Carbocyclic derivatives and their conjugate derivatives, and their use in vaccines

Abstract

To provide carba analogue polysaccharide derivatives of the Neisseria meningitidis capsular polysaccharides, having good stability, exhibiting a good immunogenic profile, and suited for the preparation of a vaccine, preferably to be formulated in liquid form, against meningitis.SOLUTION: The invention provides an oligomer of formula (Ia) or (Ib).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of vaccines and to oligomers having a selected degree of polymerization, obtained by linking many repeating carbon ring units, as well as conjugate derivatives thereof. The oligomers and conjugate derivatives thereof of the present invention also have a selected degree of acetylation. The derivatives of the present invention are useful, for example, in the preparation of immunogenic compositions in the form of vaccines. [Background technology]

[0002] Neisseria meningitidis is the leading cause of bacterial meningitis and sepsis worldwide, and can lead to outbreaks and epidemics of invasive meningococcal disease. Invasive meningococcal disease occurs globally. Incidence varies by region of the world, but infants, children, and adolescents are most vulnerable to developing invasive disease. The disease progresses rapidly and often leads to a catastrophic outcome. Based on the antigenic differences of their capsular polysaccharides, 12 serotypes of Neisseria meningitidis have been identified. Virtually all disease-associated isolates are encapsulated, and serotypes A, B, C, W, X, and Y are involved in more than 90% of invasive meningococcal infections worldwide. The distribution of these serotypes is geographically and temporally volatile.

[0003] Generally, meningococcal capsular polysaccharides (CPS) are T-cell independent antigens, meaning they can evoke an immune response without T-cell involvement. This response lacks several key characteristics that characterize T-cell-dependent immune responses, such as immunological memory, IgM-to-IgG class switching, and affinity maturation. However, when a portion of the polysaccharide is linked to a carrier protein, it triggers a cellular immune response that produces a memory effect, protecting infants. Such polysaccharides linked to carrier proteins are often referred to as complex carbohydrates and are particularly valuable as vaccines. In this regard, particularly effective vaccines (complex carbohydrate vaccines) can be made by linking the carbohydrate to a carrier protein via a linker portion (or spacer), or even by direct coupling of the carbohydrate to a selected carrier protein. In any case, complex carbohydrates can induce a T-cell-dependent immune response with memory and effect, even in infants, while non-complex CPS generally do not provide either a memory effect in adults or substantial immunogenic effects in infants.

[0004] Among meningococcal capsular polysaccharides, meningococcal serotype A capsular polysaccharide (MenA CPS) is known to be affected by intrinsic chemical instability in water (see, for example, Frasch et al., Adv. Biotechnol. Processes, 1990, 12, 123-145). MenA CPS is composed of (1→6) linked 2-acetamido-2-deoxy-α-D-mannopyranosylphosphate repeating units, and the hydrolytic instability of MenA polysaccharide is mainly due to hydrolysis of phosphodiester linkages promoted by ring oxygen and N-acetamide. In fact, both the oxygen and N-acetyl group in the ring destabilize the phosphodiester glycoside linkage, and as shown in scheme A below, the axial position of NHAc is also recognized to contribute to this mechanism (Berti et al., Vaccine, 2012, 30, 6409-6415). [ka]

[0005] The availability of hydrolysis-resistant MenA polysaccharide mimics would be highly attractive for developing more stable conjugate vaccines. Stabilization of CPS can be achieved in various ways, and MenA CPS analogs in which the ring oxygen is replaced with a methylene group have been reported in the prior art. In particular, as shown in Scheme B, replacing the oxygen in the ring with carbon prevents the destabilization described in Scheme A. [ka]

[0006] Toma et al., Org. Biomol. Chem., 2009, 7, 3734-3740, describes the preparation of O-(2-acetamido-2-deoxy-5a-carb-α-D-mannopyranosyl) phosphate, a monomer in which the pyranose oxygen in the repeating unit of MenA CPS is replaced with a methylene group. This document only refers to the chemical synthesis of the monomer itself.

[0007] Gao et al. (Org. Biomol. Chem. 2012, 10(33), 6673, and ACS Chem. Biol. 2013, 8(11), 2561) and Ramella D. et al. (Eur J. Org. Chem, 2014, 5915-5924) describe the stabilization of glycosyl 1-O-phosphate by using carba sugars in which a methylene group replaces the pyranose oxygen atom. They also report the conjugation of synthetic carba trimers to protein carriers, but do not further investigate the behavior of carba analogs with higher degrees of polymerization. There is also no mention of carba analogs with specific levels of acetylation and / or specific acetylation patterns. Furthermore, the trimers examined are unlikely to inhibit the binding of anti-MenA CPS antibodies, indicating that the described derivatives are relatively weak conjugate antigens.

[0008] Therefore, there is a need to find a carbapenem derivative that has good stability, also shows a good immunogenic profile, can be obtained according to a reliable and convenient synthetic method, and is preferably formulated in liquid form for the production of a vaccine against meningitis.

Summary of the Invention

[0009] In a first aspect, the present invention relates to an oligomer of formula (Ia) or (Ib).

Chemical formula

[0010] In a second embodiment, the present invention relates to an oligomeric conjugate antigen of formula (IIa) or (IIb). [ka] During the ceremony, n, R, R', R x and R y is as defined above in relation to the first aspect; Z is a linker or coupling; P stands for protein.

[0011] In a third embodiment, the present invention relates to an immunogenic composition comprising (a) the conjugate according to a second embodiment of the present invention; and (b) at least one pharmaceutically acceptable excipient.

[0012] In a fourth embodiment, the present invention relates to a vaccine comprising the above-described conjugate according to a second aspect of the present invention, or the above-described immunogenic composition according to a third aspect of the present invention.

[0013] In a fifth embodiment, the present invention relates to a method for treating or preventing meningitis A, C, W135 or Y in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a conjugate according to a second embodiment, an immunogenic composition according to a third embodiment of the present invention, or a vaccine according to a fourth embodiment of the present invention.

[0014] In a sixth embodiment, the present invention relates to a method for immunizing a subject against meningitis A, C, W135, or Y, comprising administering an immunologically effective amount of an immunogenic composition according to a third embodiment of the present invention or a vaccine according to a fourth embodiment of the present invention to the subject.

[0015] In a seventh embodiment, the present invention relates to a method for inducing an immune response to meningitis A, C, W135, or Y in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition according to a third embodiment of the present invention or a vaccine according to a fourth embodiment of the present invention.

[0016] In an eighth aspect, the present invention relates to the use of an immunogenic composition according to a third aspect of the present invention, or a vaccine according to a fourth aspect of the present invention, in the manufacture of a pharmaceutical for the treatment or prevention of meningitis A, C, W135, or Y.

[0017] In a ninth aspect, the present invention relates to an immunogenic composition according to a third aspect of the present invention, or a vaccine according to a fourth aspect of the present invention, for use in the prevention or treatment of meningitis A, C, W135, or Y.

[0018] In a tenth aspect, the present invention relates to an immunogenic composition according to a third aspect of the present invention, or a vaccine according to a fourth aspect of the present invention, used for inducing an immune response to meningitis A, C, W135, or Y. [Brief explanation of the drawing]

[0019] [Figure 1] This is a 1H-NMR spectral monitoring of the three-step random O-acetylation reaction of carba analog DP8, i.e., equation (Ia) with n=8. [Figure 2] This is the 1H-NMR spectrum of the final random O-acetylated carba analog DP8 (i.e., equation (Ia) for n=8), including the integral to determine the acetylation rate (%). [Figure 3]This is the 31P NMR spectrum of the final random O-acetylated carba analog DP8 (formula Ia). The spectrum shows simultaneous acetylation occurring at the C3+C4 position for approximately 44%, and acetylation occurring at either C3 or C4 for approximately 28%. 27% of the molecule remains unacetylated. [Figure 4] This figure shows the characterization of CRM197 and the SDSpage of the crude reaction, along with a diagram illustrating the conjugation scheme of the oligomer according to the present invention. [Figure 5a] These are ELISA titers after administration of two and three doses of the vaccine. The p-value indicates a comparison between the natural benchmark MenA-CRM197 and other vaccine-vaccinated groups. [Figure 5b] These are ELISA titers after administration of two and three doses of the vaccine. The p-value indicates a comparison between the natural benchmark MenA-CRM197 and other vaccine-vaccinated groups. [Figure 6] This figure shows ELISA titers measured after administration of three doses of vaccine: Anti-MenA polysaccharide IgG antibodies were evaluated using CRM197 conjugates of random O-acetylated carbamenA analog DP8, compared with CRM197 conjugates of selective 3-O-acetylated carbamenA DP8 and natural MenA-CRM197 vaccine as a benchmark (i.e., positive control). [Figure 7] This figure shows the SBA titers obtained in rabbits (rSBA) and human complement (hSBA) after administration of the two-dose and three-dose vaccines according to the present invention. [Figure 8] This figure shows the SBA titer after administration of three doses of vaccine: human complement-mediated bactericidal titer was measured in serum induced with CRM197 conjugate of random O-acetylated carbamenA analog DP8 compared with CRM197 conjugate of selective 3-O-acetylated carbamenA DP8 and natural MenA-CRM197 vaccine as a benchmark (i.e., positive control). [Figure 9]This graph compares the stability of MenA-CRM197 (i.e., natural MenA polysaccharide conjugated to CRM197) with that of the acetylated oligomer of the present invention, where n is 7 and the oligomer is conjugated to CRM197. [Modes for carrying out the invention]

[0020] To facilitate understanding of the present invention, many terms and phrases are defined below. Where not specifically stated, the following terms and phrases (including tenses such as past and present) are likely to be associated with industry-recognized synonyms or alternatives.

[0021] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise. That is, "a" means "one or more" unless otherwise specified.

[0022] The term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A", and "B". Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0023] Unless otherwise stated, the designations "A%-B%", "AB%", "A% to B% (A% to B%)", "A to B% (A to B%)", "A%-B", and "A% to B (A% to B)" are all given their usual and customary meanings. In some embodiments, these designations are synonymous.

[0024] The terms “substantially” or “substantial” mean that the described or claimed condition functions as the described standard in all important aspects. Thus, “substantially uncontaining” means that a condition functions as uncontaining in all important aspects, even if the numerical value indicates the presence of some impurities or substances. “Substantial” generally means a value greater than 90%, preferably greater than 95%, and most preferably greater than 99%. Where a particular value is used in the specification and claims, unless otherwise stated, the term “substantially” means an acceptable margin of error for that particular value.

[0025] "Effective quantity" means a quantity sufficient to produce the referenced effect or outcome. "Effective quantity" can be determined empirically and conventionally using known techniques in relation to the stated purpose.

[0026] The “immunologically effective dose” or “therapeutic effective dose” means that the amount administered to an individual, either as a single dose or as part of a series of doses, is effective in treating or preventing disease. This dose may vary depending on the health and physical condition of the individual being treated, age, the taxonomic group of the individual being treated (e.g., non-human primates, primates, etc.), the individual’s immune system’s ability to synthesize antibodies, the desired degree of protection, the vaccine formulation, the medical condition assessment by the treating physician, and other relevant factors. The dose is expected to fall within a relatively broad range that can be determined by standard testing.

[0027] The term "treatment" means any one of the following: (i) prevention of infection or reinfection, as in the case of conventional vaccines; (ii) reduction of the severity of symptoms or elimination of symptoms; (iii) delay of symptom recurrence; and (iv) substantial or complete elimination of the pathogen or disorder in question in the subject. Thus, treatment can be influenced prophylactically (before infection) or therapeutically (after infection).

[0028] The term "weight % (%w / w)" indicates, as shown, the weight percentage of a given compound relative to a different compound or to the total content of a composition.

[0029] Similarly, the term "volume % (%v / v)" indicates the volume percentage of a given compound relative to the total content of a different compound or composition, as shown.

[0030] The term "oligosaccharide," in its sense as is commonly known in the art, includes polysaccharides having 3 to 10 monosaccharide units (see, for example, https: / / en.wikipedia.org / wiki / Oligosugar).

[0031] The term "oligomer" refers to carba-related polysaccharides that provide a cyclohexane skeleton by replacing the intraring oxygen with a methylene (-CH2-) group.

[0032] The degree of polymerization (DP) indicates the number of monomers linked together to provide the final oligomer. In this invention, unless otherwise specified, DP is represented by "n" in formulas (I) and (II).

[0033] The "average degree of polymerization" (avDP) indicates the average number of repeating units that make up an oligomer.

[0034] The term "capsular polysaccharides / saccharides" (CPS) refers to sugars found on the outer surface of bacterial cells, specifically in a layer that is part of the outer layer of the bacterial cell itself. CPS are expressed on the outermost surface of a wide range of bacteria, and in some cases, in fungi as well.

[0035] Unless otherwise specified, the term "conjugation" refers to the connection or linkage of the entities in question, in particular the oligomer of the present invention having n (i.e., DP) ≥ 6 and the selected protein.

[0036] As used herein, the term “alkyl” refers to a saturated, linear, or branched hydrocarbon moiety. The term “C1-C6-alkyl” refers to an alkyl moiety containing 1 to 6 carbon atoms.

[0037] As used herein, the term "haloalkyl" refers to a saturated, linear, or branched hydrocarbon moiety in which one or more hydrogen atoms are replaced by halogen atoms. In particular, when "haloalkyl" is said, it is a reference to "fluoroalkyl" where the halogen is fluoro. The term "C1-C6-haloalkyl" refers to an alkyl moiety containing 1 to 6 carbon atoms in which one or more hydrogen atoms are replaced by halogen atoms. Examples include -CF3, -CH2F, and -CH2CF3.

[0038] As used herein, phenyl may be substituted, in particular according to the definition of Z. The phenyl group may be substituted with one or more reactive functional groups to enable conjugation such as N3, NH2, SH, etc. Other suitable groups are well known to those skilled in the art.

[0039] As used herein, the term “protecting group” refers to any suitable protecting group for a given purpose. For the selection and use of such protecting groups, and for details of their use, refer to, for example, Greene, TW and Wuts, PGM, “Protective Groups in Organic Synthesis.” Suitable protecting groups are known to those skilled in the art.

[0040] As used herein, the term “pharmaceutically acceptable phosphate counterion” means any counterion suitable for a phosphate group, i.e., a metal cation that is within reasonable medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, or other problems or complications, and that has a reasonable benefit / risk ratio. A pharmaceutically acceptable phosphate counterion may be a Group 1 or Group 2 metal. A specific example of such a pharmaceutically acceptable phosphate counterion is sodium (Na). + ) and potassium (K + For example, when the oligomer or conjugate of the present invention is in a buffer solution, the counterion is preferably sodium.

[0041] As described above, the present invention relates to polysaccharide carba analogs having a degree of polymerization of at least 6 and having a first analog monomer connected to a second analog monomer via a 1,6 linkage that connects C-1 of a first unit to C-6 of a second unit, wherein the 1,6 linkage contains a phosphonate moiety (i.e., the ring oxygen of the mannosamine unit is replaced by methylene). Notably, the derivatives of the present invention are not only capable of mimicking natural polysaccharides from the MenA serotype, but are also expected to have improved stability compared to natural CPS.

[0042] In one embodiment, the oligomer of the present invention is defined by formula (Ia). In another embodiment, the oligomeric conjugate antigen of the present invention is defined by formula (IIa).

[0043] As defined above, n is ≥ 6. In one embodiment, n is 8 to 30. In another embodiment, n is 8 to 20. In a particular embodiment, n is 8 to 15. In one embodiment, n is ≤ 15. In particular, n is 8 or 10. In one embodiment, n is 8.

[0044] In one embodiment, R is H or -P(O)(OR″)2, and at least one R″ is Na + In one embodiment, R is H.

[0045] In one embodiment, R′ is Na + The oligomer of the present invention is defined by formula (Ia') or (Ib'), preferably formula (Ia'). [ka]

[0046] Therefore, in one embodiment, the oligomeric conjugate antigen of the present invention is defined by formula (IIa') or formula (IIb'), preferably formula (IIa'). [ka]

[0047] As defined above, R x is H or -C(O)CH3, and each repeating unit may be the same or different, R y is H or -C(O)CH3, and each repeating unit may be the same or different, R x or R y At least one of these is -C(O)CH3 in at least one repeating unit, and together they are R in the oligomer. x and R y Approximately 50-90% of it is -C(O)CH3. Therefore, it should be understood that the formulas defined in square brackets according to formulas (Ia), (IIa), (Ib), and (IIb) show that each unit of the oligomer has this structure, but R x and R y Given that different options are available for each repeating unit defined by square brackets, monomer units defined by square brackets are not necessarily identical. Therefore, n and R x and R y It will be understood that different acetylation rates (%) can be achieved depending on the selection of H or -C(O)CH3 for . For example, each repeating unit of the oligomer defined by square brackets can be acetylated according to the level of acetylation, i.e., R x and R y Depending on the selection of H or -C(O)CH3 for each of these, they may be the same or different.

[0048] As defined above, in total, R in the oligomer x and R y Approximately 50-90% of it is -C(O)CH3. In other words, the total amount of acetylation of the oligomer is approximately 50-90%. In other words, in the oligomer of the present invention, R x At least one of and R y One of them is -C(O)CH3 in the same or different repeating units, and at position 3 (R y is -C(O)CH3) and 4th position (R xThe total degree of acetylation at -C(O)CH3) is approximately 50-90%. To avoid misunderstanding, as stated above, R x and R y This can be the same or different in each repeating unit of the oligomer.

[0049] In another embodiment, collectively, R in the oligomer x and R y Approximately 60-80% of it is -C(O)CH3. In other words, the total amount of acetylation of the oligomer is approximately 60-80%. To avoid misunderstanding, as stated above, R x and R y This can be the same or different in each repeating unit of the oligomer.

