NL8902361A - FIXED PHASE PEPTIDE SYNTHESIS. - Google Patents

Description

Solid phase peptide synthesis

The invention relates to solid phase peptide synthesis.

The abbreviations used in this description are in accordance with the 1983 Recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, as stated in Eur. J. Biochem, 138f 9-37 (1984).

In addition, the following abbreviations are used: TFA trifluoroacetic acid DCM dichloromethane DMF dimethylformamide NMP N-methylpyrrolidine DMAc dimethylacetamide

Amino acids and their residues have the L configuration unless otherwise indicated, so for example Ala = L-alanine, DAla = D-alanine. The term (meth) acrylic as used herein denotes either acrylic or methacrylic.

The usual solid phase peptide synthesis methods involve the following series of operations: 1. removing Boc protecting group; 2. washings; 3. neutralization of NH2 at a position; 4. washings; 5. couplings and 6. washings.

Protective groups are removed and neutralized by treatment of the resin peptide with TFA and diisopropylethylamine solutions in DCM. The same solvent is also used for intermediate washings. Whatever coupling agent is used (symmetrical anhydride, dicyclohexylcarbodiimide, hydroxybenzotriazole, etc.), the coupling reaction is carried out in either DCM or DMF.

Consequently, solid phase peptide synthesis involves large amounts of fairly expensive solvents and reagents such as TFA; therefore, the price of a peptide directly depends on the prices of DCM, NMP, DMF, DMAc and TFA used in the synthesis.

It would therefore be of particular importance if cheaper solvents and reagents are found in order to significantly reduce the preparation costs of peptides.

Applicant's European Patent No. 0079842 describes polyacrylic resins which are copolymers of three monomers, namely: (i) a first monomer providing a matrix for the copolymer, 1- (meth) acryloyl-pyrrolidine, 1- (meth) acryloyl-piperidine, 1- (meth) acryloyl-perhydroazepine, 1- (meth) acryloyl-4-methyl-piperazine, 4- (meth) acryloyl-morpholine, N, N-dimethyl- (meth) acrylamide or N, N- diethyl (meth) acrylamide, (ii) a second monomer that cross-links the copolymer, N, N'-di (meth) acryloyl-diaminomethane or Ν, Ν'-di (meth) acryloyl-1,2-diaminoethane, and (iii) a third monomer that activates the copolymer, any of the following acids 2- (meth) acrylamido-acetic acid, 3- (meth) acrylamido-propionic acid, 4- (meth) acrylamido-butyric acid, 6- (meth) acrylamido-hexanoic acid, N- (meth) acryloyl-L-alanine, N- (meth) acryloyl-L-valine, N- (meth) acryloyl-L-leucine, N- (meth) acryloyl-L-phenylalanine, N- (meth) acryloyl-L-tyrosine, N- (meth) acryloyl-L-methionine, N- (meth) acryloyl-L-lysine, o f N- (meth) acryloyl-L-proline or a methyl ester of one of these acids.

These copolymers have free carboxy or methoxycarbonyl groups from the third monomer. Applicants European Patent No. 81408 describes other polyacrylic resins in which these groups have been converted to the amide with ethylene diamide. This patent also describes the use of these other polyacrylic resins in solid phase peptide synthesis, but only in combination with the expensive solvents conventionally used with polystyrene resins, as discussed above.

The invention now provides a method for solid phase peptide synthesis, wherein a first amino acid residue is attached to a polyacrylic resin, one or more further amino acid residues are coupled to obtain the desired peptide and the peptide is detached from the resin, characterized in , the coupling protocol comprising washings of the resin in water and / or aqueous solution (s).

The method of the invention utilizes the hydrophilic properties of polyacrylic resins as opposed to polystyrene resins. The polyacrylic resins used in the process of the invention are preferably the resins described in applicants European Patent Nos. 0079842 and 0081408 mentioned above.

The first amino acid residue can be attached to the matrix via the glycolamide residue (B. Calas et al. Tetrahedron, 1985, 41, 5331). Previous attempts with other labile compounds such as the compounds of formulas 1, 2 and 3 of the enclosed formula sheet, wherein X = Br, Cl or OH, have been found to give unsatisfactory results.

