EA046159B1 - STABLE VACCINE AGAINST CLOSTRIDIUM DIFFICILE - Google Patents

Description

Technical field of the invention

The present invention relates to a synthetic saccharide of general formula (I), which refers to the cell surface polysaccharide PS-II of Clostridium difficile and its conjugate. The specified synthetic saccharide, the specified conjugate and the pharmaceutical composition containing the specified synthetic saccharide or the specified conjugate are resistant to hydrolysis, long-term stable, thermostable and are useful for the prevention and/or treatment of diseases associated with Clostridium difficile, currently called Clostridioides difficile. In addition, the synthetic saccharide of general formula (I) is useful as a marker in immunoassays for detecting antibodies to the bacteria Clostridium difficile.

Prerequisites for creating the invention

Clostridioides difficile, formerly known as Clostridium difficile, is a Gram-positive, spore-forming anaerobic bacterium that is considered the most important identifiable cause of nosocomial diarrhea. The terms Clostridioides difficile and Clostridium difficile are used interchangeably throughout this document and are both abbreviated to C. difficile. It settles in the human intestines, thus leading to Clostridium difficile infections (CDI). CDI has also become the most commonly diagnosed cause of hospital-acquired diarrhea, especially in at-risk groups that include elderly and immunocompromised patients, as well as patients receiving antibiotic treatment. C. difficile infections have become a major problem due to the rapid increase in the incidence of CDI over the past decade, largely due to the emergence of the hypervirulent and now predominant ribotype 027 strain, which causes epidemic outbreaks with increased morbidity, mortality, and high relapse rates. The cost of treatment increases significantly, especially in cases of recurrent CDI. Thus, prevention of Clostridium difficile infections is highly desirable, and vaccination of at-risk groups is the most cost-effective and powerful intervention. However, a vaccine against Clostridium difficile has not yet been developed.

Carbohydrates exposed to the surface of bacterial cells are often immunogenic and represent potential candidates for vaccine development. Compared to proteins, carbohydrates are evolutionarily more stable, and when covalently linked to a carrier protein, oligosaccharide antigens can induce long-lasting, T-cell-dependent protection.

Three different cell wall polysaccharide structures have been identified that are expressed by C. difficile cells, named PS-I, PS-II and PS-III (Expert Rev. Vaccines 2013, 12, 421). Although the expression of the PS-I saccharide may be more limited, for example expressed in ribotype 027, the PS-II saccharide was found in all C. difficile ribotypes examined, indicating that the PS-II saccharide may be a conserved cell surface antigen.

The PS-II saccharide repeating unit of C. difficile consists of:

-^6)-3-D-Glc-(l,3)-3-D-GalNAc-(l,4)-aD-Glc-(l,4)-3-D-GalNAc-(l,3) -aD-ManI(W0P0 ₃ ^ ΐ

β-D-Glc

The C. difficile PS-II saccharide is hydrolyzed in water due to the chemical lability of the (1^6) phosphodiester bond connecting the PS-II repeat unit at the anomeric position of mannose, thereby making it difficult to extract from cells with the commonly used hot acetic acid or water/phenol. Cleavage of the O1-C1 phosphodiester bond is followed by removal of the phosphomonoester group, resulting in the formation of the PS-II hexasaccharide unit. Cleavage of the phosphodiester bond of PS-II saccharide is enhanced in the presence of acids, bases or metal ions. Due to the instability of PS-II ^difficile saccharide in solution, the saccharide or its conjugate, when used as a vaccine, must be suitably buffered in a liquid formulation or lyophilized in a solid formulation that must be reconstituted before use. However, freeze-drying and cold storage of vaccines make the vaccine more expensive to produce and more difficult to ship, as it requires a functioning cold chain system to ensure optimal temperatures during transport, storage and processing. The instability of the C. difficile PS-II saccharide is well documented in the art. Thus, a new, stable vaccine against C. difficile in liquid form is required.

International patent application WO 2009/033268 A1 discloses the isolation of C. difficile cell surface PS-I and PS-II saccharide from C. difficile bacteria strains ribotype 027, MOH900 and MOH718. A synthetic approach to the cell surface saccharide PS-II of C. difficile was used by Danieli et al. (Org. Let. 2011, 13, 378-381), Costantino et al. (WO 2012/085668 A2), Seeberger (WO 2012/119769 Al) and

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Oberli et al. (Chemistry & Biology 2011, 18, 580). Monteiro (Meth Mol. Biol. 2016, 397-408) indicates the isolation of water-soluble PS-I and PS-II, as well as water- and phenol-soluble polysaccharides PS-III from C. difficile biomass by treatment with hot water - phenol.

An object of the present invention is to provide a well-defined synthetic saccharide of general formula (I) that is metabolically stable, resistant to hydrolysis and shelf stable in liquid formulations and which produces antibodies that protect against diseases caused by C. difficile. Said saccharide may be conjugated to an immunogenic carrier to provide a conjugate and pharmaceutical composition thereof that are useful for the prevention and/or treatment of diseases associated with C. difficile. In addition, the synthetic saccharide of general formula (I) is suitable as a marker in immunoassays for the detection of antibodies to the bacteria C. difficile.

The object of the present invention is achieved by examining the independent claims. Additional useful features, aspects and details of the present invention will be apparent from the dependent claims, description, figures and examples of this application.

Description of the invention

Definitions.

The term linker as used herein encompasses molecular moieties capable of connecting the reducing end of a saccharide monosaccharide to an immunogenic carrier or solid support, optionally by binding to at least one connecting molecule. Thus, the function of the linker, per se or together with the connecting molecule, is to establish, maintain and/or bridge a defined distance between the end-reducing monosaccharide and the immunogenic carrier or solid support. By maintaining a certain distance between the saccharide and the immunogenic carrier, it is possible to avoid protecting the immunogenic saccharide epitopes by the structure of the immunogenic carrier (eg, the secondary structure of the carrier protein). In addition, the linker provides higher efficiency of conjugation with saccharides by reducing steric hindrance of the reactive groups (Methods in Molecular Medicine 2003, 87, 153-174). More specifically, one end of the linker is connected to an exocyclic oxygen atom at the anomeric center of the end-reducing monosaccharide, and the other end is connected via a nitrogen atom to a linking molecule or to an immunogenic carrier or solid support.

Any linker for saccharide conjugates (eg, saccharide protein-carrier conjugate, antibody-drug conjugate) known in the art can be used within the scope of the present invention. From the large number of publications directed at saccharide protein-carrier conjugates, one skilled in the art can readily envision suitable linkers as saccharides and conjugates are disclosed herein (see Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification in Chem Soc Rev 2018, Advance Article, DOI: 10.1039/C8CS00495A; also Acc Chem Res 2017, 50, 1270-1279), since the linker used, i.e. its length and type of bond do not significantly affect the immunogenicity of the saccharide conjugate (see PLoS ONE 2017, 12 (12): e0189100; J. Immun. Meth. 1996, 191, 1-10). Such suitable linkers are innocuous (i.e., non-toxic) and non-immunogenic (i.e., do not generate non-protective antibodies upon immunization with the conjugate) and include, but are not limited to, commercially available bifunctional polyethylene glycol (Journal of Controlled Release 2013, 172, 382 -389; J. Immun. Meth. 1996, 191, 1-10), glutaric acid derivatives (J. Org. Chem. 2005, 70 (18), 7123-7132), adipic acid derivatives, squarate derivatives, alkynes, N α-hydroxysuccinimides, such as commercially available MFCO-NHS (monofluorocyclooctine Nhydroxysuccinimide ester), maleimides (as described in Acc. Chem. Res. 2017, 50, 1270-1279), or hydrophilic alkylphosphinates and sulfonyls (as described in WO 2014080251 A1 ).

As used herein, the term linking molecule refers to a bifunctional molecule containing a functional group X and a functional group Y, wherein the functional group X is capable of reacting with a terminal amino group on linker L and the functional group Y is capable of reacting with a functional group present on the immunogenic media or on a solid support. In fig. 3 shows examples of commercially available coupling molecules, but does not limit the coupling molecules that can be used in accordance with the present invention to those presented herein. It should be understood that the connecting molecule is not part of the linker or immunogenic carrier or solid support.

As used herein, the term adjuvant refers to an immunological adjuvant, i.e. a substance used in a vaccine composition that modifies or enhances the effect of each vaccine by enhancing the immune response to a given antigen contained in the vaccine without being antigenically related to it. To one skilled in the art, classically familiar examples of adjuvants include:

compositions containing minerals, including calcium salts and aluminum salts (or mixtures thereof). Calcium salts include calcium phosphate. Aluminum salts include hydroxides, phosphates, sulfates, etc., the salts taking any suitable form (eg, gel, crystalline, amorphous, etc.).

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Adsorption for these salts is preferred. The mineral containing composition may also be formulated as a metal salt particle. Adjuvants known as aluminum hydroxide and aluminum phosphate may also be used. Any of the hydroxide or phosphate adjuvants that are commonly used as adjuvants can be used in the invention. Adjuvants known as aluminum hydroxide are typically aluminum oxyhydroxide salts, which are typically at least partially crystalline. Adjuvants known as aluminum phosphate are usually aluminum hydroxyphosphates, often also containing a small amount of sulfate (eg aluminum hydroxyphosphate sulfate). They can be prepared by precipitation, with reaction conditions and concentrations during precipitation affecting the degree of hydroxyl substitution of phosphate in the salt. Mixtures of both aluminum hydroxide and aluminum phosphate can be used in the compositions in accordance with the present invention;

saponins, which are a heterologous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, stems, roots and even flowers of many plant species. Saponins from the bark of Quillaia saponaria and the Molina tree have been widely studied as adjuvants. Saponins can also be commercially obtained from Smilax ornata (sarsaparilla), Gypsophilla paniculata (tumbleweed), and Saponaria oficianalis (soaproot). Saponin adjuvant formulations include purified formulations such as QS21 as well as lipid formulations such as ISCOM. The saponin compositions were purified by HPLC and RP-HPLC. Specific purified fractions using these techniques were identified, including QS 7, QS 17, QS 18, QS21, QH-A, QH-B, and QH-C. Saponin compositions may also include a sterol, such as cholesterol. Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs). ISCOMs typically include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOM. Preferably, the ISCOM includes one or more QuilA, QHA and QHC;

microparticles (ie, particles with a diameter of 100 nm to 150 μm, more preferably with a diameter of 200 nm to 30 μm or 500 nm to 10 μm in diameter) formed from substances that are biodegradable and non-toxic. Such non-toxic and biodegradable substances include, but are not limited to, poly(α-hydroxy acid), polyhydroxybutyric acid, polyorthoester, polyanhydride, polycaprolactone;

CD1d ligands such as α-glycosylceramide, phytosphingosine-containing α-glycosylceramides, OCH, KRN7000 [DO^,4K.)-1-O-(α^-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3, 4-octadecanetriol], CRONY-101, 3-sulfo-galactosyl-ceramide;

immunostimulatory oligonucleotides, such as those containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine residue linked by a phosphate bond to a guanosine residue), or containing a CpI motif (a dinucleotide sequence containing a cytosine attached to an inosine), or double-stranded RNA, or an oligonucleotide containing a palindromic sequence , or an oligonucleotide containing a poly(dG) sequence. Immunostimulatory oligonucleotides may include nucleotide modifications/analogs, such as phosphorothioate modifications, and may be double-stranded or (other than RNA) single-stranded;

compounds containing lipids attached to a phosphate-containing acyclic backbone, such as MPLA or the TLR4 antagonist E5564;

oil emulsions (for example, Freund's adjuvant).

Theoretically, every molecule or substance that can promote or enhance a particular situation in the cascade of immunological events, which ultimately leads to a more pronounced immunological response, can be defined as an adjuvant.

In principle, using adjuvants in vaccines can:

direct and optimize immune responses suitable or desired for a vaccine;

ensure delivery of the vaccine through mucous membranes, i.e. administration that brings the vaccine into contact with a mucosal surface such as buccal or gastric or pulmonary epithelium and associated lymphoid tissue;

promote cell-mediated immune responses;

enhance the immunogenicity of weaker immunogens, such as highly purified or recombinant antigens;

reduce the amount of antigen or frequency of immunization required to provide protective immunity; and improve the effectiveness of vaccination in individuals with a reduced or weakened immune response, such as neonates, the elderly, and immunocompromised vaccine recipients.

Although little is known about their mechanism of action, adjuvants are currently believed to enhance immune responses through one of the following mechanisms:

increasing the biological or immunological half-life of antigens;

improving antigen delivery to antigen presenting cells (APCs), as well as processing and

- 3 046159 presentation of the APC antigen, for example, by allowing the antigen to cross endosomal membranes into the cytosol after receiving antigen-adjuvant APC complexes;

imitating danger signals from stressed or damaged cells that serve to initiate an immune response;

inducing the production of immunomodulatory cytokines;

a shift in the immune response towards a specific subset of the immune system; and blocking the rapid dissipation of antigen challenge.

Saccharides are known to those skilled in the art as TI-2 (T cell independent type 2) antigens and weak immunogens. Therefore, for the production of a saccharide-based vaccine, said saccharides are conjugated to an immunogenic carrier, providing a conjugate that represents increased immunogenicity compared to the saccharide. In this context, the term immunogenic carrier is defined as a structure that is conjugated to a saccharide to form a conjugate that represents increased immunity compared to the saccharide per se. Thus, conjugating saccharides to an immunogenic carrier, preferably a carrier protein, has the effect of stimulating an immune response against the saccharide without causing an immune response against the immunogenic carrier.

Therefore, the present invention is directed to the saccharide of general formula (I)

where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

T* - represents -P(=O)(OH) ₂ , -P(=O)(O ^- )(OH) or -PO ₃ ^2- ;

Z represents

E represents -NH2, -N ₃ , -CN, -O-NH2, -CH=CH ₂ , -C CH, -Br, -Cl, -I, -CO2R', -CONH-NH2, -SH, - OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

and where

- L- represents -L ^a -, -L ^a -L ^e -, -L ^a -L ^b -L ^e -, or -L ^a -L ^d -L ^e -;

- L ^a - represents -(CH ₂ ) _o -, -(CH ₂ -CH ₂ -O) _o -C ₂ H ₄ -, or -(CH ₂ -CH ₂ -O) _o -CH ₂ ;

- L ^b - represents -O-;

- L ^d - represents -(CH ₂ ) _q -, -(CH(OH)) _q -, -(CF ₂ ) _q -, -(CH ₂ -CH ₂ -O) _q -C ₂ H4-, or -(CH ₂ -CH ₂ -O) _q -CH ₂ -;

- L ^e - represents -(CH ₂ )p1-, -(CF ₂ )p1-, -C2H4-(O-CH2-CH2)p1-, -CH2-(O-CH2-CH2)p1- or -( CH ₂ )p1O-(CH2)p2-; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6;

or a pharmaceutically acceptable salt thereof.

In all general formulas (I), (II), (III), as well as all general subformulas, n is preferably an integer from 1 to 8, more preferably an integer from 1 to 6, and even more preferably 1, 2 , 3, 4, or 5, even more preferably 1, 2, 3, or 4, even more preferably 1, 2, or 3, even more preferably 1 or 2, and even more preferably 1.

Most preferably, the saccharide of formula (I) has an -O-L-E group selected from the group consisting of:

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where R' represents -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

X is -Br, -Cl, -I, -CO2H, -CN, -NO2 or -SAc.

Linker L may also include a C. difficile PS-II saccharide repeating unit or fragments thereof:

Thus, linker L is preferably selected from one of the following structures:

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^(CH ₂ ) ₂ -

(CH ₂ ) ₂ —

^(CH ₂ ) ₁₀ -

(CH ₂ ) ₁₀ - 6 046159

OH NHAc o

ABOUT

UH NHAC Q

CHSN ₂ ) ₅ -

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But but but

BUT

it

NHAc (CH ₂ ) ₄ -

ABOUT

BUT

HE

NHAc

BUT BUT

N N

BUT

(CH ₂ ) ₃ - O

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Therefore, also preferred is a saccharide of formula (I) which has the group -OL-E selected from the group consisting of the following:

BUT

nh ₂

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(CH ₂ )io—NH ₂

HO

(CH ₂ )ω—NH ₂

HO

NH ₂

%ch ₂ ) ₅ -nh ₂

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BUT

NH ₂

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or preferably a disulfide of this moiety

or preferably a disulfide of this moiety

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The saccharides according to the present invention may be hygroscopic and thus may form their various hydrates. Preferably, the molar ratio of water molecule to saccharide is in the range of 1 to 20, more preferably 1 to 10, most preferably 5 to 10.

The saccharides according to the present invention bear basic and/or acid substituents, and they can form salts with organic or inorganic acids or bases.

Examples of suitable acids for the formation of such acid addition salts are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, paminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, paminobenzoic acid, p- hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylene sulfonic acid, ptoluenesulfonic acid, naphthylsulfonic acid, sulfanilic acid, camphorsulfonic acid

- 13 046159 acid, chinese acid, mandelic acid, o-methylmandelic acid, hydrobenzenesulfonic acid, picric acid, adipic acid, d-o-tolyltartaric acid, tartronic acid, (o, m, p)toluic acid, naphthylamine sulfonic acid, and others mineral or carboxylic acids well known to those skilled in the art. Salts are prepared by contacting the free base form with enough of the desired acid to form a salt in the usual manner.

Examples of suitable inorganic or organic bases are, for example, NaOH, KOH, NH4OH, tetraalkylammonium hydroxide, lysine or arginine and the like. The salts can be prepared in the usual manner using methods well known in the art, for example by treating a compound of general formula (I) with a solution of a base selected from the group mentioned above.

It will be apparent to one skilled in the art of carbohydrate chemistry that the saccharides of general formula (I) do not contain -O-O- bonds and or sugar moieties connected or bonded to each other through their anomeric or C-1 carbon atoms.

Surprisingly, it has been found that the saccharide of general formula (I) contains an immunogenic protective epitope and is capable of inducing a protective immune response against the bacteria Clostridium difficile in a human and/or animal host. The saccharide of general formula (I) detects antibodies that, when cross-reacted with a natural saccharide on the surface of Clostridium difficile PS-II cells, specifically recognize Clostridium difficile bacteria and opsonize them for destruction by phagocytes, thereby providing protection against Clostridium difficile bacteria.

Surprisingly, it has been found that saccharides of general formula (I) are stable in acidic aqueous media, basic aqueous media, and also in suspensions containing aluminum phosphate or aluminum hydroxide, such as the commonly used adjuvant Alhydrogel. While the natural PS-II saccharide of Clostridium difficile is hydrolyzed within one day in acidic aqueous media, in basic aqueous media or in the presence of aluminum salts, the saccharides of general formula (I), as well as their conjugates, are stable for several days even at elevated temperatures. The increased stability is particularly advantageous for their use in Clostridium difficile vaccines. Thus, the saccharides of general formula (I), as well as their conjugates, are particularly suitable for shelf-stable liquid vaccine formulations against Clostridium difficile that can be stored at ambient temperatures.

The saccharides of the present invention overcome all the problems associated with saccharides derived from bacterial sources and their conjugates in terms of purity and ease of production. First, the production of cell wall saccharides requires optimization of growth conditions. Secondly, it is necessary to find depolymerization conditions under which the structural integrity of the constituent monosaccharides is maintained. Finally, it is necessary to determine purification conditions that allow the isolation of pure saccharide of a certain length and structure. In addition to the usual impurities such as cellular polysaccharides, nucleic acids, lipids and proteins, undesirable saccharides produced during the depolymerization process must also be excluded. Thus, obtaining pure saccharides of defined structure and length from bacterial sources is a tedious, almost impossible, process.

Preference is given to synthetic saccharides of the general formula (II)

where n, L, E and T* have the meanings specified herein. Thus, a saccharide of the general formula (II-a) or (II-b), wherein n, L, and E are as defined herein, is particularly preferred.

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where n, L, E and T* have the meanings specified herein. Thus, the total saccharide

Also preferred are synthetic saccharides of general formula (III), formula (III-a) or (III-b), where n, L, and E are as defined herein, is particularly preferred.

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Preferably, n is an integer selected from 1-10, more preferably 16, more preferably 1-3, and even more preferably 1-2. Therefore, a saccharide of general formula (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), where n is an integer selected from 1-2 is particularly preferred.

Preferably, the linker -L- is -La-, -La-L ^e -, -La-L ^b -L ^e - or -La-L ^d -L ^e -;

- La- is -( _CH2 ) _O- , -(CH2-CH2-O)o-C2H4- or -(CH2-CH2-O)o-CH2;

- L ^b - represents -O-;

- L ^d - represents -(CH2)q-, -(CH(OH)) _q -, -(CF2)q-, -(CH ₂ -CH ₂ -O)qC ₂ H ₄ - or -(CH ₂ -CH ₂ -O) _q -CH ₂ -;

^- L ^- represents ^-(CH 2 ⁾ p1 ^- , ^-(CF 2 ⁾ p1 ^- , “ ^C 2 ^H 4“ ^(O “ ^CH 2“ ^CH 2)p1-5 ^-CH 2 ^-(O-CH 2 ^-CH 2 ⁾ p1 ^{- or} -(CH ₂ )p ₁ -O-(CH ₂ )p ₂ -; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6.

Therefore, the saccharide is any of the general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), where

- L- is -La-, -La-L ^e -, -La-L ^b -L ^e - or -La-L ^d -L ^e -;

- La- is -(CH ₂ ) _o -, -(CH ₂ -CH ₂ -O) _o -C ₂ H ₄ - or -(CH ₂ -CH ₂ -O) _o -CH ₂ ;

- L ^b - represents -O-;

- L ^d - represents -(CH ₂ ) _q -, -(CH(OH))q-, -(CF2)q-, -(CH ₂ -CH ₂ -O) _q -C2H4- or -(CH2- CH2-O)q-CH2-;

- L ^e - represents -(CH ₂ )p _X -, -(CF ₂ )p1-, -C2H4-(O-CH2-CH2)p1-, -CH2-(O-CH2-CH2)p1- or - (CH2)p1-o-(CH2)p2-; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6 is particularly preferred.

A saccharide of any one of the general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), where

- L- is selected from: -La-, -La-L ^e -, -La-L ^b -L ^e - and -La-L ^d -L ^e -;

- La- selected from: -( _CH2 )o-, -(CH2-CH2-O)o-C2H4- -(CH2-CH2-O)o-CH2;

- L ^b - represents -O-;

- L ^d - selected from: -(CH ₂ ) _q -, -(CF ₂ ) _q -, -(CH2-CH2-O)q-C2H4- and -(CH ₂ -CH ₂ -O) _H -CH ₂ -;

^{- L -} selected from: ^-( СН2 ⁾ р1 ^- , ^-(CF 2 ⁾ pl ^- , “С2Н4“ ^(О “ ^СН 2“ ^СН 2)р1“5 ^-СН 2 ^-(О-СН 2 ^-СН 2 ⁾ p1 ^{- and -(CH} 2 ⁾ p1 ^-O-(CH 2 ⁾ p2 ^-; o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6; and n is 1 is also preferred.

Even more preferred is a saccharide of general formula (I), (II), (II-a), (II-b), (III), (III-a) or (IIIb), where -L- is -(CH ₂ ) _o - and o is an integer selected from 1, 2, 3, 4, 5 and 6.

Also preferred is a saccharide of general formula (I), (II), (II-a), (II-b), (III), (III-a) or (IIIb), where -L- is -(CH ₂ ) _o -, o is an integer selected from 1, 2, 3, 4, 5 and 6, and n is an integer selected from 1-2.

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In the most preferred embodiment of the invention, the group -O-L-E is selected from the group consisting of:

where R' represents -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl or N-succinimidyl;

X is -Br, -Cl, -I, -CO2H, -CN, -NO2 or -SAc.

Also preferred is a saccharide of general formula (I), (II), (II-a), (II-b), (III), (III-a) or (IIIb), wherein the group -O-L-E is selected from the group consisting from the following:

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(CH ₂ )io—NH ₂

HO

NH ₂

HO

nh ₂

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BUT but

BUT

He

NHAc

N

I—NH ₂ !-NH ₂ (CH ₂ ) ₂ -nh ₂

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Also preferred is a saccharide of general formula (I), (II), (II-a), (II-b), (III), (III-a) or (IIIb), where -L- is -(CH ₂ ) _o -, o represents an integer selected from 2, 3, 4, 5 and 6, and E represents an amino group.

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Preferred is the synthetic saccharide of formula (II-b) wherein n represents 1 and E represents an amino group. More preferred is a synthetic saccharide of formula (II-b) where n is 1, E is an amino group, and the linker -L- is -L ^a -, - L ^a -L ^e -, -L ^a -L ^b - L ^e -, or -L ^a -L ^d -L ^e -;

- L ^a - represents -(CH ₂ ) _o -, -(CH ₂ -CH ₂ -O) _o -C ₂ H4-, or -(CH ₂ -CH ₂ -O) _o -CH ₂ ;

- Lb- represents -O-;

- L ^d - represents -(CH ₂ )q-, -(CH(OH))q-, -(CF ₂ )q-, -(CH ₂ -CH ₂ -O\-C ₂ H4-or -( CH2-CH2-O)q-CH2-;

- L ^e - represents -(CH ₂ )p1-, -(CF2)p1-, -C2H4-(O-CH2-CH2)p1-, -CH2-(O-CH2-CH2)p1- or -(CH2 )p1-O-(CH2)p2-; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6.

Particularly preferred is the synthetic saccharide of formula (II-b) wherein n is 1, E is an amino group, the linker -L- is -(CH ₂ ) _o - and o is an integer selected from 1, 2, 3 , 4, 5, 6, 7, 8,9 and 10. Even more preferred is the synthetic saccharide of formula (II-b) wherein n is 1, E is amino, the linker -L- is -( _CH2 ) _o -, and o is an integer selected from 1, 2, 3, 4, 5 and 6.

Preferred is the synthetic saccharide of formula (II-b) where n is 2 and E is an amino group. More preferred is a synthetic saccharide of formula (II-b) where n is 2, E is an amino group, and the linker -L- is -L ^a -, -L ^a -L ^e -, -L ^a -L ^b - L ^e -, or -L ^a -L ^d -L ^e -;

- L ^a - represents -(CH ₂ ) _o -, -(CH ₂ -CH ₂ -O) _o -C ₂ H ₄ -, or -(CH ₂ -CH ₂ -O) _o -CH ₂ ;

- L ^b - represents -O-;

- L ^d - is -(CH2)q-, -(CH(OH))q-, -(CF2)q-, -(CH2-CH2-O)q-C2H4-, or -(CH2-CH2- O)q-CH2-;

- L ^e - represents -(CH ₂ ) _p1 -, -(CF ₂ ) _p1 -, -C ₂ H ₄ -(O-CH ₂ -CH ₂ ) _p i--CH ₂ -(O-CH ₂ - CH ₂ ) _p i- or -(CH2)p!-O-(CH2)p2-; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6.

Particularly preferred is the synthetic saccharide of formula (II-b) wherein n is 2, E is an amino group, the linker -L- is -(CH ₂ ) _o -, and o is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Even more preferred is the synthetic saccharide of formula (II-b) wherein n is 2, E is amino, the linker -L- is -( _CH2 ) _o -, and o is an integer selected from 1, 2, 3, 4, 5 and 6.

