Synthesis of Pseudooligosaccharides Related to the Capsular Phosphoglycan of Haemophilus influenzae Type a

Synthesis of spacer-armed pseudodi-, pseudotetra-, and pseudohexasaccharides related to the capsular phosphoglycan of Haemophilus influenzae type a, the second most virulent serotype of H. influenzae (after type b), was performed for the first time via iterative chain elongation using H-phosphonate chemistry for the formation of inter-unit phosphodiester bridges. These compounds were prepared for the design of neoglycoconjugates, as exemplified by the transformation of the obtained pseudohexasaccharide derivative into a biotinylated glycoconjugate suitable for use in immunological studies, particularly in diagnostic screening systems as a coating antigen for streptavidin-coated plates and chip slides.


Introduction
Among the numerous types of the extracellular pathogenic bacteria Haemophilus influenzae, the six encapsulated types are referred to as H. influenzae serotypes a-f [1]. Each of these bacteria has a unique glycan capsule which is associated with virulence and invasive bacterial infections [2]. In the pre-vaccine era, H. influenzae type b (Hib) contributed substantially to the burden of meningitis and other life-threatening infections in young children in the USA and Europe [3,4], while the other serotypes were considered non-invasive. After the conjugate Hib vaccine [5][6][7][8][9][10] was introduced into childhood immunization schedules, an epidemiological shift to other strains belonging to serotypes H. influenzae type a (Hia) [11][12][13][14][15][16], e [11,17], and f [11,18] was observed worldwide.
Hia was found to be the second most virulent serotype of H. influenzae (after Hib) [19]. It is a causative agent of meningitis, bacteremia, and pneumonia and affects mostly children under two years of age [15]. The rate of serotype replacement with Hia is impressive: in Brazil, a year after the implementation of Hib immunization, the surveillance for H. influenzae meningitis cases showed an eight-fold increase in the incidence of Hia meningitis [16]. Therefore, there is a clear need for a Hia testing system to monitor capsule replacement of Hia in countries where the Hib vaccine is a part of the routine childhood immunization schedule. As capsular glycans (CGs) are the principal bacterial antigens that induce high levels of serum immunoglobulins [20], this testing system is expected to be a convenient serological surveillance tool for the identification and monitoring of the disease agent.
The structure of Hia CG was published in 1977 [21] and consists of a linear phosphoglycan ( Figure 1, structure A) with a β-D-Glcp-(1→4)-D-Rib-ol-5-PO 4 →4repeating unit. In 1988, two isomeric pseudodisaccharides related to a Hia CG repeating unit [22] phosphorylated either at O-3 of the glucose residue or O-5 of the ribitol residue with triazol-activated phosphate precursors [23] were prepared. Also, the synthesis of a series of Hia CG-related pseudooligosaccharides through the use of the phosphoramidite approach for the formation A characteristic feature of the structure of Hia CG is the presence of a phosphodiester bridge between the glucose and the ribitol unit. Currently, there are two main approaches for the synthesis of carbohydrate derivatives with phosphodiester fragments: the H-phosphonate [25] and the phosphoramidite [26] methods. The latter was used in the published synthesis of a pseudodisaccharide related to a Hia CG repeating unit [22].
In the present paper, we report the synthesis of a series of spacer-armed pseudodi-, pseudotetra-, and pseudohexasaccharide derivatives (1)(2)(3), which are structurally related to one, two, and three repeating units of the Hia CG, based on a block-wise chain assembling scheme and the use of H-phosphonate chemistry [27] for the formation of inter-unit phosphodiester bridges. As compared to the phosphoramidite approach, the H-phosphonate method [27] has the following principal advantages: (a) relative simplicity and availability of reagents (phosphorous acid, pivaloyl chloride, iodine), (b) the formation of only one diasteriomeric product and not a mixture of diastereomers, and (c) no need for an additional P-deprotection step. Therefore, the H-phosphonate approach seems to be more preferable for the synthesis of molecules with more than one phosphodiester fragment. Using H-phosphonate chemistry, compounds 1-3 and a biotinylated pseudohexasaccharide derivative 4 ( Figure 1) were prepared with a view to develop an affordable and effective screening tool for fast and unambiguous detection of Hia-associated invasive diseases.

