Vitamin E-based amphipathic compound, and use thereof
11591306 · 2023-02-28
Assignee
Inventors
Cpc classification
C07H15/18
CHEMISTRY; METALLURGY
C07D311/72
CHEMISTRY; METALLURGY
C07H15/26
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a vitamin E-based amphipathic compound, a method for producing same, and a method for extracting, solubilizing, stabilizing, or crystallizing a membrane protein using same. By using a compound according to the present invention, not only is an excellent membrane protein extraction and solubilization effect achieved, but the membrane protein can be stably stored for a long period of time in an aqueous solution, and thus the compound can be utilized in analyzing the function and structure of the membrane protein. Moreover, the vitamin E-based amphipathic compounds exhibited superb properties in the visualization of protein compounds through an electron microscope. Membrane protein structure and function analysis is one of the fields of greatest interest in biology and chemistry today, and since at least half of new drugs currently being developed target membrane proteins, the vitamin E-based amphipathic compounds may be applied to the membrane protein structure research, which is closely related to the development of new drugs.
Claims
1. A compound represented by Formula 1 or 2 below: ##STR00012## where R.sup.1 and R.sup.2 are each independently hydrogen (H) or CH.sub.3; L is —CH.sub.2—, —CH.sub.2CH.sub.2—, —NHCOCH.sub.2—, —CH.sub.2OCH.sub.2CH.sub.2— or a direct linkage; X.sup.1 and X.sup.2 are each independently an oxygen-linked saccharide; Z is hydrogen (H) or —CH.sub.2—X.sup.3, and X.sup.3 is an oxygen-linked saccharide, ##STR00013## where R.sup.1 and R.sup.2 are each independently hydrogen (H) or CH.sub.3; and X.sup.4 is a glucose-centered, branched pentasaccharide.
2. The compound of claim 1, wherein each saccharide of Formula 1 is glucose or maltose.
3. The compound of claim 1, wherein R.sup.1 and R.sup.2 are CH.sub.3.
4. The compound of claim 1, wherein R.sup.1 and R.sup.2 are CH.sub.3; L is —CH.sub.2CH.sub.2— or —NHCOCH.sub.2—; and Z is hydrogen.
5. The compound of claim 1, wherein R.sup.1 and R.sup.2 are CH.sub.3; L is —CH.sub.2— or —CH.sub.2OCH.sub.2CH.sub.2—; and Z is —CH.sub.2—X.sup.3.
6. The compound of claim 1, wherein the compound is one of Formulas 3 to 7 below: ##STR00014## ##STR00015##
7. The compound of claim 1, wherein the compound is an amphipathic molecule for extracting, solubilizing, stabilizing or analyzing a membrane protein.
8. The compound of claim 1, wherein the compound has a critical micelle concentration (CMC) in an aqueous solution of 0.1 to 10 μM.
9. A composition for extracting, solubilizing, stabilizing or analyzing a membrane protein, comprising the compound of claim 1.
10. The composition of claim 9, wherein the composition is a formulation in the form of a micelle, liposome, emulsion or nanoparticle.
11. A method of preparing a compound represented by Formula 1 below, comprising: 1) introducing a linker having a —CH.sub.2—, —CH.sub.2CH.sub.2—, —NHCOCH.sub.2— or —CH.sub.2OCH.sub.2CH.sub.2— structure to a vitamin E tocopherol; 2) producing an alcohol group by reacting the product of Step 1) with 4-(bromomethyl)-methyl-2,6,7-trioxabicyclo[2,2,2]-octane or diethyl malonate and performing reduction; 3) introducing a protecting group-attached saccharide by performing glycosylation on the product of Step 2); and 4) performing deprotection on the product of Step 3): ##STR00016## where R.sup.1 and R.sup.2 are each independently hydrogen (H) or CH.sub.3; L is —CH.sub.2—, —CH.sub.2CH.sub.2—, —NHCOCH.sub.2—, —CH.sub.2OCH.sub.2CH.sub.2— or a direct linkage; X.sup.1 and X.sup.2 are each independently an oxygen-linked saccharide; Z is hydrogen (H) or —CH.sub.2—X.sup.3, and X.sup.3 is an oxygen-linked saccharide.
12. A method of preparing a compound represented by Formula 2 below, comprising repeatedly performing the steps including 1) introducing a protecting group-attached saccharide by performing glycosylation on a vitamin E tocopherol; and 2) performing deprotection on the product of Step 1): ##STR00017## where R.sup.1 and R.sup.2 are each independently hydrogen (H) or CH.sub.3; and X.sup.4 is a glucose-centered, branched pentasaccharide.
13. The method of claim 11, wherein R.sup.1 and R.sup.2 are CH.sub.3; and each saccharide is glucose or maltose.
14. A method of extracting, solubilizing, stabilizing, crystallizing or analyzing a membrane protein, comprising treating a membrane protein with the compound represented by Formula 1 or 2 below in an aqueous solution: ##STR00018## where R.sup.1 and R.sup.2 are each independently hydrogen (H) or CH.sub.3; L is —CH.sub.2—, —CH.sub.2CH.sub.2—, —NHCOCH.sub.2—, —CH.sub.2OCH.sub.2CH.sub.2— or a direct linkage; X.sup.1 and X.sup.2 are each independently an oxygen-linked saccharide; Z is hydrogen (H) or —CH.sub.2—X.sup.3, and X.sup.3 is an oxygen-linked saccharide, ##STR00019## where R.sup.1 and R.sup.2 are each independently hydrogen (H) or CH.sub.3; and X.sup.4 is a glucose-centered, branched pentasaccharide.
15. The method of claim 14, wherein R.sup.1 and R.sup.2 are each independently hydrogen (H) or CH.sub.3; L is —CH.sub.2CH.sub.2— or —NHCOCH.sub.2—; and Z is hydrogen.
16. The method of claim 14, wherein R.sup.1 and R.sup.2 are CH.sub.3; L is —CH.sub.2— or —CH.sub.2OCH.sub.2CH.sub.2—; and Z is —CH.sub.2—X.sup.3.
17. The method of claim 14, wherein the membrane protein is a complex of light harvesting-I and a reaction center (LHI-RC complex), a uric acid-xanthine/H.sup.+symporter (UapA), melibiose permease (MelB), a leucine transporter (LeuT), a G-protein coupled receptor (GPCR) or a combination of two or more thereof.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10) (a) and (c) show SDS-PAGE and Western blotting results for assessing the amounts of MelB.sub.st protein dissolved in the presence of each amphipathic compound; and
(11) (b) and (d) show a histogram expressed as percentages (%) of the total amount of MelB.sub.st protein present in a solution prepared by thermally treating the amounts of MelB.sub.st protein dissolved in the presence of individual amphipathic compounds at 45° C.
(12)
(13)
(14)
(15)
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE
(16) INVENTION
(17) Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are merely provided to exemplify the contents of the present invention, and do not limit the scope of the present invention. It will be interpreted that what can be easily inferred from the detailed description and examples of the present invention by those of ordinary skill in the art is within the scope of the present invention.
<Preparation Example 1> Method of Synthesizing VEG-1
(18) The synthetic scheme for VEG-1 is shown in
(19) <1-1> Synthesis of Compound B of
(20) A mixture of vitamin E (Compound A; DL-α-tocopherol, 1.0 equiv.) was treated with NaH (3.0 equiv.) mixed with DMF (12 mL), and the reaction mixture was stirred vigorously for 15 minutes at room temperature. 4-(bromo ethyl)-ethyl-2,6,7-trioxabicyclo[2,2,2]-octane (1.8 equiv.) dissolved in TI-IF (12 mL) was added dropwise to the reaction mixture. The resulting mixture was heated under nitrogen for 24 hours at 100° C. After the reaction was quenched with methanol, an organic solvent was removed under reduced pressure. The solid residue was dissolved in CH.sub.2Cl.sub.2, and an organic solution was washed with brine and dried over anhydrous Na.sub.2SO.sub.4. An organic solvent was concentrated, and then the residue was dissolved in a CH.sub.2Cl.sub.2/MeOH mixture. Several drops of concentrated HCl were added to this solution. The resulting mixture was heated for 4 hours at 50° C. Following neutralization with NaOH and concentration of the reaction mixture, the residue was purified by column chromatography (EtOAc/hexane), obtaining desired Compound B in 78% yield.