In yet another preferred embodiment of the invention, the saccharide in accordance with the present invention is selected from the group consisting of the following:

- 22 046159

- 23 046159

- 24 046159

- 25 046159

- 26 046159

- 27 046159

- 28 046159

- 29 046159

- 30 046159

where Z represents

Particularly preferred is the saccharide of formula (1'a-4) wherein Z is

- 31 046159

Particularly preferred is the saccharide of formula (I'b-4) wherein Z represents O II

--O-R— I θ' .

Chemical synthesis.

Method for synthesizing a saccharide of general formula (I).

where n represents 1;

T* - represents an H- or phosphate group;

Z represents

ABOUT

II --O-R-I sg

L represents a linker; And

E represents -NH2, -N3, -CN, -O-NH2, -CH=CH2, -CCH, -Br, -Cl, -I, -CO2R', -CONH-NH2, -SH, -OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

includes the following stages:

A1) providing a monosaccharide of formula 1*:

where P ¹ , P ³ , P ⁴ and P22 p represent protecting groups, C represents -L-Ep, where E _p represents a solid support or protected end group; and A2) reacting a monosaccharide of formula 1* with a compound of formula 2* to obtain compound 3*:

- 32 046159

where P ¹ , P ³ , P ⁴ - P ¹⁰ and P ²² are protecting groups, C is -L-Ep, where E _p is a solid support or protected end group E, LG ² is a leaving group, and Np represents a protected amino group; And

A3) removing the ^P5 protecting group from compound 3* to obtain compound 4*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁰ and P ²² represent protecting groups, C represents -L-Ep, where Ep represents a solid support or protected end group of E, and Np represents a protected amino group; And

A4) introducing compound 4* into the reaction with monosaccharide 5* to obtain compound 6*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁴ and P ²² represent protecting groups, C represents -L-Ep, where Ep represents a solid support or protected end group E, LG ³ represents a leaving group, and Np represents a protected amino group; And

A5) removal of the protective group P ¹³ of compound 6* to obtain compound 7*:

- 33 046159

and P ²² represent protecting groups, C represents -LE where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ where Ep represents a solid support or protected end group of E, and Np represents a protected amino group; And

A6) reacting compound 7* with monosaccharide 8* to obtain compound 9*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ - P ¹⁷ and P ²² are protecting groups, C is -LEp, where E _p is a solid support or protected end group E, LG ⁴ represents a leaving group, and Np represents a protected amino group; And

A7) removal of the protective group P ¹⁵ of compound 9* to obtain compound 10*:

and P ²² represent protecting groups, C represents where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ , P ¹⁷

-L-Ep, where E _p represents a solid support or a protected end group E, and Np represents a protected amino group; And

A8) reacting compound 10* with monosaccharide 11* to obtain compound 12*:

- 34 046159

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴¹ and P ¹⁶ - P ²² are protecting groups, C is -LEp, where E _p is a solid support or protected end group E, LG ⁵ represents a leaving group, and Np represents a protected amino group; And

A9) optionally removing the P ²¹ protecting group of compound 12* to obtain compound 13* and reacting compound 13* with a phosphorylating agent to obtain compound 14*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -LE _p where E _p is a solid support or protected end group E, and N _p represents a protected amino group; And

A10) conversion of the protected amino groups of compound 12* or 14* into the corresponding acetamido groups to obtain compound 15* or 16*:

- 35 046159

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴¹ and P ¹⁶ - P ²⁴ represent protecting groups, and C represents L-Ep, where E _p represents a solid support or protected end group; And

A11) removing all remaining protecting groups from compound 15* or 16* to obtain compound 17* or 18* of general formula (I):

Synthesis of saccharide 17* or 18* of general formula (I), wherein hexasaccharide intermediate 12* is obtained directly from compound 7* by performing step A6'.

A6') Reaction of compound 7* with disaccharide 19* to obtain compound 12*:

- 36 046159

where P ¹⁶ -P ²⁰ and P ²¹ represent protecting groups, LG ⁶ represents a leaving group, and Np represents a protected amino group.

Thus, in one embodiment, the method for synthesizing saccharide 17* or 18* of general formula (I) includes steps A1), A2), A3), A4), A5), A6'), A9), A10) and A11).

Synthesis of saccharide 17* or 18* of general formula (I), wherein hexasaccharide intermediate 12* is obtained directly from compound 1* by performing step A2'.

A2') Reaction of compound 1 with pentasaccharide 20* to obtain compound 12*:

where P ⁶ - P ¹² , P ¹⁴ and P ¹⁶ - P ²¹ are protecting groups, LG ⁷ is a leaving group, and Np is a protected amino group. Pentasaccharide 20* can be prepared by reacting compound 2* with compound 5* subsequently with compound 8* and then with compound 11* or by reacting compound 2* with compound 5* and then with compound 19*.

Thus, in one embodiment, the method for synthesizing saccharide 17* or 18* of general formula (I) includes steps A1), A2'), A9), A10) and A11).

Compound 1* can be prepared from the corresponding protected mannose donor 21* by performing steps A1a), A1b) and A1c). A1a) Providing connection 21*:

where P'-P'' ¹ represent protecting groups, and LG ¹ represents a leaving group; and converting a compound of formula 21* to an alcohol of formula 22*:

where P ¹ -P ⁴ represent protecting groups; And

A1b) reacting the compound of formula 22* with the alcohol HO-L-C in the presence of a phosphorylating agent to obtain compound 23*;

where R ^! -P ⁴ and P ²² represent protecting groups, and C represents -L-Ep, where E _p represents a solid support or protected end group E; And

Alcohol 22* in step A1a) can be prepared according to Brooks et al. (Tetrahedron 1995, 51, 7999) by reacting compound 21* with allyltrimethylsilane in the presence of a Lewis acid (J. Am. Chem Soc. 1982, 104, 4976; Tetrahedron Letters, 1985, 26, 1479), followed by isomerization with bis( benzonitrile)palladium(II) into toluene by refluxing to propenyl C

- 37 046159 mannoside, ozonolysis or Lemieux-Johnson oxidation with sodium periodate and osmium tetroxide, and reduction to alcohol 22* with sodium acetoxyborohydride (see also Org. Biomol. Chem 2016, 14, 3913).

Alternatively, alcohol 22* in step A1a) can be prepared by reacting compound 21* with (iPrO)Me ₂ SiCH ₂ MgCl in the presence of copper(1) iodide (Org. Lett. 2004, 6, 119). Additionally, alcohol 22* in step A1) can be prepared by reacting compound 21* with a vinyl Grignard reagent, which is then oxidized with osmium tetroxide and sodium periodate and reduced to alcohol 22* with a sodium borohydride reagent such as sodium acetoxyborohydride.

Alternatively, alcohol 22* is prepared from the corresponding glycoside by reaction with trimethylsulfoxonium iodide and sodium hydride (J. Org. Chem. 2002, 67, 7439) or by reaction with propargyltrimethylsilane and BF ₃ -OEt ₂ followed by ozonolysis and reduction of sodium borohydride (Synlett 2005, 7, 1147).

A1c) Removing the P ² protecting group from compound 23* to obtain compound 1*

A method for synthesizing a saccharide of general formula (I), where n is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10;

T* - represents an H- or phosphate group;

Z represents

L represents a linker; And

E represents -NH2, -N3, -CN, -O-NH2, -CH=CH2, -CCH, -Br, -Cl, -I, -CO2R', -CONH-NH2, -SH, -OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

includes the following stages: B1) providing connection 13*

are protecting groups, C is where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²²

-L-Ep, where E _p represents a solid support or a protected end group E, and Np represents a protected amino group;

and repeating the following steps n-1 times:

B1.1) reacting with a compound of formula 22* in the presence of a phosphorylating agent,

B1.2) removal of the protecting group P ² ;

B1.3) implementation of stages A2) - A8) or stages A2) - A5) and A6') or stage A2');

B1.4) removal of the protecting group P ²¹ ; or

B2.1) introducing compound 13* into the reaction with a compound of the formula

in the presence of a phosphorylating agent,

B2.2) removal of the protecting group P ²¹ ;

B2.3) optionally repeating steps B2.1 and B2.2 from one to eight times in order to synthesize the corresponding trisaccharides (n=3) to decasaccharides (n=10);

- 38 046159 to receive a 24* connection:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² are protecting groups, C is -LEp, where E _p is a solid support or protected end group E, Np is is a protected amino group, and n is an integer from 2 to 10; And

B2) Optionally reacting compound 24* with a phosphorylating agent to produce compound 25*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -LE _p where E _p is a solid support or protected end group E, N _p represents a protected amino group, and n is an integer from 2 to 10; And

B3) conversion of the protected amino groups of compound 24* or 25* into the corresponding acetamido groups to obtain compound 26* or 27*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -LE _p where E _p is a solid support or protected end group E, and n

- 39 046159 represents an integer from 2 to 10; And

B4) removing all remaining protecting groups from compound 26* or 27* to obtain compound 28* or 29* of general formula (I)

where n is an integer from 2 to 10, and L and E have the meanings specified in the document.

The method for synthesizing the saccharide of general formula (I) in this

where n represents 1;

T* - represents an H- or phosphate group;

Z represents

L represents a linker; And

E represents -NH2, -N3, -CN, -O-NH2, -CH=CH2, -CCH, -Br, -Cl, -I, -CO2R', -CONH-NH2, -SH, -OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

includes the following stages:

C1) providing a monosaccharide of formula 30*, which can be prepared in accordance with the procedure disclosed in Chem. Eur. J. 2015, 21, 7511-7519 or Synlett, 2005, 7, 1147-1151:

- 40 046159

where P ¹ , P ³ , P ⁴ and P ²² represent protecting groups, C represents -L-Ep, where E _p represents a solid support or protected end group; And

C2) reacting a monosaccharide of formula 30* with a compound of formula 2* to obtain compound 31*:

where P ¹ , P ³ , P ⁴ - P ¹⁰ and P ²² represent protecting groups, C represents -L-Ep, where Ep represents a solid support or protected end group E, LG ² represents a leaving group, and Np represents a protected amino group; And

C3) removal of the protective group P ⁵ of compound 31* to obtain compound 32*:

are protecting groups, C is -L-Ep, where E _p where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁰ and P ²² represents a solid support or protected end group E, and Np represents a protected amino group ; And

C4) reacting compound 32* with monosaccharide 5* to obtain compound 33*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁴ and P ²²

are protecting groups, C is -L-Ep, where

E _p represents a solid support or protected end group E, LG ³ represents a leaving group, and Np represents a protected amino group; And

C5) removal of the protective group P ¹³ of compound 33* to obtain compound 7*:

- 41 046159

OP ²² (34*) where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ and P ²² are protecting groups, C is -L-Ep, where Ep is a solid support or protected end group E, and Np represents a protected amino group; And

C6) reacting compound 34* with monosaccharide 8* to obtain compound 35*:

Ur Р ¹⁶ О / V\--o р ¹⁵ о-Л^А, ^N P LG ⁴ ( ^8th ) OR ¹⁷ Р ¹⁶ О / |( „ Р”О-Л ^Рр Р ¹¹ О Р ¹⁰ О-к ^{ P ⁹ OV^-O _ p8 ₀ _V^>Xi- ⁰ · ^Or6 Lg-I ) / P ⁴ O—y O ^r L, O P ³ O-'\\E°\ L and

OP ²² where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ - P ¹⁷ and P ²² are protecting groups, C is -LEp, where Ep is a solid support or protected end group E, LG ⁴ represents a leaving group, and Np represents a protected amino group; And

C7) removal of the protective group P ¹⁵ of compound 35* to obtain compound 36*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ , P ¹⁷ and P ²² represent protecting groups, C represents -L-Ep, where E _p represents a solid support or protected end group E, and Np represents a protected amino group; And

C8) Reaction of compound 36* with monosaccharide 11* to obtain compound 37*:

- 42 046159

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ and P ¹⁶ - P ²² represent protecting groups, C represents -LEp, where Ep represents a solid support or protected end group E, LG ⁵ represents is a leaving group, and Np is a protected amino group; And

C9) optionally removing the P ²¹ protecting group of compound 37* to obtain compound 38* and reacting compound 38* with a phosphorylating agent to obtain compound 39*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -L-Ep, where E _p is a solid support or a protected end group E, and Np represents a protected amino group; And

C10) Conversion of the protected amino groups of compound 37* or 39* into the corresponding acetamido groups to obtain compound 40* or 41*:

- 43 046159

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ and P ¹⁶ - P ²² represent protecting groups and C represents L-Ep, where E _p represents a solid support or protected end group; And

C11) removing all remaining protecting groups from compound 40* or 41* to obtain compound 42* or 43* of general formula (I)

HE

Synthesis of saccharide 42* or 43* of general formula (I), wherein hexasaccharide intermediate 37* is obtained directly from compound 34* by performing step A6'). Thus, in one embodiment, the method for synthesizing saccharide 42* or 43* of general formula (I) includes steps C1), C2), C3), C4), C5), A6'), C9), C10) and C11).

Synthesis of saccharide 42* or 43* of general formula (I), wherein hexasaccharide intermediate 37* is obtained directly from compound 30* by performing step A2'. Thus, in one embodiment, the method for synthesizing saccharide 42* or 43* of general formula (I) includes steps C1), A2'), C9), C10) and C11).

Thus, another method for synthesizing the saccharide of general formula (I) involves the following steps:

C1) providing monosaccharide of formula 30*:

- 44 046159

where P ¹ , P ³ , P ⁴ and P ²² represent protecting groups, C represents -L-Ep, where E _p represents a solid support or protected end group; And

C2') reacting compound 30* with pentasaccharide 20* to obtain compound 37*:

where P ⁶ -P ¹² , P ¹⁴ and P ¹⁶ -P ²¹ represent protecting groups, LG ⁷ represents a leaving group, and Np represents a protected amino group.

C9) Optionally removing the P ²¹ protecting group from compound 37* to obtain compound 38* and reacting compound 38* with a phosphorylating agent to obtain compound 39*:

- 45 046159

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -L-Ep, where E _p is a solid support or a protected end group E, and Np represents a protected amino group; And

C10) conversion of the protected amino groups of compound 37* or 39* into the corresponding acetamido groups to obtain compound 40* or 41*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ and P ¹⁶ - P ²⁴ represent protecting groups and C represents -LE _p where E _p represents a solid support or protected end group; And

C11) removing all remaining protecting groups from compound 40* or 41* to obtain compound 42* or 43* of general formula (I)

- 46 046159

this document. Compound 30* can be prepared from wherein L and E are as indicated in the corresponding protected mannose donor 21* by performing steps A1a), C1b), C1c) and C1d). C1b) Conversion of the compound of formula 22* to the corresponding halide 44*:

where P ¹ - P ⁴ represent protecting groups, and Hal is selected from -Br or -I; And

C1c) reacting the compound of formula 44* with the alcohol HO-L-C in the presence of phosphite to obtain compound 45*;

where P^P ⁴ and P ²² represent protecting groups, and C represents -L-Ep, where Ep represents a solid support or protected end group E; And

C1d) removing the P ² protecting group from compound 45* to obtain compound 30*.

The conversion of alcohol 22* to the corresponding halide 44* in step C1b) can be achieved according to standard procedures, i.e. by reacting alcohol 22* with CBr ₄ or I2 in the presence of PPh ₃ , or alternatively by converting alcohol 22* to methanesulfonate or trifluoromethanesulfonate and subsequent substitution with tetrabutylammonium bromide or tetrabutylammonium iodide.

The phosphite used in step C1c) is preferably a trialkyl phosphite, such as triethyl phosphite, which is reacted with halide 44* to form a phosphonate, and subsequently hydrolyzed to a phosphonic acid with a Lewis acid such as bromotrimethylsilane, followed by water (Tetrahedron 1995, 51, 7999). Phosphonic acid is reacted with alcohol HO-L-C in the presence of trichloroacetonitrile to obtain compound 45*.

Alternatively, the phosphite used in step C1c) may be a phosphoramidite, such as dialkyl or dibenzyl (diethylphosphoramidite), or a bis(diisopropylamino)benzyloxyphosphine, which is reacted with compound 44* in the Arbuzov reaction and with the alcohol HO-L-C in release of diethylamine.

A method for synthesizing a saccharide of general formula (I), where n is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10;

- 47 046159

T* - represents an H- or phosphate group;

Z represents

L represents a linker; And

E represents -NH2, -N3, -CN, -O-NH2, -CH=CH2, -CCH, -Br, -Cl, -I, -CO2R', -CONH-NH2, -SH, -OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

includes the following stages:

D1) providing connection 38*

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² are protecting groups,

C represents -L-Ep, where E _p represents a solid support or protected end group E, and N _p represents a protected amino group;

and repeating the following steps n-1 times:

D1.1) reacting with a compound of formula 44* in the presence of a phosphite,

D1.2) removal of the protective group P ² ;

D1.3) implementation of stages C2) - C8) or stages C2) - C5) and A6') or stage A2');

D1.4) removal of the protective group P ²¹ ; or

D2.1) reacting compound 38* with a compound of formula

in the presence of a conjugating agent,

D2.2) removal of the protective group P ²¹ ;

D2.3) optionally repeating steps D2.1 and D2.2 from one to eight times in order to synthesize the corresponding trisaccharides (n=3) to decasaccharides (n=10);

to obtain connection 46*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² represent protecting groups, C represents -L- 48 046159

Ep, where Ep represents a solid support or protected end group E, Np represents a protected amino group and n represents an integer from 2 to 10; And

D2) optionally reacting compound 46* with a phosphorylating agent to produce compound 47*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -L-Ep, where E _p is a solid support or protected end group E, Np represents a protected amino group and n represents an integer from 2 to 10; And

D3) conversion of the protected amino groups of compound 46* or 47* into the corresponding acetamido groups to obtain compound 48* or 49*:

where P ¹ , P ³ , P ⁴ , P6 - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -LE _p , where E _p is a solid support or protected a terminal group E, and n is an integer from 2 to 10; And

D4) removing all remaining protecting groups from compound 48* or 49* to obtain compound 50* or 51* of general formula (I)

- 49 046159

where n is an integer from 2 to 10, and L and E have the meanings specified herein.

A method for synthesizing a saccharide of general formula (I), where n is 1;

T* - represents an H- or phosphate group;

ABOUT

II

-O-R-I

Z represents '

L represents a linker; And

E represents -NH ₂ , -N ₃ , -CN, -O-NH ₂ , -CH=CH ₂ , -C CH, -Br, -Cl, -I, -CO ₂ R', -CONH-NH2, -SH, -OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

includes the following stages:

E1) providing monosaccharide of formula 52*:

where P ¹ , P ³ , P ⁴ and P ²⁵ represent protecting groups; And

E2) reacting a monosaccharide of formula 52* with a compound of formula 2* to obtain compound 53*:

where P ¹ , P ³ , P ⁴ -P ¹⁰ and P ²⁵ represent protecting groups, LG ² represents a leaving group, and N _p represents a protected amino group; And

E3) removing the protective group P ⁵ of compound 53* to obtain compound 54*:

where P ¹ , P ³ , P ⁴ , P ⁶ -P ¹⁰ and P ²⁵ represent protecting groups, and Np represents a protected amino group; And

E4) reacting compound 54* with monosaccharide 5* to obtain compound 55*:

- 50 046159

where P ¹ , P ³ , P ⁴ , P ⁶ -P ¹⁴ and P ²⁵ represent protecting groups, LG ³ represents a leaving group, and Np represents a protected amino group; And

E5) removing the protecting group P ¹³ from compound 55* to obtain compound 56*:

and ^P25 represent protecting groups, and Np represents a protected amino group; ^P1 , ^P3 , ^P4 , ^P6 - ^P12 , ^P14 ; And

E6) reacting compound 56* with disaccharide 19* to obtain compound 57*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ and P ¹⁶ - P ²⁵ represent protecting groups, LG ⁶ represents a leaving group, and Np represents a protected amino group; And

E7) conversion of the protected amino groups of compound 57* into the corresponding acetamido groups to obtain compound 58*:

- 51 046159 where P ¹ , P ³ , P ⁴ , P ⁶ -P ¹² , P ¹⁴ , P ²¹ and P ²⁵ are protecting groups; And

E8) removing the P ²⁵ protecting group of compound 58* to obtain compound 59* and reacting compound 59* with alcohol HO-LC in the presence of a phosphorylating agent to obtain compound 15*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - ^{P 22} represent protecting groups, and

E9) optionally removing the P ²¹ protecting group of compound 15* to obtain compound 60* and reacting compound 60* with a phosphorylating agent to obtain compound 16*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -L-Ep, where E _p is a solid support or a protected end group E; And

E10) removing all remaining protecting groups from compound 15* or 16* to obtain compound 17* or 18* of general formula (I)

- 52 046159

A method for synthesizing a saccharide of general formula (I), where n is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10; T* - represents an H- or phosphate group;

Z represents

O II -O-R-I O'

L represents a linker; And

E represents -NH2, -N3, -CN, -O-NH, -CH=CH2, -CCH. -Br, -Cl, -I, -CO2R', -CONH-NH, SH, -OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

includes the following stages:

F1) providing connection 60*

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² represent protecting groups,

C represents -L-Ep, where E _p represents a solid support or protected end group;

F2.1) reacting compound 60* with a compound of formula

- 53 046159

in the presence of a phosphorylating agent,

F2.2) removal of the protective group P ²¹ ;

F3) it is optional to repeat steps F2.1 and F2.2 n-2 times in order to synthesize the corresponding trimers (n=3) to decamers (n=10); with receiving connection 26*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² represent protecting groups, C represents -L-Ep, where E _p represents a solid support or a protected end group E, and n is an integer from 2 to 10; And

F4) optionally reacting compound 26* with a phosphorylating agent to produce compound 27*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -LE _p where E _p is a solid support or a protected end group E, and n is an integer from 2 to 10; And

F5) removing all remaining protecting groups from compound 26* or 27* to obtain compound 28* or 29* of general formula (I)

- 54 046159

where n is an integer from 2 to 10, and L and E have the meanings specified herein.

A method for synthesizing a saccharide of general formula (I), where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

T* - represents an H- or phosphate group;

Z represents

ABOUT

II

-O-R-I

ABOUT'

L represents a linker; And

E represents -NH2, -N3, -CN, -O-NH2, -CH=CH2, -CCH, -Br, -Cl, -I, -CO2R', -CONH-NH2, -SH, -OH or -SAc;

R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, or N-succinimidyl;

includes the following stages:

G1) providing monosaccharide of formula 52*:

where P ¹ , P ³ , P ⁴ and P ²⁵ represent protecting groups; And

G2) reacting a monosaccharide of formula 52* with a compound of formula 2* to obtain compound 3*:

- 55 046159 where P ¹ , P ³ , P ⁴ -P ^1C and P ²⁵ represent protecting groups, LG ² represents a leaving group, and N _p represents a protected amino group; And

G3) removing the ^P5 protecting group from compound 53* to obtain compound 54*:

where P ¹ , P ³ , P ⁴ , P ⁶ -P ^1C ' and P ²⁵ represent protecting groups, and Np represents a protected amino group; And

G4) reacting compound 54* with monosaccharide 5* to obtain compound 55*:

represent protecting groups, LG ³ represents a leaving group where P ¹ , P ³ , P ⁴ , P ⁶ -P ¹⁴ and P ²⁵ group, and N _p represents a protected amino group; And

G5) removal of the protective group P ¹³ of compound 55* to obtain compound 56*:

and ^P25 represent protecting groups, and Np represents a protected amino group; ^P1 , ^P3 , ^P4 , ^P6 - ^P12 , ^P14 ; And

G6) reacting compound 56* with disaccharide 19* to obtain compound 57*:

- 56 046159 where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ and P ¹⁶ - P ²⁵ represent protecting groups, LG ⁶ represents a leaving group, and Np represents a protected amino group; And

G7) conversion of the protected amino groups of compound 57* into the corresponding acetamido groups to obtain compound 58*:

where P ¹ , P ³ , P ⁴ , P6-P ¹² , P ¹⁴ , P ²¹ and P ²⁵ represent protecting groups; And

G8) removing the P ²⁵ protecting group of compound 58* to obtain compound 59* and reacting compound 59* with HO-LC alcohol in the presence of a phosphorylating agent to obtain compound 15*:

where P ¹ , P ³ , P ⁴ , P6 - P ¹² , P ¹⁴ , P ¹⁶ - ^{P 22} represent protecting groups, and

G9) repeating steps G9.1 and G9.2 n-1 times in order to synthesize the corresponding dimers (n=3) to decamers (n=10);

G9.1) removal of the protective group P ²¹ ; And

G9.2) reacting the product of step G9.1) with a compound of formula

in the presence of a phosphorylating agent, to obtain compound 61*:

- 57 046159

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²² represent protecting groups, C represents -L-Ep, where E _p represents a solid support or protected end group;

G10) optionally removing the P ²¹ protecting group of compound 61* or compound 15* to obtain compound 26* and reacting compound 26* with a phosphorylating agent to obtain compound 27* to provide compound 26*:

where P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are protecting groups, C is -L-Ep, where E _p is a solid support or a protected end group E, and n is an integer from 1 to 10; And

G11) removing all remaining protecting groups from compound 26* or 27* to obtain compound 28* or 29* of general formula (I):

- 58 046159

to 10, and L and E have the meanings specified in this gorge n represents an integer from the document.

Ep is a solid support or protected end group. E is -NH, -N3, -CN, -O-NH2, -CH=CH2, -CCH, -Br, -Cl, -I, -CO2R', -CONHNH2, -SH, -OH or -SAc ; and the corresponding protected end group E _p is -N(P ²⁶ )(P ²⁷ ), -N3, -CN, -ON(P ²⁶ )(P ²⁷ ), -CH=CH2, -CbC'H, -Br, -Cl, -I, -CO2R', -CONHN(P ²⁶ )(P ²⁷ ), -SP _s , or -Sac N _p represents a protected amino group. Preferably, Np is selected from -N ₃ , -NH-CO-CC13 and -NH-CO-O-CH2-CC13 (Troc).

P ¹ , P 2 , P ³ , P ⁴ , P ⁵ , P ⁶ , ^{P 7 ,} P ⁸ , P 9 , P ¹⁰ , P ¹¹ , P ¹² , P ¹³ , P ¹⁴ , P ¹⁵ , ^{P 16 ,} P ¹⁷ , P ¹⁸ , P ¹⁹ , P ²⁰ , P ²¹ , P ²² , P ²³ , P ²⁴ , P ²⁵ , P ²⁶ and P ²⁷ are protecting groups. As used herein, the term protecting group refers to commonly used groups in organic synthesis, typically used to protect hydroxyl groups, amino groups and thiols.

Preferably, the protecting group P ²¹ can be removed under conditions under which other protecting groups present in the molecule are stable.

Amine protecting groups are preferably stable under the conditions used to remove hydroxyl protecting groups present in the molecule.

Hydroxyl protecting groups, preferably other than the P ²¹ protecting group, can preferably be removed by hydrogenation.

More preferably, P ¹ , P ² , P ³ , P ⁴ , P ⁵ , P ⁶ , P ⁷ , P ⁸ , P ⁹ , P ¹⁰ , P 11, P ¹² , P ¹³ , P ¹⁴ , ^{P 15 ,} P ¹⁶ , P ¹⁷ , P ¹⁸ , P ¹⁹ , P ²⁰ , P ²¹ , P ²² , P ²³ , P ²⁴ and P ²⁵ are suitable hydroxyl protecting groups, more preferably various suitable hydroxyl protecting groups that are capable of being removed one by one another suitable unprotection sequence. Preferred protecting groups for hydroxyl groups are acetyl, phenyl, benzyl, isopropylidene, benzylidene, benzoyl, p-methoxybenzyl, p-methoxybenzylidene, p-methoxyphenyl, p-bromobenzylidene, p-nitrophenyl, allyl, acetyl, isopropyl, p-bromobenzyl, dimethoxytrityl , trityl, 2-naphthylmethyl, pivaloyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, tert-butylmethoxyphenylsilyl, triethylsilyl, trimethylsilyl, 2-trimethylsilylethoxymethyl, 9-fluorenylmethoxycarbonyl, benzyloxymethyl, methyloxymethyl, tert-butyloxymethyl, methoxyethyloxymethyl, levulino silt, naphthylidene, chloroacetyl , picoloyl, texyldimethylsilyl (TDS), (2-nitrophenyl) acetyl (NPAc), 2-(azidomethyl)benzoyl (AzmB).

