KR20180017465A - Dual stimuli-responsive oligoamine boronic acid - Google Patents

Abstract

The present invention relates to a dual stimuli-responsive oligoamine boronic acid (OAB). More specifically, the present invention relates to a biodegradable OAB patch, a method of preparing the OAB patch by using natural amino acid and 3-carboxyphenylboronic acid, and a medical use of the OAB patch, wherein the OAB patch has a specific structure containing predetermined numbers of cationic amine groups and phenyl boronic acid groups such that the OAB patch is bonded to carbon dioxide (CO_2) and monosaccharides to obtain excellent sensitivity with respect to CO_2 and monosaccharides, and which is applicable as an ideal material in various biomedical fields such as a stimulus-responsive assembly with respect to CO_2 and blood sugar in the blood, a drug delivery encapsulant, and the like since bonds of the OAB patch with CO_2 and monosaccharides not only are formed at a neutral pH value similar to in vivo conditions, but also are free from cytotoxicity. A dual stimuli-responsive OAB of the present invention is represented by chemical formula 3.

Description

DUAL STIMULI-RESPONSIVE OLIGOAMINE BORONIC ACID < RTI ID = 0.0 >

The present invention dual stimuli-responsive oligonucleotide relates to amine acid, and more particularly by jinim a specific structure containing a cationic amine group and a boronic acid group of a predetermined number, and carbon dioxide (CO ₂₎ and monosaccharides (monosaccharides Leads) and has excellent sensitivity to each of these, and this binding is carried out at a neutral pH similar to in vivo conditions and is also not cytotoxic. Thus, stimulation-sensitive assemblies for blood CO ₂ and blood glucose, encapsulating agents for drug delivery and the like Biodegradable oligoamine boronic acid (OAB) patches that can be applied as an ideal material in a variety of biomedical fields, including natural amino acids and 3-carboxyphenylboronic acid, and their pharmaceutical uses.

Phenylboronic acid can be reversibly bound to monosaccharides to form phenylboronic acid esters, which can be introduced into polyelectrolytes, polymers and dendrimers via nano- and micro-assemblies for insulin delivery, biological Which is important for the design of Glycoconjugate receptors.

However, there are still many limitations in application of such phenylboronic acids to living bodies, such as high pH conditions (> pH 9) and toxicity. In addition, in the case of the phenylboronic acid polymer, its proper structure has not yet been fully studied, and the constraint due to a large number of side conditions (strong irreversible binding, irreversible binding and aggregation induction in all the negative sites in the cell tissue) The situation is receiving. There are also some practical requirements for the production of polyelectrolyte complexes and capsules using phenylboronic acid, in addition to these restrictive side conditions.

Therefore, in the field of sensing and drug delivery, further study and development of the bioavailability and potential of phenylboronic acid materials are necessary.

In this regard, designing the phenylboronate moiety to have a non-toxic, biodegradable structure along with the amidoamine skeleton can be a good solution. In addition, when the structure is designed with a clear structure, control over the specific interaction with the negative site becomes possible, and the cell membrane can be reversibly controlled with respect to a simple external stimulus.

This structure not only allows for a more advanced smart composite structure, but also allows for controlled release of the active ingredient in a stimulus-triggered manner in various biomedical fields.

It is also expected that the amine groups in the amidoamine can lower the pKa of the phenyl boronic acid groups through intermolecular boronic acid-amine (B-N) interactions. In addition, the use of the amidoamine skeleton is expected to reduce cytotoxicity and immunogenicity, because poly (amidoamine) is usually a biocompatible polymer (macromolecule), and is suitable for living organisms. In addition, amidoamines are known to have a partial affinity for both and have the ability to form hydrogen bonds, allowing them to interact smoothly with the cell membrane.

Therefore, poly (amidoamine) is a substance that can realize multi-point interaction with phospholipid head groups, which is thought to induce endocytosis by initiating membrane curvature. to be.

If such a new material is developed, it could contribute significantly to the field of transcellular trafficking, such as the delivery of oligopeptides or protein drugs (especially insulin).

