PT91784B - PROCESS FOR THE SYNTHESIS OF SOLID PHASE PEPTIDES - Google Patents

Abstract

Solid phase peptide synthesis is accomplished on polyacrylic resins by a method which includes, in the coupling protocol, steps of washing the resin in water and/or aqueous solutions.

Description

DESCRIPTION

The present invention relates to solid phase peptide synresis. The abbreviations used in this specification are in accordance with the 1983 recommendations of the IUPAC-IU3 Joint Gommission on Biochemical Nomenclature, as described in Eur. J. Biochem, 138,

9-37 (1984). In addition, the following abbreviations were also used:

TFA trifluoroacetic acid

DCM dichloromethane

DRF dimethyl formamide

N1IP N-methyl-pyrrolidine

DMAc dimethyl acetamide

Amino acids and their residues are in the L configuration unless otherwise specified, for example, Ala = L-alanine, DAla = D-alanine. The term (meth) acrylic is used to mean acrylic or methacrylic.

The usual methods of solid phase peptide synthesis include the following sequence of operations:

- Deprotection of the Boc group;

- Washes;

- Neutralization of NH ^ in the oc position;

- Washes;

- Couplings and

- Washes.

The deprotection and neutralization steps are performed by treating the resin-peptide with TFA and con. diisopropylethylamine solutions in DCM. This same solvent is also used for intermediate washings. Regardless of the coupling agent used (symmetric anhydride, dicyclohexyl-carbo-diimide, hydroxybenzotriazole, etc.), the coupling reaction is carried out in DOM or DMF.

Consequently, solid phase peptide synthesis involves large amounts of very expensive solvents and reagents such as TFA; therefore, the cost of a peptide depends directly on the costs of DCM, NilP, DMF, DMAc and TFA used in the synthesis.

As a result, it becomes particularly interesting to find less expensive solvents and reagents in order to significantly decrease the cost of producing the peptides.

European patent 0 079 842 describes poly-acrylic resins which are copolymers of three monomers as described below:

(i) a first monomer which provides a matrix for the copolymer and which is a compound selected from the following:

l- (meth) acryloyl-pyrrolidine,

1- (meth) acryloyl-piperidine, 1- (meth) acryloyl-perhydro-azepine,

1- (meth) acryloyl-4 ~ methyl-piperazine, 1- (meth) acryloyl-morpholine,

N, N-dimethyl- (meth) acrylamide and N, N-diethyl- (meth) acrylamide (ii) a second monomer that provides the copolymer with a crosslinked bond and which is selected from one of the following compounds:

N, N'-di (meth) acryloyl-diamino-methane and Ν, Ν · -di- (meth) acryloyl-1,2-diamino-ethane, and (iii) a third monomer that activates the copolymer and is selected between one of the following acids:

2- (meth) acryl-amido-acetic acid, 3- (met) -acryl-starch-propionic acid, 4- (met) acryl-starch-butyric acid, 6- (met) acryl-starch-beoxanoic acid,

N- (met) -acryloyl-L-alanine,

N- (met) -acryloyl-L-valine,

N- (meth) acrolooyl-L-leucine,

N- (meth) acryloyl-L — phenyl-alanine,

N- (meth) acryloyl-L-methionine,

N- (meth) acryloyl-L-lysine, and

N- (meth) acryloyl-L-proline or a methyl ester of thy of these acids

These copolymers have free carboxy or methoxy — carbonyl groups derived from the third monomer

European Patent ηδ 81408 also describes polyacrylic resin in which these groups are amidated with ethylene diamine. That patent also describes the use of these polyacrylic resins for solid phase peptide synthesis, but only in conjunction with expensive solvents conventionally used with polystyrene resins, as previously described.

The invention provides a method for solid phase peptide synthesis, that method consisting of coupling a first amino acid residue to a polyacrylic resin, coupling one or more additional amino acid residues to form the desired peptide and separating the peptide from the resin, characterized by the fact that the coupling protocol covers the steps of washing resin in water and / or in an aqueous solution · the method of the present invention takes advantage of the hydrophilic properties possessed by polyacrylic resins, unlike polystyrene resins. Preferably the polyacrylic resins for use in the method of the present invention are those described in European Patent Nos. 0 79 842 and 0 081 408, as mentioned above.

first amino acid residue can be attached to the matrix via the glycolamide radical (B. Calas et al., Tetrahedron, 1985 »41» 5551) · Previous attempts made with other unstable ligands such as.

