CN116970289A - A water-soluble fluorescent dye based on oxygen-controlled enzymatic reaction, preparation method and application - Google Patents

Abstract

The invention discloses an enzyme reaction water-soluble fluorescent dye based on oxygen control, a preparation method and application thereof, wherein the water-soluble fluorescent dye product is prepared stably and efficiently by adjusting the oxygen concentration condition of an enzymatic reaction. The fluorescent dye has good light stability, heat stability, high fluorescence quantum yield and wavelength covering the whole visible spectrum region. Compared with the traditional organic chemistry synthesis method, the preparation process is simple and green, and has wide application in the fields of biology, chemical industry, medicine, electrochemistry and the like, including biological imaging, fluorescent marking, sensors, protein positioning, medicine development and the like.

Description

A water-soluble fluorescent dye based on oxygen-controlled enzyme reaction, preparation method and application

Technical Field

The present invention relates to a biomolecule containing a tyrosine group as a substrate, and an enzyme catalytic reaction regulated by oxygen to obtain a water-soluble fluorescent dye product, and a preparation method and application thereof. More specifically, the present invention relates to regulating the reaction system so that a water-soluble fluorescent dye composition and a preparation process can be stably and efficiently prepared under low oxygen conditions, and belongs to the field of biomaterials.

Background Art

Fluorescent dyes are widely used in many disciplines such as medicine, biology and environmental science. In different fields of application, corresponding requirements are put forward for the properties of fluorescent dyes. Especially in the fields of biology and medicine, fluorescent dyes are required to have: (1) biocompatibility. High biocompatibility is a necessary condition for in vivo detection of biological systems. In particular, fluorescent dyes need to have good water solubility; (2) high fluorescence quantum yield. High fluorescence quantum yield is conducive to the collection and identification of fluorescent signals and improves the sensitivity of analytical detection; (3) high stability, including chemical structure stability and environmental stability. Including light, heat and pH stability; (4) intrinsic fluorescence emission can be adjusted to visible light or even near-infrared region. On the one hand, the wavelength range of fluorophores is wide, which is conducive to the monitoring of multiple fluorescence channels. On the other hand, red and near-infrared fluorescence facilitates high signal-to-noise ratio observation in biomedicine; (5) easy synthesis and suitable for a wide range of applications. Existing water-soluble fluorescent dyes are difficult to meet the above conditions.

Nature produces colorful fur, feathers, hair, eyes, etc. through various modifications of tyrosine-based pigments. Patent CN 110325204 A provides a self-assembling peptide that can be enzymatically oxidized to form a polymer pigment. Through self-assembly and complex polymerization pathway regulation, including catalysis, templating, assembly and restricted oxidation, different melanin-like pigments are obtained, and the regulation of light absorption is initially achieved.

Recently, inspired by the chemistry of fluorescent protein chromophores in nature, peptide-based fluorescent dyes have been continuously discovered and explored, pointing the way to breakthroughs in this field. The article reported that Y-containing tripeptides can emit fluorescence in the visible light region with the help of oxidation and self/co-assembly (ChemBioChem 2019, 20, 2324-2330; ACS MacroLett. 2021, 10, 7, 825–830;); a study reported a hybridization strategy that combines some natural ring-containing biomolecules with short peptides to control their mixing equilibrium and obtain fluorescent dyes with red-shifted fluorescence (510nm) (Chem. Commun. 2020, 56 (46), 6301-6304;). It has recently been discovered that Y-rich short peptides (KYF) can be covalently assembled into fluorescent nanogels under ultraviolet light, but the fluorescence cannot be red-shifted above 410nm (Angew. Chem., Int. Ed. 2021, 60(14), 7564–7569); the above research has expanded the fluorescence properties of tyrosine-containing peptides to a certain extent from the layered assembly strategy. However, in the above system, the tuning of fluorescence depends on the nanostructure, which limits its application in molecular biology and cell biology. Moreover, the effect of regulating quantum yield and red shift only by amino acids and their sequences is limited, and the longest fluorescence obtained is 510nm (cyan). How to obtain fluorescent dyes in the entire visible light region, especially red and near-infrared fluorescent dyes, is a certain challenge.

