Synthesis of the Tetrasaccharide Motif and Its Structural Analog Corresponding to the Lipopolysaccharide of Escherichia coli O75

Background Extraintestinal pathogenic E. coli are mostly responsible for a diverse spectrum of invasive human and animal infections leading to the urinary tract infections. Bacterial lipopolysaccharides are responsible for their pathogenicity and their interactions with host immune responses. In spite of several breakthroughs in the development of therapeutics to combat urinary tract infections and related diseases, the emergence of multidrug-resistant bacterial strains is a serious concern. Lipopolysaccharides are attractive targets for the development of long-term therapeutic agents to eradicate the infections. Since the natural sources cannot provide the required amount of oligosaccharides, development of chemical synthetic strategies for their synthesis is relevant to gain access to a reservoir of oligosaccharides and their close analogs. Methodology Two tetrasaccharide derivatives were synthesized from a single disaccharide intermediate. β-d-mannoside moiety was prepared from β-d-glucoside moiety following oxidation–reduction methodology. A [2+2] stereoselective block glycosylation strategy has been adopted for the preparation of tetrasaccharide derivative. α-d-Glucosamine moiety was prepared from α-d-mannosidic moiety following triflate formation at C-2 and SN 2 substitution. A one-pot iterative glycosylation exploiting the orthogonal property of thioglycoside was carried out during the synthesis of tetrasaccharide analog. Results Synthesis of the tetrasaccharide motif (1) and its structural analog (2) corresponding to the lipopolysaccharide of Escherichia coli O75 was successfully achieved in excellent yield. Most of the reactions are clean and high yielding. Both compounds 1 and 2 were synthesized as their 4-methoxyphenyl glycoside, which can act as a temporary anomeric protecting group for further use of these tetrasaccharides in the preparation of glycoconjugates.


Introduction
Escherichia coli (E. coli) are opportunistic pathogen belong to the Gram-negative Enterobacteriaceae. Pathogenic E. coli are the major causative agents for a number of extra intestinal infections in humans and animals [1]. One of the most frequently occurring E. coli infections is urinary tract infections. Escherichia coli strains are responsible for 60 to 80% of community acquired urinary tract infections in children and adults [2,3]. Extra intestinal pathogenic E. coli are mostly responsible for a diverse spectrum of invasive human and animal infections leading to pyelonephritis in the developing and developed countries [4,5,6]. The mechanism of such kind of ascending urinary tract infections has been explained based on the interactions between E. coli adhesion and their uroepithelial receptor ligands [7]. The most of the urinary tract infections found in human are caused by a small number of E. coli O-serogroups e.g. O4, O6, O14, O22, O75 and O83 [8]. Furthermore, they have phenotypes that are epidemiologically associated with cystitis and acute pyelonephritis in the normal urinary tract [9,10]. In this context, a revised structure of the E. coli O75 lipopolysaccharide has been reported by Erbing et. al. [11] ( Figure 1). Bacterial lipopolysaccharides play vital roles for their pathogenicity and their interactions with host immune responses.
In spite of several breakthroughs in the development of therapeutics to combat urinary tract infections and related diseases, the emergence of multi drug resistant bacterial strains is a serious concern. The epidemiological data for the urinary tract infections caused by multi-drug resistant E. coli O75 and other strains in the developed and developing countries have been well documented [12][13][14]. Bacterial lipopolysaccharides and their fragments have been used to prepare several glycoconjugate derivatives towards the development of long term therapeutic agents to eradicate the infections [15][16][17]. In order to establish a clear understanding on the biological potential of the lipooligosaccharide of a particular strain and its glycoconjugates, it is essential to carry out several biological experiments which require pure oligosaccharide in large quantity. Since the natural sources can not provide the required amount of the oligosaccharides, development of chemical synthetic strategies for their synthesis are relevant to get access to a reservoir of oligosaccharides and their close analogs. As a part of the ongoing studies on the synthesis of oligosaccharides of bacterial origin for their use in the preparation of glycoconjugate derivatives, concise chemical synthetic strategies for the synthesis of tetrasaccharide repeating unit (1) (Figure 2) corresponding to the lipopolysaccharide of Escherichia coli O75 and its close tetrasaccharide analog (2) (Figure 3) are reported herein. The difference between tetrasaccharides 1 and 2 is that the Dglucosamine moiety is 1,2-trans linked in compound 1 whereas it is 1,2-cis linked in compound 2. Both tetrasaccharides 1 and 2 were synthesized as their 4-methoxyphenyl (PMP) glycosides.