(21) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.92 (s, 6H), 3.66 (s, 2H), 3.05 (br s, 3H), 2.56 (t, J=6.8 Hz, 2H), 2.16 (s, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 1.88-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.88-0.84 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 148.4, 147.1, 127.7, 125.8, 123.4, 118.0, 75.1, 64.8, 45.4, 40.2, 40.1, 39.6, 37.7, 37.6, 37.5, 32.9, 32.8, 31.4, 28.1, 25.0, 24.6, 24.0, 22.9, 22.8, 21.3, 20.8, 19.9, 19.8, 12.8, 12.0, 11.9.
(22) <1-2> Synthesis of VEG-1a Through General Procedure for Glycosylation
(23) Under a N.sub.2 atmosphere, a mixture of Compound 13 (1.0 equiv.), AgOTf (3.6 equiv.) and 2,4,6-collidine (1.0 equiv.) in anhydrous CH.sub.2Cl.sub.2 was stirred at −45° C. A solution of perbenzoylated maltosylbromide (3.6 equiv.) mixed with CH.sub.2Cl.sub.2 was added dropwise to the resulting suspension. After stirring for 30 minutes at −45° C., the reaction mixture was heated to 0° C. and stirred for 30 minutes. After the completion of the reaction (indicated by TLC), pyridine was added to the reaction mixture, followed by dilution with CH.sub.2Cl.sub.2 and filtration over Celite. The resulting filtrate was washed sequentially with a 1M Na.sub.2S.sub.2O.sub.3 aqueous solution, a 0.1M HCl aqueous solution and brine. An organic layer was dried with anhydrous Na.sub.2SO.sub.4, and the solvent was removed by a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc/hexane), obtaining VEG-1a as a glassy solid in 80% yield.
(24) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.10-7.83 (m, 7.62-7.15 (m, 42H), 5.66 (t, =9.6 Hz, 3H), 5.56 (t, =9.6 Hz, 3H), 5.41 (t, =8.0 Hz, 3H), 4.42-4.39 (m, 3H), 4.33-4.30 (m, 3H), 4.12 (d, J=8.0 Hz, 3H), 4.02 (d, J=8.0 Hz, 3H), 3.73 (d, J=8.0 Hz, 1H), 3.54 (d, J=8.0 Hz, 1H), 3.34 (m, 6H), 2.56 (t, J=6.8 Hz, 2H), 2.16 (s, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 1.88-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.88-0.84 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 166.1, 165.9, 165.1, 164.7, 147.7, 147.3, 133.6, 133.5, 133.3, 133.1, 130.0, 129.8, 129.6, 129.1, 128.9, 128.8, 128.6, 128.5, 128.4, 128.1, 126.1, 122.5, 117.3, 101.23, 74.6, 72.6, 71.9, 71.8, 69.5, 68.1, 62.9, 45.0, 39.4, 37.7, 37.5, 37.4, 32.8, 28.0, 24.9, 24.5, 23.8, 22.8, 22.7, 21.2, 19.9, 12.45, 11.8, 12.4, 11.8, 11.6.
(25) <1-3> Synthesis of VEG-1 Through Deprotection
(26) O-benzoylated VEG-1a was dissolved in MeOH and treated with a required amount of a methanol solution of 0.5M NaOMe, such that the final concentration of NaOMe was 0.05M. The reaction mixture was stirred for 6 hours at room temperature, and neutralized with Amberlite IR-120 (H.sup.+ form). The resin was removed by filtration and washed with MeOH, and a solvent was removed from the combined filtrate in vacuo. 50 mL of diethyl ether was added to the residue dissolved in a 2 mL MeOH:CH.sub.2Cl.sub.2 (1:1) mixture, obtaining VEG-1 as a white solid in 92% yield.
(27) .sup.1H NMR (400 MHz, CD.sub.3OD): δ 4.33 (d, J=8.0 Hz, 3H), 4.17 (d, J=8.0 Hz, 3H), 3.83-3.74 (m, 8H), 3.61-3.59 (m, 5H), 3.34 (t, J=8.0 Hz, 3H), 3.25-3.22 (m, 8H), 3.17 (t, J=8.0 Hz, 4H), 2.50 (t, J=8.0 Hz, 2H), 2.11 (s, 3H), 2.07 (s, 3H), 1.97 (s, 3H), 1.71-1.66 (m, 2H), 1.49-1.32 (m, 8H), 1.24-1.21 (m, 8H), 1.13-1.04 (m, 11H), 0.82-0.79 (m, 12H); .sup.13C NMR (100 MHz, CD.sub.3OD): δ 149.0, 148.9, 129.14, 127.3, 123.7, 118.7, 104.8, 78.0, 77.8, 75.7, 75.3, 72.9, 71.8, 70.0, 62.9, 46.6, 41.0, 40.6, 38.8, 38.7, 38.6, 38.5, 38.4, 34.0, 33.9, 33.8, 32.7, 29.2, 26.0, 25.5, 24.2, 24.1, 23.3, 23.2, 22.1, 21.7, 20.4, 20.3, 13.4, 12.5, 12.2; HRMS (FAB*); calcd. for C.sub.52H.sub.90O.sub.20[M+Na].sup.+ 1057.5923, observed 1057.5920.
<Preparation Example 2> Synthesis of VEG-2
(28) The synthetic scheme for VEG-2 is shown in
(29) <2-1> Synthesis of Compound C of
(30) A mixture of vitamin E (Compound A; DL-α-tocopherol, 16 mmol), methyl bromoacetate (22 mmol), anhydrous K.sub.2CO.sub.3 (35 mmol) and KI (8 mmol) in anhydrous acetone was stirred under an argon atmosphere to reflux overnight. After the removal of a solvent, the residue was dissolved in CH.sub.2Cl.sub.2, and extracted with water and brine. An organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. After complete removal of a solvent, LiAlH.sub.4 (14.0 mmol) was slowly added to the residue dissolved in THF at 0° C. The mixture was stirred for 4 hours at room temperature, and the reaction was quenched by sequentially adding MeOH, water and a 1.0N HCl aqueous solution at 0° C., followed by extraction with CH.sub.2Cl.sub.2 twice. Combined organic layers were washed with brine, and dried over anhydrous Na.sub.2SO.sub.4. The residue was purified by silica gel column chromatography (EtOAc/hexane), obtaining desired Compound C in 85% yield.
(31) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.92-3.90 (m, 2H), 3.77-3.75 (m, 2H), 2.75 (br s, 1H), 2.57 (t, =6.8 Hz, 2H), 2.17 (s, 3H), 2.13 (s, 3H), 2.08 (5, 3H), 1.88-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.88-0.84 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 148.1, 147.7, 127.8, 125.8, 123.0, 117.0, 74.9, 73.9, 62.4, 53.5, 40.3, 40.2, 39.5, 37.8, 37.7, 37.6, 37.5, 37.4, 33.0, 32.9, 32.8, 31.4, 31.3, 28.1, 25.0, 24.9, 24.6, 24.0, 22.9, 22.8, 21.2, 20.8, 19.9, 19.6, 12.8, 11.9.
