Protein and peptide-free synthetic vaccines against Streptococcus pneumoniae type 3

Abstract

The present invention provides a protein- and peptide-free conjugate comprising a synthetic carbohydrate and a carrier molecule, wherein the synthetic carbohydrate is a Streptococcus pneumoniae type 3 capsular polysaccharide related carbohydrate and the carrier molecule is a glycosphingolipid. Said conjugate and pharmaceutical composition thereof are useful for immunization against diseases associated with Streptococcus pneumoniae, and more specifically against diseases associated with Streptococcus pneumoniae type 3.

Claims

1. A conjugate of general formula (I-A) ##STR00187## wherein A is ##STR00188## B is ##STR00189## R.sup.2 is (X.sup.1).sub.p1(X.sup.2).sub.p2(X.sup.3).sub.p3X.sup.4; R.sup.3 and R.sup.4 are selected from H and OH and cannot be simultaneously H or OH; R.sup.5 is (Y.sup.1).sub.m1(Y.sup.2).sub.m2-(3).sub.m3Y.sup.4; Z represents OCH.sub.2, SCH.sub.2 or CH.sub.2CH.sub.2; X.sup.4 represents: H or ##STR00190## Y.sup.4 represents: H or -Ph; X.sup.1, X.sup.2, X.sup.3, Y.sup.1, Y.sup.2, Y.sup.3 are independently of each other selected from: CH.sub.2, ##STR00191## n2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; n1, n3 represent independently of each other an integer selected from 0 and 1; L represents -L.sup.1-NH-L.sup.2-NH-L.sup.3-; L.sup.1 represents -L.sup.1-L.sup.1-L.sup.1- or -L.sup.1-L.sup.1- or -L.sup.1-; and L.sup.3 represents -L.sup.3-L.sup.3-L.sup.3- or -L.sup.3-L.sup.3- or -L.sup.3-; and L.sup.1, L.sup.1, L.sup.1, L.sup.3, L.sup.3, and L.sup.3 are independently of each other selected from: CH.sub.2, C.sub.2H.sub.4, C.sub.3H.sub.6, C.sub.4H.sub.8, C.sub.5H.sub.10, C.sub.6H.sub.12, C.sub.7H.sub.14, C.sub.8H.sub.16, C.sub.9H.sub.18, C.sub.10H.sub.20, (CH.sub.2CH.sub.2O).sub.oCH.sub.2CH.sub.2, and (CH.sub.2CH.sub.2O).sub.oCH.sub.2; L.sup.2 is selected from: C(O), ##STR00192## R.sup.6, R.sup.7 and R.sup.8 are independently of each other selected from: H, CH.sub.3, C.sub.2H.sub.5, F, Cl, Br, OCH.sub.3 and CF.sub.3; n and o represent independently of each other an integer selected from 1, 2, 3, 4, 5 and 6; p1, p2, p3, m1, m2 and m3 represent independently of each other an integer from 0 to 10.

2. The conjugate according to claim 1 general formula (I-B) ##STR00193## wherein A is ##STR00194## B is ##STR00195## R.sup.2 is (X.sup.1).sub.p1(X.sup.2).sub.p2(X.sup.3).sub.p3X.sup.4; R.sup.3 and R.sup.4 are selected from H and OH and cannot be simultaneously H or OH; R.sup.5 is (Y.sup.1).sub.m1(Y.sup.2).sub.m2(Y.sup.3).sub.m3Y.sup.4; X.sup.4 represents: ##STR00196## Y.sup.4 represents: H or -Ph; X.sup.1, X.sup.2, X.sup.3, Y.sup.1, Y.sup.2 and Y.sup.3 are independently of each other selected from: CH.sub.2, and ##STR00197## n2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; n1 and n3 represent independently of each other an integer selected from 0 and 1; L represents -L.sup.1-NH-L.sup.2-NH-L.sup.3-; L.sup.1 represents -L.sup.1-L.sup.1-L.sup.1- or -L.sup.1-L.sup.1- or -L.sup.1-; and L.sup.3 represents -L.sup.3-L.sup.3-L.sup.3- or -L.sup.3-L.sup.3- or -L.sup.3-; and L.sup.1, L.sup.1, L.sup.1, L.sup.3, L.sup.3, and L.sup.3 are independently of each other selected from: CH.sub.2, C.sub.2H.sub.4, C.sub.3H.sub.6, C.sub.4H.sub.8, C.sub.5H.sub.10, C.sub.6H.sub.12, C.sub.7H.sub.14, C.sub.8H.sub.16, C.sub.9H.sub.18, C.sub.10H.sub.20; L.sup.2 is selected from: C(O), ##STR00198## R.sup.6, R.sup.7 and R.sup.8 are independently of each other selected from: H, CH.sub.3, C.sub.2H.sub.5, F, Cl, Br, OCH.sub.3 and CF.sub.3; n represents an integer selected from 1, 2, 3, 4, 5 and 6; p1, p2, p3, m1, m2 and m3 represent independently of each other an integer from 0 to 10.

3. The conjugate according to claim 1, wherein R.sup.3 is H and R.sup.4 is OH.

4. The conjugate according to claim 1 general formula (I-C) ##STR00199## wherein R.sup.2 is (X.sup.1).sub.p1(X.sup.2).sub.p2(X.sup.3).sub.p3X.sup.4; R.sup.5 is (Y).sub.m1(Y.sup.2).sub.m2(Y.sup.3).sub.m3Y.sup.4; X.sup.4 represents: H ##STR00200## Y.sup.4 represents H or -Ph; X.sup.1, X.sup.2, X.sup.3, Y.sup.1, Y.sup.2, Y.sup.3 are independently of each other selected from: CH.sub.2, ##STR00201## n2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; n1, n3 represent independently of each other an integer selected from 0 and 1; L.sup.1 represents -L.sup.1-L.sup.1-L.sup.1- or -L.sup.1-L.sup.1- or -L.sup.1-; and L.sup.3 represents -L.sup.3-L.sup.3-L.sup.3- or -L.sup.3-L.sup.3- or -L.sup.3-; and L.sup.1, L.sup.1, L.sup.1, L.sup.3, L.sup.3, and L.sup.3 are independently of each other selected from: CH.sub.2, C.sub.2H.sub.4, C.sub.3H.sub.6, C.sub.4H.sub.8, C.sub.5H.sub.10, C.sub.6H.sub.12, C.sub.7H.sub.14, C.sub.8H.sub.16, C.sub.9H.sub.18, C.sub.10H.sub.20, (CH.sub.2CH.sub.2O).sub.oCH.sub.2CH.sub.2 and (CH.sub.2CH.sub.2O).sub.oCH.sub.2; R.sup.6, R.sup.7 and R.sup.8 are independently of each other selected from: H, CH.sub.3, C.sub.2H.sub.5, F, Cl, Br, OCH.sub.3 and CF.sub.3; R.sup.9 to R.sup.20 represent independently of each other H, CH.sub.3, C.sub.2H.sub.5, or C.sub.3H.sub.7; o represents an integer selected from 1, 2, 3, 4, 5 and 6; n represents an integer selected from 1, 2, 3, 4, 5 and 6; p1, p2, p3, m1, m2 and m3 represent independently of each other an integer from 0 to 10.

5. The conjugate according to claim 1, wherein R.sup.2 is selected from (CH.sub.2).sub.24CH.sub.3, (CH.sub.2).sub.23CH.sub.3, (CH.sub.2).sub.22CH.sub.3, (CH.sub.2).sub.21CH.sub.3, (CH.sub.2).sub.20CH.sub.3, (CH.sub.2).sub.19CH.sub.3, (CH.sub.2).sub.18CH.sub.3, (CH.sub.2).sub.17CH.sub.3, (CH.sub.2).sub.16CH.sub.3, (CH.sub.2).sub.15CH.sub.3, (CH.sub.2).sub.14CH.sub.3, (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.8CH.sub.3, ##STR00202## and R.sup.6 is selected from: H, CH.sub.3, F, Cl, OCH.sub.3 and CF.sub.3.

6. The conjugate according to claim 1, wherein R.sup.5 is selected from (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.8CH.sub.3, (CH.sub.2).sub.7CH.sub.3, (CH.sub.2).sub.6CH.sub.3, (CH.sub.2).sub.5CH.sub.3, (CH.sub.2).sub.4CH.sub.3, (CH.sub.2).sub.2-Ph, (CH.sub.2).sub.3-Ph, (CH.sub.2).sub.4-Ph, (CH.sub.2).sub.5-Ph, (CH.sub.2).sub.6-Ph, (CH.sub.2).sub.7-Ph, (CH.sub.2).sub.8-Ph, and (CH.sub.2).sub.9-Ph.

7. The conjugate according to claim 1, wherein -L.sup.1- and -L.sup.3- are independently of each other selected from: CH.sub.2, C.sub.2H.sub.4, C.sub.3H.sub.6, C.sub.4H.sub.8, C.sub.5H.sub.10 and C.sub.6H.sub.12.

8. A pharmaceutical composition comprising a conjugate according to claim 1 together with at least one pharmaceutically acceptable acceptable carrier, excipient and/or diluent.

9. The conjugate according to claim 4, wherein R.sup.2 is selected from (CH.sub.2).sub.24CH.sub.3, (CH.sub.2).sub.23CH.sub.3, (CH.sub.2).sub.22CH.sub.3, (CH.sub.2).sub.21CH.sub.3, (CH.sub.2).sub.20CH.sub.3, (CH.sub.2).sub.19CH.sub.3, (CH.sub.2).sub.18CH.sub.3, (CH.sub.2).sub.17CH.sub.3, (CH.sub.2).sub.16CH.sub.3, (CH.sub.2).sub.15CH.sub.3, (CH.sub.2).sub.14CH.sub.3, (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.8CH.sub.3, ##STR00203## and R.sup.6 is selected from: H, CH.sub.3, F, Cl, OCH.sub.3 and CF.sub.3.

10. The conjugate according to claim 4, wherein R.sup.5 is selected from (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.8CH.sub.3, (CH.sub.2).sub.7CH.sub.3, (CH.sub.2).sub.6CH.sub.3, (CH.sub.2).sub.5CH.sub.3, (CH.sub.2).sub.4CH.sub.3, (CH.sub.2).sub.2-Ph, (CH.sub.2).sub.3-Ph, (CH.sub.2).sub.4-Ph, (CH.sub.2).sub.5-Ph, (CH.sub.2).sub.6-Ph, (CH.sub.2).sub.7-Ph, (CH.sub.2).sub.8-Ph, and (CH.sub.2).sub.9-Ph.

11. The conjugate according to claim 4, wherein -L.sup.1- and -L.sup.3- are independently of each other selected from: CH.sub.2, C.sub.2H.sub.4, C.sub.3H.sub.6, C.sub.4H.sub.8, C.sub.5H.sub.10 and C.sub.6H.sub.12.

12. The conjugate according to claim 2, wherein R.sup.3 is H and R.sup.4 is OH.

13. The conjugate according to claim 2, wherein R.sup.2 is selected from (CH.sub.2).sub.24CH.sub.3, (CH.sub.2).sub.23CH.sub.3, (CH.sub.2).sub.22CH.sub.3, (CH.sub.2).sub.21CH.sub.3, (CH.sub.2).sub.20CH.sub.3, (CH.sub.2).sub.19CH.sub.3, (CH.sub.2).sub.18CH.sub.3, (CH.sub.2).sub.17CH.sub.3, (CH.sub.2).sub.16CH.sub.3, (CH.sub.2).sub.15CH.sub.3, (CH.sub.2).sub.14CH.sub.3, (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.8CH.sub.3, ##STR00204## and R.sup.6 is selected from: H, CH.sub.3, F, Cl, OCH.sub.3 and CF.sub.3.

14. The conjugate according to claim 2, wherein R.sup.5 is selected from (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.8CH.sub.3, (CH.sub.2).sub.7CH.sub.3, (CH.sub.2).sub.6CH.sub.3, (CH.sub.2).sub.5CH.sub.3, (CH.sub.2).sub.4CH.sub.3, (CH.sub.2).sub.2-Ph, (CH.sub.2).sub.3-Ph, (CH.sub.2).sub.4-Ph, (CH.sub.2).sub.5-Ph, (CH.sub.2).sub.6-Ph, (CH.sub.2).sub.7-Ph, (CH.sub.2).sub.8-Ph, and (CH.sub.2).sub.9-Ph.

15. The conjugate according to claim 2, wherein -L.sup.1- and -L.sup.3- are independently of each other selected from: CH.sub.2, C.sub.2H.sub.4, C.sub.3H.sub.6, C.sub.4H.sub.5, C.sub.5H.sub.10 and C.sub.6H.sub.12.

16. The conjugate according to claim 3, wherein R.sup.2 is selected from (CH.sub.2).sub.24CH.sub.3, (CH.sub.2).sub.23CH.sub.3, (CH.sub.2).sub.22CH.sub.3, (CH.sub.2).sub.21CH.sub.3, (CH.sub.2).sub.20CH.sub.3, (CH.sub.2).sub.19CH.sub.3, (CH.sub.2).sub.18CH.sub.3, (CH.sub.2).sub.17CH.sub.3, (CH.sub.2).sub.16CH.sub.3, (CH.sub.2).sub.15CH.sub.3, (CH.sub.2).sub.14CH.sub.3, (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.5CH.sub.3, ##STR00205## and R.sup.6 is selected from: H, CH.sub.3, F, Cl, OCH.sub.3 and CF.sub.3.

17. The conjugate according to claim 3, wherein R.sup.5 is selected from (CH.sub.2).sub.13CH.sub.3, (CH.sub.2).sub.12CH.sub.3, (CH.sub.2).sub.11CH.sub.3, (CH.sub.2).sub.10CH.sub.3, (CH.sub.2).sub.9CH.sub.3, (CH.sub.2).sub.8CH.sub.3, (CH.sub.2).sub.7CH.sub.3, (CH.sub.2).sub.6CH.sub.3, (CH.sub.2).sub.5CH.sub.3, (CH.sub.2).sub.4CH.sub.3, (CH.sub.2).sub.2-Ph, (CH.sub.2).sub.3-Ph, (CH.sub.2).sub.4-Ph, (CH.sub.2).sub.5-Ph, (CH.sub.2).sub.6-Ph, (CH.sub.2).sub.7-Ph, (CH.sub.2).sub.8-Ph, and (CH.sub.2).sub.9-Ph.

18. The conjugate according to claim 3, wherein -L.sup.1- and -L.sup.3- are independently of each other selected from: CH.sub.2, C.sub.2H.sub.4, C.sub.3H.sub.6, C.sub.4H.sub.8, C.sub.5H.sub.10 and C.sub.6H.sub.12.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Schematic representation of the liposome containing the conjugate 43* according to the present invention.

