KR20230032321A - Amino acid specific masking agent and uses thereof - Google Patents

Abstract

The present invention relates to an amino acid-specific blocking agent and a usage thereof. More specifically, the present invention relates to a method for searching an amino acid-specific blocking agent using trypsin-specific fluorescent probe compounds, and to a lysine-specific blocking agent or an arginine-specific blocking agent screened thereby. By using the amino acid-specific blocking agents according to the present invention, arginine or lysine can be selectively blocked. Accordingly, when manufacturing a protein or peptide drug purified using enzymes such as a trypsin, the trypsin can recognize and cut off only a desired region of a protein or peptide to effectively produce and purify the protein or peptide drug.

Description

Amino acid specific blockers and their uses {AMINO ACID SPECIFIC MASKING AGENT AND USES THEREOF}

The present invention relates to amino acid specific blockers and uses thereof. More specifically, it relates to a method for searching for an amino acid-specific blocker using a trypsin-specific fluorescent probe compound and a lysine- or arginine-specific blocker screened by the method.

Insulin glargine (Lantus ^® , Sanofi) is a drug used for diabetes, and is a long-acting insulin derivative that maintains efficacy for a long time by supplementing the disadvantages of insulin with a short action time. Insulin glargine is designed to maintain long-lasting efficacy by replacing Gly ²¹ of insulin A-chain with Asn ²¹ and introducing two Arg (Arg ³¹ -Arg ³² ) to the terminal of B-chain.

Genetically recombinant insulin derivatives including insulin glargine are obtained in the form of ring-structured proinsulin through cultivation and fermentation using bacteria such as E. Coli or yeast, and are produced by proteolytic enzymes such as trypsin. It is produced by purification using The purification process of proinsulin using trypsin is obtained as a mixture of insulin glargine and by-products having various sequences, and the by-product having one Arg at the B-chain end (Arg ³¹ -Glargine) is better than glargine. It is made of many main products.

The trypsin enzyme used in the production and purification of glargine is a serine protease that hydrolyzes the C-terminal peptide bonds of positively charged amino acid residues such as lysine and arginine, except for proline.

Many peptide-based drugs are purified by the same method as insulin glargine, and the purification method using enzymes such as trypsin has the disadvantage of generating a mixture with by-products and is a factor in increasing the production cost of the drug. Accordingly, the development of technology capable of effectively and economically producing biological drugs including peptide drugs is required.

In addition, in order to produce drugs in high yield without producing peptides as by-products generated during the purification process of peptide drugs such as insulin glargine, arginine or lysine is selectively blocked so that trypsin recognizes only the desired portion of the protein or peptide. Cutting skills are required.

Under this background, the present inventors have made efforts to develop an amino acid-specific blocking agent using an activity-based fluorescent probe and to develop a high value-added technology capable of effectively producing and purifying a peptide drug using the same. As a result, an activity-based fluorescent probe capable of controlling fluorescence emission in response to protein activity and a specific amino acid-specific blocker using the same were developed. Specifically, by developing a trypsin-specific fluorescent probe into which arginine or lysine that can be recognized by trypsin is introduced and various blocking agents that inhibit the recognition of trypsin by blocking bases on the side chains of arginine or lysine using the same, the present invention invention was completed.

Korean Patent Publication No. 10-2017-0036643 (published on April 3, 2017) Republic of Korea Patent Publication No. 10-2017-0026284 (published on March 8, 2017)

One object of the present invention is acetic anhydride; benzoic acid anhydride; diethyl pyrocarbonate; p -toluene sulfonic acid anhydride; maleic anhydride; citraconic anhydride; phthalic anhydride; phenyl glyoxal; 4-(trifluoromethyl)phenyl glyoxal; 2-(trifluoromethyl)phenyl glyoxal; 4-nitro phenyl glyoxal; 4-methoxy phenyl glyoxal; 6-methoxy-2-naphthyl glyoxal; benzaldehyde; 1,2 cyclohexadione; and 2,3-butadione compounds.

Another object of the present invention is to provide a method for searching for the amino acid-specific blocker using a trypsin-specific fluorescent probe compound.

In order to achieve the above object, the present invention provides acetic anhydride; benzoic acid anhydride; diethyl pyrocarbonate; p -toluene sulfonic acid anhydride; maleic anhydride; citraconic anhydride; phthalic anhydride; phenyl glyoxal; 4-(trifluoromethyl)phenyl glyoxal; 2-(trifluoromethyl)phenyl glyoxal; 4-nitro phenyl glyoxal; 4-methoxy phenyl glyoxal; 6-methoxy-2-naphthyl glyoxal; benzaldehyde; 1,2 cyclohexadione; and 2,3-butadione compounds.

In addition, the present invention provides a method for searching for the amino acid-specific blocker using a trypsin-specific fluorescent probe compound represented by Formula 1 below:

[Formula 1]

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Arginine or lysine can be selectively blocked by using an amino acid-specific blocking agent according to the present invention, specifically, an arginine- or lysine-specific blocking agent. Accordingly, when producing a protein or peptide drug that is purified using an enzyme such as trypsin, such as insulin glargine, the protein or peptide drug can be effectively produced and purified because trypsin can recognize and cleave only a desired portion of the protein or peptide. there is.