Specifically, studies on the development of substances sensitive to carbon dioxide and blood sugar in blood have been actively carried out, and when these materials are used as a drug coating agent, they are dissociated or dissolved in the process of bonding with carbon dioxide and monosaccharides in blood And the drug release can be controlled.

In this connection, various types of materials including phenylboronic acid have been reported, but most of the materials reported so far can not be used in living organisms due to their operation at high pH or their strong toxicity.

As a result, it has been shown that a phenylboronic acid-based material having no cytotoxicity exhibits specific sensitivity to blood glucose and the like in a neutral pH region similar to a living body condition, and thus can be applied to a variety of biomedical fields such as stimuli-sensitive sensing, drug delivery, Development is required.

De Geest, B. G .; Jonas, A. M .; Demeester, J .; De Smedt, S. C. Langmuir 2006, 22, 5070.

The present invention relates to a non-cytotoxic double-stimulus-sensitive oligoamine boronic acid capable of smoothly binding to carbon dioxide and monosaccharide at a neutral pH similar to a living condition, And to provide a medicinal use thereof.

In order to achieve the above object, the present invention provides a dual stimuli-responsive oligoamine boronic acid (OAB) represented by the following formula (3).

(3)

As a result of intensive studies, the present inventors have found that a homodisperse double stimulus-sensitive oligoamine boronic acid (OAB) based on natural amino acid and 3-carboxyphenylboronic acid (so-called oligoamine patch modified with phenylboronate ) Was successfully synthesized.

The oligomainic boronic acid according to the present invention possesses a suitable number of cationic amine groups (exactly seven amine groups) and is sufficiently strong and reversible to anionic surfaces and polymers to be able to bind non-cytotoxic, . Specifically, the oligomainic boronic acid of the present invention has sensitivity to carbon dioxide (CO ₂ ), and its overall charge due to reversible CO ₂ complexation and carbamate formation is between cationic and anionic It can be converted in the usual pH range (5.5 to 7.5).

The oligoamine boronic acid according to the present invention has an appropriate number of phenylboronic acid groups (specifically, four phenylboronic acid groups) and is reacted with a monosaccharide (monosaccharide) in neutral pH conditions in a 0.1 M phosphate buffer (Colorimetry) and Wang's competitive binding assay (quantitation). The results are shown in FIG.

That is, the oligoamine boronic acid having a specific structure according to the present invention reversibly binds to a green trigger such as CO ₂ at a neutral pH condition (for example, pH 7.4) and exhibits strong binding properties to monosaccharides, (E.g., insulin) patches, stimulation-sensitive assemblies for blood carbon dioxide and blood glucose, encapsulating agents for drug delivery, drug coatings, and drug release control agents.

The present invention also relates to a process for the preparation of a compound of formula (I), which comprises coupling an oligoamine-2 compound represented by the following formula 2 with 3-carboxyphenylboronic acid activated by N, N'-diisopropylcarbodiimide Characterized in that an oligoamine boronic acid to be displayed is synthesized.

Specifically, the present invention relates to a method for preparing a compound represented by the following formula (1): S1) reacting N-methyliminodiacetic acid to which N, N'-diisopropylcarbodiimide (DIC) Synthesizing a block-1 compound; S2) reacting the above-mentioned block-1 compound with Boc-Lys (Boc) -OH to which N, N'-diisopropylcarbodiimide (DIC) was added to synthesize an oligoamine-2 compound represented by the following formula ; And S3) reacting the oligoamine-2 compound with 3-carboxyphenylboronic acid to which N, N'-diisopropylcarbodiimide (DIC) is added to synthesize an oligoamine boronic acid represented by the following formula Wherein the method comprises the steps of:

[Chemical Formula 1]

(2)

(3)

The oligoamine boronic acid according to the present invention exhibits dual stimulus-sensitivity for each of carbon dioxide and monosaccharide at neutral pH conditions similar to in vivo.

In addition, the oligomainic boronic acid according to the present invention has no cytotoxicity, is biodegradable and excellent in biocompatibility.

