Vaccine against Klebsiella pneumoniae

Abstract

The present invention relates to novel oligosaccharide-carrier protein conjugates of formula (I) and their use as pharmaceuticals, particularly as vaccines. The present invention also relates to related aspects, including oligosaccharide intermediates of formulas (II) and (III) and methods for preparing such conjugates. Furthermore, the present invention relates to pharmaceutical compositions comprising the oligosaccharide-carrier protein conjugates and to the use of oligosaccharide-carrier protein conjugates of formula (IV) in biological assays. [Formula 1] JPEG2025536393000131.jpg85168

Description

[Technical Field]

[0001] This invention was made with U.S. government support under IDSEP160030-01, IDSEP160030-02, and IDSEP160030-03 awarded by HHS / ASPR. The U.S. government has certain rights in this invention.

[0002] The present invention relates to novel oligosaccharide-carrier protein conjugates of formula (I) and their use as pharmaceuticals, particularly as vaccines. The invention also relates to oligosaccharide intermediates of formulas (II) and (III) and related aspects including methods for preparing such conjugates. Furthermore, the invention relates to pharmaceutical compositions comprising the oligosaccharide-carrier protein conjugates and to the use of oligosaccharide-carrier protein conjugates of formula (IV) in biological assays. [Background technology]

[0003] Klebsiella pneumoniae (or K. pneumoniae) is a Gram-negative, facultative anaerobic bacillus that primarily colonizes the respiratory, intestinal, and urinary tracts, as well as the skin, causing K. pneumoniae infections (KPIs). This bacterium primarily acts as an opportunistic pathogen. KPIs are a major cause of hospital-acquired infections and primarily affect immunocompromised patients. Infections caused by K. pneumoniae have become a significant challenge in healthcare settings due to the emergence and worldwide spread of strains resistant to almost all available antimicrobial agents. Infections caused by K. pneumoniae cause high morbidity and mortality. Therefore, prevention of infections caused by K. pneumoniae is highly desirable, and vaccination is the most cost-effective and powerful means of combating KPIs.

[0004] K. pneumoniae is an encapsulated bacterium that expresses lipopolysaccharide (LPS) and capsular polysaccharide (CPS, K antigen) on its outer membrane, which contribute to the virulence of this species.

[0005] LPS consists of three components: a lipid A moiety that functions as a membrane anchor, a core oligosaccharide covalently bound to lipid A, and a terminal antigenic polysaccharide with repeating sugar units that form the O antigen covalently bound to the core oligosaccharide. Extracted LPS has been shown to be pyrogenic, toxic, and capable of causing tissue damage. LPS can be coated by CPS and is usually less exposed on the surface than CPS.

[0006] CPSs are composed of repeating sugar units and form a layer on the bacterial outer surface. CPSs are usually complex, linear or branched, and have a larger molecular weight than LPS. Their high immunogenicity and surface exposure make them attractive targets for vaccine strategies. For example, WO2016156338 discloses conjugates of synthetic oligosaccharides related to carbapenem-resistant K. pneumoniae CPS.

[0007] However, Klebsiella CPS is highly diverse. Serologically, over 77 different CPS types, so-called K types, K serotypes, or K antigens, have been identified, with at least 141 K types. These additional K types are identified based on the capsule gene locus (cps-locus) or K gene locus (K-locus) and are referred to as the KL series.

[0008] On the other hand, LPS variability is low, and the currently known so-called O types, O serotypes, or O antigens are limited to 11 major groups: O1, O2a, O2ac, O2afg, O2aeh (formerly O9), O3 (including subserotypes O3, O3a, and O3b), O4, O5, O7, O8, and O12. In addition to the 11 species listed above, additional O types have been reported based on the O locus, known as the OL series. Although O antigens are less immunogenic and less exposed on the membrane surface than K antigens, they have also been considered in vaccine strategies. A recent large-scale survey of clinical isolates revealed the relative prevalence of lipopolysaccharide (LPS) serotypes, particularly in multidrug-resistant isolates. O1 antigens play a major role in K. pneumoniae infections worldwide, particularly in the Americas, Asia, and Africa. WO2019106201 discloses conjugates of synthetic oligosaccharides with the O-polysaccharides of K. pneumoniae serotypes O1, O2, O2ac and O8 and the O-polysaccharide of carbapenem-resistant K. pneumoniae ST258.

[0009] In particular, WO2019106201 describes an octasaccharide-carrier protein conjugate, i.e., CRM 197 Compound 61 bound to * (compound 61 * ) which resulted in the production of IgG in immunization experiments with mice. The resulting sera recognized the corresponding O-antigen BSA conjugate in ELISA.

[0010] However, to date, there is no approved vaccine available against K. pneumoniae, which highlights the difficulties involved in developing such a vaccine.

[0011] It remains difficult and unpredictable which CPS or LPS may be suitable candidate or model sequences for a vaccine, and in particular which short oligosaccharides are suitable for generating the desired immune response in vivo.

[0012] It has now been found that certain oligosaccharide-carrier protein conjugates exhibit superior immunological properties compared to the prior art. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1: HPLC-SEC characterization of C7-CRM197* glycoconjugates compared to CRM197. [Figure 2] Figure 2: SDS-PAGE of C7-CRM197* glycoconjugates compared to CRM197 and Marker (protein size marker is GelCode™ Blue Safe Protein Stain (Thermo Scientific)). [Figure 3] Figure 3 shows: a mouse immunogenicity study in BALB / c mice (6 mice) using a Compar-CRM197* antigen dose of 5 μg per mouse and per immunization on days 0, 14, and 28; i.e., Figure 3A shows an ELISA against the corresponding BSA glycoconjugate; Figure 3B shows an ELISA against O1 LPS isolated from strain PCM12 (Polish Collection of Microorganisms) using an LPS extraction kit (JH Science); sera were diluted as indicated on the graph. [Figure 4] Figure 4: Mouse immunogenicity study in C57BL / 6 mice (6 mice) using a C7-CRM197* antigen dose of 2.5 μg per mouse and per immunization on days 0, 14, and 28; i.e., Figure 4 shows ELISA against the corresponding BSA glycoconjugate; sera were diluted as indicated on the graph. [Figure 5]Figure 5 shows: a mouse immunogenicity study in C57BL / 6 mice (6 mice) using a C7-CRM197* antigen dose of 2.5 μg per mouse and per immunization on days 0, 14, and 28; Figure 5 shows an ELISA against isolated LPS on day 35, with pooled sera diluted 1:100. LPS was isolated from the strains Friedlaender (O1), NCTC9148 (O2a), or PCM27 (Gal III) using an LPS extraction kit (JH Science). [Figure 6] Figure 6: Rabbit immunogenicity results (ELISA against the corresponding BSA conjugate). Bars represent pooled sera from 4 animals at the serum dilutions indicated on the graph. Rabbits were immunized with a C7-CRM197* antigen dose of 2 μg per animal and per immunization on days 0, 21, and 35, and serum samples were collected on days 0, 7, 28, and 42. "Blank" is secondary antibody only (goat anti-rabbit IgG-HRP, SIGMA A4914, 1:10,000 dilution). [Figure 7] Figure 7: Binding of rabbit IgG to isolated LPS of an O1-expressing strain (PCM12) measured by ELISA. Data from four individual rabbits are shown (1:100 serum dilution); bars represent mean values. Rabbits were immunized with a C7-CRM197* antigen dose of 2 μg per animal and per immunization on days 0, 21, and 35, and serum samples were collected on days 0, 7, 28, and 42. The "blank" is secondary antibody (goat anti-rabbit IgG-HRP, SIGMA A4914, 1:10,000 dilution) only. [Figure 8]Figure 8 shows survival data from a challenge experiment in mice. CD-1 mice (10 per group) were intraperitoneally injected twice with 250 μL of rabbit antiserum raised with C7-CRM197* (obtained by immunization with a 2 μg C7-CRM197* antigen dose per rabbit and per immunization on days 0, 14, and 28, followed by collection on day 35) or with a control antiserum raised with placebo (aluminum hydroxide adjuvant (Brenntag) in buffer) at -24 h and -1 h prior to infection. At 0 h, mice were infected intraperitoneally with a lethal dose of Klebsiella pneumoniae O1-expressing strain PCM12, along with galactosamine treatment (20 mg / mouse). Mice were monitored for survival for 24 h. The survival curves show a statistically significant difference, P=0.0045 (Log-rank (Mantel-Cox test)). [Figure 9] Figure 9 shows survival data from a challenge experiment in mice. C57BL / 6 mice (8 mice per group) were immunized with a C7-CRM197* antigen dose of 2 μg per animal and per immunization or placebo (aluminum hydroxide adjuvant in buffer) at -43, -27, and -15 days prior to infection. At 0 h, mice were infected intraperitoneally with a lethal dose of Klebsiella pneumoniae O1-expressing strain PCM12 (Polish Collection of Microorganisms) in the presence of 5% mucin. Mice were monitored for survival for 24 h. Survival curves show a statistically significant difference, P = 0.0007 (Log-rank (Mantel-Cox test)). [Figure 10] Figure 10: Amino acid sequence of CRM197 SEQ ID NO:1. Summary of the Invention

[0014] Detailed Description of the Invention 1) In a first aspect, the present invention provides an oligosaccharide-carrier protein conjugate of formula (I):

[0015] [ka]

[0016] (In the formula, m is 4, 5 or 6; n is 5, 6 or 7; i is 1 to 28; -LT- represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. 197 the shortest distance between the nitrogen of the amino group of the lysine residue, and the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulfur; or a pharmaceutically acceptable salt thereof.

[0017] The definitions set forth herein apply uniformly to compounds of formula (I), (II), (III), and (IV) as defined in any one of embodiments 1) to 57), and apply mutatis mutandis throughout the specification and claims, unless a broader or narrower definition is given by a specific definition. It is to be understood that any definition or preferred definition of a term may independently (and together with) define and replace the respective term in any or all other terms or preferred definitions defined herein.

[0018] The oligosaccharide portion (i.e., the epitope or antigen portion) of compounds of formulas (I), (II), (III), and (IV) consists of D-galacto-pyranoside and D-galacto-furanoside, respectively. The configuration of each anomeric center is alpha or beta. Depending on the configuration of the anomeric center, a mixture of anomers may be formed, and the anomers may be synthesized in either the alpha or beta form, preferably as pure alpha or beta anomers. The mixture of anomers may be separated by methods known to those skilled in the art.

[0019] The term "essentially", when used in terms such as "essentially pure", is understood in the context of the present invention to mean in particular that at least 90, particularly at least 95, and especially at least 99 percent by weight of the oligosaccharides / oligosaccharide-linker compounds / oligosaccharide-linker-spacer compounds / glycoconjugates are pure oligosaccharides / oligosaccharide-linker compounds / oligosaccharide-linker-spacer compounds / glycoconjugates, respectively.

[0020] Whenever a substituent is described as optional, such substituent may be absent (i.e., the respective residue is unsubstituted with respect to such optional substituent), in which case all sites having a free valence (e.g., in an aromatic ring, ring carbon atoms and / or ring nitrogen atoms having a free valence, to which such optional substituent could be attached) are replaced with hydrogen, as the case may be. Similarly, when the term "optional" is used with respect to (ring) heteroatoms, this term means that each optional heteroatom, etc. is absent (i.e., a group has no heteroatoms / is a carbocyclic ring / etc.) or that each optional heteroatom, etc. is present as explicitly defined.

[0021] In this application, the terms "lysine residue" and "lysine site" are used interchangeably.

[0022] The oligosaccharides of the present invention are galactans, i.e. - Beta-D-galactofuranose / β-D-Galf:

[0023] [ka]

[0024] (Dotted lines indicate points of attachment (i.e., C1 and C3)); - Alpha-D-Galactopyranose / α-D-Galp:

[0025] [ka]

[0026] (Dotted lines indicate points of attachment (i.e., C1 and C3)); - Beta-D-Galactopyranose / β-D-Galp:

[0027] [ka]

[0028] (Dotted lines indicate points of attachment (i.e., C1 and C3)); It consists of:

[0029] As used herein, the term "oligosaccharide-carrier protein conjugate" is considered synonymous with the term "glycoconjugate."

[0030] CRM 197 " stands for Cross Reactive Material 197, which is a non-toxic variant of diphtheria toxin, in which a single amino acid substitution of glycine (Gly, G) at position 52 with glutamic acid (Glu, E) renders the protein non-toxic.

[0031] CRM 197 CRM is produced by C. diphtheriae infected with the nontoxigenic phage β197tox, which was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida et al., J. Biol. Chem., 1973, Vol. 245, No. 11, pp. 3838-3844). 197 Proteins are safe and effective T cell-dependent carriers of sugars. 197 CRM has been described, for example, by Giannini et al. in Nucleic Acids Research, Vol. 12, No. 10, 1984, pp. 4063-4069. 197 Further details regarding the preparation of CRM can be found, for example, in US Pat. No. 5,614,382, which is incorporated herein by reference. 197may be produced in various expression systems, such as Corynebacterium diphtheriae, Escherichia coli or Pseudomonas fluorescens (Hickey et al., J. Pharm. Sci., 2018, 107, 1806-1819).

[0032] In the present invention, "CRM 197 The term "protein" encompasses proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9% identity to the amino acid sequence SEQ ID NO:1 (preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the amino acid sequence SEQ ID NO:1; especially 95%, 96%, 97%, 98%, 99% or 99.9% identity to the amino acid sequence SEQ ID NO:1), optionally with an additional methionine (Met, M) at the N-terminus and / or optionally a CRM at the lysine site. 197 and the residue may be in a capped (i.e., inactivated) form.

[0033] CRM at the lysine site 197 The expression "residue resulting from functionalization of CRM" 197 is functionalized at a lysine site with a functional group suitable for forming a covalent bond to an epitope, i.e., a linker and / or spacer moiety attached to the oligosaccharide-linker portion of the conjugate. 197 are known to those skilled in the art. The above functional groups are particularly suitable for binding to thiols or for performing click chemistry. For example, such functional groups are groups having bromoacetamide, iodoacetamide, maleimide, azide or alkyne groups. 197optionally comprises a lysine residue functionalized with a bromo-acetamide, iodo-acetamide, maleimide, azide or alkyne group (preferably a bromo-acetamide, iodo-acetamide, maleimide group), which may be in capped form.

[0034] Preferred functionalized CRMs 197 CRM has a group bearing a bromoacetamide, iodoacetamide, or maleimide group, all of which are suitable for reaction with a thiol group provided by the oligosaccharide / linker moiety. 197 The unreacted functional groups of may then be quenched with a pharmaceutically acceptable thiol, such as L-cysteine ​​or cysteamine (2-aminoethane-1-thiol), to give the "capped form."

[0035] Preferred Lysine-Functionalized CRMs 197 is selected from the group consisting of:

[0036] [ka]

[0037] In a preferred embodiment, the CRM 197 is not functionalized as described above, meaning that "natural" lysine residues are used to directly attach oligosaccharide / linker / spacer moieties thereto, rather than being "pre-functionalized."

[0038] CRM 197 The amino acid sequence of is known to those skilled in the art and is outlined in FIG. 10 as SEQ ID NO:1.

[0039] CRM for the synthesis of glycoconjugates 197 Use of CRM 197 The above preferred binding sites have been reported (e.g., Moeginger et al., Sci. Rep. 6, 20488; doi:10.1038 / srep20488(2016)), which is incorporated herein by reference.

[0040] "-LT-" represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. 197 and the atoms of said backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulfur, and the phrase "is intended to mean that the backbone may be saturated, unsaturated, unsubstituted, or may contain oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 The ring structure may be saturated, unsaturated, or an aromatic 3- to 8-membered ring, including fused ring systems of 2 to 4 rings, and the ring atoms of the ring structure are selected from carbon, nitrogen, oxygen, and sulfur (particularly carbon and nitrogen), and the ring may be unsubstituted or may be substituted with oxo, (C 1-4 ) alkyl, halogen and (C 1-2 ) alkoxy; and optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents (particularly oxo).

[0041] For the avoidance of any doubt, the number 5 to 25 atoms relates to the number of atoms in the backbone and not to the number of atoms in the bridge.

[0042] Examples of such optional ring structures that may be part of the backbone are pyrrolidine-2,5-dione, cyclobut-3-ene-1,2-dione, triazole, isoindolin-1-one, 8,9-dihydro-1H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocine, cyclohexane, and benzene, as shown below:

[0043] [ka]

[0044] The introduction of these rings or ring systems is known to those skilled in the art of linker chemistry.

[0045] The phrase "the backbone may be unsaturated" means that the backbone chain may have one or more double bonds, which may or may not be part of a ring system.

[0046] For example, the number of atoms in a bridge having a saturated skeleton with three oxo substituents, and which skeleton is part of a ring system, is as follows:

[0047] [ka]

[0048] That is, the number of atoms forming the skeleton starts from the first atom after the oxygen of C1 and continues until the CRM 197 It ends with the last atom attached to the lysine nitrogen.

[0049] An oxygen atom in a saturated chain is preferably separated from another oxygen atom by one or more (especially 2, 3, 4 or 5, especially 2) carbon atoms.

[0050] The sulfur atoms in the saturated chain are preferably separated from another sulfur atom by one or more (especially 1, 2, 3, 4 or 5) carbon atoms.

[0051] The term "halogen" means fluorine, chlorine or bromine, preferably fluorine or chlorine, more preferably fluorine.

[0052] The term "oxo" refers to the functional group =O, ie, a substituent oxygen atom attached to another atom (preferably a carbon atom) by a double bond.

[0053] The term "alkyl," used alone or in combination, means a straight or branched saturated hydrocarbon chain having from 1 to 4 carbon atoms. x-y The term (C )alkyl (x and y are each integers) refers to an alkyl group as defined above having x to y carbon atoms. For example, (C 1-4 ) The alkyl group has 1 to 4 carbon atoms. 1-4 ) Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and tert-butyl. (C 1-2 ) Examples of alkyl groups are methyl and ethyl.

[0054] The term "alkoxy", used alone or in combination, means an alkyl-O- group, wherein the alkyl group is as previously defined. x-y The term "alkoxy" (x and y are each integers) refers to an alkoxy group as defined above having x to y carbon atoms. For example, (C 1-2 ) alkoxy group is "(C 1-2 )alkyl” has the meaning previously described; 1-2 ) alkyl-O- group. 1-2 ) Examples of alkoxy groups are methoxy and ethoxy.

[0055] 2) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m is 4 or 5.

[0056] 3) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m is 4.

[0057] 4) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), or a pharmaceutically acceptable salt thereof, wherein n is 6 or 7.

[0058] 5) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2) or 3), or a pharmaceutically acceptable salt thereof, wherein n is 6.

[0059] 6) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to embodiment 1), or a pharmaceutically acceptable salt thereof, wherein m is 4 and n is 6.

[0060] 7) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2), 3), 4), 5), or 6), or a pharmaceutically acceptable salt thereof, wherein the bridge does not have an aromatic or heteroaromatic ring.

[0061] 8) In a further embodiment, -LT- represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, the length being such that the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM are separated. 197 the shortest distance between the nitrogen of the amino group of the lysine residue, and the backbone has at most one double bond; the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; The above skeleton is oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 ) optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents independently selected from alkoxy (particularly oxo); A part of the skeleton may optionally be:

[0062] [ka]

[0063] may be a 4-, 5- or 6-membered ring moiety selected from: The present invention relates to an oligosaccharide-carrier protein conjugate according to any one of aspects 1), 2), 3), 4), 5) or 6), or a pharmaceutically acceptable salt thereof.

[0064] Here, the "maximum one double bond" is preferably a double bond of the cyclobut-3-ene-1,2-dione ring.

[0065] 9) A further embodiment is a linker L and a spacer T, in which -LT- represents a linker L and a spacer T that together form a bridge, said bridge consisting of a backbone that is a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur (particularly carbon, nitrogen and oxygen), said chain being unsubstituted or having oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 ) an oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2), 3), 4), 5), or 6), or a pharmaceutically acceptable salt thereof, which is optionally substituted by one or more (particularly 1, 2, 3, or 4) substituents (particularly oxo) independently selected from alkoxy;

[0066] This means that the bridge consists of a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur (particularly carbon, nitrogen and oxygen), and the chain can be unsubstituted or can be oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 ) alkoxy, which means that it may be optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents (particularly oxo) independently selected from alkoxy.

[0067] For the avoidance of any doubt, in this embodiment, the bridge does not have a ring structure.

[0068] 10) A further embodiment is a linker L and a spacer T, in which -LT- represents a linker L and a spacer T which together form a bridge, said bridge consisting of a backbone which is a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen and oxygen (particularly carbon and nitrogen), said chain being unsubstituted or having oxo, (C 1-4 ) alkyl, fluoro and (C 1-2) an oligosaccharide-carrier protein conjugate according to any one of embodiments 1), 2), 3), 4), 5), or 6), or a pharmaceutically acceptable salt thereof, which is optionally substituted by one or more (particularly 1, 2, 3, or 4) substituents (particularly oxo) independently selected from alkoxy;

[0069] This means that the bridge consists of a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen and oxygen (particularly carbon and nitrogen), and the chain can be unsubstituted or can be oxo, (C 1-4 ) alkyl, fluoro and (C 1-2 ) alkoxy, which means that it may be optionally substituted by one or more (particularly 1, 2, 3 or 4) substituents (particularly oxo) independently selected from alkoxy.

[0070] For the avoidance of any doubt, in this embodiment, the bridge does not have a ring structure.

[0071] 11) A further embodiment is a crosslinker in which the backbone of the crosslinker has a length of 8 to 20, preferably 8 to 16, atoms covalently linked together, the length being such that the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM are separated. 197 The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1), 2), 3), 4), 5), 6), 7), 8), 9), or 10), wherein the amino group of the lysine residue of the oligosaccharide-carrier protein conjugate forms the shortest distance between the amino group of the lysine residue of the oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof.

