Resorcinarene-based amphipathic compound and use thereof
10793589 ยท 2020-10-06
Assignee
Inventors
Cpc classification
C07C41/30
CHEMISTRY; METALLURGY
C07C39/17
CHEMISTRY; METALLURGY
G01N33/6842
PHYSICS
C07C2603/92
CHEMISTRY; METALLURGY
C07D493/22
CHEMISTRY; METALLURGY
C07C41/30
CHEMISTRY; METALLURGY
C07C39/17
CHEMISTRY; METALLURGY
C07H15/18
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a newly developed resorcinarene-based amphipathic compound, a method for producing the same, and a method for extracting, solubilizing, stabilizing, crystallizing, or analyzing a membrane protein by using the same. In addition, compared to a conventional compound, the compound can efficiently extract, from a cell membrane, membrane proteins having a greater variety of structures and characteristics, and can stably store such membrane proteins for a long time in an aqueous solution. Therefore, the compound can be used to analyze the functions and structures of such membrane proteins. Analysis of the structures and functions of membrane proteins, being closely related to the development of new drugs, is one of the fields of greatest interest in biology and chemistry today.
Claims
1. A compound represented by the following Formula 1 or Formula 2: ##STR00019## wherein R.sup.1 to R.sup.4 are each independently a substituted or unsubstituted C.sub.3-C.sub.30 alkyl group, a substituted or unsubstituted C.sub.3-C.sub.30 cycloalkyl group, or a substituted or unsubstituted C.sub.3-C.sub.30 aryl group; X.sup.1 to X.sup.8 are saccharides; and m.sup.1 to m.sup.8 are 0, 1, or 2, ##STR00020## wherein R.sup.1 to R.sup.4 are each independently a substituted or unsubstituted C2-C30 alkyl group, a substituted or unsubstituted C.sub.3-C.sub.30 cycloalkyl group, or a substituted or unsubstituted C.sub.3-C.sub.30 aryl group; X.sup.1 to X.sup.4 are saccharides; and m.sup.1 to m.sup.4 are 0, 1, or 2.
2. The compound of claim 1, wherein the saccharide is a monosaccharide or a disaccharide.
3. The compound of claim 1, wherein the saccharide is glucose or maltose.
4. The compound of claim 1, wherein R.sup.1 to R.sup.4 are each independently said substituted or unsubstituted alkyl group; X.sup.1 to X.sup.8 are glucoses or maltoses; and m.sup.1 to m.sup.8 are 1.
5. The compound of claim 1, which is a compound represented by one of the following Formulas 3 to 14: ##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
6. The compound of claim 1, wherein the compound is an amphipathic molecule for extracting, solubilizing, stabilizing, crystallizing, or analyzing a membrane protein.
7. The compound of claim 1, wherein the compound has a critical micelle concentration (CMC) of 0.0001 to 1 mM in an aqueous solution.
8. A composition for extracting, solubilizing, stabilizing, crystallizing, or analyzing a membrane protein, comprising the compound according to claim 1.
9. The composition of claim 8, wherein the composition is prepared in a form of micelles, liposomes, emulsions or nanoparticles.
10. A method of preparing a compound represented by the following Formula 1, comprising: 1) introducing an alkyl group by acid-catalyzed condensation of four 1,3-bis(2-hydroxyethoxy)benzene compounds; 2) introducing a protective group-attached saccharide by glycosylating the product of step 1); and 3) de-protecting the product of step 2): ##STR00027## wherein R.sup.1 to R.sup.4 are each independently a substituted or unsubstituted C.sub.3-C.sub.30 alkyl group, a substituted or unsubstituted C.sub.3-C.sub.30 cycloalkyl group, or a substituted or unsubstituted C.sub.3-C.sub.30 aryl group; X.sup.1 to X.sup.8 are saccharides; and m.sup.1 to m.sup.8 are 0, 1, or 2.
11. The method of claim 10, wherein R.sup.1 to R.sup.4 are each independently a substituted or unsubstituted C.sub.3-C.sub.30 alkyl group; X.sup.1 to X.sup.8 are glucoses; and m.sup.1 to m.sup.8 are 1.
12. A method of preparing a compound represented by the following Formula 2, comprising: 1) introducing an alkyl group by acid-catalyzed condensation of four methyl resorcinol compounds; 2) subjecting the product of step 1) to a methylene bridging reaction using bromochloromethane; 3) brominating a benzylic position in the product of step 2); 4) hydrolyzing or causing the product of step 3) to have ethylene glycol linkages; 5) introducing a protective group-attached saccharide by glycosylating the product of step 4); and 6) de-protecting the product of step 5): ##STR00028## wherein R.sup.1 to R.sup.4 are each independently a substituted or unsubstituted C.sub.2-C.sub.30 alkyl group, a substituted or unsubstituted C.sub.3-C.sub.30 cycloalkyl group, or a substituted or unsubstituted C.sub.3-C.sub.30 aryl group; X.sup.1 to X.sup.4 are saccharides; and m.sup.1 to m.sup.4 are 0, 1, or 2.
13. A method of extracting, solubilizing, stabilizing, crystallizing, or analyzing a membrane protein, the method comprising treating a membrane protein with a compound represented by the following Formula 1 or Formula 2 in an aqueous solution: ##STR00029## wherein R.sup.1 to R.sup.4 are each independently a substituted or unsubstituted C.sub.3-C.sub.30 alkyl group, a substituted or unsubstituted C.sub.3-C.sub.30 cycloalkyl group, or a substituted or unsubstituted C.sub.3-C.sub.30 aryl group; X.sup.1 to X.sup.8 are saccharides; and m.sup.1 to m.sup.8 are 0, 1, or 2, ##STR00030## wherein R.sup.1 to R.sup.4 are each independently a substituted or unsubstituted C.sub.2-C.sub.30 alkyl group, a substituted or unsubstituted C.sub.3-C.sub.30 cycloalkyl group, or a substituted or unsubstituted C.sub.3-C.sub.30 aryl group; X.sup.1 to X.sup.4 are saccharides; and m.sup.1 to m.sup.4 are 0, 1, or 2.
14. The method of claim 13, wherein R.sup.1 to R.sup.4 are each independently said substituted or unsubstituted alkyl group; X.sup.1 to X.sup.8 are glucoses or maltoses; and m.sup.1 to m.sup.8 are 1.
15. The method of claim 13, wherein the membrane protein is a uric acid-xanthine/Hxanthine/H.sup.+ symporter (UapA), a leucine transporter (LeuT), a human .sub.2 adrenergic receptor (.sub.2AR), a melibiose permease (MelB), or a combination of two or more thereof.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEW OF THE DRAWINGS
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DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
(13) Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are provided to exemplify contents of the present invention and the scope of the present invention is not limited to the following examples. It should be understood that examples which can be easily deduced from detailed descriptions and the following examples of the present invention by one of ordinary skill in the art are within the scope of the present invention.
<Example 1> Method of Synthesizing Resorcinarene-Based Maltosides (RMAs)
(14) A synthesis scheme of RMAs is shown in
<1-1> General Procedure for the Synthesis of octol-resorcin[4]arene (Step a of FIG. 1A and FIG. 1B)
(15) Methyl resorcinol (1.0 g, 0.0081 mol) was dissolved in a mixed solution of ethanol (6.27 mL) and 37% HCl (1.51 mL). To the resultant solution, an aldehyde (0.0081 mol) was added at 0 C. over 15 minutes. The reaction mixture was warmed at room temperature, then transferred to a preheated oil bath, and stored at 80 C. for 16 hours while stirring. After completion of the reaction (as detected by TLC), the reaction mixture was added to distilled water. An organic layer was extracted using ethyl acetate and washed twice with water and brine. The organic layer was dried with anhydrous Na.sub.2SO.sub.4, and a solvent was removed using a rotary evaporator. The residue was purified by column chromatography, thereby obtaining compound A as a yellow solid.
<1-2> General Procedure for the Synthesis of Tetramethylcavitand (Step b of FIG. 1A and FIG. 1B)
(16) K.sub.2CO.sub.3 (88 mmol) was added to a mixed solution (0.55 mmol) of dry DMF and compound A in a sealed tube. To the mixture, bromochloromethane (88 mmol) was added at room temperature, and the resultant mixture was stirred for 30 minutes and then transferred to an oil bath preheated at 80 C. The reaction mixture was stirred at 80 C. for 24 hours. After completion of the reaction (as detected by TLC), ice water was added to the reaction mixture. An organic layer was extracted using ethyl acetate and washed twice with water and brine. The organic layer was dried with anhydrous Na.sub.2SO.sub.4, and a solvent was removed using a rotary evaporator. The residue was purified by column chromatography, thereby obtaining compound B as a white solid.
