Improved carbohydrate coupling

Abstract

The present invention relates to a compound of formula (I) (G-X-NH) o-Y wherein: G is a glycan comprising n monosaccharide units linked by a glycosidic bond; n is an integer of monosaccharide units connected through a glucosidic bond, preferably n is 2 to 200, more preferably 2 to 100, and most preferably 2 to 20 monosaccharide units; o is an integer of 0 to 10, if Y is a peptide or protein, Y corresponds to an integer of 1 to 10, preferably an integer of 1 to 5, per 10 kDa of peptide or protein; x is based on an aldose, linked via a glycosidic bond to G and wherein the aldehyde group of the aldose has reacted with the primary amino group of the group Y to obtain a product of formula (I) wherein the aldehyde group of the aldose has been reduced in form in formula (I) to the corresponding secondary amino group preferably by reductive amination to obtain the secondary amino group of formula (I); and Y is selected from amino acids, peptides and proteins.

Description

Technical Field

[0001] This invention relates to a compound of formula (I). (GX-NH) o -Y (I) Wherein G is a polysaccharide, which contains 2 to 200, more preferably 2 to 100, and most preferably 2 to 20 monosaccharide units linked by glycosidic bonds; o is an integer from 1 to 10. If Y is a peptide or protein, then it is an integer from 1 to 10 corresponding to each 10 kDa peptide or protein Y, preferably 1 to 5. X is based on an aldose, linked to G via a glycosidic bond, wherein the aldehyde group of the aldose has reacted with the primary amino group of Y to give the product of formula (I), wherein the aldehyde group of the aldose has preferably been reduced to the corresponding secondary amino group in formula (I) by reductive amination to give the secondary amino group in formula (I); and Y is selected from: amino acids, peptides, and proteins. Background Technology

[0002] Immunization using non-protein targets requires covalently binding them to their respective carrier proteins, thereby providing enhanced target molecule half-life and immune stimulation to the recipient organism. Coupled hapten-like structures (such as synthetic carbohydrates) to proteins, typically by random coupling to specific groups (e.g., free SH groups), N-glycosylation sites, or primary amino groups including lysine residue side chains. Coupling with primary amines usually requires the carbohydrate to have a functional group capable of covalently coupling with an amino group. An example is a linker with a primary amine or thiol functional group. This carbohydrate functional group is typically introduced at the reducing end of the carbohydrate at the start of synthesis. The chemical reactions used to generate the covalent bond between the sugar and the carrier protein produce unnatural and, due to the chemical reactions used, often (typically highly) immunogenic structures, such as heterocyclic structures or fatty chains. Especially in the case of low immunogenic targets (such as carbohydrates), this may result in an immune response after vaccination that is predominantly based on the linker used, while the immune response against the specific target may be negligible or nonexistent (see Adamo 2014 for an overview).

[0003] The linker molecule that is frequently used and has high immunogenicity is shown in the figure below. This is obtained by coupling oligosaccharides to a carrier protein: Linker "A": Triazole group Linker "B": Aliphatic group (Wu 2004) Linker "C": Maleimide group (Buskas 2004) Currently, various methods for chemically linking non-protein targets to protein carriers are described in the literature (Berti 2018). However, glycan-protein conjugates for immunization are most commonly obtained by applying "click" chemistry, using maleimide linkers, or other organic moieties. These methods result in the presence of, for example, triazole groups (linker "A"), alkyl chains (linker "B"), or other cyclic moieties (linker "C"), which are potent immunogens due to their hydrophobic and / or rigid structures. A recent review describes other methods for conjugating carbohydrates (Mettu 2020).

[0004] The reductive amination reaction, which couples carbohydrates to carrier proteins, has been used for decades (e.g., as described in Anderson 1985) and is still being optimized in terms of reaction conditions and yields (e.g., see Gildersleeve 2008). This reaction is based on the balance between the cyclic hemiacetal (internal hemiacetal) and the open-chain aldehyde form at the reducing end of the carbohydrate molecule. The latter forms an imine ion (Schiff base) with the terminal amino group on the protein, which is then irreversibly reduced to a secondary amine, as shown in the diagram below. Therefore, the linker consists of an open-chain form of monosaccharide residues present at the reducing end of the oligosaccharide and exhibits very low immunogenicity because this open-chain structure, representing the natural monosaccharide structure, is tolerated by the immune system due to its persistent abundance. This reductive amination chemical reaction is also used in currently commercially available pneumococcal conjugate vaccines, such as Prevnar. ® Or Prevnar 13 ® (Turner 2017) demonstrates the safety and synthetic efficiency of this method. However, since the carbohydrates are isolated from pathogens, they must be functionalized. Therefore, coupling isolated pneumococcal carbohydrates to a carrier protein requires oxidizing the hydroxyl functional group to a carbonyl group via periodate, which is accompanied by ring-opening in a non-directional manner. This oxidative destruction of at least one carbohydrate unit at the reducing end reduces the size of the structure that could potentially generate relevant antibodies, and also generates unrelated new epitopes (Poolman 2011).

[0005] With capsular polysaccharides (such as Prevnar) ® In contrast to vaccines composed of numerous repeating units, small carbohydrates with very well-defined structures (such as tumor-associated carbohydrate antigens, TACAs) may have their epitopes disrupted when coupled to random monosaccharides through non-directional oxidative ring-opening.

[0006] If small carbohydrates with free reducing ends are directly subjected to reductive amination without prior oxidation, the result will be a truncated form, as the carbohydrate units at the reducing ends are converted into linkers.

