WO2003101937A1

WO2003101937A1 - Synthetic methods for the large scale production from glucose of analogs of sphingosine, azidosphingosine, ceramides, lactosyl ceramides, and glycosyl phytosphingosine

Info

Publication number: WO2003101937A1
Application number: PCT/CA2003/000832
Authority: WO
Inventors: David R. Bundle; Chang Chun Ling; Ping Zhang
Original assignee: Bundle David R; Chang Chun Ling; Ping Zhang
Priority date: 2002-05-31
Filing date: 2003-06-02
Publication date: 2003-12-11
Also published as: AU2003233725A1

Abstract

Synthetic methods are disclosed for the production from glucose of analogs of sphingosine, azidosphingosine, ceramides, lactosyl ceramides, glycosyl phytosphingosine, and enantiomeric derivatives of phytosphingosine and/or its homologues, for use in pharmaceutical applications. The disclosed syntheses are simple, direct and easily scaled.

Description

SYNTHETIC METHODS FOR THE LARGE SCALE PRODUCTION FROM

GLUCOSE OF ANALOGS OF SPHINGOSINE. AZIDOSPHINGOSINE.

CERAMIDES. LACTOSYL CERAMIDES. AND GLYCOSYL

PHYTOSPHINGOSINE

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to D-erytbro-sphingosine, azidosphingosme, ceramides, glycosyl ceramides, glycosyl phytosphingosine, and derivatives of phytosphingosine for use in pharmaceutical applications such as immunotherapy, immunomodulation and cell regulation by secondary messenger molecules. The same compounds or their close analogs also find application in cosmetic preparations.

The invention provides simple, direct and readily scaled syntheses of analogs of sphingosine, azidosphingosme, ceramides, lactosyl ceramides, glycosyl phytosphingosine, enantiomeric derivatives of phytosphingosine and or its homologues. The resultant intermediates are key synthetic intermediates in and provide facile access to lipid or glycolipid compositions with biological activities of the following types: 1) immunogenic oligosaccharide compositions based on glycosphingolipids and gangliosides structures wherein azidosphigosine analogs are employed to elaborate ceramides, lactosyl ceramides and galactosyl phytosphingosine. In particular, the compositions comprise oligosaccharides that contain a truncated sphingosine derivative that is functionalised to permit easy coupling to a protein carrier, wherein the resultant conjugate elicits a protective immunogenic response, particularly in vaccines against tumour associated carbohydrate antigens as an auxiliary therapy against cancers. 2) immunotherapeutic glycolipid compositions based on galactosyl phytosphingosine. 3) sphingosine, azidosphingosme, and ceramides compositions that may augment or inhibit secondary messengers in cell regulation. 4) sphingosine, azidosphingosme, ceramides, glycosyl ceramides, glycosyl phytosphingosine, and enantiomeric derivatives of phytosphingosine and or its homologues with potential cosmetic preparations.

Scheme 1

Truncated azidosphingosine ally, ether Truncated azidosphingosine

R = β-D-glucopyranosyl

R = β-D-galactopyranosyl

R = α-D-galactopyranosyl

R = 4-0-(β-D-galactopyranosyl)-β-D-glucopyranosyl

- al phytoceramides

State of the Art

Numerous methods have appeared in the chemical and patent literature describing the chemical synthesis of sphingosine and ceramide¹. Most are not suitable for commercial processes, due to excessive reliance on chromatography. Some involve tedious transformation sequences and in some, the starting materials or reagents are too expensive or difficult to obtain to be of use in commercial applications. The main challenge resides in the introduction of the 2-amino and 3-OH groups in the correct ^~D-eryth.ro configuration together with a 4,5-double bond in the trαns-configuration. Carbohydrates²'³ and amino acid especially L-serine^4,5'⁶'^7,8'⁹'¹⁰'¹¹'¹² have been the main sources of the chirality and the strategies to introduce the trans-double bond have involved the use of the Wittig reaction or Birch reduction of triple bond or simple elimination reactions. The first such application was reported by Reist in 1970¹³ using 3-amino-3- deoxy-l,2:5,6-diisopropylidene-α-D-allofuranose, which was followed 3 years later by Newman's synthesis of sphingosine from L-serine^4"12. Since then exhaustive studies on the use of L-serine derivatives and carbohydrates have culminated in highly efficient and diastereoselective syntheses of D-erytbro-sphingosine, the naturally occurrmg stereoisomer, and L-tbreo-sphmgosine ^" . The most noteworthy to date is Polt's synthesis of L-tbreo-sphingosine in five steps and in 60% overall yield from L- serine⁵. In 1983 Vasella¹⁴ employed the Katsuki-Sharpless asymmetric epoxidation to set the chirality of an enynol which ultimately led to a six-step synthesis of D-erythro- sphingosine in 50% overall yield. Advantageous use of homochiral cyclohexadiene-c/,s'-l,2-diol¹⁵'¹⁶'¹⁷ available by means of biocatalytic oxidation of chlorobenzene with toluene dioxygenase, has enabled the synthesis of all four enantiomerically pure C18-sphingosines.

D-erytbrosphingosine is an integral component of mammalian cell surface glycolipids (glycosphingolipids) where it is N-acylated and O-glycosylated to give complex glycosyl ceramides¹⁸'¹⁹. These may be acidic in the case of glycosyl components that contain sialic acid or neural glycosyl c eramides in the absence o f sialic acid.

Gangliosides ( sialic acid c ontaining glycosyl c eramides), as well as neutral glycosphingolipids (neutral glycosyl ceramides) such as Lewis Y are used in several cancer vaccines²⁰'²¹. To render these glycolipids immunogenic the lipid portion of the molecule, the ceramide, is cleaved at its carbon-carbon double bond by an ozonolysis procedure and the resulting aldehyde group is used to couple to protein. Although the process was adopted for use with glycolipids isolated from natural sources, the published literature on cancer vaccines also uses this approach with synthetic gangliosides. Either process is highly inefficient and wasteful of extremely costly synthetic or natural product. Scheme 1. Conjugation of glycolipids to protein

Several literature reports²²'²³'²⁴'²⁵ establish that the ceramide portion of glycosphingolipids influences the biological recognition of the cell surface displayed glycolipid by protein receptors. ' ' It is therefore reasonable to infer that therapeutic cancer vaccines should preserve the chiral centres of O-erythro- sphingosine, since this part of the molecule may influence the relative orientation of the carbohydrate chain and thereby the specificity of the immune response to synthetic vaccines.

SUMMARY OF THE INVENTION

Starting from respectively D-galactose and D-glucose, we have developed two convenient high yielding syntheses of analogs (1 and 2, Scheme 2) that correspond to the core sphingosine structure of natural ceramide (Scheme 1). The lactosyl derivatives of

Scheme 2

these sphingosines can be used in commercial processes to make glycosphingolipids or gangliosides by either chemical or chemo-enzymatic approaches (Scheme 3). When vaccine formulations are desired, the ceramide portion, after elaboration of the glycosyl component, may be established in forms that allow several different sites of attachment to protein, either via the sphingosine chains or via the aliphatic acyl chains.

Thus different chemical features of the glycolipid that may be crucial to the immunochemical properties of the glycoconjugate antigen can be preserved. For example either the amide fatty acid chain or the sphingosine chain can be used as the point of covalent attachment to protein. The functional group whereby linkage to protein is established may also be tailored to precise preferences required by the coupling chemistry used to establish the covalent bond linking glycolipid to protein carrier (Scheme 4). Scheme 4. Conjugations to peptides or proteins

I. Coupling through acyl functionalities

II. Coupling through alkene functionalities

x=

If access to naturally occurring lipid or glycolipid structures is desired, the truncated azidosphingosine 2 provides ready access to D-erytbro-sphingosine, azidosphingosine, ceramides, glycosyl ceramides, glycosyl phytosphingosine, and derivatives of phytosphingosine.

The oligosaccharide-protein (or peptide) conjugates gangliosides and glycosphingolipids of this invention may be used as vaccines, as immunogens that elicit specific antibody production or stimulate specific cell mediated and humoral immunity responses. They may also be utilized as therapeutic modalities, for example, to stimulate the immune system to recognize tumor-associated antigens; as immunomodulators, for example, to stimulate lymphokine/cytokine production by activating specific cell receptors; as prophylactic agents, for example, to block receptors on cell membrane preventing cell adhesion; as diagnostic agents, for example, to identify specific cells; and as development and/or research tools, for example, to stimulate cells for monoclonal antibody production.

DETAILED DESCRIPTION OF THE INVENTION Prior to describing this invention in detail, the following terms will first be defined:

Definitions

As used herein, "alkyl" refers to alkyl groups having from 1 to 30 carbon atoms and more preferably 1 to 26 carbon atoms. This term is exemplified by groups such as methyl, t-butyl, «-pentyl, n-decyl, and the like.

"Substituted alkyl" refers to an alkyl group having from 1 to 3, and preferably 1 to 2, substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxylaryl, carboxyl-substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.

"Alkoxy" refers to the group "alkyl-O-" which includes, byway of example, methoxy, ethoxy, n-proppxy, wo-propoxy, w-butoxy, t-butoxy, sec-butoxy, n-pentoxy and the like.

"Substituted alkoxy" refers to the group "substituted alkyl-O-".

"Acyl" refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)- cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O), heterocyclic-C(O)-, and substituted heterocyclic-C(O)- wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

"Acylamino" refers to the group -C(O)NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where each R is joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic ring wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

"Acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, alkenyl-C(O)O-, substituted alkenyl-C(O)O-, alkynyl-C(O)O-, substituted alkynyl- C(O)O-, aryl-C(O)O-, substituted aryl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, heteroaryl-C(O)O-, substituted heteroaryl-C(O)O-, heterocyclic- C(O)O-, and substituted heterocyclic-C(O)O- wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

"Alkenyl" refers to alkenyl group preferably having from 2 to 30 carbon atoms and more preferably 2 to 26 carbon atoms and having at least 1 and preferably from 1- 2 sites of alkenyl unsaturation.