X-CH.

X-CH, \ y-c ^ -co ^

X-OH,

-C ^ -CO ^

where X «Br, Cl or OH, proved to be unsatisfactory.

Then the resin can be washed with water.

following amino acid of the peptide sequence to be constructed can be added according to the following protocol (applicable when the protecting group is Boc):

1. - Washing: distilled water - 2 to 4 times, 2 minutes each time;

2. - Deprotection: HC1 (6N) in water - once for 2 minutes and again for 50 minutes;

5 «- Washing: distilled water - 4 to 6 times, 2 minutes each time;

4. - Neutralization: 1 equivalent of 12.5 mM borate buffer; pH

8.5-9.0 - once for 1 to 2 minutes and again for 1 to 2 minutes;

5 · - Washing: distilled water - 4 to 6 times, 1 to 2 minutes each time;

6. - Washing: DMF - 2 times, 1 to 2 minutes each time;

7 · - Coupling: symmetric anhydride (2 equivalents, 2 times in DMF);

8. - Washing: DMF or NMP - 2 times, 2 minutes each time; and 9 · - Washing: distilled water - 4 times, 2 minutes each time.

progress of the coupling reaction can be controlled by ninhydrin or fluoreseamine.

Alternatively, when the protection group is Fmoc, the stretching protocol can be as follows:

1. - Washing: distilled water - 4 to 6 times, 2 minutes each time;

2. - Deprotection: piperidine. or diethylamine in water;

5 · - Wash: isopropanol, 2 times, 2 minutes each time; distilled water - 4 to 6 times, 2 minutes each time;

4. - Optional washing: DMF - 1 time, 2 minutes;

5 · - Coupling: symmetrical anhydride (3 times in excess in DMF) and

6. - Washing: DMF (2 times, 2 minutes each time); distilled mare - 6 times, 2 minutes each time.

When the synthesis is complete, the peptide is separated from the matrix by selective disruption of the glycolamide bond, according to one of the following treatments:

- NaOH in isopropanol,

- NH ^ in trifluoro-ethanol or methanol or ethanol or isopropanol,

- N ^ H ^ in DMF and

- CH ^ OH in triethylamine.

Using this method, the Dorrnan reference peptide (Leu Ala Gly Val) and the LHEH analogs were obtained in a yield of about 50%.

The present invention will be better understood by describing the following examples:

Example 1

Synthesis of DTrp ^ -LHBH: piro-Glu-His-Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-Gly.

5 g of a polyacrylic resin (0.55 mmol of N ^ / g) was prepared by copolymerizing 1-acryloyl-pyrrolidine, N, Ν'-diacryloyl-1,2-diamino-ethane and 2-acryl-starch -methyl acetate, as described in example 19 of European Patent No. 0 079 842, and then treated as follows

- Washes with DOM (4 times, 2 minutes each time).

- Neutralization with 5% diisopropyl-ethyl-amine in DCM (2 times, 2 minutes each time).

- Washes with DCií (4 times, 2 minutes each time).

4.29 6 (0.0165 mol) of bromoacetic anhydride in ^r 50 ml of DCM was added to the resin. After 45 minutes of stirring, the DCM solution was removed by filtration and the brominated support was washed as follows:

- DCM (4 times, 2 minutes each time);

- DMF (4 times, 2 minutes each time) ·

BocGlyOH cesium salt (4.22 g, 0.0137 mol) was prepared according to Mery et al. (Int. J. Protein Peptide Ees. 1988, 31, 412) and dissolved in DMF (75 ml) and then this solution was added to the resin. The mixture was stirred for 2 days at room temperature. After this period of time, DMF was removed and the polymer was washed with:

- DMF (10 times, 2 minutes each time)

- Methanol (4 times, 2 minutes each time)

- DCií (4 times, 2 minutes each time)

- Diathyl ether (4 times, 2 minutes each time)

The resin was dried under an intense vacuum in the presence of KOH granules for 12 hours. The amount of bound Gly was 0.483 mmol / g, this determination was made by amino acid analysis after hydrolysis in 6N HCl in clean and sealed tubes, at 110 ° C, for 24 hours.