[0050] In one embodiment, R x and R y Both are -C(O)CH3 in at least one identical repeating unit of the oligomer, preferably in about 40-50% of the repeating unit of the oligomer; and about 10-30% of the remaining repeating unit is -C(O)CH3. x or R y It may have one of the following, and the remaining repeating units in the oligomer are R x =R y =H has

[0051] As defined above, Az is -NH(CO)R 1 , -N(R 1 ) an aza substituent selected from the group consisting of 2 and -N3, R 1 The group is independently selected from the group consisting of H, linear or branched C1-C6-alkyl and linear or branched C1-C6-haloalkyl. The nitrogen atom is directly attached to the carba-analog repeating unit.

[0052] Examples of such Az substituents include -N3, -NH2, -NH-C1-C6 alkyl, -N-(C1-C6 alkyl)2, and -NH(CO)-C1-C6 alkyl. In some embodiments, -C1-C6 alkyl is -C1-C4 alkyl, particularly -CH3. Thus, according to some embodiments, Az is -NH(CO)-C1-C6 alkyl, particularly -NH(CO)-CH3, and is also represented as -NHAc (Ac represents acetate, i.e., -C(O)CH3).

[0053] Z may have different meanings depending on whether or not the oligomer of the present invention is conjugated to a protein.

[0054] According to formula (Ia) or (Ib), the oligomer of the present invention is not conjugated to a protein. Therefore, as defined above, according to formula (Ia) or (Ib), Z is one of the following: (i) protecting group; (ii) linear or branched C1-C6 alkyl, optionally substituted aryl, -C(O)Y, or linear or branched C1-C6-alkyl-X, (iii) Functional linkers for conjugation to proteins.

[0055] Therefore, according to one embodiment, Z is a means for capping terminal sugar units so that they may be non-reactive or reactive, for example, for further chain elongation or subsequent modification.

[0056] If Z is intended to be a means for capping terminal carba-analog units, it may include a protecting group or a capping group, such as a linear or branched C1-C6 alkyl, an optionally substituted phenyl, C(O)-Y, or a linear or branched -C1-C6 alkyl-X, where X is -NH2, -N3, -C≡CH, -CH=CH2, -SH, or -SC≡N, and Y is H, a linear or branched C1-C6 alkyl, or a protecting group.

[0057] As defined herein, Z may be a functional linker for conjugation to a protein. In this case, “functional linker” refers to any linker known in the art to be used to conjugate a sugar to a protein.

[0058] In one embodiment, X is -NH2.

[0059] In one embodiment, Z according to formula (Ia) or (Ib) is selected from -(CH2)6-NH2, -(CH2)4-NH2, -(CH2)3-NH2, and -(CH2)2-NH2, and the amino group may be protected by a suitable protecting group, such as -C(O)CH3 (for the selection and use of such protecting groups, and details of their use, refer to, for example, Greene, TW and Wuts, PGM, "protective groups in organic synthesis").

[0060] The oligomers of the present invention can be produced according to known synthetic methods in organic synthesis for the production of polysaccharide carba analogs. Generally, the production of the oligomers of the present invention can be achieved by linking at least six mannosamine carba analog constituent blocks in a desired manner by forming 1,6-alpha linkages between repeating units, thereby providing oligomers having at least 6 degrees of polymerization. As shown in formula (I), their monomers are linked via alpha-(1→6) phosphate linkages, and such linkages can be made using standard polymerization techniques, such as those described, among others, in Gao et al., Org. Biomol. Chem., 2012, 10, 6673.

[0061] The mannosamine carba analogue constituent block may have an acetate at position 3 and / or 4, or a protecting group that can be replaced with acetate at any step of synthesis.

[0062] Alternatively, according to one embodiment, the present invention relates to a method for producing an oligomer of formula (I), comprising the following steps. a. Production of monomers having phosphodiester links; b. Extension reaction of the obtained monomer, for example, using phosphoramidite; c. O-acetylation of oligomers.

[0063] In one embodiment, R y If the compound is C(O)CH3, steps (b) and (c) may be reversed so that O-acetylation occurs before the extension reaction.

[0064] More specifically, the process may include the steps shown in Scheme 1. [ka]

[0065] To avoid misunderstanding, Ac is intended to refer to the acetyl group, i.e., -C(O)CH3.

[0066] Scheme 1: Method for producing oligosaccharides according to the present invention (a) TBAF, THF, 0℃ → room temperature, 92%. (b) MeONa, MeOH, room temperature, 85%. (c) DMTrCl, Et3N, DCM, room temperature, 91%. (d) 2-Cyanoethyl N,N-diisopropyl-chlorophosphoramidite, N,N-diisopropylethylamine, DCM, room temperature, 9 (94%). (e) I.11, DCI, MeCN, II.CSO, MeCN, III.TCA, DCM, H2O, 94%. (f) I.9, DCI, MeCN, II.CSO, MeCN, III.TCA, DCM, H2O, 16 (82%), 17 (95%), 18 (90%), 19 (92%), 20 (88%), 21 (86%), 22 (87%). (g) NH4OH, H2O, dioxane. (h)H2, Pd Black, H2O, AcOH, 1 (99%), 2 (76%), 3 (69%), 4 (39%), 5 (88%), 6 (83%), 7 (77%), 8 (44%), (i): (Boc)2O, NaHCO3, room temperature, 16 hours; (l): Ac2O / imidazole, 40℃, ~9 days; (NS): TFA, room temperature, 1 hour.

[0067] In particular, the use of phosphoramidite structural blocks is more effective in forming phosphodiester linkages. The inventors chose to use dimethoxytrityl (DMTr) ether to temporarily mask the function of the primary alcohol being extended. Each extension step is based on the repetition of a three-step procedure including coupling of the phosphoramidite with the growing chain alcohol, oxidation of the intermediate phosphite to the corresponding phosphodiester, and unmasking of the primary hydroxyl on the (n+1) oligomer. As shown in Scheme 1, the key structural block 9 is obtained from intermediate 10, and intermediate 10 is obtained in three steps from a known carba sugar 12 (see, e.g., Q. Gao et al., Org. Biomol. Chem., 2012, 10, 6673-6681). The latter carbamannose structural block can be prepared from commercially available 3,4,6-tri-O-acetyl-D-glucar according to prior art methodologies. Therefore, the primary silyl ether and acetyl ester were removed from compound 12 by the sequential action of tetrabutylammonium fluoride (TBAF) and NaOMe to obtain diol 14 in 85% yield. Next, the DMTr group was regioselectively introduced to obtain alcohol 10 in 91% yield. This compound was converted to the extended block phosphoramidite 9 by reaction with 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite. The target oligomer was assembled using readily available constituent blocks. Its synthesis began with the attachment of an aminohexanol spacer to alcohol 10 using a known phosphoramidite 11. The constituent blocks were coupled in a two-step one-pot reaction using dicyanoimidazole (DCI) as an activator for the phosphoramidite activation. The phosphite formed in situ was oxidized using (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). DCI (pKa 5.2) has low acidity and is suitable for use in combination with the acid-unstable DMTr group, so it is not used in the conventional tetrazole (pKa 5.2) a4.9) was preferred. CSO was used instead of iodine due to its high solubility in non-aqueous solvents such as acetonitrile. The crude phosphodiester product was treated with TCA to cleave the DMTr group. The product was purified by size exclusion chromatography (Sephadex LH-20) to obtain monomer 15 with spacers in 94% yield. All subsequent couplings were carried out according to the above procedure until a desired degree of polymerization of 8 or more was reached. For the elongation of longer oligomers, a larger amount of phosphoramidite 9 was used and the coupling reaction time was increased to ensure complete conversion of the alcohol. The yield for each elongation cycle was good to excellent, ranging from 82% to 95%. Starting from 10, octamer 22 was obtained in a total yield of 40%. Fragments 16-22 were deprotected using a two-step procedure. First, the cyanoethyl group (CE) was removed using aqueous ammonia (33%). Next, all residual protecting groups (benzyl ether and carboxybenzylcarbamate) on the phosphodiester thus formed were cleaved by hydrogenation with palladium black to obtain the target non-acetylated oligomers 1-8.

[0068] Non-acetylated oligomers 1-8 can be randomly O-acetylated at the 3rd and / or 4th positions, i.e., combined, the R in the oligomers x and R y Approximately 50-90% of these are -C(O)CH3. This can be achieved by (i) BOC protection of the free amine group; (ii) O-acetylation using, for example, Ac2O / imidazole; and (iii) deprotection to obtain acetylated oligomers 1c-8c or 1d-8d. Such acetylated oligomers are then activated with a linker group such as bis-succinimidyl adipate (also known as SIDEA) and CRM 197 It can be conjugated to proteins such as [list of proteins]. [ka]

[0069] Scheme 2.3 Process leading to the production of the -O-acetylated monomer constituent block (a) K2CO3, MeOH; (b) PMBCH(OMe)2, PPTS; (c) BnBr, NaH; (d) DIBAL-H, DCM; (e) DMP, DCM; (f) PPh3CH3I, KHMDS, THF , -78℃; (g) m_dichlorobenzene, t, μwave; (h) NaBH4, EtOH / THF; (i) TDSCl, Im, DCM; (j) OsO4, TMANO, 3:1 acetone-H2O; (l) (MeO) 3Cme, PTSA, CAN, then 80% AcOH; (m)Tf2O, DCM / py, -20°C to room temperature; then NaN3, 19:1DMF-H2O; (n)PPh3, THF, 60°C, H2O; then Ac2O, MeOH; (o)NaOMe / MeOH; (p)TBSOTf, 2,6-lutidine, DCM; (q)DDQ, then Ac2O, py; (r)HF / pyridine, THF; (s)DMTrCl, pyridine, DCM.

[0070] Alternatively, the 3-O-acetylated monomer structural blocks and the 4-O-acetylated structural blocks can be produced by the process shown in Scheme 3 below. [ka]

[0071] Scheme 3. Process leading to the production of 3-O-acetylated and 4-O-acetylated monomer constituent blocks (a') K2CO3, MeOH; (b') TDSCl, Imidazole, DMF, -30℃; (c') BnBr, NaH, DMF, 0℃; (d') TBAF, THF; (e') IBX, AcOEt; (f') PPh3CH3I, KHMDS, THF, -78℃ to room temperature; (g') 1,3-Dichlorobenzene, NaBH4, EtOH / THF, 230℃; (h (')TIPSCl, imidazole, DMF; (i')TiCl4, DCM / toluene 2:8, -70℃; (l')NapBr, NaH, DMF, 0℃; (m')Me3NO·2H2O, acetone / H2O3:1, OsO4; (n')(MeO)3CMe, PTSA, ACN; (o')Tf2O, DCM / Py, -20℃ to room temperature; then NaN3, 19:1 DMF-H2O;(p′)NaOMe, MeOH;(q′)TBSOTf, -10℃~70℃, Pyr, DMAP;(r′)Pd / C, H2, AcOH, then Ac2O, Pyr ;(s′)HFpyr, Pyr;(t′)DMTrCl, Pyr, 0℃;(r″)DDQ, DCM, H2O;(s″)PPh3, H2O, THF, then DMTrCl, Pyr.

[0072] Acetylated structural blocks 38, 55a, 55b and fully acetylated structural blocks (i.e., those having O-Ac groups at both the C3 and C4 positions of the same unit) can be converted to their oligomeric versions by conversion to phosphorymidates and subsequent coupling as described above in relation to compound 9.

[0073] A key prerequisite for the immunogenicity of the carba analogs of the present invention is their ability to mimic the corresponding MenA capsule sugar. To investigate this, competitive ELISA was performed using carba analogs with different degrees of polymerization.

[0074] The oligomers of the present invention can be introduced into a mammalian host, and preferably a human host, either alone, linked to a carrier protein, or as a homopolymer or heteropolymer of mannosecarba analog units. In certain embodiments, the oligomers of the present invention are used as protein conjugates. Accordingly, in further embodiments, the present invention includes conjugate derivatives comprising the oligomer of formula (I) of the present invention linked to a protein according to general formula (IIa) or (IIb). [ka] During the ceremony, n, R, R', R x , and R y This is as defined above; Z is a linker or bond; P stands for protein.

[0075] The oligomers of general formula (Ia) or (Ib) are particularly useful when conjugated to a protein via a Z moiety preferably connected to the C-1 carbon of the first repeating unit via a phosphate moiety. The oligomer-protein conjugate derivatives of formula (IIa) or (IIb) thus obtained may be useful in preparing compositions that can induce an immunogenic response in infants and possibly a cellular response that provides a memory effect to extend the efficacy of vaccination.

[0076] In one embodiment, the oligomeric conjugate is preferably defined by formula (IIa), i.e., the protein is conjugated at position 1 rather than position 6 of the carba analog.

[0077] The protein (or carrier protein) can influence the immunogenic response and even the precise properties of the antibody resulting from treating a mammal with one or more compounds of the present invention when delivered as a conjugate. A suitable protein has a functional group that can react with the terminal portion of the Z moiety to form the conjugate derivative of the present invention. Preferably, the functional group is selected from -NH2 and -SH and can be attached to the Z moiety that forms an amide bond or a thioether. More preferably, the protein has an -NH2 group suitable for forming an amide bond when reacted with Z.

[0078] Useful proteins are known in the art. However, in some embodiments, P is diphtheria toxoid (DT), tetanus toxoid (TT), CRM 197 P is an inactivated bacterial toxin selected from Escherichia coli (E. coli) ST and Pseudomonas aeruginosa exotoxin (rEPA), or P is a polyamino acid such as poly(lysine:glutamic acid), or P is hepatitis B virus core protein or SPR96-2021, or Neisseria meningitidis serotype B antigen fHbp-231 (i.e., a fusion protein of variants 2, 3 and I of factor H-binding protein (fHbp) as defined in WO2015 / 128480 (incorporated herein by reference)).

[0079] In one embodiment, P is TT, DT, or CRM 197 That is the case.

[0080] In certain embodiments, P is CRM 197 That is the case.

[0081] As defined above, according to formula (IIa) or (IIb), Z is a linker or bond. If Z is a linker, it can be derived from any suitable linker known in the art that is suitable for the conjugation of oligosaccharides to proteins.

[0082] In other words, Z in its unreacted form, i.e., Z when not linked to the oligomer and protein, may be a functional linker (as defined according to formulas (Ia) and (Ib)), by having a functional group that enables it to act as a linker between the oligomer and protein of the present invention. Preferably, Z is derived from a compound containing an amine, carboxylate, or hydroxyl group for coupling with a complementary group on the protein carrier, but other groups known in the art to provide methods for conjugating oligosaccharides to proteins may also be conceived.

[0083] When the oligomer of the present invention is conjugated to a protein, the preferred Z portion in formula (IIa) or (IIb) is derived from a linker which is an amine-substituted alkoxy group, which may be in a protected form. In this form, the amine is acetylated or alkylated with a bifunctional reagent, and its other end is similarly conjugated to the protein.

[0084] In one embodiment, according to formula (IIa) or (IIb), Z is derived from either a homobifunctional or heterobifunctional linker capable of linking the oligomer of the present invention to a protein. In this regard, suitable bifunctional linkers for use in the conjugate of the present invention include dicarboxylic acids, preferably malonic acid, succinic acid, adipic acid and suberic acid, or those known in the art, such as their activated forms. Alternatively, squalate esters can be used. These types of reagents are particularly convenient for linking compounds containing an amine in the spacer portion to a protein. Preferably, the bifunctional linker is derived from N-hydroxysuccinimide diester adipic acid (SIDEA) and BS(PEG)5.

[0085] In some embodiments, Z is at least two or three atomic lengths. Some non-limiting examples of linkers include -(CH2) m A, -Ph-A, -(CH2) a -Ph-(CH2) a-A and their substituted forms, etc., where each Ph represents a phenyl group that may be substituted, and a and m each independently represent an integer from 1 to 10. "A" represents a functional group or residue that can or can link proteins, such as -NH2, -OH or -SH, esters, amides or other carboxyl-containing groups, dienes or dienophiles, maleimides, alkynes, cycloalkynes, etc. Z can contain OR′, SR′ or N(R′)2, where each R′ is independently H or C1-C6-alkyl, acyl, aryl, arylalkyl, heteroacyl, heteroaryl, or heteroarylalkyl, and may further contain A.

[0086] In one embodiment, Z in formula (IIa) or (IIb) is a heterobifunctional linker having the following formula. [ka] During the ceremony, * This indicates a connection point. p is independently selected from 1 to 10; X is selected from -O-, -S-, and -NH-.

[0087] In one embodiment, Z is an equation * -(CH2)6NHCO(CH2)4CO * It holds.

[0088] In another embodiment, Z is a linker having the following formula: [ka] During the ceremony, * represents a connection point, and m is independently selected from 1 to 10.

[0089] In another embodiment, Z has the following formula: [ka]

[0090] Z-linkers are typically introduced into monomers that are linked to proteins before the extending monomers are attached, and are optionally introduced in a protective form, so they do not affect or participate in the subsequent extension reaction.

[0091] Therefore, in one embodiment, Z is a divalent linker having the following general formula. [ka] In the formula, r is an integer between 2 and 6, ( * ) represents a connection site to the oligomer, and PG represents a hydrogen atom or a protecting group, preferably selected from alkoxycarbonyl, methoxycarbonyl, t-butyloxycarbonyl, or benzyloxycarbonyl. The protein is connected via an amine.

[0092] If present, PG can be suitably removed, allowing the Z portion to react with a protein to obtain its conjugate. Alternatively, PG can be removed, and the resulting free amino group can be further functionalized, for example, by introducing an additional spacer portion suitable for linking to a protein.

[0093] In one embodiment, an oligomer conjugate is provided according to the following formula. [ka] In the formula, n, R, R′, R x , and R y This is defined as above.

