IE20070687U1 - Boc and fmoc solid phase peptide synthesis - Google Patents
Boc and fmoc solid phase peptide synthesisInfo
- Publication number
- IE20070687U1 IE20070687U1 IE2007/0687A IE20070687A IE20070687U1 IE 20070687 U1 IE20070687 U1 IE 20070687U1 IE 2007/0687 A IE2007/0687 A IE 2007/0687A IE 20070687 A IE20070687 A IE 20070687A IE 20070687 U1 IE20070687 U1 IE 20070687U1
- Authority
- IE
- Ireland
- Prior art keywords
- product
- peptide
- coupled
- resin
- amino acid
- Prior art date
Links
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 title claims abstract description 39
- IRXSLJNXXZKURP-UHFFFAOYSA-N Fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 238000010647 peptide synthesis reaction Methods 0.000 title description 7
- 239000007790 solid phase Substances 0.000 title description 5
- 150000001413 amino acids Chemical class 0.000 claims abstract description 59
- 230000002194 synthesizing Effects 0.000 claims abstract description 6
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 5
- 229920005990 polystyrene resin Polymers 0.000 claims abstract description 4
- 229920005989 resin Polymers 0.000 claims description 80
- 239000011347 resin Substances 0.000 claims description 80
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 74
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 54
- 239000000243 solution Substances 0.000 claims description 23
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 22
- 239000002253 acid Substances 0.000 claims description 21
- 238000005859 coupling reaction Methods 0.000 claims description 21
- 125000006239 protecting group Chemical group 0.000 claims description 21
- 230000001808 coupling Effects 0.000 claims description 20
- 238000010168 coupling process Methods 0.000 claims description 20
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims description 16
- 125000003277 amino group Chemical group 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 239000003960 organic solvent Substances 0.000 claims description 11
- TVFDJXOCXUVLDH-UHFFFAOYSA-N Cesium Chemical class [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 claims description 10
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 9
- BDNKZNFMNDZQMI-UHFFFAOYSA-N N,N'-Diisopropylcarbodiimide Chemical group CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 239000012026 peptide coupling reagents Substances 0.000 claims description 8
- 239000003880 polar aprotic solvent Substances 0.000 claims description 7
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 6
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical class C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- FJDQFPXHSGXQBY-UHFFFAOYSA-L Caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Carbodicyclohexylimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 230000001376 precipitating Effects 0.000 claims description 2
- 125000000467 secondary amino group Chemical class [H]N([*:1])[*:2] 0.000 claims 2
- RYPWQHONZWFXBN-UHFFFAOYSA-N dichloromethyl(methylidene)-$l^{3}-chlorane Chemical compound ClC(Cl)Cl=C RYPWQHONZWFXBN-UHFFFAOYSA-N 0.000 claims 1
- 238000010532 solid phase synthesis reaction Methods 0.000 abstract description 4
- 239000000047 product Substances 0.000 description 62
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 40
- PUDHBTGHUJUUFI-PURAGXGVSA-N CC(C)[C@@H]1NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC2=CNC3=C2C=CC=C3)NC(=O)[C@H](CC2=CC=C(O)C=C2)NC(=O)[C@H](CSSC[C@H](NC1=O)C(=O)N[C@@H]([C@@H](C)O)C(N)=O)NC(=O)[C@@H](N)CC1=CC=C2C=CC=CC2=C1 Chemical compound CC(C)[C@@H]1NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC2=CNC3=C2C=CC=C3)NC(=O)[C@H](CC2=CC=C(O)C=C2)NC(=O)[C@H](CSSC[C@H](NC1=O)C(=O)N[C@@H]([C@@H](C)O)C(N)=O)NC(=O)[C@@H](N)CC1=CC=C2C=CC=CC2=C1 PUDHBTGHUJUUFI-PURAGXGVSA-N 0.000 description 15
- 229960002437 lanreotide Drugs 0.000 description 15
- 108010021336 lanreotide Proteins 0.000 description 15
- 239000000203 mixture Substances 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- -1 Fmoc amino acids Chemical class 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 9
- 230000002829 reduced Effects 0.000 description 8
- 230000000903 blocking Effects 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000002002 slurry Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- LLHOYOCAAURYRL-RITPCOANSA-N (2S,3R)-3-hydroxy-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)OC(C)(C)C LLHOYOCAAURYRL-RITPCOANSA-N 0.000 description 5
- 150000004702 methyl esters Chemical class 0.000 description 5
- 230000002633 protecting Effects 0.000 description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 125000000415 L-cysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])S[H] 0.000 description 4
- 229920001367 Merrifield resin Polymers 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N Ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 150000003335 secondary amines Chemical class 0.000 description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 3
- 238000005755 formation reaction Methods 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000012299 nitrogen atmosphere Substances 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 150000003141 primary amines Chemical class 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- RMTDKXQYAKLQKF-INIZCTEOSA-N (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-sulfanylpropanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CS)C(=O)O)C3=CC=CC=C3C2=C1 RMTDKXQYAKLQKF-INIZCTEOSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 229960004666 Glucagon Drugs 0.000 description 2
- 108060003199 Glucagon Proteins 0.000 description 2
- MASNOZXLGMXCHN-ZLPAWPGGSA-N Glucagonum Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 2
- 102000018997 Growth Hormone Human genes 0.000 description 2
- 108010051696 Growth Hormone Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- AKHNMLFCWUSKQB-UHFFFAOYSA-L Sodium thiosulphate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 2
- 229960000553 Somatostatin Drugs 0.000 description 2
- 102000005157 Somatostatin Human genes 0.000 description 2
- 108010056088 Somatostatin Proteins 0.000 description 2
- MBYLVOKEDDQJDY-UHFFFAOYSA-N Tris(2-aminoethyl)amine Chemical compound NCCN(CCN)CCN MBYLVOKEDDQJDY-UHFFFAOYSA-N 0.000 description 2
- 239000012445 acidic reagent Substances 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000000122 growth hormone Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000000269 nucleophilic Effects 0.000 description 2
- 150000007530 organic bases Chemical class 0.000 description 2