<1-3> General Procedure for the Synthesis of tetrakis(bromomethyl)cavitand (Step c of FIG. 1A and FIG. 1B)
(17) NBS (30 mmol) was added to a solution in which compound B (1.1 mmol) and AIBN (0.6 mmol) were stirred in 10 mL of degassed benzene. The resultant solution was stirred for 30 minutes and transferred to an oil bath preheated at 80 C. The reaction mixture was stirred at 80 C. for 24 hours. After completion of the reaction (as detected by TLC), a solid precipitate (succinimide) was filtered, and benzene was evaporated using a rotary evaporator. The reaction mixture was purified by column chromatography, thereby obtaining compound C as a white crystal solid.
<1-4> General Procedure for the Synthesis of tetrakis(hydroxymethyl)cavitand (Step d of FIG. 1A)
(18) K.sub.2CO.sub.3 (2.2 mmol) was added to a mixed solution of 40 mL of acetone and water (9:1) and compound C in a sealed tube, and the sealed tube was immersed in an oil bath preheated at 80 C. The reaction mixture was stirred at 80 C. for 24 hours. After completion of the reaction (as detected by TLC), acetone was evaporated. The residue was purified by column chromatography, thereby obtaining compound D as a white solid.
<1-5> General Procedure for the Synthesis of tetrakis(hydroxyethoxymethyl)cavitand (Step e of FIG. 1B)
(19) NaH was dissolved in dry DMF while stirring, and compound D (0.8 mmol) was added to the stirred suspension. To the resultant solution, ethylene glycol was added, and the mixture was continuously stirred at room temperature for 30 minutes. The reaction mixture was transferred to an oil bath preheated at 80 C. and continuously stirred for 24 hours. After completion of the reaction (as detected by TLC), the reaction mixture was extracted using ethyl acetate and washed with water (230 mL) and brine. An organic layer was dried with anhydrous Na.sub.2SO.sub.4 and concentrated using a rotary evaporator. The residue was purified by column chromatography, thereby obtaining compound E as a white solid.
<1-6> General Procedure for Glycosylation Reaction (Step f of FIG. 1A and FIG. 1B)
(20) Glycosylation was carried out according to the synthesis method (Nat. Methods 2010, 7. 1003.) written by P. S. Chae et al. To a solution in which an alcohol derivative (compound D or E) was stirred in CH.sub.2Cl.sub.2 (15 mL), 2,4,6-collidine (maltose (3.0 equiv.) or glucose (6.0 equiv.)) was added. AgOTf (maltose (5.0 equiv.) or glucose (10.0 equiv.)) was added to the resultant mixture at 0 C. After stirring for 10 minutes, a solution of perbenzoylated maltosylbromide (5.0 equiv. or 10.0 equiv.) dissolved in CH.sub.2Cl.sub.2 was slowly added thereto. The reaction was carried out for 30 minutes while stirring. After completion of the reaction (as detected by TLC), pyridine was added to the reaction mixture, and the mixture was diluted with CH.sub.2Cl.sub.2 (20 mL) before being filtered over Celite. A filtrate was washed with a 1 M aqueous Na.sub.2S.sub.2O.sub.3 solution (40 mL), a 0.1 M aqueous HCl solution (40 mL), and brine (340 mL). Then, an organic layer was dried with anhydrous Na.sub.2SO.sub.4, and a solvent was removed using a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc/hexane), thereby obtaining a glycosylated compound as a glassy solid.
<1-7> General Procedure for De-Protection Reaction (Step g of FIG. 1A and FIG. 1B)
(21) De-protection was carried out according to the synthesis method (Nat. Methods 2010, 7. 1003.) written by P. S. Chae et al. De-O-benzoylation was carried out under Zempln's conditions. An O-protected compound was dissolved in anhydrous CH.sub.2Cl.sub.2 and then MeOH was slowly added thereto until precipitation continuously appeared. To the reaction mixture, a methanolic solution of 0.5 M NaOMe was added such that the final concentration was 0.05 M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized with an Amberlite IR-120 (H.sup.+ form) resin. The resin was removed by filtration, a filtrate was washed with MeOH, and a solvent was removed from the filtrate in vacuo. The residue was recrystallized using CH.sub.2Cl.sub.2/MeOH/diethyl ether, thereby obtaining a purer de-O-benzolyated compound as a white solid. The compounds thus obtained are RMAs which are the compounds of the present invention.
<Preparation Example 1> Synthesis of RMA-C3
<1-1> Synthesis of Compound A1
(22) According to the general procedure for the synthesis of octol-resorcin[4]arene in Example 1-1, compound A1 was synthesized in 80% yield.
<1-2> Synthesis of Compound B1
(23) According to the general procedure for the synthesis of tetramethylcavitand in Example 1-2, compound B1 was synthesized in 80% yield.
<1-3> Synthesis of Compound C1
(24) According to the general procedure for the synthesis of tetrakis(bromomethyl)cavitand in Example 1-3, compound C1 was synthesized in 81% yield.
<1-4> Synthesis of Compound D1
(25) According to the general procedure for the synthesis of tetrakis(hydroxymethyl)cavitand in Example 1-4, compound D1 was synthesized in 70% yield.
<1-5> Synthesis of RMA-C3a
(26) According to the general procedure for a glycosylation reaction in Example 1-6, RMA-C3a was synthesized in 35% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.07 (d, J=7.2 Hz, 8H), 8.01 (d, J=7.1 Hz, 8H), 7.91-7.85 (m 12H), 7.75-7.71 (m, 20H), 7.60 (d, J=7.2 Hz, 8H), 7.59-7.48 (m, 28H), 7.47-7.31 (m, 32H), 7.29-7.15 (m, 20H), 6.94 (s, 4H), 6.09 (t, J=10.0 Hz, 4H), 5.81-5.60 (m, 16H), 5.42 (d, J=7.2 Hz, 2H), 5.31-5.19 (m, 8H), 4.82 (d, J=10.2, 2H), 4.71-4.59 (m, 6H), 4.51-4.31 (m, 20H), 4.23-4.18 (m, 4H), 4.15-3.91 (m, 6H), 2.23-2.17 (m, 8H), 0.83 (t, J=7.1 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 171.2, 166.1, 165.8, 165.6, 165.5, 165.2, 165.1, 154.2, 154.1, 137.7, 137.5, 133.3, 133.2, 133.1, 130.0, 129.9, 129.8, 129.6, 129.3, 129.2, 129.0, 128.8, 128.6, 128.4, 128.2, 128.1, 122.7, 120.7, 99.9, 99.3, 77.4, 75.4, 73.2, 73.1, 72.0, 71.0, 70.0, 69.2, 69.0, 63.5, 62.4, 61.3, 60.4, 53.5, 38.5, 22.9, 21.1, 14.2, 12.3.
<1-6> Synthesis of RMA-C3
(27) According to the general procedure for a de-protection reaction in Example 1-7, RMA-C3 was synthesized in 85% yield. .sup.1H NMR (400 MHz, CD.sub.3OD): 7.32 (s, 4H), 5.89 (d, J=7.8 Hz, 4H), 5.20 (s, 5H), 4.72-4.57 (m, 10H), 4.55-4.32 (m, 12H), 3.98-3.81 (m, 16H), 3.79-3.68 (m, 12H), 3.67-3.58 (m, 16H), 3.57-5.43 (m, 8H), 3.29-3.19 (m, 4H), 2.45-2.29 (m, 8H), 0.99 (t, J=6.8 Hz, 12H); .sup.13C NMR (100 MHz, CD.sub.3OD): 155.7, 139.4, 103.0, 81.4 77.8, 75.1, 74.9, 74.2, 71.6, 64.1, 62.8, 62.4, 33.1, 22.2, 14.5. MS (MALDI-TOF): calcd. for C.sub.92H.sub.128O.sub.52 [M+Na].sup.+ 2087.7264, found 2087.7190.
<Preparation Example 2> Synthesis of Compound RMA-C4
<2-1> Synthesis of Compound A2
(28) According to the general procedure for the synthesis of octol-resorcin[4]arene in Example 1-1, compound A2 was synthesized in 81% yield.
<2-2> Synthesis of Compound B2
(29) According to the general procedure for the synthesis of tetramethylcavitand in Example 1-2, compound B2 was synthesized in 82% yield.