[0094] In one embodiment of the present invention, the following formula is used, i.e., R' is Na + An oligomeric conjugate is provided. [ka]

[0095] In the formula, n, R, Rx , and R y This is defined as above.

[0096] When this randomly acetylated oligomeric conjugate is incorporated into a vaccine composition, it exhibits higher acetylation percentage stability than the natural MenA conjugate, which is less than 5% of the acetylation that carba analogs may lose when formulated into a vaccine.

[0097] To avoid misunderstanding, it should be noted that the oligomers of the present invention can be conjugated to proteins by any suitable method known in the art, for example, according to the method reported in "The design of semi-synthetic and synthetic glycoconjugate vaccines", P. Constantino et al., Expert Opin. Drug. Discov.

[0098] The conjugation reaction can also be carried out using a conjugation method similar to that used for conjugating MenA sugars to carrier proteins, and for example, using the method described in WO2004 / 067030. In one embodiment, the oligomer of the present invention is conjugated using a conjugation procedure utilizing a di-N-hydroxysuccinimidyl adipate linker, for example, reported in Berti et al., ACSChem. Biol., 2012, 7, 1420-1428, using CRM 197 It can be coupled with [another substance]. After treatment with a selected linker in DMSO containing trimethylamine, the resulting activated oligomer can be purified by co-precipitation with acetone and used for conjugation. Therefore, CRM can be used in a 100:1 oligomer / protein molar ratio. 197By incubating overnight, the desired neoconjugate can be obtained. The conjugation can be conceivable as activation of the oligomer of formula (Ia) / (Ib), followed by conjugation to a selected protein, or activation of the relevant protein functional group, and subsequently conjugation with the oligosaccharide of the present invention, typically via the Z moiety. Accordingly, according to one embodiment, the oligomer of the present invention is first activated with a suitable activator according to methods known in the art, and then coupled with the -NH2 residue of a selected protein.

[0099] In one embodiment, the Z group is activated by reaction with the first terminal portion of the linker, thereby allowing the other end of the linker to be linked to a selected protein. For example, and according to one embodiment, the process may include obtaining an activated ester of the starting oligomer by activating the oligomer of the present invention with SIDEA in the presence of triethylamine. Then, such an activated ester is subjected to CRM in the presence of a phosphonate buffer. 197 By reacting with this, the desired conjugate can be obtained.

[0100] After conjugation, the oligomer-protein conjugate can be purified by various techniques known in the art. One goal of the purification process is to remove unbound oligomers from the oligomer-protein conjugate. Typically, the conjugates of the present invention can be purified from unreacted proteins and oligomers by any number of standard techniques, such as size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, or ammonium sulfate fractionation, as described, for example, Anderson, PW, et al. J. Immunol. (1986) 137:1181-1186 and Jennings, HJ et al., J. Immunol. (1981) 127:1011-1018.

[0101] In another embodiment, Z may be a monosaccharide, preferably a mannosamine as described below. Thus, in further embodiments, the present invention also relates to an oligomer having the following formula (III). [ka] In the formula, R, Az, and n are as defined above; Z is: [ka] and; P and the linker are defined above in relation to the definition of Z for equations (I) and (II).

[0102] For example, one example of a conjugate defined in this way is as follows: [ka]

[0103] According to this embodiment, the derivative of the present invention can be directly linked to a selected protein via the -O-linker Z moiety to produce a conjugate derivative having an -O-linker-P moiety directly attached to the carbon atom of the terminal monomer. As far as the linker is concerned, it can be any suitable divalent linker with linker Z as described above. Alternatively, Z can be an amine for conjugation to a protein derivatized with a linker having a keto or aldehyde group.

[0104] A further aspect of the present invention provides an immunogenic composition comprising (a) the above-mentioned conjugate; and (b) at least one pharmaceutically acceptable excipient.

[0105] Generally, pharmaceutically acceptable excipients can be any substance that does not induce antibody production, is not harmful to the patient receiving the composition, and can be administered without excessive toxicity. Pharmacologically acceptable carriers and excipients used in the art may include liquids such as water, saline, glycerol, and ethanol. According to the prior art, auxiliary substances such as wetting agents or emulsifiers and pH buffers may also be present in such media.

[0106] The immunogenic composition may further contain an adjuvant. The adjuvant may be an aluminum-based adjuvant such as aluminum hydroxide or aluminum phosphate.

[0107] The immunogenic composition may further comprise at least one antigen derived from meningococcal serotypes C, W135, Y, and optionally A.

[0108] The immunogenic compositions of the present invention are often administered in combination with other pharmaceutically active substances or other vaccines. The compositions for administration may also contain other types of immunogenic compounds, such as complex carbohydrates, which can induce an immune response to provide protection against other meningitis pathogens.

[0109] A further aspect of the present invention provides a vaccine comprising the above-described conjugate or immunogenic composition.

[0110] The vaccine can be formulated as a sterile, substantially aqueous mixture, a pyrogen-free buffered saline, or a phosphate-containing solution that may or may not contain a preservative. The solution may be nearly isotonic, and its isotonicity may be adjusted with agents such as sodium tartrate, sodium chloride, or propylene glycol. The concentration of the immunogenic oligomer conjugate of the present invention in the formulation can vary greatly from less than about 0.1% by weight to 20% to 50% by weight or more, and is selected mainly by fluid volume, viscosity, etc., and according to the specific mode of administration selected.

[0111] The present invention may also include a method for enhancing an immune response in vertebrates, preferably mammals, comprising administering the oligomeric conjugate or immunogenic composition of the present invention to a mammal or other vertebrate. The immune response is preferably protective and preferably involves antibodies. This method can enhance a booster response.

[0112] In one embodiment, the present invention relates to a method for treating or preventing meningitis A, C, W135, or Y in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of an oligomeric conjugate, an immunogenic composition, or a vaccine according to the present invention. Such a method may further include administration in combination with at least one serotype selected from C, W135, Y, and optionally A.

[0113] As used herein, the term “derivatives of the present invention” refers to both the oligomer and its oligomeric conjugate. The derivatives of the present invention can also be used to immunize other mammals, such as cattle, sheep, and pigs, as well as other non-mammalian vertebrates such as fish and poultry.

[0114] In another embodiment, the present invention relates to a method for immunizing a subject against meningitis A, C, W135, or Y, comprising administering an immunologically effective amount of an immunogenic composition or vaccine according to the present invention to the subject.

[0115] In another embodiment, the present invention provides a method for inducing an immune response to meningitis A, C, W135, or Y in a subject, comprising administering an immunologically effective amount of an immunogenic composition or vaccine according to the present invention to the subject.

[0116] In one embodiment, the subject is a human.

[0117] In a further embodiment, the present invention relates to the use of an immunogenic composition or a vaccine according to the present invention in the manufacture of a pharmaceutical product for the treatment or prevention of meningitis A, C, W135, or Y.

[0118] In another embodiment, the present invention relates to an immunogenic composition or vaccine according to the present invention for use in the treatment of prevention of meningitis A, C, W135 or Y, or for use in inducing an immune response to meningitis A, C, W135 or Y.

[0119] The immunogenic compositions of the present invention are generally administered directly to the target. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of tissue), or by rectal, oral, vaginal, topical, perdermal, nasal, ocular, ear, lung, or other mucosal administration. Intramuscular administration to the thigh or upper arm is preferred, for example. Injections can be performed via a needle (e.g., a subcutaneous injection needle), but needle-free injections can also be used as an alternative.

[0120] The present invention can also be used to induce systemic and / or mucosal immunity. The drug treatment can be a single-dose schedule or a multi-dose schedule. Multi-dose administration can be used in an initial immunization schedule and / or a booster immunization schedule. A booster immunization schedule can be performed after an initial immunization schedule. The appropriate timing between initial immunizations (e.g., 4 to 16 weeks) and between initial and booster immunizations can be determined by conventional methods. Since infections affect various parts of the body, the compositions of the present invention can be prepared in various forms. For example, the compositions can be prepared as injections, either as a solution or a suspension. Solid forms suitable for dissolution or suspension in a liquid medium before injection can also be prepared. The compositions can be prepared for topical administration as ointments, creams, or powders. The compositions may be prepared for oral administration as tablets or capsules, or as syrups (which may be flavored). The compositions can be prepared for pulmonary administration using fine powders or sprays, for example, as inhalers. The compositions can be prepared as suppositories or pessaries. The compositions can be prepared for nasal, ear, or ocular administration, for example, as drops. A composition suitable for parenteral injection is most preferred. The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered to, for example, pH 6 to pH 8, generally around pH 7. The composition of the present invention may be isotonic with respect to humans.

[0121] The immunogenic composition comprises an immunologically effective amount of the conjugate of the present invention, and optionally any other specific components. Therapeutic administration may be a single-dose schedule or a multi-dose schedule (e.g., including a booster immunodose). The composition may be administered in combination with other immunomodulators. Adjuvants that can be used in the composition of the present invention include, but are not limited to, insoluble metal salts, oil-in-water emulsions (e.g., MF59 or AS03, both containing squalene), saponins, non-toxic derivatives of LPS (e.g., monophosphoryl lipid A or 3-O-deacylated MPL), immunostimulatory oligonucleotides, detoxified bacterial ADP-ribosylated toxins, microparticles, liposomes, imidazoquinolones, or mixtures thereof, preferably aluminum hydroxide, phosphates, or mixtures thereof. Other substances that act as immunostimulants are disclosed, for example, in Watson, Pediatr. Infect. Dis. J. (2000) 19:331-332. These salts include oxyhydroxides and hydroxyphosphates. The salts can exist in any preferred form (e.g., gel, crystal, amorphous, etc.).

[0122] Numbered Embodiments Embodiment 1 The oligomer of the following formula (Ia) or (Ib). [ka] During the ceremony, n is ≥ 6; R is H or -P(O)(OR″)2, and R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH3, and may be the same or different in each repeating unit; R y is H or -C(O)CH3, and may be the same or different in each repeating unit; R x or R yAt least one of these is -C(O)CH3 in at least one repeating unit, and together they are R in the oligomer. x and R y Approximately 50-90% of it is -C(O)CH3; Az is -NH(CO)R 1 , -N(R 1 ) an aza substituent selected from the group consisting of 2 and -N3, R 1 This is selected from H, linear or branched C1-C6-alkyl and linear or branched C1-C6-haloalkyl; Z is (i) protecting group; (ii) Functional linkers for conjugation to proteins, (iii) A linear or branched C1-C6 alkyl, optionally substituted phenyl, -C(O)Y, or a linear or branched C1-C6-alkyl-X And, Y is H, a linear or branched C1-C6 alkyl group, or a protecting group. X is -NH2, -N3, -C≡CH, -CH=CH2, -SH, or -SC≡N.

[0123] Embodiment 2 The oligomer of Embodiment 1, as defined by formula (Ia).

[0124] Embodiment 3 An oligomer of Embodiment 1 or Embodiment 2, wherein n is 8 to 30.

[0125] Embodiment 4 An oligomer of Embodiment 1 or Embodiment 2, wherein n is 8 to 20.

[0126] Embodiment 5 An oligomer of Embodiment 1 or Embodiment 2, wherein n is 8 to 15.

[0127] Embodiment 6 The oligomer according to any one of the prior embodiments, wherein Az is -NHC(O)CH3.

[0128] Embodiment 7 The oligomer according to any one of the preceding embodiments, where n is 8.

[0129] Embodiment 8 R x and R y The oligomer according to any one of Embodiments 1 to 7, wherein both are -C(O)CH3 in at least one same repeating unit.

[0130] Embodiment 9 R x and R y The oligomer according to any one of Embodiments 1 to 8, wherein both are -C(O)CH3 in 40 - 50% of the repeating units of the oligomer.

[0131] Embodiment 10 In 10 - 20% of the remaining repeating units of the oligomer, one of R x or R y is -C(O)CH3, and the remaining repeating units in the oligomer are R x =R y =H, the oligomer according to Embodiment 9.

[0132] Embodiment 11 The oligomer conjugate antigen of formula (IIa) or (IIb).

Chemical formula

[0133] Embodiment 12 The conjugate of Embodiment 11 defined by formula (IIa). <00​​​P is an inactivated bacterial toxin selected from diphtheria toxoid (DT), tetanus toxoid (TT), CRM 197 , Escherichia coli (E. coli) ST and recombinant Pseudomonas aeruginosa exotoxin (rEPA), or P is a polyamino acid such as poly(lysine:glutamic acid), or P is the core protein of hepatitis B virus or SPR96-2021, the conjugate of embodiment 11 or 12.

[0135] Embodiment 14 P is CRM 197 The conjugate of any one of embodiments 11 to 13.

[0136] Embodiment 15 Z is a linker having the following formula, the conjugate of any one of embodiments 11 to 14.

Chemical formula

[0137] Embodiment 16 Z is a linker having the following formula, the conjugate of any one of embodiments 11 to 14.

Chemical formula

[0138] Embodiment 17 Having the following structure, the conjugate according to any one of embodiments 11 to 16.

Chemical formula

[0139] Embodiment 18 (a) A conjugate according to any one of Embodiments 11 to 17; and (b) An immunogenic composition according to any one of Embodiments 11 to 17, comprising at least one pharmaceutically acceptable excipient.

[0140] Embodiment 19 The immunogenic composition according to Embodiment 18, further comprising an adjuvant.

[0141] Embodiment 20 The immunogenic composition according to Embodiment 18 or Embodiment 19, further comprising at least one antigen derived from one of Neisseria meningitidis serogroups C, W135, Y, and optionally A.

[0142] Embodiment 21 A vaccine comprising a conjugate according to any one of Embodiments 11 to 17, or an immunogenic composition according to any one of Embodiments 17 to 18.

[0143] Embodiment 22 A method for treating or preventing Meningitis A, C, W135 or Y in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a conjugate according to any one of Embodiments 11 to 17, or an immunogenic composition according to any one of Embodiments 18 to 20, or a vaccine according to Embodiment 21.

[0144] Embodiment 23 A method for immunizing a subject against Meningitis A, C, W135 or Y, comprising administering to the subject an immunologically effective amount of an immunogenic composition according to any one of Embodiments 18 to 20 or a vaccine according to Embodiment 21.

[0145] Embodiment 24 A method for inducing an immune response to meningitis A, C, W135, or Y in a subject, comprising administering an immunologically effective amount of an immunogenic composition described in any one of Embodiments 18 to 20 or a vaccine described in Embodiment 21 to the subject.

[0146] Embodiment 25 The method according to any one of embodiments 22 to 24, wherein the subject is a human.

[0147] Embodiment 26 Use of an immunogenic composition according to any one of Embodiments 18-20, or a vaccine according to Embodiment 21, in the manufacture of a pharmaceutical product for the treatment or prevention of meningitis A, C, W135, or Y.

[0148] Embodiment 27 An immunogenic composition according to any one of Embodiments 18 to 20, or a vaccine according to Embodiment 21, for use in the treatment or prevention of meningitis A, C, W135, or Y.

[0149] Embodiment 28 An immunogenic composition according to any one of Embodiments 18 to 20, or a vaccine according to Embodiment 21, for use in inducing an immune response to meningitis A, C, W135, or Y.

[0150] The present invention will be described in more detail in the following experimental section, which aims to better illustrate the present invention without imposing limitations on its scope. [Examples]

[0151] Experiment Section General Procedure and Materials All chemicals (Acros, Biosolve, Sigma-Aldrich, and TCI) were used in their original condition upon arrival, and unless otherwise noted, all reactions were carried out under an argon atmosphere at ambient temperature (22°C). For TLC analysis, aluminum sheets (Merck, TLC Silica Gel 60 F254) were used, with the solution in H2SO4 / EtOH (20%) or (NH4)6Mo7O 24 Solutions of 4H2O (25 g / L) and (NH4)4Ce(SO4)4·2H2O (10 g / L) in 10% H2SO4 aqueous solution, or a solution of KMnO4 (2%) and K2CO3 (1%) in H2O, were sprayed and heated at approximately 140°C. 40-63 μm 60 Å silica gel (SD Screening Devices) was used for column chromatography. NMR spectra (1H, 13 C and 31 P) was recorded using a Bruker AV-400liq, Bruker AV-500, or Bruker AV-600. High-resolution mass spectra were recorded by direct injection into a mass spectrometer with an electrospray ion source (Thermo Finnigan LTQ Orbitrap) in positive mode with a resolution of R=60000, using dioctyl phthalate as the rock mass (m / z=391.28428) at m / z 400 (mass range m / z=150–2000) and R=60000 (power supply voltage 3.5kV, sheath gas flow 10, capillary temperature 250°C).

[0152] abbreviation Acetic acid (ACOH) ACN = Acetonitrile DCM = Dichloromethane DMTrCl = 4,4′-dimethoxytrityl chloride alkyl = ethyl acetate THF = Tetrahydrofuran TBAF = Tetrabutylammonium fluoride

[0153] Example 1: Preparation of the oligomer of formula (Ia) of the present invention according to Scheme 1 Acetamide-3,4-di-O-benzyl-2-deoxy-6-O-texyldimethylsilyl-5a-carba-α-domannapyranose(13) Silyl ether 12 can be prepared according to the procedure described in Q. Gao et al. Org. Biomol. Chem., 2012, 10, 6673.

[0154] Silyl ether 12 (1.6 g, 2.7 mmol) was dissolved in dry THF (20 mL). The mixture was cooled to 0°C. 0.1 M TBAF / THF solution (4.1 mL, 4.1 mmol) was slowly added. The reaction mixture was warmed to room temperature and stirred for 3 hours. AcOH (0.31 mL) was added to the reaction mixture. The solution was extracted three times with DCM and washed once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (siRNA / hexane) to obtain product 13 in 92% yield (1.1 g, 2.52 mmol). The spectral data were consistent with reported data.

