IE62008B1 - "Solid phase peptide synthesis" - Google Patents
"Solid phase peptide synthesis"Info
- Publication number
- IE62008B1 IE62008B1 IE303789A IE303789A IE62008B1 IE 62008 B1 IE62008 B1 IE 62008B1 IE 303789 A IE303789 A IE 303789A IE 303789 A IE303789 A IE 303789A IE 62008 B1 IE62008 B1 IE 62008B1
- Authority
- IE
- Ireland
- Prior art keywords
- meth
- acryloyl
- washing
- dmf
- coupling
- Prior art date
Links
- 238000010647 peptide synthesis reaction Methods 0.000 title claims abstract description 7
- 239000007790 solid phase Substances 0.000 title claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000011347 resin Substances 0.000 claims abstract description 34
- 229920005989 resin Polymers 0.000 claims abstract description 34
- 238000005406 washing Methods 0.000 claims abstract description 28
- 238000005859 coupling reaction Methods 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 17
- 230000008878 coupling Effects 0.000 claims abstract description 15
- 238000010168 coupling process Methods 0.000 claims abstract description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 21
- 239000012153 distilled water Substances 0.000 claims description 14
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 12
- 229940024606 amino acid Drugs 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 11
- 150000008064 anhydrides Chemical class 0.000 claims description 11
- 229920001577 copolymer Polymers 0.000 claims description 10
- 239000000178 monomer Substances 0.000 claims description 9
- 238000010511 deprotection reaction Methods 0.000 claims description 7
- 239000004925 Acrylic resin Substances 0.000 claims description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- 238000006386 neutralization reaction Methods 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 125000000539 amino acid group Chemical group 0.000 claims description 5
- 150000007513 acids Chemical class 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 4
- 229960004441 tyrosine Drugs 0.000 claims description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 3
- XLPJNCYCZORXHG-UHFFFAOYSA-N 1-morpholin-4-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCOCC1 XLPJNCYCZORXHG-UHFFFAOYSA-N 0.000 claims description 2
- AIHDNLMBKLUVCV-UHFFFAOYSA-N 2-(prop-2-enoylamino)hexanoic acid Chemical compound CCCCC(C(O)=O)NC(=O)C=C AIHDNLMBKLUVCV-UHFFFAOYSA-N 0.000 claims description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 2
- 239000004395 L-leucine Substances 0.000 claims description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Substances CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 2
- 229960003767 alanine Drugs 0.000 claims description 2
- 229960003136 leucine Drugs 0.000 claims description 2
- 229960004452 methionine Drugs 0.000 claims description 2
- RWRDLPDLKQPQOW-UHFFFAOYSA-N tetrahydropyrrole Substances C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 claims description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims 3
- RESPXSHDJQUNTN-UHFFFAOYSA-N 1-piperidin-1-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCCCC1 RESPXSHDJQUNTN-UHFFFAOYSA-N 0.000 claims 1
- SQOBXMJXKGLWNQ-UHFFFAOYSA-N 4,5-diaminopent-1-en-3-one Chemical compound NCC(N)C(=O)C=C SQOBXMJXKGLWNQ-UHFFFAOYSA-N 0.000 claims 1
- 125000004492 methyl ester group Chemical group 0.000 claims 1
- 229960002429 proline Drugs 0.000 claims 1
- 229960004295 valine Drugs 0.000 claims 1
- 239000007864 aqueous solution Substances 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000000243 solution Substances 0.000 description 17
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 11
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 9
- XTUSEBKMEQERQV-UHFFFAOYSA-N propan-2-ol;hydrate Chemical compound O.CC(C)O XTUSEBKMEQERQV-UHFFFAOYSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 229960004132 diethyl ether Drugs 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- VWWKKDNCCLAGRM-GVXVVHGQSA-N (2s)-2-[[2-[[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]propanoyl]amino]acetyl]amino]-3-methylbutanoic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O VWWKKDNCCLAGRM-GVXVVHGQSA-N 0.000 description 3
- -1 Boc group Chemical group 0.000 description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 108010009932 leucyl-alanyl-glycyl-valine Proteins 0.000 description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- FUKOTTQGWQVMQB-UHFFFAOYSA-N (2-bromoacetyl) 2-bromoacetate Chemical compound BrCC(=O)OC(=O)CBr FUKOTTQGWQVMQB-UHFFFAOYSA-N 0.000 description 2
