NZ230712A

NZ230712A - Solid phase peptide synthesis on polyacrylic resins characterised by the use of aqueous washes

Info

Publication number: NZ230712A
Application number: NZ230712A
Authority: NZ
Inventors: Bernard Calas; Michel Follet; Jean Mery; Hanitra Naharisoa
Original assignee: Expansia Sa
Priority date: 1988-09-24
Filing date: 1989-09-20
Publication date: 1991-07-26
Also published as: NL8902361A; SG40692G; LU87592A1; IE62008B1; JPH0768265B2; FI101474B; FI894489A0; GB8921637D0; FR2636951B1; IT8921804A0; FI101474B1; CA1333441C; ZA897151B; IE893037L; JPH02129200A; TNSN89104A1; NO175593B; SE8903121D0; AU622705B2; GB2223227A

Description

<div id="description" class="application article clearfix"> New Zealand Paient Spedficaiion for Paient Number £3071 £ 2307 1 © NO DRAWINGS Priority Date(s):.... 2^,5.:..^.:. Complete Specification rV^n: Class: (5). .4*. - Publication Date: P.O. Journai, 26 JUL G O Patents Form No. 5 PATENTS ACT 1953 COMPLETE SPECIFICATION "SOLID PHASE PEPTIDE SYNTHESIS" ,-fENT^ - 2 0SEP W89 SJ [ce\^ WE, SOCIETE D'EXPANSION SCIENTIFIQUE EXPANSIA, a joint stock company organized and existing under the laws of Framce, of 42 rue du Docteur Blanche, 75016 Paris, France, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- -1- (Followed by la) p 230712 -la- The present invention relates to solid phase peptide synthesis. The abbreviations used in this specification are in accordance with the 1983 Recommendations of the IUPAC-IUB 5 Joint Commission on Biochemical Nomenclature, as set out in Eur. J. Biochem, 138, 9-37 (1984). In addition, the following are used : TFA trifluoroacetic acid DCM dichloromethane DMF dimethylformamide NMP N-methylpyrrolidine DMAC dimethylacetamide Amino acids and their residues are of L-configuration unless otherwise specified, e.g. Ala = 15 L-alanine, DAI a = D-alanine. The term (meth) acrylic is used to indicate either acrylic or methacrylic. The usual methods of solid phase peptide synthesis comprise the following sequence of operations : 1.- Deprotection of the Boc group ; 20 2.- Washings ; 3.- Neutralization of NH2 in a position ; 4.- Washings ; 5.- Couplings and 6.- Washings. 25 Deprotection and neutralization steps are achieved 2307 12 - 2 - by treating the resin-peptide with TFA and diisopropyl-ethy 1 amine solutions in DCM. This same solvent is also used for intermediate washings. Whatever the coupling agent used (symmetrical anhydride, dicyclohexylcarbo-5 diimide, hydroxybenzotriazol, etc.), the coupling reaction is conducted either in DCM or in DMF. Consequently, solid phase peptide synthesis involves large quantities of rather expensive solvents and reagents such as TFA ; thus, the cost of a peptide 10 directly depends on the costs of DCM, NMP, DMF, DMAc and TFA used in the synthesis. Therefore, it would be particularly interesting to find cheaper solvents and reagents in order to lower significantly the manufacturing cost of peptides. 15 Our European Patent No. 0 079 842 describes poly- acrylic resins which are copolymers of three monomers as follows : (i) a first monomer which provides a matrix for the copolymer and is one of 20 1-(meth)acryloyl-pyrrolidine, 1-(meth)acryloyl-piperidine, 1- (meth) acryloyl-perhydroazepine, 1-(meth)acryloyl-4-methyl-piperazine, 4-(meth)acryloyl-morpholine, 25 N,N-dimethyl-(meth) aery 1 amide and N,N-diethyl-(meth)acrylamide (ii) a second monomer which crosslinks the copolymer and is one of N, N' -di (meth) acryloyl-diaminomethane and 30 N,N'-di(meth)acryloyl-1,2-diaminoethane, and (iii) a third monomer which activates the copolymer and is one of the following acids 2-(meth)acrylamido-acetic acid, © - 3 - 2307 12 Q O 3-(meth) aery lamido-prop ionic acid, 4- (meth) acrylamido-butyric acid, 6-(meth)acrv1amido-hexanoic acid, N-(meth) acryloyl-L-alanine, 5 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, 10 N-(meth) acryloyl-L-lysine, and N- (meth) acryloyl-L-proline or is a