[0155] 2-Acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranose(14) Alcohol 13 (1.12 g, 2.5 mmol) was dissolved in MeOH (32 mL). NaOMe (0.03 g, 0.5 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. Amberlite H+ resin was added until a neutral pH was reached. The suspension was filtered and concentrated under reduced pressure. 1 H NMR (400MHz, CDCl3)δ=1.70-1.85(m, 2H, H-5a), 1.90(s, 3H, AcNH), 2.19-2.23(m, 1H, H-5), 3.60-3.79(m, 3 H, H-6, H-1), 3.83-3.90(m, 1H, H-2), 3.91-3.99(m, 1H, H-4), 4.14-4.23(m, 1H, H-3), 4.33-4.41(m, 1H, CHH Bn), 4.54-4.72(m, 3H, CH2Bn, CHH Bn), 5.79(m, 1H, NHAc), 7.22-7.42(m, 10H, H arom ).13 C NMR (100MHz, CDCl3)δ=23.5(CH3AcNH), 30.6(CH2C-5a), 39.5(CH C-5), 53.5(CH C-3), 64.1(CH2C-6), 67.9(CH C-4), 72.4(CH2Bn), 73.8(CH2Bn), 75.5(CH C-1), 79.0(CH C-4), 127.3-128.9(CH arom ), 171.8 (C=OAcNH). HRMS:[C 23 H 29 NO5+H] + The required value was 400.21251, and the measured value was 400.21179.

[0156] 2-Acetamide-3,4-di-O-benzyl-2-deoxy-6-O-(bis(4-methoxyphenyl)(phenyl))-5-carba-α-D-mannopyranose(10) Diol 14 (0.9 g, 2.25 mmol) was dissolved in dry DCM (30 mL). Et3N (1.9 mL, 13.5 mmol) was added to the mixture. DMTrCl (1.16 g, 3.38 mmol) was added. The reaction mixture was stirred for 2 hours. H2O was added to the reaction mixture and washed once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (siRNA / hexane) to obtain product 10 in 91% yield (1.6 g, 2.04 mmol). 1 H NMR (400MHz, CD3CN) δ=1.70-1.85(m, 1H, 5a′-H), 1.91(s, 3H, AcNH), 2.00-2.21(m, 2H, 5a-H, 5-H), 3.01-3.19(m, 1H, 6′-H ), 3.27-3.37(m, 1H, 6-H), 3.51-3.67(m, 1H, H-4), 3.73(s, 7H, H-3, 2×OMe), 4.06-4.20(m, 1H, H-1), 4.22-4.32(m, 1H, CHH Bn), 4.40-4.62(m, 3H, CH2Bn, H-2), 4.65-4.73(m, 1H, CHH Bn), 6.35-6.44(m, 1H, NHAc), 6.78-7.47(m, 23H, H arom ). 13C NMR (100MHz, CD3CN)δ=23.2(CH3AcNH), 31.6(CH2C-5a), 38.6(CH C-5), 53.3(CH C-2), 55.8(2×CH3OMe), 64.6(CH2C-6), 67.6(CH C-1), 72.1(CH2Bn), 73.8(CH2Bn), 77.2(CH C-4), 79.8(CH C-3), 86.5(Cq DMTr), 113.9(CH arom ), 127.3-130.7(CH arom ), 137.2-159.4(5×Cq DMTr), 171.1(C=OAcNH). HRMS:[C 44 H 47 [NO7+Na] + The required value was 724.32501, and the measured value was 724.32483.

[0157] 1-O-((N,N-diisopropylamino)-O-2-cyanoethyl-phosphorumidite))-2-acetamide-3,4-di-O-benzyl-2-deoxy-6-O-(bis(4-methoxyphenyl)(phenyl))-5a-carba-α-D-mannopyranose(9) Alcohol 10 (1.5 g, 2.14 mmol) was removed three times with ACN and dissolved in dry DCM (22 mL). Fresh activated MS3Å and DIPEA (0.6 mL, 3.2 mmol) were added to the mixture. 2-Cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mL, 2.6 mmol) was added to the mixture. The reaction mixture was stirred for 2 hours. H2O was added to this solution and washed once with a 1:1 brine / NaHCO3 solution. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM / acetone / Et3N) to obtain product 9 in 94% yield (1.81 g, 2.0 mmol) (mixture of diastereomers). 1¹H NMR (400MHz, CD3CN) δ = 1.04-1.24 (m, 12H, 4× isopropylamino), 1.70-1.85 (m, 1H, 5α′-H), 1.92 (s, 3H, AcNH), 2.00-2.21 (m, 2H, 5α-H, 5-H), 2.55-2.75 (m, 2H, CH2 cyanoethyl), 2.98-3 .10(m, 1H, 6′-H), 3.27-3.37(m, 1H, 6-H), 3.47-3.70(m, 3H, 2×CH isopropylamino, H-4), 3.70-3.88(m, 9H, H-3, CH2 cyanoethyl, 2×OMe), 4.06-4.20(m, 1H, H-1), 4.22-4.32(m, 1H, CHH Bn), 4.40-4.62(m, 3H, CH2 Bn, H-2), 4.65-4.73(m, 1H, CHH Bn), 6.35-6.44(m, 1H, NHAc), 6.78-7.47(m, 23H, H arom ). 13 ¹¹C NMR (100MHz, CD3CN) δ = 20.7 (CH2 cyanoethyl), 22.9 (CH3 AcNH), 24.5-24.7 (2×CH3 isopropylamino), 30.6 (CH2C-5a), 38.5 (CH3 C-5), 43.7 (2×CH isopropylamino), 51.7 (CH3 C-2), 55.5 (2×CH3 OMe), 59.1 (CH2 cyanoethyl), 64.2 (CH2C-6), 70.5 (CH3 C-1), 71.5 (CH2 Bn), 74.3 (CH2 Bn), 77.8 (CH3 C-4), 79.5 (CH3 C-3), 86.2 (Cq DMTr), 113.6 (CH3 arom ), 127.3-130.7(CH arom ), 136.8-159.2(5×Cq DMTr), 170(C=OAcNH). 31 P NMR (162MHz, CD3CN) δ=146.9, 147.26.

[0158] General procedure for phosphoramidite coupling, oxidation, and detritylation at typical scales (0.03-0.3 mmol) The starting alcohol was removed three times by co-distillation with ACN, and newly activated MS3Å and DCI (0.25 M / ACN solution, 1.5 equivalents) were added. The solution was stirred for 15 minutes. Phosphoramidite reagent (0.1-0.16 M / ACN solution, 1.3-3 equivalents) was added to the mixture and stirred until the starting materials were completely converted (approximately 2 hours). Subsequently, CSO (0.5 M / ACN solution, 2 equivalents) was added to the reaction mixture and stirred for 15 minutes. The mixture was diluted with HCl and washed with a 1:1 brine / NaHCO3 solution. The aqueous layer was extracted twice with HCl. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was removed three times by co-distillation with ACN and dissolved in DCM (5-10 mL). TCA (0.18 MDCM solution) was added to this solution and stirred for 1 hour. H2O was added to the reaction mixture and stirred for 15 minutes. The reaction mixture was washed with a 1:1 brine / NaHCO3 solution. The aqueous layer was extracted three times with DCM. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM / acetone) or size exclusion chromatography (sephadex LH-20, MeOH / DCM 1:1).

[0159] 1-O-((2-acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzyl-carbamate(15) Using the general procedure described above, alcohol 10 (0.21 g, 0.3 mmol) was coupled with phosphoramidite 11 (2.5 mL 0.16 M / ACN solution, 0.45 mmol), followed by oxidation and detritylation. The crude product was purified by flash chromatography (DCM / acetone) to obtain product 15 in 94% yield (0.216 g, 0.282 mmol). 1¹H NMR (400MHz, CD3CN) δ = 1.24-1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40-1.51 (m, 2H, CH2 hexyl spacer), 1.58-1.70 (m, 2H, CH2 hexyl spacer), 1.80-1.92 (m, 4H, 5a′-H, AcNH), 1.96-2.02 (m, 2H, 5a-H, 5-H), 2.72-2.82 (m, 2H, CH2 shear) (Noethyl), 2.96 (bs, 1H, OH), 3.02-3.12 (m, 2H, CH2 hexyl spacer), 3.56-3.74 (m, 3H, H-6, H-4), 3.76-3.84 (m, 1H, H-3), 3.95-4.07 (m, 2H, CH2 hexyl spacer), 4.08-4.20 (m, 2H, CH2 cyanoethyl), 4.44-4.63 (m, 5H, H-1, H-2, CH2Bn, CHH Bn), 4.72-4.80 (m, 1H, CHH Bn), 5.03 (s, 2H, CH2Bn spacer), 5.70 (bs, 1H, NH), 6.49-6.60 (m, 1H, NHAc), 7.23-7.44 (m, 15H, H arom ). 13 ¹¹C NMR (100MHz, CD3CN) δ = 19.9 (CH2 cyanoethyl), 22.8 (CH3AcNH), 25.4 (CH2 hexyl spacer), 26.4 (CH2 hexyl spacer), 30.0 (CH2C-5a), 30.4 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 40.0 (CH2C-5), 41.0 (CH2 hexyl spacer), 51.1 (CH2C-2), 62.9 (CH2C-6), 63.0 (CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.8 (CH2 hexyl spacer), 72.2 (CH2Bn), 74.0 (CH2Bn), 75.1 (CH2C-1), 76.7 (CH2 C-4), 79.3(CHC-3), 128.1-129.1(CH arom ), 138.9-139.7(3×Cq Bn), 170.8(C=O AcNH). 31 P NMR (162MHz, CD3CN) δ=-2.40, -2.36. HRMS:[C 40 H 52 N3O 10 [P+H] +The required value was 766.34707, and the measured value was 766.34707.

[0160] 1-O-di-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(16) Using the general procedure described above, alcohol 15 (0.186 g, 0.24 mmol) was coupled with phosphoramidite 9 (2.3 mL 0.16 M / ACN solution, 0.37 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 16 in 82% yield (0.255 g, 0.199 mmol). 1 ¹H NMR (400MHz, CD3CN) δ = 1.25-1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40-1.51 (m, 2H, CH2 hexyl spacer), 1.58-1.71 (m, 2H, CH2 hexyl spacer), 1.80-1.92 (m, 8H, 2×5a′-H, 2×AcNH), 1.96-2.02 (m, 4H, 2×5a-H, 2×5-H), 2.70-2.81 (m, 4H, 2×CH2 cyanoethyl), 2.96 (bs, 1H, OH), 3.01-3.12 (m, 2 H, CH2 hexyl spacer), 3.56-3.87 (m, 6H, 2×H-6, 2×H-4), 3.94-4.28 (m, 8H, 2×H-3, CH2 hexyl spacer, 2×CH2 cyanoethyl), 4.29-4.85 (m, 12H, 2×H-1, 2×H-2, 4×CH2Bn), 5.03 (s, 2H, CH2Bn spacer), 5.75 (bs, 1H, NH), 6.52-6.62 (m, 1H, NHAc), 6.85-6.99 (m, 1H, NHAc), 7.21-7.41 (m, 25H, H arom ). 13¹³C NMR (100MHz, CD3CN) δ = 19.9-20.0 (2×CH2 cyanoethyl), 22.9-23.0 (2×CH3AcNH), 25.5 (CH2 hexyl spacer), 26.5 (CH2 hexyl spacer), 29.1-29.2 (2×CH2C-5a), 30.1 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 38.1-40.0 (2×CH C-5), 41.1 (CH2 hexyl spacer), 50.9-51.4 (2×CH C-2), 62.5-62.6 (2×CH2C-6), 63.0-63.2 (2×CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.9 (CH2 hexyl spacer), 72.1-72.3 (4×CH2Bn), 75.0-75.4 (2×CHC-1), 75.5-76.9 (2×CHC-4), 79.2-79.5 (2×CH C-3), 128.2-129.1 (CH arom ), 138.9-139.6(5×Cq Bn), 170.8(2×C=O AcNH). 31 P NMR (162MHz, CD3CN) δ=-2.60, -2.58, -2.34, -2.32, -2.22, -2.17. HRMS:[C 66 H 83 N5O 17 P2+H] + The required value was 1280.53320, and the measured value was 1280.53320.

[0161] 1-O-tri-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(17) Using the general procedure described above, alcohol 16 (0.215 g, 0.167 mmol) was coupled with phosphoramidite 9 (1.6 mL 0.16 M / ACN solution, 0.25 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 17 in 95% yield (0.285 g, 0.158 mmol). 1¹H NMR (400MHz, CD3CN) δ = 1.25-1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40-1.51 (m, 2H, CH2 hexyl spacer), 1.58-1.71 (m, 2H, CH2 hexyl spacer), 1.80-1.92 (m, 12H, 3×5a′-H, 3×AcNH), 1.96-2.30 (m, 6H, 3×5a-H, 3×5-H), 2.68-2.83 (m, 6H, 3×CH2 cyanoethyl), 2.93 (bs, 1H, OH), 3.00-3.11 (m, 2 H, CH2 hexyl spacer), 3.59-3.89 (m, 9H, 3×H-6, 3×H-4), 3.96-4.22 (m, 11H, 3×H-3, CH2 hexyl spacer, 3×CH2 cyanoethyl), 4.31-4.86 (m, 18H, 3×H-1, 3×H-2, 6×CH2Bn), 5.03 (s, 2H, CH2Bn spacer), 5.78 (bs, 1H, NH), 6.55-6.65 (m, 1H, NHAc), 6.9-7.15 (m, 2H, 2×NHAc), 7.19-7.40 (m, 35H, H arom ). 13 ¹³C NMR (100MHz, CD3CN) δ = 20.0-20.1 (3×CH2 cyanoethyl), 22.9-23.0 (3×CH3AcNH), 25.5 (CH2 hexyl spacer), 26.5 (CH2 hexyl spacer), 28.9-29.2 (3×CH2C-5a), 30.1 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 38.0-40.0 (3×CH C-5), 41.1 (CH2 hexyl spacer), 50.8-51.4 (3×CH C-2), 62.5-63.0 (3×CH2C-6), 63.0-63.3 (3×CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.4 (CH2 hexyl spacer), 72.1-74.1 (6×CH2Bn), 75.2-75.5 (3×CH C-1), 75.5-76.1 (3×CH C-4), 79.3-79.5 (3×CH C-3), 128.2-129.1 (CH arom ), 138.9-139.7(7×Cq Bn), 170.9-171.2(3×C=O AcNH). 31P NMR (162MHz, CD3CN) δ=-2.82, -2.77, -2.62, -2.58, -2.36, -2.33, -2.24, -2.20, -2.16. HRMS:[C 92 H 11 4N7O 24 P3+H] + The required value was 1795.72333, and the measured value was 1795.22333.

[0162] 1-O-tetra-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(18) Using the general procedure described above, alcohol 17 (0.267 g, 0.148 mmol) was coupled with phosphoramidite 9 (1.4 mL 0.16 M / ACN solution, 0.22 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 18 in 87% yield (0.299 g, 0.129 mmol). 1 ¹H NMR (400MHz, (CD3)2CO) δ = 1.31-1.47 (m, 4H, 2×CH2 hexyl spacer), 1.47-1.57 (m, 2H, CH2 hexyl spacer), 1.62-1.75 (m, 2H, CH2 hexyl spacer), 1.85-2.02 (m, 16H, 4×5a′-H, 4×AcNH), 2.07-2.17 (m, 8H, 4×5a-H, 4×5-H), 2.82-3.00 (m, 8H, 4×CH2 cyanoethyl ), 3.08-3.18 (m, 2H, CH2 hexyl spacer), 3.66-4.01 (m, 12H, 4×H-6, 4×H-4), 4.04-4.36 (m, 14H, 4×H-3, CH2 hexyl spacer, 4×CH2 cyanoethyl), 4.40-4.94 (m, 24H, 4×H-1, 4×H-2, 8×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.39 (bs, 1H, NH), 7.17-7.42 (m, 45H, H arom ), 7.42-7.80(m, 4H, NHAc). 13¹³C NMR (100MHz, (CD3)2CO) δ = 20.0-20.1 (4×CH2 cyanoethyl), 23.1-23.2 (4×CH3AcNH), 25.8 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-29.8 (4×CH2C-5a), 30.8 (CH2 hexyl spacer), 30.8 (CH2 hexyl spacer), 38.3-40.3 (4×CH C-5), 41.4 (CH2 hexyl spacer), 51.2-51.5 (4×CH C-2), 62.6-63.4 (4×CH2C-6), 63.4-63.6 (4×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.8 (CH2 hexyl spacer), 72.0-75.0 (8×CH2Bn), 75.6-75.8 (4×CH C-1), 76.5-77.2 (4×CH C-4), 79.7-79.8 (4×CH C-3), 128.1-129.1 (CH arom ), 139.3-140.1(9xCq Bn), 170.7-171.2(4xC=O AcNH). 31 P NMR (162MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.47, -2.42, -2.37, -2.30, -1.96, -1.91, -1.89. HRMS:[C 118 H 145 N9O 31 P4+2H] ++ The required value was 1155.45892, and the measured value was 1155.45892.