(32) <2-2> Synthesis of Compound D of
(33) Compound C was treated with NaH (3.0 equiv.) mixed with DMF (12 mL), and the react on as stirred vigorously for 15 minutes at room temperature. 4-(bromoethyl)-ethyl-2,6,7-trioxabicyclo[2,2,2]-octane (1.8 equiv.) dissolved in THF (12 mL) was added dropwise to the reaction mixture. The resulting mixture was heated under nitrogen for 24 hours at 100° C. After the reaction was quenched with methanol, an organic solvent was removed under reduced pressure. The solid residue was dissolved in CH.sub.2Cl.sub.2, and an organic solvent was washed with brine and dried over anhydrous Na.sub.2SO.sub.4. After concentration of an organic solvent, the residue was dissolved in a CH.sub.2Cl.sub.2/MeOH mixture. Several drops of concentrated HCl were added to the solution. The resulting mixture was heated for 4 hours at 50° C. After neutralization with NaOH and concentration of the reaction mixture, the residue was purified by column chromatography (EtOAc/hexane obtaining desired Compound D in 80% yield.
(34) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.78-3.76 (m, 2H), 3.70 (s, 6H), 3.60 (s, 2H), 3.50 (s, 2H), 2.55 (t, J=6.8 Hz, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 1.88-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.87-0.83 (in, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 148.1, 147.7, 127.8, 125.9, 123.1, 117.8, 75.0, 73.1, 72.0, 71.2, 64.1, 45.4, 40.4, 40.3, 39.5, 37.7, 37.6, 37.5, 37.4, 33.0, 32.9, 32.8, 31.4, 31.3, 28.1, 25.0, 24.9, 24.6, 24.0, 22.9, 22.8, 21.2, 20.8, 20.0, 19.9, 19.8, 19.7, 19.6, 12.8, 11.9.
(35) <2-3> Synthesis of VEG-2a through general procedure for glycosylation
(36) Under a N.sub.2 atmosphere, a mixture of Compound D (1.0 equiv.), AgOTf (3.6 equiv.) and 2,4,6-collidine (1.0 equiv.) in anhydrous CH.sub.2Cl.sub.2 was stirred at −45° C. A solution of perbenzoylated maltosylbromide (3.6 equiv.) mixed with CH.sub.2Cl.sub.2 was added dropwise to the resulting suspension. After stirring for 30 minutes at −45° C., the reaction mixture was heated to 0° C. and stirred for 30 minutes. After the completion of the reaction (indicated by TLC), pyridine was added to the reaction mixture, followed by dilution with CH.sub.2Cl.sub.2 and filtration over Celite. The resulting filtrate was washed sequentially with a 1M Na.sub.2S.sub.2O.sub.3 aqueous solution, a 0.1M HCl aqueous solution and brine. An organic layer was dried with anhydrous Na.sub.2SO.sub.4, and the solvent was removed by a rotary evaporator. The residue as purified by silica gel column chromatography (EtOAc/hexane), obtaining VEG-2a as a glassy solid in 85% yield.
(37) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.10-7.80 (m, 18H), 7.60-7.10 (m, 42H), 5.66 (t, J=9.6 Hz, 3H), 5.56 (t, J=9.6 Hz, 3H), 5.41 (t, J=8.0 Hz, 3H), 4.42-4.39 (m, 3H), 4.16-4.14 (m, 3H), 3.88 (d, =8.0 Hz, 3H), 3.64 (d, =8.0 Hz, 3H), 3.36-3.46 (m, 4H), 3.38-3.25 (m, 4H), 3.22-3.15 (m, 4H), 2.55 (t, J=6.8 Hz, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 1.88-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.87-0.83 (in 1211); .sup.13C NMR (100 MHz, CDCl.sub.3): δ166.1, 165.8, 165.1, 164.8, 147.9, 147.7, 133.6, 133.4, 133.3, 133.1, 130.1, 129.8, 129.7, 129.6, 129.1, 129.0, 128.9, 128.5, 128.4, 128.3, 128.0, 127.9, 126.0, 122.8, 117.6, 101.4, 74.8, 72.7, 72.0, 71.6, 70.7, 69.7, 67.6, 63.0, 45.3, 40.4, 39.5, 37.7, 37.6, 37.5, 37.4, 37.3, 32.9, 32.8, 31.4, 28.0, 25.0, 24.5, 23.8, 23.7, 22.8, 22.7, 21.7, 20.7, 19.9, 19.7, 12.8, 12.0, 11.9.
(38) <2-4> Synthesis of VEG-2 Through Deprotection
(39) O-benzoylated VEG-2a was dissolved in MeOH and treated with a required amount of a methanol solution of 0.5M NaOMe, such that the final concentration of NaOMe was 0.05M. The reaction mixture was stirred for 6 hours at room temperature, and neutralized with Amberlit IR-120 (H±form). The resin was removed by filtration and washed with MeOH, and a solvent was removed from the combined filtrate in vacuo. 50 mL of diethyl ether was added to the residue dissolved in a 2 mL MeOH:CH.sub.2Cl.sub.2 (1:1) mixture, obtaining VEG-2 as a white solid in 92l % yield.
(40) .sup.1H NMR (400 MHz, CD.sub.3OD): δ 4.35 (d, J=8.0 Hz, 3H). 4.02 (d, J=8.0 Hz, 3H), 3.83 (d, J=8.0 Hz, 4H), 3.75-3.74 (m, 4H), 3.68-3.62 (m, 8H), 3.37-3.17 (m, 15H), 2.55 (t, J=8.0 Hz, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.76-1.71 (m, 2H), 1.53-1.36 (m, 8H), 1.28-1.18 (m, 12H), 1.1.4-1.07 (m, 7H), 0.86-0.83 (in, 12H); .sup.13C NMR (100 MHz, CD.sub.3OD): δ 149.2, 149.1, 128.8, 127.0, 123.8, 118.9, 105.1, 78.1, 77.8, 75.8, 75.2, 73.5 72.0, 71.7, 70.8, 69.9, 62.8, 46.7, 41.1, 41.0, 40.6, 38.8, 38.6, 38.5, 38.4, 34.0, 33.9, 32.7, 29.2, 26.0, 25.6, 24.2, 23.3, 23.2, 22.2, 21.7, 20.4, 20.3, 13.3, 12.4, 12.2; HRMS (FAB.sup.+): calcd. for C.sub.54H.sub.94O.sub.21 [M+Na].sup.+ 1101.6185, observed 1101.6189.
<Preparation Example 3> Synthesis of VEG-3
(41) The synthetic scheme for VEG-3 is shown in
(42) <3-1> Synthesis of Compound E of
(43) A mixture of vitamin E (Compound A; DL-α-tocopherol, 16 mmol), methyl bromoacetate (22 mmol), anhydrous K.sub.2CO.sub.3 (35 mmol) and KI (8 mmol) in anhydrous acetone was stirred under an argon atmosphere to reflux overnight. After the removal of a solvent, the residue was dissolved in CH.sub.2Cl.sub.2, and extracted with water and brine. An organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure, thereby obtaining colorless oil. The oil was treated with serinol (25 mmol) dissolved in redistilled Me.sup.2SO (20 mL) and anhydrous K.sub.2CO.sub.3 (35 mmol), and stirred for 6 hours at, 25° C. The reaction mixture was diluted with water, and extracted with Et.sub.2O. An organic layer was washed with brine, and dried over anhydrous Na.sub.2SO.sub.4. After complete evaporation of a solvent, the residue was purified by fresh column chromatography (EtOAc/hexane), obtaining desired Compound E as a white solid in 85% yield.
