KR101375792B1 - Derivatives Conjugated to Cell Penetrating Peptide and Uses Thereof - Google Patents

Abstract

The present invention relates to derivatives in which target molecules are conjugated to the N-terminus or C-terminus of dipeptides with improved cell permeability and medical, food engineering and cosmetic engineering applications of these derivatives. According to the present invention, through the cell permeation performance of the dipeptide, by combining the target material that has been difficult to enter into the conventional cells can be efficiently introduced into these cells.

Description

Derivatives Conjugated to Cell Penetrating Peptide and Uses Thereof}

The present invention relates to derivatives bound to cell penetrating peptides, and more particularly, to derivatives which are bound to peptides having greatly improved cell penetrating performance and thus having improved cell penetrating performance and / or stability, and their use as industrial applications.

In order for various medicines, functional foods or functional cosmetics to treat, prevent or alleviate diseases of humans or animals in order to function efficiently in vivo, the active ingredients or active ingredients contained in these medicines, foods and cosmetics are incorporated into cells. It needs to be introduced. In general, a method of using endocytosis in a cellular system has been considered as a method for introducing macromolecules into cells. However, since some active ingredients or active ingredients do not flow into eukaryotic cells in particular, for example, by modifying the form of the preparation, a method of allowing these active ingredients or active ingredients to penetrate the cells and introduce them into the cells. Sought.

Among them, studies on cell penetrating peptides (CPP) have been made based on the fact that some virus-derived peptides improve cell penetration efficiency. CPPs are peptides that can cross biological membranes or physiological barriers, such as cell permeable peptides, protein-transduction domains (PTDs) or membrane-transition sequences (membrane- translocation sequences (MTS).

Roneun currently known CPP human immunodeficiency virus type 1 (HIV-1) protein Tat protein (Frankel, AD and Pabo, CO (1988) Cellular of the tat protein from human immunodeficiency virus Cell 55 (6):. 1189-93) , herpes virus envelope protein VP22: the (Elliott, G. and O'Hare, P. (1997) Intercellular Trafficking and protein Delivery by a herpesvirus Structural protein Cell 88 (2) 223-233.), fruit fly (Drosophila sp.) Penetratin from antennnapdeia homeoprotein (Derossi, D. et al. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes.J. Biol. Chem. 269 (14): 10444-450; Schwarze, SR and Dowdy, SF (2000) In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA.Trends.Pharmacol.Sci. 21 (2): 45-48), fibroblast growth factor (Rojas, M. et al. .... (1998) Genetic engineering of proteins with cell membrane permeability Nat Biotechnol 16: 370-75) , including reports It was. Although the cellular infiltration mechanism of these CPPs is not known precisely, the cationic amino acids arginine (Arg) and lysine (Lys) occupy a large portion of these CPPs, it is assumed that these cationic amino acids play a role.

CPPs can carry macromolecules up to several times their molecular weight into cells, and because they are efficient for a variety of cell types, they effectively penetrate into cells the active or active ingredients found in pharmaceuticals, functional foods or functional cosmetics. It is estimated to have the potential to make it possible. For example, CPP bound to the target material can enter the cell and / or cell nucleus by transposing the cell membrane in vitro or in vivo. It is known that various biological molecules such as antigenic peptides, peptide nucleic acids, antisense oligonucleotides, full length proteins, nanoparticles, liposomes can be delivered via CPP. Given that most peptides or nucleic acid-based drugs are difficult to enter into cells, limiting their development as therapeutic agents, CPP could double the efficiency of these drugs in vivo. It is expected.

For example, by combining CPP and a target substance, a target substance or a drug or a marker called a cargo or the like is introduced into an intracellular destination, or from outside the cell through the plasma membrane into the cytoplasm or in the nucleus, mitochondria, lysosomes, or the like. It can be directed to the desired location, and the target material can be guided through the barrier in the blood, mucous membranes, skin and the like. In particular, the possibility of transporting macromolecules through the brain-blood barrier using CPP has been suggested (Schwarze, SR et al. (1999) In vivo protein transduction: delivery of biologically active protein into the mouse) Science 285: 1569-72).

Regarding the derivatives conjugated with the aforementioned cell penetrating peptides, Korean Patent Publication No. 10-2003-0028232 discloses an ascorbic acid derivative in which a cell penetrating Tat peptide is introduced into a hydroxyl group of carbon 2 or 3 and the same. A cosmetic composition for skin whitening is included, and Korean Patent No. 10-0490363 discloses a fusion protein of nitroreductase, which is important in enzyme / prodrug therapy, using Tat peptide as a transport domain. In addition, Korean Patent Laid-Open No. 10-2008-0108592 discloses a camptothecin or a derivative thereof connected to the linker using a cell penetrating peptide having cysteine present at the N-terminus or C-terminus as a carrier. . In addition, Korean Patent Publication No. 10-2009-0122779 discloses a protein in a state in which a peptide including an intracellular transfer domain having a hydrophilic domain and a hydrophobic domain and a charge between a charged material is not physically conjugated with the protein. I'm suggesting delivery to me.

However, most cell-penetrating peptides developed to date use peptides derived from viruses, such as HIV-derived Tat protein or herpes virus envelope protein VP22, which may cause safety problems. Therefore, it was not easy to develop the product by applying CPP.

The present invention has been proposed to solve the above-mentioned problems, and an object of the present invention is to provide a derivative in which a target substance is bound to a cell-penetrating peptide not derived from a virus.

Another object of the present invention is to provide an application such as a drug, food, dyes and / or cosmetics containing as an active ingredient a target substance derivative combined with a cell penetrating peptide.

Other objects and advantages of the present invention will become more apparent from the drawings and accompanying drawings.

According to the present invention having the above-mentioned object, the present invention is ascorbic acid, ascorbic acid in which a part of the hydroxy group is substituted with a carboxy group as a target substance to be transported to the terminal of the dipeptide composed of glycine-proline or glycine-hydroxyproline. Derivatives, fluorescein, or fluorescein derivatives selected from the group consisting of fluorescein isothiocyanates, fluorescein amidaites and fluorescein sulfonylchlorides, possible isomers thereof, possible salts thereof or Provided are compounds in which a target substance selected from combinations thereof is conjugated.

For example, the compound is a compound represented by the following general formula (I) and includes their possible isomers or their possible salts.

In formula (I), R ₁ is hydrogen or a hydroxy group; R ₂ is hydrogen, ascorbic acid, fluorescein, or fluorescein isothiocyanate, fluorescein amidite and fluorescein sulfonyl A fluorescein derivative selected from the group consisting of chloride; R ₃ is hydrogen, an ascorbic acid derivative in which a part of a hydroxy group is substituted with a carboxy group, fluorescein, or fluorescein isothiocyanate, fluorescein amine Fluorescein derivatives selected from the group consisting of diet and fluorescein sulfonylchloride; at least one of R ₂ and R ₃ is not hydrogen)

In this case, the compound represented by the formula (I) is a compound which is a fluorescein derivative represented by the following formula (II), and may include their possible isomers or possible salts thereof.

In formula (II), R ₁ is the same as defined above; R ₄ and R ₅ are each independently hydrogen, fluorescein, or fluorescein isothiocyanate, fluorescein amidite and fluore Fluorescein derivatives selected from the group consisting of shin sulfonylchloride, at least one of R ₄ and R ₅ is not hydrogen)

The compound as a fluorescein derivative represented by the above formula (II) is a compound represented by the following formula (IIa) or the following formula (IIb), and may include their possible isomers or possible salts thereof.

In which Formula (IIa) and (IIb), R ₁ is the same as defined above

In addition, the compound represented by the said general formula (I) is a compound which is an ascorbic acid derivative represented by the following general formula (III), and includes these possible isomers or these possible salts.

(Formula (III), R ₁ is the same as defined in formula (I); R ₆ is hydrogen or ascorbic acid; R ₇ is ascorbic acid derivative in which a part of hydrogen or hydroxy group is substituted with carboxyl group, At least one of R ₆ and R ₇ is not hydrogen)

The compound represented by the above general formula (III) is a compound represented by the following general formula (IIIa) to the following general formula (IIIc), and includes possible isomers thereof and possible salts thereof.

(In Formulas (IIIa) to (IIIc), R ₁ is the same as defined above)

According to another aspect of the present invention, there is also provided a fluorescent composition comprising the compound represented by the above formula (II), preferably the above formulas (IIa) to (IIb), their possible isomers or mixtures of these isomers. do.

In addition, the present invention provides a skin whitening or skin aging containing the compound represented by the above formula (III), preferably the above formulas (IIIa) to (IIIc), their possible isomers or possible salts thereof as an active ingredient. It provides a functional cosmetic composition for prevention.

At this time, the compound represented by the formula (III), for example, the formula (IIIa) to the formula (IIIc), the possible isomers thereof or the possible salts thereof as active ingredients are 10 to 100 μg / ml in the cosmetic composition. It may be contained at a concentration of.

In addition, the present invention comprises the steps of (a) synthesizing a dipeptide of glycylproline or glycylhydroxyproline; And (b) reacting at least one of ascorbic acid and an ascorbic acid derivative in which a portion of the hydroxy group is substituted with a carboxy group at the C-terminus or N-terminus of the dipeptide using an organic solvent in the presence of a condensing agent. It provides a method for producing the ascorbic acid derivative represented by the formula (III), for example, the formula (IIIa) to the formula (IIIc) comprising a.

