METHODS FOR THE SYNTHESIS OF ARGININE-CONTAINING PEPTIDES

Abstract

Methods for the synthesis of arginine-containing peptides are provided. The methods include a deprotection step that minimizes the transfer of by-products deriving from cleaved sulfonyl-5 based side chain protecting groups from arginine to amino acids carrying electron rich side chains.

Claims

1. A method for deprotecting a peptide comprising a Mtr-, Pmc- or Pbf-side chain protected arginine and at least an amino acid carrying an electron rich aromatic side chain, said method comprising the steps of: a.) solubilizing the peptide in a solvent mixture of at least one aprotic organic solvent, a thiol scavenger, and either a protic solvent or a carboxylic acid selected from the group of formic acid, acetic acid and/or trifluoroacetic acid or a mixture thereof, wherein the amount of the carboxylic acid is selected in the range of 5 to 30%, based on the total volume of the solvent mixture, followed by b.) mixing the solution obtained in step a.) with a mixture of (i) a carboxylic acid selected from the group of formic acid, acetic acid and/or trifluoroacetic acid and (ii) a sulfonic acid selected from the group of methanesulfonic acid, trifluoromethane sulfonic acid, benzene sulfonic acid and/or p-toluene sulfonic acid, with the proviso that the total amount (by moles) of the carboxylic acid used in step a.) and b.) is higher than the total amount of the sulfonic acid.

2. The method according to claim 1, wherein the at least one electron rich aromatic side chain is selected from the group arylC.sub.1-C.sub.6 alkyl groups and/or heteroarylC.sub.1-C.sub.6 alkyl groups.

3. The method according to claim 2, wherein the arylC.sub.1-C.sub.6 alkyl group are selected from arylC.sub.1-C.sub.2 alkyl groups which are unsubstituted or substituted with one or two substituents selected from the group of F, hydroxy or nitro, preferably from phenyl(m)ethyl, 4-hydroxyphenyl(m)ethyl, 3-nitro-4-hydroxyphenyl(m)ethyl and/or naphthyl(m)ethyl, more preferably from phenylmethyl, 4-hydroxyphenyl(m)ethyl, 3-nitro-4-hydroxyphenylmethyl and/or naphthylmethyl.

4. The method according to claim 2, wherein the heteroarylC.sub.1-C.sub.6 alkyl groups are selected from unsubstituted heteroaryl(m)ethylene group, preferably from (1H-indol-3-yl)(m)ethylene, and/or (1H-imidazol-4-yl)(m)ethylene.

5. The method according to claim 1, wherein the at least one aprotic organic solvent in step a.) is selected from the group of aromatic hydrocarbons, which are liquid at ambient temperature, C.sub.1-6-halogenoalkanes and acetonitrile as well as mixtures thereof.

6. The method according to claim 1, wherein the amount of aprotic solvent(s) in step a) is selected in the range from 1 to 100 mL/g peptide, preferably from 2 to 10 mL/g peptide, even more preferably from 2.5 to 7 mL/g peptide.

7. The method according to claim 1, wherein the protic solvent in step a) is selected from the group of water and an alcohol as well as mixtures thereof, preferably of water and a C.sub.1-4-alkyl alcohols as well mixtures thereof.

8. The method according to claim 1, wherein the thiol scavenger is an aliphatic thiol, more preferably dodecanethiol.

9. The method according to claim 1, wherein the temperature in step a.) is selected in the range of 15 to 25° C.

10. The method according to claim 1, wherein the amount of the sulfonic acid in step b.) is selected in the range from 1 to 20 mol-equivalents, preferably from 2 to 12 mol-equivalents, more preferably from 3 to 10 mol-equivalents, most preferably from 5 to 8 mol equivalents based on each protected arginine moiety present in the peptide.

11. The method according to claim 1, wherein the molar ratio of the total amount acid of the carboxylic acid used in step a.) and b.) and the amount of the sulfonic acid such as preferably methane sulfonic acid used in step b.) is selected in the range of 100:1 to 10: 1, preferably in the range from 50:1 to 12:1, more preferably in the range from 30:1 to 15:1, most preferably in the range from 25:1 to 18:1.

