KR20210035501A - Synthetic method of peptide-based nitric oxide donor - Google Patents

Abstract

The present invention relates to a preparation method of a peptide which is obtained by S-nitrosylation of cysteine at the N-terminus of a peptide of 8 mer or more. By using to the preparation method of a peptide according to the present invention, a peptide of 8 mer or more, in which cysteine at the N-terminus thereof is subjected to S-nitrosylation at high yield and high purity, can be obtained through a simple preparation process.

Description

Synthetic method of peptide-based nitric oxide donor

The present invention relates to a method of preparing a nitrogen monoxide donor by performing S-nitrosylation at the N-terminus of an 8 mer or more peptide.

Recently, as the functions of Nitric Oxide (NO) in the cardiovascular, respiratory, digestive, urinary and nervous systems in the biochemistry and physiology sectors have been revealed one after another, research on nitrogen monoxide is expanding. According to recent studies, nitrogen monoxide produced in the body is a substance that plays a very important role in various physiological reactions such as vasodilation, nerve transmission, angiogenesis, phagocytosis, wound healing, prevention of thrombus formation, prevention of myocardial damage, and immune response. Is known. Due to the discovery of the importance of the physiological role of nitrogen monoxide, research on a method of stably storing nitrogen monoxide in a substance as well as accurately delivering it to a site to be delivered is also actively being conducted.

Several substances have been reported that can store or deliver nitrogen monoxide. From small single molecules to dendrimers, liposomes, nanoparticles, carbon nanotubes, porous particles, and micelles, nitrogen monoxide can be stored in a variety of materials depending on the application.

In order to deliver such nitrogen monoxide, S-nitrosothiol or the like may be used. Depending on the structure, the thiol group can act as a carrier for nitrogen monoxide (NO) or can be covalently bonded to NO. In addition, certain molecules may have some characteristics of both NO transporters and covalently bonded molecules. Certain SNO agents act as critical signaling agents in the regulation of the rate and depth of breathing (respiratory control), ventilation-perfusion matching, upper respiratory muscle tone and pulmonary vascular tone.

Some methods of modifying amino acids by subjecting S-nitrosothiol, particularly cysteine to S-nitrosylation as described above, are known. _{Methods of using NaNO 2} , administering an excessive amount of nitric oxide gas, or using S-glutathione to perform S-Nitrosylation on cysteine are known. However, these methods of performing S-nitrosylation on cysteine are to perform S-nitrosylation on a conventional short peptide of 5mer or less or S-nitrosylation on cysteine contained in the middle of the peptide. Despite this, there is no known method for performing S-Nitrosylation on long peptides.

Under this background, the present inventors developed a technique for performing S-nitrosylation at the N-terminus for long peptides of 8mer or more, and S-nitrosylation on the cysteine at the N-terminus of the 8-mer or more peptide with high yield and purity. The present invention was completed by performing (S-nitrosylation).

The present inventors have conducted research and development on a variety of known S-nitrosylation methods for performing S-nitrosylation at the N-terminus for long peptides of 8-mer or more, but synthesis itself is not performed with the majority of known methods. It was confirmed that it was not. Accordingly, after hard work, suitable solvent conditions, temperature conditions, and S-nitrosylation to allow S-nitrosylation to the thiol group of the N-terminal cysteine of a long peptide of 8-mer or more with high yield and purity. The present invention was completed by finding a suitable nitrogen source and completing the manufacturing process.

One object of the present invention is (a) dissolving a peptide having an amino acid sequence of 8-mer or more in which cysteine is bonded at the N-terminus in a solvent containing water and acetonitrile and cooling to 0 to 10°C; (b) adding t-BuNO ₂ dropwise and stirring; And (c) obtaining a peptide having S-nitrosylation at the N-terminus from the reaction product of step (b); S-nitrosylation of the cysteine at the N-terminus comprising It is to provide a method for preparing the peptide.