[0072] 12) A further aspect is L, * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) Fluoroalkylene-NH-; * -(CH2) c NHC(O)(CH2) d -NH- (c and d are independently 2 to 6); * -(CH2) e NHC(O)NH(CH2) h -NH- (e and h are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10 ) alkylene-O-NH-; represents; T, -C(O)-(C 0-10 ) alkylene-C(O)-; -C(O)-CH2CH2-(OCH2CH2) r -C(O)- (r is 1 to 5); -C(O)-CH2(CH2) f -(SCH2(CH2) f’ ) f’’ -C(O)- (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3);

[0073] [ka]

[0074] represents; or LT, * -(C 2-10 ) Alkylene-SR 1 ; represents; R 1 teeth,

[0075] [ka]

[0076] represents; The present invention relates to an oligosaccharide-carrier protein conjugate according to any one of aspects 1), 2), 3), 4), 5), 6) or 11), or a pharmaceutically acceptable salt thereof.

[0077] The " * " means that the linker is attached to the oligosaccharide at this position.

[0078] " attached to the spacer T * " means that the spacer is bonded to the linker L at this position.

[0079] R 1 The "♯" attached to the R 1 means that the bond is bonded to the sulfur.

[0080] "-(C x-y The term (C) alkylene- (x and y are each integers), used alone or in combination, refers to a bivalently bound, straight or branched saturated hydrocarbon chain having x to y carbon atoms. For example, (C 2-10 ) alkylene group has 2 to 10 carbon atoms, (C 0-10 A straight chain -(C x-y ) alkylene-, i.e., -(CH2) x-y - is preferred. (C 2-10 Representative examples of alkylene groups are ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene (particularly 1,2-ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, 1,7-heptylene, 1,8-octylene, 1,9-nonylene, and 1,10-decylene).

[0081] "(C x-yThe term "fluoroalkylene" (x and y are each integers), used alone or in combination, means a two-bonded, straight-chain or branched saturated hydrocarbon group having x to y carbon atoms in which one or more (and in some cases all) hydrogen atoms have been replaced by fluorine. x-y ) Fluoroalkylene- is preferred.

[0082] For the avoidance of any doubt, in these embodiments, the backbone length of -LT- is 5 to 25 atoms, 8 to 20 atoms, or 8 to 16 atoms. This is because the linker L and spacer T, if applicable, are in the range of R 1 together form a bridge having a backbone length of 5 to 25 (8 to 20 or 8 to 16) atoms covalently linked together, said length being such that the oxygen at C1 of the reducing end of said oligosaccharide and said carrier protein CRM 197 This means that the shortest distance between the amino group nitrogen of the lysine residue is formed.

[0083] 13) A further aspect is L, * -(CH2) l -NH-; (l is 2 to 10); * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) fluoroalkylene-NH- (fluoroalkylene is a saturated linear chain); * -(CH2) c NHC(O)(CH2) d -NH- (c and d are independently 2 to 6); * -(CH2) e NHC(O)NH(CH2) h -NH- (e and h are independently 2 to 6); * -(CH2) u -C(O)-NH-(CH2) u’ -NH-; (u is 1 to 10, and u' is 2 to 10); or * -(CH2) g -O-NH- (g is 2 to 10); represents; or LT, * -(CH2) q SR 1 (q is between 2 and 10); 12) or a pharmaceutically acceptable salt thereof;

[0084] 14) A further aspect is L, * -(CH2) l -NH-; (l is 2 to 10); * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3); * -(C 2-10 ) fluoroalkylene-NH- (fluoroalkylene is a saturated linear chain); * -(CH2) u -C(O)-NH-(CH2) u’ -NH-; (u is 1 to 10, and u' is 2 to 10); or * -(CH2) g -O-NH- (g is 2 to 10); represents; or LT, * -(CH2) q SR 1 (q is between 2 and 10); or a pharmaceutically acceptable salt thereof, according to embodiment 12), which represents:

[0085] 15) A further aspect is L, * -(CH2) l -NH-; (l is 2 to 10, preferably 2 to 6); * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3, preferably 1 or 2); * -(CH2) u -C(O)-NH-(CH2) u’ -NH-; (u is 1 to 10, preferably 1 to 6, and u' is 2 to 10, preferably 2 to 6); or * -(CH2) g -O-NH- (g is 2 to 10, preferably 2 to 6); represents; or LT, * -(CH2) q SR 1 (q is 2 to 10, preferably 2 to 6); or a pharmaceutically acceptable salt thereof, according to embodiment 12), which represents:

[0086] 16) A further aspect is L, * -(CH2) l -NH-; (l is 2 to 10, preferably 2 to 6); * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3, preferably 1 or 2); or * -(CH2) g -O-NH- (g is 2 to 10, preferably 2 to 6); or a pharmaceutically acceptable salt thereof, according to embodiment 12), which represents:

[0087] 17) A further aspect is L, * -(CH2) l -NH-; (l is 2 to 10, preferably 2 to 6); or * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3, preferably 1 or 2); or a pharmaceutically acceptable salt thereof, according to embodiment 12), which represents:

[0088] 18) A further aspect is that L is * -(CH2) l The oligosaccharide-carrier protein conjugate according to embodiment 12) or a pharmaceutically acceptable salt thereof, wherein 1 represents -NH-; (l is 2 to 10, preferably 2 to 6).

[0089] 19) A further embodiment is where L is * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH-, * -(CH2)5-NH- or * The oligosaccharide-carrier protein conjugate according to embodiment 12), or a pharmaceutically acceptable salt thereof, wherein —(CH 2 ) 6 —NH— represents

[0090] 20) A further aspect is that L is * The oligosaccharide-carrier protein conjugate according to embodiment 12), or a pharmaceutically acceptable salt thereof, wherein —(CH 2 ) 5 —NH— represents

[0091] 21) A further aspect is that L is * -(CH2CH2O) a The oligosaccharide-carrier protein conjugate according to embodiment 12) or a pharmaceutically acceptable salt thereof, wherein a represents —CH 2 CH 2 NH— (a is 1 or 2; preferably a is 1).

[0092] 22) A further aspect is T, -C(O)-(CH2)p -C(O)- (p is 0 to 10); -C(O)-CH2CH2-(OCH2CH2) r -C(O)- (r is 1 to 5); -C(O)-CH2(CH2) f -(SCH2(CH2) f’ ) f’’ -C(O)- (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3);

[0093] [ka]

[0094] The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 12) to 21), wherein

[0095] 23) A further aspect is T, -C(O)-(CH2) p -C(O)- (p is 0 to 10, preferably 0 to 6); -C(O)-CH2CH2-(OCH2CH2) r —C(O)— (r is 1 to 5, preferably 1 to 3, and more preferably 1); -C(O)-CH2(CH2) f -(SCH2(CH2) f’ ) f’’ -C(O)- (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2 or 3, preferably 1); or

[0096] [ka]

[0097] The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 12) to 21), wherein

[0098] 24) A further aspect is T, -C(O)-(CH2) p -C(O)- (p is 0 to 6); -C(O)-CH2CH2-(OCH2CH2) r -C(O)- (r is 1 or 2); or -C(O)-CH2(CH2) f -(SCH2(CH2) f’ ) f’’ -C(O)- (f is 0 or 1, f' is 0 or 1, and f'' is 1, preferably f and f' are 0 and f'' is 1); The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 12) to 21), wherein

[0099] 25) A further aspect is T, -C(O)-(CH2) p -C(O)- (p is 0, 1, 2, 3, 4, 5 or 6, preferably 4); or -C(O)-CH2CH2-(OCH2CH2) r -C(O)- (r is 1 or 2); The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 12) to 21), wherein

[0100] 26) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 12) to 21), or a pharmaceutically acceptable salt thereof, wherein T represents —C(O)—(CH2)4—C(O)—.

[0101] 27) A further aspect is T,

[0102] [ka]

[0103] The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 12) to 21), wherein

[0104] 28) A further aspect is T,

[0105] [ka]

[0106] The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 12) to 21), wherein

[0107] 29) A further aspect is R 1 but,

[0108] [ka]

[0109] or a pharmaceutically acceptable salt thereof, according to any one of embodiments 12), 13), 14), 15), 22), 27), and 28), wherein

[0110] Preferably, R 1 teeth:

[0111] [ka]

[0112] Represents.

[0113] 30) A further aspect is L, * -(CH2)2-NH-, * -(CH2)3-NH-, * -(CH2)4-NH- or * -(CH2)5-NH-; represents; T, -C(O)-(CH2)4-C(O)-; or a pharmaceutically acceptable salt thereof, according to any one of embodiments 1), 2), 3), 4), 5) or 6), which represents:

[0114] 31) A further aspect is L * represents -(CH2)5-NH- and T represents -C(O)-(CH2)4-C(O)-; or a pharmaceutically acceptable salt thereof.

[0115] 32) A preferred embodiment is an oligosaccharide-carrier protein conjugate having the formula:

[0116] [ka]

[0117] (wherein L, T and i are as defined in any one of embodiments 1) or 7) to 31); or a pharmaceutically acceptable salt thereof.

[0118] 33) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 32), or a pharmaceutically acceptable salt thereof, wherein i is 1 to 28, 1 to 25, 1 to 23; 1 to 20, 1 to 18, 3 to 25, 3 to 23, 3 to 20, 3 to 18, 5 to 23, 5 to 20, 5 to 18, 6 to 23, 6 to 20, 6 to 18, 6 to 15.

[0119] The variable i is the CRM 197The loading of epitopes / oligosaccharides on a protein carrier is described and is an integer per single molecule. However, it should be noted that if the glycoconjugate is considered as a product of more than one single molecule, the loading can be described as a statistical distribution, i.e., essentially a Gaussian distribution. The chemical process for producing the product results in a mixture of molecules with such a statistical distribution of the loadings, and in this case, the loading will be given as the average of the statistical distribution, particularly the Gaussian distribution.

[0120] If i is 2 or more, CRM via -LT- 197 The m and / or n of two or more oligosaccharides linked to may be the same or different. Preferably, all of the i oligosaccharides are represented by the same combination of m and n (i.e., have the same structure), or all of the i oligosaccharides are represented by a first combination of m and n or a second combination of m and n (i.e., have one of two structures); most preferably, all of the i oligosaccharides are represented by the same combination of m and n. The linker-spacer unit -LT- is the same among the i oligosaccharides of a particular oligosaccharide-carrier protein conjugate.

[0121] In other words, preferred oligosaccharide-carrier protein conjugates are those that have the same oligosaccharide / linker / spacer moieties, i.e., carrier CRM 197 The oligosaccharides contain only one specific type of oligosaccharide / linker / spacer residue attached to the oligosaccharide.

[0122] 34) A further embodiment relates to an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 32), or a pharmaceutically acceptable salt thereof, wherein i is 6 to 15.

[0123] 35) A preferred embodiment is the oligosaccharide-carrier protein conjugate described above, having the structure of formula (Ib):

[0124] [ka]

[0125] (wherein i is 1 to 28); or a pharmaceutically acceptable salt thereof.

[0126] For the avoidance of any doubt, the oligosaccharide-carrier protein conjugate of formula (Ib) according to this embodiment may also be depicted schematically as follows:

[0127] [ka]

[0128] (wherein i is 1 to 28) or a pharmaceutically acceptable salt thereof. 197 ' is a CRM as defined herein 197 The only difference is that in formula (Ic), the amino group of the lysine residue is specifically shown as the point of attachment for the linker / spacer moiety -LT-.

[0129] 36) A further preferred embodiment is the above oligosaccharide-carrier protein conjugate having the structure of formula (Ib):

[0130] [ka]

[0131] (wherein i is 6 to 15); or a pharmaceutically acceptable salt thereof.

[0132] For the avoidance of any doubt, the oligosaccharide-carrier protein conjugate of formula (Ib) according to this embodiment may also be depicted schematically as follows:

[0133] [ka]

[0134] (wherein i is 6 to 15) or a pharmaceutically acceptable salt thereof.

[0135] Accordingly, the present invention relates to compounds of formula (I) as defined in embodiment 1), as well as to such compounds further defined by the features of any one of embodiments 2) to 36), according to their respective subdivisions; pharmaceutically acceptable salts thereof; and to uses of such compounds as further described below. In particular, compounds of formulae (Ia), (Ib) and (Ic) are subforms of formula (I). For the avoidance of any doubt, the following embodiments in particular are possible and contemplated with respect to compounds of formula (I), and are specifically disclosed herein as individual forms: 1、2+1、3+1、4+1、4+2+1、4+3+1、5+1、5+2+1、5+3+1、6+1、7+1、7+2+1、7+3+1、7+4+1、7+4+2+1、7+4+3+1、7+5+1、7+5+2+1、7+5+3+1、7+6+1、8+1、8+2+1、8+3+1、8+4+1、8+4+2+1、8+4+3+1、8+5+1、8+5+2+1、8+5+3+1、8+6+1、9+1、9+2+1、9+3+1、9+4+1、9+4+2+1、9+4+3+1、9+5+1、9+5+2+1、9+5+3+1、9+6+1、10+1、10+2+1、10+3+1、10+4+1、10+4+2+1、10+4+3+1、10+5+1、10+5+2+1、10+5+3+1、10+6+1、11+1、11+2+1、11+3+1、11+4+1、11+4+2+1、11+4+3+1、11+5+1、11+5+2+1、11+5+3+1、11+6+1、11+7+1、11+7+2+1、11+7+3+1、11+7+4+1、11+7+4+2+1、11+7+4+3+1、11+7+5+1、11+7+5+2+1、11+7+5+3+1、11+7+6+1、11+8+1、11+8+2+1、11+8+3+1、11+8+4+1、11+8+4+2+1、11+8+4+3+1、11+8+5+1、11+8+5+2+1、11+8+5+3+1、11+8+6+1、11+9+1、11+9+2+1、11+9+3+1、11+9+4+1、11+9+4+2+1、11+9+4+3+1、11+9+5+1、11+9+5+2+1、11+9+5+3+1、11+9+6+1、11+10+1、11+10+2+1、11+10+3+1、11+10+4+1、11+10+4+2+1、11+10+4+3+1、11+10+5+1、11+10+5+2+1、11+10+5+3+1、11+10+6+1、12+1、12+2+1、12+3+1、12+4+1、12+4+2+1、12+4+3+1、12+5+1、12+5+2+1、12+5+3+1、12+6+1、12+11+1、12+11+2+1、12+11+3+1、12+11+4+1、12+11+4+2+1、12+11+4+3+1、12+11+5+1、12+11+5+2+1、12+11+5+3+1、12+11+6+1、13+12+1、13+12+2+1、13+12+3+1、13+12+4+1、13+12+4+2+1、13+12+4+3+1、13+12+5+1、13+12+5+2+1、13+12+5+3+1、13+12+6+1、13+12+11+1、13+12+11+2+1、13+12+11+3+1、13+12+11+4+1、13+12+11+4+2+1、13+12+11+4+3+1、13+12+11+5+1、13+12+11+5+2+1、13+12+11+5+3+1、13+12+11+6+1、14+12+1、14+12+2+1、14+12+3+1、14+12+4+1、14+12+4+2+1、14+12+4+3+1、14+12+5+1、14+12+5+2+1、14+12+5+3+1、14+12+6+1、14+12+11+1、14+12+11+2+1、14+12+11+3+1、14+12+11+4+1、14+12+11+4+2+1、14+12+11+4+3+1、14+12+11+5+1、14+12+11+5+2+1、14+12+11+5+3+1、14+12+11+6+1、 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28+19+11+3+1, 28+19+11+4+1, 28+19+11+4+2+1, 28+19+11+4+3+1, 28+19+11+5+1, 28+19+11+5+2+1, 28+19+11+5+3+1, 28+19+11+6+1, 28+20+1, 28+20+2+1, 28+20+3+1, 28+20+4+1, 28+20+4+2+1, 28+20+4+3+1, 28+20+ 5+1, 28+20+5+2+1, 28+20+5+3+1, 28+20+6+1, 28+20+11+1, 28+20+11+2+1, 28+20+11+3+1, 28+20+11+4+1, 28+20+11+4+2+1, 28+20+11+4+3+1, 28+20+11+5+1, 28+20+11+5+2+1, 28+20+11+5+3+1, 28+20+11+6+1, 28+21+1, 28+21+2+1, 2 8+21+3+1, 28+21+4+1, 28+21+4+2+1, 28+21+4+3+1, 28+21+5+1, 28+21+5+2+1, 28+21+5+3+1, 28+21+6+1, 28+21+11+1, 28+21+11+2+1, 28+21+11+3+1, 28+21+11+4+1, 28+21+11+4+2+1, 28+21+11+4+3+1, 28+21+11+5+1, 28+21+11+5+2+ 1, 28+21+11+5+3+1, 28+21+11+6+1, 30+1, 30+2+1, 30+3+1, 30+4+1, 30+4+2+1, 30+4+3+1, 30+5+1, 30+5+2+1, 30+5+3+1, 30+6+1, 31+1, 31+2+1, 31+3+1, 31+4+1, 31+4+2+1, 31+4+3+1, 31+5+1, 31+5+2+1, 31+5+3+1, 31+6+1, 32, 35 and 36;, In the above list, the numbers refer to the embodiments corresponding to the number, and "+" indicates a dependency from another embodiment. The various embodiments are individually separated by commas. In other words, for example, "4+2+1" means embodiment 4) which is dependent on embodiment 2) which is dependent on embodiment 1), i.e. embodiment "4+2+1" corresponds to the compound of embodiment 1) further limited by the characteristics of embodiments 2) and 4).

[0136] The ranges of i set forth in embodiments 33) and 34) shall be considered to be expressly disclosed for each of the above combinations.

[0137] When the plural forms are used for compounds, conjugates, salts, pharmaceutical compositions, diseases, etc., they are intended to refer to the singular compound, conjugate, salt, pharmaceutical composition, disease, etc.

[0138] Any reference to a compound of formula (I) as defined in any one of embodiments 1) to 36) will be understood to also refer to the salts (particularly pharmaceutically acceptable salts) of such compounds, where appropriate and appropriate.

[0139] The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the subject compound and exhibits minimal undesired toxic effects. Such salts include inorganic or organic acid and / or base addition salts, depending on the presence of basic and / or acidic groups in the subject compound. They may be used for stabilization in the form of a buffer or for lyophilized products containing a buffer. For reference, see, for example, "Handbook of Pharmaceutical Salts. Properties, Selection and Use," P. Heinrich Stahl, Camille G. Wermuth (Eds.), Wiley-VCH, 2008; and "Pharmaceutical Salts and Co-crystals," Johan Wouters and Luc Quere (Eds.), RSC Publishing, 2012.

[0140] This embodiment also includes isotopically labeled, especially 2Also included are H (deuterium) labeled compounds of formula (I), (Ia), (Ib), (Ic), (II), (IIa), (III), and (IIIa), which are identical to compounds of formula (I), (Ia), (Ib), (Ic), (II), (IIa), (III), and (IIIa) except that one or more atoms have been replaced, respectively, by an atom having the same atomic number but an atomic mass different from the atomic mass normally found in nature. 2 H (deuterium) labeled compounds of formula (I), (Ia), (Ib), (Ic), (II), (IIa), (III) and (IIIa) and salts thereof are included within the scope of this embodiment. 2 Substitution with H (deuterium) can increase metabolic stability, e.g., prolong in vivo half-life, reduce the required dose, or reduce inhibition of cytochrome P450 enzymes, thereby improving, for example, the safety profile. In one embodiment, compounds of formulas (I), (Ia), (Ib), (Ic), (II), (IIa), (III), and (IIIa) are not isotopically labeled, or they are labeled only with one or more deuterium atoms. In a subembodiment, compounds of formulas (I), (Ia), (Ib), (Ic), (II), (IIa), (III), and (IIIa) are not isotopically labeled at all. Isotopically labeled compounds of formulas (I), (Ia), (Ib), (Ic), (II), (IIa), (III), and (IIIa) may be prepared similarly to the methods described below, except for using appropriate isotopic versions of the appropriate reagents or starting materials. For example, the label may be in the linker L and / or the spacer T.

[0141] The compounds of formulae (I), (Ia), (Ib) and (Ic) as defined in any one of aspects 1) to 36) and pharmaceutically acceptable salts thereof can be used as medicines, for example in the form of pharmaceutical compositions for parenteral, enteral (such as oral) or nasal administration, in particular for parenteral administration such as intramuscular, subcutaneous and intradermal injection.

[0142] 37) Thus, one aspect of the present invention relates to a pharmaceutical composition comprising, as an active ingredient, an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 36), in particular embodiments 35) and 36), and further comprising at least one therapeutically inactive excipient.

[0143] Pharmaceutical compositions can be prepared in a manner well known to anyone skilled in the art (see, for example, Remington, The Science and Practice of Pharmacy, 23rd Edition (2021), published by Elsevier Inc., ISBN: 978-0-12-820007-0; Vaccine Development and Manufacturing, 1st edition (2014), published by John Wiley & Sons, ISBN: 9780470261941). The compounds of formula (I), (Ia), (Ib) and (Ic) or pharmaceutically acceptable salts thereof can be combined with, optionally, other therapeutically beneficial substances, and formulated into a dosage form together with suitable non-toxic, inert, therapeutically compatible solid or liquid carrier materials and, optionally, conventional pharmaceutical adjuvants.

[0144] The pharmaceutical compositions are suitable for inducing a protective immune response in a human and / or animal (particularly mammalian (including human)) host and are therefore useful for the prevention and / or treatment of diseases associated with Klebsiella pneumoniae. Preferably, the pharmaceutical compositions are suitable for use in humans.

[0145] The terms "prevention," "preventing," and / or "prophylaxis" are used interchangeably and refer to inhibiting the initial onset of a pathological process (i.e., preventing the onset of a disease, disorder, or condition in a prophylactic manner) so that the pathological process that may ultimately lead to the onset of symptoms does not progress or so that the symptoms develop at a less severe and less dangerous intensity.

