Stable hydrolysis-resistant synthetic polyribosylribitolphosphate derivatives as vaccines against <i>Haemophilus influenzae </i>type b

Abstract

The present invention provides a stable synthetic saccharide of Hib polyribosylribitol-phosphate (PRP) derivative and conjugate thereof. Said saccharide, said conjugate and pharmaceutical compositions thereof are hydrolysis-resistant, long-term stable and useful for the prevention and/or treatment of diseases associated with Haemophilus influenzae, and more specifically of diseases associated with Haemophilus influenzae type b, preferably diseases selected from meningitis, pneumonia, and epiglotitis. They have general formula (I): wherein A is formula (II) or formula (III); B is formula (IV); C is formula (V); D is formula (VI); E is formula (VII); F is formula (VIII) or formula (IX).

Claims

1. A saccharide of the formula (IV-1) ##STR00231## wherein m is an integer selected from 1 to 9; and Xa is H, F, —OCO—N(CH.sub.3).sub.2, or —OCH.sub.3.

2. A saccharide selected from the group consisting of: ##STR00232## ##STR00233## ##STR00234## ##STR00235## ##STR00236##

3. The saccharide according to claim 1, conjugated with an immunogenic carrier through the nitrogen atom of the —O—(CH.sub.2).sub.5—NH.sub.2 group.

4. A pharmaceutical composition comprising an effective amount of a saccharide of claim 1.

5. A vaccine composition comprising a saccharide according to claim 1 together with at least one pharmaceutically acceptable adjuvant, carrier, cryoprotectant, lyoprotectant, excipient and/or diluent.

6. The vaccine composition according to claim 5 further comprising at least one of diphtheria antigen, tetanus antigen, pertussis antigen, hepatitis B antigen, inactivated polio vaccine and inactivated rotavirus vaccine.

7. The saccharide according of claim 2, conjugated with an immunogenic carrier through the nitrogen atom of the —O—(CH.sub.2).sub.5—NH.sub.2 group.

8. A pharmaceutical composition comprising an effective amount of a saccharide of claim 2.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1: Commercially available interconnecting molecules according to the present invention.

(2) FIG. 2: (A) saccharide conjugated with a carrier protein; (B) saccharide conjugated on a solid support.

(3) FIG. 3: Synthetic scheme of 2′-methoxyribosylribitolphosphate tetramer 29 conjugated to CRM.sub.197 and cysteine.

(4) FIG. 4: .sup.1H NMR spectra of (A) fragmented Hib CPS after treatment with 0.1 M sodium hydroxide solution for 20 hours and (B) untreated Hib CPS as reference. The comparison shows that Hib CPS hydrolyzes completely within 20 hours under basic conditions.

(5) FIG. 5: (A) HPLC chromatogram and (B) ESI mass spectrum of fragmented Hib CPS after treatment with 0.1 M sodium hydroxide solution for 20 hours.

(6) FIG. 6: HPLC chromatogram of Hib CPS after treatment with Alhydrogel® (A) for 7 days and (B) for 2 days at 37° C. as well as (D) for 7 days and (E) for 2 days at room temperature. HPLC chromatogram of untreated Hib CPS is shown in (C).

(7) FIG. 7: HPLC chromatogram of compound 16 after treatment with (A) Alhydrogel®, (B) with aluminum phosphate, (C) with water for 7 days at 37° C. HPLC chromatograms of untreated compounds 16 and 8 are shown in (D) and (E).

(8) FIG. 8: HPLC chromatogram of compound 16 after treatment with (A) Alhydrogel®, (B) with aluminum phosphate, (C) with water for 7 days at 2-8° C. HPLC chromatograms of untreated compounds 16 and 8 are shown in (D) and (E).

(9) FIG. 9: HPLC chromatogram of compound 16 after treatment with (A) Alhydrogel®, (B) with aluminum phosphate, (C) with water for 7 days at room temperature. HPLC chromatograms of untreated compounds 16 and 8 are shown in (D) and (E).

(10) FIG. 10: HPLC chromatogram of compound 16 after treatment with (A) Alhydrogel®, (B) with aluminum phosphate, (C) with water for 7 days at 70° C. HPLC chromatograms of untreated compounds 16 and 8 are shown in (D) and (E).

(11) FIG. 11: HPLC chromatogram of compound 30 after treatment with (A) Alhydrogel®, (B) with phosphate buffer, (C) with water for 14 days at room temperature. HPLC chromatograms of untreated compounds 30, 16 and 8 are shown in (D), (E) and (F).

(12) FIG. 12: HPLC chromatogram of compound 30 after treatment with (A) Alhydrogel®, (B) with phosphate buffer, (C) with water for 14 days at 2-8° C. HPLC chromatograms of untreated compounds 30, 16 and 8 are shown in (D), (E) and (F).

(13) FIG. 13: HPLC chromatogram of (A) compound 30 after treatment with after treatment with 0.1 M sodium hydroxide solution for 3 days; (C) compound 16 after treatment with after treatment with 0.1 M sodium hydroxide solution for 3 days, HPLC chromatograms of untreated compounds 30, 16 and 8 are shown in (B), (D) and (E).

(14) FIG. 14: MALDI mass spectra of (A) compound 30 conjugated to CRM.sub.197 and (B) compound 94.

(15) FIG. 15: HPLC-SEC chromatograms of compound 94 in presence of phosphate buffer.

(16) FIG. 16: shows the structure of conjugate 94.

(17) FIG. 17: Glycan array analysis: (A) Binding of compounds 19, 24, 29, 53 and 62 to antibodies (Hib human reference serum) in the presence of Hib PRP inhibitor (grey bars) or no inhibitor (black bars) after 21 days; (B) Binding of compounds 19, 24, 29, 53 and 62 to antibodies (rabbit typing serum) in the presence of Hib PRP inhibitor (grey bars) or no inhibitor (black bars) after 21 days; Binding of compounds 19, 24, 29, 53, 62 and natural Hib PRP as reference to antibodies raised by immunization with compound 94 (C) and compound 94 with Alhydrogel (D) after 35 days. Error bars represent the standard deviation of the quadruplicate samples.

(18) FIG. 18: ELISA study: Binding of IgG antibodies from rabbits (n=4) immunized with unadjuvanted conjugate 94 (.square-solid.), with conjugate 94 adjuvanted with Alhydrogel (.box-tangle-solidup.), with PBS/Alhydrogel (.circle-solid.) and with HiberiX® (.Math.) to plates coated with native Hib PRP antigen after 0 days (A—negative control), 14 days (B), 21 days (C) and 35 days (D). Sera were diluted 5-fold with 1% BSA-PBS. Diluted sera (100 μL) were added per well of the microtiter plate which was coated with 1 μg of Hib-PRP polysaccharide, detected with a HRP conjugated goat anti-rabbit secondary antibody diluted to 1:10000 and developed using TMB. Absorbance was measured at 450 nm and the data were plotted using the graphpad prism software.

(19) FIG. 19: ELISA study: Binding of IgG antibodies from rabbits (n=4) immunized with unadjuvanted conjugate 94 (.square-solid.), with conjugate 94 adjuvanted with Alhydrogel (.box-tangle-solidup.), with PBS/Alhydrogel (.circle-solid.) and with HiberiX® (.Math.) to commercial ADi plates precoated with native Hib PRP antigen after 0 days (A—negative control), 14 days (B), 21 days (C) and 35 days (D). Sera were diluted 5-fold with 1% BSA-PBS. Diluted sera (100 μL) were added per well of the microtiter plate which was coated with 1 μg of Hib-PRP polysaccharide, detected with a HRP conjugated goat anti-rabbit secondary antibody diluted to 1:10000 and developed using TMB. Absorbance was measured at 450 nm and the data were plotted using the graphpad prism software.

(20) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples, which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments, which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

(21) Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

(22) Chemical Synthesis

(23) Abbreviations:

(24) TLC: thin layer chromatography, EtOAc: ethyl acetate, DCM: dichloromethane, RBF: round bottom flask, ACN: acetonitrile, AcOH: acetic acid, TBAF: tetrabutylammonium fluoride, BnBr: benzyl bromide, DMAP: dimethylaminopyridine, PTFE: polytetrafluoroethylene, AIBN: azobisisobutyronitrile, THF: tetrahydrofuran, NAP: 2-naphthylmethyl, Lev: levulinyl.

(25) General Information for Chemical Synthesis

(26) Commercial reagents were used without further purification except where noted. Solvents were dried and redistilled prior to use in the usual way. All reactions were performed in oven-dried glassware under an inert atmosphere unless noted otherwise. Analytical thin layer chromatography (TLC) was performed on Kieselgel 60 F254 aluminium plates precoated with a 0.25 mm thickness of silica gel. The TLC plates were visualized with UV light and by staining with Hanessian solution (ceric sulfate and ammonium molybdate in aqueous sulfuric acid) or sulfuric acid-ethanol solution. Column chromatography was performed on Fluka Kieselgel 60 (230-400 mesh). Optical rotations (OR) were measured with a Schmidt & Haensch UniPol L1000 polarimeter at a concentration (c) expressed in g/100 mL. .sup.1H and .sup.13C NMR spectra were measured with a Varian 400-MR or Varian 600 spectrometer with Me.sub.4Si as the internal standard. NMR chemical shifts (δ) were recorded in ppm and coupling constants (J) were reported in Hz. High-resolution mass spectra (HRMS) were recorded with an Agilent 6210 ESI-TOF mass spectrometer.

(27) General Procedure A) Disilyloxy Deprotection

(28) TBAF (0.21 mL, 0.21 mmol) and AcOH (7 μL, 0.11 mmol) were added to a stirred solution of starting material (0.073 mmol) in THF (1.5 mL) at room temperature in a 10 mL RBF (oven dried) under argon atmosphere. Reaction mixture was stirred at room temperature for 4 h. Reaction was monitored by TLC. After complete consumption of starting material reaction mixture was diluted with DCM (10 mL) and concentrated under vacuum to obtain the crude product. The crude product was by automated flash chromatography using EtOAc in n-hexane (20-60%) as the eluent. Concentration of solvent from test tubes containing product (based on TLC) in vacuum resulted in a colourless oil.