- 239000002798 polar solvent Substances 0.000 description 2
- 238000004237 preparative chromatography Methods 0.000 description 2
- 235000019345 sodium thiosulphate Nutrition 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- MGHMWKZOLAAOTD-XMMPIXPASA-N (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(1H-indol-3-yl)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@@H](C(=O)O)CC1=CNC2=CC=CC=C12 MGHMWKZOLAAOTD-XMMPIXPASA-N 0.000 description 1
- SWZCTMTWRHEBIN-QFIPXVFZSA-N (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(O)C=C1 SWZCTMTWRHEBIN-QFIPXVFZSA-N 0.000 description 1
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 description 1
- YRKFMPDOFHQWPI-IBGZPJMESA-N (2S)-6-azaniumyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCN)C(O)=O)C3=CC=CC=C3C2=C1 YRKFMPDOFHQWPI-IBGZPJMESA-N 0.000 description 1
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 1
- PXJBCMWIAPDWAU-UHFFFAOYSA-N 2-piperidin-4-ylethanamine Chemical compound NCCC1CCNCC1 PXJBCMWIAPDWAU-UHFFFAOYSA-N 0.000 description 1
- RDOXTESZEPMUJZ-UHFFFAOYSA-N Anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 1
- WXOFKRKAHJQKLT-UHFFFAOYSA-N Cysteinyl-Lysine Chemical compound NCCCCC(C(O)=O)NC(=O)C(N)CS WXOFKRKAHJQKLT-UHFFFAOYSA-N 0.000 description 1
- FXHOOIRPVKKKFG-UHFFFAOYSA-N DMA Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N Diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010073071 Hepatocellular carcinoma Diseases 0.000 description 1
- 206010062060 Hyperlipidaemia Diseases 0.000 description 1
- 208000001365 Hyperprolactinemia Diseases 0.000 description 1
- 206010022489 Insulin resistance Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 206010025650 Malignant melanoma Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010036832 Prolactinoma Diseases 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 230000003042 antagnostic Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000003247 decreasing Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 230000035877 hyperamylinemia Effects 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 230000003472 neutralizing Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005897 peptide coupling reaction Methods 0.000 description 1
- LWMPFIOTEAXAGV-UHFFFAOYSA-N piperidin-1-amine Chemical compound NN1CCCCC1 LWMPFIOTEAXAGV-UHFFFAOYSA-N 0.000 description 1
- LTEKQAPRXFBRNN-UHFFFAOYSA-N piperidin-4-ylmethanamine Chemical compound NCC1CCNCC1 LTEKQAPRXFBRNN-UHFFFAOYSA-N 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002952 polymeric resin Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 200000000008 restenosis Diseases 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000004083 survival Effects 0.000 description 1
- 201000010874 syndrome Diseases 0.000 description 1
- QQWYQAQQADNEIC-RVDMUPIBSA-N tert-butyl [(Z)-[cyano(phenyl)methylidene]amino] carbonate Chemical compound CC(C)(C)OC(=O)O\N=C(/C#N)C1=CC=CC=C1 QQWYQAQQADNEIC-RVDMUPIBSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/655—Somatostatins
- C07K14/6555—Somatostatins at least 1 amino acid in D-form
Abstract
ABSTRACT A solid phase method for synthesizing a peptide containing three or more amino acid residues utilizing both Boc and Fmoc protected amino acids and a chloromethylated polystyrene resin.
Description
Solid phase peptide synthesis was introduced in 1963 with the
intent to overcome many of the intermediate purification problems
associated with solution peptide synthesis. Stewart, et al., Solid Phase
Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984). During solid
phase synthesis, amino acids are assembled (i.e., coupled) into a peptide
of any desired sequence while one end of the chain (i.e., the C—terminus)
is anchored to an insoluble support. Once the desired sequence has been
linked together on the support, the peptide is then deblocked (i.e.,
cleaved) from the support. The two standard protecting groups for Ot-
amino functions of the coupled amino acids are Boo, which is removed
by treatment with a strong acid, and Fmoc, which is removed with a
base. The present invention relates to a convenient method of
manufacturing peptides using a combination of both of these ot-amino
protecting groups in a single synthesis on inexpensive chloromethylated
polystyrene resin.
In designing a synthesis of a peptide by the solid phase method
using either of the above mentioned on-amino protection schemes, it is
important that any reactive “side groups” of the constituent amino acids
be protected from unwanted chemical reactions throughout the chain
assembly. It is also desirable that the chemical groups chosen to protect
the various side groups be resistant to removal by the reagents used to
remove the on-amino protecting groups. Thirdly, it is important that the
linkage of the growing peptide chain to the resin particle be stable to the
reagents used to remove either type of ot-amino protecting group during
chain assembly. In the case of the Fmoc ot—amino protection scheme,
the side group protection functions should be resistant to the basic
reagents used to remove the Fmoc. In practice, these side chain
protecting groups are generally removed by mildly acidic reagents after
the peptide chain has been assembled. When using the Boc on-amino
protection scheme, the side chain protecting groups must be resistant to
removal by the mild acid reagent used to deprotect the Boc group at
every cycle. In practice, these side chain protecting groups for the Boc
ot—amino protection scheme are generally removed by anhydrous HF
after the peptide chain has been assembled. Therefore, in practice, the
side chain protecting groups commonly used with the Fmoc oL—amino
protection are not stable under the conditions used for Boc ot—amino
deprotection and the two types of on-amino protection schemes are not
mixed in the assembly of a peptide chain by solid phase peptide
synthesis. In addition, while the least expensive polymeric resin used in
peptide synthesis, chloromethylated polystyrene or “Merrifield resin”, is
widely used with Boc protected amino acids, the literature suggests it is
unsuitable for use with Fmoc protection on the 0t—amino group due to its
[ability in basic conditions. (see Stewart, et al., Solid Phase Peptide
Synthesis (Pierce Chemical Co., 2d ed., 1984). The present invention is
directed to a method for mixed usage of both Boc and Fmoc amino acids
on “Merrifield resin” during solid phase synthesis of certain peptides.
Lanreotide® is an analog of somatostatin and is known to inhibit
growth hormone release as well as inhibit insulin, glucagon and
pancreatic exocrine secretion.
