AU622705B2 - Solid phase peptide synthesis - Google Patents

Description

4V 75016 PARIS COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Form Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Frelated Art: 901aeof Applicant: Address of Applicant: 00 6 SOCIETE, D'EXPANSION SCIENTIFIQUE EXPANSIA 42 rue du Docteur Blanche, 75016 Paris, France Actual Inventor: BERNAFD CALAS, MICHEL FOLLET, JEAN MERY and HANITRA. NAHARISOA *A.,ddress for Service: FW Matermark Patent Trademark Attornrys QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: SOLID PHASE PEPTIDE SYNTHESIS The following statement is a full description of this invention, including the best method of performing it known to Us rN i i -i ~I 1 The present peptide synthesis.

invention relates to solid phase g e.

0 S 0 0 so 0 0 C Se 0 4, eo.

00 0 41 *0SS 00 .5 0 ee C. S 0 eg The abbreviations used in this specification are in accordance with the 1983 Recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, as set out in Eur.J. Biochem, 138, 9-37 (1984). In addition, the following are used

TFA

DCM

10 DMF

NMP

DMAc trifluoroacetic acid dichloromethane dimethylformamide N-methylpyrrolidine dimethylacetamide

V

<Iw Amino acids and their residues are of L-configuration unless otherwise specified, e.g. Ala 15 L-alanine, DAla 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 Deprotection of the Boc group Washings Neutralization of NH 2 in a position Washings Couplings and Washings.

Deprotection and neutralization steps are achieved Li__ 1 4.

I

L 10 Th r ~nn-~r 1 rr 9* r 9 9 2 by treating the resin-peptide with TFA and diisopropylethylamine solutions in DCM. This same solvent is also used for intermediate washings. Whatever the coupling agent used (symmetrical anhydride, dicyclohexylcarbodiimide, 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 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.

Our European Patent No. 0 079 842 describes polyacrylic resins which are copolymers of three monomers as follows a first monomer which provides a matrix for the copolymer and is one of l-(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-l,2-diaminoethane, and (iii) a third monomer which activates the copolymer and is one of the following acids 2-(meth)acrylamido-acetic acid, i: 1 1 1 1

C~-

u~ -qi~ i I 3 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, and N-(meth)acryloyl-L-proline or is a methyl ester of one of those acids.

These copolymers have free carboxy or methoxycarbonyl groups deriving from the third monomer. Our 15 European Patent No. 81408 describes further polyacrylic resins in which these groups are amidified with ethylene Sdiamine. It also describes the use of these further polyacrylic resins in solid phase peptide synthesis, but only in conjunction with the expensive solvents conventionally used with polystyrene resins, as discussed hereinabove.

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 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 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.

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 X-CH2 CO2H X-CH 2 CH2-CO 2

H

X-CH

2 -2 O- -CH -CO2H X-C2 2 OS wherein X Br, C1 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)

S.

10 Washing distilled water 2 to 4 times, 2 mn each Deprotection HC1 (6N) in water once, S* 2 mn and once again, 30 minutes Washing distilled water 4 to 6 times, 2 ran each 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 7'I 4' 5 S 10 4

A

Washing distilled water 4 to 6 times, 1 to 2 mn each Washing DMF twice, 1 to 2 mn each Coupling symmetrical anhydride (2 equivalents, twice in DMF) Washing DMF or NMP twice, 2 mn each and 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 Washing distilled water 4 to 6 times, 2 mn each Deprotection piperidine or diethylamine in water Washing isopropanol, twice, 2 mn each distilled water 4 to 6 times, 2 mn each Optionally washing DMF once, 2 mn Coupling symmetric anhydride (3 times in excess in DMF) and Washing DMF (twice, 2 mn each) distilled water 6 times, 2 mn each.

When the synthesis is complete, the peptide is I I

S

I

A SI 6 separated from the matrix by a selective breaking of the glycolamide bond obtained by one of the following treatments NaOH in isopropanol, NH in trifluoroethanol or methanol or ethanol or isopropanol, N2H 4 in DMF and

CH

3 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 I EXAMPLE 1 6 Synthesis of DTrp -LHRH pyro-Glu-His-Trp-Ser-Tyr-DTrp- .o Leu-Arg-Pro-Gly.

5 g of a polyacrylic resin (0.55 mmol NH 2 prepared by copolymerising 1-acryloyl-pyrrolidine, N,N'o" diacryloyl-l,2-diaminoethane and methyl 2-acrylamidoacetate 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 SDCM (2 times, 2 mn each) 3/ washes with DCM (4 times, 2 mn each) 4.29 g (0.0165 mol) of bromoacetic anhydride in 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 i i 7 1/ DCM (4 times, 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) 10 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 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.

o* BocGly-Resin (4.47 g) was washed with water (4 times, 2 mn each) and the Boc group was cleaved using 6N HCI 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 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 OC and dicyclohexylcarbodiimide (1.69 g, 8.25 mmol) in 10 ml of

L

8 DCM was added. After stirring at 0°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).

10 This protocol was used to incorporate the other amino acids of the DTrp -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.

J The peptide-resin was then treated with thiophenol f (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 0°C with 50 ml of the following precooled solution :triflucromethansulfonic acid (3.6 ml), anisole (4 ml), thioanisole (4 ml), 15 S 25 *f4 2 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 (3 times, 2 mn each), isopropanol-water (70-30) (3 times, 2 mn each). Peptide-resin was then suspended in a NH 3 saturated trifluoroethanolic solution (250 ml).

