JPS626560B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel peptide derivatives that can be used as fluorescent substrates for measuring renin activity. It is known that the enzyme renin in the kidney plays an important role in regulating blood pressure and metabolizing water and electrolytes, and quantifying this enzyme makes it possible to diagnose diseases related to this enzyme. Become. Currently, renin activity uses the proteinaceous substrate angiotensinogen as a substrate, and produces angiotensin (angiotensin).
) is measured by radioimmunoassay method.
However, methods using peptide substrates containing β-naphthylamine are also known. However, none of these methods satisfy all of the requirements of measurement sensitivity, safety, and simplicity. The present inventors conducted research to develop a measurement method that satisfies all of measurement sensitivity, safety, and simplicity, and the structural formula: We succeeded in synthesizing a new peptide derivative shown in
We discovered that this new compound serves as a fluorescent substrate that enables simple and highly sensitive measurement of renin activity.
The present invention has now been completed. The peptide derivatives of the invention include acid addition salt forms. To obtain the target compound of the present invention, for example, N.O.
-Protection-N-protecting group of 7-(N.O-protected-tyrosyl)-amino-4-methylcoumarin produced by dehydration condensation reaction of tyrosine and 7-amino-4-methylcoumarin (AMC) After 7-
(N-protected-leucyl-leucyl-valyl-0-
Protected-tyrosyl)-amino-4-methylcoumarin is obtained. Next, the N-protecting group is removed by a method commonly used in peptide synthesis, and then N-N ^G -protected-arginyl-brolyl-phenylalanyl-
By reacting histidine-azide, 7
-(N〓・N ^G -protected-arginyl-prolyl-phenylalanyl-histidyl-leucyl-leucyl-valyl-O-protected-tyrosyl)-amino-4
- Obtain methylcoumarin. The protecting group can then be removed by reacting in anhydrous hydrogen fluoride at 0°C for 40 minutes. As mentioned above, the protecting group for N–·N ^G and the hydroxyl group can be selected from the protecting groups commonly used in peptide synthesis. To measure the renin activity contained in a liquid using the peptide derivative of the present invention, a renin-containing liquid and the general formula 7-(leucyl-valyl-tyrosyl)-amino-4-methylcoumarin is then brought into contact with the peptide derivative represented by the formula, and then AMC is generated by acting on the peptide derivative such as aminopeptidase M, etc. The renin activity in the liquid can be measured with high sensitivity by quantifying the amount of free AMC produced after contacting with the enzyme. Hereinafter, the present invention will be explained in detail with reference to Reference Examples and Examples. Reference example 1 N-t-butyloxycarbonyl-0-2.6
22 g (50 mmol) of -dichlorobenzyl-L-tyrosine was dissolved in 200 ml of tetrahydrofuran, and while stirring under cooling, 5.2 g (25 mmol) of dicyclohexylcarbodiimide was added. After 1 hour, the precipitated dicyclohexylurea was removed by filtration, and the filtrate was stirred while cooling to give 4.4 g of AMC.
(25 mm) of dimethylformamide (DMF) 15
ml solution was added dropwise. After stirring for 1 day at 20°C, the solvent was distilled off, the residue was dissolved with 100 ml of ethyl acetate, and washed by shaking with 100 ml of 1N hydrochloric acid. The organic layer was washed with 100 ml each of 5% deuterated water and then water, dried over anhydrous magnesium sulfate, and then ethyl acetate was distilled off. The residue was recrystallized from 100 ml of methyl alcohol to give 7-(N-
9.8 g (yield: 65.3%) of t-butyloxycarbonyl-0-2.6-dichlorobenzyl-L-tyrosyl)-amino-4-methylcoumarin was obtained. Melting point 234-235°C (decomposition). [α] ²⁷ _D = +57.7゜ (C = 2.13, DMF) Elemental analysis: Measured values C62.23%, H5.01%, N4.54% Calculated values as C ₃₁ H ₃₀ N ₂ O ₆ Cl ₂ ,
C62.31%, H5.06%, N4.69% Reference example 2 7-(Nt-butyloxycarbonyl-0-
2,6-dichlorobenzyl-L-tyrosyl)-amino-4-methylcoumarin 1.79 g (3 mmol)
Add 10 ml of trifluoroacetic acid to the solution and stir at 20℃ for 20 minutes, then distill off the trifluoroacetic acid and add ether to the residue.
