Mannitol-based amphipathic compound and use thereof
10889609 ยท 2021-01-12
Assignee
Inventors
Cpc classification
C07H15/04
CHEMISTRY; METALLURGY
G01N33/6872
PHYSICS
Y02P20/55
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07H1/00
CHEMISTRY; METALLURGY
International classification
C07H15/04
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a mannitol-based amphipathic compound, a method of preparing the same, a method of extracting, solubilizing, stabilizing or crystallizing a membrane protein using the compound, and a method of analyzing a structure of the membrane protein under an electron microscope using the compound. When the mannitol-based compound according to the present invention is used, the membrane protein can be stably stored in an aqueous solution for a prolonged period of time and thus can be applied to analysis of functions and structures thereof. Since the analysis of the structures and functions of the membrane protein is one of the fields of most interest in biology and chemistry currently, and more than half of new drugs currently in development are targeting membrane proteins, the present invention is applicable to research on the structures of membrane proteins closely related to the development of the new drugs.
Claims
1. A composition for extracting, solubilizing, stabilizing or crystallizing a membrane protein, or analyzing a structure of the membrane protein using an electron microscope, the composition comprising a compound represented by the following Formula 1: ##STR00015## wherein R.sup.1 and R.sup.2 are each independently an unsubstituted C.sub.5-C.sub.20 alkyl group, a substituted or unsubstituted C.sub.5-C.sub.20 cycloalkyl group, or a substituted or unsubstituted C.sub.5-C.sub.20 aryl group; and X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are each independently a glucose or maltose, whereby the composition is for extracting, solubilizing, stabilizing or crystallizing a membrane protein, or analyzing the structure of the membrane protein using the electron microscope.
2. The composition of claim 1, wherein each of R.sup.1 and R.sup.2 is C.sub.7-C.sub.18 alkyl group; R.sup.1 and R.sup.2 are the same.
3. The composition of claim 1, wherein the compound is represented by the following Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, Formula 8, Formula 9, Formula 10, or Formula 11: ##STR00016## ##STR00017## ##STR00018##
4. The composition of claim 1, wherein the compound is an amphipathic molecule for extracting, solubilizing, stabilizing or crystallizing a membrane protein.
5. The composition of claim 1, wherein the compound has a critical micellar concentration (CMC) of 110.sup.4 mM to 1.0 mM in an aqueous solution.
6. The composition of claim 1, wherein the compound is an amphipathic molecule capable of forming a complex with the membrane protein for analyzing a structure of the membrane protein using an electron microscope.
7. The composition of claim 1, wherein the composition is a micelle, a liposome, an emulsion, or a nanoparticle formulation.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
(24) Hereinafter, the present invention will be described in further detail with reference to exemplary embodiments thereof. However, it should be understood that the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit or define the scope of the invention. Accordingly, it will be apparent to those skilled in the art that various changes and modifications can be made to the exemplary embodiments of the present invention without departing from the scope of the present invention, so it should be understood that the present invention covers all such changes and modifications provided they are within the scope of the appended claims and their equivalents.
<Example 1>
Synthesis Method of Mannitol-Based Amphiphiles (MNAs)
(25) A synthesis scheme of MNAs is shown in
<1-1>General Synthesis Procedure for Synthesis of 3,4-O-di-alkyl-1,2:4,5-di-O-isopropylidene-D-mannitol (Step a; Synthesis of Compound A)
(26) Compound A was synthesized using mannitol to which a protecting group was introduced (protected mannitol) as a starting material.
(27) Specifically, a starting material, (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol, was added to a cold suspension of dissolved sodium hydride (3 equivalents) in anhydrous DMF and stirred for 30 minutes. Thereafter, an alkyl bromide (2.2 equivalents) was added, and the resulting reaction mixture was stirred at room temperature for 4 hours. Sodium hydride remaining after the reaction was quenched using a few drops of methanol, and an organic compound was extracted with CH.sub.2Cl.sub.2 and washed with distilled water (320 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4 and evaporated by a rotary evaporator. The product was purified using silica gel column chromatography to obtain a pure 3,4-O-di-alkyl-1,2:4,5-di-O-isopropylidene-D-mannitol (compound A).
<1-2>General Synthesis Procedure for Synthesis of 3,4-O-di-alkyl-D-mannitol (Step b; AB)
(28) The compound A synthesized in Example 1-1 was added to a stirring solution of methanol (25 mL) and CH.sub.2Cl.sub.2 (25 mL) (1:1) with 200 mg of p-TSA and stirred overnight at room temperature. After the reaction was completed, solid NaHCO.sub.3 was slowly added and vigorously stirred to neutralize the reaction mixture. The reaction mixture was filtered and dried using a rotary evaporator, and the resulting product was purified by silica gel column chromatography (EtOAc/hexane) to obtain a 3,4-O-di-alkyl-D-mannitol (compound B).
<1-3>General Synthesis Procedure for Glycosylation Reaction (Step c; BC)
(29) Glycosylation was carried out according to a modified method defined in the article by P. S. Chae et al. (Nat. Methods 2010, 7. 1003-1008.). Specifically, the alcohol derivative (compound B) synthesized in Example 1-2 was dissolved in CH.sub.2Cl.sub.2 (15 mL) at room temperature, and molecular sieves (4 ) were added thereto. AgOTf (6.9 equivalents) was added to the mixture at 0 C., and a perbenzoylated glucosylbromide solution (6.8 equivalents) dissolved in CH.sub.2Cl.sub.2 (2 mL) was slowly added. After 15 minutes of the reaction at 0 C., the reaction mixture was warmed to room temperature and then stirred for an hour. After the reaction was completed (confirmed by TLC analysis), pyridine was added to stop the reaction. Then, the mixture was diluted with CH.sub.2Cl.sub.2 (20 mL), and filtered through celite. The resulting filtrate was successively washed with a 1 M Na.sub.2S.sub.2O.sub.3 aqueous solution (40 mL), a 0.1 M HCl aqueous solution (40 mL), and brine (340 mL). The organic layer was dried on anhydrous Na.sub.2SO.sub.4, and a solvent was removed using a rotary evaporator. The residue was purified using silica gel chromatography (EtOAc/hexane) to obtain compound C in a glassy solid state.
<1-4>General Synthesis Procedure for de-O-Benzoylation Under Zemplen's Conditions (Step d; CD)
(30) The O-benzoylated compound C synthesized in Example 1-3 was dissolved in a small amount of anhydrous CH.sub.2Cl.sub.2, and MeOH was added dropwise until precipitation occurred faintly. A required amount of a methanolic solution of 0.5 M NaOMe was slowly added so that the final concentration of NaOMe was 0.05 M. During this, methanol was added at regular intervals to prevent occurrence of precipitates. The reaction mixture was stirred at room temperature for 6 hours and then neutralized with an Amberlite IR-120 (H.sup.+ form) resin. The resin was removed by filtration and then washed with MeOH, and a solvent was removed in vacuo from the mixed filtrate. The residual product was purified using silica gel column chromatography. After an additional purification, the residue was recrystallized with CH.sub.2Cl.sub.2/MeOH/diethyl ether to obtain a de-O-benzoylated product (compound D) as a white solid. The compound D obtained thus was named MNAs.
