KR20220087385A - Novel tris(hydroxymethyl)methane core-based amphiphiles and uses thereof - Google Patents

Abstract

The present invention relates to a newly developed amphiphilic compound having a tris (hydroxymethyl) methane central structure, a method for preparing the same, and a method for extracting, dissolving, stabilizing, crystallizing, or analyzing a membrane protein using the same. In addition, this compound can efficiently extract membrane proteins with various structures and properties from the cell membrane and store them stably in aqueous solution for a long period of time, which can be utilized for functional and structural analysis. Membrane protein structure and function analysis is one of the fields of current interest in biology and chemistry as it is closely related to the development of new drugs.

Description

Amphiphilic compound having a novel tris(hydroxymethyl)methane core structure and utilization thereof {Novel tris(hydroxymethyl)methane core-based amphiphiles and uses thereof}

The present invention relates to a newly developed amphiphilic compound having a tris (hydroxymethyl) methane central structure, a method for preparing the same, and a method for extracting, dissolving, stabilizing, crystallizing, or analyzing a membrane protein using the same.

Membrane proteins play important roles in biological systems. Since these bio-macromolecules contain hydrophilic and hydrophobic moieties, amphiphilic molecules are required to extract membrane proteins from cell membranes and to solubilize and stabilize them in aqueous solutions.

For structural analysis of membrane proteins, high-quality membrane protein crystals must be obtained. For this, structural stability of membrane proteins in aqueous solution must be preceded. The number of existing amphiphilic molecules that have been used in the study of membrane proteins is more than 100, and there are many, but only about 5 of them have been actively used in the study of the structure of membrane proteins. These five amphoteric molecules are OG (n-octyl-β-D-glucopyranoside), NG (n-nonyl-β-D-glucopyranoside), DM (n-decyl-β-D-maltopyanoside), DDM (n- dodecyl-β-D-maltopyranoside), and lauryldimethylamine- N -oxide (LDAO) (Non-Patent Document 1, Non-Patent Document 2). However, since many membrane proteins surrounded by these molecules tend to quickly lose their function due to their structure being easily denatured or aggregated, there are significant limitations to the study of the function and structure of membrane proteins using these molecules. This is because conventional molecules do not exhibit various properties due to their simple chemical structure. Therefore, it is necessary to develop a new amphoteric material with new and excellent properties through a new structure.

Accordingly, the present inventors have developed an amphiphilic compound including a pendant chain having hydrophilicity and oleophobicity in a tris(hydroxymethyl)methane central structure, and completed the present invention by confirming the membrane protein stabilization properties of this compound.

S. Newstead et al., Protein Sci. 17 (2008) 466-472. S. Newstead et al., Mol. Membr. Biol. 25 (2008) 631-638.

An object of the present invention is to provide a compound represented by Formula 1 or an isomer thereof.

Another object of the present invention is to provide a composition for extraction, solubilization, stabilization, crystallization or analysis of a membrane protein containing the compound.

Another object of the present invention is to provide a method for preparing the compound.

One embodiment of the present invention provides a compound represented by the following formula (1) or formula (2) or an isomer thereof:

[Formula 1]

[Formula 2]

In Formula 1 or 2,

R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₃₀ alkyl group;

X ¹ to X ³ are each independently a saccharide; and

L is a C _1-5 alkyl group or C _1-5 alkoxy, wherein the C _1-5 alkoxy may be substituted with C _1-5 alkoxy or saccharide.

As used herein, the term “saccharide” refers to a compound having a relatively small molecule among carbohydrates and having a sweet taste when dissolved in water. Sugars are classified into monosaccharides, disaccharides, and polysaccharides according to the number of molecules constituting the sugar. Here, the saccharide may act as a hydrophilic group.

The saccharide used in the above embodiment may be a monosaccharide or a disaccharide, and specifically may be glucose or maltose, but is not limited thereto.

In Formula 1 or 2 of the present invention, the saccharide of X ¹ to X ³ may function as a hydrophilic group. In addition, the alkyl group of R ¹ to R ³ may function as a hydrophobic group. In the compound according to one embodiment of the present invention, three alkyl chains and three saccharides were introduced to optimize the hydrophilicity and hydrophobicity balance (hydrophile-lipophile balance).

In addition, the compound according to an embodiment of the present invention is an amphiphilic compound by adding a hydrophobic group or a hydrophilic group through the pendant chain, which is the fourth substituent attached to the quaternary carbon in the central structure of tris (hydroxymethyl) methane. The density of hydrophilic and hydrophobic groups can be controlled. By engineering the micellar structure by maximizing the density of hydrophobic groups through the fluidity of the molecular structure and modifying the molecular geometry, the function of the self-assembly of this nanostructure was greatly improved.

In a specific embodiment of the present invention, R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are each independently glucose or maltose; and L may be a C _1-3 alkyl group.

In another specific embodiment of the present invention, R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are each independently glucose or maltose; and L is C _1-3 alkoxy, wherein the C _1-3 alkoxy may be substituted with C _1-3 alkoxy or saccharide.

In another specific embodiment of the present invention, R ¹ to R ³ are an unsubstituted C ₃ -C ₁₅ alkyl group; X ¹ to X ³ are glucose or maltose; and L may be a methyl group, methoxyethoxy or glucoseethoxy.

The compound according to another embodiment of the present invention may be an amphiphilic molecule for extracting, solubilizing, stabilizing, crystallizing or analyzing a membrane protein, but is not limited thereto.

Specifically, the extraction may be to extract the membrane protein from the cell membrane.

As used herein, the term “amphiphilic molecule” refers to a molecule capable of having affinity for both polar and non-polar solvents by coexisting a hydrophobic group and a hydrophilic group in one molecule. Phospholipid molecules present in surfactants or cell membranes have a hydrophilic group at one end and a hydrophobic group at the other end, have amphiphilic properties, and form micelles or liposomes in aqueous solution. Although the hydrophilic group has polarity, since the non-polar group coexists, these amphiphilic molecules tend not to dissolve well in aqueous solution. However, when the concentration exceeds a certain limit concentration (critical aggregation concentration, CAC), the hydrophobic group aggregates inside due to hydrophobic interaction and the hydrophilic group aggregates in a round or oval shape exposed on the surface, greatly increasing solubility in water.

A method for measuring CAC is not particularly limited, but a method widely known in the art may be used, for example, it may be measured by a fluorescence staining method using diphenylhexatriene (DPH).

The compound according to one embodiment of the present invention may have a critical aggregation concentration (CAC) of 0.0001 to 1 mM in aqueous solution, specifically 0.0001 to 0.1 mM, more specifically 0.001 to 0.1 mM, and even more specifically 0.001 to 0.01 mM. may be, but is not limited thereto.

Compared to the case of DDM, which has been mainly used for membrane protein research, the critical aggregation concentration of 0.17 mM, the compound of the present invention has a very small CAC value. Therefore, since the compound of the present invention easily forms aggregation even at a low concentration, it can be said that it is advantageous in terms of utilization over DDM because it can effectively study and analyze membrane proteins using a small amount.

In addition, another embodiment of the present invention provides a composition for extraction, solubilization, stabilization, crystallization or analysis of a membrane protein containing the compound.

Specifically, the extraction may be to extract the membrane protein from the cell membrane.

The composition may be in the form of micelles, liposomes, emulsions, or nanoparticles, but is not limited thereto.

The micelle may have a radius of 1.0 nm to 100 nm, specifically 5.0 nm to 100.0 nm, for example, 7 nm to 90 nm, but is not limited thereto.

A method of measuring the radius of the micelles is not particularly limited, but a method widely known in the art may be used, for example, it may be measured using a dynamic light scattering (DLS) experiment.

The micelles, liposomes, emulsions or nanoparticles may bind to membrane proteins due to their hydrophobicity therein. That is, the micelles, liposomes, emulsions, or nanoparticles can be wrapped by extracting the membrane protein present in the cell membrane. Therefore, it is possible to extract, solubilize, stabilize, crystallize or analyze membrane proteins from the cell membrane by the micelles.

The composition may further include a buffer, etc. that can be helpful in extraction, solubilization, stabilization, crystallization or analysis of membrane proteins.

Specifically, the extraction may be to extract the membrane protein from the cell membrane.

As used herein, the term "membrane protein" is a generic term for proteins or glycoproteins that are introduced into the cell membrane lipid bilayer. It penetrates the entire layer of the cell membrane, is located on the surface layer, or exists in various states such as lining the cell membrane. Examples of membrane proteins include, but are not limited to, enzymes, receptors such as peptide hormones and local hormones, receptor carriers such as sugars, ion channels, and cell membrane antigens.

The membrane protein includes any protein or glycoprotein that is introduced into the cell membrane lipid bilayer, specifically LeuT (Leucine transporter), MelB (melibiose permease), β ₂ AR (human β ₂ adrenergic receptor), MOR (mouse μ- opioid receptor) or a combination of two or more thereof, but is not limited thereto.

As used herein, the term "extraction of a membrane protein" means separation of a membrane protein from a cell membrane (membrane).

As used herein, the term "solubilization of membrane proteins" means to dissolve membrane proteins insoluble in water into micelles in aqueous solution.

As used herein, the term "stabilization of membrane protein" refers to stably preserving the tertiary or quaternary structure so that the structure and function of the membrane protein are not changed.

As used herein, the term "crystallization of a membrane protein" means to form a crystal of a membrane protein in solution.

As used herein, the term "analysis of membrane protein" refers to analyzing the structure or function of a membrane protein. In the above embodiment, the analysis of the membrane protein may use a known method, but is not limited thereto, and for example, the structure of the membrane protein is analyzed using electron microscopy or nuclear magnetic resonance. can do.

In addition, another embodiment of the present invention provides a method for preparing a compound represented by Formula 1 according to Scheme 1:

1) preparing a compound of Formula 4 by introducing an alkanol through an epoxide ring opening reaction of the compound of Formula 3 (step 1); and

2) After introducing a saccharide to which a protecting group is attached to the compound of Formula 4 through a saccharification reaction, a deprotection reaction is performed (step 2).

[Scheme 1]

In Scheme 1,

R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₃₀ alkyl group;

X ¹ to X ³ are each independently a saccharide; and

L is a C _1-5 alkyl group or C _1-5 alkoxy, wherein the C _1-5 alkoxy may be substituted with C _1-5 alkoxy or saccharide.

In a specific embodiment of the present invention, R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are each independently glucose or maltose; and L may be a C _1-3 alkyl group.

In another specific embodiment of the present invention, R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are each independently glucose or maltose; and L is C _1-3 alkoxy, wherein the C _1-3 alkoxy may be substituted with C _1-3 alkoxy or glucose.

In another specific embodiment of the present invention, R ¹ to R ³ are an unsubstituted C ₃ -C ₁₅ alkyl group; X ¹ to X ³ are glucose or maltose; and L may be a methyl group, methoxyethoxy or glucoseethoxy.

In addition, another embodiment of the present invention provides a method for preparing a compound represented by Formula 2 according to Scheme 2:

1) preparing a compound of Formula 6 by attaching a protecting group Y to the compound of Formula 5 (step 1);

2) reacting the compound of formula 6 with an alkyl iodide and then removing the protecting group Y to prepare a compound of formula 7 (step 2); and

3) A method for preparing a compound represented by Formula 2, comprising the step of introducing a saccharide having a protecting group to the compound of Formula 7 through a saccharification reaction and then performing a deprotection reaction (step 3):

[Scheme 2]

In Scheme 2,

R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₃₀ alkyl group;

X ¹ to X ³ are each independently a saccharide;

L is a C _1-5 alkyl group or C _1-5 alkoxy, wherein the C _1-5 alkoxy may be substituted with C _1-5 alkoxy or saccharide; and

Y may be a protecting group.

In a specific embodiment of the present invention, R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are each independently glucose or maltose; and L may be a C _1-3 alkyl group.

In another specific embodiment of the present invention, R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are each independently glucose or maltose; and L is C _1-3 alkoxy, wherein the C _1-3 alkoxy may be substituted with C _1-3 alkoxy or glucose.

In another specific embodiment of the present invention, R ¹ to R ³ are an unsubstituted C ₃ -C ₁₅ alkyl group; X ¹ to X ³ are glucose or maltose; and L may be a methyl group, methoxyethoxy or glucoseethoxy.

In Scheme 1 or Scheme 2, the protecting group Y may be tert-butyldimethylsilyl (TBDMS), and the saccharide to which the protecting group is attached may be perbenzoylated maltose or perbenzoylated glucose.

By using the compound based on the tris (hydroxymethyl) methane structure according to the embodiments of the present invention, the membrane protein can be stored stably in an aqueous solution for a long period of time compared to the existing compound, which can be used for functional analysis and structural analysis. can

Membrane protein structure and function analysis is one of the most interesting fields in current biology and chemistry, so it can be applied to protein structure research that is closely related to drug development.

In addition, since the compounds according to the embodiments of the present invention can be synthesized in a simple manner from readily available starting materials, mass production of compounds for membrane protein research is possible.

