acyl
2671. acyl H acyl H H H acyl H acyl H
2672. acyl H acyl H H H acyl H acyl acyl
2673. acyl H acyl H H H acyl acyl H H
2674. acyl H acyl H H H acyl acyl H acyl
2675. acyl H acyl H H H acyl acyl acyl H
2676. acyl H acyl H H H acyl acyl acyl acyl
2677. acyl H acyl H H acyl H H H H
2678. acyl H acyl H H acyl H H H acyl
2679. acyl H acyl H H acyl H H acyl H
2680. acyl H acyl H H acyl H H acyl acyl
2681. acyl H acyl H H acyl H acyl H H
2682. acyl H acyl H H acyl H acyl H acyl
2683. acyl H acyl H H acyl H acyl acyl H
2684. acyl H acyl H H acyl H acyl acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2685. acyl H acyl H H acyl acyl H H H
2686. acyl H acyl H H acyl acyl H H acyl
2687. acyl H acyl H H acyl acyl H acyl H
2688. acyl H acyl H H acyl acyl H acyl acyl
2689. acyl H acyl H H acyl acyl acyl H H
2690. acyl H acyl H H acyl acyl acyl H acyl
2691. acyl H acyl H H acyl acyl acyl acyl H
2692. acyl H acyl H H acyl acyl acyl acyl acyl
2693. acyl H acyl H acyl H H H H H
2694. acyl H acyl H acyl H H H H acyl
2695. acyl H acyl H acyl H H H acyl H
2696. acyl H acyl H acyl H H H acyl acyl
2697. acyl H acyl H acyl H H acyl H H
2698. acyl H acyl H acyl H H acyl H acyl
2699. acyl H acyl H acyl H H acyl acyl H
2700. acyl H acyl H acyl H H acyl acyl acyl
2701. acyl H acyl H acyl H acyl H H H
2702. acyl H acyl H acyl H acyl H H acyl
2703. acyl H acyl H acyl H acyl H acyl H
2704. acyl H acyl H acyl H acyl H acyl acyl
2705. acyl H acyl H acyl H acyl acyl H H
2706. acyl H acyl H acyl H acyl acyl H acyl
2707. acyl H acyl H acyl H acyl acyl acyl H
2708. acyl H acyl H acyl H acyl acyl acyl acyl
2709. acyl H acyl H acyl acyl H H H H
2710. acyl H acyl H acyl acyl H H H acyl
2711. acyl H acyl H acyl acyl H H acyl H
2712. acyl H acyl H acyl acyl H H acyl acyl
2713. acyl H acyl H acyl acyl H acyl H H
2714. acyl H acyl H acyl acyl H acyl H acyl
2715. acyl H acyl H acyl acyl H acyl acyl H
2716. acyl H acyl H acyl acyl H acyl acyl acyl
2717. acyl H acyl H acyl acyl acyl H H H
2718. acyl H acyl H acyl acyl acyl H H acyl
2719. acyl H acyl H acyl acyl acyl H acyl H
2720. acyl H acyl H acyl acyl acyl H acyl acyl
2721. acyl H acyl H acyl acyl acyl acyl H H
2722. acyl H acyl H acyl acyl acyl acyl H acyl
2723. acyl H acyl H acyl acyl acyl acyl acyl H
2724. acyl H acyl H acyl acyl acyl acyl acyl acyl
2725. acyl H acyl acyl H H H H H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2726. acyl H acyl acyl H H H H H acyl
2727. acyl H acyl acyl H H H H acyl H
2728. acyl H acyl acyl H H H H acyl acyl
2729. acyl H acyl acyl H H H acyl H H
2730. acyl H acyl acyl H H H acyl H acyl
2731. acyl H acyl acyl H H H acyl acyl H
2732. acyl H acyl acyl H H H acyl acyl acyl
2733. acyl H acyl acyl H H acyl H H H
2734. acyl H acyl acyl H H acyl H H acyl
2735. acyl H acyl acyl H H acyl H acyl H
2736. acyl H acyl acyl H H acyl H acyl acyl
2737. acyl H acyl acyl H H acyl acyl H H
2738. acyl H acyl acyl H H acyl acyl H acyl
2739. acyl H acyl acyl H H acyl acyl acyl H
2740. acyl H acyl acyl H H acyl acyl acyl acyl
2741. acyl H acyl acyl H acyl H H H H
2742. acyl H acyl acyl H acyl H H H acyl
2743. acyl H acyl acyl H acyl H H acyl H
2744. acyl H acyl acyl H acyl H H acyl acyl
2745. acyl H acyl acyl H acyl H acyl H H
2746. acyl H acyl acyl H acyl H acyl H acyl
2747. acyl H acyl acyl H acyl H acyl acyl H
2748. acyl H acyl acyl H acyl H acyl acyl acyl
2749. acyl H acyl acyl H acyl acyl H H H
2750. acyl H acyl acyl H acyl acyl H H acyl
2751. acyl H acyl acyl H acyl acyl H acyl H
2752. acyl H acyl acyl H acyl acyl H acyl acyl
2753. acyl H acyl acyl H acyl acyl acyl H H
2754. acyl H acyl acyl H acyl acyl acyl H acyl
2755. acyl H acyl acyl H acyl acyl acyl acyl H
2756. acyl H acyl acyl H acyl acyl acyl acyl acyl
2757. acyl H acyl acyl acyl H H H H H
2758. acyl H acyl acyl acyl H H H H acyl
2759. acyl H acyl acyl acyl H H H acyl H
2760. acyl H acyl acyl acyl H H H acyl acyl
2761. acyl H acyl acyl acyl H H acyl H H
2762. acyl H acyl acyl acyl H H acyl H acyl
2763. acyl H acyl acyl acyl H H acyl acyl H
2764. acyl H acyl acyl acyl H H acyl acyl acyl
2765. acyl H acyl acyl acyl H acyl H H H
2766. acyl H acyl acyl acyl H acyl H H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2767. acyl H acyl acyl acyl H acyl H acyl H
2768. acyl H acyl acyl acyl H acyl H acyl acyl
2769. acyl H acyl acyl acyl H acyl acyl H H mo. acyl H acyl acyl acyl H acyl acyl H acyl
2771. acyl H acyl acyl acyl H acyl acyl acyl H mi. acyl H acyl acyl acyl H acyl acyl acyl acyl
2773. acyl H acyl acyl acyl acyl H H H H
2774. acyl H acyl acyl acyl acyl H H H acyl
2775. acyl H acyl acyl acyl acyl H H acyl H
2776. acyl H acyl acyl acyl acyl H H acyl acyl
2777. acyl H acyl acyl acyl acyl H acyl H H
2778. acyl H acyl acyl acyl acyl H acyl H acyl
2779. acyl H acyl acyl acyl acyl H acyl acyl H
2780. acyl H acyl acyl acyl acyl H acyl acyl acyl
2781. acyl H acyl acyl acyl acyl acyl H H H
2782. acyl H acyl acyl acyl acyl acyl H H acyl
2783. acyl H acyl acyl acyl acyl acyl H acyl H
2784. acyl H acyl acyl acyl acyl acyl H acyl acyl
2785. acyl H acyl acyl acyl acyl acyl acyl H H
2786. acyl H acyl acyl acyl acyl acyl acyl H acyl
2787. acyl H acyl acyl acyl acyl acyl acyl acyl H
2788. acyl H acyl acyl acyl acyl acyl acyl acyl acyl
2789. acyl acyl H H H H H H H H
2790. acyl acyl H H H H H H H acyl
2791. acyl acyl H H H H H H acyl H
2792. acyl acyl H H H H H H acyl acyl
2793. acyl acyl H H H H H acyl H H
2794. acyl acyl H H H H H acyl H acyl
2795. acyl acyl H H H H H acyl acyl H
2796. acyl acyl H H H H H acyl acyl acyl
2797. acyl acyl H H H H acyl H H H
2798. acyl acyl H H H H acyl H H acyl
2799. acyl acyl H H H H acyl H acyl H
2800. acyl acyl H H H H acyl H acyl acyl
2801. acyl acyl H H H H acyl acyl H H
2802. acyl acyl H H H H acyl acyl H acyl
2803. acyl acyl H H H H acyl acyl acyl H
2804. acyl acyl H H H H acyl acyl acyl acyl
2805. acyl acyl H H H acyl H H H H
2806. acyl acyl H H H acyl H H H acyl
2807. acyl acyl H H H acyl H H acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2808. acyl acyl H H H acyl H H acyl acyl
2809. acyl acyl H H H acyl H acyl H H
2810. acyl acyl H H H acyl H acyl H acyl
2811. acyl acyl H H H acyl H acyl acyl H
2812. acyl acyl H H H acyl H acyl acyl acyl
2813. acyl acyl H H H acyl acyl H H H
2814. acyl acyl H H H acyl acyl H H acyl
2815. acyl acyl H H H acyl acyl H acyl H
2816. acyl acyl H H H acyl acyl H acyl acyl
2817. acyl acyl H H H acyl acyl acyl H H
2818. acyl acyl H H H acyl acyl acyl H acyl
2819. acyl acyl H H H acyl acyl acyl acyl H
2820. acyl acyl H H H acyl acyl acyl acyl acyl
2821. acyl acyl H H acyl H H H H H
2822. acyl acyl H H acyl H H H H acyl
2823. acyl acyl H H acyl H H H acyl H
2824. acyl acyl H H acyl H H H acyl acyl
2825. acyl acyl H H acyl H H acyl H H
2826. acyl acyl H H acyl H H acyl H acyl
2827. acyl acyl H H acyl H H acyl acyl H
2828. acyl acyl H H acyl H H acyl acyl acyl
2829. acyl acyl H H acyl H acyl H H H
2830. acyl acyl H H acyl H acyl H H acyl
2831. acyl acyl H H acyl H acyl H acyl H
2832. acyl acyl H H acyl H acyl H acyl acyl
2833. acyl acyl H H acyl H acyl acyl H H
2834. acyl acyl H H acyl H acyl acyl H acyl
2835. acyl acyl H H acyl H acyl acyl acyl H
2836. acyl acyl H H acyl H acyl acyl acyl acyl
2837. acyl acyl H H acyl acyl H H H H
2838. acyl acyl H H acyl acyl H H H acyl
2839. acyl acyl H H acyl acyl H H acyl H
2840. acyl acyl H H acyl acyl H H acyl acyl
2841. acyl acyl H H acyl acyl H acyl H H
2842. acyl acyl H H acyl acyl H acyl H acyl
2843. acyl acyl H H acyl acyl H acyl acyl H
2844. acyl acyl H H acyl acyl H acyl acyl acyl
2845. acyl acyl H H acyl acyl acyl H H H
2846. acyl acyl H H acyl acyl acyl H H acyl
2847. acyl acyl H H acyl acyl acyl H acyl H
2848. acyl acyl H H acyl acyl acyl H acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2849. acyl acyl H H acyl acyl acyl acyl H H
2850. acyl acyl H H acyl acyl acyl acyl H acyl
2851. acyl acyl H H acyl acyl acyl acyl acyl H
2852. acyl acyl H H acyl acyl acyl acyl acyl acyl
2853. acyl acyl H acyl H H H H H H
2854. acyl acyl H acyl H H H H H acyl
2855. acyl acyl H acyl H H H H acyl H
2856. acyl acyl H acyl H H H H acyl acyl
2857. acyl acyl H acyl H H H acyl H H
2858. acyl acyl H acyl H H H acyl H acyl
2859. acyl acyl H acyl H H H acyl acyl H
2860. acyl acyl H acyl H H H acyl acyl acyl
2861. acyl acyl H acyl H H acyl H H H
2862. acyl acyl H acyl H H acyl H H acyl
2863. acyl acyl H acyl H H acyl H acyl H
2864. acyl acyl H acyl H H acyl H acyl acyl
2865. acyl acyl H acyl H H acyl acyl H H
2866. acyl acyl H acyl H H acyl acyl H acyl
