VACCINES AGAINST STREPTOCOCCUS PNEUMONIAE SEROTYPE 8
20170239341 · 2017-08-24
Inventors
- Peter H. Seeberger (Kleinmachnow, DE)
- Claney Lebev Pereira (Berlin, DE)
- Chakkumkal Anish (Den Haag, NL)
- Benjamin Schumann (Berlin, DE)
- Sharavathi Guddehalli Parameswarappa (Berlin, DE)
- Heung Sik Hahm (Charlottesville, VA)
- Subramanian Govindan (Chennai, Tamil Nadu, IN)
Cpc classification
A61K47/6415
HUMAN NECESSITIES
C07H15/04
CHEMISTRY; METALLURGY
C07K16/1271
CHEMISTRY; METALLURGY
A61K47/646
HUMAN NECESSITIES
A61K31/7028
HUMAN NECESSITIES
C07H15/18
CHEMISTRY; METALLURGY
A61K2039/6037
HUMAN NECESSITIES
International classification
A61K39/09
HUMAN NECESSITIES
Abstract
The present invention relates to synthetic saccharides of general formula (I) that are related to Streptococcus pneumoniae serotype 8 capsular polysaccharide, conjugates thereof and the use of said saccharides and conjugates for raising a protective immune response in a human and/or animal host. Furthermore, the synthetic saccharide structures of general formula (I) are useful as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae 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; ##STR00123## V*— represents H—, H—U.sub.x— or H—U.sub.x+1—U.sub.x—; R.sup.# represents —COOH or —CH.sub.2OH; L represents a linker; or a pharmaceutically acceptable salt thereof.
2. The saccharide according to claim 1, wherein R.sup.# represents —COOH.
3. The saccharide according to claim 1 or 2, wherein V*— represents H—U.sub.x+1—U.sub.x—.
4. The saccharide according to claim 1 or 2, wherein V*— represents H—U.sub.x—.
5. The saccharide according to claim 1 or 2, wherein V*— represents H—.
6. The saccharide according to any one of the claims 1-5, wherein x represents 3.
7. The saccharide according to claim 1 selected from the group consisting of: α-
8. A conjugate comprising a saccharide of general formula (I) according to any of the claims 1-7 covalently linked to an immunogenic carrier through the nitrogen atom of the —O-L-NH.sub.2 group.
9. The conjugate according to claim 7 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 2 and 18; —W— is selected from: ##STR00124## 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 any one of the claims 1-7.
10. A saccharide according to any one of the claims 1-7 or a conjugate according to claim 8 or 9 for use in raising a protective immune response in a human and/or animal host.
11. A saccharide according to any one of the claims 1-7 or a conjugate according to claim 8 or 9 for use in the prevention and/or treatment of a disease associated with bacteria containing in their capsular polysaccharide one of the following saccharide fragments: α-D-Glcp-(1.fwdarw.4)-α-D-Galp-(1.fwdarw.4)-β-D-GlcAp-(1.fwdarw.4)-β-D-Glcp, β-D-Glcp-(1.fwdarw.4)-α-D-Glcp-(1.fwdarw.4)-α-D-Galp-(1.fwdarw.4)-β-D-GlcAp, β-D-GlcAp-(1.fwdarw.4)-β-D-Glcp-(1.fwdarw.4)-α-D-Glcp-(1.fwdarw.4)-α-D-Galp, α-D-Galp-(1.fwdarw.4)-β-D-GlcAp-(1.fwdarw.4)-β-D-Glcp-(1.fwdarw.4)-α-D-Glcp.
12. The saccharide for use or the conjugate for use according to claim 11, wherein the bacteria is Streptococcus pneumoniae serotype 8.
13. The saccharide for use or the conjugate for use according to claim 11, wherein the disease associated with bacteria is selected from the group comprising: pneumonia, meningitis, otitis media, bacteremia and acute exacerbation of chronic bronchitis, sinusitis, arthritis and conjunctivitis.
14. A vaccine comprising the saccharide according to any of the claims 1-7 and/or the conjugate according to claim 8 or 9 together with at least one pharmaceutical acceptable adjuvant, cryoprotectant, lyoprotectant, excipient and/or diluent.
15. A saccharide according to any of the claims 1-7 for use as marker in immunological assays for detection of antibodies against Streptococcus pneumoniae serotype 8.
Description
DESCRIPTION OF THE FIGURES
[0656]
[0657]
[0658]
[0659]
[0660]
[0661]
[0662]
[0663]
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[0665]
[0666]
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[0671]
[0678] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples, which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments, which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
[0679] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
EXAMPLES
[0680] 1. Chemical Synthetic Experiments
[0681] General information for chemical synthesis.
[0682] All commercially available starting materials and reagents were used as received unless otherwise noted. All the reactions were performed under argon atmosphere. Solvents were dried. High-resolution mass spectra (HRMS) were recorded with an Agilent 6210 ESI-TOF mass spectrometer at the Freie Universitat Berlin.
[0683] Abbreviations
[0684] In the following schemes occurring abbreviations mean Ac (acetyl), BAIB ([bis(acetoxy)iodo]benzene), Bn (benzyl), t-Bu (tert-butyl), Bz (benzoyl), Cbz (carboxybenzyl), DCM (dichloromethane), DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone), Im (imidazole), NAP (2-naphthylmethyl), NIS (N-iodosuccinimide), Py/Pyr (pyridine), TEMPO ((2,2,6,6-tetramethyl-1-piperdinyl)oxy), Tf (trifluoromethanesulfonyl), THF (tetrahydrofurane), TMS(trimethylsilyl), TMSOTf (trimethylsilyl trifluoromethane sulfonate), p-Ts (para-tolylsulfonyl).
Synthesis of saccharide by f2+21 glycosylation approach
[0685] ##STR00048##
Example 1-1
Synthesis of Compound 3
[0686] ##STR00049##
[0687] Thioglycoside donor substrate 1 (6.0 g, 11.53 mmol) and acceptor with C-2 linker 2 (dried azeotropically using toluene in rotary evaporator, 3.93 g, 13.83 mmol) were taken in dry DCM (100 mL) and added 5 g of MW dried 4 Å MS to it and stirred at rt for 15 min and then cooled to —10° C. NIS (3.83 g, 17.29 mmol) and TfOH (0.15 mL, 1.73 mmol) were then added to RM (reaction mixture) and stirred at −10° C. to −5° C. for 1 hr. Reaction completion was monitored by TLC. RM was then quenched with 10% aq. Na.sub.2S.sub.2O.sub.3 solution (50 mL) and then extracted with EtOAc (25 ml×3). Combined organic layer was then washed with brine (10 ml), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum to get pale yellow oily compound. Crude product was purified on silica gel column chromatography using 20-30% EtOAc in hexanes to get spot which on evaporation yielded desired product 3 as pale yellow colored transparent gummy liquid (7.60 g, 89%).
[0688] .sup.1H NMR (400 MHz, CDCl.sub.3) δ =7.97 (dd, J=8.4, 1.2 Hz, 4H), 7.59-6.90 (m, 21H), 5.91-5.71 (m, 1H), 5.62-5.41 (m, 2H), 5.22-4.95 (m, 2H), 4.80 (d, J=7.7 Hz, 0.5H), 4.67 (d, J=7.7 Hz, 0.5H), 4.56-4.22 (m, 3H), 4.10-3.52 (m, 5H), 3.50-3.33 (m, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ =165.7, 165.4, 156.35, 156.2, 137.9, 136.9, 133.4, 133.2, 129.9, 129.5, 129.3, 129.1, 128.7, 128.5, 128.4, 128.3, 128.1, 127.8, 127.4, 127.2, 126.2, 101.9, 101.6, 78.9, 72.6, 72.1, 69.1, 68.7, 67.4, 67.2, 66.7, 51.7, 46.9, 45.8.
Example 1-2
Synthesis of Compound 4
[0689] ##STR00050##
[0690] Substrate 3 (7.50 g, 10.08 mmol) was taken in DCM (75 mL) under argon with activated 3 Å MS for 10 min before cooling to 0° C. Added triethylsilane (12.88 mL, 81.0 mmol) followed by TFA (4.66 mL, 60.5 mmol) drop wise and stirred the RM at rt for 16 h before quenching with water (100 mL). Extracted the aqueous with DCM (30 mL×3), combined organics were washed thoroughly with water (20 mL×3), brine (20 mL), dried over anhyd. Na.sub.2SO.sub.4, filtered, evaporated in vacuum to get colorless gummy solid. Crude product was purified by silica column chromatography using 30%-100% EtOAc in hexanes to get product 4 and evaporated in vacuum to get colorless oil (6.1 g, 81%).
[0691] .sup.1H NMR (400 MHz, CDCl.sub.3) δ =8.04-7.84 (m, 4H), 7.60-6.87 (m, 21H), 5.55-5.36 (m, 2H), 5.22-4.90 (m, 2H), 4.77-4.53 (m, 3H), 4.51-4.30 (m, 2H), 4.06-3.93 (m, 2H), 3.87-3.53 (m, 4H), 3.46-3.20 (m, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ =167.3, 165.5, 138.0, 137.7, 133.6, 130.1, 129.9, 128.6, 128.5, 128.1, 127.9, 127.8, 127.4, 101.3, 101.2, 76.7, 74.7, 73.9, 71.6, 71.5, 71.2, 70.0, 69.0, 67.4, 67.2, 51.7, 46.8, 45.8.
Example 1-3
Synthesis of Compound 5
[0692] ##STR00051##
[0693] Acceptor 4 (2.0 g, 2.68 mmol) was taken in DCM (30 mL) with activated 4 Å AWMS and stirred at rt for 30 min before cooling to 0° C. TMSOTf (0.49 μL, 0.27 mmol) was then added followed by the imidate donor 6 (Carbohydrate Res 2008, 344, 439-447.) (2.20 g, 3.89 mmol) in DCM (5 mL) over 5 min and the reaction mixture was stirred for 30 min at 0° C. Quenched the RM with Et.sub.3N (1mL), filtered and the solvents removed under vacuum. Crude product was purified by flash chromatography using EtOAc in hexanes to get product 5 (3.2 g, 98%).
[0694] .sup.1H NMR (400 MHz, CDCl.sub.3) δ =8.13-6.88 (m, 35H), 5.67-5.52 (m, 1H), 5.46-5.31 (m, 1H), 5.20 (s, 1H), 5.16-4.89 (m, 3H), 4.68 (t, J=11.2 Hz, 1H), 4.55 (d, J=8.1 Hz, 1.5H), 4.47-4.24 (m, 3.5H), 4.20-3.89 (m, 1.5H), 3.89-3.19 (m, 9.5H), 3.13 (td, J=9.7, 4.9 Hz, 1H), 2.63 (t, J=10.2 Hz, 1H), 0.63 (s, 9H), -0.12 (s, 3H), −0.19 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ =165.4, 165.36, 164.7, 138.2, 137.1, 133.4, 133.2, 129.94, 129.9, 129.2, 128.7, 128.6, 128.5, 128.4, 128.2, 128.0, 127.8, 127.3, 126.4, 101.7, 101.2, 101.1, 81.2, 75.5, 75.1, 74.6, 73.7, 73.4, 73.0, 68.9, 68.0, 67.3, 66.1, 51.7, 46.9, 25.6, 18.0, −4.1, −4.8.
Example 1-4
Synthesis of Compound 7
[0695] ##STR00052##
[0696] Substrate 5 (1.6 g, 1.317 mmol) was taken in pyridine (10 mL) at 0° C. and added HF-pyridine (3.56 mL, 39.5 mmol) to it and stirred at rt for 24 h. RM was washed with water and extracted with DCM (20 mL×3). Combined organics were then washed with dil. HCl (50 mL×2), sat. NaHCO.sub.3 solution (50 mL), brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum to get crude product which on purification using silica column chromatography using 35-40% EtOAc in hexanes yielded white colored foam 7 (1.3 g, 90%).
