KR20090097704A - Flourescent peptide probe for selective detection copper or zinc ion, and preparation method thereof - Google Patents

Abstract

A fluorescence peptide sensor for selectively detecting copper or zinc ion is provided to detect copper ion and zinc ion in aqueous environment and selectively detect the ions in a bio sample. A fluorescence peptide sensor is a peptide sensor which is able to detect copper or zinc ion in which fluorescence probing material and peptide are covalent. The fluorescence probing material is 5-dimethylamino-1-naphthalenesulfonyl group. A method for manufacturing the fluorescence peptide sensor comprises: a step of sequencially reacting amino acid and resin with amino terminal; a step of reacting the complex of resin and peptide with 5-dimethylamino-1-naphthalene sulfonyl chloride; and a step of reacting reacted material with trifluoroacetic acid.

Description

Fluorescent Peptide probe for selective detection copper or zinc ion, and preparation method

The present invention relates to a fluorescent peptide sensor for selectively detecting copper and / or zinc ions, a manufacturing method thereof, and a detection apparatus using the same.

The design and research of new sensors for key substances and ions in vivo has been actively conducted. Recent understanding and research on supramolecule chemistry has shown great potential for the design of host compounds that can selectively bind ions or various other types of guest compounds. In addition, it has been of great help in the development of fluorescent chemosensors, which can more easily observe selective binding with guest compounds using fluorescence changes.

Fluorescence is a photochemical phenomenon that occurs when a photon with a specific light wave (excitation wavelength) collides with an indicator molecule and the electron is excited by a high energy level as a result of the collision. Among the various analytical methods, fluorescence has the advantage of being able to observe signals even at 10 ^-9 M concentrations due to their excellent sensitivity. Recently, research on fluorescence chemical sensors for cations, anions and neutral organic molecules has been published using these properties.

Recently, researches to detect various kinds of potential metals by synthesizing various fluorescent peptide sensors have been actively conducted. Fluorescent peptide sensors are of great interest in several aspects as described below. Fluorescent peptide sensors comprising natural amino acids can be synthesized relatively easily in a short time using a solid phase synthesis method, and by using a combination of various amino acids, it is possible to control the selectivity and sensitivity for the desired metal ion. In addition, the melting point in an aqueous solution similar to physiological conditions can be said to be distinguished from the fluorescence chemical sensor. Furthermore, the synthesized fluorescent peptide sensor can be easily coupled to a water-insoluble solid phase device, which allows for further applications in the future. However, since the synthesized fluorescent peptide sensor is chemically very water soluble, it is more difficult to penetrate into the cell, but if additional function is given through the amide bond present in the peptide, the fluorescent peptide sensor is penetrated into the cell for further research. I can do it.

High selective detection of metal ions in physiological conditions is of great importance in the development of fluorescent chemical sensors for environmental and biological applications [ Fluorescent Chemosensors for Ion and Molecular Recognition , Czarnik, AW Ed., American Chemical Society: Washington, DC, 1993 ]. Copper ions and zinc ions are one of the most abundant transition metal ions in humans and play essential roles in gene expression, enzyme activity regulation, and neurotransmitter processes [Falchuk, KH Mol. Cell. Biochem . 1998 , 188 , 41].

As an example, zinc ions are known to affect amyloid plaque formation in the early stages of Alzheimer's disease [Bush, AI; Pattingel, WH; Multhaup, G .; Paradis, MD; Vonsattel, JP; Gusella, JF; Beyreuther, K .; Masters, CL; Tanzi, RE Science 1994 , 265 , 1464. The concentration of zinc ions present in cells ranges from nanomoles (10 ^-6 M) to millimoles (10 ^-3 M) [Lippard, SJ; Berg, JM Principles of Bioinorganic Chemistry University Science Books: Mill Valley, CA, 1994 , pp 10, 14, 78 ?? 183]. Therefore, there is a need for peptide sensors optimized for a wide range. Most peptide sensors for detecting zinc ions have been synthesized based on zinc finger domains [Walkup, GK; Imperiali, B. J. Am. Chem. Soc. 1996 , 118 , 3053]. Most of these peptide sensors consist of 24 amino acids, some of which include non-natural amino acids [Shults, MD; Pearce, DA; Imperiali, B. J. Am. Chem. Soc . 2003 , 125 , 10591] Most of these fluorescent peptide sensors have a weak detection ability at low concentrations (usually in the millimolar limit of detection), interference with other metals (Mg ²⁺ , Cd ²⁺ ), pH-dependent activity, synthesis process Difficulties, due to denaturation of the synthesized peptide (easy oxidation in the air when two or more cysteine residues are contained in a peptide) and poor solubility in physiological aqueous solution [Ajayaghosh, A .; Carol, P .; Sreejith, S. J. Am. Chem. Soc . 2004 , 127 , 14962, and reference therein].

Copper ions, another metal ions, are not only involved in promoting the reduction reactions occurring in vivo, but also play an important role in inhibiting active radicals, which are potentially dangerous substances in cells [Miranda S., Opazo. C., Larrondo LF, Muozoz FJ, Ruiz F., Leighton F. and Inestrosa NC Prog. Neurobiol. 62 , 633-648. Thus, many organisms have several mechanisms for properly controlling the concentration of copper ions in cells.

For this reason, observing the contents of copper and zinc ions in the body can be an important help in understanding the physiological phenomena occurring in vivo.

