AU771708B2 - Protecting groups for carbohydrate synthesis - Google Patents

Description

WO 00/42057 PCT/AU00/00025 1 PROTECTING GROUPS FOR CARBOHYDRATE SYNTHESIS This invention relates to methods of synthesis of glycoconjugates, and in particular to orthogonally protected carbohydrate building blocks. The invention provides collections of orthogonally protected monosaccharides as universal building blocks for the synthesis of glycoconjugates of non-carbohydrate molecules, neo-glycoconjugates and oligosaccharides. This orthogonal protection strategy allows for the specific deprotection of any substituent on the saccharide ring, and greatly facilitates targeted or library-focused carbohydrate related syntheses.

BACKGROUND OF THE INVENTION Oligosaccharides are important components of a variety of different types of biological molecules, and are involved in antigenic recognition and cell-cell interactions. In many cases, bio-molecules require conjugation with a carbohydrate component in order to be fully functional. In order to enable investigation of the biological function, and to exploit the exquisite biochemical and antigenic specificity of oligosaccharides, it is essential to have access to highly defined, specific synthetic oligosaccharides. Therefore achieving efficient, cost-effective synthesis of oligosaccharides and glycoconjugates by either solution or solid phase methods is of the utmost importance.

This task is enormously complicated by the complexity of oligosaccharides. Because of the number of sites which can carry substituents, and the number of possible ways in which two saccharide molecules can be linked, the number of permutations is enormously high.

In naturally-occurring oligosaccharides Dglucose, D-galactose L-fucose, D-mannose, D-glucosamine and D-galactosamine are among the most common sugar residues.

To construct oligosaccharides and carbohydrate conjugates PCT/AU00/00025 Received 27 February 2001 2 using these sugars, current methodologies require long, protracted syntheses, involving synthesis of as many as one hundred different specially-protected sugar donors in order to cover adequately all the possible permutations of glycosidic link formation (eg. 1-3, link type (eg.

aor P) and to include all possible branching points in the oligosaccharide.

Orthogonal protection of bi-functional molecules has been a widely used technique in organic chemistry, which provided general building blocks for selected syntheses. However, orthogonal protection in the case of molecules with a greater degree of functionalisation is quite rare. Our technology involves penta-functional monosaccharide building blocks, which require a much higher level of chemical specificity to attain the appropriate orthogonality.

Orthogonal protection has been defined by Merrifield as follows: "The principle of orthogonal stability requires that only those protecting functions should be used that can be cleaved under different reaction conditions without affecting the other functions present" (Merrifield, 1977).

Orthogonal protecting strategies and conditions are reviewed in the textbook "Protecting Groups in Organic Synthesis" by Green and Wicks 3 rd edition).

Although the use of orthogonal protection would greatly facilitate carbohydrate-related synthesis, there has been limited success in devising suitable protecting groups and methods.

Wong et al. synthesised a universal building block with chloroacetyl, p-methoxybenzyl, levulinyl and tert-butyldiphenylsilyl protecting groups, selectively removable with sodium bicarbonate, trifluoroacetic acid, hydrazine and hydrogen fluoride-pyridine respectively, on a galactopyranose ring with an aryl-thio leaving group at the glycosidic position. This building block was used solely to synthesise a 6-hexanate glycoside. The subsequent recombinant oligosaccharide library formation focused on using the 6-hexanate derivatised building block which \\melb_files\homeS\WendyS\Keep\species\PCT-AUOO-00025 Alchemia.doc 26/02/01 AMENDED SHEET

PRAIAU

WO 00/42057 PCT/AU00/00025 3 exhibits only four degrees of orthogonality (Wong et al, 1998).

Similarly Kunz and coworkers synthesised an orthogonally protected D-glucopyranose derivative, but synthetic manipulations were only performed on the aglycon.

These authors describe orthogonal protection of hydroxyl groups on a monosaccharide linked at C1 via a thioglycoside group to a solid support or to a succinimide moiety. In this case the protecting groups are acetyl or methyl at C2, allyl at C3, ethoxyethyl at C4, and tert-butyldiphenylsilyl at C6. The thioglycoside anchor functionalized in the side-chain is stated to be crucial. Again there is no suggestion that this protection system can be used for substituted sugars. Kunz's orthogonally-protected building block was not used for glycosylation or construction of glycoconjugates or neo-glycoconjugates, by directly attaching functionalitites to the pyranose ring (Wunberg et al. 1998).

In our earlier International Patent Applications No. PCT/AU97/00544, No. PCT/AU98/00131 and No. PCT/AU98/00808, we described protecting and linking groups which enabled oligosaccharides and aminooligosaccharides to be synthesised using solid phase methods of the type which for many years have been used in peptide synthesis. In addition the protecting groups, described therein were useful for solution-phase synthesis.

The entire disclosures of these specifications are incorporated herein by this reference.

We have now devised new types of building blocks which greatly facilitate the synthesis of oligosaccharides and glycoconjugates, using orthogonally-protected saccharide building blocks with five degrees of othogonality. These building blocks contain a leaving group or latent leaving group at the glycosidic position, and another four orthogonally-protected functional groups around the carbohydrate ring.

4 Using our approach with six universal building blocks based on six of the most common naturally occurring sugars, any one of the one hundred sugars referred to above may be quickly synthesised in a facile manner, using simple, well-known protecting group chemistry. The years of work and complex protection strategies required to produce these one hundred building blocks by previously-available methods can be avoided by use of our six universal building blocks, which do not require a high level of skill to use, and enable one to achieve the synthesis of a specific desired oligosaccharide or glycoconjugate much faster and more efficiently than previously possible.

SUMMARY OF THE INVENTION In its most general aspect the invention provides a universal monosaccharide building block of General Formula I or General Formula II

DX

4 0 A

EX

4 0 A 2 0 C2 XB DX 3

X

I

B

XC2 in which I H A is a leaving group selected from the group S' 25 consisting of -SR; where R is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl; halogen excluding chlorine; trichloroacetimidoyl-; sulphoxide; and -O-alkenyl; XI, X 2 and X 3 are independently selected from H, O, N, NH or N 3 with the proviso that only one of X 1

X

2 and X 3 may be H, N or N 3 in any molecule;

X

4 is H, -CH 2 O, -CH 2 N, -CH 2 NH-, -CH 3

-CH

2

N

3 or COO- with the proviso that X 4 may only be H, -CH 2 N, -CH 3 35 or -CH 2

N

3 when none of X 1 to X 3 is H; and B, C, D and E are different, and are selected from protecting groups which can be cleaved orthogonally H:\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 5 in any order such that the cleavage conditions do not compromise the stability of the other protecting or functional groups on the monosaccharide building block, and in which B or C or D or E is absent if the corresponding

X

1 to X 3 is H or N 3 or if the corresponding X 4 is H, -CH 3 or -CH 2

N

3 The following non-limiting sets have been designated as orthogonal to each other on the basis of their cleavage conditions. A protecting group is classified in a particular set according to its lability to the cleavage conditions for a particular set and its stability to the cleavage conditions required for the removal of those groups in the remaining sets. Each set is to be taken to include, but is not be limited, by the members thereof.

Of the sets defined, set 1, the 'Base Solvolysis' set, is of particular importance, because in addition to the fact that the members of this set are considered to be orthogonal to the members of the remaining sets, some members of this set are also considered to be orthogonal to each other. Where this is the case, the alternative condition of cleavage that provides orthogonality is specified in brackets following the listing of the S' 25 protecting group.

1. Base Solvolysis a) for hydroxy protection: o acyl-type protecting groups, eg. chloroacetate (also thiourea-sensitive) :bromoacetate (also pyridine-sensitive) carbonates, eg. Alloc (Pd°) S. Fmoc (p-elimination) 35 Troc p-nitrophenylsulphonylethyloxy carbonyl) levanoyl (also hydrazine sensitive) 0 H:\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 WO 00/42057 PTAO/02 PCT/AUOO/00025 -6b) for amino protection: Dde, Wow (primary amine-sensitive) tetraphthaloyl dichlorophthaloyl 2, 5-dimethyl-pyrroyl (primary amine-sensitive) benzyloxycarbonyl pentenyl 2. Fluoride Ion-Sensitive for hydroxy protection: t-butyldiphenylsilyl triisopropylsilyl trimethylsilylethyl triphenylsilylethyl (all cleavable with HF/Pyridine) 3. Reduction-Sensitive tri fluoromethyl trichloromethyloxymethyl trichloromethyloxycarbonate (all cleavable with zinc/acetic acid) 4. P-Elimi at ion- Sensitive, Base-Labile Protecting Groups ethoxyethyl cyanoethyl NSC (p -nit robenzyl -sulphonyl ethyl oxycarbonyl) p-nitrobenzyl-sulphonylethyl Hydrogenolysis -Sensitive Protecting Groups naphthylmethyl substituted naphthylmethyl PCT/AUOO/00025 Received 01 November 2000 -7- 6. Oxidation-Sensitive Protecting Groups: p-rnethoxybenzyl 3, 4-dimethoxybenzyl 2, 4,6-trimethoxybenzy.