[0697] .sup.1H NMR (400 MHz, CDCl.sub.3) δ =8.15-6.92 (m, 1H), 5.65-5.51 (m, 1H), 5.44-5.30 (m, 1H), 5.23 (s, 1H), 5.11-5.04 (m, 3H), 4.77-4.49 (m, 3H), 4.49-4.24 (m, 4H), 4.25 -3.91 (m, 2H), 3.91-3.59 (m, 4H), 3.57-3.00 (m, 7H), 2.68 (t, J=10.3 Hz,1H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ =165.4, 165.3, 156.4, 156.2, 138.2, 136.9, 133.6, 133.2, 130.3, 130.0, 129.9, 129.4, 128.7, 128.7, 128.5, 128.5, 128.4, 128.1, 128.1, 127.8, 127.4, 126.4, 101.8, 101.2, 101.1, 80.6, 75.9, 74.9, 74.7, 73.7, 73.5, 72.6, 72.0, 71.9, 68.9, 67.9, 67.4, 67.2, 66.0, 51.7, 46.9, 45.9.
Example 1-5
Synthesis of Compound 8
[0698] ##STR00053##
[0699] A mixture of (2S,3R,4S,5R,6R)-6-((benzyloxy)methyl)-2-(ethylthio)-5-hydroxytetrahydro-2H-pyran-3,4-diyl dibenzoate 9 (J Carbohydrate Chemistry 1993, 12, 309) (4.00 g, 7.654 mmol, 1.0 eq.) and (4aR,6R,7R,8aR)-8-((tert-butyldimethylsilyl)oxy)-2-phenyl-6-(2,2,2-trichloro-1-iminoethoxy)hexahydropyrano [3,2-d][1,3]dioxin-7-yl benzoate 11 (Carbohydrate Res, 2008, 344, 439.) (6.28 g, 9.95 mmol, 1.3 eq.) in DCM (140 mL) was stirred under an atmosphere of argon for 30 min. The reaction mixture was cooled (—20° C.) and TMSOTf (0.16 mL, 0.880 mmol, 0.115 eq.) was added. After stirring for 45 min, the reaction mixture was quenched by the addition of Et.sub.3N (1.0 mL). The organic solution was concentrated under vacuo.
[0700] The resulting dark yellow oil was purified by flash chromatography over silica gel (EtOAc/hexanes, 1/3, v/v) to give (2S,3R,5R,6R)-5-(((4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-8-((tert-butyldimethylsilyl)oxy)-2-phenylhexahydro pyranophenylhexahydropyrano[3,2-d][1,3]d ioxin-6-yl)oxy)-6-((benzyloxy)methyl)-2-(ethylthio)tetrahydro-2H-pyran-3,4-diyl dibenzoate 8 (6 g, 79%) as a colorless solid: R.sub.f=0.5 (EtOAc/hexanes, 3/7, v/v). .sup.1H NMR (400 MHz, CDCl.sub.3) 6=-0.19 (s, 3H), −0.11 (s, 3H), 0.63 (s, 9H), 1.20 (t, J=7.4 Hz, 3H), 2.67 (m, 2H), 3.15 (td, J=9.7 Hz, 4.9 Hz, 2H), 3.28 (t, J=9.2 Hz, 1H), 3.53-3.37 (m, 1H), 3.74-3.55 (m, 1H), 3.79 (t, J=9.0 Hz, 1H), 4.19 (t, J=9.5 Hz, 1H), 4.37 (d, J=12.2 Hz, 1H), 4.57 (d, J=10.0 Hz, 1H), 4.59 (dd, J=15.1, 9.0 Hz, 2H), 4.67 (d, J=12.2 Hz, 1H), 5.12 (dd, J=8.9, 8.2 Hz, 1H), 5.21 (s, 1H), 5.41 (t, J=9.8 Hz, 1H), 5.63 (t, J=9.3 Hz, 1H), 7.29-7.72 (m, 19H), 7.88-8.03 (m, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ =165.27, 165.07, 164.45, 138.16, 137.00, 133.14, 133.10, 132.97, 130.21, 129.79, 129.77, 129.75, 129.34, 128.99, 128.51, 128.38, 128.28, 128.22, 128.05, 128.02, 127.99, 126.21, 101.57, 101.06, 83.39, 81.05, 78.70, 77.43, 77.11, 76.80, 75.41, 74.93, 74.52, 73.49, 72.90, 70.59, 67.86, 67.45, 65.97, 25.43, 24.08, 17.80, 14.85, −4.20, −4.97.
Example 1-6
Synthesis of Compound 12
[0701] ##STR00054##
[0702] TBS substrate 8 (2.0g, 2.018 mmol, 1 equiv.) was taken in pyridine (10 mL) at 0° C. and added 70% HF-pyridine (5.45 mL, 60.5 mmo, 30 equiv.) to it and stirred at rt for 36 h.
[0703] RM was washed with water (50 mL) and extracted with DCM (50 mL×3). Combined organics were then washed with sat. NaHCO.sub.3 solution (50 mL), brine (20 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum to get crude product which on purification using silica column chromatography using 35-40% EtOAc/Hexanes to yield white colored foam 13 (1.7g, 96%).
[0704] .sup.1H NMR (400 MHz, CDCl.sub.3) δ =8.08-7.85 (m, 6H), 7.69-7.28 (m, 19H), 5.62 (t, J=9.3 Hz, 1H), 5.41 (t, J=9.8 Hz, 1H), 5.22 (s, 1H), 5.08 (dd, J=9.2, 7.9 Hz, 1H), 4.70 (d, J=7.8 Hz, 1H), 4.63 (d, J=12.1 Hz, 1H), 4.58 (d, J=10.0 Hz, 1H), 4.40 (d, J=12.1 Hz, 1H), 4.19 (t, J=9.5 Hz, 1H), 3.82 (td, J=9.2, 3.6 Hz, 1H), 3.70 (dd, J=11.2, 3.4 Hz, 1H), 3.63 (dd, J=10.6, 5.0 Hz, 1H), 3.59-3.55 (m, 1H), 3.54-3.48 (m, 1H), 3.32 (t, J=9.3 Hz, 1H), 3.15 (td, J=9.7, 5.0 Hz, 1H), 2.78-2.60 (m, 3H), 2.50 (d, J=3.6 Hz, 1H), 1.22 (t, J=7.5 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ =165.38, 165.29, 165.13, 138.02, 136.70, 133.43, 133.14, 133.09, 130.08, 129.87, 129.81, 129.71, 129.30, 129.28, 129.23, 128.50, 128.48, 128.29, 127.95, 127.86, 126.19, 101.59, 100.88, 83.42, 80.42, 78.71, 75.71, 74.67, 74.51, 73.46, 72.42, 70.48, 67.70, 67.51, 65.76, 24.11, 14.85.
[0705] Substrate 13 (1.6g, 1.824 mmol, 1 equiv.) was taken in anhydrous DCM (10 mL) at 0° C. and added pyridine (10 mL) and BzCl (0.635 mL, 5.47 mmol, 3 equiv.) to it dropwise and RM was stirred for 16 h.
[0706] RM was then evaporated in vacuum to remove solvents and then taken again in DCM (25 mL) and washed with aq.NaHCO.sub.3 solution(5mL×2). Organic layer was then dried on Na.sub.2SO.sub.4, filtered and evaporated in vacuum. which was then triturated using methanol to get off-white solid (12), filtered, dried in vacuum (1.5g, 84%).
[0707] .sup.1H NMR (400 MHz, CDCl.sub.33) 6 7.95 (ddd, J=16.9, 12.1, 7.3 Hz, 8H), 7.60-7.12 (m, 22H), 5.66 (t, J=9.3 Hz, 1H), 5.58 (t, J=9.6 Hz, 1H), 5.43 (t, J=9.8 Hz, 1H), 5.36 (dd, J=9.5, 7.9 Hz, 1H), 5.20 (s, 1H), 4.77 (d, J=7.9 Hz, 1H), 4.60 (t, J=10.3 Hz, 2H), 4.39 (d, J=12.1 Hz, 1H), 4.23 (t, J=9.5 Hz, 1H), 3.74-3.43 (m, 5H), 3.29 (td, J=9.7, 4.9 Hz, 1H), 2.83-2.55 (m, 3H), 1.22 (t, J=7.4 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 165.46, 165.25, 165.16, 164.72, 137.82, 136.62, 133.31, 133.12, 133.02, 130.05, 129.80, 129.73, 129.70, 129.27, 129.24, 128.99, 128.97, 128.63, 128.43, 128.29, 128.22, 128.17, 128.10, 127.98, 126.03, 101.12, 83.39, 78.65, 78.29, 75.85, 74.44, 73.44, 72.43, 71.96, 70.47, 67.71, 67.31, 66.16, 24.04, 14.84.
Example 1-7
Synthesis of 2,3-di-O-benzoyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucoyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (14)
[0708] ##STR00055##
[0709] To a stirred solution of alcohol 13 (400 mg, 0.36 mmol) in pyridine (5.0 mL) was added at 0° C. benzoyl chloride (63 μL, 0.55 mmol). The reaction was slowly warmed to room temperature and stirred for 16 h at that temperature. An additional 0.5 equiv. BzCl were added to drive the reaction to completion. The mixture was stirred for 2 h at room temperature, quenched with water (30 ml) and diluted with EtOAc (50 mL). After separation, the organic fraction was washed with 0.1 M HCl (20 mL) and the aqueous fraction was re-extracted with EtOAc (30 mL). The combined organic fractions were washed with sat. aq. NaHCO.sub.3 (20 mL) and brine (10 mL), dried over Na.sub.2SO.sub.4 and concentrated to give the intermediate tetrabenzoate as a yellow oil. To a stirred solution of the intermediate tetrabenzoate in CH.sub.2Cl.sub.2 (6.5 mL) were added at room temperature ethanethiol (0.36 mL, 4.9 mmol) and p-toluenesulfonic acid (12 mg, 0.06 mmol). The mixture was stirred for 2 h at that temperature, quenched with Et.sub.3N (50 μL) and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:10 to 1:8) to give diol 14 (389 mg, 0.349 mmol) as a white foam. HRMS (ESI) calcd. for C.sub.64H.sub.61NO.sub.17 (M+Na).sup.+1138.3837 found 1138.3850 m/z.
Example 1-8
Synthesis of methyl(2,3-di-O-benzoyl-β-D-glucopyranosyl)uronate-(1→4)-2,3-di-O-benzoyl-6-0-benzyl-β-D-glucoyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (15)
[0710] ##STR00056##
[0711] To a stirred solution of alcohol 14 (90 mg, 0.081 mmol) in CH.sub.2Cl.sub.2 (2.0 mL) and water (0.8 mL) were added at 0° C. TEMPO (2.5 mg, 0.016 mmol) and BAIB (55 mg, 0.170 mmol). The reaction was stirred for 20 min at that temperature and warmed to room temperature. The mixture was stirred for 2 h at that temperature and diluted with EtOAc (20 mL) and water (10 mL). After separation, the aqueous fraction was extracted with EtOAc (2×10 mL), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:2 to 1:1, then 1:1+5% AcOH) to give the intermediate carboxylic acid as a white foam.
[0712] To a stirred solution of the intermediate carboxylic acid in toluene (1.6 mL) and MeOH (0.8 mL) was added at room temperature TMS-diazomethane (0.04 mL, 0.081 mmol). The reaction was stirred for 2 h at that temperature. An additional 0.25 equiv. TMS-diazomethane was added to drive the reaction to completion. The mixture was stirred for 1 h at that temperature, quenched with AcOH (0.1mL) and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:1) to give methyl ester 15 (73 mg, 0.064 mmol, 79% over two steps) as a clear oil. HRMS (ESI) calcd. for C.sub.65H.sub.61NO.sub.18 (M+Na).sup.+1166.3786 found 1166.3762 m/z.