<2-3> Synthesis of Compound C2
(30) According to the general procedure for the synthesis of tetrakis(bromomethyl)cavitand in Example 1-3, compound C2 was synthesized in 83% yield.
<2-4> Synthesis of Compound D2
(31) According to the general procedure for the synthesis of tetrakis(hydroxymethyl)cavitand in Example 1-4, compound D2 was synthesized in 72% yield.
<2-5> Synthesis of RMA-C4a
(32) According to the general procedure for a glycosylation reaction in Example 1-6, RMA-C4a was synthesized in 35% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.06 (d, J=7.2 Hz, 8H), 8.01 (d, J=7.1 Hz, 8H), 7.89-7.79 (m 12H), 7.74-7.69 (m, 20H), 7.60 (d, J=7.2 Hz, 8H), 7.58-7.48 (m, 28H), 7.47-7.30 (m, 32H), 7.30-7.16 (m, 20H), 6.94 (s, 4H), 6.10 (t, J=10.0 Hz, 4H), 5.80-5.61 (m, 12H), 5.42 (d, J=7.2 Hz, 2H), 5.31-5.19 (m, 8H), 4.82 (d, J=10.2 Hz, 2H), 4.71-4.59 (m, 6H), 4.50-4.29 (m, 20H), 4.25-4.19 (m, 4H), 4.15-3.89 (m, 6H), 2.20-2.16 (m, 8H), 1.65 (s, 4H), 1.31-1.17 (m, 8H), 0.92 (t, J=7.1 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 171.1, 166.2, 165.9, 165.7, 165.6, 165.3, 165.2, 154.3, 154.1, 137.9, 137.8, 133.7, 133.5, 133.3, 133.2, 130.1, 130.0, 129.9, 129.7, 129.4, 129.2, 129.1, 128.9, 128.7, 128.5, 128.3, 128.2, 122.7, 120.9, 100.0, 99.4, 96.6, 73.9, 73.3, 72.1, 71.1, 70.1, 69.3, 69.1, 66.0, 63.6, 62.5, 60.6, 36.5, 32.0, 21.2, 21.1, 15.4, 14.4, 14.3.
<2-6> Synthesis of RMA-C4
(33) According to the general procedure for a de-protection reaction in Example 1-7, RMA-C4 was synthesized in 85% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 7.62 (s, 4H), 5.89 (d, J=7.0 Hz, 4H), 5.56-5.41 (m, 8H), 5.01-4.81 (m, 16H), 4.71-4.47 (m, 8H), 4.37-4.21 (m, 12H), 3.82-3.68 (m, 4H), 3.67-3.54 (m, 8H), 3.52-5.43 (m, 8H), 3.32-3.19 (m, 12H), 3.16-3.06 (m, 4H), 2.96-2.88 (m, 4H), 2.42-2.29 (m, 8H), 1.36-1.27 (m, 8H), 0.99 (t, J=7.4 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 153.7, 153.4, 137.7, 123.8, 102.4, 100.8, 79.6, 76.4, 75.3, 73.4, 73.3, 72.7, 72.4, 69.9, 60.8, 48.6, 39.9, 31.2, 20.6, 13.9; MS (MALDI-TOF): calcd. for C.sub.96H.sub.136O.sub.52 [M+Na].sup.+ 2143.7890, found 2143.7869.
<Preparation Example 3> Synthesis of RMA-C5
<3-1> Synthesis of Compound A3
(34) According to the general procedure for the synthesis of octol-resorcin[4]arene in Example 1-1, compound A3 was synthesized in 84% yield.
<3-2> Synthesis of Compound B3
(35) According to the general procedure for the synthesis of tetramethylcavitand in Example 1-2, compound B3 was synthesized in 84% yield.
<3-3> Synthesis of Compound C3
(36) According to the general procedure for the synthesis of tetrakis(bromomethyl)cavitand in Example 1-3, compound C.sub.3 was synthesized in 85% yield.
<3-4> Synthesis of Compound D3
(37) According to the general procedure for the synthesis of tetrakis(hydroxymethyl)cavitand in Example 1-4, compound D3 was synthesized in 75% yield.
<3-5> Synthesis of RMA-C5a
(38) According to the general procedure for a glycosylation reaction in Example 1-6, RMA-C5a was synthesized in 38% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.07 (d, J=7.2 Hz, 8H), 7.97 (d, J=7.1 Hz, 8H), 7.90-7.79 (m, 8H), 7.78-7.68 (m, 20H), 7.61 (d, J=7.2 Hz, 8H), 7.58-7.49 (m, 28H), 7.48-7.30 (m, 32H), 7.29-7.16 (m, 20H), 6.95 (s, 4H), 6.11 (t, J=10.0 Hz, 4H), 5.82-5.59 (m, 12H), 5.41 (d, J=7.2 Hz, 2H), 5.31-5.19 (m, 8H), 4.82 (d, J=10.2 Hz, 2H), 4.76-4.61 (m, 6H), 4.54-4.29 (m, 20H), 4.25-4.19 (m, 4H), 4.12-3.92 (m, 6H), 2.22-1.97 (m, 8H), 1.52-1.15 (m, 16H), 0.92 (t, J=7.1 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.1, 165.8, 165.6, 165.5, 165.2, 165.1, 154.2, 154.0, 137.8, 137.7, 133.5, 133.4, 133.2, 133.1, 130.0, 129.9, 129.8, 129.6, 129.3, 129.1, 129.0, 128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 122.6, 120.7, 99.9, 99.2, 96.5, 75.4, 73.1, 73.0, 72.0, 71.0, 69.9, 69.2, 69.0, 63.5, 62.3, 61.2, 60.4, 53.5, 36.7, 30.0, 29.6, 22.9, 14.2.
<3-6> Synthesis of RMA-C5
(39) According to the general procedure for a de-protection reaction in Example 1-7, RMA-C5 was synthesized in 87% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 7.60 (s, 4H), 5.89 (d, J=6.9 Hz, 4H), 5.54-5.41 (m, 8H), 5.10-4.91 (m, 16H), 4.67-4.43 (m, 8H), 4.37-4.19 (m, 12H), 3.82-3.68 (m, 4H), 3.67-3.54 (m, 8H), 3.50-5.41 (m, 8H), 3.31-3.19 (m, 12H), 3.10-3.01 (m, 4H), 2.96-2.89 (m, 4H), 2.41-2.22 (m, 8H), 1.56-1.21 (m, 16H), 0.91 (t, J=7.2 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 153.6, 153.3, 137.6, 123.7, 102.4, 100.7, 79.5, 76.4, 75.2, 73.3, 73.2, 72.6, 72.4, 69.9, 60.8, 60.6, 39.1, 29.9, 28.9, 22.1, 14.0; MS (MALDI-TOF): calcd. for C.sub.100H.sub.114O.sub.52 [M+Na].sup.+ 2199.8516, found 2199.8540.
<Preparation Example 4> Synthesis of RMA-C3E
<4-1> Synthesis of Compound A1
(40) According to the general procedure for the synthesis of octol-resorcin[4]arene in Example 1-1, compound A1 was synthesized in 80% yield.
<4-2> Synthesis of Compound B1
(41) According to the general procedure for the synthesis of tetramethylcavitand in Example 1-2, compound B1 was synthesized in 80% yield.
<4-3> Synthesis of Compound C1
(42) According to the general procedure for the synthesis of tetrakis(bromomethyl)cavitand in Example 1-3, compound C1 was synthesized in 81% yield.
<4-4> Synthesis of Compound E1
(43) According to the general procedure for the synthesis of tetrakis(hydroxyethoxymethyl)cavitand in Example 1-5, compound E1 was synthesized in 62% yield.
<4-5> Synthesis of RMA-C3Ea
(44) According to the general procedure for a glycosylation reaction in Example 1-6, RMA-C3Ea was synthesized in 40% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.07 (d, J=7.2 Hz, 8H), 7.96 (d, J=7.1 Hz, 8H), 7.85-7.59 (m, 32H), 7.56 (d, J=7.2 Hz, 8H), 7.49-7.32 (m, 32H), 7.31-7.20 (m, 28H), 7.19-7.04 (m, 20H), 6.92 (s, 4H), 6.02 (t, J=10.0 Hz, 4H), 5.70-5.49 (m, 14H), 5.31-5.13 (m, 8H), 4.92-4.59 (m, 10H), 4.57-4.41 (m, 6H), 4.40-4.35 (m, 8H), 4.25-4.19 (m, 4H), 4.17-3.90 (m, 6H), 3.88-3.79 (m, 4H), 3.62-3.49 (m, 4H), 3.47-3.30 (m, 4H), 2.19-2.12 (m, 8H), 1.65 (s, 4H), 1.31-1.17 (m, 8H), 0.86 (t, J=7.1 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 171.2, 166.3, 165.9, 165.8, 165.5, 165.3, 165.2, 165.1, 154.2, 154.1, 154.0, 153.9, 153.6, 138.0, 137.8, 133.7, 137.4, 137.2, 133.6, 133.5, 133.2, 133.1, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 129.2, 129.0, 128.8, 128.6, 128.4, 128.3, 128.2, 124.3, 124.1, 124.0, 123.4, 120.4, 101.1, 101.0, 99.5, 99.2, 96.5, 75.1, 73.2, 73.0, 72.2, 71.0, 69.7, 69.2, 63.6, 62.6, 60.5, 53.6 38.8, 23.2, 21.2, 14.3, 12.5, 10.6.