(29) General Procedure B) Benzylation Using Bu.sub.2SnO

(30) Ag.sub.2O (0.116 g, 0.5 mmol) and BnBr (8 μL, 0.07 mmol) were added to a stirred solution of starting material (0.063 mmol) in DCM (1 mL) at room temperature in a 10 mL RBF (oven dried) under argon atmosphere. Reaction mixture was kept for stirring at room temperature for 6 h. Reaction was monitored by TLC. After complete consumption of starting material reaction mixture was diluted with DCM (30 mL) and filtered through celite pad and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-50%) as the eluent. Concentration of solvent from test tubes containing the product (based on TLC) in vacuum resulted in the colourless oil.

(31) Bu.sub.2SnO (2.42 g, 9.73 mmol) was added to a clear solution of the diol starting material (4.6 g, 6.48 mmol) in toluene (50 mL) at room temperature under argon atmosphere equipped with a stir bar and stirring of 1400 rpm. Then the reaction mixture was kept under reflux at 130° C. for 6 h. After 6 h, solvent were removed under vacuum and the reaction was azeotropic dried with toluene (5×10 mL dry toluene). After complete removal of solvents acetal was dried under vacuum for 20 min. Acetal was removed from vacuum in presence of argon and dissolved in DMF (50 mL). To this solution BnBr (1.16 mL, 9.73 mmol) and TBAI (4.78 g, 12.96 mmol) were added and the reaction mixture was kept for stirring at 100° C. for 20 h. Reaction was monitored by TLC (40% EtOAc in n-hexane). After 20 h, reaction mixture was diluted with EtOAc (50 mL) and water (20 mL). The aqueous layer was separated and washed with EtOAc (2×30 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 (˜2 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography on silica gel using EtOAc in n-hexane (gradient, 0 to 30%) as the eluent. Concentration of solvent from test tubes resulted in the colourless oil (3.8 g, 73%).

(32) General Procedure C) Protection with Lev

(33) To a solution of the hydroxyl compound (0.195 mmol) in DCM (3 mL) in a 25 mL RBF under argon atmosphere was added levulinic acid (0.3 mmol, 30 μL), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.3 mmol, 58 mg) and DMAP (0.2 mmol, 24 mg). The resulting reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC. After complete consumption of the starting material, the reaction mixture was diluted with DCM (10 mL) and washed with brine (5 mL). The aqueous layer was extracted with DCM (2×5 mL). The organic layer was dried over Na.sub.2SO.sub.4 (0.2 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-50%) as the eluent. Concentration of solvent from test tubes containing the products (based on TLC) in vacuum resulted in a white oil.

(34) General Procedure D) NAP Deprotection

(35) To a solution of the NAP protected compound (0.048 mmol) in dichloromethane:H.sub.2O (1.8:0.2 mL) in a 10 mL RBF (oven dried) under argon atmosphere was added 2.3-dichloro-5,6-dicyano-1,4-benzoquinone (14 mg, 0.058 mmol) at 0° C. The reaction mixture was stirred at room temperature for 1.5 h. Reaction was monitored by TLC. Reaction was diluted with DCM (10 mL) and extracted with NaHCO.sub.3 aq. sat. solution (5 mL) and brine (5 mL). The organic layer was dried over Na.sub.2SO.sub.4 (0.2 g), filtered, and the filtrate was concentrated under vacuum for 15 min to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-80%) as the eluent. Concentration of solvent from test tubes containing the products (based on TLC) in vacuum resulted in a white oil.

(36) General Procedure E) Phosphate Coupling: Phosphoramidite Method

(37) To a solution of 3-OH compound (0.054 mmol) in DCM (1.2 mL) in a 25 mL RBF (oven dried) under argon atmosphere was added bis(diisopropylamino)-benzyloxyphosphine (0.108 mmol) and diisopropylammonium tetrazolide (0.081 mmol) and the solution stirred at room temperature for 1.5 h. Reaction was monitored by TLC. Reaction mixture was diluted with DCM (10 mL) and quenched with NaHCO.sub.3 aq. sat. solution (5 mL). The aqueous layer was extracted with DCM (2×5 mL). The combined organic layer was washed with brine (5 mL), dried over Na.sub.2SO.sub.4 (0.5 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified on silica gel column chromatography (column 2×10 cm) using EtOAc in n-hexane (0-30%) and 2% triethylamine as the eluent. Concentration of solvent from test tubes containing product (based on TLC) in vacuum resulted in a yellow oil. The product was transferred to a smaller RBF (10 mL) using toluene (5 mL) and evaporation of the solvent under vacuum for 20 min resulted in a white oil. The product was left in high vacuum for 10 min flushed with argon and used immediately for the next step. To a solution of the phosphoramidite (0.054 mmol) in DCM (1.2 mL) in a 10 mL RBF (oven dried) under argon atmosphere was added 5-OH compound (0.036 mmol) and tetrazole 0.45 M solution in ACN (0.24 mL, 0.108 mmol) and the solution stirred at room temperature for 2 h. Then, t-butyl peroxide 5.0-6.0 M solution in decane (0.015 mL, 0.072 mmol) was added at room temperature and the reaction mixture stirred for 1 h. Reaction mixture was diluted with DCM (10 mL) and quenched with NaHCO.sub.3 aq. sat. solution (5 mL). The aqueous layer was extracted with DCM (2×5 mL). The combined organic layer was washed with brine (5 mL), dried over Na.sub.2SO.sub.4 (0.3 g), filtered, and the filtrate was concentrated under vacuum at 35° C. bath temperature of rotary evaporator for 15 min to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-80%). Concentration of solvent from test tubes containing the product (based on TLC) in vacuum resulted in a colorless oil.

(38) General Procedure F) Phosphate Coupling with Linker: Phosphoramidite Method (One Pot Procedure)

(39) To a solution of the hydroxyl compound (0.016 mmol) in DCM (1.0 mL) in a 10 mL RBF (oven dried) under argon atmosphere was added bis(diisopropylamino)-benzyloxyphosphine (0.032 mmol) and diisopropylammonium tetrazolide (0.024 mmol) and the solution stirred at room temperature for 1.5 h. Then, linker (0.097 mmol) and tetrazole 0.45 M solution in ACN (0.11 mL, 0.049 mmol) and the solution stirred at room temperature for 2 h. Reaction was monitored by TLC. Then, t-butyl peroxide 5.0-6.0 M solution in decane (0.006 mL, 0.032 mmol) was added at room temperature and the reaction mixture stirred for 1 h. Reaction was monitored by TLC. Reaction mixture was diluted with DCM (5 mL) and quenched with NaHCO.sub.3 aq. sat. solution (3 mL). The aqueous layer was extracted with DCM (2×3 mL). The combined organic layer was washed with brine (3 mL), dried over Na.sub.2SO.sub.4 (0.2 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-80%). Concentration of solvent from test tubes containing the product (based on TLC) in vacuum resulted in a colorless oil.

(40) The following linkers were employed in the synthesis:

(41) ##STR00148## ##STR00149##
General Procedure G) Lev Deprotection

(42) To a solution of the Lev protected compound (0.060 mmol) in DCM (2 mL), a solution of hydrazine hydrate (0.267 mmol, 13 μL) dissolved in acetic acid (0.08 mL) and pyridine (0.12 mL) was added. The resulting reaction mixture was stirred at room temperature for 2 h. The reaction was quenched by the addition of acetone (0.5 mL) and the solvent removed under vacuum to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-80%) as the eluent. Concentration of solvent from test tubes containing the products (based on TLC) in vacuum resulted in a white oil.

(43) General Procedure H) Hydrogenation

(44) To a solution of the protected compound (20 mg) in a EtOAc:MeOH:H.sub.2O:AcOH (1.0:0.5:0.25:0.125, 1.825 mL) in a 10 mL round bottom flask equipped with a stir bar was added palladium on carbon (20 mg). Using a balloon, the solution was flushed with hydrogen for 2 minutes and stirred at room temperature under hydrogen pressure for 40 h. Reaction was filtered through PTFE filter (0.45 μM) and the flask washed using H.sub.2O:MeOH solution (1:1, 5 mL). The filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified on SepPak C18 ec (small) using 100% H.sub.2O, H.sub.2O:MeOH (1:1) and 100% MeOH as the eluent. Concentration of solvent from the vial containing the main product in lyophilizer overnight resulted in a white solid.

Example A. Glycosylation

Example A-1. Compound 4

(45) ##STR00150##

(46) 2,3,4-Tri-O-benzyl-5-O-(2-naphthalenylmethyl)-1-O-D-ribitol 2 (40 g, 71.08 mmol) was coevaporated with anhydrous toluene and dried in vacuum. A solution of 2 in anhydrous DCM (320 mL) under argon atmosphere and stirring was cooled to 0° C. and BF.sub.3.Et.sub.2O (19.3 mL, 156.38 mmol) added dropwise. After 5 min, a solution of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose 1 (15.08 g, 47.38 mmol) in toluene (480 mL) was added over 45 minutes. The reaction mixture was warmed to room temperature and stirred for 2 h. Reaction was monitored by TLC. Reaction mixture was quenched by addition of triethylamine (50 mL) and diluted with DCM (200 mL). The reaction mixture was washed with NaHCO.sub.3 aq. sat. solution (200 mL) and the aqueous layer extracted with DCM (2×150 mL). The combined organic layers were washed with NaCl aq. sat. solution (100 mL) and dried over Na.sub.2SO.sub.4 (˜20 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product 4 was purified on silica gel column chromatography using EtOAc in n-hexane (20-30%) as the eluent. Concentration of solvent from test tubes containing product in vacuum resulted in the yellow oil. The product (31.05 g, 37.82 mmol) was then redissolved in methanol (70 mL) in a 250 mL RBF (oven dried) under argon atmosphere and sodium methoxide 25% wt solution in methanol (4.09 mL, 18.91 mmol) was added. The solution was stirred at room temperature for 4 h. Reaction was monitored by TLC. Reaction mixture was neutralized (pH 7) by addition of Amberlite IR-120H (500 mg), filtered and the solvent evaporated under vacuum to obtain the crude product. The crude product was purified on silica gel column chromatography using EtOAc in n-hexane (50-100%) as the eluent. Concentration of solvent from test tubes containing product 4 in vacuum resulted in the yellow oil (11.46 g, 35% over 2 steps). HRMS (ESI.sup.+) Calcd for C.sub.42H.sub.46O.sub.9Na.sup.+[M+Na].sup.+ 717.3040, found 717.3057.