Example 1-9
Synthesis of t-hexyl 2,3,6-tri-O-benzyl-β-D-galactopyranoside (17)
[0713] ##STR00057##
[0714] To a stirred solution of benzylidene acetal 16 (J Carbohyd Chem 1996, 15 (2), 241) (1.68 g, 2.84 mmol) in CH.sub.2Cl.sub.2 (60 mL) over activated MS (3 Å-AW) were added at 0° C. triethyl silane (2.72 mL, 17.06 mmol) and trifluoroacetic acid (1.81 mL, 17.06 mmol). The reaction was slowly warmed to room temperature and stirred for 16 h at that temperature. The reaction was quenched with Et.sub.3N (2 mL), filtered through Celite and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:20 to 1:7) to give alcohol 17 (1.46 g, 2.46 mmol, 87%) as a clear oil. HRMS (ESI) calcd. for C.sub.35H.sub.48O.sub.6Si (M+Na).sup.+615.3117 found 615.3104 m/z.
Example 1-10
Synthesis of tHexyl 4-O-benzoyl-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-O-D-galactopyranoside (44)
[0715] ##STR00058##
[0716] Thioglycoside 43 (J. Org. Chem. 2012, 77 (1), 291). (667 mg, 1.11 mmol) and alcohol 17 (550 mg, 0.93 mmol) were co-evaproated with dry toluene (3×10 mL) and kept under high vacuum for 30 min. The mixture was dissolved in Et.sub.2O (14 mL) and CH.sub.2Cl.sub.2 (2.8 mL) and stirred over activated molecular sieves (3 Å-AW) for 30 min at room temperature. The solution was cooled to −20° C. and treated with NIS (250 mg, 1.11 mmol) and triflic acid (16 μL, 0.19 mmol). The mixture was stirred for 1 h and slowly warmed to −10° C. The reaction was quenched with Et.sub.3N (0.05 mL), diluted with CH.sub.2Cl.sub.2 (20 mL), filtered through Celite and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:8 to 1:6) to give disaccharide 44 (553 mg, 0,490 mmol, 53%) along with the corresponding β-anomer (231 mg, 0.205 mmol, 22%). Analytical data for 7: Clear oil. HRMS (ESI) calcd. for C.sub.69H.sub.80O.sub.12Si (M+Na).sup.+1151.5316 found 1151.5293 m/z.
Example 1-11
Synthesis of 4-O-benzoyl-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-αβ-D-galactopyranosyl trifluoro-(N-phenyl)acetimidate (45)
[0717] ##STR00059##
[0718] To a stirred solution of silyl ether 44 (470 mg, 0.416 mmol) in THF (8.3 mL) was added at 0° C. acetic acid (0.24 mL, 4.19 mmol) and TBAF (1.0 M solution in THF, 4.2 mL, 4.20 mmol). The reaction was slowly warmed to room temperature and stirred for 2 h at that temperature. Acetic acid (0.24 mL, 4.19 mmol) and TBAF (1.0 M solution in THF, 4.2 mL, 4.20 mmol) were added and the reaction was stirred for 16 h at room temperature. The mixture was diluted with Et.sub.2O (50 mL), washed with water (3×30 mL) and the aqueous phase was re-extracted with Et.sub.2O (2×20 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was filtered through a short plug of silica gel (EtOAc/hexanes 1:3 to 1:1) to give the intermediate lactol mixture as a clear oil.
[0719] To a stirred solution of the lactol mixture in CH.sub.2Cl.sub.2 (7.8 mL) were added at room temperature cesium carbonate (318 mg, 0.975 mmol) and F.sub.3CC(NPh)Cl (202 mg, 0.975 mmol). The mixture was stirred for 2.5 h at that temperature, diluted with hexanes (0.5% (v/v) Et.sub.3N, (10 mL) and filtered through Celite. The residue was purified by flash chromatography (EtOAc/hexanes 0:1+0.5% Et.sub.3N to 1:3+0.5% Et.sub.3N) to give imidate mixture 45 (404 mg, 0.349 mmol, 84% over two steps) as a clear oil. HRMS (ESI) calcd. for C.sub.69H.sub.66F.sub.3NO.sub.12 (M+Na).sup.+1180.4434 found 1180.4458 m/z.
Example 1-12
Synthesis of 4-O-benzoyl-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→4)-methyl [2,3-Di-O-benzoyl-β-D-glucopyranosyl]uronate-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucoyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (46)
[0720] ##STR00060##
[0721] Alcohol 15 (100 mg, 87 μmol) and imidate 45 (121 mg, 105 μmol) were co-evaporated with dry toluene (3×10 mL) and kept under high vacuum for 30 min. The mixture was dissolved in Et.sub.2O (3.3 mL) and CH.sub.2Cl.sub.2 (1.1 mL) and stirred over activated molecular sieves (3 Å-AW) for 30 min at room temperature. The solution was cooled to −20° C. and treated with TMSOTf (3.2 μL, 17 μmol). The mixture was stirred for 1 h and slowly warmed to 0° C. The reaction was quenched with sat. aq. NaHCO.sub.3 (10 mL), extracted with CH.sub.2Cl.sub.2 (3×20 mL) and the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes/toluene 1:3:3 to 1:2:2) to give tetrasaccharide 46 (130 mg, 62 μmol, 71%) as a clear oil. HRMS (ESI) calcd. for C.sub.126H.sub.121NO.sub.29 (M+Na).sup.+2134.7921 found 2134.7879 m/z.
Example 1-13
Synthesis of α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→4)-β-D-glucopyranosyluronic acid-(1→4)-β-D-glucoyranosyl-(1→1)-(2-amino)ethanol (10)
[0722] ##STR00061##
[0723] To a stirred solution of ester 46 (56 mg, 26 μmol) in THF (5 mL) and MeOH (1 mL) were added at 0° C. hydrogen peroxide (6% aq. solution, 265 μL, 530 μmol) and LiOH (1 M aq. solution, 265 μL, 132 mol). The reaction was stirred for 1 h and warmed to room temperature. The reaction was kept at that temperature and treated after 2 h with hydrogen peroxide (6% aq. solution, 265 μL, 530 μmol) and LiOH (1 M aq. solution, 265 μL, 132 mol). After 2 h, NaOH (15% aq. solution, 1 mL) was added and the mixture was stirred for 72 h at room temperature. The solvents were evaporated under reduced pressure, the residue was co-evaporated with toluene (2μ5 mL) and dissolved in MeOH (5 mL). The solution was treated at room temperature with sodium methoxide (143 mg, 2.65 mmol) and stirred for 96 h at that temperature. The solvent was evaporated and the residue was dissolved in water (5 mL). The solution was neutralized at 0° C. with 0.5 M aq. NaHSO.sub.4 and extracted with EtOAc (5×5 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated to give the intermediate acid as a white foam.
[0724] The intermediate acid in MeOH (2 mL) was added at room temperature to a suspension of Pd/C (50 mg) in MeOH (1 mL), water (0.1 mL) and AcOH (5 drops). The reaction was stirred under an atmosphere of H.sub.2 for 48 h, filtered and concentrated. The residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and lyophilized to give tetrasaccharide 10 (acetate salt, 13.6 mg, 18 μmol, 69% over 3 steps) as a white solid. HRMS (MALDI) calcd. for C.sub.26H.sub.45NO.sub.22 (M+Na).sup.+746.2330 found 746.2323 m/z.
Example 1-14
Synthesis of 4-O-Benzoyl-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (47)
[0725] ##STR00062##
[0726] Imidate 45 (200 mg, 0.173 mmol) and alcohol 2 (74 mg, 0.259 mmol) were co-evaproated with dry toluene (2×5 mL) and kept under high vacuum for 30 min. The mixture was dissolved in Et.sub.2O (2.8 mL) and CH.sub.2Cl.sub.2 (0.7 mL) and stirred over activated molecular sieves (3 Å-AW) for 30 min at room temperature. The solution was cooled to −40° C. and treated with TMSOTf (6.2 μL, 35 μmol). The mixture was stirred for 10 min at that temperature and then slowly warmed to −10° C. The reaction was quenched with sat. aq. NaHCO.sub.3 (5 mL), extracted with CH.sub.2Cl.sub.2 (3×20 mL) and the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:6 to 1:4) to give carbamate 47 (160 mg, 0.128 mmol, 74%) along with the corresponding β-anomer (32 mg, 0.026 mmol, 15%). Analytical data for 47: Clear oil. HRMS (ESI) calcd. for C.sub.78H.sub.79NO.sub.14 (M+Na).sup.+1276.5398 found 1276.5405 m/z.
Example 1-15
Synthesis of 2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (48)
[0727] ##STR00063##
[0728] To a stirred solution of ester 47 (126 mg, 0.100 mmol) in THF (5 mL) and MeOH (5 mL) was added at 0° C. sodium methoxide (0.5 M in MeOH, 1 mL, 0.500 mmol). The reaction was slowly warmed to room temperature and kept at that temperature for 24 h. Sodium methoxide (0.5 M in MeOH, 1 mL, 0.500 mmol) was added and the reaction was warmed to 37° C. The mixture was stirred for 7 h at that temperature, neutralized with Amberlite IR120 (H.sup.+form), filtered and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:6 to 1:4) to give alcohol 48 (98 mg, 85 μmol, 85%) as a clear oil. HRMS (ESI) calcd. for C.sub.71H.sub.75NO.sub.13 (M+Na).sup.+1172.5136 found 1172.5103 m/z.
Example 1-16
Syntheis of α-D-glucopyranosyl-(144)-α-D-galactopyranosyl-(1→1)-(2-amino)ethanol (49)
[0729] ##STR00064##
[0730] Benzyl ether 48 in EtOAc (1 mL) was added at room temperature to a suspension of Pd/C (30 mg) in MeOH (3 mL), water (0.5 mL) and AcOH (3 drops). The reaction was stirred under an atmosphere of H.sub.2 for 24 h, filtered and concentrated to give disaccharide 49 (2.9 mg, 18 μmol, 87%) as a white solid. HRMS (ESI) calcd. for C.sub.14H.sub.27NO.sub.11 (M+Na).sup.+408.1481 found 408.1499 m/z.
Example 1-17
Synthesis of 2,3-di-O-benzoyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucoyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (50)
[0731] ##STR00065##
[0732] Alcohol 48 (47 mg, 41 μmol) and thioglycoside 12 (60 mg, 61 μmol) were co-evaporated with dry toluene (2×5 mL) and kept under high vacuum for 30 min. The mixture was dissolved in CH.sub.2Cl.sub.2 (2 mL) and stirred over activated molecular sieves (3 Å-AW) for 30 min at room temperature. The solution was cooled to −10° C. and treated with NIS (13.8 mg, 61 μmol) and triflic acid (1 μL, 11 μmol). The mixture was kept for 1 h at that temperature and slowly warmed to 0° C. The reaction was quenched with Et.sub.3N (50 μL), filtered and concentrated to give the intermediate benzylidene acetal as a yellow oil.
[0733] To a stirred solution of the intermediate benzylidene acetal in CH.sub.2Cl.sub.2 (2 mL) were added at room temperature ethanethiol (0.3 mL, 4.06 mmol) and p-toluenesulfonic acid (10 mg, 0.053 mmol). The mixture was stirred for 1 h at that temperature, quenched with Et.sub.3N (20 μL) and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:3 to 1:2) to give diol 50 (78 mg, 39 μmol, 96%) as a clear oil. HRMS (ESI) calcd. for C.sub.118H.sub.117NO.sub.27 (M+Na).sup.+2002.7710 found 2002.7731 m/z.
