KR102039483B1 - Tandem neopentyl glycol-based amphiphiles and uses thereof - Google Patents

Abstract

The present invention relates to an amphiphilic compound based on tandem neopentyl glycol, a method for preparing the same, and a method for extracting, solubilizing, stabilizing or crystallizing a membrane protein using the same. By using the tandem neopentyl glycol-based compound according to the present invention, not only the membrane protein solubilization effect is excellent, but also the membrane protein can be stably stored in an aqueous solution for a long time, and thus the functional analysis and structural analysis are performed. Can be utilized. Membrane protein structure and function analysis is one of the areas of greatest interest in current biology and chemistry, and since more than half of currently developed drugs target membrane proteins, they are applied to the study of membrane protein structures closely related to drug development. This is possible.

Description

Tandem neopentyl glycol-based amphiphiles and uses thereof

The present invention relates to a newly developed amphiphilic compound based on tandem neopentyl glycol and a method for extracting, solubilizing, stabilizing, crystallizing or analyzing membrane proteins using the same.

Membrane proteins play an important role in biological systems. Since these bio-macromolecules contain hydrophilic and hydrophobic moieties, amphiphilic molecules are required to extract membrane proteins from the lipid environment and to dissolve and stabilize them in aqueous solutions.

For the structural analysis of membrane proteins, it is necessary to obtain high-quality membrane protein crystals, which requires the structural stability of membrane proteins in aqueous solution. There are more than 100 existing amphiphilic molecules that have been used for membrane protein research, but only about 5 of them have been actively used for membrane protein structure research. These five amphoteric molecules are n-octyl-β-D-glucopyranoside (OG), n-nonyl-β-D-glucopyranoside (NG), n-decyl-β-D-maltopyranoside (DM), and DDM (n- dodecyl-β-D-maltopyranoside) and LDAO (lauryldimethylamine- N- oxide) are included (nonpatent literature 1, nonpatent literature 2). However, many membrane proteins surrounded by these molecules have significant limitations in studying the function and structure of membrane proteins utilizing these molecules because their structure is easily denatured or aggregated and quickly loses their function. This is because conventional molecules do not exhibit sufficiently diverse characteristics due to their simple chemical structure.

For structural analysis of membrane proteins, it is important to maintain the structural stability of membrane proteins in aqueous solution, and membrane proteins are known as amphiphilic molecules because they have many unknown types and structural characteristics. The number of membrane proteins that can be used has been limited. Thus, in accordance with the necessity of developing various amphiphilic molecules different from the fluid alkyl chains of the existing amphiphilic molecules, the present inventors repeatedly connect two less fluid neopentyl glycols to introduce two quaternary carbons into the center of the molecule. The present invention was completed by developing a new amphiphilic compound that promotes membrane protein crystallization by greatly restricting the overall fluidity.

 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 the formula (1).

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

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

Still another object of the present invention is to provide a method for extracting, solubilizing, stabilizing, crystallizing or analyzing membrane proteins using the compounds.

One embodiment of the present invention provides a compound represented by Formula 1:

[Formula 1]

In Chemical Formula 1,

R ¹ , R ² and Each R ³ may independently be a substituted or unsubstituted C ₃ -C ₂₀ alkyl group, a substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl group, or a substituted or unsubstituted C ₃ -C ₂₀ aryl group;

X ¹ , X ² and X ³ may each independently be a saccharide linked to oxygen;

Y ¹ , Y ² and Y ³ are oxygen (O); And

M may be 0 or 1.

As used herein, the term "saccharide" refers to a compound that is relatively small molecule in carbohydrates and is sweet in water. Sugars are classified into monosaccharides, disaccharides and polysaccharides according to the number of molecules constituting the sugar.

The sugar used in the above embodiments may be monosaccharide or disaccharide, specifically glucose or maltose, and more specifically maltose, but not limited thereto. Do not.

The saccharide may act as a hydrophilic group. Compound according to an embodiment of the present invention by connecting three hydrophilic saccharides in parallel to increase the size of the hydrophilic group while minimizing the increase in length to reduce the size when forming a complex with the membrane protein. If the complex of the compound with the membrane protein is small, high quality membrane protein crystals can be obtained (GG Prive, Methods 2007, 41, 388-397). In particular, amphiphilic molecules with small hydrophilic groups, such as glucosides, can have an excellent effect on membrane protein crystallization.

In addition, the R ¹ , R ² And R ³ may act as a hydrophobic group. Compounds according to one embodiment of the present invention were introduced into three hydrophobic groups of different lengths in order to optimize the hydrophile-lipophile balance (hydrophile-lipophile balance).

Compound according to an embodiment of the present invention may be linked to a hydrophobic group and a hydrophilic group by ether (ether) bond. In other words, a linker for sufficiently securing the fluidity of the alkyl chain is introduced while maintaining the rigidity of the center of the molecule. Specifically, the ether bond may be introduced using a tandem neopentyl glycol linker.

Specifically, the R ¹ , R ² And R ³ may be a C ₇ -C ₁₄ alkyl group; R ¹ , R ² and R ³ may be the same; Y ¹ , Y ² and Y ³ may be oxygen; X ¹ to X ³ may be maltose connected by oxygen; And m may be O.

In addition, the R ¹ , R ² And R ³ may be a C ₇ -C ₁₄ alkyl group; R ¹ , R ² and R ³ may be the same; Y ¹ , Y ² and Y ³ may be oxygen; X ¹ to X ³ may be maltose connected by oxygen; And m may be 1.

In one embodiment of the invention, the R ¹ , R ² And R ³ is a C ₉ -C ₁₄ alkyl group; R ¹ , R ² and R ³ is the same; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The compound whose m is 0 or 1 was named "tandem neopentyl glycol-derived maltosides" (TNMs).

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₉ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is named 1 compound "TNM-C9L", it can be represented by the formula (2).

[Formula 2]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₀ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is named 1 compound "TNM-C10L", it can be represented by the formula (3).

[Formula 3]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₁ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is named 1 compound "TNM-C11L", it can be represented by the formula (4).

[Formula 4]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₂ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is named 1 compound "TNM-C12L", it can be represented by the formula (5).

[Formula 5]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₃ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is named 1 compound "TNM-C13L", it can be represented by the formula (6).

[Formula 6]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₄ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is named "TNM-C14L" and the compound of 1 may be represented by the following formula (7).

[Formula 7]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₁ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is a compound of 0 is named "TNM-C11S", it can be represented by the formula (8).

[Formula 8]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₂ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is a compound of 0 is named "TNM-C12S", it can be represented by the formula (9).

[Formula 9]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₃ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is a compound of 0 is named "TNM-C13S", it can be represented by the formula (10).

[Formula 10]

More specifically, in one embodiment of the present invention, the R ¹ , R ² And R ³ is a C ₁₄ alkyl group; Y ¹ , Y ² and Y ³ are oxygen; X ¹ to X ³ are maltose connected by oxygen; The m is a compound of 0 is named "TNM-C14S", it can be represented by the formula (11).

[Formula 11]

Compounds according to other embodiments of the invention may be, but are not limited to, amphiphilic molecules for extracting, solubilizing, stabilizing, crystallizing or analyzing membrane proteins.

As used herein, the term "amphiphilic molecule" refers to a molecule having a hydrophobic group and a hydrophilic group in one molecule and having affinity for both polar and nonpolar solvents. Phospholipid molecules present in surfactants and cell membranes are amphiphilic molecules with hydrophilic groups at one end and hydrophobic groups at the other end, and have the characteristic of forming micelles or liposomes in aqueous solution. Hydrophilic groups have polarity, but because the nonpolar groups coexist, their amphiphilic molecules tend to be insoluble in water. However, when the concentration is above a certain limit concentration (critical micelle concentration, CMC), hydrophobic groups are collected by hydrophobic interactions, and micelles with hydrophilic groups are formed on the surface, and solubility in water is greatly increased.

The method of measuring CMC is not particularly limited, but may be used by a method well known in the art, and may be measured by, for example, a fluorescent dye method using diphenylhexatriene (DPH).

The compound according to an embodiment of the present invention may have a critical micelle concentration (CMC) in an aqueous solution of 0.0001 mM to 1 mM, specifically, 0.0001 mM to 0.0 1 mM, more specifically, 0.0004 mM to 0.001 mM, This is not restrictive.

In the case of DDM, which is mainly used for membrane protein research, the TNMs of this embodiment had a much lower CMC value than that of DDM, compared to a critical micelle concentration of 0.17 mM. Therefore, since the micelles are easily formed in small amounts, TNMs can be effectively studied and analyzed membrane proteins using a small amount, it was confirmed that the advantage over DDM.

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

The composition may be, but is not limited to, a formulation of micelles, liposomes, emulsions or nanoparticles.

The micelles may have a radius of 2.0 nm to 60.0 nm, specifically 3.0 nm to 55.0 nm, and more specifically micelles formed by TNMs according to embodiments of the present invention have a radius of 3.0 nm to 50.0 nm. May be, but is not limited thereto.

The method of measuring the radius of the micelle is not particularly limited, but a method well known in the art may be used, and may be measured using, for example, dynamic light scattering (DLS) experiments.

The size of the micelle formed by the TNMs can be seen to be wide range.

The micelles, liposomes, emulsions or nanoparticles may comprise a membrane protein 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 by the micelles.

The composition may further include a buffer or the like that may be helpful for the extraction, solubilization, stabilization or analysis of the membrane protein.

In addition, the present invention provides a method for preparing a compound represented by the following Chemical Formula 1, comprising the following steps 1) to 7):

1) 5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane (5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane) and alcohol ( alcohol) to synthesize dialkylated diols;

2) synthesizing a trialkylated monool by adding 1-bromoalkane to the product of step 1);

3) allylating the alcohol by adding allyl iodide to the product of step 2);

4) synthesizing trialkylated monool by hydroboration oxidation of the product of step 3);

5) 4- (bromomethyl) -l-methyl-2,6,7-trioxabicyclo [2.2.2] -octane (4- (bromomethyl) -l-methyl-2 in the product of step 4) , 6,7-trioxabicyclo [2.2.2] -octane) to synthesize trialkylated triols;

6) introducing a saccharide with a protecting group by performing a glycosylation reaction on the product of step 5); And

7) performing a deprotection reaction (deprotection) reaction on the product of step 6);

[Formula 1]

In Chemical Formula 1,

R ¹ , R ² and Each R ³ is independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group, a substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl group, or a substituted or unsubstituted C ₃ -C ₂₀ aryl group;

X ¹ , X ² and X ³ are each independently a saccharide linked by oxygen;

Y ¹ , Y ² and Y ³ are oxygen (O); And

M is 1.

The compound prepared according to the method may be a compound represented by Formula 2 to Formula 7.

In addition, the present invention provides a method for preparing a compound represented by the following Chemical Formula 1, comprising the following steps 1) to 8):

1) Alkylation of 5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane (5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane) Reacting to synthesize a compound in the form of a dialkylated diol;

2) adding 1-bromoalkane to the product of step 1) to synthesize a trialkylated monool type compound;

3) allylating the alcohol by adding allyl iodide to the product of step 2);

4) hydroboration oxidation of the product of step 3) to synthesize trialkylated monool compounds;

5) synthesizing the compound of the trialkylated aldehyde form through alcohol oxidation of the product of step 4);

6) adding formaldehyde and water-soluble alkaline ethanol to the product of step 5) to synthesize a trialkylated triol form of the compound;

7) introducing a saccharide to which a protecting group is attached by performing a glycosylation reaction on the product of step 6); And

8) A process for preparing a compound represented by the following Chemical Formula 1 comprising the step of performing a deprotection reaction on the product of step 7):

[Formula 1]

In Chemical Formula 1,

R ¹ , R ² and Each R ³ is independently a substituted or unsubstituted C ₃ -C ₂₀ alkyl group, a substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl group, or a substituted or unsubstituted C ₃ -C ₂₀ aryl group;

X ¹ , X ² and X ³ are each independently a saccharide linked by oxygen;

Y ¹ , Y ² and Y ³ are oxygen (O); And

M is zero.