1 is a diagram schematically showing the concept of enzymatic cleavage of a peptide fluorescent probe according to an embodiment of the present invention by trypsin.
Figure 2 is a peptide fluorescent probe according to an embodiment of the present invention under trypsin concentration dependent treatment (0.025, 0.1, 0.25, 1, 2.5, 10, 25 μg / mL) conditions (A) Rh-Lys-Gly-Leu ( Ac) and (B) are diagrams showing fluorescence intensity after site-specific cleavage of Rh-Arg-Gly-Leu (Ac). Here, (A) Rh-Lys-Gly-Leu (Ac) is a lysine-specific fluorescent probe (LysFB), and (B) Rh-Arg-Gly-Leu (Ac) is an arginine-specific fluorescent probe (LysFB).
3 shows (A) Rh-Lys-Gly-Leu (Ac), a peptide fluorescent probe according to an embodiment of the present invention, under trypsin concentration-dependent treatment conditions (0.25, 0.45, 0.65, 0.85, and 1 μg/mL); (B) It is a diagram showing fluorescence intensity after site-specific cleavage of Rh-Arg-Gly-Leu (Ac). Here, (A) Rh-Lys-Gly-Leu (Ac) is a lysine-specific fluorescent probe (LysFB), and (B) Rh-Arg-Gly-Leu (Ac) is an arginine-specific fluorescent probe (ArgFB).
4 is a diagram schematically showing the concept of amino acid blocking using a peptide fluorescent probe according to an embodiment of the present invention and evaluation of the inhibitory activity of the blocking agent. Specifically, (A) fluorescence intensity change after site-specific cleavage of the peptide fluorescent probe with trypsin; And (B) is a diagram showing the effect of amino acid blocking on the cleavage of the peptide fluorescent probe by trypsin.
5 is a diagram showing the degree of amino acid blocking activity of acetic anhydride according to an embodiment of the present invention. Specifically, the degree of lysine and arginine blocking activity of acetic anhydride using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. is also confirmed.
6 is a diagram showing the degree of amino acid blocking activity of benzoic anhydride according to an embodiment of the present invention. Specifically, the degree of lysine and arginine blocking activity of benzoic anhydride using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. is also confirmed.
7 is a diagram showing the degree of amino acid blocking activity of diethyl pyrocarbonate according to an embodiment of the present invention. Specifically, lysine and arginine blocking in diethyl pyrocarbonate using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. It is a diagram confirming the degree of activity.
8 is a diagram showing the degree of amino acid blocking activity of p -toluenesulfonic anhydride according to an embodiment of the present invention. Specifically, using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes, respectively, in the presence of trypsin, lysine and It is a diagram confirming the degree of arginine blocking activity.
9 is a diagram showing the degree of amino acid blocking activity of maleic anhydride according to an embodiment of the present invention. Specifically, lysine and arginine blocking activity of maleic anhydride using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. It is a way to confirm the degree.
10 is a diagram showing the degree of amino acid blocking activity of citraconic anhydride according to an embodiment of the present invention. Specifically, lysine and arginine blocking of citraconic anhydride using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. It is a diagram confirming the degree of activity.
11 is a diagram showing the degree of amino acid blocking activity of phthalic anhydride according to an embodiment of the present invention. Specifically, the degree of lysine and arginine blocking activity of phthalic anhydride using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. is also confirmed.
12 is a diagram showing the degree of amino acid blocking activity of phenyl glyoxal according to an embodiment of the present invention. Specifically, the lysine and arginine blocking activity of phenylglyoxal using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. It is a way to confirm the degree.
13 is a diagram showing the degree of amino acid blocking activity of 4-(trifluoromethyl)phenyl glyoxal according to an embodiment of the present invention. Specifically, 4-(trifluoromethyl)phenyl using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. It is a diagram confirming the degree of lysine and arginine blocking activity of glyoxal.
14 is a diagram showing the degree of amino acid blocking activity of 2-(trifluoromethyl)phenyl glyoxal according to an embodiment of the present invention. Specifically, using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin, respectively, 2-(trifluoromethyl)phenyl It is a diagram confirming the degree of lysine and arginine blocking activity of glyoxal.
15 is a diagram showing the degree of amino acid blocking activity of 4-nitro phenyl glyoxal according to an embodiment of the present invention. Specifically, using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes, respectively, in the presence of trypsin, lysine and 4-nitrophenylglyoxal It is a diagram confirming the degree of arginine blocking activity.
16 is a diagram showing the degree of amino acid blocking activity of 4-methoxy phenyl glyoxal according to an embodiment of the present invention. Specifically, lysine of 4-methoxyphenylglyoxal using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin. And it is a diagram confirming the degree of arginine blocking activity.
17 is a diagram showing the degree of amino acid blocking activity of 6-methoxy-2-naphthyl glyoxal hydrate according to an embodiment of the present invention. Specifically, 6-methoxy-2-naphthyl using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin, respectively. It is a diagram confirming the degree of lysine and arginine blocking activity of glyoxal hydrate.
18 is a diagram showing the degree of amino acid blocking activity of benzaldehyde according to an embodiment of the present invention. Specifically, (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) were used as peptide fluorescent probes in the presence of trypsin to determine the degree of lysine and arginine blocking activity of benzaldehyde. It is also confirmed.
19 is a diagram showing the degree of amino acid blocking activity of 1,2 cyclohexadione according to an embodiment of the present invention. Specifically, using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin, respectively, lysine and It is a diagram confirming the degree of arginine blocking activity.
20 is a diagram showing the degree of amino acid blocking activity of 2,3-butadione according to an embodiment of the present invention. Specifically, using (A) Rh-Lys-Gly-Leu (Ac) and (B) Rh-Arg-Gly-Leu (Ac) as peptide fluorescent probes in the presence of trypsin, respectively, lysine and 2,3-butadione It is a diagram confirming the degree of arginine blocking activity.
21 is a 14-mer peptide standard (Leu-Leu-Thr-Pro-Glu-Thr-Arg-Arg-Lys-Ala-Glu-Asp-Leu-Leu-NH ₂ ; LLTPETRRKAEDLL-NH ₂ ) is a diagram conceptually showing a degradation pattern for cleavage at peptide site 1 (arginine site).
22 is a diagram conceptually showing a degradation pattern for cleavage at peptide site 2 (arginine site) of a 14-mer peptide standard material according to an embodiment of the present invention by trypsin.
23 is a diagram conceptually showing a degradation pattern for cleavage at peptide site 3 (lysine site) of a 14-mer peptide standard material according to an embodiment of the present invention by trypsin.
24 is a diagram showing the results of RP-HPLC-MS (reversed-phase-high-performance liquid chromatography-mass spectrometry) analysis of a 14-mer peptide standard material according to an embodiment of the present invention.
25 is a diagram showing the results of RP-HPLC-MS analysis of fractions obtained after digesting a 14-mer peptide standard material with trypsin according to an embodiment of the present invention.
26 is a diagram conceptually showing a degradation pattern for cleavage at peptide site 1 (arginine site) of a 14-mer peptide standard by trypsin after blocking lysine with citraconic anhydride according to an embodiment of the present invention. am.
27 is a diagram conceptually showing a degradation pattern for cleavage at peptide site 2 (arginine site) of a 14-mer peptide standard by trypsin after blocking lysine with citraconic anhydride according to an embodiment of the present invention. am.
28 is a diagram showing the results of RP-HPLC-MS analysis of fractions obtained after lysine blocking with citraconic anhydride and digestion of 14-mer peptide standard materials by trypsin according to an embodiment of the present invention.
29 is a diagram conceptually showing a degradation pattern for cleavage at peptide site 3 (lysine site) of a 14-mer peptide standard by trypsin after blocking lysine with phenyl glyoxal according to an embodiment of the present invention. .

The present invention relates to the development of trypsin-specific fluorescent probes and amino acid-specific masking agnets capable of controlling trypsin-activity that selectively blocks specific amino acids such as arginine and lysine in peptides or proteins using the same. will be. Specifically, it relates to a trypsin-specific fluorescent probe capable of controlling fluorescence emission in response to trypsin activity, and to a novel amino acid blocker that exhibits a blocking effect on arginine and lysine developed using the trypsin-specific fluorescent probe.

In one aspect, the present invention provides an amino acid specific blocker selected from the following compounds:

acetic anhydride;

benzoic anhydride;

diethyl pyrocarbonate;

p -Toluene sulfonic anhydride ( p -Toluene sulfonic anhydride);

maleic anhydride;

citraconic anhydride;

phthalic anhydride;

phenyl glyoxal;

4- (trifluoromethyl) phenyl glyoxal;

2-(trifluoromethyl)phenyl glyoxal;

4-nitro phenyl glyoxal;

4-methoxy phenyl glyoxal;

6-methoxy-2-naphthyl glyoxal;

benzaldehyde;

1,2 cyclohexadione; and

2,3-butadione.

The blocking agent may be a lysine-specific blocking agent or an arginine-specific blocking agent.

As used herein, the term "specific" means that a masking agent reacts only to a specific amino acid, specifically lysine or arginine, and may be used interchangeably with "selective".

The lysine-specific blocking agent may be selected from, but is not limited to, acetic anhydride, benzoic anhydride, diethyl pyrocarbonate, p -toluene sulfonic anhydride, maleic anhydride, citraconic anhydride, and phthalic anhydride. Preferably, it may be selected from acetic anhydride, maleic anhydride, citraconic anhydride, and phthalic anhydride.

