Efficient Synthesis of Unsaturated 1-Monoacyl Glycerols for in meso Crystallization of Membrane Proteins

Abstract

A highly efficient synthesis of unsaturated 1-monoacyl glycerols was established to fulfill the pressing need for materials that form lipidic mesophases utilized in membrane protein crystallization.

Key words

hydrogenation - lipidic cubic phase - lipids - membrane proteins - transesterification

References and Notes

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10

2-MAG isomers can be clearly identified by examining their signature ^¹H NMR and ^¹³C NMR signals.^5,6

11

Representative Procedures for the Synthesis of 9.9-MAG (Entry l)
2-[(8-Bromooctyl)oxy]tetrahydro-2 H -pyran (1l): To a stirred solution of 8-bromooctan-1-ol (10.5 g, 50 mmol) in anhyd CH₂Cl₂ (50 mL), DHP (5.2 mL, 57 mmol) and p-toluenesulfonic acid (PTSA, 430 mg, 2.5 mmol) were added at r.t. The reaction was stirred overnight and then sat. aq NaHCO₃ (50 mL) was added. The aqueous layer was extracted with CH₂Cl₂ (50 mL). The combined organic extracts were dried over Na₂SO₄ and concentrated in vacuo. Flash chromatography on silica gel using 2% EtOAc in hexane as the eluent gave 1l ^¹² as a colorless oil in 95% yield. 2-(Octadec-9-yn-1-yloxy)tetrahydro-2 H -pyran (2l): To a stirred solution of 1-decyne (2.76 g, 20 mmol) in anhyd THF (50 mL), hexamethylphosphoramide (HMPA, 10 mL) and n-BuLi (1.6 M solution in hexane, 11 mL) were slowly added at -60 ˚C. After half an hour the reaction mixture was warmed to -20 ˚C, and then 1l (4.1 g, 14 mmol) was added dropwise. The reaction mixture was slowly warmed to r.t. and stirred overnight. The reaction was quenched by addition of sat. aq NH₄Cl (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with sat. aq NaHCO₃ and brine, and then dried over Na₂SO₄ and filtered. Compound 2l ^¹³ was purified by silica gel chromatography (2% EtOAc in hexane) as a pale yellow oil in 90% yield.
Octadec-9-ynoic Acid (3l): Freshly prepared Jones reagent (2.7 M, 10 mL) was added dropwise to a solution of 2l (2.8 g, 8 mmol) in acetone (50 mL) while maintaining the reaction temperature at 0-4 ˚C. Upon the disappearance of 2l, as monitored by TLC (ca. 4 h), i-PrOH (1 mL) was added to quench excessive Jones oxidant. The chromium salt was removed by filtration and washed thoroughly with acetone (50 mL). The filtrate was concentrated in vacuo, and the residue was submitted to silica gel chromatography (10-40% EtOAc in hexane) to give 3l ^¹³ as a white solid (mp 47-48 ˚C) in 95% yield.
Methyl Octadec-9-ynoate (4l): Concentrated H₂SO₄ (0.1 mL, ca. 96%) was added to a suspension of 3l (2.0 g, 7 mmol) in MeOH (30 mL). The solution was concentrated after overnight reaction. Purification by silica gel chromatography (2-5% EtOAc in hexane) gave compound 4l as a light yellow oil in 97% yield. ^¹H NMR (400 MHz, CDCl₃): δ = 3.67 (s, 3 H), 2.30 (t, J = 7.5 Hz, 2 H), 2.13 (t,
J = 6.9 Hz, 4 H), 1.59-1.69 (m, 2 H), 1.42-1.54 (m, 4 H), 1.17-1.42 (m, 16 H), 0.88 (t, J = 6.7 Hz, 3 H).
Methyl ( Z )-Octadec-9-enoate (5l): Ni(OAc)₂˙4H₂O (250 mg, 1 mmol) was dissolved in degassed EtOH (10 mL), followed by the addition of 1,2-ethylenediamine (67 µL,