The compound prepared according to the method may be a compound represented by Formula 8 to Formula 11.

Still another embodiment of the present invention provides a method for extracting, solubilizing, stabilizing, crystallizing or analyzing membrane proteins. Specifically, the present invention provides a method for extracting, solubilizing, stabilizing, crystallizing, or analyzing a membrane protein, comprising treating the membrane protein with a compound represented by Formula 1 in an aqueous solution:

[Formula 1]

In Chemical Formula 1,

R ¹ , R ² and Each R ³ may independently be a substituted or unsubstituted C ₃ -C ₂₀ alkyl group, a substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl group, or a substituted or unsubstituted C ₃ -C ₂₀ aryl group;

X ¹ , X ² and X ³ may each independently be a saccharide linked to oxygen;

Y ¹ , Y ² and Y ³ are oxygen (O); And

M may be 0 or 1.

Specifically, the R ¹ , R ² And R ³ may be a C ₇ -C ₁₄ alkyl group; R ¹ , R ² and R ³ may be the same; Y ¹ , Y ² and Y ³ may be oxygen; X ¹ to X ³ may be maltose connected by oxygen; And m may be O.

In addition, the R ¹ , R ² And R ³ may be a C ₇ -C ₁₄ alkyl group; R ¹ , R ² and R ³ may be the same; Y ¹ , Y ² and Y ³ may be oxygen; X ¹ to X ³ may be maltose connected by oxygen; And m may be 1.

The compound may be 11 kinds of compounds represented by the following Chemical Formula 2 according to one embodiment of the present invention, but is not limited thereto.

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

The membrane protein includes any protein or glycoprotein introduced into the cell membrane lipid bilayer, specifically UapA (uric acid-xanthine / H ⁺ symporter), MelB (melibiose permease), LeuT (Leucine transporter), GPCRs (G- protein coupled receptors) or a combination of two or more thereof, but is not limited thereto.

As used herein, the term "extraction of membrane proteins" refers to the separation of membrane proteins from membranes.

As used herein, the term "solubilization" of membrane proteins means that membrane proteins that are insoluble in water are dissolved in micelles in aqueous solution.

As used herein, the term "stabilization of membrane proteins" means the stable preservation of tertiary or quaternary structures such that the structure, function of the membrane protein does not change.

As used herein, the term "crystallization of membrane proteins" means the formation of crystals of membrane proteins in solution.

As used herein, the term "analysis" of a membrane protein means to analyze the structure or function of the membrane protein. In the above embodiment, the analysis of the membrane protein may use a known method, but is not limited thereto. For example, the structure of the membrane protein may be analyzed by electron microscopy.

Using a tris- or neopentyl glycol-based compound according to embodiments of the present invention can be stored for a long time stably compared to the existing compound in aqueous solution, it can be utilized for its functional analysis and structural analysis.

Membrane protein structure and function analysis is one of the areas of greatest interest in current biology and chemistry, and since more than half of currently developed drugs are targeted to membrane proteins, it is important to study protein structure closely related to drug development. Application is possible.

Specifically, the compound according to the embodiments of the present invention has a small hydrophilic group and may not only have an excellent effect on the membrane protein crystallization, but also cause a problem that the stability of the membrane protein generated when using a small hydrophilic group is somewhat deteriorated. Shows better membrane protein stability than conventional compounds.

In addition, the compound according to the embodiments of the present invention can be synthesized from a readily available starting material by a simple method, thereby enabling mass production of the compound for membrane protein research.

1 is a diagram showing a synthesis scheme of TNMs of the present invention.
2 is a diagram showing a synthesis scheme of TNMs of the present invention.
3 is a diagram showing the chemical structure of the TNMs of the present invention.
4 is a dynamic light scattering (DLS) profile of micelles formed by (a) TNM-Ls and (b) TNM-Ss. The analysis based on the number provided information on the size of the micelles.
Figure 5 shows the structural stability of the LHI-RC complex by (a) TNM-Ls or (b) TNM-Ss used at a concentration of CMC + 0.04 wt% in aqueous solution, measured by monitoring the absorbance at 875 nm. It is also.
Figure 6 shows the structural stability of the LHI-RC complex by (a) TNM-Ls or (b) TNM-Ss used at a concentration of CMC + 0.2 wt% in an aqueous solution, measured by monitoring the absorbance at 875 nm. It is also.
Figure 7 shows the results of measuring the structural stability of the UapA protein by (a) TNM-Ls or (b) TNM-Ss used in CMC + 0.2 wt% concentration in an aqueous solution using a CPM assay.
Figure 8 is a diagram showing the results of measuring the structural stability of the UapA protein by (a) TNM-Ls or (b) TNM-Ss used in CMC + 0.04 wt% concentration in an aqueous solution using a CPM assay.
9 is a diagram showing the results of measuring the stability of the LeuT protein by TNMs used at the concentration of CMC + 0.04 wt% using a scintillation proximity assay (SPA).
10 is a diagram showing the results of measuring the stability of LeuT protein by TNMs used at the concentration of CMC + 0.2 wt% using a scintillation proximity assay (SPA).
11 is a diagram showing the results of measuring protein activity using [ ³ H] -dihydroalprenolol (DHA) at regular intervals while incubating at room temperature for 4 days to measure the effect on the stability of β ₂ AR by TNMs.
12 is a diagram showing the results of measuring protein activity using [ ³ H] -dihydroalprenolol (DHA) at regular intervals while incubating at room temperature for 4 days to measure the effect on the stability of β ₂ AR by TNM-C12L. .

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples merely illustrate the contents of the present invention and do not limit or limit the scope of the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can be easily inferred to be within the scope of the present invention.

< EXAMPLE  1> TNM - Ls  Synthetic Method

The synthesis scheme of TNM-Ls is shown in FIG. 1. Six compounds of TNM-Ls (Tandem Neopentyl Glycol Maltosides) were synthesized according to the synthesis methods of the following <1-1> to <1-7>, and are shown in FIG. 3.

<1-1> Dialkylated Of dialkylated diols  General Synthesis Procedure (Step a) of Figure 1

To a solution of NaH (4.0 equiv) mixed with DMF, each alkyl alcohol (3.0 equiv) was added dropwise under 0 ° C., N ₂ atmosphere, and the resulting mixture was stirred at room temperature for 15 minutes. After addition of 5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane (A) (1.0 equiv), the reaction mixture is warmed to 120 ° C. and at this temperature for 15 h It was left. After cooling to room temperature, the reaction was terminated with ice water and the organics were extracted three times with diethyl ether. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated on a rotary evaporator. After the solvent was completely evaporated, the residue was dissolved in a 1: 1 mixture of CH ₂ Cl ₂ and MeOH and p-toluene sulfonic acid (p-TSA) monohydrate (catalyst amount) was added and stirred at room temperature for 2 hours. The reaction mixture was neutralized with saturated aqueous NaHCO ₃ solution and the volume of solvent was reduced with a rotary evaporator. The reaction mixture was partitioned between CH ₂ Cl ₂ and H ₂ O. The collected organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. Flash column chromatography (EtOAc / hexanes) afforded an ether-containing diol (B1-B6) as a white solid (92-94% (two steps)).

<1-2> Trialkylated Monool ( trialkylated  mono- ol General synthesis procedure (step b of FIG. 1)

To a suspension of NaH (1.3 equiv) mixed with anhydrous DMF was slowly added a solution of diol derivative (B1-B6) (1.0 equiv) mixed with the anhydrous DMF at 0 ° C. under argon. After 15 min stirring, 1-bromoalkane (RBr) (1.3 equiv) was added to the mixture and the temperature was increased to 100 ° C. The reaction mixture was left at this temperature for 4 hours, then cooled to room temperature and terminated with H ₂ O. The reaction mixture was extracted twice with CH ₂ Cl ₂ , washed with brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was concentrated in vacuo and the resulting residue was purified by silica gel column chromatography (EtOAc / hexane) to give trialkyl-containing monools (C1-C6) in the form of an oily liquid (85-90%).

<1-3> General Synthesis Procedure of Arlylation (Step c of FIG. 1)

To a suspension of NaH (2.0 equiv) mixed with anhydrous DMF was slowly added a solution of trialkylated monool derivatives (C1-C6) (1.0 equiv) mixed with the anhydrous DMF at 0 ° C. under argon. After stirring for 15 minutes, allyl iodide (1.5 equiv) was added to the mixture and the temperature increased to 60 ° C. The reaction mixture was left at this temperature for 4 hours, then cooled to room temperature and terminated with H ₂ O. The reaction mixture was extracted twice with CH ₂ Cl ₂ , washed with brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was concentrated in vacuo and the resulting residue was purified by silica gel column chromatography (EtOAc / hexane) to give trialkylated allyl compound (D1-D6) in oily liquid form (90-95%).

<1-4> General Synthesis Procedure of Hydroboration (Step d of FIG. 1)

Completely distilled anhydrous THF (3 ml) and trialkylated allyl compound (D1-D6) (1.65 g, 2.77 mmol) were placed in a dry round bottom flask under N ₂ and cooled to 0 ° C. in an ice bath. 1M BH ₃ solution mixed with THF (3 ml) was slowly added to the mixture, then the reaction mixture was stirred at 0 ° C. for 10 hours. The reaction mixture was then poured into 3M NaOH solution (2 ml) and stirred for an additional 20 minutes and 30% H ₂ O ₂ solution (2 ml) was added. The mixture was stirred at rt for 2 h. After saturated with K ₂ CO ₃ , the reaction mixture was extracted with diethyl ether. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The resulting mixture was purified by silica gel column chromatography (EtOAc / hexanes) to give trialkylated hydroborated compounds (E1-E6) as oily liquids (55-60%).

<1-5> Trialkylated Of trialkylated tri-ols  General Synthesis Procedure (Step e of Figure 1)

To a solution of trialkylated hydroborated compound (E1-E6) (1.0 equiv) mixed in DMF (10 ml) was added NaH (3.0 equiv) at 0 ° C., under argon, and the reaction mixture was stirred for 12 minutes. . 4- (Bromomethyl) -1-methyl-2,6,7-trioxabicyclo [2.2.2] -octane (3.0 equiv) dissolved in THF (3 ml) was added slowly. The resulting mixture was heated at 100 ° C. for 24 hours. After quenching with water, the solvent was removed under reduced pressure. The solid residue was dissolved in diethyl ether, washed with brine and dried over anhydrous Na ₂ SO ₄ . After concentration of the organic layer by rotary evaporator, the residue was dissolved in a DCM / MeOH (10 ml / 10 ml) mixture. Several drops of concentrated HCl were added to this solution and the resulting mixture was heated at 50 ° C. for 4 hours. After neutralization with NaOH and concentration of the reaction mixture, the resulting residue was purified by column chromatography (EtOAc / hexanes) to give trialkylated triols (F1-F6) in oily liquid form (45-50%).

<1-6> General Synthesis Procedure of Glycosycosylation Reaction (Step f of FIG. 1)

This was followed by the synthesis method of Chae , PS et al. (Nat. Methods 2010, 7, 1003.). 2,4,6-collidine (1.0 equiv) was added to a stirred solution of trialkylated triol derivatives (F1-F6) mixed with anhydrous CH ₂ Cl ₂ under nitrogen. AgOTf (4.5 equiv) was added to the mixture at 0 ° C. To this solution was added 4.0 equivalents of a perbenzoylated maltosylbromide solution dissolved in CH ₂ Cl ₂ . Stir at room temperature for 10 minutes. After the reaction was completed (confirmed by TLC to complete the reaction), pyridine was added to the reaction mixture, which was diluted with CH ₂ Cl ₂ and then filtered through celite. The filtrate was washed with 1M Na ₂ S ₂ O ₃ aqueous solution, 0.1M HCl aqueous solution and brine. The organic layer was then dried over anhydrous Na ₂ SO ₄ and the solvent removed using a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc / hexane) to give a glycosylated compound (TNM-La) as a white solid (45 to 75%).

