VACCINES AGAINST CARBAPENEM-RESISTANT KLEBSIELLA PNEUMONIAE
20180078630 ยท 2018-03-22
Inventors
- Peter H. Seeberger (Kleinmachnow, DE)
- Claney Lebev Pereira (Berlin, DE)
- Guozhi Xiao (Berlin, DE)
- Naeem Khan (Berlin, DE)
- Chakkumkal Anish (Den Haag, NL)
Cpc classification
C07H15/04
CHEMISTRY; METALLURGY
A61K2039/6037
HUMAN NECESSITIES
A61K31/702
HUMAN NECESSITIES
A61K31/7028
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to synthetic saccharides of general formula (I): V*[U.sub.x+2U.sub.x+1U.sub.x].sub.nVO-L-NH.sub.2 that are related to carbapenem-resistant Klebsiella pneumoniae capsular polysaccharide and conjugates thereof. Said conjugates and pharmaceutical composition containing said conjugates are useful for prevention and/or treatment of diseases associated with carbapenem-resistant Klebsiella pneumoniae. Furthermore, the synthetic saccharides of general formula (I): V*[U.sub.x+2U.sub.x+1U.sub.x].sub.nVO-L-NH.sub.2 are useful as marker in immunological assays for detection of antibodies against carbapenem-resistant Klebsiella pneumoniae bacteria.
Claims
1. A synthetic saccharide of general formula (I)
V*[U.sub.x+2U.sub.x+1U.sub.x].sub.nVO-L-NH.sub.2(I) wherein x is an integer selected from 1, 2 and 3; n is an integer selected from 1, 2 and 3; ##STR00138## represents a bond ##STR00139## V represents a bond, U.sub.x+2 or U.sub.x+2U.sub.x+1; V* represents H, HU.sub.x- or HU.sub.x+1U.sub.x; L represents a linker; or a diastereoisomer or a pharmaceutically acceptable salt thereof.
2. The synthetic saccharide of general formula (I) according to claim 1, wherein ##STR00140## or a diastereoisomer or a pharmaceutically acceptable salt thereof.
3. The synthetic saccharide of general formula (I) according to claim 1, wherein ##STR00141## or a diastereoisomer or a pharmaceutically acceptable salt thereof.
4. The synthetic saccharide of general formula (I) according to claim 1, wherein ##STR00142## or a diastereoisomer or a pharmaceutically acceptable salt thereof.
5. The synthetic saccharide according to claim 1 of general formula (II) ##STR00143## wherein ##STR00144## and L have the meanings as defined in claim 1, or a diastereoisomer or a pharmaceutically acceptable salt thereof.
6. The synthetic saccharide according to claim 1, wherein -L- is selected from: -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, -L.sup.a-L.sup.d-L.sup.e-; -L.sup.a- is selected from: (CH.sub.2).sub.o, (CH.sub.2CH.sub.2O).sub.oC.sub.2H.sub.4, (CH.sub.2CH.sub.2O).sub.oCH.sub.2; -L.sup.b- represents O; -L.sup.d- is selected from: (CH.sub.2).sub.q, (CF.sub.2).sub.q, (CH.sub.2CH.sub.2O).sub.qC.sub.2H.sub.4, and (CH.sub.2CH.sub.2O).sub.qCH.sub.2; -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 and (CH.sub.2).sub.p1O(CH.sub.2).sub.p2; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, and 6
7. A conjugate comprising the saccharide according to claim 1.
8. The conjugate according to claim 7, of general formula (VI)
[V*[U.sub.x+2U.sub.x+1U.sub.x].sub.nVO-L-NH-T].sub.m-CRM.sub.197(VI) wherein m is comprised between 2 and 18; -T- is selected from: ##STR00145## a represents an integer from 1 to 10; b represents an integer from 1 to 4; and V*-, U.sub.x+2, U.sub.x+1, U.sub.x, x, V, n and L have the meanings as defined in any one of the claims 1-5.
9. The conjugate according to claim 7 of general formula (X) ##STR00146## wherein ##STR00147## and L have the meanings as defined in claim 1; m is comprised between 2 and 18; -T- is selected from: ##STR00148## a represents an integer from 1 to 10; and b represents an integer from 1 to 4.
10. The conjugate according to claim 8, wherein -T- represents ##STR00149## and a is an integer selected from 2, 3, 4, 5 and 6.
11. The conjugate according to claim 8, wherein -L- is selected from: -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, -L.sup.a-L.sup.d-L.sup.e-; -L.sup.a- is selected from: (CH.sub.2).sub.o, (CH.sub.2CH.sub.2O).sub.oC.sub.2H.sub.4, (CH.sub.2CH.sub.2O).sub.oCH.sub.2; -L.sup.b- represents O; -L.sup.d- is selected from: (CH.sub.2).sub.q, (CF.sub.2).sub.q, (CH.sub.2CH.sub.2O).sub.qC.sub.2H.sub.4, and (CH.sub.2CH.sub.2O).sub.qCH.sub.2; -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 and (CH.sub.2).sub.p1O(CH.sub.2).sub.p2; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, and 6.
12. A method for raising a protective immune response in a human or animal host comprising administering to the human or animal host a saccharide according to claim 1 or a conjugate comprising the saccharide.
13. A method for increasing immunogenicity against or treating a disease associated with carbapenem-resistant Klebsiella pneumoniae comprising administering to a patient a saccharide according to claim 1.
14. A pharmaceutical composition comprising the saccharide according to claim 1 and/or a conjugate comprising the saccharide together with at least one pharmaceutically acceptable adjuvant or excipient.
15. (canceled)
16. A method comprising the use of the saccharide according to claim 1 as marker in immunological assays for detection of antibodies against carbapenem-resistant Klebsiella pneumoniae.
17. A method of synthesis of a saccharide of general formula (II) ##STR00150## wherein ##STR00151## and L have the meanings as defined in claim 1, comprising the following steps: A) reacting a compound 1 of the formula: ##STR00152## wherein P.sup.1-P.sup.3 represent protecting groups and P.sup.4 is selected from: CH.sub.3, ##STR00153## with a compound 2 of the formula: ##STR00154## wherein P.sup.5-P.sup.7 represent protecting groups and LG.sup.1 represents a leaving group selected from: ##STR00155## to provide a compound 3 of formula: ##STR00156## wherein P.sup.1-P.sup.3, P.sup.5-P.sup.7 represent protecting groups and P.sup.4 is selected from: ##STR00157## and performing selective removal of protective group P.sup.2 on compound 3 to obtain compound 4 of formula: ##STR00158## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.7 represent protecting groups and P.sup.4 is selected from: ##STR00159## and reacting compound 4 with compound 5 of formula: ##STR00160## wherein P.sup.8-P.sup.11 represent protecting groups and LG.sup.2 represents a leaving group selected from: ##STR00161## to obtain compound 6 of formula: ##STR00162## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.11 represent protecting groups and P.sup.4 is selected from: ##STR00163## and converting compound 6 to compound 7 of formula: ##STR00164## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.11 represent protecting groups and LG.sup.3 represents a leaving group selected from: ##STR00165## and B1) reacting compound 2 with compound 8 of formula: ##STR00166## wherein P.sup.12 and P.sup.13 represent protecting groups to provide compound 9 of formula: ##STR00167## wherein P.sup.5-P.sup.7, P.sup.12 and P.sup.13 represent protecting groups; and performing selective removal of protective group P.sup.5 on compound 9 to provide compound 10 of formula: ##STR00168## wherein P.sup.6, P.sup.7, P.sup.12 and P.sup.13 represent protecting groups; and B2) reacting compound 2 with compound 10 to obtain compound 11 of formula: ##STR00169## wherein P.sup.5-P.sup.7, P.sup.12 and P.sup.13 represent protecting groups; and performing selective removal of protective group P.sup.5 on compound 11 to provide compound 12 of formula: ##STR00170## wherein P.sup.6, P.sup.7, P.sup.12 and P.sup.13 represent protecting groups; and B3) reacting compound 2 with compound 12 to obtain compound 13 of formula: ##STR00171## wherein P.sup.5-P.sup.7, P.sup.12 and P.sup.13 represent protecting groups; and performing selective removal of protective group P.sup.5 on compound 13 to provide compound 14 of formula: ##STR00172## wherein P.sup.6, P.sup.7, P.sup.12 and P.sup.13 represent protecting groups; and C1) reacting compound 7 obtained at step A with compound 10 obtained at step B1 to provide compound 15 of formula: ##STR00173## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.13 represent protecting groups; or C2) reacting compound 7 obtained at step A with compound 12 obtained at step B2 to provide compound 16 of formula: ##STR00174## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.13 represent protecting groups; or C3) reacting compound 7 obtained at step A with compound 14 obtained at step B3 to provide compound 17 of formula: ##STR00175## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.13 represent protecting groups; and D) performing removal of protecting groups P.sup.1, P.sup.3, P.sup.5-P.sup.13 on compounds 15, 16 and 17 to provide the saccharides of general formula (II).
18. An intermediate compound of formula (7), (10), (15), (12), (16), (14) or (17): ##STR00176## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.11 represent protecting groups and LG.sup.3 represents a leaving group selected from: ##STR00177## wherein P.sup.6, P.sup.7, P.sup.12 and P.sup.13 represent protecting groups and L has the meaning as defined in claim 1; ##STR00178## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.13 represent protecting groups and L has the meaning as defined in claim 1; ##STR00179## wherein P.sup.6, P.sup.7, P.sup.12 and P.sup.13 represent protecting groups and L has the meaning as defined in claim 1; ##STR00180## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.13 represent protecting groups and L has the meaning as defined in claim 1; ##STR00181## wherein P.sup.6, P.sup.7, P.sup.12 and P.sup.13 represent protecting groups and L has the meaning as defined in claim 1; ##STR00182## wherein P.sup.1, P.sup.3, P.sup.5-P.sup.13 represent protecting groups and L has the meaning as defined in claim 1.
19. The intermediate compound according to claim 18, wherein -L- is selected from: -L.sup.a-, -L.sup.a-L.sup.e-, -L.sup.a-L.sup.b-L.sup.e-, -L.sup.a-L.sup.d-L.sup.e-; -L.sup.a- is selected from: (CH.sub.2).sub.o, (CH.sub.2CH.sub.2O).sub.oC.sub.2H.sub.4, (CH.sub.2CH.sub.2O).sub.oCH.sub.2; -L.sup.b- represents O; -L.sup.d- is selected from: (CH.sub.2).sub.q, (CF.sub.2).sub.q, (CH.sub.2CH.sub.2O).sub.qC.sub.2H.sub.4, and (CH.sub.2CH.sub.2O).sub.qCH.sub.2; -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 and (CH.sub.2).sub.p1O(CH.sub.2).sub.p2; and o, q, p1 and p2 are independently of each other an integer selected from 1, 2, 3, 4, 5, and 6.
Description
DESCRIPTION OF THE FIGURES
[0156]
[0157]
[0158]
[0159]
[0160] (A) C57BL/6 mice (n=3) were immunized subcutaneously with 3 doses, each containing 3 g of hexasaccharide 23* in Freund's adjuvant. Hyperimmune sera were pooled and the total IgG, IgM and IgG isotypes (IgG1, IgG2a and IgG3) endpoint titer was determined by glycan microarray (B - F); respectively. Total serum IgG and IgM titer at day 49 are shown in (G and H), respectively. Antibody titers were plotted as MFI values of n=3SEM. Bars represent mean values+SEM *P0.1, **P0.05, ***P0.01 and ****P0.001.
[0161]
[0162] (A) Immunization pattern.
[0163] (B) Left panel showing printing pattern of microarray slides [0164] position 1: hexasaccharide 23* Gal(1-3)[Rha(1-4)]GalA(1-2)Rha(1-2)Rha(1-2)Rha(1-1)aminopentanol), [0165] position 2: trisaccharide Rha(1-2)Rha(1-2)Rha(1-1)aminopentanol, [0166] position 3: trisaccharide Gal(1-3)[Rha(1-4)]GalA(1-2)aminopentanol), [0167] position 4: Polysaccharide (C200, clade 1) corresponding to CPS from carbapenem-resistant strain K. pneumoniae strain 34 (CPS-K34), [0168] position 5: Polysaccharide (clade 2) corresponding to CPS from K. pneumoniae, and [0169] position 6: Polysaccharide (non typable) corresponding to CPS from K. pneumoniae. [0170] Right panel is the representative microarray scanning with pooled sera from mice (n=3) immunized with the conjugate CRM.sub.197-23* with two booster immunization.
[0171] (C) The serum cross-reactivity with oligosaccharides expressed as MFI values of n=3SEM.
[0172] (D) The serum cross-reactivity with capsular polysaccharide from carbapenem-resistant strain K. pneumoniae strain 34 (CPS-K34) expressed as MFI values of n=3SEM.
[0173]
[0174] (A) Schematic representation of immunization and sera collection.