U.S. Patent No. 4,853,371 discloses and claims Lanreotide®, a
method for making it and a method for inhibiting the secretion of
growth hormone, insulin, glucagon and pancreatic exocrine secretion.
U.S. Patent No. 5,147,856 discloses the use of Lanreotide® of
treating restenosis.
U.S. Patent No. 5,411,943 discloses the use of Lanreotide® for
treating hepatoma.
U.S. Patent No. 5,073,541 discloses the use of Lanreotide® for
treating lung cancer.
U.S. Application No. 08/089,410 filed July 9, 1993 discloses the
use of Lanreotide® for treating melanoma.
U.S. Patent No. 5,504,069 discloses the use of Lanreotide® for
inhibiting the accelerated growth of a solid tumor.
U.S. Application No. 08/854,941 filed May 13, 1997, discloses the
use of Lanreotide® for decreasing body weight.
US. Application No. 08/854,943 filed May 13, 1997, discloses the
use of Lanreotide® for treating insulin resistance and Syndrome X.
US. Patent No. 5,688,418 discloses the use of Lanreotide® for
prolonging the survival of pancreatic cells.
PCT Application No. PCT/US97/14154 discloses the use of
Lanreotide® for treating fibrosis.
U.S. Application No. 08/855,311 filed May 13, 1997, discloses the
use of Lanreotide® for treating hyperlipidemia.
US. Application No. 08/440,061 filed May 12, 1995, discloses the
use of Lanreotide® for treating hyperamylinemia.
U.S. Application No. 08/852,221 filed May 7, 1997, discloses the
use of Lanreotide® for treating hyperprolactinemia and prolactinomas.
The contents of the foregoing patents and patent applications are
incorporated herein by reference.
Summary Of The Invention
This invention features a method for preparing a peptide
comprising three or more amino acid residues having an N-terminal
amino acid, a next to last amino acid adjacent to the N—terininal amino
acid and a C-terminal amino acid, wherein said method comprises the
steps of:
(a) attaching a first amino acid to a solid support resin through an
ester bond to form a first-coupled-product, which comprises (i) reacting
an aqueous solution of cesium carbonate with an alcohol solution of the
first amino acid to form a cesium salt of the first amino acid, (ii)
obtaining a solvent free cesium salt of the first amino acid, (iii) reacting
the solid support resin with the cesium salt of the first amino acid in a
dry polar aprotic solvent to form a first-coupled product;
wherein the first amino acid corresponds to the C—terminal amino
acid of the peptide, the first amino acid’s non-side chain amino
group is blocked by a Boc and the first amino acid does not have a
side chain functionality requiring protection, and the solid support
resin is a chloromethylated polystyrene resin;
(b) deblocldng the Boc from the first—coupled—product with an acid
to form a first—deblocked-coupled-product;
(c) optionally coupling a next—amino—acid to the first—deblocked—
coupled—product, which comprises reacting the next-amino-acid with the
first-deblocked—coupled—product in an organic solvent comprising a
peptide coupling reagent to form a next—blocked-coupled-product,
wherein the next-amino-acid has a non-side chain amino group blocked
by Boc and if the next-amino-acid has one or more side chain
functionalities then the side chain functionalities do not require
protection or the side chain functionalities have protecting groups that
are stable to acid and base reagents used to deblock Boc and Fmoc,
respectively;
(d) deblocking the Boc from the next—blocked-coupled-product
which comprises reacting the next-blocked—coupled—product with a an
acid to yield a next-deblocked—coupled-product;
(e) optionally repeating steps (c) and ((1), each cycle forming an (X
+ 1)-next—deblocked-coupled-product where X is the number of desired
cycle repetitions;
(f) coupling a next-amino-acid to the first-deblocked—coupled-
product from (b), or optionally to the (X + 1)—next-deblocked—coup1ed-
product from (c), which comprises reacting the next-amino-acid with
said first-deblocked-coupled—product or said (X + l)-next-deblocked-
coupled-product in an organic solvent comprising a peptide coupling
reagent to form a next-blocked-coupled-product, wherein the next-
amino-acid has a non-side chain amino group blocked by Fmoc,
provided that if the next-amino-acid has one or more side chain
functionalities then the side chain functionalities do not require
protection or the side chain fiinctionalities have protecting groups that
are stable to base reagents used to deblock Fmoc;
(g) deblocking the Fmoc from the next-blocked-coupled-product
which comprises reacting the next—blocked-coupled-product with a
primary or secondary amine to yield a next-deblocked-coupled-product;
(h) optionally repeating steps (f) and (g), each cycle forming an (X
+ l)—next—deblocked—coupled—product where X is the desired number of
cycle repetitions, until the next to last amino acid of the peptide is
coupled and deblocked;
(i) coupling an N—terminal amino acid to the (X + 1)-next-
deblocked-coupled—product, which comprises reacting the N-terminal
amino acid with the (X + 1)-next—deblocked-coupled—product in an
organic solvent comprising a peptide coupling reagent to form a
completed-blocked-coupled—product, wherein the N-terminal-amino-acid
has a non-side chain amino group blocked by Boc or Fmoc;
(j) deblocking the Boo or Fmoc group from the completed-
blocked—coupled-product, which comprises reacting the completed-
blocked—coupled-product with an acid in the case of Boc, or a base in the
case Fmoc, to form a completed-peptide—resin-product;
(k) if side-chain functionalities are present on the completed-
peptide—resin-product then optionally deprotecting the side-chain
functionalities of the completed—peptide-resin—product, which comprises
reacting the completed-peptide-resin—product with the appropriate
deprotecting reagents to form a deprotected—completed-peptide-resin-
product; and
(1) cleaving the peptide from the solid support resin of the
completed—peptide-resin-product or the deprotected-completed—peptide-
resin-product to yield the peptide, which comprises reacting the
completed—peptide-resin-product or the deprotected-completed-peptide-
resin—product with ammonia, a primary amine or a secondary amine until
the cleavage of the peptide from the resin is substantially complete;
provided that steps (D and (g) must be carried out at least once in
the synthesis of the peptide.
