KR101684380B1 - Biomimetic adhesion molecule and self-assembly body produced by the same - Google Patents

Abstract

The present invention relates to a biomimetic molecule comprising an adhesive functional group and a self assembly made using the same, and more particularly, to a catechol group having a hydroxyl group (-OH) at both terminals, Has excellent self-assembling property to spherical shape and exhibits a strong affinity with metal ions, and is capable of adsorbing and reducing inorganic metal ions, and as a template for a drug carrier or an inorganic material, a newly synthesized biologically mimicable Bolaamphiphilic molecule And a self-assembly manufactured using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomimetic molecule comprising an adhesive functional group and a self-assembly made using the same. ≪ Desc / Clms Page number 1 >

The present invention relates to a biomimetic molecule comprising an adhesive functional group and a self assembly made using the same, and more particularly, to a catechol group having a hydroxyl group (-OH) at both terminals, Amphiphilic molecules capable of adsorbing and reducing inorganic metal ions and as a template for drug delivery or inorganic materials, exhibiting a strong affinity with metal ions, And a self-assembly manufactured using the same.

Mussel living in the sea is an aquatic creature that grows on stones, mosses and aquatic plants by a byssal thread made of adhesive protein. These mussel adhesive proteins have been attracting much attention as research subjects because they have strong adhesive force in underwater environments, unlike adhesives that have been lost in contact with water such as bonds and tapes generally used in real life. It has been known that it has a strong adhesive force on most surfaces of metal, non-metal, organic polymer, and inorganic material, including polytetrafluoroethylene (PTFE) known to be unattached, and studies on adhesion mechanisms are actively conducted.

This adhesion of mussels has been reported to result from the presence of 3,4-dihydroxyphenylalanine (3,4-dihydroxyphenylalanine, DOPA), an amino acid identified through analysis of the amino acid composition of the mussel protein. In the mussel adhesive protein, the ratio of the amino acids glycine, asparagine, tryptophan and lysine, which occupy a high percentage of protein components, is only about 10 mol% It was confirmed that DOPA was contained in a high content. This suggests that DOPA plays a very important role in the specific binding capacity of mussel proteins.

DOPA is a form of tyrosine, an amino acid that is a component of protein, hydrated by a polyphenol oxidizing agent. It can form a very strong hydrogen bond with a hydrophilic surface and can bind to a hydrophobic surface including an aromatic material or a metal surface have. Because of this, mussels can be bonded to a wide variety of surfaces, including glass, metal, plastic, and Teflon.

On the other hand, studies on nanomaterials have been actively carried out in order to synthesize amphipathic molecules and to form nanostructures through self-assembly thereof and to utilize them for various purposes.

Accordingly, the present inventor has peptide bond in a molecular structure through a bio-mimetic molecule and a self-assembly comprising it, and discloses a single hydroxy (-OH) functional group Which is excellent in hydrophilicity and spherical self-assembly properties, and which can be applied to various fields of application, and a self-assembly using the biomimetic molecule.

However, such conventional biocompatible amphipathic molecules have a somewhat lack of hydrophilic properties and thus have insufficient self-assembly ability, and it is difficult to maintain self-assembled structure due to changes in environmental conditions such as pH, And it was possible to apply it to only a limited number of fields.

Particularly, since adhesion with metal materials is insufficient, it is necessary to apply media coating such as silver when attaching or coating metal. Therefore, it is limited to be used as a mold or a molding agent for inorganic materials, It is not easy to apply to hydrophilic surface modification or cell surface treatment.

Patent Laid-Open Publication No. 2012-0044773 (Published on May 05, 2012, "Bioimpedance Molecules and Self Assemblies Comprising the Same")

Material-Independent Surface Modification Inspired by Mussel-Adhesion, Polymer Science and Technology, Vol. 23, No. 4, 396-406 Use of the Self-Assembly of Tyrosine-Containing Bolaamphiphile Molecules as a Reactive Template for Metal Deposition, J. Kwak et al., Colloids and Surfaces B, Biointerfaces 102 (2013), 70-75

SUMMARY OF THE INVENTION The present invention has been made in order to overcome the above-mentioned problems, and an object of the present invention is to provide a method for producing a peptide having a peptide bond in a molecular structure and having two hydroxy functional groups at both ends, And to provide an amphiphilic biomimetic molecule with improved adhesion.