2867. acyl acyl H acyl H H acyl acyl acyl H
2868. acyl acyl H acyl H H acyl acyl acyl acyl
2869. acyl acyl H acyl H acyl H H H H
2870. acyl acyl H acyl H acyl H H H acyl
2871. acyl acyl H acyl H acyl H H acyl H
2872. acyl acyl H acyl H acyl H H acyl acyl
2873. acyl acyl H acyl H acyl H acyl H H
2874. acyl acyl H acyl H acyl H acyl H acyl
2875. acyl acyl H acyl H acyl H acyl acyl H
2876. acyl acyl H acyl H acyl H acyl acyl acyl
2877. acyl acyl H acyl H acyl acyl H H H
2878. acyl acyl H acyl H acyl acyl H H acyl
2879. acyl acyl H acyl H acyl acyl H acyl H
2880. acyl acyl H acyl H acyl acyl H acyl acyl
2881. acyl acyl H acyl H acyl acyl acyl H H
2882. acyl acyl H acyl H acyl acyl acyl H acyl
2883. acyl acyl H acyl H acyl acyl acyl acyl H
2884. acyl acyl H acyl H acyl acyl acyl acyl acyl
2885. acyl acyl H acyl acyl H H H H H
2886. acyl acyl H acyl acyl H H H H acyl
2887. acyl acyl H acyl acyl H H H acyl H
2888. acyl acyl H acyl acyl H H H acyl acyl
2889. acyl acyl H acyl acyl H H acyl H H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2890. acyl acyl H acyl acyl H H acyl H acyl
2891. acyl acyl H acyl acyl H H acyl acyl H
2892. acyl acyl H acyl acyl H H acyl acyl acyl
2893. acyl acyl H acyl acyl H acyl H H H
2894. acyl acyl H acyl acyl H acyl H H acyl
2895. acyl acyl H acyl acyl H acyl H acyl H
2896. acyl acyl H acyl acyl H acyl H acyl acyl
2897. acyl acyl H acyl acyl H acyl acyl H H
2898. acyl acyl H acyl acyl H acyl acyl H acyl
2899. acyl acyl H acyl acyl H acyl acyl acyl H
2900. acyl acyl H acyl acyl H acyl acyl acyl acyl
2901. acyl acyl H acyl acyl acyl H H H H
2902. acyl acyl H acyl acyl acyl H H H acyl
2903. acyl acyl H acyl acyl acyl H H acyl H
2904. acyl acyl H acyl acyl acyl H H acyl acyl
2905. acyl acyl H acyl acyl acyl H acyl H H
2906. acyl acyl H acyl acyl acyl H acyl H acyl
2907. acyl acyl H acyl acyl acyl H acyl acyl H
2908. acyl acyl H acyl acyl acyl H acyl acyl acyl
2909. acyl acyl H acyl acyl acyl acyl H H H
2910. acyl acyl H acyl acyl acyl acyl H H acyl
2911. acyl acyl H acyl acyl acyl acyl H acyl H
2912. acyl acyl H acyl acyl acyl acyl H acyl acyl
2913. acyl acyl H acyl acyl acyl acyl acyl H H
2914. acyl acyl H acyl acyl acyl acyl acyl H acyl
2915. acyl acyl H acyl acyl acyl acyl acyl acyl H
2916. acyl acyl H acyl acyl acyl acyl acyl acyl acyl
2917. acyl acyl acyl H H H H H H H
2918. acyl acyl acyl H H H H H H acyl
2919. acyl acyl acyl H H H H H acyl H
2920. acyl acyl acyl H H H H H acyl acyl
2921. acyl acyl acyl H H H H acyl H H
2922. acyl acyl acyl H H H H acyl H acyl
2923. acyl acyl acyl H H H H acyl acyl H
2924. acyl acyl acyl H H H H acyl acyl acyl
2925. acyl acyl acyl H H H acyl H H H
2926. acyl acyl acyl H H H acyl H H acyl
2927. acyl acyl acyl H H H acyl H acyl H
2928. acyl acyl acyl H H H acyl H acyl acyl
2929. acyl acyl acyl H H H acyl acyl H H
2930. acyl acyl acyl H H H acyl acyl H acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2931. acyl acyl acyl H H H acyl acyl acyl H
2932. acyl acyl acyl H H H acyl acyl acyl acyl
2933. acyl acyl acyl H H acyl H H H H
2934. acyl acyl acyl H H acyl H H H acyl
2935. acyl acyl acyl H H acyl H H acyl H
2936. acyl acyl acyl H H acyl H H acyl acyl
2937. acyl acyl acyl H H acyl H acyl H H
2938. acyl acyl acyl H H acyl H acyl H acyl
2939. acyl acyl acyl H H acyl H acyl acyl H
2940. acyl acyl acyl H H acyl H acyl acyl acyl
2941. acyl acyl acyl H H acyl acyl H H H
2942. acyl acyl acyl H H acyl acyl H H acyl
2943. acyl acyl acyl H H acyl acyl H acyl H
2944. acyl acyl acyl H H acyl acyl H acyl acyl
2945. acyl acyl acyl H H acyl acyl acyl H H
2946. acyl acyl acyl H H acyl acyl acyl H acyl
2947. acyl acyl acyl H H acyl acyl acyl acyl H
2948. acyl acyl acyl H H acyl acyl acyl acyl acyl
2949. acyl acyl acyl H acyl H H H H H
2950. acyl acyl acyl H acyl H H H H acyl
2951. acyl acyl acyl H acyl H H H acyl H
2952. acyl acyl acyl H acyl H H H acyl acyl
2953. acyl acyl acyl H acyl H H acyl H H
2954. acyl acyl acyl H acyl H H acyl H acyl
2955. acyl acyl acyl H acyl H H acyl acyl H
2956. acyl acyl acyl H acyl H H acyl acyl acyl
2957. acyl acyl acyl H acyl H acyl H H H
2958. acyl acyl acyl H acyl H acyl H H acyl
2959. acyl acyl acyl H acyl H acyl H acyl H
2960. acyl acyl acyl H acyl H acyl H acyl acyl
2961. acyl acyl acyl H acyl H acyl acyl H H
2962. acyl acyl acyl H acyl H acyl acyl H acyl
2963. acyl acyl acyl H acyl H acyl acyl acyl H
2964. acyl acyl acyl H acyl H acyl acyl acyl acyl
2965. acyl acyl acyl H acyl acyl H H H H
2966. acyl acyl acyl H acyl acyl H H H acyl
2967. acyl acyl acyl H acyl acyl H H acyl H
2968. acyl acyl acyl H acyl acyl H H acyl acyl
2969. acyl acyl acyl H acyl acyl H acyl H H
2970. acyl acyl acyl H acyl acyl H acyl H acyl
2971. acyl acyl acyl H acyl acyl H acyl acyl H
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl n7, Rl n8, Rl n9, Rl
2972. acyl acyl acyl H acyl acyl H acyl acyl acyl
2973. acyl acyl acyl H acyl acyl acyl H H H
2974. acyl acyl acyl H acyl acyl acyl H H acyl
2975. acyl acyl acyl H acyl acyl acyl H acyl H
2976. acyl acyl acyl H acyl acyl acyl H acyl acyl
2977. acyl acyl acyl H acyl acyl acyl acyl H H
2978. acyl acyl acyl H acyl acyl acyl acyl H acyl
2979. acyl acyl acyl H acyl acyl acyl acyl acyl H
2980. acyl acyl acyl H acyl acyl acyl acyl acyl acyl
2981. acyl acyl acyl acyl H H H H H H
2982. acyl acyl acyl acyl H H H H H acyl
2983. acyl acyl acyl acyl H H H H acyl H
2984. acyl acyl acyl acyl H H H H acyl acyl
2985. acyl acyl acyl acyl H H H acyl H H
2986. acyl acyl acyl acyl H H H acyl H acyl
2987. acyl acyl acyl acyl H H H acyl acyl H
2988. acyl acyl acyl acyl H H H acyl acyl acyl
2989. acyl acyl acyl acyl H H acyl H H H
2990. acyl acyl acyl acyl H H acyl H H acyl
2991. acyl acyl acyl acyl H H acyl H acyl H
2992. acyl acyl acyl acyl H H acyl H acyl acyl
2993. acyl acyl acyl acyl H H acyl acyl H H
2994. acyl acyl acyl acyl H H acyl acyl H acyl
2995. acyl acyl acyl acyl H H acyl acyl acyl H
2996. acyl acyl acyl acyl H H acyl acyl acyl acyl
2997. acyl acyl acyl acyl H acyl H H H H
2998. acyl acyl acyl acyl H acyl H H H acyl
2999. acyl acyl acyl acyl H acyl H H acyl H
3000. acyl acyl acyl acyl H acyl H H acyl acyl
3001. acyl acyl acyl acyl H acyl H acyl H H
3002. acyl acyl acyl acyl H acyl H acyl H acyl
3003. acyl acyl acyl acyl H acyl H acyl acyl H
3004. acyl acyl acyl acyl H acyl H acyl acyl acyl
3005. acyl acyl acyl acyl H acyl acyl H H H
3006. acyl acyl acyl acyl H acyl acyl H H acyl
3007. acyl acyl acyl acyl H acyl acyl H acyl H
3008. acyl acyl acyl acyl H acyl acyl H acyl acyl
3009. acyl acyl acyl acyl H acyl acyl acyl H H
3010. acyl acyl acyl acyl H acyl acyl acyl H acyl
3011. acyl acyl acyl acyl H acyl acyl acyl acyl H
3012. acyl acyl acyl acyl H acyl acyl acyl acyl acyl
compound 2 nl, Rl n2, Rl n3, Rl n4, Rl n5, Rl n6, Rl nl, Rl n8, Rl n9, Rl
3013. acyl acyl acyl acyl acyl H H H H H
3014. acyl acyl acyl acyl acyl H H H H acyl
3015. acyl acyl acyl acyl acyl H H H acyl H
3016. acyl acyl acyl acyl acyl H H H acyl acyl
3017. acyl acyl acyl acyl acyl H H acyl H H
3018. acyl acyl acyl acyl acyl H H acyl H acyl
3019. acyl acyl acyl acyl acyl H H acyl acyl H
3020. acyl acyl acyl acyl acyl H H acyl acyl acyl
3021. acyl acyl acyl acyl acyl H acyl H H H
3022. acyl acyl acyl acyl acyl H acyl H H acyl
3023. acyl acyl acyl acyl acyl H acyl H acyl H
3024. acyl acyl acyl acyl acyl H acyl H acyl acyl
3025. acyl acyl acyl acyl acyl H acyl acyl H H
3026. acyl acyl acyl acyl acyl H acyl acyl H acyl
3027. acyl acyl acyl acyl acyl H acyl acyl acyl H
3028. acyl acyl acyl acyl acyl H acyl acyl acyl acyl
3029. acyl acyl acyl acyl acyl acyl H H H H
3030. acyl acyl acyl acyl acyl acyl H H H acyl
3031. acyl acyl acyl acyl acyl acyl H H acyl H
3032. acyl acyl acyl acyl acyl acyl H H acyl acyl
3033. acyl acyl acyl acyl acyl acyl H acyl H H
3034. acyl acyl acyl acyl acyl acyl H acyl H acyl
3035. acyl acyl acyl acyl acyl acyl H acyl acyl H
3036. acyl acyl acyl acyl acyl acyl H acyl acyl acyl
3037. acyl acyl acyl acyl acyl acyl acyl H H H
3038. acyl acyl acyl acyl acyl acyl acyl H H acyl
3039. acyl acyl acyl acyl acyl acyl acyl H acyl H
3040. acyl acyl acyl acyl acyl acyl acyl H acyl acyl
3041. acyl acyl acyl acyl acyl acyl acyl acyl H H
3042. acyl acyl acyl acyl acyl acyl acyl acyl H acyl
3043. acyl acyl acyl acyl acyl acyl acyl acyl acyl H
3044. acyl acyl acyl acyl acyl acyl acyl acyl acyl acyl
[0069] In a further aspect, the invention provides polyvalent antigen combinations (and conjugates thereof) representing pluralities of any of the different
oligosaccharides described herein.