3, 4-methylenedioxybenzyl acyl ami doben zyl az idobenzyl p-az ido-rn-chlorobenzyl 7. Allylic Protecting Groups Cleavable with Pd 0 complexes 8. Photolabile Protecting Groups: 0-ni trobenzyloxycarbonate o-nitrobenzyl dinitrobenzyl 2-oxo-l, 2-diphenylethyl 9. Protecting Groups Removable by Relay Deprotection methylthioethyl acyloxybenzyl benzylthioethyl.

In one preferred embodiment, the invention provides a compound of General Formula III

E

1

X

4 0 A

DIX

3

XIBI

X

2

C

1

III

\\melbfi les\home$ \WeldyS\Keep\ species\ FP- 1217 8 PCT Aichemiadoc 31/10/00 AMEZ"':jA, H'ME 8 in which A, X 1

X

2

X

3 and X 4 are as defined for General Formulae I and II, and

B

1

C

1 Di and El are orthogonal protecting groups (ie. an orthogonal set) selected from protecting group sets 1, 2, 6 and 8 as herein defined.

Another preferred embodiment provides a compound of General Formula IV

E

2 X4 O A

D

2

X

3

XIB

2 X2C2

IV

in which A, X 1

X

2

X

3 and X 4 are as defined for General Formulae I and II, and

B

2

C

2

D

2 and E 2 are selected from the members of protecting group set 1 as herein defined, the members of set 1 being orthogonal to each other, for example the carbohydrate-protecting groups levanoyl (ammonia-labile), chloroacetate (thiourea-labile), p-methoxybenzyloxycarbonyl (oxidation-labile) and 2-trimethylsilylethylcarbonate 25 (fluoride ion-labile).

This embodiment provides universal building blocks with protecting groups selected from the protecting groups of set 1.

In a third preferred embodiment the invention provides a compound of General Formula V

E

3

X

4 0 A

D

3

X

3

XIB

3 •X2C3 S V H:\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 9 in which A, X 1

X

2

X

3 and X 4 are as defined for General Formula I and II, and

B

3

C

3

D

3 and E 3 are orthogonal protecting groups selected from the members of set 1 as herein defined and from the remaining orthogonal sets 2 to 9 as herein defined.

This embodiment provides orthogonally protected building blocks, the protecting group constituents of which may be selected from within set 1 and from the remaining sets.

It will be clearly understood that the invention is not limited to use with monosaccharides, but is also applicable to any compound in which substituents are linked to a pyranose or furanose ring, such as sugar analogues.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

For the purposes of this specification "orthogonal cleavage" is defined as the regioselective cleavage of a hydroxy or amino protecting group from a carbohydrate, in which the cleavage conditions do not compromise the stability of the other protecting or 25 functional groups on the molecule. Such cleavages can be effected in any order of priority. "Cleaved orthogonally" and "orthogonal cleavage" are taken to be synonymous.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations used herein are as follows: Alloc Allyloxycarbonyl Bn Benzyl SBu Butyl S* 35 DCM Dichloromethane Dde N-l-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl H.\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 WO 00/42057 PCT/AU00/00025 10 Dde-OH

DMAP

DMF

DMTST

EEDQ

EtOAc EtOH

FAB-MS

HRMS

Fmoc

MBHA

Me MeOH

NCS

NMR

ODmab

PEG

tBu

TFA

THF

Troc 6-Hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl N,N'-Dimethylaminopyridine N,N'-Dimethylformamide Dimethyl(methylthio)sulphoniumtrifluoromethanesulphonate l-isobutyloxycarbonyl-2-isobutyloxy-1, 2-dihydroquinoline Ethyl acetate Ethanol Fast atom bombardment mass spectrometry High resolution mass spectrometry Fluoromethoxycarbonyl Methyl benzyhydryamine resin Methyl Methanol p-Nitrobenzyl-sulphonylethyloxycarbonyl Nuclear magnetic resonance 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)- 3-methylbutyl]-amino}benzyl alcohol Polyethylene glycol Tertiary-butyl Trifluoroacetic acid Tetrahydrofuran 2,2,2-Trichloroethoxycarbonyl The invention provides universal building blocks, which are useful in the solution and solid phase synthesis of oligosaccharides. The reaction scheme for synthesis of each target molecule is designed so as to specify the orthogonally-protected functional groups which must be freed for glycosylation, and those which need to be capped with a protecting group such as benzyl, benzoyl, or another such group which remains uncleaved until the end of the synthesis, in order to avoid competition during glycosylations later in the synthesis.

WO 00/42057 PCT/AU00/00025 11 When participation during the glycosylation reaction is required, the 2-hydroxyl is selectively deprotected and re-protected with a benzoyl group which, again, remains until the completion of the synthesis. In the case of 2-deoxy 2-aminosugars, if participation or stereoselectivity is required the Dde group might be removed and replaced with a tetrachlorophthaloyl or dimethylpyrrole group.

Example 1 Synthesis of an Exemplary Tetrasaccharide A strategy for synthesis of the tetrasaccharide of formula VI is set out in Scheme 1.

OH OH

O

HO

NH

2

OH

o OOH

HO

OH OH WO 00/42057 PCT/AUOO/00025 12 OB OA co

OL

P)]

coj L [2] A-D=Orthogonal 1-ydroxy Protecting Groups DN=Orhogonal Amino Protecting Group P=Pernianent Protecting Group (Benzoyl) L--Activating Group OB OA 3 L

OD

1. Deprotect A 2. Benzoyiate 0 CO&

GD

OD

1. Remove A, C OP.O 2 Benzoyiate 1 DHrtc B 3.Giycasylate 1 ertc GB OP

GD

1. Deprotect C 1.Deprotect A WO 00/42057 PCT/AU00/00025 13 In solution phase, protecting groups A and C from the first sugar residue of the target molecule (residue are selectively removed, and the sites capped by a permanent protecting group, eg. benzoyl group. The residue is then coupled to the resin, followed by selective removal of protecting group B. In solution phase, protecting group A from sugar residue is selectively removed, and the site is capped by a permanent protecting group. Residue is then linked to the resin-bound sugar residue via a glycosylation reaction. Protecting group C from the new disaccharide is removed, and residue is linked via a glycosylation. Protecting group A is finally selectively removed to regenerate the 6-hydroxyl group, which is linked with residue 1.

PCT/AUOO/00025 Received 0 1 November 2000 -14 Example 2 Synthesis of an Orthogonally Protected Thioglycoside Building Block, Methyl 6-O-(tbutyldiphenylsilyl) (p-chlorobenzoyl) -2deoxy-2- Cl- (4,4-dimethyl-2, 6-dioxocyclohex-1ylidene) ethylamino] -4-O-tetrahydropyranyl-lthio-f3-D glucopyranoside OH 0 0 HO O~N SMe 0 OSMe 0 S e 0NH NH 0 0: 0~ 0< OTBDPS OTBDPS

O

THPO HO-- H B NH PNH pCBzOX

SNH

000 43 Methyl 4, 6-O-benzylidene-2-deoxy-2- [1-(4,4-dimethyl- 2, 6dioxocyclohex-1-ylidene)ethylamino] -l-thio-p-D glucopyranoside (1) A mixture of methyl 2-deoxy-2-[l-(4,4-dimethyl- 2, 6-dioxocyclohex-l-ylidene)ethylamino] -1-thio-f3-D glucopyranoside (20 g, 54 mrmol) a- dime thoxyto luene (9.78 g, 64 minol) and p-toluenesulphonic acid (50 mg) in dry acetonitrile (100 mL), was stirred at 602C for 2 hours.

The reaction mixture was cooled to room temperature and adjusted to pH 7 with the addition of triethylamine. The solvent was removed in vacuo, the residue was taken up in

CH

2 C1 2 (200 ml) washed with brine (50 ml) with water \~ebfiles \home$S\WendyS \Keep\ species \FP- 1217 8 PCT Aichemiadoc 31/10/00 WO 00/42057 WO 0042057PCT/AUOO/00025 :15 ml) and dried over MgSO 4 The organic phase was concentrated to give a yellow solid, methyl 4, 6-O-benzylidene-2-deoxy-2- 4-dimethyl-2, 6dioxocyclohex-l-ylidene) ethylamino] -1-thio-3 -D glucopyranoside (24.5 g, 98%).

