De novo synthesis of D- and L-fucosamine containing disaccharides

Summary The availability of rare monosaccharides that cannot be isolated from natural sources is currently limiting the access to the synthesis and the biological evaluation of complex bacterial cell-surface glycans. Here, we report the synthesis of D- and L-fucosamine building blocks by a de novo approach from L- and D-Garner aldehydes. These differentially protected monosaccharide building blocks were utilized to prepare disaccharides present on the surface of Pseudomonas aeruginosa bacteria.


De novo synthesis of D-and L-fucosamine containing disaccharides
Introduction Protein functions are directly influenced by their glycosylation patterns [1,2]. Therefore, an understanding of protein glycosylation is of utmost importance in order to develop new therapeutics [3][4][5][6]. The ability of bacteria to colonize human hosts and cause diseases is directly influenced by their capacity to synthesize glycoproteins and express them on cell surfaces. This evidence makes it particularly relevant especially for the identification of novel antibacterial agents as well as vaccines [7][8][9]. Those bacterial glycans often contain unusual monosaccharides that are not present in the human body. An immune response against these cell-surface glycans is the basis for the development of new vaccine candidates against bacterial infections [10][11][12][13].
Our efforts were directed to the development of new vaccine candidates [14][15][16] to prevent bacterial infections, including glycans of the highly pathogenic bacteria Pseudomonas aeruginosa. P. aeruginosa is a nosocomial pathogen that is involved in ventilator-associated pneumonia and has become resistant to many antimicrobials. The somatic pili of P. aeruginosa are a Scheme 1: Retrosynthetic analysis of D-and L-fucosamine building blocks.
major virulence factor playing a pivotal role in the adherence and invasiveness of the bacterium. In 2001, the P. aeruginosa pilin O-linked glycans were found to be linear trisaccharides that are covalently attached to serine ( Figure 1). The O-glycans contain a D-fucosamine residue at the protein-binding site. This unusual monosaccharide is not present in eukaryotes, and therefore may be used to stimulate an antibacterial response in the host organism [17][18][19][20][21][22][23]. Access to differentially protected D-and L-fucosamine building blocks, which can be used in preparing the corresponding glycans, is instrumental for the evaluation of oligosaccharide-based vaccine candidates against this bacterium [24][25][26][27][28].
Here, a full account of the de novo synthesis of differentially protected D-and L-fucosamine building blocks is described following a recent preliminary communication [47]. The building blocks prepared by de novo synthesis were used in the assembly of two disaccharides that are found on P. aeruginosa.