1 is a diagram showing a synthesis scheme of E-GTMs according to Example 1 of the present invention.
2 is a diagram showing a synthesis scheme of M-GTMs according to Example 1 of the present invention.
3 is a diagram showing the size distribution of micelles formed by E-GTMs.
4 is a diagram showing the size distribution of micelles formed by M-GTMs.
5 is a diagram showing the size of micelles formed by GTMs according to concentration and temperature.
6 shows the results of periodically measuring LeuT (Leucine transporter) structural stability in aqueous solution by M-GTMs or DDM at CMC + 0.04 wt% (a) and CMC + 0.2 wt% (b) for 13 days.
7 shows the results of periodically measuring the structural stability of LeuT (Leucine transporter) in aqueous solution by E-GTMs or DDM at CMC + 0.04 wt% (a) and CMC + 0.2 wt% (b) for 13 days.
8 shows the results of measuring MelB protein solubilization ability according to temperature by M-/E-GTMs or DDM.
9 shows the results of periodically measuring the stability of the β ₂ AR structure in an aqueous solution by M-/E-GTMs GTMs or DDM at CMC + 0.2 wt% for 5 days.
10 shows the results of periodically measuring the MOR structure stability in aqueous solution by M-GTMs or DDM at CMC + 0.1 wt% for 3 days.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope of the present invention. What a person skilled in the art can easily infer from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention.

<Example 1> Synthesis method of E-GTM-Is

The synthesis scheme of E-GTM-Is is shown in FIG. 1 . Three compounds of E-GTM-Is were synthesized according to the following synthesis method.

<1-1> General synthetic procedure of epoxide ring opening reaction (step a in FIG. 1)

에서 24시간 동안 교반하였다. 빙냉 1M HCl을 적가하여 반응을 ?칭하였다. 희석된 반응 혼합물을 에틸아세테이트로 세척하고 혼합된 유기 분획을 염수로 세척하고 무수 Na₂SO₄로 건조시켰다. 유기 용매를 회전 증발하여 얻어진 유상 잔류물을 컬럼 크로마토그래피 정제하여 목적 화합물물 B를 수득하였다. NaH (4.5 equiv) was added to the alkanol (ROH; 6.0 equiv) under an inert atmosphere. The mixture was stirred at room temperature for 30 min, then glycidyl ether (A1; 1.0 eq.) was added dropwise. 50 of the reaction mixture

was stirred for 24 hours. The reaction was quenched by dropwise addition of ice-cold 1M HCl. The diluted reaction mixture was washed with ethyl acetate and the combined organic fractions were washed with brine and dried over anhydrous Na ₂ SO ₄ . The oily residue obtained by rotary evaporation of the organic solvent was purified by column chromatography to obtain the target compound B.

<1-2> General synthetic procedure of glycosycosylation reaction (step b of FIG. 1)

Under N ₂ atmosphere, a mixture of compound B (1 eq) and AgOTf (3.6 eq) in anhydrous CH ₂ Cl ₂ was stirred at -45 °C. Then perbenzoylated maltosylbromide (3.6 eq) dissolved in CH ₂ Cl ₂ was added to this suspension. The mixture was stirred at -45 °C for 5 min and then at 0 °C for 30 min. After completion of the reaction (analyzed by TLC), pyridine was added to the reaction mixture. The reaction mixture was diluted with CH ₂ Cl ₂ (30 mL) and then filtered through celite. The filtrate was washed successively with 1.0 M aqueous Na ₂ S ₂ O ₃ aqueous solution, 0.1 M aqueous HCl solution and brine. Then the organic layer was dried over anhydrous Na ₂ SO ₄ and the solvent was removed by rotary evaporation. The resulting residue was purified by silica gel column chromatography (EtOAc/hexane) to obtain the saccharified target compound.

<1-3> General synthetic procedure for deprotection reaction (step c of FIG. 1)

The O -benzoylated compound was dissolved with anhydrous CH ₂ Cl ₂ and then MeOH was added slowly until persistent precipitation appeared. NaOMe, a methanolic solution of 0.5 M, was added to the reaction mixture to a final concentration of 0.05 M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized using Amberlite IR-120 (H ⁺ form) resin. The resin was removed by filtration, washed with MeOH, and the solvent was removed from the filtrate under vacuum conditions ( in vacuo ). The residue was purified using silica gel column chromatography (CH ₂ Cl ₂ /MeOH) to obtain the target compound.

<Preparation Example 1> Synthesis of E-GTM-I10

<1-1> Compound B1((13R)-17-(((R)-3-(decyloxy)-2-hydroxypropoxy)methyl)-17-ethyl-11,15,19,23-tetraoxatritriacontane-13,21 -diol) synthesis

According to Example 1-1, compound B1 was synthesized in a yield of 30%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.93 (t, J = 4.0 Hz, 3H), 3.51-3.33 (m, 24H), 3.10 (s br, 3H), 1.56 (quin, J = 8.0 Hz, 6H), 1.39 (q, J = 8.0 Hz, 2H), 1.36-1.20 (m, 42H), 0.88 (t, J = 8.0 Hz, 9H), 0.84 (t, J = 8.0 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.9, 72.6, 72.2, 72.0, 71.9, 70.7, 69.4, 64.4, 43.5, 32.1, 29.8, 29.7, 29.6, 29.5, 26.3, 23.4, 22.8, 14.3.

<1-2> Synthesis of E-GTM-I10a

According to Example 1-2, E-GTM-I10a was synthesized in a yield of 66%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.13 (t, J = 6.0 Hz, 6H), 8.02 (d, J = 8.0 Hz, 6H), 7.87-7.85 (m, 12H), 7.81-7.74 (m) , 15H), 7.65-7.62 (m, 6H), 7.56-7.47 (m, 6H), 7.43-7.36 (m, 24H), 7.31-7.15 (m, 30H), 6.12 (t, J = 10.0 Hz, 3H) ), 5.75-5.67 (m, 10H), 5.39-5.22 (m, 7H), 5.10-4.90 (m, 5H), 4.78-4.62 (m, 3H), 4.49-4.41 (m, 7H), 4.40-4.35 (m, 4H), 4.32-4.22 (m, 4H), 4.16-4.05 (m, 3H), 3.97-3.74 (m, 5H), 3.43-2.91 (m, 18H), 1.42-1.38 (m, 2H) , 1.30-1.05 (m, 48H), 0.88 (t, J = 8.0 Hz, 9H), 0.65-0.52 (m, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 165.8, 165.6, 165.5, 165.1, 165.0, 133.4, 133.3, 133.1, 130.0, 129.9, 129.7, 129.6, 129.5, 129.4, 128.9, 128.8, 128.7, 128.4, 128.3, 128.2, 128.1, 100.9, 100.6, 96.5, 75.2, 73.3, 72.8, 72.6, 71.8, 71.7, 70.9, 70.7, 70.0, 69.1, 63.6, 62.5, 32.0, 29.7, 29.6, 29.4, 26.1, 26.0, 25.8, 22.7, 14.2.

<1-3> Synthesis of E-GTM-I10

According to Example 1-3, compound E-GTM-I10 was synthesized in a yield of 89%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.18 (d, J = 4.0 Hz, 3H), 4.63-4.35 (m, 3H), 4.08-4.00 (m, 3H), 3.88-3.82 (m, 12H) ), 3.68-3.51 (m, 25H), 3.49-3.44 (m, 9H), 3.39-3.30 (m, 9H), 3.29-3.25 (m, 4H) 1.57-1.51 (m, 6H), 1.42-1.20 ( m, 44H), 0.88 (t, J = 12.0 Hz, 12H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.1 102.9, 81.2, 78.3, 77.8, 76.6, 75.1, 74.8, 74.1, 72.8, 72.4, 72.0, 71.5, 70.5, 62.8, 62.3, 33.2, 30.9, 30.8, 30.7, 30.6, 27.4, 27.3, 23.8, 14.7; HRMS (MALDI ⁺ ) : calcd. for C ₈₁ H ₁₅₂ O ₃₉ [M+Na] ⁺ 1771.9803, found 1771.8369.

<Preparation Example 2> Synthesis of E-GTM-I11

<2-1> Compound B2((14R)-18-ethyl-18-(((R)-2-hydroxy-3-(undecyloxy)propoxy)methyl)-12,16,20,24-tetraoxapentatriacontane-14, 22-diol) synthesis

According to Example 1-1, compound B2 was synthesized in a yield of 33%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.93 (t, J = 4.0 Hz, 3H), 3.51-3.33 (m, 24H), 3.04 (s br, 3H), 1.56 (quin, J = 8.0 Hz, 6H), 1.39 (q, J = 8.0 Hz, 2H), 1.36-1.20 (m, 48H), 0.88 (t, J = 8.0 Hz, 9H), 0.84 (t, J = 8.0 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.9, 72.6, 72.1, 72.0, 71.9, 70.7, 69.4, 64.3, 43.5, 32.1, 29.8, 29.7, 29.6, 29.5, 26.3, 23.4, 22.8, 14.3.

<2-2> Synthesis of E-GTM-I11a

According to Example 1-2, E-GTM-I11a was synthesized in a yield of 64%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.11 (t, J = 6.0 Hz, 6H), 8.06 (d, J = 8.0 Hz, 6H), 7.86-7.84 (d, J = 8.0 Hz, 9H), 7.79-7.70 (m, 15H), 7.69-7.62 (m, 6H), 7.57-7.49 (m, 7H), 7.45-7.37 (m, 24H), 7.35-7.12 (m, 32H), 6.11 (t, J ) = 8.0 Hz, 3H), 5.79-5.75 (m, 6H), 5.69 (t, J = 10.0 Hz, 3H), 5.31-5.27 (m, 6H), 5.09-4.97 (m, 3H), 4.92 (t, J = 8.0 Hz, 3H), 4.78-4.76 (m, 3H), 4.53-4.47 (m, 6H), 4.43-4.26 (m, 6H), 4.13-4.05 (m, 4H), 3.97-3.74 (m, 3H), 3.53-2.82 (m, 23H), 1.44-1.38 (m, 2H), 1.30-1.05 (m, 54H), 0.88 (t, J = 8.0 Hz, 9H), 0.65-0.52 (m, 3H) ; ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 166.1, 165.8, 165.6, 165.5, 165.1, 133.5, 133.4, 133.3, 133.1, 133.0, 130.0, 129.9, 129.7, 129.6, 129.5, 129.4, 129.3, 128.9, 128.8, 128.7, 128.6, 128.4, 128.3, 128.2, 128.1, 100.6, 100.4, 96.5, 73.3, 72.7, 72.6, 71.8, 71.7, 70.9, 70.7, 70.0, 69.1, 63.6, 62.5, 60.4, 53.4, 32.0, 29.7, 29.6, 29.5, 29.4, 26.1, 26.0, 22.7, 21.1, 14.2.

<2-3> Synthesis of E-GTM-I11

According to Example 1-3, compound E-GTM-I11 was synthesized in a yield of 90%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.18 (s, 3H), 4.54-4.48 (m, 3H), 4.02-3.90 (m, 3H), 3.87-3.63 (m, 9H), 3.61-3.47 (m, 35H), 3.39-3.30 (m, 9H), 3.29-3.21 (m, 7H) 1.57-1.51 (m, 6H), 1.42-1.20 (m, 50H), 0.88 (t, J = 8.0 Hz, 12H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.2, 103.7, 102.9, 81.2, 78.4, 77.7, 76.7, 75.1, 74.8, 74.2, 72.9, 72.8, 72.5, 72.1, 71.5, 70.5, 62.8, 62.3, 44.8 , 44.7, 33.2, 30.9, 30.8, 30.7, 27.4, 23.9, 14.7; HRMS (MALDI ⁺ ) : calcd. for C ₈₄ H ₁₅₈ O ₃₉ [M+Na] ⁺ 1814.0272, found 1813.8971.

<Preparation Example 3> Synthesis of E-GTM-I12

<3-1> Compound B3((15R)-19-(((R)-3-(dodecyloxy)-2-hydroxypropoxy)methyl)-19-ethyl-13,17,21,25-tetraoxaheptatriacontane-15, 23 -diol) synthesis

According to Example 1-1, compound B3 was synthesized in a yield of 32%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.93 (t, J = 4.0 Hz, 3H), 3.51-3.33 (m, 24H), 2.94 (s br, 3H), 1.56 (quin, J = 8.0 Hz, 6H), 1.39 (q, J = 8.0 Hz, 2H), 1.36-1.20 (m, 54H), 0.88 (t, J = 8.0 Hz, 9H), 0.84 (t, J = 8.0 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.0, 72.9, 72.1, 72.0, 71.9, 70.7, 69.4, 64.4, 43.5, 32.1, 29.8, 29.7, 29.6, 29.5, 26.2, 23.3, 22.8, 14.3.

