DESC:FIELD OF THE INVENTION:
The present invention relates to a novel compound of Haemophilus influenzae type b (Hib) oligosaccharides. The present invention also relates to a chemical process of synthesis of the Hib oligosaccharides /oligomers and its use as a candidate for development of conjugate vaccine against Hib infections. The present invention further relates to a novel linker and its chemical synthesis.

BACKGROUND OF THE INVENTION:
Haemophilus is a gram-negative, an aerobic bacteria belonging to Pasteurellaceae family. It is a pleomorphic bacterium with a general cocci shape, which causes an array of diseases such as meningitis, epiglottitis, pneumonia, septic arthritis, and respiratory tract infections such as bronchitis and otitis media. Haemophilus has six capsular serotypes starting from (a) till (f). Of these six types, the serotype b usually designated as Haemophilus influenzae type b or Hib in short has been found to be the most invasive serotype affecting mammal especially human infants in the age group of 9 months to 18 months.

Hib poses serious health issues for many countries around the world especially the developing countries. It has been reported that it accounts for 3 million cases and 386,000 annual childhood deaths (WHO, 2006).

Immune response to any antigen may be characterised as T-cell dependent (TD) immune response or T-cell independent (TI) immune response. Proteins and peptides are known to elicit TD antigens by stimulating the helper T lymphocytes and generating memory T and B lymphocytes. In contrast, polysaccharides generate the TI antigens which do not involve T-cell activation and do not form any memory B cells which is the major drawback, while dealing with infants as they have an immature immune system.

The Hib polysaccharide capsule which is a repeating polymer of ribosyl ribitol phosphate (PRP) is responsible for the virulence of the bacteria and provides the resistance against the immune response of the host.

Vaccines based on purified polysaccharide of Hib are able to induce protective immunity in adults, but it is observed that the vaccine is not very efficient in children below two years of age. This lead to further research which explored the covalent linking (conjugating) of the polysaccharide to a protein (also known as carrier) resulting in the formation of a conjugated vaccine. Such conjugated vaccines have been found to be effective in infants. The small sized PRP (oligosaccharides) have also been successfully used in developing Hib conjugate vaccines.

Looking at the costly and cumbersome production and purification procedures of Hib PRP using bacterial fermentation, several methods have been deployed for preparing synthetic Hib- oligosaccharide (Hib oligomers). For instance, the US 6,765,091 titled “Oligosaccharides derived from ribose-ribitol-phosphate, and vaccines containing them” describes a method for the chemical synthesis of oligosaccharide mixtures derived from polyribosyl ribitol phosphate that corresponds to the Hib capsular polysaccharide with a spacer molecule. The process leads to generation of mixture of oligomers of varied chain lengths. The patent application no. EP 0320942 (A2) teaches the synthesis of oligomeric subunits of PRP found in Hib. It further teaches a method of inducing an immune response which is T-cell dependent. The study focuses on the requirement of insertion of a spacer between the synthetic PRP of dimeric or trimeric chains and the carrier proteins. The process of synthesis requires multiple deprotection steps.

Presently the oligomers have been found to be a useful tool for vaccination against Hib. But the known oligomers made so far are made using solid phase synthesis or the existing systems for the synthesis reported are either time consuming or give rise to the mixture of different sizes of oligomers.

OBJECT OF THE INVENTION:
Thus the main object of present invention is the synthesis of the Hib- oligosaccharides/ oligomers.

Another object of the present invention is the synthesis of Hib- oligosaccharides/ oligomers in a solution phase.

Yet another object of the present invention is a synthetic pathway using purified oligomers with specific chain length e.g. a tetramer that may be used to provide conjugate vaccine with better reproducibility.

Yet another object of the present invention is a linker that mimics part of the sugars to provide the maximum number of epitopic sites.

Yet another object of the present invention is to provide a process for the preparation of synthetic Hib-PRP oligomer which meets the expected physico-chemical quality standards for the purity.

Yet another object of the invention is its cost effectiveness and improved antigenicity.

SUMMARY OF THE INVENTION:
Accordingly, the present invention provides for the process of synthesizing oligosaccharides (oligomers) of ribosyl ribitol phosphate. In one embodiment, the ribitol unit which is a component of the Hib-tetramer is prepared starting from sugars such as D-ribose with good yields. The initiation and propagation units are prepared by glycosidation of tetra-acetate protected D-ribose along with suitably protected ribitol. In order to construct the final structure of Hib oligomer the initiation and propagation units are combined using tetrazole coupling reagent.

The subsequent propagation units are added by using tetrazole coupling with initiation units. Finally, linker is coupled to the oligomer using tetrazole coupling reaction. The benzyl protecting groups are removed by catalytic hydrogenation to achieve the final product.

The present invention discloses solution phase synthesis of uniform Hib oligosaccharide unit of defined length. These units when used to prepare vaccines ensure that there is a minimal batch to batch variation in size of the final conjugate during the conjugation with protein. This also ensures that there is enhanced antigenicity. Presently, vaccines reported so far, consist of a range of number of monomer units per oligo/polysaccharide antigen leading to batch to batch variation.

Further the present invention provides for a novel linker which mimics the sugars to provide the maximum number of epitopic sites. A long chain linker is used which avoid the steric hindrances. In addition, the position of linker is suitably optimized, thereby enhancing the immunogenicity as well as the yield of the conjugate vaccine.

Brief Description of Drawings:
Figure 1(a) depicts the structural analysis of the Hib-tetramer by 1H NMR.
Figure 1(b) depicts the structural analysis of Hib-tetramer by 13C NMR.
Figure 2 depicts the purity analysis of Hib-tetramer by High Performance Liquid Chromatography (HPLC).
Figure 3 depicts the graphical representation of Inhibition ELISA with various antigens.

Detailed Description of Invention:
The present invention relates to the chemical synthesis of the Hib-oligomers more particularly Hib tetramer and its use as a candidate for development of conjugate vaccine against Haemophilus influenzae type b (Hib) infections. The oligosaccharide obtained by the chemical synthesis of the present invention, comprise repeating units of phosphate–ribose-ribitol or ribosyl–ribitol-phosphate. This synthesis of oligomers is based on the structure of the Hib capsular polysaccharide, the said synthesis being accomplished in the following steps:
1. synthesis of Ribitol Unit, as shown in Scheme 1,
2. synthesis of Initiation Unit and Propagation Unit, as shown in Scheme 2,
3. synthesis of Linker Unit, as shown in Scheme 3, and
4. synthesis of Hib-Tetramer with linker also referred to as Hib oligomer, as shown in Scheme 4

The term oligosaccharides and oligomers are used interchangeably for the purposes of this specification.
The invention is not limited to the particular materials described in the examples or specific embodiments. Further, the terminology used herein is for the purpose of describing the particular embodiment only, and is not intended to limit the scope of the present invention in any way. It must be noted that unless as defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention belongs.

