Synthesis of methyl 3-amino-3,6-dideoxy- a - D galactopyranoside carrying di ﬀ erent amide substituents

Bacterial polysaccharides may contain rare sugars of di ﬀ erent stereochemistry and diverse functional groups; the repertoire can be further extended by varying the exocyclic substituents. Synthesis of four monosaccharides is described utilizing a suitably protected key intermediate obtained by regioselective acetal ring-opening reduction, dexoygenation at C6, alcohol oxidation at C3 followed by formation of an oxime, which was stereoselectively reduced by samarium diiodide to give a 3-amino-derivative having the desired galacto -con ﬁ guration. Subsequent functionalization was performed resulting in one to four carbon atoms in the amide substituent.


Introduction
Lipopolysaccharides (LPS) cover a large portion of the outer membrane of Gram-negative bacteria where they play important roles in interactions with host cells. 1 The LPS consists of three parts, namely, the lipid A which anchors it in the membrane, a core region which is the outer part in rough bacteria and the O-antigenic polysaccharide which contains the outer part in smooth type bacteria. Whereas many of the biological effects are the consequences of the interactions of lipid A with the immune system of the host, the O-antigen plays important roles in colonization of the host and resistance to its immune system.
The lipid A and the core region of different bacteria are relatively conserved within a species and usually only a few variants are observed. The O-antigen polysaccharide, on the other hand, shows large variability both with respect to the polymer synthesized and the sugar components being part of it, where to date several hundred different sugar residues have been identied as constituents. 2 Branched sugars with carbon chains extending from the cyclic ring of the monosaccharide 3,4 are rare and many sugars are uncommon only being found in nature in a few instances. 5 The monosaccharide D-Fucp3N (3-amino-3,6-dideoxy-D-galactopyranose) has been found a-linked as a side-chain to the backbone polymer in the O-antigen polysaccharide of Providencia alcalifaciens O21 (ref. 6) in which it was N-formylated, which also was the case in the O-antigen from Salmonella enterica O60. 7 The same type of substitution pattern (terminal side-chain and a-linked) was present in the O-polysaccharide from Xanthomonas campestris pv. campestris 8004, but here the amino group was N-acetylated, 8 which is also the case for the monosaccharide in the glycan chain of the S-layer protein of Aneurinibacillus thermoaerophilus L420-91T. 9 In the core part of Proteus penneri strain 16 LPS 10 the terminal Fuc3N residue carries an (R)-3hydroxybutyryl group and in the O-antigen from Pseudoalteromonas nigrifaciens strain KMM 161 the substituent is a 4hydroxybutyryl group. 11 In the O-antigens of Escherichia coli O74 and Proteus vulgaris O45 the D-Fucp3NAc residues are b-linked. 12,13 Herein, we describe the synthesis of methyl 3amino-3,6-dideoxy-a-D-galactopyranoside having the above four amide-linked groups as substituents.