Therefore, great attention is being paid to the development of a novel fluorescent peptide sensor having sufficient selectivity for the metal ion. Although many fluorescence chemical sensors have been reported to selectively recognize copper ions and zinc ions, few examples of fluorescence chemical sensors that selectively increase fluorescence in copper and zinc ions in aqueous solutions. Therefore, the inventors of the present invention, while studying a compound that selectively recognizes the copper ions and / or zinc ions, it was confirmed that it is possible to detect the copper ions and zinc ions in the aqueous solution by introducing a fluorescent Dansyl to the peptide Was completed.

It is an object of the present invention to provide a fluorescent peptide sensor for the selective detection of copper or zinc ions.

Another object of the present invention is to provide a fluorescent peptide sensor for selectively detecting copper or zinc ions from an aqueous environment or a biological sample such as groundwater or river.

It is still another object of the present invention to provide a fluorescent peptide sensor for selectively detecting copper or zinc ions in a wide range from weakly acidic aqueous solution to basic aqueous solution.

Still another object of the present invention is to provide a method of manufacturing the fluorescent peptide sensor.

Still another object of the present invention is to provide an apparatus for selectively detecting copper or zinc ions using the fluorescent peptide sensor.

The present invention provides a peptide sensor capable of detecting copper or zinc ions covalently bonded to a fluorescent substance, wherein the fluorescent substance is a 5-dimethylamino-1-naphthalenesulfonyl group, and the peptide is X1-Y1-Y2-X2-. X3 or X3-X1-Y1-Y2-X2 amino acid sequence, the sulfonyl group of the fluorescent substance and the amino terminal of the peptide is covalently bonded, wherein X1, X2 and X3 are each independently Cys, His or Glu And Y1 and Y2 are each independently Gly or Pro.

In addition, the present invention is a fluorescent peptide sensor, Y1-Y2 sequence is inserted between the amino acids X2 and X3 of the peptide consisting of X1-Y1-Y2-X2-X3, X1-Y1-Y2-X2-Y1-Y2- It is characterized in that the sequence consisting of X3.

In another aspect, the present invention is a fluorescent peptide sensor, X3 is deleted from the peptide, it is characterized in that the sequence consisting of X1-Y1-Y2-X2-Y1-Y2.

In another aspect, the present invention, a fluorescent peptide sensor, characterized in that the peptide covalently bonded to the fluorescent display material is a peptide consisting of any one amino acid sequence selected from SEQ ID NO: 1 to 8.

In another aspect, the present invention, a fluorescent peptide sensor, characterized in that the carboxy terminal of the peptide covalently bonded to the fluorescent display material is substituted with an amino group.

The present invention also provides a fluorescent peptide sensor, wherein the hydroxyl group (-OH) of the carboxy terminus (COOH) of the peptide covalently bonded to the fluorescent display material is -NH ₂ , -NHR1 (R1 is an alkyl group having 1 to 18 carbon atoms), -OR1 (R 1 is an alkyl group having 1 to 18 carbon atoms),-(OCH ₂ CH ₂ ) n-COOH (n = 2 to 5000),-(OCH ₂ CH ₂ ) n-polystyrene (n = 2 to 5000) or an amino acid It is characterized by.

The present invention also provides a fluorescent peptide sensor, wherein the carboxy terminus of the peptide covalently bonded to the fluorescent display material or the opposite end of the end of the alkyl, alkylene oxide group or alkyl ester group and the peptide is bonded to the solid support It features.

The present invention also provides a method for producing a fluorescent peptide sensor, wherein the side chain reactive to the amino terminal is sequentially reacted with an amino acid and a resin protected by a protecting group, and the resin and the peptide portion of the fluorescent peptide sensor of claim 1 or 2. Synthesizing the binder by solid phase synthesis; Reacting the combination of the resin and the peptide with 5-dimethylamino-1-naphtharensulfonyl chloride; And reacting the reaction product with a side chain protecting group and a resin removing solution.

In another aspect, the present invention is characterized by using the fluorescent peptide sensor or the fluorescent peptide sensor prepared by the method as a detection method of copper or zinc ions.

In another aspect, the present invention is a device for detecting copper or zinc ions, characterized in that it comprises a fluorescent peptide sensor or a fluorescent peptide sensor produced by the method, and a fluorescent detector.

In the fluorescent peptide sensor of the present invention, the conventional sensor was able to detect heavy metal ions mainly in organic solvents, while the compound according to the present invention was able to detect copper ions and zinc ions in an aqueous environment such as groundwater and rivers. It can be used as a fluorescent peptide sensor.

In addition, since the selectivity is excellent in the pH range of the weakly acidic to basic aqueous solution of the present invention, especially a biological sample, it can be used as a fluorescent peptide sensor for selectively detecting copper or zinc ions from the biological sample.

In addition, the fluorescent peptide sensor of the present invention may selectively detect fluorescence by selectively chemiluminizing copper ions due to chelate copper ions, or selectively amplify fluorescence only in zinc ions by chelate zinc ions, By selectively reducing fluorescence through copper ions and selectively amplifying fluorescence with zinc ions simultaneously, copper and zinc ions can be selectively detected simultaneously.

In the fluorescent peptide sensor of the present invention, the fluorescent display material is a 5-dimethylamino-1-naphthalensulfonyl group (or "Dansyl") is used, and shows a very sensitive fluorescence change even in a minute environment.

In the fluorescent peptide sensor of the present invention, the amino terminal of the peptide consisting of 5 to 7 amino acid residues and the fluorescent display is covalently bonded.