Example A-2. Compound 5

(47) ##STR00151##

(48) 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (8.2 g, 11.79 mmol) was added dropwise to a solution of triol 4 (3.53 g, 11.2 mmol) and imidazole (3.05 mg, 44.8 mmol) in acetonitrile (70 mL) in a 100 mL RBF (oven dried) under argon atmosphere. Reaction was monitored by TLC. After stirring at room temperature for 10 min, reaction mixture was quenched by addition of water (10 mL) and diluted with DCM (50 mL) and water (20 mL). The aqueous layer was separated and washed with DCM (2×30 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 (˜2 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-30%) as the eluent. Concentration of solvent from test tubes containing product 5 in vacuum resulted in a colourless oil (8.4 g, 76%). HRMS (ESI.sup.+) Calcd for C.sub.54H.sub.72N.sub.2O.sub.10Si.sub.2Na.sup.+[M+Na].sup.+ 959.4562, found 959.4590.

Example B. 2′-Methoxypolyribosylribitolphosphate

Example B-1. Compound 6

(49) ##STR00152##

(50) Ag.sub.2O (6 g, 26.0 mmol) was added to a stirred solution of the alcohol 5 (3.5 g, 3.73 mmol) in MeI (6 mL) at room temperature in a 25 mL RBF (oven dried) under argon atmosphere. Reaction mixture was stirred for 2.5 days. Reaction was monitored by TLC. After complete consumption of starting material reaction mixture was diluted with DCM (50 mL) and filtered through Celite® pad and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified on silica gel column chromatography using EtOAc in n-hexane (0-20%) as the eluent. Concentration of solvent from test tubes containing product in vacuum resulted in the colourless oil 6 (3.0 g, 85%). HRMS (ESI.sup.+) Calcd for C.sub.55H.sub.74N.sub.2O.sub.10Si.sub.2Na.sup.+[M+Na].sup.+ 973.4718, found 973.4738.

Example B-2. Compound 7

(51) ##STR00153##

(52) According to general procedure A) the silylated compound 6 was converted to diol product 7 (4.6 g, 96%). HRMS (ESI.sup.+) Calcd for C.sub.43H.sub.48O.sub.9Na.sup.+[M+Na].sup.+ 731.3196, found 731.3217.

Example B-3. Compound 8

(53) ##STR00154##

(54) According to general procedure H), the monomer 7 was converted to the deprotected monomer 8 (5 mg, 67%). HRMS (ESI.sup.+) Calcd for C.sub.11H.sub.22O.sub.9Na.sup.+[M+Na].sup.+ 321.1162, found 321.1187.

Example B-4. Compound 9

(55) ##STR00155##

(56) Benzyl bromide (77 mg, 0.45 mmol) and sodium hydride (16 mg, 0.672 mmol) were added to a stirred solution of the diol 7 (80 mg, 0.112 mmol) in THF:DMF (1.5:0.2, 1.7 mL) solution under argon atmosphere at 0° C. The reaction was warmed to room temperature and stirred overnight. The reaction mixture was quenched with ice cold water (1 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 (˜0.5 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by flash chromatography using EtOAc in n-hexane (0-50%) as the eluent. Concentration of solvent from test tubes containing product 9 in vacuum resulted in a colourless oil (75 mg, 75%). HRMS (ESI.sup.+) Calcd for C.sub.57H.sub.60O.sub.9Na.sup.+[M+Na].sup.+911.4135, found 911.4162.

Example B-5. Compound 10

(57) ##STR00156##

(58) According to general procedure D), the NAP protected intermediate 9 was converted to the 5-hydroxyl compound 10 (189 mg, 89%). HRMS (ESI.sup.+) Calcd for C.sub.46H.sub.52O.sub.9Na.sup.+[M+Na].sup.+ 771.3509, found 771.3521.

Example B-6. Compound 11

(59) ##STR00157##

(60) According to general procedure B) the diol 7 was converted to the benzylated product 11 (3.8 g, 73%). HRMS (ESI.sup.+) Calcd for C.sub.50H.sub.54O.sub.9Na.sup.+[M+Na].sup.+ 821.3666, found 821.3672.

Example B-7. Compound 12

(61) ##STR00158##

(62) According to general procedure C), the 3-hydroxyl intermediate 11 was converted to the Lev protected compound 12 (1.75 g, 97%). HRMS (ESI.sup.+) Calcd for C.sub.55H.sub.60O.sub.11Na.sup.+[M+Na].sup.+ 919.4033, found 919.4062.

Example B-8. Compound 13

(63) ##STR00159##

(64) According to general procedure D), the NAP protected intermediate 12 was converted to the 5-hydroxyl compound 13 (1.23 g, 80%). HRMS (ESI.sup.+) Calcd for C.sub.44H.sub.52O.sub.11Na.sup.+[M+Na].sup.+ 779.3407, found 779.3431.

Example B-9. Compound 15

(65) ##STR00160##

(66) According to general procedure E), the 3-hydroxyl compound 11 and the 5-hydroxyl compound 10 were converted to dimer 15 (60 mg, 95%). HRMS (ESI.sup.+) Calcd for C.sub.103H.sub.111O.sub.20PNa.sup.+[M+Na].sup.+ 1721.7304, found 1721.7393.

Example B-10. Compound 16

(67) ##STR00161##

(68) According to general procedure H), the dimer 15 was converted to the deprotected dimer 16 (5.6 mg, 77%). HRMS (ESI.sup.+) Calcd for C.sub.11H.sub.22O.sub.9Na.sup.+[M+Na].sup.+ 681.1983, found 681.2000.

Example B-11. Compound 17

(69) ##STR00162##

(70) According to general procedure D), the NAP protected dimer 15 was converted to the 5-hydroxyl compound 17 (30 mg, 60%). HRMS (ESI.sup.+) Calcd for C.sub.92H.sub.103O.sub.20Na.sup.+[M+Na].sup.+ 1581.6678, found 1581.6734.

Example B-12. Compound 18

(71) ##STR00163##

(72) According to general procedure E), the dimer 17 was coupled to the linker 5-azido-pentanol to give dimer 18 (6 mg, 42%). HRMS (ESI.sup.+) Calcd for C.sub.104H.sub.119N.sub.3O.sub.23P.sub.2Na.sup.+[M+Na].sup.+ 1863.7641, found 1863.7709.

Example B-13. Compound 19

(73) ##STR00164##

(74) According to general procedure H), the dimer 18 was converted to the deprotected dimer 19 (1.2 mg, 55%). HRMS (ESI.sup.+) Calcd for C.sub.27H.sub.50O.sub.23P.sub.2Na.sup.+[M+Na].sup.+846.2538, found 846.2540.

Example B-14. Compound 20

(75) ##STR00165##

(76) According to general procedure E), the 3-hydroxyl compound 11 and the 5-hydroxyl compound 13 were converted to dimer 20 (1.5 g, 76%). HRMS (ESI.sup.+) Calcd for C.sub.101H.sub.111O.sub.22PNa.sup.+[M+Na].sup.+ 1729.7202, found 1729.7240.

Example B-15. Compound 21

(77) ##STR00166##

(78) According to general procedure D), the NAP protected intermediate 20 was converted to the 5-hydroxyl compound 21 (386 mg, 63%). HRMS (ESI.sup.+) Calcd for C.sub.90H.sub.103O.sub.22PNa.sup.+[M+Na].sup.+ 1589.6576, found 1589.6613.

Example B-16. Compound 22

(79) ##STR00167##

(80) According to general procedure G), the dimer 20 was converted to 3-hydroxyl dimer 22 (473 mg, 74%). HRMS (ESI.sup.+) Calcd for C.sub.96H.sub.105O.sub.20PNa.sup.+[M+Na].sup.+1631.6835, found 1631.6873.

Example B-17. Compound 23

(81) ##STR00168##

(82) According to general procedure E), the 3-hydroxyl dimer 22 was coupled to the linker 5-azido-pentanol to give dimer 23 (3 mg, 30%). HRMS (ESI.sup.+) Calcd for C.sub.108H.sub.121N.sub.3O.sub.23P.sub.2Na.sup.+[M+Na].sup.+ 1912.7764, found 1912.7781.

Example B-18. Compound 24

(83) ##STR00169##

(84) According to general procedure H), the dimer 23 was converted to the deprotected dimer 24 (1.1 mg, 76%). HRMS (ESI.sup.+) Calcd for C.sub.27H.sub.50O.sub.23P.sub.2Na.sup.+[M+Na].sup.+846.2538, found 846.2540.

Example B-19. Compound 25

(85) ##STR00170##

(86) According to general procedure E), the 3-hydroxyl dimer 22 and the 5-hydroxyl dimer 21 were converted to tetramer 25 (367 mg, 86%). HRMS (ESI.sup.+) Calcd for C.sub.193H.sub.213O.sub.44P.sub.3Na.sup.+[M+Na].sup.+3350.3540, found 3350.3531.

Example B-20. Compound 26

(87) ##STR00171##

(88) According to general procedure D), the NAP protected tetramer 25 was converted to the 5-hydroxyl tetramer 26 (125 mg, 75%). HRMS (ESI.sup.+) Calcd for C.sub.182H.sub.205O.sub.44P.sub.3Na.sup.+[M+Na].sup.+ 3210.2914, found 3210.2911.

