KR20180040567A - Novel Nselia meningitidis serogroup Y oligomer and method for its synthesis - Google Patents

Abstract

The present invention relates to a novel oligomer (Men-Y oligomer) of a repeating unit of Nselia meningitidis serogroup Y clathrate polysaccharide and a method for the synthesis of a novel Men-Y oligomer. In particular, the present invention relates to the chemical synthesis of tetrameric tetrameric polysaccharide repeat units that can be used as candidates in the development of semisynthetic or fully synthetic conjugate vaccines against meningococcal serogroup Y bacterial infections.

Description

Novel Nselia meningitidis serogroup Y oligomer and method for its synthesis

The present invention relates to the use of the Neisseria meningitidis meningitidis ) serogroup Y capsular polysaccharide repeat units (hereinafter Men-Y oligomer). The present invention also relates to a novel method for the synthesis of Nyserial meningitidis serogroup Y oligomers. More particularly, the present invention relates to a method for the semi-synthetic of meningococcal serogroup Y bacterial infections and the use of a serpentine of the Nacelia meningitidis serogroup Y (hereinafter referred to as " Men-Y & ). ≪ / RTI >

Bacterial meningitis has a mortality rate of at least 5-10% in industrialized countries and 20% in developing countries and causes approximately 170,000 deaths annually. Streptococcus pneumoniae , Haemophilus influenzae type b (Hib) and N. meningitidis are the causes of most cases of bacterial meningitis worldwide. However, even though modern antibiotics are available, Nyseria meningitidis remains a major cause of bacterial meningitis and other infectious bacterial infections.

Meningococcal disease is a medical emergency requiring immediate diagnosis and treatment.

Although a total of thirteen different serogroups of AseB, C, D, 29E, H, I, K, L, W135, X, Y and Z have been identified so far, about 90% Serum groups A, B, C, Y and W135.

The following types of vaccines are available for meningococcal infections:

ㆍ Polysaccharide vaccine that has been available for over 30 years to prevent the disease. Meningococcal polysaccharide vaccines may be administered in various combinations, for example, two (serogroup A and C), three (serogroup A, C and W) or four (serogroup A, W).

The meningococcal conjugate vaccine against serogroup C has been available since 1999. The four Class A, C, Y and W conjugate vaccines are widely used and have been approved for use by children and adults in various regions of the world since 2005, including Canada, USA, Europe and India. Other combinations of the above serogroups are also available in licensed conjugate vaccines. Apart from the single serogroup A conjugate vaccine, currently available meningococcal conjugate vaccines are not available to those in need in underdeveloped countries due to high prices.

Currently, vaccines available for Men-Y use isolated polysaccharides from bacterial sources associated with a variety of risks, such as the risk of handling live bacterial culture. Also, often the isolated bacterial polysaccharides are so heterogeneous that they can not pass the desired homogeneity item. The heterogeneity of the bacterial polysaccharides disrupts the conjugate batches because it provides heterogeneity to the conjugates produced using them and induces large deformation.

Over the last decade, we have increasingly focused on the organic synthesis of bacterial antigens, including clonal oligosaccharide analogs. In view of the prior art, a meningococcal conjugate vaccine containing a synthetic Men-Y oligosaccharide is advantageous over conventional vaccines. The synthetic antigens are uniform in size and well characterized so that the heterozygosity of the conjugate is reduced and less strain is produced in each batch in the final conjugate. One of the major advantages of having synthetic antigens is that they can be manipulated with the desired linkers to not only simplify the conjugation but also produce better yields.

At present, there is no prior art describing the production and purification process of organic synthesis of Men-Y. International Patent Application No. PCT / US2013 / 042428, titled " Multivalent meningococcal conjugates and methods for preparing conjugates ", discloses that serogroups A, C, W-135 and Y To an immunoconjugate comprising at least one polysaccharide or protein conjugated to a Nyseria surface protein capable of eliciting an immune response against a meningococcal polysaccharide (PS). The above International Patent Application describes merely a general description of the preparation of the Men Y oligomer and there is no promise for the production of the Men-Y oligomer. Furthermore, existing techniques available for other serogroups of Nyseria meningitidis produce time-consuming or mixtures of oligomers of different sizes.

The main object of the present invention is to provide novel oligomers of Nyseria meningitidis serogroup Y climbing polysaccharide repeat units.

It is yet another object of the present invention to provide novel oligomers of Nyserial meningitidis serogroup Y climbing polysaccharide repeat units that can be used as candidates in the development of synthetic and semi-synthetic conjugate vaccines against meningococcal bacterial infections.

Yet another object of the present invention is to provide a novel Men-Y tetramer that can be used as a candidate in the development of semisynthetic or fully synthetic vaccines against meningococcal serogroup Y bacterial infection.

It is another object of the present invention to provide a chemical method for synthesizing a Men-Y oligomer using purified sugars having a specific chain length.

It is yet another object of the present invention to provide a chemical method for synthesizing a Men-Y oligomer having improved antigenicity capable of producing an immunoconjugate vaccine.

It is a further object of the present invention to provide a process for the preparation of synthetic Men-Y narrow-wall oligomers meeting physico-chemical quality standards for purity.

It is another object of the present invention to provide a cost-effective Men-Y oligomer that increases efficiency and shelf life when conjugated to a carrier protein.

Thus, the present invention describes the Nisserian meningitidis serogroup Y oligomer and synthetic methods for obtaining it. The Men-Y oligomers may be conjugated to suitable carrier proteins and then used as candidates in the development of semi-synthetic and synthetic conjugates for meningococcal serogroup Y bacterial infection. The vaccine may be administered by a parenteral route.