Synthesis of Ribitol Unit:
In one embodiment of the invention, the protected ribitol unit compound 9 is synthesized by a known method which is shown in Scheme 1. The process of preparing the ribitol unit which is the starting point of the present invention has been disclosed by Bencomo et al (US 6,765,091 also ‘091). The Scheme 1 shows the schematic path followed by the synthesis of protected ribitol backbone.

As disclosed in ‘091, the compound 2 shown in Scheme 1 is prepared by reacting a monosaccharide preferably D-ribose with 2,2-dimethoxy propane (2,2-DMP) in acetone. The 5-hydroxyl group at the 5’ position of compound 2 is allylated by reacting with allyl bromide and aqueous sodium hydroxide to produce compound 3. The acetonide group in compound 3 is deprotected by treatment with sulfuric acid in methanol resulting in compound 4; thereafter the benzyl protection of the resulting hydroxy groups is carried out by reaction with benzyl chloride and sodium hydride in dimethylformamide to give compound 5. The anomeric methyl group in compound 5 is hydrolysed by reacting with hydrochloric acid and the resulting hemiacetal of compound 6 is reduced using sodium borohydride in ethanol to give partially protected ribitol compound 7. The primary hydroxy group of compound 7 is selectively protected as its trityl. This is achieved by the treatment of said primary hydroxy group of compound 7 with tritylchloride and pyridine to give compound 8, which is then subjected to benzyl protection of its free hydroxy group by using benzyl chloride and sodium hydride in dimethylformamide to obtain compound 8a. Finally the trityl group deprotection in compound 8a provides selectively protected ribitol unit, compound 9. It is this protected ribitol unit, compound 9 which serves as the backbone for the synthesis of oligomeric units having repeating units of phosphate–ribose-ribitol or ribose–ribitol-phosphate.

Scheme 1. Synthesis of Ribitol Unit

Synthesis of Initiation Unit and Propagation Unit
The protected ribitol unit synthesized in Scheme 1 act as a backbone for the synthesis of the initiation unit and the propagation unit. The synthetic pathway employed in synthesizing Hib-PRP uses a different chemistry as disclosed in the prior art and provide purified saccharides which require less number of steps and thus is quick and easy to scale.

In one of the embodiment, the initiation unit and the propagation unit are prepared by the glycosylation of the acetate protected ribose compound 10 with ribitol and their subsequent transformations as shown in Scheme 2.
The protected ribitol unit compound 9 obtained from Scheme 1 is then reacted with acetyl protected ribose 10 to obtain the glycosyl ribitol preferably ß- glycosyl ribitol compound 11. The obtained ß- glycosyl ribitol is subjected to the treatment with sodium methoxide to remove the acetates resulting in compound 12. The compound 12 serves as a source / originator for both of the initiation unit as well as the propagation unit.

The initiation unit is synthesized by subjecting the hydroxy groups in compound 12 to benzylation using benzyl bromide, catalytic TBAI (Tetrabutylammonium iodide) and sodium hydride in tetrahydrofuran and dimethylformamide mixture resulting in the formation of compound 12A. Further, the allyl group in compound 12A is deprotected selectively by using catalyst such as but not limited to palladium chloride in organic solvent like methanol to give the initiation unit 15.

The propagation unit 14 is prepared by selectively protecting the hydroxy groups of compound 12 using a reagent having the property of regioselective protection such as but not limited to dibutyltin oxide and benzyl chloride to give compound 13. The allyl group in compound 13 is then deprotected using palladium chloride and the resulting hydroxy compound is protected as its dimethoxytrityl (DMT ether) to give compound 13B. This trityl derivatized compound 13B is subjected to phosphorylation which leads to the phosphorylation of the free hydroxy in compound 13B and resulting into formation of propagation unit 14.

Scheme 2. Synthesis of Initiation Unit and Propagation Unit

Once the initiation unit 15 and the propagation unit 14 are ready, the platform is set for the coupling of these two units. The coupling of these two units results in formation of dimer, trimer, tetramer and higher oligomers. The higher oligomers so formed are then linked to carrier proteins with the aid of linker. A novel linker is synthesized that mimics the natural sugars to provide the maximum number of epitopic sites by leaving a part of the Hib PRP monomeric unit in it.
This attachment of the oligomers with the linker is shown in Scheme 4.

Synthesis of Linker Unit:
In one of the embodiment as shown in Scheme 3, the azido alcohol compound 2A is reacted with a monosaccharide moiety preferably ribose compound 10 in presence of catalyst such as iodine in a solvent such as but not limited to Tetrahydrofuran (THF) to provide compound 3A. The product so obtained i.e. 3A exists in both alpha as well as beta anomeric forms. Either or both the anomeric forms or mixture thereof can be utilized for the preparation of linker unit, but preferably the beta anomers are selected, as the beta anomers mimics natural PRP orientation which results in higher yield.
The acetate groups of the beta anomer of compound 3A are deprotected using a deprotonated base followed by selective benzyl protection using dibutyltin oxide, sodium hydride, benzyl chloride and tetrabutylammonium iodide with alcohol resulting in compound 5A. The azide group present at either of the end in 5A is selectively reduced by hydrogenation using palladium over carbon as a catalyst in methanol to provide amine in compound 5B and then protected as its Cbz protection to give compound 5C. Finally alcohol moiety of 5C is phosphorylated using THF solvent providing solution phase for the reaction mixture such as but not limited to 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite to give linker 6A. This linker unit thus obtained mimics the sugars to provide the maximum number of epitopic sites in the oligomer and hence has potential for greater immunogenicity.