Example B-21. Compound 27

(89) ##STR00172##

(90) According to general procedure E), the tetramer 26 was coupled to the linker 5-azido-pentanol to give tetramer 27 (35 mg, 77%). HRMS (ESI.sup.+) Calcd for C.sub.194H.sub.221N.sub.3O.sub.47P.sub.4Na.sup.+[M+Na].sup.+3491.3844, found 3492.3787.

Example B-22. Compound 28

(91) ##STR00173##

(92) According to general procedure G), the tetramer 27 was converted to 3-hydroxyl tetramer 28 (30 mg, 90%). HRMS (ESI.sup.+) Calcd for C.sub.189H.sub.215N.sub.3O.sub.45P.sub.4Na.sup.+[M+Na].sup.+3393.3476, found 3393.3543.

Example B-23. Compound 29

(93) ##STR00174##

(94) According to general procedure H), the tetramer 28 was converted to the deprotected tetramer 29 (10 mg, 73%). HRMS (ESI.sup.+) Calcd for C.sub.49H.sub.97NO.sub.45P.sub.4Na.sup.+[M+Na].sup.+1566.4181, found 1566.4174.

Example B-24. Compound 30

(95) ##STR00175##

(96) Tetramer 30 is prepared in two steps from tetramer 26 according to general procedure G) and general procedure H).

Example B-25. Compound 31

(97) ##STR00176##

(98) According to general procedure E), the 3-hydroxyl dimer 22 and the 5-hydroxyl tetramer 26 were converted to hexamer 31 (85 mg, 85%). HRMS (ESI.sup.+) Calcd for C.sub.285H.sub.315O.sub.66P.sub.5Na.sup.+[M/2+Na].sup.+2496.9888, found 2496.0271.

Example B-26. Compound 32

(99) ##STR00177##

(100) According to general procedure D), the NAP protected hexamer 31 was converted to the 5-hydroxyl hexamer 32 (50 mg, 65%). HRMS (ESI.sup.+) Calcd for C.sub.274H.sub.307O.sub.66P.sub.5Na.sup.+[M/2+Na].sup.+2426.9677, found 2426.9977.

Example B-27. Compound 33

(101) ##STR00178##

(102) According to general procedure E), the 3-hydroxyl dimer 22 and the 5-hydroxyl hexamer 32 were converted to octamer 33 (50 mg, 77%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.86-7.62 (m, 4H), 7.51-7.35 (m, 3H), 7.37-7.06 (m, 195H), 5.10 (t, J=5.5 Hz, 1H), 5.03 (dt, J=7.6, 4.2 Hz, 7H), 4.97-4.77 (m, 22H), 4.69-4.37 (m, 60H), 4.36-4.14 (m, 23H), 3.98-3.55 (m, 47H), 3.53-3.36 (m, 13H), 3.34-3.21 (m, 24H), 2.72 (t, J=6.5 Hz, 2H), 2.65-2.54 (m, 2H), 2.16 (s, 3H).

Example B-28. Compound 34

(103) ##STR00179##

(104) According to general procedure D), the NAP protected octamer 33 was converted to the 5-hydroxyl octamer 34 (35 mg, 73%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.43-7.07 (m, 195H), 5.10 (t, J=5.4 Hz, 1H), 5.08-4.78 (m, 29H), 4.70-4.45 (m, 51H), 4.45-4.15 (m, 38H), 3.95-3.54 (m, 50H), 3.54-3.36 (m, 16H), 3.36-3.25 (m, 24H), 2.72 (t, J=6.5 Hz, 2H), 2.65-2.56 (m, 2H), 2.16 (s, 3H).

Example B-29. Compound 35—Phosphonate Chemistry

(105) ##STR00180##

(106) To a solution of 3-hydroxyl compound 11 (10 mg) and imidazole (5.96 mg, 0.087 mmol) in DCM (2 mL) in a 10 mL RBF (oven dried) under argon atmosphere was added PCl.sub.3 (4.37 μL, 0.050 mmol) and triethylamine (12.28 μL, 0.087 mmol) at 0° C. After 5 min, the reaction mixture was warmed to room temperature and stirred for 1 h. Reaction was monitored by TLC. The reaction was diluted with DCM (5 mL), quenched by the addition of NaHCO.sub.3 aq. sat. solution (5 mL) and triethylammonium buffer solution (5 mL). The aqueous layer was extracted with DCM (2×10 mL), the combined organic layers were washed with brine (5 mL). The organic layer was dried over Na.sub.2SO.sub.4 (0.25 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified on silica gel column chromatography using 10% MeOH in DCM as the eluent. Concentration of solvent from test tubes containing product 35 (based on TLC) in vacuum resulted in the yellow oil (10 mg, 83%). HRMS (ESI.sup.+) Calcd for C.sub.56H.sub.71NO.sub.11SP.sup.+[M+H].sup.+ 964.4765, found 964.4759.

Example B-30. Compound 36

(107) ##STR00181##

(108) H-phosphonate 35 (10 mg, 0.010 mmol) and compound 13 (7.85 mg, 0.010 mmol) were dissolved in 1n pyridine (1 mL). Trimethylacetyl chloride (3.83 μL, 0.031 mmol) was then slowly added. The reaction mixture was stirred for 1 h at room temperature. A solution of iodine (2.6 mg, 0.010 mmol) in pyridine-water (96:4, v/v; 67 μL) was added and the reaction was further stirred for 30 min at room temperature. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (5 mL), washed with aq. sat. Na.sub.2S.sub.2O.sub.3 (5 mL) and then with 1.0 M TEAB solution (5 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 filtered, and concentrated in vacuum. The residue was purified by flash chromatography (DCM/MeOH, 10:1, v/v) to yield 36 (5.5 g, 29%) as colorless oil. HRMS (ESI.sup.+) Calcd for C.sub.94H.sub.104O.sub.22P.sup.+[M].sup.− 1615.6762, found 1615.7056.

Example C. 2′-Deoxypolyribosylribitolphosphate

Example C-1. Compound 37

(109) ##STR00182##

(110) To a solution of the alcohol 4 (860 mg, 0.92 mmol) in 1,2-dichloroethane (5 mL) in a 25 mL RBF (oven dried) under argon atmosphere was added thiocarbonyldiimidazole (327 mg, 1.83 mmol). The solution was stirred at 60° C. for 64 h. Reaction was monitored by TLC. The solvent was evaporated under vacuum to obtain the crude product. The crude product was purified by flash column chromatography using EtOAc in n-hexane (0-30%) as the eluent. Concentration of solvent from test tubes containing the product 37 (based on TLC) in vacuum resulted in a white oil (950 mg, 98%). HRMS (ESI.sup.+) Calcd for C.sub.58H.sub.74N.sub.2O.sub.10SSi.sub.2Na.sup.+[M+Na].sup.+1069.4500, found 1069.4525.

Example C-2. Compound 38

(111) ##STR00183##

(112) To a solution of Bu.sub.3SnH (0.49 mL, 1.814 mmol) and AIBN (0.2 M in toluene, 0.46 mL, 0.091 mmol) in toluene (20 mL) in a 100 mL RBF (oven dried) under argon atmosphere at 60° C. was added dropwise a solution of the thiocarbamate 37 (950 mg, 0.907 mmol) in toluene (20 mL). The reaction mixture was stirred for 2 h. Reaction was monitored by TLC. Reaction mixture was diluted was quenched with NaHCO.sub.3 aq. sat. solution (10 mL). The aqueous layer was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with brine (10 mL), dried over Na.sub.2SO.sub.4 (0.5 g), filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-50%). Concentration of solvent from test tubes containing the product 38 (based on TLC) in vacuum resulted in a colorless oil (510 mg, 61%). HRMS (ESI.sup.+) Calcd for C.sub.58H.sub.72O.sub.9Si.sub.2Na.sup.+[M+Na].sup.+943.4613, found 943.4640.

Example C-2. Compound 39

(113) ##STR00184##

(114) According to general procedure A) the silylated compound 38 was converted to diol product 39 (330 mg, 90%). HRMS (ESI.sup.+) Calcd for C.sub.42H.sub.46O.sub.8Na.sup.+[M+Na].sup.+701.3090, found 701.3117.

Example C-3. Compound 40

(115) ##STR00185##

(116) According to general procedure B) the diol 39 was converted to the benzylated product 40 (260 mg, 69%). HRMS (ESI.sup.+) Calcd for C.sub.49H.sub.52O.sub.8Na.sup.+[M+Na].sup.+791.3560, found 791.3565.

Example C-4. Compound 41

(117) ##STR00186##

(118) According to general procedure C), the 3-hydroxyl intermediate 40 was converted to the Lev protected compound 41 (135 mg, 82%). HRMS (ESI.sup.+) Calcd for C.sub.54H.sub.58O.sub.10Na.sup.+[M+Na].sup.+889.3928, found 889.3928.

Example C-5. Compound 42

(119) ##STR00187##

(120) According to general procedure D), the NAP protected intermediate 41 was converted to the 5-hydroxyl compound 42 (95 mg, 85%). HRMS (ESI.sup.+) Calcd for C.sub.43H.sub.50O.sub.10Na.sup.+[M+Na].sup.+749.3302, found 749.3325.

Example C-6. Compound 44

(121) ##STR00188##

(122) According to general procedure E), the 3-hydroxyl compound 40 and the 5-hydroxyl compound 42 were converted to dimer 44 (160 mg, 78%). HRMS (ESI.sup.+) Calcd for C.sub.99H.sub.107O.sub.20PNa.sup.+[M+Na].sup.+1669.6991, found 1669.6981.

Example C-7. Compound 45

(123) ##STR00189##

(124) According to general procedure D), the dimer 44 was converted to 5-hydroxyl dimer 45 (53 mg, 73%). HRMS (ESI.sup.+) Calcd for C.sub.99H.sub.107O.sub.20PNa.sup.+[M+Na].sup.+1529.6365, found 1529.6397.