[0070] Compositions of the present invention include oligosaccharide structures defined by Formulas 1a and 1b, which may include a linker (L) and may optionally contain a carrier (Y is H or a carrier).
[0071] Suitable linkers comprise at one end a grouping able to enter into a covalent bonding with a reactive functional group of the carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with a hydroxyl group of an oligosaccharide according to the present invention. Between the two functional groups of the linker molecule there is a biocompatible bridging molecule of suitable length, e.g. substituted or
unsubstituted heteroalkylene, arylalkylene, alkylene, alkenylene, or (oligo)alkylene glycol groups. Linkers preferably include substituted or unsubstituted alkylene or alkenylene groups containing 1 -10 carbon atoms.
[0072] Linkers able to react with thiol groups on the carrier are, for example, maleimide and carboxyl groups; preferred groupings able to react with aldehyde or carboxyl groups are, for example, amino or thiol groups. Preferred covalent attachments between linkers and carriers include thioethers from reaction of a thiol with an a-halo carbonyl or a-halo nitrile, including reactions of thiols with maleimide; hydrazides from reaction of a hydrazide or hydrazine with an activated carbonyl group (e.g. activated NHS-ester or acid halide); triazoles from reaction of an azide with an alkyne (e.g. via "click chemistry"); and oximes from reaction of a
hydroxylamine and an aldehyde or ketone as disclosed, for example, in Lees et al., Vaccine, 24:716, 2006. Although amine-based conjugation chemistries could be used in principle for coupling linkers and/or spacers to the oligosaccharides described herein, these approaches would typically sacrifice uniformity inasmuch as the oligosaccharides of the present invention typically contain a plurality of amines bonded to second carbon of the respective monosaccharide units.
[0073] Further suitable linker molecules are known to skilled workers and commercially available or can be designed as required and depending on the functional groups present and can be prepared by known methods.
[0074] Suitable carriers are known in the art (See e.g., Remington's
Pharmaceutical Sciences (18th ed., Mack Easton, PA (1990)) and may include, for example, proteins, peptides, lipids, polymers, dendrimers, virosomes, virus-like particles (VLPs), or combinations thereof, which by themselves may not display particular antigenic properties, but can support immunogenic reaction of a host to the
oligosaccharides of the present invention (antigens) displayed at the surface of the carrier(s).
[0075] Preferably, the carrier is a protein carrier, including but are not limited to, bacterial toxoids, toxins, exotoxins, and nontoxic derivatives thereof, such as tetanus toxoid, tetanus toxin Fragment C, diphtheria toxoid, CRM (a nontoxic diphtheria toxin mutant) such as CRM 197, cholera toxoid, Staphylococcus aureus exotoxins or toxoids, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, including recombinantly produced, genetically detoxified variants thereof; bacterial outer membrane proteins, such as Neisseria meningitidis serotype B outer membrane protein complex (OMPC), outer membrane class 3 porin (rPorB) and other porins; keyhole limpet hemocyanine (KLH), hepatitis B virus core protein, thyroglobulin, albumins, such as bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin; pneumococcal surface protein A (PspA),
pneumococcal adhesin protein (PsaA); purified protein derivative of tuberculin (PPD); transferrin binding proteins, polyamino acids, such as poly(lysine:glutamic acid); peptidyl agonists of TLR-5 (e.g. flagellin of motile bacteria like Listeria); and derivatives and/or combinations of the above carriers. Preferred carriers for use in humans include tetanus toxoid, CRM 197, and OMPC.
[0076] Depending on the type of bonding between the linker and the carrier, and the structural nature of the carrier and oligosaccharide, a carrier may display on average, for example, 1 to 500, 1 to 100, 1 to 20, or 3 to 9 oligosaccharide units on its surface.
[0077] Methods for attaching an oligosaccharide to a carrier, such as a carrier protein are conventional, and a skilled practitioner can create conjugates in accordance with the present invention using conventional methods. Guidance is also available in various disclosures, including, for example, U.S. Pat. Nos.
4,356,170; 4,619,828; 5,153,312; 5,422,427; and 5,445,817; and in various print and online Pierce protein cross-linking guides and catalogs (Thermo Fisher, Rockford, IL).
[0078] In one embodiment, the carbohydrate antigens of the present invention are conjugated to CRM 197, a commercially available protein carrier used in a number of FDA approved vaccines. CRM-conjugates have the advantage of being
easier to synthesize, purify and characterize than other FDA approved carriers such as OMPC. Carbohydrate antigens may be conjugated to CRM via thiol-bromoacetyl conjugation chemistry. CRM activation may be achieved by reacting the lysine side chains with the NHS ester of bromoacetic acid using standard conditions as previously described in U.S. Pat. Appl. Publ. 2007-0134762, the disclosures of which are incorporated by reference herein. CRM may be functionalized with 10-20 bromoacetyl groups per protein (n= 10-20) prior to conjugation. Conjugation may be performed at pH=9 to avoid aggregation of CRM. Careful monitoring of pH must be employed to ensure complete CRM reaction with NHS-bromoacetate while minimizing background hydrolysis of CRM. Activated CRM may be purified by size exclusion chromatography prior to conjugation. Antigen-CRM conjugates may be synthesized by reacting thiol-terminated carbohydrate antigens with
bromoacetamide-activated CRM.
[0079] CRM conjugates may be purified via size exclusion chromatography to remove and recover any unreacted carbohydrate. MBTH (specific for GlcNAc residues) and Bradford assays may be used to determine carbohydrate:protein ratio and protein content, respectively, as previously described (Manzi et al., Curr. Prot. Mol. Biol., section 17.9.1 (Suppl. 32), 1995. In preferred embodiments, a minimum carbohydrate content of about 10% by weight for each conjugate may be generated. Typically, a conjugate may include about 3-20 antigens per protein carrier.
[0080] In another embodiment, carbohydrate antigens may be conjugated to one or more carriers suitable for development of diagnostic assays, including ELISAs and microarrays. Exemplary carriers for use in such assays include bovine serum albumin (BSA), keyhole limpet hemocyanine (KLH), biotin, a label, a glass slide or a gold surface. By way of example, synthetic carbohydrate antigens may be conjugated to BSA by a thiol-maleimide coupling procedure (FIG. 5B). Maleimide- BSA contains 15-20 maleimide groups per protein (n=15-20). Accordingly, oligosaccharide antigens may be conjugated to maleimide functionalized BSA, whereby a 20-fold molar excess of the antigen is reacted with commercially available Imject maleimide BSA (Pierce) in maleimide conjugation buffer (Pierce).
Conjugation may be performed at pH=7.2 to avoid hydrolysis of the maleimide group during conjugation.
[0081] BSA conjugates may be purified via size exclusion chromatography to remove and recover any unreacted carbohydrate. Characterization via the phenol- sulfuric acid and Bradford assays may be performed along with MALDI-MS to provide information on the carbohydrate content and valency of the conjugates. In preferred embodiments, conjugates will contain a minimum carbohydrate content of about 10% by weight per BSA conjugate and >8 antigen copies per conjugate.
[0082] Methods for Synthesizing Oligosaccharide Structures
[0083] In a further aspect, the present invention provides a method for
assembling synthetic homogenous oligosaccharide structures from N. meningitidis, including those described above from building blocks as depicted in FIG. 2.
[0084] Compositions and methods for synthesis of the above described oligosaccharides and conjugates thereof are described in the examples below.
Protecting groups employed in the synthesis of oligosaccharides 1a and 1b may include those customarily considered in sugar chemistry, for example those mentioned in "Protective Groups in Organic Synthesis", 3rd edition, T. W. Greene and P. G. M. Wuts (Ed.), John Wiley and Sons, New York, 1999.
[0085] Immunogenic and Immunoprotective Compositions and Methods of their Use
[0086] In another aspect, the present invention provides immunogenic and immunoprotective compositions containing oligosaccharides or oligosaccharide- protein carrier conjugates for inducing an immune response. The immunogenic compositions may include one or more adjuvants, as well as pharmaceutically acceptable vehicles suitable for administration to an animal or individual. An immunogenic or immunoprotective composition will include a "sufficient amount" or "an immunologically effective amount" of a oligosaccharide- protein carrier conjugate according to the present invention, as well as any of the above mentioned
components, for purposes of generating an immune response or providing protective immunity, as further defined herein.
[0087] In one embodiment, the invention provides an immunogenic composition comprising one or more oligosaccharide(s) 1a or oligosaccharide-protein carrier conjugate(s) 1 b suitable for inducing an immune response against N. meningitidis, particularly serotypes NmA, NmB, NmC and NmW-135, particularly preferably NmB.
[0088] In another embodiment, the invention provides a pharmaceutical composition comprising an oligosaccharide(s) 1a or oligosaccharide-protein carrier conjugate 1 b formulated as a vaccine for protection against N. meningitidis infections, particularly serotypes NmA, NmB, NmC and NmW-135, particularly preferably NmB.
[0089] In another embodiment, the invention provides a pharmaceutical composition comprising an oligosaccharide-protein carrier conjugate 1 b formulated as a vaccine for protecting against N. meningitidis as described herein.
