JPH05137599A - Fluorescent substrate - Google Patents

Abstract

PURPOSE:To provide a new fluorescent substrate useful for measuring the activity of an intramolecularly quenching hydrolase, especially an endopeptidase and capable of rapidly measuring the activity of the endopeptidase in high sensitivity. CONSTITUTION:A fluorescent substrate used for measuring the activity of an endopeptidase of the formula (X is a polypeptide capable of cleaving the endopeptidase). The substrate is obtained e.g. as follows: synthesizing dabsyl peptide from dabsyl chloride, glycyl-phenylalanine hydrochloride and triethylamine, reacting N-Boc-glycine and N-ethylmorpholine with aminoethylthiobimane hydrobromide synthesized from 2-aminoethanethiol and monobromobimane to produce N-Boc-glycyl-aminoethylthiobimane, and subsequently reacting the peptidylaminoethylthiobimane with the dabsyl peptide by a mixed anhydride method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel synthetic substrate used for measuring endopeptidase activity and a method for measuring activity.

[0002]

PRIOR ART 9,10-Dioxa-syn-3,4
6,7-Tetramethylbimane (bimane) is an excellent fluorophore. Biman fluorescence is quenched by intramolecular tryptophan, guanylic acid, etc., and a fluorescent enzyme substrate quenched in the molecule, that is, a biman-peptide or biman-nucleotide containing tryptophan or guanylic acid is a hydrolase, such as chymotrypsin, Aminopeptidase, carboxypeptidase A, cathepsin, angiotensin I converting enzyme, metalloendopeptidase or phosphodiesterase I, and ribonuclease T
₁ is a fluorescent substrate that can be microquantified with high sensitivity [Bioorganic Chemistry (Bioorg. Chem.
), Vol. 16, pp. 298-306 (1988), Chemical and Pharmaceutical Bulletin (Che
m. Pharm. Bull), 36, 4494-4498 (1988), 38, 2043-2044 (1990), 37, 145-147 (19).
89), Vol. 39, pp. 2146-2148 (199).
1), Journal of Biochemistry (J. Bi
ochem.), 110, 345-349 (199)
1), Journal of Pharmacobio-Dynamics (J. Pharm. Dyn.), Vol. 14, pp. 43-46 (1991)].

[0003]

The intramolecular quenching fluorescent substrate is extremely useful for the quantitative measurement of hydrolases,
The development of new fluorescent reagents with excellent quenching properties is desired. The object of the present invention is to use biman as a fluorophore and dimethylaminoazobenzenesulfonic acid (dabsyl) as a quencher.
An object of the present invention is to provide a fluorescent substrate for measuring an intramolecular quenching hydrolase, particularly an endopeptidase activity, to which a new combination is applied, and a method of using the same.

[0004]

The first aspect of the present invention is an invention relating to a fluorescent substrate for measuring endopeptidase activity, which is represented by the following general formula (Formula 1):

[0005]

[Chemical 1]

[Wherein X represents a polypeptide which can be cleaved by an endopeptidase].

A second invention of the present invention relates to a method for measuring the activity of endopeptidase, wherein in the activity measurement of endopeptidase, the fluorescence generated by reacting endopeptidase with the fluorescent substrate of the first invention is measured. It is characterized by measuring.

The inventors of the present invention have been searching for a quencher that is more effective than tryptophan for fluorescence of bimane, and have found that the fluorescence of bimane is effectively quenched by the presence of dabsyl group. The present invention has been completed by synthesizing a bimane peptide containing, and found that the substance is a fluorescent substrate useful for the measurement of endopeptidase.

In the fluorescent substrate of the present invention, the polypeptide sequence between the fluorophore and the quencher may be any sequence that can be recognized and cleaved by the endopeptidase for the purpose of measurement. The number of amino acids in a polypeptide is usually 1 rather than the efficiency of quenching.
The number may be 0 or less, preferably 2 to 8.

