METHOD FOR PRODUCING PEPTIDE COMPOUND

Abstract

The invention provides a method for producing a peptide which comprises the following steps (1) and (2): (1) a step of condensing a C-protected amino acid or a C-protected peptide to a C-terminal of an N-protected amino acid or an N-protected peptide represented by the formula (I):

##STR00001##

wherein Y represents an amino acid in which a C-terminal is unprotected or a peptide in which a C-terminal is unprotected, R.sup.1, R.sup.2 and R.sup.3 each independently represent an aliphatic hydrocarbon group which may have a substituent(s), a total number of the carbon atoms in the R.sup.1R.sup.2R.sup.3Si group is 10 or more, and the R.sup.1R.sup.2R.sup.3SiOC(O) group is bonded to the N-terminal in Y, and (2) a step of removing the protective group at the C-terminal of the peptide obtained in step (1).

Claims

1. A method for producing a peptide, which comprises the following Steps (1) and (2): (1) a step of condensing a C-protected amino acid or a C-protected peptide to a C-terminal of an N-protected amino acid or an N-protected peptide represented by the formula (I): ##STR00034## wherein, Y represents an amino acid in which a C-terminal is unprotected or a peptide in which a C-terminal is unprotected, R.sup.1, R.sup.2 and R.sup.3 each independently represent an aliphatic hydrocarbon group which may have a substituent(s), a total number of the carbon atoms in the R.sup.1R.sup.2R.sup.3Si group is 10 or more, and the R.sup.1R.sup.2R.sup.3SiOC(O) group is bonded to the N-terminal in Y, and (2) a step of removing the protective group at the C-terminal of the peptide obtained in Step (1).

2. The producing method according to claim 1, which further comprises one or more repeating the following Steps (3) and (4): (3) a step of condensing the C-protected amino acid or the C-protected peptide to the C-terminal of the peptide obtained in Step (2) or (4), and (4) a step of removing the protective group at the C-terminal of the peptide obtained in Step (3).

3. The producing method according to claim 1, which comprises a step of purifying the obtained peptide by liquid separating.

4. The producing method according to claim 1, which comprises a step of purifying the obtained peptide by liquid separating with an acidic aqueous solution or a basic aqueous solution.

5. The producing method according to claim 1, wherein the protective group at the C-terminal of the C-protected amino acid or the C-protected peptide is a C.sub.1-6 alkyl group, a C.sub.7-10 aralkyl group or a tri-C.sub.1-6 alkylsilyl group.

6. The producing method according to claim 1, wherein the protective group at the C-terminal of the C-protected amino acid or the C-protected peptide is a C.sub.1-6 alkyl group or a tri-C.sub.1-6 alkylsilyl group.

7. The producing method according to claim 1, wherein the protective group at the C-terminal of the C-protected amino acid or the C-protected peptide is a tri-C.sub.1-6 alkylsilyl group.

8. The producing method according to claim 1, wherein the protective group at the C-terminal of the C-protected amino acid or the C-protected peptide is a tri-methylsilyl group.

9. The producing method according to claim 1, wherein the condensation in Step (1) is carried out by using a condensation agent selected from the group consisting of a carbodiimide-based condensation agent, a chloroformate-based condensation agent, an acid halide-based condensation agent, a phosphonium-based condensation agent and an uronium-based condensation agent.

10. The producing method according to claim 1, wherein the condensation in Step (1) is carried out using a condensation agent selected from the group consisting of isobutyl chloroformate, pivaloyl chloride and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate.

11. The producing method according to claim 1, wherein a base is further used in Step (1).

12. The producing method according to claim 11, wherein the base is an aliphatic amine or an aromatic amine.

13. The producing method according to claim 11, wherein the base is N,N-diisopropylethylamine or N-methylmorpholine.

14. The producing method according to claim 1, wherein the deprotection conditions in Step (2) are conditions using a deprotecting agent other than the fluorine compound.

15. The producing method according to claim 1, wherein the deprotection conditions in Step (2) are conditions using water, a base or an acid, or using hydrogen and a metal catalyst.

16. The producing method according to claim 1, wherein the deprotection conditions in Step (2) are conditions using water, trifluoroacetic acid or lithium hydroxide, or using hydrogen and palladium carbon powder.

17. The producing method according to claim 7, wherein the deprotection conditions in Step (2) are conditions using water.

18. The producing method according to claim 1, which further comprises the following Step (5): (5) a step of removing the protective group at the N-terminal of the peptide obtained in Step (2) or (4) with a deprotecting agent.

19. The producing method according to claim 18, wherein the deprotecting agent using in Step (5) is a fluorine compound.

20. The producing method according to claim 19, wherein the fluorine compound is potassium fluoride or ammonium fluoride.

21. The producing method according to claim 1, which further comprises the following Steps (6) and (7): (6) a step of condensing a C-protected amino acid or C-protected peptide to the C-terminal of the peptide obtained in Step (2) or (4), and (7) a step of removing the protective group at the N-terminal of the peptide obtained in Step (6) with a deprotecting agent.

22. The producing method according to claim 21, wherein the deprotecting agent using in Step (7) is a fluorine compound.

23. The producing method according to claim 22, wherein the fluorine compound is potassium fluoride or ammonium fluoride.

24. The producing method according to claim 21, wherein the protective group at the C-terminal of the C-protected amino acid or the C-protected peptide is a C.sub.1-6 alkyl group or a benzyl group.

25. The producing method according to claim 1, wherein a total number of the carbon atoms in the R.sup.1R.sup.2R.sup.3Si group is 10 to 100.

26. The producing method according to claim 1, wherein a total number of the carbon atoms in the R.sup.1R.sup.2R.sup.3Si group is 10 to 40.

27. The producing method according to claim 1, wherein a total number of the carbon atoms in the R.sup.1R.sup.2R.sup.3Si group is 12 to 26.

28. The producing method according to claim 1, wherein two or three of R.sup.1, R.sup.2 and R.sup.3 are each independently a secondary or tertiary aliphatic hydrocarbon group.

29. The producing method according to claim 28, wherein two of R.sup.1, R.sup.2 and R.sup.3 are each independently a secondary aliphatic hydrocarbon group, and the remaining one is a tertiary aliphatic hydrocarbon group.

30. The producing method according to claim 29, wherein two of R.sup.1, R.sup.2 and R.sup.3 are each independently a secondary C.sub.3-6 alkyl group, and the remaining one is a tertiary C.sub.4-6 alkyl group.