(2) FIG. 2. Characterization of the SP3 tetrasaccharide-CRM.sub.197 conjugate 44*: (a) SDS-PAGE; (b) MALDI-TOF-MS.

(3) FIG. 3. Primary antibody response against the liposome containing conjugate 43* and SP3 tetrasaccharide-CRM.sub.197 conjugate 44* in mice: (a) representation of the printed saccharide with symbols; (b) microarray printing pattern; (c) immune response of a mouse immunized with the liposome containing the conjugate 43* (time frame: day 0 to week 2); (d), (e), (f) comparison of the primary antibody response against the liposomes containing conjugate 43* and SP3 tetrasaccharide-CRM.sub.197 conjugate 44* (averaged data of six mice of both groups).

(4) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill 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 of skill 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.

(5) 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

(6) Chemical Synthesis

(7) Abbreviations:

(8) NIS: N-iodosuccinimide;

(9) TfOH: triflic acid;

(10) hr: hour;

(11) DCM: dichloromethane;

(12) TLC: thin layer chromatography;

(13) MW: microwave

(14) rt: room temperature;

(15) RM: reaction mixture;

(16) EtOAc: ethyl acetate;

(17) MS: molecular sieves;

(18) TMS: trimethylsilyl;

(19) Tempo: 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical;

(20) BAIB: bis(acetoxy)iodobenzene;

(21) HOBt: 1-Hydroxybenzotriazole.

(22) General Information for Chemical Synthesis

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

(24) A. Synthesis of Streptococcus pneumoniae Type 3 Capsular Polysaccharide Related Carbohydrate

Example A.1: Synthesis of (2R,4aR,6R,7R,8S,8aR)-6-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxine-7,8-diyldibenzoate (1*)

(25) ##STR00078##

(26) (2R,4aR,6S,7R,8S,8aR)-6-(ethylthio)-2-phenylhexahydropyrano[3,2-d][1,3]dioxine-7,8-diyldibenzoate (6.0 g, 11.53 mmol) and benzyl benzyl(2-hydroxyethyl)carbamate 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 microwave-dried 4 MS to it and stirred at rt for 15 min and then cooled to 10 C. After addition of NIS (3.83 g, 17.29 mmol) and TfOH (0.15 mL, 1.73 mmol), the reaction mixture under stirring was warmed from 10 C. to 5 C. during 1 hr. RM was then quenched with 10% aq. Na.sub.2S.sub.2O.sub.3 solution (50 mL) and then extracted with EtOAc (25 ml3). 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 provide desired product 1* as pale yellow colored transparent gummy liquid (7.60 g, 89%).

(27) .sup.1H NMR (400 MHz, CDCl.sub.3) 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) 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 A.2: Synthesis of (2R,3R,4S,5R,6R)-2-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-6-((benzyloxy)methyl)-5-hydroxytetrahydro-2H-pyran-3,4-diyldibenzoate (2*)

(28) ##STR00079##

(29) Glucose 1* (7.50 g, 10.08 mmol) was taken in DCM (75 mL) under argon with activated 3 MS for 10 min before cooling to 0 C. Triethylsilane (12.88 mL, 81.0 mmol), followed by TFA (4.66 mL, 60.5 mmol) were added dropwise and the RM was stirred at rt for 16 h before quenching with water (100 mL). The RM was extracted with DCM (30 mL3), and the combined organic layers were washed thoroughly with water (20 mL3), brine (20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, evaporated in vacuum to get colorless gummy solid. The crude product was purified by silica column chromatography using 30%-100% EtOAc in hexanes to provide after evaporation in vacuum target compound as colorless oil (6.1 g, 81%).

(30) .sup.1H NMR (400 MHz, CDCl.sub.3) 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) 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 A.3: Synthesis of (2R,3R,4S,5R,6R)-5-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-8-((tert-butyldimethylsilyl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-2-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3,4-diyldibenzoate (3*)

(31) ##STR00080##

(32) (2R,3R,4S,5R,6R)-2-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-6-((benzyloxy)methyl)-5-hydroxytetrahydro-2H-pyran-3,4-diyldibenzoate (2.0 g, 2.68 mmol) was taken in DCM (30 mL) with activated 4 acid washed MS and stirred at rt for 30 min before cooling to 0 C. TMSOTf (0.49 L, 0.27 mmol) was then added followed by the (2R,4aR,6S,7R,8S,8aR)-8-((tert-butyldimethylsilyl)oxy)-2-phenyl-6-(2,2,2-trichloro-1-iminoethoxy)hexahydropyrano[3,2-d][1,3]dioxin-7-yl benzoate (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. The RM was quenched with Et.sub.3N (1 mL), filtered and the solvents removed under vacuum. The crude product was purified by flash chromatography using EtOAc in hexanes to get product 3* (3.2 g, 98%).

(33) .sup.1H NMR (400 MHz, cdcl.sub.3) 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) 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 A.4: Synthesis of (2R,3R,4S,5R,6R)-5-(((2R,4aR,6S,7R,8S,8aS)-7-(benzoyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-2-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3,4-diyldibenzoate (4*)

(34) ##STR00081##

(35) (2R,3R,4S,5R,6R)-5-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-8-((tert-butyl dimethylsilyl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-2-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3,4-diyldibenzoate (1.6 g, 1.317 mmol) was taken in pyridine (10 mL) at 0 C. and treated with HF-pyridine (3.56 mL, 39.5 mmol). The mixture was stirred at rt for 24 h. RM was washed with water and extracted with DCM (20 mL3). Combined organic layers were then washed with diluted HCl (50 mL2), saturated 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 after purification using silica column chromatography using 35-40% EtOAc in hexanes yielded target compound as white colored foam (1.3 g, 90%).

(36) .sup.1H NMR (400 MHz, CDCl.sub.3) 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) 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 A.5: Synthesis of (2S,3R,4S,5R,6R)-2-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-6-(((2R,3R,4S,5R,6R)-4,5-bis(benzoyloxy)-6-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-5-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-8-((tert-butyldimethylsilyl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3,4-diyldibenzoate (5*)

(37) ##STR00082##

(38) Acceptor 4* (1.0 g, 0.91 mmol), (2S,3R,4S,5R,6R)-5-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-8-((tert-butyldimethylsilyl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)-2-(ethylthio)tetrahydro-2H-pyran-3,4-diyldbenzoate (12*) (1.08 g, 1.091 mmol) and 20 g of dried 4 MS were taken in DCM (30 mL), stirred at rt for 15 min and then cooled to 10 C. NIS (0.245 g, 1.09 mmol) and TfOH (0.016 mL, 0.18 mmol) were then added and the reaction mixture was for 1 h stirred at 5 C. (2S,3R,4S,5R,6R)-5-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-8-((tert-buty ldimethylsilyl)oxy)-2-phenyl hexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)-2-(ethylthio)tetrahydro-2H-pyran-3,4-diyl dibenzoate (0.45 g, 0.454 mmol, 0.5 equiv.) and NIS (0.102 mg, 0.454 mmol, 0.5 equiv.) were added again to the reaction mixture and stirred at 5 C. for 1 h, then warmed to 5 C. After filtration through a Celite bed, the RM was quenched with 10% Na.sub.2S.sub.2O.sub.3 solution (25 mL) and then extracted with DCM (15 ml3). Combined organic layers were then washed with sat. NaHCO.sub.3 solution (15 mL), brine (10 ml), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum to get white colored fluffy solid compound. Crude product was purified by silica column chromatography using 30-35% EtOAc in hexanes to get target 5* as fluffy white solid (1.0 g, 54%).

(39) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.05-6.87 (m, 60H), 5.57-5.40 (m, 1H), 5.39-5.24 (m, 2H), 5.21-4.86 (m, 7H), 4.60 (d, J=7.9 Hz, 1H), 4.54-4.15 (m, 9H), 4.07-3.85 (m, 3H), 3.81-3.70 (m, 3H), 3.60 (dd, J=10.6, 4.8 Hz, 1H), 3.52 (dd, J=10.6, 4.9 Hz, 1H), 3.47-3.14 (m, 9H), 3.13-2.96 (m, 3H), 2.64 (t, J=10.4 Hz, 1H), 2.55 (t, J=10.3 Hz, 1H), 0.58 (s, 9H), 0.18 (s, 3H), 0.26 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 165.4, 165.1, 164.9, 164.5, 164.0, 156.4, 156.2, 138.4, 138.1, 137.1, 137.0, 133.2, 133.0, 132.6, 130.3, 130.1, 130.0, 129.96, 129.9, 129.8, 129.4, 129.1, 128.6, 128.5, 128.51, 128.4, 128.3, 128.2, 128.15, 128.1, 128.06, 128.0, 127.8, 127.3, 126.4, 126.1, 101.7, 101.5, 101.2, 101.18, 101.0, 100.2, 81.1, 79.5, 77.4, 75.9, 75.6, 75.1, 74.4, 73.7, 73.5, 73.4, 73.2, 72.4, 68.8, 67.9, 67.3, 66.2, 66.0, 51.7, 46.9, 45.9, 21.2, 17.9, 4.1, 4.9.

Example A.6: Synthesis of (2S,3R,4S,5R,6R)-2-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-6-(((2R,3R,4S,5R,6R)-4,5-bis(benzoyloxy)-6-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-5-(((2R,4aR,6S,7R,8S,8aS)-7-(benzoyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3,4-diyldibenzoate (6*)

(40) ##STR00083##

(41) Tetrasaccharide 5* (1.0 g, 0.493 mmol) was taken in pyridine (10 mL) at 0 C. and added HF-pyridine (1.33 mL, 14.78 mmol) to it and stirred at rt for 36 hrs. The RM was washed with water and extracted with DCM (20 mL3). Combined organic layers were then washed with cold diluted HCl (50 mL2), saturated 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 after purification on silica column chromatography using 50% EtOAc in hexanes yielded target compound as a white colored foam (0.71 g, 75%).

(42) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.07-6.94 (m, 60H), 5.56-5.41 (m, 1H), 5.37-5.23 (m, 2H), 5.22-5.14 (m, 2H), 5.13-5.02 (m, 2H), 5.02-4.91 (m, 3H), 4.60 (dd, J=7.9, 3.5 Hz, 2H), 4.56-4.48 (m, 1H), 4.46 (d, J=7.9 Hz, 1H), 4.43-4.24 (m, 4H), 4.20 (d, J=12.1 Hz, 2H), 4.09-3.88 (m, 3H), 3.85-3.70 (m, 3H), 3.61 (dd, J=10.6, 4.7 Hz, 1H), 3.54-3.31 (m, 5H), 3.31-3.15 (m, 5H), 3.16-2.97 (m, 3H), 2.65 (t, J=10.4 Hz, 1H), 2.56 (t, J=10.4 Hz, 1H), 2.38 (d, J=3.5 Hz, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) 165.3, 165.2, 165.0, 164.9, 164.7, 163.9, 138.2, 137.9, 136.8, 136.8, 133.4, 133.0, 132.5, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.2, 129.0, 128.5, 128.4, 128.35, 128.3, 128.1, 128.0, 127.97, 127.9, 127.8, 127.7, 127.2, 126.2, 126.0, 101.6, 101.3, 101.0, 100.9, 100.8, 100.0, 80.4, 79.3, 78.3, 77.2, 76.6, 76.0, 75.5, 74.8, 74.3, 73.6, 73.2, 72.3, 72.1, 68.7, 67.6, 67.2, 66.1, 65.7, 51.5, 46.7, 45.7.

Example A.7: Synthesis of (2S,3R,4S,5R,6R)-2-(((2R,4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-6-(((2R,3R,4S,5R,6R)-4,5-bis(benzoyloxy)-6-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)-6-((benzyloxy)methyl)-5-(((2R,4aR,6S,7R,8S,8aR)-7,8-bis(benzoyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)tetrahydro-2H-pyran-3,4-diyldibenzoate (7*)

(43) ##STR00084##

(44) Tetrasaccharide 6* (0.65 g, 0.339 mmol) was taken in pyridine (5 mL), treated with BzCl (0.79 mL, 0.679 mmol) and stirred at rt for 16 hrs. The RM was diluted with water and extracted with DCM (20 mL3). Combined organics were washed with cold diluted HCl (10 mL2), saturated NaHCO.sub.3 (10 mL2), water (10 mL), brine (20 mL), dried over Na.sub.2SO.sub.4, filtered, and concentrated in vacuum to get crude product, which was then triturated using cold MeOH (5 mL3) to get target 7* as white solid (0.65 g, 95%).

(45) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.10-6.92 (m, 65H), 5.55-5.41 (m, 2H), 5.40-5.15 (m, 4H), 5.13-4.96 (m, 4H), 4.93 (s, 1H), 4.67 (d, J=7.9 Hz, 1H), 4.61 (d, J=7.9 Hz, 1H), 4.55-4.47 (m, 1H), 4.45 (d, J=7.9 Hz, 1H), 4.43-4.16 (m, 6H), 4.10-3.86 (m, 3H), 3.84-3.72 (m, 1H), 3.65-3.34 (m, 6H), 3.33-3.15 (m, 6H), 3.11-3.06 (m, 3H), 2.67-2.59 (m, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) 165.6, 165.4, 165.2, 165.1, 164.8, 164.0, 156.3, 156.2, 138.1, 138.1, 138.0, 137.0, 136.8, 133.4, 133.1, 132.7, 130.2, 129.9, 129.9, 129.8, 129.8, 129.7, 129.5, 129.3, 129.3, 129.2, 129.1, 128.6, 128.6, 128.6, 128.5, 128.4, 128.35, 128.3, 128.2, 128.16, 128.1, 127.8, 127.3, 126.2, 126.1, 125.4, 101.5, 101.3, 101.2, 101.0, 100.1, 79.5, 78.4, 77.4, 76.3, 75.6, 74.3, 73.7, 73.5, 73.4, 73.1, 72.3, 72.3, 72.3, 68.8, 68.0, 67.8, 67.1, 66.24, 66.2, 51.7, 46.9, 45.8.

Example A.8: Synthesis of (2S,3R,4S,5R,6R)-2-(((2S,3R,4S,5R,6R)-3-(benzoyloxy)-2-(((2R,3R,4S,5R,6R)-4,5-bis(benzoyloxy)-6-(2-(benzyl((benzyloxy carbonyl)amino)ethoxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)-6-((benzyloxy)methyl)-5-(((2S,3R,4S,5R,6R)-3,4-bis(benzoyloxy)-5-hydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4-diyldibenzoate (8*)

(46) ##STR00085##

(47) Tetrasaccharide 7* (0.54 g, 0.267 mmol) was taken in DCM (5 mL) at rt, treated with PTSA (10 mg, 0.053 mmol) and EtSH (0.297 mL, 4.01 mmol), and stirred for 4 hrs. The RM was quenched with Et.sub.3N (1 mL), evaporated in vacuum and purified using 60% EtOAc in hexanes to get target 8* as white colored solid product (0.46 g, 93%).

