Vaccine against <i>Klebsiella pneumoniae</i>
11891413 ยท 2024-02-06
Assignee
Inventors
- Arun Naini (Berlin, DE)
- Daniel Knopp (Storkow, DE)
- Bopanna MONNANDA (Berlin, DE)
- Arne Von Bonin (Basel, CH)
- Claney Lebev Pereira (Berlin, DE)
Cpc classification
A61K47/6415
HUMAN NECESSITIES
C07H15/04
CHEMISTRY; METALLURGY
A61K31/335
HUMAN NECESSITIES
C07H15/26
CHEMISTRY; METALLURGY
A61K47/646
HUMAN NECESSITIES
G01N33/56916
PHYSICS
A61K47/549
HUMAN NECESSITIES
C07H15/18
CHEMISTRY; METALLURGY
A61K2039/6037
HUMAN NECESSITIES
A61K31/702
HUMAN NECESSITIES
International classification
C07H3/06
CHEMISTRY; METALLURGY
A61K31/335
HUMAN NECESSITIES
A61K31/702
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
C07H15/04
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a synthetic oligosaccharide of general formula (I): T*-[(U.sub.x+4U.sub.x+3U.sub.x+2U.sub.x+1U.sub.x).sub.m(V.sub.x+2V.sub.x+1V.sub.x).sub.1-m-T-O-L-E that is related to Klebsiella pneumoniae serotype O3, O3b and/or O5 lipopolysaccharide and conjugate thereof. Said synthetic oligosaccharide, said conjugate and pharmaceutical composition containing said synthetic oligosaccharide or said conjugate are useful for prevention and/or treatment of diseases associated with Klebsiella pneumoniae. Furthermore, the synthetic oligosaccharide of general formula (I) is useful as marker in immunological assays for detection of antibodies against Klebsiella pneumoniae serotype O3, O3b and/or O5 bacteria.
Claims
1. An oligosaccharide of general formula (II)
T*-[(U.sub.x+4U.sub.x+3U.sub.x+2U.sub.x+1U.sub.x).sub.m(V.sub.x+2V.sub.x+1V.sub.x).sub.1-m].sub.nO-L-E(II) wherein m is an integer selected from 0 and 1; x is 1; n is an integer selected from 2, 3, 4, 5, 6, 7, and 8; ##STR00220## T*- represents H; -L- represents -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, or -L.sup.a-L.sup.d-L.sup.e-; -L.sup.a- represents (CH.sub.2).sub.o, (CH.sub.2CH.sub.2O).sub.oC.sub.2H.sub.4, or (CH.sub.2CH.sub.2O).sub.oCH.sub.2; -L.sup.b- represents O, NHCONH, NHCOCH.sub.2NH, NHCO; -L.sup.d- represents (CH.sub.2).sub.q, (CH(OH)).sub.q, (CF.sub.2).sub.q, (CH.sub.2CH.sub.2O).sub.qC.sub.2H.sub.4, or (CH.sub.2CH.sub.2O).sub.qCH.sub.2; -L.sup.e- represents (CH.sub.2).sub.p1, (CF.sub.2).sub.p1, C.sub.2H.sub.4(OCH.sub.2CH.sub.2).sub.p1, CH.sub.2(OCH.sub.2CH.sub.2).sub.p1 or (CH.sub.2).sub.p1O(CH.sub.2).sub.p2; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, and 6; E represents NH.sub.2, N.sub.3, CN, ONH.sub.2, CHCH.sub.2, CCH, Br, Cl, I, CO.sub.2R, COR, CONHNH.sub.2, SH, or SAc; R represents H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, N-hydroxysuccinimidyl, -(3-sulfo-N-hydroxysuccinimidyl), or -(dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl); or a pharmaceutically acceptable salt thereof.
2. The oligosaccharide according to claim 1, wherein -L- represents (CH.sub.2).sub.o; and o is an integer selected from 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.
3. The oligosaccharide according to claim 1, wherein O-L-E is selected from the group consisting of: ##STR00221## wherein R represents H, -Me, -Et, 4-nitrophenyl, pentafluorophenyl, N-hydroxysuccinimidyl, (3- sulfo-N-hydroxysuccinimidyl), or -(dibenzocyclooctyne-sulfo-N-hydroxy succinimidyl); X represents Br, Cl, I, CO.sub.2H, or SAc, or a pharmaceutically acceptable salt thereof.
4. The oligosaccharide according to claim 1 selected from the group consisting of: ##STR00222## ##STR00223## ##STR00224## ##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229## or a pharmaceutically acceptable salt thereof.
5. The oligosaccharide according to claim 1 selected from the group consisting of: ##STR00230## or a pharmaceutically acceptable salt thereof.
6. The oligosaccharide according to claim 5, wherein n is an integer selected from 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.
7. The oligosaccharide according to claim 1, wherein E represents an amino group, or a pharmaceutically acceptable salt thereof.
8. The oligosaccharide according to claim 6, wherein E represents an amino group, or a pharmaceutically acceptable salt thereof.
9. A pharmaceutical composition comprising the oligosaccharide according to claim 1 together with at least one pharmaceutically acceptable adjuvant and/or excipient.
10. A conjugate comprising an oligosaccharide according to claim 1 covalently linked to an immunogenic carrier through the residue E of the O-L-E group, wherein the immunogenic carrier is a carrier protein selected from the group consisting of: a diphtheria toxoid, a mutated diphtheria toxoid, a modified diphtheria toxoid, a mutated and modified diphtheria toxoid, a tetanus toxoid, a modified tetanus toxoid, a mutated tetanus toxoid, non-lipidated cell-surface liporotein (protein D) of non-typeable Haemophilus influenzae, outer membrane protein (OMP) complex of Neisseria meningitidis, bovine serum albumin (BSA), keyhole limpet hemocyanine (KLH), recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A (rEPA) or cholera toxoid (CT).
11. A pharmaceutical composition comprising the conjugate according to claim 10 together with at least one pharmaceutically acceptable adjuvant and/or excipient.
12. A method for raising a protective immune response in a human and/or animal host, said method comprising administering at least one conjugate according to claim 10 to said human or animal host.
13. A method of treatment of a disease associated with Klebsiella pneumonia serotype O3, O3b and/or O5 bacteria in a human and/or animal host comprising administering at least one conjugate according to claim 10 to said human or animal host.
14. The method according to claim 13, wherein the disease associated with Klebsiella pneumonia serotype O3, O3b and/or O5 bacteria is pneumonia, bronchitis, meningitis, urinary tract infection, wound infection, osteomyelitis, bacteremia, septicemia or ankylosing spondylitis.
15. A conjugate of general formula (IV)
[T*-((U.sub.x+4U.sub.x+3U.sub.x+2U.sub.x+1U.sub.x).sub.m(V.sub.x+2V.sub.x+1V.sub.x).sub.1-m).sub.n-T-O-L-E.sub.1-W].sub.c-CP(IV) wherein m is an integer selected from 0 and 1; x is 1; n is an integer selected from 2, 3, 4, 5, 6, 7, and 8; ##STR00231## T*- represents H; -L- represents -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, or -L.sup.a-L.sup.d-L.sup.e-; -L.sup.a- represents (CH.sub.2).sub.o, (CH.sub.2CH.sub.2O).sub.oC.sub.2H.sub.4, or (CH.sub.2CH.sub.2O).sub.oCH.sub.2; -L.sup.b- represents O, NHCONH, NHCOCH.sub.2NH, NHCO; -L.sup.d- represents (CH.sub.2).sub.q, (CH(OH)).sub.q, (CF.sub.2).sub.q, (CH.sub.2CH.sub.2O).sub.qC.sub.2H.sub.4, or (CH.sub.2CH.sub.2O).sub.qCH.sub.2; -L.sup.e- represents (CH.sub.2).sub.p1, (CF.sub.2).sub.p1, C.sub.2H.sub.4(OCH.sub.2CH.sub.2).sub.p1, CH.sub.2(OCH.sub.2CH.sub.2).sub.p1 or (CH.sub.2).sub.p1O(CH.sub.2).sub.p2; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, and 6; T represents a bond; c is comprised between 2 and 18; -E.sub.1- represents a covalent bond, NH, ONH, O, S, CO, CHCH, CONH, CONHNH, ##STR00232## W is selected from: ##STR00233## a represents an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, b represents an integer selected from 1, 2, 3 and 4; and CP is a carrier protein selected from the group consisting of: a diphtheria toxoid, a mutated diphtheria toxoid, a modified diphtheria toxoid, a mutated and modified diphtheria toxoid, a tetanus toxoid, a modified tetanus toxoid, a mutated tetanus toxoid, non-lipidated cell-surface liporotein (protein D) of non-typeable Haemophilus influenzae, outer membrane protein (OMP) complex of Neisseria meningitidis, bovine serum albumin (BSA), keyhole limpet hemocyanine (KLH), recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A (rEPA) or cholera toxoid (CT).
16. The conjugate according to claim 15 of general formula (V)
[H((U.sub.x+4U.sub.x+3U.sub.x+2U.sub.x+1U.sub.x).sub.m(V.sub.x+2V.sub.x+1V.sub.x).sub.1-m).sub.nO-L-E.sub.1-W].sub.c-CRM.sub.197(V)
17. The conjugate according to claim 15, wherein -E.sub.1- represents a covalent bond, NH, CHCH, CONH, CONHNH, ##STR00234##
18. The conjugate according to claim 16, wherein -E.sub.1- represents a covalent bond, NH, CHCH, CONH, CONHNH, ##STR00235##
19. The conjugate according to claim 17, wherein W represents ##STR00236## and a represents an integer selected from 2, 3, 4, 5, and 6.
20. The conjugate according to claim 18, wherein W represents ##STR00237## and a represents an integer selected from 2, 3, 4, 5, and 6.
21. The conjugate according to claim 15, wherein the conjugate has any one of the following formula (V-1), (V-6) or (V-9) ##STR00238## ##STR00239##
22. The conjugate according to claim 21, wherein the conjugate has the following formula (V-6) ##STR00240## wherein -L- represents (CH.sub.2).sub.oand o is an integer selected from 2, 3, 4, 5, and 6; W represents ##STR00241## and a represents an integer selected from 2, 3, 4, 5, and 6.