"Substituted alkenyl" refers to alkenyl groups having from 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylalkyl,. carboxyl-substituted alkyl, carboxylaryl, carboxyl-substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.

"Alkynyl" refers to alkynyl group preferably having from 2 to 30 carbon atoms and more preferably 2 to 26 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation.

"Substituted alkynyl" refers to alkynyl groups having from 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxylaryl, carboxyl-substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.

"Amino" refers to the group -NH₂.

"Substituted amino" refers to the group -NR'R" where R' and R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R' and R" are joined, together with the nitrogen bound thereto to form a heterocyclic or substituted heterocylic group provided that R' and R" are both not hydrogen.

"Aminoacyl" refers to the groups -NRC(O)alkyl, -NRC(O)substituted alkyl, - NRC(O)cycloalkyl, -NRC(O)substituted cycloalkyl, -NRC(O)alkenyl, -NRC(O)substituted alkenyl, -NRC(O)alkynyl, -NRC(O)substituted alkynyl,

-NRC(O)aryl, -NRC(O)substituted aryl, -NRC(O)heteroaryl, -NRC(O)substituted heteroaryl, -NRC(O)heterocyclic, and -NRC(O)substituted heterocyclic where R is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

"Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2- benzoxazolinone, 2H-l,4-benzoxazin-3(4H)-one-7yl, and the like). Preferred aryls include phenyl and naphthyl.

"Substituted aryl" refers to aryl groups which are substituted with from 1 to 3 substituents selected from the group consisting of hydroxy, acyl, acylamino, acyloxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxylaryl, carboxyl-substituted aryl, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic, substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl, halo, nitro, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, and substituted heterocyclyloxy.

"Aryloxy" refers to the group aryl-O- that includes, by way. of example, phenoxy, naphthoxy, and the like.

"Substituted aryloxy" refers to substituted aryl-O- groups.

"Aryloxyaryl" refers to the group -aryl-O-aryl.

"Substituted aryloxyaryl" refers to aryloxyaryl groups substituted with from 1 to 3 substituents on either or both aryl rings as defined above for substituted aryl. "Carboxyl" refers to -COOH or salts therof.

"Carboxylalkyl" refers to -C(O)Oalkyl where alkyl is as defined herein.

"Carboxyl-substituted alkyl" refers to -C(O)O-substituted alkyl where substituted alkyl is as defined herein.

"Carboxylaryl" refers to -C(O)Oaryl where aryl is as defined herein.

"Carboxyl-substituted aryl" refers to -C(O)O-substituted aryl where substituted aryl is as defined herein.

"Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including, by way of example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.

"Cycloalkenyl" refers to cyclic alkenyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings and further having at least 1 and preferably from 1 to 2 internal sites of ethylenic (C=C) unsaturation.

"Substituted cycloalkyl" and "substituted cycloalkenyl" refers to an cycloalkyl or cycloalkenyl group, having from 1 to 5 substituents selected from the group consisting of oxo (=O), thioxo (=S), alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxylaryl, carboxyl-substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.

"Cycloalkoxy" refers to -O-cycloalkyl groups.

"Substituted cycloalkoxy" refers to -O-substituted cycloalkyl groups. "Halo" or "halogen" refers to fluoro, chloro, bromo and iodo and preferably is either chloro or bromo.

"Heteroaryl" refers to an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl, indolyl and furyl.

"Substituted heteroaryl" refers to heteroaryl groups which are substituted with from 1 to 3 substituents selected from the same group of substituents defined for substituted aryl.

"Heteroaryloxy" refers to the group -O-heteroaryl and "substituted heteroaryloxy" refers to the group -O-substituted heteroaryl.

"Heterocycle" or "heterocyclic" refers to a saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur or oxygen within the ring wherein, in fused ring systems, one or more the rings can be aryl or heteroaryl.

"Substituted heterocyclic" refers to heterocycle groups which are substituted with from 1 to 3 of the same substituents as defined for substituted cycloalkyl.

Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

"Heterocyclyloxy" refers to the group -O-heterocyclic and "substituted heterocyclyloxy" refers to the group -O-substituted heterocyclic.

"Thiol" refers to the group -SH.

"Thioacyl" refers to the groups H-C(S)-, alkyl-C(S)-, substituted alkyl-C(S)-, alkenyl-C(S)-, substituted alkenyl-C(S)-, alkynyl-C(S)-, substituted alkynyl-C(S)- cycloalkyl-C(S)-, substituted cycloalkyl-C(S)-, aryl-C(S)-, substituted aryl-C(S)-, heteroaryl-C(S)-, substituted heteroaryl-C(S)-, heterocyclic-C(S)-, and substituted heterocyclic-C(S)- wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

"Thioalkyl" refers to the groups -S-alkyl.

"Substituted thioalkyl" refers to the group -S-substituted alkyl.

"Thiocycloalkyl" refers to the groups -S-cycloalkyl.

"Substituted thiocycloalkyl" refers to the group -S-substituted cycloalkyl.

"Thioaryl" refers to the group -S-aryl and "substituted thioaryl" refers to the group -S-substituted aryl.

"Thioheteroaryl" refers to the group -S-heteroaryl and "substituted thioheteroaryl" refers to the group -S-substituted heteroaryl.

"Thioheterocyclic" refers to the group -S-heterocyclic and "substituted thioheterocyclic" refers to the group -S-substituted heterocyclic. The term "amino acid" refers to α-amino acids of the formula H₂NCH(R⁷)COOH where R⁷ is alkyl, substituted alkyl or aryl. Preferably, the α- amino acid is one of the twenty naturally occurring L amino acids.

The term "peptide" refers to compounds comprising from 2 to 25 amino acids.

The term "protein carrier" or "carrier" refers to a substance that elicits a thymus dependent immune response that can be coupled to a hapten or antigen to form a c onjugate. hi p articular, v arious p rotein a nd/or g lycoprotein a nd/or s ub-unit carriers can be used, including but not limited to, tetanus toxoid/toxin, diphtheria toxoidtoxin, bacteria outer membrane proteins, crystalline bacterial cell surface layers, serum albumin, gamma globulin or keyhole limpet hemocyanin.

The term "conjugate" refers to oligosaccharides that have been covalently coupled to a protein or other larger molecule with a known biological activity through a linker. The oligosaccharide may be conjugated through the inter-glycosidic oxygen or sulfur.

The oligosaccharide is attached though a linker to a protein carrier using conventional chemical techniques providing for linkage of the oligosaccharide to the carrier. In one embodiment, reaction chemistries well known in the art that result in covalent linkages between the linker and both the protein carrier and the oligosaccharide and are used. Such chemistries preferably involve the use of complementary functional groups on the hetero- or homo-biftmctional cross-coupling reagent. Preferably, the complementary functional groups are selected relative to the functional groups available on the oligosaccharide or protein carrier for bonding or which can be introduced onto the oligosaccharide or carrier for bonding. Again, such complementary functional groups are well known in the art. For example, reaction between a carboxylic acid of either the linker or the protein and a primary or secondary arnine of the protein or the linker in the presence of suitable, well-known activating agents results in formation of an amide bond; reaction between an amine group of either the linker or the protein and a sulfonyl halide of the protein or the linker results in formation of a sulfonamide bond covalently; and reaction between an alcohol or phenol group of either the linker or the protein carrier and an alkyl or aryl halide of the carrier or the linker results in formation of an ether bond covalently linking the carrier to the linker. Similarly these complimentary reactions can occur between the linker and the oligosaccharide to form a linkage between the oligosaccharide and the linker. The table below illustrates numerous complementary reactive groups and the resulting bonds formed by reaction there between.

COMPLEMENTARY BINDING CHEMISTRIES

FIRST REACTIVE SECOND REACTIVE

GROUP GROUP LINKAGE hydroxyl isocyanate urethane amine epoxide β-hydroxyamine sulfonyl halide amine sulfonamide carboxyl amine amide acyl azide amine amide hydroxyl alkyl/aryl halide ether epoxide alcohol β-hydroxyether epoxide sulfhydryl β-hydroxythioether maleimide sulfhydryl thioether carbonate amine carbamate

The term "heterobifunctional cross coupling reagents" refers to a reagent that is used to couple two other molecules or species together by having two different functional groups built into one reagent. Such cross coupling reagents are well known in the art and include, for example, X-Q-X', where each of X and X' are preferably independently cross coupling groups selected, for example, from -OH, -CO₂H, epoxide, -SH, -N=C=S, and the like. Preferably Q is a group covalently coupling X and X' having from 1 to 20 atoms and preferably from 1 to 20 carbon atoms.

Examples of suitable heterobifunctional cross coupling reagents include squarate derivatives, as found in the attached appendix, as well as entities derived from succinic anhydride, maleic anhydride, polyoxyalkylenes, adepic acid (CO₂H-C₆- CO₂H) and azelaic acid (CO₂H-C₉-CO H). The heterobifunctional cross coupling reagents may also be a lipid or lipid mimic, where the carbohydrate hapten may be covalently linked to the lipid or the lipid is co-administered as an immunological adjuvant. The term "homobifunctional cross coupling reagents" refers to a reagent that is used to couple two other molecules or species together by having two of the same functional groups built into one reagent. Such cross coupling reagents are well known in the art and include, for example, X-Q-X, where X and Q are as defined above. 1,2- Diaminoethane, a dicarboxylic acid chloride and diethyl squarate are examples of such homobifunctional cross coupling reagents. Homobifunctional cross coupling reagents may also be derived from lipids and lipid mimics.

The term "linker" refers to the residue produced after covalent bonding of the cross coupling reagent to the oligosaccharide and the protein carrier.

The term "carbohydrate" or "glycosyl" refers to oligosaccharides comprising from 1 to 20 saccharide units and more preferably 1 to 8 saccharide units. The particular saccharide units employed are not critical and include, by way of example, all natural and synthetic derivatives of glucose, galactose, N-acetylglucosamine, N- acetylgalactosamine, fucose, sialic acid, 3-deoxy-D,L-octulosonic acid, and the like. In addition to being in their pyranose form, all saccharide units described herein are in their D form except for fucose which is in its L form.

"Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound of Formula LTA which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

The term "vaccine" refers to a composition used to stimulate an immune response in a mammal and so confer resistance to the disease or infection in that mammal, which used herein, infers that the response has immunologic memory. .

The term "immune response" refers to the reaction of the body to foreign or potentially dangerous substances (antigens), particularly disease-producing microorganisms. The response involves the production by specialized white blood cells (lymphocytes) of proteins known as antibodies, which react with the antigens to render them harmless. The antibody-antigen reaction is highly specific. Vaccines also stimulate immune responses.

The term "immunologic memory" refers to the ability of the immune system to remember a previously encountered antigen. Antibodies are produced as a result of the first exposure to an antigen and stored in the event of subsequent exposure.

The term "immunologically effective amount" refers to the quantity of a immune response inducing substance required to induce the necessary immunological memory required for an effective Vaccine.

The synthesis of azidosphingosine 1 was carried out through 6 high yielding transformations steps from the known diol 3 which is derived from D-galactose³. The primary hydroxyl group of 3 was selectively silylated with tert-butyldiphenylsilyl chloride in pyridine (-» 4), and the secondary hydroxyl group was mesylated in-situ by adding methanesulfonyl chloride. The mesylate of compound 5 was substituted by azide and the primary hydroxyl group was revealed after removal of the silyl group using tetra-n-butylammonium fluoride in THF (-» 7). After allylation (-» 8) using standard condition, azidosphingosine 1 was obtained after the hydrolysis of the benzylidene p rotecting group. T he r eaction s equence w orked e fficiently so t hat t he sequence from compound 3 to 7 required only one column chromatographic purification, and compound 7 was obtained in overall 33% yield.

The synthesis of azidosphingosine analog 2 started from 1,2-O-acetal- -D- glucopyranose, specifically, 1,2-O-isopropylidene-α-D-glucopyranose (9), which is commercially available in kilogram quantities. Acetals protecting groups includes, but not limited to isopropylidene, methylidene, ethylidene, propylidene, cyclohexylidene, benzylidene, ortho-nitrobenzaldehyde.

Trimesylation step on a 100-gram scale was earned out in pyridine using 4.0 equivalents of methanesulfonyl chloride as reagent (Scheme 6). Unlike standard sulfonation conditions that require vigorous anhydrous conditions, we found that reagent quality pyridine (< 0.3% water content) is adequate. No chromatography was necessary as the 3,5,6-trimesylate 10 is insoluble in water, one simple precipitation in ice-water was sufficient to afford analytically pure 10 in almost quantitative yield.

The next step involved the construction of a double bond at the 5,6-position. The literature procedure²⁹ employed large quantities of ketone such as acetone as solvent and sodium iodide as reagent under refluxing conditions. We found that these conditions lead to incomplete elimination of the 5,6-mesylate and around 5-10% starting material remained untransformed even after adding more sodium iodide and prolonged reaction time (36 h). However, when we carried out the reaction in NN- dimethylformamide (DMF) and used the more readily available potassium iodide as reagent and h eating t he r eaction t o 90°, t he r eaction w ent t o c ompletion i n a s hort period of time (10 h). Most of the DMF was removed by evaporation and the rest was washed off at the extraction step. The purification of compound 11 was carried out by simple extraction between ethyl acetate and an aqueous solution of sodium thiosulfate, no chromatography was needed at this step, ΝMR confirmed the purity of the material and compound 11 was obtained in quantitative yield.

Convenient transformation of compound 11 to 15 involves four reaction sequences but only one purification step is needed. The 1,2-O-isopropylidene protecting group of 11 was removed smoothly by heating a solution of compound 11 in 50% aqueous trifluoroacetic acid at 55° C for 2 hours. TLC revealed clean deprotection. After evaporating the acid and co-evaporation with toluene, the diol 12 was oxidized with sodium periodate in a mixture of ethanol - water (4:1), the intermediate aldehyde 13 was isolated by extraction. The reduction of the aldehyde function was carried out in methanol using sodium borohydride, and the diol 14 was formed along with some minor impurities according to TLC. h fact, we have purified diol 14 by chromatography at this stage, but it was found that at the following step, the nucleophilic substitution of mesylate with azido group did not work well as the yield was notably low. In addition, having two hydroxyl groups unprotected complicated the purification step. So we have protected the diol with acetates at this stage. Therefore, after reduction, the solvent was removed and the diol was obtained by triturating the solid material with ethyl acetate. After evaporation, the mixture containing the diol 14 was acetylated and the diacetate 15 was obtained by chromatography in 74% yield. The nucleophilic substitution of mesylate in 15 with sodium azide was carried out in DMF at 95° C in moderate 56% yield. The use of tefra-ra-butyl-unmonium bromide as a catalyst improved the yield to 70%. Compound 16 was formed along with several side products that justified the need for one more chromatographic purification.

The last step is a transesterification and no chromatography is required. The diol 2 was obtained in quantitative yield.

B. Preparation of novel lactosyl ceramide The chemical synthesis of lactosyl ceramides analogs requires that the secondary hydroxyl groups of both 1 and 2 to be selectively protected. hi the case of azidosphingosine 1, we followed a protection strategy well known to chemists (Scheme 7). Thus the primary hydroxyl group of 1 was tritylated in dichloromethane using triethylamine as base (-» 17), and the secondary hydroxyl group was benzoylated in-situ (-»■ 18), after work-up, the crude 18 was de-tritylated under acidic condition to afford the desired acceptor 19 in 73% overall yield from 1.

The transformations from 1 to 19 (Scheme 7) involved the use of several reagents. By carefully tuning the reaction conditions, we have avoided column purification of the intermediates 17 and 18. However, the final reaction mixture was rather complex and 27% of material was lost during purification.

The target azidosphingosine derivative 21 was obtained in a more efficient way (Scheme 8) that involves a one-pot, two-stage-reaction. The diol 2 was first treated with trimethyl orthobenzoate³⁰ in the presence of a catalytic amount of camphor sulfonic acid in chloroform, when the starting material was consumed, the reaction mixture was evaporated and the intermediate cyclic orthoester was opened with a 90% a cetic acid - w ater s olution t o a fford a n 1 : 1 m ixture t hat c ontains t he undesired 20 and desired 21. The overall yield of the transformation was excellent and the two isomers were readily separated by column chromatography on silica gel. The undesired isomer 20 was quantitatively recycled to 2 by a simple transesterification.

The advantage of this new method is demonstrated by fact that the reaction can be finished in short time; all the reagents are volatile and the final reaction mixture is quite clean (the only by-product of the reaction is methyl benzoate). The disadvantage is that a 1:1 mixture of 20 and 21 was obtained. However, since 20 can be quantitatively recycled to 2, there is virtually no waste of material.

The glycosylation of 21 was carried out with perbenzoylated lactosyl bromide 24, which was prepared from lactose 22. The glycosylation was promoted by silver triflate in anhydrous toluene, no base was added. The reaction proceeded extremely well and the intermediate glycoside 25 was not purified but subjected to a full removal of benzoates under Zemplen transesterication condition. The crude lactosyl azidosphingosine 26 was obtained after washing the mixture with acetone. NMR analysis of the crude material revealed an almost quantitative transformation of the perbenzoylated lactosyl bromide to the desired lactoside 24. The crude 26 was carried on to the next step. A pure sample of 26 was obtained by reverse-phase chromatography on a C18 column.

In order to functionalize the lactosyl azidosphingosine 26 with stearic acid, the azido group was reduced to amine with hydrogen sulfide; and the resulting amine 27 was directly esterified with an activated ester of stearic acid 28 to afford the modified lactosyl ceramide 29. The overall yield for these two steps exceeds 87%.

Lactosyl ceramide 29 has limited solubility in water at room temperature, a property that will cause problems if enzymatic reactions are to be used to elaborate ganglioside of glycosphingolipid analogs. A water soluble compound 30, was prepared via photo- addition of cysteamine to the double bond of 29. The photo-addition was carried out in methanol under continuous UV irradiation and compound 30 was isolated as an ammonium salt.

Compound 30 was soluble in water at room temperature. Not only may compound 30 be used as an enzymatic substrate, but since it is already functionahzed, the resulting oligosaccharides after enzymatic reactions are ready for immediate conjugation, via the terminal amino group.

Another chemical methods are known in art to convert compound 29 into water soluble form. For example, one may add thioacetic acid (HSCH₂CO₂H) to the double bond of 29 using radical initiated addition. See, for example, K. Matsuoka, H. Oka, T. Koyama, N. Esumi and D. Terunuma, Tetrahedron Letters, 42, 3327-3330, 2001. Expoxidation of compound 29 with the following reaction of the epoxide with amines would give water soluble amines derivatized as different salts. Ozonolyis of the double bond of compound 29 provides an aldehyde, which may be oxidized to a carboxylic acid or its salts. The aldehyde can be subjected to reductive amination to provide a water-soluble amine. Addition of bromine to the double bond provides dibromides that may be substituted by azide to yield diazides and hence after reduction, diamines. Further elaboration of groups known to be converted to carboxylic acids or amines may be envisaged (i.e. nitriles hydrolzyed to imidates and hence caboxylates). Guanidine groups may also be introduced via an intermediate amine.

C Preparations of novel analogs of gangliosides and Gb₃

Both compounds 26 and 30 (alternate forms of the truncated lactosyl ceramide) are good substrates for enzymatic transformations using cloned and over- expressed glycosyltransferase enzymes and the appropriate sugar nucleotide donor molecules. For example, when CMP-ΝeuΝAc is used as a donor, with a α2,3- sialyltransferase, the corresponding GM₃ analogs 32 and 38 (Scheme 9 and 10) can be obtained³¹. When the same compounds are used as acceptors with a α2,6- sialyltransferase, the 2,6-isomers of GM₃ 35 and 39 are obtained³¹. Scheme 9 Enzymatic synthesis of analogs of GM₃ and its ismers from substrate 26

We also demonstrated that both 26 and 30 are good substrates for the αl,4- galactosyltransferase (schemes not shown here), which with UDP-galactose as donor, yields the corresponding analogs of Gb₃ in excellent yields.

