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

Abstract

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.

1. 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-PO₄→4- repeating 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 of inter-unit phosphodiester bridges was described in a PhD thesis in 2020 [24]. However, a convenient synthetic approach to Hia CG-related pseudooligosaccharides has not been yet reported.

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.

2. 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 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 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 ¹³C 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 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 H-phosphonate 20. Its condensation with CbzNH(CH₂)₂O(CH₂)₂OH 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.

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 ¹H, ¹³C, and ³¹P 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 Section 3.2.7, Section 3.2.12, Section 3.2.15 and Section 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 ¹H NMR spectra for all protected and deprotected derivatives. Also, for mono-, di-, and tri-phosphodiester derivatives, the corresponding signals in ³¹P NMR spectra were observed in the expected area of ~0 ppm.

3. Materials and Methods

3.1. 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) H₃PO₄ 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 CH₃COOH 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 ¹H and ¹³C 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 ¹H NMR and δ 77.0 for ¹³C 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 CHCl₃.

3.2. Synthesis of Compounds 7, 8, 10, 11, 12, 13, 1, 14, 15, 16, 17, 2, 18, 21, 3, and 4

3.2.1. 1-O-Benzoyl-5-O-(tert-butyldimethylsilyl)-2,3-O-isopropylidene-d-ribitol (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 CH₂Cl₂ and washed with 1M HCl, water, and satd NaHCO₃. The organic layer was concentrated in vacuo and the column chromatography provided 7 (3.5 g, 77%) as a colorless oil (see Supplementary Materials, p. 3). ${[\alpha ]}_D^{16}$ −18.0 (c 1, CHCl₃). R_f = 0.8 (Tol-EtOAc 2:1). ¹H NMR (300 MHz, CDCl₃) δ −0.1 (s, 6H, -Si(CH₃)₂), 0.75 (s, 9H, -C(CH₃)₃), 1.25 and 1.35 (both s, 6H, Me₂C), 2.55 (br s, 1H, OH), 3.61 (dd, 1H, J_4,5 = 4.3 Hz, J_5,6 = 5.2 Hz, H-5b), 3.69 (t, 1H, J_3,4 = J_4,5 = 7.9 Hz, H-4), 3.77 (dd, 1H, J_4,5 = 2.9 Hz, J_5,6 = 9.6 Hz, H-5a), 4.03 (dd, 1H, J_2,3 = 6.1 Hz, J_3,4 = 9.3 Hz, H-3), 4.37 (dd, 1H, J_1,2 = 7.2 Hz, J_gem = 11.5 Hz, H-1b), 4.51–4.43 (m, 1H, H-2), 4.67 (dd, 1H, J_1,2 = 3.0 Hz, J_gem = 11.5 Hz, H-1a), 8.02–7.29 (m, 5H, Ph). ¹³C NMR (75 MHz, CDCl₃) δ −5.3 and −5.4 (-Si(CH₃)₂), 25.5 and 27.9 (Me₂C), 25.9 (-C(CH₃)₃), 64.0 (C-1), 64.5 (C-5), 69.3 (C-4), 75.7 (C-2), 76.0 (C-3), 109.2 (Me₂C), 128.3, 129.8, and 132.9 (Ph), 166.5 (PhC(O)O-). HRMS (ESI): calcd m/z for [M + Na]⁺ C₂₁H₃₄O₆S 433.2017, found 433.2011.

3.2.2. 1-O-Benzoyl-4-O-(4,6-O-benzylidene-2,3-di-O-acetyl-β-d-glucopyranosyl)-5-O-(tert-butyldimethylsilyl)-2,3-O-isopropylidene-d-ribitol (8)