According to one embodiment of the present invention, there is provided a compound represented by the following general formula (1).

[Chemical Formula 1]

(Wherein X is independently an alkylene group having 1 to 4 carbon atoms, and n is an integer of 1 to 12.)

In formula (1), n may be an integer of 5 to 9, X may independently be an alkylene group having 1 to 2 carbon atoms, and a hydroxy group substituted in the phenyl group may have a para position and a meta position .

In one preferred embodiment, the compound of Formula 1 may be a compound of Formula 2 below.

(2)

According to another aspect of the present invention, there is provided a magnetic assembly formed by self-assembling a compound as described above.

At this time, it is preferable that the magnetic assembly has an adhesive property and a spherical shape, and it is more preferable to maintain a spherical shape regardless of pH change.

According to another aspect of the present invention, there is provided a magnetic assembly as described above, And magnetic nanoparticles attached to the surface of the magnetic assembly.

The magnetic nanoparticles may be nanoparticles containing at least one metal component selected from the group consisting of manganese, iron, cobalt, copper and cadmium, or nanoparticles selected from the group consisting of carbon nanotubes, carbon nanofibers, and fullerenes And is preferably a nanoparticle containing at least one carbon nano component.

In order to achieve the above object, according to another embodiment of the present invention, there is provided a magnetic assembly as described above, which is uniformly applied to a substrate surface including an organic material and an inorganic material, There is provided a composition for surface treatment for imparting compatibility.

The substrate may be at least one of polydimethylsiloxane (PDMS), glass, silicon rubber, acrylic, polytetrafluoroethylene (PTFE), cobalt, iron, copper, , A silicon wafer (Silicon wafer), and silica (SiO ₂ ).

According to another embodiment of the present invention, there is provided a process for preparing a compound of formula (I), which comprises reacting a compound of formula (III) with a salt of a compound of formula Drying the obtained reaction product (S20); And a step (S30) of deprotecting the dried reaction product.

(3)

(Wherein n represents an integer of 1 to 12).

[Chemical Formula 4]

Wherein X represents an alkylene group having 1 to 4 carbon atoms, and Y represents a protecting group.

At this time, the salt of the compound of Formula 4 may be p-toluenesulfonate salt, and the step of obtaining the reactant may be carried out in the presence of a crosslinking agent containing a carbodiimide compound , The carbodiimide compound is N, N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC) and 1-ethyl-3- (3- Carbodiimide (EDAC), and the like.

The step (S10) of obtaining the reactant is a step of reacting hydroxybenzotriazole (HOBt) 1-Hydroxy-7-azabenzotriazole (HOAt), sulfo-N-hydroxysuccin It is preferably carried out in the presence of at least one coupling agent selected from the group consisting of sulfo-NHS and N-hydroxysuccinimide (NHS).

The biomimetic molecule comprising the adhesive functional group of the present invention as described above and the self-assembly made using the biomimetic molecule of the present invention are excellent in hydrophilicity and self-assembling in a spherical shape uniformly even in the change of the environment such as pH, It can be applied to various application fields such as drug delivery materials or molds for inorganic materials.