Hereinafter, examples of chymotrypsin, collagenase and thermolysin as the endopeptidase will be described. Chymotrypsin is a typical endopeptidase, and it selectively catalyzes the hydrolysis of peptide bonds including tyrosine, phenylalanine, and tryptophan. Collagenase is a metalloproteinase, which contains zinc, requires calcium, and hydrolyzes a peptide having a specific sequence, for example, the polypeptide represented by SEQ ID NO: 1 in the sequence listing to obtain Xaa-Pro-Xaa and Gl.
Give y-Pro. [Xaa may be any amino acid: Journal of Biochemistry, No. 58
Volume, 407-416 (1965), Analytical
Biochemistry, Anal. Biochem., 107
Vol. 96-102 (1980)]. Thermolysin is a metalloendopeptidase that has specificity for peptide bonds contributed by amino acids with hydrophobic side chains in the amino group, such as leucine and phenylalanine [Archives of Biochemistry and Biophysics (Arch. .Biophys.),
146, 291-296 (1971)]. Due to these substrate specificities, fluorescent substrates 1a to 1d shown in Table 1, for example, are synthesized as fluorescent substrates for each enzyme.

[0011]

[Table 1] Table 1 ──────────────────────────────────── Dabsyl-X-NH (CH ₂ ) ₂ S-Bim → Dabsyl-Y-OH + HZ-NH (CH ₂ ) ₂ S-Bim 1 2 3 1a: X = Gly-Phe-Gly 2a: Y = Gly-Phe 3a: Z = Gly 1b: X = A 2b: Y = Gly-Pro-Leu 3b: Z = Gly-Pro 1c: X = Gly-Phe-Gly 2c: Y = Gly 3c: Z = Phe-Gly 1d: X = B 2d: Y = Phe- Gly 3d: Z = Leu-Ala ─────────────────────────────────────

[0012]

[Chemical 2]

[0013]

[Chemical 3]

That is, the fluorescent substrate 1a is a fluorescent substrate for measuring chymotrypsin activity, and X in the general formula (Formula 1) is G.
ly-Phe-Gly, and chymotrypsin
A cleavage occurs between he and Gly. The fluorescent substrate 1b is a fluorescent substrate for measuring collagenase activity, and is represented by X of the general formula (Formula 1).
Is the polypeptide A represented by SEQ ID NO: 2 in the sequence listing, and is cleaved between Leu and Gly by collagenase. 1c is the same substance as 1a and is cleaved between Gly and Phe by thermolysin. 1d is a fluorescent substrate for measuring thermolysin activity, X in the general formula (Formula 1) is the polypeptide B represented by SEQ ID NO: 3 in the sequence listing, and cleavage between Gly and Leu is caused by thermolysin.

These biman substrates (1a to d) are subjected to the usual mixed anhydride method by the dabsyl peptide (2) [Analytical Chemistry, Anal. Chem., Vol. 47, pp. 1634 to 1638 (1975)]. , And peptidylaminoethylthiobimane (3). In a substrate for endopeptidases with a new combination of biman and dabcyl groups, the N-terminal dabcyl residue acts as the quencher moiety of the biman group. The biman group also plays a dual role as a fluorophore and as a C-terminal peptide blocking group. Table 2 shows the relative fluorescence intensity (RFI) of these biman substrates.

[0016]

[Table 2] Table 2 ─────────────────────────── Substrate RFI ─────────────── ───────────── 1a 0.029 1b 0.015 1d 0.024 ───────────────────────── ──

As shown in Table 2, the relative fluorescence intensities (RFI) with respect to the fluorescence intensities of the equimolar mixtures of the corresponding hydrolyzates 3 and 2 of 1a, 1b and 1d are 0.029, respectively.
0.015 and 0.024 (excitation: 400 nm,
Emission: 480 nm).

The RFI of 1a has a concentration of 1a of 4.14 ×.
In an 80 mM Tris-hydrochloric acid buffer containing 10 ^-6 M, 100 mM CaCl ₂ , 4.3% dimethylsulfoxide and pH 7.8, RFI of 1b has a 1b concentration of 2.5 × 10 ^-6 M,
P containing 10 mM CaCl ₂ , 5% dimethyl sulfoxide
1d RF in H7.5 50 mM Tricine buffer
I is 1d concentration 2.5 × 10 ^-6 M, 2.5 mM CaC
It was measured in hydrochloric acid buffer - l _2, 50 mM Tris 6.6% dimethyl sulfoxide containing pH 7.2.

