LU87592A1 - SOLID PHASE PEPTIDE SYNTHESIS - Google Patents

Description

The present invention relates to a solid phase peptide synthesis process.

The abbreviations used below are in accordance with the 1983 recommendations of the Joint Commission on the Biochemical Nomenclature of IUPAC-IUB, as indicated in Eur.J. Biochem, 138, 9-37 (1984). The following abbreviations are also used: TFA trifluoroacetic acid DCM dichloromethane DMF dimethylformamide NMP N-methylpyrrolidine DMAc dimethylacetamide

In the absence of an express indication, the amino acids and their residues are of series L: for example

Ala = L-alanine, but DAla = D-alanine. The term (meth) acrylic is used to indicate either acrylic or methacrylic.

The usual methods of peptide synthesis in solid phase include the following sequence of operations: 1. - Deprotection of the Boc group; 2. - Washes; 3. - Neutralization of NH2 in position a; 4. - Washes; 5. - Couplings and, 6. - Washes.

The deprotection and neutralization steps are carried out by treating the peptide-resin with solutions of TFA and diisopropylethylamine in DCM. This same solvent is also used during intermediate washes. Whatever the technique used (symmetrical anhydride, dicyclohexylcarbodiimide, hydroxy-benzotriazole, etc.), the coupling is carried out either in DCM or in DMF.

Consequently, solid phase peptide synthesis requires the use of large quantities of relatively expensive solvents and reagents such as TFA, so that the cost of a peptide is directly linked to that of DCM, NMP, DMF, DMAc and TFA used in the synthesis.

It would therefore be particularly advantageous to find less expensive solvents and reagents in order to significantly lower the cost of manufacturing the peptides.

European Patent No. 0 079 842 describes polyacrylic resins which are copolymers of the three monomers defined as follows: (i) a first monomer constituting the matrix of the copolymer and chosen from the following bodies: (meth) acryloyl-1-pyrrolidine, (meth) acryloyl-l-piperidine, (meth) acryloyl-l-perhydroazepine, (meth) acryloyl-l-methyl-4-piperazine, (meth) acryloyl-4-morpholine, Ν, Ν-dimethyl- (meth) acrylamide and Ν, Ν-diethyl- (meth) acrylamide (ii) a second monomer constituting the crosslinking agent of the copolymer and chosen from the following bodies: N, N'-di (meth) acryloyl-diamino-1,2-methane and N, N '-di (meth) acryloyl-diamino-1,2-ethane, and (iii) a third monomer activating the copolymer and chosen from the following acids: (meth) acrylamido-2 acetic acid, (meth) acid acrylamido-3 propionic, acid (meth) acrylamido-4 butyric acid (meth) acrylamido-6 hexanoic, 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, and N- (meth) acryloyl-L-proline or the methyl ester of one of these acids.

These copolymers have free carboxy or methoxy-carbonic groups originating from the third monomer. European Patent No. 81408 describes other polyacrylic resins in which these groups are amidified with ethylene diamine. It also describes the use of these other polyacrylic resins during solid phase peptide synthesis, but only in relation to the expensive solvents generally used with polystyrene resins, as described above.

The invention relates to a method for solid phase peptide synthesis, this method comprising the fixing of a first amino acid residue to a polyacrylic resin, the coupling of one or more other amino acid residues to obtain the desired peptide and the separation of the peptide from the resin, characterized in that the coupling protocol includes steps of washing the resin in water and / or in an aqueous solution.

The method of the invention takes advantage of the hydrophilic properties of polyacrylic resins unlike polystyrene resins. The polyacrylic resins used in the process of the invention are preferably those described in European Patents Nos. 0 079 842 and 0 081 408.

The first amino acid residue is attached to. the matrix using the glycolamide link (B. CALAS et Al., Tetrahedron, 1985, 41, 5331). Previous tests with other labile remains such as:

in which X = Br, Cl or OH have not given satisfactory results.

The resin is then washed in water. The amino acid following the peptide sequence to be constructed is then introduced in the following manner (applicable when the protected group is Boc): 1. - Washing: distilled water - 2 to 4 times 2 min; 2. - Deprotection: HCl (6N) in solution in water - 1 time 2 min then again 30 min; 3. - Washing: distilled water - 4 to 6 times 2 min? 4. - Neutralization: 1 equivalent of 12.5 mM borate buffer pH 8.5-9.0 - 1 time, 1 to 2 min and again 1 to 2 min; 5. - Washing: distilled water - 4 to 6 times, 1 to 2 min; 6. - Washing: DMF - 2 times, 1 to 2 min; 7. - Coupling: Symmetrical anhydride (2 equivalents, 2 times in the DMF); 8. - Washing: DMF or NMP - 2 times, 2 min; and 9. - Washing: distilled water - 4 times, 2 min.