[0146] The pharmaceutical compositions of the present invention are suitable for administration to animal (particularly human) patients and therefore include both human and veterinary uses. The pharmaceutical compositions may be used in a method of raising an immune response in a patient, such method comprising administering the composition to the patient.

[0147] The pharmaceutical compositions of the present invention may be administered before a subject is exposed to Klebsiella pneumoniae and / or after a subject is exposed to Klebsiella pneumoniae, and are preferably used before a subject is exposed to Klebsiella pneumoniae.

[0148] The pharmaceutical compositions are preferably in aqueous form, particularly at the time of administration, but may also be in non-aqueous or dried form, such as gelatin capsules or freeze-dried products, etc. For example, solid powders obtained by spray drying, spray freeze drying, vacuum or air drying or freeze-drying may be reconstituted before use.

[0149] The pharmaceutical composition may contain one or more therapeutically inactive excipients, which may be selected from the group consisting of citric acid monohydrate, sodium citrate, sodium citrate dihydrate, acetic acid, sodium hydroxide, tromethamine, tromethamine hydrochloride (for pH adjustment), cholesterol, sorbitan trioleate, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and (4-hydroxybutyl)azanediylbis(hexane-6,1-diyl)bis(2-hexyldecanoate), polydimethylsiloxane (antifoaming agent), and ascorbic acid (antioxidant).

[0150] Excipients may also serve to adjust osmolality, for example sodium chloride (NaCl) may be present at 1-20 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate anhydrous, magnesium chloride, calcium chloride, etc.

[0151] The pharmaceutical composition may contain one or more excipients that function as preservatives, which may be selected from the group consisting of 2-phenoxyethanol, benzethonium chloride, EDTA (ethylenediaminetetraacetic acid), formaldehyde, phenol, and thiomersal (thimerosal). Mercury-free compositions are preferred, and preservative-free vaccines can be produced.

[0152] The pharmaceutical composition may comprise one or more excipients that function as surfactants, which may be selected from the group consisting of polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 80 (polyoxyethylene (80) sorbitan monooleate), nonylphenol ethoxylate, octoxynol-10, and sodium deoxycholate.

[0153] The pharmaceutical compositions may contain the compound (with or without insoluble metal salts) in plain water (e.g., water for injection, WFI), but typically contain one or more buffers. Typical buffers include phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer (especially with aluminum hydroxide adjuvant); or citrate buffer. Buffer salts are typically present in the range of 5-20 mM.

[0154] Pharmaceutical compositions typically have a pH between 5.0 and 9.5, for example a pH between 6.0 and 8.0.

[0155] The pharmaceutical composition may further comprise one or more stabilizers.

[0156] The pharmaceutical composition is preferably sterile and gluten-free.

[0157] 38) A further embodiment of the invention relates to a pharmaceutical composition according to embodiment 37), further comprising an adjuvant.

[0158] As used herein, the term "adjuvant" refers to an immunological adjuvant, i.e., a substance used in a vaccine composition that modifies or increases the effectiveness of a vaccine by enhancing the immune response to an antigen contained in the vaccine without antigenically contributing to it. Examples of immunological adjuvants known to those skilled in the art in the classical sense include: aluminum or calcium salt-based adjuvants, saponin or saponin-based adjuvants (e.g., Matrix-M), CpG oligodeoxynucleotide-based adjuvants (e.g., CpG1018), oil-in-water emulsions (e.g., Freund's adjuvant, MF59), activators of natural killer T cells (NKT cells) or invariant NKT cells (e.g., glycosphingolipids such as KRN7000), Toll-like receptor 1 / 2 (TLR-1 / 2) agonists (e.g., Pam3CSK4), TLR-3 agonists (e.g., Poly(I:C)), TLR-4 agonists (e.g., lipopolysaccharide), TLR-5 agonists (e.g., flagellin), TLR-7 / 8 agonists (e.g., resiquimod), immunomodulatory proteins (e.g., Escherichia coli), Detoxified heat-labile enterotoxin from coli (dmLT), TLR-4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) and monophosphoryl lipid A (MPL), non-ionic block polymers, cytokines (e.g., type 1 interferons (IFNs), granulocyte-macrophage colony-stimulating factors (GM-CSFs), interleukins), papain-like cysteine ​​proteases, and many others (e.g., AS04, AS03, AS01). Band formulations of the above adjuvants as liposomes or nanoparticles made with lipids such as DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol and / or ALC-0315, formulations as virus-like particles, and co-formulations of the above adjuvants, particularly co-formulations with aluminum or calcium salt-based adjuvants.

[0159] The adjuvant, "aluminum," "aluminum-based adjuvant," or "aluminum salt-based adjuvant" is one or more of the following: amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate (Alum).

[0160] An example of a calcium-based or calcium salt-based adjuvant is calcium phosphate.

[0161] Matrix-M is a saponin-based adjuvant consisting of nanoparticles of saponin extracted from the Quillaja saponaria (soapbark) tree, cholesterol, and phospholipids.

[0162] CpG-based adjuvants are immunostimulatory oligodeoxynucleotides bearing one or more CpG motifs (CpG ODNs), which are unmethylated cytosine-guanine dinucleotides. The methylation status of the CpG immunostimulatory motif generally relates to the cytosine residue in the dinucleotide. Immunostimulatory oligonucleotides with at least one unmethylated CpG dinucleotide have a 5' unmethylated cytosine linked to a 3' guanine by a phosphate bond and activate the immune system through binding to Toll-like receptor 9 (TLR-9).

[0163] Freund's adjuvant is a mineral oil-based oil-in-water adjuvant.

[0164] MF59 is an oil-in-water emulsion with 4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80) and 0.5% w / v sorbitan trioleate (Span 85).

[0165] Glycosphingolipids are a class of lipids that stimulate the unconventional invariant T-cell receptor on NKT cells or iNKT cells when they are presented on MHC class I-associated molecules such as CD1d.

[0166] Pam3CSK4 (Pam3CysSerLys4) is a synthetic triacylated lipopeptide that is a ligand for TLR-1 and TLR-2 and mimics the acylated amino terminus of bacterial lipopeptides.

[0167] Poly(I:C) is a polymer consisting of one strand of inosinic acid and one strand of cytidylic acid, an analog of double-stranded RNA. It stimulates TLR-3 and simulates viral infection.

[0168] Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and a stimulator of TLR-4.

[0169] Flagellin is a globular protein that forms the filaments of bacterial flagella and activates TLR-5 and TLR-11.

[0170] Resiquimod (R848; 1-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol) is an immune response modifier and small molecule that activates TLR-7 and TLR-8.

[0171] dmLT is a double mutant (and thereby detoxified) form of the heat-labile enterotoxin from Escherichia coli, and is an effective mucosal and systemic adjuvant.

[0172] GLA-SE is an oil-in-water emulsion adjuvant made by combining aqueous glucopyranosyl lipid A (GLA), a TLR-4 agonist, with squalene.

[0173] MPL (monophosphoryl lipid A), a truncated LPS, is a clinically used TLR-4 agonist.

[0174] Nonionic block polymers (NBPs) suitable as adjuvants are simple copolymers of polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP), and vary in molecular weight, POE ratio, and bonding mode between POE and POP.

[0175] Cytokines are small proteins secreted by cells that affect cell-cell interactions and communication. Typically, cytokines activate target cells, triggering the secretion of additional cytokines and signaling cascades. Cytokines are involved in the induction of innate and adaptive immunity. As adjuvants, cytokines can be used as recombinant proteins or encoded on DNA molecules such as plasmids.

[0176] Papain-like cysteine ​​proteases are derived from viruses, bacteria, yeast, protozoa, plants, or animals and contain a cysteine ​​thiol at the active site. This class of proteases can stimulate a Th2-type immune response.

[0177] AS04 (Adjuvant System 04) is a complex of MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum hydroxide or aluminum phosphate.

[0178] AS03 (Adjuvant System 03) is a squalene-in-water emulsion containing DL-alpha-tocopherol (vitamin E) and polysorbate 80.

[0179] AS01 B is a mixture of 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and saponin QS-21.

[0180] Preferred adjuvants are aluminum-based adjuvants, especially aluminum hydroxide.

[0181] 39) A further aspect of the present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 36), in particular embodiments 35) and 36), for use as a medicament, in particular as a vaccine. In other words, the present invention relates to a vaccine comprising an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 36), in particular embodiments 35) and 36). Preferably, the vaccine is used for active vaccination.

[0182] 40) A further aspect of the present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 36), in particular embodiments 35) and 36), for use in the prevention and / or treatment of K. pneumoniae infections.

[0183] 41) A further aspect of the present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of aspects 1) to 36), in particular aspects 35) and 36), for use in the prophylaxis and / or treatment of K. pneumoniae infections in individuals 50 years of age or older; hospital-acquired (i.e. nosocomial) K. pneumoniae infections, such as nosocomial pneumonia, nosocomial bloodstream infections and nosocomial urinary tract infections; community-acquired K. pneumoniae infections; and all pneumonias, bronchitis, meningitis, urinary tract infections, intra-abdominal infections, wound infections, blood infections, osteomyelitis, bacteremia, sepsis, liver abscesses and inflammatory bowel diseases (IBD) caused by K. pneumoniae infections.

[0184] A population-based vaccination strategy for individuals aged 50 years and older against K. pneumoniae infection is desirable because this population is particularly susceptible to K. pneumoniae infection, especially individuals aged 60 years and older who are at risk of exposure to K. pneumoniae and / or who are expected to have weakened immunity.

[0185] K. pneumoniae is a notorious pathogen that frequently causes nosocomial (i.e., hospital-acquired) respiratory and urinary tract infections and is the second most common cause of Gram-negative bacteremia. Drug-resistant isolates are associated with high mortality rates (over 50% in some studies) and significantly prolong hospital stays, which is particularly problematic in ICUs.

[0186] Therefore, it is desirable to prevent hospital-acquired (i.e., hospital-acquired) pneumoniae infections, especially in populations at high risk of exposure, such as patients undergoing elective surgery (e.g., joint replacement) with hospitalization exceeding 72 hours, and immunocompromised and expected to become immunocompromised (e.g., solid organ transplant recipients, patients receiving chemotherapy after non-urgent solid tumor surgery). In these populations, vaccination 2 to 8 weeks before surgery, followed by an optional booster vaccination, may be indicated.

[0187] Additionally, prevention of community-acquired infections in specific target populations, such as healthcare workers or the elderly (ages 60 and older) in long-term care facilities or nursing homes, is desirable. The term "community-acquired K. pneumoniae infection" refers to K. pneumoniae infections acquired in the general community, as opposed to nosocomial (hospital-acquired) infections.

[0188] Furthermore, the oligosaccharide-carrier protein conjugate of the present invention or a pharmaceutically acceptable salt thereof according to any one of Aspects 1) to 36), particularly Aspects 35) and 36), may be used in the prevention and / or treatment of pneumonia, bronchitis, meningitis, urinary tract infections, intra-abdominal infections, wound infections, blood infections, osteomyelitis, bacteremia, sepsis, liver abscess, and inflammatory bowel disease (IBD) caused by K. pneumoniae infection.

[0189] 42) A further aspect of the present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of aspects 1) to 36), in particular aspects 35) and 36), for use in the prevention and / or treatment of K. pneumoniae infections as described in aspects 40) and 41) above, wherein the K. pneumoniae is selected from the O-serotype having O1.

[0190] 43) For the avoidance of any doubt, the oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 36), in particular embodiments 35) and 36), as well as the pharmaceutical composition according to embodiment 37) or 38), and the vaccine according to embodiment 39) are likewise suitable for the prevention and / or treatment of K. pneumoniae infections as described in any one of embodiments 40), 41) and 42).

[0191] 44) Preferably, the oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 36), in particular embodiments 35) and 36), as well as the pharmaceutical composition according to embodiment 37) or 38), and the vaccine according to embodiment 39) are suitable for preventing K. pneumoniae infections as described in any one of embodiments 40), 41) and 42).

[0192] 45) A further aspect of the present invention relates to a method for inducing an immune response against K. pneumoniae in a human and / or animal (in particular a mammalian (including human)) host, comprising administering to said human and / or animal an effective amount of an oligosaccharide-carrier protein conjugate according to any one of aspects 1) to 36), in particular aspects 35) and 36), or a pharmaceutically acceptable salt thereof. The dosage is preferably 0.05 μg to 30 μg glycan per immunization of a human patient. The term "glycan" refers to the antigen, i.e., the oligosaccharide excluding the linker L and the spacer T. In some cases, more than one immunization may be required.

[0193] 46) Similarly, an embodiment of the present invention relates to a method for inducing an immune response against K. pneumoniae in a human and / or animal (in particular a mammalian (including human)) host, comprising administering to said human and / or animal an effective amount of a composition according to embodiment 37) or 38) and a vaccine according to embodiment 39).

[0194] 47) For the avoidance of any doubt, where an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1) to 36), in particular embodiments 35) and 36), is described as being useful for the prevention and / or treatment of K. pneumoniae infections according to any one of embodiments 40), 41) and 42), such oligosaccharide-carrier protein conjugate is likewise suitable for use in the manufacture of a medicament for the prevention and / or treatment of said K. pneumoniae infections according to any one of embodiments 40), 41) and 42).

[0195] 48) A further aspect of the present invention relates to a multivalent vaccine comprising an oligosaccharide-carrier protein conjugate according to any one of embodiments 1) to 36), preferably an oligosaccharide-carrier protein conjugate according to embodiment 35) or 36), or a pharmaceutically acceptable salt thereof.

[0196] In this regard, the term "multivalent vaccine" relates to a vaccine which comprises antigens against two or more different K. pneumoniae strains, in particular against two or more pathogenic K. pneumoniae strains.

[0197] 49) A further aspect of the present invention is an intermediate compound for producing an oligosaccharide-carrier protein conjugate according to any one of embodiments 12) to 36), the intermediate compound having formula (II):

[0198] [ka]

[0199] (In the formula, m is 4, 5 or 6, preferably 4 or 5, more preferably 4; n is 5, 6 or 7, preferably 6 or 7, more preferably 6; L 1 teeth, * -(C 2-10 ) alkylene-NH2; * -(CH2CH2O) a -CH2CH2NH2 (a is 1, 2 or 3); * -CH2CH2S-CH2CH2NH2; * -(C 2-10 ) Fluoroalkylene-NH2; * -(CH2) c NHC(O)(CH2) d -NH2 (c and d are, independently, 2 to 6); * -(CH2) e NHC(O)NH(CH2) h -NH2 (e and h are, independently, 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH2; * -(C 2-10 ) alkylene-O-NH2; or * -(C 2-10 ) alkylene-SH; represents. ); or a pharmaceutically acceptable salt thereof.

[0200] 50) A further embodiment relates to an intermediate compound according to embodiment 49), or a pharmaceutically acceptable salt thereof, wherein m is 4 and n is 6.

[0201] 51) A further aspect is L 1 but, * -(CH2) l -NH2; (l is 2 to 10); * -(CH2CH2O) a -CH2CH2NH2 (a is 1, 2 or 3); * -CH2CH2S-CH2CH2NH2; * -(C 2-10 ) Fluoroalkylene-NH2 (Fluoroalkylene is a saturated straight chain); * -(CH2) c NHC(O)(CH2) d -NH2 (c and d are, independently, 2 to 6); * -(CH2) e NHC(O)NH(CH2) h -NH2 (e and h are, independently, 2 to 6); * -(CH2) g -O-NH2 (g is 2 to 10); * -(CH2) u -C(O)-NH-(CH2) u’ -NH2; (u is 1 to 10, and u' is 2 to 10); or * -(CH2)q SH (q is 2 to 10); or a pharmaceutically acceptable salt thereof according to embodiment 49) or 50), which represents

[0202] Similarly, embodiments 14) to 21) include further preferred L having a terminal amino- or SH-group as shown in embodiment 49). 1 shall be disclosed.

[0203] 52) A further embodiment is an intermediate compound of formula (IIa) having the following structure:

[0204] [ka]

[0205] or a pharmaceutically acceptable salt thereof.

[0206] 53) A further aspect of the present invention is an intermediate compound for producing an oligosaccharide-carrier protein conjugate according to any one of embodiments 12) to 36), the intermediate compound having formula (III):

[0207] [ka]

[0208] (In the formula, m is 4, 5 or 6, preferably m is 4 or 5, most preferably 4; n is 5, 6 or 7, preferably n is 6 or 7, most preferably 6; L is * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; *-(C 2-10 ) Fluoroalkylene-NH-; * -(CH2) c NHC(O)(CH2) d -NH- (c and d are independently 2 to 6); * -(CH2) e NHC(O)NH(CH2) h -NH- (e and h are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10 ) alkylene-O-NH-; represents; T 1 teeth, -C(O)-(C 0-10 ) alkylene-C(O)X; -C(O)-CH2CH2-(OCH2CH2) r -C(O)X (r is 1 to 5); -C(O)-CH2(CH2) f -(SCH2(CH2) f’ ) f’’ -C(O)X (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3);

[0209] [ka]

[0210] represents; -C(O)X represents -C(O)OH or an activated ester; Y represents Me, Et, Bu or -(CH2CH2O)3CH3; or a pharmaceutically acceptable salt thereof.

[0211] Preferably, X is

[0212] [ka]

[0213] Represents.

[0214] The term "activated ester" refers to the CRM 197 By "carboxylic acid" is meant a functionalized carboxylic acid that has enhanced reactivity towards amines (compared to carboxylic acids) for reaction with the amino groups of lysine residues of .

[0215] Embodiments 13) to 21) are intended to disclose further preferred L's encompassed by this embodiment. Similarly, embodiments 22) to 28) disclose further preferred T's having terminal X-, OY-, or SH- groups as shown in this embodiment. 1 That is, -T- disclosed in these embodiments is intended to disclose CRM 197 For the avoidance of any doubt, the terminal "C(O)-" disclosed in the T of these embodiments refers to a T having a "C(O)X" 1 In the case of squaric acid, CRM 197 The point of attachment to R is represented as "OY" 1 is H. These preferred T 1 is deemed to be expressly disclosed.

[0216] 54) A further embodiment is an intermediate compound of formula (IIIa) having the following structure: or a pharmaceutically acceptable salt thereof:

[0217] [ka]

[0218] wherein -C(O)X represents -C(O)OH or an activated ester, and preferably X is

[0219] [ka]

[0220] represents.)

[0221] 55) A further aspect of the present invention relates to an assay having a compound of formula (IV):

[0222] [ka]

[0223] (m, n, i, L and T are as described in any one of embodiments 1) to 36), particularly embodiments 35) and 36), and CP is a carrier protein.)

[0224] In this embodiment, the carrier protein CP may be any carrier protein suitable for assays, in particular ELISAs. A preferred carrier protein is BSA.

[0225] The synthesis of compounds of the antigen of formula (IV) conjugated to BSA is described and exemplified in the experimental section, and it should be understood that this synthesis applies equally to all antigens of formula (IV) and therefore can be prepared by one skilled in the art.

[0226] The assay of this embodiment is suitable for detecting antibodies to the O1 strain of K. pneumoniae.

[0227] 56) A further aspect of the present invention is a conjugate comprising a compound of formula (III) 197 By conjugating to the lysine residue of:

[0228] [ka]

[0229] (In the formula, m is 4, 5 or 6, preferably m is 4 or 5, most preferably 4; n is 5, 6 or 7, preferably n is 6 or 7, most preferably 6; L is * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) Fluoroalkylene-NH-; * -(CH2) c NHC(O)(CH2) d -NH- (c and d are independently 2 to 6); * -(CH2) e NHC(O)NH(CH2) h -NH- (e and h are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10 ) alkylene-O-NH-; represents; a) T 1 teeth, -C(O)-(C 0-10 ) alkylene-C(O)X; -C(O)-CH2CH2-(OCH2CH2) r -C(O)X (r is 1 to 5); -C(O)-CH2(CH2) f -(SCH2(CH2) f’ ) f’’ -C(O)X (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3);

[0230] [ka]

[0231] represents; -C(O)X represents -C(O)OH or an activated ester; Y represents Me, Et, Bu or -(CH2CH2O)3CH3; or b) a compound of formula (III) comprising T 1 but,

[0232] [ka]

[0233] represents; or LT 1 but, * -(C 2-10 ) alkylene-SH; a compound of formula (III) representing 197 By combining with;

[0234] [ka]

[0235] The present invention relates to an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of aspects 12) to 36), which can be obtained or produced.

[0236] Preferably, X is

[0237] [ka]

[0238] Represents.

[0239] Embodiments 13) to 21) are intended to disclose further preferred L's encompassed by this embodiment. Similarly, embodiments 22) to 28) disclose further preferred T's having terminal X-, OY-, or SH- groups as shown in this embodiment. 1 That is, -T- disclosed in these embodiments is intended to disclose CRM 197 For the avoidance of any doubt, the terminal "C(O)-" disclosed in the T of these embodiments refers to a T having a "C(O)X" 1 In the case of squaric acid, CRM 197 The attachment point to 1 is H. These preferred T 1 is deemed to be expressly disclosed.

[0240] 57) A further aspect of the present invention relates to a method for producing an oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of aspects 12) to 36), comprising the steps of: The compound of formula (III) is 197 to a lysine residue of:

[0241] [ka]

[0242] (In the formula, m is 4, 5 or 6, preferably m is 4 or 5, most preferably 4; n is 5, 6 or 7, preferably n is 6 or 7, most preferably 6; L is * -(C 2-10 ) alkylene-NH-; * -(CH2CH2O) a -CH2CH2NH- (a is 1, 2 or 3); * -CH2CH2S-CH2CH2NH-; * -(C 2-10 ) Fluoroalkylene-NH-; * -(CH2) c NHC(O)(CH2) d -NH- (c and d are independently 2 to 6); * -(CH2) e NHC(O)NH(CH2) h -NH- (e and h are independently 2 to 6); * -(C 1-10 ) alkylene-C(O)-NH-(C 2-10 ) alkylene-NH-; or * -(C 2-10 ) alkylene-O-NH-; represents; a) T 1 teeth, -C(O)-(C 0-10 ) alkylene-C(O)X; -C(O)-CH2CH2-(OCH2CH2) r -C(O)X (r is 1 to 5); -C(O)-CH2(CH2) f -(SCH2(CH2) f’ ) f’’ -C(O)X (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3);

[0243] [ka]

[0244] represents; -C(O)X represents -C(O)OH or an activated ester; Y represents Me, Et, Bu or -(CH2CH2O)3CH3; or b) a compound of formula (III) comprising T 1 but,

[0245] [ka]

[0246] represents; or LT 1 but, * -(C 2-10 ) alkylene-SH; a compound of formula (III) representing 197 and combining the

[0247] [ka]

[0248] Preferably, X is

[0249] [ka]

[0250] Represents.