To a mixture of compound 5 (100 mg, 0.2 mmol) and acceptor 7 (70 mg, 0.17 mmol) in anhydrous CH₂Cl₂ (1 mL), TMSOTf (1.1 μL, 6 μmol, 3% mol) was added. The mixture was stirred for 20 min at rt until full conversion of the starting material (TLC control), and the mixture was then neutralized with Et₃N and concentrated in vacuo. The residue was purified by column chromatography to obtain product 8 (76 mg, 60%) as a white amorphous powder (see Supplementary Materials, p. 5). ${[\alpha ]}_D^{16}$ −0.9 (c 1, CHCl₃). R_f = 0.48 (toluene:EtOAc 5:1). ¹H NMR (300 MHz, CDCl₃) δ 0.08 and 0.09 (both s, 6H, -Si(CH₃)₂), 0.88 (s, 9H, -C(CH₃)₃), 1.36 and 1.47 (both s, 6H, Me₂C), 2.06 and 2.08 (both s, 6H, -C(O)OCH₃), 3.35–3.45 (m, 1H, H-5’), 3.64 (t, 1H, J_3,4 = J_4,5 = 9.6 Hz, H-4′), 3.69–3.83 (m, 2H, H-5b, H-6′b), 4.02–4.10 (m, 1H, H-6′a), 4.10–4.25 (m, 3H, H-5a, H-2, H-3), 4.46–4.53 (m, 1H, H-4), 4.57 (d, 1H, J_1,2 = 5.9 Hz, J_gem = 11.4 Hz, H-1b), 4.65 (dd, 1H, J_1,2 = 2.3 Hz, J_gem = 11.4 Hz, H-1a), 4.92–5.01 (m, 1H, H-2′), 5.17 (d, 1H, J_1,2 = 7.8 Hz, H-1′), 5.28 (t, 1H, J_2,3 = J_3,4 = 9.5 Hz, H-3′), 5.49 (s, 1H, PhCH), 7.25–8.20 (m, 10H, Ph). ¹³C NMR (75 MHz, CDCl₃) δ −5.5 and −5.4 (-Si(CH₃)₂), 18.2 and 20.8 (-C(O)OCH₃), 25.5 and 27.6 (Me₂C), 25.8 (-C(CH₃)₃), 63.9 (C-5), 64.4 (C-1), 66.3 (C-5’), 68.4 (C-6′), 71.9 (C-3′), 72.5 (C-2′), 74.2 and 74.8 (C-2, C-3), 75.4 (C-4), 78.4 (C-4′), 98.3 (C-1′), 108.9 (Me₂C), 126.1, 126.2, 128.2, 128.4, 129.1, 129.6, 129.8, 130.3, 132.9, and 136.9 (Ph), 166.4 (PhC(O)O-), 169.7 and 170.2 (-OC(O)CH₃). HRMS (ESI): calcd m/z for [M + Na]⁺ C₃₈H₅₂O₁₃Si 767.3069, found 767.3070.

3.2.3. 1-O-Benzoyl-4-O-(6-O-benzyl-2,3-di-O-acetyl-β-d-glucopyranosyl)-5-O-(tert-butyldimethylsilyl)-2,3-O-isopropylidene-d-ribitol (10)

A solution of 8 (1.4 g, 1.9 mmol) in THF (28 mL) was cooled to 0 °C, and Me₃N·BH₃ (562 mg, 7.7 mmol), AlCl₃ (1.52 g, 11.4 mmol), and H₂O (68 μL, 3.8 mmol) were then added. The reaction mixture was stirred at rt for 3 h, diluted with CH₂Cl₂, and washed with 1 M HCl, water, and satd NaHCO₃. The organic layer was concentrated in vacuo and flash column chromatography of the residue provided product 9 (980 mg, 64%) as a colorless oil (see Supplementary Materials, p. 7). ${[\alpha ]}_D^{16}$ −15.1 (c 1, CHCl₃). R_f = 0.22 (toluene:EtOAc 4:1). ¹H NMR (300 MHz, CDCl₃) δ 0.10 and 0.11 (both s, 6H, -Si(CH₃)₂), 0.94 (s, 9H, -C(CH₃)₃), 1.36 and 1.46 (both s, 6H, Me₂C), 2.06 and 2.08 (both s, 6H, -C(O)OCH₃), 3.13 (br s, 1H, OH), 3.45–3.51 (m, 1H, H-5’), 3.69–3.81 (m, 4H, H-4′, H-5b, H-6′ab), 4.04 (dd, 1H, J_4,5 = 11.5 Hz, J_5,6 = 1.4 Hz, H-5a), 4.07–4.20 (m, 2H, H-2, H-3), 4.47–4.64 (m, 4H, H-1b, H-4, -CH₂Ph), 4.69 (dd, 1H, J_1,2 = 11.5 Hz, J_gem = 2.3 Hz, H-1a), 4.90 (dd, 1H, J_1,2 = 7.8 Hz, J_2,3 = 9.8 Hz, H-2′), 5.02–5.08 (m, 2H, H-1′, H-3′). ¹³C NMR (75 MHz, CDCl₃) δ −5.5 and −5.4 (-Si(CH₃)₂), 20.8 and 20.9 (-C(O)OCH₃), 25.6 and 27.7 (Me₂C), 25.9 (-C(CH₃)₃), 63.8 (C-5), 64.3 (C-1), 70.6 (C-6′), 71.4 (C-4′), 73.5 (5‘), 73.8 (-CH₂Ph), 74.3 and 74.7 (C-2, C-3), 75.4 and 75.5 (C-3′, C-4), 97.9 (C-1′), 108.9 (Me₂C), 127.8, 127.9, 128.3, 128.4, 129.8, 130.3, and 132.8 (Ph), 166.4 (PhC (O)O-), 169.7 and 171.1 (-C(O)OCH₃). HRMS (ESI): calcd m/z for [M + Na]⁺ C₃₈H₅₄O₁₃Si 769.3226, found 769.3225.

