METHOD FOR PRODUCING PEPTIDE COMPOUND

Abstract

The invention provides a method for producing a peptide which includes a step of reacting a peptide in which a C-terminal carboxy group is activated which is obtained by reacting an N-protected peptide represented by the formula (I):

P-AA.sub.1-OH  (I)

a tertiary amine represented by the formula (II):

##STR00001##

and an acid halide in a flow reactor, with a silylated amino acid or peptide obtained by reacting an amino acid or peptide represented by the formula (III):

H-AA.sub.2-OH  (III)

with a silylating agent, in a flow reactor, wherein, in the formulae (I) to (III), AA.sub.1, AA.sub.2, P, R.sup.1, R.sup.2 and R.sup.3 are as defined herein.

Claims

1. A method for producing a peptide which comprises the following step (1): (1) a step of reacting a peptide in which a C-terminal carboxy group is activated which is obtained by reacting an N-protected peptide represented by the formula (I):
P-AA.sub.1-OH  (I) wherein AA.sub.1 represents a group derived from a peptide comprising a 2 to 20 amino acids, and P represents an N-terminal protective group, a tertiary amine represented by the formula (II): ##STR00025## wherein among R.sup.1, R.sub.2 and R.sup.3, one or two is/are a methyl group(s), the remainder(s) is/are an aliphatic hydrocarbon group(s) which may have a substituent(s), when one of R.sup.1, R.sup.2 and R.sup.3 is a methyl group, the remaining two may form a 6 to 7-membered ring, together with the nitrogen atom to which they are bonded, by combining them to form a C.sub.5-6 alkylene chain and, in this case, one of the alkylene chains may be substituted with O or NR.sup.8 where R.sup.8 represents an aliphatic hydrocarbon group which may have a substituent(s), and an acid halide in a flow reactor, with a silylated amino acid or peptide obtained by reacting an amino acid or a peptide represented by the formula (III):
H-AA.sub.2-OH  (III) wherein AA.sub.2 represents an amino acid or a group derived from a peptide having 2 to 20 residues, with a silylating agent, in a flow reactor.

2. The method for producing a peptide according to claim 1, wherein an amount of the tertiary amine represented by the formula (II) to be used is 0.05 mol equivalent to 1 mol equivalent based on the N-protected peptide represented by the formula (I), and further using a tertiary amine represented by the formula (IV) ##STR00026## wherein R.sup.4, R.sup.5 and R.sup.6 each independently represent an aliphatic hydrocarbon group, provided that a methyl group is excluded, which may have a substituent(s), two of R.sup.4, R.sup.5 and R.sup.6 may form a 6 to 7-membered ring, together with the nitrogen atom to which they are bonded, by combining them to form a C.sub.5-6 alkylene chain and, in this case, one of the alkylene chains may be replaced with O or NR.sup.8 where R.sup.8 represents an aliphatic hydrocarbon group which may have a substituent(s).

3. The method for producing a peptide according to claim 2, wherein a combined amount of the tertiary amine represented by the formula (IV) and the tertiary amine represented by the formula (II) to be used is 1.0 to 10 mol equivalents based on the N-protected peptide represented by the formula (I).

4. The method for producing a peptide according to claim 1, wherein the acid halide is an alkyl chloroformate, carboxylic acid chloride, sulfonyl chloride or phosphoryl chloride.

5. The method for producing a peptide according to claim 1, wherein the acid halide is a C.sub.1-6 alkyl chloroformate.

6. The method for producing a peptide according to claim 1, wherein the acid halide is isobutyl chloroformate.

7. The method for producing a peptide according to claim 1, wherein the tertiary amine represented by the formula (II) is N,N-dimethylbutylamine, N,N-dimethylbenzylamine, N-methyldiethylamine, N-methylpiperidine or N-methylmorpholine.

8. The method for producing a peptide according to claim 2, wherein the tertiary amine represented by the formula (IV) is N,N-diisopropylethylamine or tri-n-propylamine.

9. The method for producing a peptide according to claim 1, wherein the amino acid or the peptide is constituted by an α-amino acid.