(48) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.02-7.81 (m, 10H), 7.59-6.91 (m, 45H), 5.52 (t, J=9.2 Hz, 1H), 5.47-5.15 (m, 5H), 5.12-4.90 (m, 3H), 4.62 (d, J=7.6 Hz, 1H), 4.61-4.57 (m, 1H), 4.52 (d, J=7.7 Hz, 1H), 4.47-4.36 (m, 3H), 4.34-4.18 (m, 4H), 4.13-3.99 (m, 2H), 3.95-3.73 (m, 2H), 3.69 (td, J=9.1, 4.3 Hz, 1H), 3.61-3.50 (m, 3H), 3.27 (m, 1H), 3.11-2.95 (m, 3H), 2.89 (d, J=4.3 Hz, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) 167.4, 165.36, 165.23, 165.2, 165.2, 164.9, 163.8, 156.3, 156.2, 138.1, 137.3, 133.7, 133.6, 133.3, 133.0, 132.7, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.51, 129.5, 129.2, 129.0, 128.9, 128.8, 128.7, 128.69, 128.6, 128.5, 128.4, 128.3, 128.13, 128.1, 127.8, 127.3, 127.2, 101.3, 101.1, 100.8, 100.3, 85.1, 77.4, 77.0, 76.0, 75.8, 74.8, 74.7, 74.4, 73.8, 73.6, 73.3, 72.2, 71.7, 71.7, 69.4, 69.3, 68.9, 67.6, 67.1, 62.5, 61.6, 51.7, 46.9, 45.9. MALDI-TOF: calculated for C.sub.104H.sub.99NNaO.sub.30 [M+H].sup.+, 1864.61, found 1864.77.

Example A.9: Synthesis of (2S,3S,4S,5R,6R)-4,5-bis(benzoyloxy)-6-(((2R,3R,4S,5R,6S)-4,5-bis(benzoyloxy)-6-(((2R,3R,4S,5S,6S)-3-(benzoyloxy)-2-(((2R,3R,4S,5R,6R)-4,5-bis(benzoyloxy)-6-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-6-carboxy-5-hydroxytetrahydro-2H-pyran-4-yl)oxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-3-hydroxytetrahydro-2H-pyran-2-carboxylic acid (9*)

(49) ##STR00086##

(50) Tetrasaccharide 8* (0.125 g, 0.068 mmol) was taken in a mixture DCM/water (7 ml, 5:2) and cooled to 0 C. After addition of tempo (2.1 mg, 0.014 mmol), followed by BAIB (0.109 g, 0.339 mmol), the RM was stirred at 0 C. for 20 min and slowly warmed up to rt and further stirred at rt for 2 h (total 3 h). The RM was diluted with DCM (5 mL) and water (5 mL), and the layers were separated. The aqueous layer was extracted with DCM (5 mL4). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, concentrated in vacuum to provide the crude product, which was then purified on silica column using 10-15% acetone in DCM+1-2% AcOH to yield after evaporation desired product 9* as a yellowish solid (0.09 g, 71%).

(51) .sup.1H NMR (400 MHz, CD.sub.3OD) 8.00-6.52 (m, 55H), 5.61-5.29 (m, 3H), 5.27-5.05 (m, 3H), 4.99 (d, J=9.8 Hz, 1H), 4.94 (d, J=8.0 Hz, 1H), 4.88-4.83 (m, 2H), 4.76 (d, J=7.9 Hz, 1H), 4.53 (d, J=8.0 Hz, 1.5H), 4.43 (dd, J=12.1, 4.8 Hz, 1H), 4.37 (d, J=7.8 Hz, 0.5H), 4.33-4.02 (m, 7H), 3.87-3.39 (m, 11H), 3.26-3.03 (m, 4H). .sup.13C NMR (101 MHz, CD.sub.3OD) 170.4, 170.35, 167.2, 167.18, 166.9, 166.7, 166.5, 166.4, 165.7, 158.0, 157.7, 139.2, 138.8, 138.6, 134.8, 134.5, 134.4, 134.1, 134.0, 131.4, 130.8, 130.8, 130.7, 130.6, 130.5, 130.3, 130.1, 129.8, 129.7, 129.53, 129.5, 129.4, 129.3, 129.2, 129.1, 128.8, 128.6, 128.2, 102.2, 101.9, 101.8, 101.7, 84.2, 77.6, 76.7, 76.4, 75.4, 74.6, 74.4, 73.7, 73.5, 73.3, 71.5, 71.1, 69.5, 69.4, 68.5, 68.31, 68.3, 52.6, 52.5, 47.10. MALDI-TOF: calculated for C.sub.104H.sub.95NO.sub.32 [M+H].sup.+, 1892.57, found 1892.71.

Example A.10: Synthesis of (2S,3S,4S,5R,6R)-6-(((2R,3S,4R,5R,6R)-6-(2- (benzyl((benzyloxy)carbonyl)amino)ethoxy)-2-((benzyloxy)methyl)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)oxy)-4-(((2S,3R,4R,5S,6R)-6-((benzyloxy)methyl)-5-(((2R,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4-dihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,5-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (10*)

(52) ##STR00087##

(53) Tetrasaccharide 9* (0.09 g, 0.48 mmol) was taken in MeOH (5 mL), treated with 0.5 M solution NaOMe in methanol (4.81 mL, 2.405 mmol) and stirred at rt for 24 hrs. The RM was then neutralized with Amberlite 120H.sup.+ resin to give a clear solution, which was filtered through a cotton plug, washed thoroughly with MeOH and evaporated in vacuum to give a yellowish gum. The yellowish gum was taken in diethyl Et.sub.2O and triturated to provide a pale yellowish solid. The ether layer was then decanted (33 ml). The pale yellowish solid was triturated with DCM to give a white solid, which was then dried under vacuum to yield target 10* as white powder (0.05 g, 91%). .sup.1H NMR (400 MHz, CD.sub.3OD) 7.49-7.06 (m, 20H), 5.14 (d, J=9.5 Hz, 2H), 4.68-4.51 (m, 6H), 4.47-4.37 (m, 3H), 4.27-4.17 (m, 1H), 3.96-3.34 (m, 23H), 3.30-3.19 (m, 2H). LCMS (ESI): calculated for C.sub.55H.sub.66NO.sub.25 [MH].sup.+, 1140.39, found 1140.2.

Example A.11: Synthesis of (2S,3S,4S,5R,6R)-6-(((2R,3S,4R,5R,6R)-6-(2-aminoethoxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4-(((2S,3R,4R,5S,6R)-5-(((2R,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-3,5-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (11*)

(54) ##STR00088##

(55) A mixture of tetrasaccharide 10* (50 mg) and 10% Pd/C (100 mg) in MeOH (2 mL) was stirred at rt under hydrogen for 18 h. The RM was then filtered through PTFE hydrophobic filters and washed thoroughly with methanol, water-methanol, and later with NH.sub.4OH in methanol. Evaporation of the filtrate and drying under vacuum provided target 11* as white glassy film (23 mg, 71%).

(56) .sup.1H NMR (400 MHz, D.sub.2O) 4.84 (d, J=8.0 Hz, 1H), 4.56 (d, J=8.0 Hz, 2H), 4.53 (d, J=7.9 Hz, 1H), 4.14 (dt, J=11.5, 4.9 Hz, 1H), 4.04-3.92 (m, 3H), 3.89-3.75 (m, 5H), 3.72-3.49 (m, 10H), 3.43-3.34 (m, 3H), 3.29 (t, J=5.1 Hz, 2H). .sup.13C NMR (101 MHz, D.sub.2O) 175.4, 175.2, 102.3, 102.2, 102.0, 101.8, 82.7, 78.9, 78.6, 75.7, 75.2, 74.8, 74.7, 74.1, 73.1, 72.9, 72.7, 71.6, 70.1, 65.7, 60.0 39.3. HRMS (ESI): calculated for C.sub.26H.sub.44NO.sub.23 [M+H].sup.+, 738.23, found 738.27.

(57) Tetrasaccharides 11*a-11*c were synthesized by applying the procedures described in examples A.1 to A.11 to benzyl benzyl(3-hydroxypropyl)carbamate, benzyl benzyl(4-hydroxybuthyl)carbamate and benzyl benzyl(5-hydroxypentyl)carbamate.

(58) ##STR00089##

(59) HRMS (ESI): calculated for C.sub.27H.sub.45NO.sub.23 [M+H].sup.+, 752.23, found 752.21.

(60) ##STR00090##

(61) HRMS (ESI): calculated for C.sub.28H.sub.47NO.sub.23 [M+H].sup.+, 766.25, found 766.21.

(62) ##STR00091##

(63) HRMS (ESI): calculated for C.sub.29H.sub.49NO.sub.23 [M+H].sup.+, 780.26, found 780.24.

Example A.12: Synthesis of (2S,3R,5R,6R)-5-(((4aR,6S,7R,8S,8aR)-7-(benzoyloxy)-8-((tert-butyldimethylsilyl)oxy)-2-phenylhexahydropyranophenyl hexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)-2-(ethylthio)tetrahydro-2H-pyran-3,4-diyldibenzoate (12*)

(64) ##STR00092##

(65) A mixture of (2S,3R,4S,5R,6R)-6-((benzyloxy)methyl)-2-(ethylthio)-5-hydroxytetrahydro-2H-pyran-3,4-diyl dibenzoate (4.00 g, 7.654 mmol, 1.0 eq.) and (4a R,6R,7R,8a R)-8-((tert-butyldimethylsilyl)oxy)-2-phenyl-6-(2,2,2-trichloro-1-iminoethoxy)hexahydropyrano[3,2-d][1,3]dioxin-7-yl benzoate (6.28 g, 9.95 mmol, 1.3 eq.) in DCM (140 mL) was stirred under an Ar atmosphere 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. 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]dioxin-6-yl)oxy)-6-((benzyloxy)methyl)-2-(ethylthio)tetrahydro-2H-pyran-3,4-diyl dibenzoate 12* (6 g, 79%) as a colorless solid:

(66) R.sub.f=0.5 (EtOAc/hexanes, 3/7, v/v). .sup.1H NMR (400 MHz, CDCl.sub.3) 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) 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.

(67) B. Synthesis of the Glycosphingolipid

Example B.1: Synthesis of (S)-3-(tert-Butoxycarbonyl)-N-methoxy-2,2, N-trimethyloxazolidine-4-carboxamide (13*)

(68) ##STR00093##

(69) To a solution of L-Boc-serine (12.33 g, 60.1 mmol) in DCM (240 mL) were added N,O-dimethylhydroxylamine hydrochloride (6.04 g, 61.9 mmol) and N-methylmorpholine (6.8 mL, 61.9 mmol) at 0 C. To this solution was added N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (11.86 g, 61.9 mmol) portionwise over a period of 20 min. and the solution was stirred for another 1 h. Then, aqueous HCl solution (1.0 M, 30 mL) was added and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (2100 mL). The combined organic layers were washed with saturated aqueous NaHCO.sub.3 solution (30 mL) and the aqueous layer was again extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic layers were dried over MgSO.sub.4 and the solvent was removed in vacuo to obtain the corresponding Weinreb amide (14.07 g, 94%) as white solid.

(70) R.sub.f=0.3 (EtOAc);

(71) .sup.1H NMR (250 MHz, CDCl.sub.3) 5.60 (d, J=6.0 Hz, 1H), 4.77 (br s, 1H), 1.42 (s, 9H), 3.80 (d, J=3.3 Hz, 2H), 3.76 (s, 3H), 3.21 (s, 3H), 2.66 (br s, 1H).

(72) The crude product was dissolved in acetone (180 mL) to which 2,2-dimethoxypropane (57 mL) and BF.sub.3.Et.sub.2O (0.5 mL) were added. The orange solution was stirred for 90 min. at r.t. and then quenched with Et.sub.3N (1.2 mL) and solvents removed in vacuo. The crude product was purified by flash column chromatography on silica gel (gradient EtOAc/cyclohexane=1:2.fwdarw.1:1) to yield isopropylidene-protected Weinreb amide 13* (15.32 g, 89% over two steps) as a white solid. The NMR spectra consist of two sets of signals due to the presence of rotamers.

(73) [].sub.D.sup.r.t.=30.9 (c=1, CHCl.sub.3); R.sub.f=0.45 (Hexanes/EtOAc=1:1); IR (film) .sub.max 2976, 2938, 1702, 1682, 1364, 1167, 1098, 998, 848, 768, 716; .sup.1H NMR (250 MHz, CDCl.sub.3) 4.77 (dd, J=9.8, 2.8 Hz, 1H), 4.70 (dd, 7.5, 3.8, Hz, 1H), 4.18 (dd, J=7.5, 4.0 Hz, 1H), 4.15 (dd, J=7.8, 3.8 Hz, 1H), 3.95 (dd, J=9.3, 3.0 Hz, 1H), 3.91 (dd, J=9.0, 3.5 Hz), 3.72 (s, 3H), 3.68 (s, 3H), 3.19 (s, 6H), 1.68 (s, 3H), 1.66 (s, 3H), 1.54 (s, 3H), 1.50 (s, 3H), 1.47 (s, 9H), 1.39 (s, 9H); .sup.13C NMR (101 MHz, CDCl.sub.3) 171.4, 170.7, 152.2, 151.4, 95.1, 94.5, 80.6, 80.0, 66.2, 66.0, 61.3, 61.3, 57.9, 57.8, 28.5, 28.4, 25.8, 25.5, 24.8, 24.6; HR ESI Calcd for C.sub.13H.sub.24N.sub.2O.sub.5 [M+Na.sup.+]: 311.1577 found: 311.1582.

Example B.2: Synthesis of tert-Butyl (S)-4-formyl-2,2-dimethyloxazolidine-3-carboxylate (14*)

(74) ##STR00094##

(75) To a solution of Weinreb amide 13* (8.00 g, 27.7 mmol) in THF (100 mL) at 0 C. were added LiAlH.sub.4 (1.0 M in THF, 13.9 mL, 13.9 mmol) dropwise and the solution was stirred for 1 h at 0 C. After 1 h, the solution was cooled to 10 C. and KHSO.sub.4 (1M, 70 mL) was added carefully and the solution was diluted with Et.sub.2O (170 mL). The mixture was allowed to warm to r.t. and stirred for 30 min. The organic layer was separated, dried over MgSO.sub.4, filtered and the solvent was removed in vacuo to yield Garner's aldehyde 14* as a pale yellow oil (6.24 g, >95% purity by .sup.1H NMR). The NMR spectra consist of two sets of signals due to the presence of rotamers. .sup.1H NMR (250 MHz, CDCl.sub.3) 9.58 (d, J=0.8 Hz, 1H), 9.52 (d, J=2.5 Hz, 1H), 4.32 (m, 1H), 4.16 (m, 1H), 4.06 (m, 4H), 1.53-1.63 (m, 12H), 1.49 (s, 9H), 1.40 (s, 9H). All spectral data in good accordance with reported data (Synthesis 1998, 1707). The crude product was used in the subsequent reaction without further purification.