3.2.4. 1-O-Benzoyl-4-O-(6-O-benzyl-2,3,4-tri-O-acetyl-β-d-glucopyrano-syl)-2,3-O-isopropylidene-d-ribitol (11)

To a solution of 9 (500 mg, 0.67 mmol) in a mixture of Py:Ac₂O 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 CH₂Cl₂, 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 CH₂Cl₂ 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). ${[\alpha ]}_D^{16}$ −12.2 (c 1, CHCl₃). R_f = 0.19 (toluene:EtOAc 4:1). ¹H NMR (300 MHz, CDCl₃) δ 1.37 and 1.45 (both s, 6H, Me₂C), 1.88, 1.98, and 2.05 (each s, 9H, -C(O)OCH₃), 2.12–2.21 (m, 1H, OH), 3.50 (d, 2H, J_5,6 = 4.1 Hz, H-6′ab), 3.59–3.67 (m, 1H, H-5’), 3.73–3.83 (m, 1H, H-5b), 3.74–3.93 (m, 1H, H-5a), 4.00–4.07 (m, 1H, H-4), 4.27–4.34 (m, 1H, H-3), 4.41 and 4.49 (AB system, J_gem = 11.9 Hz, -CH₂Ph), 4.52–4.61 (m, 2H, H-1b, H-2), 4.66–4.75 (m, 1H, H-1a), 4.77 (d, 1H, J_1,2 = 8.0 Hz, H-1′), 4.94 (dd, 1H, J_1,2 = 8.0 Hz, J_2,3 = 9.4 Hz, H-2′), 5.05 (t, 1H, J_3,4 = J_4,5 = 9.5 Hz, H-4′), 5.17 (t, 1H, J_2,3 = J_3,4 = 9.4 Hz, H-3′), 7.05–8.10 (m, 10H, Ph). ¹³C NMR (75 MHz, CDCl₃) δ 20.60, 20.64, and 20.70 (-C(O)OCH₃), 25.5 and 27.5 (Me₂C), 62.4 (C-5), 63.9 (C-1), 68.7 (C-6′), 69.0 (C-4′), 71.7 (C-2′), 73.0 (C-3′), 73.4 (C-5’), 73.6 (-CH₂Ph), 75.4, 75.5, and 75.6 (C-2, C-3, C-4), 98.2 (C-1′), 109.2 (Me₂C), 125.8, 127.7, 127.9, 128.3, 128.4, 129.7, 130.2, 133.0, 136.5, and 137.7 (Ph), 166.3 (PhC(O)O-), 169.3, 169.4, and 170.4 (-C(O)OCH₃). HRMS (ESI): calcd m/z for [M + Na]⁺ C₃₄H₄₂O₁₄ 697.2467, found 697.2471.