<Preparative Example 1>Synthesis of MNA-1
<1-1>Synthesis of 3,4-O-di-octyl-1,2:5,6-di-O-isopropylidene-D-mannitol (Compound 1)
(31) 3,4-O-di-octyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 1) was synthesized with a yield of 90% according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and octyl bromide as an alkyl bromide. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<1-2>Synthesis of 3,4-O-di-octyl-D-mannitol (Compound 11)
(32) 3,4-O-di-octyl-D-mannitol (compound 11) was synthesized from the compound 1 with a yield of 93% according to the method of Example 1-2. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<1-3>Synthesis of MNA-1a
(33) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-1a from the compound 11 with a yield of 75%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.24-8.21 (m, 4H), 8.12-7.98 (m, 4H), 7.97-7.92 (m, 8H), 7.91-7.89 (m, 6H), 7.87-7.79 (m, 16H), 7.61-7.56 (m, 6H), 7.55-7.43 (m, 16H), 7.41-7.35 (m, 32H), 7.34-7.27 (m, 4H), 5.77-5.74 (m, 2H), 5.57-5.42 (m, 4H), 5.41-5.30 (m, 6H), 4.88-4.74 (m, 2H), 4.48-4.46 (m, 2H), 4.45-4.39 (m, 6H), 4.33-4.20 (m, 6H), 4.01-3.91 (m, 2H), 3.54-3.41 (m, 5H), 3.40-3.32 (m, 2H), 3.31-3.14 (m, 4H), 1.81-1.68 (m, 2H), 1.24-1.12 (m, 20H), 0.86 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 165.9, 165.8, 165.6, 165.1, 165.0, 164.8, 133.6, 133.4, 130.0, 129.9, 129.8, 129.7, 129.5, 129.2, 128.9, 128.5, 128.4, 128.3, 101.4, 100.3, 82.4, 73.3, 72.9, 72.8, 72.7, 72.3, 71.8, 71.6, 71.1, 69.9, 69.7, 63.1, 62.9, 32.0, 30.2, 29.6, 29.5, 26.1, 22.8, 14.2.
<1-4>Synthesis of MNA-1
(34) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-1, from which a protecting group was removed, from the compound MNA-1a with a yield of 95%. .sup.1H NMR (400 MHz, CD.sub.3OD): 4.55-4.53 (m, 2H), 4.31-4.30 (m, 2H), 4.13-4.11 (m, 2H), 4.00-3.97 (m, 4H), 3.79-3.72 (m, 4H), 3.70-3.64 (m, 3H), 3.63-3.56 (m, 10H), 3.30-3.21 (m, 15H), 3.20-3.10 (m, 5H), 1.47-1.43 (m, 4H), 1.21 (m, 20H), 0.90 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.1, 104.7, 82.5, 80.9, 78.2, 78.0, 77.8, 75.6, 75.3, 74.4, 71.6, 71.4, 71.2, 62.8, 62.6, 33.1, 31.4, 30.8, 30.6, 27.4, 23.8, 14.5; HRMS (EI): calculated for C.sub.46H.sub.86O.sub.26[M+Na].sup.+ 1054.5407, found 1054.5406. The .sup.1H NMR spectrum of MNA-1 is shown in
<Preparative Example 2>Synthesis of MNA-2
<2-1>Synthesis of 3,4-O-di-nonyl-1,2:5,6-di-O-isopropylidene-D-mannitol (Compound 2)
(35) 3,4-O-di-nonyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 2) was synthesized with a yield of 90% according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and nonyl bromide as an alkyl bromide. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Colloids surf A 2011, 377, 349-355.).
<2-2>Synthesis of 3,4-O-di-nonyl-D-mannitol (Compound 12)
(36) 3,4-O-di-octyl-d-mannitol (compound 12) was synthesized from the compound 2 with a yield of 93% according to the method of Example 1-2. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<2-3>Synthesis of MNA-2a
(37) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-2a from the compound 12 with a yield of 74%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.23-8.20 (m, 4H), 8.09-8.01 (m, 4H), 8.00-7.97 (m, 8H), 7.90-7.79 (m, 6H), 7.72-7.65 (m, 16H), 7.61-7.56 (m, 6H), 7.55-7.43 (m, 16H), 7.40-7.35 (m, 32H), 7.34-7.27 (m, 4H), 5.82-5.74 (m, 2H), 5.61-5.51 (m, 4H), 5.49-5.33 (m, 6H), 4.88-4.74 (m, 2H), 4.48-4.46 (m, 2H), 4.45-4.39 (m, 6H), 4.33-4.20 (m, 6H), 4.09-3.95 (m, 2H), 3.58-3.45 (m, 5H), 3.42-3.34 (m, 2H), 3.29-3.19 (m, 4H), 1.83-1.74 (m, 2H), 1.45-1.12 (m, 24H), 0.86 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 165.8, 165.3, 165.2, 165.0, 133.8, 133.6, 133.4, 133.2, 130.2, 130.0, 129.8, 129.7, 129.6, 129.4, 129.3, 129.2, 129.1, 128.6, 128.5, 101.5, 100.4, 82.5, 80.2, 73.4, 73.0, 72.9, 72.8, 72.4, 72.2, 72.0, 71.7, 71.2, 70.0, 69.9, 63.3, 63.1, 32.2, 30.3, 29.9, 29.8, 29.6, 26.3, 22.9.
<2-4>Synthesis of MNA-2
(38) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-2, from which a protecting group was removed, from the compound MNA-2 with a yield of 94%. .sup.1H NMR (400 MHz, CD.sub.3OD): 4.52-4.51 (m, 2H), 4.29-4.27 (m, 2H), 4.12-4.09 (m, 2H), 3.96-3.94 (m, 4H), 3.80-3.73 (m, 4H), 3.71-3.64 (m, 3H), 3.63-3.53 (m, 10H), 3.27-3.19 (m, 15H), 3.18-3.07 (m, 5H), 1.44-1.39 (m, 4H), 1.19 (m, 24H), 0.90 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.6, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.5, 71.3, 62.8, 62.7, 33.2, 31.6, 31.0, 30.9 30.6, 27.5, 23.9, 14.6; HRMS (EI): calculated for C.sub.48H.sub.90O.sub.26[M+Na].sup.+ 1082.5720, found 1082.5717. The .sup.1H NMR spectrum of MNA-2 is shown in
<Preparative Example 3>Synthesis of MNA-3
<3-1>Synthesis of 3,4-O-di-decyl-1,2:5,6-di-O-isopropylidene-D-mannitol (Compound 3)
(39) 3,4-O-di-decyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 3) was synthesized with a yield of 90% according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and decyl bromide as an alkyl bromide. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<3-2>Synthesis of 3,4-O-di-decyl-D-mannitol (compound 13)
(40) 3,4-O-di-decyl-D-mannitol (compound 13) was synthesized from the compound 3 with a yield of 92% according to the method of Example 1-2. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<3-3>Synthesis of MNA-3a
(41) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-3a from the compound 13 with a yield of 71%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.23-8.20 (m, 4H), 8.12-7.98 (m, 4H), 7.97-7.92 (m, 8H), 7.91-7.89 (m, 6H), 7.87-7.79 (m, 16H), 7.61-7.56 (m, 6H), 7.55-7.43 (m, 16H), 7.40-7.35 (m, 32H), 7.34-7.26 (m, 4H), 5.77-5.74 (m, 2H), 5.57-5.42 (m, 4H), 5.41-5.30 (m, 6H), 4.88-4.74 (m, 2H), 4.48-4.46 (m, 2H), 4.45-4.39 (m, 6H), 4.33-4.20 (m, 6H), 4.01-3.91 (m, 2H), 3.54-3.41 (m, 5H), 3.40-3.32 (m, 2H), 3.31-3.14 (m, 4H), 1.81-1.68 (m, 2H), 1.24-1.12 (m, 28H), 0.86 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.1, 166.0, 165.8, 165.3 165.2, 165.0, 133.8, 133.6, 133.5133.4, 133.2, 130.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 129.2, 129.1, 128.6, 128.5, 101.5, 100.4, 82.4, 73.4, 73.0, 72.8, 72.4, 72.0, 71.8, 71.2, 70.0, 69.9, 63.3, 63.1, 32.2, 30.3, 29.9, 29.7, 26.3, 22.9, 14.3.