<4-6> Synthesis of RMA-C3E
(45) According to the general procedure for a de-protection reaction in Example 1-7, RMA-C.sub.3E was synthesized in 85% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO) 7.58 (s, 4H), 5.89 (d, J=6.9 Hz, 4H), 5.55-5.40 (m, 8H), 5.10-4.89 (m, 16H), 4.61-4.38 (m, 8H), 4.31-4.19 (m, 8H), 4.09-3.97 (m, 16H), 3.87-3.78 (m, 4H), 3.75-3.52 (m, 8H), 3.51-5.39 (m, 16H), 3.21-3.12 (m, 16H), 3.16-2.94 (m, 8H), 2.44-2.31 (m, 8H), 0.94 (t, J=7.4 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 153.4, 153.3, 137.4, 123.7, 102.7, 100.9, 79.5, 76.4, 75.1, 73.3, 73.0, 72.4, 69.9, 60.8, 60.5, 54.8, 48.6, 38.9, 22.4, 12.2; MS (MALDI-TOF): calcd. for C.sub.100H.sub.114O.sub.56 [M+Na].sup.+ 2263.8312, found 2263. 8523.
<Preparation Example 5> Synthesis of RMA-C4E
<5-1> Synthesis of Compound A2
(46) According to the general procedure for the synthesis of octol-resorcin[4]arene in Example 1-1, compound A2 was synthesized in 81% yield.
<5-2> Synthesis of Compound B2
(47) According to the general procedure for the synthesis of tetramethylcavitand in Example 1-2, compound B2 was synthesized in 82% yield.
<5-3> Synthesis of Compound C2
(48) According to the general procedure for the synthesis of tetrakis(bromomethyl)cavitand in Example 1-3, compound C2 was synthesized in 83% yield.
<5-4> Synthesis of Compound E2
(49) According to the general procedure for the synthesis of tetrakis(hydroxyethoxymethyl)cavitand in Example 1-5, compound E2 was synthesized in 64% yield.
<5-5> Synthesis of RMA-C4Ea
(50) According to the general procedure for a glycosylation reaction in Example 1-6, RMA-C4Ea was synthesized in 42% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.07 (d, J=7.2 Hz, 8H), 7.97 (d, J=7.1 Hz, 8H), 7.97-7.79 (m, 32H), 7.65 (d, J=7.2 Hz, 8H), 7.51-7.42 (m, 32H), 7.41-7.28 (m, 28H), 7.27-7.14 (m, 20H), 6.97 (s, 4H), 6.09 (t, J=10.0 Hz, 4H), 5.81-5.49 (m, 14H), 5.38-5.21 (m, 8H), 4.93-4.65 (m, 10H), 4.64-4.59 (m, 6H), 4.56-4.32 (m, 8H), 4.30-4.18 (m, 4H), 4.17-3.97 (m, 16H), 3.96-3.79 (m, 4H), 3.69-3.57 (m, 4H), 3.51-3.36 (m, 4H), 2.21-2.16 (m, 8H), 1.42-1.25 (m, 8H), 0.97 (t, J=7.1 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 171.2, 166.2, 165.9, 165.7, 165.5, 165.2, 165.1, 165.0, 154.2, 154.0, 138.0, 137.6, 133.6, 133.5, 133.4, 133.3, 133.1, 133.0, 130.1, 129.9, 129.8, 129.7, 129.6, 129.5, 129.3, 129.1, 129.0, 128.7, 128.5, 128.4, 128.2, 128.1, 123.6, 120.1, 101.0, 99.9, 98.1, 96.4, 75.0, 73.1, 72.9, 72.2, 71.0, 69.9, 69.4, 69.1, 63.4, 62.5, 60.5, 51.1, 36.3, 32.1, 21.1, 20.9, 14.2.
<5-6> Synthesis of RMA-C4E
(51) According to the general procedure for a de-protection reaction in Example 1-7, RMA-C.sub.4E was synthesized in 87% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 7.59 (s, 4H), 5.89 (d, J=6.9 Hz, 4H), 5.54-5.38 (m, 8H), 5.09-4.78 (m, 16H), 4.67-4.38 (m, 8H), 4.31-4.19 (m, 8H), 4.09-3.97 (m, 16H), 3.88-3.79 (m, 4H), 3.75-3.52 (m, 8H), 3.51-3.37 (m, 16H), 3.21-3.09 (m, 16H), 3.06-2.94 (m, 8H), 1.41-1.23 (m, 8H), 0.99 (t, J=7.4 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 153.3, 137.5, 124.1, 121.9, 102.6, 100.7, 79.5, 76.3, 75.1, 73.3, 73.2, 72.9, 72.4, 69.9, 69.4, 67.6, 60.8, 60.5, 36.2, 31.1, 20.4, 13.8; MS (MALDI-TOF): calcd. for C.sub.104H.sub.152O.sub.56 [M+Na].sup.+ 2319.8938, found 2319.8875.
<Preparation Example 6> Synthesis of RMA-C5E
<6-1> Synthesis of Compound A3
(52) According to the general procedure for the synthesis of octol-resorcin[4]arene in Example 1-1, compound A3 was synthesized in 83% yield.
<6-2> Synthesis of Compound B3
(53) According to the general procedure for the synthesis of tetramethylcavitand in Example 1-2, compound B3 was synthesized in 84% yield.
<6-3> Synthesis of Compound C3
(54) According to the general procedure for the synthesis of tetrakis(bromomethyl)cavitand in Example 1-3, compound C.sub.3 was synthesized in 85% yield.
<6-4> Synthesis of Compound E3
(55) According to the general procedure for the synthesis of tetrakis(hydroxyethoxymethyl)cavitand in Example 1-5, compound E3 was synthesized in 64% yield.
<6-5> Synthesis of RMA-C5Ea
(56) According to the general procedure for a glycosylation reaction in Example 1-6, RMA-C5Ea was synthesized in 45% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.07 (d, J=7.2 Hz, 8H), 7.98 (d, J=7.1 Hz, 8H), 7.91-7.73 (m, 32H), 7.65 (d, J=7.2 Hz, 8H), 7.51-7.40 (m, 32H), 7.39-7.28 (m, 28H), 7.27-7.14 (m, 20H), 6.97 (s, 4H), 6.10 (t, J=10.0 Hz, 4H), 5.81-5.49 (m, 14H), 5.38-5.21 (m, 8H), 4.93-4.65 (m, 10H), 4.64-4.59 (m, 6H), 4.56-4.32 (m, 8H), 4.30-4.19 (m, 6H), 4.17-3.97 (m, 16H), 3.96-3.79 (m, 4H), 3.70-3.59 (m, 4H), 3.53-3.38 (m, 4H), 2.24-2.17 (m, 8H), 1.51-1.19 (m, 16H), 0.92 (t, J=7.1 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 171.3, 166.3, 165.9, 165.8, 165.6, 165.2, 165.1, 154.0, 137.8, 133.6, 133.5, 133.4, 133.3, 133.2, 133.1, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.4, 129.2, 129.0, 128.8, 128.7, 128.6, 128.5, 128.3, 128.2, 123.7, 120.4, 101.0, 99.4, 96.5, 75.0, 73.0, 72.9, 72.5, 71.0, 70.0, 69.9, 69.5, 69.2, 63.6, 63.2, 62.6, 60.5, 53.6, 36.8, 30.2, 29.9, 22.9, 21.2, 20.9, 14.3.