FIG. 1 shows the result of analyzing the synthesized biomimetic molecule of the present invention through an infrared spectroscope.
FIG. 2 shows the result of analyzing the synthesized biomimetic molecule of the present invention with a nuclear magnetic resonance (NMR) spectrometer.
FIG. 3 is a scanning electron microscope (SEM) photograph showing the shape of particles of biomimetic molecules of the present invention dispersed in deionized distilled water and observed with time (leftmost photograph - center and right photograph).
FIG. 4 is a result of analyzing a comparative sample including a synthesized biomimetic molecule-containing sample according to an ultraviolet-visible (UV-vis) spectroscope.
FIG. 5 is a scanning electron microscope (SEM) photograph of a self-assembly manufactured using the biomimetic molecules of the present invention, which shows a spherical shape maintained regardless of pH environment change.
FIG. 6 is a photograph of the surface of a substrate of various components (PDMS, SiO2, Silicon rubber, Acrylic, PTFE, etc.) coated with a magnetic assembly according to the present invention and measuring the contact angle of droplets on the surface (left- The right side is a coated substrate).
FIG. 7 is a scanning electron microscope (SEM) photograph of a surface of iron (Fe) coated with a magnetic assembly according to the present invention, and FIG. 8 is a three-dimensional image thereof.
FIG. 9 is an electron microscope (SEM) photograph of a surface of a silicon wafer before coating the surface of a silicon wafer with a magnetic assembly according to the present invention, and FIG. 10 is a photograph of a surface after coating.
Fig. 11 is a graph showing the results of comparison between a glass substrate (negative control) without any treatment, a glass substrate coated with polydodamine (control_p DOPA), and a glass substrate (Dopa-C7) coated with the magnetic assembly of the present invention This is a photograph of cell culture experiment.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. Prior to the description, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings and should be construed in accordance with the technical concept of the present invention.

Throughout this specification, when a member is " on " another member, this includes not only when the member is in contact with another member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including" an element, it is understood that it may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "first "," second ", and the like are intended to distinguish one element from another, and the scope of the right should not be limited by these terms. For example, the first component may be referred to as a second component, and similarly, the second component may also be referred to as a first component.

In each step, the identification code is used for convenience of explanation, and the identification code does not describe the order of the steps, and each step may be performed differently from the stated order unless clearly specified in the context. have. That is, each of the steps may be performed in the same order as described, or may be performed substantially concurrently or in the reverse order.

First, the present invention provides a compound represented by the following formula 1 according to a preferred embodiment. The compound represented by the following formula (1) is a biomimetic molecule proposed in the present invention as a building block or monomer unit of a self-assembly.

[Chemical Formula 1]

In Formula 1, X is independently an alkylene group having 1 to 4 carbon atoms, and n is an integer of 1 to 12.

As shown in Formula 1, the biomimetic molecule of the present invention has a peptide bond in the molecular structure and two hydroxy groups (-OH) are bonded to both terminal benzene rings.

As a result, compared to conventional biomimetic molecules, mussels exhibit strong adhesiveness to various materials, and properties such as hydrophilicity, biocompatibility, adhesion with inorganic materials, and self-assembly properties are greatly improved, It can be used as a useful synthetic material that can be grafted.

In Formula 1, n is an integer of 5 to 9, X is independently an alkylene group having 1 to 2 carbon atoms, and the hydroxy group substituted in the phenyl group is preferably substituted at the para position and the meta position Do.

Finally, the compound of formula (1) according to the preferred embodiment may be a compound of formula (2).

(2)

The compound of formula (2) is an amphipathic biomimetic molecule in which hydrophilic DOPA, which is contained in large amounts in mussel adhesive proteins and is known to greatly contribute to the adhesion of mussels, is bound to both ends of a hydrophobic hydrocarbon backbone through peptide bonds to be.

Meanwhile, the present invention provides a method for producing a biomimetic molecule, comprising the steps of obtaining a reactant (S10), a drying step (S20), and a deprotection step (S30) according to another preferred embodiment.

In the step (S10) of obtaining a reactant, a step of reacting a compound of the following formula (3) with a salt of a compound of the formula (4) is performed.

(3)

[Chemical Formula 4]

In Formula 3, n represents an integer of 1 to 12, X in Formula 4 represents an alkylene group having 1 to 4 carbon atoms, and Y may represent a protecting group. More preferably, n in the general formula (3) represents an integer of 5 to 9, X in the general formula (4) represents an alkylene group having 1 to 2 carbon atoms, and a hydroxy group substituted in the phenyl group has a para position and meta ) Position.

In the above formula (4), the protecting group Y may be a benzyl group, but a methyl group, a t-butyl group, a silyl group or an oxazoline group, which is known in the art as a protecting group of a carboxyl group, may be used without limitation.

The salt of the compound of Formula 4 may be, but is not limited to, p-toluenesulfonate salts.

The first step (S10) of obtaining the reactants is preferably carried out in the presence of a crosslinker comprising a carbodiimide compound. The carbodiimide compound can be prepared by reacting N, N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC) and 1-ethyl-3- (3- (EDAC), but the present invention is not limited thereto.