3.2.5. [1-O-Benzoyl-4-O-(6-O-benzyl-2,3,4-tri-O-acetyl-β-d-glucopyrano-syl-2,3-O-isopropylidene)-d-ribitol]-5-yl Hydrogenphosphonate Triethylammonium Salt (12)

To a mixture of H₃PO₃ (586 mg, 7.15 mmol) and PivCl (398 μL, 3.25 mmol) in Py (5 mL), primary alcohol 11 (438 mg, 0.65 mmol) was added. The reaction mixture was stirred at 90 °C for 1 h, diluted with CH₂Cl₂, washed with 1M HCl, water, and brine, and then concentrated in vacuo and purified with column chromatography to provide H-phosphonate 12 (324 mg, 74%) as a white amorphous powder (see Supplementary Materials, p. 11). R_f = 0.49 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). ¹H NMR (300 MHz, CDCl₃) δ 1.35 and 1.45 (both s, 6H, Me₂C), 1.85, 1.95, and 2.05 (each s, 9H, -C(O)OCH₃), 3.55 (d, 2H, J_5,6 = 3.2 Hz, H-6′ab), 3.63–3.76 (m, 1H, H-5′), 3.90–4.05 (m, 2H, H-5ab), 4.05–4.36 (m, 2H, H-4, H-3), 4.42 and 4.49 (AB system, J_gem = 12.0 Hz, -CH₂Ph), 4.52–4.58 (m, 1H, H-2), 4.62 (d, 1H, J_1,2 = 7.4 Hz, H-1b), 4.74 (d, 1H, J_1,2 = 10.9 Hz, H-1a), 4.88–5.13 (m, 3H, H-1′, H-2′, H-4′), 5.13–5.32 (m, 1H, H-3′), 5.80 (s, ½ H, H-P), 7.05–8.15 (m, 10H, Ph), 7.85 (s, ½ H, H-P). ¹³C NMR (75 MHz, CDCl₃) δ 20.6, 20.7, and 21.0 (-OC(O)CH₃), 25.6 and 27.7 (Me₂C), 64.1 (C-1), 66.8 (C-5), 68.8 (C-6′), 69.3 (C-4′), 71.7 (C-2′), 73.0 (C-3′), 73.4 (C-5′), 74.6 (-CH₂Ph), 75.4 (C-3, C-4), 76.6 (C-2), 98.2 (C-1′), 108.9 (Me₂C), 127.6, 127.8, 128.2, 128.3, 129.8, 130.2, 132.9, and 137.8 (Ph), 166.2 (PhC(O)O-), 166.4, 169.4, and 170.3 (-OC(O)CH₃). ³¹P (122 MHz, CDCl₃) δ 5.08 (d, J_P,H = 616.4 Hz). HRMS (ESI): calcd m/z for [M + H]⁺ C₃₄H₄₃O₁₆P 737.2216, found 737.2211.

3.2.6. (2-(2-(Benzyloxycarbonylamino)ethoxy)ethyl [1-O-benzoyl-4-O-(6-O-benzyl-2,3,4-tri-O-acetyl-β-d-glucopyranosyl)-2,3-O-isopropylidene-d-ribitol]-5-yl Phosphate Triethylammonium Salt (13)