Protecting groups can be divided into permanent protecting groups and temporary protecting groups. Permanent protecting groups are protecting groups that are stable throughout the synthesis and that can be effectively removed at a late stage of the synthesis. In this case, permanent protecting groups include P ¹ , P ³ , P ⁴ , P ⁶ - ^{P 12} , P ¹⁴ , P ¹⁶ - ^{P 20} , P ²² - P ²⁶ . P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - P ²⁰ and P ²² - P ²⁴ are masking hydroxyl groups throughout the synthesis, while protecting groups P ²⁶ and P ²⁷ are masking terminal amino groups present in the terminal group Er. Preferably, the protecting groups P ³ , P ⁴ , P ⁸ -P ¹² , P ¹⁴ , P ¹⁶ -P ²⁰ and P ²² -P ²⁴ are benzyl groups, the protecting group P ¹ is a benzoyl group, the protecting groups P ⁷ and P ¹⁸ are acetyl groups, the P ²⁶ protecting group is a benzyl group and the P ²⁷ protecting group is a benzyloxycarbonyl group (Cbz). Temporary protecting groups are generally orthogonal protecting groups that can be selectively removed at different levels of synthesis to free hydroxyl groups for the subsequent introduction of various substituents, including monosaccharides, other protecting groups or other residues present on the molecule. In this case, the temporary protecting groups include P ² , P ⁵ , P ¹³ , P ¹⁵ , P ²¹ and P ²⁵ .

Temporary protecting groups P ² , P ⁵ , P ¹³ , P ¹⁵ , P ²¹ and P ²⁵ are preferably selected from the following, but are not limited to: allyl, p-methoxybenzyl, 2-naphthylmethyl, tri-isopropylsilyl, tert-butyldimethylsilyl, tert-butylmethoxyphenylsilyl, triethylsilyl, trimethylsilyl, 2trimethylsilylethoxymethyl, 9-fluorenylmethoxycarbonyl, texyldimethylsilyl, (2-nitrophenyl)acetyl, 2-(azidomethyl)benzoyl, and levulinoyl. Preferably, the protecting groups P ² , P ⁵ , P ¹³ , P ¹⁵ , P ²¹ and P ²⁵ can be selectively removed in the presence of the protecting groups P ¹ , P ³ , P ⁴ , P ⁶ - P ¹² , P ¹⁴ , P ¹⁶ - R ²⁰ , R ²² -R ²⁴ . Prefer

- 59 046159 specifically, P ² , P ⁵ , P ¹³ , P ¹⁵ , P ²¹ and P ²⁵ are 9-fluorenylmethoxycarbonyl or levulinoyl. In a preferred embodiment of the invention, the protecting groups P ¹³ and P ²¹ are 9fluorenylmethoxycarbonyl, and the protecting groups P ¹ , P ⁵ and P ¹⁵ are levulinoyl. Preferably, P ²¹ is selected from tri-isopropylsilyl, tert-butyldimethylsilyl, tert-butylmethoxyphenylsilyl. Preferably, P ²⁵ is 2-naphthylmethyl.

The innovative choice of protecting groups allows access to a library of saccharides of general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), functionalized with end group for subsequent conjugation to an immunogenic carrier or solid support. In addition, the choice of leaving group affects the stereochemical outcome of glycosylation reactions at stages A1a), A2), A2'), A4), A6), A6'), A8), B1.3), C2), C4), C6) , C8), D1.3), E2), E4) and E6).

Structural elements 2*, 5*, 8*, 11*, 19*, 20* and 21* are glycosylation agents. As used herein, the term glycosylation agent refers to a monosaccharide functionalized at the anomeric position with a leaving group, which upon activation with a suitable activating agent produces an oxocarbenium intermediate capable of reacting with a nucleophile, such as a hydroxyl group. Therefore, glycosylation agents 2*, 5*, 8*, 11*, 19*, 20* and 21* are functionalized at the anomeric position with leaving groups LG ¹ , LG ² , LG ³ , LG ⁴ , LG ⁵ , LG ⁶ and LG ⁷ . Examples of leaving groups useful in the present synthesis are well known to those skilled in carbohydrate chemistry and include halides, thioesters, imidates, acetate and phosphate.

Preferably, the leaving groups LG ¹ , LG ² , LG ³ , LG ⁴ , LG ⁵ , LG ⁶ and LG ⁷ are selected from halogen (-Cl, -Br, -F, -I), -OC(=NH)-CCl3, -OC(=NPh)-CF3, -OAc, -SRL, -SO-RL, -SO-Ph, -SO-CH2-Ph, -SO-Tol, -SO-C6H4-(para-OCH3), -O -(CH2)3-CH=CH2, -OP(OR ^l )2, -O-PO(OR ^l )2, -O-CO-ORL, -O-CO-SRL, -O-CS-SRL, Me —O —O-CO-N^j — O-CS-nQj — ' ^{5 N} ' -O-CS-ORL, where R ^l can be any of alkyl or aryl groups, preferably methyl, ethyl, propyl, isopropyl , phenyl or toluyl.

Preferably, the leaving groups LG ¹ , LG ² , LG ³ , LG ⁴ , LG ⁵ , LG ⁶ and LG ⁷ are selected from the groups of leaving groups which consist of: SBox, STaz,

where thioesters may also be substituted.

As stated above, the provision of an oxocarbenium intermediate depends on the activation of the leaving group installed at the anomeric position of the glycosylation agent with a suitable or suitable activating agent. It will be known to those skilled in the art that suitable activating agents for phosphate (i.e., phosphate activating agents) and imidate (i.e., imidate activating agents) are Lewis acids such as silyl triflate or silver triflate, while suitable activating agents for thioester i.e. thioester activating agents include, but are not limited to: NIS/TfOH, NIS/TMSOTf, NIS/BF ₃ Et ₂ O, NIS/AgOTf, DMTST/Tf ₂ O, IDPC, BSP/Tf ₂ O, Ph ₂ SO/ Tf ₂ O. Examples of silyl triflate include, but are not limited to, trimethylsilyl trifluoromethanesulfonate, tert-butyldimethyl trifluoromethanesulfonate, triiospropyl trifluoromethanesulfonate.

Preferably, LG ¹ , LG ² , LG ³ , LG ⁴ , LG ⁵ , LG ⁶ and LG ⁷ are thioesters and even more preferably, LG ¹ , LG ² , LG ³ , LG ⁴ , LG ⁵ , LG ⁶ and LG ⁷ selected from a group that consists of:

It is preferable that the coupling reaction between saccharides at stages A1a), A2), A2'), A4), A6), A6'), A8), B1.3), C2), C4), C6), C8), D1 .3), E2), E4) and E6) was carried out by activation of NIS/TfOH or TMSOTf in a mixture of a non-polar solvent and a polar aprotic solvent at a temperature of 10°C to 10°C. Even more preferably, said reaction is carried out in a mixture of a non-polar solvent and a polar aprotic solvent by treatment with NIS/TfOH at a temperature of approximately 0°C.

The preferred polar aprotic solvent is tetrahydrofuran, di

- 60 046159 ethyl ether and dioxane. Preferred non-polar solvents are toluene, halogenated solvents such as chloroform and methylene chloride. Preferred mixtures of non-polar and polar aprotic solvent are: methylene chloride/tetrahydrofuran, methylene chloride/diethyl ether, toluene/diethyl ether, toluene/tetrahydrofuran.

Removal of protective groups P ¹ , P ³ , P ⁴ , P ⁶ -P ¹² , P ¹⁴ , P ¹⁶ -P ²⁰ , P ²² -P ²⁴ , P ²⁶ and P ²⁷ , which is carried out at stages A11), B4), C11 ), D4), E10) and F5), includes:

first cleavage of base-labile protecting groups by treatment with a base in the presence of hydrogen peroxide in a solvent mixture. Preferably, the base is NaOMe or LiOH; and a second cleavage of the hydrogenation sensitive protecting groups by exposing the compound to hydrogen in the presence of a palladium catalyst in a solvent mixture.

The phosphorylating agent used in steps A9), B2), C9), D2), E9) and F2.1) is a compound capable of introducing the P(O)(OH) ₂ group in its free form or as a monoester in a reactive position into the connection. Thus, the phosphate group is transferred to the hydroxyl group in steps A9), B2), C9), D2), E9) and F2.1). Preferred phosphorylating agents used in the present invention are diphenylphosphite, bis(diisopropylamino)benzyloxyphosphine, benzyl NN-diisopropylphosphonamidate or K,K-daethyl-1,5dihydro-3H-2,3,4-benzodioxaphosphepine-3-amine in combination with an activating agent. agent such as 1Htetrazole and subsequent oxidation with an oxidizing agent such as hydrogen peroxide or 3-chloroperbenzoic acid. In a preferred embodiment of the invention in steps A9), B2), C9), D2), E9) and F2.1), the phosphorylating agent is bis(diisopropylamino)benzyloxyphosphine in combination with 1Htetrazole and 3-chloroperbenzoic acid. In a preferred embodiment of the invention in steps A9), B2), C9), D2), E9) and F2.1), the phosphorylating agent is diphenylphosphite.

The phosphorylating agent used in step A1b) is preferably bis(diisopropylamino)benzyloxyphosphine, benzyl N,N-diisopropylphosphonamidate or N1,5-dihydro-3H-2,3,4-benzodioxaphosphepine-3-amine. The preferred activating agent used in step A1b) is 1H-tetrazole, 4,5-dicyanoimidazole, 2-benzylthiotetrazole, 5ethylthiotetrazole, benzimidazolium triflate or imidazolium triflate. Most preferred is 1H-tetrazole as the activating agent. The oxidation reaction is preferably carried out in the presence of an oxidizing agent such as hydrogen peroxide or 3-chloroperbenzoic acid.

Glycoconjugates.

Another aspect of the present invention relates to a conjugate that contains a saccharide of general formula (I) covalently linked or covalently attached to an immunogenic carrier through a terminal E group of the -O-L-E group. In other words, another aspect of the present invention is directed to a saccharide of any of the general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), conjugated to an immunogenic carrier through the terminal E group of the -O-L-E group. A conjugate which contains a synthetic saccharide of the general formula (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), covalently bound or covalently attached to immunogenic carrier through the terminal E group of the -O-L-E group, is also defined as a conjugate obtained by reacting a saccharide of any of the general formulas (I), (II), (II-a), (II-b), (III), ( III-a) or (III-b) with an immunogenic carrier. Surprisingly, this conjugate was effective as a vaccine for immunization against diseases associated with the bacteria Clostridium difficile.

Saccharides are commonly known to those skilled in the art as TI-2 (T-independent type 2 cell) antigens and weak immunogens. TI-2 antigens are antigens that are recognized only by mature B cells through cross-linking of surface immunoglobulin receptors. Without the help of T cells, immunological memory is not created, and neither isotype switching from IgM to other IgG subclasses nor affinity maturation of B cells occurs. Moreover, saccharides are known to be poor immunogens in humans due to structural homology with human glycolipids and glycoproteins. Because of their weak immunogenic properties, saccharides exhibit poor ability to both produce antibodies by B cells and form memory cells, features that are essential for the production of potent vaccines.

Therefore, to produce a potent saccharide-based vaccine, saccharides of general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b) are conjugated with an immunogenic a carrier to provide conjugates that present increased immunogenicity compared to the saccharide. Therefore, a conjugate which contains a saccharide moiety is also included within the scope of the present application.

- 61 046159

where n, Z and T* have the meanings specified herein, are covalently attached via an O atom to the immunogenic carrier.

Said conjugate includes at least one synthetic saccharide of general formula (I) and an immunogenic carrier to which at least one saccharide (I) is covalently linked.

Surprisingly, it has been found that immunization with a conjugate which contains a saccharide of general formula (I) covalently attached to an immunogenic carrier results in the production of high titers of antibodies specific to the carbohydrate moiety of the saccharide of general formula (I). These antibodies cross-react with the native PS-II cell wall of Clostridium difficile and exhibit opsonophagocytosis and bactericidal activity, thereby providing protection against Clostridium difficile bacteria.

In this context, the term immunogenic carrier is defined as a structure that is conjugated to a saccharide to form a conjugate that exhibits increased immunogenicity compared to the saccharide per se. Thus, conjugation of saccharides of general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b) with an immunogenic carrier has the effect of stimulating an immune response against a saccharide of general formula (I) without inducing an immune response against said immunogenic carrier.

Preferred immunogenic carriers are carrier proteins (CPs) or glycosphingolipids with immunomodulatory properties. To one skilled in the art, a carrier protein (CP) is a protein that is non-toxic and non-reactogenic and can be obtained in sufficient quantity and purity. The carrier protein is selected from the group which includes or consists of: diphtheria toxoid, such as CRM ₁₉₇ , mutated diphtheria toxoid, modified diphtheria toxoid, mutated and modified diphtheria toxoid, tetanus toxoid, modified tetanus toxoid, mutated tetanus toxoid, non-lipidated cell surface liporoprotein (protein D) non-typeable Haemophilus influenzae, outer membrane protein (OMP), Neisseria meningitidis complex, bovine serum albumin (BSA), fissurella hemocyanin (KLH) or cholera toxoid (CT). The term toxoid, as used herein, refers to a bacterial toxin (usually an exotoxin) whose toxicity has been inactivated or suppressed by either chemical (formalin) or heat treatment, while other properties, typically immunogenicity, are retained. The mutated toxoid used herein is a recombinant bacterial toxin that has been modified to be less toxic or even non-toxic by changing the amino acid sequence of the wild type. Such a mutation may be a substitution of one or more amino acids. Such a mutated toxoid presents on its surface a functional group that can react with the Y functional group of the connecting molecule to form a modified toxoid. This functionality is known to one skilled in the art and includes, but is not limited to, the primary amino functional group of a lysine residue that can react with an activated ester, isocyanate group or aldehyde in the presence of a reducing agent, the carboxylate functional group of a glutamate or aspartate residue that can be activated carbodiimides, or the thiol functional group of a cysteine residue.

Activated esters include Na-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS), succinimidyl (4-iodoacetyl)aminobenzoate (sulfo-SIAB), succinimidyl-3-(bromoacetamido)propionate (SBAP), disuccinimidyl glutarate (DSG), disuccinimidyl adipate (DSA), 2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide (PEG-4-SPDP) (see Fig. 2).

The cysteine residue on the carrier protein can be converted to the corresponding dehydroalanine, which can be further reacted with a suitable coupling molecule to produce a modified carrier protein having the X functional group of the coupling molecule on its surface.

- 62 046159

It is particularly preferred when the saccharides of general formula I are conjugated to the non-toxic mutated diphtheria toxin CRM ₁₉₇ , which represents the primary amine functional groups of a lysine residue as functional groups.

CRM ₁₉₇ , like the wild-type diphtheria toxin, is a single polypeptide chain of 535 amino acids (58 kDa), consisting of two subunits linked by disulfide bridges having a single amino acid substitution of glutamic acid for glycine. It is used as a carrier protein in a number of approved disease conjugate vaccines, such as Prevnar.

Thus, in a preferred embodiment of the invention, the carrier protein presents on its surface primary amino functional groups of lysine residues, which are capable of reacting with the Y functional group of the connecting molecule to form a modified carrier protein having on its surface the specified X functional group of the connecting molecule, which is capable of interact with the terminal amino group of the linkers of compounds of general formula (I).

Said functional group X of the connecting molecules is selected from the group which contains or consists of the following: maleimide; α-iodoacetyl; α-bromoacetyl; and Nhydroxysuccinimide ester (NHS), aldehyde, imidoester, carboxylic acid, alkyl sulfonate, sulfonyl chloride, epoxide, anhydride, carbonate (see FIG. 3).

Preferably, the saccharide of general formula I is conjugated to a non-toxic mutated diphtheria toxin CRM ₁₉₇ , which is modified with maleimide. In yet another preferred embodiment of the invention, the saccharide of general formula I is conjugated to the non-toxic mutated diphtheria toxin CRM ₁₉₇ , which is modified with α-bromoacetamide. In the most preferred embodiment of the invention, the saccharide of general formula I is conjugated to the non-toxic mutated diphtheria toxin CRM ₁₉₇ , which is modified with Nhydroxysuccinimide adipate.

Preferred is the conjugate of general formula (IV)

where c is in the range between 2 and 18;

-E1- represents a covalent bond, -NH-, -O-NH-, -O-, -S-, -CO-, -CH=CH-, -CONH-,

-CO-NHNH-,

-W- selected from:

a is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, b is an integer selected from 1, 2, 3 and 4, CP is a carrier protein ; and n, L, Z and T* have the meanings set forth herein.

Preferably E1 is a covalent bond, -NH-,

-CH=CH-, -CONH-,

- 63 046159

Preferably the CP is CRM ₁₉₇ . Thus, in one embodiment of the invention, the conjugate has the general formula (IV) wherein CP is CRM ₁₉₇ and c, -E1-, W, n, L, Z and T* are as defined herein.

Preferably, in general formula (IV), the linker -L- is selected from: -L ^a -, -L ^a -L ^e -, -L ^a -L ^b -L ^e -, and -L ^a -L ^d -L ^e - ;

- L ^a - selected from: -(CH)o-, -(CH-SSH-OX^SCH-, -(SSH-SSH-O^-SSH;

- L ^b - represents -O-;

- L - selected from: -(CH^q—, -(CF2)q-, -(CH2-CH2-O)q-C2H4—, and

- (CH2-CH2-O)q-CH2-;

- L ^e - selected from: -(CH)^, -Н)рг, ^HHO-CH^C^V, -CH2-(O-CH2-CH2)p1— and -(CH2) _Р 1-О-( CH2) _P 2—;

and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6.

Also, a conjugate of general formula (IV) wherein -W- is an integer selected from 2, 3, 4, 5 and 6 is preferred.

A conjugate of general formula (IV), wherein the linker -L- is selected from: -L ^a - -L ^a -L ^e - -L ^a -L ^b -L ^e - and -L ^a -L ^d -L ^e -;

, and a represents co-

- L ^a - selected from: -(CH)o-, -(CH2-CH2-O)o-S2Shch-, -(SShch-SShch-OXO-SShch;

- L - represents -O-;

- L ^d - selected from: -(CH2)q—, -(CF2)q— -(CH2-CH2-O)q-C2H4— and

- (CH2-CH2-O)q-CH2-;

- L ^e - selected from: -(СШ)р1-, —Н)рг, -C2H4-(O-CH2-CH2)p1—, -СННО-СШ-СШЧрг and -(СН2)р1-О-(СН2) p2-;

o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6;

-W- represents , and a represents an integer selected from 2, 3, 4, 5 and

6 is particularly preferred.

Even more preferred is a conjugate of general formula (IV), wherein n is selected from 1, 2 or 3;

the linker -L- is selected from: -L ^a - -L ^a -L ^e - -L ^a -L ^b -L ^e - and -L ^a -L ^d -L ^e -;

- L ^a - selected from: -(CH)o-, -(CH2-CH2-O)o-S2Shch-, -(SShch-SShch-OXO-SShch;

- L ^b - represents -O-;

- L ^d - selected from: -(CH2)q—, -(CF2)q—, -(CH2-CH2-O)q-C2H4—, and

- (CH2-CH2-O)q-CH2-;

^- L ^e - ^{selected from:} -(CH^rG, -(CF^pr, - ^C 2 ^H 4 ^- ( ^O-CH 2 ^_CH 2 ⁾ pr _3. ^-CH 2 ^-(O-CH 2 ^-CH 2)р1 ^{- and -(CH} 2 ⁾ p1 ^-O-(CH 2 ⁾ p2 ^-; o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6;

-W- represents , and a represents an integer selected from 2, 3, 4, 5 and 6.

Particularly preferred is the conjugate of general formula (IV) wherein the linker -L- is -(CH ₂ ) _o -, o is an integer selected from 2, 3, 4, 5 and 6;

-W- represents , and a represents an integer selected from 2, 3, 4, 5 and 6.

Particularly preferred is the conjugate of general formula (IV), where n is an integer of 1, 2 or 3;

the linker -L- is -(CH ₂ ) _o -, o is an integer selected from 2, 3, 4, 5 and 6;

-W- represents , and a represents an integer selected from 2, 3, 4, 5 and 6.

Particularly preferred is the conjugate of general formula (IV), where

N is an integer from 1, 2 or 3;

the linker -L- is -(CH ₂ ) _o -, o is an integer selected from 2, 3, 4, 5 and 6;

-W- represents , and a represents an integer selected from 2, 3, 4, 5 and 6:

- 64 046159

and Z represents

Preferably c is in the range between 2 and 18, more preferably between 5 and 15, even more preferably between 8 and 12. It is also preferable that n is 1.

More preferred is a conjugate of any of formulas (IV-1) to (IV-4):

(IV-1)

- 65 046159

where L, E1, W, c, CP, and n have the same meanings as above.

Particularly preferred is the conjugate of formula (IV-2) wherein L is -(CH ₂ ) ₅ -, E1 is -NH-, n is an integer selected from 1 or 2, and c and W are the same which are listed above.

Also preferred is the conjugate of general formula (V)

where c is in the range between 2 and 18;

- 66 046159

-E ₁ - represents a covalent bond, -NH-, -O-NH-, -O-, -S-, -CO-, -CH=CH-, -CONH-, n=nm ^{/N N} n=nn =n

..U-. -<,N-CO-NHNH-, · , ;

-W- selected from:

Oh oh

a is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; b is an integer selected from 1, 2, 3 and 4; and n, L, Z and T* have the meanings set forth herein.

A conjugate of general formula (V), wherein the linker -L- is selected from: -L ^a - -L ^a -L ^e - -L ^a -L ^b -L ^e - and -L ^a -L ^d -L ^e -;

- L ^a - selected from: -(CH ₂ )o-, -(CH2-CH2-O)o-C2H4-, -(CH2-CH2-O)o-CH2;

- L ^b - represents -O-;

- L ^d - selected from: -(CH ₂ )q-, -(CF2)q-, -(CH2-CH2-O)q-C2H4- and -(CH2-CH2-O)q-CH2-;

- L ^e - selected from: -(CH2)p1-, -(CF2)p1-, -C2H4-(O-CH2-CH2)p1-, -CH2-(O-CH2-CH2)p1- and -(CH ₂ )p _GO- (CH ₂ )p2-; o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6;

-W- represents

a is an integer selected from 2, 3, 4, 5 and 6, is particularly preferred.

Even more preferred is a conjugate of general formula (V), wherein n is selected from 1, 2 or 3;

the linker -L- is selected from: -L ^a - -L ^a -L ^e - -L ^a -L ^b -L ^e - and -L ^a -L ^d -L ^e -;

- L ^a - selected from: -(CH ₂ )o-, -(CH2-CH2-O)o-C2H4-, -(CH ₂ -CH ₂ -O)o-CH ₂ ;

- L ^b - represents -O-;

- L ^d - selected from: -(CH ₂ ) _q -, -(CF2)q-, -(CH2-CH2-O)q-C2H4-, and -(CH2-CH2-O)q-CH2-;

- L ^e - selected from: -(CH ₂ ) _pl -, -(CF2)pi-, -C2H4-(O-CH ₂ -CH ₂ ) _pr , -CH ₂ -(O-CH ₂ -CH ₂ ) _pr and -(CH ₂ )p1-O-(CH ₂ )p2-;

o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6;

-W- represents

, and a is an integer selected from 2, 3, 4, 5 and 6.

Particularly preferred is a conjugate of general formula (V) wherein the linker -L- is -(CH ₂ ) _o -, o is an integer selected from 2, 3, 4, 5 and 6;

-W- represents

, and a is an integer selected from 2, 3, 4, 5 and 6.

Particularly preferred is the conjugate of general formula (V), where

N is an integer from 1, 2 or 3;

the linker -L- is -(CH ₂ ) _o -, o is an integer selected from 2, 3, 4, 5 and 6;

Oh Oh

-W- represents '

', and a is an integer selected from 2, 3, 4, 5 and 6.

Particularly preferred is the conjugate of general formula (V), where

N is an integer from 1, 2 or 3;

the linker -L- is -(CH ₂ ) _O -, o is an integer selected from 2, 3, 4, 5 and 6;

Oh Oh

-W- represents

ζ and a is an integer selected from 2, 3, 4, 5 and 6:

- 67 046159

and Z represents

Particularly preferred is the conjugate of general formula (V), where n is an integer of 1, 2 or 3;

the linker -L- is -(CH ₂ ) _o -, o is an integer selected from 2, 3, 4, 5 and 6;

-W- represents , and a represents an integer selected from 2, 3, 4, 5 and 6:

and T* represents a phosphate group.

Also preferred is a conjugate of general formula (IV) wherein the group -O-L-E is selected from the group consisting of:

.ABOUT

HE/HE

BUT but .Oh but

BUT but he

HE/HE

O/OH but^ ? ⁿ

NHAc

ABOUT.

(CH ₂ ) ₂ -NH ₂

BUT but

NO o/OH NO

HE

BUT but _ό ^(ΟΗ ₂ )2-ΝΗ ₂ ,O

HE/HE

BUT but .Oh but

BUT but he

HE/HE

O/OH but^ ? ⁿ

NHAc

O.

(CH ₂ )io—ΝΗ ₂

BUT but

NO o/OH NO

HE

BUT but q^(CH ₂ ) ₁₀ -nh ₂

- 68 046159

BUT

NH ₂

^(CH ₂ ) ₅ -NH ₂

BUT

NH ₂

- 69 046159

BUT

NH ₂

- 70 046159

BUT

SH

- 71 046159

- 72 046159

More preferred is a conjugate of any of formulas (V-1) to (V-4):

where L, E1, W, c, and n have the same meanings as above.

More preferred is a conjugate of any of formulas (IV), (IV-1)-(IV-4), (V) and (V-1)-(V-4), where n is an integer from 1 to 3.

More preferred is a conjugate of any of formulas (IV), (IV-1)-(IV-4), (V) and (V-1)-(V-4), where c is selected from 4 to 10.

Preferably -W- is a brane of 2, 3, 4, 5 and 6.

and a represents an integer, you are thus a conjugate of the general formula (IV), (IV-1)-(IV-4), (V) and (V-1)-(V-4), where -W- represents - 73 046159

is and a is an integer selected from 2, 3, 4, 5 and is particularly preferred.

Preferably, the linker -L- is -L ^a -, -L ^a -L ^e -, -L ^a -L ^b -L ^e -, or -L ^a -L ^d -L ^e -;

- L ^a - represents -(CH ₂ ) _o -, -(CH ₂ -CH ₂ -O) _o -C ₂ H ₄ -, or -(CH ₂ -CH ₂ -O) _o -CH ₂ ;

- L ^b - represents -O-;

- L ^d - represents -(CH ₂ )q-, -(CH(OH))q-, -(CF2)q-, -(OD-OD-OX-SSHgili -(CH ₂ -CH ₂ -O) q-CH ₂ -;

- L - represents -(CH2)p1-, -(CF2)p1-, -C2H4-(O-CH2-CH2)pi--CH2-(O-CH2-CH2)p1- or ^{-(CH2 )} p1 ^-O-(CH 2 ⁾ p2 ^{-; and} o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6.