Scheme 3. Synthesis of Linker Unit

Synthesis of Tetramer and higher oligomers thereof:
In one of the embodiment, the initiation unit 15 is reacted with propagation unit 14 using tetrazole followed by oxidation using iodine, which is further subjected to acidification and mark the production of the dimer compound 16. The dimer compound 16 so obtained is now ready for further couplings. The dimer compound 16 is reacted with the propagation unit 14 using identical coupling conditions and followed by acid treatment to obtain trimer compound 17. The trimer compound 17 is further coupled with propagating unit under identical conditions followed by acid treatment provided the tetramer compound 18. The tetramer compound 18 is now ready for attachment of linker 6A.

The full scheme of the synthesis of tetramer is shown below as Scheme 4

Scheme 4. Synthesis of Tetramer
Finally the linker compound 6A is reacted with tetramer unit 18 using tetrazole coupling reagent and followed by oxidation using iodine resulting in compound 19A as shown in Scheme 4. The final task is the deprotection of protecting groups. The compound 19A is first reacted with 20% to 40% ammonium hydroxide to remove cyano ethyl groups over phosphate. The benzyl group’s deprotection is carried out using 10% to 20% palladium over carbon and 70 psi H2 pressure in mixture of methanol, ethyl acetate and water. This reaction after 36 hours just provided the partially deprotected product. For complete deprotection of benzyl groups in the compound 19A, the said compound is subjected to treatment with 20% Pd (OH)2 in methanol/water/ethyl acetate in the ratio of 3:1:1 at 70 psi H2 pressure, which provides the clean deprotection in the range of 20 to 40 hours. The crude product is purified using sephadex G-25 eluting with water which provides the pure product tetramer as Hib –tetramer 20. This strategic removal of protecting groups in one step makes the process highly efficient and provides good yield.

This purified Hib-tetramer 20 can now be conjugated with the carrier proteins which can serve as a potential candidate for vaccine against Haemophilus influenzae type-B.

Determination of antigenic properties of synthetic Hib tetramer and Hib tetramer-TT conjugates.

The antigenicity of different Hib-tetramer-TT conjugates is compared with no-antigen control in a competition enzyme-linked immunosorbent assay (ELISA). In this assay, 100 fold diluted rat serum containing anti-PRP antibodies is incubated for 1 hour at 37 °C with different antigens (Hib-tetramer and tetramer-TT conjugates) at 10 µg/mL diluted in phosphate-buffered saline (PBS) containing 0.3 % v/v Tween 20, 10 mM ethylenediamine tetraacetic acid (EDTA) and 1% BSA (Bovine serum albumin) in 96 well micro titer plate (Plate A). The serum is generated from pool of sera of rats immunized with a licensed Hib conjugate vaccine.
A separate plate (plate B) is coated with Hib oligosaccharide – Human Serum Albumin (HbO-HA) conjugate and subsequently blocked with 1% BSA after overnight incubation at 2 °C – 8 °C. To this plate B, serum-antigen mix from plate A is added and incubated for 90 minutes at room temperature. The plate is washed with PBS at pH 7.4 containing 0.05% Tween 20 and 10 mM EDTA. The plate is then incubated for 90 minutes at room temperature with peroxidase labelled anti-rat IgG antibodies in PBS with 0.3% Tween 20, 10 mM EDTA, and 1% BSA. Plates are washed again and incubated for 10 minute at room temperature with the 100 µl peroxidase substrate, 3,3’,5,5’ – tetramethylbenzidine-H2O2 in sodium acetate buffer. The reaction is stopped by adding 50 µl of 2 M H2SO4. The A450 was recorded on an ELISA reader (Tecan micro plate reader).
The antigenicity of two Hib tetramer-TT conjugates is compared with no antigen control and tetramer control by competition (inhibition) ELISA. As shown in Figure 3; both unconjugated and conjugated tetramer are able to neutralize the anti-PRP antibodies and inhibit the binding of antibodies to the coated PRP on plate indicating that the tetramer and its conjugates are antigenic and are able to neutralize the Hib antibodies in rat sera. However, conjugated tetramer showed comparatively much high inhibition as compared to the unconjugated PRP indicating the presentation of epitopes in tetramer-TT conjugate similar to the licensed vaccine against which the antibodies were raised.

The present invention is disclosed and illustrated by the way of examples below but does not limit the scope of invention.

Examples:
Synthesis of Ribitol Unit:
Step 1: Synthesis of compound 2.

In a 50 lit reactor, 25 lit of acetone is charged at room temperature. This is followed by methanol (4 lit), 2.2-dimethoxypropane (4 lit) and D-ribose (2.0 kg; 13.33 mol). The reaction mixture is cooled to 0 °C using ice-bath. In another flask, a solution of hydrochloric acid (HCl) in methanol is prepared by passing dry HCl through methanol under ice-cold condition. The flask is weighed after 4 h to check the amount of HCl gas absorbed. The HCl solution thus prepared above (2.5 lit, 2 molar) is added to the reaction mixture at 0°C. The reaction mixture is allowed to stir for 16h. The resulting orange solution is neutralized with pyridine (2 lit) (up to pH became neutral). The clear solution is evaporated under vacuum to obtain yellow oil. This oil is partitioned between water (4 lit) and ethyl acetate (10 lit). The organic layer is separated and dried over anhydrous sodium sulphate. Evaporation yielded a product 2 as pale yellow oil (2.5 kg, 60%). It is characterized by 1H nuclear magnetic resonance (1H NMR).

STEP 2: Synthesis of compound 3

In a 4 necked 10 lit round bottom flask (RBF) equipped with a mechanical stirrer, thermometer pocket and pressure equalizing addition funnel, compound 2 (2.5 kg, 12.254 mol), 50% aqueous sodium hydroxide (1875 mL) and TBAI (55 g, 0.183 mol) are added at room temperature. The reaction mixture is cooled to 15 °C and allyl bromide (1750 mL, 20.23 mol) is added drop wise maintaining temperature between 10-15 °C. The reaction mixture is stirred at RT for 16 h. It is transferred to a separating funnel and the layers are separated. Aqueous layer is extracted with ethyl acetate (2*5 lit). The combined organic layers are concentrated and the crude product is taken to next step (3 kg, 90%) without further purification. The product is characterized by 1H NMR.

