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Organic & Biomolecular Chemistry
suspension of the phosphonic acid 18a (20 mg, 0.03 mmol)
Triethylammonium (2-amino-2-deoxy-α-D-glucopyranosyl)-
obtained above and glycosyl acceptor 21 (37 mg, 0.037 mmol) (1–6)-1-O-(sn-3,4-di-O-palmitoyl-butyl-1-phosphonate)-D-myo-
in dry pyridine (1 mL) at 60 °C was added trichloroacetonitrile inositol (23a). The protected C-P GPI intermediate 22a (0.02 g,
(500 µL, 5 mmol), and the reaction mixture was stirred at 0.012 mmol) and the catalyst 20% Pd(OH)2 (0.05 g) were dis-
60 °C for 60 h. After completion, the reaction mixture was con- solved in a solvent mixture of MeOH (2 mL), CH2Cl2 (2 mL)
centrated under reduced pressure to afford a pale orange and H2O (0.1 mL). The residual and the dissolved air from the
residue. Purification of the residue by chromatography on flask were removed by repeated evacuations and the reaction
silica gel eluting with CHC13–CH3OH (9 : l) gave 40 mg (77%) mixture was stirred under a hydrogen atmosphere overnight.
of 22a as a white foam. Rf = 0.6 (CH2Cl2–MeOH, 9 : 1); [α]D
=
After completion of the reaction, the mixture was filtered
1
+25.33 (c 0.01, CHCl3); H NMR (400 MHz, CDCl3): δ 7.34–7.20 through a small celite pad, and concentrated under reduced
(m, 35H), 5.67 (d, J = 3.4 Hz, 1H), 5.08–4.98 (m, 3H), 4.93–4.81 pressure. The product was purified by quick filtration through
(m, 4H), 4.78–4.73 (m, 3H), 4.65–4.57 (m, 4H), 4.54–4.51 (m, a silica column using MeOH–CH2Cl2 (1 : 1) to provide the com-
1H), 4.48–4.44 (m, 1H), 4.39–4.32 (m, 1H), 4.13–4.01 (m, 4H), pound 23a (9.5 mg, 80%). Rf = 0.4 (EtOAc–MeOH–CH3COCH3–
3.85–3.75 (m, 3H), 3.65–3.49 (m, 4H), 3.31–3.26 (m, 1H), H2O, 7 : 1 : 1 : 1); 1H NMR (400 MHz, CDCl3–CD3OD–D2O =
2.89–2.87 (m, 6H), 2.21–2.14 (m, 4H), 1.85–1.74 (m, 4H), 4 : 4 : 1): δ 5.47 (m, 1H), 5.17 (m, 2H), 4.34 (m, 2H), 4.15–4.03
1.56–1.46 (m, 4H), 1.31–1.20 (m, 48H), 1.18 (t, J = 7.2 Hz, 9H), (m, 3H), 3.87 (m, 2H), 3.73–3.67 (m, 3H), 3.39–3.36 (m, 2H),
0.88 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, CDCl3): δ 23.48; 3.19 (q, J = 7.0 Hz, 6H), 3.14 (t, J = 5.0 Hz, 1H), 2.34–2.31 (m,
13C NMR (100 MHz, CDCl3): 173.3, 173.2, 139.5, 138.6, 138.6, 4H), 2.00–1.84 (m, 4H), 1.63–1.62 (m, 4H), 1.39–1.27 (m, 57H),
138.5, 138.4, 138.2, 138.1, 128.4, 128.3, 128.3, 128.2, 128.1, 0.89 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, CDCl3–CD3OD–
128.0, 128.0, 127.9, 127.8, 127.8, 127.6, 127.5, 127.4, 127.4, D2O = 4 : 4 : 1): δ 30.39; 13C NMR (100 MHz, CDCl3–CD3OD–
127.0, 126.9, 97.2, 84.2, 82.2, 80.8, 80.8, 80.4 (d, JCP = 6.1 Hz), D2O = 4 : 4 : 1): 175.4, 175.3, 95.4, 77.8, 73.5, 72.7, 72.6, 71.5,
78.2 (d, JCP = 5.7 Hz), 77.6 (d, JCP = 6.2 Hz), 75.7, 75.2, 75.1, 70.5, 70.4, 64.8, 64.7, 62.6, 61.3, 61.2, 54.5, 46.6, 34.3, 29.6
74.9, 73.6, 73.5, 72.3, 71.8 (d, JCP = 14.8 Hz), 71.5, 70.1, 68.3, (multiple peaks), 24.9, 22.7, 22.6, 21.8, 13.5, 8.5; HRMS (ESI):
64.4, 63.7, 45.3, 34.3, 34.3, 34.1, 31.9, 29.7, 29.7, 29.6, 29.5, calcd for C48H92NO16P (M − H)− 968.6081, found 968.6093.
