WO2011115435A2 - Thiol derivative of biotin, and analysis method of substrate specificity of serine/threonine kinase using same - Google Patents

Abstract

The present invention relates to a thiol derivative of biotin, and an analysis method of the substrate specificity of a serine/threonine kinase using the same. It is possible to screen the substrate specificity of a serine/threonine kinase by the present invention.

Description

Substrate specificity analysis method of thiol derivative of biotin and serine / threonine kinase using the same

The present invention relates to novel thiol derivatives of biotin and methods of substrate specificity analysis of serine / threonine kinases using the same. The present invention allows screening for serine / threonine kinase substrate specificity.

Kinases are enzymes that phosphorylate serine / threonine and tyrosine of proteins or peptides using adenosine triphosphate (ATP). Phosphorylation by kinases plays an important role in cellular signal transduction and vital vital mechanisms. Overexpression of kinases is involved in diseases such as cancer, and clarifying the substrate specificity of kinases plays a major role in the treatment of diseases and in biochemical and medical research.

In particular, although most of the phosphorylated peptides of the living body are serine / threonine peptides, although there are many kinds of kinases compared to tyrosine, antibodies that act on phosphorylated serine / threonine have a disadvantage in that they are less specific than antibodies that recognize phosphorylated tyrosine peptides. In fact, it is known that it is difficult to distinguish the kinase substrate.

As a method for identifying a substrate of a kinase, a method using a chelate compound of zinc ions or a method using a radioisotope has been used. However, there is a problem in that the method using zinc ions is affected by carboxyl groups or other negatively charged functional groups. In addition, the method of using ATP labeled phosphorus is cumbersome and can only be detected through radioactive equipment, so there is a limit in analyzing a large amount of samples using combinatorial chemistry and the like.

Phosphorylated serine / threonine is known to make an alkene compound by causing a β-elemination reaction under strong base conditions, and a method of analyzing serine / threonine phosphorylated peptide using the same has been tried. Among them, in order to analyze a small amount of phosphorylated peptide, ethanedithiol was introduced into the alkene group generated after the β-removal reaction using Michael addition, followed by biotin succimide. A study of mass spectrometry by reaction with derivatives has been reported (2001), and the results of selective separation of phosphorylated peptides using avidin and others (2001) have also been published (2001).

In addition, a study has been reported in which a thiol derivative of a compound exhibiting fluorescence is synthesized to react thiol with an alkene group generated after β-removal reaction of the phosphorylated peptide to fluoresce the polymer support into which the phosphorylated peptide is introduced (Shoji Akita, Organic Letters). 7 (25), 5565-5568, 2005).

In the case of fluorescence analysis can be distinguished only by microscopic analysis with a laser of the appropriate wavelength, in some cases the concentration of the fluorescent compound can be canceled effect.

The basic object of the present invention is to provide a compound of formula (I).

Formula (I)

R in formula (I) is an inert group such as H, CH ₃ , CH ₂ CH ₃ , CONH ₂ or SH; n is an integer from 1 to 30.

Still another object of the present invention is to prepare a solid support resin having a link amide introduced therein by (i) reacting a solid support resin having an amino group introduced therein with a Fmoc-link amide; (ii) introducing cysteine into the link amide; (iii) introducing a polyethylene glycol (-(OCH ₂ CH ₂ ) _n- ) derivative into the cysteine; (iv) introducing biotin into the polyethylene glycol derivative; And (v) separating the compound of formula (I) from the product of step (iv).

It is still another object of the present invention to (i) phosphorylating serine or threonine in the ladder peptide sequence by reacting a kinase with a solid support resin having a ladder peptide sequence; (ii) removing the side chain phosphate group of the serine or threonine residue of the phosphorylated peptide through a β-elimination reaction using a strong base, followed by addition of the compound of formula (I) via Michael addition reaction; (iii) binding any one selected from the group consisting of avidin, streptavidin, and neutravidin to which alkaline phosphatase or horsered peroxidase is bound, to a compound of formula (I) in step (ii) above; And (iv) analyzing the sequence of the peptide bound to the surface of the support resin by selecting a color-supported support resin by adding BCIP / NBT reagent after the reaction of step (iii). It is to provide a substrate specificity analysis method.

