The Journal of Organic Chemistry
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2′), 9.0 (C-3′); HRMS (ESI) m/z calcd for [C6H12O4 + H]+ 149.0808,
obsd 149.0809.
was maintained at −78 °C. After 30 min, the reaction mixture was
warmed to room temperature over 2 h, and the mixture was poured into
an ice−water solution (50 mL) containing KHCO3 (5 g, 50 mmol). The
aqueous layer was washed with ethyl acetate and concentrated to form a
white semisolid residue. The solids were extracted with methanol and
filtered. Concentration of the mother liquor yielded the crude potassium
cyclophosphate, which was purified using silica gel column chromatog-
raphy (EtOAc → EtOAc/MeOH, 3/1, v/v) to yield the pure
cyclophosphatidic acids as white solids.
1-O-Prop-2-enyl-2,3-O-isopropylideneglycerol. 1,2-O-Isopropyli-
deneglycerol (5.35 mL, 5.0 g, 37.8 mmol) was coevaporated with
DMF, dissolved in DMF (150 mL), and cooled to 0 °C using an ice bath.
Allyl bromide (6.4 mL, 5.0 g, 41.6 mmol, 1.1 equiv) was added and, with
continuous stirring, NaH (60% in mineral oil, 1.8 g, 45 mmol, 1.2 equiv)
was added slowly, portionwise. After stirring was continued for 1 h at
room temperature, the reaction mixture was quenched by the addition of
ice−water (100 mL) and extracted with ether (2×). The combined
ethereal extracts were washed with water and brine, dried (MgSO4),
filtered, and concentrated. Purification of the residue by silica gel column
chromatography (PE) gave the title compound as a colorless oil (5.45 g,
31.6 mmol, 84%): Rf = 0.52 (PE/EtOAc, 9/1, v/v); IR ν 3081, 2986,
2933, 2864, 1456, 1422, 1380, 1371, 1257, 1213, 1150, 1076, 1052, 999,
926, 844, 793, 737 cm−1; 1H NMR (500 MHz, CDCl3) δ 5.89 (ddt, J2′,3a′
= 17.3 Hz, J2′,3b′ = 10.5 Hz, J2′,1′ = 5.8 Hz, 1H, H-2′), 5.27 (d, J2′,3a′ = 17.4
1-O-Propanoylcyclophosphatidic Acid Potassium Salt (I). 1-O-
Propanoylglycerol (163 mg, 1.1 mmol) was subjected to the general
procedure for the synthesis of cyclophosphatidic acids to produce the
title compound I as a white solid (234 mg, 0.94 mmol, 86%): Rf = 0.29
(MeOH/EtOAc, 1/1, v/v); IR ν 3358, 2976, 2941, 1552, 1465, 1407,
1
1371, 1297, 1221, 1105, 1073, 1008, 971, 811 cm−1; H NMR (600
MHz, D2O) δ 4.64 (dp, J1a,2 = 3.5 Hz, J1b,2 = J2,3a = J2,3b = J2,P = 6.5 Hz,
1H, H-2), 4.27 (ddd, J2,3a = 6.5 Hz, J3a,3b = 9.7 Hz, J3a,P = 12.0 Hz, 1H, H-
3a), 4.26 (dd, J1a,1b = 12.2 Hz, J1a,2 = 3.5 Hz, 1H, H-1a), 4.13 (dd, J1b,2b
12.2 Hz, J1b,2 = 6.0 Hz, 1H, H-1b), 4.00 (dt, J2,3b = 7.0 Hz, J3a,3b = J3a,P
=
=
Hz, 1H, H-3a′), 5.18 (d, J2′,3b′ = 10.5 Hz, 1H, H-3b′), 4.27 (p, J1,2 = J2,3
6.0 Hz, 1H, H-2), 4.07−3.99 (m, 3H, H-3a and CH2-1′), 3.73 (t, J2,3b
=
=
9.5 Hz, 1H, H-3b), 2.38 (q, J2′,3′ = 7.6 Hz, 2H, CH2-2′), 1.03 (t, J2′,3′ = 7.6
Hz, 3H, CH3-3′); 13C NMR (150 MHz, D2O) δ 174.6 (s, CO), 71.4
(d, JP = 2.6 Hz, C-2), 63.1 (d, JP = 1.1 Hz, C-3), 61.6 (d, JP = 5.4 Hz, C-1),
24.5 (s, C-2′), 5.7 (d, JP = 6.4 Hz, C-3′); 31P NMR (120 MHz, D2O) δ
18.0 (ddd, 3JH‑2,P = 3.5 Hz, 2JH‑3a,P = 11.9 Hz, 2JH‑3b = 9.5 Hz); HRMS
(ESI) m/z calcd for [C6H10O6P]− 209.0220, obsd 209.0221.