Summary of the invention

In view of the above-mentioned deficiencies and shortcomings of the prior art, the present invention provides a biomolecule containing a tyrosine group as a substrate, and controls the oxygen content of the reaction system to regulate the catalytic reaction path of the oxidase to obtain a water-soluble fluorescent dye product. The water-soluble fluorescent dye has good photostability, thermal stability, high fluorescence quantum yield and wavelength covering the entire visible spectrum region, especially its fluorescence emission can reach the red light to near-red light range.

As an important component of the earth's environment, oxygen has played a key role in the evolution of life since its appearance. At the molecular level of peptides and amino acids, the regulation of oxygen on the chemical evolution of these peptides and protein molecules is also very important. Recently, an article reported that oxygen has a regulatory role in the chemical evolution of tyrosine molecules. By changing the oxygen content of tyrosine-containing molecules in the reaction system, biomaterials with photothermal and photoluminescent phenomena can be obtained. Under oxygen-rich conditions, the path of tyrosine groups to melanin analogs is dominant, and the resulting products have obvious photothermal effects; in an oxygen-deficient environment, tyrosine groups will preferentially transform into dityrosine to obtain pH-responsive luminescent materials (Angew. Chem. Int. Ed., 2019, 58, 5872–5876).

In addition, the conventional understanding of the oxidase reaction catalysis is that it requires oxygen, but the present invention unexpectedly discovered that under low oxygen to anaerobic conditions, the oxidase can still catalyze the reaction and efficiently obtain fluorescent materials.

In the present invention, peptide sequence design, oxygen content regulation and enzyme reaction regulation are creatively combined to obtain a water-soluble molecular fluorescent dye with excellent properties.

In view of this, the present invention adopts the following technical solutions:

In a first aspect, the present invention first relates to a product obtained by enzymatic oxidation reaction of a peptide of formula (1) or formula (2) or formula (3) or a salt or a derivative thereof under hypoxic conditions.

The formula (1) is NH ₂ -X ₁ -Tyr-COOH;

The formula (2) is NH ₂ -(A)nX ₁ -Tyr-X ₂ -COOH;

The formula (3) is NH ₂ -X ₁ -Tyr-X ₂ -(A)n-COOH;

Wherein, preferably, _X1 is an amino acid; preferably, _X1 is any one of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine and pyrrolysine;

Preferably, _X2 is an amino acid; preferably, _X2 is any one of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine and pyrrolysine;

(A)n represents a linker, an amino acid or a peptide, and n is an integer greater than or equal to 0; when n=0, (A)n represents a chemical linker; when n=1, (A)n is selected from any amino acid;

When n is greater than 1, (A) n is any peptide of length n, wherein the peptide is a molecule formed by condensation of n amino acids through peptide bonds, wherein n ≥ 2, preferably, 2 ≤ n ≤ 10;

A is an amino acid; preferably, A is any one of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine and pyrrolysine, or a combination of multiple amino acids.

In a preferred embodiment of the present invention, the peptide is characterized in that the peptide of formula (1) or formula (2) or formula (3) has one of the following sequences:

The amino acids constituting the peptides of the present invention and indicated by the term AA may be in L- and D-isomerizable configurations. Preferably, the amino acids are in the L-form.

The term "peptide" or "peptide compound" refers to a peptide chain of two or more amino acids linked to each other by peptide bonds or a peptide sequence linked together by a modified peptide chain.

"Peptide" or "peptide compound" or "peptide substrate" also refers to a natural or synthetic peptide of the present invention as described above, or at least one of its fragments, whether it is obtained by protein hydrolysis or synthesis, or any natural or synthetic peptide whose sequence consists entirely or partially of the sequence of the above-mentioned peptide.

Wherein, the salt of the peptide of formula (1) or formula (2) or formula (3) is in the form of a salt selected from one of acetate, chloride, phosphate and trifluoroacetate;

In order to improve the tolerance to degradation, it is optional to use the peptide substrate of the present invention in a protected form, that is, to form a derivative of the peptide of formula (1) or (2) or (3), which may contain a protecting group Trt, Boc, Fmoc, Cbz/Z, Allyl at the NH2 _end ; or a protecting group OFm, Otbu, OBzl, OAll, OMe, OEt at the COOH end; or a peptide derivative in which both the _NH2 end and the COOH end are protected;

The peptides of the present invention may be of natural or synthetic origin, preferably, according to the present invention, the peptides are of synthetic origin, obtained by chemical synthesis. The peptide substrates of formula (1) or formula (2) or formula (3) of the present invention may be obtained by typical chemical synthesis (in solid phase or liquid homogeneous phase) or by enzymatic synthesis from constituent amino acids.