Still another object of the present invention is to (i) phosphorylate serine or threonine in the peptide or protein by reacting a peptide or protein introduced into the solid support resin or substrate with a kinase; (ii) removing the side chain phosphate group of the phosphorylated serine or threonine residue through a β-elimination reaction using a strong base, and then adding the compound of formula (I) through a Michael addition reaction; (iii) binding any one selected from the group consisting of avidin, streptavidin and neutravidin, to which a fluorescent dye is bound, to the compound of formula (I) of step (ii); And (iv) to provide a method for analyzing the substrate specificity of the serine / threonine kinase comprising the step of selecting a fluorescent peptide or protein through the fluorescence analysis after the reaction of step (iii).

The basic object of the present invention is to provide a compound of formula (I).

Formula (I)

R in formula (I) is an inert group such as H, CH ₃ , CH ₂ CH ₃ , CONH ₂ or SH; n is an integer from 1 to 30.

Still another object of the present invention is to prepare a solid support resin having a link amide introduced therein by (i) reacting a solid support resin having an amino group introduced therein with a Fmoc-link amide; (ii) introducing cysteine into the link amide; (iii) introducing a polyethylene glycol (-(OCH ₂ CH ₂ ) _n- ) derivative into the cysteine; (iv) introducing biotin into the polyethylene glycol derivative; And (v) separating the compound of formula (I) from the product of step (iv).

It is still another object of the present invention to (i) phosphorylating serine or threonine in the ladder peptide sequence by reacting a kinase with a solid support resin having a ladder peptide sequence; (ii) removing the side chain phosphate group of the serine or threonine residue of the phosphorylated peptide through a β-elimination reaction using a strong base, followed by addition of the compound of formula (I) via Michael addition reaction; (iii) binding any one selected from the group consisting of avidin, streptavidin, and neutravidin to which alkaline phosphatase or horsered peroxidase is bound, to a compound of formula (I) in step (ii) above; And (iv) analyzing the sequence of the peptide bound to the surface of the support resin by selecting a color-supported support resin by adding BCIP / NBT reagent after the reaction of step (iii). It is to provide a substrate specificity analysis method.

Still another object of the present invention is to (i) phosphorylate serine or threonine in the peptide or protein by reacting a peptide or protein introduced into the solid support resin or substrate with a kinase; (ii) removing the side chain phosphate group of the phosphorylated serine or threonine residue through a β-elimination reaction using a strong base, and then adding the compound of formula (I) through a Michael addition reaction; (iii) binding any one selected from the group consisting of avidin, streptavidin and neutravidin, to which a fluorescent dye is bound, to the compound of formula (I) of step (ii); And (iv) to provide a method for analyzing the substrate specificity of the serine / threonine kinase comprising the step of selecting a fluorescent peptide or protein through the fluorescence analysis after the reaction of step (iii).

The thiol derivatives of biotin of the invention can be used to detect serine / threonine phosphorylated peptides. In particular, serine / threonine kinase substrate specificity can be screened using a ladder library of peptides prepared using N-acetyl amino acids.

1 is one embodiment of a method for synthesizing a derivative of the biotin thiol of the present invention (formula (I)).

Figure 2 is one embodiment of preparing the ladder-type peptide of the present invention.

Figure 3 shows the results of the MALDI-TOF analysis for the ladder-form ketide prepared in Example 2 of the present invention.

Figure 4 is a schematic diagram of the method for analyzing the substrate specificity of the serine / threonine kinase of the present invention.

Figure 5 is one embodiment of a method for preparing a ladder library peptide library of the present invention.

Figure 6 is the result of detecting the phosphorylation of serine / threonine kinase according to Example 4 of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following description of the embodiments is only intended to specifically describe the specific embodiments of the present invention, it is not intended to limit or limit the scope of the present invention to the contents described therein.

Example 1 Biotin-Thiol Derivative Synthesis

In order to introduce acid-decomposable linker (Fmoc-Rink-amide) and cysteine, tetra ethylene glycol, biotin using solid phase synthesis, polystyrene beads (0.6 mmol / g, 1 g) coated with aminomethyl groups were used as a polymer support. First, Fmoc-link-amide (2 equivalents) was added to HBTU (O-Benzotriazole-N, N, N ', N'-tetramethyl-uronium-hexafluoro-phosphate (2 equivalents), HOBt (N-Hydroxybenzotriazol , 2 equivalents), dissolved in DIEA (N, N-diisopropyl-ethyl-amine, 4 equivalents) and NMP (N-methyl-2-pyrrolidone, 10 mL), and then reacted with the amine functional group of the polystyrene beads at room temperature for 2 hours. Let's do it. The filtrate was filtered off and washed with NMP, DCM and MeOH to obtain a support resin with Fmoc-PLL bound. The Fmoc protecting group was removed by reacting with a 20% piperidine / NMP solution for 3 minutes and 17 minutes, respectively, and then washed as described above.