[0175] (B) The endpoint titer (total IgG) was determined in day 0, 14 and 21 pooled sera (n=3).
[0176] (C) The antibody response were analyzed at each time point and plotted as fold chance.
[0177] (D) The individual rabbit antibody titer at day 0 and 21. Each dot represents the individual animal. The data were plotted as geometric mean with 95% CI.
[0178]
[0179] (A) Printing pattern of microarray slides.
[0180] (B) Glycan microarray analysis after the incubation of slide(s) with pooled (n=3) anti-CRM.sub.197-23* conjugate sera (day 21).
[0181] (C) The mean fluorescence intensity (MFI) of each spot of 0.2 mM concentration.
[0182] 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.
[0183] 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.
[0184] 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
A. Chemical Synthesis
[0185] General Information:
[0186] Commercial grade solvents were used unless stated otherwise. Dry solvents were obtained from a Waters Dry Solvent System. Solvents for chromatography were distilled prior to use. Sensitive reactions were carried out in heat-dried glassware and under an argon atmosphere. Analytical thin layer chromatography (TLC) was performed on Kieselgel 60 F254 glass plates precoated with a 0.25 mm thickness of silica gel. Spots were visualized by staining with vanillin solution (6% (w/v) vanillin and 10% (v/v) sulfuric acid in 95% EtOH) or Hanessian's stain (5% (w/v) ammonium molybdate, 1% (w/v) cerium(II) sulfate and 10% (v/v) sulfuric acid in water). Silica column chromatography was performed on Fluka Kieselgel 60 (230-400 mesh). .sup.1H, .sup.13C and two-dimensional NMR spectra were measured with a Varian 400-MR spectrometer at 296 K. Chemical shifts (d) are reported in parts per million (ppm) relative to the respective residual solvent peaks (CDCl.sub.3: d 7.27 in .sup.1H and 77.23 in .sup.13C NMR; CD.sub.3OD: d 3.31 in .sup.1H and 49.15 in .sup.13C NMR). The following abbreviations are used to indicate peak multiplicities: s singlet; d doublet; dd doublet of doublets; t triplet; dt doublet of triplets; q quartet; m multiplet. Coupling constants (J) are reported in Hertz (Hz). Optical rotation (OR) measurements were carried out with a Schmidt & Haensch UniPol L1000 polarimeter at A=589 nm and a concentration (c) expressed in g/100 mL in the solvent noted in parentheses. High resolution mass spectrometry (HRMS) was performed at the Free University Berlin, Mass. Spectrometry Core Facility, with an Agilent 6210 ESI-TOF mass spectrometer. Infrared (IR) spectra were measured with a Perkin Elmer 100 FTIR spectrometer.
Abbreviations
[0187] All: allyl;
TBAI: tetrabutylammonium iodide;
Bn: benzyl;
Cbz: benzyloxycarbonyl;
EtOAc: ethyl acetate;
Lev: levulinoyl;
PMB: 4-methoxybenzyloxyl;
Tr: triphenylmethyl.
Example A-1: Synthesis of 1,2-O-Isopropylidene-3-O-allyl-5-O-triphenylmethyl--
[0188] ##STR00102##
[0189] The alcohol 1* (Chem. Eur. J. 2013, 19, 3995-4002) (21 g, 48.6 mmol) was dissolved in dry DMF (140 mL) and cooled to 0 C. 60% NaH (3.88 g, 97 mmol) was slowly added. After 5 min, AllBr (8.4 mL, 97 mmol) and TBAI (1.79 g, 4.86 mmol) were added. The mixture was allowed to stir overnight at room temperature. The mixture was quenched with sat. NH.sub.4Cl and then poured into ice water. The mixture was extracted with CH.sub.2C2. The combined organic layer was washed with sat. NH.sub.4Cl and water, dried and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=20/1 to 10/1 to 1/1) to give target compound 2* (22.9 g, quant.). [].sub.D.sup.25=2.85 (c=1.07, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.46-7.21 (m, 15H), 5.88 (d, J=4.0 Hz, 1H), 5.71 (m, 1H), 5.13 (m, 2H), 4.54 (d, J=4.0 Hz, 1H), 4.38 (m, 1H), 4.04 (dd, J=5.6, 12.8 Hz, 1H), 3.98 (d, J=2.8 Hz, 1H), 3.93 (dd, J=5.6, 12.8 Hz, 1H), 3.50 (dd, J=5.2, 8.8 Hz, 1H), 3.31 (dd, J=7.6, 8.8 Hz, 1H), 1.54 (s, 3H), 1.33 (s, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 143.9, 134.2, 128.8, 127.9, 127.1, 117.6, 111.7, 105.0, 86.9, 82.6, 81.5, 79.4, 71.3, 60.7, 26.9, 26.3; HRMS (ESI): calcd for C.sub.30H.sub.32O.sub.5Na [M+Na].sup.+: 495.2147, found: 495.2167.
Example A-2: Synthesis of 3-O-Allyl-4,5-O-isopropylidene-
[0190] ##STR00103##
[0191] Acetonide 2* (9.5 g, 20.1 mmol) was dissolved in CH.sub.2Cl.sub.2 (80.4 mL) and cooled to 0 C. EtSH (29.7 mL, 402 mmol) and ZnBr.sub.2 (22.64 g, 101 mmol) were added and the mixture was stirred at 0 C. for 75 min. The reaction was quenched by adding 5% aq. ammonium hydroxide and the resulting slurry was diluted with CH.sub.2Cl.sub.2 and 1 M aq. HCl. The phases were separated and the aqueous layer was extracted with CH.sub.2C1.sub.2. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=1/3 to EtOAc) to give the alcohol (5.44 g, 18.36 mmol). pTsOH (524 mg, 2.75 mmol) was added to a solution of the above triol (5.44 g, 18.36 mmol) in acetone (124 mL). The mixture was stirred at room temperature for 12 h and the reaction was quenched with sat aq. NaHCO.sub.3. The suspension was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel (Hexane/EtOAc=4.5/1) to give alcohol 3* (4.13 g, 61% for two steps). [].sub.D.sup.25=43.76 (c=0.98, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 5.92 (m, 1H), 5.22 (d, J=18.8 Hz, 1H), 5.11 (d, J=10.4 Hz, 1H), 4.38 (m, 2H), 4.14 (dd, J=6.8, 12.4 Hz, 1H), 4.02 (m, 2H), 3.79 (dd, J=1.6, 6.8 Hz, 1H), 3.69 (t, J=8.0 Hz, 1H), 3.43 (dd, J=1.2, 8.4 Hz, 1H), 3.10 (br, 1H), 2.56-2.75 (m, 4H), 1.40 (s, 3H), 1.34 (s, 3H), 1.25 (t, J=7.2 Hz, 3H), 1.24 (t, J=7.6 Hz, 3H); .sup.13C NMR (101 MHz, cdcl.sub.3) 135.0, 117.0, 109.3, 78.7, 77.7, 73.4, 72.2, 65.9, 55.3, 26.7, 25.5, 25.2, 24.1, 14.6, 14.5; HRMS (ESI): calcd for C.sub.15H.sub.28O.sub.4S.sub.2Na [M+Na].sup.+: 359.1327, found: 359.1331.
Example A-3: Synthesis of 2-O-Benzyl-3-O-allyl-4,5-O-isopropylidene-
[0192] ##STR00104##
[0193] The alcohol 3* (4.12 g, 12.27 mmol) was dissolved in dry THF (40 mL) and cooled to 0 C. 60% NaH (0.98 g, 24.54 mmol) was slowly added. After 5 min, BnBr (2.2 mL, 18.41 mmol) and TBAI (0.29 g, 1.23 mmol) were added. The mixture was allowed to stir overnight at room temperature. The mixture was quenched with MeOH and concentrated in vacuo. The residue was diluted with EtOAc. The mixture was washed with water, dried and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=12/1) to give target compound 4* (5.2 g, 99%). [].sub.D.sup.25=11.80 (c=0.98, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.32-7.17 (m, 5H), 5.81 (m, 1H), 5.14 (d, J=17.2 Hz, 1H), 5.04 (d, J=10.4 Hz, 1H), 4.82 (d, J=11.2 Hz, 1H), 4.65 (d, J=11.2 Hz, 1H), 4.23 (m, 1H), 4.12 (m, 1H), 4.08 (d, J=4.0 Hz, 1H), 3.86 (t, J=6.4 Hz, 1H), 3.81 (t, J=4.0 Hz, 1H), 3.71 (t, J=8.0 Hz, 1H), 3.63 (t, J=5.6 Hz, 1H), 2.52-2.75 (m, 4H), 1.35 (s, 3H), 1.26 (s, 3H), 1.20 (t, J=7.2 Hz, 3H), 1.17 (t, J=7.2 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 138.3, 134.9, 128.2, 127.9, 127.6, 116.8, 108.8, 82.7, 79.9, 76.4, 74.7, 73.8, 65.8, 52.4, 26.5, 25.7, 25.6, 25.2, 14.5, 14.4; HRMS (ESI): calcd for C.sub.22H.sub.34O.sub.4S.sub.2Na [M+Na].sup.+: 449.1796, found: 449.1802.
Example A-4: Synthesis of 2-O-Benzyl-3-O-allyl-
[0194] ##STR00105##
[0195] Acetonide 4* (5.2 g, 12.19 mmol) was dissolved in 70% aq. HOAc (121 mL). The mixture was allowed to stir at 50 C. for 3h. The mixture was concentrated in vacuo. The residue was co-evaporated twice with toluene and then purified by column chromatography on silica gel (Hexanes/EtOAc=1/1.4) to give compound 5* (4.59 g, 98%). [].sub.D.sup.25=1.60 (c=0.78, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.33-7.20 (m, 5H), 5.80 (m, 1H), 5.13 (d, J=16.0 Hz, 1H), 5.07 (d, J=10.4 Hz, 1H), 4.81 (d, J=10.8 Hz, 1H), 4.67 (d, J=11.2 Hz, 1H), 4.21 (dd, J=5.6, 12.4 Hz, 1H), 3.97-4.03 (m, 3H), 3.76 (br, 1H), 3.58-3.69 (m, 3H), 2.54-2.75 (m, 6H), 1.19 (t, J=8.4 Hz, 3H), 1.15 (t, J=7.2 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 138.3, 134.5, 128.3, 127.9, 127.6, 117.7, 83.1, 80.9, 75.3, 74.1, 71.1, 64.4, 53.1, 26.0, 25.2, 14.5, 14.5; HRMS (ESI): calcd for C.sub.19H.sub.30O.sub.4S.sub.2Na [M+Na].sup.+: 409.1483, found: 409.1488.
Example A-5: Synthesis of 2-O-Allyl-3-O-benzyl-
[0196] ##STR00106##
[0197] NaIO.sub.4 (1.508 g, 7.05 mmol) in H.sub.2O (20 mL) was added to a solution of diol 5* (2.42 g, 6.27 mmol) in THF (79 mL) at room temperature. After stirring for 2 h, the mixture was diluted with CH.sub.2Cl.sub.2 and washed with sat aq. NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4, concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=7/1) to give aldehyde 6* (1.34 g, 60%) and the starting diol 5* (474 mg, 20%). [].sub.D.sup.25=1.27 (c=34.61, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 9.79 (s, 1H), 7.28-7.20 (m, 5H), 5.81 (m, 1H), 5.16 (d, J=17.2 Hz, 1H), 5.12 (d, J=10.4 Hz, 1H), 4.76 (d, J=11.2 Hz, 1H), 4.57 (d, J=11.2 Hz, 1H), 4.13 (dd, J=6.0, 12.8 Hz, 1H), 3.98-4.07 (m, 3H), 2.51-2.70 (m, 4H), 1.20 (t, J=7.2 Hz, 3H), 1.14 (t, J=7.2 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 201.8, 137.5, 133.9, 128.5, 128.3, 128.0, 118.6, 83.1, 83.0, 74.8, 72.9, 52.3, 26.1, 25.5, 14.5, 14.3; HRMS (ESI): calcd for C.sub.18H.sub.26O.sub.3S.sub.2Na [M+Na].sup.+: 377.1221, found: 377.1220.