A preferred method of this invention is where the ammonia,
primary amine or secondary amine of step (1) is in a solvent comprising
an alcohol, and optionally, an aprotic polar solvent.
A preferred method of this invention is where step (1) further
comprises the steps of:
precipitating the cleaved peptide from the solvent;
filtering the solid support resin and the precipitated peptide; and
extracting the peptide in an acid solution to isolate the peptide.
A preferred method of this invention is where the first amino acid
is Boc-L-Thr.
A preferred method of this invention is where the first amino acid
is Boc-L—Thr-cesium salt yielding Boc-L-Thr-resin as the first-coupled-
product and H-L-Thr-resin is the first—deblocked-coupled-product.
A preferred method of this invention is where the acid used to
deblock the Boo group in step (j) is TFA.
A preferred method of the immediately foregoing method is where
the organic solvent is methylene
dimethylformamide and the
chloride,
peptide
diisopropylcarbodiimide, dicyclohexylcarbodiimide, or N-ethyl-N'—(3—
chloroform, or
coupling reagent is
dimethyl—aminopropy1) carbodiimide.
A preferred method of the immediately foregoing method
comprises carrying out steps (0 and (g) six times after the formation of
the first-deblocked-coupled-product of the formula H-L-Thr—resin
wherein the following amino acids are coupled in the order of Fmoc-L-
Cys(Acm), Fmoc-L—Val, Fmoc—L—Lys(Boc), Fmoc-D-Trp, Fmoc-L-
Tyr(O-t-Bu) and Fmoc—L-Cys-(Acm) to form H—Cys(Acm)—Tyr(O-t-Bu)-
D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin.
A preferred method of the immediately foregoing method
comprises coupling Boc-D—B-Na] to H-Cys(Acm)-Tyr(O—t—Bu)-D-Trp-
Lys(Boc)-Val-Cys(Acm)—Thr-resin according to step (c) to form Boc-D-
[3-Nal-Cys(Acm)-Tyr(O-t—Bu)-D-Trp—Lys(Boc)-Val-Cys(Acm)-Thr-
resin.
A preferred method of the immediately foregoing method
comprises simultaneously deblocking the Boc group blocking D-B-Nal,
the O-t-Bu group protecting Tyr and the Boc group protecting Lys of
Boc-D-B-Nal-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)—Val-Cys(Acm)-
Thr—resin according to step (j) to yield the completed-peptide-resin-
product of the formula H-D-B-Nal—Cys(Acm)—Tyr-D—Trp-Lys-Val-
Cys(Acm)-Thr-resin.
A preferred method of the immediately foregoing method
comprises cleaving the peptide, H-D-B—Na1—Cys(Acm)-Tyr-D-Trp-Lys-
Val-Cys(Acm)-Thr, from the solid resin by reacting H-D-B-Nal-
Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-resin with ammonia in a
solvent comprising an alcohol, and optionally, an aprotic polar solvent
until the cleavage is substantially complete to yield H-D—B—Nal—
Cys(Acm)—Tyr—D—Trp-Lys-Val—Cys(Acm)-Thr-NH;.
A preferred method of the immediately foregoing method is where
and
the alcohol is methanol
the polar aprotic solvent is
dimethylformamide.
A preferred method of the immediately foregoing method
comprises simultaneously deprotecting the Acm groups protecting Cys
and cyclizing the resulting deprotected Cys residues of the completed-
peptide-product of the formula H-D-B-Nal-Cys(Acm)—Tyr-D—Trp—Lys-
Va1—Cys(Acm)—Thr—NH2 by reacting H-D-B-Nal-Cys(Acm)-Tyr-D-Trp-
Lys-Val-Cys(Acm)-Thr-NH; with a solution of iodine in an alcohol until
the deprotecting and cyclizing is substantially complete to yield H-D—B—
Na]-[Cys—Tyr—D-Trp-Lys—Val-Cys]-Thr—NH2.
A preferred method of this invention is where the peptide is H-D-
B-Nal-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2.
A preferred method of the immediately foregoing method is where
the peptide is a somatostatin analog.
Definitions of terms used in the description of the present
invention are as follows:
“first amino acid”: encompasses any amino acid having its non-side
chain amino group protected by Boc, which are commercially available
or can be synthesized according to methods known to one of ordinary
skill in the art, e.g., Boc-L-Thr;
“first—coupled—product”: describes the product, which is attached to the
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solid support resin, resulting from the coupling of a first amino acid to
the solid support resin, e.g. Boc-L-Thr—resin;
“tirst—deblocked—c.oupled-product”: describes the product resulting from
the removal or deblocking of the Boo group from the first-coup1ed-
product, e.g., H-L-Thr-resin, where the “H” represents the available
hydrogen of the non-side chain amino group resulting from the
deblocking step;
“next-amino-acid”: describes any amino acid having its non-side chain
amino group protected by Boc or Fmoc, which are commercially
available or can be synthesized according to methods known to one of
ordinary skill in the art. Since step (c) and step (f) can be in a repeating
cycle wherein the step is carried out more than once, each time step (c)
or step (f) is carried out a next-amino—acid can be independently selected
from the group of known and synthesizable amino acids having its non-
side chain amino group protected by Boc or Fmoc, respectively;
“(X + 1)—next-blocked-coupled-product”: describes the product, which is
attached to the solid support resin, resulting from the coupling of a next-
amino-acid with the next-deblocked-coupled-product. Since steps (c) and
(d) and steps (f) and (g) can be in a repeating cycle wherein additional
ncxt—amino—acids can be coupled, the term (X + 1)-next-blocked
coupled—product is meant to represent the product resulting from each of
the previous cycle of coupling;
“(X + 1)-next—deblocked-coupled—product”: describes the product
resulting from the deblocking of the Fmoc group from the (X + l)-next-
blocked-coupled—product;
“completed-peptide—resin-product”: describes the peptide product, which
is attached to the solid support resin, after the N-terminal amino acid has
H 70587
been attached to the peptide chain and after the N-terminal amino acid’s
non—side chain amino group has been removed or deblocked but which
still has any of the side chain functionality protecting groups which were
not removed by the reaction to deblock the N-terminal amino acids non-
side chain blocking group; and
“deprotected-completed-peptide-resin—product”: describes the peptide
product, which is attached to the solid support resin, wherein any
protecting groups of the amino acids’ side chain functionalities have
been removed or deprotected.
