Validation and Application of an Innovative Protective Group Concept: Enhancing Substrate Reactivity in Glycosylations by Disrupting Intermolecular Interactions

Abstract

We have established an innovative protective approach that disrupts intermolecular interactions to enhance substrate reactivity. Specifically, diacetylimide protection of acetamide prevents the formation of hydrogen bonds, while the incorporation of tert-butyl groups on the aromatic protecting group disrupts π-stacking interactions, both of which culminate in heightened reactivity in glycosylations. We explored the synergistic implementation of these protective measures and applied them to the synthesis of 6-sulfo sialyl Lewis X.

Key words

protecting groups - intermolecular interactions - glycan synthesis - 6-sulfo sialyl Lewis X - sialic acid

Publication History

Received: 09 January 2024

Accepted after revision: 15 February 2024

Accepted Manuscript online:
15 February 2024

Article published online:
05 March 2024

• References and Notes

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• 17 Synthesis of 16: To a mixture of freeze-dried donor 11 (38.9 mg, 0.0315 mmol), acceptor 12 (30.0 mg, 0.0315 mmol), and freshly activated powder MS 4Å (70.0 mg) was added anhydrous Et₂O (0.32 mL) at r.t. To the donor and acceptor solution was then added TMSOTf (5.7 μL, 32.0 μmol) at –40 °C. After stirring for 1 h at –40 °C, the reaction was quenched with saturated aqueous NaHCO₃. The aqueous layer was extracted with CH₂Cl₂ and the organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by silica-gel column chromatography (toluene/EtOAc, 3:1 to EtOAc) to give 16 (54.4 mg, 86%) as a white powder. The stereoselectivity of the glycosylation was verified by the large coupling constant observed between Gal-H1 and Gal-H2 (J = 8.2 Hz). ¹H NMR (500 MHz, CDCl₃): δ = 8.22 (d, J = 7.5 Hz, 2 H, aromatic), 8.02 (d, J = 8.3 Hz, 2 H, aromatic), 7.99 (d, J = 7.5 Hz, 2 H, aromatic), 7.82 (s, 1 H, aromatic), 7.79–7.73 (m, 3 H, aromatic), 7.56 (t, J = 7.5 Hz, 1 H, aromatic), 7.51–7.46 (m, 6 H, aromatic), 7.38–7.29 (m, 7 H, aromatic), 7.22–7.18 (m, 12 H, aromatic), 6.10 (d, J = 8.0 Hz, 1 H, -NH-Troc), 5.67–5.60 (m, 2 H, Sia-H8, -CH₂CH=CH₂), 5.49–5.36 (m, 4 H, Sia-H4, Fuc-H1, Gal-H2, Gal-H4), 5.11 (d, J = 8.2 Hz, 1 H, Gal-H1), 5.07 (d, J = 17.2 Hz, 1 H, -CH₂CH=CH₂), 5.02–4.99 (m, 2 H, Sia-H7, -CH₂CH=CH₂), 4.93–4.86 (m, 4 H, GlcN-H1, Gal-H3), 4.76–4.70 (m, 5 H), 4.58 (dd, J = 10.2, 2.4 Hz, 1 H, Sia-H6), 4.54 (d, J = 11.6 Hz, 1 H), 4.47 (q, J = 6.4 Hz, 1 H, Fuc-H5), 4.32–4.24 (m, 4 H, Gal-H6a, Gal-H6b), 4.19 (dd, J = 12.5, 2.3 Hz, 1 H, Sia-H9a), 4.12 (t, J = 9.2 Hz, 1 H, GlcN-H4), 4.07–4.03 (m, 4 H, GlcN-H3, Fuc-H2, Fuc-H3, Gal-H5), 4.00–3.90 (m, 4 H, Sia-H5, Sia-H9b, GlcN-H6b, CH₂CH=CH₂), 3.89–3.76 (m, 6 H, GlcN-H2, GlcN-H6a, CH₂CH=CH₂, OCOCH₃), 3.59–3.57 (m, 2 H, GlcN-H5, Fuc-H4), 2.49 (dd, J = 12.4, 5.0 Hz, 1 H, Sia-H3_eq), 2.29 (s, 3 H, Ac), 2.14 (s, 3 H, Ac), 2.13 (s, 3 H, Ac), 1.96 (s, 3 H, Ac), 1.86 (s, 3 H, Ac), 1.59 (t, J = 12.4 Hz, 1 H, Sia-H3_aq), 1.44 (s, 3 H, Ac), 1.31 (d, J = 6.4 Hz, 3 H, -CH₃), 1.22 (s, 9 H, t-BuBz). ¹³C{¹H} NMR (125 MHz, CDCl₃): δ = 174.1, 173.6, 170.7, 170.5, 170.0, 169.8, 168.0, 166.2, 165.6, 164.9, 157.0, 154.2, 139.0, 138.2, 135.9, 133.6, 133.2, 133.04, 132.98, 132.85, 130.5, 130.0, 129.9, 129.8, 128.5, 128.3, 128.2, 128.12, 128.06, 127.9, 127.8, 127.6, 127.4, 127.2, 127.1, 127.0, 126.9, 126.0, 125.8, 125.6, 125.54, 125.51, 117.8, 100.1, 97.8, 96.9, 95.6, 95.5, 79.1, 78.8, 77.6, 74.9, 74.4, 73.5, 73.4, 72.9, 72.7, 72.1, 72.0, 71.4, 70.7, 69.2, 68.7, 68.5, 68.4, 67.2, 66.9, 66.8, 66.5, 62.1, 61.7, 56.1, 55.9, 53.1, 38.4, 35.0, 31.0, 28.0, 26.4, 21.4, 20.71, 20.66, 20.5, 17.0. HRMS (ESI-Orbitrap): m/z [M + Na]⁺ calcd for C₁₀₃H₁₁₃O₃₂Cl₃N₂Na: 2017.6234; found: 2017.6243.
• 18 Synthesis of 17: To a solution of 16 (50.0 mg, 25.0 μmol) in anhydrous THF (0.80 mL) was added [Ir(cod)(PPh₂Me)₂]PF₆ (1.1 mg, 1.3 μmol) in anhydrous THF (0.45 mL) at r.t., which was activated by H₂. After the reaction mixture was stirred for 18 h at r.t., to the reaction mixture were added H₂O (0.63 mL) and I₂ (10.1 mg, 40.0 μmol) at r.t. After stirring for 50 min at r.t., the reaction was quenched with saturated aqueous Na₂S₂O₃. The aqueous layer was extracted with EtOAc and the organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was roughly purified by silica-gel column chromatography (toluene/EtOAc = 1:0 to 3:2) to give hemiacetal (45.6 mg, 93%) as a white powder. This α/β mixture was used for the next reaction without further purification. To a solution of the obtained hemiacetal (45.6 mg, 23.3 μmol) in anhydrous acetone (1.2 mL) were added N-phenyltrifluoroacetimidoyl chloride (10.7 mg, 52.0 μmol) and K₂CO₃ (10.2 mg, 74.0 μmol) at r.t. After stirring the reaction mixture for 4 h at r.t., to the reaction mixture were added acetone (1.2 mL), N-phenyltrifluoroacetimidoyl chloride (11.2 mg, 53.9 μmol), and K₂CO₃ (10.9 mg, 78.9 μmol) at r.t. After stirring the reaction mixture for 2 h at r.t., insoluble materials were filtered, and concentrated in vacuo. The residue was purified by silica-gel column chromatography (toluene/EtOAc = 1:0 to 3:2) to give the donor (39.7 mg, 82%) as a white powder. This α/β mixture was used for the next reaction