31. The producing method according to claim 1, wherein the R.sup.1R.sup.2R.sup.3Si group is a di-i-propyl-t-butylsilyl group.

32. The producing method according to claim 28, wherein two of R.sup.1, R.sup.2 and R.sup.3 are each independently a secondary aliphatic hydrocarbon group, and the remaining one is a secondary aliphatic hydrocarbon group having a substituent, wherein the substituent of the secondary aliphatic hydrocarbon group exists on the carbon atom bonded to the silyl atom.

33. The producing method according to claim 32, wherein two of R.sup.1, R.sup.2 and R.sup.3 are each independently a secondary C.sub.3-6 alkyl group, and the remaining one is a secondary C.sub.3-6 alkyl group substituted by a phenyl group, wherein the phenyl group which is a substituent of the secondary C.sub.3-6 alkyl group exists on the carbon atom bonded to the silyl atom.

34. The producing method according to claim 33, wherein the R.sup.3R.sup.2R.sup.3Si group is a di-i-propylcumylsilyl group.

35. The producing method according to claim 28, wherein two of R.sup.1, R.sup.2 and R.sup.3 are each independently a tertiary aliphatic hydrocarbon group.

36. The producing method according to claim 35, wherein two of R.sup.1, R.sup.2 and R.sup.3 are each independently a tertiary C.sub.4-6 alkyl group.

37. The producing method according to claim 36, wherein the R.sup.1R.sup.2R.sup.3Si group is a di-t-butylisobutylsilyl group.

38. The producing method according to claim 36, wherein the R.sup.1R.sup.2R.sup.3Si group is a benzyl-di-t-butylsilyl group, a di-t-butyloctadecylsilyl group or a di-t-butylcyclohexylsilyl group.

39. The producing method according to claim 1, wherein the amino acid or the peptide consists of an α-amino acid.

Description

EXAMPLES

[0149] 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.

[0150] In the present specification, when the amino acid, etc., are indicated as an abbreviation, each indication is based on the abbreviation by IUPAC-IUB Commission on Biochemical Nomenclature or the conventional abbreviation in this field of the art.

[0151] Incidentally, in Synthetic Examples, “M” means mol/L.

[0152] 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 dimethyl sulfoxide solvent, and the chemical shift was shown by the δ value (ppm) when tetramethylsilane was used as the internal standard (0.0 ppm).

[0153] In the description of the NMR spectrum, “s” means singlet, “d” means doublet, “t” means triplet, “q” means quartet, “sep” means septet, “dt” means doublet of triplet, “m” means multiplet, “br” means broad, “J” means coupling constant, “Hz” means helz, and “CDCl.sub.3” means deuterated chloroform.

[0154] High performance liquid chromatography/mass analysis was measured 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.

[0155] In the description of high performance liquid chromatography/mass analysis, ESI+ means a positive mode of the electrospray ionization method, M+H means a proton adduct and M+Na means a sodium adduct.

[0156] In the description of high performance liquid chromatography/mass analysis, ESI− means a negative mode of the electrospray ionization method, and M−H means a proton deficient.

[0157] In 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 AG, silica gel 60 manufactured by Merck or PSQ60B manufactured by Fuji Silysia Chemical Ltd., was used.

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

[0158] ##STR00004##

[0159] (i) Boc-Phe-OBn (1.49 g, 4.22 mmol) and lutidine (0.68 g, 6.3 mmol) were mixed with acetonitrile (15 mL), and triisopropylsilyl trifluoromethanesulfonate (1.42 g, 4.63 mmol) was added thereto at 0° C. and the mixture was stirred for 3 hours. To the mixture were added lutidine (0.68 g, 6.3 mmol) and triisopropyl silyl trifluoromethane-sulfonate (1.42 g, 4.63 mmol) at 0° C., and the temperature of the mixture was raised to room temperature and stirred for 20 hours. The obtained reaction mixture was diluted with t-butyl methyl ether (45 mL), and washed successively with a saturated aqueous sodium hydrogen carbonate solution (15 mL), saturated aqueous ammonium chloride solution (15 mL) and water (15 mL) in this order. After concentrating the obtained organic layer, the residue was purified by silica gel column chromatography to obtain Tsoc-Phe-OBn (1.56 g, Yield: 82%) as a colorless liquid.

[0160] .sup.1H-NMR (CDCl.sub.3)

[0161] δ ppm: 1.04-1.06 (18H, m), 1.27 (3H, sep, J=7.8 Hz), 3.04-3.18 (2H, m), 4.63-4.50 (1H, m), 5.09-5.21 (3H, m), 7.02-7.63 (10H, m)

[0162] MASS (ESI+) m/z; (M+H)+456.37

[0163] (ii) Tsoc-Phe-OBn (0.383 g, 0.840 mmol) was mixed with ethyl acetate (8.0 mL), and after adding 10 wt % Pd—C (39.9 mg, 0.037 mmol) thereto, the mixture was stirred under a hydrogen gas atmosphere at room temperature for 1 hour. The reaction mixture was filtered and the obtained filtrate was concentrated to obtain Tsoc-Phe-OH (0.31 g,

[0164] Yield: 101%).

[0165] .sup.1H-NMR (CDCl.sub.3)

[0166] δ ppm: 1.05 (18H, d, J=7.5 Hz), 1.26 (3H, Sep, 7.5 Hz), 3.06-3.27 (2H, m), 4.60-4.66 (1H, m), 5.13 (2H, d, 7.5 Hz), 7.18-7.33 (5H, m)

[0167] MASS (ESI+) m/z; (M+H)+366.32

Reference Synthetic Example 2: Synthesis of Tsoc-Phe-Phe-OH

[0168] ##STR00005##

[0169] Tsoc-Phe-OH (0.222 g, 0.608 mmol) and N-methylmorpholine (0.064 g, 0.63 mmol) were mixed with tetrahydrofuran (4.0 mL), and isobutyl chloroformate (0.082 g, 0.60 mmol) was added thereto at 0° C. and the mixture was stirred for 15 minutes. To the solution was added a solution separately prepared by mixing H-Phe-OH (0.109 g, 0.66 mmol), N,O-bis(trimethylsilyl)acetamide (0.268 g, 1.32 mmol) and tetrahydrofuran (1.0 mL) and stirring at 55° C. for 1 hour, and the mixture was stirred at 0° C. for 2 hours so that disappearance of the starting materials was confirmed. The obtained reaction mixture was diluted with t-butyl methyl ether (4.0 mL), and successively washed with a 10 wt % aqueous potassium carbonate solution (2.0 mL), a 20 wt % aqueous ammonium chloride solution (2.0 mL) and a saturated aqueous sodium chloride solution (4.0 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Tsoc-Phe-Phe-OH (0.089 g, Yield: 29%) as a white solid.