The Men-Y oligomers of the present invention have improved antigenicity and improved shelf life. The Men-Y oligomers also meet physicochemical quality standards for purity and are cost effective.

The method of the present invention describes the synthesis of a Men-Y oligomer comprising two major building blocks, an initiation unit and a propagation unit. The starting unit is prepared by glycosidation of a suitably protected monosaccharide, more particularly, but not exclusively, with neuraminic acid and another suitably protected monosaccharide. Said another suitably protected monosaccharide is selected from hexose, and more particularly from glucose. The propagation unit may be formed by glycosidation of a suitably protected monosaccharide, such as but not limited to, more particularly, neuraminic acid and another suitably protected monosaccharide (more specifically, hexose, and more specifically, but not exclusively glucose) .

The initiation unit and the propagation unit are coupled together using a catalyst at a predetermined temperature to provide a saccharide or dimer unit. The catalyst used in the coupling is N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), trimethylsilyltrifluoromethane sulfonate (TMSOTf), silver trifluoromethanesulfonate (CF ₃ selected from SO ₃ Ag), but are not limited to, a glycoside reagent / Lewis acid catalyst. The dimer unit reacts with a basic reagent such as, but not limited to, sodium methoxide to promote ring opening. The thus obtained dimer unit again reacts with the propagation unit to form a tetramer. The tetramer undergoes repetitive reactions under similar conditions to provide a protected higher oligomer (Y), including tetramers, dimers, tracers, lumens, and the like.

The protected oligomers thus obtained are sequentially deprotected in a protecting group to produce a Men-Y advanced oligomer.

The thus obtained advanced oligomers of the Nisserian meningitidis serogroup Y have improved yield and high efficiency and can be used as candidates for the development of conjugate vaccines that protect against diseases caused by Men-Y infection.

All of the exemplary steps designed to synthesize the Men-Y oligomer using the new approach further increase the yield of the tetramer and enhance the antigenicity as shown in FIG. 9 with a purity of > 95% as shown in FIG.

Brief Description of the Drawings Fig. 26) shows a ¹ H-NMR of.
Figure 2 shows ¹³ C-NMR of the Men-Y tetramer.
Figure 3 shows the COSY NMR spectrum of the Men-Y tetramer.
Figure 4 shows the HMBC NMR spectrum of the Men-Y tetramer.
Figure 5 shows the HSQC NMR spectrum of the Men-Y tetramer.
Figure 6 shows the TOCSY NMR spectrum of the Men-Y tetramer.
Figure 7 shows the DEPT NMR spectrum of the Men-Y tetramer.
Figure 8 shows an HPSEC analysis of the purity of the Men-Y tetramer.
Figure 9 shows an antigenic assay of the Men-Y tetramer and the Men-Y tetramer-tetanus toxoid (TT) conjugate by an inhibitory ELISA against anti-Men-Y polyclonal sera.

Thus, the present invention is directed to novel Men-Y oligomers. The present invention further relates to a method for synthesizing a novel Men-Y oligomer. More particularly, the present invention relates to the synthesis of Men-Y tetramers that can be used as candidates in the development of semi-synthetic and synthetic vaccines against meningococcal bacterial infections. The vaccine may be administered in a suitable manner, more particularly via a parenteral route. The synthesis is accomplished in the following steps:

Step 1: Synthesis of Propagation Unit (Compound 18 as shown in Scheme I)

Step 2: Synthesis of the starting unit (compound 20 as shown in Scheme II)

Step 3: Synthesis of Advanced Oligomers (Compounds 21, 22, 23, 24, 25, d26 as shown in Scheme III)

Before describing preferred embodiments of the present invention, it should be understood that the specific materials described may be modified and thus the invention is not limited to these materials. It is also to be understood that the terminology herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention in any way.

The singular forms (as used herein with the terms "a", "an" and "the" in the original text) include plural forms unless the context clearly dictates otherwise.

In one non-limiting example of the method, the N-acetylneuraminic acid, Compound 1 , undergoes methylation in the presence of a catalyst such as Dowex 50W X8 (H ⁺ ) resin to provide compound 2 . Compound 2 thus obtained undergoes acylation in the presence of pyridine in the presence of an acylating agent such as acetic anhydride to give compound 3. [ The method aids shielding the hydroxyl group.

Compound 3 thus obtained undergoes selective halogenation, more particularly chlorination in the presence of a halogenating agent such as acetyl chloride and HCl to provide compound 4. [ Compound 4 reacts with a thiolating reagent such as p-methylphenyl thiol (TolSH) in the presence of DIPEA, also known as a whiskey base (poor nucleophile) to yield compound 5 in which the -Stol group is attached in the second position. Compound 5 thus obtained undergoes deacylation in the presence of MeOH, methanesulfonic acid (MsOH) to provide compound 6. [ Compound 6 thus obtained is reacted with a carbonylating reagent such as triphosgene, NaHCO _{3 in} the presence of MeCN / H ₂ O and undergoes an oxazolidinone ring formation reaction to provide compound 7 . Compound 7 thus obtained was protected in the presence of a protecting reagent such as tertiary butyl diphenylsilyl chloride TBDPSCl / DMF in the terminal position and then protected with acetonide using 2,2-DMP Acylation using AcCl provides compound 8 . Compound 8 thus obtained is reacted with a reaction reagent such as dibutyl phosphate (Bu ₂ PO ₄ H) in the presence of NIS, TfOH, DCM to obtain a phosphate leaving group at the terminal position, which increases the alpha selectivity as compound 9 .