[0090] In a further embodiment, the invention provides a pharmaceutical composition comprising an antibody and a physiologically acceptable vehicle for use in a method for providing passive immunity or treatment against N. meningitidis. More particularly, the invention provides an antibody preparation against one or more oligosaccharide conjugate 1 b compositions in accordance with the present invention. The antibody preparation may include any member from the group consisting of polyclonal antibody, monoclonal antibody, mouse monoclonal IgG antibody, humanized antibody, chimeric antibody, fragment thereof, or combination thereof. The invention further contemplates a hybridoma cell producing a
monoclonal antibody directed against any of the oligosaccharide described herein.
[0091] Administration of oligosaccharides or oligosaccharide-protein carrier conjugates or antibodies thereto may be carried out by any suitable means, including by parenteral administration (e.g., intravenously, subcutaneously, intradermally, or intramuscularly); by topical administration, of for example, antibodies to an airway surface; by oral administration; by in ovo injection in birds, for example, and the like.
[0092] In specific embodiments, each immunogenic or immunoprotective composition includes one or more oligosaccharide(s) 1a or 1 b or conjugates thereof in a pharmaceutically acceptable vehicle or diluent forming a substantially aqueous mixture. In preferred embodiments, the immunogenic or immunoprotective compositions includes one or more oligosaccharide-protein carrier conjugates(s) in conjunction with one or more pharmaceutically acceptable adjuvant(s), vehicles and/or protein carriers suitable for administration to an animal or individual.
[0093] Adjuvants
[0094] An oligosaccharide-protein carrier conjugate composition may further include one or more immunologic adjuvant(s). An immunologic adjuvant is a compound that, when combined with an antigen, increases the immune response to the antigen as compared to the response induced by the antigen alone so that less antigen can be used to achieve a similar response. For example, an adjuvant may augment humoral immune responses, cell-mediated immune responses, or both.
[0095] Those of skill in the art will appreciate that the terms "adjuvant," and "carrier," can overlap to a significant extent. For example, a substance which acts as an "adjuvant" may also be a "carrier," and certain other substances normally thought of as "carriers," for example, may also function as an "adjuvant." Accordingly, a substance which may increase the immunogenicity of the synthetic oligosaccharide or carrier associated therewith is a potential adjuvant. As used herein, a carrier is generally used in the context of a more directed site-specific conjugation to an oligosaccharide of the present invention, whereby an adjuvant is generally used in a less specific or more generalized structural association therewith.
[0096] Exemplary adjuvants and/or adjuvant combinations may be selected from the group consisting of mineral salts, including aluminum salts, such as aluminum phosphate and aluminum hydroxide (alum) (e.g., Alhydrogel™, Superfos, Denmark) and calcium phosphate; RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion, whereby any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2; toll-like receptor (TLR) agonists, including monophosphoryl lipid A (MPL®), 3 De-O-acylated monophosphoryl lipid A (3 D-MPL), OM-174 (E. coli lipid A derivative); OM triacyl lipid A derivative, and other MPL- or lipid A-based formulations and combinations thereof, including MPL®-SE, RC-529 (Dynavax Technologies), AS01
(liposomes+MPL+QS21 ), AS02 (oil-in-water PL + QS-21 ), and AS04 (Alum + MPL)(GlaxoSmith Kline, Pa.), CpG-oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs, double-stranded RNA, polyinosinic:polycytidylic acid (poly l:C), and other oligonucleotides or polynucleotides optionally encapsulated in liposomes; oil-in-water emulsions, including AS03 (GlaxoSmith Kline, Pa.), MF-59 (microfluidized detergent stabilized squalene oil-in-water emulsion; Novartis), and
Montanide ISA-51 VG (stabilized water-in-oil emulsion) and Montanide ISA-720 (stabilized water/squalene; Seppic Pharmaceuticals, Fairfield, NJ); cholera toxin B subunit; saponins, such as Quil A or QS21 , an HPLC purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina (STIMULON™ (Antigenics, Inc., Lexington, Mass.) and saponin-based adjuvants, including immunostimulating complexes (ISCOMs; structured complex of saponins and lipids) and other ISCOM- based adjuvants, such as ISCOMATRIX™ and AblSCO®-100 and -300 series adjuvants (Isconova AB, Uppsala, Sweden); QS21 and 3 D-MPL together with an oil in water emulsion as disclosed in U.S. Pat. Appl. No. 2006/0073171 ; stearyl tyrosine (ST) and amide analogs thereof; virus-like particles (VLPs) and reconstituted influenza virosomes (IRIVs); complete Freund's adjuvant (CFA); incomplete
Freund's adjuvant (IFA); E. coli heat-labile enterotoxin (LT); immune-adjuvants, including cytokines, such as IL-2, IL-12, GM-CSF, Flt3, accessory molecules, such as B7.1 , and mast cell (MC) activators, such as mast cell activator compound 48/80 (C48/80); water-insoluble inorganic salts; liposomes, including those made from DNPC/Chol and DC Choi; micelles; squalene; squalane; muramyl dipeptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '2'-dipalmitoyl-n-glycero-3- hydroxyphosphoryl; SAF-1 (Syntex); AS05 (GlaxoSmith Kline, Pa.); and
combinations thereof.
[0097] In preferred embodiments, adjuvant potency may be enhanced by combining multiple adjuvants as described above, including combining various delivery systems with immunopotentiating substances to form multi-component adjuvants with the potential to act synergistically to enhance antigen-specific immune responses in vivo. Exemplary immunopotentiating substances include the above- described adjuvants, including, for example, MPL and synthetic derivatives, MDP and derivatives, oligonucleotides (CpG etc), ds RNAs, alternative pathogen- associated molecular patterns (PAMPs)(E. coli heat labile enterotoxin; flagellin, saponins (QS-21 etc), small molecule immune potentiators (SMIPs, e.g., resiquimod [R848]), cytokines, and chemokines.
[0098] Pharmaceutically-acceptable delivery vehicles
[0099] Pharmaceutically-acceptable delivery vehicles, including those described above may be employed to enhance the delivery and/or control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the oligosaccharides, oligosaccharide conjugates, and/or adjuvants. Controlled delivery may be effected by selecting appropriate
macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and
poly(methylmethacylate)-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
[00100] The oligosaccharide compositions of the present invention, including oligosaccharide-protein carrier conjugate compositions, can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a
pharmaceutically acceptable vehicle (or diluents). Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences, supra. In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the above-described
compounds together with a suitable amount of protein carrier and/or vehicle.
[00101] Typically, the immunogenic or immunoprotective compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection. An
aqueous composition for parenteral administration, for example, may include a solution of the immunogenic component(s) dissolved or suspended in a
pharmaceutically acceptable vehicle or diluent, preferably a primarily aqueous vehicle. Pharmaceutically acceptable vehicles or diluents may include water, saline, including neutral saline solutions buffered with phosphate, Tris, glycerol, ethanol, and the like. An aqueous composition may be formulated as a sterile, pyrogen-free buffered saline or phosphate-containing solution, which may include a preservative or may be preservative free. Suitable preservatives include benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride, for example.
Aqueous solutions are preferably approximately isotonic, and its tonicity may be adjusted with agents such as sodium tartrate, sodium chloride, propylene glycol, and sodium phosphate. Additionally, auxiliary substances required to approximate physiological conditions, including pH adjusting and buffering agents, tonicity adjusting agents, wetting or emulsifying agents, pH buffering substances, and the like, including sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. may be included with the vehicles described herein.
[00102] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The preparation of such pharmaceutical compositions is within the ordinary skill in the art, and may be guided by standard reference books such as Remington's Pharmaceutical Science, supra, which is incorporated herein by reference.
[00103] Compositions may be formulated in a solid or liquid form for oral delivery. For solid compositions, nontoxic and/or pharmaceutically acceptable solid protein carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition may be formed by incorporating any of the normally employed excipients, including those protein carriers previously listed, and a unit
dosage of an active ingredient, that is, one or more compounds of the invention, whether conjugated to a protein carrier or not.
[00104] Topical application of antibodies to an airway surface, for example, can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatuses for administering respirable particles of pharmaceutical
formulations are well known, and any conventional technique can be employed. Oral administration may be in the form of an ingestable liquid or solid formulation.
[00105] Further, compositions may be formulated in an aerosol for nasal administration. For aerosol administration, the immunogenic compounds are preferably supplied in finely divided form along with one or more surfactant(s) and/or propellant(s). The surfactant will be nontoxic, and preferably soluble in the
propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1 %-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A protein carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
[00106] The concentration of the immunogenic oligosaccharides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1 %, usually at or at least about 0.1 % to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., and in accordance with the particular mode of administration selected. A human unit dose form of the compounds and composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable protein carrier, preferably an aqueous protein carrier, and is administered in a volume of fluid that is known by
those of skill in the art to be used for administration of such compositions to humans, and is adjusted according to commonly understood principles for a particular subject to be treated. Thus in one embodiment, the invention provides a unit dosage of the vaccine components of the invention in a suitable amount of an aqueous solution, such as 0.1 -3 ml, preferably 0.2-2 mL.
[00107] Methods of treatment
[00108] The immunogenic and immunoprotective compositions of the present invention may be administered to any animal species at risk for developing an infection by N. meningitidis.
[00109] The treatment may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of treatment may be with 1 -10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, at 1 -4 months for a second dose, and if needed, a subsequent dose(s) after several months. Examples of suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.
[00110] The amounts effective for inducing an immune response or providing protective immunity will depend on a variety of factors, including the oligosaccharide composition, conjugation to a protein carrier, inclusion and nature of adjuvant(s), the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. By way of example, the amounts may generally range for the initial immunization (that is for a prophylactic administration) from about 1 .0 pg to about 5,000 pg of carbohydrate antigen for a 70 kg patient, (e.g., 1 .0 pg, 2.0 pg, 2.5 pg, 3.0 pg, 3.5 pg, 4.0 pg, 4.5 pg, 5.0 pg, 7.5 pg, 10 pg, 12.5 pg, 15 pg, 17.5 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 75 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ,000 pg, 1 ,500 pg, 2,000 pg, 2,500 pg, 3,000 pg, 3,500 pg, 4,000 pg, 4,500 g or 5,000 pg). The actual dose administered to a subject is often determined according to an appropriate amount per kg of the subject's body weight. For example, an effective amount may be about 0.1 g to 5 pg/kg body weight.
[00111] A primary dose may optionally be followed by boosting dosages of from about 1 .0 to about 1 ,000 of carbohydrate antigen (e.g., 1 .0 pg, 2.0 pg, 2.5 pg, 3.0 pg, 3.5 pg, 4.0 pg, 4.5 pg, 5.0 pg, 7.5 pg, 10 pg, 12.5 pg, 15 pg, 17.5 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 75 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ,000 pg, 1 ,500 pg, 2,000 pg, 2,500 pg, 3,000 pg, 3,500 pg, 4,000 pg, 4,500 pg or 5,000 pg) pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific T cell activity in the patient's blood.
[00112] The present invention contemplates the use of single- and multi-valent glycoconjugate vaccines comprising any of the synthetic oligosaccharides described herein. The identification of a single oligosaccharide antigen eliciting a cross- reactive immune response can facilitate development of a single-antigen vaccine candidate active against N. meningitidis.