Results and Discussion
De novo synthesis of D-and L-fucosamine building blocks Our retrosynthetic analysis of D-fucosamine envisioned the installation of the syn-1,2-diol unit by osmium-catalysed dihydroxylation of allylic ether A. It was anticipated that the conformation adopted by the molecule would allow for the formation of the required anti relationship between C3 and C4 hydroxy groups. A in turn would be accessed by the addition of a carbon nucleophile to L-Garner aldehyde L-1 (Scheme 1) [48]. The fact that D-and L-Garner aldehydes are commercially available greatly facilitates the synthesis of both D-and L-fucosamine building blocks. The fucosamine residues are usually further elongated at the C3 position in P. aeruginosa requiring two orthogonal protecting groups (PGs) at C3 and C4 Scheme 2: (A) Synthesis of aldehydes 6a and 6b. (B) Alkyne reduction by hydrosilylation-protodesilylation sequence (see Table 1).
of the building block in order to differentiate the two hydroxy groups at a later stage.
The three carbon C4-C6 fragment of D-fucosamine was introduced by chelation-controlled addition [49,50] of commercially available propynylmagnesium bromide to L-1 (Scheme 2A) [51,52] The following E-selective alkyne reduction was accomplished by using diluted RedAl in Et 2 O [53]. This reaction proved to be highly dependent on the quality of the aluminium reagent obtained from commercial sources. Inspired by the work from the Trost group [54,55], an alternative reduction protocol based on a Ru-catalyzed hydrosilylation-protodesilylation sequence was pursued (Scheme 2B). Thus, exposure of alkyne 2 to Cp*Ru(CH 3 CN) 3 PF 6 catalyst in the presence of the appropriate trialkylsilane gave the desired silylated products 7a-c ( Table 1). The alkene geometry was confirmed as Z by nuclear Overauser effect (nOe). Treatment of 7a-c with TBAF and CuI delivered 3 in high yields. Generally, BnMe 2 SiH has been found to be optimal for these transformations [54,55]. In our hands however, the best results were achieved using the cheaper Et 3 SiH (Table 1). With a gram amount of 3 in hand, the C3 hydroxy group was protected and the acetonide removed by treatment with p-TSA. Oxidation of primary aminoalcohols 5a-b with the Dess-Martin reagent [56] yielded the desired aldehydes 6a-b, in five steps from commercially available starting materials. The two different O-protecting groups, namely naphthyl ether (Nap) [57][58][59] and benzoate ester (Bz), were introduced to gain access to two sets of electronically different and orthogonally protected derivatives (Scheme 2A).  [60][61][62]. The formation of the desired C3-C4-C5 syn,syn cyclic product was confirmed based on observation of a 3 J H3-H4 coupling of 3.5 Hz. When the same sequence of dihydroxylation-peracetylation was performed on Bz-substituted aldehyde 6b, compound D-8b was formed in 71% as a single diastereomer (Scheme 3). This product was crystallized from n-hexane/EtOAc solvent mixture, and the stereochemical assignment was confirmed by X-ray analysis (data shown in Supporting Information File 1).
Scheme 4: Epimerization of aldehyde 6a. During the optimization of the synthetic sequence we explored potentially more economic oxidation protocols to convert the primary alcohol 5a to the D-fucosamine building block. Since α-amino protected aldehydes easily undergo α-epimerization, oxidation had to proceed under mild conditions in order to avoid the formation of the undesired D-talosamine building block D-9 (Scheme 4).
Several oxidation methods were tested ( Table 2). All the reactions were performed sequentially without column-chromatographic purification of the aldehyde. Classic Swern oxidation [63] ( Table 2, entry 2) gave a 2:1 mixture of C2 epimers demonstrating the acid lability of aldehyde 6a. This ratio in favour of the desired product was improved to 8:1 by switching the base from Et 3 N to iPr 2 EtN as reported by Dondoni and co-workers ( Table 2, entry 3) [64]. Parrikh-Doering oxidation [65] and TCCA-TEMPO mediated oxidation [66] ( Table 2, entries 4 and 6, respectively) were not suitable as considerable amounts of D-talosamine building block were formed. DMP emerged as the reagent of choice for the oxidation of 5a. Interestingly, the dihydroxylation of aldehyde epi-6a resulted in the formation of talosamine D-9 as a single diastereomer while the dihydroxylation of 6a gave D-8a in 5:1 dr (Scheme 3).
Carrying out the synthetic sequence optimized for the synthesis of D-8a, on D-Garner aldehyde (D-1, commercially available) Scheme 5: Synthesis of L-fucosamine building block L-8a from D-Garner aldehyde.

Synthesis of fucosamine-containing disaccharides
To facilitate the use of the bacterial monosaccharides in the glycan microarray platform and their conjugation to carrier proteins, an appropriate linker was required at the anomeric position [67]. Placement of a C3 naphthyl ether anticipated the site for further glycosylation at this position, which typically serves as the connection to the next sugar. Therefore, building block 8a is ideal in terms of orthogonality and chemical synthesis. Hence, we tested the ability of D-8a to undergo anomeric functionalization and to effect glycosylation at the C3 hydroxy group. Due to the presence of N-acetylated D-and L-fucosamine residues in P. aeruginosa O-linked glycans, the strategic N-protecting group was evaluated. Direct glycosylation by using the building block D-8a was not possible. Hence, a direct N-Boc deprotection/N-acetylation sequence afforded D-10 in 84% yield (Scheme 6A). Direct glycosylation of D-10 by using glycosylating agent 11 [67] and BF 3  When the same synthetic sequence was performed on L-FucNAc building block L-10 the fully deprotected monomer L-13 and disaccharide 19 were obtained in similar yields and selectivities (Scheme 6B). Diagnostic 1 J C-H analysis also corroborated the formation of the β-glycosydic linkages (Scheme 6B). Linker-terminated disaccharide 19 represents the terminal unit of the P. aeruginosa serotype O11 O-linked glycan (Figure 1).

