Synthesis of a Pentasaccharide Fragment Related to the Inner Core Region of Rhizobial and Agrobacterial Lipopolysaccharides

The pentasaccharide fragment α-d-Man-(1 → 5)-[α-d-Kdo-(2 → 4)-]α-d-Kdo-(2 → 6)-β-d-GlcNAc-(1 → 6)-α-d-GlcNAc equipped with a 3-aminopropyl spacer moiety was prepared by a sequential assembly of monosaccharide building blocks. The glucosamine disaccharide—as a backbone surrogate of the bacterial lipid A region—was synthesized using an 1,3-oxazoline donor, which was followed by coupling with an isopropylidene-protected Kdo-fluoride donor to afford a protected tetrasaccharide intermediate. Eventually, an orthogonally protected manno-configured trichloroacetimidate donor was used to achieve the sterically demanding glycosylation of the 5-OH group of Kdo in good yield. The resulting pentasaccharide is suitably protected for further chain elongation at positions 3, 4, and 6 of the terminal mannose. Global deprotection afforded the target pentasaccharide to be used for the conversion into neoglycoconjugates and “clickable” ligands.


■ INTRODUCTION
The outer membrane of the Gram-negative bacterial cell wall is covered to a large extent by lipopolysaccharide (LPS) molecules, which are involved in a multitude of major immune reactions in the context of bacterial infections. 1,2 In addition to their biomedical relevance, LPS structures are also engaged in important bacteria-plant interactions, e.g., leading to the formation of nodules in leguminous roots when colonized by Rhizobia. 3 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 Oantigenic polysaccharides. 4,5 This core domain may be further divided into an outer and an inner core region, wherein a 3deoxy-D-manno-2-octulosonic acid (Kdo) usually forms the link to the lipid A part. In many bacterial genera, this Kdo unit is then extended by a lateral α-(2 → 4)-linked Kdo moiety and further elongated at position 5 by additional inner core sugars such as L-glycero-D-manno-heptose (L,D-Hep), D-glucose, Dgalacturonic acid or D-mannose. 6 Specifically, 5-O-mannosylated Kdo has been found in bacteria such as Francisella tularensis, Brucella melitensis, Agrobacterium tumefaciens, Sinorhizobium meliloti and Rhizobiaceae. 2,7 In the latter strains, defects in the mannosyl transferase LpsC impairs their nodulation activity. 8,9 Recently, the structure of an oligomannose linked to O-5 of Kdo was reported for the inner core of the lipooligosaccharide (LOS) of Rhizobium radiobacter Rv3, also termed Agrobacterium radiobacter and Agrobacterium tumefaciens ( Figure 1). 10,11 Of note is the structural resemblance of the α-(1 → 2)-linked oligomannose part to the D1 arm of mammalian high-mannose glycans, which are particularly abundant on HIV-1 gp120. 12 Rhizobium radiobacter Rv3 LOS and heat-killed cells are bound by the oligomannosespecific HIV-neutralizing antibody 2G12 and a crystal structure of the carbohydrate backbone of Rhizobium radiobacter strain Rv3 complexed to the 2G12 antibody has recently been determined. 13 In order to further exploit the natural mimicry of high-mannose carbohydrate epitopes of HIV-1 gp120 seen with this bacterial LOS scaffold, we have set out to extend the bacterial oligosaccharides in order to elicit HIV neutralizing antibodies. In a first step we have prepared the basic branched core oligosaccharide in a sufficient scale to allow for regioselective mannosylation at O-3 and O-6 of the central mannose unit, respectively, as well as for global deprotection to give the pentasaccharide core fragment equipped with a functional spacer group to allow for conversion into neoglycoconjugates.

■ RESULTS AND DISCUSSION
The pentasaccharide was assembled starting from the β-(1 → 6)-linked diglucosamine backbone to be subsequently elongated by the Kdo residues. To simplify deprotection at the final stages, both 2-amino-2-deoxy-groups were protected as N-acetyl derivatives keeping in mind difficulties encountered in several reported glycosylation reactions describing the formation of the respective imidates. 14 Although these unwanted reactions were known, the variety of protecting groups had to be limited in order to minimize loss of material in the multistep synthesis. The reducing terminus was equipped with an αconfigured 3-aminopropyl spacer glycoside amenable to eventual coupling to proteins as well as suitable for conversion into the corresponding 3-azidopropyl derivatives to be used as substrate for ensuing "click"-chemistry. 15 Starting from known 16 3-azidopropyl glycoside 1, which was prepared according to an improved procedure in a multigram scale, 17 6-O-tritylation was achieved smoothly in 90% yield by treatment of 1 with trityl chloride in pyridine and catalytic 4dimethylaminopyridine (DMAP). Subsequent introduction of the 1,3-tetraisopropyldisiloxane-1,3-diyl group by reaction with Figure 1. Chemical structure of the inner core and lipid A region of R. radiobacter Rv3 LOS. 10 Dotted lines indicate substoichiometric substitution. The targeted pentasaccharide is marked in red.