without further purification. A mixture of freeze-dried obtained donor (35.6 mg, 16.3 μmol), 2-(benzyloxycarbonylamino)-1-ethanol (12.8 mg, 65.3 μmol), and freshly activated powder MS 4Å (48 mg) was dissolved in anhydrous CH₂Cl₂ (1.6 mL). To the donor and acceptor solution were added TMSOTf (3.0 μL, 16.0 μmol) at –78 °C. After stirring for 2 h at –78 °C, the reaction was quenched with saturated aqueous NaHCO₃. The aqueous layer was extracted with CH₂Cl₂ and the organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by silica-gel column chromatography (toluene/EtOAc = 1:0 to 2:1) to give 17 (34.8 mg, quant) as a white powder. ¹H NMR (500 MHz, CDCl₃): δ = 8.21 (d, J = 7.2 Hz, 2 H, aromatic), 8.02 (d, J = 8.6 Hz, 2 H, aromatic), 7.95 (d, J = 7.3 Hz, 2 H, aromatic), 7.76–7.70 (m, 4 H, aromatic), 7.55–7.37 (m, 10 H, aromatic), 7.31–7.25 (m, 12 H, aromatic), 7.24–7.17 (m, 9 H, aromatic), 5.79 (d, J = 7.0 Hz, 1 H), 5.66–5.62 (m, 1 H, Sia-H8), 5.50–5.40 (m, 4 H, Sia-H4), 5.12 (s, 1 H), 5.05–5.01 (m, 4 H, Sia-H7), 4.97 (dd, J = 10.1, 3.7 Hz, 1 H), 4.75–4.71 (m, 4 H), 4.65–4.63 (m, 2 H), 4.61 (dd, J = 10.2, 2.5 Hz, 1 H, Sia-H6), 4.55–4.53 (m, 2 H), 4.46 (d, J = 11.8 Hz, 1 H), 4.33–4.24 (m, 5 H, Fuc-H4, Sia-H9a), 4.13–4.10 (m, 2 H, Fuc-H5), 4.06–3.93 (m, 5 H, Sia-H5, Sia-H9b), 3.87 (s, 3 H, OCOCH₃), 3.82–3.75 (m, 3 H), 3.59–3.54 (m, 2 H), 3.43 (d, J = 5.7 Hz, 1 H), 3.31 (d, J = 1.9 Hz, 1 H), 3.23–3.21 (m, 3 H), 2.51 (dd, J = 12.4, 5.4 Hz, 1 H, Sia-H3_eq), 2.29 (s, 3 H, Ac), 2.16 (s, 3 H, Ac), 2.15 (s, 3 H, Ac), 1.96 (s, 3 H, Ac), 1.86 (s, 3 H, Ac), 1.60 (t, J = 12.4 Hz, 1 H, Sia-H3_aq), 1.47 (s, 3 H, Ac), 1.28 (s, 9 H, t-BuBz), 1.17 (d, J = 6.4 Hz, 3 H, -CH₃). ¹³C{¹H} NMR (125 MHz, CDCl₃): δ = 174.1, 173.6, 170.7, 170.6, 170.0, 169.8, 168.0, 166.1, 165.6, 165.4, 157.1, 156.5, 154.1, 138.9, 138.8, 138.4, 136.7, 135.8, 133.3, 133.2, 133.0, 132.9, 130.2, 130.1, 129.9, 129.8, 129.7, 128.6, 128.5, 128.3, 128.24, 128.23, 128.19, 128.11, 128.04, 128.00, 127.9, 127.75, 127.70, 127.6, 127.4, 127.2, 127.1, 126.7, 126.1, 126.0, 125.8, 125.6, 125.5, 100.7, 100.0, 96.9, 95.5, 79.0, 78.3, 76.5, 75.1, 75.0, 74.4, 73.3, 73.0, 72.5, 71.52, 71.47, 69.3, 68.7, 68.4, 67.08, 66.98, 66.94, 66.6, 62.1, 62.0, 56.0, 53.1, 40.9, 38.3, 35.1, 31.1, 28.0, 26.5, 21.5, 20.68, 20.67, 20.5, 16.7. HRMS (ESI-Orbitrap): m/z [M + Na]⁺ calcd for C₁₁₀H₁₂₀O₃₄Cl₃N₃Na: 2154.6711; found: 2154.6721.