In the most preferred embodiment of the invention, E1 is a

tape bond, -NH-, -CH=CH-, -CONH-,

Also preferred is a conjugate of general formula (IV), (IV-1)-(IV-4), (V) and (V-1)-(V-4), wherein the group -O-L-E is selected from the group consisting of:

.0

OH/OH

BUT BUT ,Ο

BUT

BUT but

HE

OH/OH about he but^ ? ⁿ

NHAc (CH ₂ ) ₂ -NH ₂

BUT BUT

BUT

O/OH NO

HE

BUT BUT _o /(CH ₂ )2-NH ₂ ,O

HE/HE

BUT BUT

BUT

O /OH but^ ? ⁿ

BUT but

HE

NHAc

O.

(CH ₂ )ω—NH ₂

- 74 046159

- 75 046159

BUT

NH ₂

.ABOUT

HE/HE

BUT but

oh he but he .o

OH/OH he o HO ^-o NHAc n

N^/(CH ₂ ) ₄ -NH ₂

BUT but but o/HE BUT he

BUT but he

NHAc

- 76 046159

BUT he oh but

BUT

BUT

(CH ₂ ) ₂ -NH ₂

Η (CH ₂ ) ₅ -SH i-SH

HE

Jo

NHAc n no o no

-ABOUT

NHAc

- 77 046159

In another embodiment of the invention, said immunogenic carrier is preferably a glycosphingolipid with immunomodulatory properties, and more preferably (2S, 3S, 4R)-1-(α-D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol. The term glycosphingolipid with immunomodulatory properties as used herein refers to a suitable glycosphingolipid capable of stimulating an immune system response to a target antigen, but which does not itself confer immunity as defined above.

Glycosphingolipids, as used herein, are compounds containing a carbohydrate moiety α-attached to a sphingolipid. Preferably, the carbohydrate moiety is hexopyranose and most preferably is α-D-galactopyranose. For one skilled in the art, sphingolipids are a class of lipids containing a C18 amino alcohol attached via an amide bond to a fatty acid. The C18 amino alcohol is preferably mono-, di- or poly-substituted with hydroxyl groups.

Particularly preferably, the C18 amino alcohol is phytosphingosine. The fatty acid is preferably a monocarboxylic acid having a saturated alkyl chain of a number of carbon atoms ranging from 16 to 28 and more preferably from 18 to 26. Glycosphingolipids

- 78 046159 with immunomodulatory properties include, but are not limited to, (2S,3S,4R)-1-(a-Dgalactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol, which can stimulate natural killer (NK) cell activity and production cytokines by natural killer T (NKT), and exhibits potent antitumor activity in vivo (Proc. Natl Acad. Sci. USA, 1998, 95, 5690).

Conjugates of saccharides of general formula I with glycosphingolipid with immunomodulatory properties have the advantage that they are thermostable. To be suitable for conjugation, a functional group is introduced on the glycosphingolipid with immunomodulatory properties. Said functional group tends to react directly with the terminal amino group of the linker saccharides of general formula I, to provide conjugates of saccharides of formula I, or with the Y functional group of the linking molecule, to provide a modified glycosphingolipid with immunomodulatory properties.

Preferably, said functionality is introduced into the C6 carbohydrate moiety of a glycosphingolipid with immunomodulatory properties. Thus, a glycosphingolipid with immunomodulatory properties is functionalized with a functional group that is susceptible to reacting with the terminal amino group of the saccharides or the Y functional group of the linking molecule. Functional group prone to react with the amino group includes, but is not limited to, activated ester, isocyanate group, aldehyde, epoxide, imidoester, carboxylic acid, alkyl sulfonate and sulfonyl chloride. A functional group prone to react with the Y functional group of the linking molecule so as to provide a modified glycosphingolipid with immunomodulatory properties representing the X functional group of the linking molecule includes, but is not limited to, amine, alcohol, thiol, activated ester, isocyanate group, aldehyde , epoxide, vinyl, imidoester, carboxylic acid, alkyl sulfonate, sulfonyl chloride, vinyl group, alkynyl group and azido group.

Preferably, the functional group introduced into the C6 carbohydrate moiety of the glycosphingolipid with immunomodulatory properties is selected from the group which includes or contains an amine, a thiol, an alcohol, a carboxylic acid, vinyl, maleimide, α-iodoacetyl, α-bromoacetyl, Nhydroxysuccinimide ester (NHS), 2-pyridyldithiols.

Said functional group X of the linking molecules is selected from the group which contains or consists of the following: maleimide, α-iodoacetyl, α-bromoacetyl, N-hydroxysuccinimide ester (NHS), aldehyde, carboxylic acid, epoxide, alkyl sulfonate, sulfonyl chloride, anhydride, carbonate .

As used herein, the term linking molecule refers to a bifunctional molecule containing a functional group X and a functional group Y, wherein the functional group X is capable of reacting with the terminal amino group on the linker -L-, and the functional group Y is capable of reacting with the functional group , present on an immunogenic carrier or solid support.

Vaccines containing at least one conjugate in accordance with the present invention cause fewer side effects and/or non-protective immune responses compared to vaccines containing isolated (and not synthesized) mixtures of saccharides obtained by non-selective cleavage of the C. difficile capsular polysaccharide or its conjugates. Moreover, the vaccines of the invention are easier to produce under GMP regulations than vaccines containing isolated mixtures of indiscriminately degraded capsular polysaccharides and are easier to characterize, making it easier to monitor stability and purity, as well as determine the type and amount of impurities.

It has been found that a conjugate which contains a saccharide of any of the general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), and especially a conjugate of any of the general formulas (IV), (IV-1)-(IV-4), (V) and (V-1)-(V-4), elicits a protective immune response in a human and/or animal host , and is therefore useful for the prevention and/or treatment of diseases associated with the bacteria Clostridium difficile. Thus, conjugates that contain saccharides of general formula (I) conjugated to an immunogenic carrier are useful for the prevention and/or treatment of diseases associated with Clostridium difficile bacteria that contain in their cell wall saccharide one of the following saccharide moieties:

- 79 046159

- 6)-3-D-Glc-(l, 3)-3-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[3-D-Glc-(l, 3) ]^-D-GalNAc-(l,

3)-a-D-Man-(l-;

- 3)-a-D-Man-(l, 6)-3-D-Glc-(l, 3)^-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-|>D -Glc-(l, 3)]-

3-D-GalNAc-(l;

- 4)-[3-D-Glc-(l, 3)]-3-D-GalNAc-(l, 3)-a-D-Man-(l, 6)^-D-Glc-(l, 3) ^-D-GalNAc-(l,

4)-a-D-Glc-(l;

- 4)-[3-D-Glc-(l, 3)]-3-D-GalNAc-(l, 3)-a-D-Man-(l, 6)^-D-Glc-(l, 3) ^-D-GalNAc-(l,

4)-a-D-Glc-(l;

- 3)-3-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[3-D-Glc-(l, 3)]^-D-GalNAc-(l, 3) -a-D-Man-(l,

6)-3-D-Glc-(l.

Preferably, the bacterium which contains one of the following saccharide moieties in its cell wall saccharide is Clostridium difficile.

In a preferred embodiment of the invention, conjugates which contain saccharides of general formula I conjugated to an immunogenic carrier are useful for the prevention and/or treatment of diseases associated with bacteria, and especially diseases associated with bacteria containing in their cell wall polysaccharide one of the following saccharide fragments:

-6)-β-ϋGlc-(1, 3)-3-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[3-D-Glc-(l, 3)]^ -D-GalNAc-(l, 3)-a-D-Man(1-; -3)-a-D-Man-(l, 6)^-D-Glc-(l, 3)^-D-GalNAc-(l , 4)-a-D-Glc-(l, 4)-|>D-Glc-(l, 3)]P-D-GalNAc-(l; -4)-[3-D-Glc-(l, 3)] ^-D-GalNAc-(l, 3)-a-D-Man-(l, 6)-3-D-Glc-(l, 3)-βD-GalNAc-(l, 4)-a-D-Glc-(l ; -4)-[β-ϋ-01ο-(1, 3)]^-D-GalNAc-(l, 3)-a-D-Man-(l, 6)-β-ϋGlc-(1, 3)^ -D-GalNAc-(l, 4)-a-D-Glc-(l; -3)^-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-^-D-Glc(1 , 3)]^-D-GalNAc-(l, 3)-a-D-Man-(l, 6)-β-0-01ο-(1, and preferably with Clostridium difficile, where these diseases include diarrhea, pseudomembranous colitis and paralytic intestinal obstruction.

Pharmaceutical compositions.

Another aspect of the present invention is directed to a pharmaceutical composition or vaccine that contains at least one conjugate that includes a saccharide of general formula (I) conjugated to an immunogenic carrier, and/or at least one saccharide of general formula (I) together at least with one pharmaceutically acceptable adjuvant and/or excipient. Said pharmaceutical composition can be used to enhance a protective immune response in a human and/or animal host. Ideally, the pharmaceutical composition is suitable for use in humans.

In another aspect of the present invention, said pharmaceutical composition or vaccine further comprises at least a cell wall saccharide or a fragment of a cell wall saccharide and/or protein conjugates thereof of Clostridium difficile bacteria, which are selected from the group that contains or consists of Clostridium difficile strains, 027, MON718 and MON900.

As used herein, the term adjuvant refers to an immunological adjuvant, i.e. an agent used in a vaccine composition that modifies or enhances the effect of said vaccine by enhancing the immune response to a given antigen contained in the vaccine without being antigenically related to it. For those skilled in the art, classically recognized examples of immunological adjuvants include, but are not limited to, oil emulsions (eg, Freund's adjuvant), saponins, aluminum or calcium salts (eg, Alum), nonionic block polymer surfactants, and many others.

Pharmaceutical compositions are preferably in aqueous form, especially when administered, but they may also be presented in non-aqueous liquid forms or in dry forms, for example, gelatin capsules, or lyophilisates, etc.

Pharmaceutical compositions may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Formulations without mercury are preferred, and vaccines can be prepared without preservatives.

Pharmaceutical compositions may include a physiological salt, such as a sodium salt, for example to control tone. Sodium chloride (NaCl) is typical and may be present in co

- 80 046159 in quantities from 1 to 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride, calcium chloride, etc.

Pharmaceutical compositions may have an osmolality from 200 mOsm/kg to 400 mOsm/kg.

Pharmaceutical compositions may include the compounds (with or without an insoluble metal salt) in plain water (eg, water for injection), but typically include 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 included in amounts in the range of 5-20 mM.

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

The pharmaceutical compositions are preferably sterile and gluten-free.

The pharmaceutical compositions are suitable for administration to animal patients (and, in particular, humans), and thus include both human and veterinary use. They can be used in a method of enhancing the immune response in a patient, which includes the step of administering the composition to the patient.

Pharmaceutical compositions in accordance with the present invention can be administered before the subject is exposed to C. difficile and/or after the subject is exposed to C. difficile bacteria.

In another aspect of the present invention, the invention is directed to the use of at least one conjugate which comprises at least one saccharide of general formula (I) conjugated to an immunogenic carrier and/or at least one saccharide of general formula (I) for the production of said a pharmaceutical composition or said vaccine for the prevention and/or treatment of diseases associated with the bacteria C. difficile, especially diseases associated with the bacteria C. difficile selected from the group which includes or consists of diarrhea, pseudomembranous colitis and paralytic ileus.

Preferably, the present invention relates to the use of at least one saccharide of any of the general formulas (I), (II), (II-a), (II-b), (III), (III-a) or (III-b ) and/or at least one of the conjugates, which contains at least one saccharide of any of the general formulas (I), (I), (II), (II-a), (II-b), (III), (III-a) or (III-b), for the preparation of said pharmaceutical composition or said vaccine.

More preferably, the present invention relates to the use of at least one of the saccharides Ga-1 - Ga-11, Ga-1 - I'b-11 and I'c-1 - I'c-11 and/or at least one from conjugates that contains at least one of the saccharides Ga-1 - Ga-11, I'b-1 - I'b-11 and I'c-1 - I'c-11, for the preparation of the specified pharmaceutical composition or the specified vaccines.

In particular, the present invention relates to the use of at least one conjugate of any of the general formulas (IV), (IV-1) - (IV-4), (V) and (V-1) - (V-4) for the preparation said pharmaceutical composition or said vaccine.

Pharmaceutical compositions can be prepared in unit dose form. In some embodiments, a unit dose may have a volume of from 0.1 to 1.0 ml, such as about 0.5 ml.

The invention also provides a delivery device (eg, syringe, nebulizer, nebulizer, inhaler, skin patch, etc.) containing a pharmaceutical composition in accordance with the invention, eg containing a unit dose. This device can be used to administer the composition to a vertebral subject.

The invention also provides a sterile container (eg, a vessel) containing a pharmaceutical composition in accordance with the invention, eg containing a unit dose.

The invention also provides a unit dose of the pharmaceutical composition in accordance with the invention.

The invention also provides a hermetically sealed container containing a pharmaceutical composition in accordance with the invention. A suitable container includes, for example, a vessel.

Pharmaceutical compositions according to the invention can be obtained in various forms. For example, the compositions can be prepared as injections, or as liquid solutions or suspensions. Solid forms suitable for solution or suspension of liquid carriers prior to injection (eg, a lyophilized composition or a spray-frozen dry composition) may also be prepared. The composition can be prepared for topical use, for example in the form of an ointment, cream or powder. The composition may be prepared for oral administration, for example, in the form of a tablet or capsule, in the form of a spray, or in the form of a syrup (optionally flavored). The composition can be prepared for pulmonary administration, for example via an inhaler, using a fine powder or spray. You can obtain the composition in the form of a suppository. The composition can be formulated for nasal, auricular or ocular administration, for example in the form of a spray or drops. Intramuscular injections are typical.

The pharmaceutical compositions may contain an effective amount of an adjuvant, i.e. an amount which, when administered to an individual, either as a single dose or as part of a series, is effective

- 81 046159 active to enhance the immune response to the co-administered PS-II saccharide antigen of C. difficile.

This amount may vary depending on the health and physical condition of the individual being treated, the age, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection, vaccine composition, the attending physician's assessment of the medical situation and other relevant factors. The amount will be within a relatively wide range, which can be determined by routine testing.

The composition and administration of the vaccine in accordance with the present invention can be achieved in accordance with any method known in the art.

A therapeutically effective dose of one conjugate according to the present invention or one saccharide of general formula (I) refers to that amount of the compound that results in at least partial immunization against the disease. The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical, pharmacological and toxicological procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects represents the therapeutic index. The actual amount of the composition administered will depend on the subject being treated, the weight of the subject, the severity of the disease, the route of administration and the judgment of the attending physician.

Another aspect of the present invention is directed to a method of inducing an immune response against C. difficile in a human and/or animal host, which comprises administering a saccharide of general formula (I) and/or a salt thereof and/or a conjugate thereof or a pharmaceutical composition thereof to said human- and/or host animal. A method for treating or preventing diseases caused by C. difficile in a human and/or animal host in accordance with the present invention includes administering at least one saccharide of general formula (I) and/or a salt thereof and/or a conjugate thereof, or its pharmaceutical composition to a specified human and/or animal host.

Immunological tests

Another aspect of the present invention relates to a saccharide of general formula (I) for use as a marker in immunoassays for the detection of antibodies to bacteria containing in their cell wall polysaccharide one of the following saccharide moieties:

- 6)-3-D-Glc-(l, 3)-3-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[3-D-Glc-(l, 3) ]-3-D-GalNAc- (1, 3)-a-D-Man-(l-;

- 3)-a-D-Man-(l, 6)-3-D-Glc-(l, 3)-3-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[3 -D-Glc-(l,

3)]-3-D-GalNAc-(l;

- 4)-[3-D-Glc-(l, 3)]-3-D-GalNAc-(l, 3)-a-D-Man-(l, 6)-3-D-Glc-(l, 3 )-3-D-GalNAc- (1, 4)-a-D-Glc-(l;

- 4)-a-D-Glc-(l, 4)-[3-D-Glc-(l, 3)]-3-D-GalNAc-(l, 3)-a-D-Man-(l, 6)- 3-D-Glc-(l,

3)-3-D-GalNAc-(l;

- 3)-3-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[3-D-Glc-(l, 3)]-3-D-GalNAc-(l, 3 )-a-D-Man- (1, 6)-3-D-Glc-(l.

Such assays include, for example, microarray analysis and ELISA, suitable for the detection of antibodies to bacteria containing in their cell wall polysaccharide one of the above saccharide moieties, such as C. difficile.

The saccharides of the present invention can be readily conjugated to solid supports to provide immunoassays useful for detecting antibodies against C. difficile. Said solid supports present on their surface a functional group that can react with the amino group of the saccharides of general formula (I) or with the Y functional group of the connecting molecule to obtain modified solid supports presenting on their surface the functional group X of the connecting molecule, which can further react in reaction with the amino group of saccharides of general formula (I). In one embodiment of the invention, the solid supports are microarray slides having on their surface a functional group that is prone to react with the Y functional group of the linking molecule to produce modified microarray slides presenting on their surface the X functional group of the linking molecule. Examples of such microarray slides include, but are not limited to, Corning® epoxy-coated slides or Corning® GAPS™ II-coated slides.

In a preferred embodiment of the invention, the solid substrates are microarray slides on the surface of which there is a functional group that tends to

- 82 046159 to react with the amino group of saccharides of general formula (I), and more preferably with an N-hydroxysuccinimide-activated ester (NHS). Such microarray slides are, for example, CodeLink® NHS slides.

Description of the figures

Fig. 1 shows the chemical structure of the PS-II saccharide repeat unit of the C. difficile cell wall.

Fig. 2 shows examples of functional group X of a connecting molecule in accordance with the present invention.

Fig. 3 shows examples of functional group X of a connecting molecule in accordance with the present invention.

Fig. 4 shows CRM ₁₉₇ conjugate of general formula (V-2) as preferred compounds of this application.

Fig. Figure 5 shows two routes for the cleavage of compound 33 by rutem treatment with NaOH. Path I shows cleavage at the phosphate group, where the phosphate group remains in the linker portion and the compound LA, 5-aminopentyl dihydrogen phosphate, is formed. Pathway II shows cleavage at the phosphate group, where the phosphate group remains on the saccharide moiety (compound 33B) to produce compound LB, 5-aminopentan-1-ol.

Fig. 6 shows HPLC plots from bottom to top of the following compounds: Compound 33 (standard), compound 33A (control), compound 33 after one day treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 after four days treatment with 0.1 M sodium hydroxide solution sodium at room temperature, purified compound 33B, compound LB. From fig. 7 it is evident that compound 33 is completely stable under basic conditions for one day. After four days of NaOH treatment at RT, another 50% of compound 33 remains unchanged.

Fig. 7 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day of treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 (standard), compound 33 after two months at 2°C8° C in water, compound 33 after two months at 2°C-8°C in NaPi, which is synonymous with PBS (phosphate buffered saline), compound 33 after two months at 2°C-8°C in Alhydrogel and PBS. From fig. 7 it is obvious that compound 33 is completely stable at temperatures from 2°C to 8°C for two months.

Fig. 8 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day of treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 (standard), compound 33 after two months at 25 ° C in water, compound 33 after two months at 25°C in NaPi, which is synonymous with PBS (phosphate buffered saline), compound 33 after two months at 25°C in Alhydrogel and PBS. From fig. 8 it is obvious that compound 33 is completely stable at 25°C for two months.

Fig. 9 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day of treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 (standard), compound 33 after two months at 37°C in water , compound 33 after two months at 37°C in NaPi, which is synonymous with PBS (phosphate buffered saline), compound 33 after two months at 37°C in Alhydrogel and PBS. From fig. 9 it is evident that compound 33 is completely stable at 37°C for two months.

Fig. 10 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day of treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 (control), compound 92 (standard), compound 92 after a week at 2-8°C in water, compound 92 after a week at 2-8°C in NaPi, which is synonymous with PBS (phosphate buffered saline), compound 92 after a week at 2-8°C in Alhydrogel and PBS. From fig. 10 it is evident that compound 92 is completely stable at 2-8°C for one week.

Fig. 11 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day of treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 (control), compound 92 (standard), compound 92 after a week at 25°C in water, compound 92 after a week at 25°C in NaPi, which is synonymous with PBS (Phosphate Buffered Saline), compound 92 after a week at 25°C in Alhydrogel and PBS. From fig. 11 it is evident that compound 92 is completely stable at 25°C for one week.

Fig. 12 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day of treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 (control), compound 92 (standard), compound 92 after a week at 37°C in water, compound 92 after a week at 37°C in NaPi, which is synonymous with PBS (phosphate buffered saline), compound 92 after a week at 37°C in Alhydrogel and PBS. From fig. 12 it is evident that compound 92 is completely stable at 37°C for one week.

Fig. 13 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 33 after one day of treatment with 0.1 M sodium hydroxide at room temperature and purification, compound 33 (control), compound 54 (standard), compound 54 after a week

- 83 046159 at 25°C in water, compound 54 after a week at 2-8°C in water, compound 54 after a week at 37°C in water. From Figure 13 it is evident that compound 54 is completely stable at 37°C for one week.

Fig. 14 shows HPLC plots from bottom to top of the following compounds: compound 33A (control), compound 54 (standard), compound 54 after a week at 25°C in Alhydrogel, compound 54 after a week at 2-8°C in Alhydrogel, compound 54 after a week at 37°C in Alhydrogel. From fig. 14 it is evident that compound 54, when formulated with Alhydrogel, is predominantly adsorbed to aluminum hydroxide and that the expected degradation products that can be detected by HPLC in the presence of aluminum hydroxide are not formed. Thus, compound 54 is stable at 37°C for one week.

Fig. 15 shows ELISA titers on day-0, day-7 and day-42 of mixed sera from rabbits (n=4) immunized with 33-CRMi ₉₇ C. difficile saccharide formulations (36). Sera obtained from rabbits immunized with compound 36 were diluted 1:100, 1000 with 1% BSA-PBS. Diluted sera (100 μl) were added to each well of a microtiter plate that was coated with 0.5 μg of the appropriate 33-BSA conjugate (compound 37). Detection was performed using an HRP conjugated goat anti-rabbit secondary antibody, which is diluted to 1:10,000 and which is formulated using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. Absorbance was measured at 450 nm and data were plotted using Graphpad prism software. On the 42nd day from FIG. 15 a remarkable immunological response is evident.

Fig. 16 shows ELISA titers of rabbit antisera against C. difficile strain 630 (mixed sera). Rabbits (4 animals per study group) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant as indicated. . PBS with Alum served as a negative control. The immunogen was conjugate 56. Mixed sera from different time points (days 0, 7, 21, 35, 77 and 84) were tested for total IgG against formalin-inactivated C. difficile (strain 630) coated on ELISA plates. Coated ELISA plates purchased from tgcBIOMICS GmbH were blocked with 200 μl per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 h. Sera were diluted 1:100 with 1 w/v% BSA in PBS and incubated for 1 hour in a volume of 100 µl per well. Total IgG was then detected using HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, reference A4914) diluted to 1:10,000 in 1 w/v% BSA in PBS for 30 min and developed using TMB substrate. (Thermo Scientific, ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using GraphPad Prism software. From fig. 16, it is evident that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of C. difficile strain 630 bacteria. In addition, the addition of Alum adjuvant results in higher total IgG titers.

Fig. 17 shows ELISA titers of rabbit antisera against C. difficile strain 630 (individual sera). Rabbits (4 animals per study group) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg of glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant as indicated. PBS with Alum served as a negative control. The immunogen was conjugate 56. Sera from different time points (days 0, 735, 77 and 84) were tested for total IgG against formalin-inactivated C. difficile (strain 630) coated on ELISA plates. Coated ELISA plates purchased from tgcBIOMICS GmbH were blocked with 200 μl per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 h. Sera were diluted 1:300 with 1 w/v% BSA in PBS and incubated for 1 hour in a volume of 100 µl per well. Total IgG was detected using HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, reference A4914) diluted to 1:10,000 in 1 w/v% BSA in PBS for 30 min and developed using TMB substrate (Thermo Scientific , ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using GraphPad Prism software. From fig. 17 it is evident that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of C. difficile strain 630 bacteria. In addition, the addition of Alum adjuvant results in higher total IgG titers.

Fig. 18 shows ELISA titers of rabbit antisera against C. difficile strain R20291. Rabbits (4 animals per study group) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg of glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant as indicated. PBS with Alum served as a negative control. The immunogen was conjugate 56. Mixed sera from different time points (days 21, 35, 77 and 84) were tested for total IgG against formalin-inactivated C. difficile (strain R20291) coated on ELISA plates. Commercially available coated ELISA plates were blocked with 200 μl per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 h. Sera were diluted 1:100 with 1 w/v% BSA in PBS and incubated for 1 h in volume 100 µl per well. Total IgG was determined using HRP-conjugated

- 84 046159 goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, reference A4914) diluted to 1:10,000 in 1 w/v% BSA in PBS for 30 min and developed using TMB substrate (Thermo Scientific , ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using GraphPad Prism software. From fig. 18 it is evident that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of the bacterium C. difficile, strain R20291. In addition, the addition of Alum adjuvant results in higher total IgG titers.

Fig. 19A shows ELISA titers of rabbit antiserum (day 35) against C. difficile strain VPI10463. Rabbits (4 animals per study group) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg of glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant as indicated. PBS with Alum served as a negative control. The immunogen was conjugate 56. Mixed sera from day 35 were tested for total IgG against formalin-inactivated C. difficile (strain VPI10463) coated on ELISA plates. From fig. 19A, it is evident that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the surface of the bacterium C. difficile, strain VPI10463. In addition, the addition of Alum adjuvant results in higher total IgG titers.

Fig. 19B shows ELISA titers of rabbit antiserum (day 35) against isolated C. difficile PS-II polysaccharide. Rabbits (4 animals per study group) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg of glycan antigen per injection of aluminum hydroxide adjuvant (Alum) as indicated. PBS with Alum served as a negative control. The immunogen was conjugate 56. Mixed sera from day 35 were tested for total IgG against isolated PS-II polysaccharide. From fig. 19B, it is apparent that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to the isolated PS-II polysaccharide.

Fig. 19C demonstrates ELISA titers of rabbit antiserum (day 35) against C. difficile strain 630 with or without preincubation with isolated C. difficile PS-II polysaccharide. Rabbits (4 animals per study group) were immunized four times (days 0, 14, 28, 77) subcutaneously with 10 μg of glycan antigen per injection of aluminum hydroxide adjuvant (Alum). The immunogen was conjugate 56. Mixed sera (diluted 1:100 in 1 wt/vol% BSA in PBS) from day 35 were incubated on ice for 30 min with 10 or 50 μg of isolated PS-II polysaccharide or with PBS. The sera were then incubated for 1 h (100 μl/well) on commercially available coated ELISA plates (C. difficile strain 630), which were pre-blocked with 200 μl per well of commercial blocking reagent (Roche, ref. 11112589001) for 2 Total IgG was determined using an HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, reference A4914) diluted to 1:10,000 in 1 wt/vol% BSA in PBS for 30 min and developed with using TMB substrate (Thermo Scientific, ref. 34028). Absorbance was measured at 450 nm in a microplate reader and background-subtracted data were plotted using GraphPad Prism software. It is apparent from Figure 19C that the binding of rabbit antiserum to C. difficile bacteria can be blocked by PS-II polysaccharide in a dose-dependent manner, indicating that antibacterial antibody responses are specific to PS-II polysaccharide.