<3-4>Synthesis of MNA-3
(42) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-3, from which a protecting group was removed, from the compound MNA-3a with a yield of 94%. .sup.1H NMR (400 MHz, CD.sub.3OD): 6 4.52-4.51 (m, 2H), 4.29-4.27 (m, 2H), 4.12-4.09 (m, 2H), 3.96-3.94 (m, 4H), 3.80-3.73 (m, 4H), 3.71-3.64 (m, 3H), 3.63-3.53 (m, 10H), 3.27-3.19 (m, 15H), 3.18-3.07 (m, 5H), 1.43-1.38 (m, 4H), 1.19 (m, 28H), 0.90 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.6, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.5, 71.3, 62.8, 62.7, 33.2, 31.6, 31.0, 30.9 30.6, 27.5, 23.9, 14.6; HRMS (EI): calculated for C.sub.50H.sub.94O.sub.26[M+Na].sup.+ 1110.6033, found 1110.6037. The .sup.1H NMR spectrum of MNA-3 is shown in
<Preparative Example 4>Synthesis of MNA-4
<4-1>Synthesis of 3,4-O-di-undecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (Compound 4)
(43) 3,4-O-di-undecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 4) was synthesized with a yield of 90% according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yDethane-1,2-diol as a starting material and undecyl bromide as an alkyl bromide. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by S. Roy et al. (Colloids surf. A 2011, 377, 349-355.).
<4-2>Synthesis of 3,4-O-di-undecyl-D-mannitol (Compound 14)
(44) 3,4-O-di-undecyl-D-mannitol (compound 14) was synthesized from the compound 4 with a yield of 93% according to the method of Example 1-2. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<4-3>Synthesis of MNA-4a
(45) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-4a from the compound 14 with a yield of 70%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.23-8.20 (m, 4H), 8.09-8.01 (m, 4H), 8.00-7.97 (m, 8H), 7.90-7.79 (m, 6H), 7.72-7.65 (m, 16H), 7.61-7.56 (m, 6H), 7.55-7.51 (m, 16H), 7.48-7.35 (m, 32H), 7.33-7.21 (m, 4H), 5.82-5.74 (m, 2H), 5.61-5.51 (m, 4H), 5.49-5.33 (m, 6H), 4.88-4.74 (m, 2H), 4.48-4.46 (m, 2H), 4.45-4.39 (m, 6H), 4.33-4.20 (m, 6H), 4.09-3.95 (m, 2H), 3.58-3.45 (m, 5H), 3.42-3.34 (m, 2H), 3.29-3.19 (m, 4H), 1.83-1.74 (m, 2H), 1.45-1.12 (m, 32H), 0.86 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.1, 166.0, 165.7, 165.3, 165.1, 164.9, 133.8, 133.6, 133.5, 133.4, 133.2, 129.9, 129.7, 129.6, 129.4, 129.3, 129.2, 129.1, 128.6, 128.5, 101.5, 100.4, 82.5, 80.2, 73.4, 73.0, 72.8, 72.4, 72.0, 71.7, 71.2, 70.0, 69.9, 63.3, 63.0, 32.1, 30.3, 30.0, 29.8, 29.6, 26.3, 22.9, 14.3.
<4-4>Synthesis of MNA-4
(46) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-4, from which a protecting group was removed, from the compound MNA-4a with a yield of 92%. .sup.1H NMR (400 MHz, CD.sub.3OD): 4.51-4.50 (m, 2H), 4.31-4.28 (m, 2H), 4.12-4.09 (m, 2H), 3.98-3.96 (m, 4H), 3.82-3.74 (m, 4H), 3.71-3.64 (m, 3H), 3.64-3.55 (m, 10H), 3.28-3.20 (m, 15H), 3.19-3.06 (m, 5H), 1.49-1.41 (m, 4H), 1.19 (m, 32H), 0.90 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.6, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.5, 71.3, 62.8, 62.7, 33.2, 31.6, 31.0, 30.9 30.6, 27.4, 23.9, 14.6; HRMS (EI): calculated for C.sub.52H.sub.98O.sub.26[M+Na].sup.+ 1138.6346, found 1138.6341. The .sup.1H NMR spectrum of MNA-4 is shown in
<Preparative Example 5>Synthesis of MNA-5
<5-1>Synthesis of 3,4-O-di-dodecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (Compound 5)
(47) 3,4-O-di-dodecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 5) was synthesized with a yield of 90% according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and dodecyl bromide as an alkyl bromide. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<5-2>Synthesis of 3,4-O-di-dodecyl-D-mannitol (Compound 15)
(48) 3,4-O-di-dodecyl-D-mannitol (compound 15) was synthesized from the compound 5 with a yield of 92% according to the method of Example 1-2. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by J. Walton et al. (Tetrahedron Lett. 2006, 47, 737-741.).
<5-3>Synthesis of MNA-5a
(49) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-5a from the compound 15 with a yield of 65%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.24-8.21 (m, 4H), 8.12-7.98 (m, 4H), 7.97-7.92 (m, 8H), 7.91-7.89 (m, 6H), 7.87-7.79 (m, 16H), 7.61-7.56 (m, 6H), 7.55-7.43 (m, 16H), 7.41-7.35 (m, 32H), 7.34-7.27 (m, 4H), 5.78-5.74 (m, 2H), 5.57-5.42 (m, 4H), 5.41-5.30 (m, 6H), 4.88-4.74 (m, 2H), 4.48-4.46 (m, 2H), 4.45-4.39 (m, 6H), 4.33-4.20 (m, 6H), 4.01-3.91 (m, 2H), 3.54-3.41 (m, 5H), 3.40-3.32 (m, 2H), 3.31-3.14 (m, 4H), 1.78-1.65 (m, 2H), 1.30-1.10 (m, 36H), 0.86 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.0, 165.9, 165.7, 165.2, 165.1, 164.9, 133.7, 133.5, 133.4, 133.3, 133.1, 130.1, 130.0, 129.9, 129.8, 129.6, 129.5, 129.3, 129.2, 129.0, 128.9, 128.5, 128.4, 101.4, 100.4, 82.4, 80.2, 73.4, 72.9, 72.8, 72.7, 72.4, 71.9, 71.6, 71.1, 69.9, 69.8, 63.2, 63.0, 32.0, 30.3, 29.9, 29.8, 29.7, 29.5, 26.2, 22.8, 14.2.