[0163] 1-O-penta-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(19) Using the general procedure described above, alcohol 18 (0.277 g, 0.120 mmol) was coupled with phosphoramidite 9 (1.1 mL 0.16 M / ACN solution, 0.18 mmol) for oxidation and detritylation. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 19 in 92% yield (0.31 g, 0.110 mmol).1 ¹H NMR (400MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.58 (m, 2H, CH2 hexyl spacer), 1.62-1.75 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 20H, 5×5a′-H, 5×AcNH), 2.07-2.19 (m, 10H, 5×5a-H, 5×5-H), 2.82-2.97 (m, 10H, 5×CH2 cyanoethyl) ), 3.08-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.02 (m, 15H, 5×H-6, 5×H-4), 4.04-4.36 (m, 17H, 5×H-3, CH2 hexyl spacer, 5×CH2 cyanoethyl), 4.38-4.95 (m, 30H, 5×H-1, 5×H-2, 10×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.43 (bs, 1H, NH), 7.16-7.41 (m, 55H, H arom ), 7.42-7.86(m, 5H, NHAc). 13 ¹³C NMR (100MHz, (CD3)2CO) δ = 19.8-20.0 (5×CH2 cyanoethyl), 23.0-23.1 (5×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.7 (CH2 hexyl spacer), 29.2-30.0 (5×CH2C-5a), 30.7 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (5×CH C-5), 41.2 (CH2 hexyl spacer), 51.0-51.4 (5×CH C-2), 62.5-63.2 (5×CH2C-6), 63.3-63.5 (5×CH2 cyanoethyl), 66.1 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.0-75.0 (10xCH2Bn), 75.6-75.8 (5×CH C-1), 76.5-77.2 (5×CH C-4), 79.7-79.8 (5×CH C-3), 128.0-129.0 (CH arom ), 139.2-140.0(11×Cq Bn), 170.7-171.2(5×C=OAcNH). 31P NMR (162MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.47, -2.42, -2.37, -2.30, -1.96, -1.88, -1.89, -1.86, -1.84, -1.79. HRMS:[C 144 H 176 N 11 O 38 P5+2H] ++ The required value was 1412.55219, and the measured value was 1412.55219.

[0164] 1-O-Hexa-((2-Acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(20) Using the general procedure described above, alcohol 19 (0.280 g, 0.099 mmol) was coupled with phosphoramidite 9 (1.24 mL 0.16 M / ACN solution, 0.20 mmol), followed by oxidation and detritylation. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 20 in 88% yield (0.29 g, 0.087 mmol). 1 ¹H NMR (500MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.57 (m, 2H, CH2 hexyl spacer), 1.63-1.74 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 24H, 6×5a′-H, 6×AcNH), 2.07-2.30 (m, 12H, 6×5a-H, 6×5-H), 2.82-2.97 (m, 12H, 6×CH2 cyanoethyl ), 3.09-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.04 (m, 18H, 6×H-6, 6×H-4), 4.04-4.38 (m, 20H, 6×H-3, CH2 hexyl spacer, 6×CH2 cyanoethyl), 4.38-5.00 (m, 36H, 6×H-1, 6×H-2, 12×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16-7.41 (m, 65H, H arom), 7.42-7.89(m, 6H, NHAc). 13 ¹³C NMR (100MHz, (CD3)2CO) δ = 19.9-20.0 (6×CH2 cyanoethyl), 23.0-23.1 (6×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-30.2 (6×CH2C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (6×CH C-5), 41.3 (CH2 hexyl spacer), 51.0-51.4 (6×CH C-2), 62.5-63.4 (6×CH2C-6), 63.4-63.5 (6×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.2-75.6 (12×CH2Bn), 75.6-75.8 (6×CH C-1), 76.5-77.2 (6×CH C-4), 79.7-79.8 (6×CHC-3), 128.1-129.1 (CH arom ), 139.2-140.0(13×Cq Bn), 170.7-171.2(6×C=O AcNH). 31 P NMR (162MHz, CD3)2CO)δ=-2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS:[C 170 H 207 N 13 O 45 P6+NH4] + The required value was 3356.312, and the measured value was 3357.010.

[0165] To produce an oligomer with n=6, the general deprotection procedure described below can be performed after the above steps.

[0166] 1-O-epta-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(21) Using the general procedure described above, alcohol 20 (0.140 g, 0.042 mmol) was coupled with phosphoramidite 9 (0.8 mL 0.1 M / ACN solution, 0.84 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 21 in 86% yield (0.139 g, 0.036 mmol). 1 ¹H NMR (500MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.57 (m, 2H, CH2 hexyl spacer), 1.63-1.74 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 28H, 7×5a′-H, 7×AcNH), 2.07-2.30 (m, 14H, 7×5a-H, 7×5-H), 2.82-2.97 (m, 14H, 7×CH2 cyanoethyl ), 3.09-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.04 (m, 21H, 7×H-6, 7×H-4), 4.04-4.38 (m, 23H, 7×H-3, CH2 hexyl spacer, 7×CH2 cyanoethyl), 4.38-5.00 (m, 42H, 7×H-1, 7×H-2, 14×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16-7.41 (m, 75H, H arom ), 7.42-7.89(m, 7H, NHAc). 13¹³C NMR (125MHz, (CD3)2CO) δ = 19.9-20.0 (7×CH2 cyanoethyl), 23.0-23.1 (7×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-30.2 (7×CH2C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (7×CH C-5), 41.3 (CH2 hexyl spacer), 51.0-51.4 (7×CH C-2), 62.5-63.4 (7×CH2C-6), 63.4-63.5 (7×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.2-75.6 (14×CH2Bn), 75.6-75.8 (7×CH C-1), 76.5-77.2 (7×CH C-4), 79.7-79.8 (7×CH C-3), 128.1-129.1 (CH arom ), 139.2-140.0(15×Cq Bn), 170.7-171.2(7×C=O AcNH). 31 P NMR (202MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS:[C 196 H 238 N 15 O 52 P7+2H] ++ The required value was 1926,73908, and the measured value was 1926,73908.

[0167] 1-O-octa-((2-acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(22) n=8 Using the general procedure described above, alcohol 22 (0.105 g, 0.027 mmol) was coupled with phosphoramidite 9 (0.7 mL 0.1 M / ACN solution, 0.68 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 22 in 87% yield (0.103 g, 0.023 mmol). 1 ¹H NMR (500MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.57 (m, 2H, CH2 hexyl spacer), 1.63-1.74 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 32H, 8×5a′-H, 8×AcNH), 2.07-2.30 (m, 16H, 8×5a-H, 8×5-H), 2.82-2.97 (m, 16H, 8×CH2 cyanoethyl ), 3.09-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.04 (m, 24H, 8×H-6, 8×H-4), 4.04-4.38 (m, 26H, 8×H-3, CH2 hexyl spacer, 8×CH2 cyanoethyl), 4.38-5.00 (m, 48H, 8×H-1, 8×H-2, 16×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16-7.41 (m, 85H, H arom ), 7.42-7.89(m, 8H, NHAc). 13¹³C NMR (125MHz, (CD3)2CO) δ = 19.9-20.0 (8×CH2 cyanoethyl), 23.0-23.1 (8×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-30.2 (8×CH2C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (8×CH C-5), 41.3 (CH2 hexyl spacer), 51.0-51.4 (8×CH C-2), 62.5-63.4 (8×CH2C-6), 63.4-63.5 (8×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.2-75.6 (16×CH2Bn), 75.6-75.8 (8×CH C-1), 76.5-77.2 (8×CH C-4), 79.7-79.8 (8×CH C-3), 128.1-129.1 (CH arom ), 139.2-140.0(17×Cq Bn), 170.7-171.2(8×C=O AcNH). 31 P NMR (202MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS:[C 222 H 269 N 17 O 59 P8+2H] ++ The required value was 2184.33410, and the measured value was 2184.33410.

[0168] General procedure for deprotection at typical scales (5-40 μmol) The starting alcohol was dissolved in NH3 (30-33% aqueous solution, 1 mL per 10 μmol) and dioxane until completely dissolved. The reaction mixture was stirred for 2 hours. The mixture was concentrated under reduced pressure. 1 1H NMR and 311P NMR analysis showed complete conversion to a semi-protected intermediate. The crude product was dissolved in MilliQ H2O and eluted using a column containing Dowex Na+ cation exchange resin (type: 50WX4-200, stored in 0.5M NaOH / H2O solution, MilliQ H2O and MeOH flushed before use). The crude product was dissolved in MilliQ H2O (2 mL per 10 μmol). 4-5 drops of glacial acetic acid were added to the reaction mixture. The mixture was purged with Ar. 1 cup of Pd Black was added to the solution. The reaction mixture was purged with H2 for several seconds and stirred under an H2 atmosphere for 3 days. Celite was added to the mixture. The solution was filtered and concentrated under reduced pressure. The crude product was purified by size exclusion chromatography (Toyopearl HW-40). The pure compound was dissolved in MilliQ H2O, eluted using a column containing Dowex Na+ cation exchange resin (type: 50WX4-200, stored in 0.5M NaOH / H2O solution, MilliQ H2O and MeOH were flushed before use), and lyophilized.

[0169] 1-O-octa-(2-acetamide-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)-6-hexylamine(8)n=8 Alcohol 22 (23.2 μmol) was deprotected using the general procedure described above. Pure oligomer 8 was obtained in 44% yield (25.9 mg, 10.2 μmol). 1 ¹H NMR (500MHz, D2O) δ = 1.33-1.43 (m, 4H, 2×CH2 hexyl spacer), 1.57-1.69 (m, 4H, 2×CH2 hexyl spacer), 1.73-2.08 (m, 48H, 8×5a′-H, 8×5a-H, 8×5-H, 8×AcNH), 2.92-3.00 (m, 2H (CH2 hexyl spacer), 3.48-3.68 (m, 8H, 8×H-4), 3.68-3.76 (m, 2H, CH2 hexyl spacer), 3.81-4.22 (m, 24H, 8×H-3, 8×H-6), 4.25-4.36 (m, 8H, 8×H-1), 4.37-4.53 (m, 8H, 8×H-2). 13¹³C NMR (126MHz, D2O) δ = 21.9 (8×CH3AcNH), 24.4 (CH2 hexyl spacer), 25.1 (CH2 hexyl spacer), 26.6 (CH2 hexyl spacer), 28.0 (8×CH2C-5a), 29.5 (CH2 hexyl spacer), 38.6 (8×CH₃C-5), 39.4 (CH₃Hexyl spacer), 53.5 (8×CH₃C-2), 61.9 (8×CH₃C-6), 66.2 (CH₃Hexyl spacer), 70.1 (8×CH₃C-1), 70.4 (8×CH₃C-4), 71.9 (8×CH₃C-3), 174.7 (8×C=O AcNH). 31 P NMR (202MHz, D2O) δ=0.25, 0.37, 0.41, 0.44, 0.48. HRMS:[C 78 H 145 N9O 57 P8+H] ++ The required value was 1183.83071, and the measured value was 1183.83071.

[0170] Production of randomly acetylated carbaoligomers according to the present invention 1. Amine protection as a Boc derivative Dried carba analogs DP6 (n=6), DP7 (n=7), and DP8 (n=8) were dissolved in H2O:dioxane in a 1:1 volume ratio. Then, NaHCO3 (2.95 equivalents) and (Boc)2O (1.13 equivalents) were added at 4°C. The reaction mixture was then maintained overnight at room temperature under magnetic stirring. The product was then purified using a Sephadex G10 column (eluent: H2O), and the fraction containing the compound was dried.

[0171] 2. Random O-acetylation The dried Boc-protected carba analog from step 1 was resuspended in acetonitrile, and acetic anhydride (3.6 equivalents per -OH group in the molecule) and imidazole (1.8 equivalents) were added. The reaction mixture was kept at 40°C, and the acetylation reaction time was extended until the target acetylation rate (%) (approximately 75%) was reached. 1 The results were monitored by 1H-NMR. Next, the crude acetylated compound was dried.

[0172] To avoid misunderstanding, "random O-acetylation" is R x and R y This means that the number of -C(O)CH3 groups is ultimately not controlled. However, using NMR techniques, the total O-acetylation rate (%) in the oligomer can be determined.

[0173] 3.Boc deprotection The dried crude O-acetylated carba analog from step 2 was dissolved in CH2Cl2:TFA 4:1 (volume ratio), and the reaction mixture was maintained at room temperature under magnetic stirring for 1 hour. Next, the crude reaction mixture was dried, redissolved in H2O, and purified using a Sephadex G10 column (eluent: H2O).

[0174] NMR protocol for measuring acetylation rate (%) The sample was vacuum-dried, regenerated with 0.6 mL of D2O, and transferred to a 5 mm NMR tube. Proton NMR spectra were obtained using a standard one-dimensional pulse program at 400 MHz and 25 °C. Spectrum acquisition and processing were performed using TopSpin Bruker software.

[0175] The O-acetylation rate (%) in carba analogs was measured by integrating the H3+H4O-Ac (i.e., H of the acetate group) peak at 5–5.4 ppm and the triplet of CH2 adjacent to NH2 of the linker at approximately 3 ppm, where a value of 2 is given. As shown in Figure 1, assuming that the integral value of H3+H4O-Ac must be 12 in DP6 (14 in DP7 and 16 in DP8) when O-acetylation is 100%, the following proportions apply. 12:100 = 9.04:X (X = acetylation rate (%))

[0176] The final product 1 The structures were characterized by 1H-NMR to confirm their identity, and the O-acetylation rate (%) of the synthesized sugars was determined (Figure 2 and Table 1).

[0177] Figure 2 shows the final randomly acetylated carba analog. 1 This shows the 1H NMR spectrum, and also the integral of the acetylation rate (%) measurement (n=8).

[0178] [Table 1]

[0179] For the same random acetylated carba analog of equation (Ia) with n=8, the distribution of acetyl groups between the 3rd and 4th positions is as follows: 31 The results were obtained by 1P NMR spectroscopy (101 MHz, D2O). The recorded spectra are shown in Figure 3. This indicates simultaneous acetylation occurring at the C3 and C4 positions in approximately 44% of cases (i.e., R in the same repeating unit of the oligomer). x and R y Both are -C(O)CH3. ) and acetylation at C3 or C4 up to about 28% (i.e., the same repeating unit, R x is -C(O)CH3, and R y H is, or R x H is R y This indicates that -C(O)CH3, and that 27% of the repeating units are not acetylated.

[0180] Production of selectively acetylated carbamonomer constituent blocks by Scheme 2 (i.e., R x H is R y (is -C(O)CH3) D-glucar (23) [ka] A mixture of 3,4,6-tri-O-acetyl-D-glucar (10.0 g, 36.7 mmol) was mixed with 150 mL of K2CO3 (508 mg, 3.67 mmol) / dried MeOH solution, and the mixture was stirred at room temperature under N2. After 1 hour, the reaction was completed, and the reaction was stopped with acetic acid to pH 7. The solvent was removed under reduced pressure, and the crude product of D-glucar, a clear oily substance, was used directly in the next step.

[0181] 4,6-O-(4-methoxybenzylidene)-D-glucar(24) [ka] Crude compound 23 in dry DMF (100 mL) was mixed with anisaldehyde dimethyl acetal (9.40 mL, 55.1 mmol) under N2 conditions, followed by pyridine p-toluenesulfonate (922 mg, 3.67 mmol). The reaction was carried out on a rotary evaporator under reduced pressure (180 mbar) at 25-30°C for 2.5-3 hours. The DMF was then removed under reduced pressure, and the crude product was extracted with 100 mL of DCM. The organic layer was washed sequentially with 50 mL of NH4Cl, 50 mL of distilled water, and 50 mL of brine solution. Finally, the collected aqueous layer was extracted with 50 mL of DCM. The mixture was then dried over Na2SO4, and the solvent was removed under reduced pressure to obtain 4,6-O-(4-methoxybenzylidene)-D-glucar as a white powder in 45% yield.

[0182] δ 1 H(400MHz;CDCl3) 7.43(2H, td, J8.6, J4.7, 8-H), 6.90(2H, dt, J8.8, J4.9, 9-H), 6.33(1H, ddd, J6 .1, J1.6, J0.4, 1-H), 5.55 (1H, s, 7-H), 4.76 (1H, dd, J6.1, J2.0, 2-H), 4.49 (1H) , brd, J7.3, 3-H), 4.35(1H, dd, J10.3, J5.0, 5-H), 3.93-3.87(1H, m, 6-H), 3.83 -3.79(1H, m, 6-H), 3.80(3H, s, -OMe), 3.77-3.75(1H, m, 4-H), 2.47(1H, s, -OH).

[0183] δ 13 C(100MHz;CDCl3) 159.4(11-C), 143.3(1-C), 128.6(8-C), 126.7(9-C), 112.8(10-C), 102.7(2 -C), 100.9(7-C), 79.8(4-C), 68.9(5-C), 67.6(6-C), 65.7(3-C), 54.4(OMe).

[0184] 3-O-benzyloxy-4,6-O-(4-methoxybenzylidene)-D-glucar(25) [ka] To a 350 mL solution of 24 (16.05 g, 60.7 mmol) DMF at 0°C, 7.29 g, 182 mmol of 60% sodium hydride / mineral oil was added in small increments (the mineral oil from the NaH could be washed off three times beforehand with dry n-hexane). After stirring at the same temperature for 30 minutes, the ice bath was removed. Benzyl bromide (14.4 mL, 121 mmol) was added, and the reaction mixture was stirred overnight, during which time it was heated to room temperature. Next, the reaction was stopped with methanol (20 mL), and the DMF was evaporated under reduced pressure. The organic phase was extracted with 100 mL of siRNA, and then the organic layer was washed with NH4Cl, NaHCO3, and brine (50 mL each). The organic layer was dried with Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (siRNA / hexane = 3:7) using silica gel to obtain 3-O-benzyloxy-4,6-O-(4-methoxybenzylidene)-D-glucar (18.43 g, 86%) as a white powder.

[0185] δ 1 H(400MHz;CDCl3) 7.42(2H, dt, J8.5, J4.6, 8-H), 7.37-7.23(7H, m, H arom), 6.90(2H, dt, J8.9, J4.9, 9-H), 6.34(1H, dd, J6.2, J1.4, 1-H), 5.58(1H, s, 7-H), 4.81(1H, dd, J6.17, J2.06, 2-H), 4.79(1H, d, J12.110-H CH2Ph), 4.70(1H, d, J12.1, 10-H CH2Ph), 4.36-4.32(2H, m, 3-H, 6a-H), 4.00(1H, dd, J9.8, J7.4, 6b-H), 3. 88 (1H, td, J10.1, J4.7, 5-H), 3.81 (1H, t, J10.1, 4-H), 3.80 (3H, s, -OMe).