<1-7> Deprotection Vaporization  reaction ( deprotection  General synthetic procedure for the reaction (step g of FIG. 1)

This was followed by the synthesis method of Chae , PS et al. (Nat. Methods, 2010, 7, 1003.). Under Zemplen's condition to - O - benzoyl Chemistry (de- O -benzoylation) it was carried out. The O- protected compound was dissolved in anhydrous CH ₂ Cl ₂ and then MeOH and NaOMe, a 0.5 M methanolic solution, were added alternately to prevent precipitation. To the reaction mixture was added 0.5M methanolic solution, NaOMe, to a final concentration of 0.05M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized with 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 by silica gel chromatography (CH ₂ Cl ₂ / MeOH) to give a white solid compound (90 to 95%). The compound thus obtained is the compound TNM-Ls of the present invention.

Preparation Example 1 Synthesis of TNM-C9L

<1-1> Synthesis of 2,2-bis ((nonyloxy) methyl) propane-1,3-diol (Compound B1)

Compound B1 was synthesized in a yield of 92% according to the procedure of Example 1-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.65 (d, J = 4.0 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 4.0 Hz, 4H), 2.85 (t, J = 4.0 Hz, 2H), 1.56 (quin, J = 4.0 Hz, 4H), 1.28-1.26 (m, 24H), 0.88 (t, J = 7.2 Hz, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.4, 72.2, 65.7, 44.6, 32.1, 29.8, 29.7, 29.5, 26.3, 22.9, 14.1.

<1-2> 3- ( nonyloxy ) -2,2- bis ((nonyloxy) methyl) propan -One- ol (compound C1)  synthesis

Compound C1 was synthesized in the yield of 87% according to the procedure of Examples 1-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.71 (d, J = 8.0 Hz, 2H), 3.43 (s, 6H), 3.38 (t, J = 8.0 Hz, 6H), 3.17 (t, J = 4.0 Hz, 1H), 1.53 (quin, J = 4.0 Hz, 6H), 1.30-1.26 (m, 36H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.6, 71.3, 66.3, 44.7, 31.6, 29.5, 25.8, 22.6, 14.1.

<1-3> 1- (3- (allyloxy) -2,2-bis ( ( nonyloxy methyl) propoxy ) of nonane (Compound D1)  synthesis

Compound D1 was synthesized in the yield of 92% according to the procedure of Examples 1-3. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.93-5.83 (m, 1H), 5.28-5.22 (m, 1H), 5.14-5.11 (m, 1H), 3.95-3.93 (m, 2H), 3.42 ( s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 1.52 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 36H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.4, 116.1, 72.4, 72.1, 71.9, 71.7, 69.8, 69.7, 45.6, 32.1, 29.8, 29.5, 26.4, 26.3, 22.9, 14.2.

<1-4> 3- (3- ( nonyloxy ) -2,2- bis ((nonyloxy) methyl) propoxy ) propan -One- ol (compound E1)  synthesis

Compound E1 was synthesized in a yield of 55% according to the procedure of Examples 1-4. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.76 (t, J = 8.0 Hz, 2H), 3.60 (t, J = 8.0 Hz, 2H), 3.43 (s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 2.88 (br s, 1H), 1.80 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.30-1.26 (m, 36H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.8, 71.4, 70.2, 62.9, 45.3, 32.1, 31.9, 29.9, 29.7, 29.8, 29.5, 26.4, 22.9, 14.2.

<1-5> 2- ( hydroxymethyl ) -2-((3- (3- ( nonyloxy ) -2,2- bis (( nonyloxy Synthesis of) methyl) propoxy) propoxy) methyl) propane-1,3-diol (Compound F1)

Compound F1 was synthesized in the yield of 45% according to the procedure of Examples 1-5. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70 (s, 6H), 3.52 (t, J = 8.0 Hz, 2H) 3.50 (s, 2H), 3.46 (t, J = 8.0 Hz, 2H), 3.38 (quin, J = 8.0 Hz, 12H), 3.35 (s, 2H), 2.88 (br s, 3H), 1.79 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.26 (m, 36H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.3, 71.6, 69.9, 69.6, 68.8, 67.7, 64.9, 45.4, 31.9, 29.7, 29.6, 29.5, 29.4, 26.2, 22.7, 14.1.

<1-6> Synthesis of TNM-C9La

TNM-C9La in 52% yield according to the general saccharification reaction procedure of Examples 1-6 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.08 (d, J = 8.0 Hz, 6H), 7.98 (d, J = 8.0 Hz, 6H), 7.92-7.83 (m, 12H), 7.79-7.66 (m , 12H), 7.65-7.20 (m, 69H), 6.10 (t, J = 18.2 Hz, 3H), 5.75-5.62 (m, 3H), 5.43 (t, J = 16.4 Hz, 3H), 5.19-5.10 ( m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.34-4.26 (dd, J = 8.0, 12.0 Hz, 9H), 4.18 (d, J = 8.0 Hz, 3H), 3.70 (t, J = 20.0 Hz, 6H), 3.37 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 14H), 1.79 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H ), 1.31-1.25 (m, 36H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.0, 165.7, 165.5, 165.0, 164.7, 133.4, 133.1, 129.9, 129.8, 129.7, 129.6, 29.4, 129.3, 128.9, 128.8, 128.7, 128.6, 128.4, 128.2 , 100.8, 95.7, 72.1, 72.0, 71.5, 71.2, 69.7, 68.9, 68.8, 63.3, 62.3, 45.3, 31.9, 29.7, 29.5, 29.4, 26.2, 22.7, 14.2.

<1-7> Synthesis of TNM-C9L

TNM-C9L in 90% yield according to the general synthetic procedure for deprotection reactions of Examples 1-7. Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.35 (d, J = 8.0 Hz, 3H), 3.98 (d, J = 12.0 Hz, 3H), 3.84 -3.78 (m, 6H), 3.70-3.58 (m, 15H), 3.55-3.22 (m, 45H), 1.81 (quin, J = 12.0 Hz, 2H), 1.56 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 36H), 0.89 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.1, 103.1, 81.5, 77.9, 76.6, 75.2, 74.8, 74.3, 72.6, 71.5, 70.6, 62.8, 46.7, 46.6, 33.3, 31.0, 30.8, 30.6, 27.6, 23.9, 14.7; HRMS ( EI ) : calcd. for C ₇₆ H ₁₄₂ 0 ₃₈ [M + Na] ⁺ 1685.9077, found 1685.9081.

Preparation Example 2 Synthesis of TNM-C10L

<2-1> Synthesis of 2,2-bis ((decyloxy) methyl) propane-1,3-diol (Compound B2)

Compound B2 was synthesized in the yield of 92% according to the procedure of Example 1-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.65 (d, J = 4.0 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 4.0 Hz, 4H), 2.85 (t, J = 4.0 Hz, 2H), 1.56 (quin, J = 4.0 Hz, 4H), 1.28-1.26 (m, 28H), 0.88 (t, J = 7.2 Hz, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.4, 72.2, 65.7, 44.6, 32.1, 29.8, 29.7, 29.5, 26.3, 22.9, 14.1.

<2-2> 3- ( decyloxy ) -2,2- bis ((decyloxy) methyl) propan -One- ol  Synthesis of (Compound C2)

Compound C2 was synthesized in a yield of 90% according to the procedure of Examples 1-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.71 (d, J = 8.0 Hz, 2H), 3.44 (s, 6H), 3.36 (t, J = 8.0 Hz, 6H), 3.17 (t, J = 8.0 Hz, 1H), 1.52 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 42H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.6, 71.3, 66.3, 44.7, 31.8, 29.6, 29.1, 26.1, 22.6, 14.1.

<2-3> 1- (3- (allyloxy) -2,2-bis ( ( decyloxy methyl) propoxy ) of decane (compound D2)  synthesis

Compound D2 was synthesized in the yield of 92% according to the procedure of Examples 1-3. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.93-5.84 (m, 1H), 5.27-5.22 (m, 1H), 5.13-5.10 (m, 1H), 3.95-3.93 (m, 2H), 3.42 ( s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 1.52 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 42H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.5, 116.1, 72.4, 72.1, 71.9, 69.8, 69.7, 45.6, 32.1, 29.8, 29.5, 26.4, 26.3, 22.9, 14.2.

<2-4> 3- (3- ( decyloxy ) -2,2- bis ((decyloxy) methyl) propoxy ) propan -One- ol  Synthesis of (Compound E2)

Compound E2 was synthesized in the yield of 58% according to the procedure of Examples 1-4. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.76 (t, J = 8.0 Hz, 2H), 3.59 (t, J = 8.0 Hz, 2H), 3.43 (s, 2H), 3.37 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 2.88 (brs, 1H), 1.80 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.30-1.26 (m, 42H ), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.8, 71.4, 70.2, 62.9, 45.3, 32.1, 29.9, 29.8, 29.5, 26.4, 22.9, 14.2.

<2-5> 2- ( hydroxymethyl ) -2-((3- (3- ( decyloxy ) -2,2- bis ((decyloxy) methyl) Synthesis of propoxy) propoxy) methyl) propane-1,3-diol (Compound F2)

Compound F2 was synthesized in the yield of 47% according to the procedure of Examples 1-5. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.67 (s, 6H), 3.51 (t, J = 8.0 Hz, 2H) 3.49 (s, 2H), 3.46 (t, J = 8.0 Hz, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 3.35 (s, 2H), 2.68 (br s, 3H), 1.79 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.26 (m, 42H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.3, 71.6, 69.9, 68.8, 67.7, 64.9, 45.4, 31.9, 29.7, 29.6, 29.4, 26.2, 22.7, 14.1.

<2-6> Synthesis of TNM-C10La

TNM-C10La was obtained in 45% yield according to the general saccharification reaction procedure of Examples 1-6. Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.08 (d, J = 8.0 Hz, 6H), 7.98 (d, J = 8.0 Hz, 6H), 7.92-7.83 (m, 12H), 7.79-7.66 (m , 12H), 7.65-7.20 (m, 69H), 6.12 (t, J = 18.2 Hz, 3H), 5.75-5.63 (m, 3H), 5.44 (t, J = 16.4 Hz, 3H), 5.20-5.11 ( m, 6H), 4.58 (t, J = 3.2 Hz, 6H), 4.34-4.26 (dd, J = 8.0, 12.0 Hz, 9H), 4.18 (d, J = 8.0 Hz, 3H), 3.70 (t, J = 20.0 Hz, 6H), 3.36 (quin, J = 18.4 Hz, 12H), 3.24-2.99 (m, 14H), 1.79 (quin, J = 8.0 Hz, 2H), 1.53 (quin, J = 8.0 Hz, 6H ), 1.31-1.25 (m, 42H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.0, 165.7, 165.5, 165.0, 164.7, 133.4, 133.1, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 128.9, 128.8, 128.6, 128.4, 128.3, 100.8 , 95.7, 72.1, 72.0, 71.5, 71.2, 69.7, 68.8, 63.3, 62.3, 45.3, 31.9, 29.7, 29.5, 29.4, 26.3, 22.7, 14.2.