Thus, the oligomainic boronic acid according to the present invention can be used in a variety of biomedical fields to design stimulus-sensitive nano- and micro-assemblies, sensing assemblies, drug delivery capsules, drug release control agents, glycoconjugate receptors, It can be a very ideal material to do.

It is expected that the present invention can be practically applied to a living body and has a very large ripple effect on the pharmaceutical industry.

FIG. 1 (A) shows the conductivity measurement results (top) of OAB (5 mM in Millipore water, pH 7.0) and the conductivity measurement results (bottom) of pure water (Millipore water, pH 7.0) as a reference value; (B) is a photograph showing the appearance change of OAB in MeOH / (50% ether / hexane) solution (1: 1, v / v) before and after slow CO ₂ bubbling for 1 minute.
(A) of Figure 2 (a) ARS (1.44 x 10 -4 M, 0.1 M in phosphate buffer (pH 7.4)), (b ) ARS (1.44 x 10 -4 M) + OAB (1.5 x 10 -3 M), (c) ARS ( 1.44 x 10 -4 M) + OAB (1.5 x 10 -3 M) + glucose (0.5 M), (d) ARS (1.44 x 10 -4 M) + OAB (1.5 x 10 ^-3 M) + fructose (0.5 M) (* image of inset = a, b, c, d solution of each solution); (B) is a graph showing the fluorescence change at 572 nm of the ARS / OAB complex upon addition of glucose and fructose;
Figure 3 (A) shows the ¹ H NMR spectrum of OAB; (B) is a ¹³ C NMR spectrum of OAB (* MeOH-d4, 400 MHz, asterisk = residual solvent and H ₂ O peak).
Figure 4 is the result of liquid chromatography mass spectrometry (LCMS) of OAB.
FIG. 5 is a photograph showing the appearance of ARS (1.44 x ^10-4 M) + OAB (1.5 x ^10-3 M) in 0.1 M phosphate buffer, pH 7.4 (left side = no saccharide, middle = 0.5 M glucose, 0.5 M fructose).
Figure 6 (A) shows the ARS (1.44 x 10 ^-4 M) fluorescence profile (λ _ex = 468 nm, λ _em = 572 nm) with increasing concentration (0-1.0 x 10 ^-2 M) of the ARS- ; (B) is a graph showing the increase in fluorescence intensity of ARS (1.44 x 10 ^-4 M) in the presence of OAB (pH 7.4, 0.1 M phosphate buffer); (C) is 1 / ΔI _f for calculating ARS-OAB (K _eq1 ) in the presence of ARS (1.44 × 10 ^-4 M) and OAB (0.71 × 10 ^-3 to 1.0 × 10 ^-2 M). 1 / R plot (* K _eq1 = 1215.0);
Figure 7 (A) shows the ARS fluorescence profile associated with OAB (1.5 x ^10-3 M) binding according to an increase in glucose concentration (0 to 1.0 M); (B) shows the [S] / P vs. the binding of OAB (1.5 x ^10-3 M) and glucose (0.10-1.0 M) in a three-component system containing the ARS solution (1.44 x ^10-4 M). Q plot (* K _eq = 5.8).
Figure 8 (A) shows the ARS fluorescence profile associated with OAB (1.5 x ^10-3 M) binding according to increasing fructose concentration (0-1.0 M); (B) shows the effect of [S] on the binding of OAB (1.5 x 10 ^-3 M) and fructose (9.95 x 10 ^-3 to 1.0 M) in a three-component system containing ARS solution (1.44 x 10 ^-4 M) / P vs. Q plot (* K _eq = 73.4);

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example

(1) materials and equipment

(TEA, Aldrich), N, N'-diisopropylcarbodiimide (DIC, Aldrich), tris (2-aminoethyl) amine (TAEA, Aldrich), N-methyliminodiacetic acid , TCI), 3-carboxyphenylboronic acid (CPBA, TCI), MeOH (LCMS grade), trifluoroacetic acid (TFA; Aldrich) and Boc- amino acid derivatives (Boc- Was used as received.

All other reagents were purchased commercially and used as is.