<6-6> Synthesis of RMA-C5E
(57) According to the general procedure for a de-protection reaction in Example 1-7, RMA-C5E was synthesized in 88% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 7.58 (s, 4H), 5.89 (d, J=6.9 Hz, 4H), 5.15-4.98 (m, 8H), 4.95-4.85 (m, 8H), 4.67-4.46 (m, 16H), 4.34-4.12 (m, 16H), 3.92-3.78 (m, 4H), 3.74-3.65 (m, 4H), 3.64-3.51 (m, 16H), 3.50-3.41 (m, 16H), 3.28-3.18 (m, 8H), 3.12-2.98 (m, 8H), 2.41-2.29 (m, 8H), 1.51-1.19 (m, 16H), 0.91 (t, J=7.4 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 153.4, 137.6, 124.2, 102.7, 100.8, 99.4, 79.6, 76.4, 75.2, 73.5, 73.3, 73.0, 69.9, 69.5, 67.8, 62.3, 60.8, 60.5, 36.7, 30.0, 29.0, 22.2, 14.2; MS (MALDI-TOF): calcd. for C.sub.100H.sub.160O.sub.56 [M+Na].sup.+ 2375.9564, found 2375.9355.
<Example 2> Method of Synthesizing Resorcinarene-Based Glucosides (RGAs)
(58) A synthesis scheme of RGAs is shown in
<2-1> General Procedure for the Synthesis of octa-2-hydroxyethoxy Tetraalkylresorcinarene (Step h of FIG. 2)
(59) 1,3-bis(2-hydroxyethoxy) benzene (1.0 g, 0.005 mol) was added to a mixed solution of 1.5 ml of ethanol and 2 ml of concentrated HCl. To the resultant mixture, an aldehyde (0.005 mol) was added at 0 C. over 15 minutes. After stirring at room temperature for 15 minutes, the reaction mixture was transferred to an oil bath preheated at 90 C. and heated for 48 hours while stirring. After completion of the reaction (as detected by TLC), the reaction mixture was extracted using ethyl acetate and washed with water (230 mL) and brine. An organic layer was dried with anhydrous Na.sub.2SO.sub.4 and concentrated using a rotary evaporator. The residue was purified by column chromatography, thereby obtaining compound F as a white crystal solid.
<2-2> General Procedure for Glycosylation Reaction (Step f of FIG. 2)
(60) Glycosylation was carried out according to the synthesis method (Nat. Methods 2010, 7. 1003.) written by P. S. Chae et al. To a solution in which an alcohol derivative (compound F) was stirred in CH.sub.2Cl.sub.2 (15 mL), 2,4,6-collidine (maltose (3.0 equiv.) or glucose (6.0 equiv.)) was added. AgOTf (maltose (5.0 equiv.) or glucose (10.0 equiv.)) was added to the resultant mixture at 0 C. After stirring for 10 minutes, a solution of perbenzoylated maltosylbromide (5.0 equiv. or 10.0 equiv.) dissolved in CH.sub.2Cl.sub.2 was slowly added thereto. The reaction was carried out for 30 minutes while stirring. After completion of the reaction (as detected by TLC), pyridine was added to the reaction mixture, and the mixture was diluted with CH.sub.2Cl.sub.2 (20 mL) before being filtered over Celite. A filtrate was washed with a 1 M aqueous Na.sub.2S.sub.2O.sub.3 solution (40 mL), a 0.1 M aqueous HCl solution (40 mL), and brine (340 mL). An organic layer was dried with anhydrous Na.sub.2SO.sub.4, and a solvent was removed using a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc/hexane), thereby obtaining a maltose-glycosylated compound as a glassy solid.
<2-3> General Procedure for De-Protection Reaction (Step g of FIG. 2)
(61) De-protection was carried out according to the synthesis method (Nat. Methods 2010, 7. 1003.) written by P. S. Chae et al. De-O-benzoylation was carried out under Zempln's conditions. An O-protected compound was dissolved in anhydrous CH.sub.2Cl.sub.2 and then MeOH was slowly added thereto until precipitation continuously appeared. To the reaction mixture, a methanolic solution of 0.5 M NaOMe was added such that the final concentration was 0.05 M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized with an Amberlite IR-120 (H.sup.+ form) resin. The resin was removed by filtration, a filtrate was washed with MeOH, and a solvent was removed from the filtrate in vacuo. The residue was recrystallized using CH.sub.2Cl.sub.2/MeOH/diethyl ether, thereby obtaining a purer de-O-benzolyated compound as a white solid. The compounds thus obtained are RGAs which are the compounds of the present invention.
<Preparation Example 7> Synthesis of RGA-8
<7-1> Synthesis of Compound F1
(62) According to the general procedure for the synthesis of octa-2-hydroxyethoxy tetraalkylresorcinarene in Example 2-1, compound F1 was synthesized in 40% yield.
<7-2> Synthesis of RGA-8a
(63) According to the general procedure for a glycosylation reaction in Example 2-2, RGA-8a was synthesized in 47% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.20-7.95 (m, 8H), 7.94-7.89 (m, 8H), 7.88-7.77 (m, 22H), 7.76-7.71 (m, 12H), 7.70-7.60 (m, 16H), 7.56-7.49 (m, 16H), 7.48-7.39 (m, 20H), 7.38-7.32 (m, 32H), 7.31-7.12 (m, 10H), 7.11-6.90 (m, 16H), 6.21-5.92 (m, 8H), 5.91-5.73 (m, 12H), 5.72-5.48 (m, 8H), 4.78-4.59 (m, 16H), 4.58-4.38 (m, 16H), 4.37-4.09 (m, 16H), 4.02-3.55 (m, 10H), 3.51-3.15 (m, 8H), 1.81-1.41 (m, 4H), 1.24-1.12 (m, 40H), 0.83 (t, J=7.6 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 166.0, 165.4, 165.3, 133.5, 133.3, 130.0, 129.9, 129.8, 129.2, 129.1, 128.5, 101.5, 73.4, 72.4, 72.2, 72.0, 70.0, 68.6, 63.1, 60.6, 32.4, 30.4, 29.8, 23.0, 21.3, 14.4.
<7-3> Synthesis of RGA-8
(64) According to the general procedure for a de-protection reaction in Example 2-3, RGA-8 was synthesized in 87% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 6.67 (s, 4H), 6.65 (s, 4H), 5.10-4.79 (m, 24H), 4.61-4.38 (m, 12H), 4.21-4.19 (m, 8H), 4.18-3.92 (m, 12H), 3.91-3.82 (m, 8H), 3.81-3.60 (m, 16H), 3.58-3.39 (m, 8H), 3.38-3.22 (m, 16H), 3.21-3.13 (m, 16H), 3.05-2.92 (m, 4H), 1.81-1.59 (m, 8H), 1.20 (s, 40H), 0.83 (t, J=6.4 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 154.5, 103.2, 103.1, 76.4, 73.4, 70.0, 67.8, 67.4, 61.0, 31.3, 29.3, 28.6, 27.4, 22.0, 13.8; MS (MALDI-TOF): calcd. for C.sub.120H.sub.192O.sub.56 [M+Na].sup.+ 2552.2068, found 2552.1887.
<Preparation Example 8> Synthesis of RGA-9
<8-1> Synthesis of Compound F2
(65) According to the general procedure for the synthesis of octa-2-hydroxyethoxy tetraalkylresorcinarene in Example 2-1, compound F2 was synthesized in 42% yield.
<8-2> Synthesis of RGA-9a
(66) According to the general procedure for a glycosylation reaction in Example 2-2, RGA-9a was synthesized in 47% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.21-7.95 (m, 8H), 7.94-7.87 (m, 8H), 7.86-7.77 (m, 22H), 7.76-7.71 (m, 12H), 7.70-7.61 (m, 16H), 7.55-7.48 (m, 16H), 7.47-7.38 (m, 20H), 7.37-7.32 (m, 32H), 7.31-7.11 (m, 10H), 7.10-6.90 (m, 16H), 6.20-5.91 (m, 8H), 5.91-5.71 (m, 12H), 5.70-5.48 (m, 8H), 4.78-4.59 (m, 16H), 4.58-4.38 (m, 16H), 4.37-4.09 (m, 12H), 4.02-3.55 (m, 10H), 3.51-3.11 (m, 8H), 1.72-1.51 (m, 4H), 1.24-1.08 (m, 48H), 0.84 (t, J=7.6 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 166.0, 165.2, 133.4, 133.3, 130.0, 129.9, 129.8, 129.7, 129.2, 129.1, 128.5, 101.4, 73.4, 72.4, 72.2, 72.0, 69.9, 68.6, 63.1, 53.6, 32.2, 30.5, 30.1, 29.9, 22.9, 14.3.