In this way, Fmoc-Cys (Trt) is introduced after the Rink-amide. Meanwhile, Fmoc-TEG-SA was synthesized using diamino TEG, Fmoc-OSu, and succinic anhydride (SA), followed by cysteine. After reacting for 2 hours at room temperature using an ester form of biotin activated as an N-hydroxysuccinimide group (-NHS), tetraethylene glycol was introduced followed by biotin, followed by K reagent (Reagent K; TFA: phenol: H20 Thiol derivatives of biotin were cut off using: thioanisole: 1,2-ethanedithiol = 82.5: 5: 5: 5: 2.5) (see FIG. 1). The thiol derivatives of the synthesized biotin were confirmed by mass spectrometry.

Example 2. Serine / Threonine Peptide Preparation

To prepare a peptide for screening serine / threonine kinase, photodegradable linkers and spacers were introduced into core-shell polystyrene beads, and then peptides were synthesized. High core ^TM (HiCore ^TM) resin support (0.3 mmol / g, 100 mg ) was swollen after the using the NMP (N-methyl-2- pyrrolidone) in 5 mL, Fmoc- photodegradable linker (Fmoc-photolabile linker ( Fmoc-PLL), Fmoc-4- [4- (1-aminoethyl) -2-methoxy-5-nitrophenoxy] butyric acid, 2 equivalents), BOP (benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluoro-phosphate, 2 equivalents ), HOBt (2 equiv) and DIEA (4 equiv) were dissolved in 3 mL of NMP and added. After reacting at room temperature for 2 hours, the filtrate was filtered off and washed with NMP, DCM and MeOH to obtain a support resin having Fmoc-PLL bound thereto. Termination of the reaction was carried out using Kaiser ninhydrin test (Kaiser ninhydrin). Fmoc protecting groups were removed by reacting with 20% piperidine / NMP solution for 5 minutes and 17 minutes, respectively, and then washed in the manner described above. To introduce spacers, Fmoc-ε-ACA (Fmoc-ε-aminocaproic acid; 2 equivalents) and Fmoc-β-Ala (2 equivalents) were added to BLL (2 equivalents) and HOBt (2 equivalents) to the support resin to which the PLL was bound. And repeating the alternating reaction two times using DIEA (4 equivalents) to prepare a resin beads into which photodegradable linkers and spacers were introduced (see FIG. 2).

In order to confirm whether the prepared thiol derivative of biotin responds to the serine / threonine position, a kemptide known as a substrate of protein kianase A (PKA) was synthesized. In addition, for comparative experiments, phosphorylated chemtide (kemtide, LRRA (p) SLG) was synthesized using phosphorylated serine.

After serine / threonine kinase screening, peptides were synthesized as ladder peptides for sequencing via MALDI-TOF. N-acetyl glycine was used to prepare a ladder-type peptide library in a manner that controls the degree of capping of the amino acid at the bead end (see FIG. 5).

To 100 mg of the prepared resin beads into which the photodegradable linker and the spacer were introduced, Fmoc-amino acid (4 equivalents), N-acetyl glycine (0.1 equivalents), BOP (4.1 equivalents), HOBt (4.1 equivalents) and DIEA (8.2 equivalents) were added. It was dissolved in 3 mL of NMP and added. After reacting for 2 hours at room temperature, the filtrate was filtered off and washed with NMP, DCM, MeOH. The Fmoc protecting group was removed by reacting with 20% piperidine / NMP solution for 5 minutes and 17 minutes respectively, then washed in the same manner and dried in a vacuum oven for 12 hours. The synthesized ladder peptide was confirmed by mass spectrometry (FIG. 3).

Example 3. Detection of Phosphorylated Serine / Threonine Peptides

In order to confirm whether the synthesized biotin thiol derivative can distinguish the phosphorylated peptide, the synthesized chemtide resin beads were used as a control group, and the experiments were carried out using the resin beads to which chemtide containing phosphorylated serine was introduced as an experimental group. .