The arginine-specific blocker includes, but is not limited to, phenyl glyoxal, 4-(trifluoromethyl)phenyl glyoxal, 2-(trifluoromethyl)phenyl glyoxal, 4-nitro phenyl glyoxal, 4-methyl oxyphenyl glyoxal, 6-methoxy-2-naphthyl glyoxal, benzaldehyde, 1,2 cyclohexadione, and 2,3-butadione. Preferably, it is selected from phenyl glyoxal, 4-(trifluoromethyl)phenyl glyoxal, 2-(trifluoromethyl)phenyl glyoxal, 4-nitro phenyl glyoxal, and 4-methoxy phenyl glyoxal. can

In another aspect, the present invention provides the amino acid-specific blocker screening method of claim 1 using a trypsin-specific fluorescent probe compound represented by Formula 1 below:

[Formula 1]

during the ceremony,

또는

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or

am.

As used herein, the term "trypsin" specifically hydrolyzes the C-terminus of arginine or lysine, which is an amino acid constituting proteins. It is an essential enzyme to obtain the necessary peptides.

As used herein, the term "search" refers to finding or identifying a blocker for a specific amino acid, specifically lysine or arginine, and may be used interchangeably with "screening".

The blocking agent may be a lysine-specific blocking agent or an arginine-specific blocking agent.

In the present invention, the fluorescent probe compound may be represented by Formula 2 or Formula 3 below:

[Formula 2]

[Formula 3]

.

.

In addition, the fluorescent probe compound is characterized in that it exhibits a fluorescence on phenomenon by reacting with trypsin. The fluorescent probe compound is characterized by an increase in fluorescence intensity due to arginine or lysine cleavage activity by trypsin (see FIGS. 1 and 4).

In addition, the fluorescent probe compound does not react with trypsin by blocking a specific amino acid, specifically arginine or lysine, and thus does not generate fluorescence (see FIGS. 1 and 4).

In the present invention, the fluorescent probe compound represented by Chemical Formula 2 can specifically detect lysine.

In addition, the fluorescent probe compound represented by Chemical Formula 3 can specifically detect arginine.

Lysine or arginine can be selectively detected by measuring the fluorescence intensity by the reaction between the fluorescent probe compound and trypsin.

That is, the amino acid blocking agent can be screened by confirming that the fluorescent probe compound and the candidate amino acid blocking agent are not reacted with trypsin by blocking a specific amino acid, that is, fluorescence is not formed.

The specific amino acid may be, but is not limited to, lysine or arginine.

The amino acid blocker may be, but is not limited to, a lysine-specific inhibitor and an arginine-specific inhibitor.

The lysine-specific inhibitor may be, but is not limited to, an anhydride inhibitor or a cyclic anhydride inhibitor.

The anhydride inhibitor may be, but is not limited to, acetic anhydride, benzoic anhydride, diethyl pyrocarbonate or p -toluene sulfonic anhydride.

The cyclic anhydride inhibitor may be, but is not limited to, maleic anhydride, citraconic anhydride or phthalic anhydride.

The arginine-specific inhibitor may be, but is not limited to, an α,β-ketone aldehyde inhibitor, a monoaldehyde inhibitor, or an α,β-diketone inhibitor.

The α,β-ketone aldehyde inhibitors include, but are not limited to, phenyl glyoxal, 4-(trifluoromethyl)phenyl glyoxal, 2-(trifluoromethyl)phenyl glyoxal, 4-nitro phenyl glyoxal, 6-methoxy-2-naphthyl glyoxal or 4-methoxy phenyl glyoxal.

The monoaldehyde inhibitor may be, but is not limited to, benzaldehyde.

The α,β-diketone inhibitor may be, but is not limited to, 1,2 cyclohexadione or 2,3-butadione.

Arginine or lysine may be selectively blocked by using the amino acid-specific blocking agent, specifically, an arginine- or lysine-specific blocking agent. Accordingly, when producing a protein or peptide drug that is purified using an enzyme such as trypsin, the protein or peptide drug can be effectively produced and purified by trypsin recognizing and cutting only the desired portion of the protein or peptide. It can be widely applied in the field of pharmaceutical manufacturing.

The above objects, other objects, features and advantages of the present invention will be easily understood through the following preferred embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed content will be thorough and complete and the spirit of the present invention will be sufficiently conveyed to those skilled in the art.

I. Preparation of trypsin-specific fluorescent probes

Trypsin-specific fluorescent probes, specifically Lysine-responsive fluorescent probe (LysFB) and Arginine-responsive fluorescent probe (ArgFB), were performed according to the general procedures A to C below.

[Synthesis Procedure]

1. General Procedure A: Amide Coupling

To a solution of aniline or amine (1.0 equiv.) and AA (1.2 equiv.) in CH ₂ Cl ₂ was added DIC (1.2 equiv.) and HOBt (1.2 equiv.). The reaction mixture was stirred at room temperature (rt) for 2 hours. After completion of the reaction, the reaction solvent was evaporated and the resulting solid was purified by column chromatography to obtain the desired compound.

2. General Procedure B: Fluorenylmethyloxycarbonyl (Fmoc) Deprotection

To a solution of Fmoc protected compound (1.0 equiv.) was added piperidine (1.2 equiv.) in anhydrous CH ₃ CN. The reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solvent was evaporated. The resulting residue was purified by column chromatography to obtain the desired compound.

3. General Procedure C: Acid Labile Deprotection (Boc/Pbf)

To a solution of the Boc/Pbf protected compound (1.0 eq) was added TFA (10% in solvent) in anhydrous CH ₂ Cl ₂ . The reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction mixture was basified with 1N NaOH. The compound was extracted with an organic solvent mixture (DCM:MeOH = 90:10). The organic layer was dried over Na ₂ SO ₄ and evaporated under vacuum. The resulting residue was purified by column chromatography to obtain the desired compound.

Example 1. Synthesis of lysine-detection fluorescent probe [Rh-Lys-Gly-Leu (Ac)]

[Scheme 1]

The synthesis process of the compounds shown in [Reaction Scheme 1] will be described in more detail below.

Example 1-1. Synthesis of Compound 1

(9 H -fluoren-9-yl)methyl tert -Butyl ((5 R )-6-((3'-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthene]-6'-yl)amino)-6-oxohexane-1,5-diyl)dicarbamate (1)

To a solution of methoxyrodol (100 mg, 0.29 mmol) and Fmoc-Lys(Boc)-OH (163 mg, 0.35 mmol) in chloroform (8 mL) was added EEDQ (86 mg, 0.35 mmol). The reaction mixture was stirred at room temperature (rt) for 1.5 hours. After completion of the reaction, the reaction solvent was evaporated, and the resulting solid was purified by column chromatography to obtain the desired compound 1 in 82% yield (190 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.87 (s, 1H), 8.01 (dd, J = 6.6, 1.1 Hz, 1H), 7.75-7.71 (m, 3H), 7.65-7.59 (m, 2H) , 7.57–7.51 (m, 2H), 7.37–7.33 (m, 2H), 7.29–7.23 (m, 3H), 7.08 (d, J = 7.3 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H) ), 6.71 (d, J = 2.3 Hz, 1H), 6.66–6.63 (m, 2H), 6.57 (dd, J = 8.9, 2.5 Hz, 1H), 5.74 (s, 1H), 4.68 (s, 1H) , 4.38 (d, J = 4.6 Hz, 2H), 4.31 (s, 1H), 4.17 (t, J = 7.1 Hz, 1H), 3.80 (s, 3H), 3.13–3.02 (m, 2H), 1.94 ( s, 1H), 1.83 (s, 2H), 1.71 (s, 1H), 1.56-1.44 (m, 1H), 1.40 (s, 9H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 172.4, 169.2, 161.6, 156.6, 156.1, 153.1, 152.3, 151.4, 144.4, 144.3, 141.6, 141.3, 136.3, 130.8, 129.5, 129.0, 128.2, 127.6 126.3, 125.9, 125.3, 124.5, 120.6, 115.9, 113.8, 112.7, 111.3, 106.8, 101.4, 82.6, 77.9, 66.2, 56.2, 56.1, 55.4, 47.2, 23.5, 28.5, 29.8, 29.8 HRMS (ESI ⁺ ): m/z Calcd for C ₄₇ H ₄₆ N ₃ O ₉ [M+H] ⁺ : 796.3234, Found: 796.3229.