[00113] The present invention further contemplates multi-antigen glycoconjugate vaccines comprising a plurality of any of the synthetic oligosaccharides described herein so as to provide protection against N. meningitidis. Thus, in one
embodiment, for example, the invention provides a composition containing two, three, four or more different oligosaccharide antigens according to Formula 1 b.
[00114] The immunogenic compositions comprising a compound of the invention may be suitable for use in adult humans or in children, including young children or others at risk for contracting an infection caused by a N. meningitidis. Optionally such a composition may be administered in combination with other pharmaceutically active substances, and frequently it will be administered in combination with other vaccines as part of a childhood vaccination program.
[00115] Compositions for administration may beneficially include multiple oligosaccharide- or oligosaccharide conjugates that elicit an immune response to a plurality of different epitopes so as to provide increased protection against N.
meningitidis. Moreover, compositions may be administered whereby a prime immunization with one or multiple antigen conjugates is followed by boosting events with one or more cross-reactive core conjugates according to the present invention.
[00116] Antibody compositions
[00117] In another embodiment, the invention provides diagnostic antibodies, as well as pharmaceutical compositions comprising one or more anti-oligosaccharide 1a antibody(ies) or a functional fragment(s) thereof, and a physiologically acceptable vehicle. Methods for generating these antibodies are further described below.
[00118] Pharmaceutical antibody compositions may be used in a method for providing passive immunity against N. meningitidis infections. A pharmaceutical antibody composition may be administered to an animal subject, preferably a human, in an amount sufficient to prevent or attenuate the severity, extent of duration of the infection by N. meningitidis.
[00119] The administration of one or more antibodies may be either prophylactic (prior to anticipated exposure to a bacterial infection) or therapeutic (after the initiation of the infection, at or shortly after the onset of the symptoms). The dosage of the one or more antibodies will vary depending upon factors as the subject's age, weight and species. In general, the dosage of the antibody may be in a range from about 1 -10 mg/kg body weight. In a preferred embodiment, the antibody is a humanized antibody of the IgG or the IgA class. The route of administration of the one or more antibodies may be oral or systemic, for example, subcutaneous, intramuscular or intravenous.
[00120] The use of antibodies as diagnostic agents is further described below and in U.S. Pat. No. 7,595,307 and U.S. Pat. Appl. Publ. No. 2009/0155299, the discolosures of which are incorporated by reference herein.
[00121] The present invention also provides one or more kits useful for diagnosing, treating, and/or preventing a N. meningitidis infection. For example, the kits may include one or more containers holding the diagnostic or pharmaceutical compositions of the invention. The kits may also include other container(s) containing, for example, one or more solutions necessary or convenient for the particular diagnostic or pharmaceutical use. The container means can be made of glass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. The kit may also contain written information, such as procedures for carrying out the present invention or analytical information, such as the amount of reagent contained in the
container(s).
[00122] Generation of antibodies and their use in assay development
[00123] In a further aspect, the present invention provides compositions and methods for inducing production of antibodies for use in assay development, including their use as detection agents and serum screening tools.
[00124] Antisera to conjugates may be generated in New Zealand white rabbits by 3-4 subcutaneous injections over 13 weeks. A pre-immune bleed may generate about 5 ml_ of baseline serum from each rabbit. A prime injection (10 μg antigen equivalent) may be administered as an emulsion in complete Freund's adjuvant (CFA). Subsequent injections (5 μg antigen equivalent) may be given at three week intervals in incomplete Freund's adjuvant (IFA). Rabbits may be bled every two weeks commencing one week after the third immunization. Approximately 25 - 30 ml_ of serum per rabbit may be generated from each bleeding event and frozen at - 80°C. Serum may be analyzed by ELISA against the corresponding
oligosaccharide-conjugate. In addition, antisera from later bleeds may be affinity purified.
[00125] The oligosaccharides and antibodies of the present invention can be used as diagnostic reagents for detecting N. meningitidis antigens or antibodies thereagainst, which are present in biological samples. The detection reagents may be used in a variety of immunodiagnostic techniques, known to those of skill in the art, including ELISA- and microarray-related technologies. In addition, these reagents may be used to evaluate antibody responses, including serum antibody levels, to immunogenic oligosaccharide conjugates. The assay methodologies of the invention typically involve the use of labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, and/or secondary immunologic reagents for direct or indirect detection of a complex between an antigen or antibody in a biological sample and a corresponding antibody or antigen bound to a solid support.
[00126] Such assays typically involve separation of unbound antibody in a liquid phase from a solid phase support to which antibody-antigen complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates);
polyvinyl idine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
[00127] Typically, a solid support is first reacted with a first binding component (e.g., an antigen or antibody in accordance with the present invention) under suitable binding conditions such that the first binding component is sufficiently immobilized to the support. In some cases, mobilization to the support can be enhanced by first coupling the antibody or oligosaccharide to a protein with better binding properties, or that provides for immobilization of the antibody or antigen on the support without significant loss of antibody binding activity or specificity. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind antibodies to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules are well known to those of ordinary skill in the art and are described in, e.g., U.S. Pat. No. 7,595,307 and U.S. Pat. Appl. No. US 2009/0155299.
EXAMPLES
[00128] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
[00129] Materials and Methods
[00130]
[00131] List of Standard Operating Procedures:
[00132] Standard Operating Procedure 1 : Protection of hydroxyl groups by benzylidene acetals.
[00133] Standard Operating Procedure 2: Protection of hydroxyl groups by acetylation.
[00134] Standard Operating Procedure 3: Amine protection by
propionylation.
[00135] Standard Operating Procedure 4: Regio-selective cleavage of benzylidene acetals.
[00136] Standard Operating Procedure 5: Amine protection by Boc group.
[00137] Standard Operating Procedure 6: Sialylation promoted by NIS/TfOH.
[00138] Standard Operating Procedure 7: Aglycone leaving group
transformation.
[00139] Standard Operating Procedure 8: Base hydrolysis.
[00140] Standard Operating Procedure 9: Hydrogenation.
[00141] Standard Operating Procedure 10: /V-Acylation with SATP.
[00142] Standard Operating Procedure 11 : Boc cleavage.
[00143] Standard Operating Procedure 1 : Protection of hydroxyl groups by benzylidene acetal.
[00144] To a solution of the starting material (4.44 mmol) in anhydrous DMF (90 mL) were added camphorsulfonic acid (468 mg, 2.01 mmol) and benzaldehyde dimethyl acetal (3.63 mL, 24.2 mmol). The reaction mixture was stirred at room temperature overnight and quenched with Et3N (561 μί, 4.03 mmol). The solution was diluted with EtOAc and washed with brine three times. The organic solution was dried over Na2SO4, filtered and concentrated under vacuum. Purification by silica gel column chromatography (eluent: EtOAc/Heptane) afforded the desired product.
[00145] Standard Operating Procedure 2: Protection of hydroxyl groups by acetylation.
[00146] A solution of the starting material (1 .99 mmol) in anhydrous pyridine (15 mL) and AC2O (10 mL) was stirred overnight at room temperature and then concentrated under reduced pressure. The residue was co-evaporated with toluene twice. Purification via silica gel column chromatography (eluent: EtOAc/Heptane) afforded the desired acetylated product.
[00147] Standard Operating Procedure 3: Amine protection by
propionylation.
[00148] A suspension of the starting material (1 .99 mmol), EtN(/'-Pr)2 (3.5 mL, 19.9 mmol) and 4A powdered molecular sieves (1 .1 g) in anhydrous CH2CI2 (20 mL) was stirred at room temperature for 1 hr and then cooled down to 0 °C. To the cold solution was added propionyl chloride (1 .4 mL, 15.9 mmol) drop wise. After addition, the reaction mixture was stirred at 0 °C for 1 hr and then slowly warmed up to room temperature. The solution was diluted with CH2CI2, filtered through celite and concentrated under vacuum. The residue was dissolved in EtOAc, washed with aqueous saturated NaHCO3 solution and brine, dried over Na2SO4, and
concentrated under reduced pressure. Purification through silica gel chromatography (eluent: Heptane/EtOAc) afforded the desired product.
[00149] Standard Operating Procedure 4: Regio-selective cleavage of benzylidene acetals.
[00150] To a solution of the starting material (9.97 mmol) in anhydrous 1 ,4- dioxane (87.0 mL) was added a solution of AICI3/ BH3 NMe3 (1 : 1 molar ratio, 0.3 M, , 99.7 mL) in anhydrous 1 ,4-dioxane under stirring at room temperature. After being stirred at r.t. for 1 hr, the reaction mixture was diluted with EtOAc, washed with cold 1 M HCI and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. Purification by silica gel column chromatography (eluent:
EtOAc/Heptane, 30-50% EtOAc gradient) afforded the product.
[00151] Standard Operating Procedure 5: Amine protection by Boc group.
[00152] The solution of the substrate (2.94 mmol), DMAP (359 mg, 2.94 mmol) and B0C2O (963 mg, 4.41 mmol) in anhydrous CH2CI2 (50 mL) was stirred for 1 hr at room temperature. The mixture was concentrated under reduced pressure and the residue was purified through silica gel chromatography (eluent: Heptane/EtOAc) to give the product.
[00153] Standard Operating Procedure 6: Sialylation procedure promoted by NIS/TfOH.
[00154] The suspension of donor (1 .16 mmol, 1 .79 eq), acceptor (0.647 mmol, 1 eq) and activated 4A powdered molecular sieves (1 .0 g) in anhydrous CH2CI2 (10 mL) and CH3CN (5 mL) was stirred at room temperature under N2 protection for 1 hr and then cooled down to - 78 °C with dry ice/acetone bath. To the cold reaction mixture was added NIS (524 mg, 2.33 mmol, 1 .79x2 eq), followed by the injection of
TfOH (103 μΙ_, 1 .16 mmol, 1 .79 eq). After addition, the reaction mixture was stirred for 10 min at - 78 °C and then quenched with Et3N (324 μΙ_, 2.33 mmol, 1 .79x2 eq). The mixture was diluted with CH2CI2 and filtered through celite. The organic solution was washed with 10% aqueous Na2S2O3, dried over Na2SO4, and concentrated under reduced pressure. Purification of the residue through silica gel column chromatography (eluent: Heptane/EtOAc or Toluene/EtOAc) afforded the coupling product.
[00155] Standard Operating Procedure 7: Aglycone leaving group
transformation.
[00156] A suspension of the starting phenyl thioglycoside (0.616 mmol), Ph2SO (373 mg, 1 .85 mmol), 2,4,6-Tri-tert-Butyl Pyrimidine (306 mg, 1 .23 mmol) and activated 4A powdered molecular sieves (900 mg) in anhydrous CH2CI2 (8 mL) was stirred at room temperature under argon protection for 1 hr and then cooled down to - 78 °C with dry ice/acetone bath. To the cold reaction mixture was added Tf2O (124 μΙ_, 0.739 mmol) drop wise. After addition, the mixture was stirred for 10 min and then a 1 -adamantanethiol (31 1 mg, 1 .85 mmol) solution in anhydrous CH2CI2 (1 .5 mL) was injected into the mixture drop wise along the inside wall of the cold flask. After addition, the mixture was stirred at - 78 °C for 1 hr and slowly warmed up to room temperature. The reaction mixture was diluted with CH2CI2, filtered through celite, washed with aqueous saturated NaHCO3 solution and brine, dried over Na2SO4, and concentrated under reduced pressure. Purification through silica gel column chromatography (eluent: Heptane/EtOAc) afforded the desired product.