Methyl 4, 6-O-benzylidene-3-O- (p-chlorobenzoyl) -2-deoxy-2- 4-dimethyl-2, 6-dioxocyclohex-1-ylidene)ethylaminoJ -1thio-f3-D glucopyranoside (2) A mixture of methyl 4,6-O-benzylidene-2-deoxy-2-[l-(4,4dimethyl-2, 6-dioxocyclohex-l-ylidene) ethylamino] -l-thio-O D-glucopyranoside (6.3 g, 13.5 mmol), p-chlorobenzoylchloride (2.6 ml, 20 mmol) and 4-dimethylaminopyridine (2.44 g, 40 rrmol) in dry 1,2-dichioroethane (100 ml), was stirred at room temperature overnight. The resultant suspension was filtered, the filtrate diluted with chloroform (100 ml) and washed with diluted brine (3 x 50 ml, H 2 0/Brine, The organic phase was dried over MgSO 4 and the solvent removed in vacuo to give yellow solid. The residue chromatographed EtOAc/Hexane 1:1 as the mobile phase to give methyl 4,6-0benzylidene-3 (p-chlorobenzoyl) -2 -deoxy-2 4dimethyl 6 -dioxocyclohex-lI-yl idene) ethylamino]1 -1l-thio-3 D-glucopyranoside (6.4 g, Methyl 3-0- (p-chlorobenzoyl) -2-deoxy-2- [1-(4,4-dimethyl- 2, 6-dioxocyclohex-l-ylidene) ethylamino] -l-thio-3-D glucopyranoside (3) A mixture of methyl 4,6-0-benzylidene-3-O-(pchlorobenzoyl) -2-deoxy-2- 4-dimethyl-2, 6dioxocyclohex-l-ylidene) ethylainino] -1-thio-P -D WO 00/42057 WO 0042057PCT/AUOO/00025 -16 glucopyranoside (2.51 g, 4.20 mmol) and 50% aqueous solution of tetrafluoroboric acid (1 ml) in acetonitrile mL), was stirred at room temperature for 2 hours. The pH was adjusted to 7 with the addition of triethylamine and the resultant suspension concentrated. The residue was crystallised from diisopropyl ether-ethyl acetate to give methyl 3-O-(p-chlorobenzoyl) -2-deoxy-2- [l-(4,4-dimethyl- 2, 6-dioxocyclohex-1-ylidene) ethylamino] -l-thio-o-D glucopyranoside (1.7 79%).

Methyl 6-0- (t-butyldiphenylsilyl) (p-chlorobenzoyl) -2deoxy-2- [1-(4,4-dimethyl-2, 6-dioxocyclohex-1ylidene)ethylaminoJ -1-thio-3-D glucopyranoside (4) A mixture of methyl 3-O-(p-chlorobenzoyl)-2-deoxcy-2-[1- (4,4-dimethyl-2,6-dioxocyclohex-l--ylidene)-ethylamino] -1thio-P -D-glucopyranoside 00 g, 1. 95 mmrol) tbutyldiphenylsilylchloride (536 mg, 1.95) and 4-dimethylaminopyridine (238 mg, 1.95 mmol), in l,2-dichloroethane (30 mL), was stirred under ref lux for 6 hours. The reaction mixture was cooled to room temperature, diluted with chloroform (60 niL) and washed with diluted brine (3 x 50 mL, brine/water, dried over MgSO 4 The solvent was removed in vacuo and the residue was chromatographed using hexane EtOAc 1:1 as the mobile phase to give a white solid, methyl 6-O-(tbutyldiphenylsilyl) -3-O-(p-chlorobenzoyl) -2-deoxy-2- Il- 4-dimethyl-2, 6-dioxocyclohex-l-ylidene)ethylaminol -1thio-p3-D-glucopyranoside (1.1 g, WO 00/42057 WO 0042057PCT/AUOO/00025 1.7 Methyl 6-0- (t-butyldiphenylsilyl) (p-chlorobenzoyl) -2deoxy-2- [1-(4,4-dimethyl-2, 6-dioxocyclohex-lylidene) ethylamino] -4-0-tetrabydropyranyl-1 -thio-fp-D glucopyranoside A mixture of methyl 6-O-(t-butyldiphenylsilyl)-3---(pchlorobenzoyl) -2-deoxy-2- 4-dimethyl-2,6dioxocyclohex-l-ylidene) ethylamino] -l-thio-13-Dglucopyranoside (500 mg, 0.6 mmol), 3,4-dihydro-2H-pyran rnL) and p-toluenesulphonic acid (5 mg) in dry acetonitrile (10 mL) was stirred at room temperature for 1 hour. The reaction mixture was adjusted to pH 7 with the addition of triethylamine and then evaporated to dryness.

The residue was taken up in dichioromethane (30 mL) washed with water (2 x 10 mL) and the organic phase dried over MgSO 4 The solvent was removed in vacuc and the residue was chromatographed using hexane EtOAc 2:1 as the mobile phase to give methyl 6-0- (t-butyldiphenylsilyl) chlorobenzoyl) -2-deoxy-2- [I-(4,4-dirnethy1-2, 6dioxocyclohex-1-ylidene) ethylamino] 1-thio-p-D-glucopyranoside (420 mg, WO 00/42057 PCT/AUOO/00025 18 Example 3 Synthesis of an Orthogonally Protected Thioglycoside Building Block, methyl 2azido-6-O- (t-butyldiphenylsilyl) -2-deoxy-3- 0-(4-methoxybenzyl)-4-O-biphenylcarbonyl-lthio-3-D glucopyranoside Ph SMe MeO SMe OTBDPS OTBDPS

OH

M o SMe MeOBnO SMe MeOBnO SMe 11 N 3 10 N 3 9 N 3 Methyl 2-azido-4, 6-O-benzylidene-2-deoxy--thio-3-D glucopyranoside (7) A mixture of methyl 2-azido-2-deoxy-l-thio-o-D glucopyranoside (10g, 4.25 mmcl), c,a-dimethoxytoluene (9.71 g, 64 rmol) and p-toluenesulphonic acid (50 ug) in dry acetonitrile (100 mL), was stirred at 609C for 2 hours.

The reaction mixture was cooled to room temperature and adjusted to pH 7 with the addition of triethylamine. The solvent was removed in vacuo. The residue was taken up in

CH

2 Cl 2 (200 mL) washed with brine (50 mL), with water (50 mL) and dried over MgSO. The organic phase was concentrated to give a white solid, methyl 2-azido-4,6-0benzylidene-2-deoxy-l-thio-j-D glucopyranoside (10.5 g, 73%) WO 00/42057 PCT/AU00/00025 19 Methyl 2-azido-4,6-0-benzylidene-2-deoxy-3-0-(4methoxybenzyl) -1-thio-P-D glucopyranoside (8) A suspension of sodium hydride (1.0 g, 41.8 mmol) in dry DMF (50 mL) was cooled to 0 OC, and a solution of methyl 2azido-4, 6-O-benzylidene-2-deoxy-l-thio-3-D glucopyranoside (9.0 g, 27.8 mmol) in dry DMF (50 mL) was added dropwise in 30 minutes. The resulting solution was stirred at 0 °C for 30 minutes and 4-methoxybenzyl chloride (6.54 g, 41.8 mmol) was added dropwise at 0 The reaction mixture was stirred at room temperature overnight, cooled to 0 °C and dry methanol (5 mL) was added dropwise. The reaction mixture was concentrated under reduced pressure, then xylene (50 mL) was co-evaporated from the residue. The residue was taken up in CHC13 (200 mL) washed with H 2 0 (400 ml), saturated NaHCO 3 solution (200 mL) dried over MgSO 4 and evaporated to dryness. The residue was crystallized from EtOH to give methyl 2-azido-4,6-O-benzylidene-2-deoxy- 3-0-(4-methoxybenzyl)-1-thio-P-D-glucopyranoside g, 73%) as white crystalline solid.

Methyl 2-azido-2-deoxy-3-O-(4-methoxybenzyl)-l-thio-3-Dglucopyranoside (9) A mixture of methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O- (4-methoxybenzyl)-l-thio-p-D glucopyranoside (12.0 g, 27.08 mmol) and p-toluenesulphonic acid (300 mg) in MeOH MeCN 1:1 (400 mL) was stirred at 50 C" for 1 hour. The reaction mixture was evaporated, the residue was chromatographed using CHC13 EtOAc gradient to give methyl 2-azido-2-deoxy-3-0-(4-methoxybenzyl)-1-thio-p-Dglucopyranoside (8.21 g, 88%).

WO 00/42057 PCT/AU00/00025 20 Methyl 2 -azido-6-O-tert-butyldiphenylsilyl-2-deoxy-3-O-(4methoxybenzyl)-l-thio-P-D glucopyranoside A mixture of t-butyldiphenylsilyl chloride (8.66 g, 31.53 mmol), 4-dimethylaminopyridine (5.12 g, 42.04 mmol) and methyl 2-azido-2-deoxy-3-0-(4-methoxybenzyl) -1-thio-p-Dglucopyranoside (7.21 g, 21.02 mmol) in dry 1,2dichloroethane (100 mL) was stirred at 800C for 2 hours.