<3-2> Synthesis of E-GTM-I12a

According to Example 1-2, E-GTM-I12a was synthesized in a yield of 66%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.11 (t, J = 6.0 Hz, 6H), 8.07 (d, J = 8.0 Hz, 6H), 7.86-7.84 (d, J = 8.0 Hz, 9H), 7.79-7.70 (m, 12H), 7.69-7.62 (m, 6H), 7.57-7.49 (m, 6H), 7.47-7.37 (m, 28H), 7.35-7.19 (m, 32H), 6.12 (t, J ) = 8.0 Hz, 3H), 5.77-5.74 (m, 6H), 5.67 (t, J = 10.0 Hz, 3H), 5.31-5.24 (m, 6H), 5.06-4.93 (m, 3H), 4.89 (t, J = 8.0 Hz, 3H), 4.74-4.69 (m, 3H), 4.53-4.45 (m, 6H), 4.43-4.26 (m, 6H), 4.13-4.02 (m, 4H), 3.97-3.76 (m, 3H), 3.53-2.82 (m, 23H), 1.44-1.38 (m, 2H), 1.30-1.05 (m, 60H), 0.88 (t, J = 8.0 Hz, 9H), 0.65-0.52 (m, 3H) ; ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.3, 166.0, 165.8, 165.6, 165.2, 133.5, 133.2, 130.2, 130.1, 130.0, 129.9, 129.7, 129.6, 129.1, 128.9, 128.8, 128.6, 128.3, 128.2, 100.5, 100.4, 96.5, 73.4, 72.7, 72.6, 71.8, 71.7, 70.9, 70.7, 69.1, 63.6, 62.5, 60.4, 53.4, 32.0, 29.8, 29.6, 29.5, 29.4, 26.2, 26.0, 22.7, 21.2, 14.2.

<3-3> Synthesis of E-GTM-I12

According to Example 1-3, compound E-GTM-I12 was synthesized in a yield of 90%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.17 (d, J = 4.0 Hz, 3H), 4.56-4.47 (m, 3H), 4.02-3.90 (m, 3H), 3.87-3.79 (m, 9H) ), 3.69-3.51 (m, 27H), 3.49-3.40 (m, 12H), 3.39-3.34 (m, 9H), 3.27-3.25 (m, 3H), 1.57-1.51 (m, 6H), 1.42-1.20 (m, 56H), 0.88 (t, J = 8.0 Hz, 12H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.2, 103.0, 81.4, 78.2, 77.8, 76.8, 76.4, 75.2, 75.0, 74.9, 74.3, 73.1, 72.9, 71.9, 71.6, 62.9, 62.4, 33.2, 30.9 , 30.8, 30.6, 27.4, 23.9, 14.6; HRMS (MALDI ⁺ ) : calcd. for C ₈₇ H ₁₆₄ O ₃₉ [M+Na] ⁺ 1856.0742, found 1855.9846.

<Example 2> Synthesis method of E-GTM-Os

The synthesis scheme of E-GTM-Os is shown in FIG. 1 . Three types of compounds of E-GTM-Os were synthesized according to the following synthesis method.

<2-1> General procedure of TBDMS protective vaporization reaction of primary alcohol (step d in FIG. 1)

에서 이미다졸(6.0 당량)을 조금씩 첨가하였다. 5분 후, DCM 중 TBDMSCl(3.6당량)의 용액을 첨가하고 생성된 혼합물을 실온에서 1시간 동안 교반한 다음 얼음으로 ?칭하였다. 증류수로 추가 희석한 후, 반응 혼합물을 DCM으로 추출하였다. 혼합된 DCM 분획을 빙냉 HCl(0.1M), 물 및 염수로 연속적으로 세척한 다음 무수 Na₂SO₄로 건조시켰다. 반응 혼합물로부터 용매를 제거한 후 얻어진 유상 잔류물을 크로마토그래피로 정제하여 목적 화합물 C를 74% 수율로 수득하였다. ¹ H NMR (400 MHz, CDCl₃): δ 3.73 (quin, J = 4.0 Hz, 3H), 3.54 (d, J = 8.0 Hz, 6H), 3.43-3.35 (m, 6H), 3.33-3.27 (m, 6H), 2.87 (s br, 3H), 1.33 (quin, J = 8.0 Hz, 2H), 0.83 (s, 27H), 0.76 (t, J = 8.0 Hz, 3H), 0.01 (s, 18H); ¹³ C NMR (100 MHz, CDCl₃): δ 72.4, 72.3, 72.0, 70.5, 70.4, 64.0, 43.3, 43.2, 25.9, 25.7, 23.2, 18.3, 18.0, 7.7, 7.6, -4.7, -4.8, -5.4.Hexa-ol derivative (A2; 3,3'-((2-((2,3-dihydroxypropoxy)methyl)-2-ethylpropane-1,3-diyl)bis(oxy )) bis(propane-1,2-diol)) under argon 0

imidazole (6.0 equivalents) was added little by little. After 5 min, a solution of TBDMSCl (3.6 eq) in DCM was added and the resulting mixture was stirred at room temperature for 1 h and then quenched with ice. After further dilution with distilled water, the reaction mixture was extracted with DCM. The combined DCM fractions were washed successively with ice-cold HCl (0.1M), water and brine, then dried over anhydrous Na ₂ SO ₄ . After removing the solvent from the reaction mixture, the obtained oily residue was purified by chromatography to obtain the target compound C in 74% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.73 (quin, J = 4.0 Hz, 3H), 3.54 (d, J = 8.0 Hz, 6H), 3.43-3.35 (m, 6H), 3.33-3.27 (m) , 6H), 2.87 (s br, 3H), 1.33 (quin, J = 8.0 Hz, 2H), 0.83 (s, 27H), 0.76 (t, J ) = 8.0 Hz, 3H), 0.01 (s, 18H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.4, 72.3, 72.0, 70.5, 70.4, 64.0, 43.3, 43.2, 25.9, 25.7, 23.2, 18.3, 18.0, 7.7, 7.6, -4.7, -4.8, -5.4 .

<2-2> General procedure of trialkylated triol derivative synthesis (step e of FIG. 1)

에서 24시간 동안 교반하였다. 얼음을 첨가하여 반응을 ?칭한 다음, 물로 희석하였다. 희석된 반응 혼합물을 에틸아세테이트로 추출하고 혼합한 유기 분획을 염수로 세척하고 무수 Na₂SO₄로 건조시켰다. 감압하에서 용매를 제거하여 THF에 용해된 오일성 잔류물을 얻었다. 이 용액에 THF 중 1.0 M TBAF(4.5 당량)를 첨가한 다음, 실온에서 12시간 동안 교반하였다. 반응 종료 후, 반응 혼합물을 물로 희석하고 에틸아세테이트로 세척하였다. 혼합한 유기 분획을 염수로 세척하고 무수 Na₂SO₄로 건조시켰다. 감압하에서 용매를 제거하여 유상 잔류물을 얻었고, 이를 컬럼 크로마토그래피 정제하여 목적 화합물 D를 수득하였다.To an ice-cooled solution of compound C (1.0 equiv) in DMF was added NaH (4.5 equiv) under an inert atmosphere. After bubbling ceased, alkyl iodide (RI; 4.5 eq) was added dropwise and the resulting mixture was stirred at 70

was stirred for 24 hours. The reaction was quenched by adding ice and then diluted with water. The diluted reaction mixture was extracted with ethyl acetate, and the combined organic fractions were washed with brine and dried over anhydrous Na ₂ SO ₄ . The solvent was removed under reduced pressure to give an oily residue dissolved in THF. To this solution was added 1.0 M TBAF in THF (4.5 eq) and then stirred at room temperature for 12 h. After completion of the reaction, the reaction mixture was diluted with water and washed with ethyl acetate. The combined organic fractions were washed with brine and dried over anhydrous Na ₂ SO ₄ . The solvent was removed under reduced pressure to obtain an oily residue, which was purified by column chromatography to obtain the target compound D.

<2-3> General synthesis procedure of glycosycosylation reaction (step f of FIG. 1)

Under N ₂ atmosphere, a mixture of compound D (1 eq) and AgOTf (3.6 eq) in anhydrous CH ₂ Cl ₂ was stirred at -45 °C. Then perbenzoylated maltosylbromide (3.6 eq) dissolved in CH ₂ Cl ₂ was added to this suspension. The mixture was stirred at -45 °C for 5 min and then at 0 °C for 30 min. After completion of the reaction (detected by TLC), pyridine was added to the reaction mixture. The reaction mixture was diluted with CH ₂ Cl ₂ (30 mL) and then filtered through celite. The filtrate was washed successively with 1.0 M aqueous Na ₂ S ₂ O ₃ aqueous solution, 0.1 M aqueous HCl solution and brine. Then the organic layer was dried over anhydrous Na ₂ SO ₄ and the solvent was removed by rotary evaporation. The resulting residue was purified by silica gel column chromatography (EtOAc/hexane) to obtain the saccharified target compound.

<2-4> General synthetic procedure for deprotection reaction (step g in FIG. 1)

The O -benzoylated compound was dissolved with anhydrous CH ₂ Cl ₂ and then MeOH was added slowly until persistent precipitation appeared. NaOMe, a methanolic solution of 0.5 M, was added to the reaction mixture to a final concentration of 0.05 M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized using Amberlite IR-120 (H ⁺ form) resin. The resin was removed by filtration, washed with MeOH, and the solvent was removed from the filtrate under vacuum conditions ( in vacuo ). The residue was purified using silica gel column chromatography (CH ₂ Cl ₂ /MeOH) to obtain the target compound.

<Preparation Example 4> Synthesis of E-GTM-O10

<4-1> Synthesis of compound D1((2R)-3-(2,2-bis((2-(decyloxy)-3-hydroxypropoxy)methyl)butoxy)-2-(decyloxy)propan-1-ol)

According to Example 2-2, compound D1 was synthesized in a yield of 68%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70 (dd, J _1-2 = 4.0 Hz, J _1-3 = 12.0 Hz, 3H), 3.62-3.42 (m, 18H), 3.30 (s, 6H) , 2.76 (s br, 3H), 1.56 (quin, J = 6.0 Hz, 6H), 1.38-1.20 (m, 44H), 0.88 (t, J = 8.0 Hz, 9H), 0.83 (t, J = 8.0 Hz) , 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ 78.3, 78.2, 73.2, 72.5, 72.3, 72.0, 71.8, 71.5, 71.3, 70.5, 69.5, 62.9, 62.8, 43.3, 32.1, 30.2, 29.8, 29.7, 29.6, 29.5, 26.3, 23.2, 22.8, 14.3, 7.7.

<4-2> Synthesis of E-GTM-O10a

According to Example 2-3, E-GTM-O10a was synthesized in a yield of 65%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.11 (t, J = 4.0 Hz, 6H), 8.02 (d, J = 8.0 Hz, 6H), 7.86 (t, J = 8.0 Hz, 12H), 7.75 ( t, J = 4.0 Hz, 12H), 7.65 (t, J = 4.0 Hz, 6H), 7.54-7.51 (m, 3H), 7.48 (d, J = 8.0 Hz, 3H), 7.41-7.32 (m, 22H) ), 7.30-7.14 (m, 35H), 6.13 (t, J = 10.0 Hz, 3H), 5.82-5.78 (m, 6H), 5.70 (t, J = 8.0 Hz, 3H), 5.38-5.21 (m, 6H), 4.94-4.88 (m, 6H), 4.79 (d, J = 6.0 Hz, 3H), 4.56 (t, J = 10.0 Hz, 3H), 4.49 (d, J = 8.0 Hz, 3H), 4.41 ( d, J = 8.0 Hz, 3H), 4.28 (d, J = 12.0 Hz, 3H), 3.37-3.84 (m, 3H), 3.49-3.36 (m, 8H), 3.27-3.18 (m, 10H), 2.95 -2.80 (m, 6H), 1.42-1.38 (m, 2H), 1.30-1.05 (m, 50H), 0.88 (t, J = 8.0 Hz, 9H), 0.65-0.52 (m, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 165.8, 165.7, 165.5, 165.1, 133.5, 133.4, 133.3, 133.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.2, 128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 101.2, 100.9, 96.5, 75.0, 73.2, 72.9, 72.4, 71.7, 70.9, 70.5, 70.0, 69.1, 63.5, 62.5, 60.4, 32.0, 30.1, 29.7, 29.5, 29.4, 26.1, 26.0, 22.8, 21.0, 14.2.

<4-3> Synthesis of E-GTM-O10

According to Example 1-3, compound E-GTM-O10 was synthesized in a yield of 90%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.18 (d, J = 4.0 Hz, 3H), 4.33-4.29 (m, 3H), 3.95-3.78 (m, 13H), 3.71-3.44 (m, 37H) ), 3.41-3.24 (m, 13H), 1.55 (quin, J = 8.0 Hz, 6H), 1.42-1.20 (m, 44H), 0.88 (t, J = 12.0 Hz, 12H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.9, 104.7, 103.0, 81.4, 81.3, 79.4, 77.8, 76.4, 75.2, 74.9, 74.8, 74.2, 72.9, 72.6, 71.8, 71.7, 71.6, 70.5, 62.8 , 62.3, 52.8, 44.7, 33.3, 31.4, 31.0, 30.9, 30.8, 30.7, 27.4, 24.1, 23.9, 14.7; HRMS (MALDI ⁺ ) : calcd. for C ₈₁ H ₁₅₂ O ₃₉ [M+Na] ⁺ 1771.9803, found 1771.8602.