Results and Discussion
Previously described imidate 5 [28] and ribitol derivative 6 [29,30] (Scheme 1) were used as starting materials for the preparation of target compounds 1-3. Regioselective benzoylation of diol 6 transformed it into its 1-O-benzoylated derivative 7, which was then glycosylated by imidate 5 [28] in the presence of TMSOTf to obtain pseudodisaccharide product 8. β-Configuration of the newly formed glycosidic bond in compound 8 was confirmed by the characteristic value of the coupling constant (J1,2 7.8 Hz). Regioselective reductive opening of a 4,6-benzylidene acetal in glycoside 8 (δ PhCH 5.49 ppm) under the action of Me3N·BH3-AlCl3-H2O-THF [31] readily afforded the key building block 9 (δ C-4 71.4, δ C-6 70.6 ppm) at a yield of 64%. The presence of the benzyl group at O-6 and the location of the free OH-group at C-4 of the glucose unit was unambiguously confirmed by disappearance of the signals of the 4,6-O-benzylidene group, the downfield A characteristic feature of the structure of Hia CG is the presence of a phosphodiester bridge between the glucose and the ribitol unit. Currently, there are two main approaches for the synthesis of carbohydrate derivatives with phosphodiester fragments: the H-phosphonate [25] and the phosphoramidite [26] methods. The latter was used in the published synthesis of a pseudodisaccharide related to a Hia CG repeating unit [22].
In the present paper, we report the synthesis of a series of spacer-armed pseudodi-, pseudotetra-, and pseudohexasaccharide derivatives (1-3), which are structurally related to one, two, and three repeating units of the Hia CG, based on a block-wise chain assembling scheme and the use of H-phosphonate chemistry [27] for the formation of inter-unit phosphodiester bridges. As compared to the phosphoramidite approach, the H-phosphonate method [27] has the following principal advantages: (a) relative simplicity and availability of reagents (phosphorous acid, pivaloyl chloride, iodine), (b) the formation of only one diasteriomeric product and not a mixture of diastereomers, and (c) no need for an additional P-deprotection step. Therefore, the H-phosphonate approach seems to be more preferable for the synthesis of molecules with more than one phosphodiester fragment. Using H-phosphonate chemistry, compounds 1-3 and a biotinylated pseudohexasaccharide derivative 4 ( Figure 1) were prepared with a view to develop an affordable and effective screening tool for fast and unambiguous detection of Hia-associated invasive diseases.