<8-3> Synthesis of RGA-9
(67) According to the general procedure for a de-protection reaction in Example 2-3, RGA-9 was synthesized in 88% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 6.67 (s, 4H), 6.48 (s, 4H), 5.11-4.74 (m, 24H), 4.61-4.37 (m, 12H), 4.36-4.17 (m, 8H), 4.16-3.97 (m, 12H), 3.96-3.84 (m, 8H), 3.83-3.60 (m, 16H), 3.59-3.39 (m, 8H), 3.26-3.16 (m, 16H), 3.15-2.89 (m, 16H), 1.81-1.59 (m, 8H), 1.20 (s, 48H), 0.83 (t, J=6.4 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 154.5, 103.2, 103.1, 76.6, 73.4, 70.0, 67.4, 61.0, 31.2, 29.3, 28.9, 28.7, 27.4, 22.0, 13.8; MS (MALDI-TOF): calcd. for C.sub.124H.sub.200O.sub.56 [M+Na].sup.+ 2608.2694, found 2608.2463.
<Preparation Example 9> Synthesis of RGA-10
<9-1> Synthesis of Compound F3
(68) According to the general procedure for the synthesis of octa-2-hydroxyethoxy tetraalkylresorcinarene in Example 2-1, compound F3 was synthesized in 44% yield.
<9-2> Synthesis of RGA-10a
(69) According to the general procedure for a glycosylation reaction in Example 2-2, RGA-10a was synthesized in 46% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.17-7.92 (m, 8H), 7.91-7.85 (m, 8H), 7.84-7.75 (m, 22H), 7.74-7.70 (m, 12H), 7.69-7.59 (m, 16H), 7.54-7.47 (m, 16H), 7.46-7.36 (m, 20H), 7.35-7.30 (m, 32H), 7.29-7.14 (m, 10H), 7.13-6.88 (m, 16H), 6.15-5.89 (m, 8H), 5.88-5.70 (m, 12H), 5.69-5.48 (m, 8H), 5.19-4.91 (m, 10H), 4.82-4.61 (m, 8H), 4.37-4.09 (m, 16H), 4.02-3.55 (m, 10H), 3.51-3.11 (m, 8H), 1.72-1.50 (m, 4H), 1.24-1.08 (m, 56H), 0.83 (t, J=7.6 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 166.0, 165.9, 165.4, 165.3, 133.4, 133.3, 130.0, 129.9, 129.8, 129.7, 129.2, 129.1, 128.5, 101.5, 72.4, 72.2, 72.0, 70.0, 68.6, 63.1, 53.6, 35.3, 34.9, 32.2, 30.5, 30.2, 29.7, 28.4, 22.9, 22.2, 14.3.
<9-3> Synthesis of RGA-10
(70) According to the general procedure for a de-protection reaction in Example 2-3, RGA-10 was synthesized in 88% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 6.67 (s, 4H), 6.48 (s, 4H), 4.98-4.75 (m, 24H), 4.55-4.36 (m, 12H), 4.34-4.17 (m, 8H), 4.16-4.02 (m, 12H), 4.01-3.78 (m, 12H), 3.77-3.60 (m, 16H), 3.57-3.39 (m, 8H), 3.25-3.16 (m, 16H), 3.15-2.89 (m, 16H), 1.82-1.59 (m, 8H), 1.20 (s, 56H), 0.83 (t, J=7.0 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 154.5, 103.2, 103.1, 99.6, 76.7, 73.4, 70.0, 67.8, 67.4, 61.1, 35.0, 34.0, 31.2, 29.4, 29.1, 29.0, 28.6, 27.4, 22.0, 13.8; MS (MALDI-TOF): calcd. for C.sub.128H.sub.208O.sub.56 [M+Na].sup.+ 2664.3320, found 2664.3345.
<Preparation Example 10> Synthesis of RGA-11
<10-1> Synthesis of Compound F4
(71) According to the general procedure for the synthesis of octa-2-hydroxyethoxy tetraalkylresorcinarene in Example 2-1, compound F4 was synthesized in 44% yield.
<10-2> Synthesis of RGA-11a
(72) According to the general procedure for a glycosylation reaction in Example 2-2, RGA-la was synthesized in 45% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.20-7.96 (m, 8H), 7.95-7.87 (m, 8H), 7.86-7.78 (m, 22H), 7.77-7.72 (m, 12H), 7.71-7.61 (m, 16H), 7.57-7.50 (m, 16H), 7.49-7.40 (m, 20H), 7.39-7.33 (m, 32H), 7.32-7.14 (m, 10H), 7.13-6.90 (m, 16H), 6.18-5.87 (m, 8H), 5.86-5.69 (m, 12H), 5.68-5.43 (m, 8H), 5.21-4.79 (m, 8H), 4.78-4.59 (m, 10H), 4.58-4.38 (m, 8H), 4.37-4.09 (m, 16H), 4.08-3.55 (m, 16H), 3.51-3.11 (m, 4H), 1.76-1.56 (m, 4H), 1.49-1.08 (m, 64H), 0.84 (t, J=7.6 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 165.4, 133.3, 130.0, 129.9, 129.7, 129.3, 129.1, 128.5, 101.3, 73.5, 72.5, 72.3, 72.1, 69.9, 68.7, 63.1, 53.6, 32.2, 30.4, 30.0, 29.9, 22.9, 14.3.
<10-3> Synthesis of RGA-11
(73) According to the general procedure for a de-protection reaction in Example 2-3, RGA-11 was synthesized in 87% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 6.67 (s, 4H), 6.49 (s, 4H), 5.57-4.79 (m, 24H), 4.61-4.38 (m, 12H), 4.36-4.20 (m, 8H), 4.18-4.03 (m, 12H), 4.02-3.81 (m, 12H), 3.78-3.61 (m, 16H), 3.57-3.41 (m, 8H), 3.40-3.34 (m, 16H), 3.15-2.89 (m, 16H), 1.82-1.59 (m, 8H), 1.21 (s, 64H), 0.83 (t, J=6.8 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 154.6, 154.5, 125.7, 125.3, 103.2, 103.1, 99.6, 76.7, 73.4, 70.0, 68.3, 67.8, 67.4, 61.1, 35.1, 34.0, 31.2, 29.4, 29.2, 29.0, 28.7, 27.4, 22.0, 13.8; MS (MALDI-TOF): calcd. for C.sub.132H.sub.216O.sub.56 [M+Na].sup.+ 2720.3946, found 2720.4314.
<Preparation Example 11> Synthesis of RGA-12
<11-1> Synthesis of Compound F5
(74) According to the general procedure for the synthesis of octa-2-hydroxyethoxy tetraalkylresorcinarene in Example 2-1, compound F5 was synthesized in 46% yield.
<11-2> Synthesis of RGA-12a
(75) According to the general procedure for a glycosylation reaction in Example 2-2, RGA-12a was synthesized in 45% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.21-7.97 (m, 8H), 7.96-7.87 (m, 8H), 7.86-7.79 (m, 22H), 7.78-7.63 (m, 10H), 7.57-7.43 (m, 16H), 7.42-7.34 (m, 16H), 7.33-7.22 (m, 32H), 7.21-7.15 (m, 14H), 7.14-6.89 (m, 10H), 7.13-6.88 (m, 12H), 6.15-5.84 (m, 8H), 5.83-5.67 (m, 12H), 5.65-5.49 (m, 8H), 5.19-4.89 (m, 8H), 4.79-4.68 (m, 8H), 4.56-4.44 (m, 10H), 4.35-4.11 (m, 16H), 4.10-3.71 (m, 10H), 3.65-3.33 (m, 8H), 1.72-1.49 (m, 4H), 1.41-0.95 (m, 64H), 0.83 (t, J=7.6 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 166.0, 165.4, 165.3, 133.2, 130.0, 129.9, 129.2, 129.1, 128.5, 101.5, 73.4, 72.2, 72.0, 70.0, 63.2, 32.1, 30.4, 30.1, 30.0, 29.6, 22.9, 14.3.
<11-3> Synthesis of RGA-12
(76) According to the general procedure for a de-protection reaction in Example 2-3, RGA-12 was synthesized in 89% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 6.67 (s, 4H), 6.48 (s, 4H), 5.01-4.79 (m, 24H), 4.57-4.38 (m, 12H), 4.32-4.18 (m, 8H), 4.17-4.02 (m, 12H), 4.01-3.82 (m, 12H), 3.79-3.61 (m, 16H), 3.58-3.41 (m, 8H), 3.28-3.17 (m, 16H), 3.16-2.89 (m, 16H), 1.81-1.59 (m, 8H), 1.21 (s, 72H), 0.83 (t, J=6.8 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 154.6, 154.5, 125.7, 125.3, 103.2, 103.1, 76.7, 73.4, 70.0, 68.2, 67.4, 61.0, 35.1, 34.0, 31.2, 29.4, 29.2, 29.0, 28.6, 27.4, 22.0, 13.8; MS (MALDI-TOF): calcd. for C.sub.136H.sub.224O.sub.56 [M+Na].sup.+ 2776.4572, found 2776.4949.