23. The conjugate according to claim 22, wherein -E.sub.1- is NH.
24. The conjugate according to claim 23, wherein c is between 5 and 15.
25. A pharmaceutical composition comprising the conjugate according to claim 15 together with at least one pharmaceutically acceptable adjuvant and/or excipient.
26. A pharmaceutical composition comprising at least one conjugate according to claim 21 together with at least one pharmaceutically acceptable adjuvant and/or excipient.
27. A method for raising a protective immune response in a human and/or animal host, said method comprising administering at least one conjugate according to claim 15 to said human or animal host.
28. A method of treatment of a disease associated with Klebsiella pneumonia serotype O3, O3b and/or O5 bacteria in a human and/or animal host comprising administering at least one conjugate according to claim 15 to said human or animal host.
29. The method according to claim 28, wherein the disease associated with Klebsiella pneumonia serotype O3, O3b and/or O5 bacteria is pneumonia, bronchitis, meningitis, urinary tract infection, wound infection, osteomyelitis, bacteremia, septicemia or ankylosing spondylitis.
30. A method for raising a protective immune response in a human and/or animal host, said method comprising administering at least one conjugate according to claim 21 to said human or animal host.
31. A method of treatment of a disease associated with Klebsiella pneumonia serotype O3, O3b and/or O5 bacteria in a human and/or animal host comprising administering at least one conjugate according to claim 21 to said human or animal host.
32. The method according to claim 31, wherein the disease associated with Klebsiella pneumonia serotype O3, O3b, and/or O5 bacteria is pneumonia, bronchitis, meningitis, urinary tract infection, wound infection, osteomyelitis, bacteremia, septicemia or ankylosing spondylitis.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
EXAMPLES
A. Chemical Synthesis
(10) General Information:
(11) Commercial grade solvents were used unless stated otherwise. Dry solvents were obtained from a Waters Dry Solvent System. Solvents for chromatography were distilled prior to use. Sensitive reactions were carried out in heat-dried glassware and under an argon atmosphere. Analytical thin layer chromatography (TLC) was performed on Kieselgel 60 F254 glass plates precoated with a 0.25 mm thickness of silica gel. Spots were visualized by staining with vanillin solution (6% (w/v) vanillin and 10% (v/v) sulfuric acid in 95% EtOH) or Hanessian's stain (5% (w/v) ammonium molybdate, 1% (w/v) cerium(II) sulfate and 10% (v/v) sulfuric acid in water). Silica column chromatography was performed on Fluka Kieselgel 60 (230-400 mesh). .sup.1H, .sup.13C and two-dimensional NMR spectra were measured with a Varian 400-MR spectrometer at 296 K. Chemical shifts (d) are reported in parts per million (ppm) relative to the respective residual solvent peaks (CDCl.sub.3: d 7.26 in .sup.1H and 77.16 in .sup.13C NMR; CD.sub.3OD: d 3.31 in .sup.1H and 49.15 in .sup.13C NMR). The following abbreviations are used to indicate peak multiplicities: s singlet; d doublet; dd doublet of doublets; t triplet; dt doublet of triplets; q quartet; m multiplet. Coupling constants (J) are reported in Hertz (Hz). Optical rotation (OR) measurements were carried out with a Schmidt & Haensch UniPol L1000 polarimeter at =589 nm and a concentration (c) expressed in g/100 mL in the solvent noted in parentheses. High resolution mass spectrometry (HRMS) was performed at the Free University Berlin, Mass Spectrometry Core Facility, with an Agilent 6210 ESI-TOF mass spectrometer. Infrared (IR) spectra were measured with a Perkin Elmer 100 FTIR spectrometer at applicant's facility.
ABBREVIATIONS
(12) AcOH Acetic acid Alloc Allyloxycarbonyl aq. aqueous BH.sub.3 borane BBr.sub.3 boron tribromide Boc tert-Butoxycarbonyl br. broad CAS CAS Registry Number (CAS=Chemical Abstracts Service) CHCl.sub.3 chloroform cHex cyclohexane d doublet dd doublet of doublets DCM dichloromethane DEAD diethyl azodicarboxylate DIPEA N,N-diisopropyl-ethylamine DME dimethoxyethane DMF dimethylformamide DMSO dimethylsulfoxide DPPA diphenylphosphoryl azide EDCHCl N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3 diamine hydrochloride ES electrospray Et.sub.2O diethyl ether EtOAc ethyl acetate h hour HCl hydrochloric acid H.sub.2O water HOBt.H.sub.2O 1H-benzo[d][1,2,3]triazol-1-ol hydrate K.sub.2CO.sub.3 potassium carbonate m multiplet MeCN acetonitrile MeOH methanol MeI methyl iodide MgSO.sub.4 magnesium sulphate min minutes MS mass spectrometry Na.sub.2CO.sub.3 sodium carbonate NaCNBH.sub.3 sodium cyanoborohydride NaHCO.sub.3 sodium hydrogencarbonate NaH sodium hydride NaOH sodium hydroxide Na.sub.2SO.sub.4 sodium sulphate NCS N-chlorosuccinimide NIS N-iodosuccinimide NMR nuclear magnetic resonance PBS phosphate-buffered saline Pd/C palladium on carbon PPh.sub.3 triphenylphosphine q quartet rt room temperature s singlet sat. saturated sep septet t triplet TFA trifluoroacetic acid THF tetrahydrofuran TsOH tosic acid Wt weight.
Example 1: Synthesis of Monosaccharide Building Blocks
(13) ##STR00125##
(14) Compound 1* was prepared according to a procedure described in Carb. Res., 2010, 345, 10, 1316-1323.
(15) ##STR00126##
(16) Compound 2* was prepared according to a procedure described in ChemistryA European Journal, 2010, 16(44), 13163-13175.
(17) ##STR00127##
(18) Compound 3* was prepared according to a procedure described in Org. Biomol. Chem., 2018, (16) 13, 2277-2288.
(19) ##STR00128##
(20) Compound 4* was prepared according to a procedure described in J. Org. Chem., 2012, 77 (1), 108-125.
(21) ##STR00129##
(22) Compound 4* (2 g, 3.30 mmol) was dissolved in anhydrous DCM (33 mL). Benzyl bromide (1.4 g, 8.24 mmol) and Ag.sub.2O (7.64 g, 33 mmol) were added and the reaction mixture was vigorously stirred at room temperature overnight. The reaction was filtered through celite and concentrated under reduced pressure to give crude product. The crude was charged on isolute and purified using an automated purification system on silica (ethyl acetate/cyclohexane) to give the product (1.43 g, 62%). HRMS (ESI.sup.+) Calcd for C.sub.44H.sub.40O.sub.6SNa.sup.+ [M+Na].sup.+ 719.2443, found 719.2390.
(23) ##STR00130##
(24) Compound 6* was prepared according to a procedure described in J. Am. Chem. Soc., 2017, 139 (2), 1011-1018 starting from compound 9*: Compound 9* (400 mg, 1.068 mmol) was dissolved in anhydrous pyridine (5 mL). FmocCl (431 mg, 1.666 mmol) and DMAP (19.58 mg, 0.160 mmol) were added and the reaction mixture was stirred at room temperature overnight. The reaction was diluted with ethyl acetate and washed with sat. NaHCO.sub.3 and brine. The organic layer was dried using Na.sub.2SO.sub.4 and the solvent evaporated to give the crude product. The crude was charged on isolute and purified using an automated purification system using silica (ethyl acetate/cyclohexane) to give the product (360 mg, 56%). HRMS (ESI.sup.+) Calcd for C.sub.35H.sub.32O.sub.7SNa.sup.+ [M+Na].sup.+ 597.1947, found 597.1857.
(25) ##STR00131##
(26) Compound 6* (1.7 g, 2.274 mmol) was dissolved in BH.sub.3.THF (27 mL, 27 mmol) and TMSOTf (0.41 mL, 2.274 mmol) was added. The solution was stirred at room temperature for 1.5 h. The reaction was quenched with methanol (cooling with an ice/water bath) and concentrated under reduced pressure to give crude product. The crude was charged on isolute and purified using an automated purification system with ethyl acetate/cyclohexane to give the product (930 mg, 61%). HRMS (ESI.sup.+) Calcd for C.sub.41H.sub.38O.sub.7SNa.sup.+ [M+Na].sup.+ 697.2236, found 697.2188.
(27) ##STR00132##
(28) Compound 7* (930 mg, 1.37 mmol) was dissolved in anhydrous DCM (14 mL). Benzyl bromide (589 mg, 3.45 mmol) and Ag.sub.2O (3.19 g, 13.78 mmol) were added and the reaction mixture was vigorously stirred at room temperature overnight. The reaction was filtered through Celite and concentrated under reduced pressure to give crude product. The crude was charged on isolute and purified using the automated purification system using silica (ethyl acetate/cyclohexane) to give the product 8* (680 mg, 65%). HRMS (ESI.sup.+) Calcd for C.sub.48H.sub.44O.sub.7SNa.sup.+ [M+Na].sup.+ 787.2705, found 787.2653.
(29) ##STR00133##
(30) Compound 9* was prepared according to a procedure described in Chem. Eur. J. 2014, 20, 3578-3583.
Example 2: Synthesis of K. pneumoniae Serotype O3 Oligosaccharides
(31) ##STR00134##
(32) To a solution of compound 5* (550 mg, 0.789 mmol) and 5-azidopentanol (306 mg, 2.368 mmol) in anhydrous DCM (2.9 mL) was added 4 molecular sieves and the mixture let stir at room temperature for 30 min. Then, NIS (213 mg, 0.947 mmol) was added and the reaction mixture cooled to 20 C. TMSOTf (14 L, 0.079 mmol) was added and the reaction mixture stirred for 1.5 h at 0 C. Reaction mixture was filtered and washed with DCM, and the filtrate was washed with sat. Na.sub.2S.sub.2O.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and Brine (10 mL). Dried over anhydr. Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification by an automated purification system using silica (ethyl acetate/cyclohexane) afforded the product 10* after evaporation of the solvent as a colorless thick gel (51 mg, 49%).
(33) HRMS (ESI+) Calcd for C.sub.43H.sub.45N.sub.3O.sub.7Na.sup.+ [M+Na].sup.+ 738.3155, found 738.3147.