In these substrates, the viman group fluorescence is quenched intramolecularly by the dabsyl moiety attached to the substrate molecule. However, since 3 is released enzymatically, fluorescence increases. Since this increase can be easily and continuously detected, the hydrolytic activity of the enzyme can be measured.

Next, Table 3 shows the hydrolysis rate constants of the biman substrates 1a to 1d by the respective endopeptidases.

[0021]

[Table 3] Table 3 ──────────────────────────────────── Fermentation substrate Km k _cat k _cat / Km (M) (S ^-1 ) (M ^-1 S ^-1 ) ──────────────────────────────── ───── Chymotrypsin 1a 2.1 × 10 ^-5 1.1 × 10 ^-2 5.2 × 10 ² Z-Trp-Gly-NH- 1.0 × 10 ^-4 9.4 × 10 ^-3 9.4 × 10 Bim (6) Collagenase 1b 5.0 × 10 ^-5 1.1 x 10 ^-2 2.2 x 10 ² Mcc-Pro-Leu-Gly- 1.2 x 10 ^-4 2.4 x 10 ^-2 2.0 x 10 ² Pro-D-Lys (Dnp) -OH (7) Thermolysin 1c 1.7 × 10 ^-5 1.4 × 10 ^-1 8.2 × 10 ³ 1d 2.0 × 10 ^-6 2.1 1.1 × 10 ⁶ FA-Gly-Leu-NH ₂ 3.3 × 10 ^-3 2.6 × 10 7.8 × 10 ³ (8) Abz-C -Nba (9) 1.4 × 10 ^-4 2.9 × 10 ² 2.1 × 10 ⁶ ───────────────────────────────── ────

In the table, C is the polypeptide represented by SEQ ID NO: 4 in the sequence listing, Z- is a carbobenzoxy group, Mcc-.
Is 7-methoxycoumarin-3-carboxyl group, Dnp
-Is a 2,4-dinitrophenyl group, FA- is a furylacryloxy group, Abz- is an o-aminobenzoyl group, and Nb.
a-represents a p-nitrobenzylamide group, and the substrate 6 is Bioorganic Chemistry, Vol. 16, 298.
~ 306 (1988), Substrate 7 is Analytical Biochemistry, Vol. 186, 112-115 (1990), Substrate 8 is Biochemical and Biophysical Research Communications, 3rd.
2, Vol. 326-332 (1968), Biochemistry (Biochem), Vol. 9, 2784-2791 (1970), Substrate 9 refers to Journal of Biological Chemistry (J. Biol. Chem.), Vol. 255
Vol., 3482-3486 (1980).

In order to compare the characteristics of these bimane fluorescent substrates, kinetic parameters of 6 to 8 were also measured using the same lot of enzyme under the same assay conditions such as buffer, pH, temperature and organic solvent. The measured values for Substrate 9 are quoted from the literature. Regarding the kinetic parameters of substrate 1a for chymotrypsin, the Km value of 1a is smaller than that of the fluorescent substrate 6 containing biman and tryptophan.
However, k _cat values of 1a is comparable almost to the k _cat values of 6, and therefore 1a of k _cat / Km value of 6 k _cat /
Slightly larger than Km. The hydrolysis rate of 1a is at least 5.3 using a substrate concentration of 3.77 × 10 ⁻⁶ M.
It is proportional to the concentration of chymotrypsin at around 10 ^-10 . 1
Kinetic parameters of b and fluorescent substrate 7 for collagenase
In comparison with the kinetic parameters of
Both _cat values are less than the Km and k _{cat of} 7. Therefore, 1b of k _cat / Km value Most 7 of k _cat / K
Equal to the m value. A plot of fluorescence intensity vs. collagenase concentration yielded satisfactory linearity with a substrate concentration of 4.67 x
The minimum concentration of the enzyme is about 1.4 × 10 ^-9 M compared to 10 ^-6 M
Is. Hydrolysis of 1c, which is a common substrate with chymotrypsin, by thermolysin was performed, and the kinetic parameters of 1c and 8 were compared. In the comparison, the Km value of 1c and the k _cat value are the Km value of 8 and k
Both are about two orders of magnitude smaller than the _cat value. Therefore 1c k
_cat / Km value is comparable to the k _cat / Km value of almost 8. Where 8 is the most commonly used continuous spectrophotometric substrate for the thermolysin assay.

