Validation and Application of an Innovative Protective Group Concept: Enhancing Substrate Reactivity in Glycosylations by Disrupting Intermolecular Interactions
Kumpei Yano‡
a
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
,
Takuya Yoshimoto‡
a
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
,
Masato Tsutsui
a
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
,
Yoshiyuki Manabe∗
a
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
b
Forefront Research Center, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
,
Koichi Fukase∗
a
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
b
Forefront Research Center, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
c
Center for Advanced Modalities and DDS, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan
› Author AffiliationsThis work was financially supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants 20H05675, 20K05727, 20H04709, 21H05074, and 23K17372, as well as the Japan Science and Technology Agency (JST) CREST grant JPMJCR20R3, and JST FOREST Program grant JPMJFR211Z.
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
Supporting Information
Supporting information for this article is available online at https://doi.org/10.1055/a-2269-7680.
15m
Krishnamurthy VR,
Sardar MY. R,
Ying Y,
Song X,
Haller C,
Dai E,
Wang X,
Hanjaya-Putra D,
Sun L,
Morikis V,
Simon SI,
Woods RJ,
Cummings RD,
Chaikof EL.
Nat. Commun. 2015; 6: 6387
17Synthesis 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-H3eq), 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-H3aq), 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.
18Synthesis 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-H3eq), 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-H3aq), 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.
19Synthesis 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-H3eq,Sia-H3aq), 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.
20Synthesis 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 tBuOH/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-H3eq), 1.91 (s, 3 H, Ac), 1.71 (s, 3 H, Ac), 1.69 (d, J = 12.5 Hz, 1 H, Sia-H3aq), 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.
