Synthesis of trisaccharides incorporating the  -Gal antigen functionalized for neoglycoconjugate preparation

The synthesis of trisaccharides 1 and 2 , which contain the  - D -galactopyranosyl-(1  3)-  - D - galactopyranosyl (  -Gal) motif, is described. A key step in the synthesis of the trisaccharides was the glycosylation of a monosaccharide acceptor with a disaccharide trichloroacetimidate donor. Subsequent modification of the products of this [2+1] glycosylation afforded 1 and 2 , which contain an activated ester moiety suitable for reaction with, for example, proteins or amine-coated surfaces.


Introduction
The -galactopyranosyl-(13)--D-galactopyranosyl disaccharide motif, also known as the -Gal antigen, is found commonly on the surface of mammalian tissues with the exception of old world monkeys and humans. 1 This antigen has long been identified as a contraindication for xenotransplantation. 2 Although the problems associated with xenotransplantation have been largely overcome by the advent of -Gal knockout pigs, 3,4 the role of -Gal epitopes in transplantation [5][6][7] and in several disease conditions [8][9][10][11] is still under investigation.There is also growing interest in the use of bioconjugates bearing the -Gal epitope in cancer therapies [12][13][14][15] and vaccine development 16 to enhance immune responses.Access to synthetic -Gal epitopes is required to facilitate most of this research.
-(13)-Galactosyltransferase knockout (GTKO) mice have been routinely used to study the immune response to carbohydrate antigens. 17Analysis of -Gal response in GTKO mice is considered a good approximation for the study of accommodation and immune tolerance induction in transplantation of ABO-incompatible organs in humans, 18 as the -Gal epitope is found in the A and B blood groups. 19As part of a larger investigation on the preparation of glycosylated nanomaterials 20 for use in generating immune tolerance against carbohydrate antigens in neonates, we had the need for -Gal-containing oligosaccharides functionalized with an activated flexible linker, which would be used for the attachment to various surfaces.We describe here the synthesis of two such targets, trisaccharides 1 and 2 (Figure 1).

Results and Discussion
2][23] Access to these compounds via this approach involves the use of recombinant -(13)-galactosyltransferase enzymes and chemically modified substrates.Although this method is efficient and provides sufficient material for biological assays, access to larger quantities of material still favours chemical synthesis.
5][26][27][28] For example, Wang and coworkers achieved the synthesis of an -Gal pentasaccharide via a one-pot strategy using various thioglycoside donors. 24,25Schmidt and coworkers described the synthesis of a related pentasaccharide using a convergent approach and trichloroacetimidate donors. 27In another investigation, Litjens et al. reported the one-pot synthesis of an -Gal trisaccharide using thioglycoside donors. 26Most previously reported synthetic routes to the trisaccharides such as 1 and 2 involve the selective protection and glycosylation of a lactose or lactosamine acceptor.We decided instead to prepare an -Galp-(13)-Galp disaccharide donor as a precursor and then react it with a monosaccharide acceptor.This would allow us to change the acceptor thus allowing for a straightforward convergent approach to the preparation of -Gal-containing tetra-and pentasaccharides at a later stage.
The preparation of 1 and 2 was accomplished using trichloacetimidate donors developed originally by Schmidt and co-workers. 29First, the -Galp-(13)-Galp linkage was achieved in good yield and stereoselectivity by reaction of tricholoroacetimidate 3 and thioglycoside alcohol 4 using diethyl ether as a solvent (Scheme 1). 30To perform this reaction, thioglycoside 4, which was sparingly soluble in diethyl ether, was dissolved in a binary mixture of diethyl ether and dichloromethane (10:1).Reverse dropwise addition of the trichloroacetimidate donor 3 to the solution of 4 at -10 °C in the presence of TMSOTf as an activator gave exclusively the desired -linked product, 5, in 76% yield.The -stereochemistry of the newly formed linkage in 5 was readily apparent from its 1 H NMR spectrum; the value of 3 J1',2', was 3.0 Hz.This reaction was easily conducted in gram scale quantities with no change in stereoselectivity, thus providing large amounts of material for further synthesis.