Peptides covalently bonded to the fluorescent material in the present invention is a regular interval, that is, any one amino acid selected from cysteine (Cys), histidine (His) and glutamic acid (Glu) found in zinc finger domains Two amino acid residues arranged between the two amino acids selected from among cysteine, histidine, and glutamic acid, and two amino acids are glycine (Gly) -glycine (Gly) or proline (Pro) -glycine (Gly) Or glycine (Gly) -proline (Pro) or proline (Pro) -proline (Pro) is suitable for allowing the cysteine, histidine and glutamic acid to be uniformly arranged in the spatial region of the secondary structure. Therefore, the peptide covalently bonded to the fluorescent substance of the present invention comprises the amino acid sequence X1-Y1-Y2-X2-X3 or X3-X1-Y1-Y2-X2, wherein X1, X2 and X3 are each independently Cys, His or Glu, and Y1 and Y2 are each independently characterized as Gly or Pro. Even if the peptide moiety includes X 1 -Y 1 -Y 2 -X 2, if the peptide moiety has less than 5 amino acid residues, the selectivity for detecting copper or zinc ions is not good because of its poor selectivity to copper or zinc ions compared to other heavy metals. It is not preferable as a sensor, and the eighth or more amino acid residue from the fluorescent display generally has little effect on copper zinc ion sensing when the influence on the formation of the secondary structure is small.

In the present invention, when the peptide covalently bonded to the fluorescent substance consists of five amino acid residues, even if the amino acid residues are bonded to the carboxy terminus of X1-Y1-Y2-X2-X3 or X3-X1-Y1-Y2-X2, No influence Preferably X 1 is Cys or His. In addition, the amino acid bound to the carboxy terminus of X1-Y1-Y2-X2-X3 or X3-X1-Y1-Y2-X2 can be any 20 unmodified amino acids, but only metal binding ligands found in many zinc finger regions. Cys, His, Glu, Asp having a general amino acid except for amino acids, such as Pro, which affects the structure is more preferred.

In the present invention, when the peptide covalently bonded to the fluorescent substance consists of 6 or 7 amino acid residues, the Y1-Y2 sequence is repeated between X2 and X3 amino acids of the peptide of the amino acid sequence consisting of X1-Y1-Y2-X2-X3 It is preferable for the selective detection of copper or zinc ions, and in the repeating Y1-Y2 sequence, one is Gly-Gly and the other is Pro-Gly, which is more preferable since copper and zinc can be detected simultaneously.

In addition, in the present invention, when any one of X1, X2 and X3 of the peptide covalently bonded to the fluorescent display material is Cys, the other amino acid is not Cys is advantageous in terms of stability of the fluorescent peptide sensor.

Preferred compounds for the fluorescent peptide sensor of the present invention are illustrated in Tables 1 and 2 below.

division Chemical formula Remarks One Dansyl-Cys-Gly-Gly-His-Gly-Gly-Glu-CONH ₂ GG2 2 Dansyl-Cys-Pro-Gly-His-Gly-Gly-Glu-CONH ₂ - 3 Dansyl-Cys-Gly-Gly-His-Pro-Gly-Glu-CONH ₂ PG1 4 Dansyl-Cys-Pro-Gly-His-Pro-Gly-Glu-CONH ₂ PG2 5 Dansyl-Cys-Gly-Pro-His-Gly-Pro-Glu-CONH ₂ - 6 Dansyl-Cys-Gly-Pro-His-Gly-Gly-Glu-CONH ₂ - 7 Dansyl-Cys-Gly-Gly-His-Gly-Pro-Glu-CONH ₂ - 8 Dansyl-Cys-Gly-Pro-His-Gly-Pro-Glu-CONH ₂ -

SEQ ID NO: Chemical formula Remarks 9 Dansyl-Cys-Pro-Gly-His-Glu-CONH ₂ Cp1 10 Dansyl-His-Pro-Gly-Cys-Glu-CONH ₂ Cp1-His 11 Dansyl-Cys-His-Pro-Gly-Glu-CONH ₂ Cp1-Cys-Glu 12 Dansyl-Glu-Pro-Gly-Cys-His-CONH ₂ Cp1-Glu 13 Dansyl-His-His-Pro-Gly-Glu-CONH ₂ Cp1-His-His

Further, in the present invention, the hydroxyl group (-OH) of the carboxy terminus (COOH) of the peptide to which the fluorescent substance is covalently bonded is -NH ₂ , -NHR1 (R1 is a C1-18 alkyl group), -OR1 (R1 is a C1-C1). 18 alkyl group),-(OCH ₂ CH ₂ ) n-COOH (n = 2 to 5000),-(OCH ₂ CH ₂ ) n-polystyrene (n = 2 to 5000) or amino acid. The carboxy terminal-substituted peptide may be prepared by known methods such as solid phase synthesis and liquid phase synthesis, and are not particularly limited.

In addition, the carboxy terminus of the peptide of the fluorescent peptide sensor of the present invention; Or the terminal whose carboxy terminal is substituted by the amino group, the terminal of the alkylene oxide group, or the alkyl ester group is fixed to a solid support, and can be used as an immobilization sensor. The solid support may be in the form of beads, films, nanotubes, porous particles, and the like. The components of the solid support may be a silica surface, a TiO ₂ surface, or the like, and a polymer resin may be a polystyrene resin connected with polyethylene oxide. This is possible. In addition, any method known in the art may be used as long as it is suitable for the purpose of the present invention.