<2-7> Synthesis of TNM-C10L

TNM-C10L in 95% yield according to the general synthetic procedure for deprotection reactions of Examples 1-7. Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.34 (d, J = 8.0 Hz, 3H), 3.97 (d, J = 10.0 Hz, 3H), 3.84 -3.78 (m, 6H), 3.70-3.58 (m, 15H), 3.55-3.22 (m, 45H), 1.79 (quin, J = 12.8 Hz, 2H), 1.54 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 48H), 0.90 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.1, 103.1, 81.5, 77.8, 76.6, 75.1, 74.8, 74.2, 72.6, 71.5, 70.6, 70.0, 62.8, 62.2, 46.7, 46.6, 33.3, 31.0, 30.8, 30.7, 27.6, 23.9, 14.7; HRMS (EI) : calcd. for C ₈₂ H ₁₅₄ O ₃₈ [M + Na] ⁺ 1770.0016, found 1770.0011.

Preparation Example 3 Synthesis of TNM-C11L

<3-1> Synthesis of 2,2-bis ((undecyloxy) methyl) propane-1,3-diol (Compound B3)

Compound B3 was synthesized in a yield of 94% according to the procedure of Example 1-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.65 (d, J = 4.0 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 8.0 Hz, 4H), 2.85 (t, J = 4.0 Hz, 2H), 1.56 (quin, J = 4.0 Hz, 4H), 1.28-1.25 (m, 32H), 0.88 (t, J = 7.2 Hz, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.5, 72.3, 65.7, 44.6, 32.1, 29.8, 29.7, 29.6, 29.5, 26.3, 22.9, 14.1.

<3-2> 3- ( undecyloxy ) -2,2- bis ((undecyloxy) methyl) propan -One- ol  Synthesis of (Compound C3)

Compound C3 was synthesized in 85% yield according to the procedure of Examples 1-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.71 (d, J = 8.0 Hz, 2H), 3.43 (s, 6H), 3.38 (t, J = 8.0 Hz, 6H), 3.16 (t, J = 8.0 Hz, 1H), 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.7, 71.4, 62.9, 45.1, 32.1, 29.9, 29.7, 29.5, 26.4, 22.7, 14.2.

<3-3> 1- (3- ( allyloxy ) -2,2- bis ((undecyloxy) methyl) propoxy ) undecane  Synthesis of (Compound D3)

Compound D3 was synthesized in the yield of 94% according to the procedure of Examples 1-3. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.95-5.85 (m, 1H), 5.27-5.22 (m, 1H), 5.14-5.11 (m, 1H), 3.95-3.93 (m, 2H), 3.42 ( s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 1.52 (quin, J = 8.0 Hz, 6H), 1.29-1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.5, 116.1, 72.4, 72.1, 71.9, 70.0, 69.8, 69.7, 45.6, 32.1, 29.8, 29.5, 26.4, 26.3, 22.9, 14.2.

<3-4> 3- (3- ( undecyloxy ) -2,2- bis ((undecyloxy) methyl) propoxy ) propan -One- ol  Synthesis of (Compound E3)

Compound E3 was synthesized in the yield of 58% according to the procedure of Examples 1-4. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.77 (t, J = 8.0 Hz, 2H), 3.60 (t, J = 8.0 Hz, 2H), 3.43 (s, 2H), 3.37 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 2.90 (brs, 1H), 1.80 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.30-1.26 (m, 48H ), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.8, 71.5, 70.2, 63.0, 45.3, 32.1, 29.9, 29.8, 29.6, 29.5, 26.4, 22.9, 14.2.

<3-5> 2- ( hydroxymethyl ) -2-((3- (3- ( undecyloxy ) -2,2- bis ((undecyloxy) methyl) propoxy) propoxy) methyl) propane-1,3-diol (Compound F3)

Compound F3 was synthesized in the yield of 47% according to the procedure of Examples 1-5. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.67 (s, 6H), 3.50 (t, J = 8.0 Hz, 2H) 3.48 (s, 2H), 3.45 (t, J = 8.0 Hz, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 3.35 (s, 2H), 3.29 (brs, 3H), 1.79 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.31 -1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.3, 71.6, 69.9, 69.6, 68.8, 67.7, 64.9, 45.4, 44.8, 31.9, 29.7, 29.6, 29.4, 26.2, 22.7, 14.2.

<3-6> Synthesis of TNM-C11La

TNM-C11La in 48% yield according to the general saccharification reaction procedure of Examples 1-6 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.08 (d, J = 8.0 Hz, 6H), 7.98 (d, J = 8.0 Hz, 6H), 7.92-7.85 (m, 12H), 7.79-7.67 (m , 12H), 7.65-7.22 (m, 69H), 6.11 (t, J = 20 Hz, 3H), 5.75-5.63 (m, 6H), 5.44 (t, J = 16.4 Hz, 3H), 5.20-5.10 ( m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.34-4.26 (dd, J = 8.0, 12.0 Hz, 9H), 4.19 (d, J = 8.0 Hz, 3H), 3.70 (t, J = 20.0 Hz, 6H), 3.37 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 14H), 1.79 (quin, J = 8.0 Hz, 2H), 1.51 (quin, J = 8.0 Hz, 6H ), 1.30-1.25 (m, 48H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.1, 165.8, 165.5, 165.1, 164.7, 133.4, 133.1, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 128.9, 128.8, 128.7, 128.6, 128.4, 128.2 , 100.8, 95.7, 72.1, 72.0, 71.5, 71.2, 69.7, 68.9, 68.8, 63.3, 62.4, 45.3, 31.9, 29.7, 29.5, 29.4, 26.3, 22.7, 14.2.

<3-7> Synthesis of TNM-C11L

TNM-C11L in 94% yield according to the general synthetic procedure for deprotection reactions of Examples 1-7. Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.34 (d, J = 8.0 Hz, 3H), 3.97 (d, J = 10.0 Hz, 3H), 3.84 -3.78 (m, 6H), 3.70-3.58 (m, 15H), 3.55-3.22 (m, 45H), 1.79 (quin, J = 12.8 Hz, 2H), 1.54 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 48H), 0.90 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.1, 103.1, 81.5, 77.8, 76.6, 75.1, 74.8, 74.2, 72.6, 71.5, 70.6, 70.0, 62.8, 62.2, 46.7, 46.6, 33.3, 31.0, 30.8, 30.7, 27.6, 23.9, 14.7; HRMS (EI) : calcd. for C ₈₂ H ₁₅₄ O ₃₈ [M + Na] ⁺ 1770.0016, found 1770.0011.

Preparation Example 4 Synthesis of TNM-C12L

<4-1> Synthesis of 2,2-bis ((dodecyloxy) methyl) propane-1,3-diol (Compound B4)

Compound B4 was synthesized in a yield of 94% according to the procedure of Example 1-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.64 (d, J = 4.0 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 8.0 Hz, 4H), 2.85 (t, J = 4.0 Hz, 2H), 1.55 (quin, J = 4.0 Hz, 4H), 1.28-1.25 (m, 36H), 0.88 (t, J = 7.2 Hz, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.6, 72.4, 65.8, 44.6, 32.1, 30.2, 29.9, 29.8, 29.5, 26.3, 22.9, 14.2.

<4-2> 3- ( dodecyloxy ) -2,2- bis ((dodecyloxy) methyl) propan -One- ol  Synthesis of (Compound C4)

Compound C4 was synthesized in a yield of 85% according to the procedure of Examples 1-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.71 (d, J = 8.0 Hz, 2H), 3.43 (s, 6H), 3.38 (t, J = 8.0 Hz, 6H), 3.17 (t, J = 8.0 Hz, 1H), 1.53 (quin, J = 8.0 Hz, 6H), 1.30-1.26 (m, 54H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.8, 71.4, 70.2, 62.9, 45.1, 32.1, 29.9, 29.7, 29.6, 26.4, 22.7, 14.2.

<4-3> 1- (3- ( allyloxy ) -2,2- bis ((dodecyloxy) methyl) propoxy ) dodecane  Synthesis of (Compound D4)

Compound D4 was synthesized in a yield of 94% according to the procedure of Examples 1-3. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.93-5.83 (m, 1H), 5.27-5.22 (m, 1H), 5.14-5.11 (m, 1H), 3.95-3.93 (m, 2H), 3.42 ( s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 54H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.6, 116.2, 72.4, 72.1, 71.9, 69.9, 69.7, 45.6, 32.1, 29.8, 29.6, 26.4, 26.3, 22.9, 14.2.

<4-4> 3- (3- ( dodecyloxy ) -2,2- bis ((dodecyloxy) methyl) propoxy ) propan -One- ol  Synthesis of (Compound E4)

Compound E4 was synthesized in the yield of 57% following the procedure of Examples 1-4. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.77 (t, J = 8.0 Hz, 2H), 3.60 (t, J = 8.0 Hz, 2H), 3.43 (s, 2H), 3.37 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 2.90 (brs, 1H), 1.80 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.30-1.25 (m, 54H ), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 79.1, 71.4, 70.2, 62.9, 45.3, 32.1, 29.9, 29.8, 29.6, 29.5, 26.4, 22.9, 14.3.

<4-5> 2- ( hydroxymethyl ) -2-((3- (3- ( dodecyloxy ) -2,2- bis ((dodecyloxy) methyl) propoxy) propoxy) methyl) propane-1,3-diol (Compound F4)

Compound F4 was synthesized in the yield of 50% according to the procedure of Examples 1-5. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70 (s, 6H), 3.52 (t, J = 8.0 Hz, 2H) 3.50 (s, 2H), 3.46 (t, J = 8.0 Hz, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 3.35 (s, 2H), 2.91 (br s, 3H), 1.79 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.26 (m, 54H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 77.2, 71.6, 69.9, 68.9, 68.8, 67.7, 65.1, 45.4, 44.9, 31.9, 29.7, 29.6, 29.4, 26.2, 22.7, 14.2.

<4-6> Synthesis of TNM-C12La

TNM-C12La in a yield of 70% according to the general saccharification reaction procedure of Examples 1-6 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.09 (d, J = 8.0 Hz, 6H), 7.98 (d, J = 8.0 Hz, 6H), 7.92-7.83 (m, 12H), 7.79-7.66 (m , 12H), 7.65-7.20 (m, 69H), 6.11 (t, J = 18.2 Hz, 3H), 5.75-5.63 (m, 6H), 5.44 (t, J = 16.4 Hz, 3H), 5.20-5.11 ( m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.34-4.26 (dd, J = 8.0, 12.0 Hz, 9H), 4.18 (d, J = 8 Hz, 3H), 3.70 (t, J = 20.0 Hz, 6H), 3.36 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 14H), 1.79 (quin, J = 8.0 Hz, 2H), 1.53 (quin, J = 8.0 Hz, 6H ), 1.34-1.25 (m, 54H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.0, 165.7, 165.5, 165.0, 164.7, 133.4, 133.1, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 128.9, 128.7, 128.6, 128.4, 128.2, 100.8 , 95.7, 72.1, 72.0, 71.5, 71.2, 69.7, 68.9, 68.8, 63.3, 62.3, 45.4, 31.9, 29.8, 29.6, 29.4, 26.3, 22.7, 14.2.

<4-7> Synthesis of TNM-C12L

TNM-C12L in 95% yield according to the general synthetic procedure for the deprotection reaction of Examples 1-7. Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.35 (d, J = 8.0 Hz, 3H), 3.98 (d, J = 12.0 Hz, 3H), 3.84 -3.78 (m, 6H), 3.70-3.58 (m, 15H), 3.55-3.22 (m, 45H), 1.80 (quin, J = 12.0 Hz, 2H), 1.55 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 54H), 0.90 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.1, 103.1, 81.5, 77.8, 76.6, 75.1, 74.9, 74.2, 72.6, 71.5, 70.6, 62.8, 46.7, 46.6, 33.3, 31.0, 30.8, 30.7, 27.6, 23.9, 14.7; HRMS (EI) : calcd. for C ₈₅ H ₁₆₀ 0 ₃₈ [M + Na] ⁺ 1812.0485, found 1812.0480.