Example B.3: Synthesis of (4R,1Z)-3-(tert-Butoxycarbonyl)-2,2-dimethyl-4-(1-hexadecenyl)oxazolidine (15*)

(76) ##STR00095##

(77) n-BuLi (1.6 M in hexane, 25.2 mL, 40.3 mmol) was added dropwise to pentadecyl-triphenylphosphonium bromide 16* (24.03 g, 43.4 mmol) in anhydrous THF (220 mL) at 78 C. The resulting orange solution was allowed to warm to 0 C. and stirred for another 30 min. The solution was then cooled to 78 C. and Garner's aldehyde 14* (6.23 g, 27.2 mmol) in anhydrous THF (30 mL) was added slowly. After being stirred for 2 h at r.t., the reaction was diluted with sat. aq. NH.sub.4Cl solution (35 mL) and the layers were separated. The aqueous layer was extracted with CH.sub.2Cl.sub.2 (335 mL) and the combined organic extracts were washed with saturated aqueous NaCl solution (50 mL), dried over MgSO.sub.4 and concentrated in vacuo. Purification by flash column chromatography on silica (EtOAc/Hexanes=1:2) gel gave (Z)-olefin 15* as a pale yellow oil (11.27 g, 78%).

(78) [].sub.D.sup.r.t.=+45.2 (c=1, CHCl.sub.3); R.sub.f=0.40 (EtOAc/Hexanes=1:2); IR (film) .sub.max 2923, 2854, 1699, 1457, 1382, 1251, 1175, 1093, 1056, 850, 768 cm.sup.1; .sup.1H NMR (250 MHz, CDCl.sub.3) 5.27-5.40 (m, 2H), 4.58 (br s, 1H), 4.02 (dd, J=6.3, 8.8 Hz, 1H), 3.61 (dd, J=3.3, 8.5 Hz, 1H), 1.96 (br s, 2H), 1.23-1.56 (m, 39H), 0.85 (t, J=7 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3) 152.1, 130.9, 130.4, 94.1, 79.8, 69.2, 54.7, 32.1, 29.9, 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 29.4, 28.6, 28.6, 27.6, 22.8, 14.2; HR ESI Calcd for C.sub.26H.sub.49NO.sub.3 [M+Na+]: 446.3605 found: 446.3614. All spectral data were in good accordance with reported data (Synthesis 2004, 847).

(79) The desired (Z)-olefin can easily be distinguished from the undesired (E)-olefin by-product, when considering the olefinic protons in the .sup.1H NMR spectrum: Z-15* .sup.1H NMR (250 MHz, CDCl.sub.3) 4.05 (dd, J=6.3, 8.6 Hz, I H), 3.64 (dd, J=3.3, 8.6 Hz, 1H) cf. E-15* .sup.1H NMR (250 MHz, CDCl.sub.3) 4.01 (dd, J=6.1, 8.7 Hz, 1H), 3.71 (dd, J=2.1, 8.7 Hz, 1H).

Example B.4: Synthesis of pentadecyltriphenylphosphonium bromide (16*)

(80) A solution of 1-bromopentadecane (30.0 g, 103 mmol) and triphenylphosphine (27.02 g, 103 mmol) in MeCN (200 mL) was refluxed at 80 C. for five days. After removal of the solvent in vacuo, Et.sub.2O (30 mL) was added and the resulting white precipitate was filtered off, washed with Et.sub.2O and dried on high vacuum for 24 h to give pentadecyltriphenylphosphonium bromide (16*) (49.66 g, 87%) as a white powder.

Example B.5: Synthesis of (2R,3Z)-2-(tert-Butoxycarbonyl)amino-3-octadecen-1-ol (17*)

(81) ##STR00096##

(82) Para-Toluensulfonic acid (371 mg, 1.95 mmol) was added to a stirred solution of (Z)-olefin 15* (5.00 g, 12.2 mmol) in MeOH/water (50 mL total, ratio=9:1 v/v) and the mixture was stirred for 68 h. The reaction mixture was concentrated in vacuo to yield a white solid, which was re-dissolved in CH.sub.2Cl.sub.2 (100 mL). The solution was washed with brine (30 mL), dried over MgSO.sub.4 and the solvent was removed in vacuo. Purification by flash column chromatography on silica gel (gradient cyclohexane/EtOAc=4:1.fwdarw.2:1) afforded alcohol 17* as a white solid (2.71 g, 59%). All spectral data were in good accordance with reported data (Synthesis 2004, 847).

Example B.6: Synthesis of (2S,3S,4R)-2-(tert-Butoxycarbonyl)amino-1,3,4-octadecanetriol (18*)

(83) ##STR00097##

(84) Alcohol 17* (1.50 g, 3.91 mmol) was dissolved in t-BuOH/water (38 mL total, ratio 1:1) and methanesulfonamide (371 mg, 3.91 mmol) was added. The reaction mixture was cooled to 0 C. and AD-mix- (5.48 g) was added. The resulting mixture was stirred at 0 C. for 41 h and another 7 h at r.t., then it was quenched by the addition of solid Na.sub.2SO.sub.3 (6.0 g) and left to stir for 30 min. Extraction with EtOAc (340 mL) followed. The organic extracts were washed with NaOH (1 M, 20 mL), water (20 mL) and saturated aqueous NaCl solution (20 mL), dried over MgSO.sub.4 and solvents were removed in vacuo. Purification by flash column chromatography on silica gel (gradient EtOAc/cyclohexane=1:1.fwdarw.2:1) provided triol 18* as a white solid (1.05 g, 64%). All spectral data were in good accordance with reported data (Synthesis 2004, 847).

Example B.7: Synthesis of (2S,3S,4R)-2-aminooctadecane-1,3,4-triol (19*)

(85) ##STR00098##

(86) Triol 18* (60 mg, 0.14 mmol) was dissolved in TFA/H.sub.2O (20:1, 0.6 mL) and stirred at r.t. for 30 min. The solution was diluted with CH.sub.2Cl.sub.2 (1.5 mL) and then carefully neutralized (to pH 8) with saturated aqueous NaHCO.sub.3 solution (10 mL) upon which precipitation of a white solid occurred. The white solid removed by filtration, washed with water (310 mL) and dried under reduced pressure. Recrystallization from MeCN yielded phytosphingosine 19* as a white powder (38 mg, 82%). All spectral data were in good accordance with reported data (Synthesis 2004, 847).

Example B.8: Synthesis of hexacosanoic N-hydroxysuccinimidyl ester (20*)

(87) ##STR00099##

(88) To a solution of hexacosanoic acid (121 mg, 0.304 mmol) in CH.sub.2Cl.sub.2 (4 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.058 mL, 0.33 mmol) and N-hydroxysuccinimide (42 mg, 0.37 mmol). The reaction mixture was heated to 40 C., stirred for 3 h and then quenched with water (4 mL). The solution was diluted with Et.sub.2O (8 mL) and the two layers were separated. The aqueous phase was extracted with Et.sub.2O (8 mL) and the combined organic layers were washed with saturated aqueous NaCl solution (5 mL), dried over MgSO.sub.4 and filtered. After removal of the solvent in vacuo, N-hydroxysuccinimidyl ester 20* was obtained as a white solid (85 mg, 57%).

Example B.9: Synthesis of N-((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)heptacosanamide (21*)

(89) ##STR00100##

(90) To a solution of phytosphingosine 19* (15 mg, 0.047 mmol) in anhydrous THF (1 mL) was added succinimidyl ester 20* (34 mg, 0.071 mmol) and Et.sub.3N (24 L, 0.14 mmol). The solution was heated to 50 C. and stirred for 20 h. EtOAc (5 mL) was added and the resulting suspension was centrifuged (30 min., 3000 rpm). The white precipitate was removed by filtration and dried under reduced pressure to yield amide 21* (29 mg, 88%).

Example B.10: Synthesis of (2S,3S,4R)-1,3,4-Tri-t-butyl-dimethylsilyloxy-2-hexacosanoylamino-1-octadecane (22*)

(91) ##STR00101##

(92) To a stirred suspension of amide 21* (25 mg, 0.036 mmol) in CH.sub.2Cl.sub.2 (1.2 mL) was added TBSOTf (43 L, 0.18 mmol) and 2,6-lutidine (65 L, 0.054 mmol) at 0 C. The reaction mixture was stirred at r.t. for 2 h. The reaction was quenched with MeOH (0.2 mL). The mixture was diluted with Et.sub.2O (2 mL) and washed with saturated aqueous NaHCO.sub.3 solution (1 mL) and saturated aqueous NaCl solution (1 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (cyclohexane/Et.sub.2O=15:1) to give TBS protected ceramide 22* as a colorless oil (27 mg, 71%). All spectral data were in good accordance with reported data (Synthesis 2004, 847).

Example B.11: Synthesis of (2S,3S,4R)-3,4-Bis-tert-butyldimethylsilyloxy-2-hexacosanoylamino-4-octadecanol (23*)

(93) ##STR00102##

(94) To a solution of ceramide 22* (90 mg, 0.087 mmol) in THF (2 mL) was added TFA (40 L, 0.519 mmol) in water (0.5 mL, 27.8 mmol) at 10 C. The reaction mixture was left to warm to 10 C. over a period of 2 h. Then, the reaction mixture was quenched by the addition of saturated aqueous NaHCO.sub.3 solution until neutral pH was reached. The resulting mixture was diluted with Et.sub.2O (10 mL), washed with water (10 mL), saturated aqueous NaHCO.sub.3 (10 mL), saturated aqueous NaCl solution (10 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 EtOAc/cyclohexane=10:1.fwdarw.5:1) to yield alcohol 23* (68 mg, 85%) as a colorless oil.

(95) [].sub.D.sup.r.t.=11.6 (c=1, CHCl.sub.3); R.sub.f=0.3 (cyclohexane/EtOAc=4:1); IR (film) .sub.max 3285, 2920, 2851, 1645, 1465, 1253, 1034, 835, 776, 721, 680 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 6.27 (d, J=7.8 Hz, 1H), 4.21 (dd, J=11.3, 3.0 Hz, 1H), 4.06 (td, J=6.5, 3.2 Hz, 1H), 3.91 (t, J=2.8 Hz, 1H), 3.76 (td, J=6.4, 2.6 Hz, 1H), 3.59 (dd, J=11.3, 3.7 Hz, 1H), 3.15 (dd, J=9.0, 3.3 Hz, 1H), 2.20-2.16 (m, 2H), 1.67-1.47 (m, 6H), 1.45-1.16 (m, 68H), 0.92 (s, 9H), 0.90 (s, 9H), 0.87 (t, J=6.9 Hz, 6H), 0.11 (s, 6H), 0.08 (s, 6H); .sup.13C NMR (126 MHz, CDCl.sub.3) 172.62, 77.42, 76.36, 63.62, 51.3, 36.93, 34.42, 31.92, 29.80, 29.70, 29.63, 29.53, 29.48, 29.37, 26.00, 25.94, 25.81, 25.60, 22.69, 18.14, 18.12, 14.13, 3.76, 4.08, 4.53, 4.91; HR ESI Calcd for C.sub.56H.sub.117NO.sub.4Si.sub.2 [M+Na.sup.+]: 924.8594 found: 924.8604.

(96) Alcohols 23*a-23*o were synthesized according to the procedure described at example 15-23 starting from common aldehyde 14*:

(97) TABLE-US-00001 Comp. Structure HRMS 23*a 03 embedded image C.sub.35H.sub.75NO.sub.4Si.sub.2 Calc.: 631.1544 [M + H.sup.+] Found: 631.1521 23*b 04 C.sub.45H.sub.95NO.sub.4Si.sub.2 Calc.: 771.4206 [M + H.sup.+] Found: 771.4181 23*c 05 C.sub.38H.sub.73NO.sub.4Si.sub.2 Calc.: 665.1707 [M + H.sup.+] Found: 665.1733 23*d 06 C.sub.43H.sub.83NO.sub.4Si.sub.2 Calc.: 735.3038 [M + H.sup.+] Found: 735.3001 23*e 07 embedded image C.sub.37H.sub.69F.sub.2NO.sub.4Si.sub.2 Calc.: 687.1250 [M + H.sup.+] Found: 687.1212 23*f 08 C.sub.47H.sub.99NO.sub.4Si.sub.2 Calc.: 799.4738 [M + H.sup.+] Found: 799.4791 23*g 09 C.sub.48H.sub.101NO.sub.4Si.sub.2 Calc.: 813.5004 [M + H.sup.+] Found: 813.4962 23*h 0 C.sub.50H.sub.97NO.sub.4Si.sub.2 Calc.: 833.4901 [M + H.sup.+] Found: 833.4913 23*i embedded image C.sub.39H.sub.67NO.sub.4Si.sub.2 Calc.: 671.1338 [M + H.sup.+] Found: 671.1306 23*j C.sub.46H.sub.95NO.sub.4Si.sub.2 Calc.: 783.4313 [M + H.sup.+] Found: 783.4281 23*k C.sub.51H.sub.105NO.sub.5Si.sub.2 Calc.: 869.5638 [M + H.sup.+] Found: 869.5604 23*l C.sub.50H.sub.97NO.sub.4Si.sub.2 Calc.: 833.4901 [M + H.sup.+] Found: 833.4887 23*m embedded image C.sub.56H.sub.109NO.sub.4Si.sub.2 Calc.: 917.6498 [M + H.sup.+] Found: 917.6528 23*n C.sub.49H.sub.87NO.sub.4Si.sub.2 Calc.: 811.4000 [M + H.sup.+] Found: 811.4063 23*o C.sub.57H.sub.103NO.sub.4Si.sub.2 Calc.: 923.6129 [M + H.sup.+] Found: 923.6097

Example B.12: Synthesis of 6-hydroxyhexyl 4-methylbenzenesulfonate (24*)

(98) ##STR00118##

(99) To a solution of hexane-1,6-diol (10.0 g, 85 mmol) in DCM (200 mL) was added 4-methylbenzene-1-sulfonyl chloride (17.8 g, 93 mmol) dissolved in pyridine (100 mL) at 5 C. dropwise over 15 min. The reaction mixture was warmed to r.t. over the period of 5 h. Solvents were removed in vacuo and the crude was purified by silica flash column chromatography (gradient hexanes/EtOAc=1:0.fwdarw.1:1) to afford monotosylated hexanediol 24* (6.5 g, 28%) as a colorless oil.