[0251] Embodiments 13) to 21) are intended to disclose further preferred L's encompassed by this embodiment. Similarly, embodiments 22) to 28) disclose further preferred T's having terminal X-, OY-, or SH- groups as shown in this embodiment. 1 That is, -T- disclosed in these embodiments is intended to disclose CRM 197 For the avoidance of any doubt, the terminal "C(O)-" disclosed in the T of these embodiments refers to a T having a "C(O)X" 1 In the case of squaric acid, CRM 197 The attachment point to 1 is H. These preferred T 1 is deemed to be expressly disclosed.

[0252] Whenever the words "between" or "to" are used to describe a range of numerical values, the endpoints of the stated range are expressly disclosed and are intended to be included within that range. This means, for example, that when a temperature range is stated to be between 40°C and 80°C (or 40°C to 80°C), the endpoints of 40°C and 80°C are meant to be included within the range; or, when a variable is defined as an integer between 1 and 4 (or 1 to 4), it means that the variable is the integer 1, 2, 3, or 4.

[0253] However, for the avoidance of any doubt, the term "covalently linked bridge having a backbone length of 5 to 25 atoms, said length being such that the oxygen at C1 of the reducing end of said oligosaccharide is bonded to said carrier protein CRM" is also used. 197 The definition of "the shortest distance between the nitrogen of the amino group of the lysine residue and the oxygen in C1 and CRM" is 197 This means that the nitrogen of the amino group of the lysine residue in is not counted in the backbone numbering thus defined.

[0254] When not used in reference to temperature, the term "about" (or "in the vicinity of") before a numerical value "X" refers in this application to a temperature between 10% of XX and 10% of X+X, preferably between 5% of XX and 5% of X+X. In the specific case of temperatures, the term "about" (or "in the vicinity of") before a temperature "Y" refers in this application to a temperature between Y-10°C and Y+10°C, preferably between Y-5°C and Y+5°C. Furthermore, the term "room temperature" as used herein means a temperature of about 25°C.

[0255] Preparation of Compounds of Formulae (I), (Ia), (Ib), (Ic), (II), (IIa), (IIb), (III), (IIIa) and (IV) A further aspect of the present invention is a method for preparing compounds of formula (I), (Ia), (Ib), (Ic), (II), (IIa), (IIb), (III), (IIIa), and (IV). Compounds according to formula (I), (Ia), (Ib), (Ic), (II), (IIa), (IIb), (III), (IIIa), and (IV) of the present invention can be prepared from commercially available or known starting materials according to the methods described in the experimental section; by analogous methods; or according to the general reaction sequence outlined below, in which L, T, L 1 , T 1 , X and Y are as defined for formulas (I), (Ia), (Ib), (Ic), (II), (IIa), (IIb), (III), (IIIa) and (IV). Other abbreviations used herein are either explicitly defined or as defined in the experimental section.

[0256] The synthesis of the compounds of the present invention requires a protecting group strategy. Such protecting group strategies can be quite sophisticated, but the use of protecting groups is well known in the art (see, for example, "Protective Groups in Organic Synthesis," T.W. Greene, P.G.M. Buts, Wiley-Interscience, 1999). The resulting compounds may be converted into salts, particularly pharmaceutically acceptable salts thereof, by methods known per se.

[0257] Common manufacturing routes: Antigen display

[0258] [ka]

[0259] Scheme 1: O using the NHS-ester method 1 -CRM 197 Synthesis of the conjugate

[0260] [ka]

[0261] O1-antigen 1' in a suitable solvent (e.g., DMSO) is treated in a vial at room temperature with activated Bis-NHS ester 2' of a diacid (e.g., Bis-NHS adipic acid ester, which is commercially available or can be prepared by one skilled in the art using the corresponding Bis-acid and N-hydroxysuccinic acid) (Odom, OW, Biochemistry, Vol. 29, No. 48, 1990) (5-20 equivalents) in DMSO in the presence of triethylamine, and stirred at room temperature for 3 hours. Antigen-NHS ester 3' is precipitated by adding EtOAc, centrifuged, and the precipitate is then washed with EtOAc and dried under vacuum before use in the next step. Antigen-NHS ester 3' (25-100 equivalents) and CRM 197 The resulting O1-antigen-CRM solution is stirred at room temperature for 20-24 hours. 197 The conjugate 4' is washed, purified and stored in an appropriate buffer solution.

[0262] Scheme 2: O using the squaric acid ester (squarate) method 1 -CRM 197 Synthesis of the conjugate

[0263] [ka]

[0264] O1-antigen 1' in a suitable solvent (e.g., HO-EtOH, buffer) is added to the desired alkyl squarate 5' (e.g., 3,4-dibutoxy-3-cyclobutene-1,2-dione, 3,4-(di(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-3-cyclobutene-1,2-dione) (Ganesh et al., JACS, 2014, 136, 16260-16269 and Xu et al., Carbhyd R. Res., 2018, 456, 24-29) and stirred in a solvent at room temperature and an appropriate pH (7-8). The reaction mixture is neutralized with acetic acid and then concentrated under vacuum (or lyophilized). The crude product is purified using a C18 (or SEC) column with water-acetonitrile as the eluent. The product-containing fractions are frozen and lyophilized to give 6'. Antigen-squaric acid ester 6' (25-100 equivalents) and CRM 197 The resulting O1-antigen-CRM was stirred in a 0.5 M borate buffer solution at pH 9 for 24-72 h at room temperature (S. Hou et al., Carbhydr. Res., 2008, 343, 196-210). 197 The conjugate 7' is washed, purified and stored in an appropriate buffer solution.

[0265] Scheme 3: Synthesis of antigen-thiol

[0266] [ka]

[0267] O1-antigen 1' in a suitable solvent (e.g., DMSO) is treated in a vial at rt with 8' (e.g., DSP (dithiobis(succinimidyl propionate)) or DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)) to give the corresponding disulfide, which is then reduced with DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) to give antigen-thiol 9'.

[0268] Scheme 4: Antigen-thiol and functionalized CRM 197 Using O 1 -CRM197 Synthesis of the conjugate

[0269] [ka]

[0270] a) Antigen-thiol-maleimide method 1 -CRM 197 Synthesis of the conjugate Antigen-CRM functionalized with thiol 9' (25-100 equivalents) and maleimide 10' 197 (e.g. CRM 197 (This can be prepared by those skilled in the art by treating with 3-maleimido-propionic acid succinimidyl ester or any other suitable NHS ester with maleimide) (Robert M.F. van der Put et al., ACS Cent. Sci. 2022, 8, 4, 449-460) in a buffer solution containing RM is stirred at room temperature for 20-24 hours. L-cysteine ​​in the buffer is then added to the RM and stirred at room temperature for 1 hour to quench the excess maleimide groups. The resulting O1-antigen-CRM 197 The -thio-maleimide conjugate 11' is washed, purified and stored in an appropriate buffer solution.

[0271] b) Antigen-thiol-ether method 1 -CRM 197 Synthesis of the conjugate Antigen - Protein functionalized with thiol 9' (25-100 equivalents) and α-bromoacetate ester 10' (e.g., CRM 197 and CRM synthesized using SBAP (N-succinimidyl 3-(2-bromoacetamido)propanoate) or any other suitable NHS ester with α-bromoacetate ester. 197 A buffer solution containing O1-antigen-CRM (O1-BAP) (Schumann, B. et al., Chem. Sci., 2014, 5, 1992-2002) was stirred at room temperature for 24 hours. L-cysteine ​​in the buffer solution was then added to the RM, and the mixture was stirred at room temperature for 1 hour to quench the excess α-bromoacetate groups. The resulting O1-antigen-CRM 197The -thio-ether conjugate 11' is washed, purified and stored in an appropriate buffer solution.

[0272] A general retrosynthetic approach to the O1-antigen The O1-antigen can be synthesized using functionalized building blocks as shown in Scheme 5. The fully deprotected antigen RS-1 has a linker L1 at its reducing end, which is essential for conjugation to a protein carrier. RS-1 can be obtained by deprotection of fully protected RS-2. Deprotection strategies may include (acidic or basic) hydrolysis, hydrogenolysis, Birch reduction, or removal of esters, amides, imides, or carbamates via reduction of the azide group to an amine. The deprotection sequence varies depending on the protecting groups and their compatibility with the reaction conditions. One skilled in the art can successfully accomplish this. RS-2 can be obtained from glycosylation of RS-3 (Gal II moiety) as the donor and RS-4 (Gal I moiety) as the acceptor, or by glycosylation of RS-4 (Gal I) with a Gal II repeating unit smaller than RS-3 (e.g., a disaccharide or tetrasaccharide donor). RS-3 (Gal II moiety) can be synthesized from repeating unit RS-5, which in turn can be obtained by glycosylation of RS-6 and RS-7.

[0273] Removal of the LG3 group of RS-8 gives RS-4 (Gal I moiety), which is equipped with an appropriate linker handle (Lx). In this reaction, the RS-9 donor can be treated with a linker handle selected from the various linker handles listed in Table 1 below to give RS-8. RS-9 can be obtained from repeating unit RS-10, which can be obtained in turn by glycosylation of the RS-11 donor and RS-12 acceptor.

[0274] Scheme 5: Retrosynthetic approach to the O1-antigen

[0275] [ka]

[0276] [ka]

[0277] Scheme 6: Introduction of a linker handle

[0278] [ka]

[0279] The linker nucleophile Ln (e.g., 5-azidopentan-1-ol) and RS-9 donor are taken up in a RBF and azeotropically dried under vacuum with dry toluene. The mixture is taken up in an appropriate solvent (e.g., DCM) at rt, 4 Å molecular sieves are added, and the mixture is stirred for 30-45 min under a N2 atmosphere. The RM is cooled to an appropriate temperature (e.g., 0 °C to -20 °C), and an activating agent (e.g., TMSOTf, TfOH) is added to the RM and stirred for 20 min. The RM is then allowed to warm slowly to room temperature over 1 h. The reaction completion is monitored by TLC. The RM is quenched (e.g., with saturated NaHCO3, Na2SO3 solution) and extracted with a solvent (e.g., DCM, EtOAc). The combined organic portions are washed with water and brine, then dried and evaporated under vacuum to give the crude product. The crude product is purified by silica column chromatography using EA / cyclohexane as the eluent. The fractions containing the product are evaporated and dried under vacuum to give the product.

[0280] [Table 1]

[0281] The oligosaccharide portions of the claimed compounds, i.e., the length ranges claimed, may be prepared as exemplified in the experimental section below, or by methods analogous thereto. [Example]

[0282] Experimental section: Abbreviations (used in this section and above): AcOH acetic acid aq. aqueous solution Bn Benzyl BSA Bovine serum albumin CDCl3 deuterated chloroform Cs2CO3 Cesium Carbonate Cy Cyclohexane D2O deuterium oxide DCM dichloromethane DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DMAP 4-(dimethylamino)pyridine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide ELISA enzyme-linked immunosorbent assay equiv equivalent ESI Electrospray Ionization Et3N(TEA) Triethylamine EtOAc(EA) ethyl acetate EtOH ethanol EtSH Ethanethiol Fr fraction h time H2 Hydrogen H2O Water H2SO4 Sulfuric Acid HCl Hydrochloric acid HPLC High Performance Liquid Chromatography HPLC-SEC High Performance Liquid Chromatography-Size Exclusion Chromatography I2 Iodine ICU intensive care unit IPA Isopropanol LPS lipopolysaccharide M molar concentration MeOH Methanol Min MS Molecular Sieves N2 nitrogen Na sodium Na2S2O3 Sodium thiosulfate Na2SO4 Sodium Sulfate NaCl Sodium chloride NaHCO3 Sodium Bicarbonate NaOMe Sodium methoxide NaPi buffer sodium phosphate buffer NH2NH2Hydrazine NIS N-iodosuccinimide NMR nuclear magnetic resonance spectroscopy PBS Phosphate-buffered saline PBS-T: Phosphate-buffered saline containing 0.1% (v / v) Tween-20 Pd(OH)2 palladium hydroxide Pd / C Palladium Carbon py pyridine RBF Round Bottom Flask RM reaction mixture rt room temperature sat. saturation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SM starting material sol. solution TBAF Tetrabutylammonium Fluoride TBS Tris-buffered saline TDS Dimethyl-Thexylsilylchlorid TLC thin layer chromatography TMB 3,3',5,5'-tetramethylbenzidine TMSOTf Trimethylsilyl trifluoromethanesulfonate UV ultraviolet light

[0283] I. Chemistry The following examples illustrate the preparation of biologically active compounds of the present invention but are not intended to limit its scope in any way.

[0284] General Information : All reagents and solvents were used as purchased, and solvents used in reactions were anhydrous. Except for reactions involving water as a solvent, all reactions were performed under a N atmosphere in dry glassware (purchased from VWR and ROTH). It is highly recommended to azeotropically dry the acceptor and donor twice with anhydrous toluene before glycosylation. A Heidolph magnetic stirrer was used to perform the experiments. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 glass plates (Merck) or aluminum plates (VWR). Developed TLC plates were visualized under a short-wave UV lamp by heating the plates immersed in a sugar stain (3-methoxyphenol (0.225 mL), H2SO4 (6 mL), and EtOH (200 mL)). All automated flash chromatography purifications on silica gel (FlashPure Silica 40 μm irregular: BUCHI columns) were performed on Biotage Isolera and Biotage Select. A BUCHI rotary evaporator was used to evaporate solvents. Combinations of dry ice and acetone and ice / water were used to cool the reaction mixture to the desired temperature. All NMR experiments were performed on a BRUKER 400 MHz instrument.

[0285] Temperatures are given in degrees Celsius (°C). In mixtures, the proportions of solvents or eluents or mixtures of reagents in liquid form are given as volumetric relationships (v / v) unless otherwise specified.

[0286] Characterization methods used: HPLC-SEC: The glycoconjugates used for immunization were analyzed by HPLC-SEC to determine the bound and unbound CRM. 197 Mass differences between proteins were observed. Samples were diluted with 50 mM Tris, 20 mM NaCl, pH 7.2, and run on an Agilent 1100 HPLC system equipped with a Tosoh TSK G2000 column (SWx1, 7.8 mm x 30 cm, 5 μm) and a Tosoh TSK Gel Guard column (SWx1 6.0 mm x 4 cm, 7 μm). The flow rate was maintained at 1 mL / min.

[0287] SDS-PAGE: Samples were diluted in Laemmli loading buffer and heated to 95°C for 5 min. After cooling to RT for 5 min, approximately 2-2.5 μg of sample was loaded into wells of a 10% polyacrylamide gel along with approximately 5 μL of protein size marker. Samples were run at a constant voltage of 120 V for approximately 30-45 min. Staining was performed using GelCode® Blue Safe Protein Stain according to the manufacturer's instructions. Gels were washed overnight with deionized water and scanned.

[0288] Synthesis of thexyldimethylsilyl 4,6-O-benzylidene-2-O-benzyl-3-O-naphthylmethyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside A3:

[0289] [ka]

[0290] A2 (see WO2019106201, p. 164) (51.0 g, 80 mmol) and A1 (see WO2019106201, p. 162) (61.9 g, 92 mmol) were dissolved in anhydrous toluene (3 × 100 mL), azeotropically dried, and the residue was dried under high vacuum for 1 h. The dried mixture was dissolved in anhydrous toluene (750 mL) and dioxane (250 mL) at rt, 4 Å molecular sieves were added, and the mixture was stirred for 45 min under a N2 atmosphere. The RM was cooled to 0 °C using an ice-water bath, and TMSOTf (1.45 mL, 8.03 mmol) was added to the RM. The RM was stirred at 5 °C for 20 min. The RM was then allowed to warm slowly to room temperature over 1 h. TLC analysis (20% EA / cyclohexane) indicated the reaction was complete. The RM was quenched with saturated NaHCO3 (250 mL) and stirred for 10 min. Extraction with EA (200 mL x 3) was performed. The combined organic portions were washed with water (100 mL), brine (50 mL), dried (Na2SO4) and evaporated under vacuum to give the crude product. Column purification on silica was performed on a biotage using EA / cyclohexane, and the product was evaporated and dried under high vacuum to give a white sticky solid A3 (68.4 g, 76%). HRMS (ESI+) C 66 H 70O 14 NaSi + [M+Na] + The calculated value was 1137.4427 and the measured value was 1337.4432.

[0291] Synthesis of thexyldimethylsilyl 2,4-di-O-benzyl-3-O-naphthylmethyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside A4:

[0292] [ka]

[0293] A3 (47 g, 42.1 mmol) was taken up in THF (250 mL) and dried 4 Å molecular sieves were added thereto and stirred at rt for 15 min. 1 M BH3-THF solution (169 mL, 169 mmol) was added to the RM and stirred for 5 min. After that, TMSOTf (0.76 mL, 4.21 mmol) was added and stirred at rt for 16 h. TLC analysis showed the reaction was complete. The RM was slowly quenched with methanol (35 mL) at rt (be careful of foaming), stirred for 45 min, and then diluted with saturated NaHCO3 solution (250 mL) and EA (300 mL). The RM was stirred vigorously for 2 h. It was diluted with water (200 mL). The layers were separated. The aqueous layer was extracted with EA (100 mL x 3). The combined organic portions were washed with brine (100 mL), dried (NaSO), filtered, and evaporated in vacuo to give a colorless, viscous liquid. The crude product was purified by column chromatography using EtOAc / cyclohexane. The product was eluted with 20-30% EtOAc / Cy. The fractions containing the product spot were evaporated on a rotary evaporator to give A4 (29.8 g, 63%) as a colorless, viscous liquid. HRMS (ESI+) C 66 H 72 O 14 NaSi + [M+Na] + The calculated value was 1139.4584 and the measured value was 1339.4583.

[0294] Synthesis of thexyldimethylsilyl 6-O-benzoyl-2,4-di-O-benzyl-3-O-naphthylmethyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside A5:

[0295] [ka]

[0296] A4 (29.8 g, 26.7 mmol) was taken in DCM (300 mL) at rt, and pyridine (10.79 mL, 133 mmol) and DMAP (0.33 g, 2.67 mmol) were added thereto and stirred for 5 min. Then, BzCl (6.19 mL, 53.3 mmol) was added thereto and stirred for 18 h. TLC analysis (20% EA / Cy) showed the reaction was complete. The RM was diluted with NaHCO (100 mL), and the layers were separated. The aqueous layer was extracted with DCM (100 mL x 2). The combined organic layers were washed with brine solution (50 mL), dried (NaSO), filtered, and evaporated under vacuum to give a light brown residue. This was purified by biotage using a silica column and EtOAc and cyclohexane as eluents. The fractions containing the product spot were collected, evaporated under vacuum, and dried under high vacuum to give a white sticky liquid A5 (28 g, 86%). HRMS(ESI+) C 73 H 76 O 15 NaSi + [M+Na] + The calculated value was 1243.4846 and the measured value was 1243.4827.

[0297] Synthesis of thexyldimethylsilyl 6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside A6:

[0298] [ka]

[0299] Substrate A5 (14 g, 11.46 mmol) was taken in DCM (150 mL) and PBS buffer solution (300 mL) at rt. DDQ (5.2 g, 22.92 mmol) was added portionwise over 1 h. The RM turned black and then reddish-brown and was stirred for 3 h. TLC analysis (20% EA / Cy) showed a polar spot and a small amount of SM. Therefore, stirring was continued for another 2 h. The RM was quenched with NaHCO3 solution (250 mL) and extracted with DCM (100 mL x 3). The combined organic portions were washed with NaHCO3 solution (250 mL), brine solution (100 mL), dried (Na2SO4), filtered, and concentrated in vacuo to give the crude product. The crude solid was triturated with methanol and filtered. The residue was washed with methanol to give a white solid, which was dried under high vacuum (fr1, 7.5 g), and the mother liquor was evaporated under vacuum to give a yellow solid, which was triturated with methanol to give a second crop as a white solid (2.37 g). The total yield of A6 was therefore 9.85 g (80%). HRMS (ESI+) C 62 H 68 O 15 NaSi + [M+Na] + The calculated value was 1103.4220 and the measured value was 1103.4225.

[0300] Synthesis of thexyldimethylsilyl 6-O-benzoyl-2,4-di-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside A7:

[0301] [ka]

[0302] Substrate A6 (21 g, 19.17 mmol) was taken in DCM (200 mL) at rt, and LevOH (6.68 g, 57.5 mmol), DMAP (0.47 g, 3.83 mmol), and DIPEA (50.2 mL, 288 mmol) were added and stirred for 5 min. HOBt (2.94 g, 19.17 mmol) and EDC.HCl (18.38 g, 96 mmol) were then added and stirred at rt overnight. TLC analysis (20% EA / Cy) showed the presence of a polar spot. Therefore, the RM was diluted with DCM (500 mL) and washed with dilute aqueous HCl (250 mL × 2) followed by saturated NaHCO3 solution (250 mL × 2). The organic layer was washed with brine (200 mL), dried (Na2SO4), filtered, and concentrated in vacuo to give the crude product as a brown oil. Purification by silica column chromatography using EA and cyclohexane as eluents was performed, and the fractions containing the pure product spot were collected separately, evaporated under vacuum, and dried under high vacuum to give an off-white fluffy solid A7 (20.3 g, 89%). HRMS (ESI+) C 67 H 74 O 17 NaSi + [M+Na] + The calculated value was 1201.4587 and the measured value was 1201.4596.