Fluorescent peptides of the present invention selectively select copper or zinc ions by amplifying or reducing fluorescence at a wavelength of 450 to 550 nm, preferably 490 to 520 nm when the excitation wavelength is in the range of 300 to 360 nm. Can be detected.

In addition, the manufacturing method of the fluorescent peptide sensor of the present invention is a reaction of the amino terminal and the side chain reactive with the amino group and the resin is sequentially reacted, the resin and the peptide portion of the fluorescent peptide sensor of claim 1 or the combination Synthesizing by a solid phase synthesis method; Reacting the combination of the resin and the peptide with 5-dimethylamino-1-naphtharensulfonyl chloride; And reacting the reaction product with a side chain protecting group and a resin removing solution; Characterized in that it comprises a.

The protecting group used in the solid phase synthesis method is a protecting group for protecting the amino terminal, and a protecting group for protecting side chains reactive with 9-fluorenylmethyloxycarbonyl group (hereinafter referred to as "Fmoc") is triphenylmethyl (hereinafter referred to as "Trt"). Butyloxycarbonyl (hereinafter referred to as "Boc"), t-butyl ester (hereinafter referred to as "tBu"), and the like. Examples of such amino acids are Fmoc-L-Asp (Trt) -OH, Fmoc-L-His (Trt) -OH, Fmoc-L-Gly-OH, Fmoc-L-Cys (Trt) -OH, Fmoc-L- Glu (OtBu) -OH Fmoc-L-Pro-OH and the like can be used.

The resin used for peptide synthesis is preferably a linked amide methylbenzhydrylamine (MBHA) resin in which the carboxy terminus is made in the form of an amide when the peptide is cleaved from the resin.

In general, when the peptide is synthesized in the solid phase using amino acids and resins, it is preferable to use the resins and amino acids in a molar ratio of 1: 2 to 1:10, and it is economical to use the molar ratio of 1: 3 to 1: 7. For example, 0.1 mmole of resin and 0.3 mmole of amino acid can be used. Peptides are synthesized from the carboxy terminus to the amino terminus. At this time, the carboxyl group of one amino acid previously attached to the resin and the other amino acid to be attached is activated as an activator, and the protecting group at the amino terminus is piperidine (Piperidine). ), And the following synthesis can be performed. N, N`-Diisopropyl carbodiimde (hereinafter referred to as "DIPC") is used as the carboxyl activator, and N-Hydrobenxotriazole (hereinafter referred to as "HOBt") is generally used as an addition aid. The synthesis time is about 1 hour.

After the peptide synthesis was completed, the Fmoc protecting group attached to the amino terminus was removed using piperidine, and then a solution containing Dansyl chloride (5-Dimethylamino-1-naphthalenesulfonyl chloride) and DIPEA (diisopropylethylamine) was added to the resultant. For about 2 hours, the fluorescent material is peptide-bound. The side chain protecting group and the resin removing solution are reacted with a solution of triplefuoroacetic acid, preferably 90% by volume of trichloroacetic acid, 5% by volume of trimethylsilane, and 5% by volume of water at room temperature for about 3 hours to remove the side chain protecting group of the amino acid and the resin. After removing the peptide from the separation, the fluorescent peptide sensor dissolved in the resin and the solution by filtration is separated.

Trifluoroacetic acid in the filtered fluorescent peptide sensor separation solution can be removed by passing nitrogen gas, the remaining material is added to the diethyl ether solution and precipitated fluorescent peptide sensor after addition of diethyl ether cooled to -20 ℃ Can be separated.

The fluorescent peptide sensor thus separated can be purified using column chromatography, and the molecular weight of the purified peptide can be confirmed using a Maldi-TOF mass spectrometer.

The present invention also provides a method for the selective detection of copper or zinc ions present in an aqueous solution environment or a living body using the fluorescent peptide sensor.

The detection of the copper or zinc ions may be performed by confirming the chelate amplification fluorescence (CHEF) effect by exciting the complex of the fluorescent peptide sensor and the copper or zinc ion. The ion recognition ability is maintained even after the film is fixed, and in this case, copper and zinc ions at lower concentrations can be detected and recycled. In this case, the excitation wavelength for chelate amplification is preferably 330 nm.

Hereinafter, the present invention will be described in more detail with reference to Examples and drawings. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Preparation of Fluorescent Peptide Sensor

Rinked amide MBHA resin (147 mg, 0.1 mmol) was added to 3 ml dry DMF and swelled for about 30 minutes. After adding 3 ml of 50% piperidine / DMF solution to the resin for 15 minutes to remove the amino terminal Fmoc group, 0.3 mmol Fmoc-L-Glutamic acid (158 mg) and DIC (48) in 1.5 ml anhydrous DMF solvent were used. [Mu] l) and HOBt (40 mg) were added and then reacted with the solution activated for 20 minutes in advance for about 1 hour. After the reaction, the solution is filtered off, the resin is washed several times with DMF and methanol, followed by a kaiser test. If the kaiser test is positive, recreate and add the above activation solution. If negative, synthesize the next sequence of amino acids. When the peptide synthesis was completed, the amino terminal Fmoc group was removed using 50% piperidine / DMF, and a solution containing Dansyl Cholride (0.3 mmol) and DIPEA (0.6 mmol) in anhydrous DMF solvent was mixed with the resin. After stirring at room temperature for 1 hour at an appropriate rate. The fluorescent peptide containing the protecting group attached to the resin was reacted with the removal solution (90% by volume of trifluoroacetic acid, 5% by volume of trimethylsilane, and 5% by volume of water) at room temperature for 4 hours to remove the side chain protecting group of the amino acid and to remove the peptide from the resin. After filtration, the fluorescent peptide dissolved in the resin and the solution was separated. The trifluoroacetic acid in the filtered fluorescent peptide separation solution was removed by passing nitrogen gas, and the remaining material was added with diethyl ether cooled to -20 ° C, and the diethyl ether solution and precipitated fluorescent peptide were centrifuged ( 3,000 rpm, 20 min). The fluorescent peptide thus obtained was purified using reversed phase HPLC (C-18 column delta pak C18-300A, 1.9 × 30 cm) (Eluent: water / acetonitrile in 0.1% TFA (gradient): Flow Rate 2.5 mL / min). The molecular weight of the purified fluorescent peptide was confirmed using a Maldi-TOF mass spectrometer (FIG. 10).