12. The method according to claim 1, wherein the peptide is a di- to hexapeptide.

13. The method according to claim 1, wherein the peptide is selected from the group consisting of Boc-Trp-Arg(Pbf)-OH, Ac-Arg(Pbf)-His(Trt)-Phe-OH, TFA*H-Tyr(tBu)-Arg(Pbf)-Pro-OH, Boc-rac-6FTrp-Gly-Arg(Pbf)-Glu(OtBu)-OH, Boc-Tyr(Et)-Arg(Pbf)-Ala-Phe-OH, Ac-5-(OH)Trp-Ala-Arg(Pbf)-Ser(tBu)-Leu-Phe-OH, Boc-Arg(Mtr)-Tyr(tBu)-Phe-OH, Boc-2Nal-Leu-Arg(Pbf)-Phe-OH, Ac-Arg(Pbf)-Met-m(NO.sub.2)Tyr-Pro-OH and Bz-Gly-His(Trt)-D-Phe-Arg(Pbf)-D-Trp-N(Pr).sub.2.

14. The method according to claim 1, wherein the Mtr-, Pmc- or Pbf-side chain protected arginine and the amino acid carrying an electron rich side chain are separated by zero to three amino acids.

15. The method according to claim 1, wherein the peptide is a peptide comprising at least one Pbf-protected arginine and an amino acid selected from the group of histidine, tyrosine, tryptophan, 5-hydroxytryptophan and 6-fluoro tryptophan as well as mixtures thereof.

Description

EXAMPLE 1. DEPROTECTION OF BOC-TRP-ARG(PBF)-OH TO H-TRP-ARG-OH

[0101] Starting material: 142 mg of the peptide (96% UPLC purity, 0.185 mmol) per test. [0102] Cleavage time: 90 min. [0103] Reference: 31.7%. [0104] Invention: 68.0% (additional 0.08 mL PhMe and 50 μL TFA, for a total of 13.7 eq TFA, were required for full dissolution). [0105] UPLC-MS: calcd m/z for [M+H].sup.+ 361.20, found m/z 361.2

EXAMPLE 2. DEPROTECTION OF AC-ARG(PBF)-HIS(TRT)-PHE-OH TO AC-ARG-HIS-PHE-OH

[0106] Starting material: 369 mg peptide (60% UPLC purity, 0.222 mmol) per test, 180 min [0107] Cleavage time: 180 min [0108] Reference: 65.3%. [0109] Invention: 70.2% (additional 17 μL TFA, for a total of 8.2 eq TFA, were required for full dissolution). [0110] UPLC-MS: calcd m/z for [M+H].sup.+ 501.26, found m/z 501.2.

EXAMPLE 3. DEPROTECTION OF BOC-ARG(MTR)-TYR(TBU)-PHE-OH TO H-ARG-TYR-PHE-OH

[0111] Starting material: 234 mg (98% UPLC purity, 0.260 mmol) per test. [0112] Cleavage time: 90 min. [0113] Invention: 84.8%. [0114] Invention: 94.5% (additional 0.12 mL PhMe and 20 μL TFA, for a total of 8.2 eq TFA, were required for full dissolution). [0115] UPLC-MS: calcd m/z for [M+H].sup.+ 485.25, found m/z 485.1.

EXAMPLE 4. DEPROTECTION OF BOC-2NAL-LEU-ARG(PBF)-PHE-OH TO H-2NAL-LEU-ARG-PHE-OH

[0116] Starting material: 364 mg (87% UPLC purity, 0.322 mmol) per test. [0117] Cleavage time: 110 min. [0118] Reference: 89.7%. [0119] Invention: 90.5% (additional 24 μL TFA, for a total of 8.2 eq TFA, were required for full dissolution). [0120] UPLC-MS: calcd m/z for [M+H].sup.+ 632.26, found m/z 632.3.

EXAMPLE 5. DEPROTECTION OF AC-ARG(PBF)-MET-M(NO.SUB.2.)TYR-PRO-OH TO AC-ARG-MET-M(NO.SUB.2.)-TYR-PRO-OH

[0121] Starting material: 85 mg (96% UPLC purity, 0.089 mmol) per test. [0122] Cleavage time: 90 min. [0123] Reference: 93.6%. [0124] Invention: 95.3% (additional 0.02 mL PhMe and 35 μL TFA, for a total of 12.4 eq TFA, were required for full dissolution). [0125] UPLC-MS: calcd m/z for [M+H].sup.+ 653.27, found m/z 653.2.

EXAMPLE 6. DEPROTECTION OF BOC-RAC-6FTRP-GLY-ARG(PBF)-GLU(OTBU)-OH TO H-RAC-6FTRP-GLY-ARG-GLU-OH

[0126] Starting material: 126 mg HPLC-purified peptide (97% UPLC purity, 0.122 mmol) per test. [0127] Cleavage time: 90 min. [0128] Reference: 59.0%. [0129] Invention: 86.8% (additional 0.08 mL PhMe and 35 μL TFA, for a total of 12.4 eq TFA, were required for full dissolution). [0130] UPLC-MS: calcd m/z for [M+H].sup.+ 565.26, found m/z 565.2.