The enzymatic oxidation reaction uses an oxidase; preferably, the oxidase is selected from one or a mixture of horseradish catalase, peroxidase, Bacillus bisporus tyrosinase (AbTYR), human tyrosinase (hTYR), tryptophan dioxygenase, laccase, and cytochrome P450 oxidase complex.

The results of the present invention show that, for the peptide of formula (1) or formula (2) or formula (3) or its salt or derivative thereof, when _X1 is an electron-rich group containing hydroxyl, amino, guanidine and thiomethyl, the emission wavelength of the obtained water-soluble fluorescent dye is red-shifted; in particular, when _X2 is glycine, alanine and lysine, the quantum yield of the water-soluble fluorescent dye is higher; and (A)n and the terminal protecting group have little effect on the luminescent properties.

In a particular embodiment, the present invention also found that when Tyr is located at the N-terminus, the emission wavelength is blue-shifted and the quantum yield is low.

In a second aspect, a method for preparing any one of the peptide-based water-soluble fluorescent dyes described in the first aspect is provided, comprising the following preparation steps:

(1) Prepare a phosphate buffer solution of the substrate peptide.

(2) deoxygenating the solution system obtained in step (1);

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding an appropriate amount of oxidase to the sample solution of step (2);

(4) The solution of step (3) is reacted at 5-50° C. for a certain period of time to perform an enzyme-catalyzed reaction to obtain a water-soluble fluorescent dye.

In step (1), the phosphate buffer solution is a phosphate buffer solution with a pH of 7.0-10.0, wherein the concentration of phosphate is 0.01M-1M;

In step (2), the deoxygenation treatment includes but is not limited to the introduction of high-purity inert gas, freezing-degassing cycle, heating and ultrasonic treatment; preferably, by introducing high-purity inert gas into the system and freezing-degassing cycle;

In step (2), the dissolved oxygen concentration of the system after deoxygenation treatment is lower than 5 mg/L, preferably, the dissolved oxygen concentration of the system after deoxygenation treatment is lower than 2.5 mg/L; more preferably, the dissolved oxygen concentration of the system after deoxygenation treatment is lower than 2 mg/L;

In step (3), the concentration range of the oxidase is: 1-5000 UI/mg; optimally, the concentration range of the oxidase is: 2-1000 UI/mg;

In step (4), the reaction time is 12-240 hours; preferably, the reaction time is 12-120 hours; more preferably, the reaction time is 24-72 hours;

The unit UI/mg represents the titer of oxidase corresponding to each mg of substrate peptide;

In a third aspect, the present invention provides the use of the water-soluble fluorescent dye as described in any one of the first aspect and the second aspect, including but not limited to applications in the fields of biology, chemical engineering, medicine, electrochemistry, etc., including but not limited to biological imaging, fluorescent labeling, sensors, protein localization, drug development, etc.

The water-soluble fluorescent dye provided by the present invention has excellent comprehensive optical properties and good application prospects. It has at least one, multiple or all of the following beneficial effects:

1) The water-soluble fluorescent dye of the present invention has good light stability compared with the currently commercialized biological fluorescent dyes;

2) The water-soluble fluorescent dye of the present invention has good thermal stability compared with the currently commercialized biological fluorescent dyes;

3) Compared with existing peptide-based fluorescent dyes, the water-soluble fluorescent dye of the present invention has red-shifted absorption and emission wavelengths and improved fluorescence quantum yield;

4) Compared with the currently commercialized biological fluorescent dyes, the water-soluble fluorescent dye of the present invention has a simple and green preparation process and can be prepared on a large scale;

5) The water-soluble fluorescent dye of the present invention is compatible with different bioorthogonal groups without affecting its fluorescence properties, and is compatible with the labeling of polypeptides, proteins, nucleic acids, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a fluorescence emission spectrum of the water-soluble fluorescent dye solutions prepared in Examples 1-3 and Comparative Examples 1-3.

FIG. 2 is an excitation emission fluorescence spectrum of the water-soluble fluorescent dye solution prepared in Example 4.