Example 1-18
Synthesis of 2,3-di-O-benzoyl-β-D-glucopyranosyluronate-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucoyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (51)
[0734] ##STR00066##
[0735] To a vigorously stirred solution of alcohol 50 (45 mg, 23 μmol, 73%) in CH.sub.2Cl.sub.2 (2 mL) and water (0.8 mL) were added at 0° C. TEMPO (3 crystals) and BAIB (15.4 mg, 48 μmol). The reaction was stirred for 20 min at that temperature and slowly warmed to room temperature. After 1 h, TEMPO (2 crystals) and BAIB (10 mg, 31 μmol) were added and the mixture was stirred for 2 h at room temperature. The reaction was diluted with CH.sub.2Cl.sub.2 (5 mL) and quenched with 10% aq. Na.sub.2S.sub.2O.sub.3 (5 mL). Following separation, the aqueous phase was extracted with EtOAc (2×10 mL), the combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography twice (EtOAc/hexanes 0:1 to 1:2 to 8:1, then EtOAc/hexanes 1:1+1% AcOH) and co-evaporated with heptane repeatedly to give acid 51 (33 mg, 17 μmol, 74%) as a clear oil. HRMS (ESI) calcd. for C.sub.118H.sub.115NO.sub.28 (M+Na).sup.+2016.7503 found 2016.7558 m/z.
Example 1-19
Synthesis of β-D-Glucopyranosyluronic acid-(1→4)13-D-glucoyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→1)-(2-amino)ethanol (18)
[0736] ##STR00067##
[0737] To a stirred solution of ester 51 (45 mg, 23 μmol) in THF (4 mL) and MeOH (0.5 mL) were added at 0° C. NaOH (1 M aq. solution, 1 mL). The reaction was slowly warmed to room temperature and stirred for 16 h at that temperature. The solution was neutralized at 0° C. with 0.5 M aq. NaHSO.sub.4 and extracted with EtOAc (5×5 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated to give the intermediate alcohol as a white foam.
[0738] The intermediate alcohol in MeOH (3 mL) was added at room temperature to a suspension of Pd/C (20 mg) in MeOH (6 mL), water (6 drops) and AcOH (3 drops). The reaction was stirred under an atmosphere of H.sub.2 for 96 h, filtered and concentrated. Since the reaction had not proceeded to completion, the residue was subjected to the same conditions again and stirred for 72 h at room temperature. The reaction was filtered and concentrated, the residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and lyophilized to give tetrasaccharide 18 (11.3 mg, 16 μmol, 68% over 2 steps) as a white solid. HRMS (MALDI) calcd. for C.sub.26H.sub.45NO.sub.22 (M+Na).sup.+746.2330 found 746.2416 m/z.
Example 1-20
Synthesis of methyl[2,3-di-O-benzoyl-β-D-glucopyranosyl]uronate-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucoyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (52)
[0739] ##STR00068##
[0740] To a stirred solution of carboxylic acid 51 (100 mg, 50 μmol) in DMF (2.5 mL) were added at room temperature Cs.sub.2CO.sub.3 (24.5 mg, 75 μmol) and methyl iodide (10.7 mg, 75 μmol) and the reaction was stirred at that temperature. After 2 h, methyl iodide (10.7 mg, 75 μmol) was added and the mixture was stirred for another 2 h at room temperature. The reaction was quenched with sat. aq. NH4Cl (5 mL), extracted with EtOAc (4×10 mL), the combined organic extracts were dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 2:3) to give methyl ester 52 (81 mg, 40 μmol, 80%) as a white foam. HRMS (MALDI) calcd. for C119H117N028 (M+Na)+2030.7659 found 2030.7660 m/z.
Example 1-21
Synthesis of 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl-(1→4)-methyl[2,3-di-O-benzoyl-β-D-glucopyranosyl]uronate-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucoyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2 -N-benzyl-N-benzyloxycarbonylamino)ethanol (53)
[0741] ##STR00069##
[0742] Alcohol 52 (14 mg, 7 μmol) and thioglycoside 54 (J Org Chem 1990, 55, 2860.) (16.3 mg, 28 μmol) were co-evaporated with dry toluene (3×10 mL) and kept under high vacuum for 30 min. The mixture was dissolved in Et.sub.2O (1.05 mL) and CH.sub.2Cl.sub.2 (0.35 mL) and stirred over activated molecular sieves (3 Å-AW) for 30 min at room temperature. The solution was cooled to −20° C. and treated with NIS (6.3 mg, 28 pmol) and TMSOTf (1 μL, 5.5 μmol). The mixture was stirred for 1 h and slowly warmed to 0° C. The reaction was quenched with a 1:1 (v/v) mixture of sat. aq. NaHCO.sub.3 (10 mL) and 10% (w/v) Na.sub.2SO.sub.3 (5 mL) and extracted with CH.sub.2Cl.sub.2 (4×10 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:4 to 1:3) to give pentasaccharide 53 (12.5 mg, 4.9 μmol, 71%) as a clear oil. HRMS (MALDI) calcd. for C.sub.126H.sub.121NO.sub.29 (M+Na).sup.+2553.0066 found 2553.0066 m/z.
Example 1-22
Synthesis of α-D-galactopyranosyl-(1→4)-β-D-glucopyranosyluronate-(1→4)-β-D-glucoyranosyl-α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→1)-(2-amino)ethanol (55)
[0743] ##STR00070##
[0744] To a stirred solution of ester 53 (26 mg, 10.3 μmol) in THF (1 mL) and MeOH (1 mL) was added at 0° C. a 1:1 (v/v) mixture (450 μL) of hydrogen peroxide (6% (v/v) aq. solution, 397 μmol) and LiOH (0.5 M aq. solution, 113 μmol). The reaction was warmed to room temperature and stirred for 1 h at that temperature. The reaction was treated with NaOH (0.5 M aq. solution, 1 mL) and stirred for 16 h at room temperature. The solvents were evaporated under reduced pressure, the residue was co-evaporated with toluene (2×5 mL) and dissolved in MeOH (1 mL). The solution was treated at room temperature with NaOMe (0.5 M in MeOH, 1 mL) and stirred for 16 h at that temperature. The reaction was diluted with water (0.5 mL) and CH.sub.2Cl.sub.2 (0.5 mL), neutralized at 0° C. with Amberlite IR-120 (H.sup.+form), filtered and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:4 to 1:2 to 1:2 +1% (v/v) AcOH to 1:1 +1% (v/v) AcOH) to give the intermediate carboxylic acid as a clear oil.
[0745] The intermediate carboxylic acid in CH.sub.2Cl.sub.2/tBuOH/water (1:16:8, 1 mL) was purged with argon and treated at 0° C. with a suspension of Pd(OH).sub.2 on carbon (20% (w/w) loading, 20 mg) in the same solvent mixture (0.5 mL). The suspension was purged with hydrogen, stirred under hydrogen atmosphere for 16 h, filtered and concentrated. Since the reaction had not proceeded to completion, the residue was subjected to the same conditions again and stirred for 24 h at room temperature. The mixture was filtered and concentrated, the residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and lyophilized to give pentasaccharide 55 (8.1 mg, 9.1 μmol, 88% over two steps) as a white solid. HRMS (MALDI) calcd. for C.sub.32H.sub.55NO.sub.27 (M+Na).sup.+884.2883 found 884.2942 m/z. .sup.1H NMR (400 MHz, D.sub.2O) δ 5.48 (d, J=3.5 Hz, 1H), 5.02 (d, J=3.2 Hz, 1H), 4.88 (d, J=3.9 Hz, 1H), 4.51 (m, 2H), 4.20 (d, J=9.9 Hz, 1 H), 4.04 (m, 1H), 4.02-3.52 (m, 26H), 3.43-3.19 (m, 4H).
Example 1-23
Synthesis of 4-O-benzoyl-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→4)-methyl[2,3-di-O-benzoyl-β-D-glucopyranosyl]uronate-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucoyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol 56)
[0746] ##STR00071##
[0747] Alcohol 52 (50 mg, 25 μmol) and imidate 45 (72.1 mg, 62 μmol) were co-evaproated with dry toluene (3×10 mL) and kept under high vacuum for 30 min. The mixture was dissolved in Et.sub.2O (2 mL) and CH.sub.2Cl.sub.2 (0.67 mL) and stirred over activated molecular sieves (3 Å-AW) for 30 min at room temperature. The solution was cooled to −20° C. and treated with TMSOTf (2 μL, 11 μmol). The mixture was stirred for 1 h and slowly warmed to 0° C. The reaction was quenched with sat. aq. NaHCO.sub.3 (10 mL) and extracted with CH.sub.2Cl.sub.2 (4×10 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:3 to 3:7 to 1:2) to give hexasaccharide 56 (51 mg, 17 μmol, 69%) as a clear oil. HRMS (MALDI) calcd. for C.sub.180H.sub.177NO.sub.39 (M+2Na).sup.2+1511.0847 found 1511.0576 m/z.
Example 1-24
Synthesis of α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→4)-β-D-glucopyranosyluronate-(1→4)-β-D-glucoyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→1)-(2-amino)ethanol (57)
[0748] ##STR00072##
[0749] To a stirred solution of ester 56 (22 mg, 7.4 μmol) in THF (1 mL) and MeOH (1 mL) was added at 0° C. a 1:1 (v/v) mixture (296 μL) of hydrogen peroxide (6% (v/v) aq. solution, 295 μmol) and LiOH (0.5 M aq. solution, 74 μmol). The reaction was warmed to room temperature and treated after 2 h and 4 h with another 294 μL of the same LiOOH solution, respectively. The mixture was stirred for 16 h at room temperature and treated with NaOH (1 M aq. solution, 0.5 mL) and MeOH (0.5 mL). The reaction was stirred for 20 h at that temperature, quenched with 10% aq. Na.sub.2SO.sub.3 (0.8 mL) and concentrated under reduced pressure. The residue was dissolved in water (4 mL), neutralized with NaHSO.sub.4 (0.5 M aq. solution) and extracted with EtOAc (4×10 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated. The residue was treated with NaOMe (0.5 M solution in MeOH, 1 mL), warmed to 40° C. and stirred for 5 h at that temperature. The reaction was cooled to room temperature, stirred for another 16 h at that temperature and treated with water (0.5 mL). The mixture was neutralized with Amberlite IR-120 (H.sup.+form), filtered and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1:0 to 1:4+2% (v/v) AcOH to 1:1+2% (v/v) AcOH) to give the intermediate carboxylic acid as a clear oil.
[0750] The intermediate carboxylic acid in CH.sub.2Cl.sub.2/tBuOH/water (1.5:16:8, 3 mL) was purged with argon and treated at 0° C. with a suspension of Pd(OH).sub.2 on carbon (20% (w/w) loading, 30 mg) in the same solvent mixture (1 mL). The suspension was purged with hydrogen, stirred under hydrogen atmosphere for 18 h, filtered and concentrated. The residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and lyophilized to give hexasaccharide 57 (7 mg, 6.7 μmol, 86% over three steps) as a white solid. HRMS (ESI) calcd. for C.sub.38H.sub.65NO.sub.32 (M+Na).sup.+1070.3387 found 1070.3391 m/z.