Example 1-2. Synthesis of compound 2

Tert -Butyl(5-amino-6-((3'-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthen]-6'-yl)amino)-6-oxohexyl)carbamate (2)

Compound 2 (174 mg) was obtained in 96% yield via deprotection of Fmoc from Compound 1 (250 mg, 0.31 mmol) using piperidine (32 mg, 0.38 mmol) in anhydrous CH ₃ CN according to General Procedure B. synthesized

¹ H-NMR (400 MHz, CDCl ₃ ) δ 9.74 (s, 1H), 8.00 (d, J = 7.3 Hz, 1H), 7.82 (dd, J = 5.7, 1.6 Hz, 1H), 7.64 (t, J = 7.3 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.12 (d, J = 7.3 Hz, 1H), 7.06 (t, J = 6.4 Hz, 1H), 6.75 (d, J = 2.3 Hz, 1H), 6.70 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.59 (dd, J = 8.7, 2.3 Hz, 1H), 4.61 (s, 1H), 3.82 (s, 3H), 3.54 (s, 1H), 3.10 (s, 2H), 1.94 (s, 1H), 1.65-1.56 (m, 1H), 1.48 (s, 3H), 1.41 (s, 9H) ; ¹³ C-NMR (100 MHz, CDCL ₃ ) Δ 169.6, 161.5, 156.3, 153.3, 152.5, 151.9, 139.8, 135.1, 129.8, 129.0, 128.6, 126.7, 125.1, 124.0, 115.3, 114.5, 111.8, 111.0, 107.5, 100.9, 83.0, 79.3, 55.7, 55.2, 53.5, 40.0, 29.8, 28.5, 22.6; HRMS (ESI ⁺ ): m/z Calcd for C ₃₂ H ₃₆ N ₃ O ₇ [M+H] ⁺ : 574.2553, Found: 574.2546.

Example 1-3. Synthesis of compound 3

(9 H -fluoren-9-yl)methyl(2-(((2 R )-6-(( tert -butoxycarbonyl)amino)-1-((3'-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthene]-6'-yl)amino)-1-oxohexan-2-yl)amino)-2-oxoethyl)carbamate (3)

Compound 3 (130 mg) was prepared from Compound 2 (109 mg) using Fmoc-Gly-OH (62.14 mg, 0.21 mmol), DIC (28.78 mg, 0.23 mmol) and HOBT (30.80 mg, 0.23 mmol) according to General Procedure A. , 0.19 mmol) was synthesized in 80% yield through amide coupling.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.26 (s, 1H), 8.15 ₍ d, J = 7.8 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.85-7.81 (m , 3H), 7.76 (t, J = 7.5 Hz, 1H), 7.70 (d, J = 7.3 Hz, 1H), 7.66 (d, J = 7.8 Hz, 2H), 7.53 (t, J = 6.2 Hz, 1H) ), 7.36 (td, J = 7.3, 4.1 Hz, 2H), 7.27 (td, J = 7.4, 2.7 Hz, 2H), 7.22 (d, J = 7.8 Hz, 1H), 7.19-7.13 (m, 1H) , 6.93 (d, J = 1.8 Hz, 1H), 6.71–6.63 (m, 4H), 4.35 (q, J = 6.7 Hz, 1H), 4.25–4.16 (m, 3H), 3.78 (s, 3H), 3.65 (d, J = 6.4 Hz, 2H), 2.84 (d, J = 5.5 Hz, 2H), 1.67-1.64 (m, 1H), 1.57-1.55 (m, 1H), 1.33 (s, 2H), 1.29 -1.19 (m, 11H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 171.8, 169.7, 169.2, 161.6, 157.1, 156.1, 153.1, 152.3, 151.4, 144.4, 141.5, 141.2, 136.3, 130.8, 129.5, 129.0, 128.1, 127.6, 127.6, 126.3; 125.8; 125.3; 124.5; 120.6; 116.0; HRMS (ESI ₊ ): m/z Calcd for C ₄₉ H ₄₉ N ₄ O ₁₀ [M+H] ⁺ : 853.3449, Found: 853.3448.

Example 1-4. Synthesis of compound 4

Tert -Butyl ((5 R )-5-(2-aminoacetamido)-6-((3′-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthen]-6'-yl)amino)-6-oxohexyl)carbamate (4)

Compound 4 was synthesized from compound 3 (30 mg, 0.035 mmol) via deprotection of Fmoc using piperidine (3.59 mg, 0.042 mol) in anhydrous CH ₃ CN (726 μL) according to General Procedure B. The residue was purified by flash column chromatography on silica gel to give compound 4 (21 mg) in 95% yield.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.37 (d, J = 2.7 Hz, 1H), 8.11 (s _, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.81 (dd, J = 16.2, 2.1 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.3 Hz, 1H), 7.23 (dd, J = 7.8, 2.7 Hz, 1H), 7.16 (ddd , J = 13.7, 8.7, 1.8 Hz, 1H), 6.94 (d, J = 2.3 Hz, 1H), 6.72–6.63 (m, 4H), 4.40 (s, 1H), 3.78 (s, 3H), 3.12 ( s, 2H), 2.84 (q, J = 6.1 Hz, 2H), 1.71–1.64 (m, 1H), 1.62–1.51 (m, 1H), 1.30 (s, 12H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 173.2, 171.8, 169.2, 161.7, 156.1, 153.1, 152.3, 151.4, 141.5, 136.3, 130.8, 129.5, 129.0, 126.3, 125.3, 124.5, 116.0, 113.9, 112.7, 111.3, 106.9, 101.4, 82.6, 77.8, 63.3, 56.2, 55.4, 53.6, 44.9, 32.7, 29.8, 28.8, 23.1; HRMS (ESI ⁺ ): m/z Calcd for C ₃₄ H ₃₉ N ₄ O ₈ [M+H] ⁺ : 631.2768, Found: 631.2768.