<Preparation Example 5> Synthesis of E-GTM-O11

<5-1> of compound D2)(2R)-3-(2,2-bis((3-hydroxy-2-(undecyloxy)propoxy)methyl)butoxy)-2-(undecyloxy)propan-1-ol) synthesis

According to Example 2-2, compound D2 was synthesized in a yield of 67%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.69 (dd, J _1-2 = 4.0 Hz, J _1-3 = 12.0 Hz, 3H), 3.61-3.42 (m, 18H), 3.30 (s, 6H) , 2.83 (s br, 3H), 1.56 (quin, J = 6.0 Hz, 6H), 1.38-1.20 (m, 50H), 0.88 (t, J = 8.0 Hz, 9H), 0.83 (t, J = 8.0 Hz) , 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ 78.3, 73.2, 72.4, 72.3, 72.0, 71.8, 71.3, 70.4, 69.4, 62.8, 62.7, 43.2, 32.0, 30.2, 29.8, 29.7, 29.6, 29.4, 26.3, 23.2, 22.8, 14.2, 7.7.

<5-2> Synthesis of E-GTM-O11a

According to Example 2-3, E-GTM-O11a was synthesized in a yield of 66%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.11 (t, J = 6.0 Hz, 6H), 8.00 (d, J = 8.0 Hz, 6H), 7.86 (d, J = 8.0 Hz, 12H), 7.76 7.74 (m, 12H), 7.66-7.64 (m, 6H), 7.59-7.50 (m, 6H), 7.44-7.36 (m, 24H), 7.31-7.15 (m, 33H), 6.12 (t, J = 10.0) Hz, 3H), 5.82-5.78 (m, 6H), 5.69 (t, J = 8.0 Hz, 3H), 5.39-5.22 (m, 6H), 4.93-4.86 (m, 6H), 4.78 (d, J = 8.0 Hz, 3H), 4.56 (dt, J _1-2 = 4.0 Hz, J _1-3 = 10.0 Hz, 3H), 4.47 (d, J = 12.0 Hz, 3H), 4.42 (d, J = 8.0 Hz, 3H), 4.26 (d, J = 12.0 Hz, 3H), 3.37-3.84 (m, 3H), 3.49-3.36 (m, 8H), 3.27-3.18 (m, 10H), 2.95-2.80 (m, 6H) , 1.42-1.38 (m, 2H), 1.30-1.05 (m, 50H), 0.88 (t, J = 8.0 Hz, 9H), 0.65-0.52 (m, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 165.8, 165.7, 165.5, 165.1, 133.5, 133.4, 133.3, 133.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.2, 128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 101.2, 100.9, 96.5, 75.0, 73.2, 72.9, 72.4, 71.7, 70.9, 70.5, 70.0, 69.1, 63.5, 62.5, 60.4, 32.0, 30.1, 29.7, 29.5, 29.4, 26.1, 26.0, 22.8, 21.0, 14.2.

<5-3> Synthesis of E-GTM-O11

According to Example 2-3, compound E-GTM-O11 was synthesized in a yield of 92%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.18 (d, J = 4.0 Hz, 3H), 4.33-4.29 (m, 3H), 3.94-3.78 (m, 13H), 3.71-3.44 (m, 37H) ), 3.40-3.26 (m, 13H), 1.55 (quin, J = 8.0 Hz, 6H), 1.42-1.20 (m, 50H), 0.88 (t, J = 8.0 Hz, 12H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.8, 104.7, 102.9, 81.3, 81.2, 79.4, 77.7, 76.6, 75.1, 74.8, 74.7, 74.1, 72.8, 72.6, 71.7, 71.6, 71.5, 70.4, 62.8 , 62.3, 52.8, 44.6, 33.2, 31.3, 30.9, 30.8, 30.6, 27.4, 24.0, 23.9, 14.7; HRMS (MALDI ⁺ ) : calcd. for C ₈₄ H ₁₅₈ O ₃₉ [M+Na] ⁺ 1814.0272, found 1813.8898.

<Preparation Example 6> Synthesis of E-GTM-O12

<6-1> Synthesis of compound D3((2R)-3-(2,2-bis((2-(dodecyloxy)-3-hydroxypropoxy)methyl)butoxy)-2-(dodecyloxy)propan-1-ol)

According to Example 2-2, compound D3 was synthesized in a yield of 69%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70 (dd, J _1-2 = 4.0 Hz, J _1-3 = 12.0 Hz, 3H), 3.62-3.42 (m, 18H), 3.30 (s, 6H) , 2.66 (s br, 3H), 1.56 (quin, J = 6.0 Hz, 6H), 1.38-1.20 (m, 56H), 0.88 (t, J = 8.0 Hz, 9H), 0.83 (t, J = 8.0 Hz) , 3H). ¹³ C NMR (100 MHz, CDCl ₃ ): δ 78.3, 72.3, 72.1, 72.0, 71.9, 71.7, 71.4, 70.8, 69.5, 63.0, 62.9, 43.3, 32.1, 30.3, 29.9, 29.8, 29.7, 29.5, 26.3, 23.2, 22.9, 20.9, 14.3, 7.7.

<6-2> Synthesis of E-GTM-O12a

According to Example 2-3, E-GTM-O12a was synthesized in a yield of 66%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.09 (t, J = 6.0 Hz, 6H), 7.92 (d, J = 8.0 Hz, 6H), 7.83 (d, J = 8.0 Hz, 6H), 7.82 7.80 (m, 6H), 7.74 (d, J = 8.0 Hz, 12H), 7.65-7.62 (m, 6H), 7.55-7.49 (m, 6H), 7.47-7.38 (m, 24H), 7.32-7.18 ( m, 33H), 6.10 (t, J = 10.0 Hz, 3H), 5.78-5.74 (m, 6H), 5.67 (t, J = 8.0 Hz, 3H), 5.37-5.29 (m, 3H), 5.27 (dd , J _1-2 = 4.0 Hz, J _1-3 = 10.0 Hz, 3H), 4.90 (d, J = 12.0 Hz, 3H), 4.85 (d, J = 8.0 Hz, 3H), 4.76 (dd, J _{1 -2} = 4.0 Hz, J _1-3 = 12.0 Hz, 3H), 4.53 (dt, J _1-2 = 4.0 Hz, J _1-3 = 10.0 Hz, 3H), 4.46 (d, J = 12.0 Hz, 3H) ), 4.38 (d, J = 10.0 Hz, 3H), 4.25 (dd, J _1-2 = 4.0 Hz, J _1-3 = 10.0 Hz, 3H), 4.12-4.07 (m, 3H), 3.94-3.78 ( m, 3H), 3.62-3.30 (m, 9H), 3.37-3.25 (m, 3H), 3.18-3.05 (m, 6H), 3.01-2.90 (m, 3H), 2.82 (m, 3H), 1.42- 1.38 (m, 2H), 1.30-1.05 (m, 60H), 0.88 (t, J = 8.0 Hz, 9H), 0.65-0.52 (m, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.3, 166.3, 165.9, 165.7, 165.6, 165.2, 133.6, 133.5, 133.4, 133.2, 133.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.3, 129.0, 128.8, 128.7, 128.6, 128.5, 128.3, 128.2, 101.2, 100.9, 96.5, 75.2, 75.0, 73.2, 72.9, 72.4, 71.8, 71.2, 71.0, 70.7, 70.0, 69.2, 63.6, 62.6, 60.5, 32.1, 30.2, 29.8, 29.6, 29.5, 29.4, 26.2, 26.0, 22.8, 21.2, 14.3.

<6-3> Synthesis of E-GTM-O12

According to Example 2-4, compound E-GTM-O12 was synthesized in a yield of 92%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.14 (d, J = 4.0 Hz, 3H), 4.29-4.25 (m, 3H), 3.90-3.88 (m, 4H), 3.87-3.84 (s, 2H) ), 3.80-3.78 (m, 6H), 3.75-3.68 (m, 13H), 3.67-3.60 (m, 11H), 3.58-3.50 (m, 7H), 3.49-3.40 (m, 6H), 3.35-3.30 (m, 9H), 3.27-3.21 (m, 5H), 1.53 (quin, J = 8.0 Hz, 6H), 1.42-1.20 (m, 56H), 0.86 (t, J = 8.0 Hz, 12H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.8, 104.7, 102.9, 81.3, 81.2, 79.4, 77.7, 76.6, 75.1, 74.8, 74.7, 74.1, 72.8, 71.7, 71.6, 71.5, 62.8, 62.3, 52.8 , 44.6, 33.2, 31.3, 30.9, 30.8, 30.6, 27.4, 24.1, 23.9, 14.7; HRMS (MALDI ⁺ ) : calcd. for C ₈₇ H ₁₆₄ O ₃₉ [M+Na] ⁺ 1856.0742, found 1855.9741.

<Example 3> Synthesis method of M-GTM-Is

The synthesis scheme of M-GTM-Is is shown in FIG. 2 . Three types of compounds of M-GTM-Is were synthesized according to the following synthesis method.

<3-1> Synthesis procedure of compound E (FIG. 2)

에서 4시간 동안 가열하였다. NaOH로 중화한 후, 반응 혼합물을 농축하였다. 생성된 잔류물을 50% NaOH에 용해시키고 tert-뷰틸 암모늄 브로마이드(0.5 당량) 및 알릴 브로마이드(4 당량)를 후속적으로 첨가하였다. 반응 혼합물을 상온에서 5시간 동안 교반하고 55

에서 24시간 동안 열활성화시킨 후 에틸 에테르로 추출하였다. 혼합한 유기층을 염수로 세척하고, 무수 Na₂SO₄로 건조시키고, 감압하에 농축시켰다. 잔류물을 컬럼 크로마토그래피로 정제하여 목적 화합물 E를 74% 수율로 수득하였다. ¹ H NMR (400 MHz, CDCl₃): δ 5.92-5.83 (m, 3H), 5.27 (quin, J = 8.0 Hz, 1.5H), 5.22 (quin, J = 8.0 Hz, 1.5H), 5.14 (quin, J = 8.0 Hz, 1.5H), 5.11 (quin, J = 8.0 Hz, 1.5H), 3.95-3.93 (m, 6H), 3.58-3.55 (m, 2H), 3.52-3.50 (m, 2H), 3.48 (s, 6H), 3.36 (s, 3H); ¹³ C NMR (100 MHz, CDCl₃): δ 135.2, 115.9, 72.1, 71.7, 70.8, 70.1, 69.2, 58.9, 45.4.4-[(2-methoxyethoxy)methyl]-1-methyl-2,6,7-trioxabicyclo[2.2.2]octane was dissolved in DCM/MeOH (1:1). A few drops of the concentrate were added to this solution. HCl and the resulting mixture to 50

was heated for 4 hours. After neutralization with NaOH, the reaction mixture was concentrated. The resulting residue was dissolved in 50% NaOH and tert-butyl ammonium bromide (0.5 equiv) and allyl bromide (4 equiv) were subsequently added. The reaction mixture was stirred at room temperature for 5 hours and 55

After thermal activation for 24 hours, it was extracted with ethyl ether. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the target compound E in 74% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.92-5.83 (m, 3H), 5.27 (quin, J = 8.0 Hz, 1.5H), 5.22 (quin, J = 8.0 Hz, 1.5H), 5.14 (quin , J = 8.0 Hz, 1.5H), 5.11 (quin, J = 8.0 Hz, 1.5H), 3.95-3.93 (m, 6H), 3.58-3.55 (m, 2H), 3.52-3.50 (m, 2H), 3.48 (s, 6H), 3.36 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.2, 115.9, 72.1, 71.7, 70.8, 70.1, 69.2, 58.9, 45.4.

<3-2> Compound F1(2,2'-(((2-((2-methoxyethoxy)methyl)-2-((oxiran-2-ylmethoxy)methyl)propane-1,3-diyl)bis(oxy Synthesis procedure of ))bis(methylene))bis(oxirane)) (step a1 in Fig. 2)

To an ice-cold solution of compound E in DCM was added 3-chloroperbenzoic acid (4 eq). The reaction mixture was stirred at room temperature for 12 hours. After completion of the reaction, the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography (EtOAc/hexane) to obtain the target compound F1 in 76% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.69 (quin, J = 8.0 Hz, 3H), 3.58-3.55 (m, 2H), 3.52-3.49 (m, 7H), 3.47 (d, J = 8.0 Hz) , 3H), 3.4-3.36 (m, 6H), 3.13-3.09 (m, 3H), 2.77 (quin, J = 8.0 Hz, 3H), 2.59 (quin, J = 8.0 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.8, 134.3, 132.9, 131.8, 129.8, 129.7, 127.9, 71.9, 71.6, 70.7, 69.9, 69.6, 58.8, 50.7, 45.6, 44.0.