100 ml was added to form a solution. 7-(0-2.6-dichlorobenzyl-L-tyrosyl)-amino-4-methyl coumarin trifluoroacetate was collected by filtration. Dissolve this in 10 ml of DMF and give 1.13 g (3.6 mmol) of N-t-butyloxycarbonyl-L-valine-N-hydroxysuccinimide ester.
was added, triethylamine was added to adjust the pH to 7, and the mixture was stirred overnight at room temperature. Distill the solvent, dissolve in 100 ml of chloroform and 5 ml of methanol, add 100 ml of 0.5N hydrochloric acid, and then 100 ml of water.
After washing with 1.9 g (yield: 91%) of -valyl-L-tyrosyl)-amino-4-methylcoumarin was obtained. Melting point 214-215°C (decomposition). [α] ²⁷ _D = +33.7゜ (C = 1.07, DMF) Elemental analysis: Actual value C61.79%, H5.55%, N5.88% Calculated value as C ₃₆ H ₃₉ N ₃ O ₇ Cl ₂
C62.07%, H5.64%, N6.03% Reference example 3 7-(t-Butyloxycarbonyl-L-valyl-0-2,6-dichlorobenzyl-L-tyrosyl)-amino-4-methylcoumarin Add 10 ml of dichloromethane and 5 ml of acetic acid to 1.4 g (2 mmol) and dissolve to give 1.15 g (6 mmol) of toluenesulfonic acid monohydrate.
mmol) was added, and dichloromethane was distilled off at 20°C. After stirring for 1 hour at 20℃, ether
Add 100ml of 7-(L-valyl-0-2.6)
-dichlorobenzyl-L-tyrosyl)-amino-
The precipitate of 4-methylcoumarin toluenesulfonate was collected by filtration. Dissolve this in 10ml of DMF and add triethylamine.
After adjusting the pH to 7, 788 mg (2.4 mmol) of N-t-butyloxycarbonyl-L-leucine-N-hydroxysuccinimide ester was added and stirred overnight at room temperature. The solvent was distilled off, 50 ml of water was added, the resulting precipitate was collected by filtration, dissolved in 5 ml of methyl alcohol and 50 ml of chloroform, and insoluble matter was removed by filtration. Ether was added to the filtrate and recrystallized to obtain 7-(t
-Butyloxycarbonyl-L-leucyl-L-
700 mg (yield: 43%) of valyl-0-2,6-dichlorobenzyl-L-tyrosyl)-amino-4-methylcoumarin was obtained. Melting point 240-241℃ (decomposition). [α] ²⁷ _D = +25.9゜ (C = 1.85, DMF) Elemental analysis: Actual value C62.28%, H6.13%, N6.78% Calculated value as C ₄₂ H ₅₀ N ₄ O ₈ Cl ₂
C62.29%, H6.22%, N6.92% Reference example 4 7-(t-Butyloxycarbonyl-L-leucyl-L-valyl-0-2,6-dichlorobenzyl-L-tyrosyl)-amino- Add and dissolve 10 ml of dichloromethane and 3 ml of acetic acid to 647 mg (0.8 mmol) of 4-methylcoumarin, add 458 mg (2.4 mmol) of toluenesulfonic acid monohydrate, distill off dichloromethane at 20°C, and then stir at 20°C for 1 hour. Stirred. Then, 100 ml of ether was added to the resulting 7-(L-
The precipitate of leucyl-L-valyl-0-2.6-dichlorobenzyl-L-tyrosyl)-amino-4-methylcoumarin toluenesulfonate was collected by filtration. Dissolve this in 5ml of DMF and PH with triethylamine.