Results and Discussion
The synthesis of the tetrasaccharide 1 and its close structural analog 2 as their 4-methoxyphenyl glycosides was achieved by series of stereoselective glycosylations of a number of suitably functionalized monosaccharide derivatives 4, 5 [18], 6, 7, 8 [19], and 9 [20] prepared from the commercially available reducing sugars using synthetic methodologies reported earlier. Since, the preparation of b-linked D-mannose moiety from D-mannose and a-D-glucosamine moiety from D-glucosamine are challenging issues, these two moieties are successfully prepared using b-Dglucosyl moiety and a-D-mannosyl moiety respectively as the precursors after completion of the glycosylations with required stereochemical outcome. The key features of this synthetic strategy include, (a) use of a common disaccharide derivative 11 for the preparation of both 1 and 2; (b) convenient conversion of b-Dglucoside moiety to b-D-mannoside moiety using Dess-Martin periodinane oxidation of C-2 followed by sodium borohydride reduction of the keto-group; (c) [2+2] stereoselective block glycosylation; (d) use of a-D-mannosidic moiety as a precursor for a-D-glucosamine moiety; (e) triflate formation followed by S N 2 substitution by azido group at C-2 position of a-D-mannosidic moiety; (f) high yield in most of the intermediate steps.
Stereo selective glycosylation of compound 4 with glucosyl trichloroacetimidate derivative 5 [18] in the presence of trifluoromethane sulfonic acid (TfOH) [25]  The D-glucopyranosyl moiety in compound 10 was converted into a D-mannopyranosyl moiety by epimerization at C-2. For this purpose, removal of acetyl group in compound 10 using sodium methoxide and oxidation of the hydroxyl group using Dess-Martin Periodinane [26] followed by reduction of the resulting keto group using sodium borohydride [27] resulted in the b-D-mannopyranosyl moiety, which was acetylated to give compound 11 in 76% overall yield. The spectral analysis of compound 11 supported its formation [ Since the presence of b-D-glucosidic moiety in compound 10 was unambiguously confirmed from the NMR spectra and the b-D-mannosidic moiety of the compound 11 was prepared from the b-D-glucosidic moiety of compound 10 by oxidation-reduction at the C-2 center without affecting the stereochemistry at the glycosyl linkages, the glycosyl linkage of the D-mannosidic moiety in compound 11 remained as b-linked (1,2-cis). Compound 11 was treated with palladium chloride [28] to remove allyl group to give compound 12 in 76% yield. In another set of experiment, the O-acetyl group of compound 11 was transformed into benzyl group on treatment with benzyl bromide in the presence of solid sodium hydroxide [23] to give compound 13 in 90% yield, which was treated with palladium chloride [28]  Stereoselective glycosylation of thioglycoside derivative 6 with thioglycoside derivative 7 in the presence of a combination of Niodosuccinimide (NIS) and trifluoromethane sulfonic acid (TfOH)  [29,30] furnished disaccharide thioglycoside derivative 15 [31] in 77% yield exploiting the concept of ''Relative reactivity values'' of thioglycosides discussed in the earlier report [31] (Scheme S3).
Using block synthetic strategy, stereoselective glycosylation of disaccharide thioglycoside 15 with disaccharide acceptor 12 in the presence of a combination of NIS and TfOH [29,30] in the 13 C NMR spectra]. Compound 16 was subjected to a series of reactions involving (a) transformation of Nphthalimido group to acetamido group by hydrazinolysis [31] followed by N-acetylation; (b) removal of benzyl groups and benzylidene acetal by exhaustive hydrogenolysis over pearlman's catalyst [32] and finally, (c) saponification using sodium methoxide to furnish target tetrasaccharide 1 in 57% overall yield. Spectral analysis of compound 1 unambiguously supported its formation [ In another synthetic strategy for the synthesis of compound 2, a tetrasaccharide derivative 18 was prepared using a convergent reaction protocol, which involves iodonium ion promoted iterative stereoselective glycosylations in one-pot. Stereoselective condensation of thioglycoside 8 and thioglycoside 9 in the first step of the iterative glycosylation using NIS-TfOH [29,30] as promoter furnished disaccharide thioglycoside derivative 17 following the principle of ''armed-disarmed'' glycosylation concept [33,34]. Immediate reaction of the in situ generated disaccharide thioglycoside derivative 17 with the disaccharide acceptor 14 in the presence of same activator present in the reaction pot led to the formation of tetrasaccharide 18 in 71% overall yield together with a minor quantity (,8%) of other isomeric product generated from the first step of glycosylation, which was separated by column chromatography. In the first step of the iterative glycosylation, although both compound 8 and 9 are thioethyl glycosides, presence of electron donating benzyl group at C-2 makes compound 9 activated or armed to act as glycosyl donor, whereas compound 8 acted as glycosyl acceptor because of the deactivation due to the presence of electron withdrawing O-acetyl group at C-2. In the second step, required 1,2-trans glycosylated product was obtained due to the presence of O-acetyl group at C-2 of the in situ formed disaccharide donor 17. The formation of tetrasaccharide derivative 18 was unambiguously confirmed from its spectral In summary, synthesis of the tetrasaccharide motif (1) and its structural analog (2)