Example A-6: Synthesis of (R)-4-Benzyl-3-((2S,3R,4S,5R)-4-allyloxy-5-benzyloxy-6,6-bis(ethylthio)-3-hydroxy-2-(4-methoxybenzyloxy)hexanoyl)oxazolidin-2-one (8*)
[0198] ##STR00107##
[0199] A stirred solution of di-isopropylamine (0.978 mL, 6.86 mmol) in THF (11 mL) was cooled to 20 C. and n-BuLi (2.74 mL, 2.5 M in hexane, 6.86 mmol) was added dropwise and stirred for 30 min, then re-cooled to 78 C. and auxiliary 7* (2.44 g, 6.86 mmol) as a solution in 5.5 mL THF was added to the mixture over a period of 20 min. After 1.5 h, aldehyde 6* (1.2 g, 3.43 mmol) was dissolved in 9.8 mL THF and added to the mixture over a period of 30 min. The reaction was quenched after 2 h by the addition of 33 mL sat. aq. NH.sub.4Cl-solution and then warmed to r.t., where the mixture was stirred for another 30 min. The mixture was extracted with CH.sub.2Cl.sub.2 (3150 mL) and dried over MgSO.sub.4. The solvent was removed in vacuo and the crude product was purified by flash column chromatography on silica gel (gradient Hexanes/EtOAc=8:1-3:1) to afford 8* (1.78 g, d.r.=10.4/1, 73%). [].sub.D.sup.25=30.3 (c=1.09, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.31-7.10 (m, 12H), 6.79 (d, J=8.4 Hz, 2H), 5.79 (m, 1H), 5.42 (br s, 1H), 5.18 (d, J=17.2 Hz, 1H), 5.01 (d, J=10.8 Hz, 1H), 4.81 (d, J=11.2 Hz, 1H), 4.67 (d, J=11.2 Hz, 1H), 4.62 (m, 1H), 4.55 (d, J=10.4 Hz, 1H), 4.31 (d, J=10.8 Hz, 1H), 4.28 (m, 1H), 4.17-3.91 (m, 7H), 3.70 (s, 3H), 3.11 (dd, J=2.8, 13.2 Hz, 1H), 2.81 (d, J=7.6 Hz, 1H), 2.71-2.49 (m, 5H), 1.17 (t, J=7.6 Hz, 3H), 1.14 (t, J=7.6 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 171.4, 159.6, 153.1, 138.4, 135.0, 134.9, 130.2, 129.5, 129.4, 129.0, 128.3, 128.2, 128.1, 127.9, 127.4, 127.3, 116.2, 113.9, 113.8, 83.0, 78.5, 77.5, 74.7, 72.9, 72.4, 71.9, 66.9, 55.3, 54.9, 52.8, 37.8, 25.8, 25.3, 14.5, 14.5; HRMS (ESI): calcd for C.sub.38H.sub.47NO.sub.8S.sub.2Na [M+Na].sup.+: 732.2641, found: 732.2650.
Example A-7: Synthesis of (R)-4-Benzyl-3-((2S,3R,4S,5R)-4-allyloxy-5-benzyloxy-6,6-bis(ethylthio)-3-levulynoxy-2-(4-methoxybenzyloxy)hexanoyl)oxazolidin-2-one (9*)
[0200] ##STR00108##
[0201] Alcohol 8* (1.65 g, 2.32 mmol) was dissolved in CH.sub.2Cl.sub.2 (43 mL). To the stirred solution at r.t., levulinic acid (0.35 mL, 3.49 mmol), 4-dimethylaminopyridine (455 mg, 3.72 mmol) and di-isopropylcarbodiimide (0.55 mL, 3.49 mmol) were added. After 12 h, the same amount of levulinic acid, 4-dimethylaminopyridine and di-isopropylcarbodiimide were added and the mixture was stirred for additional 6 h. After filtration over celite, the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (Hexanes/EtOAc=2:1) to afford ester 9* (1.88 g, quant.) as the pale yellow oil. [].sub.D.sup.25=3.72 (c=0.96, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.32-7.15 (m, 12H), 6.77 (d, J=8.8 Hz, 2H), 5.83 (m, 1H), 5.68 (dd, J=2.0, 8.0 Hz, 1H), 5.45 (d, J=2.0 Hz, 1H), 5.25 (d, J=17.2 Hz, 1H), 5.04 (d, J=10.4 Hz, 1H), 4.70 (d, J=10.8 Hz, 1H), 4.53 (d, J=10.8 Hz, 1H), 4.45 (s, 2H), 4.36-4.26 (m, 3H), 4.11-4.00 (m, 3H), 3.94 (m, 1H), 3.74-3.64 (m, 1H), 3.69 (s, 3H), 3.16 (dd, J=2.8, 13.6 Hz, 1H), 2.68-2.44 (m, 9H), 1.96 (s, 3H), 1.18 (t, J=7.6 Hz, 3H), 1.14 (t, J=7.6 Hz, 3H); .sup.13C NMR (101 MHz, CHCl.sub.3) 206.1, 172.0, 170.1, 159.5, 153.0, 138.6, 135.9, 135.0, 130.6, 130.0, 129.4, 129.1, 128.9, 128.0, 128.0, 127.2, 127.1, 116.1, 113.9, 113.6, 81.9, 75.3, 74.5, 72.8, 72.6, 71.9, 66.8, 66.3, 55.4, 55.3, 53.4, 37.8, 37.1, 29.8, 28.1, 25.6, 24.7, 14.4, 14.4; HRMS (ESI): calcd for C.sub.43H.sub.53NO.sub.10S.sub.2Na [M+Na].sup.+: 830.3009, found: 830.2964.
Example A-8: Synthesis of (R)-4-Benzyl-3-((ethyl 3-O-allyl-2-O-benzyl-4-O-levulinoyl-1-thio--
[0202] ##STR00109##
[0203] Paramethoxybenzyl ether 9* (1.85 g, 2.29 mmol) was dissolved in a mixture of 5% TFA in CH.sub.2Cl.sub.2 (30.2 mL) and stirred at r.t. After 5 min, anhydrous anisole (0.45 mL, 4.14 mmol) was added. The reaction was quenched after 13 h by addition of sat. aq. NaHCO.sub.3-solution (195 mL) and CH.sub.2Cl.sub.2 (98 mL). The aqueous layer was separated and extracted with CH.sub.2Cl.sub.2 (3200 mL). The solvent was removed in vacuo and the crude product was purified by column chromatography on silica gel (Hexanes/EtOAc=1.2:1 to 1:1.2) to afford 10* (1.26 g, /=3.9/1, 88%) as white foam. [].sub.D.sup.25=82.49 (c=1.12, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.34-7.15 (m, 10H), 5.87-5.75 (m, 3H), 5.47 (d, J=5.6 Hz, 1H), 5.21 (d, J=15.6 Hz, 1H), 5.10 (d, J=10.4 Hz, 1H), 4.68 (d, J=12.4 Hz, 1H), 4.63 (d, J=12.4 Hz, 1H), 4.59 (m, 1H), 4.16-3.99 (m, 5H), 3.75 (dd, J=3.2, 10.0 Hz, 1H), 3.20 (dd, J=3.2, 13.6 Hz, 1H), 2.64-2.39 (m, 7H), 2.02 (s, 3H), 1.19 (t, J=7.2 Hz, 3H); .sup.13C NMR (101 MHz, CHCl.sub.3) 205.8, 172.3, 166.9, 153.3, 138.0, 135.5, 134.3, 129.3, 129.1, 128.4, 128.0, 127.8, 127.3, 117.4, 84.3, 75.6, 74.0, 72.9, 71.1, 69.5, 68.7, 67.3, 55.4, 37.9, 37.3, 29.8, 28.0, 24.2, 14.6; HRMS (ESI): calcd for C.sub.33H.sub.39O.sub.9NS.sub.2Na [M+Na].sup.+: 648.2243, found: 648.2237.
Example A-9: Synthesis of Methyl (ethyl 3-O-allyl-2-O-benzyl-4-O-levulinoyl-1-thio--
[0204] ##STR00110##
[0205] Oxazolidinone 10* (1.02 g, 1.63 mmol) was dissolved in MeOH (9.7 mL) and Sm(OTf).sub.3 (293 mg, 0.49 mmol) was added. The mixture was vigorously stirred for 60 h at r.t. The solvents were then removed in vacuo and the crude product was purified by silica gel flash column chromatography (Hexanes/EtOAc=1.2/1) to afford compound 11* (634 mg, 81%). [].sub.D.sup.25=122.62 (c=1.05, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.31-7.19 (m, 5H), 5.78 (m, 1H), 5.64 (dd, J=1.6, 3.6 Hz, 1H), 5.41 (d, J=5.6 Hz, 1H), 5.19 (d, J=17.6 Hz, 1H), 5.08 (d, J=10.4 Hz, 1H), 4.86 (br s, 1H), 4.65 (d, J=12.0 Hz, 1H), 4.59 (d, J=12.0 Hz, 1H), 4.05 (dd, J=5.2, 12.8 Hz, 1H), 3.95 (dd, J=5.6, 12.8 Hz, 1H), 3.88 (dd, J=5.6, 9.6 Hz, 1H), 3.69 (s, 3H), 3.63 (dd, J=3.6, 10.0 Hz, 1H), 2.71-2.39 (m, 6H), 2.08 (s, 3H), 1.18 (t, J=7.6 Hz, 3H); .sup.13C NMR (101 MHz, CHCl.sub.3) 205.9, 171.3, 168.0, 137.9, 134.3, 128.3, 127.9, 127.8, 117.2, 83.9, 75.4, 74.0, 72.9, 71.0, 69.0, 68.8, 52.6, 37.9, 29.7, 27.9, 24.0, 14.6; HRMS (ESI): calcd for C.sub.24H.sub.32O.sub.8NSNa [M+Na].sup.+: 503.1716, found: 503.1701.
Example A-10: Synthesis of Methyl (ethyl 3-O-allyl-2-O-benzyl-1-thio--
[0206] ##STR00111##
[0207] Levulinate ester 11* (322 mg, 0.67 mmol) was dissolved in CH.sub.2Cl.sub.2 (7 mL). Subsequently, pyridine (1.6 mL), AcOH (1.1 mL) and hydrazine hydrate (65 L) were added. After stirring at r.t. for 2.5 h, the reaction mixture was quenched by the addition of acetone (7 mL) and the solvents were removed in vacuo. The crude product was purified by silica gel flash column chromatography (Hexanes/EtOAc=1.5/1 to 1/1) to give alcohol 12* (244 mg, 95%). [].sub.D.sup.25=84.74 (c=0.23, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.31-7.20 (m, 5H), 5.85 (m, 1H), 5.39 (d, J=5.2 Hz, 1H), 5.21 (d, J=17.2 Hz, 1H), 5.13 (d, J=10.4 Hz, 1H), 4.77 (br s, 1H), 4.61 (s, 2H), 4.31 (br s, 1H), 4.19-4.06 (m, 2H), 3.97 (m, 1H), 3.75 (s, 3H), 3.58 (dd, J=3.2, 9.6 Hz, 1H), 2.58-2.42 (m, 3H), 1.20 (t, J=7.2 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 169.2, 137.8, 134.3, 128.4, 128.0, 127.8, 117.6, 83.4, 74.4, 72.7, 71.8, 70.1, 68.5, 52.5, 24.0, 14.7; HRMS (ESI): calcd for C.sub.19H.sub.26O.sub.6SNa [M+Na].sup.+: 405.1348, found: 405.1329.
Example A-11: Synthesis of Methyl 2-O-acetyl-3,4-di-O-benzyl--
[0208] ##STR00112##
[0209] Donor 13* (Eur. J. Org. Chem. 2008, 5526-5542) (885 mg, 1.66 mmol) and acceptor 12* (319 mg, 0.83 mmol) were dissolved in dry CH.sub.2Cl.sub.2 (8 mL). 4A MS (800 mg) were added. The mixture was stirred at r.t. for 20 min and then cooled to 0 C., followed by slow addition of TBSOTf (38 L, 0.16 mmol). After 1 h, the reaction was quenched with Et.sub.3N. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=3/1) to give 14* (612 mg, 98%). [].sub.D.sup.25=50.77 (c=0.25, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.34-7.15 (m, 15H), 5.84 (m, 1H), 5.44 (br, 1H), 5.39 (d, J=5.6 Hz, 1H), 5.20 (d, J=17.6 Hz, 1H), 5.09 (d, J=9.6 Hz, 1H), 5.07 (br, 1H), 4.79 (d, J=11.2 Hz, 1H), 4.76 (br, 1H), 4.65 (s, 2H), 4.55 (d, J=9.2 Hz, 1H), 4.48 (d, J=10.4 Hz, 1H), 4.38 (br s, 1H), 4.30 (d, J=12.4 Hz, 1H), 4.20-4.08 (m, 2H), 3.99 (m, 1H), 3.76 (m, 1H), 3.70 (s, 3H), 3.62 (m, 1H), 3.53 (m, 1H), 3.28 (t, J=8.0 Hz, 1H), 2.55-2.39 (m, 2H), 2.06 (s, 3H), 1.19 (m, 6H); .sup.13C NMR (101 MHz, CHCl.sub.3) 170.1, 168.8, 139.0, 138.2, 137.8, 134.6, 128.5, 128.5, 128.4, 128.2, 128.2, 128.2, 128.1, 128.0, 127.7, 127.5, 127.4, 117.0, 99.3, 84.1, 79.6, 78.1, 78.0, 75.1, 74.8, 73.7, 72.8, 72.0, 71.9, 70.4, 68.8, 68.4, 52.6, 24.2, 21.2, 18.1, 14.8; HRMS (ESI): calcd for C.sub.41H.sub.50O.sub.11SNa [M+Na].sup.+: 773.2972, found: 773.2953.