STEP 3: Synthesis of compound 4

In a 50 lit reactor, methanol (25 lit) is charged at room temperature. Compound 3 (from above step) (3 kg, 12.29 mol) is added. The reaction mixture is stirred mechanically and water (1500 mL) is added. The solution is cooled to 0°C and conc. H2SO4 (300 mL) is added drop wise. The resulting reaction mixture is warmed to room temperature and refluxed for 6 h. The reaction mixture (RM) is cooled to 0°C and quenched with saturated sodium bicarbonate solution (3 lit). The mixture is concentrated under reduced pressure and the residue is extracted with ethyl acetate. The organic layers are dried over sodium sulphate source in the above table and concentrated on rotavapour under vacuum. The crude product is obtained as brown color liquid (2.2 kg, 80%). It is taken for next step without further purification. It is characterized by 1H-NMR.

STEP 4: Synthesis of Compound 5.

To a stirred solution of compound 4 prepared in above step (2.2 kg, 10.784 mol) in dimethyl formamide (DMF) (16 lit), sodium hydride (1.207 kg, 30.196 mol) is added portion wise at 0°C. Reaction mixture (RM) is stirred at rt for 30 minutes. Benzyl chloride (3.6 lit, 2.432 mol) is added to the RM at 0°C, and stirred at room temperature for 4 h. Once the starting material is consumed, checked by thin layer chromatography (TLC); the reaction mixture (RM) is quenched with ice cold water (2 lit), and extracted with ethyl acetate (2*5 lit). Organic layers are separated, dried over sodium sulphate (Na2SO4) concentrated under reduced pressure. Crude compound is purified by flash column chromatography, eluting with 10% ethyl acetate: hexane. Compound 5 is obtained as brown color liquid (3.5 kg 80% yield). The product is characterized by 1H-NMR and mass spectrometry (MS).

STEP 5: Synthesis of Compound 6

3.5 kg of compound 5 (9.114 mol) obtained from above step and 18 lit of 1,4 dioxane are taken in a 50 L glass reactor and are charged at room temperature. 18 lit of 2N dilute HCl is then added to the glass reactor. The reaction mixture stirred mechanically and heated at 80 °C for 2 h. Reaction is monitored by TLC. The complete disappearance of starting material is checked by TLC which is an indicator of completion of the reaction. The reaction is stopped after completion (TLC) and the phases are separated. The aqueous phase extracted with ethyl acetate (2*6.5 L). The combined organic layers are washed with approximately 5 L of water and 4 L of brine. The washed organic layer is then dried over Na2SO4 (Sodium sulphate) and concentrated on rotavapour. The concentrated brown liquid is collected as crude compound 6 weighing 3.2 kg. The formation of compound 6 is then characterized by 1H-NMR.

STEP 6: Synthesis of compound 7

To a mechanically stirred solution of compound 6 (3.2 kg, 8.648 moles) in ethanol (20 lit) sodium borohydride (772 g, 20.324 mol) is added portion wise at 20°C. The reaction mixture is stirred at RT for 2 h. The reaction mixture is quenched with acetic acid (1.1 L) to pH-7. Ethanol is evaporated and the crude product is extracted in ethyl acetate (2*5 lit) and water. The combined organic layers are dried over sodium sulphate and concentrated using rotavapour. Compound 7 is obtained as brown color liquid (2.9 kg, crude). It is characterized by 1H-NMR and MS spectral data.

STEP 7: Synthesis of compound 8

To a solution of compound 7 in dichloromethane (DCM) (28 lit), pyridine (1881 mL, 23.387mol) is added at 0°C. To this solution dimethylamino pyridine (DMAP) (190 g, 1.559 mol) and trityl chloride (2.6 kg, 9.354 moles) are added at 0°C. The resulting mixture is stirred at RT for 5 h. The reaction mixture is quenched with saturated sodium bicarbonate solution (5 lit) and then two phases are separated. Organic layer is dried over sodium sulphate and concentrated. Crude compound obtained as brown color sticky oil (5.8 kg crude). The crude product 8 is taken for next step without further purification. 1H NMR and MS confirmed the product formation.

STEP 8: Synthesis of compound 8A

To a mechanically stirred solution of compound 8 (2.8 kg, 4.56 mol) in DMF (8 lit), sodium hydride (401 g, 10.032 mol) is added portion wise at 0°C. RM is stirred at RT for 30 minutes. Benzyl chloride (692 g, 5.472mol) is added to the RM at 0°C, stirred at room temperature for 4 h. After completion of reaction (by TLC), RM quenched with ice cold water (5 lit), and extracted with ethyl acetate (2*5 lit). The separated organic layers are concentrated under reduced pressure. Crude compound is purified by flash column chromatography (FCC), eluted with 10% ethyl acetate-hexane. Compound 8a is obtained as brown color liquid (2 kg, 60% yield). It is characterized by 1H-NMR.

STEP 9: Synthesis of Compound 9

To a mechanically stirred solution of compound 8a (2.5 kg, 3.55 mol) in DCM (13.5 lit) and water (1.5 lit), trifluoro acetic acid (700 mL, 10.653 mol) is added drop wise to the reaction mixture at room temperature and stirred for 14h. After completion of reaction by TLC, water is added to RM and organic layer is separated and concentrated under reduced pressure. Crude compound is purified by FCC, eluted with 15% ethyl acetate-hexane. Compound 9 is obtained as brown color liquid (700 g, 45% for 5 steps). The product is characterized by 1H and 13C NMR, MS spectra.

Synthesis of Initiation unit and Propagation Unit:
STEP 1: Synthesis of Compound 11

To a magnetically stirred solution of 9 (150 g, 324.67 mmol) in 1,2-dichloroethane (DCE) (1 lit), 4A° molecular sieves powder (120 g) is added to the reaction mixture and is cooled to 0°C. Boron trifluoride: diethyl ether complex (130 mL, 1035.6 mmol) is added to the reaction mixture at 0°C and stirred for 30 minutes. A solution of 10 (149.7 g, 470.77 mmol) in DCE (750 mL) is added to the reaction mixture at 0°C. Ice-bath is removed and the reaction is allowed to age for 4 h at room temperature. RM is neutralized (pH-7) with triethyl amine (150 mL), then diluted with saturated sodium bicarbonate (150 mL) solution and extracted with DCM (1 lit). The extract is concentrated under reduced pressure. Crude compound is purified by flash silica column chromatography (FCC), by eluting with 25% ethyl acetate and hexane. Compound 11 obtained as yellow color sticky oil (120 g, 52%). Product is confirmed by 1H-NMR and MS.