Example 1-5. Synthesis of compound 5

Tert -Butyl ((5 R )-5-(2-(( R )-2-acetamido-4-methylpentanamido)acetamido)-6-((3'-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthene]-6'-yl)amino)-6-oxohexyl)carbamate (5)

Compound 5 (20 mg) was prepared from Compound 4 (21 mg) using Ac-Leu-OH (6.34 mg, 0.037 mmol), DIC (5.04 mg, 0.04 mmol) and HOBT (5.40 mg, 0.04 mmol) according to General Procedure A. , 0.033 mmol) was synthesized in 76% yield through amide coupling.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.18 (d, J = 6.4 Hz, 1H), 8.28 (q, J = _5.8 Hz, 1H), 8.04 (dd, J = 7.5, 2.1 Hz, 1H) ), 7.99 (d, J = 7.6 Hz, 2H), 7.84 (dd, J = 17.2, 2.1 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H) ), 7.23-7.18 (m, 2H), 6.93 (d, J = 2.3 Hz, 1H), 6.74-6.63 (m, 4H), 4.29 (q, J = 7.2 Hz, 1H), 4.18 (q, J = 7.2 Hz, 1H). 7.5 Hz, 1H), 3.78 (s, 3H), 3.67 (t, J = 5.9 Hz, 2H), 2.84 (q, J = 6.6 Hz, 2H), 1.78 (d, J = 9.1 Hz, 3H), 1.66 −1.51 (m, 3H), 1.40 (t, J = 7.1 Hz, 3H), 1.34–1.30 (m, 12H), 0.81 (dd, J = 17.2, 6.2 Hz, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 173.5, 173.4, 171.7, 170.1, 169.5, 169.2, 161.6, 156.1, 153.1, 152.3, 151.3, 141.5, 136.3, 130.8, 129.5, 129.0, 126.3, 125.3 124.5,116.0,113.9,112.6,111.3,106.9,101.4,82.6,77.8,56.2,55.4,54.1,51.9,42.6,32.0,29.8,28.8,24.7,23.5,223.3; HRMS (ESI ⁺ ): m/z Calcd for C ₄₂ H ₅₂ N ₅ O ₁₀ [M+H] ⁺ : 786.3714, Found: 786.3710.

Example 1-6. Synthesis of compound 6

(2 R )-2-(2-(( R )-2-acetamido-4-methylpentanamido)acetamido)-6-amino- N -(3'-methoxy-3-oxo-3 H -spiro [isobenzofuran-1,9'-xanthen] -6'-yl) hexanamide (6)

Compound 6 was synthesized from compound 5 (40 mg, 0.050 mmol) via deprotection of Boc using TFA (10%, 0.8 mL) in anhydrous CH ₂ Cl ₂ (8 mL) according to General Procedure C. The residue was purified by flash column chromatography on silica gel to give compound 6 (16 mg) in 45% yield.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.22 (d, J = 11.0 Hz, 1H), 8.32 (q, J = _6.4 Hz, 1H), 8.08-7.97 (m, 3H), 7.85 (t , J = 2.7 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.24–7.20 (m, 2H), 6.93 (d, J = 2.1 Hz) , 1H), 6.72–6.63 (m, J = 18.9, 8.5 Hz, 3H), 4.32 (q, J = 6.7 Hz, 1H), 4.18 (q, J = 7.5 Hz, 1H), 3.78 (s, 3H) , 3.67 (s, 2H), 2.70 (t, J = 7.5 Hz, 2H), 1.78 (d, J = 10.5 Hz, 3H), 1.74–1.68 (m, 1H), 1.61–1.54 (m, 3H), 1.50-1.37 (m, 3H), 1.35-1.30 (m, 2H), 0.84-0.78 (m, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 173.5, 173.5, 171.7, 170.2, 169.5, 169.2, 161.6, 158.9, 158.6, 158.3, 158.0, 153.1, 152.3, 151.3, 141.5, 136.3, 132.2, 132.1, 130.8, 129.5, 129.2, 128.9, 126.3, 125.3, 124.5, 122.3, 119.3, 116.3, 116.1, 113.9, 112.6, 111.3, 106.9, 101.4 29.5, 29.4, 29.2, 27.1, 24.7, 23.5, 23.0, 22.8, 22.6, 22.1; HRMS (ESI ⁺ ): m/z Calcd for C ₃₇ H ₄₄ N ₅ O ₈ [M+H] ⁺ : 686.3190, Found: 686.3187.

Example 2. Synthesis of arginine-detection fluorescent probe [Rh-Arg-Gly-Leu (Ac)]

[Scheme 2]

The synthesis process of the compounds shown in [Reaction Scheme 2] will be described in more detail below.

Example 1-7. Synthesis of compound 7

(9 H -fluoren-9-yl)methyl((2 R )-1-((3'-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthene]-6'-yl)amino)-1-oxo-5-(3-((2,2,4,6,7-pentamethyl-2, 3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)pentan-2-yl)carbamate (7)

To a solution of methoxy lodol (50 mg, 0.15 mmol) and Fmoc-Arg(Pbf)-OH (122 mg, 0.19 mmol) in CH ₂ Cl ₂ (10 mL) was added EDC (111 mg, 0.58 mmol). The reaction mixture was stirred at room temperature (rt) for 1 hour. After completion of the reaction, the reaction solvent was evaporated, and the resulting solid was purified by column chromatography to obtain the desired compound 7 in 64% yield (90 mg).

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.36 (s, 1H), _8.01 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 7.3 Hz, 3H), 7.79 (t, J = 7.5 Hz, 1H), 7.72 (t, J = 7.1 Hz, 4H), 7.40 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.3 Hz, 2H), 7.26 (d, J = 7.3 Hz, 1H), 7.19–7.16 (m, 1H), 6.98 (d, J = 2.3 Hz, 1H), 6.75–6.66 (m, 4H), 4.27 (d, J = 6.9 Hz, 2H), 4.21 (t , J = 7.1 Hz, 1H), 4.13 (q, J = 7.3 Hz, 1H), 3.81 (s, 3H), 3.06 (s, 2H), 2.91 (s, 2H), 2.45 (s, 3H), 2.38 (s, 3H), 1.95 (s, 3H), 1.67-1.59 (m, 2H), 1.51-1.42 (m, 2H), 1.36 (s, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 172.1, 169.2, 161.7, 158.0, 156.6, 153.1, 152.3, 151.4, 144.4, 144.3, 141.5, 141.3, 137.8, 136.3, 130.8, 129.5, 129.0, 129.0 128.2, 127.6, 126.3, 125.8, 125.3, 124.8, 124.5, 120.6, 116.8, 116.0, 113.8, 112.7, 111.3, 106.8, 101.4, 86.8 19.4, 18.1, 12.8; HRMS (ESI ⁺ ): m/z Calcd for C ₅₅ H ₅₄ N ₅ O ₁₀ S [M+H]+: 976.3591, Found: 976.3586.

Example 1-8. Synthesis of compound 8

(2 R )-2-amino- N -(3'-methoxy-3-oxo-3 H -Spiro[isobenzofuran-1,9'-xanthen]-6'-yl)-5-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran -5-yl) sulfonyl) guanidino) pentanamide (8)

Compound 8 (90 mg) was obtained via deprotection of Fmoc from compound 7 (122 mg, 0.13 mmol) using piperidine (12.77 mg, 0.15 mmol) in anhydrous CH ₃ CN (2.6 mL) according to General Procedure B. It was synthesized in 96% yield.

^1H -NMR (400 MHz, DMSO- d6 ) δ 7.99 (d, J = 7.8 Hz, 1H), 7.90 (dd, J = 6.4, _1.8 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H) ), 7.69 (t, J = 7.3 Hz, 1H), 7.23 (d, J = 7.3 Hz, 1H), 7.20–7.16 (m, 1H), 6.95 (d, J = 2.7 Hz, 1H), 6.71–6.63 (m, 4H), 6.33 (s, 1H), 3.78 (s, 3H), 3.01 (d, J = 5.5 Hz, 2H), 2.89 (s, 2H), 2.42 (s, 3H), 2.36 (s, 3H), 1.93 (s, 3H), 1.57-1.55 (m, 1H), 1.49-1.44 (m, 1H), 1.42-1.38 (m, 2H), 1.34 (s, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 175.5, 169.2, 161.6, 157.9, 156.6, 153.1, 152.3, 151.4, 141.6, 137.8, 136.3, 132.0, 130.8, 129.5, 128.9, 126.3, 125.3, 124.8, 124.5, 116.8, 116.0, 113.7, 112.6, 111.3, 106.7, 101.4, 86.8, 82.7, 56.2, 55.9, 43.0, 28.8, 19.5, 18.1, 12.8; HRMS (ESI ⁺ ): m/z Calcd for C ₄₀ H ₄₄ N ₅ O ₈ S [M+H] ⁺ : 754.2911, Found: 754.2908.