<5-4>Synthesis of MNA-5
(50) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-5, from which a protecting group was removed, from the compound MNA-5a with a yield of 92%. .sup.1H NMR (400 MHz, CD.sub.3OD): 4.57-4.55 (m, 2H), 4.35-4.33 (m, 2H), 4.15-4.02 (m, 2H), 4.00-3.97 (m, 4H), 3.86-3.77 (m, 4H), 3.76-3.69 (m, 3H), 3.67-3.59 (m, 10H), 3.33-3.22 (m, 15H), 3.21-3.13 (m, 5H), 1.51-1.46 (m, 4H), 1.23 (m, 36H), 0.90(t, J =8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.7, 81.0, 78.2, 78.1, 77.8, 75.6, 75.3, 74.5, 71.7, 71.5, 71.3, 62.8, 62.6, 33.2, 31.6, 30.9 30.6, 27.4, 23.8, 14.6; HRMS (EI): calculated for C.sub.54H.sub.102O.sub.26[M+Na].sup.+ 1166.6659, found 1166.6658. The .sup.1H NMR spectrum of MNA-5 is shown in
<Preparative Example 6>Synthesis of MNA-6
<6-1>Synthesis of 3,4-O-di-tridecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 6)
(51) 3,4-O-di-tridecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 6) was synthesized with a yield of 92% according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and tridecyl bromide as an alkyl bromide. .sup.1H NMR (400 MHz, CDCl.sub.3): 4.25-4.20 (m, 2H), 4.19-4.05 (m, 2H), 3.97-3.91 (m, 2H), 3.61-3.51 (m, 4H), 3.34-3.31 (m, 2H), 1.57-1.53 (m, 4H), 1.41 (s, 6H), 1.34 (s, 6H), 1.25 (m, 40H), 0.88 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 108.6, 80.6, 76.0, 73.6, 67.0, 58.7, 33.0, 32.1, 30.5, 29.9, 29.8, 29.7, 26.9, 26.3, 26.0, 25.6, 22.9, 14.3.
<6-2>Synthesis of 3,4-O-di-tridecyl-D-mannitol (Compound 16)
(52) 3,4-O-di-tridecyl-D-mannitol (compound 16) was synthesized from the compound 6 with a yield of 92% according to the method of Example 1-2. .sup.1H NMR (400 MHz, CDCl.sub.3): 4.03-3.97 (m, 2H), 3.90-3.82 (m, 2H), 3.81-3.75 (m, 2H), 3.73-3.62 (m, 4H), 3.60-3.33 (m, 2H), 1.57-1.54 (m, 4H), 1.25 (m, 40H), 0.88 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 108.6, 80.4, 79.2, 76.1, 72.4, 71.7, 66.2, 63.4, 30.2, 29.8, 29.7, 29.6, 26.7, 26.3, 25.3, 22.9, 14.3.
<6-3>Synthesis of MNA-6a
(53) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-6a from the compound 16 with a yield of 60%. .sup.111 NMR (400 MHz, CDCl.sub.3): 8.23-8.21 (m, 4H), 8.20-7.98 (m, 4H), 7.97-7.92 (m, 8H), 7.91-7.89 (m, 6H), 7.87-7.79 (m, 16H), 7.61-7.56 (m, 6H), 7.55-7.43 (m, 16H), 7.40-7.35 (m, 32H), 7.34-7.27 (m, 4H), 5.77-5.74 (m, 2H), 5.57-5.42 (m, 4H), 5.41-5.30 (m, 6H), 4.88-4.74 (m, 2H), 4.48-4.46 (m, 2H), 4.45-4.39 (m, 6H), 4.33-4.20 (m, 6H), 4.01-3.91 (m, 2H), 3.54-3.41 (m, 5H), 3.40-3.32 (m, 2H), 3.31-3.14 (m, 4H), 1.81-1.34 (m, 2H), 1.24-1.12 (m, 40H), 0.86 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.0, 165.9, 165.7, 165.2, 165.1, 164.9, 133.7, 133.6, 133.4, 133.3, 133.1, 130.2, 130.1, 130.0, 129.9, 129.8, 129.6, 129.5, 129.3, 129.2, 129.0, 128.9, 128.5, 128.4, 128.1, 101.4, 100.4, 98.4, 82.4, 80.1, 73.4, 72.9, 72.7, 72.4, 71.9, 71.6, 71.4, 71.1, 69.9, 69.8, 69.5, 63.2, 63.0, 62.6, 60.5, 32.0, 30.3, 29.9, 29.8, 29.7, 29.5, 26.2, 22.8, 14.2.
<6-4>Synthesis of MNA-6
(54) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-6, from which a protecting group was removed, from the compound MNA-6a with a yield of 90%. .sup.1H NMR (400 MHz, CD.sub.3OD): 4.55-4.53 (m, 2H), 4.31-4.30 (m, 2H), 4.13-4.11 (m, 2H), 4.00-3.97 (m, 4H), 3.79-3.72 (m, 4H), 3.70-3.64 (m, 3H), 3.63-3.56 (m, 10H), 3.30-3.21 (m, 15H), 3.20-3.10 (m, 5H), 1.46-1.44 (m, 4H), 1.20 (m, 40H), 0.90 (t, J =8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.7, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.5, 71.3, 62.8, 62.7, 33.2, 31.6, 31.0, 30.9 30.6, 27.5, 23.8, 14.6; HRMS (EI): calculated for C.sub.56H.sub.106O.sub.26[M+Na].sup.+ 1194.6972, found 1194.6968. The .sup.1H NMR spectrum of MNA-6 is shown in
<Preparative Example 7>Synthesis of MNA-7
<7-1>Synthesis of 3,4-O-di-tetradecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (Compound 7)
(55) 3,4-O-di-tetradecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 7) was synthesized with a yield of 92% according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and tetradecyl bromide as an alkyl bromide. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by S. Roy et al. (Colloids surf A 2011, 377, 349-355.).
<7-2>Synthesis of 3,4-O-di-tetradecyl-D-mannitol (Compound 17)
(56) 3,4-O-di-tetradecyl-D-mannitol (compound 17) was synthesized from the compound 7 with a yield of 92% according to the method of Example 1-2. The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by S. Roy et al. (Colloids surf A 2011, 377, 349-355.).
<7-3>Synthesis of MNA-7a
(57) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-7a from the compound 17 with a yield of 54%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.24-8.23 (m, 4H), 8.12-7.98 (m, 4H), 7.97-7.93 (m, 8H), 7.91-7.89 (m, 6H), 7.88-7.83 (m, 16H), 7.61-7.56 (m, 6H), 7.55-7.43 (m, 16H), 7.41-7.35 (m, 32H), 7.34-7.27 (m, 4H), 5.79-5.77 (m, 2H), 5.57-5.54 (m, 4H), 5.49-5.43 (m, 6H), 5.30-5.21 (m, 2H), 4.90-4.79 (m, 2H), 4.60-4.39 (m, 6H), 4.33-4.20 (m, 6H), 4.01-3.91 (m, 2H), 3.56-3.49 (m, 5H), 3.40-3.32 (m, 2H), 3.31-3.15 (m, 4H), 1.51-1.32 (m, 2H), 1.27-1.12 (m, 44H), 0.86 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 166.1, 166.0, 165.9, 165.7, 165.2, 165.1, 164.9, 133.7, 133.6, 133.5, 133.4, 133.3, 133.1, 130.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.3, 129.2, 129.1, 129.0, 128.9, 128.8, 128.5, 128.4, 128.3, 128.1, 101.5, 100.4, 82.4, 80.2, 73.4, 73.0, 72.9, 72.8, 72.4, 71.9, 71.7, 71.1, 70.0, 69.9, 63.2, 63.0, 32.0, 30.3, 29.9, 29.8, 29.5, 26.2, 22.8, 14.2.