The resin can then be washed in water. The following amino acid of the peptide sequence to be built can be added according to the following protocol (applicable when the protecting group is Boe): 1. wash: distilled water - 2 to 4 times, 2 min each; 2. removing the protecting group: HCl (6N) in water - once, 2 min and again, 30 min; 3. wash: distilled water - 4 to 6 times, 2 min each; 4. Neutralization: 1 equivalent of Borate buffer 12.5 iM pH 8.5-9.0 - once, 1 to 2 min and again, 1 to 2 min; 5. wash: distilled water - 4 to 6 times, 1 to 2 min each; 6. wash: DMF - 2 times, 1 to 2 min each; 7. coupling: symmetrical anhydride (2 equivalents, 2 times in DMF); 8. wash: DMF or NMP - 2 times, 2 min each; and 9. wash: distilled water - 4 times, 2 min each.

The progress of the coupling reaction can be monitored with ninhydrin or fluorescamine.

Also, when the protecting group is Fmoc, the extension protocol can be as follows: 1. wash: distilled water - 4 to 6 times, 2 min each; 2. removing the protecting group: piperidine or diethylamine in water; 3. wash: isopropanol, 2 times, 2 min each; distilled water - 4 to 6 times, 2 min each; 4. optional wash: DMF - once, 2 min; 5. coupling: symmetrical anhydride (3 times in excess in DMF); and 6. washing: DMF (2 times, 2 min each); distilled water - 6 times, 2 min each.

When the synthesis is complete, the peptide is separated from the matrix by selectively breaking the glycolamide bond by one of the following treatments: - NaOH in isopropanol, - NH3 in trifluoroethanol or methanol or ethanol or isopropanol - in DMF, or - CHjOH in triethylamine .

By this method, the reference Dorman peptide (Leu Ala Gly Val) and LHRH analogs were obtained in yields of greater than about 50%.

The invention is now illustrated by the following examples:

Example I

Synthesis of DTrp6-LHRH: pyro-Glu-His-Trp-Ser-Tyr-DTrp-

Leu-Arg-Pro-Gly.

5 g of a polyacrylic resin (0.55 mmol NH2 / g), prepared by copolymerizing 1-acryloyl-pyrrolidine, NjN'-diacryloyl-1-diaminoethane and methyl 2-acrylamido acetate, as described in Example 19 of EP 0 079 842, was treated as follows: 1. washes with DCM (4 times, 2 min each) 2. neutralization with 5% diisopropylethylamine in DCM (2 times, 2 min each) 3. washes with DCM (4 times, 2 each min).

4.29 g (0.0165 mol) of bromoacetic anhydride in 50 ml of DCM were added to the resin. After shaking for 45 min, the DCM solution was removed by filtration. The brominated support was then washed as follows: 1. DCM (4 times, 2 min each) 2. DMF (4 times, 2 min each).

The cesium salt of BocGlyOH (4.22 g, 0.0137 mol), prepared according to Mery et al. (Int. J. Protein Peptide Res. 1988, 31, 412), was dissolved in DMF (75 ml) and this solution was added to the resin. The mixture was shaken at ambient temperature for 2 days. DMF was then decanted and the polymer was washed with: 1. DMF (10 times, 2 min each) 2. Methanol (4 times, 2 min each) 3. DCM (4 times, 2 min each) 4. Diethyl ether (4 times , each 2 min).

The resin was dried under high vacuum in the presence of KOH pellets for 12 hours. The amount of Gly coupled was 0.483 mmol / g, determined by amino acid analysis after hydrolysis in 6 N HCl at 110 ° C for 24 hours in vacuum and sealed tubes.

The BocGly resin (4.47 g) was washed with water (4 times, 2 min each) and the Boc group was cleaved using 6N aqueous HCl (2 times, first 2 min and then another 30 min) . HCl was removed by filtration and the resin was washed with water (6 times, 2 min each). Neutralization was performed with borate buffer (12.5 mmol, pH 9), the resin was treated twice with 50 ml buffer (1 min each). Ka washings with water (6 times, 2 min each) and with DMF (2 times, 2 min each) the symmetrical anhydride of BocProOH in DMF (50 ml) were added to the resin.

The symmetrical anhydride solution was prepared as follows: BocProOH (3.55 g, 0.0165 mol) was dissolved in 40 ml DMF, the solution was cooled to 0 ° C and dicyclohexylcarbodiimide (1.69 g, 8.25 mmol) ) in 10 ml DCM was added. After stirring at 0 ° C for 30 min and filtration, the solution was evaporated under high vacuum and without heating, the residue was dissolved in DMF and added to the resin. The mixture was shaken for 30 min, after which the qualitative ninhydrin test from Kaiser et al. (Anal. Biochem. 1970, 3 ±, 575) was negative, indicating a coupling yield of more than 99.6%. The DMF was then removed and the support was washed twice with DMF (2 min each) and then with water (4 times, 2 min each).