The progress of the coupling is monitored using ninhyndrin or fluorescamine tests.

Alternatively, when the protected group is Fmoc, the elongation process is as follows: 1. Washing: distilled water - 4 to 6 times, 2 min; 2. Deprotection: piperidine or diethylamine in solution in water; 3. Washing: isopropanol - 2 times, 2 min; distilled water - 4 to 6 times, 2 min; 4. Optional washing: DMF - 1 time, 2 min; 5. Coupling: symmetrical anhydride - 3 times in excess in DMF) and, 6. Washing: DMF (2 times, 2 min); distilled water -6 times, 2 minutes.

When the synthesis is complete, the peptide is separated from the matrix by selective cleavage of the glycolamide linkage obtained by one of the following treatments: - NaOH in isopropanol, - NH3 in trifluoroethanol or in methanol or in 1 ' ethanol or in isopropanol, - N2H4 in DMF and, - CHjOH in triethylamine.

With this method, one obtains, for example, the Dorman peptide (Leu Ala Gly Val) and analogues of LHRH with yields greater than 50%.

The invention will be better understood from the description of the examples which follow.

EXAMPLE 1 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 copolymerization of acryloylpyrrolidine-1, of N, N'-diacryloyl diamino-1,2 ethane and of acryl-2 amido-methyl acetate as this is described in Example 19 of European Patent 0 079 842 are treated as follows: 1. Washes with DCM (4 times, 2 min), 2. Neutralization with 5% diisopropylethylamine in DCM (2 times, 2 min), 3. Washes with DCM (4 times, 2 min).

4.29 g (0.0165 mol) of bromoacetic anhydride dissolved in 50 ml of DCM are added to the resin. After stirring for 45 min, the DCM solution is filtered and the brominated support is washed as follows: 1. DCM (4 times, 2 min) 2. DMF (4 times, 2 min).

Cesium salt of BocGlyOH (4.22 g, O, 0137 mol) prepared according to the method of Mery et al.

(Int.J. Protein Peptide Res. 1988, 31, 412) in 75 ml of DMF and this solution is added to the resin. The mixture is stirred for 2 days at room temperature. The DMF is drained and the polymer is washed with: 1. DMF (10 times, 2 min), 2. Methanol (4 times, 2 min), 3. DCM (4 times, 2 min) 4. Ethyl ether (4 times , 2 mins).

The resin is dried under high vacuum in the presence of KOH pellets for 12 hours. The total of Gly bound represents 0.483 mmol / g; it is determined by the analysis of amino acids after hydrolysis 6N HCl, in vacuum-sealed tubes at 110 ° C for 24 hours.

The BocGly resin (4.47 g) is washed with water (4 times, 2 min) and the Boc group is cleaved by treatment of 6N HCl in solution in water (2 times, once 2 min and one another time 30 mins). The HCl is removed by filtration and the resin washed with water (6 times, 2 min). For neutralization, borate buffer (12.5 mmol, pH 9) is used, the resin being treated twice 1 min with 50 ml of the buffer. After washing with water (6 times, 2 min) and DMF (2 times, 2 min), the symmetrical anhydride of BocProOH dissolved in 50 ml of DMF is added to the resin.

The symmetrical anhydride solution is prepared as follows: BocProOH (3.55 g, 0.0165 mol) dissolved in 40 ml of DMF, the solution is cooled to 0 ° C and dicyclohexylcarbodiimide (1.69 g) is added , 8.25 mmol) dissolved in 10 ml of DCM. After having left under stirring at 0 ° C for 30 min and filtration, the solution is evaporated under high vacuum without raising the temperature and the residue is dissolved in DMF and then added to the resin. The mixture is stirred for 30 min after which it is checked that the qualitative ninhydrin test by Kaiser et al. (Anal. Biochem. 1970, 34_, 575) is negative - indicating a coupling efficiency greater than 99.6% -.

The DMF is then removed and the support is washed twice with DMF (2 min) and water (4 times, 2 min).

This protocol is used to introduce the following amino acids of the DTrp6-LHRH sequence. To prepare symmetrical anhydrides, the following are used: BocArg (Mts) OH (7.52 g, 0.0165 mol), BocLeuQH (4.11 g, 0.0165 mol), BocDTrpOH (5.02 g, 0.0165 mol ), BocTyr (2.6 dichlorobenzyl) OH or BocTyr (2.6 DCB) 0H (7.26 g, 0.0165 mol), BocSer (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, 0.0165 mol), pyro-GluOH (2.13 g, 0.0165 mol) .