[0303] Synthesis of 6-O-benzoyl-2,4-di-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside A8:

[0304] [ka]

[0305] Substrate A7 (29.2 g, 24.76 mmol) was taken in DCM (250 mL) in a 1 L RBF at rt, to which AcOH (22 mL, 384 mmol) was added and stirred for 5 min. Then, 1 M TBAF in THF (371 mL, 371 mmol) was added to the RM and stirred at rt for 24 h. TLC analysis showed the reaction was complete. Therefore, it was diluted with water (200 mL) and DCM (100 mL). The layers were separated. The aqueous layer was extracted with DCM (200 mL x 2). The combined organic layers were washed with water (250 mL), saturated NaHCO3 solution (250 mL), brine solution (250 mL), dried (Na2SO4), filtered, and evaporated in vacuo. Purification by biotage using a silica column and EA and cyclohexane as eluents, fractions containing the product spot were collected, evaporated under vacuum and dried under high vacuum to give an off-white sticky solid A8 (22 g, 86%). HRMS (ESI+) C 59 H 56 O 17 Na + [M+Na] + The calculated value was 1059.3410 and the measured value was 1059.3411.

[0306] Synthesis of 6-O-benzoyl-2,4-di-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl 2,2,2-trifluoro-N-phenylacetimidate A9:

[0307] [ka]

[0308] Hemiacetal A8 (21.1 g, 20.4 mmol) was taken in DCM (100 mL) under N2 atmosphere at rt, and Cs2CO3 (26.5 g, 81 mmol) was added thereto and stirred for 5 min. Then, (E)-2,2,2-trifluoro-N-phenylacetimidoyl chloride (12.7 g, 9.7 mL, 61 mmol) was added thereto and stirred overnight. TLC analysis showed the reaction was complete, with no SM present. Therefore, the RM was filtered through Celite to remove the solid, and the residue was washed with DCM (100 mL x 4). The filtrate was concentrated under vacuum and co-evaporated with toluene (100 mL) three times. Evaporation and drying under vacuum afforded a pale yellowish fluffy solid A9 (23.11 g, 94%). HRMS (ESI+) C 67 H 64 F3N2O 17 + [M+NH4] + The calculated value was 1225.4152 and the measured value was 1225.4132.

[0309] Synthesis of thexyldimethylsilyl 6-O-benzoyl-2,4-di-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside A10:

[0310] [ka]

[0311] Acceptor A6 (17.5 g, 16.18 mmol) and imidate donor A9 (23.46 g, 19.42 mmol) were taken in DCM (270 mL) at rt, 4A molecular sieves were added, and the mixture was stirred at rt for 45 min under a N atmosphere. The RM was cooled to -10 °C using a cold acetone bath, and TMSOTf (1.23 mL, 3.24 mmol) was added to the RM. The RM was stirred at 0 °C for 15 min and then slowly warmed to 5 °C over 1 h. TLC analysis (25% EA / Cy) indicated the reaction was complete, with the absence of acceptor SM and the presence of a slight polar spot. The RM was quenched with saturated NaHCO solution (100 mL), the layers were separated, and the aqueous layer was extracted with DCM (100 mL x 2). The combined organic layers were washed with saturated NaHCO3 solution (100 mL), brine solution (100 mL), dried (Na2SO4), filtered, and evaporated in vacuo. Purification by silica gel column chromatography using EA / Cy afforded the pure product as a white fluffy solid A10 (28 g, 82%). MALDI-TOF C 121 H 122 NaO 31 Si + [M+Na] + The calculated value was 2121.7632 and the measured value was 2121.883.

[0312] Synthesis of tetrasaccharide acceptor A11:

[0313] [ka]

[0314] Lev-substrate A10 (28 g, 13.33 mmol) was taken up in DCM (162 mL)-pyridine (16 mL) at rt, to which hydrazine acetate (6.14 g, 66.7 mmol) was added and stirred at rt for 18 h. TLC showed the presence of a slightly non-polar sugar-active spot relative to the Rf value of SM in 30% EA / hexane. The RM was then quenched with acetone (10 mL) and stirred at rt for 45 min. The RM was then evaporated to dryness under vacuum. The residue was purified by silica column chromatography using EA-Cy as the eluent to obtain the sugar-active spot, which, after evaporation and drying under high vacuum, gave the desired compound as a white fluffy solid A11 (24.4 g, 91%). MALDI-TOF C 116 H 116 NaO 29 S + [M+Na] + The calculated value was 2023.7269 and the measured value was 2024.259.

[0315] Synthesis of tetrasaccharide hemiacetal A12:

[0316] [ka]

[0317] Substrate A10 (28 g, 13.33 mmol) was taken in DCM (135 mL) in a 1 L RBF at rt, and AcOH (12.3 mL, 213 mmol) was added thereto and stirred for 5 min. Then, 1 M TBAF in THF (200 mL, 200 mmol) was added to the RM and stirred at rt for 20 h. TLC analysis showed that the reaction was nearly complete. Therefore, it was diluted with water (200 mL) and DCM (100 mL). The layers were separated. The aqueous layer was extracted with DCM (50 mL x 2). The combined organic layers were washed with water (100 mL), saturated NaHCO3 solution (100 mL x 2), and brine solution (100 mL), dried (Na2SO4), filtered, and evaporated in vacuo. Purification was carried out using a silica column and EA and Cy as eluents, and the fractions containing the product spot were evaporated under vacuum and dried under high vacuum to give a white fluffy solid A12 (23 g, 88%). 113 H 104 NaO 31 + [M+Na] + The calculated value was 1979.6454 and the measured value was 1979.764.

[0318] Synthesis of tetrasaccharide imidate donor A13:

[0319] [ka]

[0320] Hemiacetal A12 (25.7 g, 13.13 mmol) was taken in DCM (130 mL) under a N atmosphere at rt, and CsCO (17.11 g, 52.5 mmol) was added thereto and stirred for 5 min. (E)-2,2,2-trifluoro-N-phenylacetimidoyl chloride (8.17 g, 6.2 mL, 39.4 mmol) was then added thereto and stirred overnight. TLC analysis indicated the reaction was complete, with a strong nonpolar spot and the absence of SM. Therefore, the RM was filtered through Celite to remove the solid, and the residue was washed with DCM (100 mL x 4). The filtrate was concentrated under vacuum and coevaporated with toluene (100 mL) three times. Evaporation and drying under vacuum gave a pale yellowish fluffy solid A13 (27.9 g, quantitative).

[0321] Synthesis of octasaccharide A14:

[0322] [ka]

[0323] Both acceptor A11 (11.9 g, 5.78 mmol) and donor A13 (13.9 g, 6.36 mmol) were taken in a RBF and azeotropically dried under vacuum with dry toluene. The mixture was taken up in DCM (130 mL) at room temperature, to which 4A molecular sieves were added, and stirred for 45 min under a N atmosphere. The RM was cooled to -10 °C, and TMSOTf (0.2 mL, 1.16 mmol) was added to the RM. The RM was stirred at -5 °C for 20 min. The RM was then allowed to warm slowly to room temperature over 1 h. TLC analysis (30% EA / Cy) showed the presence of a strong spot of slightly polar product. The RM was quenched with saturated NaHCO (250 mL), stirred for 10 min, and extracted with DCM (100 mL x 3). The combined organic portions were washed with water (100 mL), brine (100 mL), dried (Na2SO4), and evaporated in vacuo to give the crude product. Column purification was performed on a biotage using a silica column with EA / Cy. The product-containing fractions were evaporated and dried in vacuo to give the desired product as a white foamy solid A14 (21.8 g, 93%). MALDI-TOF C 229 H 218 NaO 59 Si + [M+Na] + The calculated value was 3962.3720 and the measured value was 3963.910.

[0324] Synthesis of octasaccharide hemiacetal A15:

[0325] [ka]

[0326] Substrate A14 (21.6 g, 5.48 mmol) was taken in DCM (55 mL) in a 500 mL RBF at rt, to which AcOH (4.9 mL, 85 mmol) was added and stirred for 5 min. Then, TBAF (82 mL, 82 mmol) was added. The RM was stirred at rt for 24 h. TLC analysis showed the reaction was complete. Therefore, it was diluted with water (200 mL) and DCM (100 mL). The layers were separated. The aqueous layer was extracted with DCM (100 mL x 2). The combined organic layers were washed with saturated NaHCO3 solution (100 mL x 2), brine solution (100 mL), dried (Na2SO4), filtered, and evaporated in vacuo. Purification on Biotage using a silica column with EA and Cy as eluents was performed. Fractions containing the glycosyl dye-active product spot were pooled, evaporated under vacuum, and dried under high vacuum to give an off-white solid as the desired product A15 (17.8 g, 85%). MALDI-TOF C 221 H 200 NaO 59 + [M+Na] + The calculated value was 3820.2542 and the measured value was 3821.537.

[0327] Synthesis of octasaccharide imidate donor A16:

[0328] [ka]

[0329] Hemiacetal A15 (17.6 g, 4.6 mmol) was taken in DCM (100 mL) under a N atmosphere at rt, and CsCO (6.0 g, 18.5 mmol) was added thereto and stirred for 5 min. (E)-2,2,2-trifluoro-N-phenylacetimidoyl chloride (2.9 g, 2.2 mL, 13.9 mmol) was then added thereto and stirred overnight. TLC analysis indicated the reaction was complete, with a strong nonpolar spot and the absence of SM. Therefore, the RM was filtered through Celite to remove the solid, and the residue was washed with DCM (100 mL x 4). The filtrate was concentrated under vacuum and coevaporated with toluene (100 mL) three times. Evaporation and drying under vacuum gave a pale yellowish fluffy solid A16 (18.3 g, quantitative).

[0330] Synthesis of octasaccharide with linker A17:

[0331] [ka]

[0332] 5-Azidopentan-1-ol (1.661 g, 12.86 mmol) and imidate donor A16 (17.02 g, 4.29 mmol) were placed in a RBF and azeotropically dried under vacuum with dry toluene. The mixture was taken up in DCM (425 mL) at room temperature, to which 4A molecular sieves were added, and stirred for 30 min under a N atmosphere. The RM was cooled to -7 °C, and TMSOTf (0.155 ml, 0.857 mmol) was added to the RM. The RM was stirred at -5 °C for 20 min. The RM was allowed to warm slowly to room temperature over 1 h. The RM was quenched with saturated NaHCO (250 mL), stirred for 10 min, and extracted with DCM (100 mL x 3). The combined organic portions were washed with water (100 mL), brine (100 mL), dried (NaSO), and evaporated under vacuum to give the crude product. Column purification was performed on a biotage using a silica column with EA / cyclohexane. The product-containing fractions were evaporated and dried under vacuum to give the product as a white foamy solid A17 (13.14 g, 78%). MALDI-TOF C 226H 210 N3O 59 + [M+H] + The calculated value was 3909.3519 and the measured value was 3910.123.

[0333] Synthesis of octasaccharide acceptor A18:

[0334] [ka]

[0335] To a solution of starting material A17 (7.8 g, 1.99 mmol) in DCM (50 mL) was added a solution of hydrazine hydrate (0.26 g, 7.98 mmol) dissolved in acetic acid (4.34 mL, 76 mmol) and pyridine (6.45 mL, 80 mmol). The resulting reaction mixture was stirred at rt for 18 h. The reaction was quenched by the addition of acetone (3 mL), stirred for 1 h, and the solvent was removed in vacuo to give the crude product. The crude product was purified on a silica column using automated flash chromatography with EA-cyclohexane as the eluent to give a sugar-active spot, which after evaporation and drying under high vacuum gave the desired compound as a white fluffy solid A18 (6.97 g, 92%). MALDI-TOF C 221 H 204 N3O 57 + [M+H] + The calculated value was 3811.3151 and the measured value was 3811.774.

[0336] Synthesis of B2

[0337] [ka]

[0338] To a stirred solution of compound B1 (Angew. Chem. Int. Ed., 2011, 50.7315) (72.4 g, 0.1406 mol) in DMF (724 mL) was added NaH (5.0 g, 0.2250 mol) portionwise at 0 °C, followed by dropwise addition of BnBr (38.50 g, 0.2110 mol), and the reaction mixture was stirred at rt for 14 h. After completion of the reaction was confirmed by TLC, the reaction mixture was poured into ice water, and the precipitated solid was filtered, washed with 5% ethyl acetate in hexane, and dried under vacuum to give compound B2 (79.1 g, 93%) as a colorless solid.

[0339] Synthesis of B3

[0340] [ka]

[0341] To a stirred solution of compound B2 (79.1 g, 0.1308 mol) in DCM:MeOH (1:1, 1.582 L) at RT, P-TSA (24.98 g, 0.1308 mol) was added, and the reaction mixture was stirred at RT for 12 h. After completion of the reaction was confirmed by TLC and HPLC, the reaction mixture was quenched with ice-cold water. The volatiles were removed in vacuo, and the residue was diluted with water and extracted with DCM (2 x 500 mL). The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure. The crude compound thus obtained was purified by column chromatography on silica gel eluting with 35-40% ethyl acetate in hexane to give compound B3 (50.6 g, 75%) as a viscous liquid.

[0342] Synthesis of B4

[0343] [ka]

[0344] To a stirred solution of compound B3 (50.6 g, 0.098 mol) in DCM (506 mL) at rt, pyridine (27.11 g, 0.3432 mol) was added dropwise, followed by BzCl (48.24 g, 0.3432 mol), and DMAP (2.39 g, 0.0196 mol) at 0 °C. The resulting reaction mixture was stirred at rt for 14 h. After completion of the reaction was monitored by TLC, the reaction mixture was quenched with ice-cold water and extracted with DCM (2 × 400 mL). The combined organic layers were washed with 5% citric acid solution, water, dried over NaSO, and evaporated under reduced pressure. The crude compound thus obtained was washed with 20% EtOAc in hexane to give compound B4 (57.5 g, 81%) as an off-white solid.

[0345] Synthesis of B5

[0346] [ka]

[0347] To a stirred solution of compound B4 (57.5 g, 0.0794 mol) in DCM:water (9:1, 1.15 L) was added DDQ (36 g, 0.1588 mol) portionwise at 0 °C. The reaction mixture was then stirred at rt for 6 h. After confirming the completion of the reaction by TLC and LCMS, the reaction mixture was filtered through a celite bed. The filtrate was diluted with DCM and washed with sodium thiosulfate and saturated NaHCO3 solution. The organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude product thus obtained was purified by column chromatography on silica gel eluting with 15–20% ethyl acetate in hexane to give compound B5 (40.3 g, 87%) as a viscous liquid.

[0348] B6 synthesis

[0349] [ka]

[0350] To a stirred solution of compound B5 (40.3 g, 0.690 mol) in DCM (806 mL) at 0 °C, LevOH (9.6 g, 0.0828 mol) was added dropwise, followed by DIPC (11.31 g, 0.0897 mol) and DMAP (2.53 g, 0.0207 mol) at the same temperature. The reaction mixture was stirred at rt for 5 h. After completion of the reaction was confirmed by TLC, the reaction mixture was quenched with ice-cold water and extracted with DCM. The organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude compound thus obtained was purified by column chromatography on silica gel eluting with 10–15% ethyl acetate in hexane to give compound B6 (36.27 g, 77%) as a colorless gum. 1 H NMR (400MHz, chloroform-d) δ 7.99(ddd, J=8.5, 3.1, 1.4Hz, 4H), 7.74-7.24(m, 14H), 7.07(d, J=8.0Hz, 2H), 5.74(dd, J=3. 4, 1.1Hz, 1H), 5.17(dd, J=9.6, 3.3Hz, 1H), 4.84(d, J=10.8Hz, 1H), 4.74(d, J=9.7Hz, 1H), 4. 63(d, J=10.8Hz, 1H), 4.57(dd, J=11.4, 7.0Hz, 1H), 4.34(dd, J=11.4, 5.8Hz, 1H), 4.14(ddd, J=6.9, 5.6, 1.1Hz, 1H), 3.80(t, J=9.6Hz, 1H), 2.88-2.36(m, 4H), 2.34(s, 3H), 2.07(s, 3H).

[0351] Synthesis of B7

[0352] [ka]

[0353] To a stirred solution of compound B6 (36.27 g, 0.0531 mol) in DCM:water (9:1, 725.4 mL) at 0 °C, NIS (35.85 g, 0.1593 mol) was added portionwise and stirred at rt for 3 h. After completion of the reaction was confirmed by TLC, the reaction mixture was evaporated under reduced pressure to remove volatiles. The crude product thus obtained was diluted with DCM, washed with sodium thiosulfate and brine solutions, and the organic layer was dried over Na2SO4 and evaporated under vacuum. The crude product was purified by column chromatography on silica gel eluting with 20–25% ethyl acetate in hexane to give compound B7 (25.4 g, 83%) as a pale yellow viscous liquid.

[0354] Synthesis of B8

[0355] [ka]

[0356] To a stirred solution of compound B7 (25.4 g, 0.0440 mol) in DCM (508 mL) at 0° C., DSK-457E (22.88 g, 0.1101 mol) and then cesium carbonate (42.8 g, 0.1321 mol) were added and stirred at rt for 8 h. After completion of the reaction was confirmed by TLC, the rm was filtered through a pad of Celite, and the filtrate was evaporated under reduced pressure. The crude product thus obtained was washed with hexane at −20° C. and dried to give compound B8 (23.38 g, 71%) as a pale yellow viscous liquid.

[0357] Synthesis of B9

[0358] [ka]

[0359] To a stirred solution of compound B1 (100 g, 0.1943 mol) in DCM (1 L) were added pyridine (20.38 mL, 0.2526 mol), BzCl (28.98 mL, 0.2526 mol), and then DMAP (14.22 g, 0.1165 mol) dropwise at 0 °C. The reaction mixture was then stirred at rt for 14 h. After completion of the reaction was confirmed by TLC, the reaction mixture was poured into ice water. The organic layer was separated and washed with cold 5% citric acid solution (2 × 250 mL), brine solution (250 mL), dried over Na2SO4, and evaporated under reduced pressure. The residue thus obtained was triturated with 5% ethyl acetate in hexane to give compound B9 (100.8 g, 84%) as a colorless solid.

[0360] B10 synthesis

[0361] [ka]

[0362] A stirred solution of compound B9 (100.8 g, 0.1629 mol) in DCM:water (9:1, 2 L) was cooled to 5-10 °C, and NIS (109.95 g, 0.4887 mol) was added portionwise. The mixture was warmed to rt and stirred for 4 h. After complete consumption of SM was confirmed by TLC, the reaction mixture was quenched with 10% sodium thiosulfate solution, and the layers were separated. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude compound thus obtained was dissolved in DCM (2 L) and cooled to 0 °C. TEA (227 mL, 1.6291 mol) was added dropwise, and the reaction mixture was stirred at rt for 12 h. After the completion of the reaction was confirmed by TLC, the reaction mixture was evaporated under reduced pressure to give the crude compound, which was purified by column chromatography on silica gel eluting with 35-40% ethyl acetate in hexane to give compound B10 as an off-white solid (64.6 g, 77%).

[0363] B11 synthesis

[0364] [ka]

[0365] To a stirred solution of compound B10 (64.6 g, 0.1260 mol) in DMF (517 mL) at rt, imidazole (21.45 g, 0.315 mol) was added dropwise at 0 °C, followed by TDS-Cl (44.86 g, 0.2520 mol). The reaction mixture was stirred at rt for 12 h. After completion of the reaction was confirmed by TLC, the reaction mixture was poured into ice-cold water and extracted with ethyl acetate (2 × 250 mL). The combined organic layers were washed with water and dried over NaSO. The mixture was then evaporated under reduced pressure. The crude compound thus obtained was purified by column chromatography on silica gel eluting with 12–14% EtOAc in hexane to give compound B11 (61.8 g, 75%) as an off-white solid.

[0366] B12 synthesis

[0367] [ka]

[0368] To a stirred solution of compound B11 (61.8 g, 0.0944 mol) in THF (1.23 L) at rt, BH3 (283 mL, 0.2834 mol) in THF was added dropwise at -10 °C, followed by TMSO-Tf (4.2 g, 0.0188 mol), and the mixture was stirred at the same temperature for 3 h. After confirming the completion of the reaction by TLC and LCMS, the reaction mixture was quenched by adding methanol and then TEA dropwise until the mixture was basic (pH ∼8) (foaming was observed), and the mixture was stirred at RT for 14 h. The reaction mixture was evaporated under reduced pressure, and the crude product was diluted with DCM, washed with water, dried over Na2SO4, and evaporated under reduced pressure. The crude compound thus obtained was purified by column chromatography on silica gel eluting with 15–20% ethyl acetate in hexane to give compound B12 (47.73 g, 77%) as a colorless viscous liquid.

[0369] B13 synthesis

[0370] [ka]

[0371] To a stirred solution of compound B12 (47.73 g, 0.0727 mol) in DCM (477 mL) at RT, pyridine (11.51 g, 0.1455 mol) was added, followed by dropwise addition of BzCl (15.28 g, 0.1091 mol) at 0 °C. DMAP (0.88 g, 0.0072 mol) was added to the reaction mixture, which was then stirred at RT for 12 h. After completion of the reaction was confirmed by TLC, the reaction mixture was quenched with ice-cold water and extracted with DCM (2 × 400 mL). The combined organic layers were washed with 5% acetic acid solution, dried over NaSO, and evaporated under reduced pressure. The crude compound thus obtained was purified by column chromatography on silica gel eluting with 10–15% ethyl acetate in hexane to give compound B13 (43.13 g, 78%) as a colorless solid.