Scheme 1. Synthesis of the β-(1 → 6)-Linked Diglucosamine Acceptor 11 via Two Different Routes
The Journal of Organic Chemistry Article TIPDSCl 2 in CH 2 Cl 2 and imidazole gave the crystalline 3,4-Osilyl ether 3 in 87% yield (Scheme 1). Removal of the trityl group could be achieved by treatment with silica-supported NaHSO 4 . In the process partial migration of the TIPDS-group to give the 4,6-O-substituted derivative was observed which then required cumbersome chromatography to separate the two resulting compounds. 18 Hence, as an alternative, the mild trityl deprotection using BCl 3 in CH 2 Cl 2 at −20°C and quenching of the reaction with triethylamine was carried out and afforded compound 4 in a gratifying 93% yield. 19 Next, assembly of the β-(1 → 6)-linked diglucosamine backbone was performed on a multigram-scale. In the presence of FeCl 3 the 2-chloroacetamido-2-deoxy-β-1-O-acetyl derivative 5 generates an 1,3-oxazoline intermediate and thus secures the formation of the β-anomeric product. 20 This way, the crystalline β-(1 → 6)-linked disaccharide 6 was isolated in 77% yield; the β-anomeric configuration was confirmed by the value of the homonuclear J 1,2 coupling constant (8.0 Hz). Subsequent conversion of the chloroacetamido group into the N-acetylated form was challenging. Reaction of 6 with thiourea afforded the corresponding N-acetyl derivative; the procedure, however, was difficult to reproduce and consistently led to thiocontaining byproducts which were difficult to separate from the product. 21 As an alternative, which concomitantly allowed an early stage removal of the silyl protecting group, hydrolysis of all protecting groups followed by ensuing O-and N-acetylation was performed. The five-step sequence included reaction of the 2-chloroacetamido group with pyridine 22 at 90°C, followed by ester and amide hydrolysis with aqueous NaOH. The crude product was subsequently O,N-acetylated followed by removal of the TIPDS group with TBAF in THF and O-acetylation to afford the penta-O-acetyl derivative 8 in 66% yield (over 5 steps). Having established this reaction sequence, it was decided to further simplify it by exploiting the higher reactivity of the primary hydroxyl group in N-acetyl glucosamine glycosyl acceptor derivatives. 23 Direct glycosylation of the triol glycoside 1 with donor 5 in nitromethane/DCM in the presence of FeCl 3 afforded the disaccharide 7 in acceptable yields (45%). Conversion of the chloroacetamido group into the N-acetyl group was performed similar to compound 6without the need to remove the silyl ether groupand eventually gave disaccharide 8 in 81% yield. Overall yields (Route A 37%; Route B 36%) for both pathways were comparable, but route B saved four steps in protecting group manipulation.
In order to selectively protect the primary hydroxy group of the distal glucosamine unit, compound 8 was subjected to Zempleń transesterification affording 9 in near theoretical yield. Next, the 6′-OH group was selectively protected with a TBDPS-group using TBDPSCl and Huenig's base in dry DMF, followed by a one-pot protection of the remaining OH-groups by acetylation with acetic anhydride in pyridine to deliver 10 in 85% yield. Silyl ether cleavage was carried out using HFpyridine as reagent at 0°C. This way, 4 → 6 O-acetyl migration could be minimized and the resulting glycosyl acceptor 11 was obtained in 77% yield by chromatography. The crude product, however, was of sufficient purity and could be directly subjected to glycosylation reaction with Kdo donors.
Glycosylation reactions with glycosyl donors of Kdo suffer from lack of anomeric selectivity, low reactivity due the electron-withdrawing ester group and facile formation of elimination products such as glycal ester 16. 24 A limited number of groups have successfully accomplished the challenging glycosylation of the unreactive axial 5-OH group at the proximal unit of the central bacterial Kdo disaccharide α-D-Kdo-(2 → 4)-α-D- Kdo. 25 The synthesis of the resulting 4,5disubstituted oligosaccharides is further complicated by the facile formation of 1′ → 5 interlinked Kdo lactone derivatives under acidic conditions, which would then block glycoside formation. Recently, the per-O-acetylated 3-iodo-Kdo fluoride donor 17 has successfully been employed for the assembly of oligomeric Kdo derivatives. 26 Reactions of 17 with disaccharide acceptor 11 promoted either by BF 3 ·Et 2 O or TMSO-triflate in dichloromethane, however, were not successful and mainly unreacted educts were recovered from the reaction mixtures. Hence, the known 4,5;7,8-di-O-isopropylidene donor 15 -envisaged to be sufficiently α-selectivewas selected for the glycosylation steps. 27 Donor 15 was prepared via a route different from the previously published procedure, which had been based on direct fluorination of a 4,5;7,8-di-O-isopropylidene protected Kdo hemiketal. 28 First, treatment of the peracetylated Kdo methyl ester 12 with HF-pyridine afforded known fluoride 13, 29 which was then subjected to Zempleń transesterification to give the tetraol fluoride 14, suitable for further modification and regioselective introduction of protecting groups. Compound 14 is rather unstable and was therefore immediately transformed into the bench stable donor 15 in 64% yield (over two steps from 13) using camphorsulfonic acid and 2-methoxypropene (Scheme 2).