The commercially available compound 10 β-D-glucose pentaacetate is reacted with thiocresol and (catalyst) BF ₃ : OEt _{2 in} DCM (solvent) to protect the alpha position to provide compound 11 . Compound 11 undergoes deacetylation in the presence of MeONa in MeOH to give compound 12 . Compound 12 thus obtained was reacted with a silyl protecting group such as tert-butyldiphenylsilyl chloride (TBDPS-Cl) to give compound 13 in 80% yield while protecting the -OH group at the 6-position of compound 12 do. The thus obtained compound 13 is subjected to alkylation and benzylated to protect the specific position to give compound 14 in a 76% reaction yield at 0 < 0 > C to room temperature.

The above-mentioned method is a known method.

Then, the compound 14 in tetrahydrofuran (THF) -n- from tetra-butylammonium fluoride to compound 15 by recovering one of the de-protected by reacting with reagents protected hydroxyl group of compound 14, such as (TBAF) 85% to RTI ID = 0.0 > 95%. ≪ / RTI > The compound 14 acts as an intermediate in the synthesis of the radio wave unit 18 .

The residual amount of compound 14 thus obtained undergoes glycosidation with 5-azido-hexanol to provide compound 16. [ The yield of Compound 16 thus obtained is in the range of 65% to 75%, more specifically 70%. The compound 16 is treated with a silyl group deprotecting reagent such as TBAF in a THF solvent to provide the compound 17 by removing the sterically hindered -OTBDPS group. The reaction provides Compound 17 with a reaction yield ranging from 55% to 65%, and more specifically 60%, while increasing the alpha: beta ratio overall (95: 5). Compound 17 thus obtained serves as an intermediate in the synthesis of initiation unit 20 .

Subsequently, a portion of compound 9 is reacted with compound 15 in the presence of a catalyst such as TMSOTf at low temperature, more particularly in the range of -80 캜 to -60 캜 to provide a radio wave unit (compound 18 ).

The remaining portion of compound 9 is then reacted with compound 17 in the presence of a catalyst such as TMSOTf at low temperature, more specifically in the range of -80 캜 to -60 캜 to provide compound 19 .

Compound 19 thus obtained is reacted with a basic reagent such as NaOMe in the presence of methanol (MeOH) / dichloromethane (DCM) to provide the starting unit (Compound 20 ).

The initiation unit 20 and the propagation unit 18 are coupled together using a catalyst at a low temperature, more specifically in the range of -50 ° C to -30 ° C, to provide a saccharide or dimer unit 21 . The catalyst used in the coupling is, but is not limited to, NIS / TfOH / trimethylsilyl trifluoromethanesulfonate (TMSOTf) / silver trifluoromethanesulfonate (CF ₃ SO ₃ Ag) in the presence of a solvent. The catalyst activates the thiotolyl (Stol) group of compound 18 . After activation, this is easy to remove and the Stol group is replaced by the free hydroxy group of the initiating unit ( 20 ).

The dimeric unit ( 21 ) is produced as a protected dimer unit ( 22 ) by reacting with a basic reagent, such as, but not limited to, sodium methoxide to promote the ring opening reaction. The thus obtained dimer undergoes repetitive reactions under similar conditions in the presence of the catalyst and the basic reagent, and is then subjected to repeated reactions such as trimer, tetramer, trimer, trimer, lambda, 24, 26, 28 ... n Oligomers, and the like.

The thus obtained dimer unit ( 22 ) reacts with the radio wave unit under similar conditions of temperature and pressure to form trimer ( 23, 24 ). The thus obtained trimer reacts again with the propagation unit under similar conditions to provide the tetramer unit ( 25 ). The tetramer undergoes repetitive reactions under similar conditions to provide the advanced oligomer (Y), including tetramers, pentamers, terpolymers, lighter oligomers, and the like.

Thus advanced than the oligomer obtained is BF _3: OEt _2, it performs the final deprotection of the protecting group in the presence of a deprotecting reagent, such as ACN, NaOH, MeOH, H2 / Pd (OH) 2. The protecting groups are sequentially deprotected in the presence of the deprotecting reagent to produce the deprotected Men-Y advanced oligomers (d22, d24, d26, d28 .... dn). The total time required for the process ranges from 330 hours to 400 hours, more specifically 370 hours, and the production cost is significantly reduced.

The thus obtained advanced oligomer is linked to the carrier protein by conjugation through an endogenous linker to provide a semi-conjugate vaccine against meningococcal serogroup Y bacterial infection. Such chemical synthesis methods can also be used to prepare synthetic protein / peptide to produce fully synthetic conjugate vaccines. It should be noted that this conjugation with the carrier protein produces a vaccine with good yield and enhanced antigenicity.

In one of the above embodiments, the Men-Y tetramer is tested for antigenicity by performing an inhibitory enzyme-linked immunosorbant assay (inhibition ELISA), and the conjugate of the tetramer and tetanus toxoid is tested against Men- It has been found to neutralize certain antibodies.