(100) R.sub.f=0.55 (Hexanes/EtOAc=1:1); IR (film) .sub.max 3381, 2935, 2862, 1598, 1461, 1352, 1172, 959, 921, 813, 661 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.76-7.71 (m, 2H), 7.29 (dt, J=4.3, 1.2 Hz, 2H), 3.97 (t, J=6.5 Hz, 2H), 3.55 (t, J=6.5 Hz, 2H), 2.40 (s, 3H), 1.65-1.56 (m, 2H), 1.55 (s, 1H), 1.52-1.41 (m, 2H), 1.36-1.18 (m, 4H); .sup.13C NMR (101 MHz, CDCl.sub.3) 144.7, 133.1, 129.8, 127.8, 70.5, 62.6, 32.4, 28.7, 25.1, 25.0, 21.6; HR ESI Calcd for C.sub.13H.sub.20O.sub.4S [M+Na.sup.+]: 295.0975 found: 295.0968.

Example B.13: Synthesis of 6-azidohexan-1-ol (25*)

(101) ##STR00119##

(102) 6-Hydroxyhexyl 4-methylbenzenesulfonate 24* (4.3 g, 15.79 mmol) was dissolved in DMF (23 mL) and sodium azide (1.75 g, 26.8 mmol) was added. The mixture was heated to 55 C. and after 16 h it was cooled to r.t. and diluted with water (150 mL). The mixture was extracted three times with CH.sub.2Cl.sub.2 and washed with saturated aqueous NaCl solution. The organic layer was dried over MgSO.sub.4 and solvents were removed in vacuo. The crude product was purified by silica flash column chromatography on silica gel (gradient hexanes/EtOAc=1:0.fwdarw.1:1) to afford 6-azidohexan-1-ol 25* (2.2 g, 97%) as a colorless oil.

(103) R.sub.f=0.50 (Hexanes/EtOAc=2:1); IR (film) .sub.max 3329, 2935, 2891, 2090, 1256, 1349, 1258, 1055, 910, 731 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 3.63 (t, J=6.5 Hz, 2H), 3.25 (t, J=6.9 Hz, 2H), 1.64-1.51 (m, 4H), 1.43-1.32 (m, 4H); .sup.13C NMR (101 MHz, CDCl.sub.3) 62.8, 51.5, 32.6, 28.9, 26.6, 25.4; HR ESI Calcd for C.sub.6H.sub.13N.sub.3O [M+Na.sup.+]: 166.0951 found: 166.0945.

Example B.14: Synthesis of 6-azidohexyl 4-methylbenzenesulfonate (26*)

(104) ##STR00120##

(105) To a solution of 6-azidohexan-1-ol 25* (2.7 g, 18.9 mmol) in pyridine (70 mL) was added 4-methylbenzene-1-sulfonyl chloride (4.0 g, 21.0 mmol). The reaction mixture was left to stir for 5 h at r.t. after which the solvent was removed in vacuo and the crude product was dissolved in CH.sub.2Cl.sub.2, washed with water and dried over MgSO.sub.4. Solvents were removed in vacuo and the crude product was purified by silica flash column chromatography on silica gel (gradient hexanes/EtOAc=1:0.fwdarw.1:1) to afford azide 26* (5.0 g, 89%) as a colourless oil.

(106) R.sub.f=0.50 (Hexanes/EtOAc=3:1); IR (film) .sub.max 2938, 2863, 2092, 1598, 1455, 1356, 1258, 1174, 1097, 956, 919, 813, 724, 662 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) ; 7.85-7.67 (m, 2H), 7.33 (dd, J=8.5, 0.6 Hz, 2H), 4.01 (t, J=6.4 Hz, 2H), 3.21 (t, J=6.9 Hz, 2H), 2.43 (s, 3H), 1.71-1.57 (m, 2H), 1.52 (dd, J=9.1, 4.9 Hz, 2H), 1.38-1.12 (m, 4H); .sup.13C NMR (101 MHz, CDCl.sub.3) 144.8, 133.2, 129.9, 127.9, 70.4, 51.3, 28.7, 28.7, 26.1, 25.0, 21.7; HR ESI Calcd for C.sub.13H.sub.19N.sub.3O.sub.3S [M+Na.sup.+]: 320.1045 found: 320.1057.

(107) Azides 26*a-26*f were synthesized following the procedure described in examples 24-26 starting from the corresponding commercially available diols.

(108) TABLE-US-00002 comp. structure HRMS 26*a embedded image C.sub.15H.sub.23N.sub.3SO.sub.6 Calc.: 374.4344 [M + H.sup.+] Found: 374.4388 26*b C.sub.17H.sub.19N.sub.3SO.sub.3 Calc.: 346.4259 [M + H.sup.+] Found: 346.4212 26*c C.sub.11H.sub.15N.sub.3SO.sub.3 Calc.: 270.3297 [M + H.sup.+] Found: 270.3229 26*d C.sub.19H.sub.31N.sub.3SO.sub.3 Calc.: 382.5426 [M + H.sup.+] Found: 382.5461 26*e embedded image C.sub.15H.sub.23N.sub.3S.sub.3O.sub.3 Calc.: 390.5683 [M + H.sup.+] Found: 390.5662 26*f C.sub.9H.sub.11N.sub.3S.sub.3O.sub.3 Calc.: 306.4086 [M + H.sup.+] Found: 306.4041

Example B.15: Synthesis of allyl 6-O-trityl--D-galactopyranoside (27*)

(109) ##STR00127##

(110) 1-O-Allyl-galactoside (Org. Lett. 2002, 4, 489) (4 g, 18.2 mmol) was dissolved in pyridine (18 mL). To the solution was added trityl chloride (6.58 g, 23.6 mmol) and the mixture was stirred at r.t. for 18 h after which the solvent was removed in vacuo. The crude product was purified by flash column chromatography on silica gel (CH.sub.2Cl.sub.2/MeOH=10:1) to yield pyranoside 27* (7.0 g, 83%) as colorless oil.

(111) [].sub.D.sup.r.t.=+60.0 (c=1, CHCl.sub.3); R.sub.f=0.8 (CH.sub.2Cl.sub.2/MeOH=5:1); IR (film) .sub.max 3402, 2929, 1491, 1449, 1218, 1152, 1070, 1032, 746, 703 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.51-7.18 (m, 15H), 5.99-5.88 (m, 1H), 5.25 (ddq, J=35.9, 10.4, 1.4 Hz, 2H), 4.95 (d, J=3.8 Hz, 1H), 4.25 (ddt, J=12.8, 5.4, 1.4 Hz, 1H), 4.05 (ddt, J=12.8, 6.3, 1.3 Hz, 1H), 3.96 (s, 1H), 3.89 (t, J=5.8 Hz, 1H), 3.81 (d, J=5.7 Hz, 1H), 3.75 (d, J=9.8 Hz, 1H), 3.47 (s, 1H), 3.43 (dd, J=9.8, 6.1 Hz, 1H), 3.32 (dd, J=9.8, 5.3 Hz, 1H), 2.86 (d, J=2.1 Hz, 1H), 2.71 (d, J=8.1 Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) 143.8, 133.7, 128.6, 127.8, 127.1, 117.8, 97.5, 86.9, 71.2, 69.8, 69.5, 69.5, 68.5, 63.3; HR ESI Calcd for C.sub.25H.sub.25O.sub.5 [M+Na.sup.+]: 485.1935 found: 485.1941.

Example B.16: Synthesis of allyl 2,3,4-tri-O-benzyl-6-O-trityl--D-galactopyranoside (28*)

(112) ##STR00128##

(113) To a solution of allyl 6-O-trityl-/-D-galactopyranoside 27* (3.7 g, 8.0 mmol) in DMF (32 mL) was added sodium hydride (60% in mineral oil, 1.50 g, 36.0 mmol) portionwise at r.t. After 1 h benzyl bromide (4.2 mL, 35.2 mmol) was added. The reaction mixture was left to stir for 48 h after which it was quenched by the addition of MeOH (5 mL). The mixture was diluted with Et.sub.2O and extracted twice from saturated aqueous NaHCO.sub.3. The combined organic layer was washed with water (3100 mL) and saturated aqueous NaCl solution and dried over MgSO.sub.4. The solvent was removed in vacuo and the crude product was over a plug of silica gel (hexanes/EtOAc=2:1, silica gel was neutralized with 1% NEt.sub.3) to yield the benzyl ether 28* (5.5 g) as a pale yellow oil which was used in the subsequent step without further purification.

Example B.17: Synthesis of allyl 6-(6-azidohexyl)-2,3,4-tri-O-benzyl--D-galactopyranoside (29*)

(114) ##STR00129##

(115) A solution of allyl 2,3,4-tri-O-benzyl-6-O-trityl--D-galactopyranoside 28* (5.00 g, 6.82 mmol) and triethyl silane (5.45 mL, 34.1 mmol) in CH.sub.2Cl.sub.2 (68 mL) was cooled to 0 C. To the stirred solution was added trifluoroacetic acid (2.6 mL, 34.1 mmol) dropwise. The mixture was quenched after 15 min. with saturated aqueous NaHCO.sub.3 solution and extracted with CH.sub.2Cl.sub.2. The crude product was filtered over a plug of silica gel. All silane and trityl residues were removed with 10:1 hexanes/EtOAc and the product was eluted with EtOAc to yield allyl 2,3,4-tri-O-benzyl--D-galactopyranoside (3.0 g) as a pale yellow oil, which was used without further purification in the subsequent reaction.

(116) To a solution of allyl 2,3,4-tri-O-benzyl--D-galactopyranoside (1.0 g, 2.04 mmol) in DMF (10 mL) was added sodium hydride (60% in mineral oil, 0.12 g, 3.1 mmol) at 0 C. After 15 min, the mixture was warmed to r.t. and stirred for another 1 h. Then, 6-azidohexyl 4-methylbenzenesulfonate 26* (0.9 g, 3.1 mmol) was added and the reaction mixture was stirred at r.t. for a further 8 h after which the mixture was quenched by the addition of MeOH (2 mL). After dilution with DCM, saturated aqueous NH.sub.4Cl solution was added and the mixture was extracted with DCM. The combined organic layer was washed with water and saturated aqueous NaCl solution. The organic layer was 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=1:0.fwdarw.1:1) to yield azide 29* (1.0 g, 68% over three steps) as a colorless oil. [].sub.D.sup.r.t.=+25.4 (c=1, CHCl.sub.3); R.sub.f=0.65 (Hexanes/EtOAc=4:1); IR (film) .sub.max 2933, 2863, 2094, 1497, 1454, 1358, 1177, 1098, 1059, 926, 816, 736, 697 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.94-7.16 (m, 15H), 5.95 (dddd, J=17.1, 10.3, 6.6, 5.2 Hz, 1H), 5.31 (dq, J=17.2, 1.6 Hz, 1H), 5.21 (ddd, J=10.3, 2.8, 1.1 Hz, 1H), 5.01-4.58 (m, 7H), 4.17 (ddt, J=13.0, 5.2, 1.4 Hz, 1H), 4.09-3.99 (m, 3H), 3.98-3.90 (m, 2H), 3.50-3.18 (m, 6H), 1.72-1.47 (m, 4H), 1.44-1.30 (m, 4H); .sup.13C NMR (75 MHz, CDCl.sub.3) 138.9, 138.8, 138.6, 134.0, 129.8, 128.3, 128.3, 128.2, 128.1, 128.0, 127.9, 127.6, 127.5, 127.4, 117.9, 96.3, 79.1, 76.5, 75.3, 74.7, 73.3, 73.3, 71.3, 70.3, 69.5, 69.4, 68.2, 51.4, 51.2, 29.6, 28.8, 28.7, 28.6, 26.6, 26.1, 25.7, 25.0, 21.6. HR ESI Calcd for C.sub.36H.sub.45N.sub.3O.sub.6 [M+Na.sup.+]: 638.3201 found: 638.3229.

(117) Azides 29*a-29*f were obtained starting from allyl 2,3,4-tri-O-benzyl--D-galactopyranoside and intermediates 26*a-26*f.

(118) TABLE-US-00003 comp. structure HRMS 29*a 0 embedded image C.sub.38H.sub.50N.sub.3O.sub.9 Calc.: 693.8278 [M + H.sup.+] Found: 693.8241 29*b C.sub.36H.sub.46N.sub.3O.sub.6 Calc.: 617.7764 [M + H.sup.+] Found: 617.7721 29*c C.sub.34H.sub.42N.sub.3O.sub.6 Calc.: 589.7231 [M + H.sup.+] Found: 589.7274 29*d C.sub.42H.sub.58N.sub.3O.sub.6 Calc.: 701.9361 [M + H.sup.+] Found: 701.9400 29*e embedded image C.sub.38H.sub.50N.sub.3S.sub.2O.sub.6 Calc.: 709.9618 [M + H.sup.+] Found: 709.9651 29*f C.sub.32H.sub.38N.sub.3S.sub.2O.sub.6 Calc.: 625.8021 [M + H.sup.+] Found: 625.7996

(119) In a similar manner, the analogues in glucose series 29**a-29**f were obtained starting from allyl 2,3,4-tri-O-benzyl--D-glucopyranoside and intermediates 26*a-26*f.

(120) TABLE-US-00004 comp. structure mass spec 29**a embedded image C.sub.38H.sub.50N.sub.3O.sub.9 Calc.: 693.8278 [M + H.sup.+] Found: 693.8241 29**b C.sub.36H.sub.46N.sub.3O.sub.6 Calc.: 617.7764 [M + H.sup.+] Found: 617.7721 29**c C.sub.34H.sub.42N.sub.3O.sub.6 Calc.: 589.7231 [M + H.sup.+] Found: 589.7274 29**d C.sub.42H.sub.58N.sub.3O.sub.6 Calc.: 701.9361 [M + H.sup.+] Found: 701.9400 29**e 0 embedded image C.sub.38H.sub.50N.sub.3S.sub.2O.sub.6 Calc.: 709.9618 [M + H.sup.+] Found: 709.9651 29**f C.sub.32H.sub.38N.sub.3S.sub.2O.sub.6 Calc.: 625.8021 [M + H.sup.+] Found: 625.7996

Example B.18: Synthesis of 6-(6-azidohexyl)-2,3,4-tri-O-benzyl-/-D-galactopyranose (30*)

(121) ##STR00142##

(122) Allyl 6-(6-azidohexyl)-2,3,4-tri-O-benzyl--D-galactopyranoside 29* (1.4 g, 2.3 mmol) was dissolved in MeOH (16 mL) and PdCl.sub.2 (0.21 g, 1.17 mmol) was added to the solution at r.t. The mixture was stirred at for 4 h after which the mixture was filtered over celite and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (gradient hexanes/EtOAc=1:0.fwdarw.1:1) to yield lactol 30* (1.2 g, 88%) as a colorless oil.