[0007] Therefore, it is necessary to use aldoses as linkers for smaller carbohydrates to prepare compounds while avoiding the potential drawbacks of the individual carbohydrates in terms of bioactivity. Invention Overview This invention relates to a compound of formula (I). (G n -X-NH) o -Y (I) in G is a polysaccharide, which contains n monosaccharide units linked by glycosidic bonds; n is the number of monosaccharide units linked by glycosidic bonds, preferably n is 2 to 200, more preferably 2 to 100, and most preferably 2 to 20 monosaccharide units; o is an integer from 1 to 10. If Y is a peptide or protein, then it is an integer from 1 to 10 corresponding to each 10 kDa peptide or protein Y, preferably an integer from 1 to 5. X is based on an aldose, which is linked to G via a glycosidic bond and wherein the aldehyde group of the aldose has reacted with the primary amino group of the group Y to give the product of formula (I), wherein the aldehyde group of the aldose has preferably been reduced to the corresponding secondary amino group in formula (I) by reductive amination to give the secondary amino group in formula (I). Y is selected from: amino acids, peptides, and proteins.

[0009] Furthermore, the present invention also relates to a pharmaceutical composition comprising the compound and at least one pharmaceutically acceptable carrier.

[0010] The present invention also relates to the use of compounds and pharmaceutical compositions in pharmaceuticals.

[0011] This invention provides the possibility of using an additional aldose to couple carbohydrates to amino acids, peptides, or proteins, which acts as a “sacrificial” aldose and linker, allowing coupling with other molecules via reductive amination without disrupting the cyclic structure of the terminal aldose of the associated carbohydrate itself, thereby altering its biological activity. Attached Figure Description

[0012] Figure 1 Immunological research: Printed patterns of glycan microarrays.

[0013] Figure 2A) Binding of mouse serum immunized with compound 5 or 6 to the target sTRA (13) or the truncated sTRA trisaccharide MLB-153 (17). B) Glycan array analysis results of 5 mice immunized with glycoconjugate 5 (serum dilution 1:100).

[0014] Figure 3 The structural relationship between sTRA, LSTA and TRA.

[0015] Figure 4 Printed patterns of glycan microarrays.

[0016] Figure 5 Glycan microarray analysis of serum from six animals immunized with compounds 7 and 8. Serum was tested at dilutions of 1:100, 1:1k, and 1:10k. Fluorescence signals were read at 488 nm. The secondary antibody was specific for mouse IgG.

[0017] Figure 6 MFI values ​​of serum from six mice immunized with compounds 7 and 8 were obtained from FACS analysis. The serum was diluted 1:1000. The results showed that only the serum from mice immunized with compound 8 showed binding to MCF7, thus confirming the presence of anti-Globo-H antibody. Invention Details It should be noted that, as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more such different reagents, and reference to “the method” includes equivalent steps and methods known to those skilled in the art that can modify or replace the methods described herein.

[0019] Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to determine, many equivalents of the embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered within the scope of the invention.

[0020] In this document, the term “and / or” includes the meaning of “and”, “or”, and “all or any other combination of the elements connected by the term”.

[0021] In this specification and the appended claims, unless the context otherwise requires, the word “comprise” and its variations (such as “comprises” and “comprising”) shall be understood to mean including the stated element or step or group of elements or steps, but not excluding any other element or step or group of elements or steps. When used herein, the term “comprise” may be replaced by the terms “containing” or “comprising”, or sometimes by “having”. When used herein, “consisting of” excludes any unspecified element, step, or ingredient.

[0022] This invention relates to a compound of formula (I). (G n -X-NH) o -Y (I) G is a polysaccharide containing n monosaccharide units. If n is greater than 1, these monosaccharide units are linked by glycosidic bonds.

[0023] Polysaccharides are understood as molecules in which two or more monosaccharide units are linked by glycosidic bonds.

[0024] n is an integer representing the number of monosaccharide units linked by glycosidic bonds, preferably an integer from 1 to 200, more preferably from 2 to 100, and most preferably from 2 to 20 monosaccharide units.

[0025] The definition of "linked by glycosidic bond" is well known to those skilled in the art. Preferably, "linked by glycosidic bond" refers to the formation of a bond involving oxygen (ether bond) or other heteroatoms (e.g., N or S) or even carbon atoms, which relates to the hemiketal or hemiacetal group of the sugar. Preferably, the glycosidic bond is formed in the form of an ether bond between the hemiketal or hemiacetal group of one sugar and a hydroxyl group of at least one other sugar, thereby linking at least two sugars to form a polysaccharide.

[0026] The monosaccharide units in the polysaccharide can exhibit additional functional groups attached to the heteroatom-containing groups by substituting one or more hydrogen atoms in these heteroatom-containing groups (not involved in glycosidic bonds), preferably hydroxyl, amino, or –SH groups, more preferably hydroxyl or amino. These can be selected from known protecting groups, such as –C(O)(C1-C6)alkyl, benzyl (Bn), benzoyl (Bz), benzyloxycarbonyl (Cbz), fluorenemethyloxycarbonyl (fmoc), preferably acetal (Ac).

[0027] The term "alkyl" refers to a monovalent free radical of a saturated straight-chain or branched hydrocarbon. Preferably, the alkyl group comprises 1 to 6 carbon atoms, i.e., 1, 2, 3, 4, 5, or 6 carbon atoms, more preferably 1 to 4 carbon atoms, and most preferably 1 carbon atom. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, secondary-pentyl, neopentyl, 1,2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, and secondary-hexyl.

[0028] o is an integer from 1 to 10. If Y is a peptide or protein, then it is an integer from 1 to 10 corresponding to each 10 kDa peptide or protein Y, preferably an integer from 1 to 5.