Example 1-9. Synthesis of compound 9

(9 H -fluoren-9-yl)methyl(2-(((2 R )-1-((3'-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthene]-6'-yl))amino)-1-oxo-5-(3-((2,2,4,6,7-pentamethyl-2 ,3-dihydrobenzofuran-5-yl) sulfonyl) guanidino) pentan-2-yl) amino) -2-oxoethyl) carbamate (9)

Compound 9 (90 mg) was prepared from compound 8 (90 mg) using Fmoc-Gly-OH (22.27 mg, 0.13 mmol), DIC (17.71 mg, 0.14 mmol) and HOBT (18.97 mg, 0.14 mmol) according to General Procedure A. , 0.12 mmol) was synthesized in 73% yield through amide coupling.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.29 (s, 1H) _, 8.20 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 6.8 Hz, 3H), 7.76 (t, J = 7.3 Hz, 1H), 7.71 (d, J = 7.3 Hz, 1H), 7.66 (d, J = 7.3 Hz, 2H), 7.53 (t, J = 6.2 Hz, 1H), 7.38-7.34 (m, 2H), 7.28-7.22 (m, 3H), 7.17 (d, J = 8.7 Hz, 1H), 6.93 (s, 1H), 6.72-6.63 (m, 3H) , 6.33 (s, 1H), 4.38 (q, J = 7.2 Hz, 1H), 4.24 (d, J = 6.4 Hz, 2H), 4.18 (t, J = 6.9 Hz, 1H), 3.78 (s, 3H) , 3.64 (d, J = 5.9 Hz, 2H), 3.01 (s, 2H), 2.86 (s, 2H), 2.41 (s, 3H), 2.34 (s, 3H), 1.92 (s, 3H), 1.70- 1.65 (m, 1H), 1.56-1.50 (m, 1H), 1.46-1.36 (m, 2H), 1.33 (s, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 171.6, 169.7, 161.6, 158.0, 157.1, 156.6, 153.1, 152.3, 151.4, 144.3, 141.4, 141.2, 137.8, 136.3, 132.0, 130.8, 129.5, 129.0 128.1, 127.6, 126.3, 125.8, 125.3, 124.8, 124.5, 120.6, 116.8, 116.0, 114.0, 112.7, 111.2, 101.4, 86.8, 82.6 HRMS (ESI ⁺ ): m/z Calcd for C ₅₇ H ₅₇ N ₆ O ₁₁ S [M+H] ⁺ : 1033.3806, Found: 1033.3805.

Example 1-10. Synthesis of compound 10

(2 R )-2-(2-aminoacetamido)- N -(3'-methoxy-3-oxo-3 H -Spiro[isobenzofuran-1,9'-xanthen]-6'-yl)-5-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran -5-yl) sulfonyl) guanidino) pentanamide (10)

Compound 10 was synthesized from compound 9 (85 mg, 0.082 mmol) via deprotection of Fmoc using piperidine (8.4 mg, 0.099 mmol) in anhydrous CH ₃ CN (1.7 mL) according to General Procedure B. The residue was purified by flash column chromatography on silica gel to give compound 10 (65 mg) in 97% yield.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.45 (s, 1H) _, 8.20 (s, 1H), 7.99 (d, J = 7.3 Hz, 1H), 7.82 (dd, J = 8.7, 1.8 Hz , 1H), 7.76 (t, J = 7.3 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.23 (dd, J = 7.3, 2.3 Hz, 1H), 7.18 (td, J = 7.8, 1.8 Hz, 1H), 6.95 (d, J = 2.3 Hz, 1H), 6.73–6.63 (m, 3H), 6.34 (s, 1H), 4.44 (s, 1H), 3.78 (s, 3H), 3.15 ( s, 2H), 3.01 (q, J = 6.1 Hz, 2H), 2.88 (s, 2H), 2.41 (s, 3H), 2.35 (s, 3H), 1.92 (s, 3H), 1.72-1.64 (m , 1H), 1.60-1.49 (m, 1H), 1.44-1.38 (m, 2H), 1.34 (s, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 171.6, 169.2, 161.6, 158.0, 156.6, 153.1, 152.3, 151.3, 141.4, 137.8, 136.3, 132.0, 130.8, 129.5, 129.1, 126.3, 125.3, 124.8, 124.5, 116.8, 116.0, 114.0, 112.7, 111.2, 106.9, 101.4, 86.8, 82.6, 56.2, 53.2, 44.5, 43.0, 30.4, 28.8, 19.5, 18.1, 12.8; HRMS (ESI ⁺ ): m/z Calcd for C ₄₂ H ₄₇ N ₆ O ₉ S [M+H] ⁺ : 811.3125, Found: 811.3124.

Example 1-11. Synthesis of compound 11

(2 R )-2-acetamido- N -(2-(((2 R )-1-((3'-methoxy-3-oxo-3 H -spiro[isobenzofuran-1,9'-xanthen]-6')-yl)amino)-1-oxo-5-(3-((2,2,4,6,7-pentamethyl-2 ,3-dihydrobenzofuran-5-yl) sulfonyl) guanidino) pentan-2-yl) amino) -2-oxoethyl) -4-methylpentanamide (11)

Compound 11 (45 mg) was prepared from Compound 10 (50 mg) using Ac-Leu-OH (11.75 mg, 0.068 mmol), DIC (9.39 mg, 0.074 mmol) and HOBT (10.00 mg, 0.074 mmol) according to General Procedure A. , 0.062 mmol) was synthesized in 76% yield through amide coupling.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.20 (d, J = 8.2 Hz, 1H), 8.30 (q, J = _6.1 Hz, 1H), 8.06-8.00 (m, 2H), 7.98 (d , J = 7.8 Hz, 1H), 7.84 (dd, J = 8.7, 1.8 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.3 Hz, 1H), 7.23–7.19 (m, 2H), 6.93 (d, J = 2.3 Hz, 1H), 6.72–6.63 (m, 3H), 6.32 (s, 1H), 4.32 (q, J = 6.9 Hz, 1H), 4.17 (q, J = 7.5 Hz, 1H), 3.78 (s, 3H), 3.67 (t, J = 6.6 Hz, 2H), 3.00 (pent, J = 6.3 Hz, 2H), 2.87 (s, 2H), 2.41 (s, 3H), 2.35 (s, 3H), 1.92 (s, 3H), 1.77 (d, J = 9.1 Hz, 3H), 1.71–1.53 (m, 2H), 1.41–1.37 (m, 4H), 1.33 (s , 6H), 0.80 (dd, J = 17.4, 5.9 Hz, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) δ 171.5, 169.5, 169.2, 161.7, 158.0, 157.3, 156.6, 153.1, 152.3, 151.3, 141.4, 137.8, 136.3, 132.0, 130.8, 129.5, 129.0, 126.3, 125.3, 124.8, 124.6, 124.5, 119.8, 116.1, 114.0, 112.7, 111.3, 106.9, 101.4, 86.8, 82.6, 56.2, 53.7, 43.0, 41.2, 29.5, 29.2, 28.8, 24.7, 23.8 22.6, 22.2, 19.4, 18.1, 14.5, 12.8; HRMS (ESI ⁺ ): m/z Calcd for C ₅₀ H ₆₀ N ₇ O ₁₁ S [M+H] ⁺ : 966.4072, Found: 966.4066.