Next, the comparison of the fluorescent substrate 1d for thermolysin with the continuous spectrophotometric substrate 8 and the continuous fluorescent substrate 9 is as follows.
K _cat values for 1d is approximately one order of magnitude smaller than the k _cat value of 8, about two orders of magnitude less than the k _cat value of 9. Also, the Km value of 1d is about 3 digits smaller than the Km value of 8 and about 2 digits smaller than the Km value of 9.
Therefore, 1d of k _cat / Km values are 8 of k _cat / Km
Greater than the value and approximately equal to a k _cat / Km value of 9. From these kinetic results, Substrate 1d was used to study the linear relationship between thermolysin concentration and fluorescence intensity. When the substrate concentration is 5.63 × 10 ⁻⁷ M, substrate 1d can be used to detect thermolysin to a low concentration of about 6.6 × 1.0 ⁻¹² .

The above results indicate that the dabsyl group can be used as a strong quencher in the synthesis of an intramolecular quenching fluorescent substrate bound with bimane. Further, the biman-dabcyl system has the following advantages over the conventionally known substrates for endopeptidases. (1) In comparison of relative fluorescence intensities, the dabcyl group has higher quenching efficiency than tryptophan, and thus the biman-dabcyl system has lower relative fluorescence intensity value than the biman-tryptophan system (6). Hydrolysis of 7 or 9 increases fluorescence 27-fold or 6-8-fold, respectively. Therefore, the biman-dabcyl substrate is quenched more effectively than 7 and 9. (2) As continuous fluorescent substrates for collagenase and thermolysin, 7
-Methoxycoumarin (Mcc) and o-aminobenzoyl (Abz) were reported. However, the fluorescence intensity of the biman group is higher than that of Mcc or Abz, and the emission wavelength (480 nm) of the biman system is longer than the emission wavelength of Mcc (405 nm) or Abz (415 nm). (3) Furyl acryloyl peptides have been reported as a continuous spectrophotometric method. However, this method is based on measuring the slight difference in optical density of the substrate and product after hydrolysis at 345 nm. (4) 7-aminomethylcoumarin (AM
C) is the most widespread fluorescent amine of hydrolase substrates and has been used in carboxy-endopeptidase and aminopeptidase assays. But,
It was not used for amino endopeptidases due to the difficulty in designing an enzymatically hydrolyzable bond between AMC and the amino side of the peptide. The biman-dabcyl system can provide a continuous and rapid fluorescent assay for the amino-endopeptidase thermolysin.

Due to these advantages, the new combination of biman and dabsyl groups used in the assay of endopeptidases can be widely applied to the synthesis of other enzyme substrates, and the general formula of the present invention ( By using a polypeptide that can be cleaved by each endopeptidase as X in Chemical formula 1), a substrate for each endopeptidase, for example, a substrate for measuring enqueriferinase activity, a substrate for limulus test, can be synthesized, and further for endopeptidase Application to substrates is also possible.

[0027]

EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited to the scope of the following examples.

Example 1 Synthesis of Fluorescent Substrates 1a, 1b, 1d (1) Synthesis of Dabsyl Peptides 2a, 2b, 2d Dabsyl peptides were synthesized by the method described in the above-mentioned Analytical Chemistry.

(I) 2a: dabsyl chloride (95 mg,
0.29 mmol) and glycyl-phenylalanine hydrochloride (102 mg, 0.4 mmol) and triethylamine were synthesized, and 76 mg of red powder was obtained from acetone-water with a yield of 51%. Shows its physical properties. (A) mp119 to 121 ° C. (decomposition), (b) IR (nujol): 3350, 3170, 1690, 163.
⁰ , 1610, 1585 cm ^-1 , (c) [α] _D (20
C) -6.4 ° (c = 0.4365, DMSO),
(D) Elemental analysis Calculated value for C ₂₅ H ₂₇ N ₅ O ₅ S.H ₂ O: C, 56.91; H, 5.54; N, 13.2
7; S, 6.08, analytical value: C, 57.18; H, 5.
54; N, 13.32; S, 6.33.