Example 1-25
Synthesis of β-D-Glucopyranosyl-(1→4)β-D-glucoyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→1)-(2-amino)ethanol (60)
[0751] ##STR00073##
[0752] To a stirred solution of ester 50 (20 mg, 10.1 μmol) in THF (1 mL) and MeOH (0.33 mL) was added at room temperature NaOMe (0.5 M solution in MeOH, 0.5 mL). The reaction was warmed to 40° C. and stirred for 5 h at that temperature. The mixture was cooled to room temperature and stirred for 16 h at that temperature. The reaction was neutralized with Amberlite IR-120 (H.sup.+form), filtered and concentrated. The residue was purified by size exclusion chromatography (Sephadex LH-20, CH.sub.2Cl.sub.2/MeOH 2:1) to give the intermediate hexaol as a white foam.
[0753] The intermediate hexaol in CH.sub.2Cl.sub.2/tBuOH/water (1:16:8, 1 mL) was purged with argon and treated at 0° C. with a suspension of Pd(OH).sub.2 on carbon (20% (w/w) loading, 20 mg) in the same solvent mixture (1 mL). The suspension was purged with hydrogen, stirred under hydrogen atmosphere for 18 h, filtered and concentrated. The residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and lyophilized to give tetrasaccharide 60 (6.8 mg, 9.0 μmol, 89% over two steps) as a white solid. HRMS (ESI) calcd. for C.sub.26H.sub.47NO.sub.21 (M+Na).sup.+732.2538 found 732.2504 m/z.
Example 1-26
Synthesis of 2,3-di-O-benzoyl-β-D-glucoyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→1)-(2-N-benzyl-N-benzyloxycarbonylamino)ethanol (61)
[0754] ##STR00074##
[0755] Alcohol 48 (15 mg, 13 μmol) and thioglycoside 1 (20.4 mg, 39 μmol) were co-evaporated with dry toluene (2×5 mL) and kept under high vacuum for 10 min. The mixture was dissolved in CH.sub.2Cl.sub.2 (1.3 mL) and stirred over activated molecular sieves (3 Å-AW) for 30 min at room temperature. The solution was cooled to −20° C. and treated with NIS (8.8 mg, 39 μmol) and TfOH (1 μL, 11 μmol). The mixture was stirred for 1 h at that temperature and slowly warmed to 0° C. The reaction was quenched with a 1:1 (v/v) mixture of sat. aq. NaHCO.sub.3 (10 mL) and 10% (w/v) Na.sub.2SO.sub.3 (5 mL) and extracted with CH.sub.2Cl.sub.2 (4×10 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:5 to 1:4 to 1:3) to give the intermediate benzylidene acetal as a yellow oil.
[0756] To a stirred solution of the intermediate benzylidene acetal in CH.sub.2Cl.sub.2 (2 mL) were added at room temperature ethanethiol (0.2 mL, 2.8 mmol) and p-toluenesulfonic acid (6 mg, 32 μmol). The mixture was stirred for 1 h at that temperature, quenched with Et.sub.3N (100 μL) and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 0:1 to 1:3 to 2:3) to give diol 61 (14.7 mg, 9.7 μmol, 75%) as a clear oil. HRMS (MALDI) calcd. for C.sub.26H.sub.47NO.sub.21 (M+Na).sup.+1542.6188 found 1542.6145 m/z.
Example 1-27
Synthesis of β-D-Glucoyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→1)-(2-amino)ethanol (62)
[0757] ##STR00075##
[0758] To a stirred solution of ester 61 (26 mg, 17 μmol) in CH.sub.2Cl.sub.2 (1 mL) and MeOH (1 mL) was added at room temperature NaOMe (0.5 M solution in MeOH, 0.5 mL). The reaction was stirred for 2 h at that temperature, neutralized at 0° C. with Amberlite IR-120 (H.sup.+form), filtered and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes 1:3 to 2:1) to give the intermediate tetraol as a white foam.
[0759] The intermediate tetraol in CH.sub.2Cl.sub.2/tBuOH/water (1:6:2, 5 mL) was purged with argon and treated at 0° C. with a suspension of Pd(OH).sub.2 on carbon (20% (w/w) loading, 30 mg) in the same solvent mixture (1 mL). The suspension was purged with hydrogen, stirred under hydrogen atmosphere for 24 h, filtered and concentrated. Since the reaction had not proceeded to completion, the residue was subjected to the same conditions again and stirred for 48 h at room temperature. The mixture was filtered and concentrated, the residue was purified by solid phase extraction (Chromabond C18, Macherey-Nagel) and lyophilized to give trisaccharide 62 (7.3 mg, 13 μmol, 79% over two steps) as a white solid. HRMS (ESI) calcd. for C.sub.26H.sub.47NO.sub.21 (M+Na).sup.+570.2010 found 570.2000 m/z.
Synthesis of Saccharide by Stepwise Automated Glycosylation
Example 1-28
Synthesis of Glucose Building Block 32
[0760] ##STR00076## ##STR00077##
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-3-0-benzyl-4,6-0-benzylidene-1-thio-β-D-glucopyranose (30)
[0761] (2-Methyl-5-tert-butylphenyl) 2,4,6-tri-O-acetyl-β-O-benzyl-1-thio-β-D glucopyranose (29) was dissolved in MeOH, NaOMe (1.0 eq) was added and the reaction mixture was stirred overnight. The mixture was neutralized with IR-120-H.sup.+amberlite resin, filtered off, concentrated and co-evaporated with toluene. The crude triol was dissolved in DMF. Benzaldehyde dimethyl acetal (2.0 eq) and a catalytic amount of para-toluene sulfonic acid were added and the mixture was stirred at 80° C. for 2 h. After the mixture was cooled to room temperature, saturated aqueous NaHCO.sub.3 was added. After phase separation, the organic phase was extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and concentrated. The crude was dissolved in DCM, cooled to 0° C. and Bz.sub.2O (2.0 eq), DMAP (0.5 eq) and triethylamine (4.0 eq) were added. After complete conversion, the reaction was quenched with saturated aqueous NaHCO.sub.3 and the combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and concentrated. The residue was purified by flash column chromatography on silica gel to provide compound (88% over three steps). Rf: 0.25 (Hexane:EtOAc=9:1). [β]D=68.96 (c=3.18, CHCl.sub.3).); IR (thin film, chloroform): u=2962, 1729, 1265, 1093 cm-1; 1H-NMR (400 MHz, CDCl.sub.3) δ =8.01 (ddd, J=5.8, 5.3, 0.8 Hz, 2H, H Ar), 7.60 (ddd, J=7.1, 2.6, 1.3 Hz, 1H, H Ar), 7.51 (ddd, J=6.0, 5.6, 2.3 Hz, 3H, H Ar), 7.49-7.43 (m, 2H, H Ar), 7.43-7.35 (m, 3H, H Ar), 7.19 (dt, J=6.3, 3.1 Hz, 1H, H Ar), 7.13 (dd, J=6.7, 4.0 Hz, 3H, H Ar), 7.10-7.04 (m, 3H, H Ar), 5.63 (s, 1H CHO2Ph), 5.42-5.32 (m, 1H, H-2), 4.82 (d, J=11.9 Hz, 1H, CHHPh), 4.81 (d, J=10.2 Hz, 1H, H-1), 4.69 (d, J=12.0 Hz, 1H, CHHPh), 4.39 (dd, J=10.5, 5.0 Hz, 1H, H-6′), 3.94-3.86 (m, 3H, H-3, H-4, H-6), 3.56 (dt, J=14.6, 4.9 Hz, 1H, H-5), 2.19 (s, 3H), 1.27 (s, 9H). 13C-NMR (100 MHz, CDCl.sub.3) δ 165.23 (C═O), 149.66, 137.84, 137.28, 137.03, 133.34, 132.37, 130.07, 130.04, 129.93, 129.90, 129.19, 128.51, 128.43, 128.29, 128.22, 127.71, 126.13, 125.38 (Ar), 101.44 (CHO.sub.2Ph), 88.03 (C-1), 81.64 (C-3), 79.43 (C-4), 74.36 (CH2Ph), 72.25 (C-2), 70.66 (C-5), 68.81 (C-6), 34.56 (Cq tBu thio), 31.36 (tBu), 20.36 (CH3 thio); MS ESI+-HRMS m/z [M+Na]+calcd for C.sub.38H.sub.40O.sub.6SNa 647.2462, found 647.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-3,6-di-O-benzyl-1-thio-β-D-glucopyranose (31)
[0762] To a solution of compound 1 was co-evaporated with toluene, and dissolved in DCM (6.5 mL) under an argon atmosphere. Triethylsilane (0.62 mL, 3.88 mmol) and trifluoroacetic anhydride (0.27 mL, 1.94 mmol) were added and the solution was cooled to 0° C. Trifluoroacetic acid (0.30 mL, 3.88 mmol) were added dropwise, and the reaction was stirred and allowed to warm to room temperature. After complete conversion of the starting material, the solution was diluted with DCM, and quenched with saturated aqueous NaHCO.sub.3. The combined organic layer was dried over MgSO.sub.4 and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (hexane/ethyl acetate, 9:1 to 7:3) to give a white foam (92%). MS ESI+-HRMS m/z [M+Na]+ calcd for C.sub.38H.sub.42O.sub.6SNa 649.2600 found 649.2585.
(2-Methyl-5-tert-butylphenyl) 2-O-benzoyl-3,6-di-O-benzyl-4-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranoside (32)
[0763] Compound 31 was dissolved in DCM (6.5 mL) under an argon atmosphere. 9-fluorenylmethyl chloroformate (8.3 g, 31.9 mmol) and pyridine (3.44 mL, 42.5 mmol) were added into the solution at 0° C. After complete conversion of the starting material, the solution was diluted with DCM, extracted with 1 M aqueous HCl and saturated aqueous NaHCO.sub.3. The combined organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was purified by silica gel flash column chromatography affording the title compound MS ESI+-HRMS m/z [M+Na]+ calcd for C.sub.53H.sub.52O.sub.8SNa 871.3281 found 871.3311.
Example 1-29
Synthesis of Glucose Building Block 35
[0764] ##STR00078##
(2-Methyl-5-tert-butylphenyl) 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (34)
[0765] (2-Methyl-5-tert-butylphenyl) 4,6-O-benzylidene-1-thio-β-D-glucopyranoside (33) was dissolved in DCM (6.5 mL) under an argon atmosphere. Benzyl bromide, and NaH were added into the solution at 0° C. After complete conversion of the starting material, the reaction mixture was quenched with methanol, and diluted with ether, extracted with saturated NH.sub.4Cl. The combined organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was purified by silica gel flash column chromatography affording the title compound. MS ESI+-HRMS m/z [M+Na]+ calcd for C.sub.38H.sub.42O.sub.5SNa 633.2651 found 633.2644.
(2-Methyl-5-tert-butylphenyl) 6-O-acetyl-2,3-di-O-benzyl-4-O-fluorenylmethoxycarbonyl-1-thio-β-D-galactopyranoside (35)
[0766] To the solution of (2-Methyl-5-tert-butylphenyl) 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-β-D-glucopyranoside (34) in DCM (6.5 mL) were added TFA and water. After completion, the crude was decanted with hexane to remove byproduct. The crude was used to the next reaction. To the solution of the crude was added acetic acid, 2-chloro-1-methylpyridium iodide, and DABCO at −15° C. After complete conversion of the starting material, the solution was diluted with DCM, extracted with 1 M aqueous HCl and saturated aqueous NaHCO.sub.3. The combined organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was purified by silica gel flash column chromatography affording the title compound. The crude was dissolved in DCM (6.5 mL) under an argon atmosphere. 9-fluorenylmethyl chloroformate (8.3 g, 31.9 mmol) and pyridine (3.44 mL, 42.5 mmol) were added into the solution at 0° C. After complete conversion of the starting material, the solution was diluted with DCM, extracted with 1 M aqueous HCl and saturated aqueous NaHCO.sub.3. The combined organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was purified by silica gel flash column chromatography affording the title compound. MS ESI+-HRMS m/z [M+Na]+ calcd for C.sub.48H.sub.50O.sub.8SNa 809.3124 found 809.3137.