Conclusion
The de novo synthesis of differentially protected D-and L-fucosamine building blocks from D-and L-Garner aldehyde is reported. Placement of a naphthyl ether protecting group at the C3 position allows for further elongation by glycosylation.
The key oxidation step was optimized to minimize the formation of the unwanted D-talosamine building block D-9. The fucosamine building blocks prepared by de novo synthesis enabled the preparation of monosaccharides and disaccharides for attachment to microarray surfaces. The terminal disaccharide of P. aeruginosa O11 O-linked glycan has been prepared and will be the basis for biological studies involving this pathogen.

Experimental General experimental details
All reagents were obtained from commercial suppliers and were used without further purification unless otherwise specified. . Column chromatography was performed by using Kieselgel 60 (230-400 mesh) silica gel with a typical 50-100:1 weight ratio of silica gel to crude product. For the preparation and characterization of compounds 2-10, D-12 and D-16 see [14].

L-Fucosamine monosaccharide L-12
Under the same reaction conditions reported in [14],

D-Fucosamine monosaccharide D-13
A solution of D-12 (56 mg, 80.0 mmol, 1.0 equiv) in CH 2 Cl 2 (0.9 mL) and H 2 O (90 mL) was treated with DDQ (21 mg, 94.0 mmol, 1.2 equiv) and stirred in the dark at rt for 2 h. Saturated NaHCO 3 (2 mL) and CH 2 Cl 2 (5 mL) were added and the layers were separated. The aqueous layer was extracted with CH 2 Cl 2 (2 × 3 mL). The combined organic layers were dried (MgSO 4 ), filtered and concentrated. The crude was purified by filtration over a short plug of silica gel eluting with CH 2 Cl 2 /

L-Fucosamine disaccharide 18
A solution of L-12 (60 mg, 86 mmol, 1.0 equiv) in CH 2 Cl 2 (1 mL) and H 2 O (0.1 mL) was treated with DDQ (21 mg, 95 mmol, 1.2 equiv) and stirred in the dark at rt for 2 h. Saturated NaHCO 3 (2 mL) and CH 2 Cl 2 (5 mL) were added and the layers were separated. The aqueous layer was extracted with CH 2 Cl 2 (2 × 3 mL). The combined organic layers were dried (MgSO 4 ), filtered and concentrated. The crude was purified by filtration over a short plug of silica gel eluting with CH 2 Cl 2 / MeOH (9:1) and immediately used for the next step. An ovendried flask was immediately charged with the product and 15 (77 mg, 0.12 mmol, 1.5 equiv) was added. The flask was left under high vacuum for 4 h. After refilling the flask with argon, freshly distilled CH 2 Cl 2 (1.5 mL) was added, and the mixture was cooled to −30 °C. NIS (29 mg, 0.13 mmol, 1.5 equiv) and TfOH (4 mL, 43 mmol, 0.5 equiv) were added. The mixture was stirred at −30 °C for 4 h and then it was allowed to warm to rt overnight. Saturated Na 2 SO 3 (3 mL) and CH 2 Cl 2 (3 mL) were added and the layers were separated. The aqueous layer was extracted with CH 2 Cl 2 (2 × 5 mL

L-Fucosamine disaccharide 19
A solution of 18 (29 mg, 27.0 mmol, 1.0 equiv) in MeOH (0.6 mL) and THF (0.6 mL) was treated with KOH (0.8 mg, 13.5 mmol, 0.5 equiv) and stirred at rt for 30 min. H 2 O (2 mL) was added and the solvents were removed under vacuum. CH 2 Cl 2 (5 mL) was added and the layers were separated. The aqueous layer was extracted with CH 2 Cl 2 (2 × 5 mL). The combined organic layers were dried (MgSO 4 ), filtered and concentrated. The crude product was solubilised in MeOH/H 2 O/ AcOH