[0372] B14 synthesis

[0373] [ka]

[0374] To a stirred solution of compound B13 (43.13 g, 0.0566 mol) in DCM:water (9:1, 862.6 mL) at RT, DDQ (38.59 g, 0.17 mol) was added portionwise at 0 °C. The reaction mixture was stirred at RT for 6 h. After completion of the reaction was confirmed by TLC and LCMS, the reaction mixture was filtered through a pad of Celite. The filtrate was diluted with DCM and washed with sodium thiosulfate and saturated NaHCO3 solution. The organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude compound thus obtained was purified by column chromatography on silica gel eluting with 10–15% EtOAc in hexane to give B13 (25.68 g, 73%) as a pale yellow viscous liquid. 1H NMR (400MHz, chloroform-d) δ 8.24-7.78(m, 4H), 7.82-7.27(m, 11H), 5.26(dd, J=10.1, 7.6Hz, 1H), 5.05-4.71(m, 3H), 4.54(dd, J=11.2, 7.3Hz, 1H), 4.41(dd, J=11. 2, 5.7Hz, 1H), 3.93(dd, J=3.6, 1.2Hz, 1H), 3.90-3.80(m, 2H), 1.49(hept, J=6.9Hz, 1H), 0.80-0.60(m, 12H), 0.14(s, 3H), 0.08(s, 3H).

[0375] Synthesis of B16

[0376] [ka]

[0377] BH3.THF (63 mL, 63.9 mmol) was added dropwise to a stirred solution of compound B15 (see WO2019106201, page 147) (8.0 g, 15.9 mmol) in DCM (80 mL) under a nitrogen atmosphere at 0 °C, followed by the addition of TMSOTf (1.5 mL, 7.9 mmol) over 30 min at the same temperature. The reaction mixture was allowed to warm to room temperature and stirred at rt for 5 h. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with methanol (500 mL) and TEA (20 mL) and evaporated to dryness to obtain a crude residue. The crude residue was quenched with NaHCO3 (500 mL) and extracted with DCM (3 × 500 mL). The combined organic layers were washed with water (2 × 500 mL), saturated brine (1 × 500 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound thus obtained was washed with hexane to give compound B16 as an off-white solid (5.0 g, 62%).

[0378] Synthesis of B17

[0379] [ka]

[0380] TEA (6.7 mL, 47.7 mmol) was added dropwise to a stirred solution of compound B16 (3.0 g, 5.9 mmol) in DCM (30 mL) at 0 °C under a nitrogen atmosphere, and BzCl (2.8 mL, 23.8 mmol) and then DMAP (145 mg, 1.9 mmol) were added at the same temperature. The reaction mixture was allowed to warm to room temperature and stirred at rt for 13 h. After completion of the reaction was confirmed by TLC, the reaction mixture was quenched with ice water, extracted with DCM (3 × 500 mL), and the layers were separated. The combined organic layers were washed with water (2 × 500 mL), saturated brine (1 × 500 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude compound thus obtained was further recrystallized using hexane to give compound B17 as an off-white solid (3.5 g, 83%).

[0381] Synthesis of B18

[0382] [ka]

[0383] NIS (2.3 g, 105.0 mmol) was added to a stirred solution of compound B17 (5.0 g, 7.0 mmol) in DCM:water (25 mL:5 mL) at 0 °C under a nitrogen atmosphere, and TFA (0.27 mL, 3.5 mmol) was added dropwise over 30 min at the same temperature. The reaction mixture was allowed to warm to room temperature and stirred at rt for 1 h. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with saturated NaHCO (200 mL), extracted with DCM (2 × 500 mL), and the layers were separated. The combined organic layer was washed with water (2 × 500 mL), saturated brine (1 × 500 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude compound thus obtained was purified by column chromatography on silica gel (60-120 mesh) eluted with 10-15% EtOAc in hexane to give compound B18 as a pale yellow liquid (3.4 g, 79%).

[0384] Synthesis of B19

[0385] [ka]

[0386] CClCN (3.2 mL, 32.0 mmol) was added dropwise to a stirred solution of compound B18 (2.0 g, 3.2 mmol) in DCM (20 mL) at −10° C. under a nitrogen atmosphere, followed by the dropwise addition of DBU (0.049 mL, 0.032 mmol) over a period of 30 min at the same temperature. The reaction mixture was stirred at 0° C. for 1 h. After completion of the reaction was confirmed by TLC, the reaction mixture was concentrated under reduced pressure. The crude residue thus obtained was purified by column chromatography on silica gel (60-120 mesh) eluting with 30-40% EtOAc in hexane to give compound B19 (2.3 g, 95%) as a pale yellow liquid.

[0387] Synthesis of B20

[0388] [ka]

[0389] To a stirred solution of compound B19 (2.3 g, 3.0 mmol) in DCM (25 mL), 4A molecular sieves were added and stirred for 15 min. Allyl alcohol (0.65 mL, 9.0 mmol) was then added dropwise under a nitrogen atmosphere at 0 °C, followed by the dropwise addition of TMSOTf (0.05 mL, 0.3 mmol) dissolved in DCM (25 mL). The reaction mixture was allowed to warm to room temperature and stirred at rt for 10 h. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with saturated NaHCO3 solution (50 mL), extracted with DCM (3 × 50 mL), and the layers were separated. The combined organic layer was washed with water (2 × 50 mL), saturated brine (1 × 50 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude material thus obtained was further recrystallized using hexane to give compound B20 as an off-white solid (800 mg, 42%). HRMS (ESI+) C 41 H 38 O8Na + [M+Na] + The calculated value was 681.2459 and the measured value was 681.2512.

[0390] Synthesis of B21

[0391] [ka]

[0392] DDQ (0.69 g, 3.04 mmol) was added to a stirred solution of compound B20 (800 mg, 1.21 mmol) in DCM:PBS buffer pH 7.4 (12 mL:12 mL) over 2.5 h and stirred at rt for 4 h. After completion of the reaction was confirmed by TLC, the reaction mixture was filtered through a pad of Celite, the filtrate was washed with saturated NaHCO3 solution (50 mL), extracted with DCM (2 x 50 mL), and the layers were separated. The combined organic layers were washed with water (2 x 50 mL), saturated brine (1 x 50 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to give the crude compound. The crude compound thus obtained was purified by column chromatography on silica gel eluting with EtOAc in hexane to give compound B21 as an off-white solid (0.57 g, 91%). HRMS (ESI+) C 41 H 38 O8Na + [M+Na] + The calculated value was 541.1833 and the measured value was 541.1883.

[0393] Synthesis of thexyldimethylsilyl 4,6-di-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranoside B22:

[0394] [ka]

[0395] A mixture of donor B8 (11.91 g, 15.93 mmol) and acceptor B14 (8.24 g, 13.27 mmol) was dissolved in anhydrous toluene (3 × 50 mL) and azeotropically dried. The residue was dried under high vacuum for 20 min. The residue was dissolved in anhydrous DCM (180 mL) and microwave-dried. MS4Å was added, and the resulting yellow solution was stirred at RT for 1 h. The solution was then cooled to 0 °C, and TMSOTf (0.48 mL, 2.65 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 40 min, after which TLC reaction control indicated completion of the reaction. The reaction mixture was allowed to reach RT and quenched by adding TEA (0.37 mL), stirred for an additional 5 min, and then filtered through a cotton plug covered with sea sand and plug washed with DCM (3 × 100 mL). The filtrate was concentrated under reduced pressure to give the crude product. Purification by flash column chromatography on a silica column using EA / cyclohexane as eluent gave the desired compound B22 as a white foam (13.46 g, 86%). 1 H NMR (400MHz, CDCl3) δ 8.17-7.84(m, 8H), 7.74-7.12(m, 22H), 5.68(dd, J=10.2, 7.5Hz, 1H), 5.51-5.36(m, 2H), 5.23-5.09(m, 2H), 4.89-4.57(m, 4H), 4.46(dd, J=11.3, 7.2Hz, 1H), 4.38-4.26 (m, 2H), 4.11-3.98(m, 3H), 3.96-3.87(m, 2H), 3.71(t, J=6.3Hz, 1H), 2.97-2.29(m, 4H), 2.10(s, 3H), 1.53-1.43(m, 1H), 0.82-0.60(m, 12H), 0.13(s, 3H), 0.02(s, 3H). HTMS-QTOF C 67 H 74 NaO 17 Si + [M+Na] + The calculated value was 1201.4587 and the measured value was 1201.4351.

[0396] Synthesis of allyl 4,6-di-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranoside B23:

[0397] [ka]

[0398] A mixture of donor B8 (39.8 g, 53.2 mmol) and acceptor B21 (23 g, 44.4 mmol) was taken in a 1 L RBF, and dry 4 Å molecular sieves (MS) and anhydrous DCM (590 mL) were added. It was stirred at rt for 45 min and then cooled to 0 °C. TMSOTf (1.6 mL, 8.87 mmol) was added to the reaction mixture and stirred for 30 min. It was then slowly warmed to 10 °C for 0.5 h and then stirred at rt for 1 h. The reaction was quenched at 5 °C by the addition of TEA (1.2 mL, 8.87 mmol). The crude product was purified by flash chromatography using an EtOAc / Cy gradient system. The collected fractions were concentrated under vacuum and dried under high vacuum for 16 h to give product B23 as an off-white fluffy solid (45.1 g, 94%). 1 H NMR (400MHz, CDCl3) δ 8.23-7.81(m, 8H), 7.80-6.81(m, 22H), 5.88-5.63(m, 2H), 5.53-5.33(m, 2H), 5.25-4.97(m, 4H), 4.84- 4.46(m, 5H), 4.45-4.26(m, 3H), 4.22-3.85(m, 6H), 3.78-3.67(m, 1H), 2.94-2.24(m, 4H), 2.08(s, 3H). HRMS-QTOF C 62 H 60 NaO 17 + [M+Na] + The calculated value was 1099.3723 and the measured value was 1099.3817.

[0399] Synthesis of disaccharide acceptor B24:

[0400] [ka]

[0401] B22 (13.46 g, 11.41 mmol) was dissolved in DCM (285 mL), and to this stirred, colorless solution at RT was added a mixture of hydrazine monohydrate (1.42 mL, 45.70 mmol), pyridine (36.9 mL, 457 mmol), and AcOH (24.8 mL, 434 mmol) and stirred for 2 h. The reaction was quenched by the addition of acetone (50 mL) and stirred for an additional 45 min. The reaction mixture was concentrated under reduced pressure to give the crude product as an oily residue, which was purified by automated flash column chromatography using EA / cyclohexane to give the desired compound B24 as a colorless foam (12.16 g, 99%). HRMS-QTOF C 62 H 72 NO 15 Si + [M+NH4] + The calculated value was 1098.4666 and the measured value was 1098.4429.

[0402] Synthesis of disaccharide acceptor B25:

[0403] [ka]

[0404] To a solution of B23 (15 g, 13.93 mmol) in DCM was added a solution of hydrazine hydrate (1.7 mL, 55.7 mmol) dissolved in AcOH (30.3 mL, 529 mmol) and py (45 mL, 557 mmol). The resulting reaction mixture was stirred at rt for 2 h. The reaction was quenched by the addition of acetone (50 mL) and the solvent was removed in vacuo to give the crude product. The crude product was purified by automated flash chromatography using an EtOAc / Cy gradient system as the eluent. Concentration of the solvent in vacuo from the test tube containing the product gave a white solid B25 (12.93 g, 95%). HRMS-QTOF C 57 H 54 NaO 15 + [M+Na] + The calculated value was 1001.3355 and the measured value was 1001.3309.

[0405] Synthesis of disaccharide hemiacetal B26 from B22:

[0406] [ka]

[0407] B22 (16.14 g, 13.68 mmol) was azeotropically dried with dry toluene, and the residue was dried under high vacuum for 30 min. The above material was then dissolved in anhydrous DCM (140 mL), to which AcOH (12.53 mL, 219 mmol) was added and stirred for 5 min. A 1 M solution of TBAF in anhydrous THF (205 mL, 205 mmol) was added to the RM, and the reaction mixture was stirred at RT for 24 h until the reaction was complete. The reaction was quenched by the addition of water (100 mL), the phases were separated, and the aqueous phase was extracted with DCM (2 x 100 mL). The combined organic phases were washed with saturated aqueous NaHCO (200 mL) and brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give a crude colorless oil. The crude material was purified by automated flash column chromatography using DCM / cyclohexane as eluent and the product-containing fractions were concentrated to give the desired product as a white form B26 (14.11 g, 99%). 59 H 60 NO 17 + [M+NH4] + The calculated value was 1054.3856 and the measured value was 1054.3628.

[0408] Synthesis of disaccharide hemiacetal B26 from B23:

[0409] [ka]

[0410] B23 (26 g, 24.1 mmol) and N,N'-dimethylbarbituric acid (DMBA) (9.42 g, 60.3 mmol) were taken up in MeOH (290 mL) under nitrogen (suspension), to which THF (190 mL) was added (a clear solution resulted) and heated to 45 °C for 2 min. The RM was degassed and backfilled with N three times, and then 5 mol% Pd(PPh3)4 (1.40 g, 1.2 mmol) was added. The RM was then degassed again and the reaction vessel was backfilled with N2 (twice). Heating of the yellow solution was continued at 50 °C for 16 h. The RM was cooled to rt, diluted with EtOAc (500 mL), washed with NaHCO3 solution (200 mL), brine (150 mL), dried over Na2SO4, filtered, and concentrated in vacuo. Purification on a flash chromatography system using an EtOAC / Cy gradient system gave the product B26 as a yellowish fluffy solid (21.52 g, 86%). 59 H 56 NaO 17 + [M+Na] + The calculated value was 1059.3410 and the measured value was 1059.3384.

[0411] Synthesis of disaccharide donor B27:

[0412] [ka]

[0413] B26 (21.52 g, 20.75 mmol) was dissolved in anhydrous DCM (260 mL). Cs2CO3 (13.52 g, 41.5 mmol) and 2,2,2-trifluoro-N-phenylacetimidoyl chloride (9.9 mL, 62.3 mmol) were added to this solution. The reaction mixture was stirred at rt overnight. The reaction mixture was filtered through a pad of Celite. The pad was washed with DCM (250 mL), and the filtrate was concentrated under reduced pressure, co-evaporated with toluene, and dried under high vacuum to give a pale yellow solid B27 (25 g, 100%). HRMS-QTOF C 67 H 60 F3NNaO 17 + [M+Na]+ The calculated value was 1230.3706 and the measured value was 1230.3672.

[0414] Synthesis of thexyldimethylsilyl 4,6-di-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranoside B28:

[0415] [ka]

[0416] Both acceptor B24 (6.62 g, 6.12 mmol) and donor B27 (8.51 g, 7.04 mmol) were taken up in a RBF and azeotropically dried under vacuum with dry toluene. The mixture was taken up in anhydrous DCM (120 mL) at rt, 4 Å molecular sieves were added to it, and stirred for 30 min under a N atmosphere. The RM was cooled to −2 °C using an ice-water bath, and TMSOTf (0.22 mL, 1.224 mmol) was added to the RM. The RM was stirred at 5 °C for 20 min. The RM was then allowed to warm slowly to room temperature over 1 h. TLC analysis was performed to monitor the completion of the reaction. The RM was quenched by the addition of TEA (0.2 mL), stirred for an additional 5 min, then filtered through a cotton plug covered with sea sand, the plug washed with DCM (3 × 100 mL), and concentrated under vacuum. Column purification of the crude product was carried out on silica using EA / cyclohexane on a Biotage®. The product-containing fractions were evaporated and dried under vacuum to give the desired compound as a white form B28 (10.95 g, 85%). HRMS-QTOF C 121 H 122 NaO 31 Si + [M+Na] + The calculated value was 2121.7632 and the measured value was 2121.6391.

[0417] Synthesis of allyl 4,6-di-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranoside B29:

[0418] [ka]

[0419] B27 (19.55 g, 16.18 mmol) and B25 (13.2 g, 13.48 mmol) were taken in a RBF, and dry 4 Å molecular sieves (MS) and anhydrous DCM (270 mL) were added. It was stirred at rt for 45 min and then cooled to 0 °C. TMSOTf (0.49 mL, 2.70 mmol) was added to the reaction mixture and stirred for 30 min. It was then slowly warmed to 10 °C for 0.5 h and then stirred at rt for 1 h. The reaction was quenched at 5 °C by the addition of TEA (0.38 mL, 2.70 mmol). The crude product was purified by flash chromatography using an EtOAc / Cy gradient system. The collected fractions were concentrated under vacuum and dried under high vacuum for 16 h to give the product B29 as an off-white fluffy solid (25 g, 93%). HRMS-QTOF C 116 H 108 NaO 31 + [M+Na] + The calculated value is 2019.6767 and the measured value is 2019.6693.

[0420] Synthesis of tetrasaccharide hemiacetal B30 from B28:

[0421] [ka]

[0422] Substrate B28 (13 g, 6.19 mmol) was azeotropically dried with dry toluene, and the residue was dried under high vacuum for 30 min. The above material was then dissolved in anhydrous DCM (60 mL), to which AcOH (5.67 mL, 99 mmol) was added and stirred for 5 min. A 1 M solution of TBAF in anhydrous THF (93 mL, 93 mmol) was added to the RM, and the reaction mixture was stirred at RT for 24 h until the reaction was complete. The reaction was quenched by the addition of water (50 mL) and vortexed for 5 min. The phases were separated, and the aqueous phase was extracted with DCM (2 x 50 mL). The combined organic phases were washed with saturated aqueous NaHCO (50 mL x 2) and brine (50 mL). The combined organic phases were dried over anhydrous NaSO, filtered, and concentrated in vacuo to give a crude colorless oil. The crude material was purified by automated flash column chromatography using EA / Cy as eluent to give the desired product as a white form B30 (11.3 g, 93%). 113 H 108 NO 31 + [M+NH4] + The calculated value was 1974.6900 and the measured value was 1974.7003.

[0423] Synthesis of tetrasaccharide hemiacetal B30 from B29:

[0424] [ka]

[0425] [(1,5-Cyclooctadiene)(pyridine)(tricyclohexylphosphine)-Ir(I)]PF (0.56 g, 0.661 mmol) was dissolved in THF (65 mL), and N was bubbled through the solution at rt for 2 min while the red catalyst dissolved. The solution was then purged with H for 5 min until the red solution turned colorless, and the solution was stirred under hydrogen for 15 min. The solution of the activated catalyst was then added via syringe to a solution of B29 (13.2 g, 6.61 mmol) in THF (130 mL) under N and stirred at rt for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and evaporated to give the allylic isomerized compounds (isomerization was 1 (Confirmed by H NMR). The vinyl substrate was then taken up in a mixture of THF:H2O (2:1, 130 mL:65 mL) and I2 (3.35 g, 13.21 mmol) was added at rt. The brown solution was stirred for 2 h before being quenched with a 10% solution of Na2S2O3 (250 mL). The aqueous phase was extracted with EtOAc (3 x 100 mL), and the combined organic layers were dried over Na2SO4, filtered, and the solvent was evaporated. Flash column chromatography (EtOAc / Cy gradient system) afforded the product as a yellow solid B30 (12 g, 93%). HRMS-QTOF C 113 H 104 NaO 31 + [M+Na] + The calculated value was 1979.6454 and the measured value was 1979.6497.

[0426] Synthesis of tetrasaccharide donor B31:

[0427] [ka]

[0428] B30 (7.4 g, 3.78 mmol) was dissolved in anhydrous DCM (47 mL). Cs2CO3 (2.46 g, 7.56 mmol) and 2,2,2-trifluoro-N-phenylacetimidoyl chloride (1.80 mL, 11.34 mmol) were added to the solution. The reaction mixture was stirred at rt overnight. The reaction mixture was filtered through a pad of Celite. The pad was washed with DCM (150 mL x 2), and the filtrate was concentrated under reduced pressure, co-evaporated with toluene, and dried under high vacuum to give a pale yellow solid B31 (8.0 g, 99%). HRMS-QTOF C 121 H 108 F3NNaO 31 + [M+Na] + The calculated value was 2150.6750 and the measured value was 2150.6774.

[0429] Synthesis of tetrasaccharide acceptor B32:

[0430] [ka]

[0431] To a solution of B28 (3 g, 1.428 mmol) in DCM (35 mL) was added a solution of hydrazine hydrate (0.18 mL, 5.71 mmol) dissolved in acetic acid (3.11 mL, 54.3 mmol) and pyridine (4.62 mL, 57.1 mmol). The resulting reaction mixture was stirred at rt for 2 h. The reaction was quenched by the addition of acetone (20 mL) and the solvent was removed in vacuo to give the crude product. The crude product was purified by automated flash chromatography using an EtOAc / Cy gradient system as eluent. Concentration of the solvent in vacuo from the test tube containing the product (based on TLC) gave a white solid B32 (2.5 g, 87%). HRMS-QTOF C 116 H 116 NaO 29 Si + [M+Na] + The calculated value was 2023.7264 and the measured value was 2023.6054.

[0432] Synthesis of tetrasaccharide acceptor B33:

[0433] [ka]

[0434] To a solution of B29 (11 g, 5.51 mmol) in DCM (140 mL) was added a solution of hydrazine hydrate (0.68 mL, 22.02 mmol) dissolved in acetic acid (12.0 mL, 209 mmol) and pyridine (17.81 mL, 220 mmol). The resulting reaction mixture was stirred at rt for 2 h. The reaction was quenched by the addition of acetone (20 mL) and the solvent was removed in vacuo to give the crude product. The crude product was purified by automated flash chromatography using an EtOAc / Cy gradient system as eluent. Concentration of the solvent in vacuo from the test tube containing the product (based on TLC) gave a white solid, B33 (10.1 g, 97%). HRMS-QTOF C 111 H 102 NaO 29 + [M+Na] + The calculated value was 1921.6399 and the measured value was 1921.6449.