10. The method for producing a peptide according to claim 1, wherein the protective group of the N-terminal of the N-protected peptide is a carbamate-based protective group.

11. The method for producing a peptide according to claim 1, wherein the protective group of the N-terminal of the N-protected peptide is a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group or a t-butoxycarbonyl group.

12. The method for producing a peptide according to claim 1, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide.

Description

EXAMPLES

[0090] Hereinafter, the present invention will be explained in more detail by referring to Reference Synthetic Examples and Synthetic Examples, but the present invention is not limited to these Examples.

[0091] In the present specification, when the amino acid, etc., are indicated as abbreviations, each indication is based on abbreviation by IUPAC-IUB Commission on Biochemical Nomenclature or the conventional abbreviation in this field of the art.

[0092] The proton nuclear magnetic resonance (.sup.1H-NMR) in Examples was measured by, unless otherwise specifically mentioned, JNM-ECP300 manufactured by JEOL, Ltd., or JNM-ECX300 manufactured by JEOL, Ltd., or Ascend™500 manufactured by Bruker Co., in deuterated chloroform or deuterated dimethylsulfoxide solvent, and the chemical shift was shown by the δ value (ppm) when tetramethylsilane was used as the internal standard (0.0 ppm).

[0093] In the description of the NMR spectrum, “s” means singlet, “d” means doublet, “t” means triplet, “q” means quartet, “dd” means doublet of doublet, “dt” means doublet of triplet, “m” means multiplet, “br” means broad, “J” means coupling constant, “Hz” means hertz, and CDCl.sub.3 means deuterated chloroform.

[0094] High performance liquid chromatography/mass analysis was measured by using, unless otherwise specifically mentioned, either of ACQUITY UPLC H-Class/QDa manufactured by Waters Corporation, ACQUITY UPLC H-Class/SQD2 manufactured by Waters Corporation or LC-20AD/Triple Tof5600 manufactured by Shimadzu Corporation.

[0095] In the description of the high performance liquid chromatography/mass analysis, ESI+ is a positive mode of the electrospray ionization method, M+H means a proton adduct and M+Na means a sodium adduct.

[0096] In the description of the high performance liquid chromatography/mass analysis, ESI− is a negative mode of the electrospray ionization method and M−H means a proton deficient.

[0097] For purification by silica gel column chromatography, unless otherwise specifically mentioned, either of Hi-Flash column manufactured by Yamazen Corporation, SNAP Ultra Silica Cartridge manufactured by Biotage, Silica gel 60 manufactured by Merck or PSQ60B manufactured by Fuji Silysia Chemical Ltd., was used.

[0098] Incidentally, the mixer used in the present Examples is, unless otherwise specified, a static type mixer (trade name: Comet X-01) manufactured by Techno Applications Co., Ltd.

Reference Synthetic Example 1: Synthesis of Fmoc-Phe-Phe-OH

[0099] ##STR00004##

[0100] Phenylalanine (12.8 g, 78.5 mmol) was mixed with tetrahydrofuran (75.0 g), and after adding N,O-bistrimethylsilylacetamide (31.5 g, 157 mmol), the mixture was stirred at 40° C. for 1 hour and then cooled to 0° C. (silylated amino acid solution). Fmoc-Phe-OH (25.0 g, 64.5 mmol) and N-methylmorpholine (7.83 g, 78.5 mmol) were mixed with tetrahydrofuran (125 g), the mixture was cooled to 0° C., and after adding isobutyl chloroformate (9.70 g, 71.0 mmol) at once, the mixture was stirred for 2 minutes and the silylated amino acid solution was added thereto, and the resulting mixture was stirred for 1 hour. To the obtained reaction mixture was added water (50 g), and the mixture was concentrated under reduced pressure. The concentrated liquid was extracted with ethyl acetate (130 g) twice, and washing of the obtained organic layer with water was carried out three times. The obtained organic layer was concentrated under reduced pressure, acetonitrile (150 g) was added to the residue and the mixture was again concentrated under reduced pressure, acetonitrile (130 g) was added to the residue and the mixture was stirred for 0.5 hour, and then, the mixture was filtered to obtain white solid. To the obtained solid was added acetonitrile (150 g) and the mixture was stirred for 0.5 hour and after obtaining a solid by filtration, and the solid was subjected to drying under reduced pressure to obtain Fmoc-Phe-Phe-OH (26.7 g, Yield: 78%, diastereomeric excess: 99.7 % de) as a white solid.