[0029] o indicates that each Y can have one or more items as shown in parentheses in equation (I).

[0030] G is preferably a mammalian polysaccharide, preferably composed of n monosaccharide units, which are independently selected from: arabinose, fructose, fucose, galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, N-acetylglucosamine, glucuronic acid, mannose, muramic acid, neuraminic acid, sialic acid, rhamnose, ribose, and xylose.

[0031] Mammalian polysaccharides are polysaccharides that exist in mammalian cells, preferably on the surface of mammalian cells.

[0032] In one embodiment, the glycan is present on the surface of human cancer cells.

[0033] G can be selected from:

[0034] *A common name has not yet been defined.

[0035] X is based on an aldose, preferably a D-aldose, which is linked to G via a glycosidic bond and wherein the aldehyde group of the aldose has reacted with the primary amino group of group Y to give the product of formula (I), wherein the aldehyde group has preferably been reduced to the corresponding secondary amino group in formula (I) by reductive amination to give the secondary amino group in formula (I).

[0036] Preferably, the aldose is selected from hexose, pentose, tetroose, or tricose; more preferably, i) Hexoses are selected from: glucose; galactose, mannose, allose, adroose, idole, tarose, or ii) Pentoses are selected from: ribose, arabinose, xylose, and lysol; or iii) Tuccinate is selected from erythrose and threose; iv) Triose is glyceraldehyde.

[0037] In one embodiment, X is glucose, preferably D-glucose.

[0038] Y is selected from: amino acids, peptides, and proteins.

[0039] Since all peptides and / or proteins have a primary amino group required to form the basic -NH- group in formula (I), this invention is applicable to all peptides and proteins containing a primary amino group at the N-terminus or within a lysine residue.

[0040] Amino acids, peptides, or proteins may contain natural and / or non-natural amino acids.

[0041] In one embodiment, Y is a peptide or polypeptide composed of 2 to 10,000 amino acids, preferably 20 to 30,000 amino acids, more preferably 10 to 10,000 amino acids.

[0042] In one implementation, Y is a protein with a molecular weight of 10 kDa to 400 kDa.

[0043] In one embodiment, Y is a protein selected from proteins that enhance immune responses. Specific examples include, but are not limited to, cross-reactive material 197 (CRM197) or keyhole hemocyanin (KLH).

[0044] In one embodiment, Y comprises a lysine side chain within a peptide or protein, the lysine side chain providing a primary amino group for the -NH- group in formula (I).

[0045] Y can be a protein selected from the following: CRM 197 Keyhole cyanin (KLH), diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae (Hib) H. influenza The protein D (HiD), meningococcal outer membrane protein complex (OMPC), Qβ protein, bovine serum albumin (BSA), and immunoglobulin G (IgG) are present.

[0046] The present invention also relates to a pharmaceutical composition comprising the compound described above and at least one pharmaceutically acceptable carrier.

[0047] Pharmaceutically acceptable "carriers" refer to diluents, adjuvants, excipients, or mediators administered in conjunction with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the drug composition is administered orally. Saline and glucose solutions are preferred carriers when the drug composition is administered intravenously. Saline and glucose solutions, as well as glycerol solutions, are preferably used as liquid carriers for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. This composition can be formulated as a suppository using conventional binders and carriers, such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's "Remington's Pharmaceutical Sciences". Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in a purified form, and an appropriate amount of carrier to provide a form suitable for patient administration. The formulation should be suitable for the administration method.

[0048] Furthermore, the present invention relates to the use of the compounds described above in pharmaceuticals.

[0049] Furthermore, this invention relates to the use of the compounds described above in research.

[0050] Furthermore, this invention relates to the use of the compound for generating antibodies against the compound. Antibodies can be generated by methods known to those skilled in the art (Dübel & Reichert 2014).

[0051] The compounds of the present invention can be prepared using carbohydrate, peptide, and protein synthesis procedures known to those skilled in the art, optionally including solid-phase synthesis and / or protecting group chemical reactions. Different synthetic methods can be selected to obtain the compounds of the present invention. However, it is preferred that all methods include a reductive amination step, which preferably includes the formation of an imine intermediate to link group Y to linker X. The reductive amination step includes a reducing agent that reduces the imine to the corresponding amine but not the aldehyde to the corresponding alcohol. Reducing agents exhibiting this imine reduction selectivity are, for example, NaCNBH3, sodium triacetoxyborohydride, certain BH3-amine complexes, hydrogen in combination with a suitable metal catalyst (Tripathi 2008).

[0052] The embodiments are not intended to limit the scope of the invention in any way.

[0053] Example Experimental details General Method Unless otherwise stated, all reagents and solvents were obtained from commercial sources. Anhydrous solvents were obtained using a solvent dispensing system (JC Meyer). Palladium on carbon was removed from the reaction mixture by filtration using a Rotilabo syringe filter (Roth) and a PTFE filter (pore size: 0.45 μm).

[0054] Nuclear magnetic resonance (NMR) spectra were obtained using an Ascend 400 (Bruker) and Agilent 400 MHz NMR Magnet (Agilent Technologies) spectrometer at 400 MHz (1H) and 100 MHz (13C), or a Varian 600 (Agilent) spectrometer at 600 MHz (1H) and 150 MHz (13C). Unless otherwise specified, CDCl3 or D₂O was used as the solvent, and chemical shifts (δ) were referenced to internal standards (CDCl3: 7.26 ppm 1H, 77.16 ppm 13C; D₂O: 4.79 ppm 1H). Assignments were supported by COSY and HSQC experiments. Reactions were monitored by mass spectrometry using an Agilent 1100 Series LC / MSD mass spectrometer. MALDI spectra were obtained using a Daltonics Autoflex Speed ​​spectrometer (Bruker).