methyl ester of one of those acids. These copolymers have free carboxy or methoxy— carbonyl groups deriving from the third monomer. Our 15 European Patent No. 81408 describes further polyacrylic resins in which these groups cure amidified with ethylene diamine. It also describes the use of these further polyacrylic resins in solid phase peptide synthesis, but only in conjunction with the expensive solvents conventionally 20 used with polystyrene resins, as discussed hereinabove. n ^—' The invention provides a method for solid phase peptide synthesis, the method comprising attaching a first amino acid residue to a polyacrylic resin, coupling one or more further amino acid residues to form the desired ^ 25 peptide and detaching the peptide from the resin, characterised in that the coupling protocol includes steps of washing the resin in water and/or aqueous solution(s). The method of the invention takes advantage of the hydrophilic properties possessed by polyacrylic resins 30 unlike polystyrene resins. The polyacrylic resins for use in the method of the invention are preferably those described in our European Patents Nos. 0 079 842 and 0 081 408 as above discussed. 2307 12 4 The first amino acid residue may be fixed on the matrix through the glycolamide moiety (B.Calas and Al., Tetrahedron, 1985, 41, 5331). Previous attempts with other labile binders such as wherein X = Br, CI or OH proved unsatisfactory. The resin may then be washed in water. The next amino acid of the peptide sequence to be built may be added according to the following protocol (applicable when the protecting group is Boc) : 1.- Washing : distilled water - 2 to 4 times, 2 mn each ; 2.- Deprotection : HC1 (6N) in water - once, 2 mn and once again, 30 minutes ; 3.- Washing : distilled water - 4 to 6 times, 2 mn each ; 4.- Neutralization : 1 equivalent of borate buffer 12.5 mM pH 8.5-9.0 - once, 1 to 2 mn and once again, 1 to 2 mn ; 2307 12 - 5 - 5.- Washing : distilled water - 4 to 6 times, 1 to 2 mn each ; 6.- Washing : DMF - twice, 1 to 2 mn each ; 7.- Coupling : symmetrical anhydride (2 equivalents, twice in DMF) 8.- Washing : DMF or NMP - twice, 2 mn each ; and 9.- Washing : distilled water - 4 times, 2 mn each. The progress of the coupling reaction may be controlled by ninhydrine or fluorescamine. Alternatively, when the protecting group is Fmoc, the elongation protocol may be as follows : 1.- Washing : distilled water - 4 to 6 times, 2 mn each ; 2.- Deprotection : piperidine or diethylamine in water ; 3.- Washing : isopropanol, twice, 2 mn each ; distilled water - 4 to 6 times, 2 mn each ; 4.- Optionally washing : DMF - once, 2 mn ; 5.- Coupling : symmetric anhydride (3 times in excess in DMF) and 6.- Washing : DMF (twice, 2 mn each) ; distilled water - 6 times, 2 mn each. When the synthesis is complete, the peptide is © 2307 12 - 6 - separated from the matrix by a selective breaking of the glycolamide bond obtained by one of the following treatments : NaOH in isopropanol, NH3 in trifluoroethanol or methanol or ethanol or isopropanol, N-H. in DMF and 2 4 CH^OH in triethylamine. With this method, the reference peptide of Dorman 10 (Leu Ala Gly Val) and LHRH analogs were obtained with yields of approx. over 50 %. The invention will be better understood from the description of the following examples : EXAMPLE 1 15 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 copolymerising 1-acryloyl-pyrrolidine, N,N'-diacryloyl-l, 2-diaminoethane and methyl 2-aery 1 amido-acetate as described in example 19 of EP 0 079 842, were treated as follows : 1/ washes with DCM (4 times, 2 mn each) 2/ neutralization with 5 % diisopropylethylamine in DCM (2 times, 2 mn each) 3/ washes with DCM (4 times, 2 mn each). 