At this time, the step (b) is a step of protecting a portion of the hydroxy group present in the ascorbic acid or ascorbic acid derivative in which a portion of the hydroxy group is substituted with a carboxy group with acetal or ketal and / or one end of the dipeptide The method may further include binding ascorbic acid or a derivative thereof to the other end (eg, N-terminus) of the dipeptide in a state in which ascorbic acid or a derivative thereof is bound to (eg, C-terminus).

In the present invention, it was newly identified that the dipeptide of glycylproline has a cell permeability.

The newly identified dipeptides in the present invention are easy to synthesize, and compared to those in which cell-derived peptides derived from viruses of the prior art have not been utilized as actual products, since the peptides of the present invention are not derived from viruses, the safety is improved and the living body is improved. It can also be used inside.

Therefore, the derivative of the present invention can be utilized as an active ingredient or active ingredient of a fluorescent composition or functional cosmetics for skin whitening or wrinkle improvement.

1 is a view schematically illustrating a reaction in which a derivative is synthesized by conjugation of a target molecule to the C-terminus of a cell penetrating peptide according to the present invention.
Figure 2 schematically shows a reaction in which a derivative is synthesized by conjugation of a target molecule to the N-terminus of a cell penetrating peptide according to the present invention.
3A and 3B are schematic views illustrating a reaction mechanism in which a fluorescent substance FITC, a fluorescein derivative, is bound to a terminal of a cell penetrating peptide, thereby synthesizing a derivative according to one embodiment of the present invention.
4a to 4c schematically show a reaction mechanism in which ascorbic acid is bound to the terminal of a cell penetrating peptide to synthesize a derivative according to one embodiment of the present invention, and FIG. 4a is a hydrate at position 6 of ascorbic acid. 4B shows a reaction mechanism for synthesizing a derivative in which an oxy group is ester-bonded with the C-terminus of a peptide. 4C shows a reaction mechanism in which a derivative in which a di-peptide is bound to a hydroxyl group at positions 3 and 6 of ascorbic acid is synthesized.
5A to 5C are diagrams measuring the degree of internalization of cells into which the dipeptides and fluorescein are bound according to an embodiment of the present invention. FIG. 5A is a graph showing the measurement results, and FIG. 5B. 5C are photographs observed with a fluorescence microscope.
Figure 6a is a graph showing the HPLC measurement results of the derivative coupled to the dipeptide and ascorbic acid according to an embodiment of the present invention, Figure 6b is a graph measuring the content of ascorbic acid.
Figure 7 is a graph showing the cytotoxicity measurement results of the derivative coupled to the dipeptide and ascorbic acid according to an embodiment of the present invention.
8 is a graph showing the results of the ALP measurement of the derivative coupled to the dipeptide and ascorbic acid according to an embodiment of the present invention.
Figure 9 is a graph showing the results of measurement of collagen expression of the derivative coupled to the dipeptide and ascorbic acid according to an embodiment of the present invention.

The present inventors researched and developed a solution for solving the above-mentioned problems of the prior art, and found that glycylproline composed of glycine-proline (Pro) has cell permeability and completed the present invention. It was. Hereinafter, the present invention will be described with reference to the accompanying drawings.

Ⅰ. Derivatives Associated with Cell Permeable Peptides

The present invention relates to derivatives wherein the target substance is conjugated to the N-terminus and / or C-terminus of the dipeptide of glycyl (hydroxy) proline. The inventors have found that the dipeptide of glycyl (hydroxy) proline is capable of cell permeation. Thus, the present invention makes the dipeptide a kind of carrier moiety, and the target substance bound thereto is a carrier component. It can be understood as a conjugate (delivery moiety).

The carrier component according to the invention is a peptide or analogue thereof which can promote the penetration of the carrier component into cells or tissues and improve the stability. The dipeptide according to the present invention is glycyl (hydroxy) proline, which is a derivative in which the target substance is bound to the dipeptide. As used herein, the term 'target molecule' includes chemicals, small molecules, polypeptides, nucleic acids, or viruses that can bind to and be transported into a cell-penetrating peptide. For example, a substance capable of binding to glycyl (hydroxy) proline as a cell penetrating peptide (CPP) whose efficacy has been confirmed according to the present invention may be an active ingredient such as proteins, peptides, nucleotide sequences, drugs, dyes, cosmetics, etc. It includes, but is not limited to, chemicals used. Derivatives bound to the dipeptides of the present invention penetrate the cell membrane with very high efficiency and remain in the cytoplasm and nucleus within the cell.

As used herein, the term "conjugation" or "conjugated" refers to the interaction between each component, such as covalent, ionic or hydrophobic interactions, such that the carrier component and the carrier component are linked to each other to maintain proximity. It means action.

Derivatives produced by binding to the dipeptides of the present invention should be construed to include salts, optical and / or geometric isomers, partial isomers or mixtures thereof. For example, derivatives of the invention may be in the form of possible optical and / or geometric isomers of dipeptides and / or target substances or mixtures of these isomers, or pharmaceutically, food engineering, cosmetically acceptable salts of the target substances. And may be in free or zwitterionic form or in the form of a pharmaceutically acceptable inorganic or organic base or acid added salt.

In this context the target material may bind to the dipeptide via a linker or crosslinker, which terms are used interchangeably herein and bind / link two components together and contain one or more atoms. Chain and / or ring.

"Peptide" as used herein also encompasses "peptide analogs", ie, analogues that are one or more substituted for the side chain or alpha-amino acid backbone of L-amino acids. Examples of side chain or backbone modified peptide analogs include hydroxyproline in which the pyrrolidine ring is substituted with a hydroxy group, or an N-methyl glycine "peptoid".

Small molecules capable of binding dipeptides according to the present invention include, for example, drugs, contrast agents (e.g., T1 contrast agents, T2 contrast agents such as superparamagnetics, radioisotopes, fatty acids, etc.), fluorescent markers, cosmetic ingredients, foods Components, and the like, but is not limited thereto. For example, polypeptides, including peptides and proteins, eg, proteins involved in cell immortality (eg, SV40 large T antigen and telomerase), anti-apoptotic proteins (eg, mutant p53 and BclxL), antibodies, Oncogenes (eg ras, myc, HPV E6 / E7 and adenovirus Ela), cell cycle regulatory proteins (eg cyclin and cyclin dependent kinase) or enzymes (eg green fluorescent protein, beta-galactosidase and chloramphenicol Acetyl transferase), but is not limited thereto. In addition, the nucleic acid may be, for example, RNA, DNA or cDNA, and the sequence of nucleic acids may be a coding site sequence or a non-coding site sequence (eg, an antisense oligonucleotide or siRNA). The virus can be a virus core (ie, viral nucleic acid packaged without the envelope of the virus) that includes the virus as a whole or the nucleic acid of the virus. Examples of viruses and viral cores that can be transported include, but are not limited to, papilloma virus, adenovirus, baculovirus, retrovirus core and semilky virus core. Nucleotides as target materials may be standard nucleotides (eg adenosine, cytosine, guanine, thymine, inosine and uracil) or analogs (eg phosphorothioate nucleotides), in particular antisense sequences or si-consists of phosphorothioate nucleotides RNA may be. Since the amine group and the carboxyl group exist in the terminal of the dipeptide of the present invention, the derivatives of these target materials can be prepared by combining with the aforementioned various target materials.

 For example, Figure 1 briefly illustrates a state in which the target material is bound to the N-terminus of the dipeptide according to the present invention. In (a) of FIG. 1, the carboxyl group of the dipeptide C-terminus and the hydroxy group of the target substance react to illustrate the conjugate of the dipeptide-target substance ester-bonded at the C-terminus of the dipeptide. Examples of the target substance having a hydroxyl group include alcohols, polyhydric alcohols such as dihydric alcohols such as ethylene glycol, and trihydric alcohols such as glycerol, and especially ascorbic acid used as an ingredient or antioxidant of a whitening cosmetic. Although the derivative is mentioned, this invention is not limited to this.

In (b) of FIG. 1, another hydroxy group and the C-terminus of the second dipeptide, which are already present in the target substance ester-bonded with the C-terminus of the dipeptide, form an ester bond, so that the target substance is formed with two dipeptides, respectively. The bound form is illustrated (ie, dipeptide: target substance = 1: 1). For example, ascorbic acid or a derivative thereof has a large number of hydroxyl groups, so as a first step, a hydroxyl derivative at position 6 of ascorbic acid or a derivative thereof forms a primary derivative that forms an ester bond with the C-terminus of the first dipeptide. It is possible to synthesize and to form an ester bond with the C-terminus of the second dipeptide, with a hydroxyl group at position 2 or 3 of the ascorbic acid derivative as the primary derivative.

In the figure, although the target material is shown to form an ester bond with the C-terminus of each dipeptides, the target substance binds with the N-terminus of the first dipeptides and binds with the C-terminus of the second dipeptides. It is also possible. For example, a primary derivative in which an amide bond is formed with an N-terminus of the first dipeptides after substituting a hydroxyl group at a specific position (eg, position 3) present in ascorbic acid or a derivative thereof with a carboxyl group or the like. And the hydroxy group at the 6-position of the ascorbic acid derivative as the primary derivative forms an ester bond with the C-terminus of the second dipeptides.