General methods
All reactions were monitored by thin layer chromatography over silica gel coated TLC plates. The spots on TLC were visualized by warming ceric sulphate (2% Ce(SO 4 ) 2 in 2N H 2 SO 4 ) sprayed plates in hot plate. Silica gel 230-400 mesh was used for column chromatography. 1D and 2D NMR spectra were recorded on Bruker Avance 500 and 600 MHz spectrometer using CDCl 3 and CD 3 OD as solvents and TMS as internal reference unless stated otherwise. Chemical shift value is expressed in d ppm. ESI-MS were recorded on a Micromass mass spectrometer. Elementary analysis was carried out on Carlo Erba analyzer. Optical rotations were measured at 25 uC on a Jasco-P 2000 polarimeter. Commercially available organic solvents of adequate purity are used in all reactions.

4-Methoxyphenyl 3-O-allyl-2,6-di-O-benzyl-a-Dgalactopyranoside (4)
A solution of compound 3 (5.0 g, 11.0 mmol) in 0.1 M CH 3 ONa in CH 3 OH (50 mL) was stirred at room temperature for 3 h and neutralized with Amberlite IR 120 (H + ) resin. The reaction mixture was filtered and concentrated under reduced pressure. To a solution of the crude product in anhydrous DMF (15 mL) were added 2,2-dimethoxypropane (3 mL, 24.4 mmol) and p-TsOH (250.0 mg) and reaction mixture was allowed to stir at room temperature for 12 h. The reaction mixture was cooled to 0 uC and powdered NaOH (2.0 g, 50.0 mmol) was added to it followed by benzyl bromide (2.6 mL, 21.9 mmol) and the reaction was stirred at room temperature for 4 h. The reaction mixture was diluted with water (150 mL) and the extracted with EtOAc (26100 mL). The organic layer was washed with water, dried (Na 2 SO 4 ) and concentrated. A solution of the crude benzylated product in 80% AcOH (100 mL) was stirred at 80 uC for 1.5 h. The solvents were evaporated under reduced pressure and coevaporated with toluene and the crude product was passed through a short pad of SiO 2 . To a solution of the dihydroxyl compound in anhydrous CH 3 OH (150 mL) was added Bu 2 SnO (4.0 g, 16.07 mmol) and the reaction was allowed to stir at 70 uC for 3 h. The solvents were removed under reduced pressure and the stannylidene acetal was dissolved in dry DMF (10 mL). To the solution of the crude product were added allyl bromide (1.4 mL, 16.2 mmol) and Bu 4 NBr (500 mg) and the reaction mixture was allowed to stir at 60 uC for 8 h. The reaction mixture was diluted with water and extracted with EtOAc (26100 mL). The organic layer was washed with 1 N HCl, satd. NaHCO 3 and water in succession, dried (Na 2 SO 4 ) and concentrated. The crude product was purified over SiO 2 using hexane-EtOAc (4:1) as eluant to give pure compound 4 (3.