Example A-12: Synthesis of Methyl 2-O-acetyl-3,4-di-O-benzyl--
[0210] ##STR00113##
[0211] Under an argon atmosphere, a solution of Ir[COD(PCH.sub.3Ph.sub.2).sub.2]PF.sub.6 (5.63 mg, 6.66 mol) in THF (1 mL) was degassed by vacuum and gassed with H.sub.2 (5 cycles). The reaction was stirred under H.sub.2 atmosphere at r.t. for 20 min before the solution was degassed by vacuum and gassed with argon (5 cycles). To this reaction flask, a solution of an allyl protected disaccharide 14* (25 mg, 33 mol) in THF (1 mL) was added via syringe in one portion at r.t. The reaction was stirred at r.t. for 21 h before concentrated in vacuo. The crude product was treated with p-TsOH (1.3 mg, 6.66 mol) in a mixture of CH.sub.2Cl.sub.2 and MeOH (2 mL, v/v=1/1) for 12 h at r.t. The mixture was diluted with EtOAc, washed with sat NaHCO.sub.3, dried over Mg.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2/1) to give alcohol 15* (16 mg, 68%). [].sub.D.sup.25=38.5 (c=0.16, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.34-7.19 (m, 15H), 5.55 (d, J=4.8 Hz, 1H), 5.41 (br s, 1H), 5.11 (br s, 1H), 4.80 (d, J=11.2 Hz, 1H), 4.78 (br s, 1H), 4.65 (d, J=11.2 Hz, 1H), 4.55 (d, J=11.2 Hz, 1H), 4.49 (d, J=11.2 Hz, 1H), 4.46 (d, J=11.6 Hz, 1H), 4.40 (br s, 1H), 4.34 (d, J=10.8 Hz, 1H), 3.94 (m, 1H), 3.86 (dd, J=2.4, 10.0 Hz, 1H), 3.75 (dd, J=3.2, 9.2 Hz, 1H), 3.72 (s, 3H), 3.55 (m, 1H), 3.29 (t, J=9.6 Hz, 1H), 2.40-2.57 (m, 2H), 2.06 (s, 3H), 1.20 (m, 6H); .sup.13C NMR (101 MHz, CDCl.sub.3) 170.3, 168.7, 139.0, 138.2, 137.0, 128.7, 128.4, 128.4, 128.3, 128.2, 127.7, 127.5, 127.4, 99.4, 83.2, 79.7, 78.0, 75.6, 74.9, 74.9, 71.9, 71.9, 70.7, 70.2, 68.8, 68.4, 52.6, 29.8, 24.3, 21.3, 18.1, 14.9; HRMS (ESI): calcd for C.sub.38H.sub.46O.sub.11SNa [M+Na].sup.+: 733.2659, found: 733.2680.
Example A-13: Synthesis of Methyl 2-O-acetyl-3,4-di-O-benzyl--
[0212] ##STR00114##
[0213] Donor 16* (J. Am. Chem. Soc. 2012, 134, 15556-15562) (323 mg, 0.51 mmol) and acceptor 15* (90 mg, 0.12 mmol) were dissolved in CH.sub.2Cl.sub.2 (2 mL). 4A MS (200 mg) were added. The mixture was stirred for 30 min at r.t., then cooled to 30 C., followed by slow addition of TBSOTf (9 L, 0.038 mmol). After stirring at 30 C. for 1.5 h, the reaction mixture was quenched with Et.sub.3N. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=3/1 to 2/1) to afford trisaccharide 17* (123 mg, 82%). [].sub.D.sup.25=20.48 (c=0.18, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.30-7.14 (m, 30H), 5.43 (m, 1H), 5.31 (d, J=5.6 Hz, 1H), 5.28-5.24 (m, 2H), 4.86 (d, J=12 Hz, 1H), 4.81 (d, J=11.2 Hz, 1H), 4.70-4.47 (m, 9H), 4.41-4.32 (m, 4H), 4.05 (dd, J=6.0, 10.0 Hz, 1H), 3.94 (dd, J=2.0, 10.0 Hz, 1H), 3.81 (br, 1H), 3.74 (dd, J=3.2, 9.2 Hz, 1H), 3.64 (s, 3H), 3.55 (m, 2H), 3.49-3.44 (m, 2H), 3.38 (dd, J=2.8, 10.0 Hz, 1H), 3.27 (t, J=12.0 Hz, 1H), 2.52-2.35 (m, 2H), 1.97 (s, 3H), 1.87 (s, 3H), 1.17 (m, 6H); .sup.13C NMR (101 MHz, CDCl.sub.3) 170.2, 169.3, 168.6, 163.4, 139.0, 138.5, 138.4, 137.8, 137.8, 137.4, 128.6, 128.4, 128.4, 128.4, 128.3, 128.3, 128.2, 128.1, 128.1, 127.8, 127.8, 127.7, 127.5, 127.5, 127.4, 127.3, 102.1, 98.6, 83.8, 80.0, 79.5, 78.4, 77.1, 76.1, 74.7, 74.4, 73.8, 73.7, 73.5, 72.7, 72.6, 71.9, 71.7, 71.2, 70.4, 68.6, 68.2, 68.1, 52.5, 24.0, 21.1, 20.9, 18.0, 14.7; HRMS (ESI): calcd for C.sub.67H.sub.76O.sub.17SNa [M+Na].sup.+: 1207.4701, found: 1207.4667.
Example A-14: Synthesis of 2-O-acetyl-3,4-di-O-benzyl--
[0214] ##STR00115##
[0215] To a solution of trisaccharide 17* (140 mg, 0.118 mmol) in acetone and water (4/1, 5 mL), trichloroisocyanuric acid (27 mg, 0.118 mmol) was added at 0 C. Then, the reaction mixture was gradually warmed to r.t. and stirred overnight. Acetone was evaporated in vacuo. The residue was diluted with CH.sub.2Cl.sub.2 and washed with sat. NaHCO.sub.3 and water. The organic layer was dried over anhydrous MgSO.sub.4. The residue was purified by column chromatography (Hexanes/EtOAc=1/1.5-1/2) to give the hemiacetal (90 mg, 72%). To a solution of hemiacetal (90 mg, 0.079 mmol) in acetone (4 mL) was added K.sub.2CO.sub.3 (33 mg, 0.24 mmol) and PhN=C(Cl)CF.sub.3 (49 mg, 0.24 mmol). The mixture was stirred overnight at r.t. The solution was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.5/1) to give imidate 18* (97 mg, 94%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.37-7.26 (m, 32H), 7.09 (m, 1H), 6.83 (d, J=7.6 Hz, 2H), 5.53 (br, 1H), 5.44 (br, 1H), 5.40 (dd, J=8.4, 10.0 Hz, 1H), 4.99-4.90 (m, 2H), 4.85-4.80 (m, 3H), 4.70-4.50 (m, 8H), 4.43 (s, 2H), 3.97-3.90 (m, 4H), 3.78 (s, 3H), 3.64-3.56 (m, 2H), 3.53-3.37 (m, 4H), 2.14 (s, 3H), 2.02 (s, 3H), 1.31 (d, J=8.0 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 170.2, 169.1, 167.2, 143.3, 139.1, 138.5, 138.4, 137.9, 137.8, 137.3, 128.6, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.2, 127.9, 127.9, 127.6, 127.6, 127.5, 127.5, 127.3, 124.3, 119.3, 101.6, 98.3, 79.9, 79.5, 78.2, 78.0, 77.5, 75.5, 74.8, 74.5, 74.4, 73.9, 73.8, 73.6, 72.6, 71.9, 71.8, 71.0, 68.5, 68.2, 68.2, 52.8, 21.3, 20.8, 18.0; HRMS (ESI): calcd for C.sub.73H.sub.76F.sub.3NO.sub.18Na [M+Na].sup.+: 1334.4912, found: 1334.4892.
Example A-15: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl--
[0216] ##STR00116##
[0217] Donor 13* (209 mg, 0.39 mmol) and the linker (Org. Lett. 2013, 15, 2270-2273) (99 mg, 0.3 mmol) were dissolved in dry CH.sub.2Cl.sub.2 (4 mL). 4A MS (200 mg) was added. The mixture was stirred at r.t. for 30 min and then cooled to 0 C. TBSOTf (18 L, 0.079 mmol) was added slowly to the reaction mixture. After stirring 1 h, the reaction mixture was quenched with Et.sub.3N. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.8/1) to give the intermediate monosaccharide. Said monosaccharide was dissolved in dry MeOH/CH.sub.2Cl.sub.2 (3 mL, v/v=1/1). A solution of NaOMe (0.09 mL, 0.5 M) was added and the reaction mixture was stirred overnight at r.t. Then, the reaction was concentrated in vacuo and the residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.8/1 to 1/1) to give alcohol 19* (102 mg, 53% for two steps). [].sub.D.sup.25=23.36 (c=1.02, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.27-7.09 (m, 20H), 5.07 (d, J=12.4 Hz, 2H), 4.78 (dd, J=2.4, 10.8 Hz, 1H), 4.67 (br, 1H), 4.59 (br, 2H), 4.52 (dd, J=2.4, 11.2 Hz, 1H), 4.39 (d, J=9.2 Hz, 2H), 3.92 (br, 1H), 3.73 (m, 1H), 3.61 (br, 1H), 3.50 (m, 1H), 3.36 (t, J=9.2 Hz, 1H), 3.24-3.10 (m, 3H), 1.44 (m, 4H), 1.22 (d, J=6.0 Hz, 3H), 1.18 (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) 156.7, 156.1, 138.3, 138.0, 137.9, 136.8, 136.7, 128.5, 128.5, 128.4, 128.0, 127.9, 127.8, 127.8, 127.7, 127.3, 127.2, 98.9, 80.1, 80.0, 75.4, 71.9, 68.5, 67.2, 67.2, 50.5, 50.2, 47.1, 46.1, 29.1, 27.9, 27.5, 23.4, 17.9; HRMS (ESI): calcd for C.sub.40H.sub.47NO.sub.7Na [M+Na].sup.+: 676.3250, found: 676.3265.
Example A-16: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl--
[0218] ##STR00117##
[0219] Acceptor 19* (303 mg, 0.46 mmol) and the donor 13* (369 mg, 0.69 mmol) were dissolved in dry CH.sub.2Cl.sub.2 (4.5 mL). 4A MS (600 mg) were added. The mixture was stirred at r.t. for 30 min and then cooled to 0 C. TBSOTf (21 L, 0.093 mmol) was added slowly to the reaction mixture. After stirring 1 h, the reaction was quenched with Et.sub.3N. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.8/1) to give the intermediate disaccharide. Said disaccharide was dissolved in CH.sub.2Cl.sub.2/MeOH (2 mL, v/v=1/1). NaOMe (50 mg, 0.93 mmol) was added. The mixture was stirred overnight at r.t. The reaction mixture was quenched with acid resin, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hex/EA=2/1) to afford alcohol 20* (389 mg, 86%). [].sub.D.sup.25=20.34 (c=0.88, CHCl.sub.3); ); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.27-7.09 (m, 30H), 5.07 (d, J=12.8 Hz, 2H), 5.00 (br, 1H), 4.79 (d, J=6.8 Hz, 1H), 4.77 (d, J=6.8 Hz, 1H), 4.62 (s, 2H), 4.60-4.56 (m, 3H), 4.52 (d, J=4.0 Hz, 1H), 4.39 (d, J=9.6 Hz, 2H), 4.05 (m, 1H), 3.90 (br, 1H), 3.79 (dd, J=7.2, 9.2 Hz, 1H), 3.76-3.70 (m, 2H), 3.55 (m, 1H), 3.46 (m, 1H), 3.39 (t, J=9.2 Hz, 1H), 3.29 (t, J=9.6 Hz, 1H), 3.23-3.09 (m, 3H), 1.41 (m, 4H), 1.21 (d, J=6.4 Hz, 3H), 1.19 (d, J=6.0 Hz, 3H), 1.16 (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) 156.7, 156.2, 138.5, 138.4, 138.3, 138.0, 137.9, 128.8, 128.6, 128.5, 128.4, 128.4, 128.2, 128.2, 128.0, 128.0, 128.0, 127.9, 127.8, 127.7, 127.3, 127.2, 100.7, 98.8, 80.4, 80.0, 79.9, 79.5, 75.4, 74.7, 72.2, 72.2, 68.7, 67.9, 67.8, 67.2, 50.5, 50.2, 47.1, 46.1, 29.2, 28.0, 27.6, 23.4, 18.1, 18.0; HRMS (ESI): calcd for C.sub.60H.sub.69NO.sub.11Na [M+Na].sup.+: 1002.4768, found: 1002.4791.