Example 2 Preparation of Fluorescent Peptide Sensor Supported on Solid Phase

Tentagel resin (g, 0.1 mmol) is added to 3 ml dry DMF and swelled for about 30 minutes. 0.3 mmol Fmoc-L-Glutamic acid in 1.5 ml anhydrous DMF solvent (158 mg), DIC (48 μl), and HOBt (40 mg) are added, followed by reaction for about 1 hour with the solution activated for 20 minutes in advance. After the reaction, the solution is filtered off, the resin is washed several times with DMF and methanol, followed by a kaiser test. If the kaiser test is positive, recreate and add the above activation solution. If negative, synthesize the next sequence of amino acids. When the peptide synthesis was completed, the amino terminal Fmoc group was removed using 50% piperidine / DMF, and a solution containing Dansyl Cholride (0.3 mmol) and DIPEA (0.6 mmol) in anhydrous DMF solvent was mixed with the resin. After stirring at room temperature for 1 hour at an appropriate rate. The protecting group attached to the side chain of the fluorescent peptide sensor was reacted with a removal solution (90% by volume of trifluoroacetic acid, 5% by volume of trimethylsilane, and 5% by volume of water) at room temperature for 4 hours, and then separated from the resin and the solution by filtration. Repeat the wash several times each time with and methanol to wash off the removal solution.

Experimental Example 1 Observation of Fluorescence According to Binding of Fluorescent Peptide Sensor and Heavy Metal Ions

1-1. Neon Peptide  Fluorescence titration experiment by combining sensor and heavy metal ions

The fluorescent peptide sensor was completely dissolved in water, so it was tested by dissolving in 10 mM HEPES buffer solution (pH 7.4) to which no organic solvent was added. The fluorescence emission spectrum was excited at 330 nm to observe a region of 350 nm to 650 nm. Experiments included Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Pb ²⁺ , Cu ²⁺ , Ag ⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , and Zn ²⁺ perchlorate salts (Perchlorate form) and Na ⁺ , Al ³⁺ , K ⁺ chloride salts (chlorate form) were used as heavy metal ions. FIG. 1 shows the fluorescence emission spectra after addition of GG2, PG1 , and PG2 (10 μM each) and heavy metal ions (1 eq.) Of several peptides Table 1 in 10 mM HEPES buffer solution (pH 7.4).

GG2 (Dansyl-Cys-Gly- Gly-His-Gly-Gly-Glu-NH 2) with the exception of copper ions did the fluorescence change was observed in any other metal, was placed a copper ion, fluorescence is sharply at 510 mn Reduced.

PG1 ( Dansyl-Cys-Gly-Gly-His-Pro-Gly-Glu-NH ₂ ) did not change fluorescence in any heavy metal ions except copper and zinc ions. Fluorescence increased when zinc ions were added, whereas fluorescence decreased when copper ions were added.

In FIG. 1, PG 2 ( Dansyl-Cys-Pro-Gly-His-Pro-Gly-Glu-NH ₂ ) did not show fluorescence change in any other metal including copper except zinc and cadmium ions. The addition of zinc ions increased the fluorescence at 510 mn, which means that PG2 detects zinc ions more selectively than copper ions, although copper ions and zinc ions have similar sizes and electronic configurations.

In general, it is known that fluorescence chemistry or peptide sensors comprising the Dansyl group and the imidazole group of hissteine have a high binding force with copper due to the imidazole group. [Shults MD, Pearce DA, Imperiali B: J Am Chem Soc 2003 , 125 : 10591-10597.] However, PG2 contains a Dansyl group and an imidazole group, but does not have a fluorescence change with respect to copper ions. In addition, GG2 showed higher fluorescence than PG1 when there were other metals, and PG1 showed the lowest fluorescence at the same concentration. Thus, this is an example to prove that Dansyl groups are in different environments due to the secondary structure of the peptide around them.

On the other hand, the fluorescent peptide sensor in which the amino acid Glu at the carboxy terminus was deleted in PG1, PG2, and GG2, respectively, did not differ in the pattern of fluorescence for PG1, PG2, GG2 and copper and zinc ions, respectively, except that the intensity of fluorescence was about 5 ~ 15% reduction.

Also, in order to facilitate the synthesis of the peptide moiety, the carboxy terminus of PG1, PG2, GG2 was substituted with an amino group, but in the case of a fluorescent peptide sensor in which the carboxy terminus was not substituted with an amino group in PG1, PG2, GG2, the fluorescence pattern or intensity There was no difference.