Example C-8. Compound 46

(125) ##STR00190##

(126) According to general procedure G), the dimer 44 was converted to 3-hydroxyl dimer 46 (85 mg, 91%). HRMS (ESI.sup.+) Calcd for C.sub.94H.sub.101O.sub.18PNa.sup.+[M+Na].sup.+1571.6623, found 1571.6656.

Example C-9. Compound 49

(127) ##STR00191##

(128) According to general procedure E), the 3-hydroxyl dimer 46 and the 5-hydroxyl dimer 45 were converted to tetramer 49 (96 mg, 83%). HRMS (ESI.sup.+) Calcd for C.sub.189H.sub.205O.sub.40P.sub.3Na.sup.+[M+Na].sup.+3230.3118, found 3230.3111.

Example C-10. Compound 50

(129) ##STR00192##

(130) According to general procedure D), the NAP protected tetramer 49 was converted to the 5-hydroxyl tetramer 50 (50 mg, 56%). HRMS (ESI.sup.+) Calcd for C.sub.178H.sub.197O.sub.40P.sub.3Na.sup.+[M+Na].sup.+3090.2492, found 3090.2405.

Example C-11. Compound 51

(131) ##STR00193##

(132) According to general procedure E), the tetramer 50 was coupled to the linker 5-azido-pentanol to give tetramer 51 (41 mg, 76%). HRMS (ESI.sup.+) Calcd for C.sub.190H.sub.213N.sub.3O.sub.43P.sub.4Na.sup.+[M+Na].sup.+3371.3421, found 3371.3321.

Example C-12. Compound 52

(133) ##STR00194##

(134) According to general procedure G), the tetramer 51 was converted to 3-hydroxyl tetramer 52 (36 mg, 92%). HRMS (ESI.sup.+) Calcd for C.sub.185H.sub.207N.sub.3O.sub.41P.sub.4Na.sup.+[M+Na].sup.+3273.3053, found 3273.3079.

Example C-13. Compound 53

(135) ##STR00195##

(136) According to general procedure H), the tetramer 52 was converted to the deprotected tetramer 53 (6 mg, 70%). HRMS (ESI.sup.+) Calcd for C.sub.45H.sub.89NO.sub.41H.sub.4Na.sup.+[M+Na/2].sup.+723.1879, found 723.1996.

Example D. 2′-N,N,-Dimethylaminocarbonylpolyribosylribitolphosphate

Example D-1. Compound 54

(137) ##STR00196##

(138) To a solution of alcohol 4 (2.153 g, 2.299 mmol) in DCM (10 mL) at room temperature under argon atmosphere was added 1,1′-carbonyldiimidazole (145 mg, 4.598 mmol). The reaction mixture was stirred for 20 min (when CDI was completely dissolved) and monitored by TLC until complete consumption of the starting material. The solvent was evaporated in vacuum to give the crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-100%) as the eluent. Concentration of solvent from test tubes containing the product 54 (based on TLC) in vacuum resulted in the colourless oil (2.35 g, 99%). HRMS (ESI.sup.+) Calcd for C.sub.58H.sub.75N.sub.2O.sub.11Si.sub.2.sup.+[M+H].sup.+ 1031.4909, found 1031.4936.

Example D-2. Compound 55

(139) ##STR00197##

(140) Monomer 54 (2.396 g, 2.32 mmol) was dissolved in ACN (20 mL) at room temperature under argon atmosphere and Me.sub.2NH was added (1.89 g, 23.2 mmol). The reaction mixture was stirring at room temperature for 65 h and monitored by TLC. The solution was filtered off through a pad of celite and washed with DCM (50 mL). The solvent was evaporated in vacuum. The resulting solid was solubilized in DCM (100 mL) and the organic solution was washed with brine (100 mL) and dried with Na.sub.2SO.sub.4 (˜2 g). The organic solvent was evaporated in vacuum to give to crude product. The crude product was purified by automated flash chromatography using EtOAc in n-hexane (0-100%) as the eluent. Concentration of solvent from test tubes containing the product 55 (based on TLC) in vacuum resulted in the colourless oil (2 g, 85%). HRMS (ESI.sup.+) Calcd for C.sub.57H.sub.77NO.sub.11Si.sub.2Na.sup.+[M+Na].sup.+1030.4933, found 1030.4958.

Example D-3. Compound 56

(141) ##STR00198##

(142) According to general procedure A) the silylated compound 55 was converted to diol product 56 (1.1 g, 73%). HRMS (ESI.sup.+) Calcd for C.sub.45H.sub.51NO.sub.10Na.sup.+[M+Na].sup.+788.3411, found 788.3431.

Example D-4. Compound 57

(143) ##STR00199##

(144) According to general procedure B) the diol 56 was converted to the benzylated product 57 (91 mg, 16%). HRMS (ESI.sup.+) Calcd for C.sub.52H.sub.57NO.sub.10Na.sup.+[M+Na].sup.+878.3880, found 878.3930.

Example D-5. Compound 58

(145) ##STR00200##

(146) According to general procedure C), the 3-hydroxyl intermediate 57 was converted to the Lev protected compound 58 (46 mg, 92%). HRMS (ESI.sup.+) Calcd for C.sub.57H.sub.63NO.sub.12Na.sup.+[M+Na].sup.+976.4248, found 976.4318.

Example D-6. Compound 59

(147) ##STR00201##

(148) According to general procedure D), the monomer 58 was converted to 5-hydroxyl monomer 59 (23 mg, 60%). HRMS (ESI.sup.+) Calcd for C.sub.46H.sub.55NO.sub.12Na.sup.+[M+Na].sup.+836.3622, found 836.3682.

Example D-7. Compound 60

(149) ##STR00202##

(150) According to general procedure E), the monomer 59 was coupled to the linker 5-azido-pentanol to give monomer 60 (19 mg, 70%). HRMS (ESI.sup.+) Calcd for C.sub.58H.sub.71N.sub.4O.sub.15PNa.sup.+[M+Na].sup.+1117.4551, found 1117.4628.

Example D-8. Compound 61

(151) ##STR00203##

(152) According to general procedure G), the monomer 60 was converted to 3-hydroxyl monomer 61 (12 mg, 71%). HRMS (ESI.sup.+) Calcd for C.sub.53H.sub.65N.sub.4O.sub.13PNa.sup.+[M+Na].sup.+1019.4183, found 1019.4246.

Example D-9. Compound 62

(153) ##STR00204##

(154) According to general procedure H), the monomer 61 was converted to the deprotected monomer 62 (3 mg, 60%). HRMS (ESI.sup.+) Calcd for C.sub.18H.sub.38N.sub.2O.sub.13PNa.sup.+[M+H].sup.+ 521.2112, found 521.2125.

Example E. 2′-Fluoropolyribosylribitolphosphate

Example E-1. Compound 63

(155) ##STR00205##

(156) To compound 4 in acetone was added 2,2-dimethoxypropane (1.2 equiv.) followed by camphorsulfonic acid (0.5 equiv). After 6 h of stirring, the reaction mixture was concentrated under reduced pressure which on column chromatographic purification gave the pure compound 63.

Example E-2. Compound 64

(157) ##STR00206##

(158) Compound 63 was treated at 0° C. with triflic anhydride and pyridine in DCM. After two hours, tetrabutylammonium nitrite and sodium nitrite were added and the mixture was stirred at r.t. for additional 5 hours. The product was extracted into hexane (200 mL). The extract was washed with aqueous sodium hydroxide (2N), brine, dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure to give a brown oil. Purification by flash chromatography (eluting with hexane:ether) gave 64.

Example E-3. Compound 65

(159) ##STR00207##

(160) DAST (diethylamino sulfur trifluoride) (1.2 equiv.) was added dropwise to a stirred solution of 64 in dichloromethane at −78° C. The mixture was allowed to warm to room temperature overnight with the cooling bath in place. After 20 h, the solution was poured slowly into a vigorously stirred mixture of ice and excess saturated aqueous sodium bicarbonate. When effervescence had ceased, the product was extracted into hexane (200 mL). The extract was washed with aqueous sodium hydroxide (2N), brine, dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure to give a brown oil. Purification by flash chromatography (eluting with hexane:ether) gave 65.

Example E-4. Compound 66

(161) ##STR00208##

(162) Compound 65 was taken in 80% acetic acid and stirred for 2 h at 80° C. After completion of reaction (TLC), the acidic solution was concentrated under reduced pressure to give a crude product which was purified by column chromatography to yield compound 66.

Example E-5. Compound 67

(163) ##STR00209##

(164) According to general procedure B) the diol 66 was converted to the benzylated product 67.

Example E-6. Compound 68

(165) ##STR00210##

(166) According to general procedure C), the 3-hydroxyl intermediate 67 was converted to the Lev protected compound 68.

Example E-7. Compound 69

(167) ##STR00211##

(168) According to general procedure D), the NAP protected intermediate 68 was converted to the 5-hydroxyl compound 69.

Example E-8. Compound 71

(169) ##STR00212##

(170) According to general procedure E), the 3-hydroxyl compound 67 and the 5-hydroxyl compound 69 were converted to dimer 71.

Example E-9. Compound 72

(171) ##STR00213##

(172) According to general procedure H), the dimer 71 was converted to the deprotected dimer 72.

Example E-10. Compound 73

(173) ##STR00214##

(174) According to general procedure D), the NAP protected dimer 71 was converted to the 5-hydroxyl compound 73.

Example E-11. Compound 74

(175) ##STR00215##

(176) According to general procedure E), the dimer 73 was coupled to the linker 5-azido-pentanol to give dimer 74.

Example E-12. Compound 75

(177) ##STR00216##

(178) According to general procedure H), the dimer 74 was converted to the deprotected dimer 75.