After incorporating pyro-GluOH, the resin is washed with methanol (4 times, 2 min), ethyl ether (4 times, 2 min) and is dried under high vacuum at room temperature for 48 hours.

The peptide-resin is then treated with 10 ml of thiophenol in solution in 50 ml of DMF to separate the dinitrophenyl group on the histidine side chain.

After stirring for 45 min, the thiophenol solution is drained and the resin is washed with DMF (4 times, 2 min), DCM (4 times, 2 min) and with ethyl ether (4 times , 2 mins). The resin is dried under high vacuum for 12 hours. It is treated twice 30 min at 0 ° C with 50 ml of the following solution, previously cooled: trifluoromethanesulfonic acid (3.6 ml), anisole (4 ml), thioanisole (4 ml), metacresol (4 ml) and trifluoroacetic acid (40 ml). At the end of the deprotection sequence, the resin is washed with DCM (2 times, 2 min), DCM / DMF (50/50) (2 times, 2 min), diisopropylethylamine in 5% solution in DCM (2 times, 1 min), DMF (3 times, 2 min), isopropanol / water (70/30) (3 times, 2 min). The peptide-resin is suspended in 250 ml of a trifluoroethanolic solution saturated with NH3.

After stirring the mixture at room temperature for 15 hours, the trifluoroethanolic solution containing the deprotected DTrps-LHRH is separated and the support is washed with water (4 times, 2 min), then methanol (4 times, 2 min) and again with water (6 times, 2 min). The filtrates are combined, the pH is brought to about 4 with IN hydrochloric acid and then they are concentrated in vacuo without raising the temperature. The residue is fractionated on a carboxymethyl cellulose column (Wathman CM 52, 10 x 2 cm) having a linear gradient of NaCl (10 mM AcONa pH 5.0 to 10 mM AcONa, 0.15M NaCl pH 5.0). The appropriate fractions are combined, lyophilized and desalted by filtration of the gel on a column (100 x 2.5 cm) of Sephadex G10 in 10 M HCl. The peptide fraction is then purified by HPLC treatment on a column (270 x 20 mm) of Lichrosorb RP 18 (10 μία) with 0.01% trifluoroacetic acid (TFA) 0.01% in solution in water and Acetonitrile as eluents.

Yield: 51% (based on the amino groups of the initial support).

Amino acid analysis: Glu 0.99 (1), Leu 1.0 (1), His 1.0 (1), Trp 1.89 (2), Ser 0.96 (l), Tyr 0.97 (1),

Pro 0.99 (1), Gly 1.07 (1).

For some of the amino acids which are less stable in an acid medium, the analytical values are lower than those expected due to their degradation.

The same process is used for the preparation of 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 protein kinose from the yeast cell cycle "Pombee"

Yield = 44.1% (unprocessed 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% (peptide in the raw state)

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 2 Leu-Ala-Gly-Val

1 g of the polyacrylic resin used in Example 1 is treated as follows: 1. Washes with DCM (4 times, 2 min), 2. Neutralization with 5% of diisopropylethylamine in DCM, 3. Washes with DCM (4 times, 2 min).

0.858 g of bromoacetic anhydride dissolved in 10 ml of DCM is added to the resin. After stirring for 45 min, the DCM solution is removed by filtration and the brominated support is washed as follows: 1. DCM (4 times, 2 min) 2. DMF (4 times, 2 min)

Cesium salt of FmocValOH (1.29 g, 2.75 mmol) prepared according to the method of Mery et al. (Int. J. Peptide Protein Res. 1988, 31, 412) in 15 ml of DMF and this solution is added to the resin. The suspension is stirred at room temperature for 3 days. The DMF is then drained and the polymer is washed with: 1. DMF (10 times, 2 min) 2. Methanol (4 times, 2 min) 3. DCM (4 times, 2 min) 4. Ethyl ether (4 times, 2 mins)

The resin is dried under high vacuum in the presence of KOH flakes for 12 hours. The total of bound Val represents 0.492 mmol / g; it is determined by amino acid analysis, after hydrolysis in 6N HCL in sealed tubes under vacuum for 24 hours.

The Resin-FmocVal (1.1 g) is washed with water (4 times, 2 min) and the Fmoc group is cleaved by treatment with piperidine or diethylamine (10%) in solution in water (2 times, 2 min). The resin is then washed with isopropanol (2 times, 2 min) and with water (4 times, 2 min).

Symmetrical anhydride of FmocGlyOH dissolved in 15 ml of DMF is added to the support. The symmetrical anhydride solution is prepared as follows:

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

This protocol is used to introduce the amino acids Leu and Ala. The symmetrical anhydrides are prepared from 1.16 g (3.3 mmol) of FmocLeuOH and 1.02 g (3.3 mmol) of FmocAlaOH. After the synthesis, the whole peptide-resin is washed with isopropanol (4 times, 2 min), with water (4 times, 2 min) and, finally with an isopropanol / water mixture (70/30 ) (4 times, 2 min).