[0186] δ 13 C(100MHz;CDCl3) 160.2(11-C), 144.5(1-C), 138.6(13-C), 129.9(8-C), 129.9-127.2(C arom 9, 14, 15, 16-C), 113.7(10-C), 102.4(2-C), 101.3(7-C), 80.1(5-C), 73.2(4-C), 72.1(6-C), 68.8(3-C), 68.4(12-C), 55.4(-OMe).

[0187] 3-O-benzyloxy-4-O-(4-methoxybenzyloxy)-D-glucar(26) [ka] Glucar 25 (780 mg, 2.20 mmol) was dissolved in DCM (20 mL), cooled to 0°C, and stirred at room temperature for 20 minutes. Next, 11.0 mL, 11.0 mmol, of 1 M DIBAL-H / hexane solution was added dropwise at 0°C. The mixture was stirred at 0°C for 2 hours. The reaction was stopped for 20 minutes with a distilled aqueous solution of potassium sodium tartrate tetrahydrate (1.5 g of tartrate in 7.5 mL of water), commonly known as Rochelle salt. Next, the mixture was extracted with DCM (30 mL), and the organic layer was washed twice with distilled water and brine (40 mL each). Finally, the aqueous layer was extracted with DCM (20 mL). The organic phase was collected and dried over Na2SO4. The solvent was removed under reduced pressure. The residue was purified by flash chromatography using silica gel (siRNA / hexane = 1:3) to obtain 3-O-benzyloxy-4-O-(4-methoxybenzyloxy)-D-glucar as a white solid in 84% yield.

[0188] δ 1 H(400MHz;CDCl3) 7.34-7.20 (7H, m, H arom ), 6.83(2H, dt, J8.7, J4.8, 9-H), 6.34(1H, dd, J6.1, J1.2, 1-H), 4.82(1H, dd, J6.1, J2.6, 2-H), 4.75(1H, d, J11.1, 10-H CH2Ph), 4.63(1H, d, J11.1, 10-H CH2Ph), 4.61(1H, d, J11.8, 7-H CH2Ph(4-OMe)), 4.52(1H, d, J11.8, 7-H CH2Ph(4-OMe)), 4.19(1H, ddd, J6.3, J2.4, J2.3, 3-H), 3.87(1H, dt, J8.8, J4.2, 5-H), 3. 81-3.79(2H, m, 6-H), 3.77(1H, dd, J8.7, J6.3, 4-H), 3.71(3H, s, -OMe), 2.65(1H, s, -OH).

[0189] δ 13 C(100MHz;CDCl3) 159.2(11-C), 144.4(1-C), 138.1(13-C), 130.1(8-C), 129.7-127.6(C arom9, 14, 15, 16-C), 113.7(10-C), 100.1(2-C), 77.5(5-C), 75.6(3-C), 74.1(4-C), 73.3(12-C), 70.4(7-C), 61.4(6-C), 55.1(-OMe).

[0190] 1,5-Anhydro-3-O-benzyloxy-4-O-(4-methoxybenzyloxy)-2,6,7-Trideoxy-D-arabinohepto-1,6-dienitol(28) [ka] DMP (926 mg, 2.18 mmol) was added to a 6.1 mL dry DCM solution of the aforementioned alcohol 26 (650 mg, 1.82 mmol). The mixture was then stirred at room temperature (25°C) for 1 hour.

[0191] Meanwhile, an ylide was prepared with a dry THF solution (12.0 mL) of fresh PPh3CH3I (1.48 g, 3.65 mmol) at -78°C and stirred for 25 minutes. Next, KHMDS (7.3 mL, 3.65 mmol, 0.5 M / toluene solution) was added dropwise at -78°C. The mixture was stirred at -78°C for 20 minutes, at 0°C for 50 minutes, and finally at -78°C for 30 minutes to form the ylide.

[0192] Furthermore, the oxidation reaction was stopped for 10 minutes with a solution of Na2S2O3 (30 mL) and NaHCO3 (30 mL). Next, the aldehyde was work-treated with DCM (3 times with 40 mL), dried with Na2SO4, and the DCM was removed under reduced pressure.

[0193] Next, aldehyde (11.0 mL) in dry THF was added dropwise to the ylide at -78°C. The reaction mixture was stirred overnight. The mixture was treated with NH4Cl (20 mL) and DCM (50 mL). The organic layer was then extracted again with DCM (twice with 30 mL), washed with NaCl (80 mL), and dried over Na2SO4. The residue was purified by flash chromatography (nHexane / Ã=7:3) to obtain the alkene as a yellow oil in two steps with a yield of 83%.

[0194] δ 1 H (400 MHz; CDCl3) 7.37 - 7.27 (4H, m, H arom )、7.24 (2H, dt, J 8.6, J 5.5, 9 - H)、6.86 (2H, td, J 8.7, J 5.5, 10 - H)、6.41 (1H, dd, J 6.1, J 1.3, 1 - H)、6.04 (1H, ddd, J 17.2, J 10.6, J 6.6, 6 - H)、5.43 (1H, dt, J 2.9, J 17.3, 7b - H)、5.31 (1H, dt, J 2.6, J 10.6, 7a - H)、4.88 (1H, dd, J 6.2, J 2.7, 2 - H)、4.70 (1H, d, J 10.9, 11 - H, CH2Ph)、4.64 (1H, d, J 11.7, 8 - H, CH2Ph(4 - OMe))、4.62 (1H, d, J 10.9, 11 - H CH2Ph)、4.58 (1H, d, J 11.7, 8 - H CH2Ph(4 - OMe))、4.31 (1H, dd, J 7.1, J 8.0, 5 - H)、4.19 (1H, ddd, J 6.2, J 2.5, J 1.5, 3 - H)、3.79 (3H, s, - OMe)、3.59 (1H, dd, J 8.6, J 第6.2, 4 - H)。

[0195] δ<好的 13 C (100 MHz; CDCl3) 159.4 (12 - C)、144.6 (1 - C)、138.5 (14 - C)、134.5 (6 - C)、130.3 (9 - C)、129.8 - 127.8 (C arom 10、15、16、17 - C)、118.4 (7 - C)、113.9 (11 - C)、100.5 (2 - C)、78.2 (5 - C)、78.0 (4 - C)、75.5 (3 - C)、73.6 (8 - C)、70.8 (13 - C)、55.4 (- OMe)。

[0196] (3R, 4R, 5R) - 4 - O - Benzyloxy - 3 - O - (4 - methoxybenzyloxy) - 5 - (hydroxymethyl) cyclohexene (29)

Chemical Structure

[0197] δ 1 H(400MHz;CDCl3) 7.28-7.16 (7H, m, H arom ), 6.79(2H, brd, J8.3, 14-H), 5.67-5.64(1H, m, 1-H), 5.64-5.59(1H, m, 2-H), 4.88(1H, d, J11.3, 8-H CH2Ph), 4.64(1H, d, J11.3, 8-H CH2Ph), 4.56(1H, d, J11.2, 12-H CH2Ph(4-OMe)), 4.48(1H, d, J11.7, 12-H CH2Ph(4-OMe)), 4.12(1H, brd, 4-H), 3.71(3H, s, -OMe), 3.57-3.47(3H, m, 3-H, 6-H), 2. 35(1H, s, -OH), 2.07-2.00(1H, m, 7-H), 1.97-1.88(1H, m, 5-H), 1.82-1.75(1H, m, 7-H)δ 13 C (100MHz; CDCl3).

[0198] δ 13 C(100MHz;CDCl3) 159.4(17-C), 138.5(9-C), 132.1(14-C), 130.5-128.0(C arom10, 11, 12, 15-C), 127.7(1-C), 126.1(2-C), 114.0(16-C), 82.3(3-C), 80.9(4) -C), 74.4(8-C), 71.1(13-C), 65.9(6-C), 55.4(-OMe), 40.7(5-C), 28.1(7-C).

[0199] 4-O-benzyl-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5-methylcyclohexene(30) [ka] Alcohol 29 (715 mg, 2.02 mmol) was dissolved in dry THF (17 mL) at room temperature. Imidazole (125 mg, 1.83 mmol) was added, and the mixture was stirred at room temperature for 5 minutes, then at 0°C for 10 minutes. Next, texyldimethylsilyl chloride (1.19 mL, 6.05 mmol) was carefully added dropwise to form a white precipitate. The ice bath was removed at the time of the first precipitate, and the remaining TDSCl was slowly added to the mixture. The mixture was heated to room temperature and stirred overnight. The reaction was monitored by TLC (Pent / AcOEt3:1). The organic phase was extracted with ethyl acetate and then washed with distilled water (5 times). The residue was purified by flash chromatography (nHex / AcOEt95:5) to form compound 30 as a yellow oil in quantitative yield.

[0200] δ 1 H(400MHz;CDCl3) 7.37-7.16 (7H, m, H arom), 6.88-6.84(2H, m, 14-H), 5.75(1H, ddq, J9.0, J4.3, J2.4, 1-H), 5.64(1H, brd, 2-H), 4.91(1H, d, J11.0, 8-H CH2Ph), 4.68(1H, d, J11.0, 8-H CH2Ph), 4.64(1H, d, J11.3, 12-H CH2Ph(4-OMe)), 4.60(1H, d, J11.3, 12-H CH2Ph(4-OMe)), 4.16(1H, ddq, J7.1, J3.6, J1.8, 3-H), 3.86(1H, dd, J9.8, J4.8, 6-H), 3 .79(3H, s, -OMe), 3.64(1H, dd, J10.0, J6.6, 4-H), 3.63-3.58(1H, m, 6-H), 2.28-2.16(1 H, m, 7-H), 2.10 (1H, dt, J18.4, J5.3, 7-H), 1.91 (1H, ttd, J10.5, J5.1, J2.7, 5-H), 1.64 (1H, hept, J6.9, 17-H), 0.90 (6H, d, J6.9, 18-H), 0.87 (6H, s, 16-H), 0.13 (6H, s, 15-H).

[0201] δ 13 C(100MHz;CDCl3) 159.3(14-C), 139.3(9-C), 133.8(17-C), 131.0-128.0(C arom 10, 11, 12, 15-C), 127.6(1-C), 126.3(2-C), 113.9(16-C), 81.5(3-C), 79.7(4-C), 74.7(8-C), 71.5(13-C), 62.6(6-C), 5 5.4(-OMe), 41.4(5-C), 34.3(21-C), 28.7(7-C), 25.3(19-C), 20.5-20.3(20-C), 18.8-18.7(22-C), -3.27--3.46(18-C).

[0202] 4-O-benzyl-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-glucopyranose(31) [ka] Compound 30 (230 mg, 0.46 mmol) was dissolved in a mixture of acetone (1.69 mL) and water (562 μL). A solution of OsO4 (4.5 mL of H2O and 537 μL of a solution of 250 mg of OsO4 dissolved in 18 mL of acetone) and TMANO (116 mg, 1.02 mmol) was added at room temperature. The reaction was carried out at 25 °C for 48 hours. A saturated aqueous solution of Na2S2O3 (2 mL) was added, and the mixture was stirred at room temperature to reduce OsO4. The organic phase was extracted with CHCl3 (15 mL), washed with brine (10 mL), and finally dried over Na2SO4. The crude product was purified by flash chromatography (nHex / AcOEt, 8.2) to form diol 31 as a colorless oil in 77% yield.

[0203] δ 1 H(400MHz;CDCl3) 7.37-7.15 (7H, m, H arom ), 6.87(2H, brd, J8.7, 14-H), 4.90(1H, d, J12, 8-H CH2Ph), 4.88(1H, d, J8, 12-H CH2Ph(4-OMe)), 4.69(1H, d, J10.9, 8-H CH2Ph), 4.61(1H, d, J11.1, 12-H CH2Ph(4-OMe)), 4.05(1H, brd, J2.7, 1-H), 3.96(1H, dd, J10.0, J3.3, 6-H), 3.78(3H, s, -OMe), 3.7 1(1H, t, J9.4, 3-H), 3.48(2H, t, J10.0, 6-H, 4-H), 3.43(1H, dd, J2.3, J9.4, 2-H), 2.64(1H, s, -OH) , 2.58 (1H, s, -OH), 2.09-2.03 (1H, m, 5-H), 1.77 (1H, dt, J14.5, J3.6, 7-H), 1.62 (1H, hept, J6.9, 1 7-H), 1.59-1.52(1H, m, 7-H), 0.88(6H, d, J6.9, 18-H), 0.85(6H, d, d1.2, 16-H), 0.07(6H, s, 15-H).

[0204] δ 13 C(100MHz;CDCl3) 159.5(14-C), 138.9(9-C), 130.9(17-C), 129.7-127.7(C arom 10, 11, 12, 15-C), 114.2(16-C), 83.4(3-C), 81.0(4-C), 75.1(13-C), 74.9(8-C), 74.6(2-C), 68.5(1-C), 62.1(6-C), 55 .3(-OMe), 38.9(5-C), 34.3(21-C), 30.4(7-C), 25.2(19-C), 20.5-20.4(20-C), 18.8-18.7(22-C), -3.35--3.56(18-C).

[0205] 1-O-acetyl-4-O-benzyl-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-glucopyranose(32) [ka] Compound 31 (155 mg, 0.29 mmol) was dissolved in acetonitrile (2.9 mL) under nitrogen at room temperature. Trimethyl orthoacetate (115 μL, 0.88 mmol) and PTSA (5 mg, 0.03 mmol) were added sequentially to the mixture, and the mixture was stirred under nitrogen at room temperature for 60 minutes. After the reaction was complete, an 80% AcOH solution (AcOH 2.32 mL + H2O 0.58 mL) was added. The subsequent acetylation reaction was completely completed in 60 minutes. The organic phase was extracted with DCM (5 mL), washed with water (5 mL) and NaHCO3 (5 mL), and finally dried over Na2SO4. The residue was purified by flash chromatography (nHex / AcOEt) to obtain compound 32, selectively acetylated at the pseudoanomeric position, as a colorless oil in quantitative yield.

[0206] δ 1 H(400MHz;CDCl3) 7.39-7.13 (7H, m, H arom)、6.87(2H、dt、J8.7、J5.0、14-H)、5.26(1H、dd、J5.7、J3.0、1-H)、4.91(1H、d、J10.6、8-H CH2Ph)、4.90(1H、d、J10.9、12-H CH2Ph(4-OMe))、4.70(1H、d、J10.0、8-H CH2Ph)、4.68(1H、d、J10.5、12-H CH2Ph(4-OMe))、3.95(1H、dd、J10.0、J3.5、6-H)、3.80(3H、s、-OMe)、3.75(1H、t、J9.3、3-H)、3.58(1H、brd、J9.6、2-H)、3.53(1H、dd、J9.1、J10.1、4-H)、3.50(1H、dd、J9.8、J2.4、6-H)、2.28(1H、s、-OH)、2.08(3H、s、-OAc)、1.95-1.88(1H、m、5-H)、1.85(1H、dt、J14.8、J7.6、7-H)、1.61(1H、dt、J13.8、J6.9、7-H)、1.61(1H、hept、J6.9、17-H)、0.88(6H、d、J6.8、18-H)、0.84(6H、d、J1.7、16-H)、0.07(6H、d、J4.4、15-H)。

[0207] δ 13 C(100MHz;CDCl3) 170.9(C(O)、-OAc)、159.5(14-C)、138.7(9-C)、130.8(17-C)、129.8-127.9(C arom 10、11、12、15-C)、114.8(16-C)、84.0(3-C)、80.5(4-C)、75.4(13-C)、75.3(8-C)、73.4(2-C)、71.8(1-C)、61.8(6-C)、55.4(-OMe)、39.6(5-C)、34.3(21-C)、28.8(7-C)、25.3(19-C)、21.4(CH3、-OAc)、20.5-20.4(20-C)、18.8-18.7(22-C)、-3.28--3.53(18-C)。

[0208] 1-O-acetyl-2-azide-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-mannopyranose [ka] Compound 32 (220 mg, 0.38 mmol) was dissolved in a mixture of DCM / pyridine (5:1, 0.05 M) and stirred at 10°C for 10 minutes under nitrogen. Triflate anhydrous (355 μL, 2.11 mmol) was added dropwise at -10°C. The mixture was stirred sequentially for 30 minutes to slowly reach 0°C, and then stirred at 0°C for another 30 minutes. After the reaction was complete, the organic phase was washed with NaHCO3 and brine. The organic layer was dried over Na2SO4, and the resulting crude product was used directly in the next step after co-distillation with toluene (3 times). Next, the dried crude product was dissolved in DMF / H2O (19:1, 0.2 M) at 40°C. Sodium azide (125 mg, 1.92 mmol) and 15-crown-5 (15.2 μL, 0.08 mmol) were added at room temperature, and the reaction was carried out overnight at 40°C. After the triflate intermediate had completely disappeared, the solvent was removed by distillation, and the residue was finally purified by flash chromatography (nHex / siRNA) to form the title compound azide as a colorless oil in 82% yield.

[0209] δ 1 H(400MHz;CDCl3) 7.38-7.14 (7H, m, H arom), 6.86(2H, dt, J8.6, J4.9, 14-H), 4.98-4.94(1H, m, 1-H), 4.88(1H, d, J10.7, 8-H CH2Ph), 4.66(1H, d, J19.1, 12-H CH2Ph(4-OMe)), 4.63(1H, d, J19.5, 12-H CH2Ph(4-OMe)), 4.59(1H, d, J10.9, 8-H CH2Ph), 3.87-3.84(1H, m, 2-H), 3.84(1H, dd, J6.3, J2.7, 6-H), 3.80(3H, s, -OMe), 3.82-3.75(2H, m, 4-H, 3-H), 3.52(1H, dd, J9.9, J2.1, 6-H), 2.00 (3H, s, -OAc), 1.91-1.82(2H, m, 5-H, 7-H), 1.65-1.57(2H, m, 7-H, 17-H), 0 .89(6H, d, J6.9, 18-H), 0.85(6H, d, J1.2, 16-H), 0.07(6H, d, J4.1, 15-H).