EXAMPLE 7. DEPROTECTION OF BOC-TYR(ET)-ARG(PBF)-ALA-PHE-OH TO H-TYR(ET)-ARG-ALA-PHE-OH

[0131] Starting material: 376 mg crude peptide (92% UPLC purity, 0.361 mmol) per test. [0132] Cleavage time: 90 min. [0133] Reference: 86.6%. [0134] Invention: 87.0%. (additional 0.10 mL PhMe and 135 μL TFA, for a total of 12 eq TFA, were required for full dissolution).

EXAMPLE 8. FINAL DEPROTECTION OF AC-5(OH)TRP-ALA-ARG(PBF)-SER(TBU)-LEU-PHE-OH TO AC-5(OH)TRP-ALA-ARG-SER-LEU-PHE-OH

[0135] Starting material: 262 mg (96% UPLC purity, 0.217 mmol) per test. [0136] Cleavage time: 85 min. [0137] Reference: 39.3%. [0138] Invention: 62.9% (additional 0.14 mL PhMe and 272 μL TFA, for a total of 24.1 eq TFA, were required for full dissolution). [0139] UPLC-MS: calcd m/z for [M+H].sup.+ 837.43, found m/z 837.5.

[0140] Comparative example C1. Deprotection of Boc-Tyr(Et)-Arg(Pbf)-Ala-Phe-OH to H-Tyr-Arg-Ala-Phe-OH employing a MSA in step a.) to dissolve the peptide [0141] Starting material: 351 mg (93% UPLC purity, 0.309 mmol) per test. [0142] Cleavage time: 110 min. [0143] Reference: 75.8%. [0144] Two-step method: 75.0% UPLC-MS: calcd m/z for [M+H].sup.+ 584.32, found m/z 584.3

[0145] As can be retrieved from this comparative example, no improvement of the yield is obtained.

EXAMPLE 9. (TWO-STEP) DEPROTECTION OF BZ-GLY-HIS(TRT)-D-PHE-ARG(PBF)-D-TRP-N(PR).SUB.2 .TO BZ-GLY-HIS-D-PHE-ARG-D-TRP-N(PR).SUB.2 .WITH VARYING PARAMETERS

Example 9A1. Improved Two-Step Deprotection

[0146] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL PhMe, 0.09 mL TFA (7.1 eq) and 0.163 mL DDCT (4 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL PhMe and rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 90.4%.

Example 9A2. Improved Two-Step Deprotection with Reversed Addition

[0147] The protected peptide (0.168 mmol, 93% purity) was given in a round-bottomed flask equipped with stirrer and dissolved in a mixture of 0.70 mL PhMe, 0.09 mL TFA (7.1 eq) and 0.163 mL DDCT (4 eq); to the peptide solution a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq) were added dropwise and stirred for 140 min. Results: 90.8%.

Example 9A3. Improved Two-Step Deprotection Using 95% TFA(Aq)

[0148] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL PhMe, 0.09 mL 95% TFA(aq) (6.8 eq) and 0.163 mL DDCT (4 eq); the peptide solution was added dropwise to a mixture of 1.58 mL 95% TFA(aq) (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL PhMe and rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 89.6%.

Example 9A4. Improved Two-Step Deprotection with Dissolution in 30% TFA(Aq)

[0149] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL PhMe, 0.25 mL 30% TFA(aq) (20 eq) and 0.163 mL DDCT (4 eq); the peptide solution was added dropwise to a mixture of 30% 1.35 mL TFA(aq) (107 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL PhMe and rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 90.7%.

Example 9A5. Improved Two-Step Deprotection Replacing Toluene with Dichloromethane

[0150] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL DCM, 0.09 mL TFA (7.1 eq) and 0.163 mL DDCT (4 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL DCM and rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 91.3%.

Example 9A6. Improved Two-Step Deprotection with Dissolution in Dichloromethane/Water

[0151] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 1.30 mL DCM, 24 μL H.sub.2O (7.9 eq) and 0.163 mL DDCT (4 eq); to the peptide solution a mixture of 1.60 mL TFA (127 eq) and 0.078 mL MSA (7.1 eq) was added dropwise and stirred for 140 min. Results: 91.5%.