<7-4>Synthesis of MNA-7
(58) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-7, from which a protecting group was removed, from the compound MNA-7 with a yield of 92%. a .sup.1H NMR (400 MHz, CD.sub.3OD): 4.29-4.27 (m, 2H), 4.11-4.09 (m, 2H), 3.98-3.96 (m, 2H), 3.80-3.77 (m, 4H), 3.67-3.63 (m, 4H), 3.62-3.59 (m, 3H), 3.58-3.53 (m, 10H), 3.27-3.17 (m, 15H), 3.16-3.08 (m, 5H), 1.55-1.46 (m, 4H), 1.21 (m, 44H), 0.90 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.7, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.6, 71.3, 62.8, 62.7, 33.2, 31.6, 30.9 30.6, 27.5, 23.9, 14.6; HRMS (EI): calculated for C.sub.58H.sub.110O.sub.26[M+Na].sup.+ 1222.7285, found 1222.7283. The .sup.1H NMR spectrum of MNA-7 is shown in
<Preparative Example 8>Synthesis of MNA-8
<8-1,2>Synthesis of 3,4-O-di-tetradecyl-D-mannitol (compound 8)
(59) 3,4-O-di-pentadecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 8) was synthesized according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and pentadecyl bromide as an alkyl bromide, and the next steps were then carried out without purification. 3,4-O-di-tetradecyl-D-mannitol (compound 18) was synthesized from the compound 8 with a in a yield of 76% according to the method of Example 1-2 (two steps). The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by S. Roy et al. (Colloids surf A 2011, 377, 349-355.).
<8-3>Synthesis of MNA-8a
(60) A glycosylation reaction was performed according to the method of Example
(61) 1-3 to synthesize MNA-8a from the compound 18 with a yield of 54%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.23-8.21 (m, 4H), 8.20-7.99(m, 4H), 7.98-7.93 (m, 8H), 7.92-7.88 (m, 6H), 7.87-7.78 (m, 16H), 7.56-7.38 (m, 6H), 7.37-7.34 (m, 16H), 7.33-7.29 (m, 32H), 7.27-7.25(m, 4H), 5.77-5.74 (m, 2H), 5.57-5.51 (m, 4H), 5.47-5.39 (m, 6H), 4.80-4.76 (m, 2H), 4.48-4.46 (m, 2H), 4.32-4.29 (m, 6H), 4.28-4.22 (m, 6H), 4.01-3.94 (m, 2H), 3.54-3.41 (m, 5H), 3.40-3.32 (m, 2H), 3.31-3.14 (m, 4H), 1.81-1.34 (m, 2H), 1.24-1.12 (s, 48H), 0.87 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 165.9, 165.8, 165.6, 165.1, 165.0, 164.8, 133.6, 133.4, 133.2, 133.1, 130.2, 130.0, 129.8, 129.7, 129.5, 129.2, 129.0, 128.9, 128.7, 128.5, 128.4, 101.4, 100.3, 91.4, 82.4, 80.1, 73.3, 72.9, 72.8, 72.7, 72.3, 71.8, 71.6, 71.1, 69.9, 69.8, 63.2, 62.9, 60.5, 32.0, 30.2, 29.9, 29.8, 29.7, 29.4, 26.2, 22.8, 14.2.
<8-4>Synthesis of MNA-8
(62) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-8, from which a protecting group was removed, from the compound MNA-8a with a yield of 90%. .sup.1H NMR (400 MHz, CD.sub.3OD): 4.65-4.62 (m, 2H), 4.42-4.37 (m, 2H), 4.22-4.19(m, 2H), 4.13-4.04 (m, 2H), 3.91-3.86 (m, 4H), 3.85-3.72 (m, 4H), 3.71-3.68 (m, 3H), 3.67-3.65 (m, 10H), 3.37-3.25 (m, 5H), 3.28-3.19(m, 15H), 1.56-1.48 (m, 4H), 1.28 (s, 48H), 0.89 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.7, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.5, 71.3, 62.8, 62.7, 33.2, 31.6, 31.0, 30.9 30.6, 27.5, 23.8, 14.6.
<Preparative Example 9>Synthesis of MNA-9
<9-1,2>Synthesis of 3,4-O-di-hexadecyl-D-mannitol (compound 19)
(63) 3,4-O-di-hexadecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 9) was synthesized according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and hexadecyl bromide as an alkyl bromide, and the next steps were then carried out without purification. 3,4-O-di-hexadecyl-D-mannitol (compound 19) was synthesized from the compound 9 with a yield of 76% according to the method of Example 1-2 (two steps). The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by S. Roy et al. (Colloids surf A 2011, 377, 349-355.).
<9-3>Synthesis of MNA-9a
(64) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-9a from the compound 19 with a yield of 52%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.23-8.21 (m, 4H), 8.20-7.99(m, 4H), 7.98-7.93 (m, 8H), 7.92-7.88 (m, 6H), 7.87-7.78 (m, 16H), 7.56-7.38 (m, 6H), 7.37-7.34 (m, 16H), 7.33-7.29 (m, 32H), 7.27-7.25(m, 4H), 5.77-5.74 (m, 2H), 5.57-5.51 (m, 4H), 5.47-5.39 (m, 6H), 4.80-4.76 (m, 2H), 4.48-4.46 (m, 2H), 4.32-4.29 (m, 6H), 4.28-4.22 (m, 6H), 4.01-3.94 (m, 2H), 3.54-3.41 (m, 5H), 3.40-3.32 (m, 21-1), 3.31-3.14 (m, 4H), 1.81-1.34 (m, 2H), 1.24-1.12 (s, 48H), 0.87 (t, J=7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 165.9, 165.8, 165.6, 165.1, 165.0, 164.8, 133.6, 133.4, 133.2, 133.1, 130.2, 130.0, 129.8, 129.7, 129.5, 129.2, 129.0, 128.9, 128.7, 128.5, 128.4, 101.4, 100.3, 91.4, 82.4, 80.1, 73.3, 72.9, 72.8, 72.7, 72.3, 71.8, 71.6, 71.1, 69.9, 69.8, 63.2, 62.9, 60.5, 32.0, 30.2, 29.9, 29.8, 29.7, 29.4, 26.2, 22.8, 14.2.
<9-4>Synthesis of MNA-9
(65) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-9, from which a protecting group was removed from the compound MNA-9a with a yield of 92%. .sup.1H NMR (400 MHz, CD.sub.3OD): 6 4.65-4.62 (m, 2H), 4.42-4.37 (m, 2H), 4.22-4.19(m, 2H), 4.13-4.04 (m, 2H), 3.91-3.86 (m, 4H), 3.85-3.72 (m, 4H), 3.71-3.68 (m, 3H), 3.67-3.65 (m, 10H), 3.37-3.25 (m, 5H), 3.28-3.19(m, 15H), 1.56-1.48 (m, 4H), 1.28 (s, 48H), 0.89 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.7, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.5, 71.3, 62.8, 62.7, 33.2, 31.6, 31.0, 30.9 30.6, 27.5, 23.8, 14.6.