[00157] Standard Operating Procedure 8: Base hydrolysis.
[00158] To a solution of the starting protected material (0.14 mmol) in MeOH (3 mL) was added NaOMe (0.13mL, 4M in MeOH). The reaction mixture was stirred at room temperature for 1 hour then warmed to 40°C for 1 hour. After cooling to room temperature, NaOH (1 mL, 1 M in H2O) was added and the reaction mixture was warmed to 40°C and stirred for an additional 12 hours. The reaction mixture was cooled to room temperature and neutralized to pH=7 with amberlyst A-15 resin. The solution was filtered through celite and solids were washed with MeOH (50 mL). The solution was concentrated in vacuo to afford a white solid. Purification via size exclusion chromatography on a Sephadex G-10 column (1 " x 3" column, H2O eluent,
3 mL fractions) afforded the desired product. Typical isolated yields for the product formation varied between 80-95%.
[00159] Standard Operating Procedure 9: Hydrogenation.
[00160] To a solution of the starting material (0.14 mmol) in H2O:MeOH (1 :1 , v:v, 10 mL total) were added AcOH (20 μί, 1 .2 mmol) and Pd on carbon (200 mg, 10% Pd/C, dry). The reaction mixture was purged with hydrogen 10 times and pressure was increased to 30 psi. The reaction mixture was stirred vigorously at room temperature for 3 days. After purging with N2 (10 times), the reaction mixture was filtered through celite and solids were washed with MeOH (50 mL).
Concentration in vacuo afforded the desired product as a white solid. The
hydrogenated material was used without purification in the next step.
[00161] Standard Operating Procedure 10: A/ acylation with SATP.
[00162] To a solution of the starting material (0.068 mmol) in H2O (0.5 mL) were added triethylamine (19 μί, 0.135 mmol). To the reaction mixture was added a solution of S-acetylthiopropionic acid (SATP) (33 mg, 0.135 mmol, in 0.1 mL DMF). The reaction mixture was stirred at room temperature for 30 minutes then loaded directly onto a Sephadex G-10 column (1 " x 3" column, H2O eluent, 3 mL fractions). Typical isolated yields for the desired product formation varied between 80-95%.
[00163] Standard Operating Procedure 11 : Boc cleavage.
[00164] To a solution of the starting material (0.068 mmol) in H2O:CH3CN (1 :1 , v:v, 2 mL total) was added 10% HCI(aq) (100 μί). The reaction mixture was stirred at room temperature for 15 min. The HCI addition and 15 min. wait cycle were repeated 5 times until TLC showed cleavage of the Boc group. The reaction mixture was concentrated in vacuo to afford a white solid. Purification via size exclusion chromatography on a Sephadex G-10 column (1 " x 3" column, H2O eluent, 3 mL fractions) afforded the desired product. Typical isolated yields for the product formation varied between 50-80%.
[00165] Example 1 - Synthesis of Building Blocks (See Figure 2)
[00166] Triol 1 (2.03 g, 4.44 mmol, prepared as described in J. Org. Chem. 2007, 72, 2387.) was treated using standard operating procedure (SOP) 1.
Purification by silica gel chromatography (eluent: EtOAc/Heptane, 25-75% EtOAc gradient) afforded 2 (1 .90 g, 3.48 mmol, 79% yield) as a white foam.
[00167] To a suspension of 2 (320 mg, 0.655 mmol) and activated 4A
powdered molecular sieves (500 mg) in anhydrous THF (7.5 mL) were added AICI3 (530 mg, 3.93 mmol, portion wise addition) and BH3 NMe3 (290 mg, 3.93 mmol). After addition, the mixture was stirred at room temperature over 17 hr and then diluted with EtOAc, filtered through celite, washed with cold 1 M H2SO4 and brine. The organic layer was dried over Na2SO4 and concentrated under vacuum.
Purification through silica gel chromatography (eluent: CHCI3/MeOH, 20/1 ) afforded 3 (253 mg, 0.517 mmol, 79% yield) as a white solid.
[00168] The intermediate 2 (0.970 g, 1 .99 mmol) was treated with SOP 2. The crude product was applied for the next step without purification.
[00169] The crude product 4 (1 .99 mmol) was treated with SOP 3 to give 5
(797 mg, 1 .36 mmol, 68% yield over two steps) as a white foam.
[00170] The intermediate 5 (5.84 g, 9.97 mmol) was treated with SOP 4 to give
6 (4.28 g, 7.28 mmol, 73% yield) as a white foam.
[00171] Starting material 7 (1 .44 g, 3.16 mmol, prepared as described in J. Org. Chem. 2007, 72, 7794.) was treated with SOP 2 to give 8 (1 .72 g, 2.94 mmol, 93% yield) as a white solid.
[00172] The intermediate 8 (1 .08 g, 1 .86 mmol) was treated with SOP 3 to give
9 (930 mg, 1 .45 mmol, 78% yield) as a white solid.
[00173] The intermediate 8 (1 .72 g, 2.94 mmol) was treated with SOP 5 to give
10 (1 .82 g, 2.66 mmol, 90% yield) as a white solid.
[00174] The triol 7 (2.03 g, 4.44 mmol, prepared as described in J. Org. Chem. 2007, 72, 7794.) was treated with SOP 1 to give 11 (7.98 g, 14.6 mmol, 73% yield) as a white foam.
[00175] The intermediate 11 (5.49 g, 10.1 mmol) was treated with SOP 2 to give 12 (5.22 g, 8.88 mmol, 88% yield) as a white foam.
[00176] The intermediate 12 (5.22 g, 8.88 mmol) was treated with SOP 3 to give 13 (4.7 g, 7.30 mmol, 82% yield) as a white solid.
[00177] The suspension of 13 (1 .06 g, 1 .64 mmol), 6-azidohexane-1 -ol (346 μΙ_, 2.46 mmol) and activated 4A powdered molecular sieves (1 .27 g) in anhydrous CH2CI2 (16.6 mL) and CH3CN (8.3 mL) was stirred at r.t. under N2 protection over 1 hr and then cooled down to - 78 °C with dry ice/acetone bath. To the cold mixture
was added NIS (739 mg, 3.28 mmol), followed by the injection of TfOH (145 μΙ_, 1 .64 mmol). After addition, the mixture was stirred over 1 hr at - 78 °C and then pyridine (664 μΙ_, 8.21 mmol) and AcCI (293 μΙ_, 4.10 mmol) were injected in to the reaction mixture. The mixture was warmed up to room temperature and stirred over another 1 hr, followed by dilution with CH2CI2 and filtration through celite. The organic solution was washed with 10% aqueous Na2S2O3 solution, dried over Na2SO4, and concentrated under reduced pressure. Purification of the residue through silica gel column chromatography (eluent: Heptane/EtOAc, 25-75% EtOAc gradient) afforded 14 (860 mg, 1 .39 mmol, 85% yield) as a yellow oil.
[00178] The coupling product 14 (860 mg, 1 .39 mmol) was treated with SOP 4 to give 15 (640 mg, 1 .03 mmol, 74% yield) as a yellow oil.
[00179] Donor 10 (796 mg, 1 .16 mmol, 1 .8 eq) and acceptor 15 (401 mg, 0.647 mmol, 1 eq) was treated with SOP 6 (NIS 1 .8x2 eq, TfOH 1 .8 eq, Et3N 1 .8x2 eq) to give the coupling product 16 (509 mg, 0.448 mmol, 69% yield) as a white foam.
[00180] Example 2 - Assembly of oligosaccharides from Building Blocks (Figure 3)
[00181] Donor 10 (1 .95 g, 2.85 mmol, 1 .0 eq) and acceptor 6 (2.51 g, 4.27 mmol, 1 .5 eq) were treated with SOP 6 (NIS 1 .2 eq, TfOH 1 .0 eq, Et3N 2 eq) to give the coupling product 17 (3.63 g, 3.29 mmol, 67% yield) as a white foam.
[00182] The dimer 17 (679 mg, 0.616 mmol) was treated with SOP 7 to give product 18 (449 mg, 0.387 mmol, 69% yield).
[00183] Donor 18 (416 mg, 0.358 mmol, 1 .0 eq) and acceptor 6 (316 mg, 0.537 mmol, 1 .5 eq) were treated with SOP 6 (NIS 1 .2 eq, TfOH 1 .0 eq, Et3N 2 eq) to give the coupling product 19 (253 mg, 0.160mmol, 45% yield).
[00184] The trimer 19 (231 mg, 0.146 mmol) was treated with SOP 7 to give product 20 (162 mg, 0.0989 mmol, 68% yield).
[00185] Donor 20 (468 mg, 0.285 mmol, 1 .0 eq) and acceptor 15 (303 mg, 0.488 mmol, 1 .83 eq) were treated with SOP 6 (NIS 2.0 eq, TfOH 1 .0 eq, Et3N 2 eq) to give the coupling product 21 (121 mg, 0.0579 mmol, 20% yield).
[00186] Donor 20 (1 .95 g, 1 .19 mmol, 1 .0 eq) and acceptor 6 (1 .39 g, 2.36 mmol, 1 .98 eq) were treated with SOP 6 (NIS 1 .2 eq, TfOH 1 .0 eq, Et3N 2 eq) to give the coupling product 22 (1 .18 g, 0.573 mmol, 48% yield).
[00187] The tetramer 22 (1 .18 g, 0.573 mmol) was treated with SOP 7 to give product 23 (900 mg, 0.425 mmol, 74% yield).
[00188] Donor 23 (900 mg, 0.425 mmol, 1 .0 eq) and acceptor 15 (602 mg, 0.971 mmol, 2.28 eq) were treated with SOP 6 (NIS 2.0 eq, TfOH 1 .0 eq, Et3N 2 eq). The coupling product 24 (121 mg, 0.0471 mmol, 1 1 % yield) was isolated through preparative reverse phase HPLC (CH3CN:H2O (isocratic) 85%:15%, Column:
Discovery HS C18, 568543-U, 25 cm x 21 .1 mm, 5 μηη ).
[00189] Donor 9 (303 mg, 0.474 mmol, 1 .3 eq) and acceptor 3 (178 mg, 0.364 mmol, 1 .0 eq) were treated with SOP 6 (NIS 1 .3x1 .5 eq, TfOH 1 .3 eq, Et3N 1 .3x2 eq). The crude product 25 was put into next step without purification.
[00190] The dimer 25 (in crude mixture) was treated with SOP 2 to give product 26 (293 mg, 0.292 mmol, 80% yield over two steps).