Example 1-12. Synthesis of compound 12

(2 R )-2-acetamido- N -(2-(((2 R )-5-guanidino-1-((3'-methoxy-3-oxo-3 H -Spiro[isobenzofuran-1,9'-xanthene]-6'-yl)amino)-1-oxopentan-2-yl)amino)-2-oxoethyl)-4-methylpentanamide (12)

Compound 12 was synthesized from compound 11 (45 mg, 0.047 mmol) via deprotection of Pbf using TFA (10%, 0.8 mL) in anhydrous CH ₂ Cl ₂ (8 mL) according to General Procedure C. The residue was purified by flash column chromatography on silica gel to give compound 12 (15 mg) in 45% yield.

^1H -NMR (400 MHz, DMSO- d6 ) δ 10.28 (d, J = 11.9 Hz, 1H), 8.33 (q, J = 6.4 Hz, 1H), 8.08 (t, J = 7.8 Hz _, 2H), 7.99 (d, J = 7.3 Hz, 1H), 7.86 (t, J = 2.7 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.54 ( s, 1H), 7.26–7.20 (m, 2H), 7.09 (s, 4H), 6.93 (d, J = 1.8 Hz, 1H), 6.72 (d, J = 8.7 Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 6.65 (d, J = 8.7 Hz, 1H), 4.37 (q, J = 6.6 Hz, 1H), 4.18 (q, J = 7.3 Hz, 1H), 3.78 (s, 3H), 3.68 (t, J = 5.3 Hz, 2H), 3.07 (q, J = 6.4 Hz, 2H), 1.79 (d, J = 10.1 Hz, 3H), 1.74–1.70 (m, 1H), 1.63–1.53 (m , 2H), 1.51–1.38 (m, 4H), 0.81 (ddd, J = 17.7, 6.5, 1.5 Hz, 6H); ¹³ C-NMR (100 MHz, DMSO- D ₆ ) Δ 173.5, 173.5, 171.4, 170.2, 169.6, 169.2, 161.7, 158.3, 157.3, 153.1, 152.3, 151.3, 141.4, 136.3, 130.8, 129.5, 129.3, 126.3, 126.3 125.3,124.5,116.1,114.0,112.6,111.3,106.9,101.4,82.6,56.2,55.4,53.7,51.9,49.1,42.7,29.5,29.4,25.6,24.0,223.5 HRMS (ESI ⁺ ): m/z Calcd for C ₃₇ H ₄₄ N ₇ O ₈ [M+H] ⁺ : 714.3251, Found: 714.3247.

II. Measurement of enzymatic cleavage activity of trypsin using a peptide fluorescent probe

Enzymatic cleavage activity of trypsin using a lysine-detecting fluorescent probe [Rh-Lys-Gly-Leu (Ac)] and an arginine-detecting fluorescent probe [Rh-Arg-Gly-Leu (Ac)] as peptide fluorescent probes was measured.

Example 2. Measurement of enzymatic cleavage activity of peptide fluorescent probe by trypsin

As shown in FIG. 1, the enzymatic catalytic activity of trypsin was measured based on the increase in fluorescence intensity after a specific site in the peptide fluorescent probe was cleaved. Concentration dependent trypsin catalytic activity studies were performed using a BioTek Synergy™ H1 microplate reader. A stock solution was prepared with peptide fluorescent probe (20 μM in DMSO) and trypsin (specific concentration in 50 mM HEPES buffer, pH 8.3).

The trypsin reaction was performed in the wells of a microreader 96-well plate. First, 100 μl of 50 mM HEPES buffer (pH8.3) was mixed with 10 μl of probe stock solution (final concentration 1 μM). Concentration dependent studies were performed by adding an appropriate amount of trypsin enzyme stock. The final volume was adjusted to 200 μl with 50 mM HEPES buffer (pH8.3). Microplates were incubated at 30° C. for 30 minutes with shaking and fluorescence intensity was measured every 2 minutes using a BioTek Synergy™ H1 microplate reader as λex/em = 476/516 (λmax of methylodole).

Trypsin concentration-dependent catalytic activity for site-specific cleavage in peptide fluorescent probes was evaluated using various concentrations (0.025, 0.1, 0.25, 1, 2.5, 10, 25 μg/mL) of trypsin. The results are as shown in FIG. 2 . For peptide fluorescent probes, the best results for trypsin cleavage were obtained in the range of 0.25 to 1 μg/mL.

Next, trypsin concentration-dependent catalytic activity was performed for concentrations ranging from 0.25 to 1 μg/mL (FIG. 3). In this experiment, a 1 μg/mL trypsin concentration gave the best results providing fast kinetic, high fluorescence and stable cleavage rate. Therefore, all further experiments were performed keeping the trypsin concentration at 1 μg/mL.

III. Measurement of inhibitory activity of amino acid blockers using peptide fluorescent probes

Amino acid (lysine or arginine) using a lysine-detecting fluorescent probe [Rh-Lys-Gly-Leu (Ac)] and an arginine-detecting fluorescent probe [Rh-Arg-Gly-Leu (Ac)] as a peptide fluorescent probe The inhibitory activity of the blockers was determined (see Tables 1 and 2).

Specifically, as amino acid blockers, lysine-specific inhibitors and arginine-specific inhibitors were used. Anhydride inhibitors (acetic anhydride, benzoic anhydride, diethyl pyrocarbonate, p -toluene sulfonic anhydride) and cyclic anhydride inhibitors (maleic anhydride, citraconic anhydride, phthalic anhydride) were used as lysine-specific inhibitor candidate compounds, As arginine-specific inhibitor candidate compounds, α,β-ketone aldehyde inhibitors (phenyl glyoxal, 4-(trifluoromethyl)phenyl glyoxal, 2-(trifluoromethyl)phenyl glyoxal, 4-nitrophenyl glyoxal, 4-methoxy phenyl glyoxal, 6-methoxy-2-naphthyl glyoxal), monoaldehyde inhibitors (benzaldehyde) and α,β-diketone inhibitors (1,2 cyclohexadione, 2,3-butadione) ] was used.

Example 3. Measurement of inhibitory activity of amino acid-specific blockers using peptide fluorescent probes

The inhibitory activity of amino acid-specific masking agents was evaluated using a peptide fluorescent probe as shown in FIG. 4 . Concentration dependent blocking activity was performed using a BioTek Synergy™ H1 microplate reader. The stock solution contains peptide fluorescent probe (20 μM in DMSO), trypsin (20 μg/mL in 50 mM HEPES buffer, pH8.3), inhibitor (0.001, 0.01, 0.1, 1, 10, 100, 1000 mM), NaOH ( 0.002, 0.02, 0.2, 2, 20, 200, 2000 mM).