In addition, the step of obtaining the reactant (S10) may further include a step of reacting hydroxybenzotriazole (HOBt) 1-Hydroxy-7-azabenzotriazole to suppress the side reaction and increase the reaction yield. may be carried out in the presence of at least one coupling agent selected from the group consisting of azabenzotriazole (HOAt), sulfo-N-hydroxysuccinimide (Sulfo-NHS) and N-hydroxysuccinimide have.

Next, step (S20) of drying the reactant obtained as the second step may be performed. The drying step S20 may include a freeze-drying step (S21) using a freeze dryer and a step of drying at 50 to 90 ° C in a vacuum oven.

Next, as a third step, step (S30) of deprotecting the dried reactant can be performed. The deprotection step (S30) is a step of deprotecting the protecting group Y. Specifically, when the protecting group is a methyl group or a t-butyl group, acid or base treatment is carried out. When the protecting group is a benzyl group, basic hydrolysis is performed. Acid or base treatment or an organometallic reagent may be used, and in the case of oxazoline, high temperature strong acid may be used.

Hereinafter, embodiments of the biocompatible molecule including the adhesive functional group of the present invention and the self-assembly produced using the same will be described. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents. shall.

1. Synthesis of biomimetic molecules

The biomimetic molecule of the present invention can be synthesized through an EDC / NHS coupling reaction, a base hydrolysis reaction and an acidification reaction of L-DOPA and azelaic acid.

Specifically, 0.65 g of HOBT used in the EDC / NHS reaction and 0.5 g of azelaic acid, which is a structure having seven carbon atoms and a reactive carboxylic acid at both ends, was dissolved in 100 mL of dimethylformamide (DMF).

The solution was stirred in an ice bath and mixed with 0.92 g EDAC in chloroform. After reacting for one hour, a solution of L-tyrosine benzyl ester p-toluenesulfonate salt in methanol was mixed, and 0.67 mL of triethylamine (TEA) serving as a catalyst was immediately added thereto.

After 30 minutes of reaction, they were refrigerated for about one day. The refrigerated solution was dried using a freeze dryer and refrigerated for another two days. When stored in the refrigerator, a needle-shaped precipitate forms, which is then filtered and washed with acetone to wash out the impurities.

The acetone-washed intermediate was dried in a vacuum oven at 70 < 0 > C. The dried intermediate was dissolved again in 80 mL of DMF and heated to 80 ° C with stirring. After heating, 0.1 N NaOH aqueous solution was added to perform base hydrolysis, which is a deprotecting process.

After the reaction was completed, the solution was allowed to cool to room temperature, and then an aqueous 1N HCl solution was added thereto to carry out an acidification reaction. After the reaction was completed, the solution was refrigerated for two days and then dried using a freeze dryer.

Immediately after drying, formation of a precipitate was observed, and the solution was filtered and washed with acetone to wash the impurities. The material obtained by washing with acetone was dried in a vacuum oven at 70 ° C to synthesize a biomimetic molecule of the present invention having a functional group of carboxylic acid and L-dopa at both ends of the azelaic acid skeleton.

FIG. 1 shows the result of analyzing the synthesized biomimetic molecule of the present invention through an infrared spectroscope. Referring to FIG. 1, a hydroxyl group bonded to a phenol group can be identified from the fact that a large peak is formed in the range of 3200 ^- 3600 cm ^-1 .

FIG. 2 shows the result of analyzing the synthesized biomimetic molecule of the present invention with a nuclear magnetic resonance (NMR) spectrometer. Referring to FIG. 2, it can be seen that a peak for the hydroxyl group (-OH) was observed in the region between 6-7 ppm.

It was confirmed that the chemical structure of the biomimetic molecule synthesized through a series of spectroscopic experiments agrees with that of the compound shown in Chemical Formula 2.

2. Self-assembly characteristics of biomimetic molecules

When the biomimetic molecules prepared in Example 1 were dissolved in a small amount of water, spherical particles were formed through self-assembly, and then particles formed using a centrifugal separator were observed with a scanning electron microscope (SEM) . The self-assembly of the synthesized biomimetic molecules has a spherical shape with a diameter of 200-300 nm.