MASS (ESI+) m/z; 535.34 (M+H)+

[0101] The diastereomeric excess was calculated by the analysis <Analytical condition A> using high performance liquid chromatography.

<Analytical condition A>
High performance liquid chromatography: HPLC LC-20A manufactured by SHIMADZU Corporation
Column: Poroshell 120EC-C18 (2.7 μm, 3.0×100 mm) manufactured by Agilent
Column oven temperature: 40° C. [0102] Eluent: Acetonitrile : 0.05 vol % phosphoric acid aqueous solution 45:55 (0 to 15 min), 45:55 to 95:5 (15 to 18 min), 95:5 (18 to 22 min) (v/v)
Eluent rate: 0.8 mL/min
Detection wavelength: 210 nm

Synthetic Example 1: Synthesis of Fmoc-Phe-Phe-Phe-OH using N-Methylmorpholine

[0103] ##STR00005##

Fmoc-Phe-Phe-OH (2.00 g, 3.74 mmol) and N-methylmorpholine (0.45 g, 4.49 mmol) were mixed with tetrahydrofuran (8.89 g) and acetonitrile (7.86 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.56 g, 4.11 mmol) was dissolved in acetonitrile (7.86 g), which was made Solution 2. Phenylalanine (0.74 g, 4.49 mmol) was mixed with acetonitrile (7.86 g), and after adding N,O-bistrimethyl-silylacetamide (1.83 g, 8.98 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.1 mL per a minute and Solution 2 was fed at 5.80 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.14 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (1.0 mL) had been added in advance for 1.5 minutes. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 97.6% and the diastereomeric excess was 99.8 % de.
MASS (ESI+) m/z; 682.5 (M+H)+

[0104] The quantitative yield was calculated by a quantitative analysis method based on analysis <Analytical condition B> using high performance liquid chromatography.

<Analytical condition B>
High performance liquid chromatography: HPLC LC-20A manufactured by SHIMADZU Corporation
Column: Poroshell 120EC-C18 (2.7 μm, 3.0×100 mm) manufactured by Agilent
Column oven temperature: 40° C.
Eluent: Acetonitrile : 0.05 vol % phosphoric acid aqueous solution 10:90 (0 min), 10:90 to 95:5 (0 to 11 min), 95:5 (11 to 15 min) (v/v)
Eluent rate: 0.7 mL/min
Detection wavelength: 210 nm

[0105] The diastereomeric excess was calculated by the analysis <Analytical condition C> using high performance liquid chromatography.

<Analytical condition C>
High performance liquid chromatography: HPLC LC-20A manufactured by SHIMADZU Corporation
Column: Poroshell 120EC-C18 (2.7 μm, 3.0×100 mm) manufactured by Agilent
Column oven temperature: 40° C.
Eluent: Acetonitrile : 0.05 vol% phosphoric acid aqueous solution 50:50 (0 to 15 min), 50:50 to 95:5 (15 to 18 min), 95:5 (18 to 22 min) (v/v)
Eluent rate: 0.7 mL/min
Detection wavelength: 210 nm

[0106] Quantitative analysis was carried out by the absolute calibration curve method using Fmoc-Phe-Phe-Phe-OH synthesized by the following procedure as a standard substance.

[0107] A part of the N-methylpyrrolidone solution obtained by the reaction was taken out, concentrated under reduced pressure, diluted with methylene chloride (10.0 g) and washed with water (5.0 g) twice. The obtained organic layer was concentrated under reduced pressure, acetonitrile (5.0 g) was added thereto, and the mixture was stirred and filtered to obtain a solid. Then, drying under reduced pressure was carried out to obtain Fmoc-Phe-Phe-Phe-OH (0.41 g) as a white solid.