Example 1-30
Synthesis of (2-methyl-5-Cert-butylphenyl) 3,6-di-O-acetyl-2,4-di-O-benzyl-1-thio-β-D-galactopyranoside (37)
[0767] ##STR00079##
[0768] (2-Methyl-5-tert-butyl phenyl)4,6-O-benzyl idene-2-O-benzyl-1-thio-β-D-glucopyranoside (36) (Tetrahedron Letter, 1999, 40, 6523) was co-evaporated toluene and dissolved under an Ar atmosphere in DCM (170 mL). A 1 M solution of BH.sub.3 in THF (108 mL, 108 mmol) was added and the solution was cooled to 0° C. After 10 min, trimethylsilyl triflate (1.66 mL, 9.2 mmol) was added and the reaction was stirred. After completion, the solution was diluted with DCM, and quenched with saturated aqueous NaHCO.sub.3 dropwise. The organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was used without further purification for the next step.
[0769] To a solution of the crude in DCM was added acetic anhydride (8.3 g, 31.9 mmol) and pyridine (3.44 mL, 42.5 mmol). After complete conversion of the starting material, the solution was diluted with DCM, extracted with saturated aqueous NaHCO.sub.3. The combined organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was purified by silica gel flash column chromatography affording the title compound (37). MS ESI+-HRMS m/z [M+Na]+calcd for C.sub.35H.sub.42O.sub.7SNa 629.2546 found 629.2522.
Example 1-31
Synthesis of Protected Galactose Building Blocks
[0770] ##STR00080##
[0771] Ethyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (38) was co-evaporated with toluene, and dissolved in DCM (6.5 mL) under an Ar atmosphere. Triethylsilane (0.62 mL, 3.88 mmol) and trifluoroacetic anhydride (0.27 mL, 1.94 mmol) were added and the solution was cooled to 0° C. Trifluoroacetic acid (0.30 mL, 3.88 mmol) were added dropwise, and the reaction was stirred and allowed to warm to room temperature. After complete conversion of the starting material, the solution was diluted with DCM, and quenched with saturated aqueous NaHCO.sub.3. The combined organic layer was dried over MgSO.sub.4 and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (hexane/ethyl acetate, 9:1 to 7:3) to give a white foam (94%). [Christopher E. Martin, Markus W. Weishaupt, and Peter H. Seeberger, Chem. Commun., 2011, 47, 10260-10262.]
[0772] Ethyl 2,3,6-tri-O-benzyl-1-thio-β-D-galactopyranoside was dissolved in DCM (6.5 mL) under an argon atmosphere. Acetic anhydride (8.3 g, 31.9 mmol) and pyridine (3.44 mL, 42.5 mmol) were added into the solution at 0° C. After complete conversion of the starting material, the solution was diluted with DCM, extracted with 1 M aqueous HCl and saturated aqueous NaHCO.sub.3. The combined organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was purified by silica gel flash column chromatography affording the title compound (40). (Chem. Commun., 2011, 47, 10260)
Ethyl 2,3,6-tri-O-benzyl-4-O-fluorenylmethoxycarbonyl-1-thio-β-D-galactopyranoside (41)
[0773] To a solution of compound 39 were added 9-fluorenylmethyl chloroformate (8.3 g, 31.9 mmol) and pyridine (3.44 mL, 42.5 mmol) at 0° C. After complete conversion of the starting material, the solution was diluted with DCM, extracted with 1 M aqueous HCl and saturated aqueous NaHCO.sub.3. The combined organic phase was dried over MgSO.sub.4 and the solvent was removed in vacuo. The crude product was purified by silica gel flash column chromatography affording the title compound. MS ESI-F-HRMS m/z [M+Na]+ calcd for C.sub.44H.sub.44O.sub.7SNa 739.2705 found 739.2673.
Example 1-32
Preparation of Glucuronic Acid Building Block 42
[0774] ##STR00081##
Methyl (2-methyl-5-tert-butyl-phenyl)-2-O-benzoyl-3-O-benzyl-4-O-fluorenylmethoxycarbonyl-1-thio-β-D-glucopyranosyluronate (42)
[0775] was synthesized according to the procedure described in Angew. Chem. Int. Ed. 2013, 52, 5858.
Example 1-33
Synthesis of the Functionalized Solid Support
[0776] Functionalized resin was synthesized according to the procedure described in Angew. Chem. Int. Ed. 2013, 52, 5858.
##STR00082##
Example 1-34
General Procedure 1: Automation Module Preparation of Stock Solutions
[0777] Activator Solution: N-lodosuccinimide (1.48 g, 6.66 mmol) and TfOH (60 μL, 0.66 mmol) was dissolved in a mixture of DCM (20 mL) and dioxane (20 mL).
[0778] Fmoc Deprotection Solution: A solution of 20% triethylamine in DMF (v/v) was prepared.
[0779] Thioglycoside Building Block Solution: 0.25 mmol of building block was dissolved in 2 ml of DCM.
[0780] Module 1: Glycosylation: The resin is swollen in 2 mL DCM and the temperature of the reaction vessel is adjusted to T1. For the glycosylation reaction, the DCM is drained and a solution of building block (5 eq in 1.0 mL DCM) is delivered to the reaction vessel. After the set temperature is reached, the reaction is started by the addition of activator (5 eq in 1.0 mL of solution). The glycosylation is performed for 5 min at temperature T1 and for 45 min at T2. This procedure is repeated twice. After the reaction was complete, the solution is drained and the resin is washed with DCM (six times each with 2 mL for 25 s). This procedure is repeated two times more.
[0781] Module 2: Fmoc Deprotection: The resin is washed with DMF (six time), swollen in 2 mL DMF and the temperature of the reaction vessel is adjusted to 25° C. For Fmoc deprotection the DMF is drained and 2 mL of a solution of 20% triethylamine in DMF is delivered to the reaction vessel. After 5 min the reaction solution is collected in the fraction collector of the oligosaccharide synthesizer and 2 mL of a solution of 20% triethylamine in DMF is delivered to the resin. This procedure is repeated three times. For the next glycosylation the resin is washed with DMF, THF, DCM (six times each). For Fmoc quantification the reaction solutions are combined and a 100 μL aliquot is taken. This aliquot is diluted with a solution of 20% triethylamine in DMF to 5 mL and the UV absorption at A=294 nm is determined.
[0782] Cleavage from Solid Support (Angew. Chem. Int. Ed. 2013, 52, 5858-5861):
[0783] Continuous photocleavage flow reactor-general procedure: The flow reactor set-up consists of a medium pressure Hg lamp (Hanovia) with arc lengths of 27.9 cm and power of 450 W surrounded by a UV filter (Pyrex, 50% transmittance at 305 nm) in a quartz glass cooling system connected to a chiller to maintain a reaction temperature of 25° C. A fluorinated ethylene propylene (FEP) tubing (inner diameter: 0.03 inch; volume: 12 mL) is wrapped around the cooling system. A syringe pump is connected to the FEP tubing and is used to flush solvents and resin via the inlet through the reactor. The solid support is filtered off by a frit and the product solution is pooled and the solvents are removed in vacuo. The UV lamp is located in a box that is additionally cooled by a fan.
[0784] To finish the automated synthesis process, the resin is washed with DCM (six times), swollen in 2 mL DCM and transferred into a disposable syringe (20 mL). To prepare the photoreactor, the FEP tubing is washed with 15 mL MeOH, then subsequently with 15 mL DCM using a flow rate of 4 mL.Math.min-1. For the cleavage, the resin is slowly injected from the disposable syringe (20 mL) into the reactor and pushed through the tubing with 15 mL DCM (flow rate: 500 μL.Math.min-1). To wash out remaining resin, the tubing is washed with 20 mL DCM (flow rate: 500 μL.Math.min-1). The suspension leaving the reactor is directed into a filter where the resin is filtered off and washed with DCM. The tubing is re-equilibrated with 15 mL DCM using a flow rate of 4 mL.Math.min-1. The entire procedure is performed twice. The resulting solution is evaporated in vacuo and the crude product is purified by HPLC (column: Luna silica; flow rate: 5 mL.Math.min-1).
[0785] General Procedure 2: Global Deprotection
[0786] To a solution of purified saccharide in a mixture of THF and MeOH (1.2 ml, v/v=4:1) were added 1 M LiOH-35% H.sub.2O.sub.2 (150 μL, v/v=2:1) at 0° C. The reaction was warmed up to room temperature, kept. After 4hr, 1M KOH (0.5 mL) was added and the mixture was stirred. After completion, the mixture was neutralized with IR-120-H.sup.+ amberlite resin, filtered off, concentrated. The crude was dissolved in MeOH, ethyl acetate, and acetic acid (5 mL: 0.75 mL: 0.25 mL), followed by Pd/C (20mg). The mixture was bubbled under an atmosphere of argon for 30 min, then at atmosphere of H.sub.2 for 12 hr, filtered and concentrated. The residue was purified by HPLC (Hyper-carbon) and lyophilized to give saccharide (formic acid salt) as a white solid.
[0787] General Procedure to Run Automation:
[0788] The functionalized resin of Example 1-33 (65 mg; loading 0.385 mmol/g; 0.025 mmol) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM. To start the synthesis sequence, the resin was washed consecutively with DMF, THF, then DCM (three times each with 2 mL for 25 s). Module 1 for each building block and module 2 for Fmoc deprotection were performed to produce each saccharide structure.
[0789] Purification on HPLC
[0790] After synthesis of the desired compound, the resin was cleaved from the solid support. The crude product was purified by semi-preparative HPLC (column: Luna-Silica (21×250 mm; 5 μm); flow rate: 5 mL/min; eluents: Hexane/Ethyl acetate; gradient: 20% (5 min) 60% (in 45 min) 100% (in 5 min); detection: 210 and 280 nm) affording the target oligosaccharide.
[0791] After global deprotection of the desired compound, the crude product was purified by semi-preparative HPLC (column: Luna-Hyper-Carbon (21×250 mm; 5 μm); flow rate: 5 mL/min; eluents: 0.1% formic acid in Water/0.1% formic acid in acetonitrile; gradient: 10% (5 min).fwdarw.40% (in 30 min).fwdarw.100% (in 5 min); detection: ELSD) affording the target oligosaccharide.
Example 1-34
Synthesis of N-benzyloxycarbonyl-5-amino-pentanyl methyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl-(1→4)-6-O-acetyl-2,3-di-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranoside (19a)
[0792] ##STR00083##
[0793] Tetrasaccharide 21a was prepared according to the reactions sequence:
TABLE-US-00001 Sequence Module Details Condition I 1 Building block 41 T1 = −30° C., T2 = −10° C. 2 Fmoc Removal r.t for 5 min II 1 Building block 35 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min III 1 Building block 32 T1 = −40° C., T2 = −20° C. 2 Fmoc Removal r.t for 5 min IV 1 Building block 42 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min
[0794] 8% from the resin, MS ESI+-HRMS m/z [M+Na]+ calcd for C.sub.110N.sub.117NO.sub.27Na 1906.7711, found 1906.7590.
Example 1-35
Preparation of 5-amino pentanyl β-D-glucopyranosyl uronic acid-(1→4)-β-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-galactopyranoside (19)
[0795] ##STR00084##
[0796] Tetrasaccharide 19a was subjected to the general deprotection procedure to afford tetrasaccharide 19: 36%; MS ESI+-HRMS m/z [M+H]+ calcd for C.sub.29H.sub.52NO.sub.22 766.2975, found 766.2988.