[0170] .sup.1H-NMR (CDCl.sub.3)

[0171] δ ppm: 0.90-1.19 (18H, m), 1.21-1.32 (3H, m), 2.79-3.21 (4H, m), 4.37 (1H, br), 4.58 (1H, br), 5.66 (1H, br), 5.87 (1H, br), 7.09 (10H, br)

[0172] MASS (ESI+) m/z; (M+H)+513.35

Reference Synthetic Example 3: Synthesis of Tsoc-Phe-Phe-OH

[0173] ##STR00006##

[0174] (i) Tsoc-Phe-OH (0.298 g, 0.815 mmol) and N-methylmorpholine (0.186 g, 1.84 mmol) were mixed with ethyl acetate (8.0 mL), and isobutyl chloroformate (0.126 g, 0.92 mmol) was added thereto at −30° C. To the solution was added H-Phe-OMe hydrochloride (0.201 g, 0.93 mmol), and then, the mixture was stirred at the same temperature for 30 minutes. The temperature of the reaction mixture was raised to room temperature, and after stirring the mixture for 1 hour, it was successively washed with 5 wt % an aqueous sodium hydrogen carbonate solution (3.0 mL, twice) and water (3.0 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Tsoc-Phe-Phe-OMe (0.436 g, Yield: 102%) as colorless oil.

[0175] .sup.1H-NMR (CDCl.sub.3)

[0176] δ ppm: 0.90 (1H, d, J=6.9 Hz), 1.02-1.06 (18H, m), 1.26 (3H, Sep, J=7.5 Hz), 2.97-3.10 (4H, m), 3.65 (3H, s), 4.30-4.38 (1H, m), 4.71-4.78 (1H, m), 5.21 (1H, d, J=7.8 Hz), 6.16 (1H, d, J=7.2 Hz), 6.97-7.29 (10H, m)

[0177] MASS (ESI+) m/z; (M+H)+527.33

[0178] (ii) Tsoc-Phe-Phe-OMe (0.078 g, 0.15 mmol) was mixed with methanol (1.6 mL), and 5 wt % aqueous lithium hydroxide solution (0.085 g, 0.18 mmol) was added thereto and the mixture was stirred at room temperature for 10 minutes. When the reaction mixture was analyzed by LC-MS, Tsoc-Phe-Phe-OMe was decomposed and no Tsoc-Phe-Phe-OH was formed.

Reference Synthetic Example 4: Synthesis of Tsoc-Phe-Phe-OH

[0179] ##STR00007##

[0180] (i) Tsoc-Phe-OH (0.279 g, 0.764 mmol), N-methylmorpholine (0.162 g, 1.60 mmol) were mixed with ethyl acetate (5.6 mL), and isobutyl chloroformate (0.110 g, 0.80 mmol) was added thereto at −10° C. To the solution was added H-Phe-O(t-Bu) hydrochloride (0.207 g, 0.80 mmol), and then, the mixture was stirred at the same temperature for 45 minutes. The obtained reaction mixture was successively washed with 5 wt % an aqueous sodium hydrogen carbonate solution (5.6 mL, twice) and water (5.6 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Tsoc-Phe-Phe-O(t-Bu) (0.294 g, Yield: 68%) as a white solid.

[0181] .sup.1H-NMR (CDCl.sub.3)

[0182] δ ppm: 1.05 (18H, d, J=7.2 Hz), 1.27 (3H, Sep, J=7.2 Hz), 1.35 (9H, s), 3.01-3.06 (4H, m), 4.31-4.38 (1H, m), 4.57-4.63 (1H, m), 5.22 (1H, d, J=8.1 Hz), 6.15 (1H, d, J=7.5 Hz), 7.03-7.29 (10H, m)

[0183] MASS (ESI+) m/z; (M+H)+569.38

[0184] (ii) Tsoc-Phe-Phe-O(t-Bu) (0.10 g, 0.18 mmol) was mixed with methylene chloride (2.0 mL), and trifluoroacetic acid (0.80 g, 7.0 mmol) was added thereto and the mixture was stirred for 2 hours and 30 minutes. When the reaction mixture was analyzed by LC-MS, Tsoc-Phe-Phe-O(t-Bu) was decomposed, and no Tsoc-Phe-Phe-OH was formed.

Reference Synthetic Example 5: Synthesis of BIBSoc-Phe-OH

[0185] ##STR00008##

[0186] (i) Boc-Phe-OBn (0.752 g, 2.11 mmol) and lutidine (0.34 g, 3.16 mmol) were mixed with acetonitrile (7.5 mL), di-t-butylisobutylsilyl trifluoromethanesulfonate (0.81 g, 2.32 mmol) was added thereto at 0° C. and the mixture was stirred for 2 hours. To the mixture were added lutidine (0.11 g, 1.1 mmol) and di-t-butylisobutylsilyl trifluoro-methanesulfonate (0.27 g, 0.77 mmol) at 0° C., and the temperature of the mixture was raised to room temperature and it was stirred for 16 hours. The obtained reaction mixture was diluted with t-butyl methyl ether (23 mL), and successively washed with a saturated aqueous sodium hydrogen carbonate solution (7.5 mL), a saturated aqueous ammonium chloride solution (7.5 mL) and water (7.5 mL) in this order. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain BIBSoc-Phe-OBn (0.83 g, Yield: 79%) as a colorless liquid.

[0187] .sup.1H-NMR (CDCl.sub.3)

[0188] δ ppm: 0.83 (2H, d, J=6.9 Hz), 0.94 (6H, d, J=6.6 Hz), 1.03 (18H, m), 1.99 (1H, br Sep, J=6.9 Hz), 3.03-3.17 (2H, m), 4.67 (1H, dt, J=8.4 Hz, 6.0 Hz), 5.08-5.19 (3H, m), 7.01-7.35 (10H, m)

[0189] MASS (ESI+) m/z; (M+H)+498.42

[0190] (ii) BIBSoc-Phe-OBn (0.330 g, 0.69 mmol) was mixed with ethyl acetate (5.0 mL), and after adding 10 wt % Pd—C (38.9 mg, 0.037 mmol) thereto, the mixture was stirred under a hydrogen gas atmosphere at room temperature for 1 hours and 30 minutes. The reaction mixture was filtered and the obtained filtrate was concentrated to obtain BIBSoc-Phe-OH (0.250 g, Yield: 92%).