1 (c) shows that the target material having an amine group forms an amide bond with the C-terminus of the dipeptide. As a target substance having an amine group, a drug that may be considered as an antagonist of a neurotransmitter, which is a natural amine, may be mentioned first. For example, antihistamines are used in the form of amine hydrochloride as an active ingredient in the active ingredient chlorpheniramine, an active ingredient in tranquilizers, chlorpromazine, decongestants or nasal salts. Amphetamine or methamphetamine, used as an active ingredient in ephedrine, phenylephrine, sychostimulant, and tertiary amine, atritriphylline (an active ingredient in tricyclic antidepressants) amitriptyline, imipramine, lofepramine, clomipramine, and secondary amines, nortriptyline and desipramine, active ingredients of tricyclic antidepressants ), Amoxapine and the like. Or a target substance linked with a linker having an amine group, for example fluorescein or a derivative thereof, as described below. For example, in the case of using a linker having an amine group at both ends, first, an amine formed at one end of the linker and an isothiocyanate group of fluorescein isothiocyanate (FITC) as a target material react to form a thiourea bond. After synthesizing the new isothiocyanate derivative, it can be considered that the amine group formed at the other end of the synthesized fluorescein isothiocyanate derivative (linker-FITC) forms an amide bond with the carboxy group of the dipeptide C-terminus. .

In addition, polyamines, such as putrescine, spermidine, and spermine, which are involved in immune function, synthesis of proteins or nucleic acids, metabolism, etc., have two or more amine groups in vivo. Thus, two or more dipeptides may be combined to form amide bonds with the C-terminus of each.

On the other hand, in contrast to the binding of the target material to the C-terminus of the dipeptide in Figure 1, Figure 2 schematically shows the form of the target material to the N-terminus of the dipeptide. First, as shown in (a) of FIG. 2, a target substance having a carboxyl group at one end forms an amide bond with an amine group of the dipeptide N-terminus. There are many kinds of target substances having a carboxyl group, and examples of the biological substance include fatty acids. It is preferably a higher fatty acid, more preferably a higher saturated fatty acid having 16-20 carbon atoms, most preferably 16-18 carbon atoms. In addition, polycarboxylic acid may be used as a target material for forming an amide bond with the N-terminus of the dipeptide. Or, for example, in the case of using a linker having a reactor at both ends, firstly, one end of the linker and the target material are combined, and then the dipeptide N-terminus and the other end of the linker carboxy group are bonded by an ester reaction so that the target material is linked through the linker. It may be indirectly connected.

In addition, (b) of FIG. 2 illustrates a form in which a target material having an isothiocyanate group reacts with an N-terminal amine of a dipeptide to form a thiourea bond. Phenolic isothiocyanate (phenylethyl iso), which is present in some plants and is known to inhibit carcinogenesis and tumor formation by inducing tumor cell proliferation in some plants. Drugs or drug candidates, such as thiocyanate (also known as 2-Isothiocyanatoethyl) benzene) and sulforaphane (1-Isothiocyanato-4-methylsulfinylbutane) in broccoli as an anti-cancer / anti-obesity / antimicrobial effect Or fluorescein isothiocyanate (FITC), a fluorescein derivative well known as a fluorescent substance.

On the other hand, Figure 2 (c) shows that the target substance substituted with succinimidyl ester reacts with the N-terminus of the dipeptide to form an ester amide bond. As is well known, succinimidyl esters, in particular, react with amine groups to show similar safety as natural peptide bonds. Reactors similar to succinimidyl esters may include, for example, imidazole esters and the like, which equivalents may also be used as functional groups of the target material according to the invention. As a target substance which has functional groups, such as a succinimidyl ester or an imidazole ester, Ascorbic acid derivative | substrate in which at least one part of the hydroxyl group was substituted by the imidazole ester group; Carboxy fluorescein succinimidyl esters or derivatives thereof such as 5-carboxyfluorescein succinimidyl ester (5-FAM) used as a fluorescent substance for labeling peptides and the like; 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester or a derivative thereof, which is a blue fluorescent substance used to label peptides, nucleic acids, and the like. have.

In addition, (d) of FIG. 2 shows that the target material having a sulfonyl chloride group reacts with the dipeptide N-terminus to form sulfonamide by Hinsberg reaction. Since sulfonyl chloride is highly reactive, it can bind rapidly with dipeptide N-terminal amine groups. As a target material having sulfonyl chloride, as well as fluorescein sulfonyl chloride, a fluorescent material used to label peptides, And chemicals having tosyl chloride (4-toluenesulfonyl chloride) and mesyl chloride (methanesulfonyl chloride).

When the dipeptide N-terminal amine group is reacted with isothiocyanate, succinimidyl ester, imidazole ester group or sulfonyl chloride, each of which is illustrated as a reactor of the target material in Figs. 2 (b) to (d), respectively, It functions as a nucleophile. Derivatives with thiourea bonds formed by reaction of isothiocyanate with amine groups show some stability, but since the stability may decrease over time, they react with the dipeptide N-terminal amine group to form a conjugate. It may be particularly desirable to use target materials having or substituted succinimidyl ester groups to form.

In FIG. 2, the target material binds to one dipeptide, but as shown in FIG. 1B, the same or different target material may bind to two or more dipeptides. For example, polycarboxylic acids such as oxalic acid, adipic acid, azelaic acid, maleic acid and terephthalic acid having two or more carboxyl groups are used as targets. Derivatives are also possible in which an amide bond is formed between each carboxyl group of these polycarboxylic acids and the N-terminus of each dipeptide.

1 and 2 illustrate that the target material binds to the C-terminus and the N-terminus of the dipeptide, respectively. However, if desired, two identical or different target substances may bind to the N-terminus and C-terminus of the dipeptide, respectively (target substance: dipeptide = 2: 1). For example, ascorbic acid or a derivative thereof may bind with the C-terminus of the dipeptide in an ester reaction, and fluorescein or a derivative thereof may bind with the N-terminus of the dipeptide through a thiourea reaction. In addition, if necessary, two peptides may be bound to one target substance (target substance: dipeptide = 1: 1).

As briefly described above, the target substance and the dipeptide according to the present invention may be directly bonded through respective functional groups such as carboxyl groups, amino groups, hydroxyl groups, isothiocyanate groups, and indirectly through linkers. can do. The linker may provide flexibility in the molecule or adjust the distance between the dipeptide-targeting substances to help maintain biological activity.

At this time, the functional group (reactive group) of the linker used to form a covalent bond between the linker and the dipeptide and / or between the linker and the target material is an amine group, a hydroxyl group, a hydrazino group, a thiol group, a maleimido group, a carbonyl group And carboxyl groups. Linkers having these functional groups at least at one end are, for example, straight or branched alkyl groups having 1 to 10 carbon atoms; C1-C10 linear or branched heteroalkyl group which has hetero atoms, such as O, N, and S; A cycloalkyl group having 3 to 8 carbon atoms; A cycloalkyl group having 3 to 8 carbon atoms having the aforementioned hetero atom; Aryl groups having 1-3 rings; It may be a hetero aryl group having 1-3 rings and containing 1-4 hetero atoms, such as N, O, and S.

In addition, these functional groups may be identical (homologous groups) and may have different types of functional groups (heterologous groups). Preferably, the functional group of the linker may be selected from an amine group, a carboxyl group and a maleimido group and, if necessary, may include a short sequence of 1-4 amino acid residues having a thiol group which is a medium for selectively linking the linker to the dipeptide. have.

For example, the linker can be independently substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aldehyde, acid, ester, anhydride, sulfhydryl or carboxyl group. 2-functional and multi-functional organic radicals selected from maleimido derivatives, maleimido cyclohexane derivatives, maleimido benzoic acid derivatives, maleimidocaproic acid derivatives and succinimido derivatives, or cyanogen bromide or chloride, Succinimidyl esters or sulfonyl halides or the like or combinations thereof.

Crosslinking agents can be used to induce chemical bonds when forming a combination of a dipeptide and a target material. Since the N-terminal of the dipeptide has an amine group, the formation of a conjugate by a crosslinking agent is easy. In particular, crosslinking agents which form reversible or irreversible linkages with amino groups are preferred, which may be used as 1,5-difluoro-2,4-dinitrobenzene, p, p'-difluoro-N, N ' -Dinitrodiphenylsulfone, dimethyl adiimidate, phenol-1,4-disulfonylchloride, hexamethylene diisocyanate, hexamethylene diisothiocyanate, azophenyl-p-diisocyanate, glutaraldehyde (various side chains And N-3-maleimidopropanoic acid, N-6-maleimidocaproic acid, N-11-maleimidodecanoic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxy-6 Amidocaproic acid, 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethylene glycol (BM [PEO] 4), 1-ethyl-3- [3-dimethyl aminopropyl Carbodiimide hydrochloride (EDC), m-maleimidobenzoyl-N-hydrosuccinimide ester (MBS) and its sulfonates (sulfo-MBS), Cinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and its sulfonates (sulfo-SMCC), succinimidyl-4- (N-maleimidomethyl) -cyclohexane -1-carboxy (6-amidocaproate) (LC-SMCC), N-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N-succinimidyl-3- (m-maleimido ) Propionic acid esters or sulfonates thereof, succinimidyl-4- (p-maleimidophenyl) butyric acid ester (SMPB), succinimidyl 6- [3- (2-pyridyldithio) -lopionamido] hexano SSPDP and its sulfonate flame (sulfo-SPDP) and the like, but are not limited thereto. The succinimidyl group of the crosslinker reacts with the primary amine forming an amide bond. Some of these crosslinkers have low solubility in water, and hydrophilic groups such as sulfonate groups can be added to the crosslinker to improve solubility in water.

The target substance bound to the dipeptide according to the present invention is a fluorescent label, an active ingredient such as a fluorescent substance, a drug or a cosmetic used as a phosphor in a contrast agent or a biomaterial detection sensor, as well as lipids, fatty acids, carbohydrates, vitamins, peptides, poly Biomaterials such as peptides, proteins, nucleic acids, and drugs. For example, target materials capable of binding to the dipeptides of the present invention are fluorescein, ascorbic acid or derivatives thereof, including their possible geometric and / or optical isomers and possible salts. These target substances may bind to the N-terminus or C-terminus of the dipeptide, and if necessary, two target substances, which are the same or different from each other, may respectively bind to the N-terminus and C-terminus of the dipeptide, respectively. (Target substance: dipeptide = 2: 1). Alternatively, as needed, two peptides may be bound to one target material (target material: dipeptide = 1: 2).