STEP 2: Synthesis of Compound 12

To a stirred solution of compound 11 (120 g, 167.36 mmol) in methanol (800 mL) 25% solution of sodium methoxide (10.8 mL, 50.208 mmol) is added and stirred at rt for 2h. Reaction is monitored by TLC, once reaction is completed RM is neutralized with acidic resin (200 g) (Amberlite IR-120 Hydrogen form) until pH 6-7. Then resin is filtered through celite and washed with methanol (100 mL). The filtrate is concentrated under reduced pressure. Crude compound is purified by FCC, by eluting with 45% ethyl acetate and hexane. Compound 12 is obtained as brown color sticky oil (62 g, 62%). Product is confirmed by 1H-NMR, 13C NMR and MS.

STEP 3: Synthesis of compound 12A

In a 500 mL 3 necked round bottom flask equipped with a magnetic stirrer, thermometer pocket and calcium chloride guard tube, compound 12 (10 g,16.835m. mol) is dissolved in DMF(100 mL). The reaction mixture is cooled (ice-bath) and sodium hydride (2.693 g, 67.34 mmol) is added portion wise at 0°C. This is followed by TBAI (1.863 g, 5.05 mmol). RM is stirred at room temperature for 1h. Reaction mixture is again cooled and benzyl bromide (10.07 g, 59.259 mmol) is added drop wise from a dropping funnel. The mixture is stirred at room temperature for 4h. After completion (by TLC) RM is quenched with ice cold water (100 mL), and extracted with ethyl acetate (250 mL). The organic layer is separated and concentrated under reduced pressure. Crude compound is purified by FCC, eluted with 10% ethyl acetate-hexane, compound 12A is obtained as brown color liquid (10 g, 70% yield). It is characterized by 1HNMR.

STEP 4: Synthesis of compound 15

A stirred solution of compound 12A (25 g, 28.968 mmol) in methanol (250 mL) is degassed with argon for 15 minutes, then palladium(II) chloride( 1.538g, 8.690 mmol) is added to it. It is stirred for 16 h at room temperature. The reaction mixture is monitored by TLC which showed complete consumption of the starting materials. Reaction mass is filtered through celite and the celite bed is washed with methanol. The filtrate is concentrated under reduced pressure in a rotary evaporator and the obtained crude product is purified by FCC eluting with 25% ethyl acetate and hexane. Compound 15 is obtained as yellow viscous oil (16 g, 67%). Product is characterized by 1H-NMR and MS.

STEP 5: Synthesis of Compound 13

To a stirred solution of compound 12 (140 g, 235.69 mmoles) in toluene (1400 mL), is added dibutyltin oxide (58.61 g, 235.69 mmoles) and the reaction mixture is refluxed for 4h. Reaction mixture is cooled to room temperature and sodium hydride (18.85 g, 471.38 mmol) is added portion wise and again reaction mixture is heated at 80°C for 30 minutes. To this solution, added TBAI (43.48 g, 117.84 mmol) and stirred at 80°C for 1h. Benzyl chloride (50 mL) is added to the reaction and stirred at 80°C for 6h. More benzyl chloride (25 mL) is added to the reaction mixture and the heating is continued until completion of reaction (by TLC). Reaction mixture is cooled to 0°C and quenched with 1N HCl (500 mL), and diluted with water (500 mL) and extracted with ethyl acetate (2 lit). The organic layer is separated and dried over with sodium sulphate and concentrated under reduced pressure. Crude product is purified by FCC, compound is eluted with 25% ethyl acetate: hexane, compound 13 obtained as light yellow color oil (100 g 60%). Product is confirmed by 1H-NMR and MS.

STEP 6: Synthesis of compound 13A

The stirred solution of compound 13 (70 g, 90.439 mmol) in methanol (700 mL) is degassed with argon for 15 minutes, then palladium (II) chloride (4.81g, 27.131 mmol) is added to the reaction mixture. Reaction mixture is stirred for 16h at room temperature. Progress of reaction is monitored by TLC after completion reaction mixture filtered through celite and celite bed is washed with methanol and concentrated under reduced pressure. Crude compound is purified by FCC, by eluting with 25% ethyl acetate and hexane. Compound 13A is obtained as yellow color sticky oil (45 g, 70%). Product is confirmed by 1H-NMR and MS.

STEP 7: Synthesis of compound 13B

To a solution compound 13A (45 g, 65.484 mmol) in DCM (500 mL), pyridine (16.03 g, 203.0 mmoles) is added at 0°C. To the above solution DMAP (4.17 g, 34.248 mmoles) and dimethoxy trityl chloride (9.6 g, 87.094 mmol) are added at 0°C. The resulting mixture stirred at RT for 5h. The reaction mixture is quenched with saturated sodium bicarbonate solution (100 mL) and then extracted with DCM. The organic layer is concentrated and the residue is purified by flash column chromatography using 20% EA in hexane as eluent. Compound 13B, obtained as yellow color sticky oil (40 g, 65%). Product is confirmed by 1H-NMR and MS.

STEP 8: Synthesis of compound 14

To a stirred solution of compound 13B (25g, 24.108 mmoles) in dry THF (250 mL) is added DIPEA (16.765 mL, 96.432 mmol) and 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (11.41 g, 48.216 mmol) at 0°C. Reaction mixture is stirred at room temperature for 4h. Reaction is monitored by TLC, after completion, reaction mixture is quenched with aqueous sodium bicarbonate solution (150 mL) and extracted with ethyl acetate (2*250 mL), organic layers are separated and dried over sodium sulphate. Organic layer is concentrated under reduced pressure, crude product is purified by FCC, compound is eluted with hexane: diethyl ether: triethylamine (6:3:1), compound 14 obtained as light yellow color oil (21 g, 70%). Product is confirmed by 1H-NMR and MS.

Synthesis of Linker Unit:
STEP 1: Synthesis of compound 2A:

To a stirred solution of 5-bromo-1-pentanol (50 g, 0.2994 moles) in DMSO (410 mL), sodium azide (29.19 g, 0.4491 mol) is added. The reaction mixture is stirred at room temperature for 3 days. Reaction is monitored by 1H NMR and infrared spectroscopy (IR). Reaction mixture is quenched with ice cold water (250 mL) and extracted with diethyl ether (2*250 mL), and then organic layers are separated and dried over with sodium sulphate, filtered and concentrated under reduced pressure. Compound 2A is obtained as light yellow color oil (30 g, 78%). Product is confirmed by 1H-NMR and IR.