The peptide-resin is then suspended in an isopropanol / water mixture (70/30) to which 1.1 ml of NaOH IN is added. After stirring the mixture at room temperature for 5 hours, the isopropanol-water mixture containing the Leu-Ala-Gly-Val is separated and the support is washed with water (4 times, 2 min), methanol (4 times, 2 min) and with water (4 times, 2 min). The filtrates are combined and the pH is brought to 4 with IN HCl; the solution is concentrated under vacuum at room temperature. The residue is purified by HPLC treatment on a column (250 × 20 mm) of Lichrosorb RP 18 (10 μία) with TFA

(0.1%) in solution in water and acetonitrile as eluents.

Yield: 54% (based on amino groups of the initial support).

Amino acid analysis: Leu 1.02 (1), Ala 0.99 (1), Gly 1.1 (1), Val 1.0 (1).

The same method is used for the synthesis of the following peptides: - Dorman peptide: Leu Ala Gly ValOH Yield = 46.6%

Gly = 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 - CDC 28 protein kinase from the yeast cell cycle "Pombee".

Yield = 48.6% (unprocessed 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% (unprocessed 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 (6)

1. Washing: distilled water - 4 to 6 times, 2 min; 1. Washing: distilled water - 2 to 4 times, 2 min; 1. A method for peptide synthesis in solid phase, comprising the fixing of a first amino acid residue to a polyacrylic resin, the coupling of one or more other amino acid residues to obtain the desired peptide and the separation of the resin peptide, characterized in that the coupling protocol includes, steps for washing the resin in water and / or an aqueous solution. 2. Deprotection: Piperidine or diethylamine in water; 2. Deprotection: HCl (6N) in solution in water - 1 time, 2 min and another time, 30 min; 2. Method according to claim 1 in which the polyacrylic resin is a copolymer of the three monomers defined as follows: (i) a first monomer constituting the matrix of the copolymer and chosen from the following bodies: (meth) acryloyl-1-pyrrolidine, ( meth) acryloyl-1-piperidine, (meth) acryloyl-1-perhydroazepine, (meth) acryloyl-1-methyl-4-piperazine, (meth) acryloyl-4-morpholine, N, N-dimethyl- (meth) acrylamide and , Ν, Ν-diethyl- (meth) acrylamide (ii) a second monomer constituting the crosslinking agent of the copolymer and chosen from the following bodies: N, N'-di (meth) acryloyl-diamino-1,2-methane and N, N'-di (meth) acryloyl-diamino-1,2-ethane-2, and (iii) a third monomer activating the copolymer and chosen from the following acids: (meth) acrylamido-2 acetic acid, acid ( meth) acrylamido-3 propionic, (meth) acrylamido-4 butyric acid, (meth) acrylamido-6 hexanoic acid, N- (meth) acryloyl-L-alanine, N- (meth) acryloyl-L-valine, N- ( meth) ac ryloyl-L-leucine, N- (meth) acryloyl-L-phenylalanine, N- (meth) acryloyl-L-tyrosine, N- (meth) acryloyl-L-methionine, N- (meth) acryloyl-L-lysine, and N- (meth) acryloyl-L-proline or the methyl ester of one of these acids. 3. Washing: isopropanol - 2 times, 2 min; distilled water - 4 to 6 times, 2 min; 3. Washing: distilled water - 4 to 6 times, 2 min; 3. Method according to claim 1 wherein the polyacrylic resin is a copolymer according to claim 2, amidified with ethylene diamine. 4. Optional washing: DMF - 1 time, 2 min 4. Neutralization: 1 equivalent of 12.5 mM borate buffer pH 8.5 - 9.0 - 1 time, 1 to 2 min and another time, 1 to 2 min; 4. Method according to one of the preceding claims, in which the N-protected group, used in the coupling of amino acids is Boc and the coupling protocol is: 5. Coupling: symmetrical anhydride (3 times in excess in the DMF) and 5. Method according to one of claims 1 to 3 in which the H-protected group used in the coupling of amino acids is Fmoc and the coupling protocol is: 5. Washing: distilled water - 4 to 6 times, 1 to 2 minutes; 6. Washing: DMF - 2 times, 1 to 2 min: 7. Coupling: symmetrical anhydride (2 equivalents, 2 times in DMF); 8. Washing: DMF or NMP - 2 times, 2 min; and 9. Washing: distilled water - 4 times, 2 min. 6. Washing: DMF (2 times, 2 min); distilled water - 6 times, 2 min.