Synthesis of 5-O-oligoglucosyl extended α-(2→4)-Kdo disaccharides corresponding to inner core fragments of Moraxellaceae lipopolysaccharides.

The heptose-deficient inner core of the lipopolysaccharide of several pathogenic strains of the Moraxellaceae family (Moraxella, Acinetobacter) and of Bartonella henselae, respectively, comprises an α-D-glucopyranose attached to position 5 of Kdo. In continuation of the synthesis of fragments of Acinetobacter haemolyticus LPS, the branched α-Glcp-(1 → 5)[α-Kdo-(2 → 4)]-α-Kdo trisaccharide motif was elaborated. The glycosylation of a suitably protected, α-(2 → 4)-interlinked Kdo-disaccharide was achieved in high yield and fair anomeric selectivity using a 4,6-O-benzylidene N-phenyltrifluoroacetimidate glucosyl donor. Subsequent regioselective reductive benzylidene opening afforded a trisaccharide acceptor, which was extended with β-D-glucopyranosyl and isomaltosyl residues. Global deprotection provided tri- to pentasaccharide structures corresponding to the inner core region of A. haemolyticus lipopolysaccharide.


Introduction
The Gram-negative bacterial cell wall is covered by densely packed lipopolysaccharide (LPS) molecules which trigger major immune responses of the respective host during pathogenesis. 1,2 In structural terms, these glycolipids comprise the endotoxically active lipid anchor (lipid A) and an extended oligosaccharide fraction, which harbors a core region of limited structural variability and the strain-specific, highly diverse O-antigenic polysaccharides. 3,4 The core domain may be further divided into an outer and an inner core region, wherein a 3-deoxy-D-manno-2-octulosonic acid (Kdo) usually forms the link to the lipid A part. In many bacterial genera, this Kdo unit is extended by a lateral α-(2→4)-linked Kdo moiety. 5 In a few cases -such as in some Acinetobacter strains -Kdo is nonstoichiometrically replaced by its 3-oxy analogue D-glycero-D-talo-2-octulosonic acid (Ko). 6 The α-(2→4)-Kdo disaccharide is frequently substituted at position 5 by a L-glycero-Dmanno-heptose residue, and D-mannose, D-galactosamine and Kdo itself were also occasionally identified as the branching sugar. 7 Specifically, an α-(1→5)-linked Dglucopyranosyl residue has been found in LPS of the Moraxellaceae family and was shown to elicit cross-reactive antibodies recognizing different serotypes. 8 In addition, Bartonella henselae -the causative agent of the cat-scratch disease -expresses a truncated core region being composed of α-Glcp-(1→5) ]-α-Kdo linked to lipid A. 9 Furthermore, in A. haemolyticus this basic trisaccharide pattern is elongated at the glucose unit by a β-(1→4)-linked isomaltotriose. 10 The observation of a high binding affinity of isolated inner core fragments from A. haemolyticus towards different mannose binding lectins 11 prompted us to synthesize part structures thereof in order to identify the relevant binding epitopes.
Previously, stereoselective α-(1→5)-coupling of glucosyl donors to Kdo monosaccharide acceptors was successfully performed by Oscarson using a thioglycoside donor in the presence of DMTST. 12 In a previous contribution we have capitalized on the use of an NPTFA-glucosyl donor and an orthogonal blocking group at position-4 of Kdo to allow for the introduction of the lateral Kdo at a later stage. 13 A limited number of papers has been published addressing the synthesis of 5-O-glycosylated α-Kdo-(2→4)-α-Kdo disaccharides. 14 Ichiyanagi et al. recently reported on the convenient preparation of 5-Osubstituted Kdo disaccharides applying a Kdo disaccharide acceptor for coupling with heptose, D-mannose and 2-azido-2-deoxy-D-galactose (as a precursor for D-galactosamine) donors. 15 Extending these studies, we present herein a high-yielding method to access the branched α-Glcp-(1(5) ]-α-Kdo trisaccharide with fair stereoselectivity and its subsequent elongation into tetra-and pentasaccharide derivatives.