[0435] Synthesis of octasaccharide B34:

[0436] [ka]

[0437] B31 (8.07 g, 3.79 mmol) and B33 (6.0 g, 3.16 mmol) were taken in a RBF, and dry 4 Å molecular sieves (MS) and anhydrous DCM (65 mL) were added. It was stirred at rt for 45 min and then cooled to 0 °C. TMS-OTf (0.12 mL, 0.63 mmol) was added to the reaction mixture and stirred for 30 min. It was then slowly warmed to 10 °C for 0.5 h and then stirred at rt for 1 h. The reaction was quenched at 5 °C by the addition of triethylamine (0.09 mL, 0.63 mmol). The crude product was purified by flash chromatography using an EtOAc / Cy gradient system. The collected fractions were concentrated under vacuum and dried under high vacuum for 16 h to give product B34 as an off-white fluffy solid (9.96 g, 82%). MALDI-TOF C 224 H 204 NaO 59 + [M+Na] + The calculated value was 3860.2855 and the measured value was 3861.337.

[0438] Synthesis of octasaccharide acceptor B35:

[0439] [ka]

[0440] To a solution of B34 (12 g, 3.12 mmol) in DCM (78 mL) was added a solution of NH2NH2 (0.40 mL, 12.50 mmol) dissolved in AcOH (6.80 mL, 119 mmol) and Py (10.10 mL, 125 mmol). The resulting reaction mixture was stirred at rt for 2 h. The reaction was quenched by the addition of acetone (10 mL), and the solvent was removed in vacuo to give the crude product. The crude product was purified by automated flash chromatography using an EtOAc / Cy gradient system as the eluent. Concentration of the solvent in vacuo from the test tube containing the product (based on TLC) gave a white solid, B35 (10.52 g, 90%). MALDI-TOF C 219 H 198 NaO 57 +[M+Na] + The calculated value was 3762.2487 and the measured value was 3764.559.

[0441] Synthesis of octasaccharide hemiacetal B36:

[0442] [ka]

[0443] [(1,5-Cyclooctadiene)(pyridine)(tricyclohexylphosphine)-Ir(I)]PF (0.22 g, 0.258 mmol) was dissolved in THF (25 mL), and N was bubbled through the solution at rt for 2 min while the red catalyst dissolved. The solution was then purged with H for 2 min until the red solution turned colorless, and the solution was stirred under hydrogen for 15 min. The solution of the activated catalyst was then added via syringe to a solution of B34 (9.9 g, 2.58 mmol) in THF (50 mL) under N and stirred at rt for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and evaporated to give the allylic isomerized compounds (isomerization was 1 (Confirmed by H NMR). The vinyl substrate was taken up in a mixture of THF:H2O (2:1, 50 mL:25 mL) and I2 (1.31 g, 5.16 mmol) was added at rt. The brown solution was stirred for 2 h and then quenched with a 10% solution of Na2S2O3 (100 mL). The aqueous phase was extracted with EtOAc (3 x 50 mL), and the combined organic layers were dried over Na2SO4, filtered, and the solvent was evaporated. Flash column chromatography (EtOAc / Cy gradient system) afforded the product as a yellow solid B36 (8.0 g, 82%). MALDI-TOF C 221 H 200 NaO 59 + [M+Na] + The calculated value was 3820.2542 and the measured value was 3821.524.

[0444] Synthesis of octasaccharide donor B37:

[0445] [ka]

[0446] B36 (5 g, 1.316 mmol) was dissolved in anhydrous DCM (17 mL). CsCO (0.86 g, 2.63 mmol) and 2,2,2-trifluoro-N-phenylacetimidoyl chloride (0.82 g, 3.95 mmol) were added to the solution. The reaction mixture was stirred at rt overnight. The reaction mixture was filtered through a pad of Celite. The pad was washed with DCM (250 mL), and the filtrate was concentrated under reduced pressure and azeotropically dried with toluene. The residue was dried under high vacuum for 16 h to give the product B37 as an off-white fluffy solid (5.1 g, 98%).

[0447] Synthesis of dodecasaccharide B38 using an (8+4) approach:

[0448] [ka]

[0449] B37 (1.6 g, 0.403 mmol) and B32 (0.81 g, 0.403 mmol) were taken in a RBF, and dry 4 Å MS and anhydrous DCM (6 mL) were added. It was stirred at rt for 45 min and then cooled to 0 °C. TMSOTf (15 μL, 0.081 mmol) was added to the reaction mixture and stirred for 30 min. It was then slowly warmed to 10 °C for 0.5 h and then stirred at rt for 1 h. The reaction was quenched at 5 °C by the addition of saturated aqueous NaHCO (25) and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and evaporated to give the crude product. The crude product was purified by flash chromatography using an EtOAc / Cy gradient system. The collected fractions were concentrated in vacuo and dried under high vacuum for 16 h to give the product B38 as a white fluffy solid (1.63 g, 70%).

[0450] Synthesis of dodecasaccharide B39 using a (4+8) approach:

[0451] [ka]

[0452] B31 (7.17 g, 3.37 mmol) and B35 (10.5 g, 2.81 mmol) were taken in a RBF, and dry 4 Å MS and anhydrous DCM (56 mL) were added. It was stirred at rt for 45 min and then cooled to 0 °C. TMSOTf (0.10 mL, 0.56 mmol) was added to the reaction mixture and stirred for 30 min. It was then slowly warmed to 10 °C for 0.5 h and then stirred at rt for 1 h. The reaction was quenched at 5 °C by the addition of TEA (0.08 mL, 0.56 mmol). The crude product was purified by flash chromatography using an EtOAc / Cy gradient system. The collected fractions were concentrated under vacuum and dried under high vacuum for 16 h to give the product B39 as a white fluffy solid (12.10 g, 76%). MALDI-TOF C 332 H 300 KO 87 + [M+K] + The calculated value was 5716.8682 and the measured value was 5714.291.

[0453] Synthesis of dodecasaccharide hemiacetal B40 from B38:

[0454] [ka]

[0455] B38 (1.63 g, 0.282 mmol) was azeotropically dried with dry toluene, and the residue was dried under high vacuum for 30 min. The above material was then dissolved in anhydrous DCM (3 mL), to which AcOH (0.25 mL, 4.37 mmol) was added and stirred for 5 min. A 1 M solution of TBAF in anhydrous THF (4.23 mL, 4.23 mmol) was added to the RM, and the reaction mixture was stirred at RT for 24 h until the reaction was complete. The reaction was quenched by the addition of water (100 mL) and extracted with DCM (2 x 50 mL). The combined organic phases were washed with saturated aqueous NaHCO (200 mL) and brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give a crude colorless oil. The crude material was purified by automated flash column chromatography using EtOAc / cyclohexane as eluent and the product-containing fractions were concentrated to give the desired product as a white Form B40 (1.26 g, 79%).

[0456] Synthesis of dodecasaccharide hemiacetal B40 from B39:

[0457] [ka]

[0458] [(1,5-Cyclooctadiene)(pyridine)(tricyclohexylphosphine)-Ir(I)]PF (0.12 g, 0.141 mmol) was dissolved in THF (15 mL), and N was bubbled through the solution at rt for 2 min while the red catalyst dissolved. The solution was then purged with H for 2 min until the red solution turned colorless, and the solution was stirred under hydrogen for 15 min. The solution of the activated catalyst was then added via syringe to a solution of B39 (8.0 g, 1.408 mmol) in THF (30 mL) under N and stirred at rt for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and evaporated to give the allylic isomerized compounds (isomerization was 1(Confirmed by H NMR). The vinyl substrate was taken up in a mixture of THF:H2O (2:1, 28 mL:14 mL) and I2 (0.71 g, 2.82 mmol) was added at rt. The brown solution was stirred for 2 h and then quenched with a 10% solution of Na2S2O3 (50 mL). The aqueous phase was extracted with EtOAc (3 x 5 mL), and the combined organic layers were dried over Na2SO4, filtered, and the solvent was evaporated. Flash column chromatography (EtOAc / Cy gradient system) afforded the product as a yellow solid B40 (5.7 g, 72%). MALDI-TOF C 329 H 296 NaO 87 + [M+Na] + The calculated value was 5660.8630 and the measured value was 5665.367.

[0459] Synthesis of dodecasaccharide donor B41:

[0460] [ka]

[0461] B40 (2.0 g, 0.354 mmol) was dissolved in anhydrous DCM (5 mL). CsCO (0.231 g, 0.709 mmol) and 2,2,2-trifluoro-N-phenylacetimidoyl chloride (0.17 mL, 1.063 mmol) were added to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered through a pad of Celite. The pad was washed with DCM (50 mL x 2), and the filtrate was concentrated under reduced pressure. The collected fractions were concentrated and azeotropically dried with toluene under vacuum to give the product B41 as an off-white fluffy solid (2.0 g, 99%).

[0462] 5-Azido-pentyl 4,6-di-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactopyranosyl Synthesis of furanosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)]-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside C1:

[0463] [ka]

[0464] Both acceptor A18 (2 g, 0.525 mmol) and donor B31 (1.23 g, 0.577 mmol) were taken up in a RBF and azeotropically dried under vacuum with dry toluene. The mixture was taken up in anhydrous toluene (15 mL) at room temperature, 4 Å molecular sieves were added to it, and the mixture was stirred for 30 min under a N atmosphere. The RM was cooled to -2 °C using an ice-water bath, and TMSOTf (19 μL, 0.1 mmol) was added to the RM. The RM was stirred at 5 °C for 20 min. The RM was then allowed to warm slowly to room temperature over 1 h. TLC analysis was performed to monitor the completion of the reaction. The RM was quenched with saturated NaHCO3, stirred for 10 min, and extracted with EA. The combined organic portions were washed with water, brine, dried (Na2SO4), and evaporated under vacuum to give the crude product. Column purification on silica was performed on a Biotage® column using EA / cyclohexane. The product containing fractions were evaporated and dried under vacuum to give the desired product as a fluffy white solid C1 (2.15 g, 71%). 1 H NMR (400MHz, CDCl3) δ 8.27-6.54(m, 180H), 5.92-5.42(m, 13H), 5.40-4.84(m, 17H), 4.77(d, J=3.8Hz, 1H), 4.73-3.45(m, 87H), 3.30(dt , J=9.6, 6.4Hz, 1H), 3.11(t, J=6.9Hz, 3H), 2.80-2.24(m, 4H), 2.05(s, 3H), 1.57-1.39(m, 4H), 1.36-1.17(m, 2H). MALDI-TOF C 334 H 306 N3O 87 + [M+H] + The calculated value was 5749.9607 and the measured value was 5749.992.

[0465] Synthesis of dodecasaccharide acceptor C2:

[0466] [ka]

[0467] C1 Lev-substrate (2.1 g, 0.365 mmol) was taken in DCM-pyridine (25 mL-0.5 mL) at rt, to which hydrazine acetate (0.5 g, 5.48 mmol) was added and stirred at rt for 20 h. TLC analysis indicated the reaction was complete. The RM was quenched with acetone (5 mL), stirred for 1 h, and then evaporated under vacuum to give the crude product. Purification was carried out on a Biotage® silica column using EA / Cy as the eluent. The product-containing fractions were collected and evaporated under vacuum to give the desired product as a fluffy white solid. The product fraction was applied to an LH-20 SEC column using 60% CHCl3 / methanol as the eluent to give the product SEC fraction, which was evaporated under vacuum and dried to give C2 (1.9 g, 92%) as a fluffy white solid. MALDI-TOF C 329 H 300 N3O 85 + [M+H] + The calculated value was 5651.9239 and the measured value was 5651.881.

[0468] 5-Azido-pentyl 4,6-di-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl Lactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β- D-Galactopyranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofurano Synthesis of syl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)]-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside C3:

[0469] [ka]

[0470] Both acceptor C2 (1.9 g, 0.336 mmol) and donor B31 (0.83 g, 0.386 mmol) were taken in a RBF and azeotropically dried under vacuum with dry toluene. The mixture was taken up in anhydrous toluene (20 mL) at room temperature, 4 Å molecular sieves were added, and the mixture was stirred for 30 min under a N atmosphere. The RM was cooled to −2 °C using an ice-water bath, and TMSOTf (12 μL, 0.067 mmol) was added to the RM. The RM was stirred at 5 °C for 20 min. The RM was then allowed to warm slowly to room temperature over 1 h. TLC analysis was performed to monitor the completion of the reaction. The RM was quenched with saturated NaHCO3, stirred for 10 min, and extracted with EA. The combined organic portions were washed with water, brine, dried (Na2SO4), and evaporated under vacuum to give the crude product. Column purification on silica was performed on a Biotage® column using EA / cyclohexane. The product containing fractions were evaporated and dried under vacuum to give the desired product as a fluffy solid C3 (2.15 g, 84%). 1 H NMR (400MHz, CDCl3) δ 8.29-6.55(m, 240H), 5.96-5.30(m, 16H), 5.29-5.12(m, 4H), 5.12-4.82(m, 16H), 4.75(d, J=3.8Hz , 1H), 4.72-3.03(m, 111H), 2.77-2.23(m, 4H), 2.05(s, 3H), 1.55-1.38(m, 4H), 1.37-1.19(m, 2H). MALDI-TOF C 442 H 402 N3O 115 + [M+H] + The calculated value was 7590.5695 and the measured value was 7590.489.

[0471] Synthesis of the hexadecasaccharide acceptor C4:

[0472] [ka]

[0473] Lev-substrate C3 (2.1 g, 0.276 mmol) was taken in DCM-pyridine (25 mL-0.5 mL) at rt, to which hydrazine acetate (0.38 g, 4.15 mmol) was added and stirred at rt for 20 h. The RM was quenched with acetone (5 mL), stirred for 1 h, and then evaporated under vacuum to give the crude product. Purification was carried out on a Biotage® silica column using EA / Cy as the eluent. The product-containing fractions were collected and evaporated under vacuum to give the desired product as a fluffy white solid C4 (1.91 g, 92%). MALDI-TOF C 437 H 396 N3O 113 + [M+H] + The calculated value was 7492.5327 and the measured value was 7492.520.

[0474] Using the (4+16) approach, 5-azido-pentyl 4,6-di-O-benzoyl-2-O-benzyl-3-O-levulinoyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl Lactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β- D-Galactopyranosyl-(1→3)-4,6-di-O-benzoyl-2-O-benzyl-α-D-galactopyranosyl-(1→3)-2,6-di-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl Synthesis of Lactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranosyl-(1→3)]-6-O-benzoyl-2,4-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,5,6-tri-O-benzoyl-β-D-galactofuranoside C5:

[0475] [ka]

[0476] Both acceptor C4 (1.85 g, 0.245 mmol) and donor B31 (0.6 g, 0.282 mmol) were taken in a RBF and azeotropically dried under vacuum with dry toluene. The mixture was taken up in anhydrous toluene (20 mL) at room temperature, 4 Å molecular sieves were added, and the mixture was stirred for 30 min under a N atmosphere. The RM was cooled to −2 °C using an ice-water bath, and TMSOTf (9 μL, 0.05 mmol) was added to the RM. The RM was stirred at 5 °C for 20 min. The RM was then allowed to warm slowly to room temperature over 1 h. TLC analysis was performed to monitor the completion of the reaction. The RM was quenched with saturated NaHCO3, stirred for 10 min, and extracted with EA. The combined organic portions were washed with water, brine, dried (Na2SO4), and evaporated under vacuum to give the crude product. Column purification on silica was performed on a Biotage® column using EA / cyclohexane. The fractions containing the product were evaporated and dried under vacuum to give the desired product, which was further purified on an SEC column using LH-20 resin and 60% CHCl3 in MeOH as the eluent. The fractions containing the product were evaporated and dried under vacuum to give the desired product C5 (1.35 g, 58%). MALDI-TOF C 550 H 497 N3NaO 143 + [M+Na] + The calculated value was 9453.1603 and the measured value was 9453.779.

[0477] Synthesis of C5 using the (12+8) approach:

[0478] [ka]

[0479] A18 (500 mg, 0.131 mmol) and B41 (991 mg, 0.170 mmol) were taken in DCM (25 mL) at rt, to which 4 Å MS was added and stirred for 45 min. The RM was cooled to -10 °C using a cold acetone bath, and TMSOTf (5 μL, 0.028 μmol) was added. The RM was stirred at -10 °C for 5 min and slowly warmed to 5 °C over 1 h. The reaction was monitored for completion using TLC analysis (45% EtOAc / Cy). The RM was quenched with NaHCO3 solution (30 mL), stirred for 10 min, and the layers were separated. The aqueous layer was extracted with DCM (10 mL x 2). The combined organic layers were washed with water (20 mL), brine solution (20 mL), dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified by silica column chromatography using EtOAc / Cy to obtain fractions containing the product, which upon evaporation in vacuo afforded the desired product C5 (880 mg, 71%). 1 H NMR (400MHz, CDCl3) δ 8.30-6.52(m, 300H), 5.95-5.31(m, 18H), 5.29-5.13(m, 6H), 5.09-4.79(m, 20H), 4.75(d, J=3.7Hz , 1H), 4.69-2.98(m, 139H), 2.77-2.24(m, 4H), 2.05(s, 3H), 1.58-1.39(m, 4H), 1.37-1.20(m, 2H).

[0480] Synthesis of partially protected decasaccharide C6:

[0481] [ka]

[0482] Substrate C5 (1.8 g, 0.191 mmol) was taken in 15 mL of THF at room temperature, to which excess 0.5 M NaOMe in methanol (28.6 mL, 14.31 mmol) was added, and stirring was continued at 55 °C for 20 h. Then, 0.5 mL of water was added, and stirring was continued for another day. The RM was cooled to room temperature and evaporated to dryness. Water was added to the residue and mixed well. All the colored solids dissolved, except for some off-white solids. Therefore, it was filtered through a syringe filter equipped with a PTFE bed, and the residue was washed with warm water (10 mL x 5). The solid was washed with a very dilute aqueous acetic acid solution (5 drops of AcOH in 25 mL of water) and then with hot water (10 mL x 3). The solid was dried on a rotary evaporator to give a pale yellow solid, which was analyzed by nmr and MALDI. The product was further purified on an LH-20 SEC column using methanol-CHCl3 as the eluent, and the product fractions were collected, evaporated, and dried under vacuum to give the desired product as a pale yellowish solid C6 (834 mg, 84%). 265 H 332 N3O 101 + [M+H] + The calculated value was 5172.0930 and the measured value was 5172.072.

[0483] 5-Amino-pentyl α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→3) Synthesis of -α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranosyl-(1→3)-α-D-galactopyranosyl-(1→3)-β-D-galactofuranoside C7:

[0484] [ka]

[0485] Substrate C6 (60 mg) was taken up in a mixture of IPA:EA:water (1.5:1.25:1) to give a cloudy mixture, to which AcOH (25 μL), Pd / C (30 mg), and Pd(OH) (30 mg) were added and hydrogenated under ~11 bar H atmosphere for 20 h. The RM was filtered through a PTFE filter and washed with methanol and 50% aqueous methanol. The filtrate was concentrated under vacuum to give the crude product, which was purified using a C18-sepak column with water-acetonitrile as the eluent. All fractions were frozen, lyophilized, and analyzed by nmr and Maldi. The product fractions were further purified by SEC on G-25 resin with water as the eluent. The product fractions from SEC were collected, frozen, and lyophilized to give a fluffy white solid as the desired product C7 (13.23 mg, 34%). Substrate C6 (50 mg) was taken in mL of a mixture of IPA:EA:water:PBS (3:1:1:0.5) to give a cloudy mixture. AcOH (10 μL) was added and stirred for 5 min. Pd / C (100 mg) was taken in mL of a solvent mixture of IPA:water (1:0.5) and dimethylamine hydrochloride (4 mg) was added. The mixture was mixed well and maintained at room temperature for 15 min. This Pd / C suspension was transferred to a vial containing the substrate in the solvent mixture and hydrogenated under a H2 atmosphere at ~5 bar for 20–24 h. The RM was filtered through a PTFE filter and washed with methanol and water. The filtrate was concentrated under vacuum to give the crude product, which was purified using a C18-Sepak column with water-acetonitrile as the eluent. All fractions were frozen, lyophilized, and analyzed by NMR and Maldi. The product fractions were further purified by SEC on G-25 resin using water as the eluent. The product fractions from SEC were collected, frozen, and lyophilized to give a fluffy white solid as the desired product C7 (12.9 mg, 40%). 1H NMR (400MHz, D2O) δ 5.18(s, 3H), 5.16(d, J=3.9Hz, 5H), 5.12(d, J=4.0Hz, 1H), 5.06(s, 4H), 5.01(s, 1H), 4.70-4.56(m, 6H), 4.39(s, 3H), 4.32-3.53(m, 119H), 2.98(t, J=7.6Hz, 2H), 1.72-1.59(m, 4H), 1.48-1.37(m, 2H). MALDI-TOF C 125 H 213 NNaO 101 + [M+Na] + The calculated value was 3367.1454 and the measured value was 3367.167.

[0486] Synthesis of comparative compound "Compar":

[0487] [ka]

[0488] The synthesis of the octasaccharide compound Compar is described in WO2019106201, page 178, as compound 61. * It is described as:

[0489] Synthesis of the above conjugate: Synthesis of NHS ester of compound C7 (C7-adipate-NHS)

[0490] [ka]

[0491] Compound C7 (8 mg, 2.391 μmol) was dissolved in DMSO-HO (400 μL-10 μL) in a 15 mL falcon tube at room temperature. Triethylamine (12 μL, 0.084 mmol) was added thereto. Activated adipate-NHS ester bis(2,5-dioxopyrrolidin-1-yl)adipate (16.3 mg, 0.048 mmol) in DMSO (350 μL) was added and stirred at room temperature for 2 h. Compound C7-adipate-NHS was precipitated by adding 9 mL of EtOAc, centrifuged, and the precipitate was washed with EtOAc (5 mL × 2) and dried under vacuum to give a white solid (8 mg, 94%), which was used in the next step.