BocGly-Resin (4.47 g) was washed with water (4 times, 2 minutes each time) and the Boc group was cleaved using 6N HCl in water (2 times, 1 time for 2 minutes and again for 30 minutes). The HCl was removed by filtration and the resin was washed with water (6 times, 2 minutes each time). Neutralization was carried out using borate buffer

- 7 (12.5 mmol, pH 9) 'the resin was treated twice with 50 ml of buffer (1 minute each time). After washing with water (6 times, minutes each time) and with DMF (2 times, 2 minutes each time), BocProOH symmetrical anhydride in DMF (50 ml) was added to the resin.

The symmetric anhydride solution was prepared as follows: BocProOH (5.55 Si 0.0165 mol) was dissolved in 40 ml of DMF, the solution was cooled to 0 ° C and dicyclohexyl was added. carbo-diimide (1.69 Si 8.25 mmol) in 10 ml of DCM. After stirring at 0 ° C for 50 minutes and after filtration, the solution was evaporated in an intense vacuum without heating and the residue was dissolved in DMF and added to the resin. The mixture was stirred for 50 minutes, and after that time the qualitative test of the ninidri in the second Kaiser et al. (Anal. Biochem. 1970, 54, 575) was negative indicating a coupling yield greater than 99.6%. Then the DMF was removed and the support was washed 2 times with DMF (2 minutes each time) and with water (4 times, 2 minutes each time).

This protocol was used to incorporate the other amino acids of the DTrp ^ -LHRH sequence. Symmetric anhydrates were prepared using: BocArg (Mts) OH (7 »52 g? 0.0165 mol), BccLeuOH (4.11 g; 0.0165 mol), BocIUrpOH (5» 02 g; 0.0165 mol) , BocTyr (2.6 dichloro-benzyl) 0H or BocTyr (2.6 DCB) OH (7.26 g; 0.0165 mol), BocSer (Bzl) OH (4.86 g;

0.0165 mol), BocTrpOH (5.02 g? 0.0165 mol), BooHis (dinitro-phenyl) 0H or BocHis (Dnp) OH (6.94 g; 0.0165 mol), piro-GluOE (2, 15 Sí 0.0165 mol).

After the incorporation of piro-GluOH, the resin was washed with methanol (4 times, 2 minutes each time), with diethyl ether (4 times, 2 minutes each time) and dried in an intense vacuum at room temperature for 48 hours.

- 8 '; '^' ^ XOWaweiemui * ,,

Then the peptide-resin was treated with thiophenol (10 ml) in DMF (50 ml) to remove the diuitrophenyl group from the histidine side chain.

After 45 minutes of stirring, the thiophenol solution was removed and the resin was washed with Di® (4 times, minutes each time), DCM (4 times, 2 minutes each time) and diethyl ether (4 times). times, 2 minutes at a time). The resin was dried under high vacuum for 12 hours. Was it treated twice (30 minutes each time) at 0 ° C with 50 ml of the following pre-cooled solution? trifluoro-methanesulfonic acid (3.6 ml), anisole (4 ml), thio-anisol (4 ml), metacresol (4 ml) and trifluoro-acetic acid (40 ml). After the deprotection was over, the resin was washed with DCM (2 times, 2 minutes each time), with DCM / DMF (50:50) (2 times, 2 minutes each time), with diisopropyl-ethyl-amine at % in DCM (2 times, 1 minute each time), with DMF (3 times, 2 minutes each time), with isopropanol / water (70-30) (3 times, 2 minutes each time). Then a suspension of peptide-resin in a saturated NH4 trifluoro-ethanolic solution (250 ml) was prepared.

The mixture was stirred at room temperature for 15 hours, taken up in etanóli trifluoro-fi · _u ca _.-- solution containing D-Trp-deprotected and washed LHSH support with water (4 times, 2 mn each time ), with methanol (4 times, minutes each time) and with water (6 times, 2 minutes each time). All filtrates were combined, the pH was adjusted to about 4 with 1N hydrochloric acid; then the concentration was performed in a vacuum without heating. The residue was fractionated on a carboxy methyl cellulose column (Wathman CM 52, 10 x 2 cm) with a linear gradient of NaCl (10 mM AcONa, pH 5.0 to 10 mM AcONa, 0.15 pH 5 NaCl, 0). The appropriate fractions were combined, lyophilized and desalted by gel filtration on a column (100 x 2.5 cm) of Sephadex G10 in 10M DC1. Then the peptide fraction was purified by HPLC on a column (270 x 20 mm) of Lichrosorb RP18 (10 iam) using as an eluent acid

- 9 trifluoroacetic (TFA) 0.01% in water and acetonitrile.