Examples of acids that can be used to deblock Boc are
trifluoroacetic acid (TFA), methane sulfonic acid, and organic solutions
containing HCI.
Examples of primary and secondary amines that can be used to
deblock Fmoc are 4-(aminomethyl)piperidine, piperidine, diethylamine,
DBU and tris(2-aminoethyl)amine.
Examples of non-nucleophilic bases that can be used for
neutralizing the TFA salts of the freed amino group (RNH3+CF3COO',
these salts must be converted to the “free” amine (NH2) before or during
coupling of the next amino acid or the coupling will not work) are
diisopropylethylamine (DIEA) and triethylamine (TEA).
Examples of organic solvents that can be used for the amino acid
coupling reactions are methylene chloride, chloroform, dichloroethane,
dimethylformamide, dimethylacetamide, tetrahydrofuran, ethyl acetate,
l—methyl—2-pyrrolidinone, acetonitrile, or a combination of the above
solvents.
Examples of peptide coupling agents include substituted
carbodiimides such as
diisopropylcarbodiimide,
I2 070587
dicyclohexylcarbodiimide and N-ethyl-N‘-(3—dimethyl—aminopropyl)
carbodiimide.
The carboxyl and amino groups -that participate in the formation of
the peptide amide bond are called “non-side chain” carboxyl group or
amino group, respectively. On the other hand, any functional groups of
an amino acid which are not involved in formation of a peptide amide
bond are called “side chain” functionalities.
The term "base-stable group" refers to protecting groups used to
protect functionalities of the amino acids which (1) are base stable, e.g.,
cannot be removed by bases, such as 4-aminoethyl—piperidine,
piperidine, or tris—(2-aminoethyl)amine, which are bases that are
typically used to remove the protecting group Fmoc, and (2) can be
removed by an acid, such as trifluoroacetic acid, or by other means, such
as catalytic hydrogenation.
The symbol "Fmoc" and "Boc“ are used herein and in the
appended claims to mean 9—fluorenyl methoxycarbonyl and t-
butyloxycarbonyl, respectively.
The above-described method can be used to prepare peptides,
preferably somatostatin analogs, such as the octapeptide Lanreotide®,
which has the following formula H-D-B-Na]-[Cys-Tyr-D-Trp-Lys-Vah
Cys]-Thr—NH2. When H-D-B—Na1—[Cys-Tyr-D-T1-p-Lys—Val—Cys]—Thr—
NH2 is to be synthesized, the base-stable protecting groups used to block
the side chain functionalities of Cys Lys, and Tyr can be
acetamidomethyl (Acm), Boc, and t-butyl, respectively. Acm is preferred
for Cys.
What is meant by a "somatostatin" analog is a peptide which
exhibits biological activity similar (i.e., agonist) or opposite (i.e.,
antagonist) to that of somatostatin.
In the formula H—D—B—Nal—[Cys—Tyr-D-Trp-Lys-Val-Cys]-Thr-
NH2, each of the conventional three-letter amino acid symbols (e.g., Lys)
represents a structural residue of an amino acid. For example, the symbol
Lys in the above formula represents -NH-Cl-l((CH2)4NH2)-CO-. The
symbol D-[3-Nal represents the amino acid residue Dnaphthylalaninyl.
The brackets represent a disulfide bond attaching the free thiols of the
two Cys residues of the peptide, indicating that the amino acids of the
peptide within the brackets is cyclic.
Detailed Description Of The Invention
One skilled in the art can, based on the description herein, utilize
the present invention to its fullest extent.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Also, all
publications, patent applications, patents, and other references mentioned
herein are incorporated by reference.
A peptide can be made according to a method of the present
invention according to the following procedure.
A solution of 0.5 molar equivalents of cesium carbonate in water
is slowly added to a solution of 1 molar equivalents of a Boc-AA‘
(Bachem California, Torrance, CA), wherein AA] corresponds to the C-
terminal amino acid, dissolved in an alcohol, preferably methanol. The
resulting mixture is stirred for about 1 hour at room temperature, and
then all of the alcohol and water are removed under reduced pressure
M 070687
yielding a dry powder of the cesium salt of Boc—AA1. Merrifield resin,
1.0 equivalent, (chloromethylated polystyrene; 200-400 mesh. chloride
incorporation of 1.3 meq/gram, Advanced ChemTech, Louisville,
Kentucky or Polymer Laboratories, Church Stretton, England) is rinsed
with a chlorinated solvent, preferably dichloromethane (DCM), an
alcohol, preferably methanol, and a polar aprotic solvent, preferably
dimethylformamide (DMF). The Boc—AAl cesium salt powder is
dissolved in a dry polar aprotic solvent, preferably DMF, and the
solution is combined with the above washed resin. The slurry is gently
mixed at about 45°-65° C, preferably 50°—60° C, for about 48 to 106
hours, preferably 85 to 90 hours under an inert atmosphere such as
nitrogen. The resin is filtered and rinsed well with a polar aprotic
solvent, preferably DMF, water, and finally an alcohol, such as MeOH.
The Boc-AA‘-resin is dried under reduced pressure.
The Boc—AA‘-resin is added to a glass reactor with a coarse
sintered glass filter bottom. The resin is rinsed with a chlorinated
solvent, such as DCM, deblocked with an organic acid, preferably 25%
TFA/DCM, rinsed briefly with a chlorinated solvent, such as DCM, and
an alcohol, such as MeOH, is neutralized with an organic base,
preferably triethylamine in DCM, and rinsed again with DCM and a
polar aprotic solvent, such as DMF, to yield the deblocked AA‘-resin.