Hereinafter, the melting point is a melting point measuring device Yamato MP
It was measured at -21 and corrected. The mass spectrum was measured with a mass spectrometer JMS-D300. The infrared spectrum was measured with an infrared spectrometer JASCO IRA-1. The optical rotatory power was measured with a polarimeter JASCODIP-4.

(Ii) 2b: Dove silk chloride (162 m
g, 0.5 mmol) and glycyl-prolyl-leucine hydrochloride (193 mg, 0.6 mmol) and triethylamine to obtain 194 mg of dark red powder with a yield of 68%. Shows its physical properties. (A) mp184-188 ° C., (b) IR (nujol): 1680, 1640, 1605 cm ^-1 , (c)
[Α] _D (20 ° C.)-9 ° (c = 1.2, DMSO),
(D) Elemental analysis Calculated value for C ₂₇ H ₃₆ N ₆ O ₆ S.1 / 2H ₂ O: C, 55.75; H, 6.41; N, 1
4.45; S, 5.51, analytical value: C, 55.72;
H, 6.41; N, 14.18; S, 5.51.

(Iii) 2d: Dove silk chloride (424
mg, 1.3 mmol) and phenylalanyl-glycine hydrochloride (504 mg, 2.0 mmol), dark red powder 4
66 mg was obtained with a yield of 70%. Shows its physical properties. (A) mp186 to 190 ° C., (b) IR (nujol): 1720, 1655, 1605 cm ⁻¹ , (c)
[Α] _D (20 ° C) + 84 ° (c = 0.502, DMS
O), (d) elemental analysis calculated for C ₂₅ H ₂₇ N ₅ O ₅ S.H ₂ O: C, 56.91; H, 5.54; N, 1
3.27; S, 6.08, analytical value: C, 57.14;
H, 5.63; N, 13.47; S, 6.27.

(2) Synthesis of aminoethylthio-bimanhydrobromide (4) Stirred 2-aminoethanethiol (69 mg, 0.
To a solution of 9 mmol) in methanol (3 ml) was added monobromobimane (172 mg, 0.63 mmol). After 1.5 hours, anhydrous ethyl ether was added to obtain 211 mg of a yellow precipitate with a yield of 96%. Shows its physical properties. (A) mp 161-165 ° C., (b) IR (nujol): 1740, 1650, 1620, 1580 cm ⁻¹ ,
(C) Elemental analysis Calcd for C ₁₂ H ₁₈ N ₃ O ₂ SBr: C, 41.39; H, 5.21; N, 12.07;
S, 9.21, Br, 22.94, analytical value: C, 41.
13; H, 5.40; N, 12.18; S, 9.41;
Br, 22.87.

(3) Synthesis of N-Boc-peptidyl-aminoethylthio-biman 5a, 5b, 5c, 5d N-Boc-amino acid or peptide (N-Boc-glycine 175 mg, N-Boc-glycyl-proline 272)
mg, N-Boc-phenylalanyl-glycine 322m
g, N-Boc-leucyl-alanine 302 mg, 1.0 m
mol) and N-ethylmorpholine (115 mg,
1.0 mmol) in a solution of ice-salt cooled and stirred in anhydrous tetrahydrofuran (10 ml) was added to chlorocarbonate iso-
Butyl (137 mg, 1.0 mmol) was added. 10 minutes later,
Aminoethylthio-bimanhydrobromide (4) of Example 1-2 (348 mg, 1.0 mmol) and N-ethylmorpholine (115 mg, 1.0 mmol) in anhydrous DMF (5 ml).
The solution was added dropwise, stirred at the same temperature for 2 hours, and then left at room temperature overnight. The solution was then concentrated under reduced pressure to 1/3 of the original volume, ethyl acetate was added to the concentrate, washed with saturated bicarbonate solution, saturated sodium chloride solution, 10% citric acid solution, and saturated sodium chloride solution, and finally It was dried over anhydrous sodium sulfate. After removing the solvent, a glassy material was obtained. Using a silica-gel preparation layer (20 x 20 cm),
Chromatograph and develop with ethyl acetate. The polar band was eluted with ethanol.