1-2. Measurement of stoichiometric ratio and binding constant of fluorescent peptide sensor for zinc and copper ions

Since both copper and zinc ions play an important role in several biochemical functions such as gene expression, cell death, enzyme regulation and amyloid fibril formation, in order to determine the binding constants between the fluorescent peptide sensor and copper and zinc ions, The same experiment was performed.

As shown in the calibration curve of the fluorescent peptide sensor for copper in FIG. 2, it can be seen that the fluorescence intensity of GG2 reached its maximum with 10 μM copper. Job plot analysis can be used to analyze the stoichiometry of GG2. [Chang, CT; Wu, C.-SC; Yang, JT Anal. Biochem. 1978 , 91 ] The point where the fluorescence was reduced completely indicates 0.5 mole, which means that GG2 and copper ions are in a 1: 1 complex. The binding constant was calculated using the ENZFITTER program through a fluorescence assay curve to obtain 4.2 × 10 ⁶ M ⁻¹ .

Figure 3 is the result of measuring the fluorescence change of PG2 in accordance with the concentration of zinc ion in 10 mM HEPES buffer solution (pH 7.4). It can be seen that the fluorescence intensity of the fluorescence emission spectrum increases significantly as the concentration of zinc ions increases. When approximately 9 μM of zinc ions were added, the fluorescence intensity reached the maximum point, and the job plot analysis shows that the zinc ions and PG2 form 1: 1 complexes within the micro molar range. . The binding constant was calculated using the ENZFITTER program through the calibration curve of the fluorescent peptide sensor for copper to obtain 6.0 × 10 ⁶ M ^-1 .

4 is a result of measuring the fluorescence change of PG1 according to the concentration of zinc ions in 10 mM HEPES buffer solution (pH 7.4). Unlike PG2, the fluorescence intensity of the fluorescence emission spectrum increases as the concentration of zinc ions increases, but the fluorescence decreases when the copper ions increase. Job plots show that zinc ions and copper ions form 1: 1 complexes with PG1. Binding constants were calculated using the ENZFITTER program through the calibration curve of the fluorescent peptide sensor for copper and are summarized in FIG. 9.

In addition, Table 3 shows the results of confirming the selectivity and Ka value of zinc and copper ions for the compounds in Table 2.

SEQ ID NO: Chemical formula Metal ion selectivity Ka (M ^-1 ) 9 Dansyl-Cys-Pro-Gly-His-Glu-CONH ₂ Zn (II) 690000 Cu (II) 4000000 10 Dansyl-His-Pro-Gly-Cys-Glu-CONH ₂ Zn (II) 460000 Cu (II) 500000 11 Dansyl-Cys-His-Pro-Gly-Glu-CONH ₂ Zn (II) 620000 Cu (II) 1000000 12 Dansyl-Glu-Pro-Gly-Cys-His-CONH ₂ Zn (II) 220000 Cu (II) 430000 13 Dansyl-His-His-Pro-Gly-Glu-CONH ₂ Zn (II) 710000 Cu (II) 920000

All of the peptides showed strong selectivity and fluorescence change with respect to copper zinc ions, and the binding mode was 1: 1. The calibration curve of each of the compounds is not shown.

1-3. Fluorescence titration experiment of fluorescent peptide sensor and metal ion according to pH

When copper ions and zinc are present, the following experiment was performed to measure the fluorescence change of the fluorescent peptide sensor according to the pH change.

5 is a graph showing the fluorescence emission spectrum of the fluorescent peptide sensor for the metal at pH below 5.5. The intensity of fluorescence is very weak here, probably because the dimethylamino group of the Dansyl group (p K _a- 4) is protonated. The fluorescence intensity of GG2 at pH higher than 6.5 increases with increasing pH, but the GG2-copper ionic complex shows very weak fluorescence in the region between pH 3.5 and 10.5. Differences between PG1 and PG1-zinc ion complexes at pH higher than 6.5 are not significant because of the increase in fluorescence intensity with increasing pH. However, PG1 and PG1-copper ion complexes show a lot of difference because PG1-copper ion complexes exhibit 11.5 weak fluorescence intensity at the pH of the entire region, that is, pH 3.5. The fluorescence difference between PG2 and PG2-zinc ion complex was maximum at pH 9.5. At pH 9.5, the fluorescence intensity of PG2-zinc ion complex was about 4 times stronger than that of PG2.

Overall, these results indicate that the fluorescent peptide sensors are very useful tools for detecting copper and zinc ions at weakly acidic to basic pHs, preferably at pH 6.5-12, more preferably at pH 7-11. .

1-4. Fluorescence titration experiment of fluorescent peptide sensor and copper ion and zinc ion in the presence of other metals

In general, it is known that dislocation metals are present at relatively low concentrations, while group 1 and group 2 metal elements exist in a range of millimoles (mM) in vivo. Therefore, the following experiments were carried out to determine whether the fluorescent peptide sensor works well under such conditions.

In order to make conditions similar to those in vivo, experiments were conducted under the following conditions. 10 mM HEPS buffer solution (pH 7.4) containing 1 eq GG2-copper ion complex, PG1-zinc ion complex, and PG2-zinc ion complex added 1 eq potential metal or 100 eq Group 1 and 2 metal ions Thereafter, the fluorescence change was measured. As shown in FIG. 6, the CG 2 -copper ion complex was not affected by any other metals. In the case of the PG1-zinc ion composite, the change in fluorescence was also small except when copper ions were added. As shown in FIG. 9, PG1 was found to bind better with copper ions than with zinc ions. In the case of PG2-zinc ion complexes, the fluorescence also increased with the addition of copper ions, which overcomes the fluorescence quenching phenomenon of fluorescent chemical / peptide sensors containing Dansyl and imidazole groups due to copper ions. In conclusion, even in the presence of other metal ions, GG2, PG2, and PG1 were found to be fluorescent peptide sensors that can effectively detect copper and zinc ions.