Nuclear magnetic resonance (NMR) spectra using the solvent MeOH-d ₄ was recorded on a Agilent DD2 NMR (400 MHz) spectrometer.

Mass spectra were recorded using an Agilent 6300 Ion trap LC / MS system.

UV-visible absorption spectra were obtained using a Perkin Elmer (Lambda-40) UV-vis spectrophotometer (Quartz cell, Path length: 10 mm).

Fluorescence spectra were recorded using a FluoroMate FS-2 fluorescence spectrometer.

The ARS fluorescence intensity was measured under conditions of an excitation wavelength of 468 nm and an emission wavelength of 572 nm.

Conductivity was measured using a Thermo Scientific Orion 4-star pH / conductivity meter.

(2) Block Synthesis of -1

Tris (2-aminoethyl) amine (5.3 mL, 54 mmol, 2 eq.) Was dissolved in 100 mL MeOH.

N-Methyliminodiacetic acid (4.0 g, 27 mmol, 1 eq.) Was dissolved in MeOH (35 mL) while slowly adding triethylamine (5 mL) in a separate flask.

N, N'-Diisopropylcarbodiimide (DIC) (8.4 mL, 54 mmol, 2 equiv.) Was then slowly added under ice-cooling and the solution was stirred at 0 ° C for 15 minutes.

The activated diacetic acid solution was transferred into the dropping funnel and slowly added to the amine solution with vigorous stirring.

After the addition was complete, the solution was stirred at room temperature for 24 hours.

Then, the solvent was removed under vacuum, and precipitated twice with Block-1 crude product from MeOH-Et ₂ O. (* Yield: 95%, melting point: 98 占 폚)

^{1 H-NMR (400 MHz,} MeOH-d4) δ (ppm) = 1.22-1.30 (d, 24H, H1-5), 2.52-2.55 (s, 3H, H6), 2.60-2.69 (m, 2H, H7 ), 2.72-2.87 (m, 4H, H8), 3.32-3.41 (m, 4H, H9), 3.78-3.88 (m, 4H, H10). ^{13 C-NMR (400 MHz,} MeOH-d4) δ (ppm) = 37.59 (s, C6), 39.67 (d, C2), 40.34 (d, C3), 41.91 (s, C10), 44.01 (m, C8 ), 53.10 (d, C 4), 53.90 (d, C 5), 61.65 (s, C 9), 176.97 (s, C 11). ESI-MS: m / z = 399 ([M-4]), 437 ([M + CH 3 OH + H] +), 263 ([M + 3ACN + 2H] 2+). FT-IR (KBr, cm ^-1 ): 1169 (tertiary amines), 1584 (amide II), 1620 (amide I), 2945 (N-CH ₃ ), 3300-3500 (primary amines).

(3) Oligoamine Synthesis of -2

Block-1 (403 mg, 1 mmol, 1 eq.) Was dissolved in 50 mL MeOH.

In a separate flask, Boc-Lys (Boc) -OH (1.39 g, 4 mmol, 4 eq.) Was dissolved in MeOH (10 mL) and the solution was ice-cooled.

The Boc-amino acid was obtained in the form of its dicyclohexylamine salt and was transferred to free acid according to standard protocols before use.

Then DIC (619 [mu] L, 4 mmol, 4 eq.) Was slowly added and the solution was stirred for 15 minutes.

The activated amino acid solution was transferred into a dropping funnel and slowly added to the Block-1 solution with vigorous stirring.

After the addition was complete, the solution was stirred overnight at room temperature.

The solvent was then removed under vacuum.

To the protected oligoamine crude product was added a solution of trifluoroacetic acid (TFA) (30%) in DCM and stirred for 2 hours.