(I) N-Boc-glycyl-aminoethylthio-bimane (5a): The solvent was removed and recrystallized from acetonitrile to obtain 200 mg of a yellow powder in a yield of 47%. Shows its physical properties. (A) mp 160 to 167 ° C., (b) IR (nujol): 3280, 1740, 1690, 1660 cm ⁻¹ ,
(C) Elemental analysis Calculated value for C ₁₉ H ₂₈ N ₄ O ₅ S:
C, 53.76; H, 6.65; N, 13.20; S,
7.75, analytical value: C, 53.59; H, 6.70;
N, 13.23; S, 7.46.

(Ii) N-Boc-glycyl-prolyl-
Aminoethylthio-bimane (5b): 227 mg of a pale yellow glassy substance was obtained with a yield of 44%. This was used in the next step without further purification. Shows its physical properties. (A) IR (nujol): 3320, 1730, 17
00, 1650 cm ^-1 .

(Iii) N-Boc-phenylalanyl-
Glycyl-aminoethylthio-bimane (5c): 512 mg of a pale yellow glassy substance was obtained with a yield of 79%. This was used in the next step. Shows its physical properties. (A) IR (nujor): 1735, 1690, 16
60 cm ^-1 .

(Iv) N-Boc-leucyl-alanyl-
Aminoethylthio-bimane (5d): 480 mg of pale yellow glassy substance was obtained with a yield of 87%. This was used in the next step without further purification. Shows its physical properties. (A) IR (nujor): 3300, 1735, 16
95, 1660 cm ^-1 .

(4) Preparation of 3a, 3b, 3c, 3d by deblocking the protecting group from N-Boc-peptidyl-aminoethylthio-bimane. 5a obtained in Example 1-3 (200 mg, 0.47 mmo
l), 5b (227 mg, 0.44 mmol), 5c (512 m)
g, 0.79mmol), or 5d (480mg, 0.87mmo)
l) were each treated with 98% formic acid (10 ml) for 3 hours,
Then 5N HCl-dioxane (0.2 for 5a and 5b)
ml, 5c and 5d are 0.4 ml) and the solution is evaporated,
The residue was treated twice with methanol and concentrated from methanol-ethyl acetate to precipitate a hygroscopic yellow powder.

(I) Glycyl-aminoethylthio-bimane hydrochloride (3a): 146 mg was obtained with a yield of 91%. Shows its physical properties. (A) mp225-233 ° C., (b) IR (nujol): 1730, 1650, 1620, 1590 cm ⁻¹ ,
(C) Elemental analysis Calculated value for C ₁₄ H ₂₁ N ₄ O ₃ SCl.H ₂ O: C, 44.38; H, 6.12; N, 1
4.79; S, 8.46; Cl, 9.36, analytical value:
C, 44.42; H, 6.01; N, 14.62; S,
8.16; Cl, 9.16.

(Ii) Glycyl-prolyl-aminoethylthio-bimane hydrochloride (3b): 188 mg was obtained with a yield of 59%. Shows its physical properties. (A) mp 210 to 212 ° C., (b) IR (nujol): 1730, 1665 cm ⁻¹ , (c) [α] _D (20
C) -30 ° (c = 0.717, MeOH), (d) Elemental analysis Calculated value for C ₁₉ H ₂₈ N ₅ O ₄ SCl.H ₂ O: C, 47.92; H, 6.36; N , 14.72;
S, 6.74; Cl, 7.45, analytical value: C, 47.8.
7; H, 6.49; N, 14.67; S, 7.03; C
1, 7.69.