To a solution of H-phosphonate 12 (100 mg, 0.15 mmol) and benzyl (2-(2-hydrohyethohy)ethyl)carbamate (81 mg, 0.30 mmol) in Py (2 mL), PivCl (55 μL, 0.45 mmol) and Et₃N (42 μL, 0.30 mmol) were added. The reaction mixture was stirred for 15 min at rt until the starting material disappeared (TLC control), and I₂ (56 mg, 0.22 mmol) and water (100 μL, 5.56 mmol) were then added. The reaction mixture was stirred for 15 min at rt, and it was then quenched with a 0.1N aqueous solution of Na₂S₂O₃, diluted with CH₂Cl₂, and washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo and column chromatography provided 13 (43 mg, 34%) as a white amorphous powder (see Supplementary Materials, p. 14). R_f = 0.39 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). ¹H NMR (300 MHz, CDCl₃) δ 1.05 (t, 9 H, J = 7.4 Hz, (CH₃CH₂)₃N), 1.20–1.25 (m, 2H, -NHCH₂CH₂OCH₂CH₂O-), 1.28 and 1.39 (both s, 6H, Me₂C), 1.83, 1.94, and 2.05 (each s, 9H, -C(O)OCH₃), 3.27–3.43 (m, 2H, -NHCH₂CH₂OCH₂CH₂O-), 3.43–3.72 (m, 5H, H-5′, H-6′ab, -NHCH₂CH₂OCH₂CH₂O-), 3.73–3.87 (m, 2H, -NHCH₂CH₂OCH₂CH₂O-), 3.88–4.17 (m, 3H, H-4, H-5ab), 4.25 (s, 1H, H-3), 4.34–4.46 (m, 2H, -CH₂Ph), 4.47–4.64 (m, 2H, H-1b, H-2), 4.71 (d, 1H, J_1,2 = 9.9 Hz, H-1a), 4.96–5.13 (m, 4H, H-1′, H-2′, H-4′, PhCH₂OC(O) NH-), 5.18 (t, 1H, J_2,3 = J_3,4 = 9,1 Hz, H-3′), 7.10–8.15 (m, 15H, Ph). ¹³C NMR (75 MHz, CDCl₃) δ 20.5, 20.6, and 20.7 (-C(O)OCH₃), 25.6 and 27.7 (Me₂C), 29.7 (NHCH₂CH₂OCH₂CH₂O-), 40.9 (NHCH₂CH₂OCH₂CH₂O-), 41.6 (NHCH₂CH₂OCH₂CH₂O-), 64.2 (C-1), 66.6 (C-5), 67.2 (PhCH₂OC(O) NH-), 68.9 (C-6′), 69.5 (C-4′), 69.9 (NHCH₂CH₂OCH₂CH₂O), 70.7 (C-2′), 71.6 (C-3′), 72.8 (C-5′), 73.0 (-CH₂-Ph), 73.5 and 74.7 (C-3, C-4), 75.4 (C-2), 98.3 (C-1′), 108.9 (Me₂C), 127.6, 127.8, 128.1, 128.2, 128.3, 128.5, 129.8, 130.2, 132.9, and 137.8 (Ph), 156.8 (PhC(O)O-), 169.4, 169.7, and 170.3 (-C(O)OCH₃). ³¹P (122 MHz, CDCl₃): δ 1.28. HRMS (ESI): calcd m/z for [M + H]⁺ C₄₆H₅₈O₂₀P 974.3217, found 974.3214.

3.2.7. 2-Aminoethoxyethyl (4-O-β-d-glucopyranosyl)-d-ribitol-5)-yl Phosphate Sodium Salt (1)

A suspension of 13 (43 mg, 44 μmol) in CF₃COOH 90% aq. (73 μL, 0.88 mmol) was placed in an ultrasonic bath for 10 min, diluted with EtOAc, and then coevaporated with toluene. The residue was dissolved in MeOH (440 μL), and 1N MeONa in MeOH (44 μL, 44 μmol) was then added. The reaction mixture was stirred at rt for 1 h until the starting material disappeared (TLC control). The reaction was then quenched with excess CH₃COOH (10 μL, 0.16 mmol) and P(OH)₂/C (86 mg, 200 mass%) and H₂O (440 μL) were added. The reaction mixture was stirred under H₂ atmosphere at rt for 3 h. The mixture was filtered and concentrated in vacuo. Column chromatography of the residue on TSK HW-40 with 0.1 N CH₃COOH and lyophilization provided the target disaccharide 1 (8 mg, 36%) as a white powder (see Supplementary Materials, p. 24). R_f = 0.32 (EtOH:H₂O 4:1). ¹H NMR (300 MHz, D₂O) δ 3.13–3.19 (m, 2H, NH₂CH₂CH₂OCH₂CH₂O-), 3.21–3.29 (m, 1H, H-5′), 3.30–3.48 (m, 3H, H-2′, H-4′, H-4), 3.52–3.61 (m, 1H, H-5b), 3.62–3.71 (m, 3H, H-1b, NH₂CH₂CH₂OCH₂CH₂O-), 3.72–3.86 (m, 6H, H-1a, H-2, H-3, H-5a, H-6′ab), 3.96–4.03 (m, 2H, NH₂CH₂CH₂OCH₂CH₂O-), 4.04–4.14 (m, 3H, H-3′, NH₂CH₂CH₂OCH₂CH₂O-), 4.59 (d, J_1,2 = 7.9 Hz, H-1′). ¹³C NMR (75 MHz, D₂O) δ 39.2 (NH₂CH₂CH₂OCH₂CH₂O-), 60.6 (C-1), 62.5 (C-5), 64.6 (NH₂CH₂CH₂OCH₂CH₂O-), 65.05 (NH₂CH₂CH₂OCH₂CH₂O-), 66.4 (C-6′), 69.6 (C-4′), 70.2 (NH₂CH₂CH₂OCH₂CH₂O-), 71.4 and 71.7 (C-2, C-3), 73.3 (C-5′), 75.6 and 75.8 (C-4, C-2′), 79.0 (C-3′), 102.3 (C-1′). HRMS (ESI): calcd m/z for [M + H]⁺ C₁₅H₃₂O₁₄P 480.1477, found 480.1479. ³¹P (122 MHz, D₂O): δ 0.88.