Example F. 2′-Substituted Polyribosylribitolphosphates

Example F-1. Compound 76

(179) ##STR00217##

(180) According to general procedure E), the 3-hydroxyl compound 67 and the 5-hydroxyl compound 69 were converted to dimer 76.

Example F-2. Compound 77

(181) ##STR00218##

(182) According to general procedure D), the NAP protected intermediate 76 was converted to the 5-hydroxyl compound 77.

Example F-3. Compound 79

(183) ##STR00219##

(184) According to general procedure E), the 3-hydroxyl dimer 22 and the 5-hydroxyl dimer 77 were converted to tetramer 79.

Example F-4. Compound 80

(185) ##STR00220##

(186) According to general procedure D), the NAP protected tetramer 79 was converted to the 5-hydroxyl tetramer 80.

Example F-5. Compound 81

(187) ##STR00221##

(188) According to general procedure E), the tetramer 80 was coupled to the linker 5-azido-pentanol to give tetramer 81.

Example F-6. Compound 82

(189) ##STR00222##

(190) According to general procedure G), the tetramer 81 was converted to 3-hydroxyl tetramer 82.

Example F-7. Compound 83

(191) ##STR00223##

(192) According to general procedure H), the tetramer 82 was converted to the deprotected tetramer 83.

Example F-8. Compound 85

(193) ##STR00224##

(194) According to general procedure E), the 3-hydroxyl dimer 22 and the 5-hydroxyl dimer 45 were converted to tetramer 85.

Example F-9. Compound 86

(195) ##STR00225##

(196) According to general procedure D), the NAP protected tetramer 85 was converted to the 5-hydroxyl tetramer 86.

Example F-10. Compound 87

(197) ##STR00226##

(198) According to general procedure E), the tetramer 86 was coupled to the linker 5-azido-pentanol to give tetramer 87.

Example F-11. Compound 88

(199) ##STR00227##

(200) According to general procedure G), the tetramer 87 was converted to 3-hydroxyl tetramer 88.

Example F-12. Compound 89

(201) ##STR00228##

(202) According to general procedure H), the tetramer 88 was converted to the deprotected tetramer 89.

Example G. Synthesis of a Stabilized Saccharide-CRM197—Cysteine Conjugate

Example G-1. Compound 90

(203) ##STR00229##

(204) Making CRM.sub.197 Ready for the Conjugation:

(205) CRM.sub.197 (3.0 mg, 0.0517 μmol) from the stock solution was transferred to the Amicon filter (0.5 mL, MWCO 30 KDa) and centrifuged for 3 min at 10000 RPM. 300 μL of the 1×PBS was added to the remaining protein in Amicon filter and centrifuged again for 3 min at 10000 RPM. Then 400 μL of 1×PBS buffer (pH 7.4) was added to Amicon filter and centrifuged for 3 min at 10000 RPM. Then the filter containing protein was reversed inside of a new Amicon micro centrifuge tube (1.5 mL) and centrifuged for 1 min at 1000 RPM to collect the protein in micro centrifuge tubes (volume ˜=100-120 μL).

(206) Conjugation Procedure:

(207) CRM.sub.197 in 1×PBS buffer (˜100 μL) was diluted with 1.3 mL of 1×PBS buffer in the reaction vial at r.t. equipped with a stir bar.

(208) 3-(Maleimido)propionic acid N-hydroxysuccinimide ester (0.688 mg, 2.58 μmol) was transferred to a Type I glass-2 mL reaction vial, dissolved with 20 μL of DMSO, added it to the reaction vial containing CRM197 in 1×PBS buffer. Stirred the reaction at rt for 4 h. RM was clear colorless solution.

(209) Washing Steps:

(210) The reaction mixture was transferred to the Amicon filter (0.5 mL, MWCO 30 KDa) and centrifuged for 3 min at 10000 RPM and repeated the process till whole reaction mixture was transferred and centrifuged. Then added 400 μL of 1×PBS buffer to the reaction vial and rinsed and transferred it to the Amicon filter. This washing was repeated four more times with 400 μL of the PBS buffer solution. Finally, Amicon filter containing CRM197-maleimide was reversed inside of a new Amicon micro centrifuge tube (1.5 mL) and centrifuged for 1 min at 1000 RPM to collect ˜130 μL CRM197-maleimide in micro centrifuge tubes. Added 950 μL of PBS solution (pH 7.4) to it and stored the vial at 2-8° C. (total volume=˜1.08 mL). The CRM197-Maleimide conjugate was analyzed by MALDI, SDS-page, Western blot, SEC-HPLC and protein estimation by BCA. The CRM197-Maleimide conjugate was analyzed by MALDI and found between 21-24. BCA estimation also showed the good recovery of the protein. SDS page showed the CRM-maleimide conjugate 90 was higher molecular weight than CRM.sub.197 and stable. Western blot shows that CRM anti-goat antibodies are recognizing the CRM-conjugate 90 whereas the Hib Anti-rabbit antibodies did not recognize the CRM-maleimide conjugate 90 which was without sugar. Sample was stored at 2-8° C. till further use.

Example G-2. Compound 94 Saccharide-CRM197—Cysteine Conjugate

(211) The synthetic route for compound 94 is outlined in FIG. 3.

(212) DTT Method:

(213) Compound 29 (5 mg, 0.003 mmol) was dissolved in pH 7.4 PBS buffer (5 mL) and DSP (dithiobis(succinimidyl propionate)) (20 mg, 0.049 mmol) in DMSO (2.5 mL) was added at room temperature overnight. After that, dithiothreitol (DTT) (4 mg, 0.025 mmol) was added and the solution stirred at 40° C. for further 2 h. Compound 91 was purified through Sephadex G-25 column chromatography (4.5 mg, 88%). HRMS (ESI.sup.+) Calcd for C.sub.51H.sub.99NO.sub.46P.sub.4S [M+2Na+2K−4H].sup.− 1736.2793, found 1736.279.

(214) Making Saccharide-Thiol Ready for the Conjugation: TCEP Method:

(215) 100 μL of TCEP suspension was taken in an 1.5 ml vial and added 300 μL of 1×PBS solution (pH 7.4) was added. Centrifuged, and the supernatant solution was removed. And the washing with PBS repeated once more. And finally the residue was taken in 100 μL of PBS solution and added to the Sugar-disulfide 91 (1.38 mg, 0.292 μmol) in PBS buffer (0.15 mL) in a 2 mL type I glass vial. Shaken gently using orbital shaker for 1 h at rt. The RM was transferred to vial, and glass reaction vial was rinsed with 200 μL of PBS solution and transferred to the vial. Centrifuged, collected the supernatant (˜325 μL) of PBS solution in a 2 mL type I glass vial. Added 200 μL of PBS solution to the residue and mixed well, centrifuged, the supernatant was collected and transferred to the 2 mL type I glass vial again. So, total volume of the reduced thiol 78 solution was ˜525 μL. ˜25 μL were kept for analysis.

(216) Conjugation Procedure:

(217) CRM.sub.197-maleimide 90 in 1×PBS buffer (˜850 μL) was taken in a reaction vial at rt equipped with a stir bar. The saccharide-thiol 92 in PBS solution (˜500 μL) in 1×PBS buffer was added to the CRM.sub.197-maleimide 90 solution dropwise. The vial was rinsed with 50 μL PBS solution and the solution was transferred to the reaction mixture at rt. RM was clear solution and stirred at r.t. for 20 h. [An aliquot of ˜40 μL of the reaction mixture was taken out for analysis purposes]. Then cysteine (0.14 mg) in phosphate buffer (pH 7.4, 30 μL) was added to the RM containing 93 and stirred for an hour at rt.

(218) Washing Steps:

(219) The reaction mixture containing 94 was transferred to the Amicon filter (0.5 mL, MWCO 30 KDa) and centrifuged for 3 min at 10000 RPM and repeated the process till whole reaction mixture was transferred and centrifuged. Then 400 μL of 1×PBS buffer were added to the reaction vial and rinsed and the solution was transferred to the Amicon filter. This washing was repeated four more times with 400 μL of the PBS buffer solution. Finally, the filter containing CRM.sub.197-maleimide-saccharide-cysteine 94 was reversed inside of a new Amicon micro centrifuge tube (1.5 mL) and centrifuged for 1 min at 1000 RPM to collect ˜150 μL CRM197-maleimide-sugar-Cysteine in micro centrifuge tube. Added 200 μL of PBS solution (pH 7.4) to the Amicon filter and rinsed well and transferred the solution to the micro centrifuge tube and diluted the solution with 750 μL of PBS solution (pH 7.4) and stored the vial at 2-8° C. (total volume=˜1100 μL). An aliquot of 40 μL of the conjugate 94 was washed with milliQ water using Amicon filter and analyzed using MALDI (sinapinic acid matrix) and loading between 3.4-4.2 was obtained. The conjugate 94 was further successfully analyzed using SDS-Page, western blot and SEC-HPLC. Protein estimation using BCA method showed that good recovery of the protein in the conjugate.

Example H. Stability Tests

Example H-1. Stability of Hib Capsular Polysaccharide in Basic Medium

(220) Stability study of the inventive saccharides is critical for the development of stable liquid Hib glycoconjugate vaccines. As a control we examined first the stability and cleavage of Hib polyribosylribitolphosphate capsular polysaccharide (CPS). Accordingly, Hib CPS was fragmented using standard procedures as described in Vaccine, 2000, 18, 1982-1993: 1 mg Hib CPS in 0.43 mL of 0.1 M NaOH, 20 h. Fragments obtained after purification (filtered through 30 kDa Amicon filters followed by desalting) were analyzed by .sup.1H NMR, HPLC and HRMS.

(221) ##STR00230##

(222) FIG. 4A shows the .sup.1H NMR spectrum of fragmented Hib CPS and FIG. 4B shows the .sup.1H NMR spectrum of untreated Hib CPS. FIG. 5A shows the HPLC chromatogram of fragmented Hib CPS and FIG. 5B shows the ESI mass spectrum of treated Hib CPS. This set of initial experiments demonstrated that the Hib CPS was cleaved to its smallest 2 and 3-0 phosphate fragments in basic medium.