Another object of the present invention is (a) a peptide having an amino acid sequence of SEQ ID NO: 1 to which a cysteine is bound to the N-terminus, and an amino acid sequence of SEQ ID NO: 1 to which a cysteine is bound to the N-terminus, and at least linked to the C-terminus thereof Dissolving any one of the peptides selected from one to three peptides having the amino acid sequence of SEQ ID NO: 2 in a solvent containing water and acetonitrile and cooling to 0 to 10°C; (b) adding t-BuNO ₂ dropwise and stirring; And (c) obtaining a peptide having S-nitrosylation at the N-terminus from the reaction product of step (b); S-nitrosylation of the cysteine at the N-terminus comprising It is to provide a method for preparing the peptide.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

The nomenclature used to describe the peptide follows conventional practice. Although not specifically indicated, the amino- and carboxyl-terminal groups are of the form that assumes physiological pH values unless otherwise specified. In the amino acid structural formula, each residue is generally denoted by a standard three letter or single letter designation. The amino acid sequences of the peptides presented herein are generally designated using standard single letter symbols (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I) , Isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine ).

In the present invention, the term "SNO" or "S-NO" refers to a form in which NO is bound to the S group. For example, C(SNO) means that NO is bonded to the thiol group of cysteine.

One aspect of the present invention for achieving the above object is, (a) a peptide having an amino acid sequence of 8-mer or more in which a cysteine is bonded at the N-terminus is dissolved in a solvent containing water and acetonitrile, and is 0 to 10°C. Cooling with a furnace; (b) adding t-BuNO ₂ dropwise and stirring; And (c) obtaining a peptide having S-nitrosylation at the N-terminus from the reaction product of step (b); S-nitrosylation of the cysteine at the N-terminus comprising It is to provide a method for preparing the peptide.

According to the method for preparing a peptide obtained by S-nitrosylation of the N-terminal cysteine according to the present invention, S-nitrosylation of the N-terminal cysteine with high yield and high purity through a simple manufacturing process. ), more than 8-mer peptide can be obtained.

In the present invention, the term “peptide” refers to a molecule formed by bonding amino acid residues to each other by an amide bond (or a peptide bond). The peptide may be synthesized using a gene recombination and protein expression system, and preferably may be synthesized in vitro through a peptide synthesizer or the like.

In the present invention, the term S-nitrosylation refers to the covalent addition of nitrogen monoxide to the thiol of the amino acid cysteine.

For example, as shown in Fig. 1 below, the substitution can be schematically performed. By substitution of NO in the thiol group in the N-terminal cysteine residue, a peptide containing S-nitrothiol can be prepared.

The method for preparing a peptide obtained by S-nitrosylation of an N-terminal cysteine according to the present invention includes (a) a peptide having an amino acid sequence of 8-mer or more in which cysteine is bonded to the N-terminus in water and acetonitrile. Dissolving in a solvent containing a step of cooling to 0 to 10 ℃; includes.

The peptide sequence according to the present invention is a peptide comprising an 8-mer or more amino acid sequence while including a cysteine sequence at the N-terminus. The peptide is, for example, a peptide comprising 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more amino acids.

The peptide sequence according to the present invention may preferably have the N-terminus and C-terminus protected with a protecting group. For example, the N-terminus or C-terminus is a conventional protecting group Fmoc, trityl (Trt), tert-butyloxycarbonyl (t-Boc), t-butyl (t-Bu), acetyl, -NH ₂ It can be protected by the back. Preferably, in the peptide according to the present invention, the amino group of the peptide N-terminal cysteine is protected with an acetyl group, and the C-terminus of the peptide is amidated. That is, by replacing or protecting the N-terminus and C-terminus with a protecting group as described above, it is possible to prevent the peptide from being decomposed or secondary reactions from occurring during the reaction. This peptide sequence is dissolved in a mixed solvent of water and acetonitrile as a solvent to carry out the reaction. The mixed solvent of water and acetonitrile is a mixed solvent of 1:15 to 1:25 by volume, preferably 1:17 to 1:20, and more preferably about 1:19. Only such a mixed solvent of water and acetonitrile can provide a solvent condition suitable for performing S-nitrosylation at the N-terminus of a peptide having an amino acid sequence of 8-mer or more. When the concentration of water exceeds the above range or the reaction is performed with other solvents, it is difficult to perform the desired reaction, and a mixed solvent of water and acetonitrile in the volume ratio is preferably used.

In the above step, the temperature of the solvent is preferably 0 to 10 °C, preferably 2 to 6 °C, more preferably about 4 °C. These temperature conditions provide suitable temperature conditions for the reaction with _{t-BuNO 2.} It is most preferable to perform all these steps in dark conditions.

The method for preparing a peptide obtained by S-nitrosylation of the N-terminal cysteine according to the present invention includes (b) adding t-BuNO ₂ dropwise and stirring.