FIG3 is a fluorescence emission spectrum of the water-soluble fluorescent dye solution prepared in Examples 5-8.

FIG4 is a physical picture of the red fluorescence emission of the water-soluble fluorescent dye solution prepared in Example 9 under a green laser pen.

Figure 5: (a) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 10 emitting blue light under the excitation of violet light; (b) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 11 emitting cyan light under the excitation of violet light; (c) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 12 emitting yellow-green light under the excitation of violet light; (d) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 12 emitting red light under the excitation of green light.

FIG6 is an ultraviolet absorption spectrum of the water-soluble fluorescent dye solution prepared in Example 13.

FIG7 is a fluorescence emission diagram of the water-soluble fluorescent dye solution prepared in Example 13 under 600 nm excitation.

Figure 8: (a) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 14; (b) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 14 emitting red light under the excitation of a green laser pen.

Figure 9: (a) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 15 emitting orange-red light under the excitation of a green laser pen; (b) is a physical picture of the water-soluble fluorescent dye solution prepared in Example 16 emitting red light under the excitation of a green laser pen.

FIG10 is an excitation emission fluorescence spectrum of the water-soluble fluorescent dye solution prepared in Example 17.

FIG11 is a fluorescence spectrum of the water-soluble fluorescent dye solution prepared in Example 18 at the beginning and after being placed at room temperature for 180 days.

FIG12 is a fluorescence image of the natural protein labeled with the water-soluble fluorescent dye prepared in Example 20 under 365 nm excitation.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the invention are not limited thereto. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

In the following expressions, "peptide", "peptide compound", "peptide sequence" and "peptide substrate" all refer to the peptide compound in the present invention.

Example 1

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Gly-Tyr (SEQ ID No. 1) under hypoxic conditions comprises the following steps:

(1) Prepare the phosphate buffer solution of the substrate peptide. Weigh 20 mg of dipeptide Gly-Tyr powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 0.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 24 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 1, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 573nm.

Example 2

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Met-Tyr-Gly (SEQ ID No. 12) under hypoxic conditions comprises the following steps:

(1) Prepare the phosphate buffer solution of the substrate peptide. Weigh 2 mg of Met-Tyr-Gly powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 9.0, 0.05 M) and stir to fully dissolve it;

(2) continuously introducing high-purity nitrogen into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.0 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding horseradish catalase to the sample solution of step (2), with the final concentration controlled to be 5000 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 24 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 1, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 564nm.

Example 3

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Gly-Tyr-Lys-OMe (SEQ ID No. 22) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 4 mg of Gly-Tyr-Lys-OMe (SEQ ID No. 22) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 2.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 24 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 1, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 570nm.

Example 4

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Sec-Tyr-Gly (SEQ ID No. 6) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Sec-Tyr-Gly (SEQ ID No. 6) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The solution after the reaction is green. When a laser pen with a wavelength of 365nm is used to irradiate the aqueous solution of the product, obvious red fluorescence can be observed. The result is shown in Figure 1;

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-800nm under different excitation wavelengths. The obtained excitation emission fluorescence spectrum results are shown in Figure 2, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 668nm.

Example 5

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Trp-Tyr-Gly (SEQ ID No. 9) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Trp-Tyr-Gly (SEQ ID No. 9) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 3, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 576nm.

Example 6

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Phe-Tyr-Gly (SEQ ID No. 10) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Phe-Tyr-Gly (SEQ ID No. 10) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 3, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 578nm.

Example 7

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of His-Tyr-Gly (SEQ ID No. 11) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of His-Tyr-Gly (SEQ ID No. 11) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 3, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 574nm.

Example 8

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction Phe-Phe-His-Try-Gly (SEQ ID No. 19) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Phe-Phe-His-Try-Gly (SEQ ID No. 19) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) The sample of step (2) was transferred to a sealed deoxygenated glove box, and tryptophan dioxygenase was added to the sample solution of step (2) to a final concentration of 500 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 3, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 574nm.

Example 9

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Pro-Tyr-Gly (SEQ ID No. 14) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Pro-Tyr-Gly (SEQ ID No. 14) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The solution after the reaction is light yellow. When a laser pen with a wavelength of 450nm is used to irradiate the aqueous solution of the product, obvious red fluorescence can be observed. The result is shown in Figure 4;

Example 10

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Tyr-Ser-Gly under hypoxic conditions comprises the following steps:

(1) Prepare the phosphate buffer solution of the substrate peptide. Weigh 20 mg of Tyr-Ser-Gly powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum under 365nm laser excitation. The results are listed in Table 1; when the product was excited by light of 365nm wavelength, blue fluorescence was visible, and the result is shown in Figure 5 (a).