29.5, 29.4, 29.3, 29.3, 29.1, 25.0 (d, J = 4.9 Hz), 24.8, 22.7, 14.1,
8.5; HRMS (MALDI): calcd for C97H132N3O16P (M − H)− (1–6)-1-O-(sn-2,3-di-O-palmitoyl-propyl-1-phosphonate)-D-myo-
1625.9306, found 1625.9333. inositol (23b). The protected C-P GPI intermediate 22b (0.02 g,
Triethylammonium (2-amino-2-deoxy-α-D-glucopyranosyl)-
Triethylammonium (3,4,6-tri-O-benzyl-2-azido-2-deoxy-α-D- 0.012 mmol) and the catalyst 20% Pd(OH)2 (0.05 g) were dis-
glucopyranosyl)-(1–6)-2,3,4,5-tetra-O-benzyl-1-O-(sn-2,3-di-O- solved in a solvent mixture of MeOH (2 mL), CH2Cl2 (2 mL)
palmitoyl-propyl-1-phosphonate)-D-myo-inositol (22b). To
a
and H2O (0.1 mL). The residual and the dissolved air from the
suspension of the phosphonic acid 18b (20 mg, 0.03 mmol) flask were removed by repeated evacuations and the reaction
obtained above and glycosyl acceptor 21 (37 mg, 0.037 mmol) mixture was stirred under a hydrogen atmosphere overnight.
in dry pyridine (1 mL) at 60 °C was added trichloroacetonitrile After completion of the reaction, the mixture was filtered
(500 µL, 5 mmol), and the reaction mixture was stirred at through a small celite pad and concentrated under reduced
60 °C for 60 h. After completion, the reaction mixture was con- pressure. The product was purified by quick filtration through
centrated under reduced pressure to afford a pale orange a silica column using MeOH–CH2Cl2 (1 : 1) to provide the com-
residue. Purification of the residue by chromatography on pound 23b (9.3 mg, 80%). Rf = 0.4 (EtOAc–MeOH–CH3COCH3–
silica gel eluting with CHC13–CH3OH (9 : l) gave 25 mg (50%) H2O, 7 : 1 : 1 : 1); 1H NMR (400 MHz, CDCl3–CD3OD–D2O =
of 22b as a white foam. Rf = 0.6 (CH2Cl2–MeOH, 9 : 1); [α]D
=
4 : 4 : 1): δ 5.44 (m, 2H), 4.47–4.35 (m, 2H), 4.12–3.68 (m, 9H),
1
+28.38 (c 0.01, CHCl3); H NMR (500 MHz, CDCl3): δ 7.40–7.18 3.46–3.35 (m, 2H), 3.19 (q, J = 7.0 Hz, 7H), 2.35–2.33 (m, 4H),
(m, 35H), 5.67 (d, J = 3.4 Hz, 1H), 5.06–4.97 (m, 3H), 4.92 (d, 2.19–2.14 (m, 2H), 1.63–1.62 (m, 4H), 1.38–1.28 (m, 57H), 0.89
J = 3.4 Hz, 1H), 4.85–4.75 (m, 6H), 4.65–4.56 (m, 4H), 4.48–4.44 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, CDCl3–CD3OD–D2O =
(m, 2H), 4.39–4.34 (m, 1H), 4.14–4.00 (m, 5H), 3.83–3.74 (m, 4 : 4 : 1): δ 23.53; 13C NMR (100 MHz, CDCl3–CD3OD–D2O =
3H), 3.65–3.53 (m, 3H), 3.35–3.31 (m, 1H), 2.98 (q, J = 7.0 Hz, 4 : 4 : 1): 174.4, 174.3, 95.1, 77.7, 75.6, 72.9, 72.3, 71.8, 70.4,
6H), 2.20–2.14 (m, 4H), 1.81–1.75 (m, 2H), 1.53–1.51 (m, 4H), 70.0, 66.8, 64.9, 64.8, 60.7, 60.6, 54.1, 46.2, 33.6, 31.8–29.0,
1.25–1.21 (m, 57H), 0.88 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, 28.5, 24.3, 22.1, 13.2, 7.9; HRMS (ESI): calcd for C47H90NO16P
CDCl3): δ 16.62; 13C NMR (125 MHz, CDCl3): 173.6, 173.2, (M + H)+ 956.6075, found 956.6042.
138.8, 138.8, 138.6, 138.5, 138.3, 138.2, 138.1, 128.5, 128.4,
128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1,
B. Method for the PI-PLC reaction17–19
128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7, 127.6, The sample in CHCl3 was evaporated and re-suspended in H2O
127.6, 127.5, 127.5, 127.4, 127.3, 127.2, 127.0, 98.4, 83.0, 82.0, by bath sonication (5 min) such that the final concentration of
81.5, 80.9, 80.5, 78.5, 77.5, 75.5, 75.4, 75.1, 74.9, 74.7, 73.6, the sample was 10 mM. To 5 µL of the sample (10 mM) were
73.3, 72.2, 70.8, 70.1, 68.1, 64.1, 63.32, 45.3, 34.5, 34.4, 34.2, added 5 µL of sodium deoxycholate (0.8%, w/v), 10 µL of
31.9, 29.7, 29.7, 29.6, 29.5, 29.3, 29.2, 29.2, 22.7, 14.1, 8.4; borate buffer [0.1 M (sodium borate/HCl (pH 7.5))] and 4 µL of
HRMS (MALDI): calcd for C96H130N3O16P (M − H)− 1611.9149, enzyme PI-PLC. The reaction mixture was incubated at 37 °C
found 1611.9126.
for 20 minutes and quenched with 200 µL CHCl3–MeOH–
1170 | Org. Biomol. Chem., 2014, 12, 1163–1172
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