- ZQEBQGAAWMOMAI-ZETCQYMHSA-N (2s)-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)(C)OC(=O)N1CCC[C@H]1C(O)=O ZQEBQGAAWMOMAI-ZETCQYMHSA-N 0.000 description 2
- CNBUSIJNWNXLQQ-NSHDSACASA-N (2s)-3-(4-hydroxyphenyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CNBUSIJNWNXLQQ-NSHDSACASA-N 0.000 description 2
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 2
- MXNRLFUSFKVQSK-UHFFFAOYSA-N 2-Amino-6-(trimethylazaniumyl)hexanoate Chemical compound C[N+](C)(C)CCCCC(N)C([O-])=O MXNRLFUSFKVQSK-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 2
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 2
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 2
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 2
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 2
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- CGOMLCQJEMWMCE-STQMWFEESA-N Phe-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CGOMLCQJEMWMCE-STQMWFEESA-N 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 2
- JAGGEZACYAAMIL-CQDKDKBSSA-N Tyr-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC1=CC=C(C=C1)O)N JAGGEZACYAAMIL-CQDKDKBSSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical class [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920005990 polystyrene resin Polymers 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- UTDSSTFBUGDVAI-LURJTMIESA-N (2s)-1-prop-2-enoylpyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)C=C UTDSSTFBUGDVAI-LURJTMIESA-N 0.000 description 1
- PPRBGMXQDAMDAB-BYPYZUCNSA-N (2s)-2-(prop-2-enoylamino)propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)C=C PPRBGMXQDAMDAB-BYPYZUCNSA-N 0.000 description 1
- DMBKPDOAQVGTST-LBPRGKRZSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylmethoxypropanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)COCC1=CC=CC=C1 DMBKPDOAQVGTST-LBPRGKRZSA-N 0.000 description 1
- AYMLQYFMYHISQO-QMMMGPOBSA-N (2s)-3-(1h-imidazol-3-ium-5-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CN=CN1 AYMLQYFMYHISQO-QMMMGPOBSA-N 0.000 description 1
- UHWCAAMSQLMQKV-ZETCQYMHSA-N (2s)-3-methyl-2-(prop-2-enoylamino)butanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)C=C UHWCAAMSQLMQKV-ZETCQYMHSA-N 0.000 description 1
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 1
- WLPAQAXAZQUXBG-UHFFFAOYSA-N 1-pyrrolidin-1-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCCC1 WLPAQAXAZQUXBG-UHFFFAOYSA-N 0.000 description 1
- 125000003287 1H-imidazol-4-ylmethyl group Chemical group [H]N1C([H])=NC(C([H])([H])[*])=C1[H] 0.000 description 1
- VRPJIFMKZZEXLR-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(O)=O VRPJIFMKZZEXLR-UHFFFAOYSA-N 0.000 description 1
- TZGPACAKMCUCKX-UHFFFAOYSA-N 2-hydroxyacetamide Chemical group NC(=O)CO TZGPACAKMCUCKX-UHFFFAOYSA-N 0.000 description 1
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 1
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- 239000003480 eluent Substances 0.000 description 1
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- 238000002523 gelfiltration Methods 0.000 description 1
- XLXSAKCOAKORKW-UHFFFAOYSA-N gonadorelin Chemical class C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CCCN=C(N)N)NC(=O)C(CC(C)C)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 XLXSAKCOAKORKW-UHFFFAOYSA-N 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
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- 238000004519 manufacturing process Methods 0.000 description 1
- 229940100630 metacresol Drugs 0.000 description 1
- 125000005395 methacrylic acid group Chemical group 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 1
- GPXGYCOUOXWPOW-UHFFFAOYSA-N methyl 2-(prop-2-enoylamino)acetate Chemical compound COC(=O)CNC(=O)C=C GPXGYCOUOXWPOW-UHFFFAOYSA-N 0.000 description 1
- 150000004702 methyl esters Chemical group 0.000 description 1
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- 238000003756 stirring Methods 0.000 description 1
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- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Polymerization Catalysts (AREA)
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
Amino acids and their residues are of L-configuration unless otherwise specified, e.g. Ala = 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: -21. - Deprotection of the Boc group; 2. - Washings; 3. - Neutralization of NI^ in a position; 4. - Washings; . Couplings; and 6. - Washings.