STEP 2: Synthesis of compound 3A:

To a stirred solution of compound 2A (19 g, 0.1472 mol) and beta-D-Ribofuranose 1,2,3,5-tetraacetate (46.8 g, 0.1472 moles) in acetonitrile (200 mL), iodine is added in one portion. Reaction is heated to reflux for 2 h. After completion (TLC), RM is diluted with ethyl acetate (500 mL). The resulting organic layer is washed with sodium thiosulfate solution (500 mL). Organic layer is separated, dried over Na2SO4 and concentrated under reduced pressure. Crude compound is purified by FCC, by eluting with 25% ethyl acetate and hexane. Compound 3A is obtained as oil (19 g, 35%). Product is confirmed by 1H-NMR.

STEP 3: Synthesis of compound 4A

To a magnetically stirred solution of compound 3A (19 g, 0.0509 moles) in methanol (300 mL), 25% of sodium methoxide (4.4 mL, 0.0203 mol) in methanol is added in one portion and stirred at RT for 2 h. Once the reaction is completed (TLC), the RM is neutralized with acid resin (Amberlite IR-120 Hydrogen form) until pH-7. The resin is filtered through celite and washed with methanol. The filtrate is concentrated under reduced pressure. Crude compound is purified by FCC, eluted with 35% ethyl acetate-hexane. Compound 4A is obtained as colorless sticky mass (10.5 g, 85%). Product is confirmed by 1H-NMR.

STEP 4: Synthesis of compound 5A

To a stirred solution of compound 4A (10.5 g, 0.0428 moles) in toluene (110 mL), dibutyltin oxide (10.65 g, 0.0428 mol) is added and the mixture is refluxed for 4 h. The reaction mixture is cooled to room temperature and sodium hydride (3.33 g, 0.0857 moles) is added portion wise. The mixture is again heated at 80°C for 30 minutes. To this solution TBAI (7.9 g, 0.0214moles) is added and stirred at 80°C for another 1h. Benzyl chloride (19.81 mL, 0.1714 moles) is added drop wise under heating condition to the reaction mixture. The heating is continued at 80°C for another 16 h. The reaction mixture then is cooled to 0 °C and quenched with 1N HCl (50 mL). Water and ethyl acetate are added to the mixture and the layers are separated. The ethyl acetate layer is dried over sodium sulphate and concentrated under reduced pressure. The crude product is purified by flash column chromatography, eluted with 20% ethyl acetate-hexane. Product 5A is obtained as light yellow colored oil (13 g, 50%) and is characterized by 1H-NMR.

STEP 5: Synthesis of compound 5B

To a stirred solution of compound 5A (0.5 g, 1.136 mmol) in methanol (10 mL) 10% palladium carbon (150 mg) is added under nitrogen atmosphere. Reaction is maintained under hydrogen atmosphere (balloon pressure) for 2 h. After completion of reaction (by TLC); the reaction mixture is filtered through celite; the bed is washed with methanol. Filtrate is concentrated under reduced pressure. Compound 5B is obtained as light yellow color oil (0.43 g 90%). Product is confirmed by 1H-NMR and MS.

STEP 6: Synthesis of compound 5C

A magnetically stirred mixture of compound 5B (0.765 g, 1.84 mmol) and potassium carbonate (0.303 g, 2.195 mmol) in a mixture of water (14 mL) and DCM (23 mL) is cooled to 0°C with ice-bath. Benzyl chloroformate (0.313 g, 1.84 mmol) is added to the reaction mixture. The ice-bath is removed and stirred for 1h at RT. After completion (by TLC), water (20 mL) and DCM (50 mL) are added. The layers are separated; the organic layer is washed with water (2*20 mL), dried over sodium sulphate and evaporated under reduced pressure. The crude product is purified by flash column chromatography using 50% ethyl acetate-hexanes as eluent to afford the product as viscous liquid (0.65 g, 65%); confirmed by 1H-NMR and MS.

STEP 7: Synthesis of compound 6A

A magnetically stirred solution of compound 5C (0.15g, 0.273 mmol) in dry THF (3 mL) is cooled in ice-bath. To this solution diisopropylethylamine (DIPEA) (0.14 g, 1.902 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.129 g, 0.546 mmol) are added. The ice-bath is removed and the reaction mixture is aged at room temperature for 4 h. A TLC is checked which showed that the starting material is completely consumed. The reaction mixture is quenched with saturated sodium bicarbonate solution (2 mL), and extracted with ethyl acetate (2*10 mL). Combined organic layers are dried over sodium sulphate and concentrated under reduced pressure. Crude product is purified by silica-gel chromatography eluting with 10% ethyl acetate-hexane containing 0.1% triethyl amine. Compound 6A is obtained as sticky liquid (0.15 g 66%). Product is confirmed by 1H-NMR and MS.

Synthesis of Tetramer:
STEP 1, 2 and 3: Synthesis of Compound 16

STEP-1 and 2:
A round bottom flask (500 mL single neck) containing the compound 14 (21 g, 16.99 mmol) and tetrazole (8.5 g, 121.3 mmol) is fitted with a rubber septum and a short needle is inserted through it for the purpose of vacuum drying. Another round bottom flask (250 mL single neck) containing the compound 15 (10 g, 12.135 mmol) is also fitted with the rubber septum-needle arrangement. Both flasks are kept inside a vacuum desicator containing P2O5. Vacuum is applied to the desiccator and the contents of the flask are dried for about 6 h. The desiccator is then filled with argon. A solution of the compound 15 in anhydrous acetonitrile (240 mL; Dried over P2O5, distilled at 80°C) is added in one portion to the mixture of compound 14 and tetrazole. After stirring for 24 h, reaction is monitored by TLC. When starting material is consumed; dry pyridine (12 mL, 145.6 mmol) is added followed by a 0.5 M solution of I2 in THF/Water (2:1) until the brown color persisted (30 mL). The solvent is removed under reduced pressure and the residue is dissolved in chloroform (500 mL). This solution is then washed with saturated sodium thiosulfate solution (250 mL) and brine; dried (Na2SO4) and concentrated to dryness. The crude compound is used in next step without purification.

