KR102141656B1 - Method for bioconversion of corticosterone by glucosyltransferase and corticosterone glucoside produced thereby - Google Patents

Abstract

The present invention provides a method for bioconversion of corticosterone including a step of glucosylating a 21-hydroxyl group of corticosterone using UDP-glucosyltransferase, UDP-glucosyltransferase used as an enzyme, and corticosterone 21-glucoside obtained therefrom. The corticosterone glucoside obtained from the method for bioconversion of corticosterone using the glucosyltransferase of the present invention can be usefully used as a cranial nerve cell protective agent having low cytotoxicity to brain cells in the fields of medicine and pharmacy.

Description

Method for bioconversion of corticosteroid using glucosyltransferase and corticosteroid glucoside obtained therefrom {Method for bioconversion of corticosterone by glucosyltransferase and corticosterone glucoside produced thereby}

The present invention relates to a bioconversion method of corticosterone using glucosyltransferase and a corticosteroid glucoside obtained therefrom, more specifically, it is expressed from the nucleotide sequence of SEQ ID NO: 1 by bioconversion method or of SEQ ID NO: 2 It relates to a method for bioconverting to the corticosteroid 21-glucoside by glucosylating the hydroxyl group #21 of corticosteroid using a UDP-glucosyltransferase comprising an amino acid sequence.

Drugs, poisons or debris in vivo are metabolized by the cytochrome P450 in the liver and excreted in the urine. Hydrophobic compounds are converted into hydrophilic compounds through the reaction of conjugation with glutathione or glucuronic acid using UDP-glucuronosyl-transferase (UDT). , Finally, these metabolites are excreted through the bile duct or blood.

Steroid hormones are mainly secreted by internal factors and external stimuli from the testes, ovaries, adrenals, gonads and endocrine glands, synthesized in the adrenal glands and transported to target organs. In general, steroidal hormones are classified into androgen, estrogen, and corticoid according to their physiological function and structure. In particular, corticosteroid hormones are further classified into glucocorticoids and mineralocorticoids. In addition, examples of glucocorticoids include corticosterone, cortisone and cortisol synthesized in the adrenal glands. Corticosterone is the main adrenal cortical hormone and is secreted in small amounts only in humans with low levels of activity.

Since glucocorticoids express physiological functions such as immunity, growth, emotion and cognition, they can be used as drugs having anti-inflammatory and immunomodulatory effects. Research and development of a variety of in vivo steroidal hormones or innovative drugs is important, and synthesis of standard reagents and test preparations is essential for studying the properties and action of these hormones. In particular, oxidative stress, bioenergy damage and mitochondrial disorders have been implicated in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and stroke. An increase in corticosteroid secretion by stress is known to cause damage to nerve cells and may be associated with the manifestation of behavior in depression. Corticosterone is released into the bloodstream during stress and easily enters the brain through the blood-brain-barrier (BBB) and is distributed in various brain regions.

Most of the glucuronides have glucuronic acid linked to the hydroxyl group of the 21st carbon. Corticoid glucuronide is useful in vivo and has data on synthesis methods, melting point, optical rotation properties, infrared spectroscopy (IR), ultraviolet-visible spectroscopy (UV) and nuclear magnetic resonance (NMR). Is known. However, since the adrenal cortical hormones cortisol and corticosterone are lipids, anhydrous reaction or many steps are required, and the purchase of cortisol and corticosteroid is also difficult. Like steryl, sterol and steroid glucosides, which are commonly found in plants and bacteria, corticoid glucosides are advantageous in that they have better hydrophilicity and are useful for drug development. There have been many reports of the use of corticoid glucosides in in vivo studies (Schulze, E., et al., Biomed. Biochim. Acta, 1986, 45, 1049-1056 and Csaba, G., et al., Acta Biol Med. Ger. 1978, 37, 1377-1380).

Glycosylation in oxygen that binds to the 11β-hydroxyl group of corticosteroids has been reported (Chou, FP et al., Chem. Biol. Drug Des., 2017, 89, 61-66), but binds to the 21-hydroxyl group There have been no reports of glycosylation in oxygen. On the other hand, 11-deoxycorticosterone (11-deoxycorticosterone) portion is limited to the binding position of only the 21-hydroxyl group, acetylation and 11-deoxycorticosterone portion of the 21-hydroxyl group Compounds obtained by performing glucoside compounds have been used in many in vivo studies. However, these compounds have limitations on the glycosylation site, and oxygen binds only to the 21-hydroxyl group. Therefore, it is considered that the compound obtained by performing glycosylation only on the 21-hydroxyl group, leaving 11β-hydroxyl group, is very useful for the reasons mentioned above. In addition, the interpretation of the NMR structure of corticosterone and its derivatives is insufficient compared to cortisone and cortisol.