Men-Y Tetramer  Complete reaction scheme for synthesis:

Scheme I: Synthesis of radio wave unit 18

The sialic acid Phosphate  Synthesis of Donor

Glucose  Synthesis of Receptors 15 and 17

Synthesis of constituent radio wave unit 18

Reaction Scheme II: Synthesis of Starting Unit 20

Scheme III: Synthesis of dimers, trimers and tetramers

Dimer , Trimer  And Tetramer  Synthesis of Units

The detailed description of the above method is illustrated by the following non-limiting examples:

Example :

Example  1: Step 1 - Preparation of compound 2

Procedure : Dowex 50W X8 resin is added to a stirred solution of N-acetylneuraminic acid (2.0 kg, 6.472 mol) in methanol (32 liters). The reaction is stirred at room temperature for 24 hours. The reaction is monitored by TLC (20% methanol; DCM). After completion, the reaction mixture was filtered and the resin was washed with methanol (2 liters). The filtrate was concentrated to give the required product as a white solid (2.0 kg; 95%).

Example  2: Step 2 - Preparation of compound 3

Procedure : To a mechanically stirred solution of compound 2 (1 kg, 3.095 mol) in pyridine (10 liters) was added DMAP (38 g, 0.0309 mol) at 0 占 and acetic anhydride (11.7 liters) was added. The reaction was stirred at room temperature for 24 hours. After completion, the reaction mixture was concentrated under vacuum (10 mbar). The crude compound was purified by flash column chromatography on silica-gel (230-400 mesh) eluting with 30% ethyl acetate-hexanes to 100% ethyl acetate. Compound 3 was obtained as a white foamy solid (1.4 kg; 87%).

Example  3: Step 3 - Preparation of compound 4

Procedure : A stirred solution of 3 (1.4 kg, 2.626 mol) in acetyl chloride (7.5 liters) was cooled to 0 C and a solution of the just prepared HCl in acetyl chloride (4.5 liters) was added. The resulting reaction mixture was allowed to warm to room temperature and stirred for 16 h. After completion, the reaction mixture was concentrated under reduced pressure. The obtained compound is co-distilled with toluene (2 * 2.5 liters) and the crude product is passed to the next step (1.4 kg, 99%).

Example  4: Step - Preparation of compound 5

Procedure : To a mechanically stirred solution of compound- 4 (1.35 kg, 2.652 mol) in DCM (13.5 liters) was added p-thiocresol (324 g, 2.652 mol). The solution was cooled to 0 < 0 > C and diisopropylethylamine (548 ml, 3.135 mol) was slowly added to the dropping funnel under a nitrogen atmosphere for 30 minutes. The resulting reaction mixture was allowed to warm to room temperature and stirred for 18 hours. The reaction was monitored by TLC and after completion, the reaction mixture was cooled to 0 C, diluted with DCM (5.0 liters) and washed with water (2 * 5 liters). The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated. The resulting compound was treated with 25% ethyl acetate / petroleum ether. The resulting white solid was filtered and dried (900 g, 60%).

Example  5: Step 5 - Preparation of compound 6

Procedure : Methanesulfonic acid (322 g, 3.35 mol) was slowly added to a stirred solution of compound- 5 (500 g, 0.8375 mol) in methanol (5.0 liters) at 0 < 0 > C. The resulting reaction mixture was heated to reflux for 36 hours. The reaction mixture was cooled to 0 C and quenched with triethylamine (790.0 ml). The mixture was concentrated and the residue was taken on to the next step without any further purification. LC MS purity 96%. (Crude weight: 650 g)

Example  6: Step 6 - Preparation of compound 7

Procedure: Compound-6 was dissolved in the mixture is mechanically stirred in acetonitrile (4.0 liters), water (5.0 liter) and _{NaHCO 3 (2.352 kg, 46.51 moles} ), (1.8 kg, 4.651 moles) at room temperature. The reaction mixture was cooled to 0 C using an ice bath and a solution of triphosgene (0.895 kg, 4.651 moles) in acetonitrile (1.0 liter) was added dropwise over a period of 60 minutes. The reaction was stirred at 0 < 0 > C for a further 3 h. After completion, the reaction mixture was neutralized with 10% HCl (3 liters). The product was extracted with ethyl acetate (2 * 4.0 liters). The organic layer was washed with water, brine and dried over Na ₂ SO ₄ and concentrated. The crude compound was dissolved in 2-5% methanol; Purification by flash column chromatography, eluting with DCM. Compound- 6 was obtained as an off-white solid (700 g, 56% for two steps).

Example  7: Step 7 - Preparation of compound 7A

Procedure : To a stirred solution of compound- 7 (700 g, 1.694 mol) in DMF (6.0 liters) was added imidazole (288 g, 4.237 mol). t-Butyl diphenylsilyl chloride (557 g, 2.033 mol) was slowly added thereto at 0 ° C for 10 minutes. The reaction temperature was raised to room temperature and stirred for 16 hours. The progress of the reaction was monitored by TLC. After completion, the reaction material was diluted with water (10 liters). The product was extracted with ethyl acetate (2 x 4 liters). The organic layer was separated, washed with brine and dried over Na ₂ SO ₄ and concentrated. The product was purified by flash column chromatography on silica gel using 60% ethyl acetate-hexane as eluent to give an off-white solid (1000 g, 90%).

Example  8: Step 8 - Preparation of compound 7B

Procedure : Camphorsulfonic acid (470 g, 2.036 mol) was added to a stirred solution of compound- 7A (1050 g, 1.697 mol) in 2,2-dimethoxypropane (7 liters). The mixture was stirred at room temperature for 3 hours. The progress of the reaction was monitored by TLC. After completion by TLC, the reaction mixture was quenched with triethylamine (350 mL; 2.515 mol). The reaction mixture was concentrated. The crude compound was purified by flash column chromatography on silica gel. Compound- 7B was obtained as an off-white foamy solid (1000 g, 92%).