(123) R.sub.f=0.50 (Hexanes/EtOAc=2:1); IR (film) .sub.max 3414, 2933, 2862, 2093, 1454, 1255, 1060, 910, 733, 696 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.45-7.20 (m, 30H), 5.33-5.27 (m, 1H), 5.01-4.90 (m, 3H), 4.85-4.71 (m, 7H), 4.66 (ddd, J=16.7, 11.5, 6.0 Hz, 3H), 4.18-4.09 (m, 1H), 4.05 (dd, J=9.2, 3.6 Hz, 1H), 3.96 (s, 2H), 3.93 (d, J=2.8 Hz, 1H), 3.88 (d, J=2.8 Hz, 1H), 3.78 (dd, J=9.6, 7.5 Hz, 1H), 3.63-3.52 (m, 3H), 3.52-3.37 (m, 5H), 3.37-3.28 (m, 2H), 3.28-3.21 (m, 5H), 1.65-1.49 (m, 8H), 1.42-1.24 (m, 8H); .sup.13C NMR (101 MHz, CDCl.sub.3) 138.8, 138.7, 138.5, 138.4, 128.5, 128.5, 128.4, 128.3, 128.3, 128.3, 128.3, 128.1, 127.9, 127.7, 127.7, 127.7, 127.6, 127.6, 97.9, 92.0, 82.3, 80.9, 78.8, 76.7, 75.2, 74.9, 74.8, 74.7, 73.8, 73.7, 73.6, 73.1, 73.1, 71.5, 71.4, 69.6, 69.6, 69.5, 51.5, 29.5, 28.9, 26.6, 25.8; HR ESI Calcd for C.sub.33H.sub.41N.sub.3O.sub.6 [M+Na.sup.+]: 598.2883 found: 598.2869.

Example B.19: Synthesis of 6-(6-Azidohexyl)-2,3,4-tri-O-benzyl--D-galactopyranosyl N-phenyl trifluoroacetimidate (31*)

(124) ##STR00143##

(125) To a solution of 6-(6-azidohexyl)-2,3,4-tri-O-benzyl-/-D-galactopyranose 30* (400 mg, 0.70 mmol) in DCM (7 mL) was added cesium carbonate (340 mg, 1.04 mmol). To the mixture was added 2,2,2-trifluoro-N-phenylacetimidoyl chloride (216 mg, 1.04 mmol) and the reaction mixture was stirred at r.t. for 3.5 h after which it was filtered over celite and washed with DCM. The solvent was removed in vacuo and the crude product was purified by flash column chromatography on silica gel (gradient hexanes/EtOAc=10:1.fwdarw.1:1) to yield the imidate 31* (490 mg, 94%) as a colorless oil.

(126) [].sub.D.sup.r.t.=+60.8 (c=0.4, CHCl.sub.3); R.sub.f=0.80 (Hexanes/EtOAc=2:1); IR (film) .sub.max 3064, 2934, 2865, 2094, 1717, 1598, 1454, 1321, 1207, 1099, 1027, 910, 734, 696 cm.sup.1, .sup.1H NMR (400 MHz, CDCl.sub.3) 7.45-6.60 (m, 20H), 5.56 (s, 1H), 4.90 (d, J=11.5 Hz, 1H), 4.75 (s, J=1.5 Hz, 2H), 4.68 (s, J=12.4 Hz, 2H), 4.58 (d, J=11.6 Hz, 1H), 4.00 (t, J=8.7 Hz, 1H), 3.84 (d, J=2.4 Hz, 1H), 3.58-3.39 (m, 4H), 3.34 (dt, J=9.3, 6.5 Hz, 1H), 3.23 (dt, J=9.3, 6.5 Hz, 1H), 3.14 (t, J=6.9 Hz, 2H), 1.52-1.38 (m, 4H), 1.32-1.16 (m, 4H); .sup.13C NMR (101 MHz, CDCl.sub.3) 138.6, 138.3, 138.2, 128.8, 128.6, 128.5, 128.4, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 124.3, 119.4, 82.3, 78.3, 77.4, 77.2, 76.8, 75.7, 74.9, 74.6, 73.4, 73.2, 71.4, 68.7, 51.5, 29.7, 28.9, 26.7, 25.8; HR ESI Calcd for C.sub.41H.sub.45F.sub.3N.sub.4O.sub.6 [M+Na.sup.+]: 769.3183 found: 769.3239.

(127) Starting from hemiacetals 29*a-29*f, imidate donors 31*a-31*f were synthesized according to the procedures described in examples 30 and 31.

(128) TABLE-US-00005 comp. structure HRMS 31*a embedded image C.sub.43H.sub.50F.sub.3N.sub.4O.sub.9 Calc.: 824.8834 [M + H.sup.+] Found: 824.8804 31*b C.sub.41H.sub.46F.sub.3N.sub.4O.sub.6 Calc.: 748.8320 [M + H.sup.+] Found: 748.8299 31*c C.sub.39H.sub.42F.sub.3N.sub.4O.sub.6 Calc.: 720.7788 [M + H.sup.+] Found: 720.7712 31*d C.sub.47H.sub.58F.sub.3N.sub.4O.sub.6 Calc.: 832.9917 [M + H.sup.+] Found: 832.9977 31*e embedded image C.sub.43H.sub.50F.sub.3N.sub.4S.sub.2O.sub.6 Calc.: 841.0174 [M + H.sup.+] Found: 841.0108 31*f C.sub.37H.sub.38F.sub.3N.sub.4S.sub.2O.sub.6 Calc.: 756.8577 [M + H.sup.+] Found: 756.8506

(129) In a similar manner, imidate donors in glucose series 31**a-31**f were accessed starting from the corresponding hemiacetals 29**a-29**f.

(130) TABLE-US-00006 comp. structure HRMS 31**a 0 embedded image C.sub.43H.sub.50F.sub.3N.sub.4O.sub.9 Calc.: 824.8834 [M + H.sup.+] Found: 824.8804 31**b C.sub.41H.sub.46F.sub.3N.sub.4O.sub.6 Calc.: 748.8320 [M + H.sup.+] Found: 748.8299 31**c C.sub.39H.sub.42F.sub.3N.sub.4O.sub.6 Calc.: 720.7788 [M + H.sup.+] Found: 720.7712 31**d C.sub.47H.sub.58F.sub.3N.sub.4O.sub.6 Calc.: 832.9917 [M + H.sup.+] Found: 832.9977 31**e embedded image C.sub.43H.sub.50F.sub.3N.sub.4S.sub.2O.sub.6 Calc.: 841.0174 [M + H.sup.+] Found: 841.0108 31**f C.sub.37H.sub.38F.sub.3N.sub.4S.sub.2O.sub.6 Calc.: 756.8577 [M + H.sup.+] Found: 756.8506

Example B.20: Synthesis of (2S,3S,4R)-3,4-bis-tert-butyldimethylsilyloxy-2-hexacosanoylamino-1-(6-(6-azidohexyl)-2,3,4-tri-O-benzyl)--D-galactopyranosyl)octadecane (32*)

(131) ##STR00156##

(132) Nucleophile 23* (156 mg, 0.169 mmol) and glycosylating agent 31* (189 mg, 0.253 mmol) were co-evaporated with toluene three times and dried on high vacuum for 3 h after which they were dissolved in Et.sub.2O (2 mL) and THF (0.4 mL) and cooled to 40 C. To the mixture was added TMSOTf (9.0 L, 0.051 mmol) and the solution was warmed to 10 C. over the period of 3 h. The reaction was quenched by the addition of NEt.sub.3 (0.05 mL) and solvents were removed in vacuo and the crude product was purified by silica flash column chromatography (gradient hexanes/EtOAc=10:1.fwdarw.4:1) to afford glycoside 32* (180 mg, 72% -anomer) as a white foam.

(133) [].sub.D.sup.r.t.=+18.9 (c=1, CHCl.sub.3); R.sub.f=0.46 (Hexanes/EtOAc=6.5:1); IR (film) .sub.max 3328, 2925, 2854, 2096, 1731, 1656, 1452, 1348, 1246, 1156, 1099, 1058, 835, 777, 696 cm.sup.1; .sup.1H NMR (400 MHz, CDCl.sub.3) 7.39-7.27 (m, 15H), 5.99 (d, J=7.07 Hz, 1H), 4.95 (d, J=11.5 Hz, 1H), 4.83 (d, J=3.7 Hz, 1H), 4.81-4.59 (m, 6H), 4.11-4.08 (m, 1H), 4.04 (dd, J=10.1, 3.6 Hz, 1H), 3.96-3.82 (m, 6H), 3.65 (ddd, J=7.0, 5.1, 1.85 Hz, 1H), 3.50-3.45 (m, 1H), 3.40 (dq, J=6.7, 4.0 Hz, 1H), 3.33-3.27 (m, 1H), 3.25 (t, J=6.9 Hz, 2H), 2.02-1.98 (m, 2H), 1.62-1.49 (m, 8H), 1.30-1.23 (m, 72H), 0.91-0.87 (m, 24H), 0.07 (s, 3H), 0.06 (s, 3H), 0.03 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) 173.1, 138.8, 138.7, 138.6, 128.33, 128.30, 128.2, 128.1, 127.8, 127.6, 127.50, 127.46, 127.3, 100.2, 79.1, 77.20, 76.57, 75.7, 75.6, 74.9, 74.8, 73.4, 72.9, 71.4, 69.7, 69.4, 69.0, 51.8, 51.4, 36.8, 33.2, 31.9, 29.9, 29.74, 29.71, 29.66, 29.60, 29.5, 29.4, 28.8, 26.6, 26.14, 26.09, 25.7, 25.6, 22.7, 18.3, 18.2, 14.1, 3.7, 3.9, 4.6, 4.9; HR ESI Calcd for C.sub.89H.sub.156N.sub.4O.sub.9Si.sub.2 [M+Na.sup.+]: 1505.1333 found: 1505.1388.

Example B.21: Synthesis of (2S,3S,4R)-2-hexacosanoylamino-1-(6-(6-azidohexyl)-2,3,4-tri-O-benzyl--D-galactopyranosyl)octadecane-3,4-diol (33*)

(134) ##STR00157##

(135) To a solution of bis-TBS ether 32* (16.0 mg, 10.8 mol) in THF (1 mL) was added a solution of TBAF (1 M in THF, 0.150 mL, 0.15 mmol) slowly. After 3.5 h the reaction mixture was diluted with CH.sub.2Cl.sub.2 (10 mL). Solvents were removed in vacuo and crude product was purified by silica flash column chromatography (gradient hexanes/EtOAc=1:0.fwdarw.1:1) to afford diol 33* (10.5 mg, 78%) as a clear oil.

(136) [].sub.D.sup.r.t.=+121.9 (c=0.2, CHCl.sub.3); R.sub.f=0.40 (Hexanes/EtOAc=2:1); IR (film) .sub.max 3329, 2919, 2851, 2096, 1640, 1543, 1467, 1455, 1350, 1094, 1046, 907, 730, 696 cm.sup.1;

(137) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.32-7.18 (m, 15H), 6.34 (d, J=7.91 Hz, 1H), 4.88-4.51 (m, 7H), 4.15 (m, 1H), 3.98-3.96 (m, 1H), 3.88-3.74 (m, 5H), 3.41-3.21 (m, 6H), 3.17 (t, J=6.5 Hz, 2H), 2.19-2.08 (t, J=7.05 Hz, 2H), 1.53-1.35 (m, 8H), 1.31-1.18 (m, 72H), 0.81 (m, 6H); .sup.13C NMR (101 MHz, CDCl.sub.3) 173.0, 138.5, 138.3, 137.8, 128.44, 128.39, 128.2, 128.1, 128.1, 127.9, 127.62, 127.60, 127.4, 99.1, 79.3, 76.2, 76.0, 74.7, 74.5, 74.2, 73.2, 72.7, 71.4, 69.8, 51.3, 49.5, 36.7, 31.9, 29.7, 29.5, 29.4, 29.4, 29.3, 28.8, 26.5, 25.9, 25.7, 25.7, 22.7, 14.1; HR ESI Calcd for C.sub.77H.sub.128N.sub.4O.sub.9 [M+Na.sup.+]: 1275.9574 found: 1275.9536.

Example B.22: Synthesis of (2S,3S,4R)-1-(6-(6-aminohexyl)--D-galactopyranosyl)-2-hexacosanoylaminooctadecane-3,4-diol (34*)

(138) ##STR00158##

(139) To a solution diol 33* (55 mg, 0.044 mmol) in EtOH (0.5 mL) and chloroform (0.15 mL) was added Pd(OH).sub.2 on charcoal (10% w/w, wet 38 mg). The solution was stirred at r.t. under an atmosphere of Ar for 15 min. after which H.sub.2 gas was inserted into the suspension and the mixture was hydrogenated for 12 h. The mixture was filtered over celite and thoroughly washed with CH.sub.2Cl.sub.2, THF and MeOH. Solvents were removed in vacuo and the crude was purified by silica flash column chromatography on silica gel (CH.sub.2Cl.sub.2/MeOH=4:1) to afford linker equipped glycosphingolipid 34* (38 mg, 90%) as a pale yellow powder.

(140) [].sub.D.sup.r.t.=+66.1 (c=1.0, Pyridine); R.sub.f=0.44 (CH.sub.2Cl.sub.2/MeOH=4:1);

(141) IR (film) .sub.max 3292, 2918, 2850, 1640, 1539, 1468, 1304, 1073, 1038, 970, 721 cm.sup.1;

(142) .sup.1H NMR (400 MHz, d-pyr) 8.88 (d, J=8.5 Hz, 1H), 5.54 (d, J=2.5 Hz, 1H), 5.24-5.21 (m, 1H), 4.62-4.55 (m, 3H), 4.44-4.32 (m, 5H), 4.00-3.92 (m, 2H), 3.31-3.26 (m, 2H), 2.56 (t, J=7.4 Hz, 2H), 2.22-2.18 (m, 1H), 2.00-1.90 (m, 2H), 1.90-1.78 (m, 4H), 1.73-1.60 (m, 1H), 1.55-1.47 (m, 2H), 1.44-1.20 (m, 70H), 0.87 (m, 6H); .sup.13C NMR (101 MHz, d-pyr) 173.2, 100.4 (J.sub.CH=169 Hz), 76.0, 72.2, 71.0, 70.9, 70.7, 70.6, 70.3, 69.6, 67.5, 50.4, 39.6, 36.5, 33.9, 31.8, 30.1, 29.9, 29.7, 29.68, 29.65, 29.62, 29.59, 29.5, 29.48, 29.28, 27.8, 26.3, 26.19, 26.17, 25.6, 22.6, 14.0; HR ESI Calcd for C.sub.56H.sub.112N.sub.2O.sub.9 [M+H.sup.+]: 957.8441 found: 957.8468.

(143) The following glycosphingolipids were prepared in a similar manner.