The resulting clear solution was cooled to room temperature, diluted with CHC1 3 (300 mL), washed with H 2 0 (3 x 200 mL), brine solution (200 mL), dried over MgSO 4 and evaporated. The residue was purified by chromatography using hexane ether 2:1 as the mobile phase to give methyl 2 -azido-6-O-tert-butyldiphenylsilyl-2-deoxy-3-0-(4methoxybenzyl)-1-thio-P-D glucopyranoside (10) (9.73 g, Methyl 2-azido-6-O-tert-butyldiphenylsilyl-4-0biphenylcarbonyl-2-deoxy-3-O-(4-methoxybenzyl)-1-thio-P-D glucopyranoside (11) A mixture of methyl 2-azido-6-O-tert-butyldiphenylsilyl-2deoxy-3-O-(4-methoxybenzyl)-l-thio-P-D glucopyranoside (12.7 g, 21.46 mmol), 4-dimethylaminopyridine (5.23 g, 42.92 mmol) in dry 1,2-dichloroethane (100 mL) was stirred at room temperature. Biphenylcarbonyl chloride (6'.97 g, 32.19 mmol) was added to the stirred reaction mixture in minutes. After the addition the resulting suspension was stirred under reflux for 3 hours. The reaction mixture was cooled to 10°C and filtered. The crystalline solid was washed on the funnel with dry 1,2-dichloroethane (50 mL) and filtered. The filtrates were combined, diluted with CHC1 3 (200 mL) and washed twice with diluted brine solution (water-brine 2:1) (150 mL). The organic layer was dried over MgS04 and evaporated. The residue was crystallized from EtOH (75 mL) to give methyl 2-azido-6-O-tert- WO 00/42057 PCT/AUOO/00025 21 butyldiphenylsilyl-4--biphenylcarbonyl-deoxy3o. (4 rethoxybenzyl) -1-thio-13-D-glucopyranoside (11) (12.7 g, 76%) Example 4 Synthesis of an Orthogonally Protected Thioglycoside Building Block, methyl 2-azido-6-O-(tbutyldiphenylsilyl)-2-deoxy-3-0-(4-methoxybenzyl)-4-0biphenylcarbonyl-1-thio--D-galactopyranoside (17) OOHPh Ph 0 00~ SMe 0 0 12 N 3 H SMe MeOB Se 13 N 3 14 N 3 BPCO OT3DPS OHOTBDPS OH 0

O

Me0BrO SMe MeOB SMe MeOWO We 17 N 3 16 N 3 15 N 3 Methyl 2-azido-4, 6-O-benzylidene-2-deoxy-l-thio--D galactopyranoside (13) A mixture of methyl 2-azido-2-deoxy-l-thio-p-Dgalactopyranoside (12) (3.0 g, 12.76 nmuol), a,adimethoxytoluene (2.91 g, 19.14 mmol) and ptoluenesuiphonic acid (30 mg) in dry acetonitrile (15 mL), was stirred at 709C for 20 minutes. The reaction mixture was cooled to room temperature and adjusted to pH 7 with the addition of triethylamine. The solvent was removed in vacuo and the residue was taken up in CH 2 C1 2 (100 mL), washed with brine (50 rL), with water (50 mL) and dried over MgSO 4 The organic phase was concentrated to give a WO 00/42057 PCT/AU00/00025 22 white solid, methyl 2-azido-4,6-O-benzylidene-2-deoxy-lthio-3-D-galactopyranoside (13) (3.09 g, Methyl 2-azido-4,6-0-benzylidene-2-deoxy-3-O-(4methoxybenzyl)-1-thio-P-D-galactopyranoside (14) A suspension of sodium hydride (123 mg, 4.87 mmol) in dry DMF (10 mL) was cooled to 0 OC, and a solution of methyl 2azido-4,6-0-benzylidene-2-deoxy-l-thio--Dgalactopyranoside (13) (1.05 g, 3.25 mmol) in dry DMF mL) was added dropwise in 30 minutes. The resulting solution was stirred at 0 OC for 30 minutes and 4methoxybenzyl chloride (763 mg, 4.87 mmol) was added dropwise at 0 The reaction mixture was stirred at room temperature overnight, cooled to 0 °C and dry methanol (2 mL) was added dropwise. The reaction mixture was concentrated under reduced pressure, then xylene (25 mL) was co-evaporated from the residue. The residue was taken up in CHC13 (50 mL) washed with H 2 0 (40 ml), saturated NaHCO 3 solution (50 mL) dried over MgSO 4 and evaporated to dryness. The residue was crystallized from EtOH (10 mL)to give methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(4methoxybenzyl)-1-thio--D-galactopyranoside (14) (1.0 g, as white crystalline solid.

Methyl 2-azido-2-deoxy-3-0-(4-methoxybenzyl)-1-thio-P-Dgalactopyranoside A mixture of methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-0- (4-methoxybenzyl)-1-thio-P-D-galactopyranoside (14) (500 mg, 1.12 mmol) and p-toluenesulphonic acid (10 mg) in MeOH MeCN 1:1 (50 mL) was stirred at 50 Co for 1 hour. The reaction mixture was evaporated, the residue was WO 00/42057 WO 0042057PCT/AUOO/00025 23 chromatographed using CHC13 EtOAc gradient to give methyl 2 -azido-2-deoxy--3-o- (4-methoxybenzyl) -1-thio-P-Dgalactopyranoside (15) (309 mg, Methyl 2-azido-6-O-tert-butydiphenysiyl-2-deoxy3..o-(4methoxybenzyl) -1-thio-P-D-galactopyranoside (16) A mixture of t-butyldiphenylsilyl chloride (151 mg, 0.54 mmol), 4 -dimethylaminopyri dine (90 mg, 0.73 mmol) and methyl 2-azido-2-deoxy-3-O- (4-methoxybenzyl) -l-thio-o- D-galactopyranoside (15) (130 mg, 0.36 mmol) in dry 1,2dichioroethane (8 ML) was stirred at 8000 for 2 hours. The resulting clear solution was cooled to room temperature, diluted with CHC1 3 (20 mL) washed with H 2 0 (3 x 20 mL) brine solution (20 mL), dried over MgSO 4 and evaporated.

The residue was purified by chromatography using hexane ether 2:1 as the mobile phase to give methyl 2-azido-6-Otert-butyldiphenylsilyl-2 -deoxy-3 (4-methoxybenzyl) -1thio-1 3 -D-galactopyranoside (16) (142 mg, 68%).

Methyl 2-azido-6 -O-tert-butyldiphenylsilyl-4 -0biphenylcarbonyl-2-deoxy-3-O- (4-methoxybenzyl) -1-thio-P3-D.

galactopyranoside (17) A mixture of methyl 2-azido-6-O-tert-butyldiphenylsilyl-2deoxy-3-O- (4-methoxybenzyl) -l-thio-p 3 -D-galactopyranoside (16) (213 mg, 0. 36 mmol) 4 -dime thyl aminopyri dine (67 mg, 0.55 mmol) in dry l,2-dichloroethane (10 mL) was stirred at room temperature. Biphenylcarbonyl chloride (119 mg, 0.55 mmol) was added to the stirred reaction mixture. The resulting suspension was stirred under ref lux for 3 hours.

The reaction mixture was cooled to 1000 and filtered. The crystalline solid was washed on the funnel with dry l,2-dichloroethane (5 mL) and filtered. The filtrates were PCT/AUOO/00025 Received 27 February 2001 24 combined, diluted with CHC1 3 (20 mL) and washed twice with diluted brine solution (water-brine 2:1) (15 mL). The organic layer was dried over MgSO 4 and evaporated. The residue was purified by chromatography using hexane CHC1 3 1:1 as the mobile phase to give methyl 2-azido-6-O-tertbutyldiphenylsilyl4o-biphenylcarbonyl2deoxy3Q (4methoxybenzyl) -l-thio-f-D-galactopyranoside (17) (180 mg, Example 5 Synthesis of an orthogonally Protected Thioglycoside Building Block, Methyl 6-O-(tbutyldiphenylsilyl) -2-deoxy-2- dimethyl-2,4, 6(lH, 311,51)-trioxopyrimidin-5ylidene)methylaninoj (4-methoxybenzyl) 4-O-biphenylcarbonyl-1-thio-3-D glucopyranoside (24) HO-o-4t. SMe N H

H

3 C 0 0 18

OTBDPS

MeOBno-.& S00e H 0 0 N- CH 3

H

3 C 0 23 SMe SMe \meb~ ile \om$ WendyS \Keep\ species\ PCT-AUOO-0002 Alcheznia.doc 21/02/01 AMENDED SHEET

IPMNAU

WO 00/42057 PCT/AU00/00025 25 Methyl 2-deoxy-2-[(1,3-dimethyl-2,4,6(1H,3H,5H)trioxopyrimidin-5-ylidene)methylamino]-l-thio-3-Dglucopyranoside (19) To methyl 2-deoxy-2-[1-(4,4-dimethyl-2,6-dioxocyclohex-lylidene)-ethylamino]-l-thio-p-D-glucopyranoside (18)(100 g, 268 mmol) was added conc. ammonia solution (300 mL) and the reaction mixture was stirred at 100 C° for 1 hour. The suspension was cooled to room temperature and filtered. The filtrate was washed with CHC1 3 (3x200 mL), then the aqueous phase was evaporated under reduced pressure. The residue was taken up in EtOH benzene 1:1 (250 mL) and evaporated to dryness.