Preparation Example 5 Synthesis of TNM-C13L

<5-1> Synthesis of 2,2-bis ((tridecyloxy) methyl) propane-1,3-diol (Compound B5)

Compound B5 was synthesized in a yield of 94% according to the procedure of Example 1-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.65 (d, J = 4.0 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 8.0 Hz, 4H), 2.85 (t, J = 4.0 Hz, 2H), 1.56 (quin, J = 4.0 Hz, 4H), 1.28-1.25 (m, 40H), 0.88 (t, J = 7.2 Hz, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.6, 72.5, 65.8, 44.6, 32.1, 30.3, 29.9, 29.7, 29.6, 26.3, 22.9, 14.2.

<5-2> 3- ( trideyloxy ) -2,2- bis ((trideyloxy) methyl) propan -One- ol  Synthesis of (Compound C5)

Compound C5 was synthesized in a yield of 85% according to the procedure of Examples 1-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.71 (d, J = 8.0 Hz, 2H), 3.43 (s, 6H), 3.38 (t, J = 8.0 Hz, 6H), 3.17 (t, J = 8.0 Hz, 1H), 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.25 (m, 60H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.7, 71.4, 70.2, 62.9, 45.1, 32.1, 29.9, 29.7, 29.6, 26.4, 22.7, 14.2.

<5-3> 1- (3- ( allyloxy ) -2,2- bis ((tridecyloxy) methyl) propoxy ) tridecane  Synthesis of (Compound D5)

Compound D5 was synthesized in the yield of 95% according to the procedure of Examples 1-3. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.93-5.83 (m, 1H), 5.27-5.22 (m, 1H), 5.14-5.11 (m, 1H), 3.95-3.93 (m, 2H), 3.42 ( s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 1.52 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 60H), 0.88 ((t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.6, 116.1, 72.4, 72.1, 71.9, 69.8, 69.7, 45.6, 32.1, 29.9, 29.8, 29.7, 29.6, 26.4, 26.3 , 22.9, 14.3.

<5-4> 3- (3- ( tridecyloxy ) -2,2- bis ((tridecyloxy) methyl) propoxy ) propan Synthesis of -1-ol (Compound E5)

Compound E5 was synthesized in the yield of 60% according to the procedure of Examples 1-4. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.76 (t, J = 8.0 Hz, 2H), 3.61 (t, J = 8.0 Hz, 2H), 3.43 (s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 2.90 (brs, 1H), 1.80 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.30-1.25 (m, 60H ), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 79.1, 71.4, 70.2, 62.9, 45.3, 32.1, 31.9, 30.1, 29.9, 29.8, 29.5, 26.4, 22.9, 14.3.

<5-5> 2- ( hydroxymethyl ) -2-((3- (3- ( tridecyloxy ) -2,2- bis ((tridecyloxy)  Synthesis of methyl) propoxy) propoxy) methyl) propane-1,3-diol (Compound F5)

Compound F5 was synthesized in the yield of 49% according to the procedure of Examples 1-5. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70 (s, 6H), 3.52 (t, J = 8.0 Hz, 2H) 3.50 (s, 2H), 3.46 (t, J = 8.0 Hz, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 3.35 (s, 2H), 2.67 (brs, 3H), 1.79 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.31 -1.25 (m, 60H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.14, 71.6, 69.9, 68.8, 67.7, 65.1, 45.5, 31.9, 29.7, 29.6, 29.4, 26.2, 22.7, 14.2.

<5-6> Synthesis of TNM-C13La

TNM-C13La in a yield of 65% according to the general saccharification reaction procedure of Examples 1-6 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.09 (d, J = 8.0 Hz, 6H), 7.96 (d, J = 7.2 Hz, 6H), 7.90-7.83 (m, 12H), 7.79-7.66 (m , 12H), 7.65-7.20 (m, 69H), 6.08 (t, J = 18.2 Hz, 3H), 5.75-5.63 (m, 6H), 5.41 (t, J = 16.4 Hz, 3H), 5.20-5.10 ( m, 6H), 4.56 (t, J = 3.2 Hz, 6H), 4.34-4.26 (dd, J = 8.0, 12.0 Hz, 9H), 4.18 (d, J = 8 Hz, 3H), 3.70 (t, J = 20.0 Hz, 6H), 3.36 (quin, J = 18.4 Hz, 12H), 3.20-2.96 (m, 14H), 1.60 (quin, J = 8.0 Hz, 2H), 1.50 (quin, J = 8.0 Hz, 6H ), 1.34-1.21 (m, 60H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 165.8, 165.5, 165.0, 164.7, 133.4, 133.1, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 128.9, 128.8, 128.7, 128.6, 128.4, 128.2 , 100.8, 95.7, 72.1, 72.0, 71.5, 71.2, 69.7, 68.9, 68.8, 63.3, 62.3, 45.4, 32.0, 29.7, 29.5, 29.4, 26.2, 22.8, 14.3.

<5-7> Synthesis of TNM-C13L

TNM-C13L in 93% yield according to the general synthetic procedure for deprotection reactions of Examples 1-7. Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.34 (d, J = 8.0 Hz, 3H), 3.97 (d, J = 10.0 Hz, 3H), 3.86 -3.78 (m, 6H), 3.70-3.57 (m, 15H), 3.55-3.20 (m, 45H), 1.79 (quin, J = 12.4 Hz, 2H), 1.54 (quin, J = 12.0 Hz, 6H), 1.34-1.28 (m, 60H), 0.90 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.1, 103.1, 81.5, 77.8, 76.7, 75.1, 74.8, 74.2, 72.6, 71.5, 70.6, 70.0, 62.8, 46.7, 46.6, 33.3, 31.0, 30.8, 30.7, 27.6, 23.9, 14.7; HRMS (EI) : calcd. for C ₈₈ H ₁₆₆ O ₃₈ [M + Na] ⁺ 1855.0989, found 1855.1305.

Preparation Example 6 Synthesis of RMA-C5E

<6-1> Synthesis of 2,2-bis ((tetradecyloxy) methyl) propane-1,3-diol (Compound B6)

Compound B6 was synthesized in a yield of 94% according to the procedure of Example 1-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.65 (d, J = 4.0 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 8.0 Hz, 4H), 2.85 (t, J = 4.0 Hz, 2H), 1.56 (quin, J = 4.0 Hz, 4H), 1.28-1.25 (m, 44H), 0.88 (t, J = 7.2 Hz, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.6, 72.5, 65.8, 44.6, 32.1, 30.3, 29.9, 29.7, 29.6, 26.4, 22.9, 14.3.

<6-2> 3- ( tetradecyloxy ) -2,2- bis ((tetradecyloxy) methyl) propan -One- ol  Synthesis of (Compound C6)

Compound C6 was synthesized in a yield of 85% according to the procedure of Examples 1-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.71 (d, J = 8.0 Hz, 2H), 3.43 (s, 6H), 3.38 (t, J = 8.0 Hz, 6H), 3.16 (t, J = 8.0 Hz, 1H), 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.25 (m, 66H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 71.7, 71.4, 70.3, 62.9, 45.1, 32.1, 31.9, 29.9, 29.7, 29.5, 26.4, 22.7, 14.2.

<6-3> 1- (3- ( allyloxy ) -2,2- bis ((tetradecyloxy) methyl) propoxy ) tetradecane  Synthesis of (Compound D6)

Compound D6 was synthesized in a yield of 93% according to the procedure of Examples 1-3. ¹ H NMR (400 MHz, CDCl ₃ ): δ 5.93-5.83 (m, 1H), 5.27-5.22 (m, 1H), 5.14-5.11 (m, 1H), 3.95-3.93 (m, 2H), 3.42 ( s, 2H), 3.38 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 1.52 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 66H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 135.7, 116.2, 72.5, 72.4, 72.1, 71.9, 71.7, 69.8, 69.7, 45.6, 32.1, 29.8, 29.5, 26.4, 26.4, 23.2, 14.3.

<6- 4> 3 -(3- ( tetradecyloxy ) -2,2- bis ((tetradecyloxy) methyl) propoxy ) propan Synthesis of -1-ol (Compound E6)

Compound E6 was synthesized in 58% yield following the procedure of Examples 1-4. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.77 (t, J = 8.0 Hz, 2H), 3.60 (t, J = 8.0 Hz, 2H), 3.43 (s, 2H), 3.37 (s, 6H), 3.35 (t, J = 8.0 Hz, 6H), 2.89 (brs, 1H), 1.80 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.30-1.25 (m, 66H ), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 79.1, 71.4, 70.2, 62.9, 45.3, 32.2, 31.1, 29.9, 29.8, 29.7, 29.5, 26.5, 22.9, 14.3.

<6-5> 2- ( hydroxymethyl ) -2-((3- (3- ( tetradecyloxy ) -2,2- bis ((tetrade cyloxy) methyl) propoxy) propoxy) methyl) propane-1,3-diol (Compound F6)

Compound F6 was synthesized in the yield of 50% according to the procedure of Examples 1-5. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.70 (s, 6H), 3.52 (t, J = 8.0 Hz, 2H) 3.50 (s, 2H), 3.46 (t, J = 8.0 Hz, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 3.35 (s, 2H), 2.65 (br s, 3H), 1.79 (quin, J = 8.0 Hz, 2H), 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.25 (m, 66H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.14, 71.6, 69.9, 68.8, 67.7, 65.1, 45.5, 32.1, 31.9, 29.7, 29.7, 29.4, 26.2, 22.7, 14.3.

<6-6> Synthesis of TNM-C14La

TNM-C14La in 75% yield according to the general saccharification reaction procedure of Examples 1-6 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.08 (d, J = 8.0 Hz, 6H), 7.98 (d, J = 8.0 Hz, 6H), 7.88-7.84 (m, 12H), 7.79-7.66 (m , 12H), 7.65-7.20 (m, 69H), 6.10 (t, J = 18.2 Hz, 3H), 5.75-5.62 (m, 6H), 5.43 (t, J = 16.4 Hz, 3H), 5.19-5.12 ( m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.34-4.26 (dd, J = 8.0, 12.0 Hz, 9H), 4.18 (d, J = 8.0 Hz, 3H), 3.69 (t, J = 20.0 Hz, 6H), 3.32 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 14H), 1.65 (quin, J = 8.0 Hz, 2H), 1.51 (quin, J = 8.0 Hz, 6H ), 1.31-1.21 (m, 66H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 165.8, 165.5, 165.1, 165.0, 164.7, 133.4, 133.1, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 128.9, 128.8, 128.7, 128.6, 128.4 , 128.2, 100.8, 95.7, 72.1, 72.0, 71.5, 71.2, 69.7, 68.9, 68.8, 63.3, 62.3, 45.4, 32.0, 29.7, 29.5, 29.4, 26.2, 22.8, 14.3.

<6-7> Synthesis of TNM-C14L

TNM-C14L in 95% yield according to the general synthetic procedure for the deprotection reaction of Examples 1-7. Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.34 (d, J = 7.6 Hz, 3H), 3.97 (d, J = 10.0 Hz, 3H), 3.84 -3.78 (m, 6H), 3.70-3.58 (m, 15H), 3.55-3.22 (m, 45H), 1.79 (quin, J = 12.8 Hz, 2H), 1.54 (quin, J = 12.0 Hz, 6H), 1.34-1.28 (m, 66H), 0.90 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 103.1, 81.5, 77.9, 74.9, 72.5, 71.5, 70.6, 62.8, 54.9, 46.6, 33.3, 30.8, 30.7, 27.6, 24.3, 23.9, 14.7; HRMS ( EI ) : calcd. for C ₉₁ H ₁₇₂ O ₃₈ [M + Na] ⁺ 1897.1459, found 1897.1396.

Example 2 Synthesis of TNM-Ss

The synthesis scheme of TNM-Ss is shown in FIG. 2. Four compounds of TNM-Ss were synthesized according to the synthesis methods of <2-1> to <2-7> shown in FIG. 3.