STEP-3:
To a stirred solution of above crude material (step 1 and 2) (35 g) in DCM (200 mL) is added 3% dichloro acetic acid in DCM (500 mL) at once at 0°C and stirred for 1h at room temperature. Reaction is monitored by TLC, and then reaction mixture is quenched with aqueous sodium bicarbonate solution (250 mL) after completion. Organic layer is separated and concentrated under reduced pressure. Crude compound is purified by flash column chromatography over silica-gel, eluting with 30% ethyl acetate:hexane; compound 16 is obtained as a light yellow color liquid (10 g, 50%). Compound is confirmed by 1H-NMR, 31P-NMR, MS, and 13C-NMR.

STEP 4, 5 and 6: Synthesis of compound 17
Step-4 and 5
A round bottom (RBF) flask containing the compound 14 (10.35 g, 8.368 mmol), tetrazole (4.18 g, 59.77 mmol) is fitted with a rubber septum and a short needle is inserted through it. Another RBF containing the compound 16 (10 g, 5.977 mmol) is also fitted with rubber septum-needle arrangement. Both flasks are dried under vacuum in desicator containing P2O5 for about 6 h. The flasks are then filled with argon. A solution of the compound 16 in anhydrous acetonitrile (240 mL, dried over P2O5, distilled at 80°C.) is added to the compound 14 and the tetrazole mixture. After stirring for 24 h, reaction is monitored by TLC, starting material is consumed, then dry pyridine (5.66 mL, 71.72 mmol) is added in one portion and followed by a 0.5 M of solution I2 in tetrahydrofuran (THF)/Water (2:1) until the brown color persisted (15 mL). Reaction mixture is concentrated completely on rotavapour at RT. The solvent is removed in vacuo and the residue is dissolved in chloroform, this solution is washed with saturated sodium thiosulfate (250 mL) and brine (250 mL), dried over Na2SO4 and concentrated to dryness. The crude product is used to next step without purification.

Step-6
To a stirred solution of above crude material (obtained in step 4 & 5) (25 g) in DCM (50 mL), 3% dichloroacetic acid in DCM (450 mL) is added at 0°C, and stirred for 1h at room temperature. Reaction is monitored by TLC, and then reaction mixture is quenched with aqueous sodium bicarbonate solution (250 mL). Organic layer is separated and concentrated under reduced pressure. Crude compound is purified by flash column chromatography over silica-gel, eluting with 40% ethyl acetate; hexane. Compound 17 obtained as a light yellow color liquid (8 g, 53% yield). Compound is confirmed by 1H-NMR, 31P-NMR and MS (MALDI).

STEP 7, 8 and 9: Synthesis of compound -18
Step-7and 8
A round bottom flask containing 5.82 g of compound 14 (4.716 mmol) and 2.35 g of tetrazole (33.69 mmol) is fitted with a rubber septum and a short needle is inserted through it. Another round bottom flask containing 8.5 g of compound 17 (3.369 mmol) is also fitted with rubber septum-needle arrangement. Both flasks are dried under vacuum in desicator containing P2O5 for about 6 h. The flasks are then filled with argon. A solution of the compound 17 in anhydrous acetonitrile (240 mL) is added to the mixture of compound 14 and the tetrazole at room temperature. After stirring for 24 h, reaction is monitored by TLC, starting material is consumed, and then 3.19 mL of dry pyridine ( 40.42 mmol) is added and followed by a 0.5 M of I2 in THF/Water (2:1) (15 mL) until the brown color persisted. The solvent is removed and the residue is dissolved in chloroform, this solution is then washed with saturated sodium thiosulfate (200 mL), brine, dried (Na2SO4) concentrated to dryness. The crude product is used to next step without purification.

Step-9
To a stirred solution of crude material (17 g) obtained in step 7 & 8 in DCM (100 mL), 3% dichloroacetic acid in DCM (500 mL) is added at once to the reaction mixture at 0°C, and stirred for 1h at room temperature. Reaction is monitored by TLC, then reaction mixture is quenched with aqueous sodium bicarbonate solution (250 mL), organic layer is separated and concentrated under reduced pressure. Crude compound is purified by flash column chromatography over silica-gel, eluting with 50% ethyl acetate; hexane, compound 18 obtained as a light yellow color liquid (4.5 g, 40% yield). Compound is confirmed by 1H-NMR, 31P-NMR.

STEP 10 and 11: Synthesis of compound 19A
A flask containing the compound 6A (0.466 g, 0.622 mmol) and tetrazole (0.30 g, 4.4 mmol) is fitted with a septum and a short needle is inserted through it. Another flask containing the compound 18 (1.5 g, 0.444 mmol) is also fitted with the septum-needle, arrangement. Both flasks are dried under vacuum over P2O5 for about 6 h. The flasks are then filled with argon. A solution of the tetramer compound 18 in anhydrous acetonitrile (30 mL) is added drop wise to the compound 6A and the tetrazole at room temperature. After stirring for 24 h, reaction is monitored by TLC, starting material is consumed, and then dry pyridine (0.4 mL, 4.4 mmol) is added and followed by a 0.5 M of I2 in THF/Water (2:1) until the brown color persisted (1 mL). The solvent is removed under reduced pressure at 30 °C and the residue is dissolved in chloroform, this solution is then washed with saturated sodium thiosulfate (20 mL), brine, dried (Na2SO4) concentrated to dryness. The crude product is purified by flash column chromatography on Silica-gel (grace purification) using a mixture of ethyl acetate-hexane. Compound 19A is obtained as a colorless foamy liquid (0.75 g, 45%). Compound 19A is confirmed by 1H-NMR, 31P-NMR and MS.

STEP-12: Decyanoethylation of compound 19A

To a stirred solution of compound 19A (0.75 g, 0.185 mmol) in methanol (36 mL) concentrated ammonium hydroxide (25%) solution (18 mL) and THF (30 mL) is added. The reaction mixture is stirred at room temperature for 16h. Reaction is monitored by TLC. Solvents are removed in vacuum (50 mbar), and crude compound (0.8 g, 100%) is taken to next step without purification.

STEP-13 and 14: Preparation of HIB tetramer 20
The 0.8 g of crude compound obtained (0.8 g) is dissolved in methanol/ water / ethyl acetate (150:50:50) and 20% Pd (OH)2 (2.5 g) is added. The resulting reaction mixture is stirred under hydrogen atmosphere (70 psi) for 36 h, at room temperature. After 36h, reaction is monitored by 1H-NMR, aromatic protons are absent in NMR spectra then catalyst is filtered through celite and filter cake is washed with 10 mL methanol and 10 mL water. After evaporation of methanol, ethyl acetate and water, then residue is dissolved in 10 mL water. Amberlite-IR 120 Na+ form (5 g) is added and stirred for 1h. Resin is removed by filtration (filtered through stem funnel and cotton) and filtrate is dried by lyophilization. The resultant crude compound weighing 250 mg is obtained as Hib-Tetramer.