The NMR of the standard substance is shown.
.sup.1H NMR (300 MHz, CDCl.sub.3):

δ 12.8 (1H, s), 8.35 (1H, d, J=8.1 Hz), 8.06 (1H, d, J=7.5 Hz), 7.87 (2H, d, J=7.8 Hz), 7.52-7.62 (2H, m), 7.11-7.42 (20H, m), 4.42-7.62 (2H, m), 3.92-4.22 (4H, m), 2.62-3.10 (6H, m),

Reference Synthetic Example 2: Synthesis of Fmoc-Phe-Phe-Phe-OH using N-ethylmorpholine

[0108] ##STR00006##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and N-ethylmorpholine (0.129 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 19.3%.

Synthetic Example 2: Synthesis of Fmoc-Phe-Phe-Phe-OH using N-methyl-piperidine

[0109] ##STR00007##

[0110] Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and N-methylpiperidine (0.112 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 93.0% and the diastereomeric excess was 99.6% de.

Reference Synthetic Example 3: Synthesis of Fmoc-Phe-Phe-Phe-OH using N-ethylpiperidine

[0111] ##STR00008##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and N-ethylpiperidine (0.127 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 19.3%.

Synthetic Example 3: Synthesis of Fmoc-Phe-Phe-Phe-OH using N-methyl-diethylamine

[0112] ##STR00009##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and N-methyldiethylamine (0.098 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 95.4% and the diastereomeric excess was 99.7% de.

Reference Synthetic Example 4: Synthesis of Fmoc-Phe-Phe-Phe-OH Using Triethylamine

[0113] ##STR00010##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and triethylamine (0.114 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 33.2%.

Synthetic Example 4: Synthesis of Fmoc-Phe-Phe-Phe-OH using N,N-dimethylbutylamine

[0114] ##STR00011##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and N,N-dimethylbutylamine (0.114 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetra-fluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetra-fluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 95.4% and the diastereomeric excess was 99.8% de.

Synthetic Example 5: Synthesis of Fmoc-Phe-Phe-Phe-OH Using N,N-dimethylbenzylamine

[0115] ##STR00012##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and N,N-dimethylbenzylamine (0.152 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 92.2% and the diastereomeric excess was 99.5% de.

Synthetic Example 6: Synthesis of Fmoc-Phe-Phe-Phe-OH Using N,N-diisopropylethylamine and N-methylmorpholine

[0116] ##STR00013##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol), N,N-diisopropylethylamine (0.121 g, 0.94 mmol) and N-methylmorpholine (0.019 g, 0.19 mmol) were mixed with N-methyl-pyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 97.3% and the diastereomeric excess was 99.8% de.

Reference Synthetic Example 5: Synthesis of Fmoc-Phe-Phe-Phe-OH Using N,N-diisopropylethylamine

[0117] ##STR00014##

Fmoc-Phe-Phe-OH (0.50 g, 0.94 mmol) and N,N-diisopropylethylamine (0.145 g, 1.13 mmol) were mixed with N-methylpyrrolidone (2.58 g) and chloroform (3.73 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.153 g, 1.13 mmol) was dissolved in chloroform (3.73 g), which was made Solution 2. Phenylalanine (0.185 g, 1.13 mmol) was mixed with N-methylpyrrolidone (2.58 g), and after adding N,O-bistrimethylsilylacetamide (0.457 g, 2.26 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.93 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.02 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which water (0.5 mL) had been added in advance for 18 seconds. The obtained solution was homogenized by adding N-methylpyrrolidone, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 2.0%.