By way of example, the present invention relates to a derivative in which a dipeptide and a target substance are bound by the following formula (I).

In formula (I), R ₁ is hydrogen or a hydroxy group; R ₂ is hydrogen, ascorbic acid, fluorescein, or fluorescein isothiocyanate, fluorescein amidite and fluorescein sulfonyl A fluorescein derivative selected from the group consisting of chloride; R ₃ is hydrogen, an ascorbic acid derivative in which a part of a hydroxy group is substituted with a carboxy group, fluorescein, or fluorescein isothiocyanate, fluorescein amine Fluorescein derivatives selected from the group consisting of diet and fluorescein sulfonylchloride; at least one of R ₂ and R ₃ is not hydrogen)

The derivative of the present invention may be composed of glycylproline or glycylhydroxyproline as a dipeptide, which can be largely defined as a carrier component, and a target substance as a carrier component. At this time, the target material or cargo (cargo) may be a variety of materials described above, for example, fluorescein or derivative thereof, such as Fluorescein isothiocyanate (FITC) widely used as a fluorescent dye, or Ascorbic acid or a derivative thereof widely used as an antioxidant or whitening, cosmetics for improving wrinkles, and the like. Therefore, the present invention may be defined as a conjugate of the dipeptide and the aforementioned target material. Since the dipeptides have amine groups (amino groups) and carboxyl groups at the N-terminus (amino terminus) and C-terminus (carboxy terminus), respectively, the dipeptides can bind to the above-mentioned target substances.

For example, the fluorescein derivatives of the present invention are derivatives represented by the following general formula (II) and include their possible isomers or possible salts thereof.

(Formula (II), R ₁ is the same as defined in formula (I); R ₄ and R ₅ are each independently hydrogen, fluorescein, or fluorescein isothiocyanate, fluorescein amide Fluorescein derivative selected from the group consisting of sodium and fluorescein sulfonylchloride, at least one of R ₄ and R ₅ is not hydrogen)

Since FITC, one of the fluorescein derivatives, has an isothiocyanate group, the dipeptide-linked fluorescein isothiocyanate derivatives can be synthesized by reacting with a dipeptide N-terminal amine group to form a thiourea bond. Can be. In addition, fluorescein having no substituent is first substituted with succinimidyl ester, and reacted with an N-terminal amine group to form an ester bond, thereby producing a fluorescein derivative in which a dipeptide is bound. For example, the following formula (IIa) illustrates a derivative in which FITC and dipeptide are directly bonded.

(Wherein R ₁ in formula (IIa) is the same as defined in formula (I))

In addition, fluorescein derivatives can be indirectly bound to the C-terminus or N-terminus of the dipeptide via a suitable linker. For example, using a linker having an amine group and a carboxyl group at both ends, first reacting the amine group of the linker group with the fluorescein isothiocyanate, followed by the carboxyl group and the dipeptide N at the other end of the linker linked to the fluorescein isothiocyanate. The ends may form amide bonds. In addition, a linker having two amine groups may be used to first react the amine group of the linker group with the fluorescein isothiocyanate, and the amine group at the other end of the linker may form an amide bond with the carboxy group of the dipeptide C-terminus. have. For example, the following formula (IIb) shows fluorescein isothiocyanate bound to a dipeptide via a linker.

In which formula (IIb), R ₁ is the same as defined in formula (I)

In addition, derivatives in which ascorbic acid or a derivative thereof is bound to a dipeptide may be represented by the following general formula (III), and include possible isomers thereof or possible salts thereof.

(Formula (III), R ₁ is the same as defined in formula (I); R ₆ is hydrogen or ascorbic acid; R ₇ is ascorbic acid derivative in which a part of hydrogen or hydroxy group is substituted with carboxyl group, At least one of R ₆ and R ₇ is not hydrogen)

Ascorbic acid or derivatives thereof have a large number of hydroxyl groups, which are bonded by forming an ester bond with the carboxyl group of the dipeptide C-terminus. For example, the dipeptide may be bound via any one hydroxyl group selected from positions 2, 3, 5 or 6 in ascorbic acid. Or if a number of hydroxy groups present in ascorbic acid or derivatives thereof are substituted, for example with succinimidyl ester or imidazole ester, the substituted reactor reacts with the dipeptide N-terminal amine group and ascorbic acid or derivative thereof Binds to the N-terminus of the dipeptide via an amide bond. If necessary, a derivative in which two or more peptides are bound to ascorbic acid or a derivative thereof can be prepared. In the following general formula (IIIa) to the following general formula (IIIc), the derivative which the dipeptide couple | bonded with ascorbic acid or its derivative is illustrated.

R ₁ in formulas (IIIa) to (IIIc) are the same as defined in formula (I).

Ⅱ. Manufacturing method

1. Method for producing cell permeable peptide

Dipeptide of the present invention can be used genetic engineering methods or chemical synthesis methods using a recombinant expression vector. As a genetic engineering method, recombinant expression vectors suitable for bases encoding glycine (GGA) (GGA, GGC, GGG, GGT) and bases encoding proline (Pro) (CCA, CCC, CCG, CCT), for example histidine tags. Cloning the dipeptide coding base using a PCR technique to a pET vector which is a plasmid vector having a recombinant vector to prepare a recombinant vector, followed by mass expression in the form of His-Gly-Pro peptide in the host E. coli, followed by pure purification It can be prepared through. In this case, the base encoding the dipeptide and the base encoding the other peptide may be inserted at the same time and expressed in the form of a fusion protein.

The chemical synthesis method may be a method for synthesizing a solid phase peptide using an organic synthesis device for peptide synthesis. For solid phase synthesis, a suitable resin is synthesized by coupling proline constituting the carboxy terminus of the dipeptide, followed by coupling glycine with proline. In this case, in the case of glycine, the alpha-amino group may be coupled to proline in a protected state.

First, the (hydroxy) proline constituting the carboxy terminus of the dipeptide is coupled to an appropriate resin to swell the (hydroxy) proline binding resin. Proline-bonded resins are required for solid phase synthesis, and as the resin bound to (hydroxy) proline, hydroxymethyl resin, aminomethyl resin, chloromethylated resin, or chlorotitatile resin can be used. By loading hydroxy) proline, (hydroxy) proline is ester bonded and coupled to these resins. Subsequently, a peptide coupling reaction of (hydroxy) proline and glycine is carried out through a solid phase synthesis reaction. In this case, glycine may use glycine protected with alpha-amino acid, and a well-known substance such as Fmoc (9-fluorenyl methoxyarbonyl) or Boc (t-butyloxycarbonyl) may be used as a protecting group for protecting the alpha-amino group. After the dipeptide synthesis is completed, the depeptide synthesis of glycyl (hydroxy) proline is terminated by a deprotection reaction to remove the Fmoc protecting group of glycine using piperidine or the like. If necessary, a process of separating the dipeptide from which the protecting group has been removed from the resin using an appropriate acid may be performed first, followed by a binding reaction with the target material.

2. Synthesis of Target Substance Derivatives Bound to Cell Permeable Peptides

end. Synthesis of Fluorescein-Peptide Binding Derivatives

Fluorescein or derivatives thereof basically react with the dipeptide N-terminus to form a conjugate. For example, FITCs react with dipeptide N-terminal amine groups to form thiourea bonds to synthesize dipeptide-dipeptide conjugates. Or by replacing fluorescein with succinidimyl ester or imidazole ester and reacting the fluorescein substituted with these esters with the dipeptide, a fluorescein derivative which is bonded through the amide bond with the N-terminus of the dipeptide is synthesized. . Or a fluorescein derivative indirectly bound to the dipeptide N-terminus or C-terminus using an appropriate linker. In the case of the fluorescein derivative represented by the above-mentioned formula (IIa) or formula (IIb), the starting material Gly-Pro dipeptide and FITC are reacted with triethylamine, and then synthesized using ethyl acetate and an appropriate catalyst. (See FIGS. 3A and 3B).

I. Synthesis of Ascorbic Acid-peptide Binding Derivatives

As described above, since ascorbic acid contains a large number of hydroxy groups, for example, ascorbic acid derivatives exemplified by the above general formulas (IIIa) to (IIIc) can be synthesized. For example, FIG. 4A illustrates the synthesis of an ascorbic acid derivative represented by the general formula (IIIa) as a derivative in which a hydroxyl group at position 6 of ascorbic acid is ester-bonded with the C-terminus of the peptide. First, among the one or more hydroxy groups present in ascorbic acid or derivatives thereof, the hydroxyl group at the remaining position is protected with a protecting group so as to form an ester bond with the carboxy group at the C-terminus. For example, the hydroxy group at position 6 of ascorbic acid may ester bond with the carboxyl group at the peptide C-terminus to form a conjugate with the peptide. First, the hydroxy groups at positions 5 and 6 of ascorbic acid are protected with acetyl / ketal by washing with acetone / hexane, and then benzyl bromide and potassium carbonate / dimethylformamide (DMF) are reacted. After protecting the hydroxyl group at position benzyl with strong acid such as hydrochloric acid, the positions 5 and 6 are deprotected. In this state, ester reaction with the dipeptide is carried out. At this time, it is preferable to protect the N-terminus of the dipeptide using a protecting group such as, for example, Boc.