D Chemical preparations of novel analogs of gangliosides GM₂, GM₃

(i) Synthesis of GM₂ related to azidosphingosine 1

Since GM₂ and GM₃ are all tumour associated. antigens, we were interested in synthesizing these antigens chemically, since the enzyme that is responsible for transfer of GalNAc from UDP-GalNAc to the 4'-positon of lactose in the biosynthesis of GM₂ is not yet commecially available. The analog of GM₂ bearing the ceramide cooresponding to azidosphingosine 1 was prepared first (Scheme 11). Starting from the known trisaccharide 40 and imidate 41³², tetrasaccharide 42 was obtained under the promotion of catalytic ammount of TMSOTf at room temperature. The benzyl protecting groups were removed by hydrogenation and the intermediate 43 was acetylated (- 44). After removing the anomeric TMSEt (2-trimethylsilylethyl) protecting group, the anomeric hydroxyl group was converted to imidate (-> 46).

Scheme 11 Chemical synthesis analog of GM₂ ceramide related to 1

Glycosylation of 46 with 19 (the benzoylated derivative of 1) in dichloromethane under the promotion of TMSOTf affored the desired tetrasaccharide 47 which could not be obtained in pure form. However, when all the protecting groups were removed, compound 49 was obtained in pure form. The deprotection sequence was carried out by first removing all the acetates and benzoate under the Zemplen transesterification conditions, followed by saponifying the methyl ester linkage of the sialic acid unit (-» 47); the phthalimido group was removed under the treatment with ethylenediamine in hot H-butanol, and the desired 49 was obtained after selective acetylation of the intermediate amine 48. The final tetrasaccharide ceramide 51 was obtained after reducing the azido group by hydrogen sulfide and acylating the intermediate amine with steric acid.

(ii) Synthesis of GM₂ related to azidosphingosine 2

We also carried out the synthesis of an analog of GM that is related to azidosphingosine 2. i the synthesis of GM ceramide 51, we followed a strategy commonly employed in the literature ' , this involves the incorporation of azidosphingosine at the final stage; thus the anomeric position of lactosyl unit needs to be protected with a temeporary group. After building the oligosaccharide block, the anomeric position of lactosyl unit is then deprotected, activated with imidate and coupled to azidosphingosine. Since the oligosaccharide block usually is rather large, thus less reactive, the coupling step gives moderate to poor yields (40 to 65% usually). If we count all steps that involve the installation and deprotection of the protecting group at lactosyl anomeric center, and the steps to activate the anomeric center, the inefficiency of this strategy is obvious. We should not also forget that this strategy involve the manipulation of elaborated oligosaccharide unit - already difficult to obtain, for several steps, this results tremendous loss of costy material, consequencely, the chemical synthesis of gangliosides at large sacle has not been possible. We decided to synthesize the analog of GM ceramide related to azidosphingosine 2 through a new strategy. Since we were able to prepare azidosphingosine 2 on a large scale from cheap material, and we have carried out the glycosylation step in highyiled, we decided to use 26 as the starting material, and carry on the azidosphingosine 2 all the way through the synthesis.

The synthesis started from the preparation of lactosyl acceptor 54. Thus the an isopropyhdene group was selectively installed at the 3'4'-position of 26, and the reamaining hydroxyl groups were per-benzylated. After removing the isopropyhdene, the desired acceptor 54 was obtained. In fact, we were be able to prepare 54 from perbenzoylated lactosyl bromide, without the need to purify the all the intermediates, and only one final chromatograghy is needed to afford the acceptor 54 in 30-50% overall yield.

During the installation of isopropyhdene, if kinetic conditions are applied, the 4'6'-diol was selectively protected, and after a similar transformation steps, another lactosyl acceptor bearing 4'6'-dihydroxy groups (59) was obtained. Compound 59 is a suitable acceptor for the synthesis of 2,6-linked isomeric form of GM₃.

The azido groups of both acceptor 54 and 59 could be quantitatively reduced using triphenylphosphine - water as reagent and acylated using an activated ester 28 to afford acceptors 56 and 61.

Alternatively, acceptor 54 was prepared from readily accessible donor 62. Thus under the activation of IS-TfOH, aglycon 21 was coupled to 62 to afford the glycoside 63, and the benzoates were transesterified, followed by a perbenzylation to give the intermediate 65. Acidic hydrolysis of the isopropyhdene group of 65 led to the desired acceptor 54 in over 50% yield. Purifications of all the intermediates 63-65 were not necessary.

The assembly of GM₂ was efficiently carried by coupling acceptor 54 with sialic acid donor 66³⁵ under the promotion of NIS-TfOH acetonitrile (- 67, ~50% yield). A small amount of β-isomer was obtained, but was easily separated by column chromatography. Another glycosylation with galactosamine donor 41 led to fully protected GM₂ azidosphingosine 68 in -70% yield. Following a similar reaction sequence to that described in Scheme 11, the azido group was reduced and acylated; after removing all the acetates and hydrolysing the methyl ester of the sialic acid unit, the phthalimido group was readily removed with ethylenediamine in hot «-butanol and the intermediate amine was selectively acetylated. The 6 benzyl groups was finally removed under Birch readuction conditions, the alkene functionalities survived to yield the desired analog of GM₂ related to azidosphingosine 2.

D Preparation of Glycoconjugate from analog of GM? 51

Having an alkene functionality in the oligosaccharide as a potential functional group represent two advantages, first the alkene group is inert toward most of the reaction conditions that are commonly using in carbohydrate chemistry; second, it can be readily derivatized to numerous active functinal groups such a s epoxides, amines, aldehydes and thiol etc. We have successfully prepared a PADRE (peptide- glycoconjugate of GM₂ analog 51 using epoxide as the activating group (Scheme 15). The epoxidation of 51 was quantitatiuvely carried out using benzonitrile- hydrogen peroxide³⁶ as the reagent in methanol. The intermediate epoxide 74 was relatively stable, and can be purified by reverse phase HPLC. The conjugation step was realized through the thiol group of the cytseine residue in the PADRE peptide and the conjugate 75 was prepared in overall > 50% effiency.

E Preparation of Stereoisomers of Sphingosine, Azidosphingosine,

Ceramides, Lactosyl Ceramides, Glycosyl Phytosphingosine, Enantiomeric Derivatives of Phytosphingosine or Its Homologues

Analogs 1 and 2 that are prepared by simple, direct and easily scaled reactions possess tremendous pharmaceutical value. The process to prepare 2 required only two chromatographic purification steps and most of the transformations were carried out in excellent yields. The starting material and reagents are cheap and the reaction conditions generally mild. The process can be easily scaled to the kilogram scale.

The design of 1 and 2 permits highly efficient processes to quantitatively link the new azidosphingosine to lactose viable at least to kilogram scale. The resulting lactosyl azidosphingine 26 was efficiently transformed to the new functionahzed lactosyl ceramide 30. Both 26 and 30 act as efficient enzymic substrates, and by uitilizing enzymatic transformations performed with cloned and over-expressed glycosyltransferase enzymes and the appropriate sugar nucleotide d onor m olecules, these two compounds lead to the preparations of variable new pharmaceuticals that can be readily coupled to bioactive peptides or proteins.

Compound 2 is also a versatile intermediate, which has been transformed to families of analogs with known pharmaceutical activities, such as α-Gal phytosphingosines, sphingosine phosphate and analogs, sphingosines, ceramides,

T7 phytoceramides, and dihydrosphingosines etc (Schemes 16-18). Crossed metathesis reaction applied to compound 2 or the amine derived form it provides the natural products such as 78a and 78b and 79a and 79b. The fully deprotected analog of GM₃ can be easily obtained from trisaccharide

67 (Scheme 19). If we choose disaccharide 59 as acceptor, the analog of α2,6-linked GM can be prepared (Scheme 20). Disaccharide 54 is also an intermediate which can led to the chemical synthesis of another two tumour accociated antigens GD₂ and GD₃ (Schemes 21 and 22).

Scheme 22 Synthesis of analog of GD₃ related to 2

112 R^H, R₂=Bn

113 R^H, R₂=H

EXAMPLES

Methods and Procedures

Optical rotations were measured with a Perkin-Elmer 241 polarimeter at 22±2°

C. Analytical TLC was performed on Silica Gel 60-F₂₅₄ (E. Merck, Darmstadt) with detection by quenching of fluorescence and/or by charring with sulfuric acid. Unless otherwise stated, all commercial reagents were used as supplied. Column chromatography was performed on Silica Gel 60 (Merck 40-60 μM, Darmstadt). ^H NMR spectra were recorded at Varian 600 or 500 MHz (Varian Unity 600 or 500) and first order proton chemical shifts δ_H are referenced to either internal CHC1₃ (δ_H 7.24, CDC1₃). Organic solutions were dried prior to concentration under vacuum at < 40 C (bath). Microanalyses were carried out by the analytical services at this department. Fast atom bombardment mass spectra were recorded on samples suspended in Cleland's matrix using a Kratos AEIMS9 instrument with xenon as the bombarding gas.