[0055] Monosaccharide building blocks for automated glycan assembly (AGA) were purchased from Glyco Universe GmbH & Co. KGaA, Germany. CMP-N-acetylneuraminic acid (CMP-NaNa) was obtained from Roche Diagnostics Deutschl and GmbH. Globo-H with free reducing ends and NAc-CH2-(1,4-triazole)-(CH2)2-EG3-NH2 linkers was purchased from Elicityl.

[0056] Automated Glycan Assembly (AGA) General Information All synthesis was performed using a custom synthesizer developed by the Max Planck Institute of Colloids and Interfaces. Except for dichloromethane (DCM) used to prepare the building blocks, activator, and TMSOTf solution, and dioxane used for the activator solution, all solvents used for AGA were HPLC grade. These solvents were derived from anhydrous solvent systems (JC Meyer-solvent systems). Four different building blocks were used in these synthesis.

[0057] AGA solution Module solution: Dissolve the module (96 µmol or 192 µmol) in DCM (1 mL or 2 mL).

[0058] NIS / TfOH activator solution A: for recrystallization N Iodosuccinimide (NIS, 1.35 g, 6.0 mmol) was dissolved in 40 mL of a 2:1 v / v mixture of anhydrous DCM and anhydrous dioxane. Trifluoromethanesulfonic acid (55 μL, 0.6 mmol) was then added. The solution was maintained at 0 °C during automated operation.

[0059] Fmoc deprotection solution: Prepare a 20% piperidine solution in dimethylformamide (DMF, v / v).

[0060] TMSOTf solution: Dissolve TMSOTf (0.45 mL, 2.49 mmol) in DCM (40 mL).

[0061] Capping solution: Prepare 50 mL of DCM (v / v) solution of 10% acetic anhydride and 2% methanesulfonic acid.

[0062] AGA module Preparation of Module A Resin AGA synthesis was always performed at a scale of 0.016 mmol. The resin (modified Merrifield resin, 0.35 mmol / g) was placed in a reaction vessel and swollen in DCM at room temperature for 20 min. After 20 min, the resin was washed three times with DMF, THF, and DCM (2 mL each time for 25 s), and then the synthesis was started.

[0063] Module B Acid Washing The temperature of the reaction vessel was adjusted to -20°C. During this period, the resin swelled in 2 mL of DCM. When the temperature of the reaction vessel reached -20°C, 1 mL of TMSOTf solution was added dropwise to the reaction vessel. The reaction was bubbled with argon gas for 3 min, then the solution was purged and the resin was washed with 2 mL of DCM for 25 s.

[0064] Module C1 thioglycoside glycosylation 1 cycle Module C1 was used for both BB1 and BB3. The building block solution (0.096 mmol BB in 1 mL DCM) was delivered to the reaction vessel. After reaching the set temperature, the activator solution (1.0 mL, 0.15 mmol) was added dropwise to the reaction vessel. The reaction was bubbled for 20 min, then the solution was drained, and the resin was washed with DCM, DCM / dioxane, and again with DCM (2 mL each time for 25 s). The reaction vessel temperature was set to 25 °C for the next reaction step.

[0065] Module C2 thioglycoside glycosylation 2 cycles Module C2 was used for BB2 and BB4. The building block solution (0.096 mmol BB dissolved in 1 mL dichloromethane for each glycosylation reaction) was delivered to the reaction vessel. After reaching the set temperature, the activator solution (1.0 mL, 0.15 mmol) was added dropwise to the reaction vessel. The reaction was bubbled for 20 min, then the solution was drained, and the resin was washed with DCM, DCM / dioxane, and again with DCM (twice, 2 mL each time for 25 s). The above glycosylation cycle was then repeated. After the second glycosylation was completed, the temperature of the reaction vessel was set to 25 °C for the next reaction.

[0066] Module D end cap When the reaction vessel is adjusted to 25°C, wash the resin twice with DMF (2 mL, 25 s). Incubate the resin in the reaction vessel for 1 min with pyridine solution (2 mL, 10% DMF solution). Drain the solution and wash the resin with DCM (3 times, 3 mL each time, 25 s each time). Transfer the end-capping solution (4 mL) into the reaction vessel. After reacting for 20 min, drain the solution and wash the reaction vessel with DCM (3 times, 3 mL each time, 25 s each time).

[0067] Module EFmoc unprotected When the reaction vessel is adjusted to 25°C, the resin is washed three times with DMF (2 mL, 25 s). Fmoc deprotection solution (2 mL) is then introduced into the reaction vessel. After bubbling with argon for 5 min, the solution is purged from the reaction vessel. The resin is then washed with DMF (3 times, 2 mL each time, 25 s) and DCM (5 times, 2 mL each time, 25 s). If another glycosylation is subsequently performed, the reaction is set to -20°C.

[0068] AGA Post-Module Methanol hydrolysis of module F on resin The resin was suspended in anhydrous tetrahydrofuran (THF, 4 mL). Sodium methoxide (0.5 M methanol solution, 0.4 mL) was added. The suspension was shaken at room temperature for 24 h (in a 5 mL plastic syringe). After the reaction, the resin was washed three times with methanol (4 mL) and DCM (4 mL).

[0069] Module G undergoes UV pyrolysis on a solid support The semi-protected glycans were cleaved from the solid support using a continuous flow photoreactor. As previously described [Hurevich, 2014], the glycans attached to the solid support were suspended in a DCM (10 mL) and passed through the photoreactor (mercury lamp, 450 W) at a rate of 0.5 mL / min. After the procedure, the crude glycans were concentrated in a vacuum.