Regarding the esterification reaction of ascorbic acid with a peptide, ascorbic acid and its derivatives are insoluble in water and can be ester-linked with the dipeptide using an organic solvent under anhydrous conditions. Organic solvents that can be used include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), hexamethylphosphoramide (HMPA), N-methylpyrrolidone, pyrrolidone, dimethylacetamide (DMAC), 1,3 -Dimethyl-3,4,5,6-tetrahydro-2 (1H) -limidinone (DMPU). The esterification reaction can proceed without a catalyst, but if a catalyst is used, the carboxylic acid can be activated using a known Lewis acid or by using a halide such as, for example, thionyl chloride, and then hydrate of ascorbic acid or a derivative thereof. The reaction with the oxy group is induced or the esterification reaction is induced using methanesulfonic acid or the like as a catalyst. In consideration of the fact that the esterification reaction is a reversible reaction, it is preferable to use at least 10 equivalents of ascorbic acid or a derivative thereof. N, N-dicyclohexylcarbodiimide (DCC) or N, N-carbonyl diimidazole (N, N-carbonyl diimidazol, CDI) may be used as a condensing agent, which is an substance that induces an ester reaction. As a catalyst for promoting the reaction, N, N-dimethylaminopyridine (DMAP) or 1-hydroxybenzotriazole (1-HOBT) can be commonly used.

When the esterification reaction is completed, a benzyl group, which is a protecting group at the 2nd and 3rd positions, is removed by using a catalyst such as palladium-charcoal (Pd-C) for hydrogenation and using hydrogen gas and ethanol acetate. If the N-terminus of the dipeptide is protected by Boc or the like in the esterification reaction, the protecting group is removed through a deprotection reaction.

In addition to the one shown in Figure 4a can be synthesized ascorbic acid derivatives bound to the peptide through an ester bond by reacting the hydroxy group present in the position 2 and / or position 3 of the ascorbic acid and the carboxy group on the peptide C-terminus. . With regard to the synthesis of derivatives in which the hydroxyl groups at positions 2 and / or 3 of the ascorbic acid and the carboxyl groups at the dipeptide C-terminus are ester-bonded, the hydroxyls at positions 5 and 6 of ascorbic acid are as described above. Form acetal or ketal by washing the group with acetone / hexane, and reacting the dipeptide at position 2 or 3 in the presence of a base to form an ester bond, and then the acetal at positions 5 and 6 Or a method of removing the ketal may be used.

Specifically, ascorbic acid or its derivatives are washed with acetone / hexane to protect the hydroxyl groups at positions 5 and 6 with acetal groups / ketal groups, for example, at the positions 2 and / or 3 The group can be activated to induce an ester bond with the C-terminus of the dipeptide. As a method of activating a hydroxyl group at a specific position, ascorbic acid protected at positions 5 and 6 with an acetal group / ketal group and N, N-carbonyldiimidazole (CDI) are reacted under anhydrous THF to synthesize. Or synthesized with 3-benzyl ascorbic acid using, for example, ascorbic acid cesium salt substituted with cesium (Cs) at position 3, and then treated with cesium bicarbonate salt and reacted with dipeptide to After preparing -benzyl-2-dipeptide-ascorbic acid, a derivative which is ester-bonded with the dipeptide at position 2 may be synthesized through a hydrogenation reaction.

On the other hand, if necessary, ascorbic acid derivatives (primary derivatives) in which the 6-hydroxy group is ester-bonded with the C-terminus of the dipeptides are synthesized in the same manner as described in FIG. In the state where the N-terminus is protected by BOC or the like, an ester bond is induced between a hydroxyl group at position 2 or 3 of the ascorbic acid present in the primary derivative and a carboxy group at the C-terminus of another dipeptide. Ascorbic acid derivatives each having an ester bond with the C-terminus of the two peptides represented by (IIIc) can be synthesized (that is, in this case dipeptide: ascorbic acid derivative = 2: 1).

In addition, with respect to the synthesis of ascorbic acid derivatives that bind to the N-terminus of the dipeptide as represented by the formula (IIIb), after activating ascorbic acid or a part of its derivatives in the same manner as shown in Figure 4b, N-terminal and amide conjugated conjugates of the peptides can be synthesized. As described in FIG. 4A, first, positions 5 and 6 of ascorbic acid are protected with an acetal / ketal, and, for example, carbonyl diimidazol is reacted and refluxed in THF solvent to position 3 The hydroxyl group is substituted with imidazole ester. Since substituted imidazole ester groups are particularly reactive with primary amines, such ascorbic acid derivatives substituted with hydroxy groups at specific positions and imidazole esters can react with amine groups of the dipeptide to form amide bonds. Triethylamine (TEA) can be used with THF solvent in connection with the N-terminal amide bond reaction of ascorbic acid or its derivatives with dipeptides via an imidazole ester group. After forming an amide bond between ascorbic acid and the dipeptide N-terminus, a deprotection reaction using TFA and CH ₂ Cl ₂ is performed to remove the protecting groups at positions 5 and 6, thereby removing the original hydroxyl group of ascorbic acid. Derivatives that amide bond with the N-terminal amine group of the dipeptide at specific positions can be obtained.

At this time, if necessary, ascorbic acid derivatives (primary derivatives) that amide bond with the N-terminus of the first dipeptide are synthesized similarly to those shown in FIG. 4C, and the C-terminus of the dipeptides of the primary derivatives are also synthesized. Is protected with an appropriate protecting group, thereby inducing ester bonds between the hydroxy group at position 6 of the ascorbic acid present in the primary derivative and the carboxy group at the C-terminus of the other dipeptide (second dipeptide). It is possible to synthesize a derivative in which two peptides are respectively bonded at positions 3 and 6 of ascorbic acid (ie, in this case, dipeptide: ascorbic acid derivative = 2: 1). Alternatively, if necessary, ascorbic acid derivatives (primary derivatives) that amide bond to the N-terminus of the dipeptide are first synthesized as shown in FIG. It is also possible to synthesize derivatives which form ester bonds between the hydroxyl groups of another ascorbic acid or derivatives thereof (ie dipeptide: ascorbic acid derivative = 1: 2 in this case). For example, by reacting the primary derivative with another ascorbic acid or a derivative thereof in which the hydroxyl groups at positions 2 and 3 are protected by the benzyl group in the same manner as shown in FIG. It is also possible to form an amide bond with position 3 of ascorbic acid or a derivative thereof, and the C-terminus of the dipeptide to form an ester bond with position 6 of the second ascorbic acid or derivative thereof. For example, ascorbic acid derivatives of formula (III) can be separated and purified using conventional separation and purification methods, such as recrystallization or column chromatography.

Ⅲ. Application or Use of Derivatives Associated with Peptides

1. Fluorescein-peptide binding derivatives

Fluorescein or a derivative thereof is a fluorescent substance which is widely used especially for detecting substances in vivo because it usually emits a characteristic fluorescence in the near infrared region. Fluorescein or derivatives thereof are markers that are fluorescent labels for detecting the presence of peptides, proteins, nucleic acids, etc., fluorescent components of sensors related to enzyme activity measurements, or fluorescent components in contrast agents for diagnosis of various diseases such as cancer diagnosis. Widely used. For example, fluorescein is used as fluorescein isothiocyanate (FITC) as a fluorescent label for detecting peptides or as a phosphor for contrast agents for diagnosing cancer, and fluorescein amidite Fluorescent materials used to detect the presence of complementary nucleic acids in the synthesis of oligonucleotides.

According to the present invention, for example, the dipeptide-fluorescein derivative represented by the general formula (II) significantly improves the intracellular penetration effect compared to the free fluorescein. Therefore, the fluorescent fluorescein derivative of the present invention can be used as a fluorescent material in the contrast agent for diagnosing a disease including a diagnosis of a fluorescent dye or a cancer for detecting and detecting the presence of a peptide or nucleic acid. That is, the present invention relates to fluorescent compositions containing fluorescein derivatives bound to dipeptides. In addition to containing the fluorescein derivative according to the present invention as a fluorescent material, such a fluorescent composition may include other components conventionally included in the fluorescent composition according to each application. The preparation and blending ratio of the other components to be blended in the fluorescent composition, including the fluorescein derivative, are well known in the art, and thus detailed descriptions are omitted. Fluorescein derivatives bound to the dipeptides according to the present invention increased intracellular input compared to fluorescein not bound to the dipeptides (see FIGS. 5A-5C).

2. Ascorbic acid-peptide binding derivatives

There is a growing interest in skin whitening due to abnormal pigmentation of the skin due to an increase in the aged population and increase in the amount of ultraviolet light exposure due to the destruction of the ozone layer due to environmental pollution. Human skin color depends on the amount of melanin and hemoglobin, and melanin is a key factor in determining human skin color. Melanin, which is synthesized in the pigment cells in the epidermis, functions to block ultraviolet rays, protect skin organs, and remove free radicals generated from the skin. In vivo, melanin is synthesized by an oxidation process using tyrosine as a substrate and catalyzing enzymes such as tyrosinase, and excessive production of melanin by increasing free radicals on the skin, inflammatory reactions caused by various stresses, and ultraviolet irritation. Can be. Since over-produced melanin can cause spots and freckles, it is necessary to suppress the production of such melanin pigments in connection with skin whitening.

On the other hand, vitamin C is known to have effects such as anti-wrinkle, antioxidant, skin wrinkle improvement through promoting collagen formation, strengthening immunity and the like. For example, collagen, a major protein that makes up the skin, is a fibrous protein that makes up about 90% of the dermis. It is a substance that provides skin tension and elasticity with elastin, and protects the skin from ultraviolet rays and prevents wrinkles. .