• 19 Synthesis of 18: To a solution of 17 (31.0 mg, 14.5 μmol) in CH₂Cl₂ (0.58 mL) and MeOH (0.15 mL) was added DDQ (16.5 mg, 73.0 μmol) at r.t. After stirring for 3 h at r.t., the reaction was quenched with saturated aqueous NaHCO₃. The aqueous layer was extracted with CH₂Cl₂ and the organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by silica-gel column chromatography (1st: toluene/EtOAc = 2:3, 2nd: toluene/EtOAc = 1:1) to give the crude product (16.3 mg) as an inseparable mixture. This crude mixture was used for the next reaction without further purification. To a solution of crude product (16.3 mg, 8.17 μmol) in anhydrous DMF (0.82 mL) was added SO₃·Py (13.0 mg, 81.7 μmol) at r.t. After stirring the reaction mixture for 10 h at r.t., SO₃·Py (65.0 mg, 0.409 mmol) was added at r.t.. After stirring for 17 h at r.t., the reaction mixture was concentrated in vacuo. The pyridinium salt was exchanged to the sodium salt by eluting over Dowex^®(Na⁺) ion-exchange resin, and the eluent was concentrated in vacuo. The crude product was purified by chromatography on silica-gel (CH₂Cl₂/MeOH/Et₃N = 100:0:0.1 to 93.8:6.3:0.1) to give 18 (16.1 mg, 53% in 2 steps) as a white powder. ¹H NMR (500 MHz, CD₃OD): δ = 8.17 (d, J = 7.6 Hz, 2 H, aromatic), 8.04–8.00 (m, 4 H, aromatic), 7.56–7.37 (m, 8 H, aromatic), 7.22–7.09 (m, 15 H, aromatic), 7.01–6.92 (m, 5 H, aromatic), 5.69 (s, 1 H, Gal-H4), 5.49 (t, J = 9.2 Hz, 1 H, Gal-H2), 5.36–5.29 (m, 2 H, Gal-H1, Sia-H4), 5.27 (d, J = 3.3 Hz, 1 H, Fuc-H1), 5.21 (s, 1 H), 5.03 (d, J = 7.9 Hz, 1 H), 4.97–4.94 (m, 3 H), 4.90 (dd, J = 10.1, 3.2 Hz, 1 H, Gal-H3), 4.66–4.61 (m, 3 H, Fuc-H5), 4.51–4.46 (m, 4 H, Gal-H6a), 4.41 (d, J = 11.9 Hz, 1 H), 4.37 (dd, J = 12.8 2.4 Hz, 1 H), 4.34–4.24 (m, 4 H, Gal-H5, GlcN-H1), 4.14 (d, J = 10.6 Hz, 1 H), 4.05–4.02 (m, 4 H, Fuc-H3, Gal-H6b), 3.87–3.83 (m,3 H, GlcN-H3, Sia-H5), 3.78 (dd, J = 10.2, 3.7 Hz, 1 H, Fuc-H2), 3.65 (s, 1 H), 3.57 (s, 1 H, Fuc-H4), 3.52–3.49 (m, 1 H, GlcN-H2), 3.41–3.37 (m, 1 H), 3.21 (m, 6 H), 3.15–3,12 (m, 2 H), 3.07 (q, J = 7.3 Hz, 2 H), 2.27 (s, 3 H, Ac), 2.18 (s, 3 H, Ac), 2.11–2.06 (m, 5 H, Ac, Sia-H3_eq,Sia-H3_aq), 1.90 (m, 6 H, Ac), 1.85 (s, 3 H, Ac), 1.38 (d, J = 6.4 Hz, 3 H), 1.19–1.16 (m, 4 H), 1.13 (s, 9 H, t-BuBz). ¹³C{¹H} NMR (125 MHz, CD₃OD): δ = 176.7, 176.6, 172.4, 172.1, 171.4, 169.0, 167.6, 167.5, 167.3, 166.5, 158.6, 156.7, 140.3, 140.0, 139.9, 138.3, 138.2, 134.7, 134.3, 131.3, 131.2, 131.0, 130.7, 130.0, 129.9, 129.5, 129.2, 129.14, 129.1, 129.04, 128.99, 128.85, 128.75, 128.6, 128.3, 128.1, 127.9, 126.8, 103.1, 101.7, 101.3, 100.9, 98.2, 96.9, 81.3, 79.6, 77.5, 76.9, 76.2, 75.6, 74.9, 74.7, 73.5, 73.3, 73.2, 73.1, 71.9, 71.3, 71.1, 69.6, 68.1, 67.9, 67.7, 67.5, 63.4, 62.8, 59.7, 59.3, 53.2, 48.0, 41.9, 36.0, 31.5, 27.9, 25.9, 21.3, 20.9, 20.72, 20.68, 17.5, 9.3. HRMS (ESI-Orbitrap): m/z [M + Na]⁺ calcd for C₉₉H₁₁₁O₃₇Cl₃N₃SNa: 2116.5473; found: 2116.5461.