Coupling of Kdo fluoride donor 15 (2 equiv) to disaccharide acceptor 11 was accomplished using 2.2 equiv of BF 3 ·Et 2 O as promoter in dichloromethane and in the presence of molecular sieves 4 Å. Trisaccharide 18 was obtained in 64% yield (2 steps, based on 10) after separation from minor byproducts, unreacted educt as well as glycal ester 16 by silica gel chromatography. Next the acetonide groups were hydrolyzed without affecting the acid-labile ketosidic linkage of Kdo by treatment with p-toluenesulfonic acid monohydrate (PTSA) in dry MeOH in 93% crude yield. Unfortunately silica gel chromatography did not lead to separation of the product from PTSA, and the use of basic ion-exchange resin (HCO 3 − form) led to partial saponification of the acetate groups.
Thus, the milder CeCl 3 -hydrate/oxalic acid had to be used, affording the tetraol 19 in 76% yield after column chromatography. 30 Cleavage of the isopropylidene groups released the steric strain of the skew-boat Kdo conformation in compound 18 and regenerated the 5 C 2 conformation in compound 19, which then allowed to confirm the α-anomeric configuration of the Kdo unit on the basis of 1 H NMR chemical shifts of the 3-deoxy protons as well as 13 C NMR data of C-4 and C-6, respectively. 31 In order to keep the number of protecting groups at a minimum, the side-chain diol system of Kdo was then protected by a carbonate ester group, based on previous experience in the synthesis of Kdo oligomers. 23 Conversion of 19 into the 7,8-O-carbonyl protected trisaccharide 20 had to be performed under carefully controlled conditions. A solution of diphosgene in THF was slowly added at −50°C to −40°C to a solution of 19 and symcollidine in THF. In this way compound 20 could be isolated in yields from 60 to 80% with minor formation of the 4,5;7,8dicarbonyl derivative (∼10%) and recovery of unreacted 19 (∼10%). Whereas the introduction of the first Kdo unit could be accomplished in good yields, coupling of the lateral Kdo residue to trisaccharide acceptor 20 was less straightforward (Scheme 3, Table 1).
Promotion of the glycosylation step in the presence of BF 3 · Et 2 O and molecular sieves 4 Å in dichloromethane afforded modest yields of tetrasaccharide 21. The main byproduct of the reaction was identified as the 4,5-O-substituted monoisopropylidene trisaccharide 22 arising from isopropylidene cleavage and transfer to the diol 20. Variation of the reaction time and temperature (see Table 1, entries 1−4) did not lead to any reduction of byproduct formation and even the addition of base (Table 1, entry 4, 5) just slowed down the reaction rate without affecting the product distribution.
Fortunately, byproduct 22 can be reconverted into acceptor 20 by treatment with a 1:1 mixture of DCM/90% aq TFA. Thus, despite the progress in Kdo glycosylation chemistry, generally applicable, selective and high-yielding methods still need to be developed for synthesizing complex Kdo glycosides.
The orthogonally protected mannosyl donor 26, suitable for selective deprotection of positions 3 and 6 was elaborated from known thioglycoside 23 following published procedures (Scheme 4). 32 First, compound 23 was subjected to 6-Osilylation using TBS-chloride and DMAP in pyridine which gave the silyl ether 24 in 93% yield. Next, the thioglycoside  a BF 3 ·Et 2 O was slowly added during 20 min using a syringe pump. b Reaction was carried out with 1 equiv of NEt 3 . c Reaction was carried out with 1 equiv of DIPEA.