TNM-Ss was synthesized from the trialkylated hydroborated compound (E3-E6) produced through the procedure of Examples <1-1> to <1-4> through the following procedure.

<2-1> alcohol Of alcohol oxidation  General Synthesis Procedure (Step h of Figure 2)

CH ₂ Cl ₂ Slowly add trialkylated hydroborated compound (E3-E6) (1.0 equiv) to a solution in which pyridinium chlorochromate (PCC) (2.0 equiv) and celite were mixed in a 1: 1 ratio. Added. The resulting mixture was stirred at rt for 1 day. The solution was diluted with CH ₂ Cl ₂ and then filtered over a short pad of celite. The solvent obtained after removal of the solvent at low pressure was sequentially purified by silica gel column chromatography (EtOAc / hexane) to give trialkylated aldehyde compound (H3-H6) in the form of an oil liquid (90-95%).

<2-2> Trialkylated  Triol ( trialkylated tri - ol  General Synthesis Procedure (Step i of FIG. 2)

A formaldehyde solution was added to a solution of trialkylated aldehyde (H3-H6) mixed with 50% water soluble alkaline EtOH (KOH 30%). The resulting mixture was stirred at room temperature for 2 hours and then heated at 60 ° C. for 2 days. After removing EtOH with a rotary evaporator, the resulting mixture was extracted with diethyl ether. The organic layer was dried over anhydrous Na ₂ SO ₄ , and then purified by silica gel column chromatography (EtOAc / hexane) to give trialkylated triol compound (I3-16) as an oil liquid (30 to 40%).

<2-3> General Synthesis Procedure of Glycosycosylation Reaction (Step f in Fig. 2)

This was followed by the synthesis method of Chae , PS et al. (Nat. Methods, 2010, 7, 1003.). 2,4,6-collidine (1.0 equiv) was added to a stirred solution of trialkylated triol compound (I3-I6) mixed with anhydrous CH ₂ Cl ₂ under nitrogen. AgOTf (4.5 equiv) was added to the mixture at 0 ° C. To this solution was slowly added 4.0 equivalents of a perbenzoylated maltosylbromide solution dissolved in CH ₂ Cl ₂ . Stir at room temperature for 10 minutes. After the reaction was complete (confirmed by TLC to complete the reaction), pyridine was added to the reaction mixture, which was diluted with CH ₂ Cl ₂ and then filtered through celite. The filtrate was washed with 1M Na ₂ S ₂ O ₃ aqueous solution, 0.1M HCl aqueous solution and brine. The organic layer was then dried over anhydrous Na ₂ SO ₄ and the solvent removed using a rotary evaporator. The residue was purified by silica gel column chromatography (EtOAc / hexane) to give a glycosylated compound (TNM-Sa) as a white solid (45 to 75%).

<2-4> Deprotection Vaporization  reaction ( deprotection  General synthetic procedure for the reaction (step g of FIG. 2)

This was followed by synthetic methods (Nat Meth 2010, 7, 1003.) by Chae , PS, etc. Under Zemplen's condition to - O - benzoyl Chemistry (de- O -benzoylation) it was carried out. The O- protected compound was dissolved in anhydrous CH ₂ Cl ₂ and then MeOH and 0.5M methanolic solution NaOMe were added alternately to prevent precipitation. To the reaction mixture was added 0.5M methanolic solution, NaOMe, to a final concentration of 0.05M. The reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was neutralized with 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 by silica gel chromatography (CH ₂ Cl ₂ / MeOH) to give a white solid compound (90 to 95%). The compound thus obtained is the compound TNM-Ss of the present invention.

Preparation Example 7 Synthesis of TNM-C11S

<7-1> 3- (3- ( undecyloxy ) -2,2- bis ((undecyloxy) methyl) propoxy ) propanal  Synthesis of Compound H3

Compound H3 was synthesized in a yield of 90% according to the procedure of Example 2-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 9.69 (s, 1H), 3.68 (t, J = 8.0 Hz, 2H), 3.35 (s, 6H), 3.28 (t, J = 8.0 Hz, 6H), 2.54-2.50 (m, 2H) 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ CNMR (100 MHz, CDCl ₃ ): δ 202.0, 71.7, 70.4, 69.7, 65.4, 45.5, 44.0, 32.1, 29.9, 29.8, 29.7, 29.5, 26.4, 22.9, 14.3.

<7-2> 2- ( hydroxymethyl ) -2-((3- ( undecyloxy ) -2,2- bis (( undecyloxy ) methyl) propoxy) methyl) propane-1,3-diol Synthesis of Compound I3

Compound I3 was synthesized in the yield of 37% according to the procedure of Example 2-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.67 (s, 6H), 3.46 (s, 2H) 3.42 (s, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 2.75 (brs, 3H) , 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.5, 72.5, 72.0, 70.6, 65.2, 45.4, 32.1, 29.9, 29.7, 29.5, 26.4, 22.9, 14.3.

<7-3> Synthesis of TNM-C11Sa

Following the general saccharification reaction procedure of Example 2-3, TNM-C11Sa was obtained at a yield of 45%. Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.07 (d, J = 8.0 Hz, 6H), 7.97 (d, J = 8.0 Hz, 6H), 7.90-7.83 (m, 12H), 7.78-7.72 (m , 12H), 7.66-7.22 (m, 69H), 6.11 (t, J = 20.0 Hz, 3H), 5.75-5.63 (m, 6H), 5.44 (t, J = 16.4 Hz, 3H), 5.20-5.10 ( m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.34-4.26 (dd, J = 8.0, 12.0 Hz, 9H), 4.19 (d, J = 8.0 Hz, 3H), 3.70 (t, J = 20.0 Hz, 4H), 3.37 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 12H), 1.79 (quin, J = 8.0 Hz, 2H), 1.51 (quin, J = 8.0 Hz, 6H ), 1.30-1.25 (m, 48H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 165.9, 165.6, 165.2, 165.1, 164.8, 133.5, 133.2, 130.1, 130.0, 129.9, 129.7, 129.6, 129.5, 129.1, 129.0, 128.9, 128.8, 128.5 , 128.4, 101.2, 96.0, 72.3, 71.5, 71.3, 70.0, 69.5, 69.1, 45.7, 32.1, 29.9, 29.8, 29.5, 26.4, 22.9, 14.3.

<7-4> Synthesis of TNM-C11S

TNM-C11Sa in 94% yield according to the general synthetic procedure for deprotection reaction of Examples 2-4 Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.35 (d, J = 8.0 Hz, 3H), 3.98 (d, J = 10.0 Hz, 3H), 3.87 -3.79 (m, 6H), 3.70-3.58 (m, 13H), 3.55-3.22 (m, 43H), 1.54 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 48H), 0.90 (t , J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.2, 103.1, 81.6, 77.9, 76.7, 75.2, 74.9, 74.3, 72.7, 71.6, 70.9, 62.8, 62.3, 33.2, 31.0, 30.9, 30.8, 30.7, 27.6 , 23.9, 14.6; HRMS ( EI ) : calcd. for C ₇₉ H ₁₄₈ 0 ₃₇ [M + Na] ⁺ 1711.9597, found 1711.9594.

Preparation Example 8 Synthesis of TNM-C12S

<8-1> 3- (3- ( dodecyloxy ) -2,2- bis ((dodecyloxy) methyl) propoxy ) propanal  Synthesis of (Compound H4)

Compound H4 was synthesized in a yield of 93% according to the procedure of Example 2-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 9.69 (s, 1H), 3.68 (t, J = 8.0 Hz, 2H), 3.35 (s, 6H), 3.28 (t, J = 8.0 Hz, 6H), 2.54-2.50 (m, 2H) 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 202.0, 71.7, 70.4, 69.7, 65.4, 45.5, 44.0, 32.1, 29.9, 29.7, 29.5, 26.4, 22.9, 14.3.

<8-2> 2- ( hydroxymethyl ) -2-((3- ( dodecyloxy ) -2,2- bis (( dodecyloxy Synthesis of) methyl) propoxy) methyl) propane-1,3-diol (Compound I4)

Compound I4 was synthesized in the yield of 30% according to the procedure of Example 2-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.67 (s, 6H), 3.46 (s, 2H) 3.42 (s, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 2.82 (brs, 3H) , 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.26 (m, 54H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.5, 72.5, 72.0, 70.6, 65.2, 45.4, 32.1, 29.9, 29.7, 29.5, 26.4, 22.9, 14.3.

<8-3> Synthesis of TNM-C12Sa

TNM-C12Sa in 58% yield according to the general saccharification reaction procedure of Example 2-3 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.07 (d, J = 8.0 Hz, 6H), 7.97 (d, J = 8.0 Hz, 6H), 7.90-7.83 (m, 12H), 7.78-7.72 (m , 12H), 7.66-7.22 (m, 69H), 6.11 (t, J = 20.0 Hz, 3H), 5.75-5.63 (m, 6H), 5.44 (t, J = 16.4 Hz, 3H), 5.20-5.10 ( m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.35-4.27 (dd, J = 8.0, 12.0 Hz, 9H), 4.19 (d, J = 8.0 Hz, 3H), 3.70 (t, J = 20.0 Hz, 4H), 3.37 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 12H), 1.79 (quin, J = 8.0 Hz, 2H), 1.51 (quin, J = 8.0 Hz, 6H ), 1.30-1.25 (m, 54H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 165.9, 165.6, 165.2, 165.1, 164.8, 133.5, 133.2, 130.1, 130.0, 129.9, 129.7, 129.6, 129.5, 129.1, 129.0, 128.9, 128.8, 128.7 , 128.5, 128.4, 101.2, 96.0, 72.3, 71.5, 71.3, 70.0, 69.5, 69.1, 45.7, 32.1, 29.9, 29.8, 29.5, 26.4, 22.9, 14.3.

<8-4> Synthesis of TNM-C12S

TNM-C12Sa in a yield of 90% according to the general synthetic procedure for deprotection reaction of Examples 2-4 Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.35 (d, J = 8.0 Hz, 3H), 3.98 (d, J = 10.0 Hz, 3H), 3.87 -3.79 (m, 6H), 3.70-3.58 (m, 13H), 3.56-3.23 (m, 43H), 1.54 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 54H), 0.90 (t , J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.2, 103.1, 81.6, 77.9, 76.7, 75.2, 74.9, 74.3, 72.7, 71.6, 70.9, 62.8, 62.3, 33.2, 31.0, 30.9, 30.8, 30.6, 27.6 , 23.9, 14.6; HRMS ( EI ) : calcd. for C ₈₂ H ₁₅₄ O ₃₇ [M + Na] ⁺ 1754.0067, found 1754.0071.

Preparation Example 9 Synthesis of TNM-C13S

<9-1> 3- (3- ( tridecyloxy ) -2,2- bis ((tridecyloxy) methyl) propoxy ) propanal  Synthesis of (Compound H5)

Compound H5 was synthesized in a yield of 95% according to the procedure of Example 2-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 9.69 (s, 1H), 3.68 (t, J = 8.0 Hz, 2H), 3.36 (s, 6H), 3.28 (t, J = 8.0 Hz, 6H), 2.55-2.51 (m, 2H) 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 202.0, 71.7, 70.4, 69.7, 65.4, 45.5, 44.0, 32.1, 29.9, 29.8, 29.7, 29.5, 26.4, 22.9, 14.3.

<9-2> 2- ( hydroxymethyl ) -2-((3- ( tridecyloxy ) -2,2- bis ((tridecyloxy)  Synthesis of methyl) propoxy) methyl) propane-1,3-diol (Compound I5)

Compound I5 was synthesized in the yield of 35% according to the procedure of Example 2-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.67 (s, 6H), 3.47 (s, 2H) 3.42 (s, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 2.90 (brs, 3H) , 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.26 (m, 60H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.5, 72.5, 72.0, 70.6, 65.2, 45.4, 32.1, 30.0, 29.9, 29.7, 29.5, 26.4, 22.9, 14.3.