[0070] Module H Hydrogenolysis The crude deprotected polysaccharide obtained from module G was dissolved in 4 mL of EtOAc: t In a 2:1:1 mixture of MeOH and H₂O, add a spoonful of Pd(OH)₂ / C (10-20%) to the stirred reaction solution. Purge the suspension with N₂ for 5 min, then with H₂ for 5 min, and finally stir under H₂ atmosphere. Monitor the reaction using MALDI. After the reaction is complete, filter the reaction suspension (PTFE filter, 0.45 µM). Wash the filter five times with MeOH: H₂O (1:1, 5 mL), and concentrate the filtrate under vacuum.

[0071] Polysaccharide synthesis Synthesis of TRA-C5(1)

[0072] After AGA, methanololysis, UV pyrolysis, and hydrogenolysis, the crude polysaccharide was purified by RP-HPLC (Hypercarb column, 150x10mm, 5μm) at a flow rate of 3.5 mL / min. The eluent was ACN / H2O (0.1% formic acid) [isocratic 100% H2O (0.1% formic acid) (5 min), linear gradient to 30% ACN (30 min), linear gradient to 100% ACN (5 min)]. After purification and lyophilization, the product was separated into a white solid (2.1 mg, total yield 16%).

[0073] Synthesis of TRA-Sacrificial Aldose (2)

[0074] After AGA, methanololysis, UV pyrolysis, and hydrogenolysis, the crude polysaccharide was purified by RP-HPLC (Hypercarb column, 150x10mm, 5μm) at a flow rate of 3.5 mL / min. The eluent was ACN / H2O (0.1% formic acid) [isocratic gradient: 100% H2O (0.1% formic acid) (5 min), linear gradient to 30% ACN (30 min), linear gradient to 100% ACN (5 min)]. After purification and lyophilization, the product was separated into a white solid (1.9 mg, total yield 13%, α / β mixture).

[0075] 2. Enzymatic salivation The expression of CST-1 for sialylation was performed as described in other literature [Tsai, 2019].

[0076] Synthesis of sTRA-C5(3) Add CMP-Na-Na (6.71 mg, 10.5 µmol, 1.25 equivalents) dissolved in reaction buffer (321.5 µL, 50 mM HEPES, pH 8.5, 10 mM MgCl2, 10 mM MnCl2) to a 1.5 mL Eppendorf tube (6.97 mg, 8.36 µmol, 1 equivalent). Add CST-1 (74.4 µL of 2.66 mg / mL solution) to bring the final enzyme concentration to 0.5 mg / mL. Incubate the solution at 37 °C with shaking at 300 rpm for 16 h. After incubation, add 400 µL of ethanol and incubate at 4 °C for 30 min. The mixture was centrifuged (10 min, 10000 rpm), the supernatant was diluted with water, and analyzed and purified by RP-HPLC (Hypercarb column, 150 x 10 mm, 5 μm, flow rate 3.5 mL / min, eluent ACN / H2O (0.1% formic acid) [isocratic: 100% H2O (0.1% formic acid) (5 min), linear gradient to 30% ACN (30 min), linear gradient to 100% ACN (5 min)]). After purification and lyophilization, the product was separated into a white solid (6.0 mg, yield 63%).

[0077] Analyze the data: 1 H NMR (400 MHz, D2O) δ 4.58 (d, J = 8.4 Hz, 1H), 4.37 (dd, J =7.7, 2.5 Hz, 2H), 4.32 (d, J = 7.8 Hz, 1H), 4.01 (d, J = 3.2 Hz, 1H), 3.95 (dd, J =9.9, 3.2 Hz, 1H), 3.85 (dd, J = 12.2, 2.1 Hz, 1H), 3.81 – 3.37 (m, 29H), 3.33(ddd, J = 9.8, 5.0, 2.4 Hz, 1H), 2.89 – 2.78 (m, 2H), 2.62 (dd, J = 12.4, 4.6 Hz,1H), 1.89 (s, 9H), 1.64 (t, J = 12.1 Hz, 1H), 1.60 – 1.39 (m, 4H), 1.26 (td, J =8.7, 4.4 Hz, 2H). 13C NMR (176 MHz, D2O) δ 175.0, 174.9, 174.4, 173.9, 103.4,102.9, 102.5, 101.1, 99.6, 82.1, 82.0, 78.5, 75.6, 75.2, 75.1, 74.9, 74.8,72.8, 72.4, 71.8, 70.1, 70.0, 69.1, 68.4, 68.4, 68.3, 68.0, 67.2, 62.5, 61.0,60.9, 60.5, 60.0, 55.0, 54.6, 51.6, 39.3, 27.9, 26.3, 22.3, 22.1, 22.1, 22.0.

[0078] Synthesis of sTRA-sacrificial aldose (4) Add CMP-Na-Na (1.37 mg, 2.13 µmol, 1.25 equivalents) dissolved in reaction buffer (65.8 µL, 50 mM HEPES, pH 8.5, 10 mM MgCl2, 10 mM MnCl2) to a 1.5 mL Eppendorf tube (1.55 mg, 1.70 µmol, 1 equivalent). Add CST-1 (15.2 µL of 2.66 mg / mL solution) to bring the final enzyme concentration to 0.5 mg / mL. Incubate the solution at 37 °C with shaking at 300 rpm for 16 h. After incubation, add 80 µL of ethanol and incubate at 4 °C for 30 min. The mixture was centrifuged (10 min, 10000 rpm), the supernatant was diluted with water, and analyzed and purified by RP-HPLC (Hypercarb column, 150 x 10 mm, 5 μm, flow rate 3.5 mL / min, eluent ACN / H2O (0.1% formic acid) [isocratic: 100% H2O (0.1% formic acid) (5 min), linear gradient to 30% ACN (30 min), linear gradient to 100% CAN (5 min)]). After purification and lyophilization, the product was separated into a white solid (1.1 mg, yield 55%).