(144) TABLE-US-00007 Comp. Structure HRMS 34*a embedded image C.sub.35H.sub.61F.sub.2N.sub.2O.sub.9 Calc.: 692.8730 [M + H.sup.+] Found: 692.8707 34*b 0 C.sub.52H.sub.103N.sub.2O.sub.9 Calc.: 901.3922 [M + H.sup.+] Found: 901.3958 34*c C.sub.51H.sub.87N.sub.2O.sub.12 Calc.: 921.2527 [M + H.sup.+] Found: 921.2500 34*d C.sub.45H.sub.82N.sub.2O.sub.9 Calc: 795.6020 [M + H.sup.+] Found: 795.5984 34*e embedded image C.sub.46H.sub.84N.sub.2O.sub.9 Calc: 809.6177 [M + H.sup.+] Found: 809.6157 34*f C.sub.51H.sub.94N.sub.2O.sub.9 Calc: 879.6959 [M + H.sup.+] Found: 879.6789 34*g C.sub.57H.sub.106N.sub.2O.sub.9 Calc: 963.7898 [M + H.sup.+] Found: 963.7854 34*h C.sub.61H.sub.114N.sub.2O.sub.9 Calc: 1018.8524 [M + H.sup.+] Found: 1018.8514 34*i embedded image C.sub.44H.sub.79FN.sub.2O.sub.9 Calc: 799.5770 [M + H.sup.+] Found: 799.5755 34*j C.sub.45H.sub.82N.sub.2O.sub.9 Calc: 811.5969 [M + H.sup.+] Found: 811.5932 34*k C.sub.45H.sub.79N.sub.2O.sub.9 Calc: 849.5738 [M + H.sup.+] Found: 849.5715 34*l 0 C.sub.47H.sub.85FN.sub.2O.sub.9 Calc: 841.6239 [M + H.sup.+] Found: 841.6227 34*m embedded image C.sub.45H.sub.82N.sub.2O.sub.9 Calc: 853.6439 [M + H.sup.+] Found: 853.6430 34*n C.sub.45H.sub.79N.sub.2O.sub.9 Calc: 891.6207 [M + H.sup.+] Found: 891.6198 34*o C.sub.50H.sub.92N.sub.2O.sub.9 Calc: 864.6803 [M + H.sup.+] Found: 864.6809 34*p C55H102N2O9 Calc: 934.7585 [M + H.sup.+] Found: 934.7536 34*q embedded image C.sub.45H.sub.90N.sub.2O.sub.9 Calc: 803.6646 [M + H.sup.+] Found: 803.6625 34*r C.sub.54H.sub.108N.sub.2O.sub.8S Calc: 945.7826 [M + H.sup.+] Found: 945.7812

(145) C. Synthesis of Conjugates

Example C.1: Synthesis of 2,5-dioxopyrrolidin-1-yl 5-((6-(((2R,3R,4S,5R,6S)-6-(((2S,3S,4R)-2-hexacosanamido-3,4-dihydroxyoctadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)hexyl)amino)-5-oxopentanoate (35*)

(146) ##STR00177##

(147) To glycosphingolipid 34* (10 mg, 10.44 mol) in CHCl.sub.3:MeOH:Et.sub.3N mixture (1:1:0.1, 7 ml) was added excess glutaric anhydride (14.9 mg, 131 mol) in one portion and left to stir at the r.t. After three days the completion of the reaction was indicated by the disappearance of the starting material mass on the LCMS, the reaction mixture was evaporated to dryness and the resultant residue was triturated with dichloromethane to give intermediate carboxylic acid (8 mg, 71.5%) as a white powder.

(148) IR (film) .sub.max 3300, 2918, 2850, 1718, 1637, 1539, 1466, 1304, 1073, 1038, 970, 719 cm.sup.1; .sup.1H NMR (400 MHz, Pyridine-d.sub.5) 8.45-8.33 (m, 2H), 5.53 (d, J=3.8 Hz, 1H), 5.29-5.18 (m, 1H), 4.63 (ddd, J=13.0, 9.9, 4.5 Hz, 2H), 4.46 (t, J=6.1 Hz, 1H), 4.44-4.25 (m, 5H), 4.02 (ddd, J=39.8, 9.9, 6.0 Hz, 2H), 3.46 (dq, J=13.3, 6.6 Hz, 4H), 2.66 (t, J=7.3 Hz, 2H), 2.57 (t, J=7.3 Hz, 2H), 2.44 (t, J=7.5 Hz, 2H), 2.37-2.21 (m, 3H), 1.97-1.74 (m, 4H), 1.75-1.62 (m, 1H), 1.61-1.50 (m, 4H), 1.47-1.06 (m, 65H), 0.86 (t, J=6.6 Hz, 6H); .sup.13C NMR (101 MHz, pyridine) 172.78, 101.07, 76.28, 72.14, 71.08, 70.59, 70.44, 70.36, 69.79, 68.33, 50.90, 39.26, 36.43, 35.49, 33.94, 31.76, 30.03, 29.80, 29.74, 29.68, 29.64, 29.55, 29.47, 29.42, 29.25, 26.80, 26.14, 26.04, 25.83, 22.57, 21.73, 13.91; MALDI-TOF (THAP, RN) [MH].sup. calcd 1069.861, found 1069.642.

(149) To a solution of intermediate carboxylic acid (1.45 mg, 1.36 mol) in DMSO:THF (1:1, 500 L) was added N-hydroxysuccinimide (0.18 mg, 1.61 mol) in one portion, followed by a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.3 mg, 6.78 mol). After five days, disappearance of the starting material mass in LCMS indicated the completion of the reaction. 2-Mercaptoethanol (20 L) was then added to the reaction mixture to quench the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. NHS ester activated carboxylic acid 35* was used without any purification for coupling to the SP-3 capsular polysaccharide related saccharides:

(150) HRMS (ESI) C.sub.35H.sub.122N.sub.3O.sub.14 [M+H].sup.+ calcd 1167.8921, found 1168.8931.

Example C.2: Synthesis of 4-nitrophenyl (6-(((2R,3R,4S,5R,6S)-6-(((2S,3S,4R)-2-hexacosanamido-3,4-dihydroxyoctadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)hexyl)carbamate (36*)

(151) ##STR00178##

(152) Glycosphingolipid 34* (3.9 mg, 4.1 mol) was dissolved in 0.5 mL of dry pyridine, then to it was added bis(4-nitrophenyl)carbonate (6.1 mg, 20 mol), followed by Et.sub.3N (25 l, 0.179 mmol). The resulting yellow solution was stirred at rt overnight, then concentrated in vacuo and purified by column chromatography on silica gel, using a gradient of 0-5-10-20% MeOH in DCM, yielding 3.6 mg (3.2 mol, 79% yield) of glycosphingolipid 36* as a pale yellow oil. .sup.1H NMR (400 MHz, pyridine) 9.02 (t, J=5.6 Hz, 1H), 8.49 (d, J=8.7 Hz, 1H), 8.26 (d, J=9.2 Hz, 2H), 7.54 (d, J=9.2 Hz, 2H), 5.56 (d, J=3.8 Hz, 1H), 5.26 (s, 2H), 4.67 (ddd, J=13.1, 10.0, 4.5 Hz, 2H), 4.51 (t, J=6.0 Hz, 1H), 4.41 (dd, J=9.0, 5.6 Hz, 2H), 4.35 (s, 2H), 4.10 (dd, J=9.8, 5.7 Hz, 1H), 4.02 (dd, J=9.8, 6.5 Hz, 1H), 3.52 (td, J=9.2, 2.7 Hz, 2H), 3.45 (dd, J=13.0, 6.9 Hz, 2H), 2.46 (t, J=7.2 Hz, 2H), 2.36-2.23 (m, 1H), 1.97-1.79 (m, 4H), 1.74-1.65 (m, 3H), 1.64-1.54 (m, 2H), 1.41 (d, J=7.1 Hz, 5H), 1.34-1.22 (m, 65H), 0.87 (t, J=6.7 Hz, 6H). .sup.13C NMR (101 MHz, pyridine) 171.70, 155.90, 152.65, 143.22, 123.92, 121.09, 100.02, 75.15, 71.05, 70.01, 69.98, 69.62, 69.41, 69.35, 68.71, 67.28, 49.83, 40.13, 35.35, 32.84, 30.69, 30.68, 28.95, 28.73, 28.62, 28.59, 28.57, 28.55, 28.50, 28.48, 28.46, 28.41, 28.34, 28.19, 28.18, 25.59, 25.08, 24.98, 24.75, 21.51, 12.85.

(153) HRMS: expected [M+Na].sup.+=1144.8322, found: 1444.8373.

Example C.3: Synthesis of N-((2S,3S,4R)-1-(((2S,3R,4S,5R,6R)-6-(((6-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)hexyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4-dihydroxyoctadecan-2-yl)hexacosanamide (37*)

(154) ##STR00179##

(155) To a stirred solution of glycosphingolipid 34* (11.4 mg, 12 mol) and DIPEA (5.3 L, 30 mol) in CHCl.sub.3 (1.2 mL) and MeOH (0.4 mL) was added at room temperature N-succinimidyl-3-maleimidopropionate (7.9 mg, 30 mol). The mixture was stirred for 2 h at that temperature and concentrated. The residue was purified by trituration with EtOAc (3 mL) and MeOH (3 mL) to give maleimide 37* (8.2 mg, 7.4 mol) as a white solid.

(156) .sup.1H NMR (400 MHz, CD.sub.3OD/CDCl.sub.31:3) 6.70 (s, 2H), 4.86 (d, J=3.8 Hz, 1H), 4.16-4.10 (m, 1H), 3.92-3.80 (m, 3H), 3.79-3.72 (m, 3H), 3.71-3.57 (m, 4H), 3.46 (m, 4H), 3.09 (t, J=7.1 Hz, 2H), 2.43 (t, J=7.2 Hz, 2H), 2.20-2.11 (m, 2H), 1.66-1.15 (m, 82H), 0.83 (t, J=6.8 Hz, 6H).

Example C.4: Synthesis of (2S,3S,4S,5R,6R)-methyl 6-(((2R,3S,4R,5R,6R)-6-(2-(benzyl((benzyloxy)carbonyl)amino)ethoxy)-2-((benzyloxy)methyl)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)oxy)-4-(((2S,3R,4R,5S,6R)-6-((benzyloxy)methyl)-3,4-dihydroxy-5-(((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,5-dihydroxytetrahydro-2H-pyran-2-carboxylate (38*)

(157) ##STR00180##

(158) Tetrasaccharide 10* (30.0 mg, 0.026 mmol) was taken in methanol (5 mL) and treated with Amberlite120H.sup.+ (5 mg) to it and heated to reflux for 24 h. The reaction mixture was cooled and filtered through cotton plug, washed thoroughly with MeOH (3 mL4). Combined organics were evaporated in vacuum to get pale yellow solid (30 mg, 98%) corresponding to target compounds 38*.

(159) .sup.1H NMR (400 MHz, cd.sub.3od) 7.56-6.86 (m, 20H), 5.14 (d, J=9.7 Hz, 2H), 4.70-4.33 (m, 9H), 4.22 (dd, J=32.2, 7.4 Hz, 1H), 3.99-3.72 (m, 11H), 3.70-3.40 (m, 15H), 3.39-3.32 (m, 2H), 3.29-3.15 (m, 2H).

Example C.5: Synthesis of (2S,3S,4S,5R,6R)-methyl 6-(((2R,3S,4R,5R,6R)-6-(2-aminoethoxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-4-(((2S,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,5-dihydroxytetrahydro-2H-pyran-2-carboxylate (39*)

(160) ##STR00181##

(161) Tetrasaccharide 38* (30.0 mg) was taken in methanol (5 mL) and added 10% Pd/C (30 mg) to it and stirred at 50 psi for 24 h. The reaction mixture was filtered through PTFE filter, washed thoroughly with methanol (3 mL3), and with 10% aq. methanol (3 mL3). Combined filtrate was evaporated in vacuum. 1H, HSQC nmr showed that still one benzyl group left. So, re subjected to same reaction conditions with 10 mg of Pd/C added to material in methanol and hydrogenated at 60 psi for 16 h. RM was filtered through PTFE filter, washed thoroughly with methanol (3 mL3), and with 10% aq methanol (3 mL3). Combined filtrates were evaporated in vacuum to get white solid (18 mg, 89%) corresponding to target tetrasaccharide 39*.

(162) .sup.1H NMR (600 MHz, cd.sub.3od) 4.65 (d, J=7.9 Hz, 1H), 4.56 (d, J=7.9 Hz, 1H), 4.47 (d, J=7.9 Hz, 1H), 4.38 (d, J=7.8 Hz, 1H), 4.05 (dt, J=11.5, 4.7 Hz, 1H), 4.02-3.98 (m, 1H), 3.96 (d, J=9.8 Hz, 1H), 3.94-3.84 (m, 4H), 3.84-3.76 (m, 5H), 3.65 (dd, J=5.9, 3.3 Hz, 2H), 3.58-3.44 (m, 8H), 3.41 (t, J=9.1 Hz, 1H), 3.38-3.32 (m, 3H), 3.30-3.24 (m, 2H), 3.17 (t, J=5.0 Hz, 2H).

Example C.5: Synthesis of (2S,3S,4S,5R,6R)-methyl 6-(((2R,3S,4R,5R,6R)-4,5-dihydroxy-2-(hydroxymethyl)-6-(2-(6-(4-nitrophenoxy)-6-oxohexanamido)ethoxy)tetrahydro-2H-pyran-3-yl)oxy)-4-(((2S,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,5-dihydroxytetrahydro-2H-pyran-2-carboxylate (40*)

(163) ##STR00182##

(164) Tetrasaccharide 39* (3.0 mg, 3.92 mol) and bis(4-nitrophenyl) adipate (9.89 mg, 0.025 mmol) were taken in a mixture of pyridine (1 mL) and DCM (1 mL) and stirred for 5 min, then treated with 5 L of Et.sub.3N and stirred for 20 min. Solvents were removed under vacuum. Washed with DCM (31 mL) to remove excess adipate ester, and the remaining white solid was dried to get product 40* (3.9 mg, 98%). .sup.1H NMR (400 MHz, cd.sub.3od) 8.31 (d, J=8.8 Hz, 2H), 7.39 (d, J=8.9 Hz, 2H), 4.65 (d, J=7.8 Hz, 1H), 4.54 (d, J=7.8 Hz, 1H), 4.47 (d, J=7.8 Hz, 1H),4.31 (d, J=7.7 Hz, 1H), 4.04-3.74 (m, 13H), 3.70-3.37 (m, 17H), 3.28-3.19 (m, 3H), 2.69 (t, J=10.0 Hz, 2H), 2.28 (t, J=5.9 Hz, 2H), 1.86-1.66 (m, 4H).