The residue was taken up in hot MeOH (600 mL) and 1, 3dimethyl-5-[(dimethylamino)methylene]2, 4, 6 (1H, 3H, trioxopyrimidine (Wow-reagent) (62.27 g, 294.9 mmol) in hot MeOH (120 mL) was added. /Synthesis of 1, [(dimethylamino)methylene]2, 4, 6 (1H, 3H, trioxopyrimidine (Wow-reagent): N, N-Dimethylformamide dimethyl acetal (252 g, 2.11 mol) was stirred at 0°C in CHC1 3 (750 mL). 1, 3-Dimethylbarbituric acid (300 g, 1.92 mol) in CHC13 (2100 mL) was added to the stirring acetal solution over 2 hours. The CHC13 was evaporated immediately following complete addition and the resulting residue resuspended in CHC13 (2000 mL) and washed with water (3x600 mL) and saturated brine solution (600 mL). The organic phase was dried over MgS0 4 filtered and evaporated to dryness under high vacuum. The residue was re-suspended in diethyl ether (750 mL), filtered and washed on the funnel with additional diethyl ether (500 mL) to yield 1, 3- Dimethyl-5-[(dimethylamino)methylene]2, 4, 6 (1H, 3H, trioxopyrimidine as a pale-yellow solid (271.85 g, The reaction mixture was stirred under reflux for minutes, then cooled to room temperature. The resulting suspension was filtered, the solid was washed with MeOH (150 mL), ether (150 mL), dried to give methyl 2-deoxy-2- 3 -dimethyl-2,4,6(1H,3H,5H)-trioxopyrimidin-5- WO 00/42057 PCT/AUOO/00025 26 ylidene)methylamino] -l-thio-f-D-glucopyranoside (19) (83 g, Methyl 4,6-O-benzylidene-2-deoxy-2-[(1,3-dimethyl- 2,4,6 (1H, 3H, 5H) -trioxopyrimidin-5-ylidene)methylamino] -1thio-P-D-glucopyranoside A mixture of methyl 2-deoxy-2-[(1,3-dimethyl- 2,4,6(1H,3H,5H)-trioxopyrimidin-5-ylidee)methylamino]-1thio-p-D-glucopyranoside (19) (84.64 g, 226.31 nmol), c,adimethoxytoluene (51.66 g, 339.46 mmcl) and ptoluenesuiphonic acid (500 mg) in dry acetonitrile (600 mL), was stirred at 609C for 2 hours. The reaction mixture was cooled to room temperature and filtered. The solid was washed with ether (200 mL), dried to give methyl 4,6-0-benzylidene-2-deoxy-[(1,3-dimethyl glucopyranoside (20) (80 g, 77%) Methyl 4, 6-O-benzylidene-2-deoxy-2- 3-dimethyl- 2,4,6(1H,3H,5H)-trioxopyrimidin-5-ylidene)methylamil-3-0- (4-methoxybenzyl)-l-thio-p-D-glucopyranoside (21) A suspension of sodium hydride (6.82 g, 269.97 mmol) in dry DMF (50 mL) was cooled to 0 OC, and a solution of methyl 4,6-0-benzylidene-2-deoxy-2-[(1,3-dimeth trioxopyrimidin-5-ylidene)methylamino]-l-thio-1-Dglucopyranoside (20) (50 g, 107.99 mmol in dry DMF (200 mL) was added dropwise in 30 minutes. The resulting solution was stirred at room temperature for 30 minutes and 4methoxybenzyl chloride (37.36 g, 238.56 mmol) was added dropwise at 0 The reaction mixture was stirred at room temperature overnight, cooled to 0 'C and dry methanol mL) was added dropwise. The reaction mixture was WO 00/42057 WO 0042057PCT/AUOO/00025 -27 concentrated under reduced pressure, then xylene (200 mL) was co-evaporated from the residue. The residue was taken up in CHCl 3 (1000 mL) washed with H 2 0 (1000 Ml) saturated NaHCO 3 solution (1000 mL) dried over MgSO 4 and evaporated to dryness. The residue was crystallized from EtOH to give methyl 4, 6-0-benzylidene-2-deoxy-2-[ (1,3-dimethyl- 2,4,6 (lH, 3H, 5H) -trioxopyrimidin-5-ylidene)methylamino] (4-methoxybenzyl) -l-thio-p-D-glucopyranoside (21) (52.21 g, 82%).

Methyl 2-deoxy-2-[(1,3-dimethyl-2,4,6(1H,3H,5H)- (4methoxybenzyl) -1-thio-13-D-glucopyranoside (22) A mixture of methyl 4,6-O-benzylidene-2-deoxy-2-[(1,3dimethyl-2, 3H, 5H) ylidene)methylamino] (4-methoxybenzyl) -1-thio-f3-Dglucopyranoside (21) (52.21 g, 89.55 mmol and ptoluenesulphonic acid (200 mg) in MeOH MeCN 1:1 (400 InL) was stirred at 50 for 1 hour. The reaction mixture was evaporated, the residue was chromatographed using CHC13 MeOH 10:1 as the mobile phase to give methyl 2-deoxy-2- [I(1,3-dimethyl-2,4,6(lH,3H,5H)-trioxopyrimidin-5ylidene)methylamino]-3-O- (4-methoxybenzyl) -l-thio-p3-Dglucopyranoside (22) (31.0 g, Methyl 6-0-tert-butyldiphenylsilyl-2-deoxy-2- dimethyl-2,4, 6 3H, 5H) ylidene)methylaminoJ (4-methoxybenzyl) -1-thio-f3-Dglucopyranoside (23) A mixture of t-butyldiphenylsilyl chloride (16.65 g, 60.60 mmol), 4-dimethylaminopyridine (9.85 g, 80.80 rnmol) and methyl 2-deoxy-2-[(1,3-dimethyl--2,4,6(lH,3H,5H)- WO 00/42057 PCT/AU00/00025 28 trioxopyrimidin-5-ylidene)methylamino]-3-0-(4methoxybenzyl)-1-thio-p-D-glucopyranoside (22) (20 g, 40.4 mmol) in dry 1,2-dichloroethane (200 mL) was stirred at 0 C for 2 hours. The resulting clear solution was cooled to room temperature, diluted with CHC13 (200 mL), washed with H 2 0 (3 x 500 mL), brine solution (500 mL), dried over MgSO 4 and evaporated. The residue was purified by chromatography using 1,2-dichloroethane EtOAc 10:1 as the mobile phase to give methyl 6-O-tert-butyldiphenylsilyl-2deoxy-2-[(1,3-dimethyl-2,4,6(1H,3H,5H)-trioxopyrimidin-5ylidene)methylamino]-3-O-(4-methoxybenzyl)-l-thio--Dglucopyranoside (23) (23.3 g, 79%).

Methyl 6-O-tert-butyldiphenylsilyl-4-0-biphenylcarbonyl-2deoxy-2-[(1,3-dimethyl-2,4,6(1H,3H,5H)-trioxopyrimidin-5ylidene)methylamino]-3-O-(4-methoxybenzyl)-1-thio-P-Dglucopyranoside (24) A mixture of methyl 6-O-tert-butyldiphenylsilyl-2-deoxy-2- [(l,3-dimethyl-2,4,6(lH,3H,5H)-trioxopyrimidin-5ylidene)methylamino]-3-O-(4-methoxybenzyl)-l-thio-p-Dglucopyranoside (23) (10.0 g, 13.64 mmol), 4dimethylaminopyridine (2.5 g, 20.46 mmol) in dry 1,2dichloroethane (100 mL) was stirred at room temperature.

Biphenylcarbonyl chloride (4.42 g, 20.46 mmol) was added to the stirred reaction mixture. The resulting suspension was stirred under reflux for 3 hours. The reaction mixture was cooled to 10 0 C and filtered. The crystalline solid was washed on the funnel with dry 1,2-dichloroethane (20 mL) and filtered. The filtrates were combined, diluted with CHC1 3 (100 mL) and washed twice with diluted brine solution (water-brine 2:1) (150 mL). The organic layer was dried over MgSO 4 and evaporated. The residue was purified by chromatography using hexane CHCl 3 1:1 as the mobile phase to give methyl 6-O-tert-butyldiphenylsilyl-4-0- PCT/AU0O/00025 Received 01 November 2000 29 biphenylcarbonyl-2-deoxy-2-[(1,3-dimethyl-2,4,6(iN,3H,5H)trioxopyrimidin-5-ylidene)methylamino]-3-0-(4methoxybenzyl)-l-thio-J-D-glucopyranoside (24) (9.5 g, Example 6 Synthesis of an Orthogonally Protected Thioglycoside Building Block, Methyl 6-O-(tbutyldiphenylsilyl)-2-0-(4-methoxybenzyl)-3-O-allyl-4-Oacetyl-l-thio-3-D-galactopyranoside (31) OH OH OH OTBDPS o OTBDPS SMe HO SMe 2 5 OH 2 6 H 2OH OH OTBDPS OH OTBDPS o OBhDPS ARO SMe HO ><O0 SMe OBrOMe 29 OBrOMe 28 MOW A OLDPS DIO SMe OBnOMe 31 Methyl galactopyranoside (26) A mixture of methyl 1-thio-o-D-galactopyranoside (25) (5 g, 28 mmol), chioro t-butyldiphenylsilane (5.85 g, 21 mmol) and DMAP (2.63 g, 21 mmol) in dry 1, 2-dichioroethane (130 mL) was left to stir at reflux for 2.5 h. The reaction mixture was cooled to room temperature, diluted with dichloromethane (200 mL) and washed with saturated sodium chloride solution (2 x 250 mL). The organic phase was dried over MgSO 4 and subsequently evaporated to dryness to \\melbfiles\homeS\WendyS\Keep\species\P-12178 PCT Alchemia.doc 31/10/00 AM "rE S~ET WO 00/42057 PCT/AU00/00025 30 give methyl galactopyranoside (26) (7.5 g, 81%) as a colorless oil.