3.2.8. Compound 14

To a solution of H-phosphonate 12 (100 mg, 0.14 mmol) and alcohol 9 (119 mg, 0.16 mmol) in Py (2 mL), PivCl (51 μL, 0.42 mmol) and Et₃N (39 μL, 0.28 mmol) were added. The reaction mixture was stirred for 15 min at rt until the starting material disappeared (TLC control), and I₂ (51 mg, 0.20 mmol) and H₂O (100 μL, 5.56 mmol) were then added. The reaction mixture was stirred for 15 min at rt, and it was then quenched with a 0.1N aqueous solution of Na₂S₂O₃, diluted with CH₂Cl₂, and washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo and column chromatography provided 14 (160 mg, 77%) as a white amorphous powder (see Supplementary Materials, p. 17). R_f = 0.53 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). ¹H NMR (300 MHz, CDCl₃) δ 0.07 and 0.08 (both s, 6H, Si(CH₃)₂), 0.91 (s, 9H, -C(CH₃)₃), 1.30 and 1.33 (both s, 6H, Me₂C), 1.83, 1.96, 2.03, 2.07, and 2.11 (each s, 15H, -C(O)OCH₃), 3.51 (d, 2H, J_gem = 3.8 Hz, H-6′ab), 4.55–4.64 (m, 1H, CH₂-Ph), 4.98–5.10 (m, 3H, H-1′, H-1‴, H-4′), 7.09–7.32 (m, 12H, Ph), 7.47–7.57 (m, 2H, Ph), 7.98–8.12 (m, 4H, Ph). ³¹P (122 MHz, CDCl₃): δ-1.21. HRMS (ESI): calcd m/z for [M + H]⁺ C₇₂H₉₅O₂₉PSi 1481.5382, found 1481.5370.

3.2.9. Compound 15

Transformation of tetrasaccharide 14 (160 mg, 0.11 mmol) into derivative 15 (133 mg, 90%) was performed similarly to the process described for the preparation of compound 11 from 10. R_f = 0.41 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). HRMS (ESI): calcd m/z for [M + H]⁺ C₆₆H₈₁O₂₉P 1367.4517, found 1367.4510.

3.2.10. Compound 16

Transformation of tetrasaccharide 15 (133 mg, 97 μmol) into derivative 16 (133 mg, 96%) was performed similarly to the process described for the preparation of compound 12 from 11 (see Supplementary Materials, p. 20). R_f = 0.21 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). ³¹P (122 MHz, CDCl₃): δ-1.30, 0.56. Calcd m/z for [M + H]⁺ C₆₆H₈₂O₃₁P₂ 1431.4243, found 1431.4247.

3.2.11. Compound 17

Transformation of tetrasaccharide 16 (133 mg, 93 μmol) into derivative 17 (85 mg, 54%) was performed similarly to the process described for the preparation of compound 13 from 12 (see Supplementary Materials, p. 22). R_f = 0.32 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). ³¹P (122 MHz, CDCl₃): δ-1.31, 0.50. Calcd m/z for [M + H]⁺ C₇₈H₉₇O₃₅P₂ 833.7585, found 833.7611.

3.2.12. Compound 2

Transformation of tetrasaccharide 17 (85 mg, 50 μmol) into derivative 2 (25 mg, 59%) was performed similarly to the process described for the preparation of compound 1 from 13 (see Supplementary Materials, p. 27). R_f = 0.29 (EtOH-H₂O 4:1). ¹H NMR (300 MHz, D₂O, selected data) δ 3.31–3.34 (m, 2H, NH₂CH₂CH₂OCH₂CH₂O-), 4.59 (d, 1H, J_1,2 = 7.9 Hz, H-1′), 4.61 (d, 1H, J_1,2 = 7.9 Hz, H-1‴). ¹³C NMR (75 MHz, D₂O, selected data) δ 39.1 (NH₂CH₂CH₂OCH₂CH₂O-), 102.2 (C-1′), 102.3 (C-1‴). ³¹P (122 MHz, D₂O) δ 0.42, 0.90. Calcd m/z for [M + H]⁺ C₂₆H₅₃O₂₆P₂ 856.2258, found 856.2264.