Example 9A7. Improved Two-Step Deprotection with Dissolution in Dichloromethane Containing Methanol

[0152] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 1.10 mL DCM, 0.10 mL MeCN, 0.05 mL MeOH (7.3 eq) 0.163 mL DDCT (4 eq) and 0.138 mL TIS (4 eq); to the peptide solution a mixture of 1.60 mL TFA (127 eq) and 0.078 mL MSA (7.1 eq) was added dropwise and stirred for 140 min. Results: 90.2%.

Comparative Example C2A1. Two-Step Deprotection Using TIS Instead of DDCT as a Scavenger

[0153] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL PhMe, 0.09 mL TFA (7.1 eq) and 0.140 mL TIS (4 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL PhMe, rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 76.4%.

Comparative Example C2A2. Two-Step Deprotection Using PhSMe Instead of DDCT as a Scavenger

[0154] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL PhMe, 0.09 mL TFA (7.1 eq) and 0.080 mL PhSMe (4 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL PhMe, rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 35.1%.

Comparative Example C2A3. Two-Step Deprotection Using H.SUB.2.O Alone as a Scavenger

[0155] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL PhMe, 0.18 mL TFA (14.2 eq) and 0.060 mL H.sub.2O (20 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL PhMe, rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 85.4%.

Comparative Example C2A4. Two-Step Deprotection with Dissolution in THF Instead of PhMe

[0156] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL THF, 0.09 mL TFA (7.1 eq) and 0.163 mL DDCT (4 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL THF, rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 31.7%.

Comparative Example C2A5. Two-Step Deprotection with Dissolution in DMSO Instead of PhMe

[0157] The protected peptide (0.168 mmol, 93% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL DMSO, 0.09 mL TFA (7.1 eq) and 0.163 mL DDCT (4 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL DMSO, rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 2.9%.

Comparative Example C2A6. Two-Step Deprotection with Dissolution in AcOEt Instead of PhMe

[0158] The protected peptide (0.168 mmol, 93% purity) was given in a round-bottomed flask equipped with stirrer and dissolved in a mixture of 0.70 mL AcOEt, 0.09 mL TFA (7.1 eq) and 0.163 mL DDCT (4 eq); the peptide solution was added dropwise to a mixture of 1.51 mL TFA (120 eq) and 0.078 mL MSA (7.1 eq), the vial with the peptide solution was rinsed with 0.07 mL AcOEt, rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 61.8%.

Comparative Example C2A7. Two-Step Deprotection Using Pure MSA for the Cleavage Solution

[0159] The protected peptide (0.168 mmol, 93% purity) was given in a round-bottomed flask equipped with stirrer and dissolved in a mixture of 0.75 mL PhMe, 0.40 mL 95% TFA(aq) (30 eq) and 0.163 mL DDCT (4 eq); to the peptide solution 0.22 mL MSA (20 eq) were added dropwise, the resulting mixture was stirred for 140 min. Results: 38.8%.

Example 9A8. Improved Two-Step Deprotection with AcOH as Acid in the Peptide Solution

[0160] The protected peptide (0.173 mmol, 96% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL PhMe, 0.072 mL AcOH (7.2 eq) and 0.167 mL DDCT (4 eq). The peptide solution was added dropwise under stirring to a mixture of 1.56 mL TFA (120 eq) and 0.081 mL MSA (7.1 eq); the vial with the peptide solution was rinsed with 0.07 mL PhMe, rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 92.0%.

Example 9A9. Improved Two-Step Deprotection with Using EtOH as Protic Co-Solvent

[0161] The protected peptide (0.173 mmol, 96% purity) was given in a round-bottomed flask equipped with stirrer and dissolved in a mixture of 1.1 mL PhMe, 0.10 mL EtOH and 0.167 mL DDCT (4 eq). To the peptide solution a mixture of 1.65 mL TFA (127 eq) and 0.081 mL MSA (7.1 eq) was added dropwise under stirring and stirred for 140 min. Results: 90.8%.

Comparative Example C2A8. Two-Step Deprotection —No Aprotic Solvent

[0162] The protected peptide (0.173 mmol, 96% purity) was given in a vial equipped with stirrer and dissolved in a mixture of 0.63 mL AcOH (63 eq), 25 μL H.sub.2O (8 eq) and 0.167 mL DDCT (4 eq). The peptide solution was added dropwise under stirring to a mixture of 1.65 mL TFA (127 eq) and 0.081 mL MSA (7.1 eq); the vial with the peptide solution was rinsed with 0.07 mL AcOH (7 eq), rinsing solutions were added to the reaction flask and stirred for 140 min. Results: 65.6%.