Purification:
The crude product (500 mg) purified by sephadex-G-25 (7 g), eluting with water, before purification sephedex-G25 is swelled for 4h in water, poured in to a glass column (15 cm length and 2 cm diameter). Crude compound (50 mg) is dissolved in 1 mL of water and loaded in to column, and 70 mL of water added to the column and eluted (1 drop per 4 sec). Compound fractions are identified by TLC (charred aluminum silica plate in ninhydrin or 5% sulphuric acid in methanol). All the compound fractions are pooled and lyophilized for 48 h to give the fully deprotected HIB-Tetramer as a white solid (280 mg, 51%).
,CLAIMS:We claim:
1. Synthesis of Hib oligosaccharides of the Formula I:

wherein ‘n’ is an integer = 2, and R1 = H, and R2 is linker unit (6A) with defined formula of
R2 =

wherein ‘j’ is an integer from 1 to 6 and ‘K’ is an any functional group capable of attaching with proteins, said ‘K’ being selected from NH2, CHO, SH, OH, COOH, N-hydroxysuccinimido, hydrazine, halogen.

2. The synthesis of Hib oligosaccharides as claimed in claim 1 wherein n is 4, j is 5 and K is NH2.

3. A process for preparing Hib oligosaccharides of the Formula I as claimed in claim 1 said process comprising the steps of
- synthesizing initiation unit (compound 15) and propagation unit (compound 14) using protected ribitol unit (compound 9)
- synthesizing linker unit (compound 6A),
- synthesizing Hib tetramer (compound 20) in good yield.

4. The process for preparing Hib oligosaccharides of the Formula I as claimed in claim 3 for synthesizing initiation unit (compound 15) and propagation unit (compound 14) using protected ribitol unit (compound 9) wherein
- protected ribitol unit (compound 9) obtained from known process is reacted with acetyl protected ribose (compound 10) to obtain glycosyl ribitol (compound 11)
- glycosyl ribitol (compound 11) is treated with sodium methoxide to obtain intermediate compound (compound 12) whereupon
- said initiation unit (compound 15) is synthesized by treating predetermined quantity of intermediate compound 12 with benzyl bromide, catalytic TBAI and sodium hydride in tetrahydrofuran and dimethylformamide mixture to obtain intermediate compound (compound 12A), said intermediate compound (compound 12A) being deprotected selectively by using catalyst in solvent to yield said initiation unit (compound 15), and
- said propagation unit (compound 14) is synthesized by treating predetermined quantity of intermediate (compound 12) with regioselective protection reagent to obtain compound 13, said compound 13 being deprotected using catalyst like palladium chloride and the resulting hydroxy compound is protected as its dimethoxytrityl (DMT) ether selectively to obtain compound 13B which is subjected to phosphorylation to yield said propagation unit (14).

5. The process for preparing Hib oligosaccharides of the Formula I as claimed in claim 4 wherein the glycosyl ribitol unit is ß-glycosyl ribitol.

6. The process for preparing Hib oligosaccharides of the Formula I as claimed in claim 4 wherein said regioselective reagent used alone or in combination is dibutyltin oxide and benzyl chloride.

7. The process of preparing Hib oligosaccharides as claimed in claim 1 and claim 3 wherein said linker (compound 6A) is prepared by
- reacting azido alcohol with a monosaccharide moiety preferably ribose in presence of catalyst in a solvent resulting in formation of beta anomer (compound 3A),
- said beta anomer (compound 3A) is deprotected using a deprotonated base followed by selective benzyl protection of alcohol to yield intermediate compound (compound 5A) wherein the azide group of said intermediate compound (compound 5A) is selectively reduced to provide amine and is subjected to Cbz protection to give intermediate compound 5C,
- alcohol moiety of said intermediate compound 5C is phosphorylated using phosphorylating reagent in solvent providing solution phase for the reaction mixture to yield the linker (Compound 6A).

8. The process of preparing Hib oligosaccharides as claimed in claim 7 wherein said phosphorylating reagent is 2-Cyanoethyl N,N-diisopropylchloro- phosphoramidite.

9. The process of preparing Hib oligosaccharides as claimed in claim 7 wherein said catalyst is Iodine.

10. The process of preparing Hib oligosaccharides as claimed in claim 7 wherein said solvent is TetraHydro furan (THF).

11. A process for preparing Hib oligosaccharides of the Formula I as claimed in claim 3 for synthesizing Hib tetramer comprising the steps of
- reacting said initiation unit (compound 15) with propagation unit (compound 14) in the presence of a coupling reagent and a oxidizing agent resulting in Hib dimer (compound 16),
- said Hib dimer (compound 16) being further coupled with said propagation unit (compound 14) in the presence of said coupling reagent and said oxidizing agent to yield Hib trimer (compound 17),
- said Hib trimer (compound 17) being further coupled with said propagation unit (compound 14) in the presence of said coupling reagent and said oxidizing agent to yield Hib oligomer more particularly Hib tetramer (compound 18),
- said Hib tetramer (compound 18) being coupled with linker unit (compound 6A) in the presence of said coupling reagent and said oxidizing agent to produce Hib oligomer of Formula I.

12. The process for preparing Hib oligosaccharides of the Formula I as claimed in claim 11 wherein said coupling reagent is tetrazole.

13. The process for preparing Hib oligosaccharides of the Formula I as claimed in claim 11 wherein said oxidising reagent is Iodine.

14. Synthesis of Hib oligosaccharides of the Formula II :

wherein ‘n’ is an integer = or > 2, and R2 = H, and R1 is linker with defined formula of
R1 =

wherein ‘j’ is an integer from 1 to 6 and ‘K’ is an any functional group capable of attaching with proteins, said ‘K’ being selected from NH2, CHO, SH, OH, COOH, N-hydroxysuccinimido, hydrazine, halogen.

15. The synthesis of Hib oligosaccharides as claimed in claim 14 wherein n is 4, j is 5 and K is NH2.