Results and Discussion
Previously described imidate 5 [28] and ribitol derivative 6 [29,30] (Scheme 1) were used as starting materials for the preparation of target compounds 1-3. Regioselective benzoylation of diol 6 transformed it into its 1-O-benzoylated derivative 7, which was then glycosylated by imidate 5 [28] in the presence of TMSOTf to obtain pseudodisaccharide product 8. β-Configuration of the newly formed glycosidic bond in compound 8 was confirmed by the characteristic value of the coupling constant (J 1,2 7.8 Hz). Regioselective reductive opening of a 4,6-benzylidene acetal in glycoside 8 (δ PhCH 5.49 ppm) under the action of Me 3 N·BH 3 -AlCl 3 -H 2 O-THF [31] readily afforded the key building block 9 (δ C-4 71.4, δ C-6 70.6 ppm) at a yield of 64%. The presence of the benzyl group at O-6 and the location of the free OH-group at C-4 of the glucose unit was unambiguously confirmed by disappearance of the signals of the 4,6-O-benzylidene group, the downfield displacement of the chemical shift of C-6 by 3 ppm, and the upfield displacement of the chemical shift of C-4 by 7 ppm in the 13 C NMR spectrum (see Section 3). O-Acetylation of 9 and desilylation of the thus obtained acetate 10 provided alcohol 11 at an excellent yield (97% over 2 steps).
Molecules 2023, 28, x FOR PEER REVIEW 3 of 12 displacement of the chemical shift of C-6 by 3 ppm, and the upfield displacement of the chemical shift of C-4 by 7 ppm in the 13 C NMR spectrum (see Section 3). O-Acetylation of 9 and desilylation of the thus obtained acetate 10 provided alcohol 11 at an excellent yield (97% over 2 steps). The H-phosphonate building block 12 was prepared by the treatment of primary alcohol 11 with a pyrophosphonate obtained in situ from 2 moles of phosphonic acid and 1 mole of pivaloyl chloride to avoid the formation of a diester by-product [32]. Condensation of H-phosphonate 12 with CbzNH(CH2)2O(CH2)2OH in Py in the presence of Et3N as an organic base and PivCl as a condensing agent, followed by oxidation of the intermediate product with I2 [27], provided the corresponding target pseudodisaccharide 13 at 74% yield. Removal of its isopropylidene group by hydrolysis in aq. trifluoroacetic acid followed by alkaline deacetylation and hydrogenolysis of the thus formed phosphodiester 13 afforded the spacer-armed monomer 1 at an overall 36% yield (Scheme 1).
In order to assemble compound 2, phosphonylation of the alcohol 9 with H-phosphonate 12 in the presence of PivCl/Py and subsequent oxidation were performed to obtain phosphodiester 14 at a 77% yield, which was readily desilylated to produce the primary alcohol 15 (Scheme 2). Transformation of alcohol 15 into H-phosphonate 16 and the following condensation with CbzNH(CH2)2O(CH2)2OH resulted in the formation of the spacer-armed pseudotetrasaccharide 17. Removal of the protective groups, as described above for the preparation of the pseudidisaccharide 1, provided the target pseudotetrasaccharide 2 at a 59% yield. The H-phosphonate building block 12 was prepared by the treatment of primary alcohol 11 with a pyrophosphonate obtained in situ from 2 moles of phosphonic acid and 1 mole of pivaloyl chloride to avoid the formation of a diester by-product [32]. Condensation of H-phosphonate 12 with CbzNH(CH 2 ) 2 O(CH 2 ) 2 OH in Py in the presence of Et 3 N as an organic base and PivCl as a condensing agent, followed by oxidation of the intermediate product with I 2 [27], provided the corresponding target pseudodisaccharide 13 at 74% yield. Removal of its isopropylidene group by hydrolysis in aq. trifluoroacetic acid followed by alkaline deacetylation and hydrogenolysis of the thus formed phosphodiester 13 afforded the spacer-armed monomer 1 at an overall 36% yield (Scheme 1).
In order to assemble compound 2, phosphonylation of the alcohol 9 with H-phosphonate 12 in the presence of PivCl/Py and subsequent oxidation were performed to obtain phosphodiester 14 at a 77% yield, which was readily desilylated to produce the primary alcohol 15 (Scheme 2). Transformation of alcohol 15 into H-phosphonate 16 and the following condensation with CbzNH(CH 2 ) 2 O(CH 2 ) 2 OH resulted in the formation of the spacer-armed pseudotetrasaccharide 17. Removal of the protective groups, as described above for the preparation of the pseudidisaccharide 1, provided the target pseudotetrasaccharide 2 at a 59% yield.
The preparation of compound 3 was performed using the [4+2]-coupling strategy. Condensation of pseudo-tetrasaccharide H-phosphonate 16 and alcohol 9 followed by the oxidation step provided pseudohexasaccharide 18 at a moderate yield of 45% (Scheme 2). The subsequent desilylation step afforded alcohol 19, which was transformed into Hphosphonate 20. Its condensation with CbzNH(CH 2 ) 2 O(CH 2 ) 2 OH in the presence of PivCl/Py and the following oxidation afforded the spacer-armed pseudohexasaccharide 21 (44% over three steps). Total deprotection provided the target trimer 3 at 90% yield. (e) TFA aq. ultrasonication; (f) 0.09M NaOMe/MeOH; (g) H 2 gas, Pd(OH) 2 /C, AcOH, MeOH, yield of 59% for 2 and 90% for 3 over three steps; (h) Et 3 N, DMF, 70%. Treatment of pseudohexasaccharide 3 with biotin-containing pentafluorophenyl ester 22 [33] resulted in the formation of the target neoglycoconjugate 4 at a yield of 70%. The structure of the thus synthesized compounds 1-4 was confirmed by the combination of HRMS and NMR spectroscopy. In particular, the 1 H, 13 C, and 31 P NMR spectra of these products contained full series of signals related to the spacer group, β-D-glucose, and ribitol units connected via (1→2)-bonds and the phosphodiester bridge (see Sections 3.2.7, 3.2.12, 3.2.15 and 3.2.16). β-Configuration of the glycosidic bond in Glc (1→4)Rib was confirmed by characteristic J 1,2 coupling constant values between 7.8 and 8.7 Hz in the 1 H NMR spectra for all protected and deprotected derivatives. Also, for mono-, di-, and triphosphodiester derivatives, the corresponding signals in 31 P NMR spectra were observed in the expected area of~0 ppm.