Example  9: Step 9 - Preparation of compound 8

Procedure : Diisopropylethylamine (1500 ml, 8.538 mol) was added to a stirred solution of compound-7B (590 g, 0.8538 mol) in DCM (5.0 liters). The solution was cooled to 0 < 0 > C and acetyl chloride (490 ml, 6.83 mol) was slowly added to the dropping funnel under a nitrogen atmosphere for 10 minutes. The resulting reaction mixture was allowed to warm to room temperature and stirred for 18 hours. The reaction was monitored by TLC and after completion, the reaction mixture was cooled to 0 C, diluted with DCM (3 liters) and washed with water (3 liters). The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated. 10-20% ethyl acetate as eluent; Purification via silica column chromatography using hexane (yield: 500 g, 80%).

Example  10: Step 10 - Preparation of compound 9

Procedure : To a stirred mixture of compound 8 (100 g, 0.1364 mol) and dibutyl phosphate (54 mL; 0. 2728 mol) in DCM (1 liter) under a nitrogen atmosphere was added 4A molecular sieves 100 g, wt / wt) was added under high vacuum. The mixture was stirred at room temperature for 2 hours then cooled to 0 C and NIS (61.11 g, 0.2728 mol) followed by triflic acid (3.62 mL, 0.0409 mol). The reaction temperature was maintained at 0 < 0 > C for 5 hours. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was quenched with saturated hypo solution (250 ml). It was filtered through celite and extracted. The organic layer was separated, washed, dried and concentrated using a Na ₂ SO ₄ with water. The crude compound was purified by flash column chromatography on silica gel. Compound- 9 was obtained as a sticky substance (80.0 g, 70%).

Example  11: Step 11 - Preparation of compound 11

Process: β-D- glucose penta-acetate 10 (500 g, 1.2816 mol) , p- thio-cresol (255 g, 2.050mol) and 4Å minute dichloromethane self powder (500 g) under an argon atmosphere in a flask equipped with a flame dried (5 liters). The solution was cooled to _{0 ℃, BF 3 · Et 2} O; was added (49% 1000 ml, 3.5886 mol ). The mixture was stirred for 16 hours. The reaction mixture was diluted with dichloromethane (2 liters) and washed with sodium bicarbonate (2 x 2.5 liters saturated aqueous) followed by water (3 liters). Dry the organic layer over Na ₂ SO _4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to afford 11 (417 g, 72%) as a white solid.

Example  12: Step 12 - Preparation of compound 12

Procedure : Compound 11 (417 g, 0.9182 mol) was dissolved in methanol (4.0 liters) under an argon atmosphere in a flame dried round bottom flask. A solution of sodium methoxide in methanol (39.6 ml of a 25% solution) was added and the resulting mixture was stirred for 2 hours. After completion, Amberlite IR-120 ⁺ ion exchange resin was divided into fractions until the solution became neutral. The reaction mixture was filtered to remove the resin and the filtrate was concentrated in vacuo to give compound 12 (254 g, 95%) as a white crystalline solid.

Example  13: Step 13 - Preparation of compound 13

Procedure : Imidazole (114 g, 1.678 moles) was added to a stirred solution of compound- 12 (200 g, 0.402 moles) in DMF (2.0 liters). To the solution was slowly added t-butyldiphenylsilyl chloride (269 g, 0.979 moles) at O < 0 > C for 10 min. The reaction temperature was raised to room temperature and stirred for 16 hours. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was diluted with water (4 liters). The product was extracted with ethyl acetate (2 * 1 liter). The organic layer was separated and washed with brine, dried over Na ₂ SO ₄ and concentrated. The product was purified by flash column chromatography on silica gel using 60% ethyl acetate-hexane as eluent to give an off-white solid (260 g, 72%).

Example  14: Step 14 - Preparation of compound 14

Procedure : A stirred mixture of 13 (260 g, 0.4962 mol) and benzyl bromide (273 ml, 2.2328 mol) in DMF (2.5 L) was cooled to 0 C and sodium hydride (99 g, 2.480 mol) . The reaction material was stirred at room temperature for 16 hours. TLC showed the starting material to be consumed, then the reaction was quenched with ice cold water (4 L) and extracted with ethyl acetate (2 x 1 L). The organic layer was separated and concentrated under reduced pressure. The crude compound was purified by flash column chromatography and the product was eluted with 5% ethyl acetate-hexane to give compound 14 as a colorless liquid (300 g, 76% yield).

Example  15: Step 15 - Preparation of compound 15

Procedure: dissolve in the compound 14 (145 g, 0.1826 mol) to THF (1.4 liters) was cooled to 0 ℃, it was added 1M TBAF in THF (219 mL, 0.2191 mol) . The cold bath was removed and the reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC and after completion the reaction was diluted with water and extracted with ethyl acetate (2 * 500 ml). The organic layer was dried over Na ₂ SO _4, filtered and concentrated. The crude product was purified by FCC and eluted with 20% EtOAc / hexane. The compound was obtained as a viscous liquid (90 g, 89%).