The deblocked AA1—resin is then optionally coupled with any
desired number of amino acids. If a subsequent amino acid is Fmoc
protected on the on-amino group (Fmoc-AA‘), then the side-chain group
must either not require protection (such as Fmoc—Gly, Fmoc-Ala, Fmoc-
Phe or Fmoc-Threonine) or the side chain must be protected by a group
that is resistant to removal base. A molar excess of the Fmoc-AA‘
(where x is the sequence number of the amino acid in the peptide,
counted from the C-terminal) is coupled with the deblocked AAi—resin
using a peptide coupling reagent such as diisopropylcarbodiimide (DIC)
in a mixture of DCM/DMF for about 60 minutes. The coupled resin is
rinsed with DIVE, alcohol and DCM to yield Fmoc-AA‘-AA‘-resin. The
coupling can be checked by Kaiser ninhydrin method. The Fmoc-AA"-
AA‘-resin is then rinsed once with DMF and then deblocked with a
solution of a base in an organic solvent such as piperidine in DMF to
yield AA‘-AA‘-resin. The AA"—AA'—resin is then rinsed with DMF and
then several times with both an alcohol, such as MeOH, and DCM. The
AA"—AA1-resin is rinsed once for about 3 minutes with DMF, three
times, preferably for about 2 minutes each with isopropanol (IPA), and
three times, preferably for about 2 minutes each with DCM. The resin is
then ready for further coupling with either an Fmoc protected amino acid
as described above, or a Boc amino acid as described below.
Similarly, if any subsequent amino acid which is to be coupled to
the deprotected AA‘-resin is chosen with Boc protection on the or-amino
group (Boc—AA"), then the side-chain group also must either not require
protection (such as Boc—Gly, Boc-Ala, Boc-Phe or Boc~Threonine) or the
side chain must be protected by a group that is resistant to removal by
both acid and base such as Boc-Cys(S—Acm). A Boc-AA‘, if selected, is
coupled with the same reagents and solvents as the Fmoc amino acids
described hereinabove and can be checked for coupling completion by
the Kaiser ninhydrin method. The Boc—AA"-AA‘-resin is then deblocked
with a solution of an acid in an organic solvent such as TFA in DCM to
yield CF3CO' H+AA"—AA1-resin. This resin is then rinsed with
chlorinated solvents, such as DCM, and alcohol, such as MeOH, several
687
times and neutralized with a non-nucleophilic base, such as
triethylamine, in DCM and then rinsed several times more with a
chlorinated solvent, such as DCM, to yield AA‘-AA‘-resin. The resin is
then ready for further coupling with either a Boc or an Fmoc protected
amino acid as described above.
Depending on the desired peptide sequence and the type of ot-
amino blocked amino acid used, whether Fmoc protected or Boc
protected, the appropriate combination of the foregoing coupling
procedures are executed until an amino acid is required in the sequence
with a side chain having a protecting group which can be removed by
either the base used to deblock the Fmoc on the on-amino group or by the
acid used to deblock the Boc on the on-amino group. Such a protected
amino acid may be N—ot—Boc-N’-e-Fmoc-Lysine or N—ot—Fmoc—N’—e-Boc-
Lysine. Once this occurs, all of the subsequent amino acids’ oc-amino
blocking groups chosen must be compatible with the side group
protection chosen for that position until the N-terrninal amino acid. That
is, the side chain protecting groups must be stable against the deblocking
agent used to deblock the subsequent ot—amino blocking group. For the
N-terminal amino acid, either a Boc or an Fmoc can be used as the OL-
amino blocking group since the deblocking of the N-terminal amino acid
can simultaneously deprotect certain of the protected side chains without
an adverse affect on the synthesis strategy of the peptide because no
further amino acids will be added.
The completed peptide chain, which is still attached to the resin,
must then be deprotected and deblocked. To remove any base stable
protecting groups and ot—amino blocking group of the N—temiina1 amino
acid, if applicable, the peptide-resin is treated with an acid in an organic
W 070687
solvent, such as TFA in DCM. To remove any acid stable protecting
groups and oL—amino blocking group of the N—terminal amino acid, if
applicable, the peptide-resin is treated with an organic base, such as
piperidine in DMF. Or the acid stable groups can be left on for removal
in the subsequent cleavage of the peptide by ammonia or an amine base.
The deprotected peptide-resin is then rinsed with a chlorinated solvent,
such as DCM, and an alcohol, such as MeOH, and is dried to constant
weight under reduced pressure.
The peptide is cleaved from the resin and the C—terminal converted
to an amide by suspending the peptide-resin in 3:1 MeOH/DMF. The
suspension is cooled to about <10° C under nitrogen, and anhydrous
ammonia gas is added below the solvent surface until the solution is
saturated, while the temperature is maintained at below about 10° C. The
slurry is gently mixed for about 24 hours while allowing the temperature
to increase to about 20° C. The reaction is checked for completion by
monitoring the disappearance of the methyl ester intermediate by HPLC
under appropriate conditions depending on the peptide. The reaction is
cooled, and more anhydrous ammonia is added, as needed, until the area
of the methyl ester is less than 10% of the area of the desired product
peak on the HPLC. The slurry is cooled to about less than 10° C, and
mixing is continued overnight to allow the peptide to precipitate. The
precipitate and resin are filtered and rinsed with cold MeOH. The
precipitate and resin are dried under reduced pressure, and the product is
extracted from the resin with aqueous acetic acid.
If a peptide contains protected Cys residues within its sequence,
the thiol groups of the Cys can be deprotected and cyclized according to
the following procedure. The peptide having Acm-protected Cys groups
w 07068;
is dissolved in aqueous acetic acid under nitrogen atmosphere. The
solution is stirred rapidly, and a solution of iodine in alcohol is added in
one portion. The mixture is stirred and tested by HPLC for completion of
deprotection and then quenched by titration with a 2% sodium
thiosulfate solution to a colorless end-point. The crude mixture is
purified by preparative chromatography on C8 packing with a 0.1
ammonium acetate/acetonitrile gradient buffer, desalted on C8 packing
with a 0.25N acetic acid/acetonitrile gradient, and lyophilized to give the
desired peptide.
The following example is provided to illustrate a method of the
present invention and is not to be construed as limiting the scope thereof.