Embodiment 11

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Tyr-Tyr-Ser-Leu (SEQ ID No. 18) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Tyr-Tyr-Ser-Leu (SEQ ID No. 18) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding Bacillus bisporus tyrosinase (AbTYR) to the sample solution of step (2), with the final concentration controlled to be 5 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum under 365nm laser excitation. The results are listed in Table 1; when the product was excited by light of 365nm wavelength, blue-green fluorescence was visible, and the result is shown in Figure 5 (b).

Example 12

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Ser-Tyr-Ala (SEQ ID No. 13) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Ser-Tyr-Ala (SEQ ID No. 13) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding laccase to the sample solution of step (2), with the final concentration controlled to be 1 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum under 365nm laser excitation, and the results are listed in Table 1; when the product was excited by light of 365nm wavelength, yellow-green fluorescence was visible, and the result is shown in Figure 5 (c); when the product was excited by light of 550nm wavelength, red fluorescence was visible, and the result is shown in Figure 5 (d).

Example 13

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Gln-Tyr-Gly (SEQ ID No. 16) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Gln-Tyr-Gly (SEQ ID No. 16) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding laccase to the sample solution of step (2), with the final concentration controlled to be 1 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum within the range of 300-800nm, and the results are shown in Figure 6; the fluorescence emission spectrum within the range of 300-800nm was measured, and the results are shown in Figure 7. The corresponding maximum ultraviolet absorption and fluorescence emission values are listed in Table 1. The results show that the maximum absorption peak of the solution is located at 600nm, and has a very large red-shifted fluorescence emission, which is located at 650nm and 688nm, respectively. Its fluorescence emission is red and close to the near-infrared region. It shows that the fluorescence performance of the present invention is significantly improved.

Embodiment 14

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Gln-Tyr-His (SEQ ID No. 15) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Gln-Tyr-His (SEQ ID No. 15) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding laccase to the sample solution of step (2), with the final concentration controlled to be 1 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum under 400nm laser excitation, and the results are listed in Table 1; the solution after the reaction was blue, as shown in Figure 8 (a); the product was excited by light of 550nm wavelength, and orange-red fluorescence was visible, as shown in Figure 8 (b).

Embodiment 15

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Asp-Tyr-Gly (SEQ ID No. 8) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Asp-Tyr-Gly (SEQ ID No. 8) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding laccase to the sample solution of step (2), with the final concentration controlled to be 1 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum under 400nm laser excitation. The results are listed in Table 1; the product after the reaction solution was excited with light of 550nm wavelength, and orange-red fluorescence was visible. The result is shown in Figure 9 (a).

Example 16

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction Z-Gln-Tyr-Gly-Gly (SEQ ID No. 21) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Z-Gln-Tyr-Gly-Gly (SEQ ID No. 21) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding laccase to the sample solution of step (2), with the final concentration controlled to be 1 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and the fluorescence spectrum under 400nm laser excitation, and the results are listed in Table 1; the product after the reaction was excited by light with a wavelength of 550nm, and red fluorescence was visible, as shown in Figure 9 (b). This shows that the substituents and non-ortho amino acids have little effect on the luminescent properties.

Embodiment 17

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Hyp-Tyr-Gly (SEQ ID No. 7) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Hyp-Tyr-Gly (SEQ ID No. 7) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding laccase to the sample solution of step (2), with the final concentration controlled to be 1 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-800nm under different excitation wavelengths. The obtained excitation emission fluorescence spectrum results are shown in Figure 10, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 605nm.