Korean Registered Patent No. 10-0192005 (1999.01.27.)

Chou F.P., et al, Chem Biol Drug Des, 2017, 89: 61-66. Koirala, N., Preprints 2018, 2018040247 (doi: 10.20944/preprints201804.0247.v1).

In the present invention, by using glucosyltransferase (glucosyltransferase), glucosylation was performed in the 21-hydroxyl group of corticosterone, leaving only the 11-hydroxyl group, one-dimensional (1D) ¹ H-NMR and one-dimensional ¹³ C-NMR and correlated spectroscopy (COSY), rotating-frame overhauser effect spectroscopy (ROESY), distortion-resistant reinforced-heteronuclear single quantum correlation (ROESY) Corticos through two-dimensional (2D) NMR experiments and mass spectrometry of quantum correlation-distortionless enhancement by polarization transfer (HSQC-DEPT) and heteronuclear multiple-bond correlation (HMBC) Theron glucoside structure was investigated.

After mass production and isolation of corticosterone 21-glucoside, neuroprotective activity against SH-SY5Y neuroblastoma cells induced by hydrogen peroxide (H ₂ O ₂ ) was investigated and compared to corticosteroid.

According to one aspect of the invention, there is provided a method for bioconverting corticosteroids comprising the step of glucosylating the 21-hydroxyl group of corticosteroids using UDP-glucosyltransferase.

According to another aspect of the present invention, there is provided a UDP-glucosyltransferase used as an enzyme in the bioconversion method of corticosteroid.

In one embodiment, the UDP-glucosyltransferase may be a protein expressed from the nucleotide sequence of SEQ ID NO: 1, and the UDP-glucosyltransferase may include the amino acid sequence of SEQ ID NO: 2.

According to another aspect of the present invention, a corticosteroid 21-glucoside obtained by the bioconversion method of corticosteroid is provided.

According to another aspect of the present invention, there is provided a pharmaceutical composition for protecting brain neurons comprising the corticosteroid glucoside.

According to the present invention, a method for bioconverting corticosteroid to corticosterone 21-glucoside using a UDP-glucosyltransferase expressed from a nucleotide sequence of SEQ ID NO: 1 or comprising an amino acid sequence of SEQ ID NO: 2 is revealed. lost. In addition, the structure and position of corticosterone 21-glucoside produced therefrom was found by methods such as ¹ H, ¹³ C, COSY, ROESY, HSQC-DEPT and HMQC NMR experiments and TOF-ESI HRMS. Corticosterone 21-glucoside according to the present invention has a low cytotoxicity to neurons, a protective effect against hydrogen peroxide cytotoxicity, and an inhibitory effect on ROS production, compared to the same concentration of corticosterone.

Therefore, the bioconversion method of corticosteroid using the glucosyltransferase of the present invention and the corticosteroid glucoside obtained therefrom can be usefully used as a neuronal cell protective agent having low cytotoxicity against brain cells in the pharmaceutical and pharmaceutical fields.

1 is a schematic diagram showing the response of corticosteroid to glucosyltransferase and glucurosyltransferase.
2 is an HPLC graph of a glucosylated corticosteroid product after bioconversion.
FIG. 3 is a rotating-frame overhauser effect spectroscopy (ROESY) spectrum of corticosterone 21-glucoside to allocate different hydrogen bound to the aglycone moiety H-12 and stereocenter. Is part of The intersection of the circles is a remarkable peak, and the numbers on the side indicate the hydrogen's position number in the aglycone.
Figure 4 is from ROESY experiments H-9, H-14 and ¹ H- ¹ of corticosterone 21-glucoside and form nuclei over Hauser effect (nuclear overhauser effect, NOE) confirmed by the stereochemistry of the H-18 It is a schematic diagram showing the H correlation. In addition, ¹ H- ¹³ C dinuclear multiple bond correlation method and a combination of glucose (heteronuoclear multiple-bond correlation, HMBC ) 1 H- 13 C long range (range lone) coupling Any corticosterone through the relationship with irradiation experiment position Shows. The abnormality of the glucose portion is confirmed by analysis of ¹ H- ¹ H correlated spectroscopy (COSY) experiments and ¹ H- ¹³ C HMBC experiments.
5 is a graph showing the effect of corticosteroid and corticosteroid 21-glucoside on SH-SY5Y neuroblastoma cell line. (a) is the cell viability, (b) is the ability to protect cells against hydrogen peroxide (H ₂ O ₂ ) treated cells, and (c) is the reactive oxygen species (ROS) in H ₂ O ₂ treated cells. Indicates the production capacity of each. Data are presented as a percentage of untreated control. ^* P<0.05, ^** p<0.01 and ^*** p<0.001 when compared with the result of the untreated control group, and ^# p<0.05, ^## p<0.01 when compared with the result of the H ₂ O ₂ treated cell group And ^### p<0.001.