The solvent was then reduced in vacuo and the residue was precipitated twice from MeOH-Et2O to give oligoamine- ₂ . (* Yield: 95%, melting point: 119 ~ 121 캜)

¹ H NMR (400 MHz, MeOH-d4)? (Ppm) = 1.17-1.23 (d, 20H, H1), 1.38-1.49 (m, 8H, H2), 1.62-1.73 (M, 12H, H4), 2.91-3.05 (m, 11H, H5 and H12), 3.33-3.58 (m, 12H, H6), 3.67-3.80 , 4H, H8), 4.05-4.28 (m, 4H, H9). ^{13 C-NMR (400 MHz,} MeOH-d4) δ (ppm) = 20.51 (m, C3), 25.72 (m, C5), 29.75 (m, C2), 35.61 (s, C12), 35.71 (m, C4 ), 38.22 (m, C7), 43.33 (s, C6), 52.16 (d, Cl), 56.52 (s, C9), 160.69 (m, C10), 168.30 (m, C11). ESI-MS: m / z = 525 ([M + 2MeOH + 2H] ²⁺ ), 329 ([M + 3Na] ³⁺ ). FT-IR (KBr, cm ^-1 ): 1180 (tertiary amines), 1626 (amide II), 1679 (amide I), 2980 (N-CH ₃ ), 3300-3500 (primary amines).

(4) OAB Synthesis of

Oligramine-2 (300 mg, 0.327 mmol, 1 eq.) Was dissolved in 10 mL MeOH.

In a separate flask, 3-carboxyphenylboronic acid (217 mg, 1.309 mmol, 4 eq.) Was dissolved in MeOH (10 mL) and the solution was ice-cooled.

Then DIC (204 L, 1.309 mmol, 4 eq.) Was slowly added and the solution was stirred at 0 < 0 > C for 30 min.

The activated 3-carboxyphenylboronic acid solution was slowly added to the oligoamine-2 solution while vigorously stirring.

After the addition was complete, the solution was stirred at room temperature for 24 hours.

Then, the solvent was removed under vacuum, and precipitated twice with OAB crude product from MeOH-Et ₂ O. (* Yield: quantitative)

^{1 H NMR (400 MHz, MeOH} -d4) δ (ppm) = 1.05-1.13 (d, 8H, H1a), 1.19-1.31 (m, 4H, H1), 1.45-1.60 (m, 8H, H2), 1.64 8H, H5), 3.36-3.51 (m, 4H, H6), 3.73-3.85 (m, 8H, H3), 1.85-2.00 , H7), 3.97-4.09 (m, 4H, H8), 4.19-4.50 (m, 4H, H9), 7.35-8.30 (m, 16H, H13, 15-17). ^{13 C-NMR (400 MHz,} MeOH-d4) δ (ppm) = 21.15 (m, C2), 22.24 (m, C3), 26.60 (m, C4), 38.74 (m, C5), 41.38 (s, C10 ), 43.09 (m, C7), 44.18 (m, Cl), 55.85 (m, C9), 112.34 (s, C15), 115.30 18), 161.67 (m, C13), 169.61 (s, C19), 172.25 (s, C20), 174.90 (s, C21). ESI-MS: m / z 527.0 ([M + 3Na] ³⁺ ), (OAB m / z 1508). FT-IR (KBr, cm ^-1 ): 1180 (tertiary amine), 1620 (amide II), 1679 (amide I), 2923 (N-CH ₃ ), 3030 (aromatic), 3300-3500 (primary amines).

Experimental conditions and methods

(One) ARS By law K _a  Methodology for Measurement

* Expression for association constant calculation

1 / ΔI _f = (ΔK p _o I _o K _eq1 ) ^-1 / [R] + (ΔK p _o I _o ) ^-1 (1)

(R) is the concentration of the receptor (OAB), and I _f is the fluorescence intensity). In the above equation (1),? K, p _o and I _o are parameters of the fluorescence spectrophotometer,

Q = [I] / [RI] = (I _RI - I) / (I - I _I ) (2)

(In the formula (2), Q is the measured absorbance, I _RI receptor-indicator (OAB-ARS) and the strength of the composite, I _I is the intensity of the free indicator)

P = [R ₀ ] - 1 / (QK _eq 1) - [I ₀ ] / (Q + 1)

(In the above formula (3), R ₀ is the total amount of OAB and I ₀ is the total amount of ARS)