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

Amino acid analysis: Glu 0.99 (1) »Leu 1.0 (1), His 1.0 (1), Trp 1.89 (2), Ser 0.96 (1), Tyr 0.97 (1 ), Pro 0.99 (D »Gly 1.07 (1) ·

For some amino acids that are less stable in acidic conditions, the analytical values may be lower than expected, due to their degradation.

The same method was used for the following peptides:

- Dorman's 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 ArgNH ₂ Yield · 35.6%

Ser - 0.75, Gly - 1.03, He - 0.99, Tyr - 1, Arg «0.99 ·

- Protein that is not CDC 28 of the yeast cell cycle * Pombee ** 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 - 099, Lys - 1, Pro - 1.

- Tyrosine phosphatase

Yield - 5θ, 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,

Read 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.

- 10 Example 2

Synthesis of Leu-Ala-Gly-Val

1 g of the polyacrylic resin used in example 1 was treated as follows:

- Washes with DCM (4 times, 2 minutes each time)

- Neutralization with 5% diisopropylethylamine in DCM (2 times, 2 minutes each time)

- Washes with DOM (4 times, 2 minutes each time) ·

0.858 g (3 »3 mmol) of bromoacetic ani in 10 ml of DOM was added to the resin. After 45 minutes of stirring, the DCM solution was removed by filtration and the brominated support was washed, as follows

- DOM (4 times, 2 minutes each time)

- DMF (4 times, 2 minutes each time).

Cesium salt of FmocValOH (1.29 g, 2.75 mmol) according to Mery et al. (Xnt. J. Peptide Protein fies. 1988, 3 <412) was prepared, dissolved in DMF (15 ml ) and the solution was added to the resin. The suspension was stirred at room temperature for 3 days. After this period of 'time ^r DMF was removed and the polymer washed with:

- DMF (10 times, 2 minutes each time)

- Methanol (4 times, 2 minutes each time)

- DCM (4 times, 2 minutes each time)

- Diethyl ether (4 times, 2 minutes each time).

The resin was dried under intense vacuum in the presence of KDH granules for 12 hours. The amount of Vai bound was 0.492 mmol / g, as determined by amino acid analysis, after hydrolysis in 6N HCl, in clean and sealed tubes, for 24 hours.

- 11 FmocVal-Resin (1.1 g) was washed with water (4 times, 2 minutes each time) and the Fmoc group was cleaved using 10% piperidine or diethylamine in water (2 times, 2 minutes at a time). Then the resin was washed with isopropanol (2 times, 2 minutes each time) and water (4 times, 2 minutes each time) ·

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

FmocGlyOH (0.981 g, 3.3 mmol) was dissolved in DOM (15 ml), the solution was cooled to 0 ° C and dicyclohexyl-carbo-diimide (0.339 6i 1.65 mmol) was added in DCI3 (10 ml). The cloudy mixture was stirred for 20 minutes at 0 ° C, the dicyclohexyl urea precipitate was filtered off and the filtrate was concentrated in vacuo at room temperature. The oily residue was dissolved in DMF (15 ml) and the solution was added to the resin. The mixture was stirred at room temperature for 45 minutes, and the Kaiser et al. (Anal. Biochem. 197θ, 34, 575) ninhydrin test was negative after that period of time. DMF was filtered and the support was washed with DMF (2 times, 2 minutes each time) and then with water (6 times, 2 minutes each time).

This protocol was used to incorporate the following amino acids: Leu and Ala. Symmetric anhydride was prepared from 1.16 g (3.3 mmol) of FmocLeuOH and 1.02 g (3.3 mmol) of FmocAlaOH. After the synthesis was completed, the peptide-resin adduct was washed with isopropanol (4 times, 2 minutes each time), with water (4 times, 2 minutes each time) and with isopropanol / water (70:30) (4 times, 2 minutes each time).

- 12 Then the peptide-resin was suspended in isopropanol / water (≤ D: 30) and 1.1 ml of 1 N NaOH was added. The mixture was stirred at room temperature for 5 hours, collected the isopropanol / water solution containing à »Leu-Ala-Gly-Val and the support was washed with water (4 times, 2 minutes each time), with methanol (4 times, 2 minutes each time) and with water (4 times, 2 minutes each time). The filtrates were combined, adjusted to pH 4 with HCl, IN, the solution was concentrated in vacuo and at room temperature. The residue was purified by HPLC on a column (250 x 20 mm) of Lichrosorb SP 18 (10 µm) using 0.1% TFA in water and acetonitrile as eluents.