Example  16: Step 16 - Preparation of compound 16

Procedure : To a stirred mixture of compound 14 (50 g, 0.0629 mol) and 6-azido hexanol (18 g; 0.1259 mol) in diethyl ether (500 ml) under a nitrogen atmosphere was added 4A molecular sieves 50 g, wt / wt) was added under high vacuum at 140 ° C. The mixture was stirred at room temperature for 2 hours and then cooled to -40 < 0 > C. NIS (28.2 g, 0.1259 mol) followed by triflic acid (2.8 ml, 0.0314 mol) was added and stirring was continued for 2 hours. After completion by TLC, the reaction mixture was quenched by the addition of saturated hypo solution (100 ml). The reaction mixture was filtered through celite, and the filtrate was transferred to a separatory funnel. The organic layer was separated, washed with water and dried over Na ₂ SO ₄ and concentrated. The crude compound was purified by flash column chromatography eluting with 10% ethyl acetate and hexane. Compound- 16 , anomeric mixture was obtained as a sticky substance (35 g, 70%).

Example  17: Step 17 - Preparation of compound 17

Procedure : Compound 16 (100 g, 0.123 mol) was dissolved in THF (1.0 liter), cooled to 0 C and 1M TBAF in THF (148 mL, 0.1476 mol) was added. The cold bath was removed and the mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion, the reaction was diluted with water and extracted with ethyl acetate (2 x 500 ml). Dried, filtered, the organic layer over Na ₂ SO ₄ and concentrated. The crude product was purified by flash column chromatography eluting with 20% EtOAc / hexane. The compound was obtained as a viscous liquid (55 g, 78%).

Example  18: Step 18 - Preparation of compound 18

Procedure : A mixture of compound 15 (43.3 g, 0.0778 mol) and compound 9 (84 g; 0.1307 mol) was dissolved in DCM (430 ml) and 4 Å molecular sieves (45 g) were added. The reaction mixture was stirred at room temperature for 2 hours. It was then cooled to -78 < 0 > C and TMSOTf (15.58 mL, 0.0855 mol) was added. The reaction was maintained at -78 < 0 > C and stirred for 1 hour. The progress of the reaction was monitored by TLC and after completion the reaction mixture was quenched with triethylamine (1.2 eq) and filtered through celite. The organic layer was washed with saturated sodium bicarbonate solution (300 mL), The organic layer was separated and washed with water and brine solution and dried over Na ₂ SO _4, filtered and concentrated. The crude compound was purified by flash column chromatography eluting with 30% ethyl acetate and hexanes to give compound- 18 as a white solid (85 g, 93%).

Example  19: Step 19 - Preparation of compound 19

Procedure : To a mixture of compound 17 (45 g, 0.0778 mol) and compound 9 (85 g; 0.1037 mol) in DCM (450 mL) was added 4 A molecular sieves (45 g) and the resulting reaction mixture was stirred at room temperature And stirred for 2 hours. It was then cooled to -78 < 0 > C and TMSOTf (15.66 mL, 0.0855 mol) was added. Stirring was continued at the same temperature for 2 hours. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with triethylamine (1.2 eq) and filtered through celite. Then the organic layer into a saturated sodium bicarbonate solution (500 mL) was washed with water and brine solution and dried sikimyeo filtered over Na ₂ SO ₄ and concentrated. The crude compound was purified by flash column chromatography eluting with 30% ethyl acetate and hexanes to give compound- 19 as a white foamy solid (80 g, 86%).

Example  20: Step 20 - Preparation of compound 20

Procedure : Compound 19 (80 g, 0.0676 mol) was dissolved in methanol (533 mL) and DCM (267 mL) under an argon atmosphere. A solution of sodium methoxide in methanol (14.6 mL, 25% NaOMe in methanol) was added and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the ion exchange resin (IR 120+) was divided into fractions until the solution was neutralized. The reaction mixture was filtered and the filtrate was concentrated and the crude product was purified by flash column chromatography (ethyl acetate: petroleum ether 2: 1 as eluent) to yield compound 20 (55 g, 70% As a white foamy solid.

Example  21: Step 21 - Preparation of compound 21

Procedure : To a stirred solution of compound 18 (53 g, 0.0453 mol) and compound 20 (35 g; 0.0302 mol) in diethyl ether (350 mL) was added 4A molecular sieves (35 g) Lt; / RTI > The mixture was cooled to 0 < 0 > C. NIS (13.54 g, 9.030 mmol) was added all at once to the mixture followed by triflic acid (1.34 mL, 0.0151 mol). The reaction temperature was raised to room temperature and held for 16 hours. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with saturated sodium thiosulfate solution (250 ml) and diluted with ethyl acetate (300 ml). It was filtered through celite and the organic layer was separated. Sikimyeo washing the organic layer with water, dried over anhydrous Na ₂ SO ₄ filtered and concentrated. The crude compound was purified by flash column chromatography eluting with 30% ethyl acetate and hexane. Compound- 21 was obtained as a white solid (32 g, 48%).

Example  22: Step 22 - Preparation of compound 22

Procedure : Compound 21 (10 g, 4.5454 mmol) was dissolved in methanol (65 mL) and DCM (35 mL) under an argon atmosphere in a flame dried flask. A solution of sodium methoxide in methanol (0.982 mL, 25% in methanol) was added and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture, quenched with Amberlite IR 120, was divided into fractions until the solution was neutralized. The reaction mixture was filtered and the filtrate was concentrated and purified by flash column chromatography (ethyl acetate: petroleum ether 2: 1 to 3: 1 as eluent) to give compound 22 (6.1 g, 62% Obtained as a foamy solid.