[S] / P = (K eq1 / K eq) Q + 1 (4)

(In the above formula (4), [S] is the substrate concentration)

(2) fluorescence and Absorbance  Research

For a typical ARS-OAB fluorescence measurement, 1.44 x 10 ^-3 M ARS stock solution (stored in the manufacturing and refrigerator within the last 7 days) in 0.10 M sodium phosphate monobasic buffer was diluted to 0.10 M Diluted 10-fold with sodium phosphate monobasic buffer, and adjusted to pH 7.4 with 4 M NaOH to prepare a 1.44 x 10 ^-4 M ARS solution in 0.10 M phosphate buffer (pH 7.4) (solution A).

The OAB was added to a portion of the solution to make a 1.44 x ^10-4 ARS, 1.5 x ^10-2 M OAB solution, and the pH of the solution was adjusted to 7.4 (solution B).

Solution B was titrated into Solution A to form a mixture of ARS at a constant concentration and a concentration range of 7.1 x 10 ^-4 to 1.0 x 10 ^-2 M OAB and each mixture was left for 10 min to reach equilibrium.

Then, 1 mL of the solution was slowly transferred into the cuvette for fluorescence measurement.

The emission intensity was recorded at 572 nm.

For all quantitative experiments, the excitation wavelength was set at 468 nm.

The relationship between the change in fluorescence intensity and the equilibrium constant can be expressed using Equation (1).

The association constant (K _eq1 ) for ARS-OAB is 1 / ΔI _f vs.. Calculated from the slice and slope of the slice on a 1 / [R] plot, and the mean value of K _eq was determined through two measurements.

Absorbance studies were performed in a similar manner, except that OAB at a concentration of 1.5 x 10 < ^-3 > M was used.

(3) 3- Component  Through analysis OAB And monosaccharides (Competitive binding measurement study)

The competitive binding measurement study was conducted in a similar manner to the ARS-OAB study.

1.44 x ^10-4 M ARS and 1.5 x ^10-3 M OAB solution were made to pH 7.4 in 0.10 M sodium phosphate monobasic buffer (solution B).

A portion of Solution B was added with monosaccharide to form a 1.44 x 10 ^-4 ARS, 1.5 x 10 ^-3 M OAB, 2.0 M monosaccharide solution (Solution C).

Solution C was titrated into solution B to make a mixture of constant concentrations of ARS and OAB and monosaccharides in various concentration ranges (0.18 to 1.0 M for glucose and 0.01 to 1.0 M for fructose) So that the curves can be covered.

K _eq was calculated using equations (3) and (4).

Experiment result

(One) OAB Synthesis confirmation

OAB was synthesized by coupling oligoamine-2 with 4 equivalents of 3-carboxyphenylboronic acid using N, N'-diisopropylcarbodiimide (DIC) (Figure 1).

The number of free primary amine groups in the OAB structure was calculated based on their characteristic aromatic proton signal on ¹ H NMR spectroscopy.

The OAB showed peaks for 16 protons at 7.35 to 8.30 δ ppm, which means that there are four primary amine groups in the OAB.

Liquid chromatography mass spectrometry (LCMS) showed ESI-MS: m / z 527.0 ([M + 3Na] ³⁺ ) for OAB (m / z 1508).

[Figure 1] Synthesis of oligoamine boronic acid (OAB)

(2) OAB Wow CO ₂ Of reversible binding

Reversible binding of CO ₂ to 5 mM OAB aqueous solution (pH 7.0) was investigated by conductivity measurement.

During the slow bubbling of CO ₂ or N ₂ into the solution, the change in conductivity in the OAB solution was measured over time (FIG. 1A).

During constant CO ₂ bubbling, the conductivity of the OAB solution increased sharply at the beginning and reached a stable value without significant change in the minute range.

This increase in conductivity is due to CO ₂ binding to the primary amine functional group of OAB to form carbamates (Figure 2).

Figure 2 shows the different modes involved in the bonding of CO ₂ and amine functional groups: (a) carbamate formation by covalent bonding of CO ₂ , (b) the formation of carbonic acid by solvation of CO ₂ in water, anger

After all of the primary amine functionality had been consumed, no further reaction occurred and consequently the conductivity reached a steady state value.