(44) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.68 (d, =8.0 Hz, 1H), 4.20 (br s, 2H), 4.09-4.01 (m, 1H), 3.88-3.85 (m, 2H), 3.78-3.75 (m, 2H), 2.55 (t, 6.8 Hz, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 1.88-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.87-0.83 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 170.3, 148.5, 146.8, 127.4, 125.5, 123.3, 117.8, 75.0, 71.1, 61.8, 52.3, 40.3, 40.2, 39.5, 37.7, 37.6, 37.6, 37.4, 32.9, 32.8, 31.2, 28.1, 24.9, 24.6, 23.8, 22.8, 22.7, 21.1, 20.7, 20.0, 19.8, 19.7, 19.6, 12.8, 12.0.
(45) <3-2> Synthesis of VEG-3a Through General Procedure for Glycosylation
(46) Under a N.sub.2 atmosphere, a mixture of Compound E (1.0 equiv.), AgOTf (3.6 equiv.) and 2,4,6-collidine (1.0 equiv.) in anhydrous CH.sub.2Cl.sub.2 was stirred at −45° C. A solution of perbenzoylated maltosylbromide (2.4 equiv.) mixed with CH.sub.2Cl.sub.2 was added dropwise to the resulting suspension. After stirring for 30 minutes at −45° C., the reaction mixture was heated to 0° C. and stirred for 30 minutes. After the completion of the reaction (indicated by TLC), pyridine was added to the reaction mixture, followed by dilution with CH.sub.2Cl.sub.2 and filtration over Celite. The resulting filtrate/as washed sequentially with a 1M Na.sub.2S.sub.2O.sub.3 aqueous solution, a 0.1M HCl aqueous solution and brine. An organic layer was dried with anhydrous Na.sub.2SO.sub.4, and the solvent was removed by a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc/hexane), obtaining VEG-3a as a glassy solid in 80% yield.
(47) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.09-7.93 (m, 14H), 7.89-7.86 (m, 4H), 7.82-7.80 (m, 4H), 7.76-7.71 (m, 4H), 7.64-7.58 (m, 2H), 7.53-7.16 (m, 42H), 6.18 (t, J=8.0 Hz, 211), 5.73-5.64 (m, 4H), 5.40-5.33 (m, 2H), 5.21-5.13 (m, 4H), 4.72-4.56 (m, 4H), 4.39-4.10 (m, 10H), 3.85-3.80 (m, 2H), 3.39 (d, =8.0 Hz, 1H), 3.33 (t, J=8.0 Hz, 2H), 3.07 (d, J=8.0 Hz, 2H), 2.91 (t, J=8.0 Hz, 1H), 2.51 (t, J=6.8 Hz, 2H), 2.14 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 1.86-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.87-0.84 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 168.7, 166.2, 166.1, 166.0, 165.9, 165.8, 165.6, 165.5, 165.1, 165.0, 164.9, 164.8, 148.4, 147.1, 134.0, 133.7, 133.6, 133.5, 133.4, 130.1, 130.0, 129.9, 129.8, 129.7, 129.5, 129.3, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5, 128.3, 127.5, 125.6, 123.2, 117.7, 100.9, 95.7, 74.9, 72.0, 71.4, 69.8, 69.1, 69.0, 62.6, 39.4, 37.6, 37.5, 37.4, 37.3, 32.8, 28.0, 24.9, 24.5, 24.0, 23.8, 22.8, 22.7, 21.1, 19.9, 19.8, 12.0, 11.9.
(48) <3-3> Synthesis of VEG-3 Through Deprotection
(49) O-benzoylated VEG-1a was dissolved in MeOH and treated with a required amount of a methanol solution of 0.5M NaOMe, such that the final concentration of NaOMe was 0.05M. The reaction mixture was stirred for 6 hours at room temperature, and neutralized with Amberlite IR-120 (H.sup.+ form). The resin was removed by filtration and washed with MeOH, and a solvent was removed from the combined filtrate in vacuo. 50 mL of diethyl ether was added to the residue dissolved in a 2 mL MeOH:CH.sub.2Cl.sub.2 (1:1) mixture, obtaining VEG-3 as a white solid in 90% yield.
(50) .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.13 (d, J=4.0 Hz, 2H), 4.42-4.39 (m, 1H), 4.36 (t, J=8.0 Hz, 2H), 4.14 (s, 2H), 4.04-3.98 (m, 2H), 3.91-3.86 (m, 3H), 3.81-3.74 (m, 6H), 3.68-3.57 (m, ION), 3.49 (t, J=8.0 Hz, 2H), 3.44-3.38 (m, 5H), 1.32-3.23 (m, 6H), 2.56 (t, J=8.0 Hz, 2H), 2.12 (s, 3H), 2.09 (s, 3H), 2.02 (s, 3H), 1.78-1.73 (m, 2H), 1.53-1.34 (m, 8H), 1.28-1.19 (m, 12H), 1.13-1.05 (m, 7H), 0.86-0.83 (in, 12H); .sup.13C NMR (100 MHz, CD.sub.3OD): δ 171.8, 149.6, 148.6, 128.5, 126.8, 124.1, 119.1, 104.8, 102.9, 81.3, 77.8, 76.7, 76.0, 75.1, 74.9, 74.8, 74.2, 72.3, 71.5, 69.4, 62.8, 62.3, 50.6, 41.0, 40.6, 38.6, 38.5, 38.4, 34.0, 33.9, 33.8, 32.7, 29.2, 26.0, 25.5, 24.2, 23.3, 23.2, 22.2, 21.2, 20.8, 19.9, 19.8, 19.7, 19.6, 13.0, 12.2, 12.0; HRMS (FAB.sup.+): calcd. for C.sub.58H.sub.99NO.sub.25 [M=Na].sup.+ 1232.6404, observed 1232.6410.
<Preparation Example 4> Synthesis of VEG-4
(51) The synthetic scheme for VEG-4 is shown in
(52) <4-1> Synthesis of Compound C of
(53) A mixture of vitamin E (Compound A; DL-α-tocopherol, 16 mmol), methyl bromoacetate (22 mmol), anhydrous K.sub.2CO.sub.3 (35 mmol) and KI (8 mmol) in anhydrous acetone was stirred under an argon atmosphere to reflux overnight. After the removal of a solvent, the residue was dissolved in CH.sub.2Cl.sub.2, and extracted with water and brine. An organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. After complete removal of a solvent, LiAlH.sub.4 (14.0 mmol) was slowly added to the residue dissolved in THF at 0° C. The mixture was stirred for 4 hours at room temperature, and the reaction was quenched by sequentially adding MeOH, water and a 1.0N HCl aqueous solution at 0° C., followed by extraction with CH.sub.2Cl.sub.2 twice. Combined organic layers were washed with brine, and dried over anhydrous Na.sub.2SO.sub.4. The residue was purified by silica gel column chromatography (EtOAc/hexane), obtaining desired Compound C in 85% yield.
(54) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.92-3.90 (m, 2H), 3.77-3.75 (m, 2H), 2.75 (br s, 1H), 2.57 (t, J=6.8 Hz, 2H), 2.17 (s, 3H), 2.13 (s, 3H), 2.08 (s, 3H), 1.88-1.72 (m, 2H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.88-0.84 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 148.1, 147.7, 127.8, 125.8, 123.0, 117.0, 74.9, 73.9, 62.4, 53.5, 40.3, 40.2, 39.5, 37.8, 37.7, 37.6, 37.5, 37.4, 33.0, 32.9, 32.8, 31.4, 31.3, 28.1, 25.0, 24.9, 24.6, 24.0, 22.9, 22.8, 21.2, 20.8, 19.9, 19.6, 12.8, 11.9.