This protocol was used to build in the other amino acids of the DTrp6-LHRH sequence. The symmetrical anhydrides were prepared with: BocArg (Mts) OH (7.52 g, 0.0165 mol), BocLeuOH (4.11 g, 0 0.0165 mol), BocDTrpOH (5.02 g, 0.0165 mol), BocTyr (2,6-dichlorobenzyl) 0H or BocTyr (2.6 DCB) 0H (7.26 g, 0.0165 mol), Boc-Ser (Bzl) OH (4.86 g, 0.0165 mol), BocTrpOH (5.02 g, 0.0165 mol), BocHis (dinitrophenyl) OH or BocHis (Dnp) OH (6.94 g, 0 0.0165 mol), pyro-GluOH (2.13 g, 0.0165 mol).

After incorporation of pyro-GluOH, the resin was washed with methanol (4 times, 2 min each) and dried with diethyl ether (4 times, 2 min each) and dried in high vacuum at ambient temperature for 48 hours.

The peptide resin was then treated with thiophenol (10ml) in DMF (50ml) to remove the dinitrophenyl group on the histidine side chain.

After shaking for 45 min, the thiophenol solution was poured off and the resin was washed with DMF (4 times, 2 min each), DCM (4 times, 2 min each) and diethyl ether (4 times, 2 min each). The resin was dried under high vacuum for 12 hours. The resin was then treated with 50 ml of the following pre-cooled solution 2 times (30 min each) at 0 ° C: trifluoromethanesulfonic acid (3.6 ml), anisole (4 ml), thioanisole (4 ml), metacresole (4 ml ) and trifluoroacetic acid (40 ml). After removing the protecting group, the resin was washed with DCM (2 times, 2 min each), DCM / DMF (50-50) (2 times, 2 min each), 5% diisopropylethylamine in DCM (2 times, 1 min each), DMF (3 times, 2 min each) and isopropanol water (70-30) (3 times, 2 min each). The peptide resin was then suspended in NH3 saturated tri-fluoroethanol solution (250 ml).

The mixture was shaken at ambient temperature for 15 hours, the trifluoroethanol solution with DTrp6-LHRH without protecting group was collected and the support was washed with water (4 times, 2 min each), methanol (4 times, 2 min each) and water (6 times , each 2 min). The filtrates were collected, the pH adjusted to about 4 with 1N hydrochloric acid; they were concentrated under vacuum without heating. The residue was fractionated on a carboxymethyl cellulose (Wathman CM 52, 10 x 2 cm) column with a linear NaCl gradient (10 mM AcONa pH 5.0 to 10 mM AcONa, 0.15 M NaCl pH 5.0). Suitable fractions were collected, lyophilized and desalted by gel filtration on a Sephadex GlO column (100 x 2.5 cm) in 10 M HCl. The peptide fraction was then purified by HPLC on a Lichrosorb R18 (10 Mm) column (270 x 20 mm) with 0.01% aqueous trifluoroacetic acid (TFA) and acetonitrile as running liquids.

Yield: 51% (based on the amount of amino groups of the carrier in the beginning).

Amino acid analysis: Glu 0.99 (1), Leu 1.0 (1), His 1.0 (1), Trp 1.89 (2), Ser 0.96 (1), Ty 0.97 (1), Pro 0.99 (1), Gly 1.07 (1).

For some amino acids that are less stable under acidic conditions, the analysis values may be lower than expected due to degradation of these amino acids.

The same procedure was used for the following peptides: - Dorman peptide: Leu Ala Gly ValOH Yield® 46.6%

Gly = 0.96, Ala® 0.94, Val® 1.05, Leu = 1.04 -Laminin: Tyr Ile Gly Ser ArgNH2 Yield® 35.6%

Ser = 0.75, Gly® 1.03, Ile = 0.99, Tyr = 1, Arg = 0.99.

CDC 28 kinase protein from the cell cycle of Pombee yeast

Yield: 44.1% (crude peptide)

Tyr Lys Ala Leu Asp Leu Arg Pro GlyOH

Asp = 1, Gly = 1.08, Ala = 1, Leu = 1.05 x 2, Tyr = 0.99, Arg = 0.99, Lys = 1, Pro = 1.