< Experimental Example 2> Observation of fluorescence changes due to binding of fluorescent metal immobilized on PEG-poly styrene resin synthesized in Example 2 with heavy metal ions

2 -1. Experiments on Fluorescence Change According to Binding of Immobilized Fluorescent Peptide Sensors and Heavy Metal Ions

PEG-Poly Styrene Fluorescent peptide immobilized on the resin swells well in water, but if it is not stirred, it is easily precipitated, so it is important to stir well with a magnetic stirrer when measuring fluorescence change. All fluorescence changes were measured using a fluorescence spectrophotometer equipped with a magnetic stirrer (ParkinElmer LS 55 Luminescence Spectrometer), and the fluorescence emission spectrum was excited at 330 nm to observe an area of 350 nm to 650 nm. Experiments included Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Pb ²⁺ , Cu ²⁺ , Ag ⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , and Zn ²⁺ perchlorate salts (Perchlorate form) and Na ⁺ , Al ³⁺ , K ⁺ chloride salts (chlorate form) were used as ions. First PEG-poly styrene in 2 ml of 10 mM HEPES buffer solution (pH 7.4) Immobilized fluorescent peptides PG1 and PG2 (100 nmol each) were added to the resin, and each metal ion (1 eq.) Was added thereto, followed by fluorescence emission spectrum after 30 minutes.

12, PEG-poly styrene The fluorescent peptide PG1 immobilized on the resin ( Dansyl-Cys-Gly-Gly-His-Pro-Gly-Glu-NH ₂ ) is the same as that of the peptide not immobilized on the resin and fluoresce in any heavy metal ion except copper and zinc ions. Unchanged. Fluorescence increased when zinc ions were added, whereas fluorescence decreased when copper ions were added.

In Figure 13, PEG-poly styrene The fluorescent peptide PG2 immobilized on the resin ( Dansyl-Cys-Gly-Gly-His-Pro-Gly-Glu-NH ₂ ) is the same as that of the peptide not immobilized on the resin, except for zinc and cadmium ions. Unchanged. Fluorescence increased when zinc and cadmium ions were added.

Experimental Example 3 Analysis of Two-Dimensional Structure of Peptides Using Circular Dichroism

The fluorescent peptide was dissolved in 10 mM HEPES buffer (pH 7.4) to a final concentration of 150 mg / mL, and placed in a 1 mm pathlength cell, then between 190 nm and 300 nm using Jasco J-715 spectropolarimeter (Tokyo, Japan). Circular dichroism spectra were measured at room temperature. In addition, after adding 1 equivalent of zinc or copper metal ion to the same fluorescent peptide solution, the solution was placed in a 1 mm pathlength cell after 30 minutes, and then between 190 nm and 300 nm using Jasco J-715 spectropolarimeter (Tokyo, Japan). The circular dichroism spectrum of was measured at room temperature. CD spectra are expressed as mean residue ellipticity.

14 shows that GG2 has the lowest mean residue ellipticity at 205 nm and 215 nm, and GG2 peptide has a random structure or a few screen structures. In contrast, PG1 shows a strong negative band at 230 nm, indicating that PG1 has a bending structure, and PG2 also shows a positive band characteristic at 225 nm at 225 nm. This structure of PG1 and PG2 indicates that the role of Pro having a folding structure in the one-dimensional structure is important, and that the peptide having this folding structure can commonly combine with zinc ions and have fluorescence amplification.

1 is 10mM HEPS buffer solution (pH 7.4) in the fluorescence sensor -GG2 peptide, PG2, and PG1- copper ion, zinc ion and various metal ions, one equivalent of a (10 μM) (Ca ^{+ 2,} Cd ^{+ 2,} Co ^{2 +} , Pb ²⁺ , Ag ⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Na ⁺ , Al ³⁺ , and K ⁺ )

2 is a graph showing a fluorescence assay curve and a job plot analysis of GG2 (10 μM) for copper ions in 10 mM HEPS buffer solution (pH 7.4),

FIG. 3 is a graph showing fluorescence assay curves and Job plot analysis of PG1 (10 μM) for zinc ions (top) and copper ions (bottom) in 10 mM HEPS buffer solution (pH 7.4),

4 is a graph showing a fluorescence assay curve and a job plot analysis of PG1 (10 μM) against zinc ions in 10 mM HEPS buffer solution (pH 7.4),

5 is a graph showing a fluorescence emission spectrum of a fluorescent peptide sensor (10 μM) at various pHs in the presence of one equivalent of copper or zinc ions.