<3-3> General synthetic procedure of epoxide ring opening reaction (step b of FIG. 2)

에서 24시간 동안 교반하였다. 빙냉 1M HCl을 적가하여 반응을 켄칭하였다. 희석된 반응 혼합물을 에틸아세테이트로 세척하고 혼합된 유기 분획을 염수로 세척하고 무수 Na₂SO₄로 건조시켰다. 유기 용매를 회전 증발하여 얻어진 유상 잔류물을 컬럼 크로마토그래피 정제하여 목적 화합물물 G를 수득하였다. NaH (4.5 equiv) was added to the alkanol (ROH; 6.0 equiv) under an inert atmosphere. The mixture was stirred at room temperature for 30 min, then glycidyl ether (A1; 1.0 eq.) was added dropwise. 50 of the reaction mixture

was stirred for 24 hours. The reaction was quenched by dropwise addition of ice-cold 1M HCl. The diluted reaction mixture was washed with ethyl acetate and the combined organic fractions were washed with brine and dried over anhydrous Na ₂ SO ₄ . The oily residue obtained by rotary evaporation of the organic solvent was purified by column chromatography to obtain the target compound G.

<3-4> General synthetic procedure of glycosycosylation reaction (step c in FIG. 2)

Under N ₂ atmosphere, a mixture of compound B (1 eq) and AgOTf (3.6 eq) in anhydrous CH ₂ Cl ₂ was stirred at -45 °C. Then perbenzoylated maltosylbromide (3.6 eq) dissolved in CH ₂ Cl ₂ was added to this suspension. The mixture was stirred at -45 °C for 5 min and then at 0 °C for 30 min. After completion of the reaction (detected by TLC), pyridine was added to the reaction mixture. The reaction mixture was diluted with CH ₂ Cl ₂ (30 mL) and then filtered through celite. The filtrate was washed successively with 1.0 M aqueous Na ₂ S ₂ O ₃ aqueous solution, 0.1 M aqueous HCl solution and brine. Then the organic layer was dried over anhydrous Na ₂ SO ₄ and the solvent was removed by rotary evaporation. The resulting residue was purified by silica gel column chromatography (EtOAc/hexane) to obtain the saccharified target compound.

<3-5> General synthetic procedure for deprotection reaction (step d of FIG. 2)

The O -benzoylated compound was dissolved with anhydrous CH ₂ Cl ₂ and then MeOH was added slowly until persistent precipitation appeared. NaOMe, a methanolic solution of 0.5 M, was added to the reaction mixture to a final concentration of 0.05 M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized using Amberlite IR-120 (H ⁺ form) resin. The resin was removed by filtration, washed with MeOH, and the solvent was removed from the filtrate under vacuum conditions ( in vacuo ). The residue was purified using silica gel column chromatography (CH ₂ Cl ₂ /MeOH) to obtain the target compound.

<Preparation Example 7> Synthesis of M-GTM-I10

<7-1> Compound G1 (3,3'-((2-(((( R )-2-(decyloxy)-3-hydroxypropoxy)methyl)-2-((2-methoxyethoxy)methyl)propane-1,3-diyl)bis (oxy))bis(2-(decyloxy)propan-1-ol ) synthesis

According to Example 3-3, compound G1 was synthesized in a yield of 68%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.92 (dd, J _1-2 = 4.0 Hz, J _1-3 = 12.0 Hz, 3H), 3.57-3.54 (m, 2H), 3.51-3.40 (m, 28H), 3.24 (s, 3H), 3.24 (s br, 3H), 1.56 (quin, J = 6.0 Hz, 6H), 1.35-1.20 (m, 42H), 0.88 (t, J = 8.0 Hz, 9H) , ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.8, 71.9, 71.7, 71.5, 70.7, 70.3, 70.1, 69.0, 58.7, 53.3, 45.4, 31.8, 29.5, 29.4, 29.2, 26.0, 22.5, 14.0.

<7-2> Synthesis of M-GTM-I10a

According to Example 3-4, M-GTM-I10a was synthesized in a yield of 65%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.11 (t, J = 4.0 Hz, 6H), 8.08 (d, J = 8.0 Hz, 6H), 7.86 (t, J = 8.0 Hz, 12H), 7.76 ( t, J = 4.0 Hz, 12H), 7.66-7.62 (m, 6H), 7.54-7.48 (m, 6H), 7.44-7.34 (m, 24H), 7.32-7.14 (m, 34H), 6.11 (t, J = 10.0 Hz, 3H), 5.80-5.66 (m, 9H), 5.32-5.21 (m, 8H), 5.08-4.88 (m, 6H), 4.77 (d, J = 6.0 Hz, 3H), 4.52-4.40 (m, 9H), 4.29 (d, J = 12.0 Hz, 3H), 4.11-4.05 (m, 3H), 3.91-3.85 (m, 3H), 3.47-3.27 (m, 18H), 3.22-2.86 (m) , 14H), 1.47-1.38 (m, 3H), 1.30-1.11 (m, 45H), 0.89 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.1, 165.8, 165.6, 165.5, 165.4, 165.1, 165.0, 133.4, 133.3, 133.2, 133.0, 132.9, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 100.3, 96.5, 75.2, 75.1, 73.3, 72.7, 72.6, 71.8, 71.7, 70.8, 69.9, 69.1, 63.6, 62.5, 60.4, 58.9, 53.4, 45.4, 31.9, 29.8, 29.6, 29.5, 29.4, 26.1, 26.0, 22.7, 21.0, 14.2, 14.1.

<7-3> Synthesis of M-GTM-I10

According to Example 3-5, compound M-GTM-I10 was synthesized in a yield of 89%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.17 (d, J = 4.0 Hz, 3H), 4.55-4.47 (m, 3H), 4.03-3.80 (m, 12H), 3.71-3.40 (m, 52H) ), 3.31-3.23 (m, 8H), 1.60-1.55 (m, 6H), 1.35-1.25 (m, 42H), 0.90 (t, J = 12.0 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.1, 103.7, 102.9, 81.2, 78.3, 77.8, 77.6, 76.6, 75.1, 74.8, 74.1, 73.1, 72.8, 72.7, 72.5, 72.0, 71.8, 71.5, 71.1 , 62.8, 62.3, 59.4, 47.0, 46.9, 33.1, 30.8, 30.7, 30.5, 27.3, 23.8, 14.6; HRMS (FAB ⁺ ) : calcd. for C ₈₃ H ₁₅₆ O ₄₁ [M+Na] ⁺ 1832.0020, found 1832.0012.

<Preparation Example 8> Synthesis of M-GTM-I11

<8-1> Compound G2 (3,3'-((2-(((( R )-3-hydroxy-2-(undecyloxy)propoxy)methyl)-2-((2-methoxyethoxy)methyl)propane-1,3-diyl)bis (oxy))bis(2-(undecyloxy)propan-1- ol)) synthesis

According to Example 3-3, compound G2 was synthesized in a yield of 68%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.92 (dd, J _1-2 = 4.0 Hz, J _1-3 = 12.0 Hz, 3H), 3.57-3.53 (m, 2H), 3.50-3.40 (m, 28H), 3.35 (s, 3H), 3.05 (s br, 3H), 1.55 (quin, J = 6.0 Hz, 6H), 1.33-1.22 (m, 42H), 0.89 (t, J = 8.0 Hz, 9H) , ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.8, 71.8, 71.7, 70.8, 70.5, 70.3, 69.2, 58.9, 45.4, 31.9, 29.6, 29.5, 29.3, 26.1, 22.7, 14.1.

<8-2> Synthesis of M-GTM-I11a

According to Example 3-4, M-GTM-I11a was synthesized in a yield of 64%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.11 (t, J = 4.0 Hz, 6H), 8.07 (d, J = 8.0 Hz, 6H), 7.86 (t, J = 8.0 Hz, 12H), 7.75 ( t, J = 4.0 Hz, 12H), 7.66-7.61 (m, 6H), 7.55-7.49 (m, 6H), 7.43-7.34 (m, 24H), 7.32-7.14 (m, 34H), 6.10 (t, J = 10.0 Hz, 3H), 5.80-5.65 (m, 9H), 5.32-5.19 (m, 8H), 5.08-4.85 (m, 6H), 4.77 (d, J = 6.0 Hz, 3H), 4.52-4.39 (m, 9H), 4.29 (d, J = 12.0 Hz, 3H), 4.11-4.03 (m, 3H), 3.90-3.85 (m, 3H), 3.45-3.25 (m, 18H), 3.22-2.85 (m) , 14H), 1.47-1.38 (m, 3H), 1.29-1.09 (m, 51H), 0.88 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.1, 165.8, 165.6, 165.5, 165.0, 133.5, 133.3, 133.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.3, 129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 96.5, 75.2, 72.6, 71.8, 71.7, 70.8, 70.0, 69.1, 62.5, 58.9, 53.5, 31.9, 29.7, 29.6, 29.4, 26.1, 26.0, 22.7, 14.2.

<8-3> Synthesis of M-GTM-I11

According to Example 3-5, compound M-GTM-I11 was synthesized in a yield of 89%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.18 (d, J = 4.0 Hz, 3H), 4.55-4.46 (m, 3H), 4.05-3.82 (m, 12H), 3.70-3.40 (m, 52H) ), 3.30-3.20 (m, 8H), 1.60-1.52 (m, 6H), 1.35-1.23 (m, 48H), 0.89 (t, J = 12.0 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.1, 103.7, 102.9, 81.2, 78.4, 77.8, 77.6, 76.6, 75.1, 74.8, 74.1, 73.0, 72.8, 72.7, 72.6, 72.0, 71.5, 62.7, 62.3 , 59.4, 54.9, 47.0, 46.9, 33.1, 30.9, 30.8, 30.7, 30.5, 27.3, 23.8, 14.6; HRMS (FAB ⁺ ) : calcd. for C ₈₆ H ₁₆₂ O ₄₁ [M+Na] ⁺ 1875.0523, found 1875.0497.

<Preparation Example 9> Synthesis of M-GTM-I12

<9-1> compound G3 (3,3'-((2-((( R )-2-(dodecyloxy)-3-hydroxypropoxy)methyl)-2-((2-methoxyethoxy)methyl)propane-1,3-diyl)bis (oxy))bis(2-(dodecyloxy)propan-1-ol )) synthesis

According to Example 3-3, compound G3 was synthesized in a yield of 68%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.92 (dd, J _1-2 = 4.0 Hz, J _1-3 = 12.0 Hz, 3H), 3.57-3.54 (m, 2H), 3.51-3.40 (m, 28H), 3.24 (s, 3H), 3.24 (s br, 3H), 1.56 (quin, J = 6.0 Hz, 6H), 1.35-1.20 (m, 42H), 0.88 (t, J = 8.0 Hz, 9H) , ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.8, 71.8, 71.7, 70.7, 70.3, 70.1, 69.1 58.7, 53.3, 45.4, 31.8, 29.5, 29.3, 29.2, 26.2, 22.5, 14.0.

<9-2> Synthesis of M-GTM-I12a

According to Example 3-4, M-GTM-I12a was synthesized in a yield of 65%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.11 (t, J = 4.0 Hz, 6H), 8.07 (d, J = 8.0 Hz, 6H), 7.87 (t, J = 8.0 Hz, 12H), 7.75 ( t, J = 4.0 Hz, 12H), 7.66-7.62 (m, 6H), 7.54-7.47 (m, 6H), 7.44-7.34 (m, 24H), 7.32-7.14 (m, 34H), 6.10 (t, J = 10.0 Hz, 3H), 5.80-5.64 (m, 9H), 5.32-5.21 (m, 8H), 5.06-4.86 (m, 6H), 4.77 (d, J = 6.0 Hz, 3H), 4.52-4.38 (m, 9H), 4.30 (d, J = 12.0 Hz, 3H), 4.11-4.04 (m, 3H), 3.91-3.85 (m, 3H), 3.47-3.25 (m, 18H), 3.22-2.86 (m) , 14H), 1.48-1.38 (m, 3H), 1.33-1.14 (m, 57H), 0.88 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.1, 166.0, 165.8, 165.6, 165.4, 165.1, 165.0, 133.4, 133.3, 133.2, 133.0, 132.8, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 100.5, 100.3, 96.4, 75.1, 73.3, 72.6, 72.5, 71.8, 71.7, 70.8, 69.9, 69.1, 63.6, 62.5, 31.9, 29.7, 29.5, 29.4, 26.1, 26.0, 22.7, 14.1.

<9-3> Synthesis of M-GTM-I12

According to Example 3-5, compound M-GTM-I12 was synthesized in a yield of 89%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.18 (d, J = 4.0 Hz, 3H), 4.56-4.47 (m, 3H), 4.05-3.80 (m, 12H), 3.71-3.41 (m, 52H) ), 3.31-3.20 (m, 8H), 1.62-1.53 (m, 6H), 1.32-1.20 (m, 54H), 0.89 (t, J = 12.0 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.1, 103.7, 102.9, 81.3, 78.3, 77.6, 76.7, 75.1, 74.8, 74.1, 73.0, 72.8, 72.7, 72.5, 72.0, 71.5, 62.7, 62.3, 59.3 , 33.1, 30.8, 30.7, 30.5, 27.3, 23.8, 14.5; HRMS (FAB ⁺ ) : calcd. for C ₈₉ H ₁₆₈ O ₄₁ [M+Na] ⁺ 1916.0959, found 1916.0968.