Reference Synthetic Example 6: Synthesis 1 of Fmoc-Phe-Phe-Phe-OH By Batch System

[0118] ##STR00015##

Phenylalanine (0.074 g, 0.44 mmol) was mixed with acetonitrile (1.58 g), and after adding N,O-bistrimethylsilylacetamide (0.183 g, 0.88 mmol) thereto, and the mixture was stirred at 50° C. for 1 hour and cooled to 25° C. (silylated amino acid solution). Fmoc-Phe-Phe-OH (0.20 g, 0.37 mmol) and N-methylmorpholine (0.045 g, 0.44 mmol) were mixed with tetrahydrofuran (0.89 g) and acetonitrile (0.79 g), the mixture was cooled to 0° C., and after adding isobutyl chloroformate (0.061 g, 0.44 mmol) at once, the resulting mixture was stirred for 30 minutes and the silylated amino acid solution was added thereto over 2 hours followed by stirring for 30 minutes. To the obtained reaction mixture were added water (1 g) and N-methylpyrrolidone and the mixture was homogenized. The quantitative yield of Fmoc-Phe-Phe-Phe-OH of the obtained solution was 42.0% and the diastereomeric excess was 95.6% de.

Reference Synthetic Example 7: Synthesis 2 of Fmoc-Phe-Phe-Phe-OH By Batch System

[0119] ##STR00016##

Phenylalanine (0.074 g, 0.44 mmol) was mixed with acetonitrile (1.58 g), and after adding N,O-bistrimethylsilylacetamide (0.183 g, 0.88 mmol) thereto, and the mixture was stirred at 50° C. for 1 hour and cooled to 25° C. (silylated amino acid solution). Fmoc-Phe-Phe-OH (0.20 g, 0.37 mmol) and N-methylmorpholine (0.045 g, 0.44 mmol) were mixed with tetrahydrofuran (0.89 g) and acetonitrile (0.79 g), and after adding isobutyl chloroformate (0.061 g, 0.44 mmol) at 25° C. at once, the resulting mixture was stirred for 30 minutes and the silylated amino acid solution was added thereto over 2 hours followed by stirring for 30 minutes. To the obtained reaction mixture were added water (1 g) and N-methylpyrrolidone and the mixture was homogenized, and the quantitative yield of Fmoc-Phe-Phe-Phe-OH of the thus obtained solution was 21.7% and the diastereomeric excess was 30.1% de.

Synthetic Example 7: Synthesis of Fmoc-Phe-Phe-Cys(Trt)-OH

[0120] ##STR00017##

Fmoc-Phe-Phe-OH (2.00 g, 3.74 mmol) and N-methylmorpholine (0.454 g, 4.49 mmol) were mixed with tetrahydrofuran (8.89 g) and acetonitrile (7.86 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.562 g, 4.11 mmol) was dissolved in acetonitrile (7.863 g), which was made Solution 2. S-tritylcysteine (1.632 g, 4.49 mmol) was mixed with acetonitrile (17.3 g), and after adding N,O-bistrimethyl-silylacetamide (1.827 g, 8.98 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 9.4 mL per a minute and Solution 2 was fed at 4.5 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 11.1 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which an aqueous 10% potassium carbonate solution (10 mL) had been added in advance for 125 seconds. The obtained solutions were separated, an aqueous 10% potassium carbonate solution (15 g) was added thereto again and the liquids were separated, and the organic layer was washed with an aqueous 10% sodium chloride solution. To the obtained organic layer were added an aqueous 10% sodium chloride solution (5 g) and an aqueous 10% ammonium chloride solution (5 g), and the liquids were separated twice. The obtained organic layer was concentrated and purified by silica gel chromatography to obtain Fmoc-Phe-Phe-Cys(Trt)-OH (2.74 g, Yield: 94%, diastereomeric excess: 99.2% de) as a white solid.
MASS (ESI+) m/z; 902.36 (M+Na)+

[0121] The diastereomeric excess was calculated by the analysis <Analytical condition D> using high performance liquid chromatography.