(55) <4-2> Synthesis of Compound F of
(56) A mixture of Compound C (1.0 equiv.), PPh.sub.3 (1.5 equiv.) and CBr.sub.4 (1.2 equiv.) in anhydrous CH.sub.7Cl.sub.2 was stirred at room temperature for 4 hours under argon. The resulting solution was washed with NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4 and then filtered. A product was obtained by evaporating the filtrate, and used in the subsequent step without additional purification. The product was added to a solution prepared by stirring diethyl malonate (1.0 equiv.) and NaH (1.0 equiv.) in ethanol. The resulting mixture was heated under reflux for 4 hours. After cooling to room temperature, water was added, the product was extracted with Et.sub.2O, dried over Na.sub.2SO.sub.4, and a solvent was removed using a rotary evaporator to obtain a product. The resulting product was further treated with LiAlH.sub.4 (3.5 equiv.) mixed with dry THF for 4 hours at room temperature. After the reaction was quenched, water was added dropwise to the resulting solution, and extracted with CH.sub.2Cl.sub.2 twice. The combined extracts were washed with 1.0M HCl and brine, dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane), obtaining desired Compound F in 80% yield.
(57) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.83-3.78 (m, 2H), 3.75-3.70 (m, 4H), 3.05 (br s, 2H), 2.56 (t, J=6.8 Hz, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 1.88-1.76 (m, 2H), 1.74-1.70 (m, 2H), 1.69-1.67 (m, 1H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.87-0.83 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 148.2, 148.1, 127.8, 125.9, 123.4, 117.7, 75.0, 71.3, 65.7, 40.5, 40.3, 40.2, 39.5, 37.7, 37.6, 37.5, 37.4, 33.0, 32.9, 32.8, 31.4, 31.3, 29.1, 28.1, 25.0, 24.6, 24.0, 22.9, 22.8, 21.2, 20.8, 19.9, 19.8, 19.7, 19.6, 13.0, 12.2, 12.0.
(58) <4-3> Synthesis of VEG-4a Through General Procedure for Glycosylation
(59) Under a N.sub.2 atmosphere, a mixture of Compound F (1.0 equiv.), AgOTf (3.6 equiv.) and 2,4,6-collidine (1.0 equiv.) in anhydrous CH.sub.2Cl.sub.2 was stirred at −45° C. A solution of perbenzoylated maltosylbromide (2.4 equiv.) mixed with CH.sub.2Cl.sub.2 was added dropwise to the resulting suspension. After stirring for 30 minutes at −45° C., the reaction mixture was heated to 0° C. and stirred for 30 minutes. After the completion of the reaction (indicated by TLC), pyridine was added to the reaction mixture, followed by dilution with CH.sub.2Cl.sub.2 and filtration over Celite. The resulting filtrate was washed sequentially with a 1M Na.sub.2S.sub.2O.sub.3 aqueous solution, a 0.1M HCl aqueous solution and brine. An organic layer was dried with anhydrous Na.sub.2SO.sub.4, and the solvent was removed by a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc/hexane), obtaining VEG-4a as a glassy solid in 78% yield.
(60) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.11 (d, J=8.0 Hz, 2H), 8.05-7.93 (m, 12H), 7.87-7.85 (m, 6H), 7.81-7.78 (m, 2H), 7.74-7.72 (m, 6H), 7.53-7.18 (m, 42H), 6.15 (t, J=8.0 Hz, 2H), 5.81 (t, J=4.0 Hz, 2H), 5.71-5.62 (m, 4H), 5.37-5.24 (m, 4H), 5.19-5.09 (m, 2H), 4.77-4.08 (n, 12H), 3.74-3.72 (in, 2H). 3.60-3.44 (m, 4H), 3.30-3.27 (m, 2H), 3.16 (d, J=4.0 Hz, 1H), 3.05 (d, J=8.0 Hz, 1H), 2.99 (d, J=8.0 Hz, 1H), 2.85 (d, J=8.0 Hz, 1H), 2.53 (t, J=6.8 Hz, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 1.88-1.76 (m, 2H), 1.74-1.70 (m, 2H), 1.69-1.67 (m, 1.1-1), 1.54-1.50 (m, 3H), 1.43-1.05 (in, 21H), 0.88-0.84 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 166.2, 166.1, 165.9, 165.8, 165.5, 165.2, 165.1, 164.0, 164.9, 148.3, 147.6, 133.7, 133.6, 133.5, 133.4, 133.3, 133.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 127.8, 125.8, 122.7, 117.5, 74.7, 72.3, 72.1, 71.3, 70.2, 69.8, 69.1, 69.0, 39.4, 37.5, 37.4, 37.3, 32.8, 32.7, 28.0, 25.0, 24.5, 23.9, 22.8, 22.7, 21.1, 20.6, 20.0, 19.8, 12.8, 12.0, 11.8.
(61) <4-4> Synthesis of VEG-4 Through Deprotection
(62) O-benzoylated VEG-4a was dissolved in MeOH and treated with a required amount of a methanol solution of 0.5M NaOMe, such that the final concentration of NaOMe was 0.05M. The reaction e was stirred for 6 hours at room temperature, and neutralized with Amberlite IR-120 (H.sup.+ form). The resin was removed by filtration and washed with MeOH, and a solvent was removed from the combined filtrate in vacuo. 50 mL of diethyl ether was added to the residue dissolved in a 2 mL MeOH:CH.sub.2Cl.sub.2. (1:1) mixture, obtaining VEG-4 as a white solid in 90% yield.
(63) .sup.1H NMR (400 MHz, CD.sub.3OD): 5.16 (d, J=4.0 Hz, 2H), 4.35 (d, J=8.0 Hz, 2H), 4.02-3.96 (d, J=4.0 Hz, 2H), (m, 2H), 3.90-3.58 (m, 22H), 3.52 (t, J=8.0 Hz, 2H), 3.45-3.42 (m, 2H), 3.39-3.33 (m, 4H), 3.30-3.23 (m, 6H), 2.56 (t, J=8.0 Hz, 2H), 2.26-2.22 (m, 1H), 2.13 (s, 3H), 2.09 (s, 3H), 2.02 (s, 3H), 1.91-1.85 (m, 2H), 1.78-1.73 (m, 2H), 1.58-1.35 (m, 8H), 1.29-1.19 (m, 12H), 1.16-1.05 (m, 7H), 0.87-0.84 (m, 12H); .sup.13C NMR (100 MHz, CD.sub.3OD): J 149.6, 149.0, 128.7, 126.9, 123.8, 118.8, 104.9, 104.7, 103.0, 81.4, 77.9, 76.6, 75.8, 74.8, 74.2, 72.2, 71.6, 71.3, 70.8, 62.8, 62.3, 40.9, 40.6, 38.6, 38.5, 38.4, 38.1, 38.0, 34.0, 339, 32.8, 30.2, 29.2, 26.0, 25.5, 24.3, 23.3, 23.2, 22.1, 21.7, 20.4, 20.3, 13.3, 1.2.4, 12.2; HRMS (FAB.sup.+): calcd. for C.sub.58H.sub.100O.sub.24 [M+Na].sup.+ 1203.6502, observed 1203.6504.
<Preparation Example 5> Synthesis of VEG-5
(64) The synthetic scheme for VEG-5 is shown in
(65) <5-1> Synthesis of Compound G Through Glycosylation and Deprotection
(66) Under a N.sub.2 atmosphere, a mixture of vitamin E (Compound A; DL-α-tocopherol, 1.0 equiv.), AgOTf (3.6 equiv.) and 2,4,6-collidine (1.0 equiv.) in anhydrous CH.sub.2Cl.sub.2 was stirred at −45° C. A solution of perbenzoylated maltosylbromide (1.2 equiv.) mixed with CH.sub.2Cl.sub.2 was added dropwise to the resulting suspension. After stirring for 30 minutes at −45° C., the reaction mixture was heated to 0° C. and stirred for 30 minutes. After the completion of the reaction (indicated by TLC), pyridine was added to the reaction mixture, followed by dilution with CH.sub.2Cl.sub.2 and filtration over Celite. The resulting filtrate was washed sequentially with a 1M Na.sub.2S.sub.2O.sub.3 aqueous solution, a 0.1M HCl aqueous solution and brine. An organic layer was dried with anhydrous Na.sub.7SO.sub.4, and the solvent was removed by a rotary evaporator. The O-benzoylated product was dissolved in MeOH, and treated with a required amount of a methanol solution of 0.5M NaOMe, such that the final concentration of NaOMe was 0.05M. The reaction mixture was stirred for 6 hours at room temperature, and neutralized with Amberlite IR-120 (H.sup.+ form) resin. The resin was removed by filtration and washed with MeOH, and a solvent was removed from the combined filtrate in vacuo. 50 mL of diethyl ether was added to the residue dissolved in a 2 mL MeOH:CH.sub.2Cl.sub.2 (1:1) mixture, obtaining Compound G as a white solid in 88% yield.