The present invention provides a method for bioconverting corticosteroids comprising the step of glucosylating the 21-hydroxyl group of corticosteroids using UDP-glucosyltransferase.

The UDP-glucosyltransferase may be a protein expressed from the base sequence of SEQ ID NO: 1 (see Table 1), and may include an amino acid sequence of SEQ ID NO: 2 (see Table 1). In addition, the enzyme may be included in 1 ~ 100 ㎍ / ㎖, preferably 5 ~ 50 ㎍ / ㎖, most preferably 10 ~ 30 ㎍ / ㎖. In addition, in the enzymatic reaction, UDP-glucose may be included at 0.1 to 100 mM, preferably 1 to 50 mM, most preferably 2 to 10 mM, and corticosteroid is 0.1 to 100 mM, preferably 0.2 to 50 mM, most preferably 0.4 to 10 mM. The reaction may be carried out at a temperature of 20 ~ 50 ℃, preferably 30 ~ 40 ℃, most preferably 34 ~ 38 ℃.

In addition, the present invention provides a UDP-glucosyltransferase used as an enzyme in the bioconversion method of corticosteroid.

The UDP-glucosyl transferase in the present invention may be derived from microorganisms, and may preferably be derived from Teribacillus PAMC 23288 (pre-sale from the Polar Research Institute attached to the Korea Institute of Ocean Science and Technology). The recombinant vector obtained by cloning the sequence encoding the UDP-glucosyltransferase gene (base sequence of SEQ ID NO: 1) can be prepared by using a conventional protein production method for overexpressing E. coli.

The total reaction time using the UDP-glucosyltransferase may take 1 to 5 hours, preferably 2 to 4 hours, most preferably 3 hours, and cooled organic solvent (for example, to stop the reaction) , Methanol, etc.).

In addition, the present invention provides a corticosteroid 21-glucoside obtained by the bioconversion method of corticosteroid.

The corticosteroid 21-glucoside can be confirmed by using ¹ H, ¹³ C, COSY, ROESY, HSQC-DEPT and HMQC NMR experiments and methods such as TOF-ESI HRMS. Corticosterone 21-glucoside can confirm the cell protective effect and the inhibitory effect on ROS production against cytotoxicity by hydrogen peroxide, low cytotoxicity against neurons compared to the same concentration of corticosteroid.

In addition, it provides a pharmaceutical composition for the protection of cranial nerve cells comprising the corticosteroid glucoside. The disease related to the oxidative damage of the cranial nerve cells may be one or more selected from the group consisting of stroke, dementia, Alzheimer's disease, Huntington's disease, and Parkinson's disease.

In one embodiment, the pharmaceutical composition may include the corticosterone 21-glucoside at a concentration of 1 to 1,000 µg/ml, preferably 1 to 700 µg/ml, but is not limited thereto. Age, gender, health, diet, administration time, administration method, excretion rate and disease severity may vary. In addition, the pharmaceutical composition for protecting brain neurons of the present invention may be administered orally or parenterally depending on the desired method.

In one embodiment, the pharmaceutical composition may further include at least one pharmaceutically acceptable carrier selected from the group consisting of fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, diluents and excipients.

Pharmaceutically acceptable carriers can be used by mixing one or more of these components: saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and antioxidants, if necessary. Other conventional additives such as buffers and bacteriostatic agents can be added.

In addition, the pharmaceutical composition of the present invention, in the form of aqueous solutions, suspensions, emulsions, etc., can be prepared in various dosage forms, such as injectable formulations, pills, capsules, granules, ointments, orally administered or tablets.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Materials and Methods

(1) General experimental procedure for corticosteroid 21-glucoside

1) Isolation of corticosteroid 21-glucoside

The product was analyzed with a Dionex Ultimate 3000 UHPLC+ system (Thermo Fisher Scientific, Germering, Germany) using a Mightysil reverse-phase C18 GP column (4.6×250 nm, 5 μm). The HPLC system consists of an LPG-3400SD pump, an ACC-3000 automatic sampler column compartment, and a DAD-3000 diode array detector. The mobile phase consists of solution A (in HPLC grade water) and solution B (in HPLC grade acetonitrile). The flow rate was maintained at 1.0 ml/min and the oven temperature was maintained at 30°C. To analyze the product, the gradient system is as follows: the percentage of solution B from 5% to 8% (0-4 minutes), 20% (7 minutes), 40% (10 minutes), 70%. (13 minutes), 100% (18-25 minutes), 80% (28 minutes), 50% (30-33 minutes) and 5% (38 minutes). UV detection was performed at 245 nm to identify the substrate and its products.