4.29 g (0.0165 mol) of bromoacetic anhydride in 50 ml of DCM were added to the resin. After 45 mn of shaking, the DCM solution was removed by filtration and the brominated support was washed as follows : 5 O 20 25 - 7 - 2307 12 1/ DCM (4 tines, 2 mn each) 2/ DMF (4 times, 2 mn each) 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 for two days at ambient temperature. At this time, DMF was drained and the polymer washed with : 1/ DMF (10 times, 2 mn each) 2/ Methanol (4 times, 2 mn each) 3/ DCM (4 times, 2 mn each) 4/ Diethyl ether (4 times, 2 mn each) The resin was dried under high vacuum in presence of KOH pellets for 12 hours. The amount of Gly linked was 0.483 mmol/g, determined by amino acid analysis after hydrolysis in 6N HC1 in evacuated and sealed tubes at 110*C for 24 hours. BocGly-Resin (4.47 g) was washed with water (4 times, 2 mn each) and the Boc group was cleaved using 6N HCl in water (2 times, once 2 mn and once again 30 mn). HC1 was removed by filtration and the resin washed with water (6 times, 2 mn each). The neutralization was performed using borate buffer (12.5 mmol, pH 9), the resin being treated twice with 50 ml of buffer (1 mn each). After washing with water (6 times, 2 mn each) and with DMF (2 times, 2 mn each) symmetrical anhydride of BocProOH in DMF (50 ml) was added to the resin. The solution of symmetrical anhydride was prepared as follows : BocProOH (3.55 g, 0.0165 mol) was dissolved in 40 ml of DMF, the solution was cooled at 0*C and dicyclohexylcarbodiimide (1.69 g, 8.25 mmol) in 10 ml of 2307 1 2 - 8 - DCM was added. After stirring at O'C for 30 mn and filtration, the solution was evaporated under high vacuum without heating and the residue dissolved in DMF and added to the resin. The mixture was shaken for 30 mn, at this time the qualitative ninhydrin test of Kaiser et al. (Anal.Biochem. 1970, 34, 575) was negative indicating a coupling yield higher than 99.6 %. The DMF was then removed and the support was washed twice with DMF (2 mn each) and with water (4 times, 2 mn each). This protocol was used to incorporate the other amino acids of the DTrp6-LHRH sequence. The symmetrical anhydrides were prepared using : BocArg(Mts)OH (7.52 g, 0.0165 mol), BocLeuOH (4.11 g, 0.0165 mol), BocDTrpOH (5.02 g, 0.0165 mol), BocTyr (2.6 dichlorobenzyl)OH 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), BocHis (Dinitrophenyl)OH or BocHis (Dnp)OH (6.94 g, 0.0165 mol), pyro-GluOH (2.13 g, 0.0165 mol). After the incorporation of pyro-GluOH, the resin was washed with methanol (4 times, 2 mn each), with diethylether (4 times, 2 mn each) and dried in high vacuum at ambient temperature for 48 hours. The peptide-resin was then treated with thiophenol (10 ml) in DMF (50 ml) to remove the dinitrophenyl group on the histidine side-chain. After 45 mn of shaking, the thiophenol solution was drained and the resin washed with DMF (4 times, 2 mn each), DCM (4 times, 2 mn each) and diethylether (4 times, 2 mn each). The resin was dried under high vacuum for 12 hours. It was treated twice (30 mn each) at O'C with 50 ml of the following precooled solution : trifluoromethan-sulfonic acid (3.6 ml), anisole (4 ml), thioanisole (4 ml), 2307 121 o - 9 - metacresol (4 ml) and trifluoroacetic acid (40 ml). After the end of deprotection, the resin was washed with DCM (2 times, 2 mn each), DCM/DMF (50-50) (2 times, 2 mn each), diisopropylethylamine 5 % in DCM (2 times, 1 mn each), DMF 5 (3 times, 2 mn each), isopropanol-water (70-30) (3 times, 2 mn each). Peptide-resin was then suspended in a NH^ saturated trifluoroethemolic solution (250 ml). The mixture was shaken at ambient temperature for 15 hours, the trifluoroethanolic solution containing 10 deprotected DTrp6-LHRH was collected and the support was washed with water (4 times, 2 mn each), methanol (4 times, 2 mn each) and water (6 times, 2 mn each). The filtrates were pooled, the pH was brought to about 4 with IN hydrochloric acid ; they were concentrated under vacuum 15 without heating. The residue was fractionated on a column of carboxymethylcellulose (Wathman CM 52, 10 X 2 cm) with a linear gradient of NaCl (10 mM AcONa pH 5.0 to 10 mM AcONa, 0.15M NaCl pH 5.0). Appropriate fractions were pooled, lyophilized and desalted by gel filtration on a 20 column (100 X 2.5 cm) of Sephadex G10 in 10 M HC1. The G peptide fraction was then purified by HPLC on a column (270 X 20 mm) of Lichrosorb RP18 (10 fan) using trifluoroacetic acid (TFA) 0.01 % in water and acetonitrile as eluents. O 25 Yield : 51 % (based on the starting amino groups of the support). 