<Analytical condition D>
High performance liquid chromatography: HPLC LC-20A manufactured by SHIMADZU Corporation
Column: Poroshell 120EC-C18(2.7 μm, 3.0×100mm) manufactured by Agilent
Column oven temperature: 40° C.
Eluent: Acetonitrile : 0.05 vol % phosphoric acid aqueous solution 58:42 (0-27 min), 58:42-95:5 (27-32 min), 95:5 (32-34 min) (v/v)
Eluent rate: 0.9 mL/min
Detection wavelength: 210 nm

Synthetic Example 8: Synthesis of Fmoc-Phe-Phe-Cys(Trt)-Tyr(t-Bu)-OH

[0122] ##STR00018##

Fmoc-Phe-Phe-Cys(Trt)-OH (2.00 g, 2.27 mmol) and N-methylmorpholine (0.276 g, 2.72 mmol) were mixed with tetrahydrofuran (8.89 g) and acetonitrile (7.86 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.341 g, 2.50 mmol) was dissolved in acetonitrile (7.863 g), which was made Solution 2. O-t-Bu-tyrosine (0.647 g, 2.72 mmol) was mixed with acetonitrile (7.86 g), and after adding N,O-bistrimethylsilylacetamide (1.110 g, 5.45 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.4 mL per a minute and Solution 2 was fed at 5.8 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 6.8 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected in a container to which an aqueous 10% potassium carbonate solution (10 mL) had been added in advance for 90 seconds. The obtained solutions were separated, an aqueous 10% potassium carbonate solution (15 g) was added thereto again and the liquids were separated, and the organic layer was washed with an aqueous 10% sodium chloride solution. The obtained organic layer was concentrated and purified by silica gel chromatography, and after concentrating the collected solution, hexane (40 g) was mixed therewith and the precipitated solid was filtered and dried to obtain Fmoc-Phe-Phe-Cys(Trt)-Tyr(t-Bu)-OH (1.95 g, Yield: 92%, diastereomeric excess: 99.6% de) as a white solid.
MASS (ESI+) m/z; 1121.64 (M+Na)+

[0123] The diastereomeric excess was calculated by the analysis <Analytical condition E> using high performance liquid chromatography.

<Analytical condition E>
High performance liquid chromatography: HPLC LC-20A manufactured by SHIMADZU Corporation
Column: Poroshell 120EC-C18 (2.7 μm, 3.0×100 mm) manufactured by Agilent
Column oven temperature: 40° C.
Eluent: Acetonitrile : 0.05 vol % phosphoric acid aqueous solution 70:430 (0 to 27 min), 70:30 to 95:5 (27 to 32 min), 95:5 (32 to 34 min) (v/v)
Eluent rate: 0.9 mL/min
Detection wavelength: 210 nm

Reference Synthetic Example 8: Synthesis of Fmoc-Phe-D-Phg-OH

[0124] ##STR00019##

[0125] Fmoc-Phe-OH (2.00 g, 5.16 mmol) and N-methylmorpholine (0.627 g, 6.19 mmol) were mixed with tetrahydrofuran (8.89 g) and acetonitrile (7.86 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.776 g, 5.68 mmol) was dissolved in acetonitrile (7.863 g), which was made Solution 2. D-phenylglycine (0.936 g, 6.19 mmol) was mixed with acetonitrile (7.86 g), and after adding N,O-bistrimethylsilylacetamide (3.255 g, 12.4 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 11.5 mL per a minute and Solution 2 was fed at 5.6 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was fed to the obtained mixed solution at 7.8 mL per a minute and the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected for 105 seconds in a container to which water (2 mL) had been added in advance. With the obtained solution was mixed acetonitrile (80 g), and the precipitated solid was filtered and dried to obtain Fmoc-Phe-D-Phg-OH (1.50 g, Yield: 62%) as a white solid.