(34) ##STR00135##
(35) To a solution of compound 10* (361 mg, 0.504 mmol) in DCM:PBS (2:1, 16.81 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (229 mg, 1.01 mmol) at 0 C. The reaction mixture was stirred for 2.5 h at room temperature. Reaction was monitored by TLC (EtOAc in cyclohexane, 2:1). Reaction was quenched with sat. NaHCO.sub.3 (50 mL) and extracted with DCM (250 mL). The combined organic layers were washed with brine (50 mL) and dried over Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by automated flash chromatography using silica (ethyl acetate/cyclohexane) to obtain a colorless oil of compound 11* (210 mg, 72%).
(36) HRMS (ESI+) Calcd for C.sub.32H.sub.37N.sub.3O.sub.7Na.sup.+ [M+Na].sup.+ 599.2563, found 599.2555.
(37) ##STR00136##
(38) To a solution of compound 5* (276 mg, 0.396 mmol) and compound 11* (190 mg, 0.330 mmol) in anhydrous DCM (8.4 mL) was added 4 MS and the mixture was letting stirred at room temperature for 30 min. Then, NIS (89 mg, 0.396 mmol) was added and the reaction mixture was cooled to 20 C. TMSOTf (6 L, 0.03 mmol) was added and the reaction mixture stirred for 1 h at 0 C. Reaction mixture was filtered, the filtrate was washed with sat. Na.sub.2S.sub.2O.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and brine (10 mL). After drying over anhydr. Na.sub.2SO.sub.4, the layers were concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product 12* after evaporation of the solvent as a cloudy thick gel (300 mg, 78%).
(39) HRMS (ESI+) Calcd for C.sub.70H.sub.71N.sub.3O.sub.13Na.sup.+ [M+Na].sup.+ 1184.4885, found 1184.4902.
(40) ##STR00137##
(41) To a solution of compound 12* (290 mg, 0.294 mmol) in DCM:PBS (2:1, 8.3 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (113 mg, 0.499 mmol) at 0 C. The reaction mixture was stirred for 2.5 h at room temperature and monitored by TLC (EtOAc in cyclohexane, 2:1). Reaction was quenched with sat. NaHCO.sub.3 (40 mL) and extracted with DCM (240 mL). The combined organic layers were washed with brine (20 mL), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to obtain the crude product. The crude product was purified by automated flash chromatography using silica (ethyl acetate/cyclohexane) to obtain compound 13* as a colorless oil (136 mg, 53%).
(42) HRMS (ESI+) Calcd for C.sub.59H.sub.63N.sub.3O.sub.13Na.sup.+ [M+Na].sup.+ 1044.4259, found 1044.4252.
(43) ##STR00138##
(44) To a solution of compound 8* (135 mg, 0.176 mmol) and 13* (150 mg, 0.147 mmol) in anhydrous DCM (3.8 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. Then, NIS (40 mg, 0.176 mmol) was added and the reaction mixture cooled to 20 C. TMSTOf (2.6 L, 0.015 mmol) was added and the reaction mixture stirred for 1.5 h at 0 C. Reaction mixture was filtered and washed with sat. Na.sub.2S.sub.2O.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and brine (10 mL). After drying over anhydr. Na.sub.2SO.sub.4, the crude product were purified by automated purification system using silica (ethyl acetate/cyclohexane) which afforded the product 14* after evaporation of the solvent as a cloudy thick gel (184 mg, 75%).
(45) HRMS (ESI+) Calcd for C.sub.101H.sub.101N.sub.3O.sub.20Na.sup.+ [M+Na].sup.+ 1699.6910, found 1699.6886.
(46) ##STR00139##
(47) To a solution of compound 14* (180 mg, 0.107 mmol) in DCM (2 mL) triethylamine (208 L, 1.491 mmol) was added at room temperature and stirred for 1 h. Volatiles were removed under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product 15* after evaporation of the solvent as a cloudy thick gel (138 mg, 88%).
(48) HRMS (ESI+) Calcd for C.sub.85H.sub.91N.sub.3O.sub.18Na.sup.+ [M+Na].sup.+ 1476.6195, found 1476.6198.
(49) ##STR00140##
(50) Sodium methoxide solution in MeOH (0.5M) (0.075 mL, 0.330 mmol) was added to a solution of compound 15* (24 mg, 0.016 mmol) in a mixture of MeOH:THF (2:1, 1.5 mL). The reaction was stirred at the same temperature for 20 h. The reaction was quenched by the addition of H.sub.2O (2 mL) and diluted with brine (5 mL). Reaction mixture extracted with EtOAc (210 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4, and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a colorless thick gel (18 mg, 88%).
(51) HRMS (ESI+) Calcd for C.sub.99H.sub.90O.sub.22Na.sup.+ [M+Na].sup.+ 1268.5671, found 1268.5813.
(52) ##STR00141##
(53) Compound 16* (8.6 mg, 6.90 mol) was taken in solvent mixture DCM (1 mL), tert-butanol (1 mL) and two drops of water. Pd/C was added to it and hydrogenated for 24 h at 7 bar pressure of H.sub.2 at rt. The reaction mixture was filtered through PTFE filter and the residue washed with methanol (6 mL), (50% methanol-water (6 mL). The filtrate was evaporated under vacuum to get the crude product. Crude product was clean by 1H NMR, sample recovered and freeze dried to obtain compound 17* as a white crystalline solid (3.8 mg, 93%).
(54) HRMS (ESI+) Calcd for C.sub.23H.sub.43NO.sub.16H.sup.+ [M+H].sup.+ 590.2694, found 590.2683.
(55) .sup.1H NMR (400 MHz, D.sub.2O) 5.15 (d, J=1.7 Hz, 1H), 5.11 (d, J=1.8 Hz, 1H), 4.85 (d, J=1.8 Hz, 1H), 4.24 (dd, J=3.3, 1.8 Hz, 1H), 4.06-4.12 (m, 2H), 4.03 (dd, J=9.1, 3.3 Hz, 1H), 3.86-3.97 (m, 5H), 3.71-3.86 (m, 8H), 3.61-3.71 (m, 2H), 3.51-3.61 (m, 1H), 3.01 (t, J=7.6 Hz, 2H), 1.61-1.78 (m, 4H), 1.39-1.54 (m, 2H).
(56) ##STR00142##
(57) To a solution of compound 8* (43 mg, 0.056 mmol) and compound 15* (65 mg, 0.045 mmol) in anhydrous DCM (1.94 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. Then, NIS (12 mg, 0.054 mmol) was added and the reaction mixture cooled to 20 C. TMSTOf (0.8 L, 4.47 mol) was added and the reaction mixture stirred for 35 min at 0 C. Reaction monitored by TLC until no starting material was left. Triethylamine (250 l) was added and the mixture was warmed to room temperature gradually over 1 h. Reaction mixture was filtered and washed sat. Na.sub.2S.sub.2O.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and brine (10 mL), subsequently dried over anhydr. Na.sub.2SO.sub.4 and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a cloudy thick gel (46 mg, 54%).
(58) HRMS (ESI+) Calcd for C.sub.113H.sub.119N.sub.3O.sub.23H.sup.+ [M+H].sup.+ 1909.8166, found 1909.8160.
(59) ##STR00143##
(60) To a solution of compound 3* (16.8 mg, 0.026 mmol) and compound 18* (28 mg, 0.015 mmol) in mixture of anhydrous toluene (0.9 mL) and anhydrous dioxane (0.3 mL) was added 4 MS and the mixture was stirred at room temperature for 1 h. Then, NIS (4 mg, 0.018 mmol) was added and the reaction mixture cooled to 20 C. TMSTOf (0.27 L, 1.484 mol) was added and the reaction mixture stirred for 2 h allowing to warm to room temperature. Reaction mixture was filtered and washed with sat. Na.sub.2S.sub.2O.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and brine (10 mL), subsequently dried over anhydr. Na.sub.2SO.sub.4 and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a cloudy thick gel (32 mg, 89%).
(61) HRMS (ESI+) Calcd for C.sub.147H.sub.151N.sub.3O.sub.29Na.sup.+ [M+Na].sup.+ 2445.0331, found 2445.9951.
(62) ##STR00144##
(63) Sodium methoxide solution in MeOH (25% w/w) (0.051 mL, 0.223 mmol) was added to a solution of the pentasaccharide 19* (27 mg, 0.011 mmol) in a mixture of MeOH:THF (2:1, 1.5 mL). The reaction was stirred at the same temperature for 16 h. The reaction was quenched by the addition of H.sub.2O (3 mL) and diluted with brine (5 mL). Reaction mixture extracted with EtOAc (210 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4 and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a colorless thick gel (17 mg, 72%).
(64) HRMS (ESI+) Calcd for C.sub.126H.sub.139N.sub.3O.sub.26Na.sup.+ [M+Na].sup.+ 2133.9578, found 2133.9517.
(65) ##STR00145##
(66) Compound 20* (17 mg, 8.05 mol) was taken in a solvent mixture of DCM (1 mL), tert-butanol (1 mL) and two drops of water. Pd/C was added and hydrogenated for 24 h under H.sub.2 balloon at rt. The reaction mixture was filtered through PTFE filter, washed the residue with methanol (6 mL), (50% methanol-water (6 mL). The filtrate was evaporated under vacuum to get the crude product. 1H NMR analysis showed the completion of the reaction and the presence of product. So, crude product was purified through the C18 Sepak column using water (3 mL2, fr1), 20% acetonitrile-water (3 mL2, fr2) and acetonitrile (3 mL, fr3). All the fractions were frozen and lyophilized for 24 h to get one pure fraction fr1 of compound 21* (white solid, 6.4 mg, 87%), and two impure fractions white fluffy solid (fr2, 0.4 mg) a white fluffy solid (fr.3, 0.6 mg).
(67) HRMS (ESI+) Calcd for C.sub.35H.sub.63NO.sub.26H.sup.+ [M+H].sup.+ 914:3717, found 914:3725.
(68) .sup.1H NMR (400 MHz, D.sub.2O) 5.35 (d, J=1.7 Hz, 1H), 5.28 (d, J=1.8 Hz, 1H), 5.08 (d, J=1.8 Hz, 1H), 5.02 (d, J=1.8 Hz, 1H), 4.82 (d, J=1.8 Hz, 1H), 4.20 (dd, J=3.3, 1.8 Hz, 1H), 4.02-4.11 (m, 4H), 3.92-4.01 (m, 3H), 3.80-3.92 (m, 8H), 3.50-3.80 (m, 18H), 2.93-3.04 (m, 2H), 1.58-1.76 (m, 4H), 1.37-1.58 (m, 2H).