[00191] The dimer 26 (293 mg, 0.292 mmol) was treated with SOP 5 to give product 27 (265 mg, 0.240 mmol, 82% yield).
[00192] The dimer 27 (205 mg, 0.186 mmol) was treated with SOP 7 to give product 28 (126 mg, 0.108 mmol, 58% yield).
[00193] Donor 28 (1 .30 g, 1 .12 mmol, 1 .0 eq) and acceptor 6 (1 .12 g, 1 .90 mmol, 1 .7 eq) were treated with SOP 6 (NIS 1 .3 eq, TfOH 1 .0 eq, Et3N 2 eq) to give the coupling product 29 (1 .28 g, 0.810 mmol, 66% yield).
[00194] The trimer 29 (1 .08 g, 0.683 mmol) was treated with SOP 7 to give product 30 (701 mg, 0.428 mmol, 63% yield).
[00195] Donor 30 (246 mg, 0.150 mmol, 1 .0 eq) and acceptor 15 (186 mg, 0.300mmol, 2.0eq) were treated with SOP 6 (NIS 2.0 eq, TfOH 1 .0 eq, Et3N 2 eq) to give the coupling product 31 (85.1 mg, 0.0407 mmol, 27% yield).
[00196] Donor 30 (607 mg, 0.370 mmol, 1 .0 eq) and acceptor 6 (435 mg, 0.740 mmol, 2.0 eq) were treated with SOP 6 (NIS 1 .3 eq, TfOH 1 .0 eq, Et3N 2 eq) to give the coupling products 32 (a and β anomeric mixture, not separable via silica gel column, 636 mg, 0.314 mmol, 84% yield).
[00197] The trimer 32 (636 mg, 0.309 mmol) was treated with SOP 7 to give product 33 (a isomer, 214 mg, 0.101 mmol, 33% yield over two steps from 30 to 33).
[00198] Donor 33 (214 mg, 0.101 mmol, 1 .0 eq) and acceptor 15 (226 mg, 0.365 mmol, 3.6 eq) were treated with SOP 6 (NIS 2.0 eq, TfOH 1 .0 eq, Et3N 2 eq) to
give coupling products 34 (a and β mixture, not separable via silica gel column, 199 mg, 0.0775 mmol, 76% yield). The pure a isomer of 34 was isolated through preparative reverse phase HPLC (CH3CN:H2O (isocratic) 85%:15%, Column:
Discovery HS C18, 568543-U, 25 cm x 21 .1 mm, 5 μηη )
[00199] Example 3 - Deprotection (Figure 4)
[00200] Saponification was performed as described in SOP 8 using: 4M
NaOMe/MeOH (0.13 mL, 0.52 mmol), 16 (0.163 g, 0.14 mmol), and 1 M NaOHaq (1 mL, 1 mmol). Product 35 was recovered as a white solid (0.12g, 96%).
[00201]
[00202] Hydrogenation was performed as described in SOP 9 using: 35 (0.120 g, 0.14 mmol), 10% Pd/C (dry) (200 mg), HOAc (20 μΙ_) and hydrogen (30 psi).
Product 36 was recovered as a white/grey solid (0.082 g, 76%).
[00203] /V-acylation was performed as described in SOP 10 using: 36 (0.052 g, 0.068 mmol), Et3N (19 μΙ_, 0.135 mmol) and SATP (33 mg, 0.135 mmol). Product 37 was recovered as a white solid (0.050 g, 82%).
[00204] Boc cleavage was performed as described in SOP 11 using: 37 (0.050 g, 0.055 mmol) and 10% HCIaq (500 μΙ_ total). Product 38 was recovered as a white solid (0.022 g, 52%).
[00205] Saponification was performed as described in SOP 8 using: 4M
NaOMe/MeOH (0.13 mL, 0.52 mmol), 21 (0.060 g, 0.0284 mmol), and 1 M NaOHaq (1 mL, 1 mmol). Product 39 was recovered as a white solid (0.050 g, 83%).
[00206] Hydrogenation was performed as described in SOP 9 using: 39 (0.050 g, 0.029 mmol), 10% Pd/C (dry) (100 mg), HOAc (20 μΙ_) and hydrogen (30 psi). Product 40 was recovered as a white/grey solid (0.025 g, 49%).
[00207] /V-acylation was performed as described in SOP 10 using: 40 (0.025 g, 0.0175 mmol), Et3N (4.9 μΙ_, 0.035 mmol) and SATP (8.6 mg, 0.035 mmol). Product 41 was recovered as a white solid (0.022 g, 81 %).
[00208] Boc cleavage was performed as described in SOP 11 using: 41 (0.022 g, 0.014 mmol) and 10% HCIaq (500 μΙ_ total). Product 42 was recovered as a white solid (0.0188 g, 92%).
[00209] Saponification was performed as described in SOP 8 using: 4M
NaOMe/MeOH (0.13 mL, 0.52 mmol), 24 (0.0538 g, 0.0208 mmol), and 1 M NaOHaq (1 mL, 1 mmol). Product 43 was recovered as a white solid (0.042 g, 94%).
[00210] Hydrogenation was performed as described in SOP 9 using: 43 (0.050 g, 0.023 mmol), 10% Pd/C (dry) (100 mg), HOAc (20 μΙ_) and hydrogen (30 psi). Product 44 was recovered as a white/grey solid (0.022 g, 56%).
[00211] /V-acylation was performed as described in SOP 10 using: 44 (0.022 g, 0.0127 mmol), Et3N (3.5 μΙ_, 0.025 mmol) and SATP (6.2 mg, 0.025 mmol). Product 45 was recovered as a white solid (0.021 g, 88%).
[00212] Boc cleavage was performed as described in SOP 11 using: 45 (0.021 g, 0.01 1 mmol) and 10% HCIaq (500 μί total). Product 46 was recovered as a white solid (0.0121 g, 63%).
[00213] Example 4 - Conjugation of Disaccharide Antigen 38 (Figure 5)
[00214] Conjugation Stock Solution of dimer 38: The dimer 38 (3 mg, 3.8 μηηοΙ) was dissolved in a solution of hydrazine in H2O (0.4 mL, 0.01 M N2H ). The reaction mixture was stirred at room temperature for 4 hours then concentrated in vacuo. The residue was co-evaporated with 3 x 1 mL H2O and then dissolved in H2O (360 μί). A solution of tris(2-carboxyethyl)phosphine (TCEP) in water (40 μί, 0.05 M, 1 .95 μιτιοΙ) was added and stirred for 1 hour. Imject® Conjugation Buffer (Pierce, 400 μί) was added to provide a stock solution for conjugation to KLH and BSA.
[00215] Conjugation Stock Solution of dimer thiol 38 (620 μΙ_, 3.0 μηηοΙ) was added to a solution of maleimide-activated bovine serum albumin (Imject® BSA, Pierce, Rockford, IL) (5 mg, ~ 1 .5 μιτιοΙ maleimide) in Imject® Conjugation Buffer (Pierce, 250 μί diluted with 250 μί water) and the resulting solution stirred for 18 hours at room temperature. The reaction mixture was purified by de-salting on D- Salt P-6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No. 77159), the reaction mixture was loaded onto the column and eluted with purification buffer. 1 -mL fractions were collected and analyzed for protein content by absorbance at 280 nm (A28o)- Fractions containing protein were combined and lyophilized to give the desired dimer-BSA conjugate 55.
[00216] Conjugation Stock Solution of dimer thiol 38 (180 μΙ_, 0.86 μηηοΙ) was added to a solution of maleinnide-activated keyhole limpet hemocyanin (Imject® KLH, Pierce, Rockford, IL) (5 mg, ~ 0.43 μιτιοΙ maleimide) in water (0.5 ml_) was added and the resulting solution stirred overnight at room temperature. The reaction mixture was purified by de-salting on D-Salt P-6000 10 ml_ column (Pierce,
Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No. 77159), the reaction mixture was loaded onto the column and eluted with purification buffer. 1 -mL fractions were collected and analyzed for protein content by absorbance at 280 nm (A28o)- Fractions containing protein were combined and lyophilized to give the desired dimer-KLH conjugate 56.
[00217] Example 5 - Conjugation of Tetrasaccharide Antigen 42 (Figure 5)
[00218] Conjugation Stock Solution of tetramer 42: The tetramer 42 (5.3 mg, 3.8 μιτιοΙ) was dissolved in a solution of hydrazine in H2O (0.4 mL, 0.01 M N2H ). The reaction mixture was stirred at room temperature for 4 hours then concentrated in vacuo. The residue was co-evaporated with 3 x 1 mL H2O and then dissolved in H2O (360 μί). A solution of tris(2-carboxyethyl)phosphine (TCEP) in water (40 μί, 0.05 M, 1 .95 μιτιοΙ) was added and stirred for 1 hour. Imject® Conjugation Buffer (Pierce, 400 μί) was added to provide a stock solution for conjugation to KLH and BSA.
[00219] Conjugation Stock Solution of tetramer thiol 42 (620 μί, 3.0 μηηοΙ) was added to a solution of maleimide-activated bovine serum albumin (Imject® BSA, Pierce, Rockford, IL) (5 mg, ~ 1 .5 μιτιοΙ maleimide) in Imject® Conjugation Buffer (Pierce, 250 μί diluted with 250 μί water) and the resulting solution stirred for 18 hours at room temperature. The reaction mixture was purified by de-salting on D- Salt P-6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No. 77159), the reaction mixture was loaded onto the column and eluted with purification buffer. 1 -mL fractions were collected and analyzed for protein content by absorbance at 280 nm (A28o)- Fractions containing protein were combined and lyophilized to give the desired tetramer-BSA conjugate 57.
[00220] Conjugation Stock Solution of tetramer thiol 42 (180 μί, 0.86 μηηοΙ) was added to a solution of maleimide-activated keyhole limpet hemocyanin (Imject®
KLH, Pierce, Rockford, IL) (5 mg, ~ 0.43 μηηοΙ maleimide) in water (0.5 ml_) was added and the resulting solution stirred overnight at room temperature. The reaction mixture was purified by de-salting on D-Salt P-6000 10 ml_ column (Pierce,
Rockford, IL). The column was pre-equilibrated with 30 ml_ of purification buffer (Pierce, Prod. No. 77159), the reaction mixture was loaded onto the column and eluted with purification buffer. 1 -mL fractions were collected and analyzed for protein content by absorbance at 280 nm (A28o)- Fractions containing protein were combined and lyophilized to give the desired tetramer-KLH conjugate 58.
[00221] Example 6 - Conjugation of Pentasaccharide Antigen 46 (Figure 5)
[00222] Conjugation Stock Solution of pentasaccharide 46: The
pentasaccharide 46 (6.5 mg, 3.8 μιτιοΙ) was dissolved in a solution of hydrazine in H2O (0.4 ml_, 0.01 M N2H4). The reaction mixture was stirred at room temperature for 4 hours then concentrated in vacuo. The residue was co-evaporated with 3 x 1 ml_ H2O and then dissolved in H2O (360 μΙ_). A solution of tris(2- carboxyethyl)phosphine (TCEP) in water (40 μΙ_, 0.05 M, 1 .95 μιτιοΙ) was added and stirred for 1 hour. Imject® Conjugation Buffer (Pierce, 400 μΙ_) was added to provide a stock solution for conjugation to KLH and BSA.