<Example 4> Synthesis method of M-GTM-Os

The synthesis scheme of M-GTM-Os is shown in FIG. 2 . Three types of compounds of M-GTM-Os were synthesized according to the following synthesis method.

<4-1> Synthesis procedure of compound E (FIG. 2)

에서 4시간 동안 가열하였다. NaOH로 중화한 후, 반응 혼합물을 농축하였다. 생성된 잔류물을 50% NaOH에 용해시키고 tert-뷰틸 암모늄 브로마이드(0.5 당량) 및 알릴 브로마이드(4 당량)를 후속적으로 첨가하였다. 반응 혼합물을 상온에서 5시간 동안 교반하고 55

에서 24시간 동안 열활성화시킨 후 에틸 에테르로 추출하였다. 혼합한 유기층을 염수로 세척하고, 무수 Na₂SO₄로 건조시키고, 감압하에 농축시켰다. 잔류물을 컬럼 크로마토그래피로 정제하여 목적 화합물 E를 74% 수율로 수득하였다. ¹ H NMR (400 MHz, CDCl₃): δ 5.92-5.83 (m, 3H), 5.27 (quin, J = 8.0 Hz, 1.5H), 5.22 (quin, J = 8.0 Hz, 1.5H), 5.14 (quin, J = 8.0 Hz, 1.5H), 5.11 (quin, J = 8.0 Hz, 1.5H), 3.95-3.93 (m, 6H), 3.58-3.55 (m, 2H), 3.52-3.50 (m, 2H), 3.48 (s, 6H), 3.36 (s, 3H); ¹³ C NMR (100 MHz, CDCl₃): δ 135.2, 115.9, 72.1, 71.7, 70.8, 70.1, 69.2, 58.9, 45.4.4-[(2-methoxyethoxy)methyl]-1-methyl-2,6,7-trioxabicyclo[2.2.2]octane was dissolved in DCM/MeOH (1:1). A few drops of the concentrate were added to this solution. HCl and the resulting mixture to 50

was heated for 4 hours. After neutralization with NaOH, the reaction mixture was concentrated. The resulting residue was dissolved in 50% NaOH and tert-butyl ammonium bromide (0.5 equiv) and allyl bromide (4 equiv) were subsequently added. The reaction mixture was stirred at room temperature for 5 hours and 55

After thermal activation for 24 hours, it was extracted with ethyl ether. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the target compound E in 74% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.92-5.83 (m, 3H), 5.27 (quin, J = 8.0 Hz, 1.5H), 5.22 (quin, J = 8.0 Hz, 1.5H), 5.14 (quin , J = 8.0 Hz, 1.5H), 5.11 (quin, J = 8.0 Hz, 1.5H), 3.95-3.93 (m, 6H), 3.58-3.55 (m, 2H), 3.52-3.50 (m, 2H), 3.48 (s, 6H), 3.36 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.2, 115.9, 72.1, 71.7, 70.8, 70.1, 69.2, 58.9, 45.4.

<4-2> Compound F2(3,3'-((2-((2,3-dihydroxypropoxy)methyl)-2-((2-methoxyethoxy)methyl)propane-1,3-diyl)bis(oxy) ) Synthesis procedure of bis(propane-1,2-diol) (step a2 in Fig. 2)

To an ice-cooled solution of compound E dissolved in acetone, H ₂ O and tert-BuOH (1:1:0.2), N-methylmorpholine (NMO) and OsO ₄ (4.0 wt%) were added dropwise. The resulting mixture was stirred at room temperature for 24 hours. After the reaction was complete as indicated by TLC, the solvent and volatile components were removed under reduced pressure. Column chromatography purification of the crude residue gave compound F2 in 74% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.72 (quin, J = 4.0 Hz, 3H), 3.55 (d, J = 8.0 Hz, 6H), 3.50-3.48 (m, 3H), 3.45-3.34 (m) , 18H), 3.29 (s, 3H), 2.94 (s br, 3H), 0.83 (s, 27H), 0.01 (s, 18H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.4, 71.7, 70.8, 70.5, 70.4, 70.3, 64.0, 58.8, 45.4, 25.8, 18.2.

<4-3> General procedure of TBDMS protective vaporization reaction of primary alcohol (step e of FIG. 2)

에서 이미다졸(6.0 당량)을 조금씩 첨가하였다. 5분 후, DCM 중 TBDMSCl(3.6당량)의 용액을 첨가하고 생성된 혼합물을 실온에서 1시간 동안 교반한 다음 얼음으로 켄칭하였다. 증류수로 추가 희석한 후, 반응 혼합물을 DCM으로 추출하였다. 혼합된 DCM 분획을 빙냉 HCl(0.1M), 물 및 염수로 연속적으로 세척한 다음 무수 Na₂SO₄로 건조시켰다. 반응 혼합물로부터 용매를 제거한 후 얻어진 유상 잔류물을 크로마토그래피로 정제하여 목적 화합물 H를 74% 수율로 수득하였다. ¹ H NMR (400 MHz, CDCl₃): δ 3.72 (quin, J = 4.0 Hz, 3H), 3.55 (d, J = 8.0 Hz, 6H), 3.50-3.48 (m, 3H), 3.45-3.34 (m, 18H), 3.29 (s, 3H), 2.94 (s br, 3H), 0.83 (s, 27H), 0.01 (s, 18H); ¹³ C NMR (100 MHz, CDCl₃): δ 72.4, 71.7, 70.8, 70.5, 70.4, 70.3, 64.0, 58.8, 45.4, 25.8, 18.2, -5.4Compound F2 dissolved in THF was added to 0 under argon.

imidazole (6.0 equivalents) was added little by little. After 5 min, a solution of TBDMSCl (3.6 eq) in DCM was added and the resulting mixture was stirred at room temperature for 1 h and then quenched with ice. After further dilution with distilled water, the reaction mixture was extracted with DCM. The combined DCM fractions were washed successively with ice-cold HCl (0.1M), water and brine, then dried over anhydrous Na ₂ SO ₄ . After removing the solvent from the reaction mixture, the obtained oily residue was purified by chromatography to obtain the target compound H in 74% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.72 (quin, J = 4.0 Hz, 3H), 3.55 (d, J = 8.0 Hz, 6H), 3.50-3.48 (m, 3H), 3.45-3.34 (m) , 18H), 3.29 (s, 3H), 2.94 (s br, 3H), 0.83 (s, 27H), 0.01 (s, 18H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 72.4, 71.7, 70.8, 70.5, 70.4, 70.3, 64.0, 58.8, 45.4, 25.8, 18.2, -5.4

<4-4> General procedure of trialkylated triol derivative synthesis (step f in FIG. 2)

에서 24시간 동안 교반하였다. 얼음을 첨가하여 반응을 켄칭한 다음, 물로 희석하였다. 희석된 반응 혼합물을 에틸아세테이트로 추출하고 혼합한 유기 분획을 염수로 세척하고 무수 Na₂SO₄로 건조시켰다. 감압하에서 용매를 제거하여 THF에 용해된 오일성 잔류물을 얻었다. 이 용액에 THF 중 1.0 M TBAF(4.5 당량)를 첨가한 다음, 실온에서 12시간 동안 교반하였다. 반응 종료 후, 반응 혼합물을 물로 희석하고 에틸아세테이트로 세척하였다. 혼합한 유기 분획을 염수로 세척하고 무수 Na₂SO₄로 건조시켰다. 감압하에서 용매를 제거하여 유상 잔류물을 얻었고, 이를 컬럼 크로마토그래피 정제하여 목적 화합물 I를 수득하였다.To an ice-cooled solution of compound H (1.0 equiv) in DMF was added NaH (4.5 equiv) under an inert atmosphere. After bubbling ceased, alkyl iodide (RI; 4.5 eq) was added dropwise and the resulting mixture was stirred at 70

was stirred for 24 hours. The reaction was quenched by addition of ice and then diluted with water. The diluted reaction mixture was extracted with ethyl acetate, and the combined organic fractions were washed with brine and dried over anhydrous Na ₂ SO ₄ . The solvent was removed under reduced pressure to give an oily residue dissolved in THF. To this solution was added 1.0 M TBAF in THF (4.5 eq) and then stirred at room temperature for 12 h. After completion of the reaction, the reaction mixture was diluted with water and washed with ethyl acetate. The combined organic fractions were washed with brine and dried over anhydrous Na ₂ SO ₄ . The solvent was removed under reduced pressure to obtain an oily residue, which was purified by column chromatography to obtain the desired compound I.

<4-5> General synthetic procedure of glycosycosylation reaction (step g in FIG. 1)

Under N ₂ atmosphere, a mixture of compound I (1 eq) and AgOTf (3.6 eq) in anhydrous CH ₂ Cl ₂ was stirred at -45 °C. Then perbenzoylated maltosylbromide (3.6 eq) dissolved in CH ₂ Cl ₂ was added to this suspension. The mixture was stirred at -45 °C for 5 min and then at 0 °C for 30 min. After completion of the reaction (detected by TLC), pyridine was added to the reaction mixture. The reaction mixture was diluted with CH ₂ Cl ₂ (30 mL) and then filtered through celite. The filtrate was washed successively with 1.0 M aqueous Na ₂ S ₂ O ₃ aqueous solution, 0.1 M aqueous HCl solution and brine. Then the organic layer was dried over anhydrous Na ₂ SO ₄ and the solvent was removed by rotary evaporation. The resulting residue was purified by silica gel column chromatography (EtOAc/hexane) to obtain the saccharified target compound.

<4-6> General synthetic procedure for deprotection reaction (step h in FIG. 2)

The O-benzoylated compound was dissolved with anhydrous CH ₂ Cl ₂ and then MeOH was added slowly until persistent precipitation appeared. NaOMe, a methanolic solution of 0.5 M, was added to the reaction mixture to a final concentration of 0.05 M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized using Amberlite IR-120 (H+ form) resin. The resin was removed by filtration, washed with MeOH, and the solvent was removed from the filtrate in vacuo. The residue was purified using silica gel column chromatography (CH ₂ Cl ₂ /MeOH) to obtain the target compound.

<Preparation Example 10> Synthesis of M-GTM-O10

<10-1> compound I1 ((13 R ,21 R )-17-((( R Synthesis of )-3-(decyloxy)-2-hydroxypropoxy)methyl)-17-((2-methoxyethoxy)methyl)-11,15,19,23-tetra oxatritriacontane-13,21-diol)

According to Example 4-4, compound I1 was synthesized in a yield of 30%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.71-3.68 (m, 3H), 3.61-3.40 (m, 30H), 3.37 (s, 3H), 2.74 (s br, 3H), 1.56 (quin, J = 8.0 Hz, 6H), 1.39 (q, J = 8.0 Hz, 2H), 1.36-1.20 (m, 42H), 0.88 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 78.1, 78.0, 73.1, 71.8, 71.7, 71.2, 71.1, 70.9, 70.7, 70.2, 69.2, 62.7, 62.6, 58.9, 45.3, 31.8, 30.0, 29.6, 29.5, 29.3, 26.1, 22.6, 14.1.

<10-2> Synthesis of M-GTM-O10a

According to Example 4-5, M-GTM-O10a was synthesized in a yield of 66%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.08 (t, J = 6.0 Hz, 6H), 7.99 (d, J = 8.0 Hz, 6H), 7.85-7.78 (m, 12H), 7.74-7.72 (m , 15H), 7.64-7.61 (m, 6H), 7.54-7.49 (m, 6H), 7.46-7.36 (m, 24H), 7.34-7.17 (m, 30H), 6.08 (t, J = 10.0 Hz, 3H) ), 5.79-5.62 (m, 10H), 5.35-5.23 (m, 7H), 4.90-4.83 (m, 5H), 4.77-4.73 (m, 3H), 4.54-4.42 (m, 7H), 4.39-4.36 (m, 4H), 4.32-4.21 (m, 4H), 4.13-4.06 (m, 3H), 3.89-3.78 (m, 3H), 3.58-3.54 (m, 2H) 3.46-2.98 (m, 28H), 1.34-1.05 (m, 48H), 0.87 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.1, 165.8, 165.6, 165.4, 165.4, 165.1, 133.4, 133.3, 133.2, 133.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.2, 128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 128.0, 96.4, 75.0, 73.1, 72.8, 72.3, 71.7, 71.0, 70.9, 70.6, 69.9, 69.2, 62.5, 58.9, 31.9, 30.1, 29.6, 29.5, 29.3, 26.0, 25.9, 22.7, 14.1.

<10-3> Synthesis of M-GTM-O10

According to Example 4-6, the compound M-GTM-O10 was synthesized in a yield of 89%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.21 (d, J = 4.0 Hz, 3H), 4.37-4.32 (m, 3H), 4.06-3.85 (m, 12H), 3.74-3.40 (m, 52H) ), 3.34-3.28 (m, 8H), 1.62-1.59 (m, 6H), 1.40-1.29 (m, 42H), 0.94 (t, J = 12.0 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.7, 104.6, 102.9, 81.3, 81.2, 79.3, 77.7, 76.6, 75.1, 74.8, 74.6, 74.1, 73.0, 72.7, 72.0, 71.7, 71.5, 71.3, 62.7 , 62.2, 59.4, 33.2, 31.2, 30.9, 30.8, 30.7, 30.5, 27.3, 23.8, 14.6; HRMS (FAB ⁺ ) : calcd. for C ₈₃ H ₁₅₆ O ₄₁ [M+Na] ⁺ 1832.0020, found 1832.0014.