(69) ##STR00146##
(70) Compound 22* is prepared similarly to compound 21* starting from compound 5* and azidoethanol.
(71) ##STR00147##
(72) Compound 23* is prepared similarly to compound 21* starting from compound 5* and azidodecanol.
(73) ##STR00148##
(74) Compound 24* is prepared similarly to compound 21* starting from compound 5* and 2-(2-azidoethoxy)ethanol.
(75) ##STR00149##
(76) Compound 25* is prepared similarly to compound 21* starting from compound 5* and 3-azido-2,2-difluoropropanol.
(77) ##STR00150##
(78) Compound 26* is prepared similarly to compound 21* starting from compound 5* and the corresponding azido alcohol.
(79) ##STR00151##
(80) Compound 27* is prepared similarly to compound 21* starting from compound 5* and the corresponding azido alcohol.
(81) ##STR00152##
(82) Compound 28* is prepared similarly to compound 21* starting from compound 5* and the corresponding S-benzylthio alcohol.
(83) ##STR00153##
(84) Compound 29* is prepared similarly to compound 21* starting from compound 5* and 5-hexenol.
(85) ##STR00154##
(86) Compound 30* is prepared similarly to compound 21* starting from compound 5* and 11-azido-3,6,9-trioxaundecanol.
(87) ##STR00155##
(88) Compound 31* is prepared similarly to compound 21* starting from compound 5* and azido-PEG7-alcohol.
(89) ##STR00156##
(90) Compound 32* is prepared similarly to compound 21* starting from compound 5* and 5-benzyloxypentanol.
(91) ##STR00157##
(92) Compound 33* is prepared similarly to compound 21* starting from compound 5* and 12-benzyloxydecanol.
(93) ##STR00158##
(94) Compound 34* is prepared similarly to compound 21* starting from compound 5* and methyl 6-hydroxydecanoic acid.
(95) ##STR00159##
(96) Compound 34a* is prepared similarly to compound 21* starting from compound 5* and methyl 6-hydroxydecanoic acid.
(97) ##STR00160##
(98) Compound 35* is prepared similarly to compound 21* starting from compound 5* and 1,2-dibenzyl glycerol or acetonide protected glycerol.
(99) ##STR00161##
(100) Compound 36* is prepared similarly to compound 21* starting from compound 5* and 2-(chloroethoxy)ethanol.
(101) ##STR00162##
(102) Compound 37* is prepared similarly to compound 21* starting from compound 5* and 4-penten-1-ol.
Example 3: Synthesis of K. pneumoniae Serotype O5 Trisaccharide
(103) ##STR00163##
(104) To a solution of compound 3* (490 mg, 0.758 mmol) and 5-azidopropanol (294 mg, 2.273 mmol) in a mixture of anhydrous toluene (11.4 mL) and anhydrous dioxane (3.76 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. Then, NIS (205 mg, 0.909 mmol) was added and the reaction mixture cooled to 0 C. TfOH (11.4 mg, 0.076 mmol) was added and the reaction mixture stirred for 2 h at 0 C. Reaction mixture was filtered and washed with sat. Na.sub.2S.sub.2O.sub.3 solution (25 mL) and extracted with CH.sub.2Cl.sub.2 (240 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (25 mL) and brine (10 mL) and dried over anhydr. Na.sub.2SO.sub.4, and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product 38* after evaporation of the solvent as a colorless thick gel (420 mg, 83%).
(105) HRMS (ESI+) Calcd for C.sub.39H.sub.43N.sub.3O.sub.7Na.sup.+ [M+Na].sup.+ 688.2999, found 688.3009.
(106) ##STR00164##
(107) Sodium methoxide solution in MeOH (25% w/w) (0.41 mL, 1.802 mmol) was added to a solution of the monosaccharide 38* (400 mg, 0.601 mmol) in a mixture of MeOH:THF (2:1, 12 mL). The reaction was stirred at the same temperature for 20 h. The reaction was quenched by the addition of H.sub.2O (15 mL) and diluted with brine (20 mL). Reaction mixture extracted with EtOAc (260 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4 and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a colorless thick gel (300 mg, 89%).
(108) HRMS (ESI+) Calcd for C.sub.32H.sub.39N.sub.3O.sub.6Na.sup.+ [M+Na].sup.+ 584.2737, found 584.2738.
(109) ##STR00165##
(110) To a solution of compound 3* (355 mg, 0.548 mmol) and compound 39* (280 mg, 0.499 mmol) in a mixture of anhydrous toluene (10 mL) and anhydrous dioxane (3.3 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. Then, NIS (135 mg, 0.598 mmol) was added and the reaction mixture cooled to 0 C. TfOH (7.5 mg, 0.05 mmol) was added and the reaction mixture stirred for 1 h at 0 C. Reaction mixture was filtered and washed with sat. Na.sub.2S.sub.2O.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and brine (10 mL), subsequently dried over anhydr. Na.sub.2SO.sub.4 and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a cloudy thick gel (350 mg, 64%) and starting compound 39* (62 mg, 22%).
(111) HRMS (ESI+) Calcd for C.sub.66H.sub.71N.sub.3O.sub.12Na.sup.+ [M+Na].sup.+ 1120.4935, found 1120.4922.
(112) ##STR00166##
(113) Sodium methoxide solution in MeOH (25% w/w) (0.8 mL, 3.19 mmol) was added to a solution of compound 40* (350 mg, 0.319 mmol) in a mixture of MeOH:THF (2:1, 7.5 mL). The reaction was stirred at the same temperature for 20 h. The reaction was quenched by the addition of H.sub.2O (15 mL) and diluted with brine (20 mL). Reaction mixture was extracted with EtOAc (260 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4, and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a colorless thick gel (278 mg, 88%).
(114) HRMS (ESI+) Calcd for C.sub.59H.sub.67N.sub.3O.sub.11Na.sup.+ [M+Na].sup.+ 1016.4673, found 1016.4686.
(115) ##STR00167##
(116) To a solution of compound 2* (180 mg, 0.304 mmol) in anhydrous DCM (11 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. Then, 1-(phenylsulfinyl)piperidine (69.7 mg, 0.333 mmol) and 2,4,6-tri-tert-butylpyrimidine (150 mg, 0.606 mmol) was added, reaction mixture cooled to 65 C. and stirred for 30 min. Triflic anhydride (61 L, 0.362 mmol) was added and the reaction mixture stirred for 20 min at 65 C. Reaction mixture was then cooled to 78 C. and compound 41* (275 mg, 0.277 mmol) in DCM (5 mL) was added dropwise and stirred for 6 h at 78 C. and then warmed to 0 C. over 1 h. The reaction mixture was filtered and washed with sat. NaHCO.sub.3 solution (25 mL) and extracted with CH.sub.2Cl.sub.2 (235 mL). Combined organic layers were washed with brine (10 mL) and dried over anhydr. Na.sub.2SO.sub.4. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a cloudy thick gel (220 mg, 54%).
(117) HRMS (ESI+) Calcd for C.sub.99H.sub.95N.sub.3O.sub.16Na.sup.+ [M+Na].sup.+ 1496.6610, found 1496.6623.
(118) ##STR00168##
(119) Compound 42* (8 mg, 5.99 mol) was taken in solvent mixture DCM (1 mL), tBuOH (1 mL) and two drops of water. Pd/C was added and hydrogenated under H.sub.2 balloon at rt. The reaction mixture was filtered through PTFE filter, washed the residue with methanol (6 mL), (50% methanol-water (6 mL). The filtrate was evaporated in vacuum to get the crude product. Crude product was clean by .sup.1H NMR, sample recovered and freeze dried to obtain a white crystalline solid (3.53 mg, quantitative).
(120) HRMS (ESI+) Calcd for C.sub.23H.sub.43NO.sub.16H.sup.+ [M+H].sup.+ 590.2660, found 590.2814.
(121) .sup.1H NMR (400 MHz, D.sub.2O) 5.12 (d, J=1.8 Hz, 1H), 5.07 (d, J=1.7 Hz, 1H), 4.77 (s, 1H), 4.28 (dd, J=3.4, 1.8 Hz, 1H), 4.02 (d, J=3.2 Hz, 1H), 3.81-4.00 (m, 6H), 3.48-3.81 (m, 11H), 3.36 (ddd, J=9.4, 6.7, 2.3 Hz, 1H), 2.98 (t, J=7.6 Hz, 2H), 1.58-1.74 (m, 4H), 1.36-1.51 (m, 2H).
(122) ##STR00169##
(123) Compound 44* is prepared similarly to compound 43* starting from compound 3* and azidoethanol.
(124) ##STR00170##
(125) Compound 45* is prepared similarly to compound 43* starting from compound 5* and azidodecanol.
(126) ##STR00171##
(127) Compound 46* is prepared similarly to compound 43* starting from compound 5* and 2-(2-azidoethoxy)ethanol.
(128) ##STR00172##
(129) Compound 47* is prepared similarly to compound 43* starting from compound 5* and 3-azido-2,2-difluoropropanol.
(130) ##STR00173##
(131) Compound 48* is prepared similarly to compound 43* starting from compound 5* and the corresponding azido alcohol.
(132) ##STR00174##
(133) Compound 49* is prepared similarly to compound 43* starting from compound 5* and the corresponding azido alcohol.
(134) ##STR00175##
(135) Compound 50* is prepared similarly to compound 43* starting from compound 5* and the corresponding S-benzylthio alcohol.
(136) ##STR00176##
(137) Compound 51* is prepared similarly to compound 43* starting from compound 5* and 5-hexenol.
(138) ##STR00177##
(139) Compound 52* is prepared similarly to compound 43* starting from compound 5* and 11-azido-3,6,9-trioxaundecanol.
(140) ##STR00178##
(141) Compound 53* is prepared similarly to compound 43* starting from compound 5* and azido-PEG7-alcohol.
(142) ##STR00179##
(143) Compound 54* is prepared similarly to compound 43* starting from compound 5* and 5-benzyloxypentanol.
(144) ##STR00180##
(145) Compound 55* is prepared similarly to compound 43* starting from compound 5* and 12-benzyloxydecanol.
(146) ##STR00181##
(147) Compound 56* is prepared similarly to compound 43* starting from compound 5* and methyl 6-hydroxydecanoic acid.