(Iii) Phenylalanyl-glycyl-aminoethylthio-bimane hydrochloride (3c): 376 mg was obtained with a yield of 81%. Shows its physical properties. (A) mp 190 to 195 ° C., (b) IR (nujol): 1730, 1650 cm ⁻¹ , (c) [α] _D (20
℃) + 29 ° (c = 0.354, MeOH), (d) Elemental analysis _{C 23 H 30 N 5 O 4} SCl · H 2 O · 1 / 2CH 3
Calculated for OH: C, 52.07; H, 6.32;
N, 12.92; S, 5.91; Cl, 6.54, analytical value: C, 51.82; H, 6.07; N, 12.66;
S, 5.92; Cl, 6.51.

(Iv) Leucyl-alanyl-aminoethylthio-bimane hydrochloride (3d): 338 mg was obtained with a yield of 73%. Shows its physical properties. (A) mp208 to 215 ° C., (b) IR (nujol): 3180, 1540 cm ⁻¹ , (c) [α] _D (20
C) -7.2 ° (c = 1.15, MeOH), (d) Elemental analysis Calculated value for C ₂₁ H ₃₄ N ₅ O ₄ SCl.1 / 2H ₂ O: C, 50.72; H, 7 .10; N, 14.1
0; S, 6.45; Cl, 7.14, analytical value: C, 5
0.72; H, 7.12; N, 13.84; S, 6.2
0; Cl, 6.94.

(5) Synthesis of Fluorescent Substrates 1a, 1b, 1d Dabsyl peptides 2a, 2b, 2d obtained in Example 1-1
(51 mg, 0.1 mmol) and N-ethylmorpholine (12 mg, 0.1 mmol) in a solution of ice-salt cooled stirred tetrahydrofuran (10 ml) in iso-butyl chlorocarbonate (14 mg, 0.1 mmol). ) Was added.
After 10 minutes, the corresponding biman peptide 3 obtained in Examples 1-4 (3a, 36 mg, 3b, 46 mg, 3d, 49 mg) and N-ethylmorpholine (12 mg, 0.1 mmol) in anhydrous D.
MF (2 ml) solution was added dropwise. Stirring was continued for 1 hour at the same temperature and left overnight at room temperature. After removing the solvent, ethyl acetate (100 ml) was added to the residue, washed with saturated sodium bicarbonate solution and water, and dried over anhydrous sodium sulfate.
Methanol-ethyl acetate was concentrated to obtain dark red powder products 1a, 1b, and 1d, respectively.

(I) 1a: 60 mg was obtained with a yield of 72%.
Shows its physical properties. (A) mp126 to 128 ° C., (b) IR (nujol): 3280, 1730, 1650, 1600 cm ⁻¹ ,
(C) [α] _D (20 ° C.) + 4.0 ° (c = 0.45)
1, DMSO), (d) Elemental analysis C ₃₉ H ₄₅ N ₉ O ₇ S
₂ · 2CH calcd for ₃ OH: C, 55.96;
H, 6.07; N, 14.32; S, 7.29, analytical value: C, 56.05; H, 5.78; N, 14.09;
S, 7.25.

(Ii) 1b: 70 mg was obtained with a yield of 72%.
Shows its physical properties. (A) mp138-145 ° C., (b) IR (nujol): 3280, 1730, 1635, 1600 cm ⁻¹ ,
(C) [α] _D (22 ° C) + 87 ° (c = 0.53, D
MSO), (d) Elemental analysis C ₄₆ H ₆₁ N ₁₁ O ₉ S ₂ · H
Calculated for ₂ O: C, 55.57; H, 6.39;
N, 15.50; S, 6.45, analytical value: C, 55.3.
8; H, 6.37; N, 15.20; S, 6.66.

(Iii) 1d: 70 mg was obtained with a yield of 74%. Shows its physical properties. (A) mp140 to 144 ° C., (b) IR (nujol): 3240, 1735, 1675, 1630, 16
05 cm ^-1 , (c) [α] _D (22 ° C) + 45 ° (c =
1.60, DMSO), (d) Elemental analysis C ₄₆ H ₅₈ N ₁₀
Calculated value for O ₈ S ₂ · 1 / 2H ₂ O: C, 58.0
3; H, 6.25; N, 14.71; S, 6.73, analytical value: C, 58.05; H, 6.17; N, 14.4.
8; S, 6.75.