Example A-17: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 3,4-di-O-benzyl--
[0220] ##STR00118##
[0221] Acceptor 20* (389 mg, 0.39 mmol) and donor 13* (316 mg, 0.59 mmol) were dissolved in dry CH.sub.2Cl.sub.2 (4 mL). 4A MS (650 mg) were added. The mixture was stirred at r.t. for 30 min and then cooled to 0 C., followed by slow addition of TBSOTf (18 L, 0.079 mmol). After 1 h of stirring at 0 C., the reaction mixture was quenched with Et.sub.3N. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.8/1) to give the intermediate trisaccharide. Said trisaccharide was dissolved in CH.sub.2C.sub.2/MeOH (2 mL, v/v=1/1). NaOMe (43 mg, 0.79 mmol) was added. The mixture was stirred overnight at r.t., then the reaction mixture was quenched with acid resin, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.5/1) to afford alcohol 21* (449 mg, 87%). [].sub.D.sup.25=25.55 (c=1.00, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.44-7.21 (m, 40H), 5.23 (d, J=12.4 Hz, 2H), 5.19 (br, 1H), 5.14 (br, 1H), 4.95-4.90 (m, 3H), 4.76-4.62 (m, 9H), 4.55 (d, J=9.6 Hz, 2H), 4.19 (br, 2H), 3.96-3.78 (m, 7H), 3.65-3.37 (m, 6H), 3.34-3.21 (m, 3 H), 1.56-1.45 (m, 4H), 1.42 (m, 2H), 1.36 (d, J=6.0 Hz, 3H), 1.32 (d, J=6.0 Hz, 3H), 1.27 (d, J=6.0 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 156.7, 156.2, 138.5, 138.4, 138.3, 138.3, 138.0, 137.9, 136.9, 136.8, 128.6, 128.6, 128.6, 128.5, 128.4, 128.4, 128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7, 127.6, 127.3, 127.2, 100.5, 100.5, 98.8, 80.4, 80.3, 80.1, 79.7, 79.6, 79.2, 75.5, 75.3, 74.6, 74.5, 72.4, 72.2, 72.0, 68.8, 68.4, 68.0, 67.9, 67.2, 50.5, 50.2, 47.1, 46.1, 29.7, 29.2, 28.0, 27.6, 23.4, 18.1, 18.1, 17.9; HRMS (ESI): calcd for C.sub.80H.sub.91NO.sub.15Na [M+Na].sup.+: 1328.6286, found: 1328.6217.
Example A-18: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-acetyl-3,4-di-O-benzyl--
[0222] ##STR00119##
[0223] N-phenyltrifluoroacetimidate 18* (65 mg, 0.050 mmol) and trisaccharide acceptor 21* (32 mg, 0.025 mmol) were dissolved in Tol/Et.sub.2O (1.1 mL, v/v 10/1). 4A MS (100 mg) were added. The mixture was stirred at r.t. for 20 min, and then cooled to 70 C. TBSOTf solution in toluene (0.10 mL, 0.05 M) was added slowly. After stirring at 70 C. for 3 h, the reaction mixture was slowly warmed to r.t. The reaction mixture was quenched with Et.sub.3N. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Toluene/EtOAc=10/1) to give hexasaccharide a isomer 22* (48.8 mg, 81%) and P isomer 22* (10.2 mg, 17%). Hexasaccharide a isomer 22*: [].sub.D.sup.25=15.01 (c=0.11, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.35-7.09 (m, 70H), 5.56 (br, 1H), 5.39-5.35 (m, 2H), 5.17 (d, J=10.0 Hz, 2H), 5.09 (br, 1H), 4.98 (br, 1H), 4.95-4.77 (m, 8H), 4.73-4.66 (m, 3H), 4.63-4.53 (m, 11H), 4.48-4.28 (m, 9H), 4.18-4.11 (m, 3H), 4.09 (m, 1H), 3.89-3.84 (m, 4H), 3.82 (d, J=3.2 Hz, 1H), 3.80-3.74 (m, 3H), 3.73-3.68 (m, 2H), 3.67-3.61 (m, 2H), 3.58-3.53 (m, 3H), 3.51-3.48 (m, 2H), 3.47 (s, 3H), 3.42 (dd, J=2.8, 10.0 Hz, 1H), 3.37-3.32 (m, 2H), 3.31 (m, 1H), 3.28-3.15 (m, 5H), 2.01 (s, 3H), 1.83 (s, 3H), 1.50-1.41 (m, 4H), 1.33-1.15 (m, 14H); .sup.13C NMR (151 MHz, CDCl.sub.3) 169.9, 169.2, 169.0, 139.2, 138.8, 138.6, 138.4, 138.0, 137.9, 128.6, 128.5, 128.5, 128.5, 128.5, 128.4, 128.4, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 127.3, 127.3, 113.6, 102.4, 100.6, 98.8, 98.7, 98.5, 96.9, 80.7, 80.5, 80.4, 80.3, 79.7, 79.5, 79.1, 78.5, 78.4, 76.6, 75.5, 75.3, 74.8, 74.6, 74.6, 74.4, 74.2, 73.8, 73.7, 72.9, 72.4, 72.3, 72.0, 72.0, 71.9, 71.7, 71.4, 70.6, 68.9, 68.7, 68.6, 68.4, 68.1, 67.9, 67.3, 67.3, 52.2, 32.0, 31.9, 29.8, 29.5, 29.2, 23.5, 22.8, 21.1, 20.9, 18.2, 18.1, 18.1, 14.2, 1.1; HRMS (ESI): calcd for C.sub.145H.sub.161O.sub.32NNa [M+Na].sup.+: 2451.0899, found: 2451.0881.
Example A-19: Synthesis of 5-Amino-pentanyl -
[0224] ##STR00120##
[0225] A mixture of hexasaccharide 22* (32 mg, 0.013 mmol) and Pd/C (143 mg, 10%) in MeOH/H.sub.2O/HOAc (140/10/1, v/v/v, 7.55 mL) was stirred overnight at r.t. under H.sub.2 atmosphere. After stirring 24 h, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by Sephadex LH20 column (MeOH/H.sub.2O=8/1) to give the benzyl deprotected hexasaccharide (10 mg, 77%). The above hexasaccharide (6 mg, 5.34 ummol) was dissolved in MeOH and water (1 mL, v/v=1/1) and cooled to 0 C. Premixed solution of 1M LiOH (0.27 mL, 0.27 mmol) and 30% H.sub.2O.sub.2 (0.12 mL, 1.17 mmol) was added. The reaction mixture was allowed to warm to r.t. After 8 h, the reaction was neutralized with AcOH (18 uL, 0.32 mmol) and concentrated in vacuo. The residue was purified by Sep-Pak C18 (H.sub.2O, 10% MeOH, 25% MeOH, 50% MeOH) to give target hexasaccharide 23* (5.4 mg, 99%) as a white solid. [].sub.D.sup.25=8.36 (c=0.11, H.sub.2O); .sup.1H NMR (400 MHz, D.sub.2O) 5.41 (br, 1H), 5.10 (br, 1H), 5.07 (br, 1H), 5.06 (br, 1H), 4.85 (br, 1H), 4.66 (br, 2H), 4.58 (d, J=8.0 Hz, 1H), 4.20 (dd, J=2.8, 10.8 Hz, 1H), 4.12 (m, 1H), 4.11 (m, 1H), 4.08 (m, 1H), 4.06 (m, 1H), 3.90 (m, 3H), 3.86 (m, 2H), 3.81 (m, 1H), 3.78 (m, 1H), 3.75 (m, 1H), 3.73 (m, 2H), 3.71 (m, 1H), 3.69 (m, 1H), 3.68 (m, 2H), 3.66 (m, 1H), 3.58 (m, 1H), 3.53 (m, 1H), 3.49 (m, 2H), 3.46 (m, 1H), 3.36 (t, J=9.6 Hz, 1H), 2.98 (t, J=7.6 Hz, 2H), 1.71-1.60 (m, 4H), 1.47-1.38 (m, 2H), 1.29 (m, 9H), 1.21 (d, J=6.0 Hz, 3H); .sup.13C NMR (151 MHz, D.sub.2O) 176.4, 106.4, 102.4, 101.4, 101.2, 99.9, 98.9, 81.4, 80.2, 80.1, 78.1, 77.1, 76.8, 74.2, 73. 8, 73.7, 73.6, 73.3, 73.0, 72.9, 71.9, 71.7, 71.7, 71.4, 70.9, 70.9, 70.4, 70.3, 70.3, 69.2, 68.8, 62.7, 40.9, 29.5, 28.1, 24.0, 18.2, 18.2, 18.2, 18.1; HRMS (ESI): calcd for C.sub.41H.sub.71O.sub.28NNa [M+Na].sup.+: 1048.4060, found: 1048.4002.
Example A-20: Synthesis of 2-O-acetyl-3,4-di-O-benzyl--
[0226] ##STR00121##
[0227] Alcohol 15* (136 mg, 0.19 mmol) was dissolved in CH.sub.2Cl.sub.2 (2 mL). Levulinic acid (0.1 mL, 0.96 mmol), 4-dimethylaminopyridine (140 mg, 1.15 mmol) and di-isopropylcarbodiimide (0.15 mL, 0.96 mmol) were added. The reaction mixture was stirred at r.t. overnight. The mixture was diluted with CH.sub.2Cl.sub.2, washed with brine and then concentrated in vacuo. The crude product was purified by chromatography column on silica gel (Hexanes/EtOAc=1:1) to afford the levulinate ester (150 mg, 97%). The levulinate ester (161 mg, 0.199 mmol) was dissolved in acetone and water (6.25 mL, v/v=4/1) at 0 C. TCCA (46 mg, 0.199 mmol) was added. The reaction mixture was allowed to warm to r.t. overnight. The reaction mixture was diluted with ethyl acetate, washed with sat NaHCO.sub.3 and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=1/2) to give the hemiacetal (121 mg, 79%). To a solution of hemiacetal (121 mg, 0.158 mmol) in acetone (3 mL) was added K.sub.2CO.sub.3 (66 mg, 0.48 mmol) and PhN=C(Cl)CF.sub.3 (98 mg, 0.48 mmol). The mixture was stirred overnight at r.t. The solution was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.2/1 to 1/1) to give the imidate 24* (139 mg, 94%). [].sub.D.sup.25=59.57 (c=0.25, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.31-7.25 (m, 16H), 7.19 (m, 1H), 7.09 (m, 1H), 6.73 (m, 3H), 5.42 (brs, 1H), 5.31 (dd, J=2.0, 10.8 Hz, 1H), 4.97 (br, 1H), 4.86 (d, J=10.8 Hz, 1H), 4.74-4.56 (m, 6H), 4.37 (d, J=11.2 Hz, 1H), 4.01 (dd, J=2.8, 10.4 Hz, 1H), 3.79 (s, 3H), 3.77 (m, 1H), 3.68 (m, 1H), 3.36 (t, J=9.6 Hz, 1H), 2.89-2.69 (m, 3H), 2.52 (m, 1H), 2.18 (s, 3H), 2.11 (s, 3H), 1.29 (d, J=6.4 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 206.31, 172.54, 170.44, 167.16, 143.40, 138.75, 138.08, 137.33, 128.88, 128.64, 128.53, 128.38, 128.23, 128.20, 127.89, 127.76, 127.67, 127.61, 124.44, 119.45, 100.57, 79.70, 77.76, 77.31, 75.19, 73.11, 72.07, 72.01, 71.75, 71.18, 68.94, 68.86, 52.79, 37.97, 29.81, 27.86, 21.27, 18.08; HRMS (ESI): calcd for C.sub.49H.sub.52F.sub.3NO.sub.14Na [M+Na].sup.+: 958.3238, found: 958.3303.