[0191] .sup.1H-NMR (CDCl.sub.3)

[0192] δ ppm: 0.83 (2H, d, J=6.9 Hz), 0.94 (6H, br d, J=6.6 Hz), 1.03 (18H, m), 1.99 (1H, Sep, J=6.6 Hz), 3.06-3.26 (2H, m), 4.60-4.66 (1H, m), 5.04 (1H, d, J=7.8 Hz), 7.16-7.32 (5H, m)

[0193] MASS (ESI+) m/z; (M+H)+408.33

Synthetic Example 1: Synthesis of BIBSoc-Phe-Phe-OH

[0194] ##STR00009##

[0195] BIBSoc-Phe-OH (0.151 g, 0.368 mmol) and N-methylmorpholine (0.043 g, 0.42 mmol) were mixed with tetrahydrofuran (3.0 mL), and isobutyl chloroformate (0.055 g, 0.40 mmol) was added thereto at 0° C. and the mixture was stirred for 15 minutes. To the solution was added a solution separately prepared by mixing H-Phe-OH (0.074 g, 0.44 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 g, 0.88 mmol) and tetrahydrofuran (0.75 mL) and stirring at 55° C. for 1 hour, and the mixture was further stirred while maintaining to 0° C. for 2 hours. The obtained reaction mixture was diluted with t-butyl methyl ether (3.0 mL), and successively washed with a 10 wt % aqueous potassium carbonate solution (1.5 mL), a 20 wt % aqueous ammonium chloride solution (1.5 mL) and a saturated aqueous sodium chloride solution (1.5 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain BIBSoc-Phe-Phe-OH (0.174 g, Yield: 85%) as a white solid.

[0196] .sup.1H-NMR (CDCl.sub.3)

[0197] δ ppm: 0.76-1.04 (27H, m), 2.85-3.30 (4H, m), 4.36 (1H, br), 4.60 (1H, br), 6.91-7.20 (10H, br)

[0198] MASS (ESI+) m/z; (M+H)+555.38

Synthetic Example 2: Synthesis of BIBSoc-Phe-Phe-OH

[0199] ##STR00010##

[0200] (i) BIBSoc-Phe-OH (0.299 g, 0.736 mmol) and N-methylmorpholine (0.156 g, 1.55 mmol) were mixed with ethyl acetate (6.0 mL), and isobutyl chloroformate (0.106 g, 0.77 mmol) was added thereto at −10° C. To the solution was added H-Phe-OMe hydrochloride (0.199 g, 0.92 mmol), and then, the mixture was stirred at the same temperature for 30 minutes. The obtained reaction mixture was successively washed with a 5 wt % an aqueous sodium hydrogen carbonate solution (6.0 mL, twice) and water (6.0 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain BIBSoc-Phe-Phe-OMe (0.420 g, Yield: 101%) as a white solid.

[0201] .sup.1H-NMR (CDCl.sub.3)

[0202] δ ppm: 0.81 (2H, d, J=6.3 Hz), 0.89-0.95 (6H, m), 1.02 (18H, d, J=1.8 Hz), 1.98 (1H, Sep, J=6.6 Hz), 2.97-3.09 (4H, m), 3.65 (3H, s), 4.31-4.39 (1H, m), 4.71-4.78 (1H, m), 5.13 (1H, d, J=8.1 Hz), 6.13 (1H, d, J=7.8 Hz), 6.94-7.29 (10H, m)

[0203] MASS (ESI+) m/z; (M+H)+569.38

[0204] (ii) BIBSoc-Phe-Phe-OMe (0.10 g, 0.18 mmol) was mixed with methanol (2.0 mL), and 5 wt % aqueous lithium hydroxide solution (0.10 g, 0.21 mmol) was added thereto and the mixture was stirred for 23 hours. To the obtained reaction mixture was added 4 wt % hydrochloric acid (1.0 mL) and the mixture was quenched, ethyl acetate (2.0 mL) and water (2.0 mL) were added thereto and the liquids were separated. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain BIBSoc-Phe-Phe-OH (0.082 g, Yield: 85%) as a white solid.

Synthetic Example 3: Synthesis of BIBSoc-Phe-Phe-OH

[0205] ##STR00011##

[0206] (i) BIBSoc-Phe-OH (0.159 g, 0.393 mmol) and N-methylmorpholine (0.083 g, 0.82 mmol) were mixed with ethyl acetate (3.2 mL), and isobutyl chloroformate (0.056 g, 0.41 mmol) was added thereto at −10° C. To the solution was added H-Phe-O(t-Bu) hydrochloride (0.109 g, 0.42 mmol), and then, the mixture was stirred at the same temperature for 45 minutes. The obtained reaction mixture was successively washed with a 5 wt % aqueous sodium hydrogen carbonate solution (3.2 mL, twice) and water (3.2 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain BIBSoc-Phe-Phe-O(t-Bu) (0.239 g, Yield: 100%) as a white solid.

[0207] .sup.1H-NMR (CDCl.sub.3)

[0208] δ ppm: 0.82 (2H, d, J=5.7 Hz), 0.90-0.96 (6H, m), 1.02 (18H, d, J=1.8 Hz), 1.35 (9H, s), 1.99 (1H, Sep, J=6.6 Hz), 3.00-3.05 (4H, m), 4.31-4.38 (1H, m), 4.56-4.63 (1H, m), 5.17 (1H, d, J=8.1 Hz), 6.19 (1H, d, J=7.5 Hz), 7.03-7.28 (10H, m)

[0209] MASS (ESI+) m/z; (M+H)+611.45

[0210] (ii) BIBSoc-Phe-Phe-O(t-Bu) (0.092 g, 0.15 mmol) was mixed with methylene chloride (1.9 mL), and trifluoroacetic acid (0.69 g, 6.1 mmol) was added thereto and the mixture was stirred for 4 hours and 30 minutes. To the obtained reaction mixture was added a 10 wt % aqueous sodium carbonate solution (2.0 mL) and the mixture was quenched, and the liquids were separated. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain BIBSoc-Phe-Phe-OH (0.079 g,

[0211] Yield: 94%) as a white solid.