FIG. 3 is a scanning electron microscope (SEM) photograph showing the shape of particles of biomimetic molecules of the present invention dispersed in deionized distilled water and observed with time (leftmost photograph - center and right photograph).

Referring to FIG. 3, powdery crystalline particles were observed immediately after being dispersed in the deionized distilled water. However, it can be seen that a spherical self-assembly is formed by gradually self-assembling.

Meanwhile, FIG. 4 shows the result of analyzing a comparative sample including a synthesized biomimetic molecule-containing sample according to an ultraviolet-visible (UV) viscometer. In FIG. 3, a black graph located at the uppermost position in the vicinity of the wavelength range of 280 nm is a result of measurement of dopamine as a control, and in order, a green graph represents the concentration of the biomimetic molecule of the present invention dissolved in ethanol The red graph represents the measurement result for dopa benzyl ester and the lowermost blue graph represents the measurement result of the biomimetic molecule of the present invention dissolved in deionized distilled water (DDW) . The self-assembling property of the biomimetic molecule of the present invention can be confirmed in distilled water, and the highest value can be seen in the deionized distilled water.

5 is a scanning electron microscope (SEM) photograph of a self-assembly manufactured using the biomimetic molecule of the present invention, which shows a shape maintaining a spherical shape regardless of a change in pH environment. Referring to FIG. 5, it can be seen that the magnetic assembly of the present invention maintains its spherical shape under all pH conditions under basic conditions (pH 10), neutral conditions (pH 7), and acid conditions (pH 4). As a result, it is possible to maintain a spherical shape irrespective of changes in the environment of the body (for example, pH change due to the digestive organs and the digestive juices secreted therein), so that the self assembly of the present invention can be utilized as a contrast agent or drug delivery vehicle injected into a human body Lt; / RTI >

Specifically, metal nanoparticles such as manganese, iron, cobalt, copper, and cadmium or carbon nano components such as carbon nanotubes, carbon nanofibers, and fullerenes are attached to the surface to function as a contrast agent or drug delivery vehicle It can be processed so that it can be performed.

Particularly, in the case of a conventional magnetic assembly (for example, a magnetic assembly proposed by the present inventor's previous application, Patent Application Publication No. 2012-0044773), a pretreatment process of coating an intermediate material such as silver However, the self-assembly of the present invention can greatly adhere to various metals without pretreatment.

3. Surface treatment (hydrophilic property, biocompatibility) using the magnetic assembly of the present invention

FIG. 6 is a photograph of the surface of a substrate of various components (PDMS, SiO2, Silicon rubber, Acrylic, PTFE, etc.) coated with a magnetic assembly according to the present invention and measuring the contact angle of droplets on the surface (left- The right side is a coated substrate). Referring to FIG. 6, it can be seen that the hydrophilic property is improved due to the surface treatment through the self-assembly, so that the contact angle of the droplet is reduced with respect to all substrates of various materials.

FIG. 7 is a scanning electron microscope (SEM) photograph of a surface of iron (Fe) coated with a magnetic assembly according to the present invention, and FIG. 8 is a three-dimensional image thereof. 9 is a scanning electron micrograph (SEM) photograph of the surface of a silicon wafer before coating the surface of a silicon wafer according to the present invention, and FIG. 10 is a photograph of the surface after coating.

Referring to FIGS. 7 to 10, it can be seen that the magnetic assembly of the present invention is uniformly applied to the surface of various substrates including metals, organic materials, and inorganic materials. As a result, the magnetic assembly of the present invention can be utilized as a template for an inorganic material capable of acting as a morphological mediator between an inorganic material and an organic material.

On the other hand, FIG. 11 shows the compatibility of a glass substrate (negative control) without any treatment, a glass substrate coated with polydodamine (control_p DOPA), and a glass substrate coated with the magnetic assembly of the present invention (Dopa-C7) This is a photograph of a cell culture experiment for viewing.