After adjusting the pH to 7, 328 mg (1 mmol) of N-t-butyloxycarbonyl-L-leucine-N-hydroxysuccinimide ester was added and stirred overnight at room temperature. Then, distill off the solvent and add 50ml of water.
was added, the resulting precipitate was collected by filtration, and methyl alcohol 5
ml and 30 ml of chloroform, filtered off the insoluble materials, and recrystallized by adding ether.
(N-t-butyloxycarbonyl-L-leucyl-L-leucyl-L-valyl-0-2,6-dichlorobenzyl-L-tyrosine)-amino-4-
560 mg (yield 76%) of methylcoumarin was obtained. Melting point 245-247°C (decomposition). [α] ²⁷ _D = +1.4 (C = 1.60, DMF) Elemental analysis: Actual value C62.63%, H6.51%, N7.55% Calculated value as C ₄₈ H ₆₁ N ₅ O ₉ Cl ₂
C62.46%, H6.66%, N7.59% Reference example 5 N-t-amyloxycarbonyl-N ^G -tosyl-L-arginine-66.6g (0.15 mol) and L
-Proline benzyl ester hydrochloride 40.2g
(0.165 mol) in 500 ml of dichloromethane,
25.4 g (0.164 mol) of N.N-dimethylaminopropyl-N'-ethylcarbodiimide was added dropwise. After stirring overnight at room temperature, the solvent was distilled off, the residue was dissolved in 500 ml of ethyl acetate, and the residue was dissolved in 500 ml of 1N hydrochloric acid.
It was washed by shaking it together. Next, 500 water
After washing by shaking sequentially with 500 ml of 5% heavy sour water and 500 ml of water, the organic layer was dried with magnesium sulfate, the solvent was distilled off, the residue was dissolved in methyl alcohol, and recrystallized by adding ether. −
Amyloxycarbonyl-N ^G -tosyl-L-arginyl-L-proline benzyl ester 52g
(yield 55%). Melting point 166℃～167.5℃ [α] ^23D _-34.5゜ (C=1.0, DMF) Elemental analysis: Actual value C58.58%, H6.94%, N11.26% C ₃₁ H ₄₃ O ₇ N ₅ S Calculated value as
C59.12%, H6.88%, N11.12% Reference example 6 N-t-amyloxycarbonyl-N ^G -tosyl-L-arginyl-L-proline 40 g (64 mmol) of benzyl ester in 1 part of ethyl alcohol Comb, add 5% palladium on carbon catalyst,
After the reduction reaction was carried out for 9 hours by passing hydrogen gas under stirring at room temperature and normal pressure, the catalyst was filtered off and the solvent was distilled off. The residue was solidified by adding ether and n-hexane and collected by filtration to give N-t-amyloxycarbonyl-N ^G -tosyl-L-arginyl-
34 g (yield 99%) of L-proline was obtained. This material was used in the next experiment without being purified. Reference example 7 N-t-amyloxycarbonyl-N ^G -tosyl-L-arginyl-L-proline 5.4 g (10
mmol) and trifluoroacetic acid-P-nitrophenyl ester (4.7 g (20 mmol)) were dissolved in 50 ml of dry pyridine, stirred at 50°C for 3 hours, then added with 1 ml of water and concentrated to dryness to obtain N-t-amyloxy- Carbonyl-N ^G -tosyl-L-arginyl-
An oil containing L-proline-P-nitrophenyl ester was obtained. Meanwhile, 4.5 g of N-carbobenzoxy-L-phenylalanyl-L-histidine methyl ester
(10 mmol) was added with 20 ml of 25% hydrogen bromide acetic acid solution, stirred at room temperature for 1 hour, and then 500 ml of ether was added.