Example 1-36
Synthesis of N-benzyloxycarbonyl-5-amino-pentanyl 4-O-acetyl-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl-(1→4)-6-O-acetyl-2,3-di-O-benzyl-α-D-glucopyranoside (20a)
[0797] ##STR00085##
[0798] Tetrasaccharide 20a was prepared according to the following reactions sequence:
TABLE-US-00002 Sequence Module Details Condition I 1 Building block 35 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min II 1 Building block 32 T1 = −30° C., T2 = −10° C. 2 Fmoc Removal r.t for 5 min III 1 Building block 42 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min IV 1 Building block 40 T1 = −40° C., T2 = −20° C. 2 Fmoc Removal r.t for 5 min
[0799] 16% from the resin, MS ESI+-HRMS m/z [M+Na].sup.+ calcd for C.sub.112H.sub.119NO.sub.28Na 1948.7816, found 1948.7796.
Example 1-37
Preparation of 5-amino pentanyl α-D-galactopyranosyl-(1→4)-β-D-glucopyranosyluronic acid-(1→4)-β-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (20)
[0800] ##STR00086##
[0801] Tetrasaccharide 20a was subjected to the general deprotection procedure to afford tetrasaccharide 20: 40% MS ESI+-HRMS m/z [M+H]+ calcd for C.sub.29H.sub.52NO.sub.22 766.2975, found 766.2964.
Example 1-38
Preparation of N-benzyloxycarbonyl-5-amino-pentanyl 3,6-di-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate-(1→4)-2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranoside (21a):
[0802] ##STR00087##
TABLE-US-00003 Sequence Module Details Condition I 1 Building block 32 T1 = −30° C., T2 = −10° C. 2 Fmoc Removal r.t for 5 min II 1 Building block 42 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min III 1 Building block 41 T1 = −40° C., T2 = −20° C. 2 Fmoc Removal r.t for 5 min IV 1 Building block 37 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min
[0803] 20% from the resin, MS ESI+-HRMS m/z [M+Na]+ calcd for C.sub.112H.sub.119NO.sub.25Na 1948.7816, found 1950.7906
Example 1-39
Preparation of 5-Amino pentanyl α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→4)-β-D-glucopyranosyluronic acid-(1→4)-β-D-glucopyranoside (21)
[0804] ##STR00088##
[0805] Tetrasaccharide 21a was subjected to the general deprotection procedure to afford tetrasaccharide 21: 42% MS ESI+-HRMS m/z [M+I-1]+calcd for C.sub.29H.sub.52NO.sub.22 766.2975, found 766.2977.
Example 1-40
Synthesis of N-benzyloxycarbonyl-5-amino-pentanyl 2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl-(1→4)-6-O-acetyl-3,4-di-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl uronate (22a)
[0806] ##STR00089##
[0807] Tetrasaccharide 22a was synthesized according to the following reactions sequence:
TABLE-US-00004 Sequence Module Details Condition I 1 Building block 42 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min II 1 Building block 41 T1 = −40° C., T2 = −20° C. 2 Fmoc Removal r.t for 5 min III 1 Building block 35 T1 = −30° C., T2 = 0° C. 2 Fmoc Removal r.t for 5 min IV 1 Building block 32 T1 = −30° C., T2 = −10° C. 2 Fmoc Removal r.t for 5 min
[0808] 21% from the resin, MS ESI+-HRMS m/z [M+Na].sup.+ calcd for C.sub.110N.sub.117NO.sub.27Na 1906.7711, found 1906.7624.
Example 1-41
Synthesis of 5-amino pentanyl 13-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl uronic acid (22)
[0809] ##STR00090##
[0810] Tetrasaccharide 22a was subjected to the general deprotection procedure to afford tetrasaccharide 22: 52% MS ESI+-HRMS m/z [M+H].sup.+ calcd for C.sub.29H.sub.52NO.sub.22 766.2975, found 766.2988.
[0811] Further examples of inventive saccharides according to the procedures described in
Examples 1-28 to 1-34
[0812] ##STR00091##
[0813] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.41F1.sub.71NO.sub.32 1090.3959;
[0814] 5-amino pentanyl α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4-α-D-galactopyranoside;
##STR00092##
[0815] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.42H.sub.73NO.sub.32 1104.4116;
[0816] 5-amino pentanyl galactopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-gl ucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside;
##STR00093##
[0817] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.41H.sub.69NO.sub.33 1104.3752;
[0818] 5-amino pentanyl β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)- β-D-glucopyranoside;
##STR00094##
[0819] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.41H.sub.69NO.sub.33 1104.3752;
[0820] 5-amino pentanyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid;
##STR00095##
[0821] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.42H.sub.75NO.sub.31 1090.4323;
[0822] 5-amino pentanyl α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4-α-D-galactopyranoside;
##STR00096##
[0823] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.41H.sub.73NO.sub.31 1076,4167.
[0824] 5-amino pentanyl β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside
##STR00097##
[0825] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.41H.sub.73NO.sub.31 1076.4167.
[0826] 5-amino pentanyl β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00098##
[0827] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.42H.sub.75NO.sub.31 1090.4323;
[0828] 5-amino pentanyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1-4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside (70);
##STR00099##
[0829] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.33H.sub.57NO.sub.27 890.3118;
[0830] 3-aminopropyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranoside;
##STR00100##
[0831] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.34H.sub.59NO.sub.27 914.3274;
[0832] 5-amino pentanyl α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside;
##STR00101##
[0833] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.33H.sub.57NO.sub.27 890.3118;
[0834] 3-aminopropyl β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00102##
[0835] MS ESI+-HRMS m/z [M+H].sup.+ calcd for: C.sub.35H.sub.59NO.sub.28 942.3224;
[0836] 5-amino pentanyl β-D-glucopyranosyluronic acid-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyluronic acid;
##STR00103##
[0837] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.34H.sub.61NO.sub.26 890.3482;
[0838] 4-aminobutyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)- α-D-glucopyranosyl-(1.fwdarw.4-α-D-galactopyranoside;
##STR00104##
[0839] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.36H.sub.65NO.sub.26 928.3795;
[0840] 6-amino hexanyl α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside;
##STR00105##
[0841] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.33H.sub.59NO.sub.26 886.3325;
[0842] 3-aminopropyl β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00106##
[0843] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.35H.sub.63NO.sub.26 914.3638;
[0844] 5-amino pentanyl β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00107##
[0845] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.28F1.sub.51NO.sub.21 738.2954;
[0846] 4-aminobutyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside;
##STR00108##
[0847] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.27H.sub.49NO.sub.21 724.2797;
[0848] 3-aminopropyl α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00109##
[0849] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.29H.sub.53NO.sub.21 752.3110;
[0850] 6-amino hexanyl β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00110##
[0851] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.401-1.sub.71NO.sub.31 1062.4010;
[0852] 4-amino butyl β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl -(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranoside;
##STR00111##
[0853] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.39H.sub.69NO.sub.31 1048.3854;
[0854] 3-aminopropyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside;
##STR00112##
[0855] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.39H.sub.69NO.sub.31 1048.3854;
[0856] 3-aminopropyl α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00113##
[0857] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.36H.sub.65NO.sub.26 928.3795;
[0858] 6-amino hexanyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00114##
[0859] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.33H.sub.59NO.sub.26 886.3325;
[0860] 3-aminopropyl α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4-α-D-galactopyranoside;
##STR00115##
[0861] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.34H.sub.61 NO.sub.26 900.3482;
[0862] 4-aminobutyl β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside;
##STR00116##
[0863] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.28H5.sub.1NO.sub.21 738.2954;
[0864] 4-aminobutyl β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranoside;
##STR00117##
[0865] MS ESI+-HRMS m/z [M+H]+ calcd for: C.sub.30H.sub.55NO.sub.21 766.3267;
[0866] 6-aminohexanyl α-D-galactopyranosyl-(1.fwdarw.4)-β-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranosyl-(1.fwdarw.4)-α-D-glucopyranoside.
[0867] Biological Experiments
Example 2-1
Synthesis of Microarrays using CodeLink NHS Slides
[0868] The indicated glycans were spotted onto CodeLink NHS slides using an automatic piezoelectric arraying robot (Scienion, Berlin, Germany) and incubated for 24 h (1% w/v in PBS) at room temperature. Slides were incubated in blocking buffer (100 mM ethanolamine in 50 mM NaPi pH>9) for 30 min at room temperature, washed three times each with water and ethanol, and dried. Slides were then blocked with 1% (w/v) bovine serum albumin in phosphate buffered saline for 1 h at 37° C., washed with water three times and dried.
Example 2-2
Binding Experiments using the Microarrays Synthesized According to the Procedure Described at Example 2.1
[0869] Binding experiments were performed by incubating microarray slides coated with the saccharides of general formula (I) with either a rabbit anti-SP8 typing serum or human pneumococcal reference serum 007sp (pooled sera of 287 humans immunized with Pneumovax® vaccine purchase from National Institute for Biological Standards and Control) in the dilutions indicated in the presence or absence of native SP8 polysaccharide, and using fluorescently labeled anti-rabbit (goat anti-rabbit IgG-FITC, abcam ab6717) or anti-human secondary antibodies (Alexa Fluor 488 goat anti-human IgM, Invitrogen A21215; Alexa Fluor 647 goat anti-human IgG, Invitrogen A21445).
Example 2-3
Conjugation of Synthetic Tetrasaccharides 10 and 18 to CRM.SUB.197 .using Disuccinimidyl Adipate:
[0870] ##STR00118## ##STR00119##
[0871] To a stirred solution of disuccinimidyl adipate (10 mg, 29 μmol) and triethylamine (10 μL, 72 μmol) in anhydrous DMSO (150 μL) was added at room temperature dropwise a suspension of tetrasaccharide 10 or 18 (approx. 2 mg, 2.8 μmol) in anhydrous DMSO (150 μL). The reaction was stirred for 2 h at that temperature under an Argon atmosphere and treated with a 100 mM sodium phosphate buffer pH 7.4 (NaPi, 200 μL). The mixture was extracted with chloroform (10 mL) and the phases separated by centrifugation (2 min, 1800 g, room temperature). The organic phase was discarded and the extraction step was repeated two times. The aqueous layer was clarified by centrifugation in a 1.5 mL reaction tube (1 min, 14500 g, room temperature) and added to a stirring solution of CRM.sub.197 (1 mg, 17.3 nmol) in NaPi (1 mL). The mixture was stirred for 16 h at room temperature and dialyzed using a centrifugal filter (10 kDa MWCO, Millipore, Darmstadt, Germany). The conjugate was characterized by MALDI-MS:
[0872] Conjugate 58: ca. 67000 m/z (incorporation of 10.7 tetrasaccharide molecules on average)
[0873] Conjugate 59: ca. 66000 m/z (incorporation of 9.6 tetrasaccharide molecules on average)
Example 2-4
Immunization Procedure
[0874] Mice (6-8 week old female Balb/c mice, Charles River) were immunized s. c. with CRM-Sp8 conjugates obtained at example 2.3 (corresponding to 4 pg synthetic glycan) formulated either with Freund's adjuvant (Sigma-Aldrich, St. Louis, US), Alum (Alhydrogel, Brenntag) or without adjuvant at a total volume of 100 μL at days 0, 14 and 28. The immune response was monitored by glycan array. The conjugate 59 induced an oligosaccharide-specific immune response in mouse #1160 receiving the said conjugate formulated with Freund's adjuvant. Importantly, a robust immune response was observed in immune serum from mouse #1160 against the native Sp8 polysaccharide (see
Example 2-5
Generation of Monoclonal Antibodies
[0875] Monoclonal antibodies were prepared using BM-Condimed H1 (Roche, Penzberg, Germany) according to the manufacturer's instructions. Following fusion, single clones were generated using limited dilution and two subsequent rounds of subcloning. Antibody production was monitored by glycan array and ELISA. 33 clones were eventually isolated that produced mAbs recognizing both tetrasaccharide 19 and Sp8 native polysaccharide.