(148) ##STR00182##
(149) Compound 56a* is prepared similarly to compound 43* starting from compound 5* and methyl 6-hydroxydecanoic acid.
(150) ##STR00183##
(151) Compound 57* is prepared similarly to compound 43* starting from compound 5* and 1,2-dibenzyl glycerol.
(152) ##STR00184##
(153) Compound 58* is prepared similarly to compound 43* starting from compound 5* and 2-(chloroethoxy)ethanol.
(154) ##STR00185##
(155) Compound 59* is prepared similarly to compound 43* starting from compound 5* and 4-penten-1-ol.
Example 4: Synthesis of K. pneumoniae Serotype O5 Hexasaccharide
(156) ##STR00186##
(157) To a solution of compound 42* (220 mg, 0.149 mmol) in DCM:PBS (2:1, 7.4 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (67.7 mg, 0.298 mmol) at 0 C. The reaction mixture was stirred for 4 h at room temperature and monitored by TLC (EtOAc in cyclohexane, 2:1). Reaction was quenched with sat. NaHCO.sub.3 (50 mL) and extracted with DCM (250 mL). The combined organic layers were washed with brine (25 mL) and dried over Na.sub.2SO.sub.4 to obtain the crude product. The crude product was purified by automated flash chromatography using silica (ethyl acetate/cyclohexane) to obtain compound 60* as colorless oil (125 mg, 63%).
(158) HRMS (ESI+) Calcd for C.sub.79H.sub.87N.sub.3O.sub.16Na.sup.+ [M+Na].sup.+ 1356.5984, found 1356.5983.
(159) ##STR00187##
(160) To a solution of compound 8* (160 mg, 0.209 mmol) in DCM (3 mL) was added triethylamine (0.2 mL, 1.435 mmol) at 0 C. The reaction mixture was stirred for 1 h at room temperature. Reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by automated flash chromatography using silica (ethyl acetate/cyclohexane) to obtain a colorless oil (96 mg, 85%).
(161) HRMS (ESI+) Calcd for C.sub.33H.sub.34O.sub.5SNa.sup.+ [M+Na].sup.+ 566.2025, found 566.2065.
(162) ##STR00188##
(163) To compound 61* (1.05 g, 1.623 mmol) in DCM (10 mL) and H.sub.2O (1 mL) was added N-iodosuccinimide (365 mg, 1.623 mmol) and trifluoroacetic acid (124 L, 1.623 mmol) at 0 C. and stirred for 2 h. The reaction mixture was partitioned between sat. aqu. NaHCO.sub.3 (50 mL) solution and DCM (50 mL). The organic layer was washed with sat. Na.sub.2S.sub.2O.sub.3 solution (50 mL) and dried over Na.sub.2SO.sub.4. The crude product was purified by column chromatography using EtOAc and cyclohexane as solvents to afford the desired product (730 mg, 81%) as colorless oil.
(164) HRMS (ESI+) Calcd for C.sub.34H.sub.34O.sub.7Na.sup.+ [M+Na].sup.+ 577.2202, found 577.2208.
(165) ##STR00189##
(166) Cs.sub.2CO.sub.3 (141 mg, 0.433 mmol) and 2,2,2-trifluro-N-phenyl-acetimidoyl chloride (135 mg, 0.649 mmol) were added to a solution of lactol 62* (120 mg, 0.216 mmol) in DCM (2.2 mL). The reaction mixture was stirred at room temperature and monitored by TLC. After 2 hours all the starting material was consumed, the reaction was filtered through Celite and washed with DCM (10 mL). The solvent was evaporated and the crude product (157 mg, quantitative) was used in the next step without any purification.
(167) ##STR00190##
(168) To a solution of compound 63* (157 mg, 0.216 mmol) and compound 61* (117 mg, 0.216 mmol) in anhydrous toluene (4.6 mL) and dioxane (1.5 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. TMSOTf (3.92 L, 0.022 mmol) was added and the reaction mixture stirred at 10 C. for 1 h. The reaction was quenched with sat. NaHCO.sub.3 solution (25 mL) and extracted with DCM (220 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated to give crude product. The residue was purified by column chromatography using EtOAc and cyclohexane as solvents to afford the desired product (193 mg, 83%) as colorless oil.
(169) HRMS (ESI+) Calcd for C.sub.67H.sub.66O.sub.11SNa.sup.+ [M+Na].sup.+ 1101.4224, found 1101.4073.
(170) ##STR00191##
(171) To a solution of compound 64* (116 mg, 0.108 mmol) and compound 60* (120 mg, 0.090 mmol) in a mixture of anhydrous toluene (4.5 mL) and anhydrous dioxane (1.5 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. Then, NIS (26.3 mg, 0.117 mmol) was added and the reaction mixture cooled to 0 C. TfOH (1.35 mg, 8.99 mol) was added and the reaction mixture stirred for 3 h and gradually warmed to room temp. Reaction mixture was filtered and washed with sat. Na.sub.2S.sub.2O.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and brine (10 mL) and dried over anhydr. Na.sub.2SO.sub.4. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a cloudy thick gel (110 mg, 53%).
(172) HRMS (ESI+) Calcd for C.sub.140H.sub.147N.sub.3O.sub.27Na.sup.+ [M+Na].sup.+ 2326.0153, found 2326.0177.
(173) ##STR00192##
(174) Sodium methoxide solution in MeOH (25% w/w) (0.051 mL, 0.239 mmol) was added to a solution of the pentasaccharide 65* (110 mg, 0.048 mmol) in a mixture of MeOH:THF (2:1, 3 mL). The reaction was stirred at the same temperature for 16 h. The reaction was quenched by the addition of H.sub.2O (5 mL) and diluted with brine (10 mL). Reaction mixture extracted with EtOAc (220 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product 66* after evaporation of the solvent as a colorless thick gel (95 mg, 90%).
(175) HRMS (ESI+) Calcd for C.sub.140H.sub.147N.sub.3O.sub.27Na.sup.+ [M+Na].sup.+ 2221.9891, found 2221.9960.
(176) ##STR00193##
(177) To a solution of compound 2* (23.16 mg, 0.039 mmol) in anhydrous DCM (2 mL) was added 4 MS and the mixture was stirred at room temperature for 10 min. Then, 1-(phenylsulfinyl)piperidine (8.59 mg, 0.041 mmol) and 2,4,6-tri-tert-butylpyrimidine (18.55 mg, 0.075 mmol) were added. The reaction mixture was cooled to 65 C. and stirred for 30 min. Triflic anhydride (7.55 L, 0.045 mmol) was added and the reaction mixture stirred for 15 min at 65 C. Reaction mixture was then cooled to 78 C. and compound 66* (75 mg, 0.034 mmol) in DCM (1.5 mL) was added dropwise and stirred for 6 h at 78 C. and then warmed to 25 C. within 1 h. Reaction mixture was filtered and washed with sat. NaHCO.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with brine (10 mL) and dried over anhydr. Na.sub.2SO.sub.4. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product (as / mixture) after evaporation of the solvent as a cloudy thick gel (35 mg, 38%).
(178) HRMS (ESI+) Calcd for C.sub.164H.sub.171N.sub.3O.sub.31Na.sup.+ [M+Na].sup.+ 2702.1828, found 2702.1783.
(179) ##STR00194##
(180) To compound 67* (35 mg, 0.013 mmol) in DCM (2.5 mL) was added ethane thiol (9.66 L, 0.131 mmol) and p-toluene sulfonic acid monohydrate (1.24 mg, 6.53 mol) at room temperature and the mixture was stirred for 1.5 h. Reaction mixture was quenched with triethylamine (1 mL) and concentrated under vacuum, the residue was purified by column chromatography using EtOAc and cyclohexane as solvents to afford the desired product as pure desired -isomer (16 mg, 49%) as colorless oil.
(181) HRMS (ESI+) Calcd for C.sub.150H.sub.163N.sub.3O.sub.31Na.sup.+ [M+Na].sup.+ 2526.1202, found 2526.1152.
(182) ##STR00195##
(183) Compound 68* (15 mg, 5.99 mol) was taken in solvent mixture DCM (1 mL), tBuOH (1 mL) and two drops of water. Pd/C was added and hydrogenated for 24 h under H.sub.2 balloon at r.t. The reaction mixture was filtered through PTFE filter, and the residue was washed with methanol (6 mL), (50% methanol-water (6 mL). The filtrate was evaporated under vacuum to get the crude product. .sup.1H NMR analysis showed the completion of the reaction and the presence of product. So, crude product was purified through the C18 Sepak column using water (3 mL2, fr1), 20% acetonitrile-water (3 mL2, fr2) and acetonitrile (3 mL, fr3). All the fractions were frozen and lyophilized for 24 h to get one pure fraction fr1 of compound 69* (white solid, 5.64 mg, 87%), and two impure fractions white fluffy solid (fr2, 0.2 mg) a white fluffy solid (fr.3, 0.2 mg).
(184) HRMS (ESI+) Calcd for C.sub.41H.sub.73NO.sub.31H.sup.+ [M+H].sup.+ 1076.4244, found 1076.4245.
(185) .sup.1H NMR (400 MHz, Deuterium Oxide) 5.33 (d, J=1.8 Hz, 1H), 5.14 (d, J=1.8 Hz, 1H), 5.10 (d, J=1.7 Hz, 1H), 5.06 (d, J=1.7 Hz, 1H), 4.65-4.68 (m, 2H), 4.27 (dt, J=3.2, 1.4 Hz, 2H), 4.14-4.20 (m, 1H), 4.09 (dd, J=3.4, 1.7 Hz, 1H), 3.82-4.04 (m, 12H), 3.47-3.82 (m, 23H), 3.32-3.43 (m, 2H), 2.94-3.02 (m, 2H), 1.59-1.73 (m, 4H), 1.35-1.53 (m, 2H).
Example 5: Synthesis of K. pneumoniae Serotype O5 Nonasaccharide
(186) ##STR00196##
(187) To a solution of compound 63* (720 mg, 0.978 mmol) and 4-methoxyphenol (121 mg, 0.978 mmol) in anhydrous toluene (7.3 mL) and dioxane (2.5 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. TMSOTf (18 L, 0.098 mmol) was added and the reaction mixture stirred at 10 C. for 1 h. The reaction was quenched with sat. NaHCO.sub.3 solution (35 mL) and extracted with DCM (250 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated to give crude product. The residue was purified by column chromatography using EtOAc and cyclohexane as solvents to afford the desired product (540 mg, 84%) as colorless oil.