J3a,3b = 6.8 Hz, 1H, H-3b), 3.52 (dd, J1a,2 = 5.8 Hz, J1a,1b = 9.8 Hz, 1H, H-
1a), 3.43 (dd, J1b,2 = 5.3 Hz, J1a,1b = 9.8 Hz, 1H, H-1b), 1.42 (s, 3H, CH3
iPr), 1.36 (s, 3H, CH3 iPr); 13C NMR (125 MHz, CDCl3) δ 134.5 (C-
2′), 117.3 (C-3′), 109.4 (Cq iPr), 74.7 (C-2), 72.5 (C-1′), 71.1 (C-1),
66.8 (C-3), 26.7, 25.4 (2 × CH3 iPr); HRMS (ESI) m/z calcd for
[C9H16O3 + H]+ 173.1172, obsd 173.1168.
1-O-Prop-1-enylcyclophosphatidic Acid Potassium Salt (II). 1-O-
Prop-2-enylglycerol (132 mg, 1.0 mmol) was subjected to the general
procedure for the synthesis of cyclophosphatidic acids to produce the
title compound II as a white solid with an E/Z ratio of 1.8/1 (178 mg,
0.77 mmol, 77%): Rf = 0.21 (MeOH/EtOAc, 1/1, v/v); IR ν 3336, 2947,
2835, 1450, 1411, 1213, 1103, 1071, 1019, 928, 884, 813 cm−1; HRMS
(ESI) m/z calcd for [C6H10O5P]− 193.0271, obsd 193.0273. Data for
the E isomer are as follows: 1H NMR (500 MHz, CDCl3) δ 6.17 (dq,
1-O-Prop-2-enylglycerol. 1,2-O-Isopropylideneglycerol (1.72 g, 10
mmol) was dissolved in a mixture of acetic acid (20 mL) and water (5
mL) and stirred for 4 h at 50 °C, after which the reaction mixture was
concentrated and coevaporated with toluene. Purification of the residue
by Kugelrohr distillation gave the title compound as a colorless oil (1.15
g, 8.7 mmol, 87%): Rf = 0.62 (EtOAc/MeOH, 4/1, v/v); IR ν 3343,
2940, 2833, 1450, 1398, 1269, 1111, 928, 852, 735 cm−1; 1H NMR (500
MHz, CDCl3) δ 5.81 (ddt, J2′,3a′ = 17.4 Hz, J2′,3b′ = 10.3 Hz, J2′,1′ = 5.9 Hz,
1H, H-2′), 5.20 (dd, J2′,3a′ = 17.4 Hz, J3a′,3b′ = 1.3 Hz, 1H, H-3a′), 5.13 (d,
J
1′,2′ = 12.5 Hz, 4J1′,3′ = 1.6 Hz, 1H, H-1′), 4.87 (dq, J1′,2′ = 12.5 Hz, J2,3
6.8 Hz, 1H, H-2′), 4.59−4.53 (m, 1H, H-2), 4.20 (ddd, J2,3a = 6.5 Hz,
J3a,3b = 9.3 Hz, J3a,P = 12.3 Hz, 1H, H-3a), 3.90 (ddd, J2,3b = 7.1 Hz, J3a,3b
=
J
2′,3b′ = 10.3 Hz, 1H, H-3b′), 3.93 (d, J1′,2′ = 6.0 Hz, 2H, CH2-1′), 3.77−
=
3.72 (m, 1H, H-2), 3.49 (dd, J2,3a = 4.5 Hz, J3a,3b = 11.8 Hz, 1H, H-3a),
3.46 (dd, J1a,2 = 4.0 Hz, J1a,1b = 10.7 Hz, 1H, H-1a), 3.41 (dd, J2,3b = 6.2
Hz, J3a,3b = 11.8 Hz, 1H, H-3b), 3.36 (dd, J1b,2 = 7.0 Hz, J1a,1b = 10.7 Hz,
1H, H-1b); 13C NMR (125 MHz, CDCl3) δ 133.7 (C-2′), 118.2 (C-3′),
72.0 (C-1′), 70.7 (C-1), 70.3 (C-2), 62.6 (C-3); HRMS (ESI) m/z calcd
for [C6H12O3 + H]+ 133.0859, obsd 133.0858.