• 20 Synthesis of 19: To a solution of 18 (20.0 mg 9.54 μL) in THF (2.4 mL) was added 1 M aqueous NaOH (191.0 μL) at r.t. After stirring for 12 h at r.t., 1 M aqueous NaHCO₃ (47.7 μL), H₂O (450.0 μL), and Ac₂O (4.50 μL, 47.7 μmol) were added at r.t. After stirring for 24 h at r.t., 1 M aqueous NaHCO₃ (47.7 μL), H₂O (450.0 μL), and Ac₂O (4.50 μL, 47.7 μmol) were added at r.t. After stirring for 29 h at r.t., 1 M aqueous NaHCO₃ (47.7 μL), and Ac₂O (4.50 μL, 47.7 μmol) were added at r.t. After stirring for 24 h at r.t., the reaction mixture was quenched with 1 M aqueous HCl and freeze-dried with H₂O. This crude mixture was used for the next reaction without further purification. To a solution of the obtained mixture in _t BuOH/H₂O/AcOH = 10:8:1 (950.0 μL) was added a solution of 50% Pd(OH)₂ (13.4 mg, 47.7 μmol) in H₂O (150.0 μL) at r.t. After stirring for 4 h under H₂ atmosphere (balloon) at r.t., the catalyst was removed by centrifugation. The supernatant layer was freeze-dried with H₂O. This crude mixture was used for the next reaction without further purification. To a solution of obtained mixture in H₂O (382.0 μL), and acetone (572.0 μL) were added 1 M aqueous NaHCO₃ (38.2 μL) and FmocOSu (19.3 mg, 57.2 μmol) at r.t. After stirring for 1 h at r.t., 1 M aqueous NaHCO₃ (38.2 μL) and FmocOSu (19.3 mg, 57.2 μmol) were added at r.t. After stirring for 41 h at r.t., the reaction mixture was concentrated in vacuo and freeze-dried with H₂O and purified by reverse-phase HPLC (Nacalai, COSMOSIL 5C₁₈-AR-300, 10ID × 250 mm column, 15→35% MeCN aq. containing 10 mM ammonium formate for 40 min, flow rate 4.0 mL min^–1) to give 19 as a white powder (6.3 mg, 56% in 3 steps). ¹H NMR (500 MHz, D₂O): δ = 7.81 (d, J = 7.6 Hz, 2 H, aromatic), 7.60(d, J = 7.2 Hz, 2 H, aromatic), 7.39 (t, J = 7.6 Hz, 2 H, aromatic), 7.32 (t, J = 7.2 Hz, 2 H, aromatic), 4.94 (d, J = 3.0 Hz, 1 H, anomeric), 4.69–4.52 (m, 3 H, NHCOOCH₂), 4.50–4.47 (m, 2 H, anomeric), 4.33–4.15 (m, 3 H, anomeric), 3.98 (d, J = 8.6 Hz, 1 H), 3.83–3.39 (m, 21 H), 3.06 (s, 2 H, OCH₂CH₂NH), 2.63 (dd, J = 12.5, 4.6 Hz, 1 H, Sia-H3_eq), 1.91 (s, 3 H, Ac), 1.71 (s, 3 H, Ac), 1.69 (d, J = 12.5 Hz, 1 H, Sia-H3_aq), 1.06 (d, J = 5.7 Hz, 1 H, -CH₃). ¹³C{¹H} NMR (226 MHz, D₂O): δ = 174.9, 174.0, 171.0, 158.2, 143.9, 143.8, 140.96, 140.93, 128.0, 127.50, 127.46, 125.0, 124.9, 124.6, 120.1, 101.1, 100.9, 99.5, 98.5, 75.4, 74.8, 74.5, 72.8, 72.6, 71.8, 71.3, 69.4, 69.1, 68.7, 68.4, 68.0, 67.7, 67.2, 66.7, 65.9, 65.8, 62.5, 61.4, 55.5, 51.7, 47.0, 40.2, 39.6, 22.02, 22.00, 15.3. HRMS (ESI-Orbitrap): m/z [M – Na – H]^2– calcd for C₄₈H₆₅O₂₈N₃S: 581.6743; found: 581.6750.