Embodiment 18

A method for preparing a water-soluble fluorescent dye based on an enzyme-catalyzed reaction of Gly-Tyr-Lys (SEQ ID No. 17) under hypoxic conditions comprises the following steps:

(1) Prepare a phosphate buffer solution of the substrate peptide. Weigh 20 mg of Gly-Tyr-Lys (SEQ ID No. 17) powder and place it in a 5 ml conical flask, then add 4 ml of phosphate buffer solution (pH = 8.5, 0.1 M) and stir to fully dissolve it;

(2) continuously introducing high-purity argon gas into the solution system to deoxygenate the solution, and using a dissolved oxygen tester to test the dissolved oxygen concentration of the system to be 1.5 mg/L;

(3) transferring the sample of step (2) into a sealed and deoxygenated glove box, and adding laccase to the sample solution of step (2), with the final concentration controlled to be 1 UI/mg;

(4) The solution in step (3) is subjected to an enzyme-catalyzed reaction at 37° C. to obtain a water-soluble fluorescent dye after 48 hours of reaction.

The product solution after the reaction was placed in a quartz cuvette to measure its ultraviolet absorption spectrum in the range of 300-800nm and its fluorescence spectrum in the range of 450-750nm when the excitation wavelength was 400nm. The fluorescence spectrum results are shown in Figure 11, and the corresponding maximum absorption wavelength and maximum emission wavelength are listed in Table 1. It can be seen that its fluorescence emission peak is located at 570nm.

Example 19 Stability Test of Fluorescent Dye

The product solution obtained in Example 18 was heated to 80°C for inactivation and then placed at room temperature for 180 days. The fluorescence spectrum of the product solution in the range of 450-750nm was measured again at an excitation wavelength of 400nm. The fluorescence spectrum results are shown in FIG11 . The results show that the fluorescence spectrum of the product solution has hardly changed after the sample has been placed for 180 days, indicating good stability.

Example 20 Protein labeling with fluorescent dyes

The reaction substrate of the water-soluble fluorescent dye provided by the present invention is a conventionally codable amino acid sequence, and the reaction conditions for forming fluorescent emission are mild, so it can be used for fluorescent labeling of proteins containing corresponding sequences. For example, from the protein database (PDB; http://www.rcsb.org/pdb/), a characteristic sequence containing GY in the casein sequence can be queried, so according to the conditions of Example 1, the peptide substrate is replaced with casein of equal mass; the solution after the reaction is irradiated with a laser lamp with a wavelength of 365nm to obtain an aqueous solution of the product, and obvious fluorescence can be observed. The results are shown in Figure 12; it is obvious that the preparation method of the water-soluble fluorescent dye provided by the present invention is suitable for protein labeling, which is a mild and efficient fluorescent labeling strategy.

Comparative Example 1

In Example 1, only the deoxygenation step is omitted, and the other steps remain unchanged. Under the same feed ratio conditions, the solution obtained by the reaction is regarded as Comparative Example 1;

Similarly, the product solution after the reaction was placed in a quartz cuvette to measure its fluorescence spectrum in the range of 450-750 nm. The fluorescence spectrum result is shown in FIG1 , and it can be seen that the fluorescence emission intensity is significantly reduced, and the emission peak is located at 511 nm.

Comparative Example 2

In Example 2, only the deoxygenation step is omitted, and the other steps remain unchanged. Under the same feed ratio conditions, the solution obtained by the reaction is regarded as Comparative Example 2;

Similarly, the product solution after the reaction was placed in a quartz cuvette to measure its fluorescence spectrum in the range of 450-750 nm. The fluorescence spectrum results are shown in FIG1 , and it can be seen that the fluorescence emission intensity is significantly reduced, and there is almost no fluorescence emission.

Comparative Example 3

In Example 3, only the deoxygenation step is omitted, and the other steps remain unchanged. Under the same feed ratio conditions, the solution obtained by the reaction is regarded as Comparative Example 3;

Similarly, the product solution after the reaction was placed in a quartz cuvette to measure its fluorescence spectrum in the range of 450-750 nm. The fluorescence spectrum result is shown in FIG1 , and it can be seen that the fluorescence emission intensity is significantly reduced, and the emission peak is located at 508 nm.

It can be seen from the results of Comparative Examples 1-3 above that the deoxygenation process step proposed in the present invention is one of the key steps to obtain a water-soluble fluorescent dye with high quantum yield and long wavelength fluorescence emission.

Table 1 Optical characteristics of the products obtained in the present invention (maximum ultraviolet absorption and fluorescence emission peak)

Among them, except for those with clear conditions, the data of (SEQ ID No2), (SEQ ID No3), (SEQ ID No4), (SEQ ID No5) and (SEQ ID No19) were obtained according to the conditions of Example 1.