Deprotection and neutralization steps are achieved 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 quanties 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 significantly to lower the manufacturing cost of peptides.
Our European Patent No. 79842 describes polyacrylic resins which are copolymers 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-methy1-piperazine, 4-(meth)acryloyl-morpholine, -3N,N-dimethyl-(meth)aerylamide and Ν,Ν-diethyl-(meth)aerylamide (ii) a second monomer which crosslinks the copolymer and is one of N,N’-di(meth)acryloyl-diaminomethane and N,N'-di(raeth)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, N-(meth)acryloyl-L-alanine, N-(meth)acryloyl-L-valine, N-(meth)acryloyl-L-leucine, N-(meth) aeryloy1-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 European Patent No. 81408 describes further polyacrylic resins in which these groups are 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 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 -4desired 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 79842 and 81408 as above discussed.
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-CI12 wherein X = Br, Cl or OH proved unsatisfactory. -5The 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, mn each; 2. - Deprotection : HCl (6N) in water - once, mn and once again, 30 minutes; 3. - Washing : distilled water - 4 to 6 times, mn each; 4. - Neutraliation : 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, 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 I 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: -61. - Washing : distilled water - 4 to 6 times, 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; . - Coupling : symmetric anhydride (3 times in excess in DMF) and 6. - Washing : DMF (twice, 2 ran each); distilled water - 6 times, 2 mn each.
When the synthesis is complete, the peptide is separated from the matrix by a selective breaking of the glycolamide bond obtained by one of the following treatments : - NaOH in isopropanol, - NH^ in trifluroethanol or methanol or ethanol or isopropanol, - in DMF and - CHgOH in triethylamine.
With this method, the reference peptide of Dorman (Leu Ala Gly Val) and LHRH analogs were obtained with yields of approx, over 50 %.
The following Examples illustrate the invention: EXAMPLE 1 Synthesis of DTrp^-LHRH : pyro-Glu-His-Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-Gly. -75 g of a polyacrylic resin (0.55 mmol NH^/g), prepared by copolymerising 1-acryloyl-pyrrolidine, N,Ν'-diacryloyl- 1, 2-diaminoethane and methyl 2-acrylamido-acetate as described in Example 19 of EP 0079842, were treated as follows 1/ washes with DCM (4 times,. 2 mn each) 2/ neutralization with 5% diisopropylethylamine in DCM (2 times, 2 ran 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: 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) 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 HCl in evacuated and sealed tubes at 110°C for 24 hours.
BocGly-Resin (4.47 g) was washed with water (4 times, mn each) and the Boc group was cleaved using 6N HCl in water (2 times, once 2 mn and once again 30 mn). -8HC1 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 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).
This protocol was used to incorporate the other amino acids of the DTrp^-LHRH sequence. The symmetrical anhydrides were prepared using: BocArg(Mts)0H (7.52 g, 0.0165 mol), BocLeuOH (4.11 g, 0.0165 mol), BocDTrpOH (5.02 g, 0.0165 mol), BocTyr (2.6 dichlorobenzyl)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), BocHis(Dinitrophenyl)0H or BocHis (Dnp)OH (6.94 g, 0.0165 mol), pyro-GluOH (2.13 g, 0.0165 mol).