The HPLC separation method is as follows: preparative HPLC (prep HPLC) using a C18 column (YMC-Pack ODS-AQ (250 nm×20 nm ID, 10 μm)) connected to a UV detector (245 nm). Purification of the compound was carried out. Two programs with ACN 5% to 40% (0 to 10 minutes) were used for 35 minutes, then increased to 100% (10 to 18 minutes) at a flow rate of 10 ml/min, and 100% (18 to 25 minutes) ), then reduced to 5% (25-35 minutes).

2) Nuclear magnetic resonance and mass spectrometry

Samples were prepared by dissolving the purified product on a Bruker BioSpin AVANCE II 900 MHz spectrometer (Bruker GmbH, Rheinstetten, Germany) in hexadeuterio-dimethyl-sulfoxide (DMSO- d ₆ ). One-dimensional NMR ( ¹ H NMR and ¹³ C NMR) tests and two-dimensional NMR (COSY, ROESY, HSQC-DEPT and HMBC) tests were performed to reveal the exact structure of the glucosylated steroid.

In vitro glucosylation reaction initiation The mass of substrate and product is high resolution liquid chromatography electrospray ionization quadrupole flight time difference using ACQUITY UPLC ^® (Waters Corporation, Milford, MA, USA) combined with SYNAPT G2-S (Waters Corporation) High resolution mass spectrometry (UPLC-ESI-Q-TOF-HRMS) was performed.

(2) bioconversion reaction

The present inventors derive from the Arctic soil sample, Terribacillus sp. PAMC 23288 was used by pre-sale from the Polar Research Institute (Incheon, South Korea) attached to the Korea Institute of Ocean Science and Technology, and from this, the UDP-glucosyltransferase-1 (UTG-1) gene was found. The encoding sequence of the UGT-1 gene (base sequence of 1182 bp [SEQ ID NO: 1], see Table 1) was overexpressed in Escherichia coli BL21 (DE3) after being cloned into the pET28a(+) vector. As a result of analysis, it was confirmed that the amino acid sequence of 393 (SEQ ID NO: 2, see Table 1). Purification of UGT-1 was performed by a general protein purification method.

For in vitro analysis, a final volume of 100 μl was constructed with 10 μg/ml glucosyltransferase, 100 mM Tris-HCl pH 8.5 buffer, 10 mM MgCl ₂ , 2 mM UDP-glucose and 0.4 mM corticosteroid. The reaction proceeded at 35° C. for 3 hours. When the reaction was completed, the sample was extracted with 400 μl of methanol, and then centrifuged at 12,000×g for 10 minutes. The separated supernatant was used for further analysis.

Purified glucosyltransferase (30 μg/ml), 10 mM UDP-glucose (~ 56 mg), 10 mM substrate (~ 10 mg, dissolved in DMSO), 100 mM Tris-HCl (pH 8.0) buffer and 10 The pre-scale reaction was confirmed using a 30 ml volume of mM MgCl ₂ ·6H ₂ O, which was reacted at 37° C. for 3 hours. The reaction was stopped by adding 3 times the volume of cooled methanol. The reaction mixture was simply mixed and centrifuged (12,000 rpm, 10 minutes, 4°C) to remove the denatured protein. Finally, the supernatant was concentrated by evaporation and purification.

(3) Cell analysis of corticosteroid 21-glucoside

1) Analysis of cytotoxicity and cell protection

The SH-SY5Y neuroblastoma cell line was purchased from the American Cell Line Bank (American Type Culture Collection, Manassas, VA, USA). The cells were Dulbecco's Modified Eagle's Medium (DMEM, Gibco-BRL, Karlsruhe, Germany) containing 10% fetal bovine serum (Gibco-BRL) supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL). Incubated in a 37 °C, 5% CO ₂ incubator.