The resulting precipitate was obtained by decantation. Dissolve this in 50 ml of DMF, adjust the pH to 7 with triethylamine, and add the above N-t-amyloxycarbonyl-N ^G -tosyl-L-arginyl-L-
An oil containing proline-P-nitrophenyl ester was added and stirred overnight at room temperature, and then the solvent was distilled off. Dissolve the residue in 100 ml of ethyl acetate and add 0.5
It was washed by shaking with 100 ml of normal hydrochloric acid. followed by water
After shaking and washing with 100 ml, the organic layer was dried with magnesium sulfate. The solvent was distilled off and the residue was solidified by adding 100 ml of ether to obtain Nt-t-amyloxycarbonyl-N ^G -tosyl-L-arginyl-L-prolyl-L-phenylalanyl-L-histidine methyl ester. Yield: 6.8g (yield: 81%). This material contained a by-product thought to be P-nitrophenol in silica gel thin layer chromatography using a solvent system of chloroform:methanol:acetic acid = 85:10:5, but it was not purified further and was used in the reaction. Reference example 8 N-t-amyloxycarbonyl-N-tosyl-L-arginyl-L-prolyl-L-phenylalanyl-L-histidine methyl ester
2.5 g (3 mmol) was dissolved in 50 ml of methyl alcohol, 5.6 ml (approximately 90 mmol) of hydrazine monohydrate was added, and after stirring at room temperature for one day, the solvent was distilled off. Add 10 ml of water to the residue to remove soluble materials, then add a small amount of methanol and 100 ml of ether to solidify N-t-amyloxycarbonyl-N.
^G -tosyl-L-arginyl-L-prolyl-L
-Phenylalanyl-L-histidine hydrazide was obtained. Yield: 1.35g (yield 54%). This substance is used for silica gel thin layer chromatography solvent system chloroform:methanol:acetic acid = 85:
Small amounts of two by-products were observed at 15:5, but these were used in the next reaction without further purification. Reference example 9 N-t-amyloxycarbonyl-NG-tosyl-L-arginyl-L-prolyl-L-phenylalanyl-L-histidine hydrazide 269 mg
(0.3 mmol) was dissolved in 4 ml of DMF, a 5.4 N hydrogen chloride dioxane solution was added, and 0.1 ml of isoamyl nitrite was added while stirring at -30°C. The mixture was stirred at -10°C for 30 minutes to obtain a Nt-amyloxycarbonyl-N ^G -tosyl-L-arginyl-L-prolyl-L-phenylalanyl-L-histidine azide solution []. On the other hand, 7-(Nt-butyloxycarbonyl-L-leucyl-L-leucyl-L-valyl-0
-2,6-dichlorobenzyl-L-tyrosyl)-
Add 5 ml of dichloromethane and 1 ml of acetic acid to 184.4 mg (0.2 mmol) of amino-4-methylcoumarin to obtain 191 mg (1 mmol) of toluenesulfonic acid monohydrate.
was added and dichloromethane was distilled off under reduced pressure at 20°C, followed by stirring at 20°C for 3 hours. Next, 50 ml of ether was added, and the resulting precipitate was collected by filtration and washed with ether to obtain 7-(L-leucyl-L-leucyl-
L-valyl-0-2,6 dichlorobenzyl-L-
Tyrosyl)-amino-4-methylcoumarin toluenesulfonate was obtained. Dissolve this in 5 ml of DMF and adjust the pH to 7 with triethylamine. Cool the above [] to -50°C, add 0.76 ml of triethylamine, and add []. After stirring for one day at 0°C to 2°C, the solvent was distilled off, 10ml of water was added to the residue, and the resulting precipitate was collected by filtration. Combine this with 1 ml of methyl alcohol and 10 ml of ethyl acetate.
After stirring and filtering off the insoluble matter, 20 ml of ether was added for reprecipitation and purification to give 7-(N-t-amyloxycarbonyl-N ^G -tosyl-L-arginyl-L-prolyl-L-phenylalanyl-L). -Histidyl-L-leucyl-L-valyl-0-2.