The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Sequence Listing

<110> Institute of Process Engineering, Chinese Academy of Sciences

<120> A water-soluble fluorescent dye based on oxygen-controlled enzyme reaction, preparation method and application

<130> CP2022059

<141> 2022-04-21

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2

<212> PRT

<213> Unknown

<400> 1

Gly Tyr

1

<210> 2

<211> 2

<212> PRT

<213> Unknown

<400> 2

Phe Tyr

1

<210> 3

<211> 2

<212> PRT

<213> Unknown

<400> 3

His Tyr

1

<210> 4

<211> 1

<212> PRT

<213> Unknown

<400> 4

Tyr

1

<210> 5

<211> 1

<212> PRT

<213> Unknown

<400> 5

Tyr

1

<210> 6

<211> 2

<212> PRT

<213> Unknown

<400> 6

Tyr Gly

1

<210> 7

<211> 2

<212> PRT

<213> Unknown

<400> 7

Tyr Gly

1

<210> 8

<211> 3

<212> PRT

<213> Unknown

<400> 8

Asp Tyr Gly

1

<210> 9

<211> 3

<212> PRT

<213> Unknown

<400> 9

Trp Tyr Gly

1

<210> 10

<211> 3

<212> PRT

<213> Unknown

<400> 10

Phe Tyr Gly

1

<210> 11

<211> 3

<212> PRT

<213> Unknown

<400> 11

His Tyr Gly

1

<210> 12

<211> 3

<212> PRT

<213> Unknown

<400> 12

Met Tyr Gly

1

<210> 13

<211> 3

<212> PRT

<213> Unknown

<400> 13

Ser Tyr Ala

1

<210> 14

<211> 3

<212> PRT

<213> Unknown

<400> 14

Pro-Tyr Gly

1

<210> 15

<211> 3

<212> PRT

<213> Unknown

<400> 15

Gln Tyr His

1

<210> 16

<211> 3

<212> PRT

<213> Unknown

<400> 16

Gln Tyr Gly

1

<210> 17

<211> 3

<212> PRT

<213> Unknown

<400> 17

Gly Tyr Lys

1

<210> 18

<211> 4

<212> PRT

<213> Unknown

<400> 18

Tyr Tyr Ser Leu

1

<210> 19

<211> 4

<212> PRT

<213> Unknown

<400> 19

Pro Phe Gln Gly

1

<210> 20

<211> 4

<212> PRT

<213> Unknown

<400> 20

Phe Phe His Gly

1

<210> 21

<211> 4

<212> PRT

<213> Unknown

<400> 21

Gln Tyr Gly Gly

1

<210> 22

<211> 3

<212> PRT

<213> Unknown

<400> 22

Gly Tyr Lys

1

Claims (10)

1. The water-soluble fluorescent dye of a peptide group is characterized in that the peptide of the formula (1) or the formula (2) or the formula (3) or at least one or more of the salts or derivatives thereof is subjected to enzymatic oxidation reaction under the condition of low oxygen, wherein the condition of low oxygen refers to the concentration of dissolved oxygen in a reaction system is between 0.1mg/L and 5 mg/L;

the formula (1) is NH ₂ -X ₁ -Tyr-COOH；

The formula (2) is NH ₂ -(A)n-X ₁ -Tyr-X ₂ -COOH；

The formula (3) is NH ₂ -X ₁ -Tyr-X ₂ -(A)n-COOH；

Wherein X is ₁ Is an amino acid; x is X ₂ Is an amino acid; a is an amino acid;

n is an integer of 0 or more, and when n=0, (a) n represents a chemical bond, and when n=1, (a) n is selected from any one of amino acids.

2. A class of peptidyl water soluble fluorescent dyes according to claim 1, wherein X ₁ Any one of glycine, serine, hydroxyproline, selenocysteine, glutamine, methionine, proline, alanine, valine, leucine, isoleucine, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and pyrrolysine;

preferably X ₂ Is any one of glycine, hydroxyproline, selenocysteine, glutamine, pyrrolysine, methionine, alanine, valine, leucine, isoleucine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

3. A class of peptidyl water-soluble fluorescent dyes according to claims 1-2, characterized in that the salt of the peptide of formula (1) or (2) or (3), in the form of a salt, is selected from one of acetate, hydrochloride, phosphate and trifluoroacetate;

the peptide derivative of formula (1) or (2) or (3) may comprise NH ₂ A terminal protecting group Trt, boc, fmoc, cbz/Z, allyl; or a COOH terminal protecting group OFm, otbu, OBzl, OAll, OMe, OEt; or NH ₂ Peptide derivatives with both terminal and COOH terminal protected.