<Preparative Example 10>Synthesis of MNA-10
<10-1,2>Synthesis of 3,4-O-di-heptadecyl-D-mannitol (Compound 20)
(66) 3,4-O-di-heptadecyl-1,2:5,6-di-O-isopropylidene-D-mannitol (compound 10) was synthesized according to the method of Example 1-1 using (1R,2R)-1,2-bis((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol as a starting material and heptadecyl bromide as an alkyl bromide, and the next steps were then carried out without purification. 3,4-O-di-heptadecyl-D-mannitol (compound 20) was synthesized from the compound 10 with a yield of 75% according to the method of Example 1-2 (two steps). The .sup.1H NMR spectra of the separated product were identical to the data reported in the article by S. Roy et al. (Colloids surf. A 2011, 377, 349-355.).
<10-3>Synthesis of MNA-10a
(67) A glycosylation reaction was performed according to the method of Example 1-3 to synthesize MNA-10a from the compound 20 with a yield of 50%. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.23-8.21 (m, 4H), 8.20-7.99(m, 4H), 7.98-7.93 (m, 8H), 7.92-7.88 (m, 6H), 7.87-7.78 (m, 16H), 7.56-7.38 (m, 6H), 7.37-7.34 (m, 16H), 7.33-7.29 (m, 32H), 7.27-7.25(m, 4H), 5.77-5.74 (m, 2H), 5.57-5.51 (m, 4H), 5.47-5.39 (m, 6H), 4.80-4.76 (m, 2H), 4.48-4.46 (m, 2H), 4.32-4.29 (m, 6H), 4.28-4.22 (m, 6H), 4.01-3.94 (m, 2H), 3.54-3.41 (m, 5H), 3.40-3.32 (m, 2H), 3.31-3.14 (m, 4H), 1.81-1.34 (m, 2H), 1.24-1.12 (s, 48H), 0.87 (t, J =7.6 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): 165.9, 165.8, 165.6, 165.1, 165.0, 164.8, 133.6, 133.4, 133.2, 133.1, 130.2, 130.0, 129.8, 129.7, 129.5, 129.2, 129.0, 128.9, 128.7, 128.5, 128.4, 101.4, 100.3, 91.4, 82.4, 80.1, 73.3, 72.9, 72.8, 72.7, 72.3, 71.8, 71.6, 71.1, 69.9, 69.8, 63.2, 62.9, 60.5, 32.0, 30.2, 29.9, 29.8, 29.7, 29.4, 26.2, 22.8, 14.2.
<10-4>Synthesis of MNA-10
(68) A de-O-benzoylation reaction was performed according to the method of Example 1-4 to synthesize MNA-10, from which a protecting group was removed, from the compound MNA-10a with a yield of 92%. .sup.1H NMR (400 MHz, CD.sub.3OD): 4.65-4.62 (m, 2H), 4.42-4.37 (m, 2H), 4.22-4.19(m, 2H), 4.13-4.04 (m, 2H), 3.91-3.86 (m, 4H), 3.85-3.72 (m, 4H), 3.71-3.68 (m, 3H), 3.67-3.65 (m, 10H), 3.37-3.25 (m, 5H), 3.28-3.19(m, 15H), 1.56-1.48 (m, 4H), 1.28 (s, 48H), 0.89 (t, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CD.sub.3OD): 105.2, 104.8, 82.7, 81.0, 78.3, 78.1, 77.9, 75.7, 75.4, 74.5, 71.7, 71.5, 71.3, 62.8, 62.7, 33.2, 31.6, 31.0, 30.9 30.6, 27.5, 23.8, 14.6.
Example 2
Characteristics of MNAs
(69) To determine characteristics of the MNAs of Preparative Example 1 to 10 synthesized according to the synthesis method of Example 1, the molecular weights (M.W.) and critical micellar concentrations (CMCs) of the MNAs and the hydrodynamic radii (R.sub.h) of the formed micelles were measured.
(70) Specifically, the critical micellar concentration (CMC) was measured using a hydrophobic fluorescence stain (diphenylhexatriene (DPH)), and the hydrodynamic radii (R.sub.h) of the micelles formed by each of the preparations were measured by a dynamic light scattering (DLS) experiment. The measured results were compared to those of DDM that is a conventional amphipathic molecule (a detergent), and are listed in Table 1.
(71) TABLE-US-00001 TABLE 1 Detergent M.W. CMC (mM) CMC (% by weight) R.sub.h (nm) MNA-1 1055.2 Approximately Approximately 0.016 2.3 0.13 0.15 MNA-2 1083.2 Approximately Approximately 0.0054 2.5 0.05 0.05 MNA-3 1111.3 Approximately Approximately 0.0017 2.7 0.04 0.015 MNA-4 1139.3 Approximately Approximately 0.0007 2.9 0.12 0.006 MNA-5 1167.4 Approximately Approximately 0.0005 3.0 0.01 0.004 MNA-6 1195.4 Approximately Approximately 0.0002 3.3 0.04 0.002 MNA-7 1233.5 Approximately Approximately 0.0001 3.3 0.08 0.001 MNA-8 1251.53 Approximately Approximately 0.0003 3.5 0.1 0.003 MNA-9 1279.58 Approximately Approximately 0.0001 3.8 0.1 0.001 MNA-10 1307.64 Approximately Approximately 0.0001 4.1 0.2 0.001 DDM 510.1 Approximately Approximately 0.0087 3.4 0.03 0.17
(72) The measurement results of the critical micellar concentrations (CMCs) of the MNAs showed that MNA-1 had a CMC value of 150 M (0.016% by weight), MNA-2 had a CMC value of 50 M (0.0054% by weight), MNA-3 had a CMC value of 15 M (0.0017% by weight), MNA-4 had a CMC value of 6 M (0.0007% by weight), MNA-5 had a CMC value of 4 M (0.0005% by weight), MNA-6 had a CMC value of 2 M (0.0002% by weight), MNA-7 had a CMC value of 1 M (0.0001% by weight), MNA-8 had a CMC value of 3 M (0.0003% by weight), MNA-9 had a CMC value of 1 M (0.0001% by weight), and MNA-10 had a CMC value of 1 M (0.0001% by weight). That is, it was revealed that the CMC value of MNA-1 was 150 M which was substantially similar to that of DDM, but the CMC values of MNA-2 to MNA-10 were in a range of 1 to 50 M which was smaller than that of DDM having a CMC value of 170 M. Also, the CMC values tended to decrease with an increase in lengths of the alkyl chains of the compounds. Here, it was confirmed that MNA-1 having the shortest alkyl chain (C8) had a CMC value of approximately 50 M (approximately 0.016% by weight), and MNA-7, MNA-9 and MNA-10 having long alkyl chains (C14, C16, and C17) had a CMC value of 1 M (approximately 0.0001% by weight). Therefore, it could be seen that, since the micelles were easily formed even when the MNAs were used in small quantities, the MNAs may be used in an amount smaller than that of DDM to study the membrane proteins and are more soluble in water than DDM.