(67) .sup.1H NMR (400 MHz, CD.sub.3OD): δ 4.52 (d, J=8.0 Hz, 1H), 3.77-3.74 (m, 1H), 3.65-3.62 (m, 1H), 3.52-3.48 (m, 1H), 3.44-3.40) (m, 1H), 3.31 (s, 2H), 2.58 (t, =6.8 Hz, 2H), 2.22 (s, 3H), 2.18 (s, 3H), 2.04 (s, 3H), 1.88-1.76 (m, 2H), 1.74-1.70 (m, 2H), 1.69-1.67 (m, 1H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.87-0.83 (m, 12H); .sup.13C NMR (100 MHz, CD.sub.3OD): δ 149.4, 147.4, 129.9, 129.8, 128.0, 123.5, 118.6, 106.2, 78.1, 77.9, 75.9, 75.8, 71.9, 63.0, 40.6, 38.6, 38.5, 34.1, 34.0, 33.9, 29.3, 26.0 25.6, 24.3, 23.3, 23.2, 21.8, 20.4, 20.3, 14.3, 13.4, 12.1, 12.0.
(68) <5-2> Synthesis of VEG-5a Through General Procedure for Glycosylation
(69) Under a N.sub.2 atmosphere, a mixture of Compound G (1.0 equiv.), AgOTf (3.6 equiv.) and 2,4,6-collidine (1.0 equiv.) in anhydrous CH.sub.2Cl.sub.2 was stirred at −45° C. A solution of perbenzoylated maltosylbromide (5.0 equiv.) mixed with CH.sub.2Cl.sub.2 was added dropwise to the resulting suspension. After stirring for 30 minutes at −45° C., the reaction mixture was heated to 0° C. and stirred for 30 minutes. After the completion of the reaction (indicated by TLC), pyridine was added to the reaction mixture, followed by dilution with CH.sub.2Cl.sub.2 and filtration over Celite. The resulting filtrate was washed sequentially with a 1M Na.sub.2S.sub.2O.sub.3 aqueous solution, a 0.1M HCl aqueous solution and brine. An organic layer was dried with anhydrous Na.sub.2SO.sub.4, and the solvent was removed by a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc/hexane), obtaining VEG-5a as a glassy solid in 70% yield.
(70) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.26 (d, J=8.0 Hz, 2H), 8.15-7.80 (m, 24H), 7.72-6.67 (m, 5H), 7.66-7.59 (m, 3H), 7.58-7.12 (m, 46H), 5.90-5.75 (m, 4H), 5.71-4.45 (m, 7H), 5.34 (t, J=8.0 Hz, 1H), 4.95 (d, J=8.0 Hz, 1H), 4.82-4.75 (m, 4H). 4.62-4.56 (m, 3H), 4.47-4.35 (in 4H), 4.16-4.08 (m, 2H), 4.04-4.01 (m, 1H), 3.92-3.85 (m, 3H), 3.80-3.68 (m, 2H), 3.02 (brs, 1H), 2.56 (t, J=6.8 Hz, 2H), 2.17 (s, 3H), 2.16 (s, 3H), 2.02 (s, 3H), 1.88-1.75 (m, 2H), 1.74-1.70 (m, 2H) 1.69-1.67 (m, 1H), 1.54-1.50 (m, 3H), 1.43-1.05 (m, 21H), 0.87-0.84 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 165.9, 165.3, 165.2, 165.1, 133.7, 133.5, 133.2, 130.2, 129.9, 129.8, 129.7, 129.6, 129.5, 129.3, 129.0, 128.8, 128.6, 128.5, 128.4, 128.0, 122.4, 74.8, 74.6, 74.2, 72.5, 72.4, 72.3, 72.1, 69.8, 69.1, 39.5, 37.6, 37.5, 32.9, 28.1, 25.0, 24.6, 22.9, 22.8, 19.9, 19.8, 14.2, 13.3, 12.1, 11.9.
(71) <5-3> Synthesis of VEG-5 Through Deprotection
(72) O-benzoylated VEG-5a was dissolved in MeOH and treated with a required amount of a methanol solution of 0.5M NaOMe, such that the final concentration of NaOMe was 0.05M. The reaction mixture was stirred for 6 hours at room temperature, and neutralized with Amberlite IR-120 (H.sup.+ form). The resin was removed by filtration and washed with MeOH, and a solvent was removed from the combined filtrate in vacuo. 50 mL of diethyl ether was added to the residue dissolved in a 2 mL MeOH:CH.sub.2Cl.sub.2 (1:1) mixture, obtaining VEG-5 as a white solid in 88% yield.
(73) .sup.1H NMR (400 MHz, CD.sub.3OD): δ 4.99 (d, J=8.0 Hz, 1H), 4.93 (d, J=8.0 Hz. 1H), 4.72 (d, J=8.0 Hz, 1H), 4.65 (d, J=8.0 Hz, 1H), 4.26 (d, =8.0 Hz, 1H), 4.20-4.09 (m, 4H), 3.87-3.75 (m, 5H), 3.69-3.55 (m, 5H), 3.41-3.12 (m, 0.2H), 2.57 (t, J=8.0 Hz, 2H), 2.20 (s, 3H), 2.17 (s, 3H), 2.01 (s, 3H), 1.78-1.73 (m, 2H), 1.52-1.36 (m, 8H), 1.28-1.18 (m, 12H), 1.12-1.07 (m, 7H), 0.85-0.82 (m, 12H); .sup.13C NMR (100 MHz, CD.sub.3OD): δ 149.6, 147.5, 123.7, 123.6, 118.8, 104.8, 104.5, 103.8, 103.0, 102.4, 81.4, 79.9, 78.1, 78.0, 77.9, 77.8, 75.9, 75.3, 75.2, 71.9, 71.8, 71.6, 71.5, 69.6, 62.9, 62.7, 41.1, 40.6, 38.8, 38.6, 38.5, 38.4, 34.0, 33.9, 29.2 26.0, 25.5, 24.2, 23.3, 23.2, 22.2, 21.8, 20.4, 20.3, 20.2, 14.6, 14.5, 13.7, 12.1, 12.0; HRMS (FAB.sup.+): calcd. for C.sub.59H.sub.100O.sub.27 [M+Na].sup.+ 1263.6350 observed 1263.6353.
<Example 1> Properties of VEGs
(74) To identify the properties of VEGs synthesized according to the synthetic methods of Preparation Examples 1 to e molecular weights (M.W.) and critical micellar concentrations (CMCs) of VEGs, and the hydrodynamic radii (R.sub.h) of formed micelles were measured.
(75) Specifically, the CMCs were measured using hydrophobic fluorescence staining and diphenylhexatriene (DPH), and the hydrodynamic radii (R.sub.h) of the micelles formed by each formulation were measured by dynamic light scattering (DLS). The results were compared with that of a conventional amphiphilic molecule (detergent), that is, DDM, and are shown in Table 1.