<Preparation Example 11> Synthesis of M-GTM-O11

<11-1> compound I2 ((14 R ,22 R )-18-((( R Synthesis of )-2-hydroxy-3-(undecyloxy)propoxy)methyl)-18-((2-methoxyethoxy)methyl)-12,16,20,24-tet raoxapentatriacontane-14,22-diol)

According to Example 4-4, compound I2 was synthesized in a yield of 30%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70-3.68 (m, 3H), 3.60-3.38 (m, 30H), 3.38 (s, 3H), 2.80 (s br, 3H), 1.56 (quin, J = 8.0 Hz, 6H), 1.39 (q, J = 8.0 Hz, 2H), 1.35-1.20 (m, 48H), 0.88 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 78.1, 78.0, 73.1, 71.8, 71.6, 71.2, 71.1, 70.9, 70.7, 70.2, 69.2, 62.6, 58.9, 45.3, 31.8, 30.0, 29.6, 29.4, 29.3, 26.1, 22.6, 14.0.

<11-2> Synthesis of M-GTM-O11a

According to Example 4-5, M-GTM-O11a was synthesized in a yield of 66%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.09 (t, J = 6.0 Hz, 6H), 7.99 (d, J = 8.0 Hz, 6H), 7.86-7.78 (m, 12H), 7.75-7.72 (m , 15H), 7.64-7.61 (m, 6H), 7.55-7.49 (m, 6H), 7.46-7.35 (m, 24H), 7.33-7.17 (m, 30H), 6.09 (t, J = 10.0 Hz, 3H) ), 5.80-5.62 (m, 10H), 5.35-5.23 (m, 7H), 4.91-4.83 (m, 5H), 4.77-4.73 (m, 3H), 4.54-4.41 (m, 7H), 4.39-4.36 (m, 4H), 4.32-4.21 (m, 4H), 4.13-4.05 (m, 3H), 3.89-3.77 (m, 3H), 3.58-3.54 (m, 2H) 3.46-2.99 (m, 28H), 1.34-1.06 (m, 54H), 0.88 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.1, 165.8, 165.6, 165.4, 165.1, 165.0, 133.5, 133.3, 133.2, 133.1, 133.0, 129.9, 129.8, 129.7, 129.6, 129.4, 129.2, 128.9, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 101.2, 100.9, 96.4, 75.0, 73.2, 72.8, 72.4, 72.3, 71.8, 71.0, 70.9, 70.8, 70.6, 69.9, 69.5, 69.1, 62.5, 58.9, 45.3, 31.9, 30.1, 29.6, 29.5, 29.4, 26.1, 25.9, 22.7, 14.2.

<11-3> Synthesis of M-GTM-O11

According to Example 4-6, the compound M-GTM-O11 was synthesized in a yield of 89%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.21 (d, J = 4.0 Hz, 3H), 4.37-4.31 (m, 3H), 4.06-3.85 (m, 12H), 3.73-3.38 (m, 52H) ), 3.34-3.27 (m, 8H), 1.64-1.59 (m, 6H), 1.40-1.28 (m, 48H), 0.93 (t, J = 12.0 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.8, 104.7, 102.9, 81.3, 81.2, 79.3, 77.7, 76.6, 75.1, 74.8, 74.7, 74.1, 73.0, 72.7, 72.5, 72.0, 71.7, 71.5, 71.2 , 62.7, 62.3, 59.4, 33.1, 31.2, 30.9, 30.8, 30.7, 30.5, 27.3, 23.8, 14.6; HRMS (FAB ⁺ ) : calcd. for C ₈₆ H ₁₆₂ O ₄₁ [M+Na] ⁺ 1874.0489, found 1874.0494.

<Preparation Example 12> Synthesis of M-GTM-O12

<12-1> Compound I3 ((15) R ,23 R )-19-((( R Synthesis of )-3-(dodecyloxy)-2-hydroxypropoxy)methyl)-19-((2-methoxyethoxy)methyl)-13,17,21,25-tet raoxaheptatriacontane-15,23-diol)

According to Example 4-4, compound I3 was synthesized in a yield of 30%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70-3.67 (m, 3H), 3.61-3.40 (m, 30H), 3.38 (s, 3H), 2.75 (s br, 3H), 1.56 (quin, J = 8.0 Hz, 6H), 1.39 (q, J = 8.0 Hz, 2H), 1.34-1.20 (m, 54H), 0.87 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 78.1, 78.0, 71.8, 71.6, 71.2, 71.1, 70.9, 70.7, 70.2, 69.2, 62.7, 62.6, 58.9, 45.3, 36.4, 31.9, 30.0, 29.6, 29.5, 29.3, 26.1, 22.6, 14.0.

<12-2> Synthesis of M-GTM-O12a

According to Example 4-5, M-GTM-O12a was synthesized in a yield of 66%. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.08 (t, J = 6.0 Hz, 6H), 8.01 (d, J = 8.0 Hz, 6H), 7.86-7.78 (m, 12H), 7.74-7.72 (m , 15H), 7.65-7.61 (m, 6H), 7.55-7.49 (m, 6H), 7.46-7.36 (m, 24H), 7.35-7.16 (m, 30H), 6.08 (t, J = 10.0 Hz, 3H) ), 5.79-5.60 (m, 10H), 5.35-5.23 (m, 7H), 4.90-4.80 (m, 5H), 4.77-4.73 (m, 3H), 4.54-4.42 (m, 7H), 4.39-4.34 (m, 4H), 4.32-4.21 (m, 4H), 4.11-4.06 (m, 3H), 3.88-3.78 (m, 3H), 3.58-3.54 (m, 2H) 3.47-2.98 (m, 28H), 1.34-1.04 (m, 60H), 0.87 (t, J = 8.0 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.1, 165.8, 165.6, 165.5, 165.0, 133.5, 133.3, 133.2, 133.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.4, 129.2, 128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 96.4, 75.0, 73.1, 72.8, 72.4, 71.8, 71.1, 70.9, 70.6, 70.0, 69.1, 63.5, 62.5, 58.9, 31.9, 30.1, 29.7, 29.6, 29.4, 26.1, 25.9, 22.7, 14.2.

<12-3> Synthesis of M-GTM-O12

According to Example 4-6, the compound M-GTM-O12 was synthesized in a yield of 89%. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.21 (d, J = 4.0 Hz, 3H), 4.38-4.32 (m, 3H), 4.07-3.84 (m, 12H), 3.74-3.40 (m, 52H) ), 3.34-3.27 (m, 8H), 1.62-1.58 (m, 6H), 1.41-1.28 (m, 54H), 0.94 (t, J = 12.0 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 104.8, 102.9, 81.3, 79.3, 79.2, 77.7, 76.6, 75.1, 74.8, 74.7, 74.1, 73.0, 71.7, 71.5, 71.2, 62.7, 62.2, 59.4, 33.1 , 31.2, 30.9, 30.8, 30.5, 27.3, 23.8, 14.5; HRMS (FAB ⁺ ) : calcd. for C ₈₉ H ₁₆₈ O ₄₁ [M+Na] ⁺ 1917.0992, found 1917.1056.

<Experimental Example 1> Characteristics of GTMs

In order to confirm the characteristics of the GTMs synthesized according to the synthesis method of Examples 1 to 4, the molecular weight (MW) of the GTMs, the critical aggregation concentration (CAC), and the hydrodynamic diameter of the formed micelles (hydrodynamic diameter; D _h ) was measured.

Specifically, the critical aggregation concentration (CAC) was measured using fluorescence staining and diphenylhexatriene (DPH), and the hydrodynamic diameter ( D _h ) of micelles formed by each agent (1.0 wt%) was It was measured through a dynamic light scattering (DLS) experiment. The measured results are shown in Table 1 in comparison with DDM, which is an existing amphiphilic molecule (detergent).

Detergent MW ^a
(Da) CAC (μM) D _h
(nm) ^b Solubility (wt%) E-GTM-I10 1750.1 10 32.8 ± 0.4 ~10 E-GTM-I11 1792.1 3.0 62.8 ± 0.2 ~5 E-GTM-I12 1834.2 1.5 76.0 ± 0.6 ~3 E-GTM-O10 1750.1 5.0 47.0 ± 0.4 ~10 E-GTM-O11 1792.1 2.0 77.8 ± 0.2 ~5 E-GTM-O12 1834.2 1.5 85.6 ± 1.4 ~1 M-GTM-I10 1810.1 6.0 9.2 ± 0.4 ~10 M-GTM-I11 1852.2 5.0 22.2 ± 4.0 ~10 M-GTM-I12 1894.3 4.0 35.4 ± 1.6 ~10 M-GTM-O10 1810.1 5.0 7.6 ± 0.2 ~10 M-GTM-O11 1852.2 4.0 12.6 ± 0.2 ~10 M-GTM-O12 1894.3 3.0 33.6 ± 0.6 ~10 DDM 510.6 170 6.8 ± 0.6 ~10

^a Molecular weight of the detergent. ^b Hydrodynamic diameter of the micelles determined at 1.0 wt % by dynamic light scattering. The CAC values (0.0015 to 0.010 mM) of most GTMs were significantly smaller than those of DDM (0.17 mM). Therefore, GTMs readily form micelles even at low concentrations, indicating a higher tendency for self-assembly. In addition, the CAC value of GTMs decreased as the length of the alkyl chain increased, which is thought to be due to the increase in hydrophobicity with the extension of the length of the alkyl chain. In addition, when comparing the differences according to the type of pendant, M-GTM in which hydrophilic MEM was present instead of hydrophobic ethyl pendant reduced the hydrophobicity of lipophilic groups, providing higher CAC as expected compared to E-GTM. This means that M-GTM has a more favorable molecular structure for effective aggregate formation than E-GTM. When compared in terms of isomers, in terms of regioisomeric GTMs, GTM-O gave a lower CAC than GTM-I due to the shorter interalkyl chain distance.

In addition, as a result of examining the size distribution of micelles formed by GTMs through DLS, when analyzing the population of amphiphilic compound aggregates in terms of size, the aggregates formed by M-GTM-O11 were different from those formed by E-GTM-O11. This was a general trend for all GTMs, confirming that the self-assembled aggregates formed by M-GTM showed higher homogeneity than those formed by E-GTM (Figs. 3 and 4).

In addition, the size of micelles according to the change in the concentration or temperature of the amphiphilic compound solution was analyzed. Both E-GTM-O11 and M-GTM-O11 showed a tendency to form large aggregates with increasing solution concentration and temperature (FIG. 5).

<Experimental Example 2> Evaluation of GTMs LeuT membrane protein structure stabilization ability

An experiment was conducted to measure the structural stability of the LeuT protein by GTMs. Each amphiphilic compound was used at a concentration of CMC + 0.04 wt% or CMC + 0.2 wt%, and the substrate binding properties of LeuT were measured using [ ³ H]-Leu through a scintillation proximity assay (SPA). Measurements were each performed at regular intervals during a 13-day incubation period at room temperature.

Specifically, a wild type LeuT (leucine transporter) derived from the thermophilic bacterium Aquifex aeolicus was purified by the previously described method (G. Deckert et al. Nature 1998, 392, 353-358). LeuT was expressed in E. coli C41 (DE3) transformed with pET16b encoding a C-terminal 8xHis-tagged transporter (expression plasmid was provided by Dr E. Gouaux, Vollum Institute, Portland, Oregon, USA). . Briefly, after separation of the bacterial membrane and solubilization in 1 wt% DDM, the protein was bound to Ni ²⁺ -NTA resin (Life Technologies, Denmark), 20 mM Tris-HCl (pH 8.0), 1 mM NaCl, 199 mM Eluted in KCl, 0.05% (w/v) DDM and 300 mM imidazole. Thereafter, purified LeuT (0.5 mg/ml) was prepared except for DDM and imidazole in the same buffer as above, and GTMs or DDM had a final concentration of CMC + 0.04% (w/v) or CMC + 0.2 wt% (w/ v) was diluted with buffer supplemented with Protein samples were incubated for 14 hours at room temperature, centrifuged at designated times, and protein properties were confirmed by measuring [ ³ H]-Leucine binding capacity using SPA. SPA was prepared from each protein in buffer containing 450 mM NaCl, respective GTMs (or DDM), 20 nM [ ³ H]-Leucine and 1.25 mg/ml copper chelate (His-Tag) YSi beads (Perkin Elmer, Denmark). was performed with the sample. Total [ ³ H]-Leucine binding to each sample was measured using a MicroBeta liquid scintillation counter (Perkin Elmer).

As shown in FIG. 6 , all M-GTMs had similar initial activity compared to DDM, and the effect of maintaining the substrate binding properties of LeuT during the 13-day incubation period was superior to that of DDM. In addition, as shown in FIG. 7 , it was confirmed that most of the E-GTMs exhibited superior activity compared to DDM, except for I12, 010 and 012. It was confirmed that the most excellent substrate binding activity was confirmed in the compound having a relatively short alkyl chain length, and it was confirmed that the initial activity of the compound having a relatively long alkyl chain was low.