Example 1
H2-D-B-Nab[Cvs-Tyr-D-Trp—Lvs-Val-Cvs]-Th r-N112
A) Boc-L-Thr-Resin
A solution of 2.58 grams of cesium carbonate in 2.5 ml of water
was slowly added to a solution of 3.48 grams of Boc—L—Threonine
(Bachem California, Torrance, CA) dissolved in 7 ml of methanol. The
resulting mixture was stirred for about 1 hour at room temperature, and
then all methanol and water were removed under reduced pressure
yielding a dry powder of cesium salt of Boc-L-Threonine. 10 grams of
Merrifield resin (chloromethylated polystyrene; 200-400 mesh, chloride
incorporation of 1.3 meq/gram, Advanced ChemTech, Louisville,
Kentucky) was rinsed with dichloromethane (DCM), methanol (MeOH),
and dimethylfonnamide (Dlvfi?) (2 x 70 ml each). The Boc—L-Threonine
cesium salt powder was dissolved in 60 ml of dry DMF, and the solution
was combined with the above washed resin. The slurry was gently
mixed at about 50°-60°C for about 85 to 90 hours under nitrogen
atmosphere. The resin was filtered and rinsed well with DMF, deionized
water, and finally MeOH. The Boc—Threonine resin was dried under
reduced pressure at about 40°C (Threonine incorporation = 0.85i0.l5
meq/gram of dry resin).
B) H-D-B-Nal-Cys(Acm)—Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-
Resin
.0 grams of Boc-Threonine resin from step A was added to a 50
ml glass reactor with a coarse sintered glass filter bottom (batch scale =
1.74 mmole). The resin was rinsed two times for about 5 minutes each
with DCM (20 ml), deblocked with 25% TFA/DCM (30 ml) once for
about 2 minutes and once for about 25 minutes, rinsed three times for
about 2 minutes with DCM (20 ml), isopropanol (IPA) (20 ml), and
DCM (20 ml), neutralized two times for about 5 minutes with 10%
triethylamine/DCM (20 ml), rinsed three times for about 2 minutes with
DCM, and rinsed once for about 5 minutes with DMF (20 ml).
The deblocked resin was coupled with 1.8 grams (4.35 mole, 2.5
eq.) of Fmoc-L-Cysteine(Acm) (Bachem, CA) and 683 pl (4.35 mmole,
2.5 eq) diisopropylcarbodiimide (DIC) in 14 ml of 2:1 DCM/DMF for
about 1 hour. The coupled resin was rinsed once for about 3 minutes
with DMF (20 ml), three times for about 2 minutes with isopropanol
(IPA), and three times for about 2 minutes with DCM (20 ml). The
coupling was checked by Kaiser ninhydrin method.
The coupled resin was then rinsed once with DMF and then
m 070587
deblocked with a solution of piperidine in DMF. The deblocked coupled
resin was then rinsed with DMF and several times with both MeOH and
DCM. The coupled resin was rinsed once for about 3 minutes with DMF
(20 ml), three times for about 2 minutes with isopropanol (IPA)(20 ml),
and three times for about 2 minutes each with DCM (20 ml). The
coupling was checked by Kaiser ninhydrin method.
Each of the following protected amino acids were coupled with the
rinsed resin using DIC in DMF/DCM and deblocked in the following
order as described above: Fmoc-L-Valine, Fmoc-L-Lysine(Boc), Fmoc-
D—Tryptophan, Fmoc-L—Tyrosine(O-t-Bu), and Fmoc-L-Cysteine(Acm)
(all from Bachem California), Boc-DNaphthylalanine (Synthetech,
Albany, OR).
The completed peptide chain was deblocked and deprotected twice
with 75:20:5 DCM/TFA/anisole (30 ml) for about 2 minutes and about
minutes, rinsed three times for about 2 minutes each with DCM (20
ml), IPA (10 ml), and DCM (20 ml), neutralized two times for about 5
minutes with 10% triethylamine/DCM (20 ml), and rinsed three times for
about 2 minutes with DCM (20 ml) and MeOH (20 ml). The resin was
dried under reduced pressure. Dry weight = 3.91 grams (103% of
theory).
C) H-D-B-Nal-Cys-(Acm)-Tyr-D-Trp-Lys—Val-Cys(Acm)-Thr-NH;
.93 grams of the peptide loaded resin from step B (1.3 mmoles
eq.) was suspended in 50 ml of 3:1 MeOH/DMF. The suspension was
cooled to about <10° C under nitrogen, and anhydrous ammonia gas was
bubbled until saturated while the temperature was maintained at below
about 10° C. The slurry was gently mixed for about 24 hours while
allowing the temperature to increase to about 20° C. The reaction was
checked for completion by monitoring the disappearance of the methyl
ester intermediate by HPLC (Rt. ~ 14 minutes for methyl ester vs. Rt.
~9.3 minutes for amide product on VYDAC®, 5p 100 A, C18 with 26%
CH3CN/0.1% TFA Isocratic, 1 ml/min, 220 nm). The reaction was
cooled, and more anhydrous ammonia was added until the area of the
methyl ester was less than 10% the area of the product peak on the
HPLC. The slurry was cooled to about less than 10° C, and mixing was
continued overnight to allow the peptide to precipitate. The precipitate
and resin were filtered and rinsed with 15 ml of cold MeOH. The
precipitate and resin were dried under reduced pressure, and the product
was extracted from the resin with 50% aqueous acetic acid (3 x 30 ml
portions). HPLC analysis showed 870 mg (0.70 moles) of the title
product present in the mixture (96% purity on Isocratic HPLC system).
D) H-D-B-Nal-[Cys-Tyr-D—Trp-Lys-Val-Cys]-Thr-NH;
mg (0.40 mmoles) of the peptide from step C was dissolved in
300 ml of 4% acetic acid and heated to about 55° C under nitrogen
atmosphere. The solution was stirred rapidly, and a 2% w/v solution of
iodine in 7.7 ml of MeOH (0.60 mmoles) was added in one portion. The
mixture was stirred for about 15 minutes and then quenched by titration
with a 2% sodium thiosulfate solution to a colorless end-point (~2 ml).