Cells were seeded in 96-well plates at a density of 1×10 ⁴ cells/well to confirm the cell viability due to the treatment of corticosterone and corticosteroid 21-glucoside, and cultured at 37° C. for 24 hours. Plates were treated with different concentrations (0, 5, 10, 25 and 50 μM) of corticosteroid and corticosteroid 21-glucoside, and then the plates were further incubated at 37° C. for 24 hours. Cell viability [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxy-methoxyphenyl) -2- (4-sulfophenyl) -2 H - tetrazolium inner salt (MTS) ] Was measured using CellTiter 96 ^® Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) based on reduction from formazan according to the manufacturer's instructions. After removing the medium, 200 μl of DMEM containing MTS was added to each well, and then cultured at 37° C. for 1 hour. Absorbance was measured at 490 nm using a microplate fluorescence meter (Molecular Devices, Sunnyvale, CA, USA).

Cells were plated in 96-well plates at a density of 1 x 10 ⁴ cells/well in a 96-well plate to confirm the cell protective effect of corticosterone and corticosteroid 21-glucoside and maintained at 37°C for 24 hours. Corticosterone and corticosteroid 21-glucoside were added to cells at different concentrations (0, 1, 2.5, 5 and 10 μM) and incubated at 37° C. for 24 hours. After adding 100 μM hydrogen peroxide (H ₂ O ₂ ) to the cells and incubating for 1 hour, the protective effect of the cells was measured by reducing MTS with formazan.

2) Reactive Oxygen Species (ROS) production analysis

5(6)-carboxy-2′,7′-dichlorofluorescein diacetate[5(6)-carboxy-2′,7′-dichlorofluorescein diacetate, DCF-DA, Sigma-Aldrich, St. Louis, MO, USA] The intracellular ROS was measured using a fluorescent probe. SH-SY5Y cells were plated in 96-well assay plates and test samples with different concentrations (0, 1, 2.5, 5 and 10 μM) were treated for 24 hours each. 100 μM hydrogen peroxide was added to the cells and incubated for 1 hour. Cells were incubated with 10 μM DCF-DA at 37° C. for 30 minutes and then washed twice with PBS. The fluorescence intensity of DCF was measured using a microplate reader having an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

2. Results

(1) bioconversion of corticosteroid 21-glucoside

As a result of bioconversion to corticosteroid obtained with a conversion rate of about 40% (see FIG. 1), HPLC analysis of the corticosteroid reaction mixture showed only one glucoside peak at 254 nm. The time-of-flight electrospray ionization high-resolution mass spectrometry (TOF-ESI HRMS) of the product was calculated to match m / z = 509.2747 (C ₂₇ H ₄₁ O ₉ ⁺ :) consistent with the correct mass of corticosteroid glucoside [M + 1] ^+. The exact mass value m / z = 509.2745) was shown (see Fig. 2).

NMR experiments measured ¹ H, ¹³ C, COSY, ROESY, HSQC-DEPT and HMQC and confirmed the carbon and conformation with each hydrogen by ROESY. Ciuffreda, et al. Steroids 2009, 74, 870-875] report the ¹ H- and ¹³ C-NMR chemical shifts to corticosteroid 21-glucuronide. Corticosterone 21-glucuronide and corticosterone 21-glucoside are almost identical in the aglycone moiety, the only difference being the carboxylic acid or alcohol at the 6'position of the sugar moiety as shown in FIG. Comparative corticosteroid derivatives between glucuronide and glucoside for chemical shifts of ¹³ C and ¹ H and ¹ H- ¹ H binding constants are shown in Table 2 below.

The ¹³ C and ¹ H chemical shifts of the aglycone moiety are described in Ciuffreda, et al. Steroids 2009, 74, 870-875], but the chemical shift of H-12 to α and β reversed these results. The determination of α and β of the steroid was confirmed as follows: When the ring of the steroid is marked as a protrusion on the paper surface, the formula is generally directed eastward as shown in FIG. 1. In FIG. 1, atoms or groups (groups) attached to the ring shown in the east direction are referred to as alpha (α) or beta (β) when placed on the plane of the paper. These α and β positions can be confirmed by examining the three-dimensional correlation using a method of observing the nuclear overhauser effect (NOE) between each hydrogen. ROESY or NOESY experiments are preferred because they form a complex by overlapping the ¹ H-NMR signal of other aglycone protons.

The results of the ROESY experiment showed that at δ 0.91 and δ 1.10, the correlated NOE cross peaks for each steric position hydrogen (see FIG. 3) H-9 and H-14 were in the axial and α positions, respectively, H Δ 1.55 of -12, δ 1.67 of H-15, δ 1.56 of H-16 and δ 0.97 of H-7 should be in the α position due to the presented NOE cross peak. For NOE experiment results, there is a possibility that there is a NOE correlation between δ = 1.5, H-12, H-15, H-16 and H-8 at δ = 2.05, 1.25, 2.03 and 1.87 compared to H-18 at δ = 1.78. Higher. This possibility was practically confirmed in FIG. 3. The NOE correlation is schematically illustrated in FIG. 4.