[0492] CRM 197 C7 conjugate (C7-adipate-CRM 197 or C7-CRM 197 * ) synthesis Compound C7-adipate-NHS (8 mg, 2.24 μmol) was dissolved in 0.1 M NaPi buffer (pH 7.0, 100 μL) in a 15 mL falcon tube. Freshly washed CRM in 0.1 M NaPi buffer (pH 7.0, 200 μL) in a vial 197 (Obtained from EirGenix, Inc., Taiwan, expression system E. coli) (3 mg, 0.051 μmol) was added dropwise thereto. The vial was rinsed with 0.1 M NaPi buffer (pH 7.0, 100 μL) and transferred to the reaction mixture in the falcon tube, which was then stirred at room temperature for 20 h. The resulting C7-adipate-CRM 197The solution was transferred to an Amicon Ultra vial (10 kDa, MWCO) and centrifuged for 5 minutes at 2-8°C. 300 μL of 0.1 M NaPi was added to the reaction falcon tube, rinsed, transferred to the filter, and centrifuged again. Five additional washes were performed using 1×PBS solution. After the final wash, the conjugate was sterile filtered and stored in 1.5 mL of PBS (pH 7.4) at 2-8°C. The loading obtained using MALDI-TOF MS was 13.47. The conjugate was analyzed using SDS-PAGE, BCA protein quantitation, endotoxin content, and SEC-HPLC.

[0493] BSA conjugates (C7-adipate-BSA or C7-BSA * ) synthesis The compound C7-adipate-NHS (5.96 mg, 1.67 μmol) was dissolved in 200 μL of 0.1 M NaPi buffer (pH 7.0) in a 15 mL falcon tube. Freshly washed BSA (obtained from Sigma-Aldrich, heat shock fraction, pH 7, ≥98%; product number A7906) (3 mg, 0.045 μmol) in 200 μL of 0.1 M NaPi buffer (pH 7.0) in a vial was added dropwise. The vial was rinsed with 50 μL of 0.1 M NaPi buffer (pH 7.0) and transferred to the reaction mixture in the falcon tube. The mixture was stirred at room temperature for 20 h. The resulting C7-adipate-BSA solution was transferred to an Amicon Ultra vial (10 kDa, MWCO) and centrifuged at 2-8 °C for 5 min. 300 μL of 0.1 M NaPi was added to the reaction falcon tube, rinsed, transferred to the filter, and centrifuged again. Five additional washes were performed using 1×PBS solution. After the final wash, the conjugate was sterile filtered and stored in 1.35 mL of PBS (pH 7.4) at 2-8°C. The resulting load using MALDI-TOF MS was 11.86. The conjugate was analyzed using SDS-PAGE and SEC-HPLC.

[0494] Synthesis of PNP ester of compound Compar (Compar-adipate-PNP) The synthesis of Compar-adipate-PNP is described on page 253 of WO2019106201 as compound 61 * The synthesis of PNP-esters has been described.

[0495] CRM 197 and Compar conjugate with BSA (Compar-adipate-CRM 197 or Compare-CRM 197 * , Compar-Adipate-BSA or Compar-BSA * ) synthesis Compar-CRM 197 * The synthesis of Compound 61 is described on pages 253-254 of WO2019106201. * CRM 197 or as binding to BSA.

[0496] II. Biology material: - ELISA Plate (High-binding, EIA / RIA Plate, 96-well, flat bottom with low evaporation lid, company: Costar® 3361) - Detection antibodies: Goat anti-rabbit IgG peroxidase conjugate (Sigma, #A4914) and Goat anti-mouse IgG (H+L) peroxidase conjugate (Dianova Code: 115-035-068).

[0497] - Blocking solution: Commercially available blocking solution (Roche, cat. no. 11112589001) - Antibody diluent: PBS + 1% BSA (w / v).

[0498] - Washing buffer: PBS + 0.1% Tween 20 (PBS-T) Developing solution: 1Step® Ultra TMB-ELISA developer (ThermoScientific, Cat#: 34028) - Stop solution: 2M sulfuric acid (H2SO4).

[0499] - Plate reader: Anthos HT2 or FLUOstar Omega (BMG LABTECH).

[0500] - Software: GraphPad Prism7 for data plotting and analysis.

[0501] - Alum: Aluminum Hydroxide Gel Adjuvant (Alhydrogel® 2%), Brenntag, Batch#: 5447 Exp Dt: Feb 2020.

[0502] - QuantiPro® BCA Assay Kit (SIGMA) Product: QPBCA-1KT; Lot#: SLBR7451V; Pcode: 1002296464 - Mini-PROTEAN® TGX™ Gels-10%, 10 wells (30 μL / well) Control Nr: 64175708, - GelCode(TM) Blue Safe Protein Stain;ThermoScientific;Ref:1860957;Lot#:TA260266 method : Bacterial strains and LPS Klebsiella pneumoniae (KPC) strains with and without capsules, each with different LPS (O antigen), were used to isolate and purify the corresponding LPS. The purified LPS was used as a coating antigen for enzyme-linked immunosorbent assay (ELISA). LPS was isolated using a commercially available LPS extraction kit (JH Science) according to the manufacturer's protocol.

[0503] [Table 2]

[0504] Formulation of vaccine candidates for immunization. All formulations were manufactured under sterile conditions. The drug substance (DS) and buffer (PBS or TBS) were mixed at the appropriate dilution ratio (see below) calculated in advance for the required glycan dose, excluding the required volume of aluminum hydroxide adjuvant (0.25 mg / mL). The DS-buffer mixture was gently mixed, and aluminum hydroxide adjuvant ("Aluminum") stock was added to achieve a final aluminum concentration of 0.250 mg / mL aluminum. The mixture was quickly mixed by gentle pipetting and then mixed on a horizontal shaker at 250 rpm for 2 h at RT. Aliquots were stored in type 1 glass vials at 4°C until further use.

[0505] The vaccine is prepared to contain the intended glycan dose (e.g., 2 μg of glycan per injection) as follows: The average loading factor of the glycan antigen (expressed as moles of antigen per mole of carrier protein) is determined by the CRM 197 The mass of the glycan antigen is determined by MALDI-TOF MS by subtracting the molecular weight (m / z=1) of the glycan antigen from the determined molecular weight of the DS (m / z=1), and then dividing this mass difference by the theoretical molecular weight of the glycan antigen (including the linker (here: alkyl) and spacer (here: adipoyl) moieties). The resulting loading factor is multiplied by the theoretical molecular weight of the glycan antigen excluding the linker and spacer moieties to obtain the average total mass of the glycan attached per DS molecule. The total mass of the glycan is then divided by the determined CRM. 197Dividing by the molecular weight of the protein gives the glycan-to-protein mass ratio of the DS. Multiplying this ratio by the protein concentration of the DS measured by BCA Assay Kit (Sigma) according to the manufacturer's protocol gives the glycan concentration of the DS. To obtain the dilution ratio required to dilute the DS to obtain the intended glycan dose per immunization, the glycan concentration of the DS is divided by the required glycan concentration (for example, to obtain a glycan dose of 2 μg per mouse in an injection volume of 100 μL, the glycan concentration is 20 μg / mL). Then, the DS is diluted by this dilution ratio to the final volume of the vaccine preparation.

[0506] Immunization: Female Zika rabbits were immunized via the intramuscular (im) route with an injection volume of 500 μL per dose. Female mice were immunized via the subcutaneous (sc) route with an injection volume of 100 μL per dose. Animals were maintained under specific pathogen-free conditions and provided with water and food ad libitum.

[0507] ELISA: Coat the plate with antigen: antigen-BSA glycoconjugate (C7-BSA * and Compare-BSA *) and isolated LPS were used for coating. LPS was dissolved in isopropanol to a concentration of 10 μg / mL, and 100 μL was used for coating, resulting in 1 μg of LPS per well. The LPS solution was allowed to evaporate overnight at RT in a safety cabinet. Antigen-BSA glycoconjugates were diluted to 2 μg / mL in PBS, and 50–100 μL (0.1–0.2 μg) were coated per well and incubated overnight at 4°C. Blocking: The plates were blocked with 100 μL of a commercially available blocking solution and incubated for 1 h at RT. After blocking, the plates were washed 3x with PBS containing 0.1% (v / v) Tween-20 (PBS-T). Incubation with diluted sera: Pooled or individual sera from different time points were diluted with 1% BSA (w / v) in PBS to yield their respective dilutions. 50–100 μL of diluted serum was added to ELISA wells in duplicate and incubated for 1 h at RT. 100 μL / well of 1% BSA (w / v) in PBS was used as a blank. After incubation with serum, the plate was washed 3X with PBS-T. Incubation with detection antibody: Anti-mouse or anti-rabbit IgG HRP conjugate was diluted 1:10,000 in 1% BSA (w / v) in PBS, added 100 μL / well, and incubated for 30 min at RT. After incubation with detection antibody, the plate was washed 3X with PBS-T. Substrate addition: 100 μL of TMB substrate was added to each well and incubated for approximately 15 min. The reaction was stopped by adding 50 μL / well of 2 M H2SO4. Absorbance was measured at 450 nm using a plate reader. Absorbance values ​​were analyzed using GraphPad Prism software.

[0508] Results: As shown by HPLC-SEC (Figure 1) and SDS-PAGE (Figure 2), compound C7 binds to the carrier protein CRM. 197 The comparative conjugate compound (Compar-CRM with an octasaccharide antigen) was successfully conjugated to the 197 * ) is a CRM in mice. 197Although C7-CRM was immunogenic as a conjugate (Figure 3A), the IgG induced did not bind to isolated O1 LPS (Figure 3B). 197 * was also immunogenic in mice (Fig. 4), whereas the comparison conjugate Compar-CRM 197 * In contrast, the induced IgG recognized the natural antigen, isolated LPS from O1 (Figure 5). 197 * C7-CRM197 was also immunogenic in rabbits (Fig. 6), and the IgG recognized the native LPS antigen (Fig. 7). As shown in a challenge experiment in which rabbit antisera were transferred to mice (passive immunization) and then lethally infected with an O1-expressing K. pneumoniae strain (PCM12), C7-CRM197 * The antibodies induced by C7-CRM197 were protective (Figure 8). * Mice actively vaccinated with were significantly protected from O1 K. pneumoniae infection (PCM12) (Fig. 9).

[0509] SEQ ID No.1: CRM 197 The amino acid sequence of: GADDVVDSSK SFVMENFSSY HGTKPGYVDS 30 IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY 60 DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK 90 VLALKVDNAE TIKKELGLSL TEPLMEQVGT 120 EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI 150 NNWEQAKALS VELEINFETR GKRGQDAMYE 180 YMAQACAGNR VRRSVGSSLS CINLDWDVIR 210 DKTKTKIESL KEHGPIKNKM SESPNKTVSE 240 EKAKQYLEEF HQTALEHPEL SELKTVTGTN 270 PVFAGANYAA WAVNVAQVID SETADNLEKT 300 TAALSILPG GSVMGIADGA VHHNTEEIVA 330 QSIALSSLMV AQAIPLVGEL VDIGFAAYNF 360 WATER FILTER 390 HDGYAVSWNT VEDSIIRTGF QGESGHDIKI 420 TAENTPLPIA GVLLPTIPGK LDVNKSKTHI 450 SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG 480 NGVHANLHVA FHRSSSEKIH SNEISSDSIG 510 VLGYQKTVDH TKVNSKLSLF FEIKS 535

Claims

1. Oligosaccharide-carrier protein conjugates of formula (I): 【Chemistry 1】 (In the formula, m is 4, 5 or 6, preferably m is 4 or 5; n is 5, 6 or 7, preferably n is 6 or 7; i is 1 to 28; -LT- represents a linker L and a spacer T that together form a bridge with a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. １９７ and the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen and sulfur; or a pharmaceutically acceptable salt thereof.

2. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein m is 4 and n is 6.

3. 3. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the bridge does not have an aromatic or heteroaromatic ring.

4. -LT- represents a linker L and a spacer T that together form a bridge having a covalently linked backbone of 5 to 25 atoms in length, the length being determined by the distance between the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM. １９７ the shortest distance between the nitrogen of the amino group of the lysine residue ... the atoms of the backbone are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; The skeleton is oxo, (C １－４ ) alkyl, fluoro and (C １－２ ) optionally substituted by one or more (especially 1, 2, 3 or 4) substituents independently selected from alkoxy (especially oxo); A part of the skeleton may optionally be: 【Chemistry 2】 It may be a 4-, 5- or 6-membered ring moiety selected from: The oligosaccharide-carrier protein conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

5. -LT- represents a linker L and a spacer T that together form a bridge, said bridge consisting of a backbone that is a saturated chain of 5 to 25 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur (preferably carbon, nitrogen and oxygen, more preferably carbon and nitrogen), said chain being unsubstituted or having a substituent selected from the group consisting of oxo, (C １－４ ) alkyl, fluoro and (C １－２ 3. The oligosaccharide-carrier protein conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, optionally substituted by one or more (preferably 1, 2, 3, or 4) substituents (preferably oxo) independently selected from:

6. The backbone of the bridge has a length of 8 to 20 atoms, preferably 8 to 16 atoms, covalently linked, and the length is such that the oxygen at C1 of the reducing end of the oligosaccharide and the carrier protein CRM are bonded together. １９７ The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the oligosaccharide-carrier protein conjugate forms the shortest distance between the nitrogen of the amino group of the lysine residue of

7. L, ＊ -(C ２－１０ ) alkylene-NH-; ＊ - (CH ２ CH ２ O) ａ -CH ２ CH ２ NH- (a is 1, 2 or 3); ＊ -CH ２ CH ２ S-CH ２ CH ２ NH-; ＊ -(C ２－１０ ) fluoroalkylene-NH-; ＊ - (CH ２ ) ｃ NHC(O)(CH ２ ) ｄ -NH- (c and d are each independently 2 to 6); ＊ - (CH ２ ) ｅ NHC(O)NH(CH ２ ) ｈ -NH- (e and h are independently 2 to 6); ＊ -(C １－１０ ) alkylene-C(O)-NH-(C ２－１０ ) alkylene-NH-; or ＊ -(C ２－１０ ) alkylene-O-NH-; represents; T, -C(O)-(C ０－１０ ) alkylene-C(O)-; —C(O)—CH ２ CH ２ -(OCH ２ CH ２ ) ｒ —C(O)— (r is 1 to 5); —C(O)—CH ２ (CH ２ ) ｆ - (SCH ２ (CH ２ ) ｆ’ ) ｆ’’ -C(O)- (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3); 【Transformation 3】 or L-T, ＊ -(C ２－１０ ) alkylene-S-R １ ; represents; R １ teeth, 【Chemistry 4】 Represents; The oligosaccharide-carrier protein conjugate according to claim 1 or 6, or a pharmaceutically acceptable salt thereof.

8. L, ＊ - (CH ２ ) ｌ -NH-; (l is 2 to 10); ＊ - (CH ２ CH ２ O) ａ -CH ２ CH ２ NH- (a is 1, 2 or 3); ＊ -CH ２ CH ２ S-CH ２ CH ２ NH-; ＊ -(C ２－１０ ) fluoroalkylene-NH- (fluoroalkylene is a saturated linear chain); ＊ - (CH ２ ) ｃ NHC(O)(CH ２ ) ｄ -NH- (c and d are each independently 2 to 6); ＊ - (CH ２ ) ｅ NHC(O)NH(CH ２ ) ｈ -NH- (e and h are independently 2 to 6); ＊ - (CH ２ ) ｕ -C(O)-NH-(CH ２ ) ｕ’ -NH-; (u is 1 to 10 and u' is 2 to 10); or ＊ - (CH ２ ) ｇ —O—NH— (g is 2 to 10); represents; Or, L-T, ＊ - (CH ２ ) ｑ S-R １ (q is 2 to 10); represents; T, —C(O)—(CH ２ ) ｐ -C(O)- (p is 0 to 10); —C(O)—CH ２ CH ２ -(OCH ２ CH ２ ) ｒ —C(O)— (r is 1 to 5); —C(O)—CH ２ (CH ２ ) ｆ - (SCH ２ (CH ２ ) ｆ’ ) ｆ’’ -C(O)- (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3); 【Transformation 5】 represents; R １ teeth, 【Transformation 6】 Represents; The oligosaccharide-carrier protein conjugate according to claim 7 or a pharmaceutically acceptable salt thereof.

9. L, ＊ - (CH ２ ) ｌ -NH-; (l is 2 to 10, preferably 2 to 6); ＊ - (CH ２ CH ２ O) ａ -CH ２ CH ２ NH- (where a is 1, 2 or 3, preferably 1 or 2); or ＊ - (CH ２ ) ｇ -O-NH- (g is 2 to 10, preferably 2 to 6); represents; T, —C(O)—(CH ２ ) ｐ —C(O)— (p is 0 to 10, preferably 0 to 6); —C(O)—CH ２ CH ２ -(OCH ２ CH ２ ) ｒ —C(O)— (r is 1 to 5, preferably 1 to 3, and more preferably 1); —C(O)—CH ２ (CH ２ ) ｆ - (SCH ２ (CH ２ ) ｆ’ ) ｆ’’ -C(O)- (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2 or 3, preferably 1); or 【Transformation 7】 Represents; The oligosaccharide-carrier protein conjugate according to claim 7 or a pharmaceutically acceptable salt thereof.

10. L ＊ - (CH ２ ) ５ represents —NH—; T is -C(O)-(CH ２ ) ４ represents —C(O)—; The oligosaccharide-carrier protein conjugate according to claim 1 or 7, or a pharmaceutically acceptable salt thereof.

11. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein i is 6 to 15.

12. The oligosaccharide-carrier protein conjugate according to any one of claims 1 to 10, wherein the oligosaccharide-carrier protein conjugate has a structure of formula (Ib): 【Transformation 8】 (In the formula, i is 1 to 28.)

13. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to claim 12, wherein i is 6 to 15.

14. A pharmaceutical composition comprising the oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 as an active ingredient, and further comprising at least one therapeutically inactive excipient.

15. 15. The pharmaceutical composition of claim 14, further comprising an adjuvant.

16. 14. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, for use as a medicine, in particular as a vaccine.

17. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, for use in the prevention and / or treatment of K. pneumoniae infection.

18. 18. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof for use according to claim 17, wherein the K. pneumoniae infection is selected from the group consisting of K. pneumoniae infections in individuals 50 years of age or older; nosocomial K. pneumoniae infections, such as nosocomial pneumonia, nosocomial bloodstream infections, and nosocomial urinary tract infections; community-acquired K. pneumoniae infections; and pneumonia, bronchitis, meningitis, urinary tract infections, intra-abdominal infections, wound infections, blood infections, osteomyelitis, bacteremia, sepsis, liver abscess, and inflammatory bowel disease (IBD) caused by K. pneumoniae infection.

19. The oligosaccharide-carrier protein conjugate or a pharmaceutically acceptable salt thereof for use according to claim 17 or 18, wherein the K. pneumoniae is selected from the O-serotype having O1.

20. A multivalent vaccine comprising the oligosaccharide-carrier protein conjugate according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof.

21. An intermediate compound for producing the oligosaccharide-carrier protein conjugate according to any one of claims 7 to 13, which has the formula (II): 【Chemistry 9】 (In the formula, m is 4, 5 or 6, preferably 4 or 5, more preferably 4; n is 5, 6 or 7, preferably 6 or 7, more preferably 6; L １ teeth, ＊ -(C ２－１０ ) alkylene-NH ２ , preferably ＊ - (CH ２ ) ｌ -NH ２ (l is 2 to 10, more preferably l is 5); ＊ - (CH ２ CH ２ O) ａ -CH ２ CH ２ NH ２ (a is 1, 2 or 3); ＊ -CH ２ CH ２ S-CH ２ CH ２ NH ２ ; ＊ -(C ２－１０ ) Fluoroalkylene-NH ２ ; ＊ - (CH ２ ) ｃ NHC(O)(CH ２ ) ｄ -NH ２ (c and d are independently 2 to 6); ＊ - (CH ２ ) ｅ NHC(O)NH(CH ２ ) ｈ -NH ２ wherein e and h are independently 2 to 6; ＊ -(C １－１０ ) alkylene-C(O)-NH-(C ２－１０ ) alkylene-NH ２ ; ＊ -(C ２－１０ ) alkylene-O-NH ２ or ＊ -(C ２－１０ ) alkylene-SH; represents.) or a pharmaceutically acceptable salt thereof.

22. An intermediate compound for producing the oligosaccharide-carrier protein conjugate according to any one of claims 7 to 13, having the formula (III): 【Chemistry 10】 (In the formula, m is 4, 5 or 6, preferably m is 4 or 5, most preferably 4; n is 5, 6 or 7, preferably n is 6 or 7, most preferably 6; L is, ＊ -(C ２－１０ ) alkylene-NH-, preferably ＊ - (CH ２ ) ｌ -NH- (l is 2 to 10, more preferably 5); ＊ - (CH ２ CH ２ O) ａ -CH ２ CH ２ NH- (a is 1, 2 or 3); ＊ -CH ２ CH ２ S-CH ２ CH ２ NH-; ＊ -(C ２－１０ ) fluoroalkylene-NH-; ＊ - (CH ２ ) ｃ NHC(O)(CH ２ ) ｄ -NH- (c and d are each independently 2 to 6); ＊ - (CH ２ ) ｅ NHC(O)NH(CH ２ ) ｈ -NH- (e and h are independently 2 to 6); ＊ -(C １－１０ ) alkylene-C(O)-NH-(C ２－１０ ) alkylene-NH-; or ＊ -(C ２－１０ ) alkylene-O-NH-; represents; T １ teeth, -C(O)-(C ０－１０ ) alkylene-C(O)X; —C(O)—CH ２ CH ２ -(OCH ２ CH ２ ) ｒ -C(O)X (r is 1 to 5); —C(O)—CH ２ (CH ２ ) ｆ - (SCH ２ (CH ２ ) ｆ’ ) ｆ’’ -C(O)X (f is 0 or 1, f' is 0 or 1, and f'' is 1, 2, or 3); 【Chemistry 11】 represents; -C(O)X represents -C(O)OH or an activated ester; preferably X is 【Chemistry 12】 represents; Y is Me, Et, Bu or -(CH ２ CH ２ O) ３ CH ３ represents.) or a pharmaceutically acceptable salt thereof.

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