Reference Synthetic Example 6: Synthesis of IPBS-OTf

[0212] ##STR00012##

[0213] Di-i-propyl-t-butylsilane (0.300 g, 1.74 mmol) was mixed with methylene chloride (10.0 g), and after adding trifluoromethanesulfonic acid (0.160 g, 1.07 mmol) dropwise under ice-cooling, the temperature of the mixture was raised to room temperature and the mixture was stirred for 1 hour. The formed di-i-propyl-t-butylsilyl triflate was used in the next reaction as a methylene chloride solution without isolation.

Reference Synthetic Example 7: Synthesis of IPBSoc-Phe-OH

[0214] ##STR00013##

[0215] (i) Boc-Phe-OBn (0.500 g, 1.41 mmol) and lutidine (0.326 g, 3.04 mmol) were mixed with acetonitrile (5.0 mL), and a methylene chloride solution of di-i-propyl-t-butylsilyl trifluoromethanesulfonate (1.74 mmol) was added thereto at 0° C., and then, the mixture was stirred at room temperature for 2 hours. A methylene chloride solution of di-i-propyl-t-butylsilyl trifluoromethanesulfonate (0.42 mmol) was further added at 0° C., and the mixture was stirred at room temperature for 16 hours. The obtained reaction mixture was diluted with t-butyl methyl ether (10 mL), and successively washed with a saturated aqueous sodium hydrogen carbonate solution (10 mL), a saturated aqueous ammonium chloride solution (10 mL) and water (10 mL) in this order. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain IPBSoc-Phe-OBn (0.618 g, Yield: 94%) as a colorless liquid.

[0216] MASS (ESI+) m/z; (M+H)+470.26

[0217] (ii) IPBSoc-Phe-OBn (0.161 g, 0.341 mmol) was mixed with ethyl acetate (3.2 mL), and after adding 10 wt % Pd—C (20.4 mg, 0.019 mmol) thereto, the mixture was stirred under a hydrogen gas atmosphere at room temperature for 1 hour. The reaction mixture was filtered and the obtained filtrate was concentrated to obtain IPBSoc-Phe-OH (0.138 g, Yield: 107%).

[0218] .sup.1H-NMR (CDCl.sub.3)

[0219] δ ppm: 1.02 (9H, s), 1.10-1.12 (12H, m), 1.42 (2H, Sep, 7.4 Hz), 3.06-3.27 (2H, m), 4.60-4.66 (1H, m), 5.13 (2H, d, J=8.1 Hz), 7.17-7.32 (5H, m)

[0220] MASS (ESI+) m/z; (M+H)+380.30

Synthetic Example 4: Synthesis of IPBSoc-Phe-Phe-OH

[0221] ##STR00014##

[0222] IPBSoc-Phe-OH (0.138 g, 0.365 mmol) and N-methylmorpholine (0.040 g, 0.39 mmol) were mixed with tetrahydrofuran (2.6 mL), and isobutyl chloroformate (0.051 g, 0.37 mmol) was added thereto at 0° C. and the mixture was stirred for 15 minutes. To the solution was mixed with a solution separately prepared by mixing H-Phe-OH (0.068 g, 0.41 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 g, 0.82 mmol) and tetrahydrofuran (0.7 mL) and stirring at 55° C. for 1 hour, and the mixture was further stirred while maintaining to 0° C. for 2 hours. The obtained reaction mixture was diluted with t-butyl methyl ether (2.6 mL), and successively washed with a 10 wt % aqueous potassium carbonate solution (1.3 mL), a 20 wt % aqueous ammonium chloride solution (1.3 mL) and a saturated aqueous sodium chloride solution (2.6 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain IPBSoc-Phe-Phe-OH (0.152 g, Yield: 79%) as a white solid.

[0223] .sup.1H-NMR (CDCl.sub.3)

[0224] δ ppm: 0.97-1.02 (21H, m), 1.36 (2H, br), 2.80-3.21 (4H, m), 3.79 (1H, br), 4.38-4.61 (1H, m), 7.15 (10H, br)

[0225] MASS (ESI+) m/z; (M+H)+527.35

Reference Synthetic Example 8: Synthesis of IPCS-OTf

[0226] ##STR00015##

[0227] Di-i-propylcumylsilane (0.48 g, 2.0 mmol) was mixed with methylene chloride (2.5 g), and after adding trifluoromethanesulfonic acid (0.38 g, 2.5 mmol) dropwise under ice-cooling, and the temperature of the mixture was raised to room temperature and the mixture was stirred for 0.5 hour. The formed di-i-propylcumylsilyl triflate was used in the next reaction as a methylene chloride solution without isolation.

Reference Synthetic Example 9: Synthesis of IPCSoc-Phe-OH

[0228] ##STR00016##

[0229] (i) Boc-Phe-OBn (0.37 g, 1.0 mmol) and lutidine (0.36 g, 3.4 mmol) were mixed with acetonitrile (5.0 g), and a methylene chloride solution of di-i-propylcumylsilyl triflate (2.0 mmol) was added thereto at 0° C., and then, the mixture was stirred at room temperature for 3 hours. The obtained reaction mixture was diluted with ethyl acetate, and successively washed with a saturated aqueous sodium hydrogen carbonate solution, a saturated aqueous ammonium chloride solution and saturated brine in this order. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain IPCSoc-Phe-OBn (0.518 g, Yield: 92%) as a colorless oily product.

[0230] MASS (ESI+) m/z; (M+H)+532.29

[0231] (ii) IPCSoc-Phe-OBn (0.20 g, 0.38 mmol) was mixed with ethyl acetate (4.0 g), and after adding 10 wt % Pd—C (0.03 g), the mixture was stirred under a hydrogen gas atmosphere at room temperature for 5 hours. After filtrating the reaction mixture, the obtained filtrate was concentrated to obtain IPCSoc-Phe-OH (0.17 g, Yield: 100%).

[0232] MASS (ESI+) m/z; (M+H)+442.24

Synthetic Example 5: Synthesis of IPCSoc-Phe-Lys(Boc)-OH

[0233] ##STR00017##

[0234] IPCSoc-Phe-OH (0.20 g, 0.46 mmol) and N-methylmorpholine (0.06 g, 0.59 mmol) were mixed with tetrahydrofuran (2.0 g), isobutyl chloroformate (0.08 g, 0.58 mmol) was added thereto at 0° C. and the mixture was stirred for 5 minutes. To the solution was added a solution separately prepared by mixing H-Lys(Boc)-OH (0.15 g, 0.61 mmol), N,O-bis(trimethylsilyl)acetamide (0.25 g, 1.2 mmol) and tetrahydrofuran (1.3 g) and stirring at 55° C. for 0.5 hour, and the mixture was further stirred while maintaining to 0° C. for 2 hours. The obtained reaction mixture was diluted with ethyl acetate, and successively washed with a saturated aqueous sodium hydrogen carbonate solution, a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated to obtain IPCSoc-Phe-Lys(Boc)-OH (0.32 g, Yield: 103%) as a white solid.