Aglycone for corticosteroid glucoside identified each proton and carbon. Because of the two hydroxyl groups of corticosterone, the linkage position must be determined among the 11β- and 21-hydroxyl groups. When glucose is linked to the 11β-hydroxyl group, chemical shifts to H-11 and H-21 should show different values for glucuronide and glucoside in Table 2. When the chemical shifts of glucuronide and glucose are compared to those in Table 2, the results for glucoside show the same linking sites for glucose in corticosterone. In addition, the location of the glycosidic linkage to the steroid glucoside was confirmed by a correlation between the glycosides in the ¹ H- ¹³ C HMBC spectrum, and between the signals at δ 4.15 (H-1′) and δ 73.44 (C-21). In the correlation, substitution of the aglycone by the β-glucoside moiety at the C-21 position was confirmed. In addition, the above-mentioned connection positions were confirmed through correlation to the HMBC spectrum between the signals at δ 4.20 and 4.33 (two hydrogens of H-21) and δ 102.14 (C-1').

The abnormality of the glucose portion of corticosterone 21-glucoside was confirmed through 1D ¹ H-NMR and 2D COSY experiments after assignment to the anionic proton and H-2'. The relatively large coupling constant between H-1' and H-2' values for the glucose portion (7.8 Hz, Figure 4 and Table 2) indicated the β-form of the D-glucose portion (see Table 2).

(2) Biological activity of corticosteroid 21-glucoside

Neurodegenerative disorder is closely related to abnormal neuronal cell death, and neurons in the brain are more sensitive to oxidative stress because they have a greater dependence on oxidative phosphorylation for energy than other cells. As a stress hormone, corticosteroids cause nerve damage characterized by DNA damage and cell death. In the present invention, cell death by hydrogen peroxide in SH-SY5Y cells and neuroprotective activity of corticosteroid and corticosteroid 21-glucoside against reactive oxygen species (ROS) were investigated. In addition, the difference in cytotoxicity between corticosteroid and corticosteroid 21-glucoside was first confirmed.

As shown in Figure 5a, treatment with corticosteroid (5, 7.5, 10, 25 and 50 μM) compared to the untreated control (100%), cell viability was 114.8%, 98.8%, 81.1%, 41.0%, respectively. And 20.8%. However, the cell viability by corticosteroid 21-glucoside (5, 7.5, 10, 25, 50 μM) was 109.6%, 121.5%, 121.9%, 102.4%, 82.5%, respectively, indicating that cytotoxicity of corticosteroid It was confirmed that the toxicity was not strong compared to.

To confirm the neuroprotective activity of corticosterone and corticosteroid 21-glucoside, cell protection tests were performed on cells with or without 100 μM hydrogen peroxide. Cytotoxicity (49.9% survival rate compared to untreated control) was observed when 100 μM hydrogen peroxide was treated with SH-SY5Y cells. The results of pretreatment with corticosteroid 21-glucoside (1, 2.5, 5 and 10 μM) were pretreated with corticosteroid (1, 2.5, 5 and 10 μM) (64.9%, 63.2%, 54.5% and 45.6) %) compared to 88.4%, 94.8%, 102.9% and 103.2% of cell viability, respectively (see Figure 5b). Corticosterone 21-glucoside was superior to corticosterone in that it dose-dependently inhibits hydrogen peroxide-mediated cytotoxicity (see Figure 5B).

Neurodegenerative diseases associated with disorders of mitochondrial metabolism increase reactive oxygen species (ROS) production and mitochondrial dysfunction. It is believed that the production of mitochondrial superoxides plays an important role in the reduction of cellular function. Therefore, ROS generation analysis was performed to investigate the mitochondrial peroxide scavenging ability against corticosteroids and corticosteroid 21-glucosides. When treated with hydrogen peroxide in SH-SY5Y cells, ROS production was increased to 430.7% compared to the untreated control group (100%) (see FIG. 5C ).

As a result of pretreatment with corticosteroid 21-glucoside (1, 2.5, 5 and 10 μM), the level of mitochondrial peroxide was pretreated with corticosterone (1, 2.5, 5 and 10 μM) (271.5%, 356.6%). , 378.6% and 406.1%), respectively, 121.6%, 120.6%, 102.4% and 93.8% (see Fig. 5c). Similar to the results of the neuroprotective activity test, corticosteroid 21-glucoside showed significantly greater ROS production lowering activity by down-regulating mitochondrial peroxide levels dose-dependently than corticosterone (see Figure 5c).