Scheme 1. Preparation of trisaccharides 13 and 14.
Direct coupling of the resulting thioglycoside 5 with the acceptors 8 and 9 31 could not be achieved, even in neutral conditions.We attribute this to the electrophilic by-products produced during the reaction, reacting with the alkene moiety in the acceptor.Thus, thioglycoside 5 was first transformed into the corresponding hemiacetal 6 in 84% yield, by the action of Nbromosuccinimide in acetone, and subsequently converted to trichloroacetimidate 7 by treatment with trichloroacetonitrile in DBU.
Coupling of trichloracetimidate 7 to either 8 or 9 proceeded uneventfully to give the corresponding trisaccharides 10 and 11 in 87 and 83% yield, respectively.The -stereochemistry of the newly formed bond in both molecules was confirmed by the 3 JH1,H2 in 10 and 11, which were 8.1 Hz and 8.0 Hz, respectively.The azido acceptor was chosen as an aminosugar precursor, primarily for ease of conversion to the desired N-acetyl group.This transformation could be achieved by reaction of 11 with thioacetic acid in pyridine, 32 providing the expected acetamide derivative in 83% yield.Simultaneous deprotection of the benzoyl and benzyl protecting groups in 10 and 12 was carried out using dissolving metal reduction.This was achieved by reaction of each compound with sodium metal in THF/CH3OH, conditions that allowed the removal of the benzyl groups while leaving the alkene functionality intact.In addition, the benzoate esters were cleaved under the basic conditions of the reaction.
Having in place a robust route for the preparation of 13 and 14, we investigated their conversion into 1 and 2. The octenyl linker of 13 and 14 was further functionalized by first introducing a cysteamine residue via a UV-promoted thiol-ene reaction 33 to give the corresponding amines 15 and 16 as their hydrochloride salts in 90-97% yield (Scheme 2).Further elaboration of the linker using di-p-nitrophenyl adipate 34 in dimethylacetamide gave the highly reactive p-nitrophenol (PNP) esters 1 and 2. The final products were lyophilized and could be stored at -20 °C without significant degradation over time.

Conclusions
In summary, we report an efficient route to -Gal trisaccharides 1 and 2 via a route in which a key transformation is the reaction of a disaccharide trichloroacetimidate donor with a monosaccharide acceptor.This route should be applicable to more complex glycans containing

Experimental Section
General.Reagents and solvents were purchased from commercial sources and were used without further purification, unless otherwise stated.Dry solvents were purified by successive passage through columns of alumina and copper under nitrogen.Reactions were conducted in an inert atmosphere of argon gas, unless otherwise stated.Thin layer chromatography (TLC) was performed on E. Merck Silica Gel 60 F254 aluminum-backed plates that were stained by heating (>200 °C) with either p-anisaldehyde in 5% sulfuric acid in EtOH or 10% ammonium molybdate in 10% sulfuric acid.Unless otherwise indicated, all column chromatography was performed on Silica Gel 60 (40-60 M).C18 silica gel (35-70 M) was manufactured by Toronto Research Chemicals.Optical rotations were measured at 589 nm, at 22 ± 2 °C and are in units of deg•dm -1 •cm 3 •g -1 , in all cases the concentrations are in units g/100 mL. 1 H NMR spectra were recorded at 500 MHz, chemical shifts were referenced to the peak for TMS (0.0 ppm, CDCl3) or CD3OD (3.30 ppm, CD3OD). 13C NMR (APT) spectra were recorded at 125 or 100 MHz, and 13 C chemical shifts were referenced internal CDCl3 (77.1 ppm, CDCl3) or CD3OD (49.0, CD3OD).High resolution mass spectra were obtained using electrospray ionization in the appropriate solvents.