- Tyrosine Phosphatase Yield® 58.6% (crude peptide)

Cys Ser Asp Ser Glu Lys Leu Asn Leu Asp Ser IleOH Asp = 0.95 x 3, Ser = 0.58 X 3, Glu = 0.67, Ile = 1.1, Leu = 1.1.

- Oncogene: Phe Arg Gly Thr Leu Arg Yield® 53.4%

Phe = 0.97, Arg® 2 x 1.1, Gly = 1.02, Thr = 0.99, Leu = 1 Example II

Synthesis of Leu-Ala-Gly-Val 1 g Of the polyacrylic resin used in example I was treated as follows: 1. washings with DCM (4 times, 2 min each) 2. neutralization with 5% diisopropylethylamine in DCM (2 times, each time) 2 min) 3. washes with DCM (4 times, 2 min each).

0.858 g (3.3 mmol) of bromoacetic anhydride in 10 ml of DCM was added to the resin. After shaking for 45 min, the DCM solution was removed by filtration. The brominated support was washed as follows: 1. DCM (4 times, 2 min each) 2. DMF (4 times, 2 min each).

The cesium salt of FmocValOH (1.29 g, 2.75 mmol), prepared according to Mery et al. (Int. J. Peptide Protein Res. 1988, 31, 412), was dissolved in DMF (15 ml) and the solution was added to the resin. The suspension was shaken at ambient temperature for 3 days. The DMF was then decanted and the polymer was washed with: 1. DMF (10 times, 2 min each) 2. Methanol (4 times, 2 min each) 3. DCM (4 times, 2 min each) 4. Diethyl ether (4 times, 2 min each).

The resin was dried under high vacuum in the presence of KOH pellets for 12 hours. The amount of coupled Val was 0.492 mmol / g determined by amino acid analysis, after hydrolysis in 6N HCl, vacuumed for 24 hours and sealed tubes.

The FmocVal resin (1.1 g) was washed with water (4 times, 2 min each) and the Fmoc group was cleaved using 10% piperidine or diethylamine in water (2 times, 2 min each). The resin was then washed with isopropanol (2 times, 2 min each) and with water (4 times, 2 min each).

The symmetrical anhydride of FmocGlyOH in DMF (15 ml) was added to the support. The symmetrical anhydride solution was prepared as follows:

FmocGlyOH (0.981g, 3.3mmol) was dissolved in DCM (15ml), the solution was cooled to 0 ° C and dicyclohexylcarbodiimide (0.339g, 1.65mmol) in DCM (10ml) was added. The cloudy mixture was stirred at 0 ° C for 20 min, the dicyclohexylurea precipitate was removed by filtration and the filtrate was concentrated under vacuum at room temperature. The oily residue was dissolved in DMF (15 ml) and the solution was added to the resin. The mixture was shaken at room temperature for 45 min, after which the qualitative ninhydrin test from Kaiser et al. (Anal. Biochem. 1970, 34/575) was negative. The DMF was removed by filtration and the support was washed with DMF (2 times, 2 min each) and then with water (6 times, 2 min each).

This protocol was used to build in the following amino acids: Leu and Ala. The symmetrical anhydrides were prepared from 1.16 g (3.3 mMnol) FmocLeuOH and 1.02 g (3.3 mMnol) FmocAlaOH. After the completion of the synthesis, the peptide resin adduct was washed with isopropanol (4 times, 2 min each), water (4 times, 2 min each) and isopropanol water (70-30) (4 times, each 2 min).

The peptide resin was then suspended in isopropanol water (70-30) and 1.1 ml of 1N NaOH was added. The mixture was shaken at ambient temperature for 5 hours, the isopropanol-water solution containing the Leu-Ala-Gly-Val was collected and the support was washed with water (4 times, 2 min each), methanol (4 times, 2 min each ) and water (4 times, 2 min each). The filtrates were collected, the pH was adjusted to 4 with 1N HCl, and the solution was concentrated under vacuum at room temperature. The residue was purified by HPLC on a Lichrosorb RP 18 (10 µm) column (250 x 20 mm) using 0.1% aqueous TFA and acetonitrile as running fluids.

Yield: 54% (based on the amount of amino groups of the carrier in the beginning).

Amino Acid Analysis: Leu 1.03 (1), Ala 0.99 (1), Gly 1.1 (1), Val 1.0 (1).