Example 2 Fluorescence measurement and enzyme activity measurement The ultraviolet and visible absorption spectra were measured by a spectrophotometer Hitachi 210-
It was measured at 10. The fluorescence spectrum was measured with a fluorescence spectrophotometer Hitachi 650-10.

(1) Kinetic parameters (Km,
Measurement of k _cat ) All fluorescence measurements were carried out at 25 ° C. using the following substrates, enzymes and buffers. 1a: 80 mM Tris-HCl buffer (containing 100 mM CaCl ₂ and 5% DMSO, pH 7.8), chymotrypsin [from beef pancreas; EC
3.4.4.5. Worthington Biochemical (W
orthington biochemical)]. 1b: 50 mM Tricine buffer (10 mM CaCl ₂ and 6.6% DMS)
O containing pH 7.5), collagenase type I [Clostridium histo
lyticum): EC 3.4.24.3. Sigma chemical). 1a (for thermolysin) and 1d: 50 mM Tris buffer [2.5 mM
CaCl ₂ and 6.6% (1a) or 4.5% (1d)
Containing DMSO of pH 7.2] thermolysin [Bacillus thermoproteolyticus Rocco (Bacillus ther
moproteolyticus Rokko) Protease Type X: EC 3.4.24.4. Sigma Chemical]. The wavelengths of excitation and emission of fluorescence are 400 nm and 48, respectively.
It was 0 nm. The increase in fluorescence due to hydrolysis of the substrates (1a-d) was automatically recorded over time, usually 6 minutes. From the slope of this plot, the enzyme activity was calculated by comparing the fluorescence intensity of the control compound (3a for chymotrypsin, 3b for collagenase, 3c and 3d for thermolysin). Km and k _Cat values are Lineweaver-Burk (Li
Neweaver-Burk) plots were obtained for the following substrate and enzyme concentrations. 1a: (for chymotrypsin): 1.
22 × 10 ^{−5 to} 4.06 × 10 ⁻⁵ M, chymotrypsin:
8.79 × 10 ⁻⁸ M, 1b: 0.497 × 10 ⁻⁵ to 1.
66 × 10 ⁻⁵ M, collagenase: 4.99 × 10 ⁻⁸ M,
1a (relative to thermolysin): 0.497 × 10 ⁻⁵
1.66 × 10 ⁻⁵ M, 1d: 1.69 × 10 ^{−6 to} 3.3
8 × 10 ⁻⁶ M, thermolysin: 1.17 × 10 ⁻⁷ M.

(2) Linear Relationship between Fluorescence Intensity and Enzyme Concentration Fluorescence measurement was carried out by the method described in Example 2-1 at the final concentrations of the following substrate and enzyme. 1a: 3.77 × 10
^-6 M, chymotrypsin: 5.29 × 10 ^{-10 to} 1.60
× 10 ⁻⁷ M, 1b: 4.67 × 10 ⁻⁶ M, collagenase: 1.41 × 10 ^{−9 to} 3.52 × 10 ⁻⁷ M, 1d:
5.63 × 10 ⁻⁷ M, thermolysin: 6.55 × 10
^{-12 to} 1.31 x 10 ^-9 M.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, an intramolecular quenching fluorescent substrate is provided by combining an excellent fluorophore and a strong quencher, and various endopeptidase activities are sensitively and rapidly measured using the substrate. A method was provided.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence characteristics: 1 Any amino acid 3 Any amino acid SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear SEQ ID NO: 3 Sequence length: 4 Sequence type: Amino acid Number of chains: Single chain Topology: Linear SEQ ID NO: 4 Sequence length: 4 Sequence type: Amino acid Number of chains: Single strand Topology: Linear

Claims (2)

[Claims] 1. The following general formula (Formula 1): (Wherein X represents a polypeptide that can be cleaved by endopeptidase), and a fluorescent substrate for measuring endopeptidase activity. 2. A method for measuring endopeptidase activity, which comprises reacting the fluorescent substrate according to claim 1 with endopeptidase and measuring the generated fluorescence in the measurement of endopeptidase activity.