[0876] Clones 1H8 and 1F1 were expanded in serum-free medium. MAb 1H8 was purified from the cell culture supernatant using a Protein G Antibody Purification kit (Pro-Chem, Littleton, USA) (see
Example 2-6
Enzyme-Linked Immunosorbent Assay (ELISA)
[0877] ELISA was performed using high-binding polystyrene 96-well plates (Corning, Corning, US). Plates were coated using native Sp8 polysaccharide (SSI Diagnostica, Kopenhagen) at a concentration of 10 μg/mL in PBS for 20 h at 4° C. Plates were blocked with 10% (v/v) fetal calve serum in PBS for 1 h at 37° C. and washed once with PBS containing 0.1′)/0 (v/v) Tween 20 (PBS-T). Cell culture supernatants of anti-Sp8 mAbs (50 μL) were applied. Plates were incubated for 1 h at 37° C., washed with PBS-T three times and treated with a horseradish peroxidase (HRP)-labeled secondary antibody (goat anti-mouse IgG HRP conjugate, dianova, Hamburg, Germany). Plates were washed with PBS-T three times and HRP activity was measured with TMB substrate (BD Biosciences, San Jose, US) according to the manufacturer's instructions. Monoclonal antibodies generated from #1160 specifically recognized both synthetic saccharide 19 and the native Sp8 polysaccharide, as assessed by ELISA.
Example 2-7
Surface Plasmon Resonance
[0878] Surface Plasmon Resonance was performed on a Biacore T100 instrument (GE Healthcare). Analysis was performed using the Mouse Antibody Capture Kit and Amine Coupling Kit (GE Healthcare) for immobilization. About 10000 response units (RU) of capture antibody were immobilized. A commercial mouse IgG (cat. no. 026502, Invitrogen, Carlsbad, US) was immobilized as a dummy in a reference cell (about 10000 RU). About 800 RU of mAb 1H8 were captured prior to every run using a mAb concentration of 50 μg/mL. Runs were performed using PBS as a running buffer and a flow rate of 30 μL/min with 60 s association and 120 s dissociation periods, respectively. Polysaccharides were used at a concentration of 10 μg/mL and saccharide 18 at 20 μM in PBS. Monoclonal antibody mAb 1H8 specifically recognizes both synthetic saccharide 18 and the native Sp8 polysaccharide (see
Example 2-8
Immunofluorescence of UV-inactivated S. pneumoniae
[0879] S. pneumoniae serotype 8 (ATCC 6308) or serotype 1 (ATCC 6301) (approx. 4×10.sup.8 cfu/mL) were inactivated by irradiation with 254 nm for 10 min in PBS at room temperature. Cells were harvested by centrifugation, washed once with PBS and frozen in Todd Hewitt Broth containing 0.5% (w/v) yeast extract and 20% (v/v) glycerol. For immunofluorescence, bacteria (8×10.sup.8 cfu ST8 or 4×10.sup.8 cfu ST1) were thawed, harvested by centrifugation (16800 g, 15 min, r.t.) and washed once in buffer A (50 mM NaHCO.sub.3, 100 mM NaCl, pH 7.5). Cells were resuspended in buffer A (1 mL) and treated with a fluorescein isothiocyanate (FITC, Sigma-Aldrich) solution (10 mg/mL in DMSO) to a final concentration of 0.1 mg/mL. Bacteria were labeled in the dark for 1 h at 37° C., harvested by centrifugation and washed twice with 0.25% (w/v) BSA in PBS (1 mL). Labeling was monitored by fluorescence microscopy using an Axio Imager.M2 system equipped with a LSM 700 confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Cells were suspended in 1% (w/v) BSA in PBS (1 mL for ST8, 0.5 mL for ST1) and the suspension was distributed into two aliquots. The suspensions were treated with mAb 1H8 or mAb 1E12 (IgG1) against Yersinia pestis lipopolysaccharide core trisaccharide as an isotype control to a final mAb concentration of 10 μg/mL. Bacteria were incubated in the dark for 16 h at 4° C. under agitation and washed with 1% (w/v) BSA in PBS (0.5 mL). The cells were suspended in a solution of goat anti-mouse IgG-Alexa635 conjugate (1:100 dilution in 200 μL 1% (w/v) BSA in PBS, Invitrogen), incubated in the dark for 1.5 h at room temperature and washed with 1′)/0 (w/v) BSA in PBS and PBS (0.5 mL, respectively). Fluorescently labeled bacteria were visualized by fluorescence microscopy and images were processed with using Zen 2011 software (Carl Zeiss Microscopy GmbH). As shown in
Example 2-9
Assessment of the Binding of mAbs 1H8 and 1F1 to S. pneumoniae Serotype 8 Bacteria by Flow Cytometry
[0880] S. pneumoniae serotype 8 (ATCC 6308), serotype 1 (ATCC 6301) or serotype 3 (PN36, NCTC7978) were UV-inactivated, FITC-labeled and treated with a fluorescent secondary antibody (anti-mouse IgG-Alexa635 conjugate or anti-mouse IgM-Alexa680 conjugate, Invitrogen) as described before (see Example 2.8). Flow cytometry was performed using a FACSCanto II flow cytometer (BD Pharmingen, Heidelberg, Germany) and analyzed using FlowJo software (Tree Star Inc., Ashland, Oreg., USA). Both monoclonal antibodies 1H8 and 1F1 bind to S. pneumoniae serotype 8, but do not bind to S. pneumoniae serotypes 1 or 3 (see
Example 2-10
Opsonophagocytotic Killing Assay
[0881] A opsonophagocytotic killing assay was employed as described in Romero-Steiner et al., Clin. Diagn. Lab. Immunol., 1997, 4. Briefly, HL-60 cells were differentiated for one week with N,N-dimethylformamide as reported (Romero-Steiner et al., 1997), washed twice with OPKA buffer (Hanks' buffer with 0.1% (w/v) gelatin) and diluted to a density of 10.sup.7 cells/mL in the same buffer directly before use. Bacteria were grown in growth medium (Todd-Hewitt broth+0.5% (w/v) yeast extract) at 37° C/5% CO.sub.2 to log phase (OD 0.2-0.3), diluted in freezing medium (growth medium with 15% (v/v) glycerol) to a density of approx. 10.sup.6 cells/mL and frozen in 0.5 mL aliquots at −80° C. Bacteria were diluted with OPKA buffer and aliquoted (1000 cells in 20 μL each) in a 96 well-plate. Bacterial suspensions were treated with appropriate antibody or antisera (1:4) dilutions and incubated for 15 min at 37° C. Complement source (baby rabbit complement, CedarLane, 10 μL) and differentiated HL-60 cell suspension (40 μL, phagocyte/bacteria ratio 400:1) were added and the suspensions incubated for 45 min at 37° C. with shaking (220 rpm). Opsonophagocytosis was performed in triplicates. 10% of the contents of each well were plated on Columbia Agar plates and colonies were counted after 10-12 h incubation at 37° C./5% CO.sub.2. Control wells lacked either antibody or complement sources.
[0882] Monoclonal antibody 1H8 raised against synthetic S. pneumoniae serotype 8 tetrasaccharide 18, but not an isotype control mAb, exhibited a similar opsonophagocytotic killing capacity in the presence of complement and differentiated HL-60 cells as control sera in all concentrations tested (rabbit S. pneumoniae type 8 typing serum (1:4 dilution) and human antiserum 007sp (1:4 dilution), respectively) (see
Example 2-11
Glycan Array Analysis using the Monoclonal Antibodies mAbs 1H8 and 1F1
[0883] Binding experiments were performed by incubating microarray slides synthesized as shown in Example 2.1 in the presence or absence of SP8 capsular polysaccharide and including compounds 19, 49, 90, 60, 62, 55 and 57.
[0884] As shown by
[0885] As shown in
Example 2-12
Assessment of the Binding of mAb 28H11 to the Saccharides of the Present Invention by Microarray
[0886] Glycan arrays were fabricated as shown above (Example 2-1 and 2-2), except that a murine monoclonal antibody 28H11 (IgM) raised against native Sp8 polysaccharide (Yano and Pirofski (2011), Clin. Vaccine Immunol., 18 (1), 59-66) was used for binding at different dilutions with or without addition of 10 μg/mL S. pneumoniae type 8 CPS. A Donkey anti-Mouse IgM Alexa Fluor® 594 conjugate (dianova, Hamburg, Germany) was used as a secondary antibody for detection.
[0887] MAb 28H11, a murine IgM that has been raised against native S. pneumoniae type 8 CPSs, is well-characterized and protects mice from infection with live S. pneumoniae type 8 pneumococci in various settings (Yano and Pirofski (2011), Clin. Vaccine Immunol., 18 (1), 59-66). Glycan microarray analysis revealed a robust interaction of mAb 28H11 with saccharide 19 that was specific to S. pneumoniae type 8, as shown by the ablation of binding by native S. pneumoniae type 8 CPSs of up to 10 μg/mL (see
Example 2-13
Conjugation of a Oligosaccharides to CRM.SUB.197 .using bis(4-nitrophenyl)adipate
[0888] To a stirred solution of the oligosaccharide (amount specified below; typically 0.2 pmol) in a 1:3 (v/v) mixture of anhydrous DMSO and anhydrous pyridine (100-200 μL) were added bis(4-nitrophenyl)adipate (12 equivalents relative to the amount of sugar; typically 2.4 μmol) and triethylamine (10 μL). The reaction was stirred for 2 h at room temperature under an Argon atmosphere. The mixture was shock-frozen and lyophilized. The residue was triturated with chloroform (4×0.5 mL) and dichloromethane (4×0.5 mL), transferred to a new reaction tube using DMSO as a solvent and lyophilized again. CRM.sub.197 (3 mg, 52 nmol) was dialyzed twice against 0.1 M sodium phosphate buffer pH 8.0 using a centrifugal filter (10 kDa MWCO, Millipore, Darmstadt, Germany), concentrated to approx. 300 μL and added to the activated oligosaccharide. The mixture was stirred at room temperature for 16 h and dialyzed (see above) four times against water. An aliquot was taken for characterization and the mixture was dialyzed three times against phosphate-buffered saline. The glycoconjugates were characterized by MALDI-TOF MS (see
[0889] Exact Quantities of Oligosaccharides Used:
[0890] Tetrasaccharide 18: 2.6 μmol (1.9 mg)
[0891] Tetrasaccharide 60: 2.1 μmol (1.5 mg)
[0892] Hexasaccharide 57: 1.9 μmol (2.0 mg)
[0893] Conjugate CRM.sub.197-18: ca. 65503 m/z (incorporation of 8.8 tetrasaccharide molecules on average);
[0894] Conjugate CRM.sub.197-60: ca. 68281 m/z (incorporation of 12.9 tetrasaccharide molecules on average);
[0895] Conjugate CRM.sub.197-57: ca. 63535 m/z (incorporation of 4.6 hexasaccharide molecules on average).
##STR00120## ##STR00121## ##STR00122##
Example 2-14
Evaluation of the Immune Response Against the Conjugates of Example 2-13 in Rabbits
[0896] Rabbits (n=3 per group) were immunized subcutaneously at multiple sites with glycoconjugates CRM197-18, CRM197-60 or CRM197-57 (10 μg glycan per dose) or CRM.sub.197 (100 μg) at days 0 and 14. Serum was collected at days 0, 14 and 21. The results of the immunization studies are summarized by
[0897] All rabbits immunized with conjugates show a marked immune response against S. pneumoniae type 8 CPS-related oligosaccharides and S. pneumoniae type 8 CPS. Hence, all conjugates induce immunity against S. pneumoniae bacteria in rabbits.