Fig. 20 demonstrates ELISA titers of rabbit antiserum against synthetic C. difficile 54PS-II hexasaccharide. Rabbits (4 animals per test group) were immunized four times (days 0, 14, 28, 77) subcutaneously with 2.5 μg or 10 μg glycan antigen per injection with or without aluminum hydroxide (Alum) adjuvant as indicated. PBS with Alum served as a negative control. The immunogen was conjugate 56. Mixed sera from different time points (days 0, 21, 35, 77 and 84) were tested for total IgG against the synthetic C. difficile PS-II hexasaccharide 54. From fig. 20 it is evident that vaccination of rabbits with conjugate 56 induces IgG antibodies that bind to synthetic immunogen 54. In addition, the addition of Alum adjuvant results in higher overall IgG titers.

Fig. 21 shows ELISA titers of rabbit antisera against C. difficile strain 630. Mice (7 or 8 animals per test group) were immunized twice (days 0, 14, 28) subcutaneously with either conjugate 94 or conjugate 56 at a dose of 0.5 or 2 μg of glycan antigen per injection. PBS served as a negative control, and aluminum hydroxide adjuvant (Alum) was used for all immunizations. Mixed sera from days 21 and 35 were tested for total IgG against formalin-inactivated C. difficile (strain 630) coated on ELISA plates. From fig. 21 it is evident that vaccination of mice with conjugate 94 or 56 induces IgG antibodies that bind to the surface of the bacterium C. difficile, strain 630.

Fig. 22 shows ELISA titers of rabbit antisera against C. difficile strain R20291. Mice (7 or 8 animals per test group) were immunized twice (days 0, 14, 28) subcutaneously with either conjugate 94 or 56 at a dose of 0.5 or 2 μg of glycan antigen per injection. PBS served as a negative control, and aluminum hydroxide adjuvant (Alum) was used for all immunizations. Mixed sera from days 21 and 35 were tested for total IgG against

- 85 046159 formalin-inactivated C. difficile bacteria (strain R20291) applied to ELISA plates. From fig. 22 it is evident that vaccination of mice with conjugate 94 or 56 induces IgG antibodies that bind to the surface of C. difficile strain 630 bacteria.

Fig. 23 shows ELISA titers of mouse antisera against synthetic C. difficile PS-II antigens. Mice (7 or 8 animals per test group) were immunized twice (days 0, 14, 28) subcutaneously with either conjugate 94 or conjugate 56 at a dose of 0.5 or 2 μg of glycan antigen per injection. PBS served as a negative control, and aluminum hydroxide adjuvant (Alum) was used for all immunizations. Mixed sera from days 21 and 35 were tested for total IgG against the corresponding synthetic C. difficile glycan antigen used for immunization. From fig. 23 it is evident that vaccination of mice with conjugate 94 or 56 induces IgG antibodies that bind to synthetic immunogens. In addition, the addition of Alum adjuvant results in higher total IgG titers.

Fig. 24 shows SEC chromatograms of two glycoconjugates 94 and 56 used for immunization experiments. Unconjugated CRMj ₉₇ protein served as a control.

Fig. 25 shows SDS-PAGE of C. difficile glycoconjugates 94 and 56 (2.5 μg per well) used for immunization experiments separated using a 10% polyacrylamide gel. Unconjugated CRM ₁₉₇ protein served as a control.

The following examples are included herein to demonstrate a preferred embodiment of the invention. It will be understood by those skilled in the art that the methods disclosed as examples follow the methods discovered by the inventor for the good operation of the invention in practice and thus can be considered as preferred methods for his practice. However, those skilled in the art will understand, in light of the present disclosure, that many changes may be made to the specific embodiments of the invention that are disclosed and still achieve the same or similar result without departing from the spirit and scope of the present invention.

Further modifications and alternative embodiments of various aspects in accordance with the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description should be construed as illustrative only and intended to educate those skilled in the art in the general manner of carrying out the invention. It should be understood that the forms of the invention shown and described herein are to be considered as examples of embodiments of the invention. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be used independently, all as will be apparent to one skilled in the art upon reading this specification. with invention. Changes may be made to the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

Examples

A. Chemical synthesis.

General information.

Unless otherwise stated, commercial grade solvents were used. Dry solvents were obtained from the Waters Dry Solvent System. Solvents for chromatography were distilled before use. Sensitive reactions were carried out in thermally dried glass containers and in an argon atmosphere. Analytical thin-layer chromatography (TLC) was carried out on Kieselgel 60 F254 glass plates pre-coated with 0.25 mm thick silica gel. Spots were visualized by staining with vanillin solution (6% w/v and 10% v/v sulfuric acid in 95% EtOH) or Hanessian stain (5% w/v ammonium molybdate, 1 w/v .% cerium (II) sulfate and 10 vol./vol.% sulfuric acid in water). Silica gel column chromatography was performed on Fluka Kieselgel 60 (230-400 mesh).

1H, ^13C and two-dimensional NMR spectra were measured on a Varian 400-MR spectrometer at 296 K. Chemical shifts (d) are indicated in parts per million (ppm) relative to the corresponding residual solvent peaks (CDCl ₃ : d 7.27 in 1H and 77.23 at ^13C NMR; CD3OD: d 3.31 at 1H and 49.15 at ^13C NMR).

The following abbreviations are used to indicate peak folds: s singlet; d doublet; dd doublet doublets; t triplet; dt doublet of triplets; q quartet; m multiplet. Coupling constants (J) are given in hertz (Hz). Optical rotation (OR) measurements were carried out using a Schmidt & Haensch UniPol L1000 polarimeter at λ=589 nm and concentration (c) expressed in g/100 ml in solvent as indicated in parentheses. High-resolution mass spectrometry (HRMS) was performed at the Free University Berlin, Mass Spectrometry Core Facility on an Agilent 6210 ESI-TOF mass spectrometer. Infrared (IR) spectra were measured on a Perkin Elmer 100 FTIR spectrometer.

A.I Abbreviations:

ACN - acetonitrile;

AcOH - acetic acid;

AIBN - azobisisobutyronitrile;

- 86 046159

Alhydrogel - Adjuvant-Gel aluminum hydroxide, Al: 10 mg/ml (Brenntag);

Alloc - allyloxycarbonyl;

aq. - water;

BH3 - borane;

BBr ₃ - boron tribromide;

Boc - tert-butoxycarbonyl;

BnBr - benzyl bromide;

br. - wide;

CAS - CAS registration number (CAS=Chemical Abstract Service);

CHCl3 - chloroform;

cHex - cyclohexane;

d - doublet;

dd - doublet of doublets;

DCM - dichloromethane;

DDQ - 2,3-dichloro-5,6-dicyano-1,4-benzoquinone;

DEAD - diethyl azodicarboxylate;

DIPEA - N.N-diisopropylethylamine;

DMAP - dimethylaminopyridine;

DME - dimethoxyethane;

DMF - dimethylformamide;

DMSO - dimethyl sulfoxide;

DPPA - diphenylphosphoryl azide;

EDC-HCl - N-((ethylimino)methylene)-N,N-dimethylpropane-1,3-diamine hydrochloride;

ES - electrospray;

Et ₂ O - diethyl ether;

EtOAc - ethyl acetate;

FCS - fetal calf serum;

FmocCl - 9-fluorenylmethoxycarbonyl chloride;

GSDMD - Gasdermin-D;

h - hour;

HCl - hydrochloric acid;

HEK293T - embryonic kidney fibroblastic cell line;

H ₂ O - water;

HOBI-H2O - 1H-benzo[1,2,3]triazol-1-ol hydrate;

hPBMC - human peripheral blood mononuclear cells;

IC ₅₀ - concentration of half-maximal inhibition;

K2CO3 - potassium carbonate;

LDH - lactate dehydrogenase;

LiAlH4 - lithium aluminum hydride;

m - multiplet;

MeCN - acetonitrile;

MeOH - methanol;

MeI - methyl iodide;

MgSO ₄ - magnesium sulfate;

min - minutes;

MS - mass spectrometry;

Na2CO3 - sodium carbonate;

NaCNBH3 - sodium cyanoborohydride;

NaHCO3 - sodium bicarbonate;

NaH - sodium hydride;

NaOH - sodium hydroxide;

NAP - 2-naphthylmethyl;

NapBr - 2-naphthylmethyl bromide;

NaPi - buffer phosphate buffered saline (PBS);

Na ₂ SO ₄ - sodium sulfate;

NBS - N-bromosuccinimide;

NCS - N-chlorosuccinimide;

NET - neutrophil extracellular traps;

NIS - N-iodosuccinimide;

NMR - nuclear magnetic resonance;

PBBBr - n-bromobenzyl bromide;

- 87 046159

PBS=NaPi - phosphate buffer saline solution;

Pd/C - palladium catalyst on a carbon carrier;

Pd(PPh3) ₄ - Tetrakis(triphenylphosphine)palladium(0);

PMA - phorbol 12-myristate 13-acetate;

PPh ₃ - triphenylphosphine;

PTFE - polytetrafluoroethylene;

q - quartet;

RBF - round bottom flask;

RT - room temperature;

s - singlet;

sat. - rich;

sep - septet;

t - triplet;

TBAF - tetrabutylammonium fluoride;

TFA - trifluoroacetic acid;

THF - tetrahydrofuran;

THP1 - acute monocytic leukemia cancer cell line;

TLC - thin layer chromatography;

TMSOTf - trimethylsilyl trifluoromethanesulfonate;

TsOH - tosinic acid;

wt. - weight.

A.2 Synthesis of hexasaccharide 33.

Synthesis of compound 2.

NIS (3.0 equiv.) was added to a cooled solution of compound 1 (prepared in accordance with Chem. Em. J. 2014, 20, 3578 - 3583) in THF:H ₂ O (4:1, 25 ml/1 g) at 0 °C. After 10 minutes, the reaction mixture was brought to RT and stirred for 2 hours. After complete consumption of the starting material, THF was removed under reduced pressure and the resulting crude residue was dissolved in EtOAc and washed with aq. Na2S2O3 and aq. NaHCO3. The separated organic layer was dried over Na2SO4, concentrated and the crude product was purified by automated silica gel flash column chromatography (0-60% EtOAc in cyclohexane) to give the desired hemiacetal 2 (84%) as a foam. HRMS (ESI ¹ ) was calculated for C38H ₃₈ O ₆ Na ⁺ [M+Na] ⁺ 613.2566, 613.2574 was found.

Synthesis of compound 3.

Ac ₂ O (2.0 equiv.) and trimethylamine (6.0 equiv.) were added to a pure solution of compound 2 in DCM (10 ml/1 g) and maintained with stirring at RT for 4 hours. After complete consumption of the starting material, the solvents were removed in vacuum and the crude product was purified by automated silica gel flash column chromatography (0-50% EtOAc in cyclohexane) to give the desired product 3 (94%) as a viscous liquid. MSHR (ESI ⁺ ) was calculated for C ₄₀ H ₄₀ O ₇ Na ⁺ [M+Na] ⁺ 655.2672, 655.2679 was found.

- 88 046159

Synthesis of compound 4.

AllylTMS, TMSOTf CH ₃ CN, (((((, 40 min 91%,

Allyl trimethylsilane (2.0 equiv.) was added to a pure solution of compound 3 in dry acetonitrile (20 ml/1 g) at room temperature and then TMSOTf (0.5 equiv.) was added dropwise. The flask was sealed and placed in an ultrasonic bath (frequency 80 Hz, 100% power 230 V, RT) until the reaction was completed by TLC (40 min)). After complete consumption of the starting material, the reaction mixture was quenched with aq. NaHCO ₃ , diluted with EtOAc and washed with brine. The separated organic layers were dried over Na ₂ SO ₄ , concentrated and the crude product was purified by automated silica gel flash column chromatography (0-60% EtOAc in cyclohexane) to give the desired C-glycoside 4 as an oil (91%). MSHR (ESI') was calculated for C ₄₁ H ₄₂ O ₅ Na ⁺ [M+Na] ⁺ 637.2930, 637.2929 was found.

Synthesis of compound 5.

PdCl ₂ (0.1 equiv.) was added to a degassed (30 min) solution of compound 4 in toluene (100 ml/1 g). After adding _PdCl2, the reaction mixture was degassed again for 30 min and maintained under stirring at 120°C for 2.5 days. After complete consumption of the starting material, the reaction mixture was passed through a celite pad and concentrated under reduced pressure. The crude residue was purified by automated silica gel flash column chromatography (050% EtOAc in cyclohexane) to give the compound with migrated double bond 5 (70%) as a yellowish liquid. MSHR (ESI ⁺ ) was calculated for C ₄₁ H ₄₂ O ₅ Na ⁺ [M+Na] ⁺ 637.2930, 637.2942 was found.

Synthesis of compound 6.

DDQ (1.2 equiv.) was added to a biphasic solution of compound 5 in DCM:H ₂ O (19:1, 20 ml/1 g) at 0°C. After 10 min at 0°C, the reaction mixture was warmed to room temperature and stirred at room temperature for 1 hour. After complete consumption of the starting material, the reaction mixture was diluted with DCM and extracted with aq. NaHCO3 and saline solution. The organic layer was dried over Na ₂ SO ₄ , filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by automated flash silica gel chromatography (0-80% EtOAc in cyclohexane) to give the desired product 6 as a white oil (94%). MSHR (ESI ⁺ ) was calculated for C ₃₀ H ₃₄ O ₅ Na ⁺ [M+Na] ⁺ 497.2304, 497.2312 was found.

- 89 046159

Synthesis of compound 7.

Ac2O (2.0 equiv.) and trimethylamine (6.0 equiv.) were added to a pure solution of compound 6 in DCM (10 ml/1 g) and maintained under stirring at RT for 4 h. After complete consumption of the starting material, the solvents were removed in vacuo and the crude product was purified by automated silica gel flash column chromatography (0-50% EtOAc in cyclohexane) to give the desired product 7 (90%) as a viscous liquid. MSHR (ESI+) was calculated for C ₃₂ H ₃₆ O ₆ Na ⁺ [M+Na] ⁺ 539.2410, 539.2419 was found.

Synthesis of compound 8.

Ozone was bubbled through a cooled solution of compound 7 in DCM:MeOH (1:1, 170 ml/1 g) at 78°C until the blue color remained. To remove residual Oz, pure O2 was bubbled through the reaction mixture until the solution was clear. Then, NaBH ₄ was added at -78°C, and the reaction mixture was stirred for 30 minutes at the same temperature. After complete consumption of the starting material, the reaction mixture was quenched with aq. NH ₄ Cl at -78°C and washed with DCM. The separated organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired compound 8 (60% over 2 steps) as a yellowish liquid. MSHR (ESI ⁺ ) was calculated for C ₃₀ H ₃₄ O ₇ Na ⁺ [M+Na] ⁺ 529.2202, 529.2220 was found.

Synthesis of compound 9.

To a solution of compound 8 in methanol (10 ml/1 g) was added sodium methoxide in MeOH (0.5 M, 10 ml) and the mixture was maintained with stirring at RT for 1 hour. After complete consumption of compound 8, AcOH (1 ml) was added until until the pH of the reaction mixture becomes acidic. After neutralization, the reaction mixture was concentrated and the crude residue was purified by flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired compound 9 (90%) as a paste. MSHR (ESI ⁺ ) was calculated for C ₂₈ H ₃₂ O ₆ Na ⁺ [M+Na] ⁺ 487.2097, 487.2111 was found.

Alternative synthesis of compound 9 - compound 10.

Propargyltrimethylsilane (9.11 ml, 61.5 mmol, 2.0 equiv) was added to a pure solution of compound 3 (19.5 g, 30.8 mmol) in dry acetonitrile (390 ml) at room temperature and then TMSOTf (2.8 ml, 15.4 mmol, 0.5 equiv) was added dropwise .). The flask was sealed and placed in an ultrasonic cleaning bath (frequency 80 Hz, 100% power 230 V, 5-10°C) until the reaction was completed by TLC (40 min)). After complete consumption of the starting material, the reaction mixture was quenched

- 90 046159 aq. NaHCO3, diluted with EtOAc and washed with brine. The separated organic layers were dried over Na ₂ SO ₄ , concentrated and the crude product was purified by automated silica gel flash column chromatography (0-60% EtOAc in cyclohexane) to give the desired C-glycoside 10 as an oil (16.2 g, 86%) . MSHR (ESI+) was calculated for C ₄₁ H ₄₀ O ₅ Na ⁺ [M+Na] ⁺ 635.2773, 635.2786 was found.

An alternative synthesis of compound 9 is compound 11.

DDQ (18.7 g, 82.0 mmol, 1.2 equiv.) was added to a biphasic solution of compound 10 (42 g, 68.5 mmol) in DCM:H ₂ O (19:1, 950 ml) at 0°C. After 10 min at 0°C, the reaction mixture was warmed to room temperature and stirred at room temperature for 1 hour. After complete consumption of the starting material, the reaction mixture was diluted with DCM and extracted with aq. NaHCO ₃ and brine. The organic layer was dried over Na ₂ SO ₄ , filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by automated silica gel flash chromatography (0-80% EtOAc in cyclohexane) to give the desired product 11 as a white oil (24 g, 74%, α-isomer only). MSHR (ESI ⁺ ) was calculated for C ₃₀ H ₃₂ O ₅ Na ⁺ [M+Na] ⁺ 495.2147, 495.2151 was found.

An alternative synthesis of compound 9 is compound 9.

Ozone was bubbled through a cooled solution of compound 11 (10.6 g, 22.4 mmol) in DCM:MeOH (1:1, 1 L) at -78°C until the blue color remained. To remove residual O ₃ , pure O ₂ was bubbled through the reaction mixture until the solution was clear. Then, NaBH ₄ (5.1 g, 135.0 mmol, 6.0 equiv.) was added at -78°C, and the reaction mixture was gradually brought to RT over 3 hours and stirred at RT for 45 minutes. After complete consumption of the starting material, the reaction mixture was quenched with aq. NH4Cl and washed with DCM three times. The separated organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The crude residue was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired compound 9 (8.4 g, 81% over 2 steps) as an oil (sticky white solid after drying in vacuo) . MSHR (ESI ⁺ ) was calculated for C ₂₈ H ₃₂ O ₆ Na ⁺ [M+Na] ⁺ 487.2097, 487.2106 was found.

Synthesis of compound 12.

Sodium hydride (2.0 equiv., 60% in mineral oil) was added at 0°C to a stirred solution of compound 9 in THF (20 ml/1 g). After 10 min, NapBr (1.05 eq.) was added and the mixture was stirred for 24 h at 0°C. After 24 h, the reaction mixture was quenched with MeOH, water, and extracted with AtOAc. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered, and

- 91 046159 concentrated. The resulting crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired compound 12 (54%) as a paste. MSHR (ESI') was calculated for C ₃₉ H ₄₀ O ₆ Na ⁺ [M+Na] ⁺ 627.2723, 627.2748 was found.

Synthesis of compound 14.

Et ₃ SiH (3.0 equiv.), TfOH (3.3 equiv.) were added to a cooled solution of compound 13 (prepared in accordance with Org. Lett. 2011, 73, 378 - 381) in DCM (10 ml/1 g) with freshly activated molecular sieves (4 A) at -78°C. The reaction mixture was stirred at the same temperature for 4 hours. After complete consumption of the starting material, the reaction mixture was quenched with Et ₃ N (1 ml) and diluted with DCM. The solution was washed with aq. NaHCO ₃ and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired 4-OH compound 14 (83%) as a white solid. HRMS (ESI ⁺ ) was calculated for C ₅ 1H ₅ 4Cl3NOi2NaS ⁺ [M+Na] ⁺ 1034.2300, 1034.2406 was found.

Synthesis of compound 15.

FmocCl (2.0 equiv.) and pyridine (3.0 equiv.) were added to a pure solution of compound 14 in DCM (10 ml/1 g) and maintained under stirring at RT for 3.5 h. After complete consumption of the starting material, the reaction mixture was diluted with DCM and it was washed with saline solution. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired compound 15 (93%) as a white solid. MSHR (ESI ⁺ ) was calculated for C ₆₆ H6 ₄ Cl ₃ NOi ₄ NaS ⁺ [M+Na] ⁺ 1256.2981, 1256.3125 was found.

Synthesis of compound 16.

NIS (1.4 equiv.) and TfOH (0.26 equiv.) were added to a cooled solution of acceptor 15 (1.0 equiv.) and donor 12 (1.2 equiv.) in DCM (0.06 M) in the presence of 4 A MS at -30°C. After 1.5 h, the starting material was completely consumed, then Et ₃ N (1.4 equiv.) was added and maintained with stirring at RT for 2 h. After 2 h, the reaction mixture was diluted with DCM and MS was filtered. The organic layer was washed with aq. Na ₂ S ₂ O ₃ and the separated organic layer were dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to obtain the desired acceptor three

- 92 046159 saccharide 16 (58% over 2 stages) as a white solid. MSHR (ESI ⁺ ) was calculated for C84H88Cl3NO18Na ⁺ [M+Na] ⁺ 1528.4935, 1528.5037 was found.

Synthesis of compound 18.

NIS, TfOH, toluene, 1,4 dioxane ^P 4 A MS, RT, 30 min, 76%

BnO

NIS (1.5 equiv.) and TfOH (0.4 equiv.) were added to a cooled solution of acceptor 16 (1.0 equiv.) and donor 17 (prepared in accordance with J. Org. Chem. 2016, 81, 162-184) (1.5 equiv. ) in a toluene:dioxane mixture (4:1, 0.03 M) in the presence of 4 A MS at 0°C. After 2 min, the reaction mixture was maintained at RT and stirred for 30 min. After 30 min, the reaction mixture was quenched with Et ₃ N, diluted with DCM and MS filtered. The organic layer was washed with aq. Na ₂ S ₂ O ₃ and the separated organic layer were dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired tetrasaccharide 18 (76%) as a white solid. MSHR (ESI') was calculated for C111H114Cl3NO23Na ⁺ [M+Na] ⁺ 1958.6745, 1958.6871 was found.

Synthesis of compound 19.

Et ₃ SiH (3.0 equiv.), TfOH (3.3 equiv.) were added to a cooled solution of compound 18 in DCM (10 ml/1 g) in the presence of freshly activated molecular sieves (4 A) at -78°C. The reaction mixture was stirred at the same temperature for 4 hours. After complete consumption of the starting material, the reaction mixture was quenched with Et ₃ N (1 ml) and diluted with DCM. The solution was washed with aq. NaHCO ₃ and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%,

- 93 046159

EtOAc in cyclohexane) to obtain the desired tetrasaccharide 19 (82%) as a white solid. MSHR (ESI) was calculated for C ₁₁₁ H ₁₁₆ Cl3NO ₂₃ Na ⁺ [M+Na] ⁺ 1960.6901, 1960.7024 was found.

Synthesis of compound 21.

Sodium hydride (2.0 equiv., 60% in mineral oil) was added at 0° C. to a stirred solution of compound 20 (prepared according to Tetrahedron: Asymmetry, 2000, 77, 481-492) in DMF (10 ml/1 g). After 10 min, PBBBr (1.1 eq) was added and the mixture was brought to RT. After stirring at RT for 1 hour, the reaction mixture was quenched with NH ₄ Cl and extracted with AtOAc. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered, and concentrated. The resulting crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to obtain the desired compound 21 (62%) as a paste. HRMS (ESI ⁺ ) was calculated for C ₃₆ H ₃₅ BrO ₇ NaS ⁺ [M+Na] ⁺ 713.1185, 713.1225 was found.

Synthesis of compound 22.

NBS, TMSOTf DCM, H ₂ O, 10 min 70% SEt -------------------·

NBS (1.1 equiv.) and TMSOTf (0.1 equiv.) were added to a cooled solution of compound 21 in DCM:H ₂ O (20:1, 10 ml/1 g) at 0°C. After 10 minutes, the reaction mixture was quenched with aq. NaHCO ₃ and diluted with DCM. The organic layer was washed with brine. The separated organic layer was dried over Na ₂ SO ₄ , concentrated and the crude product was purified by automated silica gel flash column chromatography (0-60% EtOAc in cyclohexane) to give the desired hemiacetal 22 (70%) as a foam. HRMS (ESI ⁺ ) was calculated for C ₃₄ H ₃₁ BrO ₈ Na ⁺ [M+Na] ⁺ 669.1100, 669.1132 was found.

Synthesis of compound 23.

Cs ₂ CO ₃ (3.0 equiv.), CF ₃ C(NPh)Cl (3.0 equiv.) were added to a stirred solution of compound 22 in DCM (10 ml/1 g) at 0°C. After 10 min, the mixture was brought to RT and stirred for 1 h. After complete consumption of compound 22, the reaction mixture was filtered and the filtrate was concentrated. The resulting crude residue was purified by automated silica gel flash column chromatography (0-60% EtOAc in cyclohexane) to obtain the desired imidate donor 23 (87%) as a foam.

Synthesis of compound 25.

Acceptor-thioglycoside 24 was synthesized according to Danieli, E.; Lay, L.; Proietti, D.; Berti, F.; Costantino, P.; Adamo, R. Org Lett. 2011, 13, 378-381.

TMSOTf in DCM (0.1 M, 0.2 equiv.) was added to a mixture of thioglycoside acceptor 24 (1.0 equiv.) and

- 94 046159 freshly dried 4 A MS in DCM at -78°C. After 2 min, a solution of imidate 23 (1.2 equiv.) in DCM was added. After 1 hour, the reaction mixture was quenched with Et ₃ N, and then filtered through a pad of celite. The filtrate was concentrated and the crude residue was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired disaccharide 25 (61%) as a solid. HRMS (ESI ⁺ ) was calculated for C ₅₆ H ₅₁ BrCl ₃ NO ₁₃ NaS ⁺ [M+Na] ⁺ 1186.1207, 1186.1314 was found.

Synthesis of compound 26.

Et ₃ SiH (3.0 equiv.), TfOH (3.3 equiv.) were added to a cooled solution of compound 25 in DCM (10 ml/1 g) with freshly activated molecular sieves (4 A) at -78°C. The reaction mixture was stirred at the same temperature for 4 hours. After complete consumption of the starting material, the reaction mixture was quenched with Et ₃ N (1 ml) and diluted with DCM. The solution was washed with aq. NaHCO ₃ and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired 4-OH compound 26 (80%) as a white solid. HRMS (ESI ⁺ ) was calculated for C ₅₆ H ₅₃ Cl ₃ NBrO ₁₃ NaS ⁺ [M+Na] ⁺ 1188.1364, 1188.1436 was found.

Synthesis of compound 27.

ACCl (2.0 equiv.) and pyridine (3.0 equiv.) were added to a pure solution of compound 26 in DCM (10 ml/1 g) at 0°C and maintained with stirring at RT for 3.5 h. After complete consumption of the starting material, the reaction the mixture was diluted with DCM and washed with brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired compound 27 (70%) as a white solid. HRMS (ESI) was calculated for C ₅₈ H ₅₅ Cl3NBrO14NaS ⁺ [M+Na] ⁺ 1230.1469, 1230.1563 was found.

Synthesis of compound 28.