250 mg (yield 77%) of 6-dichlorobenzyl-L-tyrosyl)-amino-4-methylcoumarin was obtained. Although 3 to 4 by-products and unreacted substances were observed in this substance using silica gel thin layer chromatography using a solvent system of chloroform:methanol:acetic acid=85:15:5, no further purification was performed. Example 7-(N〓-t-amyloxycarbonyl- ^NG
-tosyl-L-arginyl-L-prolyl-L-
Add 1 ml of anisole to 244 mg (0.15 mmol) of phenylalanyl-L-histidyl-L-leucyl-L-leucyl-L-valyl-0-2,6-dichlorobenzyl-L-tyrosyl)-amino-4-methylcoumarin and anhydrous fluorination. After reacting with 3 ml of hydrogen at 0°C for 40 minutes to remove the protective group, hydrogen fluoride was distilled off, and 20 ml of 1 molar acetic acid and 50 ml of ether were added to the residue for extraction. The water layer was prepared using “Dowex”1 manufactured by Dow Chemical Company.
After passing through 10 ml of ion exchange resin (acetic acid type) to remove attached hydrogen fluoride, the mixture was freeze-dried to obtain a powder. This was purified by column chromatography using gel filtration resin LH-20 (5 x 160 cm) using 1 mol acetic acid as a solvent to obtain the desired product 7-(L-arginyl-L-
Prolyl-L-phenylalanyl-L-histidyl-L-leucyl-L-leucyl-L-valyl-
L-tyrosyl)-amino-4-methylcoumarin
60 mg (yield 30%) of diacetate monohydrate was obtained. The ratio of amino acids contained in this compound was determined using an amino acid analyzer.

[Table] Elemental analysis values Actual values C59.17%, H7.04%, N14.95% C ₆₈ H ₈₄ D ₁₁ N ₁₄・2CH ₃ COOH・H ₂ O Calculated values C59.17%, H7.07 %, N14.64% [α] ²² _D = -42.4°C (C = 0.77 20% CH ₃ COOH) Cellulose thin layer chromatography solvent system n-butanol:acetic acid:water = 4:1:5
Rf=0.66 n-butanol:acetic acid:water:pyridine=
A single spot was given at 15:3:12:10 Rf=0.82 using Sakaguchi reagent, Pauli reagent, ninhydrin reagent drop and fluorescence. Experiments were conducted to demonstrate that the peptide derivatives of the present invention serve as fluorescent substrates for measuring renin activity. 7-(L-arginyl-L-prolyl-L-phenyl-alanyl-L-histidyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl)
-Amino-4-methylcoumarin Diacetate monohydrate in 25 DMF solutions (μmoles/ml) and 250 volumes of 0.05M pyrrolidate buffer (PH5.6) were mixed with pure renin.
After reacting 0.6 to 2.6μPH at 37℃ for 60 minutes,
The enzyme was inactivated by heating to 100°C for minutes. to this
10 pieces of aminopeptidase (aminopepti-
dase) M (5 U/ml water) was added and reacted at 37°C for 60 minutes, and the fluorescence intensity of the resulting reaction solution was measured using a wavelength of Em 440 nm (excited at EX 380 nm) of the fluorescence spectrum. The results for each concentration of pure renin are shown below.

[Table] From the above results, it can be seen that this substrate produces AMC in proportion to the renin concentration. On the other hand, it was also confirmed that this substrate produced AMC in proportion to the reaction time. Therefore, by applying this substrate to a sample solution according to the method described above, measuring the amount of AMC produced and comparing it with the standard curve, the amount of renin activity in the sample solution can be accurately measured, which is useful in the diagnosis of the aforementioned diseases. I understand that.

Claims (1)

[Claims] 1 Structural formula: A peptide derivative represented by