Methyl 6-O-(t-butyldiphenylsilyl)-3,4-O-isopropylidene-lthio-P-D-galactopyranoside (27) A mixture of methyl 6-0-(t-butyldiphenylsilyl)-l-thio-3-Dgalactopyranoside (26) (7.4 g, 16.5 mmol) and ptoluenesulphonic acid (20 mg) in 2,2-dimethoxypropane (100 mL) was left to stir at room temperature for 2 h. The reaction mixture was then neutralized with triethylamine (1 mL) and evaporated to dryness. The residue was dissolved in dichloromethane (250 mL), washed with water (1 x 250 mL), dried over MgSO 4 and evaporated to dryness to give methyl 6-O-(t-butyldiphenylsilyl)-3,4-O-isopropylidene-l-thio-P-Dgalactopyranoside (27) (7.0 g, 87%) as a white solid.

Methyl 6-0-(t-butyldiphenylsilyl)-2-0-(4-methoxybenzyl)- 3,4-0-isopropylidene-l-thio-P-D-galactopyranoside (28) To a suspension of sodium hydride 0.53 g, 21 mmol) in dry DMF (100 mL) at 00 Co, was added dropwise methyl (t-butyldiphenylsilyl)-3,4-O-isopropylidene-l-thio--Dgalactopyranoside (27) (6.8 g, 13.9 mmol) as a solution in dry DMF (25 mL) in 5 minutes. The resulting mixture was left to stir at 0 C O for 15 min and then at room temperature for 1 h. The mixture was then cooled to 0 Co and a solution of 4-methoxybenzyl chloride (3.27 g, 21 mmol) in dry DMF (25 mL) was added dropwise, over 5 min.

The reaction mixture was left to stir at 00 C for 15 min and then at room temperature for 16 h. After this period the reaction was neutralized with absolute ethanol (15 mL) at 00 C, and then evaporated to dryness. The residue was taken up in chloroform (400 mL), washed with water (300 mL) WO 00/42057 PCT/AU00/00025 31 and saturated sodium bicarbonate solution (300 mL). The organic phase was dried over MgSO 4 and evaporated to dryness to give the crude product as an orange oil g).

The crude material was chromatographed using EtOAc hexane 25 75 as the mobile phase to give methyl 6-O-(tbutyldiphenylsilyl)-2-O-(4-methoxybenzyl)-3,4-0isopropylidene-l-thio-P-D-galactopyranoside (28) as a pale yellow oil (6.5 g, 77%).

Methyl 6-O-(t-butyldiphenylsilyl)-2-O-(4-methoxybenzyl)-1thio-P-D-galactopyranoside (29) A suspension of methyl 6-O-(t-butyldiphenylsilyl)-2-0-(4methoxybenzyl)-3, 4 -O-isopropylidene-l-thio-P-Dgalactopyranoside (28) (6.4 g, 10.5 mmol) in acetic acid 150 mL) was left to stir at 70 Co for 1.5 h. The reaction mixture was evaporated to dryness and the remaining residue was chromatographed using EtOAc hexane 1 1) to give methyl 6-O-(t-butyldiphenylsilyl)-2-0-(4methoxybenzyl)-l-thio-3-D-galactopyranoside (29) as a pale yellow oil (3.0 g, Methyl 6-O-(t-butyldiphenylsilyl)-2-0-(4-methoxybenzyl)-3- O-allyl-l-thio--D-galactopyranoside A mixture of methyl 6-0-(t-butyldiphenylsilyl)-2-0-(4methoxybenzyl)-l-thio-P-D-galactopyranoside (29) (2.8 g, 4.9 mmol) and dibutyl tin oxide (1.6 g, 6.4 mmol) in anhydrous methanol (200 mL) was stirred at reflux for 1 h.

The reaction mixture was evaporated to dryness and the remaining residue dissolved in dry toluene (50 mL).

Tetraethylammonium bromide (1.34 g, 6.4 mmol) and allyl bromide (7.7 g, 64 mmol) were added. The reaction mixture was left to stir at reflux overnight. The reaction mixture WO 00/42057 PCT/AU00/00025 32 was cooled to room temperature and filtered. The filtrate was evaporated to dryness and the residue was purified by chromatography using EtOAc hexane 15 85 as the mobile phase to give methyl 6-O-(t-butyldiphenylsilyl)-2-O-(4methoxybenzyl)-3-O-allyl-l-thio- -D-galactopyranoside g, 50%) as a pale yellow oil.

Methyl 6-O-(t-butyldiphenylsilyl)-2-O-(4-methoxybenzyl)-3- O-allyl-4-O-acetyl-l-thio-3-D-galactopyranoside (31) To a solution of methyl 6-0-(t-butyldiphenylsilyl)-2-O-(4methoxybenzyl)-3-O-allyl-l-thio-P-D-galactopyranoside (1.4 g, 2.3 mmol) in pyridine (30 mL) was added acetic anhydride (20 g, 196 mmol) in one portion. The resulting solution was left to stir at room temperature for 72 h.

The reaction contents were then evaporated to dryness and the residue was dissolved in dichloromethane (200 mL). The solution was washed with potassium hydrogen sulphate solution (1M, 2 x 150 mL) followed by saturated sodium chloride (150 mL), dried over MgS0 4 and evaporated to dryness. The crude residue was purified by chromatography using dichloromethane as the mobile phase to give Methyl 6- O-(t-butyldiphenylsilyl)-2-O-(4-methoxybenzyl)-3-O-allyl-4- O-acetyl--thio-P-D-galactopyranoside (31) (750 mg, 48%) as a pale yellow oil.

Example 7 Selective Deprotection Etherification study using an Orthogonally Protected Thioglycoside Building Block, Methyl 2azido-6-O-tert-butyldiphenylsilyl-4-Obiphenylcarbonyl-2-deoxy-3-O-(4methoxybenzyl)-1-thio-p-D glucopyranoside (11) WO 00/42057 WO 0042057PCTAUOOOOO25 33 OIBDPS OTBDPS OTBDPS MeOBnO SMe MeOBnO-~.... SMe MeOBnO...&. SMe

SN

3 10 N 3 32

N

3 M~CI OBiCI OH'if Bn 09g BO Se MeOBnO SMe MeOBnO We

N

3

N

3 33 N 3 OBiCI OBrCI Bn 0- B Me 5

B

O~rCI H 3

W

MeMe 5 Bze O N

CHH

3 UO1 Methyl 38zd---etbtliheysll2doy3O 4 methoxybenzyl H1ti---~cprn d Sodiumo (8 g)wsratdidyMeH(0 rLte Mty -zd---etbutyldiphenylsilyl-2-deoxy-3-O- (4-ehoynyl-lhi-- D-glucopyranoside (11) (3 g, 3.88 mmol) in THE (25 mL) was added. The reaction mixture was stirred at 40 C' for minutes, then cooled to room temperature. The solution was WO 00/42057 PCT/AU00/00025 34 neutralized by Amberlite IR 120 (H 4 ion exchange resin.

The suspension was filtered, the filtrate was evaporated.

The residue was purified by chromatography using EtOAc hexane 1 4 as the mobile phase to give methyl 2-azido-6- O-tert-butyldiphenylsilyl-2-deoxy-3-O- (4-methoxybenzyl) -1thio-o-D-glucopyranoside (10) (2.1 g, 91%) Methyl 2-azido-4-0-benzyl-6-O-tert-butyldiphenylsilyl-2deoxy-3-O-(4-methoxybenzyl)-1-thio-P-D-glucopyranoside (32) A suspension of sodium hydride (196 mg, 5.1 mmol) in dry DMF (10 mL) was cooled to 0 OC, and a solution of methyl 2azido-6-O-tert-butyldiphenylsilyl-2-deoxy-3-O-(4methoxybenzyl)-l-thio-P-Dglucopyranoside (10) (2.53 g, 4.3 mmol) in dry DMF (20 mL) was added dropwise in 30 minutes.

The resulting solution was stirred at room temperature for minutes and benzyl bromide (880 mg, 5.1 mmol) was added dropwise at 0 The reaction mixture was stirred at room temperature overnight, cooled to 0 °C and dry methanol (1 mL) was added dropwise. The reaction mixture was concentrated under reduced pressure, then xylene (20 mL) was co-evaporated from the residue. The residue was taken up in CHC1 3 (100 mL) washed with H 2 0 (100 ml), saturated NaHCO3 solution (100 mL) dried over MgS0 4 and evaporated to dryness. The residue was purified by chromatography using EtOAc Hexane 1 9 as the mobile phase to give methyl 2azido-4-O-benzyl-6-O-tert-butyldiphenylsilyl-2-deoxy-3-O- (4-methoxybenzyl)-1-thio-P-D-glucopyranoside (32) (2.0 g, 68%).

Methyl 2-azido-4-O-benzyl-2-deoxy-3-O-(4-methoxybenzyl)-1thio-P-D-glucopyranoside (33) To a mixture of methyl 2-azido-4-O-benzyl-6-O-tertbutyldiphenylsilyl-2-deoxy-3-O-(4-methoxybenzyl)-l-thio-P- D-glucopyranoside (32) (1.5 g, 2.2 mmol) and anhydrous AcOH (28.8 mL) in dry THF (169 mL) hydrogen fluoride-pyridine complex (20.3 mL) was added in a polypropylene container.