MASS (ESI+) m/z; 521.3 (M+Na)+

Synthetic Example 9: Synthesis of Fmoc-Phe-D-Phg-Phe-OH

[0126] ##STR00020##

Fmoc-Phe-D-Phg-OH (0.50 g, 0.96 mmol) and N-methylmorpholine (0.117 g, 1.15 mmol) were mixed with N-methylpyrrolidone (2.58 g) and acetonitrile (1.97 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.144 g, 1.06 mmol) was dissolved in acetonitrile (1.97 g), which was made Solution 2. Phenylalanine (0.19 g, 1.06 mmol) was mixed with acetonitrile (1.97 g), and after adding N,O-bistrimethylsilylacetamide (0.469 g, 2.30 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 11.9 mL per a minute and Solution 2 was fed at 5.8 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was further fed at 7.2 mL per a minute to the obtained mixed solution and the resulting mixture was mixed with a mixer and passed through a tube made of a polytetrafluoro-ethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected for 20 seconds in a container to which water (1 mL) had been added in advance. The obtained solution was concentrated under reduced pressure and acetonitrile (25 g) was mixed therewith, and the precipitated solid was filtered and dried to obtain Fmoc-Phe-D-Phg-Phe-OH (0.45 g, Yield: 95%) as a white solid.
MASS (ESI+) m/z; 668.4 (M+Na)+

[0127] The diastereomeric excess was calculated by the analysis <Analytical condition C> using high performance liquid chromatography.

Synthetic Example 10: Synthesis of Cbz-Phe-Phe-Leu-OH

[0128] ##STR00021##

Cbz-Phe-Phe-OH (2.00 g, 4.48 mmol) and N-methylmorpholine (0.591 g, 5.38 mmol) were mixed with tetrahydrofuran (8.89 g) and acetonitrile (7.86 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.641 g, 4.93 mmol) was dissolved in acetonitrile (7.863 g), which was made Solution 2. Leucine (0.705 g, 5.38 mmol) was mixed with acetonitrile (7.86 g), and after adding N,O-bistrimethyl-silylacetamide (2.187 g, 10.8 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 12.0 mL per a minute and Solution 2 was fed at 5.8 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was further fed at 73 mL per a minute to the obtained mixed solution and the resulting mixture was mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected for 100 seconds in a container to which 5 wt % hydrochloric acid (5 g) had been added in advance. The obtained solution was concentrated under reduced pressure, ethyl acetate (30 g) was added to the residue and the liquids were separated, and the organic layer was washed with an aqueous 10 wt % sodium chloride solution (5 g) twice. The obtained organic layer was concentrated and hexane (40 g) was added thereto and the resulting mixture was stirred, and the precipitated solid was filtered and dried to obtain Cbz-Phe-Phe-Leu-OH (2.17 g, Yield: 96%, diastereomeric excess: 100% de) as a white solid.
MASS (ESI+) m/z; 560.4 (M+H)+

[0129] The diastereomeric excess was calculated by the analysis <Analytical condition A> using high performance liquid chromatography.

Reference Synthetic Example 9: Synthesis of H-Phe-Phe-Leu-OH

[0130] ##STR00022##

Cbz-Phe-Phe-Leu-OH (1.50 g, 2.68 mmol) was added to tetrahydrofuran (10 g) and methanol (10 g) and dissolved therein, and a 10 wt % palladium carbon (0.15 g, available from NE Chemcat Corporation, PE Type) was added to the solution and the resulting mixture was stirred at 21° C. by hydrogen substitution for 1 hour. The obtained solution was filtered, N,N-dimethylformamide (150 g) was added to the filtrate and the mixture was filtered to obtain Filtrate 1. To the obtained filtrate was again added N,N-dimethylformamide (150 g) and after stirring the mixture, it was filtered to obtain Filtrate 2. Filtrate 1 and Filtrate 2 were mixed and concentrated under reduced pressure, and acetonitrile (20 g) was added and after the mixture was stirred, filtration was carried out and the obtained solid was dried to obtain H-Phe-Phe-Leu-OH (1.03 g, Yield: 90%) as a gray solid.
MASS (ESI+) m/z; 426.4 (M+H)+

Synthetic Example 11: Synthesis of Fmoc-Phe-Phe-Cys(Trt)-Tyr(t-Bu)-Phe-Phe-Leu-OH