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 (1), Gly 1.07 (1). 30 For some amino acids which are less stable in acidic conditions, analytical values may be lower than the expected ones due to degradation of the same. 2307 12 - 10 - The same method 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 - Laminine : Tyr lie Gly Ser ArgNH2 yield = 35.6 % Ser = 0.75, Gly = 1.03, lie = 0.99, Tyr = 1, Arg = 0.99. - CDC 28 kinose protein from the cellular 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 = l, Pro = l. - 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, lie = 1.1, Leu = l.l. - 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 = l. EXAMPLE 2 Synthesis of Leu-Ala-Gly-Val 1 g of the polyacrylic resin used in example 1 was treated as follows : 1/ washes with DCM (4 times, 2 mn each) 2/ neutralization with 5 % diisopropylethylamine in DCM (2 times, 2 mn each) 23 07 1 2 - n - 3/ washes with DCM (4 times, 2 mn each) 0.858 g (3.3 mmol) of bromoacetic anhydride in 10 ml of DCM was added to the resin. After 45 mn of shaking, the DCM solution was removed by filtration and the brominated support was washed as follows : 1/ DCM (4 times, 2 mn each) 2/ DMF (4 times, 2 mn each) 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 three days. At this time, the DMF was drained and the polymer was washed with : 1/ DMF (10 times, 2 mn each) 2/ Methanol (4 times, 2 mn each) 3/ DCM (4 times, 2 mn each) 4/ Diethylether (4 times, 2 mn each) The resin was dried under high vacuum, in presence of KOH pellets for 12 hours. The amount of Val linked was 0.492 mmol/g, determined by amino acid analysis, after hydrolysis in 6N HC1 in evacuated and sealed tubes for 24 hours. FmocVal-Resin (1-1 g) was washed with water (4 times, 2 mn each) and the Fmoc group was cleaved using 10 % piperidine or diethylamine in water (2 times, 2 mn each) . The resin was then washed with isopropanol (2 times, 2 mn each) and with water (4 times, 2 mn each). Symmetrical anhydride of FmocGlyOH in DMF (15 ml) was added to the support. The solution of symmetrical 2307 12 - 12 - anhydride was prepared as follows : FmocGlyOH (0.981 g, 3.3 mmol) was dissolved in DCM (15 ml), the solution was cooled to O'C and dicyclohexyl-carbodiimide (0.339 g, 1.65 mmol) in DCM (10 ml). The cloudy mixture was stirred for 20 mn at O'C,- the precipitate of dicyclohexylurea 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 mn, at this time the qualitative ninhydrin test of 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 mn each) and then with water (6 times, 2 mn each). This protocol was used to incorporate the following amino acids : Leu and Ala. The symmetrical anhydride were prepared starting from 1.16 g (3.3 mmol) of FmocLeuOH and 1.02 g (3.3 mmol) of FmocAlaOH. After the completion of the synthesis the peptide-resin adduct was washed with isopropanol (4 times, 2 mn each), water (4 times, 2 mn each) and isopropanol-water (70-30) (4 times, 2 mn each). Peptide-resin was then suspended in isopropanol-water (70-30) and 1.1 ml of IN NaOH were 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 suppport washed with water (4 times, 2 mn each), methanol (4 times, 2 mn each) and water (4 times, 2 mn each) . The filtrates were pooled, the pH was brought to 4 with IN HC1, the solution was concentrated under vacuum at room temperature. The residue was purified by HPLC on a column (250 X 20 mm) of Lichrosorb RP 18 (10 A*m) using TFA 0.1 % in water and acetonitrile as eluants. </div>