(188) HRMS (ESI+) Calcd for C.sub.41H.sub.40O.sub.8Na.sup.+ [M+Na].sup.+ 683.2621, found 683.2643.
(189) ##STR00197##
(190) Sodium methoxide solution in MeOH (25% w/w) (0.52 mL, 2.406 mmol) was added to a solution of the benzoate 70* (530 mg, 0.802 mmol) in a mixture of MeOH:THF (4:1, 7.5 mL). The reaction was stirred at the same temperature for 16 h. The reaction was quenched by the addition of H.sub.2O (3 mL) and diluted with brine (25 mL). Reaction mixture was extracted with EtOAc (250 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4 and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a colorless thick gel (430 mg, 96%).
(191) HRMS (ESI+) Calcd for C.sub.34H.sub.36O.sub.7Na.sup.+ [M+Na].sup.+ 579.2359, found 579.2395.
(192) ##STR00198##
(193) To a solution of compound 3* (100 mg, 0.155 mmol) and compound 71* (86 mg, 0.155 mmol) in a mixture of anhydrous toluene (2.3 mL) and anhydrous dioxane (0.8 mL) was added 4 MS and the mixture let stir at room temperature for 30 min. Then, NIS (41.7 mg, 0.186 mmol) was added and the reaction mixture cooled to 10 C. TfOH (2.32 mg, 0.015 mmol) was added and the reaction mixture stirred for 1 h and gradually warmed to room temp. Reaction mixture was filtered and washed with sat. Na.sub.2S.sub.2O.sub.3 solution (25 mL) and extracted with CH.sub.2Cl.sub.2 (230 mL). Combined organic layers were washed with sat. NaHCO.sub.3 solution (15 mL) and brine (10 mL), dried over anhydr. Na.sub.2SO.sub.4, and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a cloudy thick gel (132 mg, 78%).
(194) HRMS (ESI+) Calcd for C.sub.66H.sub.68O.sub.13Na.sup.+ [M+Na].sup.+ 1115.4558, found 1115.4595.
(195) ##STR00199##
(196) Sodium methoxide solution in MeOH (25% w/w) (0.074 mL, 0.343 mmol) was added to a solution of the benzoate 72* (125 mg, 0.114 mmol) in a mixture of MeOH:THF (4:1, 2.3 mL). The reaction was stirred at the same temperature for 16 h. The reaction was quenched by the addition of H.sub.2O (3 mL) and diluted with brine (25 mL). Reaction mixture extracted with EtOAc (225 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4, and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a colorless thick gel (108 mg, 95%).
(197) HRMS (ESI+) Calcd for C.sub.61H.sub.64O.sub.12Na.sup.+ [M+Na].sup.+ 1011.4295, found 1011.4326.
(198) ##STR00200##
(199) To a solution of compound 2* (197 mg, 0.334 mmol) in anhydrous DCM (6 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. Then, 1-(phenylsulfinyl)piperidine (76 mg, 0.365 mmol) and 2,4,6-tri-tert-butylpyrimidine (165 mg, 0.664 mmol) were added, reaction mixture cooled to 65 C. and stirred for 30 min. Triflic anhydride (67 L, 0.397 mmol) was added and the reaction mixture stirred for 20 min at 65 C. Reaction mixture was then cooled to 78 C. and compound 73* (300 mg, 0.303 mmol) in DCM (4 mL) was added dropwise and stirred for 6 h at 78 C. and then warmed to 0 C. within 1 h. Reaction mixture was filtered and washed with sat. NaHCO.sub.3 solution (25 mL) and extracted with CH.sub.2Cl.sub.2 (225 mL). Combined organic layers were washed with brine (10 mL), dried over anhydr. Na.sub.2SO.sub.4, and concentrated under reduced pressure. Purification by automated purification system using silica (ethyl acetate/cyclohexane) afforded the product after evaporation of the solvent as a cloudy thick gel (300 mg, 67%).
(200) HRMS (ESI+) Calcd for C.sub.92H.sub.95O.sub.17Na.sup.+ [M+Na].sup.+ 1492.6266, found 1492.6232.
(201) ##STR00201##
(202) To trisaccharide 74*(289 mg, 0.197 mmol) in acetonitrile (8.7 mL) and H.sub.2O (1.1 mL) was added ceric ammonium nitrate (172 mg, 0.315 mmol) at 0 C. and stirred for 2 h. Reaction mixture was monitored by TLC, another portion of ceric ammonium nitrate (172 mg, 0.315 mmol) was added and stirred for 2 h. The reaction mixture was partitioned between sat. aq. NaHCO.sub.3 (25 mL) solution and DCM (35 mL). The aqueous layer was extracted with DCM (25 mL), combined organic layers were dried over Na.sub.2SO.sub.4 and evaporated to give crude product. The residue was purified by column chromatography using EtOAc and cyclohexane as solvents to afford the desired product (125 mg, 46%) as light-yellow oil.
(203) HRMS (ESI+) Calcd for C.sub.85H.sub.86O.sub.16Na.sup.+ [M+Na].sup.+ 1385.5814, found 1385.5885.
(204) ##STR00202##
(205) Cs.sub.2CO.sub.3 (38.2 mg, 0.117 mmol) and 2,2,2-trifluro-N-phenyl-acetimidoyl chloride (36.5 mg, 0.176 mmol) were added to a solution of lactol 75* (80 mg, 0.059 mmol) in DCM (8 mL). The reaction mixture was stirred at room temperature and monitored by TLC. After 2 hours all the starting material was consumed, the reaction was filtered through celite and washed with DCM (20 mL). The solvent was evaporated and the crude product (90 mg, quantitative) was used in the next step without any purification.
(206) ##STR00203##
(207) To a solution of compound 76* (80 mg, 0.052 mmol) and compound 60* (69.6 mg, 0.052 mmol) in anhydrous toluene (2 mL) and dioxane (0.66 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. TMSOTf (1 L, 5.21 mol) was added and the reaction mixture stirred at 10 C. for 1 h. The reaction was quenched with sat. NaHCO.sub.3 solution (20 mL) and extracted with DCM (225 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated to give crude product. The residue was purified by column chromatography using EtOAc and cyclohexane as solvents to afford the desired product (70 mg, 50%) as colorless oil.
(208) HRMS (ESI+) Calcd for C.sub.164H.sub.171N.sub.3O.sub.31Na.sup.+ [M+Na].sup.+ 2702.1828, found 2702.1853.
(209) ##STR00204##
(210) To a solution of compound 77* (60 mg, 0.022 mmol) in DCM:PBS (2:1, 5.1 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (10.2 mg, 0.045 mmol) at 0 C. The reaction mixture was stirred for 2 h at room temperature and monitored by TLC (EtOAc in cyclohexane, 2:1). A portion of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (5 mg) was added again and the mixture was stirred at room temp for 2 h. Reaction was quenched with sat. NaHCO.sub.3 (25 mL) and extracted with DCM (225 mL). The combined organic layers were washed with brine (15 mL) and dried over Na.sub.2SO.sub.4, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by automated flash chromatography using silica (ethyl acetate/cyclohexane) to obtain a colorless oil (30 mg, 53%).
(211) HRMS (ESI+) Calcd for C.sub.153H.sub.163N.sub.3O.sub.31Na.sup.+ [M+Na].sup.+ 2562.1202, found 2562.1219.
(212) ##STR00205##
(213) To a solution of compound 76* (35 mg, 0.023 mmol) and compound 78* (29 mg, 0.011 mmol) in anhydrous toluene (3 mL) and dioxane (1 mL) was added 4 MS and the mixture was stirred at room temperature for 30 min. TMSOTf (0.2 L, 1.142 mol) was added and the reaction mixture stirred at 10 C. for 1 h. The reaction was quenched with sat. NaHCO.sub.3 solution (20 mL) and extracted with DCM (225 mL). The organic layer was dried over Na.sub.2SO.sub.4 and evaporated to give crude product. The residue was purified by column chromatography using EtOAc and cyclohexane as solvents to afford the desired product (28 mg, 63%) as yellow oil.
(214) MALDI-TOF: Calcd for C.sub.238H.sub.247N.sub.3O.sub.46H.sup.+ [M+H].sup.+ 3885.722, found 3885.105.
(215) ##STR00206##
(216) Compound 79* (6.0 mg, 1.415 mol) was taken in solvent mixture DCM (1 mL), tBuOH (1 mL) and two drops of water. Pd/C was added to it and hydrogenated for 24 h under H.sub.2 balloon at rt. The reaction mixture was filtered through PTFE filter, the residue was washed with methanol (6 mL), (50% methanol-water (6 mL). The filtrate was evaporated under vacuum to get the crude product. .sup.1H NMR analysis showed the completion of the reaction and the presence of product. So, crude product was purified through the C18 Sepak column using water (3 mL2, fr1), 20% acetonitrile-water (3 mL2, fr2) and acetonitrile (3 mL, fr3). All the fractions were frozen and lyophilized for 24 h to get one pure fraction of compound 80* fr1 (white solid, 2.4 mg, 99%), and two impure fractions white fluffy solid (fr2, 0.03 mg) a white fluffy solid (fr.3, 0.4 mg).
(217) HRMS (ESI+) Calcd for C.sub.59H.sub.103NO.sub.46H.sup.+ [M+H].sup.+ 1562.5829, found 1562.5815.
Example 5: Automated Synthesis of K. pneumoniae Serotype O3 Oligosaccharides
General Methods and Materials
(218) Anhydrous* solvents used to prepare building block as well as activator, TMSOTf and capping stock solutions were taken from a solvent drying system (JC Meyer solvent systems). HPLC Grade DCM was used for washing. All other washing solvents (DMF, THF, dioxane and MeOH) were reagent grade.
(219) Building blocks are dried by co-evaporation with toluene (3) and drying under high vacuum for approximately 1 to 2 hours.
(220) All synthesis were carried out on a scale of 0.0125 mmol using a Merrifield resin modified with a photocleavable linker (loading=0.41 mmol/g). The structure of the linker loaded resin is as follows
(221) ##STR00207##
Stock Solutions
(222) Activator solution: 150 mM NIS/15 mM TfOH in DCM*:dioxane*
(223) Acid wash solution: 62 mM TMSOTf in DCM*
(224) Capping solution: 10% (v/v) Ac.sub.2O/2% (v/v) MeSO.sub.3H in DCM*
(225) pyridine pre-wash solution: 10% (v/v) pyridine in DMF
(226) Fmoc deprotection solution: 20% (v/v) piperidine in DMF
AUTOMATION MODULES
Module A: Initial Resin Swelling/Beginning of Synthesis Wash
(227) The resin is washed with DCM, DMF and THF (3, 2 mL, 25 s each) before swelling in DCM (2 mL) for 30 mins with occasional mixing using pulsed Argon bubbling.
Module B: Acidic Wash with 62 mM TMSOTf
(228) DCM (2 mL) is delivered into the reaction vessel, and the temperature is adjusted to 20 C. The DCM is drained and replaced by another 2 mL of DCM before TMSOTf solution (1 mL) is added dropwise. The mixture is incubated for 1.5 mins under Ar bubbling before draining washing with 2 mL DCM for 25 s.
(229) TABLE-US-00001 T Incubation Action Cycles Reagent Amount ( C.) Time Cooling 20 Deliver 2 DCM 2 mL 20 Deliver TMSOTf 1 mL 20 1.5 min Wash DCM 2 mL 20 25 s
Module C: Thioglycoside Coupling
(230) Building Block Stocks and Glycosylation Parameters:
(231) TABLE-US-00002 c T1 Structure Excess (mM) ( C.) T2 ( C.) A 81*
(232) Glycosylation Cycle:
(233) DCM (2 mL) is added to the resin and the temperature is set to the activation temperature T12 K. While cooling down, the building block solution is delivered to the reaction vessel. After the set temperature has stabilized at T12 K, the reaction is started by adding 1 mL of activator solution. The mixture is kept at T1 for 5 minutes before a second 20 minute incubation cycle is started during which the temperature is raised to a temperature T2. Upon completion of the incubation cycle the reaction mixture is drained and the resin is washed once each with DCM:Dioxane 1:1 (2 mL) and DCM (2 mL). The module finishes by raising the temperature to 25 C. while performing two additional DCM washes (2 mL).
(234) TABLE-US-00003 T Incubation Action Cycles Solution Amount ( C.) Time Cooling T1-2 Deliver 2 for A BB Solution 1 mL Deliver 1 for B Activator 1 mL Solution Incubation T1 5 min Incubation T2 20 min Wash 1 DCM:Dioxane 2 mL T2 25 s 1:1 Wash 1 DCM 2 mL 0 25 s Heating 25 Wash 2 DCM 2 mL 25 25 s
Module D: Capping
(235) The resin is washed with DMF (2, 25 s) and the temperature of the reaction vessel was set to 25 C. 2 mL of 10% Pyridine in DMF is delivered into the reaction vessel. After 1 min, the solution is drained and the resin is washed with DCM (3, 2 mL, 25 s). Then, 4 mL of capping solution is delivered into the reaction vessel and incubated for 20 mins under Argon bubbling. The cycle concludes by draining the reaction mixture washing the resin with DCM (3, 2 mL, 25 s).
(236) TABLE-US-00004 T Incubation Action Cycles Solution Amount ( C.) Time Heating 25 Wash 2 DMF 2 mL 25 25 s Deliver 1 10% Py in 2 mL 25 1 min DMF Wash 3 DCM 2 mL 25 25 s Deliver 1 Capping 4 mL 25 20 mins solution Wash 3 DCM 2 mL 25 25 s
Module E: FMOC Deprotection
(237) The resin is washed with DMF (3, 2 mL, 25s) and the temperature of the reaction vessel is adjusted to 25 C. 2 mL of FMOC deprotection solution is delivered into the reaction vessel. After 5 mins, the solution was drained through the UV-sensor and the resin is washed with DMF (3, 2 mL) and DCM (5, 2 mL, 60 s each). The temperature of the reaction vessel was decreased to 20 C. in preparation of the next cycle.
(238) TABLE-US-00005 T Incubation Action Cycles Solution Amount ( C.) Time Heating 25 Wash 3 DMF 2 mL 25 25 s Deliver 1 FMOC deprotection 2 mL 25 5 mins Solution Wash 1 DMF 2 mL Cooling 20 Wash 3 DMF 2 mL 25 s Wash 5 DCM 2 mL 25 s
Post-Automation Steps
Cleavage from Solid Support
(239) After automated synthesis, the oligosaccharides were cleaved from solid support using a continuous flow photoreactor. The sample (resin loaded with target oligosaccharide) is taken up in 20 mL DCM (stabilized with amylene, LC-MS grade) and injected into the reactor (Wavelength=300 nm) at the rate of 1.0 mL/min. When all of the resin is inside the reactor, fresh DCM (20 mL) is injected to retrieve the photocleaved resin. The filtrate so obtained is concentrated in-vacuo and subjected to further analysis and purification.
Purification and HPLC Analysis
(240) The crudes were dissolved in 1:1 hexane: ethyl acetate and analyzed using analytical HPLC (YMC-Diol-300 column, 1504.6 mm, ELSD Detector and DAAD, 280 nm). Method(Stop Time60.0 mins)
(241) TABLE-US-00006 Time (min) % Ethyl Acetate % Hexane Flow (mL/min) 0.00 20 80 1.000 5.00 20 80 1.000 40.00 55 45 1.000 45.00 100 0 1.000 50.00 100 0 1.000
LIST OF OLIGOSACCHARIDES OBTAINED
(242) TABLE-US-00007 Amount Structure 22 mg
Experimental Procedures for the Deprotection of Fully Protected Oligosaccharides from Automation Steps in Solution Phase Synthesis
(243) Benzoyl and Acetate Deprotection:
(244) Sodium methoxide solution in MeOH (25% w/w) (30-45 eq.) was added to a solution of benzoate 83*-87* (1 eq.) in a mixture of MeOH:THF (2:1). The reaction was stirred at the same temperature for 16 h. The reaction was quenched by the addition of H.sub.2O (1 mL) and diluted with brine (5 mL). The reaction mixture extracted with EtOAc (210 mL). Combined organic layers were dried over anhydr. Na.sub.2SO.sub.4 and concentrated under reduced pressure. Crude product 83a*-87a* after evaporation of the solvent obtained as a yellow thick gel which was used in next step without any further purification.
(245) Benzyl Deprotection:
(246) 83a*-87a* (1 eq.) was taken in solvent mixture DCM (2 mL), tBuOH (2 mL) and two drops of water. Pd/C was added and hydrogenated for 24 h under H.sub.2 balloon at r.t. The reaction mixture was filtered through PTFE filter and the residue washed with methanol (6 mL), (50% methanol-water (6 mL). The filtrate was evaporated under vacuum to get the crude product. .sup.1H NMR analysis showed the completion of the reaction and the presence of product. So, crude product was purified through the C18 Sepak column using water (3 mL2, fr1), 50% acetonitrile-water (3 mL2, fr2) and acetonitrile (3 mL, fr2). All the fractions were frozen and lyophilized for 24 h to get one pure fraction of compound 83b*-87b* fr1, and impure fraction fr2.
(247) Thus, hexasaccharide 83b*, decasaccharide 84b*, dodecasaccharide 85b* and pentadecasaccharide 86b* have been obtained using the protocol stated above. Following the similar protocol, the fully deprotected Icosasaccharide 87b* can be achieved from compound 87a*.
(248) TABLE-US-00008 Compound structure Amount Mass
B Immunization Studies of K. pneumoniae Serotype O3 and O5 Oligosaccharides
(249) Materials: ELISA plates (high-binding, EIA/RIA Plate, 96 well, flat bottom with low evaporation lid, company: Costar 3361) Detection antibody: Goat anti rabbit IgG peroxidase conjugate (Sigma, #A4914) and Goat anti-Mouse IgG (H+L) peroxidase conjugate (Dianova Code: 115-035-068). 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 solution2M sulphuric acid (H.sub.2SO4). Plate reader: Anthos HT 2. Software: WinRead 2.36 for absorbance measurements and GraphPad Prism 7 for data plotting and analysis. Alum: Aluminium Hydroxide Gel Adjuvant (Alhydrogel 2%), Brenntag, Batch #: 5447 Exp Dt: February 2020. Incomplete Freund's Adjuvant (IFA). InvivoGen; Cat: vac-ifa-10, Batch #: IFA-39-03; Exp Dt: September 2019 QuantiPro BCA Assay Kit (SIGMA) Product: QPBCA-1KT; Lot #: SLBR7451V; Pcode: 1002296464 Mini-PROTEAN TGX Gels- 10%, 10 well (30 L/well) Control Nr:64175708, Precision Plus Dual Color, Cat: 1610374; Control Nr: 641798899 GelCode Blue Safe Protein Stain; ThermoScientific; Ref: 1860957; Lot #: TA260266 Klebsiella pneumoniae LPS. SIGMA- L4268; Lot #: 116 M 4057 V
(250) Methods:
(251) 1. Bacterial Strains and LPS.
(252) Klebsiella pneumoniae (KPC) strains differing in their LPS (O-antigen) with/without the capsule were used to isolate and purify the corresponding LPS. The purified LPS were used as coating antigen in Enzyme Linked Immunosorbent Assay (ELISA). The O2a,c LPS was procured from Sigma-Aldrich.
(253) TABLE-US-00009 TABLE 1 Klebsiella pneumoniae strains used for LPS isolation. # LPS/O-antigen 1 O1 2 O2a 3 O2a,c 4 Galactan-III
(254) 2. Production of Glycoconjugate and Characterization.
(255) The KPC synthetic antigens were 21* and 69* conjugated to the carrier protein CRM.sub.197 (21*-CRM.sub.197 and 69*-CRM.sub.197) for immunization experiments and to Bovine Serum Albumin (BSA; (21*-CRM.sub.197 and 69*-CRM.sub.197)) as coating antigen for ELISA according to the procedure described below.
General Conjugation Protocol
Step 1: PNP-Ester Synthesis
(256) Compound 21* or 69* (1 eq) was dissolved in DMSO or DMSOH.sub.2O at room temperature in a 8 mL vial. Activated bis-(4-nitrophenyl) adipate (20 eq) was added to it and stirred for 5 minutes. Triethylamine (50 eq) was added and the reaction mixture was allowed to stir at room temperature for 3-5 h. The reaction mixture was frozen using liquid nitrogen and then lyophilized for 18 h to dryness to afford pale yellow colored crude product along with the excess of the reagent. The crude product was washed thoroughly with sufficient CHCl.sub.3 followed by DCM to remove excess reagent. The solid para-nitrophenyl (PNP) ester was dried and taken for the next step.