[0079] Analyze the data: 1 H NMR (600 MHz, D2O) δ 5.09 (d, J = 3.7 Hz, 1H-α), 4.63 (d, J =8.4 Hz, 1H), 4.51 (d, J = 8.0 Hz, 1H-β), 4.45 (t, J= 8.3 Hz, 1H), 4.41 (d, J = 7.8Hz, 1H), 4.36 (d, J = 7.9 Hz, 1H), 4.07 – 4.02 (m, 2H), 3.99 (dd, J = 9.7, 3.2Hz, 2H), 3.89 (dt, J = 12.2, 2.2 Hz, 1H), 3.84 (d, J = 3.2 Hz, 1H), 3.82 – 3.56(m, 20H), 3.56 – 3.51 (m, 2H), 3.51 – 3.42 (m, 5H), 3.42 – 3.33 (m, 2H), 3.32– 3.22 (m, 1H), 3.11 (dd, J = 9.4, 8.0 Hz, 1H), 2.70 – 2.61 (m, 1H), 1.95 (s, 3H), 1.93 (s, 6H), 1.70 (t, J = 12.2 Hz, 1H). 13 C NMR (151 MHz, D2O) δ 174.9,174.9, 174.6, 173.6, 103.3, 102.9, 102.5, 101.6, 99.5, 95.9 (H1-β), 92.1 (H1-α), 82.1, 81.9, 78.4, 75.7, 75.6, 75.2, 75.0, 74.9, 74.7, 74.0, 72.8, 72.3,71.7, 71.3, 70.2, 70.0, 69.5, 69.1, 68.6, 68.4, 68.3, 68.0, 67.2, 62.5, 61.0,60.9, 60.5, 60.0, 54.9, 54.5, 51.6, 39.6, 22.3, 22.2, 22.0.

[0080] Protein conjugation Glycoconjugation via DNAP cross-linking agents (Method A) As previously described [Wu, 2004], a glycan with a linker carrying a primary amine was conjugated to a protein via di-p-nitrophenyl adipate (DNAP). A 10-fold molar excess of DNAP dissolved in 300 µl of anhydrous DMSO was added to the glycan. 30 µl of triethylamine was added. The reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated by lyophilization. The solid product was washed with chloroform (5x, 1 mL) and DCM (5x, 1 mL). Thin-layer chromatography was used to ensure complete removal of excess DNAP. The purified glycan-linker conjugate was then added to the protein containing carrier CRM dissolved in sodium phosphate buffer (100 mM, pH 8, 100 µL). 197 The reaction was slowly stirred for 24 hours. The crude product was purified by washing with deionized water and reburied in PBS using a 10 kDa centrifugal filter unit (Merck Millipore). The glycan-to-protein ratio was determined by MALDI. Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop ND-1000 spectrophotometer (ThermoScientific).

[0081] Reductive amination (Method B) The method of coupling glycans with free reducing ends to proteins via reductive amination is adapted from Gildersleeve et al. [Gildersleeve, 2008]. Protein CRM 197 Approximately 75 µL of glycan was added to a 1.5 mL Eppendorf tube in deionized H₂O. Sodium sulfate (12.3 µL, 1.5 M), sodium borate (18.5 µL, 400 mM), and sodium borohydride (5.55 µL, 1.5 M) were added to the mixture. The reaction mixture was incubated at 37 °C for 96 h without stirring. The crude product was purified by washing with deionized water and reburied in PBS using a 10 kDa centrifugal filter unit (Merck Millipore). The glycan-to-protein ratio was determined using MALDI. Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop ND-1000 spectrophotometer (ThermoScientific).

[0082] Sugar conjugates Sugar conjugate CRM 197 -3 (5) Compound 3 (1.16 mg, 1.03 µmol) was coupled to CRM via method A. 197 (1 mg). A product with a polysaccharide to protein ratio of 6.5 was obtained. 180 µl of product was obtained with a concentration of 3.79 mg / ml.

[0083] Sugar conjugate CRM 197 -4 (6) Compound 4 (1.86 mg, 1.55 µmol) was coupled to CRM via method B. 197 (1.5 mg). The product showed a glycan to protein ratio of 6.5. 190 µl was obtained, with a concentration of 2.53 mg / mL.

[0084] Sugar conjugate CRM 197 -EG3-Globo-H (7) Commercially available Globo-H-NAc-CH2-(1,4-triazole)-(CH2)2-EG3-NH2 (“triazole linker”) (0.81 mg, 0.62 µmol) was coupled to CRM via method A. 197 (0.6 mg). The ratio of polysaccharide to protein is 7.3.

[0085] Sugar conjugate CRM 197 -Globo-H (8) Commercially available Globo-H (1.05 mg, 1.03 µmol) was coupled to CRM via method B. 197 (1 mg). The product showed a glycan to protein ratio of 8.7. 275 µl was obtained, with a concentration of 2.47 mg / ml.