The molar ratio of the peptide according to the present invention and t-BuNO ₂ is preferably 1:15 to 1:30, preferably 1:17 to 1:28, more preferably 1:19 to 1:26. According to an embodiment of the present invention, the molar ratio may be 1:20. According to another embodiment of the present invention, the molar ratio may be 1:25.

Within this range, it is easy to substitute NO for the S group of the cysteine at the N-terminus of the peptide. In addition, the method according to the present invention can provide a peptide obtained by S-nitrosylation of the desired N-terminal cysteine with high yield and purity, and the reaction is smoothly performed only when the dropwise addition method is used.

The step of adding and stirring t-BuNO ₂ dropwise is preferably performed for 30 minutes to 3 hours, more preferably 40 minutes to 2 hours, and even more preferably about 1 hour. Even within such a short time, S-nitrosylation can be performed quickly and a desired peptide can be obtained in high yield.

The method for preparing a peptide obtained by S-nitrosylation of the N-terminal cysteine according to the present invention is (c) S-nitrosylation at the N-terminus from the reaction product of step (b). It includes; obtaining a peptide.

According to the present invention, the reaction product of step (b) is dried to obtain a final desired product. Preferably, only the desired peptide can be obtained by using a conventional known method, for example, through a freeze-drying step.

The prepared S-nitrosylation peptide at the N-terminus may act as a nitrogen monoxide donor. That is, it can be used for various purposes by having the ability to release nitrogen monoxide. The present invention is the first to produce a nitrogen monoxide donor based on a peptide.

Another aspect of the present invention for achieving the above object is (a) a peptide having an amino acid sequence of SEQ ID NO: 1 in which a cysteine is bonded at the N-terminus, and an amino acid sequence of SEQ ID NO: 1 in which a cysteine is bonded at the N-terminus And dissolving any one of the peptides having an amino acid sequence of SEQ ID NO: 2 linked to the C-terminus thereof in a solvent containing water and acetonitrile and cooling to 0 to 10° C.;

(b) adding t-BuNO ₂ dropwise and stirring; And

(c) obtaining a peptide having S-nitrosylation at the N-terminus from the reaction product of step (b); S-nitrosylation of the cysteine at the N-terminus comprising It is to provide a method for producing a peptide.

In the present invention, the amino acid sequence of SEQ ID NO: 1 is a CGRKKRRQRRR sequence, that is, it may be a peptide (CPP) that includes a cysteine sequence at the N-terminus and has cell penetration properties.

The amino acid sequence of SEQ ID NO: 2 of the present invention is PKKKRKV, and can act as a nuclear localization sequence (NLS).

According to the present invention, the peptide used in the method for preparing a peptide obtained by S-nitrosylation of the N-terminal cysteine may be a peptide having the amino acid sequence of SEQ ID NO: 1. In addition, it may be a peptide sequence to which at least one, two, or three sequences of SEQ ID NO: 2 are further added thereto. That is, these sequences may be any one or more selected from the sequences set forth in SEQ ID NOs: 3 to 5.

SEQ ID NO: 3: CGRKKRRQRRRPKKKRKV

SEQ ID NO: 4: CGRKKRRQRRRPKKKRKVPKKKRKV

SEQ ID NO: 5: CGRKKRRQRRRPKKKRKVPKKKRKVPKKKRKV

These sequences are characterized by the ability to smoothly transfer nitrogen into the nucleus by including cell-penetrating and/or nuclear target sequences. That is, the manufacturing method of the present invention is a method of attaching nitrogen monoxide as an active ingredient to the N-terminal cysteine to an amino acid sequence having cell permeability and nuclear targeting ability. It is modified to have a delivery function. This method corresponds to an excellent synthesis method in that a sequence capable of delivering nitrogen monoxide into the nucleus was synthesized while using the sequence of the long peptide according to the present invention.

Steps (a), (b) and (c) may be similarly applied to the method of preparing a peptide obtained by S-nitrosylation of the N-terminal cysteine.

Accordingly, the present invention provides a modified peptide prepared by a method for producing a peptide obtained by S-nitrosylation of the N-terminal cysteine.

According to the method for preparing a peptide according to the present invention, an 8-mer or more peptide obtained by S-nitrosylation of the N-terminal cysteine in high yield and high purity can be obtained through a simple manufacturing process.