[0210] δ 13 C(100MHz;CDCl3) 169.8(C(O), -OAc), 159.6(14-C), 138.9(9-C), 130.2(17-C), 129.8-127.8(C arom 10, 11, 12, 15-C), 114.0(16-C), 81.1(4-C), 77.0(3-C), 75.4(8-C), 72.9(13-C), 70.6(1-C), 62.2(6-C), 61.4(2-C), 55.4(-OMe) , 39.8(5-C), 34.4(21-C), 27.1(7-C), 25.3(19-C), 21.2(CH3, -OAc), 20.6-20.5(20-C), 18.8-18.7(22-C), -3.35--3.52(18-C).

[0211] 1-O-acetyl-2-acetamide-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-mannopyranose(33) [ka] To the mixture of azide (334 mg, 0.56 mmol) described above, PPh3 (366 mg, 1.40 mmol) and a catalytic amount of pyridine (13.6 μL, 0.17 mmol) were added, and the mixture was stirred at 60°C for 24 hours. After the starting materials had disappeared, the solvent was removed from the resulting amine by distillation, and then it was dissolved in pyridine (5.6 mL). Acetyl anhydride (1.06 mL, 11.2 mmol) was added, and the solution was stirred again for 24 hours. The crude product was purified by flash chromatography (nHex / AcOEt) to obtain acetamide 33 as a yellow oil in 75% yield.

[0212] δ 1 H(400MHz;CDCl3) 7.39-7.28 (5H, m, H arom ), 7.19(2H, dt, J9.4, J4.6, 13-H), 6.86(2H, dt, J9.4, J4.8, 14-H), 5.59( 1H, d, J8.1, NHAc), 5.12 (1H, td, J7.2, J3.9, 1-H), 4.71 (1H, d, J11.3, 8-H CH2Ph), 4.56(1H, d, J11.3, 8-H CH2Ph), 4.50(1H, d, J11.2, 12-H CH2Ph(4-OMe)), 4.42(1H, td, J7.7, J4.1, 2-H), 4.36(1H, d, J11.2, 12-H CH2Ph(4-OMe)), 3.84(1H, dd, J2.4, J4.0, 3-H), 3.85-3.82(1H, m, 6-H), 3.80(3H, s, -OM e), 3.72(1H, t, J6.3, 4-H), 3.60(1H, dd, J9.9, J5.5, 6-H), 2.09-2.02(1H, m, 5-H), 2.01( 3H, s, -OAc), 1.90 (3H, s, -NHAc), 1.82 (2H, tdd, J14.2, J7.4, J4.6, 7-H), 1.66-1.57 (1H, hept, J6.9, 17-H), 0.89 (6H, d, J6.9, 18-H), 0.84 (6H, s, 16-H), 0.08 (6H, d, J6.2, 15-H).

[0213] δ 13 C(100MHz;CDCl3) 170.7(C(O), -NHAc), 170.1(C(O), -OAc), 159.6(14-C), 138.6(9-C), 130.0(15-C), 129.9(17-C), 128.6-127.8(C arom 10, 11, 12-C), 114.1(16-C), 78.7(3-C), 74.4(4-C), 73.6(8-C), 71.9(13-C), 69.6(1-C), 62.5(6-C), 55.4(-OMe), 50.6(2-C), 39. 9(5-C), 34.4(21-C), 27.1(7-C), 25.2(19-C), 23.5(CH3, -NHAc), 21.3(CH3, -OAc), 20.5(20-C), 18.8(22-C), -3.37--3.48(18-C).

[0214] 1-O-Terbutylsilyl-2-acetamide-4-O-benzyl-2-deoxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carb-α-D-mannopyranose(35) [ka] Compound 33 (582 mg, 0.95 mmol) was dissolved in MeOH (9.5 mL). NaOMe (11 mg, 0.2 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. Amberlite H was added until a neutral pH was reached. + An ion exchange resin was added. The suspension was filtered and concentrated under reduced pressure. The crude product was removed three times by co-distillation with toluene.

[0215] Under a flow of N2 gas, 4 mL of 34 (0.95 mmol) of DCM solution was placed in a flask. At 0°C, 2,6-lutidine (2.37 mmol), followed by TBSOTf (437 μL, 1.9 mmol) was added dropwise. The mixture was stirred and heated to room temperature. After completion, the reaction was cooled to room temperature, the reaction was stopped with MeOH, and the mixture was diluted with chloroform. The mixture was washed with 10% CuSO4 aqueous solution (twice), H2O, and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (nHex / Â) yielded the title compound 35 as an orange-red oil in 83% yield in two steps.

[0216] JDC Codee et al., J. Org. Chem, 2017, 82, 2, 848-868

[0217] δ 1 H(400MHz;CDCl3) 7.41-7.24 (5H, m, H arom)、7.19(2H、dt、J9.5、J4.6、13-H)、6.86(2H、dt、J9.4、J4.8、14-H)、5.57(1H、d、J5.7、NHAc)、4.93(1H、d、J10.6、8-H CH2Ph)、4.58(1H、d、J10.5、8-H CH2Ph)、4.56(1H、d、J11.1、12-H CH2Ph(4-OMe))、4.48(1H、d、J11.1、12-H CH2Ph(4-OMe))、4.27(1H、dd、J5.2、J2.3、2-H)、4.25-4.21(1H、m、1-H)、4.03(1H、dd、J9.6、J4.5、3-H)、3.97(1H、dd、J9.7、J3.6、6-H)、3.81(3H、s、-OMe)、3.54(1H、t、J9.9、4-H)、3.48(1H、dd、J9.7、J2.2、6-H)、2.09-2.02(1H、m、5-H)、2.01(3H、s、-NHAc)、1.78-1.69(1H、m、7-H)、1.69-1.59(1H、m、17-H)、1.52-1.45(1H、m、7-H)、0.93(6H、d、J6.9、18-H)、0.87(6H、s、16-H)、0.86(6H、s、20-H)、0.84(6H、s、16-H)、0.12(6H、d、J12.0、19-H)、0.09(6H、d、J9.4、15-H)。

[0218] δ 13 C(100MHz;CDCl3) 170.7(C(O)、-NHAc)、159.5(14-C)、139.1(9-C)、130.2(17-C)、130.0(15-C)、128.6-127.7(C arom 10、11、12-C)、114.0(16-C)、78.5(3-C)、77.6(4-C)、75.5(8-C)、71.4(13-C)、67.7(2-C)、62.6(6-C)、55.4(-OMe)、53.4(1-C)、38.6(5-C)、34.6(21-C)、30.4(7-C)、25.9(25-C)、25.2(19-C)、23.6(CH3-NHAc)、20.7-20.6(20-C)、18.9-18.8(22-C)、18.0(24-C)、-3.37--3.58(18-C)、-4.82--4.92(23-NS)。

[0219] 1-O-tert-butylsilyl-2-acetamide-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carb-α-D-mannopyranose [ka] A 3.4 mL solution of 14 (71 mg, 0.10 mmol) in DCM was cooled to 0°C, and freshly prepared phosphate buffer (362 μL, pH 7.5, 10 mM) was added. Freshly prepared DDQ (50.0 mg, 0.22 mmol) was added in small amounts over 1 hour, and the mixture was then warmed to room temperature and stirred for 30 minutes. The mixture was diluted with NaHCO3, and the aqueous layer was extracted twice with DCM. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. By purification by column chromatography (nHex / siRNA), compound 15 was obtained as an orange-red solid in 72% yield.

[0220] Dan Van Der Es, Thesis, 2016, Universiteit Leiden, pp160.

[0221] δ 1 H(400MHz;CDCl3) 7.41-7.27 (5H, m, H arom), 5.52 (1H, d, J5.4, NHAc), 4.73 (2H, s, 8-H CH2Ph), 4.26 (1H, brd, J2.7, 1-H), 4.16 (1H, dt, J9.0, J3.8, 3-H), 4.06 (1H, dd, J9.0, J4.5, 2-H), 3.94 (1H) , dd, J9.9, J3.7, 6-H), 3.53 (1H, dd, J10.0, J2.1, 6-H), 3.46 (1H, t, J9.5, 4-H), 2.73 (1H, s, -OH), 2.10-2.0 3(1H, m, 5-H), 2.00(3H, s, -NHAc), 1.81-1.69(1H, m, 7-H), 1.69-1.59(1H, m, 14-H), 1.51(1H, dt, J13.7, J 3.2, 7-H), 0.93 (6H, d, J6.9, 15-H), 0.88 (6H, s, 17-H), 0.87 (6H, s, 13-H), 0.14-0.04 (12H, m, 16-H, 12-H).

[0222] δ 13 C(100MHz;CDCl3) 170.1(C(O), -NHAc), 138.8(9-C), 128.7-127.7(C arom 10, 11, 12-C), 79.6(4-C), 74.8(8-C), 70.7(3-C), 67.6(1-C), 62.9(6-C), 56.5(2-C), 38.5(5-C), 34.6(16-C), 31.2(7-C), 25.9(20) -C), 25.3(14-C), 23.6(CH3, -NHAc), 20.7-20.6(15-C), 18.9-18.8(17-C), 18.0(19-C), -3.37--3.53(13-C), -4.80--4.90(18-C).

[0223] 1-O-tert-butylsilyl-2-acetamide-4-O-benzyloxy-6-O-texyldimethylsilyl-5a-carb-α-D-mannopyranose(36) [ka] The alcohol (180 mg, 0.32 mmol) indicated above was dissolved in dry DCM (3.2 mL) at room temperature under nitrogen. Pyridine (257 μL, 3.18 mmol), acetic anhydride (601 μL, 6.36 mmol), and a catalytic amount of DMAP (7.8 mg, 0.06 mmol) were added in that order, and the mixture was stirred until the reaction was complete. The solution was stopped with MeOH and then concentrated under reduced pressure. Compound 36 was formed in quantitative yield as a yellow oil by flash chromatography (nHex / Â).

[0224] δ 1 H(400MHz;CDCl3) 7.37-7.13 (5H, m, H arom ), 5.44(1H, dd, J10.3, J4.5, 3-H), 5.27(1H, d, J7.4, NHAc), 4.70(2H, d, J10.9, 8-H CH2Ph), 4.61(1H, d, J10.9, 8-H CH2Ph), 4.31(1H, dt, J7.3, J3.8, 2-H), 4.10(1H, brd, J2.7, 1-H), 3.97(1H, dd, J9.8, J3.2, 6-H), 3.61( 1H, t, J10.3, 4-H), 3.46 (1H, dd, J9.8, J2.0, 6-H), 2.18-2.11 (1H, m, 5-H), 2.00 (3H, s, -NHAc), 1.98 (3H, s, -OAc), 1.79-1.70(1H, m, 7-H), 1.70-1.61(1H, m, 14-H), 1.52(1H, dt, J14.3, J2.8, 7-H), 0.95(6H, d, J6.9, 15-H), 0.90 (6H, s, 17-H), 0.88 (6H, s, 13-H), 0.13 (6H, d, J15.1, 16-H), 0.09 (6H, d, J14.8, 12-H).

[0225] δ 13 C(100MHz;CDCl3) 170.0(C(O), -NHAc), 169.8(C(O), -OAc), 138.7(9-C), 128.6-127.6(C arom10, 11, 12-C), 76.2(4-C), 75.1(8-C), 73.2(3-C), 68.1(1-C), 62.3(6 -C), 54.0(2-C), 38.7(5-C), 34.6(16-C), 30.6(7-C), 25.8(20-C), 25. 3(14-C), 23.6(CH3, -NHAc), 21.2(CH3, -OAc), 20.7-20.6(15-C), 19.0-18.9(17-C), 18.1(19-C), -3.41--3.62(13-C), -4.90--4.99(18-C).

[0226] 2-Acetamide-4-O-benzyloxy-5a-carba-α-D-mannopyranose(37) [ka] Compound 36 (120 mg, 0.20 mmol) was dissolved in dry THF (2.0 mL) at 0°C. A 30% HF / Py solution (420 μL) was added dropwise, and the reaction mixture was slowly heated from 0°C to room temperature while stirring overnight. Next, the reaction was stopped with NaHCO3 (3 mL). The organic layer was extracted twice with siRNA, washed with brine, and dried over Na2SO4. The resulting crude compound 37 was filtered through silica to obtain a white solid in 60% yield.

[0227] δ 1 H (400MHz; CD3OD) 7.37-7.26 (5H, m, H arom ), 5.33(1H, dd, J8.4, J4.4, 3-H), 4.72(2H, d, J11.4, 8-H CH2Ph), 4.66(1H, d, J11.4, 8-H CH2Ph), 4.45(1H, t, J4.8, 2-H), 4.10(1H, brd, J2.7, 1-H), 3.87(1H, q, J4.5, 1-H), 3.78-3.73(2H, m, 4-H, 6-H), 3.68(1H, dd, J10.6, J4.2 , 6-H), 2.17-2.09(1H, m, 5-H), 2.04(1H, s, -OH), 2.03(1H, s-OH), 2.02(3H, s, -NHAc), 1.98(3H, s, -OAc), 1.83(2H, dd, J7.8, J3.8, 7-H).

[0228] δ 13 C(100MHz;CDCl3) 173.6(C(O), -NHAc), 172.0(C(O), -OAc), 140.0(9-C), 129.3-128.6(C arom 10, 11, 12-C), 77.2(4-C), 74.9(8-C), 74.7(3-C), 68.2(1-C), 63.1(6-C) , 54.0(2-C), 40.7(5-C), 30.9(7-C), 22.5(CH3, -NHAc), 21.1(CH3, -OAc).

[0229] 2-Acetamide-4-O-benzyloxy-6-O-dimethoxytrityl-5a-carba-α-D-mannopyranose(38) [ka] Compound 37 (15 mg, 42.7 μmol) was dissolved in dry DCM under nitrogen at room temperature. Dry pyridine (5.2 μL, 64.0 μmol) and DMTrCl (217 mg, 64.0 μmol) were added sequentially, and the mixture was stirred at room temperature for 3 hours. Next, H2O was added to the reaction product. The organic layer was washed once with brine, dried over Na2SO4, and concentrated under reduced pressure. By purification by flash chromatography (nHex / AcOEt, 0.1% TEA), compound 38 was obtained as a white solid in 74% yield.

[0230] δ 1 H (400MHz; CD3OD) 7.40-7.05 (14H, m, H) arom), 6.79(4H, dd, J8.9, J1.7, 13-H), 5.24(1H, dd, J7.9, J4.3, 3-H), 4.53(1H, d, J11.3, 8-H CH2Ph), 4.38(1H, t, J4.8, 2-H), 4.31(1H, d, J11.3, 8-H CH2Ph), 3.79(1H, q, J5.2, 1-H), 3.72(3H, s, -OMe), 3.72(3H, s, -OMe), 3.61(1H, t, J8.1, 4-H), 3.34-3.26(1H, m, 6-H), 3.05(1H , t, J8.3, 6-H), 2.34-2.24(1H, m, 5-H), 2.08-1.98(1H, m, 7-H), 1.95(3H, s, -NHAc), 1.86(3H, s, -OAc), 1.85-1.79(1H, m, 7-H).

[0231] δ 13 C (100MHz; CD3OD) 173.6(C(O), -NHAc), 172.0(C(O), -OAc), 160.0(17-C), 146.7(9-C), 137.6(14-C), 137.5(14-C), 137.3(9-C), 131.4(18-C), 129.9-126.3(C arom 10, 11, 12, 15, 19, 20, 21-C), 114.0(16-C), 87.2(13-C), 77.3(4-C), 74.5(3-C), 74.4(8-C), 68.0(1- C), 65.0(6-C), 55.7(-OMe), 54.1(2-C), 39.2(5-C), 31.9(7-C), 22.5(CH3, -NHAc), 21.1(CH3, -OAc).

[0232] Example: Non-acetylated oligomeric conjugate - CRM 197 -MenA DP6 (without OAC) and CRM 197 - Preparation of MenA DP8 (without OAc) The starting oligomers (DP6 and DP8) were vacuum-dried and dissolved in a 1:9 H2O:DMSO solution to a final amino group concentration of 40 mmol / mL. These were then reacted with a 12-fold molar excess of di-N-hydroxysuccinimidyl adipic acid linker (SIDEA) in the presence of triethylamine in a 5-fold molar excess compared to the amino groups. The reaction was maintained at room temperature for 3 hours with gentle stirring. The activated oligosaccharides were precipitated with 4 times the volume of ethyl acetate, and then purified by washing the pellet 10 times with 1 mL of the same solvent. Finally, the pellet was vacuum-dried, and the content of the introduced N-hydroxysuccinimidyl ester groups was determined.

[0233] The conjugate was prepared using an active ester (AE) to protein molar ratio of 40:1 in 50 mM NaH2PO4 pH 7 and allowed to stand overnight at room temperature with gentle stirring. The conjugate was purified by tangential flow filtration (Vivaspin) using a 30 kDa cutoff and PBS pH 7.2 as the buffer. The conjugate was prepared using SDS-page, and the total protein content was measured using microBCA. 2 The total sugar content was then characterized using MALDI analysis.

[0234] Sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-Page). SDS-Page was performed on precast 3-8% polyacrylamide gels (NuPAGE® Invitrogen). For electrophoresis, 5 μg of protein was loaded for each sample and run for approximately 40 minutes in Tris-acetate SDS running buffer (NuPAGE® Invitrogen) using an electrophoresis chamber at 150 V. Samples were prepared by adding 3 μL of NuPAGE® LDS sample buffer. After electrophoresis, the gels were washed three times with H2O and stained with Coomassie.