After the incorporation of the pyro-GluOH, the resin was washed with methanol (4 times, 2 mn each), with diethylether (4 times, 2 mn each) and dried in high -9vacuum 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 (3θ ran each) at 0°C with 50 ml of the following precooled solution : trifluororaethansulfonic acid (3-6 ml), anisole (4 ml), thioanisole (4 ml), metacresol (4 ml) and trifluoroacetic acid (40 ml). After the end of deprotection, the resin was washed with DCM (2 times, 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^ saturated trifluoroethanolic solution (250 ml). ’ The mixture was shaken at ambient temperature for 15 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 ran 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 (Wathwan (Trade Mark) CM 52, 1u 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 column -ΙΟί 100 X 2.5 cm) of Sephadex (Trade Mark) G1C! .in 10 M HCl. The peptide fraction was then purified by HPLC on a column (270 X 20 mm) Lichrosorb (Trade Mark) RP18 (10 pm) using trif luoroacetic 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 (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).
For some amino acids which are less table 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 lie Gly Ser ArgNH2 yield = 35.6% Ser = 0.75. Gly = 1.03, He = 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 = 1, Pro = 1.
Tyrosine phosphatase yield = 58.6% (crude peptide) -11Cys Ser Asp Ser Glu Lys Leu Asn Leu Asp Ser IleOH Asp = 0.95 x 3, Ser = 0.58 x 3, Glu = 0.67, He = 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 Synthesis of Leu-Ala-Gly-Val 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% diisopropylethylaraine in DCM (2 times, 2 mn each) 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, 21» ^Ί2), 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: -121/ 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 HCI in evacuated and sealed tubes for 24 hours.
FmocVal-Resin (1.1 g) was washed with water (4 times, 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 anhydride was prepared as follows: FmocGlyOH (0.981 g, 3.3 mmol) was dissolved in DCM (15 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 shaken at room temperature . for 45 mn, at this time the qualitative ninhydrin test of Kaiser et al. (Anal. Biochm. 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). -13This 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 1N 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 support washed with water (4 times, 2 ran 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 1N HCI, 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 pm) using TFA 0.1% in water and acetonitrile as eluants.
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).
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 lie Gly Ser ArgNH2 yield = 46.3% -14Ser = 0.79, Gly = 1.01, He = 0.96, Tyr = 0.89, Arg = 0.93.
CDC 28 kinase protein from the oellular cycle of ’'Pombee’1 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, He = 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 (7)
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.
6. A method substantially as described herein with reference to either of the Examples.
7. A peptide whenever synthesized by a method as claimed in any of the preceding claims.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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GB888822502A GB8822502D0 (en) | 1988-09-24 | 1988-09-24 | New peptide synthesis method |
Publications (2)
Publication Number | Publication Date |
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IE893037L IE893037L (en) | 1990-03-24 |
IE62008B1 true IE62008B1 (en) | 1994-12-14 |
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Application Number | Title | Priority Date | Filing Date |
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IE303789A IE62008B1 (en) | 1988-09-24 | 1989-09-22 | "Solid phase peptide synthesis" |