Example A-21: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-acetyl-3,4-di-O-benzyl--
[0228] ##STR00122##
[0229] Donor 24* (139 mg, 0.149 mmol) and the linker (49 mg, 0.149 mmol) were dissolved in a mixture toluene/1,4-dioxane (4 mL, ). 4A MS (200 mg) were added. The reaction mixture was stirred at r.t. for 20 min and then cooled to 0 C. TBSOTf (7 L, 0.03 mmol) in toluene was slowly added. The reaction was stirred at 0 C. After 3 h, the reaction was quenched with Et.sub.3N and filtered, then concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=1/1 to 1/2) to give the disaccharide 25* (119 mg, 75%, /=1.3/1). isomer 25*: []D.sub.25=33.46 (c=0.20, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.36-7.17 (m, 30H), 5.46 (m, 1 H), 5.30 (dd, J=2.8, 10.8 Hz, 1H), 5.18 (m, 3H), 4.94 (d, J=1.6 Hz, 1H), 4.89 (m, 2H), 4.67 (m, 1H), 4.61-4.55 (m, 2H), 4.51-4.41 (m, 5H), 3.88 (dd, J=3.6, 10.4 Hz, 1H), 3.84 (dd, J=3.2, 9.6 Hz, 1H), 3.75 (s, 3H), 3.68 (m, 1H), 3.61 (m, 1H), 3.54 (m, 1H), 3.36 (t, J=9.6 Hz, 1H), 3.29 (m, 1H), 3.19 (m, 1H), 2.87-2.66 (m, 3H), 2.55-2.48 (m, 1H), 2.16 (s, 3H), 2.11 (s, 3H), 1.51 (m, 4H), 1.34 (m, 2H), 1.27 (d, J=6.0 Hz, 3H); .sup.13C NMR (101 MHz, cdcl.sub.3) 206.2, 172.4, 170.4, 168.2, 138.8, 138.1, 137.9, 137.9, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.9, 127.7, 127.5, 127.4, 127.3, 100.1, 97.5, 79.7, 77.7, 77.6, 75.0, 72.9, 72.6, 71.9, 71.5, 69.6, 68.9, 68.7, 67.2, 52.5, 37.9, 32.3, 29.7, 29.1, 27.8, 23.4, 21.2, 18.0; HRMS (ESI): calcd for C.sub.61H.sub.71NO.sub.16Na [M+Na].sup.+: 1096.4671, found: 1096.4595.
Example A-22: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-acetyl-3,4-di-O-benzyl--
[0230] ##STR00123##
[0231] Levulinate ester 25* (52 mg, 48 umol) was dissolved in pyridine (2 mL) and cooled to 0 C. Subsequently, a solution of NH.sub.2NH.sub.2 (1 M in 3:2 Pyridine.AcOH, 0.2 mL) was added. The mixture was stirred at r.t. After 4 h of stirring, the reaction was quenched by the addition of acetone (2 mL) and the solvents were removed in vacuo. The crude product was purified by silica gel flash column chromatography (Hexanes/EtOAc=1.3/1) to give the product 26* (46 mg, 97%). [].sub.D.sup.25=28.84 (c=0.14, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.36-7.17 (m, 30H), 5.51 (br, 1H), 5.19 (m, 3H), 4.90 (m, 2H), 4.69-4.57 (m, 4H), 4.50-4.38 (m, 5H), 4.12 (m, 1H), 3.86 (dd, J=3.2, 9.2 Hz, 1H), 3.77 (s, 3H), 3.74 (m, 1H), 3.65-3.58 (m, 2H), 3.55 (m, 1H), 3.37 (t, J=9.6 Hz, 1H), 3.25 (m, 1H), 3.18 (m, 1H), 2.13 (s, 3H), 1.55-1.45 (m, 4H), 1.31 (m, 2H), 1.29 (d, J=6.0 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 170.3, 168.8, 138.9, 138.2, 137.9, 137.7, 128.7, 128.6, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.2, 99.4, 96.9, 79.7, 78.0, 77.3, 76.2, 75.9, 75.0, 72.8, 71.9, 70.1, 69.5, 68.8, 68.4, 67.3, 52.6, 29.8, 29.2, 23.5, 21.3, 18.1; HRMS (ESI): calcd for C.sub.56H.sub.65NO.sub.14Na [M+Na].sup.+: 998.4303, found: 998.4289.
Example A-23: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-acetyl-3,4-di-O-benzyl--
[0232] ##STR00124##
[0233] Disaccharide acceptor 26* (20 mg, 0.020 mmol) and monosaccharide donor 16* (52 mg, 0.082 mmol) were dissolved in dry toluene (1 mL). 4A MS (100 mg) were added. The reaction mixture was stirred at r.t. for 30 min, then cooled to 30 C. TBSOTf (0.1 mL, 0.08 M) in toluene was added slowly. The reaction mixture was allowed to warm to r.t. After 3 h of stirring, the mixture was quenched with Et.sub.3N. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hex/EA=1.8/1) to give the trisaccharide 27* (24 mg, 80%). [].sub.D.sup.25=6.74 (c=0.11, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.35-7.22 (m, 40H), 5.52 (br, 1H), 5.40 (br, 1H), 5.37 (dd, J=8.0, 10.0 Hz, 1H), 5.16 (m, 2H), 4.96 (d, J=12.0 Hz, 1H), 4.89 (d, J=11.6 Hz, 1H), 4.78-4.72 (m, 4H), 4.66-4.41 (m, 11H), 4.35 (m, 1H), 4.16 (m, 1H), 3.91 (m, 1H), 3.87 (dd, J=3.6, 10.4 Hz, 1H), 3.82 (dd, J=3.2, 9.2 Hz, 1H), 3.72 (s, 3H), 3.64-3.49 (m, 5H), 3.47 (dd, J=2.4, 10.0 Hz, 1H), 3.35 (m, 2H), 3.24 (m, 1H), 3.16 (m, 1H), 2.05 (s, 3H), 1.97 (s, 3H), 1.57-1.45 (m, 4H), 1.27 (m, 2H), 1.26 (d, J=5.6 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 170.1, 169.1, 168.9, 139.1, 138.7, 138.5, 138.0, 128.7, 128.6, 128.5, 128.5, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 127.9, 127.9, 127.9, 127.8, 127.6, 127.5, 127.5, 127.3, 102.2, 98.5, 97.7, 80.3, 79.6, 78.5, 77.3, 76.6, 76.2, 74.7, 74.4, 74.4, 73.7, 73.6, 73.5, 72.8, 72.0, 71.8, 71.2, 70.3, 68.7, 68.3, 68.1, 67.3, 52.6, 29.8, 23.3, 21.2, 21.0, 18.1; HRMS (ESI): calcd for C.sub.85H.sub.95NO.sub.20Na [M+Na].sup.+: 1472.6345, found: 1472.6374.
Example A-24: Synthesis of 5-amino-pentanyl -
[0234] ##STR00125##
[0235] Disaccharide 26* (13 mg, 0.013 mmol) was dissolved in MeOH and HOAc (100/1, 5.05 mL). Pd/C (56.7 mg, 10%) was added. The reaction mixture was stirred under H.sub.2 atmosphere at r.t. After 24 h of stirring, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by LH20 (MeOH) to give the product (6 mg, 94%). The above disaccharide (6 mg, 12 ummol) was dissolved in MeOH and water (2 mL, v/v=1/1) and cooled to 0 C. Premixed solution of 1M LiOH (0.62 mL, 0.62 mmol) and 30% H.sub.2O.sub.2 (0.28 mL, 2.74 mmol) was added. The reaction mixture was allowed to warm to r.t. After 8 h of stirring, the reaction was neutralized with AcOH (43 L, 0.75 mmol) and concentrated in vacuo. The residue was purified by Sep-pak C18 (H.sub.2O, 10% MeOH, 25% MeOH, 50% MeOH) to give the product 28* (3 mg, 57%). [].sub.D.sup.25=51.50 (c=0.03, H.sub.2O); .sup.1H NMR (400 MHz, D.sub.2O) 5.20 (d, J=2.4 Hz, 1H, H1), 4.93 (d, J=3.6 Hz, 1H, H1), 4.36 (br, 1H, H4), 4.19 (m, 1H, H5), 4.03 (m, 1H, H2), 4.01 (m, 1H, H3), 3.09 (m, 1H, H2), 3.79-3.72 (m, 2H, H3, H5), 3.65 (m, 1H, OCH.sub.2), 3.53 (m, 1H, OCH.sub.2), 3.32 (t, J=9.6 Hz, 1H, H4), 3.13 (m, 1H, NCH.sub.2), 2.96 (t, J=7.6 Hz, 1H, NCH.sub.2), 1.65-1.55 (m, 4H, CCH.sub.2C), 1.46-1.33 (m, 2H, CCH.sub.2C), 1.21 (d, J=6.4 Hz, 3H, H6); .sup.13C NMR (151 MHz, D.sub.2O) 177.3, 103.1, 100.9, 78.8, 74.7, 73.5, 73.3, 72.9, 72.6, 71.3, 70.6, 70.5, 41.9, 30.6, 28.9, 24.9, 19.2; HRMS (ESI): calcd for C.sub.17H.sub.31NO.sub.11Na [M+Na].sup.+: 448.1795, found: 448.1808.
Example A-25: Synthesis of 5-Amino-pentanyl -
[0236] ##STR00126##
[0237] Trisaccharide 27* (12 mg, 0.013 mmol) was dissolved in MeOH, H.sub.2O and HOAc (100/1/1, 5.1 mL). Pd/C (70.4 mg, 10%) was added. The reaction mixture was stirred under H.sub.2 atmosphere at r.t. After 24 h of stirring, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by LH20 (MeOH) to give the debenzylated product (5 mg, 88%). The debenzylated trisaccharide (5 mg, 7.3 mmol) was dissolved in MeOH and water (1.2 mL, v/v=1/1) and cooled to 0 C. Premixed solution of 1M LiOH (0.37 mL, 0.37 mmol) and 30% H.sub.2O.sub.2 (0.17 mL, 1.6 mmol) was added. The reaction mixture was allowed to warm to r.t. After 18 h, the reaction was neutralized with AcOH (25 L, 0.44 mmol) and concentrated in vacuo. The residue was purified by Sephadex LH 20 to give the product 29* (4 mg, 93%). [].sub.D.sup.25=32.62 (c=0.05, H.sub.2O); .sup.1H NMR (400 MHz, D.sub.2O) 5.41 (br, 1H), 4.96 (br, 1H), 4.63 (br, 1H), 4.55 (d, J=7.6 Hz, 1H), 4.24 (br, 1H), 4.10-3.97 (m, 3H), 3.90-3.63 (m, 8H), 3.58-3.54 (m, 2H), 3.37 (t, J=10.0 Hz, 1H), 2.99 (t, J=7.6 Hz, 2H, NCH.sub.2), 1.69-1.57 (m, 4H, CCH.sub.2C), 1.48-1.39 (m, 2H, CCH.sub.2C), 1.22 (d, J=6.0 Hz, 3H); .sup.13C NMR (151 MHz, D.sub.2O) 177.2, 107.5, 102.4, 100.7, 83.0, 79.0, 77.8, 75.1, 74.7, 73.9, 73.5, 72.9, 72.6, 71.4, 71.3, 70.5, 69.8, 63.8, 41.9, 30.7, 28.9, 24.9, 19.2; HRMS (ESI): calcd for C.sub.23H.sub.43NO.sub.16 [M+H].sup.+: 588.2504, found: 588.2517.
Example A-26: Synthesis of Methyl (ethyl 2-O-benzyl-4-O-levulinoyl-1-thio--
[0238] ##STR00127##
[0239] Under an argon atmosphere, a solution of Ir[COD(PCH.sub.3Ph.sub.2).sub.2]PF.sub.6 (29.6 mg, 0.035 mmol) in THF (2.5 mL) was degassed by vacuum and gassed with H.sub.2 balloon (5 cycles). The reaction was stirred under H.sub.2 atmosphere at r.t. for 20 min before the solution was degassed by vacuum and gassed with argon (5 cycles). To this reaction flask, a solution of monosaccharide 11* (84 mg, 0.18 mmol) in THF (2.5 mL) was added via syringe in one portion at r.t. The reaction was stirred at r.t. for 3.5h before being concentrated in vacuo. The crude product was treated with p-TsOH (7 mg, 0.035 mmol) in MeOH (2.5 mL) at r.t. After 16.5 h, the mixture was diluted with ethyl acetate, washed with sat. NaHCO.sub.3, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=1/2) to give the product 30* (59 mg, 77%). [].sub.D.sup.25=135.61 (c=0.50, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.39-7.26 (m, 5H), 5.66 (dd, J=1.2, 3.2 Hz, 1H), 5.56 (d, J=5.2 Hz, 1H), 4.93 (d, J=1.2 Hz, 1H), 4.69 (d, J=11.6 Hz, 1H), 4.59 (d, J=11.6 Hz, 1H), 4.02 (m, 1H), 3.92 (dd, J=5.2, 10.0 Hz, 1H), 3.74 (s, 3H), 2.74-2.67 (m, 3H), 2.60-2.48 (m, 4H), 2.14 (s, 3H), 1.23 (t, J=7.6 Hz, 3 H); .sup.13C NMR (101 MHz, CDCl.sub.3) 206.6, 172.0, 168.1, 137.3, 128.5, 128.4, 128.3, 128.2, 128.0, 83.4, 75.3, 72.4, 71.0, 69.0, 68.7, 52.7, 38.1, 29.8, 28.0, 24.2, 14.7; HRMS (ESI): calcd for C.sub.21H.sub.28O.sub.8SNa [M+Na].sup.+: 463.1403, found: 463.1412.