1 schematically shows a schematic diagram of performing S-nitrosylation on an N-terminal cysteine.
2 shows the results of LC/MS analysis showing the results of performing S-nitrosylation according to the present invention.
3 shows the results of LC/MS analysis showing the results of performing S-nitrosylation according to the present invention.
4 shows the results of LC/MS analysis showing the results of performing S-nitrosylation according to the present invention.
5 shows the result of confirming that synthesis is not performed by performing S-nitrosylation on cysteine using s-nitrosoglutathione (GSNO).
6 shows the result of confirming that the synthesis was not performed by performing S-nitrosylation on the cysteine located after the N-terminal γE using _{NaNO 2.}
7 shows the result of confirming that the synthesis was not performed by performing S-nitrosylation on the N-terminal cysteine using t-BuNO _{2 under DMSO solvent conditions.}
8 shows the results of measuring the nitrogen monoxide emission of the peptides according to the present invention using a NO sensor.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Peptide synthesis and separation

In order to synthesize the peptide, a general peptide solid phase synthesis method (Wang C. Chan, Perter D. White, “Fmoc Solid phase peptide synthesis”, Oxford) was carried out. More specifically, coupling was performed one by one from the N-terminus using an automatic synthesizer (ASP48S, Peptron, Inc.) using the Fmoc-SPPS (9-Fluorenylmethyloxycarbonyl solid phase peptide synthesis) method.

All monomer raw materials used for the synthesis of peptide variants have the N-terminus protected with Fmoc, and the residues are trityl (Trt), tert-butyloxycarbonyl (t-Boc), t-butyl (t-Bu), etc. Protected amino acids were used.

HBTU (2-(1H-Benzotirazloe-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate / HOBt (Hydroxybenzotriazloe) / NMM (N-Methylmorpholine) was used as the coupling reagent.

In addition, for some of the peptides used below, the N-terminus of the final peptide was acetylated (-CH ₃ CO), and the C-terminus was amidation (-NH ₂ ) to protect each terminal.

(1) Protected amino acids (8 equivalents) and coupling agent HBTU (8 equivalents) / NMM (16 equivalents) were dissolved in DMF (Dimethyl formamide) and added, followed by reaction at room temperature for 2 hours. (2) Fmoc removal was reacted twice for 5 minutes at room temperature by adding 20% (v/v) Piperidine / DMF. The reactions of (1) and (2) were repeated to produce a peptide.

Thereafter, the separation of the peptide from the resin and amino acid protecting group was performed by TFA (Trifluoroacetic acid) / EDT (1,2-ethanedithiol) / Thioanisole / TIS (Triisopropylsilane) / H ₂ O (mixing ratio (based on weight) = 90 / 2.5 / 2.5 / 2.5 / 2.5). The obtained mixed solution was subjected to an excessive amount of diethyl ether solvent stored in a refrigerator to generate a precipitate, and the obtained precipitate was centrifuged to completely precipitate, and then the excess TFA, EDT, Thioanisole, and TIS were first removed. The same procedure was repeated twice to obtain a solidified precipitate. The obtained precipitate was water containing 0.1% (v/v) TFA using a high-performance liquid chromatography instrument (Shimadzu Prominence HPLC, Japan) using a C18 column (250 mm × 22 mm, 10 μm, Vydac Everest, USA)-Acetonitrile It was separated by a liner gradient (Acetonitrile concentration: 10 to 75% (v/v)) method. The molecular weight of the purified peptide was confirmed by LC/MS (Shimadzu LC/Mass-2020, Japan), and the purified fraction was freeze-dried to obtain a white powdery peptide.

Peptides to be used in the following experiments were synthesized through the above method.

Example 2. Peptide synthesis through S-nitrosylation at the N-terminus of the peptide

Example 2-1. NO-CPP-0xNLS: Acetyl-C(SNO)GRKKRRQRRR-NH2 (SEQ ID NO: 1)

30 mg of the peptide (SEQ ID NO: 1) requiring nitration reaction in the thiol group of cysteine was dissolved in 30 ml of water and acetonitrile (1:19 volume ratio), cooled to 4°C, and then _{100 μl of t-BuNO 2} was added dropwise to 60 The reaction was carried out while stirring for a minute. Specifically, the peptide used in this example was used to protect the terminal by acetylating the cysteine at the N-terminus and amidating the C-terminus. All reactions were carried out under dark film conditions, and after the reaction, freeze-dried to obtain an off-white powdery peptide.

Specifically, the sequence, retention time, and molecular weight of the synthesized peptide are shown below and in FIG. 2.