Example  23: Step 23 - Preparation of compound 23

Procedure : To a stirred solution of compound 22 (15 g, 6.899 mmol) and compound- 18 (12 g; 10.349 mmol) in anhydrous diethyl ether (150 mL) was added 4A molecular sieves (15 g) And the mixture was stirred at room temperature for 2 hours. The mixture was cooled to 0 < 0 > C. To the mixture was added NIS (3.1 g, 13.790 mmol) followed by triflic acid (0.306 ml, 3.450 mmol). The resulting reaction mixture was stirred at room temperature for 16 hours. After completion by TLC, the reaction mixture was quenched by the addition of saturated sodium thiosulfate solution (150 ml). The reaction mixture was filtered through Celite, and the filtrate organic layer was washed with water and brine, dried over anhydrous Na ₂ SO ₄ sikimyeo filtered and concentrated. The crude compound was purified by flash column chromatography, eluting with 50% ethyl acetate and hexane. Compound- 23 was obtained as a white foamy solid (12 g, 41%).

Example  24: Step 24 - Preparation of 24

Procedure : Compound 23 (12 g, 3.732 mmol) was dissolved in methanol (80 mL) and DCM (40 mL) in an argon atmosphere in a flame dried flask. A solution of sodium methoxide in methanol (0.806 mL, 25%) was added and the mixture was stirred at 0 < 0 > C for 1 hour. After completion by TLC, the reaction mixture was quenched by dividing the ion exchange resin (IR 120+) into fractions until the solution was neutralized. The reaction mixture was filtered and concentrated and the crude product was purified by flash column chromatography (ethyl acetate: petroleum ether 2: 1 as eluent) to give 24 (7 g, 60%) as a white foamy solid .

Example  25: Step 25 - Preparation of compound 25

Procedure : 4A molecular sieves (4.1 g) were added to a stirred solution of compound 24 (4.1 g, 1.4733 mmol) and compound 18 (3.1 g; 2.6505 mmol) in diethyl ether (40 mL) Lt; / RTI > NIS (0.34 g, 1.5175 mmol) was added all at room temperature and then triflic acid (0.13 mL, 1.4733 mmol) was added. Stirring was continued at room temperature for 60 hours. The progress of the reaction was monitored by TLC (35% ethyl acetate: hexane). Upon completion, the reaction mixture was quenched with saturated sodium thiosulfate solution (40 mL). It was filtered through celite and the organic layer was separated. Sikimyeo washing the organic layer with water, dried over anhydrous Na ₂ SO ₄ filtered and concentrated. The crude compound was purified by flash column chromatography, eluting with 20-30% ethyl acetate and hexane. Compound- 25 was obtained as a white solid (2.06 g, 38%).

Example  26: Step 26 - Preparation of compound 25A

Procedure : Compound- 25 (3 g, 0.7088 mmol) was dissolved in methanol (624 mL) and DCM (12 mL) under an argon atmosphere. A solution of sodium methoxide in methanol (0.184 mL, 25%) was added and the mixture was stirred at room temperature for 1 hour. After completion by TLC, the reaction mixture was quenched using ion exchange resin Amberlite IR 120 until the solution was neutralized. The mixture was filtered and concentrated. The crude product was purified by flash column chromatography (ethyl acetate: petroleum ether 2: 1) to provide compound 25A (1.7 g, 58%) as a white solid.

Example  27: Step 27 - Preparation of compound 25B

Procedure : Compound 25A (1.7 g, 0.4040 mmol) was dissolved in acetonitrile (45 mL) and cooled to 0 <0> C. 45% BF ₃ ; OEt ₂ (7.65 mL, 24.2453 mmol) was added and stirring was continued for 3 h. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with saturated aqueous NaHCO ₃ (80 mL) followed by brine. It was then dry the organic layer over Na ₂ SO _4, filtered and concentrated. The residue was washed with hexane and the resulting solid product obtained was taken up in ethanol and water. Lithium hydroxide was added (0.17 g, 4.0408 mmol) and the reaction was stirred overnight at 80 &lt; 0 &gt; C. Removing the solvent under reduced pressure; The crude material was dissolved in water (20 mL), washed with diethyl ether (20 mL), and the aqueous layer was separated. The pH of the aqueous layer was adjusted to 5 with 1N HCl (0.4 mL) solution. The product was extracted with 10% methanol: DCM (2 * 60 mL). And dried and concentrated the organic layer over Na ₂ SO ₄ to give the compound 25B (0.8 g; 80% for two steps)

Example  28: Step 28 - Preparation of compound d26

Procedure : Compound- 25B (0.8 g, 0.2638 mmol) was dissolved in a mixture of methanol (50 mL) and water (12 mL) in a 250 mL hydrogenation flask. 20% Pd (OH) _{2 /} C (0.8 g) was added under a nitrogen atmosphere. The flask was purged with hydrogen 3 times and the contents were stirred under hydrogen atmosphere (100 psi) for 24 hours. After completion, the reaction mixture was filtered through celite and concentrated under reduced pressure at 25 &lt; 0 &gt; C. The product was purified by gel filtration through a Sephadex-G-10 column, eluting with water. The compound fractions were collected and lyophilized to give the Men-Y tetramer (d26) as a white solid (350 mg; 70%).

Example  29: Men-Y Tetramer  And Men-Y Tetramer -tetanus Toxoid ( TT ) Antigenicity analysis of conjugate

Procedure: ELISA plates were coated with the bacterial polysaccharide, which thio-ethers of different concentrations prepared using chemical Men-Y of (0.39-100 ㎍ / ml), or tetramer, or suppressed to a Men-Y tetramer -TT conjugates React with elevated polyclonal sera against unconverted anti-Men-Y bacterial capsular polysaccharide. When the reaction is developed using a HRP labeled secondary antibody, both the Men-Y tetramer and its conjugate neutralize the Men-Y specific IgGs, as shown in Figure 9, (% Inhibition) after serum dilution adhesion with different antigen concentrations.