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JP (1) | JPH0768265B2 (en) |
KR (1) | KR900004761A (en) |
AR (1) | AR244701A1 (en) |
AT (1) | AT400439B (en) |
AU (1) | AU622705B2 (en) |
BE (1) | BE1002237A3 (en) |
CA (1) | CA1333441C (en) |
CH (1) | CH679672A5 (en) |
DE (1) | DE3931731C2 (en) |
DK (1) | DK468589A (en) |
ES (1) | ES2018924A6 (en) |
FI (1) | FI101474B (en) |
FR (1) | FR2636951B1 (en) |
GB (2) | GB8822502D0 (en) |
GR (1) | GR1000564B (en) |
HK (1) | HK47792A (en) |
IE (1) | IE62008B1 (en) |
IT (1) | IT1231960B (en) |
LU (1) | LU87592A1 (en) |
MA (1) | MA21633A1 (en) |
MY (1) | MY106567A (en) |
NL (1) | NL8902361A (en) |
NO (1) | NO175593C (en) |
NZ (1) | NZ230712A (en) |
OA (1) | OA09243A (en) |
PT (1) | PT91784B (en) |
SE (1) | SE8903121L (en) |
SG (1) | SG40692G (en) |
TN (1) | TNSN89104A1 (en) |
ZA (1) | ZA897151B (en) |
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US5811241A (en) * | 1995-09-13 | 1998-09-22 | Cortech, Inc. | Method for preparing and identifying N-substitued 1,4-piperazines and N-substituted 1,4-piperazinediones |
PL205949B1 (en) | 2000-12-22 | 2010-06-30 | Ipsen Mfg Ireland Ltd | Process for the synthesis of a peptide having a tryptophan residue |
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US3951741A (en) * | 1973-07-10 | 1976-04-20 | Peter Pfaender | Process and apparatus for the synthesis of peptides by use of n-carboxyanhydrides |
US4436874A (en) * | 1981-11-19 | 1984-03-13 | Societe D'expansion Scientifique "Expansia" | Acrylic copolymers and their use in solid phase peptide synthesis |
-
1988
- 1988-09-24 GB GB888822502A patent/GB8822502D0/en active Pending
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1989
- 1989-09-18 GR GR890100587A patent/GR1000564B/en unknown
- 1989-09-19 BE BE8901003A patent/BE1002237A3/en not_active IP Right Cessation
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- 1989-09-20 AT AT0219989A patent/AT400439B/en not_active IP Right Cessation
- 1989-09-20 NZ NZ230712A patent/NZ230712A/en unknown
- 1989-09-20 AR AR89314966A patent/AR244701A1/en active
- 1989-09-21 LU LU87592A patent/LU87592A1/en unknown
- 1989-09-21 MY MYPI89001300A patent/MY106567A/en unknown
- 1989-09-21 FR FR898912394A patent/FR2636951B1/en not_active Expired - Fee Related
- 1989-09-21 NL NL8902361A patent/NL8902361A/en active Search and Examination
- 1989-09-21 ES ES8903198A patent/ES2018924A6/en not_active Expired - Lifetime
- 1989-09-21 CH CH3435/89A patent/CH679672A5/fr not_active IP Right Cessation
- 1989-09-22 CA CA000612438A patent/CA1333441C/en not_active Expired - Fee Related
- 1989-09-22 IT IT8921804A patent/IT1231960B/en active
- 1989-09-22 TN TNTNSN89104A patent/TNSN89104A1/en unknown
- 1989-09-22 PT PT91784A patent/PT91784B/en active IP Right Revival
- 1989-09-22 IE IE303789A patent/IE62008B1/en not_active IP Right Cessation
- 1989-09-22 AU AU41614/89A patent/AU622705B2/en not_active Ceased
- 1989-09-22 DE DE3931731A patent/DE3931731C2/en not_active Expired - Fee Related
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- 1989-09-22 DK DK468589A patent/DK468589A/en not_active Application Discontinuation
- 1989-09-22 FI FI894489A patent/FI101474B/en not_active IP Right Cessation
- 1989-09-22 MA MA21887A patent/MA21633A1/en unknown
- 1989-09-22 SE SE8903121A patent/SE8903121L/en not_active Application Discontinuation
- 1989-09-22 JP JP1245374A patent/JPH0768265B2/en not_active Expired - Lifetime
- 1989-09-22 OA OA59652A patent/OA09243A/en unknown
- 1989-09-23 KR KR1019890013716A patent/KR900004761A/en not_active Application Discontinuation
- 1989-09-25 GB GB8921637A patent/GB2223227B/en not_active Expired - Fee Related
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1992
- 1992-04-14 SG SG406/92A patent/SG40692G/en unknown
- 1992-07-02 HK HK477/92A patent/HK47792A/en not_active IP Right Cessation
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