The mixture was shaken at ambient temperature for hours, the trifluoroethanolic solution containing deprotected DTrp -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 without heating. The residue was fractionated on a column of carboxymethylcellulose (Wathman CM 52, 10 X 2 cm) with a linear gradient of NaCI (10 mM AcONa pH 5.0 to 10 mM AcONa, 0.15M NaCl pH Appropriate fractions were pooled, lyophilized and desalted by gel filtration on a column (100 X 2.5 cm) of Sephadex G10 in 10 M HC1. The peptide fraction was then purified by HPLC on a column (270 X 20 mm) of Lichrosorb RP18 (10 pm) using trifluoroacetic acid (TFA) 0.01 in water and acetonitrile as eluents.

Yield 51 (based on the starting amino groups of the support).

Amino acid analysis Glu 0.99 Leu 1.0 His 1.0 Trp 1.89 Ser 0.96 Tyr 0.97 Pro 0.99 Gly 1.07 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.

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 Ile Gly Ser ArgNH 2 yield 35.6% Ser 0.75, Gly =1.03, Ile =0.99, Tyr Arg =0.99.

-CDC 28 kinose protein from the cellular cycle of tiPombeei yeast yield =44. 1 (crude peptide) Lys Ala Leu Asp Leu Arg Pro GlyOH two Asp 1, Gly =1.08, Ala 1, Leu 1.05 x 2, Tyr =0.99, Arg= 0.99, Lys 1, Pro =1.

Li Tyrosine phosphatase yield =58.6 (crude peptide) Cys Ser Asp Ser Glii Lys Leu Asn Leu Asp Ser IleCH Asp =0.95 x 3, Ser 0.58 x 3, Glu 0.67, Ile =1.1, 0 0 00 Leu 1.1.

@0 -Oncogene Phe Arg Gly Thr Leu Arg yield 53.4% Phe 0. 97, Arg 2 x 1. 1, Gly 02, Thr 99, Leu 1.

0 00 7 EXAMPLE2 Synthesis of Leu-Ala-Gly-val 1 g of the polyacrylic resin used in example 1 was treated as follows 1/ washes with DOM (4 times, 2 mn each) 2/ neutralization with 5 diisopropylethylamine in DCM (2 times, 2 mn each) I ii 11 3/ washes with DCM (4 times, 2 mn each) 0.858 q (3.3 mmol) of bromoacetic anhydride in ml of DCM was added to the resin. After 45 mn of shaking, the DCM solution wias 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 10 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

S

hours.

JS

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 12 anhydride was prepared as follows

I

ii:

I

t i FmocGlyOH (0.981 g, 3.3 mmol) was dissolved in DCM ml), the solution was cooled to 0°C and dicyclohexylcarbodiimide (0.339 g, 1.65 mmol) in DCM (10 ml). The cloudy mixture was stirred for 20 mn at 0°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 10 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 15 each).

46** 0

S@S

eOS

S.

*5 *5 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).

'IS..

U S *5 0 S S

S.

*5 IY 05 Peptide-resin was then suspended in isopropanolwater (70-30) and 1.1 ml of 1N NaOH were added. The 25 mixture was shake- 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 lN 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 pm) using TFA 0.1 in water and acetonitrile as eluants.

i

I

13 Yield :54 (based on the starting amino groups of the support).

mino acid analysis :Leu 1. 02 Ala 0. 99 Gly 1. 1 Val 1. 0 The same method was used for the synthe'sis of thcfollowing peptides: Dorman peptide Leu Ala Gly ValOH yield 46.6 Gly 0.93, Ala 0.91, Val 1.01, Leu =1.

10 Laminine :Tyr Ile Gly Ser ArgNH 2 yield 46.3 Ser= 0. 79, Gly 01, Ile 96, Tyr 89, Arg =0.93.

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, too*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)

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 pol" water and/or aqueous solution(s)

2. A method according to claim 1 wherein the polyacrylic resin is a copolymer of three monomers as 10 follows a first monomer which provides a matrix for the copolymer and is one of 1-(meth)acryloyl-pyrrolidine, 2. 1 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 20 (ii) a second monomer which crosslinks the copolymer and is one of N,N'-di(meth)acryloyl-diaminomethane and N,N'-di(meth)acryloyl-l,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)acrylamido-propionic acid, 4-(meth)acrylamido-butyric acid,

6-(meth)acrylamido-hexanoic acid, S 0O 0 a 0 OS 0 I S .0 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 poly- acrylic 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 Washing distilled water 2 to 4 times, 2 mn each Deprotection HC1 (6N) in water once, 2 mn and once again, 30 minutes Washing distilled water 4 to 6 times, 2 mn each 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 Washing distilled water 4 to 6 times, 1 to 2 mn each Washing DMF twice, 1 to 2 mn each Coupling symmetrical anhydride (2 equivalents, twice in DMF) i I IL 16 8. Washing DMF or NMP twice, 2 mn each; and 9. Washing distilled water 4 times, 2 mn each. A method according to any of 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 timed, 2 mn each; 2. Deprotection :piperidine or diethylkamine in water; 3. Washing :isopropanol, twice, 2 mn each; distilled water 4 to 6 times, i 2 mneach; 4. Optionally washing DNF 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, 2mn each. 6. A method for solid phase peptide synthesis substantially as described herein with reference to Example 1 or 2. DATED this 28th day of January 1992. SOCIETE D'EXPANSION SCIENTIFIQUE EXPANSIA WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRAUA