Synthetic vaccines against <i>Streptococcus pneumoniae </i>serotype 2
10864261 · 2020-12-15
Assignee
Inventors
- Peter H. Seeberger (Kleinmachnow, DE)
- Claney Lebev Pereira (Berlin, DE)
- Chakkumkal Anish (The Hague, NL)
- Naeem Khan (Berlin, DE)
- Madhu EMMADI (Berlin, DE)
Cpc classification
C07H15/04
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
A61K31/715
HUMAN NECESSITIES
C07H1/00
CHEMISTRY; METALLURGY
A61K31/7028
HUMAN NECESSITIES
C07H15/00
CHEMISTRY; METALLURGY
A61K2039/6037
HUMAN NECESSITIES
A61K47/36
HUMAN NECESSITIES
International classification
A61K39/09
HUMAN NECESSITIES
A61K31/715
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
C07H15/00
CHEMISTRY; METALLURGY
A61K31/7028
HUMAN NECESSITIES
C07H1/00
CHEMISTRY; METALLURGY
A61K47/36
HUMAN NECESSITIES
Abstract
The present invention relates to a synthetic saccharide of general formula (I) that is related to Streptococcus pneumoniae serotype 2 capsular polysaccharide, a conjugate thereof and the use of said saccharide and conjugate for raising a protective immune response in a human and/or animal host. Furthermore, the synthetic saccharide of general formula (I) is useful as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae type 2 bacteria.
Claims
1. A saccharide of general formula (I)
V*-U.sub.x+3-U.sub.x+2-U.sub.x+1-U.sub.x-O-L-NH.sub.2 (I) wherein x is an integer selected from 1, 2, 3 and 4; ##STR00057## V*- represents H-, H-U.sub.x-, H-U.sub.x+1-U.sub.x-, H-U.sub.x+2-U.sub.x+1-U.sub.x- or H-U.sub.x+3-U.sub.x+2-U.sub.x+1-U.sub.x-; L is selected from CH.sub.2, (CH.sub.2).sub.2, (CH.sub.2).sub.3, (CH.sub.2).sub.4, (CH.sub.2).sub.5, (CH.sub.2).sub.6, (CH.sub.2).sub.7, (CH.sub.2).sub.8, (CH.sub.2).sub.9, (CH.sub.2).sub.10, CF.sub.2, (CF.sub.2).sub.2, (CF.sub.2).sub.3, (CF.sub.2).sub.4, (CF.sub.2).sub.5, (CF.sub.2).sub.6, (CF.sub.2).sub.7, (CF.sub.2).sub.8, (CF.sub.2).sub.9, (CF.sub.2).sub.10, (CH.sub.2).sub.2O(CH.sub.2).sub.2, CH.sub.2O(CH.sub.2).sub.3, (CH.sub.2).sub.3OCH.sub.2, CH.sub.2O(CH.sub.2).sub.2, (CH.sub.2).sub.2OCH.sub.2, (CH.sub.2).sub.3O(CH.sub.2).sub.2, (CH.sub.2).sub.2O(CH.sub.2).sub.3, (CH.sub.2).sub.4OCH.sub.2, CH.sub.2O(CH.sub.2).sub.4, -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, -L.sup.a-L.sup.b-L.sup.d-L.sup.c-L.sup.e-, and -L.sup.a-L.sup.d-L.sup.e-; wherein -L.sup.a- is selected from: (CH.sub.2).sub.o, (CF.sub.2).sub.o, (CH.sub.2CH.sub.2O).sub.oC.sub.2H.sub.4, (CH.sub.2CH.sub.2O).sub.oCH.sub.2, (CR.sup.10R.sup.11).sub.o, ##STR00058## -L.sup.b- and -L.sup.c- are independently of each other selected from: O, NHC(O)NH, NHC(S)NH, NHC(O), C(O)NH, NHC(O)O, NR.sup.9, NR.sup.18, SO.sub.2, ##STR00059## -L.sup.d- represents (CH.sub.2).sub.q, (CF.sub.2).sub.q, (CR.sup.12R.sup.13).sub.q, (CH.sub.2CH.sub.2O).sub.qC.sub.2H.sub.4, CH.sub.2CH.sub.2O).sub.qCH.sub.2, ##STR00060## -L.sup.e- is selected from: (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, (CH.sub.2).sub.p1O(CH.sub.2).sub.p2, (CR.sup.14R.sup.15).sub.p1, (CR.sup.14R.sup.15).sub.p1O(CR.sup.21R.sup.22).sub.q2, ##STR00061## R.sup.9 and R.sup.18 are independently of each other selected from: CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, and C(O)CH.sub.3; R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.19, R.sup.20, R.sup.21 and R.sup.22 are independently of each other selected from: H, F, Cl, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.5H.sub.9, C.sub.6H.sub.13, OCH.sub.3, OC.sub.2H.sub.5, CH.sub.2F, CHF.sub.2, CF.sub.3, C(O)NH.sub.2, SCH.sub.3, SC.sub.2H.sub.5, NHC(O)CH.sub.3, N(CH.sub.3).sub.2 and N(C.sub.2H.sub.5).sub.2; o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; or a pharmaceutically acceptable salt thereof.
2. The saccharide according to claim 1, wherein x represents 1, or a pharmaceutically acceptable salt thereof.
3. The saccharide according to claim 1, wherein ##STR00062## or a pharmaceutically acceptable salt thereof.
4. The saccharide according to claim 1, wherein ##STR00063## or a pharmaceutically acceptable salt thereof.
5. A method of synthesis of a saccharide of general formula (I) according to claim 1, wherein x represents 1, V*- represents H- and ##STR00064## comprising the following steps: A) reacting a disaccharide of general formula (II) ##STR00065## wherein P.sup.1-P.sup.3 represent protecting groups, and LG.sup.1 represents a leaving group, with a disaccharide of general formula (III) ##STR00066## wherein P.sup.4-P.sup.8 represent protecting groups and L has the meaning as defined in claim 1, to obtain a tetrasaccharide of general formula (IV) ##STR00067## wherein P.sup.1-P.sup.8 represent protecting groups; and B) subjecting the tetrasaccharide of general formula (IV) to selective deprotection to obtain a tetrasaccharide of general formula (V) ##STR00068## wherein P.sup.1-P.sup.4, P.sup.6-P.sup.8 represent protecting groups and L has the meaning defined in claim 1; and C) reacting the tetrasaccharide of general formula (V) with a disaccharide of general formula (VI) ##STR00069## wherein P.sup.9 and P.sup.10 represent protecting groups and LG.sup.2 represents a leaving group to obtain a hexasaccharide of general formula (VII) ##STR00070## wherein P.sup.1-P.sup.4, P.sup.6-P.sup.10 represent protecting groups and L has the meaning defined in claim 1; and D) performing the removal of protecting groups P.sup.1-P.sup.4, P.sup.6-P.sup.10 on the compound of general formula (VII).
6. An intermediate of general formula (V) ##STR00071## wherein P.sup.1- P.sup.4, P.sup.6-P.sup.8 represent protecting groups and L has the meaning defined in claim 1.
7. The intermediate of general formula (V) according to claim 6, wherein P.sup.1, P.sup.6 and P.sup.7 represent a benzyl group, P.sup.2, P.sup.3 and P.sup.4 are independently of each other selected from benzoyl and acetyl group, and P.sup.8 represents a benzyloxy carbonyl group.
8. A conjugate of general formula (X)
[V*-U.sub.x+3-U.sub.x+2-U.sub.x+1-U.sub.x-O-L-NH-W].sub.m-CRM.sub.197 (X) wherein m is comprised between about 2 and about 18; -W- is selected from: ##STR00072## a represents an integer from 1 to 10; b represents an integer from 1 to 4; and V*, U.sub.x+3, U.sub.x+2, U.sub.x+1, U.sub.x, x and L have the meanings as defined in claim 1.
9. A saccharide according to claim 1 for use in raising a protective immune response in a human and/or animal host.
10. A saccharide according to claim 1 for use in the prevention and/or treatment of a disease caused by Streptococcus pneumoniae type 2.
11. A vaccine comprising the saccharide and/or the pharmaceutically acceptable salt thereof according to claim 1 together with at least one pharmaceutically acceptable adjuvant and/or excipient.
12. The vaccine composition according to claim 11, further comprising at least a capsular polysaccharide of Streptococcus pneumoniae and/or a fragment of a capsular polysaccharide of Streptococcus pneumoniae and/or a conjugate of a carrier protein and a capsular polysaccharide of Streptococcus pneumoniae or a fragment of a capsular polysaccharide of Streptococcus pneumoniae, wherein Streptococcus pneumoniae is selected from the group comprising Streptococcus pneumoniae type 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F.
13. A saccharide according to claim 1 for use as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae type 2.
14. A conjugate according to claim 8 for use in raising a protective immune response in a human and/or animal host.
15. A conjugate according to claim 8 for use in the prevention and/or treatment of a disease caused by Streptococcus pneumoniae type 2.
16. A vaccine comprising the conjugate according to claim 8 together with at least one pharmaceutically acceptable adjuvant and/or excipient.
17. The vaccine composition according to claim 16, further comprising at least a capsular polysaccharide of Streptococcus pneumoniae and/or a fragment of a capsular polysaccharide of Streptococcus pneumoniae and/or a conjugate of a carrier protein and a capsular polysaccharide of Streptococcus pneumoniae or a fragment of a capsular polysaccharide of Streptococcus pneumoniae, wherein Streptococcus pneumoniae is selected from the group comprising Streptococcus pneumoniae type 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8) 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.
(9) 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.
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, 600-MR and Bruker Avance 700 spectrometer at 296 K. Chemical shifts (6) are reported in parts per million (ppm) relative to the respective residual solvent peaks (CDCl.sub.3: 7.26 in .sup.1H and 77.16 in .sup.13C NMR; D.sub.2O: 4.79 in .sup.1H 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). High resolution mass spectrometry (HRMS) was performed at the Free University Berlin, Mass Spectrometry Core Facility, with an Agilent 6210 ESI-TOF mass spectrometer.
ABBREVIATIONS
(12) Ac Acetyl AcOH Acetic acid Ac.sub.2O Acetic anhydride BAlB Bisacetyliodobenzene Bn Benzyl .sup.tBuOH t-Butanol Bz Benzoyl CAN Cericammonium nitrate Cbz Benzyloxycarbonyl Cu(OAc).sub.2 Copper(II) acetate DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC N,N-Dicyclohexylcarbodiimide DCM Dichloromethane DMAP N,N-Dimethylaminopyridine DMF N,N-Dimethylformamide ESI Electrosprayionization Et.sub.3N Triethylamine Et Ethyl EtOAc Ethyl acetate FmocCl 9-Fluorenylmethylchloroformate g Grams h Hours HRMS High resolution mass spectrometry Lev Levulinyl min Minute mL Millilitre Me Methyl MeI Methyl iodide MeOH Methanol MP p-Methoxy phenyl MS Molecular sieves NaHCO.sub.3Sodium bicarbonate NaOH Sodium hydroxide NaOMe Sodium methoxide NIS N-Iodo succinimide NMR Nuclear magnetic resonance Pd/C Palladium on charcoal Ph Phenyl Pico Picoloyl CPS Capsular polysaccharide Py Pyridine RT Room temperature TCA Trichloroacetamide TEMPO 2,2,6,6-Tetramethylpiperidinyloxy TfOH Trifluromethanesulfonic acid TMSOTf Trimethylsilyltrifluromethanesulfonate THF Tetrahydrofuran Tol p-Tolyl
Example A.1: Synthesis of Disaccharide Acceptor 8
(13) ##STR00033##
(14) Synthesis of Building Block 14
(15) ##STR00034##
(16) To a clear solution of 13 (Dhenin S. G. Y. et al., Org. Biomol. Chem. 2009, 7, 5184) (6.7 g, 14.4 mmol) in CH.sub.2Cl.sub.2 (80 mL) were added FmocCl (5.6 g, 21.62 mmol), and pyridine (2.4 mL, 28.8 mmol) and stirred at room temperature for 12 h. After complete consumption of starting material, the reaction mixture was diluted with CH.sub.2Cl.sub.2 (80 mL) and washed successively with 1 M HCl (60 mL), water (60 mL) and aq. sat. NaHCO.sub.3 (60 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (9:1) to afford the desired product 14 as white foam (9.3 g, 94%).
(17) H NMR (400 MHz, CDCl.sub.3) 8.06 (d, J=7.7 Hz, 2H), 7.78-7.67 (m, 2H), 7.63 (t, J=7.5 Hz, 1H), 7.58-7.52 (m, 2H), 7.49 (t, J=7.6 Hz, 2H), 7.44-7.05 (m, 13H), 5.89 (s, 1H), 5.49 (s, 1H), 5.29 (dd, J=9.5, 3.2 Hz, 1H), 4.87 (d, J=11.1 Hz, 1H), 4.71 (d, J=11.1 Hz, 1H), 4.61-4.48 (m, 1H), 4.41 (dq, J=12.2, 6.5 Hz, 1H), 4.28 (d, J=6.0 Hz, 2H), 3.76 (t, J=9.6 Hz, 1H), 2.32 (s, 3H), 1.42 (d, J=6.1 Hz, 3H);
(18) .sup.13C NMR (101 MHz, CDCl.sub.3) 165.6, 154.3, 143.7, 143.2, 141.4, 141.3, 138.2, 137.9, 133.6, 132.7, 130.1, 130.0, 129.7, 129.6, 128.6, 128.5, 128.1, 128.0 (2C), 127.9, 127.3, 127.2, 125.5, 125.2, 120.1 (2C), 86.2, 78.8, 76.8, 75.4, 72.1, 70.4, 69.1, 46.8, 21.3, 18.1; HRMS (ESI): Calcd for C.sub.42H.sub.38O.sub.7S [M+Na]+709.2236, found: 709.2238.
(19) Synthesis of Building Block 12
(20) ##STR00035##
(21) A solution of compound 14 (0.17 g, 0.25 mmol), aminopentyl linker 15 (0.16 g, 0.5 mmol) and 4 {acute over ()} acid washed molecular sieves (AWMS) (0.3 g) in CH.sub.2Cl.sub.2 (5 mL) were stirred at room temperature for 30 min. The solution was cooled to 20 C. and NIS (62 mg, 0.28 mmol), and TfOH (2.5 L, 0.028 mmol) were added. The reaction mixture was gradually brought to room temperature over 2 h. After complete consumption of starting material, Et.sub.3N (2 mL) was added and the reaction mixture was stirred at room temperature for another 2 h. Reaction mixture was diluted with CH.sub.2Cl.sub.2 (25 mL) and washed with aq. sat. Na.sub.2S.sub.2O.sub.3 (10 mL). Separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (4:1) to obtain the desired product 12 as colorless oil (0.135 g, 82%).
(22) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.06 (d, J=7.7 Hz, 2H), 7.59 (q, J=9.3, 8.4 Hz, 1H), 7.47 (t, J=7.6 Hz, 2H), 7.42-7.13 (m, 15H), 5.32 (s, 1H), 5.18 (d, J=12.1 Hz, 2H), 4.86 (d, J=11.1 Hz, 1H), 4.83-4.79 (m, 1H), 4.75 (d, J=11.1 Hz, 1H), 4.51 (d, J=6.4 Hz, 2H), 4.21 (s, 1H), 3.78 (d, J=8.1 Hz, 1H), 3.62 (d, J=16.6 Hz, 1H), 3.46 (t, J=9.4 Hz, 1H), 3.42-3.13 (m, 2H), 2.16 (s, 1H), 1.70-1.42 (m, 6H), 1.39 (d, J=6.2 Hz, 3H);
(23) .sup.13C NMR (101 MHz, CDCl.sub.3) 174.0, 166.4, 133.5, 130.0, 129.8, 128.7, 128.6, 128.2, 128.1, 128.0, 127.4, 125.3, 110.1, 97.4, 81.9, 75.4, 73.5, 70.7, 67.6, 67.3, 50.4, 29.2, 18.3; HRMS (ESI): Calcd for C.sub.40H.sub.45O.sub.8N [M+K]+706.2782, found: 706.2705.
(24) Synthesis of Building Block 11
(25) ##STR00036##
(26) Pyridine (0.8 mL, 10.0 mmol) was added dropwise at 0 C. to a stirred solution of 16 (Rajput V. K. J. Org. Chem. 2008, 73, 6924) (2.4 g, 6.6 mmol) and FmocCl (1.8 g, 7.0 mmol) in CH.sub.2Cl.sub.2 (50 mL). The mixture was gradually heated to room temperature over 2 h, and diluted CH.sub.2Cl.sub.2 (100 mL), washed successively with 1 M HCl (50 mL) and water (50 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (10:1 to 4:1) to obtain 24a (0.92 g, 24%) and 24b (1.3 g, 34%; 20% of 16 was recovered).
(27) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.78 (d, J=7.5 Hz, 2H), 7.71 (t, J=8.4 Hz, 2H), 7.42 (t, J=8.3 Hz, 4H), 7.38-7.28 (m, 7H), 7.13 (d, J=7.8 Hz, 2H), 5.39 (d, J=3.5 Hz, 1H), 4.86-4.71 (m, 3H), 4.56-4.42 (m, 2H), 4.36 (t, J=7.6 Hz, 1H), 3.88 (dt, J=8.2, 3.6 Hz, 1H), 3.49 (t, J=9.2 Hz, 1H), 3.41 (dd, J=9.5, 5.8 Hz, 1H), 2.34 (s, 3H), 1.46 (d, J=6.0 Hz, 3H);
(28) .sup.13C NMR (101 MHz, CDCl.sub.3) 155.8, 143.5, 141.5, 138.3, 138.2, 132.5, 130.2, 130.0, 128.8, 128.2 (2C), 128.0, 127.4, 127.3, 125.6, 125.5, 120.2, 120.1, 85.7, 80.9, 77.9, 76.2, 75.6, 74.3, 70.7, 46.9, 21.3, 18.4; HRMS (ESI): Calcd for C.sub.35H.sub.34O.sub.6S [M+Na]+605.1974, found: 609.1993.
(29) Levulinic anhydride (1.4 g, 6.69 mmol) and pyridine (0.54 mL, 6.69 mmol) were added to a stirred solution of 24b (1.3 g, 2.23 mmol) in CH.sub.2Cl.sub.2 (20 mL). After stirring at room temperature for 2 days, the reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL) and washed successively with 1 M HCl (50 mL) and aq. sat. NaHCO.sub.3(50 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (4:1) to obtain 11 as viscous oil (1.04 g, 69%).
(30) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.77 (d, J=7.6 Hz, 2H), 7.61 (dd, J=15.4, 7.5 Hz, 2H), 7.45-7.27 (m, 11H), 7.12 (d, J=7.8 Hz, 2H), 5.62 (dd, J=3.2, 1.6 Hz, 1H), 5.33 (d, J=1.6 Hz, 1H), 5.16 (dd, J=9.7, 3.3 Hz, 1H), 4.83 (d, J=11.0 Hz, 1H), 4.67 (d, J=11.0 Hz, 1H), 4.51 (dd, J=10.3, 6.7 Hz, 1H), 4.42-4.24 (m, 3H), 3.62 (t, J=9.5 Hz, 1H), 2.80-2.65 (m, 4H), 2.33 (s, 3H), 2.15 (s, 3H), 1.36 (d, J=6.2 Hz, 3H);
(31) .sup.13C NMR (101 MHz, CDCl.sub.3) 206.1, 171.8, 154.1, 143.6, 143.1, 141.3, 141.2, 138.1, 137.8, 132.6, 129.9, 129.5, 128.4, 127.9, 127.8 (2C), 127.2, 127.1, 125.2, 125.1, 120.1, 120.0, 85.8, 78.6, 76.3, 75.3, 71.7, 70.1, 68.9, 46.7, 37.9, 29.8, 28.0, 21.1, 17.8; HRMS (ESI): Calcd for C.sub.40H.sub.40O.sub.8S [M+Na]+703.2342, found: 703.2359.
(32) Synthesis of Disaccharide Acceptor 8
(33) ##STR00037##
(34) A solution of donor 11 (0.25 g, 0.37 mmol), acceptor 12 (0.165 g, 0.25 mmol) and 4 acid washed molecular sieves (AWMS) (0.3 g) in CH.sub.2Cl.sub.2 (5 mL) were stirred at room temperature for 30 min. The solution was cooled to 200C and NIS (83 mg, 0.37 mmol), TfOH (3.3 L, 0.037 mmol) were added. The reaction mixture was gradually brought to room temperature over 2 h. After complete consumption of starting material, Et.sub.3N (2 mL) was added and the reaction mixture was stirred at room temperature for another 2 h. Reaction mixture was diluted with CH.sub.2Cl.sub.2 and washed with aq. sat. Na.sub.2S.sub.2O.sub.3. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (3:1) to obtain the desired product 8 as colorless oil (0.18 g, 73%).
(35) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.05 (d, J=7.7 Hz, 2H), 7.60 (t, J=7.4 Hz, 1H), 7.48 (t, J=7.6 Hz, 2H), 7.39-7.18 (m, 20H), 5.32 (s, 1H), 5.24-5.12 (m, 3H), 4.87 (d, J=10.8 Hz, 1H), 4.83-4.76 (m, 2H), 4.76-4.55 (m, 3H), 4.50 (d, J=4.8 Hz, 2H), 4.26-4.15 (m, 1H), 3.97 (dd, J=9.5, 3.4 Hz, 1H), 3.77 (dd, J=9.7, 6.0 Hz, 2H), 3.57 (t, J=9.4 Hz, 2H), 3.24 (dd, J=19.8, 10.2 Hz, 4H), 2.75 (q, J=6.4, 5.8 Hz, 2H), 2.60 (qd, J=16.7, 8.1 Hz, 2H), 2.18 (s, 3H), 1.64-1.43 (m, 6H), 1.31 (d, J=6.2 Hz, 3H), 1.18 (d, J=6.2 Hz, 3H);
(36) .sup.13C NMR (101 MHz, CDCl.sub.3) 207.1, 172.2, 166.0, 138.4, 138.0, 133.4, 129.9, 129.8, 128.6 (2C), 128.5 (2C), 128.51, 128.4, 128.3, 128.0, 127.7 (2C), 127.3, 99.5, 97.0, 81.4, 80.5, 77.9, 77.4, 75.7, 74.1, 73.0, 69.9, 68.3, 67.8, 67.3, 50.6, 50.3, 47.2, 46.3, 38.3, 29.9, 29.2, 28.3, 23.5, 18.2, 17.9; HRMS (ESI): Calcd for C.sub.58H.sub.67O.sub.14N [M+Na]+1024.4459, found: 1024.4321.
(37) Applying the synthetic procedures described at example A.1 to linker building blocks 15a, 15b, 15c and 15d provides disaccharides 8a, 8b, 8c and 8d.
(38) ##STR00038## ##STR00039##
Example A.2: Synthesis of Disaccharide Donor 7
(39) ##STR00040##
(40) Synthesis of Building Block 9
(41) ##STR00041##
(42) To a stirred solution of compound 17 (Bourke J. Org. Biomol. Chem. 2014, 12, 1114) (0.25 g, 0.55 mmol) in CH.sub.2Cl.sub.2 (2.5 mL) were added picolinic acid (93 mg, 0.75 mmol), DCC (0.17 g, 0.8 mmol) and DMAP (13.5 mg, 0.11 mmol). After stirring at room temperature for 2.5 h, the reaction mixture was diluted with CH.sub.2H.sub.2 (25 mL) and washed successively with cold water (10 mL) and aq. sat. NaHCO.sub.3 (10 mL). The organic layer was dried over Na.sub.2SO.sub.4 filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (3:1) to give the desired product 9 as pale yellowish oil (0.307 g, quantitative).
(43) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.80 (ddd, J=4.7, 1.8, 0.9 Hz, 1H), 8.01 (dt, J=7.9, 1.1 Hz, 1H), 7.81 (td, J=7.7, 1.8 Hz, 1H), 7.49 (ddd, J=7.6, 4.7, 1.2 Hz, 1H), 7.32-7.27 (m, 2H), 7.25-7.17 (m, 4H), 7.17-7.09 (m, 4H), 5.43 (dd, J=9.5, 3.4 Hz, 1H), 5.31 (d, J=1.7 Hz, 1H), 4.85 (d, J=11.1 Hz, 1H), 4.74-4.65 (m, 2H), 4.57 (d, J=12.3 Hz, 1H), 4.27-4.14 (m, 1H), 4.11 (dd, J=3.4, 1.7 Hz, 1H), 3.90 (t, J=9.5 Hz, 1H), 2.74-2.51 (m, 2H), 1.38 (d, J=6.2 Hz, 3H), 1.27 (t, J=7.4 Hz, 3H).
(44) Synthesis of Disaccharide 18
(45) ##STR00042##
(46) NIS (0.15 g, 0.65 mmol) and TfOH (6.0 L, 0.065 mmol) were added to a cooled solution of donor 9 (0.32 g, 0.64 mmol), acceptor 10 (Bundle D. R. et al. ACS Chem. Biol. 2012, 7, 1754) (0.25 g, 0.43 mmol) and 4 acid washed molecular sieves (AWMS) (2.0 g) in CH.sub.2Cl.sub.2 (20 mL) at 40 C. Reaction mixture was gradually warmed to 20 C. over 1 h, diluted with CH.sub.2Cl.sub.2 (30 mL) and washed with aq. sat. Na.sub.2S.sub.2O.sub.3 (15 mL). The organic layer was dried over Na.sub.2SO.sub.4 filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (4:1 to 3:1) to obtain the desired product 18 as pale yellowish oil (0.23 g, 53%).
(47) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.88 (d, J=4.7 Hz, 1H), 8.15 (d, J=7.8 Hz, 2H), 8.05 (d, J=8.0 Hz, 1H), 7.87 (t, J=7.9 Hz, 1H), 7.64 (t, J=7.4 Hz, 1H), 7.59-7.44 (m, 3H), 7.42-7.17 (m, 20H), 7.04 (d, J=8.6 Hz, 2H), 6.77 (d, J=8.8 Hz, 2H), 5.59 (t, J=8.3 Hz, 1H), 5.09 (d, J=7.9 Hz, 1H), 5.03 (dd, J=9.8, 3.2 Hz, 1H), 4.88-4.82 (m, 3H), 4.76-4.58 (m, 6H), 4.54 (d, J=10.4 Hz, 1H), 4.27 (d, J=10.4 Hz, 1H), 4.11 (d, J=3.2 Hz, 1H), 4.00-3.79 (m, 4H), 3.77 (s, 3H), 3.39 (dq, J=11.9, 6.2 Hz, 1H), 1.40 (d, J=6.0 Hz, 3H);
(48) .sup.13C NMR (101 MHz, CDCl.sub.3) 165.3, 164.0, 155.4, 151.5, 150.2, 147.7, 138.7, 138.4, 138.0, 137.0, 133.4, 129.8 (2C), 128.9, 128.6, 128.4, 128.3 (3C), 128.1, 128.0, 127.8, 127.6, 127.5 (2C), 127.0, 125.2, 118.6, 114.5, 100.8, 100.5, 83.2, 78.6, 75.8, 75.7, 75.3, 74.8, 74.0, 73.5, 71.7, 70.0, 55.6, 17.9; HRMS (ESI): Calcd for C.sub.60H.sub.59O.sub.13N [M+Na]+1024.3884, found: 1024.3896.
(49) Synthesis of Disaccharide Building Block 19
(50) ##STR00043##
(51) Cu(OAc).sub.2.H.sub.2O (70 mg, 0.347 mmol) was added to a solution of 18 (0.23 g, 0.23 mmol) in CH.sub.2Cl.sub.2 (6 mL) and MeOH (3 mL). After stirring at room temperature for 1 h, reaction mixture was filtered through celite pad and the filtrate was concentrated. The crude product was dissolved in CH.sub.2Cl.sub.2 (5 mL) and to this Ac.sub.2O (1 mL), and methyl imidazole (0.2 mL) was added. After 1 h, the reaction mixture was evaporated and purified by flash chromatography using hexanes and ethyl acetate as eluent (6:1 to 5:1) to obtain the desired product 19 as colorless oil (0.193 g, 90%).
(52) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.01 (d, J=7.7 Hz, 2H), 7.51 (t, J=7.4 Hz, 1H), 7.39 (t, J=7.6 Hz, 2H), 7.33-7.01 (m, 20H), 6.89 (d, J=8.9 Hz, 2H), 6.64 (d, J=8.9 Hz, 2H), 5.42 (t, J=8.3 Hz, 1H), 4.93 (d, J=7.9 Hz, 1H), 4.71 (t, J=6.2 Hz, 2H), 4.65-4.43 (m, 7H), 4.39 (d, J=10.5 Hz, 1H), 4.15-4.07 (m, 1H), 3.83-3.67 (m, 4H), 3.65 (s, 3H), 3.63-3.41 (m, 2H), 3.19 (dq, J=12.1, 6.2 Hz, 1H), 1.87 (s, 3H), 1.23 (d, J=6.1 Hz, 3H);
(53) .sup.13C NMR (101 MHz, CDCl.sub.3) 170.2, 165.3, 155.4, 151.6, 138.7, 138.6, 138.2, 137.1, 133.4, 129.9, 129.8, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3 (2C), 128.0, 127.9, 127.8, 127.6, 127.5, 118.7, 114.5, 100.8, 100.6, 83.1, 78.7, 77.4, 76.0, 75.79, 75.76, 75.4 (2C), 75.3, 74.9, 74.0, 73.5, 71.8, 70.1, 55.7, 29.8, 21.1, 17.9; HRMS (ESI): Calcd for C.sub.56H.sub.58O.sub.13 [M+Na]+961.3775, found: 961.3841.
(54) Synthesis of Imidate Donor 7
(55) ##STR00044##
(56) Ceric ammonium nitrate (0.46 g, 0.85 mmol) was added to a solution of 19 (0.16 g, 0.17 mmol) in acetonitrile (5 mL) and H.sub.2O (1 mL). After stirring at room temperature for 1 h, Na.sub.2SO.sub.4 was added to the reaction mixture and filtered through celite pad. The filtrate was concentrated and purified by flash chromatography using hexanes and ethyl acetate as eluent (4:1) to obtain the desired hemiacetal as pale yellowish oil.
(57) The obtained hemiacetal was dissolved in CH.sub.2Cl.sub.2 (5 mL) and to this Cl.sub.3CCN (0.17 mL, 0.17 mmol), DBU (5.2 L) were added. After 30 min, hexanes (5 mL) was added to the reaction mixture and purified by flash chromatography using hexanes and ethyl acetate as eluent (5:1) to afford the desired product 7 as colorless oil (0.126 g, 76%, /=9:1).
(58) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.47 (s, 1H), 7.95 (d, J=7.8 Hz, 2H), 7.50 (t, J=7.5 Hz, 1H), 7.36 (t, J=7.7 Hz, 2H), 7.32-7.01 (m, 20H), 6.56 (d, J=3.5 Hz, 1H), 5.29 (dd, J=9.9, 3.5 Hz, 1H), 4.76 (s, 1H), 4.70-4.49 (m, 7H), 4.43 (dd, J=23.8, 11.2 Hz, 2H), 4.12 (t, J=9.3 Hz, 1H), 4.05-3.90 (m, 2H), 3.90-3.76 (m, 2H), 3.73 (dd, J=11.2, 4.8 Hz, 1H), 3.51 (t, J=9.5 Hz, 1H), 3.19 (dt, J=12.1, 6.2 Hz, 1H), 1.90 (s, 3H), 1.19 (d, J=4.9 Hz, 3H);
(59) .sup.13C NMR (101 MHz, CDCl.sub.3) 170.2, 165.5, 160.6, 138.5 (2C), 138.2, 137.2, 133.6, 129.9, 129.3, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3 (2C), 127.9, 127.8 (2C), 127.6, 127.5, 101.2, 94.1, 91.3, 79.6, 78.7, 76.0, 75.9, 75.9, 75.3, 75.0, 74.8, 73.4, 73.3, 72.9, 71.8, 68.6, 29.8, 21.2, 17.9.
Example A.3: Synthesis of Tetrasaccharide Acceptor 4
(60) Synthesis of Tetrasaccharide 20
(61) ##STR00045##
(62) To a solution of donor 7 (60 mg, 0.06 mmol), acceptor 8 (40 mg, 0.04 mmol) and 4 acid washed molecular sieves (AWMS) (100 mg) in CH.sub.2Cl.sub.2 (2 mL) at 40 C. was added TMSOTf (1.5 L, 8 mol). The reaction mixture was gradually warmed to 0 C. over 3 h. After complete consumption of donor, a drop of Et.sub.3N was added and the solvents were removed under vacuum. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (3:1 to 2:1) to afford the desired product 20 as pale yellowish oil (49 mg, 68%).
(63) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.02 (d, J=7.6 Hz, 2H), 7.82 (d, J=7.7 Hz, 2H), 7.54-7.33 (m, 6H), 7.31-7.14 (m, 36H), 6.98-6.90 (m, 2H), 6.79-6.70 (m, 2H), 5.30 (s, 1H), 5.21 (s, 1H), 5.19-5.05 (m, 3H), 4.95 (s, 1H), 4.80 (d, J=10.5 Hz, 1H), 4.76-4.62 (m, 3H), 4.61-4.45 (m, 5H), 4.43-4.37 (m, 4H), 4.35-4.21 (m, 4H), 4.12-4.09 (m, 2H), 3.80-3.69 (m, 2H), 3.67-3.57 (m, 3H), 3.55-3.37 (m, 5H), 3.30-3.03 (m, 7H), 2.63 (t, J=7.0 Hz, 2H), 2.55-2.45 (m, 2H), 2.06 (s, 3H), 1.85 (s, 3H), 1.62-1.31 (m, 6H), 1.19 (s, 3H), 1.10 (d, J=6.1 Hz, 3H), 0.91 (d, J=6.1 Hz, 3H);
(64) .sup.13C NMR (101 MHz, CDCl.sub.3) 206.9, 171.7, 170.1, 166.1, 165.0, 139.1, 138.6, 138.5, 138.2, 138.1, 138.0, 136.7, 133.3, 130.1, 129.9, 129.7, 129.6, 128.7, 128.6 (2C), 128.5 (2C), 128.4, 128.3 (2C), 128.2 (2C), 128.0, 127.9 (2C), 127.8, 127.7, 127.3, 127.2, 127.1, 126.7, 101.1, 100.9, 99.29, 97.0, 83.1, 80.4, 80.1, 78.6, 78.0, 77.4, 76.0, 75.8, 75.6, 75.3, 75.2, 74.8, 74.7, 74.1, 74.0, 73.1, 73.0, 72.4, 71.6, 69.3, 68.3, 67.8, 67.5, 67.2, 62.4, 60.5, 50.6, 50.3, 47.2, 46.2, 38.3, 29.8, 29.2, 28.3, 28.0, 27.6, 25.0, 23.4, 22.8, 22.3, 21.2, 21.1, 18.1, 17.7, 17.6; HRMS (ESI): Calcd for C.sub.107H.sub.117O.sub.25N [M+Na]+1839.7846, found: 1839.7621.
(65) Synthesis of Tetrasaccharide Acceptor 4
(66) ##STR00046##
(67) Hydrazine solution [310 L, a premixed solution of H.sub.2NNH.sub.2.H.sub.2O (50 L), pyridine (0.6 mL), AcOH (0.4 mL)] was added to a stirred solution of compound 20 (57 mg, 0.03 mmol) in CH.sub.2Cl.sub.2 (2.0 mL) and pyridine (2 mL) at 0 C. After stirring at 0 C. for 1 h, the reaction mixture was diluted with CH.sub.2Cl.sub.2 (10 mL) and washed successively with 1 M HCl (5 mL) and aq. sat. NaHCO.sub.3 (5 mL). The organic layer was dried over Na.sub.2SO.sub.4 filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (3:1 to 2.5:1) to give the desired product 4 as colorless oil (49 mg, 90%).
(68) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.98 (d, J=7.7 Hz, 2H), 7.81 (d, J=7.8 Hz, 2H), 7.52-7.32 (m, 6H), 7.31-7.13 (m, 28H), 7.12-6.94 (m, 10H), 6.77 (d, J=7.3 Hz, 2H), 5.24 (dd, J=14.9, 6.1 Hz, 2H), 5.09 (d, J=9.9 Hz, 2H), 4.90 (s, 1H), 4.81-4.67 (m, 3H), 4.65-4.38 (m, 10H), 4.36-4.21 (m, 3H), 4.20-3.96 (m, 4H), 3.85-3.63 (m, 5H), 3.60-3.39 (m, 6H), 3.35-3.07 (m, 7H), 1.86 (s, 3H), 1.56-1.38 (m, 6H), 1.23 (d, J=6.2 Hz, 3H), 1.19 (d, J=6.2 Hz, 3H), 0.85 (d, J=6.4 Hz, 3H);
(69) .sup.13C NMR (101 MHz, CDCl.sub.3) 170.2, 165.9, 165.2, 138.6, 138.5, 138.4, 138.3, 138.1, 138.0, 136.9, 133.3, 133.2, 130.2, 129.9, 129.8, 129.5, 128.8, 128.7, 128.6, 128.5 (3C) 128.4 (3C), 128.3, 128.1, 127.9 (3C), 127.8 (2C), 127.6, 127.4, 127.2, 127.1, 101.2, 100.9, 100.5, 97.1, 83.0, 82.1, 80.2, 79.2, 78.6, 78.3, 77.4, 76.0, 75.7, 75.6, 75.5, 75.3, 74.8, 74.4, 74.3, 73.6, 73.5, 72.9, 71.7, 70.1, 69.5, 68.1, 68.0, 67.6, 67.3, 50.7, 50.3, 47.2, 46.3, 21.1, 18.2, 17.9, 17.7; HRMS (ESI): Calcd for C.sub.102H.sub.111O.sub.23N [M+Na]+1741.7478, found: 1741.7240.
Example A.4: Synthesis of Disaccharide Donor 3
(70) ##STR00047##
(71) Synthesis of Glucuronic Acid Building Block 22
(72) ##STR00048##
(73) BAIB (4.34 g, 13.47 mmol) and TEMPO (0.17 g, 1.08 mmol) were added to a solution of 21 (Z. Guan et al. J. Org. Chem. 2012, 77, 8888) (3 g, 5.39 mmol) in CH.sub.2Cl.sub.2 (15 mL) and H.sub.2O (7.5 mL). The reaction mixture was stirred at room temperature for 2 h and quenched using aq. sat. Na.sub.2S.sub.2O.sub.3 solution (150 mL). The aqueous phase was extracted with EtOAc (3100 mL) and dried over Na.sub.2SO.sub.4. After concentration, the residue was purified by flash chromatography using cyclohexane and ethyl acetate as eluent (7:1 and 0.5% formic acid in eluent) to afford the acid as a white solid (2.92 g, 95%).
(74) To a stirred solution of the acid (2.92 g, 5.12 mmol) in DMF (25 mL) was added MeI (1.45 g, 10.23 mmol) and K.sub.2CO.sub.3 (1.7 g, 12.3 mmol). The solution was stirred at room temperature for 10 h and quenched by the addition of MeOH (20 mL). The reaction mixture was diluted with EtOAc (80 mL) and washed with H.sub.2O (50 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (9:1 to 6:1) to give the desired product 22 as a white solid (2.7 g, 90%).
(75) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.49-7.42 (m, 2H), 7.40-7.27 (m, 13H), 7.22 (m, 2H), 7.15-7.09 (m, 2H), 4.91-4.67 (m, 5H), 4.61 (m, 2H), 3.90 (m, 1H), 3.81 (t, J=9.3 Hz, 1H), 3.73 (s, 3H), 3.70 (t, J=8.8 Hz, 1H), 3.49 (dd, J=9.7, 8.7 Hz, 1H), 2.34 (s, 3H);
(76) .sup.13C NMR (101 MHz, CDCl.sub.3) 168.9, 138.2 (2C), 138.0, 137.8, 133.0, 129.9, 129.4, 128.6, 128.5 (3C), 128.3, 128.1, 128.0 (2C), 127.9 (2C), 127.7, 88.7, 86.0, 80.4, 79.4, 78.1, 76.0, 75.6, 75.2, 52.6, 21.3; HRMS (ESI): Calcd for C.sub.35H.sub.36O.sub.6S [M+Na]+607.2130, found: 607.2140.
(77) Synthesis of Imidate Donor 5
(78) ##STR00049##
(79) NIS (92 mg, 0.41 mmol) and TfOH (3 L, 0.34 mmol) were added at 0 C. to a solution of 22 (0.2 g, 0.34 mmol) in acetone (2 mL) and water (1 mL). After stirring at 0 C. for 4 h, the reaction mixture was quenched with Et.sub.3N (0.5 mL). Diluted the reaction mixture with CH.sub.2Cl.sub.2 (15 mL). and washed with aq. sat. Na.sub.2S.sub.2O.sub.3 (5 mL). Separated organic layer was dried over Na.sub.2SO.sub.4 filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (4:1 to 3:1) to give the hemiacetal as pale yellowish liquid (0.164 g).
(80) DBU (5 L, 0.034 mmol) and Cl.sub.3CCN (0.34 mL, 3.42 mmol) were added to a cooled solution of hemiacetal (0.164 g, 0.342 mmol) in CH.sub.2Cl.sub.2 (2 mL) 0 C. After stirring at 0 C. for 1 h, the reaction mixture was evaporated on rotor and the crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (5:1 to 4:1) to obtain the product 5 as colorless oil (0.183 g, 86%, /=2.7/1).
(81) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.60 (s, 1H), 7.33-7.08 (m, 15H), 6.45 (d, J=3.5 Hz, 1H), 5.00-4.50 (m, 5H), 4.36 (d, J=10.1 Hz, 1H), 4.11-3.95 (m, 1H), 3.82-3.66 (m, 3H), 3.65 (s, 3H);
(82) .sup.13C NMR (101 MHz, CDCl.sub.3) 169.3, 161.1, 138.4, 138.0, 137.8, 137.7, 129.2, 128.6, 128.5 (3C), 128.4, 128.3, 128.2, 128.1, 128.0 (2C), 127.9, 127.8 (2C), 125.4, 94.0, 91.1, 83.7, 80.8, 78.9, 78.8, 75.9, 75.7, 75.5, 75.2, 73.2, 72.6, 52.7; HRMS (ESI): Calcd for C.sub.30H.sub.30O.sub.7NCl.sub.3 [M+Na].sup.+644.0986, found: 644.1014.
(83) Synthesis of Disaccharide 3
(84) ##STR00050##
(85) TMSOTf (4 L, 0.02 mol) was added to a solution of donor 5 (0.14 g, 0.22 mmol), and acceptor 21 (90 mg, 0.16 mmol) in a mixture of solvents toluene (2 mL) and dioxane (6 mL) at 20 C. The reaction mixture was gradually warmed to 0 C. over 2 h. A drop of Et.sub.3N was added and the solvents were removed under vacuum. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (7:1 to 5:1) to afford the desired product 3 as pale yellowish oil (0.12 g, 73%, /=3.5:1).
(86) .sup.1H NMR (400 MHz, CDCl.sub.3, -anomer) 7.43 (d, J=7.8 Hz, 2H), 7.40-7.18 (m, 30H), 7.06 (d, J=7.8 Hz, 2H), 5.08 (d, J=3.5 Hz, 1H), 4.94 (d, J=10.9 Hz, 1H), 4.88-4.60 (m, 9H), 4.58-4.47 (m, 3H), 4.29 (d, J=10.0 Hz, 1H), 3.95 (t, J=9.3 Hz, 1H), 3.86 (dd, J=12.1, 4.3 Hz, 1H), 3.82-3.69 (m, 3H), 3.67 (d, J=4.9 Hz, 3H), 3.64-3.54 (m, 2H), 3.39 (dd, J=9.7, 3.9 Hz, 1H), 3.17 (t, J=9.3 Hz, 1H), 2.21 (s, 3H);
(87) .sup.13C NMR (101 MHz, CDCl.sub.3) 170.4, 138.6, 138.3, 138.2, 138.1 (2C), 138.0, 133.2, 129.9, 128.6 (2C), 128.5 (3C), 128.4 (3C), 128.3, 128.1 (2C), 128.0, 127.9, 127.8, 127.7, 127.6 (2C), 98.0, 88.6, 86.7, 81.1, 81.0, 79.8, 79.6, 78.9, 77.3, 75.9, 75.7, 75.6, 75.2, 75.1, 72.6, 70.4, 66.5, 52.5, 21.2; HRMS (ESI): Calcd for C.sub.62H.sub.64O.sub.11S [M+Na]+1039.4067, found: 1039.4091.
Example A.5: Synthesis of Hexasaccharide 2
(88) ##STR00051##
(89) Synthesis of Hexasaccharide 23
(90) ##STR00052##
(91) NIS (12 mg, 0.05 mmol) and TfOH (1 L) were added at 30 C. to a cooled solution of donor 3 (52 mg, 0.05 mmol), acceptor 4 (45 mg, 0.026 mmol) and 4 acid washed molecular sieves (AWMS) (0.2 g) in mixture of CH.sub.2Cl.sub.2 (1 mL) and dioxane (1 mL).
(92) Reaction mixture was gradually warmed to 10 C. over 1 h, diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with aq. sat. Na.sub.2S.sub.2O.sub.3 (5 mL). Separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by flash chromatography using hexanes and ethyl acetate as eluent (4:1 to 3:1) to obtain the desired product 23 as colorless oil (45 mg, 66%).
(93) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.05 (d, J=7.7 Hz, 2H), 7.78 (d, J=7.8 Hz, 2H), 7.50-7.32 (m, 8H), 7.30-7.01 (m, 63H), 6.98-6.89 (m, 3H), 6.86-6.83 (m, 2H), 5.25 (s, 1H), 5.19-5.12 (m, 2H), 5.07 (d, J=4.5 Hz, 2H), 4.90-4.45 (m, 20H), 4.42-4.08 (m, 12H), 4.06-3.52 (m, 16H), 3.50 (d, J=4.1 Hz, 3H), 3.48-2.95 (m, 13H), 1.87 (s, 3H), 1.58-1.36 (m, 6H), 1.14 (d, J=6.2 Hz, 3H), 1.07 (d, J=6.1 Hz, 3H), 0.88 (d, J=6.2 Hz, 3H);
(94) .sup.13C NMR (101 MHz, CDCl.sub.3) 170.6, 170.2, 166.2, 164.6, 139.4, 139.3, 138.9 (2C), 138.8, 138.7, 138.5, 138.3, 138.2, 137.0, 133.3, 133.0, 130.2, 130.0, 129.9, 128.8, 128.6 (2C), 128.5 (3C), 128.4 (3C), 128.3, 128.2 (3C), 128.1, 128.0, 127.9 (3C), 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 126.6, 101.4, 100.7, 99.3, 98.1, 97.0, 95.9, 83.2, 81.7, 81.0, 80.7, 80.4, 80.2, 79.8, 79.6, 78.7, 77.5, 77.4, 77.3, 77.2, 76.8, 76.1, 75.7, 75.2, 75.1, 75.0, 74.7, 73.8, 73.7, 73.3, 73.1, 72.7, 71.8, 71.5, 71.3, 70.4, 67.6, 67.3, 66.9, 58.6, 53.6, 52.3, 31.1, 29.8, 21.1, 18.6, 18.1, 17.8, 17.6; HRMS (ESI): Calcd for C.sub.157H.sub.167O.sub.34N [M+Na].sup.+ 2634.1301, found: 2634.0912.
Synthesis of 5-amino pentyl -L-rhamnopyranosyl-(14)--D-glucopyranosyl-(13)--L-{-D-glucopyranosyl uronate-(16)--D-glucopyranosyl-(12)}rhamnopyranosyl-(13)--L-rhamnopyranoside (2)
(95) ##STR00053##
(96) To a stirred solution of hexasaccharide 23 (6 mg, 2.3 mol) in THF (0.5 mL) and MeOH (0.5 mL), was added aq. NaOH (15%, 100 L). After stirring at room temperature for 1 h, NaOMe (6 mg) was added and allowed to stir for 12 h. After complete consumption of starting material, the reaction mixture was neutralized with Amberlite 120 H+resin, filtered, and concentrated. The crude material was purified by flash column chromatography using hexanes and ethyl acetate as eluent (1:1 to 1:2) to afford the desired deacylated product as white solid. The obtained deacylated product was dissolved in CH.sub.2Cl.sub.2 (0.5 mL), .sup.tBuOH (1 mL) and water (0.5 mL). To this solution a suspension of Pd/C (50 mg) in a mixture of .sup.tBuOH (1 mL) and water (0.5 mL) was added and stirred under hydrogen atmosphere for 36 h. Reaction mixture was then filtered, concentrated and purified by C18 column to afford the desired product 2 (0.7 mg, 30%) as a white solid.
(97) .sup.1H NMR (400 MHz, D.sub.2O) 5.07 (s, 1H), 5.02-4.94 (m, 2H), 4.91 (d, J=3.7 Hz, 1H), 4.73 (d, J=1.5 Hz, 1H), 4.63 (d, J=7.9 Hz, 1H), 4.35-4.18 (m, 3H), 4.05 (m, 4H), 3.96-3.81 (m, 3H), 3.80-3.61 (m, 8H), 3.61-3.40 (m, 8H), 3.38-3.25 (m, 3H), 3.24-3.13 (m, 1H), 2.96 (t, J=7.6 Hz, 2H), 1.66 (dt, J=15.9, 8.0 Hz, 4H), 1.43 (p, J=7.8, 7.3 Hz, 2H), 1.33-1.19 (m, 9H);
(98) HRMS (ESI): Calcd for C.sub.41H.sub.71O.sub.29N [M+Na].sup.+ 1064.4009, found: 1064.4067.
(99) Applying the synthetic procedures of example A.5 to disaccharides 15a, 15b, 15c and 15d provides hexasaccharides 2a, 2b, 2c and 2d, respectively.
(100) ##STR00054## ##STR00055##
Example A.6: Synthesis and Characterization of Conjugates
(101) General Procedure Synthesis
(102) Formation of the p-Nitro Phenyl (PNP) Amide
(103) To the saccharide of general formula (I) (1 equivalent) and diphenyl adipate (7 equivalents) in a glass vial were added a mixture of pyridine and DMSO (1:1) and the mixture let stir for 5 minutes for complete solubilization. Then, triethylamine (0.83 L, 6 mol, 10 equivalents) was added and let stir for 20 minutes. TLC indicated complete consumption of the starting material. The solvent was removed in vacuum. The residue was washed with dichloromethane (31 mL) to remove PNP ester excess and the white solid obtained was dried in vacuum.
(104) Conjugation of PNP Ester Derivatized Saccharide to CRM.sub.197
(105) 40 equivalents of lyophilized CRM.sub.197 was dissolved in 0.4 mL of sterile 0.1 M sodium phosphate, pH 8.0 and transfer into upper chamber of 10,000 Da Millipore centrifugal filter (0.5 mL). Rinse glass vial with 30.4 mL of sterile 0.1 M sodium phosphate, pH 8.0, transfer to the same centrifugal filter. Centrifuge at 10,000 rpm for 6-8 min. If needed, prolong final centrifugation step such that volume in upper chamber is 80-100 L. The CRM.sub.197 solution was then transfer into 1.5 mL tube containing lyophilized PNP ester derivatized saccharide and shake slowly (around 180-200 rpm) for 18-24 hrs at room temperature. The conjugate was washed once with 0.1M Sodium phosphate, pH 8.0 and 2-3 times with deionized, autoclaved water using 10,000 Da Millipore centrifugal filters. Take out small sample for MALDI analysis and transfer the conjugate into PBS. If needed, prolong final centrifugation step such that volume in upper chamber is about 250 L. Transfer content of upper chamber to new 1.5 mL Eppendorf tube, store at 4 C.
(106) Characterization of Glycoconjugates
(107) A. MALDI analysis: The average molecular size of conjugates were determined by Matrix-assisted laser desorption/ionization (MALDI) analysis using CRM.sub.197 as standard and calculate the average oligosaccharides attachments with per CRM.sub.197 molecule.
(108) B. SDS-PAGE: The conjugates were resolved by SDS-PAGE (10%) in denaturing condition. The samples were prepared in 6SDS-PAGE sample loading dye. The electrophoresis was carried out at 120 V and 25 mA for 1 hr 30 min in electrode buffer and gel was stained with Coomassie brilliant blue R250.
(109) Protein Estimation
(110) The protein concentration was estimated using Micro BCA Protein Assay Kit (Thermo-scientific, USA) following the manufacturer's instructions. The sample was prepared in PBS and mixed with equal volume of reagent mixture (B:C:A:24:1:25). The plate was incubated at 37 C. and the absorbance was measured at 560 nm. The standard curve was plotted with known concentration of BSA provided with the kit.
(111) Synthesis of Conjugate CRM.sub.197-Hexasaccharide 2
(112) Following the above-described procedure, conjugate CRM.sub.197-hexasaccharide 2 was synthesized. The conjugate was estimated using known amount of BSA as slandered and confirmed by 10% SDS-PAGE, showing a shift toward a higher mass of the glycoconjugates compared with unconjugated CRM.sub.197 (
(113) ##STR00056##
B. Biological Evaluation
Example B.1: Mice Immunization and Generation of Polyclonal Sera
(114) Material and Methods
(115) Mice: Six to eight week old female C57BL/6J inbred strains of mice were obtained from the Charles River, Sulzfeld (Germany). Animals were rested and handled in accordance with the Institutional Animal Ethics guidelines.
(116) Mice Immunization and Generation of Polyclonal Sera
(117) In brief, groups of 3 C57BL/6J female 6-8 week old inbred mice were immunized subcutaneously with conjugate CRM.sub.197-hexasaccharide 2 (3 g sugar per dose) emulsified with 1:1 (v/v) alum (aluminium hydroxide) adjuvants. On day 14 and 28 mice were received a booster injection with the same amount of antigen emulsified with 1:1 (v/v) alum. A group of mice were also immunized with conjugate CRM.sub.197-hexasaccharide 2 (3 g sugar per dose) only to check the immunogenicity. Mice were bled submandibular weekly using sterile single-use blood lancet. Control mice received only PBS and PBS in alum. The antibody responses were measured in both sera by glycan microarray and ELISA.
(118) Preparation of microarrays slides: The CodeLink NHS activated glass slides (Surmodics) were spotted with synthetic glycans and native polysaccharides at two different concentration (100 M and 200 M) in printing buffer (50 mM sodium phosphate, pH 8.5) by using a S3 piezoelectric microarray printer (Scienion) equipped with a type 4 coated nozzle. The relative humidity of spotted chamber was constantly maintained at 65%. The spotted slides were incubated over night at room temperature in a humidifying chamber. The unreactive groups on the slides were blocked with 50 mM sodium phosphate, 100 mM ethanolamine pH 9.0 at room temperature for one hour. Slides were subsequently washed three times for 5 min with water, dried by centrifugation at 300 g for 5 min (CombiSlide system, Eppendorf) and stored at 4 C. until use.
(119) Microarray binding assays: The printed slides were blocked with PBS-BSA (1%) for 1 h at room temperature and washed 3 times with PBS. The slides were dried by centrifugation at 1200 rpm for 5 min before use. A FlexWell 64 (Grace Bio-Labs, Bend, Oreg., USA) grid was applied to microarray slides. Slides were incubated with polyclonal sera raised in mice against conjugate CRM.sub.197-hexasaccharide 2 at multiple dilutions, diluted in 1% BSA in PBS (w/v) and incubated in a humid chamber for 1 h at room temperature. Slides were washed three times with PBST (0.1% Tween-20 in PBS) and dried by centrifugation (300g, 5 min). Slides were incubated with a fluorescence-labeled goat anti-mouse secondary antibodies (Life Technologies) diluted in 1% BSA in PBS (w/v) in a humid chamber for 1 h at room temperature, washed three times with PBST, rinsed once with deionized water and dried by centrifugation (300g, 5 min) prior to scanning with a GenePix 4300A microarray scanner (Molecular Devices, Sunnyvale, Calif., USA). Image analysis was carried out with the GenePix Pro 7 software (Molecular Devices). The photomultiplier tube (PMT) voltage was adjusted such that scans were free of saturation signals.
(120) Results:
(121) To analyze the antibody response against conjugate CRM.sub.197-hexasaccharide 2, hyperimmune sera raised in mice immunized with conjugate CRM.sub.197-hexasaccharide 2 was subjected at different dilution to microarray slides printed with synthetic oligosaccharides and polysaccharides. The microarray data confirmed that the conjugate CRM.sub.197-hexasaccharide 2 is immunogenic in mice and exhibits robust antibody response as shown by the analysis performed every week before and after immunization (
Example B.2: Evaluation of Cross-Reactivity of Antibodies
(122) ELISA: The cross-reactivity of antibodies raised in mice immunized with conjugate CRM.sub.197-hexasaccharide 2 was analyzed with capsular polysaccharide (CPS) of S. pneumoniae serotype 2 by ELISA. Ninety six well polystyrene microtiter plate (Corning, N.Y.) was coated overnight at 4 C. with CPS (50 l of 10 g/ml per well) in phosphate buffer saline, pH 7.4. The plates were washed thrice with PBS containing 0.1% Tween-20 (PBST) and blocked with PBS containing 2% BSA at room temperature for 1 hr. After washing thrice with PBST, the plate was incubated with pooled sera in two fold dilutions starting from 1 in 500 dilutions room temperature for 1 hr. The plate was washed 4 to 5 times with PBST and further incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse Ig antibody (diluted 1 in 10000 in PBS containing 0.5% BSA) followed by incubation at room temperature for 1 hr. The plate was washed thoroughly with PBST and developed using 1-Step Ultra TMB (ThermoFisher Sci. USA). The reaction was stopped by adding 2% H.sub.2SO.sub.4 and absorbance was recorded at 450 nm.
(123) Results:
(124) The ELISA data suggested that conjugate CRM.sub.197-hexasaccharide 2 with alum induced high titer of CPS specific antibodies, in contrast to mice vaccinated with conjugate CRM.sub.197-hexasaccharide 2 only or immunized with PBS (PBS+alum or PBS only) (see
Example B.3: Opsonophagocytic Killing Assay
(125) S. pneumoniae serotype 2 strain 39 (NCTC 7466) was pre-opsonized with hyperimmune sera 15 min at 37 C. and incubated with differentiated HL-60 cells (DSMZ no.:ACC 3) in 1:400 ratio (bacteria: HL-60 cells). Baby rabbit complement (Cedarlane, Canada; cat # CL3441-S) was used as complement source. The whole mixture was incubated for 45 min at 37 C. with shaking. The phagocytic activity was stopped by keeping the mixture on ice for 20 min and the survival was assessed by plating on Columbia agar with 5% sheep blood plates. Percent killing of pneumococci was calculated based on viable pneumococcal colonies obtained relative to control sera.
(126) The results suggest that anti-conjugate CRM.sub.197-hexasaccharide 2 antibodies exhibited very high bactericidal activity compared to the control groups (
Example B.4: Vaccination with Conjugate CRM.SUB.197.-Hexasaccharide 2 Provides Full Protection in Mice Against Intranasal Challenge with S. pneumoniae
(127) Seven week old female C57BL/6J mice (n=11) were immunized subcutaneously with conjugate CRM.sub.197-hexasaccharide 2 (2.2 g sugar per dose) with or without aluminium hydroxide (125 mg Al) in PBS on day 0, 14 and 28. Two control groups (n=11) received only PBS and PBS plus aluminium hydroxide (125 mg Al). Another shame group (n=11) was injected PBS only to minus the background. One week after the second booster, mice were challenged intranasally with 110.sup.7 cfu of type strain D39 per mouse. The shame group received only PBS intranasally. Animals were monitored every 12 hr. Thirty six hours post challenge mice were anesthetized with Ketamine/Xylazine and euthanized in sterile conditions. The bacterial load was analyzed in lung and blood. Flushed lungs were homogenized and single cell suspension was prepared with the help of syringe. The pneumococci from alveoli and blood were enumerated by plating on blood agar plates.
(128) The colony forming unit data (CFU) suggested that mice vaccinated with conjugate CRM.sub.197-hexasaccharide 2 reduced significantly the number of CFU against intranasal challenge compared to the PBS injected mice (see