Results and discussion
The synthesis is described from the monobenzylated 4,6-Obenzylidene acetal derivative 5 which previously has been reported in the literature. 14 Benzoylation of the hydroxyl group in position 3 gave the fully protected compound 6 (Scheme 1). The BH 3 $THF complex together with CoCl 2 (ref. 15) was used to reductively open the benzylidene acetal in a regioselective fashion 16 to obtain the 6-hydroxy derivative 7. The use of this reagent was previously shown to result in high selectivity toward producing the 6-hydroxy derivatives in several hexopyranosides and the reaction was also successfully carried out with compound 5 or its 3-O-acetyl derivative, but the highest yield (92%) was achieved with the benzoyl derivative 6.
The deoxygenative reduction of a 6-hydroxyl group was previously shown for an a-D-mannopyranoside derivative by tosylation followed by reduction with sodium borohydride in DMF, 17 but for compound 7 the procedure resulted in the bicyclic 3,6-anhydro product. Instead, bromination with CBr 4 and Ph 3 P 18 to give the 6-bromo derivative 8, followed by reduction with tributyltin hydride in the presence of AIBN 19,20 was successfully used to obtain the 6-deoxy sugar 9.
Deprotection with sodium methoxide in MeOH furnished compound 10. The oxidation of the L-enantiomer of compound 10 has been reported using 2-iodoxybenzoic acid (IBX) or pyridinium dichromate. 21 The use of IBX 22-24 gave the highest yield (92%) and was thus employed to oxidize 10 to the keto derivative 11. This was followed by reaction with hydroxylamine hydrochloride 25 to give the oxime 12.
The key step in the synthesis is the reduction of oxime 12 to the amine derivative 13 having the desired galacto-conguration. Different reducing reagents were reported earlier by Hsu et al. for the corresponding L-enantiomer, 21 where, for example, Red-AlÒ favored the gulo-conguration, but the highest stereoselectivity for the desired product was achieved by using samarium diiodide 26,27 as a single-electron donor reducing agent (the ratio between galacto-and gulo-congurations being >19 : 1). This reagent was used to reduce oxime 12 to obtain compound 13 in an isolated yield of 54%. It can be noted that for ethyl 2,4-di-O-benzyl-6-deoxy-1-thio-b-D-xylo-hexopyranosid-3-ulose (E)-oxime reduction with Red-AlÒ worked well and the amino derivative having the galacto-conguration was isolated in 80% yield, 28 highlighting the stereochemical effects of the anomeric conguration on the reduction of the oxime at position 3 of these derivatives.
The target compounds were obtained via amide coupling of 13 with activated formic acid 29 and acetic anhydride, 30 respectively, to form compounds 14 and 15, which were deprotected by catalytic hydrogenolysis over Pd/C to give 1 and 2 (Scheme 2). The acids 16 and 19 were prepared according to Toriizuka et al. 31 and Brewer et al., 32 respectively, and were coupled with 13 by using DCC as the coupling reagent 33 to obtain compounds 17 and 20, respectively. The subsequent deprotection of the silyl ethers was performed with tetra-n-butylammonium uoride (TBAF) 34 in THF to give 18 and 21, respectively. In the last deprotection step catalytic hydrogenolysis over Pd/C afforded compounds 3 and 4. The

Conclusions
The synthesis has produced four variants with differently attached amide substituents on methyl 3-amino-3,6-dideoxya-D-galactopyranoside. The synthesis methodology applied herein will be of use in formation of larger oligosaccharides containing 3-amino-3,6-dideoxy-a-D-galactopyranoside as a component and the 1 H and 13 C NMR data obtained can be utilized to improve the NMR chemical shi predictions of oligo-and polysaccharides. 37,38 Experimental section

General experimental methods
All reagents were used as delivered. Column chromatography was performed manually on silica gel with a pore size of 60Å or by using a Biotage Isolera ash purication system with KP-Sil snap chromatography cartridges. TLC was carried out on silica gel 60 F254 (20 Â 20 cm, 0.2 mm thickness), and monitored with either UV light 254 nm, sulfuric acid 8%, Cerium molybdate or KMnO 4 . NMR spectra were recorded at 25 C, except for compounds 1-4 which were recorded at 15 C, on spectrometers operating at a 1 H frequency of 400 or 500 MHz. The NMR chemical shis are reported in ppm and for 1 H referenced to TMS, sodium 3-trimethylsilyl-(2,2,3,3-2 H 4 )-propanoate (TSP), both set to 0 ppm, or the residual CHCl 3 solvent peak at 7.26 ppm as an internal standard; for 13 C the chemical shis were referenced to 1,4dioxane in D 2 O, 67.40 ppm, using an external standard or internally to the CDCl 3 solvent signal at 77.16 ppm. For compounds 1-4 1 H chemical shis and J HH coupling constants were rened from 1D 1 H NMR spectra using NMR spin simulation methodology. 39 Mass spectra were recorded on a Bruker Daltonics micrOTOF spectrometer in the positive mode.   (7). Compound 6 (3.80 g, 8.00 mmol) was dissolved in BH 3 $THF complex 1.0 M solution (120.0 mL, 120.0 mmol) followed by the addition of CoCl 2 (3.10 g, 24.00 mmol), and the reaction was stirred at r.t. for 5 h. The reaction was diluted with EtOAc and aqueous NaBH 4 (0.20 equivalent) was added, and stirred for a few min followed by washing with NaHCO 3 , water and brine.