(73) The measurement results of the hydrodynamic radii (R.sub.h) of the micelles formed by the MNAs showed that MNA-1 had a measured R.sub.h of 2.3 nm, MNA-2 had a measured R.sub.h of 2.5 nm, MNA-3 had a measured R.sub.h of 2.7 nm, MNA-4 had a measured R.sub.h of 2.9 nm, MNA-5 had a measured R.sub.h of 3.0 nm, MNA-6 had a measured R.sub.h of 3.3 nm, MNA-7 had a measured R.sub.h of 3.3 nm, MNA-8 had a measured R.sub.h of 3.5 nm, MNA-9 had a measured R.sub.h of 3.8 nm, and MNA-10 had a measured R.sub.h of 4.1 nm. Generally, the sizes of the micelles formed by the MNAs were similar to or smaller than that of DDM and tended to increase according to the lengths of the alkyl chains. MNA-1 had the smallest micelle size (2.3 nm), and MNA-10 had the highest micelle size (4.1 nm).
(74) Meanwhile, the size distributions of the micelles formed by the MNAs were measured using DLS, and the measurement results are shown in
(75) From these results, it could be seen that the micelles were easily formed even when the MNAs of the present invention were used in a small quantity since the MNAs had lower CMC values than DDM and thus had a much higher tendency toward self-assembly than DDM and that the membrane proteins were more easily separated from the micelles of the amphipathic molecules according to the present invention since the sizes of the micelles formed by the MNAs were smaller than those of DDM.
Example 3
Evaluation of abilities of MNAs to Stabilize Structure of Membrane Protein (Bor1)
(76) An experiment for measuring the structural stability of a boron transporter (Bor1) by the MNAs was performed. The structural stability of Bor1 was measured using heat fluorescence size exclusion chromatography (hFSEC), and the concentrations of the MNAs and DDM were CMC plus 0.04% by weight.
(77) Specifically, Bor1 of Arabidopsis thaliana was expressed in Saccharomyces cerevisiae FGY217 cells as a fusion protein having a C-terminal GFP-His tag. The cells were grown in a -URA medium supplemented with 0.1% glucose. Expression of proteins was induced by adding 2% galactose, and the cells were then cultured at 20 C. for 18 hours. Here, the cell culture was carried out according to the method described in the article by D. Drew et al. (Nat. Protoc. 2008, 3, 784-798.). The cells were collected to prepare membranes, and the cell collection was carried out according to the method described in the article by J. Leung et al. (Protein Expr. Purif. 2010, 72, 139-146.). The membranes including the Bor1-GFP fusion protein were diluted with phosphate-buffered saline (PBS, pH 7.4) until the final concentration of the total proteins reached 2.8 mg/mL. In this case, 1% by weight of DDM or 1% by weight of MNA (MNA-1, MNA-2, MNA-3, MNA-4, MNA-5, MNA-6, or MNA-7) was added to the PBS. Samples were incubated for an hour while shaking at 4 C., and insoluble materials were then removed by centrifugation at 14,000 g and 4 C. for an hour. The supernatant including the dissolved protein samples was heated at 40 C. for 10 minutes, and the strongly aggregated proteins were removed by centrifugation at 14,000 g and 4 C. for 10 minutes. 200 L of the supernatant of the samples was injected into a Superose 6 10/300 column equilibrated with 20 mM Tris (pH 7.5), 150 mM NaCl, and 0.03% DDM. Each of elution fractions was collected in 200 L fractions at a retention volume of 6.4 mL (e.g., 6.4 mL after elusion) in a clear bottom 96-well plate. The GFP fluorescence of each of the fractions was measured at an excitation wavelength of 470 nm and an emission wavelength of 512 nm.
(78) The measurement results are shown in
(79) From these results, it could be seen that the MNAs had a superior ability to stabilize a structure of Bor1 compared to the conventional DDM and thus was able to be used to extract or stabilize the membrane proteins.
Example 4
Evaluation of Abilities of MNAs to Stabilize Structure of Membrane Protein (LeuT)
(80) An experiment for measuring the structural stability of a leucine transporter (LeuT) by the MNAs was performed. The activities of the LeuT protein was measured by a scintillation proximity assay (SPA) using a substrate ([.sup.3H]-Leu), and the concentrations of the MNAs and DDM used were (a) CMC plus 0.04% by weight, or (b) CMC plus 0.02% by weight.
(81) Specifically, a wild-type leucine transporter (LeuT) was purified from Aquifex aeolicus according to the method disclosed in the article by G. Deckert et al. (Nature 1998, 392, 353-358.). The LeuT was expressed in E. coli C41 (DE3) transformed with pET16b coding for a C-terminal 8His-tagged transporter (an expression plasmid was kindly provided by Dr E. Gouaux, Vollum Institute, Portland, Oreg., USA). In summary, a protein was isolated, and solubilized in 1.0% by weight of DDM. Then, the protein was bound to an Ni.sup.2+-NTA resin (Life Technologies, Denmark) and eluted with 20 mM Tris-HCl (pH 8.0), 1 mM NaCl, 199 mM KCl, 0.05% DDM, and 300 mM imidazole. Subsequently, approximately 0.8 mg/ml of a protein stock was diluted with an equivalent buffer having no DDM and imidazole but supplemented with MNAs (MNA-5, MNA-6 or MNA-6) or DDM (control) so that the final concentration reached CMC plus 0.04% by weight or CMC plus 0.2% by weight. Protein samples were stored at room temperature, and centrifuged for a given period of time, and the activities of the proteins were determined by measuring [.sup.3H]-Leu binding using a scintillation proximity assay (SPA) (M. Quick et al., Proc. Natl, Acad. Sci. U.S.A. 2007, 104, 3603-3608.). SPA was performed using 5 L of each of the protein samples, 20 nM [.sup.3H]-Leu, and 1.25 mg/ml copper chelate (both commercially available from PerkinElmer, Denmark) dissolved in a buffer including 450 mM NaCl and the concentration of each of the test compounds. The [.sup.3H]-Leu binding was measured using a MicroBeta liquid scintillation counter (PerkinElmer).
(82) As shown in
(83) From these results, it could be seen that MNA-5 and MNA-6 of the present invention had a superior ability to stabilize LeuT compared to the DDM, and MNA-7 had an ability to stabilize LeuT at a level similar to DDM.
Example 5
Evaluation of Abilities of Compounds According to the Present Invention to Stabilize Structure of Membrane Protein ((.SUB.2.AR)
(84) Experiments for measuring the structural stabilities of a human 2 adrenergic receptor (.sub.2AR), a G-protein-coupled receptor (GPCR), in the MNAs were performed.
<5-1>Measurement of mBBr-.SUB.2.AR Dissolved in MNAs and DDM Micelles According to the Presence of Full Agonist (ISO) or a Combination of ISO and G-Protein
(85) Experiments for measuring the structural changes and structural stabilities of mBBr-.sub.2AR by the MNAs (MNA-5, MNA-6, MNA-7) and DDM according to the presence or absence of a full agonist (isopreoterenol (ISO)) or a combination of ISO and G-protein were performed.