(2S,3R)-2-azido-l,3-0-benzylidence-butan-l,3,4-triol (7): To a solution of diol 3 (250 mg, 1.19 mmol) in anhydrous pyridine (2 ml), was added tert- butylchlorodiphenylsilane (379 μl, 1.43 mmol), and the reaction was stirred overnight. Methanesulfonyl chloride (187 μl, 1.38 mmol) was added, the reaction was continued for 4 hours at room temperature. H O (0.5 ml) was added and the reaction was stirred for 30 min. The mixture was concentrated and the residue was dissolved in EtOAc (100 ml), washed with 2N HC1 (30 ml), 2N NaOH (30 ml) and saturated brine (30 ml), dried over anhydrous Na₂SO₄ and evaporated (- 5; ~ 650 mg). The resultant syrup was dissolved in DMF (5 ml), NaN₃ (774 mg, 11.9 mmol) was added, and the mixture was heated to 110° C for 4 hours, cooled to room temperature. EtOAc (150 ml) was added, and the organic solution was washed with 10% brine (2 x 40 ml), dried over anhydrous Na₂SO₄ and evaporated to afford crude

6 (~ 600 mg). The above mixture was dissolved in anhydrous THF (5 ml), and a solution of tetra-n-butylammonium fluoride in THF (1.0 M, 2.5 ml) was added. After 30 min, the solution was concentrated. The mixture worked up as above, and pure 7 was obtained by chromatography on silica gel using a gradient of EtOAc-hexane (10%-»30%) as eluent (92 mg, overall 33% from 3). 1H MR (CDC1₃, 500 MHz): $7.44-7.47 (m, 2H, Ph), 7.34-7.39 (m, 3H, Ph), 5.47 (s, IH, PhCH), 4.39 (dd, IH, J 5.1, 10.8 Hz, H-la), 3.94 (dd, IH, J2.5, 12.4 Hz, H-4a), 3.82 (dd, IH, J4.3, 12.4 Hz, H-4b), 3.75 (dd, IH, J5.0, 10.1 Hz, H-2), 3.66 (t, IH, J 10.7 Hz, H-lb), 3.64 (ddd, IH, J2.5, 4.4, 9.7 Hz, H-3).

(2S,3R)-4-0-AUyl-2-azido-butan-l,3,4-triol (1): To a solution of compound

7 (90 mg, 0.38 mmol) in anhydrous THF (3 ml), was added NaH (80% in mineral oil, 90 mg, 3.0 mmol), and the mixture was stirred for 20 min. Allyl bromide (132 μl, 1.52 mmol) was added, the reaction was continued for 1 hour. MeOH (0.5 ml) was added to quench the reaction, and the mixture was diluted with EtOAc (100 ml), washed with saturated brine (30 ml), dried over anhydrous Na₂SO₄, and evaporated to afford the crude 8.

The hydrolysis of benzylidene was carried out by heating a solution of crude 8 in 80% AcOH-H₂O to 100° C for 1 hour. After cooling to room temperature, the mixture was concentrated to dryness, and compound 1 was obtained by chromatography on silica gel using 7.5% MeOH-CH₂Cl₂ as eluent (67 mg, 93% yield over 2 steps). 1H NMR (CDC1₃, 360 MHz): 65.90 (m, IH, All), 5.29 (m, IH, All), 5.22 (m, IH, All), 4.03 (m, 2H, All), 3.92 (m, IH, Hla), 3.53-3.85 (m, 5H, H-lb+H- 2+H-3+H-4a+H-4b), 2.71 (d, IH, J5.7 Hz, OH-3), 2.33 (t, IH, J6.1 Hz, OH-1).

l,2-0-Isopropylidene-3,5,6-tri-0-methanesulfonyl-α-D-glucofuranose (10): 1,2-O-isopropylidene-α-D-glucofuranose 9 (100 g, 0.45 mol) was dissolved in anhydrous pyridine (1000 mL), and the mixture was cooled to 0° C. Methanesulfonyl chloride (144 mL, 1.86 mol) was added slowly to the solution and the mixture was allowed to warm up to room temperature slowly overnight. The reaction mixture was poured to a mixture of ice/water (4000 mL) with vigorous stirring. The precipitate was filtered o ff, and t he w hite s olid w as t hrown i nto w ater ( 2000 m L) again w ith vigorous stirring. The precipitate was filtered off and washed with more water (1000 mL), and air-dried. Yield 202.84 g (98% yield). 1H NMR (CDC1₃, 500 MHz): 55.92 (d, IH, J3.5 Hz, H-l), 5.08 (d, IH, J2.6 Hz, H-3), 5.04 (ddd, IH, J 1.3, 4.4, 8.9 Hz, H-5), 4.95 (d, IH, J3.5 Hz, H-2), 4.67 (d, IH, J~ 1, 12.1 Hz, H-6a), 4.46 (dd, IH, J 2.8, 8.9 Hz, H-4), 4.40 (dd, IH, J 4.6, 12.1 Hz, H-6b), 3.17 (s, 3H, Mesyl), 3.15 (s, 3H, Mesyl), 3.05 (s, 3H, Mesyl), 1.48 (s, 3H, isopropyhdene), 1.30 (s, 3H, isopropyhdene).

5,6-Dideoxy-l,2-0-isopropyIidene-3-0-methanesulfonyI-a-D- /o-hexose- 5-ene (11): The trimesylate 10 (200 g, 0.44 mol) was dissolved in DMF (1000 mL), potassium iodide (292.2 g, 1.76 mol) was added and the mixture was heated to 90° C with mechanical stirring for 10 h. TLC showed that the starting material was completely c onverted. The mixture was cooled down to room temperature and the solvent was removed under reduced pressure. The dark residue was suspended in AcOEt (3000 mL) and washed with a pre-prepared aqueous solution of Na₂S₂O₃.5H₂O (300 g dissolved in 1500 mL). The almost colorless organic solution was separated and dried over anhydrous Na₂SO₄ and evaporated to dry to give the desired alkene 11 (115.1 g, yield 99%). 1H NMR (CDC1₃, 500 MHz): 55.95 (d, IH, J 3.7 Hz, H-l), 5.83 (ddd, 1H, J6.4, 10.5, 17.3 Hz, H-5), 5.47 (ddd, 1H, J 1.3, 1.3, 17.4 Hz, H-6_trans), 5.36 (ddd, IH, J 1.2, 1.2, 10.5 Hz, H-6_cis), 4.94 (d, IH, J2.9 Hz, H- 3), 4.77 (d, IH, J 3.8 Hz, H-2), 4.75 (m, IH, H-4), 3.00 (s, 3H, Mesyl), 1.50 (s, 3H, isopropyhdene), 1.31 (s, 3H, isopropyhdene).

(2R,3R)-l,3-Di-0-acetyl-2-0-methanesulfonyl-pent-4-ene-l,2,3-triol (15): compound 11 (118.0 g, 0.45 mol) was dissolved in a mixture of trifluoroacetic acid (450 mL) and water (450 mL) and the solution was heated to 55 °C for 2 h. The mixture was concentrated under reduced pressure and co-evaporated with toluene to give the hemi-acetal as a syrup (~ 93 g).

The above syrup (91 g) was dissolved in ethanol (2800 mL). After cooling the solution to -15 °C, an aqueous solution of NaIO₄ (136.0 g dissolved in 700 ml H O, 0.64 mol) was added into the solution within 5 min and the reaction was kept at -10° - -5° C for 90 min. TLC (CH₂C1₂ : MeOH 95 : 5) indicated that the hemi-acetal was completely consumed. The insoluble material was removed by filtration and the filtrate was concentrated to afford a white solid. The solid was extratcted with EtOAc (1500 mL) and water (400 mL) and the organic solution was washed with brine (2 x 300 mL), dried over anhydrous Na₂SO₄ and concentrated to give a colorless syrup.

This above syrup was dissolved in MeOH (600 mL) and cooled to 0° C, and NaBH₄ (42.6 g, 1.13 mol) was added by small portions. The reaction was stirred at 0° C for 1.5 h, left at room temperature for 1 h, and quenched by slow addition of AcOH (100 mL). The reaction was concentrated under reduced pressure while keeping the bath t emperature b elow 30° C t o g ive a w hite s olid. T he s olid w as t riturated w ith EtOAc (3 x 500 mL) and the insoluble material was removed by filtration. The filtrate was concentrated to give a colorless syrup under reduced pressure by keeping the bath temperature below 30° C. A mixture of Ac₂O (500 mL) and pyridine (500 mL) was added to the mixture and the reaction was stirred at room temperature overnight. After concentration, AcOEt (1000 mL) was added, and the mixture was washed with brine (2 x 300 mL), dried over anhydrous Na₂SO₄ and concentrated. The compound 15 (88.8 g, yield 71.4%) was finally obtained by chromatography on silica gel using toluene - EtOAc (9 : 1) as eluent. [ ]_D ²⁵ +30° (c 1.3' CHC1₃). 1H NMR (CDC1₃, 600 MHz): 56.59 (ddd, IH, J 6.6, 10.4, 17.2 Hz, H-4), 5.48 (m, IH, H-3), 5.44 (ddd, IH, J0.9, 0.9, 17.2 Hz, H-5_trans), 5.38 (bd, IH, J 10.6 Hz, H-5_cis), 4.89 (ddd, IH, J3.1, 6.6, 7.1 Hz, H-2), 4.40 (dd, IH, J3.1, 12.5 Hz, H-la), 4.10 (dd, IH, J 7.1, 12.5 Hz, H-lb), 3.05 (s, 3H, Mesyl), 2.11 (s, 3H, OAc), 2.08 (s, 3H, OAc).

(2S,3R)-l,3-Di-0-acetyl-2-azido-pent-4-ene-l,3-diol (16): To a solution of mesylate 15 (59.5 g, 0.21 mol) in anhydrous DMF (425 mL) was added NaN₃ (54.8 g, 0.84 mol), the mixture was heated to 95° C under stirring overnight. The mixture was cooled and concentrated to ~ 50 mL and diluted with AcOEt (1000 mL), washed with brine (2 x 200 mL), dried over anhydrous Na₂SO₄ and concentrated. After chromatography on silica gel using EtO Ac-toluene (5%) as eluent, compound 16 (26.9 g, 56% yield) was obtained as a colorless syrup. 1H NMR (CDC1₃, 600 MHz): 55.80 (m, IH, H-4), 5.33 - 5.37 (m, 3H, H-3 + H-5_trans + H-5_cis), 4.14 (dd, IH, J 4.4, 11.7 Hz, H-la), 4.09 (dd, IH, J7.9, 11.7 Hz, H-lb), 3.80 (dt, IH, J4.6, 4.6, 7.9 Hz, H-2), 2.08 (s, 3H, OAc), 2.07 (s, 3H, OAc).