<Preparation Example 12> Synthesis of RGA-13
<12-1> Synthesis of Compound F6
(77) According to the general procedure for the synthesis of octa-2-hydroxyethoxy tetraalkylresorcinarene in Example 2-1, compound F6 was synthesized in 47% yield.
<12-2> Synthesis of RGA-13a
(78) According to the general procedure for a glycosylation reaction in Example 2-2, RGA-13a was synthesized in 44% yield. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.21-7.96 (m, 10H), 7.95-7.89 (m, 8H), 7.88-7.79 (m, 22H), 7.78-7.63 (m, 8H), 7.58-7.43 (m, 12H), 7.42-7.34 (m, 18H), 7.33-7.20 (m, 32H), 7.19-7.02 (m, 14H), 7.01-6.80 (m, 10H), 6.15-5.87 (m, 8H), 5.86-5.67 (m, 12H), 5.66-5.46 (m, 8H), 5.31-4.84 (m, 8H), 4.79-4.58 (m, 8H), 4.57-4.41 (m, 8H), 4.35-4.11 (m, 12H), 4.10-3.71 (m, 10H), 3.56-3.33 (m, 8H), 1.72-1.49 (m, 4H), 1.41-0.95 (m, 72H), 0.84 (t, J=7.6 Hz, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3): 166.2, 166.0, 165.3, 133.3, 130.0, 129.9, 129.1, 128.5, 101.6, 73.4, 72.0, 69.9, 63.1, 32.1, 30.4, 30.2, 30.0, 29.6, 22.9, 14.3.
<12-3> Synthesis of RGA-13
(79) According to the general procedure for a de-protection reaction in Example 2-3, RGA-13 was synthesized in 90% yield. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): 6.67 (s, 4H), 6.47 (s, 4H), 5.01-4.79 (m, 24H), 4.61-4.39 (m, 12H), 4.34-4.20 (m, 8H), 4.17-4.01 (m, 12H), 3.99-3.82 (m, 12H), 3.79-3.61 (m, 16H), 3.58-3.41 (m, 8H), 3.25-3.17 (m, 16H), 3.16-2.95 (m, 16H), 1.81-1.59 (m, 8H), 1.20 (s, 80H), 0.83 (t, J=7.0 Hz, 12H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO): 154.6, 125.3, 103.2, 76.7, 73.4, 70.0, 68.2, 67.4, 61.1, 31.2, 29.0, 28.6, 27.4, 22.0, 13.8; MS (MALDI-TOF): calcd. for C.sub.140H.sub.232O.sub.56 [M+Na].sup.+ 2832.5198, found 2832.5007.
<Experimental Example 1> Characteristics of RGAs and RMAs
(80) To investigate the characteristics of RGAs and RMAs of Preparation Examples 1 to 12 synthesized according to the synthesis method of Examples 1 and 2, molecular weights (M.W.) and critical micelle concentrations (CMCs) of RGAs and RMAs and hydrodynamic radii (R.sub.h) of formed micelles were measured.
(81) Specifically, critical micelle concentrations (CMCs) were measured using a fluorescence dye, diphenylhexatriene (DPH), and hydrodynamic radii (R.sub.h) of micelles formed of each compound (1.0 wt %) were measured by a dynamic light scattering (DLS) experiment. Measured results are shown in Table 1 in comparison with values of DDM as an existing amphipathic molecule (detergent).
(82) TABLE-US-00001 TABLE 1 Detergent M.W. CMC (M) CMC (wt %) R.sub.h (nm) RGA-C8 2530.8 ~10 ~0.025 2.8 0.1 RGA-C9 2586.9 ~7 ~0.018 2.9 0.1 RGA-C10 2643.0 ~5 ~0.013 3.5 0.2 RGA-C11 2699.1 ~3 ~0.0081 6.8 0.2 RGA-C12 2755.2 ~2.5 ~0.0069 3.0 0.2 RGA-C13 2811.3 ~2 ~0.0056 3.7 0.1 RMA-C3 2066.0 ~30 ~0.0062 5.5 0.2 RMA-C4 2122.1 ~15 ~0.0032 7.5 0.2 RMA-C5 2178.2 6.5 0.6 RMA-C3E 2242.2 ~15 ~0.0034 3.6 0.1 RMA-C4E 2298.3 ~10 ~0.0023 4.6 0.1 RMA-C5E 2354.4 ~8 ~0.0019 5.4 0.1 DDM 510.1 170 0.0087 3.4 0.0
(83) All RGAs and RMAs exhibited CMC values of 0.001 to 0.030 mM which is significantly lower than that of DDM (0.17 mM). Therefore, RGAs and RMAs easily formed micelles even at low concentrations, and thus even a smaller amount thereof may be used to exhibit the same or higher level of effects compared to DDM. In addition, the CMC values of RGAs and RMAs were decreased with an increasing alkyl chain length, which is considered to be caused by an increase in hydrophobicity due to an extending alkyl chain length. Overall, the sizes of micelles formed by RGAs and RMAs tended to increase with an increasing alkyl chain length. On the contrary, exceptionally, the sizes of micelles formed by RGA-C12 and RGA-C13 were decreased. It was confirmed that all RGAs except RGA-C11 formed micelles of smaller sizes than those formed by RMAs, which is considered to be caused by the fact that the shape of RGAs is more conical than that of RMAs. In addition, it was confirmed that all RGAs except RGA-C11 formed micelles of similar sizes to those formed by DDM, whereas most RMAs formed micelles of larger sizes than those formed by DDM.
(84) Meanwhile, size distributions of the micelles formed by RGAs and RMAs were measured using DLS. As results, micelles of all of RGAs and RMAs were measured as forming a single population, indicating that the micelles are highly homogeneous (
(85) Based on these results, it can be confirmed that, since RGAs and RMAs of the present invention have a smaller CMC value than DDM, micelles are easily formed even upon use of a small amount of RGAs and RMAs, whereby self-assembly tendencies thereof are much greater than DDM; the sizes of the micelles formed by RGAs and RMAs vary depending on the type thereof; and the micelles formed by RGAs and RMAs are highly homogeneous.
<Experimental Example 2> Evaluation of Membrane Protein (UapA) Structure Stabilization Ability of RGAs and RMAs
(86) An experiment was carried out to measure the structural stability of a uric acid-xanthine/H.sup.+ symporter (UapA) isolated from Aspergillus nidulans caused by RGAs and RMAs. The structural stability of UapA was measured using a sulfhydryl-specific fluorophore, N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl] maleimide (CPM).
(87) Specifically, UapAG411V.sub.1-11 (hereinafter, referred to as UapA) was expressed in combination with GFP in Saccharomyces cerevisiae FGY217 strains and isolated into a sample buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, and 0.6 mM xanthine) according to the method described in the paper (Mol. Membr. Biol. 2013, 30, 32-42) written by J. Leung et al. The membranes containing UapA were diluted to a final protein concentration of 2.8 mg ml.sup.1 in PBS (pH 7.4) supplemented with either 1% DDM or 1% RMAs or RGAs. The sample was incubated while gently inverting at 4 C. for 1 hour and an insoluble material was removed by centrifugation at 15,000 g at 4 C. for 1 hour. The supernatant was injected into a Superose 6 10/300 column and eluted with an elution buffer (containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.03% DDM). The individual elution fractions were transferred to a 200 l clear 96-well plate. The GFP fluorescence of each fraction was read using an excitation wavelength of 470 nm and an emission wavelength of 512 nm to evaluate the thermal stability caused by an individual amphipathic compound and DDM.
(88) As shown in
(89) Based on these results, it can be seen that RGAs and RMAs exhibited an excellent effect of maintaining UapA extracted from a cell membrane in a structurally stable state in an aqueous solution, and thus can be effectively used to stabilize a membrane protein.
<Experimental Example 3> Evaluation of Membrane Protein (LeuT) Structure Stabilization Ability of RGAs and RMAs
(90) An experiment was carried out to measure the structural stability of a leucine transporter (LeuT) caused by RGAs and RMAs. The concentrations of individual amphipathic compounds were (a) CMC+0.04 wt % or (b) CMC+0.2 wt %, and the substrate binding characteristic of LeuT was measured by scintillation proximity assay (SPA) using [.sup.3H]-Leu. The measurement was carried out at regular intervals during 10 days of incubation at room temperature.