Step 2: Conjugation to the Protein
(257) Conjugation procedure: The PNP ester of 21* or 69* in 50 L of 0.15 M NaCl in NaPi buffer was added dropwise to the reaction vial containing CRM.sub.197 or BSA in buffer (150 L). The vial was finally rinsed with 50 L of buffer solution and transferred to the reaction vial completely. Thus making the volume of the reaction in the vial 200 L. The reaction mixture became yellow in colour and stirred the reaction mixture at r.t. for 24h. The conjugate solution (21*-CRM.sub.197, 69*-CRM.sub.197, 21*-CRM.sub.197 or 69*-CRM.sub.197) was transferred to an Amicon Ultra-0.5 mL centrifugal filter, centrifuged for 6 minutes at 2-8 C. 300 L of buffer were added to the reaction vial, rinsed and transferred to the filter and centrifuged again. Additional washings were done using 1PBS solution and centrifuging till the yellow colour was gone and the conjugate became clear solution. After the final wash the conjugate was stored in 1PBS solution at 2-8 C.
(258) The conjugates were analyzed by SDS-PAGE, SEC chromatography, and MALDI analysis. The loading of the sugar on the carrier was specifically calculated by subtracting the mass between the conjugated and unconjugated protein using MALDI analysis. The protein content was estimated using the micro BCA method following manufacture protocol.
2.1 SDS-PAGE Analysis
(259) The samples were mixed in a microfuge tube and heated for 5 min at 95 C. on a thermocycler. After cooling to room temperature for 5 min, the samples at approximately 2.5 g were loaded onto the respective wells of a 10% polyacrylamide gel along with 10 L of the marker. The samples were run at a constant voltage of 120V for 1 h. Staining was done using the GelCode Blue Safe Protein Stain as per manufacture instructions. The gels were washed with deionized water overnight and scanned using the gel documentation system.
2.2 Size Exclusion Chromatography (SEC) of Glycoconjugates
(260) The glycoconjugates (21*-CRM.sub.197 and 69*-CRM.sub.197) used for immunization studies were analyzed by SEC to observe a mass difference between the conjugated and unconjugated CRM protein. The samples were diluted in 50 mM Tris, 20 mM NaCl, pH 7.2 and run on a Agilent 1100 HPLC system fitted with Tosoh TSK G2000 column (SWxl, 7.8 mm30 cm, 5 m) and a Tosoh TSKgel Guard Column (SWxl 6.0 mm4 cm, 7 m). The flow rate was kept at 1 mL/min.
3. Formulation of Vaccines for Immunization
(261) The glycoconjugates were formulated in aluminum hydroxide (alum) adjuvant for mice studies, and in Incomplete Freund's Adjuvant (IFA) for immunization in rabbits.
3.1 Formulation in Alum
(262) All the formulations were prepared under sterile conditions. The glycoconjugates (DS) and PBS were mixed in the appropriate pre-calculated ratio in a 50 mL Falcon tube corresponding to the final formulation volume leaving out the volume of alum (0.25 mg/mL) required. This formed the DS-PBS mixture. The antigen/DS dose per animal was kept at 5 g/100 L/animal. The DS-PBS mixture was gently mixed (5) using a serological pipette. To the DS-PBS mixture, the corresponding volume of stock alum (10 mg/mL) was added to give a final alum ratio of 1:40 or 0.250 mg/mL. The mixture was immediately mixed by gentle pipetting (20) using a 5 mL serological pipette. The Falcon tube was capped, wrapped with Parafilm and allowed to mix on a shaker at 250 rpm for 2 h at room temperature (RT). After the incubation time of 2 h, the formulations were brought under the clean bench, aliquoted, and further stored at 4 C. till further use.
3.2 Formulation in IFA
(263) Incomplete Freund's Adjuvant (IFA) from InvivoGen was used for formulating the vaccines for rabbit immunization studies. Protocol was followed as per manufacture. Antigen: IFA concentration was kept at 1:1. The antigen dose per animal was kept at 5 g/200 L/animal (100 L of antigen +100 L IFA). IFA at the desired calculated volume (50% of the final immunization volume) was taken in a 15 mL sterile Falcon tube. The calculated amount of the diluted antigen solution (volume adjusted with PBS to 50% of the final immunization volume) was taken in a 3 mL sterile syringe, fitted with a 20 G needle. The DS solution was added into the Falcon tube containing the IFA and immediately vortexed for 15 sec (5). The color of the formulation changes from pale-yellow to milky-white on vortexing which indicates the formation of stable emulsion. The resulting vaccine formulation was briefly vortexed and aliquoted into 2 mL sterile tubes with the desired dose volumes. Prior to immunizations, the tubes containing the vaccine formulations were vortexed and then injected into animals.
3.3 Characterization of Alum Formulations
(264) The glycoconjugates formulated in alum were characterized to determine the final alum concentration and the pH of the formulations.
4. Immunization Schedule
(265) Mice and rabbit immunizations were performed under specific pathogen-free conditions and were provided food and water ad libitum. Mice (n=6) and rabbits (n=4) were immunized sub cutaneous with the vaccine formulations (Table 2) at an injection volume of 100 L/mice, and 200 L/rabbit. The antigen dose for mice was kept at 5 g/animal except for the antigen-7 (2.5 g each of antigen 1, and -2). The antigen dose for rabbit was kept at 5 g/animal. Mice and rabbits were immunized on day 0, 14 and 28. Blood was drawn on day 1, 7, and 22 for mice and day 0, 7 and 21 for rabbits respectively, for the determination of antibody titers. On day 35, the animals were sacrificed, and blood collected.
(266) TABLE-US-00010 TABLE 2 Immunization schedule of mice (n = 6) and rabbits (n = 4). group glycoconjugate mice per group rabbits per group 1 21*-CRM.sub.197 (O3) 6 0 2 69*-CRM.sub.197 (O5) 6 4 *All values for mice sera analysis were subtracted using the values from PBS (negative control).
5. Enzyme Linked Immunosorbent Assay (ELISA) of Sera Using In-House Antigen Coated Plates
(267) Coating of Plates with Antigen:
(268) Conjugates 21*-BSA and 69*-BSA, and LPS #1-#4 were used as the coating antigen. LPS was dissolved in isopropanol at a concentration of 10/20 g/mL. 100 L was used for coating each well resulting in a coating concentration of 1-2 g/well. The LPS solutions were loaded into the well and subjected to overnight evaporation at r.t. inside the sterile bench. For conjugates 21*-BSA and 69*-BSA, the respective conjugates were dissolved at a concentration of 5 g/mL in phosphate buffered saline (PBS) pH 7.4. 100 L were coated per well and incubated overnight at 4 C. to get an antigen concentration of 0.5 g/well.
(269) Washing:
(270) After overnight adsorption of the antigen, the plates were washed 1 with PBS-T (200 L/well) and the excess fluid per well was removed by inverting the plate and tapping on a clean dry tissue towel.
(271) Blocking:
(272) The plates were blocked using 200 L of the commercial blocking solution and incubated for 2 h at RT.
(273) Washing:
(274) After blocking, the plates were washed 3 with PBS-T (200 L/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.
(275) Dilution of Sera and Incubations:
(276) Pooled sera (n=4 rabbits or n=6 mice/group) from different time-points of the different experimental groups were diluted to their respective dilutions in the antibody diluent (PBS+1% BSA). 100 L of the diluted sera samples of the different experimental groups were added in duplicates to the corresponding wells and incubated on a shaker set at 250 rpm for 2h at RT. 100 L/well of the antibody diluent (PBS+1% BSA) formed the experimental blank. After incubation with sera, the plates were washed 4 with PBS-T (200 L/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.
(277) Incubation (Detection Antibody):
(278) The corresponding detection antibody, anti-rabbit or anti-mouse IgG HRP conjugate was diluted 1:10,000 in the antibody diluent (PBS+1% BSA) and 100 L/well was added and incubated on a shaker at 250 rpm for 1h at RT. After the incubation with detection antibody, the plates were washed 5 with PBS-T (200 L/well) and the excess fluid per well was removed by inverting the plate and by tapping on a clean dry tissue towel.
(279) Substrate Addition:
(280) To each well, 100 L of the ready to use TMB (3,3,,5,5-tetramethylbenzidine) substrate (normalized to r.t. from 4 C.) was added and incubated in dark for 15 min. The blue color of the enzymatic reaction was stopped by adding 50 L/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 using a plate reader.
(281) Results:
(282) The absorption values were analyzed by plotting a graph using the GraphPad Prism software.
(283) Results.
(284) Characterization of Glycoconjugates 21*-CRM.sub.197 and 69*-CRM.sub.197.
(285) The KPC antigen glycoconjugates 21*-CRM.sub.197 and 69*-CRM.sub.197 used for the immunization studies were analyzed for the conjugation efficiency and antigen content. MALDI analysis of the glycoconjugates revealed a very good conjugation efficiency. The mass differences between the conjugated and unconjugated CRM.sub.197 protein yielded a loading from 2-15, preferably from 3-10 antigens/CRM.sub.197 molecule for the different glycoconjugates.
(286) The glycoconjugates were also analyzed by a 10% SDS-PAGE and SEC that revealed a clear mass shift as compared to the unconjugated CRM.sub.197 protein (
(287) ELISA Data.
(288) Sera from 21*-CRM.sub.197/69*-CRM.sub.197 immunized mice recognize the corresponding antigens (see
(289) The herein provided data demonstrate that after immunization with a conjugate of the present invention, functional antibodies against oligosaccharides of the present invention as well as against the natural O-polysaccharides of K. pneumoniae serotypes O3, O3b and O5 were elicited in rabbits and mice. The Antibodies do cross-react with the natural O-polysaccharides (LPS) of K. pneumoniae serotypes O3, O3b and O5 indicating the potential of these antibodies to bind to K. pneumoniae bacteria and to confer protection against K. pneumoniae infection.
(290) The ELISA data further proves that the conjugates of the present invention are immunogenic and induce high antibody titers. Hence, ELISA analysis shows that the oligosaccharides of formula (I) of the present invention are immunogenic in rabbits and mice and generate cross reactive antibodies.