Rt=8.392 min (various gradients over 20 min from 5% (v/v) to 100% (v/v) DW/Acetonitrile containing 0.01% (v/v) TFA); MS (ESI) 1568.8 m/e, [M+Na ⁺ ] = 1588

As can be seen above, it was confirmed that S-nitrosylation was made at the N-terminus through the exact mass value of the synthesized peptide.

Example 2-2. NO-CPP-1xNLS: Acetyl-C(SNO)GRKKRRQRRRPKKKRKV-NH2 (SEQ ID NO: 3)

30 mg of the peptide (SEQ ID NO: 3) requiring nitration reaction in the thiol group of cysteine was dissolved in 30 ml of water and acetonitrile (1:19 volume ratio), cooled to 4°C, and then _{100 μl of t-BuNO 2} was added dropwise to 60 The reaction was carried out while stirring for a minute. Specifically, the peptide used in this example was used to protect the terminal by acetylating the cysteine at the N-terminus and amidating the C-terminus. All reactions were carried out under blackout conditions. Then, it was freeze-dried to obtain a peptide in the form of an off-white powder.

Specifically, the sequence, retention time, and molecular weight of the synthesized peptide are shown below and in FIG. 3.

Rt= 6.158 min (various gradients over 20 min from 5% (v/v) to 100% (v/v) DW/Acetonitrile containing 0.01% (v/v) TFA); MS(ESI) 2433.4 m/e, [M+Na ⁺ ] = 2455

As can be seen above, it was confirmed that S-nitrosylation was made at the N-terminus through the exact mass value of the synthesized peptide.

Example 2-3. NO-CPP-2xNLS: Acetyl-C(SNO)GRKKRRQRRRPKKKRKVPKKKRKV-NH2 (SEQ ID NO: 4)

30 mg of the peptide (SEQ ID NO: 4) requiring a nitration reaction in the thiol group of cysteine was dissolved in 30 ml of water and acetonitrile (1:19 volume ratio), cooled to 4°C, and then _{100 μl of t-BuNO 2} was added dropwise to 60 The reaction was carried out while stirring for a minute. Specifically, the peptide used in this example was used to protect the terminal by acetylating the cysteine at the N-terminus and amidating the C-terminus. All reactions were carried out under blackout conditions. Then, it was freeze-dried to obtain a peptide in the form of an off-white powder.

Specifically, the sequence, retention time, and molecular weight of the synthesized peptide are shown below and in FIG. 4.

Rt = 5.983 min (various gradients over 20 min from 5% (v/v) to 100% (v/v) DW/Acetonitrile containing 0.01% (v/v) TFA); MS (ESI) 3298.0 m/e, [M+H+] = 3319

As can be seen above, it was confirmed that S-nitrosylation was made at the N-terminus through the exact mass value of the synthesized peptide.

Comparative Example 1. S-nitrosylation of cysteine in a peptide using s-nitrosoglutathione (GSNO)

SEQ ID NO: 6 (YGRKKRRQRRIISNASCTTN) 10 mg of the peptide and 2 mg of s-nitrosoglutathione (GSNO) _{were added at 37° C. under 200 mM NaHCO 3} buffer solvent conditions to react overnight. Then, it was freeze-dried to obtain a peptide in the form of an off-white powder.

The results are shown in FIG. 5.

As can be seen in FIG. 5, the mass of the peptide having S-nitrosylation should be confirmed as 2592, but the corresponding peak was not confirmed, and thus it was confirmed that synthesis was not performed.

Comparative Example 2. NaNO ₂ S-nitrosylation at the N-terminus using

10 mg of the peptide of SEQ ID NO: 7 (CGYGRKKRRQRRR) and _{5 mg of NaNO 2} were added at room temperature under 1M HCl conditions to react overnight. All reactions were carried out under blackout conditions. Then, it was freeze-dried to obtain a peptide in the form of an off-white powder.

The results are shown in FIG. 6.

As can be seen in FIG. 6, the mass of the peptide in which S-nitrosylation was performed should be confirmed as 1877, but the corresponding peak was not confirmed, and thus it was confirmed that synthesis was not performed.

Comparative Example 3. t-BuNO ₂ S-nitrosylation at the N-terminus using

10 mg of the peptide of SEQ ID NO: 8 (CGRKKRRQRRRPPQ) and _{20 μl of t-BuNO 2} were added at 4° C. under DMSO solvent conditions to react overnight. All reactions were carried out under blackout conditions. Then, it was freeze-dried to obtain a peptide in the form of an off-white powder.