Such collagen has a unique structure of 21% or more of proline or hydroxyproline among the total amino acids. Proline or hydroxy proline has a low rate of conversion to enzymes, and 70% to 90% can be recycled to biosynthesis into collagen. Specifically, collagen produces a dipeptide of glycylproline or glycyl (hydroxy) proline as its degradation product, and the resulting dipeptide is degraded into constituent amino acids by prolidase and recycled to collagen. In particular, hydroxyproline can be biosynthesized by an enzyme called proline hydroxylase. Vitamin C acts as a cofactor for this enzyme, which promotes collagen synthesis.

Ascorbic acid, a derivative of vitamin C, and its derivatives are used as active ingredients of whitening agents by inhibiting the activity of tyrosinase and reducing the production of melanin. However, since ascorbic acid and its derivatives are reducing substances, when stored at room temperature for a long time as a cosmetic, there is a problem that their activity is reduced or discolored when formulated. However, ascorbic acid has a hydrophilic property due to the hydroxyl groups present in the 2, 3, 5, 6 position, so that it dissolves quickly in an aqueous solution, but the solubility in the skin is limited. In addition, ascorbic acid is known to accumulate much more in the skin when administered as an external preparation than an oral administration. Since ascorbic acid does not dissolve in organic solvents commonly used in external preparations such as glycerin and propylene glycol, there is a limit to penetrating ascorbic acid into cells. have.

Ascorbic acid derivatives in which the dipeptide is bound can solve the instability of ascorbic acid in the free state, and thus can solve the above-mentioned problems. Ascorbic acid derivatives according to the invention can in particular promote bone-forming or induce collagen synthesis. Considering that ascorbic acid is effective for skin whitening and that ascorbic acid according to the present invention induces collagen synthesis, ascorbic acid derivatives in which the dipeptide is bound according to the present invention are for example, such as skin whitening or wrinkle improvement. It can be used as an active ingredient in skin anti-aging cosmetics.

Accordingly, the present invention relates to a cosmetic composition for preventing skin whitening or skin aging containing at least one or more of ascorbic acid derivatives represented by formula (III), for example, formulas (IIIa) to (IIIc) as an active ingredient. In this cosmetic composition, the ascorbic acid derivative of the present invention is contained at a concentration of 10 to 100 µg / ml, preferably at a concentration of 30 to 80 µg / ml. At lower concentrations, it is difficult to expect skin whitening or wrinkle improvement, and if exceeded, it may adversely affect safety.

To prepare a cosmetic composition for skin whitening or skin aging, ethanol, glycerin, butylene glycol, propylene glycol, glycerin-26, methylgluses-20, isocetyl myristate, isocetyloctanoate, octyldode as solvents At least one selected from silmyristate, octyldodecanol, isostearyl isostearate, cetyloctanoate and neopentyl glycol dicaprate can be used. When the cosmetic composition of the present invention is prepared using such a solvent, the solubility of the compound in the solvent is slightly different depending on the kind of the compound and the mixing ratio of the solvent. However, if a person skilled in the art to which the present invention belongs belongs, Depending on the type and the amount of the solvent can be appropriately selected. Cosmetic composition according to the present invention may be prepared in the form of cosmetics such as skin external ointment, softening longevity, nourishing cosmetics, nutrition cream, massage cream, essence, pack.

In addition to the ascorbic acid derivative represented by the general formula (III), the cosmetic composition for skin whitening or wrinkle improvement of the present invention is used in skin cosmetics for skin whitening and / or skin aging. , Solubilizers, humectants, surfactants, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents , Preservatives, blockers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, organic and inorganic pigments, organic powders, UV absorbers, preservatives, fungicides, plant extracts, pH adjusters, alcohols, pigments, flavorings, purified water or May contain adjuvants conventionally used in the art, such as any other ingredient conventionally used in cosmetics. have. These adjuvants are generally introduced in amounts commonly used in the cosmetic or dermatological arts, for example in amounts of 0 to 20% by weight of the total weight of the composition.

Hereinafter, although this invention is demonstrated with reference to an Example, this invention is not limited to the content described in these Examples.

Example 1 Glycyl (hydroxy) proline Dipeptide Synthesis

Glycosyl (hydroxy) proline dipeptide was synthesized using a solid phase peptide synthesizer. The 2-chlorotril resin was placed in a standard reaction vessel, loaded with 3 equivalents of (hydroxy) proline to couple the (hydroxy) proline to the resin, and then 1 mM of proline-coupling resin was added to the dimethylformamide solvent 10 Swelled in ml for 30 minutes at room temperature. Subsequently, peptide coupling reaction was performed using 1 mM of a glycine derivative protected with Fmoc group, peptide coupling reagent 6-chloro-1-hydroxy-1H-Benzotriazole, and diisoproply carbodiimide as a base. It was performed at room temperature for about 3 hours, and washed at room temperature for about 5 minutes using 10 ml of dimethylformamide (DMF) or dichloromethane three times before and after the peptide coupling reaction. 10 ml of a 20% concentration piperidine solution was added to the synthesized dipeptide and reacted at room temperature for about 20 minutes. The dipeptide was removed from the protecting group by repeating washing for 5 minutes at room temperature with 10 ml of DMF. Was synthesized.

Example 2 Preparation of Fluorescein Derivatives Combined with Dipeptides

(1) Synthesis of Derivative of Formula (IIa)

According to the procedure shown in Figure 3a, a derivative of the formula (IIa) (hereinafter 'FITC-GP derivative') was synthesized in the following manner. First, FITC (0.2 g, 0.51 mmol), Gly-Pro-OBn (0.16 g, 0.62 mmol), and Triethylamine (0.10 mL, 0.77 mmol) were dissolved in 10 mL DMF (dimethyl formamide), followed by stirring at room temperature for about 2 hours. . 50 mL of water was added to the reaction mixture, and the mixture was stirred vigorously, ethyl acetate (50 mL × 3) was added to separate the organic layer. The recovered organic layer was concentrated under reduced pressure to give an orange compound (1). Dissolve compound (1) in 20 mL ethyl acetate, add 10% Pd / C, stir for about 30 minutes under hydrogen gas, filter with celite, and concentrate the organic solvent under reduced pressure to give orange compound (2) (0.2). g) was obtained.

¹ H NMR (500 MHz, MeOD): δ 8.27 (s, 1 H), 7.87 (d, 1 H), 7.19 (d, 1 H), 6.71-6.69 (m, 4H), 6.58 (m, 2H), 4.69- 4.15 (m, 3H), 3.75-3.71 (m, 2H), 2.35-2.15 (m, 4H).

(2) Synthesis of Derivative of Formula (IIb)

According to the procedure shown in FIG. 3b, a FITC derivative represented by Formula (IIb) (hereinafter referred to as "FITC-aca-GP derivative") was synthesized as follows.

In a 100 mL round flask, L-Pro-OBzl (1 g, 4.14 mmol) and Boc-Gly-OH (0.87 g, 5 mmol) were dissolved in 20 mL methylene chloride solvent, EEDQ (1.22 g, 5 mmol) and TEA (0.70 mL). , 5 mmol) was added and stirred at room temperature for 12 hours. Impurities were washed several times with 10% KHSO ₃ solution and then removed, and the protective group Boc was removed by treatment with 30% TFA / MC solution. After complete removal of the remaining TFA, it is dissolved in 50 ml of methylene chloride solvent, 6-tert-Butoxycarbonylamino-hexanoic acid (1.2 g, 5 mmol), TEA (0.70 ml, 5 mmol), HOBt (0.68 g, 5 mmol), EDC ( 0.96g, 5 mmole) was added thereto and stirred at room temperature for about 12 hours. 50 ml of water was added to the reaction mixture, and the organic layer was separated and recovered. The recovered organic layer was concentrated under reduced pressure and compound (2) was obtained by column chromatography. The protecting group was removed in two steps to obtain white compound (3) (1- [2- (6-Amino-hexanoylamino) -acetyl] -pyrrolidine-2-carboxylic acid). Subsequently, 1- [2- (6- {3- [3-Carboxy-4- (6-hydroxy-3-oxo-3H-xanthen-9-) represented by compound (4) which is a FITC-aca-GP derivative. FITC (0.2g, 0.51 mmol), Compound 3 (0.17g, 0.62mmol), TEA (0.10 mL, 0.77) to synthesize yl) -phenyl] -thioureido} -hexanoylamino) -acetyl] -pyrrolidine-2-carboxylic acid mmol) was dissolved in 10 mL DMF and stirred at room temperature for about 2 hours. 50 mL of water was added to the reaction mixture, and the mixture was stirred vigorously, ethyl acetate (50 mL x 3) was added to separate the organic layer, and the mixture was recovered. The recovered organic layer was concentrated under reduced pressure and orange compound (4) was obtained by column chromatography.

¹ H NMR (500 MHz, DMSO-d ₆ ): δ 101 (s, 1H), 9.85 (s, 1H), 8.24 (s, 1H), 8.06 (s, 1H), 7.96-7.92 (m, 1H) , 7.74 (s, 1H), 6.69-6.56 (m, 6H), 4.58-3.41 (m, 7H), 2.2-1.57 (m, 12H)

Example 3 Preparation of Ascorbic Acid Derivatives Combined with Dipeptides

(1) Synthesis of Ascorbic Acid Derivative of Formula (IIIa)

Ascorbic acid derivatives of formula (IIIa) were synthesized according to the procedure shown in FIG. 4A. First, ascorbic acid (50 g, 284 mmol) was stirred for about 1 hour while suspended in 500 mL acetone. Acetyl chloride (6 mL, 85.2 mmol) was added to the suspension, followed by stirring for about 12 hours to obtain a white precipitate. The precipitate was filtered and recrystallized using acetone / hexane mixed solvent to give 5- (2,2-dimethyl- [1,3] dioxolan-4-yl) -3,4-dihydroxy as acetal-protected compound (1). -5H-furan-2-one was obtained.