The mixture was cooled to room temperature and filtered. The crude
mixture was purified by preparative chromatography on C8 packing
(YMC, lnc., Wilmington, NC) with a 0.1 ammonium acetate/acetonitrile
gradient buffer, desalted on YMC C8 packing with a 0.25N acetic
acid/acetonitrile gradient, and lyophilized to give 350 mg of the desired
peptide at 99% purity.
OTHER EMBODIMENTS
From the above description, one skilled in the art can easily
ascertain the essential characteristics of the present invention, and
without departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt it to various uses
and conditions. Thus, other embodiments are also within the claims.
Claims (5)
1. A method for preparing a peptide comprising three or more amino acid residues having an N-tenninal amino acid, a next to last amino acid adjacent to the N-terminal amino acid and a C-terminal amino acid, wherein said method comprises the steps of: (a) attaching a first amino acid to a solid support resin through an ester bond to form a first-coupled-product, which comprises (i) reacting an aqueous solution of cesium carbonate with an alcohol solution of the first amino acid to form a cesium salt of the first amino acid, (ii) obtaining a solvent free cesium salt of the first amino acid, (iii) reacting the solid support resin with the cesium salt of the first amino acid in a dry polar aprotic solvent to form a first—coupled product; wherein the first amino acid corresponds to the C—terminal amino acid of the peptide, the first amino acid’s non-side chain amino group is blocked by a Boc and the first amino acid does not have a side chain functionality requiring protection, and the solid support resin is a chloromethylated polystyrene resin; (b) deblocking the Boo from the first-coupled-product with an acid to form a first-deblocked-coupled-product; (c) optionally coupling a next-amino-acid to the first-deblocked- coupled-product, which comprises reacting the next-amino-acid with the first-deblocked-coupled-product in an organic solvent comprising a peptide coupling reagent to form a next-blocked-coupled-product, wherein the next-amino-acid has a non-side chain amino group blocked by Boc and if the next-amino-acid has one or more side chain functionalities then the side chain fiinctionalities do not require protection or the side chain functionalities have protecting groups that are stable to acid and base reagents used to deblock Boc and Fmoc, respectively; (d) deblocking the Boo from the next—blocked-coupled-product which comprises reacting the next-blocked-coupled-product with a an acid to yield a next-deblocked-coup]ed-product; (e) optionally repeating steps (c) and (d), each cycle forming an (X + 1)-next-deblocked-coupled—product where X is the number of desired cycle repetitions; (0 coupling a next—amino—acid to the first-deblocked-coupled product from (b), or optionally to the (X + 1)-next-deblocked—coup1ed— product from (e), which comprises reacting the next—amino—acid with said first-deblocked-coupled-product or said (X + 1)-next—deblocked- coupled-product in an organic solvent comprising a peptide coupling reagent to form a next-blocked-coupled-product, wherein the next- amino-acid has a non-side chain amino group blocked by Fmoc, provided that if the next—amino—acid has one or more side chain functionalities then the side chain functionalities do not require protection or the side chain functionalities have protecting groups that are stable to base reagents used to deblock Fmoc; (g) deblocking the Fmoc from the next-b1ocked-coupled-product which comprises reacting the next-blocked-coupled-product with a primary or secondary amine to yield a next—deblocked-coupled—product; (h) optionally repeating steps (f) and (g), each cycle forming an (X + 1)-next-deblocked-coupled-product where X is the desired number of cycle repetitions, until the next to last amino acid of the peptide is coupled and deblocked; (i) coupling an N-terminal amino acid to the (X + l)-next- deblocked—coupled—product, which comprises reacting the N-terminal amino acid with the (X + I)—next-deb1ocked-coupled-product in an organic solvent comprising a peptide coupling reagent to form a comp1eted-blocked-coupled-product, wherein the N-terminal-amino-acid has a non—side chain amino group blocked by Boc or Fmoc; (j) deblocking the Boc or Fmoc group from the completed- blocked-coupled-product, which comprises reacting the completed- blocked-coupled-product with an acid in the case of Boc, or a base in the case Fmoc, to form a completed—peptide-resin-product; (k) if side-chain functionalities are present on the completed- peptide-resin-product then optionally deprotecting the side-chain functionalities of the completed-peptide-resin-product, which comprises reacting the completed-peptide-resin—product with the appropriate deprotecting reagents to form a deprotected-completed-peptide-resin- product; and m 070337 (1) cleaving the peptide from the solid support resin of the comp1eted—peptide—resin—product or the deprotected-completed—peptide- resin-product to yield the peptide, which comprises reacting the completed-peptide-resin-product or the deprotected-completed-peptide- resin—product with ammonia, a primary amine or a secondary amine until the cleavage of the peptide from the resin is substantially complete; provided that steps (f) and (g) must be carried out at least once in the synthesis of the peptide.
2. A method according to claim 1, wherein step (I) further comprises the steps of: (i) precipitating the cleaved peptide from the solvent; (ii) filtering the solid support resin and the precipitated peptide; and (iii) extracting the peptide in an acid solution to isolate the peptide.
3. A method according to claim 1, wherein the first amino acid is Boc—L—Thr- cesium salt yielding Boc-L-Thr-resin as the first-coupled- product and H-L—Thr—resin is the first-deblocked—coupled-product, the acid used to deblock the Boo group in step (j) is TFA and the organic solvent is methylene chloride, chloroform, or dimethylformamide and the peptide coupling reagent is diisopropylcarbodiimide, dicyclohexylcarbodiimide, or N-ethyl—N'—(3-dimethyl—aminopropyl) carbodiimide. 0706
4. A method according to claim 1, wherein the peptide is H-D- [3-Nal—[Cys—Tyr-D—Trp—Lys—Val—Cys]—Thr-NH2, a somatostatin analog.
5. A method according to claim 1 for preparing a peptide comprising three or more amino acid residues having an N-terminal amino acid, a next to last amino acid adjacent to the N-terminal amino acid and a C—terminal amino acid substantially as hereinbefore described and exemplified.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IEIRELAND21/11/20062006/0841 |
Publications (2)
Publication Number | Publication Date |
---|---|
IE20070687U1 true IE20070687U1 (en) | 2008-05-14 |
IES84882Y1 IES84882Y1 (en) | 2008-05-14 |
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