<9-3> Synthesis of TNM-C13Sa

TNM-C13Sa in a yield of 60% according to the general saccharification reaction procedure of Example 2-3 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.07 (d, J = 8.0 Hz, 6H), 7.97 (d, J = 8.0 Hz, 6H), 7.90-7.83 (m, 12H), 7.78-7.72 (m , 12H), 7.66-7.22 (m, 69H), 6.11 (t, J = 20.0 Hz, 3H), 5.75-5.63 (m, 6H), 5.44 (t, J = 16.4 Hz, 3H), 5.20-5.10 ( m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.35-4.27 (dd, J = 8.0, 12.0 Hz, 9H), 4.19 (d, J = 8.0 Hz, 3H), 3.71 (t, J = 20.0 Hz, 4H), 3.37 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 12H), 1.79 (quin, J = 8.0 Hz, 2H), 1.51 (quin, J = 8.0 Hz, 6H ), 1.30-1.25 (m, 60H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 165.9, 165.6, 165.2, 165.1, 164.9, 133.5, 133.2, 130.1, 130.0, 129.9, 129.7, 129.6, 129.5, 129.1, 129.0, 128.9, 128.8, 128.5 , 128.4, 101.2, 96.0, 72.3, 71.5, 71.3, 70.0, 69.5, 69.1, 45.7, 32.1, 29.9, 29.8, 29.5, 26.4, 22.9, 14.3.

<9-4> Synthesis of TNM-C13S

TNM-C13Sa in 93% yield according to the general synthetic procedure for deprotection reaction of Examples 2-4 Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.35 (d, J = 8.0 Hz, 3H), 3.98 (d, J = 10.0 Hz, 3H), 3.87-3.79 (m, 6H), 3.70-3.58 (m, 13H), 3.55-3.22 (m, 43H), 1.55 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 60H), 0.90 ( t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.2, 103.1, 81.6, 77.9, 76.7, 75.2, 74.9, 74.3, 72.7, 71.6, 70.9, 62.8, 62.3, 33.2, 31.0, 30.9, 30.8, 30.6, 27.6 , 23.9, 14.6; HRMS ( EI ) : calcd. for C ₈₅ H ₁₆₀ O ₃₇ [M + Na] ⁺ 1796.0536, found 1796.0541.

Preparation Example 10 Synthesis of TNM-C14S

<10-1> 3- (3- ( dodecyloxy ) -2,2- bis ((dodecyloxy) methyl) propoxy ) propanal  Synthesis of (Compound H6)

Compound H6 was synthesized in a yield of 95% according to the procedure of Example 2-1. ¹ H NMR (400 MHz, CDCl ₃ ): δ 9.69 (s, 1H), 3.68 (t, J = 8.0 Hz, 2H), 3.36 (s, 6H), 3.28 (t, J = 8.0 Hz, 6H), 2.55-2.51 (m, 2H) 1.53 (quin, J = 8.0 Hz, 6H), 1.28-1.26 (m, 48H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 202.0, 71.7, 70.4, 69.7, 65.4, 45.5, 44.0, 32.1, 29.9, 29.8, 29.7, 29.5, 26.4, 22.9, 14.3.

<10-2> 2- ( hydroxymethyl ) -2-((3- ( dodecyloxy ) -2,2- bis (( dodecyloxy Synthesis of) methyl) propoxy) methyl) propane-1,3-diol (Compound I6)

Compound I6 was synthesized in the yield of 40% according to the procedure of Example 2-2. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.67 (s, 6H), 3.47 (s, 2H) 3.42 (s, 2H), 3.36 (quin, J = 8.0 Hz, 12H), 2.85 (brs, 3H) , 1.52 (quin, J = 8.0 Hz, 6H), 1.31-1.26 (m, 66H), 0.88 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 73.5, 72.5, 72.0, 70.6, 65.2, 45.4,32.1, 30.0, 29.9, 29.7, 29.5, 26.4, 22.9, 14.3.

<10-3> Synthesis of TNM-C14Sa

TNM-C14Sa in a yield of 55% according to the general saccharification reaction procedure of Example 2-3 Synthesized. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.07 (d, J = 8.0 Hz, 6H), 7.97 (d, J = 8.0 Hz, 6H), 7.90-7.83 (m, 12H), 7.78-7.72 ( m, 12H), 7.66-7.22 (m, 69H), 6.11 (t, J = 20.0 Hz, 3H), 5.75-5.63 (m, 6H), 5.44 (t, J = 16.4 Hz, 3H), 5.20-5.10 (m, 6H), 4.57 (t, J = 3.2 Hz, 6H), 4.35-4.27 (dd, J = 8.0, 12.0 Hz, 9H), 4.19 (d, J = 8.0 Hz, 3H), 3.71 (t, J = 20.0 Hz, 4H), 3.37 (quin, J = 18.4 Hz, 12H), 3.23-2.98 (m, 12H), 1.79 (quin, J = 8.0 Hz, 2H), 1.51 (quin, J = 8.0 Hz, 6H), 1.30-1.25 (m, 66H), 0.87 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ 166.2, 165.9, 165.6, 165.2, 165.1, 164.9, 133.5, 133.2, 130.1, 130.0, 129.9, 129.7, 129.6, 129.5, 129.1, 129.0, 128.8, 128.5, 128.4 , 101.2, 96.0, 72.3, 71.5, 71.3, 70.0, 69.5, 69.1, 45.7, 32.1, 29.9, 29.8, 29.5, 26.4, 22.9, 14.3.

<10-4> Synthesis of TNM-C14S

TNM-C14Sa in 95% yield according to the general synthetic procedure for the deprotection reaction of Examples 2-4 Synthesized. ¹ H NMR (400 MHz, CD ₃ OD): δ 5.15 (d, J = 4.0 Hz, 3H), 4.35 (d, J = 8.0 Hz, 3H), 3.98 (d, J = 10.0 Hz, 3H), 3.87-3.79 (m, 6H), 3.70-3.58 (m, 13H), 3.56-3.23 (m, 43H), 1.55 (quin, J = 12.0 Hz, 6H), 1.34-1.29 (m, 66H), 0.90 ( t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CD ₃ OD): δ 105.2, 103.1, 81.6, 77.9, 76.7, 75.2, 74.9, 74.3, 72.7, 71.6, 70.9, 62.8, 62.3, 33.2, 31.0, 30.9, 30.8, 30.6, 27.6 , 23.9, 14.6; HRMS ( EI ) : calcd. for C ₈₈ H ₁₆₆ O ₃₇ [M + Na] ⁺ 1839.1040, found 1839.0858.

Example 3 TNMs Characteristics

In order to confirm the characteristics of the TNMs synthesized according to the synthesis method of Examples 1 and 2, the molecular weight (MW), critical micellar concentration (CMC) of TNMs and the hydrodynamic radii of the micelles formed ( R) _h ) was measured.

Specifically, the critical micelle concentration (CMC) was measured using hydrophobic fluorescence staining, diphenylhexatriene (DPH), and the hydrodynamic radius ( R _h ) of micelles formed by each agent was determined by dynamic light scattering. light scattering (DLS) experiments. The measured results are shown in Table 1 in comparison with the existing amphiphilic molecules (DDM).

Detergent ^Mw CMC (mM) CMC (wt%) ^R _h  ^(nm) TNM-C9L 1646.9 ~ 0.002 ~ 0.00033 4.5 ± 0.2 TNM-C10L 1689.0 ~ 0.0015 ~ 0.00025 4.5 ± 0.1 TNM-C11L 1731.1 ~ 0.001 ~ 0.00017 29.5 ± 1.6 TNM-C12L 1773.2 ~ 0.0008 ~ 0.00016 33.8 ± 0.8 TNM-C13L 1815.2 ~ 0.0006 ~ 0.00011 45.9 ± 2.7 TNM-C14L 1857.3 ~ 0.0004 ~ 0.00007 48.1 ± 1.1 TNM-C11S 1690.0 ~ 0.0025 ~ 0.00051 5.7 ± 0.2 TNM-C12S 1732.1 ~ 0.0015 ~ 0.00035 6.5 ± 0.6 TNM-C13S 1774.2 ~ 0.001 ~ 0.00071 9.8 ± 1.7 TNM-C14S 1816.3 ~ 0.0008 ~ 0.00054 11.6 ± 0.2 DDM 510.1 ~ 0.17 ~ 0.0087 3.4 ± 0.02

The CMC value of TNMs was much smaller than that of DDM. The CMC values of TNM-C14 and TNM-C14S with the longest alkyl chains (C14) were at least 100 times smaller than DDM. Therefore, since the micelles were easily formed even in a small amount of TNMs, the solubility was better than that of DDM.

The size of micelles formed by TNMs ranged from 4.5 to 48.1 nm, forming micelles larger than DDM. The size of micelles formed by TNMs increased with increasing alkyl chain length. In addition, as a result of analyzing the size distribution based on the number of micelles formed by the TNMs, it was confirmed that the TNMs (TNM-Ls and TNM-Ss) each form micelles with one cluster (FIG. 4).

< EXAMPLE  3> Of TNMs R. capsulatus superassembly  ( LHI - RC ) Structural stabilization ability evaluation (FIG. 4)

Also by TNMs bakteo la capsule Charters (Rhodobacter experiments were conducted to evaluate the ability of the capsulatus to stabilize the photosynthetic superassembly structure. Photosynthetic assembly consists of a light-harvesting complex I (LHI) and a reaction center complex (RC). Structural stability of LHI-RC was measured by monitoring the structure of the protein for 20 days using UV-Vis spectroscopy. Amphipathic molecules were used as all TNMs of the present invention and the existing amphipathic molecules DDM and OG, the concentration of amphipathic molecules was measured at CMC + 0.04 wt% (FIG. 5A) and CMC + 0.2 wt% (FIG. 5B) The stability of LHI-RC protein according to the concentration of amphiphilic molecules was investigated.

Specifically, the stability of photosynthetic assembly of R. capsulatus was measured using the method published in our 2008 paper (PS Chae et al., Chem BioChem 2008, 9, 1706-1709.). In summary, we used membranes from R. capsulatus , U43 [pUHTM86Bgl] bacteria, which do not have a light-harvesting complex II (LHII). R. capsulatus A 10 ml aliquot of the freezing solution of the membrane was homogenized using a glass homogenizer and incubated at 32 ° C. for 30 minutes. The homogenized membrane was treated with 1.0 wt% DDM at 32 ° C. for 30 minutes. Membrane debris were ultracentrifuged at 31 ° C. at 4 ° C. for 30 minutes to collect pellets. DDM- solubilization (I in buffer containing 10 mM Tris, pH 7.8 - being balance and storage) 200 μL Ni ^{2 +} -NTA resin was added to the supernatant. The mixed solution was allowed to combine with the resin at 4 ° C. for 1 hour. The resin was collected and washed twice with 500 μL binding buffer (pH 7.8 Tris buffer solution with 1 × CMC concentration of DDM). After replacement with a new ultracentrifuge tube, the resin was treated twice with 300 μL elution buffer containing 1M imidazole (the binding buffer at pH 7.8 with 1 M imidazole added). DDM-purified superassembly was diluted with a solution (pH 7.8, 920 μL) containing each TNMs, DDM and OG at CMC + 0.04 wt%, CMC + 0.2 wt% concentrations. These protein complex samples were collected to monitor protein stability at regular intervals during the incubation period of 20 days (first 10 days at 15 or 20 ° C. and then 10 days at 32 ° C.). UV-Vis absorption spectra were measured in the range of 650 nm to 950 nm.