3.2.13. Compound 18

To a solution of H-phosphonate 16 (100 mg, 70 μmol) and alcohol 9 (63 mg, 84 μmol) in Py (2 mL), PivCl (26 μL, 0.21 mmol) and Et₃N (29 μL, 0.21 mmol) were added. The reaction mixture was stirred for 15 min at rt until the starting material disappeared (TLC control), and I₂ (26 mg, 0.10 mmol) and H₂O (100 μL, 5.56 mmol) were then added. The reaction mixture was stirred for 15 min at rt, and it was then quenched with 0.1N Na₂S₂O₃, diluted with CH₂Cl₂, and washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo and column chromatography provided 18 (69 mg, 45%) as a white amorphous powder. R_f = 0.36 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). Calcd m/z for [M + H]⁺ C₁₀₄H₁₃₄O₄₄P₂Si 2175.7420, found 2175.7415.

3.2.14. 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 CH₂Cl₂ and washed with 1M HCl and brine. The organic layer was concentrated in vacuo. The mixture of H₃PO₃ (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 CH₂Cl₂, 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 Et₃N (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 I₂ (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 Na₂S₂O₃, diluted with CH₂Cl₂, and washed with 1M HCl, water, and brine. The organic layer was concentrated in vacuo and column chromatography provided 21 (32 mg, 44%) as a white amorphous powder. R_f = 0.15 (EA:acetone:CH₂Cl₂:MeOH:H₂O 20:15:6:5:4). Calcd m/z for [M + H]⁺ C₁₁₀H₁₃₆O₅₀P₃ 787.2386, found 787.2396.

3.2.15. Compound 3

Transformation of hexasaccharide 21 (32 mg, 14 μmol) into derivative 3 (15 mg, 90%) was performed similarly to the process described for the preparation of compound 1 from 13 (see Supplementary Materials, p. 30). R_f = 0.38 (EtOH:H₂O 4:1). ¹H NMR (300 MHz, D₂O, selected data) δ 3.22–3.25 (m, 2H, NH₂CH₂CH₂OCH₂CH₂O-), 4.66 (d, 1H, J_1,2 = 8.3 Hz, H-1′), 4.67 (d, 2H, J_1,2 = 8.7 Hz, H-1‴, H-1″‴). ¹³C NMR (75 MHz, D₂O, selected data) δ 39.2 (NH₂CH₂CH₂OCH₂CH₂O-), 103.6 (C-1′), 103.7 (C-1‴), 103.69 (C-1″‴). ³¹P (122 MHz, D₂O) δ 0.42, 0.90. HRMS (ESI): calcd m/z for [M + H]⁺ C₃₇H₇₃O₃₈P₃ 1232.3029, found 1232.3004.

3.2.16. Compound 4

To a solution of 3 (1 mg, 0.8 μmol) in DMSO (150 μL), Et₃N (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 CH₃COOH, which was then followed by lyophilization to provide 4 (1 mg, 70%) (see Supplementary Materials, p. 33). R_f = 0.65 (EtOH:H₂O 4:1). ¹H NMR (600 MHz, D₂O, characteristic signals): oligosaccharide fragment: 4.63 (d, 2 H, J_1,2 = 8.2 Hz, H-1‴, H-1″‴), 4.64 (d, 1 H, J_1,2 = 8.4 Hz, H-1′); biotin fragment: 1.32–1.72 (m, 6H, H-3, H-4, H-5), 2.23 (t, 2H, J = 7.3 Hz, H-2), 2.50 (t, 2 H, J = 6.0, C(O)CH₂CH₂), 2.73 (d, 1H, J = 13.1 Hz, H-6_cis), 2.94 (dd, 1H, J = 4.8, 13.1 Hz; H-6_trans), 4.38 (dd, 1H, J = 5.0 Hz; 7.9 Hz; H-3a), 4.56 (dd, 1 H, J = 5.0 Hz; 7.9 Hz; H-6a). HRMS (ESI): calcd m/z for [M − 2H]/2²⁻ C₆₂H₁₁₇O₄₇N₄P₃S 896.2833, found 896.2908.

4. 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.