Subsequently, Compound (1) (5 g, 23 mmol), Benzyl bromide (6.1 mL, 50.1 mmol) and K ₂ CO ₃ (9.5 g, 69 mmol) were dissolved in 50 mL of DMF solvent and stirred at room temperature for 5 hours. . 100 mL of water was added to the reaction mixture, and the mixture was stirred vigorously, ethyl acetate (50 mL × 3) was added to separate the organic layer, and the mixture was recovered. The recovered organic layer was concentrated under reduced pressure, and the resulting residue was recrystallized in methanol to obtain white compound (2), 3,4-Bis-benzyloxy-5- (2,2-dimethyl- [1,3] dioxolan-4-yl). -5H-furan-2-one (6 g) was obtained. Compound (2) (6 g, 15.1 mmol) was dissolved in 50 mL THF solvent, and 3N-HCl (30 mL) was added thereto, and the mixture was stirred at room temperature for 12 hours. THF was concentrated under reduced pressure and methylene chloride (50 mL × 3) was added to separate the organic layer and recovered it. The recovered organic layer was concentrated under reduced pressure to obtain 3,4-Bis-benzyloxy-5- (1,2-dihydroxy-ethyl) -5H-furan-2-one (5 g) as a colorless liquid compound (3).

Compound (3) (1 g, 1.6 mmol) and (N) -Boc-Gly-Pro-OH (0.58 g, 2.1 mmol), and a small amount of DMAP and DCC (0.43 g) in 50 mL methylene chloride solvent , 2.1 mmol) was added and stirred at room temperature for 5 hours. The urea produced during this reaction was filtered, the filtered methylene chloride solvent was concentrated under reduced pressure, and the resulting residue was separated by silica gel chromatography (hexane / ethyl acetate = 1/4) to give 1- (2- as white compound (5). tert-Butoxycarbonylamino-acetyl) -pyrrolidine-2-carboxylic acid 2- (3,4-bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl) -2-hydroxy-ethyl ester (0.6 g ) Subsequently, Compound (5) (0.5 g, 0.82 mmol) was dissolved in 20 mL ethyl acetate solvent, 10% Pd / C was added, and the mixture was stirred for 30 minutes under a hydrogen atmosphere. Filtration was carried out using Celite, and the filtrate was concentrated to give a white compound (6). This compound was dissolved in 10 ml of a 30% TFA / MC solvent, stirred for 1 hour, and concentrated under reduced pressure, to obtain 1- (2-Amino-acetyl) -pyrrolidine-, the white final compound represented by the formula (IIIa) (7). 2-carboxylic acid 2- (3,4-dihydroxy-5-oxo-2,5-dihydro-furan-2-yl) -2-hydroxy-ethyl ester was obtained. NMR data of ascorbic acid derivatives represented by the formula (IVa) are shown below.

¹ H NMR (500 MHz, DMSO-d6): 4.68 (s, 1H), 4.43 (d, 1H), 4.41-3.54 (m, 8H), 2.22-2.19 (1H, m), 2.18-1.95 (3H, m)

(2) Synthesis of Ascorbic Acid Derivative of Formula (IIIb)

Ascorbic acid derivatives of formula (IIIb) were synthesized according to the procedure shown in FIG. 4b. In order to activate only the 3-hydroxy group of ascorbic acid protected in the form of acetal, carbonyldiimidazole, a precursor of carbamate, was reacted in anhydrous THF solvent to synthesize Compound (1) having substituted 3-hydroxy group. Since this compound is commonly used to make various derivatives, it was synthesized and stored on a scale of 10 g or more. The compound was reacted with Gly-Pro to obtain an intermediate (2) in carbamate form, and then the acetal group was removed under organic acid conditions to obtain an ascorbic acid derivative (3) as a final compound (IIIb).

(3) Synthesis of Ascorbic Acid Derivative of Formula (IIIc)

Ascorbic acid derivatives of formula (IIIc) were synthesized according to the procedure shown in FIG. 4C. In order to activate only the hydroxyl group 3 of Boc-Gly-Pro-OAA (1), which is a Boc protected compound, carbonyldiimidazole, a precursor of carbamate, was obtained by reaction in anhydrous THF solvent. The compound was reacted with Gly-Pro to obtain an intermediate (3) in carbamate form, and as a result, an ascorbic acid derivative (3) of the final compound (IIIc) was obtained by removing Boc as a protecting group under organic acid conditions. Ascorbic acid derivatives synthesized through the above-described process will be hereinafter abbreviated as 'GP-AA'.

Example 4 Measurement of Cell Internalization of FITC-GP

(1) Fluorescence intensity measurement

Each cell of HDF-N passage 7, HDF-Adult (HDF-Ad, invitrogen # C-013-5C) passage 7 was attached to a 96 well plate at 1 x 10 ⁴ cells / well and stabilized overnight. The culture medium was DMEM containing 10% FBS and antibiotics. Thereafter, FITC and FITC-acac-GP were dissolved in the same culture medium at a concentration of 50 mg / ml, and 200 μl was loaded into each well. After 24 hours of sample treatment, the plate was washed three times with PBS, and then measured for fluorescence intensity using an Enspire multimode reader (PerkinElmer # 2300) (Excitation at 488 nm, emission at 519 nm). The measurement results are shown in FIG. 5A. The control group did not process either FITC or FITC-acac-GP. Intensity was increased when FITC or FITC-acac-GP was introduced into cells, but intensity was not increased in all cells in FITC-treated group, but was significantly increased when FITC-GP was treated. In particular, the HDF-Ad, aging model significantly increased, GP confirmed that the aging model is more permeable. Comparison between the sample treatment group and the control group was verified by Student's t- test. ns (not-significant) means no significance, and * / ** / *** means that the probability of significance is 0.01, 0.001, 0.0001 or less, respectively (* p <0.05 (significant), ** p <0.01 ( highly significant), *** p <0.001 (extremely significant)).

(2) fluorescence microscope observation

The cells of HDF-N passage 6, HDF-N passage 18, and HDF-Ad passage 6 were attached to 6 well plates at 5 x 10 ⁴ cells / well and stabilized overnight. The culture medium was DMEM containing 10% FBS and antibiotics. HDF-N passage 6 (HDF-N p6) cells are normal model, HDF-N passage 18 (HDF-N p18) is passaged in vitro aging model, HDF-Ad passage 6 (HDF-AD p6) is adult The cells were used as ex vivo aging model. Thereafter, FITC and FITC-acac-GP were dissolved in the same culture medium at a concentration of 50 mg / ml, respectively, and then loaded into each well by 1 ml. After 24 hours of sample treatment, the cells were washed three times with PBS after 30-minute staining using DAPI (4 ', 6-diamidino-2-phenylindole) 10 mg / ml for cell nuclear staining after washing three times with PBS. The degree of fluorescence was observed using a fluorescence microscope (Fluorescence Inverted Microscope, Carl-Zeiss #Axio Observer D1). The results are shown in FIGS. 5B and 5C. First, as shown in FIG. 5B, there is a difference in the form of adult cells (HDF-Ad), which are aged cells, as compared with neonatal cells (HDF-N). That is, as the cell ages, the cell size increases and specifically senescence associated beta-galactosidase (SA-b-gal) is expressed. Observation of cell morpholgy using a microscope and SA-b-gal staining using a SA-b-gal detection kit (BioVision # K320-250) can be judged as a suitable senescent cell model.

On the other hand, as shown in Fig. 5c, in the FITC-treated group, no fluorescent material was observed in all cells, but in the FITC-acac-GP treated group, green fluorescent material was observed in all three types of cells. Well observed in two aging models of HDF-Ad p6. Therefore, it was confirmed that FITC combined with GP was well internalized into cells, and more particularly used in senescent cells than normal cells.

Example 5 Synthesis Confirmation of GP-AA and Measurement of Content / Binding Rate

(1) HPLC analysis

It was confirmed by HPLC (HPLC Interface, Korea) whether or not ascorbic acid was synthesized in the ascorbic acid derivative in which the C-terminus of the dipeptide was bonded to the hydroxyl group at the 6th position of the derivative synthesized in Example 4. Column: C-18ec 4.6 * 250 mm (MACHEREY-NAGEL, Germany), solvent A: DW (0.1% TFA) B: Acetonitrile (0.1% TFA), UV absorbance: 254 nm. The elution profile of GP-AA is shown in Table 1. The measurement results are shown in FIG. 6A, where the wavelength scanning results of GP-AA were observed at both the absorption wavelengths of GP and AA.

QC profile of GP-AA conjugate Time (min) Flow rate (ml / min) A (%) B (%) 0 One 80 20 20 One 20 80 20.01 One 80 20 30 One 80 20

(b) GP-AA binding rate confirmation

Ascorbic acid assay kit (Biovision # K661-100, USA) was used to quantify the content of synthesized ascorbic acid. The test method was in accordance with the manual provided. The result of measuring the content is shown in FIG. 6B. The ascorbic acid content of GP-AA was 26.514%, and the binding rate with GP was about 50% according to the following formula. In other words, ascorbic acid was bound to GP-AA 1UNIT by 0.524 unit, and 52.4% was synthesized.