4. A peptide-based water-soluble fluorescent dye according to claims 1-3, characterized in that the peptide of formula (1) or formula (2) or formula (3) is one of the following sequences:

reference numerals Sequence(s) (SEQ ID No1) Gly-Tyr (SEQ ID No2) Phe-Tyr (SEQ ID No3) His-Tyr (SEQ ID No4) Sec-Tyr (SEQ ID No5) Hyp-Tyr (SEQ ID No6) Sec-Tyr-Gly (SEQ ID No7) Hyp-Tyr-Gly (SEQ ID No8) Asp-Tyr-Gly (SEQ ID No9) Trp-Tyr-Gly (SEQ ID No10) Phe-Tyr-Gly (SEQ ID No11) His-Tyr-Gly (SEQ ID No12) Met-Tyr-Gly (SEQ ID No13) Ser-Tyr-Ala (SEQ ID No14) Pro-Tyr-Gly (SEQ ID No15) Gln-Tyr-His (SEQ ID No16) Gln-Tyr-Gly (SEQ ID No17) Gly-Tyr-Lys (SEQ ID No18) Tyr-Tyr-Ser-Leu (SEQ ID No19) Pro-Phe-Gln-Try-Gly (SEQ ID No20) Phe-Phe-His-Try-Gly (SEQ ID No21) Cbz-Gln-Tyr-Gly-Gly (SEQ ID No22) Gly-Tyr-Lys-OMe

Oxidase is used in the enzymatic oxidation reaction; preferably, the oxidase is selected from horseradish peroxidase, bisspore tyrosinase (AbTYR), human tyrosinase (hTYR), tryptophan dioxygenase, laccase, cytochrome P450 oxidase complex or a mixture of several.

5. The peptide-based water-soluble fluorescent dye according to any one of claims 1 to 4, having a fluorescence emission peak of 530nm or more, preferably 550nm or more, further preferably 560nm or more.

6. A process for the preparation of a peptide-based water-soluble fluorescent dye according to any one of claims 1-4, comprising the following preparation steps:

(1) Preparing a buffer solution of a substrate peptide;

(2) Carrying out deoxidization treatment on the solution system obtained in the step (1);

(3) Transferring the sample in the step (2) into a sealed deoxidizing box, and adding a proper amount of oxidase into the sample solution in the step (2);

(4) The solution obtained in the step (3) reacts at the temperature of 5-50 ℃ to obtain the water-soluble fluorescent dye;

preferably, the reaction time is 12 to 240 hours; further preferably, the reaction time is from 12 to 120 hours; more preferably, the reaction time is 24 to 72 hours.

7. The method for preparing a peptide-based water-soluble fluorescent dye according to claim 6, wherein the buffer solution is a phosphate buffer solution with a pH of 7.0-10.0; the concentration of the peptide is 0.1-20mg/mL; preferably, the concentration of peptide is 1-10mg/mL;

in the step (3), the concentration range of oxidase based on the amount of peptide is as follows: 1-5000UI/mg; preferably, the oxidase concentration ranges are: 2-1000UI/mg.

8. The method for preparing the peptide-based water-soluble fluorescent dye according to claim 6 or 7, wherein the deoxidizing treatment comprises the modes of high-purity inert gas introduction, freezing-degassing circulation, heating and ultrasonic treatment; preferably, the system is purged with a high purity inert gas and a freeze-degas cycle.

9. The method for preparing a peptide-based water-soluble fluorescent dye according to any one of claims 6-8, wherein the concentration of dissolved oxygen in the system after the deoxidation treatment is lower than 5mg/L, preferably the concentration of dissolved oxygen in the system after the deoxidation treatment is lower than 2.5mg/L; more preferably, the dissolved oxygen concentration of the system after the deoxidization treatment is lower than 2mg/L.

10. Use of a water-soluble fluorescent dye according to any one of claims 1-5 for bioimaging, fluorescent labeling, sensors, protein localization, drug development.