Example H-2. Stability of Hib Capsular Polysaccharide in Aluminum Hydroxide Suspension

(223) Having established the stability data of the control Hib CPS, then the stability of Hib CPS in aluminum hydroxide was investigated, where it is known to be unstable in a vaccine formulation {0.5 mg Hib CPS, 3 mL Alhydrogel® (Brentag) and 22 mL milli-Q water} at various temperatures {37° C. (A1) and rt (A3)}. At 37° C. cleavage to smallest fragments were observed after 2 days and at rt this pattern was observed after day 7 (FIG. 6). From this experiment it was confirmed that, Hib CPS decomposes to 2 and 3-O phosphate fragments in 2 to 7 days depending on temperature in Aluminum hydroxide the most common adjuvant used in commercial vaccines.

Example H-3. Stability of Compound 16

(224) Stability studies of synthesized 2′-methoxyribosylribitolphosphate dimer 16 were conducted in presence of various adjuvants. As a first step stability of 16 was studied using Al(OH).sub.3, AlPO.sub.4 and water at various temperatures {37° C. (A1), 2-8° C. (A2), rt (A3) and 70° C. (A4)}.

(225) The amount of Al(OH).sub.3 and AlPO.sub.4 used for this study was similar to the amount present in commercial Hib vaccines. Analysis of samples at different temperatures using HPLC after a week is shown in FIGS. 7-10. Compounds 16 and 8 were used as reference since decomposition of dimer 16 should lead to monomer 8. Every 24 h 40 μL of solution was aliquoted from each vial and centrifuged for 4 min at 5000 rpm. Supernatant (25 μL) was taken for HPLC analysis. Even after 7 days, no decomposition was observed. From this study it was clear that 2′-methoxyribosylribitol dimer 16 was stable at 37° C. (A1), 2-8° C. (A2), r.t. (A3) and at 70° C.

(226) The stability of dimer 16 was further investigated under aqueous basic conditions as applied in Example H-1. From the HPLC chromatogram in FIG. 13D it was concluded that dimer 16 got cleaved completely after 4 days, while the natural Hib CPS got already cleaved completely within 20 hours. This demonstrates that the 2′-methoxyribosylribitolphosphate oligomers are very stable even under extreme basic conditions compared to natural Hib CPS.

Example H-4. Stability of Compound 29

(227) Stability studies of synthesized 2′-methoxyribosylribitolphosphate tetramer bearing a linker 29 were conducted in presence of various adjuvants. As a first step stability of 29 was studied using Al(OH).sub.3, phosphate buffer (PBS) and water at various temperatures {2-8° C. (A2), and r.t. (A3)}.

(228) The amount of Al(OH).sub.3 used for this study was similar to the amount present in commercial Hib vaccines. Analysis of samples at different temperatures using HPLC after a week is shown in FIGS. 11 and 12. Compounds 29, 16 and 8 were used as reference since decomposition of tetramer 29 should lead to dimer 16 and monomer 8. Every 24 h 40 μL of solution was aliquoted from each vial and centrifuged for 4 min at 5000 rpm. Supernatant (25 μL) was taken for HPLC analysis. Even after 7 days, no decomposition was observed. From this study it was clear that 2′-methoxyribosylribitolphosphate tetramer 29 is stable in presence of Al(OH).sub.3, in phosphate buffer (PBS) and in water at 2-8° C. (A2) and r.t. (A3).

(229) The stability of tetramer 29 was further investigated under aqueous basic conditions as applied in Example H-1. From the HPLC chromatogram in FIG. 13A it was concluded that tetramer 29 got cleaved completely after 4 days, while the natural Hib CPS got already cleaved completely within 20 hours. This demonstrates that the 2′-methoxyribosylribitolphosphate oligomers are very stable even under extreme basic conditions compared to natural Hib CPS.

Example H-5. Stability of Conjugate 94 in Presence of Phosphate Buffer

(230) ˜5 μg of compound 94 in two vials were each diluted to 500 μL of PBS pH 7.4; one vial was stored at 25° C. and another at 2-8° C. The samples were analyzed by bicinchoninic acid assay (BCA) and HPLC-SEC (column: TSKgel G2000SWxl, buffer: 50 mM Tris, 20 mM NaCl, pH 7.2) after one day and after 15 days. The data showed that still after 15 days the conjugate 94 was present and stable at both temperature conditions 25° C. and 2-8° c.

Example H-6. Stability of Compound 94 in Presence of Aluminum Hydroxide

(231) Two vials containing ˜5 μg of compound 94 and Alhydrogel® (0.125 mg/dose) were each filled up to 500 μL of PBS pH 7.4; one vial was stored at 25° C. and another at 2-8° C. The samples were analyzed by bicinchoninic acid assay (BCA), HPLC-SEC column: TSKgel G2000SWxl, buffer: 50 mM Tris, 20 mM NaCl, pH 7.2), SDS-page and western blot after one day and after one week. The data showed that ˜75% of the conjugate 94 were adsorbed onto Alhydrogel® and the adsorbed conjugate was stable at both temperature conditions. No degradation products were observed during this course of time.

(232) TABLE-US-00001 TABLE 1 BCA assay of conjugate 94 in presence of phosphate buffer and aluminum hydroxide. protein final protein concentration temperature concentration at t = 0 in μg/mL in ° C. in μg/mL 94 + PBS 155 2-8 119 after 15 d 94 + PBS 155 25 107 after 15 d 94 + Alhydrogel ® 113 2-8 32 after 1 d 94 + Alhydrogel ® 113 25 36 after 1 d 94 + Alhydrogel ® 113 2-8 34 after 7 d 94 + Alhydrogel ® 113 25 39 after 7 d

Example I. Glycan Array Analysis

Example I-1. Immunization in Rabbits

(233) Immunization experiments were performed on rabbits (Chong et al. Infect. Immun., 1997, p. 4918-4925; Fernández-Santana et al. Science, 2004, 305 (5683), p. 522-525.). Rabbits were housed and handled according to international animal regulations (EU Directive 2010/63/EU) and sanctioned by governmental authorities (Landesamt für Landwirtschaft, Lebensmittelsicherheit and Fischerei Mecklenburg-Vorpommern).

(234) Four groups with four rabbits per each group were immunized in a prime-boost regime with unadjuvanted conjugate 94 containing 5 μg saccharide 29 or conjugate 94 (containing 5 μg Hib saccharide 29) adjuvanted with Alhydrogel. The negative control group received PBS/Alhydrogel only. The positive control group received the approved vaccine ActHIB® (5 μg PRP, corresponding to half the human dose), a conjugate of native PRP to tetanus toxoid (TT). Preimmune serum and antiserum of bleeding day 21 (following two immunizations) were taken and analyzed on a glycan array. The ActHIB-specific serum was obtained after three administrations in contrast to two immunisations of the HIB analogues of the present invention.

Example I-2. Glycan Array Analysis

(235) Saccharides in PBS were printed on N-Hydroxy succinimide activated glass slides (CodeLink slides, Surmodics) using an S3 microarray spotter (Scienion). Slides were incubated overnight in a humidity saturated chamber, quenched for 2 h with 100 mM ethanolamine, 50 mM sodium phosphate, pH 7.5, washed with water and dried.

(236) For incubation, slides were blocked for 1 h with 3% (w/v) BSA-PBS, washed with PBS and water, and dried by centrifugation. A 64 well incubation grid was attached. Sera were diluted in 3% BSA-PBS, 0.1% (v/v) Tween-20, incubated at 37° C. for 15 min, and centrifuged for 2 min at 3220 rpm. Serum dilutions were applied to the slide and incubated for 1 h at room temperature. Wells were washed three times with PBS+0.1% Tween-20 (PBS-T). Secondary antibodies (anti-rabbit IgG FITC, anti-rabbit IgM AlexaFluor 647, anti-human IgG-Fc AlexaFluor 488 and IgM AlexaFluor 594) were incubated on the slides for 30 min at r.t. Wells were washed twice with PBS-T, the incubation grid was removed, and the slide washed with PBS and water. After drying by centrifugation, the slide was scanned using a GenePix 4300A (Molecular Devices) microarray reader.

(237) The primary immune response was assessed by glycan array screening of serum samples retrieved at day 0, day 21 and day 35. 2′-Methoxyribosylribitolphosphate dimer 19, 2′-methoxyribosylribitolphosphate dimer 24, 2′-Methoxyribosylribitolphosphate tetramer 29, 2′-deoxyribosylribitolphosphate tetramer 53 and 2′-dimethyl-aminocarbonylribosylribitolphosphate 62 as well as natural Hib PRP were printed on NHS ester-activated microarray slides. After immunization with conjugate 94 adjuvanted with Alhydrogel and without adjuvant, antibodies of the IgG and IgM subtypes against the Hib PRP derivatives 19, 24, 29, 53 and 62 as well as against the natural Hib PRP polysaccharide in all immunized rabbits were detected.

(238) The glycan array screening also showed that anti-Hib antibodies present in human reference serum (FIG. 17A) and in rabbit typing serum (FIG. 17B) bind to the Hib PRP derivatives 19, 24, 29, 53 and 62. The presence of natural Hib PRP inhibits the binding of the Hib PRP derivatives 19, 24, 29 and 62, but not of the 2′-deoxyribosylribitolphosphate 53.

(239) Serum IgG antibodies were detectable 21 days after the first immunization with the conjugate 94 adjuvanted with Alhydrogel and 35 days after the first immunization with undajuvanted conjugate 94. Antibodies cross-reacting with the natural Hib PRP polysaccharide indicates the potential of these antibodies to bind to Haemophilus influenzae bacteria and to confer protection against Haemophilus influenzae infection.