[0235] Example 2: Preparation of the oligomer conjugate of the present invention according to formula (IIa) The randomly produced O-acetylated carba analogs were activated with di-N-hydroxysuccinimidyl adipate relinker (SIDEA), and the activation rates (%) obtained for the oligosaccharides were estimated to be 56% for DP6OAc, 79% for DP7OAc, and 84% for DP8OAc.

[0236] Activated oligosaccharides (i.e., activated O-acetylated carba analogs) were freeze-dried and prepared for the conjugation process. Conjugates were obtained by applying the chemistry reported in Figure 4 and the same figure showing the SDS-Page characterization, and the smear of the conjugates could be observed.

[0237] As shown in Table 2, purified complex carbohydrates (i.e., those containing random O-acetylated carba analogs) were characterized in terms of protein content using MicroBCA and sugar content using HPAEC-PAD.

[0238] [Table 2]

[0239] Mouse immunization and in vitro analysis of antibody responses using ELISA and serum bactericidal assays (rSBA and hSBA) Antigen preparations were prepared under sterile conditions. Ten mice (BALB / c) were immunized on days 1, 14, and 28. Blood samples were collected on day 0 (pre-immunization), day 27 (post-immunization 2), and day 42 (post-immunization 3). The vaccine was administered via saccharide dose, with a carbohydrate dose of 2 μg / mouse / dose. The adjuvant AlPO4 was added in a dose of 0.12 mg of Al. 3+ It was used in this dosage.

[0240] The vaccine formulations used in the carbaMenA conjugate were as follows: 324.96 μL of AlPO4 (4.43 mg / mL including 2 mg / mL NaCl) was added to the target conjugate. By adding PBS buffer at pH 7.2, the AlPO4 concentration was reduced to 1.2 mL and the volume to 1.2 mL. Finally, the solution was diluted 1:1 (volume ratio) with PBS to 2.4 mL, resulting in a final AlPO4 concentration of 0.6 mg / mL. 200 μL of the formulation was injected per mouse. This procedure was repeated for MenA-CRM from stock solution. 197 It was also used in the formulation of [another drug].

[0241] Serum ELISA. Antibody responses induced by complex carbohydrates are measured by ELISA. In this analysis, preimmune serum was used as a negative control. 100 μL / well of a 5 μg / mL polysaccharide solution in pH 8.2 PBS buffer was added to the plate, and then incubated overnight at 4°C to obtain HSA-DeOAc (reference). 21 (Manufactured according to the method described in) or coated with MenA CPS. HSA-DeOAc MenA CPS, CRM 197 Conjugate and CRM 197The antibodies were coated with pH 7.2 PBS buffer at a protein concentration of 2 μg / mL. The coating solution was removed from the plate by washing three times with PBS buffer (TPBS) containing 0.05% Tween 20 (Sigma). Next, a blocking step was performed by adding 100 μL / well of 3% BSA / TPBS solution and incubating the plate at 37°C for 1 hour. The blocking solution was removed from the plate by washing three times with TPBS. 200 μL / well of pre-diluted serum (1:25 for pre-immuno-negative control, 1:200-1:500 for reference serum, and 1:25-1:200 for test serum) was added to the first well of each row of the plate, and 100 μL of TPBS was dispensed into the other wells. Next, eight consecutive 2-fold dilutions were performed along each row by transferring 100 μL of serum solution from well to well. After primary antibody dilution, the plate was incubated at 37°C for 2 hours. The plates were washed three times with TPBS, and 100 μL / well of a TPBS solution containing a secondary antibody alkaline phosphatase conjugate (anti-mouse IgG1:10000, Sigma-Aldrich) was added. The plates were incubated at 37°C for 1 hour. After three further washes with TPBS, 100 μL / well of 1 mg / mL p-NPP(Sigma) in 0.5 M diethanolamine buffer pH 9.6 was added. Finally, the plates were incubated at room temperature for 30 minutes and read at 405 nm using a Spectramax 190 plate reader. Serum titer was expressed as the reciprocal of the serum dilution corresponding to a cutoff OD=1.

[0242] Each immunization group is expressed as the geometric mean (GMT) at the 95% confidence interval of the single mouse titer. Statistical and graphical analyses were performed using GraphPad Prism7 software.

[0243] Immunological evaluation To investigate the immunogenicity of conjugated carba DP6 and DP8 analogs with and without random acetylation, a group of eight BALB / c female mice were immunized with neocomplex carbohydrates. Conjugated, size-matched MenA polysaccharides were used as controls. Mice were immunized with three subcutaneous (sc) doses (2 μg carbohydrate-based) at 2-week intervals. When the anti-MenA CPS response was evaluated, the data showed no response to conjugates obtained with carba MenA glycoantigens without O-acetylation, for both glycan lengths 6 (n=6) and 8 (n=8). Conversely, carba MenA conjugates obtained after random O-acetylation of oligomers induced a significantly higher response to natural MenA CPS compared to the non-acetylated vaccine (Table 3 and Figure 5). In comparison, the response induced by the O-acetylated vaccine was better than that of the MenA-CRM benchmark for DP8, which gave a better response among the vaccines examined. 197 It was only twice as low as conjugate.

[0244] The vaccine formulations used in the CarbaMenA conjugate were as follows: 324.96 μL of AlPO4 (4.43 mg / mL including 2 mg / mL NaCl) was added to the target conjugate. By adding PBS buffer at pH 7.2, the AlPO4 concentration was reduced to 1.2 mL and the volume to 1.2 mL. Finally, the solution was diluted 1:1 (volume ratio) with PBS to 2.4 mL, resulting in a final AlPO4 concentration of 0.6 mg / mL. 200 μL of the formulation was injected per mouse. This procedure was used for MenA-CRM from stock solution. 197 It was also used in the formulation of [another drug].

[0245] Table 3 reports the ELISA responses after two and three administrations. As can be seen from Table 3, groups 2 and 3 are those according to the present invention. For group 2, n=6 and oligomeric conjugates with random acetylation as described above were used. For group 3, n=8 and oligomeric conjugates with random acetylation as described above were used. The acetylation level of the conjugates in groups 2 and 3 was approximately 75%.

[0246] [Table 3]

[0247] Figures 5a and 5b show ELISA titers after two and three doses. The p-value is the benchmark natural MenA-CRM. 197 This refers to a comparison with other groups.

[0248] A second immunological test was performed by comparing the above randomly O-acetylated carbaMenA DP8 analogs of the present invention with carbaMenA DP8 selectively O-acetylated only at position 3, where the O-acetylation rate is approximately 70%, and with MenA vaccine as a positive control, according to the method described below, and all of these were CRM 197 It was conjugated.

[0249] Three groups of 10 Balb / C mice were immunized with the conjugate described above. Mice were immunized with three subcutaneous (sc) doses (2 μg carbohydrate-based, 200 μL / mouse) at 2-week intervals. The vaccine formulation used for the carba MenA conjugate was the same as that reported above for the first immunological study. Anti-MenA CPS response was evaluated, and the data showed that the total IgG response after the third immunization with 3O-acetylated carba MenA DP8 was approximately one-tenth that of the MenA vaccine benchmark. Conversely, the random O-acetylated carba MenA DP8 conjugate of the present invention induced a significantly higher response to natural MenA CPS compared to the 3O-acetylated conjugate, and was substantially equivalent to the response of the MenA vaccine benchmark (Figure 6).

[0250] TIFF0007883010000044.tif42153

[0251] In vitro sterilization assay Functional antibodies induced by vaccine immunization were analyzed by measuring complement-mediated lysis of Neisseria meningitidis using an in vitro bactericidal assay.

[0252] Commercially available batches of infant rabbit complement were used as the source of active complement for rSBA, and human plasma obtained from volunteer donors with informed consent was used as the source of complement for hSBA. Specifically, meningococcal strains were grown overnight on chocolate agar plates at 37°C in 5% CO2. Colonies were inoculated into Mueller-Hinton broth containing 0.25% glucose to an OD600 of 0.05-0.08 and incubated at 37°C with shaking. When the OD600 of the bacterial suspension reached 0.25-0.27, the bacteria were diluted with assay buffer (DPBS containing 1% BSA and 0.1% glucose) to a working dilution ratio (approximately 10%). 4 The test serum was diluted (CFU / mL). The total volume in each well was 50 μL, with 25 μL of serial 2-fold dilution of the test serum, 12.5 μL of bacteria at the working dilution, and 12.5 μL of complement source. The test serum was pooled and heat-inactivated at 56°C for 30 minutes. Negative controls included complement serum without the test serum, complement serum containing the test serum, heat-inactivated complement, and bacteria incubated separately. Immediately after adding the baby rabbit complement, the negative controls were plated on Mueller-Hinton agar plates using the tilt method (time 0). Microtiter plates were incubated at 37°C for 1 hour, and then each sample was spot-added in double strips to Mueller-Hinton agar plates, with the controls plated using the tilt method (time 1). The agar plates were incubated overnight at 37°C, and colonies (surviving bacteria) corresponding to time 0 and time 1 were counted. Serum bactericidal titer was defined as a serum dilution in which, after incubation of bacteria in the reaction mixture for 60 minutes, the colony-forming units (CFU) / mL decreased by 50% compared to the control CFU / mL at time 0. Typically, bacteria incubated without test serum in the presence of complement (negative control) showed a 150–200% increase in CFU / mL during the 60-minute incubation period. The reference strain for meningococcal serotype A was F8238.

[0253] The results reported in Figure 7 and Table 4 demonstrate the bactericidal ability of anti-MenA antibodies against MenA strains, particularly natural MenA-CRM. 197 The vaccines obtained from the vaccine and random O-acetylated synthetic carbaenids (groups 2 and 3) maintained significant bactericidal activity even when tested with human complement. Figure 7 shows the SBA titers obtained from rabbit (rSBA) and human (hSBA) complement after two and three doses.

[0254] [Table 4]

[0255] Figure 8 shows the CRM of selectively 3-O-acetylated carba MenA DP8 and randomly acetylated carba MenA DP8 of the present invention after three administrations. 197 It shows human complement-mediated serum bactericidal titers induced by the conjugate. MenA-CRM 197 The vaccine was the positive control.

[0256] Random O-acetylated carbaMenA-CRM 197 SBA titers induced by the conjugate were statistically equivalent to the MenA vaccine benchmark after three doses, but 3O-acetylated carbaMenA-CRM 197 The conjugate induced much lower SBA titers in serum compared to the vaccine benchmark, and these were measured in both infant rabbit complement and human complement.

[0257] statistical method For data obtained from ELISA, a nonparametric t-test was performed, and the Mann-Whitney hypothesis was used to determine the two control groups (CRM). 197 -MenA avDP15 and CRM 197- The ELISA was performed using GraphPad software to compare ranks between MenA DP6OAc or DP8OAc. ELISA data were reported as geometric mean with a CI of 95%. Additionally, fixed effects such as group (all except 4 and 5), time, and group / time interactions were analyzed. 10 An analysis of variance (ANOVA) model was fitted to the antibody titers. Since identical variances were not assumed between groups, a heterogeneous variance model was used. For each endpoint, this model was used to estimate the geometric mean and its 95% confidence interval, as well as the geometric mean ratio (O-acetylated formulation vs. benchmark) and its 95% confidence interval. In contrast, for SBA data, since there was only a single observation for each group at each time point (serum pool), only graph analysis was performed.

[0258] Protocol for the quantification of hydrolyzed MenA and carbaMenA oligomers in the final conjugate. The amount of monomers released over time from the MenA and carbaMenA conjugates of the present invention was quantified using HPAEC-PAD. The titers reported in Figure 9 were obtained by hydrolyzing the sample in a dryer with HCl at a final concentration of 6 M at 110°C for 2 hours. After incubation, the sample was dried in a Speedvac system, redissolved in water, and filtered through 0.45 μm. Quantification was performed using standard curves prepared for the range of 0.5–5.0 μg / mL for carbaMenA DP7 treated as the sample, quantified by NMR. Analysis was performed on an ICS5000 system (Dionex-Themo Fisher) equipped with a guarded CarboPac PA1 column. Elution was performed using a sodium acetate gradient in the presence of 100 mM sodium hydroxide at 1.0 mL / min, and peaks were detected by pulsed integrated current measurement using a fourth-harmonic waveform for carbohydrates. Results were prepared using Chromeleon™ 7.2 Chromatography Data System (CDS) software. conclusion Based on the data obtained, it can be concluded that the carbaMenA oligomer of the present invention can be used to develop a more stable version of the MenA vaccine, and that the OAc portion combined with the oligomer length is key to inducing a functional immune response against the MenA strain. This disclosure includes the following embodiments. (1) Oligomer of formula (Ia) or (Ib). [ka] [In the formula, n is ≥ 6; R is H or -P(O)(OR″). 2 And R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH 3 And each repeating unit may be the same or different; R y is H or -C(O)CH 3 And each repeating unit may be the same or different; R x or R y At least one of them is -C(O)CH in at least one repeating unit 3 In summary, R in oligomers x and R y Approximately 50-90% of it is -C(O)CH 3 and; Az is -NH(CO)R 1 , -N(R 1 ) 2 and -N 3 an aza substituent selected from the group consisting of R 1 C is H, linear or branched. 1 -C 6 - Alkyl and linear or branched C 1 -C 6 - Independently selected from the group consisting of haloalkyl; Z is (i) protecting group; (ii) Functional linkers for conjugation to proteins, (iii) Straight or branched C 1 -C 6 Alkyl, optionally substituted phenyl, -C(O)Y, or linear or branched C 1 -C 6 -Alkyl-X And, Y is H, C for straight chain or branched. 1 -C 6 -It is an alkyl or protecting group, X is -NH 2 、-N 3 -C≡CH, -CH=CH 2 , -SH or -SC ≡ N. (2) The oligomer described in (1), defined by formula (Ia). (3) The oligomer described in (1) or (2), wherein n is 8. (4) The oligomer described in any one of (1) to (3), wherein n is between 8 and 15. (5) Az is -NHC(O)CH 3 The oligomer described in any one of items (1) to (4). (6)R x and R y Both are in at least one identical repeating unit, -C(O)CH 3 The oligomer described in any one of items (1) to (5). (7)R x and R y Both of these are present in 40-50% of the repeating units of the oligomer, -C(O)CH 3 The oligomer described in any one of items (1) to (6). (8) In 10-30% of the remaining repeating units of the oligomer, R x or R y One of them is -C(O)CH 3 The remaining repeating units in the oligomer are R x =R y The oligomer described in (7), having H. (9) Oligomer conjugate antigen of formula (IIa) or (IIb).

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Claims

1. An oligomer of formula (Ia) or (Ib). 【Chemistry 1】 [In the formula, n is ≥ 6; R is H or -P(O)(OR'') ２ And R″ is H or a pharmaceutically acceptable phosphate counterion; R' is H or a pharmaceutically acceptable phosphate counterion; R ｘ is H or -C(O)CH ３ And each repeating unit may be the same or different; R ｙ is H or -C(O)CH ３ And each repeating unit may be the same or different; R ｘ or R ｙ at least one of which is -C(O)CH ３ in at least one repeating unit, and overall, about 50-90% of the Rs ｘ and Rs ｙ in the oligomer are -C(O)CH ３ ; Az is -NH(CO)R １ , -N(R １ ) ２ and -N ３ an aza substituent selected from the group consisting of R １ H, linear or branched C １ -C ６ - Alkyl and linear or branched C １ -C ６ - Independently selected from the group consisting of haloalkyls; Z is, (i) a protecting group; (ii) Functional linkers for conjugation to proteins, (iii) Linear or branched C １ -C ６ Alkyl, optionally substituted phenyl, -C(O)Y, or linear or branched C １ -C ６ -Alkyl-X And, Y is H, a straight chain or branched C １ -C ６ - Alkyl or protecting group, X is -NH ２ , -N ３ , -C≡CH, -CH=CH ２ , -SH or -S-C≡N.

2. The oligomer according to claim 1, defined by formula (Ia).

3. The oligomer according to claim 1 or claim 2, wherein n is 8.

4. The oligomer according to claim 1 or claim 2, wherein n is 8 to 15.

5. Az is -NHC(O)CH ３ The oligomer according to any one of claims 1 to 4.

6. R ｘ and R ｙ Both are present in 40-50% of the repeating units of the oligomer, -C(O)CH ３ The oligomer according to any one of claims 1 to 5.

7. In 10-30% of the remaining repeating units of the oligomer, R ｘ or R ｙ One of them is -C(O)CH ３ The remaining repeating unit in the oligomer is R ｘ = R ｙ The oligomer according to claim 6, having H.

8. An oligomer according to any one of claims 1 to 7, for use as a vaccine.

9. An oligomer according to any one of claims 1 to 7, for use in preparing a conjugate antigen with the oligomer's protein.

10. The oligomer according to claim 9 for use in the preparation of a conjugate antigen of formula (IIa) or (IIb): 【Chemistry 2】 [In the formula, n, R, R', R ｘ and R ｙ This is as defined in any one of claims 1 to 7; Z is a linker or bond; P is diphtheria toxoid (DT), tetanus toxoid (TT), CRM １９７ P is either an inactivated bacterial toxin selected from Escherichia coli ST and Pseudomonas aeruginosa exotoxin (rEPA), or P is a polyamino acid such as poly(lysine:glutamic acid), or P is hepatitis B virus core protein or SPR96-2021, or meningococcal serotype B antigen fHbp-231.

11. The aforementioned P is CRM １９７ The oligomer according to claim 10, for use in the preparation of a conjugate antigen of formula (IIa) or (IIb).

12. An oligomer according to claim 10 or 11 for use in preparing a conjugate antigen having the following structure: 【Transformation 3】 [In the formula, n, R, R ｘ and R ｙ This is defined in any one of claims 1 to 7.

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