The results are shown in FIG. 7.

As can be seen in FIG. 7, the mass of the peptide having S-nitrosylation should be confirmed to be 1850, but the corresponding peak was not confirmed, and thus it was confirmed that synthesis was not performed. Specifically, the mass value of the peptide in the form of NO conjugation should be (1850+1)/2 = 926, but as a result of the experiment, it could be confirmed that the synthesis was not made because a value of about 21, which was 1871, was formed as Main.

Example 3. Confirmation of NO release of S-nitrosylation peptide

NO sensor (ISO-NOP, World Precision Instruments (WPI)) to release nitrogen monoxide of the peptides synthesized in Examples 2-1 to 2-3 at a concentration of 50 μM dissolved in a PBS buffer (pH 7.4) at room temperature. ), Sarasota, USA).

The results are shown in FIG. 8.

As shown in FIG. 8, the peptides according to the present invention in which the N-terminal cysteine is S-nitrosylation exhibited excellent effects as nitrogen monoxide donors by releasing nitrogen monoxide.

As the specific parts of the present invention have been described in detail above, it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> EWHA UNIVERSITY-INDUSTRY COLLABORATION FOUNDATION <120> Synthetic method of peptide-based nitric oxide donor <130> EWHA1.66P <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CPP <400> 1 Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Nuclear Localization Sequence <400> 2 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CPP-1xNLS <400> 3 Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Lys Lys Lys Arg 1 5 10 15 Lys Val <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> CPP-2xNLS <400> 4 Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Lys Lys Lys Arg 1 5 10 15 Lys Val Pro Lys Lys Lys Arg Lys Val 20 25 <210> 5 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> CPP-3xNLS <400> 5 Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Lys Lys Lys Arg 1 5 10 15 Lys Val Pro Lys Lys Lys Arg Lys Val Pro Lys Lys Lys Arg Lys Val 20 25 30 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Sequence1 <400> 6 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ile Ile Ser Asn Ala Ser 1 5 10 15 Cys Thr Thr Asn 20 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Sequence2 <400> 7 Cys Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Sequence3 <400> 8 Cys Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10

Claims (9)

(a) dissolving a peptide having an amino acid sequence of 8-mer or more to which cysteine is bound to the N-terminus in a solvent containing water and acetonitrile and cooling to 0 to 10°C; (b) adding t-BuNO ₂ dropwise and stirring; And (c) obtaining a peptide having S-nitrosylation at the N-terminus from the reaction product of step (b); S-nitrosylation of the cysteine at the N-terminus comprising Method of producing the peptide. The method of claim 1, wherein the peptide of step (a) has an amino acid sequence of 10 mer or more, wherein the N-terminal cysteine is S-nitrosylation. The method of claim 1, wherein the volume ratio of water and acetonitrile in step (a) is 1:15 to 1:25, wherein the N-terminal cysteine is S-nitrosylation. . The method of claim 1, wherein the temperature is 2 to 6° C., wherein the N-terminal cysteine is S-nitrosylation. The method according to claim 1, wherein the peptide has the amino group of the peptide N-terminal cysteine protected with an acetyl group, and the C-terminus of the peptide is amidated. The method of claim 1, wherein in step (b) t-BuNO ₂ is added dropwise at a molar ratio of 1:15 to 1:30, the N-terminal cysteine is S-nitrosylation of the peptide Manufacturing method. The preparation of a peptide obtained by S-nitrosylation of the N-terminal cysteine according to claim 1, wherein the step of obtaining the peptide in step (c) comprises freeze-drying the reactant. Way. The method of claim 1, wherein the N-terminal S-nitrosylation peptide acts as a nitrogen monoxide donor, and the N-terminal cysteine S-nitrosylation of the peptide Manufacturing method. (a) a peptide having an amino acid sequence of SEQ ID NO: 1 to which a cysteine is bound to the N-terminus, and an amino acid sequence of SEQ ID NO: 1 to which a cysteine is bound to the N-terminus, and at least one to three sequences linked to the C-terminus thereof Dissolving any one of the peptides selected from the peptides having the amino acid sequence of number 2 in a solvent containing water and acetonitrile and cooling to 0 to 10°C;
(b) adding t-BuNO ₂ dropwise and stirring; And
(c) obtaining a peptide having S-nitrosylation at the N-terminus from the reaction product of step (b); S-nitrosylation of the cysteine at the N-terminus comprising Method for producing a peptide.

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