(91) Specifically, wild type LeuT derived from Aquifex aeolicus which is a thermophilic bacterium was purified according to the method described in the paper (Nature 1998, 392, 353-358) written by G. Deckert et al. LeuT was expressed in E. coli C41 (DE3) transformed with pET16b encoding a C-terminal 8His-tagged transporter (an expression plasmid was provided by Dr. E. Gouaux (Vollum Institute, Portland, Oreg., USA)). Briefly, the bacteria membrane was isolated and solubilized in 1% (w/v) DDM, then bound to a 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% (w/v) DDM, and 300 mM imidazole. Subsequently, the purified LeuT (about 1.5 mg/ml) was diluted in the above buffer without DDM and imidazole, but supplemented with RGAs, RMAs (RMA-C3, RMA-C4, RMA-C3E, RMA-C4E, and RMA-C5E) or DDM to a final concentration of CMC+0.04% (w/v) or CMC+0.2% (w/v). The protein samples were stored at room temperature for 10 days and centrifuged at designated time points, and protein characteristics were determined by measuring the binding capacity of [.sup.3H]-Leucine using SPA. SPA was performed with 5 l of the respective protein samples in a buffer containing 450 mM NaCl and each RGA or RMA (or DDM). Also, SPA was performed in the presence of 20 nM [.sup.3H]-Leucine and 1.25 mg/ml copper chelate (His-Tag) YSi beads (Perkin Elmer, Denmark). For the individual samples, a [.sup.3H]-Leucine binding degree was determined using a MicroB eta liquid scintillation counter (Perkin Elmer).
(92) As shown in
(93) In addition, as shown in
(94) These results suggest that the overall structure of RGAs and RMAs, particularly, the entire length thereof acts as an important factor for maintaining the structural stability of LeuT.
<Experimental Example 4> Evaluation of Membrane Protein (MelB) Structure Stabilization Ability of RGAs and RMAs
(95) RGAs were selected, and an experiment was carried out to measure the structural stability of a Salmonella typhimurium melibiose permease (MelB) caused by RGAs. A MelB protein was extracted from a membrane using RGAs or DDM, and the amount and structure of the protein thus extracted was then analyzed by performing SDS-PAGE and western blotting. In this case, the concentration of the used amphipathic compound was 1.5 wt %. The protein was extracted at four temperatures (0, 45, 55, and 65 C.) and then incubated at the same temperatures for 90 minutes. Subsequently, two kinds of performance of the compound, that is, protein extraction efficiency and protein stability were evaluated at the same time by measuring the amount of a protein which remained dissolved in an aqueous solution. The amount of a protein which was extracted and stabilized by an individual amphipathic molecule was expressed as a relative value (%) with respect to the amount of the entire protein included in a membrane sample not treated with an amphipathic molecule.
(96) Specifically, a C-terminal 10-His-tagged Salmonella typhimurium melibiose permease (MelB.sub.St) was expressed in E. coli DW2 cells (melB and lacZY) using plasmid pK95AHB/WT MelB.sub.St/CH10. Cells were grown and membranes were prepared according to the method described in a paper (Nat. Commun. 2014, 5, 3009) written by A. S. Ethayathulla et al. A protein assay was carried out with a Micro BCA kit (Thermo Scientific, Rockford, Ill.). RGAs and DDM were evaluated to measure the stability of MelB.sub.St according to the protocol described in a paper (Nat. Methods 2010, 7, 1003-1008) written by P. S. Chae et al. The membrane sample (final protein concentration of 10 mg/mL) containing MelB.sub.St was incubated in a solubilization buffer containing 1.5% (w/v) DDM or RGAs (20 mM sodium phosphate, pH 7.5, 200 mM NaCl, 10% glycerol, and 20 mM melibiose) at four temperatures (0, 45, 55, and 65 C.) for 90 minutes. An insoluble material was removed by ultracentrifugation at 355,590 g at 4 C. for 45 minutes using a Beckman Optima MAX ultracentrifuge equipped with a TLA-100 rotor. A dissolved portion was separated by SDS-16% PAGE, and immunoblotting was then performed using a Penta-His-HRP antibody (Qiagen, Germantown, Md.). A membrane fraction containing 20 g of protein without treatment was used to illustrate the overall MelB, and the treated samples were loaded into each well in equal volume. MelB.sub.St was measured using the SuperSignal West Pico Chemiluminescent substrate by ImageQuant LAS 4000 Biomolecular Imager (GE Healthcare Life Sciences).
(97) As shown in
(98) However, when the temperature was raised to 55 C., RGA-C11 and RGA-C13 among RGAs efficiently extracted the MelB protein and maintained MelB solubility better than DDM. At 65 C., the MelB protein was hardly extracted in the case of all of DDM and RGAs.
(99) It can be seen that DDM generally exhibited higher protein extraction efficiency than RGAs at a low temperature (0 C.), whereas RGA-C11 and RGA-C13 exhibited similar protein extraction efficiency to that of DDM at a relatively high temperature (45 C.) and higher protein extraction efficiency than DDM at a high temperature (55 C.), indicating that although DDM is excellent in protein extraction efficiency, RGA-C11 and RGA-C13 are superior to DDM in terms of protein stability.
<Experimental Example 5> Evaluation of Membrane Protein (.SUB.2.AR) Structure Stabilization Ability of RGAs and RMAs
(100) An experiment was carried out to measure the structural stability of a human (32 adrenergic receptor (.sub.2AR) and a G protein-coupled receptor (GPCR) caused by RGAs and RMAs. That is, a receptor purified with DDM was diluted with a buffer solution containing only each RGA and RMA without cholesteryl hemisuccinate (CHS) or a buffer solution containing CHS and DDM. The final concentration of the compound was CMC+0.2 wt %, and the ligand binding characteristic of the receptor was measured using [.sup.3H]-dihydroalprenolol ([.sup.3H]-DHA) binding.
(101) Specifically, radioligand binding assay was carried out using the following method. .sub.2AR was purified using 0.1% DDM according to the method described in a paper (Science, 2007, 318, 1266-1273.) written by D. M. Rosenbaum et al. and finally concentrated to about 10 mg/ml (about 200 M). The .sub.2AR purified with DDM was used to prepare a master binding mixture containing 10 nM [.sup.3H]-dihydroalprenolol (DHA) supplemented with 0.5 mg/ml BSA in a 0.2% amphipathic compound (DDM, RGAs or RMAs). The receptor purified with DDM, RGAs or RMAs was incubated with 10 nM [.sup.3H]-DHA at room temperature for 30 minutes. The mixture was loaded into a G-50 column, the flow-through was collected with 1 ml of a binding buffer (20 mM HEPES pH 7.5, 100 mM NaCl, supplemented with 0.5 mg/ml BSA and 20CMC individual amphipathic compound, and the G-50 column was further filled with 15 ml of a scintillation fluid. Receptor-bound [.sup.3H]-DHA was measured with a scintillation counter (Beckman). The binding capacity of [.sup.3H]-DHA was measured and shown in a column graph. Each experiment was performed in triplicate.
(102) In addition, a size exclusion chromatography (SEC) test was conducted. Specifically, .sub.2AR purified with 0.1% DDM was loaded on a M1 Flag column in the presence of 2 mM CaCl.sub.2, and the column was washed with a DDM, RGA-C11 or RGA-C13 detergent buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.2% individual amphipathic compound). The receptor was eluted with 20CMC DDM, RGA-C11, or RGA-C13 with 5 mM EDTA and 0.2 mg/ml free Flag peptide. The eluate was further added to a Superdex-200 10/300 GL column (GE Healthcare) at 0.5 ml/min, and UV absorbance at 280 nm was recorded. The running buffer contained 20 mM HEPES pH 7.5, 100 mM NaCl, 20CMC individual detergent (DDM, RGA-C11 and RGA-C13).
(103) As shown in
(104) In addition, a test for identifying the effect of RGA-C11, RGA-C12, and RGA-C13 on maintaining the ligand binding of the receptor for a long period was conducted. Specifically, the ligand binding characteristic of the receptor dissolved in RGA-C11, RGA-C12, RGA-C13, or DDM was monitored at regular intervals during 2 days of incubation at room temperature, and results thereof are shown in
(105) As a result of the SEC test, as shown in
(106) As shown in