The Journal of Organic Chemistry
Article aglycon was removed by treatment of 24 with NIS/TFA to produce 25 in 92% yield, followed by subsequent introduction of the anomeric trichloroacetimidate according to R. Schmidt 33 which provided donor 26 in 88% yield. Proceeding toward the branched pentasaccharide 27, donor 26 and acceptor 22 were first reacted for 3 h with 0.1 equiv TMSOTf as promotor in the presence of molecular sieves 4 Å in dry DCM at 0°C resulting in a mixture of several isomers. One byproduct was isolated by HPLC purification and was identified as the imidate-connected pseudopentasaccharide. The structure of the imidate was proven by NMR data. One NH proton signal was missing and a low-field shifted signal was observed at 6.53 ppm which gave an HMBC-correlation to a 13 C NMR signal at 162.0 ppm being characteristic of imidate groups. 34 The nucleophilic properties of the amide oxygen of glucosamines had previously been described and higher amounts of promoter as well as higher temperatures were suggested to suppress these side reactions. 14b Hence, the mannosylation reaction was repeated at room temperature in the presence of 0.3 equiv TMSOTf affording the desired product 27 in a good yield of 70% (Scheme 5). Unreacted glycosyl acceptor 21 could be recovered in 20% yield. No βproduct was isolated and the α-anomeric configuration of the mannosyl residue was proven by the value of the heteronuclear coupling constant J C-1,H-1 (171 Hz). 35 Deprotection of the two isopropylidene groups had to be carefully established in order to avoid concomitant hydrolysis of the acid-sensitive Kdolinkages. Treatment of 27 with CeCl 3 /oxalic acid selectively    13 C NMR data of compound 30 (Table 2) could be fully assigned and were in perfect agreement (when correcting for different referencing of chemical shifts) with published data of a related pentasaccharide fragment from Agrobacterium tumefaciens A1. 7c Significant low-field shifts were observed for C-4 and C-5 of the branched Kdo residue, whereas the data of the lateral Kdo unit were consistent with similar NMR characteristics of α-(2 → 4)-linked Kdo disaccharide fragments. 36 In conclusion, a central fragment of rhizobial and agrobacterial inner core LPS has been synthesized in a limited number of steps relying mainly on ester, benzyl-type and isopropylidene protecting groups. The latter protecting group was not fully compatible with glycosylation conditions although the bis-isopropylidene protected Kdo fluoride donor was sufficiently reactive and α-selective in the glycosylation steps. Extension of this work toward the oligomannosidic fragments and neoglycoconjugates will be published in due course.
3-Azido-1-propyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1 → 6)-2-acetamido-2-deoxy-α-D-glucopyranoside (9).     13  Methyl (3-deoxy-4,5;7,8-di-O-isopropylidene-α-D-mannooct-2-ulopyranosyl)onate fluoride (15). A solution of 13 (3.65 g, 8.65 mmol, prepared from 12 according to ref 28) was dissolved in dry MeOH (90 mL) under Argon and was cooled to 0°C. Subsequently 0.1 M NaOMe (43 mL; 4.3 mmol) was added and the solution was stirred for 6 h at 0°C. The reaction was quenched by the addition of DOWEX 50 cation exchange resin (H + -form) to give pH 7.5. The resin was filtered off and the filtrate was concentrated to give unstable intermediate 14, which was dried in vacuo for 1 h. The residue was then dissolved in dry DMF (40 mL) under Ar followed by the addition of CSA (0.703 g; 3.03 mmol) and 2-methoxypropene (8.14 mL; 86.51 mmol). The solution was stirred at rt for 16 h. Aqu satd NaHCO 3 was added and the mixture was extracted with CH 2 Cl 2 (3×). The combined organic phases were dried (Na 2 SO 4 ) the solvent was removed in vacuo and the crude product was purified by silica flash chromatography (n-hexane/EtOAc 5:1 → 2.2:1) to give 15 (1.99 g; 69%) as colorless amorphous solid. Analytical data were in agreement with literature. 28 (18). A solution of 10 (1.3 g; 1.42 mmol) in dry THF (14 mL) under Argon was cooled to 0°C and a solution of HF·pyridine (2.96 mL; 14.2 mmol; 70% HF) was added dropwise. The solution was stirred for 16 h and was subsequently quenched by the addition of 10% aq KF. The solution was extracted with CH 2 Cl 2 (3×), the combined organic phases were dried (Na 2 SO 4 ) and the solvent was removed in vacuo followed by coevaporation of the crude with toluene (2 × 10 mL) and additional drying in vacuum (1.5 h). The crude product 11 and 15 (0.951 g; 2.84 mmol) were dissolved in dry CH 2 Cl 2 (5 mL) under Argon, molecular sieves 4 Å (0.5 g) were added and the suspension was stirred for 1 h at rt. The reaction mixture was cooled to 0°C followed by the addition of BF 3 ·Et 2 O (0.65 mL; 3.13 mmol). After stirring for 90 min at 0°C, the reaction was quenched by the addition of NEt 3 (1.5 mL) and the suspension was filtered over Celite. The filtrate was washed with aqu satd NaHCO 3 and the aqueous phase was extracted with CH 2 Cl 2 (2×). The combined organic phases were dried (Na 2 SO 4 ), the solvent was removed in vacuo and the crude was purified by flash chromatography (EtOAc/MeOH 20:1) to afford 18