100 μL of a saturated barium hydroxide solution or a mixed solution of 1N NaOH, DMSO, and isopropyl alcohol in a ratio of 3: 5.5: 1.5 was added to the experimental group and the control group, respectively, and stirred at 50 ° C. to 60 ° C. for 4 to 8 hours. Β-removal reaction occurred. After the reaction was washed with distilled water.

The synthesized thiol derivative of biotin was dissolved in a solution adjusted to 0.1 N NaOH: DMSO: isopropyl alcohol = 3: 5.5: 1.5 to 3 μM, and then 100 μL was added to each reaction vessel at a temperature of 20 ° C. to 30 ° C. By reacting for 1 to 4 hours, alkenes and thiols were reacted to introduce biotin derivatives. After the reaction was washed with distilled water, DMSO, isopropyl alcohol.

100 μL of a 1 U / mL streptavidin solution incorporating alkaline phosphatase was placed in the reaction mixture vessel into which the biotin was introduced, followed by reaction at a temperature of 20 ° C. to 25 ° C. for 15 minutes, followed by washing with distilled water.

In order to detect phosphorylated beads using the reaction of alkaline phosphatase, the color change of alkaline phosphatase was 0.25 μg / 0.1 mL of BCIP (5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt). ) And 0.5 μg / 0.1 mL of nitro-blue tetrazolium chloride (NBT) in Tris buffer (pH 9.5), reacted with the beads at 37 ° C for 15 minutes to 1 hour, and then washed with 0.1 N HCl aqueous solution and distilled water. The reaction was terminated. The difference between the control group and the reaction group was confirmed by color reaction. In the case of the reaction group, the color changed to dark brown after 10 to 20 minutes, and changed to purple as time passed (see FIG. 4).

Example 4. Phosphorylated Serine / Threonine Kinase Reaction and Detection

5 mg each of the chemtide-introduced resin beads synthesized in Example 3 were placed in two 1.5 mL reaction vessels, and ATP was added to 40 mM Tris-HCl (pH 7.4) buffer containing 20 mM magnesium acetate. The final concentration is 1 mM. The total volume should be 100 μL. PKA of 1 to 5 kU was added to the reaction vessel of the experimental group, and only the resin beads into which the ATP and the peptide were introduced were added to the reaction vessel of the control group, followed by stirring at 37 ° C. for 30 minutes. After the reaction was washed with 0.5% phosphoric acid aqueous solution and distilled water.

In order to distinguish between the experimental group which rapidly phosphorylated due to PKA and the control group which was phosphorylated without PKA, as described in Example 3, β- with a saturated aqueous solution of barium hydroxide or a mixed solution of 1N NaOH, DMSO, and isopropyl alcohol. After the removal reaction was performed, a biotin thiol derivative was introduced into the alkene produced. Thereafter, alkaline phosphatase bound streptavidin was bound and color change was observed through BCIP / NBT reaction. In the experimental group that reacted with PKA, it was observed that after 15 minutes compared with the control group to have a dark brown color (see FIG. 6).

Example 5. Phosphorylated Serine / Threonine Kinase Substrate Screening

Resin beads incorporating the peptide library synthesized in Example 2 were placed in two 1.5 mL reaction vessels each at 20 mg, and ATP was added to 40 mM Tris-HCl (pH 7.4) buffer containing 20 mM magnesium acetate. The final concentration was brought to 1 mM. The total volume was brought to 400 μL. 1 to 5 kU of PKA was added to the reaction vessel of the experimental group, and only the resin beads into which the ATP and the peptide were introduced were added to the reaction vessel of the control group, and reacted with stirring at 37 ° C. for 30 minutes. After the reaction was washed with 0.5% phosphoric acid aqueous solution and distilled water.

In order to detect phosphorylated peptide beads, a β-removal reaction was performed for 1 hour to 4 hours with a saturated aqueous solution of barium hydroxide or a mixed solution of 1N NaOH, DMSO, and isopropyl alcohol. Introduced. Thereafter, streptavidin bound to alkaline phosphatase was bound, and color change was observed through BCIP / NBT reaction. After 1 hour the color of the solution gradually became brown or light purple, the color of the beads became totally dark, and 200 μL of 1 N hydrochloric acid solution was added to terminate the reaction. After washing with distilled water and observing under a microscope, dark brown or dark reddish brown beads were selected. Peptides were separated from the beads by irradiation with ultraviolet rays, and then sequenced by mass spectrometry. As a result of peptide sequence analysis, positively charged amino acids (arginine, lysine, histidine) in the N-terminal direction were mainly detected and confirmed to be consistent with the literature theory.