The steady, weak increase in conductivity after reaching the steady state value may be due to the slow carboxylation of the boronic acid moiety at high CO ₂ concentrations.

It is necessary to compare the conductivity of OAB and pure water at neutral pH during CO ₂ or N ₂ bubbling.

As shown in FIG. 1A, the change in conductivity of OAB was 10 times higher than the reference value of pure water because of the formation of hydrogencarbonate species.

Figure IB is a digital photograph of OAB in a MeOH / (50% ether / hexane) (1: 1, v / v) solution.

By bubbling very slowly into CO ₂ , the turbid solution immediately turned into a clean solution in a minute range, possibly due to carbamate formation in the solution and protonation of the OAB amine group (Figure 2).

(3) OAB And monosaccharide binding assay ( UV / visible  And fluorescence spectroscopy)

UV / visible and fluorescence spectroscopy were used to evaluate the binding of monosaccharide to OAB (Figure 2).

Of the initial OAB / Alizarin Red S (ARS) complex ([OAB]: [ARS] = 10: 1) in 0.1 M phosphate buffer, pH 7.4, was added to the OAB / ARS complex solution The absorption at nm shifted to 480 nm.

The red shift in absorbance means that the monosaccharide binds to OAB and replaces OAB-binding ARS (FIG. 2A and FIG. 5).

When fructose (0.5 M) was added instead of glucose, the absorbance of the OAB / ARS complex shifted significantly to 520 nm at 465 nm.

These experimental results clearly demonstrate that OAB has a sensitive response to monosaccharides at neutral pH conditions.

Binding of the monosaccharide to OAB at neutral pH can be confirmed by colorimetric evaluation.

The formation of ARS / OAB complex changed the dark pink color of ARS into a dark yellowish color, and the color was recovered again according to the monosaccharide binding (Fig. 2A, inset).

(4) OAB And quantitative determination of monosaccharides Wang Lt; / RTI >

Based on Wang's competition binding assay, quantitative binding of OAB and monosaccharide was evaluated.

The protons in the excited state are transferred to the ketone oxygen in the phenol hydroxyl group of the ARS, resulting in fluorescence quenching of the free ARS.

Thus, when the boronic acid ester is formed, the fluorescence of the system is expected to increase through elimination of the fluorescence quenching mechanism (Figure 3).

[Figure 3] Wang's competitive binding assay (using ARS as a colorimetric reporter) on the binding of boronic acids to carbohydrates

As the OAB was added into the ARS solution to form the OAB / ARS complex, the fluorescence intensity of the ARS solution was increased 100-fold.

The association constant (K _eq ) of OAB was measured based on the fluorescence intensity of the OAB / ARS complex. Fluorescence intensity also decreased as ARS (bound to OAB) was replaced by the diol of the monosaccharide (Fig. 2B).

At pH 7.4, the K _eq for OAB and ARS binding was measured to be 1.215 x 10 ³ M ^-1 and the K _eq for binding of OAB to glucose or fructose was 5.8 M ^-1 and 73.4 M ^-1 , respectively (Table 1).

At neutral pH, the value of K _eq was comparable to the reported value for phenylboronic acid, indicating that the phenylboronic acid group contained in the OAB at neutral pH was strongly bound to the monosaccharide as well as the free phenylboronic acid It is.

[Table 1] Association constant with OAB (K _eq ) (* 0.1 M phosphoric acid buffer, pH 7.4, average value of two measurements)

Review results

The biodegradable OAB structure according to the present invention exhibited monosaccharide-responsive behavior at physiological conditions (pH 7.4) and showed reversible conversion of total charge by binding with CO ₂ .

A dual-stimulus-sensitive boronic acid-containing oligoamine patch has not been reported yet that forms a carbamate according to CO ₂ treatment at physiologically relevant pH and releases the carrier (e.g., drug) in response to the saccharide in water.