The inhibitory activity of amino acid blockers was evaluated in the wells of a microplate reader 96-well plate. First, 100 μl of 50 mM HEPES buffer (pH8.3) was mixed with 10 μl of probe stock solution (final concentration 1 μM). To this solution, 2 μl of inhibitor was added at various concentrations (final concentrations 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 mM) as stock solutions. The final volume was adjusted to 190 μl with 50 mM HEPES buffer (pH8.3). The mixture was incubated at 30° C. with shaking for 30 minutes in a microplate reader. 10 μl of trypsin (final concentration 1 μg/mL) was added to the mixture and incubated for an additional 30 minutes at 30° C. with shaking. Fluorescence intensity was measured at 30 min using a BioTek Synergy™ H1 microplate reader as λex/em = 476/516 (λmax of methylodol).

At this time, 2 μl of NaOH (final concentration 0.00002, 0.0002, 0.002, 0.02, 0.2, 2, 20 mM) was added along with inhibitors for cyclic anhydrides (i.e., citraconic anhydride, maleic anhydride and phthalic anhydride). .

The IC ₅₀ for each inhibitor was calculated by plotting a semilogarithmic plot as shown in FIGS. 5 to 20 .

IV. Evaluation of selective blocking of amino acids in peptide matrices by amino acid blockers

Selective blocking of specific amino acids in the peptide matrix by the candidate amino acid blockers was evaluated.

Example 4. Digestion Patterns of 14-mer Peptides Relative to Trypsin Cleavage Prior to Amino Acid Blocking

To evaluate the selectivity of amino acid blockers for specific amino acids, lysine or arginine, the 14-mer peptide LLTPETRRKAEDLL-NH ₂ was used as a peptide substrate. The degradation pattern of the 14-mer peptide was evaluated at an early stage. Degradation patterns before amino acid blocking are shown in FIGS. 21 to 23.

To evaluate the degradation pattern of the 14-mer peptide by trypsin, an experiment was performed as follows.

Specifically, a stock solution for research was prepared with peptide (1 mg/1 mL, 604 µM in 50 mM HEPES buffer, pH8.3), and trypsin (3 mg/mL in 50 mM HEPES buffer, pH8.3). Digestion of the peptides was performed in the wells of a microreader 96 well plate on a BioTek Synergy™ H1 microplate reader. First, 100 μl of 50 mM HEPES buffer (pH8.3) was mixed with 40 μl of peptide stock solution (final concentration 121 μM). To this solution was added 40 μl of trypsin (final concentration 600 μg). The final volume was adjusted to 200 μl with 50 mM HEPES buffer (pH8.3). The mixture was shaken for 60 minutes.

Characterization of the fractions formed on trypsin cleavage was performed by RP-HPLC-MS analysis. Analysis was performed with a gradient system of 5-35% Solvent B (Solvent A: 0.05% TFA in water and Solvent B: 0.05% TFA in AcCN) for 0-20 minutes (Column: Agilant ZORBAX Eclipse Plus C18 4.6x250 mm , 5 μM, flow rate: 1 mL/min, injection volume: 10 μL, UV wavelength: 206 nm). First, RP-HPLC-MS analysis of the 14-mer peptide standard was performed. The results are shown in FIG. 24 .

Next, RP-HPLC-MS analysis of the fractions formed by cleavage of the 14-mer peptide substrate by trypsin at different positions was evaluated, and the results are shown in FIG. 25 .

Example 5. Digestion pattern of 14-mer peptides for trypsin cleavage after amino acid blockade

Decomposition patterns after lysine blocking using citraconic acid anhydride among candidate amino acid blocking agents are shown in FIGS. 26 and 27 .

To evaluate the degradation pattern of 14-mer peptides by trypsin after blocking lysine using citraconic anhydride, an experiment was performed as follows.

Specifically, a stock solution for research was peptide (1 mg/1 mL, 604 µM in 50 mM HEPES buffer, pH8.3), trypsin (3 mg/mL in 50 mM HEPES buffer, pH8.3), citraconic acid (DMSO 2000 mM in water), NaOH (4000 mM in deionized water). Digestion of the peptides was performed in the wells of a microreader 96 well plate on a BioTek Synergy™ H1 microplate reader. First, 100 μl of 50 mM HEPES buffer (pH8.3) was mixed with 40 μl of peptide stock solution (final concentration 121 μM). To the solution was added 2 μl of citraconic anhydride and 2 μl of NaOH. The final volume was adjusted to 160 μl with 50 mM HEPES buffer (pH8.3). The mixture was shaken for 60 minutes. Finally 40 μl of trypsin was added and the mixture was shaken for a further 60 minutes.

Characterization of the fractions formed on trypsin cleavage was performed by RP-HPLC-MS analysis. Analysis was performed with a gradient system of 5-35% Solvent B (Solvent A: 0.05% TFA in water and Solvent B: 0.05% TFA in AcCN) for 0-20 minutes (Column: Agilant ZORBAX Eclipse Plus C18 4.6x250 mm , 5 μM, flow rate: 1 mL/min, injection volume: 10 μL, UV wavelength: 206 nm).

RP-HPLC-MS analysis of the fractions formed by cleavage at different positions of the 14-mer peptide substrate by trypsin after blocking lysine with citraconic anhydride was evaluated, and the results are shown in FIG. 28 .

Meanwhile, the degradation pattern after blocking arginine using phenyl glyoxal among candidate amino acid blockers is shown in FIG. 29 .

Claims (10)

Amino acid specific blockers selected from the following compounds:
acetic anhydride;
benzoic anhydride;
diethyl pyrocarbonate;
p -Toluene sulfonic anhydride ( p -Toluene sulfonic anhydride);
maleic anhydride;
citraconic anhydride;
phthalic anhydride;
phenyl glyoxal;
4- (trifluoromethyl) phenyl glyoxal;
2-(trifluoromethyl)phenyl glyoxal;
4-nitro phenyl glyoxal;
4-methoxy phenyl glyoxal;
6-methoxy-2-naphthyl glyoxal;
benzaldehyde;
1,2 cyclohexadione; and
2,3-butadione. The amino acid specific blocker according to claim 1, wherein the blocker is a lysine-specific blocker or an arginine-specific blocker. 3. The amino acid specificity of claim 2, wherein the lysine specific blocking agent is selected from acetic anhydride, benzoic anhydride, diethyl pyrocarbonate, p -toluene sulfonic anhydride, maleic anhydride, citraconic anhydride, and phthalic anhydride. enemy blocker. The method of claim 2, wherein the arginine-specific blocker is phenyl glyoxal, 4- (trifluoromethyl) phenyl glyoxal, 2- (trifluoromethyl) phenyl glyoxal, 4-nitro phenyl glyoxal, 4-methyl An amino acid specific blocker selected from oxyphenyl glyoxal, 6-methoxy-2-naphthyl glyoxal, benzaldehyde, 1,2 cyclohexadione, and 2,3-butadione. The amino acid-specific blocker screening method of claim 1 using a trypsin-specific fluorescent probe compound represented by Formula 1 below:
[Formula 1]

during the ceremony,
R is

or

am. The method of claim 5, wherein the blocking agent is a lysine-specific blocking agent or an arginine-specific blocking agent. The method of claim 5, wherein the fluorescent probe compound is represented by Formula 2 or Formula 3 below:
[Formula 2]

[Formula 3]

. The method of claim 5, wherein the fluorescent probe compound reacts with trypsin to exhibit a fluorescence turn-on phenomenon. The method of claim 7, wherein the fluorescent probe compound represented by Formula 2 is for detecting lysine. The method of claim 7, wherein the fluorescent probe compound represented by Formula 3 is for detecting arginine.

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