Example A-27: Synthesis of 2-O-acetyl-3,4,6-tri-O-benzyl--
[0240] ##STR00128##
[0241] The donor 16* (286 mg, 0.45 mmol) and the acceptor 30* (59 mg, 0.13 mmol) were dissolved in toluene (1.3 mL). 4A MS (300 mg) were added. The mixture was stirred for 30 min at r.t., then cooled to 40 C. TBSOTf (10 L, 0.046 mmol) was added. The reaction was allowed to warm to r.t. After 2.5 h, the reaction was quenched with Et.sub.3N. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=1.2/1) to give product 31* (92 mg, 75%). [].sub.D.sup.25=83.33 (c=0.63, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.36-7.28 (m, 20H), 5.62 (br, 1H), 5.41 (d, J=5.2 Hz, 1H), 5.31 (dd, J=8.0, 10.0 Hz, 1H), 4.92-4.87 (m, 2H), 4.67-4.43 (m, 8H), 4.02 (m, 1H), 3.95 (m, 2H), 3.70 (s, 3H), 3.67-3.52 (m, 3H), 3.43 (m, 1H), 2.76-2.44 (m, 6H), 2.08 (s, 3H), 1.88 (s, 3H), 1.23 (t, J=7.6 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 206.6, 171.4, 169.5, 168.0, 138.6, 138.0, 137.9, 137.7, 128.5, 128.4, 128.2, 128.2, 128.0, 128.0, 127.8, 127.8, 127.5, 127.5, 101.8, 83.5, 79.9, 75.6, 74.5, 74.5, 73.6, 73.5, 73.1, 72.6, 71.8, 71.3, 70.9, 69.0, 68.3, 52.7, 38.4, 29.7, 28.2, 23.9, 21.0, 14.6; HRMS (ESI): calcd for C.sub.50H.sub.58O.sub.14SNa [M+Na].sup.+: 937.3445, found: 937.3445.
Example A-28: Synthesis of 2-O-acetyl-3,4,6-tri-O-benzyl--
[0242] ##STR00129##
[0243] To a solution of disaccharide 31* (92 mg, 0.10 mmol) in acetone and water (4/1, 3 mL), TCCA (24 mg, 0.10 mmol) was added at 0 C. Then, the reaction was warmed gradually to r.t. After 5 h, acetone was evaporated in vacuo. The residue was diluted with ethyl acetate and washed with sat. NaHCO.sub.3 and water. The organic layer was dried over anhydrous MgSO.sub.4, filtered and concentrated in vacuo. The residue was purified by column chromatography (Hexanes/Ethyl Acetate=1/1.8) to give the intermediate hemiacetal (80 mg, 91%). To the above hemiacetal (80 mg, 0.092 mmol) in acetone (3 mL) was added K.sub.2CO.sub.3 (38 mg, 0.28 mmol) and PhN=C(Cl)CF.sub.3 (57 mg, 0.28 mmol). The mixture was stirred at r.t. for 6 h. The solution was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=2.2/1) to give the target product 32* (94 mg, 98%).
Example A-29: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-acetyl-3,4,6-tri-O-benzyl--
[0244] ##STR00130##
[0245] Donor 32* (44 mg, 0.042 mmol) and linker (14 mg, 0.042 mmol) was dissolved in Toluene/1,4-dioxane (1.2 mL, v/v=1/3). 4A MS (100 mg) was added. The reaction mixture was stirred at rt for 20 min and then cooled to 0 C. TBSOTf (0.1 mL, 0.08 M) in toluene was slowly added. The reaction mixture was stirred at 0 C. After 3 h, the reaction was quenched with Et.sub.3N and filtered, then concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=1/1 to 1/2) to give target product 33* (35 mg, 70%, /3=1/2.2). a isomer: [].sub.D.sup.25=21.93 (c=0.18, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.36-7.14 (m, 30H), 5.55 (d, J=3.6 Hz, 1H), 5.30 (dd, J=8.0, 10.0 Hz, 1H), 5.19 (m, 3H), 4.92 (d, J=11.6 Hz, 1H), 4.83 (m, 1H), 4.68 (m, 2H), 4.58 (m, 2H), 4.51 (m, 2H), 4.45 (m, 3H), 4.29 (m, 1H), 4.06 (m, 1H), 3.95-3.90 (m, 2H), 3.81 (dd, J=3.2, 9.2 Hz, 1H), 3.72 (s, 3H), 3.63-3.54 (m, 3H), 3.48 (m, 1H), 3.42 (m, 1H), 3.29 (m, 1H), 3.13 (m, 1H), 2.79-2.53 (m, 4H), 2.09 (s, 3H), 1.83 (s, 3H), 1.56-1.46 (m, 4H), 1.33-1.29 (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) 206.7, 171.6, 169.4, 167.3, 138.6, 138.4, 138.0, 128.6, 128.6, 128.5, 128.5, 128.4, 128.3, 128.1, 128.0, 127.9, 127.9, 127.7, 127.6, 127.5, 127.4, 103.3, 101.6, 79.9, 78.8, 77.3, 75.1, 74.6, 73.6, 72.6, 72.6, 71.8, 71.4, 70.5, 68.3, 67.3, 67.2, 62.8, 60.5, 52.8, 50.6, 50.2, 47.0, 46.2, 38.3, 32.4, 29.8, 29.3, 28.2, 23.4, 23.0, 21.0, 14.3; HRMS (ESI): calcd for C.sub.68H.sub.77NO.sub.17Na [M+Na].sup.+: 1202.5089, found: 1202.5129.
Example A-30: Synthesis of 5-amino-pentanyl P3-
[0246] ##STR00131##
[0247] Levulinate ester 33* (20 mg, 0.017 mmol) was dissolved in pyridine (1 mL) and cooled to 0 C. Subsequently, a solution of NH.sub.2NH.sub.2 (1 M in 3:2 Pyridine.AcOH, 0.1 mL) was added. The mixture was stirred at rt. After 5 h, the reaction was quenched by the addition of acetone (2 mL) and the solvents were removed in vacuo. The crude product was purified by silica gel flash column chromatography (Hex/EA=1.3/1) to give the alcohol. The above alcohol was dissolved in MeOH, H.sub.2O and HOAc (100/1/1, 5.1 mL). Pd/C (83 mg, 10%) was added. The reaction mixture was stirred under the atmosphere of H.sub.2 at rt. After stirring 24 h, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by Sephadex LH20 column (MeOH/H.sub.2O=10/1) to give the corresponding disaccharide. The above disaccharide was dissolved in MeOH and water (1.6 mL, v/v=1/1) and cooled to 0 C. Premixed solution of 1 M LiOH (0.51 mL, 0.51 mmol) and 30% H.sub.2O.sub.2 (0.23 mL, 2.26 mmol) was added. The reaction mixture was allowed to warm to room temperature. After 18 h, the reaction was neutralized with AcOH (35 L, 0.62 mmol), concentrated in vacuo. The residue was purified by Sephadex LH20 column to give the disaccharide 34* (2.5 mg, 38% for 3 steps) as a white solid. [].sub.D.sup.25=61.8 (c=0.02, H.sub.2O); .sup.1H NMR (400 MHz, D.sub.2O) 4.61 (d, J=7.6 Hz, 1H, H1), 4.48 (m, 1H, H4), 4.42 (d, J=8.0 Hz, 1H, H1), 4.08 (m, 1H, H5), 3.98 (m, 1H, OCH.sub.2), 3.90 (m, 1H, H4), 3.84 (m, 1H, H3), 3.79 (m, 1H, H6), 3.72 (m, 1H, H5), 3.69 (m, 1H, H6), 3.67 (m, 1H, OCH.sub.2), 3.65 (m, 1H, H2), 3.63 (m, 1H, H3), 3.59 (m, 1H, H2), 3.01 (t, J=7.6 Hz, 2H, NCH.sub.2), 1.72 (m, 2H, CCH.sub.2C), 1.64 (m, 2H, CCH.sub.2C), 1.50 (m, 2H, CCH.sub.2C); .sup.13C NMR (151 MHz, D.sub.2O) 106.9, 104.3, 85.2, 77.8, 77.5, 75.1, 73.6, 72.6, 72.2, 72.1, 71.2, 71.2, 63.6, 41.9, 30.5, 28.7, 24.5; HRMS (ESI): calcd for C.sub.17H.sub.31NO.sub.12Na [M+Na].sup.+: 464.1744, found: 464.1723.
Example A-31: Synthesis of 5-amino-pentanyl -
[0248] ##STR00132##
[0249] Levulinate ester 33a* (10 mg, 8.47 mol) was dissolved in pyridine (1 mL) and cooled to 0 C. Subsequently, a solution of NH.sub.2NH.sub.2 (1 M in 3:2 Pyridine.AcOH, 0.1 mL) was added. The mixture was stirred at rt. After 5 h, the reaction was quenched by the addition of acetone (2 mL) and the solvents were removed in vacuo. The crude product was purified by silica gel flash column chromatography (Hex/EA=1.3/1) to give the intermediate secondary alcohol. The above secondary alcohol was dissolved in MeOH, H.sub.2O and HOAc (100/1/1, 5.1 mL). Pd/C (53 mg, 10%) was added. The reaction mixture was stirred under the atmosphere of H.sub.2 at rt. After stirring for 24 h, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by Sephadex LH20 column (MeOH/H.sub.2O=10/1) to give the corresponding disaccharide. The disaccharide was dissolved in methanol and water (1.2 mL, v/v=1/1) and cooled to 0 C. Premixed solution of 1 M LiOH (0.17 mL, 0.17 mmol) and 30% H.sub.2O.sub.2 (0.077 mL, 0.75 mmol) was added. The reaction mixture was allowed to warm to room temperature. After 18 h, the reaction was neutralized with AcOH (12 uL, 0.21 mmol), concentrated in vacuo. The residue was purified by Sephadex LH20 column to give disaccharide 34* (1.1 mg, 29% for 3 steps) as a white solid. [].sub.D.sup.25=113.30 (c=0.02, H.sub.2O); .sup.1H NMR (400 MHz, D.sub.2O) 4.95 (br, 1H, H1), 4.58 (d, J=7.6 Hz, 1H, H1), 4.53 (br, 1H, H4), 4.22 (br, 1H, H5), 3.98 (m, 2H, H2, H3), 3.89 (m, 1H, H4), 3.79 (m, 1H, H6), 3.72 (m, 1H, H5), 3.68 (m, 1H, H6), 3.65 (m, 1H, OCH.sub.2), 3.63 (m, 1H, H3), 3.59 (m, 1H, H2), 3.56 (m, 1H, OCH.sub.2), 3.01 (t, J=7.6 Hz, 2H, NCH.sub.2), 1.68 (m, 2H, CCH.sub.2C), 1.60 (m, 2H, CCH.sub.2C), 1.44 (m, 2H, CCH.sub.2C); .sup.13C NMR (151 MHz, D.sub.2O) 178.1, 107.0, 100.7, 82.5, 77.8, 75.1, 73.7, 73.6, 73.1, 71.2, 70.5, 69.5, 63.6, 41.9, 30.7, 28.9, 25.8, 24.9; HRMS (ESI): calcd for C.sub.17H.sub.31NO.sub.12Na [M+Na].sup.+: 464.1744, found: 464.1737.
Example A-32: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 4,6-di-O-benzyl-2-O-benzoyl-3-O-fluorenylmethoxycarbonyl galactopyranoside (36*)
[0250] ##STR00133##
[0251] Thioglycoside 35* (117 mg, 0.16 mmol) and the linker (79 mg, 0.24 mmol) were dissolved in DCM (2 mL). 4A MS (200 mg) was added. The mixture was stirred at rt for 30 min. Then, the mixture was cooled to 20 C. NIS (47 mg, 0.21 mmol) in DCM (1 mL) was added, followed by TMSOTf (12 uL, 0.064 mmol). The reaction was allowed to warm to room temperature. After 3h, the mixture was filtered. The solution was diluted with DCM, washed with sat.NaHCO.sub.3, 1M Na.sub.2S.sub.2O.sub.3 and brine. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc=2/1) to give the monosaccharide 36* (114 mg, 72%) as a white foam. [].sub.D.sup.25=15.50 (c=0.37, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 8.02 (d, J=6.8 Hz, 2H), 7.67 (t, J=6.8 Hz, 2H), 7.48-7.06 (m, 29H), 5.71 (dd, J=8.0, 10.4 Hz, 1H), 5.14 (br, 2H), 5.04 (dd, J=2.8, 10.4 Hz, 1H), 4.79 (d, J=11.6 Hz, 1H), 4.57-4.45 (m, 4H), 4.39-4.34 (m, 2H), 4.32-4.29 (m, 1H), 4.26 (m, 1H), 4.11-4.07 (m, 2H), 3.87 (m, 1H), 3.77 (m, 1H), 3.71-3.64 (m, 2H), 3.40 (m, 1H), 3.04 (m, 1H), 2.94 (m, 1H), 1.45-1.27 (m, 4H), 1.12 (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) 165.1, 154.6, 143.3, 142.9, 141.2, 141.1, 137.8, 137.8, 133.1, 129.8, 129.8, 128.5, 128.4, 128.3, 127.9, 127.9, 127.8, 127.2, 127.1, 125.2, 125.0, 120.0, 101.5, 77.9, 75.2, 73.8, 73.6, 73.3, 70.3, 70.1, 69.7, 69.6, 68.1, 67.1, 50.5, 50.2, 47.1, 46.5, 46.1, 29.1, 27.7, 27.3, 23.1; HRMS (ESI): calcd for C.sub.62H.sub.61NO.sub.11Na [M+Na].sup.+: 1018.4142, found: 1018.4133.