Example J. ELISA Studies

(240) ELISA plates (high-binding, EIA/RIA Plate, 96 well, flat bottom with low evaporation lid, company: Costar® 3361) commercial rabbit anti-Hib-PRP IgG ELISA assembly kit (Alpha Diagnostic Intl. Inc, #980-130-PRG; Lot: 170531K5, Expiry: June 2018) antigen: Phosphoribosylphosphate (PRP) capsular polysaccharide from Haemophilius influenza b (NIBSC, Code: 12/306) test sera: supplied by BioGenes GmbH. detection antibody: goat anti rabbit IgG peroxidase conjugate (Sigma, #A4914). Phosphate Buffered Saline (PBS): Made in-house from stock (Biochrom GmbH, Cat: L182-10) blocking solution: 1% FCS (v/v) in PBS. antibody diluent: PBS+1% BSA (w/v). wash buffer: PBS+0.1% Tween 20 (PBS-T) developing solution: 1 Step™ Ultra TMB-ELISA developer. (ThermoScientific, Cat #: 34028) stop solution- 2M Sulphuric acid (H.sub.2504) (Made in-house) plate reader: Anthos ht 2. software: WinRead 2.36 for absorbance measurements and GraphPad Prism 7 for data plotting and analysis.

Example J-1. Immunization Schedule and Sera Collection

(241) All immunization experiments were performed at BioGenes GmbH Berlin. The experimental groups included i) PBS with Alhydrogel (negative control), ii) compound 94 without Alhydrogel, iii) compound 94 with Alhydrogel, and iv) the commercial vaccine HiberiX® (positive control). Briefly, rabbits (n=4) were immunized in a prime boost regime with the constructs for the respective experimental groups as mentioned in Table 1. The mice were immunized following a prime-boost regime and sera were collected on day 0 (pre-immune), day-14, day-21, and day-35.

(242) TABLE-US-00002 TABLE 2 Immunization schedule and antigen dose information of rabbits (n = 4). amount amount Immunization Sera antigen Alhydrogel schedule collection Group Rabbits [μg/dose] [mg/dose] Excipient [days] [days] 94 4 0.5 0 1X PBS d0, d14, d28 d0, d14, pH 7.4 d21, d28, d35 94 with 4 0.5 0.32 1X PBS d0, d14, d28 d0, d14, Alhydrogel pH 7.4 d21, d28, d35 PBS with 4 0 0.32 1X PBS d0, d14, d28 d0, d14, Alhydrogel pH 7.4 d21, d28, d35 HiberiX ® 4 5 — d0, d14, d28 d0, d14, d21, d28, d35
Sera Collection and Handling:

(243) The sera from different experimental groups and respective time points were stored at −20° C. Sera from the individual rabbits (20 μl) of specific experimental groups were pooled and stored at −20° C. Individual rabbit sera (20 μl) were aliquoted as a separate stock and stored at −20° C. till further use.

Example J-2. Enzyme Linked Immunosorbent Assay (ELISA) of Sera Using in-House Antigen Coated Plates

(244) Coating of Plates with Antigen:

(245) The antigen, Hib PRP capsular polysaccharide (stock concentration 1 mg/mL) was diluted to a working antigen concentration of 10 μg/mL in PBS pH 7.4. 100 μL was added in each well (1 μg antigen/well) and incubated overnight at 4° C.

(246) Washing:

(247) After overnight adsorption of the antigen, the plates were washed 3× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and tapping on a clean dry tissue towel.

(248) Blocking:

(249) The plates were blocked using 300 μL of blocking solution (PBS+1% FCS) for 2 h at RT.

(250) Washing:

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

(252) Dilution of Sera and Incubations:

(253) Pooled (n=4 rabbits/group) pre-immune and test sera of the different experimental groups and individual rabbit test sera (n=4) were diluted to their respective dilutions, in the antibody diluent (PBS-+1% BSA). 100 μL of the diluted sera samples of the different experimental groups (100 were added in duplicates to the corresponding wells and incubated on a shaker set at 250 rpm for 1 h at RT. 100 μL/well of the antibody diluent (PBS+1% BSA) formed the experimental blank. After incubation with sera, the plates were washed 5× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

(254) Incubation (Detection Antibody):

(255) The detection antibody, goat anti-rabbit IgG peroxidase was diluted 1:10,000 in the antibody diluent (PBS+1% BSA) and 100 μL was added to the well and incubated on a shaker at 250 rpm for 1 h at RT. After the incubation with detection antibody, the plates were washed 5× with PBS-T (200 μL/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.

(256) Developing:

(257) To each well, 100 μL of the ready to use TMB substrate (normalized to RT form 4° C.) was added and incubated in dark for 15 min. The blue color of the enzymatic reaction was stopped by adding 100 μL/well of 2M H.sub.2SO.sub.4 solution resulting in a yellow colored solution. The absorption of the yellow colored solution was measured at 450 nm with a correction wavelength of 630 nm using a plate reader. The absorption values were analyzed by plotting a graph using the Graphpad Prism software.

(258) Results:

(259) As seen in FIG. 18A, the pre-immune sera of all the experimental groups across all the dilutions did not show any significant response and the readings were equal to 0. In the post-immune/test sera, the day-14 HiberiX group (inverted triangles) showed an immune response considerably higher than the PBS control group (filled circles) that increased up to titers of 1:2500 in the day-21 and day-35 groups (FIGS. 18B-D). In the experimental groups immunized with adjuvanted compound 94 (triangles), the antibody titers were lower than that of the PBS group on day-14 which substantially increased to that of the HiberiX group at a titer of 1:25000 but 1-fold lower. Although the experimental group immunized with the unadjuvanted compound 94 (squares) showed immune titers comparable to the HiberiX group at day-14, no boosting response was observed at the day-21 time point where the adjuvanted group showed a boosting response. However, after the second boost, the unadjuvanted compound 94 showed an immune titer comparable to the adjuvanted group, but lower than that of the Hiberix group.

Example J-3. Enzyme Linked Immunosorbent Assay (ELISA) of Sera Using Commercial ADi Plates

(260) The rabbit anti-Hib-PRP IgG ELISA kit (ADi) detects Hib-PRP specific IgG antibodies in the sera of vaccinated animals. The kit includes a 96 well microtiter plate pre-coated with the antigen Hib-PRP polysaccharide, supplied with the wash buffers and the peroxide conjugated detection antibody. The kit also includes a set of calibrators (0.5, 1.0, 2.5, and 5.0 U/mL) and a positive control/reference sera. The calibrators provided a control of the internal assay parameters and differentiates false positives in a sera dilution of 1:50 and above. The 0.5 U/mL value becomes the lowest detectable titer below which values of tests would be a false positive. The assay was performed in a normal ELISA format by following the instructions from the manufacturer. Briefly, the sera to be tested was diluted to an appropriate dilution range of 1:100 and above in the diluent provided in the kit. 100 μL of the diluted sera along with the calibrator, positive, and negative control was added to pre-determined wells and incubated for 1 h at room temperature. The wells were then washed 4 times with the kit wash buffer and 100 μL of the detection antibody is added and further incubated for 30 min followed by 5 washes. The wells were developed by adding 100 μL of the TMB substrate and incubating for 15 min followed by stopping the reaction using 100 μL stop solution resulting in a yellow colored solution. The absorption of the yellow colored solution was measured at 450 nm with a correction wavelength of 630 nm using a microplate reader. The absorption values were analyzed by plotting a graph using the Graphpad Prism software.

(261) Results:

(262) The Hib-PRP polysaccharide (antigen) is very sensitive to hydrolysis due to pH differences and other physiochemical factors which might lead to lower detection of antigen in an ELISA. To corroborate the immune titers observed in FIGS. 18B-D and to rule out the anomalies arising due to in-house coating of the antigen, the rabbit sera were screened on a commercial diagnostic plate (Advanced Diagnostics-adi) pre-coated with the Hib-PRP polysaccharide. As described in Example J-2, the rabbit immunized sera from the different experimental groups and time-points were diluted 5-fold (1:100, 500, 2500, and 12500) and analyzed for polysaccharide specific IgG antibodies. As seen in FIG. 19A, the pre-immune sera of all the experimental groups across all the dilutions did not show any significant response and the readings were below the threshold value. The threshold refers to the absorption value got for the 0.5 U/mL calibrator run on the same plate, values below which can be assigned as negatives or false positives.

(263) In the post-immune/test sera, the day-14 HiberiX group (inverted triangles), compound 94 with (triangles) and without (squares) Alhydrogel showed an immune titer lower than the threshold value even at the highest dilution of 1:100 (FIG. 19B). However, the PBS control group (filled circles) showed a higher signal than the threshold value at the highest dilution of 1:100 only in the day-14 time point but was lower than the threshold in all other time points of day-21, and −35. This was a similar trend observed in the in-house coated antigen plates (FIG. 18B-D). The adjuvanted compound 94 (triangles) and the HiberiX group (inverted triangles) in the day-21 and -35 time points showed a saturation up to dilutions of 1:500 and 1:2500, respectively. The effect of the adjuvant and boosting response was clearly visible in the compound 94 with Alhydrogel group as the titers significantly increase from 1:2500 in the day-21 time point to 1:12500 in the day-35 time point. Clearly at day-35, the group immunized with adjuvanted compound 94 showed comparable response to HiberiX but 1-fold lower. However, in the group immunized with unadjuvanted compound 94, there were no IgG titers detectable till day-21, but significantly increased up to 1:2500 post second boost. This trend was similar to that observed in ELISA performed using the in-house antigen coated plates (FIG. 18C, D).

(264) These data demonstrate the immunogenicity of the conjugate 94. Isotype switching indicates T cell-dependent antibody responses. Serum IgG antibodies were detectable 21 days after the first immunization with the conjugate 94 adjuvanted with Alhydrogel and 35 days after the first immunization with the unadjuvanted conjugate 94. Antibodies cross-reacting with the natural Hib PRP polysaccharide indicates the potential of these antibodies to bind to Haemophilus influenzae bacteria and to confer protection against Haemophilus influenzae infection