Br

NIS, TfOH, DXM 4A MC, -20°C to 0°C,

NIS (1.8 equiv.) and TfOH (0.4 equiv.) were added to a cooled solution of acceptor 19 (1.0 equiv.) and donor 27 (1.8 equiv.) in DCM (0.025 M) in the presence of 4 A MS at -20°C. The reaction mixture was then gradually warmed to 0°C over 3 hours. After 3 hours, the reaction mixture was quenched with Et ₃ N, diluted with DCM and MS filtered. The organic layer was washed with aq. Na ₂ S ₂ O ₃ and the separated organic layer were dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired hexasaccharide 28 (65%) as a white solid. MSHR (ESI) was calculated for C ₁₆₃ H ₁₆₅ Cl ₆ N ₂ BrO ₃₇ Na ⁺ [M+Na] ⁺ 3060.8258, 3060.8275 was found.

Synthesis of compound 29.

VPO

VPO

VPO

1.Zn, AcOH, EtOAc, 3 h °C, 16 h

74% during stage 3

2. Ac ₂ O, Et ₃ N, EtOAc. 24 hours

3. NaOMe.. ΜβΟΗ,ΊΊ F

To a pure solution of 28 in EtOAc (2.0 mM), Zn (100 equiv.) and AcOH (100 equiv.) were added and the reaction mixture was kept under stirring at room temperature for 3 h. After complete consumption of the starting material, the reaction mixture was filtered through a pad of celite and concentrated. The residue obtained after removing the solvents was dissolved in EtOAc (2.0 mM), Et ₃ N (0.5 ml) and Ac ₂ O (0.5 ml) were added. After stirring at RT for 2.5 days, the reaction mixture was concentrated, and the crude material obtained after removal of solvents was dissolved in THF and methanol. To this clear solution was added 0.5 M NaOMe (3 ml) and maintained under reflux at 65°C. After 16 h, the reaction mixture was neutralized with AcOH and the solvents were removed. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired hexasaccharide 29 (74% over 3 steps) as a white solid. MSHR (ESI) was calculated for C ₁₄₃ H ₁₅ 5N ₂ BrO ₃₁ Na ⁺ [M+Na] ⁺ 2500.9708, 2500.9739 was found.

Synthesis of compound 30.

- 96 046159

Ac2O (8.0 equiv.) and trimethylamine (8.0 equiv.) were added to a pure solution of compound 29 in DCM (10 ml/1 g) and maintained under stirring at RT for 16 h. After complete consumption of the starting material, the solvents were removed in vacuo and the crude product was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired product 30 (83%) as a viscous liquid. HRMS (ESI ⁺ ) was calculated for Ci5iHi ₆₃ N2BrO ₃ 5Na ⁺ [M+Na] ⁺ 2669.0131, 2669.0407 was found.

Synthesis of compound 31.

DDQ (1.1 equiv) was added to a cooled solution of compound 30 in DCM:H2O at 0°C. After stirring the reaction mixture at the same temperature for 4 hours, the reaction mixture was diluted with DCM and extracted with aq. sat. NaHCO ₃ solution and saline solution. The organic layer was dried over Na2SO4, filtered, and the filtrate was concentrated to give the crude product. The crude product was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired product 31 (60%) as a viscous liquid. HRMS (ESI') was calculated for C ₁₄₀ H ₁₅₅ N2BrO ₃₅ Na ⁺ [M+Na] ⁺ 2527.9559, 2527.9731 was found.

- 97 046159

Synthesis of compound 32.

3. 'BuOOH'

37% over 3 stages

N ₃

To a solution of compound 31 in DCM, bis(diisopropylamino)benzyloxyphosphine (2.0 equiv) and diisopropylammonium tetrazolide (1.5 equiv) were added and the solution was stirred at RT for 1.5 h. Then 5-azidopentanol (8.0 equiv) and tetrazole (9.0) were added equiv. 0.45 M solution in CAN) and maintained with stirring at room temperature for 2 hours. After 2 hours, tert-butyl peroxide (6.0 equiv., 5.0-6.0 M solution in decane) was added and the reaction mixture was stirred for 1 hour. 1 h, the reaction mixture was diluted with DCM and quenched with aq. sat. NaHCO ₃ solution. The aqueous layer was extracted with DCM. The combined organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered, and the filtrate was concentrated in vacuo to give the crude product. The crude product was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired product 32 (37% over 3 steps) as a viscous liquid. MALDI was calculated for C ₁₅₂ H ₁₇₁ N ₅ BrO ₃₈ PH+ [M+H] ⁺ 2786.0635, 2786.870 was found.

- 98 046159

Synthesis of compound 3 3.

1. H ₂ Pd/C, EtOAc, MeOH H ₂ O, AcOH, 46 h

2. 2 M LiOH, MeOH, 3 h

80% within 2 stages

Pd/C (6 mg) was added to a pure solution of compound 32 (6 mg) in EtOAc:MeOH:H ₂ O:AcOH. The resulting heterogeneous mixture was stirred in a hydrogen atmosphere at RT for 40 hours. After 40 hours, the reaction mixture was filtered through a PTFE filter and concentrated in vacuum at 30°C in a rotary evaporator bath for 10 minutes to remove methanol, AtOAc, AcOH and water. The crude product obtained after removing the solvents was dissolved in MeOH, water and to this was added LiOH (2N in water) at 0°C. The reaction mixture was stirred at 0°C for 3 h. After 3 h, the reaction mixture was quenched with AcOH (30 μl) and the solvents were removed under reduced pressure and the resulting crude residue was purified by C18 reverse phase column chromatography using water and acetonitrile as solvents to obtain the desired final compound 33 (80% over 2 steps) as a white solid. MSHR (ESI') was calculated for C ₄₆ H ₈₂ N ₃ PO ₃₄ ⁺ [M-Na+2H] ⁺ 1252.4551, 1252.4578 was found.

- 99 046159

Synthesis of compound 34.

Pd/C (2 mg) was added to a pure solution of compound 29 in EtOAc:MeOH:H ₂ O:AcOH and the resulting heterogeneous mixture was stirred under a hydrogen atmosphere at RT for 40 hours. After 40 hours, the reaction mixture was filtered through a PTFE filter and concentrated in vacuum at 30°C rotary evaporator bath temperature for 10 minutes to remove methanol, AtOAc, AcOH and water. The crude product was purified by C18 reverse phase column chromatography using water and acetonitrile as solvents to obtain the desired final compound 34 (82%) as a white solid. MSHR (ESI) was calculated for C ₄₁ H ₇₀ N ₂ O ₃₁ ⁺ [M+Na] ⁺ 1109.3860, 1109.3853 was found.

Conjugation of compound 33 to CRM ₁₉₇ or BSA.

But

o di-N-hydroxy-succinimidyl adipate ester Et ₃ N DMSO, H ₂ O, RT, 3 h nh ₂ but

- 100 046159

RHN

R= CRM 197

R=BSA

Antigen 33 (1.0 equiv.) was dissolved in DMSO-H2O at RT in a 2 ml vessel. To this was added triethylamine (35.0 equiv.). The mixture was added to activated adipate-NHS ester (10 equiv.) in DMSO in an Eppendorf flask and stirred for 3 h at RT. The antigen-NHS ester was precipitated by adding 10 volumes of EtOAc and centrifuged, and the supernatant was carefully removed. The precipitate was washed with EtOAc (1 mlx3), dried and transferred to the next step. 1 mg of protein in NaPi buffer (~100 μl) was added dropwise into a reaction vessel containing antigen-NHS 35 ester in 50 μl of NaPi buffer (pH 7.0). The vessel was finally rinsed with 50 μL of buffer solution and completely transferred to the reaction vessel. The reaction mixture was stirred at RT for 22 hours. The antigen-protein conjugate solution was transferred to Amicon Ultra-0.5 ml and centrifuged for 6 minutes at room temperature. Add 300 µl of buffer to the reaction vessel, wash and transfer to a filter and centrifuge again. Additional washes were performed using 1x]PBS solution three more times. After the final wash, the conjugate was stored in 1x PBS solution at 2-8°C. Conjugates were analyzed using MALDI, (resulting in a loading of 4-12 antigens on the protein), SDS page, BCA assessment, SEC-HPLC.

A.Z Synthesis of hexasaccharide 54.

Synthesis of compound 41.

TBDPSCl (1.1 equiv.) and trimethylamine (2.8 equiv.) were added to a pure solution of compound 20 in CH3CN (10 ml/1 g) and maintained under stirring at RT for 10 h. After complete consumption of the starting material, the solvents were removed in vacuo and the crude product was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired product 41 (93%) as a viscous liquid. HRMS (ESI') was calculated for C45H4sO?SSiNa ⁺ [M+Na] ⁺ 783.2788, 783.2767 was found.

Synthesis of compound 42.

The procedure described for the synthesis of compound 22 was used for the synthesis of compound 42 (94%). MSHR (ESI ⁺ ) was calculated for C ₄₃ H ₄₄ O ₈ SiNa ⁺ [M+Na] ⁺ 739.2703, 739.2700 was found.

Synthesis of compound 43.

Trichloroacetonitrile (6.0 equiv.) and DBU (0.2 equiv.) were added to a cooled solution of compound 42 in DCM at 0°C. After 3 h at 0°C, the reaction was completed and the solvent was evaporated. The crude product was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired product 43 (83%) as a viscous liquid.

Synthesis of compound 44.

The procedure described for the synthesis of compound 25 was used for the synthesis of compound 44 (40%). MSHR (ESI+) was calculated for ('.JI ₍ ,iOi3SiSNCl3Na' [M+Na]+ 1256.2801, 1256.2645 was found.

Synthesis of compound 45.

The procedure described for the synthesis of compound 26 was used for the synthesis of compound 45 (60%). HRMS (ESI+) was calculated for C ₆₅ H ₆₆ O ₁₃ SiSNCl ₃ Na ⁺ [M+Na] ⁺ 1256.2987, 1256.2974 was found.

Synthesis of compound 46.

The procedure described for the synthesis of compound 27 was used for the synthesis of compound 46 (60%). HRMS (ESI ⁺ ) was calculated for C ₆₇ H ₆₈ O ₁₄ SiSNCl ₃ Na ⁺ [M+Na] ⁺ 1300.3064, 1300.3090 was found.

Synthesis of compound 47.

The procedure described for the synthesis of compound 28 was used for the synthesis of compound 47 (82%). HRMS (ESI ⁺ ) was calculated for C ₁₇₂ H ₁₇₈ O ₃₇ SiN ₂ Cl ₆ Na ⁺ [M+Na] ⁺ 3127.9728, 3127.9728 was found.

- 102 046159

Synthesis of compound 48.

1.Zn,AcOH, EtOAc,3 h

2. Ac ₂ O, Et ₃ N, EtOAc.24 H

3. NaOMe. . MeOH, THF °C, 16 h

50% over 3 stages

The procedure described for the synthesis of compound 29 was used for the synthesis of compound 48 (50%). HRMS (ESI') was calculated for C ₁₅₂ H16 ₈ O ₃₁ SiN2Na ⁺ [M+Na] ⁺ 2568.1298, 2568.1322 was found.

Synthesis of compound 49.

The procedure described for the synthesis of compound 30 was used for the synthesis of compound 49 (80%). MSHR (ESI ⁺ ) was calculated for C ₁₆₀ H ₁₇₆ O ₃₅ SiN ₂ Na ⁺ [M+Na] ⁺ 2737.1754, 2737.2001 was found.

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Synthesis of compound 50.

VPO

VPO

VPO

TBDPSO

VPO

ASO

The procedure described for the synthesis of compound 31 was used for the synthesis of compound 50 (70%). MSHR (ESI') was calculated for C ₁₄₉ H ₁₆₈ O ₃₅ SiN ₂ Na ⁺ [M+Na] ⁺ 2596.1095, 2595.9954 and 2596.9997 were found.

Synthesis of compound 51.

The procedure described for the synthesis of compound 32 was used for the synthesis of compound 51.

- 104 046159

Synthesis of compound 52. AcO /OVP TBDPSO-A L LA P L *........ 1 -....... V ^u \ AcO AcHN ^VpO ₍ VpO~^ BnO-VC°\ VpO-^ -T^AcO ORP _-O ZpO| About GU about .OVP Vpo-\| UL-o Bno-x^jQ 1 ? ^Bn TBAF, AcOH, THF NHAc < ή O—P^o o \ 51 ?

^OH AcO ^OBp

BnO^AcO

BnO o /OBn BnO-\ ⁿ ?

BnO^ BnO

OBn

O^ISo

ABOUT '

N ₃

A premixed solution of TBAF and AcOH was added to a neat solution of compound 51 in THF at RT and the reaction mixture was kept under stirring at RT for 3 hours. After complete consumption of the starting material, the reaction mixture was diluted with DCM and concentrated in vacuo to give the crude product. The crude product was purified by automated silica gel column chromatography using EtOAc in n-hexane (gradient, 0-100%) as eluent.

- 105 046159

Synthesis of compound 53. /θ ^Η AcO ^OBn ORP Acb achn ^ΒηθΑ ζ^ ΒηΟ^ O ^OBn BpO—B Bno^V^R o ^BpO A^ AcO NHAc 52 BnO _{4 z} p _z R. ^BnO <° ^Ac ? i° ^Bn /° AcO AcHN ^BnO _Br BnO^ BnO-xAs?\ BnO- ¹ -*^ AcO I ^H 1 N® nO 1. BnO _Vp ,N('Pr) ₂ f % DH1 Tsu OBn ft ^N 'N 1 ? ^{Bn Ο} O 3. 'BuOOH N ₃ iBn o ^lO O /ORP BnO-?? —O OBn NHAc < A o \

N ₃

To a solution of compound 52 in DCM, dibenzyl N,N-diisopropylphosphoramidite (2.0 equiv.) and diisopropylammonium tetrazolide (1.5 equiv.) were added and the solution was stirred at RT for 1.5 h. Then, tert-butyl peroxide (6.0 equiv., 5.0 - 6.0 M solution in decane) and the reaction mixture was stirred for 1 hour. After 1 hour, the reaction mixture was diluted with DCM and quenched with aq. sat. NaHCO3 solution. The aqueous layer was extracted with DCM. The combined organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered, and the filtrate was concentrated in vacuo to give the crude product. The crude product was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired product 53.

- 106 046159

Synthesis of compound 54.

NH ₂

The procedure described for the synthesis of compound 33 was used for the synthesis of compound 54.

Conjugation of compound 54 with CRM ₁₉₇ and BSA.

- 107 046159

CRM 197 or B SA,

ABOUT

RHN

R= CRM 197

R=BSA

The procedure described for the synthesis of glycoconjugates 36 and 37 was also used for the synthesis of compounds 56 and 57.

A.4 Alternative synthesis of hexasaccharide 54.

Synthesis of compound 58.

Ph ₂ P(O)H, pyridine, 2 h, and then Et ₃ HNHCO ₃ solution, 2 h, 90%

Diphenylphosphite was added to a pure solution of compound 48 in pyridine, and the reaction mixture was stirred at room temperature under nitrogen atmosphere for 2 hours. After 2 hours, 1 M TEAB solution was added to the reaction mixture at 0°C. After 5 minutes, the ice bath was removed and stirring was continued for another 2 hours at RT. After complete consumption of the starting material, the reaction mixture was diluted with DCM and the organic layer was washed successively with 1 M TEAB solution and concentrated under reduced pressure. The crude product was purified by automated flash column chromatography (EA:DCM:MeOH with 2% Et ₃ N) to obtain pure H-phosphonate derivative 58

- 108 046159 (90%) in the form of a viscous liquid. MSHR (ESI') was calculated for C ₁₅₅ H ₁₈₄ N ₃ PSiO ₃₇ ⁺ [M] ⁺ 2740.2189, 2740.2132 was found.

Synthesis of compound 59.

AcO ^OBn TBDPSO-\ ~ L\ n BnO-^V^R AcO-^-*7>-^ ^U — AcO AcHN BnO .OBn _-O

BnO

VlO ¹ BnOVpO| O o ί f^-θΑ

AcO •OBn VpO-\ ^P ?

_,o Bno-'VHS

NHAc

1. PivCI, BUT'

N _{3 +} °K°

HNEt ₃ 0' ^H

2.1 ₂ , ru, water

70%

AcO ..OBn tbdpso^a _. LA o BnO^VcR oAct) AcHN ^BnO

BnO^

BnO^V ² BnO- ⁴ -* .OBn

ВпО О Λ о _x OBn ВпО —\ I . °Х'°

HNEt ₃ O o

N ₃

H-phosphonate 58 (1.0 equiv.) and linker (4.0 equiv.) were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in roux and PivCl (2.0 equiv.) was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I ₂ in Py:H ₂ O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 59 (70%) as a viscous liquid. Maldi (ESI ⁺ ) was calculated for C154H178N5PNaSiO38 ⁺ [M-Et3N+Na] ⁺ 2789.1635, 2788.0 was found.

Synthesis of compound 60.

An HF solution (70% in pyridine, 0.3 ml) was added dropwise to a solution of compound 59 in DCM and pyridine at 0°C. The reaction mixture was stirred at the same temperature for 18 hours. Then, the reaction mixture was diluted with DCM, washed with saturated aqueous NaHCO ₃ solution and TEAB buffer. The organic phase was separated and dried (Na ₂ SO ₄ ) and concentrated under reduced pressure.

- 109 046159

The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to obtain the desired product 60 as a viscous liquid. Maldi (ESI ⁺ ) was calculated for C ₁₃₈ H ₁₆₀ N ₅ PNaO ₃₈ ⁺ [M-Et3N+Na] ⁺ 2550.7585, 2549.698 was found.

Synthesis of compound 54.

HO

'BuOOH

N ₃ o

1. H ₂ Pd/C, EtOAc, MeOH H ₂ O, AcOH, 46h

2. 2 M LiOH, MeOH, 3H

3. Dowex 50WX8, Na+ form

Dibenzyl diisopropylphosphoramidite (2.0 equiv.) and diisopropylammonium tetrazolide (2.0 equiv.) were added to a solution of compound 60 in DCM and the solution was stirred at room temperature for 1.5 h. Then, tert-butyl peroxide 5.0 - 6.0 M solution in decane (6.0 equiv.) was added at room temperature and the reaction mixture was stirred for 1 hour. The reaction mixture was diluted with DCM and washed with aq. sat. NaHCO ₃ solution and TEAB buffer. The aqueous layer was extracted with DCM (2x10 ml). The combined organic layer was dried over Na ₂ SO ₄ (0.5 g), filtered, and the filtrate was concentrated in vacuo to give the crude product. The crude product was purified by automated flash chromatography using EtOAc:DCM:MeOH with 2% trimethylamine to give the desired product 61 as a viscous oil (91%). The title compound 54 was obtained in 60% yield from compound 61 using the procedure described for the synthesis of compound 33. HRMS (ESI) was calculated for C ₄₆ H ₈₃ N ₃ P ₂ NaO ₃₇ ⁺ [M+Na] ^- 1354.4078, it was found 1354.9623.

A.5 Alternative synthesis of hexasaccharide 54.

Synthesis of compound 62.

Me ₃ N-BH ₃ (21.2 g, 291 mmol, 5.4 equiv.), BF ₃ -Et ₂ O (42.2 ml, 291 mmol, 5.4 equiv.) was added to a cooled solution of compound 24 (28.8 g, 54 mmol) in CH3CN (1.5 L) at 0°C. The reaction mixture was stirred at the same temperature for 1 hour. After complete consumption of the starting material, the reaction mixture was quenched with Et ₃ N (30 ml) and MeOH (50 ml). The reaction mixture was then diluted with EtOAc (1 L), washed with 1 M HCl (three times, sometimes it is difficult to see 2 layers, so then add hydrochloric acid

- 110 046159 solution to improve) and then aq. NaHCO3 until the organic layer reaches a neutral pH. The separated organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. Product 62 (22 g, 76%) as a white solid was used neat in the next step. MSHR (ESI') was calculated for C ₂₂ H ₂₄ Cl ₃ NO ₆ SNa ⁺ [M+Na] ⁺ 558.0288, 558.0332 was found.

Synthesis of compound 64.

OBn NIS, THF, water Х)Вп _p from 0°С to RT, 3 h, 85% _

BnO-^* ^, *T^' ^STo

OBn OBn

63

The procedure described for the synthesis of compound 2 was used for the synthesis of compound 64 (85%). MSHR (ESI ⁺ ) was calculated for C34H40O6N ⁺ [M+NH4] ⁺ 558.2856, 558.2976 was found.

Synthesis of compound 65.

To a stirred solution of compound 64 (24.5 g, 45.3 mmol) in anhydrous DCM (360 ml), anhydrous DMF (1 ml, 13.6 mmol, 0.30 equiv.) and (COCl) ₂ (10.3 ml, 118.0 mmol, 2.6 equiv.) was added at 0°C. After 5 minutes, the reaction mixture was brought to RT and stirred at RT for 2 hours. After complete consumption of the starting material, the reaction mixture was cooled to 0°C and quenched with Et ₃ N. The resulting salt was filtered through a short layer of celite and washed with DCM (do not wash with a large amount DXM, the salt dissolves and passes through the Celite.) The filtrate was then concentrated under reduced pressure and purified by silica gel column chromatography using ethyl acetate:cyclohexane (0-40% with 2% Et ₃ N) to give the desired glycosyl chloride 65 (24 g, 96%) as a viscous liquid. HRMS (ESI ⁺ ) was calculated for C ₃₄ H35O ₅ ClNa ⁺ [M+Na] ⁺ 581.2071, 581.2206 was found.

Synthesis of compound 66.

_Ag2O (8.8 g, 37.7 mmol, 1.5 equiv., dried in vacuum at 80°C for 3 hours before use) and 2-aminoethyl diphenylborinate (0.57 g, 2.51 mmol, 0.1 equiv.). After stirring at RT for 16 hours, the mixture was diluted with DCM (80 ml), acetone (80 ml) and filtered through celite sand and washed with DCM and acetone until the filtrate showed product. All filtrate fractions were combined and concentrated. The residue was dissolved in EtOAc (300 ml) and maintained at 55°C until the solid dissolved and became a clear solution. This clear solution was then filtered through filter paper and washed with hot EtOAc and left to recrystallize. After 1 hour, the white solid crystallized and separated from the solution to give the desired disaccharide 66 as a white solid (22 g, 83%). MSHR (ESI ⁺ ) was calculated for C5 ₆ H ₅₈ Cl ₃ NOnSNa ⁺ [M+Na] ⁺ 1080.2696, 1080.2904 was found.

Synthesis of compound 67.

- 111 046159

FmocCl (16.87 g, 63.2 mmol, 2.0 equiv.) and pyridine (7.67 mL, 95.0 mmol, 3.0 equiv.) were added to a pure solution of compound 66 (33.5 g, 31.6 mmol) in DCM (330 mL) and maintained with stirring at RT for 2 hours. After complete consumption of the starting material, the reaction mixture was diluted with DCM and washed with brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to give the desired compound 67 (34.7 g, 86%) as a white solid. MSHR (ESI) was calculated for C ₇₁ H ₆₈ Cl ₃ NO ₁₃ SNa ⁺ [M+Na] ⁺ 1302.3375, 1302.3694 was found.

Synthesis of compound 69.

The procedure described for the synthesis of compound 2 was used for the synthesis of compound 69 (80%). MSHR (ESI ⁺ ) was calculated for C ₂₉ H ₃₆ O ₇ N + [M + NH ₄ ] ⁺ 510.2492, 510.2527 was found.

Synthesis of compound 70.

To a stirred solution of compound 69 (18.0 g, 36.5 mmol) in anhydrous DCM (290 ml), anhydrous DMF (0.85 ml, 11.0 mmol, 0.30 equiv.) and (COCl) ₂ (8.3 ml, 95.0 mmol, 2.6 equiv.) was added at 0°C. After 5 min. The reaction mixture was brought to RT and stirred at RT for 2 hours. After complete consumption of the starting material, the reaction mixture was cooled to 0°C and quenched with Et ₃ N. The resulting salt was filtered through a short pad of celite and washed with DCM. The filtrate was then concentrated under reduced pressure and purified by silica gel column chromatography using ethyl acetate:cyclohexane (0-40% with 2% Et ₃ N) to give the desired glycosyl chloride 70 (16.7 g, 89%) as a viscous liquid. MSHR (ESI ⁺ ) was calculated for C ₂₉ H ₃₁ O ₆ ClNa ⁺ [M+Na] ⁺ 533.1707, 533.1752 was found.

Synthesis of compound 71.

_Ag2O (9.76 g, 42.1 mmol, 1.5 equiv dried in vacuum at 80°C for 3 hours before use) and 2-aminoethyl diphenylborinate (0.63 g, 2.81 mmol, 0.1 equiv). After stirring at RT for 16 hours, the mixture was diluted with DCM (80 ml), acetone (80 ml) and filtered through celite sand and washed with DCM and acetone until the filtrate showed product. All filtrate fractions were combined and concentrated. The residue was dissolved in EtOAc (400 ml) and maintained at 55°C until the solid dissolved and became a clear solution. This clear solution was then filtered through filter paper and washed with hot EtOAc and left to recrystallize. After 1 hour, the white solid crystallized and separated from the solution to give the desired disaccharide 71 as a white solid (23 g, 81%). MSHR (ESI ⁺ ) was calculated for C ₅₁ H ₅₄ Cl ₃ NO ₁₂ SNa ⁺ [M+Na] ⁺ 1032.2330, 1032.2423 was found.

Synthesis of compound 72.

- 112 046159

ACCl (40 ml) was added to a cloudy mixture of 71 (18.87 g, 18.66 mmol) in MeOH (200 ml) and DCM (200 ml) at 0°C. After 5 min, the ice bath was removed and maintained at RT for mixing. After stirring at room temperature for 3 hours, the reaction mixture was diluted with DCM and washed with water and aq. _NaHCO3 . The separated organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated on a rotary evaporator to yield the desired compound 72 (18.09 g, quantitative) as a white solid. MSHR (ESI') was calculated for C ₄₉ H ₅₂ Cl ₃ NO11SNa ⁺ [M+Na] ⁺ 990.2224, 990.2301 was found.

Synthesis of compound 73.

Imidazole (3.56 g, 52.3 mmol, 2.8 equiv.) and TBDPSCl (7.2 mL, 28.0 mmol, 1.5 equiv.) were added to a suspension of 72 (18.05 g, 18.6 mmol) in acetonitrile (370 ml). After 5 min the reaction mixture was completely clear and was left to stir at RT for 30 min. After 30 min, the reaction mixture was diluted with EtOAc and washed with brine. The separated organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue obtained after removal of solvents was purified by automated silica gel flash chromatography using ethyl acetate and cyclohexane as eluents to obtain the desired product 73 (20.9 g, 93%) as a solid. HRMS (ESI ⁺ ) was calculated for C ₆₅ H ₇₀ Cl ₃ NO ₁₁ SSiNa ⁺ [M+Na] ⁺ 1228.3402, 1228.3481 was found.

Synthesis of compound 74.

To a pure solution of compound 73 (18.69 g, 15.47 mmol) in DCM (200 ml) was added Et ₃ N (19 ml, 139.0 mmol, 9.0 equiv.), acetic anhydride (4.4 ml, 46.4 mmol, 3.0 equiv.) and DMAP ( 0.189 g, 1.547 mmol, 0.1 equiv.) and maintained with stirring at RT for 18 hours. After 18 hours, the reaction mixture was diluted with DCM and washed with aq. NaHCO3. The separated organic layer was dried over Na2SO4 and concentrated. The crude residue obtained after removal of solvents was purified by automated flash silica gel chromatography (cyclohexane-EtOAc) to yield the desired product 74 as a foam (17.2 g, 89%). HRMS (ESI ⁺ ) was calculated for C ₆₇ H ₇₂ Cl ₃ NO ₁₂ SSiNa ⁺ [M+Na] ⁺ 1272.3478, 1272.3530 was found.