All new compounds except TNM-C14S were superior to DDM and OG in stabilizing complexes. Among the TNMs, the compounds with the shortest alkyl chains (TNM-C9L and TNM-C11S) showed the best effect. Increasing the compound concentration from CMC + 0.04 wt% to CMC + 0.2 wt% decreases protein stability in both DDM and OG (FIGS. 5 and 6), with the longest alkyl chains such as TNMs, especially TNM-C14L and TNM-C14S. Similar results were obtained with compounds having However, at the concentration of this amphiphilic molecule (CMC + 0.2 wt%), the rest of the TNM molecules were clearly superior to DDM in stabilizing the LHI-RC complex.

<Example 4> Evaluation of membrane protein (UapA) structure stabilization ability of TNMs (Fig. 5)

Experiments were conducted to determine the structural stability of UapA (uric acid-xanthine / H ⁺ symporter) by TNMs in aqueous solution. Structural stability of UapA was measured using CPM assay, and TNMs and DDM concentrations were measured at CMC + 0.04 wt% and CMC + 0.2 wt%.

Specifically, UapA protein is Aspergillus nidulan nidulans ) is a uric acid-xanthine / H ⁺ symporter. Protein stability was determined using a fluorescence spectrophotometer using CPM ( N- [4- (7-Diethylamino-4-methyl-3-coumarinyl) phenyl] maleimide), a sulfhydryl-specific fluorophore. spectroscopy). The free sulfhydryl group (-SH) of the cysteine residue is in the center of the protein, but is exposed to the surface by protein unfolding to make the solvent accessible. CPM reacts with free thiol and becomes fluorescent, thereby acting as an annealing sensor. To measure the thermal stability, first UapAG411V _{△ 1-11} is my process as Saccharomyces celebrity non-zero (Saccharomyces cerevisiae ) expressed in GFP fusion in FGY217 strain and isolated into sample buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, 1 mM xanthine), which is described in J. Leung et al ., Mol . Membr . . followed the method described in 2013, 30, 32-42). UapA protein was concentrated to about 10 mg / ml using a 100 kDa molecular weight cut off filter (Millipore). The concentrated protein was diluted 1: 150 in a Greiner 96-well plate with buffer containing DDM or TNMs, respectively, at a concentration of CMC + 0.04 wt% or CMC + 0.2 wt%. CPM dye (Invitrogen) stored in DMSO (sigma) is diluted in dye buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, 5 mM EDTA) and 3 μl of diluted dye is tested for each protein Added to conditions. The reaction was monitored using a microplate fluorometer at 40 ° C. for 120 minutes. Relative maximum fluorescence samples were used as controls to calculate the percentage of relative folded protein remaining after 120 minutes at 40 ° C. Relative folded proteins are shown over time using GraphPad Prism.

The ability to keep the UapA protein folded at CMC + 0.2 wt% was better for all TNMs than for DDM (FIG. 7). In particular, TNM-C12L and TNM-C14S were shown to be the most effective among TNM-Ls and TNM-Ss, respectively (FIG. 7). TNMs showed similar abilities at CMC + 0.2 wt% even at CMC + 0.04 wt% (FIG. 8). From these results, it can be seen that TNMs can be effectively used to stabilize the membrane protein because it shows an excellent effect in maintaining the structurally stable state of the UapA protein extracted from the cell membrane in aqueous solution.

<Example 5> Evaluation of membrane protein (LeuT) structure stabilization capacity of TNMs

Experiments were conducted to measure the structural stability of LeuT (leucine transporter) protein by TNMs. LeuT protein activity was measured by scintillation proximity assay (SPA) using protein substrate ([ ³ H] -Leu), and the concentrations of TNMs or DDM were CMC + 0.04 wt% and CMC + 0.2 wt%.

Purification of wild type LeuT (leucine transporter) from Aquifex aeolicus was carried out according to the method published in G. Deckert et al. ( Nature 1998, 392, 353-358.). LeuT is expressed in E. coli C41 (DE3) transformed with pET16b encoding the C-terminal 8xHis-tagged transporter (expression plasmids are provided by Dr E. Gouaux, Vollum Institute, Portland, Oregon, USA ). In summary, separate the LeuT protein, after screen dissolved in 1.0 wt% DDM, and coupling a protein to the ^{Ni 2 + -NTA resin (Life Technologies} , Denmark), 20 mM Tris-HCl (pH 8.0), 1 mM NaCl Eluted with 199 mM KCl, 0.05% DDM and 300 mM imidazole. About 1.5 mg / ml protein stock was then diluted with equivalent buffer free of DDM and imidazole but supplemented with TNMs or DDM (control) to a final concentration of CMC + 0.04 wt% or CMC + 0.2 wt% . Protein samples were stored at room temperature for 10 days, centrifuged at designated times, and protein activity was determined by scintillation proximity assay (SPA) (M. Quick et al., Proc. Natl , Acad . Sci . USA . 2007, 104, 3603- 3608.) was used to measure [ ³ H] -Leu binding. The assay was performed with a buffer containing 450 mM NaCl and each of these compounds at the final concentration. SPA reactions were performed in the presence of 20 nM [ ³ H] -Leu and 1.25 mg / ml copper chelate (His-Tag) YSi beads (both purchased from PerkinElmer, Denmark). [ ³ H] -Leu binding was measured using a MicroBeta liquid scintillation counter (PerkinElmer).

As shown in FIGS. 9 and 10, LeuT solubilized by TNM-C10L in TNM-Ls and TNM-11S in TNM-Ss showed the best substrate binding ability, and the protein stabilizing effect of the compounds. Was better than DDM. In addition, it was confirmed that the shorter linker length TNM-Ss exhibited the ability of the transporter to maintain better activity than the longer TNM-Ls. The results were similar when the concentration of amphiphilic compound increased from CMC + 0.04 wt% to CMC + 0.2 wt%.

As a result, TNM-C11L among TNMs showed the ability of DDM to maintain early activity of similar solubilized receptors (FIGS. 9 and 10). However, in the long-term LeuT stabilization ability, DDM showed a rapid loss of protein activity over time, TNM-C11L was confirmed to maintain the activity of LeuT well during the 10-day incubation process (Figs. 9 and 10). Therefore, TNM-C10L and TNM-C11S showed better activity of this transporter than DDM.

Example 6 β of TNMs ₂ AR protein stabilization ability assessment

The receptor was expressed in Sf9 insect cells infected with baculovirus and solubilized in 1% DDM. Receptors solubilized in DDM were purified by alprenolol sepharose in the presence of 0.01% cholesteryl succinate (CHS). Β ₂ AR purified by DDM was diluted with buffer containing DDM or TNMs to a final concentration of CMC + 0.2 wt%. The solubilized β ₂ AR for each compound was stored at room temperature for 4 days and incubated with radioactive [ ³ H] -dihydroalprenool (DHA) at 10 nM for 30 minutes at room temperature at regular intervals. Ligand binding capacity was measured. The mixture was loaded onto a G-50 column and the supernatant collected using an amount of binding buffer (supplemented with 20 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mg / ml BSA). An additional 15 ml scintillation fluid was added. Receptor-bound [ ³ H] -DHA was measured with a scintillation counter (Beckman).

As a result, TNM-C12L among TNMs showed the ability of DDM to maintain the initial activity of similar solubilized receptors (FIG. 11). However, in the long-term receptor stabilization ability, DDM showed a rapid loss of activity over time, while receptors solubilized in TNM-C12L continued to retain the activity of the receptor for 4 days of culture (FIG. 12). Therefore, TNM-C12L could be judged to be more effective in solubilizing receptor protein studies than DDM.

Claims (22)

Compound represented by the following formula (1):
[Formula 1]

In Chemical Formula 1,
R ¹ , R ² and Each R ³ is independently an unsubstituted C ₃ -C ₂₀ alkyl group;
X ¹ , X ² and X ³ are each independently a saccharide linked by oxygen;
Y ¹ , Y ² and Y ³ are oxygen (O); And
M is 0 or 1; The method of claim 1,
The saccharide is a monosaccharide or a disaccharide (disaccharide) compound. In the first,
The saccharide is glucose or maltose The method of claim 1,
R ¹ to R ³ are each independently an unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are glucose or maltose; Y ¹ , Y ² and Y ³ are oxygen (O); And m is 1. The method of claim 1,
R ¹ to R ³ are each independently an unsubstituted C ₃ -C ₂₀ alkyl group; X ¹ to X ³ are glucose or maltose; Y ¹ , Y ² and Y ³ are oxygen (O); And m is 0. The method of claim 1,
The compound is one of the compounds represented by the formula 2 to 11:
[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

The method of claim 1,
The compound is an amphiphilic molecule for extracting, solubilizing, stabilizing, crystallizing or analyzing the membrane protein. The method of claim 1,
The compound has a micelle concentration (CMC) in the aqueous solution of 0.0001 to 1mM. Composition for extracting membrane protein comprising the compound according to claim 1. delete 1) 5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane (5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane) and alcohol ( alcohol) to synthesize dialkylated diols;
2) synthesizing a trialkylated monool by adding 1-bromoalkane to the product of step 1);
3) allylating the monool by adding allyl iodide to the product of step 2);
4) synthesizing trialkylated monool by hydroboration oxidation of the product of step 3);
5) 4- (bromomethyl) -l-methyl-2,6,7-trioxabicyclo [2.2.2] -octane (4- (bromomethyl) -l-methyl-2 in the product of step 4) , 6,7-trioxabicyclo [2.2.2] -octane) to synthesize trialkylated triols;
6) introducing a saccharide with a protecting group by performing a glycosylation reaction on the product of step 5); And
7) performing a deprotection reaction (deprotection) reaction on the product of step 6);
[Formula 1]

In Chemical Formula 1,
R ¹ , R ² and Each R ³ is independently an unsubstituted C ₃ -C ₂₀ alkyl group;
X ¹ , X ² and X ³ are each independently a saccharide linked by oxygen;
Y ¹ , Y ² and Y ³ are oxygen (O); And
M is 1. 1) Alkylation of 5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane (5,5-bis-bromomethyl-2,2-dimethyl- [1,3] dioxane) Reacting to synthesize a compound in the form of a dialkylated diol;
2) synthesizing a trialkylated monool type compound by adding 1-bromoalkane to the product of step 1);
3) allylating the monool by adding allyl iodide to the product of step 2);
4) hydroboration oxidation of the product of step 3) to synthesize trialkylated monool compounds;
5) synthesizing a compound of the trialkylated aldehyde form through alcohol oxidation of the product of step 4);
6) adding formaldehyde and water-soluble alkaline ethanol to the product of step 5) to synthesize a trialkylated triol form of the compound;
7) introducing a saccharide to which a protecting group is attached by performing a glycosylation reaction on the product of step 6); And
8) A process for preparing a compound represented by the following Chemical Formula 1 comprising the step of performing a deprotection reaction on the product of step 7):
[Formula 1]

In Chemical Formula 1,
R ¹ , R ² and Each R ³ is independently an unsubstituted C ₃ -C ₂₀ alkyl group;
X ¹ , X ² and X ³ are each independently a saccharide linked by oxygen;
Y ¹ , Y ² and Y ³ are oxygen (O); And
M is zero. delete delete delete delete Composition for solubilizing the membrane protein containing the compound according to claim 1. A composition for stabilizing a membrane protein comprising the compound according to claim 1. A composition for crystallization of a membrane protein comprising the compound according to claim 1. Composition for the analysis of membrane proteins comprising a compound according to claim 1. 21. The composition of any one of claims 9 and 17-20, wherein the composition is a formulation of micelles, liposomes, emulsions or nanoparticles. The method of claim 9 or 17 to 20, wherein the membrane protein is LHI-RC complex (light harvesting-I and the reaction center complex), UapA (Uric acid-xanthine / H + symporter), LeuT (Leucine transporter), β2AR (human β2 adrenergic receptor), MelB (Melibiose permease), or a combination of two or more thereof.

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