(2S,3R)-2-Azido-pent-4-ene-l,3-diol (2): A solution of NaOMe in MeOH (1.5 M, 5 mL) was added to a solution of compound 16 ( 40.0 g ) in anhydrous MeOH (500 mL), and the mixture was stirred at room temperature for 2 h. After neutralization with ion-exchange resin Amberlite IR-120 (H⁺), the resin was filtered off and the filtrate was concentrated under reduces pressure to give 2 (25.2 g, quantitative) as a colorless syrup. This compound should be stored in freezer. 1H NMR (CDC1₃, 600 MHz): 55.95 (ddd, IH, J6.2, 10.4, 17.0 Hz, H-4), 5.39 (ddd, IH, J 1.3, 1.3, 17.2 Hz, H-5_trans), 5.29 (ddd, IH, J 1.3, 1.3, 10.4 Hz, H-5_cis), 4.29 (m, IH, H-3), 3.80 (m, 2H, H-la + H-lb), 3.49 (ddd, IH, J5.0, 5.3, 5.3 Hz, H-2), 2.41 (b, 2H, OH-1 + OH-3).

(2S,3R)-4-0-Allyl-2-azido-3-C.-benzoyl-butan-l,3,4-triol (19): To a solution of compound 1 (400 mg, 2.14 mmol) in a mixture of anhydrous CH C1₂ (3 ml) and pyridine (1 ml), was added trityl chloride (774 mg, 2.78 mmol), the mixture was stirred overnight at room temperature. TLC revealed all the starting material was consumed; benzoyl chloride (501 μl, 4.28 mmol) was added, and the reaction was stirred at room temperature for 30 min. H₂O (0.5 ml) was added and the mixture diluted with EtOAc (100 ml), washed successively with IN HC1 (40 ml), saturated NaHCO₃ (40 ml), dried over anhydrous Na SO₄ and evaporated to afford a colorless syrup. The syrupy mixture was dissolved in acetonitrile (10 ml), a aqueous solution of 48% HBF₄ (587 Dl, 3.21 mmol) was added, the reaction was stirred for 30 min. The mixture was diluted with EtOAc (100 ml), washed with saturated NaHCO₃ (2 x 40 ml), dried over anhydrous Na₂SO₄ and evaporated. The mixture was purified by chromatography o n s ilica g el u sing 10% E tO Ac-toluene a s e luent t o yield 19 a s a colorless syrup (454 mg, 73% from 1). 1H NMR (CDC1₃, 600 MHz): 58.04 (m, 2H, Bz), 7.57 (m, IH, Bz), 7.44 (m, 2H, Bz), 5.86 (m, IH, allyl), 5.31 (d'f, IH, J4.4, 6.4 Hz, H-3), 5.26 (m, IH, allyl), 5.18 (m, IH, allyl), 4.00-4.07 (m, 2H, allyl), 3.93 (ddd, IH, J4.0, 6.2, 6.2 Hz, H-2), 3.86 (m, IH, H-la), 3.80 (dd, IH, J4.2, 10.8 Hz, H-4a), 3.75 (dd, IH, J4.2, 10.8 Hz, H-4b), 3.75 (m, IH, H-lb), 2.58 (t, IH, J5.9 Hz, OH-1).

(2S,3R)-2-azido-l-0-benzoyI-pent-4-ene-l,3-diol (20) and (2S,3R)-2-azido-

3-0-benzoyl-pent-4-ene-l,3-diol (21): To a solution containing diol 2 (7.1 g, 49.6 mmol) and trimethyl orthobenzoate (15 ml, 87.3 mmol) in chloroform (150 ml), was added camphor sulphonic acid (500 mg), and the reaction mixture was concentrated to 1/3 of its original volume using a rotavap; TLC revealed that the reaction is not finished, so more chloroform (~100 ml) was added to the mixture, and the solution was concentrated to its 1/3 volume again. This cycle was repeated until TLC indicates the complete consumption of starting material (usually 3 - 4 cycle needed). Et₃N (1 ml) was added to the reaction mixture and the solvent was removed completely; the resulting syrup was treated with a 20% AcOH-H₂O solution (140 ml). After 30 min, the reaction was concentrated to dryness. The syrupy mixture was purified by column chromatography on silica gel using 5% AcOEt-toluene to afford 20 (6.17 g, 50%) and 21 (5.47 g, 44.6%). 1H NMR (CDC1₃, 600 MHz) for 20: 58.03 (m, 2H, Bz), 7.55 (m, IH, Bz), 7.43 (m, 2H, Bz), 5.94 (ddd, IH, J6.4, 10.6, 17.0 Hz, H-4), 5.41 (ddd, IH, J 1.3, 1.3, 17.2 Hz, H-5_trans), 5.31 (ddd, IH, J 1.3, 1.3, 10.4 Hz, H-5_cis), 4.57 (dd, J 3.7, 11.9 Hz, H-la), 4.42 (dd, IH, J 7.7, 11.7 Hz, H-lb), 4.28 (m, IH, H-3), 3.80 (ddd, IH, J 3.5, 5.3, 7.7 Hz, H-2). 1H NMR (CDC1₃, 600 MHz) for 21: 58.05 (m, 2H, Bz), 7.57 (m, IH, Bz), 7.44 (m, 2H, Bz), 5.97 (ddd, IH, J 7.0, 10.6, 17.4 Hz, H-4), 5.66 (m, IH, H-3), 5.48 (ddd, IH, J 1.1, 1.1, 17.2 Hz, H- 5_trans), 5.31 (ddd, IH, J 0.9, 0.9, 10.6 Hz, H-5_cis), 3.83 (ddd, IH, J4.4, 7.1, 9.2 Hz, H-2), 3.78 (dd, J3.7, 11.9 Hz, H-la), 4.42 (dd, IH, J7.7, 11.7 Hz, H-lb),

0-(β-D-galactopyranosyl)-(l-»4)-0-(β-D-glucopyranosyl)-(l→l)-(2S,3R)- 2-azido-pent-4-ene-l,3-diol (26): Perbenzoylated lactosyl bromide 23 (13.7 g, 11.6 mmol) and selectively protected azidosphingosine 21 (3.0 g, 12.1 mmol) was dissolved in anhydrous toluene (100 ml), M.S. 4 A (6.0 g) was added, the mixture was stirred for 10 min and cooled to -78° C, AgOTf (4.5 g, 17.4 mmol) was added, and the reaction mixture was allowed to warm up to 20° C within 2 hrs. The reaction mixture was diluted with more toluene (200 ml), the insoluble material was filtered off and washed with more toluene (50 ml). The organic phase was washing with a 10% NH₃.H O aqueous solution (2 x 100 ml), and evaporated to afford a syrup. Anhydrous MeOH (300 ml) was added under a anhydrous argon atmosphere, and a solution of NaOMe in MeOH (~1.5 M, 3 ml) was added, the mixture was stirred at 60° C until T LC i ndicates a complete d ebenzoylation. T he r eaction w as c ooled t o room temperature and the mixture was neutralized with Dowex 50W (H ) resin. After the removal of MeOH, the residue was triturated with acetone to afford the desired 26 as a brownish solid. NMR revealed the crude material contains a single glycoside 26, which is contaminated with a trace amount of impurities and is used without purification. A pure sample of 26 is obtained by reverse-phase column chromatography on C18 using a 0 - 5% gradient of CH₃CN-H₂O as eluent. 1H NMR (D₂O, 600 MHz): 55.93 (ddd, IH, J6.8, 10.5, 17.3 Hz, H-4_pent), 5.39 (d'f, IH, J< 1, 17.2 Hz, H-5_trans_pent), 5.34 (d'f, IH, J < 1, 10.5 Hz, H-5_cis_pent), 4.51(d, IH, J 8.0 Hz, H-l_glc), 4.46 (d, IH, J 7.9 Hz, H-l_gal), 4.35 (m, IH, H_3_pent), 3.98 (dd, IH, J 2.2, 12.4 Hz, H-6a_glc), 3.84-3.94 (m, 4H, H-4_gal+H-2_pent+H- la_pent+H-lb_pent), 3.71-3.84 (m, 4H, H-5_gal+H-6a_gal+H-6b_gal+H-6b _glc), 3.63-3.69 (m, 3H, H-3_glc+H-4_glc+H-3_gal), 3.60 (ddd, IH, J2.1, 5.1, 12.4 Hz, H- 5_glc), 3.55 (dd, IH, J7.9, 10.0 Hz, H-2_gal), 3.36 ('f high order, IH, H-2_glc).