Synthesis of compound 76.

The procedure described for the synthesis of compound 16 was used for the synthesis of compound 76 (60% over 2 steps). HRMS (ESI ⁺ ) was calculated for C ₈₂ H ₈₆ O ₁₇ NNaCl ₃ ⁺ [M+Na] ⁺ 1574.5329, 1574.5624 was found.

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Synthesis of compound 77.

The procedure described for the synthesis of compound 18 was used for the synthesis of compound 77 (80%). MSHR (ESI') was calculated for C ₁₁₆ H ₁₂₂ O ₂₂ N2Cl ₃ ⁺ [M+NH4] ⁺ 2000.7565, 2000.7588 was found.

Synthesis of compound 78.

The procedure described for the synthesis of compound 19 was used for the synthesis of compound 78 (80%). MSHR (ESI ⁺ ) was calculated for C ₁₁₆ H ₁₂₄ O ₂₂ N ₂ Cl ₃ ⁺ [M+NH4] ⁺ 2001.7711, 2001.6469 was found.

Synthesis of compound 79.

The procedure described for the synthesis of compound 28 was used for the synthesis of compound 79 (79%). MSHR (ESI ⁺ ) was calculated for C ₁ 77H ₁₈₆ O ₃₄ N ₂ Cl ₆ Na ⁺ [M+Na] ⁺ 3147.0689, 3147.1184 was found.

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Synthesis of compound 80.

Zn (100 equiv.), AcOH (100 equiv.), Ac ₂ O were added to a pure solution of compound 79 in EtOAc (2.0 mM), and the reaction mixture was maintained with stirring at room temperature for 20 hours. After complete consumption of the starting material, the reaction mixture was filtered through a layer of celite and concentrated. The crude residue was purified by automated silica gel flash column chromatography (0-100%, EtOAc in cyclohexane) to obtain the desired hexasaccharide 80 (69%) as a white solid. MSHR (ESI') was calculated for C ₁₇₅ H ₁₈₈ N ₂ O ₃₂ Si ⁺ [M] ⁺ 2858.2948, 2858.3062 was found.

Synthesis of compound 81.

The procedure described for the synthesis of compound 31 was used for the synthesis of compound 81 (73%). MSHR (ESI ⁺ ) was calculated for C ₁₆₄ H ₁₈₀ O ₃₂ N ₂ Si ⁺ [M] ⁺ 2718.2322, 2718.2347 was found.

Synthesis of compound 82.

z. g1zM1magk>i ₃ to I. z h

87% within 2 stages

- 115 046159

The procedure described for the synthesis of compound 58 was used for the synthesis of compound 82 (87%). MSHR (ESI ⁺ ) was calculated for C164H181O34N2SiP ⁺ [M-Et3N] ⁺ 2782.2036, 2782.2077 was found.

Synthesis of compound 83.

The procedure described for the synthesis of compound 59 was used for the synthesis of compound 83 (88%). MSHR (ESI') was calculated for C ₁₆₉ H ₁₉₀ O ₃₅ N ₅ SiP ⁺ [M-Et ₃ N] ⁺ 2910.2815, 2910.2841 was found.

Synthesis of compound 84.

АСО .ОВп TBDPSO-Д ~ I\п ^Вп °Ж^о-Л^-оВпО ACHN ВпО .ОВп

HF, py, DXM, 0°С, 18 h

VPO| O

VPO _p

VSH °

VPO

ORP VpO—y ¹ ?

__about Bno-'vMQ

NHAc°\OHNETa

N ₃

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 84 (90%). MSHR (ESI ⁺ ) was calculated for C153H172O35N5P ⁺ [M-Et3N] ⁺ 2672.1638, 2672.1759 was found.

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Synthesis of compound 33.

1. H ₂ Pd/C, EtOAc, MeOH H ₂ O, AcOH, 46h 84 ---------------------►

2. 2 M LiOH, MeOH, 3 h 3. Dowex 50WX8, Na+ form

^and 11^0' ^VV 'NH ₂ for O

55% over 3 stages

The procedure described for the synthesis of compound 33 from compound 32 was used for the synthesis of compound 33 (55%). HRMS (ESI+) was calculated for C ₄₆ H ₈₂ N ₃ PO ₃₄ ⁺ [M-Na+2H] ⁺ 1252.4551, 1252.4574 was found.

Synthesis of compound 85.

To a solution of compound 84 in DCM, dibenzyl XA'-diisosyl phosphoramidite (2.0 equiv.) and diisopropylammonium tetrazolide (1.5 equiv.) were added and the solution was stirred at RT for 2.5 hours. Then tert-butyl peroxide (6.0 equiv., 5.0-6.0 M) was added solution in decane) and the reaction mixture was stirred for 1 hour. After 1 hour, the reaction mixture was diluted with DCM and quenched with aq. sat. NaHCO3 solution. cThe aqueous layer was extracted with DCM. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give the crude product. The crude product was purified by automated silica gel flash column chromatography (0-100% EtOAc in cyclohexane) to give the desired product 85 (88%). MSHR (ESI) was calculated for C ₁₆₇ H ₁₈₅ O ₃₈ N ₅ P ₂ ⁺ [M-Et ₃ N] ⁺ 2932.2240, 2932.2147 was found.

Synthesis of compound 54.

Pd/C (20 mg) was added to a pure solution of compound 85 (20 mg) in EtOAc:MeOH:H ₂ O:DCM. The resulting heterogeneous mixture was stirred under a hydrogen atmosphere at RT for 40 hours. After 40 hours, the reaction mixture was filtered through a PTFE filter and concentrated in vacuo at a 30°C rotary evaporator bath temperature for 10 minutes to remove methanol, AtOAc, DCM and water. The crude product obtained after removal of solvents was dissolved in MeOH, water and LiOH (2N in water) was added to this mixture at 0°C. The reaction mixture was stirred at 0°C for 3 hours. After 3 hours, the reaction mixture was quenched with AcOH and the solvents were removed under reduced pressure and the resulting crude residue was purified by C18 reverse phase column chromatography using water and acetonitrile as solvents to obtain the desired final compound 54 in salt form. The triethylamine salt was then replaced with Dowex resin to obtain the desired sodium salt compound (40% over 3 steps) as a white solid. MSHR (ESI) was calculated for C46H83N3P2O37+ [M-Na+H] ⁺ 1332.4214, 1332.4242 was found.

- 117 046159

Synthesis of compound 86.

The procedure described for the synthesis of compound 58 was used for the synthesis of compound 86 (94%). MSHR (ESI') was calculated for C ₁₆₅ H ₂₀₃ O ₃₇ N ₇ P ₂ ⁺ [M-2xEt ₃ N+H] ⁺ 2735.1318, 2735.1356 was found.

Synthesis of compound 87.

H-phosphonate 86 (1.0 equiv.) and benzyl alcohol (10.0 equiv.) were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in py and PivCl (5.0 equiv.) was added. The reaction mixture was kept under stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I2 in Py: _H2O (20:1) was added and the reaction mixture was kept under stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 87 (86%) as a viscous liquid. Maldi (ESI ⁺ ) was calculated for C160H179N5P2O38 ⁺ [M+H-2xEt3N] ⁺ 2842.1770, 2842.1638 was found.

Synthesis of compound 54.

Pd/C (20 mg) was added to a pure solution of compound 87 (20 mg) in EtOAc:MeOH:H ₂ O:DCM. The resulting heterogeneous mixture was stirred under a hydrogen atmosphere at RT for 40 hours. After 40 hours, the reaction mixture was filtered through a PTFE filter and concentrated in vacuo at a 30°C rotary evaporator bath temperature for 10 minutes to remove methanol, AtOAc, DCM and water. The crude product obtained after removing the solvents was dissolved in MeOH, water and to this was added LiOH (2N in water) at 0°C. The reaction mixture was stirred at 0°C for 3 hours. After 3 hours, the reaction mixture was quenched with AcOH and the solvents were removed under reduced pressure and the resulting crude residue was purified by C18 reverse phase column chromatography using water and acetonitrile as solvents to obtain the desired final compound 54 in salt form. The triethylamine salt was then replaced with Dowex resin to obtain the desired sodium salt compound (70% over 3 steps) as a white solid. MSHR (ESI ⁺ ) was calculated for C46H83N3P2O3/ [M-3Na+4H] ⁺ 1332.4214, 1332.4232 was found.

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A.6 Synthesis of dodecasaccharide 92.

Synthesis of compound 88.

1. ('Ργ) ₂ Ν

VPO

2.

ν ₃

The procedure described for the synthesis of compound 32 was used for the synthesis of compound 88, here only there is a replacement; in the second stage, instead of a linker, compound 52 was used as a nucleophile.

Synthesis of compound 89.

The procedure described for the synthesis of compound 52 was used for the synthesis of compound 89.

Synthesis of compound 90.

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1. H ₂ Pd/C, EtOAc. MeOH H ₂ b, AcOH.46 h

2. 2 M LiOH, MeOH, 3 h

h ₂ n

The procedure described for the synthesis of compound 33 was used for the synthesis of compound 90. Synthesis of compound 91.

The procedure described for the synthesis of compound 53 was used for the synthesis of compound 91. Synthesis of compound 92.

1. H ₂ Pd/C, EtOAc, MeOH H ₂ O, AcOH, 46 h

2. 2 M LiOH, MeOH.3 h

- 120 046159

The procedure described for the synthesis of compound 33 was used for the synthesis of compound 92 (60%). HRMS (ESI+) was calculated for C ₈₇ H ₁₅₁ N ₅ P ₂ O ₆₇ [(M-2Na+2H)/2] 1199.9019, 1199.8950 was found.

Conjugation of compound 92 with CRM ₁₉₇ and BSA.

di-N-hydroxy-succinimidyl adipate-ester

Et ₃ N DMSO, H ₂ O, CT, 3 i

- 121 046159

94R=CRM197 O

R= BSA NHR

The procedure described for the synthesis of glycoconjugates 36 and 37 was used for the synthesis of compounds 94 and 95.

A.7 Alternative synthesis of dodecasaccharide 92.

Synthesis of compound 96.

H-phosphonate 58 (1.2 equiv.) and acceptor 60 (1.0 equiv.) were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in roux and PivCl (1.3 equiv.) was added to this. The reaction mixture was maintained with stirring at RT for 3 hours. After 3 hours, the reaction mixture was cooled to -40 C, a freshly prepared solution of I ₂ in Py:H ₂ O (250 μl, 20:1) was added and the reaction mixture was maintained with stirring at at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 96 (70%) as a viscous liquid. MALDI (ESI+) was calculated for C ₂₈₇ H ₃₂₅ K ₂ N ₇ O ₇₅ P ₂ Si ⁺ [M-2Et ₃ N+2K] ⁺ 5237.0351, 5237.718 was found.

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Synthesis of compound 97.

HF, ru.THF, o °C, 92%

-----------------------------

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 97 (70%). Maldi (ESI+) was calculated for C ₂₇₁ H ₃₀₉ N ₇ O ₇₅ P ₂ ⁺ [M] ⁺ 4926.3745, 4926.323 was found.

Synthesis of compound 92.

The procedure described for the synthesis of compound 61 was used for the synthesis of compound 98 (89%).

- 123 046159

The procedure described for the synthesis of compound 54 was used for the synthesis of compound 92 (60%). MSHR (ESI+) was calculated for C^H^VICNI [Db/KM NH4-2H)/2] ^- 1247.8944, 1247.8791 was found.

A.8 Synthesis of hexasaccharide 112.

Synthesis of compound 99.

MsCl and pyridine (py) were added to a pure solution of the compound in DCM at 0°C. The reaction mixture was stirred at room temperature overnight and then diluted with DCM, washed with aq. NaHCO3 solution, dried over Na2SO4 and concentrated to give the crude product. The residue was purified by automated silica gel chromatography (hexane/AcOEt) to give compound 99.

Synthesis of compounds 100 and 101.

Sodium iodide was added to a pure solution of compound 99 in 2-butanone and the reaction mixture was stirred at 100°C overnight. Then, the solvent was removed and the crude residue was dissolved in DCM, washed with aq. NaISO ₃ , dried over Na ₂ SO ₄ and concentrated to obtain iodomethyl derivative 100. This iodine derivative was dissolved in freshly distilled trimethylphosphite and the solution was heated to 100°C in vacuum (water pump) for 48 hours. After concentration and chromatography on silica gel, the phosphonate derivative was obtained 101.

Synthesis of compound 102.

TEA and thiophenol were added to a pure solution of compound 101 in THF. The reaction mixture was stirred at room temperature for 24 hours. After complete consumption of the starting material, the reaction mixture was diluted with TEA and concentrated to give a crude residue, and purified by silica gel chromatography to give 102.

Synthesis of compound 103.

Phosphonate 102, linker and triphenylphosphine were dissolved in THF and the solution was cooled to 0°C and DIAD was added thereto. The mixture was stirred at room temperature for 24 hours. After 24 hours, the solution was concentrated and the crude product was purified by silica gel chromatography to give 103.

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Synthesis of compound 104.

TEA and thiophenol were added to a pure solution of compound 103 in THF. The reaction mixture was stirred at room temperature for 24 hours. After complete consumption of the starting material, the reaction mixture was diluted with TEA and concentrated to give a crude residue, and purified by silica gel chromatography to give 104.

Synthesis of compound 105.

VPO

104

Phosphonate derivative 104 was dissolved in a 0.05 M solution of NaOMe in MeOH and stirred at RT for 10 min. The reaction mixture was then quenched with AcOH and the solvents were removed in vacuo. The resulting crude residue was purified by silica gel chromatography to give compound 105.

Synthesis of compound 106.

The reaction was carried out in accordance with the synthesis of compound 16. Synthesis of compound 107.

The reaction was carried out in accordance with the synthesis of compound 18.

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Synthesis of compound 108.

The reaction was carried out in accordance with the synthesis of compound 19. Synthesis of compound 109.

The reaction was carried out in accordance with the synthesis of compound 28. Synthesis of compound 110.

The reaction was carried out in accordance with the synthesis of compound 29. Synthesis of compound 111.

110

The reaction was carried out in accordance with the synthesis of compound 30.

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Synthesis of compound 112.

The reaction was carried out in accordance with the synthesis of compound 33. Conjugation of compound 112 with SIM ₁₉₇ or BSA.

The reaction was carried out according to the conjugation of compound 33.

A.8 Synthesis of hexasaccharide 117.

Synthesis of compound 116.

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min.

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After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I ₂ in pyridine:H ₂ O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to obtain the desired product 116 as a viscous liquid.

Synthesis of compound 117.

The reaction was carried out in accordance with the synthesis of compound 31. Conjugation of compound 117 with CRM ₁₉₇ or BSA

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The reaction was carried out according to the conjugation of compound 33.

A.9 Synthesis of hexasaccharide 122.

Synthesis of compound 121.

1. PivCI,HO'

N ₃

2. I ₂ py, water + p<°

NEt ₃ O N

R'°

NEt ₃ OV

N ₃

121

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was kept under stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I2 in pyridine: _H2O (20:1) was added and the reaction mixture was kept under stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 121 as a viscous liquid.

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Synthesis of compound 122.

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step. Conjugation of compound 122 to CRM ₁₉₇ or BSA.

The reaction was carried out according to the conjugation of compound 33.

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A.10 Synthesis of hexasaccharide 127.

Synthesis of compound 126.

2 h, RU, water ^L 3'

1. PivCI, HO-(CH ₂ ) ₁₀ - _N3

126

(CH ₂ )iq—N ₃

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was kept under stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I2 in pyridine: _H2O (20:1) was added and the reaction mixture was kept under stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to obtain the desired product 126 as a viscous liquid.

Synthesis of compound 127.

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step.

- 131 046159

Conjugation of compound 127 to CRM ₁₉₇ or BSA.

The reaction was carried out according to the conjugation of compound 33.

A.11 Synthesis of hexasaccharide 132.

Synthesis of compound 131.

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C,

- 132 046159 a freshly prepared solution of I2 in pyridine:H2O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 131 as a viscous liquid.

Synthesis of compound 132.

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step. Conjugation of compound 132 to CRM197 or BSA.

The reaction was carried out according to the conjugation of compound 33.

- 133 046159

A.12 Synthesis of hexasaccharide 137.

Synthesis of compound 136.

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I ₂ in pyridine:H ₂ O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 136 as a viscous liquid.

Synthesis of compound 137.

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step.

- 134 046159

Conjugation of compound 137 to CRM ₁₉₇ or BSA.

CRM197 or VBA, NaPi, pH 7

R-NH

The reaction was carried out according to the conjugation of compound 33.

A.13 Synthesis of hexasaccharide 142.

Synthesis of compound 141.

139 R=CRM197

140 R= BSA

Et ₃ N DMSO, H ₂ O, RT, 3 h di-N-hydroxy-succinimidyl adipate ester

2. 1 ₂ , RU, water

NEt ₃ O Н _n ^(CH ₂ ) ₃ -n ₃ Н

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I ₂ in pyridine:H ₂ O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 141 as a viscous liquid.

- 135 046159

Synthesis of compound 142.

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step. Conjugation of compound 142 to CRM ₁₉₇ or BSA.

CRM197 or BSA, NaPi, pH 7

The reaction was carried out according to the conjugation of compound 33.

144R=CRM197

145 R= BSA di-I-hydroxy-succinimidyl adipate ester

Et ₃ N DMSO, H ₂ O, RT, 3 h

- 136 046159

A.14 Synthesis of hexasaccharide 147.

Synthesis of compound 146.

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I ₂ in pyridine:H ₂ O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to obtain the desired product 146 as a viscous liquid.

Synthesis of compound 142.

1. H ₂ Pd/C, EtOAc, MeOH H ₂ b, AcOH, 46 h

146 ---------------------2. 2 M LiOH, MeOH.3H

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step.

- 137 046159

Conjugation of compound 147 with SCM ₁₉₇ or BSA.

The reaction was carried out according to the conjugation of compound 33.

A.15 Synthesis of hexasaccharide 152.

Synthesis of compound 151.

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min.

- 138 046159

After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I ₂ in pyridine:H ₂ O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to give the desired product 151 as a viscous liquid.

Synthesis of compound 152.

1. H ₂ Pd/C, EtOAc, MeOH H ₂ b, AcOH, 46 h

151 -----------------------2. 2 M LiOH, MeOH.3 h

SH

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step. Conjugation of compound 152 to CRM ₁₉₇ or BSA.

SBAP (Nsuccinimidyl-3-(bromoacetamido)propionate) was added to a stirred solution of protein in sodium phosphate buffer (NaPi, pH 7.4) at room temperature. The reaction mixture was stirred for one hour at room temperature and then concentrated by membrane filtration and rebuffered in NaPi (pH 8.0). A solution of compound 152 in NaPi was added to the activated protein solution and stirred at RT for 16 hours. The glycoconjugate was then washed with sterile water and treated with l-cysteine in sterile water. Purification of the glycoconjugate was achieved by membrane filtration.

- 139 046159

A.16 Synthesis of hexasaccharide 157.

Synthesis of compound 156.

H-phosphonate 58 and the linker were evaporated together with pyridine and dried in vacuum for 30 min. After this, the resulting mixture was dissolved in pyridine and PivCl was added to this. The reaction mixture was maintained with stirring at RT for 2 hours. After 2 hours, the reaction mixture was cooled to -40°C, a freshly prepared solution of I ₂ in pyridine:H ₂ O (20:1) was added and the reaction mixture was maintained with stirring at the same temperature for 1.5 h and then brought to RT and stirred at RT for 15 min. Then, TEAB (10 ml) was added to the mixture and diluted with dichloromethane, washed successively with 10% aq. sodium thiosulfate solution, 1 M aq. solution of triethylammonium bicarbonate (TEAB), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by automated flash column chromatography (ethyl acetate:DCM:MeOH) along with 2% trimethylamine as eluents to obtain the desired product 156 as a viscous liquid.

Synthesis of compound 157.

The reaction was carried out according to the synthesis of compound 33 and the TBS deprotection step.

- 140 046159

Conjugation of compound 157 to CRM ₁₉₇ or BSA.

CRM197 or BSA, NaPi, pH 7

The reaction was carried out according to the conjugation of compound 33.

A.17 Synthesis of octadecasaccharides 162, 163, 164 and 165.

Synthesis of compound 161.

159 R=CRM197

160 R= BSA di-LH-hydroxy-succinimidyl adipate ester

Et ₃ N DMSO, H ₂ O, RT, 3 h

1. СROгкрХРгъ ОПп

m®

If Ν ^{DXM Ν} 'Ν

2.89

Η

Ιί ^Ν · ^ν -ν

3. ^f BuOOH

- 141 046159

AcO DEVP TBDPSO'A l L\ η < VpSG-\^° _x AcO AcHN ^BnO BnO^ BnO'A'Tl?'BnO-b-T' AcO o BnO-P-0--~ 1 /° AcO ,OBn AcO AcHN ^Bn0 £ BnO-^ Bno-^vti4 ΒηΟ- ^ν *'Τ^ AcO ⁰ '----- BnO -PO---- 1 z° ^Ac 9 / ^ORP OE Acb AcHN BnO— Bgo^e^. AcO ,OBn ^-0 BnOl _D _ 0 .OBn VnO-\ ^P | LVo Βηθ^ν4θ\ NHAc_____--- □Bn -0 Jnol O ^OBn BnO-y ¹ ? ΙΔ_ο BnO'''vM?\ NHAc_______--- in 0 ^OBn BnO—y? LVo Bno-vj49. ORP NHAc < A 0 161

N ₃

The procedure described for the synthesis of compound 32 was used for the synthesis of compound 161, here only with the change that in the second step, instead of the linker, compound 89 was used as a nucleophile.

Synthesis of compound 162.

where Z represents

Compound 162 was synthesized from compound 161 as described for compound 89 (removal of the TBDPS protecting group) and then as described for compound 90.

- 142 046159

Synthesis of compound 163.

where Z represents

Compound 163 was synthesized from compound 161 as described for compound 89 (removal of the TBDPS protecting group) and then as described for compounds 91 and 92.

Synthesis of compound 164.

where Z represents

Phosphonate compound 164 was synthesized as described for compound 162.

Synthesis of compound 165.

where Z represents

Phosphonate compound 165 was synthesized as described for compound 163.

- 143 046159

A.18 Alternative synthesis of octadecasaccharides 162 and 163.

Synthesis of compound 166.

2. Et ₃ NNaHCO ₃ . CT, 2 hours

166

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 166.

Synthesis of compound 167.

1.166, PivCI, ru

2. 1 ₂ , ru, N ₂ O

167

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 167.

Synthesis of compound 162.

- 144 046159

Compound 162 was synthesized from compound 167 as described for compound 33.

Synthesis of compound 168.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 168.

Synthesis of compound 169.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 169.

Synthesis of compound 170.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 170.

Synthesis of compound 163.

Compound 163 was synthesized from compound 170 as described for compound 54.

- 145 046159

A.19 Synthesis of tetracosaccharides 172 and 173.

Synthesis of compound 172.

where Z represents

Compound 172 was synthesized from dodecasaccharide 89, which was attached to dodecasaccharide 171

TBDPSO^v „

BnO^VSS o AcO-^-T^ ⁰

ASO

it according to the procedure described for compound 88, followed by deprotection of the TBDPS group as described for compound 89, and subsequently complete deprotection as described for compound 90.

Synthesis of compound 173.

Compound 173 was synthesized from dodecasaccharide 89, which was coupled to dodecasaccharide 171, according to the procedure described for compound 88, followed by deprotection of the TBDPS group as described for compound 89, phosphorylation as described for compound 91, and subsequently complete deprotection. as described for connection 92.

- 146 046159

A.20 Alternative synthesis of tetracosaccharides 172 and 173.

Synthesis of compound 174.

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 174.

Synthesis of compound 172.

Compound 172 was synthesized from compound 174 as described for compound 33.

Synthesis of compound 175.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 175.

- 147 046159

Synthesis of compound 176.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 176.

Synthesis of compound 177.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 177.

Synthesis of compound 173.

Compound 173 was synthesized from compound 177 as described for compound 54.

- 148 046159

A.21 Synthesis of triacontasaccharides 179 and 183.

Synthesis of compound 178.

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 178. Synthesis of compound 179.

nh ₂

179

Compound 179 was synthesized from compound 178 as described for compound 33.

Synthesis of compound 180.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 180.

- 149 046159

Synthesis of compound 181.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 181.

Synthesis of compound 182.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 182.

Synthesis of compound 183.

Compound 183 was synthesized from compound 182 as described for compound 54.

A.22 Synthesis of hexatriacontasaccharides 186 and 187.

Synthesis of compound 185.

Compound 185 was synthesized from octadecasaccharide 161, which was selectively deprotected with TBDPS according to the procedure described for compound 89. The deprotected trisaccharide with TBDPS was then reacted with compound 184

- 150 046159

VPO

VPO BnO

OH

184 to obtain saccharide 185.

Synthesis of compound 186.

where Z represents ^u

Compound 186 was synthesized from saccharide 185, which was converted according to the procedures described for compound 89 (TBDPS deprotection) and then for compound 90 (TBDPS deprotection).

- 151 046159

Synthesis of compound 187.

Compound 187 was synthesized from saccharide 185, which was converted following the procedures described for compound 89 (TBDPS deprotection), phosphorylation as described for compound 91, and subsequently complete deprotection as described for compound 92.

A.23 Alternative synthesis of hexatriacontasaccharides 186 and 187.

Synthesis of compound 188.

NHAc

ORP HPO

About VPO

ORP HPO

About VPO

1. 181, PivCl, ru

2. I ₂ RU, N ₂ O

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 188. Synthesis of compound 186.

- 152 046159

Compound 186 was synthesized from compound 188 as described for compound 33.

Synthesis of compound 189.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 189.

Synthesis of compound 190.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 190.

Synthesis of compound 191.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 191.

Synthesis of compound 187.

Compound 187 was synthesized from compound

191, as described for connection 54.

- 153 046159

A.24 Synthesis of oligosaccharides 193 and 197.

Synthesis of compound 192.

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 192.

Synthesis of compound 193.

Connection 193

192, as described for compound 33.

synthesized from a compound

Synthesis of compound 194.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 194.

- 154 046159

Synthesis of compound 195.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 195.

Synthesis of compound 196.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 196. Synthesis of compound 197.

Compound 197 was synthesized from compound 196 as described for compound 54.

- 155 046159

A.25 Synthesis of oligosaccharides 199 and 203.

Synthesis of compound 198.

198

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 198.

Synthesis of compound 199.

198, as described for compound 33.

Compound 199 was synthesized from compound Synthesis of compound 200.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 200.

- 156 046159

Synthesis of compound 201.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 201.

Synthesis of compound 202.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 202.

Synthesis of compound 203.

Compound 203 was synthesized from compound 202 as described for compound 54.

A.26 Synthesis of oligosaccharides 205 and 209.

Synthesis of compound 204.

- 157 046159

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 204.

Synthesis of compound 205.

205

Compound 205 was synthesized from compound 204 as described for compound 33.

Synthesis of compound 206.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 206.

Synthesis of compound 207.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 207.

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Synthesis of compound 208.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 208. Synthesis of compound 209.

Compound 209 was synthesized from compound 208 as described for compound 54.

A.27 Synthesis of oligosaccharides 211 and 215.