(3) Conclusion

The glucosylation structure and location of corticosteroid 21-glucoside was confirmed through ¹ H, ¹³ C, COSY, ROESY, HSQC-DEPT and HMQC NMR experiments and TOF-ESI HRMS data. Except for the 11β-hydroxyl group, the effect on glucosylation at the 21-hydroxyl group was clearly shown. This was almost consistent with the results of previous NMR studies, except for the difference between glucuronic acid and glucose. However, confirmation in the present invention for the hydrogen α-β structure on C-12 of the aglycone moiety was reversely verified. From the perspective of stereochemistry, structural determination in stereochemistry using NOE is important. However, this difference arises because the stereoscopic structure was not determined by NOE in the conventional reports. No detailed NMR data for corticosteroids have been reported so far, but this NMR analysis is considered to complement the steroid structural data.

It was confirmed that the cytotoxicity of corticotron 21-glucoside was not at a significant level compared to corticosteroid (114.8-20.8% compared to the control) at different concentrations (5-50 μM) (109.6-82.5% compared to the untreated control group). . Pretreatment of corticosteroid 21-glucoside (1-10 μM) compared to corticosteroid (64.9-45.6% compared to untreated control for cell viability; 271.5-406.1% for untreated control for ROS levels) To restore the cell viability (88.4-103.2% compared to the untreated control group), the intracellular ROS level (121.6-93.8% compared to the untreated control group, the case where the hydrogen peroxide treated group was 430.7%) was reduced. These results indicated that corticotron 21-glucoside lowered cytotoxicity compared to corticoterone. The neuroprotective effect observed in corticosterone 21-glucoside suggests that this compound may be useful in achieving selective neuroprotective action.

(4) Summary

Glucosylation of the 21-hydroxyl group of glucocorticoids has been shown to change the solubility from hydrophobic to hydrophilic and exhibit the same behavior, such as glucocorticoid glucuronide, a moving object in vivo. Therefore, while maintaining the 11β-hydroxyl group, glucosylation to the 21-hydroxyl group is particularly important, and the glucosylation of corticosterone is high resolution mass spectrometry and 1D ( ¹ H and ¹³ C) NMR and 2D (COSY, ROESY , HSQC-DEPT, HMBC) was confirmed by NMR. In addition, differences in bioactivity between corticosteroid and corticosteroid 21-glucoside were confirmed in vitro. Corticosterone 21-glucoside was found to have a greater neuroprotective effect on cell death and ROS induced by hydrogen peroxide compared to corticosteroid. These results indicate for the first time that bioconversion of corticosteroids through domain-selective glucosylation of new compounds may suggest structural possibilities for developing new cranial neuroprotective agents.