The reaction mixture was kept at room temperature WO 00/42057 PCT/AU00/00025 35 overnight, then diluted with EtOAc (1 The resulting solution was washed with saturated sodium hydrogen carbonate (4 x 1 saturated brine solution (1 dried over MgS0 4 and evaporated to dryness. The residue was crystallized from MeOH. The mother liquor was evaporated, the residue was treated with hexane to get more solid. The solid products were combined affording methyl 2-azido-4-Obenzyl-2-deoxy-3-O-(4-methoxybenzyl)-l-thio-3-Dglucopyranoside (33) (735 mg, Methyl 2-azido-4-0-benzyl-6-O-(4-chlorobenzyl)-2-deoxy-3-0- (4-methoxybenzyl)-l-thio-P-D-glucopyranoside (34) A suspension of sodium hydride (71 mg, 1.8 mmol) in dry DMF mL) was cooled to 0 and a solution of methyl 2azido-4-O-benzyl-2-deoxy-3-0-(4-methoxybenzyl)-1-thio-P-Dglucopyranoside (33) (680 mg, 1.5 mmol) in dry DMF (5 mL) was added dropwise in 30 minutes. The resulting solution was stirred at room temperature for 30 minutes and 4chlorobenzyl chloride (295 mg, 1.5 mmol) was added dropwise at 0 The reaction mixture was stirred at room temperature for 4.5 hours, cooled to 0 C and dry methanol (1 mL) was added dropwise. The reaction mixture was concentrated under reduced pressure, then xylene (10 mL) was co-evaporated from the residue. The residue was treated with hexane (10 mL) and filtered to give methyl 2-azido-4- O-benzyl-6-O-(4-chlorobenzyl)-2-deoxy-3-0-(4methoxybenzyl)-l-thio-3-D-glucopyranoside (34) (620 mg, 71 Methyl 2-azido-4-0-benzyl-6-0-(4-chlorobenzyl)-2-deoxy-lthio-P-D-glucopyranoside A mixture of methyl 2-azido-4-O-benzyl-6-O-(4chlorobenzyl)-2-deoxy-3-O-(4-methoxybenzyl)-l-thio-p-D glucopyranoside (34) (580 mg, 1.01 mmol) and DDQ (270 mg, 1.2 mmol) in CH 2 C12 H 2 0 9:1 (10 mL) was stirred at room temperature for 3 hours. The reaction mixture was washed with saturated NaHC03 solution (3 x 15 ml), dried over WO 00/42057 WO 0042057PCT/AUOO/00025 -36 MgSO 4 and evaporated. The residue was purified by chromatography using CHCl 3 -Hexane-MeOH 30:20:0.5 as the mobile phase to give methyl 2-azido-4-O-benzyl-6-O-(4chlorobenzyl) -2-deoxy-l-thio-fp-Dglucopyranoside (35) (300 mg, 66%).

Methyl 2-azido-4-O-benzyl-6-O- (4-chlorobenzyl) -2-deoxy-3-Opentamethylbenzyl-1-thio-p-D-glucopyranoside (36) A suspension of sodium hydride (40 mg, 1.0 mmol, 60%) in dry DMF (5 mL) was cooled to 0 0 C, and a solution of methyl 2-azido-4-O-benzyl-6-O- (4-chlorobenzyl) -2-deoxy-l-thio-p-D glucopyranoside (35) (280 mg, 0.67 mmol) in dry DMF (5 mL) was added dropwise in 30 minutes. The resulting solution was stirred at room temperature for 30 minutes and pentamethylbenzyl chloride (200 mg, 1.0 inmol) was added dropwise at 0 The reaction mixture was stirred at room temperature for 4 hours, cooled to 0 'C and dry methanol (1 mL) was added dropwise. The reaction mixture was concentrated under reduced pressure then xylene (10 mL) was co-evaporated from the residue. The residue was in EtOAc (100 rnL) washed with brine (2 x 100 mL) dried over MgSO 4 and evaporated. The resulting solid was suspended in hexane mL)and filtered to give methyl 2-azido-4-O-benzyl-6-O- (4-chlorobenzyl) -2-deoxy-3-O-pentamethylbenzyl--thio- 3

-D-

glucopyranoside (36) (290 mg, 76%).

2-E(1,3-dimethyl-2,4,6(lH,3H,5H)-trioxopyrimidin-5ylidene)methylamino] -ethyl 2-azido-4-O-benzyl-6-O- (4chlorobenzyl) -2-deoxy-3-O-pentamethylbenzyl-X, I-Dglucopyranoside (37) A mixture of methyl 2-azido-4--O-benzyl-6-O- (4chlorobenzyl) -2-deoxy-3-O-pentamethylbenzyl-l-thio-P-D glucopyranoside (36) (220 mg, 0. 36 nmol) 3-dimethyl- 2,4,6 (lH, 3H, 5H) -trioxopyrimidin-5-ylidene)methylamino] ethanol (150 mg, 0.66 mmol), molecular sieves 4A 1 g) and DMTST (138 mg, 0.66 mmol) in l,2-dichloroethane (10 mL) was stirred at room temperature for 30 minutes. The reaction WO 00/42057 WO 0042057PCT/AUOO/00025 37 mixture was neutralized with TEA (0.5 mL) and evaporated.

The residue was purified by chromatography using CHiCl 3 -MeOH mL :20 drops as the mobile phase to give dimethyl-2,4, 6(lH, 3H-,5H) ylidene)methylamino] -ethyl 2-azido-4-O-benzyl-6-O- (4chlorobenzyl) -2-deoxy-3-O-pentamethylbenzyl-P3-D glucopyranoside (37) (220 mg, 77%).

2-[(1,3-dimethyl-2,4,6(1H,3H,5H)-trioxopyrimidin-5ylidene)methylaminoJ -ethyl 2-amino-4-0-benzyl-6-0- (4chlorobenzyl) -2-deoxy-3 -0-pentamethylbenzyl-a, P-Dglucopyranoside (38) A mixture of 2-[(l,3-dimethyl-2,4,6(lH,3H,5H)- -ethyl 2-azido-4-Obenzyl-6-O- (4-chlorobenzyl) -2-deoxy-3-O-pentamethylbenzyljP-Dglucopyranoside (37) (160 mg, 0.2 mmol) and TEA (3 drops) in 1,3-propanedithiol (1 mL) was stirred at room temperature overnight. The reaction mixture was chromatographed using EtOAc hexane 1:1 then EtOAc MeOH 10:1 solvent systems as mobile phases to give dimethyl-2, 4,6 (1H, 3H, 5H) ylidene)methylamino] -ethyl 2-amino-4-O-benzyl-6-O- (4chlorobenzyl) -2-deoxy-3-O -pent amet hylben zyl fP-D glucopyranoside (38) (123 mg, 2-[Ul,3-dimethyl-2,4,6(lH,3H,SH)-trioxopyrimidin-5ylidene )methylamino] -ethyl 2- -dimethyl- 2,4,6 (1H, 3H, 5H) -trioxopyrimidin-5-ylidene)methylaminoJ benzyl (4-chlorobenzyl) -2 a, P-D glucopyranos ide (3 9) A mixture of 2-[(l,3-dimethyl-2,4,6(lH,3H,5H)- -ethyl 2-arnino-4-Obenzyl-6-O- (4-chlorobenzyl) -2-deoxy-3-O-pentamethylbenzyl- IP-Dglucopyranoside (38) (50 mg, 0.066 rnmol), 1,3-dirnethyl- 5-[(dimethylamino)methylene]2,4,6(lH,3H,5H)trioxopyrimidine (Wow-reagent) (50 mg, 0.24 mmol), TEA (0.2 mL) in CHC1 3 MeOH 3:1 (4 mL) was stirred at room 38 temperature f or 3 hours. The reaction mixture was evaporated, the resulting residue was chromatographed using EtOAc as the mobile phase to give 2-[(l,3-dimethyl- 2,4,6 (lH, 3H, 5H) -trioxopyrimidin-5-ylidene) methylamino] ethyl 2-[(l,3-dimethyl-2,4,6(lH,3H,5H)-trioxopyrimidin-5ylidene)methylamino] -4-O-benzyl-6-O- (4-chlorobenzyl) -2deoxy-3-O-pentamethylbenzyl 1-D glucopyranoside (39) mg, Example 8 Selective deprotection study using an Orthogonally Protected Thioglycoside Building Block, Methyl 2-azido-6-O-tertbutyldiphenylsilyl-4-O-biphenylcarbonyl-2 deoxy-3-O- (4-methoxybenzyl) -1-thio-p3-D glucopyranoside (11)

OTBDPS

HG~0 MeOBnO SMe

N

3

OH

BPCO 0-~ MeOBnO) SMe 40 N 3

OTBDPS

BPCOXK.-0 42

NH

2 O 0o Methyl 2-azido-6-O-tert-butyldiphenylsilyl-2-deoxy- 3 (4methoxybenzyl) -1-thio-p-D glucopyranoside Sodium (89 mg) was reacted in dry MeOH (50 mL) then a solution of methyl 2-azido-4-O-biphenylcarbonyl-6-O-tertbutyldiphenylsilyl-2-deoxy-3-O- (4-methoxybenzyl) -l-thio-p-D H:\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 WO 00/42057 PCT/AU00/00025 39 glucopyranoside (11) (3 g, 3.88 mmol) in THF (25 mL) was added. The reaction mixture was stirred at 40 Co for minutes, then cooled to room temperature. The solution was neutralized by Amberlite IR 120 ion exchange resin.