FIG. 6 shows fluorescence emission spectra generated after addition of 1 equivalent of dislocation metal or 100 equivalents of Group 1 and Group 2 metal ions to 10 mM HEPS buffer solution (pH 7.4) containing 1 equivalent of GG2-copper ion complex Is a graph showing

FIG. 7 shows the fluorescence emission spectrum generated after addition of 1 equivalent of dislocation metal or 100 equivalents of Group 1 and Group 2 metal ions to 10 mM HEPS buffer solution (pH 7.4) containing 1 equivalent of PG1-zinc ion complex Is a graph showing

FIG. 8 shows the fluorescence emission spectrum generated after addition of 1 equivalent of a potential metal or 100 equivalents of Group 1 and Group 2 metal ions to a 10 mM HEPS buffer solution (pH 7.4) containing 1 equivalent of PG2-zinc ion complex Is a graph showing

9 is a table showing the sequence, binding constant and error with copper or zinc ions of each fluorescent peptide sensor,

10 is a table showing the type, sequence, HPLC and mass spectrometry of each synthesized fluorescent peptide sensor,

11 is a table showing the type, sequence, HPLC and mass spectrometry of each synthesized solid-state fluorescent peptide sensor,

12 is PEG-poly styrene The fluorescent peptide PG1 immobilized on the resin (100 nmol) was added to 10 mM HEPS buffer solution (pH 7.4), followed by stirring. One equivalent of copper ions, zinc ions and various metal ions (Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Pb ²⁺ , Ag ⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Na ⁺ , Al ³⁺ , and K ⁺ ).

13 is PEG-poly styrene The fluorescent peptide PG2 immobilized on the resin (100 nmol) was added to 10 mM HEPS buffer solution (pH 7.4), followed by stirring, and 1 equivalent of copper ions, zinc ions and various metal ions (Ca ²⁺ , Cd ²⁺ , Co ²⁺ , Pb ²⁺ , Ag ⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Na ⁺ , Al ³⁺ , and K ⁺ ).

FIG. 14 shows that the fluorescent peptides (GG2, PG1, PG2) were dissolved in 10 mM HEPES buffer (pH 7.4) to a final concentration of 150 mg / mL, respectively, and placed in 1 mm pathlength cells, followed by Jasco J-715 spectropolarimeter (Tokyo, Japan Is a circular dichroism spectrum between 190 nm and 300 nm.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Fluorescent peptide probe for selective detection copper or zinc          ion, and preparation method <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 1 Cys Gly Gly His Gly Gly Glu   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 2 Cys Pro Gly His Gly Gly Glu   1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 3 Cys Gly Gly His Pro Gly Glu   1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 4 Cys Pro Gly His Pro Gly Glu   1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 5 Cys Gly Pro His Gly Pro Glu   1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 6 Cys Gly Pro His Gly Gly Glu   1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 7 Cys Gly Gly His Gly Pro Glu   1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 8 Cys Gly Pro His Gly Pro Glu   1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 9 Cys Pro Gly His Glu   1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 10 His Pro Gly Cys Glu   1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 11 Cys His Pro Gly Glu   1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 12 Glu Pro Gly Cys His   1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptidyl template of peptidyl fluorescent chemosensor for copper          or zinc ion <400> 13 His His Pro Gly Glu   1 5  

Claims (11)

Peptide sensor that can detect the copper or zinc ions covalently bonded to the fluorescent material and the peptide, The fluorescent display material is a 5-dimethylamino-1-naphthalenesulfonyl group, The peptide consists of an amino acid sequence of X1-Y1-Y2-X2-X3 or X3-X1-Y1-Y2-X2, The sulfonyl group of the fluorescent substance is covalently bonded to the amino terminal of the peptide, X1, X2 and X3 are each independently Cys, His or Glu, The Y1 and Y2 are each independently Gly or Pro fluorescent peptide sensor. The method of claim 1, Fluorescent peptide sensor, characterized in that the Y1-Y2 sequence is inserted between the X2 and X3 amino acids of the peptide consisting of X1-Y1-Y2-X2-X3. The method of claim 2, Fluorescent peptide sensor, characterized in that X3 is deleted from the peptide. The method of claim 2 or 3, Fluorescent peptide sensor, characterized in that one of the two Y1-Y2 sequence included in the sequence of the peptide is Gly-Gly, the other is Pro-Gly or Gly-Pro. The method of claim 4, wherein The peptide is a fluorescent peptide sensor, characterized in that the peptide consisting of any one amino acid sequence selected from SEQ ID NO: 1 to 8. The method according to any one of claims 1 to 3, Fluorine peptide sensor, characterized in that the carboxy terminal of the peptide is substituted with an amino group. The method according to any one of claims 1 to 3, The hydroxyl group at the carboxy terminus of the peptide is -NH ₂ , -NHR1 (R1 is an alkyl group having 1 to 18 carbon atoms), -OR1 (R1 is an alkyl group having 1 to 18 carbon atoms),-(OCH ₂ CH ₂ ) n-COOH (n = 2 5000),-(OCH ₂ CH ₂ ) n-polystyrene (n = 2 ~ 5000) or a fluorescent peptide sensor characterized in that substituted with an amino acid. The method according to any one of claims 1 to 3, Fluorescent peptide sensor, characterized in that the carboxy terminal of the peptide is bonded to a solid support.  A side chain reactive with an amino terminal reacts sequentially with an amino acid and a resin protected by a protecting group to synthesize a combination of the peptide portion of the fluorescent peptide sensor of any one of claims 1 to 3 and the resin by solid phase synthesis. step; Reacting the combination of the resin and the peptide with 5-dimethylamino-1-naphtharensulfonyl chloride; And Reacting the reaction product with triplefuoroacetic acid as a side chain protecting group and a resin removing solution; Method for producing a fluorescent peptide sensor comprising a. The apparatus for detecting copper or zinc ions, comprising any one of a fluorescent peptide sensor selected from claim 1 and a fluorescent detector. The method of claim 10, The apparatus for detecting copper or zinc ions, wherein the fluorescent peptide sensor is immobilized on a solid support.

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