The same procedure was used for the synthesis of the following peptides: - Dorman peptide: Leu Ala Gly ValOH yield: 46.6%

Gly 0 0.93, Ala = 0.91, Val = 1.01, Leu = 1.

-Laminin: Tyr Ile Gly Ser ArgNH2 yield: 46.3%

Ser = 0.79, Gly = 1.01, Ile = 0.96, Tyr = 0.89, Arg = 0.93. -CDG 28 kinase protein from the cell cycle of "Pombee" yeast yield = 48.6% (crude peptide)

Tyr Lys Ala Leu Asp Leu Arg Pro GlyOH

Asp = 0.97, Gly = 1.02, Ala = 0.98, Leu = 1.02 x 2, Tyr = 0.98, Arg = 0.96, Lys = 1, Pro = 0.97.

- Tyrosine phosphatase yield = 61.6% (crude peptide)

Cys Ser Asp Ser Glu Lys Leu Asn Leu Asp Ser IleOH

Asp = 0.99 x 3, Ser = 0.63 x 3, Glu = 0.73, Ile = 1.03, Leu = 1.

- Oncogene: Phe Arg Gly Thr Leu Arg yield = 49.2%

Phe = 1, Arg = 2 x 1.04, Gly = 1, Thr = 0.96, Leu = 0.98.

Claims (5)

A method for solid phase peptide synthesis, wherein a first amino acid residue is attached to a polyacrylic resin, one or more secondary amino acid residues are coupled to obtain the desired peptide and the peptide is detached from the resin, characterized in that the coupling protocol comprises washings of the resin in water and / or aqueous solution (s). 2. Process according to claim 1, characterized in that the polyacrylic resin is a copolymer of three monomers, namely: (i) a first monomer which provides a matrix for the copolymer, namely 1- (meth) acryloyl-pyrrolidine, 1 - (meth) acryloyl-piperidine, 1- (meth) acryloyl-perhydroazepine, 1- (meth) acryloyl-4-methyl-piperazine, 4- (meth) acryloyl-morpholine, N, N-dimethyl- (meth) acrylamide or Ν, Ν-diethyl (meth) acrylamide, (ii) a second monomer that cross-links the copolymer, N, N1-di (meth) acryloyl-diaminomethane or N, N'-di (meth) acryloyl-1,2 diaminoethane, and (iii) a third monomer that activates the copolymer, any of the following acids 2- (meth) acrylamido-acetic acid, 3- (meth) acrylamido-propionic acid, 4- (meth) acrylamido-butyric acid, 6- (meth) acrylamido-hexanoic acid, N- (meth) acryloyl-L-alanine, N- (meth) acryloyl-L-valine, N- (meth) acryloyl-L-leucine, N- (meth) acryloyl-L-phenylalanine , N- (meth) acryloyl-L-tyrosine, N- (meth) acryloyl-L-methionine, N- (meth) acryloyl-L-lysin e, pf N- (meth) acryloyl-L-proline or a methyl ester of one of these acids. Process according to claim 1, characterized in that the polyacrylic resin is a copolymer according to claim 2, which is amidated with ethylenediamine. Method according to any one of the preceding claims, characterized in that the N-protecting group used in the amino acid coupling is Boe and the coupling protocol consists of: 1. washing: distilled water - 2 to 4 times, 2 min each; 2. Remove the protecting group: HCl (6N) in water - once, 2 min and again, 30 min; 3. wash: distilled water - 4 to 6 times, 2 min each; 4. Neutralization: 1 equivalent of borate buffer 12.5 mM pH 8.5-9.0 - once, 1 to 2 min and again, 1 to 2 min; 5. wash: distilled water - 4 to 6 times, 1 to 2 min each; 6. wash: DMF - 2 times, 1 to 2 min each; 7. coupling: symmetrical anhydride (2 equivalents, 2 times in DMF); 8. wash: DMF or NMP - 2 times, 2 min each; and 9. wash: distilled water - 4 times, 2 min each. Process according to any one of claims 1 to 3, characterized in that the N-protecting group used in the amino acid coupling is Fmoc and the coupling protocol consists of: 1. washing: distilled water - 4 to 6 times, 2 min each; 2. removing the protecting group: piperidine or diethylamine in water; 3. wash: isopropanol, 2 times, 2 min each; distilled water - 4 to 6 times, 2 min each; 4. optional wash: DMF - once, 2 min; 5. coupling: symmetrical anhydride (3 times in excess in DMF); and 6. washing: DMF (2 times, 2 min each); distilled water - 6 times, 2 min each.