[0235] MASS (ESI+) m/z; (M+H)+670.39

Synthetic Example 6: Synthesis of IPCSoc-Phe-Lys(Boc)-Asp(Q(t-Bu))—OH

[0236] ##STR00018##

[0237] IPCSoc-Phe-Lys(Boc)-OH (0.32 g, 0.47 mmol) and N-methylmorpholine (0.06 g, 0.58 mmol) were mixed with tetrahydrofuran (4.0 g), and isobutyl chloroformate (0.08 g, 0.56 mmol) was added thereto at 0° C. and the mixture was stirred for 5 minutes. To the solution was added a solution separately prepared by mixing H-Asp(O(t-Bu))—OH (0.12 g, 0.62 mmol), N,O-bis(trimethylsilyl)acetamide (0.25 g, 1.2 mmol) and tetrahydrofuran (1.9 g) and stirring at 55° C. for 0.5 hour, and the mixture was further stirred while maintaining to 0° C. for 3 hours. The obtained reaction mixture was diluted with ethyl acetate, and successively washed with a saturated aqueous sodium hydrogen carbonate solution, a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated to obtain IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))—OH (0.38 g, Yield: 97%) as a white solid.

[0238] MASS (ESI+) m/z; (M+H)+841.48

Synthetic Example 7: Synthesis of IPCSoc-Phe-Lys(BocVAsp(O(t-Bu))-Phe-Phe-OH

[0239] ##STR00019##

[0240] IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))—OH (0.20 g, 0.24 mmol) and N-methyl-morpholine (0.03 g, 0.29 mmol) were mixed with tetrahydrofuran (2.0 g), isobutyl chloroformate (0.04 g, 0.27 mmol) was added thereto at 0° C. and the mixture was stirred for 5 minutes. With the solution was mixed a solution separately prepared by mixing H-Phe-Phe-OH (0.16 g, 0.51 mmol), N,O-bis(trimethylsilyl)acetamide (0.25 g, 1.2 mmol) and tetrahydrofuran (1.2 g), stirring at 55° C. for 20 minutes, and further adding N,O-bis(trimethylsilyl)acetamide (0.08 g, 0.41 mmol), and the mixture was further stirred while maintaining to 0° C. for 1 hour. The obtained reaction mixture was diluted with ethyl acetate, and successively washed with a saturated aqueous sodium hydrogen carbonate solution twice, a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated to obtain IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-OH (0.29 g,

[0241] Yield: 108%) as a white solid.

[0242] MASS (ESI+) m/z; (M+H)+1135.61

Synthetic Example 8: Synthesis of IPCSoc-Phe-Lys(BocVAsp(O(t-Bu))-Phe-Phe-Phe-OH

[0243] ##STR00020##

[0244] (i) IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-OH (0.25 g, 0.22 mmol) and H-Phe-OBn hydrochloride (0.08 g, 0.27 mmol) were mixed with methylene chloride (2.6 g), and after cooling to 0° C., N,N-diisopropylethylamine (0.07 g, 0.57 mmol) and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (0.12 g, 0.27 mmol) were added thereto and the mixture was stirred for 3 hours. The obtained reaction mixture was diluted with ethyl acetate, and then, successively washed with a saturated aqueous ammonium chloride solution, a saturated aqueous sodium hydrogen carbonate solution twice, a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-Phe-OBn (0.22 g, Yield: 73%) as a white solid.

[0245] MASS (ESI+) m/z; (M+H)+1372.73

[0246] (ii) IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-Phe-OBn (0.05 g, 0.04 mmol) was mixed with ethyl acetate (1.0 g), and after adding 10 wt % Pd—C (0.01 g) thereto, the mixture was stirred under a hydrogen gas atmosphere at room temperature for 5 hours. After filtrating the reaction mixture, the obtained filtrate was concentrated to obtain IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-Phe-OH (0.05 g, Yield: 99%).

[0247] MASS (ESI+) m/z; (M+H)+1282.64

Synthetic Example 9: Synthesis of H-Phe-Lys(Boc)-Asp(Q(t-Bu))-Phe-Phe-Phe-OH

[0248] ##STR00021##

[0249] IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-Phe-OH (19.7 mg, 0.02 mmol) was mixed with methanol (1.0 g), and potassium fluoride (3.4 mg, 0.06 mmol) was added thereto at room temperature and the mixture was stirred for 4 hours. The obtained reaction mixture was washed with hexane (2.0 g) three times, and a 5 wt % aqueous ammonium chloride solution was added to the obtained methanol layer to precipitate the solid and it was filtered. The obtained solid was dried to obtain H-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-Phe-OH (12.6 mg, Yield: 74%) as a white solid.

[0250] MASS (ESI+) m/z; (M+H)+1006.52

Synthetic Example 10: Synthesis of H-Phe-Lys(Boc)-Asp(Q(t-Bu))-Phe-Phe-Phe-OBn

[0251] ##STR00022##

[0252] IPCSoc-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-Phe-OBn (0.15 g, 0.11 mmol) was mixed with methanol (3.0 g) and N-methylpyrrolidone (2.0 g), and potassium fluoride (0.02 g, 0.26 mmol) was added thereto at room temperature and the mixture was stirred for 3 hours. Water (2.0 g) was added to the obtained reaction mixture, and the mixture was washed with hexane (5.0 g) three times. Ethyl acetate and saturated brine were added to the obtained aqueous layer and the liquids were separated. The obtained organic layer was concentrated, and then, water (5.0 g) was added thereto to precipitate a solid and the solid was filtered. The obtained solid was dried to obtain H-Phe-Lys(Boc)-Asp(O(t-Bu))-Phe-Phe-Phe-OBn (0.12 g, Yield: 96%) as a white solid.