Synthesis of compound 210.

210

The procedure described for the synthesis of compound 96 was used for the synthesis of compound 210.

- 159 046159

Synthesis of compound 211.

Compound 211 was synthesized from compound 210 as described for compound 33.

Synthesis of compound 212.

The procedure described for the synthesis of compound 60 was used for the synthesis of compound 212. Synthesis of compound 213.

The procedure described for the synthesis of compound 86 was used for the synthesis of compound 213.

Synthesis of compound 214.

The procedure described for the synthesis of compound 87 was used for the synthesis of compound 214.

- 160 046159

Synthesis of compound 215.

Compound 215 was synthesized from compound 214 as described for compound 54.

B. Stability studies.

Cleavage of the phosphate bond in compound 33 using NaOH.

Next, the stability of the compounds of the present invention was tested and evaluated. The objective was to find out how stable compounds 33, 54, 90, 92, 112, 117, 162, 163, 164, 165, 172 and 173 are under formulation conditions. To obtain stability in Alhydrogel, PBS buffer and water, compound 33 was treated with 0.1 M sodium hydroxide at room temperature. In this case, compound 33 was found to degrade very slowly only under very basic conditions. However, even after 4 days (10 μg of compound 33 per 200 μl), only 50% of compound 33 was degraded under these stringent conditions and 50% of compound 33 was still observed to remain intact in the HPLC chromatograms (Figures 6 and 7).

Stability of Compound 33 in Alhydrogel in PBS, PBS and Water: The stability of Compound 33 under formulation conditions was further examined. Each formulation bottle contains 30 μg of compound 33 β:ϊ) Alhydrogel in PBS or ii) PBS alone or iii) water (total volume of solution is 500 μl). NaPi is used here as a synonym for PBS. For each experiment, 60 μl of Alhydrogel containing 0.6 mg of aluminum was used. These three prepared solutions were kept at 37°C, 25°C and 2-8°C for 14 days.

Every 24 hours, 50 μl of a solution from each vessel of i) Alhydrogel in PBS, ii) PBS only and w) water at 37°C, 25°C and 2-8°C was aliquoted and analyzed by HPLC (Fig. 8, 9, 10). From these studies, it is evident that compound 33 is stable over the entire temperature range from 2°C to 37°C. In fig. 8 shows stability at 2-8°C after 4 days, FIG. 9 at 2-8°C after 14 days, Fig. 10 at 25°C after 4 days, Fig. 11 at 25°C after 14 days, Fig. 12 at 37°C after 4 days, Fig. 13 at 37°C after 14 days.

In comparison to the natural PSII polysaccharide Clostridium difficile, compounds 33, 54, 90, 92, 112, 117, 162, 163, 164, 165, 172 and 173 were found to be quite stable under the formulation conditions described above.

It has also been discovered that the natural PSII polysaccharide of Clostridium difficile, consisting of hexaglycosylphosphate repeating units as shown below,

is unstable when treated with NaOH, unstable under acidic conditions such as acetic acid, and unstable in solution at 2-8°C, 25°C and 37°C. It was found that under these conditions, natural PS II rapidly degrades to breakdown products that no longer cause an immunological effect.

Therefore, the stability experiments reported above unequivocally demonstrate that the compounds of the present invention are stable under conditions where native PSII degrades into fragments no longer useful as a vac

- 161 046159 cins, while the compounds described herein are stable in solution and do not require lyophilization and redissolution, cold storage, and do not require production and shipment using an expensive operational cold chain system.

C. Biological experiments.

SDS-PAGE analysis.

The samples were mixed in a microcentrifuge tube and heated for 5 min at 95°C on a thermal cycler. After cooling to room temperature for 5 min, approximately 2.5 μg of samples were loaded into appropriate 10% polyacrylamide gel wells along with 10 μl of marker. Samples were tested at 120 VDC for 1 hour. Staining was performed using GelCode™ Blue Safe Protein according to the manufacturer's instructions. The gels were washed with deionized water overnight and scanned using a gel documentation system.

Size exclusion chromatography (SEC) of glycoconjugates.

Glycoconjugates used for immunization studies were analyzed by SEC to detect the difference in mass between conjugated and unconjugated CRM protein. Samples were diluted in 50 mM Tris, 20 mM NaCl, pH 7.2, and run on an Agilent 1100 HPLC system equipped with a Tosoh TSK G2000 column (SWxl, 7.8 mm x 30 cm, 5 µm) and a Tosoh TSKgel® Guard Column ( SWxl 6.0 mmx4 cm, 7 µm). The flow rate was maintained at 1 ml/min.

Preparation of the glycoconjugate.

Synthetic C. difficile PS-II antigens were conjugated to CRM ₁₉₇ carrier protein for immunization experiments and to bovine serum albumin (BSA) as the coating antigen for ELISA (see A. Chemical Synthesis). The resulting conjugates were sterile filtered using a 0.2 μM membrane filter before use. Conjugates were analyzed using MALDI analysis. The saccharide loading onto the carrier protein was specifically calculated by subtracting the mass between the conjugate and unconjugate protein using MALDI analysis. Protein content was assessed using the micro-BCA method following the production protocol.

Characteristics of glycoconjugates 36 (33-CRM ₁₉₇ ), 56 (54-CRM ₁₉₇ ) and 94 (92-CRM ₁₉₇ ).

C. difficile antigen-glycoconjugates 36, 56, and 94 used for immunization studies were analyzed for conjugation efficiency and antigen content. MALDI-TOF MS analysis of the glycoconjugates revealed good conjugation efficiency. The difference in mass between conjugated and unconjugated CRM ₁₉₇ protein resulted in loading of approximately 7.5 (56) and approximately 5 (94) antigens per CRM ₁₉₇ molecule.

The glycoconjugates were also analyzed by 10% SDS-PAGE and SEC, which revealed a clear mass shift compared to the unconjugated CRM197 protein (Figures 24 and 25).

Immunization research.

Study I

Immunological evaluation of semisynthetic glycoconjugates of C. diff PS-II antigen immunized in rabbits.

1. Purpose of the study.

Evaluation of the IgG antibody response in rabbits immunized with vaccine 36 of the semisynthetic CRM ₁₉₇ conjugate of PS-II C. diff antigen.

2. Materials.

ELISA plates (high binding, EIA/RIA plate, 96-well, flat bottom with lid for low evaporation, company: costar® 3361).

Antibody detection: Goat anti-rabbit IgG peroxidase conjugate (Sigma, #A4914).

Blocking solution: 1% FCS (v/v) in PBS.

Antibody diluent: PBS+1% BSA (w/v).

Wash buffer: PBS+0.1% Tween 20 (PBS-T).

Developing solution: 1 step™ Ultra TMB-ELISA developer. (ThermoScientific, Cat #: 34028).

Stopping solution: 2M sulfuric acid (H2SO4).

Tablet reader: Anthos ht 2.

Software: WinRead 2.36 for optical density measurements and GraphPad Prism 7 for data plotting and analysis.

Incomplete Freund's adjuvant (IFA). InvivoGen; Cat: vac-ifa-10, Batch#: IFA-39-03; Exp Dt: Sept 2019.

QuantiPro™ BCA Assay Kit (SIGMA) Product: QPBCA-1KT; Lot#: SLBR7451V; Pcode: 1002296464.

3. Methods.

Preparation of vaccines for immunization.

C. difficile PS-II glycoconjugate 36 was formulated as incomplete Freund's adjuvant (IFA) for immunization of rabbits. Invivogen's Incomplete Freund's Adjuvant (IFA) was used to develop vaccines for rabbit immunization studies. The protocol was followed in accordance with production

- 162 046159 vom. Antigen: IFA concentration was maintained at a 1:1 ratio. The antigen dose per animal was maintained at 2.5 μg/200 μl/animal (100 μl antigen + 100 μl IFA). IFA at the desired calculated volume (50% of the final immunization volume) was added to a 15 ml sterile vial. The calculated amount of diluted antigen solution (volume adjusted with PBS to 50% of the final immunization volume) was added to a 3 ml sterile syringe equipped with a 20 G needle. DS solution was added to the vial containing IFA and immediately shaken for 15 s (5x). The color of the composition changes from pale yellow to milky white when shaken, indicating the formation of a stable emulsion. The resulting vaccine formulation was briefly shaken and aliquoted into 2 ml sterile tubes containing the desired dose volumes. Before immunization, the tubes containing the vaccine formulations were shaken and then administered to the animals.

Immunization schedule.

Rabbits were immunized under specific pathogen-free conditions and provided with food and water ad libitum. Rabbits (n=4) were immunized subcutaneously with vaccine formulations at an injection volume of 200 μl/rabbit. The rabbit antigen dose was maintained at 2.5 μg/animal PS-II antigen or the corresponding volume of PBS for the negative control. Rabbits were immunized on days 0, 14 and 35. On days 0, 7 and 42, blood was drawn to determine antibody titers.

4. Enzyme-linked immunosorbent assay (ELISA) of sera using tablets coated with in-house produced antigen.

Antigen coating of plates.

Antigen-BSA conjugates were used as the coating antigen. Antigen-BSA conjugates were dissolved at a concentration of 5 μg/ml in phosphate-buffered saline (PBS) at pH 7.4. Coat 100 μl per well and incubate overnight at 4°C to obtain an antigen concentration of 0.5 μg/well.

Flushing.

After antigen adsorption overnight, the plates were washed once with PBS-T (200 μl/well) and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry cloth towel.

Blocking.

The plates were blocked using 200 μl of commercial blocking solution and incubated for 2 h at RT.

Flushing.

After blocking, the plates were washed 3 times with PBS-T (200 μl/well) and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry tissue towel.

Serum dilution and incubation.

Mixed sera (n=4 rabbits) from different time points of different experimental groups were diluted to their respective dilutions in antibody diluent (PBS+1% BSA). 100 μl of diluted serum samples from different experimental groups were added in duplicate to the corresponding wells and incubated on a shaker at 250 rpm for 2 h at RT. 100 μl/well of antibody diluent (PBS+1% BSA) constituted the experiment blank. After incubation with serum, the plates were washed 4 times with PBS-T (200 μl/well), and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry cloth towel.

Incubation (detection of antibodies).

The appropriate detection antibody, HRP anti-rabbit IgG conjugate, was diluted 1:10,000 in antibody diluent (PBS+1% BSA) and 100 μl/well was added and incubated on a shaker at 250 rpm for 1 h at RT. After incubation with detection antibodies, the plates were washed 5 times with PBS-T (200 μl/well), and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry cloth towel.

Adding substrate.

100 μl of ready-to-use TMB substrate (normalized to RT 4°C) was added to each well and incubated in the dark for 15 min. The blue color of the enzymatic reaction was stopped by adding 50 μl/well of a 2M H ₂ SO ₄ solution, resulting in a yellow solution. The absorbance of the yellow-colored solution was measured at 450 nm using a plate reader.

Results.

Absorbance values were analyzed by plotting using Graphpad Prism software.

The ELISA data clearly show that sera from C. difficile PS-II conjugate-36 immunized rabbits recognize the corresponding antigens (see FIG. 15).

Study II.

Immunological evaluation of semisynthetic glycoconjugates of C. diff PS-II antigen immunized in rabbits and mice.

1. Purpose of the study.

Evaluation of IgG antibody response in rabbits and mice immunized with 56 and 94 semisynthe vaccines

- 163 046159 tic CRM ₁₉₇ PS-II antigen conjugate C. diff.

2. Materials.

ELISA plates (high binding, EIA/RIA plate, 96-well, flat bottom with lid for low evaporation, company: costar® 3361).

Antibody detection: peroxidase conjugate with goat anti-rabbit IgG (Sigma, #A4914) and anti-human IgG (H+L) -HRP, Nordic Immunology, Lot#:6276.

Blocking solution: Roche, Ref: 11112589001; Lot: 21495200, Exp.Dt: July 2019.

Antibody diluent: PBS+1% BSA (w/v).

Wash buffer: PBS+0.1% Tween 20 (PBS-T).

Developing solution: 1 step™ Ultra TMB-ELISA developer. (ThermoScientific, Cat #: 34028).

Stopping solution: 2M sulfuric acid (H ₂ SO ₄ ).

Tablet reader: Anthos ht 2.

Software: WinRead 2.36 for absorbance measurements and GraphPad Prism 7 for data plotting and analysis.

lum: aluminum hydroxide gel adjuvant (Alhydrogel 2%), Brenntag, Batch #:5447Exp Dt:Feb2020.

QuantiPro™ BCA Assay Kit (SIGMA) Product: QPBCA-1KT; Lot#: SLBR7451V; Pcode: 1002296464.

Mini-PROTEAN® TGX™ Gels - 10%, 10 wells (30 µl/well) Control No.: 64175708.

Precision Plus Dual Color, Cat: 1610374; Control No.: 641798899.

Gel Code™ Blue Safe Protein Stain; ThermoScientific; Ref: 1860957; Lot#: TA260266.

Cdiff coated ELISA plates for strains 630 (tgc BIOMICS Lot#: 630-43411) and R20291 (tgc BIOMICS Lot#: R20291-43559) Exp.Dt: 05/2020.

Patient's C.diff-positive plasma.

3. Methods.

Formulation of vaccines for immunization in aluminum hydroxide (Alum) adjuvant.

All formulations were prepared under sterile conditions. Glycoconjugates 56 and 94 (drug substances; DS) and PBS were mixed at the appropriate pre-calculated ratio in a 50 mL Falcon™ tube, which corresponds to the final formulation volume, excluding the required volume of Alum (0.25 mg/mL). This formed a DS-PBS mixture. Antigen/DS dose per animal was maintained at 2.5 μg/500 μl/animal or 10 μg/500 μl/animal (rabbit studies) or 0.5 μg/100 μl/animal or 2 μg/100 μl/animal (mice studies). The DS-PBS mixtures were mixed gently (5x) using a serological pipette. An appropriate volume of original Alum (10 mg/mL) was added to the DS-PBS mixtures to obtain a final Alum ratio of 1:40 or 0.250 mg/mL. The mixtures were immediately mixed by gentle pipetting (20x) using a 5 ml serological pipette. Falcon™ tubes were capped, wrapped in Parafilm® and allowed to mix on a shaker at 250 rpm for 2 h at room temperature (RT). After incubation for 2 h, the formulations were placed on a clean table, aliquoted, and stored at 4°C until further use. Glycoconjugates contained in Alum were characterized to determine the final concentration of Alum and the pH of the formulations.

Immunization schedule.

Mice and rabbits were immunized under specific pathogen-free conditions and provided with food and water ad libitum. Mice (n=7 or 8 per test group) and rabbits (n=4 per test group) were immunized subcutaneously with vaccine formulations at an injection volume of 100 μl/mouse and 500 μl/rabbit with different doses of antigen. Mice were immunized on days 0, 14, and 28, and blood was collected on days 21 and 35. Rabbits were immunized on days 0, 14, 28, and 77, and blood was collected on days 0, 7, 21, 35, 77, and 84. Serum was prepared from blood samples for serum antibody testing.

4. Enzyme-linked immunosorbent assay (ELISA) of sera using tablets coated with in-house produced antigen.

Antigen coating of plates.

54-BSA and 92-BSA conjugates were used as coating antigens. The corresponding conjugates were diluted to a concentration of 5 μg/ml in phosphate-buffered saline (PBS), pH 7.4. Coat 100 μl per well and incubate overnight at 4°C to obtain an antigen concentration of 0.5 μg/well. To coat the isolated PS-II polysaccharide, the polysaccharide was diluted to 50 μg/mL in PBS with 10 mM imidazole and 100 μL per well was applied at 50°C for 5 h.

Flushing.

After antigen adsorption, the plates were washed once with PBS-T (200 μl/well) and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry cloth towel.

Blocking.

The plates were blocked using 200 μl of commercial blocking solution and incubated for 2 h at RT.

- 164 046159

Flushing.

After blocking, the plates were washed 3 times with PBS-T (200 μl/well) and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry tissue towel.

Serum dilution and incubation.

Mixed sera (n=4 rabbits or n=7-8 mice/group) from different time points of different experimental groups were diluted to their respective dilutions in antibody diluent (PBS+1% BSA). 100 μl of diluted serum samples from different experimental groups were added in duplicate to the corresponding wells and incubated on a shaker at 250 rpm for 2 h at RT. For competition ELISA experiments, diluted sera were incubated on ice for 30 min with 10 or 50 μg of isolated PS-II polysaccharide or PBS before adding to ELISA plates. 100 μl/well of antibody diluent (PBS+1% BSA) constituted the experiment blank. After incubation with serum, the plates were washed 4 times with PBS-T (200 μl/well), and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry cloth towel.

Incubation with detection of antibodies.

The appropriate detection antibody, HRP conjugate with anti-rabbit or anti-mouse IgG HRP was diluted 1:10000 in antibody diluent (PBS + 1% BSA) and added 100 μl/well and incubated on a shaker at 250 rpm for 30 min at CT. After incubation with detection antibodies, the plates were washed 5 times with PBS-T (200 μl/well), and excess liquid per well was removed by inverting the plate and tapping it on a clean, dry cloth towel.

Adding substrate.

100 μl of ready-to-use TMB (3,3',5,5'-tetramethylbenzidine) substrate (normalized to RT 4°C) was added to each well and incubated in the dark for 15 min. The blue color of the enzymatic reaction was stopped by adding 50 μl/well of a 2M H ₂ SO ₄ solution, resulting in a yellow solution. The absorbance of the yellow-colored solution was measured at 450 nm using a plate reader.

Results.

Absorbance values were analyzed by plotting using Graphpad Prism software.

5. Enzyme-linked immunosorbent assay (ELISA) of sera using commercial pre-coated plates.

This procedure was identical to the ELISA protocol described above, except that the coating step was omitted.

Results.

Serum IgG from immunized rabbits recognizes the immunogen (FIG. 20), the isolated PS-II polysaccharide (FIG. 19B and 19C), and C. difficile strains 630 (FIG. 16 and 17), R20291 (FIG. 18), and VPI10463 (FIG. 19A). Serum IgG from immunized mice recognized the corresponding immunogens (Fig. 23) and C. difficile strains 630 (Fig. 21) and R20291 (Fig. 22).

The data presented herein demonstrate that, following immunization with a conjugate of the present invention, particularly conjugates 56 and 94, functional antibodies are directed to the oligosaccharides of the present invention, as well as to the natural C. difficile PS-II polysaccharide isolated and on the surface of the bacterium , have been identified in rabbits and mice. These data indicate the ability of these antibodies to provide protection against C. difficile infections.

The ELISA data further demonstrates that the conjugates of the present invention are immunogenic and induce high antibody titers. Therefore, ELISA analysis shows that the saccharides according to the present invention are immunogenic in rabbits and mice and generate cross-reactive antibodies.

Claims (25)

CLAIM 1. Saccharide of general formula (I) where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; T*- represents -P(=O)(OH) ₂ , -P(=O)(O ^- )(OH) or -POz2-; I °' ; Z represents ' E represents -NH2, -N3, -CN, -O-NH ₂ , -CH=CH2, -C=CH, -Br, -Cl, -I, -CO2R', -CONH-NH2. -SH, -OH or -SAc; R' is -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl or N-succinimidyl; and where - L- represents -L ^a -, -L ^a -L ^e -, -L ^a -L ^b -L ^e - or -L ^a -L ^d -L ^e -; - L ^a - represents -(CH ₂ ) _o -, -(CH ₂ -CH ₂ -O) _o -C ₂ H ₄ - or -(CH ₂ -CH ₂ -O) _o -CH ₂ ; - L ^b - represents -O-; - L ^d - represents -(CH ₂ ) _q -, -(CH(OH)) _q -, -(CF ₂ ) _q -, -(CH ₂ -CH ₂ -O) _q -C ₂ H4- or - (CH2-CH2-O)q-CH2-; - L ^e - represents -(CH2)pi-, -(CF2)p1-, -C2H4-(O-CH2-CH2)pi-, -CH2-(O-CH2-CH2)pi- or -( _CH2 )p1-O-(CH ₂ )p ₂ -; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6; or a pharmaceutically acceptable salt thereof. 2. The saccharide according to claim 1, wherein -L- is -(CH ₂ ) _o - and o is an integer selected from 1, 2, 3, 4, 5 and 6, or a pharmaceutically acceptable salt thereof. 3. A saccharide according to claim 1 or 2, where E represents an amino group, or a pharmaceutically acceptable salt thereof. 4. The saccharide according to claim 1, where the group -O-L-E is selected from the group consisting of: where R' represents -H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl or N-succinimidyl; X is -Br, -Cl, -I, -CO2H, -CN, -NO2 or -SAc, or a pharmaceutically acceptable salt thereof. 5. A saccharide according to claim 1, selected from the group consisting of: - 166 046159 - 167 046159 - 168 046159 - 169 046159 - 170 046159 - 171 046159 - 172 046159 - 173 046159 - 174 046159 where Z is or a pharmaceutically acceptable salt thereof. 6. Saccharide according to claim 5 of formula (I'a-4) or formula (I'b-4), - 175 046159 where Z is or a pharmaceutically acceptable salt thereof. 7. A saccharide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the pharmaceutically acceptable salt is a sodium salt. 8. A conjugate which contains a saccharide according to any one of claims 1 to 7, covalently linked to an immunogenic carrier through the E residue of group -O-L-E, and where the immunogenic carrier is a protein carrier selected from the group consisting of diphtheria toxoid, mutated diphtheria toxoid, modified diphtheria toxoid, mutated and modified diphtheria toxoid, tetanus toxoid, modified tetanus toxoid, mutated tetanus toxoid, non-lipidated cell surface lipoprotein (protein D) of non-typeable Haemophilus influenzae, outer membrane protein (OMP), Neisseria meningitidis complex, bovine serum albumin (BSA), fissurella hemocyanin (KLH) or cholera toxoid (CT), or a pharmaceutically acceptable salt thereof. 9. Conjugate of general formula (IV) where c is in the range between 2 and 18; -E1- represents a covalent bond, -NH-, -O-NH-, -O-, -S-, -CO-, -CH=CH- -CONH-, -CO-NHNH-, -W- selected from: - 176 046159 a is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, b is an integer selected from 1, 2, 3 and 4, n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; T*- represents -P(=O)(OH) ₂ , -P(=O)(O ^- )(OH) or -PO ₃ ^2- ; Z represents - L- represents -L ^a -, -L ^a -L ^e -, -L ^a -L ^b -L ^e - or -L ^a -L ^d -L ^e -; - L ^a - represents -(SShch)o-, -(SShch-SShch-O^Shch- or -(SShch-SShch-O^-SShch; - L ^b - represents -O-; - L ^d - represents -(CH ₂ ) _q -, -(CH(OH)) _q -, -(CF2) _q -, -(CH ₂ -CH ₂ -O) _q -C ₂ H ₄ - or - (CH ₂ -CH ₂ -O) _q -CH ₂ -; - L ^e - represents -(CH2)p1-, -(CF2)p1-, -C2H4-(O-CH2-CH2)p1-, -СШ-^-СШ-СШ^- or -(CH2)p1- O-(CH2)p2-; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5 and 6; And CP means a carrier protein selected from the group consisting of diphtheria toxoid, mutated diphtheria toxoid, modified diphtheria toxoid, mutated and modified diphtheria toxoid, tetanus toxoid, modified tetanus toxoid, mutated tetanus toxoid, non-lipidated cell surface lipoprotein (protein D) of non-typeable Haemophilus influenzae, outer membrane protein (OMP), Neisseria meningitidis complex, bovine serum albumin (BSA), fissurella hemocyanin (KLH) or cholera toxoid (CT), or a pharmaceutically acceptable salt thereof. 10. The conjugate according to claim 8 or 9, wherein the carrier protein is a cross-reacting material 197. 11. The conjugate according to claim 9, which has the following formula (V): Where T*- represents -P(=O)(OH) ₂ , -P(=O)(O ^- )(OH) or -PO ₃ ^2- ; n is an integer selected from 1, 2 or 3; -L- is -(CH ₂ ) _o -, where o is an integer selected from 2, 3, 4, 5 and 6; And -W- is a is an integer selected from 2, 3, 4, 5 and 6; or a pharmaceutically acceptable salt thereof. 12. Conjugate according to claim 11, where - 177 046159 E1 is a covalent bond, -NH-, -CH=CH-, -CONH-, or a pharmaceutically acceptable salt thereof. 13. The conjugate according to claim 9, which has the following formula (V-2): where n is an integer from 1-3; c is in the range between 2 and 18; -W- represents and a is an integer selected from 2, 3, 4, 5 and 6, or a pharmaceutically acceptable salt thereof. 14. The conjugate according to claim 9 or claims 11-13, which has the following formula (V-2): Where L represents -(CH ₂ ) ₅ -, E1 is -NH-, n is an integer selected from 1 or 2, or a pharmaceutically acceptable salt thereof. 15. A conjugate which has the following formula (V-2): - 178 046159 Where L is -(CH ₂ ) ₅ -, E1 is -NH-, n is 1, And -W- is a is 4, and c is in the range between 2 and 18; or a pharmaceutically acceptable salt thereof. 16. A conjugate which has the following formula (V-2): Where L is -(CH ₂ ) ₅ -, E1 is -NH-, n is 2, -W- is a is 4, and c is in the range between 2 and 18; or a pharmaceutically acceptable salt thereof. 17. A conjugate according to any one of claims 9 to 16, wherein c is in the range between 5 and 15, or a pharmaceutically acceptable salt thereof. 18. A conjugate according to any one of claims 9 to 16, where c is selected from 4 to 10, or a pharmaceutically acceptable salt thereof. 19. Conjugate formula - 179 046159 where R represents cross-reacting material 197, or a pharmaceutically acceptable salt thereof. 20. Use of a conjugate according to any one of claims 8 to 19 to enhance a protective immune response in a human and/or animal host. 21. Use of a conjugate according to any one of claims 8 to 19 for the prevention and/or treatment of diseases associated with bacteria containing in their cell wall polysaccharide one of the following saccharide fragments: - 6)-p-D-Glc-(l, 3)-P-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[p-D-Glc-(l, 3)]-3-D- GalNAc-(1, 3)-a-D-Man-(l-; - 3)-a-D-Man-(l, 6)-p-D-Glc-(l, 3)-p-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-[p-D-Glc-( l, 3)]-p-D-GalNAc-(l; - 4)-[p-D-Glc-(l, 3)]-P-D-GalNAc-(l, 3)-a-D-Man-(l, 6)-p-D-Glc-(l, 3)-p-D-GalNAc- (1, 4)-a-D-Glc-(l; - 4)-a-D-Glc-(l, 4)-|>D-Glc-(l, 3)]-p-D-GalNAc-(l, 3)-a-D-Man-(l, 6)-P-D-Glc -(l, 3)-p-D-GalNAc-(l; and - 3)-p-D-GalNAc-(l, 4)-a-D-Glc-(l, 4)-|>D-Glc-(l, 3)]-p-D-GalNAc-(l, 3)-a-D-Man - (1, 6)-P-D-Glc-(l. 22. Use of a conjugate according to claim 21, wherein the bacterium is Clostridium difficile. 23. A pharmaceutical composition which contains the conjugate according to any one of claims 8 to 19 together with at least one pharmaceutically acceptable adjuvant and/or excipient. 24. An intermediate for the preparation of a conjugate of general formula (V-2), wherein the intermediate is - 180 046159 or a pharmaceutically acceptable salt thereof. 25. The intermediate of claim 24, wherein the pharmaceutically acceptable salt is a sodium salt.