General Information
Chemicals were purchased from Acros, Fluka, or Aldrich and used without further purification. All solvents were purified according to standard protocols. All reactions involving air-or moisture-sensitive reagents were carried out using dry solvents under an Ar atmosphere. TLC was performed on Silica Gel 60 F254 plates (E. Merck), and visualization was either accomplished using UV light or via charring at~150 • C with a 20:1 mixture of 15% (v/v) H 3 PO 4 in water and 4% orcinol in ethanol. Silica gel column chromatography was performed with Silica Gel 60 (40-63 µm, E. Merck). Gel filtration was performed on a TSK HW-40 column (400 × 17 mm) through elution with 0.1N CH 3 COOH at a flow rate of 0.6 mL/min. NMR spectra were recorded on Bruker AM 300 (300 MHz) and Bruker Avance 600 spectrometers. Assignment in 1 H and 13 C NMR spectra was performed using different 2D experiments (e.g., COSY, NOESY, HSQC). Chemical shifts are presented in ppm with reference to the solvent residual peaks used as a standard (δ 7.27 for chloroform in 1 H NMR and δ 77.0 for 13 C NMR). HRMS (ESI) spectra were obtained on a MicrOTOF II (Bruker Daltonics) instrument in positive or negative modes. Optical rotations were measured using a JASCO DIP-360 polarimeter at 16 • C in CHCl 3 . 7, 8, 10, 11, 12, 13, 1, 14, 15, 16, 17, 2, 18 (7) To a solution of 6 (3.4 g, 15 mmol) in Py (20 mL), BzCl (1.9 mL, 16.5 mmol) was added. The mixture was stirred for 2 h at rt, and the reaction mixture was then diluted with CH 2 Cl 2 and washed with 1M HCl, water, and satd NaHCO 3 . The organic layer was concentrated in vacuo and the column chromatography provided 7 (3.5 g, 77%) as a colorless oil (see

1-O-Benzoyl-4-O-(6-O-benzyl-2,3,4-tri-O-acetyl-β-D-glucopyrano-syl)-2,3-Oisopropylidene-D-ribitol (11)
To a solution of 9 (500 mg, 0.67 mmol) in a mixture of Py:Ac 2 O 2:1 (1.5 mL), DMAP (10 mg, 82 µmol) was added. The reaction mixture was stirred at rt for 1 h, and it was then quenched with MeOH, diluted with CH 2 Cl 2 , and washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo to provide the crude 10. The residue was dissolved in a mixture of Py:HF (90% aq.) at a ratio of 6:1 (5.8 mL) in a plastic centrifuge tube. The reaction mixture was heated to 60 • C and stirred for 2 h until the starting material disappeared (TLC control). It was then diluted with CH 2 Cl 2 and washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo and purified with column chromatography to obtain 11 (438 mg, 97%) as a colorless oil (see Supplementary Materials, p. 9).

Compound 21
Compound 18 (69 mg, 31 µmol) was dissolved in a mixture of Py:HF (90% aq.) at a ratio of 6:1 (1 mL) in a plastic centrifuge tube. The reaction mixture was heated to 60 • C and stirred for 2 h until the starting material disappeared (TLC control). It was then diluted with CH 2 Cl 2 and washed with 1M HCl and brine. The organic layer was concentrated in vacuo. The mixture of H 3 PO 3 (28 mg, 0.34 mmol) and PivCl (20 µL, 0.16 mmol) in Py (500 µL) was added to the residue. The reaction mixture was stirred at 90 • C for 1 h, diluted with CH 2 Cl 2 , and then washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo. The residue was dissolved in Py (500 µL), then benzyl (2-(2-hydrohyethohy)ethyl)carbamate (17 mg, 62 µmol), PivCl (11 µL, 90 µmol), and Et 3 N (22 µL, 0.16 mmol) were added. The reaction mixture was stirred for 15 min at rt until the starting material disappeared (TLC control), and I 2 (12 mg, 47 µmol) and water (25 µL, 1.4 mmol) were then added. The reaction mixture was stirred for 15 min at rt, and it was then quenched with a 0.1 N aqueous solution of Na 2 S 2 O 3 , diluted with CH 2 Cl 2 , and washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo and column chromatography provided 21 ( To a solution of 3 (1 mg, 0.8 µmol) in DMSO (150 µL), Et 3 N (12 µL, 0.12 mmol) and a 62 µmol/mL solution of compound 22 in DMSO (15 µL, 0.96 µmol) were added. The reaction mixture was incubated for 1 h until the starting material disappeared (TLC control). The target biotin derivative 4 was then purified through column chromatography on the TSK HW-40 with 0.1 N CH 3 COOH, which was then followed by lyophilization to provide

Conclusions
Initial synthesis of spacer-armed pseudodi-(1), pseudotetra-(2), and pseudohexasaccharide (3) derivatives related to the Hia CG was performed efficiently using the convergent chain elongation scheme and H-phosphonate chemistry. Compounds 1-3 were obtained for application as haptens in inhibitory ELISA and conjugation with immune-tolerable protein carriers in the design of glycoconjugate vaccines. The biotinylated conjugate 4 will be used as a coating antigen in ELISA testing for immobilization on streptavidin-coated plates and chip slides. The results of immunological studies of compounds 1-4 will be published elsewhere.