4-Methoxyphenyl (2-O-acetyl-3,4,6-tri-O-benzyl-b-Dmannopyranosyl)-(1R4)-3-O-allyl-2,6-di-O-benzyl-a-Dgalactopyranoside (11)
A solution of compound 10 (2.5 g, 2.55 mmol) in 0.1 M CH 3 ONa in CH 3 OH (25 mL) was stirred at room temperature for 2 h and neutralized with Amberlite IR 120 (H + ) resin. The reaction mixture was filtered and concentrated. To a solution of the deacetylated product in anhydrous CH 2 Cl 2 (15 mL) was added Dess-Martin Periodinane (2.0 g, 4.72 mmol) and the reaction mixture was allowed to stir at room temperature for 1 h. The reaction mixture was diluted with CH 2 Cl 2 (100 mL) and the organic layer was successively washed with 5% Na 2 S 2 O 3 , satd. NaHCO 3 and water, dried (Na 2 SO 4 ) and concentrated under reduced pressure. To a solution of the crude keto product in CH 3 OH (50 mL) was added NaBH 4 (1.5 g, 39.65 mmol) and the reaction mixture was allowed to stir at room temperature for 12 h. The solvents were removed under reduced pressure and the crude mass was dissolved in CH 2 Cl 2 (100 mL). The organic layer was successively washed with 1 N HCl, satd. NaHCO 3 and water, dried (Na 2 SO 4 ) and evaporated to dryness. A solution of the crude epimerized product in acetic anhydride-pyridine (10 mL, 1:1 v/v) was kept at room temperature for 2 h. The solvents were removed under reduced pressure and the crude product was purified over SiO 2 using hexane-EtOAc

4-Methoxyphenyl (2-O-acetyl-3,4,6-tri-O-benzyl-b-Dmannopyranosyl)-(1R4)-2,6-di-O-benzyl-a-Dgalactopyranoside (12)
To a solution of compound 11 (900.0 mg, 0.92 mmol) in anhydrous CH 3 OH (5 mL) was added PdCl 2 (100.0 mg, 0.56 mmol) and the reaction mixture was allowed to stir at room temperature for 1 h. The solvents were removed under reduced pressure and the crude product was purified over SiO 2 using hexane-EtOAc (4:1) to give pure compound 12 (660.0 mg, 76%). To a solution of compound 11 (900.0 mg, 0.92 mmol) in THF (5 mL) were added powdered NaOH (0.2 g, 5.0 mmol), benzyl bromide (250 mL, 2.10 mmol) and Bu 4 NBr (25.0 mg) and reaction mixture was allowed to stir at room temperature for 3 h. The reaction mixture was diluted with CH 2 Cl 2 (50 mL) and the organic layer was washed with water, dried (Na 2 SO 4 ) and concentrated under reduced pressure. The crude product was purified over SiO 2 using hexane-EtOAc To a solution of compound 13 (800.0 mg, 0.78 mmol) in anhydrous CH 3 OH (5 mL) was added PdCl 2 (90.0 mg, 0.51 mmol) and the reaction mixture was allowed to stir at room temperature for 1 h. The solvents were removed under reduced pressure and crude product was purified over SiO 2 using hexane-EtOAc To a solution of compound 12 (600.0 mg, 0.64 mmol) and compound 15 (550.0 mg, 0.77 mmol) in anhydrous CH 2 Cl 2 (10 mL) was added MS 4 Å (1.5 g) and the reaction mixture was stirred under argon at room temperature for 30 min. The reaction mixture was cooled to 230 uC and NIS (210.0 mg, 0.93 mmol) and TfOH (2 mL) were added to it. After stirring at same temperature for 1 h the reaction mixture was filtered through a CeliteH bed and washed with CH 2 Cl 2 (50 mL). The organic layer was successively washed with 5% Na 2 S 2 O 3 , satd. NaHCO 3 and water, dried (Na 2 SO 4 ) and concentrated under reduced pressure. The crude product was purified over SiO 2 using hexane-EtOAc  (2) A solution of compound 18 (650.0 mg, 0.38 mmol) in 0.1 M CH 3 ONa in CH 3 OH (10 mL) was allowed to stir at room temperature for 1 h and neutralized with Amberlite IR 120 (H + ) resin. The reaction mixture was filtered and concentrated. To a solution of the deacetylated product in anhydrous CH 2 Cl 2 (5 mL) were added pyridine (2 mL) and triflic anhydride (200 mL, 1.19 mmol) and the reaction mixture was stirred at 210uC for 1 h. The solvents were removed under reduced pressure and triflate derivative was dissolved in HMPT-DMF (6 mL, 2:1 v/v). To the solution of the triflate derivative was added NaN 3 (500.0 mg, 7.69 mmol) and the reaction mixture was allowed to stir at 90 uC for 8 h. The reaction mixture was diluted with water and extracted with EtOAc (100 mL). The organic layer was successively washed with satd. NaHCO 3 and water, dried (Na 2 SO 4 ) and concentrated to give the crude product, which was passed through a short pad of SiO 2 . To a solution of the azido derivative in CH 3 OH (15 mL) was added 20% Pd(OH) 2 -C (200.0 mg) and the reaction mixture was stirred at room