[0253] MASS (ESI+) m/z; (M+H)+1096.57

Reference Synthetic Example 10: Synthesis of CHBS-OTf

[0254] ##STR00023##

[0255] (i) Bromobenzene (5.7 g, 36 mmol) and tetrahydrofuran (8.0 g) were mixed, and after adding the mixture to 1.55M n-butyl lithium hexane solution (35 mL, 54 mmol) at 0° C., the mixture was stirred at 25° C. for 6 hours. To the obtained mixture was added a mixed solution of t-butyldihydrosilane (2.0 g, 13.9 mmol) and tetrahydrofuran (4.0 g), and the mixture was stirred at room temperature for 15 hours. The obtained reaction mixture was successively washed with 2M hydrochloric acid (14 g) and a 5 wt % aqueous sodium chloride solution (14 g). The obtained organic layer was concentrated, and hexane and silica gel were added thereto and the mixture was filtered. The obtained organic layer was concentrated to obtain a hexane solution (6.7 g) of PhBS—H.

[0256] (ii) The hexane solution (6.7 g) of PhBS—H and hexane (21 g) were mixed, and after adding Ru—Al (0.61 g) thereto, the mixture was stirred under a hydrogen gas atmosphere at 30° C. for 19 hours. The reaction mixture was filtered and the obtained filtrate was concentrated to obtain a hexane solution (5.9 g) of CHBS—H.

[0257] (iii) The hexane solution (0.4 g) of CHBS—H was mixed with methylene chloride (1.0 g), and after adding trifluoromethanesulfonic acid (0.14 g, 0.93 mmol) dropwise thereto under ice-cooling, the temperature of the mixture was raised to room temperature and the mixture was stirred for 0.5 hour. The formed CHBS-OTf was used in the next reaction as a hexane-methylene chloride solution without isolation.

Reference Synthetic Example 11: Synthesis of CHBSoc-Phe-OH

[0258] ##STR00024##

[0259] (i) Boc-Phe-OBn (0.20 g, 0.55 mmol) and lutidine (0.12 g, 1.1 mmol) were mixed with acetonitrile (2.0 g), and after adding the hexane-methylene chloride solution of CHBS-OTf at 0° C., the mixture was stirred at room temperature for 19 hours. After adding water (1.0 g) to the obtained reaction mixture, the precipitated solid was collected by filtration to obtain CHBSoc-Phe-OBn (0.21 g, Yield: 74%) as a white solid.

[0260] MASS (ESI+) m/z; (M+H)+524.30

[0261] (ii) CHBSoc-Phe-OBn (0.20 g, 0.38 mmol) was mixed with ethyl acetate (4.0), and after adding 10 wt % Pd—C (24.1 mg) thereto, the mixture was stirred under a hydrogen gas atmosphere at room temperature for 2 hours. The reaction mixture was filtered and the obtained filtrate was concentrated to obtain CHBSoc-Phe-OH (0.17 g, Yield: 100%).

[0262] MASS (ESI+) m/z; (M+H)+434.27

Synthetic Example 11: Synthesis of CHBSoc-Phe-Ala-OH

[0263] ##STR00025##

[0264] (i) CHBSoc-Phe-OH (0.50 g, 1.2 mmol) and H-Ala-O(t-Bu) hydrochloride (0.25 g, 1.4 mmol) were mixed with methylene chloride (5.2 g), and after cooling to 0° C., N,N-diisopropylethylamine (0.38 g, 2.9 mmol) and (l-cyano-2-ethoxy-2-oxoethylidene-aminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (0.60 g, 1.4 mmol) were added thereto and the mixture was stirred for 1 hour. The obtained reaction mixture was diluted with ethyl acetate, and then, successively washed with a saturated aqueous ammonium chloride solution, a saturated aqueous sodium hydrogen carbonate solution three times, a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated to obtain CHBSoc-Phe-Ala-O(t-Bu) (0.68 g, Yield: 106%) as a pale yellowish solid.

[0265] MASS (ESI+) m/z; (M+H)+561.37

[0266] (ii) CHBSoc-Phe-Ala-O(t-Bu) (0.60 g, 1.1 mmol) was mixed with methylene chloride (12 g), and after cooling to 0° C., trifluoroacetic acid (4.9 g, 43.1 mmol) was added thereto, the temperature of the mixture was raised to room temperature and the mixture was stirred for 1 hour. The obtained reaction mixture was diluted with ethyl acetate, and then, successively washed with a saturated aqueous sodium hydrogen carbonate solution twice, a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated to obtain CHBSoc-Phe-Ala-OH (0.52 g, Yield: 97%) as a white solid.

[0267] MASS (ESI+) m/z; (M+H)+505.31

Synthetic Example 12: Synthesis of CHBSoc-Phe-Ala-Ser(t-Bu)-OH

[0268] ##STR00026##

[0269] (i) CHBSoc-Phe-Ala-OH (0.52 g, 1.0 mmol) and H-Ser(t-Bu)-OMe hydrochloride (0.26 g, 1.2 mmol) were mixed with methylene chloride (5.0 g), and after cooling to 0° C., N,N-diisopropylethylamine (0.32 g, 2.5 mmol) and (1-cyano-2-ethoxy-2-oxoethylidene-aminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (0.51 g, 1.2 mmol) were added thereto and the mixture was stirred for 2 hours. The obtained reaction mixture was diluted with ethyl acetate, and then, successively washed with a saturated aqueous ammonium chloride solution, a saturated aqueous sodium hydrogen carbonate solution three times and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated to obtain CHBSoc-Phe-Ala-Ser(t-Bu)-OMe (0.73 g, Yield: 106%) as a pale yellowish solid.

[0270] MASS (ESI+) m/z; (M+H)+662.42

[0271] (ii) CHBSoc-Phe-Ala-Ser(t-Bu)-OMe (0.40 g, 0.60 mmol) was mixed with methanol (8.0 g), and after cooling to 0° C., a 5 wt % aqueous lithium hydroxide solution (1.2 g, 2.4 mmol) was added thereto, the temperature of the mixture was raised to room temperature and the mixture was stirred for 3 hours. The obtained reaction mixture was diluted with ethyl acetate, and then, successively washed with a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated to obtain CHBSoc-Phe-Ala-Ser(t-Bu)-OH (0.36 g, Yield: 91%) as a brown solid.