Thus, the OAB according to the present invention may be an ideal material for establishing the structure of a smart complex for sensing, drug delivery and various biomedical applications.

Claims (16)

Dual stimuli-responsive Oligoamine boronic acid (OAB) represented by the following formula (3): < EMI ID =
(3)

.
The method according to claim 1,
Characterized in that the oligoamine boronic acid is simultaneously susceptible to carbon dioxide (CO ₂ ) and monosaccharides.
Dual stimuli-sensitive oligoamine boronic acid.
3. The method of claim 2,
Wherein the amine group contained in the oligomainic boronic acid is reversibly bound to carbon dioxide to have carbon dioxide-sensitive properties.
Dual stimuli-sensitive oligoamine boronic acid.
The method of claim 3,
Wherein the amine group contained in the oligomeric aminoboronic acid is combined with carbon dioxide to form a carbamate.
Dual stimuli-sensitive oligoamine boronic acid.
3. The method of claim 2,
Wherein the phenylboronic acid group contained in the oligomeric aminoboronic acid is strongly bound to the monosaccharide and has a monosaccharide-sensitive property.
Dual stimuli-sensitive oligoamine boronic acid.
6. The method of claim 5,
Wherein the binding between the phenyl boronic acid group and the monosaccharide is quantitatively determined by a colorimetric method or a competitive binding assay.
Dual stimuli-sensitive oligoamine boronic acid.
The method according to claim 6,
Characterized in that Alizarin Red S (ARS) is used as a colorimetric reporter.
Dual stimuli-sensitive oligoamine boronic acid.
3. The method of claim 2,
Characterized in that said oligoamine boronic acid is sensitive to carbon dioxide and monosaccharides under conditions of neutral pH,
Dual stimuli-sensitive oligoamine boronic acid.
9. The method of claim 8,
Characterized in that said oligoamine boronic acid binds to carbon dioxide and a monosaccharide under conditions of pH 7.4.
Dual stimuli-sensitive oligoamine boronic acid.
The method according to claim 1,
Characterized in that said oligoamine boronic acid is non-cytotoxic.
Dual stimuli-sensitive oligoamine boronic acid.
The method according to claim 1,
Characterized in that the oligomainic boronic acid is used as the material of the drug patch.
Dual stimuli-sensitive oligoamine boronic acid.
12. The method of claim 11,
Characterized in that the oligomainic boronic acid is used as a material for a stimulus-sensitive assembly for carbon dioxide and blood sugar in blood, an encapsulating agent for drug delivery, a drug coating agent or a drug release control agent.
Dual stimuli-sensitive oligoamine boronic acid.
12. The method of claim 11,
Wherein said drug is an oligopeptide or protein.
Dual stimuli-sensitive oligoamine boronic acid.
14. The method of claim 13,
Wherein the drug is insulin.
Dual stimuli-sensitive oligoamine boronic acid.
A process for preparing the double stimulus-sensitive oligomeric aminoboronic acid according to claim 1,
Coupling an oligoamine-2 compound represented by the following formula (2) with 3-carboxyphenylboronic acid activated by N, N'-diisopropylcarbodiimide (DIC) to obtain an oligo- Lt; RTI ID = 0.0 >
Preparation of dual stimuli-sensitive oligoamine boronic acid:
(2)

(3)

.
16. The method of claim 15,
S1) N-methyliminodiacetic acid to which N, N'-diisopropylcarbodiimide (DIC) is added is reacted with tris (2-aminoethyl) amine to synthesize a block- ;
S2) reacting the above-mentioned block-1 compound with Boc-Lys (Boc) -OH to which N, N'-diisopropylcarbodiimide (DIC) was added to synthesize an oligoamine-2 compound represented by the following formula ; And
S3) reacting the oligomer-2 compound with 3-carboxyphenylboronic acid to which N, N'-diisopropylcarbodiimide (DIC) is added to synthesize oligoamine boronic acid represented by the following Formula 3 ≪ / RTI >
Preparation of dual stimuli-sensitive oligoamine boronic acid:
[Chemical Formula 1]

(2)

(3)

.

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