Specifically, after polydopamine or the self-assembly of the present invention was coated on the surface of the glass substrate, the substrate was washed and dried (2 hours) (left column of FIG. 11) 11), and the surface was cleaned. (Photo in the right column of Fig. 11)

Referring to FIG. 11, since the cells are most uniformly applied to the glass substrate coated with the self-assembly of the present invention, the cells are clustered to minimize the necrosis of the cells. High hydrophilicity and biocompatibility were confirmed. On the other hand, in the case of a glass substrate without any treatment, cell coating could not be achieved. In the case of a glass substrate coated with polypodamine, the coating on the glass substrate is not uniform, I could observe the necrosis of the bundle.

The present invention is not limited to the above-described specific embodiment and description, and various changes and modifications may be made by those skilled in the art without departing from the scope of the present invention as claimed in the claims. And such modifications are within the scope of protection of the present invention.

Claims (18)

A self assembly of a compound of the formula (1)
The magnetic assembly has an adhesive property,
Wherein the self-assembly maintains a spherical shape regardless of pH changes.
[Chemical Formula 1]

(Wherein X is independently an alkylene group having 1 to 4 carbon atoms, and n is an integer of 1 to 12.) The method according to claim 1,
The biomimetic molecular self-assembly of claim 1, wherein n is an integer of 5 to 9. The method according to claim 1,
Wherein X in Formula 1 independently represents an alkylene group having 1 to 2 carbon atoms. The method according to claim 1,
Wherein the hydroxy group substituted on the phenyl group is substituted at the para position and the meta position. The method according to claim 1,
Wherein the compound of formula (1) is a compound of formula (2).
(2)

delete delete delete A biomimetic molecular self-assembly according to any one of claims 1 to 5; And
And magnetic nanoparticles attached to the surface of the magnetic assembly. 10. The method of claim 9,
The magnetic nanoparticles may be nanoparticles containing at least one metal component selected from the group consisting of manganese, iron, cobalt, copper and cadmium, or nanoparticles selected from the group consisting of carbon nanotubes, carbon nanofibers, and fullerenes Wherein the nanoparticles are nanoparticles containing at least one carbon nano component. A mold for an inorganic material comprising the biomimetic molecular self-assembly according to any one of claims 1 to 5 and acting as a morphological mediator between an inorganic material and an organic material. A biomimetic molecular self-assembly according to any one of claims 1 to 5, which is uniformly applied to a substrate surface including an organic material and an inorganic material, and is used for surface treatment for imparting hydrophilicity and biocompatibility Composition. 13. The method of claim 12,
The substrate may be made of a material selected from the group consisting of polydimethylsiloxane (PDMS), glass, silicon rubber, acrylic, polytetrafluoroethylene (PTFE), cobalt (Co), iron (Fe) A silicon wafer, and a silica (SiO ₂ ). Obtaining an intermediate product (S10) by reacting a salt of a compound of the formula (3) and a salt of a compound of the formula (4) to obtain an intermediate product;
Drying the obtained intermediate product (S20); And
(S30) of deprotecting the dried intermediate product to prepare a biomimetic molecule represented by the following formula (1)
A method of manufacturing a biomimetic molecular self-assembly capable of maintaining a spherical shape regardless of a pH change by self-assembling the biomimetic molecule,
Wherein the step (S10) of obtaining the intermediate product is performed in the presence of a crosslinking agent including a carbodiimide compound and a coupling agent.
(3)

(Wherein n represents an integer of 1 to 12).
[Chemical Formula 4]

Wherein X represents an alkylene group having 1 to 4 carbon atoms, and Y represents a methyl group, a t-butyl group, a silyl group or an oxazoline group which is a protecting group of a carboxyl group.
[Chemical Formula 1]

15. The method of claim 14,
Wherein the salt of the compound of Formula 4 is a p-toluenesulfonate salt. delete 15. The method of claim 14,
The carbodiimide compound may be selected from the group consisting of N, N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC) (EDAC). ≪ RTI ID = 0.0 > 11. < / RTI > 15. The method of claim 14,
The coupling agent may be selected from the group consisting of hydroxybenzotriazole (HOBt) 1-hydroxy-7-azabenzotriazole (HOAt), sulfo-N-hydroxysuccinimide Sulfosuccinimide (Sulfo-NHS), and N-hydroxysuccinimide (NHS).

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