The suspension was filtered, the filtrate was evaporated.

The residue was purified by chromatography using EtOAc hexane 1 4 as the mobile phase to give methyl 2-azido-6- O-tert-butyldiphenylsilyl-2-deoxy-3-0-(4-methoxybenzyl)-1thio-P-Dglucopyranoside (10) (2.1 g, 91%) Methyl 2-azido-4-O-biphenylcarbonyl-2-deoxy-3-O-(4methoxybenzyl)-1-thio-P-D-glucopyranoside To a mixture of methyl 2-azido-4-O-biphenylcarbonyl-6-Otert-butyldiphenylsilyl-2-deoxy-3-O-(4-methoxybenzyl)-1thio-o-D-glucopyranoside (11) (150 mg, 0.19 mmol) and anhydrous AcOH (2.8 mL) in dry THF (17 mL) hydrogenfluoride-pyridine complex (2 mL) was added in a polypropylene container. The reaction mixture was kept at room temperature overnight, then diluted with EtOAc (100 mL). The resulting solution was washed with saturated sodiumhydrogen carbonate (4 x 100 mL), saturated brine solution (100 mL), dried over MgSO 4 and evaporated to dryness. The residue was purified by chromatography using EtOAc hexane 2:5 as the mobile phase to give methyl 2azido-4-O-biphenylcarbonyl-2-deoxy-3-0-(4-methoxybenzyl)-1thio--D-glucopyranoside (40) (96 mg, 93%).

Methyl 2-azido-4-O-biphenylcarbonyl-6-O-tertbutyldiphenylsilyl-2-deoxy-1-thio-P-D-glucopyranoside (41) A mixture of methyl 2-azido-4-O-biphenylcarbonyl-6-O-tertbutyldiphenylsilyl-2-deoxy-3-O-(4-methoxybenzyl)-l-thio-- D-glucopyranoside (11) (150 mg, 0.19 mmol) and DDQ (52 mg, 0.23 mmol) in CH 2 C1 2

H

2 0 9:1 (5 mL) was stirred at room temperature for 3 hours. The reaction mixture was washed with saturated NaHC03 solution (3 x 3 ml), dried over MgSO 4 and evaporated. The residue was purified by chromatography using EtOAc hexane 15:85 as the mobile phase to give WO 00/42057 PCT/AUOO/00025 40 methyl 2-azido-4-O-biphenylcarbonyl-6-O-tertbutyldiphenylsilyl-2-deoxy-1-thio-3-D-glucopyranoside (41) (116 mg, 92%) Methyl 2-amino-4-O-biphenylcarbonyl-6-O-tertbutyldiphenylsilyl-2-deoxy-3-O- (4 -methoxybenzyl) -I-thio- D-glucopyranoside (42) A mixture of methyl 2-azido-4-O-biphenylcarbonyl-6-O-tertbutyldiphenylsilyl-2-deoxy-3-O- (4-methoxybenzyl) -l-thio-- D-glucopyranoside (11) (150 mg, 0.19 mmol) and TEA (3 drops) in 1,3-propanedithiol (1 mL) was stirred at room temperature overnight. The reaction mixture was chromatographed using EtOAc hexane 15:35 then EtOAc hexane 1:1 solvent systems as mobile phases to give methyl 2-amino-4-O-biphenylcarbonyl 6-0-tert-butyldiphenylsilyl- 2 deoxy-3 (4-methoxybenzyl) -l-thio-f-D-gucopyranoside (42) (130 mg, 91%) 3,4-Methylenedioxybenzyl 2-azido-4-O-biphenylcarbonyl-6-0tert-butyldiphenylsilyl-2 -deoxy-3 (4-methoxybenzyl) a, P- D-glucopyranoside (43) A mixture of methyl 2-azido-4-O-biphenylcarbonyl-6-0-tertbutyldiphenylsilyl-2-deoxy-3-0- (4-methoxybenzyl) -1-thio-P- D-glucopyranoside (11) (200 mg, 0.26 mmol), 3,4methylenedioxybenzyl alcohol 59 mg, 0.39 mmol) molecular sieves 4A (1 g) and methyltriflate (106 mg, 0.65 mmol) in l,2-dichloroethane (10 mL) was stirred at room temperature overnight. The reaction mixture was neutralized with TEA mL) and evaporated. The residue was purified by chromatography using EtOAc hexane 15:85 as the mobile phase to give 3,4-methylenedioxybenzyl 2-azido-4-0biphenylcarbonyl-6-O-tert-butyldiphenylsilyl-2-dexW 3 0 (4-methoxybenzyl)-a, -D-glucopyranoside (43) (173 mg, 76%) It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, WO 00/42057 PCT/AUOO/00025 -41 various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

References cited herein are listed below, and are incorporated herein by this reference.

WO 00/42057 WO 0042057PCT/AUOO/00025 42- REFERENC ES Merrifield, R. B.

Pept., Proc. Am. Pept. Symp., 5 1977 488.

Wong, C-H, Ye, X-S and Zhang, Z.

J. Am. Chem. Soc., 1998 120 7137-7138.

Wunberg, Kallus, Opatz,- Henke, Schmidt, W., and Kunz, H.

Angew. Chemn. Int. Ed., 1998 37 2503-2505

Claims (12)

1. A universal monosaccharide building block of General Formula I or General Formula II DX 4 O A EX 4 O0 A CX 2 XIB DX 3 XIB X 2 C I II in which A is a leaving group selected from the group consisting of -SR; where R is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl; halogen excluding chlorine; trichloroacetimidoyl-; sulphoxide; and -O-alkenyl; X 1 X 2 and X 3 are independently selected from H, O, N, NH or N 3 with the proviso that only one of X 1 X 2 and X 3 may be H, N or N 3 in any molecule; X 4 is H, -CH20, -CH 2 N, -CH 2 NH-, -CH 3 -CH 2 N 3 or COO- with the proviso that X 4 may only be H, -CH 2 N, -CH 3 20 or -CH 2 N 3 when none of XI to X 3 is H; and B, C, D and E are different, and are selected from protecting groups which can be cleaved orthogonally in any order such that the cleavage conditions do not compromise the stability of the other protecting or functional groups on the monosaccharide building block, and in which B or C or D or E is absent if the corresponding X 1 to X 3 is H or N 3 or if the corresponding X 4 is H, -CH 3 or -CH 2 N 3 H:\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 44

2. A monosaccharide building block according to claim 1, which is a compound of General Formula III E 1 X 4 D1X 3 A XJB1 III in which, A, X 1 X 2 X 3 and X 4 are as defined for General Formulae I and II, and BI, C 1 Di and El are orthogonal protecting groups selected from protecting group sets 1, 2, 6 and 8 as herein defined.

3. claim 1, A monosaccharide building block according to which is a compound of General Formula IV E 2 X4 O A D 2 X, XiB2 in which A, X 1 X 2 X 3 and X 4 are as defined for General Formulae I and II, and B 2 C 2 D 2 and E 2 are orthogonal protecting groups 20 selected from the members of protecting group set 1 as herein defined, the members of set 1 being orthogonal to each other.

4. A monosaccharide building block according to 25 claim 3, in which the members of protecting group set 1 are levanoyl, chloroacetate, p-methoxybenzyloxycarbonyl and 2- trimethylsilylethylcarbonate. H:\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 45 A monosaccharide building block according to claim 1, which is a compound of General Formula V A X 1 B3 X 2 C 3 in which Formulae selected from the defined. A, X 1 X 2 X 3 and X 4 are as defined for General I and II, and B 3 C 3 D 3 and E 3 are orthogonal protecting groups from the members of set 1 as herein defined and remaining orthogonal sets 2 to 9 as herein S. C' 0 OS S S S 55 SS

5 OS S S

6. A method of synthesis of a molecule selected from the group consisting of glycoconjugates of non-carbohydrate molecules, neo-glycoconjugates and oligosaccharides, comprising the step of using a monosaccharide building block according to any one of claims 1 to

7. A method of synthesis of a molecule selected from the group consisting of glycoconjugates of non-carbohydrate molecules, neo-glycoconjugates and oligosaccharides, comprising the step of glycosylating the molecule with a monosaccharide building block according to any one of claims 1 to

8. A method according to claim 6 or claim 7, in which the molecule comprises one or more compounds in which substituents are linked to a pyranose or furanose ring. H\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03 46

9. A method according to any one of claims 6 to 8, in which the molecule comprises a sugar analogue.

A method according to any one of claims 6 to 9, in which the synthesis is carried out in solution.

11. A method according to any one of claims 6 to 9, in which the synthesis is carried out on a solid-phase support.

12. Universal monosaccharide building blocks or methods involving them, substantially as hereinbefore described with reference to the accompanying examples. Dated this 19th day of December 2003 ALCHEMIA PTY LTD By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia *g *o oo H:\suzannet\Keep\Speci\24252-00.1 SPECI.doc 19/12/03