[0086] Immunological research Glycan array scheme Glycans with primary amine functional groups were immobilized on N-hydroxysuccinimide (NHS) activated slides (CodeLink, SurModics). The glycans (0.1 mM) were dissolved in sodium phosphate buffer (50 mM, pH 8.5) for spotting. Sixty-four identical arrays were printed on each slide using a piezoelectric spotting device (Scienion). The slides were incubated overnight in a humidified chamber at room temperature to complete the coupling via NHS, and then treated with ethanolamine (50 mM) in sodium phosphate buffer (50 mM, pH 9) for 1 h at room temperature. After quenching residual NHS groups, the slides were washed with H2O (3x) and then centrifuged to dry (300 g, 5 min).

[0087] For serum analysis, microarray slides were blocked with BSA (3%, dissolved in PBS) at room temperature for 1 h, washed twice with PBS, and once with H2O. After centrifugation and drying, the slides were placed in a FlexWell 64 grid. The slides and serum samples (diluted with PBS containing 3% BSA) were incubated in a humidified chamber at room temperature for 1 h. The wells (3x) were washed with PBS containing 0.1% Tween-20. The secondary antibody, mouse Fc IgG antibody (labeled with Alexa Fluor 488nM), was diluted 1:400 in PBS containing 3% BSA and dispensed into the wells. After incubation in a humidified chamber at room temperature for 1 h, the wells (2x) were washed with PBS containing 0.1% Tween-20. The grid was removed, and the entire slide was washed with PBS (2x) and H2O (2x), then dried by centrifugation. The slides were scanned using an InnoScan 1100AL (Synopsys). Export the fluorescence intensity (RFI) values ​​after background subtraction for further analysis (see Figure 1 ).

[0088] sTRA mouse immunization program Immunization regimen Five Swiss mice were immunized with compound 5, and five Swiss mice were immunized with compound 6. Animals were immunized every two weeks. Each animal received 10 µg of the glycoconjugate per immunization round. Five rounds of immunization were performed. Serum samples were collected from all animals on day 63 of immunization and analyzed.

[0089] Glycan array analysis Animals immunized with 6 (sacrificial aldose) showed no immune response to any glycan structure in the glycan array group. Immunization with 5 elicited an immune response against several different glycans, not all of which were associated with the sTRA structure. Figure 2 A and B). In all 5 mice, serum recognized TRA and MLB-153 in the glycan array. LSTa, which consists of the same four monosaccharide units as the non-reduced end of sTRA, was not bound by any serum. Figure 3 This demonstrates that the immune response detected in the glycan array was induced by the linker-targeting antibody. The linkers used to immobilize the glycans in the glycan array are the same as those used in glycoconjugate 5. FACS analysis confirmed that none of the mice showed a sTRA-specific immune response. No cell binding was observed (data not shown).

[0090] in conclusion Overall, sTRA showed almost no immune response in mice due to its low immunogenicity and high structural similarity to abundantly expressed mammalian cell surface glycans. Mouse immunization is likely not a suitable tool for generating anti-sTRA antibodies. However, our method demonstrated that the "sacrificial aldose linker" is effective against sTRA and CRM in mice. 197 The linker portion between them did not elicit an antibody response at all. In contrast, serum from animals immunized with sTRA conjugated via the C5 linker showed binding to many unrelated glycan structures in the glycan array, indicating the presence of antibodies against the aliphatic linker in the serum.

[0091] Globo-H Mouse Immunization Program Immunization regimen Three Balb / c mice were immunized with compound 7, and three Balb / c mice were immunized with compound 8. Animals were immunized twice at 5-week intervals. Each animal received 50 µg of the glycoconjugate per immunization round. Serum samples were collected from all animals on day 42 of immunization and analyzed by glycan microarray and FACS.

[0092] Glycan array analysis Two versions of Globo-H were immobilized in the analyzed glycan microarrays. 297 was Globo-H equipped with a C5 amino linker, and M5 was a commercially available Globo-H equipped with a triazole linker, the same linker used in the coupling of compound 7.

[0093] Serum from animals #1 and #2 immunized with 7 showed binding to Globo-H M5 but not to Globo-H 297. Both sera also recognized M1 and M4, which carry triazole linkers. This indicates that animals immunized with 7 only showed an immune response against the triazole linker, but no Globo-H specific antibodies were present in their serum. Only mouse #3 showed binding to Globo-H 297 at a 1:100 dilution, indicating the presence of a small amount of low-affinity antibody against the target.

[0094] At 1:100 and 1:1k dilutions, all sera from three animals immunized with 8 were bound to both versions of Globo-H, demonstrating the presence of Globo-H-specific antibodies. Mice #1 and #3 also bound to SSEA-4 and Gb-5, independent of the linker used in the glycan array. This suggests that reductive amination used to couple the glycan to the vector is superior to commercially available hydrophobic linkers in generating antibodies via immunization.

[0095] FACS analysis Serum samples were centrifuged to pellet the cells and diluted 1:100 in phosphate-buffered saline (PBS) containing 2% FBS. The pellet was then incubated with 1 x 10^5 MCF7 WT cells on ice for 30 min. The cells were then centrifuged again, washed with PBS-FBS, and stained with donkey anti-mouse IgG (H+L) CF488A (Biotium) (1:1000 diluted in PBS / FBS) on ice for 30 min. Subsequently, the cells were stained with the live / dead cell staining agent eFlour780 (Invitrogen) (1:1000 diluted in PBS) on ice for 10 min. The cells were then washed with PBS / FBS, and data were acquired using an Attune (Invitrogen) flow cytometer.

[0096] FACS analysis of serum binding to the Globo-H-expressing MCF7 cell line showed that the serum of mice immunized with Globo-H did not contain anti-Globo-H antibodies. As observed in the glycan array, the immune response induced by immunization with the glycoconjugate 7 targeted only the linker and vector. No cell binding to Globo-H was observed.