(76) TABLE-US-00001 TABLE 1 Detergent MW.sup.a CMC (mM) CMC (wt %) R.sub.h (nm).sup.b VEG-1 1035.3 ~0.002 ~0.0002 4.2 ± 0.1 VEG-2 1079.3 ~0.003 ~0.0003 4.7 ± 0.1 VEG-3 1210.4 ~0.002 ~0.0002 4.8 ± 0.1 VEG-4 1181.4 ~0.003 ~0.0003 5.1 ± 0.1 VEG-5 1241.4 ~0.002 ~0.0002 3.9 ± 0.1 DDM 510.1 0.170 0.0087 3.4 ± 0.0 .sup.aMolecular weight of detergents. .sup.bHydrodynamic radius of micelles was determined at 1.0 wt % by dynamic light scattering.
(77) The CMC values of VEGs were much smaller than that of DDM. Therefore, since VEGs easily formed micelles at a low concentration, it can be seen that hey tended to highly agglomerate in an aqueous solution, compared to DDM.
(78) In addition, although having different hydrophilic structures, VEGs had similar CMC values. It is determined that this is because the micelle formation of an amphipathic molecule is mainly induced by a hydrophobic effect, and all VEGs commonly contain vitamin E as a hydrophobic group.
(79) The distribution of sizes of the micelles formed by VEGs was shown in a narrow range of 3.8 to 5.2 nm, indicating that a larger micelle is formed, compared to DDM. As an additional result of analyzing the size distribution of the micelles formed by VEGs, it was confirmed that most VEGs form micelles with a uniform, size (
<Example 2> Evaluation of Ability of VEGs to Stabilize R. capsulatus Superassembly (LHI-RC) Structure (FIG. 5)
(80) An experiment was conducted to evaluate the ability of VEGs to stabilize the structure of LHI-RC. The photosynthetic superassembly consists of a complex of light-Harvesting complex I (LHI) and a reaction center (RC). The structural stability of LHI-RC was measured by a method of monitoring the structure of a protein for 20 days using UV-Vis spectroscopy. As amphipathic molecules, all VEGs of the present invention and conventional amphipathic molecules DDM and OG were used, and the concentrations of the amphipathic molecules were measured at CMC+0.04 wt % (
(81) Specifically, LHI-RC stability was measured using a method disclosed in the paper published in 2008 by the inventors (P. S. Chae et al., ChemBioChem 2008, 9, 1706-1709). Briefly, the inventors used the membrane obtained from R. capsulatus, U43 [pUHTM86Bgl] which does not have light-harvesting complex II (LHII). A 10 mL aliquot of the solution of the frozen R. capsulatus membrane was homogenized using a glass homogenizer, and incubated with gentle stirring at 32° C. for 30 minutes. The homogenized membrane was treated with 1.0 wt % DDM for 30 minutes at 32° C. Membrane debris was subjected to ultracentrifugation, thereby collecting a pellet. 200 μL of Ni.sup.2+-NTA resin (pre-equilibrated and stored in a buffer containing 10 mM Tris, pH 7.8) was added to a supernatant containing the LHI-RC complex solubilized in DDM, and incubated at 4° C. for 1 hour. The resin-containing solution was filtrated using 1.0 HisSpinTrap columns, and each column was washed twice with a 500 μL binding buffer containing 10 mM Tris (pH 7.8), 100 mL NaCl and 1×CMC DDM. Following the replacement with a new ultracentrifuge tube, the LHI-RC complex purified by DDM was eluted using a buffer containing 1M imidazole (2×300 μL). 80 μL of the protein sample was diluted with 920 μL of each of VEGs, DDM and OG so that the final concentration was CMC+0.04 wt % or CMC+0.2 t %. The LHI-RC complex produced in each detergent was incubated for 20 days at 25° C., and then the incubation temperature increased to 32° C. for 7 days. Protein stability was measured at regular intervals during the cubation by measuring UV-Vis spectra of the samples in the range of 650 to 950 nm. Protein integrity was evaluated by monitoring absorbance (A875) at 875 nm.
(82) All VEGs were superior to DDM in terms of the ability to maintain the integrity of the LHI-RC complex, and particularly, VEG-3 showed the most excellent effect (
<Example 3> Evaluation of Ability of VEGs to Stabilize Structure of UapA Membrane Protein
(83) UapAG411V.sub.Δ1-11 (hereinafter, referred to as “UapA”) was expressed in a Saccharomyces cerevisiae FGY217 strain by GFP fusion, and isolated into a sample buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, 1 mM xanthine), which was performed according to the method disclosed in the paper written by J. Leung et al. (Mol. Menthr. Biol. 2013, 30, 32-42). The protein was concentrated to be approximately 10 mg/ml using a 100 kDa molecular weight cutoff filter (Millipore). The protein was diluted 1:150 with a buffer containing DDM, VEG (VEG-1, VEG-2, VEG-3, VEG-4 or VEG-5) or LMNG at CMC+0.2 wt % in Greiner 96. CPM dye (Invitrogen) stored in DMSO (Sigma) was diluted with a dye buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, 5 mM EDTA), and 3 μL of the diluted dye was added to an individual protein sample. The reaction mixture was treated at a constant temperature of 40° C. for 120 minutes. A fluorescence emission intensity was monitored using a microplate spectrofluorometer set to excitation and emission wavelengths of 387 and 463 nm, respectively. The maximum fluorescence value was used to calculate the percentage of a protein folded during incubation. The relative amount of the folded protein was plotted over time using GraphPad Prism.
(84) The membrane containing UapA was resuspended in a pH 8.0 PBS buffer containing 10 mM imidazole, 150 mM NaCl and 10% glycerol, and a protein concentration was measured. The membrane was adjusted to a concentration of 1 mg/ml, and a 1 ml aliquot was incubated for 60 minutes with each of 1.0 mL of DDM, LMNG, VEG-3 and VEG-5, on ice under stirring. A 100 uL aliquot was obtained from each tube and subjected to ultracentrifugation for 10 minutes at 150,000 g, and then fluorescent SEC (FSEC) was performed on each sample. The remaining soluble fractions were incubated for 10 minutes at 45° C. The thermally-treated samples were applied to FSEC to monitor the integrity of a transporter. FSEC was performed using a Superose6 column (GE Healthcare) equilibrated with a buffer containing an appropriate preparation (DDM, LMNG, VEG-3 or VEG-5).
(85) As shown in
(86) As a result of performing additional FSEC comparison experiments with LMNG and DDM with respect to VEG-3 and VEG-5 having excellent effects, LMNG and DDM were superior to VEG-3 and VEG-5 in terms of the ability to extract and solubilizing a membrane protein. However, when thermally treated at 45° C., UapA solubilized by LMNG and DDM was considerably degraded, but UapA solubilized by VEG-3 and VEG-5 maintained protein integrity (
<Example 4> Evaluation of Ability of VEGs to Stabilize MelB Membrane Protein Structure
(87) An experiment was conducted to measure the structural stability of a Salmonella typhimurium melibiose per urease (MelB) protein by VEGs.
(88) Specifically, Salmonella typhimurium MelB.sub.St (melibiose permease) having a 10 His tag at the C-terminus was expressed in E. coli DW2 cells (ΔmelB and ΔlacZY) using a pK95ΔAHB/WT MelB.sub.St/CH10 plasmid. Cell growth and membrane preparation were carried out according to the methods disclosed in the paper written by A. S. Ethayathulla et al. (Nat. Commun. 2014, 5, 3009). A protein assay was performed using a Micro BCA kit (Thermo Scientific, Rockford, Ill.). MelB.sub.St stability in VEGs or DDM was evaluated using the protocol disclosed in Nat. Methods 2010, 7, 1003-1008, written by P. S. Chae et al. A MelB.sub.St-containing membrane sample (the final protein concentration was 10 mg/mL) was incubated in a solubilization buffer containing 1.5% (w/v) DDM or VEGs (50 mM sodium phosphate, pH 7.5, 200 mM NaCl, 10% glycerol, 20 mM melibiose) at two different temperatures (0 and 23° C.) for 90 minutes. To remove an insoluble material, ultracentrifugation was performed using a Beckman Optima™ MAX ultracentrifuge equipped with a TLA-100 rotor at 355,590 g and 4° C. for 30 minutes. 20 μg of the membrane protein which did not undergo ultracentrifugation was applied to an untreated membrane or the same amount of extracts of the compounds after ultracentrifugation, and the treated samples were loaded in respective wells at an equal volume. The loaded samples were analyzed by SDS-15% PAGE, and then visualized by immunoblotting with a Penta-His-HRP antibody (Qiagen, Germantown, Md.).