<110> Industry-University Cooperation Foundation Sunmoon University <120> Method for bioconversion of corticosterone by glucosyltransferase and corticosterone glucoside produced thereby <130> P190554 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1182 <212> DNA <213> Artificial Sequence <220> <223> UDP-glucosyltransferase-1 <400> 1 atgaaacagc atcatatcac aattattaat atccctgcgt atggccatgt aaaccccaca 60 cttgcattag tggagaagct ttgtgaaaaa ggacatcggg taacatacgc tacgacagag 120 gaatttgccc cggcggttca acaagcggga gcccgcccac ttttatatga aacaaccatt 180 tcggtagatc ctcagcaaat taaagaattg atggaaaaaa acgaaacgcc gcttatgttt 240 ttgaaagaat cgcttggaat cttgccacag ctagaagaat tgtataaaga tgaccagcct 300 gaccttatcg tctatgattt tatcgcactg gcgggaaaac tgttagccga taagcttcaa 360 gtgccggctg tcagactctg ttcgtcatac gctcaaaatg aatcctttca gatgggaact 420 gaggagatgc tccaacaagt caaagaagca gaggctgaat ttcaggcctt tctggagcaa 480 gaaaacttac cggctgtctc atttgaacaa ctggctatac cagaagcgtt taatattgtc 540 tttatgccgc gctcttttca gattaagaac gaaaccttcg acgaccgctt ctgttttatc 600 ggtccgtctc ttggaaaacg aagcgaacaa gagcgcctgc tgttaaaaga gaaaggcgat 660 cgcccgctta tgctgatttc actgggaacg gccttcaacg cctggccgga attctatcaa 720 atgtgtatgg aggcttttgg ggggtctgaa tggcatgtca ttatgtcggt aggaaaatca 780 attgagcctg acagtcttgg tgatattcct gacaacttca cggtccgcca gcgcgttccg 840 cagctggacg tattgacgga agctgatgta tttatttctc acggaggaat gaacagcacg 900 atggaagcga tgaacgaggg agtgccgctt gtagtgattc cgcaaatgca tgaacaggaa 960 ctcacagctc agcgtgttga agaattaggg ctgggaatct acctgccgaa agaagaggtc 1020 agtgtacctc gtttgcaaca ggcgatcggt aatatattct ctgacaaaga gattcacagc 1080 cgcgtgaaag atatgcagaa agatgtaaag gaagcaggcg gagcagaaca gggcgcggcg 1140 gagatcgaag cgtttatgaa aaagtctgtc gttccgcaat aa 1182 <210> 2 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> UDP-glucosyltransferase-1 <400> 2 Met Lys Gln His His Ile Thr Ile Ile Asn Ile Pro Ala Tyr Gly His 1 5 10 15 Val Asn Pro Thr Leu Ala Leu Val Glu Lys Leu Cys Glu Lys Gly His 20 25 30 Arg Val Thr Tyr Ala Thr Thr Glu Glu Phe Ala Pro Ala Val Gln Gln 35 40 45 Ala Gly Ala Arg Pro Leu Leu Tyr Glu Thr Thr Ile Ser Val Asp Pro 50 55 60 Gln Gln Ile Lys Glu Leu Met Glu Lys Asn Glu Thr Pro Leu Met Phe 65 70 75 80 Leu Lys Glu Ser Leu Gly Ile Leu Pro Gln Leu Glu Glu Leu Tyr Lys 85 90 95 Asp Asp Gln Pro Asp Leu Ile Val Tyr Asp Phe Ile Ala Leu Ala Gly 100 105 110 Lys Leu Leu Ala Asp Lys Leu Gln Val Pro Ala Val Arg Leu Cys Ser 115 120 125 Ser Tyr Ala Gln Asn Glu Ser Phe Gln Met Gly Thr Glu Glu Met Leu 130 135 140 Gln Gln Val Lys Glu Ala Glu Ala Glu Phe Gln Ala Phe Leu Glu Gln 145 150 155 160 Glu Asn Leu Pro Ala Val Ser Phe Glu Gln Leu Ala Ile Pro Glu Ala 165 170 175 Phe Asn Ile Val Phe Met Pro Arg Ser Phe Gln Ile Lys Asn Glu Thr 180 185 190 Phe Asp Asp Arg Phe Cys Phe Ile Gly Pro Ser Leu Gly Lys Arg Ser 195 200 205 Glu Gln Glu Arg Leu Leu Leu Lys Glu Lys Gly Asp Arg Pro Leu Met 210 215 220 Leu Ile Ser Leu Gly Thr Ala Phe Asn Ala Trp Pro Glu Phe Tyr Gln 225 230 235 240 Met Cys Met Glu Ala Phe Gly Gly Ser Glu Trp His Val Ile Met Ser 245 250 255 Val Gly Lys Ser Ile Glu Pro Asp Ser Leu Gly Asp Ile Pro Asp Asn 260 265 270 Phe Thr Val Arg Gln Arg Val Pro Gln Leu Asp Val Leu Thr Glu Ala 275 280 285 Asp Val Phe Ile Ser His Gly Gly Met Asn Ser Thr Met Glu Ala Met 290 295 300 Asn Glu Gly Val Pro Leu Val Val Ile Pro Gln Met His Glu Gln Glu 305 310 315 320 Leu Thr Ala Gln Arg Val Glu Glu Leu Gly Leu Gly Ile Tyr Leu Pro 325 330 335 Lys Glu Glu Val Ser Val Pro Arg Leu Gln Gln Ala Ile Gly Asn Ile 340 345 350 Phe Ser Asp Lys Glu Ile His Ser Arg Val Lys Asp Met Gln Lys Asp 355 360 365 Val Lys Glu Ala Gly Gly Ala Glu Gln Gly Ala Ala Glu Ile Glu Ala 370 375 380 Phe Met Lys Lys Ser Val Val Pro Gln 385 390

Claims (6)

delete delete delete A method for bioconversion of corticosteroid comprising the step of glucosylating the 21-hydroxyl group of corticosteroid using UDP-glucosyltransferase comprising the amino acid sequence of SEQ ID NO: 2. Corticosterone 21-glucoside obtained by the bioconversion method of corticosteroid of claim 4. A pharmaceutical composition for the protection of brain neurons comprising the corticosteroid 21-glucoside of claim 5.

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