0-(β-D-galactopyranosyl)-(l- 4)--0-(β-D-glucopyranosyl)-(l→l)-(2S,3R)-

2-octadecyIamido-pent-4-ene-l,3-diol (29): The fully deprotected disaccharide 26 (15 mg, 0.032 mmol) was dissolved in a mixture of pyridine-H₂O-Et₃N (10 ml/1 ml/0.2 ml), and H₂S gas was bubbled into the solution overnight. The solution was evaporated to dryness. The residue was dissolved in pyridine (5 ml), and activated ester 28 (37 mg, 0.097 mmol) was added, the mixture was stirred for 2 hours at 60° C. The reaction was cooled and the solvent was removed under reduced pressure. The residue was pre-purified on a C18 Sep-Pak and finally purified by HPLC on a reverse phase C8 column using a 0 - 80% gradient of MeOH-H₂O as eluent to afford compound 29 which was lyophilized (19.8 mg, 87% yield from 26). 1H NMR (CD₃OD, 500 MHz): 55.84 (ddd, IH, J6.7, 10.4, 17.1 Hz, H-4_pent), 5.25 (d'f, IH, J 1.5, 17.2 Hz, H-5_trans_pent), 5.11 (d'f, IH, J< 1.5, 10.5 Hz, H-5_cis_pent), 4.33 (d, IH, J 7.6 Hz, H-l_gal), 4.28 (d, IH, J 7.8 Hz, H-l_glc), 4.12-4.17 (m, 2H, H_3_pent+H-la_pent), 4.02 (ddd, IH, J3.5, 4.8, 7.7 Hz, H-lb_pent), 3.90 (dd, IH, J 2.6, 12.1 Hz, H-6a_glc), 3.82 (dd, IH, J4.3, 12.1 Hz, H-6a_glc), 3.81 (dd, IH, J<1, 3.3 Hz, H-4_gal), 3.76 (dd, IH, J 7.5, 11.5 Hz, H-6a_gal), 3.69 (dd, IH, J4.7, 11.4 Hz, H-6b_gal), 3.51-3.61 (m, 5H, H-2_gal+H-3_glc+H-4_glc+H-2_pent), 3.47 (dd, IH, J 3.3, 9.8 Hz, H-3_gal), 3.41 (m, IH, H-5_glc), 3.28 (dd, IH, J 8.0, 8.9 Hz, H- 2_glc), 2.18 (t, IH, J 7.4 Hz, COCH₂), 1.55-1.63 (m, 2H, COC_i7H₃₅), 1.22-1.43 (m, 28H, COC₁₇H₃₅), 0.89 (t, 3H, J6.8 Hz, COCι₇H₃₅).

0-(β-D-galactopyranosyl)-(l→4)-C.-(β-D-glucopyranosyl)-(l→l)-(2S,3R)- 8-amino-2-iV-octadecylamido-6-thia-octane-l,3-diol, acetic acid salt (30): A solution of alkene 29 (10 mg, 0.014 mmol) and cysteamine hydrochloride (10 mg, 0.86 mmol) in de-gazed methanol (0.7 ml) was irradiated under UN at wavelength 254 nm for 24 hours. The solution was evaporated and the residue was purified by HPLC on a reverse phase C8 column using a 0 — 80% gradient of 0.3% AcOH/MeOH-0.3% AcOH/H₂O as eluent to afford compound 30 as acetic acid salt which was lyophilized (11.2 mg, 94%). Selected 1H ΝMR (CD₃OD, 600 MHz): 54.34 (d, IH, J7.7 Hz, H-l_gal), 4.31 (d, IH, J7.9 Hz, H-l_glc), 4.17 (dd, IH, J4.4, 10.2 Hz, H-la_oct), 3.90 (dd, IH, J2.5, 12.2 Hz, H-6a_glc), 3.84 (dd, IH, J4.3, 12.1 Hz, H-6a_glc), 3.81 (br d, IH, J <1, 3.2 Hz, H-4_gal), 3.75-3.80 (m, 2H, H-6a_gal+H- 3_oct), 3.70 (dd, IH, J4.6, 11.5 Hz, H-6a_gal), 3.47 (dd, IH, J3.2, 9.7 Hz, H-3_gal), 3.42 (m, IH, H-5_glc), 3.27 ('f , IH, J 8.3 Hz, H-2_glc), 3.04 (br t, 2H, J 5.8 Hz, H- 8_oct), 2.71-2.79 (m, 3H, H-7+H-5a), 2.62 (m, IH, H-5b), 2.22 (t, J 7.4 Hz, IH, COCH₂), 1.81 (m, IH, H-3a), 1.68 (m, IH, H-3b), 1.57-1.65 (m, 2H, COC₁₇H₃₅), 1.24-1.40 (m, 28H, COC₁₇H₃₅), 0.89 (t, 3H, J7.1 Hz, COC₁₇H₃₅). References

1 For a review, see Koskinen, P.M. and Koskinen, A.M.P. (1998) Synthesis, 1075- 1091 and references therein.

2 Schmidt. R.R. and Zimmerman, P. (1986) Tetrahedron Lett. 27(4), 481-484.

3 Zimmerman, P. and Schmidt. R.R. (1988) Liebigs Ann. Chem., 663-667.

4 Newman, H. (1973) J. Am. Chem. Soc. 95, 4096.

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8 Boutin, R. H.; Rapoport, H. J. Org. Chem. 1986, 51, 5320.

9 Herald, P. Helv. Chim. Ada 1988, 71, 354.

10 Nimkar, S.; Menaldino, D.; Merrill, A. H; Liotta, D. Tetrahedron Lett. 1988, 29, 3037.

11 Gamer, P.; Park, J. M.; Malecki, E. J. Org. Chem. 1988, 53, 4395.

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13 Reist, E. J. and Christie, P. H. (1970) J. Org. Chem. 35, 4127.

14 Bernet, B. and Vasella, A. (1983) Tetrahedron Lett. 24, 5491.

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18 Hakomori, S. Sphingolipid Biochemistry. In Handbook of Lipid Research; Kanfer, J. N., Hakomori, S., Eds.; Plenum: New York, 1983; Vol. 3, p 1.

19 For a review, see Nankar, Y.D. and Schmidt. R.R. (2000) Chem. Soc. Rev. 29, 201-216.

20 For a review, see Danishefsky, S. J. and Allen, J. R. (2000) Angew. Chem. Int. Ed. Engl. 39(5), 836-863.

21 Helling, F., Shang, A., Calves, M., Zhang, S., Ren, S., Yu, R. K., Oettgen, H. F. and Livingston, P. O. (1994) Cancer Res. 54, 197-203. 22 Kawano, T., Cui, J., Yasuhiko Koezuka, Y., Isao Toura, I., Yoshikatsu Kaneko, Y., Kazuhiro Motoki, K., Ueno, H., Nakagawa, R., Sato, H., Kondo, E., Koseki, H. and Taniguchi, M.(1997) Science 278, 1626-1629

23 Prigozy, T. I ., Naidenko, O., Qasba, P., Elewaut, D., Brossay, L ., Khurana, A., Natori, T., Koezuka, Y., Kulkarni, A. and Kronenberg M. (2001) Science 291, 664-667

24 Moody, D. B., Reinhold, B. B., Guy, M. R., Beckman, E. M., Frederique, D. E., Furlong, S. T., Ye, S., Reinhold, V. N., Sieling, P. A., Modlin, R. L., Besra, G. S. and Porcelli, S. A. (1997) Science 278, 283-286.

25 Miyamoto, K, Miyake, S. and Yamamura, T (2001) Nature 413, 531 - 534.

26 Karlsson, K.-A., et al. 1992. in T.K. Korhonen, ed. "Molecular Recognition in Host Parasite Interactions", Plenum Press, New York, ppl 15-132.

27 Lingwood, C. A. (1996) Glycoconj. J. 13, 495-503.

28 Lloyd, K. O., Gordon, C. M., Thampoe, I. J., and Dibenedetto, C. (1992) Cancer Res. 52, 4948-4953.

29 Gracza, T., Hasenoehrl, T., Stahl, U. and Jaeger, V. (1991) Synthesis 1108-1118.

30 Josephson, S. and Bundle, D. R. (1980) J. C. S., Perkin 1297-301.

31 Yamada, K., Fujita, E. and Nishimura, S-I. (1998) Carbohydr. Res., 305, 443-461.

32 Leung, O., Douglas, S. P., Whitfield, D. M., Pang, H. Y. S. and Krepinsky, J. J. (1994) New J. Chem. 18, 349-363.

33 Hasegawa, A., Νagahama, T., Ohki, H. and Kiso, M. (1992) J. Carbohydr. Chem., 11(6), 699-714.

34 Castro-Palomino, J. C, Ritter, G, Fortunato, S. R., Reinhardt, S., Old, L. J. and Schmidt, R. R. (1997) Angew. Chem. Int. Ed. Engl. 36, 1998-2001.

35 Demchenko, AN. and Boons, G-J. (1999) Chem. Eur. J., 5(4), 1278-1283.

36 Payne, G. B., Deming, P. H. and Williams, P. H. (1961) J Org. Chem., 26, 659- 663.

37 Connon, S. J. and Blechert, S. (2003) Angew. Chem. Int. Ed., 42, 1900-1923.

All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

What is claimed is:

1. A compound of Formula I:

wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, substituted phosphoryl, peptidyl, and glycosyl.

2. A compound of Formula II:

3. A method of preparing a compound of Formula I

I

wherein R is hydrogen, the method comprising: a) blocking C-4 hydroxyl group of 1,3-O-diprotected butan-l,2,3,4-tetraol to form a 1,3, 4-O-triprotected butan-l,2,3,4-tetraol of formula III:

wherein Pg, Pg' and Pg" are independently protecting groups wherein Pg" can be differentially removed relative to Pg and Pg'; b) converting C-2 hydroxyl group of the 1,3,4-O-triprotected butan- 1,2,3,4- tetraol of formula III into an azido group of the opposite stereoconfiguration to form 2-azido- 1,3, 4-O-triprotected butan-l,3,4-triol of formula IN:

l₃

^^ ^{^} OPg" OPg'

IV

wherein Pg, Pg' and Pg" are as defined above; c) removing the Pg" group to form 2-azido- 1,3-O-diprotected butan- 1,3,4-triol of formula N:

V wherein Pg and Pg' are as defined above; d) allylatmg the C-4 hydroxyl group of the compound of formula N to form 4-O-allyl-l,3-O-diprotected butan-l,3,4-triol of formula VI:

VI

e) removing Pg and Pg' to form the compound of formula I wherein R is hydrogen.

4. The method of Claim 3 wherein Pg" is tert-butyldiphenylsilyl.

5. The method of Claim 3 wherein converting C-2 hydroxyl group of l,3,4-O-protected butan-l,2,3,4-tetraol into azido-group comprises: a) mesylating the C-2 hydroxyl group by adding methanesulfonyl chloride; and b) replacing the mesylated C-2 hydroxyl group with an azide group.