Claims (14)

As a novel method for synthesizing oligomers of Neisseria meningitidis serogroup Y clathrate polysaccharide repeat units,
(a) synthesizing the radio wave unit 18 ,
(b) synthesizing the initiator unit ( 20 )
(c) coupling the starting unit ( 20 ) with the propagation unit ( 18 ) in the presence of at least one catalyst and at least one basic reagent to synthesize a more advanced oligomer,
(d) repeating the reaction of step (c) in the presence of the catalyst and the basic reagent to obtain more advanced synthetic oligomers (22, 24, 26, 28 ..., n)
(e) the high-grade synthetic oligomer of step (d) is deprotected sequentially in the presence of at least one deprotecting reagent to form a deprotected Men-Y advanced oligomer (d22, d24, d26, d28 .... dn) &Lt; / RTI &gt;
Lt; / RTI &gt;
Thereby producing novel advanced synthetic oligomers with higher yields and higher purity. A novel method of synthesizing an oligomer of repeating unit of Nseleria meningitidis serogroup Y clathrate polysaccharide according to claim 1, wherein said high-grade synthetic oligomer (Y) is a novel tetramer ( d26 ). A novel method for synthesizing an oligomer of a repeating unit of Nselia meningitidis serogroup Y clathrate polysaccharide according to claim 2, wherein the time required for synthesizing the tetramer is in the range of 330 to 400 hours, more preferably 370 hours . 2. The apparatus according to claim 1, wherein said initiation unit (20)
(a) by a known method, by methylated monosaccharides compound 1 in the presence of a catalyst to give compound 2, by acylating the compound 2 in the presence of an acylating reagent to give the compound 3 and halide in the presence of a halogenating agent with Compound 3 to give the compound 4, by reacting the compound 4 with a thiol reagent to yield a compound 5, and to the compound 5 reacts with the deacylation reagent to give the compound 6, it is reacted with a carbonylation reagent to compound 6 to yield compound 7, and compound 7 the reaction and the protecting reagent to obtain a compound 8 and by phosphorylation reaction of the compound 8 to give compound 9;
(b) and by a known method, a commercially available compound 10 is reacted with an acceptor and a catalyst to give the compound 11, and by treatment of the compound 11 as a deacetylation reagent to give compound 12, and treating the compound 12 with a protecting group of the compound 13 And alkylating compound 13 to obtain compound 14 ;
(c) glycosidating compound 14 with an alcohol in the presence of at least one catalyst to obtain compound 16 ;
(d) treating compound 16 with at least one deprotecting reagent to obtain compound 17 ;
(e) reacting Compound 17 and Compound 9 in the presence of the at least one catalyst to obtain Compound 19 ; And
(f) treating compound 19 with at least one basic reagent to obtain the disclosure unit compound 20
A novel method for synthesizing an oligomer of repeating unit of Nselia meningitidis serogroup Y cymolytic polysaccharide, The method of claim 1, wherein the spread unit 18 is
(a) treating compound 14 with at least one deprotecting reagent to obtain compound 15 and
(b) reacting the compound 15 with the compound 9 in the presence of the at least one catalyst to obtain a radio wave unit 18
A novel method for synthesizing an oligomer of repeating unit of Nselia meningitidis serogroup Y cymolytic polysaccharide, 6. A novel method of synthesizing an oligomer of a Nisseria meningitidis serogroup Y clathrate polysaccharide repeat unit according to any one of claims 1, 4 and 5, wherein the catalyst is selected from NIS, TfOH, TMSOTf. 5. The novel method of claim 4, wherein the alcohol is 6-azidohexanol, an oligomer of repeating unit of Nselia meningitidis serogroup Y-capped polysaccharide. The method of claim 1, wherein the deprotecting reagent is selected from BF ₃ : OEt ₂ , ACN, NaOH, MeOH, H ₂ / Pd (OH) ₂ , oligomers of repeating units of Nselia meningitidis serogroup Y- Lt; / RTI &gt; The novel method of claim 4, wherein the monosaccharide is neuraminic acid, more particularly N-acetylneuraminic acid. The novel method according to claim 4 or 5, wherein the deprotecting reagent is selected from TABF, THF, CSA, HCl, PTSA, oligomers of repeating units of Nselia meningitidis serogroup Y condensing polysaccharide. 5. The novel method of claim 1 or 4, wherein the basic reagent is selected from NaOMe, NaOEt, KOtBu, and oligomers of repeating units of Nselia meningitidis serogroup Y clathrate polysaccharide. The novel method of claim 1, wherein the novel high-molecular-weight synthetic oligomer (Y) is a synthetic oligomer having high purity and improved antigenicity. The oligomer of claim 1, wherein the novel high-grade synthetic oligomer (Y) is a tetrahedra ( d26 ) having a purity of more than 95% and an improved efficiency of the formula &Lt; / RTI &gt;

The present invention relates to a novel high grade of Nyserial meningitidis serogroup Y clathrate polysaccharide repeat unit, prepared by the method according to claim 1, which can be used as a candidate in the development of a semisynthetic or fully synthetic conjugate vaccine against meningococcal serogroup Y bacterial infection Synthetic oligomers.

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