(89) To assess the thermal stability of MelB.sub.St in various amphipathic molecules, MelB.sub.St extracted with individual amphipathic molecules at 23° C. was subjected to additional thermal treatment at three different temperature (45, 55 and 65° C.) and ultracentrifugation, followed by SDS-15% PAGE and Western blotting. MelB.sub.St was detected using a SuperSignalWest Pico chemiluminescent substrate by an ImageQuant LAS 4000 Biomolecular Imager (GE Healthcare Life Science).
(90) As the result shown in
(91) However, when the temperature increased to 45° C., DDM and VEGs solubilized the MelB.sub.St protein with almost similar efficiencies, and at a higher temperature (55° C.), the MelB.sub.St solubilization efficiency of DDM showed a significant decrease, whereas most VEGs did not show a significant decrease in MelB.sub.St solubilization efficiency at 55° C. Particularly, the efficiency of VEG-4 and VEG-5 remained intact. At 65° C., all VEGs showed excellent MelB.sub.St solubilization ability, compared to DDM, and particularly, VEG-3 and VEG-4 showed very excellent protein solubilization ability even at a high temperature.
(92) Overall, at low temperatures (0° C. and 23° C.), DDM showed higher protein extraction efficiency than VEGs, but in a protein thermal stability experiment performed at elevated temperatures, VEGs (particularly, VEG-3 and VEG-4) showed, a higher transporter solubilization ability than DDM, indicating excellent protein stabilization ability.
<Example 5> Evaluation of Ability of VEGs to Stabilize β.SUB.2.AR Protein
(93) <5-1> Measurement of Long-Term Stability
(94) A receptor was expressed in Sf9 insect cells infected with Baculovirus and solubilized in 1% DDM. The DDM-solubilized receptor was purified by alprenolol-sepharose in the presence of 0.01% cholesteryl succinate (CHS). β.sub.2AR purified by DDM was diluted with a buffer containing DDM or VEGs (VEG-1, VEG-2, VEG-3 and VEG-5) to reach the final concentration of CMC+0.2 wt %. β.sub.2AR solubilized in each compound was stored for 10 days at room temperature, and the ligand binding ability of the receptor was measured at regular intervals by incubating the receptor with 10 nM radioactive [.sup.3H]-dihydroalprenolol (DHA) for 30 minutes at room temperature. The mixture was loaded into a G-50 column, and a supernatant was collected using a certain amount of binding buffer (supplemented with 20 nM HEPES pH 7.5, 100 mM NaCl, 0.5 mg/ml BSA). In addition, a 15 ml scintillation fluid was added. Receptor-binding [.sup.3H]-DHA was measured using a scintillation counter (Beckman).
(95) As a result, VEGs (VEG-1, VEG-2, VEG-3 and VEG-5) showed the ability to maintain initial activity of the solubilized receptor, which was similar to DDM (
(96) <5-2> Purification and Measurement of Stability of β.sub.2AR-G.sub.s Complex Solubilized in VEG-3
(97) 100 μM β.sub.2AR solubilized in 0.1% DDM mixed with 120 μM G.sub.s heterotrimer for 30 minutes at room temperature. 0.5-unit apyrase (NEB) and 2 mM MgCl.sub.2 were added to facilitate complex formation, followed by further incubation for one hour. Subsequently, 1.0% VEG-3 was added such that the final concentration reached 0.2%, and the sample was further incubated for 30 minutes to change DDM to VEG-3. The protein solution was loaded into a M1 Flag column, washed with a series of buffers with different molar ratios of 0.1% DDM buffer to 0.5% VEG-3 buffer to completely change DDM to VEG-3, and the receptor-G.sub.s complex was finally eluted with a 0.05% VEG-3 buffer. Preparative gel filtration was performed to purify the β.sub.2AR-G.sub.s complex with a running buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.005% DTM-A6, 1 mM BI, 100 mM TCEP). To measure the stability of the β.sub.2AR-G.sub.s complex in VEG-3, analytical gel filtrations were performed using the running buffer as above, but after 3 and 15-day incubation, performed without VEG-3 (compound-free condition).
(98) As the result shown in
(99) <5-3> Negative Stain EM Analysis of NAR-G.sub.s Complex Solubilized in VEG-3
(100) A β.sub.2AR-G.sub.s protein complex was prepared for electron microscopy using a conventional negative staining protocol, and imaged at room temperature using a Tecnai T12 electron microscope operated at 120 kV according to a low-dose procedure. Images recorded at a magnification of 71,138× and a defocus value of approximately −1.1 urn on a Gatan US4000 CCD camera. All images were binned (2×2 pixels) to obtain a pixel size of 4.16 Å at a specimen level. Particles were manually removed using e2boxer (part of the EMAN2 software suite). 2D reference-free alignment and classification of particle projections were performed using ISAC. 23,035 projections of β.sub.2AR-G.sub.s were subjected to ISAC producing 19 classes consistent in two-way matching and 5401 particle projections.
(101) As a result, it was seen that particles generated from the β.sub.2AR-G.sub.s complex purified by VEG-3 are highly homogeneous, different from the aggregation of particles observed in the DDM-purified complex in the previous study. In addition, in representative 2D class images, individual components (β.sub.2AR, G.sub.αs and G.sub.βγ) of the complex were clearly distinguished (
<Example 6> Evaluation of Ability of VEGs to Stabilize Membrane Protein (LeuT) Structure
(102) Wild-type LeuT derived from Aquifex aeolicus was purified according to the method disclosed in the paper written by G. Deckert et al. (Nature 1998, 392, 353-358). LeuT is expressed in E. coli C41 (DE3) transformed with pET16b encoding a C-terminal 8xHis-tagged transporter (the expression plasmid was provided by Dr E. Gouaux, Vollum Institute, Portland, Oreg., USA). Briefly, a LeuT protein was isolated and solubilized in 1.0 wt % DDM, and then the protein was bound to Ni.sup.2+-NTA resin (Life Technologies, Denmark), followed by elution with 20 mM Tris-HCl (pH 8.0), 1 mM NaCl, 199 mM KCl, 0.05% DDM and 300 mM imidazole. Afterward, approximately 1.5 mg/nal of a protein sample (stock) was diluted 10-fold with an identical buffer which does not include DDM or imidazole, but is supplemented with each of VEGs or DDM (control) to obtain a final concentration of CMC+0.04 wt %. The protein sample was stored for 10 days at room temperature, and then centrifuged at regular intervals during the incubation prior to the measurement of protein activity. Protein activity was determined by measuring [.sup.3H]-Leu binding using SPA (M. Quick et al., Proc. Natl, Acad. Sci. U.S.A. 2007, 104, 3603-3608). The assay was performed on samples containing 450 mM NaCl and each compound at the final concentration. In the presence of 20 nM [.sup.3H]-Leu and 1.25 mg/mL of copper chelate (His-Tag) YSi beads (both purchased from PerkinElmer, Denmark), a SPA reaction was perform. [.sup.3H]-Leu binding was measured using a MicroBeta liquid scintillation counter (PerkinElmer).
(103) As the result shown in