Approach A: Using an α-Glc-(1→5)-Kdo acceptor
Previously, α-Glc-(1→5)-Kdo disaccharide 1 has been prepared with excellent stereoselectivity and in a high yield. 13 The orthogonal protecting group pattern allowed for selective cleavage of the p-methoxybenzyl group (PMB) affording the 4-OH Kdo acceptor 2. Starting from 2 we envisaged to introduce the lateral Kdo moiety using the Kdo fluoride donors 3 and 4, which were recently reported to yield only α-anomeric products without significant elimination side reaction. 16 Specifically, the per-O-acetyl protected Kdo donor 3 proved to be an efficient glycosyl donor for regioselective coupling to a 4,5-diol Kdo acceptor yielding the α-(2→4)-interconnected Kdo disaccharide as a single isomer in high yield. 16a The donor, however, was unreactive in the presence of base -such as triethylamine or sym-collidine -which were added to capture hydrogen fluoride. Since the 7,8-Odisiloxane-1,3-diyl (TIPDS) and the 4,6-O-acetal group in acceptor 2 were not compatible with glycosylation conditions lacking a HF-scavenger, these protecting groups were replaced by acetyl groups. Accordingly, acid hydrolysis of the benzylidene group, followed by cleavage of the silyl ether, O-acetylation and DDQ-oxidation of the PMB group furnished the disaccharide acceptor 5 in 40% overall yield (Scheme 1). Attempted coupling of 5 with acetyl-protected donor 3 under BF 3 ·Et 2 O promotion (2 eq.) in dichloromethane in the presence of 3 Å molecular sieves at room temperature, however, failed and trisaccharide formation was not detected.
Next, the armed benzylated donor 4 was tried, which had been shown to react with 2propanol under BF 3 ·Et 2 O promotion also in the presence of triethylamine, although higher temperatures were necessary. 16b First, when using donor 4 in the absence of base and under conditions as used for donor 3, degradation of acceptor 2 was observed. In the presence of triethylamine, acceptor 2 was unaffected, but glycosylation did not take place. In a last attempt we intended to exploit the high reactivity of 4 and the compatibility of glycosyl acceptor 5 towards base-free conditions. Again, however, glycoside formation could not be achieved. NMR analysis of 5 revealed a through-space interaction between the axial hydrogen at position-3 of Kdo with H-5′ of the glucosyl residue (Fig. 1). This would indicate that the glucose unit is in close proximity to the Kdo ring and thus sterically shields the 4-OH group towards the incoming electrophile.
In addition to the Kdo-fluoride based glycosylation attempts, coupling of acceptor 2 with peracetylated Kdo bromide under Helferich conditions was also tested but did not lead to significant product formation. Previously, this approach had successfully been performed for the introduction of a lateral Kdo-unit onto a L-glycero-D-manno-heptosyl-(1→5)-Kdo glycosyl acceptor in 65% yield. 17