(86) Specifically, .sub.2AR dissolved in 0.1% DDM was purified according to the method disclosed in the article by D. M. Rosenbaum et al. (Science 2007, 318, 1266-1273) and then concentrated to a concentration of approximately 1 mg/mL. 0.5 L of non-ligand BI (agonist)-coupled monobromobimane (mBBr)-labeled .sub.2AR dissolved in 50 M of 0.1% DDM was diluted with a buffer of the MNAs (MNA-5, MNA-6 or MNA-7) which were present at a content of 500 L CMC plus 0.04% by weight or a content of CMC plus 0.2% by (finally to a 50 nM concentration of the receptor) at room temperature for 10 minutes. The mBBr-labeled .sub.2AR was cultured for 30 minutes, and the mBBr spectra thereof were measured and compared to the spectra of the mBBr-labeled receptor dissolved in 0.1% DDM. The fluorescence of the mBBr-labeled .sub.2AR was measured at 370 nm and thus determined by emission from 430 to 510 nm at 1-nm increments with 1 nm s.sup.1 integration using a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon Inc.) which was run in a photon counting mode through a 4-nm emission bandwidth pass. mBBr dissolved in DDM was used as the positive control.
(87) Meanwhile, a G protein coupling test was performed using the following method. A change in fluorescence influenced by a local structural change at a proximal portion of transmembrane helix 6 (TM6) was measured using .sub.2AR labeled with monobromobimane (mBBr) (mainly at Cys.sup.265). This followed the method proposed by S. E. Mansoor et al. (Biochemistry 2002, 41, 2475-2484.). 0.5 L of 50 M non-ligand mBBr-labeled receptor was diluted with 500 L of a 20CMC MNA-5, MNA-6 or MNA-7 buffer (finally to a 50 nM concentration of the receptor) at room temperature for 10 minutes. Then, 2 M isopreoterenol (ISO) was added, and the resulting mixture was cultured again for 10 minutes. 250 nM Gs was additionally added, the resulting mixture was cultured at room temperature for 15 minutes, and the mBB-.sub.2AR fluorescence was then measured at 370 nm. Thus, the mBB-.sub.2AR fluorescence was determined by emission from 430 to 510 nm at 1-nm increments with 1 nm s.sup.1 integration using a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon Inc.) which was run in a photon counting mode set at a 4-nm emission bandwidth pass. mBBr dissolved in DDM was used as the positive control. Data was presented as an average of three separate experiments performed in triplicate.
(88) As shown in
(89) These results showed that the structure and functions of the receptor were maintained effectively when MNA-5, MNA-6, or MNA-7 was used. As such, a decrease in the fluorescence intensity and a change in the maximum emission wavelengths in the presence of ISO and G-protein mean the occurrence of a structural change from an inactive to active state of the receptor by binding of such molecules (ISO and G-protein), indicating that the structure of .sub.2AR dissolved in MNA-5, MNA-6, or MNA-7 behaved in a pattern similar to the receptors present in the cell membranes.
<5-2>Measurement of mBBr-.SUB.2.AR at Concentration Below CMC
(90) Experiments for comparing the structural changes of the .sub.2AR protein by the MNAs and DDM at a concentration below CMCs of the amphipathic molecules were performed.
(91) Specifically, 0.5 L of a non-ligand mBBr-labeled receptor dissolved at 50 NI in a 20CMC concentration of MNA-5, MNA-6, or MNA-7 was diluted with 500 L of an NH buffer (20 mM HEPES pH 7.5, and 100 mM NaCl). The proteins were cultured for 30 minutes, and the bimane spectra thereof were measured. The receptor dissolved in 0.1% DDM was diluted with the NH buffer, and used as the control. Data was presented as an average of three separate experiments performed in triplicate.
(92) As shown in
<5-3>Measurement of Ligand (DHA) Binding Activity of mBBr-.SUB.2.AR Using Radioactive Ligand Binding Test
(93) The activity of the receptor (mBBr-.sub.2AR) dissolved in DDM or MNAs (MNA-5, MNA-6, or MNA-7) was measured by binding of [.sup.3H]-dihydroalprenolol ([.sup.3H]-DHA).
(94) Specifically, a radioactive ligand binding test was performed using the following method. The .sub.2AR dissolved in 0.1% DDM was purified according to the method disclosed in the article by D. M. Rosenbaum et al. (Science 2007, 318, 1266-1273.) until the final concentration reached approximately 5 mg/mL (approximately 100 M). The purified .sub.2AR was loaded onto an M1 Flag column in the presence of 2 mM CaCl.sub.2, and the column was then washed with an MNA buffer (20 mM HEPES pH 7.5, 100 mM NaCl, and 0.2% MNA). Thereafter, the receptor was eluted into a 20CMC concentration of MNA-5, MNA-6, or MNA-7 using 5 mM EDTA and 0.2 mg/ml of free Flag peptides. 0.1 pmol of the purified .sub.2AR dissolved in DDM or MNAs (MNA-5, MNA-6, or MNA-7) was cultured with 10 nM of radioactive DHA [.sup.3H]-dihydroalprenolol (DHA) at room temperature (RT) for 30 minutes. The mixture was loaded onto a G-50 column, and a flow-through solution was collected in a binding buffer (20 mM HEPES supplemented with 0.5 mg/ml BSA pH 7.5, 100 mM NaCl), and filled with 15 mL of a scintillation fluid. The receptor-coupled [.sup.3H]-DHA was measured using a scintillation counter (Beckman). Non-specific binding of the [.sup.3H]-DHA was measured by adding 2 M of alprenolol (Sigma) in the same binding reaction. The binding ratio of the [.sup.3H]-DHA was determined on a column graph. Each experiment was performed in triplicate.
(95) As shown in
(96) From these results, it could be seen that .sub.2AR dissolved in MNA-6 or MNA-7 had its protein functions maintained well during a substitution of the amphipathic molecules.
<5-4>Size Exclusion Chromatography (SEC) Analysis
(97) An SEC analysis was performed to determine the size of the protein-amphipathic molecule complex (i.e., a protein-detergent complex (PDC)) formed by DDM, MNA-6, or MNA-7.
(98) Specifically, the .sub.2AR dissolved in DDM, MNA-6 or MNA-7 was used as a test sample in the same manner as used in Example 5-3. The sample was loaded onto a Superdex-200 10/300 GL column (GE Healthcare) at 0.5 ml/min, an innate tryptophane fluorescence signal was measured at an excitation wavelength of 295 nm and an emission wavelength of 345 nm. A running buffer includes 20 mM HEPES pH 7.5, 100 mM NaCl, and 20CMC MNA-6 or MNA-7. A difference in retention volume of the protein samples means a difference in size of the protein-detergent complexes (PDCs) formed in the presence of the amphipathic molecules.
(99) As shown in
<5-5>Electron Microscopy (EM) Analysis
(100) A .sub.2AR protein analysis test was performed for DDM, MNA-6, or MNA-7 using an electron microscope.
(101) Specifically, samples were prepared using the conventional negative staining protocol disclosed in the article by M. Ohi et al. (Biol. Proced. Online 2004, 6, 23-34.). In summary, 3 L of .sub.2AR dissolved in DDM, MNA-6, or MNA-7 was pipetted onto a glow-discharged carbon-coated grid, and stained with 1% (w/v) uranyl formate. Thereafter, the .sub.2AR was imaged at room temperature by running a Morgagni 268(D) transmission electron microscope (FEI Company) at 100 kV. The images were recorded at 30,416calibrated magnification using an Orius SC200W CCD camera (Gatan Inc.).
(102) As shown in
(103) From such results, it could be seen that the MNAs are able to be used to analyze a structure of the membrane protein under an electron microscope (EM).