9.2 Hz, J3b,P = 18.3 Hz, 1H, H-3b), 3.76−3.71 (m, 2H, CH2-1), 1.41 (dd,
4
J2′,3′ = 6.8 Hz, J1′,3′ = 1.5 Hz, 3H, CH3-3′); 13C NMR (125 MHz,
CDCl3) δ 145.3 (s, C-1′), 103.2 (s, C-2′), 74.7 (d, JP = 1.9 Hz, C-1), 69.4
(d, JP = 5.8 Hz, C-2), 65.7 (s, C-3), 11.4 (C-3′). Data for the Z isomer are
as follows: 1H NMR (500 MHz, CDCl3) δ 5.97 (dq, J1′,2′ = 6.2 Hz, 4J1′,3′
= 1.7 Hz, 1H, H-1′), 4.59−4.53 (m, 1H, H-2), 4.87 (app p, J1′,2′ = J2,3
=
E-/Z-1-O-Prop-1-enylglycerol. A mixture of 1-O-prop-2-enylglycerol
(132 mg, 1.0 mmol), tris(triphenylphosphine)rhodium(I) chloride (60
mg, 65 μmol, 6.5 mol %), and DBU (200 mg, 1.3 mmol, 1.3 equiv) in
ethanol/H2O (20 mL, 9/1, v/v) was refluxed for 4 h, after which TLC
analysis (EtOAc) showed complete conversion of the starting material
into a slightly higher running product. The mixture was concentrated in
vacuo and coevaporated with ethanol to remove traces of water, and the
residue was purified by silica gel column chromatography (PE → PE/
EtOAc, 3/1, v/v) to give the title compound as a mixture of E and Z
isomers in a 5/2 ratio (112 mg, 0.85 mmol, 85%): Rf = 0.31 (EtOAc); IR
ν 3876, 3361, 2987, 2943, 2911, 1453, 1406, 1393, 1310, 1251, 1182,
1118, 1066, 1050, 999, 928, 900, 869, 723 cm−1; HRMS (ESI) m/z calcd
for [C6H12O3 + H]+ 133.0859, obsd 133.0858. Data for the E-isomer are
as follows: 1H NMR (500 MHz, CDCl3) δ 6.15 (d, J1,2 = 12.4 Hz, 1H, H-
1′), 4.71 (dq, J1,2 = 12.4 Hz, J2,3 = 6.6 Hz, 1H, H-2′), 3.86−3.80 (m, 1H,
H-2), 3.66−3.49 (m, 4H, CH2-1 and CH2-3), 1.46 (d, J2′,3′ = 6.6 Hz, 3H,
CH3-3′); 13C NMR (125 MHz, CDCl3) δ 146.2 (C-1′), 99.0 (C-2′),
70.5, 69.9, 63.6 (C-1, C-2, and C-3), 12.4 (C-3′). Data for the Z isomer
are as follows: 1H NMR (500 MHz, CDCl3) δ 5.89 (bd, J1,2 = 6.1 Hz, 1H,
H-1′), 4.33 (p, J1,2 = J2,3 = 6.8 Hz, 1H, H-2′), 3.86−3.80 (m, 1H, H-2),
3.68−3.49 (m, 4H, CH2-1 and CH2-3), 1.49 (d, J2′,3′ = 6.9 Hz, 3H, CH3-
3′); 13C NMR (125 MHz, CDCl3) δ 145.6 (C-1′), 101.5 (C-2′), 72.7,
70.8, 63.5 (C-1, C-2 and C-3), 9.1 (C-3′).