Approach B: Using an α-Kdo-(2→4)-Kdo acceptor
As an alternative approach, the sequence of glycosylation steps was inverted by attaching a suitably protected glucose donor to a preassembled α-Kdo-(2→4)-Kdo disaccharide acceptor. Thus, we could capitalize on the previously elaborated route towards Kdo 2 7, which was obtained in high yields and without any formation of β-products. 16a Recently, Ichiyanagi et al. successfully coupled a Kdo 2 5-OH acceptor with heptose and mannose trichloroacetimidate donors exploiting the trans-directing effect of C-2 ester groups. 15 First, the 5-OH acceptor 8 was obtained from iodo-precursor 7 by hydrogen atom transfer 18 in cyclohexane catalyzed by lauroyl peroxide. Previously, dehalogenation depended on fully blocked compounds to guarantee sufficient solubility in cyclohexane for smooth conversion. 16a Notably, using a mixture of cyclohexane and 1,2-dichloroethane 18 allowed for direct and neat dehalogenation of disaccharide 7 which provided acceptor 8 in high yield (92%).
In previous experiments, N-phenyltrifluoroacetimidate glucose donor 6 13 afforded excellent α-selectivity in the glycosylation of a 5-OH Kdo monosaccharide due to the directing effect of the 4,6-O-benzylidene group. 19 Coupling of 5-OH acceptor 8 with donor 6 in CH 2 Cl 2 under TMSOTf catalysis in the presence of ground 4 Å molecular sieves provided traces of both αand β-trisaccharides 9α and 9β. By exploiting the known 20 1,2-cis directing effect of ether solvents, the α/β-ratio could be increased significantly (from α/β = 1:1.2 in CH 2 Cl 2 to 2.7:1 in Et 2 O in preliminary experiments). The observation that glycoside formation ceased after a short initial phase led to the assumption that TfOH formed by partial hydrolysis of TMSOTf was the active promotor, which would then be rapidly scavenged by 4 Å molecular sieves. Indeed, applying TfOH as activator in the presence of 5 Å molecular sieves improved the total yield to 31%. This still disappointing outcome was attributed to donor degradation that proceeded faster than glycoside formation. Eventually, using inverse glycosylation conditions 21 in diethyl ether containing molecular sieves (5 Å) afforded a mixture of anomers 9α and 9β in a total yield of 91% (α/β = 4:1) which could be conveniently separated on an HPLC column (isolated yield of α-product 9α: 67%). The αanomeric configuration was readily assigned on the basis of the coupling constant J 1″,2″ (3.8 Hz). Lactone formation between C-1′ of the lateral Kdo unit and the free 5-hydroxyl group was also observed but to a low degree only. Sequential removal of benzyl (Pd/C, H 2 ) and acetyl (NaOMe) groups from trisaccharide 9α and ensuing methyl ester saponification under alkaline conditions (NaOH) gave trisaccharide 10 in 94% yield (Scheme 2).

Synthesis of oligoglucosyl fragments
Proceeding towards the oligoglucosyl core structures, regioselective opening of the benzylidene group in compound 9α to provide the 6-OBn protected derivative 11 was foreseen, to serve as an acceptor for the extended glucosyl core structure of A. haemolyticus.
Hence, treatment of trisaccharide 9α with triethylsilane/BF 3 ·Et 2 O 22 in dry dichloromethane afforded the desired 4-OH acceptor derivative 11 as the major product (72%) together with its 6-OH regioisomer 12 (5%) and traces of diol 13 (2%). By using the peracetylated glucopyranosyl NPTFA donor 14 23 under TfOH promotion (5 mol% for 15, 12.5 mol% for 18), tetrasaccharide 15 was obtained (Scheme 3). The isolated yield for the β-tetrasaccharide 14, however, did not exceed 46%, since several purification steps by HPLC were needed to produce a pure tetrasaccharide. Using the same strategy with the isomaltosyl donor 17, the pentasaccharide 18 was readily accessible in a 3+2 approach in a good yield (71%). Global deprotection afforded the oligosaccharide ligands 16 (99%) and 19 (90%) in excellent yields. The 13 C NMR data of the oligosaccharide (Table 1) are in good agreement with the LPS-oligosaccharides isolated from B. henselae ATCC 49882 T* and A. haemolyticus respectively. 9,10 Deviating assignments were only noted for C-7 of both Kdo units. 9

Conclusions and outlook
In summary, a straightforward method to prepare the α-Glcp-(1→5) ]-α-Kdo trisaccharide capitalizing on a Kdo disaccharide acceptor was established. Good αselectivity relied on diethyl ether as a solvent and high yields were obtained by slowly adding the donor to a pre-stirred mixture of the Kdo 2 acceptor and TfOH as a promotor. The 4,6-O-benzylidene blocking group of the donor eventually provided access to extended oligosaccharides after regioselective reductive opening. Subsequent elongation with 2-Oacetyl protected N-phenyl trifluoroacetimidate glucosyl donors allowed for the introduction of β-Glcp-(1→4)as well as α-Glcp-(1→6)-β-Glcp-(1→4)units. Thus, three fragments of the A. haemolyticus inner core were obtained which serve as ligands in binding studies with C-type collectins.
MeOH (Merck) and dry THF (Sigma-Aldrich) were purchased. Cation exchange resin DOWEX 50 H + was regenerated by consecutive washing with HCl (3 M