Claims (13)

A compound of formula (I)

Formula (I) R in formula (I) is H, CH ₃ , CH ₂ CH ₃ , CONH ₂ or SH; n is an integer from 1 to 30. (i) reacting the Fmoc-link amide with the solid-phase support resin into which the amino group is introduced to prepare a solid-phase support resin into which the link amide is introduced; (ii) introducing cysteine into the link amide; (iii) introducing a polyethylene glycol (-(OCH ₂ CH ₂ ) _n- ) derivative into the cysteine; (iv) introducing biotin into the polyethylene glycol derivative; And (v) separating the compound of formula (I) from the product of step (iv):

Formula (I) R in formula (I) is H, CH ₃ , CH ₂ CH ₃ , CONH ₂ or SH; n is an integer from 1 to 30. The method for preparing a compound of formula (I) according to claim 2, wherein the solid support resin is selected from the group consisting of polystyrene resins, epoxy resins and silica resins. The method for preparing a compound of formula (I) according to claim 2, wherein the thiol group of the cysteine of step (ii) is protected with a trityl group or tert-butoxide. The method according to claim 2, wherein Fmoc-NHC ₃ H ₆ (OCH ₂ CH ₂ ) _n CH ₂ NHCOC ₂ H ₄ -COOH prepared using polyethylene glycol or zeph amine whose end is substituted with an amine group in step (iii) A process for preparing a compound of formula (I) characterized by the introduction of-(OCH ₂ CH ₂ ) _n- . The method of preparing a compound of formula (I) according to claim 2, wherein the biotin of step (iv) is activated with a -NHS group. (i) reacting a solid phase support resin having a ladder peptide sequence with a kinase to phosphorylate serine or threonine in the ladder peptide sequence; (ii) removing the side chain phosphate group of the serine or threonine residue of the phosphorylated peptide through a β-elimination reaction using a strong base, followed by addition of a compound of formula (I) via a Michael addition reaction; (iii) binding any one selected from the group consisting of avidin, streptavidin, and neutravidin to which alkaline phosphatase or horsered peroxidase is bound, to a compound of formula (I) in step (ii) above; And (iv) adding a BCIP / NBT reagent after the reaction of step (iii) to select a color-changed support resin and analyzing the sequence of the peptide bound to the support resin surface, the substrate of serine / threonine kinase Specificity Assay:

Formula (I) R in formula (I) is H, CH ₃ , CH ₂ CH ₃ , CONH ₂ or SH; n is an integer from 1 to 30. 8. The method of claim 7, wherein the solid support resin is selected from the group consisting of polystyrene resins, epoxy resins and silica resins. The method of claim 7, wherein the strong base of step (ii) is selected from the group consisting of NaOH, KOH, Ca (OH) ₂ , and Ba (OH) ₂ . (i) phosphorylating serine or threonine in the peptide or protein by reacting a peptide or protein introduced into a solid support resin or substrate with a kinase; (ii) removing the side chain phosphate group of the phosphorylated serine or threonine residue through a β-removal reaction using a strong base, and then adding a compound of formula (I) through a Michael addition reaction; (iii) binding any one selected from the group consisting of avidin, streptavidin and neutravidin, to which a fluorescent dye is bound, to the compound of formula (I) of step (ii); And (iv) a method of analyzing the substrate specificity of serine / threonine kinase, comprising selecting a fluorescent peptide or protein by fluorescence analysis after the reaction of step (iii):

Formula (I) R in formula (I) is H, CH ₃ , CH ₂ CH ₃ , CONH ₂ or SH; n is an integer from 1 to 30. 12. The method of claim 10, wherein the solid support resin is selected from the group consisting of polystyrene resins, epoxy resins and silica resins. The method of claim 10, wherein the substrate is selected from the group consisting of glass, silicone and polyacrylic. The method of claim 10, wherein the strong base of step (ii) is selected from the group consisting of NaOH, KOH, Ca (OH) ₂ , and Ba (OH) ₂ .

Images