[0131] ##STR00023##

Fmoc-Phe-Phe-Cys(Trt)-Tyr(t-Bu)-OH (0.55 g, 0.50 mmol) and N-methylmorpholine (0.066 g, 0.60 mmol) were mixed with tetrahydrofuran (2.45 g) and acetonitrile (2.16 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.075 g, 0.55 mmol) was dissolved in acetonitrile (2.16 g), which was made Solution 2. H-Phe-Phe-Leu-OH (0.255 g, 0.60 mmol) was mixed with acetonitrile (2.16 g), and after adding N,O-bistrimethylsilylacetamide (0.244 g, 1.20 mmol) thereto, the mixture was stirred at room temperature and homogenized, which was made Solution 3. Solution 1 was fed at 12.3 mL per a minute and Solution 2 was fed at 5.8 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was further fed at 6.9 mL per a minute to the obtained mixed solution and the resulting mixture was mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected for 20 seconds in a container to which water (1 g) had been added in advance. With the obtained solution was mixed water (4 g) and the mixture was stirred, and the precipitated solid was filtered and dried to obtain Fmoc-Phe-Phe-Cys(Trt)-Tyr(t-Bu)-Phe-Phe-Leu-OH (0.43 g, Yield: 84%, diastereomeric excess: 99.4% de) as a white solid.
MASS (ESI+) m/z; 1507(M+H)+

[0132] The diastereomeric excess was calculated by the analysis <Analytical condition F> using high performance liquid chromatography.

<Analytical condition F>
High performance liquid chromatography: HPLC LC-20A manufactured by SHIMADZU Corporation
Column: Poroshell 120EC-C18 (2.7 μm, 3.0×100 mm) manufactured by Agilent
Column oven temperature: 40° C.
Eluent: Acetonitrile : 0.05 vol % phosphoric acid aqueous solution 80:20 (0 to 27 min), 80:20 to 95:5 (27 to 32 min), 95:5 (32 to 34 min) (v/v)
Eluent rate: 0.9 mL/min
Detection wavelength: 210 nm

Synthetic Example 12: Synthesis of Fmoc-Phe-Phe-MeGly-OH

[0133] ##STR00024##

Fmoc-Phe-Phe-OH (2.00 g, 3.74 mmol) and N-methylmorpholine (0.454 g, 4.49 mmol) were mixed with tetrahydrofuran (8.89 g) and acetonitrile (7.86 g), and homogenized, which was made Solution 1. Isobutyl chloroformate (0.562 g, 4.11 mmol) was dissolved in acetonitrile (7.863 g), which was made Solution 2. Sarcosine (0.667 g, 7.48 mmol) was mixed with acetonitrile (7.86 g), and after adding N,O-bistrimethyl-silylacetamide (3.04 g, 15.0 mmol) thereto, the mixture was stirred at 50° C. for 1 hour and homogenized, and the temperature of the mixture was cooled to room temperature, which was made Solution 3. Solution 1 was fed at 11.7 mL per a minute and Solution 2 was fed at 5.6 mL per a minute, respectively, the solutions were mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 0.3 m, and Solution 3 was further fed at 7.7 mL per a minute to the obtained mixed solution and the resulting mixture was mixed with a mixer and passed through a tube made of a polytetrafluoroethylene resin having an inner diameter of 1 mm and a length of 1.0 m, and the obtained solution was collected for 107 seconds in a container to which 5 wt % hydrochloric acid (10 g) had been added in advance. The obtained solution was concentrated under reduced pressure, ethyl acetate (30 g) was added thereto and the liquids were separated, and the organic layer was washed with an aqueous 10 wt % sodium chloride solution (10 g) twice. The obtained organic layer was concentrated and purified by silica gel chromatography to obtain Fmoc-Phe-Phe-MeGly-OH (2.06 g, Yield: 96%, diastereomeric excess: 99.7% de) as a white solid.
MASS (ESI+) m/z; 606.4 (M+H)+

[0134] The diastereomeric excess was calculated by the analysis <Analytical condition A> using high performance liquid chromatography.

UTILIZABILITY IN INDUSTRY

[0135] According to the present invention, a method for producing a peptide with high efficiency can be provided.