Binding rate calculation

GP-AA Molecular Weight: AA Molecular Weight * Number of Units = GP-AA Concentration: AA Concentration

348.3: (176.12 * x) = 1000 μg / ml: 265.14 μg / ml

x = 0.524 unit

Example 6 GP-AA Cytotoxicity Measurement

5 x 10 ³ cells / well were attached to the HDF-N 96 well plate and stabilized overnight. The culture medium was DMEM containing 10% FBS and antibiotics. After the concentration of each of the GP-AA, AA in the same culture medium in the range of 10 ~ 0 ㎎ / ㎖, and 200 ㎕ each well loaded in each well. After 24 hours of sample treatment, cell viability was confirmed at OD 450 nm using WST-1. The measurement results are shown in Figure 7, GP-AA showed no cytotoxicity above 1 mg / ㎖ concentrations AA is toxic.

Example 7: Confirmation of GP-AA activity: ALP assay

Ascorbic acid is known as a bone induction marker. Alkaline phosphatase activity (ALP) analysis was performed as an easily identifiable method among well-known functions to confirm the activity of ascorbic acid in GP-AA. MC3T3-E1 (Lonza # CC-2538) cells, which are mouse osteoblast cell lines, were attached to 96 well plates to be 5 x 10 ³ cells / well and stabilized overnight. The culture medium used α-MEM (JBI # LM-008-53) containing 10% FBS and antibiotics. No treatment as a control (Negative), dipeptide 50 ㎍ / ㎖ (GP), ascorbic acid 50 ㎍ / ㎖ (AA), dipeptide and ascorbic acid mixed 50 ㎍ / ㎖ each (GP + AA ), Using the dipeptide-ascorbic acid derivative of the present invention 50 ㎍ / ㎖ (GP-AA), 2-OD derivative used as a raw material of ascorbic acid 2-glucoside cosmetics to compare the activity with GP-AA 50 μg / ml (AA-2G) of 2-O-α-D-glucopyranosyl-L-ascorbic acid was used, and an external phosphate source as a bone inducer as a positive control. A sample of 1 μg / ml of glycerophosphate (β-GP) and 50 μg / ml of ascorbic acid was used. After 200 μL of each well was treated as described above, the ALP activity assay was performed on 3 and 7 days.

For ALP assay, wash twice with PBS, and then in each well 140 μl of 2-amino 2-methyl 1 propanol (AMP, sigma # A9226) buffer and 10 μl of PNPP substrate (225 mM p-Nitrophenyl phosphate disodium salt, sigma). # N2765) was reacted at 37 ° C. for 30 minutes. Absorption was measured at 405 nm after completion of the reaction with 0.25 M NaOH. The measurement results are shown in FIG.

Since the dipeptide GP does not affect ALP activity, the increase of ALP activity by GP-AA can be interpreted as ascorbic acid. The effect is very significant when treated with GP-AA, but a smaller increase than when treated with free state ascorbic acid (AA) can be explained with AA2G used as a positive control. In other words, the increased activity of ascorbic acid by derivative synthesis seems to decrease the activity of free ascorbic acid in in vitro assays, but it is not easily oxidized when the product is applied. Therefore, ascorbic acid can be used more than free state (Yamamoto, I. et. al. (1992) J. Nutr. 122 (4): 871-7).

Example 8 GP-AA activity: collagen assay

The effect of GP-AA on collagen expression was examined. In Example 5, after measuring ascorbic acid content through the ascorbic acid color development, the same concentration was treated to treat the same amount of ascorbic acid between each experimental group. HDF-N cells were attached to 6 well plates at 5 x 10 ⁴ cells / well and stabilized overnight in DMEM medium containing 10% FBS and antibiotics. After removing the medium, three times wash with PBS, serum starvation period in DMEM without FBS for 24 hours.

As a control, a group treated with nothing (Negative), a group using 30 μg / ml of dipeptide (GP), and a group using AA (10 μg / ml) of ascorbic acid were used, and 40 μg / ml of the derivative of the present invention was used. (GP-AA). After treatment with Serum free-medium (DMEM), the conditioned medium was collected after 48 hours, and measured according to the manual provided using the collagen ELISA method (mdbioproducts # M036007). The measurement results are shown in FIG. Ascorbic acid and GP increased collagen levels, respectively. Collagen levels also increased with GP-AA treatment, but no synergistic effects were observed between AA and GP. However, as shown in the above ALP assay results, it is expected to increase the collagen synthesis by increasing the stability in vivo, as well as decreasing the activity in vitro by stabilization of AA.

Claims (13)

Ascorbic acid, an ascorbic acid derivative in which a portion of the hydroxy group is substituted with a carboxyl group, fluorescein, or fluorescein isothiocia Conjugation of a target substance selected from fluorescein derivatives selected from the group consisting of nates, fluorescein amidaites and fluorescein sulfonylchlorides, their possible isomers, their possible salts or combinations thereof One compound.
The compound according to claim 1, wherein the compound to which the target substance is bound is a compound represented by the following general formula (I) and includes a possible isomer thereof or a possible salt thereof.

In formula (I), R ₁ is hydrogen or a hydroxy group; R ₂ is hydrogen, ascorbic acid, fluorescein, or fluorescein isothiocyanate, fluorescein amidite and fluorescein sulfonyl A fluorescein derivative selected from the group consisting of chloride; R ₃ is hydrogen, an ascorbic acid derivative in which a part of a hydroxy group is substituted with a carboxy group, fluorescein, or fluorescein isothiocyanate, fluorescein amine Fluorescein derivatives selected from the group consisting of diet and fluorescein sulfonylchloride; at least one of R ₂ and R ₃ is not hydrogen)
The compound according to claim 2, wherein the compound represented by the formula (I) is a compound represented by the following formula (II), and includes the possible isomers thereof or the possible salts thereof.

In formula (II), R ₁ is the same as defined in claim 2; R ₄ and R ₅ are each independently hydrogen, fluorescein, or fluorescein isothiocyanate, fluorescein amidite And a fluorescein derivative selected from the group consisting of fluorescein sulfonyl chloride, and at least one of R ₄ and R ₅ is not hydrogen.
4. The compound according to claim 3, wherein the compound represented by the formula (II) is a compound represented by the following formula (IIa) or the following formula (IIb), and includes a possible isomer thereof or a possible salt thereof.

(In formula (IIa) and (IIb), R ₁ is the same as defined in claim 3)
The compound according to claim 2, wherein the compound represented by the formula (I) is a compound represented by the following formula (III), and includes a possible isomer thereof or a possible salt thereof.

In formula (III), R ₁ is the same as defined in claim 2; R ₆ is hydrogen or ascorbic acid; R ₇ is an ascorbic acid derivative in which a part of hydrogen or a hydroxy group is substituted with a carboxyl group, R At least one of ₆ and R ₇ is not hydrogen)
6. The compound according to claim 5, wherein the compound represented by the formula (III) is a compound represented by the following formulas (IIIa) to (IIIc) and includes possible isomers thereof or possible salts thereof.

(In Formulas (IIIa) to (IIIc), R ₁ is the same as defined in claim 5).
A fluorescent composition comprising a compound represented by the following formula (II), possible isomers thereof or a mixture of these isomers.

In formula (II), R ₁ is hydrogen or a hydroxy group; R ₄ and R ₅ are each independently hydrogen, fluorescein, or fluorescein isothiocyanate, fluorescein amidaite and fluore Fluorescein derivatives selected from the group consisting of shin sulfonylchloride, at least one of R ₄ and R ₅ is not hydrogen)
8. The fluorescent composition according to claim 7, wherein the compound represented by the formula (II) comprises a compound represented by the following formula (IIa) or the following formula (IIb), possible isomers thereof, or a mixture of these isomers.

(In formula (IIa) and (IIb), R ₁ is the same as defined in claim 7)
A functional cosmetic composition for preventing skin whitening or skin aging containing a compound represented by the following formula (III), possible isomers thereof, or a possible salt thereof as an active ingredient.

In formula (III), R ₁ is hydrogen or a hydroxy group; R ₆ is hydrogen or ascorbic acid; R ₇ is an ascorbic acid derivative in which a portion of the hydrogen or hydroxy group is substituted with a carboxyl group, and R ₆ and R At least one of ₇ is not hydrogen)
10. The skin whitening or skin preparation according to claim 9, wherein the compound represented by formula (III) comprises ascorbic acid derivatives represented by the following formulas (IIIa) to (IIIc), possible isomers thereof, or possible salts thereof Functional cosmetic composition for anti-aging.

(In Formulas (IIIa) to (IIIc), R ₁ is the same as defined in claim 9)
The functional cosmetic composition for preventing skin whitening or skin aging according to claim 9, wherein the compound represented by the formula (III) is contained at a concentration of 10 to 100 µg / ml in the cosmetic composition.
(a) synthesizing a dipeptide of glycylproline or glycylhydroxyproline; And
(b) reacting at least one of ascorbic acid and an ascorbic acid derivative in which a portion of the hydroxy group is substituted with a carboxy group at the C-terminus or N-terminus of the dipeptide using an organic solvent in the presence of a condensing agent. A method for producing an ascorbic acid derivative represented by the following general formula (III).

In formula (III), R ₁ is hydrogen or a hydroxy group; R ₆ is hydrogen or ascorbic acid; R ₇ is an ascorbic acid derivative in which a portion of the hydrogen or hydroxy group is substituted with a carboxyl group, and R ₆ and R At least one of ₇ is not hydrogen)
13. The method of claim 12, wherein step (b) further comprises protecting a portion of the hydroxy group present in the ascorbic acid or an ascorbic acid derivative in which a portion of the hydroxy group is substituted with a carboxy group with acetal or ketal. Process for preparing ascorbic acid derivatives.

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