[0097] References 1. Adamo R., Hu Q.-Y., Torosantucci A., Crotti S., Brogioni G., AllanM., Chiani P., Bromuro C., Quinn D., Tontinia M. and Bertia F. Deciphering the structure–immunogenicity relationship of anti-Candida glycoconjugatevaccines. Chem. Sci., 2014, 5, 4302. 2. Anderson P., Pichichero ME and Insel RA ImmunogensConsisting of Oligosaccharides from the Capsule of Haemophilus influenzae Typeb Coupled to Diphtheria Toxoidor the Toxin Protein CRM197. J. Clin. Invest .,1985, 76, 52. 3. Berti F. and Adamo R. Antimicrobial glycoconjugate vaccines: anoverview of classic and modern approaches for protein modification. Chem. Soc. Rev ., 2018, 47, 9015. 4. Buskas T., Li Y., and Boons G.-J. The Immunogenicity of the Tumor-Associated Antigen Lewisy May Be Suppressed by a Bifunctional Cross-LinkerRequired for Coupling to a Carrier Protein. Chem. Eur. J., 2004, 10, 3517. 5. Dübel, S.,&Reichert, J. M. (Eds.). (2014). Handbook of therapeutic antibodies . John Wiley&Sons. 6. Gildersleeve J. C., Oyelaran O., Simpson J. T. and Allred B.Improved Procedure for Direct Coupling of Carbohydrates to Proteins viaReductive Amination. Bioconjug Chem. 2008, 19, 1485. 7. Mettu R., Chen C.-Y. and Wu1 C.-Y. Synthetic carbohydrate-basedvaccines: challenges and opportunities. J. Biomed. Sci., 2020, 27, 9. 8. Poolman J., Frasch C., Nurkka A., Käyhty H., Biemans R., SchuermanL. Impact of the conjugation method on the immunogenicity of Streptococcuspneumoniae serotype 19F polysaccharide in conjugate vaccines. Clin. Vaccine Immunol ., 2011, 18, 327. 9. Turner A. E.B., Gerson J. E., So H. Y., Krasznai D. J., Hilaire A.J. St., Gerson D. F. Novel polysaccharide-protein conjugates provide animmunogenic 13-valent pneumococcal conjugate vaccine for S.pneumoniae. Synthetic and Systems Biotechnology, 2017, 2, 49. 10. Tripathi R. P., Verma S. S., Pandey J. and Tiwari V. K. RecentDevelopment on Catalytic Reductive Amination and Applications. Curr. Org. Chem. , 2008, 12, 1093. 11. Wu X., Ling C.-C., and Bundle D. R. A New Homobifunctional p-Nitro Phenyl Ester Coupling Reagent for the Preparation ofNeoglycoproteins. Organic Letters 6, 4407.

Claims

1. Compounds of formula (I) (G-X-NH) o -Y (I) in, G is a polysaccharide, which contains n monosaccharide units linked by glycosidic bonds; n is an integer representing monosaccharide units linked by glycosidic bonds, preferably n is 1 to 200, more preferably 2 to 100, and most preferably 2 to 20 monosaccharide units; o is an integer from 1 to 10. If Y is a peptide or protein, then it is an integer from 1 to 10 corresponding to each 10 kDa peptide or protein Y, preferably an integer from 1 to 5. X is based on an aldose, which is linked to G via a glycosidic bond, and wherein the aldehyde group of the aldose has reacted with the primary amino group of the group Y to obtain the product of formula (I), wherein the aldehyde group of the aldose has preferably been reduced to the corresponding secondary amino group in formula (I) by reductive amination to obtain the secondary amino group in formula (I). Y is selected from: amino acids, peptides, and proteins.

2. The compound according to claim 1, wherein G is a mammalian polysaccharide, preferably a polysaccharide composed of n monosaccharide units, wherein the n monosaccharide units are independently selected from: arabinose, fructose, fucose, galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, N-acetylglucosamine, glucuronic acid, mannose, muramic acid, neuraminic acid, sialic acid, rhamnose, ribose, and xylose.

3. The compound according to claim 1 or 2, wherein the polysaccharide is present on the surface of human cancer cells.

4. The compound according to any one of claims 1 to 3, wherein G is selected from: *A common name has not yet been defined.

5. The compound according to any one of claims 1 to 4, wherein the aldose is preferably selected from: hexose, pentose, tetroose, or triose, more preferably, i) The hexose is selected from: glucose, galactose, mannose, allose, adroose, idole, tarose, or ii) The pentose is selected from: ribose, arabinose, xylose, and lysolose; or iii) The teuses are selected from erythrose and threose; iv) The triose is glyceraldehyde.

6. The compound according to any one of claims 1 to 5, wherein X is based on D-glucose.

7. The compound according to any one of claims 1 to 6, wherein Y comprises a lysine side chain within a peptide or protein, said lysine side chain providing a primary amino group for the -NH- group in formula (I).

8. The compound according to any one of claims 1 to 6, wherein Y is a protein selected from CRM. 197 Keyhole cyanin (KLH), diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae (Hib) H. influenza Protein D (HiD), meningococcal outer membrane protein complex (OMPC), Qβ protein, bovine serum albumin (BSA), and immunoglobulin G (IgG).

9. A pharmaceutical composition comprising the compound of any one of claims 1 to 8 and at least one pharmaceutically acceptable carrier.

10. The compound according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9, for use in a pharmaceutical remedy.

11. Use of the compound according to any one of claims 1 to 8 in the study.

12. Use of the compound of any one of claims 1 to 11 for generating specific antibodies against the compound.

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