6. The method of Claim 3 wherein Pg and Pg' are combined to form a 1,3- O-benzylidene group.

7. A method of preparing a compound of Formula II

wherein R is hydrogen, the method comprising: a) mesylating 1,2-O-diprotected-α-D-glucopyranose to form 3,5,6-0- trimesylated-l,2-O-diprotected-α-D-glucopyranose of formula Nil:

VII wherein Pg and Pg' are independently protecting groups;

b) converting 5,6-position of 3,5,6-O-trimesylated-l,2-O- diprotected -α- D-glucopyranose of formula Nil to a double bond to form 5,6-dideoxy-3-O- mesylated-l,2-O-diprotected-α-D-;ey/o-hexose-5-ene of formula NIII:

VIII

wherein Pg and Pg' are as defined above; c) removing Pg and Pg' to form a compound of formula IX;

d) oxidizing C-l and C-2 hydroxyl groups of the compound of formula IX to form a compound of formula X;

e) reducing aldehyde functionalities of the compound of formula X to form a compound of formula XI;

f) blocking C-l and C-3 hydroxyl groups of the compound of formula XI to form (2R,3R)-l,3-O-diprotected-2-O-methansulfonyl-pent-4-ene-l,2,3-triol of formula XII:

OMs

OPg'" XII

wherein Pg' ' and Pg" ' are independently protecting groups; g) converting 2-O-methansulfonyl group of XII into an azido group of the opposite stereoconfiguration to form (2R,3R)-l,3-O-diprotected-2-azido-pent-4-ene- 1,3-diol of formula XIII:

XIII wherein Pg" and Pg'" are as defined above; h) removing Pg' ' and Pg' ' ' to form the compound of Formula II wherein R is hydrogen.

8. The method of Claim 7 wherein Pg and Pg' are combined to form a 1,2- O-acetal group.

9. The method of Claim 7 wherein Pg' ' and Pg' ' ' are acetyl groups.

10. The method of Claim 7 wherein converting 5,6-position of 3,5,6-0- trimesylated-l,2-O-diprotected -α-D-glucopyranose of formula Nil to a double bond to form 5,6-dideoxy-3-O-mesylated-l,2-O-diprotected-a-D-^_y/o-hexose-5-ene of formula NIII comprises heating 3,5,6-O-trimesylated-l,2-O-diprotected -α-D- glucopyranose of formula Nil in Ν,Ν-dimethylformamide with potassium iodide at 90°C for 10 hours.

11. A compound of Formula XIV:

wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, substituted phosphoryl, peptidyl, and glycosyl; and

R' is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, and substituted phosphoryl.

12. A method of preparing a compound of Formula XIN

XIV

R' is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, and substituted phosphoryl; the method comprising: a) providing a compound of Formula I:

wherein R is hydrogen; b) selectively protecting a secondary hydroxyl group of the compound of Formula I to form a compound of Formula XV:

XV

c) esterifying a primary hydroxyl group of the compound of Formula XV to form a compound of Formula XVI:

XVI wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, substituted phosphoryl, peptidyl, and glycosyl; and d) reducing an azido group of the compound of Formula XVI into an amino group to form a compound of Formula XVII:

e) esterifying the amino group of the compound of Formula XVII to form a compound of Formula X-IV.

13. A compound of Formula XVIII:

14. A method of preparing a compound of Formula XNIII:

R' is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, and substituted phosphoryl; the method comprising: a) providing a compound of Formula II:

wherein R is hydrogen; b) selectively protecting a secondary hydroxyl group of the compound of Formula II to form a compound of Formula XIX:

c) esterifying a primary hydroxyl group of the compound of Formula XIX to form a compound of Formula XX:

wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, substituted phosphoryl, peptidyl, and glycosyl; and d) reducing an azido group of the compound of Formula XX into an amino group to form a compound of Formula XXI:

e) esterifying the amino group of the compound of Formula XXI to form a compound of Formula XVIII.

15. A compound of Formula XXII:

R' is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, and substituted phosphoryl; and

X^" is selected from the group consisting of halides, sulfate, nitrate, carbonate, phosphate, alkylcarboxylates, arylcarboxylates, oxalate, alkylsulfonates, and arylsulfonates.

16. A method of preparing a conjugate of a compound of Formula XIN or Formula XNIII with a peptide or a protein, the method comprising: a) providing the compound of Formula XIV or of Formula XVIII:

R' is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, and substituted phosphoryl; b) activating an alkene group of the compound of Formula XIV or of Formula XVIII; c) reacting an activated alkene group of the compound of Formula XIV or of Formula XVIII with the peptide or protein to form the conjugate of the compound of Formula XIV or Formula XVIII with the peptide or protein.

17. The method of Claim 16 wherein the alkene group of the compound of Formula XIV or of Formula XVIII in step b) is activated by a method selected from the group consisting of epoxidation, ozonolysis, and addition of thiol, alkylthiol, substituted alkylthiol, arylthiol, or substituted arylthiol by photochemical activation or radical activation.

18. A conjugate of Formula XIX :

XIX wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, substituted phosphoryl, peptidyl, and glycosyl;

R' is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl, substituted acyl, thioacyl, substituted thioacyl, phosphoryl, and substituted phosphoryl,

L is a linker; and

P.C. is a protein or peptide.

19. An anti-cancer vaccine comprising the conjugate of Claim 18 and a pharmaceutically acceptable adjuvant.

20. A method of preventing or ameliorating cancer, the method comprising administering to a mammal an immunogenic effective amount of an anti-cancer vaccine comprising the conjugate of Claim 18 and a pharmaceutically acceptable adjuvant.

21. A method of preparing stereoisomers of sphingosine, azidosphingosine, ceramides, lactosyl ceramides, glycosyl phytosphingosine, enantiomeric derivatives of phytosphingosine or its homologues, the method comprising: a) providing a compound of Formula I or of Formula II:

I II wherein R is hydrogen; b) activating an alkene group of the compound of Formula I or of Formula II; c) reacting an activated alkene group with C_nH_2n+ιX, wherein X is selected from the group consisting of halogen, hydroxyl, thiol, amino, MgBr, LiCu, Li; andn=l-25; d) reducing an azido group into an amino group; and e) acylating the amino group to form the stereoisomers of sphingosine, azidosphingosine, ceramides, lactosyl ceramides, glycosyl phytosphingosine, enantiomeric derivatives of phytosphingosine and or its homologues.

22. A method of preparing a compound of Formula XX or Formula XXI:

XXI wherein R\ and R₂ are selected from the group consisting of alkyl, substituted alkyl, alkylsilyl, substituted alkylsilyl, acyl;

R₃ is selected from the group consisting of hydrogen, acyl, alkyl, subsitituted alkyl;

R4 is selected from the group consisting of alkyl, substituted alkyl;

R₅ is selected from the group consisting of alkyl, substituted alkyl, alkylsilyl, substituted alkylsilyl, acyl, derivatized sialic acid unit; the method comprising: a) providing a compound of Formula XXII or Formula XXIII:

XXIII wherein Ri is defined as above; b) reacting the compound of Formula XXII or Formula XXIII with a compound of Formula XXIV;

XXIV wherein R₂, R₃, R₄ and R₅ are defined as above; and

X is selected from the group consisting of halogen, alkylsulfide, substituted alkyl sulfide, aryl sulfide, substituted aryl sulfide, alkyl phosphite, substituted alkyl phosphite, aryl phosphite, substituted aryl phosphite, alkyl phosphate, substituted alkyl phosphate, aryl phosphate, substituted aryl phosphate; to form the compound of Formula XX or Formula XXI.

23. A method of preparing a compound of Formula XXV or Formula XXVI

wherein Ri, R₂, Re, R₇, R₈ and R₉ is selected from the group consisting of alkyl, substituted alkyl, alkylsilyl, substituted alkylsilyl, acyl;

R₃ is selected from the group consisting of hydrogen, acyl, alkyl, subsitituted alkyl; ι is selected from the group consisting of alkyl, substituted alkyl;

R₅ is selected from the group consisting of alkyl, substituted alkyl, alkylsilyl, substituted alkylsilyl, acyl, derivatized sialic acid unit;

the method comprising: reacting derivatives of Formula XX and Formula XXI with a derivative of Formula

XXVII:

XXVII wherein X is selected from the group consisting of halogen, trichloroacetimidate, alkylsulfide, substituted alkyl sulfides, aryl sulfide, substituted aryl sulfide, alkyl phosphite, substituted alkyl phosphite, aryl phosphite, substituted aryl phosphite, alkyl phosphate, substituted alkyl phosphate, aryl phosphate, substituted aryl phosphate; and

Re, R , Rg and R₉ are defined as above; to form the compound of Formula XXV or Formula XXVI.

24. Crossed metathesis reaction with compounds of formula XXVIII

R HO.

OH XXVIII wherein R is selected from the group of amine, amide, azide, substituted amine, substituted amide with an alkene of formula XXIX

^C_nH_2n+ . XXIX where n is from 2 to 25 to produce compounds of formula XXX

XXX wherein R is selected from the group of amine, amide, azide, substituted amine, substituted amide.

Reduction of the double bond of compounds of formula XXX yields compounds of formula XXXI

XXXI wherein R is selected from the group of amine, amide, azide, substituted amine, substituted amide.

25. Epoxidation of compounds of formula XXXII

R. ^R ₂θ^Λ^^

OH XXXII wherein R is selected from the group of amine, amide, azide, substituted amine, substituted amide and R₂ is selected from the group of hydrogen, glycosyl residues, phoshoryl, substituted phosphoryl leading to compounds of formula XXXIII

XXXIII wherein R is selected from the group of amine, amide, azide, substituted amine, substituted amide and R₂ is selected from the group of hydrogen, glycosyl residues, phoshoryl, substituted phosphoryl .

Opening of the epoxide of formula XXXIII with a Grignard type reagent to yield compounds of formula XXXIV

xxxrv wherein Ri is selected from the group of amine, amide, azide, substituted amine, substituted amide and R₂ is selected from the group of hydrogen, glycosyl residues, phoshoryl, substituted phosphoryl , and n is from 2 to 25.