These results suggest that the type of structure and the length of the alkyl chain of GTMs acted as important factors in maintaining the structural stability of LeuT.

<Experimental Example 3> Evaluation of MelB membrane protein structure stabilization ability of GTMs

Plasmid pK95βAHB/WT MelB _St /CH10 was used to express Salmonella typhimurium MelB _St (melibiose permease) with a 10-His tag at the C-terminus in E. coli DW2 cells (ΔmelB and ΔlacZY). Cell growth and membrane preparation were performed according to the method described in AS Ethayathulla et al. ( Nat. Commun . 2014, 5 , 3009). Protein assays were performed with the Micro BCA kit (Thermo Scientific, Rockford, IL). P.S. Chae et al. Nat. GTMs or DDMs were evaluated for MelB _St stability using the protocol described in Methods 2010, 7, 1003-1008. Membrane samples containing MelB _St (final protein concentration of 10 mg/mL) were mixed with a dissolution buffer (20 mM sodium phosphate, pH 7.5, 200 mM NaCl, 10%) containing 1.5% (w/v) DDM, or GTMs. glycerol, 20 mM melibiose) for 90 min at 4 temperatures (0, 45, 55, 65 °C). To remove insoluble materials, ultracentrifugation was performed at 355,590 g in a Beckman Optima ^TM MAX ultracentrifuge equipped with a TLA-100 rotor at 4° C. for 45 minutes. Lysed portions were separated by SDS-15% PAGE and then immunoblotted with HisProbe-HRP antibody (Thermo Scientific). Membrane fractions containing 20 μg of protein without treatment were used to represent total MelB, and treated samples were loaded into each well in equal volumes. MelB _St was measured by ImageQuant LAS 4000 Biomolecular Imager (GE Health Care Lifer Science) using SuperSignal West Pico chemiluminescent substrate.

As shown in FIG. 8a , most GTMs at 0° C. showed less MelB protein extraction efficiency than DDM.

However, when the temperature was raised to 45 °C, most of the GTMs showed an increase in MelB protein solubilization ability compared to 0 °C, and GTMs (M-GTM-I10 and M-GTM-O10) were similar to DDM proteins. showed the ability to maintain the solubilized state.

When the temperature was raised to 55 °C, DDM markedly lowered protein solubilization ability, but most GTMs except for GTMs having long alkyl chains showed superior ability to maintain MelB protein solubilization state than DDM. At a temperature of 65 °C, neither GTMs nor DDM solubilized MelB protein was identified.

Overall, at a low temperature (0℃), DDM showed similar or higher protein extraction efficiency than GTMs, but it was confirmed that the amount of MelB protein available by GTMs increased more than DDM as the temperature increased (45℃ and 55℃). , It was confirmed that DDM was somewhat better in protein extraction efficiency, but GTMs had better ability to maintain protein solubilization, that is, protein stabilization ability.

Additionally, Trp → D ² G FRET analysis using a right side out (RSO) vesicle was performed. Specifically, RSO membrane vesicles were prepared by osmosis from E. coli DW2 cells containing MelB _St or MelB _Ec . RSO membrane vesicles mixed in buffer (pH 7.5) containing 100 mM KPi and 100 mM NaCl at a protein concentration of 1 mg/mL were treated with 1.0% DDM or GTMs at 23 °C for 60 min and 4 °C for 45 min. At 300,000 g or more, ultracentrifugation was performed using a TLA 120.2 rotor. The supernatant was applied to the Trp→D ² G FRET test using an Amico-Bowman Series 2 (AB2) Spectrofluorometer. The tryptophan residue was excited at 290 nm, and Trp→D ² G FRET was recorded at 465 nm and 490 nm for MelB _Ec and MelB _St , respectively. 10 μM D ² G and excess melibiose or equal volume of water were added at 1 and 2 min points, respectively, in time tracking.

As shown in FIG. 8b , the addition of D ² G to the active MelB _St provides a strong fluorescence signal, and when melibiose, which competes with D ² G, is added, ligand-substrate exchange occurs at the binding site and the fluorescence signal is reduced. do. While DDM solubilized MelB _St showed an adequate response to the sequential addition of D ² G and melibiose, the protein's function was completely lost when the less stable homolog MelB _EC obtained from E. coli was used under the same conditions. was observed to be In contrast, in the case of both MelB homologues solubilized with M-GTMs, their functions were well preserved. Therefore, it was confirmed that these M-GTMs are superior to DDM in maintaining MelB in a functional form.

<Experimental Example 4> β of GTMs ₂ Evaluation of AR membrane protein structure stabilization ability

An experiment was conducted to measure the structural stability of human β ₂ adrenergic receptor (β ₂ AR) and G-protein coupled receptor (GPCR) by GTMs. That is, the receptor purified with DDM was diluted with a buffer solution containing only each GTMs without CHS (cholesteryl hemisuccinate) or a buffer solution containing CHS and DDM to perform amphipathic molecular exchange. The final concentration of the amphipathic molecule was CMC+0.2 wt%, and the ligand binding properties of the receptor were determined by binding of [ ³ H]-dihydroalprenolol ([ ³ H]-DHA).

Specifically, the radioligand binding test was performed using the following method. β ₂ AR was purified using 0.1% DDM (DM Rosenbaum et al., Science , 2007, 318, 1266-1273.), and finally concentrated to about 10 mg/ml (about 200 μM). A master binding mixture containing 10 nM [ ³ H]-Dihydroalprenolol (DHA) supplemented with 0.5 mg/ml BSA in 0.2% amphiphilic compounds (DDM or GTMs) was prepared using β ₂ AR purified with DDM. Receptors purified with DDM or GTMs were incubated with 10 nM [ ³ H]-DHA at room temperature for 3 or 5 days. The mixture is loaded onto a G-50 column, the flow-through is collected with 1 ml binding buffer (20 mM HEPES pH 7.5, 100 mM NaCl supplemented with an amphiphilic compound of 0.5 mg/ml BSA and 20xCMC each), and 15 ml Filled with scintillation fluid. Receptor-bound [ ³ H]-DHA was measured with a scintillation counter (Beckman). The degree of binding of [ ³ H]-DHA was shown as a column graph.

As shown in FIG. 9 , as a result of performing a test to confirm the ligand binding retention properties of the initial receptor, most GTMs, except for E-GTM-O12 and E-GTM-I12 having long alkyl chains, were more receptor ligands than DDM. It was confirmed that the binding ability was excellent.

As shown in FIG. 10 , as a result of performing a test to confirm the ligand-binding retention properties of the receptor for a long time, it was confirmed that most GTMs are superior to the DDM in maintaining the ligand-binding ability of the receptor for a long period of time. In particular, the receptor solubilized in M-GTM-O12 was the best by maintaining 90% or more ligand binding ability after 5 days of culture.

<Experimental Example 5> MOR thermal stability test

To perform long-term stability assays, MOR was mixed with a buffer solution (20 mM HEPES pH 7.5, 100 mM NaCl) containing amphiphilic compounds (DDM or M-GTMs) with final concentrations of 20 nM and 0.1% of the final receptor and amphiphilic compounds, respectively. diluted as much as possible. Specific ligand binding by storing MOR solubilized in each amphiphilic compound (DDM or GTMs) for 6 days at 4 °C and incubating the receptor with 30 nM of radioactive [3H]-diprenorphine (DPN) for 60 min at room temperature ability was measured. Non-specific binding was determined by incubating the receptors with 30 nM [ 3 H]-DPN and 100 μM Naloxone for 60 min at room temperature. After incubation the mixture was loaded into a Zeba™ 96-well spin desalting plate, 40K MWCO. The flow-through containing the ligand-bound receptor was collected via centrifugation. After adding 5 ml of scintillation fluid, radioactivity was measured with a scintillation counter (Beckman). Three specific binding groups and one non-specific binding group were obtained for each amphiphilic compound. The final binding capacity was calculated by subtracting the radioactivity of the non-specific binding group from the specific binding group.

As a result, the receptor solubilized in DDM rapidly lost activity over time, and the residual activity was only 10% after 8 hours of incubation. In contrast, all M-GTMs tested were significantly more effective than DDM in preserving receptor stability over the course of 3 days incubation. Similar to what was observed with β ₂ AR, M-GTMs appeared to be more effective than DDMs in maintaining MOR receptor stability for a long period of time ( FIG. 10 ).

Claims (19)

A compound represented by Formula 1 or 2 or an isomer thereof:
[Formula 1]

[Formula 2]

In Formula 1 or 2,
R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₃₀ alkyl group;
X ¹ to X ³ are each independently a saccharide; and
L is a C _1-5 alkyl group or C _1-5 alkoxy, wherein the C _1-5 alkoxy may be substituted with C _1-5 alkoxy or saccharide.
According to claim 1, wherein the saccharide is a monosaccharide (monosaccharide) or disaccharide (disaccharide) compound or an isomer thereof.
The compound or isomer thereof according to claim 1, wherein the saccharide is glucose or maltose.
According to claim 1, wherein R ^One To R ³ Each independently represents a substituted or unsubstituted C ₃ -C ₂₀ Alkyl group; X ¹ to X ³ are each independently glucose or maltose; and L is a C _1-3 alkyl group, or an isomer thereof.
According to claim 1, wherein R ^One To R ³ Each independently represents a substituted or unsubstituted C ₃ -C ₂₀ Alkyl group; X ¹ to X ³ are each independently glucose or maltose; And L is C _1-3 alkoxy, wherein the C _1-3 alkoxy is a compound substituted with C _1-3 alkoxy or glucose, or an isomer thereof.
The method according to claim 1, wherein R ¹ to R ³ are an unsubstituted C ₃ -C ₁₅ alkyl group; X ¹ to X ³ are glucose or maltose; and L is a methyl group, methoxyethoxy, or glucose ethoxy, or an isomer thereof.
The compound or isomer thereof according to claim 1, wherein the compound is an amphiphilic molecule for extracting, solubilizing, stabilizing, crystallizing or analyzing a membrane protein.
The compound or isomer thereof according to claim 1, wherein the compound has a critical aggregation concentration (CAC) of 0.0001 to 1 mM in aqueous solution.
A composition for extracting a membrane protein comprising the compound according to claim 1, or an isomer thereof.
A composition for solubilizing a membrane protein comprising the compound according to claim 1 or an isomer thereof
A composition for stabilizing a membrane protein comprising the compound according to claim 1 or an isomer thereof
A composition for crystallization of a membrane protein comprising the compound according to claim 1 or an isomer thereof.
A composition for analysis of a membrane protein comprising the compound according to claim 1, or an isomer thereof.
14. The composition according to any one of claims 9 to 13, wherein the composition is a formulation of micelles, liposomes, emulsions or nanoparticles.
The method according to any one of claims 9 to 13, wherein the membrane protein is LeuT (Leucine transporter), MelB (melibiose permease), β ₂ AR (human β ₂ adrenergic receptor), MOR (mouse μ-opioid receptor) or A composition that is a combination of two or more thereof.
1) preparing a compound of Formula 4 by introducing an alkanol through an epoxide ring opening reaction of the compound of Formula 3 (step 1); and
2) A method for preparing a compound represented by Formula 1, comprising the step of introducing a saccharide having a protecting group to the compound of Formula 4 through a saccharification reaction and then performing a deprotection reaction (Step 2):
[Scheme 1]

In Scheme 1,
R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₃₀ alkyl group;
X ¹ to X ³ are each independently a saccharide; and
L is a C _1-5 alkyl group or C _1-5 alkoxy, wherein the C _1-5 alkoxy may be substituted with C _1-5 alkoxy or saccharide.
17. The method of claim 16, wherein R ¹ to R ³ are an unsubstituted C ₃ -C ₁₅ alkyl group; X ¹ to X ³ are glucose or maltose; and L is a methyl group, methoxyethoxy or glucoseethoxy.
1) preparing a compound of Formula 6 by attaching a protecting group Y to the compound of Formula 5 (step 1);
2) reacting the compound of formula 6 with an alkyl iodide and then removing the protecting group Y to prepare a compound of formula 7 (step 2); and
3) A method for preparing a compound represented by Formula 2, comprising the step of introducing a saccharide having a protecting group to the compound of Formula 7 through a saccharification reaction and then performing a deprotection reaction (step 3):
[Scheme 2]

In Scheme 2,
R ¹ to R ³ are each independently a substituted or unsubstituted C ₃ -C ₃₀ alkyl group;
X ¹ to X ³ are each independently a saccharide;
L is a C _1-5 alkyl group or C _1-5 alkoxy, wherein the C _1-5 alkoxy may be substituted with C _1-5 alkoxy or saccharide; and
Y is a protecting group.
19. The method of claim 18, wherein R ¹ to R ³ are an unsubstituted C ₃ -C ₁₅ alkyl group; X ¹ to X ³ are glucose or maltose; and L is a methyl group, methoxyethoxy or glucoseethoxy.

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