[0272] MASS (ESI+) m/z; (M+H)+648.40

Synthetic Example 13: Synthesis of H-Phe-Ala-Ser(t-Bu)-Phe-OBn

[0273] ##STR00027##

[0274] (i) CHBSoc-Phe-Ala-Ser(t-Bu)-OH (0.35 g, 0.54 mmol) and H-Phe-OBn hydrochloride (0.21 g, 0.72 mmol) were mixed with methylene chloride (4.0 g), and after cooling to 0° C., N,N-diisopropylethylamine (0.20 g, 1.5 mmol) and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (0.31 g, 0.72 mmol) were added thereto and the mixture was stirred for 2 hours. The obtained reaction mixture was diluted with ethyl acetate, and then, successively washed with a saturated aqueous ammonium chloride solution, a saturated aqueous sodium hydrogen carbonate solution twice and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain CHBSoc-Phe-Ala-Ser(t-Bu)-Phe-OBn (0.35 g, Yield: 73%) as a white solid.

[0275] MASS (ESI+) m/z; (M+H)+885.51

[0276] (ii) CHBSoc-Phe-Ala-Ser(t-Bu)-Phe-OBn (0.10 g, 0.11 mmol) was mixed with methanol (2.0 g), and ammonium fluoride (0.05 g, 1.2 mmol) was added thereto under room temperature and the mixture was stirred for 4 hours. The obtained reaction mixture was washed with heptane (2.0 g) three times, and ethyl acetate and saturated brine were added to the obtained methanol layer and the liquids were separated. The obtained organic layer was concentrated to obtain H-Phe-Ala-Ser(t-Bu)-Phe-OBn (0.07 g, Yield: 98%) as a white solid.

[0277] MASS (ESI+) m/z; (M+H)+617.33

Synthetic Example 14: Synthesis of H-Phe-Ala-Ser(t-Bu)-Phe-OBn

[0278] CHBSoc-Phe-Ala-Ser(t-Bu)-Phe-OBn (0.15 g, 0.17 mmol) obtained in Step (i) of Synthetic Example 13 was mixed with tetrahydrofuran (3.1 g), and a 1M tetrabutylammonium fluoride-tetrahydrofuran solution (0.2 mL, 0.20 mmol) was added thereto under room temperature and the mixture was stirred for 2 hours. The obtained reaction mixture was diluted with ethyl acetate, and then, successively washed with a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. The obtained organic layer was concentrated, and after mixing with acetonitrile (3.0 mL), the mixture was washed with heptane (3.0 mL) twice. The obtained organic layer was concentrated to obtain H-Phe-Ala-Ser(t-Bu)-Phe-OBn (0.10 g, Yield: 95%) as a white solid.

[0279] MASS (ESI+) m/z; (M+H)+617.33

Reference Synthetic Example 12: Synthesis of H-Phe-Phe-OMe

[0280] ##STR00028##

[0281] BIBSoc-Phe-Phe-OMe (0.10 g, 0.17 mmol) obtained in Step (i) of Synthetic Example 2 was mixed with methanol (2.0 g), potassium fluoride (0.02 g, 0.35 mmol) was added thereto under room temperature and the mixture was stirred for 5 hours. The obtained reaction mixture was washed with hexane (3.0 g) three times, and ethyl acetate and saturated brine were added to the obtained methanol layer and the liquids were separated. The obtained organic layer was concentrated to obtain H-Phe-Phe-OMe (0.07 g, Yield: 86%) as a white solid.

[0282] MASS (ESI+) m/z; (M+H)+327.16

Test Example 1: Comparison of Yields of Peptide Elongation Step and Deprotection Step of C-Terminal by N-Protected Amino Acid in which Silylcarbamate-Based Protective Group Having Specific Structure Introduced at N-Terminal

[0283] ##STR00029##

[0284] [Test Compound]

[0285] N-protected amino acids (hereinafter also referred to as starting material) in which a silylcarbamate-based protective group having a specific structure was introduced into the N-terminal described in the following Tables 1 and 2 were used. Incidentally, the starting materials can be obtained by the method described in Journal of Organic Chemistry, 1999, vol. 64, pp. 3792-3793, Journal of the American Chemical Society, 2005, vol. 127, pp. 13720-13725 and Reference Synthetic Examples 1, 5, 6 and 7, and a method equivalent thereto.

TABLE-US-00001 TABLE 1 Starting material (1) BBSoc-Phe-OH IPCSoc-Phe-OH Structural formula [00030] [00031] MASS (M + H) + 442.24 (M + H) + 442.24 (ESI+) m/z;

TABLE-US-00002 TABLE 2 Starting material (1) CHBSoc-Phe-OH ODBSoc-Phe-OH Structural formula [00032] [00033] MASS (M + H) + 434.27 (M + H) + 604.46 (ESI+) m/z;

[0286] [Test Method]

[0287] Each starting material (170 mg) and N-methylmorpholine (1.2 equivalent) were mixed with tetrahydrofuran (10-fold mass), and after cooling to 0° C., isobutyl chloroformate (1.1 equivalent) was added thereto and the mixture was stirred for 15 to 30 minutes. To the solution was added a solution separately prepared by mixing H-Phe-OH (1.2 equivalent), N,O-bis(tiimethylsilyl)acetamide (2.4 equivalent) and tetrahydrofuran (6-fold mass) and stirring at 50 to 55° C. for 0.5 to 1 hour, and the mixture was further stirred while maintaining to 0° C. for 1 to 3 hours. The obtained reaction mixture was diluted with t-butyl methyl ether (20-fold mass), and successively washed with a 10 wt % aqueous potassium carbonate solution (10-fold mass), a 20 wt % aqueous ammonium chloride solution (10-fold mass) and a saturated aqueous sodium chloride solution (10-fold mass). The obtained organic layer was concentrated and purified by silica gel column chromatography, and the yield of the objective material was calculated.

[0288] [Test Results]

[0289] Yields of the peptide elongation step and the deprotection step of the C-terminal using the respective starting materials were markedly improved as compared with the yield of Reference Synthetic Example 2.

TABLE-US-00003 TABLE 3 Objective material (3) BBSoc-Phe-Phe-OH IPCSoc-Phe-Phe-OH Yield 94% 93% State White solid White solid MASS (M + H) + 589.31 (M + H) + 589.3 (ESI+) m/z;

TABLE-US-00004 TABLE 4 Objective material (3) CHBSoc-Phe-Phe-OH ODBSoc-Phe-Phe-OH Yield 95% 92% State White solid Colorless oily product MASS (M + H) + 581.31 (M + H) + 751.54 (ESI+) m/z;

UTILIZABILITY IN INDUSTRY

[0290] According to the present invention, it can provide a method of producing a peptide with high efficiency.