6.9 Hz, 1H, H-2′), 4.21 (ddd, J2,3a = 6.5 Hz, J3a,3b = 9.3 Hz, J3a,P = 12.3 Hz,
1H, H-3a), 3.90 (ddd, J2,3b = 7.1 Hz, J3a,3b = 9.2 Hz, J3b,P = 18.3 Hz, 1H,
H-3b), 3.82 (d, J1,2 = 5.2 Hz, 1H, CH2-1), 1.43 (dd, J2′,3′ = 7.0 Hz, 4J1′,3′
=
1.7 Hz, 3H, CH3-3′); 13C NMR (125 MHz, CDCl3) δ 145.0 (s, C-1′),
101.7 (s, C-2′), 75.0 (d, JP = 1.9 Hz, C-1), 71.5 (d, JP = 5.7 Hz, C-2), 65.6
(s, C-3), 8.4 (C-3′).
1-O-Propen-2-ylcyclophosphatidic Acid Potassium Salt (III). 1-O-
Allylglycerol (132 mg, 1 mmol) was subjected to the general procedure
for the synthesis of cyclophosphatidic acids to produce the title
compound III as a white solid (187 mg, 0.81 mmol, 81%): Rf = 0.21
(MeOH/EtOAc, 1/1, v/v); IR ν 3364, 2934, 1567, 1494, 1406, 1349,
1
1289, 1225, 1077, 1031, 974, 803, 769 cm−1; H NMR (500 MHz,
CDCl3) δ 5.82 (ddt, J2′,3a′ = 17.3 Hz, J2′,3b′ = 10.5 Hz, J2′,1′ = 5.9 Hz, 1H,
H-2′), 5.22 (d, J2′,3a′ = 17.3 Hz, 1H, H-3a′), 5.15 (d, J2′,3b′ = 10.5 Hz, 1H,
H-3b′), 4.52 (h, J1,2 = J2,3 = J2,P = 6.0 Hz, 1H, H-2), 4.19 (ddd, J2,3a = 6.4
Hz, J3a,3b = 9.3 Hz, J3a,P = 12.8 Hz, 1H, H-3a), 3.98 (d, J1′,2′ = 6.1 Hz, 1H,
CH2-1′), 3.84 (q, J2,3b = J3a,3b = J3b,P = 8.5 Hz, 1H, H-3b), 3.55 (d, J1,2
=
5.3 Hz, 1H, CH2-1); 13C NMR (125 MHz, CDCl3) δ 133.4 (s, C-2′),
118.6 (s, C-3′), 75.1 (d, JP = 2.4 Hz, C-1), 72.1 (s, C-1′), 70.0 (d, JP = 5.8
Hz, C-2), 65.8 (d, JP = 1.4 Hz, C-3); 31P NMR (120 MHz, D2O) δ 18.3;
HRMS (ESI) m/z calcd for [C6H10O5P]− 193.0271, obsd 193.0275.
Immunological Methods. All extracted compounds were con-
firmed to be free of endotoxin, as determined by the Limulus amebocyte
lysate (LAL) assay.38
General Procedure for the Synthesis of Cyclophosphatidic
Acids I−III. To a solution of POCl3 (0.93 mL, 1.5 g, 10 mmol, 10 equiv)
in dry THF (5 mL) at −78 °C, under an argon atmosphere, was slowly
added pyridine (0.81 mL, 0.79 g, 10 mmol, 10 equiv). After the mixture
was stirred for 5 min at −78 °C, a solution of the diol (1 mmol) in dry
THF (5 mL) was added dropwise over 10 min, while the temperature
Macrophage Assay. Bone marrow derived macrophages were
generated as previously described.25 Briefly, bone marrow cells from
C57Bl/6 male mice were cultured in complete IMDM (2.5 × 105 cells/
mL) supplemented with 10 ng/mL of GM-CSF. On days 2, 5, and 7 half
7339
dx.doi.org/10.1021/jo501016c | J. Org. Chem. 2014, 79, 7332−7341