Yield: 5 ²¹ % (based on the initial support amino groups) ·

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

The same method was 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.

- Laminine: Tyr Ile Gly Ser ArgNH ^

Yield - 46.3%

Ser - 0.79, Gly - 1.01, Ile - 0.96, Tyr - 0.89, Arg «0.93.

- Protein kinase CDC 28 of the yeast cell cycle Pombee Yield - 48.6% (crude peptide)

Tyr Lys Ala Leu Asp Leu Arg Pro QlyOH

Asp 0.97, Gly 1.02, Ala - 0.98, Leu - 1.02 x 2, Tyr w 0, 'Arg - 0.96, Lys - 1, Pro - 0.97 ·> 8

- Tyrosine phosphatase

Yield '61.6% (crude peptide)

Cys Ser Asp Ser Glu Lys 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 (8)

- IS Process for the synthesis of a solid phase peptide, comprising the attachment of a first amino acid residue to a polyaryl resin, the joining of one or more amino acid residues to form the desired peptide and disconnecting the peptide from the resin, characterized in that the connection protocol includes steps for washing the resin with water or aqueous solutions · 2. A process according to claim 1, characterized in that the polyacrylic resin is a copolymer of three monomers as follows: i) a first monomer that provides a matrix for the copolymer and is selected from: 1- (meth) acryloyl-pyrrolidine, 1- (meth) acryloyl) -piperidine, 1- (meth) acryloyl-perhydro-azepine, 1- (meth) acryloyl-4-methyl-piperazine, 1- (meth) acrjloyl-morpholine, N, N -dimethyl- (meth) acrylamide and - 14 - N, N-diethyl- (meth) acrylamide ii) a second monomer that cross-links the copolymer and is selected from among N di (meth) acryloyl diaminomethane and N, N-di (meth) acryloyl 1,2-diaminoethane, and iii) a third monomer that activates the copolymer and is selected from the following acids: 2- (met) acrylamido-acetic acid, 3- (πΐ6 · δ) ηοιϋ £ αη1άο-ρΓθρίόη1οο, 4- (met) acrylamido-but £ rich acid, 6- (met) acrylamido-hexanoic acid, N- (meth) acryloyl-L-alanine, N- (met) acae & oil -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, is N- (meth) acryloyl-L-proline or is a methyl ester of one of those acids, where the term (meth) acrylic is used to indicate either acrylic or methacrylic. 36. A process according to claim 1, characterized in that the polyacrylic resin is a copolymer according to claim 2 amidated with ethylene diamine. - Process according to any of the previous claims, characterized by the protective group of The amino acid link is not used BOG and the link protocol is: 1. - Washing: distilled water - 2 to 4 times, 2 m each; 2. - Deprotection: HC1 (6N) in water - once, 2 m and again, 50 minutes; 5 · - Washing: distilled water - 4 ê 6 times, 2 minutes each; 4. - Neutralization: 1 equivalent of 12.5 mM borate buffer pH 8.5-9.0 - once, 1 to 2 minutes and again, 1 to 2 minutes; 5 · - Washing: distilled water - 4 to 6 times, 1 to 2 m each; 6 · - Washing: DMF - twice, 1 to 2 m each 7 · - Connection: symmetric anhydride (2 equivalent, twice in DMF) 8. - Washing: DMF or NMP - twice, 2 m each; and 9 · - Washing: distilled water - 4 times, 2 m each, where DMF «dimethylformamide NMP» N - methyl - pyrrolidine - Method 5 ^is accrodo to any of claims 1 to 5 wherein the N - protecting group used in linking the amino acid to be Fmoc and the connection protocol is: 1. - Washing: distilled water - 4 to 6 times, 2 m given; 2. - Deprotection: piperidine or diethylamine in waterj 5 · - Washing: isopropanol, twice, 2 m each; distilled water - 4 to 6 times, 2 m each; 4. - Eventual washing: DMF - once, 2 minutes; 5 · - Connection: symmetric anhydride (5 times in excess in DMF) and 6th - · - 16 6. - Washing: DMF (twice, 2 minutes each); distilled water - 6 times, 2 minutes each.