[00223] Conjugation Stock Solution of pentasaccharide thiol 46 (620 μΙ_, 3.0 μιτιοΙ) was added to a solution of maleimide-activated bovine serum albumin
(Imject® BSA, Pierce, Rockford, IL) (5 mg, ~ 1 .5 μιτιοΙ maleimide) in Imject®
Conjugation Buffer (Pierce, 250 μί diluted with 250 μί water) and the resulting solution stirred for 18 hours at room temperature. The reaction mixture was purified by de-salting on D-Salt P-6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No. 77159), the reaction mixture was loaded onto the column and eluted with purification buffer. 1 - mL fractions were collected and analyzed for protein content by absorbance at 280 nm (A28o)- Fractions containing protein were combined and lyophilized to give the desired pentamer-BSA conjugate 59.
[00224] Conjugation Stock Solution of pentasaccharide thiol 46 (180 μΙ_, 0.86 μιτιοΙ) was added to a solution of maleimide-activated keyhole limpet hemocyanin (Imject® KLH, Pierce, Rockford, IL) (5 mg, ~ 0.43 μιτιοΙ maleimide) in water (0.5 mL) was added and the resulting solution stirred overnight at room temperature. The
reaction mixture was purified by de-salting on D-Salt P-6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No. 77159), the reaction mixture was loaded onto the column and eluted with purification buffer. 1 -mL fractions were collected and analyzed for protein content by absorbance at 280 nm (A28o)- Fractions containing protein were combined and lyophilized to give the desired pentamer-KLH conjugate 60.
[00225] Conjugate Characterization Data:
[00226] It is intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that it is the following claims, including all equivalents, that are intended to define the spirit and scope of this invention.
Claims
1 . A synthetic oligosaccharide 1a
where R1 and R2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R1 can be the same or different; and X is H or a protecting group.
2. The synthetic oligosaccharide of claim 1 , which is a compound selected from compound nos. 1001 -3044 as identified in Tables 1 -9.
3. The synthetic oligosaccharide of claim 2, wherein each acyl is propionyl.
4. The synthetic oligosaccharide of claim 2, wherein each acyl is acetyl.
5. A synthetic oli osaccharide comprising an antigen 1 b:
where R1 and R2 are each independently H or acyl, provided at least one is H; n is an integer from 1 to 14, where each R1 can be the same or different; and L is a linker and Y is H or a carrier.
6. The synthetic oligosaccharide of claim 5, wherein n is an integer from 2 to 12.
7. The synthetic oligosaccharide of claim 5, wherein n is an integer from 3 to 9.
8. The synthetic oligosaccharide of any one of claims 5 to 7, where L is an alkylene thiol linker.
9. The synthetic oligosaccharide of any one of claims 5 to 8, where Y is a carrier selected from the group consisting of proteins, peptides, lipids, polymers, dendrimers, virosomes, and virus-like particles or combination thereof.
10. The synthetic oligosaccharide of claim 9, where the carrier is a carrier protein.
11. The synthetic oligosaccharide of claim 10, where the carrier protein is selected from the group consisting of bacterial toxoids, toxins, exotoxins, and nontoxic derivatives thereof.
12. The synthetic oligosaccharide of claim 11 , wherein the carrier protein is selected from the group consisting of tetanus toxoid, tetanus toxin Fragment C, diphtheria toxoid, CRM, cholera toxoid, Staphylococcus aureus exotoxins or toxoids, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, genetically detoxified variants thereof; bacterial outer membrane proteins, serotype B outer membrane protein complex (OMPC), outer membrane class 3 porin (rPorB), porins; keyhole limpet hemocyanine (KLH), hepatitis B virus core protein, thyroglobulin, albumins, and ovalbumin; pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA); purified protein derivative of tuberculin (PPD); transferrin binding proteins, peptidyl agonists of TLR-5; and derivatives and/or combinations of the above carriers.
13. The synthetic oligosaccharide of claim 12, wherein the carrier protein is selected from the group consisting of CRM 197, Neisseria meningitidis, bovine serum albumin (BSA), human serum albumin (HSA), poly(lysine:glutamic acid), flagellin of motile bacteria, and derivatives and/or combinations thereof.
14. The synthetic oligosaccharide of claim 12, wherein the carrier protein is selected from the group consisting of tetanus toxoid, CRM 197, and OMPC.
15. The synthetic oligosaccharide of any of claims 5 to 14, where L is- (CH2)pNH(CO)(CH2)oS-, where p and o are each independently an integer from 2 to 10, and Y is H.
16. The synthetic oligosaccharide of any of claim 5 to 14, where L is - (CH2)pNH(CO)(CH2)oS-, where p and o are each independently an integer from 2 to 10, and Y is a carrier.
17. The synthetic oligosaccharide of claim 5, where antigen 1 b is:
18. The synthetic oligosaccharide of claim 5, where antigen 1 b is: 
19. The synthetic oligosaccharide of claim 5, where antigen 1 b is: 
20. The synthetic oligosaccharide of claim 5, where antigen 1 b is: 
21 . A pharmaceutical composition comprising at least one oligosaccharide of any one of claims 5 to 14 in an effective amount to stimulate an immune response, optionally further comprising a pharmaceutically acceptable carrier.
22. The pharmaceutical composition of claim 15, further comprising an adjuvant.
23. The pharmaceutical composition of any one of claims 15 or 16 wherein the immune response is an antigen-specific immune response.
24. A composition comprising a synthetic oligosaccharide of any one of claims 5 to 14 and a pharmaceutically acceptable vehicle.
25. The composition of claim 24, comprising a plurality of different oligosaccharides, where each oligosaccharide is an antigen 1 b.
26. The composition of claims 24 or 25, further comprising an adjuvant.
27. The composition of claim 26, where the adjuvant is selected from the group consisting of aluminum salts, RIBI, toll-like receptor agonists, AS01 AS02 AS03, AS04, AS05, CpG-oligodeoxynucleotide, MF-59, Montanide ISA-51 VG , Montanide ISA-720, Quil A, QS21 , synthetic saponins, immunostimulating complexes, stearyl tyrosine, virus-like particles, reconstituted influenza
virosomes, cytokines, mast cell activator compound 48/80, liposomes, muramyl dipeptides, SAF-1 , and combinations thereof.
28. The composition of any of claims 24 to 27, comprising an amount of at least one oligosaccharide sufficient to confer immunity against N. meningitidis.
29. A composition comprising an oligosaccharide of any one of claims 5 to 14 as a vaccine.
30. An antibody preparation comprising antibody specific to an antigen
1 b:
31 . An antibody preparation against an oligosaccharide according to any one of claims 5 to 14.
32. The antibody preparation of claim 31 , where the antibody
preparation comprises at least one member from the group consisting of polyclonal antibody, monoclonal antibody, mouse monoclonal IgG antibody, humanized antibody, chimeric antibody, single chain antibodies, fragment thereof, or combination thereof.
33. A method of treating or preventing a N. meningitidis infection in a patient in need thereof comprising administering an effective amount for inducing an immune response against N. meningitidis of a synthetic oligosaccharide 1 a of any of claims 5 to 14 or an antibody thereto.
34. The method of claim 33, wherein N. meningitidis is NmA, NmB, NmC or NmW-135.
35. The method of claim 33, wherein N. meningitidis is NmB.
36. A method of treating a disease associated with N. meningitidis infection, comprising administering effective amount for inducing an immune response against N. meningitidis of an oligosaccharide of any of claims 5 to 14 or antibody thereto.
37. A method of treating a disease associated with N. meningitidis infection, comprising administering to a patient in need thereof a composition of any of claims 24 to 28.
38. The method of claim 36 or 37, wherein the patient is human.
39. A method for producing antibodies comprising:
(a) administering to a subject an effective amount of at least one oligosaccharide of any one of claims 5 to 14, for producing antibodies specific for N. meningitidis) optionally further comprising an adjuvant.
(b) isolating antibodies from the subject.
40. A method for producing monoclonal antibodies comprising: (a) administering to a subject an effective amount of at least one oligosaccharide of any one of claims 5 to 14, for producing antibodies specific N. meningitidis;
(b) isolating antibodies from the subject;
(c) fusing antibody producing cells from the subject to myeloma cells, and
(d) harvesting antibodies produced from a fusion subclone.
41 . The method of claim 39 or 40, wherein the subject is a rabbit.
42. The method of claim 39 or 40, wherein the subject is a human.
43. An antibody producing cell obtainable by performing steps (a) to (c) of claim 40.
44. An antibody obtainable by performing steps (a) to (d) of claim 40.
45. A method of diagnosing the presence of N. meningitidis in a sample, comprising contacting the sample with an antibody of claim 31 , 32, 26, 43, or 44.
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| WO2013034664A1 (en) * | 2011-09-06 | 2013-03-14 | Glycovaxyn Ag | Bioconjugate vaccines made in prokaryotic cells |
| WO2015128797A1 (en) * | 2014-02-25 | 2015-09-03 | Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. | Novel synthetic oligomers of neisseria meningitis serogroup x and process of preparing them |
| US9359394B2 (en) | 2013-04-08 | 2016-06-07 | Trustees Of Tufts College | Stereoselective glycosylation reactions |
| WO2017017509A1 (en) * | 2015-07-28 | 2017-02-02 | Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. | Novel neisseria meningitidis serogroup y oligomer and process for synthesizing thereof |
| US11420993B2 (en) | 2014-05-24 | 2022-08-23 | Biological E Limited | Semi-synthetic meningococcal conjugate vaccine |
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| US9359394B2 (en) | 2013-04-08 | 2016-06-07 | Trustees Of Tufts College | Stereoselective glycosylation reactions |
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| CN106061986A (en) * | 2014-02-25 | 2016-10-26 | 默沙东和惠康基金会合资的希勒曼实验室私人有限公司 | Novel synthetic oligomers of neisseria meningitis serogroup x and process of preparing them |
| JP2017512203A (en) * | 2014-02-25 | 2017-05-18 | エムエスディー ウェルカム トラスト ヒルマン ラボラトリーズ プライベート リミテッドＭｓｄ Ｗｅｌｌｃｏｍｅ Ｔｒｕｓｔ Ｈｉｌｌｅｍａｎ Ｌａｂｏｒａｔｏｒｉｅｓ Ｐｖｔ．Ｌｔｄ． | Novel synthetic oligomers of Neisseria meningitidis serotype X and methods for their preparation |
| US10183962B2 (en) | 2014-02-25 | 2019-01-22 | Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. | Synthetic oligomers of Neisseria meningitis serogroup X and process of preparing them |
| AU2015221820B2 (en) * | 2014-02-25 | 2019-01-31 | Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. | Novel synthetic oligomers of Neisseria meningitis serogroup X and process of preparing them |
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| CN107709341A (en) * | 2015-07-28 | 2018-02-16 | Msd韦尔科姆信托希尔曼实验室私人有限公司 | New Neisseria meningitidis serum group Y oligomer and its synthetic method |
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