Claims

c 2307 12 - 13 - o Yield : 54 % (based on the starting amino groups of the support). Amino acid analysis : Leu 1.02 (1), Ala 0.99 (1), Gly 1.1 (1), Val 1.0 (1). 5 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. 10 Laminine : Tyr lie Gly Ser ArgNH^ yield = 46.3 % Ser = 0.79, Gly = 1.01, He = 0.96, Tyr = 0.89, Arg = 0.93 . 15 CDC 28 kinase protein from the cellular 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. 20 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, lie = 1.03, Leu =1. 25 - 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. - 14 - 2307 12 fflHflT VIE CLAIM IS:

1. A method for solid phase peptide synthesis, the method comprising attaching a first amino acid residue to a polyacrylic resin, coupling one or more further amino acid residues to form the desired peptide and detaching the peptide from the resin, characterised in that the coupling protocol includes steps of washing the resin in water and/or aqueous solution(s).

2. A method according to claim 1 wherein the polyacrylic resin is a copolymer of three monomers as follows : (i) a first monomer which provides a matrix for the copolymer and is one of 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 and N,N-diethyl-(meth)acrylamide (ii) a second monomer which crosslinks the copolymer and is one of N, N' -di (meth) acryloyl-diaminomethane and N, N' -di (meth) acryloyl-1,2-diaminoethane, and (iii) a third monomer which activates the copolymer and is one of the following acids 2-(meth)acrylamido-acetic acid, 3-(meth)aerylamido-propionic acid, 4-(meth)acrylamido-butyric acid, 6-(meth)acrylamido-hexanoic acid, 2307 12 - 15 - 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 is a methyl ester of one of those acids.

3. A method according to claim 1 wherein the polyacrylic resin is a copolymer according to claim 2 which has been amidified with ethylene diamine.

4. A method according to any preceding claim in which the N-protecting group used in the amino acid coupling is Boc and the coupling protocol is : 1.- Washing : distilled water - 2 to 4 times, 2 mn each ; 2.- Deprotection : HCl (6N) in water - once, 2 mn and once again, 30 minutes ; 3.- Washing : distilled water - 4 to 6 times, 2 mn each ; 4.- Neutralization : 1 equivalent of borate buffer 12.5 mM pH 8.5-9.0 - once, 1 to 2 mn and once again, 1 to 2 mn ; 5.- Washing : distilled water - 4 to 6 times, 1 to 2 mn each ; 6.- Washing : DMF - twice, 1 to 2 mn each ; 7.- Coupling : symmetrical anhydride (2 equivalents, twice in DMF) 230712 - IS - 8.- Washing : DMF or NMP - twice, 2 mn each ; and 9.- Washing : distii.lea water - 4 times, 2 mn each. one

5. A method according to anyy\pf claims 1 to 3 in which the N-protecting group used in the amino acid coupling is Fmoc and the coupling protocol is : 1.- Washing : distilled water - 4 to 6 times, 2 mn each ; 2.- Deprotection : piperidine or diethylamine in water ; 3.- Washing : isopropanol, twice, 2 mn each ; distilled water - 4 to 6 times, 2 mn each ; 4.- Optionally washing : DMF - once, 2 mn ; 5.- Coupling : symmetric anhydride (3 times in excess in DMF) and 6.- Washing : DMF (twice, 2 mn each) ; distilled water - 6 times, 2 mn each. 2307 12

6. A method for solid phase peptide synthesis substantially as hereinbefore described with reference to either of the Examples. SOCIETE D7EXPANSION SCIENTIFIQUE EXPANSIA by their authorized agents P.L. BERRY & ASSOCIATES