Example C.6: Synthesis of 4-nitrophenyl 6-((6-(((2R,3R,4S,5R,6S)-6-(((2S,3S,4R)-2-hexacosanamido-3,4-dihydroxyoctadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)hexyl)amino)-6-oxohexanoate (41*)

(165) ##STR00183##

(166) Glycosphingolipid 34* (13.0 mg, 0.014 mmol) and bis(4-nitrophenyl) adipate (26.0 mg, 0.068 mmol) were taken in a solvent mixture of pyridine (1 mL) and DCM (1 mL) at rt and stirred for 1 h, then treated with 5 L of Et.sub.3N and stirred for 15 min. Solvents were removed under vacuum to obtain a yellow solid. Crude product was purified the by flash chromatography using DCM and MeOH as eluent to obtain the compound 41* as white solid (8.5 mg, 52%).

(167) .sup.1H NMR (400 MHz, cd.sub.3od) 8.23 (d, J=8.9 Hz, 2H), 7.25 (d, J=8.9 Hz, 2H), 4.85 (d, J=3.4 Hz, 1H), 4.12 (s, 1H), 3.94-3.79 (m, 3H), 3.78-3.53 (m, 4H), 3.51-3.39 (m, 4H), 3.13 (t, J=6.6 Hz, 2H), 2.60 (t, J=6.5 Hz, 2H), 2.16 (dt, J=15.1, 7.3 Hz, 4H), 1.82-1.10 (m, 84H), 0.82 (t, J=6.5 Hz, 6H).

Example C.7: Synthesis of (2S,3S,4S,5R,6R)-methyl 4-(((2S,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-(((2R,3S,4R,5R,6R)-6-(2-(6-((6-(((2R,3R,4S,5R,6S)-6-(((2S,3S,4R)-2-hexacosanamido-3,4-dihydroxyoctadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)hexyl)amino)-6-oxohexanamido)ethoxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,5-dihydroxytetrahydro-2H-pyran-2-carboxylate (42*)

(168) ##STR00184##

(169) Tetrasaccharide 39* (4.6 mg, 6.01 mol) and glycosphingolipid 41* (4.8 mg, 3.98 mol) were dissolved in pyridine (1 mL)DMSO (0.5 mL) solvent mixture and stirred for 15 min. HOBt (0.92 mg, 5.97 mol) and triethylamine (30 L) were added and the stirring was continued for 18 h. The reaction mixture was dried in vacuum and was purified by C18 Sep-Pak column using water-MeOHCHCl.sub.3 solvent combinations to get target product 42* as white solid (3.0 mg, 41%).

(170) .sup.1H NMR (600 MHz, cd.sub.3od) 4.86 (d, J=3.7 Hz, 1H), 4.52 (d, J=7.9 Hz, 1H), 4.49 (d, J=7.8 Hz, 1H), 4.40 (d, J=8.0 Hz, 1H), 4.26 (d, J=7.8 Hz, 1H), 4.18-4.08 (m, 1H), 3.98-3.74 (m, 12H), 3.74-3.36 (m, 21H), 3.29-3.25 (m, 1H), 3.20-3.07 (m, 2H), 2.23-2.08 (m, 6H), 1.66-1.08 (m, 92H), 0.84 (t, J=7.0 Hz, 6H). MALDI-TOF: calculated for C.sub.90H.sub.165N.sub.3NaO.sub.34 [M+Na].sup.+, 1856, found 1857.

Example C.8: Synthesis of (2S,3S,4S,5R,6R)-4-(((2S,3R,4R,5S,6R)-5-(((2R,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-(((2R,3S,4R,5R,6R)-6-(2-(6-((6-(((2R,3R,4S,5R,6S)-6-(((2S,3S,4R)-2-hexacosanamido-3,4-dihydroxyoctadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methoxy)hexyl)amino)-6-oxohexanamido)ethoxy)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,5-dihydroxytetrahydro-2H-pyran-2-carboxylic acid (43*)

(171) ##STR00185##

(172) Conjugate 42* (0.55 mg, 0.296 mmol) was taken in methanol (0.5 mL)THF (0.25 mL) at rt, treated with 30 L of freshly prepared 0.05 M aq. NaOH solution, and stirred at rt for 1.5 h. The reaction mixture was neutralized with Amberlite 120H.sup.+, then filtered through filter syringe, washed thoroughly with methanol-CHCl.sub.3 solvent mixture (2 mL3), and the filtrate was evaporated in vacuum to get white colored solid which was washed with CHCl.sub.3 and decanted twice. The remaining white solid was dried in vacuum to get desired product 43* (0.41 mg, 77%).

(173) .sup.1H NMR (400 MHz, cd.sub.3od) 4.86 (d, J=3.3 Hz, 1H), 4.60 (d, J=8.0 Hz, 1H), 4.49 (d, J=8.0 Hz, 1H), 4.41 (d, J=7.9 Hz, 1H), 4.28 (d, J=7.9 Hz, 1H), 4.19-4.11 (m, 1H), 3.96-3.76 (m, 9H), 3.73-3.37 (m, 19H), 3.14 (d, J=6.6 Hz, 2H), 2.28-2.09 (m, 6H), 1.79-1.06 (m, 92H), 0.85 (t, J=6.7 Hz, 6H). MALDI-TOF: calculated for C.sub.88H.sub.160N.sub.3NaO.sub.34 [M+Na2H].sup.+, 1826, found 1827.

(174) D. Biological Evaluation

Example D.1: Synthesis of Liposomes (see FIG. 1)

(175) Mixing of lipids and storage. A solution of liposome precursor was made by mixing conjugate 43* (0.2 mg, 0.111 mol), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (3.36 mg, 4.25 mol) and cholesterol (1.1 mg, 2.85 mol) in 2.2 mL of a solution of 1:4 chloroform:methanol. Then, this stock solution of 45 doses was split evenly in 5 glass vials of 5 cc volume, and each solution was evaporated in vacuo to a thin film on the glass, and stored under argon at 20 C. Each vial is meant to contain 9 doses of the liposomal formulation, reconstituted by hydrating the liposomes in 900 L of phosphate buffer saline (PBS), so that each dose is 100 L of the solution. One dose=4.4 g (2.5 nmol) of conjugate 43*, which is equal to 1.7 g of SP3 tetrasaccharide antigen (not including the linker).

(176) Rehydration of the lipid films. On the day before each immunization, one frozen vial was thawed. Lipid film was rehydrated by adding 900 L of sterile phosphate buffer saline (PBS) to the glass storage vial and stirring at 60 C. (T.sub.m of DSPC=55 C.) for one 30 min. (used a rotavap to stir the flask and the heat bath to control the temperature), yielding a mildly opalescent solution constituted of large multilamellar vesicles (LMV).

(177) Lipid extrusion into defined liposomes. After rehydration of the lipid films, the resulting opalescent solution was taken up in a glass syringe and slowly extruded through a pre-heated (to 60 C.) lipid mini-extruder system equipped with the appropriate track-etch polycarbonate membrane between the two syringes (400 m). Each solution was passed a minimum number of 31 times through the membrane.

(178) Mini-extruder: Avanti Polar Lipids, inc. http://avantilipids.com/index.php?option=com_content&view=article&id=185&Item id=193

(179) Membrane: Whatman, Nucleopore, product #800282 (0.4 m).

(180) Analysis of liposomes. An aliquot of the liposomal solutions was loaded into a plastic UV cell and analyzed using dynamic light scattering (Malvern instruments, Zetasizer V). The analysis confirmed the size and population distribution of each liposomal formulation. A uniform population with an observed diameter between 200 and 250 nm was observed.

Example D.2: Synthesis of CRM197 Conjugate (44*)

(181) ##STR00186##

(182) Tetrasaccharide 11* (2 mg, 2.8 nmol) solubilized in 100 L anhydrous dimethyl sulfoxide (DMSO) was added drop-wise to a stirred solution of di-N-hydroxy-succinimidyl adipate ester in 10-fold molar excess solubilized in 200 L anhydrous DMSO with 10 L triethylamine (Et.sub.3N) and reacted for 2 h at room temperature. Then 0.4 ml of 100 mM Na-phosphate buffer, pH 7.4, was added and unreacted di-N-hydroxy-succinimidyl adipate ester was extracted twice with chloroform. The aqueous phase was recovered and reacted with 1 mg CRM.sub.197 (Pfnex Inc., San Diego, Calif., USA) solubilized in 1 mL 100 mM Na-phosphate buffer, pH 7.4, at room temperature for 12 h. The reaction product was desalted and concentrated using 10 kDa centrifugal filters (Millipore). Protein concentration was determined with the Micro BCA Protein Assay Kit (Pierce) according to the manufacturer's recommendations.

(183) SDS-PAGE

(184) Samples were dissolved in Lammli buffer (0.125 M Tris, 20% (v/v) glycerol, 4% (w/v) SDS, 5% (v/v) beta-mercaptoethanol, bromophenol blue, pH 6.8) and boiled at 95 C. for 10 min. Samples were run in 10% polyacrylamide gels and stained with 0.025% Coomassie Brilliant Blue R-250 in an aqueous solution containing 40% (v/v) methanol and 7% (v/v) acetic acid. Characterization of the conjugate 44* by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed an expected shift of the glycoconjugate towards higher masses and band broadening compared to unconjugated CRM.sub.197 carrier protein (see FIG. 2a).

(185) MALDI-TOF Mass Spectrometry

(186) MALDI-TOF mass spectra of CRM.sub.197 and SP3 tetrasaccharide CRM.sub.197 conjugate 44* were obtained using an Autoflex Speed instrument (Bruker Daltonics, Bremen, Germany). The mass spectrometer was operated in positive linear mode. Spectra were acquired over an m/z range from 30,000 to 210,000 and data was analyzed with the FlexAnalysis software provided with the instrument. 2,4-dihydroxyacetophenone (DHAP) was used as matrix, samples were spotted using the dried droplet technique. MALDI-TOF-MS measurement reveals that conjugate 44* presents an antigen loading of 6.5 molecules of SP3 tetrasaccharide per molecule CRM.sub.197 on average (see FIG. 2b).

Example D.3: Immunizations

(187) Two groups (six mice each) of six to eight-weeks old female C57BL/6 mice (purchased from Charles River, Germany) were immunized subcutaneously (s.c.) with SP3 tetrasaccharide liposomes obtained at example D.1 (1.7 g of SP3 tetrasaccharide antigen per mouse) or with SP3 tetrasaccharide-CRM.sub.197 conjugate 44* prepared at example D.2 corresponding to 1.7 g SP3 tetrasaccharide antigen per mouse with Alum adjuvant. The conjugate 44* was mixed with Alhydrogel (Brenntag) (1 L Alhydrogel per g protein) and incubated at 4 C. at least 12 h prior to the immunizations. Blood was collected in one-week intervals via the tail vein. Sera were separated from erythrocytes by centrifugation.

Example D.4: Preparation of Microarrays

(188) Oligosaccharides bearing an amine linker, or proteins, were immobilized on CodeLink N-hydroxyl succinimide (NHS) ester activated glass slides (SurModics Inc., Eden Prairie, Minn., USA) with a piezoelectric spotting device (S3; Scienion, Berlin, Germany). Microarray slides were incubated in a humid chamber to complete reaction for 24 h, quenched with 50 mM aminoethanol solution, pH 9 for 1 h at 50 C., washed three times with deionized water, and stored desiccated until use.

Example D.5: Microarray Binding Assays

(189) Slides were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (w/v) for 1 h at room temperature, washed three times with PBS and dried by centrifugation (300g, 5 min.). A FlexWell 64 (Grace Bio-Labs, Bend, Oreg., USA) grid was applied to microarray slides. Resulting 64 wells were used for 64 individual experiments. Slides were incubated with serum, diluted 1:200 with 1% BSA in PBS (w/v) in a humid chamber for 1 h at room temperature, washed three times with 0.1% Tween-20 in PBS (v/v) and dried by centrifugation (300g, 5 min.). Slides were incubated with fluorescence-labeled secondary antibodies diluted in 1% BSA in PBS (w/v) in a humid chamber for 1 h at room temperature, washed three times with 0.1% Tween-20 in PBS (v/v), 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. Background-subtracted mean fluorescence intensity (MFI) values were exported to Microsoft Excel for further analyses. Secondary antibodies used were: Alexa Fluor 594 Goat Anti-Mouse IgG1 (1) (Life Technologies) diluted 1:400, Alexa Fluor 647 Goat Anti-Mouse IgG2a (2a) (Life Technologies) diluted 1:200, Alexa Fluor 488 Goat Anti-Mouse IgG3 (3) (Life Technologies) diluted 1:200.

(190) The primary immune response was assessed by glycan microarray screening of serum samples retrieved at weeks 0, 1 and 2. SP3 oligosaccharides, natural SP3 polysaccharide as well as S. pneumoniae cell wall polysaccharide (CWPS) as negative control were printed on NHS ester-activated microarray slides (see FIG. 3b). Representative microarray scans of one mouse immunized with SP3 liposomes are shown in FIG. 3c, indicating that IgG3 antibodies were elicited against the immunogen, the SP3 tetrasaccharide, as well as smaller substructures and the natural SP3 polysaccharide. No antibodies against the CWPS were detected in any of the mice, demonstrating the specificity of the antibody response towards SP3-related antigens. Cross-reactivity to the natural SP3 polysaccharide gives a first indication that the antibodies are capable of binding to the surface of S. pneumoniae bacteria so that to promote protection. After immunization with SP3 liposomes containing conjugate 43*, antibodies of the IgG1, IgG2a and IgG3 subtypes against the SP3 tetrasaccharide as well as against the natural SP3 polysaccharide in all immunized mice were detected (FIGS. 3d, 3e, 3f show averaged data of six mice of both groups). IgG2a and IgG3 antibodies were only detected in mice immunized with SP3 liposomes containing conjugate 43*, not in those immunized with the SP3-CRM.sub.197 conjugate 44*. The SP3-CRM.sub.197 conjugate 44* elicited IgG1 antibodies only two weeks after the immunization. Antibody responses after immunization with SP3 liposomes containing conjugate 43* were faster and were detectable already one week after the immunization.

(191) These data demonstrate the immunogenicity of the conjugate 43* formulated as liposomes. Serum IgG responses in mice were superior to the SP3-CRM.sub.197 conjugate 44* in terms of kinetics and IgG2a and IgG3 production. Isotype switching indicates T cell-dependent antibody responses. Serum IgG antibodies were detectable one week after the first immunization with the conjugate 43* formulated as liposomes. Antibodies cross-reacting with the natural SP3 polysaccharide indicates the potential of these antibodies to bind to S. pneumoniae bacteria and to confer protection against pneumococcal infection.

Protein and peptide-free synthetic vaccines against Streptococcus pneumoniae type 3

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