1 mmol). Sodium borohydride (38 mg dissolved in 950 µL EtOH and 50 µL of 2 N aq NaOH, 1 mmol) was added and the solution turned from blue to black in color. The catalyst was ready for use after cessation of hydrogen release.
A flask containing 4l (1.7 g, 6 mmol) was flushed with hydrogen. The above freshly prepared P-2 Ni-ethylene-diamine catalyst (6 mL) was introduced by a gas-tight syringe. Upon the disappearance of 4l, as monitored by TLC, the flask was flushed with air to stop the reaction. The reaction mixture was diluted with EtOAc (6 mL), filtered through a short pad of silica gel, and concentrated in vacuo. The residue was purified by silica gel chromatography (2-5% EtOAc in hexane) to provide 5l ^¹4 as a light yellow oil in 99% yield. The ratio of cis-5 to the over-reduced alkane and the corresponding trans isomer was analyzed by GC-MS (Agilent G1800C, HP-5MS column, 30 m × 0.25 mm × 0.25 µm). ^¹H NMR (400 MHz, CDCl₃): δ = 5.30-5.40 (m, 2 H), 3.67 (s, 3 H), 2.30 (t, J = 7.5 Hz, 2 H), 1.95-2.06 (m, 4 H), 1.56-1.70 (m, 2 H), 1.20-1.38 (m, 20 H), 0.88 (t, J = 6.8 Hz, 3 H).
2,3-Dihydroxypropyl ( Z )-Octadec-9-enoate (9.9-MAG): Compound 5l (1.5 g, 5 mmol) was added to a two-necked flask containing DMSO (10 mL). Glycerol (2.3 g, 25 mmol) and P1 phosphazene (137 mg, 0.5 mmol) were subsequently added. One neck of the reaction flask was connected to a vacuum line (ca. 60 mmHg) installed in the fume hood. The reaction mixture was stirred at r.t. overnight. Saturated aq NaHCO₃ (50 mL) was added; then the mixture was extracted with EtOAc (3 × 50 mL). The combined organic extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was submitted to purification by silica gel chromatography (20-60% EtOAc in hexane) to give a white solid containing 1-MAG as the major product, accompanied with 3% of corresponding 2-MAG, in a total yield of 87%.
The crude product was dissolved in MeCN at a concentration of 100 mg/mL (slight heating if needed) and then left in the refrigerator (4 ˚C) for crystallization. The precipitant was filtered to give 9.9-MAG with over 99% purity. The mixture can also be purified by preparative RP-HPLC (Atlantis dC18 column, 5 µm, 30 × 150 mm; MeCN-H₂O). Three authentic samples corresponding to the major by-products in this case: 1,3-dihydroxypropyl (Z)-octadec-9-enoate (major 2-MAG by-product), 2,3-dihydroxypropyl (E)-octadec-9-enoate and over-reduced 2,3-dihydroxypropyl octadecanoate have been prepared and analyzed to reassure effective HPLC analysis and purification. The chemical purity of final products were also examined by ^¹H NMR and ^¹³C NMR spectroscopic analyses.^{5,6,¹5 ¹}H NMR (400 MHz, CDCl₃): δ = 5.29-5.39 (m, 2 H), 4.14 (d, J = 5.5 Hz, 2 H), 3.87-3.95 (m, 1 H), 3.68 (dd, J = 11.6, 3.5 Hz, 1 H), 3.57 (dd, J = 11.6, 6.1 Hz, 1 H), 3.49 (br, 2 H), 2.34 (t, J = 7.6 Hz, 2 H), 1.95-2.07 (m, 4 H), 1.56-1.67 (m, 2 H), 1.19-1.38 (m, 20 H), 0.88 (t, J = 6.7 Hz, 3 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 174.51, 130.17, 129.84, 70.40, 65.18, 63.58, 34.30, 32.08, 29.94, 29.88, 29.70, 29.50 (2 × C), 29.37, 29.30 (2 × C), 27.39, 27.34, 25.05, 22.86, 14.29.

15

The E-double bond isomers, if present, could be detected on the ^¹³C NMR spectra taken in CDCl₃. Their olefinic carbons appeared in the downfield region relative to those of Z-configured 1-MAGs (ca. 0.4-0.6 ppm difference).