Example A-33: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 4,6-di-O-benzyl-2-O-benzoyl galactopyranoside (37*)
[0252] ##STR00134##
[0253] Monosaccharide 36* (37 mg, 0.037 mmol) was dissolved in DCM (1.5 mL). Et.sub.3N (0.4 mL) was added. The mixture was stirred at rt. After 5 h, the mixture was diluted with toluene and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hex/EtOAc=1.8/1) to give target alcohol 37* (28 mg, 97%) as a foam. [].sub.D.sup.25=8.52 (c=0.27, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 8.00 (d, J=7.2 Hz, 2H), 7.49-7.10 (m, 23H), 5.24 (dd, J=8.0, 9.6 Hz, 1H), 5.12 (br, 2H), 4.73 (s, 2H), 4.54-4.43 (m, 3H), 4.38-4.36 (m, 2H), 3.94 (d, J=3.2 Hz, 1H), 3.83 (m, 1H), 3.78 (m, 1H), 3.69-3.66 (m, 3H), 3.41 (m, 1H), 3.06 (m, 1H), 1.48-1.33 (m, 4H), 1.16-1.09 (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) 166.6, 156.6, 156.0, 138.0, 137.6, 133.1, 129.8, 129.7, 128.5, 128.4, 128.2, 128.0, 127.8, 127.7, 127.1, 101.0, 76.5, 75.4, 74.1, 73.5, 73.4, 73.1, 69.6, 69.5, 68.2, 67.0, 50.4, 50.1, 47.0, 46.0, 29.6, 29.0, 27.7, 27.3, 23.1; HRMS (ESI): calcd for C.sub.47H.sub.51NO.sub.9Na [M+Na].sup.+: 796.3462, found: 796.3464.
Example A-34: Synthesis of N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl 2-O-acetyl-3,4-di-O-benzyl--
[0254] ##STR00135##
[0255] Acceptor 37* (25 mg, 0.032 mmol) and donor 13* (34 mg, 0.065 mmol) were dissolved in toluene (1 mL). 4A MS (100 mg) was added. The mixture was stirred at rt for 30 min, then cooled to 0 C. TBSOTf (0.1 mL, 0.06 M) in toluene was slowly added. After 3 h, the reaction was quenched with Et.sub.3N. The mixture was filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hexanes/EtOAc=1.8/1 to 1/1) to give disaccharide 38* (22 mg, 60%) as a white foam. [].sub.D.sup.25=10.42 (c=0.22, CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) 8.02 (d, J=7.6 Hz, 2H), 7.46-7.07 (m, 33H), 5.67 (dd, J=8.0, 10.0 Hz, 1H), 5.21 (m, 1H), 5.11 (m, 2H), 4.93-4.82 (m, 3H), 4.56-4.50 (m, 2H), 4.47-4.40 (m, 3H), 4.37-4.33 (m, 3H), 4.15 (d, J=11.2 Hz, 1H), 3.97 (m, 1H), 3.89 (m, 1H), 3.86-3.81 (m, 2H), 3.75 (dd, J=3.2, 9.2 Hz, 1H), 3.67 (m, 1H), 3.61 (m, 1H), 3.39-3.30 (m, 2H), 3.04 (m, 1H), 2.92 (m, 1H), 1.83 (s, 3H), 1.38 (m, 4H), 1.29 (d, J=6.0 Hz, 3H), 1.13 (m, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) 169.4, 165.0, 138.5, 138.3, 137.8, 137.8, 133.1, 129.8, 129.7, 128.5, 128.4, 128.3, 128.3, 128.2, 128.0, 127.9, 127.9, 127.7, 127.2, 101.5, 99.2, 79.6, 78.6, 77.5, 77.3, 75.4, 75.2, 74.9, 73.8, 73.7, 72.4, 71.5, 69.0, 68.5, 68.3, 67.1, 50.5, 50.2, 47.1, 46.1, 29.8, 29.1, 23.1, 21.2, 20.7, 18.2; HRMS (ESI): calcd for C.sub.69H.sub.75NO.sub.14Na [M+Na].sup.+: 1164.5085, found: 1164.5069.
Example A-35: Synthesis of 5-amino-pentanyl -
[0256] ##STR00136##
[0257] The disaccharide 38* (22 mg, 0.019 mmol) was dissolved in DCM and MeOH (1.2 mL, v/v=1/1). Sodium methoxide (5.2 mg, 0.096 mmol) was added. The mixture was stirred at rt overnight, then the mixture was neutralized with acid resin, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (Hex/AcOEt=1/1.5) to give the intermediate secondary alcohol, which was further dissolved in MeOH/H.sub.2O/HOAc (5.55 mL, v/v/v=100/10/1) and treated with Pd/C (101 mg, 10%). The mixture was stirred under H.sub.2 at rt. After 18 h, the mixture was filtered and concentrated. The residue was purified by Sep-pak C18 (H.sub.2O, 10% MeOH, 20% MeOH, 30% MeOH) to obtain disaccharide 39* (7.6 mg, 96% for two steps) as a white solid. [].sub.D.sup.25=173.72 (c=0.01, H.sub.2O);
[0258] .sup.1H NMR (400 MHz, D.sub.2O) 4.99 (br, 1H, H1), 4.42 (d, J=8.0 Hz, 1H, H1), 4.04 (m, 1H, H2), 3.97 (m, 1H, H4), 3.91 (m, 1H, OCH.sub.2), 3.82 (m, 1H, H5), 3.79 (m, 1H, H6), 3.74 (m, 1H, H3), 3.71 (m, 1H, H5), 3.67-3.64 (m, 3H, OCH.sub.2, H6, H3), 3.59 (m, 1H, H2), 3.43 (t, J=9.6 Hz, 1H, H4), 2.97 (t, J=7.6 Hz, 2H, NCH.sub.2), 1.69 (m, 2H, CCH.sub.2C), 1.63 (m, 2H, CCH.sub.2C), 1.43 (m, 2H, CCH.sub.2C), 1.26 (d, J=6.4 Hz, 3H, H6); .sup.13C NMR (101 MHz, D.sub.2O) 102.3, 102.2, 80.4, 74.9, 71.7, 70.0, 69.9, 69.9, 69.8, 69.0, 68.2, 60.7, 39.2, 28.0, 26.2, 21.9, 16.4; HRMS (ESI): calcd for C.sub.17H.sub.35NO.sub.10 [M+H].sup.+: 412.2183, found: 412.2180.
B. Biological Experiments
Example B-1: Conjugation of the Inventive Saccharides to CRM.SUB.197
[0259] The saccharide (1 equivalent) and bis-(4-nitrophenyl) adipate (7 equivalents) were added to a solution of pyridine and DMSO (1:1). The resulting mixture was stirred for 5 minutes for complete solubilization. Then, triethylamine (0.83 L, 6 mol, 10 equivalents) was added and the mixture was stirred for further 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 the excess of bis-(4-nitrophenyl) adipate and the white solid obtained was dried in vacuum.
[0260] 40 equivalents of lyophilized CRM.sub.197 was dissolved in 0.4 mL of sterile 0.1M sodium phosphate, pH 8.0 and transferred into upper chamber of 10,000 Da Millipore centrifugal filter (0.5 mL). The glass vial was rinsed with 30.4 mL of sterile 0.1M sodium phosphate, pH 8.0 and the rinsing solution was transferred to the same centrifugal filter. Following centrifugation at 10,000 rpm for 6-8 min (if needed, the centrifugation step is prolonged such that volume in upper chamber is 80-100 L), the CRM.sub.197 solution was then transferred into 1.5 mL tube containing the lyophilized saccharide derivatized with PNP ester and shacked slowly (around 180-200 rpm) for 18-24 h 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. The average molecular size of the conjugate was determined by MALDI-MS analysis using CRM.sub.197 as standard and calculate the average oligosaccharides attachments with per CRM.sub.197 molecule.
[0261] Following the procedure, the hexasaccharide 23* was conjugated to CRM.sub.197. Conjugate CRM.sub.197-23*: ca. m/z 65705.7 (incorporation of 8 hexasaccharide molecules on average);
##STR00137##
Example B-2: Evaluation of the Immune Response Against the Conjugate CRM.SUB.197.-23* in Mice
[0262] Preparation of Microarrays Slides:
[0263] The CodeLink NHS activated glass slides (Surmodics) were spotted with synthetic glycans, polysaccharides, protein (CRM.sub.197) and BSA-Spacer-dimannose 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 (see
[0264] Mice:
[0265] 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.
[0266] Mice Immunization and Generation of Polyclonal Sera
[0267] Groups of 3 C57BL/6J female 6-8 week old inbred mice were immunized subcutaneously with conjugate CRM.sub.197-23* emulsified with 1:1 (v/v) Freund's adjuvant (3 g of hexasaccharide 23*). On day 14 and 35 mice received a booster injection with the same amount of antigen emulsified with 1:1 (v/v) Freund's adjuvant. Mice were bled submandibularly weekly using sterile single-use blood lancet. Control mice received only alum in PBS. The antibody responses were measured by glycan microarray.
[0268] The hexasaccharide 23*-specific endpoint antibody titers of IgG and IgM in pooled sera were analyzed by glycan microarray for every week after the immunization as expressed in MFI (
[0269] The polyclonal sera (1 in 200 dilutions in 1% BSA-PBS) were further analyzed using microarray slides printed with oligosaccharides related to the capsular polysaccharides from carbapenem-resistant K. pneumoniae and polysaccharides (capsular polysaccharides of K. pneumoniae bacteria).
[0270] 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 the conjugate CRM.sub.197-hexasaccharide 23* 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.
[0271] The microarray data show that the conjugate CRM.sub.197-23* is immunogenic in mice and exhibits robust antibody response (see
Example B-3: Evaluation of the Immune Response Against the Conjugate CRM.SUB.197.-23* in Rabbits
[0272] Rabbit Immunization and Generation of Polyclonal Sera:
[0273] Ten to twelve week old female ZIKA rabbits (n=3) were subcutaneously immunized at four different sites with CRM.sub.197-23* conjugate in alhydrogel (aluminum hydroxide) formulation at day 0 and boosted at day 14 and sera were collected at day 0, 14 and 21. The antibody response was analyzed by glycan microarray and ELISA.
[0274] ELISA:
[0275] The antibody titer of rabbit sera were analyzed by ELISA. The high binding ninety six well polystyrene microtiter plates (Corning, USA) were coated overnight at 4 C. with capsular polysaccharide of carbapenem-resistant K. pneumoniae strains (Carbohydr. Res. 2013, 369, 6-9) (50 l of 10 g/ml per well) in PBS, pH 7.2. The plates were washed thrice with PBS containing 0.1% Tween-20 (PBST) and blocked with 10% FCS in PBS at room temperature for 1 hr. After washing thrice with PBST, the plate was incubated with the individual and pooled rabbit serum (n=3) at different dilutions in duplicate at room temperature for 1 hr. The plate was washed 4-5 times with PBST and incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit total Ig antibodies (diluted 1 in 10,000 in 10% FCS-PBS) followed by incubation at room temperature another 1 hr. The plate was washed thoroughly with PBST and developed using HRP substrate 3, 3, 5, 5-tetramethylbenzidine (Thermo Scientific, USA). The reaction was stopped by adding 2% H.sub.2SO.sub.4 and absorbance were recorded at 450 nm.
[0276] Glycan Microarray Analysis:
[0277] Glycan microarray analysis was performed as described in Example B-2 using the same printed slides. Mean fluorescence intensity of spots was plotted.
[0278] Analysis of the Antibody Response by ELISA
[0279] The antibody response was analyzed by ELISA in rabbit sera (n=3) immunized with 10 g of glycan's equivalent conjugate on day 0 and boosted at day 14 subcutaneously.
[0280] The immune response was analyzed at different time points. The specific immunoglobulins by end point titer were quantified (
[0281] Analysis of the Antibody Response by Microarray
[0282] To analyze the hexasaccharide 23* specific immune response, the microarray slide(s) were incubated with rabbit sera (n=3) collected at different time points before and after immunization with CRM.sub.197-23* conjugate. The microarray data shows that the hexasaccharide 23* specific antibodies are cross-reactive with native polysaccharide and other synthetic glycans (